Carbonic anhydrase inhibitors: 2-Substituted-1,3,4-thiadiazole-5-sulfamides act as powerful and selective inhibitors of the mitochondrial isozymes VA and VB over the cytosolic and membrane-associated carbonic anhydrases I, II and IV

Article abstract of DOI:10.1016/j.bmcl.2008.10.093

A series of 2-substituted-1,3,4-thiadiazole-5-sulfamides was prepared and assayed as inhibitors of several carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, the membrane-associated CA IV and the mitochondrial CA VA and VB. The new compounds showed weak inhibitory activity against hCA I (K_Is of 102 nM-7.42 μM), hCA II (K_Is of 0.54-7.42 μM) and hCA IV (K_Is of 4.32-10.05 μM) but were low nanomolar inhibitors of hCA VA and hCA VB, with inhibition constants in the range of 4.2-32 nM and 1.3-74 nM, respectively. Furthermore, the selectivity ratios for inhibiting the mitochondrial enzymes over CA II were in the range of 67.5-415, making these sulfamides the first selective CA VA/VB inhibitors.

Full text of DOI:10.1016/j.bmcl.2008.10.093

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6332–6335
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Carbonic anhydrase inhibitors: 2-Substituted-1,3,4-thiadiazole-5-sulfamides
act as powerful and selective inhibitors of the mitochondrial isozymes VA and
VB over the cytosolic and membrane-associated carbonic anhydrases I, II and IV
Fatma-Zohra Smaine a, Fabio Pacchiano b, Marouan Rami a, Véronique Barragan-Montero a, Daniela Vullo b,
b,
Andrea Scozzafava b, Jean-Yves Winum a, , Claudiu T. Supuran
*
*
a Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CNRS-UM1-UM2 Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier,
8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France
b Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-substituted-1,3,4-thiadiazole-5-sulfamides was prepared and assayed as inhibitors of sev-
eral carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, the membrane-associated
CA IV and the mitochondrial CA VA and VB. The new compounds showed weak inhibitory activity against
Received 1 October 2008
Revised 20 October 2008
Accepted 20 October 2008
Available online 1 November 2008
hCA I (KIs of 102 nM–7.42 lM), hCA II (KIs of 0.54–7.42 lM) and hCA IV (KIs of 4.32–10.05 lM) but were
low nanomolar inhibitors of hCA VA and hCA VB, with inhibition constants in the range of 4.2–32 nM and
1.3–74 nM, respectively. Furthermore, the selectivity ratios for inhibiting the mitochondrial enzymes
over CA II were in the range of 67.5–415, making these sulfamides the first selective CA VA/VB inhibitors.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Carbonic anhydrase
Sulfamide
Isozyme VA and VB
Mitochondria
Isozyme-selective inhibitor
Among the sixteen
a
-carbonic anhydrase (CA, EC 4.2.1.1) iso-
acetate is unable to cross the mitochondrial membrane, its decar-
boxylation regenerates pyruvate which can be then transported
into the mitochondria by means of the pyruvate transporter
(Fig. 1). The acetyl-CoA 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-CoA, the conver-
sion between CO2 and bicarbonate being assisted by CA II. Subse-
quent steps involving the sequential transfer of acetyl groups
lead to longer chain fatty acids.1–5 Therefore, several CA isozymes
are critical to the entire process of fatty acid biosynthesis: VA
and/or VB within the mitochondria (to provide enough substrate
to PC), and CA II within the cytosol (for providing sufficient sub-
strate to ACC). Inhibition of CAs by clinically used sulfonamides
such as acetazolamide AZA, zonisamide ZNS or the sulfamate topi-
ramate, TPM, can decrease lipogenesis in adipocytes in cell cul-
ture.1–3 Furthermore, among some of the side effects of these
drugs is also the weight loss,6 which may in fact lead to novel anti-
obesity therapies.7
forms found in animals, two CA isozymes, VA and VB, are present
in mitochondria.1–3 These isozymes are involved in several biosyn-
thetic processes, such as ureagenesis, gluconeogenesis, and lipo-
genesis, both in vertebrates as well as in invertebrates.1,4,5 The
provision of enough of the substrate, bicarbonate, in several bio-
synthetic processes involving pyruvate carboxylase (PC), acetyl-
CoA carboxylase (ACC), and carbamoyl phosphate synthetases I
and II, is assured mainly by the catalysis involving the mitochon-
drial isozymes CA VA and CA VB, probably assisted by the high
activity cytosolic isozyme CA II, as shown schematically in Figure
1.1,2 CAs thus play a key role in fatty acid biosynthesis. Mitochon-
drial pyruvate carboxylase (PC) is needed for efflux of acetyl groups
from the mitochondria to the cytosol where fatty acid biosynthesis
takes place.1 Pyruvate is carboxylated to oxaloacetate in the pres-
ence of bicarbonate and under the catalytic influence of the mito-
chondrial isozymes CA VA and/or CA VB. The mitochondrial
membrane is impermeable to acetyl-CoA which reacts with oxalo-
acetate, leading to citrate, which is thereafter translocated to the
cytoplasm by means of the tricarboxylic acid transporter. As oxalo-
In previous contributions from our laboratories8–10 we have shown
that both CA VA and CA VB are druggable targets. Rather large
libraries of various sulfonamides and sulfamates have been as-
sayed as inhibitors of these mitochondrial enzymes, with several
low nanomolar inhibitors being detected.8–10 However, an impor-
tant drawback of most of these compounds is represented by the
rather low selectivity for inhibiting the mitochondrial CAs over
* Corresponding authors. Tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.
Supuran).
E-mail addresses: jean-yves.winum@univ-montp2.fr (J.-Y. Winum), claudiu.
supuran@unifi.it (C.T. Supuran).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.093

F.-Z. Smaine et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6332–6335
6333
O
S
NH2
N
N
SO2NH2
O
O
O
O
CH3CONH
SO2NH2
N
S
O
O
O
ZNS
AZA
O
TPM
the cytosolic/membrane-bound ubiquitous isoforms such as CA I, II
(cytosolic) and CA IV (extracellular, membrane-associated iso-
zyme).1,8–10 Considering the interest in CA VA/VB—selective inhib-
itors which might be developed as antiobesity agents, we explore
here a less investigated class of CA inhibitors (CAIs) for obtaining
compounds targeting the mitochondrial CAs, that is, the sulfa-
mides.11,12 Indeed, aromatic, heterocyclic and sugar sulfamides
have been reported earlier to generate effective inhibitors of sev-
eral CA isozymes (mainly CA I, II and IX),11,12 and the X-ray crystal
structures for two such compounds complexed within the CA II ac-
tive site are also available,12c,12d proving the sulfamide moiety to
be an effective zinc binding group for obtaining CAIs, similarly to
the bioisosteric sulfonamide and sulfamate ones.11
Here we report the synthesis of 1,3,4-thidiazole sulfamides pos-
sessing various 2-substitutents.13,14 This scaffold has been chosen
as it is present in one of the most investigated and powerful CAI,
acetazolamide AZA, used clinically since 1956,1 and also because
its binding to the enzyme is effective, as shown for many AZA
derivatives for which the X-ray crystal structure has been resolved
in adduct with different CA isoforms15 (Scheme 1).
N
N
N
N
i
R
NHSO2NH2
R
NH2
ii
S
S
1a-1j
2a-2j
Scheme 1. Synthesis of 2-substituted-1,3,4-thiadiazole-5-sulfamide 2a–2j:
Reagents and conditions: (i) t-BuOH, ClSO2NCO, NEt3, CH2Cl2. (ii) TFA 95% in CH2Cl2.
class of CAIs. Thus, starting with the unsubstituted parent com-
pound (R = H, 2a), both aliphatic (Et, t-Bu, etc), aromatic (Ph,
substituted phenyl, etc) as well as sulfide- and sulfone incorporat-
ing such moieties have been introduced in this position. X-ray
crystallographic data on many CA—sulfonamide/sulfamate/sulfam-
ide adducts revealed that in addition to the zinc binding group (a
sulfamide one for derivatives 2) and organic scaffold (1,3,4-thiadi-
azole for 2), the tails present in CAIs are critical both for the
isozyme inhibition profile as well as for modulationg the phys-
ico-chemical properties of such inhibitors, which may thus lead
to various pharmacological applications.1,15–18 This explains the
choice of the tail groups present in derivatives 2a–j reported here.
Inhibition data against five physiologically relevant CA iso-
zymes, that is, the cytosolic hCA (human CA) I and II, the mem-
brane-associated hCA IV as well as the two mitochondrial
isoforms hCA VA and VB, are presented in Table 1.19 Three standard
CAIs, that is, AZA, ZNS and TPM, have been assayed in the same
conditions in order to allow a better understanding of the inhibi-
Starting with the commercially available 2-substituted-5-ami-
no-1,3,4-thiadiazoles 1, which have been sulfamoylated with
in situ generated sulfamoyl chloride, by a method reported earlier
by one of this groups,14 the sulfamides 2a–j have been prepared
with excellent yields.13 The various substituents in position 2 of
the heterocyclic ring of the new compounds 2a–j were chosen in
such a way as to have a rather comprehensive SAR insight for this
Figure 1. The transfer of acetyl groups from the mitochondrion to the cytosol (as citrate) for the provision of substrate for de novo lipogenesis.1 All steps involving bicarbonate
need the presence of at least two CA isozymes: CA VA/VB in the mitochondrion and CA II in the cytosol (see discussion in the text).

6334
F.-Z. Smaine et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6332–6335
Table 1
against hCA VB, respectively, Table 1). For hCA VA, the sub-
stitution patterns of the heterocyclic ring leading to the best
inhibitors included the trifluoromethyl, thioethyl-, aryl and
methylsulfonyl moieties (KIs < 10 nM) whereas the remain-
ing ones generated slightly less effective inhibitors. For
hCA VB, only three compounds (2a, 2b and 2f) showed
KIs > 10 nM, all the other substitution patterns leading to
derivatives with excellent activity (KIs < 7.5 nM). These are
in fact the compounds with the best inhibitory activity ever
reported against the mitochondrial enzymes CA VA and CA
VB.
Inhibition data of human CA isozyms I, II (cytosolic), IV (membrane-associated) and
VA, and VB (mitochondrial) with compounds 2a–2j and standard inhibitors (aceta-
zolamide AZA, zonisamide ZNS and topiramate TPM), by
a stopped-flow, CO2
hydration assay.19
N
N
R
NHSO2NH2
2a-2j
S
.
No
R
KI*
hCA
Ia(
M)
hCA
l
hCA
IVb(
hCA
hCA
(v) Another very interesting property of the newly described
sulfamides 2 is related to their selective inhibition of the
mitochondrial isozymes (CA VA and VB) over the cytosolic
and membrane asociated isoforms (CA I, II and IV). Especially
CA II constitutes a problem when designing various CAIs tar-
geting other izoymes, because CA II has generally a very high
affinity for sulfonamides, sulfamates and sulfamides, and it
is also a ubiquitoous enzyme in vertebrates, including
humans.1–3 As seen from data of Table 1, the three clinically
used compounds mentioned here (but also the other CAIs in
clinical use)1 are generally much better CA II than CA VA/VB
inhibitors (except for zonisamide against CA VA). For exam-
ple the selectivity ratio of AZA for inhibiting CA VA over CA II
is of 0.19, and for inhibiting CA VB over CA II is of 0.22. Basi-
cally AZA has a much higher affinity for the cytosolic isoform
CA II than for the mitochondrial ones. However, all com-
pounds 2 reported here showed a much better inhibitory
activity against the mitochondrial isozymes than against
the cytosolic (or membrane-associated) ones. Thus, for
example, 2h has a selectivity ratio for inhibiting CA VA over
CA II of 67.5, and for inhibiting CA VB over CA II of 415. For
2i, these ratios are of 195 and of 182, respectively. Thus,
these compounds are 67.5–415-times better inhibitors of
the mitochondrial over the cytosolic isozymes, which is a
very interesting result, never evidenced earlier for any other
class of CAIs.
l
IIa(
M)
lM)
VAc(
lM)
VBc(
lM)
2a
2b
2c
2d
2e
2f
H
Et
t-Bu
CF3
MeS
EtS
7.42
7.01
5.54
6.86
2.40
1.89
1.66
0.102
0.97
0.95
0.90
1.08
0.92
1.13
0.87
0.54
8.91
28.3
18.7
10.4
7.3
32
9.3
74
63
8.64
8.40
4.32
8.30
7.58
5.54
8.76
2.8
3.9
2.9
23.1
7.5
1.3
2g
2h
Ph
4-
9.2
8.0
MeOC6H4
4-Br-
C6H4
MeSO2
–
2i
5.85
0.82
10.05
4.2
4.5
2j
0.103
0.250
0.056
0.250
0.94
6.10
0.074
8.59
4.90
8.7
63
20
2.7
54
6033
30
AZA
ZNS
TPM
0.012
0.035
0.010
–
–
63
*
Errors in the range of 5–10% of the shown data, from three different assays, by a
CO2 hydration stopped-flow assay.19
a
Human, recombinant isozymes.
Truncated cloned human isoform lacking the first 20 aminoterminal amino
b
acids.
Full length, recombinant human isoforms.8–10
c
tion profile of the new compounds 2a–2j investigated here. Data of
Table 1 allow the following SAR to be evidenced for the new sulfa-
mides 2 reported here:
(i) Against hCA I, the sulfamides 2 showed a moderate-weak
inhibitory activity, with inhibition constants in the range
of 102 nM–7.42
l
M, bing thus, with two exceptions (com-
In conclusion, we prepared a small series of 2-substituted-1,3,4-
thiadiazole-5-sulfamides and assayed them for the inhibition of
five physiologically relevant isozymes, the cytosolic CA I and II,
the membrane-associated CA IV and the mitochondrial CA VA
and VB. The new compounds showed weak inhibitory activity
pounds 2h and 2j) much weaker inhibitors as compared to
the clinically used drugs AZA-TPM (KIs in the range of 56–
250 nM). The least active hCA I inhibitor was the parent,
unsubstituted compound 2a, whereas introduction of vari-
ous substituents in position
2
of the thiadiazole ring
against hCA I (KIs of 102 nM–7.42 M), hCA II (KIs of 0.54–
l
enhances activity. The groups leading to best activity were
4-methoxyphenyl and methylsulfonyl (2h and 2j).
(ii) Isozyme hCA II was also weakly inhibited by the new sulfa-
7.42 M) and hCA IV (KIs of 4.32–10.05 lM) but were low nanomo-
l
lar inhibitors of hCA VA and hCA VB, with inhibition constants in
the range of 4.2–32 nM and 1.3–74 nM, respectively. Furthermore,
the selectivity ratios for inhibiting the mitochondrial enzymes over
CA II were in the range of 67.5–415, making these sulfamides the
first selective CA VA/VB inhibitors.
mides 2, with KIs in the range of 0.54–1.13 lM, whereas the
clinically used drugs were much stronger inhibitors of this
ubiquitous isoform (KIs in the range of 10–35 nM). It may
be observed a very flat SAR for sulfamides 2 in inhibiting
hCA II, with the nature of groups substituting in 2 the thia-
diazole ring, having a small influence on the inhibitory
power (Table 1).
References and notes
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(iii) Sulfamides 2 act as quite weak inhibitors of the membrane-
associated isoforms hCA IV, with KIs in the range of 4.32–
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10.05
l
M, unlike AZA which is a strong inhibitor (KI of
M).
74 nM) but similarly to ZNS and TPM (KIs of 4.90–8.59
l
(iv) Excellent inhibitory properties were evidenced for deriva-
tives 2 against the two mitochondrial CA iozymes, hCA VA
and hCA VB. Indeed, these compounds showed KIs in the
range of 4.2–28.3 nM against hCA VA, and of 1.3–74 nM
against hCA VB, respectively. It may be observed that com-
pounds 2 are much better inhibitors of the mitochondrial
CAs as compared to the three clinically used drugs (KIs in
the range of 20–63 nM against hCA VA, and of 30–6033 nM
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NMR (400 MHz, DMSO-d6) d 7.72 (d, 2H), 7.06 (d, 2H), 6.82 (s, 2H), 3.81 (s, 3H),
3.16 (s, 1H). 2i: mp 173–176 °C; MS (ESI+, 20 eV): m/z 359.06 [M+Na]+; 1H NMR
(400 MHz, DMSO-d6) d 7.74 (m, 4H), 6.89 (s, 2H), 3.16 (s, 1H). 2j: 154–156 °C;
MS (ESI+, 20 eV): m/z 281.15 [M+Na]+; 1H NMR (400 MHz, DMSO-d6) d 7.14 (s,
2H), 3.5 (s, 3H), 3.16 (s, 1H).
14. Winum, J. Y.; Toupet, L.; Barragan, V.; Dewynter, G.; Montero, J. L. Org. Lett.
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19. Khalifah, R.G. J. Biol. Chem., 1971, 246, 2561. An Applied Photophysics (Oxford,
UK) stopped-flow instrument has been used for assaying the CA catalysed
CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) has been
used as indicator, working at the absorbance maximum of 557 nm, with
10 mM Hepes (pH 7.5) as buffer, 0.1 M Na2SO4 (for maintaining constant the
ionic strength), following the CA-catalyzed CO2 hydration reaction.18 The CO2
concentrations ranged from 1.7 to 17 mM for the determination of the kinetic
parameters and inhibition constants. For each inhibitor at least six traces of
the initial 5–10% of the reaction have been used for determining the initial
velocity. The uncatalyzed rates were determined in the same manner and
subtracted from the total observed rates. Stock solutions of inhibitor (10 mM)
were prepared in distilled-deionized water with 10–20% (v/v) DMSO (which
is not inhibitory at these concentrations) and dilutions up to 0.1 nM were
done thereafter with distilled-deionized water. Inhibitor and enzyme
solutions were preincubated together for 15 min at room temperature prior
to assay, in order to allow for the formation of the E–I complex. The
inhibition constants were obtained by non-linear least-squares methods
using PRISM 3, and represent the mean from at least three different
determinations.
13. General procedure for the preparation of thiadiazole-sulfamides 2: To
solution of 2-substituted-5-amino-1,3,4-thiadiazole 1 in methylene chloride
and 1.1 equiv of triethylamine was added dropwise solution of tert-
a
a
butoxycarbonylamino sulfonyl chloride (prepared ab initio by reacting
1 equiv of tert-butanol and 1 equiv of chlorosulfonyl isocyanate in methylene
chloride at 0 °C).14 The mixture was stirred 1 h at room temperature, and then
concentrated under vacuum. The residue is purified on silica gel column
chromatography using ethyl acetate-petroleum ether 7–3 as eluent to give the
Boc-protected sulfamide in good yield (75–80%). This compound was then
deprotected using a solution of trifluoroacetic acid in methylene choride 50–50
v–v. 2a: mp 151–154 °C; MS (ESI+, 20eV): m/z 203.17 [M+Na]+, 383.10
[2M+Na]+ 1H NMR (400 MHz, DMSO-d6) d 8.63 (s, 1H), 6.77 (s, 2H), 3.16 (s,
1H). 2b: mp 100–110 °C; MS (ESI+, 20eV): m/z 231.1 [M+Na]+; 1H NMR
(400 MHz, DMSO-d6) d 6.71 (s, 2H), 3.16 (s, 1H), 2.78 (q, 2H), 1.2 (t, 3H).2c: mp
150–153 °C; MS (ESI+, 20eV): m/z 259.23 [M+Na]+; 1H NMR (400 MHz, DMSO-
d6) d 6.73 (s, 2H), 3.16 (s, 1H), 1.30 (s, 9H). 2d: mp 179-180°C; MS (ESI-, 20eV):
m/z 247.06 [MÀH]À; 1H NMR (400MHz, DMSO-d6) d 7.23 (s, 2H), 3.16 (s, 1H).
2e: mp 150–153 °C; MS (ESI+, 20 eV): m/z 249.09 [M+Na]+; 1H NMR (400 MHz,
DMSO-d6) d 6.85 (s, 2H), 3.16 (s, 1H), 2.61 (s, 3H). 2f: mp 145–148 °C; MS (ESI+,
20 eV): m/z 263.20 [M+Na]+; 1H NMR (400 MHz, DMSO-d6) d 6.86 (s, 2H), 3.16
(s, 1H), 3.12 (q, 2H), 1.31 (t, 3H). 2 g: mp 170–172 °C; MS (ESI+, 20 eV): m/z
279.15; [M+Na]+; 1H NMR (400 MHz, DMSO-d6) d 7.78 (s, 2H), 7.53 (s, 3H), 6.88
(s, 2H), 3.16 (s, 1H). 2h: 171–174 °C; MS (ESI+, 20 eV): m/z 309.17 [M+Na]+; 1
H