(R)-/(S)-10-Camphorsulfonyl-substituted aromatic/heterocyclic sulfonamides selectively inhibit mitochondrial over cytosolic carbonic anhydrases

Article abstract of DOI:10.1016/j.bmcl.2011.01.050

A series of aromatic and heterocyclic sulfonamides incorporating R- and S-camphorsulfonyl moieties were synthesized and investigated for the inhibition of several mammalian isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The new sulfonami

Full text of DOI:10.1016/j.bmcl.2011.01.050

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1334–1337
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
(R)-/(S)-10-Camphorsulfonyl-substituted aromatic/heterocyclic sulfonamides
selectively inhibit mitochondrial over cytosolic carbonic anhydrases
⇑
Alfonso Maresca, Claudiu T. Supuran
Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Firenze, 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 aromatic and heterocyclic sulfonamides incorporating R- and S-camphorsulfonyl moieties
were synthesized and investigated for the inhibition of several mammalian isoforms of the zinc enzyme
carbonic anhydrase (CA, EC 4.2.1.1). The new sulfonamides selectively inhibited the mitochondrial iso-
zymes hCA VA and VB (h = human isoform) over the cytosolic, off-target ones hCA I and II, with inhibition
constants in the low nanomolar range. The chirality and position of the groups substituting the sulfon-
amide scaffold greatly influenced CA inhibitory properties. These compounds are excellent leads for
designing isoform-selective enzyme inhibitors targeting mitochondrial CAs involved in lipogenesis and
obesity.
Received 1 December 2010
Revised 11 January 2011
Accepted 13 January 2011
Available online 18 January 2011
Keywords:
Carbonic anhydrase
Mitochondrial isoforms
Sulfonamide
Ó 2011 Elsevier Ltd. All rights reserved.
Camphorsulfonyl
Obesity
Isoform-selective enzyme inhibitor
Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) of the sul-
fonamide type, such as acetazolamide AAZ, methazolamide MZA,
ethoxzolamide EZA or dichlorphenamide DCP, are clinically used
for decades, for various classes of diuretics and systemically acting
antiglaucoma agents.1–4 In the last years novel applications
emerged for this class of pharmacological agents, such as the top-
ically acting antiglaucoma agents, anticonvulsants, antiobesity,
catalytic activity), their rather diffuse localization in many tissues/
organs, and the lack of isozyme selectivity of the presently avail-
able inhibitors of the sulfonamide/sulfamate type.3,8–10 Thus, there
is a stringent need of CAIs with a more selective inhibition profile
compared to the sulfonamides mentioned above (first generation
CAIs) or their isosteres (such as sulfamates, of which topiramate
TPM is a clinically used antiepileptic drug).11
N
N
N
N
N
SO2NH2
CH3CONH
SO2NH2
CH3CON
SO2NH2
S
S
S
EtO
AAZ
MZA
EZA
SO2NH2
OSO2NH2
SO2NH2
O
O
N
O
Cl
SO2NH2
O
O
Cl
O
DCP
ZNS
TPM
antipain and antitumor drugs/diagnostic tools.3–7 However critical
barriers to the use of CAIs as therapeutic agents are related to the
high number of isoforms in humans (i.e., 16 CAs, of which 13 have
Two of the 16 mammalian CA isoforms are mitochondrial
ones, CA VA and VB.12 They were shown to be involved among
others in several biosynthetic processes, such as ureagenesis,12a
⇑
Corresponding author. Tel.: +39 055 457 3005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.050

A. Maresca, C. T. Supuran / Bioorg. Med. Chem. Lett. 21 (2011) 1334–1337
1335
8'
9'
4'
7'
3'
8
9
4
5'
7
NH2
n
2'
3
1'
5
6'
O
2
i
S
1
6
O
NH
O
O
H2NO2S
S
O
O
Cl
n
H2NO2S
8: (1IR), n=0, p-NH2
1: (1R)
3: n=0, p-NH2
9: (1IS), n=0 p-NH2
10: (1IR), n=1, p-NH2 11: (1IS), n=1 p-NH2
12: (1IR), n=2, p-NH2 13: (1IS), n=2 p-NH2
14: (1IR), n=0, m-NH2 15: (1IS), n=0 m-NH2
2: (1S
)
4: n=1, p-NH2
5: n=2, p-NH2
6: n=0, m-NH2
8'
9'
4'
7'
8
9
3'
5'
6'
7
4
2'
3
5
1'
SO2NH2
S
i
2
O
HN
HCl
1
S
O
6
SO2NH2
S
N
N
O
O
N
S
O
N
O
N
Cl
16: (1IR)
17: (1IS)
1: (1R)
2: (1S
7
)
Scheme 1. Synthesis of sulfonamide derivatives 8–17. Reagents and conditions: (i) dry DMF, dry TEA, N2, 0° to rt.
gluconeogenesis,13 and lipogenesis, both in vertebrates (rodents)
as well as invertebrates (locust).14,15 Indeed, in several important
biosynthetic processes involving pyruvate carboxylase, acetyl
CoA carboxylase, and carbamoyl phosphate synthetases I and II,
bicarbonate, it is not carbon dioxide the real substrate for these
carboxylating enzymes, but its hydration product, bicarbonate,
and the provision of enough bicarbonate is assured by the catalysis
involving the mitochondrial isozymes CA VA and VB.3,13–15
As obesity represents a serious medical problem,7 mitochon-
drial CA inhibition has been proposed as an anti-obesity approach
for designing novel pharmacological agents.3,7 Indeed, topiramate
TPM and zonisamide ZNS (another clinically used antiepileptic
drug),3 showed a steadfast and important weight loss as ‘side-ef-
fects’ in obese epileptic patients.16 It is actually considered that
this effect is due to the inhibition of lipogenesis mediated by these
agents, as a consequence of mitochondrial CA inhibition.3,7 How-
ever, both TPM and ZNS, but also sulfonamides AAZ–DCP men-
tioned above, do not show any selectivity for inhibiting the
mitochondrial over the ubiquitous cytosolic CA isoforms. In this
Letter, we report a novel class of lipophilic CAIs incorporating R-
and S-10-camphorsulfonyl moieties, which show high selectivity
and excellent potency for the inhibition of the mitochondrial iso-
forms CA VA and CA VB.
horsulfonyl moieties. Such derivatizations (the ‘tail approach’)
have been investigated earlier extensively by this group,17 being
shown that they may lead to effective CAIs. All new compounds
8–17 have been thoroughly characterized by physico-chemical
methods which confirmed their structure.18
The new sulfonamides reported here, of types 8–17, as well as
the classical CAIs in clinical use AAZ–ZNS, have been assayed19
for the inhibition of four physiologically relevant CA isoforms,
the cytosolic, off-target hCA I and II, as well as the mitochondrial
hCA VA and hCA VB (Table 1). The following should be observed
regarding CA inhibition data with these compounds:
(i) Sulfonamides 8–17 incorporating the lipophilic moieties of
the R- and S-10-camphorsulfonyl type, behaved as weak inhibitors
Table 1
hCA I, II, VA and VB inhibition data with sulfonamides 8–17 and the standard
inhibitors AAZ–ZNS, by a stopped-flow, CO2 hydration assay method19
Compound
KI (nM)c
hCA VAb
hCA Ia
hCA IIa
hCA VBb
8
9
10
11
12
13
14
15
1241
3130
920
1207
1165
1563
2459
963
2254
78.9
2304
1773
398
12
17.1
68.6
16.6
27.5
64.5
33.2
35.3
48.7
5.9
21.0
63
65
25
630
63
26.4
30.0
40.3
67.3
36.2
54.1
38.4
44.3
7.8
7.3
54
62
19
Sulfonamides1–3 and sulfamates3,11 are among the most potent
CAIs reported up to now against all the physiologically relevant CA
isozymes, including the mitochondrial ones CA VA and VB.3,12
Thus, we decided to explore new series of aromatic as well as het-
erocyclic sulfonamides which should also incorporate lipophilic
moieties in their molecule, in order to endow them with good
membrane permeability and thus access to mitochondria, where
these isoforms are located. We have considered the reactions of
(1R)-(ꢀ)-10-camphorsulfonyl chloride 1 and (1S)-(+)-10-camp-
horsulfonyl chloride 2 with aromatic/heterocyclic sulfonamides
incorporating amino or imino moieties, as a facile way to generate
such new compounds (Scheme 1).
1042
4964
5513
9711
81.5
5246
4382
250
50
25
1200
250
56
16
17
AAZ
MZA
EZA
DCP
TPM
ZNS
14
8
38
10
21
30
6033
35
20
Data for the standard inhibitors are from Ref. 3a.
a
Indeed, reaction of sulfonyl chlorides 1 and 2 with amino/imi-
no-sulfonamides 3–7 afforded a series of chiral new sulfonamides
of type 8–17, which incorporate the highly lipophilic camp-
Full length, cytosolic recombinant isoform.
Full length, mitochondrial recombinant enzyme.
Errors in the range of 5–10% of the reported value, from three different assays.
b
c

1336
A. Maresca, C. T. Supuran / Bioorg. Med. Chem. Lett. 21 (2011) 1334–1337
of the cytosolic, slow isoform hCA I, with inhibition constants in
the micromolar range (KIs of 0.92–9.71 M). Only one of these
fonamides/sulfamates are much better hCA I/II inhibitors than
hCA VA/VB inhibitors, whereas exactly the reverse is true for the
new sulfonamides 8–17 reported here.
l
derivatives, 15, showed more efficient inhibition of this isoform,
with KI of 81.5 nM (Table 1). It is interesting to note the huge dif-
ference in inhibitory power between the two enantiomers 14 and
15, with the last one being approximately 120 times a better hCA
I inhibitor compared to 14. Thus, the nature of the moiety on which
the sulfonamide group is grafted (aromatic or heterocyclic) as well
as the spacer between this moiety and the camphorsulfonyl group,
do not influence markedly the activity of these compounds. It is
however difficult to explain the rather good inhibitory activity of
15, but recent work showed a very variable binding pattern within
the enzyme active site even for structurally very similar congeners
belonging to the sulfonamides.20 It may be observed on the other
hand that the clinically used derivatives AAZ–ZNS showed more
potent inhibitory activities against these isoforms, with inhibition
Work is in progress to evaluate pharmacologically these com-
pounds as lipogenesis inhibitors. Their lipophilic character makes
them interesting candidates for such an action, and it should be
mentioned that their inhibition of other CA isoforms (e.g., CA IV,
VI, IX and XII, data not shown) with important physiological roles
is not in the low nanomolar range, being thus more similar to that
of CA II (described here). Thus, there would be of little concern
their activity against these off-target CAs.
In conclusion, we report in the present paper a series of aro-
matic and heterocyclic sulfonamides incorporating R- and S-10-
camphorsulfonyl moieties, which were synthesized by reaction of
the sulfonyl chlorides with amino/imino-containing sulfonamides.
The new derivatives were investigated for the inhibition of several
mammalian isoforms the zinc enzyme carbonic anhydrase, such as
CA I, II, VA and VB. The new sulfonamides selectively inhibited the
mitochondrial isozymes hCA VA and VB over the cytosolic, off-tar-
get ones hCA I and II, with inhibition constants in the low nanomo-
lar range (5.9–68.6 nM). The nature, chirality and position of the
groups substituting the sulfonamide greatly influenced the CA
inhibitory properties. These compounds are excellent leads for
designing isoform-selective enzyme inhibitors targeting mitochon-
drial CAs involved in lipogenesis and obesity.
constants in the range of 25 nM–1.2 lM (Table 1).
(ii) A rather similar inhibition pattern with compounds 8–17 (as
for hCA I) has been also observed against the ubiquitous, physio-
logically dominant3 isoform hCA II (Table 1). Thus, weak inhibition,
in the micromolar range (KIs of 0.96–2.45 lM) was observed for all
these compounds except 14 and 17, which were more effective
hCA II inhibitors (KIs of 78.9–398 nM). Again important differences
of activity have been observed for diverse enantiomers of the same
sulfonamide (e.g., 14 vs 15, or 16 vs 17), and this has been docu-
mented by crystallographic work on different classes of chiral sul-
fonamides.21 The clinically used derivatives were on the other
hand low nanomolar inhibitors of this isoform, with KIs in the
range of 8–38 nM (Table 1).
(iii) The mitochondrial isoform hCA VA was much more suscep-
tible to inhibition by compounds 8–17 compared to the cytosolic
ones discussed above. Indeed, KIs in the range of 5.9–68.6 nM have
been measured for these derivatives (Table 1). SAR is here more
intriguing compared to the inhibition of the cytosolic isoforms.
Thus, generally the R-enantiomer was more effective as a hCA VA
inhibitor compared to the corresponding S-enantiomer (except
for the pair 12–13 where the reverse was true). The most effective
inhibitor was thus the heterocyclic derivative 16 (KI of 5.9 nM),
which as far as we know, is the most effective hCA VA inhibitor
ever reported. Its enantiomer, 17, was 3.5 times less effective an
inhibitor, being anyhow equipotent to ZNS, one of the best hCA
VA inhibitors among the clinically used drugs (Table 1 and Ref.
3). The benzenesulfonamide derivatives 8–15 showed rather com-
parable activity irrespective whether the two substituents on the
ring were in meta (14 and 15) or para (8–13) positions, and of
the length of the spacer separating the benzenesulfonamide and
the camphorsulfonamide moieties.
(iv) The second mitochondrial isoform, hCA VB was also signif-
icantly inhibited by sulfonamides 8–17, with inhibition constants
in the range of 7.3–67.3 nM, orders of magnitude better than the
inhibition of the cytosolic isoforms hCA I and II discussed above.
Again the best inhibitors were the heterocyclic sulfonamides 16
and 17, and generally the R-enantiomer was more active compared
to the corresponding S one, as for the inhibition of hCA VA dis-
cussed above (the only exception is the pair 16 and 17).
(v) The selectivity ratios for the inhibition of the mitochondrial
over the cytosolic isoforms, for the new compounds reported here,
are very good. For example, 16, a low nanomolar hCA VA and VB
inhibitor, had a selectivity ratio of 889 for the inhibition of hCA
VA over hCA I, and of 300.5 for the inhibition of hCA VA over
hCA II. The selectivity ratios for the inhibition of hCA VB over
hCA I and of hCA VB over hCA II are of 672 and 227, respectively.
For ethoxzolamide EZA, one of the best classical mitochondrial
CA inhibitors, these parameters are of 1 (hCA VA over hCA I),
0.32 (hCA VA over hCA II), 1.31 (hCA VB over hCA I) and 0.42
(hCA VB over hCA II), respectively. Thus, most of the classical sul-
Acknowledgment
This research was financed in part by a grant of the 7th FP of EU
(Metoxia project).
References and notes
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Compound 13 was synthesised by reacting (1S)-(+)-10-camphorsulfonyl
chloride 2 (1.19 mmol, 300 mg) with 4-(2-Aminoethyl)benzenesulfonamide 5
(1.19 mmol, 238.3 mg) following the general procedure mentioned above. The
crude was purified by silica gel column chromatography eluting with 2.5%
MeOH in DCM to afford compounds 13 as white solid in 70% yield. Mp 140–
142 °C, ½a 2D0 +11.1, silica gel TLC Rf 0.27 (MeOH/DCM 2.5%), mmax (KBr) cmꢀ1
,
ꢂ
3250 (N–H), 2954 (C–H), 1741 (C@O), 1599 (aromatic), 1328 (SO2–NH), dH
(400 MHz, DMSO-d6) 0.83 (3H, s, 9-H3), 1.03 (3H, s, 8-H3), 1.39–1.44 (1H, m, 6-
H), 1.49–1.56 (1H, m, 7-H), 1.92–1.93 (1H, m, 4-H), 1.94–2.01 (1H, m, 6-H),
2.06–2.08 (1H, m, 4-H), 2.31–2.38 (1H, m, 7-H), 2.32–2.39 (1H, m, 5-H), 2.86–
2.92 (3H, m, 20-H2, 10-H), 3.26–3.32 (3H, m, 10-H2, 10-H), 7.29 (2H, s, SO2NH2,
exchange with D2O), 7.46–7.50 (2H, m, 2 ꢁ 40-H), 7.78–7.80 (2H, m, 2 ꢁ 50-H),
dC (100 MHz, DMSO-d6) 215.6 (C-1), 144.2 (ipso), 143.1 (ipso), 130.3 (C-40),
126.8 (C-50), 58.7 (C-2), 48.5 (C-10), 48.4 (C-3), 44.5 (C-10), 42.9 (C-4), 42.8 (C-
5), 36.5 (C-20), 27.2 (C-6), 25.3 (C-7), 20.3 (C-8), 20.3 (C-9), m/z (ESI-) 413.1
([MꢀH]ꢀ 100%), 827.3 ([2MꢀH]ꢀ 18%), m/z (ESI+) 415.2 ([M+H]+ 100%), 829.3
([2M+H]+ 50%).
(Z)-5-(((1R)-7,7-Dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methylsulfonylimino)-
4-methyl-4,5-dihydro-1,3,4-thiadiazole-2-sulfonamide (16)
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8
9
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7
5
4
1
6
2
10
O
S
2' SO2NH2
S
O
N
O
1'
N
N
16. (a) Picard, F.; Deshaies, Y.; Lalonde, J.; Samson, P.; Richard, D. Obes. Res. 2000, 8,
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3'
17. (a) Supuran, C. T.; Ilies, M. A.; Scozzafava, A. Eur. J. Med. Chem. 1998, 33, 739; (b)
Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.; Mincione, G.; Supuran,
C. T. J. Med. Chem. 2000, 43, 4542; (c) Ilies, M.; Supuran, C. T.; Scozzafava, A.;
Casini, A.; Mincione, F.; Menabuoni, L.; Caproiu, M. T.; Maganu, M.; Banciu, M.
D. Bioorg. Med. Chem. 2000, 8, 2145.
Compound 16 was synthesised by reacting (1R)-(ꢀ)-10-camphorsulfonyl
chloride
(0.08 mmol, 200 mg) with derivative 71 (0.08 mmol, 185 mg)
1
following the general procedure mentioned above. The crude was purified by
silica gel column chromatography eluting with 3% MeOH in DCM to afford
compounds 16 as white solid in 46% yield. Mp 248–250 °C, ½a D20
ꢀ47.5, silica
ꢂ
18. Sulfonamides 3–6 and sulfonyl chlorides 1, 2 used for the synthesis were
commercially available from Sigma–Aldrich (Milan, Italy), and were used
without further purification. The synthesis of compound 7 was carried out as
reported in literature.1 All CA isozymes were recombinant ones produced and
purified in our laboratory as described earlier.2,3 Reactions of (1R)-(ꢀ)-10-
camphorsulfonyl chloride 1 and (1S)-(+)-10-camphorsulfonyl chloride 2 (0.3 g,
1.0 equiv) with amino derivatives 3–7 (1.0 equiv) were carried out at 0° to
room temperature in Schotten–Baumann conditions under nitrogen
atmosphere, in the presence of a stoichiometric amount of dropwised dry
TEA (1.0 equiv for 3, 5, 6 and 2.0 equiv for 4, 7), in dry DMF as solvent (2 ml).
When complete (monitoring by TLC), reactions were quenched with crushed
ice, extracted with ethyl acetate (20 ml), washed with 1 N HCl (2 ꢁ 10 ml) and
brine (2 ꢁ 10 ml). The collected organic phase was dried on anhydrous Na2SO4,
filtered and evaporated under vacuum. The crude was purified by silica gel
column chromatography eluting with n-hexane/ethyl acetate or DCM/
methanol to afford compounds 8–17 as white solids in medium yields.
Examples of several compounds prepared in this way are provided below:
4-(2-(((1S)-7,7-Dimethyl-2-oxobicyclo[2.2.1]heptan-1-
gel TLC Rf 0.27 (MeOH/DCM 3%), mmax (KBr) cmꢀ1, 3265 (N–H), 2962 (C–H),
1736 (C@O), 1654 (C@N), 1372 (SO2–NH), dH (400 MHz, DMSO-d6) 0.82 (3H, s,
9-H3), 1.06 (3H, s, 8-H3), 1.42–1.47 (1H, m, 6-H), 1.59–1.66 (1H, m, 7-H), 1.94–
1.98 (1H, m, 4-H), 1.94–2.02 (1H, m, 6-H), 2.07–2.10 (1H, m, 4-H), 2.32–2.44
(1H, m, 7-H), 2.33–2.43 (1H, m, 5-H), 3.14–3.17 (1H, d, J 14.8, 10-H), 3.43–3.47
(1H, d, J 14.8, 10-H), 3.74 (1H, s, 30-H), 8.60 (2H, s, SO2NH2, exchange with D2O),
dC (100 MHz, DMSO-d6) 215.4 (C-1), 165.7 (C-20), 157.0 (C-10), 58.5 (C-2), 50.5
(C-10), 48.7 (C-3), 42.9 (C-4), 42.8 (C-5), 38.6 (C-30), 27.2 (C-6), 25.2 (C-7), 20.1
(C-8), 20.1 (C-9), m/z (ESI-) 407.0 ([MꢀH]ꢀ 100%), 815.0 ([2MꢀH]ꢀ 8%), m/z
(ESI+) 409.1 ([M+H]+ 100%), 817.2 ([2M+H]+ 5%).
19. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-
flow instrument has been used for assaying the CA catalysed CO2 hydration
activity.18 Phenol red (at
a concentration of 0.2 mM) has been used as
indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes
(pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant the ionic
strength), following the initial rates of the CA-catalyzed CO2 hydration reaction
for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for
the determination of the kinetic parameters and 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, in triplicate measurements. The
uncatalyzed rates were determined in the same manner and subtracted from
the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in
distilled-deionized water and dilutions up to 0.01 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
yl)methylsulfonamido)ethyl)benzene sulfonamide (13)
8
9
3
7
4
5
6
1
2
10
O
were obtained by non-linear least-squares methods using PRISM 3, as reported
S
11,17
O
earlier,
and represent the mean from at least three different
NH
O
determinations. CA isoforms were recombinant ones obtained in house as
reported earlier.11,17
1'
2'
20. Pacchiano, F.; Aggarwal, M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.;
McKenna, R.; Supuran, C. T. Chem. Commun. 2010, 46, 8371.
21. (a) D’Ambrosio, K.; Masereel, B.; Thiry, A.; Scozzafava, A.; Supuran, C. T.; De
Simone, G. Chem. Med. Chem. 2008, 3, 473; (b) Güzel, Ö.; Temperini, C.;
Innocenti, A.; Scozzafava, A.; Salman, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2008, 18, 152.
3'
4'
5'
6'
SO2NH2