Carbonic anhydrase inhibitors. Phenacetyl-, pyridylacetyl- and thienylacetyl-substituted aromatic sulfonamides act as potent and selective isoform VII inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.04.123

A series of aromatic/heterocyclic sulfonamides incorporating phenyl(alkyl), halogenosubstituted-phenyl- or 1,3,4-thiadiazole-sulfonamide moieties and thienylacetamido; phenacetamido- and pyridinylacetamido tails were prepared and assayed as inhibitors of cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I, II and VII. The new compounds showed moderate inhibition of the two ubiquitous isoforms I and II (K_Is of 50-390 nM) and excellent inhibitory activity against the brain associated hCA VII (K_Is in the range of 4.7-8.5 nM). Isoform VII highly selective inhibitors are being detected for the first time, with selectivity ratios for inhibiting CA VII over CA II of 11-75, and for inhibiting CA VII over CA I of 10-49, which may be useful for understanding the role of CA VII in epileptogenesis and other physiologic processes.

Full text of DOI:10.1016/j.bmcl.2009.04.123

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3170–3173
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Carbonic anhydrase inhibitors. Phenacetyl-, pyridylacetyl- and thienylacetyl-
substituted aromatic sulfonamides act as potent and selective isoform VII
inhibitors
Özlen Güzel a,b, Alessio Innocenti b, Andrea Scozzafava b, Aydın Salman a, Claudiu T. Supuran b,
*
a Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34116 Beyazıt, Istanbul, Turkey
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 aromatic/heterocyclic sulfonamides incorporating phenyl(alkyl), halogenosubstituted-phenyl-
or 1,3,4-thiadiazole-sulfonamide moieties and thienylacetamido; phenacetamido- and pyridinylacetam-
ido tails were prepared and assayed as inhibitors of cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1)
isoforms hCA I, II and VII. The new compounds showed moderate inhibition of the two ubiquitous iso-
forms I and II (KIs of 50–390 nM) and excellent inhibitory activity against the brain associated hCA VII
(KIs in the range of 4.7–8.5 nM). Isoform VII highly selective inhibitors are being detected for the first
time, with selectivity ratios for inhibiting CA VII over CA II of 11–75, and for inhibiting CA VII over CA
I of 10–49, which may be useful for understanding the role of CA VII in epileptogenesis and other phys-
iologic processes.
Received 23 February 2009
Revised 22 April 2009
Accepted 25 April 2009
Available online 3 May 2009
Keywords:
Carbonic anhydrase
Cytosolic isoform
Isozyme VII
Epileptogenesis
Sulfonamide
Ó 2009 Elsevier Ltd. All rights reserved.
Tail approach
There are 16
a
-carbonic anhydrase (CA, EC 4.2.1.1) isoforms
expression is highly increased intrapyramidally at around post-
natal day 12 (in mice, but presumably in other mammals too),
being hypothesized that this developmental expression promotes
excitatory response evoked by intense GABAergic activity, which
is related to the epileptiform activity.12,13 The ionic mechanism
of the GABAergic excitation is dependent on bicarbonate, which
is the only physiological ion in addition to chloride able to medi-
ate a current through channels coupled to GABAA receptors.12,13
CA VII, through its presence mainly in the brain, is currently
considered to be involved in the mechanism of GABAergic exci-
tation.12,13 For example, ethoxzolamide EZA, a lipophilic, mem-
brane-permeant CA inhibitor (CAI), acting at low nanomolar
level against CA VII,14 was shown to prevent GABAergic excita-
tions, while membrane-impermeant CAIs had no such effects.13
Such results clearly showed the involvement of the cytosolic iso-
form CA VII in the neuronal excitation and its inhibition as a
possible antiepileptic mechanism.15,16 Furthermore, some clini-
cally used drugs, such as acetazolamide AZA, zonisamide ZNS
and topiramate TPM, which act as broad-spectrum CAIs,1 also
show anticonvulsant activity and are used as antiepileptic drugs,
although their mechanism of action is rather complex (at least
for the last two agents).15,16
expressed in mammals, CA I–CA XV, five of which, CA I–III, VII
and XIII, being cytosolic ones.1–4 These isozymes show a very
different distribution in various tissues and organs as well as
quite diverse catalytic properties for the physiologic reaction,
that is, hydration of carbon dioxide to bicarbonate and a pro-
ton.1–4 Indeed, CA II and VII possess very high efficiency as cat-
alysts for hydration of carbon dioxide, CA I and XIII are 10–50
times less active as compared to CA II and VII, whereas CA III
is a very poor catalyst for this reaction, showing around 1% of
the catalytic activity of CA II, the most active mammalian CA (to-
gether with CA IX) and one of the best catalysts known in nat-
ure.4–6 On the other hand, CA I and II are widely distributed in
many tissues/cell types in humans,7 CA III is present only in
muscles and liver,8 whereas CA VII and XIII show a rather lim-
ited distribution only in some organs/tissues, such as the brain
for CA VII,9 and the reproductive tract for CA XIII.10 The precise
physiologic role of some of these isozymes7–10 is not clearly
understood at this moment, with the least investigated and
understood one being just CA VII. This isoform was shown to
be highly expressed in the cortex, hippocampus and thalamus
regions within the mammalian brain.11,12 Furthermore, its
There are several reports14,17 in which the inhibition of CA VII
with various classes of sulfonamides and sulfamates has been eval-
uated. Although several low nanomolar or subnanomolar CAIs
were detected (some of them also showing in vivo anticonvulsant
* Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.123

Ö. Güzel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3170–3173
3171
ray crystallographic work on such enzyme-inhibitor adducts.22,23
In that region of the active site are present the amino acid residues
which are less conserved among the various mammalian CA iso-
forms,1,2 and their interactions with the tails incorporated in the
inhibitors explain why most of the novel generation inhibitors usu-
ally show a better (i.e., more isozyme-selective) inhibition profile
as compared to the classical sulfonamides, of which acetazolamide
AZA is the best studied representative.1,2 X-ray crystal works
showed that both favorable interactions as well as clashes with
particular amino acid residues present only in some CA isozymes24
are critical for the inhibition profile and isozyme selectivity issues
of the sulfonamides and their bioisosteres such as the sulfamates
and the sulfamides. Reaction of 2-thienylacetyl chloride 1 or phe-
nyl/pyridyl–acetyl halides/carboxylic acids 3 with aminosulfona-
mides 2 and 4, led to the acylated sulfonamides 5–8 by non-
exceptional procedure (Scheme 1). In addition to sulfanilamide,
homosulfanilamide, 4-aminoethyl-benzenesulfonamide and halo-
genated sulfanilamides 2, the heterocyclic derivative 5-amino-
1,3,4-thiadiazole-2-sulfonamide 4 was included in the study.25
The following structure–activity relationship (SAR) can be
evidenced by considering data of Table 1:26
N
N
N
S
SO2NH2
CH3CONH
SO2NH2
S
O
EZA
AZA
O
NH2
S
O
O
SO2NH2
O
O
N
O
O
O
O
ZNS
TPM
activity17a) the main draw-back of such compounds is related to
their low selectivity for inhibiting isoforms CA VII over other cyto-
solic (CA I, II, XIII) or membrane-associated isoforms (CA IV, IX, XII,
XIV). In fact, the mammalian brain expresses in addition to CA VII,
several other CA isoforms, such as CA I, II, III, VB, XII and XIV,15,16
and the inhibitors investigated up to now14,17 generally showed a
very high activity towards most of them. Thus, a net discrimination
between the effects of such drugs on the various isoforms and their
relationship to the anticonvulsant activity is difficult if not impos-
sible to achieve with broad spectrum CAIs which interact with
many of these CAs.18 It is thus of great importance to design com-
pounds showing selective inhibition of different physiologically
relevant CAs, and in particular, CA VII-selective inhibitors. Here,
we report such a study which led to the discovery of the most
CA VII-selective compounds reported to date.
(i) Isoform hCA I was moderately inhibited by sulfonamides 5–8
reported here. Thus, several derivatives, such as5b, 7a and 7f,
showed medium inhibitory activity, with inhibition constants
in the range of 108–263 nM, in the same range as the clinically
used compounds acetazolamide and topiramate (KI of 250 nM
for both compounds against this isoform, Table 1). The
remaining new sulfonamides were more effective hCA I inhib-
itors as compared to derivatives discussed above, with KIs in
the range of 60–84 nM. The best hCA I inhibitor was the clin-
ically used compound zonisamide ZNS (KI of 56 nM). Obvi-
ously both the aromatic sulfonamide head as well as the
aryl/hetaryl–acetyl moiety influence the biological activity
of these hCA I inhibitors. It may be observed that efficient
inhibitors incorporate both sulfanilamide, halogenated
sulfanilamide, homosulfanilamide, 4-aminoethyl-benzene-
sulfonamide and 5-amino-1,3,4-thiadiazole-2-sulfonamide
moieties, whereas the tail present in the acylating agent prob-
ably modulates and fine-tunes the binding. Except for the
three less active compounds mentioned above (5b, 7a and
7f) which incorporate a 2-thienylacetyl (5b) or a phenacetyl
moiety (7a and 7f), the remaining compounds showed a quite
compact behavior of moderately-efficient hCA I inhibitors.
Thus, the aryl/hetaryl–acetamido moieties present in these
compounds lead to significant hCA I inhibition, but all these
compounds possess KIs > 50 nM, being thus only moderately
active.
(ii) A rather similar situation was observed for the inhibition of
hCA II (Table 1), a physiologically dominant and highly rele-
vant isoform.1 Indeed, again the same three compounds (5b,
7a and 7f) showed weaker hCA II inhibitory activity, with KIs
in the range of 107–395 nM whereas all the remaining deriv-
atives behave as moderate inhibitors (KIs in the range of 50–
97 nM). It should be noted that the clinically used com-
pounds (AZA, EZA, ZNS and TPM) or the orphan drug benzo-
lamide BZA show much more potent hCA II inhibitory
activity, with KIs in the range of 8–35 nM (Table 1). It should
be also noted that the compounds investigated earlier,19
having a CH2 moiety less than the present ones, showed
much better hCA II inhibitory activity, with KIs in the range
of 3–12 nM (but they were less effective as hCA I inhibitors,
with KIs in the range of 120–365 nM).19 These finding clearly
illustrate that a very small variation in the structure of a CAI
(such as the presence of an additional CH2 moiety, in this
N
N
( )n
N
H
N
H
SO2NH2
S
X
X
O
O
SO2NH2
A
B
X = O, S, NH
n = 0, 1, 2
We used sulfonamides A and B reported earlier by our group19
as lead molecules for the design of the new sulfonamides reported
here. Indeed, sulfonamides of the type A and B, incorporating phe-
nyl(alkyl), halogeno-phenyl- or 1,3,4-thiadiazole moieties as well
as furan-2-ylcarboxamido; thien-2-ylcarboxamido or pyrrol-2-
ylcarboxamido tails, were easily prepared from the corresponding
amino sulfonamides by reactions with heterocyclic acyl halides or
carboxylic acids in the presence of carbodiimides, by the tail ap-
proach.19–21 The in vitro and in vivo biological activity of such com-
pounds was interesting, with some of them showing low
nanomolar inhibitory profiles against isoforms CA I, II and IV, as
well as antiglaucoma activity in an animal model of this disease.19
The most active compounds were the thienyl derivatives, and here
we extend the earlier work,19 focusing on this substitution pattern.
However, some structurally related derivatives incorporating
phenylacetamido- and pyridyl-acetamido moieties were also pre-
pared and assayed as CAIs, in order to understand the role that
the terminal part of the tail plays in modulating the isoform selec-
tivity profile of such compounds. The sulfonamides reported here
(Scheme 1) differ of the previously investigated ones by the incor-
poration of the aryl-/hetarylacetamido moiety in their molecules
instead of the hetarylcarboxamido one. Thus, they posess longer
tails as compared to the derivatives investigated earlier.19 The
rationale for this modification resides in the fact that the tails pres-
ent in CAIs interact with amino acid residues situated towards the
exit of the CA active site or on its edge, as shown by extensive X-

3172
Ö. Güzel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3170–3173
O
O
(CH2)n
NH
S
5
SO2NH2
Y
N
O
O
N
H2N (CH2)n
SO2NH2
SO2NH2
S
Cl
Z
NH
S
S
Y
n=0, Y=H, F, Cl, Br
n=1, Y=H
n=2, Y=H
6
1
2
X2
O
+
method A, B
(CH2)n
X2
X1
NH
N
N
+
X1
7
SO2NH2
Y
H2N
SO2NH2
S
-
N
N
X1=X2=CH, Z=Cl
X1=N, X2=CH, Z=OH
X1=CH, X2=N, Z=OH
X2
O
Cl
SO2NH2
4
S
X1
NH
3
8
Scheme 1. Synthesis of the new sulfonamides 5–8. Method A: Z = Cl, NEt3/MeCN; Method B: Z = OH, N-(3-dimethylpropyl)-N0-ethylcarbodiimide, DMAP/dioxane.
it may be observed that the selectivity ratios for inhibiting
CA I over CA VII are in the range of 8.45–49.62, whereas that
form inhibiting CA II over CA VII in the range of 7.22–74.52.
This means that all these compounds have much higher
affinity for CA VII than for CA II and I, which are ubiquitous,
cytosolic isoforms.
Table 1
Inhibition data of isoforms hCA I, II and VII with sulfonamides 5–8 and clinically used
sulfonamides/sulfamates EZA—TPM, by a stopped flow CO2 hydrase assay26
No.
n,Y
X1
X2
KI* (nM)
Selectivity ratio
hCA Ia
hCA IIa
hCA VIIa
I/VII
9.83
23.00
11.15
11.66
8.45
II/VII
5a
5b
5c
5d
5e
5f
0, H
0, F
0, Cl
0, Br
1, H
2, H
—
0, H
1, H
2, H
0, F
0, Cl
0, Br
0, H
0, H
2, H
—
—
—
—
—
—
—
—
—
—
—
—
—
—
CH
CH
CH
CH
CH
CH
CH
N
N
CH
—
—
—
61
161
77
84
60
68
72
108
75
60
67
61
263
75
71
73
63
25
250
56
50
390
50
52
52
53
51
107
54
67
61
58
395
69
74
97
51
8
6.2
7.0
6.9
7.2
7.1
7.6
6.9
4.7
6.8
5.5
5.4
6.9
5.3
7.7
7.9
8.5
6.1
0.8
2.5
117
0.9
8.06
55.71
7.24
7.22
7.32
In conclusion, we report here series of aromatic/heterocyclic
sulfonamides incorporating phenyl(alkyl), halogenosubstituted-
phenyl- or 1,3,4-thiadiazole-sulfonamide moieties and thienylace-
tamido; phenacetamido- and pyridinylacetamido tails, which were
prepared and assayed as inhibitors of cytosolic human isoforms
hCA I, II and VII. The new compounds showed moderate inhibition
of the two ubiquitous isoforms I and II (KIs of 50–390 nM) and
excellent inhibitory activity against the brain associated hCA VII
(KIs in the range of 4.7–8.5 nM). Isoform VII highly selective inhib-
itors are being detected thus for the first time, with selectivity ra-
tios for inhibiting CA VII over CA II of 11–75, and for inhibiting CA
VII over CA I of 10–49, which may be useful for understanding the
role of CA VII in epileptogenesis and other physiologic processes.
8.94
6.97
6
7ª
10.43
22.97
11.02
10.90
12.40
8.84
7.39
22.76
7.94
12.18
11.29
8.40
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
—
7b
7c
7d
7e
7f
7g
7h
7i
49.62
9.74
74.52
8.96
8.98
9.36
8.58
10.32
31.25
100
0.47
277.7
11.41
8.36
10.00
4.80
0.29
11.11
8
EZA
AZA
ZNS
TPM
—
—
—
—
—
—
—
12
35
10
Acknowledgments
—
250
*
This research was financed in part by a grant of the 6th Frame-
work Programme of the EU (DeZnIT project), by a 7th Framework
Programme EU project (METOXIA) and by an Italian FIRB project
(MIUR/FIRB RBNE03PX83_001).
Errors in the range of 5–10% of the shown data, from three different assays, by a
CO2 hydration stopped-flow assay.26
a
Human, recombinant isozymes.
References and notes
case) may have drastic consequences for the enzyme inhib-
itory activity and selectivity profile against various isozymes
of such derivatives.
1. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Supuran, C. T.;
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(iii) The brain-associated isoform hCA VII was highly inhibited
by all sulfonamides 5–8 investigated here, with KIs in the
range of 4.7–8.5 nM (Table 1). All these new compounds
were highly active, irrespective of the aryl/hetaryl sulfon-
amide head and the heterocyclic/aromatic-acetamido tails
present in their molecules. SAR is thus very simple and flat,
and also difficult to rationalize as the X-ray crystal structure
of CA VII is not known for the moment. However, this is a
remarkable finding, considering that few compounds with
such a strong inhibitory activity against this isoform have
been reported up until now.
2. Carbonic Anhydrase—Its Inhibitors and Activators; Supuran, C. T., Scozzafava, A.,
Conway, J., Eds.; CRC Press: Boca Raton (FL), USA, 2004; pp 1–376. and
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aminosulfonamide 2 or 4 was suspended/dissolved in 20–30 mL anhydrous
MeCN and 0.78 mL (0.56 g, 5.5 mmol) triethylamine was added under stirring.
The mixture was cooled to 0–5 °C, then a solution of 5 mmol phenylacetyl
chloride/2-thienylacetyl chloride 1 or 3 dissolved in 3 mL MeCN was added
dropwise during 10 min. (immediately, precipitate appeared). The reaction was
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solvent was evaporated in vacuum and the resulted product was treated with
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from ethanol.Method B. Five millimoles of aminosulfonamide 2 or 4 dissolved in
30 mL anhydrous dioxane were treated, under stirring, with 0.95 g (5 mmol) N-
(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride and 0.06 g
(0.5 mmol) dimethylaminopyridine (DMAP); the reaction mixture was kept
under nitrogen and 0.86 g (5 mmol) of pyridine-2-yl-acetic acid/pyridine-4-yl-
acetic acid 3 (Z = OH) was added. The obtained homogeneous colorless solution
turned orange-cream and in around 20 min a precipitate appeared. The stirring
was continued overnight, then the precipitate was filtered, washed with ethanol
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(C@O), 1160, 1309 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.94 (2H, s,
CH2CO), 7.01 (2H, s, thiophene C3,4-H), 7.29 (2H, s, SO2NH2), 7.42 (1H, s,
thiophene C5-H), 7.78 (4H, s, phenyl C2,3,5,6-H), 10.57 (1H, s, CONH).
26. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An SX.18MV-R Applied
Photophysics (Oxford, UK) stopped-flow instrument has been used to assay
the catalytic/inhibition of various CA isozymes. 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 7.4) as buffer, 0.1 M Na2SO4 or NaClO4 (for
maintaining constant the ionic strength; these anions are not inhibitory in the
used concentration),19 following the CA-catalyzed CO2 hydration reaction for a
period of 5–10 s. Saturated CO2 solutions in water at 25 °C were used as
substrate. Stock solutions of inhibitors were prepared at a concentration of
10 mM (in DMSO–water 1:1, v/v) and dilutions up to 0.01 nM done with the
assay buffer mentioned above. At least seven different inhibitor concentrations
have been used for measuring the inhibition constant. Inhibitor and enzyme
solutions were preincubated together for 10 min at room temperature prior to
assay, in order to allow for the formation of the E–I complex. Triplicate
experiments were done for each inhibitor concentration, and the values
reported throughout the paper are the mean of such results. The inhibition
constants were obtained by non-linear least-squares methods using PRISM 3,
as reported earlier,19 and represent the mean from at least three different
determinations. All CA isozymes used here were recombinant proteins
obtained as reported earlier by our group.19–21