Carbonic anhydrase inhibitors. Aromatic/heterocyclic sulfonamides incorporating phenacetyl, pyridylacetyl and thienylacetyl tails act as potent inhibitors of human mitochondrial isoforms VA and VB

Article abstract of DOI:10.1016/j.bmc.2009.06.006

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 four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and hCA II, and the mitochondrial hCA VA and hCA VB. The new compounds showed moderate inhibition of the two cytosolic isoforms (K_Is of 50-390 nM) and excellent inhibitory activity against the two mitochondrial enzymes, with many low nanomolar inhibitors detected (K_Is in the range of 5.9-10.2 nM). All substitution patterns explored here lead to effective hCA VA/VB inhibitors. Some hCA VA/VB selective inhibitors were also detected, with selectivity ratios for inhibiting the mitochondrial over the cytosolic isozymes of around 55.5-56.9. As hCA VA/VB are involved in several biosynthetic processes catalyzed by pyruvate carboxylase, acetyl CoA carboxylase, and carbamoyl phosphate synthetases I and II, providing the bicarbonate substrate to these carboxylating enzymes involved in fatty acid biosynthesis, their selective inhibition may lead to the development of antiobesity agents possessing a new mechanism of action.

Full text of DOI:10.1016/j.bmc.2009.06.006

Bioorganic & Medicinal Chemistry 17 (2009) 4894–4899
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Carbonic anhydrase inhibitors. Aromatic/heterocyclic sulfonamides
incorporating phenacetyl, pyridylacetyl and thienylacetyl tails act
as potent inhibitors of human mitochondrial isoforms VA and VB
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 pyridinylacetami-
do tails were prepared and assayed as inhibitors of four physiologically relevant carbonic anhydrase (CA,
EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and hCA II, and the mitochondrial hCA VA and hCA VB.
The new compounds showed moderate inhibition of the two cytosolic isoforms (KIs of 50–390 nM) and
excellent inhibitory activity against the two mitochondrial enzymes, with many low nanomolar inhibi-
tors detected (KIs in the range of 5.9–10.2 nM). All substitution patterns explored here lead to effective
hCA VA/VB inhibitors. Some hCA VA/VB selective inhibitors were also detected, with selectivity ratios
for inhibiting the mitochondrial over the cytosolic isozymes of around 55.5–56.9. As hCA VA/VB are
involved in several biosynthetic processes catalyzed by pyruvate carboxylase, acetyl CoA carboxylase,
and carbamoyl phosphate synthetases I and II, providing the bicarbonate substrate to these carboxylating
enzymes involved in fatty acid biosynthesis, their selective inhibition may lead to the development of
antiobesity agents possessing a new mechanism of action.
Received 17 February 2009
Revised 28 May 2009
Accepted 5 June 2009
Available online 9 June 2009
Keywords:
Carbonic anhydrase
Cytosolic isoforms I and II
Mitochondrial isozymes VA and VB
Sulfonamide
Antiobesity
Isoform-selective inhibitor
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
There are 16
a-carbonic anhydrase (CA, EC 4.2.1.1) isoforms
expressed in mammals, CA I–CA XV, two of which, CA VA and CA
VB, being present in mitochondria.1–3 These two isozymes are
involved in several biosynthetic processes, such as ureagenesis, glu-
coneogenesis, and lipogenesis.1,4,5 The provision of enough bicar-
bonate as substrate for several biosynthetic processes catalyzed by
pyruvate carboxylase (PC), acetyl CoA carboxylase (ACC), and car-
bamoyl phosphate synthetases I and II, is assured mainly by the
catalysis involving these mitochondrial 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. Mitochondrial pyruvate carboxylase (PC) is
needed for efflux of acetyl groups from the mitochondria to the cyto-
sol where fatty acid biosynthesis takes place.1 Pyruvate is carboxyl-
ated to oxaloacetate in the presence of bicarbonate and under the
catalytic influence of the mitochondrial isozymes CA VA and/or CA
VB. The mitochondrial membrane is impermeable to acetyl-CoA
which reacts with oxaloacetate, leading to citrate, which is thereaf-
ter translocated to the cytoplasm by means of the tricarboxylic acid
Fig. 1. The transfer of acetyl groups from the mitochondrion to the cytosol (as
citrate) for the provision of substrate for de novo lipogenesis. 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). Inhibition of the
CA-mediated processes with sulfonamides lead to inhibition of lipogenesis.1
* Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.006

Özlen Güzel et al. / Bioorg. Med. Chem. 17 (2009) 4894–4899
4895
transporter. As oxaloacetate is unable to cross the mitochondrial
2. Results and discussion
2.1. Chemistry
membrane, its decarboxylation 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 pres-
ence of ACC and bicarbonate, with formation of malonyl-CoA, the
conversion between CO2 and bicarbonate being assisted by CA II.
Subsequent 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: CA VA
and/or VB within the mitochondria (to provide enough substrate
to PC), and CA II within the cytosol (for providing sufficient substrate
to ACC). Inhibition of CAs by clinically used sulfonamides such as
acetazolamide AZA, zonisamide ZNS or the sulfamate topiramate,
TPM, can decrease lipogenesis in adipocytes in cell culture.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 antiobesity
therapies.7
Sulfonamides of the type RCONH-A-SO2NH2 (where A is a pheny-
l(alkyl), halogeno-phenyl- or 1,3,4-thiadiazole moiety and R = fur-
an-2-yl; thien-2-yl or pyrrol-2-yl group) were previously reported
by this group,8 being easily prepared from the corresponding amino
sulfonamides bytheirreactionswithheterocyclicacylhalidesorcar-
boxylic acids in the presence of carbodiimides, by the tail
approach.8–11 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 antiglau-
coma activity in an animal model of this disease.8 Thus, compounds
of type RCONH-A-SO2NH2 have been used as lead molecules in the
presentwork. Asmanyof thethiophene-carboxamidesreportedear-
lier8 were among the most potent CAIs identified in the study, we
focused on this substitution pattern for the compounds reported
here, but some structurally related derivatives incorporating pheny-
lacetamido-andpyridyl-acetamidomoietieswerealsopreparedand
assayedas CAIs, inorderto understandtherolethatthetermianlpart
of the tail plays in modulating the isoform selectivity profile of such
compounds. The sulfonamides reported here (Scheme 1) differ of the
previously investigated ones by the incorporation of the aryl-/
hetarylacetamido moiety in their molecules instead of the hetaryl-
carboxamido one. Thus, they possess longer tails as compared to
the derivatives investigated earlier.8 The rationale for this modifica-
tionresidesinthefactthatthetailspresentinCAIsinteractwithami-
no acid residues situated towards the exit of the CA active site or on
its edge, as shown by extensive X-ray crystallographic work on such
enzyme-inhibitor adducts.12,13 On the other hand, these are the ami-
no acid residues which are less conserved among the various mam-
malian CA isoforms,1,2 and their interactions with the tails
incorporated in the inhibitors explain why most of the novel gener-
ation inhibitors usually 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 structures and homology modeling are available for
the interaction of many sulfonamides with isoforms hCA I, II, and
XIII,1–3 whereas fewer such reports for hCA IX9 and hCA VA14 were
published. All these works showed that both favorable interactions
as well as clashes with particular amino acid residues present only
in some CA isozymes15 are critical for the inhibition profile and
isozyme selectivity issues of the sulfonamides and their bioisosteres
such as the sulfamates and the sulfamides.
O
NH2
S
N
N
SO2NH2
O
O
O
O
CH3CONH
SO2NH2
N
S
O
O
O
ZNS
AZA
O
TPM
In earlier drug design studies of CA inhibitors (CAIs) from our group,8
we have explored the preparation of aromatic/heterocyclic sulfona-
mides incorporating furan-, thiophene- and pyrrole-carboxamide
moieties of the type RCONH-A-SO2NH2 (where A is a phenyl(alkyl),
halogenosubstituted-phenyl- or 1,3,4-thiadiazole moiety and
R = furan-2-yl; thien-2-yl or pyrrol-2-yl group). Such compounds
have been tested as inhibitors of three physiologically relevant mam-
malian isoforms, that is, hCA I and II and bCA IV (h = human, b = bo-
vine isoform), and were also active in vivo in an animal glaucoma
model.8 Some of the best such inhibitors were those incorporating
thiophene-2-carboxamide moieties. Here we report a study dealing
with the design of new sulfonamides possessing some structural sim-
ilarity with the previously investigated derivatives,8 prepared for tar-
geting the mitochondrial isoforms hCA VA and Vb. Compounds of the
type R-CH2–CONH-A-SO2NH2 (R = thien-2-yl; Ph- and pyridinyl; A is
a phenyl(alkyl), halogenosubstituted-phenyl- or 1,3,4-thiadiazole
moiety) were obtained and assayed as inhibitors of four physiologi-
cally relevant isoforms, the cytosolic hCA I and II, as well as the mito-
chondrial hCA VA and hCA VB.
O
(CH2)n
NH
S
S
5
SO2NH2
Y
N
N
O
O
H2N (CH2)n
SO2NH2
SO2NH2
S
Cl
NH
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
O
X1
NH
N
N
+
H2N
X1
Z
7
SO2NH2
Y
SO2NH2
S
-
N
N
X2
O
X1=X2=CH, Z=Cl
Cl
X1=N, X2=CH, Z=OH
X1=CH, X2=N, Z=OH
SO2NH2
4
S
X1
NH
3
8
Scheme 1. Synthesis of the new sulfonamides 5–8. Method A: Z = Cl, Et3N/MeCN; Method B: Z = OH, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide, DMAP/dioxane.

4896
Özlen Güzel et al. / Bioorg. Med. Chem. 17 (2009) 4894–4899
Reaction of 2-thienylacetyl chloride 1 or phenyl-/pyridyl-acetyl
nated sulfanilamide, homosulfanilamide, 4-aminoethyl-ben-
zenesulfonamide and 5-amino-1,3,4-thiadiazole-2-
halides/carboxylic acids 3 with aminosulfonamides 2 and 4, led to
the acylated sulfonamides 5–8 by the procedure reported earlier8
(Scheme 1). In addition to sulfanilamide, homosulfanilamide, 4-
aminoethyl-benzenesulfonamide and halogenated sulfanilamides
2, the heterocyclic derivative 5-amino-1,3,4-thiadiazole-2-sulfon-
amide 4 was also included in the study. All these aminosulfona-
mides have been shown earlier to lead to effective CAIs by
derivatization reactions employing the tail approach.8–10 The new
compounds 5–8 have been characterized by spectroscopic and
analytic methods which confirmed their structures (see Experi-
mental or details).
sulfonamide moieties, whereas the tail present in the acylat-
ing agent probably 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 inhibi-
tion, 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 three clinically used
compounds (AZA, ZNS and TPM) show much more potent
hCA II inhibitory activity, with KIs in the range of 10–
35 nM (Table 1). It should be also noted that the compounds
investigated earlier,8 having a CH2 moiety less than the pres-
ent 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).8 The-
ses 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 case) may have drastic consequences for the
enzyme inhibitory activity and selectivity profile against
various isozymes of such derivatives.
(iii) The first mitochondrial isoform, hCA VA, showed excellent
inhibition with the new compounds 5–8 investigated here,
as all of them possessed low nanomolar affinity for it (KIs
in the range of 6.7–10.2 nM, Table 1). Basically SAR is quite
flat again, since all the substitution patterns present in the
new compounds investigated here are beneficial for obtain-
ing potent hCA VA inhibitors. This is a quite unexpected
finding, but such a behavior was in fact observed earlier
for some 1,3,4-thiadiazole-sulfamides,17 which showed very
good hCA VA/VB inhibitory activity and selectivity for the
mitochondrial over cytosolic isoforms, although the struc-
turally related 1,3,4-thiadiazole-sulfonamides are devoid of
these interesting properties, being much better hCA II than
hCA VA/VB inhibitors.1–3 It should be also noted that the
clinically used compounds (except ZNS, KI of 20 nM) are
much weaker hCA VA inhibitors compared to derivatives
5–8 reported here. Indeed, both AZA and TPM show KIs of
63 nM for inhibiting hCA VA.
(iv) The second mitochondrial isoform, hCA VB, has an inhibition
profile with derivatives 5–8 very similar to that of hCA VA.
Thus, all these compounds are very good hCA VB inhibitors,
with KIs in the range of 5.9–9.3 nM, Table 1, being much bet-
ter inhibitors compared to the clinically used drugs which
show KIs in the range of 30–6033 nM.
(v) A very interesting property of compounds 5–8 reported here,
compared to most sulfonamides and sulfamates investigated
earlier,18,19 regards their selectivity for inhibiting the mito-
chondrial (hCA VA/VB) over cytosolic isoforms (hCA I/II).
Indeed, all sulfonamides and sulfamates investigated up to
now as CAIs generally showed a much higher affinity for
hCA II (a ubiquitous and catalytically very efficient iso-
form)1–3 compared to hCA VA and hCA VB.18,19 This can be
also observed for the three standard inhibitors present in
Table 1, AZA, ZNS and TPM. On the contrary, many of the
compounds 5–8 reported here show selectivity ratios for
2.2. Carbonic anhydrase inhibition
Inhibition data against four catalytically active human (h) CA
isoforms, that is, hCA I, hCA II (cytosolic) and hCA VA and hCA
VB (mitochondrial enzymes) with the new sulfonamides 5–8
reported here, as well as the standard compounds AZA, ZNS and
TPM are shown in Table 1.
The following structure–activity relationship (SAR) can be
drawn by considering data of Table 1:16
(i) Isoform hCA I was moderately inhibited by sulfonamides 5–
8 reported here. Thus, several derivatives, such as 5b, 7a and
7f, showed medium inhibitory activity, with inhibition con-
stants 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
inhibitors as compared to derivatives discussed above, with
KIs in the range of 60–84 nM. The best hCA I inhibitor was
the clinically used compound zonisamide ZNS (KI of
56 nM). Obviously both the aromatic sulfonamide head as
well as the aryl/hetaryl-acetyl moiety influence the biologi-
cal activity of these hCA I inhibitors. It may be observed that
efficient inhibitors incorporate both sulfanilamide, haloge-
Table 1
Inhibition data of human CA isozyms I, II (cytosolic), VA and VB (mitochondrial) with
compounds 5–8 and standard inhibitors (acetazolamide AZA, zonisamide ZNS and
topiramate TPM), by a stopped-flow, CO2 hydration assay16
No.
n,Y
X1
X2
KI* (nM)
hCA VAb
hCA Ia
hCA IIa
hCA VBb
5a
5b
5c
5d
5e
5f
0, H
0, F
0, Cl
0, Br
1, H
2, H
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
61
161
77
84
60
50
390
50
52
52
53
51
107
54
67
61
58
395
69
74
97
51
12
35
10
7.2
6.9
7.7
7.5
9.1
10.2
7.6
6.7
8.6
8.1
8.3
8.2
7.1
6.8
7.3
7.6
8.4
63
7.0
7.9
8.6
8.7
7.2
8.0
7.4
7.9
8.3
8.2
8.1
8.1
5.9
6.8
9.0
9.3
6.1
54
68
72
6
7a
7b
7c
7d
7e
7f
7g
7h
7i
0, H
1, H
2, H
0, F
0, Cl
0, Br
0, H
0, H
2, H
—
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
—
CH
CH
CH
CH
CH
CH
CH
N
N
CH
—
—
—
108
75
60
67
61
263
75
71
73
63
8
AZA
ZNS
TPM
—
—
—
250
56
250
—
—
20
63
6033
30
*
Errors in the range of 5–10% of the shown data, from three different assays, by a
CO2 hydration stopped-flow assay.16
a
Human, recombinant isozymes.
b
Full length, recombinant human isoforms.8–10

Özlen Güzel et al. / Bioorg. Med. Chem. 17 (2009) 4894–4899
4897
inhibiting hCA VA over hCA II of 56.5 (compound 5b), 55.6
(7f) and 15.9 (7a) whereas most of them have this parameter
in the range of 5.2–6.9. On the other hand, the selectivity
ratio for inhibiting hCA VA over hCA II with AZA is of 0.19,
clearly showing that the standard drug has a much higher
affinity for hCA II than for hCA VA. The same situation is true
for the selectivity ratio of inhibiting hCA VB over hCA II.
Indeed, the most selective inhibitors were again 5b, 7a and
7f, possessing selectivity ratios in the range of 13.5–66.9.
Most of the other compounds 5–8 have this parameter of
around 5.8–10.9, being anyhow much more isozyme V-
selective as compared to the clinically used drugs (selectiv-
ity ratios in the range of 0.05–0.33 for AZA, ZNS and TPM).
Thus, we have detected here a new class of isozyme VA/VB
selective inhibitors, which may be useful for the investiga-
tion of the antiobesity activity of this class of derivatives.
reasonable conversion was reached (TLC control). The solvent
was evaporated in vacuum and the resulted product was treated
with 15–20 mL cold water. The crude solid product was filtered,
washed with water and air dried. The obtained compounds were
further purified by recrystallisation from ethanol.
4.1.1.2. 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-ethylcarbo-
diimide hydrochloride and 0.06 g (0.5 mmol) dimethylaminopyri-
dine (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 precipi-
tate was filtered, washed with ethanol and further purified by
recrystallisation from ethanol.
3. Conclusions
4.1.1.3. 4-[2-(2-Thienyl)acetamido]benzenesulfonamide
5a. Yield 40%; mp 207–208 °C; IR(KBr) (m
, cmꢀ1), 1663 (C@O), 1160,
A library 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 four physiologically relevant CA isoforms, the cytosolic hCA I
and hCA II, and the mitochondrial hCA VA and hCA VB. The new com-
pounds show moderate inhibition of the two cytosolic isoforms and
excellentinhibitoryactivityagainstthetwomitochondrialenzymes,
with many low nanomolar inhibitors detected (KIs in the range of
5.9–10.2 nM). All substitution patterns explored here lead to effec-
tive hCA VA/VB inhibitors. Some hCA VA/VB selective compounds
were also detected, with selectivity ratios for inhibiting the mito-
chondrial over the cytosolic isozymes of >50. As hCA VA/VB are
involved in several biosynthetic processes catalyzed by pyruvate
carboxylase, acetyl CoA carboxylase, and carbamoyl phosphate syn-
thetases I and II, providing the bicarbonate substrate to these car-
boxylating enzymes involved in fatty acid biosynthesis, their
selective inhibition may lead to the development of new antiobesity
agents possessing a totally new mechanism of action.
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). Elem. Anal. Calcd for C12H12N2O3S2 (296.36): C, 48.63;
H, 4.08; N, 9.45. Found: C, 48.51; H, 4.32; N, 9.18.
4.1.1.4. 4-[2-(2-Thienyl)acetamido]-3-fluorobenzenesulfona-
mide 5b. Yield 69%; mp 176–177 °C; IR(KBr) (m
, cmꢀ1), 1676 (C@O),
1151, 1320 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 4.04 (2H,
s, CH2CO), 7.01 (2H, s, thiophene C3,4–H), 7.44 (3H, s, thiophene C5–
H and SO2NH2), 7.62–7.74 (2H, m, phenyl C2,6–H), 8.20 (1H, d,
J = 6.00 Hz, phenyl C5–H), 10.31 (1H, s, CONH). Elem. Anal. Calcd
for C12H11FN2O3S2 (314.35): C, 45.85; H, 3.53; N, 8.91. Found: C,
45.68; H, 3.65; N, 8.82.
4.1.1.5. 4-[2-(2-Thienyl)acetamido]-3-chlorobenzenesulfona-
mide 5c. Yield 65%; mp 188–189 °C; IR(KBr) (m
, cmꢀ1), 1683 (C@O),
1166, 1334 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 4.03 (2H, s,
CH2CO), 6.99–7.05 (2H, m, thiophene C3,4–H), 7.43 (1H, dd, J = 5.10,
1.20 Hz, thiophene C5–H), 7.49 (2H, s, SO2NH2), 7.76 (1H, dd,
J = 8.40, 2.10 Hz, phenyl C6–H), 7.90 (1H, d, J = 2.10 Hz, phenyl C2–
H), 8.03 (1H, d, J = 8.40 Hz, phenyl C5–H), 10.20 (1H, s, CONH). Elem.
Anal. Calcd for C12H11ClN2O3S2 (330.81): C, 43.57; H, 3.35; N, 8.47.
Found: C, 43.86; H, 3.19; N, 8.50.
4. Experimental
4.1. Chemistry
Buffers, sulfonamides 2, 4 and other chemicals (e.g., 1 and 3)
were from Sigma–Aldrich (Milan, Italy) of highest purity available,
and were used without further purification. Halogenosulfanila-
mides were prepared as reported earlier.20 All CA isozymes were
recombinant ones produced and purified in our laboratory as
described earlier.9,12–15 All melting points were measured in open
capillary tubes with a Buchi 540 instrument and are uncorrected.
The compounds were checked for purity by TLC on silica gel
HF254 (E.Merck, Darmstadt, Germany). IR (KBr) spectra were
recorded using a Perkin–Elmer 1600 FTIR spectrophotometer and
4.1.1.6. 4-[2-(2-Thienyl)acetamido]-3-bromobenzenesulfona-
mide 5d. Yield 30%; mp 191–192 °C; IR(KBr) (m
, cmꢀ1), 1670 (C@O),
1163, 1334 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 4.04 (2H,
s, CH2CO), 7.01–7.05 (2H, m, thiophene C3,4–H), 7.42–7.44 (1H, m,
thiophene C5–H), 7.49 (2H, s, SO2NH2), 7.79 (1H, dd, J = 8.70,
2.10 Hz, phenyl C6–H), 7.93 (1H, d, J = 8.70 Hz, phenyl C5–H), 8.06
(1H, d, J = 2.10 Hz, phenyl C2–H), 9.90 (1H, s, CONH). Elem. Anal.
Calcd for C12H11BrN2O3S2 (375.26): C, 38.41; H, 2.95; N, 7.47.
Found: C, 38.73; H, 3.12; N, 7.21.
all values are expressed as
m .
max, cmꢀ1 1H NMR spectra were
recorded on a Varian 300 MHz spectrometer using DMSO-d6 as sol-
vent, and chemical shifts are given in ppm with TMS as standard.
4.1.1.7. 4-[2-(2-Thienyl)acetamidomethyl]benzenesulfonamide
5e. Yield 43%; mp196–197 °C; IR(KBr) (m
, cmꢀ1), 1644 (C@O), 1162,
1335 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.75 (2H, s,
CH2CO), 4.36 (2H, s, NHCH2), 6.97 (2H, s, thiophene C3,4–H), 7.34
(2H, s, SO2NH2), 7.43 (3H, d, J = 6.60 Hz, thiophene C5–H and phe-
nyl C3,5–H), 7.78 (2H, d, J = 6.90 Hz, phenyl C2,6–H), 8.65 (1H, s,
CONH). Elem. Anal. Calcd for C13H14N2O3S2 (310.39): C, 50.30; H,
4.55; N, 9.03. Found: C, 50.39; H, 4.27; N, 8.84.
4.1.1. Preparation of sulfonamides 5–8
4.1.1.1. Method A. An amount of 5 mmol 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 phen-
ylacetyl chloride/2-thienylacetyl chloride 1 or 3 dissolved in 3 mL
MeCN was added dropwise during 10 min. (immediately, precipi-
tate appeared). The reaction was stirred overnight or until a
4.1.1.8. 4-[2-(2-Thienyl)acetamidoethyl]benzenesulfonamide
5f. Yield 31%; mp 174–175 °C; IR(KBr) (m
, cmꢀ1), 1631 (C@O), 1156,

4898
Özlen Güzel et al. / Bioorg. Med. Chem. 17 (2009) 4894–4899
1336 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 2.79 (2H, t,
J = 7.20 Hz, NHCH2CH2), 3.38 (2H, s, CH2CO), 3.60 (2H, d,
J = 6.30 Hz, NHCH2), 6.87 (1H, d, J = 3.60 Hz, thiophene C3–H),
6.96 (1H, t, J = 3.60 Hz, thiophene C4–H), 7.33 (2H, s, SO2NH2),
7.35–7.37 (3H, m, thiophene C5–H and phenyl C3,5–H), 7.72 (2H,
d, J = 8.10 Hz, phenyl C2,6–H), 8.22 (1H, s, CONH). Elem. Anal. Calcd
for C14H16N2O3S2 (324.41): C, 51.83; H, 4.97; N, 8.63. Found: C,
52.05; H, 4.80; N, 8.39.
dd, J = 8.40, 2.10 Hz, phenyl C6–H), 7.89 (1H, d, J = 8.40 Hz, phenyl
C5–H), 8.05 (1H, d, J = 2.10 Hz, phenyl C2–H), 9.80 (1H, s, CONH).
Elem. Anal. Calcd for C14H13BrN2O3S (369.23): C, 45.54; H, 3.55;
N, 7.59. Found: C, 45.27; H, 3.68; N, 7.26.
4.1.1.16. 4-(2-Pyridin-2-ylacetamido)benzenesulfonamide
7g. Yield 30%; mp 219–220 °C; IR(KBr) (m
, cmꢀ1), 1666 (C@O),
1161, 1333 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 4.28
(2H, s, CH2CO), 7.30 (2H, s, SO2NH2), 7.74–7.79 (5H, m, phenyl
C2,3,5,6–H and pyridine C5–H), 7.88 (1H, d, J = 7.80 Hz, pyridine
C3–H), 8.34 (1H, t, J = 7.50 Hz, pyridine C4–H), 8.81 (1H, d,
J = 5.40 Hz, pyridine C6–H), 10.99 (1H, s, CONH). Elem. Anal. Calcd
for C13H13N3O3S (291.32): C, 53.60; H, 4.50; N, 14.42. Found: C,
53.86; H, 4.33; N, 14.28.
4.1.1.9. 5-[2-(2-Thienyl)acetamido]-1,3,4-thiadiazole-2-sulfon-
amide 6. Yield 76%; mp 248–249 °C; IR(KBr) (m
, cmꢀ1), 1686 (C@O),
1169, 1371 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 4.10 (2H,
s, CH2CO), 6.99–7.03 (2H, m, thiophene C3,4–H), 7.43–7.45 (1H, m,
thiophene C5–H), 8.34 (2H, br s, SO2NH2). Elem. Anal. Calcd for
C8H8N4O3S3 (304.36): C, 31.57; H, 2.65; N, 18.41. Found: C,
31.43; H, 2.90; N, 18.24.
4.1.1.17. 4-(2-Pyridin-4-ylacetamido)benzenesulfonamide
7h. Yield 33%; mp 144–145 °C; IR(KBr) (
1155, 1331 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 4.14
(2H, s, CH2CO), 7.29 (2H, s, SO2NH2), 7.78 (4H, s, phenyl C2,3,5,6–
m
, cmꢀ1), 1707 (C@O),
4.1.1.10. 4-(2-Phenylacetamido)benzenesulfonamide 7a. Yield
35%; mp 205–206 °C; IR(KBr) (
m
, cmꢀ1), 1691 (C@O), 1170, 1323
(SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.70 (2H, s, CH2CO),
7.26 (2H, s, SO2NH2), 7.35 (5H, d, J = 4.50 Hz, Ar-H), 7.77 (4H, d,
J = 3.60 Hz, Ar-H), 10.50 (1H, s, CONH). Elem. Anal. Calcd for
C14H14N2O3S (290.33): C, 57.92; H, 4.86; N, 9.65. Found: C, 58.09;
H, 4.64; N, 9.61.
H), 8.00 (2H, d, J = 6.00 Hz, pyridine C3,5–H), 8.86 (2H, d,
J = 5.40 Hz, pyridine C2,6–H), 11.00 (1H, s, CONH). Elem. Anal. Calcd
for C13H13N3O3S (291.32): C, 53.60; H, 4.50; N, 14.42. Found: C,
53.45; H, 4.74; N, 14.13.
4.1.1.18. 4-(2-Pyridin-4-ylacetamidoethyl)benzenesulfonamide
4.1.1.11. 4-(2-Phenylacetamidomethyl)benzenesulfonamide
7i. Yield 32%; mp 133–134 °C; IR(KBr) (m
, cmꢀ1), 1651 (C@O), 1156,
7b. Yield 46%; mp 204–205 °C; IR(KBr) (
m
, cmꢀ1), 1624 (C@O),
1333 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 2.73 (2H, s,
NHCH2CH2), 3.02 (2H, s, CH2CO), 3.45 (2H, s, NHCH2), 7.21 (2H, s,
SO2NH2), 7.34 (2H, s, pyridine C3,5–H), 7.38 (2H, d, J = 7.20 Hz, phe-
nyl C3,5–H), 7.74 (2H, d, J = 7.20 Hz, phenyl C2,6–H), 8.31 (1H, s,
CONH), 8.47 (2H, s, pyridine C2,6–H). Elem. Anal. Calcd for
C15H17N3O3S (319.37): C, 56.41; H, 5.37; N, 13.16. Found: C,
56.62; H, 5.19; N, 13.04.
1151, 1324 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.48
(2H, s, CH2CO), 4.31 (2H, d, J = 5.70 Hz, NHCH2), 7.24 (2H, s,
SO2NH2), 7.28 (5H, m, Ar-H), 7.37 (2H, d, J = 8.40 Hz, phenyl C3,5
–
H), 7.73 (2H, d, J = 8.40 Hz, phenyl C2,6–H), 8.60 (1H, s, CONH).
Elem. Anal. Calcd for C15H16N2O3S (304.36): C, 59.19; H, 5.30; N,
9.20. Found: C, 59.36; H, 4.98; N, 9.04.
4.1.1.12. 4-(2-Phenylacetamidoethyl)benzenesulfonamide
4.1.1.19. 5-(2-Phenylacetamido)-1,3,4-thiadiazole-2-sulfon-
7c. Yield 77%; mp 184–185 °C; IR(KBr) (
m
, cmꢀ1), 1626 (C@O), 1156,
amide 8. Yield 68%; mp 246–247 °C; IR(KBr) (m
, cmꢀ1), 1688 (C@O),
1334 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 2.79 (2H, t,
J = 6.90 Hz, NHCH2CH2), 3.29 (2H, s, CH2CO), 3.40 (2H, d,
J = 7.80 Hz, NHCH2), 7.21–7.29 (5H, m, Ar-H), 7.32 (2H, s, SO2NH2),
7.36 (2H, d, J = 8.40 Hz, phenyl C3,5–H), 7.74 (2H, d, J = 8.40 Hz, phe-
nyl C2,6–H), 8.14 (1H, s, CONH). Elem. Anal. Calcd for C16H18N2O3S
(318.39): C, 60.36; H, 5.70; N, 8.80. Found: C, 60.51; H, 5.49; N, 8.53.
1170, 1372 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.90 (2H,
s, CH2CO), 7.29 (2H, d, J = 6.00 Hz, phenyl C2,6–H), 7.35 (3H, s, phe-
nyl C3,4,5,–H), 8.35 (2H, s, SO2NH2). Elem. Anal. Calcd for
C10H10N4O3S2 (298.34): C, 40.26; H, 3.38; N, 18.78. Found: C,
39.90; H, 3.46; N, 18.54.
4.2. CA inhibition assay
4.1.1.13. 4-(2-Phenylacetamido)-3-fluorobenzenesulfonamide
7d. Yield 65%; mp 190–191 °C; IR(KBr) (
m
, cmꢀ1), 1675 (C@O),
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the catalytic/inhibition of various
CA isozymes as reported by Khalifah.16 Phenol Red (at a concentra-
tion of 0.2 mM) has been used as indicator, working at the absor-
bance 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 inhibitoryin the used concentration),8
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 sub-
strate. 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 inhibi-
tion constant. Inhibitor and enzyme solutions were preincubated to-
gether 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,9 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.12–15
1150, 1338 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.82
(2H, s, CH2CO), 7.29 (1H, d, J = 3.90 Hz, Ar-H), 7.35 (4H, d,
J = 4.80 Hz, Ar-H), 7.44 (2H, s, SO2NH2), 7.62 (1H, d, J = 8.40 Hz,
phenyl C6–H), 7.68 (1H, d, J = 2.10 Hz, phenyl C2–H), 8.18 (1H, t,
J = 8.40 Hz, phenyl C5–H), 10.24 (1H, s, CONH). Elem. Anal. Calcd
for C14H13FN2O3S (308.32): C, 54.54; H, 4.25; N, 9.09. Found: C,
54.40; H, 3.91; N, 8.83.
4.1.1.14. 4-(2-Phenylacetamido)-3-chlorobenzenesulfonamide
7e. Yield 36%; mp 174–175 °C; IR(KBr) (m
, cmꢀ1), 1672 (C@O),
1162, 1334 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.83
(2H, s, CH2CO), 7.30–7.38 (5H, m, Ar-H), 7.47 (2H, s, SO2NH2),
7.76 (1H, dd, J = 8.70, 3.00 Hz, phenyl C6–H), 7.91 (1H, d,
J = 3.00 Hz, phenyl C2–H), 8.00 (1H, d, J = 8.70 Hz, phenyl C5–H),
9.92 (1H, s, CONH). Elem. Anal. Calcd for C14H13ClN2O3S (324.78):
C, 51.77; H, 4.03; N, 8.63. Found: C, 52.06; H, 3.94; N, 8.57.
4.1.1.15. 4-(2-Phenylacetamido)-3-bromobenzenesulfonamide
7f. Yield 30%; mp 180–181 °C; IR(KBr) (m
, cmꢀ1), 1667 (C@O), 1164,
1338 (SO2); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.80 (2H, s,
CH2CO), 7.28–7.36 (5H, m, Ar-H), 7.47 (2H, s, SO2NH2), 7.79 (1H,

Özlen Güzel et al. / Bioorg. Med. Chem. 17 (2009) 4894–4899
4899
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Acknowledgments
This research was financed in part by a grant of the 6th Frame-
work Programme of the European Union (DeZnIT project). Ö.G. is
grateful to TUBITAK (Ankara, Turkey) for the providing financing
under the Contract No. 2219/2008.
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