Sulfapyridine-like benzenesulfonamide derivatives as inhibitors of carbonic anhydrase isoenzymes I, II and VI

Article abstract of DOI:10.3109/14756366.2011.617745

The inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I, II and human serum isozyme VI, with a series of tosylited aromatic amine derivatives was investigated. The KI ranges of compounds 1-14 and acetazolamide against hCA I ranged between 1.130 and- 448.2 μM, against hCA II between 0.103 and- 14.3 μM, and against hCA VI ranged between 0.340 and- 42.39 μM. Tosylited aromatic amine derivatives are thus interesting hCA I, II and VI inhibitors, and might be used as leads for generating enzyme inhibitors eventually targeting other isoforms which have not been assayed yet for their interactions with such agents.

Full text of DOI:10.3109/14756366.2011.617745

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(6): 818–824
2012 Informa UK, Ltd.
©
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.617745
ReseaRꢀh aRꢁIꢀle
Sulfapyridine-like benzenesulfonamide derivatives as
inhibitors of carbonic anhydrase isoenzymes I, II and VI
1
,2
1
1
1,3
1
Cemalettin Alp , Şeyda Özsoy , Nurdan Alcan Alp , Deryanur Erdem , Mehmet Serdar Gültekin ,
1
1,4
5
Ömer İrfan Küfrevioğlu , Murat Şentürk , and Claudiu T. Supuran
1
2
Ataturk University, Science Faculty, Department of Chemistry, Erzurum, Turkey, Erzincan University, Çayırlı
3
Vocational School, Erzincan, Turkey, Aksaray University, Faculty of Arts and Science, Chemistry Department, Aksaray,
Turkey, A g˘ rı Ibrahim Çeçen University, Science and Art Faculty, Chemistry Department, A g˘ rı, Turkey, Università degli
Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Sesto Fiorentino (Florence), Italy
4
˙
5
abꢂtrꢃct
The inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I, II and human serum isozyme
VI, with a series of tosylited aromatic amine derivatives was investigated. The K ranges of compounds 1–14 and
I
acetazolamide against hCA I ranged between 1.130 and- 448.2 μM, against hCA II between 0.103 and- 14.3 μM, and
against hCA VI ranged between 0.340 and- 42.39 μM. Tosylited aromatic amine derivatives are thus interesting hCA I,
II and VI inhibitors, and might be used as leads for generating enzyme inhibitors eventually targeting other isoforms
which have not been assayed yet for their interactions with such agents.
Kꢄywordꢂ: Carbonic anhydrase, inhibition, benzenesulfonamides
Introduction
Carbonic anhydrase (CA, EC 4.2.1.1) enzymes are
involved in important physiological and pathological
Carbonic anhydrase inhibitors or activators have
several medical applications, such as in the treatment
of glaucoma, as diuretics, in the management of several
neurological disorders, including epilepsy, possibly in
the treatment of Alzheimer’s disease, whereas several
agents are in clinical evaluations as antiobesity or antitu-
functions, such as pH and CO homeostasis, respira-
2
−
tion and transport of CO /HCO between metabolizing
2
3
tissues and the lungs, ion secretion in different tissues/
organs and biosynthetic reactions (such as gluconeogen-
esis, lipogenesis and ureagenesis). Among the sixteen 16
isoenzymes, described up to now, CAI and CAII are pres-
ent at high concentrations in the cytosol in erythrocytes,
3
–7
mor drugs/diagnostic tools .
A class of derivatives which showed very promis-
ing applications among the various CAIs reported by
Supuran’s group in the last years, were the thioureas
obtained from isothiocyanato sulfonamides (such as
e.g., 4-isothiocyanatobenzenesulfonamide) and amines,
1
–8
and CAII has the highest turnover rate of all the CAs .
Carbonic anhydrase VI (CA VI) is a secretory enzyme that
was initially described in the ovine parotid gland, saliva
8
22–24
and normal human serum .
hydrazines or amino acids . Such compounds gener-

e interactions between specific enzymes and vari-
ally showed potent inhibitory activity against the cyto-
solic isozyme hCA II as well as the transmembrane,
tumor-associated isozyme hCA IX, being thus interesting
candidates for developing anti-glaucoma/anti-tumor
ous types of inhibitors are of great importance and cor-
responding investigations have become very popular in
the recent years. ese substances are sometimes useful
in medicinal applications, whereas they have unde-
sirable effects on both human metabolism and other
2
2–24
therapies based on them
.
Many sulfonamide derivatives have been widely used
as pro-drugs or drugs. For instance, sulfadiazine is used
9
–21
organisms
.
Address for Correspondence: Dr. Murat Şentürk, Ağrı İbrahim Çeçen University, Science and Art Faculty, Chemistry Department,
4100-Ağrı, Turkey. Tel: +90 442 2314414. Fax: +90 442 2314388. E-mail: senturkm36@gmail.com; msenturk@agri.edu.tr
0
(
Received 03 August 2011; revised 22 August 2011; accepted 23 August 2011)
8
18

Sulfapyridine-like benzenesulfonamide derivatives 819
as an antibiotic, sulfapyridine is mainly used for treat-
ment of bacterial infections, acetazolamide is mainly
used anti-glaucoma agent.
63.13; H, 5.30; N, 5.66; S, 12.97. Found: C, 62.90; H, 5.40;
N, 5.64; S, 12.72.
Our groups recently investigated the interaction of
CA isozymes I, II with salicylic acid derivatives, some
antioxidant phenolic compounds, some purine analogue
Synthesis of 4-Methyl-N-(naphthalen-2-yl)
benzenesulfonamide (3)
In %98 yield, Compound 3 was recrystallized from
9–13,25,26
1
drugs, organic nitrates, and etc.
. We would like to
CH Cl -hexane solution. M.P.: 153–155°oC. H -NMR
2
2
extend these earlier investigations to some benzenesul-
fonamide derivatives in order to discover novel powerful
CAIs which might have implications in medicine.
In this work, toward the discovery of CA inhibi-
tors, we synthesized benzenesulfonamide derivatives.
Compounds were evaluated for their ability to inhibit
(400 MHz, CDCl , δ, ppm): δ= 2.32 (s, 3H in NH-Ts), 7.14
3
(d, 2H, J= 8.24 Hz, AA’ part of AA’BB’ system in NH-Ts),
7.25–7.46 (m, 4H in Naph.), 7.64 (d, 2H, J= 8.24 Hz, BB’
part of AA’BB’ system in NH-Ts), 7.70 (d, 1H, J= 6.95 Hz
in Naph.), 7.79 (m, 1H in Naph.), 7.85 (d, 1H, J= 8.05 Hz
1
3
in Naph.). C-NMR (100 MHz, CDCl , ppm): 21.7, 121.7,
3
human CA-I, II and VI. Inhibition is reported as K (µM)
and the results are the average of at least three indepen-
dent experiments.
122.9, 125.6, 126.5, 126.8, 127.4, 127.6, 128.6, 129.1, 129.7,
I
−-1
131.7, 134.5, 136.7, 144.0. IR (KBr, cm ): 3264, 1597, 1454,
1410, 1344, 1314, 1160, 1091. Anal. calcd. for C H NO S
17
15
2
(
6
297.37) C, 68.66; H, 5.08; N, 4.71; S, 10.78. Found: C,
8.46; H, 5.20; N, 4.72; S, 10.89.
Mꢃtꢄriꢃꢅꢂ ꢃnd mꢄtꢆodꢂ
Chemicals
Synthesis of 4-Methyl-N-(2,4,6-trimethyl-phenyl)-
benzenesulfonamide (4)
All chemicals and solvents are commercially available
and were used after distillation or treatment with drying
agents. Column chromatography (CC): silica gel (SiO2;
In %95 yield, Compound 4 was recrystallized from
1
CH Cl -hexane solution. M.P.: 166–168°C. H-NMR
2
2
6
0 mesh, Merck-Darmstadt, Germany). Preparative
(400 MHz, CDCl , δ, ppm): δ= 1.98 (s, 6H in Ph-CH ),
3
3
thick layer chromatography (PLC): 1 mm of SiO 60 PF
2.24 (s, 3H in Ph-CH ) 2.41 (s, 3H in NH-Ts), 5.92 (brs, 1H,
2
3
(Merck) on glass plates. Mp: cap. melting-point appara-
-NH), 6.81(s, 2H in Ph-CH ), 7.24 (d, 2H, J= 8.23 Hz, AA’
3
tus (BUCHI 530 - Flawil, Switzerland); uncorrected. IR
Spectra: solns. in 0.1 mm cells with a Mattson 1000 FT-IR
spectrophotometer (Cambrige, England). H- and C-
NMR spectra: 200 (50) and 400 (100)-MHz Varian spec-
part of AA’BB’ system in NH-Ts), 7.60 (d, 2H, J= 8.23 Hz,
BB’ part of AA’BB’ system in NH-Ts). C-NMR (100 MHz,
1
3
1
13
CDCl , ppm): 18.8, 21.1, 21.7, 127.5, 129.7, 129.8, 130.2,
3
−1
137.7, 137.8, 138.2, 143.7. IR (KBr, cm ): 3282, 1597, 1481,
1400, 1328, 1289, 1161, 1091. Anal. calcd. for C H NO S
trometer (Danbury, USA); δ in ppm; Me Si as the internal
4
16 19
2
standard. Elemental analyses: Leco CHNS-932 apparatus
(289.39) C, 66.41; H, 6.62; N, 4.84; S, 11.08. Found: C,
66.24; H, 6.80; N, 4.85; S, 10.97.
(Michigan, USA).
General procedure for the synthesis of
N-aryl-ptoluenesulfonamides
Synthesis of 4-Methyl-N-o-tolyl-benzenesulfonamide (5)
In%96yield,Compound5wasrecrystallizedfromCH Cl -
2
2
1
To a mixture of aryl amine derivative (10 mmol) and
pyridine (10 mL, 120 mmol) in dichloromethane (50 mL)
was added slowly p-toluenesulfonyl chloride (2.29 g, 12
mol, 1.2 eq.). e reaction mixture was stirred at room
hexane solution. M.P.: 102–104°C. H-NMR (400 MHz,
CDCl , δ, ppm): δ= 2.01 (s, 3H in Ph-CH ),2.38 (s, 3H in
3
3
NH-Ts), 6.60 (brs, 1H, -NH), 7.06 (m, 2H (ortho, para)
in Ph-CH ), 7.12 (m, 2H (meta) in Ph-CH ), 7.21 (d, 2H,
3
3
27
temperature for 16 h . e reaction mixture was washed
with 5% HCl. en the organic layer was washed with
J= 8.26 Hz, AA’ part of AA’BB’ system in NH-Ts), 7.31 (d,
1H, J= 8.06 Hz (meta) in Ph-CH ), 7.62 (d, 2H, J= 8.26 Hz,
3
1
3
NaHCO solution and dried over sodium sulfate, filtered,
BB’ part of AA’BB’ system in NH-Ts). C-NMR (100 MHz,
3
and evaporated to afford a residue. Purification of the
residue by silica gel column chromatography with eth-
ylacetate/hexane provided the corresponding N-aryl-p-
toluenesulfonamide 2–11 in 67–100% yields.
CDCl , ppm): 17.8, 21.7, 124.6, 126.4, 127.1, 127.4, 129.8,
3
−
1
131.0, 131.6, 134.8, 137.0, 144.0. IR (KBr, cm ): 3278,
1598, 1495, 1403, 1330, 1290, 1162, 1092. Anal. calcd. for
C H NO S (261.34) C, 64.34; H, 5.79; N, 5.36; S, 12.27.
1
4
15
2
Found: C, 64.18; H, 5.84; N, 5.34; S, 12.25.
Synthesis of 4-methyl-N-p-tolylbenzenesulfonamide (2):
In Quantitative yield, Compound 2 was recrystallized
Synthesis of N-(2-cyanophenyl)-
4-methylbenzenesulfonamide (6)
1
from CH Cl -hexane solution. M.P.: 97–99°C. H -NMR
2
2
(
400 MHz, CDCl , δ, ppm): δ= 2.36 (s, 3H in NH-Ts), 7.06
In %67 yield, Compound 6 was recrystallized from
3
1
(s, 1H, -NH), 7.10 (d, 2H, J= 8.23 Hz, AA’ part of AA’BB’ sys-
CH Cl -hexane solution. M.P.: 129–131°C. H-NMR
2
2
tem in NH-Ts), 7.22 (m, 5H in Ph), 7.68 (d, 2H, J= 8.23 Hz,
BB’ part of AA’BB’ system in NH-Ts). C-NMR (100 MHz,
(400 MHz, CDCl , δ, ppm): δ= 2.38 (s, 3H in NH-Ts), 7.17
3
13
(m, 1H (meta) in Ph-CN), 7.25 (d, 2H, J= 8.42 Hz, AA’
part of AA’BB’ system in NH-Ts), 7.46 (dd, 1H (meta) in
Ph-CN, J= 7.7, 1.5 Hz), 7.52 (brdt, 1H(para) in Ph-CN,
J= 8.8, 1.5 Hz), 7.70 (d, 2H, J= 8.42 Hz, BB’ part of AA’BB’
CDCl , ppm): 21.8, 121.7, 125.4, 127.5, 129.5, 129.9, 136.0,
3
−
1
1
1
36.8, 144.1. IR (KBr, cm ): 3257, 1599, 1496, 1412, 1336,
303, 1159, 1092. Anal. calcd. for C H NO S (247.31) C,
13
13
2
©
2012 Informa UK, Ltd.

8
20 C. Alp et al.
system in NH-Ts), 7.72 (d, 1H(ortho) in Ph-CN, J= 8.80
Synthesis of N-(4-cyanophenyl)-
4-methylbenzenesulfonamide (10)
In %72 yield, Compound 10 was recrystallized from
1
3
Hz). C-NMR (100 MHz, CDCl , ppm): 21.8, 104.5, 115.9,
3
1
21.9, 125.4, 127.6, 130.2, 133.0, 134.4, 135.7, 139.6, 145.0.
−1
1
IR (KBr, cm ): 3241, 2230, 1599, 1493, 1455, 1413, 1342,
163, 1091. Anal. calcd. for C H N O S (272.32) C, 61.75;
CH Cl -hexane solution. M.P.: 177–179°C. H-NMR
2
2
1
(400 MHz, CDCl , δ, ppm): δ= 2.39 (s, 3H in NH-Ts),
14
12
2
2
3
H, 4.44; N, 10.29; S, 11.77. Found: C, 61.64; H, 4.54; N,
0.36; S, 11.61.
7.18 (d, 2H, J= 9.0 Hz, AA’ part of AA’BB’ system (meta)
in Ph-CN), 7.27 (d, 2H, J= 8.2 Hz, AA’ part of AA’BB’ sys-
tem in NH-Ts), 7.51 (d, 2H, J= 9.0 Hz, BB’ part of AA’BB’
system (ortho) in Ph-CN), 7.76 (d, 2H, J= 8.2 Hz, BB’ part
1
Synthesis of 4-Methyl-N-(2-nitrophenyl)
benzenesulfonamide (7)
1
3
of AA’BB’ system in NH-Ts). C-NMR (100 MHz, CDCl ,
3
In %75 yield, Compound 7 was recrystallized from
ppm): 21.8, 107.9, 118.7, 119.5, 127.5, 130.2, 133.8, 135.8,
141.3, 145.0. IR (KBr, cm ): 3237, 2226, 1607, 1508, 1462,
1
−1
CH Cl -hexane solution. M.P.: 108–110°C. H-NMR
2
2
(
400 MHz, CDCl , δ, ppm): δ= 2.37 (s, 3H in NH-Ts), 7.14
1402, 1342, 1159, 1090. Anal. calcd. for C H N O S
3
14 12
2
2
(
2
ddd, 1H (meta) in Ph-NO , J = 8.4, 7.3, 1.5 Hz), 7.25 (d,
H, J = 8.2 Hz, AA’ part of AA’BB’ system in NH-Ts), 7.57
(272.32) C, 61.75; H, 4.44; N, 10.29; S, 11.77. Found: C,
61.63; H, 4.46; N, 10.28; S, 11.70.
2
(
ddd, 1H (para) in Ph-NO , J = 8.4, 7.3, 1.5 Hz), 7.72 (d,
2
2
H, J = 8.2 Hz, BB’ part of AA’BB’ system in NH-Ts), 7.83
Synthesis of 4-Methyl-N-(4-nitrophenyl)
benzenesulfonamide (11)
In %71 yield, Compound 11 was recrystallized from
(dd, 1H (meta) in Ph-CN, J = 8.4, 1.5 Hz), 8.09 (dd, 1H
1
3
(
ortho) in Ph-CN, J = 8.4, 1.5 Hz), 9.84 (brs, 1H, -NH). C-
1
NMR (100 MHz, CDCl , ppm): 21.8, 121.2, 124.0, 126.4,
CH Cl -hexane solution. M.P.: 181–183°C. H-NMR
3
2
2
1
27.5 (2C), 130.2, 134.2, 135.9, 136.1, 145.0. IR (KBr,
(400 MHz, CDCl , δ, ppm): δ= 2.40 (s, 3H in NH-Ts), 7.20
(d, 2H, J= 9.2 Hz, AA’ part of AA’BB’ system (meta) in
3
−
1
cm ): 3286, 1610, 1529, 1487, 1393, 1348, 1275, 1169,
1
4
090. Anal. calcd. for C H N O S (292.31) C, 53.42; H,
.14; N, 9.58; S, 10.97. Found: C, 53.31; H, 4.17; N, 9.56;
Ph-NO ), 7.29 (d, 2H, J= 8.3 Hz, AA’ part of AA’BB’ system
1
3
12
2
4
2
in NH-Ts), 7.77 (d, 2H, J= 8.3 Hz, BB’ part of AA’BB’ sys-
tem in NH-Ts), 8.12 (d, 2H, J= 9.2 Hz, BB’ part of AA’BB’
S, 10.90.
1
3
system (meta) in Ph-CN). C-NMR (100 MHz, CDCl3,
Synthesis of 4-Methyl-N-p-tolyl-benzenesulfonamide (8)
ppm): 21.8, 118.9, 125.6, 127.5, 130.3, 135.8, 142.9, 145.
−1
In Quantitative yield, Compound 8 was recrystallized
IR (KBr, cm ): 3254, 1596, 1520, 1497, 1342, 1296, 1161,
1090. Anal. calcd. for C H N O S (292.31) C, 53.42; H,
1
from CH Cl -hexane solution. M.P.: 115–117°C. H-
2
2
13 12
2
4
NMR (400 MHz, CDCl , δ, ppm): δ= 2.24 (s, 3H in
4.14; N, 9.58; S, 10.97. Found: C, 53.27; H, 4.16; N, 9.63;
S, 11.03.
3
Ph-CH ), 2.35 (s, 3H in NH-Ts), 6.89 (brs, 1H, -NH), 7.00
3
(d, 2H, J= 4.21 Hz, AA’ part of AA’BB’ system (ortho) in
Ph-CH ), 7.18 (d, 2H, J= 8.23 Hz, AA’ part of AA’BB’ sys-
tem in NH-Ts), 7.21 (d, 2H, J= 4.21 Hz, BB’ part of AA’BB’
Purification of carbonic anhydrase isozymes from
human blood by affinity chromatography
3
system (meta) in Ph-CH ), 7.67 (d, 2H, J= 8.23 Hz, BB’ part
Purification of hCA I and hCA II were previously
3
13
18
of AA’BB’ system in NH-Ts). C-NMR (100 MHz, CDCl ,
described: hCA I and hCA II in . Serum were purified
3
ppm): 21.0, 21.7, 122.4, 127.5, 129.8, 130.0, 134.1, 135.4,
from fresh human blood obtained from the Blood Center
of the Research Hospital at Atatürk University. e blood
samples were centrifuged at 5000 rpm for 15 min and pre-
cipitant were removed. e serum was isolated. e pH
−
1
1
1
6
36.4, 143.9. IR (KBr, cm ): 3258, 1598, 1511, 1450, 1393,
331, 1160, 1092. Anal. calcd. for C H NO S (261.34) C,
14
15
2
4.34; H, 5.79; N, 5.36; S, 12.27. Found: C, 64.12; H, 5.86;
9–11
N, 5.38; S, 12.35.
of the hemolysate was adjusted to 8.7 with solid Tris
.
Sepharose-4B-aniline-sulfanylamide affinity column
equilibrated with 25 mM Tris-HCl/0.1M Na SO (pH 8.7).
Synthesis of N-(4-ethylphenyl)-
2
4
4
-methylbenzenesulfonamide (9)
e affinity gel was washed with 25 mM Tris-HCl/22 mM
Na SO (pH 8.7). e human carbonic anhydrase (hCA-
In%98yield,Compound9wasrecrystallizedfromCH Cl -
hexane solution. M.P.: 90–92°C. H-NMR (400 MHz,
2
2
2
4
1
VI) isozyme were eluted with 0.25 M H NSO H/25 mM
2 3
CDCl , δ, ppm): δ= 1.17 (t, 3H, J= 7.7 Hz in Ph-C H ), 2.37
Na HPO (pH = 6.7). All procedures were performed at
3
2
5
2
9
4
–12
(
s, 3H in NH-Ts), 2.55 (q, 2H, J= 7.7 Hz in Ph-C H ), 6.76
4°C
.
2
5
(
brs, 1H, -NH), 6.98 (d, 2H, J= 8.6 Hz, AA’ part of AA’BB’
system (ortho) in Ph-C H ), 7.05 (d, 2H, J= 8.60 Hz, BB’
Esterase activity assay
2
5
part of AA’BB’ system (meta) in Ph-C H ), 7.21 (d, 2H,
Carbonic anhydrase activity was assayed by following the
change in absorbance at 348nm of 4-nitrophenylacetate
(NPA) to 4-nitrophenylate ion over a period of 3min at
25°C using a spectrophotometer (CHEBIOS UV-VIS)
2
5
J= 8.05 Hz, AA’ part of AA’BB’ system in NH-Ts), 7.64 (d,
1
3
2
H, J= 8.05 Hz, BB’ part of AA’BB’ system in NH-Ts). C-
NMR(100 MHz, CDCl , ppm):15.6, 21.7, 28.4, 122.5, 127.5,
3
−1
28
1
3
28.8, 129.8, 134.2, 136.5, 141.9, 143.9. IR (KBr, cm ):
259, 1598, 1512, 1457, 1397, 1333, 1160, 1092. Anal.
according to the method described by Verpoorte et al. .
e inhibitory effects of compounds 1–14 and AZA were
examined. All compounds were tested in triplicate at
each concentration used. Control cuvette activity in the
calcd. for C H NO S (275.37) C, 65.43; H, 6.22; N, 5.09; S,
1
5
17
2
1
1.64. Found: C, 65.29; H, 6.20; N, 5.12; S, 11.65.
Journal of Enzyme Inhibition and Medicinal Chemistry

Sulfapyridine-like benzenesulfonamide derivatives 821
Figure 1. Structures of the compounds.
absence of inhibitor was taken as 100%. For each inhibitor
an Activity%- [Inhibitor] graph was drawn. To determine
Recently, Supuran’s group investigated the interac-
tions of some methoxy-benzenesulfonamide derivatives
and some of its substituted derivatives with isozymes, CA
K values, three different inhibitor concentrations were
I
3
2
tested;. In these experiments, 4-nitrophenylacetate was
used as substrate at five different concentrations (0.15–
I, II and IX , evidencing some low micromolar/submi-
cromolar inhibitors as well as the possibility to design
isozyme selective CAIs. Indeed, the inhibition profile of
various isozymes with this class of agents is very variable,
with inhibition constants ranging from the millimolar to
the submicromolar range for some methoxy-benzenesul-
29
0
.75mM). e Lineweaver-Burk curves were drawn .
Regression analysis graphs were drawn for IC50 using
inhibition % values by a statistical package (SPSS-for win-
dows; version 10.0) on a computer (Student’s t-test; n: 3).
3
2
fonamide or acid hydroxamide derivatives . It appeared
7
,9–14
Protein determination
thus of interest to extend the previous studies
, includ-
Protein during the purification steps was determined spec-
trophotometrically at 595nm according to the Bradford
method, using bovine serum albumin as the Standard .
ing in this research some benzenesulfonamides with
clinical applications, sulfapyridine 13, sulfadiazine 14, as
well as some of its substituted derivatives incorporating
amino, cyano, methyl and nitro moieties as substituents
at the aromatic ring in different positions.
30
SDS polyacrylamide gel electrophoresis
SDS polyacrylamide gel electrophoresis was performed
after purification of the enzymes. It was carried out in
e purification of human erythrocyte CA I and II
isoenzymes were performed using simple single-step
method by Sepharose-4B-anilin-sulfanilamide affinity
gel chromatography . We report here the first study on
the inhibitory effects of benzenesulfonamides (1–14)
and AZA on the CA esterase activity. Data of Table 1
show the following regarding inhibition of hCA-I, II and
VI with compounds 1–15:
1
0% and 3% acrylamide for the running and the stack-
1
8
ing gel, respectively, containing 0.1% SDS according to
3
1
Laemmli .
Rꢄꢂuꢅtꢂ ꢃnd diꢂcuꢂꢂion
Benzenesulfonamide derivatives (1–14) investigated
for the inhibition of CA I, II and CA VI isozymes.
Compounds 1, 12–14 and AZA are clinically used
compounds (Figure. 1). We (commercially available)
have also been assayed compound (1, 12–14), since no
such data are available in the literature.
(i) Against the slow cytosolic isozyme hCA I, sulfon-
amides 2–5 and 9 behave as weak inhibitors, with KI
values in the range of 148.27–448.2 μM. Isoform hCA I
was moderately inhibited by sulfonamides 1, 6, 8, 12–15
reported here. us, several derivatives, such as 1, 6 and
8, showed medium inhibitory activity, with inhibition
©
2012 Informa UK, Ltd.

8
22 C. Alp et al.
Table 1. hCA I, II and VI inhibition data with new sythetizedsynthesized sulfanilamine derivatives 1-12, sulfapyridine (13), sulfadiazine
(
14) and acetazolamide (15), by an esterase assay with 4-nitrophenylacetate as substrate.
K (µμM)
I
Compound
hCA I
22.120
210.3
322.0
249.12
148.27
18.24
5.380
50.02
448.2
1.480
1.130
42.87
26.19
28.38
36.2
hCA II
0.715
14.3
hCA VI
18.970
13.44
42.39
32.18
29.58
8.430
4.710
24.28
26.53
1.230
1.112
1.214
11.63
15.52
0.340
4
4
4
4
4
-Methylbenzenesulfonamide (1)
-Methyl-N-phenylbenzenesulfonamide (2)
-Methyl-N-(naphthalen-2-yl)benzenesulfonamide (3)
-Methyl-N-(2,4,6-trimethyl-phenyl)-benzenesulfonamide (4)
-Methyl-N-o-tolyl-benzenesulfonamide (5)
0.612
0.425
0.407
0.213
0.103
1.015
0.977
0.120
0.139
0.628
0.341
0.276
0.37
N-(2-cyanophenyl)-4-methylbenzenesulfonamide (6)
4
4
-Methyl-N-(2-nitrophenyl) benzenesulfonamide (7)
-Methyl-N-p-tolyl-benzenesulfonamide (8)
N-(4-ethylphenyl)-4-methylbenzenesulfonamide (9)
N-(4-cyanophenyl)-4-methylbenzenesulfonamide (10)
4-Methyl-N-(4-nitrophenyl) benzenesulfonamide (11)
Benzenesulfonamide (12)
4
4
-Amino-N-(pyridin-2-yl) benzenesulfonamide (Sulfapyridine) (13)
-Amino-N-(pyrimidin-2-yl) benzenesulfonamide (Sulfadiazine) (14)
Acetazolamide (AZA) (15)
constants in the range of 18.24–50.02 μM, in the same
and AZA) and 1, 3–6, 9–12 show much more potent
range as the clinically used compounds sulfapyridine
hCA II inhibitory activity, with K -s in the range of
I
(
2
13), sulfadiazine (14) and acetazolamide (15) (K of
6.19–36.2 μM for both compounds against this isoform,
0.103–0.715 μM (Table 1). eses finding clearly illustrate
that a very big variation in the structure of a CAI (such as
the presence of an additional CN, NO and CH moiety, in
I
Table 1). e remaining sulfonamides were more effec-
2
3
tive hCA I inhibitors as compared to derivatives discussed
this case) may have drastic consequences for the enzyme
inhibitory activity and selectivity profile against various
isozymes of such derivatives. us, except for the one less
active compound 2, all derivatives showed moderate or
powerful hCA-II inhibitory activity (Table 1).
above, with K -s in the range of 1.130–5.380 μM. Obviously,
I
both the aromatic sulfonamide head as well as the aryl/
hetaryl-acetyl moiety influence the biological activity of
6
these hCA I inhibitors . It may be observed that efficient
inhibitors incorporate both sulfanilamide, 4-Methyl-
N-(4-nitrophenyl) benzenesulfonamide and N-(4-
cyanophenyl)-4-methylbenzenesulfonamide. Except for
the five less active compounds mentioned above (2-5
and 9) which incorporate methylbenzenesulfonamide (1
or 8), cyanophenyl (6), 4-amino-pyridin (13) or amino-
pyrimidin (14) moiety, the remaining compounds showed
a quite compact behavior of moderately-efficient hCA I
inhibitors. us, benzenesulfonamides lead to significant
(iii) Sulfapyridine 13 and some of its congeners such
as 1–9, and 14 are also weak inhibitors of the secreted
isozyme hCA VI, with K -s of 4.710–42.39 μM. However,
I
-
again the compounds 10–12 are medium potency inhi
bitors (K of 1.112–1.230 μM), and AZA (15) show a
I
higher affinity for this isozyme, with inhibition constant
in the range of 0.340 μM (Table 1).
3
. ꢀoncꢅuꢂionꢂ
hCA I inhibition, but all these compounds possess K -s
I
>
0.100μM, being thus only moderately active. Kinetic
Benzenesulfonamide derivatives 1–14 used in this study
affect the activity of CA isozymes due to the presence of
the different functional groups (CH , CH CH , CN, NO
2
investigations (Lineweaver Burk plots, data not shown)
indicate that similarly to sulfonamides and inorganic
3
2
3
33–38
anions , all the investigated compounds act as noncom-
petitive inhibitors with 4-NPA as substrate, i.e., they bind
in different regions of the active site cavity as compared to
the substrate. However, the binding site of 4-NPA itself is
unknown, but it is presumed to be in the same region as
and NH ) moieties present in their aromatic scaffold. It
2
has been determined in our study that compounds 1–14
are effective inhibitors of hCA-II compared to AZA, which
is used as reference inhibitor for carbonic anhydrase.
Our findings here indicate thus the well-known class of
possible CAIs of interest, sulfonamides/sulfamates/sul-
famides. Indeed, some benzenesulfonamide derivatives
investigated here showed effective CA I, II and VI inhibi-
tory activity, in the low micromolar range, by the esterase
39
that of CO , the physiological substrate of this enzyme .
2
(ii) A rather strong activity of these compounds has
been observed also for the inhibition of the rapid cyto-
solic isozyme hCA-II (Table 1). Indeed, again the same
one compound (2) showed weaker hCA II inhibitory
method which usually gives K -s an order of magnitude
I
13
activity, with K in the range of 14.3 μM, whereas com-
higher as compared to the CO hydrase assay. Although
I
2
pounscompounds 7, 8 behave as moderate inhibi-
benzenesulfonamide derivatives used in this study has
previously synthesized, there was not such detailed spec-
troscopic data about these substances in previous works.
tors (K -s in the range of 0.977–1.015 μM). It should be
I
noted that the three clinically used compounds (13, 14
Journal of Enzyme Inhibition and Medicinal Chemistry

Sulfapyridine-like benzenesulfonamide derivatives 823
Also, most of these derivatives could not be obtained in
one-step with high yields. ese findings point out that
substituted benzenesulfonamide derivatives may be
used as leads for generating potent CAIs eventually tar-
geting other isoforms which have not been assayed yet
for their interactions with such agents.
15. Erdogan O, Ceyhun SB, Ekinci D, Aksakal E. Impact of δmethrin
exposure on mRNA expression levels of metallothionein A, B and
cytochrome P450 1A in rainbow trout muscles. Gene 2011;484:13–17.
1
1
1
1
2
6. Ekinci D, Sentürk M, Beydemir S, Küfrevioglu OI, Supuran CT. An
alternative purification method for human serum paraoxonase
1 and its interactions with sulfonamides. Chem Biol Drug Des
2010;76:552–558.
7. Aksakal E, Ceyhun SB, Erdogan O, Ekinci D. Acute and long-term
genotoxicity of δmethrin to insulin-like growth factors and growth
hormone in rainbow trout. Comp Biochem Physiol C Toxicol
Pharmacol 2010;152:451–455.
8. Ceyhun SB, Aksakal E, Ekinci D, Erdogan O, Beydemir S. Influence
of cobalt and zinc exposure on mRNA expression profiles of
metallothionein and cytocrome P450 in rainbow trout. Biol Trace
Elem Res. Doi: 10.1007/s12011-011-9068-z.
9. Cakmak R, Durdagi S, Ekinci D, Sentürk M, Topal G. Design,
synthesis and biological evaluation of novel nitroaromatic
compounds as potent glutathione reductase inhibitors. Bioorg
Med Chem Lett 2011;21:5398–5402.
Dꢄcꢅꢃrꢃtion of intꢄrꢄꢂt

is study was financed by Turkish Republic Prime
Ministry State Planning Organization (DPT), (project
no: 2010K120440) and Agri Ibrahim Cecen University
Scientific Research Council, (project no: Agri BAP-2010/
K-10) for (MS).
Rꢄfꢄrꢄncꢄꢂ
0. Aksakal E, Ekinci D, Erdogan O, Beydemir S, Alim Z, Ceyhun
SB. Increasing stocking density causes inhibition of metabolic-
antioxidant enzymes and elevates mRNA levels of heat shock
protein 70 in rainbow trout. Livest Sci 2011. Doi:10.1016/j.
livsci.2011.07.006.
1
. Ozensoy O, Arslan O, Sinan SO. A new method for purification
of carbonic anhydrase isozymes by affinity chromatography.
Biochemistry Mosc 2004;69:216–219.
2
3
. Supuran CT. Carbonic anhydrases: Novel therapeutic applications
for inhibitors and activators. Nat Rev Drug Discov 2008;7:168–181.
. Hilvo M, Baranauskiene L, Salzano AM, Scaloni A, Matulis D,
Innocenti A et al. Biochemical characterization of CA IX, one
of the most active carbonic anhydrase isozymes. J Biol Chem
21. Siktar E, Ekinci D, Siktar E, Beydemir S, Gulcin I, Gunay M.
Protective role of L-carnitine supplementation against exhaustive
exercise-induced oxidative stress in rats. Eur J Pharmacol 2011.
Doi: 10.1016/j.ejphar.2011.07.032.
2
008;283:27799–27809.
22. Casini A, Scozzafava A, Mincione F, Menabuoni L, Ilies MA,
Supuran CT. Carbonic anhydrase inhibitors: Water-soluble
4-sulfamoylphenylthioureas as topical intraocular pressure-
4
5
6
. Supuran CT, Scozzafava A, Conway J. Carbonic Anhydrase-Its
Inhibitors and Activators. CRC Press: Boca Raton, New York,
London, 2004. pp. 1–363.
. Winum JY, Rami M, Scozzafava A, Montero JL, Supuran C. Carbonic
anhydrase IX: A new druggable target for the design of antitumor
agents. Med Res Rev 2008;28:445–463.
. Güzel O, Innocenti A, Scozzafava A, Salman A, Parkkila S, Hilvo
M et al. Carbonic anhydrase inhibitors: Synthesis and inhibition
studies against mammalian isoforms I-XV with a series of
lowering agents with long-lasting effects.
2000;43:4884–4892.
J Med Chem
23. Cecchi A, Winum JY, Innocenti A, Vullo D, Montero JL, Scozzafava
A et al. Carbonic anhydrase inhibitors: Synthesis and inhibition of
cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and
IX with sulfonamides derived from 4-isothiocyanato-benzolamide.
Bioorg Med Chem Lett 2004;14:5775–5780.
2
-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-
24. Innocenti A, Casini A, Alcaro MC, Papini AM, Scozzafava A,
Supuran CT. Carbonic anhydrase inhibitors: e first on-resin
screening of a 4-sulfamoylphenylthiourea library. J Med Chem
2004;47:5224–5229.
25. Balaydin HT, Durdagi S, Ekinci D, Senturk M, Goksu S, Menzek
A. Inhibition of human carbonic anhydrase isozymes I, II and VI
with a series of bisphenol, metoxy and bromophenol compounds.
J Enzym Inhib Med Ch 2011. Doi:10.3109/14756366.2011.596836.
26. Senturk M, Kufrevioglu OI. In vitro effects of some nucleoside
analogue drugs on enzyme activities of carbonic anhydrase
isozymes I and II from human erythrocytes. Hacettepe J Biol Chem
2009;37:289–294.
27. Park KK, Lee JJ, Ryu J. Photo-Fries rearrangement of
N-arylsulfonamides to aminoaryl sulfone derivatives. Tetrahedron
2003;59:7651–7659.
28. Verpoorte JA, Mehta S, Edsall JT. Esterase activities of human
carbonic anhydrases B and C. J Biol Chem 1967;242:4221–4229.
29. Lineweaver H, Burk D. e determination of enzyme dissocation
constants. J Am Chem Soc 1934;56:658–666.
30. Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-
dye binding. Anal Biochem 1976;72:248–254.
31. Laemmli UK. Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 1970;227:680–685.
32. Nuti E, Orlandini E, Nencetti S, Rossello A, Innocenti A, Scozzafava
A et al. Carbonic anhydrase and matrix metalloproteinase
inhibitors. Inhibition of human tumor-associated isozymes IX and
cytosolic isozyme I and II with sulfonylated hydroxamates. Bioorg
Med Chem 2007;15:2298–2311.
sulfonamides. Bioorg Med Chem 2008;16:9113–9120.
7
. Durdagi S, Sentürk M, Ekinci D, Balaydin HT, Göksu S, Küfrevioglu
ÖI et al. Kinetic and docking studies of phenol-based inhibitors
of carbonic anhydrase isoforms I, II, IX and XII evidence a new
binding mode within the enzyme active site. Bioorg Med Chem
2
011;19:1381–1389.
8
9
1
1
. Kivelä J, Parkkila S, Waheed A, Parkkila AK, Sly WS, Rajaniemi H.
Secretory carbonic anhydrase isoenzyme (CA VI) in human serum.
Clin Chem 1997;43:2318–2322.
. Sentürk M, Ekinci D, Göksu S, Supuran CT. Effects of dopaminergic
compounds on carbonic anhydrase isozymes I, II, and VI. J Enzyme
Inhib Med Chem 2011. (doi: 10.3109/14756366.2011.591290).
0. Ekinci D, Cavdar H, Talaz O, Sentürk M, Supuran CT. NO-releasing
esters show carbonic anhydrase inhibitory action against human
isoforms I and II. Bioorg Med Chem 2010;18:3559–3563.
1. Ekinci D, Ceyhun SB, Sentürk M, Erdem D, Küfrevioglu OI,
Supuran CT. Characterization and anions inhibition studies of an
a-carbonic anhydrase from the teleost fish Dicentrarchus labrax.
Bioorg Med Chem 2011;19:744–748.
2. Alp C, Ekinci D, Gültekin MS, Sentürk M, Sahin E, Küfrevioglu OI. A
novel and one-pot synthesis of new 1-tosyl pyrrol-2-one derivatives
and analysis of carbonic anhydrase inhibitory potencies. Bioorg
Med Chem 2010;18:4468–4474.
3. Ekinci D, Ceyhun SB, Aksakal E, Erdogan O. IGF and GH mRNA
levels are suppressed upon exposure to micromolar concentrations
of cobalt and zinc in rainbow trout white muscle. Comp Biochem
Physiol C Toxicol Pharmacol 2011;153:336–341.
1
1
1
4. Ceyhun SB, Sentürk M, Ekinci D, Erdogan O, Ciltas A, Kocaman
EM. δmethrin attenuates antioxidant defense system and induces
the expression of heat shock protein 70 in rainbow trout. Comp
Biochem Physiol C Toxicol Pharmacol 2010;152:215–223.
33. Briganti F, Pierattelli R, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors. Part 37. Novel classes of isozymes I
and II inhibitors and their mechanism of action. Kinetic and
©
2012 Informa UK, Ltd.

8
24 C. Alp et al.
spectroscopic investigations on the native and cobalt-substituted
enzymes. Eur J Med Chem 1996;31:1001–1010.
4. Sentürk M, Gülçin I, Dastan A, Küfrevioglu OI, Supuran CT.
Carbonic anhydrase inhibitors. Inhibition of human erythrocyte
isozymes I and II with a series of antioxidant phenols. Bioorg Med
Chem 2009;17:3207–3211.
5. Balaydin HT, Soyut H, Ekinci D, Goksu S, Beydemir S, Menzek A,
Sahin E. Synthesis and carbonic anhydrase inhibitory properties
of novel bromophenols including natural products. J Enzym Inhib
Med Ch 2011. Doi: 10.3109/14756366.2011.574131.
6. Sly WS, Hu PY. Human carbonic anhydrases and carbonic
anhydrase deficiencies. Annu Rev Biochem 1995;64:375–401.
37. Pastorekova S, Parkkila S, Pastorek J, Supuran CT. Carbonic
anhydrases: Current state of the art, therapeutic applications and
future prospects. J Enzyme Inhib Med Chem 2004;19:199–229.
38. Ceyhun SB, Sentürk M, Yerlikaya E, Erdogan O, Küfrevioglu OI,
Ekinci D. Purification and characterization of carbonic anhydrase
from the teleost fish Dicentrarchus labrax (European seabass) liver
and toxicological effects of metals on enzyme activity. Environ
Toxicol Pharmacol 2011;32:69–74.
39. Nair SK, Ludwig PA, Christianson DW. Two-site binding of
phenol in the active site of human carbonic anhydrase II:
Structural implications for substrate association. J Am Chem Soc
1994;116:3659–3660.
3
3
3
Journal of Enzyme Inhibition and Medicinal Chemistry