Carbonic anhydrase inhibitors. Regioselective synthesis of novel 1-substituted 1,4-dihydro-4-oxo-3-pyridinesulfonamides and their inhibition of the human cytosolic isozymes i and II and transmembrane cancer-associated isozymes IX and XII

Article abstract of DOI:10.1016/j.ejmech.2010.05.011

A series of 1-substituted 1,4-dihydro-4-oxo-3-pyridinesulfonamide (2-16) have been synthesized and investigated as inhibitors of four isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), that is the cytosolic ubiquitous CA I and II, and cancer-associated isozymes CA IX and XII. Against the human isozymes hCA I the new compounds showed inhibition constants in the range of 1.09-12.1 μM, against hCA II in the range of 50.5-172 nM, against hCA IX in the range of 5.2-118 nM, and against hCA XII in the range of 8.7-381 nM, respectively. Compounds 2, 3, 5-9, 11, 13 and 14 showed excellent hCA IX inhibitory efficacy, with K_Is = 5.2-11.0 nM, being much more effective as compared to the clinically used sulfonamides AAZ, MZA, EZA, DCP and IND (K_Is = 24-50 nM). Compounds 2, 3, 5-9, 11 and 13 were also very effective hCA XII inhibitors (K_Is = 8.7-45.2 nM) which are comparable or more effective than those clinically used EZA and DCP (K_Is = 22 and 50 nM), respectively. A novel 1-substituted 1,4-dihydro-4-oxo-3- pyridinesulfonamides have been synthesized and investigated as inhibitors of carbonic anhydrase isozymes hCA I, II, IX and XII. Some of them showed excellent hCA IX and XII inhibitory efficacy.

Full text of DOI:10.1016/j.ejmech.2010.05.011

European Journal of Medicinal Chemistry 45 (2010) 3656e3661
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Carbonic anhydrase inhibitors. Regioselective synthesis of novel 1-substituted
1,4-dihydro-4-oxo-3-pyridinesulfonamides and their inhibition of the human
cytosolic isozymes I and II and transmembrane cancer-associated isozymes
IX and XII
a
a
,
b
b
*
ꢀ
Zdzis1aw Brzozowski , Jaros1aw S1awinski , Alessio Innocenti , Claudiu T. Supuran
a
ꢀ
ꢀ
Department of Organic Chemistry, Medical University of Gdansk, Al. Gen. J. Hallera 107, 80-416 Gdansk, Poland
b Dipartimento di Chimica, Universita 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 1-substituted 1,4-dihydro-4-oxo-3-pyridinesulfonamide (2e16) have been synthesized and
investigated as inhibitors of four isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), that is the
cytosolic ubiquitous CA I and II, and cancer-associated isozymes CA IX and XII. Against the human
Received 20 March 2010
Received in revised form
4 May 2010
Accepted 5 May 2010
Available online 12 May 2010
isozymes hCA I the new compounds showed inhibition constants in the range of 1.09e12.1 mM, against
hCA II in the range of 50.5e172 nM, against hCA IX in the range of 5.2e118 nM, and against hCA XII in the
range of 8.7e381 nM, respectively. Compounds 2, 3, 5e9, 11, 13 and 14 showed excellent hCA IX
inhibitory efficacy, with KIs ¼ 5.2e11.0 nM, being much more effective as compared to the clinically used
sulfonamides AAZ, MZA, EZA, DCP and IND (KIs ¼ 24e50 nM). Compounds 2, 3, 5e9, 11 and 13 were also
very effective hCA XII inhibitors (KIs ¼ 8.7e45.2 nM) which are comparable or more effective than those
clinically used EZA and DCP (KIs ¼ 22 and 50 nM), respectively.
Keywords:
1,4-Dihydro-4-oxo-3-pyridinesulfonamides
Synthesis
Carbonic anhydrase isozymes I, II, IX and XII
inhibitors
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
III (Chart 1), and their inhibitory activities against human isozymes
hCA I, II, IX, XII and XIV [24]. Some of these compounds exhibited
The carbonic anhydrases (CAs, EC 4.2.1.1) [1e4] constitute inter-
esting targets for the design of pharmacological agents useful in the
treatment or prevention of a variety of disorders such as glaucoma,
acid-base disequilibria, epilepsy and other neuromuscular diseases,
altitude sickness, edema, and obesity [5,6]. A quite new and unex-
pected application of the CA inhibitors (CAIs) regards their potential
use in the management (imaging and treatment) of hypoxic tumors
[7e14], since at least two CA isozymes of the 15 presently known in
humans, thatisCA IXand XII, arepredominantlyfound in tumorcells
and lack (or are present in very limited amount) in normal tissues
[15e18]. The potential use of CAIs as antitumor agents opens thus
a new important research direction [19,20].
excellent hCA IX inhibitory efficacy, with inhibition constants of
4.6e12.0 nM, being much more effective as compared to the clinically
used sulfonamides AAZ, MZA, EZA, DCP and IND (in clinical devel-
opment drug). These findings prompted us to synthesize and inves-
tigate of the related 3-pyridinesulfonamides of type IV (Chart 1).
2. Results and discussion
2.1. Chemistry
The previously described method was applied to the synthesis
of 4-methoxy-3-pyridinesulfonamide 1 [24]. The synthesis of the
target 1,4-dihydro-4-oxo-3-pyridinesulfonamides 2e15 were ach-
ieved with yields in the 59e97% range by a convenient one-step
procedure starting from 4-methoxy-3-pyridinesulfonamide 1 and
the appropriate activated aliphatic or aromatic halides in dry
acetonitrile. The proposed mechanism leading to the formation of
the products 2e15 is outlined in Schemes 1 and 2. The initial step is
believed to be formation of intermediate 1-pyridinium salts A
(Scheme 1) or B (Scheme 2) which undergoes SN2-substitution on
the methyl by halide to form the 4-pyridones 2e15. The reaction
Recently, we have reported on the strong inhibition of human
cytosolic isozymes I and II and tumor-associated isozymes IX and XII
with
some
S-substituted
4-chloro-5-or
6-methyl-
benzenesulfonamidesIandII[21e23](Chart1). Wealsodescribedthe
syntheses a number of 4-substituted 3-pyridinesulfonamides of type
* Corresponding author. Tel.: þ48 58 349 31 40; fax: þ48 58 349 31 45.
ꢀ
E-mail address: jaroslaw@gumed.edu.pl (J. S1awinski).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.011

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 3656e3661
3657
2.2. CA inhibition studies
The compounds 2e11 and 13e16 as well as standard, clinical used
CAIs, such as acetazolamide AAZ, methazolamide MZA, ethoxzola-
mide EZA, dichlorophenamide DCP, and indisulam IND (Chart 1),
have been tested for the inhibition of two cytosolic ubiquitous
isozymes of human origin, that is, hCA I and II, and two trans-
membraneisoforms hCAIXandXII(cancer-associated). Thefollowing
shouldbenoted regardingCAinhibitory dataof Table 1: (i) against the
slow cytosolic isoform hCA I, the sulfonamides investigated here
showed high toweak inhibitory properties. Thus, derivatives 4,10e13
and 16 showed weak inhibitor of this isoform, with KIs in the range of
Chart 1.
5.88e14.2 mM, being thus much weaker inhibitors as compared to the
clinical used compounds AAZeIND (Table 1). The compounds 2, 3,14
and 15 were slightly more effective inhibitors against hCA I with KIs in
products were clean and did not need further purification. Impor-
tantly, no traces of O-alkyl products were detected in any of the
reactions. Selective methods either for N-alkylation of 2-pyridones
[25] or for conversion of 2-methoxypyridines to the corresponding
N-alkyl-2-pyridones [26] were described by Curran et al. It is worth
noting however, that the analogous mechanism of the formation of
2-pyridones (yields in the 43e86% range) by the reactions of 2-
methoxypyridine with activated bromides (RCH2Br) and NaI in
boiling acetonitrile has been described by Bowman and Bridge [27].
Similarly, a plausible mechanism leading to the formation of 1,4-
dihydro-1-(2-methoxycarbonylaminobenzoyl)-4-oxo-3-pyridine
sulfonamide 16 is depicted in Scheme 2. The nucleophilic attack of
pyridinesulfonamide 1 on the carbon atom C-4 of the 2H-3,1-ben-
zoxazin-2,4-(1H)-dione ring lead to the formation of pyridinium
rangeof1.83e4.16
mM.However, thefivecompound5e9showedhCA
I inhibitory activity (KIs ¼ 1.09e1.18
mM) in the same range as the
clinically used compounds MZA and DCP (Table 1); (ii) against the
ubiquitous and dominant rapid cytosolic isozymes hCA II, all the
compounds acting as weaker inhibitors (KIs in the range of
50.5e172 nM) (Table 1); (iii) a quite larger variation of inhibitory
activity was observed for the inhibition of the tumor-associated iso-
form, hCA IX (Table 1). Thus, the compounds 4, 10, 15 and 16 showed
moderate CA IX inhibitory activity, with KIs in range of 64e118 nM,
whereas the remaining derivatives 2, 3, 5e9, 11, 13 and 14 showed
excellent hCA IX inhibitory efficacy, with inhibition constants equal
5.2e11.9 nM, being much more effective as compared to the clinically
usedsulfonamidesAAZ, MZA, EZA, DCPandIND(Table 1);(iv)against
the second tumor-associated isoform hCA XII, the sulfonamides
investigated here showed high or weak inhibitory properties. The
sulfonamides4,10 and 14e16 showedweak inhibition of thisisoform,
with KIs in the range of 75e381 nM, whereas remaining derivatives 2,
3, 5e9, 11 and 13 were very effective hCA XII inhibitors (KIs in the
range of 8.7e45.2 nM), which are comparable or more effective than
those clinically used sulfonamides EZA and DCP, respectively.
salt
C which undergoes SN2-substitution on the methyl by
carboxylate (COOꢀ) group of the intermediate C resulting in the
formation of the neutral sulfonamide 16.
The structures of the newly obtained compounds 2e16 were
confirmed by spectroscopic data and elemental analyses (C, H, N)
presented in the Experimental section.
3. Conclusions
We have developed methods for the regioselective synthesis of
1-substituted 1,4-dihydro-4-oxo-3-pyridinesulfonamides. The
fourteen new sulfonamide have been assayed for the inhibition of
four physiological relevant CA isozymes, such as CA I and II, and the
tumor-associated isozymes CA IX and XII. Against the human
isozyme hCA I the new sulfonamide showed inhibition constants in
the range of 1.09e14.2 mM; against hCA II in the range 50.5e172 nM,
against hCA IX in the range 5.2e96 nM, and against hCA XII in the
range 8.7e381 nM, compounds 5e9 having an activity against hCA I
(KIs ¼ 1.09e1.18
m
M) which is comparable to the clinical used
sulfonamide DCP (KIs ¼ 1.20
mM). Several of new compounds
including 2, 3, 5e9,11,13 and 14 showed excellent hCA IX inhibitory
efficacy, with inhibition constants of 5.2e11.9 nM, being much
more effective as compared to the clinically used AAZ, MZA, EZA,
DCP and IND (KIs ¼ 24e50 nM), compound 2, 3, 5e9 and 11 were
also very effective hCA XII inhibitors, with inhibition constants
8.7e45.2 nM, comparable or more effective to the clinically used
sulfonamides EZA (KIs ¼ 22 nM) or DCP (KIs ¼ 50 nM), respectively.
4. Experimental protocols
Scheme 1. Synthesis of 1-(R1-methyl)-1,4-dihydro-4-oxo-3-pyridinesulfonamides
4.1. Synthesis
2e10. Reagents and conditions: a) methyl iodide (1.015 M equiv.) acetonitrile, 0e2 ꢁ
C
1 h, room temp. 12 h, reflux 20 h; b) appropriate alkyl halides: benzyl bromide, 2-
bromoacetamide, 4-chlorophenacyl bromide, 4-fluorophenacyl bromide, 3,4-
dichlorophenacyl bromide, 4-nitrophenacyl bromide, 3-nitrophenacyl bromide or
bromomethyl-2-naphthyl ketone (1.015 M equiv.) dry acetonitrile, room temp. 24 h,
reflux 48 h.
The following instruments and parameters were used: melting
ꢀ1
}
points Buchi 535 apparatus; IR spectra: KBr pellets, 400e4000 cm
Perkin Elmer 1600 FTIR spectrometer; 1H and 13C NMR: Varian Gemini
200 apparatus at 200 and 50 MHz, respectively; chemical shifts are

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Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 3656e3661
Scheme 2. Synthesis of 1-(aryl or heteroaryl)-1,4-dihydro-4-oxo-3-pyridinesulfonamides 11e15 and 1,4-dihydro-1-(2-methoxycarbonylaminobenzoyl)-4-oxo-3-pyr-
idinesulfonamide 16. Reagents, conditions and yields: a) 1-chloro-2,4-dinitrobenzene or 1-chloro-2,6-dinitrobenzene (1.015 M equiv.), dry acetonitrile room temp. 12 h, reflux 44 h,
71 and 59% respectively; b) 4-chloro-3-pyridinesulfonamide (1.015 M equiv.), dry acetonitrile, room temp. 12 h, reflux 44 h, 82%; c) 4-chloro-7-nitrofurazone (1.015 M equiv.), dry
acetonitrile, room temp. 12 h, reflux 44 h, 84%; d) 6-chloropurine (1.015 M equiv.), room temp 12 h, reflux 120 h, 63%; e) 4H-3,1-benzoxazine-2,4-(1H)-dione (1.15 M equiv.), dry
acetonitrile, reflux 22 h, 90%.
expressed in parts per million (ppm) relative to TMS as internal stan-
dard. The results of elemental analyses for C, H and N werewithin ꢂ0.4%
of theoretical values. The starting 4-methoxy-3-pyridinesulfonamide 1
was prepared according to the method described previously [24].
6.81 (s, 2H, SO2NH2), 7.38 (s, 5H, Ph), 7.95 (dd, Jortho ¼ 7.6 Hz,
Jmeta ¼ 2.2 Hz,1H, H-6, pyrid.), 8.47 (d, Jmeta ¼ 2.2,1H, H-2) ppm; 13
C
NMR (DMSO-d6)
d 58.90, 120.48, 128.17,128.78, 129.30, 130.45,
136.50, 141.45, 142.63, 172.51 ppm. Anal. (C12H12N2O3S) C, H, N.
4.1.1. 1,4-Dihydro-1-methyl-4-oxo-3-pyridinesulfonamide (2)
4.1.2.2. 1-(Carbamoylmethyl)-1,4-dihydro-4-oxo-3-pyridinesulfona-
mide (4). Starting from 2-bromoacetamide (0.98 g), the title
compound 4 was obtained (1.4 g, 86%): m.p. 268e269 ꢁC; IR (KBr)
3395, 3330, 3230 (SO2NH2 and CONH2), 1680 (O]CeNH2), 1650
To an ice-cold suspension of 4-methoxy-3-pyridinesulfonamide
1 (1.32 g, 7 mmol) in dry acetonitrile (13 ml) was added with stirring
methyl iodide (1.0 g, 7.1 mmol). After 0.5 h the ice-bath was removed
and the reaction mixture was stirred at room temperature for 12 h,
followed at reflux for 20 h. After cooling to room temperature and
standing overnight the precipitate was filtered off washed with
acetonitrile (3 ꢃ 1 ml) and dried. Yield 1.2 g (91%), m.p. 246e247 ꢁC;
IR (KBr) 3350, 3155 (SO2NH2),1650 (C]O),1330,1160 (SO2) cmꢀ1;1H
(C]O), 1390, 1160 (SO2) cmꢀ1 1H NMR (DMSO-d6)
; d 4.74 (s, 2H,
CH2), 6.34 (d, J ¼ 7.3 Hz, 1H, H-5), 6.82 (s, 2H, SO2NH2), 7.40 (s, 1H,
CONHA), 7.62 (s, 1H, CONHB), 7.72 (d, J ¼ 7.3 Hz,1H, H-6), 8.26 (s, 1H,
H-2) ppm. Anal. (C7H9N3O4S) C, H, N.
NMR (DMSO-d6)
d
3.73 (s, 3H, CH3), 6.35 (d, J ¼ 7.5 Hz,1H, H-5), 6.79
4.1.2.3. 1-(4-Chlorophenacyl)-1,4-dihydro-4-oxo-3-pyridinesulfona-
mide (5). Starting from 4-chlorophenacyl bromide (1.66 g), the title
compound 5 was obtained (2.1 g, 91%): m.p. 279e280 ꢁC dec.; IR
(KBr) 3280, 3165, 3125 (SO2NH2), 1690 (C]O), 1650 (C]O), 1320,
(s, 2H, SO2NH2), 7.76 (dd, Jortho ¼ 7.5 Hz, Jmeta ¼ 2.2 Hz,1H, H-6), 8.29
(d, Jmeta ¼ 2.2, 1H, H-2) ppm.; 13C NMR (DMSO-d6)
d 43.65, 120.04,
129.93, 142.19, 143.45, 172.34 ppm. Anal. (C6H8N2O3S) C, H, N.
1160 (SO2) cmꢀ1 1H NMR (DMSO-d6)
; d 5.78 (s, 2H, CH2), 6.42 (d,
4.1.2. Procedure for the preparation of 1-R-1,4-dihydro-4-oxo-3-
pyridinesulfonamides (3e10)
J ¼ 7.3 Hz, 1H, H-5, pyrid.), 6.87 (s, 2H, SO2NH2), 7.70 (d, J ¼ 2.8 Hz,
2H, 4-ClPh), 7.76 (dd, Jortho ¼ 7.3 Hz, Jmeta ¼ 2.0 Hz, 1H, H-6, pyrid.),
8.01 (d, J ¼ 8.8 Hz, 2H, 4-ClPh), 8.38 (d, Jmeta ¼ 2.0 Hz, 1H, H-2,
To a solution of the corresponding bromide derivatives R1CH2Br
(7.1 mmol) in dry acetonitrile (30 ml) 4-methoxy-3-pyr-
idinesulfonamide 1 (1.32 g 7 mmol) was added. The reaction
mixture was stirred at room temperature for 24 h, followed at
reflux for 48 h. After cooling to room temperature, the precipitate of
the adequate 1,4-dihydro-3-pyridinesulfonamide was collected by
filtration, washed with acetonitrile (4 ꢃ 2.5 ml) and dried. In this
manner the following sulfonamides were obtained.
pyrid.) ppm. 13C NMR (DMSO-d6)
d 61.95, 119.73, 129.37, 130.09,
130.19, 133.15, 139.28, 142.86, 143.78, 172.69, 192.54 ppm. Anal.
(C13H11Cl N2O4S) C, H, N.
4.1.2.4. 1-(4-Fluorophenacyl)-1,4-dihydro-4-oxo-3-pyridinesulfona-
mide (6). Starting from 4-fluorophenacyl bromide (1.54 g), the title
compound 6 was obtained (2.0 g, 92%): m.p. 266e267 ꢁC dec.; IR
(KBr) 3270, 3160 (SO2NH2), 1695 (C]O), 1650 (C]O), 1320, 1160
4.1.2.1. 1-Benzyl-1,4-dihydro-4-oxo-3-pyridinesulfonamide
(3).
(SO2) cmꢀ1 1H NMR (DMSO-d6)
; d 5.79 (s, 2H, CH2), 6.43 (d,
Starting from benzyl bromide (1.21 g), the title compound 3 was
J ¼ 7.6 Hz, 1H, H-5, pyrid.), 6.97 (s, 2H, SO2NH2), 7.43e7.52 (m, 2H,
4-FPh), 7.77 (dd, Jortho ¼ 7.6 Hz, Jmeta ¼ 2.1 Hz, 1H, H-6, pyrid.),
8.07e8.14 (m, 2H, 4-FPh), 8.40 (d, Jmeta ¼ 2.1 Hz, H-2, pyrid.) ppm.
Anal. (C13H11FN2O4S) C, H, N.
obtained (1.5 g, 81%): m.p. 158e159 ꢁC; IR (KBr) 3310, 3250
(SO2NH2), 1645 (C]O), 1330, 1185, 1155 (SO2) cmꢀ1 1H NMR
;
(DMSO-d6)
d
5.24 (s, 2H, CH2), 6.39 (d, J ¼ 7.6 Hz, 1H, H-5, pyrid.),

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 3656e3661
3659
Table 1
Carbonic anhydrase inhibition data for compounds 2e10 and 11, 13e16 and standard inhibitors against human isozymes hCA I, II, IX and XII, by a stopped-flow, CO2 hydration
assay [28].
O
O
R1
N
N
Ar
SO2NH2
SO2NH2
11,13-16
2-10
Compound
R1 or Ar
KIa
hCA Ib
(m
M)
hCA IIb (nM)
hCA IXc (nM)
hCA XIIc (nM)
AAZ
MZA
EZA
DCP
IND
2
0.31
0.78
0.025
1.20
0.031
3.44
12
14
8
38
15
77
25
27
34
50
24
7.1
5.7
3.4
22.0
50.0
3.4
H
28.9
3
4
4.12
5.88
80
7.8
45.2
O
H2N
172
64.0
146.0
O
5
6
7
8
1.13
1.17
1.18
1.09
112
108
114
106
11.8
9.7
35.6
32.5
25.4
31.8
Cl
F
O
O
Cl
Cl
8.6
O
10.1
O2N
O2N
O
9
1.10
14.2
105
103
11.9
12.2
O
10
118.0
127.0
11
13
O2N
N
NO2
6.1
97
5.8
5.2
34.6
8.7
SO2NH2
7.13
50.5
O2N
N
14
15
4.16
1.83
56
95
6.9
75.0
N
O
N
N
N
68.0
113.0
HN
MeO NH O
O
16
12.1
102
96.0
381.0
a
Errors in the range of ꢂ5e10% of the reported value (from 3 different assays).
Human (cloned) isozymes, by CO2 hydration method.
Catalytic domain of human, cloned isozymes [29,30], by the CO2 hydration method.
b
c

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Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 3656e3661
4.1.2.5. 1-(3,4-Dichlorophenacyl)-1,4-dihydro-4-oxo-3-pyridinesul-
fonamide (7). Starting from 3,4-dichlorophenacyl bromide (1.9 g),
the title compound 7 was obtained (2.4 g, 95%): m.p. 279e280 ꢁC
dec.; IR (KBr) 3275, 3175, 3135 (SO2NH2), 1680 (C]O), 1645 (C]O),
(KBr) 3405, 3305 (SO2NH2),1645 (C]O),1605,1595 (NO2),1345,1170
(SO2) cmꢀ1; 1H NMR (DMSO-d6)
6.53 (s,1H, H-5, pyrid.), 7.05 (s, 3H,
d
SO2NH2 and H-5, 2,6-di O2NPh), 8.04 (s,1H, H-6, pyrid), 8.12(s,1H, H-
2, pyrid) 8.66 (s, 2H, H-4 and H-6, 2,6-di O2NPh) ppm; 13C NMR
1320, 1160 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
d
5.79 (s, 2H, CH2), 6.44
(DMSO-d6) d 120.37, 129.19, 131.20, 131.66, 133.37, 142.54, 143.26,
(d, J ¼ 7.6 Hz, 1H, H-5, pyrid.), 6.88 (s, 2H, SO2NH2), 7.75 (dd,
Jortho ¼ 7.6 Hz, Jmeta ¼ 2.0 Hz, 1H, H-6, pyrid.), 7.95 (d, J ¼ 10.0 Hz,
2H, 3,4-diClPh), 8.24 (s, 1H, 3,4-diClPh), 8.39 (d, Jortho ¼ 2.0 Hz, 1H,
H-2, pyrid.) ppm. Anal. (C13H10Cl2N2O4S) C, H, N.
147.06, 173.30 ppm. Anal. (C11H8N4O7S) C, H, N.
4.1.3.3. 1,4-Dihydro-4-oxo-1-(3-sulfamoylpyrid-4-yl)-3-pyridiensu-
lfonamide (13). Starting from 4-chloro-3-pyridinesulfonamide
(1.36 g), the title compound 13 was obtained (1.9 g, 82%): m.p.
247e249 ꢁC dec.; IR (KBr) 3320, 3220 (SO2NH2),1645 (C]O),1570, (C]
4.1.2.6. 1,4-Dihydro-1-(4-nitrophenacyl)-4-oxo-3-pyridinesulfona-
mide (8). Starting from 4-nitrophenacyl bromide (1.73 g), the title
compound 8 was obtained (2.2 g, 93%): m.p. 254e255 ꢁC dec.; IR
(KBr) 3405, 3335, 3165 (SO2NH2), 1705 (C]O), 1650 (C]O), 1520
N), 1340, 1160 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
d
6.45 (d, J ¼ 7.8 Hz, 1H,
H-5, pyridone), 6.94 (s, 2H, SO2NH2, pyridone), 7.80(d, J¼ 5.2 Hz,1H, H-
5, pyridine) 7.89 (dd, Jortho ¼ 7.8 Hz, Jmeta ¼ 2.1 Hz, 1H, H-6, pyridone),
8.09 (s, 2H, SO2NH, pyridine), 8.28 (d, Jortho ¼ 2.1 Hz, H-2, pyridone),
8.99 (d, J ¼ 5.2 Hz, 1H, H-6, pyridine), 9.20 (s, 2H, H-2, pyridine) ppm;
(NO2),1355,1155 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
d 5.83 (s, 2H, CH2),
6.43 (d, J ¼ 7.6 Hz, 1H, H-5, pyrid.), 6.87 (s, 2H, SO2NH2), 7.76 (dd,
Jortho ¼ 7.6 Hz, Jmeta ¼ 2.1 Hz,1H, H-6, pyrid.), 8.22 (d, J ¼ 8.7 Hz, 2H, 4-
O2NPh), 8.40 (d, Jmeta ¼ 2.1 Hz,1H, H-2, pyrid.) 8.44 (d, J ¼ 8.7 Hz, 2H,
4-O2NPh) ppm. Anal. (C13H11N3O6S) C, H, N.
13C NMR (DMSO-d6)
d 119.40, 124.25, 130.19, 135.19, 141.68, 142.66,
146.08, 149.56, 155.26, 172.92 ppm. Anal. (C10H10N4O5S2) C, H, N.
4.1.3.4. 1,4-Dihydro-1-(7-nitrofurazon-4-yl)-4-oxo-3-pyridinesulfon-
amide (14). Starting from 4-chloro-7-nitrobenzofurazone (1.42 g),
the title compound 14 was obtained (2.0 g, 84%): m.p. 249e250 ꢁC
dec.; IR (KBr) 3380, 3280 (SO2NH2), 1640 (C]O), 1595, 1549 (C]N
4.1.2.7. 1,4-Dihydro-1-(3-nitrophenacyl)-4-oxo-3-pyridinesulfonamide
(9). Starting from 3-nitrophenacyl bromide (1.73 g), the title
compound 9 was obtained (2.3 g, 97%): m.p. 285e286 ꢁC dec.; IR (KBr)
3340, 3240 (SO2NH2), 1700 (C]O), 1650 (C]O), 1530 (NO2), 1360,
and NO2), 1340, 1155 (SO2) cmꢀ1 1H NMR (DMSO-d6)
; d 6.66 (d,
1150 (SO2) cmꢀ1
;
1H NMR (DMSO-d6)
d
5.86 (s, 2H, CH2), 6.44 (d,
J ¼ 5.2 Hz,1H, H-5, pyrid.), 7.14 (s, 2H, SO2NH) 8.00 (d, J ¼ 8.0 Hz,1H,
H-5, benzofurazonyl), 8.39 (dd, Jortho ¼ 7.8 Hz, Jmeta ¼ 2.5 Hz, 1H, H-
6, pyrid.), 8.84 (d, Jmeta ¼ 2.5 Hz, 1H, H-2, pyrid.), 8.87 (d, J ¼ 8.0 Hz,
J ¼ 7.6 Hz,1H, H-5, pyrid.), 6.87 (s, 2H, SO2NH2), 7.60 (d, J ¼ 7.6 Hz,1H,
H-6, pyrid.), 7.93 (t, J ¼ 7.9 Hz, 1H, H-5, 3-O2NPh), 8.40 (s, H-2, pyrid.)
8.42 (d, J ¼ 7.9 Hz,1H, H-6, 3-O2NPh) 8.57 (d, 1H, H-4, 3-O2NPh), 8.71
(s, 1H, H-2, 3-O2NPh) ppm. Anal. (C13H11N3O6S) C, H, N.
1H, H-6, benzofurazonyl) ppm; 13C NMR (DMSO-d6)
d 120.67,
124.22, 131.34, 133.31, 135.24, 135.95, 140.63, 141.13, 144.06, 147.12,
173.11 ppm. Anal. (C11H7N5O6S) C, H, N.
4.1.2.8. 1,4-Dihydro-1-(2-naphthoylmethyl)-4-oxo-3-pyridinesul-
fonamide (10). Starting from bromomethy-2-naphthyl ketone
(1.77 g), the title compound 10 was obtained (2.2 g, 92%): m.p.
266e267 ꢁC dec.; IR (KBr) 3350, 3180, 3160 (SO2NH2), 1690 (C]O),
4.1.3.5. 1,4-Dihydro-4-oxo-1-(6-purinyl)-3-pyridinesulfonamide (15).
Starting from 6-chloropurine (1.1 g), the title compound 15 was
obtained (1.3 g, 63%): m.p. 298e299 ꢁC dec.; IR (KBr) 3380, 3300,
3190 (SO2NH2 and NH), 1650 (C]O), 1605, 1575 (C]N), 1330, 1310,
1645 (C]O),1320,1165 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
d 5.94 (s, 2H,
CH2), 6.44 (d, J ¼ 7.6 Hz, 1H, H-5, pyrid.), 6.87 (s, 2H, SO2NH2),
7.63e7.77 (m, 2H, naphthyl), 7.81 (dd, Jortho ¼ 7.6 Hz, Jmeta ¼ 2.2 Hz,1H,
H-6, pyrid.), 7.98e8.20 (m, 4H, naphthyl) 8.44 (d, Jmeta ¼ 2.2 Hz,1H, H-
2, pyrid), 8.74 (s,1H, H-1, naphthyl) ppm. Anal. (C17H14N2O4S) C, H, N.
1160,1145 (SO2)cmꢀ1; 1HNMR (DMSO-d6)
d
6.66 (d, J ¼ 8.1 Hz,1H, H-
5, pyrid.), 7.09 (s, 2H, SO2NH2), 8.78(s,1H, H-2, purine), 8.88 (s,1H, H-
8, purine), 9.39 (dd, Jortho ¼ 8.1 Hz, Jmeta ¼ 2.2 Hz, 1H, H-6, pyrid.),
10.20 (Jmeta ¼ 2.2 Hz,1H, H-2, pyrid.),14.11 (s,1H, NH) ppm; 13C NMR
(DMSO-d6)
d 120.36, 122.18, 131.32, 137.67, 137.84, 138.00, 145.82,
4.1.3. Procedure for the preparation of 1-aryl-1,4-dihydro-4-oxo-3-
pyridinesulfonamides (11e15)
151.42, 155.44, 173.78 ppm. Anal. (C10H8N6O3S) C, H, N.
A mixture of 4-methoxy-3-pyridinesulfonamide 1 (1.32 g,
7 mmol) and the corresponding activated aromatic or hetero-
aromatic halides (7.105 mmol) in dry acetonitrile (20 ml for 11e14
or 45 ml for 15) was stirred at room temperature for 12 h, followed
at reflux for 44 h (compd 11e14) or 120 h (compd 15). After cooling
to room temperature and standing overnight, the precipitate was
filtered off, washed with acetonitrile (4 ꢃ 1.5 ml) and dried. In this
manner the following sulfonamides were obtained.
4.1.4. 1,4-Dihydro-1-(2-methoxycarbonylaminobenzoyl)-4-oxo-3-
pyridinesulfonamide (16)
A mixture of 4-chloro-3-pyridinesulfonamide 1 (1.88 g, 0.01 mol)
and 2H-3,1-benzoxazine-2,4-(1H)-dione (1.88 g, 0.0115 mol) in dry
acetonitrile (25 ml) was refluxed with stirring for 22 h. The small
amountof insoluble side products was filtered outwhenhotand filtrate
was left overnight. The title compound 16 thus obtained was collected
by filtration, washed with acetonitrile and dried. Yield 2.8 g (79.7%): m.
p. 152e153 ꢁC dec.; IR (KBr) 3350, 3315, 3245 (NH and SO2NH2), 1770,
4.1.3.1. 1,4-Dihydro-1-(2,4-dinitrophenyl)-4-oxo-3-pyridinesulfona-
mide (11). Starting from 1-chloro-2,4-dinitrobenzene (1.44 g), the
title compound 11 was obtained (1.7 g, 71%): m.p. 250e251 ꢁC dec.; IR
(KBr) 3370, 3270 (SO2NH2),1645 (C]O),1610,1600 (NO2), 1345, 1160
1720, 1620 (C]O),1365,1170 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
d 3.98 (s,
3H, CH3OeC]O), 7.16 (t, J ¼ 7.9 Hz, 1H, H-4, PhC]O), 7.25 (d, J ¼ 8.2 Hz,
1H, H-3, PhCO), 7.27 (d, J ¼ 5.8 Hz, 1H, H-6, pyridone ring), 7.38 (s, 2H,
SO2NH2), 7.71 (t, J ¼ 8.2 Hz,1H, H-5, PhC]O), 7.92 (d, J ¼ 7.9 Hz, 1H, H-6,
PhC]O), 8.63 (d, J ¼ 5.8 Hz, 1H, H-5, pyridone), 8.71 (s, 1H, H-2, pyr-
(SO2) cmꢀ1; 1H NMR (DMSO-d6)
d
6.52 (d, J ¼ 7.8 Hz, 1H, H-5, pyrid.),
7.02 (s, 2H, SO2NH2), 8.02 (dd, Jortho ¼ 7.8 Hz, Jmeta ¼ 2.3 Hz, 1H, H-6,
pyrid.) 8.18 (d, J¼ 8.7Hz,1H, H-6,2,4-diO2NPh), 8.52(d, Jmeta ¼ 2.3Hz,
1H, H-2, pyrid), 8.74 (dd, Jortho ¼ 8.7 Hz, Jmeta ¼ 2.6 Hz, 1H, H-5, di
O2NPh) 8.98 (d, Jmeta ¼ 2.6 Hz, 1H, H-3, 2,4-di O2NPh) ppm. Anal.
(C11H8N4O7S) C, H, N.
idone), 11.73 (s, 1H, NH) ppm; 13C NMR (DMSO-d6)
d 56.72, 108.63,
110.51, 115.59, 123.77, 128.07, 129.19, 137.19, 141.65, 147.36, 148.07,
155.13, 160.14, 162.40 ppm. Anal. (C14H13N3O6S) C, H, N.
4.2. CA inhibition assay
4.1.3.2. 1,4-Dihydro-1-(2,6-dinitrophenyl)-4-oxo-3-pyridinesulfona-
mide (12). Starting from 1-chloro-2,6-dinitrobenzene (1.44 g), the
title compound 12 was obtained (1.4 g, 59%): m.p. 263e265 ꢁC dec.; IR
An Applied Photophysics (Oxford, UK) stopped-flow instrument
has been used for assaying the CA-catalyzed CO2 hydration activity
[28]. Phenol red (at a concentration of 0.2 mM) has been used as

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 3656e3661
3661
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Chem. 19 (2004) 199e229.
[6] C.T. Supuran, Expert Opin. Ther. Pat. 13 (2003) 1545e1550.
[7] E. Svastova, A. Hulicova, M. Ratajova, M. Zatovicova, A. Gibadulinova, A. Casini,
A. Cecchi, A. Scozzafava, C.T. Supuran, J. Pastorek, S. Pastorekova, FEBS Lett.
577 (2004) 439e445.
[8] C.T. Supuran, Expert Opin. Investig. Drugs 12 (2003) 283e287.
[9] C.T. Supuran, A. Scozzafava, A. Casini, Med. Res. Rev. 23 (2003) 146e189.
[10] A. Scozzafava, T. Owa, A. Mastrolorenzo, C.T. Supuran, Curr. Med. Chem. 10
(2003) 925e953.
[11] D. Vullo, M. Franchi, E. Gallori, J. Pastorek, A. Scozzafava, S. Pastorekova, C.
T. Supuran, J. Enzym. Inhibit. Med. Chem. 18 (2003) 403e406.
[12] D. Vullo, M. Franchi, E. Gallori, J. Pastorek, A. Scozzafava, S. Pastorekova, C.
T. Supuran, Bioorg. Med. Chem. Lett. 13 (2003) 1005e1009.
[13] J.Y. Winum, D. Vullo, A. Casini, J.L. Montero, A. Scozzafava, C.T. Supuran, J. Med.
Chem. 46 (2003) 5471e5477.
[14] J.Y. Winum, D. Vullo, A. Casini, J.L. Montero, A. Scozzafava, C.T. Supuran, J. Med.
Chem. 46 (2003) 2197e2204.
[15] C. Potter, A.L. Harris, Cell Cycle 3 (2004) 164e167.
[16] S. Pastorekova, A. Casini, A. Scozzafava, D. Vullo, J. Pastorek, C.T. Supuran,
Bioorg. Med. Chem. Lett. 14 (2004) 869e873.
[17] M. Rafajova, M. Zatovicova, R. Kettmann, J. Pastorek, S. Pastorekova, Int. J.
Oncol. 24 (2004) 995.
[18] N. Robertson, C. Potter, A.I. Harris, Cancer Res. 64 (2004) 6160e6165.
[19] A. Thiry, J.M. Donge, B. Masereel, C.T. Supuran, Trends Pharmacol. Sci. 27
(2006) 566e573.
indicator, working at the absorbance maximum of 557 nm, with
10 mM Hepes (pH 7.5) as buffer, 0.1 M Na2SO4 (for maintaining
constant the ionic strength), following the CA-catalyzed CO2
hydration reaction for a period of 10e100 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 5e10% of the reaction have been used for
determining the initial velocity. The uncatalyzed rates were deter-
mined in the same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (1 mM) were prepared in distilled-
deionized water with 10e20% (v/v) DMSO (which is not inhibitory at
these concentrations) and dilutions up to 0.1 nM were done there-
after 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 EeI complex. The inhi-
bition constants were obtained by non-linear last-squares methods
using PRISM 3, as reported earlier [16,29e31], and represent the
mean from at least three different determinations.
Acknowledgments
[20] J.Y. Winum, M. Rami, A. Scozzafava, J.L. Montero, C.T. Supuran, Med. Res. Rev.
28 (2008) 445e463.
[21] F. Sa˛czewski, J. S1awinski, A. Kornicka, Z. Brzozowski, E. Pomarnacka,
This research was financed in part by a grant of the 6th
Framework Programme of the European Union (DeZnIT project)
and by a grant of the 7th FP of EU (Metoxia project) to CTS.
ꢀ
A. Innocenti, A. Scozzafava, C.T. Supuran, Bioorg. Med. Chem. Lett. 16 (2006)
4846e4851.
ꢀ
[22] F. Sa˛czewski, A. Innocenti, Z. Brzozowski, J. S1awinski, E. Pomarnacka,
A. Kornicka, A. Scozzafava, C.T. Supuran, J. Enzym. Inhibit. Med. Chem. 21
Appendix. Supplementary data
(2006) 536e568.
ꢀ
[23] F. Sa˛czewski, A. Innocenti, J. S1awinski, A. Kornicka, Z. Brzozowski,
E. Pomarnacka, A. Scozzafava, C. Temperini, C.T. Supuran, Bioorg. Med. Chem.
Supplementary data associated with this article can be found in
the on-line version, at doi:10.1016/j.ejmech.2010.05.011.
Lett. 16 (2008) 5e13.
ꢀ
[24] Z. Brzozowski, J. S1awinski, F. Sa˛czewski, A. Innocenti, C.T. Supuran, Eur. J.
Med. Chem. 45 (2010) 2396e2404.
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