Carbonic anhydrase inhibitors. Synthesis, molecular structures, and inhibition of the human cytosolic isozymes i and II and transmembrane isozymes IX, XII (cancer-associated) and XIV with novel 3-pyridinesulfonamide derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.07.011

A series of novel 3-pyridinesulfonamide derivatives (2-5, 9-11 and 13-15) have been synthesized and investigated as inhibitors of five isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), that is, the cytosolic ubiquitous CA I and II, and isozymes

Full text of DOI:10.1016/j.ejmech.2011.07.011

European Journal of Medicinal Chemistry 46 (2011) 4403e4410
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Carbonic anhydrase inhibitors. Synthesis, molecular structures, and inhibition
of the human cytosolic isozymes I and II and transmembrane isozymes IX, XII
(cancer-associated) and XIV with novel 3-pyridinesulfonamide derivatives
a
a
,
b
c
c
*
ꢀ
Zdzis1aw Brzozowski , Jaros1aw S1awinski , Maria Gdaniec , Alessio Innocenti , Claudiu T. Supuran
a
ꢀ
ꢀ
Department of Organic Chemistry, Medical University of Gdansk, Al. Gen. J. Hallera 107, 80-416 Gdansk, Poland
b
ꢀ
Faculty of Chemistry, A. Mickiewicz University, 60-780 Poznan, Poland
c 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 novel 3-pyridinesulfonamide derivatives (2e5, 9e11 and 13e15) have been synthesized and
investigated as inhibitors of five isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), that is, the
cytosolic ubiquitous CA I and II, and isozymes CA IX and XII (cancer-associated), and XIV. Against the
Received 27 April 2011
Received in revised form
29 June 2011
Accepted 2 July 2011
Available online 8 July 2011
human isozyme hCA I the new compounds showed KIs in the range of 0.089e251 mM, whereas toward
hCA II, KIs ¼ 50.5e487 nM. Isozyme hCA IX was inhibited with KIs in the range of 5.2e18.3 nM, while hCA
XII with KIs ¼ 6.0e16.4 nM, and hCA XIV with KIs ¼ 76.4e152.0 nM. All of the new compounds 2e5, 9e11
and 13e15 showed excellent hCA IX inhibitory efficacy, with KIs ¼ 5.2e18.3 nM, being much more
effective as compared to the clinically used AAZ, MZA, EZA, DCP and IND (KIs ¼ 24e50 nM).
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
3-Pyridinesulfonamides
Synthesis
X-ray structure
Carbonic anhydrase isozymes I, II, IX, XII and
XIV inhibitors
1. Introduction
target for anticancer therapy. Thus, agents that can selectively
inhibit CA IX activity may have therapeutic value and offer oppor-
The connections between carbonic anhydrase (CA, EC 4.2.1.1)
and cancer is known for approximately fifteen years since two
tumor-associated membrane carbonic anhydrase isozymes (CA IX
and CA XII) have been identified cloned, and sequenced [1e3].
Many reports have been published on the role of CA IX in tumor
physiology [4e6]. In addition its role in the control of tumor pH,
there are evidences that CA IX can also influence other processes in
the cell microenvironment that promote cell proliferation, invasion,
and metastasis [7]. Upregulated expression of CA IX was reported in
carcinoma cells derived from various organs including breast,
cervix, bladder, esophagus, lung, kidney, colon and rectum, head
and neck. This protein constitutes an endogenous marker of cellular
hypoxia, a natural phenotype of solid tumors [8] and its predicative
and prognostic potential has been demonstrated in various clinical
studies [9]. Considering the abnormally high expression of CA IX in
may hypoxic tumors and its demonstrated role in the tumor acid-
ification processes and oncogenesis, CA IX constitutes a candidate
tunities for the treatment or prevention of a variety of cancers. The
potential use of CA inhibitors as antitumor agents opens thus a new
important research direction [10e12].
For example, it has recently been demonstrated that sulfon-
amide or coumarin potent CA IX inhibitors have a potent effect in
inhibiting the growth of both primary tumors and metastases in an
animal model of breast cancer [13,14].
Recently, we have reported on the strong inhibition of human
cytosolic isozymes CA I and II and tumor-associated isozymes IX
and XII with some 4-chloro-5-methyl-2-(R-thio)benzenesulfona-
mides of type I [15e17] (Fig. 1). We also described the syntheses
a number of 3-pyridinesulfonamides of type II and III (Fig. 1), and
their inhibitory activities against human isozymes hCA I, II, IX, XII
and XIV [18,19]. Numerous of these compounds exhibit 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
(KIs ¼ 24e50 nM). These findings prompted us to synthesize and
investigate of the related 3-pyridinesulfonamides of types II and
IVeVI (Fig. 1).
* 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 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.011

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Z. Brzozowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4403e4410
Fig. 1. General structures of known IeIII and novel IVeVI arylsulfonamides, and clinically used sulfonamides AAZ, MZA, EZA, DCP and IND (standard CA inhibitors).
2. Results and discussion
procedures starting from commercially available 4-chloro-3-
pyridinesulfonamide 1. First, the reactions of 1 with pyridine, 2-
methylpyridine, 4-methylpyridine or 4-dimethylpyridine carried
outinboilingacetonitrileledtotheformationof1-(3-sulfamoylpyrid-
4-yl)pyridinium chlorides 2e5 in a very good yields (87e95%). Then,
upon treatment of compd. 2e4 with NaOH (1.2 M equiv.) in water at
2.1. Chemistry
As shown in Scheme 1, the syntheses of the target 3-pyr
idinesulfonamide derivatives 2e9 were achieved by convenient
Scheme 1. Synthesis and proposed mechanism of the formation of 3-pyridinesulfonamide derivatives 2e9. Reagents, conditions and yields: (a) pyridine, 2-methylpyridine,
4-methylpyridine or 4-dimethylaminopyridine (2 M equiv.), dry acetonitrile, reflux 18 h, 87e95%; (b) NaOH (1.2 M equiv.), water room temperature, 3 h, 60e85%; (c) NaOH
(2.0 M equiv.) water, 0e10 ꢀC, 1 h, room temperature, 36 h; (d) hydrochloric acid to pH 2.5, room temperature, 24 h, 70%.

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4403e4410
4405
room temperature, the desired dipyrido[2,1-c: 40,30-e][1,2,4]thiadia-
zine derivatives 6e8 were obtained in 60e85% yield. The attempted
transformation of 5 into analogous pyridodithiazine by this route
failed. However, upon treatment of 5 with an excess of NaOH
(2 M equiv.) in water at temperatures 0e20 ꢀC corresponding 4-(1,4-
dihydro-4-oxopyrid-1-yl)-3-pyridinesulfonamide 9 was obtained. In
this conditions the pyridinium chloride 5 was converted successively
to the unstable ammonium hydroxide B and the addition product of
typeC, whichundergoesaspontaneouseliminationofMe2NHleading
to the formation of the salt D, which upon acidification afforded free
sulfonamide 9 in 70% yield. Compound 9 was also obtained in 68%
yield by a convenient procedure (method B, Scheme 2) starting from
4-chloro-3-pyridinesulfonamide 1 and 4-methoxypyridine in dry
acetonitrile. The initial step is believed to be formation of interme-
diate pyridinium salt E which undergoes SN2 substitution on the
methyl by chloride to form the target sulfonamide 9. Analogously, by
reaction with 4-methoxy-3-pyridinesulfonamide was prepared the
target sulfonamide 10 as shown in Scheme 2. However, we found that
analogue reaction of 4-chloro-3-pyridinesulfonamide 1 with 2-
methoxypyridine led to the formation a mixture of 4-substituted 3-
pyridinesulfonamide 11 and the adduct of 4-(1,2-dihydro-2-
oxopyrid-1-yl)-3-pyridinesulfonamide with 2-pyridone 12, which
were separated in 12.6 and 38.5% yields. We propose a reaction
sequence for the transformation as shown in Scheme 2. The initial
step is believed to be the formation of intermediate 1-(3-
sulfomoypyrid-4-yl)-2-methoxpyridinium chloride
G
which
undergoes elimination of hydrochloride or methyl chloride to form
the mixture of intermediate dipyrido[2,1-c:40, 30-e][1,2,4]thiadiazine
H
and 4-(1,2-dihydro-2-oxopyrid-1-yl)-3-pyridinesulfoamide I,
respectively. Then, thereactionofintermediateHwithIledtothefinal
product 11 and 2-pyridone J which undergoes addition to the inter-
mediate I to form the adduct 12. Then upon treatment of adduct 12
Scheme 2. Synthesis and proposed mechanism of the formation of 3-pyridinesulfonamide derivatives 9e13. Reagents, conditions and yields: (a) 4-methoxypyridine (1.2 M equiv.)
dry acetonitrile, room temperature 24 h, reflux 45 h, 68%; (b) 4-methoxy-3-pyridinesulfonamide (1.0 M equiv.) dry acetonitrile, reflux 26 h 82%; (c) 2-methoxypyridine
(1.3 M equiv.) dry acetonitrile, room temperature 24 h, reflux 45 h, 12.6% for 11 and 38.5% for 12; (d) methanol, reflux 0.5 h water, room temperature 16 h, 91%.

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Z. Brzozowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4403e4410
with methanol and water at reflux, the free sulfonamide 13 was
obtained in 91% yield.
2.2. CA inhibition studies
We have found in our studies that the reaction of 4-chloro-3-
pyridinesulfonamide 1 with urea led to the formation of the
expected uronim chloride K, which in the presence of methanol
undergoes a further transformation. The proposed mechanism
leading to the formation of the products 14 and 15 is outlined in
Scheme 3. The initial step is believed to be formation of unstable
1Hþ-pyridinium chloride L, which after spontaneous elimination of
urea or 2-methyluronium chloride gives two intermediate prod-
ucts: 4-methoxy-3-pyridinesulfonamide hydrochloride M and 4-
hydroxy-3-pyridinesulfonamide N, respectively. In the next stages
The compounds 2e5, 9e11 and 13e15 as well as standard,
clinical used CAIs, such as acatazolamide AAZ, methazolamide
MZA, ethoxzolamide EZA, dichlorophenamide DCP, and indisulam
IND (Fig. 1) have been tested for the inhibition of two cytosolic
ubiquitous isozymes of human origin, that is, hCA I and hCA II, and
two transmembrane isoforms (cancer-associated) hCA IX, XII, and
hCA XIV isozyme (Table 1). The following should be noted
regarding CA inhibitory data of Table 1: (a) against the slow cyto-
solic izoform hCA I, the sulfonamides investigated here showed
high to weak inhibitory properties. Thus, derivatives 2, 3, 9, 10 and
15 exhibited weak inhibition of this izoform, with KIs in the range of
of reaction the intermediate
M and N reacts with starting
compound 1 led to formation of pyridinium chloride O, which
undergoes cyclocondensation to afford the hydrochloride P of final
product 14, and hydrochloride R of final product 15, respectively.
Then, upon neutralization of the salts P and R with NaOH in water
the desired free sulfonamides 14 and 15 were obtained in 66% and
24% yield, respectively.
All final products were characterized by IR and NMR spec-
troscopy as shown in experimental protocols. Elemental analyses
(C, H, N) were in accordance with the proposed structure. X-ray
crystallography was undertaken in order to investigate in detail
molecular structures of the adduct 12 and its free sulfonamide 13
(Figs. 2 and 3).
5.35e251.0 mM, being thus much weaker inhibitors as compared to
the clinically used sulfonamides AAZeIND (Table 1). The
compounds 4, 5 and 11 were slightly more effective inhibitors
against hCA I, with KIs in the range of 2.46e4.56
the one compound 14 acted as strong hCA
M), with a comparable potency as the references
mM. However, only
a
I
inhibitor
(KIs ¼ 0.089
m
compounds (i.e., EZA or IND); (b) against the ubiquitous and
dominant rapid cytosolic isozyme hCA II, all compounds 2e5, 9e11
and 13e15 acting as weaker inhibitors (KIs in the range
50.5e487.0 mM)(Table 1); (c) all of the new compounds 2e5, 9e11
and 13e15 showed excellent hCA IX inhibitory efficacy, with inhi-
bition constants of 5.2e18.3 nM, being much more effective as
Scheme 3. Proposed mechanism of the formation of 3-pyridinesulfonamide derivatives 14 and 15 in the reaction of 4-chloro-3-pyridinesulfonamide with urea in boiling methanol.

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4403e4410
4407
3. Conclusions
We have developed methods for the preparation of the novel
series of 3-pyridinesulfonamide derivatives. The several new
sulfonamides have been assayed for the inhibition of the physio-
logically relevant CA isozymes, such as CA I and II, the tumor-
associated isozymes CA IX and XII, as well as membrane-associ-
ated CA XIV. Toward the human isozyme CA I the new compounds
showed inhibition constants in the range of 0.089e251 mM, while
against hCA II in the range of 50.5e487 nM. Moreover, hCA IX was
inhibited with KIs in the range of 5.2e18.3 nM, while second
membrane-associated isozyme hCA XII was inhibited with KIs in the
range of 6.0e16.4 nM. Compound 14 has a distinctive activityagainst
hCA I (KIs ¼ 0.089
m
M) comparable with the clinical used sulfon-
amides AAZ, MZA, EZA, DCP and IND (KIs ¼ 0.025e1.20
mM). Is
noteworthy, that all of the new compounds 2e5, 9e11 and 13e15
showed excellent hCA IX inhibitory efficacy, with inhibition
constants of 5.2e18.3 nM, being much moreeffective as compared to
the clinically used AAZ, MZA, EZA, DCP and IND (KIs ¼ 24e50 nM).
4. Experimental protocols
4.1. Synthesis
The following instruments and parameters were used: melting
points Buchi 535 apparatus, IR spectra: KBr pellets, 400e4000 cmꢁ1
PerkineElmer FT IR spectrophotometer; 1H and 13C NMR: Varian
Gemini 200 apparatus at 200 and 50 MHz, respectively; chemical
shifts are expressed in parts per million (ppm) relative to TMS as
internal standard. The results of elemental analyses for C, H and N
were within ꢂ0.3% of theoretical values.
Fig. 2. ORTEP [23] representation of molecular structure of 12. Displacement ellipsoids
are shown at the 50% probability level.
compared to the clinical used AAZ, MZA, EZA, DCP and IND
(KIs ¼ 24e50 nM); (d) a quite good inhibition profile of the second
tumor-associated isoform, that is, hCA XII has also been observed
for all investigated sulfonamides of types: 2e5 (KIs ¼ 8.2e16.4 nM);
9,10 and 13 (KIs ¼ 6.1e9.1 nM); 11 and 14 (KIs ¼ 7.5e7.8 nM), and 15
(KIs ¼ 6.0 nM) (Table 1); (e) all of the investigated sulfonamides
2e5, 9e11 and 13e15 showed moderate inhibition properties
toward hCA XIV isozyme with KIs in the range of 76.4e152.0 nM
(Table 1).
4.1.1. Procedures for the preparation of 1-(3-sulfamoylpyrid-4-yl)
pyridinium chlorides (2e5)
A mixture of the appropriate pyridine derivative (24 mmol) and
4-chloro-3-pyridienesulfonamide 1 (2.31 g, 12 mmol) in acetoni-
trile (15 ml) was refluxed with stirring for 18 h and left to stand at
room temperature overnight. The precipitate was filtered off,
washed with acetonitrile (5 ꢃ 2 ml) and dried. In this manner the
following aminium chlorides were obtained.
4.1.1.1. 1-(3-Sulfamoylpyrid-4-yl)pyridinium chloride (2). Starting
from pyridine (1.9 g), the title compound 2 was obtained (3.1 g, 95%):
m.p. 201e202 ꢀC dec.; IR (KBr) 3190, 3125 (SO2NH2), 1630, 1565
(pyridine rings) 1335, 1165 (SO2) cmꢁ1; 1H NMR (DMSO-d6)
d 8.08 (d,
J ¼ 5.2 Hz, 1H, H-5, pyridine), 8.35 (t, J ¼ 7.3 Hz, 2H, H-3 and H-5,
pyridinium), 8.47 (s, 2H, SO2NH2), 8.88 (t, J ¼ 7.3 Hz, 1H, H-4 pyr-
idinium), 9.17 (d, J ¼ 5.2 Hz, 1H, H-6, pyridine), 9.36 (s, 1H, H-2 pyri-
dine), 9.39 (dd, Jortho ¼ 7.3 Hz, Jmeta ¼ 1.3 Hz, 2H, H-2 and H-6,
pyridinium)ppm;13CNMR(DMSO-d6)
d122.99,127.78,134.23,145.27,
146.58, 149.55 149.99, 155.14 ppm. Anal. (C10H10ClN3O2S) C, H, N.
4.1.1.2. 2-Methyl-1-(3-sulfamoylpyrid-4-yl)pyridinium
chloride
(3). Starting from 2-methylpyridine (2.2 g) the title compound 3
was obtained (3.1 g, 90%): m.p. 207e208 ꢀC dec.; IR (KBr) 3190,
3140 (SO2NH2), 1635, 1565 (pyridine rings), 1345, 1185 (SO2) cmꢁ1
;
1H NMR (DMSO-d6)
d
2.59 (s, 3H, CH3), 8.07 (d, J ¼ 5.2 Hz, 1H, H-5,
pyridine), 8.24e8.27 (m, 1H, H-4, pyridinium), 8.44 (s, 2H,
SO2NH2), 8.74 (d, J ¼ 8.0 Hz, 1H, H-3 pyridinium), 9.18 (d,
J ¼ 5.2 Hz, 1H, H-6, pyridine), 9.22 (d, J ¼ 8.0 Hz, 1H, H-5 pyr-
idinium), 9.32e9.34 (m, 1H, H-6, pyridinium), 9.37 (s, 1H, H-2,
pyridine) ppm. Anal. (C11H12ClN3O2S) C, H, N.
4.1.1.3. 4-Methyl-1-(3-sulfamoylpyrid-4-yl)pyridinium chloride (4).
-Starting from 4-methylpyridine (2.2 g), the title compound 4 was
Fig. 3. ORTEP [23] representation of molecular structure of 13. Displacement ellipsoids
are shown at the 50% probability level.

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Z. Brzozowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4403e4410
Table 1
Carbonic anhydrase inhibition data for compounds 2e5, 9e11 and 13e15 and standard inhibitors against human isozymes hCA I, II, IX, XII and XIV by a stopped-flow, CO2
hydration assay [23].
OMe
MeO
Cl
R
R1
S
N
S
N
S
N
S
SO NH2
SO NH2
2
2
N
N
N
NH2
N
N
N
NH2
H
O O
O O
11
O O
O O
N
2-5
9,10,13,15
14
Compd
R
R1
KIa
hCA Ib
(
m
M)
hCA IIb (nM)
hCA IXc (nM)
hCA XIIc (nM)
hCA XIVd (nM)
AAZ
MZA
EZA
DCP
IND
0.31
0.78
0.025
1.20
12
14
8
38
15
25
27
34
50
24
5.7
3.4
22
50
3.4
41
43
25
345
104
0.031
2
3
4
5
H
e
e
e
e
7.60
5.45
4.56
2.91
102
336
391
149
7.6
7.4
10.1
9.0
16.4
8.2
9.0
152
113
104
87.5
2-Me
4-Me
4-Me2N
9.9
O
O
9
e
6.12
98.3
18.3
6.1
98.5
N
N
10
11
13
14
e
e
e
e
7.13
2.46
5.38
0.089
50.5
220.0
487.0
90.0
5.2
8.7
8.7
7.5
9.1
7.8
93.8
76.4
80.6
86.1
SO2NH2
e
O
10.2
13.5
N
e
N
O
15
e
251.0
54.6
9.0
6.0
127.0
SO2NH2
a
Errors in the range of ꢂ5e10% of the reported value (from 3 different assays).
b
c
Human (cloned) isozymes, by CO2 hydration method.
Catalytic domain of human, cloned isozymes [24,25], by the CO2 hydration method.
Full-length human (cloned) isozyme [26].
d
obtained (3.2 g, 93%): m.p. 226e227 ꢀC dec.; IR (KBr) 3180, 3115
(SO2NH2), 1640, 1575 (pyridine rings), 1360, 1175 (SO2) cmꢁ1 1H
NMR (DMSO-d6)
pyridine), 8.20 (d, J ¼ 6.5 Hz, 2H, H-3 and H-5, pyridinium), 8.46 (s,
2H, SO2NH2), 9.17 (d, J ¼ 4.9 Hz, 1H, H-2, pyridine), 9.29 (d,
J ¼ 6.5 Hz, 2H, H-2 and H-6 pyridinium), 9.37 (s, 1H, H-2, pyridine)
ppm. Anal. (C11H12ClN3O2S) C, H, N.
4.1.2. Procedures for the transformations of pyridinium chlorides
2e4 into 6,6a-dihydrodipyridio[2,1-c:40,30-e][1,2,4]thiadiazine 5,5-
dioxide derivatives (6e8)
To a solution of the appropriate pyridinium chloride 2, 3 or 4
(4 mmol) in water (5 ml), solution of NaOH (0.19 g, 4.8 mmol) in
water (5 ml) was added. The reaction mixture was stirred at room
temperature for 3 h, and the resulting precipitate was collected by
filtration, washed successively with water (3 ꢃ 2 ml) and acetoni-
trile (2 ꢃ 1 ml), and dried. In this manner the following pyr-
idothiadiazines were obtained.
;
d
2.76 (s, 3H, CH3), 8.05 (d, J ¼ 4.9 Hz, 1H, H-5,
4.1.1.4. 4-Dimethylamino-1-(3-sulfamoylpyrid-4-yl)pyridinium chlo-
ride (5). Starting from 4-dimethylaminopyridine (3.4 g) the title
compound 5 was obtained (3.3 g, 87%): m.p. 242e243 ꢀC dec.; IR
(KBr) 3400, 3150 (SO2NH2), 1655, 1575 (pyridine rings), 1355, 1170
4.1.2.1. 6,6a-Dihydrodipyridio[2,1-c:40,30-e][1,2,4]thiadiazine
5,5-
(SO2) cmꢁ1
;
1H NMR (DMSO-d6)
d
3.28 (s, 6H, CH3eNeCH3), 7.18
dioxide (6). Starting from pyridinium chloride 2 (1.09 g), the title
compound 6 was obtained (0.8 g, 85%): m.p. 228e230 ꢀC dec.; IR
(KBr) 3120 (NH), 1660, 1585 (C]N and C]N), 1330, 1170 (SO2)
(d, J ¼ 7.5 Hz, 2H, H-3 and H-5 pyridinium), 7.86 (d, J ¼ 5.1 Hz, 1H,
H-5, pyridine), 8.34 (s, 2H, SO2NH2), 8.43 (d, J ¼ 7.5 Hz, 2H, H-2
and H-6 pyridinium), 9.07 (d, J ¼ 5.1 Hz, 1H, H-6, pyridine), 9.31 (s,
cmꢁ1; 1H NMR (DMSO-d6)
H-6a,7,8 and H-9), 7.04 (d, J ¼ 7.7 Hz, 1H, H-10), 7.42 (d, J ¼ 6.1 Hz,
1H, H-1), 8.48 (d, J ¼ 6.1 Hz,1H, H-2), 8.74 (s,1H, H-4), 8.99 (s,1H, H-
6) ppm. Anal. (C10H9ClN3O2S) C, H, N.
d
5.28e5.46 and 6.10e6.25 (2m, 2 ꢃ 2H,
1H, H-2, pyridine) ppm; 13C NMR (DMSO-d6)
d
107.19, 123.98,
135.26, 142.84, 145.45, 149.83, 155.10, 156.69 ppm. Anal.
(C12H15ClN4O2S) C, H, N.

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4403e4410
4409
4.1.2.2. 10-Methyl-6,6a-dihydrodipyridio[2,1-c:40,30-e][1,2,4]thiadia-
4.1.5. One-pot synthesis of 2-[10-methoxy-5,5-dioxo-6,6a-dihydrod
zine 5,5-dioxide (7). Starting from pyridinium chloride 3 (1.14 g),
the title compound 7 was obtained (0.7 g, 70%): m.p. 238e240 ꢀC
dec.; IR (KBr) 3115 (NH), 1670, 1590 (C]N and C]N), 1330, 1175
ipyrido[2,1-c:40,30-e][1,2,4]thiadiazin-6-yl]benzenesulfonamide (11)
and the adduct of 4-(1,2-dihydro-2-oxopyrid-1-yl)-3-pyrid
inesulfonamide with 2-pyridone (12)
(SO2) cmꢁ1
;
1H NMR (DMSO-d6)
d
1.80 (s, 3H, CH3), 5.27 (s, 1H, H-
A mixture of 4-chloro-3-pyridinesulfonamide 1 (5. 78 g,
6a), 5.95 (s, 1H, H-8), 6.08 (s, 1H, H-7), 6.94 (d, J ¼ 6.9 Hz, 1H, H-9),
7.44 (d, J ¼ 5.8 Hz, 1H, H-1), 8.49 (d, J ¼ 5.8 Hz, 1H, H-2), 8.78 (s, 1H,
H-4), 8.84 (s, 1H, H-6) ppm. Anal. (C11H11N3O2S) C, H, N.
30 mmol) and 2-methoxypyridine (4.26 g, 39 mmol) in dry aceto-
nitrile (60 ml) was stirred at room temperature for 24 h, followed at
reflux for 45 h. After cooling to room temperature a pitchy side
products was filtered out with charcoal. The solvent was partially
evaporated under normal pressure and the suspension (w15 g) was
left overnight. The precipitate of adduct 12 was collected by
filtration, washed with acetonitrile (3 ꢃ 1.5 ml) (Filtrate A was left
4.1.2.3. 8-Methyl-6,6a-dihydrodipyridio[2,1-c:40,30-e][1,2,4]thiadia-
zine 5,5-dioxide (8). Starting from pyridinium chloride 4 (1.14 g),
the title compound 8 was obtained (0.6 g, 60%): m.p. 210e212 ꢀC
dec.; IR (KBr) 31150 (NH), 1675, 1585 (C]N and C]N), 1325, 1170
for further work-up) and dried. Yield 2.0
g (38.5%), m.p.
(SO2) cmꢁ1; 1H NMR (DMSO-d6)
d
1.80 (s, 3H, CH3), 5.21 (s, 2H, H-6a
165e166 ꢀC; IR (KBr), 3310, 3290, 3240, 3125 (NH and SO2NH2),
and H-7), 6.14 (d, J ¼ 5.9 Hz, 1H, H-9), 7.05 (d, J ¼ 5.9 Hz, 1H, H-10),
7.43 (d, J ¼ 5.8 Hz, 1H, H-1), 8.48 (d, J ¼ 5.8 Hz, 1H, H-2), 8.75 (s, 1H,
H-4), 8.88 (s, 1H, H-6) ppm. Anal. (C11H11N3O2S) C, H, N.
1700, 1660 (C]O), 1620 (C]N), 1350, 1175 cmꢁ1; 1H NMR (DMSO-
d6)
d
6.14 (t, J ¼ 6.2 Hz,1H, arom.), 6.28e6.40 (m, 2H, arom.), 6.50 (d,
J ¼ 9.8 Hz, 1H, arom.), 7.33e7.44 (m, 2H, arom.), 7.53e7.66 (m, 5H,
SO2NH2 and 3H arom.), 8.95 (d, J ¼ 5.0 Hz, 1H, H-6, PyeSO2), 9.17 (s,
1H, H-2, PyeSO2), 11.55 (br.s, 1H, HNeC]O 4 N]CeOH of 2-
4.1.3. Synthesis of 4-(1,4-dihydro-4-oxopyrid-1-yl)-3-pyridinesulfo
namide 9 from pyridinium chloride 5 (method A), and from 4-
chloro-3-pyridinesulfonamide 1 (method B)
pyridone moiety) ppm; 13C NMR (DMSO-d6)
d 105.11, 106.17,
120.30, 120.48, 125.07, 135.76, 136.13, 138.72, 141.22, 141.92, 145.30,
149.72, 154.73, 161.65, 162.65 ppm. Anal. (C15H14N4O4S) C, H, N.
The filtrate A was evaporated under reduced pressure. To the
resulting oily residue 2-propanol (60 ml) and charcoal (0.3 g) was
added and the resulting mixture was heated under reflux for 1 h.
The charcoal and side products were separated by suction and the
hot filtrate was left at room temperature for 24 h. The precipitate of
11 was collected by filtration, washed with 2-propanol (3 ꢃ 2 ml)
and dried, Yield 0.8 g (12.6%), m.p. 201e203 ꢀC dec.; IR (KBr), 3360,
3295, 3190 (SO2NH2), 1645, 1565 (C]N and C]C), 1345, 1165 (SO2)
Method A (Scheme 1). To an ice-cold solution of NaOH (0.32 g,
8 mmol) in water (15 ml), pyridinium chloride 5 (1.26 g, 4 mmol)
was added. The reaction solution was stirred at 0e10 ꢀC for 1 h,
followed at room temperature for 36 h. A small amount of insoluble
side product was filtered out together with charcoal added. The
filtrate (pH w11) was acidified with 1% hydrochloric acid to pH 2.5.
After 24 h of stirring the title product 9 was collected by filtration,
washed with water (3 ꢃ 2 ml) and dried at temperatures gradually
ꢀ
increasing to 100 ꢀC. Yield 0.7 g (70%), m.p. 235e236 C; IR (KBr)
3310, 3190 (SO2NH2), 1640 (C]O), 1340, 1165 (SO2) cmꢁ1; 1H NMR
cmꢁ1
;
1H NMR (DMSO-d6)
d
3.75 (s, 3H, CH3O), 6.46 (d, J ¼ 7.7 Hz,
(DMSO-d6)
d
6.19 (d, J ¼ 7.6 Hz, 2H, H-3 and H-5 pyridone), 7.70 (d,
1H, H-1), 6.85e7.1 (m, 2H, H-6a and H-7), 7.40e7.68 (m, 2H, H-8
and H-9), 7.80 (d, J ¼ 5.2 Hz, H-5 of PyeSO2NH2), 7.91 (dd,
Jortho ¼ 7.7 Hz, Jmeta ¼ 2.4 Hz, 1H, H-2), 8.12 (s, 2H, SO2NH2), 8.29 (d,
Jmeta ¼ 2.4 Hz, 1H, H-4), 9.00 (d, J ¼ 5.2 Hz, 1H, H-6 of PyeSO2NH2),
9.22 (s, 1H, H-2 of PyeSO2NH2) ppm. Anal. (C16H15N5O5S2) C, H, N.
J ¼ 5.1 Hz, 1H, H-5, PyeSO2), 7.75 (d, J ¼ 7.6 Hz, 2H, H-2 and H-6,
pyridone), 8.01 (s, 2H, SO2NH2), 8.96 (d, J ¼ 5.1 Hz, 1H, H-6,
PyeSO2), 9.20 (s, 1H, H-2, PyeSO2) ppm; 13C NMR (DMSO-d6)
d
117.37, 123.90, 135.18, 141.28, 146.88, 149.77, 155.02, 177.85 ppm.
Anal. (C10H9N3O3S) C, H, N.
Method B (Scheme 2). A mixture of 1 (1.93 g, 10 mmol) and
4-methoxypyridine (1.3 g, 12 mmol) in dry acetonitrile (35 ml) was
stirred a room temperature for 24 h, followed at reflux for 45 h. After
cooling to room temperature the suspension was left overnight. The
precipitate was collected by filtration, washed successively with
acetonitrile (3 ꢃ 1.5 ml), water (5 ꢃ 3 ml) and acetonitrile (3 ꢃ 2 ml),
the dried at temperatures gradually increasing to 100 ꢀC. Yield 1.7 g
(68%), m.p. 234e236 ꢀC; IR and 1H NMR were identical with
authentic sample 9 described in method A.
4.1.6. Transformation of adduct 12 to free 4-(1,2-dihydro-2-oxopyr
id-1-yl)-3-pyridinesulfonamide (13)
A solution of adduct 11 (1.38 g, 40 mmol) in methanol (35 ml)
was refluxed with stirring for 0.5 h, and then water (7 ml) was
added. After cooling to room temperature the suspension was left
overnight. The precipitate was collected by filtration washed with
methanol (2 ꢃ 1 ml) and dried at temperatures gradually increasing
to 90 ꢀC. Yield 0.92 g (91%), m.p. 206e207 ꢀC dec.; IR (KBr) 3240,
3180 (SO2NH2), 1655 (C]O) 1580, 1560, 1530 (pyridine rings) 1335,
1160 (SO2) cmꢁ1 1H NMR (DMSO-d6)
; d 6.30e6.42 (m, 1H, arom.),
4.1.4. Synthesis of 4-(1,4-dihydro-4-oxo-3-sulfomoylpyridin-1-yl)-
3-pyridinesulfonamide (10)
6.45e6.58 (m, 1H, arom.), 7.58 (br.s, 5H, SO2NH2 and 3H arom.),
8.95 (t, J ¼ 3.4 Hz, 1H, H-6 of PyeSO2NH2), 9.17 (d, J ¼ 2.0 Hz, H-2 of
A
solution of
1
(1.35 g,
7
mmol) and 4-methoxy-3-
PyeSO2NH2) ppm; 13C NMR (DMSO-d6)
d 106.18, 120.48, 125.08,
pyridinesulfonamide (1.32 g, 7 mmol) in dry acetonitrile was
refluxed with stirring for 26 h. After cooling to room temperature
the suspension was left overnight. The precipitate was collected by
filtration, washed with acetonitrile (4 ꢃ 1.5 ml) and dried at
temperatures gradually increasing to 100 ꢀC. Yield 1.9 g (82%), m.p.
247e249 ꢀC; IR (KBr) 3325, 3220, 3150 (SO2NH2), 1645 (C]O), 1575
136.13, 138.72, 141.93, 145.31, 149.72, 154.74, 161.66 ppm. Anal.
(C10H9N3O3S) C, H, N.
4.1.7. One-pot synthesis of 2-[8-methoxy-5,5-dioxo-6,6a-dihydrodi
pyrido[2,1-c:40,30-e][1,2,4]thiadiazine]sulfonamide (14) and 4,40-oxy
dipyridine-3-sulfonamide (15) by the reaction of 4-chloro-3-pyr
idinesulfonamide (1) with urea in boiling methanol
(C]N), 1340, 1160 (SO2) cmꢁ1 1H NMR (DMSO-d6)
; d 6.45 (d,
J ¼ 7.8 Hz, 1H, H-5 of 4-pyridone ring), 6.94 (s, 2H, SO2NH2 of
pyridone ring), 7.80 (d, J ¼ 5.2 Hz,1H, H-5 of pyridine ring), 7.89 (dd,
Jortho ¼ 7.8 Hz, Jmeta ¼ 2.1 Hz, 1H, H-6 of pyridone ring), 8.09 (s, 2H,
SO2NH2 of pyridine ring), 8.28 (d, J ¼ 2.1 Hz, 1H, H-2 of pyridone
ring), 8.99 (d, J ¼ 5.2 Hz, 1H, H-6 of pyridine ring), 9.20 (s, 1H, H-2 of
A solution of 4-chloropyridine-3-sulfonamide 1 (1.93 g,
10 mmol) and urea (0.63 g, 10.5 mmol) in methanol (10 ml) was
stirred at reflux for 5 h, then methanol was evaporated under
reduced pressure. The solid residue was dissolved in water (5 ml,
20 ꢀC) then neutralized pH 7 with 1% aqueous NaOH solution. The
precipitate of compound 14 thus obtained was filtered off, washed
with water (5 ꢃ 2 ml) and methanol (6 ꢃ 2 ml) (filtrate B was left
for further work-up), and dried. Yield 1.14 g (66%), m.p. 206e208 ꢀC
pyridine ring) ppm; 13C NMR (DMSO-d6)
d 119.41, 124.25, 130.19,
135.19, 141.68, 142.66, 146.08, 149.56, 155.26, 172.92 ppm. Anal.
(C10H10N4O5S2) C, H, N.

4410
Z. Brzozowski et al. / European Journal of Medicinal Chemistry 46 (2011) 4403e4410
dec.; IR (KBr) 3380, 3265, 3115 (SO2NH2), 1660, 1475 (C]C and C]
N) 1350, 1335, 1160 (SO2) cmꢁ1 1H NMR (DMSO-d6)
3.64 (s, 3H,
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
determined 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 thereafter with distilled-deionized water. Inhibitor and
enzyme solutions were preincubated together for 15 min at room
temperature prior to assay, in order to allow for the formation
of EeI complex. The inhibition constants were obtained by non-
linear last-squares methods using PRISM 3, as reported earlier
[24e26], and represent the mean from at least three different
determinations.
;
d
CH3O), 4.69 (d, J ¼ 3.3 Hz, 1H, H-6a), 6.40 (s, 1H, H-9), 6.97 (s, 2H,
SO2NH2), 7.48 (d, J ¼ 5.7 Hz, 1H, H-10), 7.53 (d, J ¼ 5.7 Hz, 1H, H-1),
8.62 (d, J ¼ 5.7 Hz, 1H, H-2), 8.87 (s, 1H, H-4), 9.20 (br.s, 1H, NH)
ppm; 13C NMR (DMSO-d6)
d 55.63, 69.07, 86.77, 112.22, 115.31,
126.12, 131.93, 145.01, 146.54, 149.83, 153.43 ppm. Anal.
(C11H12N4O5S2) C, H, N.
The wateremethanol filtrate mixture B was evaporated under
reduced pressure. To the residue obtained a solution of acetic acid
(1.5 ml) in water (8 ml) was added and stirred at room temperature
for 2 h. The title product 15, which precipitated, was collected by
filtration, washed successively with water (2 ꢃ 1 ml) and methanol
(1 ml), and dried at temperatures gradually increasing to 105 ꢀC.
Yield 0.4 g (24%), m.p. 248e249 ꢀC; IR (KBr), 3380, 3330, 3150
(SO2NH2), 1645, 1580, 1570 (C]N and C]C), 1340, 1325, 1160 (SO2)
cmꢁ1; 1H NMR (DMSO-d6)
d
6.45 (d, J ¼ 7.7 Hz, 1H, H-5), 6.94 (s, 2H,
Appendix. Supplementary material
SO2NH2), 7.89 (dd, Jortho ¼ 7.7 Hz, Jmeta ¼ 2.4 Hz, 1H, H-6), 8.27 (d,
Jmeta ¼ 2.4 Hz, 1H, H-2), and 7.80 (d, J ¼ 5.2 Hz, 1H, H-50), 8.09 (s, 2H,
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.07.011.
0
SO2NH2 ), 8.89 (d, J ¼ 5.2 Hz, 1H, H-60), 9.20 (s, 1H, H-20) ppm. Anal.
(C10H10N4O5S2) C, H, N.
References
4.2. X-ray crystal structure analysis
[1] J. Pastorek, S. Pastorekova, I. Callebuant, J.P. Mornon, V. Zelnik, R. Opavsky,
M. Zatovicova, S. Liao, D. Portetelle, E.J. Stanbridge, J. Zavada, A. Burny,
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The diffraction data for single crystals of 12 and 13 were
collected at 100K with Oxford Diffraction XCaliburE diffractometer
using Mo Ka radiation. The intensity data were collected and pro-
cessed using CrysAlisPro Software. The structures were solved by
direct methods with the program SHELXS-97 [20] and refined by
full-matrix least-squares method on F2 with SHELXL-97 [21,22].
Hydrogen atoms from NeH groups were freely refined and those
bonded to C atoms refined as riding on their carriers.
Crystal data for 12: C10H9N3O3S$C5H5NO, triclinic, space group
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S. Pastorekova, Exp. Cell Res. 290 (2003) 332e345.
Pꢁ1, a ¼ 8.8829 (4), b ¼ 10.0844(6), c ¼ 10.5080 (7) Å,
a
¼ 112.601
(6),
b
¼ 112.306 (5),
g
¼ 95.617 (4)ꢀ, V ¼ 770.55 (10) Å3, Z ¼ 2,
T ¼ 100 K, dx ¼ 1.493 g cmꢁ3
,
m
(Mo K
a
) ¼ 0.239 mmꢁ1, 8998 data
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(2006) 566e573.
were collected up to qmax ¼ 28.97ꢀ (Rint ¼ 0.0200, R
¼ 0.0242).
s
Final R indices for 3091 reflections with I > 2
s
(I) and 163 refined
[11] J.Y. Winum, M. Rami, A. Scozzafava, J.L. Montero, C.T. Supuran, Med. Res. Rev.
28 (2008) 445e463.
[12] C.T. Supuran, Nat. Rev. Drug Discov. 7 (2008) 168e181.
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C.T. Supuran, J. Med. Chem. 54 (2011) 1896e1902.
parameters are: R1 ¼ 0.0311, wR2
¼ 0.0829 (R1 ¼ 0.0362,
wR2 ¼ 0.0842 for all 3559 data).
Crystal data for 13: C10H9N3O3S, monoclinic, space group P21/c,
a ¼ 9.6132 (8), b ¼ 7.7939(8), c ¼ 14.4521 (12) Å,
b
¼ 98.822 (9)ꢀ,
V ¼ 1070.00 (17) Å3, Z ¼ 4, T ¼ 100 K, dx ¼ 1.560 g cmꢁ3
, m(Mo
[14] Y. Lou, P.C. McDonald, A. Oloumi, S.K. Chia, C. Ostlund, A. Ahmadi, et al., Cancer
Res. (2011) 3364.
K
a
) ¼ 0.302 mmꢁ1, 11855 data were collected up to qmax ¼ 29.03ꢀ
ꢀ
[15] F. Sa˛czewski, J. S1awinski, A. Kornicka, Z. Brzozowski, E. Pomarnacka,
(Rint ¼ 0.0203, R
s
¼ 0.0162). Final R indices for 2336 reflections
A. Innocenti, A. Scozzafava, C.T. Supuran, Bioorg. Med. Chem. Lett. 16 (2006)
4846e4851.
with I > 2
s(I) and 163 refined parameters are: R1 ¼ 0.0292,
ꢀ
[16] F. Sa˛czewski, A. Innocenti, J. S1awinski, E. Pomarnacka, A. Kornicka,
wR2 ¼ 0.0808 (R1 ¼ 0.0327, wR2 ¼ 0.0818 for all 2590 data).
Crystallographic data for compounds 12 and 13 have been
deposited with the Cambridge Crystallographic Data Centre, with
the deposition numbers CCDC 821393 and 821394.
A. Scozzafava, C.T. Supuran, J. Enzym, Inhibit. Med. Chem. 21 (2006) 536e568.
ꢀ
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A. Kornicka, A. Scozzafava, C.T. Supuran, Bioorg. Med. Chem. Lett. 16 (2008)
5e13.
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ꢀ
[19] Z. Brzozowski, J. S1awinski, A. Innocenti, C.T. Supuran, Eur. Med. Chem. 45
4.3. CA inhibition assay
(2010) 3656e3661.
[20] CrysAlis Pro Software, ver. 1.171.33. Oxford Diffraction Ltd, Yarnton,
Oxfordshire, UK, 2009.
An Applied Photophysics (Oxford, UK) stopped-flow instrument
has been used for assaying the CA-catalyzed CO2 hydration activity
[23]. Phenol red (at a concentration of 0.2 mM) has been used as
indicator, working at the absorbance maximum of 557 nm, with
10 mM Hepes (pH 7.5) 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
[21] G.M. Sheldrick, Acta Cryst A64 (2008) 112e122.
[22] Ortep-3 for Windows: L.J. Farrugia J. Appl. Cryst. 30 (1997) 565.
[23] R.G. Khalifah, J. Biol. Med. 246 (1971) 2561e2573.
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J. Med. Chem. 47 (2004) 2337e2347.
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