Carbonic anhydrase inhibitors: Synthesis and inhibition of the human cytosolic isozymes I and II and transmembrane isozymes IX, XII (cancer-associated) and XIV with 4-substituted 3-pyridinesulfonamides

Article abstract of DOI:10.1016/j.ejmech.2010.02.020

A series of novel 4-substituted-3-pyridinesulfonamides (2-27 and 31-33) 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 isozymes CA I and II, and transmembrane isozymes CA IX, XII (cancer-associated) and XIV. Against the human isozymes hCA I, the new compounds showed inhibition constants in the range of 0.078-11.7 μM, against hCA II in the range of 9.9-140 nM, against hCA IX in the range of 4.6-313 nM, against hCA XII in the range of 3.4-21.6 nM, and against hCA XIV in the range of 50.9-160 nM, respectively. Compounds 4, 6, 7, 9, 11-14, 19, 20, 22-24, 26, 27, 31 and 32 showed excellent hCA IX inhibitory efficacy, with inhibition constants of 4.6-12.0 nM, being much more effective as compared to the clinically used sulfonamides AAZ, MZA, EZA, DCP and IND. 4-[N′-(6-Chloro-7-cyano-1,1-dioxo-1,4,2-benzodithiazin-3-yl)hydrazino]-3-pyridinesulfonamide (31) is the prominent of the compounds due to its remarkable inhibitory activity toward hCA I (K_Is = 0.078 μM), hCA IX (K_Is = 7.2 nM) and hCA XII (K_Is = 3.4 nM).

Full text of DOI:10.1016/j.ejmech.2010.02.020

European Journal of Medicinal Chemistry 45 (2010) 2396e2404
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Carbonic anhydrase inhibitors: Synthesis and inhibition of the human cytosolic
isozymes I and II and transmembrane isozymes IX, XII (cancer-associated) and XIV
with 4-substituted 3-pyridinesulfonamides
a
b
,
a
c
*
ꢀ
Zdzis1aw Brzozowski , Jaros1aw S1awinski , Franciszek Sa˛czewski , Alessio Innocenti ,
Claudiu T. Supuran c
a
ꢀ
ꢀ
Department of Chemical Technology of Drugs, Medical University of Gdansk, Al. Gen. J. Hallera 107, 80-416 Gdansk, Poland
b
ꢀ
ꢀ
Department of Organic Chemistry, Medical University of Gdansk, Al. Gen. J. Hallera 107, 80-416 Gdansk, Poland
c Dipartimento di Chimica, Universita degli Studi do 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 4-substituted-3-pyridinesulfonamides (2e27 and 31e33) 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 isozymes CA I and II, and transmembrane isozymes CA IX, XII (cancer-associated)
and XIV. Against the human isozymes hCA I, the new compounds showed inhibition constants in the
Received 21 December 2009
Received in revised form
3 February 2010
Accepted 4 February 2010
Available online 12 February 2010
range of 0.078e11.7 mM, against hCA II in the range of 9.9e140 nM, against hCA IX in the range of
4.6e313 nM, against hCA XII in the range of 3.4e21.6 nM, and against hCA XIV in the range of
50.9e160 nM, respectively. Compounds 4, 6, 7, 9, 11e14, 19, 20, 22e24, 26, 27, 31 and 32 showed
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. 4-[N0-(6-Chloro-7-cyano-
1,1-dioxo-1,4,2-benzodithiazin-3-yl)hydrazino]-3-pyridinesulfonamide (31) is the prominent of the
Keywords:
3-Pyridinesulfonamides
Synthesis
Carbonic anhydrase isozymes I, II, IX, XII and
XIV inhibitors
compounds due to its remarkable inhibitory activity toward hCA I (KIs ¼ 0.078
m
M), hCA IX (KIs ¼ 7.2 nM)
and hCA XII (KIs ¼ 3.4 nM).
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
Membrane-associated human carbonic anhydrase (hCAs)
isozymes IX, XII and XIV [10,12] like other hCAs regulate pH and
carbon dioxide (CO2): bicarbonate anion (HCOꢀ3 ) homeostasis,
through catalysis of the reversible hydration of CO2 to give HCO3ꢀ
and proton (Hþ). The expression level of isozymes hCA IX and XII is
elevated in response to hypoxia and research on the involvement of
these isozymes in cancer has progressed considerably in recent
years, particularly for hCA IX [12e16]. It has been confirmed that
hCA IX is a high activity CA isozyme responsible for the extracel-
lular acidification (pHe) of the tumor microenvironment. Multiple
downstream effects of this reduced pHe are associated with tumor
progression and poor prognosis [14,15]. Aromatic sulfonamide
compounds have been shown to reverse the effect of tumor acidi-
fication, to inhibit the growth of cancer cells and to suppress tumor
invasion mediated by these CAs [12e16]. Thus, the data from these
many physiological studies appear to have identified a CA medi-
ated, hypoxic tumor-specific pathway. This provides firm grounds
for exploring the effects of this class of compounds as a novel
approach to discriminate between healthy cells and cancerous cells,
specifically targeting hypoxic-tissues e an attractive attribute that
is lacking in many existing cancer therapies [17,18].
There are many connections between carbonic anhydrase (CA,
EC 4.2.1.1) and cancer [1e5]. It is well known, that some CA
isozymes are predominantly found in cancer cells and are lacking
from their normal counterparts [3e5], these are two trans-
membrane isozymes CA IX and CA XII. Isozyme CA XIV was the last
one to be discovered among the 15 CA isoforms of this widespread
metalloprotein known up to now in human [6]. Parkkila's and Sly's
groups [7e9] revealed CA XIV distribution in the human body as
well as potential physiological/pathological roles. It has been
observed that hCA XIV is highly abundant in the brain, kidney,
colon, small intestine, urinary bladder, liver and spinal cord [7e11].
Similar to isozymes CA IX and CA XII, CA XIV is a transmembrane
protein with the active site oriented extracellularly, but unlike the
first two proteins, isozyme XIV is not associated with tumor cells
[3,7e9,11].
* 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.2010.02.020

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 2396e2404
2397
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-
benzenesulfonamides of type I and II (Chart 1) [19e21]. Some of
those compounds also showed a certain degree of selectivity for the
inhibition of the tumor-associated over the cytosolic CA isoforms
[19e21]. These findings prompted us to synthesis and investigate
the inhibitory activity of the related 4-substituted 3-pyr-
idinesulfonamides of type III (Chart 1).
under reflux in methanol furnished the expected 4-(N0-hetero-
arylhydrazino)-3-pyridinesulfonamides 31e33 in 89e92% yields.
The structures of the newly obtained compounds 2e27 and
31e33 were confirmed by elemental analysis (C, H, N) and spec-
troscopic data presented in the experimental section.
2.2. CA inhibition studies
The compounds 4e7, 9e14, 16, 18e27 and 31e33 as well as
standard, clinical used CAIs, such as acetazolamide AAZ, meth-
azolamide MZA, ethoxzolamide 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 three transmembrane isoforms hCA IX, XII
(cancer-associated) and hCA XIV isozyme (Table 1). The
following should be noted regarding CA inhibitory data of Table
1: (i) against the slow cytosolic isoform hCA I, the sulfonamides
investigated here showed high to weak inhibitory properties.
Thus, derivatives 7, 9, 10, 14, 16, 18e24 and 33 showed weak
inhibition of this isoform, with KIs in the range of
2. Results and discussion
2.1. Chemistry
Syntheses of the target pyridinesulfonamides 4e14 were ach-
ieved in 76e94% yields by the convenient two-step procedure
starting from commercially available 4-chloro-3-pyridinesulfomide
1 and the corresponding hydroxyl or thiol compounds, as shown in
Schemes 1 and 2.
We have found in our studies, that the reaction of 1 with thiouera
led to the formation of the expected thiouronium chloride A which in
the presence of methanol undergoes a further transformation. The
proposed mechanism leading to the formation of the products 15 and
16 is outlined in Scheme 3. The initial step is believed to be formation
of unstable 1Hþ-pyridinium chloride B, which after spontaneous
elimination of urea or 2-methyluronium chloride gives two products
15 and intermediate C, respectively. In the final stage of the reaction
the non-isolable intermediate C reacts with starting compound 1 to
afford the corresponding sulfide 16 in 53% yield (Scheme 3).
In the different conditions however, the expected thiouronium
chloride A (17) was successfully obtained in excellent yield by the
reaction of 1 with thiourea in boiling acetonitrile (Scheme 4). Then,
upon treatment of thiouronium chloride 17 with NaOH and cor-
responding alkyl halides in tetrahydrofuranewater solution at
temperature 0e20 ꢁC, the desired 4-(R-methylthio)-3-pyr-
idinesulfonamide 18e22 were obtained in 81e88% yields. The
desired 4-(amino-or hydrazine)-3-pyridinesulfonamide derivatives
22e27 were obtained in 79e85% yields by reacting the 4-chloro-3-
pyridinesulfomide 1 with an excess of corresponding amines or
hydrazines in methanol at reflux (Scheme 5). Then, the reaction of
26 (Scheme 6) with appropriate methylthio compounds 28e30
8.32e11.77 mM, being thus much weaker inhibitors as compared
to the clinically used compounds AAZeIND (Table 1). The
compounds 4e6, 11e13, 25e26 and 33 were slightly more
effective inhibitors against hCA
5.62e7.91 M. However, the other three compounds 27, 31 and
32 showed hCA I inhibitory activity (KIs ¼ 0.078e1.52 M) in the
I with KIs in range of
m
m
same range as the clinically used compounds AAZeIND (Table
1); (ii) against the ubiquitous and dominant rapid cytosolic
isozyme hCA II, compounds 4e7, 9e14, 16, 18e20, 22e27 and
31e33 acting as weaker inhibitors (KIs in the range
45.8e140.0 nM) (Table 1), but the other only compound 21
acting as a stronger CA II inhibitors, with KIs ¼ 9.9 nM, of the
some order magnitude as must of the clinically used compounds
(Table 1); (iii) a quite larger variation of inhibitory activity was
observed for the inhibition of the tumor-associated isoform, hCA
IX (Table 1). Thus, the compounds 5 and 10 showed weak CA IX
inhibitory activity, with KIs in range of 300e313 nM. Another
groups of compounds such as 16, 18, 21, 25 and 33, showed
moderate CA IX inhibition, with KIs range 15.8e47.3 nM,
whereas the remaining derivatives 4, 6, 7, 9, 11e14, 19, 20,
22e24, 26, 27, 31 and 32 were very effective hCA IX inhibitors
Chart 1.

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Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 2396e2404
Scheme 1. Synthesis of 4-(alkoxy and aryloxy)pyridine-3-sulfonamides 2e10.
(KIs in range of 4.6e12.0 nM, Table 1); (iv) a quite good inhibi-
tion profile of the second tumor-associated isoform, hCA XII has
also been observed for all investigated sulfonamides of type
4e33 (Table 1). Thus, compounds 11, 14, 19, 20, 22e24, 26 and
27 were the moderate inhibitors (KIs in the range of
10.1e21.6 nM), whereas the remaining compounds were very
effective hCA XII inhibitors (KIs in the range of 3.4e8.9 nM,
Table 1); (v) all of the investigated sulfonamides 5, 9, 10, 16,
18e27 and 31e33 showed moderate to weak inhibition prop-
erties towards hCA XIV isozyme with KIs in the range of
50.9e160.0 nM (Table 1).
3. Conclusions
We have developed methods for the preparation of novel series
of 4-substiuted 3-pyridiesulfonamides (ethers, sulfides, amines and
hydrazines). The 24 new sulfonamides have been assayed for the
inhibition of five physiologically relevant CA isozymes, such as CA I
and II, the tumor-associated isozymes CA IX, XII as well as trans-
membrane CA XIV. Against the human isozyme hCA I the new
compounds showed inhibition constants in the range of
0.078e11.7 mM, against hCA II in the range 9.9e140.0 nM, against
hCA IX in the range 4.6e313 nM, against hCA XII in the range
Scheme 3. Proposed mechanism of the formation of the 4-mercapto-3-pyr-
idinesulfonamide derivatives 15 and 16 in the reaction of 4-chloro-3-pyr-
idinesulfonamide with thiourea in boiling methanol.
Scheme 2. Synthesis of 4-(heteroarylthio)pyridine-3-sulfonamides 11e14.

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 2396e2404
2399
Scheme 4. Synthesis of 2-(3-sulfamoyl-4-pyridyl)thiouronium chloride 17 and its
application to the synthesis of sulfides 18e22. Reagents, conditions and yields: (a)
thiourea (1.02 molar equiv.), acetonitrile, reflux, 3 h, 98%; (b) NaOH (2.14 molar equiv.),
water, tetrahydrofuran, 0e5 ꢁC; (c) alkyl halide ReCH2X (1.14 molar equiv.), tetrahy-
drofuran, 0e7 ꢁC, 0.5 h, room temperature, 5 h, 81 88%.
Scheme 6. Synthesis of 4-(N0-heteroarylhydrazino)-3-pyridinesulfonamides 31e33.
3.4e21.6 nM and against hCA XIV in the range 50.9e160 nM,
respectively. Compounds 27e32 having an activity against hCA I
4.1.1. Procedure for the preparation of 4-alkoxy-3-pyridine-
sulfonamide hydrochlorides (2, 3) and their transformation to free
sulfonamides (4 and 5)
(KIs ¼ 0.075e1.52
m
M) comparable the clinical used sulfonamides
AAZ, MZA, EZA, DCP or IND (KIs ¼ 0.025e1.2
m
M). Several of the
new compounds including 4, 6, 7, 9, 11e14, 19, 20, 22e24, 26, 27, 31
and 32 showed excellent hCA IX inhibitory efficacy, with inhibition
constants of 4.6e12.0 nM, being much more effective as compared
to the clinically used AAZ, MZA, EZA, DCP and IND
(KIs ¼ 24e50 nM).
4.1.1.1. A. Hydrochlorides 2 and 3. A solution of 4-chloro-3-pyr-
idinesulfonamide 1 (3.85 g, 0.02 mol) in the corresponding alcohol
(30e35 ml) was refluxed with stirring for 8 h. After cooling to room
temperature the suspension was left overnight. The precipitate was
collected by filtration, washed with the corresponding alcohol
(3 ꢃ 5 ml) and dried at temperatures gradually increasing to 90 ꢁC.
4.1.1.1.1. 4-Methoxy-3-pyridinesulfonamide hydrochloride (2).
Starting from anhydrous methanol (30 ml), the title compound 2
was obtained (4.25 g, 94%); m.p. 168e169 ꢁC; IR (KBr) 3305, 3140
(SO2NH2), 2715, 2615, 2135, 2060, 1910 (NHþ), 1630, 1590, 1505
4. Experimental protocols
4.1. Synthesis
(C]N and C]C), 1340, 1175 (SO2) cmꢀ1 1H NMR (DMSO-d6)
;
The following instruments and parameters were used:
d
4.20 (s, 3H, CH3O), 7.78 (d, J ¼ 6.8 Hz, 1H, H-5), 7.97 (s, 2H,
}
melting points Buchi 535 apparatus; IR spectra: KBr pellets,
SO2NH2), 8.90 (s, 1H, H-2), 8.98 (d, J ¼ 6.8 Hz, 1H, H-6) ppm; 13C
400e4000 cmꢀ1 Perkin Elmer 1600 FTIR spectrometer; 1H and
13C NMR: Varian Gemini 200 apparatus at 200 and 50 MHz,
NMR (DMSO-d6)
d
58.96, 111.38, 129.94, 141.95, 148.20,
167.23 ppm. Anal. (C6H9ClN2O3S) C, H, N.
respectively; chemical shifts are expressed at
d values relative to
4.1.1.1.2. 4-Ethoxy-3-pyridinesulfonamide hydrochloride (3).
Starting from anhydrous ethanol (35 ml), the title compound 3
was obtained (4.4 g, 92%); m.p. 167e168 ꢁC; IR (KBr) 3325, 3190
(SO2NH2), 2760, 2670, 2560, 2040, 1995 (NHþ), 1630, 1590, 1525
Me4Si as standard. The results of elemental analyses for C, H and
N were within ꢂ0.3% of theoretical values. According to the
methods described previously the following substrates were
obtained: 6-chloro-7-(cyano- and methyl)-3-methylthio-1,4,2-
benzodithiazine 1,1-dioxides 28 [22,23], 29 [24], and 3-methyl-
thiopyrido[4,3-e]-1,4,2-dithiazine 1,1-dioxide 30 [25].
(C]N and C]C), 1340, 1165 (SO2) cmꢀ1 1H NMR (DMSO-d6)
;
d
1.44 (t, J ¼ 7.0 Hz, CH3), 4.57 (q, J ¼ 7.0 Hz, 2H, CH2), 7.78 (d,
J ¼ 6.5 Hz, 1H, H-5), 7.84 (s, 2H, SO2NH2), 8.90 (s, 1H, H-2), 8.94
(d, J ¼ 6.5 Hz, 1H, H-6) ppm. Anal. (C7H11ClN2O3S) C, H, N.
4.1.1.2. B. Free sulfonamides 4 and 5. To a stirred solution of the
appropriate hydrochlorides 2 or 3 (0.01 mol) in water (8 ml) was
added dropwise a solution of 1% NaOH in water to pH 7.8. The
suspension obtained was stirred at room temperature for an
additional 4 h. The precipitate was filtered off, washed with water
(5 ꢃ 2 ml), and acetonitrile (3 ꢃ 1 ml), and dried at temperatures
gradually increasing to 100 ꢁC.
4.1.1.2.1. 4-Methoxy-3-pyridinesulfonamide (4). Starting from
hydrochloride 2 (2.25 g), the title compound 4 was obtained (1.5 g,
79%); m.p. 163e165 ꢁC; IR (KBr) 3315, 3275, 3225 (SO2NH2), 1590,
1565(C]N and C]C), 1340, 1160 (SO2) cmꢀ1 1H NMR (DMSO-d6)
;
d
3.98 (s, 3H, CH3O) 3.98 (s, 3H, CH3O), 7.27 (d, J ¼ 5.8 Hz, 1H, H-5),
7.38 (s, 2H, SO2NH2), 8.63 (d, J ¼ 5.8 Hz, 1H, H-6), 8.71 (s, 1H, H-2)
ppm; 13C NMR (DMSO-d6)
56.72, 108.65, 128.08, 148.07, 155.14,
d
162.39 ppm. Anal. (C6H8N2O3S) C, H, N.
4.1.1.2.2. 4-Ethoxy-3-pyridinesulfonamide (5). Starting from
hydrochloride 3 (2.38 g), the title compound 5 was obtained (1.9 g,
94%); m.p. 179e180 ꢁC; IR (KBr) 3310, 3145 (SO2NH2), 1590, 1550
Scheme 5. Synthesis of 4-(amino and hydrazino)-3-pyridosulfonamide derivatives
23e27.
(C]N and C]C), 1335, 1165 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
d 1.38

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Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 2396e2404
(t, J ¼ 7.0 Hz, 3H, CH3), 4.30 (q, J ¼ 7.0 Hz, 2H, OCH2), 7.20 (d,
J ¼ 5.8 Hz, 1H, H-5), 7.32 (s, 2H, SO2NH2), 8.58 (d, J ¼ 5.8 Hz, 1H, H-
6) ppm. Anal. (C7H10N2O3S) C, H, N.
(d, J ¼ 8.3 Hz, 1H, H-4, quinoline), 8.53 (d, J ¼ 4.9 Hz, 1H, H-6,
pyrid.), 8.90 (s, 1H, H-2, pyrid.) ppm. Anal. (C15H13N3O3S) C, H, N.
4.1.3. Procedure for the preparation of 4-heteroarylthiopyridine-3-
sulfonamides (11e14)
4.1.2. General procedure for the preparation of 4-aryloxy-3-
A mixture of 4-chloropyridine-3-sulfonamide 1 (1.93 g, 10 mmol)
and the corresponding heteroarylthiol (12 mmol) in acetonitrile
(25 ml)wasstirred atroom temperaturefor2 h, followed byrefluxfor
24 h. After cooling to room temperature the resulting suspensionwas
left overnight. The precipitate of the adequate crude hydrochloride of
11e14 was collected by filtration, washed with acetonitrile (3 ꢃ 2 ml)
and dried, then treated with water (10 ml). The resulting suspension
(pH 1) was slowlyadjusted to pH 8 with 1% solution of NaOH inwater.
After stirring at room temperature for 3 h, the precipitate of the
adequate sulfonamides 11e14 was filtered off, washed successively
with water (4 ꢃ1.5 ml) and acetonitrile (3 ꢃ 1.5 ml), and dried at
temperatures gradually increasing 100 ꢁC. In this manner the
following sulfonamides were obtained.
pyridinesulfonamies (5e10)
A
mixture of 4-chloropyridine-3-sulfonamide
1
(2.89 g,
0.015 mol) and the corresponding phenols or hydroxy derivatives
of heterocyclic compounds (0.018 mol) in acetonitrile (20 ml) was
refluxed with stirring for 38 h. After cooling to room temperature
and standing overnight the precipitate was collected by filtration,
washed with acetonitrile (2 ꢃ1.5 ml) and dried. The adequate
crude hydrochlorides obtained was dissolved in water (15e25 ml)
and adjusted to pH 7.5 with 1% solution of NaOH in water. After 6 h
of stirring, the precipitate of the adequate title products was filtered
off, washed successively with water (4 ꢃ 3 ml) and acetonitrile
(2 ꢃ1.5 ml), and dried at temperatures gradually increasing to
100 ꢁC. In this manner the following sulfonamide were obtained.
4.1.3.1. 4-(N-Oxide-2-pyridylthio)pyridine-3-sulfonamide (11). Start-
ing from 2-mercapto- pyridine N-oxide (1.53 g), the title compound
11 was obtained (2.3 g, 81%); m.p. 183e185 ꢁC dec.; IR (KBr) 3190,
4.1.2.1. 4-(4-Cyanophenoxy)-3-pyridinesulfonamide (6). Starting from
4-cyanophenol (2.14 g), the title compound 6 was obtained (3.2 g,
77%); m.p. 208e210 ꢁC; IR (KBr) 3360, 3255 (SO2NH2), 2235 (C^N),
1640, 1575 (C]N and C]C), 1340, 1160 (SO2) cmꢀ1; 1H NMR (DMSO-
3100 (SO2NH2), 1635, 1570 (C]N and C]C), 1345, 1170 (SO2) cmꢀ1
;
1H NMR (DMSO-d6)
d
7.28 (d, J ¼ 5.2 Hz, 1H, H-5, pyr-
d6)
d
7.03 (d, J ¼ 5.5 Hz, H-5, pyrid.), 7.39 (d, J ¼ 8.1 Hz, 2H, PhCN), 7.73
idinesulfonamide), 7.34e7.49 (m, 3H, H-3, H-4 and H-5, pyridine N-
oxide), 7.84 (s, 2H, SO2NH2), 8.43 (d, J ¼ 6.2 Hz, 1H, H-6, pyridine N-
oxide), 8.64 (d, J ¼ 5.2 Hz, 1H, pyridinesulfonamide), 9.04 (s, 1H, H-
(s, 2H, SO2NH2), 7.87 (d, J ¼ 8.1 Hz, 2H, PhCN), 8.66 (d, J ¼ 5.5 Hz,1H, H-
6, pyrid.) 8.93 (s, 1H, H-2, pyrid.) ppm. Anal. (C12H9N3O3S) C, H, N.
2, pyridinesulfonamide) ppm; 13C NMR (DMSO-d6)
d 125.66,
4.1.2.2. 4-(4-Fluorophenoxy)-3-pyridinesulfonamide (7). Starting
from 4-fluorophenol (2.02 g), the title compound 7 was obtained
(3.1 g, 77%); m.p. 182e184 ꢁC; IR (KBr) 3330, 3172 (SO2NH2), 1635,
125.96, 127.12, 129.03, 139,17, 139.42, 141.41, 145.23, 148.45,
152.77 ppm. Anal. (C10H9N3O3S2) C, H, N.
1600, 1575 (C]N and C]C), 1325, 1175, 1165 (SO2) cmꢀ1 1H NMR
;
4.1.3.2. 4-(1-Methyl-5-tetrazolylthio)pyridine-3-sulfonamide (12).
Starting from 5-mercapto-1-methyltetrazole (1.4 g), the title
compound 12 was obtained (2.3 g, 84%); m.p. 189e191 ꢁC dec.; IR
(KBr) 3360, 3135 (SO2NH2), 1570, 1540 (C]N and C]C), 1340, 1165
(DMSO-d6)
d
6.80 (d, J ¼ 5.4 Hz, 1H, H-5, pyrid.), 7.27e7.32 (m, 2H,
PhF), 7.37 (t, J ¼ 8.8 Hz, 2H, PhF) 7.68 (s, 2H, SO2NH2), 8.58 (d,
J ¼ 5.4 Hz, 1H, H-6, pyrid.), 8.87 (s, 1H, H-2, pyrid.) ppm. Anal.
(C11H9FN2O3S) C, H, N.
(SO2) cmꢀ1 1H NMR (DMSO-d6)
; d 4.07 (s, 3H, CH3), 6.84 (d,
J ¼ 5,5 Hz, 1H, H-5), 8.07 (s, 2H, SO2NH2), 8.54 (d, J ¼ 5,5 Hz, 1H, H-
4.1.2.3. 4-(3-Chloro-4-fluorophenoxy)-3-pyridinesulfonamide (8).
Starting from 3-chloro-4-fluorophenol (2.64 g), the title compound
8 was obtained (3.3 g, 72%); m.p. 210e211 ꢁC; IR (KBr) 3315, 3160
6), 8.97 (s, 1H, H-2) ppm. Anal. (C7H8N6O2S2) C, H, N.
4.1.3.3. 4-(1,3,4-Thiadiozol-2-ylthio)pyridine-3-sulfonamide (13).
Starting from 2-mercapto-1,3,4-thiadiazole (1.42 g), the title
compound 13 was obtained (2.1 g, 76%); m.p. 163e164 ꢁC dec.; IR
(KBr) 3265, 3165 (SO2NH2), 1570, 1530 (C]N and C]C), 1335, 1165
(SO2NH2), 1580, 1500 (C]N and C]C), 1340, 1165 (SO2) cmꢀ1 1H
;
NMR (DMSO-d6)
d
6.94 (d, J ¼ 5.7 Hz, 1H, H-5, pyrid.), 7.24e7.31 (m,
1H, 3-Cl-4-FPh), 7.53e7.61 (m, 2H, 3-Cl-4-FPh), 7.67 (s, 2H,
SO2NH2), 8.60 (d, J ¼ 5.7 Hz,1H, H-6, pyrid.), 8.72 (s,1H, H-2, pyrid.)
ppm. Anal. (C11H8ClFN2O3S) C, H, N.
(SO2) cmꢀ1; 1H NMR (DMSO-d6)
8.01 (s, 2H, SO2NH2), 8.57 (dd, Jortho ¼ 5.2 Hz, Jmeta ¼ 1.7 Hz, 1H, H-6,
pyrid.), 8.96 (d, Jmeta ¼ 1.7 Hz, 1H, H-2, pyrid.), 9.92 (s, 1H, H-5,
thiadiazole) ppm. Anal. (C7H6N4O2S3) C, H, N.
d
7.11 (d, J ¼ 5,2 Hz, 1H, H-5 pyrid.),
4.1.2.4. 4-(Quinolinoxy)-3-pyridinesulfonamide (9). Starting from
8-quinolinol (2.61 g), the title compound 9 was obtained (3.6 g,
79%); m.p. 218e219 ꢁC; IR (KBr) 3315, 3150 (SO2NH2), 1590, 1570,
4.1.3.4. 4-(6-Nitrobenzothiazol-2-ylthio)pyridine-3-sulfonamide
(14). Starting from 2-mercapto-6-nitrobenzothiazole (2.55 g), the
title compound 14 was obtained (2.8 g, 76%); m.p. 263e265 ꢁC dec.;
IR (KBr) 3235, 3135 (SO2NH2), 1570, 1515 (C]N and C]C), 1340,
1500 (C]N and C]C), 1340, 1330, 1160 (SO2) cmꢀ1 1H NMR
;
(DMSO-d6)
d
6.60 (d, J ¼ 5.6 Hz, 1H, H-5, pyrid.) 7.60e7.66 (m, 1H,
quinolyl), 7.70e7.75 (m, 2H, quinolyl), 7.77 (s, 2H, SO2NH2),
8.01e8.06 (m, 1H, quinolyl), 8.43 (d, J ¼ 5.6 Hz, 1H, H-6, pyrid.), 8.53
(d, J ¼ 8.4 Hz, 1H, quinolyl), 8.83 (d, J ¼ 4.0 Hz, 1H, quinolyl), 8.88
(s, 1H, H-2, pyrid.) ppm. Anal. (C14H11N3O3S) C, H, N.
1165 (SO2) cmꢀ1
;
1H NMR (DMSO-d6)
d
7.81 (d, J ¼ 3.9 Hz, 1H, H-5,
pyrid.), 8.04 (s, 2H, SO2NH2), 8.20 (d, J ¼ 8.3 Hz, 1H, H-4, 6-nitro-
benzothiazole), 8.37 (d, J ¼ 8.3 Hz, 1H, 6-nitrobenzothiazole), 8.74
(d, J ¼ 3.9 Hz, 1H, H-6, pyrid.), 9.18 (s, 1H, H-2, pyrid.), 9.23 (s, 1H, H-
7, 6-nitrobenzothiazole) ppm. Anal. (C12H8N4O4S3) C, H, N.
4.1.2.5. 4-(2-Methyl-8-quinolinoxy)-3-pyridinesulfonamide (10).
Starting from 2-methyl-8-quinolinol (2.87 g), the title compound
10 was obtained (3.6 g, 76%); m.p. 243e245 ꢁC dec.; IR (KBr) 3300,
3180 (SO2NH2), 1625, 1600 1580, 1560 (C]N and C]C), 1350, 1165
4.1.4. Synthesis of 4-methylthio-3-pyridinesulfonamide
hydrochloride (15) and 4,40-thiodipyridine-3-sulfonamide (16) by
the reaction of 4-chloro-3-pyridinesulfonamide with thiouera in
boiling methanol
(SO2) cmꢀ1 1H NMR (DMSO-d6)
; d 2.56 (s, 3H, CH3), 6.89 (d,
J ¼ 4.9 Hz, 1H, H-5, pyrid.), 7.53 (d, J ¼ 8.3 Hz, 1H, H-3, quinoline),
7.68 (t, J ¼ 7.0 Hz, 1H, H-6, quinoline), 7.82 (d, J ¼ 7.0 Hz, 1H, H-5
quinoline), 7.97 (br s, 3H, SO2NH2 and H-7, quinoline), 8.41
A
mixture of 4-chloro-3-pyridinesulfonamide
1
(4.81 g,
0.025 mol) and thiourea (1.92 g, 0.0252 mol) in methanol (15 ml)
was stirred at reflux for 5 h. After cooling to room temperature the

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 2396e2404
2401
Table 1
Carbonic anhydrase inhibition data for compounds 4e7, 9e14,16,18e27 and 31e33 and standard inhibitors against human isozymes hCA I, II, IX, XII and XIV, by a stopped-flow,
CO2 hydration assay [26].
R
N
SO2NH2
4-7, 9-14, 16, 18-27 and 31-33
Compound
R
KIa
hCA Ib
(
m
M)
hCA IIb (nM)
hCA IXc (nM)
hCA XIIc (nM)
hCA XIVd (nM)
AAZ
MZA
EZA
DCP
IND
4
0.31
0.78
0.025
1.20
0.031
7.34
6.10
12
14
8
38
15
93.8
76.7
25
27
34
50
24
5.7
3.4
22
41
43
25
345
104
NT
50
3.4
5.8
5.9
eOMe
eOEt
5.7
300.0
5
52.2
O
N
6
7
6.64
9.16
94.5
94.4
7.2
7.9
6.3
6.9
NT
NT
O
F
O
N
9
10.32
10.32
7.62
105.0
80.4
84.5
45.8
9.1
313.0
7.5
6.8
6.7
59.7
70.0
NT
O
Me
N
10
11
12
S
S
N
O
13.8
4.2
N
N
N
N
Me
6.53
4.6
NT
N
N
S
S
13
14
6.67
8.84
56.9
94.8
7.7
9.6
5.1
NT
NT
N
S
S
21.6
S
N
16
11.17
65.0
47.3
4.7
160.0
SO2NH2
18
19
eSeMe
9.03
40.1
91.1
47.0
11.7
6.7
98.3
N
S
11.71
15.5
140.0
S
20
21
8.32
9.09
108.0
9.9
6.3
15.6
6.5
56.4
S
30.4
68.2
(continued on next page)

2402
Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 2396e2404
Table 1 (continued )
Compound
R
KIa
hCA Ib
(
mM)
hCA IIb (nM)
131.0
hCA IXc (nM)
11.8
hCA XIIc (nM)
15.9
hCA XIVd (nM)
142.0
O
S
22
23
9.51
NH2
H
N
8.86
133.0
140.0
12.0
7.8
16.9
15.7
143.0
152.0
Me
N
24
10.30
N
H
N
Me
N
25
26
5.62
7.91
87.2
85.6
15.8
4.9
8.9
95.7
93.4
H
H
N
NH2
10.1
H
N
27
31
1.52
52.0
88.3
7.5
7.2
15.1
3.4
149.0
92.3
SO2NH2
O O
S
CN
Cl
N
H
0.078
N
N
H
S
O O
S
Me
Cl
N
H
N
32
0.101
8.90
46.1
5.8
8.1
4.6
50.9
83.7
N
H
S
O O
S
N
N
H
N
33
117.0
16.1
N
H
S
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 [16,27], by the CO2 hydration method.
Full-length human (cloned) isozyme [30].
d
suspension was left overnight. The precipitate of title compound 16
was filtered off, washed with methanol (3 ꢃ 1 ml) and dried. Yield
2.3 g (53%), m.p. 216e218 ꢁC dec.; IR (KBr) 3300, 3205 (SO2NH2),
1635,1560 (C]N and C]C),1335,1160 (SO2) cmꢀ1; 1H NMR (DMSO-
J ¼ 6.2 Hz, 2H, H-5), 8.03 (s, 2H, SO2NH2), 8.73 (d, J ¼ 6.2 Hz, 1H, H-
6), 8.86 (s, 1H, H-2), 11.75 (br s, 1H, NHþ) ppm; 13C NMR (DMSO-d6)
d
15.06, 121.87, 136.81, 141.29, 144.68, 158.41 ppm. Anal.
d6)
d
7.25 (d, J ¼ 5,5 Hz, 2H, H-5 and H-50), 7.87 (s, 4H, 2ꢃ SO2NH2),
8.65 (d, J ¼ 5,5 Hz, 2H, H-6 and H-60), 9.16 (s, 2H, H-2 and H-20) ppm.
4.1.5. 2-(3-Sulfamoyl-4-pyridyl)thiouronium chloride (17)
A suspension of 1 (9.63 g, 0.05 mol) and thiourea (3.88 g,
0.051 mol) in acetonitrile (85 ml) was stirred at reflux for 3 h
and left to stand at room temperature overnight. The precipitate
was filtered off, washed with acetonitrile (4 ꢃ 4 ml) and dried.
Yield 13.2 g (98%), m.p. 149e151 ꢁC dec.; IR (KBr) 3540, 3355,
3200, 3160 (SO2NH2 and CeNH2), 2990, 2720, 2630 (NHþ), 1650,
Anal. (C10H10N4O4S3) C, H, N.
The methanolefiltrate mixture was evaporated to 1/3 volume at
diminished pressure. After cooling to room temperature the
suspension was left overnight. The precipitate of compound 15 was
filtered off, washed with methanol (2 ꢃ 1 ml) and dried. Yield 1.1 g
(18%), m.p. 238e239 ꢁC dec.; IR (KBr) 3285, 3170 (SO2NH2), 2715,
2520, 2325, 2030 (NHþ),1620,1585,1530 (pyridine ring),1335,1165
1565, 1535 (NH2 and pyridine ring), 1335, 1165 (SO2) cmꢀ1
;
1H
(SO2) cmꢀ1 1H NMR (DMSO-d6)
; d 2.69 (s, 3H, CH3), 7.80 (d,
NMR (DMSO-d6)
d
7.80 (d, J ¼ 5,4 Hz, 1H, H-5), 8.02 (s, 2H,

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 2396e2404
2403
SO2NH2), 8.81 (d, J ¼ 5,4 Hz, 1H, H-6), 9.08 (s, 1H, H-2), 9.63 and
9.71 [2s, 2ꢃ 2H, SeC(NH2)2] ppm. Anal. (C6H9ClN4O2S2) C, H, N.
dropwise and stirred at room temperature for 4 h. The precipitate
thus obtained was collected by filtration, washed with water
(5 ꢃ 4 ml) and methanol (3 ꢃ1 ml), and dried.
4.1.6. Procedure for the preparation of 4-(R-methylthio)pyridine-3-
sulfonamides (18e22)
4.1.7.1. 4-(Allylamino)-3-pyridinesulfonamide (23). Starting from
allylamine the title compound 23 was obtained (1.8 g, 84%); m.p.
150e151 ꢁC; IR (KBr) 3370, 3305 (SO2NH2), 1600, 1560, 1520 (C]N
To an ice-cold solution of NaOH (0.6 g, 15 mmol) in water (3 ml)
and tetrahydrofuran (15 ml) was added with stirring thiouronium
chloride 17 (1.88 g, 7 mmol) and solution of the appropriate alkyl
halide (8 mmol) in tetrahydrofuran (8 ml). After 0.5 h the ice bath
was removed and the reaction mixture was stirred at room
temperature for 5 h. The precipitate of the adequate sulfides
obtained was filtered off, washed successively with water
(6 ꢃ 2 ml) and methanol (3 ꢃ 1.5 ml), and dried. In this manner the
following sulfides were obtained.
and C]C), 1340, 1145 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
d 3.85e4.00
(m, 2H, NCH2eCH]CH2), 5.12e5.25 (m, 2H, NCH2eCH]CH2),
5.79e5.98 (m, 1H, NCH2eCH]CH2), 6.62 (t, J ¼ 5.4 Hz, 1H,
NHeCH2), 6.68 (d, J ¼ 5.9 Hz, 1H, H-5), 7.54 (s, 2H, SO2NH2), 8.19 (d,
J ¼ 5.9 Hz, 1H, H-6), 8.50 (s, 1H, H-2) ppm; 13C NMR (DMSO-d6)
d
44.36, 107.04, 116.31, 122.03, 134.26, 148.63, 149.47, 152.53 ppm.
Anal. (C8H11N3O2S) C, H, N.
4.1.6.1. 4-Methylthiopyridine-3-sulfonamide (18). Starting from
methyl iodide (1.14 g), the title compound 18 was obtained (1.26 g,
88%); m.p. 222e223 ꢁC; IR (KBr) 3295, 3160 (SO2NH2), 1570vs (C]
4.1.7.2. 4-(5-Methyl-2-pirazolino)-3-pyridinesulfonamide (24). Start-
ing from 5-methyl-2-pirazoline the title compound 24 was obtained
(2.0 g, 83%); m.p. 206e207 ꢁC; IR (KBr) 3295, 3110 (SO2NH2), 1590,
N and C]C), 1345, 1165 (SO2) cmꢀ1 1H NMR (DMSO-d6)
; d 2.55 (s,
1525 (C]N and C]C), 1340, 1155 (SO2) cmꢀ1 1H NMR (DMSO-d6)
;
3H, CH3S), 7.45 (d, J ¼ 5,5 Hz, 1H, H-6), 7.60 (s, 2H, SO2NH2), 8.53
(d, J ¼ 5,5 Hz, 1H, H-6), 8.79 (s, 1H, H-2) ppm. Anal. (C6H8N2O2S2)
C, H, N.
d
1.13 (d, J ¼ 6.1 Hz, 3H, CH3), 2.60e2.69 (m, 1H, HA-4, pyrazoline),
3.11e3.25 (m, 1H, HB-4, pyrazoline), 4.67 (sext, 1H, H-5, pyrazoline),
7.01 (d, J ¼ 5.8 Hz, 1H, H-5, pyrid.), 7.20 (s, 2H, SO2NH2), 7.24 (s, 1H,
N]CH, pyrazoline), 8.34 (d, J ¼ 5.8 Hz, 1H, H-6, pyrid.), 8.82 (s, 1H,
4.1.6.2. 4-(Cyanomethylthio)pyridine-3-sulfonamide (19). Starting
from bromoacetonitrile (0.96 g), the title compound 19 was
obtained (1.3 g, 81%); m.p. 208e209 ꢁC dec.; IR (KBr) 3340, 3170
(SO2NH2), 2250 (C^N), 1570vs (C]N and C]C), 1340, 1170
H-2 pyrid.) ppm.; 13C NMR (DMSO-d6)
d 18.60, 41.28, 53.97, 110.60,
125.79, 146.23, 146.62, 150.59, 152.44 ppm. Anal. (C9H12N4O2S)
C, H, N.
(SO2NH2) cmꢀ1 1H NMR (DMSO-d6)
; d 4.53 (s, 2H, CH2), 7.61 (d,
Method B (for compounds 25 and 26). To a solution of methyl-
hydrazine (2.8 g, 60 mmol) in methanol (10 ml) 4-chloro-3-pyr-
idinesulfonamide (1.93 g, 10 mmol) was added. The reaction
mixture was stirred at room temperature for 16 h, followed by
reflux for 4 h. After cooling to room temperature the precipitate
was collected by filtration, washed with methanol (4 ꢃ 2 ml) and
dried at temperatures gradually increasing to 100 ꢁC.
J ¼ 5,4 Hz, 1H, H-5), 7.84 (s, 2H, SO2NH2), 8.71 (d, J ¼ 5,4 Hz, 1H, H-
6), 8.91 (s, 1H, H-2) ppm. Anal. (C7H7N3O2S2) C, H, N.
4.1.6.3. 4-(2-Propynylthio)pyridine-3-sulfonamide (20). Starting
from propargyl bromide (0.95 g), the title compound 20 was
obtained (1.3 g, 81%); m.p. 210e211 ꢁC dec.; IR (KBr) 3335, 3260
(SO2NH2), 2120 (C^CH), 1570vs (C]N and C]C), 1340, 1165 (SO2)
cmꢀ1;1HNMR (DMSO-d6)
d
3.34(s,1H, C^CH), 4.08(s, 2H, CH2), 7.54
(d, J ¼ 5,5 Hz,1H, H-5), 7.70 (s, 2H, SO2NH2), 8.59 (d, J ¼ 5,5 Hz,1H, H-
2), 8.84 (s, 1H, H-2) ppm; 13C NMR (DMSO-d6)
19.11, 74.94, 79.14,
4.1.7.3. 4-(Methylhydrazino)-3-pyridinesulfonamide (25)
Starting from methylhydrazine the title compound 25 was
obtained (1.6 g, 79%); m.p. 211e212 ꢁC dec.; IR (KBr) 3335, 3285,
3245, 3185 (SO2NH2 and NHeNH),1635,1585 (C]N and C]C),1320,
d
120.56, 135.98, 146.96, 151.64 ppm. Anal. (C8H8N2O2S2) C, H, N.
1150 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
d 3.18 (s, 3H, NCH3), 5.13 (s, 2H,
HNeNH), 6.76 (d, J ¼ 5.9 Hz, 1H, H-5), 7.27 (s, 2H, SO2NH2), 8.24 (d,
4.1.6.4. 4-Benzythiopyridine-3-sulfonamide (21). Starting from
benzyl bromide (1.37 g), the title compound 21 was obtained (1.6 g,
81%); m.p. 229e230 ꢁC; IR (KBr) 3380, 3170 (SO2NH2), 1575vs (C]N
J ¼ 5.9 Hz,1H, H-6), 8.74 (s,1H, H-2) ppm. Anal. (C6H10N4O2S) C, H, N.
and C]C), 1335, 1165 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
d 4.43 (s, 2H,
4.1.7.4. 4-Hydrazino-3-pyridinesulfonamide (26)
Starting from hydrazine hydrate the title compound 26 was
obtained (1.6 g, 85%); m.p. 202e203 ꢁC dec.; IR (KBr) 3365, 3315,
3270, 3165 (SO2NH2 and NHeNH2), 1635, 1595 (C]N and C]C),
CH2), 7.30 (t, J ¼ 7.3 Hz, 1H, H-4, Ph), 7.37 (t, J ¼ 7.3 Hz, 2H, H-3 and
H-5, Ph), 7.51 (d, J ¼ 7.8 Hz, 2H, H-2 and H-6, Ph), 7.58 (d, J ¼ 5.4 Hz,
1H, H-5, pyrid.), 7.66 (s, 2H, SO2NH2), 8.53 (d, J ¼ 5.4 Hz, 1H, H-6,
pyrid.), 8.82 (s, 1H, H-2, pyrid.) ppm. Anal. (C12H12N2O2S2) C, H, N.
1315, 1135 (SO2) cmꢀ1 1H NMR (DMSO-d6)
; d 4.69 (s, 2H, NeNH2),
7.22 (d, J ¼ 5.9 Hz, 1H, H-5), 7.52 (s, 3H, SO2NH2 and NHeN), 8.24 (s,
J ¼ 5.9 Hz,1H, H-6), 8.46 (s,1H, H-2) ppm. Anal. (C5H8N4O2S) C, H, N.
4.1.6.5. 4-(Carbamolymethylthio)pyridine-3-sulfonamide
(22).
Starting from 2-bromoacetamide (1.1 g), the title compound 22 was
obtained (1.4 g, 81%); m.p. 198e200 ꢁC dec.; IR (KBr) 3400, 3315,
4.1.7.5. 4-[2-(4-Sulfamoylphenyl)ethylamino]-3-pyridinosulfonamide
(27) (Method C).
3170 (NH2), 1690, 1660 (C]O), 1635 (C]N) 1330, 1160 (SO2) cmꢀ1
;
1H NMR (DMSO-d6)
d 3.86 (s, 2H, CH2), 7.33 (s, 1H, CONHA), 7.50 (d,
A solution of triethylamine (2.13 g, 21 mmol), 4-(2-aminoethyl)
benzenesulfonamide (2.0 g, 10 mmol) and 4-chloro-3-pyr-
idinesulfonamide (1.93 g,10 mmol) in methanol (25 ml) was stirred
at reflux for 8 h. After cooling to room temperature water (40 ml)
was added and stirred for 3 h. The crude product 27 was collected
by filtration, washed with water, dried and purified by crystalliza-
tion from methanol. Yield 2.6 g (73%), m.p. 249e251 ꢁC dec.; IR
(KBr) 3415, 3335, 3255 (SO2NH and NH), 1600, 1570 (C]N and C]
J ¼ 4.4 Hz,1H, H-5), 7.65 (s, 2H, SO2NH2), 7.70 (s,1H, CONHB), 8.56 (d,
J ¼ 4.4 Hz,1H, H-6), 8.82 (s,1H, H-2) ppm. Anal. (C7H9N3O3S2) C, H, N.
4.1.7. Procedures for the preparation of 4-(amino and hydrazino)-3-
pyridinesulfonamide derivatives (23e27), (Methods A, B and C)
Method A (for compounds 23 and 24). To a solution of allylamine
(2.85 g, 50 mmol) or 5-methyl-2-pyrazoline (4.2 g, 50 mmol) in
methanol (20 ml) 4-chloro-3-pyridinesulfonamide
1
(1.93 g,
C), 1350, 1320, 1150 (SO2) cmꢀ1 1H NMR (DMSO-d6)
; d 2.96 (t,
10 mmol) was added. The reaction mixture was stirred at reflux for
7 h. The solvent and excess of amine was evaporated under reduced
pressure. To the resulting oily residue water (25 ml) was added
J ¼ 4.9 Hz, 2H, CH2Ph), 3.52 (t, J ¼ 4.9 Hz, 2H, NeCH2), 6.50 (s, 1H,
NH), 6.83 (d, J ¼ 5.6 Hz, 1H, H-5, pyrid.), 7.36 (s, 4H, 2ꢃ SO2NH2),
7.47 (d, J ¼ 7.6 Hz, 2H, H-3 and H-5, PhSO2), 7.75 (d, J ¼ 7.6 Hz, 2H,

2404
Z. Brzozowski et al. / European Journal of Medicinal Chemistry 45 (2010) 2396e2404
H-2 and H-6, PhSO2), 8.21 (d, J ¼ 5.6 Hz, 1H, H-6, pyrid.), 8.49 (s, 1H,
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 [16,27e30], and represent the mean from at least
three different determinations.
H-2, pyrid.) ppm. Anal. (C13H16N4O4S2) C, H, N.
4.1.8. Procedure for the preparation of 4-(N0-heteroarylhydrazino)-
3-pyridinosulfonamides (31e33)
A mixture of 26 (0.76 g, 4 mmol) and the appropriate 3-meth-
ylthia-1,4,2-ditiazine 28, 29 or 30 (4 mmol) in anhydrous methanol
(35 ml) was stirred at reflux until the evolution of MeSH had ceased
(14e36 h) (Caution: because of high toxicity, MeSH should be
trapped in aqueous NaOH solution). After cooling to room
temperature the precipitate was collected by filtration, washed
with methanol (4 ꢃ 2 ml) and dried. In this manner the following 3-
pyridinesulfonamides were obtained.
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2010.02.020.
4.1.8.1. 4-[N0-(6-Chloro-7-cyano-1,1-dioxo-1,4,2-benzodithiazin-3-yl)
hydrazino]-3-pyridinesulfonamide (31). Starting from 6-chloro-7-
cyano-3-methylthio-1,4,2-benzodithiazine 1,1-dioxide 28 (1.22 g),
the title compound 31 was obtained (1.6 g, 89%); m.p. 390e393 ꢁC
dec.; IR (KBr) 3340, 2280, 3210 (SO2NH2 and HNeNH), 2225 (C^N),
1650,1580 (C]N and C]C),1360,1150 (SO2) cmꢀ1; 1H NMR (DMSO-
References
[1] C.T. Supuran, Nat. Rev. Drug Discov. 7 (2008) 168e181.
[2] C.T. Supuran, A. Scozzafava, Expert Opin. Ther. Pat. 10 (2000) 575e600.
[3] J. Pastorek, S. Pastorekova, I. Callebaut, J.P. Marnon, V. Zelnik, R. Opavsky,
M. Zatovicova, S. Liao, D. Portetelle, E.J. Stanbridge, J. Zavada, A. Burny,
R. Kettmann, Oncogene 9 (1994) 2877e2888.
[4] S. Pastorekova, S. Parkkila, A.K. Parkkila, R. Opavsky, V. Zelnik, J. Saarnio,
J. Pastorek, Gastroenterology 112 (1997) 398e408.
[5] W.R. Chegwidden, I.M. Spencer, C.T. Supuran, The roles of carbonic anhydrase
in cancer. in: G. Xue, Y. Xue, Z. Xu, G.L. Hammond, A.H. Lim (Eds.), Gene
Families: Studies of DNA, RNA, Enzymes, and Proteins. World Scientific,
Singapore, 2001, pp. 157e169.
d6)
d
7.42 (d, J ¼ 7.1 Hz, 1H, H-5, pyridine), 8.03 (s, 1H, H-5, benzo-
dithiazine), 8.09 (s, 2H, SO2NH2), 8.18 (d, J ¼ 7.1 Hz, 1H, H-6, pyri-
dine), 8.27 (s, 1H, H-8, benzodithiazine), 8.50 (s, 1H, H-2, pyridine),
10.80 (s, 1H, NH), 13.31 (br.s, 1H, NH) ppm. Anal. (C13H9ClN6O4S3) C,
H, N.
4.1.8.2. 4-[N0-(6-Chloro-7-methyl-1,1-dioxo-1,4,2-benzodithiazin-3-
yl)hydrazyno]-3-pyridinesulfonamide (32). Starting from 6-chloro-
[6] C.T. Supuran, A. Scozzafava, I. Conway, Carbonic Anhydrase. Its Inhibitors and
Activators. CRC, Boca Raton, New York, London, 2004, pp. 1e363.
[7] K. Kaunisto, S. Parkkila, H. Rajaniemi, A. Waheed, J. Grubb, W.S. Sly, Kidney Int.
61 (2002) 2111e2118.
7-methyl-3-methylthio-1,4,2-benzodithiazine
1,1-dioxide
29
[8] S. Parkkila, A.J. Kivele, K. Kaunisto, A.K. Parkkila, J. Hakkola, H. Rajaniemi,
A. Waheed, W.S. Sly, BMC Gastroenterol. 2 (2002) 13e20.
[9] S. Parkkila, A.K. Parkkila, H. Rajaniemi, G.N. Shah, J.H. Grubb, A. Waheed, W.
S. Sly, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 1918e1923.
[10] K. Fujikawa-Adachi, I. Nishimori, T. Taguchi, S. Onishi, Genomics 61 (1999)
74e81.
[11] S. Ashida, I. Nishimori, M. Tanimura, S. Onishi, T. Shuin, J. Cancer Res. Clin.
Oncol. 128 (2002) 561e568.
(1.18 g), the title compound 32 was obtained (1.6 g, 92%); m.p.
321e322 ꢁC dec.; IR (KBr) 3425, 3340, 3260, 3210 (SO2NH2 and
HNeNH), 1650, 1570 (C]N and C]C), 1345, 1320, 1140 (SO2) cmꢀ1
;
1H NMR (DMSO-d6)
d
2.40 (s, 3H, CH3), 7.43 (d, J ¼ 7.4 Hz, 1H, H-5,
pyridine), 7.65 (s, 1H, H-5, benzodithiazine), 7.84 (s, 1H, H-8, ben-
zodithiazine), 8.10 (s, 3H, SO2NH2 and NH), 8.17 (d, J ¼ 7.4 Hz, 1H, H-
6, pyridine), 8.49 (s, 1H, H-2, pyridine), 10.94 (s, 1H, NH) ppm. Anal.
(C13H12ClN5O4S3) C, H, N.
[12] O. Türeci, U. Sahin, E. Vollmar, S. Siemer, E. Göttert, G. Seitz, A.K. Parkkila, G.
N. Shah, J.H. Grubb, M. Pfreundschuh, W.S. Sly, Proc. Natl. Acad. Sci. U.S.A. 95
(1998) 7608e7613.
[13] C.C. Wykoff, J.N. Beasley, H.P. Watson, K.J. Turner, J. Pastorek, A. Sibtain, D.
G. Wilson, H. Turley, K.L. Talks, H.P. Maxwell, W.C. Pugh, J.P. Ratcliffe, L.
A. Harris, Cancer Res. 60 (2000) 7075e7083.
[14] S. Parkkila, H. Rajaniemi, A.K. Parkkila, J. Kivelä, A. Waheed, S. Pastorekova,
J. Pastorek, W.S. Sly, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 2220e2224.
[15] E. Svastova, A. Hulikova, M. Rafajova, M. Zatovicova, A. Gibadulinova, A. Casini,
A. Cecchi, A. Scozzafava, C.T. Supuran, J. Pastorek, S. Pastorekova, FEBS Lett. 577
(2004) 439e445.
4.1.8.3. 4-[N0-(1,1-Dioxopyrido[4,3-e]-1,4,2-dithiazin-3-yl)hydrazino]-
3-pyridinesulfonamide (33). Staring from 3-methylthiopyrido[4,3-e]-
1,4,2-dithiazine 1,1-dioxide 30 (0.99 g), the title compound 33 was
obtained (1.4 g, 90%); m.p. 302e303 ꢁC dec.; IR (KBr) 3370, 3270,
3220 (SO2NH2 and NH),1665,1620,1580 (C]N and C]C),1360,1320,
1150 (SO2) cmꢀ1 1H NMR (DMSO-d6)
; d 3.20 (s, 1H, NH), 7.45 (d,
[16] A. Cecchi, A. Hulikova, J. Pastorek, S. Pastorekova, A. Scozzafava, J.-Y. Winum, J.-
L. Montero, C.T. Supuran, J. Med. Chem. 48 (2005) 4834e4841.
[17] A.I. Michinton, I.F. Tannock, Nat. Rev. Cancer 6 (2006) 583e592.
[18] A. Kamb, Nat. Rev. Drug Discov. 4 (2005) 161e165.
J ¼ 7.1 Hz, 1H, H-5, pyridine), 7.59 (d, J ¼ 5.1 Hz, 1H, H-5, pyr-
idodithiazine), 8.11 (s, 2H, SO2NH2), 8.21 (d, J ¼ 7.1 Hz, 1H, H-6, pyri-
dine), 8.53 (s, 1H, H-2, pyridine), 8.60 (d, J ¼ 5.1 Hz, H-6,
pyridodithiazine), 8.88 (s, 1H, H-8, pyridodithiazine), 10.94 (s, 1H,
NH) ppm. Anal. (C11H10N6O4S3) C, H, N.
ꢀ
[19] F. Sa˛czewski, J. S1awinski, A. Kornicka, Z. Brzozowski, E. Pomarnacka,
A. Innocenti, A. Scozzafava, C.T. Supuran, Bioorg. Med. Chem. Lett. 16 (2006)
4846e4851.
ꢀ
[20] F. Sa˛czewski, A. Innocenti, Z. Brzozowski, J. S1awinski, E. Pomarnacka,
A. Kornicka, A. Scozzafava, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 21
4.2. CA inhibition assay
(2006) 563e568.
ꢀ
[21] F. Sa˛czewski, A. Innocenti, J. S1awinski, A. Kornicka, Z. Brzozowski,
E. Pomarnacka, A. Scozzafava, C. Temperini, C.T. Supuran, Bioorg. Med. Chem.
An Applied Photophysics (Oxford, UK) stopped-flow instru-
ment has been used for assaying the CA-catalyzed CO2 hydration
activity [26]. 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 main-
taining constant the ionic strength), following the CA-catalyzed
16 (2008) 3933e3940.
[22] Z. Brzozowski, J. S1awinski, Acta Pol. Pharm. 41 (1984) 5e13.
[23] Z. Brzozowski, F. Gajewski, J. S1awinski, E. Pomarnacka, Acta Pol. Pharm. Drug
ꢀ
ꢀ
Res. 50 (1993) 199e203.
ꢀ
[24] Z. Brzozowski, J. S1awinski, Acta Pol. Pharm. 41 (1984) 133e139.
ꢀ
[25] Z. Brzozowski, F. Sa˛czewski, J. S1awinski, J. Heterocycl. Chem. 45 (2008)
1407e1413.
[26] R.G. Khalifah, J. Biol. Med. 246 (1971) 2561e2573.
[27] J.R. Casey, P.E. Morgan, D. Vullo, A. Scozzafava, A. Mastrolorezo, C.T. Supuran, J.
Med. Chem. 47 (2004) 2337e2347.
[28] S. Pastorekova, A. Casini, A. Scozzafora, D. Vullo, J. Pastorek, C.T. Supuran,
Bioorg. Med. Chem. Lett. 14 (2004) 869e873.
[29] D. Vullo, A. Innocenti, I. Nishimori, J. Pastorek, A. Scozzafora, S. Pastorekova, C.
T. Supuran, Bioorg. Med. Chem. Lett. 15 (2005) 963e969.
[30] J. Nishimori, D. Vullo, A. Innocenti, A. Scozzafava, A. Mastrolorenzo, C.
T. Supuran, Bioorg. Med. Chem. Lett. 15 (2005) 3828e3833.
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 determined in the same manner and subtracted from the
total observed rates. Stock solutions of inhibitor (1 mM) were