Synthesis of a new series of N4-substituted 4-(2-aminoethyl) benzenesulfonamides and their inhibitory effect on human carbonic anhydrase cytosolic isozymes i and II and transmembrane tumor-associated isozymes IX and XII

Article abstract of DOI:10.1016/j.ejmech.2014.06.074

A series of novel N⁴-substituted 4-(2-aminoethyl) benzenesulfonamides 5-17 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 CA I and II, and tumor-associated isozymes CA IX and XII. Against the human CA I investigated compounds displayed K_I values from 96.3 to 3520 nM, toward hCA II at range of 18.1-2055 nM, while against hCA IX ranging from 5.9 to 419 nM and against hCA XII in the range of 4.0-414 nM. The very good inhibitory activity against tumor-associated hCA IX and hCA XII was found. The six new compounds displayed a powerful inhibitory potency toward hCA IX (K _I = 5.9-10.7 nM) in comparison with the clinically used CAIs AAZ, MZA, EZA, DCP and IND (24-50 nM). The most potent hCA IX and hCA XII inhibitors 11 and 12 (K_I: 5.9 and 6.2 nM for hCA IX and 4.3 and 4.0 nM for hCA XII, respectively) belonged to the compounds with cationic character and presented meaningful affinity to the transmembrane isoforms hCA IX and XII than to physiologically dominant isozymes hCA I and II with the selectivity ratios hCA IX versus hCA II and hCA XII versus hCA II for 11 and 12 in the range of 10-15.

Full text of DOI:10.1016/j.ejmech.2014.06.074

European Journal of Medicinal Chemistry 84 (2014) 59e67
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of a new series of N4-substituted 4-(2-aminoethyl)
benzenesulfonamides and their inhibitory effect on human carbonic
anhydrase cytosolic isozymes I and II and transmembrane tumor-
associated isozymes IX and XII
a
,
*
a
a
a
_
ꢀ
ꢀ
Jarosław Sławinski , Zdzisław Brzozowski , Beata Zołnowska , Krzysztof Szafranski ,
Aneta Pogorzelska a, Daniela Vullo b, Claudiu T. Supuran b
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
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
A series of novel N4-substituted 4-(2-aminoethyl)benzenesulfonamides 5e17 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 CA I and II, and tumor-associated isozymes CA IX and XII. Against the human CA I investigated
compounds displayed KI values from 96.3 to 3520 nM, toward hCA II at range of 18.1e2055 nM, while
against hCA IX ranging from 5.9 to 419 nM and against hCA XII in the range of 4.0e414 nM. The very good
inhibitory activity against tumor-associated hCA IX and hCA XII was found. The six new compounds
displayed a powerful inhibitory potency toward hCA IX (KI ¼ 5.9e10.7 nM) in comparison with the
clinically used CAIs AAZ, MZA, EZA, DCP and IND (24e50 nM). The most potent hCA IX and hCA XII
inhibitors 11 and 12 (KI: 5.9 and 6.2 nM for hCA IX and 4.3 and 4.0 nM for hCA XII, respectively) belonged
to the compounds with cationic character and presented meaningful affinity to the transmembrane
isoforms hCA IX and XII than to physiologically dominant isozymes hCA I and II with the selectivity ratios
hCA IX versus hCA II and hCA XII versus hCA II for 11 and 12 in the range of 10e15.
Article history:
Received 9 December 2013
Accepted 29 June 2014
Available online 5 July 2014
Keywords:
Sulfonamide
Synthesis
Carbonic anhydrase isozymes I, II, IX and XII
inhibitors
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
hypoxia-inducible enzyme overexpressed by cancer cells has gotten
a lot of attention as a new target for the design of antitumor agents
Carbonic anhydrases (CAs, EC 4.2.1.1.) are metalloenzymes pre-
dominating in higher vertebrates and dividing into 16 isoforms
including cytosolic (I, II, III, VII, XIII), membrane-bound (IV, IX, XII,
XIV, XV), mitochondrial (VA and VB), and secreted (VI) forms. Three
cytosolic forms, the CA related proteins (CARP) (VIII, X and XI) have
also been identified, but they are acatalytic [1]. The major function
of CA is to catalyze the reversible hydratation of carbon dioxide to
bicarbonate and a proton (CO2 þ H2O 4 HCOꢀ3 þ Hþ) and act as
regulators of respiration, acidebase regulation, bone resorption
and calcification, and biosynthetic processes [2]. Furthermore, CAs
constitute an important research objects for the design agents to
treat a variety of pathological states such as edema, glaucoma,
obesity, cancer, epilepsy or osteoporosis [3]. In last years CA IX, a
[4,5]. It has been known, that CA IX and CA XII are associated with
tumor progression, metastasis, and response to therapy [6]. An
important role of CA IX and CA XII as major tumor prosurvival pH-
regulating enzymes was suggested in genetic study. Genetic
depletion of CA IX, together with CA XII in LS147Tr colorectal cancer
xenografts led to an 85% reduction in tumor growth [7]. Because the
CA XII is more spread in some healthy tissues than CA IX [6], it is
less interesting target than CA IX in the cancer therapy. From the
therapeutic point of view, inhibition of CA IX in tumor cells de-
creases slightly alkaline intracellular pH and increases acidic
extracellular pH thus negatively influences on tumor cell survival
[5].
Classical carbonic anhydrase inhibitors belong to primary sul-
fonamides and are initially developed as diuretics and systemically
acting antiglaucoma drugs AAZ, MZA, DCP, EZA and IND [8]. Lack of
selectivity of these classical inhibitors toward CA IX and CA XII
caused the progress in design of new inhibitors targeting tumor-
* Corresponding author.
ꢀ
E-mail address: jaroslaw@gumed.edu.pl (J. Sławinski).
http://dx.doi.org/10.1016/j.ejmech.2014.06.074
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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J. Sławinski et al. / European Journal of Medicinal Chemistry 84 (2014) 59e67
60
associated isozymes without side effects associated with inhibition
of physiologically dominant isozymes such as CA I or CA II. Among
strategies of reaching a high selectivity toward CA IX the design of
positively charged molecules and the addition of bulky parts such
as fluorescent tags or sugar moieties which limit transport across
the plasma membrane should be mentioned. Other methods
concern the use of bioreductive prodrugs that are activated by
hypoxia in tumor cells and development of distinct chemotypes of
sulfonamides and coumarins [6]. It should be also indicated the
important role of CA IX inhibitors in increasing of uptake of
chemotherapeutic agent, doxorubicin in cancer therapy. The cyto-
specificity for the inhibition of the tumor-associated isoforms
versus the remaining CA isozymes CA IeVII and XIIeXV found in
mammalians. So far, a lot of effort has been made in solving this low
selectivity problem. This fact among others prompted us to syn-
thesize and investigate of novel N4-substituted 4-(aminoethyl)
benzenesulfonamides of type VI (Fig. 1), to recognize on the
structural features contributing to the carbonic anhydrase
inhibitors.
2. Results and discussion
toxic efficiency of doxorubicin in presence of 1 mM CA IX inhibitor in
hypoxic conditions in experiment on breast cancer MDA-MB-231
cell line was restored at 20% level [9].
2.1. Chemistry
As shown in Schemes 1e4, the synthesis of the target N4-
substituted 4-(2-aminoethyl)benzenesulfonamides 5e17 were
achieved by convenient procedures starting from commercially
available 4-(2-aminoethyl)benzenesulfonamide 4 or its derivative 3
previously described [18]. The condensation reactions of 4 with 3-
methylthio-1,4,2-benzodithiazines 1aec (Scheme 1), carried out in
boiling methanol led to the formation of 4-[2-(1,4,2-
benzodithiazin-3-ylamino)ethylene]benzenesulfonamides 5e7 in
very good yield (89e93%). Similarly, by addition reactions of 4 to 7-
Recently, we have reported on the inhibition of human isozymes
CA I, II [10e13] CA IX [11,12,14], CA VA and VB [13] with some 4-(R-
aminoalkyl)benzenesulfonamides of type I (Fig. 1). We have re-
ported 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)benzenesulfonamides of type II
[15e17] (Fig. 1). We also described the synthesis a number of pyr-
idine-3-sulfonamides of types III and IV [18e20] and V [21,22] and
inhibitory activities against isozymes hCA I, II, IX, XII and XIV
[10,11,23]. Numerous of these compounds exhibit excellent hCA IX
inhibitory efficacy with inhibition constants 4.6e12.0 nM, being
thus much more effective as compared to the clinically used sul-
fonamides AAZ, MZA, EZA, DCP and IND (KI ¼ 24e50 nM).
Generally, different aryl/heteroarylsulfonamides do not show
oxo-5-thia-3,6,11b-triaza-benzo[c]fluorene-5,5-dioxides
2aec
(Scheme 2), carried out in boiling tetrahydrofuran, the target 3-
[2(4-sulfamoylphenyl)ethylamino]-4H-pyrido[4,3-e]-1,2,4-
thiadiazine derivatives 8e10 were obtained in 89e94% yields. The
desired pyridinium salt 11 was obtained in 93.6% yield by the re-
action of 3 with methyl iodide in acetonitrile. Then the reaction of
Fig. 1. General structures of known IeV and novel VI arylsulfonamides, and clinically used sulfonamides AAZ, MZA, DCP, EZA and IND (standard CA inhibitors).

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J. Sławinski et al. / European Journal of Medicinal Chemistry 84 (2014) 59e67
61
Scheme 1. Synthesis of 4-[2-(6-chloro-1,1-dioxo-1,4,2-benzodithiazin-3-ylamino)ethylene] benzenesulfonamides 5e7.
Scheme 2. Synthesis of 4-(2-hydroxyphenyl)-3-[2-(4-sulfamoylphenyl)ethylamino]-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides 8e10.
Scheme 3. Synthesis of 3-sulfamoyl-4-[2-(4-sulfamoylphenyl)ethylamino]-1-methylpyridinium iodide 11 and its transformation to betaine 12.
range 96.3e3520 nM. Among them a distinctive compound was
N,N0-bis[2-(4-sulfamoylphenyl)ethyl]sulfamide (15) with the
highest inhibitory potency KI ¼ 96.3 nM.
11 with NaOH in water furnished the betaine 12 in 90.4% yield. The
desired compounds 13e15 were prepared in satisfactory yields
(71e92%) by the nucleophilic reactions of 4 with the carbaldehydes
or sulfamide (Scheme 3). The sulfonamides 16e17 (Scheme 3)
bearing the arylacetamido moiety in their structures were obtained
in good yield (81e82%) by reactions of 4 with the appropriate N-(4-
R-phenyl)chloroacetamides in the presence of triethylamine in
tetrahydrofuran solution. All finale products 5e17 were character-
ized by IR and NMR spectroscopy as shown in the experimental
protocols. Elemental analyses (C, H, N) were in accordance with the
proposed structures.
2. Toward the second cytosolic isoform hCA II, sulfonamides con-
taining 1,4,2-benzodithiazinyl fragment (5e7) showed rather
low inhibitory activity ranged from 113 nM to 243 nM, whereas
derivatives of 4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide
(8e10) were inactive KI: 1315e2055 nM. The other com-
pounds 11e17 inhibited hCA II with moderate potency KI:
18.1e63.7 nM that was lower than for reference drugs AAZ,
MZA, EZA, IND.
3. Inhibitory activities of newly synthesized compounds against
the transmembrane tumor-associated hCA IX were diversified
(KI from 5.9 to 419 nM) and depended on structural nature of
substituent R. Thus, compounds with 1,4,2-benzodithiazinyl
moiety (5e7, KI: 83.2e108 nM) were stronger inhibitors than
2.2. CA inhibition studies
The compounds 5e17 as well as standard, clinically used CAIs,
such as acetazolamide 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 II, and two transmembrane tumor-
associated isozymes: hCA IX and XII. The following should be noted
regarding CA inhibitory data of Table 1:
derivatives
group (8e10, KI: 328e419 nM), but weaker than substances
with single aromatic ring in substituent (11e17, KI:
containing
4H-pyrido[4,3-e]-1,2,4-thiadiazinyl
R
5.9e38.5 nM). It should be noted, that the most potent in-
hibitors of hCA IX (11 and 12) belonged to the compounds with
cationic nature and exhibited a powerful potency (5.9 and
6.2 nM, respectively) in comparison to the reference sulfon-
amides AAZeIND (KI in the range of 25e50 nM). Furthermore,
1. Against the slow isoform hCA I, almost all tested sulfonamide
derivatives exhibited weak inhibitory activity with KI in the

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J. Sławinski et al. / European Journal of Medicinal Chemistry 84 (2014) 59e67
62
Scheme 4. Synthesis of N4-substituted 4-(2-aminoethyl)benzenesulfonamides 13e17. Reagents and conditions: (a) aryl- or heteroarylaldehyde (1.12 molar equiv.), ethanol, reflux,
11 h; (b) sulfamide (0.5 molar equiv.), p-dioxane, reflux, 48 h; (c) appropriate N-(4-R-phenyl)chloroacetamide (1.0 molar equiv.), THF, r.t. 24 h, TEA (1.2 molar equiv.), r.t. 24 h, reflux
18 h, water, r.t., 1% hydrochloric acid to pH 2.
13e15 and 17 also were better inhibitors than above-mentioned
clinically used drugs.
single aromatic ring in substituent R. Moreover, compounds 11e15
and 17 (KI: 5.9e10.7 nM) were better hCA IX inhibitors when
compared to the reference sulfonamides AAZeIND (KI: 25e50 nM).
In the case of hCA XII the inhibition constants were in the range of
4.0e414 nM. The compounds 5e7 and 11e17 showed promising
inhibitory activity (KI: 4.0e33.1 nM) comparable with the reference
sulfonamides AAZeIND (KI: 3.4e50 nM). The research revealed
that the most potent inhibitors of both hCA IX and XII were the
cationic compounds 11 and 12 (KI: 5.9 and 6.2 nM for hCA IX and 4.3
and 4.0 nM for hCA XII, respectively). These derivatives presented
meaningful affinity to the hCA IX and XII than to the hCA I and II
with the selectivity ratios hCA IX versus hCA II, and hCA XII versus
hCA II in the range of 10e15, respectively. This result confirms the
positively charged sulfonamides may be used in the design of new
inhibitors targeting tumor-associated isozymes.
4. Similar tendency in inhibitory activity of tested sulfonamides
was observed for the second tumor-associated isoform, hCA XII.
Among them, ten compounds 5e7 and 11e17 showed promising
inhibitory potency (KI of 4.0e33.1 nM) while three 4H-pyrido
[4,3-e]-1,2,4-thiadiazine derivatives 8e10 were relatively inac-
tive (KI of 179e414 nM). It is noteworthy, that 11 (KI ¼ 4.3 nM)
and 12 (KI ¼ 4.0 nM), the most potent inhibitors of hCA XII,
demonstrated inhibitory potency better or comparable to the
references AAZeIND. As was shown in Table 1, some compounds
presented meaningful affinity to the transmembrane isoforms
hCA IX and XII than to physiologically dominant isozymes hCA I
and II. The selectivity ratios hCA IX versus hCA II and hCA XII
versus hCA II for 11 and 12 were in the range of 10e15 and this
advantage confirms possibility to use of positively charged sul-
fonamides for designing of new inhibitors targeting tumor-
associated isozymes. This cationic character should reduce
transport across the plasma membrane.
4. Experimental protocols
3. Conclusions
4.1. Synthesis
We have developed methods for the synthesis of novel series
N4-substituted 4-(2-aminoethyl)benzenesulfonamides 5e17. The
13 compounds have been assayed for the inhibition of four physi-
ological CA isozymes, such as hCA I, II, IX and XII. A relatively weak
inhibitory activity against the human CA I was observed for all
investigated compounds with KI values from 96.3 to 3520 nM. To-
ward the second physiological isoform hCA II, all tested compounds
5e17 showed lower activity (KI: 18.1e2055 nM) in comparison with
reference AAZ, MZA, EZA, IND (KI: 8e15 nM). Against hCA IX the
new compounds showed inhibition constants in the range of
5.9e419 nM. It was shown that inhibitor activity toward hCA IX
increased for compounds 11e17 (KI: 5.9e38.5 nM) containing
The following instruments and parameters were used: melting
points Buchi 535 apparatus; IR spectra: KBr pellets,
400e4000 cmꢀ1 Perkin Elmer FTIR spectrophotometer; 1H and 13
C
NMR: Varian Gemini 200 apparatus at 200 MHz, Varian Unity 500
plus apparatus at 500 MHz; 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 0.4% of theo-
retical values. The commercially unavailable substrates were ob-
tained according to the following methods described previously: 3-
methylthio-1,4,2-benzodithiazines 1a [25] and 1bec [26], 7-oxo-5-
thia-3,6,11b-triaza-benzo[c]fluorene-5,5-dioxides 2aec [27] and 4-
[2-(4-sulfamoylphenyl)ethylamino]-3-pyridinesulfonamide 3 [18].

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J. Sławinski et al. / European Journal of Medicinal Chemistry 84 (2014) 59e67
63
Table 1
Carbonic anhydrase inhibition data for compounds 5e17 and standard inhibitors against human isozymes hCA I, II, IX and XII by a stopped-flow CO2 hydrase assay [24].
Compd
R
KIa (nM)
hCA Ib
hCA IIb
hCA IXc
hCA XIIc
AAZ
MZA
EZA
DCP
IND
250
780
25
1200
31
12
14
8
38
15
25.0
27.0
34.0
50.0
24.0
5.7
3.4
22.0
50.0
3.4
5
6
675
113
267
91
12.5
33.1
1450
108
7
8
1715
2640
243
83.2
25.4
1315
328
414
9
3520
2055
419
361
10
2755
1504
327
179
11
12
923
887
61.3
60.1
5.9
6.2
4.3
4.0
13
184
24.8
10.7
12.5
(continued on next page)

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64
J. Sławinski et al. / European Journal of Medicinal Chemistry 84 (2014) 59e67
Table 1 (continued )
Compd
R
KIa (nM)
hCA Ib
hCA IIb
19.1
hCA IXc
8.2
hCA XIIc
5.9
14
15
16
176
96.3
340
63.7
50.2
18.1
10.2
38.5
9.0
6.4
20.1
6.3
17
298
a
Mean from 3 different determinations (errors were in the range of 5e10% of the reported values, data not shown).
Human (cloned) isozymes, by CO2 hydration method.
Catalytic domain of human, cloned isozymes, by the CO2 hydration method.
b
c
4.1.1. Procedure for the preparation of 4-[2-(1,4,2-benzodithiazin-3-
ylamino)ethyl]benzenesulfonamide (5e7)
mixture of the appropriate
4.1.1.3. 4-{2-[6-Chloro-7-(4-chlorophenylcarbamoyl)-1,1-dioxo-
1,4,2-benzodithiazin-3-ylamino]ethyl}benzenesulfonamide (7).
A
3-methylthio-1,4,2-
Starting from 3-methylthiobenzodithiazine 1c (2.17 g), the title
compound 7 was obtained (2.70 g, 92.2%): m.p. 214e215 ꢂC; IR
(KBr): 3360, 3305, 3260, 3200 (NH and SO2NH2), 2925, 2850
benzodithiazine 1,1-dioxide 1a, 1b or 1c (5 mmol) and 4-(2-
aminoethyl)benzenesulfonamide 4 (1.06 g, 5.3 mmol) in anhy-
drous methanol (40 ml) was refluxed with stirring until of MeSH
had ceased (12e16 h) (caution: because of high toxicity, MeSH
should be trapped in an aqueous NaOH solution). After cooling to
room temperature, the reaction mixture was left overnight. The
precipitate was collected by filtration, washed with methanol
(4 ꢁ 2 ml) and dried. In this manner, the following sulfonamides
were obtained.
(CH2CH2), 1665 (NeC]O), 1600 (C]N), 1315, 1160 (SO2) cmꢀ1 1H
;
NMR (DMSO-d6)
d
2.97 (t, J ¼ 6.8 Hz, 2H, NeCH2CH2ePh), 3.65 (t,
J ¼ 6.8 Hz, 2H, NeCH2CH2ePh), 7.32 (s, 2H, SO2NH2), 7.40e7.47 (m,
4H, arom.), 7.70e7.79 (m, 4H, arom.), 8.10 (s, 1H, H-5 benzodithia-
zine), 8.14 (s,1H, H-8 benzodithiazine), 9.94 (s,1H, NH),10.82 (s,1H,
HNC]O) ppm. Anal. (C22H18Cl2N4 O5S3) C, H, N.
4.1.2. Procedure for the preparation of 4-substituted 3-[2-(4-
sulfamoylphenyl)ethylamino]-4H-pyrido[4,3-e]-1,2,4-thiadiazine
1,1-dioxides (8e10)
4.1.1.1. 4-{2-[(6-Chloro-1,1-dioxo-7-methyl-1,4,2-benzodithiazin-3-
yl)amino]ethyl}benzenesulfonamide
(5). Starting
from
3-
methylthiobenzodithiazine (1a) (1.47 g), the title compound 5
A solution of 4-(2-aminoethyl)benzenesulfonamide 4 (0.44 g,
2.2 mmol) and appropriate 7-oxa-5-thia-3,6,11b-triaza-benzo[c]
fluorene 2a, 2b or 2c (2 mmol) in tetrahydrofuran (25 ml) was
refluxed with stirring for 6 h. After cooling to room temperature,
the suspension was left overnight. The precipitate was collected by
filtration, washed successively with tetrahydrofuran (1.5 ml),
methanol (2 ꢁ 1.5 ml) and acetonitrile (1.5 ml), and dried. In this
manner the following sulfonamides were obtained:
was obtained (2.0 g, 89.7%): m.p. 244e245 ꢂC; IR (KBr): 3330, 3235
(NH and SO2NH2), 1625 (C]N), 1325, 1295, 1155 (SO2) cmꢀ1 1H
;
NMR (DMSO-d6)
d
2.42 (s, 3H, CH3), 2.97 (t, J ¼ 6.8 Hz, 2H,
NeCH2CH2ePh), 3.63 (t, J ¼ 6.8 Hz, 2H, NeCH2CH2ePh), 7.33 (s, 2H,
SO2NH2), 7.43 (d, J ¼ 8.3 Hz, 2H, PhSO2), 7.77 (d, J ¼ 8.3 Hz, 2H,
PhSO2), 7.89 (s, 1H, H-5, benzodithiazine), 7.99 (s, 1H, H-8, benzo-
dithiazine), 8,79 (s, 1H, NH) ppm; 13C NMR (DMSO-d6, 50 MHz)
19.57, 33.56, 44.38, 126.08, 126.70, 127.72, 128.29, 129.43, 131.60,
137.32, 137.49, 142.6, 142.93, 162.66 ppm. Anal. (C16H16ClN3 O4S3) C,
H, N.
4.1.2.1. 4-(2-Hydroxyphenyl)-3-[2-(4-sulfamoylphenyl)ethylamino]-
4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide (8). Starting from
pyrido[4,3-e]-1,4,2-thiadiazine 2a (0.55 g), the title compound 8
was obtained (0.85 g, 89.7%): m.p. 234e235 ꢂC; IR (KBr): 3500 (OH)
3375, 3345, 3285 (NH and SO2NH2), 2955, 2920, 2855 (CH2CH2)
4.1.1.2. 4-{2-[6-Chloro-1,1-dioxo-7-(4-methylphenylcarbamoyl)-
1,4,2-benzodithiazin-3-ylamino]ethyl}benzenesulfonamide
(6).
Starting from 3-methylthiobenzodithiazine 1b (2.06 g), the title
compound 6 was obtained (2.50 g, 93.8%): m.p. 178e180 ꢂC; IR
(KBr): 3330, 3250 (NH and SO2NH2), 1655 (NeC]O), 1600 (C]N),
1595 (C]N), 1345, 1160 (SO2) cmꢀ1
;
1H NMR (DMSO-d6)
5.5 Hz, 2H,
d 2.89 (t,
J
¼
5.5 Hz, 2H, NeCH2CH2ePh), 3.42 (t,
J
¼
1320, 1305, 1155 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
d
2.28 (s, 3H, CH3),
NeCH2CH2ePh), 6.23 (d, J ¼ 5.9 Hz, 1H, H-5 pyridodithiazine),
7.00e7.23 (m. 3H, arom.), 7.24 (s, 1H, OH) 7.29 (s, 2H, SO2NH2), 7.35
(d, J ¼ 8.1 Hz, 2H, H-3 and H-5, PhSO2), 7.49 (t, J ¼ 7.5 Hz, 1H, arom.),
7.74 (d, J ¼ 8.1 Hz, 2H, H-2 and H-6, PhSO2), 8.48 (d, J ¼ 5.9 Hz, 1H,
H-6 pyridodithiazine), 8.89 (s, 1H, H-8 pyridodithiazine), 10.46 (s,
1H, NH) ppm; 13C NMR (DMSO-d6, 50 MHz) 34.32, 42.93, 110.55,
118.32, 120.16, 121.38, 126.06, 129.48, 130.71, 132.70, 142.45, 143.38,
2.97 (t, J ¼ 6.8 Hz, 2H, NeCH2CH2ePh), 3.65 (t, J ¼ 6.8 Hz, 2H,
NeCH2CH2ePh), 7.17 (d, J ¼ 7.9 Hz, 2H, H-3 and H-5, 4-MePh), 7.32
(s, 2H, SO2NH2), 7.44 (d, J ¼ 7.5 Hz, 2H, H-3 and H-5, PhSO2), 7.57 (d,
J ¼ 7.9 Hz, 2H, H-2 and H-6, 4-MePh), 7.77 (d, J ¼ 7.5 Hz, 2H, H-2,6,
PhSO2), 8.08 (s, 2H, H-5,8 benzodithiazine), 9.91 (s, 1H, NH), 10.58
(s, 1H, HNC]O) ppm. Anal. (C23H21ClN4 O5S3) C, H, N.

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J. Sławinski et al. / European Journal of Medicinal Chemistry 84 (2014) 59e67
65
144.39, 145.23, 150.31, 152.75, 154.44 ppm. Anal. (C20H19N5 O5S2) C,
H, N.
J ¼ 7.4 Hz, 1H, H-5 pyrid.), 6.85 (br s, 2H, SO2NH2 and SO2NH), 7.49
(d, J ¼ 8.1 Hz, 2H, H-3 and H-5, PhSO2), 7.74 (d, J ¼ 8.1 Hz, 2H, H-2
and H-6, PhSO2), 7.84 (dd, Jortho ¼ 7.4 Hz, Jmeta ¼ 1.4 Hz, 1H, H-6
pyrid.), 8.20 (d, Jmeta ¼ 1.4 Hz, 1H, H-2 pyrid.) ppm; 13C NMR
(DMSO-d6, 50 MHz) 35.23, 43.82, 45.80, 106.86, 125.87, 129.02,
129.69, 139.22, 142.35, 142.41, 143.76, 152.47 ppm. Anal. (C14H18N4
O4S2) C, H, N.
4.1.2.2. 4-(2-Hydroxy-5-methylphenyl)-3-[2-(4-sulfamoylphenyl)
ethylamino]-4H-pyrido[4,3-e]-1,2,4-thiadiazine
1,1-dioxide
(9).
Starting from pyrido[4,3-e]-1,2,4-thiadiazine 2b (0.58 g), the title
compound 9 was obtained (0.92 g, 94.3%): m.p. 276e277 ꢂC; IR
(KBr): 3500 (OH), 3360, 3305, 3280 (NH and SO2NH2), 1600 (C]N),
1335, 1160, 1150 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
d 2.24 (s, 3H CH3)
4.1.4. Procedure for preparation of 4-[2-(arylmethylideneamino)
ethyl]benzenesulfonamide (13e14)
2.86 (t, J ¼ 5.0 Hz, 2H, NeCH2CH2ePh), 3.39 (t, J ¼ 5.0 Hz, 2H,
NeCH2CH2ePh), 6.25 (d, J ¼ 5.2 Hz, 1H, H-5 pyridodithiazine),
6.93e7.19 (m, 3H, arom.), 7.26 (br s, 3H, OH and SO2NH2), 7.35 (d,
J ¼ 7.4 Hz, 2H, H-3 and H-5, PhSO2), 7.74 (d, J ¼ 7.4 Hz, 2H, H-2 and
H-6, PhSO2), 8.47 (d, J ¼ 5.2 Hz, 1H, H-6 pyridodithiazine), 8.88 (s,
1H, H-8 pyridodithiazine), 10.08 (s, 1H, NH) ppm; 13C NMR (DMSO-
d6, 50 MHz) 20.22, 34.29, 42.93, 110.66, 118.08, 119.69, 120.14,
126.03,129.53, 130.42,133.23, 142.43,143.43,144.36,145.16,150.35,
152.00, 152.75 ppm. Anal. (C21H21N5 O5S2) C, H, N.
A mixture of the appropriate arylaldehyde (9 mmol) and 4-(2-
aminoethyl)benzenesulfonamide (1.6 g,
8 mmol) in ethanol
(15 ml) was refluxed with stirring for 11 h and left to stand at room
temperature overnight. The precipitate was filtered off, washed
with ethanol (3 ꢁ 2 ml) and acetonitrile (2 ꢁ 2 ml), and dried. In
this manner the following Schiff bases were obtained.
4.1.4.1. 4-[2-(4-Chloro-3-nitrobenzylideneamino)ethyl]benzenesulfo-
namide (13). Starting from 4-chloro-3-nitrobenzaldehyde (1.67 g),
the title compound 13 was obtained (2.7 g, 91.7%): m.p.158e159 ꢂC;
IR (KBr): 3365, 3255 (SO2NH2), 2890, 2840 (CH2CH2), 1645 (C]N),
4.1.2.3. 4-(5-Chloro-2-hydroxyphenyl)-3-[2-(4-sulfamoylphenyl)eth-
ylamino]-4H-pyrido[4,3-e]-1,2,4-thiadiazine
1,1-dioxide
(10).
Starting from pyrido[4,3-e]-1,2,4-thiadiazine 2c (0.62 g), the title
compound 10 was obtained (0.94 g, 92.5%): m.p. 272e273 ꢂC; IR
(KBr): 3490 (OH) 3355, 3285 (NH and SO2NH2), 2950, 2920, 2860
(CH2CH2), 1600 (C]N), 1340, 1160 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
1340, 1325, 1165, 1145 (SO2) cmꢀ1
;
1H NMR (DMSO-d6)
7.8 Hz, 2H,
d 3.02 (t,
J
¼
7.8 Hz, 2H, NeCH2CH2ePh), 3.87 (t,
J
¼
NeCH2CH2ePh), 7.27 (s, 2H, SO2NH2), 7.43 (d, J ¼ 7.8 Hz, 2H, H-3
and H-5, PhSO2), 7.72 (d, J ¼ 7.8 Hz, 2H, H-2 and H-6, PhSO2), 7.84 (d,
J ¼ 8.3 Hz, 1H, H-5 Ph), 7.99 (dd, Jortho ¼ 8.3 Hz, Jmeta ¼ 1.4 Hz, 1H, H-
6 Ph), 8.34 (d, Jmeta ¼ 1.4 Hz, 1H, H-2 Ph), 8.38 (s, 1H, HC]N) ppm;
13C NMR (DMSO-d6, 50 MHz) 36.52, 61.37, 124.55, 125.89, 127.07,
129.59, 132.50, 132.65, 136.47, 142.26, 144.23, 148.07, 159.00 ppm.
Anal. (C15H14ClN3 O4S) C, H, N.
d
2.90 (t, J ¼ 5.0 Hz, 2H, NeCH2CH2ePh), 3.39 (t, J ¼ 5.0 Hz, 2H,
NeCH2CH2ePh), 6.32 (d, J ¼ 5.9 Hz, 1H, H-5 pyridodithiazine), 7.15
(d, J ¼ 9.5 Hz, 2H, H-3 and H-4, Ph), 7.28 (s, 2H, SO2NH2), 7.37 (d,
J ¼ 8.1 Hz, 2H, H-3 and H-5, PhSO2), 7.52 (s, 1H, OH), 7.55 (s, 1H, H-6
pyridodithiazine), 7.75 (d, J ¼ 8.1 Hz, 2H, H-2 and H-6, PhSO2), 8.49
(d, J ¼ 5.9 Hz, 1H, H-6 pyridodithiazine), 8.89 (s, 1H, H-8 pyr-
idodithiazine), 10.75 (s, 1H, NH) ppm. Anal. (C20H18ClN5 O5S2) C, H,
N.
4.1.4.2. 4-[2-(5-Nitro-2-thienylideneamino)ethyl]benzenesulfona-
mide (14). Starting from 5-nitrothiophene-2-carboxyaldehyde
(1.41 g), the title compound 14 was obtained (2.5 g, 92.0%): m.p.
203e204 ꢂC; IR (KBr): 3365, 3255 (SO2NH2), 2955, 2845 (CH2CH2),
4.1.3. Synthesis of 3-sulfamoyl-4-[2-(4-sulfamoylphenyl)
ethylamino]-1-methylpyridinium iodide (11) and its transformation
to the 4-[2-(4-sulfamoylphenyl)ethylamino]-1-methylpyridinium-
3-sulfonamidate (12)
1335, 1150 (SO2) cmꢀ1; 1H NMR (DMSO-d6)
d
3.09 (t, J ¼ 7.1 Hz, 2H,
4.1.3.1. Compound 11. A mixture of 4-[2-(4-sulfamoylphenyl)eth-
ylamino]-3-pyridinesulfonamide 3 (1.09 g, 3 mmol) and methyl
iodide (0.57 g, 4 mmol) in dry acetonitrile (20 ml) was stirred at
room temperature for 90 h, followed at reflux for 8 h. After cooling
to room temperature the precipitate was collected by filtration,
washed with acetonitrile (3 ꢁ 1.6 ml) and dried. Yield 1.4 g, (93.6%):
m.p. 272e273 ꢂC; IR (KBr): 3325, 3285, 3160 (NH and SO2NH2),
NeCH2CH2ePh), 3.85 (t, J ¼ 7.1 Hz, 2H, NeCH2CH2ePh), 7.27 (s, 2H,
SO2NH2), 7.42 (d, J ¼ 8.3 Hz, 2H, H-3 and H-5, PhSO2), 7.51 (d,
J ¼ 4.4 Hz, 1H, H-4 thiophene), 7.72 (d, J ¼ 8.3 Hz, 2H, H-2 and H-6,
PhSO2), 8.10 (d, J ¼ 4.4 Hz, 1H, H-3 thiophene), 8.45 (s, 1H, HC]N)
ppm. Anal. (C13H13N3 O4S2) C, H, N.
1650 (C]Nþ), 1350, 1340, 1160 (SO2) cmꢀ1
;
1H NMR (DMSO-d6)
4.1.5. Synthesis of N,N0-bis[2-(4-sulfamoylphenyl)ethyl]sulfamide
(15)
d
3.01 (t, J ¼ 6.8 Hz, 2H, NeCH2CH2ePh), 3.79 (t, J ¼ 6.3 Hz, 2H,
NeCH2CH2ePh), 3.93 (s, 3H, CH3Nþ), 7.33 (s, 2H, PhSO2NH2), 7.35
(d, J ¼ 7.3 Hz, 1H, H-5 pyrid.), 7.49 (d, J ¼ 7.8 Hz, 2H, H-3 and H-5,
PhSO2), 7.74 (d, J ¼ 7.8 Hz, 2H, H-2 and H-6, PhSO2), 7.80 (t,
J ¼ 6.3 Hz, 1H, NHCH2), 8.02 (s, 2H, Pyrid.-SO2NH2), 8.33 (d,
J ¼ 7.3 Hz, 1H, H-6 pyrid.), 8.73 (s, 1H, H-2 pyrid.) ppm. Anal.
(C14H19IN4 O4S2) C, H, N.
A mixture of 4-(2-aminoethyl)benzenesulfonamide 4 (4.0 g,
20 mmol) and sulfamide (0.97 g, 10 mmol) in dry p-dioxane (45 ml)
was refluxed with stirring until of NH3 had ceased (48 h). After
cooling to room temperature, the precipitate of side products
(z0.3e0.5 g) was filtered out together with charcoal added, and
the solvent was evaporated in reduced pressure. The oily residue
was triturated with water (70 ml) and precipitate was filtered off,
washed successively with water (5 ꢁ 5 ml), ethanol (3 ꢁ 1.5 ml) and
acetonitrile (3 ꢁ 3 ml), and dried. Yield 3.3 g (71.3%): m.p.
186e187 ꢂC; IR (KBr): 3400, 3325, 3270, 3250 (SO2NH2 and
HNeSO2eNH), 2953, 2865 (CH2CH2), 1600, 1560, 1450 (benzene
4.1.3.2. Compound 12. To
a
stirred suspension of 1-
methylpyridinium iodide 11 (1.02 g, 2 mmol) in water (10 ml)
was added dropwise a solution of 0.5% NaOH in water to pH 9.5. The
reaction mixture was stirred at room temperature for an additional
2 h. The precipitate was filtered off, washed with water (5 ꢁ 2 ml)
and dried at temperature gradually increasing to 110 ꢂC. Yield
0.67 g, (90.4%): m.p. 248e249 ꢂC; IR (KBr): 3355, 3310, 3285 (NH
and SO2NH2), 2945, 2925, 2855 (CH3 and CH2CH2), 2685 (SO2NH),
1645 (C]Nþ), 1350, 1315, 1155, 1150 (SO2), cmꢀ1; 1H NMR (DMSO-
ring), 1320, 1150 (SO2) cmꢀ1
; d 2.81 (t,
1H NMR (DMSO-d6)
J ¼ 7.0 Hz, 4H, 2x NHeCH2CH2ePh), 3.00 (q, J ¼ 7.0 Hz, 4H, 2ꢁ
NeCH2CH2ePh), 7.00 (t, J ¼ 5.9 Hz, 2H, 2ꢁ NHeCH2), 7.28 (s, 4H, 2ꢁ
SO2NH2), 7.39 (d, J ¼ 7.8 Hz, 4H, H-3, H-30, H-5 and H-50, PhSO2),
7.74 (d, J ¼ 7.8 Hz, 4H, H-2, H-20 and H-6, H-60, PhSO2) ppm; 13C
NMR (DMSO-d6, 50 MHz) 35.08, 43.47, 125.93, 129.46, 142.36,
143.74 ppm. Anal. (C16H22N4 O6S3) C, H, N.
d6)
d
2.95 (t, J ¼ 6.9 Hz, 2H, NeCH2CH2ePh), 3.40 (s, 1H, NH), 3.57 (t,
J ¼ 6.3 Hz, 2H, NeCH2CH2ePh), 3.77 (s, 3H, CH3Nþ), 6.77 (d,

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J. Sławinski et al. / European Journal of Medicinal Chemistry 84 (2014) 59e67
66
4.1.6. Procedure for the preparation of 2-[(4-sulfamoylphenyl)ethyl]
aminoacetamides (16e17)
complex. The inhibition constants were obtained by non-linear last-
squares methods using PRISM 3, as reported earlier [28e30], and
represent the mean from at least three different determinations.
The suspension of 4-(2-aminoethyl)benzenesulfonamide
4
(2.0 g, 10 mmol) and the appropriate N-aryl-chloroacetamide
(10 mmol) in tetrahydrofuran (50 ml) was stirred at room tem-
perature for 24 h. Then, a solution of triethylamine (1.21 g,
12 mmol) in tetrahydrofuran was added. The reaction mixture was
further stirred at room temperature for 24 h, followed at reflux for
18 h. After cooling to room temperature, the precipitate of trie-
thylamine hydrochloride was filtered off, washed with tetrahy-
drofuran (5 ml) and dried (yield 1.1 g, 80%). The filtrate was
evaporated under reduced pressure. To the resulting dry residue
water (40 ml) was added and slowly acidified to pH 2 with 1%
hydrochloric acid. After 3 h of stirring, the precipitate was collected
by filtration, washed with water (3 ꢁ 5 ml) and dried. The crude
product was treated with methanol (30 ml) and then refluxed with
stirring for 1 h and left to stand overnight. The precipitate was
filtered off, washed with methanol (3 ꢁ 1.5 ml) and dried. In this
manner the following aminoacetamides were obtained.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.074.
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2855 (CH2CH2), 1675 (O]CeN), 1585, 1525 (benzene rings), 1340,
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3.30 (s, 1H, NH), 3.40 (s, 2H, CH2eC]O), 7.30 (s, 2H, SO2NH2), 7.35
(d, J ¼ 8.9 Hz, 2H, H-2 and H-6, 4-ClPh), 7.42 (d, J ¼ 8.2 Hz, 2H, H-3
and H-5, PhSO2), 7.54 (d, J ¼ 8.9 Hz, 2H, H-3 and H-5, 4-ClPh), 7.78
(d, J ¼ 8.2 Hz, 2H, H-2 and H-6, PhSO2), 9.81 (s, 1H, HNeC]O) ppm.
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(2.48 g), the title compound 17 was obtained (3.4 g, 82.4%): m.p.
251e252 ꢂC dec.; IR (KBr): 3340, 3250, 3110, (NH and SO2NH2),
2930, 2870 (CH2CH2), 1685 (O]CeN), 1330, 1150 (SO2) cmꢀ1 1H
;
NMR (DMSO-d6) d 2.89e2.99 (m, 3H, HNeCH2CH2ePh), 3.34 (s, 2H,
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An Applied Photophysics (Oxford, UK) stopped-flow instrument
has been used for assaying the CA-catalyzed CO2 hydration activity
[24]. 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 hy-
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determining the initial velocity. The uncatalyzed rates were deter-
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J. Sławinski et al. / European Journal of Medicinal Chemistry 84 (2014) 59e67
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