Carbonic anhydrase inhibitors. Synthesis of a novel series of 5-substituted 2,4-dichlorobenzenesulfonamides and their inhibition of human cytosolic isozymes i and II and the transmembrane tumor-associated isozymes IX and XII

Article abstract of DOI:10.1016/j.ejmech.2014.05.039

A series of novel 5-substituted 2,4-dichlorobenzenesulfonamides 5a-c, 6a-d, 7a-j and 10a-i 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 tumo

Full text of DOI:10.1016/j.ejmech.2014.05.039

Accepted Manuscript
Carbonic anhydrase inhibitors. Synthesis of a novel series of 5-substituted 2,4-
dichlorobenzenesulfonamides and their inhibition of human cytosolic isozymes I and II
and the transmembrane tumor-associated isozymes IX and XII
Jarosław Sławiński , Aneta Pogorzelska , Beata Żołnowska , Kamil Brożewicz ,
Daniela Vullo , Claudiu T. Supuran
PII:
S0223-5234(14)00453-X
10.1016/j.ejmech.2014.05.039
EJMECH 6995
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 September 2013
Revised Date: 16 December 2013
Accepted Date: 8 May 2014
Please cite this article as: J. Sławiński, A. Pogorzelska, B. Żołnowska, K. Brożewicz, D. Vullo,
C.T. Supuran, Carbonic anhydrase inhibitors. Synthesis of a novel series of 5-substituted 2,4-
dichlorobenzenesulfonamides and their inhibition of human cytosolic isozymes I and II and the
transmembrane tumor-associated isozymes IX and XII, European Journal of Medicinal Chemistry
(2014), doi: 10.1016/j.ejmech.2014.05.039.
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Graphical Abstract
Carbonic anhydrase inhibitors. Synthesis of
a
novel series of 5-substituted 2,4-
dichlorobenzenesulfonamides and their inhibition of human cytosolic isozymes I and II and the
transmembrane tumor-associated isozymes IX and XII
Jarosław Sławiński*, Aneta Pogorzelska, Beata Żołnowska, Kamil Brożewicz, Daniela Vullo, Claudiu T.
Supuran
A series of novel 5-substituted 2,4-
dichlorobenzenesulfonamides have been
synthesized. Some of them showed good
carbonic anhydrase CA I, II and XII
inhibitory efficiency. The twenty one
new compounds displayed a powerful
inhibitory potency toward hCA IX.

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Original article
Carbonic anhydrase inhibitors. Synthesis of a novel series of 5-substituted
2,4-dichlorobenzenesulfonamides and their inhibition of human cytosolic
isozymes I and II and the transmembrane tumor-associated isozymes IX
and XII
Jarosław Sławiński a,*, Aneta Pogorzelska a, Beata Żołnowska a, Kamil Brożewicz a, Daniela
Vullo b, Claudiu T. Supuran b
aDepartment of Organic Chemistry, Medical University of Gdańsk, Al. Gen. J. Hallera 107, 80-416 Gdańsk, Poland
bDipartimento 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
BSTRACT
A series of novel 5-substituted 2,4-dichlorobenzenesulfonamides 5a-c, 6a-d, 7a-j and 10a-i
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
349 to 7355 nM, toward hCA II at range of 6.9-164 nM, while against hCA IX ranging from
2.8 to 76 nM and against hCA XII in the range of 2.7-95 nM. The excellent inhibitory
activity against tumor-associated hCA IX was found. The twenty one new compounds
displayed a powerful inhibitory potency toward hCA IX (KI = 2.8 - 21.7 nM) in comparison
with the clinically used CAIs AAZ, MZA, EZA, DCP and IND (24 - 50 nM). Among them
the most potent hCA IX inhibitor 7b (KI = 2.8 nM) was 8.5-fold stronger than IND (KI = 24
nM). Toward tumor-associated hCA XII compounds 6c and 10a (KI = 2.7 and 2.8 nM,
respectively) showed a better inhibitory potency than reference sulfonamides MZA and IND
(KI = 3.4 nM).
Keywords:
2,4-Dichlorobenzenesulfonamides; Synthesis; Carbonic anhydrase isozymes I, II, IX and XII
inhibitors
_________________
*Corresponding author. Tel.: +48 58 349 10 98; fax: +48 58 349 12 77.
E-mail address:jaroslaw@gumed.edu.pl (J. Sławiński).
1. Introduction
The innovative anticancer treatment strategies are based on the design of agents aimed
at the non-classical performance targets, such as proteins and enzymes of the process of
tumorigenesis, cell cycle regulators or oncogenes [1]. Carbonic anhydrases (CAs) are a family
of enzymes found in a diversity of organisms and primarily responsible for catalyze the
reversible hydratation of carbon dioxide. Currently at least fifteen humans CA isoforms
belonging to the α-CA class are known [2]. The CAs are involved in many physiological and

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pathological processes, including pH homeostasis, electrolyte secretion in various tissues and
organs, gluconeogenesis, lipogenesis, ureagenesis, bone resorption calcification and
tumorigenicity [2-12] making that the inhibitors of these proteins can be considered as a
therapeutic agents in prevention and treatment of various disease.
At the end of the last century, the new tumor-associated membrane carbonic anhydrase
isozymes CA IX and CA XII have been identified [13-15]. Since then the function of CA IX
in tumor physiology has been widely cognized [16-20]. This metalloenzyme is mainly
involved in the regulation of pH dynamics in solid tumors [20-21]. However, the CA IX
contributes to other cell processes essential for cancer such as adhesion, migration and
proliferation [22]. Moreover the CA IX is overexpressed in a broad spectrum of hypoxic
human tumors [23] and the relationship between CA IX expression and poor patient prognosis
in many kind of cancer is now well-established [22]. Because of the undeniable role of CA IX
in promote tumor cell survival and invasion, search for compounds selectively inhibit the CA
IX is an important point of the development of new anticancer therapies.
Since discovered that sulfanilamide inhibits the activity of CA [25] many sulfa drugs
were revealed as antiglaucoma agents [17, 26-28], anti-thyroid drugs [27], the hypoglicemic
sulfonamides [29] and, finally, novel types of anticancer agents [30]. It is believed that the
latter is due to the inhibition of tumor-associated CA IX and CA XII [24, 31].
The first reports about inhibition of CA IX applied for a series of aromatic and
heterocyclic sulfonamides including clinically used derivatives acetazolamide AAZ,
methazolamide MZA, ethoxzolamide EZA and dichlorophenamide DCP (Chart 1) [9].
Further showed that indisulam (E7070) IND, a novel sulfonamide anticancer agent in clinical
development for the treatment of solid tumors [32], act as a strong CA II and CA IX inhibitor
[33]. In the last decade many of inhibitors with strong selectivity toward tumor-associated
hCA IX have been detected (structures U-104, CAI17and I-III, Chart 1) [34-37]. The potency
of most of them compared to that of indisulam was several times higher. Moreover, it has
been shown that U-104 and CAI17 [36, 38-39] displayed in vivo significant inhibition of
tumor growth constituting an interesting candidates for the development of novel antitumor
agents. In addition it should be emphasized that this inhibitors have the ability to specifically
target CA IX-expressing tumors [38].
In our previous study we examined the inhibition of hCA I, II, IX and XII with some
S-substituted 4-chloro-2-mercapto-5- R- or 6-R1-benzenesulfonamides [40-42]. Some of those
compounds showed both strong acting as CAIs and the selectivity for the inhibition of the
tumor-associated over the cytosolic CA isoforms [40-42]. This findings prompted us to

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further investigation and therefore, we reported herein on the design, synthesis and
evaluations of inhibitory activity of novel class of 2,4-dichlorobenzensulfonamides
derivatives of type IV-VII which was modified in 5 position by moieties with a similar nature
as well-known CA IX inhibitors (Chart 1).
2. Results and discussion
2.1. Chemistry
The synthesis of the desired new compounds 5a-c, 6a-d, 7c-j and 10a-i were presented
at Schemes 1 and 2. Following the Scheme 1, semi- or thiosemicarbazides 5a-c were
obtained with good yield by the convenient one-step reaction of 2,4-dichloro-5-
sulfamoylbenzhydrazide 4 with appropriate isothio- or isocyanate in dry tetrahydrofuran
either at room temperature (5a, 5c) or at reflux (5b).
In turn, starting from 2,4-dichloro-5-sulfamoylbenzoyl chloride 2 and the
corresponding hydrazide the expected 2,4-dichloro-5-hydrazinecarbonylbenzenesulfonamides
6a-d were prepared in 65-75% yields through a simple addition-elimination reaction. The
reactions were carried out in dry tetrahydrofuran with the addition of 0.5-1 eq. triethylamine
at room temperature for 40-90 h.
The synthetic pathways of new 2,4-dichlorobenzenesulfonamides 7c-j, modified at the
position 5 by the azole-containing residues, were depicted in Scheme 1. As it was shown the
compounds 7c-j were synthesized according to four different methods (marked as f-g, i or j).
Conversion of 4 into the final 2,4-dichloro-5-(1,2,4-triazol-3-yl)benzenesulfonamides
7c-d with the appropriate ethyl benzimidiate hydrochloride (Method f) was carried out in
refluxing dry methanol in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).
The 5-(arylamino)-1,3,4-oxadiazole derivatives 7e-f were obtained as a result of
desulfurization of thiosemicarbazides 5a-b, respectively, with 4-toluenesulfonyl chloride in
the presence of pyridine (1.75 eq.) as a base (Method g). The excellent reaction yields (above
90%) can be explained by both, a selective and rapid sulfonation of thiocarbonyl group of the
5a-b, and the high reactivity of the resulting carbodiimide intermediate.
Alternatively, thionyl chloride (Method i) was used as the dehydrating agent required
to accomplish the dehydration of 5-(N'-benzoylhydrazinecarbonyl)benzenesulfonamide
derivatives 6b-d resulting in the formation of the desired 1,3,4-oxadiazole derivatives 7g-i.
In turn, the 1,3,4-thiadiazole derivative 7j was prepared by the reaction of
diacylhydrazine with the Lawesson's reagent (LR, Method j). Initially formed unstable

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derivative of 6b (i.e., mono- or dithiocarbonylhydrazine) was dehydrated or desulfurized to
form five-membered heterocyclic ring of 7j as shown in Scheme 1.
As shown in Scheme 2 the synthesis of 2,4-dichloro-5-ureidobenzenesulfonamides
10a-i was achieved by three-stage process starting from 2,4-dichloro-5-
sulfamoylbenzhydrazide 4. At first, treatment of hydrazide 4 with nitrous acid (NaNO2/HCl)
at -5 °C afforded the corresponding azide 8, which was converted to the isocyanate 9 by
Curtius rearrangement. Then, the isocyanate 9 was reacted with the appropriate primary or
secondary amines in refluxing dry toluene to furnish the desired urea derivatives 10a-i.
The structures of all new compounds were confirmed by IR and NMR data and
elemental analyses (see Experimental protocols).
2.2. CA inhibition studies
The compounds 5a-c, 6a-d, 7a-j and 10a-i as well as standard, clinically used CAIs,
such as acetazolamide AAZ, methazolamide 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 two transmembrane tumor-
associated isozymes: hCA IX and XII. The following structure-activity relationship (SAR)
can be drawn from CA inhibitory data of Table 1:
a) The investigated 2,4-dichlorobenzenesulfonamides 5a-c, 6a-d, 7a-j and 10a-i displayed
rather weak inhibitory properties against the slow cytosolic isoform hCA I with KI in the
range of 349-7355 nM . Thus, derivatives 5a-c and 10g-i showed weak inhibition of this
isoform, with KI in the range of 5430 - 7355 nM, being thus the weakest inhibitors among
tested compounds, including the clinically used compounds AAZ - IND (Table 1). The
compounds 7a-b, 7g-i and 10c had a slightly increased affinity to the hCA I and stand out
as the most active derivatives (KI of 349-718 nM) in comparison with the other tested 2,4
-dichlorobenzenesulfonamides (Table 1).
b) The hCA II inhibitory activity (KI in the range of 6.9-164 nM) was comparable to the
reference compounds AAZ - IND (Table 1). However, low KI values (8.7-29 nM)
observed for derivatives 7a-b and 7g-i suggest that the insertion the oxadiazole ring
system in meta position to the sulfamoyl moiety increase the inhibitory properties.
Similarly, the substitution of benzenesulfonamide by meta-hydrazinocarbonyl group in
the series 6a-d resulted in increased hCA II inhibitory activity (KI of 10.1-25 nM). In
addition, it should be emphasized, that compound 10c with KI = 6.9 was the best hCA II

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inhibitor even in comparison with EZA (KI = 8 nM) with the highest inhibitory activity in
the reference group.
c) An excellent inhibition profile of the isoform hCA IX was found for novel 2,4-
dichlorobenzenesulfonamides. All compounds, with the exception of 10i, inhibited the
activity of this isozyme similar or better (KI of 2.8-31 nM) than the references AAZ -
IND (KI of 24-50 nM). The best inhibitory properties (KI values in the range of 2.8-15.1
nM) was observed for derivatives of 6 and 7 series possessing hydrazinocarbonyl group
(6a-d), oxa- or thiadiazoles (7a-b, 7e-i) and triazoles (7c-d) moieties in meta position of
benzenesulfonamide system. It should be pointed out that the twenty one new 2,4-
dichlorobenzenesulfonamides were characterized by higher activity compared with IND,
the most effective clinically used hCA IX inhibitor (see Table 1). Moreover, the most
potent hCA IX inhibitor in the tested series was 2,4-dichloro-5-(5-methyl-1,3,4-
oxadiazol-2-yl)benzenesulfonamide (7b, KI = 2.8 nM) being thus 8.5-fold stronger than
IND (KI = 24 nM).
d) Similar results were obtained for the second tumor-associated isoform hCA XII. The
inhibitory properties of tested compounds were comparable with the references AAZ -
IND with the exception of 10i, with the higher inhibition constant KI = 95 nM. As in the
case of the hCA IX, the good hCA XII inhibitory activity was observed mainly for the
compounds of series 6 and 7 with KI in the range of 2.7-13.6 nM. The most potent
inhibitor 6c with KI = 2.7 nM was characterized by better hCA XII inhibition in
comparison with clinically used AAZ - IND. However, the most promising inhibitory
properties was found for 2,4-dichloro-5-[3-(4-R-phenyl)ureido]benzenesulfonamides
10a-c (R = H, Cl, SO2NH2) with KI: 2.8-3.6 nM. Moreover, compound 10a exerted the
highest selectivity toward hCA XII versus hCA II (hCA II / hCA XII = 15.7). Relatively
good selectivity ratios towards isoform hCA XII and hCA II displayed also compound 7j
(hCA II / hCA XII = 12.6).
e) It should be noted that the presence of 4-R-phenyl group (R = H, Cl, SO2NH2) attached
directly to the nitrogen atom N-3 of the urea moiety in the series 10a-i resulted in
increase of inhibitory potency against all tested CA isoforms. The rather significant
decrease of inhibition of hCA I, II, IX and XII was found for 10h-i possessing either 3-
(3-morpholin-4-ylpropyl)ureido moiety (10h) or 4-(benzodioxol-5-ylmethyl)piperazino-
1-carboxamide substituent (10i) in meta position to the sulfamoyl group. Moreover, the
relatively low activity against isoforms hCA I, II, IX and XII exhibited
thiosemicarbazides 5a-b (see Table 1).

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3. Conclusions
We have developed methods for the preparation of novel series of 5-substituted 2,4-
dichlorobenzenesulfonamides (thio- or semicarbazides, hydrazinocarbonyls, oxa- or
thiadiazoles, triazoles and ureas). The 26 new sulfonamides have been assayed for the
inhibition of four physiologically relevant CA isozymes, such as CA I and II, the tumor-
associated isozymes CA IX and XII. A weak inhibitory activity against the human CA I was
observed for all investigated compounds with KI values from 349 to 7355 nM. However, in
the case of the second physiological isoform hCA II the inhibitory activity of most of the
tested benzenesulfonamides was comparable with reference compounds and their KI values
were in the range from 6.9 to 164 nM. Likewise, the inhibition of tumor-associated hCA XII
by the tested benzenesulfonamides (KI = 2.7 - 95 nM) was quite similar to the clinically used
CA inhibitors AAZ - IND. It should be noted, however, that compounds 6c and 10a, with KI
= 2.7 and 2.8 nM, respectively, exhibited a better inhibitory potency than the best inhibitors in
the references, MZA and IND (KI = 3.4 nM). Moreover, compound 10a exerted the highest
selectivity ratios toward hCA XII versus hCA II (hCA II/hCA XII = 15.7). On the other hand,
the excellent inhibitory activity against hCA IX should be stressed; this isoform was inhibited
with KI values from 2.8 to 76 nM. The twenty one new compounds displayed a powerful
inhibitory potency toward hCA IX (KI = 2.8 - 21.7 nM) in comparison with the clinically used
CA inhibitors AAZ - IND (24 - 50 nM). Among them the most potent inhibitor 7b (KI = 2.8
nM) was 8.5-fold stronger than IND (KI = 24 nM). The present studies indicate that the
currently synthesized 2,4-dichloro derivatives with bulky and highly functionalized
substituents at the 5 position of the benzenesulfonamide scaffold display excellent inhibition
profile of the isoforms hCA IX and XII, similar or much better in comparison to the 2-
mercapto-5- R- or 6-R1-benzenesulfonamide analogs [40-42].
4. Experimental protocols
4.1. Synthesis
Melting points were determined with a Boethius PHMK apparatus. Infrared (IR)
spectra were recorded with a Thermo Mattson Satellite FTIR spectrophotometer. 1H and 13C
nuclear magnetic resonance (NMR) experiments were carried out on a Varian Unity Plus 500

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MHz or 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 mass spectra
were acquired on a Bruker Biflex III MALDI-TOF spectrometer after deposition on a 2,5-
dihydroxybenzoic acid (DHB) matrix. The results of elemental analyses for C, H and N were
in agreement with the theoretical values within ±0.4% range. The starting 2,4-dichloro-5-
sulfamoylbenzoyl chloride 2 and 2,4-dichloro-5-sulfamoylbenzhydrazide 4 were obtained
from commercially available 2,4-dichloro-5-sulfamoylbenzoic acid 1 according to methods
described previously [43,44]. 2,4-Dichloro-5-(1,3,4-oxadiazol-2-yl)benzenesulfonamide 7a
and 2,4-dichloro-5-(5-methyl-1,3,4-oxadiazol-2-yl)benzenesulfonamide 7b were obtained
from 4 by known method [45].
4.1.1. Procedures for the preparation of N-(2,4-dichloro-5-sulfamoylbenzoyl)-
thiosemicarbazides (5a-b)
To a suspension of 4 (1 mmol) in dry tetrahydrofuran (5 ml) corresponding
isothiocyanate (1.05 mmol) was added dropwise. The reaction mixture was stirred at room
temperature for 1.5 h (5a) or heated at reflux for 3 h (5b). The desired products were filtered
off and dried.
4.1.1.1. N-(2,4-dichloro-5-sulfamoylbenzoyl)-N"-(phenyl)thiosemicarbazide (5a). Starting
from 4 (1.0 g) and phenyl isothiocyanate (0.5 g) the desired 5a was obtained (1.065 g, 72%):
m.p. 193-195 °C; IR (KBr) 3318 (NH2, NH), 1690 (C=O), 1587, 1561, 1496 (C=C), 1324,
1164 (SO2) cm-1; 1H NMR (500 MHz, DMSO-d6) δ 7.19-7.21 (m, 1H, H arom.), 7.36-7.39
(m, 2H, H arom.), 7.48 (s, 2H, NH2), 7.82 (s, 2H, H arom.), 7.99 (m, 1H, H-3), 8.31 (s, 1H, H-
6), 9.89 (s, 2H, 2NH), 10.71 (s, 1H, NH) ppm; MALDI-TOF m/z obsd: 420.9, [M + H]+,
442.9, [M + Na]+ calcd: 419.9; Anal. (C14H12Cl2N4O3S2) C, H, N.
4.1.1.2. N-(2,4-dichloro-5-sulfamoylbenzoyl)-N"-(4-chlorophenyl)thiosemicarbazide (5b).
Starting from 4 (1.0 g) and 4-chlorophenyl isothiocyanate (0.6 g) the desired 5b was obtained
(1.053 g, 66%): m.p. 203-205 °C; IR (KBr) 3319 (NH2, NH), 1695 (C=O), 1552, 1493 (C=C),
1321, 1164 (SO2) cm-1; 1H NMR (500 MHz, DMSO-d6) δ 7.43(d, J = 8.79 Hz, 2H, H arom.),
7.51 (s, 2H, NH2), 7.77-7.83 (m, 2H, H arom.), 7.99 (m, 1H, H-3), 8.34 (s, 1H, H-6), 10 (s,
2H, 2NH), 10.73 (s, 1H, NH) ppm; 13C NMR (DMSO-d6) δ 128.31, 128.49, 130.33, 132.45,
132.97, 133.51, 135.22, 138.38, 139.97, 164.19, 164.23. Anal. (C14H11Cl3N4O3S2) C, H, N.

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4.1.2. Preparation of N-(2,4-dichloro-5-sulfamoylbenzoyl)-N"-tosylsemicarbazide (5c)
To a suspension of 4 (0.5 g) in dry tetrahydrofuran (8 ml) p-toluenesulfonyl isocyanate
(0.5 g) was added. The reaction mixture was stirred at room temperature for 0.5 h and then
evaporated under reduced pressure. 2% Hydrochloric acid solution (10 ml) and methanol (10
ml) were added, the mixture was cooled and the precipitation of N-(2,4-dichloro-5-
sulfamoylbenzoyl)-N"-tosylsemicarbazide 5c was collected by filtration, washed with water
and dried (0.7 g, 81%): m.p. 214-216 °C; IR (KBr) 3320 (NH2, NH), 1697, 1673 (C=O),
1582, 1547, 1505, 1452 (C=C), 1353, 1168 (SO2) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 2.4
(s, 3H, CH3), 7.42 (d, J = 8.05 Hz, 2H, H arom.), 7.77-7.83 (m, 5H, NH2, H arom.), 7.97 (s,
1H, H-6), 8.84 (s, 1H, NH), 10.39 (s, 1H, NH), 11.2 (s, 1H, SO2NH) ppm; MALDI-TOF m/z
obsd: 482.8, [M + H]+ calcd: 481.9; Anal. (C15H14Cl2N4O6S2) C, H, N.
4.1.3. Procedures for the preparation of 2,4-dichloro-5-
hydrazinecarbonylbenzenesulfonamides (6a-d)
To a stirred suspension of the appropriate R-hydrazide (1 eq.) in dry tetrahydrofuran,
triethylamine (0.5-1 eq.) was added and the mixture was cooled to 5 °C. A solution of 2 (1
eq.) in dry tetrahydrofuran was added dropwise and the reaction mixture was stirred at room
temperature for 40-90 h. The solvents were evaporated under reduced pressure and the
products 6a-d were isolated as described below.
4.1.3.1. 2,4-Dichloro-5-[(2-(furan-2-carbonyl)hydrazinecarbonyl]benzenesulfonamide (6a).
Starting from furane-2-carbohydrazide (1.261 g, 10 mmol) in THF (20 ml), Et3N (0.29 g, 5
mmol) and 2 (1.443 g, 5 mmol) in THF (20 ml), the crude 6a (1.486 g) was obtained through
the treatment of evaporated mixture by 18% hydrochloric acid (20 ml) and filtration of the
precipitation. The crude 6a was crystallized from methanol/water (1/9) and the title compound
6a (1.323 g, 70%) was isolated: m.p. 218 – 219 °C; IR (KBr) 3385, 3302 (NH2), 3242
(NHNH), 1710 (C=O), 1686 (C=O), 1350, 1167 (SO2) cm-1; 1H NMR (200 MHz, DMSO-d6)
δ 6.67-6.7 (m, 1H, H-furyl-4), 7.27-7.29 (m, 1H, H-furyl-3), 7.88 (s, 2H, NH2), 7.93-7.95 (m,
1H, H-furyl-5), 8.01 (s, 1H, H-3), 8.05 (s, 1H, H-6), 10.61 (s, 1H, NH), 10.64 (s, 1H, NH)
ppm; MALDI-TOF m/z obsd: 379.8, [M + H]+, 401.8, [M + Na]+ calcd: 378.9; Anal.
(C12H9Cl2N3O5S) C, H, N.

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4.1.3.2. 2,4-Dichloro-5-(2-benzoylhydrazinecarbonyl)benzenesulfonamide (6b). Starting from
benzhydrazide (14.9 g, 0.11 mol) in THF (40 ml), Et3N (6.5 g, 0.11 mol) and 2 (15.25 g,
0.055 mol) in THF (40 ml), the crude 6b was obtained through the treatment of evaporated
mixture by water, acidified with 18% hydrochloric acid and filtration of the precipitation. The
crude 6b washed with 10% aqueous solution of sodium bicarbonate (3×20 ml), ethanol and
crystallized from DMF/water (1/1) and the title compound 6b (15.35 g, 75%) was isolated:
m.p. 279 – 281 °C; IR (KBr) 3390, 3291 (NH2), 3182 (NHNH), 1674 (C=O), 1608 (C=O),
1358, 1164 (SO2) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 7.49-7.65 (m, 3H, H arom.), 7.9-
7.95 (m, 4H, H arom., NH2), 8.02 (s, 1H, H-3), 8.11 (s, 1H, H-6), 10.69 (s, 1H, NH), 10.72 (s,
1H, NH) ppm; 13C NMR (DMSO-d6) δ 127.79, 128.82, 129.62, 132.31, 132.43, 132.67,
132.97, 133.93, 134.89, 140.22, 164.32, 165.78. Anal. (C14H11Cl2N3O4S) C, H, N.
4.1.3.3. 2,4-Dichloro-5-[2-(4-chlorobenzoyl)hydrazinecarbonyl]benzenesulfonamide (6c).
Starting from 4-chlorobenzhydrazide (1.5 g, 8.8 mmol) in THF (15 ml), Et3N (0.26 g, 4.4
mmol) and 2 (1.27 g, 4.4 mmol) in THF (15 ml), the crude 6c was obtained through the
treatment of evaporated mixture by water and filtration of the precipitation. The crude 6c
crystallized from ethanol/water (7/3) and the title compound 6c (1.209 g, 65%) was isolated:
m.p. 287 – 290 °C; IR (KBr) 3355, 3251 (NH2), 3183 (NHNH), 1687 (C=O), 1655 (C=O),
1321, 1165 (SO2) cm-1; 1H NMR (500 MHz, DMSO-d6) δ 7.62 (d, J = 8.3 Hz, 2H, H arom.),
7.89 (s, 2H, NH2), 7.95 (d, J = 8.79 Hz, 2H, H arom.), 8.03 (s, 1H, H-3), 8.09 (s, 1H, H-6),
10.72 (s, 1H, NH), 10.82 (s, 1H, NH) ppm; MALDI-TOF m/z obsd: 423.8, [M + H]+, 446.8,
[M + Na]+ calcd: 422.9; Anal. (C14H10Cl3N3O4S) C, H, N.
4.1.3.4. 2,4-Dichloro-5-2'-(4-methylbenzoyl)hydrazinecarbonyl]benzenesulfonamide (6d).
Starting from 4-methylbenzhydrazide (1.501 g, 10 mmol) in THF (30 ml), Et3N (0.29 g, 5
mmol) and 2 (1.443 g, 5 mmol) in THF (20 ml), the crude 6d was obtained through the
treatment of evaporated mixture by water and filtration of the precipitation. The crude 6d
crystallized from methanol/water (4/3) and the title compound 6d (1.45 g, 72%) was isolated:
m.p. 272 – 274 °C; IR (KBr) 3346, 3186 (NH2), 1682 (C=O), 1647 (C=O), 1325, 1164 (SO2)
cm-1; 1H NMR (200 MHz, DMSO-d6) δ 2.38 (s, 3H, CH3), 7.33 (d, J = 8.35 Hz, 2H, H arom.),
7.845 (d, J = 8.26 Hz, 2H, H arom.), 7.89 (s, 2H, NH2), 8.02 (s, 1H, H-3), 8.1 (s, 1H, H-6),
10.64 (s, 2H, NHNH) ppm. Anal. (C15H13Cl2N3O4S) C, H, N.

ACCEPTED MANUSCRIPT
4.1.4. Procedures for the preparation of 2,4-dichloro-5-(5-aryl-1,2,4-triazol-3-
yl)benzenesulfonamides (7c-d)
To a suspension of 4 (1.76 mmol) in dry methanol (10 ml) corresponding ethyl
benzimidate hydrochloride (1.9 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 3.52
mmol) were added. The reaction mixture was stirred and heated at reflux for 22-24 h. The
solvents were evaporated under reduced pressure and the residue was dissolved in
dichloromethane and acidified with 2% hydrochloric acid (7c) or glacial acetic acid (7d). The
precipitation of appropriate 2,4-dichloro-5-(5-aryl-1,2,4-triazol-3-yl)benzenesulfonamide was
filtered off and dried.
4.1.4.1. 2,4-Dichloro-5-(5-phenyl-1,2,4-triazol-3-yl)benzenesulfonamide (7c).
Starting from 4 (0.5 g) and ethyl benzimidate hydrochloride (0.35 g) the 7c was obtained
(0.36 g, 53%): m.p. 301-303 °C; IR (KBr) 3339, 3249 (NH2), 3183 (NH), 3096, 3012, 2930
(C-H sp2, sp3), 1542, 1468 (C=C, C=N), 1349, 1171 (SO2) cm-1; 1H NMR (200 MHz, DMSO-
d6) δ 7.55-7.58 (m, 3H, H arom.), 7.85 (s, 2H, NH2), 8.03-8.09 (m, 3H, H arom.), 8.6 (s, 1H,
H-6), 14.82 (s, 1H, NH) ppm. Anal. (C14H10Cl2N4O2S) C, H, N.
4.1.4.2. 2,4-Dichloro-5-[5-(4-chlorophenyl)-1,2,4-triazol-3-yl]benzenesulfonamide (7d).
Starting from 4 (0.5 g) and ethyl 4-chlorobenzimidate hydrochloride (0.43 g) the 7d was
obtained (0.36 g, 51%): m.p. 288-290 °C; IR (KBr) 3380, 3273 (NH2), 3092, 2924 (C-H sp2,
sp3), 1543, 1482 (C=C, C=N), 1346, 1174 (SO2) cm-1; 1H NMR (500 MHz, DMSO-d6) δ 7.66
(m, 2H, H arom.), 7.87 (s, 2H, NH2), 8.07 (m, 2H, H arom.), 8.6 (s, 1H, H-6), 14.4 (s, 1H,
NH) ppm; 13C NMR (DMSO-d6) δ 127.58, 128.14, 128.75, 129.43, 131.21, 131.54, 133.33,
134.76, 135.35, 140.32, 156.41, 159.99. Anal. (C14H9Cl3N4O2S) C, H, N.
4.1.5. Procedures for the preparation of 2,4-dichloro-5-[5-(arylamino)-1,3,4-oxadiazol-2-
yl]benzenesulfonamides (7e-f)
To a suspension of 5a-b (1 mmol) in dry tetrahydrofuran (5 ml) 4-toluenesulfonyl
chloride (1.2 mmol) and dry pyridine (2.1 mmol) were added and heated at reflux for 3-8 h.
The solvents were evaporated under reduced pressure and the residue was treated with ethanol
(5-10 ml), stirred and cooled for 30 min. The precipitation of adequate 2,4-dichloro-5-[5-

ACCEPTED MANUSCRIPT
(arylamino)-1,3,4-oxadiazol-2-yl]benzenesulfonamides 7e-f was collected by filtration,
washed with water and ethanol and dried.
4.1.5.1. 2,4-Dichloro-5-[5-(phenylamino)-1,3,4-oxadiazol-2-yl]benzenesulfonamide (7e).
Starting from 5a (0.42 g) the desired compound 7e was obtained (0.37 g, 96%): m.p. 280-281
°C; IR (KBr) 3371, 3265 (NH, NH2), 3092, 2926, 2855 (C-H sp2, sp3), 1680 (N-H), 1602,
1590, 1556 (C=C, C=N), 1343, 1163 (SO2) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 7.04-7.07
(m, 1H, H arom.), 7.38-7.41 (m, 2H, H arom.), 7.63-7.64 (m, 2H, H arom.), 7.93 (s, 2H,
NH2), 8.14 (s, 1H, H-3), 8.5 (s, 1H, H-6), 10.89 (s, 1H, NH) ppm. Anal. (C14H10Cl2N4O3S) C,
H, N.
4.1.5.2. 2,4-Dichloro-5-[5-(4-chlorophenylamino)-1,3,4-oxadiazol-2-yl]benzenesulfonamide
(7f). Starting from 5b (0.45 g) the desired compound 7f was obtained (0.37 g, 92%): m.p.
279-281 °C; IR (KBr) 3300 (NH, NH2), 3099, 2928 (C-H sp2, sp3), 1648 (N-H), 1624, 1586,
1556 (C=C, C=N), 1337, 1163 (SO2) cm-1; 1H NMR (500 MHz, DMSO-d6) δ 7.44 (d, J = 8.79
Hz, 2H, H arom.), 7.63 (d, J = 8.79 Hz, 2H, H arom.), 7.92 (s, 2H, NH2), 8.12 (s, 1H, H-3),
8.47 (s, 1H, H-6), 11.06 (s, 1H, NH) ppm; 13C NMR (DMSO-d6) δ 119.06, 122.04, 126.10,
129.28, 130.11, 133.29, 134.02, 135.06, 137.59, 140.66, 154.88, 160.28. Anal.
(C14H9Cl3N4O3S) C, H, N.
4.1.6. Procedures for the preparation of 2,4-dichloro-5-(5-aryl-1,3,4-oxadiazol-2-
yl)benzenesulfonamides (7g-i)
The required 2,4-dichloro-5-hydrazinecarbonylbenzenesulfonamides 6b-d (2.5 mmol)
were heating with thionyl chloride (25 ml) at reflux for 6-7 h. After evaporation the crushed
ice was added and the precipitated desired compounds 7g-i were filtered off and crystallized.
4.1.6.1. 2,4-Dichloro-5-(5-phenyl-1,3,4-oxadiazol-2-yl)benzenesulfonamide (7g). Starting
from 6b (0.971 g), the title compound 7g was obtained (0.461 g, 56%) and crystallized from
methanol/water (2/1) : m.p. 216-218 °C; IR (KBr) 3382, 3280 (NH2), 1589, 1550 (C=C,
C=N), 1357, 1172 (SO2) cm-1; 1H NMR (500 MHz, DMSO-d6) δ 7.63-7.69 (m, 3H, H arom.),
7.98 (s, 2H, NH2), 8.09. (d, J = 6.84 Hz, 2H, H arom.), 8.19 (s, 1H, H-3), 8.66 (s, 1H, H-6)
ppm; 13C NMR (DMSO-d6) δ 121.84, 123.19, 127.09, 129.84, 130.93, 132.71, 134.15,
134.41, 135.85, 140.86, 161.24, 164.89 ppm. Anal. (C14H9Cl2N3O3S) C, H, N.

ACCEPTED MANUSCRIPT
4.1.6.2. 2,4-Dichloro-5-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl]benzenesulfonamide (7h).
Starting from 6c (0.634 g), the title compound 7h was obtained (0.254 g, 42%) and
crystallized from ethanol: m.p. 240-241 °C; IR (KBr) 3369, 3261 (NH2), 1589, 1542, 1483
(C=C, C=N), 1343, 1172 (SO2) cm-1; 1H NMR (500 MHz, DMSO-d6) δ 7.74 (d, J = 8.79 Hz,
2H, H arom.), 7.99 (s, 2H, NH2), 8.12 (d, J = 8,3 Hz, 2H, H arom.), 8.21 (s, 1H, H-3), 8.67 (s,
1H, H-6) ppm; 13C NMR (DMSO-d6) δ 121.72, 122.10, 128.89, 130.03, 130.95, 134.18,
134.49, 135.87, 137.46, 140.87, 161. 37, 164.14. Anal. (C14H8Cl3N3O3S) C, H, N.
4.1.6.3. 2,4-Dichloro-5-[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]benzenesulfonamide (7i).
Starting from 6d (1.0 g), the title compound 7i was obtained (0.851 g, 42%) and crystallized
from methanol: m.p. 194-196 °C; IR (KBr) 3385, 3288 (NH2), 3088, 2923 (C-H sp2, sp3),
1585, 1556, 1496 (C=C, C=N), 1357, 1172 (SO2) cm-1; 1H NMR (200 MHz, DMSO-d6) δ
2.41 (s, 3H, CH3), 7.45 (d, J = 8,06 Hz, 2H, H arom.), 7.96-7.99 (m, 4H, H arom., NH2), 8.17
(s, 1H, H-3), 8.65 (s, 1H, H-6) ppm; MALDI-TOF m/z obsd: 385.9, [M + H]+, 407.8, [M +
Na]+ calcd: 384.9; Anal. (C15H11Cl2N3O3S) C, H, N.
4.1.7. 2,4-Dichloro-5-(5-phenyl-1,3,4-thiadiazol-2-yl)benzenesulfonamide (7j).
The 6b (0.505 g, 1.3 mmol) was dissolved in dry tetrahydrofuran (15 ml), Lawesson's
reagent (LR, 0.526 g) was added and stirred at room temperature for 24 h. The solvent was
evaporated under reduced pressure, residue was washed with water and petroleum ether,
treated with tetrahydrofuran and evaporated again. Ethanol (20 ml) was added and heated at
reflux for 5 min., after cooling to room temperature the precipitate of 7j (0.273 g, 54%) was
filtered off and recrystallized from methanol/DMF (4/1): m.p. 231-233 °C; IR (KBr) 3294,
3171 (NH2), 3091, 3071, 2925 (C-H sp2, sp3), 1573, 1536, 1458 (C=C, C=N), 1362, 1174
(SO2) cm-1; 1H NMR (500 MHz, DMSO-d6) δ 7.58-7.62 (m, 3H, H arom.), 7.96 (s, 2H, NH2),
8.08 (d, J = 7.32 Hz, 2H, H arom.), 8.19 (s, 1H, H-3), 8.84 (s, 1H, H-6) ppm; MALDI-TOF
m/z obsd: 387.8, [M + H]+, 409.8, [M + Na]+ calcd: 386.9; Anal. (C14H9Cl2N3O2S2) C, H, N.
4.1.8. 2,4-Dichloro-5-sulfamoylbenzoyl azide (8)
The 4 (2.0 g, 7.04 mmol) was dissolved in 2% hydrochloric acid (26 ml). The reaction
mixture was cooled and 3.8 M aqueous solution of sodium nitrite (2 ml) was added dropwise.

ACCEPTED MANUSCRIPT
The precipitation of 2,4-dichloro-5-sulfamoylbenzoyl azide 8 was filtered off and washed
with icy water (1.53 g, 74%): m.p. 127-130 °C; IR (KBr) 3385, 3276 (NH2), 2145 (N3), 1704
(C=O), 1337, 1170 (SO2) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 7.91 (s, 2H, NH2), 8.08 (s,
1H, H-3), 8.86 (s, 1H, H-6) ppm. Anal. (C7H4Cl2N4O3S) C, H, N.
4.1.9. 2,4-Dichloro-5-isocyanatobenzenesulfonamide (9)
The 8 (1.0 g, 3.4 mmol) was dissolved in dry toluene (40 ml) and the reaction
mixture was heated at reflux until the nitrogen bubbles had disappeared (about 1 h). After
cooling the precipitation of 2,4-dichloro-5-isocyanatobenzenesulfonamide 9 was filtered off
(0.786 g, 87%): m.p. 143-145 °C; IR (KBr) 3350, 3274 (NH2), 2267 (NCO), 1349, 1177
(SO2) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 7.72 (s, 2H, NH2), 7.88 (s, 1H, H-3), 8.88 (s,
1H, H-6) ppm. Anal. (C7H4Cl2N2O3S) C, H, N.
4.1.10. Preparation of 2,4-dichloro-5-ureidobenzenesulfonamides (10a-i)
To the stirring solution of isocyanate 9 (0.75 mmol) in dry toluene (10 ml) the
adequate amine (0.75 mmol) was added. The reaction mixture was heated at reflux for 0.5-6
h. After cooling, the precipitation of desired 2,4-dichloro-5-ureidobenzenesulfonamide was
filtered off and crystallized, with the exception of compounds 10b and 10e-f which were
isolated as described below.
4.1.10.1. 2,4-Dichloro-5-(3-phenylureido)benzenesulfonamide (10a). Starting from 9 (0.2 g)
and aniline (0.071 g) the title compound 10a was obtained (0.2 g, 73%): m.p. 232-235 °C; IR
(KBr) 3377 (NH, NH2), 1699 (C=O), 1373, 1162 (SO2) cm-1; 1H NMR (200 MHz, DMSO-d6)
δ 7.1 (m, 1H, H arom.), 7.27-7.35 (m, 2H, H arom.), 7.16-7.2 (m, 2H, H arom.), 7.71 (s, 2H,
NH2), 7.81 (s, 1H, H-3), 8.55 (s, 1H, NH), 8.98 (s, 1H, H-6), 9.53 (s, 1H, NH) ppm; 13C
NMR (DMSO-d6) δ 118.63, 120.75, 122.77, 123.09, 125.16, 129.22, 131.51, 135.65, 139.29,
140.4, 152.07 ppm; MALDI-TOF m/z obsd: 391.9, [M + H]+, 413.8, [M + Na]+ calcd: 391.0;
Anal. (C13H11Cl2N3O3S) C, H, N.
4.1.10.2. 2,4-Dichloro-5-[3-(4-chlorophenyl)ureido]benzenesulfonamide (10b). Starting from
9 (0.2 g) and p-chloroaniline (0.1 g) the title compound 10b was obtained by evaporating of
solvents, heating the residue with 50% methanol and filtration the precipitation of desired

ACCEPTED MANUSCRIPT
compound (0.21 g, 71%): m.p. 242-245 °C; IR (KBr) 3337, 3259 (NH, NH2), 1700 (C=O),
1371, 1160 (SO2) cm-1; 1H NMR (500 MHz, DMSO-d6) δ 7.35 (d, J = 8.79 Hz, 2H, H arom.),
7.49 (d, J = 8.79 Hz, 2H, H arom.), 7.7 (s, 2H, NH2), 7.84 (s, 1H, H-3), 8.6 (s, 1H, NH), 8.94
(s, 1H, H-6), 9.63 (s, 1H, NH) ppm; MALDI-TOF m/z obsd: 495.8, [M + H]+, 417.8, [M +
Na]+ calcd: 394.9; Anal. (C13H10Cl3N3O3S) C, H, N.
4.1.10.3. 2,4-Dichloro-5-[3-(4-sulfamoylphenyl)ureido]benzenesulfonamide (10c). Starting
from 9 (0.2 g) and sulfanilamide (0.13 g) the title compound 10c was obtained and
crystallized from 70% DMF (0.15 g, 48%): m.p. 261-264 °C; IR (KBr) 3363, 3279 (NH,
NH2), 1703 (C=O), 1319, 1161 (SO2) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 7.25 (s, 2H,
NH2), 7.61-7.66 (m, 2H, H arom.), 7.71 (s, 2H, NH2), 7.75-7.79 (m, 2H, H arom.), 7.87 (s,
1H, H-3), 8.7 (s, 1H, NH), 8.97 (s, 1H, H-6), 9.88 (s, 1H, NH) ppm. Anal. (C13H12Cl2N4O5S2)
C, H, N.
4.1.10.3. 5-(3-Benzylureido)-2,4-dichlorobenzenesulfonamide (10d). Starting from 9 (0.2 g)
and benzylamine (0.078 g) the title compound 10d was obtained (0.11 g, 40%): m.p. 178-180
°C; IR (KBr) 3398 (NH, NH2), 1684 (C=O), 1371, 1161 (SO2) cm-1; 1H NMR (500 MHz,
DMSO-d6) δ 4.31 (d, J = 5.37 Hz 2H, CH2), 7.24-7.27 (m, 1H, H arom.), 7.31-7.36 (m, 4H,
H arom.), 7.59 (t, J = 5.37 Hz, 1H, NH), 7.64 (s, 2H, NH2), 7.77 (s, 1H, H-3), 8.4 (s, 1H, NH),
8.98 (s, 1H, H-6) ppm; 13C NMR (DMSO-d6) δ 43.16, 120.31, 122.27, 124.39, 127.24,
127.58, 128.70, 131.35, 136.28, 139.80, 140.32, 154.64. Anal. (C14H13Cl2N3O3S) C, H, N.
4.1.10.5. 5-[3-(Benzyloxy)ureido]-2,4-dichlorobenzenesulfonamide (10e). Starting from 9 (0.2
g) and O-benzylhydroxylamine hydrochloride (0.12 g) with addition of dry pyridine (0.06
ml) the title compound 10e was obtained by evaporating the solvent and precipitating with
water (0.14 g, 48%): m.p. 150-153 °C; IR (KBr) 3382, 3280 (NH, NH2), 1680 (C=O), 1374,
1163 (SO2) cm-1; 1H NMR (500 MHz, DMSO-d6) δ 4.88 (s, 2H, OCH2), 7.36-7.49 (m, 5H, H
arom.), 7.73 (s, 2H, NH2), 7.88 (s, 1H, H-3), 8.4 (s, 1H, NH), 8.71 (s, 1H, H-6), 10.15 (s, 1H,
ONH) ppm. Anal. (C14H13Cl2N3O4S) C, H, N.
4.1.10.6. 5-(2-(Benzoylhydrazinecarbonyl)-2,4-dichlorobenzenesulfonamide (10f). Starting
from 9 (0.2 g) and benzohydrazide (0.1 g) the title compound 10f was obtained by
evaporating the solvent under reduced pressure, heating the residue with 50% methanol and
filtration the desired compound (0.15 g, 50%): m.p. 231-233 °C; IR (KBr) 3357, 3251 (NH,

ACCEPTED MANUSCRIPT
NH2), 1708 (C=O), 1310, 1160 (SO2) cm-1; 1H NMR (500 MHz, DMSO-d6) δ 7.49-7.52 (m,
2H, H arom.), 7.57-7.6 (m, 1H, H arom.), 7.69 (s, 2H, NH2), 7.85 (s, H, H-3), 7.89 (d, J =
7.81 Hz, 2H, H arom.), 8.65 (s, 1H, NH), 8.826 (s, 1H, NH), 9.0 (s, 1H, H-6), 10.45 (s, 1H,
NH) ppm, 13C NMR (DMSO-d6) δ 121.23, 123.59, 127.76, 128.76, 131.61, 132.23, 132.54,
135.45, 140.42, 155.06, 166.65 ppm. Anal. (C14H12Cl2N4O4S) C, H, N.
4.1.10.7. 5-[3-(1H-Indol-1-yl)ureido]-2,4-dichlorobenzenesulfonamide (10g). Starting from 9
(0.15 g) and 1-aminoindole (0.074 g) the title compound 10g was obtained and crystallized
from ethanol (0.075 g, 33%): m.p. 243-244 °C; IR (KBr) 3368, 3317, 3270 (NH, NH2), 1662
(C=O), 1372, 1165 (SO2) cm-1; 1H NMR (200 MHz, DMSO-d6) δ 6.49 (d, 1H, J = 3.24 Hz,
H-3 indole), 7.05-7.23 (m, 2H, H arom.), 7.33 (d, 1H, J = 7.76 Hz, H arom.), 7.41 (d, 1H, J =
3.24 Hz, H-2 indole), 7.58 (d, 1H, J = 7.76 Hz), 7.68 (s, 2H, NH2), 7.88 (s, 1H, H-3), 8.7 (s,
1H, H-6), 8.84 (s, 1H, NH), 10.24 (s, 1H, NH) ppm. Anal. (C15H12Cl2N4O3S) C, H, N.
4.1.10.8. 2,4-Dichloro-5-[3-(3-morpholinopropyl)ureido]benzenesulfonamide (10h). Starting
from 9 (0.2 g) and 3-morpholinopropan-1-amine (0.11 g) the title compound 10h was
obtained and crystallized from water (0.27 g, 88%): m.p. 230-232 °C; IR (KBr) 3386 (NH,
NH2), 1692 (C=O), 1371, 1161 (SO2) cm-1; 1H NMR (500 MHz, DMSO-d6) δ 1.56-1.61 (m,
2H, propyl), 2.27-2.32 (m, 6H, morpholine, propyl), 3.1-3.14 (m, 2H, propyl), 3.55-3.56 (m,
4H, morpholine), 7.12 (s, 1H, NH), 7.6 (s, 2H, NH2), 7.75 (s, 1H, H-3), 8.28 (s, 1H, NH), 8.94
(s, 1H, H-6) ppm; 13C NMR (DMSO-d6) 18.83, 26.67, 37.64, 53.63, 55.98, 56.30, 66.46,
120.30, 122.07, 124.27, 131.30, 136.37, 140.28, 154.58; MALDI-TOF m/z obsd: 412.9, [M +
H]+ calcd: 412.0; Anal. (C14H20Cl2N4O4S) C, H, N.
4.1.10.9. 4-(Benzodioxol-5-ylmethyl)-N-(2,4-dichloro-5-sulfamoylphenyl)piperazine-1-
carboxamide (10i). Starting from 9 (0.267 g) and 1-piperonylpiperazine (0.22 g) the title
compound 10i was obtained and crystallized from ethanol (0.268 g, 55%): m.p. 183-185 °C;
IR (KBr) 3466, 3248 (NH, NH2), 1646 (C=O), 1362, 1165 (SO2) cm-1; 1H NMR (500 MHz,
DMSO-d6) δ 2.34-2.35 (m, 4H, 2CH2), 3.41 (s, 2H, CH2N), 3.43-3.44 (m, 4H, 2CH2), 5.98 (s,
2H, OCH2O), 6.75-6.76 (m, 1H, H arom.), 6.84-6.85 (m, 1H, H arom.), 6.87 (s, 1H, H arom.),
7.7 (s, 2H, NH2), 7.81 (s, 1H, H-3), 8.1 (s, 1H, H-6), 8.48 (s, 1H, NH) ppm. Anal.
(C19H20Cl2N4O5S) C, H, N.
4.2. CA inhibition assay

ACCEPTED MANUSCRIPT
An Applied Photophysics (Oxford, UK) stopped-flow instrument has been used for
assaying the CA-catalyzed CO2 hydration activity [46]. 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 10-100 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 5-10% 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 10-20% (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 E-I complex.
The inhibition constants were obtained by non-linear last-squares methods using PRISM 3, as
reported earlier [47-49] and represent the mean from at least three different determinations.
Appendix. Supplementary material
Supplementary data associated with this article can be found on the online version at
doi:...............................
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Figure and scheme captions
Chart 1. General structures of known clinically used sulfonamides AAZ, MZA, EZA, DCP and IND (standard CA inhibitors), highly
potent CA inhibitors U-104, CAI17 and sulfonamides I-III and novel 5-substituted 2,4-dichlorobenzenesulfonamides IV-VII
Scheme 1. Synthesis of N-(2,4-dichloro-5-sulfamoylbenzoyl)-N"-R-semi- or thiosemicarbazides 5a-c, 2,4-dichloro-5-(2-R-
hydrazinecarbonyl)benzenesulfonamides 6a-d, 2,4-dichloro-5-(5-R-1,3,4-oxa- or thiadiazol-2-yl)benzenesulfonamides 7a-b and 7e-j, and
2,4-dichloro-5-(5-R-1,2,4-triazol-3-yl)benzenesulfonamides 7c-d. Reagents and conditions: a) SOCl2, reflux, 4 h; b) dry MeOH, reflux, 16-
18 h; Et3N, H2O, 1 h; c) 99% hydrazine hydrate (5 M eq.), EtOH, reflux, 4 h; d) RNCX, dry THF r.t. (5a, 5c) or reflux (5b), 0.5-3 h; e)
RC(OEt)3 (6 M eq.), glacial AcOH, reflux, 7-12 h (7a-b); f) RC(OEt)NH HCl, DBU, dry MeOH, reflux, 22-24 h (7c-d); g) TsCl, Py,
acetonitrile, reflux, 3-8 h (7e-f); h) RCO(NHNH2), Et3N (1-2 M eq.), dry THF, r.t., 40-90 h; i) SOCl2, reflux, 6-7 h (7g-i); j) LR, dry THF,
r.t., 24 h (7j).
Scheme 2. Synthesis of 2,4-dichloro-5-ureidobenzenesulfonamides 10a-i. Reagents and conditions: a) NaNO2, HCl (aq.), 0-5 °C; b) dry
toluene 110 °C, 1 h; c) RH, dry toluene, 110 °C, 0.5-6 h.

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Table 1.
Carbonic anhydrase inhibition data for compounds 5a-c, 6a-d, 7a-j and 10a-i and standard inhibitors against human isozymes hCA I, II, IX
and XII by a stopped-flow, CO2 hydration assay [46].
KIa (nM)
Compd
R
X
hCA Ib
250
780
25
1200
31
6230
5490
7200
3470
2750
3025
2400
643
hCA IIb
12.0
14.0
8.0
hCA IXc hCA XIIc
25.0
27.0
34.0
50.0
24.0
31.0
27.0
15.3
3.9
5.7
3.4
22.0
50.0
3.4
24.0
30.0
8.4
AAZ
MZA
EZA
DCP
IND
5a
5b
5c
6a
6b
6c
6d
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
10a
10b
10c
10d
10e
38.0
15.0
123.0
87.0
63.0
13.5
24.0
10.1
25.0
13.1
8.7
47.0
54.0
41.0
33.0
28.0
15.1
29.0
68.0
44.0
39.0
6.9
PhNH
S
S
4-ClPhNH
4-MePhSO2NH
2-furyl
Ph
4-ClPh
4-MePh
H
Me
Ph
4-ClPh
PhNH
4-ClPhNH
Ph
O
O
O
O
O
O
O
NH
NH
O
O
O
O
O
S
4.7
10.2
4.8
5.0
2.7
5.5
8.1
3.1
7.6
718
2.8
6.3
1350
1175
3200
2340
568
9.7
6.6
12.5
8.9
13.8
15.1
13.2
10.9
4.7
7.1
6.4
8.0
13.6
8.2
4-ClPh
4-MePh
Ph
671
349
1320
2340
3200
573
1165
934
13.6
7.0
5.4
2.8
PhNH
4-ClPhNH
4-H2NSO2PhNH
BnNH
7.1
4.3
15.8
21.7
18.9
3.6
23.0
33.0
57.0
40.0
BnONH
1260
79.0
12.5
24.0
10f
5430
84.0
26.0
32.0
10g
N
N
H
6400
7355
115.0
164.0
38.0
76.0
44.0
95.0
10h
10i
O
N
N
O
a Errors in the range of ±5-10% of the reported value (from 3 different assays).
b Human (cloned) isozymes, by CO2 hydration method.
c Catalytic domain of human, cloned isozymes [47], by the CO2 hydration method.

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New series of 5-substituted 2,4-dichlorobenzenesulfonamides have been synthesized
Compounds were evaluated for human CA isoforms I, II, IX and XII inhibitory activity
Twenty one compounds were better hCA IX inhibitors than clinically used sulfonamides
Two compounds were stronger hCA XII inhibitors than clinically used sulfonamides

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Supplementary material
Carbonic anhydrase inhibitors. Synthesis of a novel series of 5-substituted 2,4-
dichlorobenzenesulfonamides and their inhibition of human cytosolic isozymes I and II
and the transmembrane tumor-associated isozymes IX and XII
a
a
a
Jarosław Sławiński a,*, Aneta Pogorzelska , Beata Żołnowska , Kamil Brożewicz , Daniela
Vullo b, Claudiu T. Supuran b
aDepartment of Organic Chemistry, Medical University of Gdańsk, Al. Gen. J. Hallera 107, 80-416 Gdańsk,
Poland
bDipartimento di Chimica, Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica
Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
*Corresponding author: jaroslaw@gumed.edu.pl
Table of contents
Spectrum 1. 1H NMR of compd 5b (500 MHz, DMSO-d6).
Spectrum 2. 13C NMR of compd 5b (50 MHz, DMSO-d6).
Spectrum 3. 1H NMR of compd 6c (500 MHz, DMSO-d6).
Spectrum 4. 1H NMR of compd 7d (500 MHz, DMSO-d6).
Spectrum 5. 13C NMR of compd 7d (50 MHz, DMSO-d6).
Spectrum 6. 1H NMR of compd 7f (500 MHz, DMSO-d6).
Spectrum 7. 1H NMR of compd 7g (500 MHz, DMSO-d6).
Spectrum 8. 1H NMR of compd 7h (500 MHz, DMSO-d6).
Spectrum 9. 13C NMR of compd 7h (50 MHz, DMSO-d6).
Spectrum 10. 1H NMR of compd 10b (500 MHz, DMSO-d6).
Spectrum 11. 1H NMR of compd 10d (500 MHz, DMSO-d6).
Spectrum 12. 13C NMR of compd 10d (50 MHz, DMSO-d6).
Spectrum 13. 1H NMR of compd 10f (500 MHz, DMSO-d6).
Spectrum 14. 1H NMR of compd 10h (500 MHz, DMSO-d6).
Appendix A: Elemental analyses of new compounds