Benzenesulfonamides with benzimidazole moieties as inhibitors of carbonic anhydrases I, II, VII, XII and XIII

Article abstract of DOI:10.3109/14756366.2012.757223

A series of benzenesulfonamide derivatives, bearing benzimidazole moieties, were designed and synthesized as inhibitors of carbonic anhydrases (CAs). Their binding affinities to recombinant human CA isozymes I, II, VII, XII and XIII were determined by the

Full text of DOI:10.3109/14756366.2012.757223

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(1): 124–131
2014 Informa UK Ltd. DOI: 10.3109/14756366.2012.757223
!
Benzenesulfonamides with benzimidazole moieties as inhibitors
of carbonic anhydrases I, II, VII, XII and XIII
1
1,2
1
1
Asta Zubriene , Edita Capkauskaite , Joana Gylyte , Migle Kisonaite , Sigitas Tumkevicius , and
2
ˇ
_
_
_
_
ˇ
_
ˇ
Daumantas Matulis1
2
1Department of Biothermodynamics and Drug Design, Institute of Biotechnology, and Department of Organic Chemistry, Faculty of Chemistry,
Vilnius University, Vilnius, Lithuania
Abstract
Keywords
A series of benzenesulfonamide derivatives, bearing benzimidazole moieties, were designed
and synthesized as inhibitors of carbonic anhydrases (CAs). Their binding affinities to
recombinant human CA isozymes I, II, VII, XII and XIII were determined by the thermal shift
assay. A group of compounds containing a benzimidazole substituent in the para position of
the benzenesulfonamide ring was found to exhibit higher binding potency toward tested CAs
than meta-substituted benzenesulfonamides. Some of these compounds exhibited nanomolar
affinities and selectivity toward the CA isozymes tested.
Benzenesulfonamide, carbonic anhydrase,
N-alkylated benzimidazoles, thermal shift
assay, ThermoFluorÕ
History
Received 18 October 2012
Revised 6 December 2012
Accepted 6 December 2012
Published online 28 January 2013
Introduction
epilepsy, dandruff, cancer and as antiviral compounds15,16
.
The majority of clinically used CA inhibitors are also primary
sulfonamides1. To date, CA inhibitors such as acetazolamide
(AZM), ethoxzolamide (EZA) and methazolamide are widely
used as systemic antiglaucoma drugs17; sultiame and zonisamide
as antiepileptic drugs18; and chlorthalidone, indapamide and
furosemide as diuretics19 (Figure 1). The largest numbers of new
Carbonic anhydrases (CAs) are widely investigated enzymes due
to their involvement in various physiological and pathological
processes1–4. There are a total of 15 CA isoforms found in
humans. However, three of them have no catalytic activity (CA
VIII, X and XI). The 12 active isozymes (CA I–IV, VA, VB, VI,
VII, IX, XII and XIV) are metalloenzymes, which participate in
the regulation of acid–base balance and ion transport in many
tissues and organs. The enhanced activity of CAs is often related
to various diseases. The isoforms CA IX and CA XII were
intensively investigated enzymes during the last decade due to
their overexpression in various human cancers5–9. Much work has
been done to design selective CA IX and CA XII inhibitors. Some
compounds such as glycosyl coumarins, N-b-glycosyl sulfamides
and ureido-substituted sulfamates were found to be low
nanomolar CA IX and XII inhibitors, while showing weak
CA inhibitors are benzenesulfonamides1,14
.
Benzenesulfonamides bind to the active site of CAs in their
deprotonated form, with the nitrogen atom of the sulfonamide
moiety coordinated to the Zn(II) ion. Depending on the nature of
the substituent, additional interactions with hydrophobic and
hydrophilic amino acids in the CA active center can be assigned.
Therefore, a variety of substitutions on the benzenesulfonamide
head group can be used for the development of novel inhibitors.
Recently, we have investigated the interaction of several
CA isozymes with pyrimidine derivatives such as [(2-pyrimidi-
nylthio)acetyl]benzenesulfonamides20, 4-[N-(substituted 4-pyrimi-
dinyl)amino]benzenesulfonamides21 and thiadiazole derivatives22.
We have also reported on the synthesis of 2-substituted
benzimidazoles N-alkylated with 2-chloro-5-bromoacetylbenze-
nesulfonamide (Figure 2, compounds 4(a–j)) and their binding to
four CA isozymes, namely CA I, CA II, CA VII and CA XIII23.
We extended these earlier investigations to new sulfonamides
containing a benzimidazole group (compounds of series 1–3).
Here we present the binding data of 4(a–j) to the catalytic
domain of CA XII, an anticancer drug target. The N-alkylated
benzimidazoles 4(a–j) exhibited dissociation constants in the
range of 0.3–10 mM against tested CAs. Furthermore, we
compared series 4 to compounds of series 1–3, which are 4-
[(2-substituted-1H-benzimidazol-1-yl)acetyl]benzenesulfonamide
1(a–j), 2-chloro-4-[(2-substituted-1H-benzimidazol-1-yl)acetyl]-
benzenesulfonamide 2(a–j) and 3-[(2-substituted-1H-benzimida-
zol-1-yl)acetyl]benzenesulfonamide 3(a–j). Their binding was
inhibition toward cytosolic isoforms CA I and CA II10–12
.
Numerous inhibitors of various CA isoforms were reviewed by
Alterio et al.1. The design of CA inhibitors as drugs has been
impeded by the large number of isoforms, their diffusion among
many tissues/organs and the lack of isozyme-selective inhibitors
among existing sulfonamides13. As the differences between the
active sites of the 12 catalytically active human CAs are very
subtle, CAs have proved to be a challenging drug target for the
development of isozyme-specific inhibitors2,14
.
Primary sulfonamides present an important class of com-
pounds which have been studied as drugs and drug candidates
against diseases such as glaucoma, inflammation, edema,
_
Address for correspondence: Asta Zubriene, Department of Biothermo-
dynamics and Drug Design, Vilnius University Institute of Biotechnol-
ogy, Graiciuno 8, Vilnius LT-02241, Lithuania. E-mail: astzu@ibt.lt,
asta.zubriene@bti.vu.lt

DOI: 10.3109/14756366.2012.757223
Benzenesulfonamides with benzimidazole moieties 125
spectra, respectively) as the internal standard. Thin-layer
chromatography was performed with silica gel 60 F254 aluminum
plates (Merck, Darmstadt, Germany) and visualized with UV
light. High-resolution mass spectra (HRMS) were recorded on a
Dual-ESI Q-TOF 6520 mass spectrometer (Agilent Technologies,
Santa Clara, CA). The purities of target compounds were analyzed
using an HPLC system with UV detection.
General procedure for the synthesis of 1–3(a–j)
A mixture of the corresponding compounds 1–3 (0.360 mmol),
appropriate 2-substituted benzimidazole (a–j) (0.540 mmol) and
sodium acetate (33.9 mg, 0.414 mmol) in tetrahydrofuran (2 ml)
were stirred at r.t. for 24 h. The reaction mixture was poured into
water. The precipitate was filtered out and then washed with water
and diethyl ether to yield 1–3(a–j).
Figure 1. Chemical structures of some clinically used sulfonamides.
4-(1H-benzimidazol-1-ylacetyl)benzenesulfonamide (1a).
Yield 60%, m.p. 242–244 ꢀC. IR (ꢀ, cmꢁ1): 3358 (NH2), 1701
(CO). H NMR ꢁ ppm: 6.11 (2H, s, CH2CO), 7.24–7.27 (2H, m,
1
0
C5 ,6 –H), 7.57–7.60 (1H, m, C7 –H), 7.67 (2H, s, NH2), 7.70–7.73
0
0
0
(1H, m, C4 –H), 8.07 (2H, d, J ¼ 8.4 Hz, C2,6–H), 8.24 (1H, s,
C2 –H), 8.31 (2H, d, J ¼ 8.1 Hz, C3,5–H). 13C NMR ꢁ ppm: 51.82,
0
111.38, 119.97, 122.32, 123.14, 126.80, 129.66, 135.31, 137.46,
143.60, 145.57, 149.09, 193.67. HRMS Calcd for C15H13N3O3S
([M þ H]þ): 316.0750, Found: 316.0746.
4-(1H-benzimidazol-1-ylacetyl)-2-chlorobenzenesulfonamide
(2a). Yield 72%, m.p. 234–236 ꢀC. IR (ꢀ, cmꢁ1): 3379 (NH2),
1705 (CO). 1H NMR ꢁ ppm: 6.10 (2H, s, CH2CO), 7.23–7.26 (2H,
0
m, C5 ,6 –H), 7.57–7.60 (1H, m, C7 –H), 7.70–7.72 (1H, m,
0
0
Figure 2. The structure of N-alkylated benzimidazole derivatives.
0
0
C4 –H), 7.93 (2H, s, NH2), 8.19–8.23 (3H, m, C2 ,5,6–H), 8.33 (1H,
s, C3–H). 13C NMR ꢁ ppm: 51.88, 111.37, 120.05, 122.27, 123.09,
127.72, 130.18, 131.75 (2C), 135.33, 138.83, 143.77, 145.50,
145.67, 192.98. HRMS Calcd for C15H12ClN3O3S ([M þ H]þ):
350.0361, Found: 350.0363.
then assayed with five CA isozymes CA I, CA II, CA VII, CA XII
and CA XII. The compounds 2(a–j) were found to be the most
potent CA inhibitors exhibiting nanomolar affinities toward CA I,
II, VII and XIII. Detailed structure–activity correlation could be
drawn by comparing the affinities and selectivity of these
compounds.
3-(1H-benzimidazol-1-ylacetyl)benzenesulfonamide (3a).
Yield 83%, m.p. 247–249 ꢀC. IR (ꢀ, cmꢁ1): 3270 (NH2), 1698
(CO). H NMR ꢁ ppm: 6.13 (2H, s, CH2CO), 7.22–7.28 (2H, m,
1
0
C5 ,6 –H), 7.57–7.61 (1H, m, C7 –H), 7.64 (2H, s, NH2), 7.69–7.75
0
0
0
(1H, m, C4 –H), 7.88 (1H, t, J ¼ 7.8 Hz, C5–H), 8.19 (1H, d,
0
J ¼ 7.8 Hz, C4–H), 8.22 (1H, s, C2 –H), 8.40 (1H, d, J ¼ 7.8 Hz,
Materials and methods
C6–H), 8.50 (1H, s, C2–H). 13C NMR ꢁ ppm: 15.71, 111.38,
120.04, 122.23, 123.08, 125.63, 130.60, 131.18, 132.26, 135.41,
135.74, 143.81, 145.60, 145.69, 193.48. HRMS Calcd for
C15H13N3O3S ([M þ H]þ): 316.0750, Found: 316.0752.
Syntheses
Synthesis of 4-(bromoacetyl)benzenesulfonamide (1) and
3-(bromoacetyl)benzenesulfonamide (3) was achieved from
commercially available 1-(4-aminophenyl)ethanone and 1-(3-
aminophenyl)ethanone, respectively, as described by Fujikura
et al.24. Synthesis of 4-(bromoacetyl)-2-chlorobenzenesulfona-
mide (2) was accomplished from 1-(4-amino-3-chloropheny-
l)ethanone as described by Fujikura et al.24. 1-(4-Amino-3-
chlorophenyl)ethanone was synthesized by the chlorination of 1-
(3-aminophenyl)-N ethanone with N-chlorosuccinimide in acet-
onitrile25,26. 2-Substituted benzimidazoles (b–i) were prepared
from 1,2-benzenediamine and the appropriate carboxylic acids
according to Phillips’ procedure27. Benzimidazole (a) and
2-methylthiobenzimidazole (j) are commercially available and
were used without further purification. All ingredients were
purchased from Sigma-Aldrich (Milwaukee, WI) and Alfa Aesar
GmbH (Karlsruhe, Germany).
The melting points of the compounds were determined in open
capillaries on a Thermo Scientific 9100 Series apparatus
(Rochford, UK) without further correction. IR spectra were
obtained on a Perkin-Elmer (Upplands Vasby, Sweden) FT-IR
spectrophotometer Spectrum BX II in KBr. 1H and 13C NMR
spectra were recorded on a Varian Unity Inova spectrometer (Palo
Alto, CA) (300 and 75 MHz, respectively) in DMSO-d6 using
residual DMSO signals (2.52 and 40.21 ppm for 1H and 13C NMR
4-[(2-Methyl-1H-benzimidazol-1-yl)acetyl]benzenesulfona-
mide (1b). Yield 63%, m.p. 146–148 ꢀC. IR (ꢀ, cmꢁ1): 3336
(NH2), 1698 (CO). 1H NMR ꢁ ppm: 2.45 (3H, s, CH3), 6.06
0
(2H, s, CH2CO), 7.14–7.21 (2H, m, C5 ,6 –H), 7.46–7.49 (1H, m,
0
0
0
C7 –H), 7.57–7.60 (1H, m, C4 –H), 7.66 (2H, s, NH2), 8.07 (2H, d,
J ¼ 8.4 Hz, C2,6–H), 8.32 (2H, d, J ¼ 8.1 Hz, C3,5–H). 13C NMR
ꢁ ppm: 14.00, 50.84, 110.60, 118.80, 122.06, 122.23, 126.74,
129.79, 136.53, 137.47, 142.88, 149.21, 153.31, 193.81. HRMS
Calcd for C16H15N3O3S ([M þ H]þ): 330.0907, Found: 330.0909.
2-Chloro-4-[(2-methyl-1H-benzimidazol-1-yl)acetyl]benze-
nesulfonamide (2b). Yield 74%, m.p. 204–206 ꢀC. IR (ꢀ, cmꢁ1):
3385, 3332 (NH2), 1707 (CO). 1H NMR ꢁ ppm: 2.45 (3H, s, CH3),
0
6.09 (2H, s, CH2CO), 7.14–7.20 (2H, m, C5 ,6 –H), 7.47–7.50
0
0
0
(1H, m, C7 –H), 7.57–7.61 (1H, m, C4 –H), 7.94 (2H, s, NH2),
8.20 (2H, s, C5,6–H), 8.37 (1H, s, C3–H). 13C NMR ꢁ ppm: 13.97,
51.01, 110.75, 118.71, 122.19, 122.32, 127.80, 130.09, 131.71,
132.06, 136.41, 138.72, 142.59, 145.78, 153.29, 193.02. HRMS
Calcd for C16H14ClN3O3S ([M þ H]þ): 364.0517, Found:
364.0517.
3-[(2-Methyl-1H-benzimidazol-1-yl)acetyl]benzenesulfona-
mide (3b). Yield 62%, m.p. 295–297 ꢀC. IR (ꢀ, cmꢁ1): 3280
(NH2), 1699 (CO). 1H NMR ꢁ ppm: 2.46 (3H, s, CH3), 6.09

126 A. Zubriene_ et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 124–131
(2H, s, CH2CO), 7.14–7.21 (2H, m, C5 ,6 –H), 7.47–7.51 (1H, J ¼ 7.8 Hz, C6–H), 8.41 (1H, s, C2–H). 13C NMR ꢁ ppm: 33.45,
0
0
0
0
m, C7 –H), 7.57–7.60 (1H, m, C4 –H), 7.63 (2H, s, NH2), 7.89 50.76, 111.01, 119.22, 122.14, 122.22, 125.67, 127.18, 129.06,
(1H, t, J ¼ 7.8 Hz, C5–H), 8.20 (1H, d, J ¼ 8.1 Hz, C4–H), 8.44 129.63, 130.43, 131.16, 132.37, 135.63, 136.67, 137.44, 142.98,
(1H, d, J ¼ 7.8 Hz, C6–H), 8.51 (1H, s, C2–H). 13C NMR ꢁ ppm: 145.55, 154.87, 193.01. HRMS Calcd for C22H19N3O3S
14.03, 50.78, 110.62, 118.83, 122.03, 122.21, 125.70, 130.57, ([M þ H]þ): 406.1220, Found: 406.1223.
131.31, 132.51, 135.66, 136.58, 142.96, 145.66, 153.33, 193.59.
4-[(2-Ethyl-1H-benzimidazol-1-yl)acetyl]benzenesulfona-
HRMS Calcd for C16H15N3O3S ([M þ H]þ): 330.0907, Found: mide (1e). Yield 71%, m.p. 236–238 ꢀC. IR (ꢀ, cmꢁ1): 3334
330.0903.
(NH2), 1699 (CO). 1H NMR ꢁ ppm: 1.31 (3H, t, J ¼ 7.5 Hz, CH3),
4-{[2-(Hydroxymethyl)-1H-benzimidazol-1-yl]acetyl}ben- 2.79 (2H, q, J ¼ 7.5 Hz, CH2), 6.07 (2H, s, CH2CO), 7.17–7.20
zenesulfonamide (1c). Yield 57%, m.p. 200–202 ꢀC. IR (2H, m, C5 ,6 –H), 7.47–7.50 (1H, m, C7 –H), 7.61–7.64 (1H, m,
0
0
0
1
(ꢀ, cmꢁ1): 3375, 3314 (NH2, OH), 1702 (CO). H NMR ꢁ ppm: C4 –H), 7.67 (2H, s, NH2), 8.07 (2H, d, J ¼ 8.1 Hz, C2,6–H), 8.33
0
4.70 (2H, s, CH2OH), 5.64 (2H, br s, OH), 6.09 (2H, s, CH2CO), (2H, d, J ¼ 8.1 Hz, C3,5–H). 13C NMR ꢁ ppm: 12.16, 20.50, 50.63,
0
7.20–7.26 (2H, m, C5 ,6 –H), 7.53–7.56 (1H, m, C7 –H), 7.64–7.68 110.70, 118.91, 122.12, 122.37, 126.73, 129.82, 136.49, 137.44,
0
0
0
(3H, m, C4 –H, NH2), 8.06 (2H, d, J ¼ 8.4 Hz, C2,6–H), 8.31 142.62, 149.21, 157.44, 193.89. HRMS Calcd for C17H17N3O3S
(2H, d, J ¼ 8.4 Hz, C3,5–H). 13C NMR ꢁ ppm: 51.04, 57.59, ([M þ H]þ): 344.1063, Found: 344.1062.
111.02, 119.59, 122.31, 123.01, 126.76, 129.72, 137.00, 137.54,
2-Chloro-4-[(2-ethyl-1H-benzimidazol-1-yl)acetyl]benzene-
142.33, 149.04, 154.76, 193.49. HRMS Calcd for sulfonamide (2e). Yield 66%, m.p. 236–238 ꢀC. IR (ꢀ, cmꢁ1):
C16H15N3O4S([M þ H]þ): 346.0856, Found: 346.0859.
3333 (NH2), 1706 (CO). H NMR ꢁ ppm: 1.30 (3H, t, J ¼ 7.5 Hz,
1
2-Chloro-4-{[2-(hydroxymethyl)-1H-benzimidazol-1-yl]ace- CH3), 2.78 (2H, q, J ¼ 7.5 Hz, CH2), 6.09 (2H, s, CH2CO),
tyl}benzenesulfonamide (2c). Yield 52%, m.p. 195–197 ꢀC. IR 7.14–7.21 (2H, m, C5 ,6 –H), 7.47–7.50 (1H, m, C7 –H), 7.60–7.63
0
0
0
1
(ꢀ, cmꢁ1): 3370, 3267 (NH2, OH), 1707 (CO). H NMR ꢁ ppm: (1H, m, C4 –H), 7.93 (2H, s, NH2), 8.17–8.23 (2H, m, C5,6–H),
0
4.69 (2H, d, J ¼ 4.8 Hz, CH2OH), 5.60 (1H, br s, OH), 6.08 (2H, s, 8.38 (1H, s, C3–H). 13C NMR ꢁ ppm: 12.19, 20.46, 50.76,
0
0
0
CH2CO), 7.20–7.25 (2H, m, C5 ,6 –H), 7.50–7.57 (1H, m, C7 –H), 110.76, 118.90, 122.14, 122.36, 127.79, 130.06, 131.71,
0
7.60–7.65 (1H, m, C4 –H), 7.92 (2H, s, NH2), 8.19 (2H, s, C5,6
–
132.10, 136.44, 138.70, 142.59, 145.78, 157.41, 193.16. HRMS
H), 8.33 (1H, s, C3–H). 13C NMR ꢁ ppm: 51.11, 57.54, 110.97, Calcd for C17H16ClN3O3S ([M þ H]þ): 378.0674, Found:
119.66, 122.27, 122.33, 127.74, 130.15, 131.05, 131.81, 137.01, 378.0670.
138.88, 142.50, 145.63, 154.71, 192.80. HRMS Calcd for
3-[(2-Ethyl-1H-benzimidazol-1-yl)acetyl]benzenesulfona-
mide (3e). Yield 66%, m.p. 292–294 ꢀC. IR (ꢀ, cmꢁ1): 3288
C16H14ClN3O4S ([M þ H]þ): 380.0466, Found: 380.0463.
3-{[2-(Hydroxymethyl)-1H-benzimidazol-1-yl]acetyl}ben- (NH2), 1699 (CO). 1H NMR ꢁ ppm: 1.31 (3H, t, J ¼ 7.5 Hz, CH3),
zenesulfonamide (3c). Yield 60%, m.p. 220–222 ꢀC. IR 2.79 (2H, q, J ¼ 7.5 Hz, CH2), 6.09 (2H, s, CH2CO), 7.14–7.21
(ꢀ, cmꢁ1): 3364, 3272 (NH2, OH), 1703 (CO). H NMR ꢁ ppm: (2H, m, C5 ,6 –H), 7.47–7.50 (1H, m, C7 –H), 7.62–7.69 (1H, m,
1
0
0
0
0
4.69 (2H, d, J ¼ 3.3 Hz, CH2OH), 5.63 (1H, br s, OH), 6.12 (2H, s, NH2, C4 –H), 7.89 (1H, t, J ¼ 7.8 Hz, C5–H), 8.20 (1H, d,
0
0
0
CH2CO), 7.21–7.24 (2H, m, C5 ,6 –H), 7.54–7.57 (1H, m, C7 –H), J ¼ 8.1 Hz, C4–H), 8.45 (1H, d, J ¼ 7.8 Hz, C6–H), 8.51 (1H, s,
7.62–7.67 (3H, m, C4 –H, NH2), 7.87 (1H, t, J ¼ 7.8 Hz, C5–H), C2–H). 13C NMR ꢁ ppm: 12.20, 20.53, 50.53, 110.65, 119.01,
0
8.18 (1H, d, J ¼ 7.5 Hz, C4–H), 8.40 (1H, d, J ¼ 7.8 Hz, C6–H), 121.99, 122.27, 125.71, 130.56, 131.31, 132.54, 135.66, 136.60,
8.49 (1H, s, C2–H). 13C NMR ꢁ ppm: 50.98, 57.64, 111.02, 142.92, 145.66, 157.46, 193.72. HRMS Calcd for C17H17N3O3S
119.63, 122.26, 122.97, 125.65, 130.59, 131.16, 132.33, 135.74, ([M þ H]þ): 344.1063, Found: 344.1065.
137.07, 142.45, 145.63, 154.75, 193.20. HRMS Calcd for
4-[(2-Propyl-1H-benzimidazol-1-yl)acetyl]benzenesulfona-
mide (1f). Yield 65%, m.p. 245–247 ꢀC. IR (ꢀ, cmꢁ1): 3300
C16H15N3O4S ([M þ H]þ): 346.0856, Found: 346.0857.
4-[(2-Benzyl-1H-benzimidazol-1-yl)acetyl]benzenesulfo- (NH2), 1700 (CO). 1H NMR ꢁ ppm: 0.97 (3H t, J ¼ 7.5 Hz, CH3),
namide (1d). Yield 62%, m.p. 241–243 ꢀC. IR (ꢀ, cmꢁ1): 3284 1.78 (2H, sextet, J ¼ 7,5 Hz, CH2), 2.75 (2H, t, J ¼ 7.2 Hz, CH2),
1
(NH2), 1703 (CO). H NMR ꢁ ppm: 4.26 (2H, s, CH2Ph), 6.06 6.08 (2H, s, CH2CO), 7.14–7.22 (2H, m, C5 ,6 –H), 7.45–7.48 (1H,
0
0
0
0
0
0
(2H, s, CH2CO), 7.15–7.28 (7H, m, C5 ,6 –H, Ph–H), 7.44–7.47 m, C7 –H), 7.60–7.63 (1H, m, C4 –H), 7.69 (2H, s, NH2), 8.07
(1H, m, C7 –H), 7.62–7.65 (1H, m, C4 –H), 7.68 (2H, s, NH2), (2H, d, J ¼ 8.4 Hz, C2,6–H), 8.33 (2H, d, J ¼ 8.7 Hz, C3,5–H). 13C
8.04 (2H, d, J ¼ 8.4 Hz, C2,6–H), 8.22 (2H, d, J ¼ 8.7 Hz, C3,5–H). NMR ꢁ ppm: 14.47, 20.95, 28.84, 50.66, 110.75, 118.88, 122.12,
13C NMR ꢁ ppm: 33.42, 50.83, 111.03, 119.17, 122.28, 122.59, 122.32, 126.71, 129.85, 136.38, 137.38, 142.66, 149.19, 156.32,
126.63, 127.19, 129.06, 129.60 (2C), 129.70, 136.60, 137.36, 193.85. HRMS Calcdfor C18H19N3O3S([M þ H]þ): 358.1220,
142.80, 149.03, 154.83, 193.15. HRMS Calcd for C22H19N3O3S Found: 358.1222.
0
0
([M þ H]þ): 406.1220, Found: 406.1217.
2-Chloro-4-[(2-propyl-1H-benzimidazol-1-yl)acetyl]benze-
4-[(2-Benzyl-1H-benzimidazol-1-yl)acetyl]-2-chlorobenze- nesulfonamide (2f). Yield 71%, m.p. 240–242 ꢀC. IR (ꢀ, cmꢁ1):
1
nesulfonamide (2d). Yield 71%, m.p. 210–212 ꢀC. IR (ꢀ, cmꢁ1): 3354 (NH2), 1702 (CO). H NMR ꢁ ppm: 0.97 (3H, t, J ¼ 7.5 Hz,
3382, 3294 (NH2), 1705 (CO). 1H NMR ꢁ ppm: 4.27 (2H, s, CH3), 1.78 (2H, sextet, J ¼ 7.5 Hz, CH2), 2.74 (2H, t, J ¼ 7.5 Hz,
0
CH2Ph), 6.07 (2H, s, CH2CO), 7.19–7.26 (7H, m, C5 ,6 –H, Ph– CH2), 6.09 (2H, s, CH2CO), 7.13–7.20 (2H, m, C5 ,6 –H),
0
0
0
0
0
0
0
H), 7.45–7.49 (1H, m, C7 –H), 7.61–7.64 (1H, m, C4 –H), 7.93 7.45–7.48 (1H, m, C7 –H), 7.59–7.62 (1H, m, C4 –H), 7.93 (2H,
(2H, s, NH2), 8.07 (1H, d, J ¼ 8.1 Hz, C5–H), 8.18 (1H, d, s, NH2), 8.20 (2H, s, C5,6–H), 8.39 (1H, s, C3–H). 13C NMR
J ¼ 8.1 Hz, C6–H), 8.25 (1H, s, C3–H). 13C NMR ꢁ ppm: 33.40, ꢁ ppm: 14.49, 21.00, 28.87, 50.78, 110.74, 118.96, 122.05,
50.91, 111.01, 119.21, 122.25, 122.56, 127.15, 127.66, 129.03, 122.24, 127.79, 130.05, 131.71, 132.13, 136.41, 138.69, 142.90,
129.62, 129.98, 131.59, 131.87, 136.61, 137.41, 138.67, 142.91, 145.78, 156.31, 193.14. HRMS Calcd for C18H18ClN3O3S
145.63, 154.77, 192.46. HRMS Calcd for C22H18ClN3O3S ([M þ H]þ): 392.0830, Found: 392.0827.
([M þ H]þ): 440.0830, Found: 440.0836.
3-[(2-Propyl-1H-benzimidazol-1-yl)acetyl]benzenesulfona-
3-[(2-Benzyl-1H-benzimidazol-1-yl)acetyl]benzenesulfona- mide (3f). Yield 87%, m.p. 275–277 ꢀC. IR (ꢀ, cmꢁ1): 3314
mide (3d). Yield 88%, m.p. 146–148 ꢀC. IR (ꢀ, cmꢁ1): 3340 (NH2), 1701 (CO). 1H NMR ꢁ ppm: 0.97 (3H, t, J ¼ 7.5 Hz, CH3),
1
(NH2), 1702 (CO). H NMR ꢁ ppm: 4.27 (2H, s, CH2Ph), 6.08 1.79 (2H, sextet, J ¼ 7.5 Hz, CH2), 2.75 (2H, t, J ¼ 7.5 Hz, CH2),
0
(2H, s, CH2CO), 7.17–7.36 (7H, m, C5 ,6 –H, Ph–H), 7.45–7.52 6.09 (2H, s, CH2CO), 7.13–7.21 (2H, m, C5 ,6 –H), 7.46–7.49 (1H,
0
0
0
0
(1H, m, C7 –H), 7.64–7.69 (1H, m, C4 –H, NH2), 7.85 (1H, t, m, C7 –H), 7.60–7.62 (3H, m, C4 –H, NH2), 7.89 (1H, t,
0
0
0
J ¼ 7.8 Hz, C5–H), 8.18 (1H, d, J ¼ 7.8 Hz, C4–H), 8.34 (1H, d, J ¼ 7.8 Hz, C5–H), 8.20 (1H, d, J ¼ 8.1 Hz, C4–H), 8.45 (1H, d,

DOI: 10.3109/14756366.2012.757223
Benzenesulfonamides with benzimidazole moieties 127
J ¼ 7.8 Hz, C6–H), 8.50 (1H, s, C2–H). 13C NMR ꢁ ppm: 14.50, 7.14–7.22 (2H, m, C5 ,6 –H), 7.46–7.49 (1H, m, C7 –H), 7.62–7.63
0
0
0
0
20.98, 28.91, 50.58, 110.69, 118.99, 122.00, 122.22, 125.71, (3H, m, C4 –H, NH2), 7.89 (1H, t, J ¼ 7.8 Hz, C5–H), 8.20 (1H, d,
130.56, 131.30, 132.58, 135.64, 136.50, 142.97, 145.65, 156.35, J ¼ 7.8 Hz, C4–H), 8.47 (1H, d, J ¼ 7.8 Hz, C6–H), 8.51 (1H, s,
193.66. HRMS Calcd for C18H19N3O3S ([M þ H]þ): 358.1220, C2–H). 13C NMR ꢁ ppm: 22.44, 26.19, 50.53, 110.87, 119.02,
Found: 358.1219.
122.13, 122.33, 125.72, 130.55, 131.32, 132.63, 135.61, 136.27,
4-[(2-Butyl-1H-benzimidazol-1-yl)acetyl]benzenesulfona- 142.67, 145.64, 161.07, 193.80. HRMS Calcd for C18H19N3O3S
mide (1 g). Yield 67%, m.p. 260–262 ꢀC. IR (ꢀ, cmꢁ1): 3299 ([M þ H]þ): 358.1220, Found: 358.1221.
(NH2), 1699 (CO). 1H NMR ꢁ ppm: 0.90 (3H, t, J ¼ 7.5 Hz, CH3),
4-[(2-Isobutyl-1H-benzimidazol-1-yl)acetyl]benzenesulfo-
1.39 (2H, sextet, J ¼ 7.5 Hz, CH2), 1.75 (2H, quintet, J ¼ 7.5 Hz, namide (1i). Yield 66%, m.p. 234–236 ꢀC. IR (ꢀ, cmꢁ1): 3333
CH2), 2.77 (2H, t, J ¼ 7.5 Hz, CH2), 6.08 (2H, s, CH2CO), (NH2), 1703 (CO). 1H NMR ꢁ ppm: 0.96 (6H, d, J ¼ 6.6 Hz,
0
0
0
7.14–7.21 (2H, m, C5 ,6 –H), 7.45–7.48 (1H, m, C7 –H), 7.60–7.63 (CH3)2), 2.12–2.25 (1H, m, CH), 2.68 (2H, d, J ¼ 6.9 Hz, CH2),
0
0 0
6.07 (2H, s, CH2CO), 7.13–7.22 (2H, m, C5 ,6 –H), 7.43–7.46 (1H,
(1H, m, C4 –H), 7.69 (2H, s, NH2), 8.07 (2H, d, J ¼ 8.4 Hz, C2,6
–
H), 8.34 (2H, d, J ¼ 8.4 Hz, C3,3–H). 13C NMR ꢁ ppm: 14.48, m, C4 –H), 7.61–7.64 (1H, m, C7 –H), 7.66 (2H, s, NH2), 8.07
22.50, 26.63, 29.65, 50.66, 110.70, 118.93, 122.06, 122.26, (2H, d, J ¼ 8.4 Hz, C2,6–H), 8.33 (2H, d, J ¼ 8.4 Hz, C3,5–H). 13C
126.73, 129.84, 136.45, 137.42, 142.82, 149.20, 156.46, 193.89. NMR ꢁ ppm: 23.08, 27.67, 35.76, 50.73, 110.82, 118.92, 122.12,
HRMS Calcd for C19H21N3O3S ([M þ H]þ): 372.1376, Found: 122.29, 126.72, 129.84, 136.34, 137.44, 142.77, 149.22, 155.71,
0
0
372.1376.
193.77. HRMS Calcd for C19H21N3O3S ([M þ H]þ): 372.1376,
4-[(2-Butyl-1H-benzimidazol-1-yl)acetyl]-2-chlorobenze- Found: 372.1377.
nesulfonamide (2 g). Yield 75%, m.p. 201–203 ꢀC. IR (ꢀ, cmꢁ1):
2-Chloro-4-[(2-isobutyl-1H-benzimidazol-1-yl)acetyl]ben-
1
3333 (NH2), 1708 (CO). H NMR ꢁ ppm: 0.90 (3H, t, J ¼ 7.2 Hz, zenesulfonamide (2i). Yield 74%, m.p. 234–236 ꢀC. IR (ꢀ,
CH3), 1.38 (2H, sextet, J ¼ 7.5 Hz, CH2), 1.74 (2H, quintet, cmꢁ1): 3378 (NH2), 1706 (CO). H NMR ꢁ ppm: 0.95 (6H, d,
1
J ¼ 7.5 Hz, CH2), 2.76 (2H, t, J ¼ 7.5 Hz, CH2), 6.09 (2H, s, J ¼ 6.3 Hz, (CH3)2), 2.12–2.24 (1H, m, CH), 2.67 (2H, d,
0
0
0
CH2CO), 7.13–7.20 (2H, m, C5 ,6 –H), 7.45–7.48 (1H, m, C7 –H), J ¼ 6.9 Hz, CH2), 6.10 (2H, s, CH2CO), 7.13–7.24 (2H, m,
0
0
0
0
0
7.59–7.61 (1H, m, C4 –H), 7.94 (2H, s, NH2), 8.20 (2H, s, C5 ,6 –H), 7.45–7.47 (1H, m, C7 –H), 7.60–7.62 (1H, m, C4 –H),
C5,6–H), 8.39 (1H, s, C3–H). 13C NMR ꢁ ppm: 14.47, 22.50, 7.93 (2H, s, NH2), 8.20 (2H, s, C5,6–H), 8.39 (1H, s, C3–H). 13C
26.60, 29.66, 50.78, 110.74, 118.93, 122.06, 122.24, 127.77, NMR ꢁ ppm: 23.07, 27.67, 35.66, 50.87, 110.88, 118.86, 122.17,
130.06, 131.71, 132.12, 136.42, 138.69, 142.85, 145.78, 156.44, 122.30, 127.79, 130.03, 131.69, 132.16, 136.24, 138.64, 142.67,
193.15. HRMS Calcd. for C19H20ClN3O3S ([M þ H]þ): 406.0987, 145.78, 155.69, 193.00. HRMS Calcd for C19H20ClN3O3S
Found: 406.0981.
([M þ H]þ): 406.0987, Found: 406.0989.
3-[(2-Butyl-1H-benzimidazol-1-yl)acetyl]benzenesulfona-
3-[(2-Isobutyl-1H-benzimidazol-1-yl)acetyl]benzenesulfo-
mide (3 g). Yield 61%, m.p. 214–216 ꢀC. IR (ꢀ, cmꢁ1): 3322 namide (3i). Yield 68%, m.p. 216–218 ꢀC. IR (ꢀ, cmꢁ1): 3312
(NH2), 1702 (CO). 1H NMR ꢁ ppm: 0.90 (3H, t, J ¼ 7.2 Hz, CH3), (NH2), 1703 (CO). 1H NMR ꢁ ppm: 0.96 (6H, d, J ¼ 6.6 Hz,
1.39 (2H, sextet, J ¼ 7.2 Hz, CH2), 1.75 (2H, quintet, J ¼ 7.5 Hz, (CH3)2), 2.19 (1H, septet, J ¼ 6.9 Hz, CH), 2.68 (2H, d,
CH2), 2.77 (2H, t, J ¼ 7.5 Hz, CH2), 6.09 (2H, s, CH2CO), J ¼ 7.2 Hz, CH2), 6.10 (2H, s, CH2CO), 7.13–7.21 (2H, m,
0
0
0
0
0
0
0
7.13–7.20 (2H, m, C5 ,6 –H), 7.45–7.48 (1H, m, C7 –H), 7.60–7.69 C5 ,6 –H), 7.45–7.47 (1H, m, C7 –H), 7.63–7.69 (3H, m, C4 –H,
0
(3H, m, C4 –H, NH2), 7.89 (1H, t, J ¼ 7.8 Hz, C5–H), 8.20 (1H, d, NH2), 7.89 (1H, t, J ¼ 7.8 Hz, C5–H), 8.20 (1H, d, J ¼ 7.5 Hz, C4–
J ¼ 7.8 Hz, C4–H), 8.45 (1H, d, J ¼ 8.1 Hz, C6–H), 8.50 (1H, s, H), 8.46 (1H, d, J ¼ 7.8 Hz, C6–H), 8.50 (1H, s, C2–H). 13C NMR
C2–H). 13C NMR ꢁ ppm: 14.47, 22.50, 26.64, 29.65, 50.57, ꢁ ppm: 23.11, 27.65, 35.77, 50.65, 110.79, 118.98, 122.06,
110.67, 118.98, 121.99, 122.20, 125.70, 130.56, 131.30, 132.56, 122.23, 125.72, 130.56, 131.30, 132.62, 135.63, 136.40, 142.96,
135.66, 136.52, 142.99, 145.65, 156.48, 193.67. HRMS Calcd for 145.65, 155.75, 193.56. HRMS Calcd for C19H21N3O3S
C19H21N3O3S ([M þ H]þ): 372.1376, Found: 372.1377.
([M þ H]þ): 372.1376, Found: 372.1375.
4-[(2-Isopropyl-1H-benzimidazol-1-yl)acetyl]benzenesulfo-
4-{[2-(Methylsulfanyl)-1H-benzimidazol-1-yl]acetyl}benze-
namide (1 h). Yield 65%, m.p. 258–260 ꢀC. IR (ꢀ, cmꢁ1): 3341 nesulfonamide (1j). Yield 62%, m.p. 213–215 ꢀC. IR (ꢀ, cmꢁ1):
(NH2), 1706 (CO). 1H NMR ꢁ ppm: 1.30 (6H, d, J ¼ 6.6 Hz, 3319 (NH2), 1702 (CO). 1H NMR ꢁ ppm: 2.72 (3H, s, CH3), 5.98
0
0
(CH3)2), 3.18 (1H, septet, J ¼ 6.6 Hz, CH), 6.09 (2H, s, CH2CO), (2H, s, CH2CO), 7.15–7.23 (2H, m, C5 ,6 –H), 7.50–7.52 (1H, m,
0
0
0
0
0
7.13–7.22 (2H, m, C5 ,6 –H), 7.44–7.47 (1H, m, C4 –H), 7.61–7.64 C4 –H), 7.61–7.63 (1H, m, C7 –H), 7.68 (2H, s, NH2), 8.07 (2H, d,
(1H, m, C7 –H), 7.66 (2H, s, NH2), 8.07 (2H, d, J ¼ 8.4 Hz, J ¼ 8.1 Hz, C2,6–H), 8.34 (2H, d, J ¼ 8.1 Hz, C3,5–H). 13C NMR
0
C2,6–H), 8.34 (2H, d, J ¼ 8.4 Hz, C3,5–H). 13C NMR ꢁ ppm: 22.41, ꢁ ppm: 15.20, 50.96, 110.32, 118.26, 122.37(2C), 126.82, 129.82,
26.20, 50.57, 110.82, 119.04, 122.12, 122.32, 126.72, 129.85, 137.18, 173.72, 143.58, 149.29, 153.70, 193.05. HRMS Calcd for
136.28, 137.44, 142.71, 149.23, 161.05, 194.03. HRMS Calcd for C16H15N3O3S2 ([M þ H]þ): 362.0628, Found: 362.0628.
C18H19N3O3S ([M þ H]þ): 358.1220, Found: 358.1218.
2-Chloro-4-{[2-(methylthio)-1H-benzimidazol-1-yl]acetyl}-
2-Chloro-4-[(2-isopropyl-1H-benzimidazol-1-yl)acetyl]ben- benzenesulfonamide (2j). Yield 53%, m.p. 195–197 ꢀC. IR
zenesulfonamide (2 h). Yield 57%, m.p. 149–151 ꢀC. IR (ꢀ, cmꢁ1): 3362, 3256 (NH2), 1707 (CO). H NMR ꢁ ppm: 2.72
1
(ꢀ, cmꢁ1): 3375, 3278 (NH2), 1708 (CO). H NMR ꢁ ppm: 1.29 (3H, s, CH3), 6.01 (2H, s, CH2CO), 7.14–7.23 (2H, m, C5 ,6 –H),
1
0
0
0
0
(6H, d, J ¼ 6.3 Hz, (CH3)2), 3.16–3.24 (1H, m, CH), 6.13 (2H, s, 7.48–7.51 (1H, m, C7 –H), 7.60–7.63 (1H, m, C4 –H), 7.92 (2H, s,
CH2CO), 7.10–7.26 (2H, m, C5 ,6 –H), 7.48–7.50 (1H, m, C7 –H), NH2), 8.21 (2H, s, C5,6–H), 8.39 (1H, s, C3–H). 13C NMR ꢁ ppm:
0
0
0
0
7.61–7.63 (1H, m, C4 –H), 7.93 (2H, s, NH2), 8.20 (2H, s, 15.22, 51.09, 110.34, 118.26, 122.34, 122.38, 127.80, 130.17,
C5,6–H), 8.40 (1H, s, C3–H). 13C NMR ꢁ ppm: 22.39, 26.11, 131.86, 132.04, 137.64, 138.43, 143.56, 145.91, 153.72, 192.37.
50.78, 111.00, 118.88, 122.33, 122.48, 127.81, 130.04, 131.71, HRMS Calcd for C16H14ClN3O3S2 ([M þ H]þ): 396.0238, Found:
132.19, 136.08, 138.64, 142.25, 145.81, 161.03, 193.22. HRMS 396.0241.
Calcd for C18H18ClN3O3S ([M þ H]þ): 392.0830, Found:
3-{[2-(Methylthio)-1H-benzimidazol-1-yl]acetyl}benzene-
sulfonamide (3j). Yield 54%, m.p. 242–244 ꢀC. IR (ꢀ, cmꢁ1):
392.0827.
3-[(2-Isopropyl-1H-benzimidazol-1-yl)acetyl]benzenesulfo- 3288 (NH2), 1706 (CO). 1H NMR ꢁ ppm: 2.72 (3H, s, CH3), 6.99
namide (3 h). Yield 51%, m.p. 267–269 ꢀC. IR (ꢀ, cmꢁ1): 3319 (2H, s, CH2CO), 7.15–7.23 (2H, m, C5 ,6 –H), 7.50–7.41(1H, m,
0
0
(NH2), 1706 (CO). 1H NMR ꢁ ppm: 1.30 (6H, d, J ¼ 6.6 Hz, C7 –H), 7.60–7.63 (3H, m, C4 –H, NH2), 7.88 (1H, t, J ¼ 7.5 Hz,
0
0
(CH3)2), 3.19 (1H, septet, J ¼ 6.9 Hz, CH), 6.12 (2H, s, CH2CO), C5–H), 8.19 (1H, d, J ¼ 7.5 Hz, C4–H), 8.45 (1H, d, J ¼ 7.8 Hz,

128 A. Zubriene_ et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 124–131
Scheme 1. Synthesis of compounds 1–3(a–j).
C6–H), 8.50 (1H, s, C2–H). 13C NMR ꢁ ppm: 15.24, 50.90,
110.36, 118.28, 122.28, 122.37, 125.69, 130.69, 131.43, 132.47,
135.45, 137.76, 143.62, 145.72, 153.65, 192.83. HRMS Calcd for
C16H15N3O3S2 ([M þ H]þ): 362.0628, Found: 362.0627.
Table 1. Dissociation constants for compounds binding to five human
recombinant CA isoforms, determined by TSA (37 ꢀC, pH 7.0).
Dissociation constants
Kd (nM) to CA isoforms
Inhibitors
R3
CA I CA II CA VII CA XII CA XIII
Protein preparation
1a
2a
3a
4a
1b
2b
3b
4b
1c
2c
3c
4c
1d
2d
3d
4d
1e
33.0
12.0
4350
4.0
8.3
4340
20.0
4.0
6670
1000
28.0
11.0
909.0
833.0
12 500 2380
2080
769.0
200.0
83.0
19.0
Expression and purification of CA I, II, VII, XII and XIII has
been previously described: CA I by Baranauskiene et al.28, CA II
by Cimmperman et al.29, CA VII and XIII by Sudzius et al.21 and
CA XII by Jogaite et al.30.
H
11 100 1560
670.0
76.9
23.2
10.0
5.88
9090
6.3
10.5
7690
Me
Determination of compound binding to CAs by thermal
shift assay
16 670 16 670 2000
10 000 2000
2500
20.0
5.0
5710
400.0
6250
1050
1000
400.0
62.5
66.7
8.33
10.0
5000
7140
4.17
14.3
4000
1000
The thermal shift assay (TSA) measurements were performed in a
Corbett Rotor-Gene 6000 (QIAGEN Rotor-Gene Q, Sydney,
Australia) instrument using the blue channel (excitation 365 ꢂ 20,
detection 460 ꢂ 15 nm). Samples contained 10 mM protein, 0–
200 mM compound, 50 mM solvatochromic dye ANS (8-anilino-1-
naphthalene sulfonate) and 50 mM phosphate buffer containing
100 mM NaCl at pH 7.0, with the final DMSO concentration at
2%. The applied heating rate was 1 ꢀC/min. Data analysis was
performed as previously described31.
CH2OH
12 500 2000
5560
8 330
167.0
333.0
286.0
6.67
1110
1000
50.0
143.0 100.0 20.0
C6H5CH2 8.33
2.86
1667
1560
5.0
6.25
7690
556.0 1670
5.56
3.57
6670
8.33
12 500 5000
3330
20.0
7.69
2500
8330
8.33
4.76
4760
7140
7.14
2.86
1000
769.0
556.0
66.7
2e
3e
4e
1f
2f
3f
Et
11.5
25 000 14 300 3330
1820
667.0
62.5
400.0
28.6
5.56
17.2
4.17
Results
Pr
25 000 11 100 1000
A series of benzenesulfonamides with a benzimidazole moiety
1–4(a–j) were designed as CA inhibitors (Figure 2). The
procedures for N-alkylation of 2-substituted benzimidazoles
(a–j) with 5-(bromoacetyl)-2-chlorobenzenesulfonamide (4)
have been described previously23. N-substituted benzimidazole
derivatives 1–3(a–j) were prepared by N-alkylation of benzimi-
dazoles a–j with 4-(bromoacetyl)benzene-sulfonamide (1),
4-(bromoacetyl)-2-chlorobenzenesulfonamide (2) and 3-(bromoa-
cetyl)benzenesulfonamide (3) (Scheme 1).
The dissociation constants for compounds 1–4(a–j) binding to
five CA isozymes CA I, II, VII, XII and XIII are listed in Table 1.
AZM and EZA were used as controls. The binding of these
compounds was determined by the TSA. Figures 3 and 4 show
representative binding data obtained by TSA. The data in Table 1
and Figure 5 show that the position of the sulfonamide group on
the benzene ring significantly influences the binding strength to
the various CA isoforms. In most cases, the para-substituted
benzenesulfonamide (compounds 1–2(a–j)) had higher binding
affinity than its meta-substituted benzenesulfonamide equivalent
(compounds 3–4(a–j)) (Figure 3). All 1–2(a–j) derivatives were
nanomolar inhibitors of CA I, II, VII and XIII (dissociation
constants (Kds) in the range of 1.6–286 nM). The binding of
1–2(a–j) to CA XII was weaker (Kd ranged from 62.5 nM to
8.3 mM). Most compounds of the 3–4(a–j) series were micromolar
inhibitors of all investigated CA isoforms (Kds in the range of
0.1–100 mM). Compounds bearing a chlorine atom in the ortho
4f
10 000 833.0 2500
1430
1000
112.0
1430
50.0
2.50
1g
2g
3g
4g
1h
2h
3h
4h
1i
2i
3i
4i
1j
11.1
3.33
4.17
2.70
20.0
3.33
Bu
667.0 2860 25 000 11 100 667.0
10 000 454.0 3 330
2860
476.0
333.0
400.0
33.3
40.0
4.0
5.0
5.56
14.3
20.0
5.0
i-Pr
i-Bu
SCH3
2500
4540
8.33
6.67
16 700 100 000 20 000 5000
667.0 2000
588.0
833.0
500.0
370.0
33.3
40.0
1430
2860
38.4
5.56
14.3
5.56
5.0
125.0 5 000 25 000 8330
3330
3.57
1.67
1670
5000
384.0 1000
2000
2000
66.7
4.0
18.2
4.54
2j
3j
4j
3.33
3450
1000
9.09
25 000 5000
833.0
1110
100.0
13.0
1670
1430
1400
36.0
Indapamide
EZA
AZM
10 000 300.0 300.0
14
1400
0.71
17.0
0.71
17.0
133.0
50.0
Average standard deviation for TSA data was below 25%.
position of the benzenesulfonamide ring were in most cases more
effective CA inhibitors than compounds without a chlorine
substitution in this position: (2(a–j) versus 1(a–j) and 4(a–j)
versus 3(a–j)). For example, compound 2g exhibited 25 times
greater affinity than 1g to CA XIII whereas compound 4h was 34

DOI: 10.3109/14756366.2012.757223
Benzenesulfonamides with benzimidazole moieties 129
70
60
50
40
30
20
10
and e–j for CA I varied only in the narrow nanomolar range (from
3.5 to 11.1 nM). The Kds for CA I were higher for compounds 1a
and 1d, 33 and 143 nM, respectively. CA II was best inhibited
(Kd ¼ 4–100 nM), while CA XII (Kd ¼ 0.48–8.33 mM) least
inhibited. Compounds bearing iso-propyl 1 h and S-methyl 1j
substitution on benzimidazole ring were good inhibitors of CA I
and CA II (Kds were about 4–5 nM), whereas iso-butyl-substituted
compound 1i potently bound CA VII (Kd ¼ 5.56 nM).
Benzyl-substituted 1d had the weakest binding affinity, especially
to CA XII (Kd ¼ 8.33 mM), but it was selective for CA VII
(Kd ¼ 20 nM).
2d
0
200
100
80
60
40
20
0
4d
0
N-alkylated benzimidazoles (2(a–j)) were the best inhibitors of
CA I, II, VII and XIII exhibiting affinities in the nanomolar range.
These compounds were weaker CA XII inhibitors by about one
order of magnitude. Compounds with methyl, ethyl, propyl and
butyl substituents had a high affinity for CA I (Kd ranged from 2.9
to 5.9 nM), CAII (Kd from 10.5 to 2.70 nM), CA VII (Kd from 11.1
to 3.33 nM) and CA XIII (Kd from 23.3 to 2.50 nM).
Benzimidazoles that contained a branched chain aliphatic tail
(compounds 2 h, 2i) had four to eight times reduced affinity for
CA II (Kds were 14.3 nM for both compounds), CA XII (Kds were
333 and 500 nM, respectively) and CA XIII (Kds were 40 nM for
both compounds), as compared to compounds 2f and 2g which
contained an unbranched tail. Benzimidazole with an S-methyl
substituent, 2j, bound to CA isoforms similarly as the propyl-
substituted compound, 2f.
200
50
60
70
T, °C
2d
70
60
1d
4d
3d
3(a–j) N-benzimidazoles were the weakest CA inhibitors (Kds
in the range of 0.67–100 mM). Only three of them (3g, i, j) had a
higher affinity for CA I and XIII, than for the other tested CAs.
For example, 3i binds to CA I 40 times stronger than CA II, 200
times stronger than CA VII, 66 times stronger than CA XII and 10
times stronger than CA XIII.
4(a–j) N-substituted benzimidazoles (Kds in the range of
0.33–11.1 mM) were slightly better inhibitors than the 3(a–j)
compounds. They behaved as weak inhibitors against CA I (Kds
are in the micromolar range 3.3–11 mM). Six derivatives, namely
4a–c, e, g and h, were better inhibitors of CA XIII, with Kds in the
range of 0.33–0.67 mM. Compound 4b, which is structurally the
most similar to the clinically used indapamide, was slightly less
potent. The binding affinity of 4b and indapamide to CA I was
similar, but 4b bound about seven times weaker to CA II and CA
VII and four times weaker to CA XII and CA XIII than
indapamide.
−5
−4
−3
0
10
10
10
L , M
t
Figure 3. TSA data of 1–4d binding to CA XIII. The top panels compare
denaturation curves observed by fluorescence at 0–200 mM added
compound concentrations. The bottom panel shows the dependence of
the protein melting temperatures Tm on compound concentrations.
64
CA XII
K (2j) = 66.7 nM
d
62
2d
2h
60
58
56
54
52
50
K (2a) = 833 nM
d
Discussion
Indapamide, which is a clinically used diuretic, may act as an
inhibitor of various CA isozymes present in the kidneys and blood
vessels, thus lowering the blood pressure in patients with
hypertension and type-2 diabetes32. It has been reported
previously by Supuran’s group that the inhibition constants (Kis)
of indapamide for several CAs were: 51 900 nM for hCA I,
2520 nM for hCA II, 0.23 nM for hCA VII, 10 nM for CA XII and
13 nM for murine CA XIII19. The TSA data confirmed that
indapamide was a weak inhibitor of the isoform CA I
(Kd ¼ 10 000 nM). However, the Kd values toward other CAs
differed from Ki values (Kds for CA II and VII were 300 nM, CA
XII ¼ 1400 nM and CA XIII ¼ 100 nM). These discrepancies
could be observed due to the different techniques utilized for
compound binding and inhibition measurements. Usually, the
TSA and other biophysical binding techniques, such as isothermal
titration calorimetry, yield dissociation constants that are close to
the inhibition constants determined by the stop-flow carbon
0
−6
−5
−4
−3
10
10
10
10
L M
t
Figure 4. TSA data of 2a, 2d, 2h and 2j binding to CA XII.
times more potent than 3h for CA VII. In contrast, CA I bound
4(a–j) weaker than 3(a–j). The highest difference was observed
between compounds 4i and 3i, with the latter being 27 times
stronger CA I inhibitor than 4i.
The binding of most benzenesulfonamides of types 1–4(a–j) to
CA I, II, VII, XII and XIII did not strongly depend on the
substituent (R3) on the benzimidazole ring. By comparing the
series of compounds bearing methyl 1–4b, hydroxymethyl 1–4c,
ethyl 1–4e, propyl 1–4f and butyl 1–4g substituents, we showed
that the tail length did not affect the binding affinity by more than
two times to any of the tested CAs. For example, the Kds for 1b, c
dioxide hydration assay28,33
.
Numerous indapamide analogues were designed in search of
new, more potent and selective CA inhibitors. A diminished

130 A. Zubriene_ et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 124–131
Figure 5. The binding constants of 1i, 2i, 3i
and 4i to CAI, II, VII, XII and XIII.
CA I CA II CA VII CA XII CA XIII
1.E+09
1.E+08
1.E+07
1.E+06
1.E+05
1.E+04
1i
2i
3i
4i
4. Supuran CT. Carbonic anhydrases – an overview. Curr Pharm Des
2008;14:603–14.
affinity of derivatives of both series 3(a–j) and 4(a–j), which are
most structurally related to indapamide when compared with the
corresponding inhibitors 1(a–j) and 2(a–j), towards isozyme I, II,
VII, XII and XIII was observed. This is thought to occur due to
the benzimidazole group in the meta position in relation to the
benzenesulfonamide moiety, which is present in the first series of
compounds. This could lead to steric hindrance when binding to
the Zn ion within the enzyme active site.
It was interesting to notice that the chlorine atom in the ortho
position of the sulfonamide group of the compounds 2(a–j) and
4(a–j) increased the affinity to all CAs, with the exception of CA
I. All compounds of the series 4(a–j) with a chlorine substitution
in the ortho position possessed a lower binding potency with CA I
5. Nordfors K, Haapasalo J, Korja M, et al. The tumour-associated
carbonic anhydrases CA II, CA IX and CA XII in a group of
medulloblastomas and supratentorial primitive neuroectodermal
tumours: an association of CA IX with poor prognosis. BMC
Cancer 2010;10:148–58.
6. Chien MH, Ying TH, Hsieh YH, et al. Tumor-associated
carbonic anhydrase XII is linked to the growth of primary oral
squamous cell carcinoma and its poor prognosis. Oral Oncol
2012;48:417–23.
7. Swietach P, Hulikova A, Vaughan-Jones RD, Harris AL. New
insights into the physiological role of carbonic anhydrase IX in
tumour pH regulation. Oncogene 2010;29:6509–21.
8. Said HM, Supuran CT, Hageman C, et al. Modulation of carbonic
anhydrase 9 (CA9) in human brain cancer. Curr Pharm Des
2010;16:3288–99.
9. Pastorekova S, Parkkila S, Zavada J. Tumor-associated carbonic
anhydrases and their clinical significance. Adv Clin Chem
2006;42:167–216.
10. Winum JY, Carta F, Ward C, et al. Ureido-substituted sulfamates
show potent carbonic anhydrase IX inhibitory and antiproliferative
activities against breast cancer cell lines. Bioorg Med Chem Lett
2012;22:4681–85.
than compounds 3(a–j) without
a chlorine substitution.
Compound 3i, bearing a bulky iso-butyl substituent in the
benzimidazole moiety, behaved as a selective CA I inhibitor,
having more than 10 times higher affinity against CA I isozyme as
compared to other CAs.
Conclusions
11. Rodriguez OM, Maresca A, Tempera CA, et al. N-b-glycosyl
sulfamides are selective inhibitors of the cancer associated carbonic
anhydrase isoforms IX and XII. Bioorg Med Chem Lett
2011;21:4447–50.
12. Touisni N, Maresca A, McDonald PC, et al. Glycosyl coumarin
carbonic anhydrase IX and XII inhibitors strongly attenuate the
growth of primary breast tumors. J Med Chem 2011;54:8271–7.
13. Supuran CT. Carbonic anhydrase inhibition with natural products:
novel chemotypes and inhibition mechanisms. Mol Divers
2011;15:305–16.
Benzenesulfonamides bearing benzimidazole substituents were
synthesized and assayed as inhibitors of five CA isozymes, CA I,
II, VII, XII and XIII. 4-[(2-Substituted-1H-benzimidazol-1-
yl)acetyl]benzenesulfonamides 1(a–j) were more than two
orders of magnitude stronger CA inhibitors than 3-[(2-substi-
tuted-1H-benzimidazol-1-yl)acetyl]benzenesulfonamides 3(a–j).
Compounds of the series 1(a–j) and 2(a–j) were nanomolar
inhibitors of CA I, II, VII and XIII (Kds in the range of
1.6–286 nM).
14. Supuran CT, Scozzafava A, Conway J. Carbonic anhydrase – its
inhibitors and activators. Boca Raton, FL: CRC Press; 2004:1–363.
15. Supuran CT, Innocenti A, Mastrolorenzo A, Scozzafava A. Antiviral
sulfonamide derivatives. Mini Rev Med Chem 2004;4:189–200.
16. Carta F, Scozzafava A, Supuran CT. Sulfonamides: a patent review
(2008–2012). Expert Opin Ther Pat 2012; 22:747–58.
17. Carta F, Supuran CT, Scozzafava A. Novel therapies for glaucoma: a
patent review 2007–2011. Expert Opin Ther Pat 2012; 22:79–88.
18. Thiry A, Dogne JM, Supuran CT, Masereel B. Anticonvulsant
sulfonamides/sulfamates/sulfamides with carbonic anhydrase inhi-
bitory activity: drug design and mechanism of action. Curr Pharm
Des 2008;14:661–71.
Declaration of interest
The authors report no conflicts of interest.
This research was funded by a grant (No. LIG-09/2012) from the
Research Council of Lithuania. The authors also acknowledge FP7-
REGPOT-2009-1 grant ‘‘MoBiLi’’, agreement No.: 245721, and the
COST projects TD0905 and CM0804. This work was partly supported by
the Lithuanian Science Council Student Research Fellowship Award (No.
SMT12P-079, student – MK).
19. Temperini C, Cecchi A, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors. interaction of indapamide and related diuretics
with 12 mammalian isozymes and X-ray crystallographic studies for
the indapamide-isozyme II adduct. Bioorg Med Chem Lett
2008;18:2567–73.
20. Capkauskaite E, Zubriene A, Baranauskiene L, et al. Design of [(2-
pyrimidinylthio)acetyl]benzenesulfonamides as inhibitors of human
carbonic anhydrases. Eur J Med Chem 2012;51:259–70.
References
1. Alterio V, Fiore AD, D’Ambrosio K, et al. Multiple binding modes
of inhibitors to carbonic anhydrases: how to design specific drugs
targeting 15 different isoforms? Chem Rev 2012;112:4421–68.
2. Aggarwal M, Kondeti B, McKenna R. Insights towards sulfonamide
drug specificity in a-carbonic anhydrases. Bioorg Med Chem 2012;
doi: 10.1016/j.bmc.2012.08.019.
3. Supuran CT. Carbonic anhydrases as drug targets – an overview. 21. Sudzius J, Golovenko D, Matuliene J, et al. 4-[n-(substituted
Curr Top Med Chem 2007;7:825–33. 4-pyrimidinyl)amino]benzenesulfonamides as inhibitors of carbonic

DOI: 10.3109/14756366.2012.757223
Benzenesulfonamides with benzimidazole moieties 131
anhydrase isozymes I, II, VII and XIII. Bioorg Med Chem 2010;18: 28. Baranauskiene L, Hilvo M, Matuliene J, et al. Inhibition and binding
7413–21.
studies of carbonic anhydrase isozymes I, II and IX with
benzimidazo[1,2-c][1,2,3]thiadiazole-7-sulphonamides.
Inhib Med Chem 2010;25:863–70.
22. Dudutiene V, Baranauskiene L, Matulis D. Benzimidazo[1,2-
c][1,2,3]thiadiazole-7-sulfonamides as inhibitors of carbonic anhy-
drase. Bioorg Med Chem Lett 2007;17:3335–8.
23. Capkauskaite E, Baranauskiene L, Golovenko D, et al. Indapamide-
like benzenesulfonamides as inhibitors of carbonic anhydrases I, II,
VII, and XIII. Bioorg Med Chem 2010;18:7357–64.
J Enzyme
29. Cimmperman P, Baranauskiene L, Jachimoviciute S, et al.
A quantitative model of thermal stabilization and destabilization
of proteins by ligands. Biophys J 2008;95:3222–31.
30. Jogaite V, Zubriene A, Michailoviene V, et al. Characterization of
human carbonic anhydrase XII stability and inhibitor binding.
Bioorg Med Chem 2012; doi: 10.1016/j.bmc.2012.10.016.
24. Fujikura T, Miigata K, Hashimoto S, et al. Studies on benzene-
sulfonamide derivatives with alpha- and beta-adrenergic antagonis-
tic and antihypertensive activities. Chem Pharm Bull (Tokyo) 31. Kazlauskas E, Petrikaite V, Michailoviene V, et al. Thermodynamics
1982;30:4092–101.
25. Zanka A, Kubota A. Practical and efficient chlorination of
of aryl-dihydroxyphenyl-thiadiazole binding to human Hsp90. PLoS
ONE 2012;7:e36899.
deactivated anilines and anilides with ncs in 2-propanol. Synlett 32. Supuran CT. Diuretics: from classical carbonic anhydrase inhibitors
1999;12:1984–6.
to novel applications of the sulfonamides. Curr Pharm Des
2008;14:641–8.
33. Zubriene A, Kazlauskas E, Baranauskiene L, et al. Isothermal
titration calorimetry and thermal shift assay in drug design.
European pharmaceutical review 2011;16:56–9.
26. Nickson TE, Roche-Dolson CA. A convenient procedure for the
chlorination of deactivated anilines. Synthesis 1985;6/7:669–70.
27. Phillips MA. The formation of 2-substituted benzimidazoles.
J Chem Soc 1928;CCCXVII:2393–9.