Indapamide-like benzenesulfonamides as inhibitors of carbonic anhydrases I, II, VII, and XIII

Article abstract of DOI:10.1016/j.bmc.2010.09.016

A series of novel 2-chloro-5-[(1-benzimidazolyl- and 2- benzimidazolylsulfanyl)acetyl]benzene-sulfonamides were designed and synthesized. Their binding to recombinant human carbonic anhydrase (hCA) isozymes I, II, VII, and XIII was determined by isothermal titration calorimetry and thermal shift assay. The designed S-alkylated benzimidazole derivatives exhibited stronger binding than the indapamide-like N-alkylated benzimidazoles, with the K_dreaching about 50-100 nM with drug-targeted hCAs VII and XIII. The cocrystal structures of selected compounds with hCA II were determined by X-ray crystallography, and structural features of the binding event were revealed.

Full text of DOI:10.1016/j.bmc.2010.09.016

Bioorganic & Medicinal Chemistry 18 (2010) 7357–7364
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Indapamide-like benzenesulfonamides as inhibitors of carbonic anhydrases
I, II, VII, and XIII
Edita Capkauskaite a,b, Lina Baranauskiene a, Dmitrij Golovenko c, Elena Manakova c,
ˇ
˙
˙
c
b
a,
⇑
ˇ
ˇ
Saulius Grazulis , Sigitas Tumkevicius , Daumantas Matulis
a
ꢀ
ˇ
Laboratory of Biothermodynamics and Drug Design, Institute of Biotechnology, Graiciuno 8, Vilnius LT-02241, Lithuania
b Department of Organic Chemistry, Faculty of Chemistry, Vilnius University, Naugarduko 24, Vilnius LT-03225, Lithuania
c
ꢀ
ˇ
Laboratory of Protein–DNA Interactions, Institute of Biotechnology, Graiciuno 8, Vilnius LT-02241, Lithuania
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 May 2010
Revised 2 September 2010
Accepted 7 September 2010
Available online 15 September 2010
A
series of novel 2-chloro-5-[(1-benzimidazolyl- and 2-benzimidazolylsulfanyl)acetyl]benzene-
sulfonamides were designed and synthesized. Their binding to recombinant human carbonic anhydrase
(hCA) isozymes I, II, VII, and XIII was determined by isothermal titration calorimetry and thermal shift assay.
The designed S-alkylated benzimidazole derivatives exhibited stronger binding than the indapamide-like
N-alkylated benzimidazoles, with the Kd reaching about 50–100 nM with drug-targeted hCAs VII and XIII.
The cocrystal structures of selected compounds with hCA II were determined by X-ray crystallography, and
structural features of the binding event were revealed.
Keywords:
Carbonic anhydrase isozymes I, II, VII, and
XIII
Ó 2010 Elsevier Ltd. All rights reserved.
Isothermal titration calorimetry
Thermal shift assay
ThermoFluorÒ
Sulfonamides
X-ray crystallography
1. Introduction
Inhibitor binding potency depends on the acidity of the sulfon-
amide group. Therefore, inhibitors with electron-withdrawing
Carbonic anhydrases (CAs, also known as carbonate dehydrata-
ses, EC 4.2.1.1) are ubiquitous zinc-containing metalloenzymes
present in prokaryotes and eukaryotes.1 They catalyze the hydra-
tion of carbon dioxide to bicarbonate and the corresponding dehy-
dration of bicarbonate in acidic medium, to regenerate CO2.2 CA
isoforms are found in a variety of tissues, where they participate
in numerous important biological processes such as pH balance,
CO2 homeostasis, respiration, carbon dioxide and ion transport,
ureagenesis, gluconeogenesis, lipogenesis, electrolyte secretion,
bone resorption, calcification and tumorigenicity.1,3–5
Many of these isozymes are important targets for the design of
inhibitors with clinical applications. The most prominent class of
CA inhibitors consists of aromatic/heterocyclic sulfonamides,
which have been studied for their use as antiglaucoma, antitumor,
antiobesity or anticonvulsant drugs.6,7 However, there are several
critical problems in the design of CA inhibitors with pharmacolog-
ical applications: the high number of isoforms (12 active forms in
human), their rather diffuse localization in many tissues/organs,
and the lack of isozyme selectivity for the presently available
inhibitors.8–10
groups that increase the sulfonamide acidity, such as chlorine and
carbonyl groups, also increase inhibitor potency. Substituents on
the benzene ring could also improve inhibitor selectivity. Despite
the progress made in the understanding of quantitative struc-
ture–activity relationships of CA inhibitors,7,11 most correlations
have been drawn based on activity-inhibition measurements. How-
ever, it is important to also measure binding by using biophysical
thermodynamic techniques.12 Here, we report the use of isothermal
titration calorimetry (ITC) and thermal shift assay (TSA, also called
ThermoFluorÒ, differential scanning fluorimetry) to measure
inhibitor binding to CAs. ITC has been routinely used to measure
ligand–protein binding thermodynamics.13,14 However, it is not
appropriate for determining weak (millimolar) or extremely tight
dissociation constants (subnanomolar Kds require displacement
ITC15) and consumes extensive amounts time and protein. In con-
trast, TSA is a rapid screening method used in pharmaceutical
industry for the identification of specific binders and requires lesser
amounts of protein.16,17 The method is based on the protein melting
temperature (Tm) shift that occurs upon ligand binding; the Tm is
observed by following intrinsic or extrinsic fluorescence changes
upon heat-induced protein unfolding. The employment of two
techniques to determine binding reactions reduces the uncertainty
of the measurements.
⇑
Corresponding author.
E-mail address: matulis@ibt.lt (D. Matulis).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.016

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7358
Indapamide (Fig. 1) has been used for the treatment of patients
Using the same reaction conditions employed for the syntheses
of 3a–k, thiones 2l–o reacted with compound 1 to form the corre-
sponding S-alkylated products 3l–o. Structural assignments were
made based upon their 1H and 13C NMR spectra. In the 1H NMR
spectra of N-alkylated benzimidazoles 3a–k, a singlet was ob-
served that corresponded to the NCH2CO group in the region of
5.97–6.08 ppm. The corresponding 1H signal for the SCH2CO group
of compounds 3l–o was observed at 5.02–5.16 ppm; in comparison
to the NCH2CO group, the signal is shifted upfield due to the lower
electronegativity of the sulfur atom. An analogous difference in the
chemical shifts for the corresponding carbons was also observed in
the 13C NMR spectra: for compounds 3a–k, the NCH2 signal ap-
pears in the region 50.53–55.96 ppm, whereas the SCH2 signal
for compounds 3l–o is observed at 39.66–40.25 ppm. The structure
of 3m is shown in Figure 1.
with hypertension and type-2 diabetes. The beneficial effect might
be due to its potent inhibition of CA isoforms present in kidneys
and blood vessels, which would thus explain both the blood pres-
sure lowering effects as well as the organ-protective activity of the
drug.18 It has been reported that the Kis of indapamide to a series of
CAs are as follows: 0.23 nM with hCA VII, 51,900 nM for hCA I,
2520 nM with hCA II, and 13 nM with murine CA XIII.19 Our ITC
and TSA measurements confirm that hCA I is poorly inhibited by
indapamide. However, the potency towards hCA VII is only about
300 (TSA) to 1800 (ITC) nM. This discrepancy may be due to the
different nature of the techniques employed. Therefore, we believe
that it is important to confirm the potency of inhibitors by numer-
ous techniques to avoid technique-specific effects.
Several our synthetic compounds reported here, 3l, 3m, and 3n,
were found to be more potent than indapamide towards the tested
CAs, making them potential candidates for further development.
Thione 2m was prepared according to a previously reported
procedure.30 The synthesis of 5-bromo-1,3-dihydro-2H-benzimid-
azole-2-thione (2n) was accomplished via the bromination of
2-nitroaniline31 and subsequent reduction and cyclization.32 1,3-
Dihydro-2H-imidazo[4,5-c]quinoline-2-thione (2o) was obtained
by the reaction of 3,4-quinolinediamine33 with carbon disulfide.
2. Results and discussion
2.1. Chemistry
A series of substituted benzene sulfonamides were designed as
CA inhibitors. The chemistry employed for the synthesis of the
compounds reported is shown in Scheme 1.
2.2. Binding studies
The binding affinities of indapamide-like benzenesulfonamides
to recombinant human carbonic anhydrase isozymes I, II, VII, and
XIII were determined by thermal shift assay and isothermal titra-
tion calorimetry. Figure 2 shows typical thermal shift assay data
for inhibitor 3f binding to hCA II. Panel A shows fluorescence
profiles of CA heat-induced denaturation. The midpoints of the
unfolding transitions are plotted in panel B, giving compound dos-
ing curves as a function of the concentration of added compound.
Figure 3 shows typical ITC data for compound 3l binding to hCA
II. Panel A shows a raw ITC data curve, and Panel B shows the
integrated ITC curve. Fitting both TSA and ITC dosing curves
yielded the binding constants for the binding of all tested
compounds to the CAs. The observed dissociation constants for
compounds 3a–o are listed in Table 1.
2-Substituted benzimidazoles 2a–i were prepared from 1,2-
benzenediamine and the appropriate carboxylic acids according
to a Phillips procedure.20 The physical characteristics of the
2-substituted benzimidazoles corresponded with the reported
data.20–24 Compound 2j was synthesized from 2-(chloromethyl)-
1H-benzimidazole via nucleophilic substitution of the chloride with
morpholine.25 2-Methylthiobenzimidazole (2k) was obtained from
1,3-dihydro-2H-benzimidazole-2-thione (2l)26 according to a pre-
viously reported procedure.27 Synthesis of 5-(2-bromoacetyl)-
2-chlorobenzenesulfonamide (1) was performed from commercially
available 1-(4-chlorophenyl)ethanone as reported previously.28,29
N-Alkylation of 2a–k with 5-(bromoacetyl)-2-chlorobenzene-
sulfonamide (1) to give N-substituted benzimidazole derivatives
3a–k was carried out in the presence of sodium acetate in tetrahy-
drofuran at room temperature (Scheme 1). Using sodium methox-
ide as a base and performing the reaction in boiling methanol led
to an inseparable mixture of products. It should be noted that bet-
ter results in the N-alkylation reaction were obtained when a slight
excess of the corresponding benzimidazole 2a–k was used.
Conversely, employing an excess of compound 1 results in the dial-
kylation of the benzimidazole ring.
The S-alkylated imidazo derivatives 3l–o have dissociation
constants in the range of 0.02–3.1
alkylated benzimidazoles 3a–k, which have dissociation constants
in the range of 0.3–40 M, as determined by TSA. Compound 3l
bound hCA VII more tightly than other CAs. However, compounds
3m–o bound CA II most strongly (Table 1).
There was some systematic discrepancy between TSA and ITC
data observed with hCA II and hCA VII, which was not observed
with the other two CAs. Both ITC and TSA experiments were re-
peated several times, and the discrepancy appeared to be out of
the range of the standard deviations for both methods.
lM and bind stronger than N-
l
O
O
HN
N
H
N
Indapamide inhibition constants (Ki) have been previously
determined for most CA isozymes.19 Kis for CAs I, II, VII, and XIII
OH
Cl
were reported to equal 51.9, 2.52, 0.00023, and 0.013 lM, respec-
Cl
SO2NH2
tively. These numbers differ from our values measured by ITC and
TSA (Table 1). CAs I and II appear to bind indapamide slightly tigh-
ter than previously determined, whereas CAs VII and XIII appear to
bind indapamide significantly weaker than previously determined.
The largest difference was observed for hCA VII. Our determined Kd
value is about 1000 times weaker than previously determined.
Despite a general agreement between the three techniques,12 quite
often there is a discrepancy between activity and binding measure-
ments, likely due to the significant differences between the tech-
niques: ITC is done at constant temperature, thermal shift assay
is performed by heating the sample, and the inhibition measure-
ments involve stop-flow kinetics, the only method to follow enzy-
matic activity.
SO2NH2
Chlorthalidone
Indapamide
O
S
N
N
O
H
Cl
SO2NH2
O
3m
Figure 1. The chemical structure of indapamide, chlorthalidone, and 3m.

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E. Capkauskaite˙ et al. / Bioorg. Med. Chem. 18 (2010) 7357–7364
7359
R
N
O
N
N
i
R
Cl
Cl
N
H
2a-k
3a-k
SO2NH2
O
O
Br
H
S
N
R'
R'
N
i
+
S
HN
R'
Cl
N
H
2l-n
SO2NH2
SO2NH2
O
R'
N
3l-n
1
S
N
H
N
i
HN
S
Cl
N
N
H
SO2NH2
3o
2o
a: R=H;
b: R=Me;
h: R=CHMe2;
i: R=CH2CHMe2;
c: R=CH2OH;
d: R=CH2Ph;
e: R=CH2Me;
f: R=(CH2)2Me;
g: R=(CH2)3Me;
j: R=CH2N(CH2CH2)2O;
k: R=SMe;
l: R'=H;
m:R'+R'=OCH2CH2O;
n: R'=Br, R'=H.
Scheme 1. Synthesis of compounds 3a–o. Reagents and conditions: (i) NaOAc (1.15 equiv), THF, room temperature, 24 h.
0.02
A
B
A
60
50
40
30
20
10
0
0.00
-0.02
-0.04
-0.06
-0.08
-0.10
-0.12
40
50
60
t,ºC
70
80
0
20
40
60
80
100
Time,min
64
62
60
58
56
54
1
0
B
-1
-2
-3
-4
-5
-6
1.E-07
1.E-06
1.E-05
Lt, M
1.E-04
1.E-03
0
0.5
1
1.5
2
Molar ratio
Figure 2. Thermal shift assay data of compound 3f binding to hCA II. Panel A—raw
data: addition of different compound concentrations increases the protein melting
Figure 3. ITC data of 3l binding to hCA II. Panel A—raw data, panel B—integrated
data.
temperature (}—200
lM, h—39 lM, 4—17 lM, ꢀ—7 lM, and +—no ligand). Panel
B—integrated compound dosing curve.
II were solved by X-ray crystallography (Fig. 4). Comprehensive
structural analysis would require comparison of binding data with
structural data for all tested CAs. However, crystallization of other
CAs bound with the compounds has been unsuccessful to date. The
2.3. Crystallography
To determine the structural features of compound binding to
CAs, the structures of compounds 3m, 3n, and 3o bound to hCA

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E. Capkauskaite˙ et al. / Bioorg. Med. Chem. 18 (2010) 7357–7364
7360
Table 1
Dissociation constants (in
compounds revised by isothermal titration calorimetry (ITC values given in brackets)
at 37 °C
3n and 3m is similar. Such conformation of inhibitors 3n and 3m
lM) determined by thermal shift assay and for several
could be maintained through the hydrophobic contacts which their
large aromatic multiring systems make with the protein pocket.
The structural data show that hCAII-bound ligands 3m–o have lar-
ger hydrogen bond pattern and bigger area of hydrophobic interac-
tions than indapamide (Table 3).
hCA I
hCA II
hCA VII
hCA XIII
3a
3b
11
10
1.6
2.0
1.0
2.5
0.7
0.4
3c
3d
7.1
8.3
1.0
1.6
0.4
3.3
0.3
1.0
3. Conclusions
3e
7.1
0.6
1.7
0.4
3f
3g
3h
3i
3j
3k
3l
3m
10
10
4.5
3.3
40
5.0
1.0 (1.3)
3.1 (2.2)
1.3 (1.4)
1.3
0.8
0.5
0.7
0.4
4.2
1.0
0.1 (0.1)
0.02 (0.18)
0.06
0.03
0.3 (0.2)
2.5
3.3
2.0
1.0
10.0
1.7
0.03 (0.1)
0.1 (1.1)
0.1
1.4
0.4
0.4
2.9
12.5
1.1
0.2 (0.1)
0.1 (0.3)
0.1 (0.2)
0.1
A series of novel benzenesulfonamides with benzimidazole moi-
ety have been synthesized and evaluated against four hCA isoforms
using isothermal titration calorimetry and thermal shift assay. The
S-alkylated benzimidazole derivatives 3l–o bind stronger than
indapamide-like N-alkylated benzimidazoles 3a–k. The S-alkylated
benzimidazole K ds reach about 50–100 nM with CAs VII and XIII.
3n
3o
4. Experimental
4.1. Syntheses
0.1
0.3 (1.8)
Indapamide
10
0.1 (0.2)
Average standard deviations for both methods were below 25%.
Melting points of the compounds were determined in open cap-
illaries on a Thermo Scientific 9100 Series and are uncorrected. IR
spectra were run on a Perkin–Elmer FT-IR spectrophotometer
Spectrum BX II in KBr. 1H and 13C NMR spectra were recorded on
a Varian Unity Inova spectrometer (300 and 75 MHz, respectively)
in DMSO-d6 using residual DMSO signals (2.52 and 40.21 ppm for
1H and 13C NMR spectra, respectively) as the internal standard.
TLC was performed with Silica Gel 60 F254 aluminum plates
(Merck) and visualized with UV light. High-resolution mass spectra
(HRMS) were recorded on a Dual-ESI Q-TOF 6520 mass spectrom-
eter (Agilent Technologies).
data refinement statistics for the crystal structures are listed in
Table 2.
The analysis of hCA II in complex with 3m–o revealed a con-
served network of bonds commonly formed upon binding of a sul-
fonamide moiety. The positions of Zn2+, His94, His96, and His119
are similar to observed in previously reported structures. The
2-chlorobenzensulfonamide ring of the compounds is fixed by
steric constraints imposed by residues Gln92, Leu198, Val121
and Val143. The chlorine atom is found in a hydrophobic pocket
formed by residues Leu141, Val143, Val207, Val121 and Leu198.
The orientation of the 2-chlorobenzensulfonamide ring within
the hCAII active site is identical to previous reports of chlorthali-
done and indapamide (Fig. 5).19,34
4.1.1. 1,3-Dihydro-2H-imidazo[4,5-c]quinoline-2-thione (2o)
To a solution of 3,4-quinolinediamine (100 mg, 0.628 mmol) in
The linker between the chlorobenzensulfonamide and the sec-
ond benzimidazole ring is an important part of ligand binding to
hCA II. The S atom likely accepts the hydrogen bond from the
Nd2 atom of Asn62 (Fig. 4). The carbonyl group participates in
methanol (5 ml)
a solution of sodium hydroxide (0.028 g,
0.691 mmol) and carbon disulfide (0.053 g, 0.691 mmol) in water
(1 ml) was added dropwise. The reaction mixture was heated at
reflux for 3 h and then stirred at rt for 24 h. The reaction mixture
was acidified with 0.5 M HCl, solvents evaporated under reduced
pressure and the residue suspended in water. The precipitate ob-
tained was filtered off and washed with water to give compound
hydrogen bond with N
e
2 of Gln92 (3m, 3n) or in two hydrogen
2 of His64 (3o). The only multir-
bonds with O 1 of Thr200 and N
c
e
ing system of 3m has additional direct and water mediated hydro-
gen bonds with the protein (Fig. 4). The conformation of inhibitors
2o. Yield 79%, mp 324–326 °C. IR
m
cmꢁ1: 3044 (NH), 1498 (C@S).
Figure 4. View of compounds 3m (a), 3n (b), and 3o (c) bound in the active center of hCA II. The Zn atom, His94, His96, and His119 are shown as transparent, inhibitors are
shown in orange. Picture is generated using MOLSCRIPT44 and Raster3D.45

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7361
Table 2
X-ray crystallographic data collection and refinement statistics
Compound
PDB ID
3m
3M67
3n
3M96
3o
3MYQ
Temperature, K
Space group
Unit cell
100
P21
100
P21
100
P21
a = 41.9722, b = 41.0069, c = 71.6806 Å,
a = 42.0496, b = 40.9205, c = 71.4681 Å,
a = 41.9796, b = 40.9099, c = 71.6071 Å,
a
=
c
= 90, b = 104.4303
a
=
c
= 90, b = 104.1442
a = c = 90, b = 104.3360
Resolution, Å (final shell)
Reflections unique (total)
Completeness (%) overall
(final shell)
40.66–1.80 (1.90–1.80)
22,019 (165,478)
99.3 (99.0)
28.88–1.40 (1.48–1.40)
46,297 (431,844)
99.2 (98.1)
28.84–1.35 (1.42–1.35)
50,807 (131,001)
98.1 (97.0)
I/rI overall (final shell)
Rmerge overall (final shell)
B(iso) from Wilson, (Å2)
31.5 (7.0)
0.052 (0.222)
19.3
22.9 (4.4)
0.095 (0.253)
14.3
16.9 (3.6)
0.057 (0.229)
13.5
Refinement
Number of atomsa
Number of solvent molecules 158
Number of bounded buffer
molecule atoms
2294
2362
239
8
2436
264
20
8
Test set size, %
10
10
10
Rcryst (Rfree
)
0.181 (0.228)
0.027(2.061)
17.3231
19.0262
25.8868
10.01
0.180 (0.213)
0.032 (2.587)
12.9173
15.857
24.8892
7.21
0.139 (0.181)
0.024 (2.187)
13.454
16.252
26.458
RMS bonds/angles
Average B factors (Å2), total
Main chain
Side chains
Solvent
8.29
Ions
Cofactors
Inhibitor
30.665
31.5714
31.5714
25.6413
21.6448
21.6448
22.9765
24.6336
24.6336
P P
P P
P
nh
nh
Rmerge
¼
i¼1jhIhi ꢁ Ihij=
i¼1jIhij, where Ihi is an intensity value of the i-th measurement of reflection h, h = (h, k, l), sum
runs over all measured reflections, and hIhi
h
h
h
is an average measured intensity of the reflection h. The nh is the number of measurements of reflection h.
a
Including atoms in alternative positions.
1H NMR d ppm: 7.67 (2H, m, C7,8-H), 8.04 (1H, d, J = 9.3 Hz, C9-H),
8.30 (1H, d, J = 9.6 Hz, C6-H), 8.77 (1H, s, C4-H), 13.18 (1H, br s, NH),
13.80 (1H, br s, NH). 13C NMR d ppm: 115.36, 122.15, 126.44,
127.59, 128.40, 130.21, 133.82, 135.04, 144.48, 169.00. HRMS calcd
for C10H7N3S ([M+H]+): 202.0433, found: 202.0434.
4.1.2.3.
yl]acetyl}benzenesulfonamide (3c). Yield 49%, mp 253–255 °C.
IR
cmꢁ1: 3385, 3285 (OH, NH2), 1706 (C@O). 1H NMR d ppm:
4.69 (2H, d, J = 4,2 Hz, CH2O), 5.67 (1H, br s, OH), 6.07 (2H, s,
2-Chloro-5-{[2-(hydroxymethyl)-1H-benzimidazol-1-
m
0
0
0
CH2CO), 7.21–7.24 (2H, m, C5 ,6 -H), 7.52–7.54 (1H, m, C7 -H),
0
7.63–7.66 (1H, m, C4 -H), 7.91 (2H, s, NH2), 7.95 (1H, d, J = 8.1 Hz,
4.1.2. General procedure for the synthesis of 3a–k
A mixture of the corresponding 2-substituted benzimidazole
(2a–k) (0.832 mmol), compound 1 (200 mg, 0.640 mmol) and so-
dium acetate (60.3 mg, 0.736 mmol) in tetrahydrofuran (5 ml)
was stirred at rt for 24 h. The reaction mixture was poured into
water. The precipitate was filtered off, washed with water and then
with diethyl ether.
C3-H), 8.38 (1H, d, J = 7.8 Hz, C4-H), 8.56 (1H, s, C6-H). 13C NMR d
ppm: 50.87, 57.51, 111.00, 119.61, 122.38, 123.08, 128.85,
133.04, 133.62, 133.92, 136.52, 136.96, 142.29, 154.70, 192.55.
HRMS calcd for
380.0472.2
C
16H14ClN3O4S ([M+H]+): 380.0466, found:
4.1.2.4. 5-[(2-Benzyl-1H-benzimidazol-1-yl)acetyl]-2-chloro-
benzenesulfonamide (3d). Yield 30%, mp 128–130 °C. IR
cmꢁ1
3401 (NH2), 1705 (C@O). 1H NMR d ppm: 4.26 (2H, s, CH2Ph), 6.05
m
:
4.1.2.1. 5-(1H-Benzimidazol-1-ylacetyl)-2-chlorobenzenesulf-
onamide (3a). Yield 83%, mp 196–198 °C. IR
m
cmꢁ1: 3382
0
0
(2H, s, CH2CO), 7.20–7.25 (m, 7H, C5 ,6 -H, C6H5), 7.45 (d, 1H,
(NH2), 1705 (C@O). 1H NMR d ppm: 6.08 (2H, s, CH2CO), 7.23–
0
0
J = 6.9 Hz, C7 -H), 7.62 (1H, d, J = 6.9 Hz, C4 -H), 7.89 (2H, s, NH2),
7.95 (1H, d, J = 8.1 Hz, C3-H), 8.31 (1H, d, J = 8.4 Hz, C4-H), 8.47
(1H, s, C6-H). 13C NMR d ppm: 33.43, 50.68, 111.01, 119.24,
122.22, 122.55, 127.19, 128.88, 129.07, 129.62, 132.85, 133.64,
133.88, 136.48, 136.67, 137.43, 142.26, 142.99, 154.84, 192.39.
0
0
0
7.26 (2H, m, C5 ,6 -H), 7.51–7.57 (1H, m, C7 -H), 7.68–7.71 (1H, m,
0
C4 -H), 7.91 (2H, s, NH2), 7.95 (1H, d, J = 8.1 Hz, C3-H), 8.19 (1H, s,
0
C2 -H), 8.35 (1H, dd, J = 2.1 Hz, J = 8.1 Hz, C4-H), 8.55 (1H, d,
J = 2.1 Hz, C6-H). 13C NMR d ppm: 51.60, 111.39, 119.97, 122.37,
123.19, 128.75, 133.06, 133.56, 133.90, 135.26, 136.54, 142.32,
143.59, 145.53, 192.80. HRMS calcd for C15H12ClN3O3S ([M+H]+):
350.0361, found: 350.0367.
HRMS calcd for
440.0837.
C
22H18ClN3O3S ([M+H]+): 440.0830, found:
4.1.2.5. 2-Chloro-5-[(2-ethyl-1H-benzimidazol-1-yl)acetyl]ben-
4.1.2.2. 2-Chloro-5-[(2-methyl-1H-benzimidazol-1-yl)acetyl]
m :
cmꢁ1
benzenesulfonamide (3b). Yield 84%, mp 266–268 °C. IR
m :
cmꢁ1
zenesulfonamide (3e). Yield 70%, mp 245–247 °C. IR
3281 (NH2), 1705 (C@O). 1H NMR d ppm: 1.29 (3H, t, J = 7.5 Hz,
CH3), 2.78 (2H, q, J = 7.5 Hz, CH2), 6.07 (2H, s, CH2CO), 7.17–7.19
3393, 3285 (NH2), 1704 (C@O). 1H NMR d ppm: 2.44 (3H, s, CH3),
0
0
6.07 (2H, s, CH2CO), 7.15–7.18 (2H, m, C5 ,6 -H), 7.47–7.49 (1H, m,
0
0
0
0
(2H, m, C5 ,6 -H), 7.47–7.50 (1H, m, C7 -H), 7.60–7.62 (1H, m, C4 -
H), 7.88 (2H, s, NH2), 7.98 (1H, d, J = 8.4 Hz, C3-H), 8.41 (1H, d,
J = 8.4 Hz, C4-H), 8.58 (1H, s, C6-H). 13C NMR d ppm: 12.16, 20.43,
50.53, 110.75, 118.85, 122.17, 122.40, 128.93, 132.96, 133.81,
133.82, 136.44, 136.67, 142.37, 142.48, 157.47, 193.01. HRMS calcd
for C17H16ClN3O3S ([M+H]+): 378.0674, found: 378.0677.
0
0
C7 -H), 7.56–7.58 (1H, m, C4 -H), 7.90 (2H, s, NH2), 7.98 (1H, d,
J = 8.4 Hz, C3-H), 8.40 (1H, d, J = 8.4 Hz, C4-H), 8.58 (1H, s, C6-H).
13C NMR d ppm: 14.01, 50.73, 110.66, 118.80, 122.06, 122.22,
128.92, 132.97, 133.80, 133.87, 136.51, 136.65, 142.35, 142.88,
153.34, 192.94. HRMS calcd for
364.0517, found: 364.0518.
C
16H14ClN3O3S ([M+H]+):

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E. Capkauskaite˙ et al. / Bioorg. Med. Chem. 18 (2010) 7357–7364
7362
Figure 5. Superposition of the hCA II-indapamide [PDB ID 3BL1] (magenta), hCA II-chlorthalidone [PDB ID 3F4X] (cyan) with hCA II-2-chlorobenzenesulfonamide adducts
(orange) 3m (a) or 3n (b). Zinc ion is shown as pink sphere, His94, His96, His119, and protein secondary structures are in green.
4.1.2.9. 2-Chloro-5-[(2-isobutyl-1H-benzimidazol-1-yl)acetyl]
Table 3
benzenesulfonamide (3i). Yield 75%, 223–225 °C. IR
m :
cmꢁ1
The comparison of ligand (3m–o) and indapamide interaction to hCA II
3316 (NH2), 1704 (C@O). 1H NMR d ppm: 0.96 (6H, d, J = 6.6 Hz,
(CH3)2), 2.11–2.24 (1H, m, CH), 2.67 (2H, d, J = 7.2 Hz, CH2), 6.07
Inhibitor
Number of hydrogen bonds
(excluding those of sulfonamide
moiety)
Buried surface area in the
hCAII-inhibitor complex,
(Å2)
0
0
0
(2H, s, CH2CO), 7.12–7.20 (2H, m, C5 ,6 -H), 7.43–7.45 (1H, m, C7 -
0
H), 7.60–7.62 (1H, m, C4 -H), 7.87 (2H, s, NH2), 7.97 (1H, d,
Indapamide
1
4
2
3
655.1
728.0
719.5
709.2
3m
3n
3o
J = 8.1 Hz, C3-H), 8.43 (1H, dd, J = 1.5 Hz, J = 8.1 Hz, C4-H), 8.58
(1H, d, J = 1.5 Hz, C6-H). 13C NMR d ppm: 23.09, 27.65, 35.73,
50.60, 110.80, 118.97, 122.08, 122.24, 128.92, 132.94, 133.85,
136.34, 136.66, 142.38, 142.93, 155.75, 192.89. HRMS calcd for
C
19H20ClN3O3S ([M+H]+): 406.0987, found: 406.0994.
4.1.2.6. 2-Chloro-5-[(2-propyl-1H-benzimidazol-1-yl)acetyl]
benzenesulfonamide (3f). Yield 73%, 247–249 °C. IR
m :
cmꢁ1
4.1.2.10.
zimidazol-1-yl]acetyl}benzenesulfona-mide (3j). Yield 63%,
199–201 °C. IR
cmꢁ1: 3435 (NH2), 1703 (C@O). 1H NMR d ppm:
2.24 (4H, br s, (CH2)2), 3.10 (4H, br s, (CH2)2), 3.79 (2H, s, CH2N),
2-Chloro-5-{[2-(4-morpholinylmethyl)-1H-ben-
3304 (NH2), 1706 (C@O). 1H NMR d ppm: 0.97 (3H, t, J = 7.5 Hz,
CH3), 1.78 (2H, sextet, J = 7.5 Hz, CH2), 2.75 (2H, t, J = 7.5 Hz,
m
0
0
CH2), 6.07 (2H, s, CH2CO), 7.13–7.20 (2H, m, C5 ,6 -H), 7.45–7.47
0
0
(1H, m, C7 -H), 7.59–7.62 (1H, m, C4 -H), 7.88 (2H, s, NH2), 7.98
(1H, d, J = 8.4 Hz, C3-H), 8.42 (1H, dd, J = 1.8 Hz, J = 8.4 Hz, C4-H),
8.59 (1H, d, J = 1.8 Hz, C6-H). 13C NMR d ppm: 14.48, 20.97, 28.87,
50.54, 110.73, 118.96, 122.06, 122.26, 128.93, 132.95, 133.86,
136.43, 136.66, 142.38, 142.86, 156.36, 192.99. HRMS calcd for
0
0
6.01 (2H, s, CH2CO), 7.19–7.26 (2H, m, C5 ,6 -H), 7.53–7.56 (1H, m,
0
0
C7 -H), 7.62–7.65 (1H, m, C4 -H), 7.91 (2H, s, NH2), 7.98 (1H, d,
J = 8.4 Hz, C3-H), 8.43 (1H, dd, J = 1.8 Hz, J = 8.1 Hz, C4-H), 8.60
(1H, d, J = 1.8 Hz, C6-H). 13C NMR d ppm: 50.57, 53.42, 55.96,
66.20, 110.89, 119.50, 122.17, 123.00, 128.82, 133.09, 133.46,
134.48, 136.35, 137.60, 142.40, 142.57, 151.50, 192.47. HRMS calcd
for C20H21ClN4O4S ([M+H]+): 449.1045, found: 449.1049.
C
18H18ClN3O3S ([M+H]+): 392.0830, found: 392.0834.
4.1.2.7. 5-[(2-Butyl-1H-benzimidazol-1-yl)acetyl]-2-chloroben-
zenesulfonamide (3g). Yield 74%, mp 221–223 °C. IR
m :
cmꢁ1
4.1.2.11. 2-Chloro-5-{[2-(methylsulfanyl)-1H-benzimidazol-1-
3363, 3302 (NH2), 1705 (C@O). 1H NMR d ppm: 0.90 (3H, t,
J = 6.9 Hz, CH3), 1.35–1.41 (2H, m, CH2), 1.71–1.75 (2H, m, CH2),
2.76 (2H, t, J = 6.9 Hz, CH2), 6.07 (2H, s, CH2CO), 7.16 (2H, br s,
yl]acetyl}benzenesulfonamide (3k). Yield 75%, 239–240 °C. IR
m
cmꢁ1: 3362, 3271 (NH2), 1710 (C@O). 1H NMR d ppm: 2.71 (3H,
0
0
s, CH3), 5.97 (2H, s, CH2CO), 7.14–7.23 (2H, m, C5 ,6 -H), 7.48–7.51
0
0
0
0
C
5 ,6 -H), 7.46 (1H, d, J = 6.6 Hz, C7 -H), 7.60 (1H, d, J = 6.6 Hz, C4 -
0
0
(1H, m, C4 -H), 7.59–7.62 (1H, m, C7 -H), 7.85 (2H, br s, NH2), 7.97
(1H, d, J = 8.4 Hz, C3-H), 8.42 (1H, d, J = 8.4 Hz, C4-H), 8.56 (1H, s,
C6-H). 13C NMR d ppm: 15.25, 50.86, 110.35, 118.25, 122.34,
122.41, 128.82, 133.11, 133.65, 133.77, 136.85, 137.66, 142.47,
143.52, 153.69, 192.20. HRMS calcd for C16H14ClN3O3S2 ([M+H]+):
396.0238, found: 396.0237.
H), 7.88 (2H, s, NH2), 7.98 (1H, d, J = 8.1 Hz, C3-H), 8.42 (1H, d,
J = 7.2 Hz, C4-H), 8.58 (1H, s, C6-H). 13C NMR d ppm: 14.46, 22.48,
26.57, 29.62, 50.53, 110.72, 118.92, 122.06, 122.25, 128.91,
132.94, 133.84, 133.87, 136.43, 136.65, 142.37, 142.81, 156.50,
193.01. HRMS calcd for C19H20ClN3O3S ([M+H]+): 406.0987, found:
406.0990.
4.1.3. General procedure for the synthesis of 3l–o
4.1.2.8. 2-Chloro-5-[(2-isopropyl-1H-benzimidazol-1-yl)acetyl]
A mixture of the corresponding benzimidazole-2-thione 2l–o
(0.320 mmol), compound 1 (100 mg, 0.320 mmol) and sodium ace-
tate (30.2 mg, 0.368 mmol) in tetrahydrofuran (3 ml) was stirred at
rt for 24 h. The reaction mixture was poured into water. The pre-
cipitate was filtered off, and subsequently washed with water
and then diethyl ether.
benzenesulfonamide (3h). Yield 65%, 265–267 °C. IR
m :
cmꢁ1
3339 (NH2), 1711 (C@O). 1H NMR d ppm: 1.28 (6H, d, J = 6 Hz,
(CH3)2), 3.12–3.27 (1H, m, CH), 6.10 (2H, s, CH2CO), 7.17 (2H, br
0
0
0
s, C5 ,6 -H), 7.46 (1H, d, J = 7.2 Hz, C7 -H), 7.61 (1H, d, J = 6.6 Hz,
0
C4 -H), 7.88 (2H, s, NH2), 7.98 (1H, d, J = 8.1 Hz, C3-H), 8.44 (1H, d,
J = 6.9 Hz, C4-H), 8.58 (1H, s, C6-H). 13C NMR d ppm: 27.16, 30.88,
55.20, 115.61, 123.78, 126.89, 127.08, 133.67, 137.69, 138.60,
138.65, 140.98, 141.43, 147.12, 147.44, 165.84, 197.91. HRMS calcd
for C18H18ClN3O3S ([M+H]+): 392.0830, found: 392.0837.
4.1.3.1. 5-[(1H-Benzimidazol-2-ylsulfanyl)acetyl]-2-chloroben-
zenesulfonamide (3l). Yield 91%, mp 167–169 °C. IR
m :
cmꢁ1
3379, 3327, 3266 (NH2, NH), 1673 (C@O). 1H NMR d ppm: 5.07

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E. Capkauskaite˙ et al. / Bioorg. Med. Chem. 18 (2010) 7357–7364
7363
0
0
0
0
(2H, s, CH2CO), 7.13–7.24 (2H, m, C5 ,6 -H), 7.42–7.43 (2H, m, C4 ,7
-
typical experiment consisted of 25–30 injections (10
ll each) with-
H), 7.85 (2H, s, NH2), 7.89 (1H, d, J = 8.7 Hz, C3-H), 8.33 (1H, d,
J = 8.1 Hz, C4-H), 8.56 (1H, s, C6-H), 12.67 (1H, br s, NH). 13C NMR
(DMSO-d6 with three drops TFA) d ppm: 41.50, 114.01, 125.28,
129.05, 133.02, 133.80, 134.46, 136.67, 142.37, 150.87, 191.57.
HRMS calcd for
382.0088.
in 3–4 min intervals. Variation of the time period between injec-
tions enabled the detection and elimination of possible kinetic
effects that may distort the binding data. Experiments were carried
out at 37 °C in a phosphate buffer containing 50 mM NaCl at pH 7.0
and with a final DMSO concentration between 0.5% and 2%.
C
15H12ClN3O3S2 ([M+H]+): 382.0081, found:
4.4. Crystallography
4.1.3.2. 2-Chloro-5-[(6,7-dihydro-1H-[1,4]dioxino[2,3-f]ben-
zimidazol-2-ylsulfanyl)acetyl]ben-zenesulfonamide
4.4.1. Crystallization
(3m). Yield 89%, mp 219–221 °C. IR
m
cmꢁ1: 3348, 3293 (NH2,
Crystals of hCAII were prepared by sitting drop method. Protein
was concentrated to 20–60 mg/ml by ultrafiltration in a buffer con-
taining 20 mM Na-Hepes pH 7.5 and 50 mM NaCl. A crystallization
drop was prepared by mixing equal volumes of protein solution
and crystallization buffer. Crystallization buffers were prepared
by mixing the following solutions: 1 M Na-Bicine, pH 9, to a con-
centration 0.1 M; 2.7 M Na-Malonate pH 7–7.55 to a final concen-
tration range of 1.5–2.2 M; and 3.5 M ammonium sulfate to a
concentration from 0 to 0.2 M. Crystals appeared in a few days
and belong to the space group P21. Crystals were soaked for several
days with the 0.5 mM inhibitor solution in a reservoir buffer.
NH), 1686 (C@O). 1H NMR d ppm: 4.22 (4H, s, O(CH2)2O), 5.02
0
0
(2H, s, CH2CO), 6.89 (2H, s, C4 ,9 -H), 7.84 (2H, s, NH2), 7.88 (1H,
d, J = 8.1 Hz, C3-H), 8.30 (1H, dd, J = 8.4 Hz, J = 2.1 Hz, C4-H), 8.56
(1H, d, J = 1.8 Hz, C6-H), 12.32 (1H, br s, NH). 13C NMR (DMSO-d6
with three drops TFA) d ppm: 41.81, 64.72, 101.02, 127.79,
129.03, 132.99, 133.76, 134.25, 136.72, 142.37, 143.32, 148.82,
191.44. HRMS calcd for
found: 440.0144.
C
17H14ClN3O5S2 ([M+H]+): 440.0136,
4.1.3.3. 5-{[(5-Bromo-1H-benzimidazol-2-yl)sulfanyl]acetyl}-2-
chlorobenzenesulfonamide (3n). Yield 64%, mp 188–190 °C. IR
m
cmꢁ1: 3367, 3327, 3278 (NH2, NH), 1690 (C@O). 1H NMR d ppm:
4.4.2. Data collection and structure determination
0
5.10 (s, 2H, CH2CO), 7.27 (d, J = 8.4 Hz, 1H, C5 -H), 7.39 (d,
Diffraction data from the complexes of hCAII with 3a, 3e, 3m,
and 3n were collected at the EMBL X12 and X13 beam lines at
the DORIS storage ring (DESY, Hamburg, Germany). MOSFLM37,38
and SCALA39 were used for image processing. Initial phases were
obtained by molecular replacement with the protein moiety from
PDB entry 3HLJ12 as an initial model. Structures were refined using
REFMAC,40 and COOT41 was used for model inspection. Atomic
coordinates of ligands were generated by DSVisualizer 1.742 (Accel-
rys). Topology and parameters for structure refinement were gen-
erated by LIBREFMAC.43 Data collection and refinement statistics
are presented in Table 2. Coordinates and structure factors were
deposited under PDB ID 3M67 (3m), 3M96 (3n), 3M98 (3a), and
3MYQ (3o).
0
0
J = 8.4 Hz, 1H, C7 -H), 7.63 (s, 1H, C4 -H), 7.87 (s, 2H, NH2), 7.90
(d, J = 8.4 Hz, 1H, C3-H), 8.33 (d, J = 8.1 Hz, 1H, C4-H), 8.56 (s, 1H,
C6-H), 12.73 (br s, 1H, NH). 13C NMR (DMSO-d6, with three drops
TFA)
d ppm: 67.72, 115.12, 115.84, 117.25, 125.70, 129.05,
133.00, 133.78, 134.83, 136.36, 137.69, 140.43, 142.30, 151.98,
192.42. HRMS calcd for C15H11BrClN3O3S2 ([M+H]+): 461.9164,
found: 461.9170.
4.1.3.4. 2-Chloro-5-[(1H-imidazo[4,5-c]quinolin-2-ylsulfanyl)
acetyl]benzenesulfonamide (3o). Yield 92%, mp 218–220 °C. IR
m
cmꢁ1: 3406, 3310 (NH2, NH), 1693 (C@O). 1H NMR d ppm: 5.16
0
0
(2H, s, CH2CO), 7.64–7.75 (2H, m, C7 ,8 -H), 7.85 (2H, s, NH2), 7.92
0
(1H, d, J = 8.4 Hz, C3-H), 8.07–8.09 (1H, m, C9 -H), 8.23–8.26 (1H,
0
m, C6 -H), 8.38 (1H, dd, J = 8.4 Hz, J = 1.8 Hz, C4-H), 8.60 (1H, d,
Acknowledgments
J = 1.8 Hz, C6-H), 9.08 (1H, s, C4 -H), 13.88 (1H, br s, NH).13C NMR
0
(DMSO-d6 with three drops TFA) d ppm: 40.54, 117.23, 121.76,
123.30, 126.51, 129.07, 129.54, 129.62, 132.05, 132.64, 133.08,
133.79, 134.89, 135.08, 136.48, 142.36, 158.46, 192.12. HRMS calcd
for C18H13ClN4O3S2 ([M+H]+): 433.0190, found: 433.0192.
The project was supported in part by EEA and Norway Grants
2004-LT0019-IP-1EEE and the Lithuanian Government. E. Cap-
kauskaite and L. Baranauskiene acknowledge the Lithuanian Sci-
˙
ence Council for a Student Research Fellowship Award. Crystal
diffraction data were collected at the EMBL/DESY, Hamburg. D.
Golovenko’s access to the measurement facilities was funded from
the European Community’s Seventh Framework Programme (FP7/
2007-2013) under grant agreement No. 226716. E. Manakova’s
and S. Grazulis’ travels to DESY, Hamburg were supported by
2004-LT0019-IP-1EEE and the Research Council of Lithuania. We
thank our local contacts at the EMBL beamlines, Dr. Gleb Bouren-
kov and Dr. Michele Cianci, for their help with beamline operation.
We thank Dr. Fernando Ridoutt for his help with the beamline
cryosystems.
4.2. Protein preparation
Expression and purification of hCA I, hCA II, hCA VII, and hCA
XIII was previously described: hCA I in,12 hCA II in,35 and hCA VII
and XIII in.36
4.3. Determination of compound binding to CAs
4.3.1. Thermal shift assay
Thermal shift assay experiments were performed in a Corbett
Rotor-Gene 6000 (QIAGEN Rotor-Gene Q) instrument using blue
channel (excitation 365 20, detection 460 15 nm). Samples con-
References and notes
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