4-[N-(Substituted 4-pyrimidinyl)amino]benzenesulfonamides as inhibitors of carbonic anhydrase isozymes I, II, VII, and XIII

Article abstract of DOI:10.1016/j.bmc.2010.09.011

A series of 4-[N-(substituted 4-pyrimidinyl)amino]benzenesulfonamides were designed and synthesised. Their binding potencies as inhibitors of selected recombinant human carbonic anhydrase (hCA) isozymes I, II, VII, and XIII were measured using isothermal

Full text of DOI:10.1016/j.bmc.2010.09.011

Bioorganic & Medicinal Chemistry 18 (2010) 7413–7421
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
4-[N-(Substituted 4-pyrimidinyl)amino]benzenesulfonamides as inhibitors
of carbonic anhydrase isozymes I, II, VII, and XIII
a
b
c
b
b
ꢀ
ˇ
Jurgis Sudzius , Lina Baranauskiene , Dmitrij Golovenko , Jurgita Matuliene , Vilma Michailoviene ,
˙
˙
˙
b
b
c
c
c
ˇ
Jolanta Torresan , Jelena Jachno , Rasa Sukackaite , Elena Manakova , Saulius Grazulis ,
Sigitas Tumkevicius , Daumantas Matulis
a Department of Organic Chemistry, Faculty of Chemistry, Vilnius University, Naugarduko 24, LT-03225 Vilnius, Lithuania
Laboratory of Biothermodynamics and Drug Design, Institute of Biotechnology, V.A. Graiciuno 8, LT-02241 Vilnius, Lithuania
Laboratory of Protein—DNA Interactions, Institute of Biotechnology, Graiciuno 8, LT-02241 Vilnius, Lithuania
˙
a
b,
⇑
ˇ
b
ꢀ
ˇ
c
ꢀ
ˇ
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-[N-(substituted 4-pyrimidinyl)amino]benzenesulfonamides were designed and synthesised.
Their binding potencies as inhibitors of selected recombinant human carbonic anhydrase (hCA) isozymes
I, II, VII, and XIII were measured using isothermal titration calorimetry and the thermal shift assay. To
determine the structural features of inhibitor binding, the crystal structures of several compounds in
complex with hCA II were determined. Several compounds exhibited selectivity towards isozymes I, II,
and XIII, and some were potent inhibitors of hCA VII.
Received 12 May 2010
Revised 27 August 2010
Accepted 2 September 2010
Available online 8 September 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Carbonic anhydrase isozymes I, II, VII, and
XIII
Isothermal titration calorimetry
Thermal shift assay
ThermoFluorÒ
Benzenesulfonamide
Pyrimidine
1. Introduction
CAs. ITC has been routinely used to measure ligand–protein
binding thermodynamics.10,11 However, it has difficulty determin-
Carbonic anhydrases (CAs, EC 4.2.1.1) are prominent targets of
drug design, as previously reviewed.1 A well-known class of CA
inhibitors is aromatic/heterocyclic sulfonamides that have been
studied for the development of antiglaucoma, antitumour, antiobe-
sity, and anticonvulsant drugs.2,3 Despite the progress achieved in
the development of isozyme-specific inhibitors,4–7 there remains a
great need of new potent CA inhibitors with one or more of the fol-
lowing advantages: improved activity, selectivity, solubility, and
reduced toxicity and side effects.
Significant progress has been made in the understanding of
quantitative structure–activity relationships of CA inhibitors.3,8
However, most correlations were drawn based on inhibition mea-
surements. It is important to extend the binding measurements by
using biophysical thermodynamic techniques in addition to inhibi-
tion measurements.9 Here, we use isothermal titration calorimetry
(ITC) and the thermal shift assay (TSA, also called ThermoFluorÒ,
differential scanning fluorimetry) to measure inhibitor binding to
ing weak (millimolar) or extremely tight (subnanomolar) dissocia-
tion constants and is quite time and protein consuming. TSA is a
rapid and relatively low protein consuming screening method for
identification of specific binders (hit compounds) used in the phar-
maceutical industry.12,13 The method is based on protein melting
temperature (Tm) shifts upon ligand binding where the Tm is ob-
served by following intrinsic or extrinsic fluorescence changes
upon protein unfolding induced by heat. Determination of the
binding reactions using two techniques reduces the uncertainty
of the measurements.
Several 4-(2-substituted amino-4-pyrimidinylamino)benzene-
sulfonamides have been shown to possess significant affinity for
hCA II and hCA IV.14 Continuing the search for more potent and
selective hCA inhibitors among heterocyclic moieties containing
sulfonamides,15 here we present the synthesis of 4-N-(4-pyrimidi-
nyl)aminobenzenesulfonamides and the results of their binding to
human recombinant CA (hCA) isozymes hCA I, hCA II, hCA VII, and
hCA XIII. To increase the interaction of the tail pyrimidine moiety
with the hydrophilic part of the active site of CA, we have designed
compounds with electron-withdrawing groups in the pyrimidine
moiety (Fig. 1). Isozyme hCA VII is highly expressed in the brain
⇑
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.2010.09.011

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J. Sudzius et al. / Bioorg. Med. Chem. 18 (2010) 7413–7421
7414
R''
N
heating reagents at 100 °C in dimethylformamide for 3–12 h. For
the synthesis of 10b,c, an economic ‘one-pot’ method from 1 has
been accomplished. Compound 11b was synthesised by treatment
of 5b with water in THF solution at room temperature.
R'
R = H, SMe;
N
R' = NO2, CN, CHO;
R'' = Cl, OMe, PhCH2NH
n = 0, 1, 2
R
N
H
n
O
O
S
2.2. Binding
NH2
The compounds shown in Figure 1 and Scheme 1 are good bind-
ers of CAs. To determine compound affinity and specificity towards
the four tested CA enzymes (hCA I, hCA II, hCA VII, and hCA XIII),
compound binding was studied using two biophysical equilibrium
techniques: TSA and ITC. The binding constants obtained by the
two methods are listed in Table 1.
Figure 1. The general structure of 4-N-(4-pyrimidinyl)aminobenzenesulfonamides.
and inhibitors have been designed for it.16,17 Isozyme hCA XIII has
also been recently characterized and shown possibly to have a role
in maintaining the acid–base balance in the kidneys and the gas-
trointestinal and reproductive tracts.18 Several of our compounds
show selectivity towards hCA XIII over other isozymes and may
be useful for the development of therapeutic compounds.
Figure 2 shows several typical CA melting curves, determined
by TSA in the presence of increasing concentration of a ligand.
The curves were fit as previously described9 and the resultant
melting temperatures (Tm) were plotted as a function of added li-
gand concentration (Fig. 3). These dosing curves were analyzed
as previously described9 yielding the binding constants at 37 °C.
ITC is a method more widely used for determination of binding
affinity between proteins and low molecular weight ligands than
TSA, but ITC consumes more protein and time than TSA. To obtain
a more reliable affinity ranking of the compounds, both methods
were used as extensively as possible. Figure 4 shows a typical ITC
raw data curve. Figure 5 shows several ITC integrated data curves
comparing 5a binding to all tested CAs, whereas Figure 6 compares
several compound binding to hCA XIII. The compounds bound stoi-
chiometrically to each tested CA isoform, as seen from the ITC
curves.
Despite some discrepancy between TSA and ITC data and the
tendency to overestimate the Kb by TSA as compared to the ITC
method, a number of correlations between compound structure
and binding energetics can be drawn from Table 1. Compounds
5a and 11b appear to bind hCA XIII significantly stronger than
any other tested CA. Compounds 7b and 10b bound hCA I signifi-
cantly stronger than other tested CAs. Compound 8c had selectivity
towards hCA II over other three CAs.
2. Results and discussion
2.1. Chemistry
A series of benzene sulfonamide derivatives were designed and
synthesised (Fig. 1, Scheme 1). Pyrimidines with chloro substitu-
ents in positions 2, 4, and 6 possess high reactivity towards various
nucleophiles. Extensive work has been done in this area to gain
control over sequential introduction of various functionalities on
the pyrimidine templates by using halopyrimidines as electro-
philes.19–25 Therefore, for the synthesis of the target compounds
reported here, the nucleophilic substitution of the corresponding
4,6-dichloropyrimidines 1–3 was applied. The choice was also sug-
gested by the reports of successful synthesis of 4-[N-(4,6-substi-
tuted
1,3,5-triazin-2-yl)amino]benzenesulfonamides
using
regioselective nucleophilic substitution of cyanuric chloride.26,27
The desired monosubstitution products 5–7 were formed when
compounds 1–3 were allowed to react with an equivalent amount
of the corresponding 4-(amino substituted)benzenesulfonamides
4a–c in the presence of potassium carbonate or triethylamine. It
should be noted that the reaction of 1 and 2 with 4-(aminometyl)-
or 4-(2-aminoethyl)benzenesulfonamides (4b,c) to give 5b,c and
6b proceeded at room temperature, whereas the best results of
syntheses 5a, 6a,c, and 7a–c were obtained at the reflux tempera-
ture of tetrahydrofuran (THF). The substitution of the second chlo-
rine group in 5a–c and 6a with benzylamine required elevated
temperatures: compounds 8a–c and 9a were synthesised by
The 4-[N-(substituted 4-pyrimidinyl)amino]benzenesulfona-
mides can be divided into several groups according to their affinity
towards the four tested CAs. Compounds 5a, 6b, 6c, 7b, 7c, 10b,
10c, and 11b were the most potent, with affinities in the submi-
cromolar range. Compounds 6a, 8a, 8b, and 8c had affinities in
the micromolar range. Compound 9a was the least potent and its
affinity was in the millimolar range towards all four CAs.
O
O
S
Cl
N
Cl
N
NH
n
NH2
H2N
R1
Cl
R1
R1
4a-c
PhCH2NH2
N
N
N
K2CO3 or Et3N,
THF
R
K2CO3, DMF
R
N
H
n
R
N
N
H
n
100 oC
O
O
1-3
O
O
S
NH2
8a-c, 9a
S
NH2
5a-c, 6a-c, 7a-c
5b, H2O, THF, r.t.
1. 4b,c, K2CO3, THF, r.t.
2. MeOH, Δ
O
OMe
NO2
NO2
HN
N
R = H, R1 = NO2 (1, 5, 8, 10)
R = SMe, R1 = CN (2, 6, 9)
R = SMe, R1 = CHO (3, 7)
a: n=0;
b: n=1;
c: n=2.
N
N
H
N
N
H
n
O
O
O
S
S
NH2
11b
10b,c
O
NH2
Scheme 1.

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J. Sudzius et al. / Bioorg. Med. Chem. 18 (2010) 7413–7421
7415
Table 1
0.02
Dissociation constants determined by TSA and ITC in
l
M at 37 °C
0.00
-0.02
-0.04
-0.06
-0.08
-0.10
-0.12
Compound
hCA I
ITC
hCA II
hCA VII
hCA XIII
TSA
TSA
ITC
TSA
ITC
TSA
ITC
5a
6a
6b
6c
7a
7b
7c
8a
8b
8c
9a
10b
10c
11b
AZM
TFM
0.13
1.4
0.26
20
0.09
0.07
0.17
0.42
0.17
0.17
0.22
ND
ND
0.32
0.13
0.83
0.1
0.77
1.7
ND
0.002 0.014
0.091 0.12
0.1
ND
ND
2.8
0.14
0.10
ND
ND
ND
ND
0.33
1.0
0.1
4.0
ND
ND
0.10
0.77
ND
0.007 0.083 0.024 0.043 0.1
0.028 0.13
0.033 0.189
0.1
0.48
ND
0.11
0.17
0.1
0.35
ND
ND
1.0
10
0.07
0.5
0.33
0.5
ND
ND
ND
ND
0.025 ND
4.2
1.4
3300
ND
ND
ND
0.63
100
ND
ND
0.016 ND
0
20
40
60
80
100
0.05
0.07
0.2
ND
100
Time,min
0.013 0.11
0.067 0.28
0.056 0.83
0.15
ND
0.83
0.44
ND
0.067 0.24
0.13
0.25
0.13
0.02
0.23
ND
Figure 4. Example of raw ITC data—10c binding to hCA VII.
0.17
1.4
0.05
ND
0.78
ND
0.017 0.018 ND
0.13
ND
0.050 0.065
0.091 0.036 0.029 0.020 0.027
ND—not determined mostly due to insufficient compound solubility.
AZM—acetazolamide, TFM—trifluoromethanesulfonamide.
Average standard deviations for both methods were about 25%.
0
-2
-4
-6
-8
60
50
40
30
20
10
0
0
0.5
1
1.5
2
Molar ratio
Figure 5. Integrated ITC data—comparison of 5a binding to hCA I (s), hCA II (h),
hCA VII (}), and hCA XIII (4).
35
45
55
65
75
t, °C
0
-2
-4
-6
-8
Figure 2. TSA data of 10c binding to hCA II. Increasing ligand concentrations
elevate melting temperatures (s—no ligand, +—3
l
M, ꢀ—7
l
M, 4—17 lM,
h—59
lM, }—200 lM ligand; protein concentration 10 l
M).
70
65
60
55
0
0.5
1
1.5
2
Molar ratio
Figure 6. Integrated ITC data—comparison of 5a (h), 6a (}), 7c (4), and 10c (s)
binding to hCA XIII.
50
All crystallographically characterized ligands (Fig. 7, Table 2) can
be divided into three groups according to the length of the linker
connecting the pyrimidine and benzene rings: 5a and 7a have a
short linker between the benzene and pyrimidine rings consisting
of just the amino group; 7b, 8b, 10b, and 11b have a longer linker
with a methylene group attached to amino group; the ligand 10c
linker is the longest and has two connecting methylene groups.
The benzenesulfonamide ring of all inhibitors in all crystal
structures is found in the same orientation. The ring is fixed by sul-
fonamide bound to catalytic Zn. Rotations of the ring are restricted
by van der Waals contacts, with residues forming an active site
cavity. Side chains of Val121 and Thr200 check the mobility from
1.E-07
1.E-06
1.E-05
1.E-04
1.E-03
Lt, M
Figure 3. 10c binding to hCAs by TSA. Symbols (s—hCA I, 4—hCA II, h—hCAVII,
}—hCA XIII) show experimental values, corresponding solid lines were fit according
to the model.
2.3. Crystallography
Crystal structures of compounds 5a, 7a, 7b, 8b, 10b, 10c, and
11b bound to hCA II were determined by X-ray crystallography.

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J. Sudzius et al. / Bioorg. Med. Chem. 18 (2010) 7413–7421
7416
of the pyrimidine ring, which could be explained by partial hydro-
lysis (or radiation damage) of the Cl group.
Compounds that contain Cl in the pyrimidine ring, especially 5b
and 5c, would hydrolyze in aqueous solution by exchanging Cl for
an OH group due to the adjacent electron-withdrawing nitro
group. Some hydrolysis was also observed with other Cl-containing
compounds in crystal structures after prolonged exposure to aque-
ous solution during compound soaking. However, the hydrolysis
was only partial, and it should not occur in ITC and TSA binding as-
says because compound solutions were freshly prepared and the
assays lasted about 1 h.
The structure of hCA II complexed with compound 7a is solved
with 1.15 Å resolution (see Table 2 for refinement statistics). An
excellent electron density for the inhibitor is visible in a composite
omit map. The pyrimidine ring of 7a is fixed by hydrophobic inter-
action with Pro202, Val135, Trp5, His64, and Phe131. Similar ori-
entation of the pyrimidine ring as in 7a is observed in the crystal
structure of hCAII complexed with inhibitor 11b, which belongs
to the second group of ligands with the medium length linker. Sim-
ilarly to 7a, the pyrimidine ring of 11b appears to be trapped in this
orientation mostly by the van der Waals interactions with Pro202,
Phe131, and Val135. In both crystal structures, the DMSO mole-
cule, which is obtained from the stock solution of the inhibitor, is
found between Phe131 and Gln92. Interestingly, compound 11b
has an alternative non-specific binding site near the N-terminus
of the protein. The second binding site could be a result of crystal
packing, because thermodynamic data do not support the binding
of two molecules of this inhibitor by hCAII.
Figure 7. View of 5a, 6a, and 7a, located in the active center of hCA II. The Zn atom,
coordinated by His94, His96, and His119, is shown as an orange sphere. The
electron density map, contoured at 0.8
r. The pictures are generated using
MOLSCRIPT,38 Raster3D,39 and BOBSCRIPT.40
the sides, whereas Leu198 supports the ring plane. The pyrimidine
ring is observed in three different orientations in the crystal
structures.
Compounds 5a, 8b, 10b, 10c, and 11b make an intramolecular
hydrogen bond-like connection between the nitro group at the
C(5) atom of the pyrimidine ring and the amino group of the linker.
Figure 8 shows the arrangement of 10b in the active site of hCA II
with closest protein–ligand interatomic distances as green lines.
Electron density of the pyrimidine ring in ligands 5a, 8b, and 10c
is shifted towards the nitro group due to its electron-withdrawing
effect. Negative difference electron density was found in com-
plexes with ligands 5a, 7a, and 7b containing Cl at the C(6) position
Figure 9 shows the position of several superimposed ligands.
The pyrimidine rings of ligands 5a, 7b, 8b, and 10b form a plane,
Table 2
X-ray crystallographic data collection and refinement statistics
Compound:
5a
7a
7b
8b
10b
10c
11b
PDB ID
3M40
3MHO
3M5E
3MHM
3MHL
3M3X
3MHI
Temperature
Spacegroup
Unit cell (Å)
100 K
P21
a = 42.18,
b = 41.16,
c = 72.48,
100 K
P21
a = 42.13,
b = 41.21,
c = 72.08,
100 K
P21
a = 42.42,
b = 41.49,
c = 72.43,
100 K
P21
a = 42.25,
b = 41.17,
c = 72.24,
100 K
P21
a = 41.71,
b = 40.68,
c = 70.72,
100 K
P21
a = 42.04,
b = 41.05,
c = 71.80,
100 K
P21
a = 42.24,
b = 41.24,
c = 71.95,
a
=
c
= 90,
a
=
c
= 90,
a
=
c
= 90,
a
=
c
= 90,
a
=
c
= 90,
a
=
c
= 90,
a = c = 90,
b = 104.54
b = 104.30
b = 104.49
b = 104.10
b = 104.13
b = 104.39
b = 104.43
Resolution, Å (final
shell)
1.60
1.15
1.70
1.50
1.90
1.77
1.70
Unique reflections
(total)
Completeness (%)
overall
30,141
(176,541)
94.4
85,194
(566,937)
99.9
27,116
(314,528)
100.0
37,716
(154,392)
97.3
18,388
(280,542)
100.0
26,749
(299,614)
98.3
26,639
(251,427)
99.9
(90.2)
(99.7)
(100.0)
(96.1)
(100.0)
(97.6)
(99.3)
(final shell)
I/rI overall
(final shell)
10.2
(6.4)
16.1
(20.2)
2410
234
20.3
(4.1)
6.7
(32.3)
2468
291
32.8
(13.0)
5.4
(11.7)
2488
332
25.4
(5.8)
3.3
(15.7)
2500
294
37.9
(7.5)
7.6
(25.2)
2202
120
50.5
(17.8)
3.9
(7.9)
2504
295
31.2
(10.7)
4.5
(18.5)
2437
282
Rmerge overall
(final shell)
Number of atoms
Number of solvent
molecules
Number of bound
buffer molecules
Test set size
Rcryst
0
2
0
0
0
0
0
10%
15.2
10%
17.5
10%
16.4
10%
13.3
10%
16.2
10%
16.2
10%
16.2
(Rfree
)
(22.4)
(19.2)
(20.2)
(18.8)
(22.1)
(22.1)
(21.5)
RMS bonds/angles
Average B-factors (Å2)
Main chain:
Side chains:
Solvent:
0.026/1.947
14.491
11.6688
14.2259
28.0151
6.32
0.006/1.182
11.401
9.461
10.726
20.6237
5.53
0.011/1.304
12.68
13.2454
11.441
24.1527
4.98
0.028/2.117
15.419
12.427
15.0619
26.1515
7.67
0.023/1.993
25.434
23.7181
26.1502
32.7987
16.33
0.028/2.197
16.159
13.2707
15.7152
27.4072
7.11
0.029/2.234
14.065
11.1913
13.8444
22.7711
9.3
Ions:
Co-factors:
20.8162
12.7227
17.7765
31.499
34.152
34.3517
28.8634
P P
P P
P
nh
nh
Rmerge
¼
i¼1jhIhi ꢁ Ihij=
i¼1jIhij; where Ihi is an intensity value of the ith measurement of reflection h, h = (h, k, l), sum
runs over all measured reflections, and hIhi is
h
h
h
an average measured intensity of the reflection h. The nh is the number of measurements of reflection h.

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J. Sudzius et al. / Bioorg. Med. Chem. 18 (2010) 7413–7421
7417
Figure 8. The view of 10b bound to the active site of hCA II. Several selected distances between the ligand and protein atoms, shown as lines. The picture was generated using
Accelrys DS visualiser.
Figure 9. Left panel: View of compounds 7a (yellow) and 11b (blue) located in the active center of hCAII. Right panel: Compounds 5a (cyan), 7b (pink), 8b (purple), and 10b
40
(green) bound in active center of hCAII. Protein residues are shown in gray. The pictures are generated using MOLSCRIPT,38 Raster3D,39 and BOBSCRIPT
.
that is, nearly orthogonal to that formed by 7a and 11b. This con-
formation is fixed in the protein active center by a stacking inter-
action with Phe131 and by a hydrogen bond between the amino
group of Gln92 and a nitro or formyl group at C(5) of the pyrimi-
dine ring (in 5a it is the N(3) atom of the pyrimidine). Phe131 re-
strains the rotation of ligands. An inferred intramolecular
hydrogen bond between the nitro group and the amino group of
the linker can render molecules more rigid because it can prevent
rotation of the pyrimidine ring.
The benzylamino moiety at position C(6) of the pyrimidine in 8b
is packed tightly between residues 130–132 and residues 235–236
of the symmetrically equivalent protein molecule, so this confor-
mation could be a result of crystal packing. This conformation is
considered to be dominant, albeit the electron density is forked at
the first methylene group of the linker. This suggests that there
might be another orientation of the pyrimidine ring and benzyl-
amino moiety, but with our present data, it could not be modeled
with certainty.
Compound 10c has the longest linker with two CH2 groups, and
therefore higher intrinsic flexibility could be expected. Neverthe-
less, it is resolved very well in the crystal structure of the corre-
sponding complex with hCA II. It appears that in the binding of
this ligand, hydrophobic interactions play the most important role.
Therefore, a nitro group is exposed to bulk solvent without con-
tacts with the protein. The linker and the pyrimidine ring adopt a
conformation that places them equidistantly between hydrophobic

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J. Sudzius et al. / Bioorg. Med. Chem. 18 (2010) 7413–7421
7418
side chains (Pro202, Val135, and Phe131). Orientation of the
pyrimidine ring of 10c differs from that of other inhibitors.
To determine structural features of compound selectivity to-
wards particular CA isozymes, we would need to obtain co-crystal
structures of the compounds with all CAs and not just with hCA II.
Further development of more potent and more selective com-
pounds through various techniques, including crystallography
with other CAs, is underway.
Ar–H), 7.78 (d, J = 8.3 Hz, 2H, Ar–H), 8.46 (s, 1H, CH), 9.11 (t, 1H,
NH, J = 5.9 Hz). 13C NMR (DMSO-d6) d (ppm): 44.7, 126.5, 128.1,
128.9, 142.9, 143.5, 151.7, 155.1, 158.9. Anal. Calcd for
C11H10ClN5O4S: C, 38.43; H, 2.93. Found: C, 38.54; H, 2.86.
4.1.3. 4-{2-[N-(6-Chloro-5-nitropyrimidin-4-yl)amino]ethyl}
benzenesulfonamide (5c)
Compound 5c was synthesised by the procedure described for
compound 5b. Yield 64%. Mp 189 °C. IR: 3267, 3349, 3396 cmꢁ1
(NH, NH2). 1H NMR (DMSO-d6) d (ppm): 2.97 (t, J = 7.2 Hz, 2H,
CH2) 3.69–3.76 (m, 2H, CH2), 7,33 (s, 2H, NH2), 7.43 (d,
J = 8.2 Hz, 2H, Ar–H), 7.77 (d, J = 8.2 Hz, 2H, Ar–H), 8.49 (s, 1H,
CH), 8.57 (t, J = 5.5 Hz, 1H, NH). 13C NMR (DMSO-d6) d (ppm):
34.7, 43.0, 126.4, 128.8, 129.9, 143.0, 143.8, 151.5, 154.9, 158.9.
Anal. Calcd for C12H12ClN5O4S: C, 40.28; H, 3.38. Found: C,
40.46; H, 3.59.
3. Conclusion
A series of 4-[N-(substituted 4-pyrimidinyl)amino]benzenesulf-
onamides bind CAs with submicromolar to nanomolar potency.
Several compounds exhibited selectivity towards one CA out of
four tested CAs: 5a and 11b towards hCA XIII, 7b and 10b towards
hCA I, and 8c towards hCA II. Compound 7b was the most potent
but was non-selective towards hCA VII.
4.1.4. 4-[N-(6-Chloro-5-cyano-2-methylthiopyrimidin-4-yl)
amino]benzenesulfonamide (6a)
To a solution of 0.22 g (1 mmol) of 4,6-dichloro-2-methylthio-
pyrimidine-5-carbonitrile (2) and 0.17 g (1 mmol) of 4-aminoben-
4. Experimental
4.1. Synthesis
zenesulfonamide (4a) in 10 mL of THF 278 lL (2 mmol) Et3N was
added dropwise. The reaction mixture was refluxed for 24 h. Then
solvent was removed under reduced pressure and the residue was
purified by column chromatography using diethyl ether as eluent
to give 0.30 g (84%) of compound 6a. Mp 217 °C (dec). IR: 3326,
3272 (NH, NH2), 2222 cmꢁ1 (CN). 1H NMR (DMSO-d6) d (ppm):
2.47 (s, 3H, SCH3), 7.36 (s, 2H, NH2), 7.80 (m, 4H, Ar–H), 10.44 (s,
1H, NH). 13C NMR (DMSO-d6) d (ppm): 14.6, 87.6, 114.3, 124.1,
126.8, 140.9, 141.0, 160.2, 162.1, 175.5. HRMS calcd for
Melting points were determined in open capillaries on a Ther-
mo Scientific 9100 Series apparatus 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) using residual
DMSO signals (2.52 ppm and 40.21 ppm for 1H and 13C NMR spec-
tra, respectively) as internal standards. Elemental analyzes were
performed at the Elemental Analysis Laboratory of the Department
of Organic Chemistry of Vilnius University. TLC was performed
with silica gel 60 F254 aluminum plates (Merck) and visualized
with UV light. For column chromatography, silica gel 60 (0.040–
0.063 mm) (Merck) was used. High-resolution mass spectra
(HRMS) were recorded on an Dual-ESI Q-TOF 6520 mass spectrom-
eter (Agilent Technologies).
C
12H10ClN5O2S2 [M+H]+ = 356.0037, found 356.0033.
4.1.5. 4-{[N-(6-Chloro-5-cyano-2-methylthiopyrimidin-4-yl)
amino]methyl}benzenesulfonamide (6b)
A mixture of 0.22 g (1 mmol) 4,6-dichloro-2-methylthiopyrim-
idine-5-carbonitrile (2), 0.22 g (1 mmol) of 4-(aminomethyl)ben-
zenesulfonamide hydrochloride (4b) and 0.28 g (2 mmol) of
anhydrous K2CO3 in 8 mL THF was stirred at room temperature
for 12 h and then poured into 50 mL of water. The precipitate
was filtered off and recrystallised to give 0.14 g (38%) of com-
pound 6b. Mp 140 °C (from methanol). IR: 3340, 3250 (NH,
NH2), 2222 cmꢁ1 (CN). 1H NMR (DMSO-d6) d (ppm): 2.37 (s, 3H,
SCH3), 4.69 (d, J = 5.8 Hz, 2H, CH2), 7.35 (s, 2H, NH2), 7.51 (d,
J = 8.1 Hz, 2H, Ar–H), 7.78 (d, J = 8.1 Hz, 2H, Ar–H), 9.13 (t,
J = 5.8 Hz, 1H, NH). 13C NMR (DMSO-d6) d (ppm): 14.5, 44.9,
85.7, 114.6, 126.4, 128.3, 143.2, 149.5, 161.1, 161.4, 175.3. Anal.
Calcd for C13H12ClN5O2S2: C, 42.22; H, 3.27. Found: C, 42.43; H,
3.42.
Synthesis of the starting materials 1–3 was accomplished as
previously described.28,29
4.1.1. 4-[N-(6-Chloro-5-nitropyrimidin-4-yl)amino]benzenesul-
fonamide (5a)
A mixture of 0.20 g (1.03 mmol) of 4,6-dichloro-5-nitropyrimi-
dine (1), 0.18 g (1.03 mmol) of 4-aminobenzenesulfonamide (4a),
and 278 lL (2 mmol) of Et3N in 10 mL THF was refluxed for 16 h.
Solvent was removed under reduced pressure and the residue
was purified by column chromatography using EtOAc/n-hexane
(1:1 v/v) as the eluent to give 0.24 g (71%) of compound 5a. Mp
200 °C (dec). IR: 3375, 3340, 3270 cmꢁ1 (NH, NH2). 1H NMR
(DMSO-d6) d (ppm): 7.38 (s, 2H, NH2), 7.72–7.85 (m, 4H, Ar–H),
8.61 (s, 1H, CH), 10.36 (s, 1H, NH). 13C NMR (DMSO-d6) d (ppm):
117.9, 125.8, 126.8, 140.8, 141.5, 152.6, 155.5, 156.7. HRMS calcd
for C10H8ClN5O4S [MꢁH]ꢁ = 327.9913, found 327.9912.
4.1.6. 4-{2-[N-(6-Chloro-5-cyano-2-methylthiopyrimidin-4-yl)
amino]ethyl}benzenesulfonamide (6c)
A mixture of 0.220 g (1 mmol) of 4,6-dichloro-2-methylthiopyr-
imidine-5-carbonitrile (2), 0.200 g (1 mmol) of 4-(aminoethyl)ben-
zenesulfonamide (4c) and 0.144 g (1.04 mmol) of anhydrous K2CO3
in 10 mL THF was refluxed for 3 h, then cooled to room tempera-
ture and poured into 70 mL of water. The precipitate was filtered
off, dried in air and recrystallised to give 0.280 g (73%) of com-
pound 6c. Mp 213 °C (from methanol). IR: 3366, 3264 (NH, NH2),
2218 cmꢁ1 (CN). 1H NMR (DMSO-d6) d (ppm): 2.53 (s, 3H, SCH3),
2.97 (t, J = 7.1 Hz, 2H, CH2), 3.65–3.72 (m, 2H, CH2), 7.33 (s, 2H,
NH2), 7.43 (d, J = 8.2 Hz, 2H, Ar–H), 7.77 (d, J = 8.2 Hz, 2H, Ar–H),
8.61 (t, J = 6.1 Hz, 1H, NH). 13C NMR (DMSO-d6) d (ppm): 14.6,
34.8, 43.0, 85.3, 114.6, 126.5, 129.9, 142.9, 143.9, 161.1, 161.3,
175.3. Anal. Calcd for C14H14ClN5O2S2: C, 43.80; H, 3.68. Found:
C, 44.12; H, 3.59.
4.1.2. 4-{[N-(6-Chloro-5-nitropyrimidin-4-yl)amino]methyl}
benzenesulfonamide (5b)
A mixture of 0.291 g (1.5 mmol) of 4,6-dichloro-5-nitropyrimi-
dine (1), 0.335 g (1.5 mmol) of 4-(aminomethyl)benzenesulfon-
amide hydrochloride (4b) and 0.345 g (2.5 mmol) of anhydrous
K2CO3 in 15 mL THF was stirred at room temperature for 8 h. The
solution was filtered to remove inorganic salts, solvent was evapo-
rated under reduced pressure and the residue was purified by col-
umn chromatography using ethyl acetate:n-hexane (1:1 v/v) as
eluent to give 0.394 g (76%) of compound 5b. Mp 166 °C. IR:
3294, 3370 cmꢁ1 (NH, NH2). 1H NMR (DMSO-d6) d (ppm): 4.76
(d, J = 5.9 Hz, 2H, CH2), 7.34 (s, 2H, NH2), 7.50 (d, J = 8.3 Hz, 2H,

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7419
4.1.7. 4-[N-(6-Chloro-5-formyl-2-methylthiopyrimidin-4-yl)
amino]benzenesulfonamide (7a)
7.23–7.37 (m, 7H, Ph, NH2), 7.51 (d, J = 8.2 Hz, 2H, Ar–H), 7.78 (d,
J = 8.2 Hz, 2H, Ar@H), 8.04 (s, 1H, CH), 9.88 (t, J = 6.1 Hz, 1H, NH),
9.96 (t, J = 6.0 Hz, 1H, NH). 13C NMR (DMSO-d6) d (ppm): 44.6,
44.9, 113.1, 126.4, 127.6, 128.0, 128.1, 129.0, 139.5, 143.2, 143.8,
157.4, 157.5, 160.0. Anal. Calcd for C18H18N6O4S: C, 52.17; H,
4.38. Found: C, 52.43; H, 4.59.
Compound 7a was synthesized from 4,6-dichloro-2-methyl-
thiopyrimidine-5-carbaldehyde (3) and 4-aminobenzenesulfona-
mide (4a) according to a procedure described for the synthesis of
6a. The reaction time was 36 h, yield 30%. Mp was 227 °C (from
methanol). IR: 3318, 3236 (NH, NH2), 1638 cmꢁ1 (CHO). 1H NMR
(DMSO-d6) d (ppm): 2.57 (s, 3H, SCH3), 7.38 (s, 2H, NH2), 7.89
(m, 4H, Ar–H), 10.25 (s, 1H, NH), 11.26 (s, 1H, CHO). 13C NMR
(DMSO-d6) d (ppm): 14.9, 105.9, 115.8, 123.2, 127.3, 140.4, 140.9,
158.4, 164.5, 191.6. Anal. Calcd for C12H11ClN4O3S2: C, 40.17; H,
3.09. Found: C, 40.27; H, 3.16.
4.1.12. 4-{2-[N-(6-Benzylamino-5-nitropyrimidin-4-yl)
amino]ethyl}benzenesulfonamide (8c)
Compound 8c was prepared according to procedure described
for the synthesis 8a. The reaction time was 3 h, yield 35%. Mp
was 211 °C. IR: 3274, 3335 cmꢁ1 (NH, NH2). 1H NMR (DMSO-d6) d
(ppm): 3.00 (t, J = 6.2 Hz, 2H, CH2), 3.79–3.86 (m, 2H, CH2), 4.80
(d, J = 5.9 Hz, 2H, CH2), 7.22–7.35 (m, 7H, Ph, NH2), 7.46 (d,
J = 8.2 Hz, 2H, Ar–H), 7.77 (d, J = 8.2 Hz, 2H, Ar–H), 8.12 (s, 1H,
CH), 9.49 (t, J = 5.8 Hz, 1H, NH), 9.86 (t, J = 5.9 Hz, 1H, NH). 13C
NMR (DMSO-d6) d (ppm): 35.1, 42.8, 44.9, 112.9, 126.5, 127.6,
128.0, 129.0, 129.9, 139.5, 142.9, 144.0, 157.4, 157.5, 160.2. HRMS
calcd for C19H20N6O4S [M+H]+ = 429.1340, found 429.1339.
4.1.8. 4-{[N-(6-Chloro-5-formyl-2-methylthiopyrimidin-4-
yl)amino]methyl}benzenesulfonamide (7b)
A mixture of 0.22 g (1 mmol) of 4,6-dichloro-2-methylthiopyrim-
idin-5-carbaldehyde(3), 0.22 g (1 mol) of 4-(aminomethyl)benzene-
sulfonamide hydrochloride (4b) and 0.28 g (2.1 mmol) of anhydrous
K2CO3 in 8 mL THFwasrefluxedfor10 h, cooled toroom temperature
and poured into 50 mL of water. Resulting precipitate was filtered
off, dried in air and recrystallised to give 0.26 g (70%) of compound
7b. Mp 195 °C (from 2-propanol). IR: 3480, 3276 (NH, NH2),
1648 cmꢁ1 (CO). 1H NMR (DMSO-d6) d (ppm): 2.39 (s, 3H, SCH3),
4.83 (d, J = 6.2 Hz, 2H, CH2), 7.33 (s, 2H, NH2), 7.50 (d, J = 8.3 Hz,
2H, Ar–H), 7.78 (d, J = 8.3 Hz, 2H, Ar–H), 9.82 (t, J = 6.2 Hz, 1H, NH),
10.18 (s, 1H, CHO). 13C NMR (DMSO-d6) d (ppm): 14.6, 44.5, 105.3,
126.5, 128.3, 143.3, 143.5, 160.1, 164.1, 176.1, 190.5. Anal. Calcd
for C13H13ClN4O3S2: C, 41.88; H, 3.51. Found: C, 41.87; H, 3.58.
4.1.13. 4-[N-(6-Benzylamino-5-cyano-2-methylthiopyrimidin-
4-yl)amino]benzenesulfonamide (9a)
Compound 9a was synthesized from 4-[N-(6-chloro-5-cyano-2-
methylthiopyrimidin-4-yl)amino]benzenesulfonamide (6a) and
benzylamine according to procedure described for the synthesis
of 8a. Compound 9a was purified by column chromatography
using diethyl ether as eluent. The reaction time was 12 h, yield
54%. Mp was 194 °C. IR: 3417 (NH, NH2), 2198 cmꢁ1 (CN). 1H
NMR (DMSO-d6) d (ppm): 2.34 (s, 3H, SCH3), 4.59 (s, 2H, CH2),
7.24–7.34 (m, 7H, 2NH, Ph), 7.66 (m, 4H, Ar–H), 8.10 (s, 1H, NH).
HRMS calcd for C19H18N6O2S2 [M+H]+ = 427.1005, found 427.1011.
4.1.9. 4-{2-[N-(6-Chloro-5-formyl-2-methylthiopyrimidin-4-yl)
amino]ethyl}benzenesulfonamide (7c)
Compound 7c was synthesized from 4,6-dichloro-2-methyl-
thiopyrimidine-5-carbaldehyde (3) and 4-(aminoethyl)benzene-
sulfonamide (4c) according to a procedure described for the
synthesis of 7b. The reaction time was 3 h, yield 61%. Mp was
182 °C (from 2-propanol). IR: 3256 (NH, NH2), 1646 cmꢁ1 (CO).
1H NMR (DMSO-d6) d (ppm): 2.56 (s, 3H, SCH3), 3.01 (t, J = 7.2 Hz,
2H, CH2), 3.79–3.86 (m, 2H, CH2), 7.34 (s, 2H, NH2), 7.46 (d,
J = 8.0 Hz, 2H, Ar–H), 7.78 (d, J = 8.0 Hz, 2H, Ar–H), 9.38 (t,
J = 5.8 Hz, 1H, NH), 10.13 (s, 1H, CH). 13C NMR (DMSO-d6) d
(ppm): 14.6, 35.0, 42.4, 105.0, 126.5, 129.9, 143.0, 143.8, 160.1,
4.1.14. 4-{[N-(6-Methoxy-5-nitropyrimidin-4-yl)amino]
methyl}benzenesulfonamide (10b)
A mixture of 0.194 g (1 mmol) 4,6-dichloro-5-nitropyrimidine
(1), 0.223 g (1 mmol) 4-(aminomethyl)benzenesulfonamide hydro-
chloride (4b) and 0.290 g (2.1 mmol) of anhydrous K2CO3 in 10 mL
THF was stirred for 8 h at room temperature. The solution was fil-
tered and the solvent was removed under reduced pressure. To the
obtained residue 20 mL MeOH was added and the mixture was re-
fluxed for 30 min. The hot solution was filtered and cooled to room
temperature. The precipitate was filtered off and dried in air to give
0.20 g (57%) of compound 12b. Mp 174 °C. IR: 3270, 3374 cmꢁ1
(NH, NH2). 1H NMR (DMSO-d6) d (ppm): 4.00 (s, 3H, OCH3), 4.80
(d, J = 6.1 Hz, 2H, CH2), 7.33 (s, 2H, NH2), 7.50 (d, J = 8.1 Hz, 2H,
Ar–H), 7.78 (d, J = 8.1 Hz, 2H, Ar–H), 8.31 (s, 1H, CH), 9.14 (t,
J = 6.1 Hz, 1H, NH). 13C NMR (DMSO-d6) d (ppm): 44.8, 55.9,
116.7, 126.4, 128.0, 143.3, 143.8, 156.6, 159.3, 163.8. Anal. Calcd
for C12H13N5O5S: C, 42.47; H, 3.86. Found: C, 42.29; H, 4.01.
164.2, 176.2, 190.7. HRMS calcd for
C14H15ClN4O3S2
[M+H]+ = 387.0347, found 387.0340.
4.1.10. 4-[N-(6-Benzylamino-5-nitro-4-yl)amino]benzenesul-
fonamide (8a)
A mixture of 0.120 g (0.364 mmol) 4-[N-(6-chloro-5-nitropyr-
imidin-4-yl)amino]benzenesulfonamide (5a), 40 lL (0.364 mmol)
of benzylamine, and 0.055 g (0.40 mmol) of anhydrous K2CO3 in
5 mL DMF was heated at 100 °C for 12 h. Then cooled to room tem-
perature and poured into 50 mL of water. Resulting precipitate was
purified by column chromatography using ethyl acetate/n-hexane
(1:1 v/v) as eluent to give 0.088 g (60%) of compound 8a. Mp
195 °C. IR: 3347 cmꢁ1 (NH). 1H NMR (DMSO-d6) d (ppm): 4.84 (d,
J = 6.0 Hz, 2H, CH2), 7.25–7.37 (m, 7H, NH2, Ph), 7.83 (s, 4H, Ar–
H), 8.18 (s, 1H, CH), 9.91 (t, J = 6.0 Hz, 1H, NH), 11.03 (s, 1H, NH).
13C NMR (DMSO-d6) d (ppm): 45.0, 113.7, 124.8, 126.9, 127.6,
128.0, 129.0, 139.4, 140.9, 141.2, 156.1, 157.2, 159.9. Anal. Calcd
for C17H16N6O4S: C, 50.99; H, 4.03. Found: C, 50.94; H, 4.23.
4.1.15. 4-{2-[N-(6-Methoxy-5-nitropyrimidin-4-yl)amino]
ethyl}benzenesulfonamide (10c)
Compound 10c was synthesized and purified by the procedure
described for compound 10b. Yield 47%. Mp 208 °C. IR: 3256,
3358 cmꢁ1 (NH, NH2). 1H NMR (DMSO-d6) d (ppm): 2.98 (t,
J = 7.2 Hz, 2H, CH2), 3.75–3.81 (m, 2H, CH2) 4.00 (s, 3H, OCH3),
7.33 (s, 2H, NH2), 7.45 (d, J = 8.2 Hz, 2H, Ar–H), 7.77 (d, J = 8.2 Hz,
2H, Ar–H), 8.38 (s, 1H, CH), 8.63 (t, J = 5.7 Hz, 1H, NH). 13C NMR
(DMSO-d6) d (ppm): 35.2, 42.9, 55.8, 120.7, 126.5, 129.9, 142.9,
144.0, 156.5, 159.4, 163.8. Anal. Calcd for C13H15N5O5S: C, 44.19;
H, 4.28. Found: C, 44.25; H, 4.38.
4.1.11. 4-{[N-(6-Benzylamino-5-nitropyrimidin-4-yl)amino]
methyl}benzenesulfonamide (8b)
Compound 8b was prepared according to a procedure described
for the synthesis 8a. The reaction time was 3 h, yield 34%. Mp was
162 °C. IR: 3339, 3273 cmꢁ1 (NH, NH2). 1H NMR (DMSO-d6) d
(ppm): 4.80 (d, J = 6.1 Hz, 2H, CH2), 4.85 (d, J = 6.0 Hz, 2H, CH2),
4.1.16. 4-{[(5-Nitro-6-oxo-1,6-dihydro-4-pyrimidinyl)
amino]methyl}benzenesulfonamide (11b)
To a solution of 0.160 g (0.466 mmol) of 4-{[N-(6-chloro-5-
nitropyrimidin-4-yl)amino]methyl}benzenesulfonamide (5b) in

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J. Sudzius et al. / Bioorg. Med. Chem. 18 (2010) 7413–7421
7420
THF (5 mL) water (150
lL) was added. The mixture was left at
4.4. Crystallography
room temperature for 3 days. The solid formed was filtered off,
washed with THF (3 ꢀ 5 mL) and dried in an air to give 0.125 g
(83%) of compound 11b. Mp >300 °C (dec) IR: 1676 (CO), 3066,
3191, 3274, 3324 cmꢁ1 (NH, NH2). 1H NMR (DMSO-d6) d (ppm):
4.86 (d, J = 6.2 Hz, 2H, CH2), 7.33 (s, 2H, NH2), 7.49 (d, J = 8.2 Hz,
2H, Ar–H), 7.78 (d, J = 8.2 Hz, 2H, Ar–H), 8.09 (s, 1H, CH), 10.0 (t,
1H, NH, J = 6.2 Hz), 12.47 (s, 1H, NH). 13C NMR (DMSO-d6) d
(ppm): 45.1, 116.3, 126.4, 128.1, 143.4, 143.5, 152.7, 155.6,
158.9. HRMS calcd for C11H11N5O5S [M+H]+ = 326.0554, found
326.0554. Anal. Calcd for C11H11N5O5S: C, 40.61; H, 3.41. Found:
C, 40.94; H, 3.64.
4.4.1. Crystallization
hCA II was concentrated prior to crystallization to 20–60 mg/mL
by ultrafiltration in 20 mM Na-Hepes pH 7.5 and 50 mM NaCl.
Crystallization was started by mixing equal volumes of protein
solution with reservoir buffer. Crystallization buffers were pre-
pared by mixing 1 M Na-Bicine pH 9 to the concentration 0.1 M,
2.7 M Na-Malonate pH 7–7.55 to the final concentration ranged
from 1.5 to 2.2 M, and 3.5 M ammonium sulfate to the final con-
centration ranged from 0 to 0.2 M. Crystals belonging to the P21
space group were grown over several days. Crystals were soaked
in a 0.5–1.0 mM solution of the ligand of interest, prepared by mix-
4.2. Construction of expression vectors and protein purification
ing 100 mM ligand solution in DMSO with 50
buffer used in crystallization.
lL of the reservoir
Expression and purification of hCA I and hCA II were previously
described: hCA I in Ref. 9 and hCA II in Ref. 30.
4.4.2. Data collection and structure determination
The hCA VII gene was obtained from RZPD Deutsches Ressour-
cenzentrum für Genomforschung (Berlin, Germany) in the pCMV-
SPORT6 vector and cloned into the pET-15b vector (Novagen, Mad-
ison, WI) for expression. The nearly full-length CA VII nucleotide
sequence encoding residues 3–264 was inserted into the BamHI
site of the pET-15b vector fusing the His-tag sequence to the N-ter-
minus of CA VII.
The hCA XIII gene was obtained from RZPD Deutsches Ressour-
cenzentrum für Genomforschung (Berlin, Germany) in the pOTB7
vector and cloned into the pET-15b vector for expression. The
nucleotide sequence encoding full-length CA XIII (residues 1–
262) was inserted into the pET-15b vector (Novagen, Madison,
WI) via NcoI and XhoI sites. The cloning procedure resulted in
the removal of the His-tag sequence from the vector, thus the
pET-15b-CA XIII plasmid contains an open reading frame for full-
length hCA XIII protein, with one additional methionine at the N-
terminus.
Diffraction data from all complexes of hCA II with inhibitors, ex-
cept 11b, were collected at the EMBL X11, X12, and X13 beam lines
at the DORIS storage ring (DESY, Hamburg). The dataset of hCA II
with 11b was measured by the Institute of Biotechnology (Vilnius,
Lithuania) with an RU-H3R diffractometer (Rugaku, Japan). MOS-
FLM31,32 and SCALA33 were used for image processing. Initial
phases were obtained by molecular replacement with the protein
moiety from PDB entry 3HLJ9 as an initial model. Structures were
refined using REFMAC34 and COOT35 was used for model inspec-
tion. Atomic co-ordinates of ligands were generated by DSVisualiz-
er 1.736 (Accelrys). Topology and parameters for structure
refinement were generated by LIBREFMAC.37 Data collection and
refinement statistics are presented in Table 2. Co-ordinates and
structure factors were deposited in the RCSB Protein Data Bank
and the PDB IDs are listed in Table 2.
Acknowledgments
Recombinant proteins were expressed in Escherichia coli strain
BL21 (DE3). Expression plasmid-transformed cells were grown at
37 °C in LB media containing 0.1 mg/mL ampicillin and 60 lM
The project was supported in part by EEA and Norway Grants
2004-LT0019-IP-1EEE and the Lithuanian Government. Diffraction
data were collected at the EMBL/DESY, Hamburg, except data for
the complex with 11b, which was measured at the Institute of Bio-
technology. D. Golovenko’s access to the measurement facilities
were funded by the European Community’s Seventh Framework
Programme (FP7/2007-2013) under Grant agreement 226716. E.
Manakova’s and S. Grazulis’ travel to DESY, Hamburg was sup-
ported by 2004-LT0019-IP-1EEE and by the Research Council of
Lithuania. We thank our local contacts at the EMBL beamlines,
Dr. Gleb Bourenkov and Dr. Michele Cianci, for help with beamline
operation. We thank Dr. Fernando Ridoutt for help with the beam-
line cryosystems.
ZnSO4 to OD550 0.5–0.8. After induction with 1 mM IPTG and
0.5 mM ZnSO4, cells were cultured at 30 °C for 4 h. Cells were har-
vested by centrifugation and lysed by sonication. Soluble protein
was purified using an affinity column (Ni+2 Chelating Sepharose,
FF GE Healthcare Bio-Sciences, Uppsala, Sweden), followed by an-
ion exchange chromatography (CM-Sepharose, FF, GE Healthcare
Bio-Sciences, Uppsala, Sweden). Eluted protein was dialyzed
against storage buffer (20 mM HEPES, pH 7.5, 50 mM NaCl) and
stored at ꢁ80 °C.
4.3. Compound binding to CA
4.3.1. Isothermal titration calorimetry (ITC)
References and notes
ITC experiments were performed using a VP-ITC instrument
(Microcal, Inc.) using 5–20
50–200 M ligand solution in the syringe. A typical experiment
consisted of 25–30 injections 10 L each within 3–4 min intervals.
Experiments were carried out at 37 °C in phosphate buffer contain-
ing 50 mM NaCl at pH 7.0, with a final DMSO concentration of 0.5–
lM protein solution in the cell and
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6000 (QIAGEN Rotor-Gene Q) instrument using the blue channel
(excitation 365 20, detection 460 15 nm). Samples contained
9. Baranauskiene, L.; Hilvo, M.; Matuliene, J.; Golovenko, D.; Manakova, E.;
˙
˙
10 lM protein, 0–200 lM ligand, 50 lM solvatochromic dye ANS
Dudutiene, V.; Michailoviene, V.; Torresan, J.; Jachno, J.; Parkkila, S.; Maresca,
˙
˙
(8-anilino-1-naphthalene sulfonate), and phosphate buffer con-
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of 2%. The applied heating rate was 1 °C/min.
ˇ
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