Design of [(2-pyrimidinylthio)acetyl]benzenesulfonamides as inhibitors of human carbonic anhydrases

Article abstract of DOI:10.1016/j.ejmech.2012.02.050

A series of [(2-pyrimidinylthio)acetyl]benzenesulfonamides were designed and synthesized. Their binding affinities as inhibitors of several recombinant human carbonic anhydrase (CA) isozymes were determined by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). A group of compounds containing a chlorine atom in the benzenesulfonamide ring were found to exhibit higher selectivity but lower binding affinity toward tested CAs. The crystal structures of selected compounds in complex with CA II were determined to atomic resolution. Docking studies were performed to compare the binding modes of experimentally determined crystallographic structures with computational prediction of the pyrimidine derivative binding to CA II. Several compounds bound to select CAs with single-digit nanomolar affinities and could be used as leads for inhibitor development toward a select CA isozyme.

Full text of DOI:10.1016/j.ejmech.2012.02.050

European Journal of Medicinal Chemistry 51 (2012) 259e270
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design of [(2-pyrimidinylthio)acetyl]benzenesulfonamides as inhibitors of human
carbonic anhydrases
a
,
b
a
a
c
c
ꢀ
_
_
_
_
_
*
Edita Capkauskaite , Asta Zubriene , Lina Baranauskiene , Giedre Tamulaitiene , Elena Manakova ,
d
c
b
a,
ꢀ
ꢀ
Visvaldas Kairys , Saulius Grazulis , Sigitas Tumkevicius , Daumantas Matulis
a
ꢀ ꢁ
Vilnius University, Institute of Biotechnology, Department of Biothermodynamics and Drug Design, Graiciuno 8, Vilnius LT-02241, Lithuania
b Vilnius University, Faculty of Chemistry, Department of Organic Chemistry, Naugarduko 24, Vilnius LT-03225, Lithuania
c
ꢀ ꢁ
Vilnius University, Institute of Biotechnology, Department of Protein e DNA Interactions, Graiciuno 8, Vilnius LT-02241, Lithuania
Vilnius University, Institute of Biotechnology, Department of Bioinformatics, Graiciuno 8, Vilnius LT-02241, Lithuania
d
ꢀ ꢁ
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of [(2-pyrimidinylthio)acetyl]benzenesulfonamides were designed and synthesized. Their binding
affinities as inhibitors of several recombinant human carbonic anhydrase (CA) isozymes were determined
by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). A group of compounds containing
a chlorine atom in the benzenesulfonamide ring were found to exhibit higher selectivity but lower binding
affinity toward tested CAs. The crystal structures of selected compounds in complex with CA II were
determined to atomic resolution. Docking studies were performed to compare the binding modes of
experimentally determined crystallographic structures with computational prediction of the pyrimidine
derivative binding to CA II. Several compounds bound to select CAs with single-digit nanomolar affinities
and could be used as leads for inhibitor development toward a select CA isozyme.
Received 14 September 2011
Received in revised form
23 February 2012
Accepted 24 February 2012
Available online 3 March 2012
Keywords:
Carbonic anhydrase isozymes
Isothermal titration calorimetry
Thermal shift assay
Ó 2012 Elsevier Masson SAS. All rights reserved.
ThermoFluorÒ
Sulfonamides as CA inhibitors
1. Introduction
development of new therapeutic agents. Classical CA inhibitors
already described are characterized by the presence of aromatic/
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous zinc-
containing metalloenzymes present in prokaryotes and eukaryotes
that catalyzethereversible hydration ofcarbon dioxideto bicarbonate
[1]. There are 15 different isozymes of human carbonic anhydrases
(CA), with different tissue distributions, subcellular locations, and
expression levels [2]. CAs are involved in numerous important bio-
logical processes related to respiration, pH balance, CO2 homeostasis,
electrolyte secretion in a variety of tissues/organs, gluconeogenesis,
lipogenesis, ureagenesis, bone resorption, calcification, tumorige-
nicity, and many other physiologic or pathologic processes [2e5].
Many CA isozymes constitute valid targets for the design and
development of CA inhibitors for clinical applications. There are at
least 30 CA inhibitors in clinical development or clinically used
drugs [2]. Diffuse localization of CA isoforms in many tissues/organs
limits potential pharmacological applications [6e8]. Hence, the
design of isozyme-specific inhibitors is the current challenge in the
heterocyclic sulfonamide or sulfamate/sulfamide scaffolds [7,9].
Recently, we have reported on the synthesis of benzimidazoles N-
and S-alkylated with 2-chloro-5-bromoacetylbenzenesulfona
mide (I, II) (Fig. 1) and the study of their CA inhibitory activity
toward CA I, CA II, CA VII, and CA XIII [10]. It was found that S-alky-
lated benzimidazole derivatives bind more strongly to the tested CAs
than N-alkylated benzimidazoles and the structurally related inda-
pamide. Moreover, some S-alkylated derivatives were found to
exhibit selectivity to CA II and CA VII. The study of cocrystal struc-
tures of the most active S-alkylated derivatives of benzimidazole
with CA II revealed that the CA II-bound S-alkylated ligands have
larger hydrogen bonding patterns due to hydrogen bonds between
the S and Nd2 atoms of Asn62 and water-mediated hydrogen bonds
of the endocyclic nitrogen atoms of benzimidazole with the protein.
The study of binding details of (4-pyrimidinyl)benzenesulfona-
mides (III) with CA II showed that in the binding of sulfonamides, in
which pyrimidine and benzenesulfonamide moieties are separated
with a NHCH2CH2 linker, hydrophobic interactions of the ligand
with the protein play the most important role [11]. We designed [(2-
pyrimidinylthio)acetyl]benzenesulfonamides 1aej, 2aej (Scheme
1) in the search for more potent and selective CA inhibitors.
Abbreviations: c, cyclic form; CA, carbonic anhydrase; ITC, isothermal titration
calorimetry; o, open chain form; TSA, thermal shift assay.
* Corresponding author. Tel.: þ370 5 269 1884; fax: þ370 5 260 2116.
E-mail addresses: matulis@ibt.lt, daumantas.matulis@bti.vu.lt (D. Matulis).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.050

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E. Capkauskaite et al. / European Journal of Medicinal Chemistry 51 (2012) 259e270
260
R1
O
N
NH2
O S
O
O
S
N
N
H
N
n
HN
R2
Cl
H2N
N
Cl
H2N
S O
O
R3
N
S O
O
III
II
I
R
Fig. 1. Structures of N- and S-alkylated (benzimidazolyl)benzenesulfonamides I, II, and (4-pyrimidinyl)benzenesulfonamides III.
Furthermore, docking studies indicate that computational
two techniques, there were some discrepancies between the TSA
and ITC data observed with CA I and, to a lesser extent, for the other
three isozymes. Both the ITC and TSA experiments were repeated
several times, and the discrepancies appeared to be out of the range
of the standard deviations for both methods. The discrepancies
appeared to be random, but the TSA data appeared to be more
reliable, especially for the tightest binding cases where ITC curves
were too steep to be fitted precisely.
modeling is consistently improving in its predictive powers in
compound affinities ranking and the estimation of the structural
features of the proteineligand complex. The structures of several
predicted compoundeprotein complexes closely matched the
crystallographic structures. The predictive method is increasingly
useful in the design of novel CA inhibitors.
2. Results and discussion
The binding affinity of all compounds in this study ranged from
approximately 4.5
mM for 1j to approximately 1.9 nM for 2d, both
2.1. Chemistry
toward CA I. The ranges were smaller for other tested CAs:
9.1e200 nM (2k and 1a) for CA II, 20e710 nM (1d and 1k) for CAVII,
and 3.5e360 nM (2d and 2a) for CA XIII.
S-alkylation of aej with
u-bromoacetylbenzenesulfonamides 1
and 2 to give pyrimidine derivatives 1aej and 2aej was carried out
in tetrahydrofuran at room temperature in the presence of sodium
acetate (Scheme 1). For comparison, 2-chloro-5-[(phenylthio)
acetyl]benzenesulfonamides 1k and 2k have also been synthesized
by the alkylation of thiophenol (k) with benzenesulfonamides 1
and 2 using the same reaction conditions employed for the
synthesis of 1aej and 2aej, respectively (Scheme 2).
In most cases, the 2-Cl-benzenesulfonamide headgroup
(compounds 1) had lower binding affinity than benzenesulfona-
mide (compounds 2). For example, 1a bound to CA I approximately
12 times more weakly than 2a, as shown by both TSA and ITC. The
same was true for CA II, though to a lesser extent, approximately 3
times. However, CA VII bound Cl-bearing compounds 1 even
stronger than compounds 2. For example, compound 1d bound CA
VII about 9 times more strongly than 2d.
2.2. Binding studies
Various aromatic rings attached to the headgroup of benzene-
sulfonamides have been previously tested. In a search for more
selective compounds, we have synthesized a series of pyrimidine-
bearing compounds. Comparison of the affinities of the
pyrimidine-containing compounds (1j and 2j) with compounds
bearing the benzene ring (1k and 2k) shows that benzene has 5e10
times stronger affinity to CA I, 1.5e2 times stronger to CA II, 1e1.3
times stronger to CA VII, and 1.3e1.5 times stronger to CA XIII.
Therefore, inclusion of the pyrimidine appears to have weakened
the affinity. However, pyrimidine-bearing compounds are often
more selective. For example, 1j binds CA II, CA VII, and CA XIII 6e14
times stronger than to CA I, while 1k binds CA II, CA VII, and CA XIII
only 1.3e3 times stronger than to CA I.
The binding affinities of the [(2-pyrimidinylthio)acetyl]benze-
nesulfonamides (1aej and 2aej) and [(2-phenylthio)acetyl]benze-
nesulfonamides (1k and 2k) to recombinant human CA isozymes
CA I, CA II, CA VII, and CA XIII were determined by thermal shift
assay (TSA) and isothermal titration calorimetry (ITC). The
observed dissociation constants for all compounds are listed in
Table 1. Fig. 2 shows representative binding data obtained by TSA
(isozymes CA I, CA II, and CA VII), while Fig. 3 shows representative
binding data obtained by ITC (isozymes CA I and CA XIII). Both
techniques were used for the measurements to obtain the most
precise binding data possible. Despite good agreement between the
O
O
SH
S
N
R3
R4
Br
NaOAc, THF
r.t., 24h
N
N
+
N
R1
R1
R5
R3
R2
R5
R2
R4
1a-j,
2a-j
a-j
1: R1=Cl, R2=SO2NH2,
2: R1=SO2NH2, R2=H,
f: R3=Me, R4=H, R5=Me,
g: R3=H, R4=Et, R5=H,
h: R3=H, R4=Pr, R5=H,
i: R3=H, R4=Bu, R5=H,
j: R3=H, R4=H, R5=H,
a: R3=OH, R4=H, R5=Me,
b: R3=OH, R4=Bn, R5=Me,
c: R3=OH, R4=H, R5=Pr,
d: R3=OH, R4=H, R5=t-Bu,
e: R3=OH, R4=CO2Et, R5=H,
Scheme 1. Synthesis of compounds 1aej and 2aej.

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E. Capkauskaite et al. / European Journal of Medicinal Chemistry 51 (2012) 259e270
261
O
compounds bound to CA II are shown in Fig. 4. The presence of
chlorine determines the position of the first ring. The 2-
chlorobenzenesulfonamide rings coincide perfectly in both crystal
structures (Fig. 5). The benzenesulfonamide ring position in the
crystal structures with inhibitors that do not contain Cl also overlap
well (Fig. 6), but the planes of the first rings in both cases differ by
an angle of approximately 30ꢀ (Fig. 7). Pyrimidine rings of ligands
that do not contain Cl are nearly co-planar to each other.
O
SH
S
Br
NaOAc, THF
r.t., 24h
+
R1
R1
R2
R2
1k, 2k
k
1: R1=Cl, R2=SO2NH2,
2: R1=SO2NH2, R2=H,
Scheme 2. Synthesis of compounds 1k and 2k.
The orientation of the first ring is defined by the tight binding of
the sulfonamide group to the Zn in the active center of the protein.
The Cl is trapped between hydrophobic residues Leu198, Leu141,
Val143, Val121, and Val207. This binding pattern is very similar to
that observed in 3M67, 3M96 and 3MYQ, as described in [10].
The conformation of the linker connecting 2-chlorobenzenesu
lfonamide in 1f and 1g is fixed in both crystal structures by
hydrogen bonds of oxygen with Gln92 and Asn67 side chains.
Sulfur participates in a hydrogen bond with the amino group of
Asn62. There are DMSO molecules trapped between the pyrimidine
moiety of 1f or 1g and the protein, which was carried over from the
solution of inhibitors used for soaking CA II crystals.
The data reported in Table 1 also show that the nature of the
substituent on the pyrimidine ring significantly influences the
binding affinity against CA I, CA II, CA VII, and CA XIII isoforms. For
example, the addition of the third ring (compounds 1a and 2a
versus 1b and 2b, respectively) did not appreciably benefit the
binding to CA I, CA II, and CA VII but increased the binding to CA XIII
by about 5e30 times. Comparing the series of compounds bearing
aliphatic tails on the pyrimidine ring (ethyl g, propyl h, and butyl i)
shows that the tail length did not affect the binding affinity by 2
times to any of the tested CAs.
The position of the pyrimidine ring in both compounds was very
similar. These parts of the molecules were found in the same plane
as the benzimidazole in the 3M67 and 3M96 structures. This
moiety is in a van der Waals contact with protein side chains that
form a substrate-binding cavity.
Although ligands 2a, 2i, 2f and 2j did not contain Cl in the
benzenesulfonamide ring, the ring was in a very similar position in
all four structures. This position is probably defined by hydrophobic
contacts with protein side chains. Leu198 supports the benzene
ring, while Thr200 and Val121 restrain its mobility from both sides.
The linker is positioned in such a way that carbonyl is too far away
to make hydrogen bonds with polar amino acids of the substrate-
binding cavity of CA II. The interaction of this group of ligands
observed in crystals is mainly hydrophobic. The cyclic form II of
compound 2f was not found in the crystal structure.
The pyrimidine ring of inhibitor 2a showed alternative confor-
mations in the crystal structure. The linker in 2a was forked after
the carbonyl group, and two alternative conformations were
modeled to explain the observed electron density. The pyrimidine
rings in both variants overlapped, and two alternate positions for
the sulfur atom of the linker were found in this structure. The
electron density of the pyrimidine of 2i was rather poor, which
means that this ring was not fixed in the crystal structure, although
the main conformation seems to be the modeled one.
It should be noted that the dissociation constants listed in
Table 1 were only observed at particular conditions, such as pH 7.0.
These dissociation constants were not corrected for the linked
protonation effects occurring upon ligand binding to CA. To deter-
mine an intrinsic binding constant, it is important to measure
ligand association with CA at a series of pH values and in at least
two buffers. Because the goal was to compare the observed disso-
ciation constants occurring at physiological conditions, no such
measurements were performed.
2.3. Crystallography
The structures of compound-CA II complex were solved to
atomic resolution for compounds 1f, 1g, 2a, 2f, 2i, and 2j. The six
compounds that were selected for soaking to CA II crystals could be
divided into two groups, compounds containing Cl in the benze-
nesulfonamide ring (1f and 1g) and compounds that do not have Cl
in the ring (2a, 2f, 2i, and 2j). The electron density maps of several
Table 1
Synthetic compound dissociation constants to four human recombinant CA isoforms
as determined by TSA and confirmed by ITC (given in brackets), both determined at
37 ꢀC, pH 7.0.
Compound Dissociation constants Kd
CA I CA II
0.83 (2.5)
(
m
M) to CA isoforms
CA VII
CA XIII
0.20 (0.10)
2.4. Docking studies
1a
2a
1b
2b
1c
2c
1d
2d
1e
2e
1f
0.20 (0.13)
0.070 (0.050)
0.033 (0.050)
0.025 (0.093)
0.13 (0.32)
0.17 (0.63)
0.14 (0.57)
0.14 (0.38)
0.067 (0.18)
0.45 (0.69)
0.033 (0.10)
0.67 (0.75)
0.017 (0.087)
0.10 (0.24)
0.0019 (0.020)
1.2 (0.42)
0.36 (0.71)
0.042 (0.17)
0.013 (0.071)
0.056 (0.081)
0.063 (0.068)
0.025 (0.062)
0.0035 (0.037)
0.13 (0.13)
0.29 (0.32)
0.33 (0.98)
0.083 (0.13)
0.11 (0.11)
0.029 (0.080)
0.067 (0.82)
0.023 (0.020)
0.067 (0.16)
0.029 (0.019)
0.33 (0.46)
Compound docking simulations to CA II were performed to
validate and improve computational procedures for the prediction
of compound binding efficiency and structural arrangement. The
docking simulations were performed using Vdock software [12].
Initially, ligands 1f and 2j were docked into corresponding CA II
X-ray structures to verify if the experimental binding mode can be
reproduced. Because of the sulfonamide nitrogen coordination with
zinc in the CA binding site, the docking was expedited by fixing the
sulfonamide nitrogen at the X-ray position. This was accomplished
in Vdock by setting the relevant nitrogen atom as the translational
center and placing it inside a zero-sized (0 ꢁ 0 ꢁ 0) translational
box. This procedure removed translational degrees of freedom from
the docking task and ensured the correct binding mode of the
sulfonamide group. As a result, the benzenesulfonamide fragment
docked very similarly to the X-ray structure.
0.067 (0.079) 0.056 (0.074)
0.083 (0.061)
0.05 (0.10)
0.029 (0.027)
0.050 (0.037) 0.025 (0.14)
0.25 (0.14)
0.020 (0.092)
0.18 (0.34)
0.017 (0.10)
1.0 (0.85)
0.018 (0.030)
0.22 (0.15)
0.20 (0.10)
0.33 (0.46)
0.13 (0.26)
0.33 (0.43)
2f
0.022 (0.10)
1.8 (0.88)
0.030 (0.11)
1.0 (1.9)
0.033 (0.20)
1.4 (0.56)
0.042 (0.27)
4.5 (0.99)
0.033 (0.052)
0.14 (0.27)
0.013 (0.057) 0.056 (0.12)
0.10 (0.20) 0.50 (0.48)
0.013 (0.037) 0.067 (0.080)
0.067 (0.18)
0.014 (0.026)
0.33 (0.43)
1g
2g
1h
2h
1i
0.20 (0.36)
0.10 (0.20)
0.71 (0.66)
2i
1j
2j
1k
2k
0.067 (0.11)
0.83 (0.64)
0.0067 (0.10)
0.017 (0.012) 0.067 (0.083)
0.25 (0.11) 0.71 (0.31)
0.0091 (0.077) 0.050 (0.14)
0.12 (0.077)
0.25 (0.26)
0.067 (0.28)
However, the flexible, partly hydrophilic “tail” of the ligand was
mostly incorrectly binding to the hydrophilic parts on the CA surface,
but not to the hydrophobic pocket lined by the residues Phe131,
Average standard deviations for both methods were below 25%.

Panel A
Panel B
75
70
2d
1d
2g
70
65
60
55
65
60
55
2d
1j
1j
10-5
10-4
10-3
10-5
10-4
10-3
0
0
L , M
t
L , M
t
80
60
40
20
80
60
40
20
2d
1d
2d
1j
0
0
160
130
0
0
60
80
0
0
60
40
20
0
40
20
0
160
130
50
2a
60
o
70
50
60
70
o
T, C
T, C
Panel C
65
2d
60
55
1j
10-5
10-4
10-3
0
L , M
t
60
40
20
2d
1j
0
130
0
100
0
80
60
40
20
130
50
60
70
o
T, C
Fig. 2. TSA data of selected compound binding to CA I (Panel A), CA II (Panel B), and CA VII (Panel C). Panels on the bottom compare denaturation curves observed by fluorescence at
0 to 130 or 160 M added ligand concentrations. Panels on the top show the dependence of the protein melting temperatures Tm on ligand concentrations.
m

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E. Capkauskaite et al. / European Journal of Medicinal Chemistry 51 (2012) 259e270
263
Val135, Leu198, and Pro202. Vdock allows for constraining the
selected dihedrals of the ligand. We constrained five rotatable
dihedrals of the ligands, starting from the sulfonamide NH2 to the S of
the thioether. The constrained dihedrals were allowed a ꢂ15ꢀ vari-
ationfrom theoriginalX-ray values. By usingthese constraints, the X-
ray binding modes of docked 1f and 2j were reproduced with heavy
atom distance root-mean-square deviation (RMSD) equal to 0.28 and
0.96 Å, respectively (Fig. 8, 1f and 2j). Compounds 1g, 2a, 2f and 2i
were also docked into CA II, with the resulting geometries fairly close
to the solved X-ray structures, with 0.56 (1g), 0.45 (conformation B of
2a), 0.26 (2f), and 1.79 (2i) Å RMSD (Fig. 8). Fig. 9 shows compounds
1aei and 2aei docked using the described constraints into the CA II
from the complexes with 1f and 2j, respectively.
The inhibitors docked closely into the hydrophobic groove on the
CA surface in the X-ray structures. This geometry can be used as
a basis for further application of more advanced energy-scoring
functions. Fig. 10 illustrates the reasonably good correlations
obtained between the experimentally observed binding energies
and the calculated apparent binding affinities obtained using the
PoissoneBoltzmann surface area (PBSA) approach [13]. Interest-
ingly, the correlations were better for chlorine-containing series
1aei than the 2aei series. The outcome is likely due to 1aei sticking
out into the solution more than 2aei; hence, the solvent exposed
surface area approximation as a part of a continuum solvation model
could be more accurate in the case of the former.
Δ
An interesting question is why the chlorine-containing
compounds bind generally more poorly than the compounds
without Cl in the first ring. Scott and co-workers found that the
scaffold constituent of 2-Cl-benzenesulfonamide has a lesser CA II
binding affinity compared to benzenesulfonamide [14]. Apparently,
the chlorine-containing ligand tails reach less deeply into the
hydrophobic pocket of CA II than the compounds without the
chloride. This also explains the greatest difference between the
affinities of the two scaffold types for ligands 1k and 2k: the most
hydrophobic tail (benzene) of molecule 2k (very similarly to 2j,
Fig. 9) makes good contacts within the hydrophobic pocket, while
1k, similar to 1f, barely reaches the groove entrance between
residues Phe131 and Pro202.
These docking and PBSA results merit use in future studies,
including use with different isoforms, to design isoform-specific
inhibitors.
3. Conclusions
A series of aromatic sulfonamide inhibitors were designed with
nanomolar affinities toward CA isozymes I, II, VII, and XIII. X-ray
crystallographic cocrystal structures and computational docking
studies provided structural details of inhibitor binding to CA II and
demonstrated
a correlation between the experimental and
computed structures. Some inhibitors exhibited up to 50-times
selectivity toward one or another CA isozyme. Application of two
biophysical methods to measure compound binding affinities (TSA
and ITC) provided a possible binding evaluation that was as accu-
rate as possible and enabled energetics-structure correlations, with
the goal of designing novel compounds with desired properties.
Δ
4. Experimental protocols
4.1. Syntheses
Synthesis of 5-(2-bromoacetyl)-2-chlorobenzensulfonamide (1)
and 4-(bromoacetyl)benzenesulfonamide (2) was accomplished
from commercially available 1-(4-chloro-3-nitrophenyl)ethanone
and 1-(4-aminophenyl)ethanone, respectively, as described in [15]
and [16]. 2-Mercaptopyrimidines a, e, f, h, and j are commercially
Fig. 3. ITC data of 2d binding to CA I (Panel A) and 2j binding to CA XIII (Panel B).

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E. Capkauskaite et al. / European Journal of Medicinal Chemistry 51 (2012) 259e270
264
Fig. 4. View of compounds 1f, 1g, 2f, 2j, and 2i in the active center of CA II. Zn2þ coordinated by His94, His96 and His119 is shown as an orange sphere. The electron density map
. The picture was generated using MOLSCRIPT [37], Raster3D [38] and BOBSCRIPT [39]. (For interpretation of the references to colour in this figure
j2Fobs ꢃ Fcalcj is contoured at 0.8
s
legend, the reader is referred to the web version of this article.)
available. 6-alkyl-2-thioxo-2,3-dihydro-4(1H)-pyrimidinones b [17],
c [18], and d [18] were synthesized by condensation of an appro-
priately substituted b-keto ester with thiourea. 5-alkyl-2(1H)-pyr-
was stirred at room temperature for 24 h. The reaction mixture was
poured into water. The precipitate was filtered off and then washed
with water and then with diethyl ether to yield 1aek or 2aek.
imidinethiones g and i were prepared from thiourea and 2-alkyl-
1,1,3,3-tetraethoxypropane as described previously [19]. All ingre-
dients were purchased from SigmaeAldrich and Alfa Aesar GmbH.
The melting points of the compounds were determined in open
capillaries on a Thermo Scientific 9100 Series apparatus without
further correction. IR spectra were obtained on a PerkineElmer 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 ppm 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
6520massspectrometer(AgilentTechnologies). The purities of target
compounds were analyzed using an HPLC system with UV detection.
4.1.1.1. 2-Chloro-5-{[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]
acetyl}benzenesulfonamide (1a). Yield 78%, mp 211e213 ꢀC. IR
n
cmꢃ1: 3344, 3220 (NH2, NH), 1698 (CO), 1634 (CONH). 1H NMR
ppm: (1:0.32) 1.94 (3H, s, CH3, open chain form (o)), 2.18 (0.96H, s,
d
CH3, cyclic form (c)), 3.58 (0.32H, d, J ¼ 12.9 Hz, CH2COH, c), 3.66
(0.32H, d, J ¼ 12.3 Hz, CH2COH, c), 4.74 (2H, s, CH2CO, o), 5.94 (0.32H,
0
0
s, C5 eH, c), 5.99 (1H, br s, C5 eH, o), 7.63 (0.64H, s, C3,4eH, c), 7.69
(0.64H, s, NH2, c), 7.85 (2H, s, NH2, o), 7.88 (1H, d, J ¼ 8.7 Hz, C3eH, o),
8.09 (0.32H, s, C6eH, c), 8.31 (1H, dd, J ¼ 1.8 Hz, J ¼ 8.1 Hz, C4eH, o),
8.53 (1H, d, J ¼ 1.5 Hz, C6eH, o), 10.73 (1H, br s, NH, o). HRMS calcd.
for C13H12ClN3O4S2 ([M þ H]þ): 374.0031, found: 374.0032.
4.1.1.2. (4-{[(4-Methyl-6-oxo-1,6-dihydro-2-pyrimidinyl)sulfanyl]ace-
tyl}benzenesulfonamide) (2a). Yield 75%, mp 183e185 ꢀC. IR
n
cmꢃ1
ppm:
:
3439, 3329, 3199 (NH2, NH),1699 (CO),1649 (CONH).1H NMR
d
4.1.1. General procedure for the syntheses of 1aek and 2aek
A mixture of the corresponding pyrimidine (aej) or thiophenol
k (0.539 mmol), appropriate compound 1 or 2 (0.539 mmol), and
sodium acetate (49.5 mg, 0.604 mmol) in tetrahydrofuran (3 ml)
(1:0.31) 1.96 (3H, s, CH3, o), 2.18 (0.93H, s, CH3, c), 3.56 (0.31H, d,
J ¼ 12.3 Hz, CH2COH, c), 3.63 (0.31H, d, J ¼ 12.3 Hz, CH2COH, c), 4.76
0
0
(2H, s, CH2CO, o), 5.94 (0.31H, s, C5 eH, c), 5.98 (1H, br s, C5 eH, o),
7.40 (0.62H, s, NH2, c), 7.59e7.63 (2.62H, m, NH2, o, C2,6eH, c), 7.83
Fig. 5. View of 1f (gold) and 1g (magenta) located in the active center of CA II. Zn2þ is
shown as an orange transparent sphere. The picture was generated using MOLSCRIPT
[37] and Raster3D [38]. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
Fig. 6. View of 2f (blue) and 2j (red) located in the active center of CA II. Zn2þ is shown
as an orange transparent sphere. The picture was generated using MOLSCRIPT [37] and
Raster3D [38]. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

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265
193e195 ꢀC. IR
n
cmꢃ1: 3341, 3223 (NH2, NH), 1693 (CO), 1636
d ppm: (1:0.31) 1.92 (3H, s, CH3, o), 2.20 (0.93H, s,
(CONH). 1H NMR
CH3, c), 3.57e3.70 (3.24H, m, CH2COH, c, CH2Ph, o, c), 4.73 (2H, s,
CH2CO, o), 7.14e7.27 (6.55H, m, PheH, o, c), 7.64 (0.62H, s, C3,4eH, c),
7.69 (0.62H, s, NH2, c), 7.86 (2H, s, NH2, o), 7.87 (1H, d, J ¼ 8.7 Hz,
C3eH, o), 8.16 (0.31H, s, C6eH, c), 8.31 (1H, dd, J ¼ 1.8 Hz, J ¼ 8.4 Hz,
C4eH, o), 8.54(1H, d, J ¼ 1.5 Hz, C6eH, o),12.68(1H, brs, NH, o). HRMS
calcd. for C20H18ClN3O4S2 ([M þ H]þ): 464.0500, found: 464.0493.
4.1.1.4. 4-{[(5-Benzyl-4-methyl-6-oxo-1,6-dihydro-2-pyrimidinyl)sul-
fanyl]acetyl}benzenesulfonamide (2b). Yield 86%, mp 181e183 ꢀC. IR
n
cmꢃ1: 3315, 3243 (NH2, NH), 1700 (CO), 1638 (CONH). 1H NMR
ppm: (1:0.32) 1.93 (3H, s, CH3, o), 2.21 (0.96H, s, CH3, c), 3.59
d
(0.32H, d, J ¼ 12.0 Hz, CH2COH, c), 3.65 (0.32H, d, J ¼ 12.0 Hz,
CH2COH, c), 3.70 (2.64H, s, CH2Ph, o, c), 4.76 (2H, s, CH2CO, o),
7.15e7.28 (6.60H, m, PheH, o, c), 7.44 (0.64H, s, NH2, c), 7.61e7.65
(2.64H, m, NH2, o, C2,6eH, c), 7.84 (0.64H, d, J ¼ 8.1 Hz, C3,5eH, c),
7.80 (2H, d, J ¼ 7.5 Hz, C2,6eH), 8.23 (2H, d, J ¼ 8.1 Hz, C3,5eH), 12.84
(1H, br s, NH, o). HRMS calcd. for C20H19N3O4S2 ([M þ H]þ):
430.0890, found: 430.0895.
4.1.1.5. 2-Chloro-5-{[(6-oxo-4-propyl-1,6-dihydropyrimidin-2-yl)thio]
acetyl}benzenesulfonamide (1c). Yield 78%, mp 202e204 ꢀC. IR
n
cmꢃ1: 3329, 3184 (NH2, NH),1702 (CO),1637 (CONH).1H NMR
d ppm:
(1:0.31) 0.66 (3H, t, J ¼ 6.8 Hz, CH3, o), 0.92 (0.93H, t, J ¼ 6.8 Hz, CH3,
c),1.18e1.29 (2H, m, CH2, o), 1.57e1.68 (0.62H, m, CH2, c), 2.11 (2H, t,
J ¼ 6.6 Hz, CH2, o), 2.40 (0.62H, t, J ¼ 7.5 Hz, CH2, c), 3.59 (0.31H, d,
J ¼ 12.6 Hz, CH2COH, c), 3.66 (0.31H, d, J ¼ 12.6 Hz, CH2COH, c), 4.72
Fig. 7. View of 1f (gold) and 2f (blue) located in the active center of CA II. Zn2þ
coordinated by His94, His96 and His119 is shown as an orange transparent sphere. The
picture was generated using MOLSCRIPT [37] and Raster3D [38]. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)
0
(2H, s, CH2CO, o), 5.93 (1.31H, br s, C5 eH, o, c), 7.63 (0.62H, s, C3,4eH,
c), 7.69 (0.62H, s, NH2, c), 7.84 (2H, s, NH2, o), 7.88 (1H, d, J ¼ 8.4 Hz,
C3eH, o), 8.10 (0.31H, s, C6eH, c), 8.33 (1H, d, J ¼ 8.4 Hz, C4eH, o), 8.53
(1H, s, C6eH, o), 12.60 (1H, br s, NH, o). HRMS calcd. for
C15H16ClN3O4S2 ([M þ H]þ): 402.0344, found: 402.0342.
(0.62H, d, J ¼ 8.7 Hz, C3,5eH, c), 8.00 (2H, d, J ¼ 8.4 Hz, C2,6eH), 8.22
(2H, d, J ¼ 8.4 Hz, C3,5eH), 12.53 (1H, br s, NH, o). HRMS calcd. for
C13H13N3O4S2 ([M þ H]þ): 340.0420, found: 340.0418.
4.1.1.6. 4-{[(6-Oxo-4-propyl-1,6-dihydro-2-pyrimidinyl)sulfanyl]ace-
4.1.1.3. 5-{[(5-Benzyl-4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)
thio]acetyl}-2-chlorobenzenesulfonamide (1b). Yield 81%, mp
tyl}benzenesulfonamide (2c). Yield 78%, mp 168e170 ꢀC. IR
n
cmꢃ1
d ppm:
:
3360, 3269 (NH2, NH), 1699 (CO), 1657 (CONH). 1H NMR
(1:0.32) 0.66 (3H, t, J ¼ 7.2 Hz, CH3, o), 0.92 (0.96H, t, J ¼ 7.2 Hz, CH3,
c), 1.27 (2H, sextet, J ¼ 6.9 Hz, CH2, o), 1.57e1.69 (0.64H, m, CH2, c),
2.13 (2H, t, J ¼ 7.2 Hz, CH2, o), 2.41 (0.64H, t, J ¼ 7.2 Hz, CH2, c), 3.57
(0.32H, d, J ¼ 12.6 Hz, CH2COH, c), 3.64 (0.32H, d, J ¼ 12.3 Hz,
0
CH2COH, c), 4.75 (2H, s, CH2CO, o), 5.91e5.93 (1.32H, m, C5 eH, o, c),
7.42 (0.64H, s, NH2, c), 7.61 (2.64H, s, NH2, o, C2,6eH, c), 7.83 (0.64H,
d, J ¼ 8.4 Hz, C3,5eH, c), 8.00 (2H, d, J ¼ 8.4 Hz, C2,6eH), 8.24 (2H, d,
J ¼ 8.4 Hz, C3,5eH), 12.58 (1H, br s, NH, o). HRMS calcd. for
C15H17N3O4S2 ([M þ H]þ): 368.0733, found: 368.0733.
4.1.1.7. 5-{[(4-tert-Butyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]acetyl}-
2-chlorobenzenesulfonamide (1d). Yield 86%, mp 201e203 ꢀC. IR
n
cmꢃ1: 3333, 3181 (NH2, NH), 1699 (CO), 1643 (CONH). 1H NMR
d ppm:
(1:0.32) 0.97 (9H, s, (CH3)3, o), 1.21 (2.79H, s, (CH3)3, c), 3.57 (0.31H, d,
J ¼ 12.6 Hz, CH2COH, c), 3.68 (0.31H, d, J ¼ 12.3 Hz, CH2COH, c), 4.82
0
0
(2H, s, CH2CO, o), 5.92 (0.31H, s, C5 eH, c), 5.98 (1H, br s, C5 eH, o),
7.63 (0.62H, s, C3,4eH, c), 7.69 (0.62H, s, NH2, c), 7.84 (2H, s, NH2, o),
7.89 (1H, d, J ¼ 8.1 Hz, C3eH, o), 8.11 (0.31H, s, C6eH, c), 8.34 (1H, d,
J ¼ 8.4 Hz, C4eH, o), 8.53 (1H, s, C6eH, o), 12.64 (1H, br s, NH, o).
HRMS calcd. for C16H18ClN3O4S2 ([M þ H]þ): 416.0500, found:
416.0495.
4.1.1.8. 4-{[(4-tert-Butyl-6-oxo-1,6-dihydro-2-pyrimidinyl)sulfanyl]
acetyl}benzenesulfonamide (2d). Yield 85%, mp 175e177 ꢀC. IR
n
cmꢃ1: 3303, 3178 (NH2, NH), 1707 (CO), 1636 (CONH). 1H NMR
ppm: (1:0.25) 0.96 (9H, s, (CH3)3, o), 1.22 (2.25H, s, (CH3)3, c), 3.59
d
Fig. 8. Docked ligand poses (red) superposed on the X-ray structures (yellow). The
constrained docking protocol was used (see text). (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
(0.25H, d, J ¼ 12.9 Hz, CH2COH, c), 3.66 (0.25H, d, J ¼ 12.0 Hz,
0
CH2COH, c), 4.83 (2H, s, CH2CO, o), 5.92 (0.25H, s, C5 eH, c), 5.97 (1H,

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266
Fig. 9. Superposed compounds 1aei (a) and 2aei (b) docked to CA II (white ribbons/sticks). Docking was performed into the receptors taken from the complexes with 1f (a) and 2j
(b) using dihedral and translational constraints (see text). The tails of the ligands extend more into the binding site in the case of non-chlorinated compounds (b).
0
br s, C5 eH, o), 7.42 (0.50H, s, NH2, c), 7.62 (2.50H, s, NH2, o, C2,6eH,
c), 7.83 (0.50H, d, J ¼ 8.4 Hz, C3,5eH, c), 8.00 (2H, d, J ¼ 8.4 Hz,
(1H, d, J ¼ 1.5 Hz, C6eH, o), 13.44 (1H, br s, NH, o). HRMS calcd. for
C15H14ClN3O6S2 ([M þ H]þ): 432.0085, found: 432.0084.
C
2,6eH), 8.26 (2H, d, J ¼ 8.4 Hz, C3,5eH),12.65 (1H, br s, NH, o). HRMS
calcd. for C16H19N3O4S2 ([M þ H]þ): 382.0890, found: 382.0886.
4.1.1.10. Ethyl 2-({2-[4-(aminosulfonyl)phenyl]-2-oxoethyl}sulfanyl)-
6-oxo-1,6-dihydropyrimidine-5-carboxylate (2e). Yield 81%, mp
4.1.1.9. Ethyl 2-({2-[3-(aminosulfonyl)-4-chlorophenyl]-2-oxoethyl}
180e182 ꢀC. IR cmꢃ1: 3312, 3223 (NH2, NH), 1726 (COOAc),
n
thio)-6-oxo-1,6-dihydropyrimidine-5-carboxylate (1e). Yield 87%,
1709(CO), 1684 (CONH). 1H NMR
d ppm: (1:0.28) 1.19e1.27 (3.84H,
mp 191e193 ꢀC. IR
n
cmꢃ1: 3306 (NH2),1727 (COOAc),1700 (CO),1694
m, CH3, o, c), 3.65 (0.28H, d, J ¼ 12.6 Hz, CH2COH, c), 3.73 (0.28H, d,
J ¼ 12.6 Hz, CH2COH, c), 4.16e4.23 (2.56H, m, CH2, o, c), 4.94 (2H, s,
CH2CO, o), 7.43 (0.56H, s, NH2, c), 7.62 (2H, s, NH2, o), 7.70 (0.56H, d,
J ¼ 8.4 Hz, C2,6eH, c), 7.85 (0.56H, d, J ¼ 8.1 Hz, C3,5eH, c), 8.01 (2H,
d, J ¼ 8.1 Hz, C2,6eH), 8.23 (2H, d, J ¼ 8.4 Hz, C3,5eH), 8.32 (1H, s,
(CONH).1H NMR
d ppm: (1:0.28) 1.19e1.26 (3.84H, m, CH3, o, c), 3.64
(0.28H, d, J ¼ 12.9 Hz, CH2COH, c), 3.76 (0.28H, d, J ¼ 12.6 Hz, CH2COH,
c), 4.16e4.22 (2.56H, m, CH2, o, c), 4.92 (2H, s, CH2CO, o), 7.64 (0.28H,
d, C3eH, c), 7.70e7.74 (0.84H, m, NH2, c, C4eH, c), 7.85 (2H, s, NH2, o),
7.88 (1H, d, J ¼ 8.4 Hz, C3eH, o), 8.17 (0.28H, d, J ¼ 1.8 Hz, C6eH, c),
0
0
C4 eH, o), 8.47 (0.28H, s, C4 eH, c), 13.45 (1H, br s, NH, o). HRMS
8.28e8.31 (2H, m, C4 eH, o, C4eH, o), 8.45 (0.28H, s, C4 eH, c), 8.53
calcd. for C15H15N3O6S2 ([M þ H]þ): 398.0475, found: 398.0470.
0
0
Fig. 10. Correlation between the calculated by PBSA and experimental CA II binding energies (B. E.) for the hydroxy-pyrimidine (squares) and pyrimidine (diamonds) subseries for
compounds 1aei (a) and 2aei (b).

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4.1.1.11. 2-Chloro-5-{[(4,6-dimethyl-2-pyrimidinyl)sulfanyl]acetyl}ben-
4.1.1.18. 4-{[(5-Butyl-2-pyrimidinyl)sulfanyl]acetyl}benzenesulfona-
mide (2i). Yield 70%, mp 147e149 ꢀC. IR cmꢃ1: 3275 (NH2), 1706
(CO). 1H NMR
zenesulfonamide (1f). Yield 93%, mp 187e189 ꢀC. IR
n
cmꢃ1: 3268
ppm: 2.23 (6H, s, 2CH3), 4.70 (2H, s,
n
(NH2), 1702 (CO). 1H NMR
d
d
ppm: 0.89 (3H, t, J ¼ 7.2 Hz, CH3), 1.28 (2H, sextet,
0
CH2CO), 6.94 (1H, s, C5 eH), 7.84 (2H, s, NH2), 7.88 (1H, d, J ¼ 8.4 Hz,
J ¼ 7.2 Hz, CH2),1.52 (2H, quintet, J ¼ 7.5 Hz, CH2), 2.48e2.55 (2H, m,
C3eH), 8.33 (1H, dd, J ¼ 2.1 Hz, J ¼ 8.1 Hz, C4eH), 8.54(1H, d, J ¼ 2.1 Hz,
CH2), 4.83 (2H, s, CH2CO), 7.59 (2H, s, NH2), 8.00 (2H, d, J ¼ 8.4 Hz,
C6eH). 13C NMR
d
ppm: 23.86 (2C), 38.03, 116.80, 128.92, 132.84,
C
2,6eH), 8.23 (2H, d, J ¼ 8.4 Hz, C3,5eH), 8.47 (2H, s, C4 ,6 eH). 13C
0
0
133.55,135.79,136.12,142.19,167.74 (2C),169.30,194.05. HRMS calcd.
NMR d ppm: 14.31, 22.26, 29.14, 32.95, 39.12, 126.74 (2C), 129.68
for C14H14ClN3O3S2 ([M þ H]þ): 372.0238, found: 372.0238.
(2C), 131.60, 139.16, 148.57, 158.09 (2C), 167.69, 194.12. HRMS calcd.
for C16H19N3O3S2 ([M þ H]þ): 366.0941, found: 366.0940.
4.1.1.12. 4-{[(4,6-Dimethyl-2-pyrimidinyl)sulfanyl]acetyl}benzenesul-
fonamide (2f). Yield 78%, mp 174e176 ꢀC. IR
n
cmꢃ1: 3346 (NH2),1698
4.1.1.19. 2-Chloro-5-[(2-pyrimidinylsulfanyl)acetyl]benzenesulfonamide
(CO).1H NMR
d
ppm:2.22(6H, s, 2CH3), 4.71 (2H, s, CH2CO), 6.92 (1H, s,
(1j). Yield 90%, mp 195e197 ꢀC. IR
n
cmꢃ1: 3325 (NH2), 1708 (CO). 1H
0
0
C5 eH), 7.56 (2H, s, NH2), 7.99 (2H, d, J ¼ 8.1 Hz, C2,6eH), 8.22 (2H, d,
NMR
d
ppm: 4.85 (2H, s, CH2CO), 7.23 (1H, t, J ¼ 4.8 Hz, C5 eH), 7.86
J ¼ 7.8 Hz, C3,5eH). 13C NMR
d
ppm: 23.85 (2C), 38.43, 116.72, 126.62
(2H, s, NH2), 7.89 (1H, d, J ¼ 8.1 Hz, C3eH), 8.33 (1H, dd, J ¼ 2.1 Hz,
(2C), 129.56 (2C), 139.74, 148.40, 167.65 (2C), 169.39, 194.83. HRMS
J ¼ 8.1 Hz, C4eH), 8.56 (1H, d, J ¼ 2.1 Hz, C6eH), 8.60 (2H, d, J ¼ 4.8 Hz,
calcd. for C14H15N3O3S2 ([M þ H]þ): 338.0628, found: 338.0622.
C
4 ,6 eH). 13C NMR
d ppm: 38.87, 118.15, 128.97, 133.00, 133.66, 135.43,
0 0
136.08, 142.26, 158.50 (2C), 170.49, 193.05. HRMS calcd. for
4.1.1.13. 2-Chloro-5-{[(5-ethyl-2-pyrimidinyl)sulfanyl]acetyl}benze-
C12H10ClN3O3S2 ([M þ H]þ): 343.9925, found: 343.9926.
nesulfonamide (1g). Yield 72%, mp 151e153 ꢀC. IR
n
cmꢃ1: 3300
(NH2), 1705 (CO). 1H NMR
d
ppm: 1.16 (3H, t, J ¼ 7.5 Hz, CH3), 2.54
4.1.1.20. 4-[(2-Pyrimidinylsulfanyl)acetyl]benzenesulfonamide
(2H, q, J ¼ 7.5 Hz, CH2), 4.81 (2H, s, CH2CO), 7.83 (2H, s, NH2), 7.88
(1H, d, J ¼ 8.4 Hz, C3eH), 8.31 (1H, dd, J ¼ 2.1 Hz, J ¼ 8.1 Hz, C4eH),
(2j). Yield 79%, mp 186e188 ꢀC. IR
n
cmꢃ1: 3313, 3290 (NH2), 1693
(CO).1H NMR
d
ppm:4.87(2H, s, CH2CO), 7.22 (1H, t, J ¼ 4.8 Hz, C5 eH),
0
8.48 (2H, s, C4 ,6 eH), 8.56 (1H, d, J ¼ 1.8 Hz, C6eH). 13C NMR
d
ppm:
7.62 (2H, s, NH2), 8.01 (2H, d, J ¼ 8.4 Hz, C2,6eH), 8.25 (2H, d, J ¼ 8.4 Hz,
0
0
3,5eH), 8.59(2H, d,J ¼ 4.8Hz, C4 ,6 eH).13C NMR
d ppm:39.22,118.12,
0
0
15.59, 22.86, 38.78, 128.98, 132.96, 133.62, 135.50, 136.03, 142.26
(2C), 157.81 (2C), 167.54, 193.19. HRMS calcd. for C14H14ClN3O3S2
([M þ H]þ): 372.0238, found: 372.0243.
C
126.75 (2C), 129.73 (2C), 139.06, 148.57, 158.48 (2C), 170.61, 193.93.
HRMS calcd. for C12H11N3O3S2 ([M þ H]þ): 310.0315, found: 310.0313.
4.1.1.14. 4-{[(5-Ethyl-2-pyrimidinyl)sulfanyl]acetyl}benzenesulfona-
4.1.1.21. 2-Chloro-5-[(phenylsulfanyl)acetyl]benzenesulfonamide
mide (2g). Yield 79%, mp 180e182 ꢀC. IR
n
cmꢃ1: 3245 (NH2), 1706
(1k). Yield 78%, mp 122e124 ꢀC. IR
n
cmꢃ1: 3363, 3257 (NH2), 1689
(CO). 1H NMR
d
ppm: 1.16 (3H, t, J ¼ 7.5 Hz, CH3), 2.54 (2H, q,
(CO). 1H NMR
d
ppm: 4.70 (2H, s, CH2CO), 7.20e7.24 (1H, m, C4 eH),
0
0 0 0 0
J ¼ 7.5 Hz, CH2), 4.83 (2H, s, CH2CO), 7.58 (2H, s, NH2), 8.00 (2H, d,
7.29e7.35 (4H, m, C2 ,3 ,5 ,6 eH), 7.82e7.86 (3H, m, NH2, C3eH), 8.27
J ¼ 8.7 Hz, C2,6eH), 8.22 (2H, d, J ¼ 8.7 Hz, C3,5eH), 8.48 (2H, s,
(1H, d, J ¼ 8.1 Hz, C4eH), 8.48 (1H, s, C6eH). 13C NMR
d ppm:
0
0
C
4 ,6 eH). 13C NMR
d
ppm: 15.58, 22.86, 39.10, 126.73 (2C), 129.68
(38.87e41.05 e superposed with DMSO), 127.03, 129.12, 129.37(2C),
129.76(2C),132.82,134.04,134.67,135.40,136.10,142.27,193.34.HRMS
calcd. for C14H12ClNO3S2 ([M þ H]þ): 342.0020, found: 342.0023.
(2C), 132.97, 139.17, 148.57, 157.78 (2C), 167.68, 194.12. HRMS calcd.
for C14H15N3O3S2 ([M þ H]þ): 338.0628, found: 338.0624.
4.1.1.15. 2-Chloro-5-{[(5-propyl-2-pyrimidinyl)sulfanyl]acetyl}benze-
4.1.1.22. 4-[(Phenylsulfanyl)acetyl]benzenesulfonamide (2k). Yield
nesulfonamide (1h). Yield 79%, mp 173e175 ꢀC. IR
n
cmꢃ1: 3348
75%, mp 145e147 ꢀC. IR
n
cmꢃ1: 3382, 3282 (NH2), 1681 (CO). 1H
(NH2),1704 (CO).1H NMR
d
ppm: 0.87 (3H, t, J ¼ 7.5 Hz, CH3),1.56 (2H,
NMR
d
ppm: 4.71 (2H, s, CH2CO), 7.23e7.30 (1H, m, C4 eH),
0
0 0 0 0
sextet, J ¼ 7.5 Hz, CH2), 2.48 (2H, t, J ¼ 7.5 Hz, CH2), 4.81(2H, s, CH2CO),
7.85 (2H, s, NH2), 7.88 (1H, d, J ¼ 8.1 Hz, C3eH), 8.31 (1H, dd, J ¼ 2.1 Hz,
7.33e7.41 (4H, m, C2 ,3 ,5 ,6 eH), 7.58 (2H, s, NH2), 7.98 (2H, d,
J ¼ 8.4 Hz, C2,6eH), 8.20 (2H, d, J ¼ 8.4 Hz, C3,5eH). 13C NMR
d ppm:
0
0
J ¼ 8.4 Hz, C4eH), 8.47 (2H, s, C4 ,6 eH), 8.55 (1H, d, J ¼ 2.1 Hz, C6eH).
(39.45e41.11 e superposed with DMSO),126.65 (2C),126.98,129.39
(2C), 129.75 (2C), 129.97 (2C), 135.58, 138.36, 148.62, 194.40. HRMS
calcd. for C14H13NO3S2 ([M þ H]þ): 308.0410, found: 308.0404.
13C NMR
d ppm: 14.08, 24.10, 31.46, 38.81, 128.97, 131.42, 132.97,
133.65,135.48,136.05,142.25,158.17(2C),167.63,193.19. HRMS calcd.
for C15H16ClN3O3S2 ([M þ H]þ): 386.0394, found: 386.0392.
4.1.2. Open and cyclic forms of the pyrimidinones
4.1.1.16. 4-{[(5-Propyl-2-pyrimidinyl)sulfanyl]acetyl}benzenesulfona-
An investigation of the 1aej and 2aej compound structures by
NMR spectrocopy showed that the 1H and 13C NMR spectra of
pyrimidinones 1aee, 2aee in DMSO-d6 solution contained two sets
of signals. These results led to a suggestion that this phenomenon
could be due to the existence of compounds 1aee and 2aee in two
forms: open chain I and cyclic II (Scheme 3).
mide (2h). Yield 75%, mp 165e167 ꢀC. IR
n
cmꢃ1: 3260 (NH2), 1706
(CO). 1H NMR
d
ppm: 0.88 (3H, t, J ¼ 7.2 Hz, CH3), 1.56 (2H, sextet,
J¼ 7.5 Hz, CH2), 2.49 (2H, t, J¼ 7.8 Hz, CH2), 4.83 (2H, s, CH2CO), 7.59 (2H,
s, NH2), 8.00 (2H, d, J ¼ 8.4 Hz, C2,6eH), 8.24 (2H, d, J ¼ 8.1 Hz, C3,5eH),
8.47 (2H, s, C4 ,6 eH). 13C NMR
d ppm: 14.06, 24.06, 31.46, 39.13, 126.73
0
0
(2C), 129.67 (2C), 131.38, 139.15, 148.56, 158.12 (2C), 167.74, 194.10.
Especially large differences were observed for signals of SCH2
group protons in the 1H NMR spectra (Table 2). This signal of open
chain form I was observed as a singlet in the 4.72e4.94 ppm region,
while SCH2 protons of cyclic form II appeared as doublets with
J ¼ 12e13 Hz at 3.56e3.73 ppm. The geminal-type spinespin
coupling of SCH2 protons arises from their non-equivalency in the
cyclic form II. 13C NMR spectra of compounds 1aee and 2aee gave
additional evidence for the existence of equilibria between open
chain and cyclic forms of compounds 1aee and 2aee in a solution.
The main differences in the 13C NMR spectra, as expected, were
observed for carbon atoms taking part in the transformation
(Table 2). Thus, the signal for carbon of the SCH2 group in open
chain form I was observed at 37.34e39.27 ppm. In the 13C NMR
HRMS calcd. for C15H17N3O3S2 ([M þ H]þ): 352.0784, found: 352.0781.
4.1.1.17. 5-{[(5-Butylpyrimidin-2-yl)thio]acetyl}-2-chlorobenzenesulf-
onamide (1i). Yield 84%, mp 124e126 ꢀC. IR
n
cmꢃ1: 3322 (NH2),
1702 (CO). 1H NMR
d
ppm: 0.88 (3H, t, J ¼ 7.5 Hz, CH3), 1.28 (2H,
sextet, J ¼ 7.5 Hz, CH2), 1.52 (2H, quintet, J ¼ 7.5 Hz, CH2), 2.48e2.53
(2H, m, CH2), 4.82 (2H, s, CH2CO), 7.85 (2H, s, NH2), 7.88 (1H, d,
J ¼ 8.7 Hz, C3eH), 8.32 (1H, dd, J ¼ 1.8 Hz, J ¼ 8.1 Hz, C4eH), 8.47 (2H,
s, C4 ,6 eH), 8.56 (1H, d, J ¼ 1.5 Hz, C6eH). 13C NMR
d
ppm: 14.33,
0
0
22.28, 29.16, 32.98, 38.81, 128.99, 131.64, 132.97, 133.65, 135.48,
136.05, 142.26, 158.13 (2C), 167.58, 193.20. HRMS calcd. for
C16H18ClN3O3S2 ([M þ H]þ): 400.0551, found: 400.0550.

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E. Capkauskaite et al. / European Journal of Medicinal Chemistry 51 (2012) 259e270
268
S
HO
O
N
N
S
N
O
R3
R4
R3
HN
R1
O
R1
R4
R2
R2
II
I
1a: R1=Cl, R2=SO2NH2, R3=Me, R4=H,
2a: R1=SO2NH2, R2=H, R3=Me, R4=H,
1b: R1=Cl, R2=SO2NH2, R3=Me, R4=Bn,
2b: R1=SO2NH2, R2=H, R3=Me, R4=Bn,
1c: R1=Cl, R2=SO2NH2, R3=Pr, R4=H,
2c: R1=SO2NH2, R2=H, R3=Pr, R4=H,
1d: R1=Cl, R2=SO2NH2, R3=t-Bu, R4=H,
2d: R1=SO2NH2, R2=H, R3=t-Bu, R4=H,
1e: R1=Cl, R2=SO2NH2, R3=H, R4=CO2Et,
2e: R1=SO2NH2, R2=H, R3=H, R4=CO2Et,
Scheme 3. Open chain and cyclic forms of compounds 1aee and 2aee.
spectra of cyclic form II, a signal for this carbon was slightly shifted
to region of lower fields and was observed in the
dye ANS (8-anilino-1-naphthalene sulfonate), and 50 mM phos-
phate 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 described [21,23]
a
42.36e43.03 ppm region. Moreover, the open chain forms of
compounds 1aee and 2aee were well characterized by the C]O
group carbon signal ranging from 192.25 to 194.11 ppm. The CeOH
group carbon signal of cyclic form II was observed in the
95.90e97.81 ppm range. Thus, the obtained NMR spectral data
showed the presence of two forms of compounds (1aee and 2aee)
in DMSO-d6 solution. The ratio I:II was 1.0:0.3, as calculated from
integral intensities of SCH2 signals of 1H NMR spectra. A similar
ratio of 1.0:0.3 was previously observed for similar compounds, 2-
(carbonylmethylthio)-4-pyrimidinones [20].
4.3.2. Isothermal titration calorimetry
ITC experiments were performed using ITC200 or VP-ITC instru-
ments (Microcal, Inc., Northampton, USA) with 5e20
solution in the cell and 50e200 M of the ligand solution in the
syringe. A typical experiment consisted of 18 or 25 injections (2 or
10 l each) within 2 or 3 min intervals. Variation of the time period
mM protein
m
m
betweeninjectionsenabled thedetectionandelimination ofpossible
kinetic effects that may distort the binding data. Experiments were
carried out at 37 ꢀC in a 50 mM phosphate buffer containing 100 mM
NaCl at pH 7.0, with a final DMSO concentration of 2%.
4.2. Protein preparation
Expression and purification of CA I, CA II, CA VII, and CA XIII was
previously described: CA I in, [21], CAIIin[22] and CAVII and XIII in [11].
4.4. Crystallography
4.3. Determination of compound binding to CAs
4.4.1. Crystallization
Crystallization of CA II that was concentrated to 40e60 mg/ml
was started by mixing equal volumes of protein solution with
reservoir buffer in sitting drops. Crystallization solutions were
prepared by mixing 1 M Na-Bicine, pH 9 to final concentration
0.1 M with 2.7 M Na-Malonate, pH 7, so that the final concentration
of malonate ranged from 1.2 to 2.1 M. Crystals belonging to the P21
4.3.1. Thermal shift assay
Thermal shift assay experiments were performed in a Corbett
Rotor-Gene 6000 (QIAGEN Rotor-Gene Q) instrument using the
blue channel (excitation 365 ꢂ 20, detection 460 ꢂ 15 nm). Samples
contained 10 mM protein, 0e200 mM ligand, 50 mM solvatochromic
Table 2
Characteristic chemical shifts of 1H and 13C NMR spectra of open chain I and cyclic II forms of compounds 1aee and 2aee in DMSO-d6 solution.
Compound
13C NMR spectra
1H NMR spectra
SCH2CO in I
SCH2C]O in I
SCH2OH in II
SCH2C]O in I
SCH2COH in II
SCH2 in II
1a
2a
1b
2b
1c
2c
1d
2d
1e
2e
37.94
38.30
37.73
38.14
37.75
38.14
37.35
37.34
38.93
39.27
42.37
42.36
42.56
42.60
42.38
42.41
42.37
42.39
42.97
43.03
193.24
194.11
193.22
194.07
193.15
193.90
192.25
192.98
192.16
193.03
96.08
96.70
96.37
96.97
96.05
96.61
95.90
96.45
97.20
97.81
4.74 (2H, s)
4.76 (2H, s)
4.73 (2H, s)
4.76 (2H, s)
4.72 (2H, s)
4.75 (2H, s)
4.82 (2H, s)
4.83 (2H, s)
4.92 (2H, s)
4.94 (2H, s)
3.58 (0.32H, d, J ¼ 12.9 Hz), 3.66 (0.32H, d, J ¼ 12.3 Hz)
3.56 (0.31H, d, J ¼ 12.3 Hz), 3.63 (0.31H, d, J ¼ 12.3 Hz)
3.57e3.70 (3.24H, m, CH2COH, CH2Ph, o, c)
3.59 (0.32H, d, J ¼ 12.0 Hz), 3.65 (0.32H, d, J ¼ 12.0 Hz)
3.59 (0.31H, d, J ¼ 12.6 Hz), 3.66 (0.31H, d, J ¼ 12.6 Hz)
3.57 (0.32H, d, J ¼ 12.6 Hz), 3.64 (0.32H, d, J ¼ 12.3 Hz)
3.57 (0.31H, d, J ¼ 12.6 Hz), 3.68 (0.31H, d, J ¼ 12.3 Hz)
3.59 (0.25H, d, J ¼ 12.9 Hz), 3.66 (0.25H, d, J ¼ 12.0 Hz)
3.64 (0.28H, d, J ¼ 12.9 Hz), 3.76 (0.28H, d, J ¼ 12.6 Hz)
3.65 (0.28H, d, J ¼ 12.6 Hz), 3.73 (0.28H, d, J ¼ 12.6 Hz)

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E. Capkauskaite et al. / European Journal of Medicinal Chemistry 51 (2012) 259e270
269
Table 3
X-ray crystallographic data collection and refinement statistics. All datasets are collected at 100 K. All crystals belong to P21 space group.
Ligand
1f
1g
2a
2i
2f
2j
PDB ID
3S9T
3SAX
3S8X
3SAP
3SBH
3SBI
Parameters of the unit cell, Å
a ¼ 42.01,
b ¼ 41.06,
c ¼ 71.84,
a ¼ 42.22,
b ¼ 41.17,
c ¼ 72.20,
a ¼ 42.21,
b ¼ 41.18,
c ¼ 71.92,
a ¼ 42.02,
b ¼ 40.96,
c ¼ 71.74,
a ¼ 42.09,
b ¼ 41.09,
c ¼ 71.84,
a ¼ 42.35,
b ¼ 41.31,
c ¼ 72.24,
a
¼
g
¼ 90ꢀ
b
¼ 104.15ꢀ
b
¼ 104.20ꢀ
b
¼ 104.29ꢀ
b
¼ 104.08ꢀ
b
¼ 104.17ꢀ
b
¼ 104.25ꢀ
Resolution, Å
1.30
1.10
1.30
1.75
1.65
1.40
Reflections unique (total)
Completeness (%) Overall (outer shell)
I/sI Overall (outer shell)
Rmerge overall (outer shell)
Number of atoms (if alt. conformations
are present, then only one is included)
Number of solvent molecules
Number of bound buffer molecule atoms
Rcryst (Rfree) Test set size 10%
RMS bonds/angles
58,642 (409,726)
100.0 (100.0)
14.0 (2.7)
0.129 (0.368)
2406
92,709 (668,605)
93.4 (79.2)
15.5 (4.8)
0.068 (0.275)
2458
56,499 (431,988)
95.9 (93.6)
26.2 (3.5)
0.064 (0.380)
2339
23,825 (171,710)
99.0 (98.6)
30.3 (7.9)
0.058 (0.266)
2292
28,165 (213,598)
97.7 (96.8)
30.7 (9.7)
0.058 (0.278)
2418
46,467 (277,941)
97.1 (95.7)
25.2 (4.6)
0.043 (0.344)
2419
309
19
0.143 (0.192)
0.034 (2.64)
14.2
377
19
0.126 (0.158)
0.029 (2.52)
12.4
248
19
0.142 (0.179)
0.028 (2.35)
16.2
195
23
0.182 (0.229)
0.015 (1.49)
17.6
303
27
0.172 (0.228)
0.029 (2.34)
15.5
320
19
0.133 (0.183)
0.029 (2.23)
14.6
Average B-factors (Å2)
Main chain
11.3
9.6
13.5
15.6
12.6
11.7
Side chains
14.3
12.0
16.5
17.5
15.1
14.2
Solvent
24.7
23.7
25.6
24.7
24.8
25.2
Ions
7.2
5.4
8.1
10.2
8.0
6.2
Inhibitor
14.3
15.0
16.8
34.4
24.5
12.2
Cofactors
23.6
20.9
27.9
37.5
33.1
29.2
P P
P P
nh
nh
Rmerge
¼
i¼1jhIhi ꢃ Ihij=
i¼1jIhij, where Ihi is an intensity value of the i-th measurement of reflection h, h ¼ (h, k, l), sum Sh runs overall measured reflections, and hIhi
h
h
is an average measured intensity of the reflection h. The nh is the number of measurements of reflection h.
ꢀ
ꢁ
space group grew within several days. Crystals were soaked with
a 0.5 mM solution of the ligand prepared by mixing of 50 mM
D
G
bindzDa Vel þ VPB
þ
bDVvdw
þ
gDSASA þ
d
where Vel
-
electrostatic interaction energy, VPB
-
solution of the ligand in DMSO with 50
the crystallization plate.
ml of reservoir buffer from
PoissoneBoltzmann electrostatic energy correction due to solva-
tion, Vvdw - van der Waals interaction energy, SASA - solvent
accessible surface area, and a, b, g, d are fitted parameters.
4.4.2. Data collection and structure determination
To compute PoissoneBoltzmann (PB) and Solvent Accessible
Surface Area for the PBSA calculations, the APBS v. 1.2.1 [35] and
SIMS [36] programs were used. For the PB calculation, protein and
solvent dielectric constants were 2 and 78, respectively. For the
SASA calculation, solvent probe radius was 1.4 Å and smoothing
radius was 0.4 Å.
Diffraction data from all complexes of CA II with compounds
selected for crystallographic studies were collected at the EMBL
X11, X12 and X13 beamlines at the DORIS storage ring (DESY,
Hamburg). Datasets were processed using MOSFLM [24,25] and
SCALA [26]. Initial phases for molecular replacement were
calculated with 3HLJ PDB entry [21] omitting heteroatoms and
ligands. REFMAC [27] and COOT [28] were used for structure
refinement and model building. Inhibitors were modeled with the
help of DSVisualizer 1.7 [29] (Accelrys), and corresponding
topology and parameters for refinement were generated using
LIBREFMAC [30]. The statistics of data collection and refinement
are presented in Table 3. Coordinates and structure factors are
deposited in the RCSB Protein Databank, and PDB IDs are given in
Table 3.
5. Associated content
PDB accession codes for the crystal structures of CA II with
compounds 1f, 1g, 2a, 2i, 2f, and 2j are 3SAX, 3S8X, 3SAP, 3SBH, and
3SBI, respectively.
Acknowledgments
This research was funded by a grant (No. LIG-16/2010) from the
Research Council of Lithuania. Diffraction data were collected at the
EMBL/DESY, Hamburg, X11, X12, X13 beamlines. Access to the
measurement facilities was funded from the European Commun-
ity’s Seventh Framework Programme (FP7/2007-2013) under grant
agreement no. 26716. EM travel to DESY, Hamburg was supported
from the Research Council of Lithuania (project No. C-01/2008,
registration No. C-08027), SG and GT travel was supported by the
FP7-REGPOT-2009-1 program (project 245721 MoBiLi). The authors
4.5. Computational docking details
The scaffolds for building the compounds were obtained from
the PDB entries 3M96 [10] (series of chlorinated compounds) and 2j
(compounds without chlorine). The construction and minimization
of the non-scaffold part of the molecule was performed using the
Avogadro program [31], keeping the scaffold frozen. Oxo-tautomer
was used for the hydroxy-pyrimidine series.
Docking was performed with the Vdock program using the
default settings [12]. CHARMM22 force field [32] was used for the
proteins, and the modified Dreiding force field [33] and VeraChem’s
partial atomic charges [34] were applied for the small molecules.
The solvent effect was modeled with the distance-dependent
dielectric approach, εij ¼ 4rij. PoissoneBoltzmann surface area
(PBSA) approach was used to evaluate the binding affinities of
ligands to CA II (Gibbs free energy of binding), employing the
following equation:
ꢀ ꢁ _
thank Zita Liutkeviciute for help with the mass spectrometry
analysis of the synthetic compounds and local contacts at the EMBL
beamlines for their help with beamline operation.
Appendix. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2012.02.050.

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270
E. Capkauskaite et al. / European Journal of Medicinal Chemistry 51 (2012) 259e270
[20] H.F. Andrew, C.K. Bradsher, A new synthesis of thiazolo[3.2-a]pyrimidinones,
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