4-Substituted-2,3,5,6-tetrafluorobenzenesulfonamides as inhibitors of carbonic anhydrases I, II, VII, XII, and XIII

Article abstract of DOI:10.1016/j.bmc.2013.01.008

A series of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides were synthesized and their binding potencies as inhibitors of recombinant human carbonic anhydrase isozymes I, II, VII, XII, and XIII were determined by the thermal shift assay, isothermal

Full text of DOI:10.1016/j.bmc.2013.01.008

Bioorganic & Medicinal Chemistry 21 (2013) 2093–2106
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
4-Substituted-2,3,5,6-tetrafluorobenzenesulfonamides as inhibitors
of carbonic anhydrases I, II, VII, XII, and XIII
Virginija Dudutiene a, Asta Zubriene a, Alexey Smirnov a, Joana Gylyte a, David Timm a, Elena Manakova b,
˙
˙
˙
b
a,
⇑
ˇ
Saulius Grazulis , Daumantas Matulis
a
ꢀ
ˇ
Department of Biothermodynamics and Drug Design,Vilnius University Institute of Biotechnology, Graiciuno 8, Vilnius LT-02241, Lithuania
Department of Protein–DNA Interactions, Vilnius University Institute of Biotechnology, Graiciuno 8, Vilnius LT-02241, Lithuania
b
ꢀ
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-substituted-2,3,5,6-tetrafluorobenezenesulfonamides were synthesized and their binding
potencies as inhibitors of recombinant human carbonic anhydrase isozymes I, II, VII, XII, and XIII were
determined by the thermal shift assay, isothermal titration calorimetry, and stop-flow CO2 hydration
assay. All fluorinated benzenesulfonamides exhibited nanomolar binding potency toward tested CAs
and fluorinated benzenesulfonamides posessed higher binding potency than non-fluorinated compounds.
The crystal structures of 4-[(4,6-dimethylpyrimidin-2-yl)thio]-2,3,5,6-tetrafluorobenzenesulfonamide in
complex with CA II and CA XII, and 2,3,5,6-tetrafluoro-4-[(2-hydroxyethyl)sulfonyl]benzenesulfonamide
in complex with CA XIII were determined. The observed dissociation constants for several fluorinated
compounds reached subnanomolar range for CA I isozyme. The affinity and the selectivity of the com-
pounds towards tested isozymes are presented.
Received 12 November 2012
Revised 29 December 2012
Accepted 3 January 2013
Available online 16 January 2013
Keywords:
Carbonic anhydrase inhibitors
Isothermal titration calorimetry
Thermal shift assay
ThermoFluorÒ
Ó 2013 Elsevier Ltd. All rights reserved.
Fluorinated benzenesulfonamide
X-ray crystallography
1. Introduction
The major class of CAIs is aromatic and heterocyclic compounds
possessing a primary sulfonamide group.11 Sulfonamide-class CAIs
The human carbonic anhydrases (CA) are family of zinc metal-
loenzymes that catalyze the reversible hydration of CO2.1,2 Iron
and other metals are sometimes found in bacterial and other
non-human CAs.3,4 This reaction regulates a broad range of physi-
ological functions such as respiration, CO2/bicarbonate transport
between lungs and metabolizing tissues, pH and CO2 homeostasis,
electrolyte secretion in many tissues/organs, etc.5,6 There are 15
carbonic anhydrase isozymes identified in humans—12 catalyti-
cally active and 3 inactive, the so called CA-related proteins. The
12 active isoforms have different subcellular localization: 5—
cytosolic (I, II, III, VII, and XIII), 3—transmembrane (IX, XII, and
XIV), 1—membrane bound (IV), 2—mitochondrial (VA and VB),
and 1—secreted (VI). Some of these isozymes (e.g., CA II, IV, VA,
VB, VII, IX, XII, XIII and XIV) constitute targets for the development
of antiglaucoma, diuretic, antiobesity, anticonvulsant or anticancer
drugs.7–10 Despite the large number of different CA inhibitors
(CAIs) that have been synthesized to date, the available pharma-
ceutical agents have a number of shortcomings. One of them is
the non-selective inhibition of many CA isozymes throughout the
human body, resulting in various side effects. Therefore, there is
a need to improve the inhibition and selectivity profile of the
developed CAIs.
are widely used as therapeutic agents for the treatment of various
diseases. Benzenesulfonamides are the most abundant CAI class
possessing a sulfonamide group. Introduction of substituents in
various positions of the benzene ring of benzenesulfonamides have
been largely investigated from diverse points of view.9,11,12 General
approaches for increasing affinity of benzenesulfonamides for CA
are the enhancement of the influence of the arylsulfonamide head
group (SO2NH2) on their binding and the amplification of interac-
tions between the substituted aryl ring and the hydrophobic pock-
et of CA.1
The contribution towards the affinity of the sulfonamide head
group can be explained by the influence of the pKa of the arylsulf-
onamide. It is known that the highest-affinity arylsulfonamides
should have a pKa near the pH of buffered medium (ꢀ7.4)1 while
nonsubstituted benzenesulfonamide possess pKa = 10.1.13 The
presence of electronegative substituents decreases the pKa of sul-
fonamide and this correlates with an increase in the CA inhibitory
properties. Introduction of halogen atoms as substituents on the
benzene ring of benzenesulfonamides is one of the possible
choices.11,12
Fluorine has been used widely in protein ligands. Extensive use
of fluorinated compounds in medicinal chemistry is due to the un-
ique properties of fluorine.14 The physical properties of fluorine
that have contributed to its relevance in medicinal chemistry in-
clude its slightly larger size compared to hydrogen (fluorine’s van
⇑
Corresponding author. Tel.: +370 5 269 1884; fax: +370 5 260 2116.
E-mail addresses: matulis@ibt.lt, daumantas.matulis@bti.vu.lt (D. Matulis).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.008

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V. Dudutiene˙ et al. / Bioorg. Med. Chem. 21 (2013) 2093–2106
der Waals radius is 1.47 Å while hydrogen 1.2 Å, the bond lengths
of CH3–F = 1.382 Å and of CH3–H = 1.087 Å), its high electronega-
tivity, low polarizability, and the contribution towards increased
lipophilicity. These features of fluorine enable the synthesis of
compounds possessing unique properties that cannot be attained
using any other element.
resolution since the first CA structure.43 Here we describe several
structures of CA II, XII, and XIII with bound fluorinated ligands.
2. Results and discussion
2.1. Chemistry
Numerous previous studies have examined the binding of fluo-
rinated arylsulfonamide ligands to CA. Part of them concentrated
on the investigation of a fluorinated ‘tail’.15–24 Another part of
them investigated the influence of fluorine incorporated directly
into the benezenesulfonamide scaffold. Monofluorination was
common approach in the synthesis of CA inhibitors.21,25–35 How-
ever introduction of several fluorine atoms was not as com-
mon.13,36–39
A
series of 4-substituted-2,3,5,6-tetrafluorobenzenesulfona-
mides were designed and synthesized (Scheme 1). The starting com-
pound for these investigations was pentafluorobenzenesulfonamide
which readily underwent nucleophilic substitution reaction and
formed the 4-substituted-2,3,5,6-tetrafluorobenzenesulfonamides.
Such compounds bearing cycloalkylamino or alkylamino groups in
the 4-position were described as anticonvulsants44 but the connec-
tion between the anticonvulsant properties and the CA inhibitory
properties was not explained.
The study by Olander et al. investigated the binding of pentaflu-
orobenzenesulfonamide (PFBSA) to bovine CA II.36
Gerig and co-workers investigated the binding of fluorinated
benzenesulfonamides to CA I and CA II by using 19F NMR spec-
troscopy. Their results proposed a binding stoichiometry of 2:1
for 2-fluoro-, 3-fluoro-, 4-fluoro-, and 2,5-difluorobenzenesulfona-
mides to CA I and II.35,37 However, the binding stoichiometry of
2,3,5,6-Tetrafluorobenzenesulfonamide (compound 1) was syn-
thesized from 1,2,4,5-tetrafluorobenzene (purchased from Alfa Ae-
sar). First, the 1,2,4,5-tetrafluorobenzene was converted to 2,3,5,6-
tetrafluorobenzenesulfonyl chloride.16 Reaction of obtained sulfo-
nyl chloride with aqueous ammonia yielded the sulfonamide 1. Sim-
ilarly, the synthesis of pentafluorobenzenesulfonamide (compound
2) via amination of pentafluorobenzenesulfonyl chloride (purchased
from Alfa Aesar) with aqueous ammonia was obtained. The synthe-
sis of penta- and 2,3,5,6-tetrafluorobenzenesulfonamides from
appropriate sulfonyl chloride were reported in the literature,45 but
substantial amounts of disulfonimideswere obtained as byproducts.
We found that the formation of imide can be avoided by keeping the
reaction medium near a neutral pH. The formation of imides is due to
low pKa of penta- and 2,3,5,6-tetrafluorobenzenesulfonamides.
Deprotonation of sulfonamide occurs in basic medium and deproto-
nated sulfonamide reacts readily with the starting material—sulfo-
nyl chloride.
4-Substituted-2,3,5,6-tetrafluorobenzenesulfonamides 3a, c, e–
i, k–o, and q–w were obtained from compound 2 by using the
appropriate nucleophile in ethanol, methanol or DMSO in the pres-
ence of Et3N or K2CO3 (excess of nucleophile was used in several
cases instead of the mentioned bases). The compound 3d was pre-
pared by oxidation of 3c with CH3COOHnH2O2. Oxidation of the
compounds 3i, 3o with CrO3 gave 3j, 3p. The reaction of 3a with
benzaldehyde led to the formation of 3b.
1:1 for 3,5-difluorobenzenesulfonamide to human CA
I was
observed.38
Whitesides and co-workers observed 1:1 binding for fluorinated
benzenesulfonamides by using biophysical and X-ray crystallo-
graphic techniques.13 Thermodynamic parameters for the
association of 2-fluoro-, 3-fluoro-, 4-fluoro-, 2,6-difluoro-, 3,5-di-
fluoro-benzenesulfonamides and PFBSA with bovine CA II were
investigated in this study. Their results highlighted the importance
of examining the thermodynamic parameters for the association of
both the ligand and protein in their active forms. Most other stud-
ies have only discussed the experimentally observed thermody-
namic parameters without the dissection of intrinsic part and the
contribution of linked protonation effects. Furthermore, they con-
cluded that fluorine is the best choice for electron-withdrawing
substituents because it decreases the pKa and also increases hydro-
phobicity of the ligand.
Supuran and co-workers investigated the binding of haloge-
nated benzenesulfonamides incorporating one or two (equal or dif-
ferent) halogens.33 Monofluorinated compounds generally were
less active than other monohalogenated derivatives. Combination
of fluorine with another halogen yielded potent CA IX inhibitors.
A study of binding thermodynamics of benzenesulfonamides
bearing chlorine or fluorine atoms was reported by Scott et al.34
They noted that similar substitutions (e.g., Cl or F in positions 2,
3 or 4) resulted in significantly different changes to the binding
thermodynamics, which would be impossible to predict from the
structural analysis alone. The presence of fluorine was more favor-
able than chlorine for the inhibition of CA.
In order to compare with fluorine-free compounds, the 4-
substituted benzenesulfonamides 6a–c were synthesized accord-
ing to the literature25 (Scheme 2). 4-Bromobenzenesulfonamide
(4) was converted to compound 5 with N,N-dimethylformamide
dimetyl acetal in acetonitrile. Treatment of protected sulfonamide
with thiols led to the replacement of bromine. The deprotection of
the sulfonamide group was accomplished by using aqueous so-
dium hydroxide and thus compounds 6a–c were obtained.
Here we describe the synthesis of benzenesulfonamides pos-
sessing as many fluorine atoms as possible in the benzenesulfon-
amide scaffold. It was expected that fluorine atoms would
contribute favorably to CA binding and that highly fluorinated
benzenesulfonamides would have a good capacity for nucleophilic
aromatic substitution reactions. It has been reported previously
that polyfluorobenzenes undergo nucleophilic aromatic substitu-
tion reactions40 and that fluorine is one of the best leaving groups
for the substitution. Nucleophilic aromatic substitution reactions
of fluorinated benzenes bearing one or several fluorines can be
accelerated by electron-withdrawing groups.41,42 These features
allow for the synthesis of a diverse group of compounds from
fluorinated benzenes.
2.2. Binding and inhibition studies
The binding affinities of fluorinated benzenesulfonamides to
five carbonic anhydrase isozymes (I, II, VII, catalytic domain of
CA XII, and CA XIII) were measured by the thermal shift assay
(TSA) and isothermal titration calorimetry (ITC). The dissociation
constants for synthesized compounds are listed in Tables 1 and
2. The data for three reference compounds (BSA—benzenesulfon-
amide, EZA—ethoxzolamide, and AZM—acetazolamide) is provided
at the end of the Table 1. Figures 1 and 2 show representative bind-
ing data obtained by TSA with isozyme CA I and CA XIII. Figure 3
shows representative binding data obtained by ITC (isozymes CA
I and CA XIII). The Kd measured by ITC in most cases was slightly
higher than by TSA, especially in the case of very tight binding. This
discrepancy could be explained by, as shown in Figure 3a, the
Herein, we report the synthesis of 4-substituted-2,3,5,6-tetra-
fluorobenzenesulfonamides and their investigation as inhibitors
of carbonic anhydrase isozymes I, II, VII, XII, and XIII. There are
many carbonic anhydrase crystal structures solved to atomic

V. Dudutiene˙ et al. / Bioorg. Med. Chem. 21 (2013) 2093–2106
2095
SO2NH2
F
SO2NH2
F
SO2NH2
F
F
F
F
F
F
F
F
F
F
F
R
(a-w)
3
2
1
R= NHNH2(a), NHN=CHPh(b), SCH2CH2OH (c), SO2CH2CH2OH(d), SCH2CH2CH3(e), SCH2COOH(f),
SCH2CH2COOH(g), NH(CH2)5COOH (h), SPh(i), SO2Ph(j), OPh(k), SCH2Ph(l), NHCH2Ph(m),
NHCH2CH2PhpOH(n), SCH2CH2Ph(o), SO2CH2CH2Ph(p),
S
N
S
N
N
(u),
S
(t),
NH
N
O (q),
(s),
S
(r),
H
N
N
S
(w)
N
(v),
N
N
S
S
Scheme 1. Structures of synthesized compounds 1, 2, 3a–w.
CHN(CH3)2
N
SO2NH2
SO2NH2
SO2
1. RH, NaH, DMFA
(CH3O)2CHN(CH3)2, CH3CN
2. NaOH, CH3OH
Br
4
R
6
Br
5
(a-c)
R= SCH2CH2OH (a) SCH2CH2CH3(b), SCH2CH2Ph(c).
Scheme 2. Synthesis of compounds 6a–c.
integrated curve of the binding of 3d to CA I, being too steep to be
fitted precisely. It was previously discussed46 that the c value
(c = C ꢁ Kb, where C is the molar concentration of the protein,
and Kb is the binding constant) for an ITC titration must be be-
tween 5 and 500. However, in our ITC experiments the concentra-
Comparing the fluorinated with nonfluorinated compounds
bearing SCH2CH2OH (3c vs 6a), SCH2CH2CH3 (3e vs 6b), and
SCH2CH2Ph (3o vs 6c) substituents showed that the fluorination
significantly increased the binding affinity to the tested CAs. For
example, the Kds for 3e and 6b for CA I varied by more than
150 times (from 0.03 to 4.5 nM), for CA II—6.5 times, for CA VII—
17 times, CA XII—10 times, and for CA XIII—29 times.
The influence of the substituent being in para position of the
benzenesulfonamide ring to binding strength was also evident
for compounds of the series 6a–c. Compounds 6a–c had signifi-
cantly higher binding affinity than benzenesulfonamide (BSA). A
significant difference in Kds was observed between compounds
6a and 6b with the first one being much weaker than the latter. Be-
cause the changes were only in the hydroxy (6a) and methyl group
(6b) at the end of substituent tail, it is supposed that additional
hydrophobic contacts with the enzyme hydrophobic pocket could
be formed for compound 6b.
All compounds used in this study bound to all the tested CAs
with 1:1 stoichiometry as demonstrated by ITC and the dosing
curves of TSA. This strongly indicated that all inhibitors bound
exclusively to the active site of the enzymes. However, direct
observation of the inhibition of the CO2 hydration by the com-
pounds would determine the Ki and confirm that the compounds
were inhibitors of the CA. However, the stop-flow hydration assay
was significantly more laborious than with TSA. Therefore, the
tion of CA I was 6 lM, and the binding constant of 3d was shown
by TSA to be 3 ꢁ 1010 Mꢂ1. Thus the c value would be 180,000,
much higher than ITC limits. The shape of the fitted curve of the
ITC isotherm was suitable only for the value of Kb 68.3 ꢁ l07 Mꢂ1
(Kd P12 nM). Therefore TSA data was more appropriate to deter-
mine tight binding than ITC.
The data in Table 1 shows that fluorine atom substitution and
the nature of the substituent on the para position of benzenesul-
fonamide ring significantly influences the binding affinity against
CA I, II, VII, XII, and XIII. The 4-substituted-2,3,5,6-tetrafluoroben-
zenesulfonamides (compounds 3a–w) bound CA I, II, VII, and XIII
with low nanomolar and subnanomolar affinity, exhibiting espe-
cially strong binding to CA I (Kd in the range of 0.05–14 nM). The
Kd ranges for CA II were 1.1–91 nM (3t and 3a), for CA VII 0.22–
465 nM (3t and 3a), and for CA XIII were 0.22–140 nM (3o and
3a). The weakest binding was observed for CA XII (Kd in the range
of 6.67–769 nM as measured by TSA). The most potent CA I inhib-
itors 3e, 3j, 3r and 3u bound with Kd in the range of 0.033–
0.067 nM. Compound 3a bearing NHNH2 group in para position
had the weakest binding affinity for all CAs.

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V. Dudutiene˙ et al. / Bioorg. Med. Chem. 21 (2013) 2093–2106
Table 1
Compound dissociation constants for five human recombinant CA isoforms, determined by TSA (37 °C, pH 7.0)
Compound
R
Dissociation constants Kd (nM), CA isoforms:
CA I
CA II
CA VII
CA XII
CA XIII
1
2
H
F
2.4
2.2
8.3
4.0
29
27
91
2.9
6.7
17
2.0
11
11
25
465
43
46
333
500
769
667
220
250
40
50
91
200
20
17
20
40
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
NHNH2
140
2.2
8.3
29
1.7
6.7
20
NHN@CHPh
SCH2CH2OH
SO2CH2CH2OH
SCH2CH2CH3
SCH2COOH
SCH2CH2COOH
NH(CH2)5COOH
SPh
SO2Ph
OPh
SCH2Ph
NHCH2Ph
NHCH2CH2PhpOH
SCH2CH2Ph
SO2CH2CH2Ph
0.11
0.20
0.033
0.20
0.40
0.10
0.13
0.050
0.10
0.25
0.20
0.17
0.20
0.25
7.1
1.1
5.0
10
147
12
0.67
1.0
1.3
133
0.50
0.83
1.3
20
67
14
4.0
3.3
2.5
1.3
9.1
1.7
1.7
1.3
1.5
3.3
1.0
0.40
5.0
0.40
0.22
0.40
50
6.7
333
154
110
77
3q
N
S
0.13
18
83
500
25
O
3r
0.067
3.3
2.5
100
3.3
N
N
S
3s
2.5
10
10
290
2.5
S
S
3t
1.1
1.1
5.0
0.22
1.0
200
17
0.25
0.5
N
NH
3u
0.050
N
3v
0.40
6.7
6.7
13
110
670
2.5
2.0
N
N
S
S
S
H
N
3w
14
1.7
N
6a
6b
6c
BSA
EZA
AZM
SCH2CH2OH
SCH2CH2CH3
SCH2CH2Ph
200
4.5
5.0
7140
14
1400
130
13
25
1790
1.1
38
170
17
40
6670
0.71
17
2500
400
330
12,500
36
133
1400
50
290
10,000
13
50
inhibitory effect Ki on CA II was determined only for the com-
pounds 3c and 6a. The compounds were dosed in a stepwise 2ꢁ
dilution beginning from 1000 nM and ending with a 2 nM concen-
tration of the inhibitor. CA II concentration was 20 nM. These com-
pounds inhibited CA II and the dosing curves exhibited the typical
sigmoidal shape with the fitted IC50 equal to 10 nM and 60 nM,
respectively for 3c and 6a. Assuming that the KM of CA II was
9.3 mM and the half concentration of saturated CO2 solution at
25 °C was 17 mM, the Kis were equal to 3.5 and 21 nM, respec-
tively. These values are slightly lower than the ones determined
by TSA and ITC. The discrepancy could be partially explained by
Table 2
Dissociation constants of selected compounds for five human recombinant CA
isoforms, determined by ITC (37 °C, pH 7.0)
Compound
Dissociation constants Kd (nM), CA isoforms:
CA I
CA II
CA VII
CA XII
CA XIII
1
18
42
121
59
37
88
510
588
94
143
150
1500
66
18
74
612
1000
3c
3d
3s
6a
612
612
612
790
612
612
612
330

V. Dudutiene˙ et al. / Bioorg. Med. Chem. 21 (2013) 2093–2106
2097
the difference in temperatures—the stop-flow assay was per-
50
40
30
formed at 25 °C and the ITC at 37 °C. However, there was a general
agreement between all three methods that both compounds were
tight inhibitors of CA II and the fluorinated compound was signif-
icantly more potent than the non-fluorinated one.
CA I - 3c
A detailed binding study of fluorinated benzenesulfonamides
(no other substituents were included, general formula C6HnF5–
nSO2NH2) with bovine carbonic anhydrase II (BCA) has been previ-
ously reported by Whitesides group.13 They assumed that the
thermodynamic parameters of binding could be partitioned into
the three different structural interactions between the fluorinated
ligand and BCA: the Zn(II) cofactor–sulfonamide bond, the hydro-
gen bonds between ligand and BCA, and the hydrophobic contacts
between the phenyl ring of the ligand and BCA. In this study PFBSA
was a 30 times more potent inhibitor than BSA primarily due to
their different pKa (8.2 and 10.1, respectively). The authors as-
sumed that a pKa of the ligand of about 7.4 should give the lowest
observed dissociation constant Kd.
20
80
CA I - 6a
60
40
20
0
50
60
T
70
80
m (oC)
As the pKas of 4-substituted-2,3,5,6-tetrafluorobenzenesulfona-
mides (compounds 3a–w) should be near that of PFBSA, the addi-
tional contacts between the substituent (R) on the
benzenesulfonamide ring and CA could result in the increased
binding affinity of most compounds of the series 3a–w, as com-
pared to PFBSA.
75
70
65
60
55
Kd(3c) = 0.11 nM
m
T
Kd(6a) = 200 nM
2.3. Crystallography
-6
0
10
10-5
10-4
10-3
Three crystal structures of CAs bound with the fluorinated
inhibitors were solved to atomic resolution: CA II and CA XII com-
plexed with compound 3s and CA XIII with compound 3d. Data col-
lection and refinement statistics are summarized in Table 3 and the
electron densities of the ligands in crystal structures are shown in
Figure 4.
Lt (M)
Figure 1. TSA data of representative compound binding to CA I. Panels on the top
show thermal shift assay raw melting curves at several added concentrations of 3c
and 6a. Panel on the bottom shows the dependence of the protein melting
temperatures Tm on ligand concentrations. Datapoints are from the experimental
values from the upper panel and the curves are simulated according to the model.
The CA XII with compound 3s structure (PDB ID 4HT2) con-
tained four protein chains in the asymmetric unit. The ligand
was observed in all subunits. The superposition of the four sub-
units showed that the position of the benzene ring of the ligand
is nearly identical in the three monomers (‘main’ orientation),
while the conformation in the fourth (subunit A) was rotated by
ꢀ80° (‘rotated’ orientation, Figs. 4C and 5).
tetrafluorobenzene ring of 3s (PDB ID 4HT0) was bound in the
same ‘main’ orientation as in these two crystal structures.
The second dimethylpyrimidine ring of the ligand 3s was lo-
cated in the mainly hydrophobic environment. It contacted
hydrophobically Phe131, Val135, Pro202 and Leu204 in CA II
(Fig. 6A). Phe131 in CA XII is replaced by Ala129. This variation
resulted in different conformations of the bound ligand. So, the
bond angle on the sulfur atom connecting the two rings is dis-
torted from the perfect tetrahedral 104° in CA XII, to 146° in
CA II, where the second ring should avoid a steric clash with
Phe131 (Figs. 6A,B and 7A). In CA XII the second ring also made
The CA XIII with compound 3d structure (PDB ID 4HU1) con-
tained two protein chains (two monomers) in the asymmetric unit.
Orientation of the ligand in both subunits in the asymmetric unit of
CA XIII was nearly identical to the ‘rotated’ orientation of 3s in CA
XII chain A (Figs. 5 and 7B).
The ‘main’ orientation of the first benzene ring was also ob-
served in the structure of 3s bound to CA II (PDB ID 4HT0). The
structure contained only one protein chain in the asymmetric
unit. The first benzene ring fit into the pocket between the side
chains of Leu198 and Thr200 in CA II (corresponding residues in
CA XII are Leu197 and Thr199). The fluorine atoms made hydro-
gen bonds with the protein backbone. The opposite side of the
fluorinated benzene ring was restricted by Val121 (Val119 in CA
XII) and Gln92 in CA II (Gln89 in CA XII). The fluorinated benzene
ring also had hydrophobic contacts with His91 in CA XII and
Phe131 in CA II. Generally, one side of the benzene ring was stick-
ing to the hydrophobic wall of the protein pocket formed by
Val121 (Val119 in CA XII), Leu141 (Leu139 in CA XII), Leu198
(Leu197 in CA XII), and Phe131 in CA II.
The orientation of the fluorinated benzene ring in the ligand
binding pocket of CA II was similar to the position of benzenesul-
fonamide (PDB ID 2WEJ). In 2WEJ, the structure of benzenesul-
fonamide made van der Waals contacts with Val121, Leu198,
His94 and Thr200. There are two crystal structures of the tetraflu-
orobenzenesulfonamide derivatives bound to CA II available in
the PDB (PDB IDs: 3P25 and 3P29) from McKenna group. The
78
CA XIII
76
74
72
70
68
66
64
62
60
58
56
Kd(3n) = 0.4 nM
m
T
Kd (6a) = 1 400 nM
-6
0
10
10-5
10-4
10-3
Lt (M)
Figure 2. TSA data of 3n, 3s, 3c, 6b, 3a, and 6a binding to CA XIII. The binding
affinity of selected compounds ranged from 0.4 nM for 3n to 1.4 M for 6a.
l

2098
V. Dudutiene˙ et al. / Bioorg. Med. Chem. 21 (2013) 2093–2106
Time (min)
Time (min)
0
10 20 30 40 50 60 70 80 90 100 110
0
10 20 30 40 50 60 70 80 90 100
0.0
-0.1
-0.2
0.0
-0.1
-0.2
CA I - 3d
CA XIII - 3d
a
b
2
0
2
0
-2
-4
-2
-6
-4
N
K
ΔH
0.85
1.36E7
-1.06E4
-8
N
K
ΔH
0.80
9.59E8
-1.63E4
-10
-12
-14
-16
-18
-6
-8
-10
-12
0.0
0.5
1.0
Molar Ratio
1.5
2.0
0.0
0.5
1.0
Molar Ratio
1.5
2.0
Figure 3. ITC data of 3d binding to CA I (a) and CA XIII (b) at 37 °C.
Table 3
X-ray crystallographic data collection and refinement statistics. All datasets were collected at 100 K, test set size was 10%
Protein-compound:
CAXII-3s
CAXIII-3d
CAII-3s
PDB ID
4HT2
4HU1
4HT0
Spacegroup
Unit cell (Å)
P1
P212121
a = 56.27, b = 57.39, c = 159.55
P21
a = 46.71, b = 67.26, c = 80.69
a = 42.02, b = 40.90, c = 71.60
a
= 81.78°,b = 84.01°
c
= 86.48°
a
= b =
c
= 90°
a
= c = 90°, b = 104.04°
Number of chains
4
2
1
Resolution, (Å) (final shell)
Reflections unique (total)
Completeness (%) overall (final shell)
I/rI overall (final shell)
Rmerge Overall (final shell)
Number of atoms
1.45
1.95
1.60
166,179 (656,979)
97.1 (95.6)
14.1 (4.8)
0.039 (0.219)
10,438
38,614 (283,080)
100.0 (100.0)
9.0 (3.6)
0.135 (0.486)
4839
30,222 (227,077)
96.5 (97.0)
21.8 (12.1)
0.061 (0.123)
2446
Number of solvent molecules
Number of bounded buffer molecule atoms
1503
15
553
8
270
2
Rcryst (Rfree
)
0.152 (0.186)
0.025 (2.255)
13.1
0.169 (0.235)
0.024 (2.026)
19.6
0.168 (0.210)
0.029 (2.562)
15.5
RMS bonds/angles
Average B-factors (Å2)
Main chain:
10.0
17.5
12.5
Side chains:
12.1
19.0
15.1
Solvent:
23.2
28.0
26.6
Cofactors:
11.1
19.5
32.8
mostly van der Waals contacts with residues (corresponding CA
II residues are shown in parenthesis) Ser130 (Gly132), Ser133
(Val135), and Leu139 (Leu141).
The ‘rotated’ conformation was found as an alternative con-
formation of compound 3s in CA XII (Figs. 6D and 7B) and it
was the only orientation of compound 3d in CA XIII (Figs. 6C
and 7B). The fluorinated benzene ring was fixed in the CA XIII
binding pocket by mostly hydrophobic interactions with
Leu200 (Leu197 in CA XII), Val202 (Thr199 in CA XII), Val145
(Val141 in CA XII), and Phe133 (no contact with Ala129 in CA
XII). The other side of the ring was buttressed by the catalytic
His96 (His91 in CA XII), Val123 (Val119 in CA XII), and Gln94
(Gln89 in CA XII). Fluorine atoms at one edge were in van der
Waals contact with the hydrophobic wall of the pocket. At the
opposite edge of the ring there was a net of fixed solvent mole-
cules between the ligand and the protein moiety.
Benzene rings were in the same plane, but in CA XII the ring is
rotated ꢀ10° (Fig. 7B). The probable explanation could be the van
der Waals interaction of the dimethylpyrimidine ring with residues
129–130 and 133 that form the edge of the ligand binding pocket

V. Dudutiene˙ et al. / Bioorg. Med. Chem. 21 (2013) 2093–2106
2099
Figure 4. View of the electron density of the compounds 3d and 3s located in the active center of CA. The catalytic Zn(II) coordinated by active center histidine residues are
shown as blue spheres. The electron density difference maps Fo are contoured at 3 in B and D, 1 in A, and 2 in C. A, the compound 3s bound to CA II. B, the ‘main’
orientation of compound 3s in protein chain B of CA XII. C, two orientations of compound 3s bound to CA XII, chain A.
r
r
r
In the ‘rotated’ orientation of ligand 3s found in CA XII, the
hydrogen bonds with fluorine were absent. Predominantly hydro-
phobic binding was observed also for compound 3d in CA XIII,
which also exhibited the ‘rotated’ position of the benzene ring as
well. This orientation was fixed in CA XIII by several hydrogen
bonds made by the sulfonyl group of the linker.
3. Conclusions
A series of 4-substituted-2,3,5,6-tetrafluorobenezenesulfona-
mides were synthesized that possess nanomolar affinities toward
CA isozymes I, II, VII, catalytic domain XII, and XIII. Most of the
compounds showed better affinity for CA I than for CA II, a rela-
tively rare feature among most aromatic sulfonamides. The binding
potency of fluorinated benzenesulfonamides was greater than non-
fluorinated derivatives. Polyfluorinated benzenesulfonamide scaf-
fold could be used as a tool for CA inhibitor development. X-ray
crystallographic cocrystal structures of two fluorinated benzene-
sulfonamides with CA II, XII, and XIII showed the presence of two
possible orientations of these ligands.
Figure 5. Two orientations of compounds 3d and 3s in the active sites of the three
crystal structures. All subunits were superimposed by Zn(II) and the three active
center histidine residues. Active center residues considered to be most important
for binding to CA II are shown transparent. The compound 3s in chain B of CA XII is
colored gold. The compound 3d in CA XIII and the second ‘rotated’ orientation of
compound 3s in the chain A of CA XII are shown in blue and cyan, respectively. The
ligand 3s bound by CA II is colored red.
4. Experimental part
4.1. Chemistry
All starting materials and reagents were commercial products
and were used without further purification. Melting points of the
compounds were determined in open capillaries, Thermo Scien-
tific 9100 Series, and are uncorrected. Column chromatography
was performed using silica gel 60 (0.04–0.063 mm, Merck). 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, respec-
tively) with TMS as an internal standard, and proton, carbon
chemical shifts were expressed in parts per million (ppm) in
the indicated solvent. 19F NMR spectra were recorded on a Var-
ian Unity Inova spectrometer (282 MHz) with CFCl3 as an inter-
nal standard, and fluorine chemical shifts were expressed in
parts per million (ppm) in the indicated solvent. Multiplicity
was defined as s (singlet), d (doublet), t (triplet), q (quartet),
dd (double doublet), ddd (double double doublet), m (multiplet),
br s (broad singlet), br d (broad doublet), or br t (broad triplet).
TLC was performed using silica gel 60 F254 aluminum plates
(Merck) and visualized with UV light. High-resolution mass spec-
tra (HRMS) were recorded on a Dual-ESI Q-TOF 6520 mass spec-
trometer (Agilent Technologies).
in CA XII. The van der Waals interactions with the side chains of
Leu139, Leu197, Asn203, and Pro201 also participated in the
binding.
The sulfonyl group of ligand 3d made water mediated hydrogen
bonds with Arg93 and Asn69 and van der Waals bond with Gln94
(Fig. 6C).
In conclusion, the orientation of the benzene ring resulted in
a distinct mode of ligand binding in the pocket of the CA active
site. Compound 3s bound in all cases mostly hydrophobically to
the inner surface of the ligand binding pockets. This compound
in its ‘main’ orientation found in CA II and CA XII also made
hydrogen bonds with the protein backbone as a result of the
fluorine atoms of the benzene ring. The second dimethylpyrimi-
dine ring adopted the conformation that made as many hydro-
phobic contacts as possible. In CA II, the steric clash with
Phe131 could be the reason for more elongated conformation
of the compound 3s than in CA XII.

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V. Dudutiene˙ et al. / Bioorg. Med. Chem. 21 (2013) 2093–2106
Figure 6. Ligand positions in the active sites of CAs. Protein atoms that are in van der Waals contact with the compounds are shown as CPK. Zn(II) and water molecules are
shown as blue and red spheres, respectively. Dashed lines connect the atoms that presumably make hydrogen bonds. (A) the compound 3s (red) in CA II. (B) the compound 3s
(gold) in CA XII in the ‘main’ orientation (protein chain B). (C) the compound 3d (blue) in CA XIII, protein chain A. (D) the compound 3s (cyan) in CA XII in ‘rotated’ orientation
as modeled in chain A.
4.1.1. General information about 13C NMR spectra
example of a 13C NMR spectrum is given in the Figure 1 of the Sup-
plementary data.
NMR spectra of fluorinated aromatic compounds have been de-
scribed in the literature.47 The 13C NMR spectra of investigated com-
pounds were complicated by the fluorine coupling to carbon. Signals
of C1 and C4 of 4-substituted-2,3,5,6-tetrafluorobenezenesulfona-
mides were detected as triplets (the coupling constants were in
the range 13–21 Hz) due to ortho-couplingof two identical fluorines.
Signals of C2, C6 or C3, C5 were detected as ddd, dd or d. The majority
of such compounds had signals of d (additional couplings are poorly
visible). The first coupling was the coupling of fluorine to the ipso po-
sition with the coupling constant J (19F–13C) = 228–258 Hz. Other
couplings were observed due to the couplings of fluorine to the ortho
and meta positions (the coupling constants were in the range of 12–
19 Hz, 4–5 Hz). In the case of 2,3,5,6-tetrafluoro-4-morpholin-4-
ylbenzenesulfonamide (3q), even the coupling of morpholine
N(CH2)2 group was observed. The signal of the N(CH2)2 group was
detected as a triplet with the coupling constant of 3.6 Hz. An
4.1.2. 2,3,5,6-Tetrafluorobenzenesulfonamide (1)
The mixture of 2,3,5,6-tetrafluorobenzenesulfonyl chloride (1 g,
4.02 mmol) and THF (60 mL) was cooled to ꢀꢂ10 °C and aqueous
ammonia (ꢀ1.3 mL, 25%) was added dropwise while stirring until
the solution was at pH ꢀ7. After stirring for an additional 0.5 h,
THF was evaporated in vacuum and the resultant precipitate was
washed with cold H2O. Recrystallization was accomplished from
H2O. Yield: 0.78 g (85%), mp 142–143 °C was close to the value in
the literature, mp 145 °C.45 1H NMR (300 MHz, DMSO-d6): 8.1–8.3
(1H, m, ArH), 8.41 (2H, s, SO2NH2). 13C NMR (75 MHz, DMSO-d6):
110.9 (C4, t, J (19F–13C) = 23 Hz), 124.5 (C1, t, J (19F–13C) = 15 Hz),
143.3 (C3, C5, d,
(
ꢂ137.92 (2F, m), ꢂ139.4: ꢂ139.65 (2F, m).
J ( J
19F–13C) = 250 Hz), 146.5 (C2, C6, d,
19F–13C) = 240 Hz). 19F NMR (282 MHz, DMSO-d6): ꢂ137.7:

V. Dudutiene˙ et al. / Bioorg. Med. Chem. 21 (2013) 2093–2106
2101
Figure 7. Two orientations of ligands in the active centers of CAs. (A) superposition of active sites of CA II-3s complex (pink, the compound 3s is in red) and CA XII-3s, chain B
(green, the compound 3s is in gold). The ligand 3s is in the ‘main’ orientation. Amino acids of CA II important for ligand binding are labeled. B, superposition of active sites of
CA XIII complex with ligand 3d (pink, the compound 3d is in blue) and CA XII with alternative orientation of 3s, chain A (green, 3s is in cyan). Here ligands are in ‘rotated’
orientation. CA XIII residues important for ligand binding are labeled.
4.1.3. Pentafluorobenzenesulfonamide (2)
DMSO-d6): 111.7 (C1, t, J (19F–13C) = 14 Hz), 127.1 (Ar), 128.5 (C4,
t, J (19F–13C) = 13 Hz), 129.5 (Ar), 130.1 (Ar), 135.1 (Ar), 136.3
The mixture of pentafluorobenzenesulfonyl chloride (1 g,
3.75 mmol) and THF (60 mL) was cooled toꢀ ꢂ10 °C and aqueous
ammonia (ꢀ1.2 mL, 25%) was added dropwise while stirring until
the solution was at pH ꢀ7. After stirring for an additional 0.5 h,
THF was evaporated in vacuum and the resultant precipitate was
washed with cold H2O. Recrystallization was accomplished from
H2O. Yield: 0.84 g (90%), mp 156–157 °C close to the value in the
literature, mp 156 °C.45 1H NMR (300 MHz, DMSO-d6): 8.48 (2H,
(C3, C5, d,
19F–13C) = 251 Hz), 144.7 (CH). 19F NMR (282 MHz, DMSO-d6):
ꢂ141.6 (2F, d, J = 16.4 Hz), ꢂ156.6 (2F, d, J = 18 Hz). HRMS for
13H9F4N3O2S [(M+H)+]: calcd 348.0424, found 348.0433.
J
(
19F–13C) = 244 Hz), 144.5 (C2, C6, d,
J
(
C
4.1.6. 2,3,5,6-Tetrafluoro-4-[(2-
hydroxyethyl)sulfonyl]benzenesulfonamide (3d)
s, SO2NH2). 13C NMR (75 MHz, DMSO-d6): 119.9 (C1, t,
J
The mixture of 2,3,5,6-tetrafluoro-4-[(2-hydroxyethyl)thio]-
benzenesulfonamide (compound 2c) (0.1 g, 0.33 mmol), CH3COOH
(2 mL), H2O (1 mL) was heated at 70 °C for 22 h. H2O2 was added
by portions (0.2 mL) every 4 h (overall amount 1 mL). The progress
of reaction was monitored by TLC. The solvent was then removed
in vacuum and crude product was purified by crystallization from
(
19F–13C) = 14 Hz), 138.2 (C3, C5, d, J (19F–13C) = 247 Hz), 143.5
(C4, d, J (19F–13C) = 255 Hz), 144 (C2, C6, d, J (19F–13C) = 252 Hz).
19F NMR (282 MHz, DMSO-d6): ꢂ139.5 (F2, F6, dd, 1J = 19 Hz,
2J = 6 Hz), ꢂ149.39 (F4, t, J = 22 Hz), ꢂ161.03 (F3, F5, t, J = 20 Hz).
4.1.4. 2,3,5,6-Tetrafluoro-4-hydrazinobenzenesulfonamide (3a)
The mixture of pentafluorobenzenesulfonamide (compound 2)
(0.32 g, 1.295 mmol), NH2NH2ꢁH2O (0.126 mL, 2.59 mmol), and
EtOH (10 mL) was stirred at ambient temperature for 24 h. EtOH
was evaporated in vacuum and the resultant precipitate was
washed with H2O. Recrystallization was accomplished from H2O.
H2O. Yield: 0.067 g, 61%, mp 139–140 °C. IR m
cmꢂ1: 3326, 3481
(NH2). 1H NMR (300 MHz, DMSO-d6): 3.75 (2H, t, J = 5.4 Hz,
SO2CH2CH2), 3.88 (2H, t, J = 5.4 Hz, SO2CH2CH2), 5.01 (1H, br s,
OH), 8.65 (2H, s, SO2NH2). 13C NMR (75 MHz, DMSO-d6): 55.9
(SO2CH2CH2), 60.2 (SO2CH2CH2), 123.4 (C1 or C4, t,
19F–13C) = 15 Hz), 128 (C1 or C4, t, J (19F–13C) = 16 Hz), 143.5
(C3, C5 or C2, C6, dd, 1J (19F–13C) = 254 Hz, 2J (19F–13C) = 18 Hz),
J
(
Yield: 0.2 g, 60%, decomp. at 160–161 °C. IR
m
cmꢂ1: 3343, 3260
(NH2). 1H NMR (300 MHz, DMSO-d6): 4.68 (2H, s, NH2), 7.75 (1H,
s, NH), 7.98 (2H, s, SO2NH2). 13C NMR (75 MHz, DMSO-d6): 108.6
(C1, t, J (19F–13C) = 14 Hz), 135 (C4, t, J (19F–13C) = 16 Hz), 136
145 (C3, C5 or C2, C6, dd, 1J
(
19F–13C) = 248 Hz, 2J
(
19F–13C) = 15 Hz). 19F NMR (282 MHz, DMSO-d6): ꢂ136.7 (2F, dd,
1J = 26 Hz, 2J = 12 Hz), ꢂ137.6 (2F, dd, 1J = 26 Hz, 2J = 12 Hz). HRMS
(C3, C5, d,
(
J
(
19F–13C) = 240 Hz), 144.1 (C2, C6, d,
J
for C8H7F4NO5S2 [(MꢂH)ꢂ]: calcd 335.9629, found 335.9635.
19F–13C) = 240 Hz). 19F NMR (282 MHz, DMSO-d6): ꢂ143.1 (2F, d,
J = 15.8 Hz), ꢂ160.2 (2F, d, J = 17.2 Hz). HRMS for C6H5F4N3O2S
4.1.7. General procedure for the syntheses of 3c, e–h, l–o, r, v
The mixture of pentafluorobenzenesulfonamide (compound 2)
(0.25 g, 1 mmol), MeOH (10 mL), Et3N (0.141 mL, 1.01 mmol) and
the appropriate nucleophile (1.1 mmol) was refluxed. Compounds
3c, g, h, m, o, r were obtained after 8 h, compound 3e was obtained
after 10 h, compounds 3f and 3n were obtained after 15 h, com-
pound 3m was obtained after 4 h, and compound 2v was obtained
after 1 h. MeOH was evaporated in vacuum and the resultant pre-
cipitate was washed with H2O (except 3f, g, h).
[(M+H)+]: calcd 335.9782, found 335.9780.
4.1.5. 4-(2-Benzylidenehydrazino)-2,3,5,6-
tetrafluorobenzenesulfonamide (3b)
The mixture of 2,3,5,6-tetrafluoro-4-hydrazinobenzenesulfona-
mide (compound 3a) (0.13 g, 0.5 mmol), benzaldehyde (0.051 mL,
0.5 mmol), and MeOH (10 mL) was stirred at ambient temperature
for 4 h. MeOH was evaporated in vacuum. Recrystallization was
accomplished from iPrOH. Yield: 0.14 g, 82%, decomp. at 272–
273 °C. IR
m
cmꢂ1: 3355, 3240 (NH2). 1H NMR (300 MHz, DMSO-
4.1.7.1. 2,3,5,6-Tetrafluoro-4-[(2-hydroxyethyl)thio]benzenesul-
d6): 7.35–7.55 (3H, m, ArH), 7.6–7.75 (2H, m, ArH), 8.12 (2H, s,
fonamide (3c). Recrystallization was accomplished from H2O.
SO2NH2), 8.25 (1H, s, NH), 10.96 (1H, s, CH). 13C NMR (75 MHz,
Yield: 0.22 g, 71%, mp 111–112 °C. IR m : 3343, 3474
cmꢂ1

2102
V. Dudutiene˙ et al. / Bioorg. Med. Chem. 21 (2013) 2093–2106
(NH2). 1H NMR (300 MHz, DMSO-d6): 3.16 (2H, t, J = 6 Hz,
SCH2CH2), 3.6 (2H, k, J = 6 Hz, SCH2CH2), 4.97 (1H, t, J = 3.6 Hz,
OH), 8.43 (2H, s, SO2NH2). 13C NMR (75 MHz, DMSO-d6): 37.3
(SCH2CH2), 61.4 (SCH2CH2), 119.95 (C1, t, J (19F–13C) = 14 Hz),
J = 18.3 Hz). HRMS for C12H14F4N2O4S [(MꢂH)ꢂ]: calcd 357.0538,
found 357.0542.
4.1.7.6. 4-(Benzylthio)-2,3,5,6-tetrafluorobenzenesulfonamide
122.9 (C4, t,
J
(
19F–13C) = 15 Hz), 143.2 (C2, C6, ddd, 1J
(3l). Recrystallization was accomplished from iPrOH. Yield:
(
19F–13C) = 254 Hz, 2J (19F–13C) = 17 Hz, 3J (19F–13C) = 4 Hz), 147
0.23 g, 64%, mp 184 °C. IR m
cmꢂ1: 3395, 3267 (NH2). 1H NMR
(C3, C5, ddd, 1J
(
19F–13C) = 228 Hz, 2J
(
19F–13C) = 14 Hz, 3J
(300 MHz, DMSO-d6): 4.3 (2H, s, CH2), 7.1–7.5 (5H, m, ArH), 8.44
(2H, s, SO2NH2). 13C NMR (75 MHz, DMSO-d6): 38.6 (CH2), 118.5
(C1, t, J (19F–13C) = 21 Hz), 123.6 (C4, t, J (19F–13C) = 16 Hz), 128.4
(Ar), 129.3 (Ar), 129.5 (Ar), 137.4 (Ar), 143.1 (C2, C6, dd, 1J
(
19F–13C) = 4 Hz). 19F NMR (282 MHz, DMSO-d6): ꢂ133.5 (2F, dd,
1J = 26.8 Hz,
2J = 13.8 Hz),
ꢂ140
(2F,
dd,
1J = 26.2 Hz,
2J = 12.7 Hz). HRMS for C8H7F4NO3S2 [(MꢂH)ꢂ]: calcd 303.9731,
found 303.9729.
(
(
19F–13C) = 254 Hz, 2J (19F–13C) = 17 Hz), 147.3 (C3, C5, dd, 1J
19F–13C) = 248 Hz, 2J
(
19F–13C) = 17 Hz). 19F NMR (282 MHz,
4.1.7.2. 2,3,5,6-Tetrafluoro-4-(propylthio)benzenesulfonamide
DMSO-d6): ꢂ132.8 (2F, dd, 1J = 27 Hz, 2J = 11 Hz), ꢂ139.6 (2F, dd,
1J = 26 Hz, 2J = 10 Hz). HRMS for C13H9F4NO2S2 [(MꢂH)ꢂ]: calcd
349.9938, found 349.9940.
(3e). Recrystallization was accomplished from EtOH:H2O (2:1).
Yield: 0.25 g, 81%, mp 120 °C. IR
m
cmꢂ1: 3349, 3253 (NH2). 1H
NMR (300 MHz, DMSO-d6): 0.96 (3H, t, J = 7.2 Hz, CH3), 1.55 (2H,
sext, J = 7.2 Hz, CH2), 3.04 (2H, t, J = 7.2 Hz, CH2), 8.41 (2H, s,
SO2NH2). 13C NMR (75 MHz, DMSO-d6): 13.3 (CH3), 23.5 (CH2),
36.4 (CH2), 119.2 (C1, t, J (19F–13C) = 20 Hz), 123.1 (C4, t, J
4.1.7.7.
4-(Benzylamino)-2,3,5,6-tetrafluorobenzenesulfona-
mide (3m). Recrystallization was accomplished from H2O:EtOH
(1:1). Yield: 0.21 g, 62%, mp 132–133 °C. IR
m
cmꢂ1: 3415, 3280
(
(
(
19F–13C) = 16 Hz), 143.2 (C2, C6, dd, 1J (19F–13C) = 254 Hz, 2J
19F–13C) = 17 Hz), 147.2 (C3, C5, dd, 1J (19F–13C) = 244 Hz, 2J
19F–13C) = 17 Hz). 19F NMR (282 MHz, DMSO-d6): ꢂ133.9 (2F, dd,
(NH, NH2). 1H NMR (300 MHz, DMSO-d6): 4.56 (2H, d, J = 6.3 Hz,
CH2), 7.2–7.6 (6H, m, NH, ArH), 7.98 (2H, s, SO2NH2). 13C NMR
(75 MHz, DMSO-d6): 47.9 (CH2), 109.1 (C1, t, J (19F–13C) = 17 Hz),
1J = 25 Hz, 2J = 11 Hz), ꢂ139.6 (2F, dd, 1J = 25 Hz, 2J = 14 Hz). HRMS
127.2 (Ar), 127.7 (Ar), 129.2 (Ar), 131.7 (C4, t,
J
for C9H9F4NO2S2 [(MꢂH)ꢂ]: calcd 301.9938, found 301.9940.
(
19F–13C) = 16 Hz), 136.5 (C2, C6, d, J (19F–13C) = 244 Hz), 140.5
(Ar), 144.3 (C3, C5, d, J (19F–13C) = 241 Hz). 19F NMR (282 MHz,
DMSO-d6): ꢂ142.5 (2F, d, J = 18.3 Hz), ꢂ160.4 (2F, d, J = 18.3 Hz).
4.1.7.3. {[4-(Aminosulfonyl)-2,3,5,6-tetrafluorophenyl]thio}ace-
tic acid (3f). The product was purified by chromatography on a
column of silica gel (0.04–0.063 mm) with ethyl acetate. Yield:
HRMS for
335.0472.
C
13H10F4N2O2S [(M+H)+]: calcd 335.0472, found
0.12 g, 38%, mp 158–159 °C. IR
m : 3338, 3237 (NH2),
cmꢂ1
2925 (OH), 1723 (CO). 1H NMR (300 MHz, DMSO-d6): 3.9 (2H,
s, CH2), 8.44 (2H, s, SO2NH2), 12.8 (1H, br s, COOH). 13C NMR
(75 MHz, DMSO-d6): 36 (CH2), 118.8 (C1, t, J (19F–13C) = 20 Hz),
4.1.7.8.
2,3,5,6-Tetrafluoro-4-{[2-(4-hydroxyphenyl)ethyl]-
amino}benzenesulfonamide (3n). Recrystallization was accom-
plished from EtOH:H2O (1:2). Yield: 0.25 g, 68%, decomp. at
123.3 (C4, t,
J
(
19F–13C) = 16 Hz), 143.1 (C2, C6, dd, 1J
100 °C. IR m
cmꢂ1: 3397 (NH2). 1H NMR (300 MHz, DMSO-d6):
(
(
19F–13C) = 254 Hz, 2J
(
19F–13C) = 16 Hz), 147 (C3, C5, d,
J
2.74 (2H, t, J = 7.8 Hz, CH2), 3.45–3.6 (2H, m, CH2), 6.7 (2H, d,
J = 8.4 Hz, ArH), 7.01 (2H, d, J = 8.4 Hz, ArH), 7.96 (2H, s, SO2NH2),
9.23 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6): 36.5 (CH2), 46.8
(CH2), 108.4 (C1, t, J (19F–13C) = 16 Hz), 115.9 (Ar), 129.4 (Ar),
130.3 (Ar), 131.9 (C4, t, J (19F–13C) = 16 Hz), 136.5 (C2, C6, d, J
19F–13C) = 249 Hz), 170.3 (COOH). 19F NMR (282 MHz, DMSO-
d6): ꢂ133.6 (2F, dd, 1J = 25 Hz, 2J = 10 Hz), ꢂ139.8 (2F, dd,
1J = 25 Hz, 2J = 12 Hz). HRMS for C8H5F4NO4S2 [(MꢂH)ꢂ]: calcd
317.9523, found 317.9525.
(
19F–13C) = 240 Hz), 144.4 (C3, C5, d, J (19F–13C) = 240 Hz), 156.5
4.1.7.4. 3-{[4-(Aminosulfonyl)-2,3,5,6-tetrafluorophenyl]thio}-
propanoic acid (3g). The product was purified by chromatogra-
phy on a column of silica gel (0.04–0.063 mm) with ethyl acetate.
(Ar). 19F NMR (282 MHz, DMSO-d6): ꢂ142.6 (2F, d, J = 19 Hz),
ꢂ161.4 (2F, d, J = 18 Hz). HRMS for C14H12F4N2O3S [(MꢂH)ꢂ]: calcd
363.0432, found 363.0435.
Yield: 0.2 g, 59%, mp 168–169 °C. IR
m
cmꢂ1: 3372, 3272 (NH2),
2928 (OH), 1702 (CO). 1H NMR (300 MHz, DMSO-d6): 2.59 (2H, t,
J = 6.9 Hz, CH2), 3.21 (2H, t, J = 6.6 Hz, CH2), 8.42 (2H, s, SO2NH2),
12.4 (1H, br s, COOH). 13C NMR (75 MHz, DMSO-d6): 30.1 (CH2),
35.4 (CH2), 118.8 (C1, t, J (19F–13C) = 20 Hz), 123.4 (C4, t, J
4.1.7.9. 2,3,5,6-Tetrafluoro-4-[(2-phenylethyl)thio]benzenesul-
fonamide (3o). Recrystallization was accomplished from EtOH:-
H2O (2:1). Yield: 0.24 g, 71%, mp 121 °C. IR
m
cmꢂ1: 3404, 3290
(NH2). 1H NMR (300 MHz, DMSO-d6): 2.91 (2H, t, J = 7.2 Hz,
SCH2CH2), 3.37 (2H, t, J = 7.2 Hz, SCH2CH2), 7.1–7.4 (5H, m, ArH),
8.41 (2H, s, SO2NH2). 13C NMR (75 MHz, DMSO-d6): 35.4
(
(
(
19F–13C) = 16 Hz), 143.2 (C2, C6, dd, 1J (19F–13C) = 254 Hz, 2J
19F–13C) = 17 Hz), 147.3 (C3, C5, dd, 1J (19F–13C) = 241 Hz, 2J
19F–13C) = 19 Hz), 173.1 (COOH). 19F NMR (282 MHz, DMSO-d6):
(SCH2CH2), 36.6 (SCH2CH2), 119.1 (C1, t,
122.9 (C4, t, J (19F–13C) = 16 Hz), 127.2 (Ar), 128.9 (Ar), 129.3
(Ar), 139.7 (Ar), 143.2 (C2, C6, dd, 1J 19F–13C) = 257 Hz, 2J
J (
19F–13C) = 20 Hz),
ꢂ133.3 (2F, dd, 1J = 25 Hz, 2J = 10 Hz), ꢂ139.6 (2F, dd, 1J = 25 Hz,
2J = 10 Hz). HRMS for C9H7F4NO4S2 [(MꢂH)ꢂ]: calcd 331.968, found
331.9683.
(
(
(
19F–13C) = 17 Hz), 147 (C3, C5, dd, 1J 19F–13C) = 249 Hz, 2J
(
19F–13C) = 17 Hz). 19F NMR (282 MHz, DMSO-d6): ꢂ133.5 (2F, dd,
4.1.7.5.
6-{[4-(Aminosulfonyl)-2,3,5,6-tetrafluorophenyl]-
1J = 26.5 Hz, 2J = 13.5 Hz), ꢂ139.8 (2F, dd, 1J = 25.6 Hz,
2J = 10.7 Hz). HRMS for C14H11F4NO2S2 [(MꢂH)ꢂ]: calcd 364.0095,
found 364.0100.
amino}hexanoic acid (3h). The product was purified by chroma-
tography on a column of silica gel (0.04–0.063 mm) with ethyl ace-
tate. Yield: 0.15 g, 42%, mp 142–144 °C. IR
m
cmꢂ1: 3422, 3380,
3286 (NH, NH2), 2945 (OH), 1702 (CO). 1H NMR (300 MHz,
DMSO-d6): 1.2–1.6 (8H, m, (CH2)4), 2.22 (2H, t, J = 7.2 Hz, CH2),
6.7 (1H, s, NH), 7.93 (2H, s, SO2NH2), 12.01 (1H, s, COOH). 13C
NMR (75 MHz, DMSO-d6): 24.9 (CH2), 26.3 (CH2), 30.7 (CH2), 34.3
(CH2), 44.7 (CH2), 108.2 (C1, t, J (19F–13C) = 15 Hz), 132 (C4, t, J
4.1.7.10. 2,3,5,6-Tetrafluoro-4-[(mesitylmethyl)thio]benzene-
sulfonamide (3r). Recrystallization was accomplished from
EtOH. Yield: 0.29 g, 73%, mp 213–214 °C. IR
m
cmꢂ1: 3342, 3264
(NH2). 1H NMR (300 MHz, DMSO-d6): 2.23 (3H, s, CH3), 2.36 (6H,
s, 2CH3), 4.31 (2H, s, CH2), 6.89 (2H, s, ArH), 8.47 (2H, s, SO2NH2).
13C NMR (75 MHz, DMSO-d6): 19.6 (CH3), 21.3 (CH3), 34.3 (CH2),
119.2 (C1, t, J (19F–13C) = 21 Hz), 123.6 (C4, t, J (19F–13C) = 18 Hz),
129.4 (Ar), 129.7 (Ar), 137.86 (Ar), 137.93 (Ar), 143.3 (C2, C6, d, J
(
19F–13C) = 16 Hz), 136.4 (C2, C6, d, J (19F–13C) = 238 Hz), 144.5
(C3, C5, d,
(282 MHz, DMSO-d6): ꢂ142.7 (2F, d, J = 16.6 Hz), ꢂ161.7 (2F, d,
J (
19F–13C) = 244 Hz), 175.1 (COOH). 19F NMR

V. Dudutiene˙ et al. / Bioorg. Med. Chem. 21 (2013) 2093–2106
2103
(
19F–13C) = 253 Hz), 147.4 (C3, C5, d, J (19F–13C) = 243 Hz). 19F NMR
(75 MHz, DMSO-d6): 116.4 (Ar), 120.4 (C1, t, J (19F–13C) = 14 Hz),
125 (Ar), 130.9 (Ar), 136.3 (C4, t, J (19F–13C) = 14 Hz), 141.9 (C3,
C5, d, J (19F–13C) = 250 Hz), 144.4 (C2, C6, d, J (19F–13C) = 256 Hz),
157 (Ar). 19F NMR (282 MHz, DMSO-d6): ꢂ139.6 (2F, d, J = 16 Hz),
ꢂ154 (2F, d, J = 16 Hz). HRMS for C12H7F4NO3S [(MꢂH)ꢂ]: calcd
320.001, found 320.0008.
(282 MHz, DMSO-d6): ꢂ133.1 (2F, dd, 1J = 25 Hz, 2J = 10 Hz),
ꢂ139.5 (2F, dd, 1J = 25 Hz, 2J = 11 Hz). HRMS for C16H15F4NO2S2
[(MꢂH)ꢂ]: calcd 392.0408, found 392.0412.
4.1.7.11. 3-{[4-(Aminosulfonyl)-2,3,5,6-tetrafluorophenyl]thio}-
[1,2,3]thiadiazolo[3,4-a]benzimidazole (3v). The obtained com-
pound was washed with MeOH. Yield: 0.13 g, 30%, decomp. at 233–
4.1.11. 2,3,5,6-Tetrafluoro-4-[(2-phenylethyl)sulfonyl]-
benzenesulfonamide (3p)
234 °C. IR
m
cmꢂ1: 3356 (NH2). 1H NMR (300 MHz, DMSO-d6): 7.33
(1H, t, J = 7.8 Hz, ArH), 7.57 (1H, t, J = 8.4 Hz, ArH), 7.81 (1H, d,
J = 8.7 Hz, ArH), 8.2 (1H, d, J = 8.1 Hz, ArH), 8.52 (2H, br s, SO2NH2).
The mixture of 2,3,5,6-tetrafluoro-4-[(2-phenylethyl)thio]ben-
zenesulfonamide (3o) (0.1 g, 0.27 mmol), CrO3 (0.082 g,
0.82 mmol), CH3COOH (10 mL), H2O (0.2 mL) was heated at 60 °C
for 4 h. The resultant precipitate was filtered, washed with H2O.
13C NMR (75 MHz, DMSO-d6): 118.4 (Ar), 120.2 (C1, t,
J
J
(
(
19F–13C) = 15 Hz), 125.9 (Ar), 126 (Ar), 127.7 (C4, t,
19F–13C) = 16 Hz), 130 (Ar), 133.2 (Ar), 133.4 (Ar), 148 (C2, C6, d,
Yield: 0.07 g, 64%, mp 248–249 °C. IR m
cmꢂ1: 3356, 3251 (NH2).
J (19F–13C) = 254 Hz), 152 (C3, C5, d, J (19F–13C) = 236 Hz), 158.7
(Ar), 158.8 (Ar). 19F NMR (282 MHz, DMSO-d6): ꢂ127.2 (2F, dd,
1J = 25.9 Hz, 2J = 11.3 Hz), ꢂ134 (2F, dd, 1J = 25.9 Hz, 2J = 10.7 Hz).
1H NMR (300 MHz, DMSO-d6): 3.12 (2H, t, J = 7.2 Hz, SO2CH2CH2),
3.97 (2H, t, J = 7.8 Hz, SO2CH2CH2), 7.1–7.4 (5H, m, ArH), 8.66
(2H, s, SO2NH2). 13C NMR (75 MHz, DMSO-d6): 28.6 (SO2CH2CH2),
58 (SO2CH2CH2), 121.5 (C1 or C4, t, J (19F–13C) = 15 Hz), 127.5
(Ar), 128.2 (C1 or C4, t, J (19F–13C) = 16 Hz), 128.9 (Ar), 129.2 (Ar),
137.4 (Ar), 143.5 (C2, C6 or C3, C5, dd, 1J (19F–13C) = 258 Hz, 2J
HRMS for
434.9667.
C
14H6F4N4O2S3 [(M+H)+]: calcd 434.9662, found
4.1.8. 2,3,5,6-Tetrafluoro-4-(phenylthio)benzenesulfonamide
(3i)
(
(
19F–13C) = 17 Hz), 144.8 (C2, C6 or C3, C5, dd, 1J
19F–13C) = 255 Hz, 2J
(
19F–13C) = 17 Hz). 19F NMR (282 MHz,
The mixture of pentafluorobenzenesulfonamide (compound 2)
(0.28 g, 1.13 mmol), MeOH (10 mL), Et3N (0.158 mL, 1.13 mmol)
and HSPh (0.116 mL, 1.13 mmol) was stirred at ambient tempera-
ture for 2 h. MeOH was evaporated in vacuum and the resultant
precipitate was washed with H2O. Recrystallization was accom-
DMSO-d6): ꢂ136 (2F, dd, 1J = 25 Hz, 2J = 12 Hz), ꢂ137.1 (2F, dd,
1J = 25 Hz, 2J = 12 Hz). HRMS for C14H11F4NO4S2 [(MꢂH)ꢂ]: calcd
395.9993, found 395.9996.
4.1.12. 2,3,5,6-Tetrafluoro-4-morpholin-4-ylbenzenesulfo-
namide (3q)
plished from EtOH:H2O (2:1). Yield: 0.28 g, 74%, mp 139 °C. IR
m
cmꢂ1: 3400, 3288 (NH2). 1H NMR (300 MHz, DMSO-d6): 7.2–7.8
(5H, m, ArH), 8.47 (2H, s, SO2NH2). 13C NMR (75 MHz, DMSO-d6):
117.7 (C1, t, J (19F–13C) = 20 Hz), 124.5 (C4, t, J (19F–13C) = 16 Hz),
128.7 (Ar), 130.3 (Ar), 130.4 (Ar), 132.4 (Ar), 143.5 (C2, C6, ddd,
The mixture of pentafluorobenzenesulfonamide (compound 2)
(0.2 g, 0.809 mmol), MeOH (10 mL), morpholine (0.141 mL,
1.62 mmol) was refluxed for 8 h. MeOH was evaporated in vacuum
and the resultant precipitate was washed with H2O. Recrystalliza-
tion was accomplished from EtOH:H2O (1:1). Yield: 0.13 g, 52%, mp
233ꢂ234 °C close to the values in the literature, mp 226–227 °C.44
1H NMR (300 MHz, DMSO-d6): 3.32 (4H, br s, 2CH2), 3.7 (4H, br s,
2CH2), 8.2 (2H, s, SO2NH2). 13C NMR (75 MHz, DMSO-d6): 51.2
(N(CH2)2, t, J (19F–13C) = 3.6 Hz), 67.2 (O(CH2)2), 115.4 (C1, t, J
1J
( ( (
19F–13C) = 255 Hz, 2J 19F–13C) = 17 Hz, 3J 19F–13C) = 5 Hz),
147.2 (C3, C5, ddd, 1J (19F–13C) = 240 Hz, 2J (19F–13C) = 17 Hz, 3J
(
19F–13C) = 5 Hz). 19F NMR (282 MHz, DMSO-d6): ꢂ132.6 (2F, dd,
1J = 24.5 Hz, 2J = 11.6 Hz), ꢂ138.8 (2F, dd, 1J = 24.5 Hz,
2J = 10.4 Hz). HRMS for C12H7F4NO2S2 [(MꢂH)ꢂ]: calcd 335.9782,
found 335.9780.
(
19F–13C) = 19 Hz), 133.3 (C4, t, J (19F–13C) = 16 Hz), 141.7 (C3, C5,
d, J (19F–13C) = 244 Hz), 144.3 (C2, C6, d, J (19F–13C) = 256 Hz). 19F
NMR (282 MHz, DMSO-d6): ꢂ141.6 (2F, d, J = 15.8 Hz), ꢂ151.2
(2F, d, J = 16.6 Hz).
4.1.9. 2,3,5,6-Tetrafluoro-4-(phenylsulfonyl)-
benzenesulfonamide (3j)
The mixture of 2,3,5,6-tetrafluoro-4-(phenylthio)benzenesul-
fonamide (compound 3i) (0.2 g, 0.59 mmol), CrO3 (0.18 g,
1.8 mmol), CH3COOH (10 mL), H2O (0.5 mL) was heated at 70 °C
for 2 h. The solvent was then removed in vacuum and the resultant
precipitate was washed with H2O. Recrystallization was accom-
4.1.13. General procedure for the syntheses of 3s, 3t, 3w
The mixture of pentafluorobenzenesulfonamide (compound 2)
(0.1 g, 0.404 mmol), K2CO3 (0.056 g, 0.406 mmol), DMSO (2 mL)
and appropriate nucleophile (0.404 mmol) was stirred at ambient
temperature for 5 h. The mixture was then diluted with H2O
(20 mL) and extracted with EtOAc (2 ꢁ 10 mL). The combined or-
ganic phase was dried over Na2SO4 and evaporated in vacuum.
plished from EtOH. Yield: 0.17 g, 77%, mp 266–267 °C. IR
m :
cmꢂ1
3389, 3284 (NH2). 1H NMR (300 MHz, DMSO-d6): 7.74 (2H, t,
J = 7.8 Hz, ArH), 7.87 (1H, t, J = 7.5 Hz, ArH), 8.09 (2H, d,
J = 7.8 Hz, ArH), 8.59 (2H, s, SO2NH2). 13C NMR (75 MHz, DMSO-
d6): 123 (C1, C4, t, J (19F–13C) = 13 Hz), 128.4 (Ar), 130.7 (Ar),
136.2 (Ar), 140.4 (Ar), 143.8 (C2, C6 or C3, C5, dd, 1J
4.1.13.1. 4-[(4,6-Dimethylpyrimidin-2-yl)thio]-2,3,5,6-tetraflu-
orobenzenesulfonamide (3s). Recrystallization was accom-
plished from EtOH:H2O (2:1). Yield: 0.12 g, 80%, mp 131–132 °C.
(
19F–13C) = 258 Hz, 2J (19F–13C) = 18 Hz), 144.4 (C2, C6 or C3, C5,
dd, 1J (19F–13C) = 258 Hz, 2J (19F–13C) = 18 Hz). 19F NMR (282 MHz,
DMSO-d6): ꢂ136.6 (4F, s). HRMS for C12H7F4NO4S2 [(MꢂH)ꢂ]: calcd
367.968, found 367.9684.
IR m
cmꢂ1: 3317 (NH2). 1H NMR (300 MHz, DMSO-d6): 2.34 (6H,
s, 2CH3), 7.15 (1H, s, ArH), 8.58 (2H, s, SO2NH2). 13C NMR
(75 MHz, DMSO-d6): 24 (CH3), 114.1 (C1, t, J (19F–13C) = 20 Hz),
118.5 (Ar), 125.5 (C4, t, J (19F–13C) = 14 Hz), 143.3 (C2, C6, d, J
4.1.10. 2,3,5,6-Tetrafluoro-4-phenoxybenzenesulfonamide (3k)
The mixture of pentafluorobenzenesulfonamide (compound 2)
(0.2 g, 0.81 mmol), sodium phenoxide trihydrate (0.145 g,
0.85 mmol) and DMSO (1 mL) was stirred at ambient temperature
for 4 h. The mixture was then diluted with H2O (20 mL) and the
resultant precipitate was filtered, washed with H2O. Recrystalliza-
tion was accomplished from EtOH:H2O (1:2). Yield: 0.07 g, 27%, mp
(
19F–13C) = 255 Hz), 147.7 (C3, C5, d, J (19F–13C) = 248 Hz), 166.3
(Ar), 169 (Ar). 19F NMR (282 MHz, DMSO-d6): ꢂ130.7 (2F, dd,
1J = 25.4 Hz, 2J = 10.7 Hz), ꢂ139.3 (2F, dd, 1J = 26.2 Hz, 2J = 11 Hz).
HRMS for
368.0142.
C
12H9F4N3O2S2 [(M+H)+]: calcd 368.0145, found
4.1.13.2. 4-(1,3-Benzothiazol-2-ylthio)-2,3,5,6-tetrafluoroben-
164–165 °C. IR
m
cmꢂ1: 3363, 3277 (NH2). 1H NMR (300 MHz,
zenesulfonamide (3t). Recrystallization was accomplished from
DMSO-d6): 7.0–7.6 (5H, m, ArH), 8.44 (2H, s, SO2NH2). 13C NMR
EtOH:H2O (2:1). Yield: 0.11 g, 69%, mp 171 °C. IR
m
cmꢂ1: 3426,

2104
V. Dudutiene˙ et al. / Bioorg. Med. Chem. 21 (2013) 2093–2106
3299 (NH2). 1H NMR (300 MHz, DMSO-d6): 7.44 (1H, t, J = 8.1 Hz,
ArH), 7.51 (1H, t, J = 8.1 Hz, ArH), 7.9 (1H, d, J = 7.5 Hz, ArH), 8.06
(1H, d, J = 8.4 Hz, ArH), 8.65 (2H, s, SO2NH2). 13C NMR (75 MHz,
DMSO-d6): 113.8 (C1, t, J (19F–13C) = 20 Hz), 122.7 (Ar), 122.9
(Ar), 126.2 (Ar), 126.5 (C4, t, J (19F–13C) = 16 Hz), 127.5 (Ar), 136
(Ar), 143.5 (C2, C6, dd, 1J (19F–13C) = 254 Hz, 2J (19F–13C) = 12 Hz),
147.6 (C3, C5, dd, 1J (19F–13C) = 250 Hz, 2J (19F–13C) = 16 Hz),
153.1 (Ar), 162.4 (Ar). 19F NMR (282 MHz, DMSO-d6): ꢂ130.5 (2F,
dd, 1J = 24.8 Hz, 2J = 11.6 Hz), ꢂ137.9 (2F, dd, 1J = 24.8 Hz,
2J = 11.6 Hz). HRMS for C13H6F4N2O2S3 [(MꢂH)ꢂ]: calcd 392.9455,
found 392.9457.
heated at 95 °C for 1 h. After removal of the DMF under vacuum,
MeOH (0.5 mL) and 10% aqueous NaOH solution (0.5 mL) were
added to the residue. This solution was refluxed for 1 h. MeOH
was evaporated in vacuum and the resultant precipitate was di-
luted with H2O. The obtained solution was washed with petroleum
ether and acidified with 10% HCl.
4.1.16.1. 4-[(2-Hydroxyethyl)thio]benzenesulfonamide (6a)
The mixture was extracted with EtOAc (3 ꢁ 10 mL). The com-
bined organic phase was dried over Na2SO4 and evaporated in vac-
uum. The product was purified by chromatography on a column of
silica gel (0.04–0.063 mm) with EtOAc, Rf = 0.63. Yield: 0.04 g, 50%,
mp 109–110 °C close to values in the literature mp 111–112 °C.25
1H NMR (300 MHz, DMSO-d6): 3.15 (2H, t, J = 7 Hz, CH2), 3.62
(2H, br t, CH2), 5.04 (1H, br s, OH), 7.34 (2H, s, SO2NH2), 7.48
(2H, d, J = 8.7 Hz, ArH), 7.73 (2H, d, J = 8.7 Hz, ArH).
4.1.13.3. 4-[(4,5-Diphenyl-1H-imidazol-2-yl)thio]-2,3,5,6-tetra-
fluorobenzenesulfonamide (3w). Recrystallization was accom-
plished from EtOH:H2O (2:1). Yield: 0.12 g, 63%, mp 221–222 °C.
IR
m
cmꢂ1: 3292 (NH2). 1H NMR (300 MHz, DMSO-d6): 7.2–7.5
(10H, m, ArH), 8.51 (2H, s, SO2NH2), 13.2 (1H, br s, NH). 13C NMR
(75 MHz, DMSO-d6): 116.6 (C1, t, J (19F–13C) = 15 Hz), 124.1 (C4,
t, J (19F–13C) = 16 Hz), 128.3 (br s, Ar), 129.3 (br s, Ar), 133.9 (Ar),
4.1.16.2. 4-Propylthiobenzenesulfonamide (6b)
The resultant precipitate was filtered and washed with H2O.
Recrystallization was accomplished from toluene. Yield: 0.05 g,
143.3 (C2, C6, d,
J ( J
19F–13C) = 253 Hz), 146.7 (C3, C5, d,
(
19F–13C) = 250 Hz). 13C NMR (75 MHz, DMSO-d6, CF3COOH):
63%, mp 124–125 °C. IR m
cmꢂ1: 3349, 3251 (NH2). 1H NMR
115.1 (C1, t, J (19F–13C) = 15 Hz), 125 (C4, t, J (19F–13C) = 16 Hz),
128.7 (Ar), 129.3 (Ar), 129.5 (Ar), 130.2 (Ar), 133.4 (Ar), 134.2
(Ar), 143.5 (C2, C6, d, J (19F–13C) = 255 Hz), 146.9 (C3, C5, d, J
(300 MHz, DMSO-d6): 1.01 (3H, t, J = 7.5 Hz, CH3), 1.65 (2H, sext,
J = 7.2 Hz, CH2), 3.05 (2H, t, J = 7.2 Hz, CH2), 7.34 (2H, s, SO2NH2),
7.47 (2H, d, J = 8.4 Hz, ArH), 7.74 (2H, d, J = 8.4 Hz, ArH). 13C NMR
(75 MHz, DMSO-d6): 13.9 (CH3), 22.4 (CH2), 33.6 (CH2), 126.9
(Ar), 127.3 (Ar), 141.2 (Ar), 142.8 (Ar). HRMS for C9H13NO2S2
[(MꢂH)ꢂ]: calcd 230.0315, found 230.0316.
(
19F–13C) = 255 Hz). 19F NMR (282 MHz, DMSO-d6): ꢂ133.5 (2F,
dd, 1J = 24.8 Hz, 2J = 10.2 Hz), ꢂ139.1 (2F, dd, 1J = 24.8 Hz,
2J = 10.2 Hz). HRMS for C21H13F4N3O2S2 [(M+H)+]: calcd 480.0458,
found 480.0449.
4.1.16.3. 4-[(2-Phenylethyl)thio]benzenesulfonamide (6c)
The resultant precipitate was filtered and washed with H2O.
Recrystallization was accomplished from toluene. Yield: 0.07 g,
4.1.14. 4-(1-Adamantylamino)-2,3,5,6-tetrafluorobenzenesulfo-
namide (3u)
The mixture of pentafluorobenzenesulfonamide (compound 2)
(0.2 g, 0.81 mmol), Et3N (0.226 mL, 1.62 mmol), adamantanamine
hydrochloride (0.15 g, 0.81 mmol) and DMSO (2 mL) was stirred
at ambient temperature for 48 h. The mixture was then diluted
with H2O (20 mL) and extracted with EtOAc (2 ꢁ 10 mL). The com-
bined organic phase was dried over Na2SO4 and evaporated in vac-
uum. The product was purified by chromatography on a column of
silica gel (0.04–0.063 mm) with EtOAc:CHCl3 (1:4), Rf = 0.63. Yield:
70%, mp 120–121 °C. IR m
cmꢂ1: 3302, 3239 (NH2). 1H NMR
(300 MHz, DMSO-d6): 2.93 (2H, t, J = 7.2 Hz, CH2), 3.35 (2H, t,
J = 7.2 Hz, CH2), 7.1-7.4 (7H, m, ArH, SO2NH2), 7.51 (2H, d,
J = 8.4 Hz, ArH), 7.76 (2H, d, J = 8.4 Hz, ArH). 13C NMR (75 MHz,
DMSO-d6): 33.1 (CH2), 35 (CH2), 127 (Ar), 127.1 (Ar), 127.3 (Ar),
129.1 (Ar), 129.3 (Ar), 140.5 (Ar), 141.3 (Ar), 142.5 (Ar). HRMS for
C
14H15NO2S2 [(MꢂH)ꢂ]: calcd 292.0471, found 292.0475.
0.04 g, 12%, mp 122–123 °C. IR
m
cmꢂ1: 3394, 3357, 3255 (NH,
4.2. Protein preparation
NH2). 1H NMR (300 MHz, CDCl3): 1.6–1.8 (6H, m, adamantane),
1.8–2 (6H, m, adamantane), 2.1–2.25 (3H, m, adamantane), 4.05
(1H, s, NH), 5.59 (2H, s, SO2NH2). 13C NMR (75 MHz, CDCl3): 30.1
(adamantane), 36.1 (adamantane), 43.6 (adamantane), 54.8 (ada-
mantane), 110.2 (C1), 130.4 (C4, t, J (19F–13C) = 14 Hz), 138.2 (C2,
C6, d, J (19F–13C) = 244 Hz), 144.5 (C3, C5, d, J (19F–13C) = 251 Hz).
19F NMR (282 MHz, CDCl3): ꢂ141.5 (2F, d, J = 16 Hz), ꢂ152.4 (2F,
Expression and purification of CA I, II, VII, XII and XIII was per-
formed as previously described: CA I in48 CA II in,49 CA VII and XIII
in,50 and CA XII in.51
4.3. Determination of compound binding and inhibition of CAs
d, J = 17.5 Hz). HRMS for
C
16H18F4N2O2S [(MꢂH)ꢂ]: calcd
4.3.1. Thermal shift assay
377.0952, found 377.0952.
Thermal shift assay experiments were performed in a Corbett
Rotor-Gene 6000 (QIAGEN Rotor-Gene Q) RT-PCR instrument using
the blue channel (excitation 365 20, detection 460 15 nm).
4.1.15. 4-Bromo-N-[(dimethylamino)methylene]benzenesulfo-
namide (5)
Sample volume was 20
ll containing 5–10 lM protein, 0–200 lM
The compound 5 was synthesized according to the literature.25
A solution of dimethyl N,N-dimethylformamide dimetyl acetal
(1.65 g, 14 mmol) in CH3CN (5 mL) was added dropwise to a sus-
pension of 4 (2.83 g, 12 mmol) in CH3CN (15 mL). The mixture
was stirred for 1 h and the solvent was removed in vacuum. The
resultant precipitate was washed with H2O. Yield: 3.44 g, 99%,
mp 139–140 °C close to values in the literature mp 141–143 °C.25
ligand, 50 M solvatochromic dye ANS (8-anilino-1-naphthalene
l
sulfonate), and 50 mM sodium phosphate buffer containing
100 mM NaCl at pH 7.0, with the final DMSO concentration at
2%. The samples were heated at a constant rate of 1 °C/min. Data
analysis was performed as previously described.48
4.3.2. Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) experiments were per-
formed using ITC200 or VP-ITC instruments (Microcal, Inc., North-
4.1.16. General procedure for the syntheses of 6a–c
The compounds 6a–c were synthesized according to the litera-
ture.25 A solution of appropriate thiol (0.68 mmol) was added
dropwise to a stirred mixture of NaH (0.033 g, 50% oil dispersion,
0.69 mmol) in anhydrous DMF (1 mL). When gas evolution was
complete, 5 (0.1 g, 0.34 mmol) was added and the mixture was
ampton, USA) with 5–20
200 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, respectively for the two calorimeters. Experiments were
performed at 37 °C in a 50 mM sodium phosphate buffer containing
lM protein solution in the cell and 50–
l
l

V. Dudutiene˙ et al. / Bioorg. Med. Chem. 21 (2013) 2093–2106
2105
ꢀ
ˇ
100 mM NaCl at pH 7.0, with a final DMSO concentration of 2%,
equal in the syringe and the cell.
the stop-flow hydration assay. The authors thank Zita Liutkeviciute
˙
for help with the mass spectrometry analysis of the synthetic com-
pounds. Crystallographic measurements were performed at the
EMBL DESY beamlines in Hamburg. The authors thank local con-
tacts, Dr. Gleb Bourenkov and Dr. Michele Cianci, for the help with
beamline operation. SG and EM travels to Hamburg were sup-
ported in part by the European Community’s Research Infrastruc-
ture Action under the Sixth Framework Programme ‘Structuring
the European Research Area Specific Programme’ (contract Num-
ber RII3-CT-2004-506008) and by the MoBiLi project. AS acknowl-
edges support by the project ‘Promotion of Student Scientific
Activities’ (VP1-3.1-ŠMM-01-V-02-003) from the Research Council
of Lithuania.
4.3.3. CO2 hydration assay
The carbon dioxide hydration activity of the recombinant hu-
man CA II was measured using Applied Photophysics SX.18MV-R
stop-flow spectrometer according to.9,52 Reaction velocities were
measured by recording the absorbance of bromothymol blue dye
(40 lM). The reaction buffer contained 10 mM NaCl, 10 mM Hepes,
pH 7.4. Saturated CO2 solution was made by the bubbling of gas in
milli-Q water at 25 °C for 1 h. CA II was incubated with inhibitors
for 15 min at room temperature. CA II concentration was 20 nM
and the final DMSO concentration was less than 0.04%. IC50 value
was determined by fitting the sigmoidal curve of the data points
and then Ki was calculated using the Cheng–Prusoff equation
assuming that the KM = 9.3 mM and half-saturated [CO2] = 17 mM,
according to.9
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.008.
4.4. Crystallography
References and notes
4.4.1. Crystallization
Human carbonic anhydrases were concentrated to 20–60 mg/
ml by ultrafiltration in 20 mM Na-Hepes, pH 7.5, containing
50 mM NaCl. Crystallization by sitting drop method was started
by mixing equal volumes of protein solution with reservoir buffer.
Reservoir buffer for CA II contained 0.1 M of Na-Bicine, pH 9.0,
0.2 M ammonium sulfate, and 2.0 M Na-malonate, pH 7.5. Reser-
voir buffer for CA XII contained 0.1 M ammonium citrate, pH 5.0
and 16% PEG4000. Reservoir buffer for CA XIII contained 0.1 M so-
dium citrate, pH 5.5, 0.1 M sodium acetate pH 4.5 and 26%
PEG4000. Crystals were soaked with 1 mM of inhibitor in reservoir
buffer for several days. For data collection, the crystals of CA XII
and CA XIII were transferred in cryo protecting buffer made of
the reservoir mixed with 15–20% ethyleneglycol and flash-cooled
after a few minutes.
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Acknowledgements
This research was funded by a grant (No. MIP-067/2011) from
the Research Council of Lithuania. The authors also acknowledge
FP7-REGPOT-2009-1 grant ‘MoBiLi’, agreement No. 245721, the
COST projects TD0905 and CM0804, and Professor Claudiu Supuran
and his group for teaching the measurement of CA inhibition by

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