A class of sulfonamides with strong inhibitory action against the α-carbonic anhydrase from trypanosoma cruzi

Article abstract of DOI:10.1021/jm400418p

Trypanosoma cruzi, the causative agent of Chagas disease, encodes for an α-carbonic anhydrase (CA, EC 4.2.1.1) possessing high catalytic activity (TcCA) which was recently characterized (Pan et al. J. Med. Chem. 2013, 56, 1761-1771). A new class of sulfonamides possessing low nanomolar/subnanomolar TcCA inhibitory activity is described here. Aromatic/heterocyclic sulfonamides incorporating halogeno/methoxyphenacetamido tails inhibited TcCA with K _Is in the range of 0.5-12.5 nM, being less effective against the human off-target isoforms hCA I and II. A homology model of TcCA helped us to rationalize the excellent inhibition profile of these compounds against the protozoan enzyme, a putative new antitrypanosoma drug target. These compounds were ineffective antitrypanosomal agents in vivo due to penetrability problems of these highly polar molecules that possess sulfonamide moieties.

Full text of DOI:10.1021/jm400418p

Article
pubs.acs.org/jmc
A Class of Sulfonamides with Strong Inhibitory Action against the
α‑Carbonic Anhydrase from Trypanosoma cruzi
†
,‡
§
∥
§
̈
Ozlen Guzel-Akdemir, Atilla Akdemir,* Peiwen Pan, Alane B. Vermelho, Seppo Parkkila,
̈
Andrea Scozzafava,⊥ Clemente Capasso,# and Claudiu T. Supuran*,⊥,▽
†Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul University, 34116, Beyazit, Istanbul, Turkey
‡Department of Pharmacology, Faculty of Pharmacy, Bezmialem Vakif University, Vatan Cadd., Fatih, Istanbul, Turkey
§Institute of Biomedical Technology, Fimlab Ltd., School of Medicine and BioMediTech, University of Tampere and Tampere
University Hospital, Medisiinarinkatu 3, 33520 Tampere, Finland
∥Laborator
́
io Proteases de Microrganismos, Instituto de Microbiologia Paulo de Goe
́
s, Universidade Federal do Rio de Janeiro −
UFRJ, CCS; Bloco I, Sala 32, Cidade Universitar
́
ia, Av. Carlos Chagas Filho, 373, Ilha do Fundao; Rio de Janeiro R.J., Brazil
̃
⊥Laboratorio di Chimica Bioinorganica, Polo Scientifico, Universita
̀
degli Studi di Firenze, Room 188, Via della Lastruccia 3, 50019
Sesto Fiorentino (Florence), Italy
#Istituto di Biochimica delle Proteine − CNR, Via P. Castellino 111, 80131 Napoli, Italy
▽Sezione di Scienze Farmaceutiche, NEUROFARBA Department, Universita
(Florence), Italy
̀
degli Studi di Firenze, 50019 Sesto Fiorentino
ABSTRACT: Trypanosoma cruzi, the causative agent of Chagas disease, encodes for an α-
carbonic anhydrase (CA, EC 4.2.1.1) possessing high catalytic activity (TcCA) which was
recently characterized (Pan et al. J. Med. Chem. 2013, 56, 1761−1771). A new class of
sulfonamides possessing low nanomolar/subnanomolar TcCA inhibitory activity is described
here. Aromatic/heterocyclic sulfonamides incorporating halogeno/methoxyphenacetamido tails
inhibited TcCA with KIs in the range of 0.5−12.5 nM, being less effective against the human off-
target isoforms hCA I and II. A homology model of TcCA helped us to rationalize the excellent
inhibition profile of these compounds against the protozoan enzyme, a putative new
antitrypanosoma drug target. These compounds were ineffective antitrypanosomal agents in
vivo due to penetrability problems of these highly polar molecules that possess sulfonamide
moieties.
from this parasite showed effective antimalarial action in vivo.9
We thus investigated whether T. cruzi may also possess such
enzymes encoded in its genome.
INTRODUCTION
■
Chagas disease affects more than 10 million people, mostly in
South and Central Americas, and is spreading also in Europe
and North America.1 It is caused by the protozoan parasite
Trypanosoma cruzi, and only two drugs are clinically used for its
treatment, the nitroazoles, benznidazole and nifurtimox, which
are not always effective in eradicating the chronic infection.2
As the carbonic anhydrases (CAs, EC 4.2.1.1)3−7 are
metalloenzymes involved in a variety of physiologic and
pathologic processes, in many types of organisms, we recently
started to explore them as possible antiinfective drug targets.8
Indeed, representatives of the α- and or β-CA class have been
investigated in pathogenic bacteria such as Brucella spp.,
Mycobacterium tuberculosis, Streptococcus pneumoniae, Salmonella
enterica serovar Typhimurium, Vibrio cholerae, and Helicobacter
pylori, as well as in the pathogenic fungi Candida albicans, C.
glabrata, and Cryptococcus neoformans.8 Only one protozoan
parasite has been investigated up until now for the presence
and drugability of CAs, the malaria-provoking organism
Plasmodium falciparum.9 It has also been observed that some
sulfonamides with potent inhibitory activity against the enzyme
We have recently reported10 that an α-CA with very high
catalytic activity is present in this organism, which was
denominated TcCA, and that this enzyme is susceptible to
inhibition by sulfonamides and thiols,10 two of the main classes
of CA inhibitors (CAIs).3−7 Furthermore, some of the thiol
CAIs also showed antitrypanosomal activity in vivo.10 However,
the sulfonamides investigated previously were not effective in
vitro TcCA inhibitors.10
The main class of CAIs is represented by the primary
sulfonamides incorporating the SO2NH2 moiety which by
coordinating to the metal ion from the enzyme active site
impairs the formation of the zinc hydroxide nucleophile species
of the enzyme, responsible for the hydration of CO2 to
bicarbonate, with a very high efficiency.3,4,11A large number of
such agents, mostly benzenesulfonamide derivatives, have been
Received: March 21, 2013
Published: July 1, 2013
© 2013 American Chemical Society
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Scheme 1. Preparation of the 4-[2-(Substituted phenyl)acetamido]benzenesulfonamides 3a−x and 5-[2-(Substituted
phenyl)acetamido]-1,3,4-thiadiazole-2-sulfonamide Derivatives 5a−d
reported in recent years, as some of them were proven to
possess strong antitumor activity in vivo, in animal models of
the diseases, with effective inhibition of growth of both primary
tumors and metastases,7 whereas other such agents show
antiepileptic, antiobesity, and antiglaucoma activities and are
clinically used.3,4,11
Table 1. Inhibition Constants of Isozymes hCA I and II and
TcCA with Sulfonamides 3a−x and 5a−d for the CO2
Hydration Reaction (stopped-flow assay) at 20 °C14
Here we report the synthesis of a new class of sulfonamide
derivatives obtained by the tail approach12 and their subsequent
testing for CA inhibition. These compounds potently inhibit
TcCA and have a lower activity against the off-target human
isoforms hCA I and II.
a
KI (nM)
selectivity ratios
hCA I/
TcCA
hCA II/
TcCA
no.
X, Y, n
hCA I hCA II
TcCA
3a
3b
3c
3d
3e
3f
4-F, H, 0
102
346
4.8
7.9
8.5
2.7
8.9
8.0
8.3
1.2
1.6
4.1
7.0
7.7
7.8
7.9
9.0
7.1
10.9
7.2
3.7
12.5
9.0
7.4
9.1
11.8
3.3
7.5
7.6
0.95
0.83
0.51
61.6
12.0
128.1
0.5
0.9
38.5
0.1
0.1
1.0
5.8
1.2
0.2
1.4
0.9
0.1
0.1
1.0
0.1
0.1
0.1
2.5
0.1
0.1
0.1
0.4
0.5
0.2
0.1
0.4
0.7
0.8
1.5
0.2
RESULTS AND DISCUSSION
■
4-Cl, H, 0
2-Br, H, 0
4-MeO, H, 0
4-F, F, 0
104
0.96
0.79
8.7
Chemistry. Here we report a new class of sulfonamide
derivatives obtained by the tail approach.12 We have attached
halogeno/methoxyphenacetamido tails to the molecules of
aromatic or heterocyclic sulfonamides such as sulfanilamide, 3-
halogenosulfanilamides, 4-aminomethyl/ethylbenzenesulfona-
mide, or 5-amino-1,3,4-thiadiazole-2-sulfonamide, as illustrated
in Scheme 1. Reaction of the substituted phenacetyl chlorides
1a−d with the amino-containing aromatic sulfonamides 2a−f
afforded the aromatic derivatives 3a−x, whereas the same
reaction involving the heterocyclic derivative 4 afforded the
substituted 1,3,4-thiadiazole-2-sulfonamides 5a−d. We have
chosen various acyl chlorides 1a−d to generate chemical
diversity in this new class of sulfonamides, because we have
observed previously that similar compounds, incorporating
thienylacetamido, phenacetamido, and pyridinylacetamido tails,
do show potent and isoform-selective inhibition profiles against
some mammalian CA isoforms, such as CA VA and VB among
others.13
Inhibition Data of TcCA and Human CA Isoforms I and
II with the New Sulfonamides. The new sulfonamides 3 and
5, as well as acetazolamide (AZ, 5-acetamido-1,3,4-thiadiazole-
2-sulfonamide, a clinically used drug)3,4 have been tested in
enzyme inhibition assays against TcCA (Table 1) using the
stopped-flow, CO2 hydrase Khalifah method.14 As seen from
data of Table 1, AZ was a medium-potency CAI against TcCA
(KI of 61.6 nM),10 whereas the new sulfonamides reported here
acted as highly potent low nanomolar or subnanomolar
inhibitors of the protozoan enzyme, with KIs in the range of
0.51−12.5 nM. The structure−activity relationship (SAR) for
TcCA inhibition with the new sulfonamides reported here is
rather straightforward:
116
104
236
8.6
14.5
12.5
196.7
5.4
4-Cl, F, 0
2-Br, F, 0
4-MeO, F, 0
4-F, Cl, 0
4-Cl, Cl, 0
2-Br, Cl, 0
4-MeO, Cl, 0
4-F, Br, 0
4-Cl, Br, 0
2-Br, Br, 0
4-MeO, Br, 0
4-F, H, 1
6.9
3g
3h
3i
1.9
106
254
9.8
0.78
9.5
25.9
36.3
1.3
3j
7.3
3k
3l
4.1
0.91
0.74
8.9
0.5
8.5
1.1
3m
3n
3o
3p
3q
3r
246
166
6.5
27.3
23.4
0.6
0.94
0.80
0.69
9.3
8.7
1.2
109
109
4.3
29.5
8.7
4-Cl, H, 1
2-Br, H, 1
4-MeO, H, 1
4-F, H, 2
0.81
0.77
0.88
3.8
3s
0.5
3t
4.9
0.7
3u
3v
3w
3x
5a
5b
5c
5d
AZ
101
9.7
11.1
0.8
4-Cl, H, 2
2-Br, H, 2
4-MeO, H, 2
4-F, −, −
4-Cl, −, −
2-Br, −, −
4-MeO, −, −
−
6.4
8.2
0.72
0.81
3.2
2.5
3.7
0.5
223
4.9
29.3
5.2
0.70
0.70
0.76
12
7.6
9.2
2.9
5.7
250
4.1
a
Errors in the range of 5−10% of the reported values (data not
shown).
substituted 1,3,4-thiadiazolesulfonamides 5b−d showed
inhibition constants in the range of 0.51−0.95 nM,
whereas most of the corresponding benzenesulfonamides
incorporating the same tails were around 1 order of
magnitude less efficient as TcCA inhibitors (Table 1)
(i) The 1,3,4-thiadiazole-2-sulfonamides 5a−d were gener-
ally more effective CAIs compared to the benzenesulfo-
namides 3a−x possessing similar scaffolds as tails. For
example the 4-chloro-, 2-bromo-, and 4-methoxyphenyl-
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Docking Studies. To rationalize these results, and because
no X-ray crystal structure is available so far for this new
protozoan enzyme, we used a homology modeling strategy to
understand the binding of sulfonamides 3a−x and 5a−d to
TcCA. The homology model was built based on the Neisseria
gonorrheae NgCA X-ray crystal structure (PDB: 1KOP),16
which shows 22.9% sequence identity to the TcCA. The three
zinc-binding histidines and the two threonines near the zinc
atom are conserved among NgCA and TcCA (His92, His94,
His111, Thr177, and Thr178; NgCA numbering). In Figure 1,
(ii) All tails present in these compounds (irrespective of
whether the halogenophenacetamido possessed a fluo-
rine, chlorine, or bromine in the 2- or 4-positions, or
whether it was a 4-methoxyphenacetamido moiety) were
equally effective in generating very strong TcCA
inhibitors (Table 1).
(iii) For the benzenesulfonamide derivatives 3a−x, the
fluorosulfanilamides 3e−h were generally more inhib-
itory compared to the corresponding chlorosulfanila-
mides 3i−l, the corresponding bromosulfanilamides
3m−p, and the corresponding unsubstituted sulfanila-
mides 3a−d, respectively, but the differences in activity
were rather small. The increase in the spacer (n) between
the benzenesulfonamide head and the tail (as in
compounds 3q−t, n = 1, and 3u−x, n = 2) did not
dramatically influence the TcCA inhibitory activity
compared to the corresponding sulfanilamides 3a,b (n
= 0), Table 1.
The human isoforms hCA I and II were generally effectively
inhibited by sulfonamides 3 and 5, with KIs in the range of 2.9−
346 nM (for hCA I) and of 0.70−104 nM (for hCA II). This is
obviously not unexpected because most aromatic/heterocyclic
sulfonamides possessing this type of substituted phenacetamido
tail show relevant inhibitory activities against these two hCAs,13
which may be considered as off-targets. However, the reversible
binding of a drug to the blood hCA I and II was recently shown
to constitute a possibility to enhance the bioavailability of the
drug and its slow release to organs not always reachable by
sulfonamides.15 Inhibition of hCA I and II may be associated
with side effects due to the ubiquity of these isoforms, but such
phenomena are transient and are dose-dependent, being
possible to find treatment windows at which the side effects
are minimized.3,4,7
The compounds in general showed selective inhibition of
TcCA compared to hCA I. A subset of 17 compounds showed
at least 5-fold selectivity toward TcCA over hCA I (Table 1).
Compounds 3b and 3f showed the highest selectivity (more
than 100 fold) against this isoform. However, 10 compounds
showed similar inhibition values for hCA I and TcCA (hCA I/
TcCA ratio: 0.5−1.3).
Figure 1. A homology model of TcCA based on the Neisseria
gonorrheae enzyme (NgCA) X-ray crystal structure.16 hCA I (orange),
hCA II (purple), and the homology model of TcCA (turquoise). The
region where the backbone of TcCA has a different fold compared to
hCA I and hCA II (Glu115-Ser122 and Thr183-Ala200 regions of
TcCA) are depicted in dark blue (the sulfamate ligand topiramate is
also shown).
The compounds showed similar KIs for hCA II and TcCA
(Table 1). Only three compounds showed selectivity toward
TcCA, i.e., derivatives 3b (38.5-fold), 3f (5.8-fold), and 3q (2.5-
fold). Sulfonamide 3b showed a marked selectivity toward
TcCA compared to hCA I and hCA II. Replacement of the p-
chlorine substituent by a p-fluorine (compound 3a) or a p-
methoxy substituent (compound 3d) abolished the selectivity
toward TcCA over hCA II, while it results in a ∼10-fold drop in
selectivity toward TcCA over hCA I (Table 1). Addition of a
fluorine atom to the meta-position (with respect to the
sulfamoyl moiety), to yield compound 3f, increased the
selectivity toward TcCA compared to hCA I, whereas it
lowered the selectivity toward TcCA compared to hCA II
(Table 1). Interestingly, adding a chlorine atom (compound 3j)
or a bromine atom (compound 3n) instead of the fluorine
lowered selectivity toward TcCA compared to both hCA I and
hCA II (Table 1). Finally, increasing the spacer length between
the amide bond and the aromatic ring (n in the general formula
of the new sulfonamides reported here) with a methylene
(compound 3r) or ethylene moiety (compound 3v) lowered
the selectivity toward TcCA compared to both human CA
isozymes (Table 1).
a superposition of the obtained TcCA model with hCA I and II
(as determined by X-ray crystallography)11 is shown. The
folding of TcCA is similar to that of hCA I and II (RMSD:
2.174 Å on Cα-atoms), but the protozoan enzyme possesses
some extra loops (among which the Thr183-Ala200) which are
absent in the mammalian enzymes. The counterpart of this
loop is the Ser125-Val143 region of hCA I. The backbone of
TcCA was folded outward from the binding pocket (compared
to the other α-CA isozymes), and hence the binding pocket is
larger (Figure 1) compared to those of the mammalian
enzymes hCA I and II.17−20 The region between Glu115 and
Ser122 in TcCA also had a different folding compared to hCA I
and hCA II, being situated slightly closer toward the active site.
Sulfonamides 3a−x and 5a−d have been docked into the
active sites of hCA I, hCA II, and TcCA, and all ligands were
forced to position their sulfonamide groups in a way similar to
that of topiramate in the hCA I crystal structure (PDB:
3LXE).17 Docking studies revealed that almost all these
sulfonamides were able to adopt docking poses that allowed
the formation of hydrogen bonds and cation−π interactions
with Arg124 of TcCA. Furthermore, some of the ligands also
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formed hydrogen bonds with Thr257. In addition, the ligands
participated in hydrophobic interactions with Leu255. For
example, compound 3b formed both a hydrogen bond and a
cation−π interaction with the side chain of Arg124 from TcCA
(Figure 2). In addition, the p-chlorine substituent of the ligand
Figure 3. Docking poses of compound 3p (magenta) within the active
site of hCA II. The methoxyphenyl moiety of the ligand forms
hydrophobic stacking with Phe131. The residues that can form
hydrogen bonds with several of the docked ligands are indicated in
green. For clarity, Zn2+ and the zinc-binding residues His94, His96,
and His119 are shown as well.
Figure 2. Docking poses of compound 3b (brown, magenta, and
turquoise) within the active site of TcCA. The ligand forms hydrogen
bonding to Arg124 (dashed red line). Also, the distance between the
ligand phenyl moiety and the cationic group of Arg124 is close enough
to form cation−π interaction (distance: ∼3.5 Å; green double arrow;
distance between atom CZ of Arg124 and the centroid of the ligand
phenyl ring). The distance between the p-chlorine substituent of the
ligand and the cationic nitrogen atom of Lys113 is ∼5.62 Å (black
dashed line), but this can decrease because of the Lys113 side chain
flexibility.
of Trp5 and Asn67. Interestingly, the compounds interacted
with Phe131 through their halogenophenyl or methoxyphenyl
moieties. This residue was shown (by means of X-ray
crystallography)22 to form stackings (edge-to-face and face-to-
face) with many sulfonamides and hence introduces a new
stabilizing interaction with the bound inhibitors. Figure 3 shows
the binding pose of compound 3p within the active site of hCA
II to illustrate this interaction between the ligand phenyl moiety
and Phe131. Phe131 is absent in the other two isozymes (Table
2). Its counterpart in hCA I is Leu131, whereas the
corresponding backbone in TcCA is folded outward (the
Thr183-Ala200 region of TcCA has a different backbone fold
than the corresponding Ser125−Val143 region of hCA I and
hCA II). In hCA I, Phe91 is present close to the region were
Phe131 in hCA II is located. However, Phe91 (hCA I) is not
available for stackings interactions with ligands because it seems
to be shielded from the ligands by Gln92, Ala121, and
Leu131.22
Docking studies for hCA I also showed that compounds 3
and 5 adopt a near linear docking pose (data not shown).
However, the absence of the Arg124 (TcCA) and Phe131
(hCA II) counterparts from hCA I could explain the higher KI
values for hCA I compared to hCA II and TcCA (Table 2).
The docking procedures did not always give clear and
definitive explanations for the observed binding and/or
inhibition data. Several reasons exist for this: (1) protein and
binding pocket flexibility that are not taken into account by the
docking procedures; (2) residues that do not directly contact
the ligands can nevertheless have influences on their binding;
(3) entropy effects that are not accurately taken into account in
the docking procedures. Nevertheless, this docking study has
resulted in a closer insight into the general inhibition data of
the ligands reported here for hCA II (the presence of Phe131
that enables hydrophobic stacking interactions with the ligands)
and TcCA (presence of Arg124 that enables cation−π
could point in different directions, one of them toward the side
chain of Lys113 and could probably form favorable electrostatic
interactions with this residue. The distance between the Lys113
ε-nitrogen atom and the ligand chlorine atom was of 5.62 Å;
however, this distance could decrease from the expected
flexibility of Lys113. The residues Lys113, Arg124, and Thr257,
which are important in the binding of ligands for TcCA, are on
the other hand not present in hCA I and II (Table 2). In
Table 2. Amino Acid Residues of hCA I and II and TcCA
Involved in the Binding of the Sulfonamide Inhibitors 3 and
5
hCA I (PDB: 3LXE) hCA II (PDB: 3B4F) TcCA (homology model)
Glu58
Asn69
Phe91
Gln92
Ala121
Leu131
Thr199
Arg58
Glu69
Ile91
Lys113
Arg124
Val155
Gln92
Val121
Phe131
Thr199
Ser156
Val179
different local fold
Thr256
addition, Glu69 is located in the hCA II binding site (its
counterpart in TcCA is Arg124; see Table 2) close to the
region were the chlorine substituent of the ligand was located,
and this could cause unfavorable electrostatic interactions
between the inhibitor and the enzyme.
Docking studies for hCA II showed that compounds 3 and 5
adopted an almost linear docking pose (see Figure 3 for a
representative pose) and that hydrogen bonds were formed
with the side chains of Gln92 and also occasionally with those
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interactions and hydrogen bonding with the new sulfonamides)
compared to hCA I. However, the homology model of TcCA
and the crystal structure of NgCA superposed very well on their
backbone atoms (1.591 Å, Cα-atoms) as shown in Figure 4.
were designed.23 Both of these agents are highly effective and
widely used as antiglaucoma agents.24 A similar issue was
encountered more recently for a sulfonamide with potent
inhibitory action (in vitro) against the nematode α-CA from
Caenorhabditis elegans.25 Although low nanomolar compounds
were obtained, they were ineffective in vivo as antinematoide
agents because of penetration problems. Thus, even if the in
vivo data were negative with these sulfonamides, we estimate
that our research may highlight the importance of this new
antitrypanosomal drug target, TcCA, and the fact that focused
drug design campaigns may lead to compounds which will also
be active in vivo.
CONCLUSIONS
■
We report here a new class of sulfonamides which potently
inhibit TcCA, the enzyme from the protozoan that causes
Chagas disease. An interesting SAR was evidenced for the
inhibition of the protozoan and human off-target isoforms hCA
I and II with these new sulfonamides. Homology modeling and
docking studies allowed us to rationalize the particularly potent
TcCA inhibition profile observed with some of these
derivatives. As some CAIs were recently shown to possess
antitrypanosomal activity in vivo, we estimate that this new
class of inhibitors may allow interesting hints regarding the role
of TcCA in the parasite life cycle and eventually be considered
as lead molecules for new drug design studies against this
disease. The new sulfonamides reported here were ineffective as
antitrypanosomal agents probably because of their highly polar
nature and inability to cross biological membranes to inhibit the
parasite enzyme in vivo.
EXPERIMENTAL SECTION
■
Figure 4. Overlay of the crystal structure of NgCA (turquoise) and the
homology model of TcCA (brown) on their backbone Cα-atoms
(RMSD: 1.591 Å). The three zinc-binding histidines and the two
threonines are depicted for clarity (His92, His94, His111, Thr177, and
Thr178; NgCA numbering) as well as the Zn2+ ions.
Chemistry. All melting points were measured in open capillary
tubes with Buchi 540 and are uncorrected. The compounds were
checked for purity by TLC on silica gel HF254 (E. Merck, Darmstadt,
Germany). IR (KBr) spectra were recorded using a Perkin-Elmer 1600
FTIR spectrophotometer, and all values are expressed as νmax cm−1. 1H
NMR and 13C NMR (HSQC-2D) were recorded on Varian 300 MHz
and VarianUNITY INOVA 500 spectrophotometers using DMSO-d6 as
solvent, and chemical shifts are given in ppm with TMS as a standard.
All compounds were >99% pure as analyzed by HPLC.
General Procedure for the Preparation of Compounds 3 and
5. An amount of 5 mmol of aminosulfonamide was suspended/
dissolved in 20−30 mL of anhydrous MeCN, and 0.78 mL (0.56 g, 5.5
mmol) of triethylamine was added under stirring. The mixture was
cooled to 0−5 °C, and then a solution of 5 mmol of substituted
phenylacetyl chloride in 3 mL of MeCN was added dropwise during
10 min (immediately a precipitate appeared). The reaction was stirred
overnight or until a reasonable conversion was reached (TLC control).
The solvent was evaporated in vacuum, and the resulting product was
treated with 15−20 mL of cold water. The crude solid product was
filtered, washed with water, and air-dried. The obtained compounds
were further purified by recrystallization from ethanol.
Obviously, the protozoan enzyme has some supplementary
loops which are absent in the bacterial enzyme, but they are
generally far away from the active site and this should influence
the binding of inhibitors substantially.
In Vivo Inhibition Studies. Some of the most active in
vitro CAIs, such as compounds 3f, 3g, 3w, 5b, 5g, and 5d, were
investigated in vivo, for their antitrypanosomal effects, using
epimastigotes of T. cruzi strains DM28 and Y, by the procedure
of Rolon et al.22 The test compounds were used at
concentrations of 256, 128, and 64 μM, and benznidazole B
was used as standard drug. Although we have shown earlier that
thiols acting as TcCA inhibitors do have antitrypanosomal
effects in vivo,10 no such activity was detected for the
compounds from the present paper (data not shown), when
working in the concentration range of 64−256 μM CAI.
This lack of activity in vivo may be due to penetration
problems of the sulfonamide compounds that, being highly
polar molecules, have difficulties to cross biological mem-
branes.23a This problem was first encountered when the topical
antiglaucoma sulfonamide CAIs were developed, as for more
than 30 years it was impossible to obtain compounds effective
via the topical route.23b−d However, these efforts eventually led
to the discovery of two clinically used drugs, dorzolamide and
brinzolamide, when compounds with a balanced lipo- and
hydrosolubility (as well as strong CA inhibitory properties)
4-[2-(4-Fluorophenyl)acetamido]benzenesulfonamide (3a). Yield
76%; mp 223−224 °C; IR (KBr) (ν, cm−1), 1690 (CO), 1149, 1325
(SO2); 1H NMR (DMSO-d6, 500 MHz) δ (ppm): 3.67 (2H, s,
CH2CO), 7.13 (2H, t, J = 8.78 Hz, 4-FPh C3,5-H), 7.22 (2H, s,
SO2NH2), 7.33−7.36 (2H, m, 4-FPh C2,6-H), 7.73 (4H, s, phenyl
C2,3,5,6-H), 10.47 (1H, s, CONH); 13C NMR (HSQC-2D, DMSO-d6,
125 MHz) δ (ppm): 42.93 (CH2CO), 115.71 (d, JC,F = 21.08 Hz, 4-
FPh C3, C5), 119.39 (Ph C3, C5), 127.38 (Ph C2, C6), 131.74 (d, JC,F
=
8.14 Hz, 4-FPh C2, C6), 132.39 (4-FPh C1), 139.09 (Ph C1), 142.70
(Ph C4), 161.86 (d, JC,F = 242.5 Hz, 4-FPh C4), 170.28 (CONH).
4-[2-(4-Chlorophenyl)acetamido]benzenesulfonamide (3b). Yield
90%; mp 231−232 °C; IR (KBr) (ν, cm−1), 1670 (CO), 1154, 1333
(SO2); 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.72 (2H, s,
5777
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Journal of Medicinal Chemistry
Article
CH2CO), 7.26 (2H, s, SO2NH2), 7.39 (4H, d, J = 5.10 Hz, 4-ClPh
C2,3,5,6-H), 7.76 (4H, s, phenyl C2,3,5,6-H), 10.53 (1H, s, CONH).
4-[2-(2-Bromophenyl)acetamido]benzenesulfonamide (3c). Yield
83%; mp 244−245 °C; IR (KBr) (ν, cm−1), 1688 (CO), 1153, 1351
(SO2); 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.93 (2H, s,
CH2CO), 7.27 (3H, d, J = 1.80 Hz, SO2NH2 and 2-BrPh C4-H), 7.40−
7.45 (2H, m, 2-BrPh C5,6-H), 7.64 (1H, d, J = 6.30 Hz, 2-BrPh C3-H),
7.78 (4H, d, J = 4.50 Hz, phenyl C2,3,5,6-H), 10.60 (1H, s, CONH).
4-[2-(4-Methoxyphenyl)acetamido]benzenesulfonamide (3d).
Yield 82%; mp 210−211 °C; IR (KBr) (ν, cm−1), 1673 (CO),
4-[2-(2-Bromophenyl)acetamido]-3-chlorobenzenesulfonamide
(3k). Yield 92%; mp 220−221 °C; IR (KBr) (ν, cm−1), 1678 (CO),
1
1162, 1324 (SO2); H NMR (DMSO-d6, 300 MHz) δ (ppm): 4.01
(2H, s, CH2CO), 7.25 (1H, t, J = 7.80 Hz, 2-BrPh C4-H), 7.39 (2H, t,
J = 7.80 Hz, 2-BrPh C5,6-H), 7.47 (2H, s, SO2NH2), 7.65 (1H, d, J =
8.10 Hz, 2-BrPh C3-H), 7.76 (1H, dd, J = 8.40, 2.10 Hz, phenyl C6-H),
7.91 (1H, d, J = 2.10 Hz, phenyl C2-H), 8.02 (1H, d, J = 8.40 Hz,
phenyl C5-H), 9.98 (1H, s, CONH).
4-[2-(4-Methoxyphenyl)acetamido]-3-chlorobenzenesulfona-
mide (3l). Yield 91%; mp 176−177 °C; IR (KBr) (ν, cm−1), 1673
(CO), 1164, 1335 (SO2); 1H NMR (DMSO-d6, 300 MHz) δ
(ppm): 3.72−3.80 (5H, m, CH2CO and CH3O), 6.93 (2H, d, J = 7.80
Hz, 4-CH3OPh C3,5-H), 7.31 (2H, d, J = 8.40 Hz, 4-CH3OPh C2,6-H),
7.48 (2H, s, SO2NH2), 7.75 (1H, d, J = 8.40 Hz, 3-ClPh C6-H), 7.90
(1H, s, 3-ClPh C2-H), 8.02 (1H, d, J = 8.40 Hz, 3-ClPh C5-H), 9.84
(1H, s, CONH).
1
1154, 1331 (SO2); H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.63
(2H, s, CH2CO), 3.75 (3H, s, CH3O), 6.92 (2H, d, J = 6.60 Hz, 4-
CH3OPh C3,5-H), 7.27 (4H, s, SO2NH2 and 4-CH3OPh C2,6-H), 7.77
(4H, s, phenyl C2,3,5,6-H), 10.47 (1H, s, CONH).
4-[2-(4-Fluorophenyl)acetamido]-3-fluorobenzenesulfonamide
(3e). Yield 81%; mp 196−198 °C; IR (KBr) (ν, cm−1), 1673 (CO),
1
4-[2-(4-Fluorophenyl)acetamido]-3-bromobenzenesulfonamide
1150, 1340 (SO2); H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.80
(3m). Yield 77%; mp 154−155 °C; IR (KBr) (ν, cm−1), 1658 (CO),
(2H, s, CH2CO), 7.19 (2H, t, J = 8.40 Hz, 4-FPh C3,5-H), 7.32−7.49
(4H, m, SO2NH2 and 4-FPh C2,6-H), 7.53 (1H, s, 3-FPh C2-H), 7.61
(1H, s, 3-FPh C6-H), 8.50 (1H, d, J = 4.80 Hz, 3-FPh C5-H), 10.24
(1H, s, CONH).
1
1156, 1341 (SO2); H NMR (DMSO-d6, 500 MHz) δ (ppm): 3.70
(2H, s, CH2CO), 7.14 (2H, t, J = 7.32 Hz, 4-FPh C3,5-H), 7.32 (1H, d,
J = 7.81 Hz, 3-BrPh C6-H), 7.36 (2H, s, SO2NH2), 7.38 (2H, td, J =
7.32, 1.95 Hz, 4-FPh C2,6-H), 7.57 (1H, dd, J = 7.81, 0.98 Hz, 3-BrPh
C5-H), 7.64 (1H, d, J = 0.98 Hz, 3-BrPh C2-H), 9.57 (1H, s, CONH);
13C NMR (HSQC-2D, DMSO-d6, 125 MHz) δ (ppm): 42.78
(CH2CO), 115.72 (d, JC,F = 21.08 Hz, 4-FPh C3, C5), 121.90 (3-
4-[2-(4-Chlorophenyl)acetamido]-3-fluorobenzenesulfonamide
(3f). Yield 65%; mp 218−219 °C; IR (KBr) (ν, cm−1), 1672 (CO),
1
1151, 1339 (SO2); H NMR (DMSO-d6, 500 MHz) δ (ppm): 3.76
(2H, s, CH2CO), 7.35 (2H, d, J = 2.93 Hz, 4-ClPh C2,6-H), 7.37 (2H,
s, 4-ClPh C3,5-H), 7.39 (2H, s, SO2NH2), 7.62 (1H, d, J = 1.95 Hz, 3-
FPh C2-H), 8.13 (1H, d, J = 8.30 Hz, 3-FPh C6-H), 8.43 (1H, d, J =
6.83 Hz, 3-FPh C5-H), 10.20 (1H, s, CONH); 13C NMR (HSQC-2D,
BrPh C3), 127.75 (3-BrPh C5), 128.68 (3-BrPh C6), 131.77 (d, JC,F
=
8.14 Hz, 4-FPh C2, C6), 132.32 (4-FPh C1), 132.60 (3-BrPh C2),
136.81 (3-BrPh C1), 144.20 (3-BrPh C4), 161.84 (d, JC,F = 242.03 Hz,
4-FPh C4), 162.84 (CONH).
DMSO-d6, 125 MHz) δ (ppm): 42.41 (CH2CO), 113.90 (d, JC,F
=
4-[2-(4-Chlorophenyl)acetamido]-3-bromobenzenesulfonamide
22.00 Hz, 3-FPh C2), 122.07 (3-FPh C5), 124.02 (3-FPh C6), 127.13
(d, JC,F = 12.94 Hz, 3-FPh C4), 128.94 (4-ClPh C3, C5), 131.76 (4-
ClPh C2, C6), 135.23 (4-ClPh C4), 135.30 (4-ClPh C1), 141.83 (d, JC,F
= 3.36 Hz, 3-FPh C1), 154.50 (d, JC,F = 250.30 Hz, 3-FPh C3), 170.24
(CONH).
(3n). Yield 99%; mp 214−215 °C; IR (KBr) (ν, cm−1), 1661 (CO),
1
1161, 1345 (SO2); H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.74
(2H, s, CH2CO), 7.41 (4H, s, 4-ClPh C2,3,5,6-H), 7.48 (2H, s,
SO2NH2), 7.60 (1H, d, J = 8.40 Hz, 3-BrPh C6-H), 7.80 (1H, d, J =
8.40 Hz, 3-BrPh C5-H), 8.06 (1H, s, 3-BrPh C2-H), 9.69 (1H, s,
CONH).
4-[2-(2-Bromophenyl)acetamido]-3-fluorobenzenesulfonamide
(3g). Yield 86%; mp 213-214 °C; IR (KBr) (ν, cm−1), 1669 (CO),
4-[2-(2-Bromophenyl)acetamido]-3-bromobenzenesulfonamide
1
1149, 1339 (SO2); H NMR (DMSO-d6, 300 MHz) δ (ppm): 4.00
(3o). Yield %; mp 158−159 °C; IR (KBr) (ν, cm−1), 1661 (CO),
(2H, s, CH2CO), 7.25 (1H, t, J = 7.20 Hz, 2-BrPh C4-H), 7.38 (2H, t,
J = 7.20 Hz, 2-BrPh C5,6-H), 7.44 (2H, s, SO2NH2), 7.63 (1H, d, J =
8.10 Hz, 2-BrPh C3-H), 7.70 (1H, s, 3-FPh C2-H), 8.20 (1H, d, J =
7.50 Hz, 3-FPh C6-H), 8.51 (1H, d, J = 7.50 Hz, 3-FPh C5-H), 10.29
(1H, s, CONH).
1
1172, 1342 (SO2); H NMR (DMSO-d6, 500 MHz) δ (ppm): 3.89
(2H, s, CH2CO), 7.11 (1H, td, J = 7.81, 1.47 Hz, 2-BrPh C4-H), 7.20
(1H, td, J = 7.81, 1.47 Hz, 2-BrPh C5-H), 7.34 (2H, s, SO2NH2), 7.37
(1H, d, J = 7.32 Hz, 3-BrPh C6-H), 7.44 (1H, dd, J = 8.83, 1.47 Hz, 2-
BrPh C6-H), 7.59 (1H, dd, J = 7.81, 0.97 Hz, 2-BrPh C2-H), 7.62 (1H,
dd, J = 6.83, 0.98 Hz, 3-BrPh C5-H), 7.65 (1H, d, J = 0.98 Hz, 3-BrPh
C2-H), 9.57 (1H, s, CONH); 13C NMR (HSQC-2D, DMSO-d6, 125
MHz) δ (ppm): 43.50 (CH2CO), 125.29 (2-BrPh C2), 127.42 (3-BrPh
C5), 127.64 (2-BrPh C4), 128.38 (3-BrPh C6), 128.69 (3-BrPh
C3),129.60 (2-BrPh C5), 132.89 (2-BrPh C6), 133.04 (2-BrPh C3),
133.34 (3-BrPh C2), 135.70 (2-BrPh C1), 136.22 (3-BrPh C1), 136.88
(3-BrPh C4), 168.84 (CONH).
4-[2-(4-Methoxyphenyl)acetamido]-3-bromobenzenesulfona-
mide (3p). Yield 89%; mp 148−149 °C; IR (KBr) (ν, cm−1), 1666
(CO), 1167, 1338 (SO2); 1H NMR (DMSO-d6, 300 MHz) δ
(ppm): 3.66 (2H, s, CH2CO), 3.75 (3H, s, CH3O), 6.92 (2H, d, J =
6.60 Hz, 4-CH3OPh C3,5-H), 7.31 (2H, d, J = 6.90 Hz, 4-CH3OPh
C2,6-H), 7.47 (2H, s, SO2NH2), 7.65 (1H, d, J = 7.50 Hz, 3-BrPh C6-
H), 7.80 (1H, d, J = 7.50 Hz, 3-BrPh C5-H), 8.05 (1H, s, 3-BrPh C2-
H), 9.50 (1H, s, CONH).
4-[2-(4-Methoxyphenyl)acetamido]-3-fluorobenzenesulfonamide
(3h). Yield 55%; mp 149−150 °C; IR (KBr) (ν, cm−1), 1674 (CO),
1
1150, 1339 (SO2); H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.71
(2H, s, CH2CO), 3.76 (3H, s, CH3O), 6.90 (2H, d, J = 7.80 Hz, 4-
CH3OPh C3,5-H), 7.26 (2H, s, 4-CH3OPh C2,6-H), 7.41 (2H, s,
SO2NH2), 7.60 (1H, s, 3-FPh C2-H), 8.18 (1H, d, J = 6.00 Hz, 3-FPh
C6-H), 8.46 (1H, d, J = 6.00 Hz, 3-FPh C5-H), 10.08 (1H, s, CONH).
4-[2-(4-Fluorophenyl)acetamido]-3-chlorobenzenesulfonamide
(3i). Yield 98%; mp 213-214 °C; IR (KBr) (ν, cm−1), 1669 (CO),
1
1161, 1335 (SO2); H NMR (DMSO-d6, 500 MHz) δ (ppm): 3.78
(2H, s, CH2CO), 7.15 (2H, t, J = 7.81 Hz, 4-FPh C3,5-H), 7.36−7.39
(2H, m, 4-FPh C2,6-H), 7.42 (2H, s, SO2NH2), 7.71 (1H, dd, J = 8.78,
1.95 Hz, 3-ClPh C6-H), 7.87 (1H, d, J = 1.95 Hz, 3-ClPh C2-H), 7.96
(1H, d, J = 8.78 Hz, 3-ClPh C5-H), 9.87 (1H, s, CONH); 13C NMR
(HSQC-2D, DMSO-d6, 125 MHz) δ (ppm): 42.29 (CH2CO), 115.75
(d, JC,F = 21.08 Hz, 4-FPh C3, C5), 125.59 (3-ClPh C6), 126.03 (3-
ClPh C5), 127.49 (3-ClPh C2), 131.82 (d, JC,F = 8.14 Hz, 4-FPh C2,
C6), 132.33 (3-ClPh C3), 132.36 (4-FPh C1), 138.52 (3-ClPh C1),
141.73 (3-ClPh C4), 161.89 (d, JC,F = 242.03 Hz, 4-FPh C4), 170.47
(CONH).
4-[2-(4-Fluorophenyl)acetamidomethyl]benzenesulfonamide
(3q). Yield 69%; mp 156−157 °C; IR (KBr) (ν, cm−1), 1636 (CO),
1
1159, 1338 (SO2); H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.50
(2H, s, CH2CO), 4.34 (2H, d, J = 5.40 Hz, NHCH2), 7.15 (2H, t, J =
8.70 Hz, 4-FPh C3,5-H), 7.28−7.36 (4H, m, SO2NH2 and 4-FPh C2,6-
H), 7.39 (2H, d, J = 7.80 Hz, phenyl C3,5-H), 7.76 (2H, d, J = 8.10 Hz,
phenyl C2,6-H), 8.65 (1H, s, CONH).
4-[2-(4-Chlorophenyl)acetamido]-3-chlorobenzenesulfonamide
(3j). Yield 60%; mp 242−243 °C; IR (KBr) (ν, cm−1), 1665 (CO),
1
1161, 1331 (SO2); H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.84
4-[2-(4-Chlorophenyl)acetamidomethyl]benzenesulfonamide
(3r). Yield 62%; mp 186−187 °C; IR (KBr) (ν, cm−1), 1635 (CO),
(2H, s, CH2CO), 7.42 (4H, s, 4-ClPh C2,3,5,6-H), 7.48 (2H, s,
SO2NH2), 7.77 (1H, d, J = 7.80 Hz, 3-ClPh C6-H), 7.91 (1H, s, 3-
ClPh C2-H), 8.00 (1H, d, J = 7.80 Hz, 3-ClPh C5-H), 9.98 (1H, s,
CONH).
1
1155, 1342 (SO2); H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.51
(2H, s, CH2CO), 4.33 (2H, d, J = 4.80 Hz, NHCH2), 7.32 (4H, s,
SO2NH2 and 4-ClPh C2,6-H), 7.36−7.42 (4H, m, 4-ClPh C3,5-H and
5778
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Journal of Medicinal Chemistry
Article
phenyl C3,5-H), 7.76 (2H, d, J = 8.40 Hz, phenyl C2,6-H), 8.66 (1H, s,
CONH).
5-[2-(2-Bromophenyl)acetamido]-1,3,4-thiadiazole-2-sulfona-
mide (5c). Yield 59%; mp 247−248 °C; IR (KBr) (ν, cm−1), 1680
(CO), 1179, 1359 (SO2); 1H NMR (DMSO-d6, 300 MHz) δ
(ppm): 4.11 (2H, s, CH2CO), 7.29 (1H, t, J = 6.90 Hz, 2-BrPh C4-H),
7.42−7.47 (2H, m, 2-BrPh C5,6-H), 7.67 (1H, d, J = 7.50 Hz, 2-BrPh
C3-H), 8.37 (2H, s, SO2NH2).
5-[2-(4-Methoxyphenyl)acetamido]-1,3,4-thiadiazole-2-sulfona-
mide (5d). Yield 64%; mp 256−157 °C; IR (KBr) (ν, cm−1), 1685
(CO), 1166, 1359 (SO2); 1H NMR (DMSO-d6, 500 MHz) δ
(ppm): 3.72 (3H, s, CH3O), 3.79 (2H, s, CH2CO), 6.88 (2H, dd, J =
6.40, 1.83 Hz, 4-CH3OPh C3,5-H), 7.23 (2H, dd, J = 6.86, 1.83 Hz, 4-
CH3OPh C2,6-H), 8.29 (2H, s, SO2NH2), 13.19 (1H, s, CONH); 13C
NMR (HSQC-2D, DMSO-d6, 125 MHz) δ (ppm): 41.26 (CH2CO),
55.77 (4-OCH3), 114.63 (4-CH3OPh C3, C5), 126.67 (4-CH3OPh
C1), 131.09 (4-CH3OPh C2, C6), 159.07 (4-CH3OPh C4), 161.90 (C-
thiadiazole), 165.15 (C-thiadiazole), 171.29 (CONH).
4-[2-(2-Bromophenyl)acetamidomethyl]benzenesulfonamide
(3s). Yield 69%; mp 193−194 °C; IR (KBr) (ν, cm−1), 1649 (CO),
1
1153, 1341 (SO2); H NMR (DMSO-d6, 500 MHz) δ (ppm): 3.67
(2H, s, CH2CO), 4.34 (2H, d, J = 5.86 Hz, NHCH2), 7.18 (1H, td, J =
7.81, 1.95 Hz, 2-BrPh C4-H), 7.28 (2H, s, SO2NH2), 7.31 (1H, td, J =
7.81, 1.46 Hz, 2-BrPh C5-H), 7.36 (1H, dd, J = 7.32, 1.95 Hz, 2-BrPh
C6-H), 7.43 (2H, d, J = 8.29 Hz, phenyl C3,5-H), 7.57 (1H, dd, J =
7.81, 0.97 Hz, 2-BrPh C2-H), 7.76 (2H, d, J = 7.32 Hz, phenyl C2,6-H),
8.59 (1H, s, CONH); 13C NMR (HSQC-2D, DMSO-d6, 125 MHz) δ
(ppm): 42.67 (CH2CO), 42.96 (NHCH2), 125.14 (2-BrPh C2),
126.32 (Ph C2, C6), 128.20 (Ph C3, C5), 128.27 (2-BrPh C5), 129.38
(2-BrPh C4), 132.79 (2-BrPh C6), 132.96 (2-BrPh C3), 136.30 (2-
BrPh C1), 143.31 (Ph C1), 144.30 (Ph C4), 169.72 (CONH).
4-[2-(4-Methoxyphenyl)acetamidomethyl]benzenesulfonamide
(3t). Yield 56%; mp 176−177 °C; IR (KBr) (ν, cm−1), 1626 (CO),
CA Inhibition Assay. An Applied Photophysics stopped-flow
instrument was used for assaying the CA-catalyzed CO2 hydration
activity.1 Phenol red (at a concentration of 0.2 mM) was used as
indicator, working at the absorbance maximum of 557 nm, with 20
mM HEPES (pH 7.5) as buffer and 20 mM Na2SO4 (for maintaining
constant ionic strength), following the initial rates of the CA-catalyzed
CO2 hydration reaction for a period of 10−100 s. The CO2
concentrations ranged from 1.7 to 17 mM for the determination of
the kinetic parameters and inhibition constants. For each inhibitor, at
least six traces of the initial 5−10% of the reaction were used for
determining the initial velocity. The uncatalyzed rates were
determined in the same manner and subtracted from the total
observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in
distilled−deionized water, and dilutions up to 0.01 nM were
performed thereafter with the assay buffer. Inhibitor and enzyme
solutions were preincubated together for 15 min to 6 h at room
temperature (15 min) or 4 °C (6 h) prior to assay, to allow for the
formation of the E−I complex. The inhibition constants were obtained
by nonlinear least-squares methods using PRISM 3, as reported
earlier,2 and represent the mean from at least three different
determinations. All CA isofoms were recombinant ones obtained in-
house as reported earlier.3−6
Construction of TcCA Homology Models. Homology models of
TcCA were constructed using the crystal structure of Neisseria
gonorrheae CA (ngCA, PDB: 1KOP, 1.90 Å) as a template. Ten
models were constructed using the MOE software package (version
2010, CCG, Montreal, Canada), and the model with both the lowest
RMSD value (Cα-atoms and all residues) and lowest contact energy
was chosen for further optimization. A steepest descent energy
minimization procedure (MMFF94x forcefield) was performed using
default settings and with constraints on all heavy backbone atoms,
His158, His160, His177, Thr256 (TcCA numbering; analogue of
Thr199 in hCA I), and Zn2+. Afterward, a second round of energy
minimization was performed with similar settings but without any
constraints. The protein model was saved as a mol2 file prior to
docking procedures. The homology model of TcCA and the crystal
structure of NgCA superpose well on their backbone atoms (1.591 Å,
Cα-atoms; supplementary Figure 4). The crystal structures of hCA I
(PDB: 3LXE), hCA II (PDB: 3B4F) and the homology model of
TcCA superpose well on their Cα-atoms (RMSD: 2.174 Å for 256
residues). The backbone folding near the binding pocket is similar for
all CA isozymes except for Thr183-Ala200 of TcCA (counterpart of
Ser125-Val143 region of hCA I). Here the backbone of TcCA is folded
outward from the binding pocket compared to the other CA isozymes,
and hence the binding pocket is larger (as shown Figure 1). Also, the
region between Glu115 and Ser122 of TcCA has a different fold
compared to the hCA I and hCA II and is situated slightly closer
toward the active site.
1
1152, 1327 (SO2); H NMR (DMSO-d6, 300 MHz) δ (ppm): 3.43
(2H, s, CH2CO), 3.75 (3H, s, CH3O), 4.34 (2H, s, NHCH2), 6.90
(2H, d, J = 6.00 Hz, 4-CH3OPh C3,5-H), 7.21 (2H, d, J = 7.50 Hz, 4-
CH3OPh C2,6-H), 7.33 (2H, s, SO2NH2), 7.40 (2H, d, J = 7.50 Hz,
phenyl C3,5-H), 7.76 (2H, d, J = 6.00 Hz, phenyl C2,6-H), 8.58 (1H, s,
CONH).
4-[2-(4-Fluorophenyl)acetamidoethyl]benzenesulfonamide (3u).
Yield 50%; mp 150−151 °C; IR (KBr) (ν, cm−1), 1630 (CO), 1157,
1
1342 (SO2); H NMR (DMSO-d6, 300 MHz) δ (ppm): 2.79 (2H, s,
NHCH2CH2), 3.38 (2H, s, CH2CO), 3.44 (2H, s, NHCH2CH2), 7.14
(2H, d, J = 8.70 Hz, 4-FPh C3,5-H), 7.23 (2H, s, SO2NH2), 7.32−7.37
(4H, m, 4-FPh C2,6-H and phenyl C3,5-H), 7.74 (1H, d, J = 7.50 Hz,
phenyl C2,6-H), 8.14 (1H, s, CONH).
4-[2-(4-Chlorophenyl)acetamidoethyl]benzenesulfonamide (3v).
Yield 52%; mp 168−169 °C; IR (KBr) (ν, cm−1), 1629 (CO), 1157,
1339 (SO2); 1H NMR (DMSO-d6, 300 MHz) δ (ppm): 2.80 (2H, t, J
= 6.00 Hz, NHCH2CH2), 3.40 (2H, s, CH2CO), 3.44 (2H, s,
NHCH2CH2), 7.25 (2H, s, SO2NH2), 7.33−7.39 (6H, m, 4-ClPh
C2,3,5,6-H and phenyl C3,5-H), 7.75 (2H, d, J = 7.20 Hz, phenyl C2,6-H),
8.18 (1H, s, CONH).
4-[2-(2-Bromophenyl)acetamidoethyl]benzenesulfonamide (3w).
Yield 95%; mp 170−171 °C; IR (KBr) (ν, cm−1), 1651 (CO), 1153,
1
1328 (SO2); H NMR (DMSO-d6, 300 MHz) δ (ppm): 2.84 (2H, t,
J=6.60 Hz, NHCH2CH2), 3.40 (2H, s, CH2CO), 3.58 (2H, s,
NHCH2), 7.21 (1H, t, J = 6.00 Hz, 2-BrPh C4-H), 7.33 (4H, s,
SO2NH2 and 2-BrPh C5,6-H), 7.43 (2H, d, J = 7.80 Hz, phenyl C3,5-
H), 7.60 (1H, d, J = 8.10 Hz, 2-BrPh C3-H), 7.77 (2H, d, J = 7.80 Hz,
phenyl C2,6-H), 8.16 (1H, s, CONH).
4-[2-(4-Methoxyphenyl)acetamidoethyl]benzenesulfonamide
(3x). Yield 90%; mp 156−157 °C; IR (KBr) (ν, cm−1), 1623 (CO),
1
1157, 1336 (SO2); H NMR (DMSO-d6, 500 MHz) δ (ppm): 2.75
(2H, t, J = 7.32 Hz, NHCH2CH2), 3.26−3.30 (4H, m, CH2CO and
NHCH2CH2), 3.71 (3H, s, CH3O), 6.83 (2H, dd, J = 6.84, 1.95 Hz, 4-
CH3OPh C3,5-H), 7.09 (2H, dd, J = 6.83, 1.95 Hz, 4-CH3OPh C2,6-H),
7.27 (2H, s, SO2NH2), 7.32 (2H, d, J = 8.78 Hz, phenyl C3,5-H), 7.70
(2H, dd, J = 8.29, 1.95 Hz, phenyl C2,6-H), 8.01 (1H, t, J = 5.37 Hz,
CONH); 13C NMR (HSQC-2D, DMSO-d6, 125 MHz) δ (ppm):
35.40 (NHCH2CH2), 39.70 (NHCH2CH2), 42.22 (CH2CO), 55.72
(4-OCH3), 114.33 (4-CH3OPh C3, C5), 126.32 (phenyl C2, C6),
128.95 (4-CH3OPh C1), 129.82 (phenyl C3, C5), 130.59 (4-CH3OPh
C2, C6), 142.70 (phenyl C1), 144.37 (phenyl C4), 158.58 (4-CH3OPh
C4), 171.18 (CONH).
5-[2-(4-Fluorophenyl)acetamido]-1,3,4-thiadiazole-2-sulfona-
mide (5a). Yield 59%; mp 273−274 °C; IR (KBr) (ν, cm−1), 1684
(CO), 1168, 1375 (SO2); 1H NMR (DMSO-d6, 300 MHz) δ
(ppm): 3.91 (2H, s, CH2CO), 7.19 (2H, d, J = 8.10 Hz, 4-FPh C3,5-
H), 7.39 (2H, s, 4-FPh C2,6-H), 8.35 (2H, s, SO2NH2).
Preparation of Protein Crystal Structures. Crystal structures
hCA I (PDB: 3LXE; 1.90 Å) and hCA II (PDB: 3B4F; 1.89 Å) were
obtained from the protein databank. Only one protein chain (chain A)
and its corresponding Zn2+ ion and ligand (CA inhibitor) was retained
per PDB file, and all other protein chains, ions, buffer molecules, and
water molecules were deleted. Hydrogen atoms and charges were
5-[2-(4-Chlorophenyl)acetamido]-1,3,4-thiadiazole-2-sulfona-
mide (5b). Yield 59%; mp 285−286 °C; IR (KBr) (ν, cm−1), 1689
(CO), 1170, 1372 (SO2); 1H NMR (DMSO-d6, 300 MHz) δ
(ppm): 3.91 (2H, s, CH2CO), 7.37 (2H, d, J = 8.70 Hz, 4-ClPh C2,6-
H), 7.42 (2H, d, J = 8.70 Hz, 4-ClPh C3,5-H), 8.35 (2H, s, SO2NH2).
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Journal of Medicinal Chemistry
Article
added to the protein using the protonate 3D tool of the MOE software
package (version 2010, CCG, Montreal, Canada), and a steepest
descent energy minimization was applied (MMFF94x forcefield). All
proteins were superposed (including the TcCA homology model) on
the hCA I structure using their Cα-atoms (RMSD: 2.174 Å), and the
proteins with and without the CAI ligands were saved as mol2 files.
Preparation of Ligand Files. Three-dimensional coordinates of
compounds 3 and 5 were prepared using the MOE software package
(version 2010, CCG, Montreal, Canada). Subsequently, all strong
bases were protonated, all strong acids were deprotonated, and the
ligands were energy minimized according to a steepest descent
protocol (MMFF94x forcefield) and finally saved as a multi-mol2 file.
Docking Studies. Compounds 3 and 5 were docked into the hCA
I and hCA II protein models as well as the TcCA homology model
using the GOLD Suite docking package (version 5.1, CCDC,
Cambridge, UK)21 and the ChemScore scoring function (25 docking
per ligand). ChemScore was chosen because of its speed and correct
prediction of the binding poses as observed in crystal structures in
redocking experiments.21 The binding pocket was defined as all
residues within 12 Å of the central carbon atom of the CA inhibitor
topiramate (atom CAL of topiramate in complex with hCA-I; PDB:
3LXE). No restrictions were applied for the ligand conformations
during the docking procedure except for the position of its
sulfonamide scaffold. All compounds were forced to position their
sulfonamide moieties in a way similar to that of topiromate in complex
with hCA-I (PDB: 3LXE), and a hydrogen bond with Thr199 (or its
counterparts in other CA isozymes) was also required (using the
“scaffold constraints” and “protein HBond” tools in the GOLD
docking program).
Several crystal structures of α-type CA isozymes in complex with
structurally diverse inhibitors were investigated prior to the docking
procedure, and all these inhibitors positioned their sulfonamide groups
in a way very similar to that observed in the topiramate−hCA I (PDB:
3LXE) complex. Therefore, compounds 3 and 5 were docked into the
active sites of hCA I, hCA II, and TcCA, and all ligands were forced to
place their sulfonamide groups in a way similar to that of topiramate in
the hCA I crystal structure (PDB: 3LXE). Therefore, all docked
compounds interact through their sulfonamide tail with Thr199 and
Zn2+ of hCA I and their counterparts in hCA II and TcCA.
In Vivo Antitrypanosomal Activity. A quantitative colorimetric
assay using the oxidation−reduction indicator resazurin was employed
to measure cytotoxicity of compounds 26−33 against two strains of T.
cruzi.22 The method is based on the detection of colorimetric changes
caused by the oxidation (blue) and reduction (pink) capabilities of the
resazurin dye, as an indicator for metabolic cell function. The
inoculum of T. cruzi epimastigotes, the resazurin concentration, and
the incubation time of different inocula of parasites were as described
previously. A log-phase culture ranging from 0.25 to 9 × 106/mL was
seeded in different culture tubes for 24 h at 28 °C. Epimastigotes were
then seeded on 96-well microtiter plates (Sarstedt, Sarstedt, Inc.) at
200-μL/well volumes and incubated for another 48 h at 28 °C. After
the incubation, 20 μL of various dilutions of resazurin solutions
ranging from 0.5 to 3 mM for each inoculum was added, and the plates
were returned to the incubator. To choose the optimal duration of
incubation for cultures in the presence of resazurin solution, the plates
were incubated for periods ranging from 1 to 6 h to allow optimal
oxidation−reduction. The absorbance values were read by dual
wavelength using an EL × 800 enzyme-linked immunosorbent assay
(ELISA) reader (Bio-Tek Instruments Inc.) at 490 and 595 nm.
Background was subtracted. All experiments were performed three
times each in triplicate at concentrations of test compound of 256,
128, and 64 μM. Benznidazole was used as standard drug.
Notes
The authors declare the following competing financial
interest(s): A patent application with these compounds has
been filed.
ACKNOWLEDGMENTS
■
This research was financed by two EU projects (Metoxia and
Dynano) to CTS, by grants from the Academy of Finland,
́
Sigrid Juselius Foundation, and Competitive Research Funding
of Tampere University Hospital (9N054) to S.P. and by
Coordenaca
(CAPES), Conselho Nacional de Desenvolvimento Cientifico e
Tecnolog
Amparo a
́
̧
o de Aperfeico
̧
amento Pessoal de Nivel Superior
̃
́
́
ico (MCT/CNPq), Fundaca̧ o Carlos Chagas Filho de
Pesquisa do Estado do Rio de Janeiro (FAPERJ), to
̃
̀
A.B.V.. We thank Alfonso Maresca for technical support.
ABBREVIATIONS USED
■
CA, carbonic anhydrase; hCA I, human carbonic anhydrase I;
hCA II, human carbonic anhydrase II; TcCA, Trypanosoma
cruzi carbonic anhydrase; NgCA, Neisseria gonorrheae carbonic
anhydrase; CAI, carbonic anhydrase inhibitors; AZ, acetazola-
mide; KI, inhibition constant
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AUTHOR INFORMATION
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Corresponding Author
*E-mail: aakdemir@bezmialem.edu.tr, tel: +90 212 523 22 88
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3005, fax: +-39-055-457-3385, e-mail: claudiu.supuran@unifi.it
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