Appraisal of anti-protozoan activity of nitroaromatic benzenesulfonamides inhibiting carbonic anhydrases from Trypanosoma cruzi and Leishmania donovani

Article abstract of DOI:10.1080/14756366.2019.1626375

Chagas disease and leishmaniasis are neglected tropical disorders caused by the protozoans Trypanosoma cruzi and Leishmania spp. Carbonic anhydrases (CAs, EC 4.2.1.1) from these protozoans (α-TcCA and β-LdcCA) have been validated as promising targets for chemotherapic interventions. Many anti-protozoan agents, such as nitroimidazoles, nifurtimox, and benznidazole possess a nitro aromatic group in their structure which is crucial for their activity. As a continuation of our previous work on N-nitrosulfonamides as anti-protozoan agents, we investigated benzenesulfonamides bearing a nitro aromatic moiety against TcCA and LdcCA, observing selective inhibitions over human off-target CAs. Selected derivatives were assessed in vitro in different developmental stages of T. cruzi and Leishmania spp. A lack of significant growth inhibition has been found, which has been connected to the low permeability of this class of derivatives through cell membranes. Further strategies necessarily need to be designed for targeting Chagas disease and leishmaniasis with nitro-containing CA inhibitors.

Full text of DOI:10.1080/14756366.2019.1626375

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Appraisal of anti-protozoan activity of
nitroaromatic benzenesulfonamides inhibiting
carbonic anhydrases from Trypanosoma cruzi and
Leishmania donovani
Alessio Nocentini, Sameh M. Osman, Igor A. Almeida, Veronica Cardoso,
Fatmah Ali S. Alasmary, Zeid AlOthman, Alane B. Vermelho, Paola Gratteri &
Claudiu T. Supuran
To cite this article: Alessio Nocentini, Sameh M. Osman, Igor A. Almeida, Veronica Cardoso,
Fatmah Ali S. Alasmary, Zeid AlOthman, Alane B. Vermelho, Paola Gratteri & Claudiu T. Supuran
(2019) Appraisal of anti-protozoan activity of nitroaromatic benzenesulfonamides inhibiting carbonic
anhydrases from Trypanosomaꢀcruzi and Leishmaniaꢀdonovani, Journal of Enzyme Inhibition and
Medicinal Chemistry, 34:1, 1164-1171, DOI: 10.1080/14756366.2019.1626375
To link to this article: https://doi.org/10.1080/14756366.2019.1626375
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1164–1171
https://doi.org/10.1080/14756366.2019.1626375
RESEARCH PAPER
Appraisal of anti-protozoan activity of nitroaromatic benzenesulfonamides
inhibiting carbonic anhydrases from Trypanosoma cruzi and Leishmania donovani
Alessio Nocentinia, Sameh M. Osmanb, Igor A. Almeidac, Veronica Cardosod, Fatmah Ali S. Alasmaryb,
Zeid AlOthmanb, Alane B. Vermelhod , Paola Gratteria and Claudiu T. Supurana
aDepartment of Neuroscience, Psychology, Drug Research and Child’s Health, Section of Pharmaceutical and Nutraceutical Sciences, University
b
c
of Florence, Sesto Fiorentino, Italy; Department of Chemistry, College of Science, King Saud University, Riyadh, Saudi Arabia; Department of
d
Natural Products and Food, School of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; BIOINOVAR – Biotechnology
Laboratories: Biocatalysis, Bioproducts and Bioenergy, Institute of Microbiology Paulo de Goes, Federal University of Rio de Janeiro, Rio de
ꢀ
Janeiro, Brazil
ABSTRACT
ARTICLE HISTORY
Received 28 April 2019
Revised 26 May 2019
Accepted 28 May 2019
Chagas disease and leishmaniasis are neglected tropical disorders caused by the protozoans Trypanosoma
cruzi and Leishmania spp. Carbonic anhydrases (CAs, EC 4.2.1.1) from these protozoans (a-TcCA and
b-LdcCA) have been validated as promising targets for chemotherapic interventions. Many anti-protozoan
agents, such as nitroimidazoles, nifurtimox, and benznidazole possess a nitro aromatic group in their struc-
ture which is crucial for their activity. As a continuation of our previous work on N-nitrosulfonamides as
anti-protozoan agents, we investigated benzenesulfonamides bearing a nitro aromatic moiety against
TcCA and LdcCA, observing selective inhibitions over human off-target CAs. Selected derivatives were
assessed in vitro in different developmental stages of T. cruzi and Leishmania spp. A lack of significant
growth inhibition has been found, which has been connected to the low permeability of this class of
derivatives through cell membranes. Further strategies necessarily need to be designed for targeting
Chagas disease and leishmaniasis with nitro-containing CA inhibitors.
KEYWORDS
Carbonic anhydrase; Chagas
disease; Leishmania;
Trypanosoma;
nitroaromatics
1. Introduction
The enzymes carbonic anhydrases (CAs, EC 4.2.1.1) identified in
these protozoans, TcCA in T. cruzi and LdcCA in L. donovani (a
parasite from the Leishmania complex, causing visceral leishmania-
sis) have recently been recognised as suitable targets to fight
these infections6,8,9. CAs are natural catalysts that speed up the
rate of CO2 conversion to bicarbonate and proton. This reaction
was shown to be basic in the growth and virulence of pathogenic
microorganisms9. TcCA and LdcCA were both cloned and charac-
terised in 201310–12. Many inhibitors of these isoforms have been
identified, which represent potential anti-protozoan agents acting
by a new mechanism of action which is probably devoid of cross-
resistance to the existing drugs.
TcCA is an a-class enzyme that contains the three highly con-
served histidines (His94, His96, and His119) coordinating to zinc
ion in the enzyme active site, and glutamic acid (Glu 106) as the
gate-keeping residue10. Measurement of the catalytic activity of
TcCA in CO2 hydration showed a kcat of 1.21 ꢀ 106 s–1, Km of
8.1 ꢀ 10ꢁ3 M and kcat/Km of 1.49 ꢀ 108 M–1 s–1 10. TcCA is inhibited
in the nanomolar range by many CA inhibitory chemotypes such
as aromatic/heterocyclic sulfonamides10,13,14, sulfamates10, thiols10,
anions15, dithiocarbamates15, hydroxamates16, benzoxaboroles17,
and N-nitrosulfonamides18. Thiols, hydroxamates, and N-nitrosulfo-
namides show in vitro anti-trypanosomal activity, deterring mul-
Chagas disease (American trypanosomiasis) and leishmaniasis are
potentially life-threatening illnesses that have been included in
the list of neglected tropical diseases (NTDs) by the World Health
Organization (WHO). These infections belong to the vector-borne
diseases affecting 20 million people and killing more than 50,000
every year and are caused by parasites of the kinetoplastida family
(Trypanosoma cruzi and Leishmania sp.)1. Kissing bugs of the
Triatoma and Rhodnius genera naturally transmit T. cruzi that is
primarily diffused in Latin America. Chagas disease progresses by
damaging organs in the cardiac, digestive, or nervous systems1.
The bite of infected phlebotomines instead is the main cause of
Leishmania transmission and potentially generates skin or visceral
fatal damages. Leishmaniasis is the first-in-class NTD in terms of
mortality and morbidity1.
To date, a limited arsenal of anti-protozoan agents is available
for the treatment of these NTDs. These drugs are marked by high
toxicity and limited efficacy, and resistance phenomena are con-
stantly increasing worldwide2–4. The poor interest shown by the
pharmaceutical industry in searching new effective drugs for NTDs
treatment is related to high costs and expected low financial
return. On the contrary, it should be considered a priority to find
new approaches in the treatment of these parasitosis2,5. Large-
scale analysis on the completely known genome sequence of tiple phases in the life cycle of the pathogen10,16,18
.
LdcCA is a b-class CA whose catalytic activity evaluation
both protozoans have recently provided the identification of new
enzymatic targets6,7
.
reported a kcat of 9.35 ꢀ 105 s–1, Km of 15.8 ꢀ 10ꢁ3 M, and kcat/Km
CONTACT Paola Gratteri
paola.gratteri@unifi.it
Department of Neuroscience, Psychology, Drug Research and Child's Health, Section of Pharmaceutical and
Nutraceutical Sciences, University of Florence, via Ugo Schiff 6, Sesto Fiorentino 50019, Italy; Claudiu T. Supuran
Neurofarba, University of Florence, via Ugo Schiff 6, Sesto Fiorentino 50019, Italy
claudiu.supuran@unifi.it
Department of
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1165
of 5.9 ꢀ 107 M–1 s–1 12. LdcCA was shown to be efficiently inhib- least three different determinations23–26. All CA isoforms were
ited by sulfonamides, heterocyclic thiols, and N-nitrosulfonamides recombinant ones obtained in-house as reported earlier27,28
with nanomolar inhibition constants12,18. Some compounds of the
.
two latter classes showed in vitro anti-leishmanial activity in pre-
2.3. Biological assays
liminary assays, causing the reduction of the parasites growth and
their death12,18
.
2.3.1. Cell cultures
N-Nitrosulfonamides have been designed by us based on the
presence of the nitro group in the structure of many anti-proto-
zoan agents, such as the nitroimidazoles, this moiety being pivotal
for the drug mechanism of action18,19. For instance, nifurtimox
and benznidazole (Bzn) have been the first effective drugs for
treating acute-phase human Chagas infection, with the first being
no longer available on the market because of undesirable side
effects6. Considering that sulfonamides are the most effective CAIs
known to date20, we first attached the nitro group on the sulfona-
mide itself, providing the N-nitro derivatives as a new chemotype
exhibiting a selective inhibition of protozoan CAs over human ubi-
quitous isoforms18. As second design strategy, we report herein,
consists in the incorporation of the nitro group on the benzene
scaffold bearing the sulfonamide, driven by the aromatic character
shown by the nitro moieties present in many anti-protozoan
agents, mentioned above. A set of 3-nitrobenzenesulfonamide
bearing a variety of substituents on the main scaffold has thus
been reported. This set has been recently evaluated also for the
inhibition of the human tumour-associated CA IX and XII over the
ubiquitous CA I and II and for hypoxia-enhanced anti-proliferative
activity on tumour cell lines21. In fact, nitroaromatic groups are
subjected to bioreduction processes in hypoxic tissues, which can
be exploited to selectively generate cytotoxins against tumour
cells21. Here, the set of nitro-benzenesulfonamides has been
screened for the inhibition of TcCA and LdcCA and the most
effective derivatives were studied in vitro against different species
of Leishmania and T. cruzi.
2.3.1.1. Trypansoma cruzi and Leishmania parasites cultures.
Epimastigote forms of the T. cruzi clone Dm28c29 and T. cruzi Y30
strain were obtained from the Laboratory of Cellular
Ultrastructure, FIOCRUZ. L. infantum MHOM/BR/1974/PP75 and
L. amazonensis IFLA/BR/1967/PH8 were donated by the Leishmania
Type Culture Collection (LTCC) of Oswaldo Cruz Institute/Fiocruz
(Rio de Janeiro, Brazil). The parasites were maintained by weekly
subcultures in PBHIL medium supplemented with 10% foetal
bovine serum (FBS) at 28 ꢂC16.
2.3.1.2. RAW 264.7 macrophage cell line cultures. RAW 264.7 mur-
ine macrophages were obtained from the National Institute of
Metrology, Quality and Technology (Instituto Nacional de
Metrologia, Qualidade e Tecnologia, INMETRO, Rio de Janeiro,
Brazil) and maintained in DMEM medium supplemented with 10%
FBS at 37 ꢂC in a 5% controlled CO2 atmosphere. Cell maintenance
was performed every 48–72 h, time necessary for cells to achieve
confluent monolayers.
2.3.2. Inhibitory activity on epimastigotes of Trypanosoma cruzi
and promastigotes of Leishmania
The evaluation of anti-parasites activity was performed in 96 well
plates where the synthetic compounds were serially diluted in the
PHBIL medium supplemented with 10% FBS in concentrations
ranging from 2 to 400 mM. Then, parasites (1.8 ꢀ 106) were added
to each well and the plates incubated for 48 h at 28 ꢂC. The
experiment controls were: negative control (culture medium with-
out parasite) and positive culture (culture medium with parasite).
Benznidazole and amphotericin B (Amp) were used as reference
drugs of T. cruzi and Leishmania, respectively. The minimum
inhibitory concentration (MIC) for epimastigotes (T. cruzi DM28c
and Y) and promastigotes (L. amazonensis and L. infantum) was
performed using resazurin (125 mM) as an indicator of cellular
metabolic function. MIC was determined as the lowest concentra-
tion of the inhibitor capable of inhibiting in vitro growth of the
2. Materials and methods
2.1. Chemistry
The synthesis of 3-nitro-4-hydroxy-sulfonamides 4–24 was
reported earlier by our group21.
2.2. Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used parasites by spectrophotometric analysis at 490 and 59531. The
for assaying the CA-catalysed CO2 hydration activity22. Phenol red concentration of drug which reduces parasites number by 50%
(at a concentration of 0.2 mM) has been 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 the
ionic strength), following the initial rates of the CA-catalysed CO2
hydration reaction for a period of 10–100 s. The CO2 concentra-
tions ranged from 1.7 to 17 mM for the determination of the kin-
etic parameters and inhibition constants. For each inhibitor, at
least six traces of the initial 5–10% of the reaction have been
used for determining the initial velocity. The uncatalysed 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-deionised water and dilutions up to 0.01 nM
were done thereafter with the assay buffer. Inhibitor and enzyme
solutions were preincubated together for 15 min at room tempera-
ture prior to assay, in order to allow for the formation of the E–I
complex. The inhibition constants were obtained by nonlinear
(IC50) was determined by regression analysis using Microsoft
Excel 2013.
2.3.3. Cytotoxicity assay in macrophages
Cytotoxicity was performed using tetrazolium dye (MTT) colorimet-
ric assay. RAW 264.7 macrophages cells were harvested after con-
fluent monolayer achievement32. The cells were washed twice
with PBS and a cellular suspension of 106 cells/ml was prepared in
fresh DMEM culture medium. Aliquots of 100 ml of the cellular sus-
pension were placed into polystyrene 96-well plates, and then
incubated at 37 ꢂC in a 5% CO2 atmosphere for 6 h in order to
allow macrophage adherence. After this period, the adherent cells
were subjected to treatment with several concentrations of the
drugs (2–256 mM), and then incubated for additional 48 h. Finally,
20 ml of MTT solution (5 mg/ml) were added to each well and the
least-squares methods using PRISM 3 and the Cheng–Prusoff plates incubated for 4 h. Macrophage viability was determined
equation, as reported earlier, and represent the mean from at after formazan crystals solubilisation with DMSO followed by the

1166
A. NOCENTINI ET AL.
absorbance measurement at 570 nm using a SpectraMax M5 spec- decomposition to sulfonic acid that occurs in the nitrating condi-
tions. Both mono- and di-nitro derivatives were obtained in differ-
ent yields and deprotected in acidic media (compounds 4 and 6).
3,5-Dinitro compound 6 was benzoylated to afford 7. A key inter-
trophotometer (Molecular Devices, Sunnyvale, CA).
mediate, 3-amino-4-hydroxy-5-nitro-benzenesulfonamide
8 was
2.3.4. Determination of selectivity index
achieved by reduction of a unique nitro group of 5 with Na2S2O4
and sequential sulfonamide deprotection in acidic media.
Intermediate 8 was subjected to several functionalisation reac-
tions. Acylation reactions produced the di-benzoyl compounds 9
and 10. The pyridinium salt 11 was prepared by the reaction of 8
with the proper pyrylium salt. The light-sensitive derivative 12
was obtained by diazonium salt formation and N2 release in aque-
ous NaNO221. A set of ureas (13–24) was prepared by the reaction
of 8 with commercially available isocyanates, in addition to the
freshly prepared one obtained from 1,3,4,6-tetra-O-acetyl-glucosa-
mine21. Compound 24 was de-acetylated with sodium methoxide
The selectivity index (SI) of tested drugs was calculated as a ratio
of RAW 264.7 macrophages CC50 to parasites IC50. Benznidazole
(Sigma-Aldrich, Milan, Italy) and Amp were used as refer-
ence drugs.
3. Results and discussion
3.1. Chemistry
A set of variably substituted 3-nitrobenzenesulfonamides was
prepared starting from 4-hydrobenzenesulfonamide 121. The
sulfonamide moiety was protected (compound 2) to avoid to give the glycoside 25 (Schemes 1 and 2).
Scheme 1. Synthetic routes to 3-nitrobenzenesulfonamides 3–2421.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1167
3.2. Carbonic anhydrase inhibition
LdcCA inhibition profiles show analogies with those against
TcCA. Again, the simplest derivatives 4, 6, 8, and 12 act as the
best LdcCA inhibitors with KIs of 0.21, 0.34, 0.46, and 0.39 mM,
respectively. Benzoylation of the hydroxy moiety at position 4
markedly reduced the LdcCA inhibitory properties of 7, 9, and 10
(KIs of 4.68, 3.87, and 8.49 mM) as well as incorporation of the
charged pyridinium portion as in 11 (KI of 6.57 mM). Ureido deriva-
tives 13–25 inhibited LdcCA in a rather flat range spanning from
0.86 to 3.65 mM.
As a general trend, most compounds were more effective
against TcCA than CA I, with an SI from 2 to >150, with the
exception of 9 and 10 (Table 2). On the other hand, only few
derivatives (8, 12, 13, 18, 19, 22, and 23) inhibited TcCA more
efficiently than CA II, with SI spanning between 2.5 and 6. LdcCA
was found to be better inhibited than hCA I by most compound,
though the SIs were lower than those TcCA/CA I, and spanned in
the range of 2–50. Most compounds inhibited CA II better than
LdcCA. All compounds inhibited the screened isoforms worse than
the standard AAZ, but the latter did not show selectivity for the
target TcCA and LdcCA compared to the ubiquitous hCAs
(Table 2).
The TcCA and LdcCA inhibitory profiles of compounds 4–25 were
evaluated by applying a stopped flow carbon dioxide hydrase
assay22 in comparison to AAZ as standard CAI and compared to
those against the human off-target CA I and II. The following SAR
can be built from the inhibition data shown in Table 1.
TcCA was effectively inhibited by most 3-nitrobenzenesulfona-
mides investigated here. Inhibition constants (KIs) span in medium
nanomolar to low micromolar range between 0.08 and 10.7 mM.
The derivative showing the lowest steric hindrance, namely 4, acts
as the most potent TcCA inhibitor with a KI of 80 nM. The incorp-
oration of a nitro, amino or hydroxy moiety in position 5 of com-
pound 4 as in 6, 8, and 11 lowered the inhibition efficacy to 160,
240, and 110 nM, respectively. Lowering of inhibition potency was
observed by benzoylation of 6 as in 7 (KI of 2.5 mM) or dybenzoy-
lation of 8 as in 9 and 10 (KIs of 3.5 and 4.8 mM). Hence, enhance-
ment of steric hindrance at position 4 has a deleterious effect on
the compounds binding to TcCA. Incorporation of a positively
charged moiety, such as pyridinium at position 5 in compound
11, caused an evident drop of inhibitory efficacy (KI of 10.7 mM).
Within the set of ureas, the unsubstituted phenyl derivative 13
and the pentafluorinated one 17 showed the most effective inhib-
ition, with KIs of 0.32 and 0.28 mM. All other substitutions on the
ureido-aromatic ring negatively affect the inhibition to
0.35–1.35 mM. Insertion of aliphatic linkers between the urea and
the outer aromatic portion also has a negative outcome on the
KIs of 22 and 23, which increase to 0.74 and 0.4 mM. The glyco-
3.3. Anti-protozoan activity
3.3.1. Trypanosoma cruzi strain DM28c and Y
Ten selected derivatives bearing different substituents at the 3-
nitrobenzenesulfonamide scaffold (4, 6, 8, 10, 11, 17, 18, 19, 21,
sidic derivatives 24 and 25 are micromolar TcCA inhibitors with and 24) were screened for their inhibition activity different species
KIs of 2.47 and 2.14 mM.
of Leishmania and Trypanosoma cruzi.
Scheme 2. Synthesis of derivative 25.
Table 1. Inhibition data of TcCA, LdCA and human CA I and II with sulfonamides reported here and the standard inhibitor acetazola-
mide (AAZ) by a stopped flow CO2 hydrase assay.
KI (lM)a
Compound
R
TcCA
LdCA
hCA I
hCA II
4
6
7
8
0.08
0.16
2.52
0.24
3.54
4.79
10.7
0.11
0.32
0.46
0.51
0.38
0.28
0.91
1.02
0.69
1.35
0.74
0.41
2.47
2.14
0.06
0.21
0.34
4.68
0.46
3.87
8.49
6.57
0.39
1.06
0.98
2.34
2.96
1.36
0.95
2.03
1.86
3.65
0.86
1.02
3.64
2.97
0.09
0.91
4.35
4.79
6.18
1.38
2.92
>50
6.21
>50
5.39
5.20
7.58
0.69
8.21
>50
8.33
5.99
9.29
>50
5.67
4.92
0.25
0.24
0.18
0.84
0.61
0.39
0.46
1.81
0.64
2.78
0.53
0.24
0.21
0.27
5.15
4.33
0.45
1.72
3.08
2.53
1.89
0.86
0.012
9
C6H5
4-F-C6H4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
AAZ
C6H5
4-F-C6H4
4-CF3-C6H4
4-F-3-CH3-C6H3
C6F5
3-CH3O-C6H4
3,4-(OCH2O)-C6H3
3,5-CH3-C6H3
3,5-CF3-C6H3
CH2CH2C6H5
CH2-(2-furyl)
2,4,5-triacetoxy-6-acetoxymethyl-tetrahydro-pyran-3-yl
2,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-3-yl
–
aMean from three different assays, by a stopped flow technique (errors were in the range of 5–10% of the reported values).

1168
A. NOCENTINI ET AL.
The MIC and IC50 values against T. cruzi epimastigote forms of macrophages cells compared to Amp, that has a CC50 of 1 mM
these compounds are shown in Table 3. The experiments showed against both strains.
that no compounds significantly affect the growth of the patho-
gen below 256 mM. The reference drug Bzn showed IC50 values out to be ineffective in vitro against strains of T. cruzi and
against Dm28c clone and strains of 16.56 1.51 and Leishmania. The lack of activity is not a totally new issue in the
Unfortunately, the tested 3-nitrobenzenesulfonamides turned
Y
field of sulfonamide CAIs against pathogens. For instance, some
sulfonamide derivatives demonstrated remarkable in vitro efficacy
in inhibiting the b-CA from the yeast Malassezia globosa, arousing
6.54 1.82 mM, respectively. The assessment of the toxicity of the
selected 3-nitrobenzensulfonamides for Raw 267.4 macrophages
cells showed that most derivatives were less toxic than Bzn (CC50
of 115.14 9.48 mM) with CC50 above 172.65 10.44 mM. anyhow complications in vivo because of permeability problems
through biological membranes33.
Compounds 4 and 6 showed instead comparable toxicity with Bnz
In the context of T. cruzi and Leishmania, some previously
tested sulfonamides showed an absence of anti-protozoan effi-
cacy, which has been related to the lack of permeability through
the biological membranes of the pathogen34–36. Hence, a formula-
tion of such sulfonamides in nano-emulsions (NEs) of clove oil
was attempted to enhance their bioavailability and penetrability
through membranes34,35. The drugs–NEs formulations potently
inhibited the growth of T. cruzi and Leishmania in vitro, with a
huge increase of efficacy over the sulfonamide CAI alone. NEs
turned out as a novel vehicle for the delivery of such hydro-
philic drugs.
Indeed, it should be noted that 3-nitro-4-hydroxybenzenesulfo-
namides reported here are even more hydrophilic, which can
cause difficulties for the compounds to cross the protozoa cell
membrane and inhibit the cytoplasmatic CAs or exert further
actions due to the nitro group. Hence, formulation to enhance the
compounds bioavailability, such as NEs, is being prepared to
evaluate the real anti-protozoan efficacy of these set of nitroaro-
matic CAIs.
with CC50 values of 97.65 11.13 and 100.21 17.27 mM.
3.3.2. L. amazonensis and L. infantum
The MIC and IC50 values of the selected compounds against two
Leishmania species are shown in Table 4. The experiments on
L. amazonensis and L. infantum strains did not show MIC values
below 400 mM. Despite the compounds showed micromolar inhib-
ition of LdcCA, their efficacy turned out to be insignificant when
translated in vitro against the pathogen cell cultures. The refer-
ence drug Amp exhibited IC50 values against the two strains of
1.65 0.28 and 1.77 0.35 mM, respectively. The tested sulfona-
mides showed anyhow remarkably minor toxicity for Raw 267.4
Table 2. Selectivity index (SI) for target protozoan CAs over hCA I and II.
SI (KI1/KI2)
Compound
CA I/TcCA
CA II/TcCA
CA I/LdcCA
CA II/LdcCA
4
6
7
8
11.4
27.2
1.9
25.8
0.4
3.0
1.1
0.3
2.5
0.1
0.1
0.2
5.8
8.7
1.2
0.4
0.6
1.0
5.7
4.2
0.7
1.3
4.2
6.3
0.8
0.4
4.3
12.8
1.0
13.4
0.4
1.1
0.5
0.2
1.3
0.1
0.1
0.3
1.6
2.6
0.5
0.1
0.1
0.2
5.4
2.1
0.2
0.5
3.6
2.5
0.5
0.3
4. Conclusions
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
0.6
0.3
We proposed here nitroaromatic sulfonamides for the treatment
of Chagas disease and leishmaniasis based on CA inhibition. As a
continuation of a previous work of us on N-nitrosulfonamides as
anti-protozoan agents, we studied here benzenesulfonamides
(4–24) bearing a nitro moiety on the aromatic scaffold against
TcCA from T. cruzi, responsible of Chagas disease, and LdcCA from
Leishmania spp. The compounds reported valuable micromolar
inhibition of these two enzymes, in some cases even selective for
>4.7
56.5
>156
11.7
10.2
19.9
2.5
>7.6
15.9
>47.2
5.5
2.2
2.6
0.5
8.6
>24.6
4.5
1.6
9.0
>49.0
12.1
4.4
12.6
>125
2.3
the target CAs over the human ubiquitous CA
I and II.
Unfortunately, a selected set of such derivatives tested in vitro
against multiple strains of T. cruzi and Leishmania did not produce
growth inhibition of the parasites. The lack of anti-protozoan effi-
cacy of sulfonamide type derivatives had been already reported
10.8
>49.0
1.6
2.3
1.7
Table 3. Minimum inhibitory concentration (MIC), IC50 values derived from growth inhibition assays of T. cruzi Dm 28c and Y, determination of cytotoxicity (CC50),
selectivity index (SI50) of compounds 4, 6, 8, 10, 11, 17, 18, 19, 21, and 24.
T. cruzi Dm 28c
IC50 (mM)b
T. cruzi Y
Compound
MIC (mM)a
SIc
MIC (mM)a
IC50 (mM)b
SIc
CC50 (mM)d
4
6
8
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
32
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
32
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
97.65 11.13
100.21 17.27
>256
179.93 21.31
>256
>256
>256
172.65 10.44
>256
10
11
17
18
19
21
24
Bzn
n.d.
n.d.
n.d.
n.d.
>256
115.14 9.48
16.56 1.51
6.54 1.82
18.45 0.32
6.168 0.81
aMinimum inhibitory concentration.
bmM – concentration which reduced the proliferation of epimastigotes by 50%.
cSelectivity index of 50% ¼ CC50/IC50
.
dmM – concentration cytotoxic which reduced 50% of RAW 267.4 cells.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1169
Table 4. Minimum inhibitory concentration (MIC), IC50 values derived from growth inhibition assays of L. amazonensis and L. infantum, determination of cytotoxicity
(CC50) and selectivity index (SI50) of compounds 4, 6, 8, 10, 11, 17, 18, 19, 21, and 24.
L. amazonensis
L. infantum
Compound
MIC (mM)a
IC50 (mM)b
SIc
MIC (mM)a
IC50 (mM)b
SIc
CC50 (mM)d
4
6
8
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
8
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.60
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
8
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.56
97.65 11.13
100.21 17.27
>256
10
11
17
18
19
21
24
Amp
179.93 21.31
>256
>256
>256
172.65 10.44
>256
>256
1.0
n.d.
n.d.
1.65 0.28
1.77 0.35
aMIC – minimum inhibitory concentration.
bIC50 mM – concentration which reduced the number of promastigotes by 50%.
cSI50–selectivity index of 50% ¼ CC50/IC50
.
dCC50 mM – cytotoxic concentration which reduced 50% of RAW 267.4 viability.
by us and justified by low permeability of this class of derivatives
through the cell membranes. The use of carriers such as nanoe-
mulsions allowed to overcome this issue. The application of this
approach has been being carried out for 3-nitrosulfonamides
4–24 to elucidate whether the combination of CA inhibition and
further anti-protozoan actions related to the nitro group could be
a winning anti-infective strategy.
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overcome the global impact of neglected tropical diseases.
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9789241564540/en/.
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drases from Trypanosoma and Leishmania as anti-protozoan
drug targets. Bioorg Med Chem 2017;25:1543–55.
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Disclosure statement
No potential conflict of interest was reported by the authors.
8. Supuran CT. Inhibition of carbonic anhydrase from
Trypanosoma cruzi for the management of Chagas disease:
an underexplored therapeutic opportunity. Future Med
Chem 2016;8:311–24.
Funding
This work was financed in part by a DSFP Project to Profs. Z.
AlOthman and C.T. Supuran from King Saud University, Riyadh,
9. (a) Capasso C, Supuran CT. An overview of the alpha-, beta-
and gamma-carbonic anhydrases from Bacteria: can bacterial
carbonic anhydrases shed new light on evolution of bac-
teria? J Enzyme Inhib Med Chem 2015;30:325–32. (b) Del
Prete S, Vullo D, Fisher GM, et al. Discovery of a new family
of carbonic anhydrases in the malaria pathogen Plasmodium
falciparum – the g-carbonic anhydrases. Bioorg Med Chem
Lett 2014;24:4389–96. (c) Supuran CT. Structure-based drug
discovery of carbonic anhydrase inhibitors. J Enzyme Inhib
Med Chem 2012;27:759–72. (d) Ozensoy Guler O, Capasso C,
Supuran CT. A magnificent enzyme superfamily: carbonic
anhydrases, their purification and characterization. J Enzyme
Inhib Med Chem 2016;31:689–94. (e) Capasso C, Supuran CT.
Bacterial, fungal and protozoan carbonic anhydrases as drug
targets. Expert Opin Ther Targets 2015;19:1689–704.
10. Pan P, Vermelho AB, Capaci GR, et al. Cloning, characteriza-
tion, and sulfonamide and thiol inhibition studies of an a-
carbonic anhydrase from Trypanosoma cruzi, the causative
agent of Chagas disease. J Med Chem 2013;56:1761–71.
11. De Menezes DR, Calvet CM, Rodrigues GC, et al. Hydroxamic
acid derivatives: a promising scaffold for rational compound
optimization in Chagas disease. J Enzyme Inhib Med Chem
2016;31:964–73.
~
Saudi Arabia, by the Coordenac¸ao de Aperfeic¸oamento Pessoal de
ꢀ
Nıvel Superior-Brasil (CAPES), Grant Code 001, by grants from
Fundac¸ao de Amparo a Pesquisa do Estado do Rio de Janeiro
(FAPERJ, Ed. 124/2013; Ed. 15/2015), Brazil, and by Conselho
Nacional de Desenvolvimento Cientıfico e Tecnologico (MCTI-
~
ꢁ
ꢀ
ꢀ
CNPq, produtividade em pesquisa 303265/2015-9), Brazil.
ORCID
Alane B. Vermelho
Claudiu T. Supuran
http://orcid.org/0000-0001-5926-4172
http://orcid.org/0000-0003-4262-0323
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