Trypanosoma cruzi Malic Enzyme Is the Target for Sulfonamide Hits from the GSK Chagas Box

Article abstract of DOI:10.1021/acsinfecdis.1c00231

Chagas disease, an infectious condition caused by Trypanosoma cruzi, lacks treatment with drugs with desired efficacy and safety profiles. To address this unmet medical need, a set of trypanocidal compounds were identified through a large multicenter phen

Full text of DOI:10.1021/acsinfecdis.1c00231

pubs.acs.org/journal/aidcbc
Article
Trypanosoma cruzi Malic Enzyme Is the Target for Sulfonamide Hits
from the GSK Chagas Box
Gustavo F. Mercaldi,∥ Amanda G. Eufrásio,∥ Americo T. Ranzani, Jessica do Nascimento Faria,
Sabrina G. R. Mota, Michelle Fagundes, Marjorie Bruder,* and Artur T. Cordeiro*
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ABSTRACT: Chagas disease, an infectious condition caused by
Trypanosoma cruzi, lacks treatment with drugs with desired efficacy
and safety profiles. To address this unmet medical need, a set of
trypanocidal compounds were identified through a large multicenter
phenotypic-screening initiative and assembled in the GSK Chagas Box.
In the present work, we report the screening of the Chagas Box against
T. cruzi malic enzymes (MEs) and the identification of three potent
inhibitors of its cytosolic isoform (TcMEc). One of these compounds,
TCMDC-143108 (1), came out as a nanomolar inhibitor of TcMEc, and
14 new derivatives were synthesized and tested for target inhibition and
efficacy against the parasite. Moreover, we determined the crystallo-
graphic structures of TcMEc in complex with TCMDC-143108 (1) and
six derivatives, revealing the allosteric inhibition site and the determinants of specificity. Our findings connect phenotypic hits from
the Chagas Box to a relevant metabolic target in the parasite, providing data to foster new structure−activity guided hit optimization
initiatives.
KEYWORDS: Trypanosoma cruzi, malic enzyme, sulfonamides, GSK Chagas Box, Tres Cantos anti-kinetoplastids set,
target-inhibitor structure
cell dead.9 In bacteria and protozoan parasites, particularities in
INTRODUCTION
■
structure and allosteric regulation suggest that ME might be
Malic enzymes (MEs) are preserved in several living
explored for the development of new antimicrobial agents.
organisms, from fungi to mammals, and catalyze the oxidative
While the human mitochondrial ME2 is activated by fumarate
decarboxylation of malate to pyruvate with the concomitant
and inhibited by ATP,10 in Trypanosoma cruzi5 and Leishmania
reduction of NAD+ or NADP+. In humans, there are three ME
mexicana6 the cytosolic isoform is allosterically activated by L-
isoforms: the cytosolic NADP+-dependent ME1 (EC
aspartate. Moreover, these protozoan parasites have only two
1.1.1.40),1 the mitochondrial NADP+-dependent ME3 (EC
ME isoforms, one in the cytosol (MEc) and another in the
1.1.1.40),2 and the mitochondrial ME2 (EC 1.1.1.38), which
mitochondrion (MEm), and both are NADP+-dependent.
can use both NAD+ and NADP+ as the cofactor, but with
Crystal structures are presently available for ME111,12 and
preference for the first one3. Moreover, ME2 is the only
ME213−15 but not for ME3. Human MEs are all tetrameric
isoform in humans which is allosterically activated by fumarate
proteins, and monomers are formed by four domains, named
and inhibited by ATP. Allosteric activation is not an exclusive
A−D, from the N- to C-terminal. The characterization of ME2
property of the mammalian ME2, as similar enzymatic
complexes with its usual ligands allowed Chang and Tong to
behavior is also observed in bacterial,4 protozoan,5,6 round-
propose a molecular mechanism for its allosteric activation by
worm,7 and plant8 homologous enzymes.
fumarate and inhibition by ATP.16 According to the proposed
In general, the biological relevance of ME is associated with
model, the tetramer may exist in an equilibrium among four
NADPH production and its essential role in biosynthetic
distinct forms (open I, open II, closed I, and closed II), which
pathways and neutralization of reactive oxygen species.
Interfering with ME enzymatic activity has been considered
an attractive strategy toward drug discovery against different
Received: April 26, 2021
diseases. In cancer, pancreatic adenocarcinoma cells recur-
rently have the ME2 gene deleted because of passenger
deletions of other tumor suppressor genes. ME3 depletion in
ME2-null pancreatic adenocarcinoma cells brakes redundancy
for NADPH production in mitochondria and induces selective
https://doi.org/10.1021/acsinfecdis.1c00231
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A

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Figure 1. Screening of the Chagas Box trypanocidal compounds reveals potent inhibitors of the parasitic cytosolic malic enzyme. 218 Compounds
from Chagas Box were tested at 5 μM in duplicate against the cytosolic (A) and mitocondrial (B) isoforms of T. cruzi malic enzymes (TcMEc and
TcMEm, respectively). Compounds TCMDC-143108 (1) (C), TCMDC-143209 (2) (D), and TCMDC-143194 (3) (E) had their activity
confirmed and IC50 values determined in dose−response assays performed with resupplied, ready to use sample plates. Percentage of activity (%
Act) was obtained by normalization of data using positive and negative controls. *EC50 values were obtained from previously reported studies.18
can be interconverted through small rearrangements of its
domains. The reaction is catalyzed by the approximation of
malate and NADP+ binding domains (active site closure),
which can happen in the presence or absence of fumarate. The
binding of fumarate to the ME2 dimeric interface facilitates
domain rearrangement, which promotes the transition from
state I to II and favors catalysis.
its ligand free form and in complex with hit compound 1 and
six analogues. To the best of our knowledge, TCMDC-143108
(1) is the first hit from the GSK Chagas Box to be reported as
a T. cruzi metabolic enzyme inhibitor and to appear in a
crystallographic complex with its target. This data can now be
explored for structure-based hit-to-lead optimization of the
trypanocidal compounds.
Our initial hypothesis was that structural and functional
differences between human and protozoan parasites could be
explored for the identification of selective inhibitors of MEs
from T. cruzi, the causative agent of Chagas disease. In our
previous work, we had identified sulfonamides as nanomolar
inhibitors, with high selectivity toward the cytosolic T. cruzi
ME isoform (TcMEc).17 Moreover, a single concentration
screening for TcMEc inhibitors against the Chagas Box, a
collection of 222 hit compounds organized by GSK after a
phenotypic high throughput screen of 1.8 million com-
pounds,18 indicated the presence of new TcMEc inhibitors in
this collection of anti-T. cruzi hits.
In the present work, we report the results from the screening
of the Chagas Box against T. cruzi cytosolic and mitochondrial
ME isoforms and the identification of three compounds with
remarkable activity over TcMEc. In addition, we describe the
synthesis of TCMDC-143108 (1), the most potent TcMEc
inhibitor, as well as of 14 new derivatives of this molecule. This
new series was assayed for TcMEc inhibition as well as for their
efficacy through an image-based assay against T. cruzi
intracellular forms infecting H9c2 rat cardiomyocytes. More-
over, we determined crystallographic structures for TcMEc in
RESULTS AND DISCUSSION
■
In recent decades, there has been a growing trend for the use
of phenotypic-based screening to identify bioactive molecules,
particularly in drug discovery programs for infectious
diseases.19−23 As such, this strategy has been employed for
the identification of compounds active against protozoan
parasites.18,19,21 Although successful for identifying novel hits
with distinct chemical scaffolds, an intrinsic drawback of this
approach is the lack of information about the primary mode of
action of the selected molecules, creating the need for target
deconvolution or identification.22 Aiming to help filling this
gap, we decided to test Chagas Box compounds18 against the
T. cruzi malic enzyme, a key enzyme in energetic metabolism
and a promising target for Chagas disease under investigation
in our group.17,24,25
T. cruzi Malic Enzymes are Inhibited by Small
Molecules from the Chagas Box. Some of the previously
reported ME inhibitors17 share common structural features
with the compounds present in the Chagas Box,18 which
prompted us to screen this collection for inhibitors of cytosolic
and mitochondrial T. cruzi ME isoforms (Figure 1A and B).
B
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Table 1. Data Collection and Refinement Statistics of Structures of TcΔMEc in Complex with Inhibitors
ligand
1
31
41
40
33
39
34
-
data collection statistics
resolution (Å)
47.50−2.14
47.69−1.88
(1.92−1.88)
47.69−1.70
(1,73−1.70)
47.29−2.10
(2.16−2.10)
47.4−1.9
47.40−1.78
(1.82−1.78)
47.43−1.95
(2.00−1.95)
29.22−1.55
(1.58−1.55)
(2.20−2.14)
(1.94−1.90)
space group
P43212
P43212
P43212
P43212
P43212
P43212
P43212
P43212
cell parameters
a = b; c (Å) 73.5; 233.7
73.8; 234.5
90
73.8; 234.5
90
73.2; 233.1
90
73.4; 233.6
90
73.4; 233.2
90
73.4; 233.8
90
73.3; 233.8
90
α = β = γ
90
(deg)
X-ray source
W01B-MX2
(LNLS)
W01B-MX2
(LNLS)
W01B-MX2
(LNLS)
W01B-MX2
(LNLS)
W01B-MX2
(LNLS)
W01B-MX2
(LNLS)
W01B-MX2
(LNLS)
I03 (diamond)
wavelength (Å) 1.46
1.46
1.46
1.46
1.46
1.46
1.46
0.98
no. of unique
36 499 (2879) 53 637 (3114) 72 505 (3730) 35 984 (2272) 50 906 (3215) 61 897 (3463) 47 202 (2994) 93 475 (4549)
reflections
a
Rmeas
0.14 (1.18)
99.9 (98.8)
0.09 (1.03)
99.4 (91.9)
0.06 (0.64)
99.9 (99.0)
0.08 (0.58)
95.1 (75.0)
0.09 (0.83)
99.3 (98.6)
0.06 (0.95)
99.7 (99.9)
0.10 (0.70)
99.0 (90.8)
0.09 (1.68)
100.0 (100.0)
completeness
(%)
⟨I/σ(I)⟩
CC1/2
multiplicity
13.0 (1.3)
19.4 (1.9)
24.6 (2.7)
14.4 (2.4)
12.6 (1.6)
20.1 (1.9)
9.2 (1.1)
17.9 (1.7)
0.999 (0.613)
14.4 (12.5)
19.7
11.1 (4.8)
11.1 (7.5)
22.2
11.7 (6.5)
18.1
6.1 (4.7)
32.0
6.0 (3.8)
24.2
6.0 (5.5)
23.4
5.2 (2.6)
31.6
Wilson B factor 35.7
(Å2)
refinement statistics
Rwork
Rfree
amino acid
residues
0.21 (0.34)
0.24 (0.39)
545
0.19 (0.30)
0.22 (0.33)
544
0.17 (0.23)
0.20 (0.24)
543
0.18 (0.26)
0.20 (0.29)
540
0.18 (0.28)
0.20 (0.28)
544
0.18 (0.27)
0.20 (0.31)
542
0.19 (0.33)
0.21 (0.33)
544
0.18 (0.27)
0.20 (0.29)
547
heteroatoms (n) 61
58
54
48
46
47
47
28
solvent (n)
74
337
525
226
318
332
269
343
avg B factor
(Å2)
protein
ligand
citrate
HEPES
water
42.8
77.7
60.0
51.4
37.3
29.3
38.8
52.6
31.7
33.1
23.3
19.8
39.4
26.7
31.4
38.9
34.8
31.5
32.4
29.7
26.7
35.4
43.7
24.1
30.6
29.4
29.7
43.6
40.1
35.5
34.1
33.2
33.4
40.2
36.4
RSCC/RSR
ligand
citrate
HEPES
0.92/0.20
0.92/0.13
0.93/0.10
0.95/0.12
0.93/0.13
0.95/0.09
0.98/0.06
0.90/0.15
0.94/0.09
0.98/0.08
0.96/0.09
0.98/0.08
0.95/0.09
0.98/0.07
0.95/0.09
0.97/0.11
0.96/0.08
0.94/0.10
0.94/0.07
validation
Ramachandran
favored
allowed
PDB ID
529 (97%)
14 (3%)
6W29
529 (97%)
12 (2%)
6W2N
530 (98%)
10 (2%)
6W49
524 (98%)
10 (2%)
6W53
535 (98%)
9 (2%)
6W56
522 (97%)
14 (3%)
6W57
524 (97%)
17 (3%)
6W59
534 (98%)
10 (2%)
7MF4
a
Rmeas = Σhkl[N/(N − 1)]1/2 Σi|Ii(hkl) − ⟨I(hkl)⟩|/ΣhklΣiIi(hkl).
Among the trypanocidal compounds tested, three molecules
(TCMDC-143108 (1), TCMDC-143209 (2), and TCMDC-
143194 (3)) completely abolished TcMEc activity while only
showing a weak effect over TcMEm at the concentration of 5
μM. In addition, these molecules were shown to be potent
inhibitors of TcMEc in confirmatory variable concentration
assays (Figure 1C−E). Notably, the sulfonamide 1 which is
known to be active against T. cruzi intracellular amastigotes
with an EC50 of 0.5 μM,18 showed a remarkable inhibitory
activity over TcMEc with an IC50 value of 80 nM.
Although we have previously reported potent TcMEc
inhibitors with a diaryl-sulfonamide scaffold, these molecules
showed weak or no effects over T. cruzi epimastigote growth.17
Further assays with a subset of 64 of the previously identified
molecules, having TcMEc IC50 values between 6.2 μM and 3.2
nM, showed that these molecules were not effective against
intracellular amastigote forms infecting H9c2 cardiomyocytes
(Supporting Information, Table S1). Thus, compound 1 is a
new TcMEc inhibitor with a better in vitro activity profile,
showing trypanocidal activity in distinct assay formats, low
host-cell toxicity, and no activity over CYP51.18
A Crystal Structure of the TcMEc:Inhibitor Complex
Reveals the Target Allosteric Site. To establish the
determinants of binding and mechanism of inhibition of 1,
we prepared sufficient quantities thereof (please refer to the
synthesis section for further details) in order to solve the
crystal structure of TcMEc in complex with the inhibitor
(Table 1). Bipyramidal crystals of the truncated TcMEc
(TcΔMEc, residues Q12-L565), with dimensions of up to 0.4
mm, were obtained after 1−2 weeks incubation at 18 °C.
These crystals were soaked with 1 for 24 h before collection of
diffraction data using synchrotron radiation. TcΔMEc crystals
C
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Figure 2. Crystal structure of T. cruzi malic enzyme (TcMEc) in complex with the trypanocidal compound TCMDC-143108 (1). (A) Overall fold
of TcMEc showing domain organization. (B) TcMEc dimer built by crystallographic symmetry operations, showing symmetry mate macromolecule
and sulfonamide inhibitor (1) in light colors and with the location of the putative catalytic center indicated. (C) Inspection of the solvent accessible
surface of TcMEc dimer shows that the inhibitors are buried in a pocket found at the dimer interface, as revealed by the sliced view in the middle of
the macromolecule. (D) TcMEc residues forming the allosteric binding site of 1 (sticks with magenta carbons). Side chains are represented as
sticks, and carbon atoms are colored according to domains organization.
belonged to the tetragonal system and molecular replacement
placed one polypeptide chain in the assimetric unit. In the
refined structure (PDB 6W29), each polypeptide chain
contained one bound molecule of 1, in addition to highly
ordered HEPES and citrate molecules that were present in the
crystallization solution.
symmetry related inhibitor (1) molecules are bound to
separated but identical sites (Figure 2B).
Inspection of the solvent accessible surface showed that the
ligand’s binding site is completely buried into the dimer
interface, without evident access to the solvent and distant
from the substrate or cofactor binding sites, as described for
other MEs12−16,28,29,31 (Figure 2C). Consequently, local
structural adjustments would be required for the ligand to
access its binding pocket. Notably, in both human mitochon-
drial and Ascaris suum MEs, the site of the allosteric activator
fumarate has also been mapped at the dimer interface.15,16,26,30
Comparison of the TcMEc structure with that of the human
mitochondrial isoform (HsME2, PDB 1GZ3) shows that
ligand 1 superposes with fumarate (Figure S3). This
observation indicates that the sulfonamide inhibitor (1)
could be binding to the allosteric site in TcMEc, which is
activated by aspartate.5 Previous studies with the mitochon-
drial isoform of the human ME (HsME2) have shown that the
enzyme could be allosterically inhibited by fumarate analogues
or by embonic acid.35,36 Despite crystallographic structures for
HsME2 in complex with allosteric inhibitors not yet being
available, site directed mutagenesis was used to demonstrate
The TcMEc protomer has an overall fold that resembles the
structures of MEs found in eukaryotes, like hu-
mans,11,13−15,26,27 pigeons,12 plants,28 and nematodes,29,30
and some prokaryotes like Mycobacterium tuberculosis, all
organized in four domains named A to D (Figure 2A).
Contrary to the majority of the eukaryotic large unit MEs,
TcMEc does not have N- nor C-terminal extensions that are
known to stabilize the tetrameric form of the enzyme14,16,28,31
(Figure S1). Further, TcMEc showed an estimated molecular
mass equivalent to a dimer in size exclusion chromatography
(SEC) experiments (Figure S2), which is in agreement with
previous studies.32,33 The TcMEc biological unit, which
corresponds to the dimer, can be built by crystallographic
symmetry operations (e.g., symmetry mates X, Y, Z and Y, X,
−Z). The dimeric interface is formed exclusively by residues
from domains A and B, it buries an area of 4580 Å2,34 and two
D
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Scheme 1. Synthesis of Sulfonamides 1 and 30−43
that modifications in the allosteric site causes a desensitization
of the enzyme toward fumarate and negatively impact the
binding affinity of embonic acid.35,36 For instance, mutations of
HsME2 residues R67 and R91 generate mutants insensitive to
embonic acid inhibition and also fumarate activation. In
TcMEc, residues R55 and T79, which correspond to R67 and
R91 of HsME2 (Figure S1), are among the residues forming
the binding pocket of 1 and R55 makes direct interactions with
the inhibitor (Figure 2D). Eventually, mutations in the TcMEc
allosteric pocket could prevent the binding of inhibitors and
result in acquisition of drug resistance. However, if these
mutations compromise the enzyme activation by aspartate, that
can have negative effects on parasite viability and growth.
These are relevant issues that need to be clarified by further
investigation.
follows a similar allosteric activation model to that proposed
for human ME2,16 both TcMEc structures are in an open state.
Therefore, compound 1 may lock the enzyme in an inactive
open conformation, preventing aspartate binding and domains
reorganization.
The allosteric inhibition pocket in TcMEc is formed by
residues from domains A and B, from two distinct polypeptide
chains (Figure 2D). The main interactions of 1 with the
protein involve a π-stacking between the central phenyl ring of
the inhibitor and residue F116, a hydrogen bond between the
oxygen from the carboxamide with residue N23, and a network
of hydrogen bonds between the sulfonamide group and
residues Q52, R55, and N80. It is worth noting that residue
T26, near Q52, is at a distance that would also allow for
molecular interactions with the sulfonamide group of the
inhibitor. Finally, the difluorophenyl and methoxyphenyl
moieties appear to be engaged mainly in van der Waals
interactions.
Superposition of the TcMEc structures of the ligand-free
form and the complex with compound 1 shows that these two
structures have the same conformation and domain arrange-
ment (Figure S4). Presuming that the trypanosomal enzyme
E
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c
Table 2. Target Inhibition, Binding, and Anti-T. cruzi activity of Compound 1 and Derivatives
a
b
c
Melting temperature (Tm) for TcMEc apo form is equal to 52.2 °C. Benznidazole EC50 = 2,2
0.4. n.d.: not detected at highest assay
concentration of 32 μM. n.a.: not assayed.
Synthesis of Compound 1 and Derivatives. Following
the identification of TcMEc as a target for TCMDC-143108
(1), the synthesis of the original molecule and derivatives was
projected. Despite 1 already being a potent inhibitor of
TcMEc, new derivatives were planned to gain a deeper
understanding on the determinants of binding, so as to allow
for future modifications of the ligand to address potential
druglike property issues.
The synthesis was devised in three steps consisting of (1)
the formation of a diaryl sulfonamide (6−17) from the
corresponding 3-nitrobenzenesulfonyl chlorides (4 (X = H)
and 5 (X = Cl)) and an aniline; (2) subsequent reduction of
the nitro group and; (3) coupling of the resulting aniline (18−
29) with a carboxylic acid or acyl chloride, which would yield
the desired target compounds (Scheme 1). Compound 1
(TCMDC-143108) was prepared following this route to
achieve the protein crystal complex. At first, we set out to
explore different groups at the sulfonamide’s aniline moiety,
maintaining the 3,5-difluorobenzoic acid moiety. Overall, the
synthesis of the target compounds proceeded smoothly, giving
compound 1 and derivatives 30−35. However, on reacting 3,5-
difluorobenzoic acid chloride with chlorinated intermediates
24 (X = Cl, R1 = 4-OMe) and 26 (X = Cl, R1 = 2-F-4-OMe),
an unexpected switch in reactivity favored acylation of the
sulfonamide nitrogen, instead of the aniline nitrogen, as
verified by NMR analyses. One could propose that a
combination of aniline nucleophilicity reduction due to the
neighboring chlorine atom and the electron-donating feature of
the methoxy group para to the sulfonamide nitrogen
accounted for the resulting products 35 and 36. The four
other chlorinated derivates (25, 27−29), on the other hand,
reacted as planned at the aniline nitrogen, yielding target
compounds 37−40. In order to also probe the binding pocket
of the 3,5-difluorobenzamide moiety of compound 1, three
more compounds were prepared. First, a heterocyclic group
was introduced under amino acid coupling conditions with
aniline 19, yielding pyrazine derivative 41. Second, 3-fluoro-4-
methoxybenzoic acid chloride was reacted with aniline 18 to
afford compound 42. Finally, we decided to prepare a
compound lacking the amide functionality to assess its
importance for ligand binding. Intermediate 19 was thus
reacted with 4-methoxybenzaldehyde under reductive amina-
tion conditions to furnish benzylamine derivative 43.
Effect of the Target Compounds on TcMEc Activity
and Stability. The newly synthesized compounds were
subjected to concentration-dependent enzymatic inhibition
and differential scanning fluorimetry (nanoDSF) assays in
order to calculate IC50 and melting temperatures (Tm) values
over recombinant TcMEc (Table 2).
F
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The in house synthesized compound 1 and most of its
derivatives returned IC50 values below 80 nM in the TcMEc
inhibition assay. Compounds 30−33, 37, and 38, with
variations at the sulfonamide aniline moiety, but all bearing
the 3,5-difluorobenzamide group, were usually better than
reference hit 1 (IC50 = 29.0 nM), especially the ones with a 2-
fluoro substitution pattern (30, 31, and 37), which displayed
IC50 values below 10 nM. Only the trifluoromethoxylated
analogues 34 and 40 proved to be less potent against TcMEc.
Derivative 41, bearing an electron-rich pyrazine moiety instead
of the hydrophobic and electron-deficient 3,5-difluorobenza-
mide moiety still had an IC50 value similar to 1, possibly
enabled by the 2-fluorine atom on the sulfonamide aniline.
Moving from a difluorobenzamide (1) to a 3-fluoro-4-
methoxybenzamide (42) did not alter the IC50 value.
Compounds 35 and 36 which were the products of
sulfonamide acylation rather than aniline acylation in the
final step, thus having a quite different structure, did not inhibit
TcMEc activity and neither did benzylamine derivative 43,
indicating that the amide bond strongly contributes to binding
interactions with the protein.
NanoDSF studies were also performed to evaluate the effect
of the compounds on TcMEc thermal stability. In the absence
of ligands, TcMEc returned a Tm of 52 °C. As observed in
Table 2, compounds with IC50 values below 100 nM caused a
marked stabilization of TcMEc, increasing its Tm by more than
12 °C. In line with the enzymatic inhibition data, compounds
35 and 36 had no effect over TcMEc thermal stability (ΔTm <
1 °C). Interestingly, compounds 37−40, which have a chlorine
atom on the central phenyl ring, showed even higher ΔTm
values in comparison with their nonchlorinated pairs, 30 and
32−34, respectively (Figure 3). The increase in ΔTm among
these four pairs of compounds varied from 3.6 to 5.4 °C.
Altogether, these compounds indicate that chlorine atoms
contribute with key interactions in stabilizing the TcMEc−
inhibitor complexes, the nature of which could be revealed by
crystallographic structures of TcMEc in complex with ligands
33, 34, 39, and 40.
Figure 3. Inhibition and thermal stability of T. cruzi cytosolic malic
enzyme by compound 1 and novel derivatives. IC50 values were
calculated from concentration response inhibition assays. Melting
temperatures (Tm) were calculated from nanoDSF denaturation
curves. Dashed lines connect pairs of compounds that differ by the
presence of a chlorine atom in the central phenyl ring. Symbols were
colored according to n-octanol/water partition coefficient (cLogP)
calculated using DataWarrior.37 Inset plot shows ΔTm values for 30
and 32−34 (gray circles, nonchlorinated molecules) and 37−40
(black circles, chlorinated compounds), with means and standard
deviations represented for each group. P value calculated using paired
t test in GraphPad Prism 6.00.
Crystal Structures of TcMEc in Complex with New
Sulfonamides and Structure−Activity Relationships.
Crystal structures for six of the sulfonamide derivatives were
obtained to support our proposals of structure−activity
relationships (Table 1). Superposition of all complexes showed
that all ligands occupy the same allosteric inhibition site with
identical binding modes to that observed for compound 1
(Figure 4).
Looking first at complex TcMEc-31 (Figure 5A), compound
31 (IC50 = 9.4 nM) bearing an additional fluorine atom at the
2-position of the aniline moiety in comparison to 1 (IC50
=
Figure 4. Superposition of the seven distinct sulfonamides for which a
crystal structure was obtained in complex with TcMEc. All inhibitors
(sticks with carbons in magenta) occupy the allosteric inhibition site
located at the dimer interface through a similar binding mode.
Molecules represented in the structure: 1, 31, 33, 34, and 39−41.
PDB IDs of complexes used to prepare the figure are reported in
Table 1.
29.0 nM), the 3-fold increase in potency could be rationalized
by the proximity of acidic protons of residues R55 and Q59
which seem to be engaging in hydrogen bonding with the
electronegative fluorine atom (Figure 5B). These additional
interactions are also featured in the TcMEc-41 complex
(Figure 5C); however, in this case, they seem to counteract a
potential loss of potency due to the polar pyrazine ring at the
other extremity of the ligand (IC50 (41) = 33.6 nM). In fact,
nearby water molecules are found to connect the heterocyclic
ring of ligand 41 to protein residues Y122 and R41 (Figure
5D), potentially imparting an entropy loss due to the solvation
penalty. Next, a group of four protein−ligand complexes is
worth discussing, namely, the ones with compounds 33, 34
and 39, 40. Their aniline moieties are substituted with
difluoromethoxyl and trifluoromethoxyl groups, respectively,
and each pair was also prepared with an additional chlorine
atom on the central phenyl ring. Although the position of
compounds 33 and 34 in their respective binding site is
identical (Figure 5E and F, respectively), and no major residue
shifts were noted, a slight decrease in potency was noted
moving from the difluoromethoxy to the trifluoromethoxy
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Figure 5. Binding mode of the TcMEc sulfonamide inhibitors revealed by crystal structures. Specific interactions of TcMEc residues with inhibitors
are shown for compound 31 (A and B), 41 (C and D), 33 (E), 34 (F), 39 (G), and 40 (H and I). Amino acid residues have been colored
according to the domain architecture (see Figure 2), and inhibitors are shown as sticks with magenta carbons.
substitution. Their chlorinated counterparts (39 and 40 in
Figure 5G and H, respectively), however, cause the expulsion
of a water molecule near the Y122 residue in order to establish
an interaction compatible with a halogen bond with Y122
(Figure 5I). In fact, the recorded geometric data (d[O···Cl] =
3.5 Å; angle O···Cl−C = 160.5°) are in line with this
assumption.38
As mentioned earlier, while the addition of this chlorine
atom did not necessarily increase the potency of these
compounds in enzymatic assays, a clear contribution regarding
protein stabilization was noted in nanoDSF assays (Table 2
and Figure 3). This makes sense, given that the halogen bond
occurs between the ligand, which is mainly “tethered” by
residues from one molecule of the dimer, and the Y122 residue
from the other polypeptide chain forming the dimer. Finally,
complexes of TcMEc with distinct derivatives (Figure 5) show
that the carbonyl of the amide group of the inhibitors interacts
with the protein residue N23, while the nitrogen interacts with
a water molecule that forms an interaction network with
residues Y122 and R41, helping to explain the loss of activity of
compound 43 (Table 2) and reinforcing the relevance of this
amide moiety for ligand binding.
of the EC50 value previously reported for TCMDC-143108
(see Figure 1C) and our synthetic version (1) (Table 2) shows
that the results are in close agreement, allowing for the
comparison of data from both reports. Among the new set of
TcMEc inhibitors with IC50 < 100 nM, none of the derivatives
were cytotoxic to host cells (rat cardiomyocytes) at 32 μM, the
highest assayed concentration (HAC) in the phenotypic
assays. Compounds 31−34 showed good dose−response
profiles (Figure 6A), with calculated EC50 values equivalent
to or better than that of benznidazole (BNZ), the reference
drug in Chagas disease (Table 2). Compound 30, however,
bearing a 2-fluoroaniline moiety, was not active at 32 μM
(HAC). The chlorinated compounds 37, 38, and 40 were
effective against the parasite but worse than BNZ, with EC50’s
higher than 10 μM. Despite being good inhibitors of TcMEc,
compounds 39 and 41 were not effective against the parasite at
the HAC, while compound 42 was slightly less active than
BNZ. Overall, derivatives 30 and 37−40 bearing an halogen at
the aniline or central sulfone moiety, despite accounting for
stronger protein binding or stabilization, showed a decreased
or absence of in vitro efficacy. A similar trend was observed for
compounds that had a more electron-rich amide moiety (41
and 42), in comparison to 3,5-difluorobenzamide. Thus, these
observations suggest that electronic effects may modulate the
anti-T. cruzi effectiveness of these sulfonamides. Whether it
affects compound solubility, membrane permeability, or
metabolic stability still needs to be investigated.
In Vitro Activity of Sulfonamides against T. cruzi and
Host Cells. The in vitro activity of TCMDC-143108 (1) was
initially assessed by Pen
̃
a and collaborators18 in an image-based
assay against intracellular T. cruzi (Y strain) amastigote forms
infecting H9c2 rat cardiomyocytes. Here we used the same
parasite strain, host cell line, and assay format to validate
compound 1 and derivatives synthesized in house. Comparison
Looking further into the biological activity of the best
compounds, image analyses of H9c2 cells infected by T. cruzi
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Figure 6. Concentration−response curves and image analyses of T. cruzi (Y strain) intracellular assays. (A) Normalized concentration−response
curves for quantification of host cells (black circles) and intracellular T. cruzi (white circles). Dashed red line indicates the correspondent EC50 for
benznidazole (BNZ). Box plots for percentage of infected cells (B) and total host cells (C) per image (N = 15). (D) Representative images (left)
and zoom from blue boxes (right) for DMSO, BNZ, and compounds 1, 33, and 34. Host cell nuclei are colored in yellow, and T. cruzi amastigotes
appear as white spots in the cytoplasm of infected cells (indicated by blue arrows).
(Y strain) and treated for 72 h with 1 μM 1 and 3.1 μM 33 and
34 showed a significant reduction in the percentage of infected
cells, a similar phenotype requiring 10 μM BNZ (Figure 6B).
Moreover, at those concentrations, compounds had no
significant impact over the count of total cells, which further
supports the absence of cytotoxicity in rat cardiomyocytes
(Figure 6C). Nevertheless, a detailed inspection of the images
permits one to view cells with putative remaining parasites
(Figure 6D). Whether these are dormant T. cruzi amastigotes39
that could lead to the recovery of infection after removing drug
pressure remains to be investigated.
nifurtimox, with toxicity issues and low efficacy for the chronic
stage of the disease. Therefore, new anti-Chagas drug discovery
initiatives should prioritize molecules that are effective against
the intracellular forms of the parasite which are associated with
chronic Chagas disease. Recently, high-throughput phenotypic
screenings revealed a significant number of compounds active
against intracellular T. cruzi forms. Characterization of the
mode of action and target identification for phenotypic hits can
accelerate their development into new drug candidates. In the
present work, we demonstrate that the T. cruzi cytosolic malic
enzyme (TcMEc) is the target for three different compounds
from the Chagas Box. One of these new TcMEc inhibitors,
TCMDC-143108 (1), and 14 new analogues where synthe-
sized and tested for target inhibition, efficacy against T. cruzi
amastigotes, and host cell cytotoxicity. Overall, four com-
pounds of this series showed nanomolar target inhibition, low
CONCLUSIONS
■
According to the World Health Organization, 6−7 millon
people worldwide are infected with T. cruzi. The current
chemotherapy relies on only two drugs, benznidazole or
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micromolar efficacy against the intracellular parasite, and no
cytotoxicity for the host cells. Moreover, the TcMEc crystal
structure and complexes with seven new inhibitors were
described here for the first time.
the following conditions: 20 μM NADP+, 2 mM MnCl2, 10
μM resazurin, 0.4 mM aspartate, 2 U/mL diaphorase, 0.3 nM
TcMEc, and 1.3 mM malate. Similarly, for TcMEm, the assays
were set as follows: 10 μM NADP+, 2 mM MnCl2, 10 μM
resazurin, 2 U/mL diaphorase, 0.8 nM TcMEm, and 1.0 mM
malate. The reactions for both enzymes were performed in an
assay buffer containing 50 mM Tris-HCl, 50 mM NaCl, and
0.01% triton X-100, pH 7.5. Reactions were always started by
the addition of malate and followed by measuring the
The target−inhibitor complexes reveal that these sulfona-
mides bind to the aspartate allosteric activation site, located at
the enzyme’s dimeric interface, blocking it in an inactive state.
The new inhibitors all bind in a very similar fashion, allowing
the identification of the most relevant interactions and the
establishment of clear structure−activity relationships. It is
worth noting that sulfonamides are present in several FDA-
approved drugs and are readily accessible from a synthetic
point of view, thus making the new TcMEc inhibitors a
promising starting point for further drug development in
Chagas disease. Moreover, the here reported TcMEc inhibitors
could now be used in chemical biology studies to investigate
target engagement and TcMEc essentiality for distinct T. cruzi
forms.
formation of resorufin (λExc 545
20 nm; λEm 600
40
nm) with a plate reader (CLARIOstar, BMG LabTech).
Velocities (RFU/min) of positive (100% activity; DMSO) and
negative (0% activity, no enzyme) controls were used for
sample data normalization.
Dose−Response Enzyme Inhibition Assays. IC50 values
for compounds were determined using the ME−diaphorase−
resorufin coupled assay by measuring the reaction velocities at
varying inhibitors concentrations. Serial dilutions of com-
pounds were prepared in microplates using Versette (Thermo-
Fisher) with DMSO as the solvent. Reactions were prepared in
quadruplicate in 384-well plates by transferring 45 μL of a
reagent mix (0.48 nM TcMEc or 1.78 nM TcMEm; 22.2 μM
NADP+, 1.11 U/mL diaphorase; 444 μM aspartate; 2.22 mM
MnCl2, and 11.1 μM resazurin in reaction buffer containing 50
mM Tris-HCl, 50 mM NaCl, and 0.01% triton X-100, pH 7.5)
with Multidrop combi (ThermoFisher); 1 μL of the
compound solutions with Versette; and 4 μL of malate 16.25
mM for TcMEc and 12.5 mM for TcMEm with Versette.
Positive controls (100% activity) were prepared with all
reaction components except compounds (DMSO only). The
enzymatic activity was followed measuring the formation of
resorufin with a CLARIOstar plate reader (BMG LabTech) in
the fluorescent mode (λExc 545 20 nm; λEm 600 40 nm).
The velocities obtained were normalized by the controls, and
the IC50 values were calculated by nonlinear regression of the
data using GraphPad Prism software.
Nanoscale Differential Scanning Fluorimetry
(nanoDSF) Assays. A Prometheus NT.48 nanoDSF instru-
ment (nanoTemper Technologies, Munich, Germany) was
employed in the study of thermal stabilization effects of
TcΔMEc inhibitors. Samples were prepared by diluting
purified TcΔMEc to 10 μM in a buffer containing Tris 10
mM (pH 8.0), NaCl 30 mM, and inhibitors at 20 μM. Because
the small molecules were diluted in DMSO, a final
concentration of 1.25% of the solvent was obtained. Capillaries
filled with samples were placed on the sample holder, and a
temperature gradient from 15 to 95 °C (1 °C/min steps) was
applied. Intrinsic protein fluorescence was recorded at 330 and
350 nm. The fluorescence of the small molecules was checked
before measurements. Melting temperatures (Tm) were
obtained by analysis of the melting curves using the software
PR.Stability Analysis, supplied by the instrument manufacturer.
X-ray Diffraction and Structure Determination. Data
for ligand free TcΔMEc was collected on beamline I03 at the
Diamond Light Source (UK), while sets for TcΔMEc in
complex with sulfonamide inhibitors were collected at the
Brazilian Synchrotron Light Laboratory (LNLS) beamline
W01B-MX2. All data sets were acquired at 100° K and
integrated using XDS.42 Using CCP4 suite,43 data were scaled
with AIMLESS44 and TcMEc structures were determined by
molecular replacement with PHASER45 using a combination of
different domains as search models, as follows: domain-A,
residues 11−128 (PDB 1GZ3),26 domain-B, residues 129−274
MATERIALS AND METHODS
■
Reagents. Chagas Box samples were provided by GSK
(Tres Cantos, Spain). TcMEc and TcMEm were expressed and
purified as previously described.5,17. Resazurin, NADP+,
malate, aspartate, MnCl2, tris(hydroxymethyl)aminomethane,
NaCl, paraformaldehyde (PFA), DMSO, and Dulbecco’s
modified Eagle’s medium (DMEM) were supplied by Merck-
Sigma-Aldrich. Clostridium kluyveri diaphorase was bought
from Worthington Biochemical. Triton X-100 was obtained
from Serva. Fetal bovine serum (FBS) was bought from
Vitrocell. Penicillin streptomycin solution (10,000 U/mL and
10 ,000 μg/mL, respectively) and Hoechst 33342 were ordered
from ThermoFisher. Microplates were obtained from Greiner
Bio-One.
Cloning. The DNA fragment encoding for the TcMEc
residues ranging from Q12 to L565 (TcΔMEc) was amplified
from pET28_TcMEc5 using the primers 5′-GCGGATCCC-
ATATGCAAGGAAGAGCAATCCTGACG-3′ and 5′-GCT-
AGTTATTGCTCAGCGGTG-3′and subcloned into the
BamHI and EcoRI restriction sites of pET-SUMO.
Protein Production, Purification, and Crystallization.
Production and purification of TcMEc and TcΔMEc followed
the protocols described previously,5,17 with the exception that
for TcΔMEc the His-SUMO tag was cleaved with ULP-1
protease for 16 h at 4 °C before the size exclusion
chromatography. Crystals of TcΔMEc were grown by the
vapor diffusion method at 18 °C by mixing 3 μL of protein (5
mg/mL, in 10 mM TRIS-HCl pH 8.0, 30 mM NaCl, 2 mM
MnCl2, 10 mM aspartate) with 3 μL reservoir solution (100
mM HEPES pH 7.0, 1.4 M trisodium citrate). For complexes,
24 h previous to data collection, TcΔMEc crystals were
transferred to drops containing 1.8 μL of reservoir solution and
0.2 μL of inhibitors stock solution (10 mM in 100% DMSO).
Screening of Chagas Box against TcMEc and TcMEm.
A total of 218 compounds from the Chagas Box were provided
by GSK preplated in 384-well microplates at 1 mM in 100%
DMSO. Plates contained either 100 or 200 nL of compounds
per well. These plates were used to screen the compounds in
duplicates, at concentrations of 5 and 10 μM, against TcMEc
and TcMEm. ME reactions were coupled to diaphorase, that
uses NADPH to produce the fluorescent molecule resorufin
from resazurin.17,40,41 Each well of the plates was filled using a
Multidrop Combi reagent dispenser (Thermo-Fisher) to a final
volume of 20 μL. For TcMEc, the reactions were performed in
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(PDB 3WJA),11 domain-C, residues 275−467 (PDB 3WJA),
and domain-D, residues 468−539 (PDB 1GQ2).12 Structures
were refined with Phenix,46 REFMAC,47 and COOT.48
pdb_extract49 was used to prepare files for deposition in the
Protein Data Bank (PDB). Crystallographic statistics and PDB
codes of deposited structures are reported in Table 1.
Molecular figures were prepared in PYMOL v.1.8 (Schro-
dinger, LLC, New York, NY, USA).
T. cruzi Intracelullar Amastigote Image-Based Assays.
Methodology for cell-based assays of intracellular T. cruzi have
been described elsewhere.50 Briefly, LLC-MK2 (green monkey
kidney epithelial cells) and H9c2 (rat cardiomyocytes) were
cultivated in DMEM supplemented with 10% heat-inactivated
fetal bovine serum (FBS) and antibiotics (100 U/mL
penicillin; 100 μg/mL streptomycin). LLC-MK2 infected
with T. cruzi (Y strain) was maintained in DMEM medium
supplemented with 2% FBS and antibiotics. Trypomastigotes
were collected from supernatant after 4−5 days of infection.
Cells were always incubated in T-flasks at 37 °C, 5% CO2, and
>95% humidity.
uncorrected. H NMR and 13C NMR data were recorded on
a Varian 500 MHz or Varian 600 MHz spectrometer using
TMS as the internal standard, or the residual nondeuterated
solvent. Chemical shifts (δ) were expressed in ppm, and
multiplicities were reported as singlet (s), broad signal (bs),
doublet (d), double doublet (dd), triplet (t), apparent triplet
(at), double triplet (dt), quartet (q), double apparent quartet
(daq), apparent quartet (aq), quintuplet (quint), heptuplet
(hept), multiplet (m), and triple triplet (tt). Coupling
1
2
constants (J) are expressed in Hertz (Hz) and refer to JHH
or JHH, unless specified otherwise. High-resolution electro-
spray ionization mass spectrometry (HRMS-ESI) and target
compound purity assessments were performed on a Waters
Acquity H-Class UPLC system coupled to a PDA detector and
a Bruker Impact II mass spectrometer.
3
Method A. General Procedure for the Synthesis of
Sulfonamides (6−17). To a solution of nitrobenzenesulfonyl
chloride (1.13 mmol; 1.0 equiv) and aniline (1.35 mmol; 1.2
equiv) in anhydrous dichloromethane (3.8 mL; 0.3M) was
added anhydrous pyridine (2.26 mmol; 2.0 equiv) dropwise at
room temperature over an argon atmosphere. TLC analysis
showed complete consumption of the starting sulfonyl chloride
after 3 h. The reaction mixture was diluted with chloroform (7
mL), washed with a 1 M HCl solution (10 mL) and brine (10
mL), dried over MgSO4, and filtered, and the solvent was
removed in vacuo. When necessary, the dark red oil was taken
up in methanol (10 mL) and passed through a pad of activated
charcoal, yielding a clear to light yellow solution, which after
drying in vacuo furnished the desired sulfonamide without
further purification.
Method B. General Procedure for the Synthesis of Amines
(18−29). A suspension of the nitro derivative (0.71 mmol; 1.0
equiv) and tin(II) chloride dihydrate (3.57 mmol; 5.0 equiv)
in ethyl acetate (7.1 mL; 0.1M) was heated at reflux for 2 h.
After cooling, the reaction mixture was diluted with water (5
mL), and the pH was adjusted to 7−8 by the addition of a
saturated aqueous NaHCO3 solution (approximately 40 mL).
The product was extracted into ethyl acetate (2 × 50 mL),
washed with water (2 × 50 mL) and brine (50 mL), dried over
MgSO4, and filtered, and the solvent was removed in vacuo to
deliver the desired amine, which was used without further
purification, unless stated otherwise. N.B.: The aqueous layers
containing tin residues were discarded in a separate container
and disposed of as hazardous waste.
Method C. General Procedure for the Synthesis of Target
Compounds (1, 30−40). To a solution of the amine derivative
(0.18 mmol; 1.0 equiv) and 3,5-difluorobenzoyl chloride (0.27
mmol; 1.5 equiv) in an anhydrous solvent (DMF, MeCN, or
CH2Cl2) (0.9 mL; 0.2M) was added N-methylmorpholine
(0.54 mmol; 3.0 equiv) at room temperature under an argon
atmosphere. Usually, the reaction was completed after 1−2 h,
upon which time the mixture was diluted with ethyl acetate (4
mL), washed with 1 M HCl (2−5 mL) and brine (2 × 2−5
mL), dried over MgSO4, and filtered, and the solvent was
removed in vacuo. Purification of the desired product was
achieved either through crystallization or by column
chromatography, as described for each compound.
N-(4-Methoxyphenyl)-3-nitrobenzenesulfonamide (6).
Method A. Yield: 68%; off white solid; mp 132−133 °C
(135 °C51); δH (500 MHz; CDCl3) 8.58 (1 H, t, J 1.9), 8.40 (1
H, daq, J 8.2, 1.0), 7.98 (1 H, dt, J 8.7, 1.2), 7.65 (1 H, t, J 8.0),
7.00 (2 H, d, J 8.9), 6.79 (2 H, d, J 9.0), 3.77 (3 H, s); δC
(125.5 MHz; CDCl3) 158.6, 148.2, 141.1, 132.9, 130.3, 127.7,
Compounds to be used in cellular image-based assays were
dissolved and/or serial diluted in DMSO. Serial dilutions were
performed applying a half-log factor. Intermediary plates were
prepared diluting compounds in fresh DMEM medium using a
proportion of 1:99.
T. cruzi trypomastigotes (5 × 105 cells) collected from
infected LLC-MK2 cells were used to infect H9c2 rat
cardiomyocytes (1 × 106 cells) overnight (16−18 h), in 75
cm2 T-flasks. Then, cells were washed with phosphate buffered
solution (PBS) to remove extracellular parasites, trypsinized,
and resuspended in fresh DMEM medium to a concentration
of 2.2 × 104 cells/mL. A volume of 45 μL of this cellular
suspension was dispensed into 384-well assay plates (1 × 103
cells per well) using a Multidrop Combi system (Thermo
Fisher Science). Plates were incubated for addition 48 h for T.
cruzi differentiation into intracellular amastigotes. Subse-
quently, 30 μL of samples from intermediary plates was
transferred to assay plates in such a way that the DMSO
concentration was reduced to 0.4%. After 72 h of incubation
(treatment period), cells were fixed with 4% paraformaldehyde
(PFA) and stained with Hoechst 33342 (4 μg/mL in PBS).
Five images per well were acquired with the fluorescence
microscope Operetta (PerkinElmer, Hamburg, DE) using a
20× long WD objective and processed in Columbus software
(PerkinElmer, Hamburg, DE) to identify host cell nuclei,
cytoplasm area, and spots in the cytoplasm. Infected cells were
defined as cells with at least three spots in the cytoplasm area.
The Infection ratio corresponds to infected cells/total cells per
well. Total cells, infected cells, and infection ratio data were
analyzed and plotted with GraphPad Prism software.
Synthetic Chemistry. Reagents and anhydrous solvents
were purchased from Sigma-Aldrich Brasil Ltd.a for carrying
out the reactions and from Labsynth Produtos para
́
Laboratorios Ltd.a (analytical grade) for workup, and were
used without further purification unless specified otherwise.
Anhydrous pyridine was distilled from and stored over
potassium hydroxide. Reactions were monitored by thin-layer
chromatography (TLC) on silica gel 60 F254 aluminum sheets
and exposed to UV radiation, followed by treatment with
adequate stains and heating. Chromatographic separations
were carried out on Merck 60 silica gel (230−400 mesh).
Melting points (mp) were recorded on a PF 1500 FARMA
apparatus with a heating rate of 5 °C/min and were
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4
3
2
127.3, 126.0, 122.5, 114.8, 55.5; HRMS-ESI calcd for
C13H16N3O5S+ [M + NH4]+: 326.0805, found: 326.0811.
N-(2-Fluorophenyl)-3-nitrobenzenesulfonamide (7).
Method A. Yield: 76%; off white solid, mp 121 °C; δH (500
MHz; CDCl3) 8.61 (1 H, t, J 2.0), 8.41 (1 H, daq, J 8.2, 0.9),
8.07 (1 H, daq, J 7.9, 0.9), 7.67 (1 H, t, J 8.0), 7.64−7.60 (1 H,
m), 7.19−7.13 (1 H, m), 6.99−6.95 (1 H, m), 6.86 (1 H, bs);
125.0 (d, JCF 3.7), 124.0, 123.3 (d, JCF 12.9), 116.3 (d, JCF
19.7); H
RMS-ESI calcd for C12H8ClFN2NaO4S+ [M + Na]+:
352.9770, found: 352.9762.
4-Chloro-N-(2-fluoro-4-methoxyphenyl)-3-nitrobenzene-
sulfonamide (14). Method A. Yield: 67%; off white solid; mp
118−119 °C; δH (500 MHz; CDCl3) 8.18 (1 H, d, J 2.1), 7.81
(1 H, dd, J 8.4, 2.2), 7.64 (1 H, d, J 8.4), 7.45 (1 H, t, J 9.0),
6.72 (1 H, daq, J 8.9, 1.3), 6.58 (1 H, bs), 6.54 (1 H, dd, J 11.7,
1
δC (125.5 MHz; CDCl3) 154.4 (d, JCF 245.5), 148.2, 140.1,
3
4
132.6, 130.5, 127.7, 127.57 (d, JCF 7.6), 125.15 (d, JCF 3.9),
3
2
2
2.7), 3.78 (3 H, s); δC (125.5 MHz; CDCl3) 159.94 (d, JCF
10.5), 156.6 (d, JCF 246.6), 147.6, 139.1, 132.8, 132.0, 131.1,
124.6, 123.35 (d, JCF 12.4), 122.5, 115.72 (d, JCF 19.4);
HRMS-ESI calcd for C12H9FN2NaO4S+ [M + Na]+: 319.0159,
found: 319.0148.
1
2
3
128.39 (d, JCF 1.7), 124.6, 114.84 (d, JCF 13.4), 110.61 (d,
4JCF 3.2), 102.05 (d, JCF 23.2), 55.8; HRMS-ESI calcd for
2
N-(2-Fluoro-4-methoxyphenyl)-3-nitrobenzenesulfona-
mide (8). Method A. Yield: 98%; dark brown solid; mp 104
°C; δH (500 MHz; CDCl3) 8.57 (1 H, t, J 1.9), 8.41 (1 H, daq,
J 8.8, 1.0), 8.01 (1 H, d, J 7.8), 7.66 (1 H, t, J 8.1), 7.48 (1 H, t,
J 9.0), 6.72 (1 H, daq, J 8.9, 1.3), 6.58 (1 H, bs), 6.49 (1 H, dd,
J 11.9, 2.8), 3.76 (3 H, s); δC (125.5 MHz; CDCl3) 159.7 (d,
C13H10ClFKN2O5S+ [M + K]+: 398.9615, found: 398.9620.
N-(Benzo[d][1,3]dioxol-5-yl)-4-chloro-3-nitrobenzenesul-
fonamide (15). Method A. Yield: 88%; brown solid; mp 129−
130 °C; δH (500 MHz; CDCl3) 8.22 (1 H, d, J 2.0), 7.81 (1 H,
dd, J 8.5, 2.0), 7.65 (1 H, d, J 8.5), 6.80 (1 H, bs), 6.69 (1 H,
s), 6.69−6.67 (2 H, m), 6.46 (1 H, dd, J 8.2, 1.0), 5.98 (2 H,
s); δC (125.5 MHz; CDCl3) 148.5, 147.7, 146.9, 139.0, 132.2,
132.0, 131.3, 128.5, 124.6, 117.8, 108.5, 106.1, 101.9; HRMS-
ESI calcd for C13H13ClN3O6S+ [M + NH4]+: 347.0208, found:
374.0199.
1
3JCF 10.4), 156.5 (d, JCF 246.4), 148.1, 141.0, 132.7, 130.3,
128.2 (d, 2JCF 1.7), 127.5, 122.5, 115.2 (d, 3JCF 13.3), 110.5 (d,
2
4JCF 3.1), 102.0 (d, JCF 23.2), 55.7; HRMS-ESI calcd for
C13H11FN2NaO5S+ [M + Na]+: 349.0265, found: 349.0281.
N-(Benzo[d][1,3]dioxol-5-yl)-3-nitrobenzenesulfonamide
(9). Method A. Yield: 81%; off white solid; mp 143−144 °C;
δH (500 MHz; DMSO-d6) 10.26 (1 H, s), 8.46−8.44 (1 H, m),
8.06 (1 H, daq, J 7.9, 0.9), 7.86−7.83 (1 H, m), 6.77 (1 H, d, J
8.3), 6.66 (1 H, d, J 2.1), 6.48 (1 H, dd, J 8.3, 2.1), 5.96 (2 H,
s); δC (125.5 MHz; DMSO-d6) 147.9, 147.6, 145.0, 140.7,
132.7, 131.3, 130.4, 127.5, 121.5, 115.7, 108.4, 104.2, 101.5;
HRMS-ESI calcd for C13H10N2NaO6S+ [M + Na]+: 345.0152,
found: 345.0153.
4-Chloro-N-(4-(difluoromethoxy)phenyl)-3-nitrobenzene-
sulfonamide (16). Method A. Yield: 99%; orange oil; δH (500
MHz; CDCl3) 8.26 (1 H, d, J 2.1), 7.82 (1 H, dd, J 8.5, 1.8),
7.66 (1 H, d, J 8.5), 7.12 (2 H, d, J 4.7), 7.08 (2 H, d, J 4.7),
2
6.94 (1 H, bs), 6.48 (1 H, t, JHF 73.3); δC (125.5 MHz;
3
CDCl3) 149.4 (t, JCF 3.0), 147.8, 140.8, 138.9, 133.1, 132.3,
132.2, 131.2, 124.6, 124.5, 121.0, 115.5 (t, 1JCF 261.4); HRMS-
ESI calcd for C13H13ClF2N3O5S+ [M + NH4]+: 396.0227,
found: 396.0215.
N-(4-(Difluoromethoxy)phenyl)-3-nitrobenzenesulfona-
mide (10). Method A. Yield: 81%; white fluffy solid; mp 106−
107 °C; δH (500 MHz; DMSO-d6) 10.59 (1 H, s), 8.48 (1 H, t,
J 1.8), 8.46 (1 H, daq, J 8.2, 1.1), 8.11 (1 H, daq, J 7.9, 0.9),
7.86 (1 H, t, J 8.1), 7.12 (1 H, t, 2JHF 74.1), 7.13 (2 H, d, J 9.2),
7.08 (2 H, d, J 9.0); δC (125.5 MHz; DMSO-d6) 147.96 (t,
3JCF 3.2), 147.91, 140.8, 133.8, 132.6, 131.4, 127.7, 123.0,
4-Chloro-N-(4-(trifluoromethoxy)phenyl)-3-nitrobenzene-
sulfonamide (17). Method A. Yield: 96%; yellow oil; δH (500
MHz; CDCl3) 8.29 (1 H, d, J 2.1), 7.83 (1 H, dd, J 8.3, 2.2),
7.67 (1 H, d, J 8.4), 7.18−7.14 (4 H, m), 7.02 (1 H, bs); δC
3
(125.5 MHz; CDCl3) 147.9, 147.4 (q, JCF 1.9), 138.9, 133.7,
133.1, 132.5, 131.1, 124.6, 123.9, 122.4, 120.3 (q, 1JCF 257.9);
HRMS-ESI calcd for C13H9ClF3N2O5S+ [M + H]+: 396.9867,
found: 396.9855.
1
121.4, 119.9, 116.2 (t, JCF 257.9); HRMS-ESI calcd for
C13H10F2N2NaO5S+ [M + Na]+: 367.0171, found: 367.0169.
N-(4-(Trifluoromethoxy)phenyl)-3-nitrobenzenesulfona-
mide (11). Method A. Yield: 80%; white fluffy solid; mp 134−
135 °C; δH (500 MHz; DMSO-d6) 10.59 (1 H, s), 8.49−8.45
(2 H, m), 8.14 (1 H, daq, J 7.9, 0.8), 7.87 (1 H, t, J 8.0), 7.28
(2 H, d, J 8.4), 7.20 (2 H, d, J 9.1); δC (150 MHz; DMSO-d6)
3-Amino-N-(4-methoxyphenyl)benzenesulfonamide (18).
Method B. Yield: 94%; off white solid; mp 179−181 °C (180−
181 °C52); δH (600 MHz; DMSO-d6) 9.74 (1 H, s), 7.11 (1 H,
t, J 7.9), 6.98 (2 H, d, J 8.9), 6.89 (1 H, t, J 2.0), 6.81−6.78 (3
H, m+d, J 9.0), 6.69 (1 H, dd, J 8.0, 2.2), 5.53 (2 H, s), 3.67 (3
H, s); δC (150 MHz; DMSO-d6) 156.3, 149.2, 140.1, 130.5,
129.4, 123.0, 117.3, 114.2, 113.3, 111.3, 55.1; HRMS-ESI calcd
for C13H15N2O3S+ [M + H]+: 279.0798, found: 279.0801.
3-Amino-N-(2-fluorophenyl)benzenesulfonamide (19).
Method B. Yield: 92%; off white solid; mp 161−162 °C; δH
(500 MHz; DMSO-d6) 9.95 (1 H, bs), 7.22 (1 H, t, J 8.5),
7.17−7.12 (3 H, m), 7.11−7.08 (1 H, m), 6.93 (1 H, t, J 2.0),
6.82 (1 H, daq, J 7.7, 0.9), 6.73 (1 H, daq, J 8.1, 1.1), 5.55 (2
3
148.0, 145.07 (q, JCF 1.8), 140.7, 136.1, 132.6, 131.5, 127.8,
122.4, 122.3, 121.4, 120.0 (q, 1JCF 256.1); HRMS-ESI calcd for
C13H9F3N2NaO5S+ [M + Na]+: 385.0076, found: 385.0085.
4-Chloro-N-(4-methoxyphenyl)-3-nitrobenzenesulfona-
mide (12). Method A. Yield: 65%; off white solid; mp 78−79
°C; δH (500 MHz; CDCl3) 8.20 (1 H, d, J 2.1), 7.76 (1 H, dd,
J 8.5, 2.2), 7.62 (1 H, d, J 8.4), 7.01 (2 H, d, J 9.0), 6.81 (2 H,
d, J 9.0), 3.78 (3 H, s); δC (125.5 MHz; CDCl3) 158.8, 147.7,
139.1, 132.8, 131.9, 131.3, 127.3, 126.2, 124.6, 114.9, 55.5;
HRMS-ESI calcd for C13H15ClN3O5S+ [M + NH4]+: 360.0415,
found: 360.0429.
1
H, bs); δC (125.5 MHz; DMSO-d6) 155.4 (d, JCF 247.2),
149.3, 140.6, 129.5, 126.82 (d, 2JCF 7.5), 125.8, 124.79 (d, 3JCF
4
2
12.8), 124.5 (d, JCF 3.8), 117.6, 115.93 (d, JCF 19.6), 113.2,
111.0; HRMS-ESI calcd for C12H12FN2O2S+ [M + H]+:
267.0598, found: 267.0599.
3 - A m i n o - N - ( 2 - fl u o r o - 4 - m e t h o x y p h e n y l ) -
benzenesulfonamide (20). Method B. Yield: 96%; light violet
solid; mp 149−150 °C; δH (500 MHz; CDCl3) 9.58 (1 H, bs),
7.13 (1 H, t, J 7.9), 7.02 (1 H, t, J 9.0), 6.87 (1 H, t, J 2.0),
6.80−6.75 (2 H, m), 6.72 (1 H, daq, J 8.0, 1.0), 6.72 (1 H, daq,
4-Chloro-N-(2-fluorophenyl)-3-nitrobenzenesulfonamide
(13). Method A. Yield: 87%; light orange solid; mp 132−133
°C; δH (500 MHz; DMSO-d6) 10.57 (1 H, s), 8.38 (1 H, d, J
2.1), 8.01 (1 H, d, J 8.4), 7.97 (1 H, dd, J 8.5, 2.1), 7.30−7.16
1
(4 H, m); δC (125.5 MHz; DMSO-d6) 156.2 (d, JCF 247.9),
3
147.3, 140.1, 133.2, 131.3, 129.9, 128.5 (d, JCF 7.7), 127.7,
L
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Article
J 8.9, 1.2), 5.53 (2 H, bs), 3.71 (3 H, s); δC (125.5 MHz;
3-Amino-N-(benzo[d][1,3]dioxol-5-yl)-4-chlorobenzene-
sulfonamide (27). Method B. Yield: 86%; light brown solid;
mp 131−132 °C; δH (500 MHz; DMSO-d6) 9.92 (1 H, bs),
7.34 (1 H, d, J 6.3), 7.127 (1 H, d, J 2.2), 6.81 (1 H, dd, J 8.3,
2.2), 6.77 (1 H, d, J 8.3), 6.648 (1 H, d, J 2.1), 6.476 (1 H, dd,
J 8.3, 2.3), 5.96 (2 H, s), 5.84 (2 H, bs); δC (125.5 MHz;
DMSO-d6) 147.4, 145.1, 144.4, 138.7, 131.5, 129.7, 120.7,
114.7, 114.1, 113.0, 108.3, 103.4, 101.3; HRMS-ESI calcd for
C13H12ClN2O4S+ [M + H]+: 327.0201, found: 327.0204.
3-Amino-4-chloro-N-(4-(difluoromethoxy)phenyl)-
benzenesulfonamide (28). Method B. Yield: 78%; light brown
solid; mp 109−111 °C; δH (500 MHz; DMSO-d6) 10.26 (1 H,
bs), 7.35 (1 H, d, J 8.41), 7.18 (1 H, d, J 2.2), 7.12−7.07 (4 H,
m), 7.10 (1 H, t, 2JHF 74.2), 6.85 (1 H, dd, J 8.3, 2.2), 5.87 (2
3
1
CDCl3) 158.6 (d, JCF 10.2), 157.4 (d, JCF 247.8), 149.1,
2
3
140.6, 129.4, 128.8 (d, JCF 2.5), 117.4, 116.7 (d, JCF 13.4),
4
2
113.3, 111.2, 110.1 (d, JCF 3.1), 102.0 (d, JCF 23.7), 55.7;
HRMS-ESI calcd for C13H14FN2O3S+ [M + H]+: 297.0704,
found: 297.0702.
3 - A m i n o - N - ( b e n z o [ d ] [ 1 , 3 ] d i o x o l - 5 - y l ) -
benzenesulfonamide (21). Method B. Yield: 98%; brown
solid; mp 128−130 °C; δH (500 MHz; DMSO-d6) 9.82 (1 H,
s), 7.13 (1 H, t, J 7.9), 6.90 (1 H, t, J 2.0), 6.81 (1 H, daq, J 7.7,
0.9), 6.76 (1 H, d, J 8.4), 6.71 (1 H, daq, J 8.1, 1.0), 6.64 (1 H,
d, J 2.1), 6.68 (1 H, dd, J 8.3, 2.1), 5.95 (2 H, s), 5.55 (2 H,
bs); δC (125.5 MHz; DMSO-d6) 149.2, 147.3, 144.1, 139.9,
131.8, 129.5, 117.5, 114.3, 113.3, 111.3, 108.42, 103.1, 101.2;
HRMS-ESI calcd for C13H13N2O4S+ [M + H]+: 293.0591,
found: 293.0581.
3
H, bs); δC (125.5 MHz; DMSO-d6) 147.4 (t, JCF 3.2), 145.3,
1
138.7, 134.8, 129.8, 121.9, 120.9, 116.4 (t, JCF 257.7), 114.0,
3 - A m i n o - N - ( 4 - ( d i fl u o r o m e t h o x y ) p h e n y l ) -
benzenesulfonamide (22). Method B. Yield: 84%; white/pink
solid; mp 102−104 °C; δH (500 MHz; DMSO-d6) 10.18 (1 H,
bs), 7.15 (1 H, t, J 7.9), 7.12 (2 H, d, J 9.1), 7.10 (1 H, t, 2JHF
76.3), 7.07 (2 H, d, J 9.1), 6.96−6.95 (1 H, m), 6.85 (1 H, daq,
J 7.7, 0.9), 6.72 (1 H, daq, J 8.1, 1.0), 5.58 (2 H, bs); δC (125.5
12.8; HRMS-ESI calcd for C13H13ClF2N2O3S+ [M + H]+:
349.0220, found: 349.0222.
3-Amino-4-chloro-N-(4-(trifluoromethoxy)phenyl)-
benzenesulfonamide (29). Method B. Yield: 90%; light brown
solid; mp 123−126 °C; δH (500 MHz; DMSO-d6) 10.47 (1 H,
bs), 7.36 (1 H, d, J 8.4), 7.28 (2 H, d, J 8.4), 7.21 (1 H, d, J
2.2), 7.17 (2 H, d, J 9.1), 6.87 (1 H, dd, J 8.4, 2.3), 5.89 (2 H,
3
MHz; DMSO-d6) 149.4, 147.08 (t, JCF 3.2), 140.0, 135.2,
1
3
129.6, 121.5, 119.8, 117.6, 116.4 (t, JCF 257.6); HRMS-ESI
bs); δC (125.5 MHz; DMSO-d6) 145.3, 144.38 (q, JCF 1.9),
calcd for C13H13F2N2O3S+ [M + H]+: 315.0609, found:
315.0618.
138.7, 137.0, 129.9, 122.1, 121.3, 121.0, 120.3 (q, JCF 256.0),
1
113.9, 112.7; HRMS-ESI calcd for C13H11ClF3N2O3S+ [M +
H]+: 367.0126, found: 367.0116.
3 - A m i n o - N - ( 4 - ( t r i fl u o r o m e t h o x y ) p h e n y l ) -
benzenesulfonamide (23). Method B. Yield: 97%; light brown
solid; mp 105−106 °C; δH (500 MHz; DMSO-d6) 10.37 (1 H,
bs), 7.25 (1 H, d, J 8.5), 7.18−7.13 (3 H, m), 6.96 (1 H, t, J
2.0), 6.86 (2 H, daq, J 8.1, 0.9), 6.72 (2 H, daq, J 8.1, 1.0), 5.59
3,5-Difluorobenzoyl Chloride. 3,5-Difluorobenzoic acid
(1.0 g, 6.32 mmol) was dissolved in a mixture of chloroform
(1M; 6.3 mL) and thionyl chloride (1.0 mL, 14.53 mmol), and
the mixture was heated at reflux for 8 h under an argon
atmosphere. While removing the solvent and excess reagent in
vacuo, the product was collected in the antisplash bulb as a
colorless liquid, which was stored in a vial, with the latter being
kept in a molecular sieve containing jar at 5−8 °C. The acyl
chloride easily hydrolyzed back to the acid upon contact with
3
(2 H, bs); δC (125.5 MHz; DMSO-d6) 149.5, 144.15 (q, JCF
1
1.9), 139.9, 137.3, 129.7, 122.0, 120.8, 120.1 (q, JCF 255.9),
117.8, 117.0, 113.2, 111.0; HRMS-ESI calcd for
C13H12F3N2O3S+ [M + H]+: 333.0515, found: 333.0523.
3 - A m i n o - 4 - c h l o r o - N - ( 4 - m e t h o x y p h e n y l ) -
benzenesulfonamide (24). Method B. Yield: 87%; off white
thick oil; δH (500 MHz; CDCl3) 7.26 (1 H, d, J 8.3), 7.12 (1
H, d, J 2.2), 6.99 (2 H, d, J 8.9), 6.96 (1 H, dd, J 8.4, 2.2), 6.77
(2 H, d, J 9.0), 6.63, (1 H, bs), 4.27, (2 H, bs), 3.76 (3 H, s);
δC (125.5 MHz; CDCl3) 158.1, 143.5, 138.2, 129.8, 128.5,
125.7, 123.5, 117.4, 114.5, 113.8, 55.4; HRMS-ESI calcd for
C13H14ClN2O3S+ [M + H]+: 313.0408, found: 313.0412.
3 - A m i n o - 4 - c h l o r o - N - ( 2 - fl u o r o p h e n y l ) -
benzenesulfonamide (25). Method B. Yield: 87%; off white
solid; mp 119−121 °C; δH (500 MHz; DMSO-d6) 10.08 (1 H,
bs), 7.35 (1 H, d, J 8.3), 7.22 (1 H, at, J 8.0), 7.20−7.10 (4 H,
m), 6.83 (1 H, dd, J 8.3, 2.2), 5.84 (2 H, bs); δC (125.5 MHz;
humid air: δH (500 MHz; CDCl3) 7.68 (2 H, m); 7.16 (1 H, tt,
1,3
J 8.3, 2.4); δC (125.5 MHz; CDCl3) 166.4, 162.8 (dd,
J
CF2
252.5, 11.9), 136.2 (at, 3JCF2 9.0), 114.25 (dd, 2,4JCF2 20.9, 7.1),
3
110.8 (at, JCF2 25.2).
3,5-Difluoro-N-(3-(N-(4-methoxyphenyl)sulfamoyl)-
phenyl)benzamide (1). Method C. Purification: the crude
solid was recrystallized from a 9:1 chloroform/ethyl acetate
mixture; yield: 35%; HPLC purity: 98%; white fluffy solid; mp
205−206 °C; δH (600 MHz; DMSO-d6) 10.61 (1 H, bs),
10.01 (1 H, s), 8.29 (1 H, t, J 1.9), 7.95 (1 H, dd, J 8.2, 1.2),
7.71−7.68 (2 H, m), 7.553 (1 H, tt, J 9.2, 2.3), 7.558 (1 H, t, J
8.1), 7.43 (1 H, d, J 8.3), 7.00 (1 H, d, J 9.0), 6.81 (1 H, d, J
9.0), 3.66 (3 H, s); δC (150 MHz; DMSO-d6) 163.16, 162.2
1
DMSO-d6) 155.7 (d, JC4F 247.3), 154.7, 139.5, 129.7, 127.15
2
3
1,3
3
(d, JCF 7.4), 126.4 (d, JCF 0.6), 124.6 (d, JCF 3.8), 124.5,
(dd,
J
CF2
247.5, 12.8), 156.5, 140.1, 139.2, 137.8 (t, JCF2
2
120.7, 116.0 (d, JCF 19.8), 114.0, 112.8; HRMS-ESI calcd for
8.6), 130.0, 129.6, 124.0, 123.5, 122.1, 118.3, 114.3, 111.25
C12H11ClFN2O2S+ [M + H]+: 301.0208, found: 301.0209.
3-Amino-4-chloro-N-(2-fluoro-4-methoxyphenyl)-
benzenesulfonamide (26). Method B. Yield: 94%; off white
solid; mp 97−99; δH (500 MHz; CDCl3) 7.42 (1 H, t, J 9.0),
7.27 (1 H, d, J 5.7), 7.11 (1 H, d, J 2.2), 6.96 (1 H, dd, J 8.4,
2.2), 6.66 (1 H, daq, J 9.0, 1.0), 6.53 (1 H, dd, J 11.8, 2.8), 6.46
(1 H, bs), 4.26 (2 H, bs), 3.76 (3 H, s); δC (125.5 MHz;
(dd,
J
CF2
20.4, 6.6), 107.3 (t, JCF2 25.9), 55.1; HRMS-ESI
2,4
2
calcd for C20H20F2N3O4S+ [M + NH4]+: 436.1137, found:
436.1133.
3,5-Difluoro-N-(3-(N-(2-fluorophenyl)sulfamoyl)phenyl)-
benzamide (30). Method C. Purification: the crude solid was
suspended in chloroform, allowing for the excess benzoyl
chloride to be removed; yield: 30%; HPLC purity: >99%;
white solid; mp 237−238 °C; δH (500 MHz; DMSO-d6) 10.62
(1 H, bs), 10.23 (1 H, bs), 8.30 (1 H, t, J 1.9), 8.00 (1 H, dat, J
8.2, 0.9), 7.69 (1 H, dd, J 8.1, 2.2), 7.58−7.53 (2 H, m), 7.47
3
1
CDCl3) 158.93 (d, JCF 10.2), 156.1 (d, JCF 246.1), 143.4,
138.1, 129.9, 127.04 (d, JCF 1.9), 123.7, 117.0, 116.4 (d, JCF
13.3), 113.7, 110.14 (d, JCF 3.3), 101.9 (d, JCF 23.3), 55.7;
HRMS-ESI calcd for C13H13ClFN2O3S+ [M + H]+: 331.0314,
found: 331.0316.
2
3
4
2
(1 H, d, J 8.3), 7.25 (1 H, dt, J 8.0, 1.7), 7.22−7.11 (3 H, m);
1,3
δC (125.5 MHz; DMSO-d6) 163.17, 162.20 (dd,
J
CF2
247.6,
M
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12.8), 155.7 (d, 1JCF 247.4), 140.6, 139.2, 137.78 (t, 3JCF2 7.7),
2JCF2 26.0); HRMS-ESI calcd for C20H17F5N3O4S+ [M +
NH4]+: 490.0854, found: 490.0869.
2
4
129.7, 127.43 (d, JCF 7.3), 126.6, 124.69 (d, JCF 3.9), 124.2,
124.26 (d, 3JCF 12.9), 121.9, 118.1, 116.06 (d, 2JCF 9.7), 111.25
N-((3-Amino-4-chlorophenyl)sulfonyl)-3,5-difluoro-N-(4-
methoxyphenyl)-benzamide (35). Method C. Purification:
the crude oil was purified on silica gel eluting with 100%
chloroform; yield: 25%; HPLC purity: 95%; green waxy solid;
δH (500 MHz; CDCl3) 7.42 (1 H, d, J 2.1), 7.36 (1 H, d, J
8.4), 7.11 (1 H, dd, J 8.4, 2.1), 7.06−7.03 (2 H, m), 6.98−6.93
(2 H, m), 6.82−6.79 (2 H, m), 6.75 (1 H, tt, J 8.5, 2.3), 4.35
(2 H, bs), 3.78 (3 H, s); δC (125.5 MHz; CDCl3) 167.31 (at,
4JCF2 3.1), 162.23 (dd, 1,3JCF2 251.1, 12.1), 160.3, 143.3, 136.84
2,4
2
(dd,
J
CF2
20.6, 6.6), 107.35 (t, JCF2 26.7); HRMS-ESI calcd
for C19H14F3N2O3S+ [M + H]+: 407.0672, found: 407.0673.
3,5-Difluoro-N-(3-(N-(2-fluoro-4-methoxyphenyl)-
sulfamoyl)phenyl)benzamide (31). Method C. Purification:
the crude solid was recrystallized from a 9:1 chloroform/ethyl
acetate mixture; yield: 35%; HPLC purity: 97%; white solid;
mp 218−220 °C; δH (500 MHz; DMSO-d6) 10.61 (1 H, bs),
9.88 (1 H, bs), 8.24 (1 H, s), 8.03 (1 H, dd, J 8.1, 1.1), 6.70 (2
H, d, J 6.0), 7.57−7.53 (2 H, m), 7.42 (1 H, d, J 7.9), 7.07 (1
H, t, J 9.0), 6.80 (1 H, dd, J 12.0, 2.7), 6.72 (1 H, dd, J 8.9,
2.4), 3.71 (3 H, s); δC (125.5 MHz; DMSO-d6) 163.14, 162.20
3
(at, JCF2 8.9), 136.7, 131.3, 129.6, 128.8, 124.7, 118.9, 116.0,
2,4
2
114.6, 111.45 (dd,
J
20.7, 6.9), 107.21 (at, JCF2 25.2),
CF2
55.5; HRMS (ESI) calcd for C20H16ClF2N2O2S+ [M + H]+:
453.0482, found: 452.9969.
1,3
3
1
(dd,
J
CF2
247.4, 12.7), 158.9 (d, JCF 10.2), 157.6 (d, JCF
247.9), 140.7, 139.1, 137.8 (t, 3JCF2 8.6), 129.6, 129.35 (d, 2JCF
N-((3-Amino-4-chlorophenyl)sulfonyl)-3,5-difluoro-N-(2-
fluoro-4-methoxy-phenyl)benzamide (36). Method C. Puri-
fication: the crude oil was purified on silica gel eluting with
hexane/chloroform/ether (5:4:1 to 4.5:3.5:2); yield: 52%;
HPLC purity: >99%; off-white powder; δH (500 MHz; CDCl3)
7.49 (1 H, d, J 2.2), 7.38 (1 H, d, J 8.4), 7.23 (1 H, t, J 8.8),
7.20 (1 H, dd, J 8.4, 2.1), 6.94−6.92 (2 H, m), 6.77 (1 H, tt, J
8.6, 2.3), 6.68 (1 H, dd, J 8.9, 2.8), 6.55 (1 H, dd, J 11.6, 2.8),
4.36 (2 H, bs), 3.78 (3 H, s); δC (125.5 MHz; CDCl3) 167.1
3
2,4
2.3), 124.0, 122.0, 116.16 (d, JCF 13.5), 111.24 (dd,
J
CF2
20.4, 6.7), 110.27 (d, 4JCF 2.9), 107.3 (t, 2JCF2 26.0), 102.06 (d,
2JCF 23.8), 55.7; HRMS-ESI calcd for C20H16F3N2O4S+ [M +
H]+: 437.0777, found: 437.0783.
N-(3-(N-(Benzo[d][1,3]dioxol-5-yl)sulfamoyl)phenyl)-3,5-
difluorobenzamide (32). Method C. Purification: the crude oil
was taken up in ethyl acetate (0.1 mL), followed by chloroform
(1.9 mL), and the precipitate was isolated by filtration; yield:
62%; HPLC purity: 98%; white fluffy solid; mp 196−198 °C;
δH (500 MHz; DMSO-d6) 10.61 (1 H, bs), 10.08 (1 H, s),
8.29 (1 H, t, J 1.9), 7.96 (1 H, daq, J 8.2, 1.0), 7.71−7.67 (2 H,
m), 7.56−7.51 (2 H, m), 7.46 (1 H, daq, J 7.9, 1.0), 6.76 (1 H,
d, J 8.3), 6.67 (1 H, d, J 2.1), 6.51 (1 H, dd, J 8.4, 2.2), 5.95 (2
4
1,3
2
(at, JCF2 3.0), 162.25 (dd,
J
251.3, 12.0), 162.20 (d, JCF
CF2
1
3
10.8), 159.0 (d, JCF 251.7), 143.3, 137.0, 136.56 (at, JCF2
8.8), 132.5 (d, JCF 1.2), 129.7, 124.9, 118.8, 111.63 (d, JCF
3.2), 116.0, 111.58 (dd,
107.16 (at, JCF2 25.1), 102.32 (d, JCF 23.5), 55.8; HRMS
(ESI) calcd for C20H15ClF3N2O4S+ [M + H]+: 471.0388,
found: 471.0392.
4
3
2,4
3
J
CF2
20.8, 7.0), 110.76 (d, JCF 2.9),
2
2
H, s); δC (125.5 MHz; DMSO-d6) 163.21 (t, 4JCF2 3.0), 162.22
1,3
(dd,
J
CF2
247.5, 12.6), 147.4, 144.5, 139.9, 139.2, 137.81 (t,
3JCF2 8.6), 131.3, 129.7, 124.1, 122.1, 118.4, 114.8, 111.27 (dd,
N-(2-Chloro-5-(N-(2-fluorophenyl)sulfamoyl)phenyl)-3,5-
difluorobenzamide (37). Method C. Purification: the crude oil
was purified on silica gel eluting with hexane/ethyl acetate (4:1
to 7:3); yield: 20%; HPLC purity: > 99%; white solid; mp
140−141 °C; δH (500 MHz; DMSO-d6) 9.06 (1 H, bs), 8.34
(1 H, bs), 7.62 (1 H, dt, J 7.9, 1.9), 7.49 (2 H, s), 7.43−7.38 (2
2,4
2
J
20.3, 6.7), 108.3, 107.36 (t, JCF2 25.9), 103.5, 101.3;
CF2
HRMS-ESI calcd for C20H18F2N2O5S+ [M + NH4]+: 450.0930,
found: 450.0925.
N-(3-(N-(4-(Difluoromethoxy)phenyl)sulfamoyl)phenyl)-
3,5-difluorobenzamide (33). Method C. Purification: the
crude oil was taken up in ethyl acetate (0.1 mL), followed by
chloroform (1.9 mL), and the precipitate was isolated by
filtration; yield: 60%; HPLC purity: 98%; white solid; mp
199−201 °C; δH (500 MHz; DMSO-d6) 10.62 (1 H, bs),
10.42 (1 H, bs), 8.35 (1 H, t, J 1.9), 7.95 (1 H, daq, J 8.0, 1.0),
7.70−7.67 (2 H, m), 7.57−7.49 (3 H, m), 7.14 (2 H, d, J 9.1),
H, m), 7.15−7.03 (3 H, m), 7.01−6.97 (2 H, m); δC (125.5
1,3
MHz; DMSO-d6) 163.20 (dd,
J
CF2
252.2, 12.1), 162.7 (t,
4JCF2 3.1), 154.1 (d, JCF 245.2), 138.9, 137.06 (t, JCF2 8.3),
1
3
2
4
134.9, 129.6, 127.8, 126.54 (d, JCF 7.5), 124.92 (d, JCF 1.8),
2
3
124.12 (d, JCF 12.4), 123.57 (d, JCF 11.3), 120.0, 115.54 (d,
2,4
2
2JCF 19.4), 110.47 (dd,
J
20.0, 7.0), 108.0 (t, JCF2 25.2);
CF2
2
HRMS-ESI calcd for C19H16ClF3N3O3S+ [M + NH4]+:
458.0548, found: 458.0542.
7.09 (1 H, t, JHF 74.1), 7.08 (2 H, d, J 9.0); δC (125.5 MHz;
DMSO-d6) 163.24 (t, 4JCF2 3.0), 162.2 (dd, 1,3JCF2 247.4, 12.8),
3
3
147.38 (t, JCF2 3.3), 140.0, 139.4, 137.8 (t, JCF2 8.6), 134.7,
N-(5-(N-(Benzo[d][1,3]dioxol-5-yl)sulfamoyl)-2-chloro-
phenyl)-3,5-difluoro-benzamide (38). Method C. Purifica-
tion: the crude oil was purified on silica gel eluting with
chloroform/ethyl acetate (9:1); yield: 53%; HPLC purity:
>99%; white fluffy solid; mp 186−187 °C; δH (500 MHz;
DMSO-d6) 8.98 (1 H, d, J 2.1), 8.97 (1 H, bs), 7.47 (1 H, d, J
8.4), 7.43 (1 H, dd, J 8.4, 2.1), 7.41−7.37 (2 H, m), 7.12 (1 H,
s), 7.05 (1 H, tt, J 8.5, 2.3), 6.74 (1 H, d, J 2.1), 6.64 (1 H, d, J
8.3), 6.54 (1 H, dd, J 8.3, 2.2), 5.93 (2 H, s); δC (125.5 MHz;
DMSO-d6) 163.20 (dd, 1,3JCF2 252.1, 12.1), 163.0 (t, 4JCF2 3.0),
1
129.8, 124.3, 122.0, 119.9, 118.25, 116.4 (t, JCF2 257.8),
2,4
2
111.26 (dd,
J
CF2
20.3, 6.6), 107.38 (t, JCF2 25.8); HRMS-
ESI calcd for C20H18F4N3O4S+ [M + NH4]+: 472.0949, found:
472.0959.
3,5-Difluoro-N-(3-(N-(4-(trifluoromethoxy)phenyl)-
sulfamoyl)phenyl)benzamide (34). Method C. Purification:
the crude oil was taken up in ethyl acetate (0.1 mL), followed
by chloroform (1.9 mL), and the precipitate was isolated by
filtration; yield: 78%; HPLC purity: > 99%; white solid; mp
208−210 °C; δH (500 MHz; DMSO-d6) 10.63 (2 H, bs), 8.38
(1 H, t, J 1.8), 7.95 (1 H, daq, J 8.0, 1.1), 7.71−7.66 (2 H, m),
7.58−7.51 (3 H, m), 7.26 (2 H, d, J 9.6), 7.20 (2 H, d, J 9.1);
3
148.1, 146.2, 138.1, 137.06 (t, JCF2 8.3), 134.7, 129.7, 129.4,
127.9, 124.2, 120.2, 117.3, 110.53 (dd, 2,4JCF2 20.1, 7.2), 108.3,
2
108.1 (t, JCF2 25.2), 105.8, 101.6; HRMS-ESI calcd for
4
C20H17ClF2N3O5S+ [M + NH4]+: 484.0540, found: 484.0539.
N-(2-Chloro-5-(N-(4-(difluoromethoxy)phenyl)sulfamoyl)-
phenyl)-3,5-difluorobenzamide (39). Method C. Purification:
the crude oil was purified on silica gel eluting with chloroform/
δC (125.5 MHz; DMSO-d6) 163.23 (t, JCF2 3.0), 162.16 (dd,
1,3JCF2 247.4, 12.6), 144.46 (q, 3JCF3 2.2), 139.9, 139.4, 137.8 (t,
3JCF2 8.6), 136.9, 129.9, 124.4, 122.1, 122.0, 121.4, 120.0 (q,
2,4
1JCF3 256.0), 118.2, 111.25 (dd,
J
CF2
20.4, 6.6), 107.37 (t,
N
https://doi.org/10.1021/acsinfecdis.1c00231
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Article
ethyl acetate (93:7); yield: 25%; HPLC purity: >99%; off
white solid; mp 161−163 °C; δH (500 MHz; DMSO-d6) 8.98
(1 H, bs), 8.37 (1 H, bs), 7.49−7.46 (3 H, m), 7.40−7.35 (2
H, m), 7.16 (2 H, d, J 9.1), 7.05 (1 H, tt, J 8.5, 2.3), 7.00 (2 H,
d, J 9.1), 6.44 (1 H, t, 2JHF 74.1); δC (125.5 MHz; DMSO-d6)
163.21 (dd, 1,3JCF2 252.3, 12.1), 163.09 (t, 4JCF2 3.1), 148.88 (t,
203 °C; δH (500 MHz; acetone-d6) 9.68 (1 H, bs), 8.78 (1 H,
bs), 8.35 (1 H, s), 8.00 (1 H, daq, J 7.9, 1.1), 7.87 (1 H, daq, J
8.6, 1.0), 7.81 (1 H, dd, J 12.2, 2.1), 7.46 (1 H, t, J 7.9), 7.43 (1
H, dt, J 8.1, 0.9), 7.27 (1 H, t, J 8.6), 7.13 (2 H, d, J 9.0), 6.81
(2 H, d, J 9.0), 3.97 (3 H, s), 3.72 (3 H, s); δC (125.5 MHz;
4
1
acetone-d6) 164.03 (d, JCF 2.2), 157.5, 151.6 (d, JCF 245.4),
3
3JCF2 2.9), 138.7, 136.99 (t, JCF2 8.4), 134.8, 133.4, 129.7,
2
150.83 (d, JCF 10.7), 140.6, 140.0, 130.2, 129.2, 127.16 (d,
128.1, 124.1, 124.0, 120.7, 120.2, 115.8 (t, 1JCF2 260.6), 111.52
3
3JCF 5.6), 124.61 (d, JCF 3.4), 124.3, 123.6, 122.1, 118.4,
2,4
2
2
4
(dd,
J
CF2
20.2, 7.1), 108.16 (t, JCF2 25.2); HRMS-ESI calcd
115.16 (d, JCF 19.9), 114.2, 113.01 (d, JCF 2.0), 55.8, 54.7;
HRMS-ESI calcd for C21H23FN3O5S+ [M + NH4]+: 448.1337,
found: 448.1348.
for C20H17ClF4N3O4S+ [M + NH4]+: 506.0559, found:
506.0551.
N-(2-Chloro-5-(N-(4-(trifluoromethoxy)phenyl)sulfamoyl)-
phenyl)-3,5-difluorobenzamide (40). Method C. Purification:
the crude oil was purified on silica gel eluting with chloroform/
ethyl acetate (93:7); yield: 14%; HPLC purity: >99%; off
white solid; mp 169−171 °C; δH (500 MHz; DMSO-d6) 9.05
(1 H, s), 8.38 (1 H, bs), 7.49−7.47 (3 H, m), 7.41−7.35 (2 H,
N-(2-Fluorophenyl)-3-((4-methoxybenzyl)amino)-
benzenesulfonamide (43). To a solution of aniline 19 (50 mg,
0.19 mmol), 4-methoxybenzaldehyde (31 mg, 0.23 mmol), and
acetic acid (13 μL, 0.23 mmol) were added crushed 3 Å
molecular sieves (50 mg) in anhydrous dichloromethane (1.9
mL, 0.1M). After stirring at room temperature for 30 min,
sodium triacetoxyborohydride (80 mg, 0.38 mmol) was added
and the reaction mixture stirred for another 16 h, upon which
time it was diluted with chloroform (3 mL) and centrifuged to
remove the molecular sieves. After washing with water (3 mL),
the organic layer was dried over MgSO4 and filtered and the
solvent was removed in vacuo. The crude product was purified
on silica gel eluting with 100% chloroform to yield the title
product (46 mg; 63%) as an off white solid; HPLC purity: 97%;
mp 109−110 °C; δH (500 MHz; CDCl3) 7.57−7.54 (1 H, m),
7.21 (2 H, d, J 8.6), 7.17 (1 H, t, J 8.0), 7.07−7.02 (3 H, m),
6.99−6.95 (2 H, m), 6.87 (2 H, d, J 8.7), 6.73−6.69 (2 H, m),
4.18 (3 H, bs), 3.80 (3 H, s); δC (125.5 MHz; CDCl3) 159.0,
153.8 (d, 1JCF 244.3), 148.4, 139.6, 130.1, 129.8, 128.8, 125.81
(d, 3JCF 7.4), 124.86 (d, 2JCF 12.3), 124.66 (d, 3JCF 3.9), 122.9,
m), 7.20 (2 H, d, J 9.0), 7.10 (2 H, d, J 8.5), 7.06 (1 H, tt, J 8.4,
1,3
2.3); δC (125.5 MHz; DMSO-d6) 163.23 (dd,
J
252.4,
CF2
12.2), 163.09 (t, 4JCF2 3.1), 146.72 (q, 3JCF3 1.9), 138.7, 136.96
(t, JCF2 8.3), 134.8, 129.7, 128.1, 124.0, 123.4, 123.3, 122.1,
3
1
2,4
120.36 (q, JCF3 257.3), 120.0, 111.52 (dd,
J
20.3, 7.1),
CF2
108.2 (t, JCF2 25.2); HRMS-ESI calcd for C20H16ClF5N3O4S+
2
[M + NH4]+: 524.0465, found: 524.0471.
N-(3-(N-(2-Fluorophenyl)sulfamoyl)phenyl)pyrazine-2-
carboxamide (41). To a solution of aniline 19 (60 mg, 0.23
mmol), pyrazine-2-carboxylic acid (56 mg, 0.46 mmol), EDCI
(65 mg, 0.34 mmol), and HOBt (52 mg, 0.34 mmol) in
anhydrous DMF (1.1 mL, 0.2M) at 0 °C was added
triethylamine (94 μL, 0.68 mmol) dropwise under an argon
atmosphere. The reaction mixture was left to stir overnight at
room temperature, diluted with ethyl acetate (3 mL), and
filtered. The resulting solution was washed with brine (2 × 5
mL), 1 M HCl (5 mL), saturated aqueous NaHCO3 (2 × 5
mL), brine (5 mL), dried over MgSO4, and filtered, and the
solvent was removed in vacuo. The crude product was purified
on silica gel eluting with hexane/ethyl acetate (1:1) to yield
the title product (44 mg; 52%) as white solid; HPLC purity:
97%; mp 205−206 °C; δH (500 MHz; DMSO-d6) 11.09 (1 H,
bs), 10.24 (1 H, bs), 9.31 (1 H, d, J 1.4), 8.95 (1 H, d, J 2.5),
8.25 (1 H, aq, J 1.3), 8.55 (1 H, at, J 1.7), 8.07 (1 H, dd, J 8.2,
1.0), 7.56 (1 H, t, J 8.0), 7.48 (1 H, d, J 8.1), 7.27 (1 H, dt, J
1.4, 8.0), 7.21−7.11 (3 H, m); δC (125.5 MHz; DMSO-d6)
2
119.6, 117.4, 115.6, 115.32 (d, JCF 19.5), 114.1, 110.2, 55.3,
47.4; HRMS-ESI calcd for C20H20FN2O3S+ [M + H]+:
387.1173, found: 387.1164.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsinfecdis.1c00231.
Sequence alignment of eukaryotic malic enzymes; SEC
and oligomeric state analysis of T. cruzi malic enzymes;
superposition of malic enzymes from T. cruzi (cytosolic
isoform) with human mitochondrial isoform (HsME2);
activity of previously reported TcMEc sulfonamide
1
162.2, 155.6 (d, JCF 247.4), 147.9, 144.8, 144.2, 143.3, 140.6,
2
4
138.8, 129.6, 127.3 (d, JCF 7.4), 126.5, 124.66 (d, JCF 3.8),
3
2
124.5, 124.32 (d, JCF 12.8), 122.1, 118.4, 116.03 (d, JCF
19.3); HRMS-ESI calcd for C17H14FN4O3S+ [M + H]+:
373.0765, found: 373.0767.
1
inhibitors over intracellular form of T. cruzi; H and
13C NMR spectra for all compounds synthesized (PDF)
3-Fluoro-4-methoxy-N-(3-(N-(4-methoxyphenyl)-
sulfamoyl)phenyl)benza-mide (42). To a solution of aniline
18 (72 mg, 0.26 mmol), 3-fluoro-4-methoxy-benzoyl chloride
(108 mg, 0.56 mmol) and DMAP (16 mg, 0.14 mmol) in
anhydrous acetonitrile (1.3 mL, 0.2M) at room temperature
was added N-methylmorpholine (85 μL; 0.78 mmol; 3.0
equiv) dropwise under an argon atmosphere. The reaction
mixture was left to stir overnight at room temperature, and the
solvent was removed in vacuo. The crude oil was diluted with
ethyl acetate (2 mL), washed with 1 M HCl (2 mL), saturated
aqueous NaHCO3 (2 mL), brine (2 mL), dried over MgSO4,
and filtered and the solvent was removed in vacuo.
Purification: the crude solid was recrystallized several times
from a 9:1 methanol/acetone mixture to yield the title product
(18 mg; 16%) as a white solid; HPLC purity: 95%; mp 202−
AUTHOR INFORMATION
■
Corresponding Authors
Artur T. Cordeiro − Brazilian Biosciences National
Laboratory, Brazilian Center for Research in Energy and
Materials, Campinas, SP 13083-970, Brazil; Faculty of
Pharmaceutic Sciences, University of Campinas, Campinas,
SP 13083-871, Brazil; orcid.org/0000-0002-5676-2456;
Email: artur.cordeiro@lnbio.cnpem.br
Marjorie Bruder − Brazilian Biosciences National Laboratory,
Brazilian Center for Research in Energy and Materials,
Campinas, SP 13083-970, Brazil; orcid.org/0000-0003-
4236-848X; Email: marjorie.bruder@lnbio.cnpem.br
O
https://doi.org/10.1021/acsinfecdis.1c00231
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
pubs.acs.org/journal/aidcbc
Article
Human Mitochondrial NADP+-Dependent Malic Enzyme. Biochem. J.
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Activation of DPN-Linked Malic Enzyme from Escherichia Coli by
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Enzyme: A New Regulatory Enzyme. FEBS Lett. 33 (2), 251−255.
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Chan, N. L., Liu, J. H., and Hung, H. C. (2014) Structural
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Authors
Gustavo F. Mercaldi − Brazilian Biosciences National
Laboratory, Brazilian Center for Research in Energy and
Materials, Campinas, SP 13083-970, Brazil
Amanda G. Eufrásio − Brazilian Biosciences National
Laboratory, Brazilian Center for Research in Energy and
Materials, Campinas, SP 13083-970, Brazil; Faculty of
Pharmaceutic Sciences, University of Campinas, Campinas,
SP 13083-871, Brazil
Americo T. Ranzani − Brazilian Biosciences National
Laboratory, Brazilian Center for Research in Energy and
Materials, Campinas, SP 13083-970, Brazil; Institute of
Biology, University of Campinas, Campinas, SP 13083-970,
Brazil
Jessica do Nascimento Faria − Brazilian Biosciences National
Laboratory, Brazilian Center for Research in Energy and
Materials, Campinas, SP 13083-970, Brazil
Sabrina G. R. Mota − Brazilian Biosciences National
Laboratory, Brazilian Center for Research in Energy and
Materials, Campinas, SP 13083-970, Brazil; Institute of
Biology, University of Campinas, Campinas, SP 13083-970,
Brazil
Michelle Fagundes − Brazilian Biosciences National
Laboratory, Brazilian Center for Research in Energy and
Materials, Campinas, SP 13083-970, Brazil
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsinfecdis.1c00231
Author Contributions
∥G.F.M and A.E.G contributed equally to this work.
Notes
The authors declare no competing financial interest.
Structures of TcMEc were obtained in a ligand free form (PDB
7MF4) and in complexes with compounds 1 (PDB 6W29); 31
(PDB 6W2N); 33 (PDB 6W56); 34 (PDB 6W59); 39 (PDB
6W57); 40 (PDB 6W53); and 41 (PDB 6W49). Atomic
coordinates and experimental data are available at Protein Data
Bank.
(13) Yang, Z., Floyd, D. L., Loeber, G., and Tong, L. (2000)
Structure of a Closed Form of Human Malic Enzyme and
Implications for Catalytic Mechanism. Nat. Struct. Biol. 7 (3), 251−
257.
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Crystal Structure of Human Mitochondrial NAD(P)+-Dependent
Malic Enzyme: A New Class of Oxidative Decarboxylases. Structure 7
(8), 877−889.
(15) Tao, X., Yang, Z., and Tong, L. (2003) Crystal Structures of
Substrate Complexes of Malic Enzyme and Insights into the Catalytic
Mechanism. Structure 11 (9), 1141−1150.
(16) Chang, G. G., and Tong, L. (2003) Structure and Function of
Malic Enzymes, A New Class of Oxidative Decarboxylases.
Biochemistry 42, 12721.
ACKNOWLEDGMENTS
■
Authors thank GSK and Open Lab Foundation (Tres Cantos,
Spain) for supplying the GSK Chagas box and performing the
phenotypic assays that generated the data present in Table S1
in the Supporting Information. This study was supported by
the Sao Paulo Research Foundation (FAPESP) (Grants 2013/
̃
03983-5 and 2018/22202-8) and by the Coordination for the
Improvement of Higher Education Personnel (CAPES) (Grant
7775/2014-98). S.G.R.M. (Process No. 2014/15590-0 and
2016/19141-1) and A.T.R. (Process No. 2012/23682-7 and
2015/03336-5) were recipients of FAPESP fellowships. A.G.E.
was recipient of FAPESP (2017/00674-2) and CAPES
(88887.480232/2020-00) fellowships. The authors also thank
the I03 beamline from the Diamond Light Source and the
CNPEM facilities LEC, ROBOLAB, LBE, LPP, and RMN
from LNBio, and UVX/MX2 beamline from LNLS.
(17) Ranzani, A. T., Nowicki, C., Wilkinson, S. R., and Cordeiro, A.
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Malic Enzyme Isoforms by Target-Based HTS. SLAS Discovery 22 (9),
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(18) Pena, I., Pilar Manzano, M., Cantizani, J., Kessler, A., Alonso-
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Padilla, J., Bardera, A. I., Alvarez, E., Colmenarejo, G., Cotillo, I.,
Roquero, I., De Dios-Anton, F., Barroso, V., Rodriguez, A., Gray, D.
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Three Kinetoplastid Parasites: An Open Resource. Sci. Rep. 5 (8771),
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