Toward the development of dual-targeted glyceraldehyde-3-phosphate dehydrogenase/ trypanothione reductase inhibitors against trypanosoma brucei and trypanosoma Cruzi

Article abstract of DOI:10.1002/cmdc.201300399

A significant improvement in the treatment of trypanosomiases has been achieved with the recent development of nifurtimox- eflornithine combination therapy (NECT). As an alternative to drug combinations and as a means to overcome most of the antitrypanosomatid drug discovery challenges, a multitarget drug design strategy has been envisaged. To begin testing this hypothesis, we designed and developed a series of quinone-coumarin hybrids against glyceraldehyde-3-phosphate dehydrogenase/trypanothione reductase (GAPDH/TR). These enzymes belong to metabolic pathways that are vital to Trypanosoma brucei and Trypanosoma cruzi, and have thus been considered promising drug targets. The synthesized molecules were characterized for their dual-target antitrypanosomal profile, both in enzyme assays and in in vitro parasite cultures. The merged derivative 2-{[3-(3-dimethylaminopropoxy)- 2-oxo-2H-chromen-7-yl]oxy}anthracene- 1,4-dione (10) showed an IC50 value of 5.4 mm against TbGAPDH and a concomitant Ki value of 2.32 mm against TcTR. Notably, 2-{4-[6-(2-dimethylaminoethoxy)- 2-oxo-2H-chromen-3-yl]phenoxy}anthracene-1,4- dione (compound 6) displayed a remarkable EC50 value for T. brucei parasites (0.026 mm) combined with a very low cytotoxicity toward mammalian L6 cells (7.95 mm). This promising low toxicity of compound 6 might be at least partially due to the fact that it does not interfere with human glutathione reductase.

Full text of DOI:10.1002/cmdc.201300399

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DOI: 10.1002/cmdc.201300399
Toward the Development of Dual-Targeted
Glyceraldehyde-3-phosphate Dehydrogenase/
Trypanothione Reductase Inhibitors against Trypanosoma
brucei and Trypanosoma cruzi
Federica Belluti,[a] Elisa Uliassi,[a] Giacomo Veronesi,[a] Christian Bergamini,[a] Marcel Kaiser,[b, c]
Reto Brun,[b, c] Angelo Viola,[a] Romana Fato,[a] Paul A. M. Michels,[d] R. Luise Krauth-Siegel,[e]
Andrea Cavalli,[a, f] and Maria Laura Bolognesi*[a]
A significant improvement in the treatment of trypanosomias-
es has been achieved with the recent development of nifurti-
mox–eflornithine combination therapy (NECT). As an alterna-
tive to drug combinations and as a means to overcome most
of the antitrypanosomatid drug discovery challenges, a multi-
target drug design strategy has been envisaged. To begin test-
ing this hypothesis, we designed and developed a series of
quinone–coumarin hybrids against glyceraldehyde-3-phos-
phate dehydrogenase/trypanothione reductase (GAPDH/TR).
These enzymes belong to metabolic pathways that are vital to
Trypanosoma brucei and Trypanosoma cruzi, and have thus
been considered promising drug targets. The synthesized mol-
ecules were characterized for their dual-target antitrypanoso-
mal profile, both in enzyme assays and in in vitro parasite cul-
tures. The merged derivative 2-{[3-(3-dimethylaminopropoxy)-
2-oxo-2H-chromen-7-yl]oxy}anthracene-1,4-dione (10) showed
an IC50 value of 5.4 mm against TbGAPDH and a concomitant Ki
value of 2.32 mm against TcTR. Notably, 2-{4-[6-(2-dimethylami-
noethoxy)-2-oxo-2H-chromen-3-yl]phenoxy}anthracene-1,4-
dione (compound 6) displayed a remarkable EC50 value for
T. brucei parasites (0.026 mm) combined with a very low cyto-
toxicity toward mammalian L6 cells (7.95 mm). This promising
low toxicity of compound 6 might be at least partially due to
the fact that it does not interfere with human glutathione re-
ductase.
Introduction
The trypanosomiases are very serious so-called neglected tropi-
cal diseases (NTD).[1] In sub-Saharan Africa, Trypanosoma brucei
causes sleeping sickness or human African trypanosomiasis
(HAT),[2] and, primarily in Latin America, Trypanosoma cruzi
causes Chagas disease.[3] Both trypanosomiases are difficult to
treat, with drugs and vaccines not yet available.[4] Current in-
sufficient antiparasite chemotherapy regimens rely mostly on
single-target drugs that are often plagued by toxic side effects,
lack of efficacy, and development of resistance. A significant
improvement in the treatment of HAT has been recently ach-
ieved by the introduction of the first combination regimen,
based on the co-administration of oral nifurtimox and of a de-
creased dose of intravenous eflornithine (referred to as nifurti-
mox–eflornithine combination therapy, or NECT).[5] Thanks to
its improved efficacy, shortened duration, and diminished side
effects, it represents a major advance in terms of making the
antitrypanosomatid treatment safer, less expensive, and easier
to administer.[6]
[a] Prof. Dr. F. Belluti, E. Uliassi, G. Veronesi, Dr. C. Bergamini, A. Viola,
Prof. Dr. R. Fato, Prof. Dr. A. Cavalli, Prof. Dr. M. L. Bolognesi
Department of Pharmacy & Biotechnology, University of Bologna
Via Belmeloro 6 / Via Irnerio 48, 40126 Bologna (Italy)
E-mail: marialaura.bolognesi@unibo.it
[b] Dr. M. Kaiser, Prof. Dr. R. Brun
Swiss Tropical & Public Health Institute
Socinstrasse 57, 4002 Basel (Switzerland)
[c] Dr. M. Kaiser, Prof. Dr. R. Brun
As an alternative to drug combinations, an innovative phar-
macological strategy in the field could involve designing multi-
target ligands[7] (MTL), that is, single small molecules that can
modulate multiple vital targets in parasites’ metabolic path-
ways.[8] As for combinations, a multitarget approach improves
the chances of better efficacy and preventing the development
of drug-resistant parasites.[8] Moreover, it would be advanta-
geous over a combined administration of individual drugs
given the simplification of the therapeutic regimen and the
fact that it would also obviate possible drug–drug interac-
tions.[7,8] This latter aspect is especially relevant for young chil-
dren, and malnourished, immunocompromised, or debilitated
University of Basel, Petersplatz 1, 4003 Basel (Switzerland)
[d] Prof. Dr. P. A. M. Michels
Institute of Structural & Molecular Biology
School of Biological Sciences, University of Edinburgh
Mayfield Road, Edinburgh EH9 3JR (UK)
[e] Prof. Dr. R. L. Krauth-Siegel
Biochemie-Zentrum, Universitꢀt Heidelberg
Im Neuenheimer Feld 328, 69120 Heidelberg (Germany)
[f] Prof. Dr. A. Cavalli
Department of Drug Discovery & Development
Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300399.
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patients which are most commonly affected by NTD in devel-
oping countries.[9] However, although a multitarget drug dis-
covery strategy offers advantages in principle, it still represents
a challenging task for medicinal chemists.[10]
ble starting point.[18] Remarkably, it was shown in retrospect to
inhibit GAPDH[19] and TR[20] in the micromolar range. Further-
more, it exhibited trypanocidal potency in the nanomolar
range (EC50 value of 80 nm against T. brucei rhodesiense) and
a promising selectivity index (SI of 74), as assessed in experi-
ments using in vitro cultured parasites and human cell lines.[18]
Based on these findings, we designed and synthesized eight
quinone–coumarin hybrid compounds and characterized them
for their multitarget antitrypanosomal profile, both in enzyme
assays and in in vitro cultures of parasites.
In this study, we aimed to develop dual-targeted inhibitors
against T. brucei and T. cruzi glyceraldehyde-3-phosphate dehy-
drogenase/trypanothione reductase (TR/GAPDH). These two
enzymes have been identified as validated targets for antitry-
panosomatid drug discovery.[11] The fact that the bloodstream
form of African trypanosomes relies on glycolysis as the sole
source of energy supply[12] and the unusual compartmentaliza-
tion of this pathway inside glycosomes,[13] make the develop-
ment of selective GAPDH inhibitors (which should only exhibit Results and Discussion
minimal affinity for the human enzyme) particularly attracti-
ve.[11b] GAPDH catalyzes the oxidative phosphorylation of
Chemistry
d-glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. An-
other metabolic peculiarity is that trypanosomatid protists pos-
sess a unique thiol metabolism.[14] The discovery that an un-
usual low-molecular-weight thiol, namely N1,N8-bisglutathionyl-
spermidine (or trypanothione), rather than glutathione, is the
major redox reactive metabolite in trypanosomatids[15] has ele-
vated the enzymes involved in its metabolism to paradigms of
antiparasitic drug targets.[16] In this pathway, TR,[17] an NADPH-
dependent disulfide oxidoreductase, is responsible for reduc-
ing trypanothione disulfide (TS2) to dihydrotrypanothione
[T(SH)2] and for maintaining a reducing intracellular milieu.
In the search for TR/GAPDH inhibitors, we considered the
previously identified hit compound B6 (1 in Table 1) as a suita-
The synthesis of the two series of quinone–coumarin hybrids
(3–6 and 7–10) was accomplished as illustrated in Schemes 1–
3. To obtain the desired coumarin intermediates 11, 16, and
18, we used two different synthetic approaches. In detail, the
reaction of 2,5-dihydroxybenzaldehyde and sodium 2-(4-me-
thoxyphenyl)acetate in the presence of acetic anhydride and
triethylamine gave, upon alkaline hydrolysis, 6-hydroxy-3-(4-
methoxyphenyl)-2H-chromen-2-one (11) in good yield[21]
(Scheme 1). Following the same conditions, reaction of 2,4-di-
hydroxybenzaldehyde and sodium 2-(4-hydroxyphenyl)acetate
acetic furnished 7-hydroxy-3-(4-hydroxyphenyl)-2H-chromen-2-
one (16, Scheme 2). Reaction of 2-hydroxy-4-methoxybenzalde-
hyde and N-acetylglycine, in the presence of acetic anhydride
and sodium acetate,[22] pro-
vided the N-(7-methoxy-2-oxo-
Table 1. Antitrypanosomal activity against T. b. rhodesiense (Tbr) and T. cruzi (Tc), and cytotoxicity in a rat skele-
tal myoblast cell line (L6) of derivatives 3–10 and reference compounds.[a]
2H-chromen-3-yl)acetamide (17)
that was subjected to acidic hy-
drolysis with methanolic HCl, to
obtain the desired 3-hydroxy-7-
methoxy-2H-chromen-2-one[23]
(18, Scheme 3). The hydroxy de-
rivatives 11 and 18 were then
reacted with the selected w-
chloroalkyldimethylamines
in
N,N-dimethylformamide (DMF)
and in the presence of Cs2CO3
as the base,[24] to obtain com-
pounds 12, 13, 19, and 20. The
methoxy group was then re-
moved by treatment with BBr3
or HBr solution (33% in AcOH)
in dichloromethane as solvent,
to afford the phenol com-
pounds 14, 15 and 21, 22, re-
spectively. Finally, the quinone
moieties were introduced by
a nucleophilic substitution reac-
tion, firstly treating the phenol
intermediates 14–16 and 21, 22
with Cs2CO3 or K2CO3 and then
with the selected bromoqui-
none (2-bromoanthraquinone
Compd
Ar
n
EC50 [mm]
Tc
Selectivity Index
Tbr Tc
Tbr
L6
1
2
3
4
5
6
7
8
–
–
–
2
–
1
1
1
1
2
2
0.08
0.05
0.95
2.84
2.51
0.026
4.56
0.14
9.18
0.25
0.01[20]
–
1.26[18]
4.66[18]
1.26
5.92[18]
1.00[18]
4.14
13.42
9.62
7.95
4.27
4.5
74.0[18]
20.0[18]
4.32
4.72
3.83
301.8
0.93
32.03
1.46
4.70
C4H4
–
0.21
3.26
0.51
6.55
0.25
0.50
0.15
0.31
–
–
26.3
1.47
31.23
8.46
29.86
43.15
ND[b]
–
C4H4
–
C4H4
–
9
10
13.43
1.30
C4H4
5.23
–
melarsoprol
benznidazole
podophyllotoxin
18.3[20]
1830.0[20]
–
–
>225[20]
–
1.70[20]
–
>384[20]
–
0.017[20]
–
[a] IC50 values represent the concentration of a compound that causes 50% growth inhibition and are the
mean of two independent determinations that varied by less than a factor of two. The experimental error is
within ꢀ50%. SI=(IC50 for L6)/(IC50 for the respective protozoan parasite).[b] Not determined due to cytotoxic-
ity in L6 cells.
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or 2-bromonaphthoquinone), to afford the final compounds 3–
10.
Design rationale and biological studies
Our strategy to discover novel dual inhibitors started from the
finding that 1 inhibited both targets at similar concentration
ranges.[19,20] As a first step, we calculated IC50 values of 7.2 and
9.0 mm, for GAPDH and TR, respectively. Although the inhibito-
ry potencies were in the micromolar range, a balanced affinity
against the targets of interest was obtained, which is a prereq-
uisite in multitarget drug discovery.
With these data in hand, we looked for a strategy to im-
prove the potencies of the starting hit through chemical opti-
mization. On the basis of the criteria of the so-called “rule of
three” it is evident that 1 can be truly considered a fragment.[25]
In fact, it has a molecular weight of 250.25 Da, fewer than
three hydrogen bond acceptors/donors, a clogP value of 2.92,
a polar surface area (PSA) of 43.37 ꢁ2, and ligand efficiencies
for GAPDH and TR of 0.31 and 0.24, respectively. Recently,
a fragment-based approach[7] has been considered very effec-
tive in multitarget drug discovery.[26] In a typical embodiment,
once a fragment binding to two proteins has been identified,
a library of analogues with additional functionality could be
made, with the goal of increasing the affinity toward both tar-
gets while concomitantly maintaining balanced activities. Fol-
lowing this strategy, 1 was firstly investigated by docking simu-
lation and its resulting predicted mode of association with
GAPDH and TR binding sites is shown in Figure 1. In detail,
1 binds to GAPDH at the active site with the catalytic Cys166
properly positioned to possibly give a nucleophilic attack to
the C2 carbon of the naphthoquinone ring. This would allow it
to act as a covalent inhibitor of GAPDH as previously hypothe-
sized[19] and herein partially supported by biochemical experi-
ments (see below). Further interactions with His194 and
Cys166 backbone contributed to stabilize 1 into the binding
pocket of GAPDH. Docking simulations of 1 with TR showed
that this fragment could be accommodated into the wide TR
catalytic pocket. In particular, one of the two carbonyls of the
naphthoquinone ring established a hydrogen bond interaction
with the Leu399 backbone, while Phe396 and Lys21 of TR in-
teracted with 1 by means of p–p and cation–p interactions.
Remarkably, these interactions could allow our compounds to
display selectivity over human glutathione reductase (hGR).[27]
These initial docking simulations showed that 1 could bind
both enzymes by establishing favorable interactions with
active site amino acids, in support of our strategy to use 1 as
starting fragment for GAPDH/TR dual-inhibitor discovery.
Scheme 1. Synthesis of compounds 3, 5, and 6. Reagents and conditions:
a) Ac2O, anhydrous NEt3, 1808C, 18 h, then K2CO3, RT, 6 h; b) w-chloroalkyldi-
methylamine hydrochloride, Cs2CO3, DMF, N2, 110 8C, 8 h; c) BBr3, CH2Cl2,
ꢁ708C, 1 h, then RT, 6 h; d) 2-bromoquinone, Cs2CO3, DMF, RT, 3 h.
Scheme 2. Synthesis of compound 4. Reagents and conditions: a) Ac2O, anhy-
drous NEt3, 1808C, 18 h, then K2CO3, RT, 6 h; b) 2-bromonaphthoquinone,
Cs2CO3, DMF, RT, 3 h.
Starting from the molecular information, we expanded
1 through a framework combination approach with the inten-
tion to develop more potent, bigger, dual-target inhibitors.
Among the reported inhibitors of GAPDH, chalepin (IC50
64 mm), a coumarin-containing natural product extracted from
the plant Pilocarpus spicatus, is the one with potential drug-
like properties. In addition, the crystallographic structure of the
chalepin–TcGAPDH complex has been available since 2002.[28]
Scheme 3. Synthesis of compounds 7–10. Reagents and conditions: a) Ac2O,
AcONa, 1208C, 5 h; b) MeOH, HCl 3n, 1208C, 10 h; c) w-chloroalkyldimethyl-
amine hydrochloride, Cs2CO3, DMF, N2, 110 8C, 8 h; d) HBr solution (33% in
AcOH), 808C, 14 h; e) 2-bromoquinone, K2CO3, DMF, RT, 3 h.
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Figure 1. Docking pose of 1 at the a) TbGAPDH (2X0N) and b) TbTR (2WPF) binding sites.
Regarding a rational drug design of an inhibitor of TR, a dis-
tinctive feature of this enzyme is its extremely wide and nega-
tively charged binding site for TS2. This has been exploited for
obtaining selectivity against hGR, the closest related counter-
part in the mammalian host, which has a positively charged
binding site for glutathione disulfide (GSSG).[17] Building on
these considerations, we expanded the structure of 1 by incor-
porating a coumarin and an amino functionality. So far, two
subsets of compounds were generated through merging or
fusing approaches,[7] as depicted in Figure 2. In detail, in com-
pounds 3–6, the coumarin framework has been linked at posi-
tion 4 of the phenoxy ring of 1; in 3 and 5, an amino function
has been introduced at position 6 of the coumarin through
ethoxy or propoxy linkers, whereas the introduction of
a second quinone moiety led to 4. In addition, to enlarge the
chemical diversity and based on the antitrypanosomal activity
of the higher homologue 2 (Figure 2),[18] the anthraquinone 6
was also designed. Similarly, by integrating the selected moiet-
ies to a greater extent, the subset 7–10, in which the benzene
ring of 1 and 2 and that of coumarin overlaps, was obtained.
The designed 3–10 were then synthesized, and their activity
profile evaluated toward the two enzymes and in whole cell
parasite assays. First, we tested the inhibitory potency of 3–10
against TbGAPDH using a spectrophotometric method, as de-
scribed by Wiggers et al.[29] with minor modifications (see the
Experimental Section). In brief, TbGAPDH activity was mea-
sured by following NAD reduction at 340 nm using GAP (glyc-
eraldehyde 3-phosphate) as a substrate.
Compounds 3–10 were assayed at a concentration of 10 mm,
and the percentages of residual enzymatic activity measured,
in comparison with 1 and 2 as parent compounds. As shown
in Figure 3, all of the newly developed hybrids (either belong-
ing to the merged or fused series) displayed remaining activi-
ties ranging from 10 to 50%, which in most cases were higher
than those observed when adding 1 or 2. The most active
compound was the merged derivative 10, which has been ana-
lyzed at different concentrations (ranging from 1 to 50 mm,
data not shown), obtaining an IC50 value of 5.4 mm, only slight-
ly higher than that of 1 (IC50 =3.3 mm). When analyzing the
reported data, the structure–activity relationship (SAR) ap-
peared quite “flat”, as the major changes made to the structure
Figure 3. Effect of compounds 1–10 (at 10 mm) on TbGAPDH activity. The ac-
tivities were measured spectrophotometrically, and data are the means ꢀSD
of triplicate determinations.
Figure 2. Design strategy leading to the fused and merged quinone–cou-
marin hybrids 3–10 (see Table 1 for individual structures).
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of 1 did not result in significant activity differences. The “flat”
SAR within the compound series might point toward a very
similar binding mode. One possible explanation may be that,
in analogy with what we observed for 1,[19] the 2-aryloxynaph-
thoquinone moieties of 3–10 could react with Cys166 of the
GAPDH active site by forming a covalent bond. We hypothe-
sized that 1 may react with the thiol group of Cys166, and a re-
action at C2 with the phenate displacement as leaving group
may take place.[19] Indeed, by analyzing the structures of our
hybrids, it is evident that they might undergo the same reac-
tion as they also carry a phenoxy leaving group.
To support this hypothesis, we exploited the peculiar spec-
tral features of our derivatives. They contain a fluorophore (the
coumarin scaffold; likely the leaving group of the covalent in-
teraction), although it displays only a very low fluorescence
due to the quenching effect of the quinone moiety. Nonethe-
less, the nucleophilic attack on the quinone by the thiol group,
causing formation of a thioether bond, should result in a disso-
ciation of the coumarin moiety that then becomes more fluo-
rescent. Indeed, an increase in fluorescence could be detected
in spectrofluorimetric experiments, by following fluorescence
emission at 485 nm (with excitation at 335 nm). We treated
compound 10 with molecules carrying a thiol group (for exam-
ple, dithiothreitol (DTT) and N-acetylcysteine (NAC)), then we
compared its fluorescence emission spectra with that of 3-(3-
aminopropoxy)-7-hydroxycoumarin 22 (the actual leaving
group). Indeed, Figure 4a shows the fluorescence emission
spectra of compound 10 (curve 1 from the bottom) and frag-
ment 22 (curve 4 from the bottom), whereas curves 2 and 3
refer to the emission of 10 treated with DTT and NAC, respec-
tively. These latter curves are clearly similar to those obtained
for the coumarin fragment alone. The same behavior could be
observed when treating 10 with GAPDH (Figure 4b), support-
ing the idea that a nucleophilic substitution reaction between
10 and a thiol nucleophile could occur.
In parallel, 3–10 were studied in comparison with 1 and 2 in
the TR enzymatic assay, as described previously.[20] As evident
from the graph of Figure 5a, the merged hybrids 7, 8, and 10
proved to be more effective inhibitors of TR than 2, and nearly
as potent as 1. In fact, at a fixed inhibitor concentration of
10 mm, and either 100 or 40 mm TS2, the remaining activity
ranged from 25 to 62% for the merged hybrids, and between
23 and 65% for compounds 1 and 2. In contrast, at 10 mm, the
fused series did not display any significant activity. Thus, we
studied the effect of 3–6 at concentrations of 50–100 mm (Fig-
ure 5b). This activity trend was difficult to rationalize, as pur-
posely addressed docking studies were not informative in this
respect (data not shown).
Figure 4. a) Comparison of the emission scan (measured as excitation of
335 nm) of compound 10 in the absence (first curve from bottom) or pres-
ence of thiols DTT and NAC (second and third curves, respectively), and that
of the coumarin fragment 22 at an equimolar concentration. The fluores-
cence maxima of 10 treated with the thiol nucleophiles were similar to that
of 22, suggesting that a nucleophilic displacement with the release of the
coumarin leaving group might occur. b) Emission scans of 10 in the pres-
ence of GAPDH.
To assess the TR/GR selectivity, the compounds were studied
at a fixed concentration against both enzymes (40 mm for 3–6
and 5 mm for 7–10) and the results graphically depicted in
Figure 7. Despite the presence in 3 and 5–10 of the protonata-
ble amino group as a putative determinant of selectivity, we
could not verify the desired profile. In fact, all compounds,
except 6, were more effective toward human GR than trypano-
somal TR. The same applied for 4, which does not carry the
amino functionality. Intriguingly, for compound 6 only a mar-
ginal inhibition of GR was observed at a concentration of
40 mm.
Concerning the mechanism of inhibition, we speculated that
it may be similar to that proposed for other quinone inhibi-
tors.[20,30] These ligands are known to easily shuttle between
hydroquinone and quinone forms. It is therefore feasible that
in addition to the observed noncompetitive inhibition, 10 may,
Taken together, the most active of the newly synthesized
compounds was 10, which was also the most powerful GAPDH
inhibitor. Notably, no difference in the degree of inhibition at
the two TS2 concentrations was observed for 7–10, 3, or 5,
suggesting an inhibition mechanism independent from the
substrate concentration. Indeed, the kinetic analysis revealed
that 10 inhibits TR noncompetitively with a Ki value of 2.7 mm
(Figure 6). Thus, in principle, TR and GAPDH are modulated by
10 at the same concentration range.
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Figure 6. Lineweaver–Burk plots for the inhibition of TcTR by 10. The assays
contained 100 mm NADPH and two fixed concentrations of inhibitor as indi-
cated in the graph, and the TS2 concentration was varied.
a redox cycling might also apply to human GR and in this case
it would lead to an unwanted cytotoxicity; however, the intrin-
sic oxidase activity of human GR is much lower than that of
TR.[32] Thus, our derivatives were studied for their ability to
induce the intrinsic oxidase activity of TR and GR by following
the consumption of NADPH in the absence of the respective
disulfide substrate (Figure 8). The oxidase activity of TR was in-
creased up to nearly a 100-fold in the presence of 3, 5, and 9,
which all share a naphthoquinone moiety. Encouragingly, the
oxidase activity of GR remained much lower than that of TR.
The other derivatives were much less effective as subversive
substrates for both enzymes, with 6 being completely inactive
for GR. This behavior is very promising in terms of selectivity
against the host.
Figure 5. Inhibition of TR by a) 7–10, 1 and 2, and b) 3–6. The activity was
measured following the NADPH consumption at 340 nm as described in the
Experimental Section. The assays contained a fixed concentration of 100 or
40 mm TS2 and the indicated inhibitor concentration. The controls contained
the same amount of DMSO used to dissolve the compounds. The remaining
percent activity refers to that of the enzyme in the presence of DMSO.
To detect the generation of radical species in the reaction of
TR with 3, 5, and 9, the oxidase assay was coupled to the one-
electron reduction of oxidized cytochrome c (Cytc–Fe3+). By
following the absorption increase at 550 nm, the single-elec-
tron reduction of the heme protein was detected (Table 2). To
substantiate the putative generation of superoxide radicals in
at least in part, act as a redox
cycler. In this process, TR re-
Table 2. Detection of radical species in the reaction of trypanothione reductase (TR) with compounds 3, 5, and
9 using an oxidase assay.[a]
duces the quinone to a semiqui-
none (one-electron reduction) or
to a hydroquinone (two-electron
reduction), causing futile con-
sumption of NADPH. The semi-
quinone formed can then react
with molecular oxygen, produc-
ing superoxide anion radicals.
The combination of TR inhibition
and redox cycling might be
beneficial, as it should lead
to stronger trypanocidal pro-
perties.[20,30,31] Of course, such
Compd
NADPH
cons. [Umgꢁ1
Cytc–Fe3+ red. [Umgꢁ1
]
NADPH ox./
Cytc red.[b]
1eꢁ red.[c]
[%]
Inh. (SOD)[d]
[%]
]
ꢁSOD
+SOD
3
5
9
1.93ꢀ0.13
1.24ꢀ0.06
0.55ꢀ0.06
3.07ꢀ0.24
1.86ꢀ0.06
0.80ꢀ0.05
0.47ꢀ0.04
0.36ꢀ0.01
0.23ꢀ0.02
1:1.6
1:1.5
1:1.45
79.5
75
73
84.5
81
71
[a] The oxidase activity of TR in the presence of compounds 3, 5, and 9 was assessed by measuring the con-
sumption of NADPH and by coupling the reaction to cytochrome c reduction. The latter reaction was carried
out in the absence or presence of superoxide dismutase (SOD) to distinguish between a direct and superox-
ide-anion-mediated single-electron reduction (1eꢁ red.) of Cytc–Fe3+. Data represent the mean ꢀSD of a tripli-
cate determination. [b] Ratio of NADPH oxidation/Cytc reduction (NADPH ox./Cytc red.); stoichiometry=1:2.
[c] Single-electron reduction (1eꢁ red.) is expressed as a percentage of overall reduction. [d] Percent inhibition
by SOD.
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Figure 8. Increase of NADPH oxidation by hGR (white columns) and TcTR
(gray columns) in the absence of the natural substrates, but in the presence
of compounds 3 and 5–10 at 40 mm acting as subversive substrates. The ac-
tivity of the enzymes, evaluated by following NADPH consumption at
340 nm, is expressed in Umgꢁ1 and compared with their intrinsic oxidase ac-
tivity (IOA), measured in the presence of the same amount of DMSO used to
dissolve the compounds. Data are the means ꢀSD of triplicate determina-
tions.
partially reduced directly by the quinone radical, as also previ-
ously observed for menadione-based subversive substrates of
TR.[30]
Despite the moderate in vitro potency of these compounds
against the two enzymes, we were quite encouraged by ex-
periments performed with intact T. brucei and T. cruzi parasites
and mammalian cells. We evaluated the activity of 3–10
against T. b. rhodesiense (bloodstream stage, STIB 900 strain)
and T. cruzi (intracellular amastigote stage, Tulahuen C2C4
strain) parasites, in comparison to the reference drugs melarso-
prol and benznidazole. Furthermore, their cytotoxicity against
mammalian cells was assessed using the L6 rat skeletal myo-
blast cell line. All the newly synthesized compounds displayed
moderate to high activity (Table 1). They were generally more
efficient against T. brucei than T. cruzi. The most effective com-
pounds were 6, 8, and 10, with 6 inhibiting the proliferation of
African trypanosomes at a concentration similar to that of the
drug melarsoprol. Remarkably, against T. cruzi, 3 and 5 dis-
played EC50 values that were even lower than that of the corre-
sponding reference drug benznidazole. As 10 was the most
active compound in the enzymatic assays, whereas 6 was one
of the weakest inhibitors of GAPDH/TR, it clearly emerged that
the in vitro whole cell data did not follow a trend similar to
that of the in vitro enzyme data, with decreases in the IC50
values not being always mirrored by corresponding decreases
in the EC50 values. The promising data obtained for 6 could
suggest that this compound has additional off-targets, or is se-
lectively concentrated/metabolically activated in the parasite,
or a combination of these. Interestingly, 6 displayed a very low
cytotoxicity toward L6 cells (7.95 mm) relative to its EC50 value
for T. brucei parasites (0.026 mm). Thanks to this profile, 6
Figure 7. Comparison of the inhibition of hGR (black columns) and TcTR
(gray columns) by a) compounds 7–10 at 5 mm, and b) compounds 3–6 at
40 mm. The activity of both enzymes was measured by following NADPH
consumption at 340 nm as described in the Experimental Section. The
assays for TR activity contained a fixed concentration of 100 mm TS2. The
assays for hGR activity contained a fixed concentration of 1 mm GSSG. The
controls contained the same amount of DMSO used to dissolve the com-
pounds. The remaining percent activity refers to that of the enzyme in the
presence of DMSO. Data are the means ꢀSD of triplicate determinations.
the overall reaction, superoxide dismutase (SOD) was added to
the assay, as previously reported.[30] In the presence of SOD,
the rate of Cytc–Fe3+ reduction was lowered by 71 to 85% as
expected if superoxide anions are formed, because the dismu-
tation of superoxide radicals by SOD proceeds much faster
than the corresponding reduction to molecular oxygen by
Cytc–Fe3+. The remaining activity indicates that Cytc–Fe3+ is
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dicated with element symbols, analytical results obtained for those
elements are within 0.4% of the theoretical values. The IR spectra
were recorded on a Jasco FT/IR-4100. Chromatographic separations
were performed on silica gel columns by a flash method (Kieselgel
40, 0.040–0.063 mm, Merck). Reactions were followed by thin layer
chromatography (TLC) on pre-coated silica gel plates (Merck Silica
Gel 60 F254) and then visualized with a UV lamp. Compounds
were named following IUPAC rules as applied by Beilstein-Institute
AutoNom (version 2.1), a PC-integrated software package for sys-
tematic names in organic chemistry.
showed a remarkable SI>300. This compound fulfills the “hit
criteria” of activity and selectivity against T. b. rhodesiense
(IC50 <0.2 mgmLꢁ1; SI>100) as defined by the drug-screening
network of TDR,[33] and is eligible for further in vivo investiga-
tion.
Conclusions
In NTD, a multitarget drug discovery approach may lead to
more effective, affordable, and sustainable medicines. Toward
this goal, we designed and synthesized novel GAPDH/TR dual
inhibitors by expanding fragment 1.
6-Hydroxy-3-(4-methoxyphenyl)-2H-chromen-2-one (11): A mix-
ture of 2,5-dihydroxybenzaldehyde (1.38 g, 10 mmol), sodium 2-(4-
methoxyphenyl)acetate (1.88 g, 10 mmol), freshly distilled acetic
anhydride (3 mL), and anhydrous triethylamine (3 mL) was heated
at 1808C for 18 h. After cooling, a K2CO3 saturated aqueous solu-
tion (10 mL) was added and the resulting mixture was stirred at RT
for 6 h. The reaction mixture was then acidified and the precipitate
was filtered off and crystallized from EtOH to afford a yellow solid
(2.15 g, 80%), mp: 1328C. 1H NMR (CDCl3, 300 MHz): d=3.82 (s,
3H), 6.96–6.99 (m, 4H), 7.25 (d, J=8.8 Hz, 1H), 7.69–7.71 (m, 3H),
7.87 ppm (br, 1H).
From a practical perspective, the obtained data reveal that
against GAPDH, the most potent derivative 10 showed an IC50
value of 5.3 mm, making it among the most effective trypano-
somal GAPDH inhibitors developed so far. Compound 10
showed a concomitant Ki of 2.32 mm against TR, which is en-
couraging in terms of dual activity. The molecules were then
tested in whole parasite growth assays and displayed remark-
able EC50 values against the trypanosomes. Compounds 3, 5,
and 6 showed activities similar to that of the reference drugs
benznidazole and melarsoprol. However, the lack of correlation
between GAPDH/TR inhibition and the trypanocidal effects for
most of the derivatives, suggested that other targets might be
involved. Interestingly, we discovered that 6 was not very toxic
against the host cell (at levels >300-fold the EC50 value against
the T. brucei parasite). The specificity of this derivative was
much higher when compared with the parent molecules 1 and
2 and with all the newly synthetized hybrids. In fact, a general
cytotoxicity was the major concern for the drug-likeness of
several quinone-based hybrids.[18,20] We speculate that the
lower toxicity of 6 might be, at least in part, attributable to the
fact that it does not interfere with human GR, neither acting as
an inhibitor nor as a subversive substrate. All in all, these data
are promising and warrant further investigation of 6 as a lead
candidate against African and American trypanosomiases.
From a conceptual perspective, the data collected in this
work emphasize that obtaining a multitarget ligand is still a dif-
ficult endeavor for medicinal chemists, and some might even
assert impossible.[10] However, in our opinion, the high possible
reward of these molecules may make their development a risk
well worth taking.
7-Hydroxy-3-(4-hydroxyphenyl)-2H-chromen-2-one (16): A mix-
ture of 2,4-dihydroxybenzaldehyde (1.38 g, 10 mmol), sodium 2-(4-
hydroxyphenyl)acetate (1.74 g, 10 mmol), freshly anhydrous acetic
anhydride (3 mL), and anhydrous triethylamine (3 mL) was heated
at 1808C for 18 h. After cooling, a K2CO3 saturated aqueous solu-
tion (10 mL) was added and the resulting mixture was stirred at RT
for 6 h. The mixture was then acidified and the precipitate was fil-
tered off and crystallized from EtOH to afford a yellow solid
(1.27 g, 50%); mp: 1848C. 1H NMR ([D6]DMSO, 300 MHz): d=6.96
(d, J=8.8 Hz, 4H), 7.25 (d, J=8.8 Hz, 1H), 7.69 (d, J=8.8 Hz, 2H),
7.70 (s, 1H), 7.87 (br, 1H, OH), 8.15 ppm (br, 1H, OH).
N-(7-Methoxy-2-oxo-2H-chromen-3-yl)acetamide (17): A mixture
of 2-hydroxy-4-methoxybenzaldehyde (7.00 g, 46 mmol), N-acetyl-
glycine (5.39 g, 46 mmol), freshly distilled acetic anhydride
(21.7 mL, 230 mmol), and sodium acetate (15.10 g, 184 mmol) was
heated at 1208C for 5 h. After cooling, a mixture of ice/water
(100 mL) was added and the precipitate was filtered off and crys-
tallized from EtOH to afford a yellow solid (2.84 g, 26.5%); mp:
1
230–2318C. H NMR (CDCl3, 300 MHz): d=2.22 (s, 3H), 3.85 (s, 3H),
6.91 (d, J=2.8 Hz, 1H), 6.95 (dd, J=2.8, 8.8 Hz, 1H), 7.40 (d, J=
8.8 Hz, 1H), 8.03 (br, 1H), 8.66 ppm (s, 1H).
3-Hydroxy-7-methoxy-2H-chromen-2-one
(18):
17
(1.2 g,
5.14 mmol) was dissolved in MeOH (36 mL) and 3N HCl (200 mL),
the solution was heated at 1108C for 10 h, and the precipitate was
1
filtered off, obtaining a yellow solid (0.75 g, 74%). H NMR (CDCl3,
300 MHz): d=3.85 (s, 3H), 6.87 (d, J=2.8 Hz, 1H), 6.89 (dd, J=2.8,
8.8 Hz, 1H), 8.04 (s, 1H), 7.33 (d, J=8.8 Hz, 1H), 7.94 ppm (br, 1H).
Experimental Section
Chemistry
General procedures for compounds 12, 13, 19, and 20
High-grade commercial products were used as starting materials,
unless otherwise specified in this section. Solvents were of analyti-
cal grade. Melting points were determined in open glass capillaries,
CsCO3 (0.65 g, 1.8 mmol) was added to a solution of hydroxycou-
marin derivative (0.50 g, 1.8 mmol) in DMF (5 mL), and the mixture
was stirred at RT and under N2 atmosphere for 30 min. Subsequent-
1
using a Bꢂchi apparatus and are uncorrected. H NMR and 13C NMR
spectra were recorded on Varian Gemini spectrometers and chemi-
cal shifts are reported as parts per million (ppm d value) relative to
the peak for tetramethylsilane (TMS) as internal standard. Standard
abbreviations indicating spin multiplicities are given as follows:
s (singlet), d (doublet), t (triplet), br (broad), q (quartet), or m (mul-
tiplet). Mass spectra were recorded on a Waters ZQ 4000 apparatus
operating in electrospray mode (ES). Wherever analyses are only in-
ly,
a
mixture of w-chloroalkyldimethylamine hydrochloride
(2.0 mmol), CsCO3 (0.65 g, 1.8 mmol) in DMF (5 mL) was added.
The resulting reaction mixture was heated at 1108C, under N2 at-
mosphere, for 8 h. On cooling, water was added (100 mL) and the
resulting solid was collected by filtration. The crude product was
purified by flash column chromatography on silica gel using a mix-
ture of CH2Cl2/MeOH (95:05) as eluent.
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6-(2-(Dimethylaminoethoxy)-7-hydroxy-3-(4-methoxyphenyl)-2H-
chromen-2-one (12): Starting from 11 and 2-chloro-N,N-dimethyl-
ethanamine hydrochloride (0.29 g), compound 12 was obtained
8.8 Hz, 2H), 7.21–7.30 (m, 2H), 7.45 (s, 1H), 7.67 (d, J=8.8 Hz, 2H),
8.03 ppm (br, 1H).
3-(2-Dimethylaminoethoxy)-7-hydroxy-2H-chromen-2-one (21):
Following procedure B and starting from 19 (0.34 g), compound
1
(0.48 g, 75%); mp: 122.28C. H NMR (CDCl3, 300 MHz): d=3.10 (s,
6H), 3.81 (t, J=6.4 Hz, 2H), 4.59 (t, J=6.4 Hz, 2H), 6.96–6.99 (m,
4H), 7.25 (d, J=8.8 Hz, 1H), 7.69 (d, J=8.8 Hz, 2H), 7.72 ppm (s,
1H).
1
21 was obtained (0.12 g, 40%), mp: 143–1458C. H NMR (CD3OD,
400 MHz): d=2.47 (s, 6H), 2.93 (t, J=5.4 Hz, 2H), 4.17 (t, J=5.4 Hz,
2H), 6.71 (d, J=2.6 Hz, 1H), 6.82 (dd, J=2.4, 8.4 Hz, 1H), 7.24 (s,
1H), 7.38 ppm (d, J=8.4 Hz, 1H).
6-(3-(Dimethylaminopropoxy)-3-(4-methoxyphenyl)-2H-chro-
men-2-one (13): Starting from 11 and 3-chloro-N,N-dimethylpro-
pan-1-amine hydrochloride (0.31 g), compound 13 was obtained
(0.45 g, 71%), mp: 129.38C. 1H NMR (CDCl3, 300 MHz): d=1.95–
2.05 (m, 2H), 2.29 (s, 6H), 2.50 (t, J=6.4 Hz, 2H), 3.85 (s, 3H), 4.06
(t, J=6.4 Hz, 2H), 6.96–7.06 (m, 4H), 7.25 (d, J=8.8 Hz, 1H), 7.64 (s,
1H), 7.66 ppm (d, J=8.8 Hz, 2H).
3-(3-Dimethylaminopropoxy)-7-hydroxy-2H-chromen-2-one (22):
Following procedure B and starting from 20 (0.34 g), compound
1
21 was obtained (0.12 g, 40%), mp: 143–1458C. H NMR (CD3OD,
400 MHz): d=2.22–2.05 (m, 2H), 2.37 (s, 6H), 2.63 (t, J=5.4 Hz,
2H), 4.02 (t, J=5.4 Hz, 2H), 7.00 (d, J=2.6 Hz, 1H), 6.73 (dd, J=2.4,
8.4 Hz, 1H), 7.11 (s, 1H), 7.31 ppm (d, J=8.4 Hz, 1H).
3-(2-Dimethylaminoethoxy)-7-methoxy-2H-chromen-2-one (19):
Starting from 18 (0.34 g) and 2-chloro-N,N-dimethylethanamine hy-
drochloride (0.29 g), compound 19 was obtained (0.46 g, 97%),
mp: 121–1238C. 1H NMR (CDCl3, 300 MHz): d=2.80 (t, J=5.8 Hz,
2H), 2.25 (s, 6H), 3.85 (s, 3H), 4.09 (t, J=5.8 Hz, 2H), 6.79 (d, J=
2.8 Hz, 1H), 6.82 (dd, J=2.8, 8.8 Hz, 1H), 6.86 (s, 1H), 7.28 (d, J=
8.8 Hz, 1H), 7.94 (br, 1H), 8.33 ppm (s, 1H).
General procedure for the synthesis of compounds 3–10
The phenol derivative (14–16, 20, 21) (0.1 g, 0.29 mmol) was dis-
solved in anhydrous DMF (7 mL) and a base (Cs2CO3 or K2CO3) was
added, the mixture was stirred at RT for 1 h. Then, 2-bromoanthra-
cene-1,4-dione or 2-bromonaphthalene-1,4-dione was added and
the reaction mixture was stirred at RT for 3 h. The resulting solu-
tion was put into an ice/water mixture (15 mL) and the solid
formed was collected by vacuum filtration. The crude was purified
by flash column chromatography using a mixture of CH2Cl2/MeOH
(95:5) as eluent.
3-(3-Dimethylaminopropoxy)-7-methoxy-2H-chromen-2-one (20):
Starting from 18 (0.34 g) and 3-chloro-N,N-dimethylpropan-1-
amine hydrochloride (0.31 g), compound 20 was obtained (0.46 g,
1
97%), mp: 125–1268C. H NMR (CDCl3, 300 MHz): d=2.00–2.04 (m,
2H), 2.22 (s, 6H), 2.45 (t, J=5.8 Hz, 2H), 3.79 (s, 3H), 4.02 (t, J=
5.8 Hz, 2H), 6.74 (d, J=2.8 Hz, 1H), 6.78 (dd, J=2.8, 8.8 Hz, 1H),
6.82 (s, 1H), 7.22 ppm (d, J=8.8 Hz, 1H).
2-{4-[6-(3-Dimethylaminopropoxy)-2-oxo-2H-chromen-3-yl]phe-
noxy}naphthalene-1,4-dione (3): Starting from 15, Cs2CO3 (0.10 g,
0.29 mmol),
and
2-bromonaphthalene-1,4-dione
(0.07 g,
0.29 mmol), compound 3 was obtained (0.07 g, 65%), mp: 129–
1308C. 1H NMR (CDCl3, 400 MHz): d=1.96–2.24 (m, 2H), 2.29 (s,
6H), 2.45 (t, J=6.4 Hz, 2H), 4.11 (t, J=6.4 Hz, 2H), 5.30 (s, 1H), 6.05
(s, 1H), 6.96 (d, J=8.8 Hz, 2H), 7.21–7.30 (m, 2H), 7.45 (s, 1H), 7.67
(d, J=8.8 Hz, 2H), 7.75–7.85 (m, 2H), 8.01–8.06 (m, 1H), 8.09–
8.12 ppm (m, 1H); 13C NMR (CDCl3, 400 MHz): d=27.51, 45.74,
57.91, 68.54, 111.27, 112.22, 113.32, 113.45, 118.01, 122.55, 126.66,
127.84, 133.48, 135.20, 140.75, 150.22, 152.32, 156.88, 157.33,
160.77, 179.10, 181.66 ppm; IR (Nujol) n˜ =1722, 1680, 1645, 1595,
1584 cmꢁ1; MS (ESI+) m/z: 496 [M+H]+.
General procedure for methyl ether cleavage
Procedure A (compounds 14, 15): A 1.0m CH2Cl2 solution of BBr3
(2.6 mL, 2.6 mmol) was slowly added to a solution of the methoxy
derivative (0.48 g, 1.3 mmol) in dry CH2Cl2 at ꢁ708C and under N2
atmosphere. The reaction mixture was then stirred at the same
temperature for 1 h and then at RT for 6 h. The resulting solution
was added to a mixture of ice and aqueous NaHCO3 (10 mL) and
extracted with EtOAc (3ꢃ50 mL). The solvent was dried (Na2SO4)
and evaporated under reduced pressure. The crude residue was
purified by chromatography on silica gel using a mixture of
CH2Cl2/MeOH/NH4OH (90:9.9: 0.1) as eluent.
2-{4-[6-(2-Dimethylaminoethoxy)-2-oxo-2H-chromen-3-yl]phe-
noxy}naphthalene-1,4-dione (5): Starting from 14, Cs2CO3 (0.10 g,
0.29 mmol),
and
2-bromonaphthalene-1,4-dione
(0.07 g,
0.29 mmol), compound 5 was obtained (0.1 g, 89%), mp: 127–
1298C. H NMR (CDCl3, 400 MHz): d=2.37 (s, 6H), 2.78 (t, J=6.4 Hz,
Procedure
B (compounds 21, 22): The methoxy derivative
1
(1.3 mmol) was dissolved in HBr solution (33% in AcOH, 28 mL)
and the solution was heated at 808C for 14 h. The reaction mixture
was allowed to stand overnight, ethyl ether was then added and
the solid formed was collected by vacuum filtration. The crude resi-
due was purified by chromatography on silica gel using a mixture
of CH2Cl2/EtOH/NH4OH (90:9.9: 0.1) as eluent.
2H), 4.12 (t, J=6.4 Hz, 2H), 5.35 (s, 1H), 6.06 (s, 1H), 7.03 (d, J=
2.8 Hz, 1H), 7.15–7.28 (m, 4H), 7.65–7.88 (m, 4H) 8.02–8.13 (m, 1H),
8.18–8.23 ppm (m, 1H); 13C NMR (CDCl3, 400 MHz): d=45.74, 59.91,
70.12, 111.33, 112.01, 113.27, 113.45, 118.84, 122.55, 126.64, 127.22,
133.95, 135.80, 140.43, 151.72, 155.72, 160.18, 177.33, 161.71,
178.90, 183.61 ppm; IR (Nujol) n˜ =1719, 1677, 1636, 1589,
1570 cmꢁ1; MS (ESI+) m/z: 482 [M+H]+.
6-(2-(Dimethylaminoethoxy)-3-(4-hydroxyphenyl)-2H-chromen-2-
one (14): Following procedure A and starting from 12 (0.48 g,
1.3 mmol), compound 14 was obtained (0.28 g, 66%), mp: 122–
1238C. 1H NMR (CD3COCD3, 400 MHz): d=3.10 (s, 6H), 3.79–3.85
(m, 2H), 4.55–4.62 (m, 2H), 6.96 (d, J=8.8 Hz, 2H), 7.29–7.45 (m,
3H), 7.62 (d, J=8.8 Hz, 2H), 8.94 ppm (br, 1H).
2-{4-[6-(2-Dimethylaminoethoxy)-2-oxo-2H-chromen-3-yl]phe-
noxy}anthracene-1,4-dione (6): Starting from 14, Cs2CO3 (0.10 g,
0.29 mmol), and 2-bromoanthracene-1,4-dione (0.09 g, 0.29 mmol),
1
compound 6 was obtained (0.11 g, 71%), mp: 158–1608C. H NMR
(CDCl3, 400 MHz): d=2.40 (s, 6H), 2.65 (t, J=6.4 Hz, 2H), 4.10 (t,
J=6.4 Hz, 2H), 6.06 (s, 1H), 6.99 (d, J=1.8 Hz, 1H), 7.10 (dd, J=2.8,
8.8 Hz, 1H), 7.22 (d, J=8.8 Hz, 2H), 7.260(s, 1H), 7.26 (d, J=8.8 Hz,
1H), 7.75 (m, 4H), 7.82 (d, J=8.8 Hz, 2H), 8.06–8.08 (m, 1H), 8.19–
8.21 ppm (m, 1H); 13C NMR (CDCl3, 400 MHz): d=45.45, 59.33,
70.41, 11.35, 112.55, 113.47, 117.68, 118.99, 122.22, 124.68, 129.33,
6-(3-(Dimethylaminopropoxy)-3-(4-hydroxyphenyl)-2H-chromen-
2-one (15): Following procedure A and starting from 13 (0.45 g,
1.3 mmol), compound 14 was obtained (0.32 g, 72%), mp: 129–
1308C. 1H NMR (CD3COCD3, 400 MHz): d=1.96–2.08 (m, 2H), 2.39
(s, 6H), 2.47 (t, J=6.4 Hz, 2H), 4.06 (t, J=6.4 Hz, 2H), 6.96 (d, J=
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130.77, 131.73, 131.99, 137.25, 138.46, 140.32, 149.85, 151.32,
158.66, 157.64, 160.81, 178.36, 186.54 ppm; IR (Nujol) n˜ =1711,
1659, 1625, 1597, 1574 cmꢁ1; MS (ESI+) m/z: 532 [M+H]+.
(s, 1H), 7.09 (dd, J=2.4 and 8.4 Hz, 1H), 7.11 (d, J=2.4 Hz, 1H),
7.48 (d, J=8.4 Hz, 1H), 7.72–7.74 (m, 2H), 8.06–8.12 (m, 2H), 8.61
(s, 1H), 8.77 ppm (s, 1H); 13C NMR (CDCl3, 400 MHz): d=20.25,
46.13, 58.34, 68.56, 109.72, 113.96, 114.11, 117.22, 118.67, 126.56,
127.75, 128.69, 131.43, 131.51, 131.55, 131.84, 131.99, 132.30,
133.76, 134.43, 137.12, 137.66, 144.33, 150.50, 152.85, 159.82,
168.32, 176.57, 184.79 ppm; IR (Nujol) n˜ =1720, 1656, 1636, 1590,
1575 cmꢁ1; MS (ESI+) m/z: 470 [M+H]+.
2-{4-[7-(1,4-Dioxo-1,4-dihydronaphthalen-2-yloxy)-2-oxo-2H-
chromen-3-yl]phenoxy}naphthalene-1,4-dione (4): Starting from
16, Cs2CO3 (0.20 g, 0.56 mmol), and 2-bromonaphthalene-1,4-dione
(0.14 g, 0.56 mmol), compound 4 was obtained (0.055 g, 35%),
mp: 171–1738C. 1H NMR (CD3COCD3, 400 MHz): d=6.01(s, 1H),
6.09 (s, 1H), 7.05 (dd, J=8.4 Hz e 1.8 Hz, 1H), 7.12 (d, J=1.8 Hz,
1H), 7.61 (d, J=8.4 Hz, 1H), 7.71–7.83 (m, 8H), 8.02–8.13 (m, 2H),
8.18–8.23 ppm (m, 2H); 13C NMR (CD3COCD3, 400 MHz): d=103.11,
112.22, 115.99, 117.62, 118.41, 122.98, 124.32, 126.42, 127.88,
127.95, 130.54, 131.21, 132.72, 133.69, 135.41, 139.71, 154.22,
156.97, 157.11, 157.82, 160.41, 178.25, 183.45 ppm; IR (Nujol): n˜ =
1734, 1701, 1682, 1673, 1656, 1585, 1565 cmꢁ1; MS (ESI+) m/z: 567
[M+H]+.
Computational studies
The three-dimensional structures of TbGAPDH (PDB ID: 2X0N) and
TbTR (PDB ID: 2WPF) were downloaded from the Protein Data
Bank.[34] Then, the structures were prepared using the Protein Prep-
aration Wizard[35] workflow as follows: assigning bond orders,
adding hydrogen atoms, filling in missing loops or side chains, de-
leting water, cofactors, ligands, metals, and ions. Following this
step, a geometric optimization of the whole system to a RMSD of
0.3 ff was performed using OPLS-2005 force field.
2-{[3-(2-Dimethylaminoethoxy)-2-oxo-2H-chromen-7-yl]oxy}-
naphthalene-1,4-dione (7): Starting from 21 (0.072 g), K2CO3
(0.04 g, 0.29 mmol), and 2-bromoanthracene-1,4-dione (0.09 g,
0.29 mmol), compound 7 was obtained (0.013 g, 11%), mp: 164–
The 3D molecular structures of compounds were built with the
Schrçdinger software. The energy minimization was carried out
using the OPLS-2005 force field. Then, all the compounds were
prepared by LigPrep module 2.4.[36] Docking simulations were per-
formed with Glide 5.6[37] in the extra-precision mode within
a region centered on Cys166 of GAPDH and Pro398 of TR. The de-
fault grid size was adopted from the Glide program and the scaling
factor for protein van der Waals radii was set as 0.8 in the receptor
grid generation. No constraints were applied for all the docking
studies. For each compound, a maximum of ten poses were saved
after the docking process. Finally, structures with lowest Glide XP
scores were selected for further analysis.
1
1658C. H NMR (CDCl3, 400 MHz): d=2.36 (s, 6H), 2.84 (t, J=6.0 Hz,
2H), 4.15 (t, J=6.0 Hz, 2H), 6.04 (s, 1H), 6.88 (s, 1H), 7.04 (dd, J=
2.4 and 8.4 Hz, 1H), 7.12 (d, J=2.4 Hz, 1H), 7.47 (d, J=8.4 Hz, 1H),
7.77–8.06 (m, 4H), 8.07–8.09 (m, 1H), 8.20–8.21 ppm (m, 1H);
13C NMR (CDCl3): d=46.23, 57.70, 68.16, 109.53, 113.35, 114.54,
117.85, 118.32, 126.56, 127.18, 128.15, 131.23, 132.08, 133.96,
134.83, 144.29, 150.66, 152.80, 159.94, 168.21, 176.73, 184.85 ppm;
IR (Nujol) n˜ =1721, 1676, 1651, 1593, 1574 cmꢁ1; MS (ESI+) m/z:
406 [M+H]+.
2-{[3-(2-Dimethylaminoethoxy)-2-oxo-2H-chromen-7-yl]oxy}-
anthracene-1,4-dione (8): Starting from 21 (0.072 g), K2CO3 (0.04 g,
0.29 mmol), and 2-bromoanthracene-1,4-dione (0.09 g, 0.29 mmol),
compound
8 was obtained (0.030 g, 29%), mp: 181–1838C.
1H NMR (CDCl3, 400 MHz): d=2.33 (s, 6H), 2.87 (t, J=6.0 Hz, 2H),
4.17 (t, J=6.0 Hz, 2H), 6.16 (s, 1H), 6.90 (s, 1H), 7.09 (dd, J=2.4,
8.4 Hz, 1H), 7.16 (d, J=2.4 Hz, 1H), 7.49 (d, J=8.4 Hz, 1H), 7.70–
7.72 (m, 2H), 8.06–8.12 (m, 2H), 8.60 (s, 1H), 8.75 ppm (s, 1H);
13C NMR (CDCl3, 400 MHz): d=46.23, 58.70, 68.11, 109.54, 113.37,
114.34, 117.65, 118.29, 126.56, 127.12, 128.21, 131.57, 131.97,
131.99, 132.08, 133.96, 134.83, 144.33, 150.51, 152.80, 159.82,
168.32, 176.73, 184.79 ppm; IR (Nujol) n˜ =1728, 1676, 1645, 1594,
1583 cmꢁ1; MS (ESI+) m/z: 456 [M+H]+.
Biological assays
Kinetic analysis of GAPDH
Recombinant TbGAPDH was expressed and purified in the Fato
laboratory, FaBiT Department, University of Bologna, as previously
described.[19] TbGAPDH activity was assayed spectrophotometrically
by following NAD+ reduction at 340 nm in triethanolamine (TEA)
buffer (10 mm TEA, 1.7 mm NaHCO3, 100 mm KCl, 5 mm MgSO4,
and 1 mm EDTA) pH 7.6 at 258C, as reported by Wiggers et al.[29]
2-{[3-(3-Dimethylaminopropoxy)-2-oxo-2H-chromen-7-yl]oxy}-
naphthalene-1,4-dione (9): Starting from 22 (0.072 g), K2CO3
(0.04 g, 0.29 mmol), and 2-bromoanthracene-1,4-dione (0.09 g,
0.29 mmol) compound 7 was obtained (0.059 g, 50%), mp: 165–
1678C. 1H NMR (CDCl3, 400 MHz): d=3.00–2.12 (m, 2H), 2.11 (s,
6H), 2.50 (t, J=6.0 Hz, 2H), 4.08 (t, J=6.0 Hz, 2H), 6.02 (s, 1H), 6.87
(s, 1H), 6.93 (dd, J=2.4 and 8.4 Hz, 1H), 7.05 (d, J=2.4 Hz, 1H),
7.43 (d, J=8.4 Hz, 1H), 7.77–7.88 (m, 4H), 8.00–8.10 (m, 1H), 8.17–
8.19 ppm (m, 1H); 13C NMR (CDCl3, 400 MHz): d=20.20, 46.23,
57.70, 68.00, 109.63, 113.67, 114.12, 117.34, 118.87, 126.12, 127.43,
128.26, 131.78, 132.00, 134.17, 134.27, 144.46, 150.76, 152.92,
160.24, 163.91, 176.76, 184.88 ppm; IR (Nujol) n˜ =1716, 1659, 1625,
1591, 1573 cmꢁ1; MS (ESI+) m/z: 420 [M+H]+.
The reaction mixtures contained TEA Buffer, pH 7.6, TbGAPDH
(ranging from 200 to 300 mg of protein), 400 mm NAD+, 500 mm
KH2PO4, 300 mm GAP and varying concentrations of inhibitors in
a total volume of 1 mL. Stock solutions of the inhibitors were pre-
pared in DMSO. Specific activity was expressed as mmolminꢁ1 mgꢁ1
of purified enzyme and inhibition data were presented as percent-
age of activity compared with the controls without inhibitor.
Experiments were carried out in triplicate.
Fluorescence experiments
Fluorescence experiments were carried out with a Fluorimeter
Jasco FP 777 at 258C, in TEA buffer, using 10 mm of compound 10
and of the fragment 3-(3-aminopropoxy)-7-hydroxycoumarin, in
the presence and absence of 50 mm DTT and N-acetylcysteine
(NAC). Wavelength values of excitation (335 nm) and emission
(485 nm) were acquired experimentally in the specific conditions of
buffer and temperature described in the previous paragraph. In
a different set of experiments 0.030 mgmLꢁ1 of purified TbGAPDH
2-{[3-(3-Dimethylaminopropoxy)-2-oxo-2H-chromen-7-yl]oxy}-
anthracene-1,4-dione (10): Starting from 22 (0.072 g), K2CO3
(0.04 g, 0.29 mmol), and 2-bromoanthracene-1,4-dione (0.09 g,
0.29 mmol), compound 8 was obtained (0.076 g, 74%), mp: 196–
1978C. 1H NMR (CDCl3, 400 MHz): d=2.01–2.12 (m, 2H), 2.29 (s,
6H), 2.53 (t, J=6.0 Hz, 2H), 4.13 (t, J=6.0 Hz, 2H), 6.16 (s, 1H), 6.91
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was added to 10 mm of compound 10 dissolved in TEA buffer. Fluo-
rescence spectra were recorded every minute for 15 min.
For assays of T. cruzi growth inhibition the Tulahuen strain C2C4
containing the b-galactosidase (Lac Z) gene and the standard drug
benznidazole were used. Rat skeletal myoblasts (L6 cells) were
seeded in 96-well microtiter plates containing RPMI 1640 medium
(100 mL) with 10% FBS. After 24 h the medium was removed and
replaced by 100 mL medium containing 5000 trypomastigote forms
per well. After 48 h, the medium was removed from the wells and
replaced by 100 mL fresh medium with or without a serial drug di-
lution of seven threefold dilution steps. After 96 h of incubation,
the substrate CPRG/Nonidet (50 mL) was added to all wells. A color
reaction developed within 2–6 h and could be read photometrical-
ly at 540 nm. Data were transferred into the graphic program Soft-
max Pro (Molecular Devices), which calculated EC50 values.
Kinetic analysis of trypanothione reductase and glutathione
reductase
The pET3a-TbTR plasmid was a kind gift of Dr. Alan Fairlamb
(Dundee, UK).[38] Both recombinant TcTR and TbTR were prepared
following the procedure described by Sullivan and Walsh.[39]
A
sample of recombinant human GR (hGR) was a kind gift of Dr.
Heiner Schirmer (Heidelberg, Germany). Trypanothione disulfide
(TS2) was generated enzymatically as described previously.[40] TR ac-
tivity was measured in a total volume of 1 mL of 40 mm HEPES,
1 mm EDTA, pH 7.5, containing 100 mm NADPH and varying con-
centrations of TS2 and/or inhibitors as well as ~10 mU TR. The ab-
sorption decrease due to NADPH oxidation was followed at
340 nm and 258C. Stock solutions of the inhibitors were prepared
in DMSO. The Ki value was determined using Equation (1).
Rat skeletal myoblasts (L6 cells) and the standard compound podo-
phyllotoxin were used for the assays, which were performed in 96-
well microtiter plates. Each well contained 100 mL of RPMI 1640
medium supplemented and 10% fetal bovine serum, and 4ꢃ104
L6 cells. Serial drug dilutions of seven threefold dilution steps were
prepared. The assay was run and evaluated following the same
protocol as used for T. b. rhodesiense.
½Iꢂ
Vmax ðobsÞ
Ki ¼
ð1Þ
Vmax
ꢁ 1
Acknowledgements
The ability of the compounds to induce the oxidase activity of TR
was measured in the presence of 100 mm NADPH, 40 mm of the
compounds (or an equivalent volume of 10 mL of DMSO for
blanks), and 1.29 U of TR in a total volume of 1 mL. Under these
conditions the spontaneous NADPH oxidation resulted in an ab-
sorption decrease of ꢃ0.0026 min. This value was subtracted from
the enzyme-catalyzed rate.
This research was supported by grants from the University of Bo-
logna (M.L.B.). G.V. thanks the COST Action CM-0801 “New Drugs
for Neglected Diseases” of the EC for a grant that enabled him to
work for two months in the laboratory of R.L.K.-S. at the Bio-
chemistry Center (BZH) in Heidelberg.
In the second type of oxidase assays, the reaction mixture also
contained 25 mm of Cytc (Fe3+). The absorption increase at
550 nm due to the single-electron reduction of Cytc was followed.
Finally, 67 mg of SOD was added to the assay mixture to prevent
cytochrome c reduction by superoxide anions.
Keywords: framework combinations
phosphate dehydrogenase · multitarget ligands · neglected
tropical diseases · trypanothione reductase
·
glyceraldehyde-3-
[1] M. P. Barrett, R. J. S. Burchmore, A. Stich, J. O. Lazzari, A. C. Frasch, J. J.
Cazzulo, S. Krishna, Lancet 2003, 362, 1469–1480.
[2] P. G. E. Kennedy, Lancet Neurol. 2013, 12, 186–194.
[3] F.-X. Lescure, G. Le Loup, H. Freilij, M. Develoux, L. Paris, L. Brutus, G.
Pialoux, Lancet Infect. Dis. 2010, 10, 556–570.
[4] P. J. Hotez, B. Pecoul, PLoS Neglected Trop. Dis. 2010, 4, e718.
[5] G. Priotto, S. Kasparian, W. Mutombo, D. Ngouama, S. Ghorashian, U.
Arnold, S. Ghabri, E. Baudin, V. Buard, S. Kazadi-Kyanza, M. Ilunga, W.
Mutangala, G. Pohlig, C. Schmid, U. Karunakara, E. Torreele, V. Kande,
Lancet 2009, 374, 56–64.
The activity of hGR was measured in 1 mL of 20.5 mm KH2PO4,
26.5 mmK2HPO4, EDTA 1 mm, KCl 200 mm, pH 7.5 containing satu-
rating substrate concentrations of 100 mm NADPH and 1 mm GSSG;
50 mU and 17 U GR were used for the inhibition and redox cycling
experiments, respectively.
Trypanosome growth inhibition assays
[6] a) A. R. Renslo, J. H. McKerrow, Nat. Chem. Biol. 2006, 2, 701–710;
b) M. L. Bolognesi, Curr. Top Med. Chem. 2011, 11, 2824–2833.
[7] R. Morphy, Z. Rankovic, Drug Discovery Today 2007, 12, 156–160.
[8] a) A. Cavalli, M. L. Bolognesi, J. Med. Chem. 2009, 52, 7339–7359;
b) P. M. Njogu, K. Chibale, Curr. Med. Chem. 2013, 20, 1715–1742.
[9] M. L. Bolognesi, Curr. Med. Chem. 2013, 20, 1639–1645.
[10] B. L. Roth, ACS Med. Chem. Lett. 2013, 4, 316–318.
[11] a) M. A. Comini, L. FlohØ in Trypanosomatid Diseases, Wiley-VCH, Wein-
heim, 2013, pp. 167–199; b) M. Gualdrón-López, P. A. M. Michels, W.
QuiÇones, A. J. Cµceres, L. Avilµn, J.-L. Concepción in Trypanosomatid
Diseases, Wiley-VCH, Weinheim, 2013, pp. 121–151.
[12] A. F. Coley, H. C. Dodson, M. T. Morris, J. C. Morris, Mol. Biol. Int. 2011,
2011, Article ID 123702.
[13] P. A. M. Michels, F. Bringaud, M. Herman, V. Hannaert, Biochim. Biophys.
Acta Mol. Cell Res. 2006, 1763, 1463–1477.
[14] R. L. Krauth-Siegel, M. A. Comini, T. Schlecker, Subcell. Biochem. 2007,
44, 231–251.
[15] R. L. Krauth-Siegel, M. A. Comini, Biochim. Biophys. Acta Gen. Subj. 2008,
1780, 1236–1248.
The in vitro growth inhibition activity of compounds against T. b.
rhodesiense and T. cruzi as well as the cytotoxicity against rat skele-
tal myoblasts (L6 cells) were determined as described previously.[41]
Briefly, T. b. rhodesiense, STIB 900 strain, and the standard drug,
melarsoprol, were used for the assay. Minimum Essential Medium
(50 mL) with 15% heat inactivated horse serum was added to each
well of a 96-well microtiter plate. Serial drug dilutions of seven
threefold dilution steps were prepared. Then 104 bloodstream
forms in 50 mL were added to each well and the plate was incubat-
ed at 378C under a 5% CO2 atmosphere for 72 h. Alamar Blue
(10 mL) was then added to each well and incubation continued for
a further 2–4 h. Then, the plates were read with a Spectramax
Gemini XS microplate fluorimeter (Molecular Devices Cooperation,
Sunnyvale, CA, USA) using an excitation wavelength of 536 nm
and an emission wavelength of 588 nm. Data were analyzed using
the microplate reader software Softmax Pro (Molecular Devices Co-
operation, Sunnyvale, CA, USA).
[16] A. Schmidt, R. L. Krauth-Siegel, Curr. Top Med. Chem. 2002, 2, 1239–
1259.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 13
&
11
&
ÞÞ
These are not the final page numbers!

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
[17] R. L. Krauth-Siegel, O. Inhoff, Parasitol. Res. 2003, 90, S77–S85.
[18] M. L. Bolognesi, F. Lizzi, R. Perozzo, R. Brun, A. Cavalli, Bioorg. Med.
Chem. Lett. 2008, 18, 2272–2276.
[30] L. Salmon-Chemin, E. Buisine, V. Yardley, S. Kohler, M. A. Debreu, V.
Landry, C. Sergheraert, S. L. Croft, R. L. Krauth-Siegel, E. Davioud-Char-
vet, J. Med. Chem. 2001, 44, 548–565.
[19] S. Pieretti, J. R. Haanstra, M. Mazet, R. Perozzo, C. Bergamini, F. Prati, R.
Fato, G. Lenaz, G. Capranico, R. Brun, PLoS Neglected Trop. Dis. 2013, 7,
e2012.
[20] F. Lizzi, G. Veronesi, F. Belluti, C. Bergamini, A. Lopez-Sanchez, M. Kaiser,
R. Brun, R. L. Krauth-Siegel, D. G. Hall, L. Rivas, M. L. Bolognesi, J. Med.
Chem. 2012, 55, 10490–10500.
[31] B. Stump, M. Kaiser, R. Brun, R. L. Krauth-Siegel, F. Diederich, ChemMed-
Chem 2007, 2, 1708–1712.
[32] K. Blumenstiel, R. Schçneck, V. Yardley, S. L. Croft, R. L. Krauth-Siegel,
Biochem. Pharmacol. 1999, 58, 1791–1799.
[33] R. Pink, A. Hudson, M.-A. Mouries, M. Bendig, Nat. Rev. Drug Discovery
2005, 4, 727–740.
[21] S. Rizzo, A. Cavalli, L. Ceccarini, M. Bartolini, F. Belluti, A. Bisi, V. Andrisa-
no, M. Recanatini, A. Rampa, ChemMedChem 2009, 4, 670–679.
[22] A. A. Kudale, J. Kendall, C. C. Warford, N. D. Wilkins, G. J. Bodwell, Tetra-
hedron Lett. 2007, 48, 5077–5080.
[23] K. Takaishi, M. Izumi, N. Baba, K. Kawazu, S. Nakajima, Bioorg. Med.
Chem. Lett. 2008, 18, 5614–5617.
[24] A. Cavalli, F. Lizzi, S. Bongarzone, R. Brun, R. L. Krauth-Siegel, M. L. Bo-
lognesi, Bioorg. Med. Chem. Lett. 2009, 19, 3031–3035.
[25] D. C. Rees, M. Congreve, C. W. Murray, R. Carr, Nat. Rev. Drug Discovery
2004, 3, 660–672.
[26] a) G. Bottegoni, A. D. Favia, M. Recanatini, A. Cavalli, Drug Discovery
Today 2012, 17, 23–34; b) X. H. Ma, Z. Shi, C. Tan, Y. Jiang, M. L. Go,
B. C. Low, Y. Z. Chen, Pharm. Res. 2010, 27, 739–749.
[34] RCSB Protein Data Bank, http://pdb.org/pdb/home/home.do (accessed
December 13, 2013).
[35] Schrçdinger Suite 2010 Protein Preparation Wizard: Epik version 2.1,
Impact version 5.6, and Prime version 2.2, Schrçdinger LLC, New York,
NY (USA), 2010.
[36] LigPrep, Schrçdinger LLC, New York, NY (USA), 2010.
[37] Glide, Schrçdinger LLC, New York, NY (USA), 2010.
[38] D. C. Jones, A. Ariza, W.-H. A. Chow, S. L. Oza, A. H. Fairlamb, Mol. Bio-
chem. Parasitol. 2010, 169, 12–19.
[39] F. X. Sullivan, C. T. Walsh, Mol. Biochem. Parasitol. 1991, 44, 145–147.
[40] M. A. Comini, N. Dirdjaja, M. Kaschel, R. L. Krauth-Siegel, Int. J. Parasitol.
2009, 39, 1059–1062.
[41] I. Orhan, B. S¸ener, M. Kaiser, R. Brun, D. Tasdemir, Mar. Drugs 2010, 8,
47–58.
[27] K. V. Santhosh, D. V. Kumar S. K. Venkatesan, V. K. Dubey, Sci. World J.
2012, 2012, Article ID 963658 .
[28] F. Pav¼o, M. S. Castilho, M. T. Pupo, R. L. A. Dias, A. G. Correa, J. B. Fer-
nandes, M. F. G. F. da Silva, J. Mafezoli, P. C. Vieira, G. Oliva, FEBS Lett.
2002, 520, 13–17.
Received: October 9, 2013
Revised: December 5, 2013
Published online on && &&, 0000
[29] H. J. Wiggers, J. Cheleski, A. Zottis, G. Oliva, A. D. Andricopulo, C. A.
Montanari, Anal. Biochem. 2007, 370, 107–114.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Mind the GAPDH/TR: The trypanoso-
miases are very serious yet neglected
tropical parasitic diseases that are diffi-
cult to treat, as effective drugs and vac-
cines are not yet available. Starting with
a quinone–coumarin hybrid scaffold,
the design and biological evaluation of
single small molecules that can modu-
late multiple vital targets in the para-
sites’ metabolic pathways is presented.
F. Belluti, E. Uliassi, G. Veronesi,
C. Bergamini, M. Kaiser, R. Brun, A. Viola,
R. Fato, P. A. M. Michels,
R. L. Krauth-Siegel, A. Cavalli,
M. L. Bolognesi*
&& – &&
Toward the Development of Dual-
Targeted Glyceraldehyde-3-phosphate
Dehydrogenase/Trypanothione
Reductase Inhibitors against
Trypanosoma brucei and Trypanosoma
cruzi
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