Rational design of nitrofuran derivatives: Synthesis and valuation as inhibitors of Trypanosoma cruzi trypanothione reductase

Article abstract of DOI:10.1016/j.ejmech.2016.10.055

The rational design and synthesis of a series of 5-nitro-2-furoic acid analogues are presented. The trypanocidal activity against epimastigote forms of Trypanosoma cruzi and the toxic effects on human HeLa cells were tested. Between all synthetic compounds, three of thirteen had an IC₅₀value in the range of Nfx, but compound 13 exhibited an improved effect with an IC₅₀of 1.0?±?0.1?μM and a selective index of 70 in its toxicity against HeLa cells. We analyzed the activity of compounds 8, 12 and 13 to interfere in the central redox metabolic pathway in trypanosomatids, which is dependent of reduced trypanothione as the major pivotal thiol. The three compounds behaved as better inhibitors of trypanothione reductase than Nfx (Ki values of 118?μM, 61?μM and 68?μM for 8, 12 and 13, respectively, compared with 245?μM for Nfx), all following an uncompetitive enzyme inhibition pattern. Docking analysis predicted a binding of inhibitors to the enzyme-substrate complex with binding energy calculated in-silico that supports such molecular interaction.

Full text of DOI:10.1016/j.ejmech.2016.10.055

Accepted Manuscript
Rational design of nitrofuran derivatives: Synthesis and valuation as inhibitors of
Trypanosoma cruzi trypanothione reductase
D.G. Arias, F.E. Herrera, A.S. Garay, D. Rodrigues, P.S. Forastieri, L.E. Luna,
M.D.L.M. Bürgui, C. Prieto, A.A. Iglesias, R.M. Cravero, S.A. Guerrero
PII:
S0223-5234(16)30917-5
10.1016/j.ejmech.2016.10.055
EJMECH 9020
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 August 2016
Revised Date: 28 September 2016
Accepted Date: 23 October 2016
Please cite this article as: D.G. Arias, F.E. Herrera, A.S. Garay, D. Rodrigues, P.S. Forastieri, L.E. Luna,
M.D.L.M. Bürgui, C. Prieto, A.A. Iglesias, R.M. Cravero, S.A. Guerrero, Rational design of nitrofuran
derivatives: Synthesis and valuation as inhibitors of Trypanosoma cruzi trypanothione reductase,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.10.055.
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ACCEPTED MANUSCRIPT
Rational design of nitrofuran derivatives: synthesis and valuation as inhibitors of
Trypanosoma cruzi trypanothione reductase
Arias D.G.1, 2, Herrera F.E. 2, Garay A.S.2, Rodrigues D. 2, Forastieri P.S. 3, Luna L.E.3, Bürgui M.D.L.M.2,
Prieto C.2, Iglesias A.A. 1, 2, Cravero R.M.3, Guerrero S.A. 1, 4 *
1 Instituto de Agrobiotecnología del Litoral (CONICET-UNL).
2 Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral.
3 Instituto de Química Rosario (CONICET) - FCByF- Universidad Nacional de Rosario.
4 Facultad Regional Santa Fe, Universidad Tecnológica Nacional (UTN).
*Corresponding Author: Sergio A. Guerrero, Instituto de Agrobiotecnología del Litoral (IAL), Centro
Científico Tecnológico Santa Fe (CCT), Colectora Ruta Nac. Nº168, Paraje el Pozo s/n,
3000 Santa Fe – Argentina. saguerrero@santafe-conicet.gov.ar
.
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ABSTRACT
The rational design and synthesis of a series of 5-nitro-2-furoic acid analogues are presented. The
trypanocidal activity against epimastigote forms of Trypanosoma cruzi and the toxic effects on human
HeLa cells were tested. Between all synthetic compounds, three of thirteen had an IC50 value in the
range of Nfx, but compound 13 exhibited an improved effect with an IC50 of 1.0 ± 0.1 µM and a selective
index of 70 in its toxicity against HeLa cells. We analyzed the activity of compounds 8, 12 and 13 to
interfere in the central redox metabolic pathway in trypanosomatids, which is dependent of reduced
trypanothione as the major pivotal thiol. The three compounds behaved as better inhibitors of
trypanothione reductase than Nfx (Ki values of 118 µM, 61 µM and 68 µM for 8, 12 and 13, respectively,
compared with 245 µM for Nfx), all following an uncompetitive enzyme inhibition pattern. Docking
analysis predicted a binding of inhibitors to the enzyme-substrate complex with binding energy
calculated in-silico that supports such molecular interaction.
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INTRODUCTION
The unicellular parasite Trypanosoma cruzi is the causative agent of Chagas’ disease. As aerobic
organisms, this parasite and all trypanosomatids, are inevitably exposed to many reactive oxygen and
nitrogen species (ROS, and RNS respectively), namely superoxide anions, hydrogen peroxide,
peroxynitrite and myeloperoxidase products. These chemical species are generated during host defence
reaction and also as by-products of aerobic metabolism. The ability of trypanosomatids to cope with such
oxidative stress conditions appears noticeably weak; because even when they possess an iron-
containing superoxide dismutase [useful to scavenge phagocyte-derived superoxide anions, see [1]],
they lack catalase and have a glutathione peroxidase-like system that exhibits low efficiency [2;3]. On
the other hand, it is know the efficiency of host catalase and selenocysteine-containing glutathione
peroxidases for hydroperoxide detoxifying [4;5]. In members of the family Trypanosomatidae, peroxide
metabolism mainly involves trypanothione [N1,N8-bis (glutathionyl)-spermidine, (T(SH)2)], a glutathionyl
derivative of spermidine. In these organisms it is present a system able to catalyze the T(SH)2-
dependent hydroperoxide removal, which involves three distinctive oxidoreductases: (i) trypanothione
reductase (TR), homologous to glutathione reductase [6-9]; (ii) a thioredoxin-related protein called
tryparedoxin (TXN); (iii) tryparedoxin peroxidases (TXNPx); and (iv) glutathione peroxidase-type proteins
[2;10-15]. It has been proposed that these redox components operate in the parasite instead of the host
specific thioredoxin (TRX)-thioredoxin reductase (TRXR) system [6]. In fact, TRXR is absent in
trypanosomatids [16-18].
After more than 100 years from the discovery of Chagas’ disease, the only drugs with a proven efficacy
against the infection are nifurtimox (Nfx) and benznidazole (Bnz) (nitrofuran and nitroimidazole,
respectively). These two drugs have been used to treat Chagas’ disease for over 40 years; even when
they can cause not only a large variety of unwanted side effects (sometimes forcing the patient to
abandon the treatment), but also presents a variable therapeutic efficacy and high cost [19-22]. In this
framework, the characterization of relevant biochemical targets in T. cruzi and rational design of specific
inhibitors constitute critical issues to develop new chemotherapeutic drugs, more effective and less toxic
than Bnz or Nfx. The action of Nfx through a redox cycling mechanism has been questioned [22-24]. In
fact, this drug require bioactivation trough a reduction step that is catalyzed by specific nitroreductases of
the parasite (TcrNTR) [25-27]. Type I TcrNTR, mediates a 2 electrons reduction of nitroaromatic
compounds to yield aromatic amines able to interact with DNA and other essential macromolecules, thus
affecting cellular functionality. Besides, a type II TcrNTR functions catalyzing a 1 electron reduction of
nitro compounds to render nitro radical anions [26;28]. As it emerges from previous works [29], TR is a
metabolic bottleneck of interest for rational design of new inhibitors with potential trypanocide activity
(Fig. 1). The enzyme from T. cruzi (TcrTR), which has been well characterized, is closely related to
human glutathione reductase (hGR) [8;9]. Both enzymes have a homodimeric structure (subunits
molecular mass of ~52 kDa) and catalyze reduction equivalents transference from NADPH to their
respective disulfide substrates through FAD and a redox active cysteine disulfide. Although TcrTR has
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40% sequence identity with hGR [8;9], the former uses T(SH)2 but not glutathione (GSH) as a substrate.
The three dimensional structure of TcrTR [30-32] has been solved, which is of great relevance for
molecular docking studies [33-36]. A recent review from Maya and co-workers [37] resume all
compounds that were designed based in TcrTR 3D structure, most of them without a trypanocide
improved effect.
Herein, the synthesis and characterization of a family of new synthetic compounds derivatives from 5-
nitro-2-furoic acid (5-NFA) is presented, being their potential as trypanocide drugs evaluated. All
compounds were rationally designed based in our search of hypothetical compounds capable of
interfering with TcrTR in the transference of reduction equivalents from NADPH to generate T(SH)2
(Fig. 1). Synthetic compounds were designed containing a nitrofuran structure, amide bonds with linear
and cyclic amines, diamines and aromatic amines, according to an analogy with Nfx and Bnz. In general,
synthetic structures were able to delocalize reduction electrons in the oxidoreductases cascade above
described. For the amide bond formation we have developed different synthetic methodologies from the
5-NFA directly or by transformation of its derivative acid chloride, Fig 2.
2. RESULTS AND DISCUSSION
Rational design of TcrTR inhibitors
Currently available literature clearly points out that TR is a key enzyme for transferring reducing
equivalents from NADPH to various cellular acceptors, through T(SH)2 (Fig. 1). It was thought in
designing compounds that could interfere in such a flow of reducing equivalents, thus disturbing the
normal performance of the oxidoreductases cascade. Accordingly, different derivatives of the 5-NFA
were synthesized by substituting the carboxylic group in position 2 of 5-NFA by an amide group with
varied amines to obtain synthetic species able to trap reducing equivalents. By combination of regions of
functional groups with progressive degree of complexity, a series of secondary and tertiary amides
structurally related to Nfx and Bnz (named compounds 1-13, see Fig. 2) were synthesized and also
optimized with good yields. All these compounds were identified by NMR spectroscopy (analyses
detailed in Materials and Methods and Supplemental Material) for further bioassays. The results
obtained after bioactivity and computational docking studies support the design of appropriate chemical
analogues after combining molecular structures of compounds with recognized trypanocidal activity.
Antiparasitic activity of 5-NFA derivatives on T. cruzi epimastigotes
In vitro assays testing antiproliferative effect of the new compounds were performed on
T. cruzi Dm28c epimastigotes. Axenic cultures of the parasite were incubated with increasing amounts of
each compound to determine the respective 50% inhibitory concentration (IC50) of 48 h of cells growth.
The results indicate that all 5-NFA derivatives evaluated showed antiparasitic activity, each one with
different potency (Table 1). Conversely, 5-NFA exhibited no effect on T. cruzi cultures (Fig. 3). Nfx, one
referent drug for the treatment of Chagas´s disease [38], having a low IC50
(
4.0 µM), was used as
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experimental control. Among all 5-NFA derivatives evaluated, compounds 1, 2, 8, 12 and 13 displayed
activity against epimastigotes with IC50 values between 1 to 19 µM, causing morphological alterations in
the parasite (Fig. 3). In order to analyze the toxicity of all synthetic compounds on mammalian cells, the
cytotoxicity on HeLa cells of those derivatives with low IC50 values for T. cruzi epimastigotes (less than
25 µM) were determined. Eight compounds were studied in their toxic activity toward HeLa cells.
Mammalian cells were treated with different inhibitor concentrations, and after 24 h living cells were
visualized with crystal violet staining. HeLa cells revealed generally higher EC50 values, which resulted in
selectivity indexes of 4 or higher (Table 1). Interestingly, compound 13 exhibited the highest value of
both potency against T. cruzi cells and selectivity index in comparison with the other derivatives. The
behavior of compound 13 is similar to that identified for Nfx, which might indicate that both compounds
could follow the same action mechanism. Some interesting structure–activity relationships for compound
13 emerged after compilation of the results (Table 1). A comparison of the series of compounds derived
from 5-NFA indicates that the introduction of aliphatic tertiary amines or benzylamide moieties apparently
enhances the trypanocide activity.
Inhibition of TcrTR activity by 5-NFA derivatives
TR activity was not affected by 5-NFA (Figure 4); however, 5-NFA derivatives (8, 12, and 13) showed an
inhibitory effect on TR activity, following a pattern of linear reversible uncompetitive inhibition regarding
TS2 substrate (Fig. 4 A). Thus, the inhibition takes place and according to the following mechanism:
Where Ks is the dissociation constant for enzyme–substrate complex (ES), kcat is the catalytic constant
and Ki is the dissociation constant for enzyme–substrate-inhibitor complex (EIS). Values of Ki and
inhibition type were calculated as detailed in Table 1, and the behavior of 5-NFA derivatives as inhibitors
of TR was compared with previous reports for other synthetic uncompetitive inhibitors, such as 1,4-
naphthoquinone derivatives [39]. Interestingly, the results indicate that Nfx exhibited a relatively low
inhibitory effect on the enzyme, but many of the compounds we synthesized from 5-NFA exhibited a
valuable inhibitory effect (Table 1). The lack of correlation between TR inhibition and cytotoxic effect on
T. cruzi epimastigotes for several 5-NFA derivatives and Nfx, suggest that the trypanocide effect could
be not due only to the interaction with TR, but also to other enzymes. However, the synthetic inhibitor 13,
between all the compounds studied herein, fitted the best correlation between inhibitory effect on the
enzyme activity (low Ki value) and high potency against T. cruzi cells with respect to other derivatives.
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Computational docking studies of synthetic compounds against TcrTR
An energetic evaluation of binding affinity was first performed, followed by a structural analysis of the
results. Such affinities for the best poses obtained for each of the docking simulation are shown in
Table 2 for interactions with TcrTR. For the structure of the enzyme interacting with trypanothione, Nfx
and compound 12 have the best affinity for the protein (with similar binding energies) and following them
are the 13 and 8 molecules. Structurally, the four molecules bind at the same position on the T. cruzi
enzyme structure with trypanothione at region R1 (Fig. 5), indicating that all the molecules are located on
the TS2 moiety. At region R2, the four molecules interact directly with the TS2, as in R1, but there are two
distinctive regions of interaction located close to both extremes of the substrate structure (Fig. 6). This
different behavior of the molecules on both regions could be related to the respective changes in
conformation observed for the TS2 molecule on each region (R1 and R2). The carboxylic group of the
trypanothione on region R2 is pointing away from the binding site (Fig. 7) and because of that some of
the molecules interact directly with this group (12 and Nfx). We observed that in the 3D structure of
TcrTR (without trypanothione), the four molecules interact with the protein with acceptable predicted
score for their binding affinity, but since they are smaller, they interacts only with the part of the cavities
that is usually occupied by the terminal portion (the glutamic acid residue) of the TS2 (when it is bound to
TcrTR).
CONCLUSIONS
Nfx, is thought to affect the viability of trypanosomes by the formation of both reactive oxygen species
and highly toxic hydroxylamine derivatives. The treatment of American Trypanosomiasis with Nfx or Bnz
has variable efficacy and severe side effects. It is clear the relevance that nowadays represents the
evaluation of new chemotherapeutic agents in order to improve efficacy of antichagasic therapy. In this
work, a series of 5-NFA analogues were rationally designed and synthesized. Their trypanocidal activity
against epimastigote forms of T. cruzi was tested. The toxic effects of synthetic compounds were tested
on human HeLa cells. Between all the compounds synthesized, three of thirteen could overcome Nfx in
their trypanocidal activity. Moreover, compound 13 was able to improve it (IC50 value much lower than
Nfx). Compound 13 showed a selective index of 70 after evaluation the toxicity against HeLa cells and
an IC50 four-fold lower than that determined for Nfx. Its activity to interfere in the flux of reducing
equivalents from NADPH to oxidizing substrates (using reduced trypanothione as the major pivotal thiol)
was tested. Hence, trypanothione reductase was selected as a critical target for this study, analyzing in
vitro the enzyme inhibition by Nfx and compounds 8, 12 and 13. All compounds showed a better
inhibition parameter than Nfx. In all inhibition reaction the mechanism for enzyme inhibition followed an
uncompetitive pattern. The introduction of aliphatic tertiary amines or benzylamide substituents improved
the inhibitory potency of the series of 5-NFA derivatives, being the ability of such compounds to inhibit
TcrTR activity and parasite growth dependent exclusively on the structure and nature of their
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substituents. Molecular docking analysis enabled us to study the type of interaction between
TcrTR and four of the synthetic inhibitors (Nfx, 8, 12 and 13). First, and very important, docking
assays showed that Nfx can be able of binding to TcrTR. That could be another possibility of the
drug to affect the viability of the parasite in addition to those reported previously and related to
nitroreductases activities. In the same way, molecular docking shows that in T. cruzi the
molecules of inhibitors can bind directly to the complex TcrTR-trypanothione, which strongly
supports our previous experimental findings of a pattern of linear reversible uncompetitive
inhibition. Figure 6 shows the analog 13 (which have rotation free due to methylene of the
benzyl group), with the phenyl groups located in parallel with a probable pi interaction that it is
not possible in the compound 12. Conversely, the incorporation of an N-amide inserted into a
ring such as the analogs 10 and 11, produces a structural rigidity that could cause the loss of
bioactivity in respect with 13.
Current results about properties of compound 13 for affecting the viability of T. cruzi epimastimote cells,
its activity as uncompetitive inhibitor of TcrTR and its evidenced capability to interact with the complex
TcrTR-TS2 highlight this synthetic molecule as relevant to develop new and effective drugs. Further
studies are necessary to reach such an advance in the fight against diseases caused by
trypanosomatids. Moreover, TR is an enzyme conserved in all trypanosomatids, so our results could be
of relevance for extending this study to the development of inhibitors for TR from Leishmania spp. or
Trypanosoma brucei spp.
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MATERIALS AND METHODS
Materials
Bacteriological media. Trypanothione disulfide (TS2) was acquired from Bachem. All other reagents and
chemicals were of the highest quality commercially available from Sigma Aldrich, Merck, Invitrogen and
Promega.
Trypanosoma cruzi cultures
T. cruzi Dm28c epimastigote cells were cultivated axenically at 28 ºC in LIT media supplemented with
10 % (w/v) bovine fetal serum and 20 µg·ml-1 hemin, as previously reported [40].
Chemistry, General
All reagents and solvents were purchased in Sigma-Aldrich and Sintorgan and were used directly as
purchased or purified according to standard procedures. All reactions involving air or moisture-sensitive
materials were carried out under nitrogen atmosphere. All reaction products were purified by flash
chromatography which was performed with 300-400 mesh silicagel under slight nitrogen pressure, with
increasing gradients of solvent mixtures. IR spectra were recorded with a Shimadzu, Prestige 21 Model
spectrophotometer, with samples as liquid films in NaCl for oils and KBr disks for solids. NMR
1
experiments were run in CDCl3 at 300.13 MHz for H NMR and at 75.4 MHz for 13C NMR with a
Bruker Avance-300 MHz NMR spectrometer and the corresponding solvent as internal reference
standard. High-resolution mass spectrometry was carried out with a LC-QTOf Bruker MicroTOF QII.
GC-MS spectra were recorded with a Shimadzu QP-2010 plus spectrometer (see supplemental
information).
Synthesis and characterization of compounds
A stirred mixture of 5-nitrofuroic acid (5-NFA 100 mg; 0.64 mmol) and SOCl2 (3 mL) was refluxed in a
silicone bath for 1 h until gas evolution stops. The reaction mixture was left to take room temperature
and, with the addition of anhydrous toluene (1 mL), the excess of SOCl2 was evaporated in vacuo. Then,
a solution of selected amine (0.86 mmol in 0.5 mL of anhydrous pyridine) was dropwise added to the
acid chloride (obtained as oil) dissolved in anhydrous acetone (2.2 mL) and the mixture was maintained
at room temperature for 18-22 h. The reaction mixture was diluted with EtOH, concentrated in vacuo and
dried to give a residue which was purified by chromatographic column. Yield: 62-95%.
1
5-nitro-2-furoic acid (5-nitrofuran-2-carboxilic acid, 5-NFA), commercial starting material: H NMR
(CDCl3) δ (ppm): 7.31 (d, 1H, J= 3.7 Hz, HC=CC=O), 7.23 (d, 1H, J= 3.8 Hz, HC=C-NO2), 7.42 (br s, 1H,
COOH).
5-Nitro-furan-2-carboxylic acid propylamide (1): Two chromatographic columns with solvent mixture
(CH2Cl2/MeOH, 99:1). Yield: 93 mg, 0.47 mmol, 73%. Crystalline, m.p.= 97.1-99.3 ºC. IR (film) ν (cm-1):
3304 (N-H), 3119 (CH furane), 2967, 2875 (CH alkane), 1681 (C=O), 1584, 1485, 1385 (C=C furane),
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1556 (C-N), 1352 (NO2), 1288 (C-N), 1016 (C-O). 1H NMR (CDCl3) δ (ppm): 7.35 (d, 1H, J= 3.8 Hz, H-3),
7.23 (d, 1H, J= 3.8 Hz, H-4), 6.68 (br s, 1H, NH), 3.41 (c, 2H, J= 6.3 Hz, NHCH2-), 1.65 (dt, 2H, J= 7.3,
14.8 Hz, NHCH2CH2CH3), 0.97 (t, 3H, J= 7.4 Hz, NHCH2CH2CH3). 13C NMR (CDCl3) δ (ppm): 156.3
(C=O amide), 151.3 (C-5), 148.3 (C-2), 115.7 (C-4), 112.5 (C-3), 41.3 (NHCH2-), 22.7 (NHCH2CH2CH3),
11.3 (NHCH2CH2CH3). EIHRMS calculated mass for C8H10N2O4Na (M + Na+) = 221.05328; found
221.05294.
5-Nitro-furan-2-carboxylic acid isopropylamide (2): Solvent mixture for purification (CH2Cl2/MeOH,
99:1). Yield: 77 mg, 0.40 mmol, 63%. White solid, m.p.= 117.7-118.1ºC. IR (film) ν (cm-1): 3294 (N-H),
3136 (CH furane), 2976, 2875 (CH), 1651 (C=O), 1584, 1557, 1485, 1386 (C=C furane), 1352 (NO2),
1279 (C-N amide), 1171, 1146 (gem-CH3). 1H NMR (CDCl3) δ (ppm): 6.40 (br s, 1H, NH), 7.35 (d, 1H, J=
3.8 Hz, H-3), 7.23 (d, 1H, J= 3.8 Hz, H-4), 6.40 (br s, 1H, NH), 4.26 (m, 1H, J= 6.7 Hz, (CH3)2CHN-),
13
1.28 (d, 6H, J= 6.6 Hz, CH3). C NMR (CDCl3) δ (ppm): 155.4 (C-5), 151.2 (C=O), 148.4 (C-2), 115.7
(C-4), 112.5 (C-3), 42.0 ((CH3)2CHN-), 22.6 ((CH3)2CHN). EIHRMS calculated mass for C8H10N2NaO4 (M
+ Na+)= 221.05328; found 221.05374.
5-Nitro-furan-2-carboxylic acid butylamide (3): Solvent mixture for purification (CH2Cl2/MeOH, 99:1).
Yield: 89 mg, 0.42 mmol, 66%. Ambar oil. IR (film) ν (cm-1): 3304 (N-H), 3138 (CH alkane), 2959, 2872
(CH), 1651 (C=O amide), 1584, 1485, 1390 (C=C), 1556, 1352 (NO2), 1286 (C-N amide), 1016 (C-O). 1H
NMR (CDCl3) δ (ppm): 7.35 (m, 1H, H-3), 7.24 (d, 1H, J= 3.6 Hz, H-4), 6.65 (br s, 1H, NH), 3.45 (c, 2H,
J= 6.6 Hz, NHCH2-), 1.61 (q, 2H, J= 7.3 Hz,CH2CH2CH2), 1.39 (m, 2H, J= 7.1 Hz, CH2CH3), 0.95 (t, 3H,
J= 7.3 Hz, CH2CH3). 13C NMR (CDCl3) δ (ppm): 156.3 (C=O amide), 151.1 (C-5), 148.3 (C-2), 115.8 (C-
4), 112.5 (C-3), 39.4 (NHCH2CH2CH2), 31.5 (NHCH2CH2CH2), 20.0 (CH2CH2CH2), 13.7 (CH2CH3).
EIHRMS calculated mass for C9H12N2O4Na (M + Na+) = 235.06893; found 235.06967.
5-Nitro-furan-2-carboxylic acid sec-butylamide (4): Solvent mixture for purification (CH2Cl2). Yield: 91
mg, 0.43 mmol, 68%. Yellow oil. IR (film) ν (cm-1): 3123 (CH furane), 2968 (CH), 1653 (C=O), 1553,
1
1485 (C=C furane), 1526, 1352 (NO), 1387 (CN), 1287 (CN), 1016 (CO furane), 810 (CN). H NMR
(CDCl3) δ (ppm): 7.34 (d, 1H, J= 7.7 Hz, H-3), 7.22 (d, 1H, J= 7.9 Hz, H-4), 6.39 (br s, 1H, NH), 4.08 (m,
1H, CH), 1.59 (m, 2H, J= 7.3 Hz, CH2CH3), 1.24 (d, 3H, J= 6.6 Hz, CHCH3), 0.94 (t, 3H, J= 7.4 Hz,
CH2CH3). 13C NMR (CDCl3) δ (ppm): 155.7 (C=O), 151.1 (C-5), 148.4 (C-2), 115.7 (C-4), 112.6 (C-3),
47.3 (CH), 29.5 (CH2CH3), 20.3 (CH3), 10.4 (CH2CH3). EIHRMS calculated mass for C9H12N2NaO4(M +
Na+) = 235.06893, found 235.06975.
5-Nitro-furan-2-carboxylic acid-dipropyl-amide (5): Solvent mixture for purification (Hexane/ CH2Cl2,
30:70). Yield: 129 mg, 0.54 mmol, 85%. Yellow oil, IR (film) ν (cm-1): 3132 (CH furane), 2966, 2874 (CH),
1636 (C=O), 1578, 1423 (C=C furane), 1381 (CN), 1350 (NO), 1254 (CN), 1018 (CO furane), 814 (CN
nitro). 1H NMR (CDCl3) δ (ppm): 7.33 (d, 1H, J= 3.8 Hz, H-3), 7.20 (d, 1H, J= 3.8 Hz, H-4), 3.53 (t, 2H, J=
7.8 Hz, NCH2), 3.43 (t, 2H, J= 7.7 Hz, NCH2), 1.74 (m, 2H, CH2CH3), 1.66 (m, 2H, CH2CH3), 0.97 (m, 3H,
CH2CH3). 13C NMR (CDCl3) δ (ppm): 157.5 (C=O), 151.1 (C-5), 149.3 (C-2), 118.0 (C-4), 111.7 (C-3),
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50.5, 49.3 (NCH2), 22.9, 20.6 (CH2CH3), 11.3, 10.9 (CH3). EIHRMS calculated mass for C11H16N2NaO4
(M + Na+) = 263.10078, found 263.10023.
5-Nitro-furan-2-carboxylic acid tert-butylamide (6): Solvent mixture for purification (Hexane/ CH2Cl2,
30:70). Yield: 102 mg, 0.48 mmol, 75%. Yellow solid, m.p.= 103.5-104.4 ºC. IR (film) ν (cm-1): 3317 (NH
amide), 3136 (C-H furane), 2970, 2873 (CH), 1678 (C=O), 1589, 1485, 1389 (C=C furane), 1550, 1354
(NO2), 1292 (CN), 1219 (t-butyl), 813 (CN nitro). 1H NMR (CDCl3) δ (ppm): 7.33 (d, 1H, J= 3.8 Hz, H-3),
7.18 (d, 1H, J= 3.8 Hz, H-4), 6.35 (br s, 1H, NH), 1.47 (s, 9H, CH3). 13C NMR (CDCl3) δ (ppm): 155.5 (C-
5), 150.9 (C=O), 148.8 (C-2), 115.4 (C-4), 112.6 (C-3), 52.4 (C(CH3)3), 28.7 (CH3). EIHRMS calculated
mass for C9H12N2NaO4 (M + Na+) = 235.0695, found 235.06902.
5-Nitro-furan-2-carboxylic acid (2-dimethylamino-ethyl)-amide (7): Solvent mixture for purification
(CH2Cl2/MeOH, 90:10). Yield: 88 mg, 0.39 mmol, 62%. Yellow oil. IR (film) ν (cm-1): 3394 (CH furane),
3120 (CH), 2920, 2780 (C-H), 1660 (C=O), 1581, 1531, 1481 (C=C furane), 1555, 1354 (N-O), 1389,
1
1290 (C-N amine), 1022 (CO furane), 814 (CN nitro). H NMR (CDCl3) δ (ppm): 7.66 (br s, 1H, J= 5.5
Hz, N-H), 7.38 (d, 1H, J= 3.9 Hz, H-3), 7.25 (d, 1H, J= 3.9 Hz, H-4), 3.54 (c, 2H, J= 5.5 Hz, NCH2), 2.56
(t, 2H, J= 6.2 Hz, NCH2 CH2), 2.31 (s, 6H, NCH3). 13C NMR (CDCl3) δ (ppm): 156.4 (C=O), 151.3 (C-5),
148.3 (C-2), 115.7 (C-4), 112.4 (C-3), 57.5 (NCH2), 45.1 (CH3), 36.8 (NHCH3). EIHRMS calculated mass
for C9H13N2NaO4 (M + Na+)= 250.0804, found 250.07983.
5-Nitro-furan-2-carboxylic acid (3-dimethylamino-propyl)-amide (8): Solvent mixture for purification
(CH2Cl2/MeOH, 94:6). Yield: 90 mg, 0.41 mmol, 64%. White solid, m.p.= 131.5-135.2ºC. IR (film) ν (cm-
1): 3300 (N-H), 3125 (CH furane), 2949, 2922, 2860, 2822, 2781 (C-H), 1661 (C=O), 1582 (C=C), 1531
(C=C furane), 1552, 1352 (NO2), 1290 (CN), 1016 (C-O), 968, 812. 1H NMR (CDCl3) δ (ppm): 9.59 (br s,
1H, NH), 7.33 (d, 1H, J= 3.8 Hz, H-3), 7.21 (d, 1H, J= 3.7 Hz, H-4), 3.56 (dd, 2H, J= 5.4 Hz, (CH3)2NCH2-
), 2.62 (t, 2H, J= 5.9 Hz, NHCH2CH2CH2), 2.41 (s, 6H, gem-CH3), 1.80 (q, 2H, J= 5.8 Hz,
13
NHCH2CH2CH2). C NMR (CDCl3) δ (ppm): 156.3 (C-5), 151.2 (C=O), 156.3 (C-5), 148.9 (C-2), 115.0
(C-4), 112.3 (C-3), 58.8 (NHCH2CH2CH2), 44.7 (gem-CH3), 40.1 (CH2CH2CH2N(CH3)2), 24.4
(NHCH2CH2CH2). EIHRMS calculated mass for C10H16N3O4 (M + H+)= 242.1135; found 242.1136.
5-Nitro-furan-2-carboxylic acid (3-diethylamino-propyl)-amide (9): Solvent mixture for purification
(CH2Cl2/MeOH, 96:4). Yield: 164 mg, 0.61 mmol, 95%. Ambar oil. IR (film) ν (cm-1): 3279 (N-H), 3140
(CH furane), 2970, 2940, 2816 (CH), 1666 (C=O), 1578, 1555, 1350 (NO nitro), 1535, 1481 (C=C
furane), 1288 (CN amide), 1018 (CO furane), 814 (C-N nitro group). 1H NMR (CDCl3) δ (ppm): 9.67 (br s,
1H, NH), 7.30 (d, 1H, J= 3.8 Hz, H-3), 7.20 (d, 1H, J= 3.8 Hz, H-4), 3.51 (t, 2H, J= 5.4 Hz, NHCH2-), 2.66
(m, 4H, J= 7.0 Hz, Et2NCH2CH2CH2, NCH2CH3), 1.76 (q, 2H, J= 6.0 Hz, NHCH2CH2CH2), 1.07 (t, 6H, J=
7.1 Hz, NHCH2CH3). 13C NMR (CDCl3) δ (ppm): 156.2 (C=O), 151.2 (C-5), 149.0 (C-2), 115.1 (C-4),
112.4 (C-3), 52.5 (Et2NCH2CH2CH2), 46.7 (CH2CH3), 40.4 (NHCH2CH2CH2), 24.0 (NHCH2CH2CH2), 10.9
(CH2CH3). EIHRMS: calculated mass for C12H20N3O4 (M + H+)= 270.14483; found 270.14478.
10

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(5-Nitro-furan-2-yl)-pyrrolidin-1-yl-methanone (10): Solvent mixture for purification (CH2Cl2/MeOH,
94:6). Yield: 105 mg, 0.50 mmol, 78%. Pale crystalls, m.p.= 160.8-161.8 ºC. IR (film) ν (cm-1): 3128 (CH
furane), 2978 (CH), 1625 (C=O), 1582, 1493 (C=C furane), 1520, 1389 (CN), 1358 (NO), 1045 (CO
furane), 814 (C-N). 1H NMR (CDCl3) δ (ppm): 7.35 (d, 1H, J= 3.8 Hz, H-3), 7.26 (d, 1H, J= 3.8 Hz, H-4),
3.95 (t, 2H, J= 6.8 Hz, NCH2), 3.67 (t, 2H, J= 6.8 Hz, NCH2), 2.07 (m, 2H, J= 6.8 Hz, -CH2), 1.94 (m, 2H,
13
J= 6.8 Hz, -CH2). C NMR (CDCl3) δ (ppm): 155.7 (C=O), 151.4 (C-5), 149.3 (C-2), 117.6 (C-4), 111.8
(C-3), 47.7 (NCH2), 47.6 (NCH2), 26.5 (NCH2CH2), 23.6 (NCH2CH2). EIHRMS calculated mass for
C9H11N2O4(M + H+) = 211.0713, found 211.0705.
(5-Nitro-furan-2-yl)-piperidin-1-yl-methanone (11): Solvent mixture for purification (CH2Cl2/MeOH,
94:6). Yield: 121 mg, 0.54 mmol, 84%. White crystalls, m.p.= 96.5-96.7 ºC. IR (film) ν (cm-1): 3125 (CH
furane), 2935 (CH), 1631 (C=O), 1578, 1435 (C=C furane), 1531, 1350 (NO), 1381 (CN), 1265 (CN),
1014 (CO furane), 810 (CN). 1H NMR (CDCl3) δ (ppm): 7.33 (d, 1H, J= 3.7 Hz, H-3), 7.09 (d, 1H, J= 3.7
Hz; H-4), 3.70 (m, 4H, NCH2); 1.70 (m, 6H, CH2-). 13C NMR (CDCl3) δ (ppm): 156.8 (C=O), 151.1 (C-5),
149.1 (C-2), 117.1 (C-4), 111.7 (C-3), 47.8 (C-2´), 44.3 (C6´), 26.7 (C-3´), 25.5 (C-5´), 24.4 (C-4´).
EIHRMS calculated mass for C10H13N2O4 (M + H+) 225.08698, found 225.08736.
5-Nitro-furan-2-carboxylic acid benzylamide (12): Solvent mixture for purification (Hexane/AcOEt,
70:30). Yield: 140 mg, 0.57 mmol, 89%. White crystalls, m.p.= 89.2-91.9 ºC. IR (film) ν (cm-1): 3296 (N-
H), 3136 (CH alkene), 2929 (CH alkane), 1651 (C=C, C=O), 1556 (asymmetric C-N), 1485 (C=C furane),
1
1352 (symmetric C-N), 1286 (C-O), 1016 (C-H), 968 (C-N), 812, 738. H NMR (CDCl3) δ (ppm): 7.38-
7.28 (complex signal, 7H, Ar-H, H-3, H-4), 6.89 (br s, 1H, NH),4.64 (d, 2H, J= 6.0 Hz, CH2-Ar). 13C NMR
(CDCl3) δ (ppm): 156.3 (C=O amide), 151.2 (C-5), 148.0 (C-2), 137.1 (1-ArH), 128.9 (3,5-ArH), 128.0
(2,6-ArH), 127.9 (4-ArH), 116.1 (C-4), 112.5 (C-3), 43.6 (-CH2Ar). EIHRMS calculated mass for
C12H11N2O4 (M + H+) 247.0713, found 247.0711.
5-Nitro-furan-2-carboxylic acid dibenzylamide (13): Solvent mixture for purification (Hexane/AcOEt,
85:15). Yield: 151 mg, 0.45 mmol, 70%. Oil, Hex: EtOAc (85:15). IR (film) ν (cm-1): 3132 (C-H furane),
3063, 3032 (C-H aromatic) 2924, 2851 (C-H alkane), 1736 (C=O), 1643 (asymmetric C-N), 1535 (C=C
1
furane), 1350 (symmetric C-N), 1280, 1258 (C-O), 1018 (C-H), 968 (C-N), 814, 748, 702. H NMR
(CDCl3) δ (ppm): 7.37-7.26 (complex signal H-Ar), 7.28 (br s, 11H, Ar-H, H-3), 7.17 (d, 1H, J= 3.8 Hz, H-
4), 4.77 (br s, 2H, CH2Ar), 4.71 (br s, 2H, CH2Ar). 13C NMR (CDCl3) δ (ppm): 158.6 (C=O amide), 151.4
(C-5), 148.4 (C-2), 136.5, 135.8 (1-ArH), 128.9 (3,5-ArH), 128.0 (2,6-ArH), 127.9 (4-ArH), 118.0 (C-4),
111.6 (C-3), 50.8, 49.4 (CH2Ar). EIHRMS calculated mass for C19H16N2NaO4 (M + Na+) 359.10078, found
359.10023.
Determination of TR activity
TR activity was measured by monitoring DTNB reduction at 405 nm (ε = 13.8 mM-1cm-1 at 405 nm) by
means of a coupled assay that guaranteed the oxidation of T(SH)2 [41]. All enzyme assays were
11

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performed at 30 °C, in a final volume of 50 µl, and using a Multiskan Ascent one channel vertical light-
path filter photometer (Thermo Electron Co.). The reaction mixture contained 100 mM Tris–HCl, pH 7.5,
2 mM EDTA, 200 µM NADPH, 4 nM TcrTR, 10-100 µM T(SH)2, 1 mM DTNB, and the respective
nitrofuroic acid (5-NFA) based synthetic compound (1–100 µM). Reactions were started by addition of
NADPH. As control assay, the 5-NFA related compounds were omitted in the reaction mixture. Kinetic
constants are the means of at least three independent sets of data, and they were reproducible within
±10%.
Toxicity assays in cultures of T. cruzi epimastigotes
Suspensions of logarithmic-phase T. cruzi Dm28c epimastigotes at 1x106 cell/ml were prepared in PBS
plus 2% (w/v) glucose and transferred in aliquots of 200 µl to 96 well plates. Derivatives of the 5-NFA
were added to different wells to get 0.1-100 µM final concentrations. The parasites were incubated for
24-48 h to evaluate epimastigote viability using resazurin assay [29] . Negative controls were performed
replacing the addition of the inhibitors by an equal proportion of DMSO (solvent). As technical control,
resazurin was incubated in the presence of each of the evaluated compounds (at different
concentrations) but in the absence of parasites [29]. Calculated IC50 values are the means of at least
three independent experiments, which were reproduced within ±10%.
Toxicity assays in HeLa cell cultures
The toxicity of the synthetic compounds was determined using the crystal violet staining, as described
elsewhere [42]. Cells were seeded at 2.5 x 104 cell/ well in 0.1 ml DMEM medium (supplemented with
2% fetal bovine serum) and incubated at 37°C and 5% CO2 during 24 h. Supernatants were discarded
and renewed with fresh medium supplemented with the synthetic inhibitors. Eight serial dilutions of each
compound were evaluated by duplicates. Cells were incubated for 24 h at 37°C and 5% CO2. The
medium was discarded and cells were fixed and stained simultaneously with a solution of 0.75% (w/v)
crystal violet in 40% (v/v) methanol. After 10 min, plates were washed with water and the remaining dye
was solubilized in 20% (v/v) acetic acid. The plates were read at 540 nm with a microtiter plate reader.
The signal intensity of each dilution was reported as the mean of the absorbance measured in triplicates.
Untreated cells were considered as negative control. The non-toxic limit concentration was calculated as
the concentration of compound which produced the same color intensity than that of the negative control.
Computational docking studies
The molecules 8, 12, and 13 along with Nfx were computationally tested (docked) against TcrTR
structure (pdb code: 1BZL (5)) in order to evaluate their in vitro activities and their theoretical binding
affinities. TcrTR structure is a homodimer crystallized in complex with the oxidized form of the
trypanothione molecule (TS2) in a well known binding region [43] that is also found using different in-
silico binding site predictors such as COACH [44], RaptorX Binding [45] and others (8,25,28). After the
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latter and also because we are interested in molecules that interfere the reductase activity, the region
used to explore the possible binding modes (binding site) of these molecules was chosen to be the
binding region of the TS2. Docking simulations were performed using the AutoDock Vina 1.1.2 program
[46]. Simulation parameters were chosen in a way ensuring a full coverage of the binding site, using a
cubic search space of 24,389 Å3 (29Å x 29Å x 29Å) centered at mass center of the TS2 molecule. The
molecules were simulated as flexible while the protein was rigid. Since TcrTR conforms a homodimers
with two binding regions (one per subunit), these two regions were used for the docking simulations and
each one was labeled as Region 1 (R1) located in chain A and Region 2 (R2) located in chain B.
Furthermore, the docking simulation was done in the presence and absence of the TS2 molecule, using
the same search space. This allowed us to predict and analyze the binding affinity of synthetic inhibitors
whether they joined into the same place of the substrate, or to the already bound TS2 into the binding
site. Therefore, the 4 molecules were docked on each region of the protein (8 dockings), with and
without the TS2 substrate (16 dockings) in both proteins.
ACKNOWLEDGMENTS
This work was supported by grants from UNL (CAI+D’11), CONICET (PIP112-2011-0100439, PIP114-
2011-0100168, PIP GI 112-200801-00796), ANPCyT (PICT-2014-3256) and UNR (1BIO303 19/B450).
PSF is fellows from CONICET. LEL is investigator from National University of Rosario (UNR). SAG, AAI,
RMC, FEH, DR and DGA are investigator career members from CONICET.
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LEGENDS TO THE FIGURES
Figure 1: Trypanothione dependent redox metabolism. TRox, oxidized trypanothione reductase; TRred, reduced
trypanothione reductase. In this system, reduced trypanothione can transfers reduction equivalents to several
metabolic pathways [29].
Figure 2: Synthesis of amides 1-13: a) 5-NFA (0.64 mmol), SOCl2 (3 mL), reflux 1h; b) acetone (2.2 mL), selected
amine (0.86 mmol) in pyridine (0.5 mL), RT. 20 h.
Figure 3: Cytotoxic effect of 5-NFA derivatives on T. cruzi epimastigotes cultures. Suspensions of logarithmic-
phase T. cruzi Dm28c epimastigotes (106 cell/ml) were incubated for 48 h in LIT-supplement medium in absence or
in presence of the 5-NFA derivatives were added.
Figure 4: Inhibition of trypanothione reductase activity by 5-NFA derivatives. A) Time-dependent inhibition of
TcrTR by nitro-derivative compounds. Assays were carried out at 30 °C, pH 7.5 and the reaction mixture included
4 nM TcrTR, 0.2 mM NADPH, 0.5 mM DTNB and 5 µM TS2 and 100 µM of 5-NFA, 8, 12 or 13. B) Plot of initial rate
(v) from progress curves as a function of TS2 concentration.
Figure 5: Binding poses of the four molecules at the region R1 of TcrTR (with TS2). Color code: 8 (pink), 12
(green), Nfx (yellow) and 13 (blue).
Figure 6: Binding poses of the four molecules at the region R2 of TcrTR (with TS2). Color code: 8 (pink), 12
(green), Nfx (yellow) and 13 (blue).
Figure 7: Superposition of the conformations of the TS2 molecule in regions R1 and R2 in TcrTR. The circle shows
the most important change between both.
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TcTR inhibition
T. cruzi growth
inhibition
IC50 (µM)
17 ± 3
HeLa cells
Selectivity
index
HeLa/T. cruzi
(regarding to TS2)*
Ki (µM)
336 (U)
414 (U)
315 (U)
1180 (U)
183 (U)
313 (U)
136 (U)
118 (U)
128 (U)
187 (U)
282 (U)
61 (U)
toxicity
EC50 (µM)
150 ± 22
211 ± 19
152 ± 23
165 ± 18
---
---
---
74 ± 7
100 ± 6
---
Drug
1
2
3
4
5
6
7
8
9
10
11
12
13
Nfx
8.8
11.7
5.8
7.2
---
---
---
3.9
4.0
---
18 ± 2
26 ± 3
23 ± 4
19.6 ± 0.8
35 ± 5
36 ± 3
19 ± 3
25 ± 2
64 ± 11
103 ± 25
15 ± 2
1.1 ± 0.1
4.0 ± 0.8
---
---
123 ± 11
78 ± 9
272 ± 70
8.2
70.1
68.0
68 (U)
245 (U)
TABLE 1: In vitro enzyme inhibition and cellular toxicity activity of nitro-derivate compounds. (*) at pH 7.5 and
30 °C.
(U)= classic reversible uncompetitive inhibition.
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T. cruzi
With Trypanothione Without Trypanothione
R1
R2
R1
R2
8
-6.1
-6.9
-6.7
-6.4
-6.0
-6.7
-7.2
-6.6
-6.0
-7.3
-7.3
-7.8
-6.1
-7.0
-7.0
-7.3
12
Nfx
13
TABLE 2: Binding energies in Kcal/mol for the interactions of each molecule with the trypanothione reductase from
Trypanosoma cruzi. The calculations were done with AutoDock Vina.
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Highlights
• Three synthetic compounds had an IC50 value in the range of Nfx.
• Compound 13 exhibited an IC50 of 1 ± 0.1 µM and a selective index of 70.
• Synthetic compounds exhibited inhibitor activity against TR.
• Docking analysis predicted a binding of inhibitors to the enzyme-substrate complex.