Optimization of antitrypanosomatid agents: Identification of nonmutagenic drug candidates with in vivo activity

Article abstract of DOI:10.1021/jm500018m

Chagas disease, caused by Trypanosoma cruzi parasite, was described thousands of years ago. Currently, it affects millions of people, mostly in Latin America, and there are not suitable drugs for treating it. As an attempt to find appropriate drugs to dea

Full text of DOI:10.1021/jm500018m

Article
pubs.acs.org/jmc
Optimization of Antitrypanosomatid Agents: Identification of
Nonmutagenic Drug Candidates with in Vivo Activity
†
†
†
†
‡
́
Guzman Alvarez, Javier Varela, Pablo Marquez, Martín Gabay, Carmen Elena Arias Rivas,
́
́
Karina Cuchilla,‡ Gustavo A. Echeverría,§ Oscar E. Piro,§ Marlus Chorilli,∥ Sandra M. Leal,⊥
Patricia Escobar,⊥ Elva Serna,# Susana Torres,# Gloria Yaluff,# Ninfa I. Vera de Bilbao,#
Mercedes Gonzalez,*,† and Hugo Cerecetto*,†
́
†Grupo de Química Medicinal, Laboratorio de Química Organ
Republica, 11400 Montevideo, Uruguay
‡Centro Nacional de Investigaciones Científicas de El Salvador, San Salvador, El Salvador
§Departamento de Física, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, and Institute IFLP (CONICET, CCT-La
Plata), C.C. 67, 1900 La Plata, Argentina
∥Departament of Drugs and Pharmaceuticals, School of Pharmaceutical Sciences, UNESP, Araraquara, Brazil
⊥Centro de Investigaciones de Enfermedades Tropicales, Departamento de Ciencias Bas
́
ica, Facultad de Ciencias−Facultad de Química, Universidad de la
́
́
icas, Universidad Industrial de Santander,
Bucaramanga, Colombia
#Departamento de Medicina Tropical, Instituto de Ciencias de la Salud, Universidad Nacional de Asuncion
́
́
, Asuncion, Paraguay
S
* Supporting Information
ABSTRACT: Chagas disease, caused by Trypanosoma cruzi
parasite, was described thousands of years ago. Currently, it
affects millions of people, mostly in Latin America, and there
are not suitable drugs for treating it. As an attempt to find
appropriate drugs to deal with this problem, we report here on
the design, synthesis, and characterization of 82 new
compounds. Trypanosomicidal behavior in vitro showed
more than 20 outstanding derivatives with anti-Trypanosoma
cruzi activity. Furthermore, we studied the nonspecific toxicity
against mammalian cells determining their selectivity and also
performed mutagenicity studies. Proof of concept, in vivo
studies, was conducted with two of the most promising
derivatives (77 and 80). They were identified as candidates
because they have (i) very simple and cost-effective syntheses; (ii) activity against different stages and strains of the parasite
showing excellent in vivo behavior during the acute phase of Chagas disease; and (iii) neither nonspecific toxicity nor mutagenic
activity.
against all Trypanosoma cruzi (T. cruzi) strains, have
significantly low efficacy in long-term chronic infections,5 and
also are mutagenics.6 Both drugs act via the reduction of the
nitro group, mediated by a type 1 nitroreductase, followed by
the fragmentation of the heterocyclic ring, which resulted in the
production of toxic metabolites.7 Side effects of Nifurtimox
results from the oxidative damage in the host’s tissues and is
thus inextricably linked to its antiparasitic activity. Despite these
limitations, we have recently shown that 5-nitrofuryl and 5-
nitrothienyl derivatives (E)-4-allyl-1-(5-nitrofurylidene)-
thiosemicarbazide (I), (E)-4-allyl-1-[3-(5-nitrofuryl)-2E-
propenylidene]thiosemicarbazide (II), and (E)-4-allyl-1-(5-
nitrothienyl)thiosemicarbazide (III) (Figure 1a) possess high
INTRODUCTION
■
Chagas disease, or American Trypanosomiasis, remains the
major parasitic disease burden in Latin America, despite recent
advances in the control of its vectorial and transfusional
transmission.1−3 Although excellent chemotherapeutic ap-
proaches have been described,4 still the used pharmacology
to control this parasitic infection remains unsatisfactory.5 The
current specific treatments are based on old and quite
nonspecific drugs, namely, ( )-3-methyl-N-[(1E)-5-nitro-2-
furylidene]thiomorpholin-4-amine 1,1-dioxide (nifurtimox, re-
cently discontinued by Bayer, Nfx, Figure 1a) and N-benzyl-2-
(2-nitro-1H-imidazol-1-yl)acetamide (benznidazole, Roche,
Bnz) associated with long-term treatments that may give rise
to severe side effects. In fact, although Nfx and Bnz are able to
eliminate established parasitemia and to reduce serological
titers in acute and early chronic infections, they are not active
Received: November 19, 2013
Published: April 21, 2014
© 2014 American Chemical Society
3984
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
Figure 1. (a) Nifurtimox and other derivatives previously described as T. cruzi growth inhibitors. (b) Structural features initially selected for
modifications to generate new active anti-T. cruzi agents.
and selective anti-T. cruzi activity in vitro and in vivo.8
Furthermore, we have demonstrated that they induce oxidative
stress9 and squalene accumulation in the parasite, hence
suggesting that the squalene epoxidase activity inhibition is
one of the operative mechanisms of action.8 However, (I) and
(II) were positive in the Ames test, using Salmonella
typhimurium TA 98 strain in the presence and absence of S9
from rat liver.10 Derivative (III) was then assessed to confirm
the negative results in the other Salmonella strains (TA 100, TA
102, TA 1535 and TA 1537) finding mutagenicity against TA
1535 and TA 1573 after treatment with S9. However, the
thienyl-analogue (E)-4-allyl-1-thienylthiosemicarbazide (IV)
(Figure 1a) with good in vitro anti-T. cruzi activity displayed
nonmutagenic properties against the five strains recommended
by the Organization for Economic Cooperation and Develop-
ment (OECD).11
Here, we describe the structural modifications done taking as
template compound (III) (Figure 1b) and the biological studies
against different T. cruzi strains and stages. The nonspecific
toxicity against mammalian cells was studied in order to
evaluate the selectivity to the parasites. For the “Hit-to-Lead”
phase we performed a series of studies that allowed sieving the
best compounds and ensure their use as anti-T. cruzi drugs, i.e.,
mutagenicity, formulation, and stability. Together with the
parent compounds (III) and (IV), two new derivatives were
studied in vivo.
are selected among those compounds with improved biological
activities, both trypanosomicidal activity, IC50 in amastigote
forms < 20.0 μM, and nonspecific cytotoxicity, selectivity index
(mammal cells/amastigotes) > 100 and absence of mutage-
nicity. Finally, in Step 4, the leads are consolidated by in vivo
evaluation. At all points of the procedure, we redesigned the
chemicals in a positive feedback between the synthesis and
biological activity trying to revert problems like synthesis,
solubility, lipophilicity, and stability.
Synthesis. Initially in order to vary the lipophilic properties
of the new synthesized derivatives, template-(III) was modified
at thiosemicarbazone-N4-nitrogen level (modification (a),
Figure 1b), generating derivatives (E)-1-(5-nitrothienyl)-
thiosemicarbazide, 1, and (E)-4-phenyl-1-(5-nitrothienyl)-
thiosemicarbazide, 2 (Table 1), and at the thiophene-
thiosemicarbazone linker (modification (d)), generating
derivative (E)-4-allyl-1-[3-(5-nitrothiophen-2-yl)-2E-
propenylidene)thiosemicarbazide, 3 (Table 1). The synthetic
procedures (Scheme 1)12,13 generated the desired compounds
in good yields. Similar modifications using compound (IV) as
template (modification (e)) produced compounds 4−7 (Table
1, Scheme 1). The other structural modification involved
thiosemicarbazone transformation to thiazole system (mod-
ification (b)). For this approach different α-haloacetyl reactants
were used to generate 4-thiazolidinones (8−16, Table 1),14 4-
methylthiazoles (17−21, Table 1),15 and 4-arylthiazoles 22−38
(Table 1 and Scheme 1).15 Transformation of thiosemicarba-
zones into thiadiazoles was accomplished by heating at reflux in
acetic anhydride to produce derivatives 39−42 (modification
(c), Table 1 and Scheme 1) and in the presence of FeCl3 to
produce derivative N-allyl-5-(5-nitrothiophen-2-yl)-1,3,4-thia-
diazol-2-amine, 43 (modification (c), Table 1 and Scheme 1) in
good yields (see procedures in Supporting Information).15
Given that in the first developed derivatives some
compounds do not possess nitro moiety but retained very
good biological activity, i.e., derivatives (E)-4-allyl-1-[5-
METHODS AND RESULTS
■
“Active-to-Hit” Phase. Design. The design of the new
derivatives was performed following the biological steps
detailed below. They are divided into three stages that involve
the preclinical development from synthesis to in vivo evaluation
(see Figure S1 in Supporting Information). Step 1 corresponds
to the identification of molecular prototypes by in vitro assay in
epimastigote forms of the parasite, using as criteria IC50 < 4.2
μM to surpass to the following step. In Steps 2 and 3, the leads
3985
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
Table 1. First Series of Structural Modifications of Parent Compounds (III) and (IV) and in vitro T. cruzi Antiproliferative
Activity against Epimastigotes of Tulahuen 2 Strain
a
b
Each compound concentration was evaluated in duplicate. This compound is shown as the base form; however, it was isolated and evaluated as the
hydrobromide form (see text).
3986
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
Scheme 1. Synthetic Procedures Used to Prepare the New Derivatives (See Procedures in Supporting Information); Y* = Range
of Yields
(thiophen-2-yl)penta-2E,4E-dienylidene]thiosemicarbazide, 6,
and (E)-4-phenyl-1-[5-(thiophen-2-yl)penta-2E,4E-
dienylidene)thiosemicarbazide, 7 (with IC50s lower than the
corresponding value for (III), Table 1), we decided to
investigate the relevance of the 5-nitrothienyl group as
pharmacophore for anti-T. cruzi activity. For that purpose we
planned a new series of derivatives where the main modification
was the substitution of 5-nitrothienyl moiety by different aryl
groups. Additionally, modifications (a), (b), and (d) (Figure
1b) were also included in these series of studies (Table 2).
Compounds 44−74 were synthesized following the general
procedures shown in Scheme 1.
(nomenclature according to Figure 2a) were either the most
abundant or the exclusive isolated species. In the case of
derivative 1-(3-allyl-4-phenylthiazol-2(3H)-ylidene)-2-(5-nitro-
2-thienyl)hydrazine, 22, each isomer, EE and EZ, was isolated,
hence confirming the NOE-diff analysis (see Figure S2 in
Supporting Information). Stereochemistry around chiral center
in derivatives 39−42 and 81 was not determined.
According to spectroscopic data, when -R = -H (derivatives
2-[2E-(5-nitro-2-thienyl)hydrazinyl]thiazol-4(5H)-one, 9, and
[4-(4-chlorofenyl)]-2-[2E-(5-nitro-2-thienyl)hydrazinyl]-
thiazol-4(5H)-one, 28), the thiazole system existed as NH
exocyclic tautomeric form (Figure 2b). The thiazoles that
precipitated from the mixture of reactions (21, 25, 26, 33, 34,
36−38, 48, 57, 62, 71, 76−78, and 80) were obtained as the
corresponding hydrobromides. Adequate single crystals were
obtained for derivative 1Z-[3-allyl-4-(4-chlorophenyl)thiazol-
2(3H)-ylidene]-2E-[3-(2-furyl)-2E-propenylidene] hydrazine,
77, to analyze its structure by X-ray diffraction methods
(Figure 2c). In the solid state, this compound existed at the 2-
aminothiazolium tautomeric form (Figure 2d).
During the analysis of biological activities, we observed that
derivative 1Z-[3-allyl-4-phenylthiazol-2(3H)-ylidene]-2E-[3-(2-
furyl)-2E-propenylidene]hydrazine, 62, a 2-furyl containing 4-
phenylthiazole derivative, displayed excellent in vitro activity.
This observation encouraged us to develop a third series of
compounds (75−81, Table 3) using this derivative as a new
structural prototype.
1
All the compounds were characterized by H NMR and 13C
NMR spectroscopy and also by MS (see results in Supporting
Information). The purity was established by TLC and
microanalysis. Stereochemistry of the exocyclic double bond
(Figure 2a) for thiazole derivatives (8−38, 45, 47, 48, 52, 53,
56−59, 60−62, 64−66, 68, 71, and 73−80) were established
using NOE-diff experiments. In general, the EZ isomers
Biological Characterization. In Vitro Anti-T. cruzi Activity.
The new developed derivatives were initially tested in vitro
against the epimastigote form of T. cruzi, Tulahuen 2 strain.
The existence of the epimastigote form of T. cruzi as an obligate
mammalian intracellular stage has been reviewed and
confirmed.17 The compounds were incorporated into the
3987
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
Table 2. Second Series of Structural Modifications from Parent Compound (III) and Derivatives 6 and 7 and in Vitro T. cruzi
Antiproliferative Activity against Epimastigotes of Tulahuen 2 Strain
a
Cpd.
44
structure type
-Ar
n
-R″
-H
-R
-R′
-Ph
IC50 (μM)
(A)
(C)
(A)
(C)
-Ph
-Ph
-Ph
0
0
1
-Ph
-Ph
19
2
45
46
47
48
49
50
51
52
53
54
55
-H
-H
>25
3
-CH2CHCH2
6.6 0.7
-CH3
-Ph
>25
>25
>25
10
3
3
3
b
(A)
(A)
(A)
(B)
(C)
(A)
(A)
(A)
(C)
-p-OHPh
-p-(SCH3)Ph
-p-ClPh
0
0
0
-H
-H
-H
-CH2CHCH2
-CH2CHCH2
-CH2CHCH2
1
1
11
>25
>25
>25
>25
>25
>25
>25
>25
>25
4
4
3
4
3
3
3
3
3
-p-ClPh
-3,4-(−OCH2O−)Ph
-5-bromo-3-pyridinyl
2-furyl
0
0
0
-H
-H
-H
-CH2CHCH2
-CH2CHCH2
-CH2CHCH2
c
(V)
56
-CH3
-Ph
b
57
58
59
(C)
-5-(SO3H)-2-furyl
2-furyl
-CH3
-Ph
c
(VI)
60
(A)
(B)
(C)
1
-H
-CH2CHCH2
7.0 0.7
>25
>25
3
3
61
-CH3
-Ph
b
d
62
3.2 0.4
63
64
65
(A)
(B)
(C)
-Ph
11
1
>25
25
3
-CH3
-Ph
3
b
66
5.0 0.6
>25
14.6 0.3
>25
5.4 0.5
67
68
69
70
(A)
(C)
(A)
(A)
(C)
(A)
(A)
(B)
(C)
-3-thienyl
0
0
0
0
0
-H
-CH2CHCH2
-CH2CHCH2
-CH2CHCH2
-CH2CHCH2
-CH2CHCH2
3
-3-thienyl
-H
-p-ClPh
-2-benzo[b]furyl
-2-naphtyl
-CH3
-H
3
b
71
-4-quinolinyl
-H
-Ph
>25
17
3
72
2
e
(VII)
73
-2,3-dimethyl-1,4-dioxide-6-quinoxalinyl
0
-H
-CH2CHCH2
>25
>25
15
3
3
74
-p-ClPh
2
a
b
Each compound concentration was evaluated in duplicate. This compound is shown as the base form; however, it was isolated and evaluated as the
hydrobromide form (see text). Product previously described.8b The compound was also evaluated against T. cruzi epimastigote Dm 28c clone
c
d
(IC50 of 4.0 0.3 μM). Product previously described.16
e
biological milieu at 25 μM, and their ability to inhibit growth of
the parasite was evaluated in comparison with that of the
control (no drug added to the milieu) on day 5 (growth
exponential phase). Nfx and Bnz were used as the
trypanosomicidal reference drugs. The IC50 concentrations
(50% inhibitory concentration) were assessed for Nfx and Bnz,
propenylidene]hydrazine, 31 (IC50 = 3.3
nitrothienyl-thiadiazole N-[4-acetyl-5-(5-nitrothiophen-2-yl)-
4,5-dihydro-1,3,4-thiadiazol-2-yl]-N-phenylacetamide, 40 (IC50
0.5 μM) or the
= 2.9 0.3 μM), and the thienyl-thiosemicarbazone 6 (IC50
=
0.4
0.60
0.06 μM) or the furyl-thiazole 62 (IC50 = 3.2
μM).
8
1 and 7
1 μM, respectively, and the most active
From the analysis of the biological response of the first 74
studied derivatives, the previous described compounds (III)−
(VII), (Tables 1 and 2), and the results against amastigotes
forms (Table 4), we redirected our synthetic strategies trying to
improve the activity of derivative 62. This derivative, without
the potential mutagenophore nitro-moiety, was twice as much
more active against epimastigotes than the corresponding
parent compound (VI) (Table 2). The cyclization of (VI) to
the thiazolidinone 60, to the methylthiazole 1E/Z-[3-allyl-4-
methylthiazol-2(3H)-ylidene]-2E-[3-(2-furyl)-2E-
propenylidene]hydrazine, 61, or to the thiadiazole N-[4-acetyl-
derivatives (Tables 1, 2, and 3). From the first and second
series of chemical modifications, more active derivatives, than
the nitrothienyl-, (III), and the thienyl-, (IV), parent
compounds (IC50 of 4.2 0.6 and 7.9 0.8 μM, respectively),
were obtained. For example, the nitrothienyl-thiosemicarba-
zone 3 (IC50 = 1.2 0.2 μM), the nitrothienyl-thiazolidinone
2Z-[3-(5-nitrothiophen-2-yl)-2E-propen-1E-ylidenehydrazo-
no]-3-allylthiazolidin-4-one, 11 (IC50 = 1.3
nitrothienyl-thiazole 1E/Z-[3-allyl-4-(4-nitrophenyl)thiazol-
2(3H)-ylidene]-2E-[3-(5-nitrothiophen-2-yl)-2E-
0.2 μM), the
3988
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
Table 3. Third Series of Structural Modifications of Structural Prototype 62 and in Vitro T. cruzi Antiproliferative Activity
against Epimastigotes of Tulahuen 2 Strain
a
b
Each compound concentration was evaluated in duplicate. This compound is shown as the base form; however, it was isolated and evaluated as the
hydrobromide form (see text).
1.3
0.2 μM), and 1Z-[3-allyl-4-(2-naphthyl)thiazol-2(3H)-
ylidene]-2E-[3-(2-furyl)-2E-propenylidene]hydrazine, 80 (IC50
= 2.4 0.3 μM), were more active than the new prototype
phenyl-thiazole 62 (Table 3).
The above studies were followed (according to Figure S1,
Supporting Information) by the assessment of the activity of
selected derivatives against the parasitic relevant form for the
disease, namely, the amastigote form, using Sylvio X-10 strain
(Table 4).18 Acetyl-derivatives 39−42 were not included in this
study due to the low stability in biological milieu (see, as an
example, the results with the active derivative 40; Figure S3,
Supporting Information). Except for some derivatives (6, 7, 10,
22EZ, 26, 27, 29, 32, 37, and 61), the developed compounds
showed similar behavior in both forms of the parasite. Sixteen
in 26, 61.5%, possessed the same tendency of activities in both
parasitic forms (Figure S4, Supporting Information). Addition-
ally, 87.5% of the most active derivatives, IC50 ≤ 20 μM, in
amastigote form possessed IC50 ≤ 20 μM in the epimastigote
one (Figure S4, Supporting Information). The most active
derivatives were the furyl containing aryl-thiazole 62, 77, and
80. They eradicated near to 100% of the amastigotes parasites
at doses at least one and a half lower than the corresponding
doses for Nfx (see IC90 values in Table 4) having derivative 77
with the best behavior with an IC90 in amastigotes of 310 nM.
In fact, these compounds were able to eradicate the amastigotes
form into Vero cells after 72 h of treatment (Figure 3).
Additionally, derivative 77 was tested in vitro against the
bloodstream trypomastigote form of T. cruzi, Y strain. The
compound was incorporated into the biological milieu at 25
μM producing 70% of parasite growth inhibition, in
comparison to that of the control (no drug added to the
milieu), after 48 h of incubation. Bnz, used as the
trypanosomicidal reference drug, produced 90% of parasite
growth inhibition in the same conditions.
Figure 2. (a) Possible configurations around exocyclic double bond for
thiazole derivatives. (b) Tautomeric form observed (right structure),
in solution, for derivatives 9 and 28. (c) Photographs of derivative 77
single crystals and drawing of the corresponding molecular model
showing the labeling of the non-H atoms and their displacement
ellipsoids at the 30% probability level. Dashed lines indicate
intermolecular H-bonds. (d) 2-Aminothiazolium tautomeric form of
derivative 77 observed in the solid state.
5-[2-(furan-2-yl)-1E-ethenyl]-4,5-dihydro-1,3,4-thiadiazol-2-
yl]-N-allylacetamide, 81 (Table 3), was conducted to
compounds with lower anti-T. cruzi activity. However, the
arylthiazoles 76−78 were more active than (VI) where
derivatives 1Z-[3-allyl-4-(4-chlorophenyl)thiazol-2(3H)-yli-
In Vitro Nonspecific Mammal Cytotoxicity. To explore the
selectivity of these new derivatives against T. cruzi we evaluated
the in vitro nonspecific mammal cytotoxicity, using VERO
monkey kidney epithelial cells, human red blood cells, and in
dene]-2E-[3-(2-furyl)-2E-propenylidene]hydrazine, 77 (IC50
=
3989
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
some cases J774.1 mouse macrophages (Table S1, Supporting
Information). The new developed compounds were selected
according to their anti-T. cruzi activity and trying to cover a
wide range of structural characteristics. The selectivity indexes
(SI, Table 4) were expressed as the ratio between IC50 in
mammal cells and IC50 in T. cruzi amastigotes Sylvio X-10
strain.19 Derivatives 62, 77, and 80 displayed low cytotoxicity
against mammal cells with adequate selectivity indexes toward
the parasite. The worst selective derivative and most cytotoxics
against VERO cells were the nitro-thienyl derivatives 22EZ, [4-
(4-chlorophenyl)]-2-[2E-(5-nitro-2-thienyl)hydrazinyl]thiazol-
4(5H)-one (28), and 40.
“Hit-to-Lead” Phase. Mutagenicity Studies. The method
of direct incubation in plate20 using culture of Salmonella
typhimurium TA 98 strain was initially performed on some
selected derivatives and Nfx. The influence of metabolic
activation was tested by adding S9 fraction of mouse liver.
Positive controls of 4-nitro-o-phenylendiamine and 2-amino-
fluorene were run in parallel. The revertant number was
manually counted and compared to the natural revertant (Table
5). The compound is considered mutagenic when the number
Table 4. Biological Characterization of Developed
Derivatives Against Amastigotes Form, Sylvio X-10 Strain,
and Mammal Cells
a
a
b
c
d
Cpd.
(III)
IC50 (μM)
IC90 (μM)
>25
14.4 0.4
4.06 0.15
SIV
SIR
SIJ
19.4 2.2
4.1 0.1
4
100
20
e
2
3
1.29 0.04
60
82
21
f
4
6
>25
>25
>25
>25
4
4
4
4
>25
>25
>25
>25
4
4
4
4
7
8
10
11
17
18
19
21
22EZ
26
27
28
29
30
31
32
37
40
43
61
62
77
80
Bnz
Nfx
AmpB
2.8 0.3
9.9 4.3
80
5
28
2.55 0.06
10.5 2.8
9.30 0.24
21.2 2.4
>25
4
>25
4
f
<7
25
4
>25
>25
>25
g
4
4
4
>25
>25
g
4
4
>25
4
>25
4
Table 5. Mutagenic Properties of New Derivatives and Nfx
on TA98 S. typhimurium Strain
16.9 0.7
8.2 0.6
8.0 1.2
25.36 0.25
>25
10.2 0.5
4
13
50
ab
,
mutagenicity
without S9
c
>25
4
>25
4
Cpd.
D
with S9
f
g
g
3
3
3, 9, 27, 83, 250
(+)
(−)
(−)
(+)
(+)
(−)
(−)
(−)
(+)
(−)
( )
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(+)
(−)
(+)
(+)
(−)
(−)
(−)
(+)
(−)
(−)
(−)
(+)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(+)
(−)
(+)
5.0 0.5
15.9 9.6
51 22
5
5, 15, 44, 133, 400
6, 19, 56, 167, 500
0.02, 0.07, 0.2, 0.7, 2
5, 15, 44, 133, 400
0.005, 0.01, 0.04, 0.13, 0.4
0.005, 0.01, 0.04, 0.13, 0.4
0.3, 1, 4, 13, 40
6.3 0.6
6
<0.24 0.02
<0.25 0.02
<0.21 0.02
3.3 0.3
1.30 0.02
0.31 0.02
1.4 0.2
>1000
>1000
>1000
300
>196
>234
>120
>200
>120
11
14
17
18
21
31
37
40
43
61
62
75
76
77
78
79
80
Nfx
0.45 0.01
1.1 0.1
2.4 0.2
200
150
10
200
h
0.04, 0.1, 0.4, 1.3, 4
3.1, 9.3, 28, 83, 250
0.005, 0.01, 0.04, 0.13, 0.4
0.05, 0.1, 0.4, 1.3, 4
6, 19, 56, 167, 500
3.1, 9.3, 28, 83, 250
0.04, 0.1, 0.4, 1.3, 4
5, 15, 44, 133, 400
5, 15, 44, 133, 400
5, 15, 44, 133, 400
5, 15, 44, 133, 400
6, 19, 56, 167, 500
0.5, 1, 3, 10, 30
a
b
Each compound concentration was evaluated in quadruplicate. SIV:
c
selectivity index, IC50,VERO cells/IC50,T. cruzi (amastigotes, Sylvio X‑10)
.
SIR:
d
selectivity index, IC50,human blood red cells/IC50,T. cruzi (amastigotes, Sylvio X‑10).
d
S I J : s e l e c t i v i t y i n d e x , I C 5 0 , J 7 7 4 . 1
/
m o u s e
e
m a cfr o p h a g e s
IC50,T. cruzi (amastigotes, Sylvio X‑10). Blank cells = not studied. Selectivity
index defined as IC50,mammal cells/IC50,T. cruzi (epimastigotes, Tulahuen 2)
g
. The
derivative was not evaluated in this assay due to its high cytotoxicity
against VERO cells (more than 50% of VERO-growth inhibition at the
h
lower evaluated concentration, 25 μM). AmpB: amphotericin B.
a
The results are the means of two independent experiments.
b
Mutagenicity, according to ref 21 (see text). (+), mutagenic; (−),
c
d
nonmutagenic. D: studied doses (in μg/plate). ( ): At the higher
studied dose the revertant level is twice the spontaneous frequencies.
of revertant colonies is at least twice the spontaneous revertant
frequencies for at least two consecutive dose levels.21 The
maximum assayed doses were determined according to toxic
effect on S. typhimurium. Fifty-six percent of the nitro
containing derivatives were mutagenic in at least one
experimental condition. The nitro group is located in the
heterocyclic-moiety like in 3, 2Z-[3-(5-nitrothiophen-2-yl)-2E-
propen-1E-ylidenehydrazono]-3-allylthiazolidin-4-one, 11, N-
[4-acetyl-5-(5-nitrothiophen-2-yl)-4,5-dihydro-1,3,4-thiadiazol-
2-yl]-N-phenylacetamide, and Nfx or in another position of the
Figure 3. (a) Optic microscopy of VERO cells infected with T. cruzi
(Sylvio X-10 strain), amastigotes (red arrow), and typomastigotes
(blue arrow) are visualized. (b) Optic microscopy of treated cells with
5 μM of derivative 77 after 72 h of incubation. The cells are free of
infection and undergoing cellular division (black arrow).
3990
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
Table 6. Mutagenic Properties of Prototype 61 and Derivatives 62, 77, and 80 on S. typhimurium Strains Recommended by
OECD
ab
,
ab
,
ab
,
ab
,
TA100
(−) S9
TA102
(−) S9
TA1535
(−) S9
TA1537
(−) S9 (+) S9
c
Cpd.
D
(+) S9
(+) S9
(+) S9
d
61
62
77
80
0.003, 0.007, 0.02, 0.06, 0.2
0.06, 0.19, 0.5, 1.7, 5
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
e
3.09, 9.26, 27.77, 83.33, 250
6, 19, 56, 167, 500
a
b
c
The results are the means of two independent experiments. Mutagenicity, according to ref 21 (see text). (+), mutagenic; (−), nonmutagenic. D:
studied doses (in μg/plate). For TA 100, the used doses were 3.09, 9.26, 27.77, 83.33, and 250 μg/plate. For TA 100, the used doses were 0.003,
0.007, 0.02, 0.06, 0.2 μg/plate.
d
e
structure, like in 1Z-[3-allyl-4-(4-nitrophenyl)thiazol-2(3H)-
ylidene]-2E-[3-(2-furyl)-2E-propenylidene]hydrazine, 79, or in
both, like in 31. Only 8% of derivatives without nitro-moiety
was mutagenic in both conditions: derivative 3-allyl-2E/Z-[2E-
[3-(thiophen-2-yl)-2E-propenylidene]hidrazono] thiazolidin-4-
one, 14.
To complete the OECD recommendations, some of the
most relevant derivatives, 61, 62, 77, and 80, were evaluated
against S. typhimurium TA 100, TA 102, TA 1535, and TA 1537
strains without and with metabolic activation (Table 6).
Positive controls, 4-nitro-o-phenylendiamine and 2-amino-
fluorene for TA 100, sodium azide, and 2-aminoantracene for
TA 102, TA 1535, and TA 1537, were run in parallel. The four
studied compounds fulfilled the OECD requirements.
Formulation Studies. For the in vivo studies the compounds
were initially suspended in a mixture of saline/Tween 80 (4:1,
v:v) as vehicle (V1) in order to employ an aqueous-like
Figure 4. Stability of selected compounds in simulated physiological
formulation. However, the use of different doses was limited by
conditions and in storage formulation. For the studies in aqueous
milieu and in the presence of cytosolic (cyt) and microsomal (mic)
fractions, the assays were conducted over a period of 1 h, at 37 °C,
while the storage studies on the vehicle (V2) were conducted over a
the low suspension ability of V1; additionally, the biological
results were very erratic using this vehicle.
Thus, studies to improve vehicle system to dispose
adequately of the compounds were undertaken. To this
purpose, we selected microemulsion as vehicle.22 This is a
mixing of water and oil stabilized by a surfactant systemwhich is
thermodynamically stable and, depending on composition,
exhibits a wide range of nanostructures.
Consequently, the second vehicle (V2) was a microemulsion
composed of cholesterol (10%) as oil phase, the surfactant
(soya phosphatidylcholine/sodium oleate/polyoxyl-40 hydro-
genated castor oil (3:6:8, 10%)), and phosphate buffer pH 7.4
(80%) as aqueous phase.23 This vehicle generated a
homogeneous and stable suspension for, at least, both one
month at 4 °C and more than 20 days at 30 °C.
Stability Studies. We analyzed the stability, of selected
derivatives, i.e., 1Z-(3-allyl-4-methylthiazol-2(3H)-ylidene)-2E-
(2-thienyl)hydrazine (19), 61, 62, 1Z-[3-allyl-4-(4-
methoxyphenyl)thiazol-2(3H)-ylidene]-2E-[3-(2-furyl)-2E-
propenylidene]hydrazine (76), 77, 1Z-[3-allyl-4-(4-bromo-
phenyl) thiazol-2(3H)-ylidene]-2E-[3-(2-furyl)-2E-
propenylidene]hydrazine (78), and 80, on simulated physio-
logical conditions, namely, aqueous solution at different pHs,19c
in the presence of hepatic rat cytosolic and microsomal
fractions,24 and in the vehicle V2 (Figure 4). Except for
thiophene derivative 19, all the compounds were stable or
partially stable, remaining more than 95% of the derivative at
the end of the assay, during 1 h on aqueous solution pH 6.0,
7.4, and 9.0. On aqueous solution at pH 1.0, 61, 62, and 77
derivatives showed some degree of instability, remaining near to
90% of the derivative at the end of the assay, and no attempt to
isolate the decomposition products was done. Derivatives 19,
period of 1 week, at 4 °C.
77, and 80 were stable on liver metabolic fraction for 1 h at 37
°C. Additionally, derivatives 62, 77, and 80 were stable in V2
during storage at 4 °C.
Proof of Concept: in Vivo anti-T. cruzi Studies. Oral Effects
on Uninfected Mice. Parent compounds (III) and (IV) and
thiazoles 77 and 80 were given orally and daily, by intragastric
gavage, to uninfected female BALB/c mice (n = 2/group) at
100 mg/kg body weight/day, in V1, for 14 days. Mice were
weighted and monitored daily, and at the end of the
experiments the animals were sacrificed and necropsies were
done in order to evaluate macroscopically the organ status.
Neither mortality nor signs associated with toxicity were
observed.
Treatment of T. cruzi Infected Mice. Parent compounds
(III) and (IV) and thiazoles 77 and 80 were evaluated in vivo
in murine models of acute Chagas’ disease. Bnz was used as the
in vivo active reference drug.
The in vivo results with parent compounds (III) and (IV)
were in agreement with their in vitro behaviors, poor capability
to decrease parasitemia and to shift the peak of maximum
parasitemia (for details see the corresponding section in
Supporting Information Material, in vivo studies of parent
compound (III) and (IV)). Additionally, some potential
immune-suppressive properties in the treatment with the
parent compounds were evidenced in the dose vs response
studies (see Figure S5b in Supporting Information), as was
3991
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
□
△
Figure 5. Parasitemia of mice treated with 107 μmol/kg b.w./day of 77 ( ), group treated with 192 μmol/kg b.w./day of Bnz ( ), and control
⧫
group ( ). (a) experiment 1: compound 77 was suspended in V1 (n = 8/group). (b) experiment 2: compound 77 was suspended in V2 (n = 7/
group). Highlighted regions () correspond to the period of treatment (between days 6 and 20 postinfection).
Table 7. Summary of in Vivo Studies
a
experiment
treatment with
vehicle
doses (μmol/kg b.w./day)
peak of maximum parasitemia (day)
percentage of survival (%)
1
V1
Bnz
77
26
55
26
28
56
35
28
56
35
26
42
42
75
V1
V1
192
107
100
75
2
3
4
V2
Bnz
77
67
V1
V2
192
107
100
100
67
V2
Bnz
77
V1
V2
192
214
100
100
50
V2
Bnz
80
V1
V2
192
107
100
100
a
At the end of the studies (day 65).
previously observed, for nitrofuran analogues;12a this means
significant parasitemia recrudescences were observed.
In a third experiment (experiment 3) the dose of derivative
77 was increased to 100 mg/kg b.w./day (214 μmol/kg b.w./
day) closer to Bnz dose. In these conditions, derivative 77 was
able to maintain the parasitemia of treated animals under the
curve of untreated ones throughout the study period (Figure
6a). After day 35, the maximum of parasitemia for treated
animals, the bloodstream form drops sharply. Additionally, in
this case we determined the level of anti-T. cruzi antibodies at
days 30 and 60 postinfection (Figure 6(b). Derivative 77 was
able to significantly decrease, with respect to untreated animals,
the antibodies at day 60. Furthermore, in this experiment the
survival animals treated with 77 was 100% (summary in Table
7).
Lastly, derivative 80 was also in vivo evaluated (experiment
4) using similar conditions as those of experiment 2, i.e.,
vehicle V2 and orally administration of 50 mg/kg b.w./day (107
μmol/kg b.w./day). The modification involved a different
treatment schedule, receiving the derivative 80 during 14 days,
in a 19 day schedule with 4 days of resting and 5 days of
administration followed by 2 days of rest, repeating this
administration scheme two more times (Figure 7a). Even
before the first peak of parasitemia derivative 80 appeared to
For the thiazoles, in the first approach (experiment 1) male
BALB/c mice infected with CL Brener clone were treated
orally, by intragastric gavage, during 14 days with derivative 77
(50 mg/kg b.w./day, 107 μmol/kg b.w./day) or Bnz (50 mg/
kg b.w./day, 192 μmol/kg b.w./day), both in V1.19b,25 The
course of infection was monitored by counting blood parasites
in a hemocytometer, and animal survival was followed for 65
days postinfection (Figure 5a and summary in Table 7).
Because of the low stability in acid milieu, the very variable
treatment vs response between mice (data not shown), and the
low V1 suspension ability of compound 77, we performed a
second study (experiment 2) varying only the vehicle, using in
this case the microemulsion V2. With this vehicle the
intermouse results were more accurate and robust being the
parasitemia and animal survival representative of the in vitro
results (Figure 5b and summary in Table 7). In this condition,
derivative 77 was able to delay the maximum of parasitemia
shifting it 7 days relative to vehicle treated mice (Table 7).
With this result, we selected V2 as formulation in the rest of the
in vivo studies.
3992
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
Figure 6. Results of in vivo experiment 3. (a) Parasitemia of mice (n = 7/group) treated with 107 (experiment 2, Figure 5a, and 214 μmol/kg b.w./
⧫
□
■
△
day of 77, and , respectively), group treated with 192 μmol/kg b.w./day of Bnz ( ), and control group ( ). (b) Anti-T. cruzi antibody levels at
(
(
(
days 30 and 60. Lower line represents the cutoff (antibody levels for healthy animals). *) p < 0.10, **) p < 0.05, ***) p < 0.01, with respect to
untreated animals (only V2). RDB: relative doses to Bnz.
○
Figure 7. Results of in vivo experiment 4. (a) Parasitemia of mice (n = 6/group) treated with 107 μmol/kg b.w./day of 80 ( ), group treated with
⧫
△
192 μmol/kg b.w./day of Bnz ( ), and control group ( ). Highlighted regions () correspond to the period of treatment schedule (between days
6 and 25 postinfection). (b) Anti-T. cruzi antibody levels at day 60. Lower line represents the cutoff (antibody levels for healthy animals).
work adequately, however, after passing the bloodstream valley
(day 34) it was not able to keep the trypomastigotes below the
corresponding levels for untreated animals. That may be the
result of the administration schedule of derivative 80, which
could modify the amount of compound availability during the
treatment in comparison with Bnz or derivative 77
administration schedules. Despite that the animal survival rate
was 100% (Table 7), therefore, anti-T. cruzi antibodies were
evaluated at day 60, which were not significantly different from
the untreated control (Figure 7b).
DISCUSSION
■
We reported the in vitro biological activity against different
forms of T. cruzi of 82 new compounds developed using
compounds (III) and (IV) as partially- and nonmutagenic
templates. Derivatives 3, 6, 7, 11, 40, 77, 78, and 80 were the
most active against epimastigotes Tulahuen 2 strain with 3, 6, 7,
11, and 77 at least 4 times more active than the template (III),
Nfx, and Bnz (Tables 1−3).
Derivatives 3, 11, 62, 77, and 80 were also active against
amastigotes Sylvio X-10 being 62, 77, and 80, equipotent to
Nfx (Table 4). Concommitantly, derivative 77 displayed
activity against the circulating form of T. cruzi, trypomastigote
3993
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
Figure 8. Summary of the tree-structural modifications performed and in vitro bioactivities obtained (green check, active against epimastigotes; blue
check, active against amastigotes; red ×, mutagenic in both absence and presence of S9; pink ×, mutagenic in the absence or presence of S9; black
check, nonmutagenic).
form. Along with these findings, derivatives 62, 77, and 80 also
possess selectivity to T. cruzi (Table 4) being 5 times more
selective than Nfx when Vero cell cytotoxicity was used as a
comparison.
abolishing the second maximum parasitemia peak that appeared
in the untreated animals at day 42. These data clearly showed
that thiazole 77 affected in vivo trypomastigotes, depleting the
blood stream forms corroborated by the shifting of first
parasitemia peak, and also the amastigote forms, corroborated
by the absence of the second parasitemia peak. These results
were in agreement with the values of anti-T. cruzi antibodies
where derivative 77 showed significantly low levels at 60 days
postinfection (Figure 6b).
However, derivative 80 was able to diminish the levels of
bloodstream forms previous to the first parasitemia maximum
peak, but after that levels were raised with respect to the
untreated controls (Figure 7a). These data could indicate the
lower in vivo effect of thiazole 80 on amastigotes that could be
corroborated by the antibody level findings (Figure 7b);
however, pharmacokinetic behavior of 80 different from 77
could not be ruled out.
From a chemical point of view, these compounds are
synthesized by simple, scalable, and cost-effective procedures.
In comparison to the reference drug or other compounds
currently studied as anti-T. cruzi agents, our thiazoles have the
following advantages (Table 8): (i) lower production cost and
(ii) lower molecular complexities.
These good findings, i.e., absence of mutagenicity and in vivo
activity, promote further thorough studies, modifying doses,
administration routes, extending the in vivo treatment until
after the maximum peak of parasitemia, and combinations with
other drugs.
Among the compounds with the best antiparasitic activities
(3, 6, 7, 11, 40, 62, 77, 78, and 80, Figure 8) the nitro
derivatives 3, 11, and 40 displayed mutagenic effects in some of
the experimental conditions, -S9 or +S9, while 62, 77, and 80
were not mutagenic in the Ames test against the five S.
typhimurium strains recommended by OECD (Tables 6 and 7).
According to their biological behaviors, furan derivatives 77
and 80 were candidates (Figure 8) for pharmacological studies.
Consequently, lead compounds were further evaluated for
stability and solubility. Although, despite being salts, they
displayed very poor solubility in aqueous milieu and they could
be little stable in the gastrointestinal tract (Figure 4); therefore,
it was decided to formulate them as microemulsions for in vivo
studies. Microemulsioned systems could improve the solubility
and stability of drugs,23 in addition to becoming long-acting,
increasing their bioavailability and decreasing its dose, and
generating vectoring differential to certain tissues or organs in
the body.
The pharmacological proofs of concept were done with
parent compounds (III) and (IV) and derivatives 77 and 80.
The results of parent compounds were in agreement with the in
vitro behavior. In the case of compound (III) the in vivo
activity was adequate, in one of the conditions, but together
with compound (IV) showed clear immune-suppressive
behavior, evidenced by the exacerbation of the levels of the
bloodstream forms, without modification in the anti-T. cruzi
antibody levels. Moreover, the leads 77 and 80 displayed in
vivo activity against the infected animals that probed the
concept. In the in vivo experiments 2−4, the animals treated
with them showed 100% survival rate at the end of the assays.
The blood study findings showed that derivative 77, at a dose
near to a half of the dose used for Bnz (experiment 2),
exhibited a particular biological profile, shifting the parasitemia
maximum to 7 days with respect to the control (Table 7) and
CONCLUSIONS
■
We have identified new thiazoles as excellent antitrypanoso-
matid agents. Observing the lack of mutagenic properties of
them, the absence of toxicity, and the in vivo antitrypanosoma
results, these compounds could be considered for further
preclinical studies in order to generate new anti-Chagas drugs.
Currently, we are performing studies related to identifying the
mechanism of action (i.e., oxidative stress and squalene
3994
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
developed by alternated cycles of Fourier methods and full-matrix
least-squares refinement on F2 with SHELXL-97.28 Refinement
showed evidence for the presence of strong overlap between
reflections from both twins. Therefore, it was resorted to the data
reduction of twin crystals facility implemented in CrysAlisPro to
generate a data set including all the reflections from both domains with
the overlapping ones flagged for further structure development
(expansion) and refinement with SHELXL-97. With these data all
hydrogen atoms were found in a difference Fourier map phased on the
heavier atoms. However, the organic molecule H atoms were
positioned stereochemically and refined with deriding model. The
water H atoms were refined with isotropic displacement parameters at
their found positions with Ow−H and H···H distances restrained to
target values of 0.86(1) and 1.36(1) Å, respectively. The solid state
molecular model of 77 was drawn with ORTEP.29
Table 8. Summary of Production Characteristics of 77
Compared to Commercial Drugs
steps in the synthetic
procedures
other
observations
a
Cpd
price per g
b
Bnz
ravuconazole
104.0
3
c
350,000.0
300,000.0
more than 5
chiral centers
chiral centers
c
posaconazole
77
more than 5
de
,
3.0
2
3
geometric
isomers
ef
,
2.6
a
The price per gram of compounds were obtained from Sigma-Aldrich
retail prices (http://www.sigmaaldrich.com/catalog) for Bnz or from
http://www.scbt.com for ravuconazole and posaconzale and are
b
expressed in $ (United States dollar). From commercial 2-
c
nitroimidazole. Antifungal drugs recently used in clinical trials for
Appearances, yields, spectroscopic data, elemental microanalyses,
and crystallographic data of the products are collected in the
Supporting Information.
d
e
the treatment of Chagas disease. From furylacrolein. Calculated only
considering the reactants using retail prices of them (http://www.
f
sigmaaldrich.com/catalog). From furfural.
General Procedure for the Preparation of Derivatives 1−7, 46,
49−51, 54, 55, 63, 67, 69, 70, and 72. A mixture of the
corresponding aldehyde (1.05 equiv), the corresponding thiosemi-
carbazide (1.00 equiv), catalytic amount of p-toluenesulfonic acid, and
dry toluene (1 mL per 100 mg of aldehyde) was stirred at room
temperature until disappearance of the aldehyde (12−24 h, checked by
TLC, SiO2, and petroleum ether/EtOAc 70:30). After that, the
precipitate was filtered off and washed with petroleum ether. The solid
was crystallized from ethanol.
General Procedure for the Preparation of Derivatives 8−16, 52,
60, 64, and 73. A mixture of the corresponding thiosemicarbazide
(1.0 equiv), ethyl bromoacetate (2.0 equiv), anhydrous sodium acetate
(4.0 equiv), and dry ethanol (1 mL per 100 mg of thiosemicarbazide)
was heated at reflux until disappearance of the thiosemicarbazide (4−
10 h, checked by TLC, SiO2, and petroleum ether/EtOAc 70:30).
After that, the mixture was cooled at room temperature, and the
precipitate was filtered off and washed with ethanol/water (80:20).
The solid was crystallized from ethanol or ethanol/water.
General Procedure for the Preparation of Derivatives 17−38, 45,
47, 48, 53, 56−59, 61, 62, 65, 66, 68, 71, 74, and 76−80. A mixture
of the corresponding thiosemicarbazide (1.0 equiv), the α-haloketone
(2.0 equiv), and dry ethanol (1 mL per 100 mg of thiosemicarbazide)
was heated at reflux until disappearance of the thiosemicarbazide (4−
10 h, checked by TLC, SiO2, and petroleum ether/EtOAc 70:30).
After that, the mixture was cooled at room temperature, and the
precipitate was filtered off and washed with ethanol/water (80:20).
The solid was crystallized from ethanol or ethanol/water.
Preparation of Derivative 75 Using Sodium Dithionite (via 1). A
mixture of derivative 79 (0.1 g), sodium dithionite (0.7 g, added
during 70 min in seven portions of 0.1 g), THF (10 mL), and water
(10 mL) was stirred at reflux under nitrogen atmosphere for 8 h. After
that, the solvent was evaporated in vacuo and the residue was
partitioned between CH2Cl2 (30 mL) and aqueous sodium
bicarbonate (5%, 2 × 15 mL). The organic layer was dried with
anhydrous Na2SO4 and evaporated in vacuo. The desired product was
purified by column chromatography (Al2O3 and petroleum ether/
EtOAc 0 to 30%).
Preparation of Derivative 75 Using Stannous Chloride (via 2). A
mixture of derivative 79 (0.1 g), SnCl2 (10 equiv), DMF (0.5 mL),
catalytic amount of acetic acid, and dried toluene (10 mL) was stirred
at 90 °C under nitrogen atmosphere for 2 h. After that, the solvent was
evaporated in vacuo and the residue was partitioned between CH2Cl2
(30 mL) and aqueous sodium bicarbonate (5%, 4 × 15 mL). The
organic layer was dried with anhydrous Na2SO4 and evaporated in
vacuo. The desired product was purified by column chromatography
(Al2O3 and petroleum ether/EtOAc 0 to 30%).
General Procedure for the Preparation of Derivatives 39−42 and
81. A mixture of the corresponding thiosemicarbazide (1 equiv) and
acetic anhydride (10 mL per mmol of thiosemicarbazide) was heated
at reflux until disappearance of the thiosemicarbazide (2−4 h, checked
by TLC, SiO2, and petroleum ether/EtOAc 70:30). After that, the
mixture was partitioned between EtOAc (30 mL) and aqueous sodium
accumulation), pharmacokinetic studies, and new pharmaco-
logical schedules.
EXPERIMENTAL SECTION
■
Chemistry. All starting materials were purchased from Sigma-
Aldrich Co. (USA) and Acros Organics (Janssen Pharmaceutical, Geel,
Belgium). All solvents were dried and distilled prior to use. All the
reactions were carried out in a nitrogen atmosphere. All of the
synthesized compounds were chemically characterized by thin layer
chromatography (TLC), nuclear magnetic resonance (1H NMR, 13C
NMR), and elemental microanalyses (CHN). Alugram SIL G/UV254
(Layer: 0.2 mm) (Macherey-Nagel GmbH & Co. KG., Duren,
̈
Germany) was used for TLC, and silica gel 60 (0.040−0.063 mm,
Merck) was used for flash column chromatography. The NMR spectra
were recorded on a Bruker DPX 400 (400 MHz for 1H and 100 MHz
for 13C), using TMS as the internal standard and with the indicated
deuterated solvent; the chemical shifts are reported in ppm (δ) and
coupling constants (J) values are given in Hertz (Hz). Signal
multiplicities are represented by s (singlet), d (doublet), dd (double
doblet), t (triplet), tt (triple triplet), and m (multiplet). Structural
assignments were corroborated by HMBC and HSQC experiments.
Mass spectrometry experiments were performed on a HEWLETT
PACKARD MSD 5973 or a LC/MSD-Serie 100 using electronic
impact (EI) or electrospray ionization (ESI), respectively. To
determine the purity of the compounds, elemental microanalyses
obtained on a Carlo Erba Model EA1108 elemental analyzer from
vacuum-dried samples were used. The analytical results for C, H, and
N were within 0.4 of the theoretical values. Melting points were
recorded on ELECTROTHERMAL IA-9100 equipment, and they
were not corrected.
The measurements for single crystal derivative 77 were performed
on an Oxford Xcalibur Gemini Eos CCD diffractometer with graphite-
monochromated CuKα (λ = 1.54178 Å) radiation. X-ray diffraction
intensities were collected (ω scans with ϑ and κ-offsets), integrated,
and scaled with CrysAlisPro26 suite of programs. Most of the
diffraction pattern was interpreted in terms of two crystal domains
(twins) related to each other through a rotation of 180° around the
reciprocal b* axis of the strongest diffracting crystal domain (hereafter,
twin #1). The unit cell parameters for both twins were equal to within
experimental accuracy. They were obtained by least-squares refine-
ment based on the angular settings for all collected, nonoverlapping
reflections with intensities larger than seven times the standard
deviation of measurement errors using CrysAlisPro. The reflections
were indexed in the reciprocal unit cell of the corresponding domain.
Data were corrected empirically for absorption employing the
multiscan method implemented in CrysAlisPro. The diffraction data
indexed in the reciprocal unit cell of twin #1 (with about 62% of
diffracting power) was employed to solve the structure by direct
methods using SHELXS-9727 and the corresponding molecular model
3995
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
bicarbonate (5%, 4 × 15 mL). The organic layer was dried with
anhydrous Na2SO4 and evaporated in vacuo. The desired product was
purified by column chromatography (SiO2 and petroleum ether/
EtOAc 0 to 40%).
Preparation of Derivative 43. A mixture of 3 (3 mmol) and ferric
chloride (1.2 mmol) in anhydrous ethanol (3 mL) was heated at reflux
for 8 h. After that, the mixture was partitioned between EtOAc (30
mL) and aqueous sodium bicarbonate (5%, 4 × 15 mL). The organic
layer was dried with anhydrous Na2SO4 and evaporated in vacuo. The
desired product was purified by column chromatography (SiO2 and
petroleum ether/EtOAc 0 to 40%).
°C for 48 h. Each solution was examined microscopically (OLYMPUS
BH2) for parasite counting. The activity (% of parasite reduction) was
compared with the untreated infected blood. Standard drug, GV,
produced 100% reduction.
Nonspecific Mammalian in Vitro Cytotoxicity: VERO Cell
Cytotoxicity Assay. The cytotoxicity against VERO cells (normal
African green monkey kidney epithelial cells) at a maximum
concentration of 2000 μM was determined for selected derivatives
actives against the amastigote form of T. cruzi. The compounds
dissolved in DMSO were evaluated at serial dilutions of the maximum
concentration in fresh culture milieu. The culture was incubated at 37
°C, 5% CO2 for 48 h. A negative control with DMSO (<0.1%) was
included in each experiment. The percentage inhibition of cell growth
was determined colorimetrically using Thiazole Blue (MTT; 3-[4,5-
dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) (Aldrich, St.
Louis MO) and reading of absorbance at 570 nm in a VersaMax Micro
microplate reader. Nfx was used as positive control.
Biology. Antitrypanosomatid in Vitro Evaluation. Anti-T. cruzi in
Vitro Test Using Epimastigotes of Tulahuen 2 Strain. Trypanosoma
cruzi epimastigotes (Tulahuen 2 strain) were grown at 28 °C in an
axenic milieu (BHI-Tryptose) as previously described,19 supplemented
with 5% fetal bovine serum (FBS). Cells from a 5−7 day old culture
(exponential phase) were inoculated into 50 mL of fresh culture milieu
to give an initial concentration of 1 × 106 cells/mL. Cell growth was
followed by measuring the absorbance of the culture at 600 nm every
day. At day 5, the exponential phase of the growth, the parasites were
inoculated with the indicated quantity of the drug from a stock
solution in DMSO. The final concentration of DMSO in the culture
milieu never exceeded 0.4%, and the control was run in the presence of
0.4% DMSO and in the absence of drugs. No effect on epimastigote
growth was observed due to the presence of up to 1% DMSO in the
culture milieu. Each compound concentration was evaluated in
duplicate. The percentage of inhibition (PGI) was calculated as
follows: PGI (%) = {1 − [(Ap − A0p)/(Ac − A0c)]} × 100, where Ap =
A600 of the culture containing the drug at day 5; A0p = A600 of the
culture containing the drug just after addition of the inocula (day 0);
Ac = A600 of the culture in the absence of drugs (control) at day 5; A0c
= A600 in the absence of the drug at day 0. In order to determine IC50
values, 50% inhibitory concentrations, parasite growth was followed in
the absence (control) and presence of increasing concentrations of the
corresponding drug. At day 5, the absorbance of the culture was
measured and related to the control. The IC50 value was taken as the
concentration of drug needed to reduce the absorbance ratio to 50%.
Anti-T. cruzi in Vitro Test Using Amastigotes of Sylvio X-10
Strain.30 VERO cells (ATCC), diluted to 3 × 105 cells/mL in RPMI
1640 milieu plus 10% hiFCS, were plated in 16-well Lab-tekTM tissue
culture chamber slides (Life Technologies, Paisley, UK) and allowed
to adhere for 24 h at 37 °C in a 5% CO2/95% air mixture. Adherent
cells were infected with tissue derived trypomastigotes (TDT) at a
ratio of parasites to cells of 10:1. The cultures were maintained in a 5%
CO2/95% air mixture at 37 °C. TDT were obtained by successive
serial infections in VERO cells with released parasites from culture
supernatants. After 24 h, free parasites were removed by washing, and
infected cultures were incubated for 72 h with 3-fold Nfx (0.3−87.0
μM) and Bnz (1.2−96.0 μM) and detest compounds (at five doses)
for 5 days at 37 °C, in a 5% CO2/95% air mixture. The cultures were
then fixed with methanol and stained with Giemsa. Each compound
concentration was evaluated in quadruplicate, and control cultures
were maintained without drug. The results are the means of three
independent experiments. Drug activity was determined by the
percentage of infected cells in treated and untreated cultures by direct
counting cells infected in a total of 300 cells using microscopy. Results
were expressed as the biological activity concentrations 50 and 90
(IC50 and IC90) by sigmoidal regression using XLfit4 software.
Anti-T. cruzi in Vitro Test Using Trypomastigotes of Y Strain.19b,25
BALB/c mice infected with T. cruzi were used 7 days after infection.
Blood was obtained by cardiac puncture using 3.8% sodium citrate as
anticoagulant in a 7:3 blood/anticoagulant ratio. The parasitemia in
infected mice ranged from 1 × 105 to 5 × 105 parasites/mL. The
products, 77 and Bnz, were dissolved in minimal quantity of DMSO
and added to PBS to give a final concentration of 25 μM. Aliquots (10
μL) of solution in triplicate were mixed in microtiter plates with 100
μL of infected blood containing parasites at a concentration near to
106 parasites/mL. Infected blood and infected blood containing
gentian violet (GV) at 250 μg/mL concentration were used as control.
The plates were shaken for 10 min at room temperature and kept at 4
Red Blood Cell Lysis Assay.31 Human blood collected in sodium
citrate solution (3.8%) was centrifuged at 200g for 10 min at 4 °C. The
plasma supernatant was removed and erythrocytes were suspended in
ice cold PBS. The cells were again centrifuged at 200g for 10 min at 4
°C. This procedure was repeated two more times to ensure the
removal of any released hemoglobin. Once the supernatant was
removed after the last wash, the cells were suspended in PBS to 2% w/
v red blood cell solution. A volume of 400 μL of compound to be
analyzed, in PBS (final doses 50, 100, and 200 μM), negative control
(solution of PBS), or AmpB (final dose 1.5 μM), was added to the 2%
w/v red blood cell solution. Ten replicates for each concentration were
done and incubated for 24 h at 37 °C prior to analysis. Complete
hemolysis was attained using neat water yielding the 100% control
value (positive control). After incubation, the tubes were centrifuged
and the supernatants were transferred to new tubes. The release of
hemoglobin into the supernatant was determined spectrophotometri-
cally at 405 nm using an EL 301 MICROWELL STRIP READER.
Results are expressed as percentage of total hemoglobin released in the
presence of the compounds. This percentage was calculated using the
equation percentage hemolysis (%) = [(A1 − A0)/(A1water)] × 100,
where A1 is the absorbance at 405 nm of the test sample at t = 24 h, A0
is the absorbance at 405 nm of the test sample at t = 0 h, and A1water is
the absorbance at 405 nm of the positive control (water) at t = 24 h.
The experiments were done by quintuplicate. AmpB was used as
positive control.
J774.1 Murine Macrophage Cell Cytotoxicity Assay. J774.1
murine macrophage cells (ATCC, USA) were grown in DMEM
culture milieu containing 4 mM L-glutamine and supplemented with
10% heat-inactivated fetal calf serum. The cells were seeded in a 96
well plate (5 × 104 cells in 200 mL culture milieu) and allowed to
attach for 48 h in a humidified 5% CO2/95% air atmosphere at 37 °C.
Afterward, cells were exposed for 48 h to the compounds (12.5−400.0
μM) or vehicle for control, and additional controls (cells in milieu)
were used in each test. Three replicates for each concentration were
done. Cell viability was then assessed by measuring the mitochondrial-
dependent reduction of MTT to formazan. For this propose, MTT in
sterile PBS (0.2% glucose) pH 7.4 was added to cells to a final
concentration of 0.1 mg/mL and cells were incubated at 37 °C for 3 h.
After removing the milieu, formazan crystals were dissolved in 180 mL
of DMSO and 20 mL MTT buffer (0.1 M glycine, 0.1 M NaCl, 0.5
mM EDTA, 10.5 pH), and the absorbance at 560 nm was read using a
microplate spectrophotometer. The IC50 is determined as the
concentration that reduces absorbance by 50% compared to control
treated with the solvent of the compounds and was determined by
linear regression analysis.
“Hit-to-Lead” Phase Studies. Mutagenicity Assay. The method of
direct incubation in plate was performed. Culture of S. typhimurium
TA 98, TA 100, TA 102, TA 1535, and TA 1537 strains in the agar
minimum glucose milieu-agar solution, Vogel Bonner E 50× and 40%
glucose solution, was used. First, the direct toxicity of the compounds
under study against the bacteria was assayed. DMSO solutions of
studied compounds at five consecutive dilutions in third (starting at
the highest doses without toxic effects) were assayed in triplicate.
3996
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
Positive controls of 4-nitro-o-phenylendiamine (20.0 μg/plate, in the
run without S9 activation) and 2-aminofluorene (10.0 μg/plate, in the
cases of S9 activation) for TA98, 4-nitro-o-phenylendiamine (20 μg/
plate, in the run without S9 activation) and 2-aminofluorene (10 μg/
plate, in the run without S9 activation) for TA100, or sodium azide (2
μg/plate, in the run without S9 activation) and 2-aminoantracene (2
μg/plate, in the run without S9 activation) for TA102, TA1535, and
TA1537 and negative control of DMSO were run in parallel. The
influence of metabolic activation was tested by adding 500 μL of S9
fraction of mouse liver treated with Aroclor, obtained from Moltox,
Inc. (Annapolis, MD, USA). The revertant number was counted
manually. The sample was considered mutagenic when the number of
revertant colonies was at least double that of the negative control for at
least two consecutive dose levels.
Formulation Studies. Vehicle 1: composed of saline (80%) and
Tween 80 (20%) (v/v). Formulation: compounds previously
pulverized in a mortar were added to the V1, and the mixture was
homogenized by a stirring system ultrasound chamber (ultasonic
Cleaner, Hwashin instruments, sonic Power 405) for 10 min at
maximum power; in some cases 10% DMSO was used. The resulting
suspension was stirred gently for 24 h in a horizontal shaker.
Vehicle 2: composed of surfactant (10%), comprising Eumulgin
HRE 40, sodium oleate, and soya phosphatidylcholine (8:6:3), oil
phase (10%), comprising cholesterol, and phosphate buffer (pH = 7.4)
(80%), according to Formariz et al.23 with little changes. To prepare
10 mL of V2, 1.0 g of surfactant (460 mg polyoxyl-40 hydrogenated
castor oil/Eumulgin HRE 40/360 mg of sodium oleate/180 mg of
soya phosphatidylcholine), 1.0 g of cholesterol, and sufficient volume
of phosphate buffer to 10 mL were used. Formulation: compounds,
previously pulverized in a mortar, cholesterol, phosphatidylcholine,
and polyoxyl-40 hydrogenated castor oil were dissolved in chloroform,
and the solvent was evaporated under vacuum to dryness. To ensure
complete removal of the chloroform, a stream of N2 was passed for 5
min. In parallel, sodium oleate was dissolved in phosphate buffer and
left for orbital shaking for 12 h at room temperature. This solution was
then evaporated, and the mixture was homogenized and immersed in
an ultrasonic bath at full power for 30 min; if it was not a
homogeneous solution or did not have the desired consistency, it was
reimmersed in an ultrasonic bath for another 30 min.
following day (6th day). Bnz or derivative 77 was administered orally,
using vehicle V1 or V2, at 50 or 100 mg/kg b.w./day, during 14 days.
Derivative 80 was administered orally, using vehicle V2, at 50 mg/kg
b.w./day according to the following administration-schedule: (i) 5
days of dosing; (ii) 2 days of resting; (iii) 5 days of dosing; (iv) 2 days
of resting; (v) and finally 4 days of dosing. Parasitaemia in the control
and treated mice was determined once a week after the first
administration, for 60 days, in tail-vein blood; the mortality rate was
recorded. All the sera obtained after centrifugation of the blood that
was extracted from infected mice were tested twice by ELISA (enzyme
linked immuno assay) at 30 and 60 days postinfection. A locally
produced ELISA kit (Chagas test, IICS, Asuncion
following the procedure recommended by the manufacturer (IICS
Production Department, Asuncion-Paraguay). The optical density
́
, Paraguay) was used
́
values were obtained in an ELISA plate reader (Titerek Unistan I).
Wilconxon test was used in order to compare the levels of anti-T. cruzi
antibodies between experimental groups. The experimental protocols
with animals were evaluated and supervised by the local Ethics
Committee, and the research adhered to the Principles of Laboratory
Animal Care.33
ASSOCIATED CONTENT
* Supporting Information
■
S
Tree of decision in the design-process (Figure S1), NOE-diff
experiments for both geometric isomers of derivative 22
(Figure S2), example of stability studies of acetyl-derivatives
(Figure S3), relationship between activities against epimasti-
gotes and amastigotes (Figure S4), cytotoxic behaviors against
mammal cells (Table S1), in vivo biological results for III and
IV derivatives (Figure S5, Table S2, and Figure S6), synthetic
procedures and spectroscopic data for all developed com-
pounds, crystal data, and structure refinement results for
derivative 77 (Table S3), corresponding fractional coordinates
and equivalent isotropic displacement parameters of the non-H
atoms (Table S4), bond lengths and angles (Table S5), atomic
anisotropic displacement parameters (Table S6), hydrogen
atoms positions (Table S7), and hydrogen bond distances and
angles (Table S8) This material is available free of charge via
the Internet at http://pubs.acs.org.
Stability Studies.32 For pH stability study, we utilized the following
aqueous solutions: (i) KCl·HCl buffer, pH 1.0; (ii) citrate buffer, pH
6.0; (iii) Tris·HCl buffer, pH 7.4, and (iv) borax·HCl buffer, pH 9.0.
The stock solutions of compounds were prepared in DMSO, and the
final concentration in the aqueous milieu was 1 mM. The solutions
were further homogeneizated and incubated at 37 °C for 1 h. After
that, thin layer chromatographies were done with the ethyl acetate
extracts in order to confirm or discard the presence of decomposition
products.
Accession Codes
Crystallographic structural data for derivative 77 have been
deposited at the Cambridge Crystallographic Data Centre
(CCDC). Any request to the Cambridge Crystallographic Data
Centre for this material should quote the full literature citation
and the reference number CCDC 965369.
For the determination of liver fraction stability, we used rat liver
microsomal and cytosolic proteins prepared following previously
described methodology.24 Briefly, homogenated livers were centri-
fuged for 30 min at 9000g at 4 °C; the pellet was discarded, and the
supernatant was further centrifuged at 100 000g for 1 h at 4 °C. The
pellet was discarded, and the supernatant fraction was further
centrifuged at 100 000g for 1 h at 4 °C. The cytosolic and microsomal
fractions, supernatant and pellet, respectively, were recovered. The
pellet was washed twice by resuspension in the above Tris·HCl buffer
solution, resedimented by centrifugation for 1 h at 100 000g at 4 °C,
and finally resuspended in the Tris·HCl buffer solution. Metabolic
assays were carried out with microsomes and cytosol either fresh or
frozen in Tris·HCl buffer and stored at −80 °C. The protein content
of the microsomal and cytosolic fractions was determined by the
bicinchoninic acid assay from Sigma, as suggested by the manufacturer.
In Vivo Anti-T. cruzi Activity (Acute Model). BALB/c male mice
(30 days old, 25−30 g) bred under specific pathogen-free conditions
were infected by intraperitoneal injection of 103 blood trypomastigotes
Y. One group of animals was used as control (inoculated orally with
the vehicle V1 or V2), and two groups of animals were treated with
each studied derivative and Bnz, respectively. First parasitemia was
carried out 5 days postinfection (week 1), and the treatment began the
AUTHOR INFORMATION
Corresponding Authors
*(M.G.) Phone: 598 25258618 (ext. 216). Fax: 598 25250749.
E-mail: megonzal@fq.edu.uy.
*(H.C.) Phone: 598 25250800. E-mail: hcerecetto@cin.edu.uy.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
■
The authors would like to thank the Comision
́
Sectorial de
́
Investigacion
́
Cientifica (CSIC)-UdelaR (CSIC-N° 661) of
Uruguay and the CONICET (PIP1529) and the ANPCyT
(PME06 2804 and PICT06 2315) of Argentina. Collaborative
work was performed under the auspices of the Iberoamerican
Program for Science and Technology (CYTED), network
́
RIDIMEDCHAG, Viceministerio de Ciencia y Tecnologia (El
Salvador), and Agencia Uruguaya de Cooperacion Internacional
(Uruguay). G.A., J.V., and Ma.Ga. thank ANII (Uruguay) for
́
3997
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
Wilkinson, S. R. Activation of Benznidazole by Trypanosomal Type I
Nitroreductases Results in Glyoxal Formation. Antimicrob. Agents
Chemother. 2012, 56, 115−123.
their scholarships. G.A.E. and O.E.P. are Research Fellows of
CONICET. M.C. thanks the Conselho Nacional de Desenvol-
́
vimento Cientifico e Tecnolog
́
ico (CNPq, Brazil), Fundaca̧ o de
̃
(8) (a) Gerpe, A.; Odreman-Nunez, I.; Draper, P.; Boiani, L.; Urbina,
̃
Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), and
̀
̃
́
J. A.; Gonzalez, M.; Cerecetto, H. Heteroallyl-containing 5-Nitro-
́
Programa de Apoio ao Desenvolvimento Cientifico (PADC-
furanes as New Anti-Trypanosoma cruzi Agents with a Dual
Mechanism of Action. Bioorg. Med. Chem. 2008, 16, 569−577.
FCF-UNESP) for a fellowship.
́
(b) Gerpe, A.; Alvarez, G.; Benítez, D.; Boiani, L.; Quiroga, M.;
ABBREVIATIONS USED
■
́ ́
Hernandez, P.; Sortino, M.; Zacchino, S.; Gonzalez, M.; Cerecetto, H.
Nfx, nifurtimox; Bnz, benznidazole; OECD, Organization for
Economic Cooperation and Development; GV, gentian violet;
V1, saline/Tween 80 (4:1, v:v); V2, microemulsion composed
of cholesterol (10%) as oil phase, the surfactant (soya
phosphatidylcholine/sodium oleate/polyoxyl-40 hydrogenated
castor oil (3:6:8, 10%)), and phosphate buffer pH 7.4 (80%) as
aqueous phase vehicle; RDB, relative doses to benznidazole;
FBS, fetal bovine serum; BHI, brain and heart infusion; PGI,
percentage of inhibition; TDT, tissue derived trypomastigotes;
AmpB, amphotericin B
5-Nitrofuranes and 5-Nitrothiophenes with Anti-Trypanosoma cruzi
Activity and Ability to Accumulate Squalene. Bioorg. Med. Chem. 2009,
17, 7500−7509.
́
(9) Aravena, M. A.; Olea, C.; Cerecetto, H.; Gonzalez, H.; Maya, J.
D.; Rodríguez-Becerra, J. Potent 5-Nitrofuran Derivatives Inhibitors of
Trypanosoma cruzi Growth: Electrochemical, Spectroscopic and
Biological Studies. Spectrochim. Acta, Part A 2011, 79, 312−319.
(10) Arbillaga, L.; San Miguel, L.; Lop
L. A.; Gil, A. G.; Cerecetto, H.; Gonzal
Evaluation of the Genotoxicity of Nine Compounds with Significant in
́
ez, M.; Dav
́
ila, M. J.; Corcuera,
ez de Cerain, A.
́
ez, M.; Lop
́
vitro Antichagasic Activity. Toxicol. Lett. 2010, 196, S169.
(11) Test No. 471: Bacterial Reverse Mutation Test. OECD Guidelines
for the Testing of Chemicals. Section 4. The Organisation for Economic
Co-operation and Development, 21-July 1997. http://www.oecd-
ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-
section-4-health-effects_20745788 (accessed March 19, 2014).
REFERENCES
■
(1) Dias, J. C.; Silveira, A. C.; Schofield, C. J. The Impact of Chagas
Disease Control in Latin America: A Review. Mem. Inst. Oswaldo Cruz
2002, 97, 603−612.
(2) Schofield, C. J.; Jannin, J.; Salvatella, R. The Future of Chagas
Disease Control. Trends Parasitol. 2006, 22, 583−588.
(3) World Health Organization/TDR. Report of the Scientific Working
Group on Chagas Disease, Buenos Aires 2005; WHO: Geneva, Italy,
2006.
(4) (a) Magalhaes Moreira, D. R.; Manso Costa, S. P.; Zaldini
Hernandes, M.; Montenegro Rabello, M.; Bezerra de Oliveira Filho,
G.; Moutinho Lagos de Melo, C.; Ferreira da Rocha, L.; de Simone, C.
A.; Salgado Ferreira, R.; Rodrigues Barbosa Fradico, J.; Santana Meira,
́
(12) (a) Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Risso, M.;
Sagrera, G.; Seoane, G.; Denicola, A.; Peluffo, G.; Quijano, C.;
Stoppani, A. O. M.; Paulino, M.; Olea-Azar, C.; Basombrío, M. A.
Synthesis and Antitrypanosomal Evaluation of E-Isomers of 5-Nitro-2-
furaldehyde and 5-Nitrothiophene-2-carboxaldehyde Semicarbazone
Derivatives. Structure-activity Relationships. Eur. J. Med. Chem. 2000,
́
35, 343−350. (b) Aguirre, G.; Cerecetto, H.; Gonzalez, M.; Gambino,
D.; Otero, L.; Olea-Azar, C.; Rigol, C.; Denicola, A. In vitro Activity
and Mechanism of Action Against the Protozoan Parasite
Trypanosoma cruzi of 5-Nitrofuryl Containing Thiosemicarbazones.
Bioorg. Med. Chem. 2004, 12, 4885−4893. (c) Aguirre, G.; Cabrera, E.;
̂
C.; Teixeira Guimaraes, E.; Mohan Srivastava, R.; Rego Alves Pereira,
̃
V.; Botelho Pereira Soares, M.; Lima Leite, A. C. Structural
Investigation of Anti-Trypanosoma cruzi 2-Iminothiazolidin-4-ones
Allows the Identification of Agents with Efficacy in Infected Mice. J.
Med. Chem. 2012, 55, 10918−10936. (b) Gunatilleke, S. S.; Calvet, C.
M.; Johnston, J. B.; Chen, C. K.; Erenburg, G.; Gut, J.; Engel, J. C.;
Ang, K. K.; Mulvaney, J.; Chen, S.; Arkin, M. R.; McKerrow, J. H.;
Podust, L. M. Diverse Inhibitor Chemotypes Targeting Trypanosoma
cruzi CYP51. PLoS Negl. Trop. Dis. 2012, 6, e1736. (c) Recher, M.;
Barboza, A. P.; Li, Z. H.; Galizzi, M.; Ferrer-Casal, M.; Szajnman, S.
H.; Docampo, R.; Moreno, S. N.; Rodriguez, J. B. Design, Synthesis
and Biological Evaluation of Sulfur-Containing 1,1-Bisphosphonic
Acids as Antiparasitic Agents. Eur. J. Med. Chem. 2013, 60, 431−440.
(d) Choi, J. Y.; Calvet, C. M.; Gunatilleke, S. S.; Ruiz, C.; Cameron, M.
D.; McKerrow, J. H.; Podust, L. M.; Roush, W. R. Rational
Development of 4-Aminopyridyl-Based Inhibitors Targeting Trypano-
soma cruzi CYP51 as Anti-Chagas Agents. J. Med. Chem. 2013, 56,
7651−7668.
́
Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Seoane, G.; Duffaut, A.;
Denicola, A.; Gil, M. J.; Martínez-Merino, V. Design, Synthesis and
Biological Evaluation of New Potent 5-Nitrofuryl Derivatives as Anti-
Trypanosoma cruzi Agents. Studies of Trypanothione Binding Site of
Trypanothione Reductase as Target for Rational Design. Eur. J. Med.
Chem. 2004, 39, 421−431. (d) Aguirre, G.; Boiani, M.; Cabrera, E.;
́
Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Denicola, A.; Sant’Anna, C.
M. R.; Barreiro, E. J. New Potent 5-Nitrofuryl Derivatives as Inhibitors
of Trypanosoma cruzi Growth. 3D-QSAR (CoMFA) Studies. Eur. J.
Med. Chem. 2006, 41, 457−466.
(13) (a) Keskin, H.; Miller, R.; Nord, F. F. Studies of the Chemistry
of Heterocyclics. XII.′ Preparation of Acetylenic Derivatives of
Thiophene. J. Org. Chem. 1951, 16, 199−206. (b) Carrara, G.;
Ettorre, R.; Fava, F.; Rolland, G.; Testa, E.; Vecchi, A. 4-Nitrocinnamic
and β-(5-Nitro-2-thienyl)-acrylic Derivatives. J. Am. Chem. Soc. 1954,
76, 4391−4395.
(14) Omar, A.-M. M. E.; Chaaban, I.; AboulWafa, O. M.; Hassan, A.
M.; Abou-Shleib, H.; Ismail, K. A. Novel Thiosemicarbazones,
Thiazolines and Thiazolidines Derived from Chalcones: Synthesis,
Antimicrobial and Anticancer Properties. Alexandria J. Pharm. Sci.
1989, 3, 211−216.
́
(5) (a) Cerecetto, H.; Gonzalez, M. Synthetic Medicinal Chemistry
in Chagas’ Disease: Compounds at The Final Stage of “Hit-To-Lead”
Phase. Pharmaceuticals 2010, 3, 810−838. (b) Guedes, P. M.; Silva, G.
K.; Gutierrez, F. R.; Silva, J. S. Current Status of Chagas Disease
Chemotherapy. Expert Rev. Anti. Infect. Ther. 2011, 9, 609−620.
́
(c) Gonzalez, M.; Cerecetto, H. Novel Compounds to Combat
(15) (a) Medne, A. Y.; Saldabols, N. O. Synthesis and Trans-
formations of Furan Derivatives. V. Synthesis of 4-Methylthiazolyl-(2)-
hydrazones of Aldehydes and Ketones of the Furan Series. Khim.
Geterotsikl. Soedin. 1965, 1, 629−631. (b) Bekhit, A. A.; Ashour, H. M.;
Abdel Ghany, Y. S.; Bekhit, A.-D.; Baraka, A. Synthesis and Biological
Evaluation of Some Thiazolyl and Thiadiazolyl Derivatives of 1H-
Pyrazole as Anti-inflammatory Antimicrobial Agents. Eur. J. Med.
Chem. 2008, 43, 456−463.
Trypanosomatid Infections: A Medicinal Chemical Perspective. Expert
Opin. Ther. Pat. 2011, 21, 699−715.
(6) Cabrera, M.; Lavaggi, M. L.; Hernan
́
dez, P.; Merlino, A.; Gerpe,
ez de Cerain,
ez, M.; Cerecetto, H. Cytotoxic, Mutagenic and Genotoxic
A.; Porcal, W.; Boiani, M.; Ferreira, A.; Monge, A.; Lop
A.; Gonzal
́
́
Effects of New anti-T. cruzi 5-Phenylethenylbenzofuroxans. Contribu-
tion of Phase I Metabolites on the Mutagenicity Induction. Toxicol.
Lett. 2009, 190, 140−149.
(7) (a) Hall, B. S.; Bot, C.; Wilkinson, S. R. Nifurtimox Activation by
Trypanosomal Type I Nitroreductases Generates Cytotoxic Nitrile
Metabolites. J. Biol. Chem. 2011, 286, 13088−13095. (b) Hall, B. S.;
(16) Gerpe, A.; Boiani, L.; Hernan
Gonzalez, M.; Cerecetto, H. Naftifine-analogues as Anti-Trypanosoma
cruzi Agents. Eur. J. Med. Chem. 2010, 45, 2154−2164.
́
dez, P.; Sortino, M.; Zacchino, S.;
́
3998
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999

Journal of Medicinal Chemistry
Article
(17) (a) Faucher, J. F.; Baltz, T.; Petry, K. G. Detection of an
“epimastigote-like” Intracellular Stage of Trypanosoma cruzi. Parasitol.
Res. 1995, 81, 441−443. (b) Almeida-de-Faria, M.; Freymuller, E.;
Colli, W.; Alves, M. J. Trypanosoma cruzi: Characterization of an
Intracellular epimastigote-like Form. Exp. Parasitol. 1999, 92, 263−
274.
(27) Sheldrick, G. M. SHELXS-97. Program for Crystal Structure
Resolution; Univ. of Gottingen: Gottingen, Germany, 1997. See also:
̈
̈
Sheldrick, G. M. Acta Crystallogr. 1990, A46, 467−473.
(28) Sheldrick, G. M. SHELXL-97. Program for Crystal Structures
Analysis; Univ. of Gottingen: Gottingen, Germany, 1997. See also:
̈
̈
Sheldrick, G. M. Acta Crystallogr. 2008, A 64, 112−122.
(29) Johnson, C. K. ORTEP-II. A Fortran Thermal-Ellipsoid Plot
Program, Report ORNL-5318; Oak Ridge National Laboratory: Oak
Ridge, TN, 1976.
(18) (a) Filardi, L. S.; Brener, Z. Susceptibility and Natural
Resistance of Trypanosoma cruzi Strains to Drugs used Clinically in
Chagas Disease. Trans. R. Soc. Trop. Med. Hyg. 1987, 81, 755−759.
(b) Molina, J.; Brener, Z.; Romanha, A. J.; Urbina, J. A. In vivo Activity
of the Bis-triazole D0870 Against Drug-susceptible and Drug-resistant
Strains of the Protozoan Parasite Trypanosoma cruzi. J. Antimicrob.
Chem. 2000, 46, 137−140.
́
(30) Luna, K. P.; Hernandez, I. P.; Rueda, C. M.; Zorro, M. M.;
Croft, S. L.; Escobar, P. In vitro Susceptibility of Trypanosoma cruzi
Strains from Santander, Colombia, to Hexadecylphosphocholine
(Miltefosine), Nifurtimox and Benznidazole. Biomedica 2009, 29,
448−455.
(19) (a) Porcal, W.; Hernan
A.; Chidichimo, A.; Cazzulo, J. J.; Olea-Azar, C.; Gonzal
́
dez, P.; Boiani, L.; Boiani, M.; Ferreira,
ez, M.;
(31) (a) Fernandez, L.; Calderon
́
, M.; Martinelli, M.; Strumia, M.;
́
Cerecetto, H.; Gonzalez, M.; Silber, J. J.; Santo, M. Evaluation of a
́
Cerecetto, H. New Trypanocidal Hybrid Compounds from the
Association of Hydrazone Moieties and Benzofuroxan Heterocycle.
Bioorg. Med. Chem. 2008, 16, 6995−7004. (b) Benitez, D.; Cabrera,
New Dendrimeric Structure as Prospective Drugs Carrier for
Intravenous Administration of Antichagasic Active Compounds. J.
Phys. Org. Chem. 2008, 21, 1079−1085. (b) Merlino, A.; Benitez, D.;
M.; Hernan
Serna, E.; Torres, S.; Ferreira, M. E.; Vera de Bilbao, N.; Torres, E.;
Perez-Silanes, S.; Solano, B.; Moreno, E.; Aldana, I.; Lopez de Cerain,
A.; Cerecetto, H.; Gonzalez, M.; Monge, A. 3-Trifluoromethylquinoxa-
́
dez, P.; Boiani, L.; Lavaggi, M. L.; Di Maio, R.; Yaluff, G.;
Chavez, S.; Da Cunha, J.; Hernan
́
dez, P.; Tinoco, L. W.; Campillo, N.
ez, M. Development of Second
E.; Paez, J. A.; Cerecetto, H.; Gonzal
́
́
́
́
́
Generation Amidino-hydrazones, Thio- and Semicarbazones as
Trypanosoma cruzi-inhibitors Bearing Benzofuroxan and Benzimida-
zole 1,3-Dioxide Core Scaffolds. Med. Chem. Commun. 2010, 1, 216−
́
line N,N′-Dioxides as Anti-trypanosomatid Agents. Identification of
Optimal anti-T. cruzi Agents and Mechanism of Action Studies. J. Med.
228. (c) Merlino, A.; Benitez, D.; Campillo, N. E.; Pae
́
z, J. A.; Tinoco,
Chem. 2011, 54, 3624−3636. (c) Hernan
́
dez, P.; Rojas, R.; Gilman, R.
ez, M.; Cerecetto,
L. W.; Gonzalez, M.; Cerecetto, H. Amidines Bearing Benzofuroxan or
́
H.; Sauvain, M.; Lima, L. M.; Barreiro, E. J.; Gonzal
́
Benzimidazole 1,3-Dioxide Core Scaffolds as Trypanosoma cruzi-
Inhibitors: Structural Basis for their Interactions with Cruzipain. Med.
Chem. Commun. 2012, 3, 90−101.
H. Hybrid Furoxanyl N-Acylhydrazone Derivatives as Hits for the
Development of Neglected Diseases Drug Candidates. Eur. J. Med.
Chem. 2013, 59, 64−74.
(32) Kerns, H. E.; Li, D. Drug Like Properties: Concepts, Structure,
Design and Methods. From ADME to Toxicity Optimization; Academic
Press: Oxford, U.K., 2008; pp 353 − 358.
(33) Morton, D. B.; Griffiths, P. H. M. Guidelines on the Recognition
of Pain, Distress and Discomfort in Experimental Animal and a
Hypothesis for Assessment. Vet. Rec. 1985, 116, 431−436.
(20) Maron, D. M.; Ames, B. N. Revised Methods for the Salmonella
Mutagenicity Test. Mutat. Res. 1983, 113, 173−215.
(21) (a) Chu, K. C.; Patel, K. M.; Lin, A. H.; Tarone, R. E.; Linhart,
M. S.; Dunkel, V. C. Evaluating Statistical Analyses and Reproduci-
bility of Microbial Mutagenicity Assays. Mutat. Res. 1981, 85, 119−
132. (b) Claxton, L. D.; Allen, J.; Auletta, A.; Mortelmans, K.;
Nestmann, E.; Zeiger, E. Guide for the Salmonella typhimurium/
Mammalian Microsome Tests for Bacterial Mutagenicity. Mutat. Res.
1987, 189, 83−91.
(22) Sintov, A. C.; Shapiro, L. New Microemulsion Vehicle
Facilitates Percutaneous Penetration in vitro and Cutaneous Drug
Bioavailability in vivo. J. Controlled Release 2004, 95, 173−183.
(23) (a) Formariz, T. P.; Chiavacci, L. A.; Scarpa, M. V.; Silva-Junior,
́
A. A.; Egito, E. S.; Terrugi, C. H.; Franzini, C. M.; Sarmento, V. H.;
Oliveira, A. G. Structure and Viscoelastic Behavior of Pharmaceutical
Biocompatible Anionic Microemulsions Containing the Antitumoral
Drug Compound Doxorubicin. Colloids Surf., B 2010, 77, 47−53.
(b) Hu, L.; Yang, J.; Liu, W.; Li, L. Preparation and Evaluation of
Ibuprofen-loaded Microemulsion for Improvement of Oral Bioavail-
ability. Drug Delivery 2011, 18, 90−95. (c) Hu, L.; Jia, Y.; Niu, F.; Jia,
Z.; Yang, X.; Jiao, K. Preparation and Enhancement of Oral
Bioavailability of Curcumin using Microemulsions Vehicle. J. Agric.
Food Chem. 2012, 60, 7137−7141. (d) Bshara, H.; Osman, R.;
Mansour, S.; El-Shamy, A. H. Chitosan and Cyclodextrin in Intranasal
Microemulsion for Improved Brain Buspirone Hydrochloride
Pharmacokinetics in Rats. Carbohydr. Polym. 2014, 99, 297−305.
(24) Boiani, M.; Merlino, A.; Gerpe, A.; Porcal, W.; Croce, F.;
́
Depaula, S.; Rodríguez, M. A.; Cerecetto, H.; Gonzalez, M. o-
Nitroanilines as Major Metabolic Products of anti-Trypanosoma cruzi
5-Phenylethenylbenzofuroxans in Microsomal and Cytosolic Fractions
of Rat Hepatocytes and in Whole Parasitic Cells. Xenobiotica 2009, 39,
236−248.
(25) Boiani, M.; Boiani, L.; Denicola, A.; Torres de Ortiz, S.; Serna,
E.; Vera de Bilbao, N.; Sanabria, L.; Yaluff, G.; Nakayama, H.; Rojas de
Arias, A.; Vega, C.; Rolan, M.; Gom
́
ez-Barrio, A.; Cerecetto, H.;
Gonzalez, M. 2H-Benzimidazole 1,3-Dioxide Derivatives: A New
́
Family of Water-Soluble Anti-Trypanosomatid Agents. J. Med. Chem.
2006, 49, 3215−3224.
(26) CrysAlisPro, version 1.171.36.20; Oxford Diffraction Ltd.:
Abingdon, U.K., 2012.
3999
dx.doi.org/10.1021/jm500018m | J. Med. Chem. 2014, 57, 3984−3999