Design, synthesis and biological evaluation of new aryl thiosemicarbazone as antichagasic candidates

Article abstract of DOI:10.1016/j.ejmech.2013.04.022

The present work reports on the synthesis, biological assaying and docking studies of a series of 12 aryl thiosemicarbazones, which were planned to act over two main enzymes, cruzain and trypanothione reductase. These enzymes are used as targets of trypanocidal activity in Chagas disease control with a minimal mutagenic profile. Three p-nitroaromatic thiosemicarbazones showed high activity against Trypanosoma cruzi in in vitro assays (IC₅₀ < 57 μM), and no mutagenic profile was observed in micronucleous tests. Although the in vitro inhibition test showed that 10-μM doses of eight compounds inhibited cruzain activity, no correlation was found between cruzain inhibition and trypanocidal activity.

Full text of DOI:10.1016/j.ejmech.2013.04.022

Accepted Manuscript
Design, synthesis and biological evaluation of new aryl thiosemicarbazone as
antichagasic candidates
Lorena Blau, Renato Farina Menegon, Gustavo H.G. Trossini, Joã o Vitor Dutra
Molino, Drielli Gomes Vital, Regina Maria Barretto Cicarelli, Gabriela Duó Passerini,
Priscila Longhin Bosquesi, Chung Man Chin
PII:
S0223-5234(13)00254-7
10.1016/j.ejmech.2013.04.022
EJMECH 6132
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 January 2013
Revised Date: 29 March 2013
Accepted Date: 8 April 2013
Please cite this article as: L. Blau, R.F. Menegon, G.H.G. Trossini, J.V. Dutra Molino, D.G. Vital, R.M.
Barretto Cicarelli, G.D. Passerini, P.L. Bosquesi, C.M. Chin, Design, synthesis and biological evaluation
of new aryl thiosemicarbazone as antichagasic candidates, European Journal of Medicinal Chemistry
(2013), doi: 10.1016/j.ejmech.2013.04.022.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of new aryl
thiosemicarbazone as antichagasic candidates
Highlights
1. Twelve thiosemicarbazones were synthesized and assayed against Trypanosoma cruzi
2. They were planned as potential inhibitors of cruzain and trypanothione reductase
3. The "in vivo" micronucleus test indicated no genotoxic effect of the compounds
4. Cruzain inhibition was assessed through docking study and biochemical assay

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of new aryl thiosemicarbazone as
antichagasic candidates
Lorena Blau1, Renato Farina Menegon2, Gustavo H. G. Trossini3, João Vitor Dutra
Molino3,Drielli Gomes Vital3, Regina Maria Barretto Cicarelli4, Gabriela Duó
Passerini4, Priscila Longhin Bosquesi1,Chung ManChin1
1.Departamento de Fármacos e Medicamentos, Faculdade de Ciências Farmacêuticas,
Universidade Estadual Paulista, Rodovia Araraquara-Jaú Km 01, 14801-902,
Araraquara, SP, Brazil
2. Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraíba, Avenida
Shishima Hifume 2911, 12244-000, São José dos Campos, SP, Brazil
3. Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de
São Paulo, Professor Lineu Prestes 580, 05508-900, São Paulo, SP, Brazil.
4.Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas,
Universidade Estadual Paulista, Rodovia Araraquara-Jaú Km 01, 14801-902,
Araraquara, SP, Brazil
Abstract
The present work reports on the synthesis, biological assaying and docking studies of a
series of 12 aryl thiosemicarbazones, which were planned to act over two main
enzymes, cruzain and trypanothione reductase. These enzymes are used as targets of
trypanocidal activity in Chagas disease control with a minimal mutagenic profile. Three
p-nitroaromatic thiosemicarbazones showed high activity against T. cruzi in in vitro
assays (IC50<57 µM), and no mutagenic profile was observed in micronucleous tests.
Although the in vitro inhibition test showed that 10-µM doses of eight compounds
inhibited cruzain activity, no correlation was found between cruzain inhibition and
trypanocidal activity.
Keywords: Chagas disease; thiosemicarbazone; oxidative stress; cruzain; docking;
mutagenicity

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1. Introduction
Chagas disease or American trypanosomiasis is still one of the major endemic
diseases in Latin America [1]. The 100-year anniversary of the discovery of Chagas
disease by Brazilian sanitarian physician Dr. Carlos J. R. Chagas was celebrated in
2009. Despite the historical landmark that this discovery represents for Brazilian
Science, Chagas disease completed its first century without any effective treatment,
especially against its chronic form [2]. Currently, an estimated 8-10 million people are
infected with trypanosomes worldwide, and around 100 million people are at risk. The
World Health Organization estimates that Chagas disease is responsible for the death of
over 10,000 people per year [3,4]. The numbers of deaths and new cases attributed to
Chagas disease in 2000 were 21,000 and 200,000, respectively [5].
Over the years, numerous chemical classes against T. cruzi have become
available; however, there is still no effective treatment for all phases of the disease [6].
Recent research pointed to the biosynthesis pathway of ergosterol as a biochemistry
target [7,8]. Azole derivatives, i.e. posaconazole and ravuconazole, have been tested and
presented trypanocidal activity, but they are not available for therapeutics yet [6, 9-12].
Despite the efforts of many investigators in the research of a new antichagasic, only two
drugs are currently used to treat Chagas disease, nifurtimox and benznidazole (Figure
1) [6]. Both drugs cause severe side-effects, which include peripheral sensitive
polyneuropathy, agranulocytosis and thrombocytopenic purpura, leading to treatment
interruption in some cases [12, 13].
Both nifurtimox and benznidazole are nitroheterocyclic compounds. The
reduction of this group is classically related to mutagenicity and carcinogenicity;
however, the nitro group plays an essential role in the biological activity of many
antimicrobials and antiprotozoan drugs [14-16], acting as a redox cycling agent and
leading to oxidative stress in parasite cells [17-19]. Nevertheless, it is incorrect to state
that all nitrocompounds exhibit mutagenic and carcinogenic properties. Lopes de
Compadre et al. [20, 21] suggest that these properties are related to some compounds'
specific characteristics, such as the potential of nitro reduction to hydroxylamines and
molecule hydrophobicity.

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A number of organic groups act over other relevant enzymes, for example,
cruzain, a cysteine protease member of the papain-like family. It is involved in immune
evasion and parasite penetration into host cells [22-27]. Du et al. [28] synthesized over
100 thiosemicarbazone derivatives without a nitro group. The structures of the
compounds exhibited a specific and consistent relationship with their activities.
Greenbaum et al. [29], Fujii et al.[30] and Siles et al. [31] further investigated the
aromatic ring substitution profile in aryl thiosemicarbazones. Neither
thiosemicarbazones with fused aromatic rings nor benzophenone thiosemicarbazones
have a nitro group and both are potent cruzain inhibitors. In 2010, Trossini and co-
workers investigated the activity of semicarbazone derivatives against cruzain using
molecular modeling and cruzain inhibition assays. They found that the carbonyl group
and the catalytic amino acid Cys 25 of the enzyme interacted [32]. Semicarbazone and
thiosemicarbazone are excellent prototypes for the design and development of novel
antichagasic agents.
In this work, we synthesized 12 thiosemicarbazones, with and without an
aromatic nitro group, and evaluated the influence of the presence and the position of the
nitro group on the trypanocidal activity against T.cruzi. A docking study and cruzain
inhibition assays were conducted in an attempt to understand the mechanism of action
of the compounds on the enzyme target. An amino group was intentionally introduced
to allow further amide and carbamate derivation of the compounds and the easy
introduction of a parasite-targeting group. The mutagenic profile was obtained to ensure
the safety of the nitrocompounds.
2. Results and Discussion
2.1. Chemistry
Twelve thiosemicarbazone derivatives were synthesized by classical
condensation of thiosemicarbazide to a specific acetophenone. This approach led to the
formation of the desired acetophenone thiosemicarbazone with mean yields from 60 to
80%. Scheme 1 depicts the simplest derivatives obtained.
Aromatic nitration was carried out from -5 to 0 ºC employing a mixture of
concentrated sulfuric and nitric acids. Proper temperature control was necessary for

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obtaining acceptable yields of the desired isomer (schemes 2 and 3). Prior to the
nitration of aromatic aminoacetophenones, the amino group was protected by
acetylation with acetic anhydride. This protection was necessary to prevent diazonium
salt formation during the nitration step (scheme 3).
Aliphatic-substituted acetophenone bromination of the ethyl moiety of
acetophenone yielded α-brome-cetophenone, which was treated either with
hexamethylenetetramine in acidic conditions, rendering α-aminoacetophenone, or with
silver nitrate in water/acetone mixture, rendering α-hydroxyacetophenone.
Hemisuccinate derivatives were prepared by treatment with succinic anhydride (scheme
4). Specific features of these reactions are described in section 5 (experimental section).
2.2. Anti-trypanosomal activity in vitro test
All synthesized compounds were in vitro tested against epimastigote forms of T.
cruzi (table 1). Albeit epimastigote is not an infective form of the parasite, the anti-
trypanosoma in vitro test against this parasite form is valid as a screening of potential
trypanocidal compounds. Several works demonstrated that the most active compounds
against epimastigote form frequently correspond to the most active compounds against
trypomastigote form too, despite the great variance of IC50 values between them [33-
35].
The presence of a nitro group may be an indication that these compounds may
act on the trypanothione reductase enzyme and produce a nitro radical, which seems to
be important for its inhibition and disruption of the intracellular protection against the
oxidative stress promoted by free radicals delivered by the host cells [36].
It is notorious that the 3 most active compounds (9, 10 and 11) present a nitro
group at para position on the aromatic ring. Compound 9 (IC50=6.3 µM; para-nitro
compound) was about five times more potent than the standard benznidazole (IC50=34.6
µM). Other structural features may be explored by comparing IC50 values presented in
Table 1. The relative position of the nitro group on the aromatic ring, as well as its
presence or absence, produces great changes in biological activity, as observed by the
analysis of pairs 7 (inactive) and 8 (IC50=60.60 µg/mL), 6 (3% of inhibition at 50

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µg/mL) and 9 (IC50=2.13 µg/mL), and 5 (3% of inhibition at 5 µg/mL) and 4
(IC50=37.68 µg/mL). Here, the meta position results in less active compounds.
The amine group does not seem to be a good substituent regarding trypanocidal
activity. The fact that compound 4 is more active than 1, 2 and 3, exemplifies the
negative effect of its presence on the molecule. However, this effect can be overcome
by introducing a nitro group at para position, as shown by the comparison of the
relative activities of compounds 9 (para), 6 (meta) and 3 (without a nitro group).
The derivatization of compounds 9 and 10 led to less active compounds, which
was more pronounced in 10 than in 9. In fact, the potency of 12 is over four-fold smaller
than the potency of 10, while the activity of 9 is about two-fold that of 11.
2.3. Mutagenicity assay – micronucleus test
Once the three most active compounds had been selected, we started the
genotoxicity study through a micronucleus test. The observed micronucleus frequency
of each compound is shown in Table 2. All data are presented as arithmetic means.
One-Way ANOVA/Tukey test was used to compare frequencies at P < 0.05
significance.
As a parameter of acceptable genotoxicity, a dose of 100 mg/kg of benznidazole
to body weight (kg bwt) was employed, as it is currently the only drug available for
Chagas disease treatment. All tested compounds were less mutagenic than benznidazole
at the same dose. No statistical difference was observed between the three tested
compounds, all of which were less mutagenic then the positive control
(cyclophosphamide), even in the highest dose (250 mg/ kg bwt).
2.4. Docking studies
To discuss the possible action of the tested compound on cruzain, we studied the
interaction between these new aryl thiosemicarbazones (ligands) and cruzain (receptor)
by computational docking and evaluated the best compound poses for the nucleophilic

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attack by cruzain. These studies were based on the proposed mechanism of action of
this class of compounds [28,32].
The docking study results suggested a good interaction between the ligands and
the cruzain active site. Figure 2 shows the best docking poses of compounds 7 and 8,
which presented 55 and 65% inhibition of cruzain, respectively, at a dose of 10 µM
dose. All best docking poses ranked showed aromatic groups in the S2 subpocket of the
cruzain active site. This result corroborates with literature data near the S2 region, with
a lipophilic character for this region in the recognition of aromatic groups [37]. The
distance between para-substituents in aromatic groups and the Glu205 residue, 3.2 Å in
compound 7 and 2.5 Å in compound 8, suggests the existence of a hydrogen bond
favorable to the interaction in the S2 subpocket (Figure 2).
Some other interesting information provided by the docking studies is the pose
of the lateral chain of the aryl thiosemicarbazone derivatives located in S1 subpocket. In
this position, the thiocarbonyl carbon is near the cruzain catalytic residue Cys25. This
pose corroborates with the mechanism of action proposed for this class of compounds
by nucleophilic attack of Cys25 in the carbonyl carbon of thiosemicarbazone or
semicarbazone compounds [28, 32].
Additionally, the best docking pose of the hemisuccinate derivatives, such as
compound 11, 2-amino-1-p-nitrophenylethan-1-one thiosemicarbazone hemisuccinate
amide (figure 3A), showed a hydrogen bond of the terminal carbonyl of the
hemisuccinate amide with cruzain Asp158 and the thiosemicarbazone groups located
near the Cys25 residue. This assignment corroborated with the experimental data that
showed a cruzain inhibition of 50%. On the other hand, compound 12, 2-amino-1-p-
nitrophenylethan-1-one thiosemicarbazone hemisuccinate ester, presented inversion of
the groups’ position in docking studies, which suggests that the hemisuccinate ester
group positioned between S1 and S1’ subpockets of the cruzain active site and the
thiosemicarbazone group is located in solvent-exposed position, probably due to the
hydrophilic character of the nitrogen atoms. The carbon atom of the carbonyl ester that
might interact with the catalytic residue and promote inhibition was 3.9 Å from Cys25,
a distance greater than those of the other compounds, which impairs the nucleophilic
attack and justifies the lower cruzain inhibition by this compound (10%). These results
agree with the cruzain inhibition assay data (Table 1).

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2.5. Cruzain inhibition assay
To validate the docking studies, we performed cruzain inhibition assays with the
compounds with single dose final concentrations of 10 µM. However, when we
consider the percent inhibition of cruzain in in vitro inhibition assay, pronounced
activity, up to 65%, was found for some compounds. We can observe in Table 1 that
some compounds presented major trypanocidal activity in addition to minimal activity
against cruzain. Compound 9 showed IC50=6.3 µM (trypanocidal activity in vitro test)
and only 20% inhibition of cruzain, similar to compound 10, which had IC50=37.7 µM
and just 10% cruzain inhibition. However, some compounds presented more balanced
cruzain inhibition and trypanocidal activity, such as compounds 11 (IC50=12.0 µM and
50% cruzain inhibition) and 8 (IC50=239.3 µM and 65% cruzain inhibition). This is an
important indication that there is no direct correlation between cruzain inhibition and
trypanocidal activity, which leads us to propose that these compounds may be acting by
some biochemical pathway other than the inhibition of cruzain. Due to the presence of
the nitro group in these molecules, oxidative stress may be involved in the global action
[18].
Therefore, despite the reduced number of compounds synthesized , we can infer
some features of the relationship of the structure and in vitro trypanocidal activity for
series: 1st. Aromatic nitro group: the para position is essential for good activity, and its
absence is preferable over the meta position, 2nd. Amino group: its absence is preferable,
but if present, an aliphatic amine is better than an aromatic amine, and 3rd. Alkyl chain:
no substitution in the alkyl chain is preferable to amino or hydroxyl substitution. A great
substituent on the side chain may lead to a decrease in the activity of the compound.
Therefore, a labile group may be an important feature for the development of a latent
prodrug against T. cruzi .
3. Conclusion
The planned compounds are potent thiosemicarbazone derivatives that may
inhibit enzyme cruzain and generate oxidative stress in T. cruzi parasite cells. The main
goal of this work was the development of potent compounds without a genotoxicity
profile, which antichagasic drugs often present.

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4. Experimental Section
Melting points were determined in open capillaries with an Electrothermal 9200
melting point apparatus. Infrared spectra were recorded in a Shimadzu FTIR-8300
1
spectrophotometer. H NMR spectra was obtained with a Bruker DPX300 NMR
spectrometer with DMSO-d6 as a solvent. The splitting patterns were as follows: s,
singlet; br s, broad singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet.
Coupling constants (J) are given in Hertz.
4.1. Materials
m-aminoacetophenone (Flucka), p-aminoacetophenone (Flucka), phenacylamine
(Flucka),
α-brome-p-nitroacetophenone
(Acros),
acetophenone
(Flucka),
thiosemicarbazide (Acros), 99.5% nitric acid (Synth), acetic anhydride (Merck),
concentrated sulfuric acid (Synth), hexamethylenetetramine (Synth), succinic anhydride
(Merck), N,N-dimethyl-4-aminopyridine (DMAP) (Flucka), benznidazole (Aldrich),
carboxymethylcellulose (Synth), Tween 40 (Synth),
cyclophosphamide (Aldrich),
acridine orange (Acros), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (Aldrich), phenazine methosulfate (PMS) (Aldrich). All reagents are ACS grade
or more than 97% of purity and were used with no further treatment, except for
hexamethylenetetramine (Synth) that was recrystallized from methanol before use.
Solvents were distilled and kept with molecular sieve.
4.2. General procedures
4.2.1. Preparation of thiosemicarbazones
A mixture of 0.01 mol of appropriate acetophenone and 0.01 mol of
thiosemicarbazide in 25 mL of ethanol was acidified with 2 mL of acetic acid to pH 3-4
and maintained at room temperature for 24 h. The solvent was evaporated under
reduced pressure and the residue was washed with a warm saturated sodium bicarbonate
solution to eliminate the start material and recrystallized from methanol. Mean yield of
approximately 90%.

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4.2.2. Protection of amino group
Acetylation
40 mmol of the appropriated amine was added carefully over 50 mL of ice
cooled acetic anhydride to avoid the temperature surpasses 30 ºC. After 1 hour, 200 mL
of cold distilled water was added, and the precipitated was filtered out and recrystallized
from 40 mL of ethanol. Mean yield of approximately 60%.
Remove of acetyl group
3 mmol of acetylated aminoacetophenone was added in 10 mL of 6N
hydrochloric acid. After 4 hours under reflux, the reaction was left to cool to room
temperature. The pH was neutralized by adding 50% NaOH dropwise in ice bath to
promote the precipitation. In some cases, precipitation is formed by cooling the
solution. Mean yield of approximately 70%.
4.2.3. Preparation of hemisuccinates derivatives
2 mmol of the appropriated thiosemicarbazone, 2 mmol of succinic anhydride
and 0,1 mmol of DMAP were dissolved in 70 mL of dry acetonitrile, and left stirring for
48 h at room temperature. The solvent was evaporated under reduced pressure and the
residue recrystallized from water. Mean yield of approximately 80%.
4.3. Specific compounds
4-aminoacetophenone thiosemicarbazone (CAS 7410-53-9) (1)
Prepared according to the general procedure (section 4.2), from 4-
aminoacetophenone.
mp: 150-151 ºC (153 °C [38]); IR (KBr, cm-1): 3447-3150,1622,1590, 1489, 1281,
1186, 1099. 1H NMR (δ): 2.19 (s, 3H), 5.46 (s, 2H), 6.53 (d, 2H, J = 8.0), 7.62 (d, 2H, J
= 8.0), 7.72 (s, 1H), 8.10 (s, 1H), 9.97 (s, 1H).

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3-aminoacetophenone thiosemicarbazone (CAS 87743-92-8) (2)
Prepared according to the general procedure (section 4.2), from 3-
aminoacetophenone.
mp: 185-186 ºC (184 – 186 °C [39]); IR (KBr, cm-1): 3440-3144, 1616, 1582, 1485,
1
1281, 1246, 1101. H NMR (δ):10.20 (s, 1H), 8.31 (s, 1H), 7.68 (s, 1H), 7.10 (s, 1H),
7.03 (d, 2H, J = 8.0), 6.6 (t, 1H, J = 8.0), 5.05 (s, 2H), 2.22 (s, 3H).
2-amino-1-phenylethan-1-one thiosemicarbazone (3)
Prepared according to the general procedure (section 4.2), from phenacylamine.
mp: 179-180 ºC; IR (KBr, cm-1): 3389-3163, 1595, 1541, 1308, 1244, 1018.1H NMR
(δ): 9.35 (s, 1H), 8.54 (br s, 5H), 7.42-7.57 (m, 5H), 3.99 (s, 2H).
acetophenone thiosemicarbazone (CAS 2302-93-4)(4)
Prepared according to the general procedure (section 4.2), from acetophenone
mp: 120-121 ºC (120-121 °C [40]); IR (KBr, cm-1): 3410-3148, 1589, 1512, 1288,
1096; 1H NMR (δ): 10.17 (s, 1H), 8.25 (s, 1H), 7.91 (m, 2H),7.89 (s, 1H), 7.37 (m, 3H),
2.29 (s, 3H).
3-nitroacetophenone thiosemicarbazone (CAS 132898-27-2) (5)
m-nitroacetophenone
8 ml of acetophenonewas added over 20 mL of concentrated sulfuric acid
(d=1.84 g/mL) pre-cooled to 0 ºC with ice/ethanol bath, the temperature should be kept
under 5 ºC. The resulting solution was cooled to -5 ºC and then was treated with a cold
solution of 8 mL of concentrated sulfuric acid (d=1.84 g/mL) was added on 5.3 mL of
concentrated nitric acid (d=1.42 g/mL), keeping the temperature below 0 ºC. After 15

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minutes, a mixture of 100 g of ice and 150 mL of water was added to promote the
precipitation. A yellow precipitate of 3-nitroacetophenone was filtered out and
recrystallized from ethanol. Yield: 55%.
The compound 5 was obtained by condensation of m-nitroacetophenone with
thiosemicarbazide, as described in section 4.2 (general procedure).
1
mp: 224-226 ºC; IR (KBr, cm-1): 3395-3148, 1597, 1512, 1350, 1296, 1096; H NMR
(δ):10.35 (s, 1H), 8.54 (t, 1H, J = 2.2), 8.4 (dd, 1H, J = 2.2, 8.0), 8.35 (s, 1H), 8.20 (dd,
1H, J = 2.2, 8.0), 8.12 (s, 1H), 7.66 (t, 1H, J = 8.0), 2.35 (s, 3H).
2-amino-1-(3-nitrophenyl)ethan-1-one thiosemicarbazone (6)
α-amine-m-nitroacetophenone
48 mmol of m-nitroacetophenone was dissolved in 300 mL of glacial acetic acid
and warmed to 55 ºC. 48 mmol of bromine was then added drop-to-drop allowing the
discoloration of the solution before add next drop. After 30 minutes at 55 ºC, 101 g of
crushed ice was added to the reaction and the formed precipitated filtered out, washed
with water and recrystallized from ethanol/petroleum ether, rendering the compound 2-
brome-1-(m-nitrophenyl)ethan-1-one. Yield: 73%.
6.15 mmol of the brominated compound was dissolved in 7 mL of chloroform
and treated with a solution of 6.15 mmol of hexamethylenetetramine in 6 mL of
chloroform. After 24 h at room temperature, the reaction was cooled with an ice bath
and the formed precipitated washed with 15 mL of chloroform and 10 mL of ethanol,
rendering the hexamethylenetetramine salt. The desired amine was obtained treating the
salt with a solution of 1:2 of concentrated hydrochloric acid and ethanol (Yield: 87%).
The reaction was kept for 72 h under room temperature; the precipitated was filtered out
and washed with water and then ethanol, rendering hydrochloric salt of α-amine-m-
nitroacetophenone (m-nitrophenacylamine) (Yield: 85%).
The final compound was obtained by condensation with thiosemicarbazide.

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mp: 213-214 ºC; IR (KBr, cm-1): 3389-3215, 1589, 1539, 1443, 1356, 1285, 1111,
1
1043; H NMR (δ):9.57 (s, 1H), 9.01 (t, 1H, J = 1.5), 8.70 (dd, 1H, J = 1.5, 7.2), 8.38
(m, 2H), 8.25 (m, 3H), 7.86 (m, 2H), 4.23 (s, 1H), 4.05 (s, 1H).
3-nitro-4-aminoacetophenone thiosemicarbazone (7)
m-nitro-p-aminoacetophenone
4.5 mmol of 4-N-acetylamino acetophenone, synthesized according to general
procedure (section 4.2) from 4-aminoacetophenone, was added carefully over a becker
containing 10 mL of cooled concentrated nitric acid (d=1.42 g/mL) to avoid the
temperature surpass 5 ºC. After 1 hour, the reaction was poured in a flask containing 60
g of water/ice mixture. The formed precipitated was filtered out and washed with cold
water. The product was purified through silica gel chromatography, with a mixture of
dichloromethane (DCM) 95% DCM and 5% ethyl acetate as mobile phase (Yield:
73%).
After the removal of acetyl group, the resulting compound was condensed with
thiosemicarbazide rendering the final compound.
mp: 231-232 ºC; IR (KBr, cm-1): 3466-3155, 1634, 1589, 1508, 1468, 1339, 1246,
1
1178, 1086; H NMR (δ):10.17 (s, 1H), 8.21-8.25 (m, 3H), 7.91 (s, 1H), 7.68 (s, 2H),
7.00 (d, 1H, J = 9.00), 2.24 (s, 3H).
3-amino-4-nitroacetophenone thiosemicarbazone (8)
5 mL of concentrated nitric acid (d=1.42 g/mL) was carefully added dropwise
with stirring over 10 mL of cold acetic anhydride, avoiding temperature surpass 10 ºC.
Once the temperature returned to 0 ºC, 3-N-acetylamino acetophenone (synthesized
according to section 4.2) was added dropwise keeping the temperature in a range of 5-
10 ºC. After complete solubilization, the reaction was kept at the same temperature and
then poured in a flask containing 60 g of water/ice mixture. The formed precipitated
was filtered out and washed with cold water. The product was purified through silica gel

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chromatography, using a mixture of 50% chloroform and 50% of ethyl ether (yield:
76%).
After the remove of acetyl group, the resulting compound was condensed with
thiosemicarbazide
thiosemicarbazone.
rendering
the
desired
3-amino-4-nitroacetophenone
1
mp: 219-220 ºC; IR (KBr, cm-1): 3315-3365, 1630, 1605, 1516, 1468, 1327; H NMR
(δ): 10.39 (s, 1H), 8.45 (s, 1H), 7.9 (s, 1H), 7.89 (d, 1H, J = 9.20), 7.44 (d, 1H, J =
2.00), 7.36 (s, 2H), 7.31 (dd, 1H, J = 2.00, 9.20), 2.24 (s, 3H).
2-amino-1-(4-nitro)acetophenone thiosemicarbazone (9)
α-amine-p-nitroacetophenone
6.15 mmol of α-brome-4-nitroacetophenone was dissolved in 7 mL of
chloroform and treated with a solution of 6.15 mmol of hexamethylenetetramine in 6
mL of chloroform. After 24 h at room temperature, the reaction was cooled with ice
bath and the formed precipitated washed with 15 mL of chloroform and 10 mL of
ethanol, rendering the hexamethylenetetramine salt. The desired amine was obtained
treating the salt with a solution of 1:2 of concentrated hydrochloric acid and ethanol
(Yield: 84%). The reaction was maintained for 72 h at room temperature; the
precipitated was filtered out and washed with water and then ethanol, rendering
hydrochloric salt of α-amine-p-nitroacetophenone (Yield: 80%).
The final compound was obtained by condensation with thiosemicarbazide.
1
mp: 170-173 ºC; IR (KBr, cm-1): 3487-2986, 1596, 1512, 1342, 1288, 1096; H
NMR(δ): 10.29 (s, 1H), 8.29-8.09 (m, 6H), 7.71-7.66 (m, 2H), 4.05-3.99 (br s, 2H).
2-hydroxy-1-(4-nitro)acetophenone thiosemicarbazone (10)
α-hydroxy-p-nitroacetophenone
20 mmol of α-brome-p-nitroacetophenone and 30 mmol of silver nitrate
(AgNO3) were dissolved in a mixture of 250 mL of water and 150 mL of acetone. The

ACCEPTED MANUSCRIPT
reaction was refluxed for 4 hours. After cooling, the insoluble material was filtered out,
and the hydroxylated compound was extracted from the solution with 2 x 150 mL
portions of dichloromethane. The solvent was dried and evaporated under reduced
pressure, and the residue recrystallized from warmed 50% ethanol, yielding 54%.
The final compound was prepared by condensation with thiosemicarbazide.
1
mp: 211-212 ºC; IR (KBr, cm-1): 3402-3163, 1604, 1512, 1342, 1265, 1126, 1064; H
NMR (δ):11.04 (s, 1H), 8.56 (s, 1H), 8.17 (s, 4H), 8.20 (s, 1H), 6.08 (t, 1H, J = 5.3),
4.77 (d, 2H, J = 5.3).
2-amino-1-(4-nitro) acetophenone thiosemicarbazone hemisuccinate amide (11)
Prepared according to the general procedure (section 4.2), from 9.
mp: 187-190 ºC; IR (KBr, cm-1): 3493-3067, 1624, 1591, 1521, 1492, 1338, 1295,
1126; 1H NMR (δ): 11.13 (s, 1H), 8.77 (t, 1H, J = 6.0), 8.52 (s, 1H), 8.11-8.26 (m, 6H),
4.33 (d, 2H, J = 6.0), 2.31-2.46 (m, 4H).
2-hidroxy-1-(4-nitro) acetophenone thiosemicarbazone hemisuccinate ester (12)
Prepared according to the general procedure (section 4.2), from 10.
mp: 179-180 ºC; IR (KBr, cm-1): 3433-3163, 1624, 1612, 1512, 1465, 1342, 1250,
1172; 1H NMR (δ): 10.93 (s, 1H), 8.61 (s, 1H), 8.25 (s, 1H), 8.13-8.20 (m, 5H), 5.27 (s,
2H), 2.42-2.47 (m, 4H).
4.4. Anti-trypanosomal activity in vitro test
Trypanocidal activity in vitro test was performed on sterile 96-well plates with
cover using T. cruzi Y strain in a Liver-Infusion Tryptose (LIT) medium supplemented
with 10% fetal calf serum. Percent death for each administrated dose was determined by
the colorimetric method in a spectrophotometer employing MTT/formazan, according to
Dutta et al. [41]. The 7-day culture epimastigote form (log phase of growth) was used in

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this test. The compounds were dissolved with DMSO, and diluted with the same solvent
to final concentrations of 100, 50, 25, 10, 5, 2.5 and 1 µg/mL.
Amounts of 3 µL of each compound were mixed with 97 µL of LIT medium
containing 107 epimastigotes/mL and incubated for 72h at 28 ºC. 10 µL of a 2.5-mg/mL
MTT-PMS (phenazine methosulfate) solution was added to each well and maintained in
the dark at 28 ºC for 75 min for reduction of MTT to formazan by the action of
mitochondrial enzyme succinate dehydrogenase. A 10% (100 µL) solution of sodium
dodecyl sulfate (SDS) was added to the previous solution and maintained in the dark at
room temperature for 30 min. Optical density was assessed at 595 nm using an ELISA
reader. IC50, the concentration that kills 50% of the parasites, was calculated by linear
regression of the optical densities. All measurements were made in triplicate and the
Probit method was employed for statistical analysis [42].
4.5. Mutagenicity assay – micronucleus test
The present study was carried out according to Hayashi et al. [43], following the
Declaration of Helsinki (1964) and was approved by the institutional experimentation
committee (CEP/CFC/Car no. 38/2008), in agreement with Resolution No. 196/96 of
the Brazilian National Health Council.
In vivo mutagenicity was assayed in Swiss albino mice, weighing about 30 g.
Three doses (50, 100 and 250 mg/kg bwt) of each drug were given through gavage;
benznidazole at 100 mg/kg bwt (gavage) was used for comparative purposes. A positive
control group was treated intraperitoneally with cyclophosphamide (50 mg/kg bwt). A
negative control group received 0.3 mL of 1% carboxymethylcellulose (CMC) and
0.2% Tween 40 aqueous solution (gavage). Water or internal control was performed
using only water to determine the mean basal frequency of micronucleus. Peripheral
blood samples (5 µL) were collected from the tail 30 h after treatment and inoculated
onto microscope slides pre-treated with acridine orange. The slides were incubated in
the dark for at least 24 h at -20 ºC prior to the cytologic study. Micronucleus frequency
in 1,000 reticulocytes of each animal was determined by fluorescence microscopy and
combined blue and yellow light filters.

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4.6. Docking Studies
Molecular docking and scoring protocols were used as implemented in GOLD version
4.0 (CCDC, Cambridge, UK) to investigate the possible ligand binding conformations
within the cruzain binding pocket. X-ray crystallographic data for cruzain complexed
with the peptide-like inhibitor 1-(1-benzyl-3-hydroxy-2-oxo-propylcarbamoyl)-2-
phenyl-ethyl-carbamic acid benzyl ester determined at 1.2 Å (PDB ID 1ME4) used in
the docking simulations were retrieved from the Protein Data Bank (PDB). The ligand
and water molecules were removed from the binding pocket and hydrogen atoms were
added in standard geometry using the Biopolymer module implemented in SYBYL 8.0.
Histidine, glutamine, and asparagine residues within the binding sites were manually
checked for possible flipped orientation, protonation, and tautomeric states with Pymol
1.3 (DeLano Scientific, San Carlos, USA) side-chain wizard script. The binding sites
were defined as all the amino acid residues encompassed within an 10.0-Å radius sphere
centered on catalytic residue Cys25.
4.7. Cruzain inhibition assays
Cruzain truncated in the C-terminal extension obtained from Escherichia coli (strain
DH5-α containing the expression plasmid) was kindly supplied by J. H. McKerrow,
from the University of California, San Francisco, USA. Substrate Bz-PheArg-AMC was
purchased from Bachem (Torrance, CA, USA). Recombinant cruzain enzyme (0.23 nM)
in 1 mL of buffer pH 6.5 (Na2PO4 50 mM; EDTA 2.5 mM) was incubated for 5 min
either in the presence or not of inhibitors. Next, 5 µL of substrate (Bz-Phe-Arg-AMC)
stock solution (170 µM) were added and the enzyme activity was determined through
fluorimetric measurements under magnetic stirring in
a
Hitachi F4500
spectrofluorimeter (excitation: 380 nm; emission: 460 nm) at 37 °C.
Single-dose inhibitor concentrations were set to 10 µM. Measurements were
performed in triplicate for 5 min.
Acknowledgements
The authors thank grants from FAPESP, Project 2011/11499-0, Prof. Do
Amaral, A. T., PhD, IQ-USP, for the use of the spectrofluorimeter, and PhD student
Erika Pisccirillo for assistance in spectrofluorimetric measurements.

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Reference and Notes
[1] M. Goldbaum, M.L. Barreto, Commentary: The contribution and example of Carlos
Chagas, Int. J. Epidemiol. 37 (2008) 697-698.
[2] J.R. Coura, L.C. Dias, Epidemiology, control and surveillance of Chagas disease:
100 years after its discovery, Mem. Inst. Oswaldo Cruz 104(S1) (2009) 31-40.
[3] WHO - Word Health Organization – Chagas Disease: Special programme for
research and training in tropical disease, TDR, 2008.
[4] DNDi – Drugs for Neglected Disease initiative, 2008.
[5] G.A. Schmunis, Epidemiology of Chagas Disease in non-endemic countries: the role
of international migration, Mem. Inst. Oswaldo Cruz 102 (2007) 75-85.
[6] J.A. Urbina, Specific chemotherapy of Chagas disease: relevance, current limitations
and new approaches, Acta Trop. 115 (2010) 55-68.
[7] V.G. Duschak, A.S. Couto, An Insight on targets and pantented drugs for
chemotherapy of Chagas Disease, Recent Patents Anti-Infect. Drug Discov. 2 (2007)
19-51.
[8] J.H. Mckerrow, P.J. Rosenthal, R. Swenerton, P. Doyle, Development of protease
inhibitors for protozoan infections, Curr. Opin. Infect. Dis. 21 (2008) 668-672.
[9] J.A. Urbina, Chemotherapy of Chagas disease, Curr. Pharm. Des. 8 (2002) 287-195.
[10] J.A. Urbina, R. DoCampo, Specific chemotherapy of Chagas disease: controversis
and advances. Trends Pasitol. 19 (2003) 495-501.
[11] G. Le Loup, G. Pialoux, F.X. Lescure, Update in treatment of Chagas disease,
Curr. Opin. Infect. Dis. 24 (2011) 428-434.
[12] P. Veiga-Santos, E.S. Barrias, J.F. Santos, T.L. De Barros Moreira, T.M. De
Carvalho, J.A. Urbina, W. De Souza, Effects of amiodarone and posaconazole on the
growth and ultrastructure of Trypanosoma cruzi, Int. J. Antimicrob. Agent 40 (2012)
61-71.
[13] N. Chamond, N. Coatnoan, P. Minoprio, Immunotherapy of Trypanosoma cruzi
infections, Curr. Drug Targets Immune Endocr. Metabol. Disord. 2 (2002) 247-254.

ACCEPTED MANUSCRIPT
[14] H. Tokiwa, Y. Ohnishi, Mutagenicity and carcinogenicity of nitroarenes and their
sources in the environment, CRC Crit. Rev. Toxicol. 17 (1986) 23-60.
[15] H.S. Rosenkranz, R.O. Mermelstein, All nitro-containing chemicals were not
created equal, Mutat. Res. 114 (1983) 217-267.
[16] P.L. Olive, Cancer Res. Correlation between Metabolic Reduction Rates and
Electron Affinity of Nitroheterocycles, 39 (1979) 4512-4515.
[17] S.N. Moreno, R.P. Mason, R.P.A. Muniz, F.S. Cruz, R. Docampo, Generation of
free radicals from metronidazole and other nitroimidazoles by Trichomonas foetus, J.
Biol. Chem. 158 (1983) 4051-4054.
[18] M.A. La-Scalea, G.H.G. Trossini, C.M.S. Menezes, M.C. Chung, E.I. Ferreira,
Electrochemical Reduction Using Glassy Carbon Electrodein Aqueous Medium of a
Potential Anti-Chagas Drug: NFOH J. Eletroc. Soc. 157 (2009) F93-F97.
[19] B. Bonnet, D. Soullez, S. Girault, L. Maes, E. Davioud-Charvet, Trypanothione
reductase inhibition/trypanomicidal activity relationships in
aminopropyl)piperazine series, Bioorg. Med. Chem. 8 (2000) 95-103.
a
1,4-bis(3-
[20] R.L. Lopez De Compadre, A.K. Debnath, A.J. Shusterman, C. Hansch, LUMO
energies and hydrophobicity as determinants of mutagenicity by nitroaromatic
compounds in Salmonella typhimurium, Environ. Mol. Mutagen. 15 (1990) 44-55.
[21] R.L. Lopez De Compadre, A.J. Shusterman, C. Hansch, The role of hydrophobicity
in the Ames test. The correlation of the mutagenicity of nitropolycyclic hydrocarbons
with partition coefficients and molecular orbital indices, Int. J. Quantum Chem. 34
(1988) 91-101.
[22] J.J. Cazzulo, M.C.I. Cazzulo Franke, J. Martinez, B.M. Franke De Cazzulo, Some
kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi,
Biochim. Biophys. Acta. 1037 (1990) 186-191.
[23] J.J. Cazzulo, V. Hellman, R. Couso, A.J. Parodi, Amino acid and carbohydrate
composition of a lysosomal cysteine proteinase from Trypanossoma cruzi. Absence of
phosphorylated mannose residues, Mol. Biochem. Parasitol. 38 (1990) 41-48.
[24] L.C. Alves, R.L. Melo, M.H.S. Cezari, S.J. Sanderson, J.C. Mottram, G.H.
Coombs, L. Juliano, M.A. Juliano, Analysis of the S-2 subsite specificities of the
recombinant cysteine proteinases CPB of Leishmaniamexicana; and cruzain of

ACCEPTED MANUSCRIPT
Trypanosoma cruzi using fluorescent substrates containing non-natural basic amino
acids, Mol. Biochem. Parasitol. 117 (2001) 137-143.
[25] M.E. Mcgrath, A.E. Eakin, J.C. Engel, J.H. Mckerrow, C.S. Craik, R.J.
Fletterick, The crystal struture of cruzain: a therapeutic target for Chagas disease, J.
Mol. Biol. 247 (1995) 251-259.
[26] E. Del Nery, M.A. Juliano, A.P. Lima, J. Scharfstein, L. Juliano, Kininogenase
activity by the major cysteinyl proteinase (cruzipain) from Trypanosoma cruzi, J. Biol.
Chem. 272 (1997) 25713-25718.
[27] A. Bocedi, L. Gradoni, E. Menegatti, P. Ascenzi, Kinetics of parasite cysteine
proteinase inactivation by NO-donors. Biochem. Biophys. Res. Commun. 315 (2004)
710-718.
[28] X. Du, C. Guo, E. Hansell, P.S. Doyle, C.R. Caffrey, T.P. Holler, J.H. Mckerrow,
F.E. Cohen, Synthesis and structure-activity relationship study of potent trypanocidal
thiosemicarbazone inhibitors of the trypanosomal cysteine protease cruzain, J. Med
Chem. 45 (2002) 2695-2707.
[29] D.C. Greenbaum, Z. Mackey, E. Hansell, P. Doyle, J. Gut, C.R. Caffrey, J.
Lehrman, P.J. Rosenthal, J.H. Mckerrow, K. Chibale, Synthesis and structure−activity
relationships of parasiticidal thiosemicarbazone cysteine protease inhibitors against
Plasmodium falciparum, Trypanosoma brucei, and Trypanosoma cruzi J. Med. Chem,
47 (2004) 3212-3219.
[30] N. Fujii, J.P. Mallari, E.J. Hansell, Z. Mackey, P. Doyle, Y.M. Zhou, P.J.
Rosenthal, J.H. Mckerrow, R.K. Guy, Discovery of potent thiosemicarbazone inhibitors
of rhodesain and cruzain, Bioor. Med. Chem. Lett. 2005, 15, 121.
[31] R. Siles, S-E. Chen, M. Zhou, K.G. Pinney, M.L. Trawick, Design, synthesis, and
biochemical evaluation of novel cruzain inhibitors with potential application in the
treatment of Chagas disease, Bioor. Med. Chem. Lett. 16 (2006) 4405-4409.
[32] G.H.G. Trossini, A. Malvezzi, A. T-Do Amaral, C.O. Rangel-Yagui, M.A. Izidoro,
M.H. Cezari, L. Juliano, M.C. Chung, C.M.S. Menezes, E.I. Ferreira, Cruzain inhibition
by hydroxymethylnitrofurazone and nitrofurazone: investigation of a new target in
Trypanosoma cruzi, J. Enzyme Inhib. Med. Chem. 25 (2010) 62-67.

ACCEPTED MANUSCRIPT
[33] M.E. Caputto, A. Ciccarelli, F. Frank, A.G. Moglioni, G.Y. Moltrasio, D. Vega, E.
Lombardo, L.M. Finkielsztein. Synthesis and biological evaluation of some novel 1-
indanone thiazolylhydrazone derivatives as anti-Trypanosoma cruzi agents. Eur. J. Med.
Chem. 55 (2012) 155-163
[34] P.F. Carneiro, S.B. do Nascimento, A.V. Pinto, M.do C.F.R. Pinto, G.C. Lechuga,
D.O. Santos, H.M. dos Santos Júnior, J.A.L.C Resende, S.C. Bourguignon, V.F.
Ferreira. New oxirane derivatives of 1,4-naphthoquinones and their evaluation against
T. cruzi epimastigote forms. Bioorg. Med. Chem. 20 (2012) 4995–5000.
[35] S.A. Carvalho, L.O. Feitosa, M. Soares, T.E.M.M. Costa, M.G. Henriques, K.
Salomão, S.L. de Castro, M. Kaiser, R. Brun, J.M. Wardell, S.M.S.V. Wardell, G.H.G.
Trossini, A.D. Andricopulo, E.F. da Silva, C.A.M. Fraga. Design and synthesis of new
(E)-cinnamic N-acylhydrazones as potent antitrypanosomal agents. Eur. J. Med. Chem.
54 (2012) 512-521
[36] J.D. Maya, S. Bollo, L.J. Nuñez-Vergara, J.A. Squella, Y. Repetto, A. Morello, J.
Périé, G. Chauvière, Trypanosoma cruzi: effect and mode of action of nitroimidazole
and nitrofuran derivatives, Biochem Pharmacol. 65 (2003) 999-1006.
[37] F. Lecaille, J. Cotton, J.H. Mckerrow, M. Ferrer-Di Martino, E. Boll-Bataille, F.
Gauthier, G. Lalmanach, Reversible inhibition of cathepsin L-like proteases by 4-mer
pseudopeptides, FEBS Lett. 507 (2001) 362-366.
[38] R. N. Prabhu, D. Pandiarajan, R. Ramesh. Ruthenium(II) mediated C-H activation
of substituted acetophenone thiosemicarbazones: Synthesis, structural characterization,
luminescence and electrochemical properties. Journal of Organometallic Chemistry. 694
(2009) 4170-4177
[39] S. Witek, J. Bielawski, A. Bielawska. New pesticides and intermediates. Part VII.
Some azaalkenyl derivatives of N-phenylurea and N-phenylcarbamic acid. Polish
Journal of Chemistry. 55 (1981) 2589-600
[40] H. R. Fatondji, F. Gbaguidi, S. Kpoviessi. J. Bero, V. Hannaert, J. Quetin-Leclercq,
J. Poupaert, M. Moudachirou, G. C. Accrombessi. Synthesis, characterization and
trypanocidal activity of some aromatic thiosemicarbazones and their 1,3,4-
thiadiazolines derivatives. African Journal of Pure and Applied Chemistry. 5 (2011) 59-
64

ACCEPTED MANUSCRIPT
[41] A. Dutta, S. Bandyopadhyay, C. Mandal, M. Chatterjee, Development of a
modified MTT assay for screening antimonial resistant field isolated of Indian visceral
leishmaniosis, Parasitol. Internat. 54 (2005) 119-122.
[42] S. Muelas-Serrano, J. Nogal-Ruiz, A. Gómez-Barrio. Setting of a colorimetric
method to determine de viability of Trypanosoma cruzi epimastigotes. Parasitol. Res. 86
(2000) 999-1002.
[43] M. Hayashi, T. Morita, Y. Kodama, T. Sofuni, M.Jr. Ishidate, The micronucleus
assay with mouse peripheral blood reticulocytes using acridine orange-coated slides,
Mutat. Res. 245 (1990) 245-249.

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Figures Capitation
Figure 1. Chemical structure of nifurtimox and benznidazole
Scheme 1. Synthesis of thiosemicarbazone derivatives by direct reaction with
thiosemicarbazide
Scheme 2. Synthesis of nitro derivatives of thiosemicarbazone from acetophenones
Scheme 3. Synthesis of nitro derivatives of thiosemicarbazone from aromatic
aminoacetophenones
Scheme 4. Synthesis of nitro derivatives of aliphatic amino-thiosemicarbazone and
hemisuccinate derivatization
Figure 2. Docking pose results of the new aryl thiossemicarbazone derivatives
indicating the major interactions in cruzain active site. a) Compound 7; b) Compound 8
Figure 3. Docking pose results of the hemisuccinate derivatives show a good interaction
of the aromatic groups in S2 subpocket, interaction of the thiosemicarbazone of the
compound 11 and Cys25 of the cruzain and inversion of the groups positions in
compound 12. a) Compound 11; b) Compound 12.

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Table 1. IC50 values for in vitro assay against T.cruzi epimastigotes
and percentage of cruzain inhibition at 10 ꢀM dose
IC50
% inhibition
of cruzain
Compound
Structure
Against T. cruzi
S
NH2
N
NH
1
> 100 µg/mL
H2N
25
(>480.1 µM)
4-aminoacetophenone
thiosemicarbazone
S
NH2
N NH
> 100 µg/mL
2
inactive
(>480.1 µM)
H2N
3-aminoacetophenone
thiosemicarbazone
S
NH2
N NH
> 100 µg/mL
3
4
inactive
inactive
.HCl
NH2
(>408.6 µM)
2-amino-1-phenylethan-1-one
thiosemicarbazone
S
NH2
37.7 µg/mL
N NH
(195.1 µM)
acetophenone
thiosemicarbazone
S
NH2
N NH
> 100 µg/mL
5
5
(>419.7 µM)
O2N
3-nitroacetophenone
thiosemicarbazone
S
NH2
N NH
NH2
> 100 µg/mL
.HBr
6
(>299.2 µM)
inactive
O2N
2-amino-1-(3-nitrophenyl)ethan-1-one
thiosemicarbazone

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S
NH2
N
NH
H2N
O2N
7
inactive
55
4-amino-3-nitroacetophenone
thiosemicarbazone
S
NH2
N NH
O2N
H2N
60.6 µg/mL
8
65
(239.3 µM)
3-amino-4-nitroacetophenone
thiosemicarbazone
S
NH2
N NH
2.1 µg/mL
O2N
9
20
(6.3 µM)
BrH.H2N
2-amino-1-(4-nitro)acetophenone
thiosemicarbazone
S
NH2
NH
OH
N
9.59 µg/mL
10
10
(37.7 µM)
O2N
2-hidroxy-1-(4-nitro) acetophenone
thiosemicarbazone
S
NH2
N NH
O2N
OH
HN
O
4.25 µg/mL
11
50
(
12.0 µM)
O
2-amino-1-(4-nitro)acetophenone
thiosemicarbazone hemisuccinate
amide
S
NH2
NH
N
O
O
OH
41.09 µg/mL
116.0 µM
O
O2N
12
10
(
)
2-hidroxy-1-(4-nitro)acetophenone
thiosemicarbazone hemisuccinate
ester

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N
NO2
N
H
9.00 µg/mL
34,6 µM
N
control
not tested
(
)
O
benznidazole
Table 2. Number of micronucleus found by 1.000 analyzed cell
Treatment
Mean*
SD
(mg/Kg bwt)
Control +
47a
0.8b
0.7b
14.29
0.78
0.67
Control -
Control water
benznidazole
100
4.8c
2.25
compound 9
50
1.05b
2.2d
3.7e
0.85
1.03
1.89
100
250
compound 10
50
1.9b,f
2.2d
2.3e
1.73
1.55
1.83
100
250
compound 11
50
2.5f
2.7d
2.8e
1.18
1.49
1.14
100
250
Control (+): ciclophosphamide 50 mg/kg bwt; Control (–): CMC (1%)/Tween (0.2%)
* Same letter means no difference between arithmetic mean (P > 0.05)

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Figure 1.
O
O
O
O
N+
S
N N
N
N
H
O-
O-
O
N+
O
N
nifurtimox
benznidazole
Scheme 1.
R2
R2
H
R1
N
NH2
S
R1
H2N
S
N NH
R3
NH2
O
R3
1: R1=-NH2; R2=-H; R3=-H
2: R1=-H; R2=-NH2; R3=-H
3: R1=-H; R2=-H; R3=-NH2
4: R1=-H; R2=-H; R3=-H
R1=-NH2; R2=-H; R3=-H - 4-aminoacetophenone
R1=-H; R2=-NH2; R3=-H - 3-aminoacetophenone
R1=-H; R2=-H; R3=-NH2 - phenacylamine
R1=-H; R2=-H; R3=-H - acetophenone
Scheme 2.
S
O
N N
H
O
NH2
HNO3 conc.
thiosemicarbazide
acetophenone
5
NO2
NO2
Br2
55 ºC
S
N N
O
NH2
H
1. hexamine
NH2
Br
2. thiosemicarbazide
6
NO2
NO2

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Scheme 3.
R2
R1
O
H
NO2
H
O
N
O
N
O
O
O
HNO3 conc.
O
O
R1= -NH2; R2=-H - 4-aminoacetophenone
R1= -H; R2=-NH2 - 3-aminoacetophenone
HCl 6N
NO2
R2
R1
H
S
N
NH2
H2N
H2N
N
7: R1= -NH2; R2=-H
8: R1= -H; R2=-NH2
S
N
NH2
H
O
Scheme 4.
S
S
N
NH
NH2
O
N
NH
X
NH2
succinic anhydride
O
O
thiosemicarbazide
a
X
O2N
OH
Br
O2N
X
O
O2N
11: X = -NH-
12: X= -O-
9: X = -NH2
10: X=-OH
for compounds 9 and 11: a=hexamine
for compounds 10 and 12: a=silver nitrate