Synthesis and structure-activity relationship study of a new series of antiparasitic aryloxyl thiosemicarbazones inhibiting Trypanosoma cruzi cruzain

Article abstract of DOI:10.1016/j.ejmech.2015.06.048

The discovery of new antiparasitic compounds against Trypanosoma cruzi, the etiological agent of Chagas disease, is necessary. Novel aryloxy/aryl thiosemicarbazone-based conformationally constrained analogs of thiosemicarbazones (1) and (2) were developed

Full text of DOI:10.1016/j.ejmech.2015.06.048

Accepted Manuscript
Synthesis and Structure–Activity Relationship Study of a New Series of Antiparasitic
Aryloxyl Thiosemicarbazones Inhibiting T. cruzi cruzain
José Wanderlan Pontes Espíndola, Marcos Veríssimo de Oliveira Cardoso, Gevanio
Bezerra de Oliveira Filho, Dayane Albuquerque Oliveira e Silva, Diogo Rodrigo
Magalhaes Moreira, Tanira Matutino Bastos, Carlos Alberto de Simone, Milena
Botelho Pereira Soares, Filipe Silva Villela, Rafaela Salgado Ferreira, Maria Carolina
Accioly Brelaz de Castro, Valéria Rego Alves Pereira, Silvane Maria Fonseca Murta,
Policarpo Ademar Sales Junior, Alvaro José Romanha, Ana Cristina Lima Leite
PII:
S0223-5234(15)30127-6
10.1016/j.ejmech.2015.06.048
EJMECH 7975
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 April 2015
Revised Date: 25 June 2015
Accepted Date: 26 June 2015
Please cite this article as: J.W. Pontes Espíndola, M.V. de Oliveira Cardoso, G.B. de Oliveira Filho, D.A.
Oliveira e Silva, D.R. Magalhaes Moreira, T.M. Bastos, C.A. de Simone, M.B.P. Soares, F.S. Villela,
R.S. Ferreira, M.C.A.B. de Castro, V.R.A. Pereira, S.M.F. Murta, P.A. Sales Junior, A.J. Romanha,
A.C.L. Leite, Synthesis and Structure–Activity Relationship Study of a New Series of Antiparasitic
Aryloxyl Thiosemicarbazones Inhibiting T. cruzi cruzain, European Journal of Medicinal Chemistry
(2015), doi: 10.1016/j.ejmech.2015.06.048.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and Structure–Activity Relationship Study of a New Series of
Antiparasitic Aryloxyl Thiosemicarbazones Inhibiting T. cruzi cruzain
a, *
a
José Wanderlan Pontes Espíndola , Marcos Veríssimo de Oliveira Cardoso , Gevanio
a
a
Bezerra de Oliveira Filho , Dayane Albuquerque Oliveira e Silva , Diogo Rodrigo
Magalhaes Moreira , Tanira Matutino Bastos , Carlos Alberto de Simone , Milena
b
b
c
b,e
d
d
Botelho Pereira Soares , Filipe Silva Villela , Rafaela Salgado Ferreira , Maria
f
f
Carolina Accioly Brelaz de Castro , Valéria Rego Alves Pereira , Silvane Maria Fonseca
g
g
g
Murta , Policarpo Ademar Sales Junior , Alvaro José Romanha and Ana Cristina Lima
a, *
Leite
a
Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde,
Universidade Federal de Pernambuco, 50740-520, Recife, PE, Brazil.
b
Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, 40296-750, Salvador,
BA, Brazil
c
Departamento de Física e Informática, Instituto de Física, Universidade de São Paulo,
1
3560-970, São Carlos-SP (Brazil)
Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais,
1270-901, Belo Horizonte, MG, Brazil.
d
3
e
Centro de Biotecnologia e Terapia Celular, Hospital São Rafael, 41253-190,
Salvador, BA, Brazil.
f
Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, 50670-420, Recife,
PE, Brazil
g
Laboratório de Parasitologia Celular e Molecular, Centro de Pesquisas René Rachou,
Fundação Oswaldo Cruz, 30.190-002, Belo Horizonte, MG, Brazil.
*
2
To whom correspondence should be addressed: phone +55 81-30370976, fax +55 81-
1268510; e-mail: acllb2003@yahoo.com

ACCEPTED MANUSCRIPT
Abstract
The discovery of new antiparasitic compounds against Trypanosoma cruzi, the
etiological agent of Chagas disease, is necessary. Novel aryloxy/aryl
thiosemicarbazone-based
conformationally
constrained
analogues
of
thiosemicarbazones (1) and (2) were developed as potential inhibitors of the T. cruzi
protease cruzain, using a rigidification strategy of the iminic bond of (1) and (2). A
structure-activity relationship analysis was performed in substituents attached in both
aryl and aryloxy rings. This study indicated that apolar substituents or halogen atom
substitution at the aryl position improved cruzain inhibition and antiparasitic activity in
comparison to unsubstituted thiosemicarbazone. Two of these compounds displayed
potent inhibitory antiparasitic activity by inhibiting cruzain and consequently were able
to reduce the parasite burden in infected cells and cause parasite cell death through
necrosis. In conclusion, we demonstrated that conformational restriction is a valuable
strategy in the development of antiparasitic thiosemicarbazones.
Key words: Chagas disease, T. cruzi, conformationally constrained analogues,
thiosemicarbazone, cruzain, necrosis.

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Highlights
Conformational restriction was employed for synthesizing new antiparasitic
thiosemicarbazones.
The compounds presented a broad, potent and selective antiparasitic activity against T.
cruzi.
Mechanistically, compounds impair protease cruzain activity and subsequently cause
necrotic parasite death.

ACCEPTED MANUSCRIPT
1
. Introduction
Chagas disease, caused by Trypanosoma cruzi, represents a serious and alarming health
problem[1]. It affects approximately 5-10% of the population of Latin America, but
even after more than 100 years since its discovery, the specific treatment of T. cruzi
remains uncertain[2,3]. Benznidazole, the drug of choice for the treatment of Chagas
disease, is able to eliminate the parasite during the acute phase; however, it is poorly
effective during the chronic phase, even after long-term administration, and it causes a
high dropout rate during treatment due to side effects[4,5]. Human vaccination against
T. cruzi infection is not available; thus, other therapies aiming to control infection or
reduce clinical symptoms are being investigated[6–8].
Knowledge of molecular targets related to various parasite processes has significantly
contributed to this investigation. Key enzymes, such as cysteine proteases, are among
the preferred targets for the development of new drugs. The structural and functional
biochemistry of cruzain, however, is by far the most studied of the proteases in T. cruzi
infections [9]. Cruzain belongs to the family of cysteine proteases (papain-like enzymes
known as clan CA) and is closely related to cathepsins L and S, which are also
associated with other pathologies in humans[10]. This enzyme is involved in the
invasion, differentiation and proliferation of parasites in host cells[11], and it is a useful
molecular target for the rational design of anti-trypanosomal agents[12].
Regarding the identification of cruzain inhibitors, most of the efforts have been
conducted through the investigation of peptides and peptide-like compounds, such as
ureas[10,13], hydrazones[14–17], triazoles[18,19] and thiosemicarbazones[20–22].
Thiosemicarbazones were originally developed as potential inhibitors of cathepsin-L,
one of the principal proteases involved in cancer cells[20]. However, based on the
homology and similar biochemical properties between cathepsin-L and cruzain,
thiosemicarbazones were investigated as a potential class of cruzain inhibitors. Later on,
aryl thiosemicarbazones were found to be a class of anti-T. cruzi compounds that inhibit
cruzain activity[21,22].
The general binding mode for aryl thiosemicarbazones with cruzain involves covalent
binding of a thioamide group to a Cys25 amino acid, as well as the location of the aryl
group within a deep hydrophobic pocket[22,23]. Based on this model, a number of
molecular modifications have been investigated, including bis-thiosemicarbazones,

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heterocyclic-derived thiosemicarbazones, thiosemicarbazones containing bioreductive
groups and metallic complexes[24–29].
Recently, our group evaluated the anti-T. cruzi activity of a series of aryl
thiosemicarbazones obtained by a homologation strategy. This led to the identification
of new potent anti-T. cruzi agents, such as compounds (1) and (2) (Figure 1), which
significantly inhibited the proliferation of epimastigotes (IC50 = 71.5 and 2.2 ꢀM,
respectively) and demonstrated toxicity to trypomastigotes (CC = 1.4 and 2.2 ꢀM,
5
0
respectively) but was not cytotoxic against spleen cells. In addition, these compounds
induced T. cruzi cell death by an apoptotic process; however, they did not exhibit
inhibitory activity for cruzain[30].
PLEASE, INSERT FIGURE 1 HERE
In parallel to thiosemicarbazones, our group observed that thiazolidinone 18 inhibits
cruzain activity but not its homologous proteins in mammalian cells (cathepsin L)[31].
However, it was several times less potent than KB2, a high-efficient cruzain
inhibitor[19]. A comparison of cruzain docking between thiazolidinone 18 and KB2 has
revealed that the major difference is that KB2 assumes a T-shaped conformation.
Therefore, thiazolidinones presenting a T-shape conformation displayed enhanced
activity than thiazolidinone (18) family. Thus, our findings were suggestive that cruzain
inhibitors displaying a conformation resembling a T-shape presented in KB2 compound
warrant activity (Figure 2).
PLEASE, INSERT FIGURE 2 HERE
Based on this, we sought that disrupting planarity and symmetry in aryl
thiosemicarbazones (1) and (2) would lead to T-shape conformational compounds and
potentially increase cruzain and antiparasitic activity. Among the chemical
modifications to achieve this[32–34], we consider attaching an aryl group in the imine
group which could change dihedral angles and twist the position of aryl rings,
producing new conformationally constrained compounds (Figure 3).
PLEASE, INSERT FIGURE 3 HERE

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New aryl thiosemicarbazones (8a-l, 9a-t) were synthesized and evaluated as anti-T.
cruzi agents as well as cruzain inhibitors. First, compound 8a was prepared, which
contains a phenyl ring without a substituent attached to the iminic carbon, for the
purpose of comparison with substituted derivatives 8b-h. Thereafter, substituents
attached to the phenyl ring were examined, such as alkyl, alkoxy, halogen atoms and
phenyl rings, generating biphenyl and naphthyl groups. In addition, we investigated a
wide range of substituents bonded to the phenoxyl ring (9a-t) to assess its functions in
terms of electronic and steric contributions to anti-T. cruzi activity. We prepared 4-
substituted phenyl derivatives, followed by the preparation of derivatives with one
substituent in meta- or ortho- positions in the phenyl ring. Derivatives containing two
substituents attached to the phenyl ring were synthesized, as well as the replacement of
a phenyl by a naphthyl ring. Finally, aryl thiosemicarbazones were synthesized
containing methyl or phenyl groups in N4 (8i-l). Evaluation of the anti-T. cruzi and
cruzain activity for these compounds (8a-l, 9a-t) led to the identification of important
structure-activity relationships.
2
. Results and discussion
2
.1. Synthesis
Obtaining aryl thiosemicarbazones (8a-l, 9a-t), as shown in Scheme 1, was performed in
two steps from different phenols and halo-substituted acetophenones, both purchased
commercially. Initially, various 1-phenoxy-2-acetophenones (5a-h; 6a-t) were obtained
from the O-alkylation of various phenols (3a-t) with 2-halo-acetophenones (4a-h) in a
basic medium and temperature environment, similar to a protocol previously
described[29]. After 3-4 h of reaction, 1-phenoxy-2-acetophenones compounds were
obtained with yields varying from 77 to 95%. These intermediate compounds (5a-h; 6a-
t) were then reacted with some thiosemicarbazides (7a-c) and catalytic HCl in an
ultrasound bath at room temperature[35]. After 2.5-3 h, thiosemicarbazones (8a-l, 9a-t)
precipitated in the reaction mixture and were collected by simple filtration. All
thiosemicarbazones were recrystallized and obtained at an acceptable purity (>95%) in
1
13
yields ranging from 69 to 90%. The structures were determined by H and C NMR,
DEPT and IR.

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PLEASE, INSERT SCHEME 1 HERE
1
In the H NMR spectra of most compounds, it was possible to observe the presence of
signals correspondent to the existence of isomers or rotamers. However, two signals for
the methylene hydrogens had a chemical shift difference of approximately 0.3 ppm; in
the case of rotamers, generally, two signals almost overlapped. These observations were
thus suggestive of the formation of a mixture of stereoisomers (Z) and (E).
Next, we aimed to define the configuration of the major isomer by crystallographic
analysis. A single crystal suitable for X-ray analysis was collected from
thiosemicarbazones 8e and 9h. As shown in the ORTEP-3 representation of
thiosemicarbazones 8e and 9h (Figure 4), the N2 and sulfur atoms are on the same side,
1
characterizing a Z configuration for the C1=N2 bond. These compounds showed H
NMR chemical shifts similar to thiosemicarbazones (8a-l, 9a-t); therefore, it is fair to
suggest that the major isomer for these thiosemicarbazones has a Z configuration about
this bond.
PLEASE, INSERT FIGURE 4 HERE
In contrast, in previous works developed by our group[30,31] aryl-thiosemicarbazones
were obtained that exhibited E configuration for this bond. The main structural feature
that differentiates the thiosemicarbazones (8a-l, 9a-t) from those previously
obtained[30,31] is the presence of a phenyl group at C1. It appears that this reversal in
the configuration around that bond is related to the presence of this group (Figure 5).
PLEASE, INSERT FIGURE 5 HERE
The ratio of the stereoisomers Z/E was estimated by considering the relative intensity of
1
the signals of the methylene protons in H NMR spectra and varied according to the
compound (supplementary material). In the spectrum of compound 9c, for example, only
one signal was detected, suggesting that it was obtained in the form of a single
stereoisomer (Z) or that the ratio of the minor isomer (E) was so small that was not
detected during analysis. In relation to the compounds 8i, 8l, 9a, 9n and 9p, the relative

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proportion of stereoisomers (Z:E) was: 8i (89.2: 10.8), 8l (91.5: 8.5), 9a (93.5: 6:5), 9n
(96.5: 3:5), 9p (95.2: 4.8), respectively.
2
.2. Antiparasitic activity against extracellular forms and cytotoxicity against host cells
First, compounds 8a-l and 9a-t were evaluated against epimastigotes and
trypomastigotes of T. cruzi. The antiparasitic activity was determined by counting the
parasite number in a Neubauer chamber and calculating the concentration of the test
compound resulting in 50% inhibition (IC , epimastigotes) or 50% cytotoxicity (CC ,
5
0
50
trypomastigotes). Cytotoxicity in host cells was determined in mouse splenocytes,
3
measured by the incorporation of [ H]-thymidine, and results were expressed as the
highest non-cytotoxic concentration (HNC). Benznidazole and nifurtimox were used as
reference antiparasitic drugs and exhibited CC50 values of 6.2 and 2.7 µM against
trypomastigotes, respectively. As a standard, compounds with CC values ≤ 6.2 µM
50
against trypomastigotes were considered potent anti-T. cruzi compounds. The results are
reported in Table 1.
PLEASE, INSERT TABLE 1 HERE
We first analysed the antiparasitic activity against the trypomastigote form. In analysing
the activity of aryl thiosemicarbazones 8a-h, in which the structural variations were
made in the para-position of the phenyl ring from the 2-haloacetophenone (Ar'), it was
observed that, in general, substitutions in the phenyl ring did not favour trypanocidal
activity. The compound 8a (CC = 2.4 µM), which has no substitution in the phenyl
50
ring, was one of the most potent compounds in the (8a-h) series, showing higher
activity than reference drugs BDZ and NFX (CC = 6.2 and 2.7 µM, respectively).
5
0
Comparing the activity of the substituted aryl-thiosemicarbazones (8b-h) with BDZ, all
compounds exhibited a greater toxicity against trypomastigotes than this reference drug,
with the exception of the compound 8h (CC = 14.7 ꢀM). The attachment of electron
50
donors groups, such as methyl (8b, CC = 3.1 ꢀM) and methoxy (8c, CC = 5.0 ꢀM),
5
0
50
to the phenyl ring at the para-position decreased the antiparasitic activity. Substitutions
in the para-position of the phenyl ring by the halogen atoms -F (8d, CC = 3.6 ꢀM), -
5
0
Cl (8e, CC50 = 5.3 ꢀM) and -Br (8f, CC50 = 3.9 ꢀM) also resulted in less active

ACCEPTED MANUSCRIPT
compounds than the unsubstituted derivative (8a). In addition, there was no correlation
between the size of the halogen atomic radius and the antiparasitic activity. On the other
hand, the attachment of a second phenyl ring at the para-position led to a slight increase
in activity against trypomastigotes, as can be observed for compound 8g (CC = 2.1
50
ꢀ
M). These data showed similarity to those obtained in the study by Moreira et al., in
which the 4-thiazolinone containing a phenyl at position 4 of the phenyl ring derived
from 2-haloacetophenones are among the two most active against the parasite[36].
Interestingly, compound 8h (CC = 14.7 ꢀM), which contains a bulky and hydrophobic
5
0
α-naphthyl group like the biphenyl of compound 8g, showed the lowest activity among
the thiosemicarbazones 8a-h.
By evaluating the trypanocidal activity of thiosemicarbazones 8i-l, it can be noted that
the substitution at position N4 reduced the toxicity against this evolutionary form in
most cases. For example, directly comparing the activity of compounds 8k (N4-Me,
CC = 7.4 ꢀM) and 8l (N4-Ph, CC = 15.6 ꢀM) to the unsubstituted analogue (8d,
50
50
CC = 3.6 ꢀM), a large reduction of activity is noted after this substitution.
50
Regarding the toxicity against trypomastigotes of aryl-thiosemicarbazones 9a-t, in
which the structural variations occur in the phenoxy ring (Ar), they all exhibited higher
or similar activity in relation to reference drug benznidazole, with the exception of
compounds 9q (CC50 = 28.3 ꢀM) and 9r (CC50 = 12.3 ꢀM). In addition, the
functionalization of the phenoxy ring was generally beneficial to the activity against
trypomastigote forms, as the compound 9a (CC50 = 6.6 ꢀM), an unsubstituted
derivative, was less active than substituted compounds (except compounds 9q and 9r).
In comparison to 9a, anti-T. cruzi activity was enhanced when an alkyl group was
attached to the para-position of the phenoxy ring. Thiosemicarbazone 9b (CC = 1.8
5
0
ꢀ
M), with an ethyl group, was about three times more potent. Compounds 9c (CC =
50
2
.9 ꢀM) and 9d (CC = 2.2 ꢀM), which have branched alkyl groups, also demonstrated
50
increased antiparasitic activity, although with slightly less intensity than the linear alkyl
chain analogue (9b). The attachment of methoxy groups to the phenyl ring also
generated more active compounds, as can be observed for thiosemicarbazones 9e (CC50
=
1.2 ꢀM) and 9f (CC50 = 3.7 ꢀM), substituted at the meta- and para-positions,
respectively. However, the compound 3-methoxy substituted (9e) was the most potent
of all of the thiosemicarbazones assessed in this study, approximately five times more

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active than BDZ. Binding of an acetamide group in the para-position of the phenoxy
ring (9g, CC = 3.5 ꢀM) increased activity compared with 9a. However, this increase
5
0
was not as significant as what was observed in previous work from our group, in which
the derivative containing such a substituent was among the most active[30].
We also evaluated the antiparasitic activity against trypomastigotes by
thiosemicarbazones containing different halogens attached to the phenoxy ring. All
derivatives containing halogens were more active than the unsubstituted
thiosemicarbazone 9a, revealing the importance of this structural modification for
antiparasitic activity. By comparing the activity of compounds 9p (CC = 2.1 ꢀM), 8f
50
(
CC = 3.9 ꢀM) and 9l (CC = 5.7 ꢀM), which show the atoms -I, -Br and -Cl at
50 50
position 4 of the phenoxy ring, respectively, a potential increase of antiparasitic activity
was observed according to the increase of the atomic radius of bound halogen. This
trend was also observed in a previous study by our group[30]. We also analysed the
effect of binding chlorine atoms in different positions of the phenoxy ring, ortho- (9j,
CC = 6.0 ꢀM), meta- (9k, CC = 1.6 ꢀM) and para- (9l, CC = 5.7 ꢀM). The meta-
50
50
50
substitution generated the most active chlorine derivative and one of the most active
compounds in this study. Moreover, di-substitutions were also evaluated in the phenoxy
ring by halogen atoms, giving rise to compounds 9i (3-Cl,4-F-Ph), 9m (2,3-diCl-Ph)
and 9n (3,4-diCl-Ph). These compounds were more active in relation to compound 9a,
especially thiosemicarbazone 9i, one of the most trypanocidal of the series with a CC50
=
1.7 ꢀM, higher than even the reference drug.
The anti-trypanosomal activity for compounds 9p, 8f and 9l increased with the atomic
radius of their halogen atoms, suggesting the steric effect is important for activity.
Based on this, we evaluated thiosemicarbazones 9q and 9r, which possess a second
phenyl ring attached to the phenoxy ring in the positions 3 and 4, respectively.
Compounds 9s and 9t, fitted with a naphthyl ring attached to the imine carbon in α and
β positions, respectively, were also obtained. The thiosemicarbazones containing
biphenyl 9q (CC50 = 28.3 ꢀM) and 9r (CC =12.3 ꢀM) were inactive, whereas the
50
compounds containing the naphthyl group 9s (CC = 3.4 ꢀM) and 9t (CC = 5.0 ꢀM)
5
0
50
only maintained the antiparasitic activity, with the replacement in the position α most
favourable. As observed, not only the steric and hydrophobic effects of the substituents
in the phenoxy ring are responsible for the potentiation of trypanocidal activity.

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Once the toxic activity of all compounds (8a-l, 9a-t) for the T. cruzi trypomastigotes
had been determined, we investigated the antiproliferative effect for T. cruzi
epimastigotes. BDZ, one of the reference drugs used in the experiments, exhibited an
IC of 48.8 ꢀM while NFX presented an IC of 5.7 ꢀM on this parasite form. All
5
0
50
thiosemicarbazones evaluated in this study were more active than the reference drug
BDZ.
Regarding the cytotoxicity to splenocytes, most aryl thiosemicarbazones evaluated in
this study (8a-l, 9a-t) caused higher toxicity than the reference drug benznidazole, with
the exception of compound 9a, which was non-toxic at concentrations up to 137.3 ꢀM,
and compound 9c, which was non-toxic to the highest concentration tested (246.0 ꢀM).
Taking into account the selectivity, the aryl thiosemicarbazone 9c stood out among all
compounds evaluated, displaying excellent trypanocidal activity for both parasite forms
of T. cruzi and lower toxicity to splenocytes than the reference drugs. In addition,
Lipinski et al present four criteria that are important to study the pharmacokinetics and
drug development. A molecule that agree with three criteria, would present a good
pharmacokinetic and became a drug candidate[37]. It is interesting to note that the
calculated values in Table 2 show that thiosemicarbazone 9c as well as the
unsubstituted derivative (9a) meet the criteria of Lipinski. On the other hand,
structurally-simple thiosemicarbazones present limited aqueous solubility.
PLEASE, INSERT TABLE 2 HERE
2
.3. Anti-T. cruzi activity against amastigote and trypomastigote forms infecting a
vertebrate cell
Due to the good activity shown against epimastigote and trypomastigote forms of T.
cruzi, the compounds were also assayed in vitro against the T. cruzi amastigote form,
which represents the intracellular form of the parasite. For this, we used a tissue cell
culture system that allows evaluating the activity against both amastigote and
trypomastigote forms simultaneously because the lengthy time of the assay (seven days)
allows compounds to act on trypomastigotes released starting from four days post-

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infection. This method is considered the in vitro method of choice in screening drugs for
activity against T. cruzi because it mimics the lifecycle of the parasite[38,39].
Results were expressed as the percent of inhibition of parasite growth after treatment
with each compound. A cell viability test was performed to evaluate the cytotoxicity of
the compounds against the murine fibroblast L-929 cell line[39,40]. The concentrations
that caused 50% inhibition of the parasite growth (IC ) were evaluated for the more
5
0
potent compounds (those presenting more than 70% inhibition in the initial screening at
00 µM) (Table 3).
1
PLEASE, INSERT TABLE 3 HERE
Most of the compounds exhibited activity against these parasite forms of T. cruzi, with
2 compounds (8a-b, 8f-h, 9-b, 9e-f, 9i, 9n, 9p) showing an IC50 ≤ 10 µM.
1
Furthermore, thiosemicarbazones 8g, 8h and 9a (IC50 = 3.3, 4.8 and 5.4 ꢀM,
respectively) showed trypanocidal activity comparable to the benznidazole.
Analysing the activity of thiosemicarbazones 8a-h, it was observed that, relative to
compound 8a (IC = 10.2 ꢀM), the presence of a methyl group in position 4 of the
5
0
phenyl ring (8b) slightly improved activity. Thiosemicarbazone 8c, which also contains
an electron donor group (OCH ) at the same position, was less active (IC = 11.9 ꢀM).
3
50
The halogen substitution in the phenyl ring (4-F, 8d; 4-Cl; 8e; 4-Br, 8f) generally did
not contribute to the activity against intracellular and trypomastigote forms, except for
thiosemicarbazone 8f (IC = 8.1 ꢀM) a bromine derivative. Compound 8g (IC = 3.3
50
50
ꢀ
M), which has a phenyl ring at the same position, was the more active compound
tested, suggesting that the presence of bulky and hydrophobic substituents, as bromine
and phenyl, may be associated with improved activity. In addition, thiosemicarbazone
8
h (IC = 4.8 ꢀM), also containing a bulky and hydrophobic grouping (naphthyl), is
50
among the most active of the series, suggesting the steric nature of these groups
positively influences activity against these forms of the parasite.
Considering the anti-T. cruzi activity of thiosemicarbazones 9a-t against these parasite
forms, it is noted that substitutions in the phenoxy ring (Ar) gave less active compounds
compared with the unsubstituted derivative 9a (IC50 = 5.4 ꢀM). However, some
structure-activity relationships were identified in this series. Comparing the activity of

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compounds 9b, 9c and 9d (IC = 6.4, 12.3 and 15.2 ꢀM, respectively), a reduction in
5
0
the trypanocidal activity was observed with increased branching of the alkyl chain
attached to position 4 in the phenoxy ring. A potentiation of the trypanocidal activity
was observed with an increased halogen atomic radius linked to the same ring position
by the order: 9h (4-F, IC = 15.7 ꢀM) < 9l (4-Cl, IC = 11.2 ꢀM) < 8f (4-Br, IC = 8.1
5
0
50
50
ꢀ
M) ~ 9p (4-I, IC = 8.6 ꢀM) (Figure 6).
50
PLEASE, INSERT FIGURE 6 HERE
Furthermore, the presence of a halogen at position 4 of the ring contributed more
significantly to the activity than position 3, as is seen in the comparison of compounds
9
l (4-Cl, IC = 11.2 ꢀM), and 8f (4-Br, IC = 8.1 ꢀM) with compounds 9k (3-Cl, IC
50 50 50
=
25.1 ꢀM) and 9o (3-Br, IC = 149.4 ꢀM), respectively.
50
The cytotoxicity of the active compounds on uninfected fibroblasts was established in
vitro to evaluate the selectivity of their antiparasitic effects. The selectivity index (SI)
was calculated as the ratio of the CC value in uninfected cells (cytotoxicity) to the IC
5
0
50
in parasite cells (Table 3). Although most of the compounds exhibited low selectivity,
thiosemicarbazones 9a, 9f and 9t showed moderate selectivity (SI> 10), suggesting that
these compounds could be prototypes for new trypanocidal drugs.
2
.4. Cruzain inhibition activity
To investigate a possible mechanism of action, compounds were tested against the
enzyme cruzain of the T. cruzi. The inhibition of cruzain enzymatic activity by all
compounds was measured using a competition based assay with the substrate Z-Phe-
Arg-aminomethylcoumarin (Z-FR-AMC)[41]. All compounds were screened at 50 ꢀM,
and only compounds having an inhibition value of >70% were chosen to determine IC50
values. However, the IC50 values of many compounds exhibiting an inhibition
percentage above this value (70%) could not be established due to their low solubility,
which made it impossible to achieve cruzain inhibition high enough (> 85%) for a good

ACCEPTED MANUSCRIPT
curve determination. Therefore, in some cases, the discussion was performed in terms of
the percentage of cruzain inhibition (Table 3).
Analysing the potential for inhibition of thiosemicarbazones 8a-h, it was observed that
the substitution of methoxy group and the halogens -F, -Cl and -Br held in position 4 of
the phenyl ring Ar' of compound 8a did not favour inhibitory activity. On the other
hand, the compounds 8b (4-CH ), 8g and 8h showed good inhibition percentages (70.9,
3
8
8.9 and 73.6%, respectively), and 8g showed an IC of 0.6 ± 1.5 µM. A common
50
feature of the latter two compounds is the presence of bulky and hydrophobic groups,
biphenyl (8g) and naphthyl (8h), attached to C1; previous docking models demonstrated
interaction with the subsite S3 enzyme[31].
The 8i-l thiosemicarbazone showed little activity against cruzain, suggesting that
replacements at N4, either methyl or phenyl grouping, are not conducive to enzyme
inhibition. These results confirm data obtained in the study conducted by Du et al.[20]
in which it was observed that the binding of various alkyl and aryl radicals in this
position generated inactive compounds.
By observing the inhibitory potential of aryl thiosemicarbazones 9a-t, in which the
structural variations were made in phenoxy ring Ar, it is noted that several of them (9b,
9
d, 9f-i, 9l-o, 9t) had percentages of inhibition greater than 70%. Relative to compound
a, substitutions at position 4 of the phenyl ring by alkyl substituents ethyl (9b) and
9
tert-butyl (9d) had increased activity, while thiosemicarbazone 9c (4-i-propyl) was less
active. The addition of methoxy substituents at positions 3 and 4 of the phenoxy ring
(9e and 9f, respectively) slightly favour enzyme inhibition, so that the compound
substituted at position 4 (9f) was more active. The work carried out by Du et al.[20] and
Siles et al.[22] explained that the presence of halogens in the phenyl ring of some aryl
thiosemicarbazones contributes to the inhibition of cruzain. In fact, thiosemicarbazones
containing halogens (F, Cl, Br, and I) in the phenyl ring (8f, 9h-p) generally showed
good percentages of inhibition. These values generally decreased according to the size
of the atomic radius of the halogen attached to the phenyl ring at position 4, as can be
observed in compounds 9h (4-F)> 9l (4-Cl)> 8f (4-Br) to 84.0 (IC = 0.4 ± 2.1 µM),
5
0
7
3.6 and 11.8%, respectively. Furthermore, the phenyl ring disubstituted by these
halogens increased the inhibition percentage, as demonstrated by thiosemicarbazones 9i
3-Cl, 4-F) 9m (2,3-diCl) and 9n (3,4-diCl), with inhibition percentages of 93.7, 81.8
and 99.1%, respectively. On the other hand, compounds 9i and 9n are the most potent
(

ACCEPTED MANUSCRIPT
compounds of the present study and exhibited IC values of 0.07 ± 1.2 and 0.008 ± 1.6
5
0
µM, respectively. Corroborating with this work, Du et al. demonstrated that the aryl-
thiosemicarbazone containing chlorine at the 3 and 4 positions in the phenyl ring was
the most active among the tested derivatives[20]. The other thiosemicarbazone that
stood out in the studies conducted by the authors and Siles et al. was that which
contained a bromine atom at position 3 of the phenyl ring. Similarly, compound 9o,
which contains the same substitution in the phenoxy ring, exhibited an inhibition
percentage of 81.3%[20,22].
Considering all evaluated aryl thiosemicarbazones (8a-l, 9a-t), compounds 8g, 9d, 9g-i,
and 9m-o inhibited over 80% of T. cruzi cruzain activity in the screening at 50 µM.
However, the IC was determined only for compounds 8g, 9h-i and 9n. Moreover,
5
0
thiosemicarbazones 9i and 9n are noteworthy because they present an IC of 0.07 ± 1.2
5
0
and 0.008 ± 1.6 µM, respectively, comparable to the most potent cruzain inhibitors
described in the literature, such as compound K11777 (IC = 0.004 µM)[42].
5
0
Thiosemicarbazones 9i and 9n have not been the most active in vitro against the
parasite, although the thiosemicarbazone 9i (IC = 1.7 µM) is among the three most
5
0
active front trypomastigote form. This may be related to the fact that the in vitro test
front cruzain was done directly in the isolated enzyme, avoiding the effect of the
parasite biological barriers, such as permeability cell. Moreover, it is probably that our
compounds act by other mechanisms of action, since some of them exhibited modest
cruzain inhibition percentage and good in vitro activity against the parasite.
The fact is that the strategy of disruption of the planarity mentioned above (Figure
3
)[30], providing a T-shaped conformation by inserting aryl groups at C1 appears to
have been effective. Directly comparing the percentage of cruzain inhibition by
compound (1) with compound 9g, it is observed that compound (1) did not inhibit the
enzyme even at a concentration of 100 µM. Thiosemicarbazone 9g, containing a T-
shaped conformation, inhibited the enzyme with a percentage of approximately 80.7%
using a concentration of 50 µM, half of the former. In addition to compounds (1) and
(2), none of the thiosemicarbazones evaluated in a previous study[30] demonstrated
activity against cruzain.

ACCEPTED MANUSCRIPT
2
.5. Flow cytometry analysis
After confirming that these thiosemicarbazones were able to kill T. cruzi parasites, our
next step was to understand how they lead to cell death. Therefore, we treated Y strain
trypomastigotes with 0.5 ꢀM (average IC values) or 5.0 (10x IC values) of each
5
0
50
thiosemicarbazone and incubated them for 24 h. Cells were then stained with propidium
iodide (PI) and examined by flow cytometry. As shown in Table 4, the positive control
(Triton X) led to 67% of cells positively stained for PI. When compared with untreated
cells, most of the parasite cells treated with thiosemicarbazones positively stained for
PI. Thiosemicarbazone 9c was the most potent among them, exhibiting concentration-
dependent activity (Figure 7). Moreover, compound 9c at 5.0 ꢀM was more efficient in
inducing parasite cell death than Benznidazole at its optimal concentration (25 ꢀM).
Therefore, we suggest that thiosemicarbazone-based treatment causes parasite cell death
through necrosis.
PLEASE, INSERT TABLE 4 HERE
PLEASE, INSERT FIGURE 7 HERE
3
. Conclusions
Using a simple and fast method, 32 aryl thiosemicarbazones were synthesized. The
trypanocidal activity of these compounds was evaluated for three life stages of the
parasite. Most of the compounds exhibited superior activity over the reference drug
BDZ against epimastigote and trypomastigote forms of T. cruzi. Compounds 9a and 9c
showed broad and selective antiparasitic activity against T. cruzi. Compound 9c was
most selective against the extracellular forms of the parasite and showed non-toxicity on
mouse splenocytes at the highest concentration tested. Among all compounds tested, 14
inhibited cruzain at rates higher than 70%, demonstrating that changes in the molecular
conformational and planarity in thiosemicarbazones increases the affinity to the cruzain

ACCEPTED MANUSCRIPT
binding site. Thiosemicarbazones 9i and 9n were shown to be potent inhibitors,
comparable to the compound K11777, a highly potent cruzain inhibitor.
4
. Experimental section
4
.1. General
Most the chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), Merck
Berlin, Germany) or Alfa-Aesar (Ward Hill, MA, USA). Reactions in ultrasound bath
(
were performed in a Unique EM-804 TGR instrument, with a frequency of 40 kHz and
a nominal power of 180 W, and without external heating. Precoated aluminum sheets
(silica gel 60 F254, Merck) were used for thinlayer chromatography (TLC) and spots
were visualized under UV light. Elemental analysis was performed with a Carlo Erba
instrument model E-1110. IR spectra in KBr pellets were acquired at Bruker FT-IR
1
13
spectrophotometer. H and C NMR were recorded on a UnityPlus 400 MHz and
Bruker AMX-300 MHz spectrometer, using DMSO-d as a solvent and trimethylsilane
6
(TMS) as the internal standard. Splitting patterns were defined as; s, singlet; d, doublet;
dd, double doublet; t, triplet; q, quartet; m, multiplet. Chemical shift values were given
in ppm. DEPT was employed to confirm the carbon assignment.
4
.2. Synthesis of 1-phenoxy-2-acetophenones (5a-h; 6a-t). Example for 2-(4-
bromophenoxy)-1-(4-methoxyphenyl)ethan-1-one (5c)
In a round bottom flask with a capacity of 100 ml, were added 13.2 mmol (2.3 g) of 4-
bromophenol, 35 mL of acetone, 13.2 mmol (1.8 g) of powdered K CO and KI in
2
3
catalytic amount. The mixture was kept under magnetic stirring at room temperature.
After 30 minutes, 8.8 mmol (2.0 g) of 2-bromo-1-(4-methoxyphenyl)ethan-1-one are
added in portions. The reaction was kept under magnetic stirring at room temperature
for 3.0 h. After completion of the reaction, the reaction mixture was filtered, so that the
supernatant was evaporated under reduced pressure and the precipitate (K CO )
2
3
discarded. After evaporation, a solid was obtained, which was resuspended using
diethyl ether and then filtered. The crystal obtained was transferred to a desiccator and

ACCEPTED MANUSCRIPT
placed under vacuum SiO . The resulting product was used in the next step without
2
further purification.
4
.3. Synthesis of thiosemicarbazones (8a-l, 9a-t). Example for 2-(4-bromophenoxy)-1-
(4-methoxyphenyl)ethan-1-one thiosemicarbazone (8c)
In a round bottom flask for 100 mL, 8.1 mmol (2.6 g) of 1-phenoxy-2-acetophenone 5c
were dissolved in 30mL EtOH, following by the addition of four drops HCl. The flask
was placed in an ultrasound bath (40 kHz, 180W) and under sonication, 10.5 mmol (1.0
g) of thiosemicarbazide 7a were added in portions to the reaction. After 2.5 h, the
mixture was cooled at 0 ºC and the precipitate was filtered in a Büchner funnel with a
sintered disc filter, washed with coldwater, ethanol and then dried over SiO . After
2
drying, the product was recrystallized from toluene.
4
.3.1. 2-(4-bromophenoxy)-1-phenylethan-1-one thiosemicarbazone (8a)
Recrystallization in toluene afforded beige crystals, yield = 88%. M.p. (ºC): 144-146. IR
-1
1
(
KBr): 3423 and 3314 (NH ), 3235 (N-H), 1599 (C=N) cm . H NMR (300 MHz,
2
DMSO-d ): δ 5.32 (s, 2H, CH ), 6.96 (dd, 2H, J = 9.30, 2.10 Hz, Ar-H), 7.38–7.41 (m,
6
2
3
H, Ar-H), 7.49 (dd, 2H, J = 9.30, 2.10 Hz, Ar-H), 7.90–7.92 (m, 2H, Ar-H), 8.09 (s
1
3
broad, 1H, NH ), 8.48 (s broad, 1H, NH ), 10.83 (s broad, 1H, NH). C NMR (75.5
2
2
MHz, DMSO- d ): δ 61.3 (CH ), 112.7 (Cq Ar), 117.1 (CH Ar), 127.0 (CH Ar), 128.2
6
2
(CH Ar), 129.2 (CH Ar), 132.1 (CH Ar), 135.6 (Cq Ar), 143.7(C=N), 157.0 (C–O, Ar),
1
79.2 (C=S). Anal. Calcd. For C H BrN OS: C, 49.46; H, 3.87; N, 11.54. Found: C,
15 14 3
4
9.88; H, 3.86; N, 11.97.
4
.3.2. 2-(4-bromophenoxy)-1-(p-tolyl)ethan-1-one thiosemicarbazone (8b)
Recrystallization in toluene afforded yellow crystals, yield = 73%. M.p. (ºC): 138. IR
-1
1
(
KBr): 3417 and 3322 (NH ), 3239 (N-H), 1601 (C=N) cm . H NMR (300 MHz,
2
DMSO-d ): δ 5.29 (s, 2H, CH ), 6.94 (dd, 2H, J = 6.90, 2.40 Hz, Ar-H), 7.19 (d, 2H, J =
6
2
8
.10 Hz, Ar-H), 7.48 (dd, 2H, J = 6.90, 2.40 Hz, Ar-H), 7.80 (d, 2H, J = 8.10 Hz, Ar-
1
3
H), 8.05 (s broad, 1H, NH ), 8.45 (s broad, 1H, NH ), 10.76 (s broad, 1H, NH). C
2
2
NMR (75.5 MHz, DMSO- d ): δ 20.8 (CH ), 61.3 (CH ), 112.7 (Cq Ar), 117.1 (CH Ar),
6
3
2
1
26.9 (CH Ar), 128.9 (CH Ar), 132.1 (CH Ar), 132.8 (Cq Ar), 138.9 (Cq Ar), 143.8

ACCEPTED MANUSCRIPT
(
C=N), 156.9 (C–O, Ar), 179.0 (C=S). Anal. Calcd. For C H BrN OS: C, 50.80; H,
1
6
16
3
4
.26; N, 11.11. Found: C, 50.40; H, 4.25; N, 11.55.
4
.3.3. 2-(4-bromophenoxy)-1-(4-methoxyphenyl)ethan-1-one thiosemicarbazone (8c)
Recrystallization in toluene afforded colorless crystals, yield = 76%. M.p. (ºC): 109-
-1 1
1
10. IR (KBr): 3414 and 3334 (NH ), 3247 (N-H), 1600 (C=N) cm . H NMR (300
2
MHz, DMSO-d ): δ 5.29 (s, 2H, CH ), 6.93 (dd, 2H, J = 8.70, 1.20 Hz, Ar-H), 6.95 (dd,
6
2
2
8
1
1
H, J = 9.00, 1.50 Hz, Ar-H), 7.49 (dd, 2H, J = 9.00, 1.50 Hz, Ar-H), 7.88 (dd, 2H, J =
.70, 1.20 Hz, Ar-H), 8.04 (s broad, 1H, NH ), 8.41 (s broad, 1H, NH ), 10.71 (s broad,
2
2
1
3
H, NH). C NMR (75.5 MHz, DMSO- d ): δ 55.2 (CH ), 61.3 (CH ), 112.7 (Cq Ar),
6
3
2
13.6 (CH Ar), 117.2 (CH Ar), 127.9 (Cq Ar), 128.6 (CH Ar), 132.1 (CH Ar), 143.7
(
C=N), 156.9 (C–O, Ar), 160.2 (C Ar - OCH ), 178.9 (C=S). Anal. Calcd. For
3
C H BrN O S: C, 48.74; H, 4.09; N, 10.66. Found: C, 48.53; H, 3.95; N, 10.73.
16
16
3
2
4
.3.4. 2-(4-bromophenoxy)-1-(4-fluorophenyl)ethan-1-one thiosemicarbazone (8d)
Recrystallization in toluene afforded colorless crystals, yield = 69%. M.p. (ºC): 160-
-1 1
1
61. IR (KBr): 3413 and 3339 (NH ), 3241 (N-H), 1606 (C=N) cm . H NMR (300
2
MHz, DMSO-d ): δ 5.30 (s, 2H, CH ), 6.94 (dd, 2H, J = 6.90, 2.10 Hz, Ar-H), 7.17–
6
2
7
8
.23 (m, 2H, Ar-H), 7.48 (dd, 2H, J = 6.90, 2.10 Hz, Ar-H), 7.95–8.00 (m, 2H, Ar-H),
1
3
.13 (s broad, 1H, NH ), 8.47 (s broad, 1H, NH ), 10.84 (s broad, 1H, NH). C NMR
2
2
(
75.5 MHz, DMSO- d ): δ 61.2 (CH ), 112.8 (Cq Ar), 115.1 (CH Ar), 117.1 (CH Ar),
6 2
1
29.4 (CH Ar), 132.1 (CH Ar), 142.9 (C=N), 156.9 (C–O, Ar), 161.1 (Cq Ar), 164.4
(
Cq Ar), 179.2 (C=S). Anal. Calcd. For C H BrFN OS: C, 47.13; H, 3.43; N, 10.99.
15 13 3
Found: C, 47.61; H, 2.87; N, 10.80.
4
.3.5. 2-(4-bromophenoxy)-1-(4-chlorophenyl)ethan-1-one thiosemicarbazone (8e)
Recrystallization in toluene afforded colorless crystals, yield = 71%. M.p. (ºC): 158-
-1 1
1
60. IR (KBr): 3413 and 3336 (NH ), 3249 (N-H), 1597 (C=N) cm . H NMR (300
2
MHz, DMSO-d ): δ 5.30 (s, 2H, CH ), 6.94 (dd, 2H, J = 8.70, 1.80 Hz, Ar-H), 7.42 (dd,
6
2
2
8
1
H, J = 8.70, 1.80 Hz, Ar-H), 7.48 (dd, 2H, J = 8.40, 1.80 Hz, Ar-H), 7.94 (dd, 2H, J =
.40, 1.80 Hz, Ar-H), 8.15 (s broad, 1H, NH ), 8.50 (s broad, 1H, NH ), 10.88 (s broad,
2
2
1
3
H, NH). C NMR (75.5 MHz, DMSO- d ): δ 61.0 (CH ), 112.8 (Cq Ar), 117.1 (CH
6
2
Ar), 128.2 (CH Ar), 128.9 (CH Ar), 132.1 (CH Ar), 133.9 (Cq Ar), 134.5 (Cq Ar),

ACCEPTED MANUSCRIPT
1
4
42.6 (C=N), 156.9 (C–O, Ar), 179.3 (C=S). Anal. Calcd. For C H BrClN OS: C,
1
5
13
3
5.19; H, 3.29; N, 10.54. Found: C, 44.95; H, 2.83; N, 10.54.
4
.3.6. 2-(4-bromophenoxy)-1-(4-bromophenyl)ethan-1-one thiosemicarbazone (8f)
Recrystallization in toluene afforded colorless crystals, yield = 82%. M.p. (ºC): 149-
-1 1
1
50. IR (KBr): 3415 and 3333 (NH ), 3251 (N-H), 1597 (C=N) cm . H NMR (300
2
MHz, DMSO-d ): δ 5.30 (s, 2H, CH ), 6.94 (dd, 2H, J = 8.70, 1.50 Hz, Ar-H), 7.49 (dd,
6
2
2
9
1
H, J = 8.70, 1.50 Hz, Ar-H), 7.57 (dd, 2H, J = 9.00, 1.80 Hz, Ar-H), 7.88 (dd, 2H, J =
.00, 1.80 Hz, Ar-H), 8.16 (s broad, 1H, NH ), 8.52 (s broad, 1H, NH ), 10.90 (s broad,
2
2
1
3
H, NH). C NMR (75.5 MHz, DMSO- d ): δ 61.0 (CH ), 112.8 (Cq Ar), 117.1 (CH
6
2
Ar), 122.8 (Cq Ar), 129.1 (CH Ar), 131.1 (CH Ar), 132.1 (CH Ar), 134.8 (Cq Ar),
1
42.7 (C=N), 156.9 (C–O, Ar), 179.3 (C=S). Anal. Calcd. For C H Br N OS: C,
15 13 2 3
4
0.65; H, 2.96; N, 9.48. Found: C, 41.09; H, 3.55; N, 9.11.
4
.3.7. 1-([1,1'-biphenyl]-4-yl)-2-(4-bromophenoxy)ethan-1-one thiosemicarbazone (8g)
Recrystallization in toluene afforded yellow crystals, yield = 70%. M.p. (ºC): 127-128.
-1 1
IR (KBr): 3424 and 3330 (NH ), 3236 (N-H), 1597 (C=N) cm . H NMR (300 MHz,
2
DMSO-d ): δ 5.36 (s, 2H, CH ), 6.99 (d, 2H, J = 9.00 Hz, Ar-H), 7.38-7.51 (m, 5H, Ar-
6
2
H), 7.67-7.72 (m, 4H, Ar-H), 8.02 (d, 2H, J = 8.70 Hz, Ar-H), 8.16 (s broad, 1H, NH2),
1
3
8
.51 (s broad, 1H, NH ), 10.88 (s broad, 1H, NH). C NMR (75.5 MHz, DMSO- d ): δ
2 6
6
2.0 (CH ), 112.3 (Cq Ar), 120.3 (CH Ar), 122.8 (CH Ar), 124.5 (CH Ar), 126.7 (CH
2
Ar), 128.4 (CH Ar), 130.3 (CH Ar), 133.1 (CH Ar), 134.6 (Cq Ar), 138.5 (Cq Ar),
39.9 (Cq Ar), 143.9 (C=N), 157.1 (C–O, Ar), 179.6 (C=S). Anal. Calcd. For
C H BrN O: C, 57.28; H, 4.12; N, 9.54. Found: C, 57.11; H, 3.75; N, 9.35.
1
21
18
3
4
.3.8. 2-(4-bromophenoxy)-1-(naphthalen-1-yl)ethan-1-one thiosemicarbazone (8h)
Recrystallization in toluene afforded yellow crystals, yield = 78%. M.p. (ºC): 77-80. IR
-1
1
(
KBr): 3417 and 3332 (NH ), 3248 (N-H), 1610 (C=N) cm . H NMR (400 MHz,
2
DMSO-d ): δ 5.45 (s, 2H, CH ), 7.58 (d, 2H, Ar-H), 7.48-7.54 (m, 4H, Ar-H), 7.88-7.96
6
2
(
m, 3H, Ar-H), 8.24 (d, 2H, Ar-H), 8.40 (s broad, 1H, NH ), 8.58 (s broad, 1H, NH ),
2 2
1
3
1
0.88 (s broad, 1H, NH). C NMR (100 MHz, DMSO- d ): δ 61.7 (CH ), 113.3 (Cq
6 2
Ar), 117.7 (CH Ar), 124,9 (CH Ar), 126.8 (CH Ar), 127.2 (CH Ar), 127.3 (CH Ar),
27.9 (CH Ar), 128.1 (CH Ar), 129.0 (CH Ar), 132.6 (CH Ar), 133.1 (Cq Ar), 133.5
1

ACCEPTED MANUSCRIPT
(Cq Ar), 133.7 (Cq Ar), 143.9 (C=N), 157.5 (C–O, Ar), 179.6 (C=S). Anal. Calcd. For
C H BrN OS: C, 55.08; H, 3.89; N, 10.14. Found: C, 54.80; H, 4.07; N, 10.01.
19
16
3
4
.3.9. 2-(4-bromophenoxy)-1-(4-methoxyphenyl)ethan-1-one N-methyl
thiosemicarbazone (8i)
Recrystallization in toluene afforded yellow crystals, yield = 80%. M.p. (ºC): 143-146.
-1 1
IR (KBr): 3332 (N-H), 3254 (N-H), 1554 (C=N) cm . H NMR (400 MHz, DMSO-d ):
6
δ 3.05 (d, 3H, J = 4.39 Hz, CH ), 3.79 (s, 3H, OCH ), 5.28 (s, 2H, CH ), 6.94 (d, 2H, J
3
3
2
=
8.39 Hz, Ar-H), 6.94 (d, 2H, J = 8.79 Hz, Ar-H), 7.47 (d, 2H, J = 8.79 Hz, Ar-H),
7
.87 (d, 2H, J = 8.39 Hz, Ar-H), 8.62 (q, 1H, J = 4.39 Hz, NH), 10.73 (s broad, 1H,
1
3
NH). C NMR (100 MHz, DMSO- d ): δ 31.1 (CH ), 55.2 (OCH ), 61.4 (CH ), 112.7
6
3
3
2
(
Cq Ar), 113.6 (CH Ar), 117.1 (CH Ar), 128.0 (Cq Ar), 128.6 (CH Ar), 132.1 (CH Ar),
43.4 (C=N), 156.9 (C–O, Ar), 160.2 (Cq Ar), 178.5 (C=S). Anal. Calcd. For
C H BrN O S: C, 50.01; H, 4.44; N, 10.29. Found: C, 54.80; H, 4.57; N, 10.01.
1
17
18
3
2
4
.3.10. 2-(4-bromophenoxy)-1-(4-methoxyphenyl)ethan-1-one N-phenyl
thiosemicarbazone (8j)
Recrystallization in toluene afforded colorless crystals, yield = 79%. M.p. (ºC): 159-
-1
1
1
62. IR (KBr): 3255 (N-H), 3210 (N-H), 1550 (C=N) cm . H NMR (400 MHz,
DMSO-d ): δ 3.79 (s, 3H, CH ), 5.36 (s, 2H, CH ), 6.95 (d, 2H, J = 8.79 Hz, Ar-H),
6
3
2
6
.99 (d, 2H, J = 8.39 Hz, Ar-H), 7.22 (t, 1H, J = 7.19 Hz, Ar-H), 7.38 (dd, 2H, J = 7.99,
.19 Hz, Ar-H), 7.50 (d, 2H, J = 8.79 Hz, Ar-H), 7.57 (d, 2H, J = 7.99 Hz, Ar-H), 7.96
7
1
3
(
d, 2H, J = 8.39 Hz, Ar-H), 10.14 (s broad, 1H, NH), 11.05 (s broad, 1H, NH-Ar). C
NMR (100 MHz, DMSO- d ): δ 55.2 (CH ), 61.6 (CH ), 112.8 (Cq Ar), 113.7 (CH Ar),
6
2
2
1
17.2 (CH Ar), 125.4 (CH Ar), 125.9 (CH Ar), 127.8 (Cq Ar), 128.1 (CH Ar), 128.9
CH Ar), 132.1 (CH Ar), 139.0 (C–NH, Ar), 144.6 (C=N), 156.9 (C–O, Ar), 160.4 (Cq
Ar), 176.9 (C=S). Anal. Calcd. For C H BrN O S: C, 56.18; H, 4.29; N, 8.93. Found:
(
2
2
20
3
2
C, 59.88; H, 4.49; N, 9.05.
4
.3.11.
thiosemicarbazone (8k)
Recrystallization in toluene afforded colorless crystals, yield = 83%. M.p. (ºC): 128-
2-(4-bromophenoxy)-1-(4-fluorophenyl)ethan-1-one
N-methyl
-1
1
1
30. IR (KBr): 3345 (N-H), 3249 (N-H), 1561 (C=N) cm . H NMR (400 MHz,
DMSO-d ): δ 3.05 (d, 3H, J = 4.39 Hz, CH ), 5.30 (s, 2H, CH ), 6.93 (d, 2H, J = 8.79
6
3
2

ACCEPTED MANUSCRIPT
Hz, Ar-H), 7.22 (dd, 2H, J = 7.99, 5.59 Hz, Ar-H), 7.47 (d, 2H, J = 8.79 Hz, Ar-H),
7
1
1
.96 (dd, 2H, J = 7.99, 5.59 Hz, Ar-H), 8.62 (q, 1H, J = 4.39 Hz, NH), 10.87 (s broad,
1
3
H, NH). C NMR (100 MHz, DMSO- d ): δ 31.2 (CH ), 61.3 (CH ), 112.8 (Cq Ar),
6
3
2
15.1 (CH Ar), 117.1 (CH Ar), 129.3 (CH Ar), 132.1 (CH Ar), 142.7 (C=N), 156.9 (C–
O, Ar), 161.5 (Cq Ar), 163.9 (Cq Ar), 178.7 (C=S). Anal. Calcd. For C H BrFN OS:
1
6
15
3
C, 48.50; H, 3.82; N, 10.60. Found: C, 48.78; H, 3.94; N, 10.25.
4
.3.12.
thiosemicarbazone (8l)
Recrystallization in toluene afforded colorless crystals, yield = 82%. M.p. (ºC): 174-
2-(4-bromophenoxy)-1-(4-fluorophenyl)ethan-1-one
N-phenyl
-1
1
1
75. IR (KBr): 3284 (N-H), 3238 (N-H), 1552 (C=N) cm . H NMR (400 MHz,
DMSO-d ): δ 5.38 (s, 2H, CH ), 6.98 (d, 2H, J = 8.39 Hz, Ar-H), 7.23 (dd, 2H, J = 8.79,
6
2
6
7
5
.79 Hz, Ar-H), 7.24 (t, 1H, J = 6.79 Hz, Ar-H), 7.39 (dd, 2H, J = 8.39, 5.19 Hz, Ar-H),
.50 (d, 2H, J = 8.39 Hz, Ar-H), 7.55 (d, 2H, J = 8.79 Hz, Ar-H), 8.06 (dd, 2H, J = 8.39,
1
3
.19 Hz, Ar-H), 10.21 (s broad, 1H, NH), 11.18 (s broad, 1H, NH-Ar). C NMR (100
MHz, DMSO- d ): δ 61.6 (CH ), 112.8 (Cq Ar), 115.1 (CH Ar), 117.1 (CH Ar), 125.5
6
2
(CH Ar), 126.1 (CH Ar), 128.1 (CH Ar), 129.7 (CH Ar), 132.1 (CH Ar), 139.0 (C–NH,
Ar), 143.7 (C=N), 156.9 (C–O, Ar), 161.6 (Cq Ar), 164.1 (Cq Ar), 177.3 (C=S). Anal.
Calcd. For C H BrFN OS: C, 55.03; H, 3.74; N, 9.17. Found: C, 55.38; H, 3.49; N,
2
1
17
3
9
.15.
4
.3.13. 1-(4-bromophenyl)-2-phenoxyethan-1-one thiosemicarbazone (9a)
Recrystallization in toluene afforded yellow crystals, yield = 87%. M.p. (ºC): 173-176.
-1 1
IR (KBr): 3419 and 3371 (NH ), 3249 (N-H), 1609 (C=N) cm . H NMR (400 MHz,
2
DMSO-d ): δ 5.31 (s, 2H, CH ), 6.93–6.99 (m, 3H, Ar-H), 7.29–7.34 (m, 2H, Ar-H),
6
2
7
.56 (d, 2H, J = 8.79 Hz, Ar-H), 7.89 (d, 2H, J = 8.79 Hz, Ar-H), 8.15 (s broad, 1H,
1
3
NH ), 8.51 (s broad, 1H, NH ), 10.87 (s broad, 1H, NH). C NMR (100 MHz, DMSO-
2
2
d ): δ 60.9 (CH ), 114.7 (CH Ar), 121.3 (CH Ar), 122.7 (Cq Ar), 129.1 (CH Ar), 129.4
6
2
(CH Ar), 131.1 (CH Ar), 134.9 (Cq Ar), 143.2 (C=N), 157.5 (C–O, Ar), 179.2 (C=S).
Anal. Calcd. For C H BrN OS: C, 49.46; H, 3.87; N, 11.54. Found: C, 49.36; H, 3.91;
1
5
14
3
N, 11.66.
4
.3.14. 1-(4-bromophenyl)-2-(4-ethylphenoxy)ethan-1-one thiosemicarbazone (9b)

ACCEPTED MANUSCRIPT
Recrystallization in toluene afforded yellow crystals, yield = 72%. M.p. (ºC): 142-144.
-1 1
IR (KBr): 3419 and 3369 (NH ), 3248 (N-H), 1610 (C=N) cm . H NMR (400 MHz,
2
DMSO-d ): δ 1.13 (t, 3H, J = 7.59 Hz, CH ), 2.53 (q, 2H, J = 7.59 Hz, CH ), 5.27 (s,
6
3
2
2
H, CH ), 6.87 (d, 2H, J = 8.39 Hz, Ar-H), 7.13 (d, 2H, J = 8.39 Hz, Ar-H), 7.56 (d, 2H,
2
J = 8.79 Hz, Ar-H), 7.89 (d, 2H, J = 8.79 Hz, Ar-H), 8.15 (s broad, 1H, NH ), 8.51 (s
2
1
3
broad, 1H, NH ), 10.83 (s broad, 1H, NH). C NMR (100 MHz, DMSO- d ): δ 15.9
2
6
(CH ), 33.8 (CH ), 61.1 (O-CH ), 114.6 (CH Ar), 122.7 (Cq Ar), 128.6 (CH Ar), 129.1
3 2 2
(CH Ar), 131.1 (CH Ar), 134.9 (Cq Ar), 136.7 (Cq Ar), 143.3 (C=N), 155.5 (C–O, Ar),
1
79.1 (C=S). Anal. Calcd. For C H BrN OS: C, 52.05; H, 4.62; N, 10.71. Found: C,
17 18 3
5
1.57; H, 5.00; N, 10.80.
4
.3.15. 1-(4-bromophenyl)-2-(4-isopropylphenoxy)ethan-1-one thiosemicarbazone (9c)
Recrystallization in toluene and posteriorly in ethanol (80%) afforded colorless crystals,
yield = 77%. M.p. (ºC): 175-177. IR (KBr): 3419 and 3337 (NH ), 3250 (N-H), 1611
2
-1 1
(
C=N) cm . H NMR (400 MHz, DMSO-d ): δ 1.16 (d, 6H, J = 6.80 Hz, CH ), 2.83 (m,
6
3
1
H, J = 6.80 Hz, CH), 5.27 (s, 2H, CH ), 6.88 (d, 2H, J = 8.39 Hz, Ar-H), 7.16 (d, 2H, J
2
=
8.39 Hz, Ar-H), 7.56 (d, 2H, J = 8.79 Hz, Ar-H), 7.90 (d, 2H, J = 8.79 Hz, Ar-H),
1
3
8
.17 (s broad, 1H, NH ), 8.52 (s broad, 1H, NH ), 10.85 (s broad, 1H, NH). C NMR
2 2
(
100 MHz, DMSO- d ): δ 24.9 (CH ), 32.6 (CH), 61.0 (CH ), 114.6 (CH Ar), 122.8 (Cq
6 3 2
Ar), 127.1 (CH Ar), 129.2 (CH Ar), 131.1 (CH Ar), 134.9 (Cq Ar), 141.4 (Cq Ar),
1
43.3 (C=N), 155.6 (C–O, Ar), 179.1 (C=S). Anal. Calcd. For C H BrN OS: C, 53.21;
18 20 3
H, 4.96; N, 10.34. Found: C, 52.85; H, 5.07; N, 10.36.
4
.3.16. 1-(4-bromophenyl)-2-(4-(tert-butyl)phenoxy)ethan-1-one thiosemicarbazone
(9d)
Recrystallization in toluene and posteriorly in ethanol (80%) afforded colorless crystals,
yield = 73%. M.p. (ºC): 176-177. IR (KBr): 3421 and 3330 (NH ), 3251 (N-H), 1610
2
-1 1
(
C=N) cm . H NMR (400 MHz, DMSO-d ): δ 1.24 (s, 9H, CH ), 5.27 (s, 2H, CH ),
6 3 2
6
.88 (d, 2H, J = 8.39 Hz, Ar-H), 7.31 (d, 2H, J = 8.39 Hz, Ar-H), 7.56 (d, 2H, J = 8.79
Hz, Ar-H), 7.90 (d, 2H, J = 8.79 Hz, Ar-H), 8.17 (s broad, 1H, NH ), 8.53 (s broad, 1H,
2
1
3
NH ), 10.85 (s broad, 1H, NH). C NMR (100 MHz, DMSO- d ): δ 31.3 (CH ), 33.8 [C
2
6
3
(
CH )], 60.9 (CH ), 114.2 (CH Ar), 122.8 (Cq Ar), 126.1 (CH Ar), 129.2 (CH Ar),
3 2
1
31.1 (CH Ar), 134.9 (Cq Ar), 143.3 (C=N), 143.6 (Cq Ar), 155.3 (C–O, Ar), 179.2

ACCEPTED MANUSCRIPT
(
C=S). Anal. Calcd. For C H BrN OS: C, 54.29; H, 5.28; N, 10.00. Found: C, 53.98;
19
22
3
H, 5.54; N, 9.94.
4
.3.17. 1-(4-bromophenyl)-2-(3-methoxyphenoxy)ethan-1-one thiosemicarbazone (9e)
Recrystallization in toluene and posteriorly in ethanol (80%) afforded yellow crystals,
yield = 80%. M.p. (ºC): 186-187. IR (KBr): 3384 and 3333 (NH ), 3261 (N-H), 1596
2
-1 1
(
C=N) cm . H NMR (400 MHz, DMSO-d ): δ 3.71 (s, 3H, CH ), 5.30 (s, 2H, CH ),
6 3 2
6
.53 (d, 2H, J = 8.39 Hz, Ar-H), 6.56 (s, 1H, Ar-H), 7.19 (t, 1H, , J = 8.39 Hz, Ar-H),
.57 (d, 2H, J = 8.39 Hz, Ar-H), 7.90 (d, 2H, J = 8.39 Hz, Ar-H), 8.17 (s broad, 1H,
7
1
3
NH ), 8.53 (s broad, 1H, NH ), 10.87 (s broad, 1H, NH). C NMR (100 MHz, DMSO-
2
2
d ): δ 55.1 (CH ), 60.9 (CH ), 100.9 (CH Ar), 107.1 (CH Ar), 122.8 (Cq Ar), 129.2 (CH
6
3
2
Ar), 129.9 (CH Ar), 131.1 (CH Ar), 132.7 (CH Ar), 134.8 (Cq Ar), 143.2 (C=N), 158.7
(
C–O, Ar), 160.4 (C Ar - OCH ), 179.2 (C=S). Anal. Calcd. For C H BrN O S: C,
3
16 16
3
2
4
8.74; H, 4.09; N, 10.66. Found: C, 48.13; H, 4.24; N, 10.88.
4
.3.18. 1-(4-bromophenyl)-2-(4-methoxyphenoxy)ethan-1-one thiosemicarbazone (9f)
Recrystallization in toluene and posteriorly in ethanol (80%) afforded colorless crystals,
yield = 78%. M.p. (ºC): 161. IR (KBr): 3417 and 3327 (NH ), 3254 (N-H), 1611 (C=N)
2
-1 1
cm . H NMR (400 MHz, DMSO-d ): δ 3.69 (s, 3H, CH ), 5.25 (s, 2H, CH ), 6.86 (d,
6
3
2
2
H, J = 9.20 Hz, Ar-H), 6.90 (d, 2H, J = 9.20 Hz, Ar-H), 7.56 (d, 2H, J = 8.39 Hz, Ar-
H), 7.89 (d, 2H, J = 8.39 Hz, Ar-H), 8.16 (s broad, 1H, NH ), 10.81 (s broad, 1H, NH);
2
1
3
8
.52 (s broad, 1H, NH ). C NMR (100 MHz, DMSO- d ): δ 55.4 (CH ), 61.7 (CH ),
2 6 3 2
1
14.6 (CH Ar), 115.8 (CH Ar), 122.8 (Cq Ar), 129.2 (CH Ar), 131.1 (CH Ar), 134.9
(
Cq Ar), 143.4 (C=N), 151.4 (C Ar - OCH ), 154.0 (C–O, Ar), 179.1 (C=S). Anal.
3
Calcd. For C H BrN O S: C, 48.74; H, 4.09; N, 10.66. Found: C, 48.42; H, 4.36; N,
1
6
16
3
2
1
0.66.
4
.3.19. N-(4-(2-(4-bromophenyl)-2-oxoethoxy)phenyl)acetamide thiosemicarbazone
(9g)
Recrystallization in toluene afforded colorless crystals, yield = 69%. M.p. (ºC): 187-
-1 1
1
90. IR (KBr): 3424 and 3368 (NH ), 3263 (N-H), 1616 (C=N) cm . H NMR (400
2
MHz, DMSO-d ): δ 2.00 (s, 3H, CH ), 5.26 (s, 2H, CH ), 6.89 (d, 2H, J = 8.39 Hz, Ar-
6
3
2
H), 7.49 (d, 2H, J = 8.39 Hz, Ar-H), 7.56 (d, 2H, J = 7.99 Hz, Ar-H), 7.88 (d, 2H, J =
.99 Hz, Ar-H), 8.16 (s largo, 1H, NH ), 8.52 (s broad, 1H, NH ), 9.90 (s broad, 1H,
7
2
2

ACCEPTED MANUSCRIPT
1
3
NH), 10.84 (s broad, 1H, N-NH). C NMR (100 MHz, DMSO- d ): δ 23.8 (CH ), 61.2
6
3
(
CH ), 114.9 (CH Ar), 120.3 (CH Ar), 122.8 (Cq Ar), 129.2 (CH Ar), 131.1 (CH Ar),
2
1
33.4 (Cq Ar), 134.9 (Cq Ar), 143.3 (C=N), 153.1 (C–O, Ar), 167.8 (C=O), 179.2
(
C=S). Anal. Calcd. For C H BrN O S: C, 48.46; H, 4.07; N, 13.30. Found: C, 48.10;
17
17
4
2
H, 3.84; N, 12.83.
4
.3.20. 1-(4-bromophenyl)-2-(4-fluorophenoxy)ethan-1-one thiosemicarbazone (9h)
Recrystallization in toluene and posteriorly in ethanol (80%) afforded yellow crystals,
yield = 71%. M.p. (ºC): 156-158. IR (KBr): 3431 and 3348 (NH ), 3254 (N-H), 1607
2
-1 1
(
C=N) cm . H NMR (400 MHz, DMSO-d ): δ 5.30 (s, 2H, CH ), 6.98-7.01 (m, 2H,
6 2
Ar-H), 7.11-7.16 (m, 2H, Ar-H), 7.56 (dd, 2H, J = 8.39, 2.00 Hz, Ar-H), 7.87 (dd, 2H, J
8.39, 2.00 Hz, Ar-H), 8.05 (s broad, 1H, NH ), 8.39 (s broad, 1H, NH ), 10.74 (s
=
2
2
1
3
broad, 1H, NH). C NMR (100 MHz, DMSO- d ): δ 61.8 (CH ), 115.6 (CH Ar), 116.2
6
2
(CH Ar), 122.5 (Cq Ar), 128.9 (CH Ar), 130.9 (CH Ar), 134.7 (Cq Ar), 142.9 (C=N),
1
4
53.7 (Cq Ar), 158.0 (C–O, Ar), 179.2 (C=S). Anal. Calcd. For C H BrFN OS: C,
15 13 3
7.13; H, 3.43; N, 10.99. Found: C, 47.09; H, 3.13; N, 11.20.
4
.3.21.
1-(4-bromophenyl)-2-(3-chloro-4-fluorophenoxy)ethan-1-one
thiosemicarbazone (9i)
Recrystallization in toluene afforded yellow crystals, yield = 74%. M.p. (ºC): 129-130.
-1 1
IR (KBr): 3463 and 3341 (NH ), 3219 (N-H), 1596 (C=N) cm . H NMR (400 MHz,
2
DMSO-d ): δ 5.31 (s, 2H, CH ), 6.94-6.98 (m, 1H, Ar-H), 7.23-7.25 (m, 1H, Ar-H),
6
2
7
.32-7.37 (m, 1H, Ar-H), 7.57 (d, 2H, J = 8.79 Hz, Ar-H), 7.87 (d, 2H, J = 8.79 Hz, Ar-
1
3
H), 8.06 (s broad, 1H, NH ), 8.39 (s broad, 1H, NH ), 10.78 (s broad, 1H, NH). C
2
2
NMR (100 MHz, DMSO- d ): δ 61.7 (CH ), 115.5 (CH Ar), 116.4 (CH Ar), 117.0 (CH
6
2
Ar), 122.6 (Cq Ar), 128.9 (CH Ar), 130.9 (CH Ar), 134.7 (Cq Ar), 142.3 (C=N), 150.9
Cq Ar), 153.3 (Cq Ar), 154.0 (C–O, Ar), 179.3 (C=S). Anal. Calcd. For
C H BrClFN OS: C, 43.24; H, 2.90; N, 10.08. Found: C, 42.94; H, 2.97; N, 9.91.
(
15
12
3
4
.3.22. 1-(4-bromophenyl)-2-(2-chlorophenoxy)ethan-1-one thiosemicarbazone (9j)
Recrystallization in toluene afforded colorless crystals, yield = 75%. M.p. (ºC): 159-
-1 1
1
62. IR (KBr): 3420 and 3350 (NH ), 3256 (N-H), 1600 (C=N) cm . H NMR (400
2
MHz, DMSO-d ): δ 5.37 (s, 2H, CH ), 7.00 (t, 1H, J = 7.99 Hz, Ar-H), 7.24 (d, 1H,
6
2
7
.59 Hz, Ar-H), 7.35 (t, 1H, J = 7.59 Hz, Ar-H), 7.42 (d, 1H, J = 7.99 Hz, Ar-H), 7.57

ACCEPTED MANUSCRIPT
(d, 2H, J = 8.79 Hz, Ar-H), 7.91 (d, 2H, J = 8.79 Hz, Ar-H), 8.19 (s broad, 1H, NH2),
1
3
8
6
.54 (s broad, 1H, NH ), 10.96 (s broad, 1H, NH). C NMR (100 MHz, DMSO- d ): δ
2 6
2.1 (CH ), 114.4 (CH Ar), 121.5 (Cq Ar), 122.2 (CH Ar), 122.6 (Cq Ar), 127.9 (CH
2
Ar), 129.0 (CH Ar), 129.7 (CH Ar), 130.9 (CH Ar), 134.8 (Cq Ar), 142.3 (C=N), 152.8
(
C–O, Ar), 179.3 (C=S). Anal. Calcd. For C H BrClN OS: C, 45.19; H, 3.29; N,
15 13 3
1
0.54. Found: C, 45.24; H, 3.13; N, 10.55.
4
.3.23. 1-(4-bromophenyl)-2-(3-chlorophenoxy)ethan-1-one thiosemicarbazone (9k)
Recrystallization in toluene afforded colorless crystals, yield = 83%. M.p. (ºC): 171. IR
-1
1
(
KBr): 3418 and 3346 (NH ), 3247 (N-H), 1610 (C=N) cm . H NMR (400 MHz,
2
DMSO-d ): δ 5.32 (s, 2H, CH ), 6.92 (d, 1H, J = 7.99 Hz, Ar-H), 7.05 (d, 1H, J = 6.79
6
2
Hz, Ar-H), 7.09 (s, 1H, Ar-H), 7.33 (dd, 1H, J = 7.99, 6.79 Hz, Ar-H), 7.56 (d, 2H, J =
8
1
1
.39 Hz, Ar-H), 7.89 (d, 2H, J = 8.39 Hz, Ar-H), 8.16 (s broad, 1H, NH ), 8.51 (s broad,
2
13
H, NH ), 10.93 (s broad, 1H, NH). C NMR (100 MHz, DMSO- d ): δ 60.9 (CH ),
2
6
2
14.1 (CH Ar), 114.7 (CH Ar), 121.1 (CH Ar), 122.8 (Cq Ar), 129.2 (CH Ar), 130.8
(CH Ar), 131.1 (CH Ar), 131.1 (CH Ar), 133.6 (Cq Ar), 134.9 (Cq Ar), 143.5 (C=N),
1
1
58.6 (C–O, Ar), 179.3 (C=S). Anal. Calcd. For C H BrClN OS: C, 45.19; H, 3.29; N,
15 13 3
0.54. Found: C, 44.98; H, 3.40; N, 10.89.
4
.3.24. 1-(4-bromophenyl)-2-(4-chlorophenoxy)ethan-1-one thiosemicarbazone (9l)
Recrystallization in toluene afforded colorless crystals, yield = 81%. M.p. (ºC): 107-
-1 1
1
10. IR (KBr): 3420 and 3341 (NH ), 3252 (N-H), 1609 (C=N) cm . H NMR (400
2
MHz, DMSO-d ): δ 5.30 (s, 2H, CH ), 6.97 (d, 2H, J = 8.39 Hz, Ar-H), 7.36 (d, 2H, J =
6
2
8
.39 Hz, Ar-H), 7.56 (d, 2H, J = 8.39 Hz, Ar-H), 7.88 (d, 2H, J = 8.39 Hz, Ar-H), 8.17
1
3
(
s broad, 1H, NH ), 8.53 (s broad, 1H, NH ), 10.91 (s broad, 1H, NH). C NMR (100
2 2
MHz, DMSO- d ): δ 61.0 (CH ), 116.6 (CH Ar), 122.8 (Cq Ar), 125.3 (CH Ar), 129.2
6
2
(CH Ar), 131.1 (CH Ar), 134.8 (Cq Ar), 142.7 (C=N), 156.5 (C–O, Ar), 179.2 (C=S).
Anal. Calcd. For C H BrClN OS: C, 45.19; H, 3.29; N, 10.54. Found: C, 45.59; H,
1
5
13
3
3
.60; N, 10.17.
4
.3.25. 1-(4-bromophenyl)-2-(2,3-dichlorophenoxy)ethan-1-one thiosemicarbazone
(9m)
Recrystallization in toluene and posteriorly in ethanol (80%) afforded colorless crystals,
yield = 79%. M.p. (ºC): 172-173. IR (KBr): 3417 and 3327 (NH ), 3265 (N-H), 1594
2

ACCEPTED MANUSCRIPT
-1 1
(
C=N) cm . H NMR (400 MHz, DMSO-d ): δ 5.40 (s, 2H, CH ), 7.24 (d, 1H, J = 8.39
6 2
Hz, Ar-H), 7.26 (d, 1H, J = 8.39 Hz, Ar-H), 7.38 (t, 1H, J = 8.39 Hz, Ar-H), 7.56 (d,
H, J = 7.59 Hz, Ar-H), 7.91 (d, 2H, J = 7.59 Hz, Ar-H), 8.19 (s broad, 1H, NH ), 8.53
2
2
1
3
(
s broad, 1H, NH ), 11.00 (s broad, 1H, NH). C NMR (100 MHz, DMSO- d ): δ 62.0
2 6
(
CH ), 112.8 (CH Ar), 120.1 (Cq Ar), 122.6 (CH Ar), 122.8 (Cq Ar), 128.3 (CH Ar),
2
1
29.2 (CH Ar), 131.1 (CH Ar), 132.2 (Cq Ar), 134.7 (Cq Ar), 141.8 (C=N), 154.6 (C–
O, Ar), 179.3 (C=S). Anal. Calcd. For C H BrCl N OS: C, 41.59; H, 2.79; N, 9.70.
1
5
12
2
3
Found: C, 41.34; H, 2.28; N, 9.69.
4
.3.26. 1-(4-bromophenyl)-2-(3,4-dichlorophenoxy)ethan-1-one thiosemicarbazone (9n)
Recrystallization in toluene and posteriorly in ethanol (80%) afforded colorless crystals,
yield = 76%. M.p. (ºC): 162-165. IR (KBr): 3474 and 3352 (NH ), 3167 (N-H), 1590
2
-1 1
(
C=N) cm . H NMR (400 MHz, DMSO-d ): δ 5.32 (s, 2H, CH ), 6.97 (dd, 1H, J =
6 2
2
,80 Hz, J = 8.79 Hz, Ar-H), 7.30 (d, 1H, J = 2.80 Hz, Ar-H), 7.56 (d, 1H, J = 8.79 Hz,
Ar-H), 7.57 (d, 2H, J = 8.39 Hz, Ar-H), 7.89 (d, 2H, J = 8.39 Hz, Ar-H), 8.18 (s broad,
1
3
1
H, NH ), 8.53 (s broad, 1H, NH ), 10.96 (s broad, 1H, NH). C NMR (100 MHz,
2 2
DMSO- d ): δ 61.1 (CH ), 116.1 (CH Ar), 116.7 (CH Ar), 122.8 (Cq Ar), 123.1 (Cq
6
2
Ar), 129.2 (CH Ar), 130.9 (CH Ar), 131.2 (CH Ar), 131.5 (Cq Ar), 134.8 (Cq Ar),
1
4
42.1 (C=N), 157.2 (C–O, Ar), 179.3 (C=S). Anal. Calcd. For C H BrCl N OS: C,
15 12 2 3
1.59; H, 2.79; N, 9.70. Found: C, 41.62; H, 2.76; N, 9.63.
4
.3.27. 2-(3-bromophenoxy)-1-(4-bromophenyl)ethan-1-one thiosemicarbazone (9o)
Recrystallization in toluene afforded colorless crystals, yield = 90%. M.p. (ºC): 183. IR
-1
1
(
KBr): 3417 and 3319 (NH ), 3222 (N-H), 1603 (C=N) cm . H NMR (400 MHz,
2
DMSO-d ): δ 5.02 (s, 2H, CH ), 6.98 (d, 1H, J = 7.99 Hz, Ar-H), 7.12 (d, 1H, J = 7.99
6
2
Hz, Ar-H), 7.18 (s, 1H, Ar-H), 7.22 (t, 1H, J = 7.99 Hz, Ar-H), 7.33 (d, 2H, J = 8.39 Hz,
Ar-H), 7.70 (d, 2H, J = 8.39 Hz, Ar-H), 8.09 (s broad, 1H, NH ), 8.48 (s broad, 1H,
2
1
3
NH ), 9.73 (s broad, 1H, NH). C NMR (100 MHz, DMSO- d ): δ 60.8 (CH ), 114.4
2
6
2
(
CH Ar), 117.7 (CH Ar), 122.0 (Cq Ar), 123.2 (Cq Ar), 124.1 (CH Ar), 130.0 (CH Ar),
30.5 (Cq Ar), 131.2 (CH Ar), 132.1 (CH Ar), 134.8 (Cq Ar), 142.4 (C=N), 158.6 (C–
O, Ar), 179.2 (C=S). Anal. Calcd. For C H Br N OS: C, 40.65; H, 2.96; N, 9.48.
1
1
5
13
2
3
Found: C, 40.72; H, 2.86; N, 9.39.
4
.3.28. 1-(4-bromophenyl)-2-(4-iodophenoxy)ethan-1-one thiosemicarbazone (9p)

ACCEPTED MANUSCRIPT
Recrystallization in toluene afforded colorless crystals, yield = 78%. M.p. (ºC): 181-
-1 1
1
83. IR (KBr): 3417 and 3325 (NH ), 3252 (N-H), 1605 (C=N) cm . H NMR (400
2
MHz, DMSO-d ): δ 5.28 (s, 2H, CH ), 6.80 (d, 2H, J = 8.79 Hz, Ar-H), 7.56 (d, 2H, J =
6
2
8
.39 Hz, Ar-H), 7.62 (d, 2H, J = 8.79 Hz, Ar-H), 7.87 (d, 2H, J = 8.39 Hz, Ar-H), 8.16
1
3
(
s broad, 1H, NH ), 8.52 (s broad, 1H, NH ), 10.90 (s broad, 1H, NH). C NMR (100
2 2
MHz, DMSO- d ): δ 60.8 (CH ), 84.1 (Cq Ar), 117.6 (CH Ar), 122.8 (Cq Ar), 129.2
6
2
(CH Ar), 131.1 (CH Ar), 134.8 (Cq Ar), 137.9 (CH Ar), 142.8 (C=N), 157.5 (C–O, Ar),
1
3
79.2 (C=S). Anal. Calcd. For C H BrIN OS: C, 36.76; H, 2.67; N, 8.57. Found: C,
15 13 3
6.22; H, 2.26; N, 8.47.
4
.3.29. 2-([1,1'-biphenyl]-3-yloxy)-1-(4-bromophenyl)ethan-1-one thiosemicarbazone
(9q)
Recrystallization in toluene afforded colorless crystals, yield = 70%. M.p. (ºC): 161-
-1 1
1
63. IR (KBr): 3419 and 3337 (NH ), 3294 (N-H), 1605 (C=N) cm . H NMR (400
2
MHz, DMSO-d ): δ 5.02 (s, 2H, CH ), 6.98 (d, 1H, J = 7.99 Hz, Ar-H), 7.12 (d, 1H, J =
6
2
7
7
1
1
.99 Hz, Ar-H), 7.18 (s, 1H, Ar-H), 7.58 (d, 2H, J = 8.39 Hz, Ar-H), 7.64 (d, 2H, J =
.19 Hz, Ar-H), 7.95 (d, 2H, J = 8.39 Hz, Ar-H), 8.20 (s broad, 1H, NH ), 8.55 (s broad,
2
13
H, NH ), 10.93 (s broad, 1H, NH). C NMR (100 MHz, DMSO- d ): δ 60.8 (CH ),
2
6
2
12.7 (CH Ar), 114.3 (CH Ar), 119.7 (CH Ar), 122.8 (Cq Ar), 126.7 (CH Ar), 127.6
(
CH Ar), 128.9 (CH Ar), 129.2 (CH Ar), 130.0 (CH Ar), 131.1 (CH Ar), 134.9 (Cq Ar),
39.8 (Cq Ar), 139.8 (Cq Ar), 141.6 (Cq Ar), 143.2 (C=N), 157.9 (C–O, Ar), 179.3
C=S). Anal. Calcd. For C H BrN OS: C, 57.28; H, 4.12; N, 9.54. Found: C, 56.91; H,
1
(
21
18
3
4
.27; N, 9.60.
4
.3.30. 2-([1,1'-biphenyl]-4-yloxy)-1-(4-bromophenyl)ethan-1-one thiosemicarbazone
(9r)
Recrystallization in toluene afforded colorless crystals, yield = 84%. M.p. (ºC): 208-
-1 1
2
10. IR (KBr): 3419 and 3347 (NH ), 3294 (N-H), 1608 (C=N) cm . H NMR (400
2
MHz, DMSO-d ): δ 5.36 (s, 2H, CH ), 7.05 (d, 2H, J = 8.39 Hz, Ar-H), 7.31 (t, 1H, J =
6
2
7
.19 Hz, Ar-H), 7.42 (dd, 2H, J = 8.39, 7.19 Hz, Ar-H), 7.58 (d, 2H, J = 8.39 Hz, Ar-
H), 7.59 (d, 2H, J = 7.99 Hz, Ar-H), 7.62 (d, 2H, J = 8.39 Hz, Ar-H), 7.92 (d, 2H, J =
.99 Hz, Ar-H), 8.19 (s largo, 1H, NH ), 8.54 (s broad, 1H, NH ), 10.93 (s broad, 1H,
7
2
2
1
3
NH). C NMR (100 MHz, DMSO- d ): δ 61.4 (CH ), 115.7 (CH Ar), 123.2 (Cq Ar),
6
2
1
26.7 (CH Ar), 127.3 (CH Ar), 128.2 (CH Ar), 129.3 (CH Ar), 129.6 (CH Ar), 131.6