Phenoxyacetohydrazones against Trypanosoma cruzi

Article abstract of DOI:10.1007/s00044-021-02768-9

Herein, we reported the design, synthesis, antitrypanosomal and cytotoxic evaluation of a new phenoxyacetohydrazones series. All derivatives were assayed against bloodstream trypomastigote forms of T. cruzi (Y strain) and intracellular amastigotes using the model of L-929 cells infected with trypomastigotes of the Tulahuen strain. Compound (E)-N′-(3.4-dihydroxybenzylidene)-2-phenoxyacetohydrazide (11) showed activity against trypomastigotes (IC₅₀/24 h = 10.3 μM) equivalent to that of benznidazole and with selectivity index (SI) = 46. Against infected cultures, (E)-N′-((5-nitrofuran-2-yl) methylene)-2-phenoxyacetohydrazide (19) was active at the nanomolar range (IC₅₀/96 h = 40 nM), being about 38-fold more active than the standard drug and with SI equal to 2500. Thus, derivatives 11 and 19 could be considered a good prototypes for the development of new candidates for Chagas disease therapy. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-021-02768-9

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:1703–1712
https://doi.org/10.1007/s00044-021-02768-9
ORIGINAL RESEARCH
Phenoxyacetohydrazones against Trypanosoma cruzi
1
,2 ●
4 ●
2 ●
3 ●
3 ●
4 ●
Camila Capelini
Kátia R. de Souza Juliana M. C. Barbosa Kelly Salomão Policarpo A. Sales Junior
Silvane M. F. Murta Solange M. S. V. Wardell James L. Wardell Edson F. da Silva Samir A. Carvalho1
5 ●
6 ●
1 ●
Received: 13 April 2021 / Accepted: 12 July 2021 / Published online: 27 July 2021
©
The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021, corrected publication 2021
Abstract
Herein, we reported the design, synthesis, antitrypanosomal and cytotoxic evaluation of a new phenoxyacetohydrazones
series. All derivatives were assayed against bloodstream trypomastigote forms of T. cruzi (Y strain) and intracellular
amastigotes using the model of L-929 cells infected with trypomastigotes of the Tulahuen strain. Compound (E)-N′-(3.4-
dihydroxybenzylidene)-2-phenoxyacetohydrazide (11) showed activity against trypomastigotes (IC /24 h = 10.3 μM)
5
0
equivalent to that of benznidazole and with selectivity index (SI) = 46. Against infected cultures, (E)-N′-((5-nitrofuran-2-
yl) methylene)-2-phenoxyacetohydrazide (19) was active at the nanomolar range (IC /96 h = 40 nM), being about 38-fold
5
0
more active than the standard drug and with SI equal to 2500. Thus, derivatives 11 and 19 could be considered a good
prototypes for the development of new candidates for Chagas disease therapy.
Graphical Abstract
Introduction
[1, 2]. It is estimated that 752,000 working days per year are
lost due to premature deaths and U$ 1.2 billion in lost
productivity in seven southernmost American countries.
Brazilian absenteeism of workers affected by Chagas dis-
ease represented an estimated minimum of $5.6 million per
year. Due to the migration of T. cruzi-infected people to
other continents, Chagas disease is also reported in non-
endemic regions, such as the European continent [3–5].
The treatment of Chagas disease is restricted to the nitro
derivatives nifurtimox and benznidazole and both are
effective in reducing the duration and clinical severity of the
Chagas disease is a parasitic illness resulting from infection
by the hemoflagellate parasite Trypanosoma cruzi. It is
endemic in 21 countries in Latin America and affects 6–7
million people worldwide, causing 10,000 deaths per year
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1007/s00044-
0
21-02768-9.
3
*
Samir A. Carvalho
Laboratório de Biologia Celular, Instituto Oswaldo Cruz,
samircar@gmail.com
Fundação Oswaldo Cruz, 21040-900 Rio de Janeiro, RJ, Brazil
4
Instituto René Rachou—Fundação Oswaldo Cruz, 30190002
Belo Horizonte, MG, Brazil
1
2
Instituto de Tecnologia em Fármacos—Farmanguinhos, Fundação
Oswaldo Cruz, 21041-250 Rio de Janeiro, RJ, Brazil
5
CHEMSOL, 1 Harcourt Road, Aberdeen AB15 5NY, Scotland
Laboratório de Físico-Química de Materiais, Seção de Engenharia
Química, Instituto Militar de Engenharia, Praça General Tibúrcio
6
Department of Chemistry, University of Aberdeen, Old Aberdeen
8
0, 22290-270 Rio de Janeiro, Brazil
AB24 3UE, Scotland

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Medicinal Chemistry Research (2021) 30:1703–1712
Fig. 1 Design of
phenoxyacetohydrazones
derivatives 3–19
disease. The available chemotherapy is unsatisfactory as a
result of severe side effects and restricted efficacy in the
chronic phase of the disease [6–8].
could influence the antiparasitic profile of this new series.
Derivatives substituted in the para position (4–9) were
planned due to the different lipophilic and electronic effects
on the ring in order to allow the evaluation of the different
contributions to the trypanocidal activity (Table 1). Deri-
vative (10) was planned due to the ability of the o-hydro-
xybenzylidene N-acylhydrazone framework to form
an electrophilic quinone methide intermediate that could
interact with nucleophilic sites in target enzymes of T. cruzi,
e.g., cysteine proteases [10]. Derivatives (12, 15, and 16)
were planned due to the recognized ability of these deri-
vatives to have antiprotozoal activity [16]. Both intracellular
amastigotes and circulating trypomastigotes forms of T.
cruzi are relevant in drug development assays since they are
present in the vertebrate host during the acute and chronic
phases of Chagas’ disease. Then, we have elected two types
of in vitro assays, the direct effect on bloodstream trypo-
mastigote form after 24 h of treatment and the effect on the
intracellular proliferation of amastigotes after 96 h. Benz-
nidazole was used as a positive control against T. cruzi and
the cytotoxicity was determined in mouse peritoneal
macrophages (24 h) and mouse fibroblast L929 cells (96 h).
The use N-acylhydrazones (NAH) plays a strategic role
in medicinal chemistry since the imine vicinal to the amide
group allows the insertion of different substituents to create
a library of bioactive compounds, which can assay against
different targets as described by some come cysteinyl pro-
teases inhibitors [9–12]. In this context, compound 1 dis-
played an excellent cruzain inhibitory activity (IC =
5
0
2
00 nM) and a considerable in vitro potency against T. cruzi
(
IC = 16.2 μM) [13]. Our group reported compound 2
5
0
containing a hydrazone-cathecol subunit which demon-
strated an important profile over T. cruzi, possibly inter-
fering in the oxidative metabolism [14].
In our effort to develop new potent trypanocidal com-
pounds, we constructed a new class of phenoxyacetohy-
drazones designed by molecular hybridization between a
potent cruzain inhibitor represented by the 2-
phenoxyacetamide derivative (1) (IC = 200 nM) [13]
50
and the 1,3,4-thiadiazole-2-arylhydrazone derivative (2)
(
IC = 5.3 μM) [14]. The design concept of these com-
5
0
pounds generates a NAH subunit with the aim to poten-
tialize the cruzain inhibitor framework of (1) and explore
the introduction of a cathecol moiety a potential radical
scavenger group [15]. The planned compounds were syn-
thesized with substituents with different stereoelectronic
properties attached to the phenyl subunit A (3–16) (Fig. 1)
and heterocycles derivatives (17–19). The variations of
substituents in the phenyl subunit A were introduced in
order to assess how the electronic effect of the substituents
Results and discussion
Chemistry
The synthetic route planned to achieve the NAH targets
compounds (3–19) consists of three steps. To a suspen-
sion of phenol (20) and potassium carbonate in

Medicinal Chemistry Research (2021) 30:1703–1712
1705
Table 1 In vitro activity, cytotoxicity, physical and spectral properties of the phenoxyacetohydrazones (3–19)
Comp.
R1
R2
R3
R4
Conf. sp/apa
IC50/24 h (μM)b,c,d
LC50/24 h (μM)e
IC50/96 h (μM)b,f,d
LC50/96 h (μM)g
3
4
5
6
7
8
9
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
0.65:1.0
0.70:1.0
0.65:1.0
0.65:1.0
0.70:1.0
0.60:1.0
0.70:1.0
0.70:1.0
0.80:1.0
0.80:1.0
0.80:1.0
0.80:1.0
0.80:1.0
0.60:1.0
0.55:1.0
1.0:1.0
>1000
ND
IN
ND
H
Cl
H
>1000
ND
IN
ND
H
Br
H
>1000
ND
>100
97.6
ND
H
F
H
>1000
ND
>100
ND
H
OCH3
NO2
OH
H
H
>1000
ND
>100
82.3
H
H
>1000
ND
>250h
>100
ND
H
H
53.3 ± 7.6
89.6 ± 7.6
10.3 ± 1.4
405.0 ± 68.8
279.4 ± 19.9
686.1 ± 61.4
>1000
IN
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
H
H
109.7 ± 11.5
472.0 ± 25.46
>250h
22.7
<100
>200
ND
OH
OCH3
OCH3
OH
OCH3
H
OH
OCH3
OH
OCH3
OCH3
H
157.6
>100
>100
IN
H
H
346.0 ± 14.1
797.3 ± 55.5
ND
ND
H
ND
OCH3
>100
17.5
ND
O–CH2–O
>1000
ND
>50
4-pyridine
114.0 ± 6.1
175.4 ± 8.0
19.11 ± 2.2
8.8 ± 1.1
>1000
>200h
47.7
>100
ND
5-bromo-2-thiophene
5-nitro-2-furane
Benznidazole
IN
0.90:1.0
–
54.2 ± 9.5
>1000
0.04 ± 0.014
1.5 ± 0.4
100
2113 ± 271.5
IN inactive, ND not determined
aData obtained at 400 MHz, using DMSO-d6 as solvent
bMean ± standard deviation of at least four separated experiments
cTreatment for 24 h at 37 °C of trypomastigotes of T. cruzi (Y strain)
dStatistical difference between compounds (p < 0.05)
eTreatment for 24 h at 37 °C of non-infected peritoneal macrophages
f
Treatment for 96 h at 37 °C of L929 cells infected with T. cruzi (Tulahuen strain expressing the β-galactosidase gene)
Treatment for 96 h at 37 °C of the non-infected cell (L929)
g
hUp to this concentration because in higher concentrations they precipitated on the cells thus masking the result
acetonitrile, was added methyl bromoacetate (21) and the
reaction was warmed till reflux to furnish methyl 2-
phenoxyacetate (22) in 96% yield [17]. Compound (22)
was confirmed by the spectra in the infrared region
through the disappearance of the absorption band at
(3–19), were obtained by addition to carbonyl followed
by the elimination of water between the N-acylhydrazide
(23) and the corresponding aromatic aldehydes (Het/
ArCHO) in water, using hydrochloric acid as catalyst
described in Scheme 1 [19, 20], confirmed by the signal
−
1
13
3
313 cm referents to phenol and the appearance of the
on C NMR of the imine carbon for the conformers at the
range of 131.2–144.7 (sp) and 135.7–148.3 (ap).
−
1
absorption bands at 1758 and 1738 cm of carbonyl
ester. The hydrazinolysis of the ester furnished the key N-
acylhydrazide intermediate (23) in 95% yield [18], con-
The HPLC and the ¹H NMR analysis was consistent with
only one geometric isomer at the imine bond level, which
X-ray diffraction data indicated as having the relative con-
figuration (E) (Fig. 2), according to previous results
[19, 21]. Phenoxyacetohydrazones NAH exists in equili-
brium between the two stable conformers synperiplanar
1
firmed by H NMR spectra through the disappearance of
a signal of the methyl group at 3.70 ppm and the
appearance of the signal of NH and NH at 4.33 ppm and
2
9
.33 ppm, respectively. Finally, the target compounds

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Medicinal Chemistry Research (2021) 30:1703–1712
Scheme 1 Synthetic route for
the preparation of the new
phenoxyacetohydrazones
(
3–19). a Acetonitrile, K CO ,
2 3
reflux, 4 h (90%). b Ethanol,
NH2NH2.H2O (80% aq.), reflux,
h (95%). c H2O, hydrochloric
3
acid 37% (cat.), aldehyde,
reflux, 30 min–2 h, (50–86%)
Fig. 2 A ORTEP representation
of compound 3 in the solid-state:
probability ellipsoids are drawn
at the 50% level. B View of the
conformation of compound 3
looking across the phenyl ring
containing C13
(
sp) and antiperiplanar (ap) in DMSO solution. It is
unit have a cis arrangement, which allows the formation of
centrosymmetric dimers, formed from pairs of classical N1-
H1---O1 hydrogen bonds. Additional contacts between
molecules are generated from C–H---O hydrogen bonds and
π(C = N)---π(Ph) stacking interactions.
described that the signal from the imine hydrogen being
more down shield matches the ap conformer [22].
Crystallography
Compound 3 was recrystallized from ethanol solution by
slow evaporation at room temperature. Atomic coordinates,
bond lengths and angles, and thermal parameters have been
deposited at the Cambridge Crystallographic Data Center,
deposit number 2050640. The angle between the two phe-
nyl rings is 69.33(8)°. The geometry of the exo-C = N bond
is (E), while the H and O1 atoms in the H1–N1–C8=O1
Antitrypanosomal activity
We started our analysis by evaluating the effect of the
compounds against bloodstream trypomastigotes of T. cruzi
(Y strain), a representative of Discrete Typing Unit (DTU)
II. The in vitro screening using trypomastigote revealed 11,
with a 3,4-dihydroxyphenyl group attached to the imine

Medicinal Chemistry Research (2021) 30:1703–1712
1707
unit, as the most active compound with an IC /24 h =
infective forms of T. cruzi, bloodstream trypomastigotes,
and intracellular amastigotes. The synthetic methodology
practiced was reproducible, with global yields ranging from
43% to 74%. The assay against trypomastigote form of T.
50
1
0.3 μM, equivalent to benznidazole, the reference drug. In
order to evaluate the contribution of its hydroxyl groups at
C-3 and at C-4, we compared its activity with different
analogs [4-OH (9); 3,4-OCH (12); 3-OCH , 4-OH (13); 3-
cruzi reported compound 11 (IC /24 h = 10.3 μM)
3
3
50
OH, 4-OCH (14) and –O–CH –O– (16)]. We observed an
equivalent to benznidazole with the selectivity index (SI) =
46. The assay against T. cruzi-infected cultures presented
compound 19 (IC /96 h = 40 nM) with the selectivity
index (SI) = 2,500. Then phenoxyacetohydrazones deriva-
tives 11 and 19 furnishes supporting data for forwarding
in vivo studies of these compounds in pertinent animal
models for Chagas disease.
3
2
important decrease in the activity, indicating that the two
hydroxyl groups in the catechol subunit have an important
characteristic against T. cruzi.
Among the phenyl substituted derivatives, the most
active four derivatives presented a hydroxyl group (9, 10,
5
0
1
1, and 13), three of them in the para position. An attractive
bioprofile was also identified for 5-nitrofurane derivative
19), which showed an IC /24 h = 19.11 μM. The para-
(
50
substituted derivatives (4–9) presenting different electronic
and lipophilic properties and the trisubstituted derivative
Materials and methods
(
15) showed no trypanocidal activity, except for the 4-OH
General experimental details
derivative (9) with an IC /24 h = 53.3 μM (Table 1).
50
In addition, to expand this study, another strain of
T. cruzi was employed, Tulahuen (DTU VI), considering its
susceptibility to nitro derivatives. All phenoxyacetohy-
drazones were also evaluated on intracellular amastigote
forms for Tulahuen strain (Table 1). The most active phe-
noxyacetohydrazone was 5-nitrofurane derivative (19),
which showed an IC /96 h = 40 nM, thirty-eight times
Melting points were determined on a Buchi (B-545). The
gas chromatography coupled to the mass detector (GC-MS)
was performed using the Agilent chromatograph model
6890 with Agilent masses model 5973 at 70 eV. The frag-
mentation and molecular ion values were expressed in terms
of mass/charge (m/z). The Agilent column was used DB-
5MS (5% diphenyl: 95% dimethylpolysiloxane). The
chromatographic analyzes were performed by split injection
method by using a ratio of 10:1 and temperature ramp of
50 °C to 325 °C in the rate 10 °C/min for 35 min with an
initial flow rate of 0.5 mL/min. The infrared spectrums (IR)
were obtained in a spectrophotometer from the Thermo
Scientific brand, model Nicolet 6700 FT-IR by ATR
(Attenuated Total Reflectance). The absorption values were
50
more potent than benznidazole. The para substituted com-
pounds 4–9 and 17 and the disubstituted ones, 12–14 and
1
6, presented only moderated activity. The trisubstituted
derivative (15) was inactive and o-OH derivative (10),
planned to increase the interaction with nucleophilic sites in
the cruzain, presented moderate activity (IC /96 h =
50
2
2.7 μM) (Table 1). Although the intracellular location of
−
1 1
the amastigote form difficult the access of a given drug, in
this study we observed that the best results were observed in
amastigotes forms. The distinct behavior of the Tulahuen
and Y parasites could be also due to intrinsic differences
between both strains and to the time of treatment of the
standardized protocols [23], requiring the assay with the
intracellular form 96 h of contact with the drug.
The cytotoxic results of the most active compounds 11
for bloodstream trypomastigotes and 19 for intracellular
amastigotes were determined and the selectivity indexes
reported in wavelength (λ) whose unit is cm . H NMR
1
3
and C NMR spectra were recorded at room temperature
on Bruker Avance 500 and Bruker Avance 400 spectro-
1
meters operating at 500 and 400 MHz ( H)/125 and
1
3
100 MHz ( C), respectively. Chemical shifts (δ) are
reported in parts per million (ppm) downfield from tetra-
methylsilane (TMS) used as an internal standard and cou-
pling constant (J) values are given in Hertz (Hz). The
chromatographic purity of the final products was deter-
mined by a Shimadzu (VP) apparatus with model LC-
20ADXR pumps, DGU-20A5R degasser, CBM20A con-
troller, and SPD-M20A model photodiode array detector
(DAD). Data acquisition and control were performed using
Shimadzu Labsolutions. Chromatographic analyzes were
monitored by scanning from 225 nm to 489 nm. In the
analyzes, the mobile phase used as eluent (A) water, pH 5.8
adjusted with 0.025 Mol/L ammonium acetate, and eluent
(B) methanol, isocratic elution for 70 min at 30 °C; the flow
of the mobile phase was 1 mL/min and the volume injected
was 1000 μL. The separation was obtained on a Supelcosil
LC 18-3 reverse phase column, 200 × 4.6 mm, with a
(
SI) (LC50 for cytotoxicity divided by IC50 for anti-
trypanosomal activity) were 46 and 2500 respectively. In
this context, the two compounds with SI ≥ 50 were con-
sidered good candidates for subsequent studies on anti-
trypanosomal activity in a murine model [23].
Conclusions
We described the synthesis, antitrypanosomal and cytotoxic
evaluation of new phenoxyacetohydrazones against both

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Medicinal Chemistry Research (2021) 30:1703–1712
particle diameter of 5 μm. The progress of all reactions was
monitored by TLC (Thin Layer Chromatography). which
was performed on 2.0–6.0 cm aluminum sheets precoated
with silica gel 60 (HF254. Merck) to a thickness of
cooled e the precipitate was collected by vacuum filtration
and re-crystallized in distilled water. The products were
obtained in yields that varied 50–86%.
(E)-N′-Benzylidene-2-phenoxyacetohydrazide (3) [24].
0
.25 mm. The developed chromatograms were viewed
White solid (H O); yield 80%; m.p. 156.2–57.0 °C; IR
2
−
1
under ultraviolet light (254 and 365 nm).
(ATR. cm ): ν 1678 (C = Oamide); 1403 (C = C ); 1259
max Ar
1
(
N = C); 1219 and 1086 (C–Oether); H NMR (400 MHz.
General procedures for the preparation of methyl
phenoxyacetate (22)
DMSO‑d ): δ 4.66 and 5.14 (2H; s; H2); 6.92–7.01 (3H; m;
H6; H7; H8); 7.27–7.35 (2H; m; H5; H9); 7.42–7.47 (3H;
6
m; H13; H14; H15); 7.69–7.72 (2H; m; H12; H16); 8.02
To a suspension of 20 (6.68 g; 0.071 mol; 1.01 eq.) and
potassium carbonate (11.61 g; 0.084 mol; 1.2 eq.) in
and 8.34 (1H; s; H10); 11.57 and 11.60 (1H; s; N–H); 13
C
NMR (100 MHz. DMSO‑d ): δ 64.4 and 66.3 (C2); 114.3
6
1
0
00 mL of acetonitrile was added slowly 21 (10.71 g;
.07 mol; 1.0 eq.). The reaction mixture was stirred under
and 114.5 (C6; C8); 120.6 and 121.2 (C7); 126.8 and 127.0
(C12; C16); 128.7 (C13; C15); 129.3 and 129.4 (C5; C9);
129.8 and 130.1 (C14); 133.8 and 134.0 (C11); 143.7 and
147.8 (C10); 157.6 and 158.1 (C4); 164.2 and 168.9 (C1);
HPLC-UV nm (%): 254 (100). ESI-MS: m/z 277.0947 [M
reflux for 4 h and after the complete reaction was cooled and
added distilled water until solubilized the mixture. The
solution was extracted with ethyl acetate (3 × 50 mL) and
the organic phase was dried over anhydrous Na SO , fil-
+
+ Na] , calcd. for C H N NaO : 277.0946; MW: 254.28.
2
4
15 14
2
2
tered, and concentrated in vacuo providing the ester used in
the next step.
(E)-N′-(4-Chlorobenzylidene)-2-phenoxyacetohydrazide
(4) [25]. White solid (H O); yield 50%; m.p.
2
−
1
Methyl 2-phenoxyacetate 22. Light brown oil (AcOEt);
175.7–177.8 °C; IR (ATR. cm ): ν
1677 (C = Oamide);
max
−
1
9
0% yield; IR (ATR. cm ): ν
1758 and 1738 (C =
1401 (C = C ); 1259 (N = C); 1218 and 1088 (C–Oether);
max
Ar
1
Oester); 1599 (C = C ); 1494 (C–H ); 1200 and 1173
H NMR (400 MHz. DMSO‑d ): δ 4.68 and 5.14 (2H; s;
Ar
Ar
6
1
(
C–Oether); H NMR (400 MHz. DMSO‑d ): δ 3.70 (3H; s;
H2); 6.92–7.01 (3H; m; H6; H7; H8); 7.27–7.34 (2H; m;
H5; H9); 7.49–7.52 (2H; m; H13; H15); 7.71–7.75 (2H; m;
H12; H16); 8.02 and 8.35 (1H; s; H10); 11.73 (1H; s; N–H);
6
O–CH ); 4.79 (2H; s; H2); 6.90–6.98 (3H; m; H6; H7; H8);
3
13
7
.27–7.32 (2H; m; H-5; H9); C NMR (100 MHz.
1
3
DMSO‑d ): δ 51.7 (O–CH ); 64.3 (C2); 114.3 (C5; C9);
C NMR (100 MHz. DMSO‑d ): δ 64.5 and 66.3 (C2);
6
3
6
1
21.1 (C7); 129.4 (C6; C8); 157.4 (C4); 169.2 (C1).
114.4 and 114.5 (C6; C8); 120.6 and 121.1 (C7); 128.5 and
128.6 (C13; C15); 128.8 and 128.8 (C12; C16); 129.3 and
General procedure for the preparation of 2-
phenoxyacetohydrazide (23)
129.4 (C5; C9); 132.8 and 133.0 (C11); 134.2 and 134.5
(C14); 142.4 and 146.4 (C10); 157.6 and 158.0 (C4); 164.3
and 169.0 (C1); HPLC-UV nm (%): 254 (100); ESI-MS:
+
To an ethanolic solution of 22 (9.97 g; 0.06 mol; 1.0 eq.)
was added slowly hydrazine hydrate 80% (aq.) (15 g;
m/z 311.0543 [M + Na] , calcd. for C H ClN NaO :
1
5
13
2
2
311.0557; MW: 288.73.
0
.3 mol; 5.0 eq.) and the reaction mixture was stirred for 3 h
(E)-N′-(4-Bromobenzylidene)-2-phenoxyacetohydrazide
under reflux. The mixture was cooled, the precipitate was
collected by vacuum filtration, washed with cold ethanol,
and dried under vacuum.
(5). White solid (H O); yield 50%; m.p. 173.7–174.8 °C; IR
2
−
1
(ATR. cm ): ν 1677 (C = Oamide); 1398 (C = C ); 1260
max
Ar
1
(N = C); 1219 and 1068 (C–Oether); H NMR (400 MHz.
2
-phenoxyacetohydrazide (23). White crystal (EtOH);
DMSO‑d ): δ 4.67 and 5.13 (2H; s; H2); 6.92–7.00 (3H; m;
H6; H7; H8); 7.27–7.34 (2H; m; H5; H9); 7.62–7.68 (4H;
6
−
1
9
3
1
5% yield; m.p. 113.5–114.2 °C; IR (ATR. cm ): ν
max
400 (N–H ); 1598 (C = O
069 (C–Oether); H NMR (400 MHz. DMSO‑d ): δ 4.33
); 1496 (C = C ); 1241 and
m; H12; H13; H15; H16); 7.99 and 8.32 (1H; s; H10); 11.68
2
amide
Ar
1
13
(1H; s; N–H); C NMR (100 MHz. DMSO‑d ): δ 64.5 and
6
6
(
7
N–H ); 4.47 (2H; s; H2); 6.94–6.97 (3H; m; H6; H7; H8);
.27–7.31 (2H; m; H5; H9); 9.33 (1H; s; N–H); C NMR
66.3 (C2); 114.4 and 114.5 (C6; C8); 120.6 and 121.1 (C7);
123.0 and 123.3 (C14); 128.7 and 128.9 (C13; C15); 129.3
and 129.4 (C5; C9); 131.7 and 131.7 (C12; C16); 133.2 and
133.3 (C11); 142.5 and 146.5 (C10); 157.6 and 158.0 (C4);
2
13
(
(
100 MHz. DMSO‑d ): δ 66.0 (C2); 114.5 (C6; C8); 121.0
6
C7); 129.3 (C5; C9); 157.7 (C4); 166.5 (C1).
1
64.3 and 169.0 (C1); HPLC-UV nm (%): 254 (100); ESI-
+
General procedure for the preparation of NAH (3–19)
MS: m/z 355.0022 [M + Na] , calcd. for C H BrN NaO :
15 13 2 2
3
55.0052; MW: 333.18.
(E)-N′-(4-Fluorobenzylidene)-2-phenoxyacetohydrazide
(6). White solid (H O); yield 50%; m.p. 155.7–159.3 °C; IR
To a solution of 23 (0.400 g; 2.41 mmol; 1.0 eq.) in 30 mL
of water, were added the corresponding aromatic aldehydes
2
−
1
(
2.43 mmol; 1.01 eq.) and hydrochloric acid in catalytic
(ATR. cm ): ν 1674 (C = Oamide); 1495 (C = C ); 1220
max Ar
1
quantity. After the complete reaction, the mixture was
(N = C); 1210 and 1086 (C–Oether); H NMR (400 MHz.

Medicinal Chemistry Research (2021) 30:1703–1712
1709
DMSO‑d ): δ 4.66 and 5.13 (2H; s; H2); 6.92–7.01 (3H; m;
H6; H7; H8); 7.26–7.34 (4H; m; H5; H9; H13; H15);
H16); 7.91 and 8.22 (1H; s; H10); 9.91 (1H; s; O–H); 11.36
6
1
3
and 11.39 (1H; s; N–H); C NMR (100 MHz. DMSO‑d6):
δ 64.4 and 66.3 (C2); 114.3 and 114.5 (C6; C8); 115.5 and
115.6 (C13; C15); 120.6 and 121.1 (C7); 124.9 and 124.9
(C11); 128.5 and 128.8 (C12; C16); 129.3 and 129.4 (C5;
C9); 144.0 and 148.1 (C10); 157.7 and 158.1 (C4); 159.2
and 159.4 (C14); 163.7 and 168.5 (C1); HPLC-UV nm (%):
254 (96.6); 274 (99.8); 292 (99.8); ESI-MS: m/z 293.0899
7
1
.74–7.80 (2H; m; H12; H16); 8.02 and 8.35 (1H; s; H10);
1.62 (1H; s; N–H); 13C NMR (100 MHz. DMSO‑d . ppm):
6
δ 64.5 and 66.3 (C2); 114.4 and 114.5 (C6; C8); 115.7 and
115.9 (C13; C15; d; JC-F = 6.1 Hz); 120.6 and 121.1 (C7);
129.0 and 129.2 (C12; C16; d; JC-F = 8.4 Hz); 129.3 and
129.4 (C5; C9); 130.5 and 130.6 (C11; d; JC-F = 2.9 Hz);
142.5 and 146.7 (C10); 157.6 and 158.1 (C4); 161.7 and
164.2 (C14; d; JC-F = 14.0 Hz); 164.2 and 169.0 (C1);
+
[M + Na] , calcd. for C H N NaO : 293.0896; MW:
1
5
14
2
3
270.28.
HPLC-UV nm (%): 254 (100); ESI-MS: m/z 295.0830
(E)-N′-(2-Hydroxybenzylidene)-2-phenoxyacetohy-
+
[
M + Na] , calcd. for C H FN NaO : 295.0853; MW:
drazide (10) [24]. White solid (H O); yield 77%; m.p.
15
13
2
2
2
−
1
2
72.27.
E)-N′-(4-Methoxybenzylidene)-2-phenoxyacetohy-
drazide (7) [24]. White solid (H O); yield 80%; m.p.
172.6–174.9 °C; IR (ATR. cm ): ν
1673 (C = Oamide);
max
1
(
1413 (C = C ); 1219 (N = C); 1086 (C–O ); H NMR
Ar ether
(400 MHz. DMSO‑d ): δ 4.69 and 5.10 (2H; s; H2);
2
6
−
1
1
36.1–138.3 °C; IR (ATR. cm ): ν
1671 (C = Oamide);
6.85–6.91 (3H; m; H6; H7; H8); 6.93–7.02 (2H; m; H13;
H15); 7.22–7.35 (4H; m; H5; H9; H14; H16); 7.52 (1H; d;
J = 6.88 Hz; O–H); 7.71 (1H; d; J = 6.44 Hz; O–H); 8.32
max
1
487 (C = C ); 1245 (N = C); 1204 and 1031 (C–Oether);
Ar
1
H NMR (400 MHz. DMSO‑d ): δ 3.79 and 3.80 (1H; s;
6
1
3
H17); 4.64 and 5.11 (2H; s; H2); 6.92–6.96 (2H; m; H13;
H15); 6.99–7.02 (3H; m; H6; H7; H8); 7.27–7.34 (2H; m;
H5; H9); 7.63–7.66 (2H; m; H12; H16); 7.96 and 8.28 (1H;
and 8.57 (1H; s; H10); 11.49 (1H; s; N–H); C NMR
(100 MHz. DMSO‑d ): δ 64.5 and 66.2 (C2); 114.3 and
6
114.6 (C6; C8); 116.0 and 116.3 (C13); 118.5 and 119.2
(C15); 119.9 and 126.3 (C16); 120.6 and 121.2 (C7); 129.1
(C14); 129.3 and 129.4 (C5; C9); 131.1 and 131.4 (C11);
141.3 and 148.1 (C10); 156.4 and 157.3 (C12); 157.6 and
158.1 (C4); 164.1 and 168.6 (C1); HPLC-UV nm (%): 254
13
s; H10); 11.44 and 11.47 (1H; s; N–H); C NMR
100 MHz. DMSO‑d ): δ 55.2 (C17); 64.5 and 66.3 (C2);
(
6
1
14.2 (C6; C8); 114.3 and 114.6 (C13; C15); 120.6 and
1
21.1 (C7); 126.5 and 126.5 (C11); 128.4 and 128.7 (C12;
+
C16); 129.3 and 129.4 (C5; C9); 143.6 and 147.7 (C10);
(99.5); ESI-MS: m/z 271.1075 [M + H] , calcd. for
+
1
1
57.7 and 158.1 (C4); 160.6 and 160.8 (C14); 163.9 and
C H N O : 271.1077; m/z 293.089428 [M + Na] , calcd.
1
5
15
2
3
68.7 (C1); HPLC-UV nm (%): 254 (100); 274 (100); ESI-
for C H N NaO : 293.0896; MW: 270.28.
15 14 2 3
+
MS: m/z 307.1038 [M + Na] , calcd. for C H N NaO :
(E)-N′-(3,4-Dihydroxybenzylidene)-2-phenoxyacetohy-
16
16
2
3
3
07.1053; MW: 284.31.
E)-N′-(4-Nitrobenzylidene)-2-phenoxyacetohydrazide
8). White solid (H O); yield 83%; m.p. 186.5–188.3 °C; IR
drazide (11). White solid (H O); yield 50%; m.p.
2
1
−
(
129.9–133.2 °C; IR (ATR. cm ): ν
3521 and 3257
max
(
(
(O–H); 1653 (C = Oamide); 1590 (C = C ); 1299 (N = C);
2
Ar
−
1
1
ATR. cm ): ν 1676 (C = Oamide); 1587 and 1339 (Aryl-
1257 and 1062 (C–Oether); H NMR (400 MHz. DMSO‑d ):
max
6
1
NO ); 1517 (C = C ); 1256 (N = C); 1089 (C–O ); H
NMR (400 MHz. DMSO‑d ): δ 4.71 and 5.19 (2H; s; H2);
6
δ 4.62 and 5.08 (2H; s; H2); 6.77 (1H; d; J = 8.1 Hz; H15);
6.89–7.00 (4H; m; H6; H7; H8; H16); 7.15 and 7.19 (1H; d;
J = 2.0 Hz; H12); 7.27–7.34 (2H; m; H5; H9); 7.83 and
8.13 (1H; s; H10); 9.19 and 9.27 (1H; s; O–H); 9.40 (1H; s;
2
Ar
ether
6
.94–7.02 (3H; m; H6; H7; H8); 7.28–7.35 (2H; m; H5;
H9); 7.95–8.00 (2H; m; H12; H16); 8.12 and 8.45 (1H; s;
H10); 8.26–8.31 (2H; m H13; H15); 11.90 (1H; s; N–H);
1
3
O–H); 11.32 and 11.35 (1H; s; N–H); C NMR (100 MHz.
1
3
C NMR (100 MHz. DMSO‑d ): δ 64.5 and 66.3 (C2);
DMSO‑d ): δ 64.4 and 66.3 (C2); 112.6 and 112.6 (C15);
6
6
1
1
1
14.4 and 114.6 (C6; C8); 120.7 and 121.2 (C7); 123.9 and
24.0 (C13; C15); 127.8 and 128.0 (C12; C16); 129.3 and
29.5 (C5; C9); 140.2 and 140.3 (C11); 141.3 and 145.3
114.3 and 114.5 (C6; C8); 115.4 and 115.5 (C12); 120.0
and 121.1 (C7); 120.5 and 120.6 (C16); 125.3 and 125.4
(C11); 129.3 and 129.4 (C5; C9); 144.3 and 148.3 (C10);
145.5 and 145.6 (C13); 147.7 and 147.9 (C14); 157.7 and
158.1 (C4); 163.7 and 168.4 (C1); HPLC-UV nm (%): 254
(
C10); 147.7 and 147.8 (C14); 157.6 and 158.0 (C4); 164.7
+
and 169.4 (C1); ESI-MS: m/z 322.0810 [M + Na] , calcd.
for C H N NaO : 322.0798; MW: 299.28.
+
(100); 274 (100); ESI-MS: m/z 309.0837 [M + Na] , calcd.
1
5
13
3
4
(
E)-N′-(4-Hydroxybenzylidene)-2-phenoxyacetohy-
for C H N NaO 309.0845; MW: 286.28.
1
5
14
2
4
drazide (9). White solid (H O); yield 50% (H O); m.p.
(E)-N′-(3,4-Dimethoxybenzylidene)-2-phenoxyacetohy-
2
2
−
1
2
15.9–216.7 °C; IR (ATR. cm ): ν
3316 (O–H); 2988
drazide (12) [26]. White solid (H O); yield 53%; m.p.
max
2
−
1
(
CH2sp3); 1672 (C = Oamide); 1604 (C = C ); 1256 (N =
153.6– 155.2 °C; IR (ATR. cm ): ν
1675 (C = Oamide);
Ar
max
1
2
Cimine); 1245 and 1220 (C–Oether); H NMR (400 MHz.
1498 (C = C ); 1406 (C-Hsp Ar); 1262 (N = C); 1162
(C–Oether); H NMR (400 MHz. DMSO‑d ): δ 3.79–3.80
Ar
1
DMSO‑d ): δ 4.62 and 5.09 (2H; s; H2); 6.82 (2H; dd; J =
6
6
8
.6 Hz; H13; H15); 6.91–7.00 (3H; m; H6; H7; H8);
(6H; m; H17; H18); 4.65 and 5.14 (2H; s; H2); 6.91–7.03
7
.27–7.34 (2H; m; H5; H9); 7.53 (2H; dd; J = 8.6 Hz; H12;
(4H; m; H6; H7; H8; H15); 7.17–7.21 (1H; m; H16);

1710
Medicinal Chemistry Research (2021) 30:1703–1712
7
.27–7.34 (3H; m; H5; H9; H12); 7.93 and 8.26 (1H; s;
H18); 3.81 and 3.82 (6H; s; H17; H19); 4.66 and 5.15 (2H;
s; H2); 6.91–7.02 (5H; m; H6; H7; H8; H12; H16); 7.27 –
7.35 (2H; m; H5; H9); 7.93 and 8.26 (1H; s; H10); 11.55
13
H10); 11.44 and 11.50 (1H; s; N–H); C NMR (100 MHz.
DMSO‑d ): δ 55.3 and 55.4 (C17; C18); 64.5 and 66.3
6
1
3
(
C2); 108.1 and 108.5 (C15); 111.3 and 111.4 (C12); 114.3
and 114.6 (C6; C8); 120.6 and 121.8 (C7); 121.1 and 121.1
C16); 126.6 and 126.7 (C11); 129.3 and 129.4 (C5; C9);
and 11.63 (1H; s; N–H); C NMR (100 MHz. DMSO‑d6):
δ 55.8 (C17; C19); 60.0 (C18); 64.6 and 66.3 (C2); 104.1
and 104.2 (C12 and C16); 114.3 and 114.6 (C6; C8); 120.6
and 121.2 (C7); 129.3 and 129.4 (C5; C9); 129.4 and 129.5
(C11); 138.9 and 139.2 (C14); 143.5 and 147.8 (C10);
153.0 and 153.1 (C13; C15); 157.6 and 158.1 (C4); 164.1
and 169.0 (C1); HPLC-UV nm (%): 254 (98.5); 274 (99.5);
(
1
1
43.7 and 148.0 (C10); 148.9 and 148.9 (C13); 150.5 and
50.7 (C14); 157.7 and 158.1 (C4); 163.9 and 168.8 (C1);
HPLC-UV nm (%): 254 (100); 274 (100); ESI-MS: m/z
+
3
37.1185 [M + Na] , calcd. for C H N NaO 337.1158;
17 18 2 4
+
MW: 314.34.
ESI-MS: m/z 367.1266 [M + Na] , calcd. for
(
E)-N′-(4-Hydroxy-3-methoxybenzylidene)-2-phenox-
C H N NaO : 367.1264; MW: 344.36.
1
8
20
2
5
yacetohydrazide (13). White solid (H O); yield 65%; m.p.
(E)-N′-(Benzo[d][1,3]dioxol-5-ylmethylene)-2-phenox-
2
−
1
1
56.3 –158.7 °C; IR (ATR. cm ): ν
3200 (O–H); 1670
yacetohydrazide (16). White solid (H O); yield 84%; m.p.
max
2
2
−1
(
C = Oamide); 1586 (C = C ); 1511 (C-Hsp Ar); 1295 (N =
198.9 –200.8 °C; IR (ATR. cm ): ν
1670 (C = Oamide);
Ar
max
1
1
C); 1240 and 1211 (C–Oether); H NMR (400 MHz.
1447 (C = C ); 1250 (N = C); 1204 (C–O ); H NMR
Ar ether
DMSO‑d ): δ 3.80 and 3.81 (3H; s; H17); 4.63 and 5.12
(400 MHz. DMSO‑d ): δ 4.64 and 5.11 (2H; s; H2); 6.08
and 6.09 (2H; s; H17); 6.92–7.00 (4H; m; H6; H7; H8;
6
6
(
2H; s; H2); 6.81–6.84 (1H; m; H15); 6.90–7.00 (3H; m;
H6; H7; H8); 7.05–7.10 (1H; m; H12); 7.27–7.34 (3H; m;
H13); 7.12–7.16 (1H; m; H16); 7.25–7.35 (3H; m; H5; H9;
H5; H9; H16); 7.90 and 8.21 (1H; s; H10); 9.51 and 9.56
H12); 7.92 and 8.24 (1H; s; H10); 11.47 and 11.48 (1H; s;
1H; s; O–H); 11.38 and 11.43 (1H; s; N–H); 13C NMR
N–H); C NMR (100 MHz. DMSO‑d ): δ 64.5 and 66.3
13
(
(
6
100 MHz. DMSO‑d ): δ 55.4 and 55.4 (C17); 64.5 and
(C2); 101.4 and 101.5 (C17); 105.0 and 105.1 (C15); 108.2
and 108.4 (C12); 114.4 and 114.5 (C6; C8); 120.6 and
121.1 (C7); 123.1 and 123.3 (C16); 128.3 and 128.4 (C11);
129.3 and 129.4 (C5; C9); 143.4 and 147.9 (C10); 147.6
(C13); 148.8 and 149.1 (C14); 157.6 and 158.1 (C4); 164.0
and 168.8 (C1); HPLC-UV nm (%): 254 (100); 274 (99.6);
6
6
6.3 (C2); 115.3 and 115.4 (C15); 114.3 and 114.6 (C6;
C8); 108.9 and 109.4 (C12); 120.5 and 122.1 (C7); 121.1
and 121.2 (C16); 125.3 and 125.3 (C11); 129.3 and 129.4
(
C5; C9); 144.1 and 148.3 (C10); 147.8 and 147.9 (C13);
1
1
48.7 and 149.0 (C14); 157.7 and 158.1 (C4); 163.7 and
+
68.6 (C1); HPLC-UV nm (%): 254 (98.2); 274 (98.6);
ESI-MS: m/z 321.0834 [M + Na] , calcd. for
+
ESI-MS: m/z 323.0996 [M + Na] , calcd. for
C H N NaO : 321.0845; MW: 298.29.
1
6
14
2
4
C H N NaO : 323.1002; MW: 300.31.
(E)-2-Phenoxy-N′-(pyridin-4-ylmethylene)
acetohy-
1
6
16
2
4
(
E)-N′-(3-Hydroxy-4-methoxybenzylidene)-2-phenox-
drazide (17). White solid (H O); yield 50%; m.p.
2
−
1
yacetohydrazide (14). White solid (H O); yield 60%; m.p.
43.9–145.9 °C; IR (ATR. cm ): ν
496 (C = C ); 1269 (N = C); 1254 and 1216 (C–Oether);
92 and 754 (Armono); H NMR (400 MHz. DMSO‑d ): δ
141.3–143.4 °C; IR (ATR. cm ): ν
1676 (C = Oamide);
max
2
−
1
1
1
7
3
(
7
1678 (C = Oamide);
1598 (C = C ); 1225 (N = C); 1089 (C–Oether); 816 (pyr-
max
Ar
1
idin 4-subs); H NMR (400 MHz. DMSO‑d ): δ 4.71 and
Ar
6
1
5.18 (2H; s; H2); 6.94–7.01 (3H; m; H6; H7; H8);
7.28–7.33 (2H; m; H5; H9); 7.63–7.68 (2H; d; j = 5.8 Hz;
H12; H15); 8.00 and 8.34 (1H; s; H10); 8.64 (2H; d; j =
6
.80 (3H; s; H17); 4.63 and 5.09 (2H; s; H2); 6.91–7.04
4H; m; H6; H7; H8; H15); 7.20–7.23 (1H; m; H12);
.27–7.34 (3H; m; H5; H9; H16); 7.87 and 8.18 (1H; s;
H10); 9.23 and 9.30 (1H; s; O–H); 11.38 and 11.42 (1H; s;
N–H); C NMR (100 MHz. DMSO‑d ): δ 55.5 (C17); 64.4
and 66.3 (C2); 111.7 (C15); 112.1 and 112.2 (C12); 114.3
and 114.6 (C6; C8); 119.9 and 120.3 (C16); 120.6 and
21.1 (C7); 126.7 and 126.8 (C11); 129.3 and 129.4 (C5;
C9); 144.0 and 147.9 (C10); 146.7 and 146.7 (C13); 149.5
and 149.8 (C14); 157.7 and 158.1 (C4); 163.8 and 168.6
C1); HPLC-UV nm (%): 254 (100); 274 (100); ESI-MS: m/
5.1 Hz; H13. H14); 11.86 and 11.88 (1H; s; N–H); 13
C
NMR (100 MHz. DMSO‑d ): δ 64.5 and 66.3 (C2); 114.4
6
1
3
and 114.6 (C6; C8); 120.7 and 121.2 (C7); 120.8 and 120.9
(C11); 129.3 and 129.5 (C5; C9); 141.0 and 145.4 (C10);
141.2 and 141.2 (C12; C15); 150.1 and 150.2 (C13; C14);
157.6 and 158.0 (C4); 164.7 and 169.4 (C1); HPLC-UV nm
(%): 254 (100); 274 (100); ESI-MS: m/z 256.1100
6
1
+
[M + H] , calcd. for C H N O : 256.1080; m/z 278.0920
1
4
14
3
2
+
(
z
[M + Na] , calcd. for C H N NaO : 278.0899; MW:
14 13 3 2
+
323.1022 [M + Na] , calcd. for C H N NaO :
255.27.
16
16
2
4
3
23.1002; MW: 300.31.
E)-2-phenoxy-N′-(3,4,5-trimethoxybenzylidene) acet-
ohydrazide (15). White solid (H O); yield 86%; m.p.
(E)-N′-((5-Bromothiophen-2-yl) methylene)-2-phenox-
(
yacetohydrazide (18). White solid (H O); yield 81%; m.p.
2
−
1
151.8–154.1 °C; IR (ATR. cm ): ν
1672 (C = Oamide);
2
max
−
1
1
1
62.2–163.1 °C; IR (ATR. cm ): ν
1679 (C = Oamide);
1425 (C = C ); 1221 (N = C); 1085 (C–O ); H NMR
max
Ar ether
1
411 (C = C ); 1232 (N = C); 1127 and 1003 (C–Oether);
(400 MHz. DMSO‑d ): δ 4.64 and 5.02 (2H; s; H2);
6.89–7.00 (3H; m; H6; H7; H8); 7.25–7.34 (4H; m; H5; H9;
Ar
6
1
H NMR (400 MHz. DMSO‑d ): δ 3.69 and 3.70 (3H; s;
6

Medicinal Chemistry Research (2021) 30:1703–1712
1711
H12; H13); 8.10 and 8.48 (1H; s; H10); 11.61 and 11.65
(ATCC® CCL-1™) were infected with trypomastigotes of
Tulahuen strain, expressing Escherichia coli β-galactosidase
as reporter gene according to the method described previously
[23]. Briefly, for the bioassay, 4000 L929 cells were added to
each well of a 96-well microtiter plate. After overnight
incubation, 40,000 trypomastigotes were added to the cells
and incubated for 2 h. The culture was maintained for 24 h to
establish the infection and then it was treated with the com-
pounds at serial decreasing dilutions for a further 96 h at
37 °C. After this period the chlorophenol red-β-D-galacto-
pyranoside (CPRG) reagent (Roche) was added in Nonidet
P40 solution (Sigma-Aldrich) and the plate was incubated for
another 18 h and the absorbance was measured at 570 nm.
Controls with uninfected cells, untreated infected cells, and
infected cells treated with benznidazole at 3.8 μM (positive
control) or DMSO 1% were used. The results were expressed
as the percentage of T. cruzi growth inhibition in compound-
tested cells as compared to the infected cells and untreated
cells. The IC50 values were calculated by linear interpolation.
Quadruplicates were run on the same plate, and the experi-
ments were repeated at least once. The active compounds
were tested in vitro for the determination of cellular toxicity
against uninfected L-929 cells and primary cultures of peri-
toneal macrophages obtained from Albino Swiss mice, using
the AlamarBlue® dye [23]. The cells were exposed to com-
pounds at increasing concentrations starting at IC50 value for
T. cruzi. After 24 or 96 h of incubation, the AlamarBlue® was
added and the absorbance at 570 and 600 nm was measured
after 4–6 h for L-929 cells and after 2 h for peritoneal mac-
rophages. The cell viability was expressed as the percentage
of difference in the reduction between treated and untreated
cells being the LC value, corresponding to the concentration
1
3
(
1H; s; N–H); C NMR (100 MHz. DMSO‑d ): δ 64.2 and
6
6
6.3 (C2); 114.2 and 114.8 (C14); 114.3 and 114.6 (C6;
C8); 120.7 and 121.2 (C7); 129.3 and 129.4 (C5; C9); 130.9
and 131.6 (C10 ou C13); 131.0 and 131.6 (C10 ou C13);
1
(
37.9 (C12); 140.5 and 140.6 (C11); 131.2 and 142.2
C10); 157.6 and 157.9 (C4); 164.2 and 168.6 (C1); HPLC-
UV nm (%): 254 (100); 274 (100); ESI-MS: m/z 360.9623
+
[
M + Na] , calcd. for C H BrN NaO S 360.9616; MW:
13
11
2
2
3
39.21.
(
E)-N′-((5-Nitrofuran-2-yl)
methylene)-2-phenox-
yacetohydrazide (19). White solid (H O); yield 60%; m.p.
2
−
1
1
1
(
69.9 – 172.7 °C; IR (ATR. cm ): ν 1695 (C = Oamide);
max
2
587 (C = C ); 1479 (C-Hsp ); 1350 (Aryl-NO ); 1239
N = C); 1116 (C–Oether); H NMR (400 MHz. DMSO‑d ):
Ar
Ar
2
1
6
δ 4.72 and 5.12 (2H; s; H2); 6.91–7.01 (3H; m; H6; H7;
H8); 7.25–7.35 (3H; m; H5; H9; H13); 7.78–7.81 (1H; m;
1
3
H12); 7.97 and 8.32 (1H; s; H10); 11.99 (1H; s; N–H);
C
NMR (100 MHz. DMSO‑d ): δ 64.3 and 66.3 (C2); 114.4
6
and 114.6 (C6; C8); 114.5 and 114.8 (C13); 115.5 (C12);
1
1
20.8 and 121.3 (C7); 129.3 and 129.5 (C5; C9); 131.9 and
35.7 (C10); 151.3 and 151.4 (C11); 151.7 and 151.9
(
C14); 157.5 and 157.9 (C4); 164.9 and 169.3 (C1); HPLC-
UV nm (%): 254 (98.1); 274 (98.3); ESI-MS: m/z 312.0577
+
[
M + Na] , calcd. for C H N NaO : 312.0590; MW:
13 11 3 5
2
89.24.
Experimental procedure for biological activity
The use of animals in our trial was performed in accordance
with the Brazilian Law 11.794/2008 and regulations of the
National Council of Animal Experimentation Control under
the license L038/2018 from the Ethics Committee for Animal
Use of the Oswaldo Cruz Institute (CEUA/IOC). The assays
were carried out using the bloodstream trypomastigotes of Y
strain obtained from infected mice at peak parasitemia [27].
The stock solutions of the phenoxyacetohydrazones and
benznidazole were prepared in dimethyl sulfoxide, with the
final concentration of the solvent never exceeding 1%, which
has no deleterious effect on the parasite. The compounds were
diluted, in series 1:2, in decreasing concentrations, with an
initial concentration of 1 mM. The assays were performed
5
0
that leads to lysis of 50% of the mammalian cells [29]. The
selectivity index (SI) was determined based on the ratio of the
LC50 value in the host cell and the IC50 value of the parasite.
Quadruplicates were run on the same plate, and the experi-
ments were repeated at least twice.
Statistical analysis
The results of IC /96 h are presented as mean and standard
5
0
deviation and were analyzed by Kruskal–Wallis followed
by comparison with the Tukey test using GraphPad Prism
3.06 software. The significance level was p < 0.05.
6
with the parasites (5 × 10 cells/mL) incubated for 24 h at
3
7 °C and 5% CO atmosphere [19, 28]. Untreated and
2
benznidazole-treated parasites were used as controls. The
parasites were quantified in Neubauer’s chamber. The results
were analyzed by plotting % lysis of T. cruzi against the
concentration of the test compound and the activity of com-
pounds was expressed as the IC /24 h corresponding to the
concentration that led to 50% lysis of the parasite and is
summarized in Table 1. Next, to evaluate the effects of the
compounds on intracellular parasites, L929 fibroblasts
Acknowledgements This study was financed in part by the Coorde-
nação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil
(CAPES)—Finance Code 001. The authors also thank the National
Crystallographic Service, Southampton, for the X-ray data collection
and the Program for Technological Development of Tools for Health-
PDTIS-FIOCRUZ for use of its facilities.
50
Conflict of interests The authors declare no competing interests.

1712
Medicinal Chemistry Research (2021) 30:1703–1712
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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Open Access This article is licensed under a Creative Commons
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