Synthesis and biological evaluation in vitro and in silico of N-propionyl-N′-benzeneacylhydrazone derivatives as cruzain inhibitors of Trypanosoma cruzi

Article abstract of DOI:10.1007/s11030-020-10156-5

Abstract: An N-acylhydrazone scaffold has been used to develop new drugs with diverse biological activities, including trypanocidal activity against different strains of Trypanosoma cruzi. However, their mechanism of action is not clear, although in T. cr

Full text of DOI:10.1007/s11030-020-10156-5

Molecular Diversity
https://doi.org/10.1007/s11030-020-10156-5
ORIGINAL ARTICLE
Synthesis and biological evaluation in vitro and in silico of N‑pro
pionyl‑N′‑benzeneacylhydrazone derivatives as cruzain inhibitors
of Trypanosoma cruzi
Timoteo Delgado‑Maldonado1 · Benjamín Nogueda‑Torres2 · José C. Espinoza‑Hicks3 · Lenci K. Vázquez‑Jiménez1 ·
Alma D. Paz‑González1 · Alfredo Juárez‑Saldívar1 · Gildardo Rivera1
Received: 3 June 2020 / Accepted: 4 November 2020
© Springer Nature Switzerland AG 2020
Abstract
An N-acylhydrazone scafold has been used to develop new drugs with diverse biological activities, including trypanocidal
activity against diferent strains of Trypanosoma cruzi. However, their mechanism of action is not clear, although in T.
cruzi it has been suggested that the enzyme cruzain is involved. The aim in this work was to obtain new N-propionyl-N′-
benzeneacylhydrazone derivatives as potential anti-T. cruzi agents and elucidate their potential mechanism of action by a
molecular docking analysis and efects on the expression of the cruzain gene. Compounds 9 and 12 were the most active
agents against epimastigotes and compound 5 showed better activity than benznidazole in T. cruzi blood trypomastigotes.
Additionally, compounds 9 and 12 signifcantly increase the expression of the cruzain gene. In summary, the in silico and
in vitro data presented herein suggest that compound 9 is a cruzain inhibitor.
Graphic abstract
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-020-10156-5) contains
supplementary material, which is available to authorized users.
Extended author information available on the last page of the article
Vol.:(0123456789)
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Molecular Diversity
Keywords N-propionyl-N′-benzeneacylhydrazone · Molecular
docking · Cruzain · Trypanosoma cruzi
Introduction
The protozoan parasite Trypanosoma cruzi (T. cruzi)
is the causal agent of American trypanosomiasis, also
called Chagas disease, which is technically transmitted
enzootically [1, 2]. Chagas disease is endemic in 21 Latin
American countries, but recently diferent cases have been
reported in Canada, Australia, Japan and Europe, suggest-
ing that it has become a global health problem [3–5].
According to the World Health Organization (WHO)
and the Center for Disease Control and Prevention (CDC),
Chagas disease afects 8 to 10 million people worldwide
[6], with an annual mortality of 10,000 people, which
translates into economic losses that exceed millions of
dollars in control and prevention eforts [7].
Fig. 1 Structure of NAH derivatives tested as trypanocidal agents
In 2017, Elizondo-Jiménez et al. [20] reported the synthe-
sis and biological evaluation of new benzenesulfonylhydra-
zones. Interestingly, compound 4 (Fig. 2) was active against
both NINOA and INC-5 strains of T. cruzi (LC50 =26.3 and
4.68 µM, respectively), and compound 11 (Fig. 2) that incor-
porates an N-acyl group presented LC50 values below 2 µM
against trypomastigotes of the INC-5 strain. Therefore, the
importance of sulfonyl and/or acyl groups is not clear. Also,
the authors suggest that these compounds could be binding
with cruzain by molecular docking. Based on the above,
in this work, it was decided to change the sulfonyl group
(–SO2) by an acyl group (–CO–) to obtain new N-propionyl-
N′-benzeneacylhydrazone derivatives and evaluate their
trypanocidal activity. Additionally, their mode of binding on
the active site of cruzain and their efect on the expression of
the cruzain gene in epimastigotes of T. cruzi were evaluated
to elucidate its possible mechanism of action.
The only drugs available are nifurtimox (Nfx) and ben-
znidazole (Bzn). However, Nfx and Bzn are not adequate
drugs because they cause severe adverse efects and have
poor efcacy in the chronic phase. Also, there are strains
resistant to these drugs, which are not over-the-counter [8,
9]. Therefore, there is a need to develop new molecules
with biological activity against T. cruzi. In that sense,
small molecules (molecular weight < 500) of easy synthe-
sis have been reported as new trypanocidal agents [10, 11].
In the last two decades, one of the most relevant strate-
gies is the development of inhibitors of pharmacological
targets exclusive of the parasite; such is the case of the
cruzain enzyme, which is a cysteine-protease abundant
in T. cruzi, and essential for its metabolism. Currently,
cruzain continues to be one of the most relevant targets
for the design of new anti-Chagas drugs, since it has been
validated through biochemical studies and in vivo models
[12–14].
Materials and methods
In medicinal chemistry, the chemical moiety of N-acylhy-
drazone (NAH, Fig. 1) is recognized as a privileged structure
because it has diverse biological activities toward diferent
therapeutic targets [15–17]. In previous studies, N-acylhy-
drazone derivatives have been used as new approaches in
developing drugs to combat Chagas disease; for example,
Romeiro et al. [18] designed and synthesized quinoxaline-
N-acylhydrazone derivatives using classical bioisoster-
ism; LASSBio-1016 (Fig. 1A) had trypanocidal activity
(IC50 = 15.9 µM) against epimastigotes of the Tulahuen 2
strain of T. cruzi. In another study carried out by Massarico
et al. [19], hybrid derivatives of N-acylhydrazone and furo-
xanes were evaluated as new trypanocidal agents; compound
6 (Fig. 1B) showed trypanocidal activity in vitro against
amastigotes (Tulahuen C2C4 strain). Also, this compound
was shown to be a cruzain inhibitor with IC50 values of
11.2 0.2 μM.
Chemistry
All reagents were purchased from Sigma Aldrich Chemi-
cal Co. and used without further purification. Reaction
progress was monitored using thin-layer chromatography
(TLC) aluminum oxide matrix Z234214-1PAK sheets from
Sigma Aldrich®. Column chromatography was performed
using silica gel 60A (230–400 mesh) and solvents employed
were of analytical grade. Infrared (IR) spectra were recorded
on an FT-IR Bruker Tensor 27 spectrophotometer coupled
to Bruker platinum ATR cell. Mass spectrometry ESI–MS
analyses were conducted on an Ultra Performance Liquid
Cromatography-Mass Spectrometer (UPLC-MS) waters.
Nuclear magnetic resonance (NMR) spectra (1H and 13C)
were measured on a Bruker Ultrashield-400 spectrom-
eter, using DMSO-d6 as a solvent and some cases TMS
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Molecular Diversity
Fig. 2 Structural modifcation
to obtain new benzene-sulfonyl-
hydrazide analog derivatives
as reference. Chemical shifts are presented on the δ scale
(ppm). Multiplicities are indicated as follows: s (singlet), d
(doublet), t (triplet), m (multiplet) or br (broad).
to a fresh medium. At all times, work was carried out under
sterile conditions.
The trypanocidal evaluation of BAH and BAH-N series
was performed against epimastigotes of T. cruzi in triplicate
assays. BHI medium supplemented with 10% FBS and 1%
penicillin/streptomycin (500 μg/mL) was prepared for this
purpose. After, 90 μL of epimastigotes 1×106 cells/mL and
10 μL of each compound at 50 μg/mL dissolved in dimethyl-
sulfoxide (DMSO) at 1% were added to a 96-well plate.
Negative control of untreated epimastigotes and the refer-
ence drugs Nfx and Bzn were included as positive controls.
The plate was incubated at 28 °C for 24 h, then 10 μL of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (MTT) at 2.5 mg/mL in 1X phosphate-bufered saline
(PBS) was added and incubated for 4 h at 37 °C. Subse-
quently, 100 μL of sodium dodecyl sulfate-hydrochloric acid
(SDS-HCl; 10%-0.01 N) were added to dissolve the MTT
crystals and again incubated at 28 °C for 24 h. The optical
density (OD) was determined at 595 nm and all absorbances
were corrected for a blank (medium BHI+FBS+Antibiotic)
by subtracting its absorbance value from each of the samples
(OD595 blank—OD595 sample). The mortality percentage
was calculated using the following formula: 100−(OD(t)/
OD(c)×100), where OD(t) is the optical density of the cul-
ture after exposure to the compound evaluated and OD(c)
is the optical density of the untreated control culture. The
half maximal inhibitory concentration (IC50) was determined
from fve concentrations serial two-fold (50 μg/mL to 3.12
μg/mL) for each compound using the Probit statistical tool.
Later the results were converted to micromolar units.
The general procedure for the preparation of N′-[(E)-
phenylmethylidene]benzeneacylhydrazone derivatives (BAH
series) was as follows: in a 50-mL round-bottomed fask
30 mL of ethanol, benzhydrazide (1 mmol) and aldehyde
derivative (1 mmol) were added at room temperature and in
constant agitation, HCl drops (37%) were used as a catalyst.
After, the mixture was refuxed for 3–6 h and the progress
of the reaction was monitored by TLC. Finally, the reac-
tion product was purifed by column chromatography using
n-hexane: ethyl acetate (98:2).
The synthesis of N-propionyl-N′-[(E)-phenylmethylidene]
benzeneacylhydrazone derivatives (BAH-N series), 30 mL
of dry dichloromethane, the previously obtained benzenea-
cylhydrazone derivative (1 mmol) and dry triethylamine
(3 mmol) were added drop by drop at 0 °C and the mixture
was stirred for 1 h. After, propionyl chloride (1.5 mmol) was
added dropwise and stirring was continued for additional
24–48 h at room temperature. The reaction mixture was puri-
fed by column chromatography using n-hexane: ethyl ace-
tate (98:2) and crystallized into dichloromethane/n-hexane.
Biological activity
Epimastigotes
The NINOA strain of epimastigotes T. cruzi was cultured
and preserved in Brain Heart Infusion (BHI) medium sup-
plemented with 10% Fetal Bovine Serum (FBS) and 1%
penicillin/streptomycin at 500 μg/mL incubated at 28 °C. To
keep the strain viable, transfers were made every 5–7 days
Trypomastigotes
Blood trypomastigotes of NINOA strain of T. cruzi were
obtained from CD1 mice by cardiac puncture after 15 days
post-infection when parasitemia was highest. To keep the
1 3

Molecular Diversity
trypomastigotes viable, transfers were made to CD1 mice
of 20 days of age with a weight of 24–26 g. At all times
we worked under sterile conditions and in accordance with
Law NOM-062-ZOO-1999 [21]. Animals were sacrifced in
accordance with international guidelines on the care and use
of animals in research.
Total RNA Isolation System (Promega®) according to the
manufacturer’s instructions. Total RNA quantifcation was
performed on a Thermo Scientifc® Nanodrop 2000.
Synthesis of cDNA from epimastigotes NINOA strain
BAH and BAH-N series, as well as Nfx and Bzn controls,
were evaluated in triplicate. After, 90 μL of blood contain-
ing blood trypomastigotes (4.5×105 cells/mL) and 10 μL of
each derivative to 50 μg/mL dissolved in 1% DMSO were
added to a 96-well plate. An untreated negative control was
included. The plate was incubated at 4 °C for 24 h, and then
the trypomastigotes were quantifed according to Brener’s
method [22]. The trypanocidal activity was expressed as a
percentage of lysis with respect to untreated control. The
half maximal lytic concentration (LC50) was determined
from fve concentrations (100, 50, 40, 20 and, 10 μg/mL)
for each compound using the Probit statistical tool. Later the
results were converted to micromolar units.
Previously, 2 ng/µL of total RNA was incubated at 70 °C for
10 min, then centrifuged for 30 s and kept on ice. cDNA syn-
thesis was performed according to Promega® commercial
transcription protocol A3500. Reverse transcription reac-
tions were prepared to contain 25 mM MgCl2, 10X reverse
transcription bufer, 10 mM dNTPs, 1 U/µL ribonuclease
inhibitor, 25 U/μL reverse transcriptase, 0.5 μg/μL oligonu-
cleotide (dT)15 and adjusted to a fnal volume of 20 µL with
nuclease-free water. Finally, the reactions were incubated
in a SimpliAmp Thermal Cycler® at 42 °C for 15 min, then
heated to 95 °C for 5 min and incubated at 5 °C for 5 min.
Analysis of the expression of the cruzain gene
The expression levels of the cruzain gene in the epimas-
tigotes (NINOA strain) exposed to compounds 9 and 12,
respectively, at the concentration of 50 μg/mL (time 0, 2, 4,
6, 8, 12, 24 and 48 h) were compared with time zero in all
cases. Primers for amplify cruzain gene and reaction condi-
tions previously reported by Mejía-Jaramillo et al. [27] were
used. The gapdh gene was used as a normalizing gene since
its expression is constitutive and maintains similar levels
of expression in the three forms of T. cruzi [28, 29]. Each
reaction mixture contained 1 µL cDNA, 5 µL Green Super-
mix, 1 µL of each specifc primer (GADPH-F: 5′-ACGGGC
ATGTCTTTTCGTGT-3′ and GADPH-R: 5′-TGGAGCTGC
GGTTGTCATT-3′) and the fnal volume was adjusted to
10 µL with sterile MiliQ water. The amplifcation was run
on the CFX96 Real-Time System, BIO RAD® equipment.
A standard deviation of 1 and a signifcance level of 95%
(p < 0.05) was considered for relative quantifcation using
the BIO RAD CFX Manager Version 3.1 software. The data
was analyzed in GraphPad Prism 6 and the comparison of
means by Tukey was made for those data that were signif-
cant (p < 0.05).
Molecular docking
The molecular binding analysis of BAH and BAH-N series
on the active site of cruzain was carried out using the
software AutoDock Vina 1.1.2 [23]. The crystallographic
structure of cruzain in complex with vinyl sulfone derived
inhibitor (WRR-483) was retrieved from the Protein Data
Bank (PDB ID: 3LXS). Then, the structure of the cruzain
was prepared using the software UCSF Chimera [24], which
removes all ligands and water molecules of the receptor
structure, also repairs missing side chains and adds polar
hydrogens. On the other hand, the co-crystallized ligand
WRR-483 and the BAH and BAH-N series were prepared
using UCSF Chimera, this tool minimized the structures of
each ligand and adds polar hydrogens to them. After, ligands
and the receptor, both were converted to pdbqt format for run
vina, using MGLTools 1.1.6 [25]. The docking search space
had values of size X=60, Y=60, Z=60 and was centered
at X=52.15, Y=−3.633 and Z=21.779. The interactions
between cruzain and the docked poses were calculated using
Protein–Ligand Interaction Profler (PLIP) [26].
RNA extraction from epimastigotes
Results and discussion
BHI medium supplemented with 10% FBS and 1% peni-
cillin–streptomycin 500 μg/mL was prepared and 90 μL of
epimastigotes (1×106 cells/mL) and 10 μL of compound 9
and 12, respectively, were added to a fnal concentration of
50 μg/mL to a 96-well plate. Aliquots were taken in trip-
licate at diferent times: 0, 2, 4, 6, 8, 12, 24 and 48 h, and
the plate was kept in incubation at 28 °C for 48 h. Total
RNA extraction was performed using the commercial kit SV
Chemistry
Synthesis of the benzeneacylhydrazone (BAH series) deriva-
tives (compounds from 1 to 7) was carried out with high
yields after the condensation of benzhydrazide with the
appropriate aldehyde in ethanol using drops of hydrochloric
acid as a catalyst. In the spectrum of the 1H-NMR analysis,
1 3

Molecular Diversity
the presence of a signal corresponding to the N=CH bond
and a characteristic signal belonging to the N–H bond
was observed, this was consistent for each derivative (1H
(400 MHz) and 13C (101 MHz) NMR spectra were recorded
with TMS as internal standard. Assignment of the NMR sig-
nals was made by HMQC and HMBC 2D methods). Also, all
derivatives were structurally confrmed by IR, NMR, UPLC-
MS m/z, and their melting point was determined. The com-
pounds 1–7 previously have been reported and their char-
acterization data were consistent with the previous reports
[30–35].
N′‑[(4‑methoxyphenyl)methylidene]benzeneacylhydrazone
(4) This compound was obtained in 93.08% from ben-
zhydrazide and 4-methoxybenzaldehyde. FT-IR (v cm−1):
3191 (w, N–H), 1639 (s, C=O), 1601 (s, C=N). 1H NMR
(400 MHz, DMSO-d6) δ 3.81 (s, 3H, OCH3), 7.02 (d,
J=8.6 Hz, 2H, H-2′′), 7.51 (t, J =7.3 Hz, 2H, H-3′), 7.58
(t, J = 7.2 Hz, 1H, H-4′), 7.67 (d, J = 7.4 Hz, 2H, H-3″),
7.90 (d, J=7.2 Hz, 2H, H-2′), 8.41 (s, 1H, H-4), 11.63 (s,
1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 55.2 (OCH3),
114.3 (C-2″), 126.9 (C-1″), 127.4 (C-2′), 128.3 (C-3′), 128.5
(C-2″), 131.4 (C-4′), 133.6 (C-1′), 147.7 (C-4), 160.8 (C-4″),
162.9 (C-1). m/z calculated: 254.28 Da, found: 255.14
[M + H]+ Da; Retention time: 0.911 min. Melting point:
143 °C.
N′‑[benzylidene]benzeneacylhydrazone (1) This compound
was obtained in 47.63% from benzhydrazide and benzalde-
hyde. FT-IR (v cm−1): 3175 (w, N–H), 1639 (s, C=O), 1600
(s, C=N). 1H NMR (400 MHz, DMSO-d6) δ 7.38–7.49 (m,
3H, H-3″, H-4″), 7.51 (tt, J=7.8, 1.3 Hz, 2H, H-3′), 7.59 (t,
J=7.3 Hz, 1H, H-4′), 7.73 (s, 2H, H-2″), 7.92 (d, J=7.2 Hz,
2H, H-2′), 8.49 (s, 1H, H-4), 11.77 (s, 1H, NH). 13C NMR
(101 MHz, DMSO-d6) δ 126.9 (C-2″), 127.5 (C-2′), 128.3
(C-3′), 128.7 (C-3″), 129.9 (C-4″), 131.5 (C-4′), 133.4
(C-1′), 134.3 (C-1″), 147.8 (C-4), 163.1 (C-1). m/z calcu-
lated: 224.09 Da, found: 225.15 [M + H]+ Da; Retention
Time: 0.820 min. Melting Point: 211 °C.
N′‑[(4‑fluorophenyl)methylidene]benzeneacylhydrazone
(5) This compound was obtained in 91.59% from benzhy-
drazide and 4-fuorobenzaldehyde. FT-IR (v cm−1): 3058 (w,
N–H), 1631 (s, C=O), 1600 (s, C=N). 1H NMR (400 MHz,
DMSO-d6) δ 7.01 (t, J=8.2 Hz, 2H, H-3″), 7.31–7.41 (m,
2H, H-3′), 7.43–7.50 (m, 1H, H-4′), 7.63–7.73 (m, 2H,
H-2″), 7.87 (t, J=7.2 Hz, 2H, H-2′), 8.36 (s, 1H, H-4), 11.49
(s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 115.0 (d,
J = 21.8 Hz, C-3″), 127.2 (C-2′), 127.7 (C-3′), 128.8 (d,
J=8.3 Hz, C-2″), 129.9 (C-1″), 131.1 (C-4′), 133.2 (C-1′),
146.7 (C-4), 163.1 (d, J=249.8 Hz, C-4″), 163.9 (C-1). m/z
calculated: 242.25 Da, found: 243.12 [M+H]+ Da; Reten-
tion time: 0.938 min. Melting point: 186 °C.
N′‑[(4‑ethylphenyl)methylidene]benzeneacylhydrazone
(2) This compound was obtained in 67.12% from ben-
zhydrazide and 4-ethylbenzaldehyde. FT-IR (v cm−1):
3186 (w, N–H), 1643 (s, C=O), 1604 (s, C=N). 1H NMR
(400 MHz, DMSO-d6) δ 1.20 (t, J=7.6 Hz, 3H, H-6″), 2.64
(q, J = 7.9 Hz, 2H, H-5″), 7.29 (d, J = 7.9 Hz, 2H, H-3″),
7.51 (t, J = 7.3 Hz, 2H, H-3′), 7.54–7.59 (m, 1H, H-4′),
7.59–7.67 (m, 2H, H-2″), 7.87–7.98 (m, 2H, H-2′), 8.48 (s,
1H, H-4), 11.75 (s, 1H, NH). 13C NMR (101 MHz, DMSO-
d6) δ 15.1 (C-6″), 28.07 (C-5″), 127.0 (C-2″), 127.5 (C-2′),
128.1 (C-3″), 128.3 (C-3′), 131.5 (C-4′), 131.8 (C-1″), 133.5
(C-1′), 146.0 (C-4″), 163.0 (C-1). m/z calculated: 252.31 Da,
found: 253.15 [M + H]+ Da; Retention time: 1.095 min.
Melting point: 127 °C.
N′‑[(2‑nitrophenyl)methylidene]benzeneacylhydrazone
(6) This compound was obtained in 72.20% from ben-
zhydrazide and 2-nitrobenzaldehyde. FT-IR (v cm−1):
3252 (w, N–H), 1673 (s, C=O), 1644 (s, C=N), 1522 (s,
NO2). 1H NMR (400 MHz, DMSO-d6) δ 7.51–7.57 (m, 2H,
H-3′), 7.58–7.65 (m, 1H, H-4′), 7.66–7.71 (m, 1H, H-5″),
7.82 (t, J = 7.5 Hz, 1H, H-4″), 7.91–7.99 (m, 2H, H-2′),
8.05–8.10 (m, 1H, H-6′′), 8.14 (s, 1H, H-3″), 8.92 (s, 1H,
H-4), 12.20 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6)
δ 124.4 (C-6″), 127.5 (C-2′), 127.7 (C-3″), 127.8 (C-1′),
128.3 (C-3′), 128.5 (C-1″), 130.5 (C-5″), 131.7 (C-4′), 133.5
(C-4″), 142.7 (C-4), 148.2 (C-2″), 165.8 (C-1). m/z calcu-
lated: 269.26 Da, found: 270.08 [M + H]+ Da; Retention
time: 0.940 min. Melting point: 202 °C.
N′‑[(4‑hydroxyphenyl)methylidene]benzeneacylhydrazone
(3) This compound was obtained in 72.65% from ben-
zhydrazide and 4-hydroxybenzaldehyde. FT-IR (v cm−1):
3184 (w, N–H), 1598 (s, C=O), 1547 (s, C=N). 1H NMR
(400 MHz, DMSO-d6) δ 6.85 (d, J=8.9 Hz, 2H, H-3″), 7.50
(t, J = 7.3 Hz, 2H, H-3′), 7.56 (d, J = 7.0 Hz, 2H, H-2″),
7.91 (d, J=6.9 Hz, 2H, H-2′), 8.39 (s, 1H, H-4), 11.60 (s,
1H, NH). 13C NMR (101 MHz, DMSO-d6) δ 115.6 (C-3′)
127.4 (C-2′), 128.3 (C-3′), 128.7 (C-2″), 131.4 (C-1′), 133.6
(C-1″), 148.2 (C-4), 159.3 (C-4′), 162.8 (C-1). m/z calcu-
lated: 240.26 Da, found: 241.10 [M + H]+ Da; Retention
time: 0.810 min. Melting point: 228 °C.
N′‑[(4‑nitrophenyl)methylidene]benzeneacylhydrazone
(7) This compound was obtained in 80.70% from benzhy-
drazide and 4-nitrobenzaldehyde. FT-IR (v cm−1): 3182 (w,
1
N–H), 1716 (s, C=O), 1644 (s, C=N), 1518 (s, NO2). H
NMR (400 MHz, DMSO-d6) δ 7.54 (t, J=7.4 Hz, 2H, H-3′),
7.61 (t, J=7.3 Hz, 1H, H-4′), 7.93 (d, J=7.6 Hz, 2H, H-2′),
7.98 (d, J=8.8 Hz, 2H, H-2″), 8.29 (d, J=8.4 Hz, 2H, H-3″),
8.56 (s, 1H, H-4), 12.05 (s, 1H, NH). 13C NMR (101 MHz,
DMSO-d6) δ 123.9 (C-3″), 127.6 (C-2′), 127.8 (C-2″), 128.3
1 3

Molecular Diversity
(C-3′), 131.7 (C-4′), 133.1 (C-1′), 140.6 (C-1″), 145.1 (C-4),
147.8 (C-4″), 163.2 (C-1). m/z calculated: 269.26 Da, found:
270.05 [M + H]+ Da; Retention time: 0.942 min. Melting
point: 241 °C.
(s, C=N), 1283 (m, C–N). 1H NMR (400 MHz, DMSO-d6)
δ 1.15 (t, J = 7.5 Hz, 3H, H-3‴), 2.62 (q, J = 7.5 Hz, 2H,
H-2‴), 7.23 (d, J=8.3 Hz, 2H, H-2″), 7.53 (t, J=7.3 Hz,
2H, H-3′), 7.56–7.62 (m, 1H, H-4′), 7.78 (d, J=7.8 Hz, 2H,
H-3″), 7.93 (d, J=7.4 Hz, 2H, H-2′), 8.50 (s, 1H, H-4), 11.85
(s, 1H, OH). 13C NMR (101 MHz, DMSO-d6) δ 8.7 (C-3‴),
26.9 (C-2‴), 122.2 (C-3″), 127.5 (C-2′), 128.1 (C-2″), 128.4
(C-3′), 131.6 (C-1″), 131.9 (C-4′), 133.4 (C-1′), 146.9 (C-4),
151.7 (C-4′′), 163.1 (C-1), 172.3 (C-1‴). m/z calculated:
296.32 Da, found: 297.06 [M + H]+ Da; Retention time:
0.965 min. Melting point: 180 °C.
The N-propionyl-N′-benzeneacylhydrazone (BAH-N
series) derivatives (compounds from 8 to 14) were obtained
with moderate yields, from the hydrazinolysis of propionyl
chloride using compounds from 1 to 7 and triethylamine
as a catalyst in dry dichloromethane and at 0 °C. In the
spectrum of the 1H NMR analysis, the absence of the sig-
nal for the N–H bond and the appearance of a new signal
for the N-propionyl chain were observed. Additionally, all
derivatives were confrmed structurally by IR spectroscopy,
UPLC-MS (m/z) and their melting point were determined.
N‑propionyl‑N′‑[(4‑methoxyphenyl)methylidene]benzeneacyl‑
hydrazone (11) This compound was obtained in 6.0% yield
from compound 4 and propionyl chloride. FT-IR (v cm−1):
2924 (m, C–H), 1723 (s, C=O), 1618 (s, C=O), 1506 (s,
C=N), 1247 (m, C–N). 1H NMR (400 MHz, CDCl3) δ 1.24
(t, J=7.4 Hz, 3H, H-3‴), 2.93 (q, J=7.4 Hz, 2H, H-2‴),
3.84 (s, 3H, OCH3), 6.87–6.93 (m, 2H, H-3″), 7.39–7.47
(m, 2H, H-3′), 7.50–7.57 (m, 1H, H-4′), 7.59–7.64 (m, 2H,
H-2″), 7.74–7.81 (m, 2H, H-2′), 8.17 (s, 1H, H-4). 13C NMR
(101 MHz, CDCl3) δ 8.9 (C-3‴), 29.4 (C-2‴), 55.5 (OCH3),
114.4 (C-3″), 126.5 (C-1″), 128.7 (C-3′), 129.6 (C-2′), 129.7
(C-2″), 133.2 (C-4′), 134.6 (C-1′), 153.9 (C-4), 162.3 (C-4″),
172.7 (C-1), 176.4 (C-1‴). m/z calculated: 310.13 Da,
found: 311.05 [M + H]+ Da; Retention time: 1.666 min.
Melting point: 166 °C.
N‑propionyl‑N′‑[phenylmethylidene]benzeneacylhydrazone
(8) This compound was obtained in 4.17% yield from
compound 1 and propionyl chloride. FT-IR (v cm−1): 2938
(m, C–H), 1690 (s, C=O), 1670 (s, C=O), 1598 (s, C=N),
1234 (m, C–N). 1H NMR (400 MHz, DMSO-d6) δ 1.13 (t,
J=7.4 Hz, 3H, H-3‴), 2.91 (q, J=7.4 Hz, 2H, H-2‴), 7.41–
7.50 (m, 3H, H-3″ and H-4″), 7.50–7.57 (m, 2H, H-3′), 7.62–
7.68 (m, 1H, H-4′), 7.76 (dd, J = 7.7, 1.8 Hz, 2H, H-2″),
7.80 (dd, J=8.4, 1.3 Hz, 2H, H-2′), 8.29 (s, 1H, H-4). 13
C
NMR (101 MHz, DMSO-d6) δ 8.4 (C-3‴), 28.1 (C-2‴),
127.5 (C-2″), 128.6 (C-3′), 128.7 (C-3″), 129.2 (C-2′), 131.0
(C-4″), 133.3 (C-4′), 133.4 (C-1″), 133.7 (C-1′), 153.4 (C-4),
172.3 (C-1), 175.6 (C-1‴). m/z calculated: 280.32 Da,
found: 281.10 [M + H]+ Da; Retention time: 1.824 min.
Melting point: 25–30 °C.
N‑propionyl‑N′‑[(4‑fuorophenyl)methylidene]benzeneacyl‑
hydrazone (12) This compound was obtained in 88.58%
yield from compound 5 and propionyl chloride. FT-IR (v
cm−1): 2980 (m, C–H), 1707 (s, C=O), 1687 (s, C=O), 1601
(s, C=N), 1198 (m, C–N). 1H NMR (400 MHz, DMSO-d6)
δ 1.11 (t, J = 7.4 Hz, 3H, C-3‴), 2.90 (q, J = 7.4 Hz, 2H,
C-2″′), 7.30 (t, J = 8.9 Hz, 2H, H-3″), 7.49–7.56 (m, 2H,
H-3′), 7.62–7.69 (m, 1H, H-4′), 7.76—7.98 (m, 4H, H-2″
and H-2′), 8.31 (s, 1H, H-4). 13C NMR (101 MHz, DMSO-
d6) δ 8.47 (C-3‴), 28.11 (C-2‴), 115.93 (d, J= 22.1 Hz,
C-3″), 128.72 (C-3′), 129.29 (C-2′), 129.9 (d, J = 8.8 Hz,
C-2″) 130.1 (d, J = 3.0 Hz, C-1″), 133.46 (C-4′), 133.76
(C-1′), 152.30 (C-4), 163.68 (d, J=249.2 Hz, C-4″), 172.37
(C-1), 175.75 (C-1‴). m/z calculated: 298.11 Da, found:
299.08 [M + H]+ Da; Retention time: 2.171 min. Melting
point: 82 °C.
N‑propionyl‑N′‑[(4‑ethylphenyl)methylidene]benzeneacylhy‑
drazone (9) This compound was obtained in 8.47% yield
from compound 2 and propionyl chloride. FT-IR (v cm−1):
2960 (m, C–H), 1693 (s, C=O), 1617 (s, C=O), 1598 (s,
C=N), 1210 (m, C–N). 1H NMR (400 MHz, DMSO-d6) δ
1.11 (t, J=7.4 Hz, 3H, H-3‴), 1.18 (t, J=7.6 Hz, 3H, H-6″),
2.64 (q, J = 7.5 Hz, 2H, H-5″), 2.89 (q, J = 7.4 Hz, 2H,
H-2‴), 7.30 (d, J =8.2 Hz, 2H, H-2″), 7.48–7.56 (m, 2H,
H-3′), 7.60–7.70 (m, 3H, H-4′ and H-3″), 7.78 (dd, J=8.4,
1.3 Hz, 2H, H-2′), 8.26 (s, 1H, H-4). 13C NMR (101 MHz,
DMSO-d6) δ 8.5 (C-3‴), 15.2 (C-6″), 28.1 (C-5″), 28.2
(C-2‴), 127.7 (C-2″), 128.2 (C-3″), 128.7 (C-3′), 129.2
(C-2′), 130.9 (C-1″), 133.3 (C-4′), 133.9 (C-1′), 147.4
(C-4″), 154.4 (C-4), 172.3 (C-1), 175.6 (C-1‴). m/z cal-
culated: 308.15 Da, found: 309.13 [M+H]+ Da; Retention
time: 1.300 min. Melting point: 58 °C.
N‑propionyl‑N′‑[(2‑nitrophenyl)methylidene]benzeneacyl‑
hydrazone (13) This compound was obtained in 3.31%
yield from compound 6 and propionyl chloride. FT-IR (v
cm−1): 2976 (m, C–H), 1710 (s, C=O), 1691 (s, C=O),
1
N‑propionyl‑N′‑[(4‑hydroxyphenyl)methylidene]benzenea‑
cylhydrazone (10) This compound was obtained in 60.06%
yield from compound 3 and propionyl chloride. FT-IR (v
cm−1): 2977 (m, C–H), 1747 (s, C=O), 1650 (s, C=O), 1541
1584 (s, C=N), 1507 (s, NO2), 1243 (m, C–N). H NMR
(400 MHz, DMSO-d6) δ 1.12 (t, J = 7.4 Hz, 3H, H-3‴),
2.91 (q, J = 7.4 Hz, 2H, H-2‴), 7.52–7.61 (m, 2H, H-3′),
7.67–7.75 (m, 2H, H-4″ and H-5″), 7.78–7.86 (m, 3H, H-2′
1 3

Molecular Diversity
and H-4′), 8.04 (ddd, J = 11.6, 8.0, 1.3 Hz, 2H, H-3″ and
H-6″), 8.48 (s, 1H, H-4). 13C NMR (101 MHz, DMSO-d6)
δ 8.4 (C-3″′), 27.5 (C-2‴), 124.5 (C-3″ or C-6″), 127.8
(C-1″), 128.9 (C-3″ or C-6″), 129.0 (C-3′), 129.6 (C-2′),
131.3 (C-4″ or C-5″), 132.9 (C-1′), 133.6 (C-4′), 134.2 (C-4″
or C-5″), 145.8 (C-4), 172.6 (C-1), 176.0 (C-1‴). m/z cal-
culated: 325.10 Da, found: 326.03 [M+H]+ Da; Retention
time: 1.875 min.
calculated: 325.10 Da, found: 326.05 [M+H]+ Da; Reten-
tion time: 1.865 min. Melting point: 101 °C.
Trypanocidal activity
The anti-T. cruzi activity of the BAH and BAH-N series
(compounds from 1 to 14) were evaluated against epimas-
tigotes and trypomastigotes of the NINOA strain (Table 1).
The IC50 was determined in those compounds that presented
the highest percentage of mortality in epimastigotes. While
for the most active compounds against trypomastigotes, the
LC50 was calculated.
N‑propionyl‑N′‑[(4‑nitrophenyl)methylidene]benzeneacyl‑
hydrazone (14) This compound was obtained in 6.91%
yield from compound 7 and propionyl chloride. FT-IR (v
cm−1): 2976 (m, C–H), 1710 (s, C=O), 1692 (s, C=O),
1
1585 (s, C=N), 1506 (s, NO2), 1245 (m, C–N). H NMR
(400 MHz, DMSO-d6) δ 1.13 (t, J=7.4 Hz, 3H, H-3‴), 2.98
(q, J = 7.4 Hz, 2H, H-2‴), 7.51–7.61 (m, 2H, H-3′), 7.71
(tt, J=7.0, 1.3 Hz, 1H, H-4′), 7.85 (dd, J=8.4, 1.2 Hz, 2H,
H-2′), 7.98–8.05 (m, 2H, H-3″), 8.23–8.33 (m, 2H, H-2″),
8.38 (s, 1H, H-4). 13C NMR (101 MHz, DMSO-d6) δ 8.3
(C-3‴), 27.6 (C-2‴), 123.9 (C-2″), 128.4 (C-3″), 129.0
(C-3′), 129.6 (C-2′), 133.1 (C-1′), 134.1 (C-4′), 139.7 (C-1″),
147.5 (C-4), 148.4 (C-4″), 172.7 (C-1), 176.2 (C-1‴). m/z
Epimastigotes
Compounds 1 and 2 of the BAH series showed low activity
against T. cruzi epimastigotes, 10.4 and 10.8% mortality,
respectively. However, compound 3 substituted with a –OH
group at para-position on the phenyl ring, enhanced biologi-
cal activity. This efect may be because the hydroxyl group is
Table 1 In vitro trypanocidal activity of benzeneacylhydrazone and N-propionyl-N′-benzeneacylhydrazone derivatives against T. cruzi NINOA
strain
Compound
R
R′
% Mortality at
50 µg/mL
Epimastigotes IC50 % Mortality at
Trypomastigotes
LC50 (µM)b
(µM)b
50 µg/mL
1
H
H
H
H
H
H
H
H
10.4
10.8
24.5
5.2
0
0
0
5.1
61.4
16.3
0
41.6
0
29.9
84.8
82.8
ND
ND
ND
ND
ND
ND
ND
ND
78.66*
ND
ND
166.28*
ND
ND
0.24
17.29
31.2
40.1
29.0
28.7
68.3
33.0
46.2
50.0
25
28.2
30.2
40.7
38.9
18.9
52.1
46.2
ND
ND
ND
ND
202.68*
ND
455.09*
238.29*
ND
ND
ND
309.38*
ND
ND
182.01
227.55
2
p-CH2–CH3
p-OH
p-OCH3
p-Fluorine
o-NO2
p-NO2
H
p-CH2–CH3
p-OH
p-OCH3
p-Fluorine
o-NO2
3
4
5
6
7
8
–CO–CH2–CH3
–CO–CH2–CH3
–CO–CH2–CH3
–CO–CH2–CH3
–CO–CH2–CH3
–CO–CH2–CH3
–CO–CH2–CH3
9
10
11
12
13
14
p-NO2
Nifurtimox
Benznidazole
aND: not determined
bThe results are means of three experiments
cBold values indicates signifcant results (p < 0.05)
1 3

Molecular Diversity
highly reactive and participates in the formation of reactive
oxygen species (ROS) [36]. Subsequently, the incorporation
of a methoxy group in compound 4 at para-position was
unfavorable. Finally, the compounds 5 (para-F phenyl), 6
(ortho-NO2 phenyl) and 7 (para-NO2 phenyl) were inactive
against T. cruzi epimastigotes from the NINOA strain, due
to solubility problems in the culture medium.
(para-fuorophenyl) had the highest level of activity against
trypomastigotes, even higher than the reference drugs
Nfx and Bzn. This can be attributed to the presence of a
fuorine atom (electron-withdrawing group) at para posi-
tion on the phenyl ring, which is similar to that reported
by Pontes-Espíndola et al. [37] who mention that the steric
efect exerted by the size of the atomic radius of the halogen
atom is important for the activity of the compounds against
trypomastigotes.
The N-acylation of compound 1 to obtain compound 8
(BAH-N series) caused a drastic loss of activity. Compounds
10 (para-hydroxy phenyl) and 11 (para-methoxy phenyl)
also had a decrease in activity. However, the N-acylation of
compound 2 to obtain 9 signifcantly enhances its biological
activity. Additionally, the trypanocidal efect of compounds
12 and 14 (para-F and para-NO2 phenyl, respectively) con-
siderably increased after N-acylation. However, compound
13 (ortho-NO2-phenyl) proved to be inactive, although the
compound has a nitro group associated with trypanocidal
activity by ROS generation.
Unexpectedly, the BAH-N series (N-acylation) did not
show a greater efect than that of the BAH series (except
compound 8, 50% mortality). In particular, compounds 9,
12, and 14 showed a loss of activity, compounds 10 and
11 remained with almost the same percentage of lysis, and
compounds 8 and 13 had enhanced trypanocidal activity.
Therefore, the structural modifcation did not improve tryp-
anocidal activity against trypomastigotes of NINOA strain.
Molecular docking
Trypomastigotes
The molecular docking analysis aimed to identify the most
favorable binding pose and interaction profle of each com-
pound on the active site of cruzain. Initially, a re-docking of
the cruzain complex co-crystallized with WRR-483 (PDB
ID: 3LXS) was performed and the root mean square devia-
tion (RMSD) was calculated to validate the methodology. A
free energy of binding (FEB) value of − 9.8 kcal/mol was
obtained for the WRR-483 and an RMSD value equal to
0.4576 Å, which was considered acceptable to perform the
molecular modeling of the fourteen derivatives of the BAH
and BAH-N series.
In general, the compounds of the BAH series had good
activity against trypomastigotes of T. cruzi (NINOA strain);
in particular, compound 5 showed a higher level of activity
than the reference drugs (Table 1).
Compound 1 produced 31.25% lysis against trypomas-
tigotes. Compound 2 substituted with an ethyl group at
para-position on the phenyl ring slightly increased its activ-
ity. However, compounds 3 with a highly reactive group
(para-hydroxyphenyl) and 4 with weak electron-donating
(para-methoxyphenyl) were not very active. Compound 5
Table 2 The pattern of
Compound
FEB (Kcal/mol)
Interaction typea
interactions between BAH and
BAH-N series derivatives on
the cruzain active site
Hydrogen bond
Hydrophobic
π-stacking
1
−6
Gln19, Gly20, Cys22
Trp184
Trp184
Trp184
Trp184
Trp184
Trp184
–
Trp184
Trp184
Trp184
Trp184
Trp184
Trp184
–
2
3
4
5
6
7
8
9
10
11
12
13
−6.8
−6.5
−6.6
−6
−6.5
−6.6
−6.4
−6.8
−6.3
−6.5
−6.5
−6.5
−6.7
−9.8
Gln19
Gln19
Gln19
Gln19, Gly20, Cys22
Asp18
Gln19
Gln19
Gln19
Gln19
Gln19
Gln19
Gln19
Gln19
Gln19, Asp161, Trp184,Thr185
Gln19
Gln19, Trp184
Trp184
Lys17, Phe28, Tyr91, Thr185
Gln19, Trp184
Trp184
Trp184, Thr185
Ala141, His162, Trp184
Gln19, Trp184
Ala141, His162
Ala141, His162, Trp184
Gln19, Trp184
His162
14
Trp184
His162
WRR-483
Gln19, Gly66, Asp161
aInteractions were obtained using the PLIP online server
1 3

Molecular Diversity
In general, the BAH series (compounds 1–7) contains
an FEB value between −6 and − 6.8 kcal/mol. While the
BAH-N series (compounds 8–14) submitted values between
−6.3 and −6.8 kcal/mol. Table 2 describes the FEB values
and the interaction profle for each compound.
In general, the compounds of the BAH series in the
molecular docking showed no infuence of the substituents
on the phenyl ring to interact on the active site of cruzain.
However, the addition of the N-propionyl chain favored the
formation of new hydrogen bridges with residues from cru-
zain subsites.
Furthermore, a visual inspection of the mode of cruzain
binding of the highest-scoring compounds in molecular
docking was performed. Compound 2 (para-ethylphenyl)
obtained the most favorable FEB value in the BAH series
and had hydrophobic interaction with Gln19, Asp161,
Trp184 and Trp185, respectively. In addition, the oxygen of
a carbonyl group acted as a hydrogen acceptor by forming
a hydrogen bond with the nitrogen from Gln19. Finally, a
π-stacking interaction with the indole group of Trp184 was
detected (Fig. 3a). On the other hand, compound 4 (para-
methoxyphenyl) showed interactions with Gln19 and Trp184
residues (Fig. 3b). The nitrogen from Gln19 formed a hydro-
gen bond with the oxygen in the carbonyl group. Likewise,
the nitrogen from Trp184’s indole bonded with the oxygen
from the carbonyl group by a hydrogen bond. Phenyl of
the hydrazone moiety had a hydrophobic interaction with
Trp184 and an interaction π-stacking.
The highest scoring compounds in the molecular docking
were 9 and 14 with an FEB value of − 6.8 and − 6.7 kcal/
mol, respectively, followed by compounds 11, 12, and 13
(−6.5 kcal/mol, respectively). Visual inspection of the cru-
zain-compound 9 (Fig. 3c) showed that the benzene-acyl
moiety was oriented opposite to the Cys25 residue; there-
fore, it only formed hydrophobic contacts with Trp184 and
Thr185. The N-propionyl chain was arranged in subsite S1
forming hydrogen bridges with Gln19 and the indol nitrogen
from the Trp184 residue. The putative pose that compound
14 adopted was in subsite S1′ interacting hydrophobically
with Gln19 and Trp184. In addition, oxygen from the N-acyl
chain favored interaction with Gln19 to form hydrogen
bridges.
On the other hand, compound 11 adopted a conformation
in subsite S1′ to interact with Gln19 and Trp184 through
hydrogen bridges between the oxygen of the carbonyl of
the N-propionyl chain. Compound 12 (Fig. 3d) presented
a conformation in subsite S1′ similar to compound 11 but
interacted hydrophobically with two new residues (Ala141
and His162). Again, the N-acyl chain oxygen functioned as a
hydrogen acceptor forming bonds with Gln19. Finally, com-
pound 13 was coupled on subsite S1′ interacting hydrophobi-
cally with Ala141, His162, and Trp184, and the oxygen of
Compound 7 (para-nitrophenyl) of the BAH series
(−6.6 kcal/mol) interacted with Gln19, His162 and Trp184
forming a hydrogen bridge, respectively, with the oxygen of
the carbonyl. It also interacted hydrophobically with Gln19
and Trp184 as well as an interaction π-stacking. However,
these compounds showed poor or null biological activity
against epimastigotes; therefore, there is no correlation
between the FEB value and trypanocidal activity.
Fig. 3 The putative binding
pose of BAH and BAH-N deriv-
atives on the active site of the
cruzain enzyme. a Compound
2, b compound 4, c compound
9, d compound 12. Hydrogen
bonds are shown as blue lines
and interaction hydrophobic as
gray dashed lines. Green dashed
lines represent π-stacking
interactions
1 3

Molecular Diversity
Fig. 4 Relative expression
levels of the cruzain gene after
treatment with compounds
9 and 12, respectively. The
dotted line represents the basal
expression of the gapdh normal-
izer gene. ANOVA, p < 0.05.
a−gComparison of means by
Tukey
the N-acyl chain interacted with Gln19 through hydrogen
Gln19 and Trp184 but not with Cys25 on the active site of
cruzain. Finally, compounds 9 and 12 cause a signifcant
increase in the expression of the cruzain gene at 48 h of
exposure. These results display that the mechanism of action
of compounds 9 and 12 could be by cruzain inhibition.
bridges.
Analysis of cruzain gene expression
An analysis to determine the expression levels of the cru-
zain gene in epimastigotes of T. cruzi after exposure to the
derivatives 9 and 12 (50 μg/mL) was carried out to indirectly
elucidate the possible mechanism of inhibition of the cruzain
enzyme by these compounds. This according to Sajid et al.
[38], who mentions that an increase in the expression levels
of the cruzain gene, can occur in response to inhibition of
the enzyme.
Acknowledgements This research was funded by Secretaria de
Investigacion y Posgrado del Instituto Politecnico Nacional (SIP-
20200491). Gildardo Rivera Sánchez and Benjamin Nogueda-Torres
holds a scholarship from the “Comisión de Operación y Fomento de
Actividades Académicas” (COFAA-IPN) and “Programa de Estímulos
al Desempeño de los Investigadores” (EDI-IPN). José Carlos Espinoza-
Hicks is thankful to the Consejo Nacional de Ciencia y Tecnología
(CONACYT,Mexico) for a grant to purchase the NMR instrument
(INFR-2014-01-226114).
Relative quantifcation was used in the trial and the com-
parative method ΔCt was applied to establish the levels of
expression of the cruzain gene with respect to the control.
The determination of the expression of the constitutive gene
gapdh allowed comparison with the expression of the cru-
zain gene after treatment with compound 9 and 12, respec-
tively. Thus, an increase of 0.01 relative units of expres-
sion could be observed from 2 h and the most pronounced
increase was from 0.03 to 48 h for both treatments. This
fnding was signifcant (p < 0.05) when contrasted with time
zero and with the gapdh gene (Fig. 4).
Compliance with ethical standards
Conflict of interest None to declare.
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Molecular Diversity
Afliations
Timoteo Delgado‑Maldonado1 · Benjamín Nogueda‑Torres2 · José C. Espinoza‑Hicks3 · Lenci K. Vázquez‑Jiménez1 ·
Alma D. Paz‑González1 · Alfredo Juárez‑Saldívar1 · Gildardo Rivera1
3
* Gildardo Rivera
Laboratorio de Química II, Departamento de Química
Orgánica, Facultad de Ciencias Químicas, Universidad
Autónoma de Chihuahua, Circuito Universitario, Campus
Universitario, Apartado Postal 669, Chihuahua, Chih.,
Mexico
gildardors@hotmail.com
1
Laboratorio de Biotecnología Farmacéutica, Centro de
Biotecnología Genómica, Instituto Politécnico Nacional,
88710 Reynosa, Mexico
2
Departamento de Parasitología, Escuela Nacional de
Ciencias Biológicas, Instituto Politécnico Nacional,
11340 Ciudad de México, Mexico
1 3