Synthesis, Biological Activity, and Mechanism of Action of 2-Pyrazyl and Pyridylhydrazone Derivatives, New Classes of Antileishmanial Agents

Article abstract of DOI:10.1002/cmdc.201800328

In this work, we report the antileishmanial activity of 23 compounds based on 2-pyrazyl and 2-pyridylhydrazone derivatives. The compounds were tested against the promastigotes of Leishmania amazonensis and L. braziliensis, murine macrophages, and intracellular L. amazonensis amastigotes. The most potent antileishmanial compound was selected for investigation into its mechanism of action. Among the evaluated compounds, five derivatives [(E)-3-((2-(pyridin-2-yl)hydrazono)methyl)benzene-1,2-diol (2 b), (E)-4-((2-(pyridin-2-yl)hydrazono)methyl)benzene-1,3-diol (2 c), (E)-4-nitro-2-((2-(pyrazin-2-yl)hydrazono)methyl)phenol (2 s), (E)-2-(2-(pyridin-2-ylmethylene)hydrazinyl)pyrazine (2 u), and (E)-2-(2-((5-nitrofuran-2-yl)methylene)hydrazinyl)pyrazine (2 v)] exhibited significant activity against L. amazonensis amastigote forms, with IC₅₀ values below 20 μm. The majority of the compounds did not show any toxic effect on murine macrophages. Preliminary studies on the mode of action of members of this hydrazine-derived series indicate that the accumulation of reactive oxygen species (ROS) and disruption of parasite mitochondrial function are important for the pharmacological effect on L. amazonensis promastigotes.

Full text of DOI:10.1002/cmdc.201800328

DOI: 10.1002/cmdc.201800328
Full Papers
Synthesis, Biological Activity, and Mechanism of Action of
2-Pyrazyl and Pyridylhydrazone Derivatives, New Classes
of Antileishmanial Agents
Elaine S. Coimbra,[c] Luciana M. R. Antinarelli,[c] Mariana de A. Crispi,[c] Thais C. M. Nogueira,[a]
Alessandra C. Pinheiro,[a] and Marcus V. N. de Souza*[a, b]
In this work, we report the antileishmanial activity of 23 com-
pounds based on 2-pyrazyl and 2-pyridylhydrazone derivatives.
The compounds were tested against the promastigotes of
Leishmania amazonensis and L. braziliensis, murine macrophag-
es, and intracellular L. amazonensis amastigotes. The most
potent antileishmanial compound was selected for investiga-
tion into its mechanism of action. Among the evaluated com-
pounds, five derivatives [(E)-3-((2-(pyridin-2-yl)hydrazono)me-
thyl)benzene-1,2-diol (2b), (E)-4-((2-(pyridin-2-yl)hydrazono)me-
thyl)benzene-1,3-diol (2c), (E)-4-nitro-2-((2-(pyrazin-2-yl)hydra-
zono)methyl)phenol (2s), (E)-2-(2-(pyridin-2-ylmethylene)hydra-
zinyl)pyrazine
(2u),
and
(E)-2-(2-((5-nitrofuran-2-
yl)methylene)hydrazinyl)pyrazine (2v)] exhibited significant ac-
tivity against L. amazonensis amastigote forms, with IC50 values
below 20 mm. The majority of the compounds did not show
any toxic effect on murine macrophages. Preliminary studies
on the mode of action of members of this hydrazine-derived
series indicate that the accumulation of reactive oxygen spe-
cies (ROS) and disruption of parasite mitochondrial function
are important for the pharmacological effect on L. amazonensis
promastigotes.
Introduction
Leishmaniasis is a group of diseases caused by several different
protozoan parasites of the genus Leishmania. The World
Health Organization considers leishmaniasis as one of the
major neglected tropical diseases in the world, with only ma-
laria receiving greater attention. Although this disease is wide-
spread and found in nearly 100 countries across Europe, Africa,
Asia, and America, over 90% of newly reported cases are con-
centrated in only six countries: Brazil, Ethiopia, India, Somalia,
South Sudan, and Sudan.[1] In addition, approximately 350 mil-
lion people worldwide are vulnerable to leishmaniasis, and
there are an estimated 0.9–1.7 million newly infected individu-
als each year.[2] The infection is more prevalent in poor popula-
tions of developing and underdeveloped countries, where
most patients do not have access to treatment or do not com-
plete the entire treatment due to high cost, low availability, in-
vasive routes of administration, and long treatment duration,
all of which significantly increase the probability of drug resist-
ance.[3]
Current treatment options (Figure 1) for the various clinical
forms of leishmaniasis are limited and lack sufficient efficacy
due to incomplete elimination of parasites from infected indi-
viduals.[4] Owing to the problems associated with existing che-
motherapies, there is an urgent need to discover and develop
[a] Dr. T. C. M. Nogueira, Dr. A. C. Pinheiro, Dr. M. V. N. de Souza
Instituto de Tecnologia em Fꢀrmacos-Far Manguinhos, Fundażo Oswaldo
Cruz, Rua Sizenando Nabuco 100, Manguinhos, Rio de Janeiro, RJ, 21041-
250 (Brazil)
E-mail: mvndesouza@gmail.com
[b] Dr. M. V. N. de Souza
Instituto de Quꢁmica, Universidade Federal do Rio de Janeiro, CP 68563, Rio
de Janeiro, RJ, CEP 21945-970, (Brazil)
[c] Dr. E. S. Coimbra, Dr. L. M. R. Antinarelli, M. de A. Crispi
Instituto de CiÞncias Biolꢂgicas, Universidade Federal de Juiz de Fora, Juiz
de Fora, Minas Gerais, CEP 36036-900 (Brazil)
The ORCID identification number(s) for the author(s) of this article can
be found under:
https://doi.org/10.1002/cmdc.201800328.
Figure 1. Drugs used in current antileishmanial therapy.
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novel antileishmanial drugs. In this context, six-membered aro-
matic heterocycles containing nitrogen, such as pyridine and
pyrazine, have received attention from the scientific communi-
ty due to their broad spectrum of biological activities,[5–8] in-
cluding antineoplastic, antibacterial, antifungal, anticonvulsant,
antiviral, and anti-inflammatory. The aim of this work was to
synthesize and evaluate the biological activity and mechanism
of action of 2-pyrazyl and 2-pyridylhydrazone derivatives, new
classes of antileishmanial agents.
The compounds were identified on the basis of their spectral
data, including 1H NMR, 13C NMR, MS, and IR spectra. The
1H NMR spectra of pyridylhydrazone derivatives 2a–n showed
four pyridine protons at 8.17–6.71 ppm, whereas those of ana-
logues 2o–w presented three pyrazine protons at 8.70–
7.94 ppm. The 1H NMR spectra of all prepared compounds
showed proton signals ranging from 7.97 to 8.37 ppm and a
characteristic IR band at approximately 1580 cmꢀ1, indicating
the presence of N=CH functional groups.
Antileishmanial activity
The cytotoxic activity of hydrazone derivatives 2 against L. am-
azonensis and L. braziliensis promastigotes, L. amazonensis in-
tramacrophage amastigotes, and murine macrophages was
tested in vitro. The results are listed in Tables 2 and 3. Most
compounds showing activity against promastigotes of L. ama-
zonensis are also active against L. braziliensis species; neverthe-
less, the behavior of compound 2j was unexpected, as it is
active against the former but not the latter.
Results and Discussion
Synthesis and characterization
For the synthesis of (E)-2-pyridylhydrazone[9] derivatives 2a–n,
commercial 2-pyridylhydrazine and the appropriate aromatic
and heteroaromatic aldehydes were used, whereas compounds
2o–w were prepared the same way as above but by using
commercial 2-pyrazylhydrazine as the starting material. After
stirring for 5 min–78 h with ethanol at room temperature
(Scheme 1), the compounds were obtained at 35–89% yields
(Table 1).
These results provided important information to better un-
derstand of the mechanism of action, and these compounds
could be useful leads for rational drug design. The aldehydes
selected in this study were based on our previous medicinal
chemistry study on other hydrazone classes and diseases, such
as tuberculosis, malaria, and cancer. For phenolic ring deriva-
tives, dihydroxy substitution is critical for biological activity, be-
cause the monohydroxy group at the ortho position in both
series 2a and 2o are inactive. Notably, however, this position
is important for the disubstituted derivatives. For the 2,3-dihy-
droxy groups, 2b and 2p pyridyl groups exhibited better activ-
ity than their respective pyrazyl counterparts (Figures 2 and 3,
Tables 2 and 3). When we changed the position of one hydroxy
group (2,3 for 2,4 position) in 2c and 2q, the activity against
promastigote forms decreased relative to the activity of 2b
and 2p, respectively. An opposite trend was observed for
L. amazonensis intracellular amastigotes, with the 2,4-hydroxy
group (2c, IC50 =11.98 mm) being more active than the 2,3-hy-
droxy group (2b, IC50 =15.10 mm) (Figure 2 and Table 3). Fol-
lowing modification of the inactive 2,5-dihydroxy in 2r, i.e., re-
placing 5-hydroxy with a nitro group, the antileishmanial activ-
ity against L. amazonensis amastigote forms was found to im-
prove (2s, IC50 =11.09 mm). Upon comparing compounds 2 f
(2-OH, 3-OCH3) and 2g (2-OH, 3-OCH3) with their respective di-
hydroxy counterparts 2b and 2c for activity against L. amazo-
nensis amastigote forms, a decrease in activity was observed
(2b IC50 =15.10 mm vs. 2 f IC50 =inactive; 2c IC50 =11.98 mm vs.
2g IC50 =23.86 mm), indicating that the hydroxy group is im-
portant for antileishmanial activity. Of the synthesized classes
of hydrazones obtained using heteroaromatic aldehydes, com-
pound 2u possessed antileishmanial activity against L. amazo-
nensis amastigote forms (IC50 =15.25 mm), indicating that the
position of the nitrogen atom is critical for biological activity,
with the pyrazyl group being more active than the pyridyl
group. In the case of five-membered aldehydes, the nitrofuran-
yl group is critical for antileishmanial activity against L. amazo-
nensis amastigote forms (2v IC50 =16.10 mm relative to the in-
Scheme 1. Synthesis of 2-pyridyl/pyrazylhydrazone derivatives 2a–w.
Table 1. 2-Pyridylhydrazone (2a–n) and 2-Pyrazylhydrazone (2o–w) deriv-
atives (see Scheme 1 for scaffold structure).
Compd
X
Ar/Het
2-OH
mp [8C]
Yield [%]
clogP[a]
2a
2b
2c
2d
2e
2 f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
N
N
N
N
N
215–216
194–196
221–222
250–252
220
160–161
160–162
184–185
275
178–180
180–181
202–204
201
260–261
220–222
242–245
274–275
279–280
250–251
222–224
206–207
244–245
280–281
65
65
81
81
64
72
44
89
71
52
70
74
37
78
60
80
87
75
78
86
72
35
52
2.27
1.57
1.77
2.21
2.70
1.88
2.30
2.35
2.90
1.16
1.09
1.04
1.67
2.31
1.59
0.88
1.08
1.08
1.52
2.01
0.48
0.99
1.63
2,3-diOH
2,4-diOH
2-OH, 5-NO2
2-OH, 4-CH3
2-OH, 3-OCH3
2-OH, 4-OCH3
2,6-diOCH3
3-NO2, 4-Cl
2-Py[b]
3-Py
4-Py
5-NO2-Fur[c]
5-NO2-Thi[d]
2-OH
2,3-diOH
2,4-diOH
2,5-diOH
2-OH, 5-NO2
2-OH, 4-CH3
2-Py
2s
2t
2u
2v
2w
5-NO2-Fur
5-NO2-Thi
[a] Calculated with Molinspiration online software: www.molinspiration.-
com. [b] Py=pyridyl. [c] Fur=furanyl. [d] Thi=thienyl.
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Table 2. Activity of series 2 compounds against Leishmania species pro-
mastigotes.
Compound
IC50 [mm][a]
L. amazonensis WT[b]
L. braziliensis
2a
2b
2c
2d
2e
2 f
2g
2h
2i
>100.0
>100.0
11.55ꢁ1.28
17.71ꢁ0.53
22.11ꢁ0.51
7.48ꢁ0.20
10.45ꢁ0.97
30.68ꢁ0.37
24.17ꢁ1.41
>100.0
36.60ꢁ1.10
12.50ꢁ0.16
27.47ꢁ1.73
14.62ꢁ1.37
19.59ꢁ2.12
>100.0
>100.0
>100.0
2j
2k
2l
2m
2n
18.22ꢁ0.66
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
2o
2p
2q
41.06ꢁ1.54
>100.0
50.67ꢁ9.08
>100.0
2r
2s
2t
50.84ꢁ4.70
>100.0
>100.0
>100.0
>100.0
>100.0
2u
2v
2w
miltefosine
17.28ꢁ1.80
17.36ꢁ1.31
>100.0
21.39ꢁ0.5
33.30ꢁ1.70
25.86ꢁ4.26
>100.0
29.7ꢁ0.04
[a] Values are the meanꢁSD of three independent experiments per-
formed in duplicate with similar results, and were calculated with GraFit
version 5 software (Erithacus Software Ltd., Horley, UK). [b] Leishmania
amazonensis wild-type (IFLA/BR/1967/PH8).
Figure 2. Synthesized compounds and their respective activities based on
aromatic aldehydes.
(IC50 =20.31 and 23.86 mm, respectively) and showed low cyto-
toxicity in murine macrophages (CC50 =123.36 and 141.70 mm,
respectively), demonstrating high selectivity for the intracellu-
lar parasite.
Because the new classes of 2-pyrazyl and pyridylhydrazone
derivatives were found to have a promising effect on Leishma-
nia promastigotes and intramacrophage amastigotes, it is es-
sential to understand the mechanism of action of these com-
pounds. Compound 2u showed the strongest effect against
L. amazonensis promastigotes and intracellular amastigotes,
with IC50 values of 17.28 and 15.25 mm, respectively. No cyto-
toxicity against murine macrophages (CC50 >150 mm) was ob-
served. We therefore selected this compound to evaluate the
mechanism of action in Leishmania death.
Initial studies suggested mitochondria to play a role in the
mechanism underlying the activity of compound 2u. This
single organelle of the Leishmania parasite is considered an at-
tractive pharmacological target because of differences in struc-
ture and function from mammalian mitochondria.[10,11]
Figure 3. Synthesized compounds and their respective activities based on
heteroaromatic aldehydes.
active 2w), indicating that oxygen is critical for biological activ-
ity, with the pyrazyl group being more active than the pyridyl
group.
Based on this evidence, we assessed the possibility of mito-
chondrial oxidative damage in L. amazonensis promastigotes
caused by compound 2u. Compound 2u induced a dose-de-
pendent increase in the production of reactive oxygen species
(ROS) at all concentrations tested. As shown in Figure 4, the
treatment of parasites with compound 2u increased ROS
levels by 36.18% and 44.83% at 17.28 mm (1ꢁIC50) and
Analysis of the cytotoxicity of the compounds against
murine macrophages revealed that the majority of the com-
pounds are not toxic (CC50 >150 mm), indicating a favorable
toxicity profile (Table 3). Compounds 2d and 2g were active
against intramacrophage amastigotes at lower concentrations
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Considering the increase in ROS production, we investigated
the mitochondrial membrane potential (DYm) in promastigotes
treated with compound 2u using MitoTracker, a fluorescent
marker that accumulates in active mitochondria. Fluorimetry
showed that compound 2u decreases DYm, but a dose–re-
sponse relationship was not observed (Figure 5). However, a
marked decrease in DYm was observed in promastigotes treat-
ed with both doses of the compound (26.17 and 29.13% at
17.28 and 34.56 mm, respectively), relative to that observed
with the positive control, carbonyl cyanide-4-(trifluoromethox-
y)phenylhydrazone (FCCP, 20.73%; Figure 5).
Table 3. Effect of series 2 compounds on mammalian macrophages and
Leishmania amazonensis intracellular amastigotes.
Compound
Macrophage
CC50 [mm][a]
L. amazonensis RFP[b]
IC50 [mm][c]
2a
2b
2c
2d
2e
2 f
2g
2h
2i
>150.0
>150.0
>150.0
123.36ꢁ26.91
>150.0
81.65ꢁ0.65
141.70ꢁ21.97
>150.0
>100.0
15.10ꢁ1.64
11.98ꢁ1.33
20.31ꢁ0.08
28.62ꢁ0.36
>100.0
23.86ꢁ0.76
>100.0
>150.0
NT[d]
2j
>150.0
>100.0
2k
>150.0
>100.0
2l
>150.0
>100.0
2m
2n
>150.0
>150.0
>100.0
>100.0
2o
>150.0
>100.0
2p
>150.0
>100.0
2q
>150.0
>100.0
2r
>150.0
>100.0
2s
2t
>150.0
>150.0
11.09ꢁ0.05
>100.0
2u
2v
2w
miltefosine
>150.0
>150.0
>150.0
138.62ꢁ10.06
15.25ꢁ2.16
16.10ꢁ0.59
>100.0
12.74ꢁ0.96
[a] Cytotoxic concentration of 50% of macrophages. [b] L. amazonensis
RFP (IFLA/BR/1967/PH8). [c] Inhibitory concentration of 50% of intracellu-
lar amastigotes. CC50 and IC50 values are the meanꢁSD of at least three
independent experiments performed in duplicate, and were calculated
with GraFit version 5 software (Erithacus Software Ltd., Horley, UK).
[d] Not tested.
Figure 5. Mitochondrial membrane potential analysis of L. amazonensis spe-
cies promastigote forms treated with compound 2u. Parasites were untreat-
ed (negative control) or treated with compound 2u at 17.28 or 34.56 mm
and after 24 h were probed with MitoTrackerꢂ Red. FCCP at 20.0 mm was
used as positive control. Analyses of labelling were performed in a fluorime-
try microplate reader; ***p<0.001, **p<0.01, significant difference com-
pared with negative control. Data are expressed as the means of three dif-
ferent experiments performed in triplicate.
Preliminary evidence concerning the mechanism of action
for compound 2u on L. amazonensis strongly suggests the in-
volvement of parasite mitochondria, reflected by the accumu-
lation of ROS through the electron transport chain that triggers
mitochondrial membrane permeabilization, culminating in mi-
tochondrial collapse and death of the parasite. Complementary
studies are being performed to better identify the parasite
death pathway involved in the mechanism of action of this
series.
Conclusions
Figure 4. ROS production in L. amazonensis promastigote forms treated with
compound 2u. Parasites were untreated (negative control) or treated with
compound 2u at 17.28 or 34.56 mm and after 24 h were probed with
H2DCFDA. Miltefosine (42.0 mm) was used as positive control. Fluorescence
was measured fluorimetrically, and the data are expressed as the fluores-
cence intensity in arbitrary units (a.u.) of three independent experiments
with similar results; ***p<0.001, **p<0.01, statistically significant difference
compared with untreated parasites.
In this work, new classes of antileishmanial agents based on 2-
pyrazyl and pyridylhydrazone derivatives were identified, with
compound 2u [(E)-2-(2-(pyridin-2-ylmethylene)hydrazinyl)pyra-
zine)] being the lead compound, suggesting that this nucleus
is more active than pyridine. The biological activities of these
classes provided important information, with compound 2u
selected for studies into the mechanism of action against
L. amazonensis. This study strongly suggests the involvement
of parasite mitochondria reflected by the accumulation of ROS
through the electron transport chain that triggers mitochondri-
34.56 mm (2ꢁIC50), respectively. Miltefosine, used as positive
control, increased the fluorescence intensity by 21.89%
(Figure 4).
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struments, Inc., Winooski, VT, USA) set at 540 nm excitation/
600 nm emission. The results are expressed as percent inhibition
relative to controls represented by L. amazonensis-infected macro-
phages. Miltefosine (1.6–25.0 mm) was used as the reference drug.
Results were obtained from three independent experiments per-
formed in duplicate. The experimental protocol was approved by
the Ethical Committee for Animal Research (012/2015-CEUA).
al membrane permeabilization, culminating in mitochondrial
collapse and death of the parasite. Further studies will provide
a better understanding of the mechanism of action, and stud-
ies in animal models are in progress.
Experimental Section
Analysis of ROS production: L. amazonensis promastigotes (1ꢁ107
cellsmLꢀ1) were untreated or treated with 17.28 or 34.56 mm com-
pound 2u for 24 h at 258C in a BOD incubator in the dark. The
parasite concentration was adjusted to 4ꢁ107 promastigotes in
200 mL PBS and incubated with 2’,7’-dichlorodihydrofluorescein di-
acetate (H2DCFDA; Invitrogen Molecular Probes, OR, USA) at 20 mm
for 30 min in dark at room temperature. ROS generation was fluori-
metrically quantified at an excitation wavelength of 485 nm and an
emission wavelength of 528 nm. Promastigotes incubated with mil-
tefosine (42.0 mm) were used as a positive control.[14]
Biological assays
Parasite cultivation: L. amazonensis wild-type (IFLA/BR/1967/PH8),
L. amazonensis (IFLA/BR/1967/PH8) transfected with the gene of
red fluorescent protein (RFP) and L. braziliensis (MHOM/Br/75/
M2903) strains were used in this study. L. amazonensis wild-type
and L. braziliensis were cultured in Warren’s medium (brain heart
infusion (BHI) plus hemin and folic acid, pH 7.2), supplemented
with 10% inactivated fetal bovine serum (FBS), 0.1% of the suspen-
sion of antibiotics penicillin G (100 UImLꢀ1) and streptomycin
(0.1 mgmLꢀ1) at 258C in a BOD incubator. L. amazonensis transfect-
ed with RFP was grown in M199 medium containing 10% FBS,
40 mm HEPES, 0.0005% hemin, 0.1 mm adenine, 0.0002% biotin,
0.1% of the antibiotic, and maintained at 258C in a BOD incuba-
tor.[12]
Determination of DYm: L. amazonensis promastigote forms (1ꢁ
107 cellsmLꢀ1) were untreated or treated with 17.28 or 34.56 mm
compound 2u at 258C in a BOD incubator. After 24 h, the parasites
were washed with PBS and incubated with 500 nm MitoTrackerꢂ
Red (Life Technologies, CA, USA) for 40 min at 258C in the dark in
a BOD incubator. Subsequently, the parasites were washed twice
with PBS, and data acquisition was performed using a fluorimeter
(FLx800), at excitation and emission wavelengths of 540 and
600 nm, respectively. Promastigotes incubated with 20.0 mm car-
bonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, Sigma–
Aldrich) were used as positive control.[14]
Antipromastigote assay: Promastigotes in logarithmic growth
phase were suspended at 2ꢁ106 cellsmLꢀ1 (L. amazonensis WT) or
3ꢁ106 cellsmLꢀ1 (L. braziliensis) and incubated in the presence of a
serial dilution of each individual compound (100.0–6.25 mm) in 96-
well culture plates for 72 h at 258C. The viability of promastigotes
was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide (MTT) (Sigma–Aldrich, St. Louis, MO, USA) colorimetric
method and the absorbance was measured at 570 nm (Multiskan
MS microplate reader, LabSystems Oy, Helsinki, Finland). Miltefosine
(supplied by Cayman Chemical Company, Ann Arbor, MI, USA) was
used as the reference drug.[12]
Statistical analysis: Data were analyzed using one-way ANOVA,
and significant differences among means were identified using
Dunnett’s test.
Chemistry
Cytotoxicity on macrophages: Murine peritoneal macrophages
were obtained by peritoneal lavage of female BALB/c mice previ-
ously inoculated with 3% sodium thioglycolate. The cells were
plated at 2ꢁ105 cells per well in 96-well culture plates in RPMI
1640 medium (Cultilab, SP, Brazil) with 10% inactivated FBS (Culti-
lab) and incubated at 338C and 5% CO2 atmosphere. Adherent
macrophages were incubated with a serial dilution of the com-
pounds (9.4–150.0 mm) for 72 h under the same conditions. Unex-
posed macrophages were used as negative controls (100% viable
macrophages). Viability of the macrophages was determined by
MTT assay, as described above, and was confirmed by comparing
the morphology with that of the control group via light microsco-
py. The concentration required to decrease 50% of viable macro-
phages (CC50) was obtained from three independent experiments
performed in duplicate. The experimental protocol was approved
by the Ethical Committee for Animal Research (013/2015-CEUA).
Melting points were determined using an MQAPF-302 apparatus
(MicroQuꢃmica Ltd., Santa Catarina, Brazil). NMR spectra were ana-
lyzed with a 400 or 500 MHz AC spectrometer (Bruker, MA, USA)
with tetramethylsilane as an internal standard. IR spectra were ob-
tained with a Thermo Nicolet 6700 spectrometer. High-resolution
mass spectra were acquired on a Bruker compact-ToF instrument.
The progress of the reactions was monitored by thin-layer chroma-
tography (TLC) on 2.0ꢁ6.0 cm aluminum sheets (silica gel 60, HF-
254, Merck) with a thickness of 0.25 mm, using ultraviolet light irra-
diation. For column chromatography, Merck silica gel (70–230 or
230–400 mesh) was used. Solvents and reagents were used with-
out further purification.
General procedure for the synthesis of 2-pyridylhydrazone de-
rivatives 2a–n: To
a stirred solution of 2-pyridylhydrazine
(1.0 mmol) in ethanol (10 mL) the appropriate aromatic or hetero-
aromatic aldehyde (0.9 mmol) was added, and the reaction mixture
was stirred for 5 min to 78 h at room temperature. The reaction
mixture was concentrated under reduced pressure, and the residue
was purified by washing with cold water (3ꢁ10 mL), yielding the
2-pyridylhydrazone derivatives 2a–n as solids at 37–89% yield.
Antiamastigote activity: The assay of activity against intracellular
amastigotes of L. amazonensis RFP (IFLA/BR/1967/PH8) was per-
formed as previously described.[12,13] In summary, murine macro-
phages were adhered to coverslips placed in 24-well tissue culture
plates for 16 h. Adherent macrophages were infected with L. ama-
zonensis RFP promastigotes in stationary growth phase at 10:1
(parasite/macrophage) ratio for 4 h at 338C in 5% CO2. Afterward,
the infected macrophages were treated with various concentra-
tions of compounds (6.25, 12.5, 25.0, 50.0, and 100.0 mm) and incu-
bated for 72 h at 338C in 5% CO2. Subsequently, the parasite
burden was determined by measurement of fluorescence intensity
of the parasites using a plate reader fluorimeter (FLx800, BioTek In-
General procedure for the synthesis of 2-pyrazylhydrazone de-
rivatives 2o–w: To
a stirred solution of 2-pyrazylhydrazine
(1.0 mmol) in ethanol (10 mL) the appropriate aromatic or hetero-
aromatic aldehyde (1.05 mmol) was added, and the reaction mix-
ture was stirred for 2–48 h at room temperature. The reaction mix-
ture was concentrated under reduced pressure, and the residue
was purified by washing with cold water (3ꢁ10 mL) followed by
ChemMedChem 2018, 13, 1 – 9
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cold ether (3ꢁ10 mL) or by column chromatography (2j, 2m, 2n,
2p, and 2v) using hexane/ethyl acetate (50!100%) as an eluent,
thus affording the 2-pyrazylhydrazone derivatives 2o–w at 35–
87% yield. These products showed purity greater than 95% when
assessed by GC and NMR.
(E)-5-methoxy-2-((2-(pyridin-2-yl)hydrazono)methyl)phenol (2g):
mp: 160–1628C; 1H NMR (400 MHz, [D6]DMSO): d=10.81 (s, 1H),
10.76 (s, 1H), 8.21 (s, 1H), 8.11 (d, J=4.2 Hz, 1H), 7.63 (m, 1H), 7.44
(d, J=8.6 Hz, 1H), 6.96 (d, J=8.4 Hz, 1H), 6.74 (m, 1H), 6.48 (d, J=
8.6 Hz, 1H), 6.46 (s, 1H), 3.75 ppm (s, 3H) (1H NMR lit.[19]); 13C NMR
(100 MHz, [D6]DMSO): d=160.8, 157.6, 156.3, 148.0, 139.8, 138.0,
128.8, 114.7, 113.4, 106.1, 105.8, 101.0, 55.2 ppm; IR (KBr): n˜ =
1570 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C13H14N3O2+: 244.1081,
found: 244.1072
(E)-2-((2-(pyridin-2-yl)hydrazono)methyl)phenol (2a): mp: 215–
2168C (lit.: 2168C[15]); 1H NMR (400 MHz, [D6]DMSO): d=10.91 (s,
1H), 10.51 (s, 1H), 8.29 (s, 1H), 8.13 (dd, J=4.9 Hz and J=1.9 Hz,
1H), 7.64 (ddd, J=8.4 Hz, J=7.2 Hz and J=1.9 Hz, 1H), 7.58 (dd,
J=7.6 Hz and J=1.6 Hz, 1H), 7.18 (ddd, J=8.3 Hz, J=7.6 Hz and
J=1.6 Hz, 1H), 7.03 (dd, J=8.4 Hz and J=1.0 Hz, 1H), 6.88 (dd, J=
8.3 Hz and J=1.0 Hz, 1H), 6.87 (dt, J=7.6 Hz and J=1.0 Hz, 1H),
6.76 ppm (ddd, J=7.2 Hz, J=4.9 Hz and J=1.0 Hz, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=156.3, 155.8, 147.8, 138.5, 137.9, 129.6,
(E)-2-(2-(2,6-dimethoxybenzylidene)hydrazinyl)pyridine
(2h):
mp: 184–1858C; 1H NMR (400 MHz, [D6]DMSO): d=10.61 (s, 1H),
8.22 (s, 1H), 8.06 (dd, J=4.9 Hz and J=1.8 Hz, 1H), 7.59 (dt, J=
8.5 Hz and J=1.8 Hz, 1H), 7.26 (t, J=8.4 Hz, 1H), 7.13 (d, J=8.5 Hz,
1H), 6.71 (m, 1H), 6.70 (d, J=8.4 Hz, 2H), 3.81 ppm (s, 6H);
13C NMR (100 MHz, [D6]DMSO): d=158.1, 158.1, 157.3, 147.5, 137.6,
127.1, 120.4, 119.2, 115.9, 114.9, 106.0 ppm; IR (KBr): n˜ =1585 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C12H12N3O+: 214.0975, found:
133.8, 129.5, 114.3, 112.0, 106.2, 104.4, 104.4, 55.9 ppm; IR (KBr):
n˜ =1592 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C13H16N3O2
:
+
214.0980
258.1237, found: 258.1232
(E)-3-((2-(pyridin-2-yl)hydrazono)methyl)benzene-1,2-diol (2b):
1
mp: 194–1968C (lit.: 195–1978C[16]); H NMR (400 MHz, [D6]DMSO):
(E)-2-(2-(4-chloro-3-nitrobenzylidene)hydrazinyl)pyridine
(2i):
1
d=10.92 (s, 1H), 10.19 (s, 1H), 9.24 (s, 1H), 8.26 (s, 1H), 8.14 (dd,
J=4.8 Hz and J=1.4 Hz, 1H), 7.64 (dt, J=8.6 Hz and J=1.4 Hz,
1H), 6.99 (m, 1H), 6.98 (d, J=7.7 Hz, 1H), 6.78 (m, 2H), 6.69 ppm (t,
J=7.7 Hz, 2H); 13C NMR (100 MHz, [D6]DMSO): d=156.2, 148.0,
145.4, 144.6, 139.6, 138.0, 120.6, 119.1, 118.0, 115.8, 115.0,
106.1 ppm; IR (KBr): 3304, 1572 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd
for C12H12N3O2+: 230.0924, found: 230.0916
mp: 2758C; H NMR (400 MHz, [D6]DMSO): d=11.22 (s, 1H), 8.31 (d,
J=1.7 Hz, 1H), 8.13 (d, J=4.1 Hz, 1H), 8.00 (s, 1H), 7.93 (dd, J=
8.4 Hz and J=1.7 Hz, 1H), 7.75 (d, J=8.4 Hz, 1H), 7.66 (m, 1H),
7.32 (d, J=8.4 Hz, 1H), 6.81 ppm (m, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=156.6, 148.2, 147.8, 138.0, 136.2, 135.0, 131.8, 130.3,
123.5, 121.9, 115.7, 106.7 ppm; IR (KBr): n˜ =1581, 1525, 1348 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C12H10ClN4O2+: 277.0487, found:
277.0492
(E)-4-((2-(pyridin-2-yl)hydrazono)methyl)benzene-1,3-diol
(2c):
mp: 221–2228C (lit.: 220–2218C[17]); H NMR (400 MHz, [D6]DMSO):
d=10.67 (s, 2H), 9.71 (s, 1H), 8.17 (s, 1H), 8.10 (d, J=3.2 Hz, 1H),
7.61 (m, 1H), 7.31 (d, J=8.0 Hz, 1H), 6.93 (d, J=8.2 Hz, 1H), 6.72
(m, 1H), 6.32 (m, 1H), 6.31 ppm (s, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=159.2, 157.6, 156.3, 147.8, 140.3, 137.8, 128.9, 114.4,
111.8, 107.4, 105.6, 102.4 ppm; IR (KBr): n˜ =3238, 1574 cmꢀ1; HRMS-
ESI m/z [M+H]+ calcd for C12H12N3O2+: 230.0924, found: 230.0918
(E)-2-((2-(pyridin-2-yl)hydrazono)methyl)pyridine (2j): mp: 178–
1
1808C (lit.: 179–1808C[20]); H NMR (400 MHz, [D6]DMSO): d=11.16
1
(s, 1H), 8.54 (dd, J=4.8 Hz and J=1.4 Hz, 1H), 8.14 (dd, J=4.9 Hz
and J=1.0 Hz, 1H), 8.08 (s, 1H), 7.96 (d, J=8.0 Hz, 1H), 7.80 (dt,
J=8.0 Hz and J=1.4 Hz, 1H), 7.67 (ddd, J=8.4 Hz, J=7.3 Hz and
J=1.0 Hz, 1H), 7.30 (dd, J=8.4 Hz and J=1.0 Hz, 1H), 7.28 (m,
1H), 6.81 ppm (ddd, J=7.3 Hz, J=4.9 Hz and J=1.0 Hz, 1H);
13C NMR (100 MHz, [D6]DMSO): d=156.6, 154.2, 149.1, 147.7, 139.1,
(E)-4-nitro-2-((2-(pyridin-2-yl)hydrazono)methyl)phenol (2d): mp:
250–2528C; 1H NMR (400 MHz, [D6]DMSO): d=11.16 (s, 1H), 8.55
(d, J=2.9, 1H), 8.32 (s, 1H), 8.14 (m, 1H), 8.07 (dd, J=9.0 and J=
2.9, 1H), 7.69 (dt, J=8.5 and J=1.8, 1H), 7.17 (d, J=8.5, 1H), 7.06
(d, J=9.0, 1H), 6.82 ppm (m, 1H); 13C NMR (100 MHz, [D6]DMSO):
d=161.1, 156.2, 147.8, 140.0, 138.1, 134.1, 124.9, 121.9, 121.2,
116.4, 115.4, 106.4 ppm; IR (KBr): n˜ =1571, 1518, 1341 cmꢀ1; HRMS-
ESI m/z [M+H]+ calcd for C12H11N4O3+: 259.0826, found: 259.0825
137.9, 136.4, 122.8, 118.7, 115.5, 106.4 ppm; IR (KBr): n˜ =1571 cmꢀ1
HRMS-ESI m/z [M+H]+ calcd for C11H11N4+: 199.0978, found:
;
199.0986
(E)-2-((2-(pyridin-3-ylmethylene)hydrazinyl)pyridine (2k): mp:
ꢀ180–1818C (lit.: 179–1808C[21]); H NMR (400 MHz, [D6]DMSO): d=
1
11.07 (s, 1H), 8.81 (d, J=1.8 Hz, 1H), 8.50 (dd, J=4.7 Hz and J=
1.5 Hz, 1H), 8.12 (m, 1H), 8.08 (ddd, J=8.0 Hz, J=1.8 Hz and J=
1.5 Hz, 1H), 8.04 (s, 1H), 7.65 (dt, J=8.4 Hz and J=1.8 Hz, 1H),
7.41 (dd, J=8.0 Hz and J=4.7 Hz, 1H), 7.29 (d, J=8.4 Hz, 1H),
6.79 ppm (m, 1H); 13C NMR (100 MHz, [D6]DMSO): d=156.8, 149.0,
(E)-5-methyl-2-((2-(pyridin-2-yl)hydrazono)methyl)phenol
(2e):
1
mp: 2208C; H NMR (400 MHz, [D6]DMSO): d=10.84 (s, 1H), 10.50
(s, 1H), 8.24 (s, 1H), 8.12 (m, 1H), 7.64 (dt, J=8.4 Hz and J=1.8 Hz,
1H), 7.43 (d, J=7.7 Hz, 1H), 7.00 (d, J=8.4 Hz, 1H), 6.76 (m, 1H),
6.71 (s, 1H), 6.69 (m, 1H), 2.26 ppm (s, 3H); 13C NMR (100 MHz,
[D6]DMSO): d=156.3, 155.9, 148.0, 139.7, 139.2, 138.0, 127.3, 120.3,
117.7, 116.4, 114.9, 106.0, 21.1 ppm; IR (KBr): n˜ =1584 cmꢀ1; HRMS-
ESI m/z [M+H]+ calcd for C13H14N3O+: 228.1131, found: 228.1133
147.7, 147.5, 137.9, 135.4, 132.2, 131.2, 123.7, 115.2, 106.4 ppm; IR
(KBr): n˜ =1591 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C11H11N4
199.0978, found: 199.0984
+
:
(E)-2-(2-(pyridin-4-ylmethylene)hydrazinyl)pyridine (2l): mp:
202–2048C (lit.: 202–2048C[16]); 1H NMR (400 MHz, [D6]DMSO): d=
11.27 (s, 1H), 8.56 (d, J=5.9 Hz, 2H), 8.15 (m, 1H), 7.98 (s, 1H), 7.69
(dt, J=8.5 Hz and J=1.7 Hz, 1H), 7.60 (d, J=5.9 Hz, 2H), 7.32 (d,
J=8.5 Hz, 1H), 6.84 ppm (m, 1H); 13C NMR (100 MHz, [D6]DMSO):
d=156.5, 149.9, 149.9, 147.7, 142.4, 138.0, 135.6, 119.9, 119.9,
115.7, 106.6 ppm; IR (KBr): n˜ =1576 cmꢀ1; HRMS-ESI m/z [M+H]+
calcd for C11H11N4+: 199.0978, found: 199.0985
(E)-2-methoxy-6-((2-(pyridin-2-yl)hydrazono)methyl)phenol (2 f):
mp: 160–1618C; 1H NMR (400 MHz, [D6]DMSO): d=10.91 (s, 1H),
9.97 (s, 1H), 8.30 (s, 1H), 8.13 (dd, J=4.8 Hz and J=1.8 Hz, 1H),
7.64 (dt, J=8.6 Hz and J=1.8 Hz, 1H), 7.21 (dd, J=7.9 Hz and J=
1.2 Hz, 1H), 7.06 (d, J=8.6 Hz, 1H), 6.93 (dd, J=7.9 Hz and J=
1.2 Hz, 1H), 6.80 (t, J=7.9 Hz, 1H), 6.77 (m, 1H), 3.81 ppm (s, 3H)
(1H NMR lit.[18]); 13C NMR (100 MHz, [D6]DMSO): d=156.4, 147.8,
147.8, 145.3, 138.2, 137.9, 120.8, 118.9, 118.6, 114.9, 112.0, 106.0,
55.7 ppm; IR (KBr): n˜ =1573 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for
C13H14N3O2+: 244.1081, found: 244.1072
(E)-2-(2-((5-nitrofuran-2-yl)methylene)hydrazinyl)pyridine (2m):
mp: 2018C (lit.: 2028C[22]); H NMR (400 MHz, [D6]DMSO): d=11.56
1
(s, 1n), 8.17 (m, 1H), 7.97 (s, 1H), 7.79 (d, J=4.0 Hz, 1H), 7.71 (ddd,
J=8.4 Hz, J=7.4 Hz and J=1.8 Hz, 1H), 7.25 (d, J=8.4 Hz, 1H),
&
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Full Papers
7.10 (d, J=4.0 Hz, 1H), 6.88 ppm (m, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=155.9, 153.5, 151.1, 147.8, 138.2, 126.3, 116.4, 115.5,
111.8, 106.8 ppm; IR (KBr): n˜ =1601, 1572, 1360 cmꢀ1; HRMS-ESI m/
z [M+H]+ calcd for C10H9N4O3+: 233.0669, found: 233.0663
140.1, 134.6, 130.5, 126.9, 120.3, 117.5, 116.4 ppm; IR (KBr): n˜ =
3191, 1567 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C12H13N4O+:
229.1084, found: 229.1085
(E)-2-(2-(pyridin-2-ylmethylene)hydrazinyl)pyrazine (2u): mp:
1
(E)-2-(2-((5-nitrothiophen-2-yl)methylene)hydrazinyl)pyridine
206–2078C (lit.: 2088C[24]); H NMR (500 MHz, [D6]DMSO): d=11.44
1
(2n): mp: 260–2618C (lit.: 2608C[23]); H NMR (400 MHz, [D6]DMSO):
(s, 1H), 8.70 (d, J=1.2, 1H), 8.56 (dd, J=4.2 and J=1.3, 1H), 8.16
(dd, J=2.6 and J=1.2, 1H), 8.12 (s, 1H), 8.06 (J=2.6, 1H), 8.04 (m,
1H), 7.83 (dt, J=7.8 and J=1.3, 1H), 7.35 ppm (ddd, J=7.8, J=4.2
and J=1.0, 1H); 13C NMR (125 MHz, [D6]DMSO): d=153.8, 152.5,
d=11.52 (s, 1H), 8.17 (m, 2H), 8.08 (d, J=4.4 Hz, 1H), 7.71 (m, 1H),
7.35 (d, J=4.4 Hz, 1H), 7.22 (d, J=8.4 Hz, 1H), 6.88 ppm (m, 1H);
13C NMR (100 MHz, [D6]DMSO): d=155.9, 148.9, 148.7, 147.8, 138.2,
131.6, 130.9, 126.3, 116.3, 106.7 ppm; IR (KBr): n˜ =1593, 1567,
149.3, 142.0, 141.3, 136.6, 135.6, 130.8, 123.4, 119.3 ppm; IR (KBr):
1363 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C10H9N4O2S+:
n˜ =1557 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C10H10N5
:
+
249.0441, found: 249.0436
200.0931, found: 200.0931
(E)-2-((2-pyrazin-2-yl)hydrazono)methyl)phenol (2o): mp: 220–
2228C; 1H NMR (400 MHz, [D6]DMSO): d=11.20 (s, 1H), 10.36 (s,
1H), 8.48 (d, J=1.5, 1H), 8.36 (s, 1H), 8.13 (dd, J=2.6 and J=1.5,
1H), 7.99 (d, J=2.6, 1H), 7.69 (dd, J=7.7 and J=1.7, 1H), 7.21 (dt,
J=8.3 and J=1.7, 1H), 6.89 ppm (m, 2H); 13C NMR (100 MHz,
[D6]DMSO): d=156.0, 152.4, 142.0, 139.9, 134.9, 130.6, 130.2, 126.9,
120.4, 119.4, 116.1 ppm; IR (KBr): n˜ =3153, 1558 cmꢀ1; HRMS-ESI m/
z [M+H]+ calcd for C11H11N4O+: 215.0927, found: 215.0926
(E)-2-(2-((5-nitrofuran-2-yl)methylene)hydrazinyl)pyrazine (2v):
1
mp: 244–2458C (lit.: 2858C[25]); H NMR (400 MHz, [D6]DMSO): d=
11.83 (s, 1H), 8.61 (s, 1H), 8.20 (d, J=2.3, 1H), 8.12 (d, J=2.3, 1H),
8.03 (s, 1H), 7.80 (d, J=3.8, 1H), 7.20 ppm (d, J=3.8, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=153.0, 151.9, 151.4, 142.1, 136.4, 131.0,
128.6, 115.3, 112.7 ppm; IR (KBr): n˜ =1575, 1354 cmꢀ1; HRMS-ESI m/
z [M+H]+ calcd for C9H8N5O3+: 234.0622, found: 234.0623
(E)-2-(2-((5-nitrothiophen-2-yl)methylene)hydrazinyl)pyrazine
(2w): mp: 280–2818C; 1H NMR (400 MHz, [D6]DMSO): d=11.75 (s,
1H), 8.57 (s, 1H), 8.22 (s, 1H), 8.19 (m, 1H), 8.11 (d, J=2.5, 1H),
8.08 (d, J=4.3, 1H), 7.43 ppm (d, J=4.3, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=151.8, 149.4, 147.9, 142.0, 136.3, 133.9, 130.7, 130.7,
127.2 ppm; IR (KBr): n˜ =1563 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd
for C9H8N5O2S+: 250.0393, found: 250.0388
(E)-3-((2-(pyrazin-2-yl)hydrazono)methyl)benzene-1,2-diol (2p):
mp: 242–2458C; 1H NMR (400 MHz, [D6]DMSO): d=11.22 (s, 1H),
9.87 (s, 1H), 9.35 (s, 1H), 8.43 (d, J=1.5, 1H), 8.34 (s, 1H), 8.13 (dd,
J=2.6 and J=1.5, 1H), 7.99 (d, J=2.6, 1H), 7.10 (dd, J=7.8 and
J=1.5, 1H), 6.79 (dd, J=7.8 and J=1.5, 1H), 6.70 ppm (t, J=7.8,
1H); 13C NMR (100 MHz, [D6]DMSO): d=152.2, 145.5, 144.8, 142.1,
140.9, 134.9, 130.6, 120.6, 119.2, 117.6, 116.0 ppm; IR (KBr): n˜ =
3193, 1580 cmꢀ1; HRMS-ESI m/z [M+H]+ calcd for C11H11N4O2
:
+
231.0877, found: 231.0883
Acknowledgements
(E)-4-((2-(pyrazin-2-yl)hydrazono)methyl)benzene-1,3-diol (2q):
mp: 274–2758C; 1H NMR (400 MHz, [D6]DMSO): d=10.98 (s, 1H),
10.46 (s, 1H), 9.75 (s, 1H), 8.37 (d, J=1.4, 1H), 8.24 (s, 1H), 8.09
(dd, J=2.6 and J=1.4, 1H), 7.94 (d, J=2.6, 1H), 7.44 (dd, J=9.2
and J=2.6, 1H), 6.32 ppm (m, 2H); 13C NMR (100 MHz, [D6]DMSO):
d=159.6, 157.7, 152.3, 141.9, 141.4, 134.3, 130.3, 128.6, 111.8,
107.6, 102.4 ppm; IR (KBr): n˜ =3172, 1595 cmꢀ1; HRMS-ESI m/z [M+
H]+ calcd for C11H11N4O2+: 231.0877, found: 231.0883
The authors are grateful to the Brazilian agency Conselho Nacio-
nal de Desenvolvimento Cientꢁfico e Tecnolꢂgico (CNPq), Funda-
żo de Amparo a Pesquisa de Minas Gerais (FAPEMIG), and Uni-
versidade Federal de Juiz de Fora (UFJF) for fellowships and fi-
nancial support.
Conflict of interest
(E)-2-((2-(pyrazin-2-yl)hydrazono)methyl)benzene-1,4-diol (2r):
mp: 279–2808C; 1H NMR (400 MHz, [D6]DMSO): d=11.13 (s, 1H),
9.56 (s, 1H), 8.86 (s, 1H), 8.45 (d, J=1.5, 1H), 8.28 (s, 1H), 8.12 (dd,
J=2.6 and J=1.5, 1H), 7.98 (d, J=2.6, 1H), 7.10 (d, J=2.9, 1H),
6.71 (d, J=8.7, 1H), 6.63 ppm (dd, J=8.7 and J=2.9, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=152.5, 150.0, 148.9, 142.0, 139.6, 134.8,
The authors declare no conflict of interest.
Keywords: 2-pyrazylhydrazones
antileishmanial activity · mitochondria
·
2-pyridylhydrazones
·
130.5, 120.7, 117.7, 116.9, 111.6 ppm; IR (KBr): n˜ =3108, 1579 cmꢀ1
;
HRMS-ESI m/z [M+H]+ calcd for C11H11N4O2+: 231.0877, found:
231.0880
[1] World Health Organization (WHO), Leishmaniasis: http://www.who.int/
leishmaniasis/en/ (accessed November 22, 2017).
[2] Drugs for Neglected Diseases initiative (DNDi), About Leishmaniasis:
https://www.dndi.org/diseases-projects/leishmaniasis/ (accessed No-
vember 22, 2017).
[3] S. Sundar, J. Chakravarty, Expert Opin. Pharmacother. 2013, 14, 53–63.
[4] V. S. Amato, F. F. Tuon, H. A. Bacha, V. A. Neto, A. C. Nicodemo, Acta
Trop. 2008, 105, 1–9.
(E)-4-nitro-2-((2-(pyrazin-2-yl)hydrazono)methyl)phenol (2s): mp:
250–2518C; H NMR (400 MHz, [D6]DMSO): d=11.76 (s, 1H), 11.43
1
(s, 1H), 8.60 (d, J=2.9, 1H), 8.56 (d, J=1.0, 1H), 8.37 (s, 1H), 8.16
(dd, J=2.5 and J=1.0, 1H), 8.10 (dd, J=9.0 and J=2.9, 1H), 8.04
(d, J=2.5, 1H), 7.06 ppm (d, J=9.0, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=161.4, 152.2, 142.0, 140.1, 136.2, 135.4, 130.8, 125.5,
121.6, 121.5, 116.6 ppm; IR (KBr): n˜ =3164, 1567, 1511, 1337 cmꢀ1
HRMS-ESI m/z [M+H]+ calcd for C11H10N5O3+: 260.0778, found:
[5] M. Asif, Int. J. Bioorg. Chem. 2017, 2, 146–152.
[6] M. Sahu, N. Siddiqui, Int. J. Pharm. Sci. 2016, 8, 8–21.
;
[7] S. B. Ferreira, C. R. Kaiser, Expert Opin. Ther. Pat. 2012, 22, 1033–1051.
[8] A. Chaubey, S. N. Pandeya, Asian J. Pharm. Clin. Res. 2011, 4, 5–8.
[9] N. R. L. Correira, T. C. M. Nogueira, A. C. Pinheiro, M. V. N. De Souza, J. L.
Wardell, S. M. S. V. Wardell, Z. Kristallogr. 2016, 231, 271–284.
[10] L. M. Fidalgo, L. Gille, Pharm. Res. 2011, 28, 2758–2770.
[11] R. F. Menna-Barreto, S. L. de Castro, BioMed. Res. Int. 2014, 614014.
[12] E. S. Coimbra, L. M. Antinarelli, N. P. Silva, I. O. Souza, R. S. Meinel, M. N.
Rocha, R. P. Soares, A. D. da Silva, Chem. Biol. Interact. 2016, 260, 50–57.
260.0766
(E)-5-methyl-2-((2-(pyrazin-2-yl)hydrazono)methyl)phenol
(2t):
mp: 222–2248C; 1H NMR (400 MHz, [D6]DMSO): d=11.13 (s, 1H),
10.33 (s, 1H), 8.44 (d, J=1.3, 1H), 8.31 (s, 1H), 8.12 (dd, J=2.6 and
J=1.3, 1H), 7.98 (d, J=2.6, 1H), 7.54 (d, J=7.8, 1H), 6.70 ppm (m,
2H); 13C NMR (100 MHz, [D6]DMSO): d=156.0, 152.2, 141.9, 140.4,
ChemMedChem 2018, 13, 1 – 9
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Full Papers
[13] P. H. F. Stroppa, L. M. R. Antinarelli, A. M. L. Carmo, J. Gameiro, E. S.
Coimbra, A. D. da Silva, Bioorg. Med. Chem. 2017, 25, 3034–3045.
[14] L. M. Antinarelli, I. O. Souza, N. Glanzmann, A. D. Almeida, G. N. Porcino,
E. G. Vasconcelos, A. D. da Silva, E. S. Coimbra, Exp. Parasitol. 2016, 171,
10–16.
[20] F. Lions, K. V. Martin, J. Am. Chem. Soc. 1958, 80, 3858–3865.
[21] F. D. Popp, Eur. J. Med. Chem. 1989, 24, 313–315.
[22] D. G. Berge, J. Chem. Eng. Data 1983, 28, 431–432.
[23] S. M. Sondhi, S. Jain, M. Dinodia, A. Kumar, Med. Chem. 2008, 4, 146–
154.
[15] N. Syeda, V. R. Srinivasan, Indian J. Chem. 1965, 3, 162.
[16] U. Sandbhor, S. Padhye, D. Billington, D. Rathbone, S. Franzblau, C. E.
Anson, A. K. Powell, J. Inorg. Biochem. 2002, 90, 127–136.
[17] M. Cushman, D. Nagarathnam, D. Gopal, R. L. Geahlen, Bioorg. Med.
Chem. Lett. 1991, 1, 215–218.
[24] F. H. Case, J. Chem. Eng. Data 1976, 21, 124–125.
[25] K. Kakemi, T. Uno, T. Arita, H. Nakano, I. Shimada, Y. Ikegami, S. Kitazawa,
Yakugaku Zasshi 1961, 81, 1609–1614.
Manuscript received: May 15, 2018
[18] S. A. Patil, C. Weng, P. Huang, F. Hong, Tetrahedron 2009, 65, 2889–
2897.
Accepted manuscript online: May 22, 2018
[19] K. Li, A. Tong, Sens. Actuators B 2013, 184, 248–253.
Version of record online: && &&, 0000
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ÝÝ These are not the final page numbers!

FULL PAPERS
Hope against Leishmania parasites: In
this work, the antileishmanial activity of
23 compounds based on 2-pyrazyl and
2-pyridylhydrazone derivatives was eval-
uated. Among the evaluated com-
pounds, five derivatives exhibited signif-
icant activity against L. amazonensis
amastigote forms, with IC50 values
below 20 mm. The majority of the com-
pounds did not show any toxic effect
on murine macrophages.
E. S. Coimbra, L. M. R. Antinarelli,
M. de A. Crispi, T. C. M. Nogueira,
A. C. Pinheiro, M. V. N. de Souza*
&& – &&
Synthesis, Biological Activity, and
Mechanism of Action of 2-Pyrazyl and
Pyridylhydrazone Derivatives, New
Classes of Antileishmanial Agents
ChemMedChem 2018, 13, 1 – 9
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ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ