Novel dialkylphosphorylhydrazones: Synthesis, leishmanicidal evaluation and theoretical investigation of the proposed mechanism of action

Article abstract of DOI:10.1016/j.ejmech.2015.06.014

As part of a program to develop new drugs for the treatment of neglected diseases, new dialkylphosphorylhydrazones were synthesized and evaluated against the trypanosomatid parasites Leishmania braziliensis and Leishmania amazonensis. The synthesis of these compounds proved satisfactory with yields ranging from moderate to good. The most active compounds against L. braziliensis presented IC₅₀ values in the 10^-2 μM range, similar to that of the reference drug pentamidine. Two compounds, 4m and 4n, showed a significant dose dependent decrease in the infection index of L. amazonensis infected macrophages and caused a complete healing of nodules and ulcers when tested in vivo against L. amazonensis-infected mice, but the control of parasite burden at the inoculation site was statistically significant only in the case of treatment with 4n. A target fishing (reverse docking) approach using molecular docking with 15 enzymes of L. braziliensis indicated that the probable target of the active compounds was hexokinase, the first enzyme of the glycolytic pathway.

Full text of DOI:10.1016/j.ejmech.2015.06.014

European Journal of Medicinal Chemistry 101 (2015) 1e12
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel dialkylphosphorylhydrazones: Synthesis, leishmanicidal
evaluation and theoretical investigation of the proposed mechanism
of action
a
a
a
Carolina Barbosa Brito da Matta , Aline Cavalcanti de Queiroz , Mariana Silva Santos ,
a
b, c
Magna Suzana Alexandre-Moreira , Vinicius Tomaz Gonçalves
Catarina de Nigris Del Cistia , Carlos Mauricio R. Sant'Anna , Jo a~ o Batista N. DaCosta
LaFI e Laborat oꢀ rio de Farmacologia e Imunidade, Instituto de Ci ^e ncias Biol oꢀ gicas e da Saúde, Universidade Federal de Alagoas, Macei oꢀ , AL, Brazil
Universidade Federal Rural do Rio de Janeiro, Departamento de Química, Rio de Janeiro, Serop ꢀe dica, Brazil
,
d
b
b, *
a
b
c
CEFET/RJ e Centro Federal de Educaç a~ o Tecnol oꢀ gica Celso Suckow da Fonseca, Itaguaí, RJ, Brazil
Universidade Federal Rural do Rio de Janeiro, Departamento de Matem aꢀ tica, Rio de Janeiro, Serop ꢀe dica, Brazil
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 December 2014
Received in revised form
As part of a program to develop new drugs for the treatment of neglected diseases, new dia-
lkylphosphorylhydrazones were synthesized and evaluated against the trypanosomatid parasites
Leishmania braziliensis and Leishmania amazonensis. The synthesis of these compounds proved satis-
factory with yields ranging from moderate to good. The most active compounds against L. braziliensis
24 April 2015
Accepted 5 June 2015
Available online 9 June 2015
ꢀ
2
presented IC50 values in the 10
mM range, similar to that of the reference drug pentamidine. Two
compounds, 4m and 4n, showed a significant dose dependent decrease in the infection index of
L. amazonensis infected macrophages and caused a complete healing of nodules and ulcers when tested
in vivo against L. amazonensis-infected mice, but the control of parasite burden at the inoculation site was
statistically significant only in the case of treatment with 4n. A target fishing (reverse docking) approach
using molecular docking with 15 enzymes of L. braziliensis indicated that the probable target of the active
compounds was hexokinase, the first enzyme of the glycolytic pathway.
Keywords:
Dialkylphosphorylhydrazones
Leishmanicidal activity
Glycolytic pathway
Molecular docking
©
2015 Elsevier Masson SAS. All rights reserved.
1
. Introduction
One of the main problems in leishmaniasis treatment is the
limited number of available drug options, along with the adverse
effects they can cause, including death [4]. In addition, there are
reports of treatment failures due to increased parasite resistance to
the drugs of first choice, the antimonials [5,6]. Second-choice drugs,
such as amphotericin B, pentamidine, paromomycin, and more
recently, miltefosine, also have toxic effects, and their use requires
hospital supervision [4,7]. Therefore, there is an urgent need for the
development of safer and more effective drugs against this parasite.
Leishmaniasis is one of the world's most neglected diseases,
with a major impact among the poorest individuals, mainly in
developing countries. The number of leishmaniasis cases is
increasing worldwide [1,2]. Leishmaniasis transmission is endemic
in 98 countries and 3 territories on 5 continents. According to the
World Health Organization, 350 million people are considered at
risk of contracting this disease, and some 2 million new cases occur
each year [1,3]. Each year approximately 58,000 cases of visceral
leishmaniasis and 220,000 cutaneous cases are officially reported.
However, it is thought that only approximately two-thirds of
countries actually report incidence data, with the sparsest data
from Africa [3].
Screening tests implemented with
a series of new dia-
lkylphosphorylhydrazones (DAPH) synthesized by our group were
indicative of promising activity profiles against Trypanosoma cruzi
and Leishmania amazonensis [8]. New compounds were then syn-
thesized and added to the series. In the present work, we show the
synthesis of the entire DAPH series and a detailed evaluation of the
compounds' leishmanicidal activity against L. amazonensis and
Leishmania braziliensis in comparison with known leishmanicidal
agents. Although the information available from these in vivo tests
*
Corresponding author.
E-mail address: dacosta@ufrrj.br (J.B.N. DaCosta).
http://dx.doi.org/10.1016/j.ejmech.2015.06.014
223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
0

2
C.B.B. da Matta et al. / European Journal of Medicinal Chemistry 101 (2015) 1e12
is essential for the identification of new leishmanicidal agents, an
understanding of the observed effects is a prerequisite for
improving the selectivity and potency of the investigated com-
pounds. In an attempt to identify probable targets for the active
compounds prepared in the present work, we also implemented a
strategy based on molecular docking of the compounds into a set of
candidate target enzymes.
7.88e8.26 ppm; and to the phosphoramidic hydrogen, P(O)NH,
with a chemical shift in the 9.74e10.21 ppm range and showing a
doublet signal with a coupling constant ranging between 27 and
31 Hz. The literature reports that this coupling occurs between 23
and 53 Hz [21]. Compounds 4i and 4j also presented an additional
doublet in the region approximately 12.8 ppm, with a coupling
constant of approximately 34 Hz, which is characteristic of an
intramolecular hydrogen bond.
The presence of the (R'O)
these molecules could act as inhibitors of enzymes that have, as
substrates, molecules containing the (R'O) P(O)OR group. There are
2
P(O)NHR group in DAPH suggests that
NOEDIF experiments were used to determine the configuration
of the synthetized compounds. The results clearly show, according
2
1
a huge number of such enzymes, so the target identification pro-
cedure should be based on some criteria to reduce the number of
possibilities to be explored. Some parasite enzymes have been
shown to be inhibited by phosphorous-containing molecules, such
as farnesyl pyrophosphate synthase (FPPS) [9,10] and hexokinase
to H-NMR spectroscopy, that all molecules have the E configura-
tion, except for compounds 4i and 4j which were obtained as a
diastereoisomeric mixture with E/Z ratio of 80:20 and 85:15,
respectively.
In the 13C NMR spectrum of DAPH, the signal that characterizes
these compounds is related to the iminic carbon, (eNHN]CHeAr),
which has a chemical shift in the 136e145 ppm range and is
observed as a doublet because it is coupled with the phosphorus
atom, with a coupling constant in the 18e21 Hz range. The same
(
HK) [11], an enzyme of glycolysis metabolic pathway in which
glucose is converted into pyruvate and the free energy released is
used to form the high-energy compounds ATP and NADH. In addi-
tion to FPPS and the enzymes of the glycolytic pathway, promising
targets also include the enzymes of the pentose phosphate pathway
x 2
feature can be observed with the alkoxide groups, (RCH O) P(O)-,
(
PPP), which serves to convert glucose-6-phosphate to ribose-5-
where the methylene hydrogens, neighbors to the ester oxygen
atoms, have chemical shifts in the range from 3.7 to 4.5 ppm.
phosphate. The PPP has been proposed to have crucial roles in the
protection of trypanosomatids against oxidative stress, as well as in
the production of nucleotide precursors [12]. Each of the enzymes of
the PPP has been identified and specific activities measured for one
of the Leishmania species, Leishmania Mexicana [13].
2.2. Biological evaluation
An initial screening was carried out to evaluate and compare the
in vitro leishmanicidal profiles of the 18 DAPH and 2 standard
drugs, miltefosine and pentamidine, against the promastigote
forms of L. braziliensis and L. amazonensis. The maximum effects
and the IC50 values (concentrations causing 50% inhibition of
growth of the promastigotes) were used as the parameters for
leishmanicidal activity (Table 1).
2
. Results and discussion
2.1. Synthesis of the dialkylphosphorylhydrazones
The synthesis of the dialkylphosphites (1) and dia-
lkylphosphorylhydrazines (2) were performed using the synthetic
route previously used by our research group [14e18], according to
the synthetic route shown in Scheme 1.
The synthesis of the new DAPH (4a-o) occurred through a
condensation reaction, catalyzed in an acidic medium, between the
respective dialkylphosphorylhydrazines (2) and different alde-
hydes (3) at room temperature, as shown in Scheme 2.
After 48 h of incubation, most of the compounds were signifi-
cantly active against L. braziliensis. Among these, those that showed
efficacy greater than 70% were as follows: 4b (IC50: 5.1 ± 0.5
m
M), 4d
M), 4g (IC50: 0.4 ± 0.3 M),
M), 4m (IC50
M).
(IC50: 0.06 ± 0.0
4h (IC50: 40.7 ± 3.5
3.6 ± 0.3
m
M), 4f (IC50: 5.4 ± 0.2
m
m
m
M), 4j (IC50: 0.06 ± 0.0
m
:
mM), 4n (IC50: 5.2 ± 0.7
m
M) and 4o (IC50: 0.03 ± 0.02
m
However, not all of these compounds were active against
L. amazonensis as they were for L. braziliensis. The compounds 4b,
4d, 4g and 4h showed high specificity against the L. braziliensis
species. The compounds were active against L. amazonensis with
The infrared spectra of the DAPH synthesized show the char-
acteristic absorption bands. The main absorption bands correspond
to the stretching frequencies of the P]O, PeOeC and C]N bonds.
In pentavalent phosphorus compounds containing a bond between
a phosphorus atom and a nitrogen atom, the stretching frequency
efficacies greater than 70% were as follows: 4f (IC50: 53.3 ± 2.9
4j (IC50: 6.2 ± 1.7 M), 4m (IC50: 26.3 ± 2.0 M), 4o (IC50:
26.0 ± 7.8 M) and 4n (IC50: 0.001 ± 0.1 M). Compound 4n was as
effective as miltefosine (IC50: 3.4 ± 0.4 M) and pentamidine (IC50:
1.8 ± 1.1 M) and was approximately 2000 times more potent than
mM),
m
m
-
1
range of the P]O bond is generally from 1198 to 1274 cm [19], the
m
m
-
1
PeOeC bond absorbs in the 950-1018 cm range, and the C]N
m
-
1
bond has a stretching frequency in the 1580-1690 cm range [20].
These frequencies were observed for all products, which confirm
the expected reactions.
m
these standard drugs.
An important criterion in the search for new substances with
leishmanicidal activity is that they should not be toxic to
mammalian cells, a requirement for further clinical development.
Therefore, the cytotoxic potential of these substances on J774
1
In the H-NMR analyses, two characteristic signals confirm that
the DAPH were obtained. These signals correspond to the iminic
hydrogen eNHN]CH-Ar, with a chemical shift (d) in the range of
1
2
) 0 °C, 10 min
) 50 °C, 2 h
PCl3
+
3 ROH
(RO) P(O)H
RCl 2 HCl
+ +
2
(
1)
NaOH, CCl4
EtOH / H O
2
(
2 2 2
CHCl3
+ + +
NaCl
RO) P(O)NHNH H O
(
RO) P(O)H
+
NH NH
2
2
2
1
) 0 °C, 10 min
) 60 °C, 3 h
(
2)
2
R = Et, i-Pr, Bu, i-Bu and sec-Bu
Scheme 1. Synthetic route for dialkylphosphites (1) and dialkylphosphorylhydrazines (2).

C.B.B. da Matta et al. / European Journal of Medicinal Chemistry 101 (2015) 1e12
3
O
R'
5
6
CHO
P
N
Ar
EtOH, HCl
room temp.
RO
RO
NH
H O
2
+
(
RO) P(O)NHNH
+
4
N1
2
2
(
2)
2
(4a-o)
H
3
(
3)
R = Et, i-Pr, Bu, i-Bu and sec-Bu;
R' = H, Cl, Br
Scheme 2. Synthetic route for DAPH(4a-o).
Table 1
Activity of DAPH against promastigote forms of L. braziliensis and L. amazonensis.
A
150
Substance
L. braziliensis
IC50 M)
L. amazonensis
IC50 M)
125
100
(
m
Efficacy (%)
(
m
Efficacy (%)
7
5
Miltefosine
Pentamidine
0.7 ± 0.6
0.06 ± 0.02
>100
5.1 ± 0.5
>100
84.5 ± 0.3
95.7 ± 0.5
NA
77.2 ± 4.2
NA
3.4 ± 0.4
1.8 ± 1.1
>100
99.3 ± 0.7
>100
>100
>100
53.3 ± 2.9
>100
83.3 ± 11.6
>100
6.2 ± 1.7
>100
98.3 ± 1.2
26.3 ± 2.0
<0.001
96,3 ± 0.3
93.8 ± 0.7
NA
45.5 ± 3.2
NA
NA
NA
85.6 ± 0.5
NA
52.8 ± 5.6
NA
69.5 ± 4.2
NA
40.7 ± 6.1
93.8 ± 0.3
95.3 ± 0.4
92.4 ± 0.1
***
50
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
***
25
***
***
***
*
**
***
***
***
10
0
1
1
1
0.06 ± 0.0
>100
84.2 ± 2.6
NA
10
10
1
00
100
100
Control
4
m
4n
Miltefosine
5.4 ± 0.2
0.4 ± 0.3
40.7 ± 3.5
7.5 ± 0.8
0.06 ± 0.0
0.7 ± 0.1
>100
3.6 ± 0.3
5.2 ± 0.7
0.03 ± 0.02
87.9 ± 1.1
75.9 ± 6.6
81.6 ± 0.4
62.1 ± 3.4
76.3 ± 8.7
56.3 ± 2.7
NA
92.2 ± 0.4
80.1 ± 2.6
74.1 ± 0.3
B
200
150
100
***
***
***
5
0
***
***
26.0 ± 7.8
***
***
*
**
***
0
Data are reported as the mean ± standard error of the mean, S.E.M. Differences with
an **p < 0.01 were considered significant in relation to the 0.1% DMSO group. IC50 is
the concentration required to give 50% inhibition; NA: compound is not active.
1
1
1
10
10
10
100
100
100
Control
4
m
4n
Miltefosine
C
100
macrophages (murine cell line) was determined by the colorimetric
MTT method, which was originally described by Mossmann [22]. In
this assay, all substances, including the reference drugs miltefosine
and pentamidine, showed low cytotoxicity against J774 macro-
80
60
40
phages with LC50 values >100 mM, except for 4o, which was toxic to
20
approximately 20.5 ± 0.1% of the treated cells. It is important to
stress that the reference drug pentamidine was toxic to 28.8 ± 0.1%
of the cells at 100 mM.
0
1
1
1
1
0
10
10
100
100
100
Control
From these results, some substances have been selected for tests
on intracellular amastigotes and in vivo model. The selection
criteria took into account the potency, the efficacy against both
species and the cytotoxicity of each compound. Among the sub-
stances tested, only 4f, 4j, 4m, 4o and 4n showed activity against
both species with efficacies greater than 70%. However, 4m and 4n
best fulfilled the requirements and, therefore, have been chosen for
the subsequent tests.
4m
4n
Miltefosine
Fig. 1. Activity of 4m, 4n and miltefosine against intracellular parasites 48 h post
infection. A: infection index. B: number of parasites per macrophages infected. C:
percentage of macrophages infected. Data are reported as the mean ± standard error.
Differences with an ***p < 0.01 were considered significant in relation to the control
group.
With respect to activity against intracellular amastigotes 48 h
post infection, the results showed that the treatment with 4m and
compared to the control after treatment with 4m and 4n (p < 0.01).
The percentage of infected macrophages (Fig. 1C) was also reduced
4
n resulted in a significant (p < 0.01), dose dependent decrease in
infection index of L. amazonensis infected macrophages with an
inhibition of 99.9% and 97.9% (100 M), respectively (Fig. 1A).
by both substances, with exception of 4m at 1
no significant difference compared to the control. 4m and 4n
showed higher percentages of inhibition of infection at 100 mM
mM, which showed
m
Infected cultures were also treated with the highest concentration
of DMSO used for substances solubilization (0.01%) and the viability
and infection of macrophages were not reduced (data not shown).
Miltefosine was also significantly different compared to the control
(99.3 and 82.2%, respectively).
Using an established mouse model of cutaneous leishmaniasis
that mimics human leishmaniasis [23], BALB/c mice were infected
5
in the ear dermis with 10 stationary phase L. amazonensis pro-
(
p < 0.01), with an inhibition of 100%.
The average of the number of parasites per macrophage and the
mastigotes, and lesion development and parasite burden were
quantified. Infected mice were treated orally for 28 consecutive
days with 4m or 4n or standard drug. The untreated group was
used as a control and resulted in an average lesion size of
0.2831 mm (S.E.M ¼ 0.1022). The treated groups had significantly
percentage of infected macrophages, which influence the calcula-
tion of the index of infection, were also analyzed (Fig.1B). The mean
number of intracellular parasites per macrophage decreased

4
C.B.B. da Matta et al. / European Journal of Medicinal Chemistry 101 (2015) 1e12
reduced lesion size in the ear, the averages being
sequence identity; GMQE (Global Model Quality Estimation), which
is a quality estimation that is expressed as a number between zero
and one, reflecting the expected accuracy of a model built with that
alignment and its template [24]; QMEAN4, which is a composite
scoring function for the estimation of the global and local model
quality, consisting of four structural descriptors [25]; and the
quaternary structure information for the models. Ramachandran
plots were generated with the Rampage server [26] and are avail-
able as supplementary material.
0
4
.0527 mm (S E.M ¼ 0.03482) and 0.0804 mm (S E.M ¼ 0.0392) in
m- and 4n-treated groups, respectively. These results were similar
to the miltefosine group, with an average lesion size of
0
.0231 mm (S.E.M ¼ 0.0021) (Fig. 2).
To investigate if there was a correlation between lesion devel-
opment and parasite replication, parasite load was estimated at both
the inoculation site and the draining lymph nodes. In the ear dermis
(Fig. 3A), 4n treatment controlled parasite burden in a statistically
significant manner, corroborating the therapeutic improvement in
lesions of the ears. However, 4m was not able to reduce the parasite
load, although it caused a reduction in the ear lesion.
However, 4m and 4n did not reduce the parasite burden in the
draining lymph node (Fig. 3B). This result indicates that either the
dose (30 mg/kg/day) was not sufficient to control the infection
systemically or, in addition to leishmanicidal activity, such com-
pounds also have anti-inflammatory activity. Parasite load was
reduced at the inoculation site and in the draining lymph nodes
after miltefosine treatment.
The sequence identities between the models and their tem-
plates were above 30%, with the exception of the ENO and PGM
enzymes. The Ramachandran plots for ENO and PGM enzyme
models presented 4.7% and 5.5% residues in the outlier region,
respectively, whereas all of the remaining models have at least 98%
of their residues in the favored and/or allowed regions. The
resulting models obtained for these two enzymes presented GMQE
values lower than 0.5 and very low values of QMEAN4. Attempts to
improve the models' quality were unsuccessful, and these enzymes
were excluded from the subsequent target fishing procedure. As
seen in Table 2, excluding PGM, a monomer resulted from the
modeling procedure in five cases. This was not a problem for mo-
lecular docking in RPE, TAL, HK, and PGK, because the active site of
these enzymes involves residues of a single chain, but this was not
the case for RPI. Analysis of the tetrameric co-crystal structure of
Trypanosoma cruzi RPI and its substrate (PDB code 3K7S) revealed
that the active site of RPI shares residues from two adjacent chains.
3
. Molecular modeling
3.1. Homology models
Table 2 presents details about models of L. braziliensis and
L. amazonensis enzymes: PDB code and resolution of the template;
Fig. 2. In vivo leishmanicidal effect of 4m and 4n in BALB/c Leishmania-infected mice. Mice were infected with 105 L. amazonensis promastigotes, and the course of lesion
development was monitored for 28 days during treatment with 4m (A), 4n (B) and miltefosine (C). Lesion size (mm) is expressed as the mean ± SEM of a representative experiment
(
n ¼ 5 mice in each experimental group) (Unpaired t-test, 5 weeks, ***p < 0.0001). (D) Images of lesions after the end of treatment of BALB/c mice infected with L. amazonensis. In
the vehicle control group (I), the lesions showed an intense swelling and were ulcerated. In groups treated with miltefosine (II), 4m (III) and 4n (IV) at doses of 30 mg/kg/day, the
pictures reveal a complete healing of the nodules and ulcers.

C.B.B. da Matta et al. / European Journal of Medicinal Chemistry 101 (2015) 1e12
5
B
Lymph nodes
A
Ears
Control
Miltefosine
5
4
3
2
1
0
8
6
4
2
0
4
m
Control
4n
Miltefosine
4
4
m
n
*
*
*
*
*
*
3
0 mg/kg/day
30 mg/kg/day
Fig. 3. Parasite load in the ear and draining lymph node were determined at 4 weeks post-infection via a limiting dilution assay. (A) Ear parasite load data (normal distribution
following log transformation) represent the mean ± SEM (n ¼ 5) (Dunnett's test, **p ¼ 0.05). (B) Lymph node parasite load data were not normally distributed, even after log
transformation, and are expressed as the median ± SEM (n ¼ 5) (Dunnett's test, **p ¼ 0.05).
Table 2
amounts to apply experimental procedures, such as in the case of
the many parasites associated with neglected diseases. We
explored molecular docking methodology as a way to implement
small-scale target fishing for the active compounds identified in the
bioassays. To improve the chances of identifying the best docking
solution for each enzyme, docking runs were performed as dupli-
cates, and the solution with the highest fitness score in each case
was selected for further analysis. GOLD fitness scores are dimen-
sionless, but the scale of the score provides a guide as to how good a
ligand pose is - the higher the score for a specific function, the
better the docking result is likely to be.
L. braziliensis and L. amazonensis enzyme models.
Enzyme PDB code of Template Sequence GMQE QMEAN4 Quaternary
model
template
resolution identity
structure of
model
6
PGDHa 1pgj(A)
2.82
2.85
1.40
1.87
2.45
2.22
1.61
2.35
2.40
1.80
1.83
2.60
1.60
1.75
2.80
2.35
1.94
2.82
2.85
72.54
65.89
49.36
45.98
59.43
84.95
37.59
88.81
70.81
91.87
88.84
91.39
48.33
21.43
23.33
92.49
62.12
72.38
65.16
0.92
0.77
0.81
0.71
0.80
0.96
0.71
0.96
0.81
0.98
0.98
0.99
0.61
0.21
0.47
0.99
0.81
0.92
0.77
ꢀ1.76
ꢀ2.20
ꢀ1.87
ꢀ2.10
ꢀ2.84
ꢀ0.15
ꢀ5.50
ꢀ1.22
ꢀ1.00
ꢀ0.28
ꢀ0.29
ꢀ0.31
ꢀ4.08
ꢀ10.22
ꢀ18.22
0.81
dimer
a
G6PDH
4e9i(A)
3k7p(A)
1h1y(A)
1f05 (A)
1r9j(A)
3o4w(A)
1t10 (A)
2hig(A)
1epx(A)
1amk(A)
1i32 (C)
16pk (A)
3f3k(A)
1iyx(B)
tetramer
monomer
monomer
monomer
dimer
monomer
dimer
tetramer
tetramer
dimer
tetramer
monomer
monomer
dimer
tetramer
dimer
a
RPI
a
RPE
TAL
a
a
TKT
a
HK
a
PGI
The limitations of scoring functions to produce data that
correlate with activity data is well known, but docking programs
have demonstrated the ability to identify active compounds from a
pharmaceutically relevant pool of decoy compounds [30]. Based on
the IC50 data (Table 1), it can be concluded that the most active
compounds against L. braziliensis were 4d, 4j and 4o, all presenting
a
PFK
a
FBPA
TPI
a
a
GPDH
a
PGK
a
PGM
ENO
a
ꢀ2
a
activities in the 10
mM range. Therefore, our analysis was focused
PYK
3pp7(A)
3dyh(A)
FPPSa
ꢀ2.06
ꢀ1.76
ꢀ2.77
on identifying enzymes for which these three compounds pre-
sented an improved binding profile in comparison with the
remaining molecules, as measured by their docking fitness scores.
As seen in Tables 3 and 4, 4d, 4j and 4o were classified as the three
best ligands only in the case of the docking procedure with
L. braziliensis HK as the target. HK was validated as a target for
6
PGDHb 1pgj(A)
dimer
tetramer
b
G6PDH
4e9i(A)
a
L. braziliensis.
L. amazonensis.
b
To obtain a structure with a complete active site, we constructed a
dimer with Swiss PDB-Viewer 4.01 by superposition of the mono-
meric model of L. braziliensis RPI with both chains of its dimeric
template 3K7P. This dimeric model of L. braziliensis RPI, after energy
minimization with GROMOS96 [27], presented a very low RMSD
value (0.34 Å) when superimposed on 3K7P and was used for the
subsequent docking study.
Table 3
ChemPLP score results from docking into L. braziliensis and L. amazonensis PPP
enzymes.
a
L. braziliensis
L. amazonensis
6
PGL 6PGDH G6PDH RPE RPI
TAL
TKT 6PGDH G6PDH
4
4
a
b
47.7
51.4
47.7
50.2
44.1
50.3
47.0
47.1
42.6
49.9
43.7
46.8
45.0
55.1
46.5
53.7
44.1
54.4
48.2
55.7
46.5
55.9
46.4
54.1
50.3
51.9
47.6
45.2
55.8
46.0
48.3
46.2
53.8
45.0
48.3
43.8
53.7
43.2
47.8
45.0
47.3
48.5
48.6 43.6 43.6 50.8 45.9
51.5 54.0 46.7 58.7 53.9
48.5 45.9 48.1 56.0 47.7
49.5 50.9 46.3 57.8 53.1
53.3 45.8 42.0 44.9 45.4
55.5 55.9 46.6 61.3 56.3
45.2 45.0 49.3 58.2 50.7
50.5 54.5 48.3 57.0 61.3
50.0 41.9 41.4 47.0 46.2
51.9 52.2 44.5 60.6 55.8
42.6 42.7 46.4 47.5 46.4
48.8 50.2 43.4 58.4 54.4
40.2 48.1 48.5 52.3 53.2
45.4 50.6 40.0 55.8 54.4
35.8 43.8 44.1 52.4 51.9
44.3
52.1
46.3
48.7
41.5
52.6
42.6
49.4
44.1
49.1
42.2
47.2
48.2
50.2
45.2
3.2. Molecular docking
4c
4
4
4
4
d
e
f
The identification of the biochemical target of a group of com-
pounds with experimentally determined bioactivities is a very
difficult task. In fact, the observed in vivo effects can involve several
biochemical targets simultaneously. Together with virtual
screening approaches to identify candidate ligands for a specific
target, the availability of a great number of 3D structures of proteins
has stimulated the development of theoretical methodologies to
search for the targets of bioactive ligands, a process called target
fishing or reverse docking [28,29]. The method is particularly useful
in cases where purified enzymes are not available in sufficient
g
4h
4
4
4
4
i
j
k
l
4m 42.8
4n
48.4
41.3
4o
a
Bold values indicate the three highest score values.

6
C.B.B. da Matta et al. / European Journal of Medicinal Chemistry 101 (2015) 1e12
Table 4
that the overall identity between the two sequences is 65.8% (471
residues), which is suggestive that similar inhibition mechanisms
may operate in HK from both species.
In the case of L. amazonensis, the inhibition profile was not the
same as with L. braziliensis, and there is only one compound with
activity in the submicromolar range, 4n. The docking analysis was,
in this case, unfortunately limited to a pair of enzymes, and 4n was
not classified by the docking procedure as the best ligand with any
of these enzymes. Therefore, it was not possible to identify the
probable biochemical target for this Leishmania species.
After identification of HK as the most probable target of the
bioactive DAPH against L. braziliensis, we compared the interaction
profiles of the best poses obtained for the most active compounds
with the co-crystallized structure of the HK reaction product,
glucose-6-phosphate (G6P) (PDB code 1CZA). As an example,
Fig. 4A and B presents the superposition of the structure 1CZA with
the complex between compound 4d and L. braziliensis HK. It can be
observed that compound 4d can occupy the same site as G6P in HK,
but one of its iso-butyl groups is inserted into an adjacent binding
site, which is occupied by a glucose molecule in the coecrystal
structure. It can also be observed that the G6P binding site is
smaller and the neck connecting both sites is narrower in the hu-
man enzyme (Fig. 1A) than in the L. braziliensis enzyme (Fig. 1B).
ChemPLP score results from docking into L. braziliensis glycolysis and FPPS
enzymes.
a
HK
PGI
PFK
FBPA
TPI
GPDH
PGK
PYK
FPPS
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
42.6
49.6
44.3
51.4
40.9
51.4
45.2
49.8
45.1
54.2
44.5
50.6
50.5
49.3
52.9
43.6
53.9
45.6
51.0
45.5
56.0
45.9
50.4
43.6
52.6
40.6
47.6
49.4
51.3
43.2
47.4
62.6
52.8
59.8
48.6
61.3
52.3
59.7
48.4
61.7
52.9
58.6
60.6
64.3
61.7
51.3
58.4
55.9
59.0
53.4
63.1
57.6
59.9
52.8
58.7
57.6
56.0
60.2
54.5
53.8
51.3
58.6
50.2
57.4
51.3
60.1
53.3
57.6
53.7
60.9
56.4
53.2
56.4
62.3
62.1
45.8
57.2
49.2
56.7
48.2
57.3
49.9
56.3
47.2
59.7
50.6
58.8
56.3
56.0
54.8
45.5
53.1
44.5
50.7
42.3
51.6
43.6
49.3
41.8
52.5
44.6
46.1
49.9
48.4
49.6
48.8
57.9
50.0
56.6
47.3
56.0
48.5
54.0
49.9
53.7
49.9
53.3
49.6
53.8
53.0
68.4
78.8
70.0
67.9
65.5
90.6
69.7
78.7
66.5
76.3
70.6
69.5
69.7
68.2
66.6
a
Bold values indicate the three highest score values.
another trypanosomatid, T. cruzi, in experiments involving some
phosphorus-containing compounds [11,31]. Comparison of
L. braziliensis (UniProtKB/Swiss-Prot code A4HBM3) and T. cruzi
(
Q4DQ27) HK sequences with the SIM alignment tool [32] revealed
Fig. 4. (A and B) Superposition of the best-ranked pose of DAPH 4d (carbon atoms in cyan, stick representation) into L. braziliensis hexokinase (carbon atoms in cyan) with the
co-crystal structure of human hexokinase (carbon atoms in green) containing glucose-6-phosphate (G6P, stick representation) and glucose (Glc, stick representation) (PDB code
1CZA). In A it is shown the molecular surface of human hexokinase and in B the molecular surface of L. braziliensis hexokinase. (C) L. braziliensis hexokinase residues (carbon atoms
in green) interacting with 4d (carbon atoms in cyan). Color code for the remaining atoms: oxygen, red; nitrogen, blue, hydrogen, white, phosphorus, orange. Figures were generated
with PyMOL. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

C.B.B. da Matta et al. / European Journal of Medicinal Chemistry 101 (2015) 1e12
7
This observation suggests the 4d molecule would probably have
difficulties to be accommodated in the human HK enzyme, which is
indicative of some level of selectivity of 4d for the parasite enzyme.
Fig. 1C presents the L. braziliensis HK amino acid residues inter-
acting with 4d, including residues from G6P and glucose binding
sites. Four of these residues have correspondent residues in the
human enzyme that are interacting directly with G6P Asp187
acid was used as the external reference. In each case, the deuterated
solvents are specified, the chemical shifts are measured in ppm and
the coupling constants are in Hertz (Hz).
5.2. General procedure
In a 50 mL flask, the dialkylphosphorylhydrazine and the spe-
cific aldehyde in a1:1 M ratio, ethanol, and three drops of 37% hy-
drochloric acid as the reaction catalyst were added. The reaction
(
Asp209 in human HK), Thr210 (Thr232), Ser394 (Ser415) and
Ser429 (Ser449).
ꢂ
mixture was stirred at room temperature (25 C) forapproximately
4
. Conclusion
4 h. At the end of the reaction, sodium bicarbonate (10%) was added
to neutralize the reaction mixture. The resulting solution was
transferred to a beaker containing 20 mL of cold distilled water in
an ice bath. After approximately 20 min, a solid material was
observed that was subsequently filtered and dried at room tem-
perature. Compounds that did not precipitate in water were
transferred to a separatory funnel with the aid of an appropriate
amount of methylene chloride. After separation of the resulting
layers, anhydrous magnesium sulfate was added to the organic
phase for complete removal of residual water. Filtration was carried
out for removal of the drying agent, and the solvent was evaporated
under vacuum, after which an oily product was obtained.
The synthesis of new dialkylphosphorylhydrazones proved
satisfactory, with yields ranging from moderate to good. An excel-
lent advantage of these compounds is that they have a fairly simple
synthesis methodology. The compounds had their in vitro leish-
manicidal profile evaluated, and some active compounds could be
identified with efficacy greater than 70% against L. braziliensis (4b,
4
4
d, 4f, 4g, 4h, 4j, 4m, 4n, and 4o) and against L. amazonensis (4f, 4j,
m, 4n, and 4o). The compounds with the greatest efficacy against
both species, 4m and 4n, were tested in vivo against L. amazonensis-
infected mice. Both of the compounds completely healed the
nodules and ulcers, but control of parasite burden at the inocula-
tion site was statistically significant only in the case of 4n
treatment.
6. Characterization of the products
0
Comparing the corresponding IC50 values, the only compounds
6.1. Phosphorohydrazidic acid, N -[(1E)-4-pyridinylmethylene]-
ꢀ
2
with IC50 against L. braziliensis in the 10
mM range (the same
diethyl ester (4a)
range for pentamidine activity) were 4d, 4j, and 4o.Compound 4n
was the only one with an IC50 value in the submicromolar range
against L. amazonensis, which was even lower than that for the
reference compounds pentamidine and miltefosine. A target fishing
procedure based on docking methodology using 15 different en-
zymes from L. braziliensis and two from L. amazonensis was
implemented to identify probable biochemical targets for the active
compounds. After comparison with the IC50 values, it was
concluded that hexokinase is the most probable target in
L. braziliensis, but it was not possible to identify a target for
L. amazonensis because of the limited number of candidate en-
zymes available for this species.
Aspect: orange oil. yield: 53%. IR (NaCl): 3425.7 (st, NH); 3149.6
(st, CHaromatic); 2985.7, 2925.9 and 2821.7 (stass, CH
1597.0 (st, C]N); 1475.5 ( ass,.CH and CH ); 1407.9 (
1240.1 (st P]O); 1163.0 (st, PeN); 1031.8 (st, PeOeC). NMR
3
and CH
2
);
);
H
d
2
3
dsim,.CH
3
1
(DMSO): 9.95 [d, (PeNeH), JHP ¼ 29.0 Hz, 1H]; 8.56 [d, (H2 and H6),
J
J
J
ortho ¼ 6.27 Hz, 2H]; 7.89 [s, (N]CH), 1H]; 7.48 [d, (H3 and H5),
ortho ¼ 6.27 Hz, 2H]; 4.03 [m, (CH CH OP), 4H]; 1.23 [t, (CH CH OP),
HH ¼ 7.41 Hz, 6H]. NMR C (DMSO): 150.13 [s, (C2 and C6), 2C];
3
2
3
2
13
141.76 [s, (C4), 1C]; 141.46 [d, (N]C), JCP ¼ 20.35 Hz, 1C]; 120.23
[s, (C3 and C5), 1C]; 62.57 [d, (CH
OP), Jcp ¼ 5.30 Hz, 2c]; 16.06
[d, (CH CH
OP), JCP ¼ 6.26 Hz, 2c]. NMR P (DMSO): 1.33 [dq,
2
31
3
2
(
PeNeH), JHP ¼ 29.35 Hz, (CH OP), JHP ¼ 7.91 Hz]. m/Z: 45(8%),
2
5
. Experimental section
81(61%), 98(100%), 126(87%), 133(3%), 153(22%), 184(3%), 200(2%),
þ
2
28(1%), 258(3%). HR-MS(ESI) calc. for
C
10
H
16
N
3
O
3
P
(M) :
þ
þ
5.1. Chemistry
257.092928, found: (M þ H) : 258.100204, (M þ Na) : 280.082149.
0
All solvents used in reactions and purification methods were
6.2. Phosphorohydrazidic acid, N -[(1E)-4-pyridinylmethylene]-
distilled prior to use and, when necessary, treated and dried ac-
cording to the usual methods described in the literature [33]. Thin
layer chromatography was performed using 0.2 mm-thick,
aluminum-backed plates containing Kieselgel 60 F254, and visu-
alized under UV light at 254 nm.
dibutyl ester (4b)
Aspect: orange oil. yield: 55%. IR (NaCl): 3437.0 (st, NH); 3101.4
(st, CHaromatic); 2960.6, 2931.7 and 2879.6 (stass, CH
1591.2 (st, C]N); 1471.6 ( ass.,CH and CH ); 1394.4 (dsim.CH
3
and CH
2
);
);
d
2
3
3
The devices used for the characterization of the compounds
were as follows: (a) PerkineElmer infrared spectrometer, model
1240.1 (st P]O); 1110.9 (st PeN); 1029.9 (st PeOeC).
1
NMR H(DMSO): 9.96 [d, (PeNeH), JHP ¼ 28.10 Hz, 1H]; 8.57 [d, (H2
1600 FT. The spectra were obtained using a film on NaCl cells in the
and H6), Jortho ¼ 6.21 Hz, 2H]; 7.88 [s, (N]CeH), 1H]; 7.48 [d, (H3
case of liquid samples, and KBr pellets in the case of solid samples.
and H5), Jortho ¼ 6.21 Hz, 2H]; 3.83 [m, (CH
2
OP), 4H]; 1.57 [qui,
ꢀ1
The absorptions were measured in reciprocal centimeters (cm );
b) gas chromatograph coupled to a mass spectrometer, Varian
Saturn model 2000. The analysis conditions were VF-5ms column
(CH
2
CH
2
OP), JHH ¼ 6.47 Hz, 4H]; 1.33 [sex, (CH
(CH
OP), JHH ¼ 7.55 Hz, 6H]. NMR
C (DMSO): 150.13 [s, (C2 and C6), 2C]; 141.80 [s, (C4), 1C]; 141.34
[d, (N]C), JCP ¼ 20.01 Hz, 1C]; 120.18 [s, (C3 and C5), 1C]; 66.04 [d,
(CH CH OP), JCP ¼ 6.57 Hz, 2C];
OP), JCP ¼ 5.35 Hz, 2C]; 31.74 [d, (CH
18.18 [s, (CH CH CH OP), 2C]; 13.37 [s, (CH CH CH CH OP), 2C].
NMR P (DMSO): 1.33 [dq, (PeNeH), JHP ¼ 27.56 Hz, (CH OP),
2 2 2
(CH ) OP),
(
J
HH ¼ 7.02 Hz, 4H]; 0.84 [t, (CH
3
2 3
)
1
3
ꢂ
ꢂ
(
30 ꢁ 0.25 ꢁ 0.25 mm), oven temperature 150e290 C/10 C/min,
ꢂ
ꢂ
ꢂ
ꢂ
injector 270 C, MS trap 220 C, manifold 60 C, transfer line 250 C,
EI ionization (70 eV); (c) NMR spectra were performed on a Bruker
AC 200 spectrometer ( H NMR: 200 MHz, C NMR: 50.3 MHz,
2
2
2
2
2
2
3
2
2
2
1
13
31
31
P
2
1
NMR: 81.0 MHz) and Bruker AVANCE II 400 spectrometer ( H NMR:
J
HP ¼ 7.35 Hz]. Coupled. m/Z: 41(13%), 80(4%), 98(100%), 124(4%),
31
4
00 MHz, 13C NMR: 100.6 MHz, P NMR: 161.9 MHz). The spectra
154(22%), 202(2%), 214(<2%), 258(<2%), 270(<2%), 314(<2%). HR-
MS (ESI) calc. for
(M þ H) : 314.162805, (M þ Na) : 336.144749.
þ
were obtained using tetramethylsilane (TMS) or the solvent itself as
C
14
H N
24 3
O
3
P
(M) : 313.155528, found:
1
13
31
þ
þ
a reference for H and C spectra. For P spectra, 85% phosphoric

8
C.B.B. da Matta et al. / European Journal of Medicinal Chemistry 101 (2015) 1e12
0
0
6
.3. Phosphorohydrazidic acid, N -[(1E)-4-pyridinylmethylene]-
6.6. Phosphorohydrazidic acid, N -[(1E)-3-pyridinylmethylene]-
diisopropyl ester (4c)
dibutyl ester (4f)
ꢂ
Aspect: orange solid. MP: 156e158 C. yield: 52%. IR (NaCl):
Aspect: orange oil. yield: 55%. IR (NaCl): 3431.2 (st, NH); 3101.4
3
431.2 (st, NH); 3116.8 (st, CHaromatic); 2979.9, 2933.6 and 2823.6
(st, CHaromatic); 2960.6, 2931.7 and 2873.8 (stass., CH
1600.8 (st, C]N); 1465.8 ( ass., CH and CH ); 1419.5 (
1242.1 (st, P]O); 1066.6 (st, PeN); 1029.9 (st, PeOeC). NMR
(DMSO): 9.77 [d, (PeNeH), JHP ¼ 28.02 Hz, 1H]; 8.70 [d, (H2),
3
and CH
dsim., CH
2
);
);
H
(
1
stass., CH
386.7 (
3
and CH
2
); 1597.0 (st, C]N); 1481.2 (dass., CH
2
and CH
3
);
d
2
3
3
1
d
sim., CH
3
); 1236.3 (st, P]O); 1114.8 (st, PeN); 1014.5 (st,
1
PeOeC). NMR H (DMSO): 9.87 [d, (PeNH), JHP ¼ 28.92 Hz, 1H];
8
7
1
J
1
(
(
.57 [d, (H2 and H6), Jortho ¼ 6.77 Hz, 2H]; 7.88 [s, (N]CeH), 1H];
.50 [d, (H3 and H5), Jorthom ¼ 6.77 Hz, 2H]; 4.55 [m, (CHOP), 2H];
.27 [d, ((CH CHOP),
CHOP), JHH ¼ 6.42 Hz, 6H]; 1.22 [d, ((CH
HH ¼ 6.42 Hz, 6H]. NMR C (DMSO): 149.80 [s, (C2 and C6), 2C];
42.25 [s, (C4), 1C]; 140.83 [d, (N]C), Jcp ¼ 21.02 Hz, 1C]; 120.20 [s,
C3 andC5), 1C]; 71.00 [d, (CHOP), JCP ¼ 5.97 Hz, 2c]; 23.56 [d,
(CH CHOP), 4.41 Hz, 2c]; 23.36 [d, ((CH CHOP),
P (DMSO): ꢀ0.79 [dt, (PeNeH),
J
HH ¼ 1.70 Hz, 1H]; 8.52 [dd, (H6), Jortho ¼ 4.74 Hz and 1.58 Hz, 1H];
7.93 [s, (N]CH), 1H]; 7.92 (td (H4), Jortho ¼ 6.45 Hz, Jmeta ¼ 1.94 Hz,
3
)
2
3 2
)
1H]; 7.41 [dd, (H5), Jortho ¼ 4.61 Hz and 7.83 Hz, 1H]; 3.98 [m,
13
(CH
2
OP), 4H]; 1.57 [ m, (CH
2
CH
2
OP), 4H]; 1.33 [sex, (CH
(CH
C (DMSO): 149.83 [s, (C6), 1C]; 147.72 [s, (C2), 1C]; 140.90 [d, (N]
C), JCP ¼ 20.84 Hz, 1C]; 132.63 [s, (C4), 1C]; 130.56 [s, (C3), 1C];
123.90 [s, (C5), 1C]; 65.95 [d, (CH
OP), JCP ¼ 6.64 Hz, 2C]; 31.71
[d, (CH CH CH CH OP), 2C];
OP), JCP ¼ 6.64 Hz, 2C]; 18.20 [s, (CH
13.38 [s, (CH OP), 2C]. NMR P (DMSO): 1.73 [dq,
(PeNeH), JHP ¼ 27.47 Hz, (CH OP), JHP ¼ 7.84 Hz]. coupled. m/Z:
41(11%), 80(5%), 98(100%), 124(7%), 154(21%), 184(2%), 214(1%),
58(1%), 270(<1%), 314(1%). HR-MS (ESI) calc. for C14
2 2 2
(CH ) OP),
J
HH ¼ 7.39 Hz, 4H]; 0.84 [t, (CH
3
2
)
3
OP), JHH ¼ 7.20 Hz, 6H]. NMR
1
3
3
)
2
J
CP
¼
3 2
)
31
J
CP ¼ 4.41 Hz, 2C]. NMR
2
JHP ¼ 29.52 Hz, (CHOP), JHP ¼ 7.64 Hz]. coupled. m/Z: 43(27%),
2
2
2
2
2
31
8
2
2
1(11%), 98(100%), 120(4%), 140(17%), 184(6%), 200(1%), 228(5%),
42(2%), 286(<2%). HR-MS (ESI) calc. for C12
85.124228, found: (M þ H) : 286.131504, (M þ Na) : 308.113449.
3 2 2 2
CH CH CH
þ
H
20
N
3
O
3
P (M) :
2
þ
þ
2
(
3
24 3 3
H N O P
þ
þ
þ
M) : 313.155528, found: (M þ H) : 314.162805, (M þ Na) :
36.144749.
0
6.4. Phosphorohydrazidic acid, N -[(1E)-4-pyridinylmethylene]-
diisobutyl ester (4d)
0
6
.7. Phosphorohydrazidic acid, N -[(1E)-3-pyridinylmethylene]-
diisopropyl ester (4g)
ꢂ
Aspect: yellow solid. MP: 110e112 C. yield: 43%. IR (NaCl):
3
453.1 (st, NH); 3097.5 (st, CHaromatic); 2956.7, 2925.9 and 2821.7
Aspect: orange oil. yield: 55%. IR (NaCl): 3431.2 (st, NH); 3099. 5
(
stass., CH
409.9 (
PeOeC). NMR H (DMSO): 9.98 [d, (PeNeH), JHP ¼ 29.0 Hz, 1H];
.57 [d, (H2 and H6), Jortho ¼ 6.27 Hz, 2H]; 7.88 [s, (N]CeH), 1H];
.48 [d, (H3 and H5), Jortho ¼ 6.27 Hz, 2H]; 3.77 [m, (CH OP), 4H];
.88 [m, (CHCH CHCH OP),
OP), JHH ¼ 6.69 Hz, 2H]; 0.88 [d, ((CH
HH ¼ 6.97 Hz, 12H]. NMR C (DMSO): 150.10 [s, (C2 and C6), 2C];
41.84 [s, (C4), 1C]; 141.31 [d, (N]C), JCP ¼ 18.90 Hz, 1C]; 120.16
s, (C3 and C5), 1C]; 72.26 [d, (CH
OP), JCP ¼ 6.28 Hz, 2C]; 28.57
d, (CHCH CHCH OP),
OP), JCP ¼ 6.76 Hz, 2C]; 18.51 [d, ((CH
P (DMSO): 1.14 [dq, (PeNeH),
OP), JHH ¼ 8.00 Hz]. Coupled. m/Z: 57(16%),
2(3%), 98(100%), 138(3%), 154(10%), 202(2%), 214(1%), 242(3%),
3
and CH
2 2 3
); 1597.0 (st, C]N); 1475.5 (dass.,CH and CH );
(
1
1
(
J
7
7
st, CHaromatic); 2979.9, 2925.9 and 2817.9 (stass., CH
604.7 (st, C]N); 1475.5 ( ass., CH and CH ); 1380.9 (
245.9 (st, P]O); 1097.4 (st, PeN); 1028.0 (st, PeOeC). NMR
3
and CH
dsim., CH
2
);
);
H
1
dsim.,CH ); 1245.9 (st, P]O); 1105.1 (st, PeN); 1033.8 (st,
3
d
2
3
3
1
1
8
7
1
J
1
[
[
DMSO): 9.67 [d, (PeNeH), JHP ¼ 28.32 Hz, 1H]; 8.70 [d, (H2),
HH ¼ 1.75 Hz, 1H]; 8.52 [dd, (H6), Jortho ¼ 4.92 Hz and 1.66 Hz, 1H];
.94 [m, (N]CH) and (H4), 2H]; 7.41 [dd, (H5), Jortho ¼ 4.72 Hz and
.94 Hz, 1H]; 4.54 [m, (CHOP), 2H]; 1.23 [d, (CH CHOP),
HH ¼ 6.22 Hz, 6H]; 1.19 [d, (CH CHOP), JHH ¼ 6.22 Hz, 6H]. NMR
C (DMSO): 149.57 [s, (C6), 1C]; 147.51 [s, (C2), 1C]; 140.43 [d, (N]
C), JCP ¼ 19.15 Hz, 1C]; 132.63 [s, (C4), 1C]; 130.71 [s, (C3), 1C];
23.93 [s, (C5), 1C]; 70.82 [d, (CHOP), JCP ¼ 5.32 Hz, 2C]; 23.58 [d,
CH CHOP), 4.52 Hz, 2C]; 23.34 [d, (CH CHOP),
CP ¼ 4.52 Hz, 2C]. NMR P (DMSO): ꢀ0.15 [dt, (PeNeH),
2
2
3
)
2
2
13
3 2
)
J
3 2
)
2
1
3
2
3
)
2
2
31
J
CP ¼ 3.97 Hz, 4C]. NMR
1
(
JHP ¼ 29.0 Hz, (CH
2
3
)
2
J
CP
¼
3 2
)
9
31
J
J
8
2
2
þ
24 3 3
314(<1%). HR-MS (ESI) calc. for C14H N O P (M) : 313.155528,
HP ¼ 27.54 Hz, (CHOP), JHP ¼ 7.36 Hz]. coupled. m/Z: 43(22%),
þ
þ
found: (M þ H) : 314.162805, (M þ Na) : 336.144749.
1(11%), 98(100%), 120(19%), 140(16%), 184(6%), 200(1%), 228(3%),
þ
42(1%), 285(1%). HR-MS (ESI) calc. for
C
12
H
20
N
3
O
3
P
(M) :
þ
þ
85.124228, found: (M þ H) : 286.131504, (M þ Na) : 308.113449.
0
6.5. Phosphorohydrazidic acid, N -[(1E)-3-pyridinylmethylene]-
0
diethyl ester (4e)
6.8. Phosphorohydrazidic acid, N -[(1E)-3-pyridinylmethylene]-
diisobutyl ester (4h)
Aspect: yellow oil. yield: 55%. IR (NaCl): 3427.3 (st, NH); 3165.0
ꢂ
(
st, CHaromatic); 2985.7 and 2927.8 (stass., CH
3
and CH
2
); 1604.7 (st,
Aspect: white solid. MP: 92e94 C. yield: 47%. IR (NaCl): 3435.1
C]N); 1475.5 (
d
ass., CH
2
and CH
3
); 1413.7 (
d
sim.,CH
3
); 1238.2 (st, P]
(st, NH); 3097.5 (st, CHaromatic); 2956.7, 2927.8 and 2823.6 (stass.
CH and CH ); 1606.6 (st, C]N); 1465.8 ( ass., CH and CH ); 1398.3
); 1247.9 (st, P]O); 1093.6(st, PeN); 1031.8 (st, PeOeC).
,
1
O); 1163.0 (st, PeN); 1031.8 (st, PeOeC). NMR H (DMSO): 9.77 [d,
3
2
d
2
3
(
PeNeH), JHP ¼ 28.48 Hz, 1H]; 8.69 [d, (H2), JHH ¼ 1.71 Hz, 1H]; 8.52
(d
sim., CH
3
1
[
dd, (H6), Jortho ¼ 4.83 Hz, Jmeta ¼ 1.71 Hz, 1H]; 7.94 [m, (N]CH and
NMR H (DMSO): 9.78 [d, (PeNeH), JHP ¼ 28.52 Hz, 1H]; 8.70 [d,
(H2), JHH ¼ 1.86 Hz, 1H]; 8.52 [dd, (H6), Jortho ¼ 4.76 Hz and 1.65 Hz,
1H]; 7.93 [s, (N]CH), 1H]; 7.92 (td (H4), Jortho ¼ 7.04 Hz,
H4), 2H]; 7.40 [dd, (H5), Jortho ¼ 4.71 Hz and 7.90 Hz, 1H]; 4.04 [m,
13
(
CH
3
CH
2
OP), 4H]; 1.23 [t, (CH
3
CH
2
OP), JHH ¼ 6.90 Hz, 6H]. NMR
C
(
DMSO): 149.82 [s, (C6), 1C]; 147.77 [s, (C2), 1C]; 141.01 [d, (N]C),
J
meta ¼ 1.86 Hz, 1H]; 7.42 [dd, (H5), Jortho ¼ 4.26 Hz and 7.65 Hz, 1H];
J
CP ¼ 19.02 Hz, 1C]; 132.65 [s, (C4), 1C]; 130.53 [s, (C3), 1C]; 123.89
3.77 [m, (CH
[d, ((CH CHCH
2
OP), 4H]; 1.88 [m, (CHCH
2
OP), JHH ¼ 6.50 Hz, 2H]; 0.88
13
[
(
(
s, (C5), 1C]; 62.45 [d, (CH
CH CH
OP), JCP ¼ 5.48 Hz, 2C]. NMR P (DMSO): 1.61 [dq,
OP), JHP ¼ 7.35 Hz]. Coupled. m/Z:
2
OP), JCP ¼ 5.48 Hz, 2C]; 16.03 [d,
3
)
2
2
OP), JHH ¼ 7.66 Hz, 12H]. NMR C (DMSO): 149.76
3
1
3
2
[s, (C6), 1C]; 147.64 [s, (C2), 1C]; 140.84 [d, (N]C), JCP ¼ 19.30 Hz,
PeNeH), JHP ¼ 29.89 Hz, (CH
2
1C]; 132.61 [s, (C4), 1C]; 130.58 [s, (C3), 1C]; 123.91 [s, (C5), 1C];
5
1(5%), 65(38%), 92(96%), 120(100%), 133(2%), 155(2%), 184(2%),
72.12 [d, (CH
2
OP), JCP ¼ 6.37 Hz, 2C]; 28.57 [d, (CHCH
CHCH
NMR P (DMSO): 1.64 [dq, (PeNeH), JHP ¼ 30.5 Hz, (CH
2
OP),
2
00(<1%), 229(<1%), 257 (8%). HR-MS (ESI) calc. for C10
H
16
N
3
O
3
P
J
CP ¼ 6.37 Hz, 2C]; 18.53 [d, (CH
3
)
2
2
OP), JCP ¼ 3.70 Hz, 4C].
þ
þ
þ
31
(
M) : 257.092928, found: (M þ H) : 258.100204, (M þ Na) :
2
OP),
2
80.082149.
JHP ¼ 7.56 Hz]. coupled. m/Z: 57(13%), 92(4%), 98(100%), 138(4%),

C.B.B. da Matta et al. / European Journal of Medicinal Chemistry 101 (2015) 1e12
9
1
C
54(11%), 184(3%), 214(1%), 242(1%), 313(<1%). HR-MS (ESI) calc. for
1.28 [d, (CH
3
)
2
CHOP), JHH ¼ 6.35 Hz, 6H]. NMR 13C (DMSO): 153.64
þ
þ
H N
14 24 3
O
3
P (M) : 313.155528, found: (M þ H) : 314.162805,
[s, (C2), 1C]; 149.25 [s, (C5), 1C]; 144.12 [d, (N]C), JCP ¼ 18.42 Hz,
þ
(
M þ Na) : 336.144749.
1C]; 136.71 [s, (C6), 1C]; 123.52 [s, (C4), 1C]; 118.77 [s, (C3), 1C];
7
0.90 [d, (CHOP), JCP ¼ 5.51 Hz, 2C]; 23.55 [d, (CH
CP ¼ 4.82 Hz, 2C]; 23.34 [d, (CH CHOP), JCP ¼ 4.82 Hz, 2C]. NMR
P (DMSO): ꢀ0.36 [dt, (PeNeH), JHP ¼ 27.43 Hz, (CHOP),
HP ¼ 7.34 Hz]. Coupled. m/Z: 43(16%), 65(20%), 92(56%), 120(100%),
147(5%), 184(5%), 201(1%), 228(2%), 243(23%), 285(5%). HR-MS (ESI)
3 2
) CHOP),
0
6.9. Phosphorohydrazidic acid, N -[(1E,Z)-2-pyridinylmethylene]-
J
3 2
)
3
1
diethyl ester (4i)
J
Aspect: dark oil. yield: 36%. IR (NaCl): 3433.1(st, NH); 3140.0 (st,
CHaromatic); 2983.7, 2931.7 and 2912.4 (stass., CH and CH ); 1585.4
); 1240.1 (st,
þ
þ
3
2
calc. for C12
286.131504, (M þ Na) : 308.113449.
20
H N
3
O
3
P (M) : 285.124228, found: (M þ H) :
þ
(
st, C]N); 1461.9 (
d
ass., CH
2
and CH
3
); 1394.4 (
d
sim., CH
3
1
P]O); 1164.9 (st, PeN); 1028.0 (st, PeOeC). NMR H (DMSO): 9.84
0
[
J
(
d, (PeNeH), JHP ¼ 27.94 Hz, 1H]; 8.53 [ddd, (H6), Jortho ¼ 4.93 Hz,
meta ¼ 1.82 Hz, Jpara ¼ 0.64 Hz, 1H]; 7.96 [s, (N]CH), 1H]; 7.79 [dt,
H4), Jortho ¼ 7.64 Hz, Jmeta ¼ 1.96 Hz, 1H]; 7.75 [td, (H3),
6.12. Phosphorohydrazidic acid, N -[(1E)-2-pyridinylmethylene]-
diisobutyl ester (4l)
ꢂ
J
J
(
1
1
ortho ¼ 7.82 Hz, Jmeta ¼ 1.10 Hz, 1H]; 7.33 [ddd, (H3), Jortho ¼ 7.52 Hz,
Aspect: brown solid. MP: 90e92 C. yield: 50%. IR (NaCl):
ortho ¼ 5.37, Jmeta ¼ 1.28 Hz, 1H]; 4.03 [m, (CH
2
OP), 4H]; 1.24 [t,
OP), JHH ¼ 7.23 Hz, 6H]. NMR C (DMSO): 153.49 [s, (C2),
C]; 149.30 [s, (C5), 1C]; 144.60 [d, (N]C), JCP ¼ 19.15 Hz, 1C];
36.74 [s, (C6), 1C]; 123.66 [s, (C4), 1C]; 119.03 [s, (C3), 1C]; 62.56
d, (CH CH
C]. NMR P (DMSO): 1.53 [dq, (PeNeH), JHP ¼ 28.25 Hz, (CH
HP ¼ 7.70 Hz]; 0.97 [td, (PeNeH), JHP ¼ 34.01 Hz, (CH
3438.9 (st, NH); 3134.2 (st, CHaromatic); 2958.7, 2875.7 (stass., CH
and CH ); 1585.4 (st, C]N); 1461.9 ( ass., CH and CH ); 1371.3
); 1228.6 (st, P]O); 1103.2 (st, PeN); 1018.3 (st, PeOeC).
3
13
CH
3
CH
2
2
d
2
3
(dsim., CH
3
1
NMR H (DMSO): 9.84 [d, (PeNeH), JHP ¼ 28.90 Hz, 1H]; 8.52 [ddd,
(H6), Jortho ¼ 4.82 Hz, Jmeta ¼ 1.92 Hz, Jpara ¼ 0.63 Hz, 1H]; 7.95 [s,
(N]CH), 1H]; 7.80 [dt, (H4), Jortho ¼ 7.75 Hz, Jmeta ¼ 1.64 Hz, 1H];
7.72 [td, (H3), Jortho ¼ 7.90 Hz, Jmeta ¼ 1.16 Hz, 1H]; 7.32 [ddd, (H5),
[
2
2
OP), JCP ¼ 5.67 Hz, 2C]; 16.04 [d, (CH
3
2
OP), JCP ¼ 6.60 Hz,
31
2
OP),
OP),
J
J
1
2
HP ¼ 7.70 Hz]. Coupled; m/Z: 51(5%), 65(39%), 92(94%), 120(100%),
J
ortho ¼ 7.51 Hz, Jortho ¼ 6.11 Hz, Jmeta ¼ 1.36 Hz, 1H]; 3.78
33(2%), 155(2%), 184(2%), 200(<1%), 229(<1%), 257(7%). HR-MS
[m, (CH
(CH CHCH
(C2), 1C]; 149.28 [s, (C5), 1C]; 144.46 [d, (N]C), JCP ¼ 19.80 Hz, 1C];
36.71 [s, (C6), 1C]; 123.61 [s, (C4), 1C]; 118.76 [s, (C3), 1C]; 72.15
[d, (CH OP), JCP ¼ 6.25 Hz,
OP), JCP ¼ 6.51 Hz, 2C]; 28.51 [d, (CHCH
2C]; 18.50 [d, (CH CHCH
OP), JCP ¼ 3.50 Hz, 4C]. NMR
2
OP), 4H]; 1.88 [n, CHCH
2
OP), JHH ¼ 6.61 Hz, 2H]; 0.87 [d,
þ
þ
13
(
ESI) calc. for C10
H
16
N
3
O
3
P (M) : 257.092928, found: (M þ H) :
3
)
2
2
OP), JHH ¼ 6.98 Hz, 12H]. NMR C (DMSO): 153,54 [s,
þ
2
58.100204, (M þ Na) : 280.082149.
1
0
6.10. Phosphorohydrazidic acid, N -[(1E,Z)-2-pyridinylmethylene]-
2
2
31
dibutyl ester (4j)
3
)
2
2
P
(
DMSO): 1.43 [dq, (PeNeH), JHP ¼ 30.05 Hz, (CH OP), JHP ¼ 7.12 Hz].
2
Aspect: dark oil. yield: 45%. IR (NaCl): 3429.3 (st, NH); 3136.1 (st,
Coupled. m/Z: 65(17%), 92(61%), 120(100%), 123(1%), 161(5%),
202(4%), 214(<1%), 258(4%), 313(2%). HR-MS (ESI) calc. for
CHaromatic); 2960.6 and 2877.7 (stass., CH
N); 1463.9 (
3
and CH
2
); 1585.4 (st, C]
þ
þ
dass., CH
2
and CH
3
); 1386.7 (
d
sim., CH
3
); 1240.1 (st, P]O);
C
14
H
24
N
3
O
3
P (M) : 313.155528, found: (M þ H) : 314.162805,
1
þ
1110.9 (st, PeN); 1028.0 (st, PeOeC). NMR H (DMSO): 9.85 [d,
(M þ Na) : 336.144749.
(
J
(
J
J
PeNeH), JHP ¼ 28.72 Hz, 1H]; 8.53 [ddd, (H6), Jortho ¼ 5.04 Hz,
meta ¼ 1.84 Hz, Jpara ¼ 0.96 Hz, 1H]; 7,95 [s, (N]CH), 1H]; 7.79 [dt,
H4), Jortho ¼ 7.73 Hz, Jmeta ¼ 1.89 Hz, 1H]; 7.74 [td, (H3),
ortho ¼ 7.87 Hz, Jmeta ¼ 1.10 Hz, 1H]; 7.34 [ddd, (H5), Jortho ¼ 7.37 Hz,
ortho ¼ 5.28 Hz, Jmeta ¼ 1.32 Hz, 1H]; 3.98 [m, (CH OP), 4H]; 1.57
m,(CH CH OP), 4H]; 1.34 [sex, (CH (CH
OP), JHH ¼ 7.28 Hz, 4H];
.84 [t, (CH (CH
OP), JHH ¼ 7.25 Hz, 6H]. NMR C (DMSO): 153.54
0
6.13. Phosphorohydrazidic acid, N -[(1E)-(5-bromo-3-pyridinyl)
methylene]-disecbutyl ester (4m)
ꢂ
2
Aspect: white solid. MP: 121e122 C. yield: 62%. IR (NaCl):
3440.9 (st, NH); 3101.4 (st, CHaromatic); 2972.2, 2931.7 and 2827.5
[
0
2
2
2
2
)
2
13
3
2
)
3
(stass., CH
3
and CH
2
); 1600.8 (st, C]N); 1471.6 (dass., CH
2
and CH
3
);
H
1
[
1
s, (C2), 1C]; 149.29 [s, (C5), 1C]; 144.49 [d, (N]C), JCP ¼ 19.11 Hz,
1242.1 (st, P]O); 1095.5 (st, PeN); 1018.3 (st, PeOeC). NMR
C]; 136.68 [s, (C6), 1C]; 123.63 [s, (C4), 1C]; 118.85 [s, (C3), 1C];
5.98 [d, (CH CH OP),
OP), JCP ¼ 5.49 Hz, 2C]; 31.76 [d, (CH
CH CH OP), 2C]; 13.36 [s,
OP), 2C]. NMR P (DMSO): 1.54 [dq, (PeNeH),
(DMSO): 9.91 [d, (PeNeH), JHP ¼ 28.62 Hz, 1H]; 8.70 [s, (H6), 1H];
6
2
2
2
8.64 [s, (H2), 1H]; 8.12 [s, (N]CH), 1H]; 7.89 [s, (H4), 1H]; 4.34
J
(
CP ¼ 6.34 Hz, 2C]; 18.19 [s, (CH
2
2
2
[m, (CHOP), 2H]; 1,55 [m, (CH
HH ¼ 5.45 Hz, 3H]; 1.21 [d, (CH
[t, (CH CH
CHOP), JHH ¼ 7.80 Hz, 3H]; 0.84 [t, (CH
2
CHOP), 4H]; 1.27 [d, (CH
CHOP), JHH ¼ 5.45 Hz, 3H]; 0.88
CH CHOP),
3
CHOP),
31
CH
3
CH
2
CH
2
CH
2
J
3
J
J
9
2
HP ¼ 29.41 Hz, (CH
2
OP), JHP ¼ 7.66 Hz]; 1.05 [td, (PeNeH),
3
2
3
2
13
HP ¼ 34.83 Hz, (CH
2
OP), JHP ¼ 7.66 Hz]. coupled. m/Z: 65(19%),
J
HH ¼ 7.80 Hz, 3H]. NMR C (DMSO): 150.00 [s, (C6), 1C]; 145.90 [s,
2(67%), 120(100%), 133(2%), 161(4%), 184(1%), 208(2%), 258(1%),
70(<1%), 313(1%). HR-MS (ESI) calc. for C14
(C2), 1C]; 138.68 [d, (N]C), Jcp ¼ 19.85 Hz, 1C]; 134.62 [s, (C4), 1C];
þ
H
24
N
3
O
3
P (M) :
132.70 [s, (C3), 1C]; 120.48 [s, (C5), 1C]; 75.55 [t, (CHOP),
þ
þ
313.155528, found: (M þ H) : 314.162805, (M þ Na) : 336.144749.
J
(
cp ¼ 5.76 Hz, 2C]; 29.84 [d, (CH
CH
CHOP), Jcp ¼ 6.00 Hz, 1C]; 21,10 [d, (CH
1C]; 20.82 [d, (CH CHOP), 2.63 Hz, 1C]; 9.32 [d,
(CH CH
CHOP), Jcp ¼ 6.72 Hz, 2C]. NMR P (DMSO): ꢀ0.30 [dt,
PeNeH), JHP ¼ 27.97 Hz, (CH OP), JHP ¼ 8.13 Hz]; Coupled. m/Z:
57(17%), 91(4%), 98(100%), 137(1%), 154(5%), 200(5%), 262(4%),
3 3
280(1%), 306(2%), 392(<1%). HR-MS (ESI) calc. for C14 23BrN O P
2
CHOP), Jcp ¼ 6.00 Hz, 1C]; 29.68 [d,
2
3
CHOP), Jcp ¼ 2.63 Hz,
0
6.11. Phosphorohydrazidic acid, N -[(1E)-2-pyridinylmethylene]-
3
J
cp
¼
31
diisopropyl ester (4k)
3
2
(
2
ꢂ
Aspect: white solid. MP: 171e173 C. yield: 54%. IR (NaCl):
3
427.3 (st, NH); 3136.1 (st, CHaromatic); 2981.8, 2943.2 and 2839.1
H
þ
þ
þ
(
1
stass., CH
382.9 (
3
and CH
2
); 1583.5 (st, C]N); 1456.2 (
dass., CH
2
and CH
3
);
(M) : 391.066041, found: (M þ H) : 392.073317, (M þ Na) :
dsim., CH
3
); 1228.6 (st, P]O); 1110.9 (st, PeN); 1024.1
414.055262.
1
(
1
1
st, PeOeC). NMR H (DMSO): 9.74 [d, (PeNeH), JHP ¼ 27.83 Hz,
0
H]; 8.53 [ddd, (H6), Jortho ¼ 5.24 Hz, Jmeta ¼ 1.95 Hz, Jpara ¼ 0.86 Hz,
H]; 7.96 [s, (N]CH), 1H]; 7.79 [dt, (H4), Jortho ¼ 7.91 Hz,
6.14. Phosphorohydrazidic acid, N -[(1E)-(2-bromo-3-pyridinyl)
methylene]-disecbutyl ester (4n)
J
1
meta ¼ 1.88 Hz, 1H]; 7.74 [td, (H3), Jortho ¼ 7.65 Hz, Jmeta ¼ 1.25 Hz,
H]; 7.32 [ddd, (H5), Jortho ¼ 7.45 Hz, Jortho ¼ 5.43 Hz, Jmeta ¼ 1.51 Hz,
H]; 4.55 [m, (CHOP), 2H]; 1.34 [d, (CH
CHOP), JHH ¼ 6.35 Hz, 6H];
Aspect: yellow oil. yield: 64%. IR (NaCl): 3435.1 (st, NH); 3134.2
(st, CHaromatic); 2974.1, 2937.5 and 2879.6 (stass., CH and CH );
3 2
1
3
)
2

10
C.B.B. da Matta et al. / European Journal of Medicinal Chemistry 101 (2015) 1e12
1
577.7 (st, C]N); 1460.0 (
d
ass., CH
2
and CH
3
); 1392.6 (
d
sym., CH
3
);
H
absorbance was measured using an ELISA reader at 540 nm [22].
Each concentration was assayed in triplicate, and the correspond-
ing cell growth controls were used in each measurement. The trials
were also performed in duplicate.
1
1234.4 (st, P]O); 1105.2 (st, PeN); 1002.9 (st, PeOeC). NMR
(
DMSO): 10.00 [d, (PeNeH), JHP ¼ 29.20 Hz, 1H]; 8.35 [dd, (H6),
J
J
m
¼ 2.12 Hz, J
¼ 1.94 Hz, J
H]; 1.55 [m, (CH
H]; 1.20 [d, (CH
o
¼ 4.87 Hz, 1H]; 8.17 [s, (N]CH), 1H]; 8.06 [dd, (H4),
m
o
¼ 7.77 Hz, 1H]; 7.50 [m, (H5), 1H]; 4.35 [m, (CHOP),
2
3
2
CHOP), 2H]; 1.25 [d, (CH
3
CHOP), JHH ¼ 6.08 Hz,
7.3. Parasite culture
3
CHOP), JHH ¼ 6.08 Hz, 3H]; 0.87 [t, (CH
CH
CHOP), JHH ¼ 7.44 Hz, 3H]. NMR
C (DMSO): 150.36 [s, (C6), 1C]; 141.21 [s, (C2), 1C]; 140.02 [d, (N]
C), Jcp ¼ 18.22 Hz,1C]; 134.53 [s, (C4),1C]; 131.10 [s, (C3),1C]; 123.96
s, (C5), 1C]; 75.66 [m, (CHOP), 2C]; 29.84 [d, (CH CHOP),
3 2
CH CHOP),
J
HH ¼ 7.44 Hz, 3H]; 0.84 [t, (CH
3
2
L. amazonensis [MHOM/BR/87/BA125] and L. braziliensis [MHOM/
BR/01/BA788] strains were kindly provided by Val eꢀ ria Borges,
PhD (Fundaç a~ o Oswaldo Cruz e BA/Brazil) and were maintained
1
3
ꢂ
[
2
in vitro as proliferating promastigotes at 26 C in Schneider's insect
J
(
1
(
cp ¼ 4.19 Hz, 1C]; 29.73 [d, (CH
2
CHOP), Jcp ¼ 4.19 Hz, 1C]; 21.10 [d,
medium supplemented with 10% heat-inactivated fetal calf serum,
CH
3
CHOP), Jcp ¼ 3.60 Hz, 1C]; 20.83 [d, (CH
3
CHOP), Jcp ¼ 3.60 Hz,
gentamycin (1 mg/L),
urine.
L-glutamine (2 mM) and 2% sterile human
31
C]; 9,38 [d, (CH
DMSO): ꢀ0.46 [dt, (PeNeH),
3
CH
2
CHOP), Jcp ¼ 6.47 Hz, 2C]. NMR
P
J
HP
¼
29.45 Hz, (CHOP),
J
1
HP ¼ 8.12 Hz]; coupled. m/Z: 41(28%), 57(20%), 73(10%), 98(100%),
7.4. Leishmanicidal activity
03(6%), 120(7%), 154(4%), 185(5%), (392(<1%). HR-MS (ESI) calc. for
þ
þ
C
(
14
H
23BrN
3
O
3
P (M) : 391.066041, found: (M þ H) : 392.073317,
Leishmania parasites were harvested at a late exponential phase
þ
M þ Na) : 414.055262.
of growth, resuspended in fresh medium, counted in a Neubauer's
5
chamber and adjusted to a concentration of 10 parasites/well in
0
6
.15. Phosphorohydrazidic acid, N -[(1E)-(3,5-dichloro-4-pyridinyl)
the presence of the corresponding concentration of compounds in a
96-microwell plate. They were allowed to proliferate in the pres-
methylene]-disecbutyl ester (4o)
ꢂ
ence of the compounds at 26 C for 48 h for promastigotes. Then,
Aspect: yellow oil. yield: 64%. IR (NaCl): 3423.5 (st, NH); 3128.4
the parasites were counted using an automatic cell counter [34]. All
of the assays were performed in triplicate, and the experiments
were repeated at least twice. The inhibition caused by each com-
pound was expressed as a percentage relative to the control cells
(treated with the vehicle, DMSO). The LD50 and their standard
errors were calculated using GraphPad Prism 5.0 software.
(
st, CHaromatic); 2974.1 and 2929.7 (stass., CH
3
and CH
2
); 1600.8 (st,
); 1238.2 (st,
C]N); 1465.2 ( ass., CH and CH ); 1388.7 (
d
2
3
d
sym., CH
3
1
P]O); 1109.3 (st, PeN); 1004.8 (st, PeOeC). NMR H (DMSO): 10.16
[
(
[
J
[
d, (PeNeH), JHP ¼ 29.74 Hz, 1H]; 8.63 [s, (H2 and H6), 2H]; 8.13 [s,
N]CH), 1H]; 4.36 [m, (CHOP), 2H]; 1.56 [m, (CH CHOP), 4H]; 1.24
d, (CH CHOP), 6.07 Hz, 3H]; 1.20 [d, (CH CHOP),
CH
CHOP), JHH ¼ 7.29 Hz, 3H]; 0.83
CHOP), JHH ¼ 7.29 Hz, 3H]. NMR C (DMSO): 148.87 [s,
C2 andC6), 2C]; 137.64 [d, (N]C), Jcp ¼ 20.19 Hz, 1C]; 136.80 [s, (C3
2
3
J
HH
¼
3
HH ¼ 6.07 Hz, 3H]; 0.86 [t, (CH
3
2
7.5. Antiamastigote assay
13
3 2
t, (CH CH
(
In vitro model of the amastigotes, inflammatory macrophages
were obtained from BALB/c mice previously inoculated by the
intraperitoneal route with 3% thyoglicollate medium (Sigma).
andC5), 2C]; 130,33 [s, (C4), 1C]; 76.17 [m, (CHOP), 2C]; 30.30 [d,
CH CHOP), Jcp ¼ 5.62 Hz,
CHOP), Jcp ¼ 5.62 Hz, 1C]; 30.14 [d, (CH
C]; 21.41 [d, (CH CHOP),
cp ¼ 3.00 Hz, 1C]; 9.70 [s, (CH
DMSO): ꢀ1.54 [dt, (PeNeH),
(
1
J
(
J
2
for
3
2
2
5
3
CHOP), Jcp ¼ 3.00 Hz, 1C]; 21.30 [d, (CH
3
Briefly, peritoneal macrophages were plated at 4 ꢁ 10 cell/well on
31
3
CH
2
CHOP), 2C]. NMR
P
coverslips (Ø 13 cm) previously arranged in a 24-well plate in
DMEM medium supplemented with 10% (v/v) heat-inactivated FBS
and allowed to adhere overnight. Adherent macrophages were
infected with L. amazonensis promastigote using a ratio of 1:10 at
J
HP
¼
29.97 Hz, (CHOP),
HP ¼ 8.12 Hz]; Coupled. m/Z: 57(15%), 98(100%), 124(2%), 154(5%),
52(2%), 296(2%), 326(<1%), 352(<1%), 382(<1%). HR-MS (ESI) calc.
þ
þ
ꢂ
C
14
H22Cl
2
N
3
O
3
P
(M) : 381.077584, found: (M
82.084860, (M þ Na) : 404.066805.
þ
H) :
37 C for 4 h. Non-internalized promastigotes were eliminated, and
þ
solutions of tested compounds were added in different concen-
ꢂ
trations and maintained at 37 C in 5% CO
2
for 48 h. Slides were
7
. Biological evaluation
fixed with methanol, stained with Giemsa and intracellular amas-
tigotes were counted (two hundred macrophages were evaluated
per assay). The results were analyzed using GraphPad Prism 5.0
software and expressed as infection index (percentage of infected
macrophages multiplied by the average number of amastigotes per
macrophage). Miltefosine was used as reference drug.
7.1. Macrophage culture
The J774 cell line-derived murine macrophage cells were pro-
vided by the Rio de Janeiro Cell Bank-HUCFF-UFRJ. The cells were
grown in DMEM medium containing -glutamine (2 mM), buffered
L
with HEPES (10 mM), and supplemented with 10% heat-inactivated
7.6. In vivo infection in a murine model
fetal calf serum and gentamicin (1 mg/L) in a humidified incubator
ꢂ
(
5% CO
2
) at 37 C. The cells were passaged twice per week.
Female BALB/c mice, 6e8 weeks old, were obtained from the
Federal University of Ribeir a~ o Preto, Brazil. The animals were
7
.2. Cytotoxicity assay in macrophages
maintained under
a
12-h light/dark cycle in
a
controlled-
ꢂ
temperature room (22 ± 2 C) with free access to water and pel-
let food. All of the procedures were performed after the approval of
the protocol by the Ethics Committee - UFAL for animal handling
(No: 2013.02).
5
J774 macrophages were seeded (1.5 ꢁ 10 cells/well) in 96-well
flat-bottom microplates with 100
allowed to attach to the bottom of the well for 24 h at 37
mL of medium. The cells were
ꢂ
C
and then exposed to the different concentrations of compounds
100, 10 and 1 M) for 48 h. Afterwards, the cells were washed with
PBS and incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-
A murine model closely resembling human pathology was pre-
viously described [23]. Briefly, the right ear dermis of BALB/c mice
(
m
was inoculated with stationary-phase promastigotes of
5
diphenyltetrazolium bromide (MTT) (100
mL/well) for 1 h in the
L. amazonensis (MHOM/BR/87/BA125), with 10 parasites in 10
mL of
ꢂ
dark at 37 C. The MTT solution was removed, the cells were
sterile saline, using a 27.5-gauge needle. After the second week post-
resuspended in 100
mL of dimethylsulfoxide (DMSO), and the
infection, a group of mice (n ¼ 5) was treated daily (28 days) with

C.B.B. da Matta et al. / European Journal of Medicinal Chemistry 101 (2015) 1e12
11
4
m, 4n or miltefosine (positive control) by oral administration at
ChemScore [40,41], GoldScore [42], and ChemPLP [43], the latter is
claimed to be generally more effective than the other scoring
functions for both pose prediction and virtual screening. ChemPLP
is the default scoring function for GOLD 5.2 and was employed by
us for the docking procedure.
Hydrogen atoms were added to protein structures based on
ionization and tautomeric states defined by the program. Serine,
threonine and tyrosine hydroxyl groups and lysine amino groups
were free to rotate during the docking procedure to allow the
best orientation of hydrogen bonds involving these groups. The
number of genetic operations (crossover, migration, mutation) for
each run was set to 100,000 during the searching procedure. The
radius of the binding sites for the enzymes was set to 10 Å around
atoms selected after superposition with available, related, ligand-
containing crystallographic structures.
3
0 mg/kg/day, diluted in water for administration. A negative control
group was treated orally with water by injection (n ¼ 5). Lesion size,
which was defined as the difference in thickness (in millimeters)
between the infected ear and the non-infected contralateral ear,
was monitored twice weekly using a digital caliper.
7.7. Parasite load quantification
Parasite load was determined using a quantitative limiting
dilution assay as previously described [35]. Briefly, infected ears
and draining lymph nodes were aseptically excised after treatment
and homogenized in Schneider medium. The homogenates were
serially diluted in Schneider medium with 10% FCS and seeded into
9
6-well plates.
The data are reported as the average ± standard error of the
mean (M ± S.E.M) after statistical analysis employing one-way
ANOVA followed by Dunnett's test. Differences between means
were considered significant when p < 0.05 when compared to the
control group. Calculations were performed using GraphPad 5.0
software.
Acknowledgments
The authors would like to thank CNPq, FAPERJ, INCT-INOFAR,
and CAPES for fellowship and financial support.
Appendix A. Supplementary data
7.8. Molecular modeling
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.06.014.
According to the reasons presented above, we chose FPPS and
the enzymes of the glycolytic and pentose phosphate pathways as
possible targets in the molecular docking study. Glycolysis is a
process catalyzed by ten enzymes: hexokinase (HK), glucose-6-
phosphate isomerase (G6PI), phosphofructokinase (PFK), fructose
bisphosphate aldolase (FBPA), triose phosphate isomerase (TPI),
glyceraldehyde-3-phosphate dehydrogenase (GPDH), phospho-
glycerate kinase (PGK), phosphoglycerate mutase (PGM), enolase
ENO) and pyruvate kinase (PYK). The PPP is subdivided into two
branches e the oxidative branch, comprising the enzymes glucose-
-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase
6PGL) and 6-phosphogluconate dehydrogenase (6PGDH); and the
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