Synthesis, biological activity, and mechanism of action of new 2-pyrimidinyl hydrazone and N-acylhydrazone derivatives, a potent and new classes of antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2019.111742

In this work, we report the antileishmanial activity of 15 compounds based on 2-pyrimidinyl hydrazone and N-acylhydrazone derivatives, being 13 new compounds. All compounds were tested against promastigotes and Leishmania amazonensis-GFP amastigotes, as well as murine macrophages. Besides, studies about the mechanism of action of the best antileishmanial compounds and in silico physicochemical and pharmacokinetic properties were performed. Studies about the mechanism of action of representative compounds of each class showed slight differences in mode of action and both are able to cause mitochondrial depolarization and increase of intracellular ROS levels. Through computational tool and further analysis of the physicochemical and pharmacokinetic parameters, the results indicating good oral bioavailability. These results confirm the potential of 2-pyrimidinyl derivatives as lead compounds in antileishmanial drug discovery.

Full text of DOI:10.1016/j.ejmech.2019.111742

Journal Pre-proof
Synthesis, biological activity, and mechanism of action of new 2-pyrimidinyl
hydrazone and N-acylhydrazone derivatives, a potent and new classes of
antileishmanial agents
Elaine Soares Coimbra, Marcus Vinícius Nora de Souza, Mariana Sequetto Terror,
Alessandra Campbell Pinheiro, Juliana da Trindade Granato
PII:
S0223-5234(19)30894-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.111742
EJMECH 111742
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 August 2019
Revised Date: 12 September 2019
Accepted Date: 26 September 2019
Please cite this article as: E.S. Coimbra, Marcus.Viní. Nora de Souza, M.S. Terror, A.C. Pinheiro, J.
da Trindade Granato, Synthesis, biological activity, and mechanism of action of new 2-pyrimidinyl
hydrazone and N-acylhydrazone derivatives, a potent and new classes of antileishmanial agents,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111742.
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© 2019 Published by Elsevier Masson SAS.

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Synthesis, Biological Activity, and Mechanism of
Action of New 2-Pyrimidinyl Hydrazone and N-
acylhydrazone Derivatives, a Potent and New
Classes of Antileishmanial Agents
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Elaine Soares Coimbraa, Marcus Vinícius Nora de Souza*,b, Mariana Sequetto
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Terrora, Alessandra Campbell Pinheirob, Juliana da Trindade Granatoa
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aDepartamento de Parasitologia, Microbiologia e Imunologia, Instituto de
Ciências Biológicas, Universidade Federal de Juiz de Fora, 36036-900, Juiz de
Fora, Minas Gerais, Brazil.
bFundação Oswaldo Cruz, Instituto de Tecnologia em Fármacos-Far
Manguinhos, 21041-250, Rio de Janeiro, RJ, Brazil.
*Corresponding author
e-mail address: mvndesouza@gmail.com

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ABSTRACT
In this work, we report the antileishmanial activity of 15 compounds
based on 2-pyrimidinyl hydrazone and N-acylhydrazone derivatives, being 13
new compounds. All compounds were tested against promastigotes and L.
amazonensis-GFP amastigotes, as well as murine macrophages. Besides,
studies about the mechanism of action of the best antileishmanial compounds
and in silico physicochemical and pharmacokinetic properties were performed.
Studies about the mechanism of action of representative compounds of each
class showed slight differences in mode of action and both are able to cause
mitochondrial depolarization and increase of intracellular ROS levels. Through
computational tool and further analysis of the physicochemical and
pharmacokinetic parameters, the results indicating good oral bioavailability.
These results confirm the potential of 2-pyrimidinyl derivatives as lead
compounds in antileishmanial drug discovery.
Keywords: pyrimidinyl nucleus; hydrazone and N-acylhydazones; mechanism
of action; antileishmanial activity; mitochondrial depolarization.

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INTRODUCTION
According to the World Health Organization, leishmaniasis as one of
the world's most neglected diseases. It is estimated that approximately 700,000
to 1 million new cases and 26,000 to 65,000 deaths occur each year [1].
Leishmaniasis is distributed among tropical and subtropical countries
worldwide and has as etiological agent protozoan of the genus Leishmania,
which is transmitted during the bite of infected sandflies. Leishmaniasis can
manifest in different clinical forms: cutaneous, mucocutaneous, and visceral
leishmaniasis [2,3].
Chemotherapy is still an important ally in controlling leishmaniasis
despite the limited arsenal of drugs available for use. In some countries,
pentavalent antimonials are used as first-line drugs, but the treatment is
contraindicated for use in patients over 50 years old, patients with
cardiopathies, liver diseases, nephropathies and pregnant women [4]. In this
way, Amphotericin B and its liposomal formulations, Pentamidine, and
paromomycin are used as alternative treatment [5]. These drugs present high
toxicity and many adverse effects or high cost [6]. Miltefosine, developed
initially as an antitumor drug, is the only oral drug used to leishmaniasis
treatment, but there have been common reports problems related to treatment
failure due to multifactorial characteristics [7]. Also, this drug is associated
with gastrointestinal problems and teratogenicity [8].
Pyrimidines are known for the richness of biological potentials. These
molecules are heterocyclic compounds formed by two nitrogen atoms replacing
two carbon atoms in the benzene ring. Compounds containing this functional
group have been widely reported, among other activities, as antitumoral [9,10],
antibacterial [11], antiplasmodial [12] and antileishmanial agents [13,14]. In
this study, we used pyrimidines coupled with hydrazones, another functional
group characterized by the structure R1-C=N-NH-R2, where R2 and R1 may
be conjugated to distinct functional groups conferring different functionalities
to the molecule. This chemical group has aroused the great interest of the
scientific community since they have potential chemical, therapeutic,
biological, and industrial properties [15]. Concerning biological properties,
recent studies involving derived from this class of compounds have shown
several important activities, such as anti-inflammatory [16], antituberculosis

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[17,18], antitrypanosomal [19], antiplasmodial [20], and antileishmanial effects
[21,22].
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In the search for new drugs with promising antileishmanial activity, our
studies focused on designing and synthesizing new pyrimidinylhydrazone and
pyrimidinyl-N-acylhydrazone derivatives, in order to evaluate the potential for
in vitro antileishmanial activity and elucidate its mechanism of action.
Recently, much attention has been done to hydrazones and acyl
hydrazones formation with different nucleus against the disease Leishmaniasis.
In this context, López and co-workers synthesized 4-chloro-1-phthalazinyl
hydrazones derivatives against Leishmania braziliensis [23], and Álvarez and
co-workers developed a multi-anti-parasitic activity based on thiazolidene
hydrazones derivatives [24]. Another work is the preparation of acyl hydrazone
derivatives of thiochroman-4-ones made by Quinõnes and co-workers [25] and
finally, our research group also investigated 2-pyrazyl and pyridyl hydrazones
derivatives against L. amazonensis forms [26].
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Besides, in silico studies addressing its physicochemical and
pharmacokinetic properties were conducted.
MATERIAL AND METHODS
Chemistry
General Procedures
Melting points were determined using an MQAPF-302 (MicroQuímica
Ltd, Santa Catarina, Brazil) instrument and are uncorrected. NMR spectra were
obtained using 400 or 500 MHz Bruker AC spectrometers with
tetramethylsilane as an internal standard. Infrared spectra were obtained using
a Thermo Nicolet 6700 spectrometer. High-resolution mass spectra were
acquired on a Bruker compact-Tof. The progress of the reactions was
monitored by thin-layer chromatography (TLC) on 2.0 cm x 6.0 cm aluminum
sheets (silica gel 60, HF-254, Merck) with a thickness of 0.25 mm, using
ultraviolet light irradiation. For column chromatography, Merck silica gel
(70e230 or 230e400 mesh) was used. Solvents and reagents were used without
further purification.
General Procedure for the synthesis of 2-pyrimidinyl-hydrazone derivatives 2

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Compounds
2
were prepared by the reaction between 2-
hydrazinylpyrimidine 1 (0.04 g, 0.36 mmol) and the appropriate aromatic or
heteroaromatic aldehyde (1.0 eq., 0.36 mmol) in ethanol (3.0 mL) [27]. The
reaction mixture was stirred for between 20 minutes to 5 hours at room
temperature. After rotary evaporation, the product was purified by washing
with cold ethanol (2.0 mL) and cold diethyl ether (2.0 mL), leading to the pure
derivatives 2a-f as solid in 27-75% yields.
Synthesis of pyrimidine-2-carbohydrazide 5
Thionyl chloride (1.49 mL, 20.0 mmol) was slowly added to methanol
(20.0 mL) at 0°C under nitrogen and stirred for 30 minutes. Pyrimidine-2-
carboxylic acid 3 (0.25 g, 2.0 mmol) was added and the mixture was stirred at
room temperature for 24 hours, concentrated under reduced pressure, and the
residue was neutralized with a saturated aqueous solution of sodium
bicarbonate [28]. The layers were separated and the aqueous phase was
extracted with dichloromethane (3 x 15 mL). The organic phases were
combined, dried with anhydrous magnesium sulfate and then concentrated
under vacuum to yield 4 in 62 %. The crude product was used in the next step
without further purification.
To a solution of methyl pyrimidine-2-carboxylate 4 (0.275 mg, 1.99
mmol) in ethanol (3.0 mL) was added an aqueous solution of hydrazine hydrate
(0.9 eq., 1.8 mmol, 0,16 mL, aqueous solution 55%). After stirring for 3 hours
at room temperature, the resulting mixture was concentrated under reduced
pressure and the residue was purified by washing with cold ethanol (2 x 3.0
mL) and diethyl ether (2 x 3.0 mL), leading to the compound 5 as pure white
solid in 98%.
General Procedure for the synthesis of 2-pyrimidinyl-N-acylhydrazone
derivatives 6
The desired compounds 6 were prepared by reaction between
pyrimidine-2-carbohydrazide 5 (0.05 g, 0.36 mmol) and the appropriate
aromatic or heteroaromatic aldehyde (1.0 eq., 0.36 mmol) in ethanol (3.0 mL)

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[28]. The reaction mixture was stirred for 20 minutes to 24 hours at room
temperature. The product was purified by washing with cold diethyl ether (3.0
mL), leading to the pure derivatives 6a-i as solid in 40-88% yields.
(E)-2-((2-(pyrimidin-2-yl)hydrazono)methyl)phenol (2a)
m.p.: 212-214°C (m.p. lit.: 213-2170C, [29]). IR (cm-1, KBr) νmax 3051
1
(OH), 1576 (C=N). H-NMR (400MHz, DMSO-d6) δ (ppm): 11.64 (1H, s,
OH); 11.53 (1H, s, NH); 8.51 (2H, d, J= 4.8, H2’ and H4’); 8.31 (1H, s,
N=CH); 7.40 (1H, dd, J= 8.2 and J= 1.6, H5); 7.24 (1H, dt, J= 7.8 and J= 1.6,
H3); 6.89 (3H, m, H2, H4 and H3’) ppm. 13C-NMR (100 MHz, DMSO-d6)
δ(ppm): 159.2; 158.4; 158.4; 156.9; 142.9; 130.0; 129.2; 119.0; 118.9; 116.2;
113.3 ppm. HR-MS (ESI): Calcd for C11H11N4O+ (M+H+): 215.0927; Found:
215.0955.
(E)-3-((2-(pyrimidin-2-yl)hydrazono)methyl)benzene-1,2-diol (2b)
m.p.: 198-2000C. IR (cm-1, KBr) νmax 3278 (OH); 1571 (C=N). 1H-
NMR (400MHz, DMSO-d6) δ (ppm): 11.72 (1H, s, OH); 11.55 (1H, s, NH);
9.02 (1H, s, OH); 8.51 (2H, d, J= 4.8, H2’ and H4’); 8.28 (1H, s, N=CH); 6.89
(1H, t, J= 4.8, H3’); 6.83 (1H, dd, J= 7.7 and J= 1.6, H5); 6.79 (1H, dd, J= 7.7
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and J= 1.6, H3); 6.71 (1H, t, J= 7.7, H4) ppm. C-NMR (100 MHz, DMSO-
d6) δ(ppm): 159.2; 158.4; 158.4; 145.4; 145.3; 143.3; 119.6; 118.9; 118.8;
116.3; 113.2 ppm. HR-MS (ESI): Calcd for C11H10N4O2 (M + H+): 231.0877;
Found: 231.0874.
(E)-2-((2-(pyrimidin-2-yl)hydrazono)methyl)benzene-1,4-diol (2c)
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m.p.: 252-2540C. IR (cm-1, KBr) νmax 3274 (OH); 1577 (C=N). H-
NMR (400MHz, DMSO-d6) δ (ppm): 11.42 (1H, s, NH); 10.76 (1H, s, OH);
8.92 (1H, s, OH); 8.49 (2H, d, J= 4.8, H2’ and H4’); 8.23 (1H, s, N=CH); 6.87
(1H, t, J= 4.8, H3’); 6.82 (1H, d, J= 2.8, H5); 6.72 (1H, d, J= 8.7, H2); 6.66
(1H, dd, J= 8.7 and J= 2.8, H3) ppm. 13C-NMR (100 MHz, DMSO-d6)
δ(ppm): 159.3; 158.4; 158.4; 149.6; 149.6; 142.5; 119.1; 117.4; 116.7; 114.0;
+
113.1 ppm. HR-MS (ESI): Calcd for C11H10N4NaO2 (M + Na+): 253.0696;
Found: 253.0684.
(E)-5-methyl-2-((2-(pyrimidin-2-yl)hydrazono)methyl)phenol (2d)
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m.p. 249-2500C; IR (cm-1, KBr) νmax 3045 (OH); 1575 (C=N). H-NMR
(400MHz, DMSO-d6) δ (ppm): 11.62 (1H, s, OH); 11.46 (1H, s, NH); 8.50

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(2H, d, J= 4.8, H2’ and H4’); 8.27 (1H, s, N=CH); 7.26 (1H, d, J= 7.6, H5);
6.87 (1H, t, J= 4.8, H3’); 6.73 (1H, s, H2); 6.71 (1H, d, J= 7.6, H4); 2.28 (3H,
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s, CH3) ppm. C-NMR (100 MHz, DMSO-d6) δ(ppm): 159.3; 158.4; 158.4;
156.9; 143.2; 140.1; 129.2; 120.0; 116.6; 116.3; 113.1; 21.0 ppm. HR-MS
(ESI): Calcd for C12H12N4O (M + H+): 229.1084; Found: 229.1079
(E)-5-nitro-2-((2-(pyrimidin-2-yl)hydrazono)methyl)phenol (2e)
m.p.: 300-3030C. IR (cm-1, KBr) νmax 2968 (OH); 1576 (C=N); 1565 and
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1338 (NO2). H-NMR (400MHz, DMSO-d6) δ (ppm): 12.59 (1H, s, OH);
11.76 (1H, s, NH); 8.53 (2H, d, J= 4.8, H2’ and H4’); 8.49 (1H, d, J= 2.9, H2);
8.43 (1H, s, N=CH); 8.11 (1H, dd, J= 9.0 and J= 2.9, H4); 7.08 (1H, d, J= 9.0,
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H5); 6.93 (1H, t, J= 4.8, H3’) ppm. C-NMR (100 MHz, DMSO-d6) δ(ppm):
162.1; 159.2; 158.5; 158.5; 139.8; 139.2; 125.3; 123.8; 120.1; 116.9; 113.7
+
ppm. HR-MS (ESI): Calcd for C11H10N5O3 (M + H+): 260.0778; Found:
260.0802.
(E)-2-(2-(pyridin-2-ylmethylene)hydrazinyl)pyrimidine (2f)
m.p.: 209-2110C (MP lit.: 208-2090C, [30]) IR (cm-1, KBr) νmax 1569 (C=N).
1H-NMR (400MHz, DMSO-d6) δ (ppm): 11.51 (1H, s, NH); 8.56 (1H, dd, J=
4.8 and J=1.4, H2); 8.51 (2H, d, J= 4.8, H2’ and H4’); 8.20 (1H, s, N=CH);
7.93 (1H, d, J= 7.8, H5); 7.82 (1H, dt, J= 7.8 and J= 1.4, H4); 7.34 (1H, m,
H3), 6.90 (1H, t, J= 4.8, H3’) ppm. 13C-NMR (100 MHz, DMSO-d6) δ(ppm):
159.6; 158.3; 158.3; 154.0; 149.2; 142.0; 136.5; 123.2; 119.1; 113.5 ppm. HR-
MS (ESI): Calcd for C10H10N5 (M + H+): 200.0931; Found: 200.0939.
+
(E)-N’-(2-hydroxybenzylidene)pyrimidine-2-carbohydrazide (6a)
m.p. :200-2020C. IR (cm-1, KBr) νmax 3312 (OH); 1685 (CO); 1567 (C=N).
1H-NMR (400MHz, DMSO-d6) δ (ppm): 12.52 (1H, s, NH); 11.26 (1H, s,
OH); 9.03 (2H, d, J= 4.8, H2’ and H4’); 8.82 (1H, s, N=CH); 7.75 (1H, t, J=
4.8, H3’); 7.70 (1H, m, H3); 7.52 (1H, d, J= 7,5, H5); 7.32 (1H, d, J= 7.5, H4);
6.94 (1H, d, H2) ppm. 13C-NMR (100 MHz, DMSO-d6) δ(ppm): 166.8; 158.7;
157.8; 157.6; 157.3; 150.0; 131.6; 128.6; 123.4; 119.3; 118.5; 116.4 ppm. HR-
MS (ESI): Calcd for C12H10N4O2 (M + Na+): 265.0701; Found: 265.0699.
(E)-N’-(2,3-dihydroxybenzylidene)pyrimidine-2-carbohydrazide (6b)
m.p..: 233-2360C. IR (cm-1, KBr) νmax 3235 (OH); 1694 (CO); 1568
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(C=N). H-NMR (500MHz, DMSO-d6) δ (ppm): 12.61 (1H, s, NH); 11.18
(1H, s, OH); 9.24 (1H, s, OH); 9.04 (2H, d, J= 4.8, H2’ and H4’); 8.78 (1H, s,

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N=CH); 7.76 (1H, t, J= 4.8, H3’); 6.94 (1H, d, J= 7.6, H5); 6.88 (1H, d, J= 7.6,
H3); 6.76 (1H, t, J= 7.6, H4) ppm. 13C-NMR (125 MHz, DMSO-d6) δ(ppm):
158.7; 157.8; 157.8; 157.2; 150.6; 146.2; 145.5; 123.4; 120.2; 119.1; 118.6;
117.5 ppm. HR-MS (ESI): Calcd for C12H10N4O3 (M + Na+): 281.0651; Found:
281.0656.
(E)-N’-(2,4-dihydroxybenzylidene)pyrimidine-2-carbohydrazide (6c)
m.p.: 176-1790C. IR (cm-1, KBr) νmax 3126 (OH); 1670 (CO); 1562
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(C=N). H-NMR (500MHz, DMSO-d6) δ (ppm): 12.40 (1H, s, NH); 11.48
(1H, s, OH); 10.02 (1H, s, OH); 9.02 (2H, d, J= 4.8, H2’ and H4’); 8.68 (1H, s,
N=CH); 7.75 (1H, t, J= 4.8, H3’); 7.28 (1H, d, J= 8.4, H5); 6.37 (1H, dd, J= 8.4
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and J=2.0, H4); 6.33 (1H, d, J= 2.0, H2) ppm. C-NMR (125 MHz, DMSO-
d6) δ(ppm): 160.8; 159.6; 158.3; 157.7; 157.7; 157.4; 150.8; 131.5; 123.3;
110.4; 107.7; 102.6 ppm. HR-MS (ESI): Calcd for C12H10N4O3 (M + Na+):
281.0651; Found: 281.0642.
(E)-N’-(2,5-dihydroxybenzylidene)pyrimidine-2-carbohydrazide (6d)
m.p.: 232-2340C. IR (cm-1, KBr) νmax 3268 (OH); 1689 (CO); 1566
1
(C=N). H-NMR (400MHz, DMSO-d6) δ (ppm): 12.46 (1H, s, NH); 10.39
(1H, s, OH); 9.03 (2H, d, J= 4.8, H2’ and H4’); 9.00 (1H, s, OH); 8.74 (1H, s,
N=CH); 7.75 (1H, t, J= 4.8, H3’); 6.96 (1H, d, J= 1.8, H5); 6.76 (2H, m, H2
13
and H3) ppm. C-NMR (100 MHz, DMSO-d6) δ(ppm): 158.7; 157.9; 157.9;
157.5; 150.4; 149.9; 149.4; 123.4; 119.2; 118.9; 117.2; 114.0 ppm HR-MS
(ESI): Calcd for C12H10N4NaO3 (M + Na+): 281.0651; Found: 281.0632.
(E)-N’-(2-hydroxy-4-methylbenzylidene)pyrimidine-2-carbohydrazide (6e)
m.p.: 201-2030C; IR (cm-1, KBr) νmax 3374 (OH); 1703 (CO); 1563 (C=N).
1H-NMR (500MHz, DMSO-d6) δ (ppm): 12.54 (1H, s, NH); 11.28 (1H, s,
OH); 9.04 (2H, d, J= 4.8, H2’ and H4’); 8.77 (1H, s, N=CH); 7.76 (1H, t, J=
4.8, H3’); 7.38 (1H, d, J= 7.8, H5); 6.78 (1H, s, H2); 6.76 (1H, d, J= 7.8, H4);
2.30 (3H, s, CH3) ppm. 13C-NMR (125 MHz, DMSO-d6) δ(ppm): 158.6;
157.8; 157.8; 157.5; 157.3; 150.2; 141.9; 129.7; 123.4; 120.4; 116.8; 115.9;
21.1 ppm. HR-MS (ESI): Calcd for C13H12N4O2 (M + Na+): 279.0858; Found:
279.0863
(E)-N’-(2-hydroxy-5-nitrobenzylidene)pyrimidine-2-carbohydrazide (6f)
m.p.: 284-2860C. IR (cm-1, KBr) νmax 3258 (OH); 1701 (CO); 1568 (C=N);
1
1560 and 1333 (NO2). H-NMR (500MHz, DMSO-d6) δ (ppm): 12.74 (1H, s,

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NH); 12.22 (1H, s, OH); 9.04 (2H, d, J= 4.7, H2’ and H4’); 8.92 (1H, s,
N=CH); 8.57 (1H, d, J= 2.6, H5); 8.19 (1H, dd, J= 9.0 and J= 2.6, H3); 7.77
(1H, t, J= 4.7, H3’); 7.12 (1H, d, J= 9.0, H2) ppm.13C-NMR (125 MHz,
DMSO-d6) δ(ppm): 163.2; 159.6; 158.4; 158.4; 158.0; 146.4; 140.4; 127.3;
124.2; 124.0; 120.5; 117.7 ppm. HR-MS (ESI): Calcd for C12H9N5O4
(M + Na+): 310.0552; Found: 310.0551
(E)-N’-(pyridin-2-ylmethylene)pyrimidine-2-carbohydrazide (6g)
m.p.: 143-1440C. IR (cm-1, KBr) νmax 3258 (OH); 1701 (CO); 1568 (C=N);
1
1560 and 1333 (NO2). H-NMR (400MHz, DMSO-d6) δ (ppm): 12.58 (1H, s,
NH); 9.05 (2H, d, J= 4.9, H2’ and H4’); 8.66 (1H, s, N=CH); 8.63 (1H, dd, J=
4.8 and J=1.5, H2); 8.02 (1H, dd, J= 7.6 and J= 1.1, H5); 7.90 (1H, dt, J= 7.6
and J= 1.5, H4); 7.7 (1H, t, J= 4.9, H3’); 7.44 (1H, ddd, J= 7.6, J= 4.8 and J=
13
1.1, H3) ppm. C-NMR (100 MHz, DMSO-d6) δ(ppm): 161.4; 158.1; 157.7;
157.4; 153.1; 149.5; 149.4; 136.8; 124.5; 123.4; 120.0 ppm. HR-MS (ESI):
Calcd for C11H9N5O (M + Na+): 250.0705; Found: 250.0694
(E)-N’-((5-nitrofuran-2-yl)methylene)pyrimidine-2-carbohydrazide (6h)
m.p..: 252-2540C. IR (cm-1, KBr) νmax 1690 (CO); 1560 (C=N); 1550 and
1355 (NO2). 1H-NMR (500MHz, DMSO-d6) δ (ppm): 12.76 (1H, s, NH); 9.04
(2H, d, J= 4.8, H2’ and H4’); 8.60 (1H, s, N=CH); 7.81 (1H, d, J= 3.8, H3);
7.78 (1H, t, J= 4.8, H3’) 7.30 (1H, d, J= 3.8, H4); ppm. 13C-NMR (125 MHz,
DMSO-d6) δ(ppm): 159.2; 158.0; 158.0; 157.1; 152.0; 151.6; 137.0; 123.7;
115.4; 114.6 ppm. HR-MS (ESI): Calcd for C10H7N5O4 (M + Na+): 284.0396;
Found: 284.0383
(E)-N’-((5-nitrothiophen-2-yl)methylene)pyrimidine-2-carbohydrazide (6i)
m.p.: 220-2220C. IR (cm-1, KBr) νmax 1702 (CO); 1570 (C=N); 1560 and
1332 (NO2). 1H-NMR (500MHz, DMSO-d6) δ (ppm): 12.71 (1H, s, NH); 9.05
(2H, d, J= 4.8, H2’ and H4’); 8.83 (1H, s, N=CH); 8.14 (1H, d, J= 4.3, H3);
7.77 (1H, t, J= 4.8, H3’); 7.57 (1H, d, J= 4.3, H4) ppm. 13C-NMR (125 MHz,
DMSO-d6) δ(ppm): 159.0; 157.8; 157.8; 157.1; 151.0; 146.3; 142.5; 130.4;
129.9; 123.5 ppm. HR-MS (ESI): Calcd for C10H7N5O3S (M + Na+): 300.0167;
Found: 300.0169
Biological assays
Parasites

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Leishmania amazonensis wild-type - WT (IFLA/BR/67/PH8) and L.
amazonensis (WHOM/BR/75/Josefa) transfected with the green fluorescent
protein (GFP) gene were used. Leishmania amazonensis-GFP promastigotes
were provided by Dr. Bartira Rossi-Bergman, Federal University of Rio de
Janeiro (Brazil). Cultures were maintained in BHI (brain and heart infusion)
medium supplemented with bovine hemin (0.1mg/mL), folic acid (0.01mg/mL)
and 10% fetal bovine serum (FBS), in a BOD incubator at 25 °C.
Effect of the compounds on L. amazonensis promastigotes
The activity of the compounds was evaluated by the MTT (Sigma-
Aldrich, St. Louis, MO, USA), which consists of reducing the tetrazolium salt
in formazan crystals by the cellular dehydrogenases and reductases [31]. Thus,
L. amazonensis (IFLA/BR/67/PH8) promastigotes (2 x 106 cells/mL) were
plated in 96-well plates and incubated in the absence or presence of varying
concentrations of the compounds (1.56 - 100 µM) for 72 h at 25 °C. Then, 10µl
of MTT (5mg/ml) was added to each well, and after 4h, the plates were read in
a spectrophotometer (Multiscan MS, LabSystem Oy, Helsinki, Finland) at 540
nm. The compounds activity was expressed as IC50 (inhibitory concentration of
50% of cell growth). Miltefosine (1.56 - 100 µM) was used as reference drug.
The IC50 values were calculated by the average of three independent
experiments performed in duplicates.
Cytotoxicity in peritoneal macrophages
The cytotoxicity of the compounds was determined using murine
macrophages obtained from BALB/c mice previously stimulated with 3% of
sodium thioglycollate for 72 h. After, the cells were adjusted to a concentration
of 2x106/mL and seeded into 96-well plates in RPMI-1640 medium
supplemented with 10% FBS, for 72 h at 33ºC and 5% CO2. After 24h, the
macrophages were washed and treated with the compounds (0.58-150 µM) for
72 h at 33°C. The cytotoxicity of the compounds was reported as CC50
(cytotoxic concentration for 50% of cells). Miltefosine (0.58-150 µM) was
used as reference drug and CC50 values were calculated by the average of three
independent experiments performed in duplicates.

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Effect on L. amazonensis- infected macrophages
The activity of the compounds against intracellular amastigotes was
evaluated by fluorimetry technique using a strain of L. amazonensis- GFP [28].
In brief: murine peritoneal macrophages were collected as described above,
seeded in 24-well plates, infected with L. amazonensis-GFP promastigotes
o
(MOI 20:1 for 4 h) in the stationary growth phase at 33 C in 5% CO2. After
washing, infected macrophages were treated with varying concentrations of the
compounds (0-100 µM) for 72h at 33 °C. Then, the supernatant was discarded,
the cells were lysed with distilled water and the contents of the wells were
transferred to 96-well plates and read in spectrofluorimeter at 485 and 528 nm
excitation and emission, respectively. Miltefosine was used as the reference
drug, and the IC50 values were calculated by the average of three independent
experiments performed in duplicates.
Mitochondrial membrane potential (∆ᴪm)
Aiming to evaluate if the compounds alter the mitochondrial
membrane potential, two fluorescent probes, Mitotracker® Red CM-H2XROS
(Molecular Probes- Eugene, OR, USA) and JC-1 (Sigma-Aldrich, St. Louis,
MO, USA) were used. The Mitotracke (Mtk) diffuses passively through the
plasma membrane of the cell and accumulates in the active mitochondria of the
parasite. The JC-1 probe has dual emission and its accumulation in the
mitochondria is dependent on that of the ∆ᴪm. At low ∆ᴪm, JC-1is a monomer
and emits green fluorescence. At increased potentials, the JC-1 forms J-
aggregates and emits red fluorescence. The mitochondrial membrane potential
is given by the red/green fluorescence ratio. Therefore, promastigotes of L.
amazonensis-WT were incubated without or with 2f (9.66 and 19.32 µM) and
6h (24.28 and 48.56 µM) for 24 h at 25ºC. Then the cells (5 x 106) were
washed, and incubated with Mtk (500nM) or JC-1 (5ug/ml) for 40 and 30
minutes, respectively. After incubation, the cells were washed three times, and
the reading performed by spectrofluorimeter (FLx800) at 540/600 nm
excitation and emission to Mtk. For JC-1, the wavelengths of 485/600nm for J-
aggregate and 485/528nm for monomers were used. Carbonyl cyanide-4-
(trifluoromethoxy)phenylhydrazone (FCCP) at 10 µM was used as a positive
control.

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Production of reactive oxygen species (ROS)
The levels of intracellular ROS in L. amazonensis-WT promastigotes
were evaluated using the non-fluorescent substance
2
', 7'-
dichlorodihydrofluorescein diacetate (H2DCFDA) [32]. The substance crosses
the plasma membrane of the cells and is oxidized by ROS produced, thereby
emitting fluorescence. Therefore, promastigotes were incubated or not with 2f
(9.66 and 19.32 µM) and 6h (24.28 and 48.56 µM) for 24 h at 25 ºC. The cells
were washed and adjusted to a concentration of 1.107 and incubated with 4 µl
ofH2DCFDA (1mM) for 30 minutes at room temperature in the dark. The
plates were read fluorimeter at 485/528 nm excitation and emission,
respectively. Miltefosine (44µM) was used as a positive control.
Analysis of the plasma membrane integrity after treatment
In order to clarify the mode of action of the compounds, and based on
the biological activity, the compounds 2f and 6h were selected to continue the
studies. Then, promastigotes of L. amazonensis-WT (1 x 107 cells/mL) were
not or incubated with 9.66 and 19.32 µM of 2f and 24.28 and 48.56 µM of 6h
(concentrations corresponding to 1 and 2 times the IC50 value in promastigote
forms) for 24 h at 25ºC. Then, the cells were washed in PBS, adjusted to a
concentration of 5 x 106 cells, labeled with propidium iodide (PI), and after 15
minutes incubation with the fluorescent probe, samples were read in
spectrofluorimeter at 540/600 nm excitation and respectively. Heated parasites
(60 ºC) were used as a positive control.
In silico analyses
Aiming to approach the physicochemical and pharmacokinetic
properties of the most relevant compounds, analysis using computational tools
were done. Therefore, the ADMET properties and molecular descriptors
covering the Lipinski's "Rules-of-five," including molecular weight (MW),
octanol/water partition coefficient (LogP), number of hydrogen bond donor
(HBD) and hydrogen bond acceptor (HBA) we verified. The chemical
structures of the compounds were designed by using the ACD/ChemSketch
Freeware software 2016.2.2, and below, the SMILE notation was generated

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and
inserted
in
the
free
online
platform
http://biosig.unimelb.edu.au/pkcsm/prediction.
Results
Synthesis and Characterization
The syntheses of the 2-pyrimidinyl-hydrazone, 2, and 2-pyrimidinyl-N-
acylhydrazone, 6, derivatives were obtained as shown in Schemes 1 and 2,
respectively, in moderate to high yields, see Tables 1 and 2.
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Scheme 1. Synthesis of 2-pyrimidinyl-hydrazone derivatives 2a-f
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Scheme 2. Synthesis of 2-pyrimidinyl-N-acylhydrazone derivatives 6a-i. Reagents and
conditions: (a) MeOH, SOCl2, rt, 24h; (b) NH2NH2.H2O (80%), EtOH, rt, 3h (c) EtOH,
ArCHO/HetCHO, rt, 20 min-24h.
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Table 1. Studied 2-pyrimidinyl-hydrazone derivatives, 2a-f, their m.p.s and
synthetic yields
Compound Substituent(s) in the
phenyl (aryl) ring
Melting point Yield (%)
(0C)
2a
2b
2c
2d
2e
2f
2-OH
2,3-diOH
2,5-diOH
2-OH, 4-CH3
2-OH, 5-NO2
2-Pya
212-214b
198-200
252-254
249-250
300-303
214-216c
63
47
69
27
75
52
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419
a2-py: aryl group = pyrindin-2-yl; b[29]; c[30]

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As can be seen in Tables 1 and 2, 2-hydroxybenzaldehydes and 5-
nitrofuran-2-carbaldehyde and - 5-nitrothien-2-carbaldehyde were used in the
syntheses. The selected choice of these aldehydes was based on the high
activities found for hydrazone and acylhydrazone heteroaromatic compounds,
built from these aldehydes [33-38].
Table 2. Studied 2-pyrimidinyl-N-acylhydrazone derivatives, 6a-i, their m.p.s,
and synthetic yields
Compound
Substituent(s) in
the phenyl (aryl)
ring
Melting point
(0C)
Yield
(%)
2-OH
200-202
233-236
276-279
232-234
201-203
284-286
143-144
252-254
220-222
64
85
88
69
71
83
43
55
40
6a
6b
6c
6d
6e
6f
2,3-diOH
2,4-diOH
2,5-diOH
2-OH, 4-CH3
2-OH, 5-NO2
2-Pya
5-NO2-Furb
5-NO2-Thioc
6g
6h
6i
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b
c
a2-py: aryl group = pyrindin-2-yl; 5-NO2-Fur: aryl group = 5-nitrofuran-2-yl; 5-NO2-Thio:
aryl group = 5-nitrothien-2-yl
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All compounds, including the novel substances 2b-e and 6a-i were
1
generally identified by H and 13C NMR and IR spectroscopy, and mass
1
spectrometry. In general, for compounds 2, the H NMR spectra showed the
three pyrimidine protons within the range of 6.83-8.54, the N=CH hydrazone
proton in the range from 8.09 to 8.43 and NH proton at 11.29-11.82 ppm.
Compounds 6 exhibited pyrimidine protons at 7.70-9.05, N=CH proton at 8.60-
8.92 and NH proton at 12.40-12.76 ppm. In the 13C NMR spectra, for
13
compounds 2, δ(C2) was observed in the range 159.2-162.1, and an IR band

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at approximately 1585 cm-1 relative to N=CH functional group was observed in
both series of compounds.
Antileishmanial effect on L. amazonensis and cytotoxicity on peritoneal
macrophages
The compounds were initially tested on L. amazonensis
promastigotes, and the cellular viability was measured by the MTT technique
after 72h of treatment. After, all compounds were assayed against the
intracellular amastigotes. In general, the 2-pyrimidinyl-hydrazone derivatives
(Table 3) showed more activity against L. amazonensis than the 2-pyrimidinyl-
N-acylhydrazone derivatives (Table 4).
As shown in Table 3, all 2-pyrimidinyl-hydrazone derivatives were
active against the promastigote forms of the parasite. Among the compounds,
the most actives are 2b (IC50 7.95 µM), 2f (IC50 9.66 µM) and 2e (IC50 10.52
µM), exhibiting IC50 values lower than the miltefosine (IC50 22.0 µM), used as
a reference drug. Regarding, amastigotes of L. amazonensis (Table 3), the
trend was like to the observed against promastigote forms, since also all
compounds showed activity against the intracellular stage (IC50 values ranging
from 4.07 µM to 57.94 µM). Among this series, the most active are 2f (IC50
4.07 µM), 2e (IC50 10.05 µM) and 2b (IC50 14.51 µM), being the compound 2f
as active as miltefosine (IC50 4.05 µM). The majority of 2-pyrimidinyl-
hydrazone derivatives showed negligible toxicity on macrophages (Table 3),
and only the best antileishmanial compounds (2e and 2f) exhibit a mild toxic
effect on macrophages (CC50 23.75 and 24.6 µM, respectively). However, these
derivatives were more destructive to the intracellular parasites than the host
cell, with an SI of 2.36 and 6.04, respectively.
Regarding 2-pyrimidinyl-N-acylhydrazone derivatives (Table 4), the
most of compounds showed low activity against promastigotes of L.
amazonensis, with IC50 values ranging from 24.28 µM to 90.00 µM. Against
the intracellular stage, only the compounds 6i and 6h showed significant
activity (IC50 of 8.41 µM and 10.08 µM, respectively). Concerning the effect of
the compounds on mammalian cells (Table 4), the majority of the compounds
did not show a toxic effect on macrophages or showed negligible toxicity
against these cells, except the compound 6h (CC50 of 29.05 µM).

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Table 3. Biological activities of 2-pyrimidinyl-hydrazone derivatives (2a-f).
Compound
Antileishmanial activity
IC50 (µM)a
Peritoneal
Macrophages
CC50 (µM)b
Selectivity
index c
Promastigotes
Amastigotes
28.12 + 2.96
7.95 + 1.1
57.94 + 2.8
14.51 + 0.42
45.68 + 1.50
52.06 + 1.6
10.05 + 0.24
4.07 + 0.62
4.05 + 1.79
84.15 + 5.22
>150
1.45
>10.33
1.95
2a
2b
27.47 + 0.32
55.98 + 4.68
10.52 + 1.59
9.66 + 0.20
22.0 + 0.5
89.41 + 2.29
54.75 + 0.99
23.75 + 2.44
24.60 + 3.43
131.9 + 3.9
2c
1.05
2d
2.36
2e
2f
6.04
Miltefosined
32.56
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495
b
aValues of inhibitory concentration of 50% of parasites (IC50); IC50 + Std. Error. Values of
c
citotoxicity concentration of 50% of macrophages (CC50); IC50 + Std. Error. SI = selectivity
index (CC50/IC50). dMiltefosine was used as reference drug.
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Table 4. Biological activities of 2-pyrimidinyl-N-acylhydrazone derivatives
(6a-i)
Compound
Antileishmanial activity
IC50 (µM)a
Peritoneal
Macrophages
CC50 (µM)b
>150
Selectivity
index
Promastigotes
Amastigotes
>100
90.0 + 19.71
27.42 + 0.95
>100
>1.5
>4.42
>1.5
6a
33.91 + 0.81
>100
>150
6b
>150
6c
39.26 + 2.02
52.02 + 3.97
28.96 + 0.99
>100
73.87 + 1.56
35.78 + 1.46
69.1 + 6.05
>100
>150
>2.03
>4.19
0.91
6d
>150
6e
63.43 + 0.91
>150
6f
>1.5
6g
24.28 + 0.9
>100
10.8 + 0.63
8.41 + 0.45
4.05 + 1.79
29.05 + 0.25
>150
2.68
6h
6i
>17.83
32.56
Miltefosined
22.0 + 0.5
131.9 + 3.9
499

b
500
501
502
503
aValues of inhibitory concentration of 50% of parasites (IC50); IC50 + Std. Error. Values of
c
citotoxicity concentration of 50% of macrophages (CC50); IC50 + Std. Error. SI = selectivity
index (CC50/IC50). dMiltefosine was used as reference drug.
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Figure 4. Compounds selected for the study of the mechanism of action
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Both pyrimidinylhydrazone-2f and pyrimidinyl-N-acylhydrazone-6h
derivatives can induce depolarization of ∆ψm.
Aiming to elucidate the mode of action of the compounds tested, two
compounds of each series were chosen based on their IC50 values in both
promastigote and amastigote forms of L. amazonensis. With this in mind, the
compounds 2f and 6h were chosen to representing the 2-pyrimidinyl-
hydrazone and the 2-pyrimidinyl-N-acylhydrazone derivatives, respectively.
The first step was to evaluate the effect of the compound on the mitochondria,
since this organelle plays a vital role in the survival of parasites. So, two
fluorescent probes, Mtk and JC-1, were used for studies about mitochondrial
dysfunction. As can be observed in Figure 1, the treatment with both
compounds induces depolarization in ∆ψm. By using Mtk, only parasites treated
the highest concentration of the compound 2f, corresponding 2X IC50 value
obtained after 72 h of treatment (19.32 µM), significantly decreases the
fluorescence intensity (30.27%) when compared to the negative control (Figure
1A). Regarding the treatment with the compound 6h, the results showed a
decrease in fluorescence intensity in promastigotes treated with both
concentrations 24.28 µM (54.94%) and 48.56 µM (55.48%) when compared to
the negative control (Figure 1C). When the parasites were staining with by JC-
1, the same pattern of results occurred (Figures B and D). Parasites treated with
2f show that only the highest concentration of treatment can decrease (10.97%)
the ratio 600/52 (Figure 1B). Figure 1D shows that treatment with the

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compound 6h (24.28 and 48.56 µM) induced a decrease in the ratio of 600/528
by 36% and 49.75%, respectively.
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Figure 1: Analysis of the ∆ψm promastigotes treated with the compounds. (A and B) ∆ψm
of cells treated with 2f (9.66 and 19.32 µM), using Mtk and JC-1 probes, respectively. (C and
D) ∆ψm of cells treated with 6h (24.28 and 48.56 µM) using the Mtk and JC-1 probes,
respectively. FCCP (10 µM) was used as a positive control. NC: negative control (without
treatment). The parasites were analyzed by fluorimetry. The results were shown as the mean of
three independent experiments performed in triplicates. Statistical analyses were performed
using GraphPad Prism 5.0 software, applying the analyses of variance (One-Way ANOVA)
followed Dunnett´s post-test: (*) P< 0.05 and (***) P< 0.001.
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Both compounds induce an increase in the intracellular ROS levels
Aiming to investigate whether treatment with 2f and 6h induces an
increase in intracellular ROS levels of the promastigotes of L. amazonensis, the
non-fluorescent substance H2CFDA was used. This substance, inside the cell,
reacts with ROS, is oxidized and emits fluorescence. Figure 2 shows that both
compounds are capable of inducing a substantial increase in the intracellular
ROS levels in all concentrations used. The compound 2f showed an increase in
fluorescence intensity of 111.1% with the lowest concentration of treatment
(9.66 µM) and 88.13% in the treatment with the highest concentration (19.32
µM) (Figure 2A). Treatment with 6h (24.28 and 48.56µM) increased the
fluorescence intensity by 47.66% and 74.11%, respectively, compared to the
untreated control (Figure 2B).

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Figure 2: Effect of the treatment on the promastigotes intracellular ROS levels.
(A) The compound 2f at 9.66 and 19.32 µM; (B) The compound 6h at 24.28 and 48.56 µM.
Miltefosine (44 µM) was used as a positive control. NC: negative control (without treatment).
The parasites were analyzed by fluorimetry. The results were shown as the mean of three
independent experiments performed in triplicates. Statistical analyses were performed using
GraphPad Prism 5.0 software, applying the analyses of variance (One-Way ANOVA) followed
Dunnett´s post-test: (*) P< 0.05; (**) P< 0.01 and (***) P< 0.001.
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The compound 6h but not 2f alters membrane integrity in L. amazonensis
The first step was to evaluate the plasma membrane integrity of
treated promastigotes. In the results, the 2-pyrimidinyl-hydrazone derivative 2f
(9.66 and 19.32 µM) did not induce an increase in fluorescence intensity,
suggesting that the plasma membrane of the treated parasites remains
integrated after treatment (Figure 3A). In contrast, the treatment with 2-
pyrimidinyl-N-acylhydrazone derivative 6h (24.28 and 48.56 µM), induced a
significant increase of 82.9% and 143%, respectively, in the fluorescence
intensity (Figure 3B). These results indicate that the compound 6h induces
changes in the plasma membrane since propidium iodide is a dye that is
excluded from the cell with an intact membrane.

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Figure 3: Analysis of the membrane integrity of L. amazonensis promastigotes. (A)
Treatment with the compound 2f (9.66 and 19.32 µM). (B) Treatment with the compound 6h
(24.28 and 48.56 µM). After treatment with the compounds and PI labeling, the parasites were
analyzed by fluorimetry. Heated parasites were used as a positive control, and not treated
parasites were used as a negative control (NC). The results were shown as the mean of three
independent experiments performed in triplicates. Statistical analyses were performed using
GraphPad Prism 5.0 software, applying the analyses of variance (One-Way ANOVA) followed
Dunnett´s post-test: (*) P< 0.05, (**) P< 0.01.
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Physicochemical and pharmacokinetic properties of the compounds
Some compounds presented a strong effect in vitro against amastigote
forms of L. amazonensis encouraging procedures to test theirs in vivo
effectiveness. However, it is important that before the procedures using the
animal model, the theoretical analysis of the in silico physicochemical and
pharmacokinetic properties can be performed. In this study, the properties of
some compounds were evaluated using the PKCSM computational tool.
According to the results presented in Table 5, the compounds did not violate
any of the Lipinski's rules (LogP ≥5; MW ≥ 500; HBA ≥5; HBD ≥ 10), which
is predictive that the compounds are likely to have good bioavailability for oral
administration [39]. Table 5 shows that these compounds are poorly absorbed
in Caco2 cells, but they showed a good profile for intestinal absorption. Except
for the compound 6i, predicted CYP4503A4 substrate, none of the other
compounds analyzed were predicted as a substrate of the CYP450 enzymes
used in this study. The compounds 2b, 2e, 6h, and 6i compounds were
predicted CYP4501A2 inhibitors, indicating the need for studies to elucidate

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the interaction with drugs that are metabolized by this enzyme. In the
parameters used for toxicity, the compounds were predicted with low toxicity.
Also, none of the compounds were considered inhibitors of hERG Ie II,
potassium channels whose inhibition could lead to fatal ventricular arrhythmia.
Table 5. In silico physicochemical and pharmacokinetic properties of the best
antileishmanial compounds
Model name
Predicted Value
Unit
2b
2e
2f
6h
6i
Water
solubility
Caco2
-2.878 -3.692
0.795
-3.555
-3.648
Numeric (log
mol/L)
0.742
0.115
0.795
0.795
0.081
0.218
Numeric (log
Papp in 10-
6 cm/s)
permeability
Intestinal
absorption
(human)
90.531 83.448
-3.073 -2.867
76.705
77.965
Numeric (%
Absorbed)
Skin
-2.979
No
-2.741
No
-2.734
No
Numeric (log
Kp)
Permeability
P-glycoprotein
substrate
Yes
No
No
Yes
No
Categorical
(Yes/No)
Categorical
(Yes/No)
Categorical
(Yes/No)
Numeric (log
L/kg)
P-glycoprotein
I inhibitor
No
No
No
P-glycoprotein
II inhibitor
No
No
No
No
VDss (human) -0.303
-0.45
0.214
-0.403
0.328
-0.851
0.276
-0.689
0.157
Fraction
unbound
(human)
BBB
0.323
Numeric (Fu)
-1.099 -0.911
-2.998 -2.692
-0.372
-2.487
No
-1.106
-3.109
No
-1.067
-2.81
No
Numeric (log
BB)
permeability
CNS
Numeric (log
PS)
permeability
CYP2D6
substrate
CYP3A4
substrate
No
No
No
No
Categorical
(Yes/No)
Categorical
(Yes/No)
No
No
Yes

CYP1A2
inhibitor
CYP2C19
inhibitior
CYP2C9
inhibitior
CYP2D6
inhibitior
CYP3A4
inhibitior
Total
Yes
No
Yes
No
No
No
Yes
No
Yes
No
Categorical
(Yes/No)
Categorical
(Yes/No)
No
No
No
No
No
Categorical
(Yes/No)
No
No
No
No
No
Categorical
(Yes/No)
No
No
No
No
No
Categorical
(Yes/No)
0.518
No
0.658
No
0.636
No
0.981
No
0.148
No
Numeric (log
ml/min/kg)
Categorical
(Yes/No)
Clearance
Renal OCT2
substrate
AMES
No
Yes
0.659
No
No
Yes
0.728
No
Yes
0.77
No
Categorical
(Yes/No)
toxicity
Max. tolerated 0.484
dose (human)
0.477
No
Numeric (log
mg/kg/day)
Categorical
(Yes/No)
hERG I
inhibitor
hERG II
inhibitor
Oral Rat
Acute
No
No
No
No
No
No
Categorical
(Yes/No)
2.021
2.234
2.302
2.29
2.081
Numeric
(mol/kg)
Toxicity
(LD50)
Oral Rat
Chronic
1.226
1.779
1.303
0.8
2.11
Numeric (log
mg/kg_bw/day)
Toxicity
(LOAEL)
Hepatotoxicity
No
No
No
No
No
No
No
No
No
No
Categorical
(Yes/No)
Skin
Sensitisation
T. pyriformis
toxicity
Categorical
(Yes/No)
0.303
2.499
0.849
1.364
0.56
2.278
0.526
1.433
0.775
0.795
Numeric (log
ug/L)
Minnow
Numeric (log
mM)
toxicity
606
607

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609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
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635
636
637
638
639
640
Discussion
As no effective vaccine has been developed for the prevention of
leishmaniasis, the treatment is based on chemotherapy. Current treatment for
leishmaniasis is a significant public health problem, not only due to the scarcity
of available drugs but also to the toxicity and treatment failure problems [40].
In this way, the search for new substances with the potential for a new
antileishmanial agent is essential. In this study, the antileishmanial activity of
2-Pyrimidinylhydrazone and 2-pyrimidinyl-N-acylhydrazone was assayed
against L. amazonensis. This Leishmania species presents great
epidemiological importance in Americas and responsible to a broad spectrum
of clinical manifestations that can range from a single skin lesion that cures
spontaneously or too severe infections such as mucocutaneous leishmaniasis or
diffuse cutaneous in immunocompromised patients that is refractory to
treatment [41,42].
Aiming at investigating the mechanisms that confer the antileishmanial
activity of the 2-Pyrimidinylhydrazone and 2-pyrimidinyl-N-acylhydrazone
derivatives, the best compounds in each of the series (2f and 6h, respectively)
were selected and their effects on the main targets of L. amazonensis
promastigotes explored. Unlike mammals, trypanosomatids have single and
branched mitochondria that occupy a high volume of the cytosol. The organelle
plays a vital role in the vital maintenance of these microorganisms and is,
therefore, an essential target in drug development [43]. The ROS are naturally
generated during mitochondrial respiration by the transport chain of electrons,
present in the internal membrane of the mitochondria. In this process, part of
the oxygen is reduced to superoxide, which is disputed to hydrogen peroxide
and hydroxyl radical [44]. The levels in the production of these substances can
be increased in response to different stimuli, especially drugs with a pro-
oxidative capacity [45]. Many studies indicate that the generated ROS can lead
to various cellular disorders such as membrane lipoperoxidation, DNA
damage, and ∆ψm alterations [22, 46, 47]. In this study, the exposure of L.
amazonensis to 2f and 6h led to an increase of ROS production and ∆ψm
changes. The compound 6h can induce both ROS and ∆ψm alterations in all
concentrations after 24 h of treatment.

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Interestingly, compound 2f at the concentration corresponding to IC50 (9.66
µM) despite inducing a significant increase of ROS (> 100%) after 24 h of
treatment, was not able to reach the ∆ψm of the parasite in this time. This event
is observed only when the parasites were treated during 24 h at 2X IC50 value
(19.32 µM). Since the IC50 value was determined after 72 h of treatment, our
hypothesis is that (i) the time of exposure of 24 h to the drug was not sufficient
to induce mitochondrial membrane depolarization; or (ii) the compound is able
to induce increased levels of ROS, but this event is not initially associated with
the mitochondrial membrane depolarization. Although the mitochondrial
respiratory chain is considered as one of the primary sources of ROS, in
principle, ROS can also originate from several other mitochondrial sources or
even an increase of metabolic activities or reduced anti-oxidative defenses
[48]. Whatever the case, the ROS-induced oxidative stress can lead to
deleterious and irreversible cellular effects compromising the survival of this
pathogen.
The plasma membrane is an essential target in several microorganisms,
including Leishmania spp. Several drugs currently used in the treatment of
leishmaniasis such as amphotericin B and miltefosine cause changes in the
plasma membrane that accompany other disorders leading to the death of the
parasite [40]. The integrity of the plasma membrane of L. amazonensis
promastigotes was studied using the dye PI, able to cross structurally damaged
membranes and to intercalate to nucleic acids. Our results show that the
treatment of 2f does not involve damage to the parasite's plasma membrane. In
contrast, 6f lead to a gradual increase in fluorescence intensity, indicating that
membrane integrity was affected in a manner permitting permeation of PI to
the interior of the cell. Although not fully understood, elevated levels of ROS
can lead to changes that may compromise maintenance and cellular functions,
including disruption of cell membranes [49]. With this mind, may the elevated
ROS levels observed after treatment of compound 6f is related to damage of
the plasma membrane. Further studies are necessary to understand this event
better.
In silico analysis of physicochemical and pharmacokinetic properties
can predict the behavior of substances in living organisms. This technology is
an essential ally in reducing costs and also the use of animals in experimental

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studies. Analysis of the physicochemical properties shows that none of the
compounds violated the Lipinski 5 rule (LogP ≤ 5; MW ≤ 500; HBA ≤5; HBD
≤ 10), which indicates that they have good bioavailability to be administered
orally [39].
Regarding pharmacokinetic properties, intestinal absorption is a
complex process involving many factors. Although the compounds did not
provide a good prediction for absorption in Caco2 cells, the compounds were
shown to be highly absorbed (greater than 75%) by the human small intestine.
Cytochrome P450 enzymes are responsive to drug metabolism. However, some
drugs may inhibit the functioning of some isoform of this enzyme. These drugs
may present problems when co-administered [50]. Our study presents a
prediction model for the two most common isoforms of CYP450 (CYP2D6 and
CYP3A4), present in a large part of the population [51]. However, by the
results obtained, none of the compounds were classified as a substrate for these
isoforms of the enzyme. Concerning inhibition, except for compound 2f, all
compounds were predicted as inhibitors of CYP1A2 and/or CYP3A4. Thus, it
is essential to emphasize the need for studies regarding drug interaction in co-
administration with drugs metabolized by this enzyme. Some drugs may inhibit
potassium channels (hERG), which is one of the causes of ventricular
arrhythmia and sudden cardiac death [52]. Therefore, it is advantageous that
none of the compounds were classified as inhibitors of hERG I and hERG II.
Besides, the compounds were not predicted as hepatotoxic or skin sensitizers.
698 Conclusion
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In this study, a new and potent class of leishmanicidal agents was
identified based on 2-pyrimidinyl hydrazone and N-acylhydrazone derivatives.
Studies about the mechanism of action of representative compounds of each
group (2f and 6h) showed slight differences in mode of action between the two
compounds after treatment of promastigote forms: only the compound 6h
induces a change in plasma membrane integrity, but both are able to cause
mitochondrial depolarization and increase of intracellular ROS levels. The
physicochemical, pharmacokinetic, and theoretical studies of these new classes
also indicating good oral bioavailability, being a good start point for the
development of new antileishmanial drugs.

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The authors confirm that this article content has no conflict of interest.
Acknowledgments
The authors thank the fellowship by CNPq (E.S. Coimbra), CAPES
(J.T. Granato) and FAPEMIG (M.S. Terror). The authors are grateful to
Reproduction Biology Center (CBR) of the Federal University of Juiz de Fora
for the supply of animals.
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