N, N′ N″-trisubstituted guanidines: Synthesis, characterization and evaluation of their leishmanicidal activity

Article abstract of DOI:10.1016/j.ejmech.2019.03.032

Leishmaniasis is a group of diseases caused by protozoan parasites from the genus Leishmania. There are estimated 1.3 million new cases annually with a mortality of 20,000–30,000 per year, when patients are left untreated. Current chemotherapeutic drugs available present high toxicity and low efficacy, the latter mainly due to the emergence of drug-resistant parasites, which makes discovery of novel, safe, and efficacious antileishmanial drugs mandatory. The present work reports the synthesis, characterization by ESI-MS, ¹H and ¹³C NMR, and FTIR techniques as well as in vitro and in vivo evaluation of leishmanicidal activity of guanidines derivatives presenting lower toxicity. Among ten investigated compounds, all being guanidines containing a benzoyl, a benzyl, and a substituted phenyl moiety, LQOF-G2 (IC_50-ama 5.6 μM; SI = 131.8) and LQOF-G7 (IC_50-ama 7.1 μM; SI = 87.1) were the most active against L. amazonensis intracellular amastigote, showing low cytotoxicity to the host cells according to their selectivity index. The most promising compound, LQOF-G2, was further evaluated in an in vivo model and was able to decrease 60% of the parasite load in foot lesions at a dose of 0.25 mg/kg/day. Moreover, this guanidine derivative demonstrated reduced hepatotoxicity compared to other leishmanicidal compounds and did not show nephrotoxicity, as determined by the analyses of biomarkers of hepatic damage and renal function, which make this compound a potential new hit for therapy against leishmaniasis.

Full text of DOI:10.1016/j.ejmech.2019.03.032

European Journal of Medicinal Chemistry 171 (2019) 116e128
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
N, N0, N00-trisubstituted guanidines: Synthesis, characterization and
evaluation of their leishmanicidal activity
,
b
,
1
c, 1
Rafael Dias do Espírito Santo a , Angela María Arenas Velasquez
,
ꢀ
ꢀ
Luana Vitorino Gushiken Passianoto a b, Alex Arbey Lopera Sepulveda c,
,
Leandro da Costa Clementino c, Renata Pires Assis c, Amanda Martins Baviera c,
d
e
e
ꢀ
ꢀ
Predrag Kalaba , Fabio Neves dos Santos , Marcos Nogueira Eberlin ,
f
g
h
c, **
ꢀ
ꢀ
Gil Valdo Jose da Silva , Martin Zehl , Gert Lubec , Marcia Aparecida Silva Graminha
,
a, b, *
ꢀ ꢀ
ꢀ
Eduardo Rene Perez Gonzalez
a
ꢀ
^
^
Laboratorio de Química Organica Fina, Departamento de Química e Biologia, Faculdade de Ciencias e Tecnologia, Universidade Estadual Paulista e UNESP,
Campus de Presidente Prudente, Rua Roberto Simonsen, 305, 19060-900, Presidente Prudente, SP, Brazil
b
ꢀ
~
^
~
Programa de Pos-Graduaçao em Ciencia e Tecnologia de Materiais (POSMAT), Universidade Estadual Paulista e UNESP, Sao Paulo, Brazil
c
ꢀ
^
^
Departamento de Analises Clínicas, Faculdade de Ciencias Farmaceuticas, Universidade Estadual Paulista e UNESP, Campus de Araraquara, Rodovia
Araraquara-Jaú, km1, 14800-903, Araraquara, SP, Brazil
d Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna, Althanstraße 14, 1090, Vienna, Austria
e
ꢀ
Laboratorio ThoMSon de Espectrometria de Massas, Instituto de Química, Universidade de Campinas e UNICAMP, Campinas, 13083-970, SP, Brazil
f
^
~
~
Departamento de Química, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo e USP, Avenida dos Bandeirantes, 3900,
~
14040-901, Ribeirao Preto, SP, Brazil
g
€
Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Wahringer Straße 38, 1090, Vienna, Austria
h Department of Neuroproteomics, Paracelsus Medical University, Salzburg, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Leishmaniasis is a group of diseases caused by protozoan parasites from the genus Leishmania. There are
estimated 1.3 million new cases annually with a mortality of 20,000e30,000 per year, when patients are
left untreated. Current chemotherapeutic drugs available present high toxicity and low efficacy, the latter
mainly due to the emergence of drug-resistant parasites, which makes discovery of novel, safe, and
efficacious antileishmanial drugs mandatory. The present work reports the synthesis, characterization by
ESI-MS, 1H and 13C NMR, and FTIR techniques as well as in vitro and in vivo evaluation of leishmanicidal
activity of guanidines derivatives presenting lower toxicity. Among ten investigated compounds, all
being guanidines containing a benzoyl, a benzyl, and a substituted phenyl moiety, LQOF-G2 (IC50-ama
Received 12 February 2019
Received in revised form
13 March 2019
Accepted 14 March 2019
Available online 21 March 2019
Keywords:
Leishmaniasis
5.6
m
M; SI ¼ 131.8) and LQOF-G7 (IC50-ama 7.1 M; SI ¼ 87.1) were the most active against
m
Leishmania amazonensis
N, N0, N00-trisubstituted guanidine
derivatives
In vivo leishmanicidal activity
Hepatoprotective effect
L. amazonensis intracellular amastigote, showing low cytotoxicity to the host cells according to their
selectivity index. The most promising compound, LQOF-G2, was further evaluated in an in vivo model
and was able to decrease 60% of the parasite load in foot lesions at a dose of 0.25 mg/kg/day. Moreover,
this guanidine derivative demonstrated reduced hepatotoxicity compared to other leishmanicidal
compounds and did not show nephrotoxicity, as determined by the analyses of biomarkers of hepatic
damage and renal function, which make this compound a potential new hit for therapy against
leishmaniasis.
© 2019 Elsevier Masson SAS. All rights reserved.
Abbreviations: ACN, acetonitrile; Bi(NO3)3$5H20, bismuth(III) nitrate pentahydrate; Et3N, triethylamine; DMF, dimethylformamide; NMR, Nuclear Magnetic Resonance;
DEPT, Distortion less enhancement by polarization transfer; CDCl3, deuterated chloroform; ESI-MS, Electrospray Ionization Mass Spectrometry; FTIR, Fourier-transform
infrared spectroscopy; AmpB, Amphotericin B; MNRET, Micronucleated reticulocytes; PBS, Phosphate Buffered Saline; ALT, alanine aminotransferase; AST, aspartate
aminotransferase; ALP, alkaline phosphatase.
ꢀ
^
^
* Corresponding author. Laboratorio de Química Organica Fina, Departamento de Química e Biologia, Faculdade de Ciencias e Tecnologia, Universidade Estadual Paulista e
UNESP, Campus de Presidente Prudente, Rua Roberto Simonsen, 305, 19060-900, Presidente Prudente, SP, Brazil.
** Corresponding author.
ꢀ
E-mail addresses: marcia.graminha@unesp.br (M.A.S. Graminha), eduardo.gonzalez@unesp.br (E.R.P. Gonzalez).
1
ꢀ
ꢀ
Rafael Dias do Espírito Santo and Angela María Arenas Velasquez contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2019.03.032
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

R.D. do Espírito Santo et al. / European Journal of Medicinal Chemistry 171 (2019) 116e128
117
1. Introduction
Leishmaniasis is a complex of diseases that comprises a wide
spectrum of clinical manifestations, including visceral, cutaneous
and mucocutaneous leishmaniasis. It is caused by more than 20
species of the genus Leishmania (Protozoa: Kinetoplastida: Trypa-
nosomatidae) that are transmitted through sandflies (Diptera:
Psychodidae: Phlebotominae). These parasitic diseases affect
mainly poor people in Africa, Asia and Latin America, and the
different disease manifestations are determined by the parasite
species and the host immune system. There are estimated 1.3
million new cases annually resulting in 20e30 000 deaths [1].
Pentavalent antimonials, paromomycin, amphotericin B and
miltefosine are the commonly used drugs, which require long pe-
riods of administration and can have serious side effects. Moreover,
drug resistant parasites also contributes to the ineffectiveness of
these few therapeutic options [2,3], calling urgently for the devel-
opment of new, safe and effective drugs against leishmaniasis [4,5].
Guanidines are frequently found in nature and used [6] for
syntheses of organic compounds including quinazolines, oxazoli-
dinones, lactones, carbonates and urethanes [7e19].
Guanidine derivatives show several biological effects [20e23],
such as cardiovascular dilatation [24], as well as antihistaminic
[25], anti-inflammatory [26,27], antidiabetic [28], antibacterial
[29], antifungal [29], antiprotozoal/antiparasitic [30e33] and anti-
viral activity [34e37]. Several compounds with guanidine-moiety
are also studied for the treatment of neglected tropical diseases
[38], and some studies have tested the effectiveness of guanidines
against leishmaniasis [31e33,38e42].
Scheme 2. Synthesis of guanidines LQOFG-1 to LQOFG-10. aReagents and conditions:
(a) Bi(NO3)3$5H2O, Et3N, DMF, 130 ꢀC, 24 h.
LQOFG-10 using the reported procedure [20,43e45]. In order to
form compounds LQOFG-1 to LQOFG-10, the thioureas were reac-
ted with benzylamine (Scheme 2).
2.1.1. Nuclear magnetic resonance spectroscopy (NMR) analysis
1H NMR spectra of guanidines LQOFG-1 to LQOFG-10 showed
the aromatic hydrogens of the benzoyl moiety, H10, H11 and H12,
around 8.3, 7.4 and 7.5 ppm, respectively (except for H10 of LQOFG-
10, which was at 7.94 ppm). The aromatic hydrogens of the p-
substituted N-phenyl showed chemical shifts of 7.0e7.4 (H4) and
6.9e8.2 ppm (H5), depending on the nature of the p-substituent
group. The chemical shifts of the two NH hydrogens were usually
observed at ~5 and 12 ppm as broad signals with low intensity
resulting from the rapid configurational interconversion between
E/Z isomers [20,44].
The present work presents synthesis, structural characteriza-
tion, in vitro and in vivo antileishmanial activity of ten N, N0, N00-
trisubstituted guanidine derivatives.
2. Results and discussion
2.1. Synthesis
Other evidence of the guanidine formation is the appearance of
methylene and aromatic hydrogens of the benzyl moiety. Table 1
shows representative chemical shifts (ppm) in the 1H NMR
spectra for compounds LQOFG-1 to LQOFG-10. Fig. 1 shows the 1H
In a first step, thioureas were synthetized (Scheme 1) as in-
termediates for the subsequent synthesis of guanidines LQOFG-1 to
Table 1
Representative chemical shifts (ppm) obtained by 1H NMR (400 MHz; CDCl3) of
compounds LQOFG-1 to LQOFG-10. (More detailed data is described in Experi-
mental part).
X
H1
H2
H4
H5
X
H10
H12
H13
H15
NO2
Br
Cl
5.99
5.45
5.44
5.26
5.18
5.27
5.16
5.06
5.08
4.76
12.63
12.15
12.16
12.22
12.05
12.09
12.21
11.93
12.09
9.16
7.36
7.14
7.21
7.30
7.20
7.19
7.02
7.19
7.09
7.43
8.23
7.53
7.42
7.44
7.16
7.40
7.70
6.92
7.25
8.12
8.29
8.29
8.29
8.30
8.28
8.27
8.25
8.28
8.26
7.94
7.50
7.56
7.56
7.50
7.47
7.47
7.49
7.46
7.46
7; 61
4.92
4.85
4.85
4.82
4.78
4.80
4.76
4.77
4.77
4.59
7.42
7.41
7.40
7.38
7.34
7.36
7.36
7.35
7.34
7.37
H
7.42
2.35
1.31
CH3
t-bu
I
OCH3
F
3.81
CF3
a
Scheme 1. Synthesis of thioureas. Reagents and conditions: (a) ACN, 75 ꢀC, 1 h; (b)
ACN, 75 ꢀC, 2 h.
Obs. Data of compounds LQOFG-1 to LQOFG-10 in the successive order.

118
R.D. do Espírito Santo et al. / European Journal of Medicinal Chemistry 171 (2019) 116e128
Fig. 1. 1H NMR spectrum of LQOFG-6.
NMR spectrum of LQOFG-6 as a representative of this series of
compounds. All 1H NMR spectra are available in supplementary
material section.
phenyl-N-(benzyl)carbodiimide. Fragment ions corresponding to
the neutral loss of benzamide (m/z 227) shown in Fig. 4 are also
present at lower intensities for these compounds (Scheme 4).
13C NMR spectra of compounds LQOFG-1 to LQOFG-10 also
clearly proof the formation of the guanidines by displaying signals
related to all three moieties inserted into the molecules. Additional
evidence of the compound's formation is the observation of the
signals around 158 ppm, characteristic for guanidine carbon (C7).
The aromatic carbons were observed in the 115e150 ppm region
and the carbonyl carbon (C8) showed the characteristic shift of
around 178 ppm. Table 2 shows representative chemical shifts
(ppm) in the 13C NMR spectra for compounds LQOFG-1 to LQOFG-
10. Figs. 2 and 3 show 13C NMR and DEPT 135 spectra of LQOFG-6,
2.1.3. Fourier-transform infrared spectroscopy (FTIR) measurements
Formation of the compounds LQOFG-1 to LQOFG-10 was also
confirmed by FTIR spectra through the disappearance of the ab-
sorption band at ~ 1262 cmꢁ1 associated to v (C]S) stretch of the
starting thioureas and by appearance of the absorption band
at ~ 1566 cmꢁ1 characteristic of v (C]N) stretch.
Furthermore, the compounds showed absorption bands in the
region of 3200e3400 cmꢁ1 typical of the v (NeH) stretch. In
addition, bands were also observed in the 3010-3100 cmꢁ1 and
1590-1610 cmꢁ1 regions, associated to aromatic v (CeH) and v
(CeC) stretch, respectively. Finally, the absorption bands in the
1630-1730 cmꢁ1 region were assigned to v (C]O) stretch of
carbonyl group [20].
respectively, as examples for this series of compounds. All other 13
NMR spectra are available in supplementary material section.
C
2.1.2. Electrospray ionization mass spectrometry (ESI-MS) analysis
Compounds LQOFG-1 to LQOFG-10 were also studied by mass
spectrometry with electrospray ionization in the positive ion mode
(ESI(þ)-MS). All compounds were identified by the detection of
their intact protonated molecules, which were selected and further
fragmented via ESI(þ)-MS/MS experiments. Fig. 4 shows the HRESI
(þ)-MS and ESI(þ)-MS/MS spectra of the compound LQOFG-9 (m/z
2.2. Evaluation of anti-leishmanial in vitro activity and in vivo
mutagenicity [46,47] and activity
2.2.1. In vitro activity evaluation of compounds LQOFG-1 to LQOFG-
10
348.1519 [MþH]þ) as
a
prototype spectra of this series of
Several guanidine derivatives have been investigated and anti-
parasitic, antibacterial, antifungal and anti-HIV effects have been
demonstrated [23,31e33,38e42,48e51]. The anti-leishmanial po-
tential of the ten compounds LQOFG-1 to LQOFG-10 was evaluated
against promastigote and intracellular amastigote forms of
L. amazonensis, and their cytotoxic effect on host macrophages was
tested. All tested compounds showed anti-promastigote activity
compounds.
Compounds LQOFG-1 to LQOFG-10 yielded fragment ions at m/z
77, 91, 105 and 122, corresponding to phenyl, tropylium, and ben-
zoyl cations, as well as protonated benzamide, respectively (Fig. 4).
The fragment ion at m/z 91 is generated by protonation at the
alkyl nitrogen, followed by the simple neutral loss of the respective
N-(p-substituted)phenyl-Ne (benzoyl)guanidines (Scheme 3), and
yields the base peak for all compounds with benzyl moiety (with an
exception of LQOFG-1, where the observed base peak was m/z 122).
The fragment ions at m/z 105 and 122 are generated by pro-
tonation of the amide nitrogen. Formation of benzoyl cation (m/z
105) is associated with neutral loss of the N-(p-substituted)phenyl-
N-(benzyl)guanidine, and the protonated benzamide (m/z 122) in-
volves elimination of a neutral molecule of N-(p-substituted)
ranging from 7 to 26 mM. Except for LQOFG-4, the other nine
compounds showed activity against the intracellular amastigotes,
the most clinically relevant stage of Leishmania parasites. Only
LQOFG-4 and LQOFG-10 showed a cytotoxic effect on the host cells,
while all others showed no cytotoxicity against macrophages (half-
maximal cytotoxicity concentration >500
Considering the anti-amastigote activity, the data showed that
compounds LQOFG-2 (IC50-ama ¼ 5.6 M) and LQOFG-7 (IC50-
mM).
m

R.D. do Espírito Santo et al. / European Journal of Medicinal Chemistry 171 (2019) 116e128
119
Table 2
Representative chemical shifts (ppm) obtained by13C NMR (400 MHz; CDCl3) of compounds LQOFG-1 to LQOFG-10. (More detailed data is described in Experimental part).
X
C3
C4
C5
X
C7
C8
C10
C12
C13
C15
C16
NO2
Br
Cl
142.6
142.2
134.1
133.6
136.0
133.2
133.2
e
123.6
127.4
127.1
125.7
130.8
125.5
127.9
122.2
129.1
127.5
125.8
133.1
131.5
127.1
125.9
127.2
127.3
115.3
127.0
125.7
156.9
158.1
158.2
158.7
159.1
159.1
158.4
158.7
159.0
156.5
177.7
177.5
177.5
177.7
177.8
177.9
178.0
177.7
177.9
177.5
129.0
128.9
130.1
129.2
129.3
129.3
129.3
129.2
129.2
128.9
132.0
131.6
132.5
131.4
131.3
131.4
131.5
131.1
131.3
131.7
45.1
44.8
44.8
45.4
45.3
45.3
45.4
45.2
45.3
45.6
127.8
127.7
127.7
127.7
127.7
127.7
127.7
127.7
127.7
127.9
127.9
128.8
128.9
128.9
128.9
128.9
129.0
128.8
128.8
128.7
H
CH3
t-bu
I
OCH3
F
21.2
34.8
55.5
57.0
128.2
e
CF3
Obs. Data of compounds LQOFG-1 to LQOFG-10 in the successive order. (ꢁ) Signal not observed.
Fig. 2. 13C NMR spectrum of LQOFG-6.
¼ 7.1
m
M) were the most efficacious in macrophage parasite
clearance (Table 3, Fig. 5) and present similar potency when
compared to pentamidine (IC50-ama ¼ 5.1 M) and AmpB (IC50-
¼ 4.9 M). Regarding the selectivity towards the parasite
amastigote activity (IC50-ama > 19
criteria herein adopted (IC50-ama < 10
LQOFG-4, showed high toxicity to macrophages and no anti-
amastigote activity.
Regarding the infection index (percentage of infected cells/
number of the intracellular parasites per infected macrophage),
m
mol Lꢁ1) does not meet the hit
M). Finally, compound
ama
m
m
m
ama
rather than to the macrophages, LQOFG-2 (SIama ¼ 131.8) and
LQOFG-7 (SIama ¼ 87.1) were twenty-eight- and eighteen-times
more selective than AmpB (SIama ¼ 4.7). These results indicate
that LQOFG-2 and LQOFG-7 could be developed into a therapeutic
alternative for the treatment of leishmaniasis that could spare the
patients the severe side effects presented by the currently used
drugs. Although the good selectivity index of compounds LQOFG-1,
LQOFG-3, LQOFG-5, LQOFG-6, LQOFG-8 and LQOFG-9, makes them
promising candidates for leishmaniasis treatment, their anti-
treatment with 25 mM concentration of LQOFG-2, LQOFG-7,
LQOFG-9 and LQOFG-3 caused a reduction of 86, 78, 70 and 19%,
respectively, in the number of intracellular amastigotes (Fig. 5).
LQOFG-2 was able to reduce the infection as effective as pentam-
idine (81%) and AmpB (84%). These data suggest the high potential
of LQOFG-2, LQOFG-7 and LQOFG-9 in reducing the number of
parasites in Leishmania infected macrophages. The compounds

120
R.D. do Espírito Santo et al. / European Journal of Medicinal Chemistry 171 (2019) 116e128
Fig. 3. DEPT 135 spectrum of LQOFG-6.
Fig. 4. ESI(þ)-MS (up) and ESI(þ)-MS/MS (down) spectra of LQOFG-9.
containing halogen atoms displayed the ability to reduce macro-
phage infection following the below potency order (the most active
to the less active):
G2(Br) 5.6 y G7(I) 7.1 > G6(t-Bu) 17.2 y G9(F) 19.0 y G8(CH3O)
21.1 y G10(CF3) 21.4 y G1(NO2) 22.1 y G5(CH3) 24.2 > G3(Cl) 41.2
Considering compound LQOFG-4 (X ¼ H) as a reference, it is
reasonable to assume that the presence of a substituent in the
fourth position of the aniline moiety is crucial for the anti-
leishmanial activity of the investigated N1-benzoyl-N2-benzyl
guanidines, with Br and I being favorable for a higher anti-
amastigote activity.
LQOFG-2 (Br) > LQOFG-7 (I) > LQOFG-9 (F) > LQOFG-3 (Cl)
In addition, for all studied compounds the anti-amastigote ac-
tivity (IC50
-mM-data in bold) suggests that the substituent (X) might
be contributing to the potency as follows:

R.D. do Espírito Santo et al. / European Journal of Medicinal Chemistry 171 (2019) 116e128
121
Scheme 3. Formation of the fragment ion at m/z 91.
Scheme 4. Proposal for the fragmentation pathway of the compounds LQOFG-1 to LQOFG-10 (example for LQOFG-9).
Table 3
Antiparasitic activities (IC50-pro/ama, half-maximal inhibitory concentration,
m
M), mammalian cell toxicity (CC50, half-maximal cytotoxicity concentration,
mM) and safety index
(SI ¼ CC50/IC50) of the guanidine compounds. The SI values ꢂ 10 in bold indicate promising compounds, i.e., those with low mammalian toxicity and high toxicity to parasites.
Each value is the mean of three experiments performed in triplicate standard deviation. pro: promastigote, ama: amastigote.
Leishmania amazonensis
Macrophage
CC50
Safety Index
promastigote
Compound
X
IC50-pro
SD
IC50-ama
SD
SD
amastigote
LQOFG-1
LQOFG-2
LQOFG-3
LQOFG-4
LQOFG-5
LQOFG-6
LQOFG-7
LQOFG-8
LQOFG-9
LQOFG-10
Pentamidine
AmpB
NO2
Br
Cl
11.5
19.6
19.7
22.6
20.9
25.9
15.9
25.4
21.9
6.8
0.7
0.2
0.1
1.1
0.5
0.9
0.9
1.5
1.8
0.2
0.1
0.1
22.1
5.6
41.2
ND
24.2
17.2
7.1
21.1
19.0
21.4
5.07
4.92
3.3
2.5
4.1
801.9
737.9
827.4
35.9
876.1
1118.9
618.7
836.6
865.5
18.6
0.3
1.4
1.5
5.6
69.7
37.7
42.0
1.6
41.9
43.2
38.9
32.9
39.5
2.7
36.4
131.8
20.1
ND
36.2
65.0
87.1
39.6
45.5
0.8
H
CH3
t-Bu
I
CH3O
F
5.2
2.1
0.8
3.6
4.6
0.6
1.14
0.14
1.7
105.4
70.2
1.6
1.7
2.9
CF3
7.6
3.2
35.7
23.1
6.8
2.5
4.7
7.2
7.0
4.7
For the compound LQOFG-2 (Br), herein the best anti-
leishmanial candidate, a strong predominance of a ‘Z’ isomer was
observed by NMR [20]. This was, however, not the case with
compound LQOFG-1 (NO2), where equal populations of isomers ‘Z’
and ‘E’ were found [20].
Therefore, the presence of these groups in the structure of
guanidines, can probably modify the binding strength to possible
targets in the parasite by charge and conformational effects.
Guanidines as well as some analogues of marine guanidine
alkaloids showed a broad spectrum of action for the treatment of
different infectious diseases including for leishmaniasis
[20,22,23,31e33,39e42,48]. Indeed, the polymeric guanidine
AKACID Plus® was able to decrease skin infections caused by
methicillin-resistant Staphylococcus aureus through topical appli-
cation [52] and several bisguanidine-indenes have exhibited anti-
Trypanosoma brucei activity in vivo mouse models [51,53], rein-
forcing the potential of guanidine compounds. Thus, in order to
further evaluate the in vivo leishmanicidal potential of the

122
R.D. do Espírito Santo et al. / European Journal of Medicinal Chemistry 171 (2019) 116e128
all tested concentrations, compared to negative control. However,
in the animal group exposed to the genotoxic agent cyclophos-
phamide, the frequency of micronuclei in mice reticulocytes was
significantly higher (24 micronucleated reticulocytes) in compari-
son to the negative control group (5 micronucleated reticulocytes).
The substance is considered to be mutagenic when the mutagenic
index (MI) is equal to or greater than 2 for at least one of the tested
doses [46]. In case of LQOFG-2 and LQOFG-7, the MI was less than 2
in all tested doses and therefore, no mutagenic properties were
observed (Table 4).
The mutagenicity evaluation is essential to determinate the drug
safety for human use and it is recommended by regulatory
agencies. Typically, the test can detect lesions in the structure of the
chromosomes as well as changes in the distribution of these
chromosomes during the process of cell division. Both lesions are
induced by chemicals, through the formation of micronuclei with
fragments of DNA or whole chromosomes. During normal eryth-
ropoiesis, micronuclei are formed at low frequency, and micro-
nuclei containing erythrocytes are removed rapidly and efficiently
from peripheral blood circulation. The presence of micronuclei
concomitantly with immature erythrocytes in circulation is an in-
dicator of chromosomal damage [46,47].
Fig. 5. In vitro effect of LQOFG-2, LQOFG-7, LQOFG-9 and LQOFG-3 on L. amazonensis
intracellular amastigotes. The infection index was calculated after 72 h of treatment
with 25
treated L. amazonensis intracellular amastigotes. Data are expressed as average SD.
*, : Statistically significant difference relative to negative control (*: p < 0.001;
p < 0.05). : Statistically significant difference relative to LQOFG-3 (p < 0.001).
mM of each guanidines, pentamidine and AmpB. The negative control is un-
a
a:
b
2.2.3. In vivo activity investigation of the best candidate LQOFG-2
L. amazonensis infected BALB/c mice (six weeks post infection)
were daily treated for 15 days with two different doses of LQOFG-2
(0.13 and 0.25 mg/kg/day) or AmpB (2 mg/kg/day) by intraperito-
neal administration. The animals treated with LQOFG-2 reduced
the parasite burden by 46 and 60% at 0.13 and 0.25 mg/kg/day,
respectively. AmpB, given at much higher dosage, reduced the
parasite load only by 41%, similarly to that obtained with the lowest
dose of the tested guanidine compound (Fig. 7).
The LQOFG-2 treatment also decreased the size of the lesion in
both doses in comparison with untreated infected animals and
infected animals treated with PBS, and similarly to AmpB at 2 mg/
kg/day (Fig. 8). On the other hand, no statistically significant change
of weight was observed during the treatment in all analyzed
groups.
In order to evaluate the potential toxic effect of LQOFG-2, the
plasma levels of creatinine and urea (biomarkers of kidney func-
tion), AST, ALT, and ALP (biomarkers of hepatic damage), and bili-
rubin (marker of hepatic function) were monitored. No changes
were observed in creatinine (Fig. 9A) and urea plasma levels
(Fig. 9B) of all animals, suggesting that both the infection with
L. amazonensis and/or the treatment with LQOFG-2 did not cause
renal dysfunction. The plasma levels of bilirubin were significantly
increased in animals infected with L. amazonensis, treated or not
with AmpB or with LQOFG2 (Fig. 9C). Considering that the in-
creases in bilirubin levels were similar among all groups of infected
animals, treated or not with LQOFG-2, it can be suggested that this
guanidine compounds developed herein, LQOFG-2 was chosen
based on the previously-mentioned hit criteria and DNDi recom-
mendations [54] (IC50 < 10
m
mol Lꢁ1 and SI ꢂ 10).
2.2.2. In vivo mutagenicity [46,47]of the two compounds (LQOFG-2
and LQOFG-7) with higher activity against intracellular amastigotes
Determination of in vivo mutagenicity was performed for com-
pounds LQOFG-2 and LQOFG-7, the most potent anti-leishmanial
guanidine derivatives. Fig. 6 shows the frequency of micro-
nucleated reticulocytes (MNRET) per 2000 cells obtained from an-
imals treated either with hydroxyethylcellulose 0.5% (negative
control), cyclophosphamide (positive control) or 25, 50 and
100 mg/kg of LQOFG-2 and LQOFG-7.
The results demonstrated that LQOFG-2 and LQOFG-7, admin-
istered at different concentrations, have not induced a significant
increase in the frequency of micronuclei in reticulocytes of mice at
Table 4
Mutagenic index (MI) of LQOFG-2 and LQOFG-7 at different doses tested against
peripheral mice blood cells. The MI was calculated as the ratio between the
micronucleated reticulocytes (MNRET) of tested drug and MNRET of negative con-
trol (hydroxyethylcellulose 0.5%).
Compound
Dose (mg/kg)
MNRET
MI
Negative control
cyclophosphamide
LQOFG-2
e
5
24
7
8
6
3
5
6
3
e
50
25
50
100
25
50
100
3
4.8
1.4
1.6
1.2
0.6
1.0
1.2
3
3
3
1
1
2
Fig. 6. LQOFG-2 and LQOFG-7 do not increase the frequency of micronucleated re-
ticulocytes (MNRET). Average frequency and standard deviation of MNRET per
2000 cells from mice treated with cyclophosphamide (positive control), hydrox-
yethylcellulose (negative control) and 25 mg/kg, 50 mg/kg and 100 mg/kg of guanidine
derivatives. *: Statistically significant difference relative to the animals treated with
50 mg/kg of cyclophosphamide (p < 0.05).
LQOFG-7

R.D. do Espírito Santo et al. / European Journal of Medicinal Chemistry 171 (2019) 116e128
123
doses of LQOFG-2. It is worth mentioning that, in infected animals
treated with 0.13 or 0.25 mg/kg/day LQOFG-2, the ALP and ALT
levels were similar those values found in uninfected animals,
highlighting the protective effect of LQOFG-2 against the harmful
consequences of L. amazonensis infection on liver integrity.
Our data indicate that LQOFG-2 is very efficient due to its
excellent biological activity against L. amazonensis and might be
considered a promising drug candidate for treatment of cutaneous
leishmaniasis since LQOFG-2 showed a high selectivity index, good
capacity to decrease parasite burden in in vivo experiments and no
toxicological/mutagenic effects.
3. Conclusion
Fig. 7. Parasite load after treatment of L. amazonensis-infected BALB/c mice with
LQOFG-2. Five groups containing five BALB/c mice infected with L. amazonensis were
treated daily with LQOFG-2 or AmpB for 15 days. Quantification of tissue parasite load
in skin lesions post-treatment was determined by the limiting dilution method. The
data are expressed as average SD. *: Statistically significant difference relative to the
untreated infected group (p < 0.001). **: Statistically significant difference relative to
In this study, ten benzoyl e benzyl guanidine derivatives were
synthetized, out of which compounds LQOFG-2 and LQOFG-7 have
been found to show highest activity against L. amazonensis intra-
cellular amastigote forms. In vivo experiments showed that the
parasite burden in L. amazonensis-infected BALB/c mice was more
strongly reduced by LQOFG-2 when compared to AmpB, the com-
mercial drug used in the treatment of Leishmania, although LQOFG-
2 was tested at lower doses than AmpB. Additionally, a reduction in
the parasite-induced hepatotoxicity and absence of nephrotoxicity
were observed in mice treated with two doses of LQOFG-2 in vivo.
LQOFG-2 and LQOFG-7 are not mutagenic as evaluated by the
micronucleus test. Therefore, the results of the biological evalua-
tion demonstrated that these compounds can be considered as
suitable candidates in the search for new drugs for the therapy of
leishmaniasis. This is very important considering the high selective
index and low toxicity of the synthetic candidate LQOFG-2 and in
addition the high infectivity by Leishmania in several countries, as
in the case of Brazil, and the inconvenience of the conventional
therapy.
the vehicle group e PBS (p < 0.001). b,g: Statistically significant difference relative to
the infected group treated with 0.25 mg/kg/day of LQOFG-2 (p < 0.01; p < 0.25).
4. Experimental section
4.1. Synthesis and analytics
Commercial benzoylchloride, ammonium isothiocyanate, ani-
line,
4-nitroaniline,
4-bromoaniline,
4-chloroaniline,
4-
Fig. 8. Reduction of foot lesions in L. amazonensis-infected BALB/c mice treated with
LQOFG-2. Groups of five BALB/c mice infected whit L. amazonensis were treated with
LQOFG-2 or AmpB for 15 days. (a) Values of volume of foot lesions were monitored
every 5 days to validate the in vivo efficacy of LQOFG-2. Data points represent the
average measurements for 7 groups of 5 mice each. The data are expressed as average
of volume (v ¼ D x d x e; D: larger diameter, d: minor diameter, e: thickness) SD.
fluoroaniline, 4-iodoaniline, 4-methoxyaniline, toluidine, 4-t-
butylaniline, benzylamine, 4-aminobutanol, triethylamine, penta-
hydrated bismuth nitrate and the solvents N,N-dimethylforma-
mide, acetonitrile and dichloromethane were used without previ-
ous purification.
Thin layer chromatography (TLC) was performed on silica gel
plates and the compounds visualized using iodine reagent.
1H and 13C NMR spectra were recorded on a Bruker Avance III e
400 MHz and a Bruker Avance III e 600 MHz (Bruker, Bremen,
Germany). The resonance frequency for 1H NMR was 400.13 MHz
and for 13C NMR 100.61 MHz. Chemical shifts for 1H NMR and 13C
NMR were referenced to TMS, analysis was performed in CDCl3 and
all chemical shifts were reported in ppm. Data are presented as
following: chemical shift, multiplicity (s ¼ singlet, d ¼ doublet,
dd ¼ doublet of doublet, t ¼ triplet, qua ¼ quadruplet, qu ¼ quin-
tuplet, m ¼ multiplet, br s ¼ broad singlet), integration, and
coupling constants (in Hertz).
a
,
b
,
g
:
Statistically significant difference relative to the untreated infected group
(p < 0.001; p < 0.01; p < 0.05). a,b: Statistically significant difference relative to the
vehicle group e PBS (p < 0.001; p < 0.01). *,**, : Statistically significant difference
relative to the infected group treated with 2 mg/kg/day of AmpB (p < 0.001; p < 0.01;
p < 0.05). : Statistically significant difference relative to the infected group treated
D
j
with 0.13 mg/kg/day of LQOFG-2 (p < 0.05).
increase in bilirubin may be due to the harmful impacts of
L. amazonensis infection on liver function, having no relationship
with drug hepatotoxicity. Corroborating the findings about the
detrimental impacts of L. amazonensis infection in liver, increased
plasma levels of ALP, AST, and ALT were observed in infected ani-
mals, treated or not with PBS (Fig. 9DeF), indicating that the
infection caused damage in hepatic cells, as previously observed
[55]. The treatment of infected animals with AmpB decreased the
plasma levels of ALP and ALT, suggesting that this treatment
partially prevented liver damage. Treatments with LQOFG-2 also
decreased the hepatic damage like AmpB: although the treatment
was not effective in reducing the AST levels (Fig. 9E), reductions in
ALP (Fig. 9D) and in ALT (Fig. 9F) levels were observed in all tested
To obtain ESI(þ)-MS data, the samples were diluted in 1 mL of
acetonitrile and 1 mL of the solution was injected using an auto-
sampler LC Agilent 1290 (Agilent, California, USA) into the elec-
trospray source of the mass spectrometer Agilent 6550 iFunnel Q-
TOF LC/MS (Agilent, California, USA). The mobile phase of the LC
was an acetonitrile solution containing 0.1% of formic acid at a flow
rate of 0.1 mL/min. The electrospray ionization source operated in
positive mode under the following conditions: nebulization gas

124
R.D. do Espírito Santo et al. / European Journal of Medicinal Chemistry 171 (2019) 116e128
Fig. 9. Plasma levels of biomarkers of renal function and liver damage in BALB/c mice noninfected and infected with L. amazonensis and treated with 0.13 or 0.25 mg/kg/day of
LQOFG2. (A) Creatinine levels; (B) Urea levels; (C) Total bilirubin levels; (D) ALP levels; (E) AST levels; (F) ALT levels. The data are expressed as averages SD. *: Statistically
significant difference relative to the uninfected animals (healthy animals) (p < 0.05);
Statistically significant difference relative to the vehicle group - PBS (p < 0.05).
a
: Statistically significant difference relative to the untreated infected group (p < 0.05); b:
temperature: 290 ꢀC, capillary voltage: 3500 V, Nozzle Voltage
320 V, dry gas flow: 14 mL/min, nebulization gas pressure: 45 psig,
gas auxiliary temperature: 350 ꢀC and auxiliary gas flow: 12 mL/
min. The quadrupole analyzer operated in the range of m/z
100e1000, fragmentor voltage: 100 V, octupole voltage: 750 V, and
acquisition of 1 spectrum/s was obtained with high resolution in
TOF up to the fifth decimal place. Formulas were assigned a 1 ppm
error using Agilent Mass Hunter Workstation software.
(SmartFormula algorithm).
The purity of the compounds was determined by HPLC method
on UFLC XR Prominence device (Shimadzu Corporation, Tokyo,
Japan). Separation was carried out on an Acclaim 120C18,
4.6 ꢄ 250 mm, 5
mm HPLC column (Thermo Fisher Scientific) using
LC-MS-grade water and acetonitrile as mobile phase A and B,
respectively. The sample components were separated and eluted
with a linear gradient from 10% to 90% B in 30 min followed by
30 min isocratic step with 90% B and a re-equilibration step. The
flow rate was 0.6 mL/min and the column oven temperature was
set to 25 ꢀC.
HRESIMS spectra were obtained on a maXis UHR ESI-Qq-TOF
mass spectrometer (Bruker Daltonics, Bremen, Germany). Sam-
ples were dissolved to 20
mg/mL in MeOH and directly infused into
the ESI source at a flow rate of 3
m
L/min with a syringe pump. The
FT-IR data were recorded on a Vector 22 instrument (Bruker,
Bremen, Germany) and collected at 25 ꢀC room temperature with
124 scans and spectral resolution of 4 cmꢁ1. Samples mixed in a
1mg/20 mg ratio with KBr and pressed to tablets.
Melting points (m.p.) were obtained using a WRS-2 Micro Pro-
cessor Melting-point apparatus. The samples were placed in a
capillary tube, and the pre-heating and final ramp temperatures
ESI ion source was operated as follows: capillary voltage: 4.5 kV,
nebulizer: 0.4 bar (N2), dry gas flow: 4 L/min (N2), and dry tem-
perature: 180 ꢀC. Mass spectra were recorded in the range of m/z
50e1900 in the positive-ion mode. The sum formulas were deter-
mined using Bruker Compass DataAnalysis 4.1 based on the mass
accuracy
(D
m/z ꢃ 5 ppm) and isotopic pattern matching

R.D. do Espírito Santo et al. / European Journal of Medicinal Chemistry 171 (2019) 116e128
125
were selected for 60 ꢀC and 250 ꢀC, respectively. The heating rate
used was 4.0 ꢀC/min.
4.1.1.4. (Z)eN-((benzylamino) (phenylamino)methylene)benzamide
(LQOFG-4). Yield 89%. 1H NMR (400.13 MHz, CDCl3)
d
¼ 12.22 (br,
1H), 8.30 (br, 2H), 7.50 (t, J ¼ 6,8 Hz, 1H), 7.44 (m, 5H), 7.37 (d, 4H),
7.33e7.30 (m, 3H), 5.26 (br, 1H), 4.82 (br, 2H). 13C (100.61 MHz,
4.1.1. General procedure for the synthesis of guanidines
[20,25,43,45].
CDCl3)
CH),130.1 (CeH),129.2 (2CH),128.9 (2CH),128.0 (2CH),127.7 (2CH),
127.6 (p-CH), 127.1 (2CH), 125.7 (2CH), 45.4 (NeCH2). FT-IR (KBr)
d
¼ 177.7 (C]O), 158.7 (N]C), 138.6 (C), 136.0 (C), 131.4 (p-
Benzoyl chloride (1.40 g, 10 mmol) was added to a solution of
ammonium isothiocyanate (0.76 g, 10 mmol) in acetonitrile (20 mL)
and the resulting mixture was refluxed for 1 h. Then, the reaction
solution was filtered for removal of ammonium chloride, and
10 mmol of respective aniline was added. The reaction mixture was
refluxed for additional 2 h. The crude product was filtered and
washed with cold acetonitrile to generate thioureas. To a solution of
1 mmol of thiourea dissolved in 5 mL of N,N-dimethylformamide
was added 2 mmol of benzylamine or 4-aminobutanol, 4 mmol of
triethylamine and then 1 mmol of Bi(NO3)3$5H2O. The solution
became black after a few minutes and the mixture was heated for
24 h at ~100 ꢀC. After this time, the suspension was filtered through
a pad of celite, which was washed with 20 mL of di-chloromethane.
The impurity of filtrate was removed via extraction with water
(4 ꢄ 15 mL), the organic phase dried over anhydrous MgSO4 and
evaporated. The crude residue was crystallized from Et2O/petro-
leum ether.
n
3413, 3168, 3067, 2918, 1601, 1566, 1355 e 1198 cmꢁ1. HRESIMS m/z
330.1601 [MþH]þ (calcd for C21H19N3Oþ, 330.1601,
D
¼ ꢁ0.1 ppm).
Ret. time 35.12 min, purity 93.5%. m.p. 94.4e94.9 ꢀC.
4.1.1.5. (Z)eN-((benzylamino) (p-tolylamino)methylene)benzamide
(LQOFG-5). Yield 53%. 1H NMR (400.13 MHz, CDCl3)
d
¼ 12.05 (br,
1H), 8.27 (d, J ¼ 7,1 Hz, 2H), 7.46 (t, J ¼ 7,0 Hz, 1H), 7.41 (t, J ¼ 7,3 Hz,
2H), 7.33 (d, J ¼ 4,5 Hz, 4H), 7.29 (dd, J ¼ 8,7, 4,5 Hz, 1H), 7.20 (d,
J ¼ 8,1 Hz, 2H), 7.15 (d, J ¼ 8.0 Hz, 2H), 5.18 (br, 1H), 4,78 (br, 2H),
2,35 (s, 3H). 13C NMR (100.61 MHz, CDCl3)
d
¼ 177.8 (C]O), 159.1
(N]C), 138.7 (C), 137.3 (C), 133.2 (C), 131.3 (p-CH), 130.8 (2CH),
129.3 (2CH), 128.9 (2CH), 128.0 (2CH), 127.7 (2CH þ p-CH), 125.9
(2CH), 45.3 (NeCH2), 21.2 (p-CH3). FT-IR (KBr)
n 3412, 3212, 3027,
2818, 1651, 1601, 1556, 1358 e 1201 cmꢁ1. HRESIMS m/z 344.1758
[MþH]þ (calcd for C22H22N3Oþ, 344.1757,
D
¼ ꢁ0.1 ppm). Ret. time
36.67 min, purity 96.0%. m.p. 91.2e91.7 ꢀC.
4.1.1.1. (Z)eN-((benzylamino)
((4-nitrophenyl)amino)methylene)
4.1.1.6. (Z)eN-((benzylamino) ((4-(tert-butyl)phenyl)amino)methy-
lene)benzamide (LQOFG-6). Yield 70%. 1H NMR (400.13 MHz, CDCl3)
1
benzamide (LQOFG-1). Yield 66%. H NMR - 60 ꢀC (400.13 MHz,
CDCl3)
d
Z isomer ¼ 12.63 (s, 1H), 5.99 (t, 1H), 4.92 (d, 2H).
d
E
d
¼ 12,09 (br,1H), 8,27 (d, J ¼ 7,1 Hz, 2H), 7,48e7,44 (m,1H), 7,42 (m,
isomer ¼ 11.33 (t, 1H), 8.73 (s, 1H), 4.64 (d, 2H). 13C NMR
2H), 7,40 (m, 2H), 7,35 (m, 4H), 7,29 (m, 1H), 7,19 (d, J ¼ 8,5 Hz, 2H),
(100.61 MHz, CDCl3)
(CeNO2), 142.6 (C), 137.8 (C), 134.8 (C), 132.0 (p-CH), 129.0 (2CH),
128.9 (2CH), 127.9 (2CH), 127.8 (2CH), 127.0 (p-CH), 125.8 (2CH),
d
Z isomer ¼ 177.7 (C]O), 156.9 (N]C), 144.2
5,27 (br, 1H), 4.79 (d, J ¼ 5.3 Hz, 2H), 1.31 (s, 9H); 13C NMR
(100.61 MHz, CDCl3)
d
¼ 177.9 (C]O), 159.1 (N]C), 150.4 (C), 138.8
(C), 133.2 (C), 131.4 (p-CH), 129.3 (2CH), 128.9 (2CH), 128.0 (2CH),
123.6 (2CH), 45.1 (NeCH2).
144.1 (CeNO2), 142.2 (C), 137.2 (C), 129.5 (C), 129.3 (p-CH), 128.3
(2CH), 128.2 (2CH), 127.9 (2CH), 126.9 (2CH), 126.1 (p-CH), 124.7
d
E isomer ¼ 178.1 (C]O); 157.7 (N]C),
127.7 (2CH þ p-CH), 127.2 (2CH), 125.5 (2CH), 45.3 (NeCH2), 34.8
[p-t-bu (C)], 31.5 [p-t-bu (CH3)]. FT-IR (KBr) n 3323, 3060, 2959,
1696, 1595, 1566, 1364 e 1209 cmꢁ1. HRESIMS m/z 386.2227
(2CH), 121.8 (2CH), 45,5 (NeCH2). FT-IR (KBr)
n
3423, 3061, 3027,
[MþH]þ (calcd for C25H28N3Oþ, 386.2227,
D
¼ ꢁ0.0 ppm). Ret. time
2930, 1630, 1601, 1566, 1505, 1329 e 1109 cmꢁ1 HRESIMS m/z
41.04 min, purity 95.8%. m.p. 124.4e125.7 ꢀC.
375.1449 [MþH]þ (calcd for C21H19N4Oþ3 , 375.1452,
Ret. time 34.95 min, purity 95.3%. m.p. 136.6e137.8.
D
¼ 0.7 ppm).
4.1.1.7. (Z)eN-((benzylamino)
((4-iodophenyl)amino)methylene)
benzamide (LQOFG-7). Yield 50%. 1H NMR (400.13 MHz, CDCl3)
d
¼ 12.21 (br, 1H), 8.25 (br, 2H), 7.71 (d, J ¼ 7.9 Hz, 2H), 7.49 (t,
4.1.1.2. (Z)eN-((benzylamino) ((4-bromophenyl)amino)methylene)
J ¼ 6.8 Hz,1H), 7.42 (t, J ¼ 7.6 Hz, 2H), 7.35 (m, 4H), 7.30 (m, 1H), 7.02
1
benzamide (LQOFG-2). Yield 71%. H NMR - 60 ꢀC (400.13 MHz,
(br, 2H), 5.16 (br, 1H), 4.76 (br, 2H); 13C NMR (100.61 MHz, CDCl3)
CDCl3)
d
Z isomer ¼ 12.15 (s, 1H), 8.29 (d, 2H), 7.54e7.48 (m, 5H),
d
¼ 178.0 (C]O), 158.4 (N]C), 139.2 (C), 138.3 (C), 131.5 (p-CH),
129.3 (2CH), 129.0 (2CH), 128.0 (2CH), 127.9 (2CH), 127.7 (2CH þ p-
CH), 127.3 (2CH), 45.4 (NeCH2). FT-IR (KBr) 3353, 3055, 2931,
7.41e7.35 (m, 5H), 7.14 (d, 2H), 5.45 (t, 1H), 4.85 (d, 2H).
d
E iso-
mer ¼ 11.16 (s, 1H). 13C NMR (100.61 MHz, CDCl3)
d
¼ 177.5 (C]O),
n
158.1 (C]N),138.2 (C), 137.6 (C), 134.1 (C),133.1 (2CH),131.6 (p-CH),
128.9 (2CH), 128.8 (2CH), 128.1 (2CH), 127.7 (2CH þ p-CH), 127.4
1654, 1603, 1566, 1508, 1354 e 1207 cm- [1]. HRESIMS m/z 456.0566
[MþH]þ (calcd for C21H19IN3Oþ, 456.0567,
D
¼ ꢁ0.3 ppm). Ret. time
(2CH), 120.6 (CeBr), 44.8 (NeCH2). FT-IR (KBr)
n 3432, 3062, 2930,
38.35 min, purity 90.9%. m.p. 99.6e101.0 ꢀC.
1601, 1559, 1363 e 1196 cmꢁ1 HRESIMS m/z 408.0701 [MþH]þ
(calcd for C21H19BrN3Oþ, 408.0706,
D
¼ 1.2 ppm). Ret. time
4.1.1.8. (Z)eN-((benzylamino) ((4-methoxyphenyl)amino)methylene)
benzamide (LQOFG-8). Yield 85%. 1H NMR (400.13 MHz, CDCl3)
37.52 min, purity 96.5%. m.p. 94.8e96.1 ꢀC.
d
¼ 11.93 (br, 1H), 8.27 (d, J ¼ 7.4 Hz, 2H), 7.46 (m, 1H), 7.41 (m, 2H),
4.1.1.3. (Z)eN-((benzylamino) ((4-chlorophenyl)amino)methylene)
7.33 (m, 4H), 7.29 (m,1H), 7.19 (d, J ¼ 8.6 Hz, 2H), 6.92 (dt, J ¼ 8.9 Hz,
1
benzamide (LQOFG-3). Yield 69%. H NMR -60 ꢀC (400.13 MHz,
2H), 5.06 (br, 1H), 4.77 (br, 2H), 3.81 (s, 3H); 13C NMR (100.61 MHz,
CDCl3)
d
Z isomer ¼ 12.16 (s, 1H), 8.29 (d, 2H, J ¼ 7,6 Hz), 7.56 (t, 1H,
CDCl3)
d
¼ 177.7 (C]O), 159.4 (C), 158.7 (C]N), 138.6 (C), 131.1 (p-
J ¼ 7,2 Hz), 7.48 (t, 2H, J ¼ 7,5 Hz), 7.42e7.37 (m, 6H), 7.31 (m, 1H),
CH), 129.2 (2CH), 128.8 (2CH), 128.2 (C), 127.8 (p-CH), 127.7 (2CH),
127.5 (2CH), 122.2 (2CH), 115.3 (2CH), 55.5 (p-OCH3), 45.2 (NeCH2).
7.21 (d, 2H, J ¼ 8,5 Hz), 5.44 (t, 1H, J ¼ 5,8 Hz), 4.85 (d, 2H,
J ¼ 5,8 Hz).
d
E isomer ¼ 11.20 (t, 0,1H, J ¼ 5,1 Hz), 10.00 (s, 0,1H). 13
C
FT-IR (KBr) n 3376, 3065, 2933, 1652, 1602, 1565, 1510, 1355 e
NMR (100.61 MHz, CDCl3)
d
¼ 177.5 (C]O), 158.2 (N]C), 138.2 (C),
1205 cmꢁ1. HRESIMS m/z 360.1711 [MþH]þ (calcd for C22H22N3Oþ2 ,
137.6 (C), 133.6 (C), 132.5 (p-CH), 131.5 (2CH), 130.1 (2CH), 128.9
(2CH), 128.7 (2CH), 128.0 (p-CH), 127.6 (2CH), 127.1 (2CH), 44.8
360.1707,
D
¼ 1.4 ppm). Ret. time 34.83 min, purity 93.7%. m.p.
107.1e107.6 ꢀC.
(NeCH2). FT-IR (KBr)
n 3435, 3064, 2931, 1601, 1558, 1361 e 1198
cm-1. HRESIMS m/z 364.1208 [MþH]þ (calcd for C21H19ClN3Oþ,
4.1.1.9. (Z)eN-((benzylamino) ((4-fluorophenyl)amino)methylene)
benzamide (LQOFG-9). Yield 73%. 1H NMR (400.13 MHz, CDCl3)
364.1211,
D
¼ 0.8 ppm). Ret. time 36.95 min, purity 88.5%. m.p.
87.3e88.5 ꢀC.
d
¼ 12.09 (s, 1H), 8.26 (d, J ¼ 7.3 Hz, 2H), 7.46 (m, J ¼ 7.2 Hz, 1H), 7.40

126
R.D. do Espírito Santo et al. / European Journal of Medicinal Chemistry 171 (2019) 116e128
(m, J ¼ 7.3 Hz, 2H), 7.34 (m, 4H), 7.31e7.27 (m, 1H), 7.24 (dd, J ¼ 8.8,
18 h at 37 ꢀC in 5% CO2 to allow parasite multiplication. Then,
infected cells were treated with different concentrations of com-
pounds, AmpB or pentamidine for 24 h. The cells were fixed with
methanol and stained with Giemsa and the number of amastigotes
were counted under optical microscopy. The infection index was
obtained by multiplying the percentage of infected macrophages by
the average number of amastigotes per infected macrophages. The
concentration that caused a 50% decrease of growth inhibition
compared to the control was also determined by regression anal-
2H), 7.09 (t, J ¼ 8.5 Hz, 2H), 5.08 (s, 1H), 4.77 (d, J ¼ 4.6 Hz, 2H).); 13
C
NMR (100.61 MHz, CDCl3)
131.8 (C), 131.3 (p-CH), 129.2 (2CH), 129.1 (2CH), 128.8 (2CH), 127.9
d
¼ 177.9 (C]O), 159.0 (N]C), 138.4 (C),
(p-CH), 127.7 (2CH), 127.5 (2CH), 127.0 (2CH), 45.3 (NeCH2). FT-IR
(KBr)
n 3353, 3055, 2931, 1654, 1603, 1566, 1508, 1354 e 1207
cm-1. HRESIMS m/z 348.1509 [MþH]þ (calcd for C21H19FN3Oþ,
348.1507,
D
¼ ꢁ0.8 ppm). Ret. time 34.97 min, purity 95.1%. m.p.
123.2e123.6 ꢀC.
ysis and expressed as IC50-ama in m .
mol Lꢁ1
4.1.1.10. (Z)eN-((benzylamino) ((4-(trifluoromethyl)phenyl)amino)
methylene)benzamide
(400,13 MHz, CDCl3)
(LQOFG-10). Yield
¼ 9.29 (s, 1H), 9.16 (t, J ¼ 5.2 Hz, 1H), 8.12 (d,
42%.
1H
NMR
4.2.5. Evaluation of cytotoxicity on murine macrophages
d
Murine macrophages were used for evaluation of compounds
cytotoxicity as previously described [58]. Briefly, macrophages
were seeded in 96-well flat-bottom plates (TPP, Trasadingen,
J ¼ 8.6 Hz, 2H), 7.93 (d, J ¼ 7.2 Hz, 2H), 7.61 (t, J ¼ 7.4 Hz, 1H), 7.52 (d,
J ¼ 7.9 Hz, 2H), 7.43 (m, 2H), 7.36 (m, 4H), 7.30 (m, 1H), 4.76 (s, 1H),
4.59 (d, J ¼ 5.9 Hz, 2H); 13C NMR (100,61 MHz, CDCl3)
d
¼ 177.5 (C]
Switzerland) at a density of 1 ꢄ 105 cells/well (100
mL/well) in RPMI
O), 156.5 (C]N), 131.7 (p-CH), 129.1 (2CH), 128.9 (2CH), 128.7(2CH),
(2CH þ CH), 127.6 (p-CH), 127.5 (2CH), 125.7 (2CH), 57.0 (CF3), 45.5
(NeCH2). HRESIMS m/z 398.1488 [MþH]þ(calcd for C22H18F3N3Oþ,
1640 medium supplemented with 10% heat-inactivated FBS, 25 mM
HEPES and 2 mM L-glutamine and incubated for 4 h at 37 ꢀC in a 5%
CO2-air mixture, following the addition of the tested compounds or
reference drugs at different concentrations. After that, plates were
incubated for 24 h at 37 2 ꢀC in a 5% CO2-air mixture. For the
398.1480,
D
¼ 2.0 ppm) m.p. dec.
4.2. Biological assays
determination of the half-maximal cytotoxic concentrations (CC50
,
mM), the MTT colorimetric assay was carried out, accordingly to our
4.2.1. Parasites
previous work [57].
Promastigotes of L. amazonensis MPRO/BR/1972/M1841-LV-79
strain was maintained at 28 ꢀC in liver-infusion tryptose medium
(LIT) [56] supplemented with 10% heat-inactivated fetal bovine
serum (FBS) (Gibco).
4.2.6. Selectivity index
The cytotoxicity for host cells and antiprotozoal activity for
promastigotes or intracellular amastigotes forms of L. amazonensis
were compared as the ratio of the CC50/IC50 and expressed as
selectivity index (SI).
4.2.2. Compounds
The guanidine compounds LQOFG1 e LQOFG10, and controls
(pentamidine and amphotericin B e AmpB (Sigma-Aldrich)) were
dissolved in dimethyl sulfoxide, DMSO, (stock solution, 16.7 mg/
mL) and added to the parasite suspension, which furnished ten
concentrations ranging from 0.5 to 100
pound (final DMSO concentration of 0.1%).
4.2.7. Anti-leishmanial in vivo evaluation
To evaluate in vivo leishmanicidal activity of LQOFG-2, female
BALB/c mice (20 4 g) 4-weeks-old (CEMIB, UNICAMP) were sub-
cutaneously inoculated at the left hind-footpad with 1 ꢄ 107 para-
sites/mL of infective promastigotes of L. amazonensis. Six weeks
later, after lesion development, the animals were randomly sepa-
rated in six groups containing five animals each. The infected ani-
mals received daily LQOFG-2 intraperitoneal doses at 0.13 and
0.25 mg/kg/day for 15 days. Stock solutions of LQOFG-2 were pre-
pared daily in PBS 1X after solubilization in DMSO (final concen-
tration of 0.1%). A group of mice infected were treated with the
reference drug AmpB (2 mg/kg/day ꢁ diluted in sterile water ac-
cording to the manufacturer's instructions) or the vehicle e PBS.
Additionally, infected and untreated mice as well as uninfected and
untreated mice were also evaluated. Infection was monitored every
five days by measuring the thickness of the foot lesions with a dial
caliper (Mitutoyo Corp., Japan). Treatment efficacy was determined
by the parasite burden of infected feet using limiting dilution
methodology [59,60].
m
mol Lꢁ1 for each com-
4.2.3. Evaluation of in vitro anti-promastigote activity
In order to determine the half maximal inhibitory concentration
(IC50, mM), the MTT colorimetric assay was used as previously
described [57,58]. Briefly, L. amazonensis promastigote forms were
plated in 96-well plates (TPP, Trasadingen, Switzerland) at a density
of 1 ꢄ 107 parasites/mL (in a final volume of 100
mL) and incubated
at 28 ꢀC in the presence of increasing concentrations of guanidine
compounds or the reference drugs AmpB or pentamidine for 72 h.
Following the incubation period, the MTT assay was done and the
formazan crystals were solubilized with a SDS-HCl mix. Then, the
absorbance was read at 570 nm (Tecan Infinite M200 PRO, plate
reader). The assays were carried out in triplicates and data analysis
was determined by nonlinear regression in order to calculate the
IC50 value.
4.2.8. Toxicity assay for BALB/c mice
4.2.4. Evaluation of in vitro activity against L. amazonensis
intracellular amastigote
Plasma levels of total bilirubin, aspartate (AST) and alanine (ALT)
aminotransferases, alkaline phosphatase (ALP), urea, and creatinine
were determined in blood plasma samples collected to BALB/c mice
at the end of the treatments, using commercial kits (Labtest Diag-
nostica S.A., Brazil) [55].
To determine the antileishmanial activity of the guanidine de-
rivatives against intracellular amastigote of L. amazonensis, murine
peritoneal macrophages obtained as previously described [58]
were plated at a density of 3 ꢄ 105 cells/well on coverslips (13-mm
diameter) previously arranged in a 24-well plate containing RPMI
1640 medium supplemented with 10% inactivated FBS and allowed
to adhere for 4 h at 37 ꢀC in 5% CO2. Adherent macrophages were
infected with Leishmania promastigotes in the stationary growth
phase at a ratio of 5:1 parasite per macrophage at 37 ꢀC in 5% CO2
for 4 h. The non-internalized parasites were removed by washing
and infected cultures were incubated in RPMI 1640 medium for
4.2.9. Evaluation of the mutagenicity by micronucleus test in
peripheral blood cells of mice
For the evaluation of in vivo mutagenicity of compounds pe-
ripheral blood cells of male Mus musculus (Swiss albino) mice
(30 4 g) was used for micronucleus test according to previously
described methodology [46]. Briefly, for the treatments, five ani-
mals were used per group and each group received 25 mg/kg,

R.D. do Espírito Santo et al. / European Journal of Medicinal Chemistry 171 (2019) 116e128
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0.3 mL of hydroxyethylcellulose 0.5%. Pre-stained laminas by acri-
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Animal experiments were approved by the Ethics Committee for
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All in vitro anti-parasitic assays were carried out in triplicates.
The statistical differences between groups were evaluated using
the One-way analysis of variance (ANOVA) test followed by
Student-Newman-Keuls Multiple Comparisons Test (Graph Pad
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Declaration of interest
The authors declare no competing financial interest.
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The authors would like to thank the Brazilian Research Foun-
dations FAPESP (2013/24487-6, 2013/08248-1, 2016/19289-9 and
2017/03552-5), CNPq and CAPES for research fellowships and
financial assistance. Authors are thankful to Prof. Chung Man Chin
and PhDs Diego Chiba and Daniela Hardman for important help
with performance of mutagenicity experiments. We are also
grateful to the post-graduation programs of the Sao Paulo State
University (Unesp), “Science and Technology of Materials”, “Phar-
maceutical Sciences” and “Biotechnology”, for scientific support.
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