Novel steroid derivatives: synthesis, antileishmanial activity, mechanism of action, and in silico physicochemical and pharmacokinetics studies

Article abstract of DOI:10.1016/j.biopha.2018.07.056

The search for new drugs for the treatment of leishmaniasis is an important strategy for improving the current therapeutic arsenal for the disease. There are several limitations to the available drugs including high toxicity, low efficacy, prolonged paren

Full text of DOI:10.1016/j.biopha.2018.07.056

Biomedicine & Pharmacotherapy 106 (2018) 1082–1090
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Novel steroid derivatives: synthesis, antileishmanial activity, mechanism of
action, and in silico physicochemical and pharmacokinetics studies
a
b
a
Juliana da Trindade Granato , Juliana Alves dos Santos , Stephane Lima Calixto ,
a
c
b
Natália Prado da Silva , Jefferson da Silva Martins , Adilson David da Silva ,
a,⁎
Elaine Soares Coimbra
a
Departamento de Parasitologia, Microbiologia e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, 36036-900, Juiz de Fora, Minas
Gerais, Brazil
b
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Juiz de Fora, 36036-900, Juiz de Fora, Minas Gerais, Brazil
Departamento de Física, Instituto de Ciências Exatas, Universidade Federal de Juiz de Fora, 36036-900, Juiz de Fora, Minas Gerais, Brazil
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
The search for new drugs for the treatment of leishmaniasis is an important strategy for improving the current
therapeutic arsenal for the disease. There are several limitations to the available drugs including high toxicity,
low efficacy, prolonged parenteral administration, and high costs. Steroids are a diverse group of compounds
with various applications in pharmacology. However, the antileishmanial activity of this class of molecules has
not yet been explored. Therefore, in the present study, we investigated the antileishmanial activity and cyto-
toxicity of novel steroids against murine macrophages with a focus on the derivatives of cholesterol (CD), cholic
acid (CA), and deoxycholic acid (DA). Furthermore, the mechanism of action of the best compound was assessed,
and in silico studies to evaluate the physicochemical and pharmacokinetic properties were also conducted.
Among the sixteen derivatives, schiffbase2, CD2 and deoxycholic acid derivatives (DOCADs) were effective
against promastigotes of Leishmania species. Despite their low toxicity to macrophages, the majority of DOCADs
were active against intracellular amastigotes of L. amazonensis, and DOCAD5 exhibited the best biological effect
against these parasitic stages (IC50 = 15.34 μM). Neither the CA derivatives (CAD) nor DA alone inhibited the
intracellular parasites. Thus, the absence of hydroxyl in the C-7 position of the steroid nucleus, as well as the
modification of the acid group in DOCADs were considered important for antileishmanial activity. The treat-
ment of L. amazonensis promastigote forms with DOCAD5 induced biochemical changes such as depolarization
of the mitochondrial membrane potential, increased ROS production and cell cycle arrest. No alterations in
parasite plasma membrane integrity were observed. In silico physicochemical and pharmacokinetic studies
suggest that DOCAD5 could be a good candidate for an oral drug. The data demonstrate the potential antil-
eishmanial effect of certain steroid derivatives and encourage new in vivo studies.
Leishmaniasis
Antileishmanial activity
Steroids
Cholesterol
Colic acid
Deoxycholic acid
Apoptosis
1. Introduction
immunosuppressed and those who are not treated in the initial phase of
the disease [3].
Leishmaniasis is a complex of infectious diseases caused by more
Chemotherapy is used for the treatment of leishmaniasis, and a
limited number of drugs is available. In some countries, the first choice
drugs are pentavalent antimonials [4,5]; however, these medications
cause several side effects such as myalgia, anorexia, fever, malaise,
headache, metallic taste sensation, and lethargy [3–5]. Furthermore,
these drugs present toxic effects such as high cardiotoxicity and ne-
phrotoxicity. If the response to pentavalent antimonials is not sa-
tisfactory or there are any restrictions on their use, the option is to use
than 20 protozoan species of the genus Leishmania. These parasites are
transmitted to humans through the bite of infected Diptera females of
the genera Phlebotomus (Old World) and Lutzomyia (New World) during
their blood meal [1,2]. The disease can be asymptomatic or present a
great variety of clinical manifestations, which are classically divided
into cutaneous, mucocutaneous, and visceral forms [3]. The latter is the
most severe form, which may lead to the death of patients who are
⁎
Corresponding author at: Departamento de Parasitologia, Microbiologia e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Juiz de For a, Rua
José Lourenço Kelmer, s/n- Campus Universitário Bairro São Pedro, 36036-900, Juiz de Fora, Minas Gerais, Brazil.
E-mail address: elaine.coimbra@ufjf.edu.br (E.S. Coimbra).
https://doi.org/10.1016/j.biopha.2018.07.056
Received 12 May 2018; Received in revised form 9 July 2018; Accepted 10 July 2018
0753-3322/ © 2018 Published by Elsevier Masson SAS.

J. da Trindade Granato et al.
Biomedicine & Pharmacotherapy 106 (2018) 1082–1090
second-choice drugs such as amphotericin B, paromomycin, and pen-
tamidine. However, these drugs also present serious toxic effect. Mil-
tefosine, originally developed as an anticancer drug, is only adminis-
tered orally [6]. This drug was initially approved for treatment of
leishmaniasis in India and Germany. Subsequently, Bangladesh, Latin
America (Argentina, Bolivia, Colombia, Ecuador, Guatemala, Honduras,
Mexico, Paraguay and Peru), Israel, Germany and USA also approved
the use of this drug for treat leishmaniasis [7]. Although toxicity is not
very common, miltefosine is associated with gastrointestinal problems
and teratogenicity [3].
have been described in the literature by our research group [17–19].
2.1.2. Preparation of cholic (CAD) and deoxycholic acids derivatives
(DOCAD)
2.1.2.1. General procedure for the synthesis of the amides CAD1, DOCAD1
and CAD2, DOCAD2. Bile amides syntheses were performed according
to the procedure previous described by our research group [17]. For
amides CAD2 and DOCAD2, however, DMAP catalysis was used.
CAD2. Beige solid. Yield: 82%. M.p.: 153.2–154.0 °C. I.R. (KBr) v:
3390, 3135, 2936, 2868, 1664, 1552, 1400, 1386. 1H-NMR (300 MHz,
Steroids are a group of diverse compounds that include corticos-
teroids, progestins, estrogens, androgens, vitamin D, and cholesterol
DMSO-d
19−CH
CH skeleton and 23−CH
7β); 3.80 (s, 1H, H-12β); 4.04 (d, 1H, 7−OH, J = 3.0); 4.14 (d, 1H,
12−OH, J = 3.0); 4.36 (d, 1H, 3−OH, J = 4.2); 5.02 (bs, 2H, -NH );
6
), δ (ppm), J (Hz): 0.59 (s, 3H, 18−CH
); 0.97 (d, 2H, 21−CH , J = 6.0); 1.26–2.29 (m, 24H, CH
); 3.17 (d, 1H, H-3β, J = 4.8); 3.62 (s, 1H, H-
3
); 0.81 (s, 3H,
3
3
2
and
[
8]. These compounds are of great interest to the pharmaceutical in-
2
dustry because of their varied pharmacological properties and strong
ability to permeate the lipid bilayer of cells. In addition, small changes
in the structure of these molecules are known to likely result in diverse
biological effects [9]. Cholesterol is one of the most important steroids,
as it is one of the constituents of the lipid bilayer of mammalian cells. In
addition, it is a precursor of several other important compounds in
organisms such as sex hormones, vitamin D, and bile acids [10]. There
are two important classes of bile acids: primary bile acids, produced in
hepatocytes through the modification of cholesterol and secondary bile
acids, produced through modification by bacterial intestinal micro-
biota. Cholic (CA) and deoxycholic (DA) acids are themajor primary
and secondarybile acids, respectively [11]. In addition to the biological
importance of bile acids in organisms, they are expected to lead to great
achievements in the field of pharmacology, such as the development of
new drugs or improvement of the pharmacokinetics of existing drugs
2
6.22 (d, 1H, H-4′, J = 7.8); 6.66 (d, 1H, H-6′, J = 7.8); 6.88 (t, 1H, H-
5′, J = 7.9); 6.93 (s, 1H, H-2′); 9.55 (s, 1H, -NH). 13C-NMR (75 MHz,
DMSO-d
(CH and CH esqueleto); 66.2 (C-7); 70.5 (C-3); 71.0 (C-12); 104.9 (C-
2’); 107.2 (C-4’); 109.1 (C-6’); 128.7 (C-5’); 139.9 (C-1’); 148.8 (C-3’);
6
), δ (ppm): 12.3 (C-18); 17.1 (C-21); 22.6 (C-19); 22.8-46.2
2
+
171.4 (C-24). MS (MALDI): m/z Calc. for [C30
47 2 4
H N O ] [M+H] Calc.
(499.3536) found 499.4271.
DOCAD2. Beige solid. Yield: 21%. M.p.: 136.5–138.0 °C. I.R. (KBr)
v: 3335, 3149, 2935, 2862, 1666, 1551, 1450. 1H-NMR (300 MHz,
DMSO-d
19−CH
CH skeleton); 2.17–2.28 (m, 2H, 23−CH
1H, 12−OH); 4.51 (s, 1H, 3−OH); 5.02 (bs, 2H, -NH
4′, J = 7.5); 6.66 (d, 1H, H-6′, J = 7.5); 6.87 (t, 1H, H-5′, J = 8.1); 6.93
(s, 1H, H-2′); 9.54 (s, 1H, -NH). 13C-NMR (75 MHz, DMSO-d
), δ (ppm):
12.5 (C-18); 17.1 (C-21); 23.1 (C-19); 23.5–46.3 (CH and CH skeleton);
6
), δ (ppm), J (Hz): 0.59 (s, 3H, 18−CH
); 0.95 (d, 2H, 21−CH , J = 4.8); 1.20–1.78 (m, 23H, CH
); 3.80 (s, 1H, H-12β); 4.22 (s,
); 6.22 (d, 1H, H-
3
); 0.84 (s, 3H,
3
3
2
and
2
2
[
12,13].
Recent studies have shown that the activity of compounds such as
6
derivatives of aminoquinolines, natural cinchona alkaloids and 6-thi-
purine derivatives containing 1,2,3-triazole was effectively improved
when these molecules were conjugated to bile acids such as CA, litho-
cholic acid, and chenodeoxycholic acid [14–16]. Considering the im-
portance of this class of molecules and the undeniable need for alter-
native treatments for leishmaniasis, this study aimed to determine the
activity of novel steroids against different species of Leishmania, with an
emphasis on the derivatives of cholesterol, CA, and DA. The mechanism
of action and the in silico physicochemical and pharmacokinetic prop-
erties of the most promising compound were evaluated.
2
47.5 (C-7); 70.0 (C-3); 71.1 (C-12); 104.9 (C-2’); 107.2 (C-4’); 109.1 (C-
6’); 128.8 (C-5’); 140.0 (C-1’); 148.8 (C-3’); 171.4 (C-24). MS (MALDI):
+
m/z Calc. for [C30
H
47
N
2
O
3
] [M] Calc. (482.3508) found 482.3510.
2.1.2.2. General procedure for the synthesis of the aldehydes CAD5 and
DOCAD5. Bile acid (2.45 mmol) was partially dissolved in 50 mL of
CH Cl and to that reaction mixture were added 1.05 equimolar
2 2
amount of 4-hydroxybenzaldehyde, 1.05 equimolar amount of DCC
and catalytic amount of DMAP. The mixture was refluxed and stirred
for 24 h, when the formations of the desired aldehydes were found by
thin layer chromatography (TLC). A simple filtration was carried out to
remove the byproduct (DCU) from the reaction medium and the
residual organic phase was evaporated. The solid obtained was then
subjected to purification on CCS using as eluent a mixture of solvents
2. Material and methods
2
2
.1. Chemistry
.1.1. General methods
CH
2
Cl
CAD5. White solid. Yield: 30%. M.p.: 194.7–196.0 °C. I.R. (KBr) v:
3429, 3326, 2927, 2850, 1764, 1699, 1625, 1575. 1H-NMR (500 MHz,
DMSO-d ), δ (ppm), J (Hz): 0.58 (s, 3H, 18−CH ); 0.61 (s, 3H,
19−CH ); 0.81 (d, 3H, 21−CH , J = 4.5); 0.84–2.20 (m, 24H, CH and
CH skeleton and 23−CH
2
and MeOH.
Melting points (m.p.) were determined using
a MQAPF-301-
Microquimica digital apparatus and are uncorrected. Infrared spectra
−
1
(
wave numbers in cm ) were recorded on a Shimadzu 8400 series
6
3
1
13
FTIR instrument. H NMR and C NMR spectra were recorded on a
Bruker AC-300 or a BRUKER AVANCE DRX300 HD 500. The chemical
shifts (δ) are given in parts per million relative to tetramethylsilane
3
3
2
2
); 3.17 (d, 3H, J = 4.1); 3.20 (bs, 1H, H-3β);
3.60 (s, 1H, H-7β); 3.80 (s, 1H, H-12β); 4.02 (s, 1H, 7−OH); 4.20 (s,
1H, 12−OH); 4.40 (s, 1H, 3−OH); 7.36 (d, 2H, H-2’ and H-6’, J = 8.0);
7.98 (d, 2H, H-3’ and H-5’, J = 8.0); 9.99 (s, 1H, HCO). C-NMR
(75 MHz, DMSO-d ), δ (ppm): 12.3 (C-18); 16.8 (C-21); 22.6 (C-19);
22.7–45.9 (CH and CH skeleton); 66.2 (C-7); 70.4 (C-3); 70.9 (C-12);
(
TMS). All MALDI spectra were obtained using a time-of-flight mass
1
3
spectrometer. Matrix-assisted laser desorption/ionization time-of-flight
MALDI-TOF) mass spectrometry experiments were performed using a
(
6
pulsed nitrogen laser with a wavelength of 337 nm of a Shimadzu
Biotech Axima Performance MALDI-TOF. Elementary analysis data was
performed in a Perkin-Elmer CHN analyzer model 2400. Column
chromatography was performed on Merck silica gel (70–230 mesh).
Reagents and materials were obtained from commercial suppliers and
were used without purification. All reagents used were analytical re-
agent grade.
2
122.6 (C-2’ and C-6’); 131.0 (C-3’ and C-5’); 133.8 (C-4’); 155.1 (C-1’);
171.8 (C-24); 191.9 (HCO).
DOCAD5. White oil. Yield: 30%. I.R. (KBr) v: 3406, 3136, 2936,
1
2862, 1741, 1703. H-NMR (300 MHz, DMSO-d
6
), δ (ppm), J (Hz): 0.58
); 0.83 (d, 3H, 21-CH , J = 4.5);
and CH skeleton and 23-CH ); 3.78 (bs, 1H, H-
(s, 3H, 18-CH
3
); 0.61 (s, 3H, 19-CH
0.87-2.30 (m, 25H, CH
3
3
2
2
Compounds cholic acid derivative 1 (CAD1), deoxycholic acid de-
rivative 1 (DOCAD1), cholic acid derivative 3 (CAD3), deoxycholic acid
derivative 3 (DOCAD3), cholic acid derivative 4 (CAD4), deoxycholic
acid derivative 4 (DOCAD4), schiffbase1 and schiffbase2 (Scheme 1)
12β); 4.23 (m, 1H, 12-OH); 4.50 (s, 1H, 3-OH); 7.35 (d, 2H, H-2’ and H-
6’, J = 8.4); 7.97 (d, 2H, H-3’ and H-5’, J = 8.4); 9.98 (s, 1H, HCO).
13
C-NMR (75 MHz, DMSO-d
6
), δ (ppm): 12.0 (C-18); 16.8 (C-21); 23.1
(C-19); 23.5-45.9 (CH and CH skeleton); 47.5 (C-7); 70.0 (C-3); 71.0
2
1083

J. da Trindade Granato et al.
Biomedicine & Pharmacotherapy 106 (2018) 1082–1090
Scheme 1. Synthetic route to preparation of cholesterol and bile acids derivatives. Reagents and conditions: (i) 1,3- or 1,4-phenylenediamine, DCC, THF, r.t.; (ii) 4-
hydroxybenzaldehyde, DCC, DMAP, CH Cl , reflux; (iii) aromatic aldehydes, MeOH, r.t; (iv) 4-formylbenzoic acid, DCC, DMAP, CH Cl , r.t.; (v) aromatic amine,
2 2 2 2
EtOH, reflux; (vi) EtOH, r.t.
(
(
C-12); 122.7 (C-2’ and C-6’); 131.0 (C-3’ and C-5’); 133.8 (C-4’); 155.1
C-1’); 171.8 (C-24); 191.9 (HCO).
4−CH
−OH); 6.94 (t, 1H, H-10’, J = 7.2); 7.05 (d, 1H, H-12’, J = 7.5); 7.25
t, 1H, H-11’, J = 8.2); 7.35 (d, 1H, H-9’, J = 7.8); 7.98 (d, 2H, H-3’and
H-5’, J = 8.1); 8.16 (d, 2H, H-2’and H-6’, J = 8.1); 8.75 (s, 1H, H-7’).
2
, J = 7.2); 4.90 (bs, 1H, 3-H); 5.46 (bs, 1H, 6-H); 6.78 (bs, 1H,
(
2
.1.3. Preparation of cholesterol derivatives (CD)
1
3
CD1. Cholesterol (0.70 g; 1.8 mmol) was dissolved in CH
2
Cl
2
C-NMR (75 MHz, CDCl
3
), δ (ppm): 12.1 (C-18); 18.9 (C-21); 19.6 (C-
and CH
(
40 mL). After, 4-formylbenzoic acid (0.27 g; 1.8 mmol) and a catalytic
19); 21.3(C-11); 22.8 and 23.0 (C-26 and C-27); 24.1–56.9 (CH
2
amount of DMAP were added and the resulting mixture was stirred
during few minutes, when a solution of dicyclohexylcarbodiimide
skeleton); 75.3 (C-3); 115.5 (C-9’); 116.0 (C-12’); 120.4 (C-6); 123.1 (C-
10’); 128.5 (C-3’ and C-5’); 129.8 (C-11’); 130.2 (C-2’ and C-6’); 133.5
(C-1’); 135.2 (C-1’); 139.6 (C-5); 152.8 (C-13’); 155.9 (C-7’); 165.6
(
2 2
DCC) (0.41 g; 1.9 mmol) solved of CH Cl was added. The mixture was
stirred for 20 h at room temperature until no starting material was
detected. The precipitated byproduct N,N’-dicyclohexylurea (DCU) was
filtered off and the solution was concentrated to give a white solid was
(COO). Elemental analysis for C41
2.30; Found: C 80.46, H 9.19, N 2.40. MS (MALDI): m/z Calc. for
[C41 56NO
] [M+H]+ Calc. (610.4260) found 610.4174.
3
H55NO : calcd. C 80.74, H 9.09, N
H
3
finally purified by column chromatography using CH
2
Cl
2
as eluent.
CD3. Yellow solid. Yield: 76%. M.p.: 163.0–164.0 °C. I.R. (KBr) v:
2947, 2864, 1712, 1626, 1466, 1382, 1277. H-NMR (500 MHz,
1
13
1
Yield: 92%. Compound CD1 was characterized by H and C NMR
spectroscopy, IR spectroscopy and melting point and were in accord
with data in the literature [20].
CDCl
and 27−CH
2H, 19−CH
3
), δ (ppm), J (Hz): 0.72 (s, 3H, 18−CH
, J = 2.0); 0.96 (d, 3H, 21−CH
and CH and CH skeleton); 2.52 (d, 2H, 4−CH
2
3
); 0.89 (2xd, 6H, 26−CH
, J = 6.5); 1.02–2.07 (m,
, J =
3
3
3
3
3
2
2
.1.3.1. General procedure for the synthesis of the compounds cholesterol
derivative 2 (CD2), cholesterol derivative 3 (CD3) and cholesterol derivative
(CD4). Aldehyde CD1 (0.10 g; 0.20 mmol) was dissolved under reflux
8.0); 4.91 (bs, 1H, 3-H); 5.45 (d, 1H, 6-H, J = 3.9); 7.26–7.28 (m, 3H,
H-10’, H-11’ and H-12’); 7.42–7.45 (m, 2H, H-9’ and H-13’); 7.99 (d,
2H, H-3’and H-5’, J = 8.5); 8.16 (d, 2H, H-2’and H-6’, J = 8.0); 8.54 (s,
4
1H, H-7’). 13C-NMR (75 MHz, CDCl
), δ (ppm): 12.1 (C-18); 18.9 (C-
21); 19.6 (C-19); 21.3(C-11); 22.8 and 23.0 (C-26 and C-27); 24.1–56.9
(CH and CH skeleton); 75.2 (C-3); 121.1 (C-9’ and C-13’); 123.0 (C-6);
in ethanol (2 mL). After, 0.20 mmol (equimolar amount) an aromatic
amine (2-hydroxyaniline to give CD2 or aniline to give CD3) or
phenylhydrazine (to give CD4) was added and the resulting mixture
was refluxed for 6 h. Stirring and heating were then stopped and the
mixture was allowed to stand for about 12 h. After this time, the
precipitate was filtered, washed with ethanol and dried in an oven.
CD2. Yellow solid. Yield: 98%. M.p.: 174.1–176.0 °C. I.R. (KBr) v:
3
2
126.6 (C-11’); 128.8 (C-3’ and C-5’); 129.4 (C-2’ and C-6’); 130.1 (C-10’
and C-12’); 133.3 (C-1’); 139.8 and 140.0 (C-5’and C-4’); 151.8 (C-8’);
159.3 (C-7’); 165.6 (COO).
These spectroscopic data are in agreement with the literature [21]
CD4. Yellow solid. Yield: 70%. M.p.: 214.3–215.0 °C. I.R. (KBr) v: 3464,
3291, 2936, 2867, 1697, 1605, 1582, 1535, 1266. 1H-NMR (300 MHz,
−
1 1
3
(
2
1
420, 2938, 2865, 1715, 1626, 1589, 1486, 1276, 1118 cm . H-NMR
300 MHz, CDCl ), δ (ppm), J (Hz): 0.71 (s, 3H, 18-CH ); 0.89 (d, 6H,
and 27-CH , J = 6.3); 0.94 (d, 3H, 21−CH , J = 6.3);
.10–2.03 (m, 32H, 19−CH and CH and CH skeleton); 2.51 (d, 2H,
3
3
6-CH
3
3
3
CDCl
and 27−CH
3
), δ (ppm), J (Hz): 0.67 (s, 3H, 18−CH
3 3
); 0.89 (d, 6H, 26−CH
, J = 7.0); 0.95 (d, 3H, 18−CH , J = 6.5); 1.01–2.03 (m,
3
2
3
3
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J. da Trindade Granato et al.
Biomedicine & Pharmacotherapy 106 (2018) 1082–1090
3
2H, 19−CH
3
and CH
2
and CH skeleton); 2.46 (d, 2H, 4−CH
2
, J =
blank, and the fluorescence intensity was obtained after using spec-
trofluorimeter (FLx 800, Biotek Instruments, Winooski, USA) at 540 nm
excitation/600 nm emission. The activity of the compounds was ex-
pressed as IC50, i.e., the concentration that inhibit 50% intracellular
parasites compared to untreated infected macrophages. Results were
obtained of three independents experiments conducted in duplicates.
All procedures involving the animals were previously approved by the
Ethical Committee for animal handling of UFJF (# 012/2015-CEUA).
Amphotericin B and miltefosine were used as the reference drugs.
7.8); 4.85 (m, 1H, 3-H); 5.40 (d, 1H, 6-H, J = 4.2); 6.89 (t, 1H, H-11’,
J = 7.3); 7.12 (m, 2H, H-10’ and H-12’); 7.25–7.30 (m, 2H, H-9’ and H-
3’); 7.65 (s, 2H, H-3’and H-5’); 7.68 (s, 1H, H-7’); 8.01 (d, 2H, H-2’ and
1
13
H-6’). C-NMR (75 MHz, CDCl
9.6 (C-19); 21.3(C-11); 22.8 and 23,0 (C-26 and C-27); 24.1–56.9 (CH
and CH skeleton); 74.9 (C-3); 113.2 (C-9’ and C-13’); 120.9 (C-6); 123.0
C-11’); 125.9 (C-3’ and C-5’); 129.6 (C-10’ and C-12’); 130.1 (C-2’ and
C-6’); 130.4 (C-1’); 135.9 (C-4’); 139.8 and 139.9 (C-5’and C-7’); 144.4
C-8’); 166.0 (COO).
3
), δ (ppm): 12.1 (C-18); 18.9 (C-21);
1
2
(
(
2.2.5. Evaluation of plasma membrane integrity
2
2
.2. Biological assays
To assess whether treatment with compound DOCAD5 alters the
integrity of the cell membrane, promastigotes of L. amazonensis
7
.2.1. Parasites
(1 × 10 ) were incubated in the presence or absence of this compound
6
Promastigotes of L. amazonensis (IFLA/Br/67/PH8) were cultured in
(54.4 and 108.8 μM) for 24 h at 25 °C. Therefore, the cells (5 × 10 )
Brain heart infusion (BHI - Himedia Mumbai, India) medium supple-
mented with hemin (0.01 mg/mL) and folic acid (0.01 mg/mL).
Leishmania braziliensis MHOM/BR/75/M2903) and L. major (MRHO/
SU/59/P) were cultured in BHI medium supplemented with L-gluta-
mine. In this work, L. amazonensis (IFLA/Br/67/PH8) transfected with
plasmid red fluorescent protein (RFP) B5947 piR1SAT-LUC(a)
DsRed2(b) integrated into the genome was used [22]. Leishmania
amazonensis-RFP promastigotes were kindly provided by Dr. Rodrigo P.
Soares (Centro de Pesquisas René Rachou- FIOCRUZ/MG- Brazil).
Leishmania infantum (MCAN/BR/2008/1112) and L. amazonensis-RFP
were cultured in 199 medium. Under all cultivation conditions were
added 10% of Fetal Bovine Serum (FBS) and the parasites were main-
tained at 25 °C in BOD incubator.
were washed with PBS and seeded in 96-well clear bottom black mi-
croplate. Then, the parasites were incubated with propidium iodide (PI
- Sigma-Aldrich, St. Louis, MO, USA) (1 μg/mL) during 15 min at the
room temperature in the dark. The intensity of fluorescence was mea-
sured by spectrofluorimeter (FLx800) at 540/600 nm of excitation and
emission, respectively. Data were obtained from at the three in-
dependent experiments performed in duplicates. Parasites warmed at
65 °C for 10 min were used as positive control.
2.2.6. Evaluation of the mitochondrial potential membrane (Δψ
m
)
7
To evaluate the Δψ , promastigote forms of L. amazonensis (1 × 10 )
m
treated with the compound DOCAD5 (54.4 and 108.8 μM) were in-
®
cubated with 500 nM of Mitotracker Red CM-H2XROS (MtR) for
4
0 min at 26 °C protect from the light. Untreated parasites were used as
6
2.2.2. Antileishmanial activity on promastigote forms of Leishmania species
negative control. Then, the cells (5 × 10 ) were washed in PBS, and
placed in 96-well clear bottom black microplate. Fluorescent intensity
was measured by spectrofluorimeter (FLx800), wavelengths of 485/
528 nm of excitation and emission, respectively. FCCP (Sigma-Aldrich,
St. Louis, MO, USA) (20.0 μM) was used as a positive control.
The antipromastigote assay was performed by using the MTT
(
Sigma-Aldrich, St. Louis, MO, USA) colorimetric method [23]. There-
6
fore, promastigote forms of L. amazonensis (2 × 10 cells/mL), L. bra-
ziliensis, L. infantum and L. major (3 × 10 cells/mL) in stationary phase
6
were placed in 96-well culture plates in the presence of steroids deri-
vatives (3.12–100.00 μM). After 72 h, the viability of cells was mea-
sured using spectrophotometer microplate reader (Multiscan MS, Lab-
System Oy, Helsink, Finland), at 540 nm. The results were expressed as
concentration needed to inhibit 50% the cellular growth (IC50). The
IC50 values were obtained of three independent experiments conducted
in duplicates. Amphotericin B and miltefosine were used as the re-
ference drugs.
2.2.7. Evaluation of ROS levels
Intracellular ROS levels in L. amazonensis promastigotes was eval-
2
uated using H DCFDA, as previously reported [26]. The parasites were
untreated or treated with the compound DOCAD5 at 54.4 and 108.8
μM, at 25 °C. After 24 h of incubation, the parasites (20 × 10 ) were
harvested, washed in PBS and incubated with 2’,7’-dichlorodihydro-
6
2
fluorescein diacetate - H DCFDA (1 mM) for 30 min in dark at the room
temperature (22 ∼ 24 °C). The ROS levels were evaluated fluorime-
trically (FLx800, BioTek Instruments, Inc., Winooski, VT, USA) at 485/
528 nm of excitation and emission, respectively. Miltefosine (44.0 μM)
was used as a control.
2
.2.3. Cytotoxicity on macrophages
The cytotoxicity on murine macrophages was performed in ac-
cordance with Carmo and colleagues [24], with some modifications.
Murine peritoneal macrophages were obtained from BALB/c mice
previously stimulated with sodium thioglycollate (3%). In brief: the
2.2.8. Cell cycle analysis
6
7
macrophages (2 × 10 cells/mL) were placed in 96-well culture plates
Leishmania amazonensis promastigotes (1 × 10 ) were untreated
in RPMI-1640 medium supplemented with 10% FBS. After 24 h, the
non-adherent cells were removed and the adherent cells were un-
exposed (negative control) or exposed to the presence of steroidal de-
(negative control) or treated with the compound DOCAD5 at 54.4 and
108.8 μM for 24 h. Then, the parasites were fixed in 70% ethanol, re-
ssuspended in RNAse (Sigma-Aldrich, St. Louis, MO, USA) (200 μg/mL)
for 60 min, and incubated with PI (1 μg/mL) for 15 min in dark at the
room temperature (22 ∼ 24 °C). Data acquisition were performed using
a Cytoflexflow cytometer (Beckman Coulter, Indianapolis, IN 46268
United States) equipped with CytExpert 2.0 software (Beckman Coulter,
Indianapolis, IN 46268 United States). A total of 10,000 events were
acquired using PI610ND-A channel. Miltefosine (40.0 μM) was used as a
positive control.
2
rivatives (4.68–150.00 μM) for 72 h at 33 °C and 5% CO . Cell viability
was determined by the MTT method, as previously described. Cyto-
toxicity concentration to reduce 50% of viable cells (CC50) was ob-
tained of three independent experiments conducted in duplicates. The
procedures involving the animals were previously approved by the
Ethical Committee for animal handling of UFJF (# 013/2015-CEUA).
2.2.4. Antileishmanial activity on amastigote forms of L. amazonensis
Murine peritoneal macrophages were obtained as described above.
2.3. In silico analysis
Adherent macrophages were infected with promastigote forms of L.
amazonensis-RFP in stationary phase as previously reported [25]. Then,
the infected macrophages were untreated (negative control) or treated
with different concentrations of steroidal derivatives (3.12–100.00 μM),
To evaluate the possible oral effectiveness DOCAD5, physico-
chemical and pharmacokinetic proprieties of molecule were analyzed as
determined Lipinski’s rules (RO5) [27], including octanol/water parti-
tion coefficient (LogP), molecular weight (MW), number of hydrogen
2
at 33 °C and 5% CO for 72 h. Uninfected macrophages were used as a
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J. da Trindade Granato et al.
Biomedicine & Pharmacotherapy 106 (2018) 1082–1090
bond donor (HBD), and number of hydrogen bond acceptor (HBA),
using Molinspiration software. In addition, the ADMET (absorption,
distribution, metabolism, excretion and toxicity) properties were eval-
uated using the admetSAR software.
species examined in this study (Table 1). The precursor of compound
CD2, schiffbase2, exhibited better antileishmanial activity against
promastigotes of L. amazonensis, L. braziliensis, and L. major (IC50 ∼2
μM) and amastigotes of L. amazonensis (IC50 = 35.45 μM). Cholesterol
only inhibited the growth of promastigotes of L. braziliensis (IC50, 27.9
μM). None of the compounds tested were toxic to murine macrophages
at concentrations up to the maximum tested (150 μM), and the com-
pounds CD1, ShiffBase2 and CD2 exhibited a selectivity index (SI)
above 1 (Table 1).
The antileishmanial effect of CAD and DOCADs is shown in Table 2,
and only DOCADs exhibited relevant biological activity. Most DOCADs
inhibited the growth of all promastigotes of Leishmania species, and
their activities were comparable among the species. The majority of
DOCADs were active on intracellular amastigotes, with IC50 values
between 15.34 and 40.43 μM, being DOCAD5 the best compound. The
tested CADs were not toxic to mammalian cells (CC50 > 150 μM), but
only three DOCAD compounds (DOCAD2, DOCAD3, and DOCAD4)
showed low toxicity to these cells. The compound DOCAD5 showed the
best SI, being ∼10 times more destructive to intracellular parasites
than to the macrophages (Table 2).
2.4. Statistical analysis
All data were obtained from three independent experiments per-
formed in duplicates. The 50% inhibitory concentration (IC50) were
calculated by Grafit® (Erithacus Software Ltd) software. The data were
statically analyzed using one or two-way-ANOVA (analyzes of variance)
followed by the Dunnett or Bonferroni post-test. All statistical analyses
were performed using GraphPad Prism version 5 (GraphPad software,
San Diego, CA, USA). Values were considered as significant when
P ≤ 0.05(*); P ≤ 0.001 (**) and P ≤ 0.0001 (***).
3. Results
3.1. Chemistry
Bile acids and cholesterol derivatives (CD) were obtained from
commercially available CA, DA, and cholesterol (Scheme 1).
CA derivatives (CAD) and DA derivatives (DOCAD) were subjected
to an amide-forming reaction with aromatic amines (1,3- and 1,4-
phenylenediamine), producing the biliar amides CAD1, DOCAD1,
CAD2, and DOCAD2.Furthermore, from the biliar amides CAD1 and
DOCAD1, the conjugates CAD3/DOCAD3 and CAD4/DOCAD4 were
obtained through the condensation of aromatic aldehydes in ethanol,
with good yields. Bile aldehydes CAD5 and DOCAD5 were obtained
directly from CA and DA moiety, respectively, using a Steglich ester-
ification between 4-formylbenzoic acid and a hydroxyl group of the C-3
steroidal nucleus of CA or DA. Conjugates CD2, CD3, and CD4, derived
from cholesterol, were synthesized via C-3 condensation between the
intermediate aldehyde CD1 and aromatic amines with good yields.
Schiffbase1 and schiffbase2 were prepared by condensation of equi-
molar amounts of aromatic aldehydes and amines in ethanol as shown
in Scheme 1.
3
.3. DOCAD5 induced biochemical alterations in promastigotes of L.
amazonensis, but plasma membrane integrity of parasites remained
intact
The DOCADs exhibited promising biological activity, and the most
active compound (DOCAD5) was selected to further study its me-
chanism of action. First, we sought to determine if the plasma mem-
brane of the parasite was intact after treatment. Incubation of
DOCAD5-treated parasites with PI did not significantly increased the
fluorescence intensity, which indicated that the plasma membrane of
the parasites remained intact after treatment with this compound, like
the untreated group (Fig. 1). As expected, parasites heated at 65 °C
(
(
positive control) showed an increase in fluorescence intensity
83.21%).
Leishmania mitochondria, a single organelle in trypanosomatids, is
considered a good drug target; therefore, the effect of the compound
DOCAD5 on this organelle was evaluated. First, the mitochondrial
function in promastigotes was evaluated using the MtR dye. A decrease
in fluorescence intensity, as shown in Fig. 2, indicated the depolariza-
3.2. Antileishmanial activity of steroid derivatives on Leishmania sp
All compounds were assayed against promastigote and amastigote
m
tion of the mitochondrial membrane potential (Δψ ) in L. amazonensis
forms of Leishmania species and their toxicity on macrophages was
evaluated. Evaluation of the antipromastigote activity of the CDs re-
vealed that only CD2 was active against at least two of the Leishmania
treated with DOCAD5 at 54.4 μM (48.83%) and 108.8 μM (50.71%).
Treatment with FCCP, used as the positive control, decreased the
fluorescence intensity by 61.58% relative to the negative control. To
Table 1
Effect of cholesterol derivatives and Schiff bases on promastigotes and amastigotes of Leishmania sp and murine macrophages.
a
Compounds
Antileishmanial activity IC50 (μM)
Macrophages
CC50 (μM)
Selectivity
Index (SI)
b
***
Promastigotes
Amastigotes
L. ama5**
L.ama1*
L. braz2
L. maj3
L. inf4
CD1
> 100
> 100
1.81 ± 0.29
> 100
> 100
> 100
> 100
1.86 ± 0.17
7.00 ± 0.61
> 100
> 100
> 100
1.93 ± 1.93
23.89 ± 2.9
> 100
> 100
> 100
> 100
> 100
69.18 ± 13.76
> 100
35.45 ± 0.74
97.97 ± 20.87
> 100
> 150
> 150
> 150
> 150
> 2.16
–
> 4.23
> 1.53
ShiffBase1
ShiffBase2
CD2
CD3
> 100
> 150
–
CD4
C
AmB
Miltefosine
> 100
> 100
0.15 ± 0.02
22.0 ± 0.5
> 100
> 100
> 100
1.31 ± 0.07
20.0 ± 0.52
> 100
> 100
1.08 ± 0.004
2.64 ± 0.29
> 100
> 100
0.71 ± 0.22
12.52 ± 0.83
> 150
> 150
85.8+ 30.4
131.9+ 3.9
–
–
c
27.9 ± 3.9
1.24 ± 0.06
29.66 ± 0.02
d
120.8
10.53
d
a
Values of inhibitory concentration of 50% of parasites (IC50).
b
c
Values of citotoxicity concentration of 50% of macrophages (CC50).
C (cholesterol) and AmB (amphotericin B). 1L. ama (L. amazonensis), 2L. braz (L. braziliensis), 3L. maj (L. major), 4L. inf (L. infantum). *Leishmania amazonensis
d
wild-type (IFLA/BR/1967/PH8). **L. amazonensis-RFP (IFLA/BR/1967/PH8). *** SI = CC50/ IC50 (amastigotes).
d
Miltefosine and AmB (amphotericin B) were used as reference drugs.
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Biomedicine & Pharmacotherapy 106 (2018) 1082–1090
Table 2
Effect of colic and deoxycholic acid derivatives on promastigotes and amastigotes of Leishmania sp and murine macrophages.
a
Compounds
Antileishmanial activity IC50 (μM)
Macrophages
CC50 (μM)
Selectivity
Index (SI)
b
***
Promastigotes
Amastigotes
L. ama5**
L.ama1*
L. braz2
L. maj3
L. inf4
CAD1
> 100
> 100
> 100
> 100
> 100
> 150
–
DOCAD1
CAD2
32.81 ± 0.76
> 100
19.33 ± 1.36
> 100
31.67 ± 0.61
> 100
29.33 ± 3.07
> 100
23.89 ± 1.99
> 100
> 150
> 150
> 6.27
–
DOCAD2
CAD3
33.31 ± 0.24
> 100
42.28 ± 2.26
> 100
33.54 ± 0.90
> 100
21.88 ± 4.08
> 100
23.33 ± 4.05
> 100
80.81+ 0.32
> 150
3.46
–
DOCAD3
CAD4
29.06 ± 0.53
> 100
15.83 ± 0.29
> 100
31.80 ± 0.46
> 100
28.88 ± 2.33
> 100
40.43 ± 2.97
> 100
53.13+ 3.36
> 150
1.31
–
DOCAD4
CAD5
> 100
> 100
56. 10 ± 8.89
> 100
56.17 ± 0.15
> 100
52.33 ± 0.14
> 100
> 100
> 100
89.85+ 7.97
> 150
< 0.89
–
DOCAD5
54.40 ± 2.17
> 100
> 100
0.15 ± 0.02
22.0 ± 0.5
78.67 ± 11.99
> 100
> 100
1.24 ± 0.06
29.66 ± 0.02
41.19 ± 0.58
> 100
> 100
1.31 ± 0.07
20.0 ± 0.52
48.49 ± 0.52
> 100
> 100
1.08 ± 0.004
2.64 ± 0.29
15.34 ± 1.16
> 100
> 100
0.71 ± 0.22
12.52 ± 0.83
> 150
> 150
> 150
85.8+ 30.4
131.9+ 3.9
> 9.77
–
–
120.8
10.53
c
CA
DOCA
AmB
d
e
Miltefosinee
a
Values of inhibitory concentration of 50% of parasites (IC50).
Values of citotoxicity concentration of 50% of macrophages (CC50).
b
c
d
CA (cholic acid), DOCA (deoxycholic acid) and.
d
AmB (amphotericin B). L. ama (L. amazonensis), 2L. braz (L. braziliensis), 3L. maj (L. major), 4L. inf (L. infantum). *Leishmania amazonensis wild-type (IFLA/BR/
1
1
967/PH8). **L. amazonensis-RFP (IFLA/BR/1967/PH8). *** SI = CC50/ IC50 (amastigotes).
Miltefosine and AmB (amphotericin B) were used as reference drugs.
e
Fig. 1. DOCAD5 does not induce loss of integrity of the plasma membrane of L.
amazonensis promastigotes. To evaluate the integrity of the plasma membrane
of L. amazonensis treated with DOCAD5. Promastigotes were incubated with
Fig. 2. Treatment with DOCAD5 decreases the mitochondrial membrane po-
tential (Δψm) of L. amazonensis promastigotes. Parasites were treated with 54.4
and 108.8 μM of the compound. The cells were then washed and incubated with
Mitotracker Red CM-H2XROS® (500 nM). Fluorescence intensity was de-
termined by fluorimetry using the wavelengths of 540 nm and 600 nm of ex-
citation and emission, respectively. FCCP (20.0 μM) was used as a positive
control. Statistical analyzes were performed using GraphPad Prism 5.0 software
using analysis of variance (One-way ANOVA), followed by Dunnett's post-test:
p < 0.05 (*); p < 0.01 (**). Data were expressed as the mean of three in-
dependent experiments.
54.4 and 108.8 μM of the compound for 24 h. then the cells were washed and
stained with PI. The parasites were analyzed by spectrofluorimetry. Heated
promastigotes (65 °C) were used as positive control. Statistical analyzes were
performed by GraphPad Prism 5.0 software by analysis of variance (One-Way
ANOVA) and statistical differences were analyzed by the Dunnett's test:
p < 0.001 (***). Data were expressed as the mean of three independent ex-
periments.
verify the ROS production in L. amazonensis promastigotes treated with
DOCAD5, the parasites were incubated with H
2
DCFDA. The data
used as a positive control, increase the proportion to 48.88% of cells in
the sub-G0/G1 phase. These results show that treatment of promasti-
gotes with the compound significantly changed the parasite cell cycle.
showed that the treatment with DOCAD5 (54.4 and 108.8 μM) in-
creased ROS production by 50.5% and 60.9%, respectively, relative to
the untreated control (Fig. 3). In parasites treated with miltefosine
(
44.0 μM), the ROS production was increased by 98.25%.
To evaluate changes in the promastigote cell cycle induced by
3.4. In silico physicochemical and pharmacokinetic analyses indicate
compound DOCAD5 is a good oral drug
treatment with DOCAD5, the parasites were stained with PI and ana-
lyzed using flow cytometry. Fig. 4 shows that the proportion of para-
sites in the sub-G0/G1 phase increased to 15.12% and 18.55%, re-
spectively, compared with the untreated control (9.27%). Miltefosine,
The data of the in silico analysis using the molinspiration and
admetSAR software packages are shown in Table 3. Thus, DOCAD5
violated only one of Lipinski’s “rule of five” (LogP ≤ 5, molecular mass
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Table 4
Pharmacokinetic properties of DOCAD5.
Model
DOCAD5
Absorption
Blood-brain barrier
Human intestinal absorption
Caco-2 Permeability
Metabolism
+
+
–
CYP450 2C9 Substrate
CYP450 2D6 Substrate
CYP450 3A4 Substrate
CYP450 1A2 Inhibitor
CYP450 2C9 Inhibitor
CYP450 2D6 Inhibitor
CYP450 2C19 Inhibitor
CYP450 3A4 Inhibitor
CYP Inhibitory promiscuity
Excretion
NS
NS
S
NI
NI
NI
NI
NI
low
Renal Organic Cation Transporter
Toxicity
NI
AMES toxicity
Carcinogens
Acute Oral Toxicity
NT
NC
III
Fig. 3. Treatment with DOCAD5 significantly increases ROS production in L.
amazonensis promastigotes. The parasites were treated with 54.4 and 108.8 Mm
of DOCAD5. Then, the cells were washed and incubated with H2DCFDA
+
(positive absorption); - (negative absorption); NS (non-substrate);
S (substrate); NI (non-inhibitor); NT (non-toxic); NC (non-carcin-
genic).
(1 Mm). Fluorescence intensity was determined by fluorimetry, using 485 nm
excitation wavelengths and 528 nm emission. Miltefosine (44.0 μM) was used as
a positive control. Statistical analyzes were performed using GraphPad Prism
not classified as a cytochrome P450 (CYP) inhibitor. In addition, this
compound did not show carcinogenic effect or AMES test toxicity and
was classified as risk class III for acute oral toxicity (Table 4).
5
.0 software using analysis of variance (One-way ANOVA), followed by
Dunnett's post-test: p < 0.001 (***). Data were expressed as the mean of three
independent experiments.
4. Discussion
Steroids are an important and diverse class of compounds that
perform a variety of functions in eukaryotic organisms and some pro-
karyotes. The ability to easily penetrate the plasma membrane and bind
to cellular receptors makes this group of compounds an interesting
target for the development of new drugs [9]. The results presented in
this work provide information on the antileishmanial activity of steroid
derivatives.
At least 20 Leishmania species are considered to be infective to hu-
mans, and the chemotherapies used to treat leishmaniasis have varied
responses to Leishmania species. Therefore, an initial drug screen was
performed against promastigotes of four Leishmania species: L. amazo-
nensis, L. braziliensis, L. major, and L. infantum. The first three are related
to cutaneous leishmaniasis, whereas the fourth is associated with
visceral leishmaniasis. The promastigotes of L. infantum were less sen-
sitive to CD, including the Schiffbase2, which showed anti-
promastigote activity against the cutaneous leishmaniasis-inducing
species. In addition, no CADs affected promastigotes of Leishmania
species, but, serendipitously, most DOCADs exhibited biological ac-
tivity against these parasitic stages. The differences in sensitivity to the
compounds among Leishmania species may reflect the biochemical and
molecular peculiarities of each species, which would affect the drug
effectiveness with important implications in disease treatment [28].
Leishmania amazonensis was chosen as a model for the anti-
amastigote assays due to the variety of clinical forms of leishmaniasis
that this species can cause, ranging from localized cutaneous leishma-
niasis (LCL) to anergic cutaneous diffuse leishmaniasis [29]. Recently,
this species was isolated from dogs that presented classic clinical
symptoms of visceral leishmaniasis, but this is still under observation
[30]. The data indicate that (i) DOCA alone did not show an inhibitory
effect against intracellular parasites, (ii) DOCADs were effective against
these parasitic stages; and (iii) none of the CADs were active against
amastigotes. These results showed that the absence of hydroxyl in the
C-7 position of the steroid nucleus, as well as the modification of the
acid group, is important to antileishmanial activity. DOCAD com-
pounds are more lipophilic than CADs are, which facilitates their
Fig. 4. Treatment with DOCAD5 at 108.8 μM induces DNA fragmentation in L.
amazonensis promastigotes. Cells were incubated with compound DOCAD5
(54.4 and 108.8 μM) for 24 h. After this period, the parasites were permeabi-
lized with 70% ethanol, and incubated with PI (μg/mL). Evaluation of the cells
cicle was performed using flow cytometry. Graphical representation of the cells
present at each stage of the cell cycle. Statistical analyzes were performed using
GraphPad Prism 5.0 software using two-way ANOVA, followed by the
Bonferroni post-test: p < 0.05 (*) and p < 0.001 (***).Data were expressed as
the mean of three independent experiments.
Table 3
Physicochemical properties of DOCAD5.
Compound
DOCAD5
logP
5.76
MW
nHBA
5
nHBD
2
496.69
LogP (Partition coefficient between n-octanol and water; MW (Molecular
weight); nHBA (number of hydrogen bond acceptor); nHBD (number of hy-
drogen bond donor).
≤
500, number of hydrogen bond acceptor groups ≤ 5, and number of
hydrogen bond donor groups ≤ 10), with a logP of 5.76, which is not
considered to limit the bioavailability of a compound [27]. In addition,
the prediction of ADMET properties suggested that DOCAD5 could
cross biological barriers such as the blood-brain barrier and human
intestinal epithelium. Furthermore, metabolically, the compound was
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Biomedicine & Pharmacotherapy 106 (2018) 1082–1090
permeation into the cellular lipid bilayer [31]. However, this physico-
chemical characteristic may also have contributed, although only
slightly, to the cytotoxicity of some DOCADs. Studies of aminoquino-
line conjugates of CA revealed the increased antileishmanial activity of
compounds, confirming the importance of this bile acid as a drug car-
rier [14].The amphiphilic molecular structure of bile acids encourages
its wide use as a drug carrier owing to the ease of permeating cell
membranes [31].
exhibited promising antileishmanial activity by altering important or-
ganelle functions that are essential for complete cellular development.
Interesting, the treatment did not alter membrane integrity of pro-
mastigotes, excluding necrosis.
In a recent review, the authors showed that many lipids, including
sterols, play important roles in numerous cellular pathways such as cell
signaling and transcriptional regulation [39]. These authors related the
importance of steroids in responses to mitochondrial stress and the
maintenance of mitochondrial homeostasis. In addition, they associated
the effects of DA on the membrane with the activation of the signal
cascades that trigger apoptotic cell death. Some authors also provided
evidence that DA had major effects on the plasma membrane, such as
increased membrane cholesterol and changes in the membrane fluidity
of HTC116 cells [40]. More studies would be necessary to clarify the
effect of DOCAD5 on the plasma membrane of L. amazonensis, including
potential interactions with lipids and cholesterol. However, we did not
exclude the possibility that DOCAD5 acts as an exogenous toxic lipid
that alters the organization or composition of the plasma and organelle
membranes, thereby interfering with signaling pathways and mi-
tochondrial homeostasis, triggering changes that lead to cell death.
In silico studies of physicochemical and pharmacokinetic properties
can be used to predict the behavior of chemicals substances in humans.
Some structural parameters of the molecule provide information that
can be used for the theoretical prediction of whether a drug is a good
candidate for oral administration [27,41]. Thus, we evaluated the
physicochemical properties of DOCAD5, and the results suggest that it
is suitable for oral administration, since it only violated one of Lipinski’s
rules, which does not represent a limitation for oral administration
[27]. The pharmacokinetic characteristics of DOCAD5 indicate that this
compound showed favorable permeation properties through the human
blood-brain barrier and intestinal epithelium. The blood-brain barrier
absorption may be advantageous for the treatment of visceral leish-
maniasis because amastigotes of Leishmania species are sometimes
found in tissues of the central nervous system [42]. The metabolic
characteristics exhibited by the compound indicate that it was meta-
bolized by CYP3A4, the enzyme responsible for metabolizing most
currently available drugs [43]. Furthermore, based on the AMES test,
the compound has been classified as non-toxic and non-carcinogenic,
being assigned to the risk class III for acute oral toxicity, which, ac-
cording to the U.S. Environmental Protection Agency (EPA), are sub-
stances that present median lethal dose (LD50) above 500 mg/kg
[44,45]. Interestingly, in silico physicochemical and pharmacokinetic
studies with the compounds Schiffbase 2, CD2, DOCAD1 and DOCAD2
presented results very similar to that of the compound with the best
antileishmanial activity DOCAD5 (data not shown). Based on these
data, the compounds exhibited satisfactory characteristics that allow
their classification as good candidates for orally administered drugs.
In summary, the DOCADs showed better antileishmanial effect than
those of cholesterol and CADs did, and the most effective compound
was DOCAD5. Treatment with this compound strongly targeted several
important cellular organelles. In addition, studies of the physicochem-
ical and pharmacokinetic properties showed that this compound is
suitable for oral administration.
Regarding structure-activity relationship analysis based on biolo-
gical activity, the organization of the steroidal derivatives was into two
groups: those with CD and those bile acid derivatives. Among the CDs
(
CD1–CD4), only the CD2 conjugate showed leishmanicidal activity,
specifically against promastigote forms of L. braziliensis and L. major.
Notably, the IC50 value was 7.00 μM in L. braziliensis, which was ap-
proximately four times more potent than miltefosine was (IC50, 28.07
μM in L. braziliensis). In contrast, the comparison of the biological ac-
tivity of the active conjugate CD2 with that of the free Schiff base
(
schiffbase2) suggests that the conjugation decrease the activity, and
the unrelated imine had a lower IC50 value than the conjugate did. The
effect of the CDs on amastigotes of L. amazonensis-RFP was similar to
that observed in promastigote forms. The relationship between the
chemical structures of bile acid derivatives, including amides, alde-
hydes, and conjugates of steroid-Schiff bases, and their anti-
promastigote activity indicates a specific trend in the antipromastigote
profile of synthetic derivatives. Specifically, only the DOCADs showed
antileishmanial activity. Therefore, the absence of a hydroxyl group at
the C-7 position of the steroidal skeleton contributed positively to the
antileishmanial activity of this class of molecules. Among the steroid-
Schiff bases conjugates (CAD3, DOCAD3, CAD4, and DOCAD4) the
DOCAD3 conjugate was the most active synthetic compound in the
series against L. braziliensis promastigotes (DOCAD3 IC50 = 15.83 μM).
Finally, of all compounds in the series, the biliar aldehyde DOCAD5
showed the most relevant activity against the intracellular form of the
parasite.
The best compound, DOCAD5, has shown itself ∼10 times more
destructive to amastigotes than to the host cell. A good or remarkable SI
value for new compounds (natural or synthetic origin) is controversial,
ranging from 1 to 20 [32–35]. Although most of the compounds did not
show remarkable antileishmanial activity and SI values, the fact that
they are not toxic to macrophages makes them selective and, therefore,
interesting.
Numerous studies of new compounds with antileishmanial effects
have been conducted; however, the mechanisms of action of these
compounds have not been well investigated, leading to gaps in the
literature. In this study, the L. amazonensis death-inducing mechanism
of the most promising compound investigated, DOCAD5, was eval-
uated. Mitochondria is an important drug target in trypanosomatids
owing to the unique nature of the organelle in these protozoa. In ad-
dition, this organelle is the main site of the production of ROS, the
byproducts of oxidative phosphorylation generated in the electron
transport chain complex (ETC) during mitochondrial respiration. High
concentrations of ROS are damaging to cells, and the maintenance of
the ΔΨ
avoid high levels of ROS [36–38]. Thus, ROS production and changes in
ΔΨ are related events. Our results showed that treatment with
DOCAD5 depolarized the ΔΨ and significantly increased ROS pro-
m
is critical to the proper function of the ETC to generate ATP and
Conflict of interest
The authors declare that there is no conflict of interest regarding
this work.
m
m
duction. These results suggest that treatment of the parasites with
DOCAD5 affected mitochondrial function, culminating in oxidative
stress. The functional impairment of the mitochondria in the treated
parasites is sufficient to generate irreversible damage, leading to cell
death. However, in association with this event, we also confirmed that
the cycle cell of the parasites treated with DOCAD5 was altered; the
population of cells in the sub-G0/G1 phase was higher than that in the
untreated cells was. As the total DNA of the parasite was analyzed, it
was not possible to determine if the affected DNA was nuclear or mi-
tochondrial. These data presented here demonstrate that DOCAD5
Acknowledgments
This work was supported by grants from FAPEMIG and UFJF. JTG
and SLC are grant recipients of CAPES. ESC and ADS are grant re-
cipients of CNPq.
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