A nature-inspired design yields a new class of steroids against trypanosomatids

Article abstract of DOI:10.3390/molecules24203800

Chagas disease and Leishmaniasis are neglected endemic protozoan diseases recognized as public health problems by the World Health Organization. These diseases affect millions of people around the world however, efficient and low-cost treatments are not available. Different steroid molecules with antimicrobial and antiparasitic activity were isolated from diverse organisms (ticks, plants, fungi). These molecules have complex structures that make de novo synthesis extremely difficult. In this work, we designed new and simpler compounds with antiparasitic potential inspired in natural steroids and synthesized a series of nineteen steroidal arylideneketones and thiazolidenehydrazines. We explored their biological activity against Leishmania infantum, Leishmania amazonensis, and Trypanosoma cruzi in vitro and in vivo. We also assayed their genotoxicity and acute toxicity in vitro and in mice. The best compound, a steroidal thiosemicarbazone compound 8 (ID_1260) was active in vitro (IC₅₀ 200 nM) and in vivo (60% infection reduction at 50 mg/kg) in Leishmania and T. cruzi. It also has low toxicity in vitro and in vivo (LD₅₀ >2000 mg/kg) and no genotoxic effects, being a promising compound for anti-trypanosomatid drug development.

Full text of DOI:10.3390/molecules24203800

molecules
Article
A Nature-Inspired Design Yields a New Class of
Steroids Against Trypanosomatids
Elena Aguilera 1,†, Cintya Perdomo 2,†, Alejandra Espindola 2, Ileana Corvo 2, Paula Faral-Tello 3,
Carlos Robello 3,4 , Elva Serna 5, Fátima Benítez 5,6, Rocío Riveros 5,6, Susana Torres 5,
Ninfa I. Vera de Bilbao 5, Gloria Yaluff 5,* and Guzmán Alvarez 2,
*
1
Grupo de Química Medicinal-Laboratorio de Química Orgánica, Facultad de Ciencias, Universidad de la
República, Montevideo C.P. 11400, Uruguay; elepao168@gmail.com
Laboratorio de Moléculas Bioactivas, CENUR Litoral Norte, Universidad de la República, Ruta 3 (km 363),
Paysandú C.P. 60000, Uruguay; cuquis266@gmail.com (C.P.); ale.espindola1991@gmail.com (A.E.);
ilecorvo@gmail.com (I.C.)
2
3
4
5
Unidad de Biología Molecular, Institut Pasteur de Montevideo, Montevideo C.P. 11400, Uruguay;
pfaral@pasteur.edu.uy (P.F.-T.); robello@pasteur.edu.uy (C.R.)
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República,
Montevideo 11200, Uruguay
Departamento de Medicina Tropical, Instituto de Investigaciones en Ciencias de la Salud, Universidad
Nacional de Asunción, San Lorenzo C.P. 2169., Paraguay; elvsern@gmail.com (E.S.);
fatimaapodaca14@gmail.com (F.B.); r.riverosmaidana@gmail.com (R.R.); susitorres1@hotmail.com (S.T.);
nverabilbao@gmail.com (N.I.V.d.B.)
Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Asunción,
San Lorenzo C.P. 2169., Paraguay
6
*
†
Correspondence: gloriayaluff@yahoo.com (G.Y.); guzmanalvarezlqo@gmail.com (G.A.)
Both authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Valeria Patricia Sülsen and Albertina Moglioni
Received: 17 August 2019; Accepted: 12 October 2019; Published: 22 October 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Chagas disease and Leishmaniasis are neglected endemic protozoan diseases recognized as
public health problems by the World Health Organization. These diseases affect millions of people
around the world however, efficient and low-cost treatments are not available. Different steroid
molecules with antimicrobial and antiparasitic activity were isolated from diverse organisms (ticks,
plants, fungi). These molecules have complex structures that make de novo synthesis extremely
difficult. In this work, we designed new and simpler compounds with antiparasitic potential
inspired in natural steroids and synthesized a series of nineteen steroidal arylideneketones and
thiazolidenehydrazines. We explored their biological activity against Leishmania infantum, Leishmania
amazonensis, and Trypanosoma cruzi in vitro and in vivo. We also assayed their genotoxicity and
acute toxicity in vitro and in mice. The best compound, a steroidal thiosemicarbazone compound
8
(ID_1260) was active in vitro (IC50 200 nM) and in vivo (60% infection reduction at 50 mg/kg) in
Leishmania and T. cruzi. It also has low toxicity in vitro and in vivo (LD50 >2000 mg/kg) and no
genotoxic effects, being a promising compound for anti-trypanosomatid drug development.
Keywords: anti-T. cruzi activity in vitro and in vivo; anti-Leishmania spp. activity in vitro and in vivo
;
cytotoxicity and genotoxicity
Molecules 2019, 24, 3800; doi:10.3390/molecules24203800
www.mdpi.com/journal/molecules

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1. Introduction
Chagas disease and Leishmaniasis are neglected endemic protozoan diseases recognized as
public health problems by the World Health Organization [ ]. Chagas disease, caused by the parasite
Trypanosoma cruzi, is found mainly in Latin America and is estimated to affect 6–7 million people and at
least 90–100 million people are at risk of infection in endemic areas [ ]. Important advances in vector
1
2
and transfusion controls of Chagas disease have been achieved by the Chagas Control Initiatives in
the Americas; however, there is still a large number of chronic patients for whom treatment is not
accessible or effective [
are at risk of infection for being exposed to the parasite [
3
]. Leishmaniasis affects 12 million people in 98 countries, and a billion people
]. There are four clinical forms of the disease
3
caused by more than 20 Leishmania species: Visceral Leishmaniasis (VL) (also called Kala-azar), dermal
Leishmaniasis (post-kala-azar), Cutaneous Leishmaniasis (CL), and Mucocutaneous Leishmaniasis
(MCL). The most common form is CL, however, VL can be fatal if untreated [4].
It is important to note that no vaccines are available for any of these diseases to prevent human
infection and the drugs currently used for their treatment have high toxicity [5]. Benznidazole
(commercial names Radanil® Abarax® or Rochagan®) and Nifurtimox (Lampit®) are highly effective
for Chagas disease treatment during the acute phase and in congenital cases, which represent
between 5% and 10% of the cases; however, the efficacy of these drugs decreases during the chronic
phase [6]. The drugs currently used for the treatment of CL and VL have quite serious adverse
reactions, like teratogenic and cardiotoxic effects. For the treatment of CL, parenteral administration
of pentavalent antimony (sodium stibogluconate or pentostam® antimoniate and meglumine or
glucantime®) is carried out for 20 days; and in the case of VL, for 28 days. This must be done under
supervised medical control, greatly increasing its cost. An alarming resistance against these drugs
has been observed, while other drugs, such as pentamidine, rifampicin, amphotericin B, allopurinol,
Miltefosine, and ketoconazole, also used to treat Leishmaniasis have limited therapeutic efficacy and
also resistance problems [7]. It is then an urgent need to develop new, accessible, and effective drugs to
control these diseases [7,8].
A steroid molecule isolated from the eggshell of the tick Rhipicephalus microplus,
HIT1 (N-(3-sulfoxy-25-cholest-5-en-26-oil)-L-isoleucine) was identified as an antimicrobial agent.
This compound is a cholesterol sulfate attached to a residue of L-isoleucine via an amide bond
(Figure 1) [9]. HIT2 (cholest-4,20,24-trien-3-one), a cholesterol derivative extracted from Pentalinon
andrieuxii roots exhibited potent activity against Leishmania mexicana promastigote and amastigote forms.
In addition, a similar compound from Trametes versicolor was characterized with anti-Leishmania
activity with an IC50 in L. mexicana amastigotes of 15
similar to the synthetic HIT3 (Figure 1) were described with antibacterial, antifungal, antiparasitic,
and anticarcinogenic activity [ 12 14]. These compounds are less toxic to mammalian cells than
µM [10,11] Furthermore, steroid molecules
9, –
nitroaromatic compounds used as antiparasitic drugs. Steroid compounds have proven to be less
likely to induce resistance in addition to its greater bioavailability due to their ability to traverse
cell membranes [15–17]. However, no mechanism of action was proposed in any of the models
described [18].

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Figure 1. Boophiline (HIT1) is a steroid molecule present in R. microplus eggs that protects them against
microbes. A similar steroid was reported with moderate antiparasitic activity from P. andrieuxii roots
and T. versicolor (HIT2). In addition, HIT3 was previously reported as a simple steroid compound with
diverse biological activities [18,19]. Brackets show where structural modifications were made for the
design of new steroid derivatives.
Many natural substances are potentially useful for the development of new antiparasitic drugs.
Among these, sterols are an important and ubiquitous class of compounds that provide promising
scaffolds to design and develop new drugs for the treatment of antiparasitic infections like Chagas
disease and Leishmaniasis. However, their structural complexity is a concern for its synthetic
production [10,20–23]. Here, we synthesized and chemically characterized a series of nineteen steroidal
arylideneketones and thiazolidenehydrazines by simple synthesis procedures. We designed three
groups of new compounds: (1) Arylideneketones as HIT3 analogs, (2) thiosemicarbazide derivatives
similar to HIT2, and (3) thiazolidenehydrazines derivatives as HIT1 analogs. We explored their
biological activity against L. infantum, L. braziliensis, and T. cruzi in vitro and in vivo. We also studied
their genotoxicity and acute toxicity in vitro and in mice and we explored their pharmacokinetic profiles.
2. Results and Discussion
2.1. Synthesis of Steroidal Arylideneketones and Thiazolidenehydrazines
As part of our ongoing program in drug development for Chagas disease, we use simple
reagents and simple conditions to get new molecules. The key intermediate was synthesized
from commercially available pregnenolone using the same procedures previously reported
(Figure 2) [24
(3 -hydroxyl-5-pregnen-20-one, 5-pregnen-3
different features of HIT1 , and structures, using simple chemical procedures and low-cost reactants.
–
26]. The stereochemistry of the compounds was determined by pregnenolone
β
β-ol-20-one). For compounds design we combined
,
2
3

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Figure 2. Synthetic procedure for steroidal arylideneketones and thiazolidenehydrazines. RT: Room
temperature (25 ◦C), CDi: 1,1’-carbonyldiimidazole.
We synthesized nineteen new compounds with good to excellent reaction yields. For the
arylideneketones derivatives, we used a classical aldol reaction following the twelve green chemistry
principles [27]. All of the new compounds were characterized by 1H-NMR, 13C-NMR, COSY, HSQC,
HMBC experiments and Mass Spectrometry. The compounds, according to the H-H coupling constants
and the NOE-diff experiments were obtained as the Z-isomers in the case of the thiazolidenehydrazines
at the double bond generated from cyclization (thiazolidene ring). The purity of the synthesized
compounds was assayed by TLC and LC-MS.
2.2. In Vitro Biological Studies
To evaluate their antiparasitic activity, we performed a phenotypic screening using the promastigote
form of L. infantum, L. braziliensis, L. amazonensis, and the epimastigote form of T. cruzi, Tulahuen
2 strain (genotype TcVI) [28] (Table 1) at an initial concentration of 25
µM. We defined as active
compounds those with IC50<25 M. The solubility in water of the compounds was poor so they were
µ
tested at a maximum dose of 25 µM.
For the three compounds derived from the condensation of pregnenolone with thiosemicarbazides,
all of the chemical motifs used yielded active molecules. In contrast, the arylideneketones derivatives
did not show any activity at the evaluated doses. We showed a qualitative correlation between the N
substituent in the thiosemicarbazides with the lipophilicity or size, because compounds with a phenyl
group were less active than those with allyl substituent and no-substitute derivative consecutively.
Compound
8 (ID_1260) was the most active compound in all species of parasites. In vitro it was
35 times more active than Nfx and Bnz, and five times more active than Miltefosine against T. cruzi and
Leishmania, respectively. Moreover, this compound was active in a Leishmania infantum strain isolated
from infected dogs in Uruguay [29] (Table 1). What is more, compound
8 showed an IC50 of less than
200 nM in human macrophages infected with two different strains of L. infantum (the reference strain
and the isolated from dogs), being able to clean the amastigotes from the cell’s cytoplasm (Figure 3).

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This is in good correlation with the activity against promastigotes that we have also seen. Our results
suggest the strong ability of this molecule to affect different forms and species of kinetoplastids.
Table 1. Trypanocidal activity against T. cruzi (epimastigotes), L. amazonensis (promastigotes) and
L. infantum (promastigotes).
IC50 ± %SD
(µM)
T. cruzi
IC50 ± %SD
(µM)
L. amazonensis
IC50 ± %SD
(µM)
L. infantum
IC50 ± %SD (µM)
STRUCTURE
No *
IDENTIFIER #
L. infantum .uy **
Nifurtimox®
Glucantime®
Miltefosine®
Pregnenolone
7 ± 2
Nd
Nd ##
18 ± 2
nd
6 ± 2
20 ± 9
0.9 ± 0.2
>25
10±2
nd
8 ± 2
>25
5 ± 2
>25
>25
1
2
3
1257
1259
1256
>25
12 ± 3
>25
>25
23 ± 5
>25
>25
>25
>25
>25
>25
>25
4
5
6
1417
1288
1279
20 ± 5
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
7
8
1289
1260
>25
>25
<22
>25
>25
1.2 ± 0.3
0.2 ± 0.1
0.2 ± 0.1
9
1154
1291
8 ± 2
nd
nd
>25
>25
>25
>25
10
25 ± 3
11
12
1272
1261
>25
16 ± 3
>25
>25
>25
>25
>25
>25
nd
13
14
1317
1263
nd
nd
>25
>25
>25
>25
8 ± 2
#
* Compounds 15
—
20 were inactive, total data in Table S1 Compounds numbers in our chemical collection **
##
parasites isolated from two dogs in the first Leishmaniasis outbreak in Uruguay [29]. no determined.

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Figure 3. Effect of compound 8 at 200 nM and 5 µM over human macrophages infected with L. infantum.
The infectivity index is the ratio between the number of amastigotes per cell before and after treatment.
A fluorescence microscopy image with DAPI (2-(4-amidinophenyl)-1H -indole-6-carboxamidine)
staining of human macrophages infected with L. infantum is shown (0
side, cells treated with 0.2 M of compound ).Amastigotes are shown with black arrows in the Control
condition around the nucleus.
µM: Control and at the right
µ
8
An interesting finding was that compounds 2, 4, 9, and 14 were active against Leishmania infantum
from Fiocruz collection but not when tested against the strains isolated from Uruguayan dogs. We need
more studies in order to explain this differential behavior but it is important to bear in mind that the
pharmacological response of a drug might differ between strains from different geographic regions.
Only compound 8 was consistently active against all strains.
We evaluated the selectivity of the most active compounds by nonspecific cytotoxicity experiments
against mammalian cells using J774.1 murine macrophages. The selectivity indexes were calculated
as the ratio between IC50 for mammalian cells and IC50 for the parasites (Table 2). Compound
the most selective compound, even if we compare it with their analogs and 10. However, these
two molecules were poorly soluble in the conditions that mammalian cells were assayed, then the
maximum dose tested was 25 M and this influenced the low selectivity indexes we observed. We also
studied the cytotoxicity of compound using a primary culture of macrophages from mice bone
8 was
9
µ
8
marrow and the same results were obtained.
We are the first to report this kind of biological activity for compound 8. Its synthesis is a one-step
reaction with excellent yields that can be done using green chemistry methods and requires low-cost
reactants. In contrast, the amide synthesis (compound 12 for example) has more steps and expensive
reactants. Then, compound
8 can be easily scaled up at low-cost and high yields, which makes it a
promising drug for the treatment of neglected diseases.

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Table 2. Nonspecific cytotoxicity test was carried out with J774.1 murine macrophages and selectivity
indexes were calculated for L. amazonensis (promastigotes), L. infantum (promastigotes), and T. cruzi
(epimastigotes).
IC50 ± SD (µM)
SI *
SI
SI
Compound
Murine
J774.1/L. amazonensis J774.1/L. infantum
J774.1/T. cruzi
Macrophages
2
8
9
10
100 ± 10
50 ± 3
25 ± 2
25 ± 5
100 ± 8
15 ± 1
50 ± 7
400 ± 4
4
>2
nd#
nd
nd
1
nd
250
nd
nd
nd
0.5
56
8
42
3
1
14
13
nd
6
Glucantime
Miltefosine
Benznidazole
nd
nd
nd
57
* SI Selectivity index is the ratio between IC50 in murine macrophages and the IC50 in the parasite. #no determined.
2.3. Toxicology In Vivo
We chose compound
8 for toxicology in vivo assays because it was the most potent and selective
in vitro. To evaluate genotoxicity we performed the micronucleus assay in mice (Table 3). The exposition
of mice to a single oral dose of 150 mg/Kg of the compound did not induce chromatin damage on bone
marrow cells, as the generation of multimicronucleated cells was similar to the negative control.
Table 3. Micronucleus test using 150 mg/Kg of compound 8 in mice.
Treatment *
Number of MnPE **
Number of PEC +
Media of MnPE ±SD ++
Control
8
Cyclophosphamide ***
(50 mg/kg)
19
24
5000
5000
4 ± 1
5 ± 1
36 ± 2
180
5000
* Five identical tests were performed at independent times, ** Sum of the micronucleated polychromatic erythrocytes
(MnPE) found in the five trials, + Total number of polychromatic erythrocytes observed, ++ Media of MnPE per
mouse ± standard deviation *** by intraperitoneal administration.
Interestingly, compound
8 has no acute toxicity in mice, as the LD50 estimated in an up and down
experiment was >2000 mg/kg of body weight, the maximum dose that OECD guidelines recommend
to test. In accordance, the in silico prediction of LC50 using the ProTox-II software was even higher
(Figure 4). The software also predicted that compound
(Figure 4).
8 is not hepatotoxic, mutagenic, or cytotoxic
2.4. Proof of Concept in Vivo
We evaluated compound
8 in an in vivo model of acute Chagas disease and Cutaneous
Leishmaniasis (Figure 5). To compare the efficacy of our compound we incorporated the reference drugs
Benznidazole for Chagas disease and Glucantime for Leishmaniasis. We chose oral administration
because it is the desirable route of administration for vulnerable populations where these neglected
diseases are more frequent. As compound
8 showed poor solubility in water, we developed a new
vehicle to favor the solubilization of lipophilic drugs to increase gastrointestinal absorption (see details
in the experimental section).

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Figure 4. We show the output screen obtained from the open-access toxicity prediction software
ProTox-II using compound 8, (-) indicates no toxicity. The structural similarity of the compound with
those in the software database is around 50%.
Figure 5. In vivo efficacy of compound 8 in mice models for Chagas disease (left) and Leishmaniasis
(right). The black arrow indicates the treatment period where a dose of 50 mg/kg was administered once
a day. In the left graph, the parasite number per mL of blood (parasitemia) is shown at different days
post-infection. The black line indicates 50% of parasitemia relative to the control without treatment. In
the right graph, the paw diameter is represented over time post-infection and the number of parasites
in the lesion at the end of the experiment is indicated.
The in vivo efficacy in both models was similar or better than the reference drugs (Figure 5 and
Table 4). For the cutaneous model of Leishmaniasis, compound
8 was better than Glucantime at less
than half the dose (127 compared to 273 mol/kg). A similar result was obtained in the acute model of
µ
Chagas disease, where our compound showed 62% reduction of parasitemia compared to 96% with
Benznidazole, but administering almost half the dose (127 compared to 200 µmol/kg).
Finally, we performed metabolic stability studies using microsomal and cytosolic hepatic fractions
and used the open-access software to predict pharmacokinetic parameters (Table 5). Compound
8
was stable in a cytosolic and microsomal liver fraction for 4 h (supporting information). Moreover,
the pharmacokinetic parameters are similar or even better than those of available drugs as seen for
Miltefosine and Glucantime [30,31].

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Table 4. In vivo results for the oral administration of 50 mg/kg of compound
Chagas and Leishmaniasis diseases.
8 in mice models of acute
Doses
mg/kg
Doses
µmol/kg
Number of
Parasites
% of
Reduction
Treatment
% of Survivals
Chagas
(0.38 ± 0.02)×106 *
Control
8
Benznidazole
0
50
50
0
127
200
0
62
96
60
100
100
(0.14
(0.01
±
±
0.03)
0.01)
×
×
106 *
106 *
Leishmaniasis
(29 ± 2) × 106
(21 ± 4) × 106
(25 ± 6) × 106
Control
8
Glucantime
0
50
100
0
127
273
0
27
12
100
100
100
* number of parasites per mL blood in the peak of parasitemia.
Table 5. Pharmacokinetic parameters predicted using SwissADME.
Skin
Penetration
Solubility
(mg/mL)
Gastrointestinal
Abortion
Brain
Permeability
Bioavailability
Score
Compound
Lipophilicity
(cm/s)
1.9 × 10−3
2.2 × 103
2.3
Miltefosine
Glucantime
Benznidazole
8
low
low
high
No
No
No
No
−4.0
−11.3
−7.2
−5.8
0.55
0.55
0.55
0.55
3.8
−2.9
0.5
9.7 × 10−3
high
3.9
3. Experimental Section
3.1. General
Reagents were purchased from Aldrich and used without further purification. Melting points
were performed using an Electrothermal Engineering Ltd. melting point apparatus, and the results
were not corrected. 1H NMR and 13C NMR spectra were recorded in the indicated solvent with
a Bruker DPX 400-MHz spectrometer. Chemical shifts are quoted in parts per million downfield
from tetramethylsilane (TMS), and the coupling constants are in Hertz. Structural assignments were
corroborated by COSY, HMBC, and HSQC experiments. All solvents were dried and distilled prior
to use. All of the reactions were carried out in a nitrogen atmosphere. Reactions were monitored by
thin-layer chromatography (TLC) using commercially available precoated plates (Merck Kieselgel 60
F254 silica), and the developed plates were examined under UV light (254 nm) or as iodine vapor stains.
Column chromatography was performed using a 200 mesh silica gel. Mass spectrometry experiments
were performed on a HEWLETT PACKARD MSD 5973 or an LC/MSD-Serie 100 using electronic
impact (EI) or electrospray ionization (ESI), respectively. To determine the purity of the compounds,
microanalyses were done on a Fisons EA 1108 CHNS–O instrument from vacuum-dried samples and
were within
±
0.4 of the values obtained by calculating their compositions. The compounds were
prepared following synthetic procedures previously reported [12,15–17].
3.1.1. General Synthetic-Procedure for Arylidene Ketones
To a solution of pregnenolone (0.316 g, 1.0 equivalent (eq.)) in ethanol (10 mL) was added a
concentrated aquoeus solution of KOH (2.0 eq.). Then, the corresponding aldehyde (1.2 eq.) was
added into the reaction mixture to get the corresponding benzylidine derivative. After completion,
as revealed by the thin-layer chromatography (TLC) in an average span of around 1 h, the reaction
mixture was precipitated using water because of the limited solubility. The precipitate was filtered,
dried and purity was monitored through TLC. It revealed just a single spot which proved the presence
of a single product. For further purification, the product was recrystallized from EtOH to obtain it as a
solid [13].

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Compound 5
(2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(β)-hydroxy-10,13-dimethyl-
1H-cyclopenta[a]phenan-thren-17( )-yl)-3-phenylprop-2-en-1-one (1288).[13] Yield: 86%, white
powder, mp: 128–131 ◦C; 1H RMN (400 MHz, CDCl3)
(ppm): 0.63 (s, 3H), 1.00 (s, 3H), 1.61–1.90 (m,
6H), 2.20–2.38 (m, 3H), 2.82 (t, J = 8.80, 1H); 3.51 (m, 1H); 6.78 (d, J = 16.0 Hz, 1H), 7.39 (m, 3H), 7.55 (m,
3H); 13C RMN (100 MHz, CDCl3)
(ppm): 13.3, 19.5, 21.4, 23.9, 25.4, 31.7, 32.2, 32.8, 37.4, 42.1, 45.7,
β
δ
δ
49.6, 49.2, 49.6, 49.9, 49.9, 50.5, 57.4, 62.3, 71.4, 121.2, 127.7, 128.4, 129.5, 130.2, 135.3, 141.3, 142.4, 201.7
C28H35O2. ESI-MS (m/z): 404.27 (100.0%), 405.27 (30.3%), 406.28 (5.0%). Elemental analysis: C, 83.12;
H, 8.97; O, 7.91.
Compound 3
(2E)-3-(4-Chlorophenyl)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(
hydroxy-10,13-dimethyl-1H-cyclo- penta[a]phenanthren-17( )-yl)prop-2-en-1-one (1256). Yield: 85%,
white powder, mp: 159–161 ◦C; 1H RMN (400 MHz CDCl3)
(ppm): 0.63 (s, 3H), 0.99 (s, 3H), 2.83
(t, 1H, J = 8.5 Hz), 3.52 (m, 1H), 5.35 (t, 1H, J = 2.5 Hz), 6.73 (d, 1H, J = 16.0 Hz), 7.35 y 7.47 (d, 2H,
J = 8.0 Hz, y d, 2H, J = 8.0 Hz), 7.49 (d, 1H, J = 16.0 Hz). 13C RMN (100 MHz CDCl3)
(ppm): 13.2, 19.5,
β)-
β
δ
δ
21.7, 23.4, 25.4, 31.7, 32.3, 32.8, 37.4, 37.9, 39.2, 42.2, 45.4, 50.5, 57.3, 62.4, 71.7, 121.2, 127.3, 129.3, 129.4,
133.7, 136.1, 140.1, 141.5, 200.2 C28H35ClO2. ESI-MS (m/z): 438.23 (100.0%), 440.23 (32.0%), 439.24
(30.8%), 441.23 (9.7%), 440.24 (5.0%), 442.24 (1.5%). Elemental analysis: C, 76.60; H, 8.04; Cl, 8.08; O,
7.29.
Compound 2
2E)-3-(2-Chlorophenyl)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(
hydroxy-10,13-dimethyl-1H-cyclo-penta[a]phenanthren-17( )-yl)prop-2-en-1-one (1259). Yield: 86%
white powder, mp 149–151 ◦C; 1H RMN (400 MHz CDCl3)
(ppm): 0.63 (s, 3H), 0.99 (s, 3H), 2.83 (t,
1H, J = 8.5 Hz), 3.52 (m, 1H), 5.35 (t, 1H, J = 2.5 Hz), 6.73 (d, 1H, J = 16.0 Hz), 7.49 (m, 4H). 13C RMN
(100 MHz CDCl3) (ppm): 13.3, 19.3, 21.4, 23.6, 25.2, 32.3, 32.5, 32.9, 36.2, 37.2, 39.1, 42.7, 45.9, 50.4,
β)-
β
δ
δ
57.5, 62.3, 72.4, 121.2, 127.4, 129.4, 129.4, 133.2, 136.7, 140.4, 141.4, 200.2 C28H35ClO2. ESI-MS (m/z):
438.23 (100.0%), 440.32 (31.0%), 439.21 (29.8%), 441.50 (8.7%), 440.24 (5.0%), 442.24 (1.5%). Elemental
analysis: C, 76.60; H, 8.04; Cl, 8.08; O, 7.29.

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Compound 4
2E)-3-(3-bromophenyl)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(
hydroxy-10,13-dimethyl-1H-cyclo-penta[a]phenanthren-17( )-yl)prop-2-en-1-one (1417). Yield: 96%
white powder, mp 159–161 ◦C; 1H RMN (400 MHz CDCl3)
(ppm): 0.63 (s, 3H), 0.99 (s, 3H), 2.83 (t,
1H, J = 8.5 Hz), 3.52 (m, 1H), 5.35 (t, 1H, J = 2.5 Hz), 6.73 (d, 1H, J = 16.0 Hz), 7.49 (m, 3H), 7.52 (s, 1H).
13C RMN (100 MHz CDCl3)
(ppm): 13.3, 19.3, 21.4, 23.6, 25.2, 32.3, 32.5, 32.9, 36.2, 37.2, 39.1, 42.7,
β)-
β
δ
δ
45.9, 50.4, 57.5, 62.3, 72.4, 121.2, 128.4, 130.4, 130.8, 136.2, 140.7, 143.4, 144.4, 200.2 C28H35BrO2. ESI-MS
(m/z): 482.18 (100.0%), 484.18 (97.3%), 483.19 (30.8%), 485.18 (29.5%), 486.19 (4.5%). Elemental analysis:
C, 69.56; H, 7.30; Br, 16.53; O, 6.62.
Compound 1
2E)-3-(2-bromopyridin-3-yl)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(
hydroxy-10,13-dimethyl-1H-cyclo-penta[a]phenanthren-17( )-yl)prop-2-en-1-one (1257). Yield: 86%
white powder, mp 193–195 ◦C; 1H RMN (400 MHz CDCl3)
(ppm): 0.63 (s, 3H), 0.99 (s, 3H), 2.83 (t,
1H, J = 8.5 Hz), 3.52 (m, 1H), 5.35 (t, 1H, J = 2.5 Hz), 6.73 (d, 1H, J = 16.0 Hz) 7.5 (t, 1H, J = 8.0 Hz), 7.8
(d, 1H, J = 8.0 Hz), 8.0 (d, 1H, J = 8.0 Hz). 13C RMN (100 MHz CDCl3)
(ppm): 13.3, 19.4, 21.2, 22.8,
β)-
β
δ
δ
24.3, 31.4, 31.8, 32.2, 36.4, 37.7, 39.5, 42.1, 45., 50.2, 57.4, 62.4, 72.4, 121.7, 123.5, 132.7, 136.6, 139.4, 140.5,
142.4, 148.7, 204.2 C27H34BrNO2. ESI-MS (m/z): 485.18 (100.0%), 483.18 (98.1%), 484.18 (29.1%), 486.18
(28.5%), 487.18 (4.5%). Elemental analysis: C, 66.94; H, 7.07; Br, 16.49; N, 2.89; O, 6.60.
Compound 15
2E)-3-(4-(methylthio)phenyl)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3(
β
)-
hydroxy-10,13-dimethyl-1H-cyclo-penta[a]phenanthren-17(
β)-yl)prop-2-en-1-one (1262). Yield: 98%
yellow powder, mp 124–126 ◦C; 1H RMN (400 MHz CDCl3) δ (ppm): 0.63 (s, 3H), 0.99 (s, 3H), 2.53 (s,
3H), 2.83 (t, 1H, J = 8.5 Hz), 3.52 (m, 1H), 5.35 (t, 1H, J = 2.5 Hz), 6.73 (d, 1H, J = 16.0 Hz), 7.35 y 7.47 (d,
2H, J = 8.0 Hz, y d, 2H, J = 8.0 Hz), 7.49 (d, 1H, J = 16.0 Hz). 13C RMN (100 MHz CDCl3)
δ (ppm): 13.2,
15.4, 19.3, 21.1, 23.2, 25.4, 32.2, 32.6, 32.9, 37.2, 37.3, 39.8, 42.1, 45.4, 50.1, 57.5, 62.1, 71.2, 121.2, 127.8,
129.4, 129.8, 133.2, 136.5, 140.1, 140.5, 200.3 C29H38O2S. ESI-MS (m/z): 450.26 (100.0%), 451.26 (32.2%),
452.26 (10.2%), 453.26 (1.4%). Elemental analysis: C, 77.29; H, 8.50; O, 7.10; S, 7.11.
O
H
H
HO
H
Compound 6
(2E,4E)-1-((8S,9S,10R,13S,14S,17S)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-5-phenylpenta-2,4-dien-1-one (1279). Yield:
99% yellow powder, mp 116–119 ◦C; 1H RMN (400 MHz, CDCl3)
δ
(ppm): 0.63 (s, 3H), 1.00 (s, 3H),
1.61–1.90 (m, 6H), 2.20–2.38 (m, 3H), 2.82 (t, J = 8.80, 1H); 3.51 (m, 1H); 6.78 (d, J = 16.0 Hz, 1H), 6.73 (t,

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1H, J = 11.0 Hz), 7.39 (m, 3H), 7.55 (m, 3H), 7.49 (t, 1H, J = 11.0 Hz). 13C RMN (100 MHz, CDCl3)
δ
(ppm): 13.1, 19.2, 21.3, 22.1, 24.3, 31.0, 31.5, 31.9, 37.2, 41.2, 45.4, 48.1, 48.5, 48.7, 49.2, 49.6, 50.1, 57.5,
62.2, 71.2, 121.2, 125.5, 126.5, 128.7, 129.5, 130.2, 134.3, 140.2, 141.6, 142.2, 201.3 C30H38O2. ESI-MS
(m/z): 430.29 (100.0%), 431.29 (33.0%), 432.29 (5.5%). Elemental analysis: C, 83.67; H, 8.89; O, 7.43.
Compound 7
(2E,4E)-5-(furan-2-yl)-1-((8S,9S,10R,13S,14S,17S)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,
14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)penta-2,4-dien-1-one (1289). Yield:
98% yellow powder, mp 100–103 ◦C; 1H RMN (400 MHz, CDCl3)
δ (ppm): 0.63 (s, 3H), 1.00 (s, 3H),
1.61–1.90 (m, 6H), 2.20–2.38 (m, 3H), 2.82 (t, J = 8.80, 1H); 3.51 (m, 1H); 6.33 (d, J = 16.0 Hz, 1H), 6.52 (m,
1H), 6.65 (d, J = 16.0 Hz, 1H), 6.71 (m, 1H), 6.75 (m, 1H), 6.78 (m, 1H), 6.95 (d, J = 8 Hz, 1H), 7.45 (m,
1H), 7.75 (d, J = 8, 1H). 13C RMN (100 MHz, CDCl3)
δ (ppm): 13.3, 19.2, 21.2, 23.2, 25.4, 31.7, 32.2, 32.5,
37.5, 42.2, 45.4, 49.2, 49.5, 49.8, 49.9, 50.1, 50.5, 57.2, 62.5, 71.4, 109.4, 109.5, 112.4, 121.7, 130.4, 139.2,
140., 144.5, 147.4, 151.2, 200.3 C28H36O3 ESI-MS (m/z): 420.27 (100.0%), 421.27 (30.8%), 422.27 (5.1%).
Elemental analysis: C, 79.96; H, 8.63; O, 11.41.
Compound 16
(2E,4E)-5-(furan-2-yl)-1-((8S,9S,10R,13S,14S,17S)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,
14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-4-methylpenta-2,4-dien-1-one (1287).
Yield: 96% yellow powder, mp: 110–113 ◦C; 1H RMN (400 MHz, CDCl3)
δ (ppm): 0.63 (s, 3H), 1.00 (s,
3H), 1.61–1.90 (m, 6H), 2.20–2.38 (m, 6H), 2.82 (t, J = 8.80, 1H); 3.51 (m, 1H); 6.33 (d, J = 16.0 Hz, 1H),
6.52 (m, 1H), 6.65 (d, J = 16.0 Hz, 1H), 6.75 (m, 1H), 6.78 (m, 1H), 6.95 (d, J = 8 Hz, 1H), 7.45 (m, 1H),
7.75 (d, J = 8, 1H). 13C RMN (100 MHz, CDCl3)
δ (ppm): 13.5, 14.2, 19.1, 21.0, 23.2, 25.4, 31.3, 32.3, 32.9,
37.2, 42.1, 45.4, 49.1, 49.4, 49.7, 49.9, 50.1, 50.5, 57.2, 62.1, 71.0,109.4, 109.8, 112.2, 121.5, 130.5, 139.8,
140.2, 144.3, 147.6, 151.2, 200.3 C29H38O3 ESI-MS (m/z): 434.28 (100.0%), 435.29 (31.9%), 436.29 (5.5%).
Elemental analysis: C, 80.14; H, 8.81; O, 11.04.
Compound 17
(2E,4E)-5-(thiofen-2-yl)-1-((8S,9S,10R,13S,14S,17S)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,12,
13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)penta-2,4-dien-1-one (1319). Yield:
53% yellow powder, mp 158–164 ◦C; 1H RMN (400 MHz, CDCl3)
δ (ppm): 0.63 (s, 3H), 1.00 (s, 3H),
1.61–1.90 (m, 6H), 2.20–2.38 (m, 3H), 2.82 (t, J = 8.80, 1H); 3.51 (m, 1H); 6.33 (d, J = 16.0 Hz, 1H), 6.52 (m,
1H), 6.65 (d, J = 16.0 Hz, 1H), 6.71 (m, 1H), 6.75 (m, 1H), 6.78 (m, 1H), 7.04 (d, J = 8 Hz, 1H), 7.17 (m,

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1H), 7.69 (d, J = 8, 1H). 13C RMN (100 MHz, CDCl3)
δ
(ppm): 13.2, 19.3, 21.2, 23.2, 25.4, 31.3, 32.1, 32.5,
37.6, 42.5, 45.5, 49.2, 49.4, 49.6, 49.8, 49.9, 50.2, 57.5, 62.2, 71.2, 121.3, 121.5, 128.2, 129.5, 130.5, 139.4,
140.5, 144.7, 147.9, 151.5, 200.2 C30H38O2S. ESI-MS (m/z): 436.24 (100.0%), 437.25 (30.8%), 438.25 (9.7%),
439.24 (1.4%). Elemental analysis: C, 77.02; H, 8.31; O, 7.33; S, 7.34.
3.1.2. General Synthetic-Procedure for Thiosemicarbazones
A mixture of pregnenolone (1.0 eq.), the corresponding thiosemicarbazides (1.0 eq.), catalytic
amount of p-toluenesulfonic acid, and dry toluene (1 mL per 100 mg of reactants) was stirred at 80 ◦C
until disappearance of the pregnenolone (5–8 h, checked by TLC, SiO2, and petroleum ether/EtOAc
70:30). After that, the precipitate was filtered off and washed with petroleum ether. The solid was
crystallized from ethanol [16,17,25,32].
Compound 8
1-(3
mp: 245–248 ◦C; 1H RMN (400 MHz CDCl3)
3.52 (m, 1H), 5.35 (t, 1H, J = 2.5 Hz), 8.56 (s, 2H). 13C RMN (100 MHz CDCl3)
β
-Hydroxy-pregn-5-ene-20E-ylidene)thiosemicarbazone (1260). Yield: 95%, white powder,
(ppm): 0.63 (s, 3H), 0.99 (s, 3H), 2.83 (t, 1H, J = 8.5 Hz),
(ppm): 13.2, 19.5, 21.2,
δ
δ
23.5, 25.2, 31.4, 32.6, 32.9, 37.2, 37.8, 39.5, 42.5, 45.5, 50.7, 57.5, 62.4, 71.2, 121.4, 165.4, 181.2 C22H35N3OS.
ESI-MS (m/z): 389.25 (100.0%), 390.25 (25.7%), 391.25 (10.0%), 392.25 (1.1%). Elemental analysis: C,
67.82; H, 9.05; N, 10.79; O, 4.11; S, 8.23.
Compound 9
1-(3
powder, mp 229–233 ◦C; 1H RMN (400 MHz CDCl3)
J = 8.5 Hz), 3.52 (m, 1H), 3.35 (m, 1H), 5.21 (d, 1H, J = 16 Hz), 5.32 (d, 1H, J = 10 Hz), 5.35 (m, 2H),
5.77 (m, 1H), 8.56 (s, 1H). 13C RMN (100 MHz CDCl3)
(ppm): 13.2, 19.3, 21.5, 23.4, 25.8, 31.9, 32.2,
β
-Hydroxy-pregn-5-ene-20E-ylidene)-4-Allylthiosemicarbazone (1154). Yield 95%, white
δ
(ppm): 0.63 (s, 3H), 0.99 (s, 3H), 2.83 (t, 1H,
δ
32.5, 37.8, 37.4, 39.7, 42.3, 45.5, 47.8, 50.2, 57.6, 62.3, 71.2, 117.6, 121.2, 134.5, 165.6, 181.2. C25H39N3OS.
ESI-MS (m/z): 429.28 (100.0%), 430.28 (28.9%), 431.28 (5.1%), 431.29 (3.9%), 432.28 (1.2%). Elemental
analysis: C, 69.88; H, 9.15; N, 9.78; O, 3.72; S, 7.46.
Compound 10
1-(3
powder, mp: 215–217 ◦C; 1H RMN (400 MHz CDCl3)
J = 8.5 Hz), 3.52 (m, 1H), 3.35 (m, 1H), 6.81 (m, 1H), 7.20 (m, 2H), 7.7 (d, 2H, J = 8.5 Hz). 13C RMN
(100 MHz CDCl3) (ppm): 13.2, 19.3, 21.5, 23.7, 25.8, 31.8, 32.2, 32.9, 37.5, 37.5, 39.4, 42.4, 45.5, 50.5,
β
-Hydroxy-pregn-5-ene-20E-ylidene)-4-phenylthiosemicarbazone (1291).[17] Yield 19% white
δ
(ppm): 0.63 (s, 3H), 0.99 (s, 3H), 2.83 (t, 1H,
δ

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57.5, 62.2, 71.8, 121.7, 126.5, 128.7, 1298, 138.2, 165.5, 181.7 C28H39N3OS. ESI-MS (m/z): 465.28 (100.0%),
466.28 (32.2%), 467.28 (5.1%), 467.29 (4.8%), 468.28 (1.4%). Elemental Analysis: C, 72.21; H, 8.44; N,
9.02; O, 3.44; S, 6.89.
3.1.3. General Synthetic-Procedure for Thiazolylidene Hydrazines
A mixture of the corresponding thiosemicarbazone [25] (1.0 eq.), the α- haloketone (1.2 eq.),
and dry ethanol (1 mL per 100 mg of thiosemicarbazone) was heated at reflux until the disappearance
of the thiosemicarbazide (4–10 h, checked by TLC, SiO2, petroleum ether:EtOAc 70:30). After that,
the mixture was cooled to room temperature, and the precipitate was filtered off and washed with
ethanol: Water (80:20). The solid was crystallized from ethanol or ethanol: Water.
Compound 18
(Z)-2-((E)-(1-((8S,9S,10R,13S,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethylidene)hydrazono)-2,3-dihydrothiazole-4-
carboxylic acid (1258). Yield 99% yellow powder, mp: 281–283 ◦C; 1H RMN (400 MHz CDCl3)
δ
(ppm):
0.63 (s, 3H), 0.99 (s, 3H), 2.83 (t, 1H, J = 8.5 Hz), 3.52 (m, 1H), 5.35 (t, 1H, J = 2.5 Hz), 7.76 (s, 1H). 13
RMN (100 MHz CDCl3) (ppm): 13.2, 19.6, 21.4, 23.5, 25.5, 31.2, 32.2, 32.5, 37.4, 37.8, 39.2, 42.5, 45.5,
C
δ
50.5, 57.5, 62.5, 71.2, 117.5, 121.5, 146.5, 146.5, 158.5, 163.5, 165.8, 180.2. C25H35N3O3S. ESI-MS (m/z):
457.24 (100.0%), 458.24 (29.1%), 459.24 (9.4%), 460.24 (1.3%). Elemental analysis: C, 65.61; H, 7.71; N,
9.18; O, 10.49; S, 7.01.
Compound 19
(Z)-3-allyl-2-((E)-(1-((8S,9S,10R,13S,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,
14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethylidene)hydrazono)-2,3-
dihydrothiazole-4-carboxylic acid (1146). Yield 99% yellow powder, mp: 145–149 ◦C; 1H
RMN (400 MHz CDCl3)
(m, 3H), 5.21 (d, 1H, J = 16Hz), 5.32 (d, 1H, J = 10Hz), 5.77 (m, 2H), 7.76 (s, 1H). 13C RMN (100 MHz
CDCl3) (ppm): 13.2, 19.4, 21.3, 23.4, 25.6, 31.2, 32.3, 32.6, 37.5, 37.9, 39.2, 42.3, 45.5, 47.4, 50.2, 57.7,
δ (ppm): 0.63 (s, 3H), 0.99 (s, 3H), 2.83 (t, 1H, J = 8.5 Hz), 3.52 (m, 1H), 5.35
δ
62.3, 71.3, 117.5, 117.9, 121.2, 134.5, 146., 146.9, 158.2, 163.5, 165.8, 180.3. C28H39N3O3S. ESI-MS (m/z):
497.27 (100.0%), 498.27 (32.2%), 499.28 (10.2%), 500.27 (1.5%). Elemental analysis: C, 67.57; H, 7.90; N,
8.44; O, 9.64; S, 6.44.

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Compound 20
1-(3
β
-Hydroxy-pregn-5-ene-20E-ylidene)-2-(3-allyl-4-(4-chloro-pheny)thiazol-2(3H)-ylidene)azine
(ppm): 0.63 (s, 3H),
(
1144). Yield 52% yellow powder, mp: 140–144 ◦C; 1H RMN (400 MHz CDCl3)
δ
0.99 (s, 3H), 2.83 (t, 1H, J = 8.5 Hz), 3.52 (m, 1H), 5.35 (m, 3H), 5.21 (d, 1H, J = 16Hz), 5.32 (d, 1H,
J = 10Hz), 5.77 (m, 2H), 7.32 (d, 2H, J = 10Hz), 7.44 (d, 2H, J = 10Hz), 7.76 (s, 1H). 13C RMN (100 MHz
CDCl3) δ (ppm): 13.4, 19.8, 21.3, 23.4, 25.6, 31.3, 32.2, 32.6, 37.5, 37.9, 39.2, 42.4, 45.4, 47.6, 50.2, 57.5, 62.3,
71.3, 117.5, 117.9, 120.2, 121.4, 128.2, 128.5, 133.7, 134.4, 146.6, 146.5, 158.7, 163.5, 165.2. C33H42ClN3OS.
ESI-MS (m/z): 563.27 (100.0%), 565.27 (41.9%), 564.28 (38.2%), 566.27 (14.7%), 567.28 (4.4%). Elemental
analysis: C, 70.25; H, 7.50; Cl, 6.28; N, 7.45; O, 2.84; S, 5.68.
Compound 11
1-(3
β
-Hydroxy-pregn-5-ene-20E-ylidene)-2-(3-allyl-4-(4-bromo-pheny)thiazol-2(3H)-ylidene)azine
(ppm): 0.63 (s, 3H),
(
1272). Yield 85%, yellow powder, mp: 179–183 ◦C; 1H RMN (400 MHz CDCl3)
δ
0.99 (s, 3H), 2.83 (t, 1H, J = 8.5 Hz), 3.52 (m, 1H), 5.35 (m, 3H), 5.21 (d, 1H, J = 16Hz), 5.32 (d, 1H,
J = 10Hz), 5.77 (m, 2H), 6.94 (s, 1H), 7.27 (d, 2H, J = 10Hz), 7.55 (d, 2H, J = 10Hz). 13C RMN (100 MHz
CDCl3) δ (ppm): 13.2, 19.5, 21.3, 23.4, 25.5, 31.4, 32.5, 32.9, 37.2, 37.8, 39.1, 42.5, 45.6, 47.7, 50.2, 57.5, 62.2,
71.5, 117.2, 117.6, 120.1, 121.5, 128.2, 128.8, 133.2, 134.4, 146.5, 146.5, 158.4, 163.2, 165.5 C30H38BrN3OS.
ESI-MS (m/z): 609.22 (100.0%), 607.22 (97.8%), 610.22 (38.4%), 608.23 (36.4%), 609.23 (6.7%), 611.23
(11.6%), 612.22 (1.6%). Elemental analysis: C, 65.12; H, 6.96; Br, 13.13; N, 6.90; O, 2.63; S, 5.27.
3.1.4. General Procedure for the Preparation of Amides
A mixture of the corresponding carboxylic acid (1.0 eq.), carbonyl diimidazole (2.0 eq.), and dry
THF (1 mL per 100 mg of acid) was stirred at room temperature for 2 h. After that, the reaction mixture
was cooled at 0 ◦C and the corresponding amine (2.0 eq.) and triethylamine (1.0 eq.) were added.
The mixture was stirred at room temperature until the disappearance of the activated acid (12–24 h,
checked by TLC, Al2O3, petroleum ether:EtOAc 70:30). After that, the solvent was evaporated in vacuo
and the residue was partitioned between methylene dichloride and saturated aqueous solution of
sodium bicarbonate. The organic layer was washed with aqueous phosphate buffer (pH 4–5), dried
with anhydrous sodium sulfate, and evaporated in vacuo. The desired product was purified from the
residue of evaporation by column chromatography (Al2O3, petroleum ether:EtOAc 0% to 40%) [26].
Compound 14
Ethyl-2-((Z)-3-allyl-2-((E)-(1-((8S,9S,10R,13S,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,
12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethylidene)hydrazono)-2,3-
1
dihydrothiazole-4-carboxamido)acetate (1263). Yield 30%, orange oil. HRMN (400 MHz CDCl3)
δ
(ppm): 0.63 (s, 3H), 0.99 (s, 3H), 1.29 (m, 3H), 2.83 (t, 1H, J = 8.5 Hz), 3.52 (m, 1H), 4.13 (m, 2H), 4.23
(s, 2H), 5.35 (m, 3H), 5.21 (d, 1H, J = 16Hz), 5.32 (d, 1H, J = 10Hz), 5.77 (m, 2H), 7.76 (s, 1H), 8.03 (s,

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1H). 13C RMN (100 MHz CDCl3)
δ
(ppm): 13.4, 14.5, 19.6, 21.5, 23.5, 25.4, 31.3, 32.5, 32.9, 37.2, 37.8,
39.2, 40.2, 42.4, 45.5, 47.4, 50.2, 57.7, 62.2, 66.4, 71.2, 117.5, 117.8, 121.4, 134.4, 146.2, 146.2, 158.4, 163.2,
165.3, 169.2. C32H46N4O4S. ESI-MS (m/z): 582.32 (100.0%), 583.33 (37.3%), 584.33 (12.2%), 585.32 (2.6%).
Elemental analysis: C, 65.95; H, 7.96; N, 9.61; O, 10.98; S, 5.50.
Compound 12
((Z)-3-allyl-2-((E)-(1-((8S,9S,10R,13S,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,
15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethylidene)hydrazono)-2,3-
dihydrothiazol-4-yl)(4-cinnamylpiperazin-1-yl)methanone (1261). Yield 21%, orange oil. 1H
RMN (400 MHz CDCl3) δ (ppm): 0.63 (s, 3H), 0.99 (s, 3H), 2.83 (t, 1H, J = 8.5 Hz), 3.52 (m, 1H), 2.79 (dd,
4H, J = 1.6 y 4 Hz), 3.02 (s, 2H), 3.33 (dd, 4H, J = 1.6 y 4 Hz), 5.35 (m, 3H), 5.21 (d, 1H, J = 16 Hz), 5.32
(d, 1H, J = 10 Hz), 5.77 (m, 2H), 6.19 (m, 1H), 6.56 (m, 1H), 7.24 (m, 2H), 7.33 (m, 1H), 7.41 (m, 2H), 7.76
(s, 1H). 13C RMN (100 MHz CDCl3)
δ (ppm): 13.2, 193, 21.2, 23.3, 25.5, 31.3, 32.2, 32.6, 37.2, 37.8, 39.2,
42.3, 45.5, 47.5, 50.2, 50.8, 55.1, 57.2, 62.4, 71.4, 117.2, 117.5, 121.3, 127.7, 128.2, 134.2, 136.5, 146.5, 146.9,
158.2, 163.5, 165.5, 180.3 C40H55N5O2S. ESI-MS (m/z): 681.41 (100.0%), 682.41 (47.9%), 683.41 (15.7%),
684.41 (4.3%). Elemental analysis: C, 72.21; H, 8.13; N, 10.27; O, 4.69; S, 4.70.
Compound 13
(2-((E)-2-(1-((8S,9S,10R,13S,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethylidene)hydrazinyl)thiazol-4-yl)(4-((Z)-3-
1
phenylallyl)piperazin-1-yl)methanone (1317). Yield 15%, orange oil, H RMN (400 MHz CDCl3)
δ
(ppm): 0.63 (s, 3H), 0.99 (s, 3H), 2.83 (t, 1H, J = 8.5 Hz), 3.52 (m, 1H), 2.79 (dd, 4H, J = 1.6 y 4 Hz), 3.02
(s, 2H), 3.33 (dd, 4H, J = 1.6 y 4 Hz), 5.35 (m, 3H), 6.19 (m, 1H), 6.56 (m, 1H), 7.24 (m, 2H), 7.33 (m, 1H),
7.41 (m, 2H), 7.76 (s, 1H). 13C RMN (100 MHz CDCl3)
δ (ppm): 13.3, 19.5, 21.3, 23.6, 25.2, 31.2, 32.2,
32.9, 37.5, 37.9, 39.2, 42.4, 45.5, 47.5, 50.2, 50.8, 55.2, 57.2, 62.6, 71.2, 117.2, 117.6, 121.2, 127.2, 128.8,
134.4, 136.5, 146.1, 146.5, 158.2, 163.2, 165.6, 180.8 C40H55N5O2S. ESI-MS (m/z): 641.38 (100.0%), 682.41
(47.9%), 683.41 (15.2%), 684.41 (5.9%). Elemental analysis: C, 72.21; H, 8.13; N, 10.27; O, 4.69; S, 4.70.
3.2. Anti-Parasitic Test In Vitro
L. amazonensis and L. infantum (MHOM/BR/2002/LPC-RPV) were obtained from Fiocruz (Collection
of Oswaldo Cruz Foundation, Rio de Janeiro, Brazil), and promastigotes were cultured as described
with some modifications [33
ornithine, 0.4% fructose, 0.6% malate, 0.05% fumarate, and 0.06% succinate, 20% Fetal bovine serum,
,
34], at 28 ◦C in an RPMI medium supplemented with 0.7% glucose, 0.1%

Molecules 2019, 24, 3800
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vitamins, and amino acids solution (Gibco). Briefly, all solids (for 1 L) including RPMI were dissolved
in 600 mL of distilled water and afterward amino acids and vitamin solutions were added, pH was
adjusted to 7.2 with NaOH. Finally, water is added to 800 mL final volume, sterilized by filtration
with 0.22
section with T. cruzi epimastigotes could be used as a low-cost alternative for Leishmania spp. culture).
Assays were performed using 2
106 promastigotes per well cultivated in 96-well plastic plates.
Compounds were dissolved in dimethylsulfoxide (DMSO). Different serial dilutions (25, 12.5, 6.25,
3.13, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05) M of the compounds with a final volume up to 200 L were added.
After 48 h at 26 ◦C, 20
L of a 2 mM resazurin solution was added, and the oxidation-reduction was
quantified at 570 and 600 nm. The solution of resazurin was prepared at 2.5 mM in phosphate-buffered
solution (PBS), pH 7.4, and filtered through 0.22 m membranes prior to use. Resazurin sodium
µ
m pore filter and stored at 4 ◦C until use (the same culture media described in the next
×
µ
µ
µ
µ
salt was obtained from Sigma-Aldrich (St. Louis, MO, USA) and stored at 4 ◦C protected from light.
The efficacy of each compound was estimated by calculating the IC50 values using OriginLab8.5®
sigmoidal regression. Each anti-proliferative experiment was done in duplicate, and each concentration
was tested in triplicate.
For the in vitro anti-T. cruzi activity, we used epimastigotes of the Tulahuen 2 strain (genotype
TcVI) grown in an axenic milieu (BHI-Tryptose). Cells from a 5–7-days-old culture were inoculated in
fresh culture milieu to give an initial concentration of 106 cells/mL. The absorbance at 600 nm of the
cells in culture was measured every day. At day five, the milieu was inoculated with different doses
of the compounds (25, 12.5, 6.25, 3.13, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05) µM from a stock solution in DMSO
(DMSO concentration in the culture milieu never exceeded 0.4%). Control parasites were cultivated
in 0.4% DMSO. Each concentration of the compound was evaluated in duplicate. At five days, the
absorbance of the culture was measured and compared to the control and the IC50 values calculated for
each compound using OriginLab8.5® sigmoidal regression. Each experiment was done in duplicate,
and each concentration was tested in triplicate.
For assessing the amastigote form of all Leishmania strains were cultured in the RPMI described
above. Parasites were seeded at 1
obtain a growth curve. For IC50 experiments, parasites were seeded at 3
with serial dilutions of compounds starting from 100 g/mL Amphotericin B, 50
×
106 cells/mL and at each time point counted at least five times to
×
106 cells/mL and incubated
M nifurtimox,
µ
µ
25
µ
M miltefosine. Control conditions of par◦asites without drug (100% growth) and medium without
parasites were included. After 72 h at 28 C parasite viability was determined by the resazurin
method described [35]. Infection with L. infantum. THP-1 monocytes (ATCC®TIB-202
™) were grown
following ATCC recommendations and seeded at 30,000 cells/well onto 18-mm round glass coverslips
in 12 wells. For stimulation cells were incubated with 100 nM of PMA for 48 h, PMA was washed and
cells left with growth media for 24 h more after incubation with parasites. 30,000 parasites/well were
added and left to interact. After 48 h, coverslips were washed with PBS, fixed with 95% (v/v) ethanol,
and stained with Fluoroshield
™ with DAPI (Sigma). Infectivity was assessed considering invasion
and replication capacity counting infected cells and parasites per infected cell, respectively. Infectivity
index calculation = (% of infected cells X (amastigotes per cell))/number of total counted cells. Image
acquisition was made with a Leica TCS-SP5 laser scanning confocal microscope (Leica Microsystems
GmbH, Wetzlar, Germany) using the LASAF v.2 software (Leica Microsystems, Wetzlar, Germany) and
observed using a 356 nm laser excitation line and an emission bandwidth from 461 nm under a 20
water immersion objective. Further image analysis, including quantitation and processing of figures
×
were made using Fiji (http://fiji.sc/) [35].
3.3. Nonspecific In Vitro Cytotoxicity of Mammalian Cells
J774.1 murine macrophages (ATCC, USA) were grown in a DMEM culture milieu containing 4 mM
L-glutamine and supplemented with 10% FCS. Cells were seeded in a 96-well plate (5.00 × 104 cells
in 200 µ
L culture medium) and incubated at 37 ◦C in a 5% CO2 atmosphere for 48 h, to allow cell
adhesion prior to drug testing. Afterward, cells were exposed for 48 h to the compounds (25–400 µM)

Molecules 2019, 24, 3800
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or the vehicle for control (0.4% DMSO), and additional controls (cells in medium) were used in each
test. Cell viability was then assessed by measuring the mitochondria-dependent reduction of MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan. For this purpose, MTT in
sterile PBS (0.2% glucose), pH 7.4, was added to the macrophages to achieve a final concentration of 0.1
mg/mL, and the cells were incubated at 37 ◦C for 3 h. After removing the medium, formazan crystals
were dissolved in 180 µL of DMSO and 20 µL of MTT buffer (0.1 M glycine, 0.1 M NaCl, 0.5 mM EDTA,
pH 10.5), and the absorbance at 560 nm was measured. The IC50 was defined as the drug concentration
at which 50% of the cells were viable, relative to the control (no drug added), and was determined by
analysis using OriginLab8.5® sigmoidal regression (% of viable cells compared to the logarithm of
the compound concentration). Tests were performed in triplicate. For the primary culture of mice
macrophages, were taken from the mice bones with PBS, then resuspended in an RPMI culture media,
*106 cells per well, incubated immediately with the compound (25
MTT assay was performed [26,33].
µM), then at 48 h of incubation in an
3.4. Vehicles/Formulation Preparation
Compound 8 was disposed in a mixture composed of a surfactant (10%), containing Eumulgin HRE
40 (polyoxyl-40hydrogenated castor oil), sodium oleate, and soya phosphatidylcholine (8:6:3), and an oil
phase (10%) containing cholesterol and phosphate buffer (pH 7.4) (80%). For formulation, cholesterol,
Eumulgin HRE 40, and phosphatidylcholine previously pulverized in mortar were dissolved in
chloroform and the solvent was evaporated under vacuum to dryness. In parallel, sodium oleate was
dissolved in phosphate buffer and left in an orbital shaker for 12 h at room temperature. The latter was
then added to the evaporated residue, and the mixture was homogenized and placed in an ultrasonic
bath at full power for 30 min and kept at room temperature until use [26].
3.5. In Vivo Micronucleus Test
For the in vivo micronucleus test, approximately three-month-old CD-1 male mice were housed
in polycarbonate cages at RT (25 ◦C) and a photoperiod of 12 h throughout the study. Compound
8
and vehicle were orally administered twice, at days one and two, to groups of five mice at a dose
of 150 mg/kg of body weight. Mice were sacrificed 24 h after the last administration, and the bone
marrow was prepared for evaluation with slight modifications of the method reported by Schmid [36].
At least two slides of the cell suspension per animal were made. The air-dried slides were stained
with Giemsa stain (5% in phosphate buffer, pH 7.4) and examined at 1000x magnification. Small round
or oval bodies, the size of which ranged from about 1/5 to 1/20 of the diameter of a polychromatic
erythrocyte (PCE), were counted as micronuclei. A total of 1000 PCEs were scored per animal by
the same observer for determining the frequencies of micronucleated polychromatic erythrocytes
(MNPCEs). Cyclophosphamide, 50 mg/kg, administered intraperitoneally (i.p.) 24 h before mouse
sacrifice, was used as a positive control. For statistical analysis, the homogeneity of variances of data
was tested by the analysis of variance (ANOVA) test (P < 0.05) using the EpiInfo (3.5.1) software [25].
3.6. In Vivo Acute Oral Toxicity in Mice
The in vivo 50% lethal dose (LD50) for compound 8 was determined according to the guidelines
of the Organization for Economic Cooperation and Development (OECD). Briefly, healthy young
adult male B6D2F1 mice (30 days old, 25 to 30 g) were used in this study. Initially, the compound
was dissolved in the vehicle described above (3.4 section) and was administered at 2000 mg/kg,
by orogastric cannula, to one animal. The animal was fasted, maintained, and observed for 48 h.
If the mouse survives, another animal receives the same dose, and 48 h later, a third animal. If there
are no toxicity signs the experiment is finished 14 days after administration, with the euthanasia
of the animals according to the OECD guidelines. Observations of the general status of the organs
were performed after sacrifice. The PROTOX software was used to predict the LD50 of compound
8
(http://tox.charite.de/protox_II/) [26].

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3.7. In Vivo Anti-T. Cruzi Studies (Acute Model)
BALB/c male mice (30 days old, 25 to 30 g) bred under specific pathogen-free conditions were
infected by intraperitoneal injection with 1x103 blood trypomastigotes of the Y strain. The mice were
divided into three groups. One group of animals, n = 8, was used as a control (treated orally with
the vehicle), and two groups of animals, n = 8 each, were treated with compound
parasitemia was counted five days postinfection (Week 1), and the treatment was begun the following
day (6th day). Bnz (at 50 mg/kg body weight [BW]/day, for 14 days) or (at 50 mg/kg BW/day, for 14
8 and Bnz. Initial
8
days) were administered orally, using the described vehicle. Parasitemia in the control and treated
mice were determined once a week after the first administration, for 60 days after the beginning of
treatment, in tail vein blood. Additionally, the mortality rate was recorded [26].
3.8. In Vivo Anti-Leishmania Studies in Cutaneous Mice Model
Female and male BALB/c mice were supplied by the IFFA-CREDO, Lyon, France, and were
bred at the Instituto de Investigaciones en Ciencias de la Salud, Asuncion, Paraguay. Golden
hamsters (Mesocritus auratus) were used to maintain the parasites. For the infection L. amazonensis
(IFLA/BR/1967/PH8) were used. The parasites were maintained by passage every six to eight weeks in
hamsters. BALB/c mice (n = 8) were inoculated in the right hind footpad with 2
×
106 amastigotes
obtained from donor hamsters. The parasites were delivered in 100 L of phosphate-buffered saline
µ
(PBS). Disease progression was monitored by the measurement of lesion diameters weekly for up to
seven to 12 weeks. In all experiments, treatment was initiated one or two weeks after inoculation,
when the infection was well established and lesions were obvious. Two days before the administration
of the drug, the mice were randomly divided into groups of eight. N-Methylglucamine antimonate
was administered to the BALB/c mice 100 mg/Kg of body weight daily for 20 days by the subcutaneous
route. Compound
8 was administered orally at 50 mg/Kg of bodyweight. The animals were sacrificed
two weeks after the cessation of treatments to assess parasitological loads in the infected footpad.
Briefly, the mice were sacrificed, and the lesions of the infected footpad were excised, weighed, and
homogenized in a tissue glass grinder and then homogenized in 5
France) tissue culture medium supplemented with 10% FCS, 1 mL of glutamine (29.4
penicillin (100 U/mL), and streptomycin (100
g/mL). After seven days of incubation at 27 ◦C, the plates
µl of RPMI 1640 (GIBCO, Paris,
µg/mL; GIBCO),
µ
(25 cm2 Falcon T) were examined with an inverted microscope (Olympus) at a magnification of 400x.
The number of parasites per gram in the lesion was calculated by the following equation: Parasite
burden = geometric mean of the number of parasites in each duplicate/(number of microscope field
counted x weight of lesion x (25000) hemocytometer correction factor). Statistical analysis: The mean
and standard deviation were calculated by using Microsoft EXCEL software. Comparisons of parasite
suppression in the infected footpads of the untreated and drug-treated groups were done by analysis of
variance (ANOVA) and Student’s T-test. Data were considered statistically significant at P < 0.05 [37].
3.9. Calculation of the Pharmacokinetic Parameters
The predictions were made with the open-access SwissADME software (http://www.swissadme.
ch), a tool that allows the prediction of different pharmacokinetic parameters such as water
solubility, gastrointestinal absorption, skin penetrability, lipophilicity, bioavailability, etc (Figure S1).
The SwissADME software input uses the SMILES codes of the molecules, which were generated with
the ChemBioOffice 2010 program [38].
3.10. Liver fraction Stability Studies
For the determination of liver fraction stability, rat liver microsomal and cytosolic proteins were
used. They were prepared according to the previously described methodology [39]. The protein
content of the microsomal and cytosolic fractions was determined by the bicinchoninic acid assay from
Sigma, as suggested by the manufacturer. The final concentration in the aqueous medium of 8 was

Molecules 2019, 24, 3800
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400 µM and was prepared from a stock solution in DMSO. The solutions were further homogenized
and incubated at 37 ◦C for 1 to 4 h. After that, thin-layer chromatography of ethyl acetate extracts was
performed in order to evaluate the presence of decomposition products (Figure S2).
4. Conclusions
In this work, we introduced a new class of steroids as anti-kinetoplastid agents. We evaluated
the trypanosomicidal effect in T. cruzi and Leishmania spp. in vitro and in vivo, and also studied
the toxicological profile of the active compounds. Nineteen new compounds were synthesized and
characterized, six of them demonstrating antiparasitic activity. The most active derivative, compound
8
has similar or better in vitro and in vivo activity compared to the drugs Benznidazole, Glucantime,
and Miltefosine currently used for the treatment of these diseases. This compound has a good
toxicological profile, high selectivity index, and promising pharmacokinetic parameters since good
absorption and distribution in vivo are predicted. These compounds can be orally administered and
have a low-cost production, which makes them promising candidates for the development of drugs for
the control of neglected diseases.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/20/3800/s1,
Table S1. Total data from the in vitro assays with the parasites for 20 compounds, Figure S1. Pharmacokinetic r
profiles data output from SwissADME, Figure S2 TLC from the hepatic metabolic stability assay in vitro.
Author Contributions: G.A. (designed and coordinated the research and experiments, analyzed the data, and wrote
the paper); C.P. and E.A. (contributed to the in vitro experiments with Leishmania spp, T. cruzi, cytotoxicity on
macrophages, in vitro metabolism stability assay); I.C. (analyzed the data and wrote the paper); P.F.-T. and C.R.
(performed experiments with Leishmania clinical isolates and amastigotes); E.S., S.T., F.B., R.R., G.Y., and N.I.V.d.B.
(in vivo studies, genotoxicity, analyzed the data). All authors read and approved the final manuscript.
Funding: This research was funded by CONACYT Programa PROCIENCIA con recursos del Fondo para la
Excelencia de la Educaci
ó
n e Investigaci
ó
n – FEEI del FONACIDE. Contrato N
º
020/2015-14-INV-154, Paraguay.
This research was funded by CSIC (Comisi
ó
n Sectorial de Investigaci n Cient
ó
í
fica) for the travel grant. GA is
thankful to the CSIC I+D 2016 program for the support grants ID435 (Investigación y Desarrollo de antiparasitarios
de amplio espectro (T. cruzi, Leishmania spp. y T. brucei) And The APC was funded by CSIC.
Acknowledgments: Paraguayan authors are thankful to CONACYT Programa PROCIENCIA–FEEI/ FONACIDE,
Paraguay. C.P. is thankful to CSIC (Comisi
ó
n Sectorial de Investigaci
ó
n Cient
í
fica) for the travel grant. GA is
thankful to the CSIC I+D 2016 program for the support grants ID435 (Investigaci
ón y Desarrollo de antiparasitarios
de amplio espectro (T. cruzi, Leishmania spp. y T. brucei) and Agencia Uruguaya de Cooperación Internacional for
travel grants (Paraguay-Uruguay).
Conflicts of Interest: The authors declare no conflict of interest.
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