Antiprotozoal activity of triazole derivatives of dehydroabietic acid and oleanolic acid

Article abstract of DOI:10.3390/molecules22030369

Tropical parasitic diseases such as Chagas disease and leishmaniasis are considered a major public health problem affecting hundreds of millions of people worldwide. As the drugs currently used to treat these diseases have several disadvantages and side effects, there is an urgent need for new drugs with better selectivity and less toxicity. Structural modifications of naturally occurring and synthetic compounds using click chemistry have enabled access to derivatives with promising antiparasitic activity. The antiprotozoal activity of the terpenes dehydroabietic acid, dehydroabietinol, oleanolic acid, and 34 synthetic derivatives were evaluated against epimastigote forms of Trypanosoma cruzi and promastigotes of Leishmaniabraziliensis and Leishmania infantum. The cytotoxicity of the compounds was assessed on NCTC-Clone 929 cells. The activity of the compounds was moderate and the antiparasitic effect was associated with the linker length between the diterpene and the triazole in dehydroabietinol derivatives. For the oleanolic acid derivatives, a free carboxylic acid function led to better antiparasitic activity.

Full text of DOI:10.3390/molecules22030369

molecules
Article
Antiprotozoal Activity of Triazole Derivatives of
Dehydroabietic Acid and Oleanolic Acid
Mariano Walter Pertino 1,*, Celeste Vega 2, Miriam Rolón 2, Cathia Coronel 2,
Antonieta Rojas de Arias 2 and Guillermo Schmeda-Hirschmann 1
1
Laboratorio de Química de Productos Naturales, Instituto de Química de Recursos Naturales,
Universidad de Talca, 3460000 Talca, Chile; schmeda@utalca.cl
Centro para el Desarrollo de la Investigación Científica (CEDIC), Manduvirá 635 entre 15 de Agosto y
2
O’Leary, Barrio La Encarnación 1255, 2511 Asunción, Paraguay; mcvegagomez@gmail.com (C.V.);
rolonmiriam@gmail.com (M.R.); cathiacoronel@gmail.com (C.C.); rojasdearias@gmail.com (A.R.d.A.)
*
Correspondence: mwalter@utalca.cl; Tel.: +56-71-2418866
Academic Editor: Jean-Marc Sabatier
Received: 23 January 2017; Accepted: 21 February 2017; Published: 28 February 2017
Abstract: Tropical parasitic diseases such as Chagas disease and leishmaniasis are considered a
major public health problem affecting hundreds of millions of people worldwide. As the drugs
currently used to treat these diseases have several disadvantages and side effects, there is an urgent
need for new drugs with better selectivity and less toxicity. Structural modifications of naturally
occurring and synthetic compounds using click chemistry have enabled access to derivatives with
promising antiparasitic activity. The antiprotozoal activity of the terpenes dehydroabietic acid,
dehydroabietinol, oleanolic acid, and 34 synthetic derivatives were evaluated against epimastigote
forms of Trypanosoma cruzi and promastigotes of Leishmaniabraziliensis and Leishmania infantum.
The cytotoxicity of the compounds was assessed on NCTC-Clone 929 cells. The activity of the
compounds was moderate and the antiparasitic effect was associated with the linker length between
the diterpene and the triazole in dehydroabietinol derivatives. For the oleanolic acid derivatives, a
free carboxylic acid function led to better antiparasitic activity.
Keywords: cytotoxicity; dehydroabietic acid; Leishmania spp.; oleanolic acid; triazole derivatives;
Trypanosoma cruzi
1. Introduction
Chagas is endemic in 21 countries in Latin America. It is a zoonotic parasitosis considered one
of the most important neglected diseases in Latin America with a total of 520,043 DALYs (years of
disability-adjusted life) [
contracting Chagas disease and eight million are infected by the flagellate protozoan Trypanosoma cruzi
with approximately 12,000 deaths and 56,000 new cases per year [ ]. An estimated 5.4 million people
will develop cardiac Chagas disease [ ], while 900,000 will have megaesophagus and megacolon [ ].
1–4]. Currently, it was estimated that 90–100 million people are at risk of
5
1,6
1
Given the globalization of our days, the disease exists in at least 10 non-endemic countries. Currently,
more than two million people infected with Chagas disease live in Spain and other European
countries [7], and even in other continents [8].
There are two phases of Chagas disease. The first acute phase last around four months and is
characterized by large numbers of parasites circulating in the patient blood. However, just 5% of
patients are detected during the acute phase and can be cured [9]. In the chronic phase, the parasitemia
is low, and the pathognomonic manifestation could appear as chronic cardiomyopathy, megas in the
digestive tract, as well as damage to the central nervous system. Two major groups of the T. cruzi
parasite strains have been described elsewhere (TcI and TcII). TcI is associated with the sylvatic cycle of
Molecules 2017, 22, 369; doi:10.3390/molecules22030369
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transmission while TcII is associated with the domestic cycle [10
,11]. It was observed that a specific
geographical distribution of these two major groups. TcI strains predominate in the Amazon basin and
the northern countries of South America, Central America, and Mexico, while TcII is commonly found
in the southern part of South America. The megas are usually found in the south. By this fact, TcII
strains are incriminated as more pathogenic than TcI strains [11].
Chances of being cured in the chronic phase, by conventional treatments, are very low. This disease
is considered, together with leishmaniasis and African Trypanosomiasis, an extremely neglected
disease. In the case of the poorest countries in the developing world, they are characterized by the fact
that they affect poor populations in developing countries, which do not add services, being excluded
socioeconomically and, consequently, pharmaceutical markets [12].
Leishmaniasis is generally endemic in 98 countries and territories in the world, and is responsible
for a disease burden of 2.35 million DALYs, 2.3% of which occur in the Americas (50,668) [4]. More
than 12 million people are infected with leishmaniasis, and 350 million are at risk. It is estimated that
75% of all cases of cutaneous leishmaniasis are concentrated in 10 countries, four of which are in the
Americas: Brazil, Colombia, Peru, and Nicaragua. Brazil is one of the six countries where 90% of the
cases of visceral leishmaniasis have been found; the others are Ethiopia, India, Bangladesh, Sudan,
and South Sudan. In the Americas, an average of 60,000 cases of cutaneous and mucosal leishmaniasis
and 4000 cases of visceral leishmaniasis are diagnosed each year, with a mortality rate of 7% [13,14].
One of the most widely distributed species in Latin America is Leishmania braziliensis that can
produce both cutaneous and mucocutaneous leishmaniasis. Another important species is L. infantum
that can produce visceral leishmaniasis, the most common species identified in animals especially
dogs [15]. These two species were selected to perform the in vitro leishmanicidal activity of the
compounds tested in this study. The life cycle of both parasites spends an important development
inside the reservoir or in the human body. The trypomastigote forms of T. cruzi enter the organism
through the skin or mucous membranes, then pass into the cells of the endothelial reticulum or
into the circulatory or lymphatic torrent [16,17]. When the parasite enters the cells, it is transformed
into amastigotes, which multiply until it completely invades the cell and becomes epimastigotes.
Afterwards, the cell in the trypomastigotes is broken, and the trypomastigotes are released into the
circulatory torrent. At this moment, the trypomastigotes can be detected by a fresh examination,
and they later invade new tissues and become amastigotes, thus closing the cycle. A similar cycle
is observed in Leishmania spp. Promastigotes are phagocytized by macrophages and other types of
mononuclear phagocytic cells, and transformation allows them to reach amastigotes, the tissue stage
of the parasite, which multiply by simple division and proceed to infect other mononuclear phagocytic
cells [18,19].
The promastigotes and epimastigotes are the extracellular dividing forms of the parasite inside
the insect vectors of Leishmania spp. and T. cruzi, respectively. As they are easy to cultivate under
laboratory conditions, these models are a good choice for preliminary in vitro screening [20,21].
Despite the progress in the chemotherapy of the mentioned parasitic diseases, there is an urgent
need for new drugs. Although Chagas disease can be treated with drugs such as nifurtimox and
benznidazole, there are reports on which these treatments fail [22]. The drugs currently used by
leishmaniasis have discreet effectiveness, long treatment times, high cost, and adverse effects, and the
parasites rapidly develop resistance to drugs [23]. Therefore, the discovery of new drugs is a priority.
Just 3.8% of drugs approved between 2000 and 2011 were for neglected diseases such as leishmaniasis,
Chagas disease, and sleeping sickness, which together represent more than 10% of the global burden
of diseases [24].
Several natural products of plants such as phenolic, alkaloids, saponins, and terpenes
show potent and selective bioactivity for the treatment of tropical diseases [25]. Abietane-type
diterpenes isolated from roots of Salvia cilicica (Lamiaceae), and triterpenes such as ursolic acid
and betulinaldehyde, display significant in vitro antileishmanial activity. Recently, different research
groups have used the Cu(I) catalyzed 1,3-dipolar-cycloaddition reaction of azide and alkyne (CuAAC)

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to generated 1,2,3-triazoles on the search for antiparasitic drugs. Using this reaction, they obtained
triazol-derivatives of sugars, benznidazoles, naphthoquinones, and sterols with excellent antiparasitic
properties [26
dehydroabietic acid (DHA) coupled with 1,2,3-triazoles (
acid (OA) derivatives (17 34). Most of the compounds showed low antiproliferative activity on tumor
and normal cells. Therefore, they should be regarded as compounds with low toxicity, which can
be an advantage when looking for selective drugs [20]. In the present work, compounds 34 were
–
30]. In previous work, we reported the synthesis and antiproliferative activity of
1–
16) and 1,2,3-triazole-substituted oleanolic
–
1–
examined for a trypanocidal effect on T. cruzi and in vitro activity against two promastigote stages of
Leishmania spp.
2. Results and Discussion
From the starting terpenes OA, DHA, and dehydroabietinol, 34 synthetic derivatives were
prepared (Figure 1) and assessed against epimastigote forms of T. cruzi, promastigotes from Leishmania
spp. as well as for cytotoxicity. The results are summarized in Table 1.
Figure 1. The structure of compounds 1–34 synthesized and evaluated as antiprotozoal agents in vitro.

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Table 1. Antiprotozoal activity of the starting terpenes dehydroabietic acid (DHA), oleanolic acid (OA),
and synthetic derivatives 34 towards Leishmania promastigotes, Trypanosoma cruzi epimastigotes, and
cytotoxicity on NCTC-Clone 929 cells. The results are presented as IC50 data in (µg/mL).
1–
Leishmanicidal Activity on
Promastigotes (µg/mL)
Antiepimastigote Activity of
NCTC-Clone
929 Fibroblasts
Compound
T. cruzi (µg/mL)
(µg/mL)
L. braziliensis
L. infantum
T. cruzi
DHA
Dehydroabietinol
44
63
72
40
212
66
-
-
1
2
3
4
53
60
46
>256
>256
>256
189
>256
>256
64
>256
>256
64
>256
>256
61
5
6
7
8
9
10
11
12
218
193
223
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
232
>256
>256
>256
>256
>256
>256
>256
242
>256
>256
>256
>256
>256
>256
>256
>256
>256
53
>256
>256
>256
>256
>256
>256
>256
>256
>256
69
13
14
15
>256
89
16
OA
17
18
19
20
21
22
23
24
25
26
27
28
29
30
71
73
199
>256
252
129
-
196
>256
>256
>256
109
>256
>256
>256
155
>256
>256
>256
>256
>256
>256
>256
>256
>256
43
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
-
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
3.3
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
128
61
>256
>256
>256
>256
>256
>256
-
31
32
33
>256
>256
>256
3.3
-
-
-
-
34
Pentamidine
Benzimidazole
-
-
15.0
2.1. Dehydroabietic Acid Derivatives
The activity on T. cruzi of DHA, dehydroabietinol, and compounds
comparing with DHA, the effect was stronger for dehydroabietinol, with IC50 of 66
with 212 g/mL for DHA. The most active products were the derivatives , and 15 with IC50 values
of 46, 61, and 69 g/mL, respectively. Compound has a CH2 linker between the diterpene and the
triazole, while (IC50 > 256 g/mL) bears two CH2 units in the linker. However, when the triazole
presents a methyl phenyl sulfide (compounds and ), the effect is stronger for the compound with
two CH2 units in the linker (>256 vs. 61 g/mL). The same trend was observed for compounds 15
and 16, with IC50 values of 69 and 199 g/mL, respectively. The most active product 15 shows two
CH2 units in the linker while 16 presents four CH2 units in the linker, respectively. These results are
1–
16 was moderate. When
g/mL compared
µ
µ
1, 4
µ
1
2
µ
3
4
µ
µ

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showing the importance of linker length in the activity against T. cruzi in this type of compounds.
Subsequent studies including linkers of different lengths would be necessary to identify the ideal
chain length to obtain derivatives with potential activity against T. cruzi. Inhibition of Leishmania
proliferation in vitro was tested against two promastigote strains. DHA, dehydroabietinol, and their
derivatives,
values of 44, 63, 53, 64, 89, and 71
1
,
4
,
15, and 16, were the most active compounds against both species evaluated with IC50
g/mL against L. braziliensis and 72, 40, 60, 64, 53, and 73 g/mL
µ
µ
against L. infantum, respectively. These compounds except the derivative 16 were also active against
T. cruzi. The same structure–activity trend discussed for T. cruzi is observed for Leishmania. When
comparing the evaluated derivatives versus the reference compound (pentamidine, IC50: 3.3 µg/mL),
a marked difference in activity against Leishmania was observed. However, pentamidine has several
side effects including nephrotoxicity, so alternative drugs are still needed. The cytotoxicity of the
compounds was evaluated on NCTC-Clone 929 fibroblasts. Among the evaluated derivatives only
compounds
129–232 g/mL. With the exception of compound 17, these dehydroabietic derivatives are those that
have shown the best antiparasitic activity in this study ( 15, and 16), so it could be deduced that the
4, 15, 16, and 17 showed moderate cytotoxic activity with IC50 values in the range of
µ
4
,
cytotoxicity is relatively non-specific, and the molecules could be acting indistinctly on the cells and
the parasites.
Natural abietane diterpenes have shown interesting antiparasitic activities. For example,
Samoylenko et al. [31] reported the antileishmanial activity of ferruginol and totarol against L. donovani
promastigotes with IC50 values of 3.5
respectively. Another naturally occurring abietane with antileishmanial activity is 12-methoxycarnosic
acid with an IC50 value of 0.75 M against axenically grown L. donovani amastigotes. Recently,
µg/mL for both compounds and IC90 values of 7.0 and 6.9 µg/mL,
µ
Olmo et al. [32] reported five abietane diterpenoids as candidates for treating Chagas disease. These
compounds were synthesized from abietic acid, essentially all of them being dehydroabietic acid
derivatives. In vitro and in vivo activity and the possible mechanism of action of the compounds were
studied. In vitro trypanocidal evaluation of the compounds against different forms of the parasite
showed better effects than the reference drug. Two compounds were the best candidates, being 29- and
52-fold more active than benznidazole against amastigotes forms. Additionally, they exhibit good
activity in vivo and low toxicity.
In recent studies, dehydroabietic acid was treated with different amino acid in search of
antiprotozoal activity [33]. The derivatives were evaluated against L. donovani amastigotes and
T. cruzi amastigotes. Several of them showed good activity—in some cases, higher than the reference
compound benznidazole. In continuation of this study, the same group reported the preparation of new
amides, but this time starting from dehydroabietylamine [34]. The derivatives were evaluated against
L. donovani and T. cruzi. The most promising compound showed IC50 values of 0.37 µM and 0.6 µM, with
a selectivity index value of 63 and 58 against L. donovani axenic amastigotes and T. cruzi, respectively.
Subsequent to this work, Dea-Ayuela et al. [35] reported new dehydroabietylamine derivatives
with antileishmanial activity against promastigotes of L. donovani, L. infantum, L. amazonensis, and
L. guyanensis with IC50 values in the range of 2.2–46.8 µM.
2.2. Oleanolic Acid Derivatives
For the OA derivatives (compounds 17
–34), only two compounds (27 and 28) showed a relevant
effect on T. cruzi with IC50 values of 43 and 61
µg/mL, respectively. They differ in the free (27) or
methylated COOH function of the triterpene (28) but show the same p-toluensulfonyl moiety in the
triazole ring. On the other hand, the results on leishmanicidal activity show that only compounds 19
and 31 have a slight effect. Compounds 19 presents activity against both L. braziliensis and L. infantum
promastigotes, with IC50 values of 109
activity only against L. infantum promastigote with an IC50 of 128
be regarded as inactive on Leishmania promastigotes (IC50 > 256 µg/mL).
µg/mL and 155
µg/mL, respectively. Compound 31 showed
µg/mL. All other compounds should

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Several natural pentacyclic triterpenoid such as epi-oleanolic acid, betulinic acid, and
betulinaldehyde, among others display leishmanicidal activity against different Leishmania
species [36,37]. Torres-Santos et al. [38] reported the antileishmanial activity of ursolic acid and
oleanolic acid. Ursolic acid showed IC50 values of 5 and 27
µg/mL, and oleanolic acid displayed
IC50 values of 10 and 11 g/mL against the promastigote and amastigote stage of L. amazonensis,
µ
respectively. Recently, Melo et al. [39] determined the activity of oleanolic acid against three different
Leishmania species (L. braziliensis, L. amazonensis, and L. infantum). The reported leishmanicidal activity
against promastigotes and amastigote shows IC50 values in the range of 30–66
µM and 38–69
µM,
respectively. The results differ from our study, where the IC50 values of the OA was >256
µg/mL.
This difference can be explained by protozoal strains, isolated from different sources and the low
solubility in water of OA.
Rodríguez-Hernández et al. [40] reported the synthesis and leishmanicidal activity of hederagenin
derivatives prepared by click chemistry. Hederagenin is a triterpene which differs from oleanolic acid
by an additional hydroxyl group at C-23. Unlike the OA derivatives reported in this paper, where
esters were at the C-3 position, the Hederagenin derivatives were prepared on the carboxylic acid
in C-28. Hederagenin esters and amides containing 1,2,3-triazole derivatives were evaluated against
amastigote forms of L. infantum. The four additional active derivatives showed IC50 values of 2
9.7 µM, 11 µM, and 12 µM.
µM,
Taking together, the synthetic strategy of coupling a diterpene (DHA) or a triterpene (OA) with a
triazole using click chemistry allowed us to obtain structural diversity for structure–activity studies.
Some of the derivatives presented better antiprotozoal effects than did the starting compounds,
suggesting that further modifications might lead to better antiprotozoal effects for this group
of compounds.
3. Materials and Methods
3.1. Chemistry
Dehydroabietic acid (DHA) was obtained from commercial rosin as described in [41]. Oleanolic
acid (OA) was isolated from the aerial parts of Fabiana imbricata [42]. The triazole derivatives were
prepared as described previously (Figure 1) [41,42]. Briefly, DHA was methylated with CH2N2 to
afford dehydroabietic acid methyl ester. Reduction of the methyl ester with LiAlH4 in THF gave the
corresponding alcohol (dehydroabietinol). Starting from DHA and its alcohol, 16 abietane-triazole
compounds were obtained using click chemistry [41]. The alkyl esters of DHA and its alcohol
were prepared using N,N-dicyclohexylcarbodiimide (DCC)/ dimethylaminopyridine (DMAP) and
appropriate alcohol or acid with alkyl function. The alkynyl esters were treated with different azides
in t-BuOH/H2O using CuSO4.5H2O and sodium ascorbate to obtain the derivatives 1–16 (Figure 1).
Oleanolic acid (OA) derivatives contained 1,2,3-triazoles linked to the 3-O function and a
free or methylated COOH function at C-28 were prepared as reported [42]. OA was treated with
the appropriate alkyne acid/DCC/DMAP to afford the alkynyl esters 17–20. The alkynyl esters
and different azides were dissolved in CH2Cl2/H2O (1:1), followed by click chemistry conditions
(CuSO4·5H2O/sodium ascorbate) to afford compounds 21–34 (Figure 1).
3.2. Antiprotozoal Assays
For in vitro studies of T. cruzi, the clone CL-B5 was used (donated by Faculty of Pharmacy,
Complutense University, Madrid, Spain). Parasites were stably transfected with the Escherichia coli
◦
β
-galactosidase gene (lacZ). Epimastigotes were grown at 28 C in liver infusion tryptose broth (Difco,
Detroit, MI, USA) with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), penicillin (Ern S.A.,
Barcelona, Spain), and streptomycin (Reig Jofré S.A., Barcelona, Spain), as described previously [20],
and harvested during the exponential growth phase. Briefly, the screening assay was performed in
96-well microplates (Sarstedt, Inc., Newton, NC, USA) with cultures that had not reached the stationary

Molecules 2017, 22, 369
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phase. Epimastigotes were seeded at 2
×
105 parasites/mL in 200
µ
L growth media. The plates were
then incubated with the respective compounds at 28 ◦C for 72 h; afterwards, 50
µL of chlorophenol
red-
β
-D-galactopyranoside (CPRG) solution was added, resulting in a final concentration of 200 M.
µ
The plates were incubated at 37 ◦C for an additional 4 h and then read at 595 nm. Each concentration
was tested in triplicate [21]. The efficacy of each compound was estimated by calculating the IC50
values. These values were calculated by the sigmoidal dose–response curve adjustment using the
statistical software program Graph-Pad Prims 3.0 (GraphPhad, San Diego, CA, USA). Benznidazole
was used as the reference drug.
Culture of L. braziliensis (MHOM/BR/75/M2904), and L. infantum (MHOM/FR/91/LEM2259V)
was obtained from the Facultad de Farmacia (Universidad Complutense de, Madrid, Spain).
The maintenance of the strains, the form of cultivation, and the isolation of shape promastigota were
performed following the procedures described by Roldos et al. [20]. The promastigotes were grown at
◦
22 C in Schneider’s Drosophila medium supplemented with 20% FBS. The assay was performed using
a modification of a previous method [43]. Promastigotes (2
96-well plastic plates. Compounds were dissolved in dimethylsulfoxide (DMSO). Different dilutions
of the compounds with a final volume up to 200
×
106 parasites/well) were cultured in
◦
µ
L were added. After 48 h at 26 C, 20
µL 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
µ
m prior to use. All tests were carried out in triplicate. Resazurin sodium 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.
3.3. Cytotoxicity
The cell line used was NCTC-Clone 929, which was grown in Minimal Essential Medium (Sigma)
supplemented with 10% heat-inactivated FBS, penicillin G (100 U/mL), and streptomycin (100 µg/mL).
Cell cultures were maintained at 37 ◦C in a humidified 5% CO2 atmosphere. The procedure for cell
viability measurement was evaluated with resazurin by a colorimetric method. The cells were plated
in 96-microtiter plates at 3
overnight at 37 C, 5% CO2. Thereafter, the medium was removed and the compounds were added in a
×
104 cells per well in a 100
µL growth medium. The cells were grown
◦
200 µL medium for 24 h. After incubation, 20 µL of a 2 mM resazurin solution was added to each well.
The plates were incubated for 3 h to allow optimal oxidation-reduction. The reduction of resazurin
was determined by dual wavelength absorbance measurement at 490 and 595 nm. Background was
subtracted. Each concentration was assayed three times. Medium and drug controls were used in each
test as blanks [44].
4. Conclusions
In the present work, 34 terpene-triazole derivatives were synthesized starting from the natural
products dehydroabietic acid, dehydroabietinol, and oleanolic acid. The antiprotozoal activity of the
compounds was evaluated against epimastigote forms of T. cruzi, promastigotes of L. braziliensis and
L. infantum, and cytotoxicity on NCTC-Clone 929 cells. The compounds that showed moderate activity
were the most active diterpene DHA, dehydroabietinol, and the derivatives 1, 4, 15, 16, 27, and 28.
The antiparasitic effect was associated with the linker length between the diterpene and the triazole in
dehydroabietinol derivatives. For the oleanolic acid derivatives, a free carboxylic acid function led to
better antiparasitic activity. Further preclinical studies on both protozoa could be carried out on the
tissue phase of both parasites (amastigotes), as well as in vivo assays in experimentally infected mice,
in order to obtain more information about their potential use of its antiprotozoal activity.
Acknowledgments: M.W.P. thanks Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT), Chile
(grant 1161092). We thank the Programas de Investigación de Excelencia Interdisciplinaria (PIEI-QUIM-BIO),
Universidad de Talca, for additional financial support. Paraguayan researchers benefit from the Programa Nacional
de Incentivo al Investigador (PRONII), Consejo Nacional de Ciencia y Tecnología, (CONACYT), Paraguay.

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Author Contributions: M.W.P. was responsible for the synthesis and wrote the manuscript, C.V., M.R., C.C.,
and A.R.d.A. carried out the biological assays, and G.S.-H. contributed with valuable discussion and revision of
the manuscript.
Conflicts of Interest: The authors report no conflict of interest.
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Sample Availability: Samples of the compounds 1–34 are available from the authors.
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