Synthesis and evaluation of novel prenylated chalcone derivatives as anti-leishmanial and anti-trypanosomal compounds

Article abstract of DOI:10.1016/j.bmcl.2015.05.072

Abstract Chalcones form a class of compounds that belong to the flavonoid family and are widely distributed in plants. Their simple structure and the ease of preparation make chalcones attractive scaffolds for the synthesis of a large number of derivatives enabling the evaluation of the effects of different functional groups on biological activities. In this Letter, we report the successful synthesis of a series of novel prenylated chalcones via Claisen-Schmidt condensation and the evaluation of their effect on the viability of the Trypanosomatidae parasites Leishmania amazonensis, Leishmania infantum and Trypanosoma cruzi.

Full text of DOI:10.1016/j.bmcl.2015.05.072

Accepted Manuscript
Synthesis and Evaluation of Novel Prenylated Chalcone Derivatives as Anti-
leishmanial and Anti-trypanosomal Compounds
Thais Gaban Passalacqua, Luiz Antonio Dutra, Letícia de Almeida, Angela
Maria Arenas Velásquez, Fabio Aurelio Esteves Torres, Paulo Renato
Yamasaki, Mariana Bastos dos Santos, Luis Octavio Regasini, Paul A.M.
Michels, Vanderlan da SilvaBolzani, Marcia A.S. Graminha
PII:
S0960-894X(15)00546-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.05.072
BMCL 22762
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
16 March 2015
19 May 2015
21 May 2015
Please cite this article as: Passalacqua, T.G., Dutra, L.A., de Almeida, L., Arenas Velásquez, A.M., Esteves Torres,
F.A., Yamasaki, P.R., dos Santos, M.B., Regasini, L.O., Michels, P.A.M., da SilvaBolzani, V., Graminha, M.A.S.,
Synthesis and Evaluation of Novel Prenylated Chalcone Derivatives as Anti-leishmanial and Anti-trypanosomal
Compounds, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.05.072
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Synthesis and Evaluation of Novel Prenylated Chalcone
Derivatives as Anti-leishmanial and Anti-trypanosomal
Compounds
a; b†
Thais Gaban Passalacqua , Luiz Antonio Dutra , Letícia de Almeida , Angela Maria
c; d†
a; b
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Arenas Velásquez , Fabio Aurelio Esteves Torres , Paulo Renato Yamasaki , Mariana Bastos
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dos Santos , Luis Octavio Regasini , Paul A. M. Michels , Vanderlan da Silva Bolzani and
c*
Marcia A. S. Graminha
a
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Instituto de Química, UNESP/ Araraquara-SP. 14800-060, Brazil.
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Programa de Pós Graduação em Biotecnologia
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Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, UNESP, Araraquara-SP
14801-902, Brazil.
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Programa de Pós Graduação em Ciências Farmacêuticas
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Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista, UNESP, São
José do Rio Preto-SP, 15054-000, Brazil.
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Institute of Structural and Molecular Biology, University of Edinburgh, UK
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† These two authors have equally contributed to this work.
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* Author to whom correspondence should be addressed: graminha@fcfar.unesp.br;
Tel.: +55-16-3301- 6957; Fax: +55-16-3301-6940.
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Received: / Accepted: / Published:
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Abstract: Chalcones form a class of compounds that belong to the flavonoid family and
are widely distributed in plants. Their simple structure and the ease of preparation make
chalcones attractive scaffolds for the synthesis of a large number of derivatives enabling
the evaluation of the effects of different functional groups on biological activities. In this
paper, we report the successful synthesis of a series of novel prenylated chalcones via
Claisen – Schmidt condensation and the evaluation of their effect on the viability of the
Trypanosomatidae parasites Leishmania amazonensis, Leishmania infantum and
Trypanosoma cruzi.
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Keywords: Prenylated chalcone; leishmanicidal activity; trypanocidal activity; Leishmania
amazonensis; Leishmania infantum; Trypanosoma cruzi; drug discovery
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Neglected Tropical Diseases (NTDs) have a higher prevalence in tropical and subtropical regions
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and affect more than one billion people worldwide . A list of 17 NTDs includes the insect vector-
borne diseases leishmaniasis and Chagas´ disease. Leishmaniasis is a widespread disease caused by
parasites belonging to more than 20 species of Leishmania, which are transmitted by phlebotomine
sandflies after injection of promastigote forms into mammals during feeding. These infective stages

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are phagocytized by macrophages and other types of mononuclear phagocytic cells. The internalized
promastigotes transform into the tissue-stage amastigotes, which multiply by simple division and
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proceed to infect other macrophages . The disease affects 12 million people around the world with
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about 1–2 million estimated new cases occurring every year . About 8-10 million people are currently
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infected with Trypanosoma cruzi, the parasite that causes Chagas’ disease, mostly in Latin America .
Chagas’ disease is transmitted by triatomine insects, which release trypomastigote forms in their feces
on the skin of a host. The trypomastigotes enter the host through the bite wound made by the blood-
feeding insect or mucous membranes. Inside the host, the trypomastigotes invade cells near the site of
entrance, where they differentiate into proliferating amastigotes and then again to trypomastigotes
which are released back into the bloodstream to infect other cells. The ingested trypomastigotes
transform into epimastigotes in the vector´s midgut where the parasites multiply and differentiate into
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metacyclic trypomastigotes .
The current treatment of leishmaniasis is based on chemotherapy and includes pentavalent
antimonials (sodium stibogluconate or meglumine antimoniate), the polyene amphotericin B (AmpB)
(as the deoxycholate salt or its liposomal formulation), the alkylphosphocholine miltefosine and the
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aminoglycoside paromomycin . The chemotherapy for Chagas’ disease involves two drugs, nifurtimox
(nitrofuran) or benznidazol (nitroimidazole), which are efficient only during the acute and initial period
of the subsequent chronic phase of the disease. Unfortunately, the toxicity of all currently available
drugs to treat these Trypanosomatidae-caused diseases, the inconvenience of parenteral administration,
the lack of a guaranteed drug supply and the increasing incidence of treatment failure, make the
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development of new therapies against them urgent .
Chalcones are molecules consist of open-chain flavonoids in which the two aromatic rings are
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joined by a three-carbon α, β-unsaturated carbonyl system . Their simple structure and the ease of
preparation make chalcones attractive scaffolds for the synthesis of a large number of derivatives
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enabling the evaluation of the effect of different functional groups on biological activities . Indeed,
several synthetic chalcones have shown a number of biological effects such as anti-inflammatory,
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antibacterial, antifungal, antiviral and antiprotozoal activities . Some natural prenylated chalcones
such as bartericina, medicanegina and licochalcone are promising compounds for designing new drugs
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against neglected diseases . Here, we present the effect of new synthetic prenylated chalcones on
the viability of the Trypanosomatidae parasites L. amazonensis, L. infantum and T. cruzi.
The chalcones 4, 5 and 10 were synthesized by Claisen-Schmidt condensation between
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acetophenone (1) and benzaldehyde derivatives (2 and 3)
; these compounds were used as
precursors for the synthesis of four novel prenylated chalcones, 6-9 as shown in figure 1. For the
preparation of prenylated chalcones 6 and 7, O-isoprenyl and O-farnesyl groups were respectively
added at position C-2, whereas for 8 and 9, O-geranyl or O-farnesyl were respectively added at
position C-3. Synthetic reactions in order to produce chalcones and their prenylated derivatives were
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followed by Nuclear Magnetic Resonance spectroscopy analysis (NMR). The H-NMR spectra
displayed a coupling constant range (J) for H-α and H-β of 15.0 Hz, corresponding to an (E)-chalcone
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scaffold . The data obtained by NMR analyses for molecules 4 - 9 are provided as Supplementary
Material, which confirmed the identity of prenylated chalcones.

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The novel synthetic prenylated chalcones 6 - 9 as well as their precursor molecules, were used in
screens of in vitro growth inhibition assays of promastigote and amastigote forms of L. amazonensis
and L. infantum, as well as epimastigotes of T. cruzi. The cytotoxicity of all molecules for peritoneal
murine macrophages was also tested, allowing the determination of the selectivity index (SI)
parameter. The inhibitory activity of these compounds was evaluated at several concentrations ranging
from 1.0 to 250 µM. The results expressed as the compound concentrations corresponding to 50 % of
parasite growth inhibition and 50 % macrophage cytotoxicity values (EC = effective concentration;
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the concentration giving 50 % effect) are summarized in Table 1. Since the O-prenylated chalcones 6-9
are novel, no reports of the leishmanicidal activities of such compounds have been published yet (see
methology in Supplementary Material).
Figure 1. General scheme for the synthesis of chalcones.
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Table 1. Leishmanicidal, trypanocidal and cytotoxic activities of chalcone and its prenylated derivatives in µM.
L. amazonensis (EC50 L. infantum (EC50 T. cruzi (EC50
Cpds
)
)
)
Swiss (EC50)
Promastigotes
333.70±23.60 (0.43)
411.60±17.70 (0.35)
4.38±0.07 (25.41)
6.47±0.05 (21.74)
8.92±0.25 (9.18)
9.25±0.47 (6.00)
13.2±1.21 (0.70)
10.19±0.85 (3.50)
3.22±0.03 (7.17)
n.d.
Amastigotes
n.d.
Promastigotes
91.74±6.70 (1.56)
184.82±13.88 (0.96)
4.69±0.14 (23.73)
10.21±0.19 (13.78)
7.81±0.14 (10.49)
5.98±0.02 (9.22)
8.89±1.91 (1.06)
67.71±8.11 (0.53)
0.92±0.01 (25.11)
n.d.
Amastigotes
n.d.
Epimastigotes
75.89±1.88 (1.88)
68.30±0.63 (2.09)
21.58±1.23 (5.16)
69.37±4.25 (2.03)
26.25±3.11 (3.12)
23.79±1.89 (2.32)
102.64±6.11 (0.09)
n.d.
Macrophages
143.00±0.16
143.00±0.63
111.30±0.34
140.65±3.62
81.94±8.11
55.14±5.60
9.50±0.03
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n.d.
n.d.
19.93±1.34 (5.58)
2.90±0.19 (48.50)
25.81±5.05 (3.17)
21.89±0.47 (2.52)
n.d.
10.41±0.99 (10.69)
2.24±0.86 (62.79)
63.89±5.89 (1.28)
32.55±1.89 (1.69)
n.d.
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AmpB
Benz
6.25±0.58 (5.71)
4.92±0.14 (4.70)
n.d.
19.77±0.52 (1.81)
2.98±0.38 (7.75)
n.d.
35.69±6.84
23.10±2.52
n.d.
n.d.
4.07± 0.31
The selectivity indices (SI) are shown in parentheses
n.d. not determined
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Although chalcone (10) presented a mild leishmanicidal activity, it showed high cytotoxicity with
SI values of 0.7 and 1.06 for L. amazonensis and L. infantum, respectively. Thus, structural changes
were considered in order to obtain more effective and less cytotoxic chalcone derivatives. The
intermediate molecules 4 and 5, which present hydroxyl groups at positions C-2 and C-3, respectively,
did not show antileishmanial activity and their cytotoxicity appeared to be very high. In contrast, the
prenylation of 4 and 5 resulting in the derivatives 6 - 9 exhibited variable in vitro activity against the
evaluated parasites. For both Leishmania species tested, L. amazonensis and L. infantum, compounds 6
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00 - 9 were very active on the promastigote forms, with 6 and 7 being the most selective ones to the
01 parasites (SI values of 25.41 and 21.74, respectively) (Table 1).
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For the amastigote form of Leishmania, the clinically most relevant stage of these parasites,
03 compound 7 was the most active and selective prenylated chalcone, for both L. amazonensis (EC =
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04 2.90 µM ± 0.19; SI= 48.50) and L. infantum (EC50 = 2.24 µM ± 0.86; SI= 62.79). Compound 7 showed
05 the most outstanding results for L. infantum when compared to pentamidine, with respect to both its
06 anti-amastigote activity and its selectivity to the parasite; these values were about 9- and 35-fold higher
07 than those of the reference drug. Regarding L. amazonensis, despite its low activity (only two–fold
08 higher than pentamidine), this compound (7) still displayed an eight times higher selectivity index.
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A reasonable explanation for the good antileishmanial activity of 6 – 9 over 4 – 5 and 10 could be
10 the presence of prenyl moieties influencing the lipophilicity of the compounds. Indeed, there is some
11 evidence in the literature that prenylation increases lipophilicity and, consequently, causes an
12 improvement of the biological activity. It has been reported that prenylated flavonoids presented
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13 enhanced effects against bacteria, fungi and viruses . The prenylated chalcones evaluated in this work
14 contain O-isoprenyl (6) and O-farnesyl (7) at C-2 or O-farnesyl (9) and O-geranyl (8) at the C-3
15 positions; thus these prenyl moieties may result in an increased lipophilicity of the compounds that is
16 responsible for the augmented leishmanicidal activity by facilitating the passage of the molecules
17 through the cell-membrane barriers of the macrophages and parasites. A close relation between
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18 lipophilicity and leishmanicidal activity was also reported by Maciel-Rezende and coworkers (2013) ,
19 when they evaluated the leishmanicidal activity of a series of O-alkyl substituted benzophenones. With
20 regard to other prenylated chalcones, Dal Picolo and coworkers showed that adunchalcone, a
21 prenylated dihydrochalcone (possessing an isoprenyl chain), displayed good antileishmanial activity
22 against promastigote forms of L. amazonensis, but lacked any effect against its intracellular
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23 amastigotes . Gupta and co-workers reported that geranyl chalcones were more potent than isoprenyl
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24 chalcones against amastigote forms of L. donovani ; these findings could be attributed to a better drug
25 delivery into host cells as a result of the size of the prenyl moiety that affects the potency of the
26 molecules on intact cells.
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In fact, for L. infantum and L. amazonensis, 7 was 4.65 and 6.87 more active than 6, respectively.
28 This effect should possibly be attributed to the presence of the 15 carbons prenyl chain of O-farnesyl in
29 7 compared to the 5 carbons prenyl chain of O-isoprenyl in 6. The long carbon skeleton of O-farnesyl
30 contributes to an increased lipophilicity, as confirmed by the calculated ClogP values (see table 2 in
31 Supplementary Material); the ClogP of 7 was found to be 7.75 whereas for 6 it was 4.69. The partition
32 coefficient logP is an important chemical parameter that determines the tendency of a given compound
33 to be distributed in a biphasic system composed of two phases (octanol/water) determining its

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34 lipophilicity / hydrophilicity
. All prenylated chalcones 6 – 9 presented calculated ClogP values
35 higher than those obtained for 4, 5 and 10. Indeed, the anti-amastigote activity of 7 is at least five times
36 higher than that of 6, in agreement with its ClogP, which reinforces the hypothesis that the larger the
37 lipophilicity, the better the antileishmanial activity.
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It was also observed that the presence of the O-farnesyl group at position C-2 in the chalcone
39 skeleton rendered compound 7 more active than both reference drugs tested; furthermore, considering
40 the observed SI values, compound 7 exhibited at least eight times less cytotoxicity than the standard
41 drug Amp B. On the other hand, the O-farnesyl group at position C-3 (compound 9) resulted in a
42 strong loss of leishmanicidal activity as well as an increased cytotoxicity profile suggesting that the
43 position of the O-farnesyl group (C-2 or C-3) influences both the leishmanicidal and cytotoxicity
44 activities. These observations suggest that the position of prenyl moieties is important for the correct
45 interaction of the compound with its potential target molecule in the parasite, which supports the
46 notion that the position of the prenyl groups as well as the presence or absence of other substituents in
47 the B ring are important for the leishmanicidal activity.
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For T. cruzi, compounds 6 (EC = 21.58 µM ± 1.23; SI= 5.16), 8 (EC = 26.25 µM ± 3.11; SI=
50 50
49 3.12) and 9 (EC50 = 23.79 µM ± 1.89; SI= 2.32) presented similar potency ranging from 21 to 26 µM,
50 five times less active than benznidazol; moreover, their low SI values rendered these molecules
51 unattractive for further evaluation against these parasites, at least the available epimastigote stage.
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Four prenylated chalcone derivatives (6 – 9) were synthesized and exhibited good antipromastigote
53 activity as well as good selectivity for both L. amazonensis and L. infantum. The most promising
54 results were obtained for compound 7 that showed the best antiamastigote activity, the clinically most
55 relevant parasite form. This compound showed the best relationship between the leishmanicidal effect
56 and the cytotoxicity activity against murine macrophages, which represents a good indication for its
57 therapeutic index for further drug development. Variation in lipophilicity of the evaluated compounds
58 seems to be an important requisite for leishmanicidal activity. Further studies should be conducted to
59 better understand the mechanism of action of these molecules. Thus, these series of prenylated
60 chalcones could be further explored as potential new leishmanicidal drugs.
161 Acknowledgments
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The authors thank Isabel Martinez for her technical support. They are also grateful to the Fundação de
Amparo à Pesquisa do Estado de São Paulo (FAPESP: n° 2010/52327-5), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq: n° 407588/2013-2), Fundaçao Coordenaçao de
Aperfeiçoamento de Pessoal de Nivel Superior (CAPES) and PROPe (Pro-Reitoria de Pesquisa da UNESP) and
the FUNDUNESP for financial support.
68 Author Contributions:
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Conceived and designed the experiments: LOR, VSB, MASG. Performed the experiments: TGP, LAD, LA,
AMAV, FAET, MBS. Analyzed the data: PRY, MASG. Contributed reagents/materials/analysis tools: LOR,
VSB, MASG. Wrote the paper: TGP, LAD, LA, PAMM, MASG.

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73 Supplementary Material
74 Supplementary material associated with this article can be found, in the online version, at doi:
75 Conflicts of Interest
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The authors declare no conflict of interest.
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214 Sample Availability: Samples of the compounds 6-9 are available from the authors.
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15 © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
16 distributed under the terms and conditions of the Creative Commons Attribution license
17 (http://creativecommons.org/licenses/by/3.0/).
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(
E)-2-O-farnesyl-chalcone (7)
Antileishmanial
activity