Effect of substituents in the A and B rings of chalcones on antiparasite activity

Article abstract of DOI:10.1002/ardp.202000157

Chalcones are a group of natural products with many recognized biological activities, including antiparasitic activity. Although a lot of chalcones have been synthetized and assayed against parasites, the number of structural features known to be involved in this biological property is small. Thus, in the present study, 21 chalcones were synthesized to determine the effect of substituents in the A and B rings on the activity against Leishmania braziliensis, Trypanosoma cruzi, and Plasmodium falciparum. The compounds were active against L. braziliensis in a structure-dependent manner. Only one compound was very active against T. cruzi, but none of them had a significant antiplasmodial activity. The electron-donating substituents in ring B and the hydrogen bonds at C-2′ with carbonyl affect the antiparasitic activity.

Full text of DOI:10.1002/ardp.202000157

Received: 15 May 2020
Revised: 25 July 2020
Accepted: 29 July 2020
|
|
DOI: 10.1002/ardp.202000157
F U L L P A P E R
Effect of substituents in the A and B rings of chalcones on
antiparasite activity
1
1,2
3
1
Luis A. González
| Yulieth A. Upegui
| Luis Rivas
| Fernando Echeverri
1
|
1
2
Gustavo Escobar
| Sara M. Robledo
| Wiston Quiñones
1
Grupo de Química Orgánica de Productos
Naturales (QOPN), Facultad de Ciencias
Exactas y Naturales, Instituto de Química,
Universidad de Antioquia, Medellín, Colombia
Abstract
Chalcones are a group of natural products with many recognized biological activities,
including antiparasitic activity. Although a lot of chalcones have been synthetized
and assayed against parasites, the number of structural features known to be
involved in this biological property is small. Thus, in the present study, 21 chalcones
were synthesized to determine the effect of substituents in the A and B rings on the
activity against Leishmania braziliensis, Trypanosoma cruzi, and Plasmodium falciparum.
The compounds were active against L. braziliensis in a structure‐dependent manner.
Only one compound was very active against T. cruzi, but none of them had a
significant antiplasmodial activity. The electron‐donating substituents in ring B and
the hydrogen bonds at C‐2′ with carbonyl affect the antiparasitic activity.
2
PECET‐Facultad de Medicina, Universidad
de Antioquia, Medellín, Colombia
3
Grupo de Investigación en Péptidos
Antibióticos Eucarióticos, Centro de
Investigaciones Biológicas, Madrid, España
Correspondence
Wiston Quiñones, Grupo de Química Orgánica
de Productos Naturales, QOPN, Facultad de
Ciencias Exactas y Naturales, Instituto de
Química, Universidad de Antioquia, Calle 70
No. 52–21, A A 1226, Medellín, Colombia.
Email: wiston.quinones@udea.edu.co
Funding information
K E Y W O R D S
Ministerio de Ciencia, Tecnología e Innovación
antiplasmodial, B ring electron‐donating substituents, C‐2′ hydrogen bond, leishmanicidal,
–
Minciencias; Universidad de Antioquia,
trypanocidal
Proyect: Síntesis de análogos de xantonas,
potenciales agentes antiparasitarios
y evaluación de su actividad in vitro,
Grant/Award Number: 111571249860 and
061‐2016
1
| INTRODUCTION
treatment, pentavalent antimonial (sodium stibogluconate and
meglumine antimoniate), miltefosine, amphotericin B, and penta-
Leishmaniasis, trypanosomiasis, and malaria are vector‐borne para-
sitic diseases with the highest global impact. Besides being fatal, they
seriously affect the quality of life of millions of people by causing
deterioration of health and social stigma. These diseases have
become one of the challenges of the World Health Organization
midine isethionate are approved drugs (Figure 1). All these drugs
have associated toxicity problems with considerable adverse side
[
2]
effects and high costs, mainly due to hospital care ; all these
drawbacks cause low adherence to treatment. For American
trypanosomiasis (Chagas disease), only two drugs are available,
nifurtimox and benznidazole, both with bioavailability and efficacy
problems during the chronic phase of the disease, reduced toler-
(WHO) and their treatment is essential to achieve the goals of
[1]
sustainable development in 2030.
[
3]
In the absence of a vaccine and the difficulties of implementing
a vector control program, current antiparasitic control tools have
focused on chemotherapeutic treatments. The need for new
pharmacological alternatives for the control of parasitic diseases
caused by Leishmania spp., Trypanosoma cruzi, and Plasmodium fal-
ciparum is associated with different factors, including the few drugs
available, the reduced effectiveness due to microbial resistance,
extensive treatments, and high toxicity. In the case of leishmaniasis
ance, and moderate or severe adverse reactions. In the case of
malaria, quinolinic derivatives and artemisinin are recommended
for treatment, and their activity is being affected by the emergence
[
4]
of resistant Plasmodium strains.
For these reasons, the con-
tinuous work for the discovery of biologically active molecules
against these parasites is still essential.
In search of antiparasitic drugs, several natural products have
been previously reported. Particularly, chalcone‐type compounds
Arch Pharm. 2020;e2000157.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
|
1 of 10
https://doi.org/10.1002/ardp.202000157

2
of 10
GONZÁLEZ ET AL.
|
FIGURE 1 Chemical structures of the main drugs used as alternatives in the treatment of leishmaniasis (I), malaria (II), and Chagas disease (III)
(
1,3‐diaryl‐2‐propen‐1‐one) exhibited activity against Leishmania
TABLE 1 The synthesis of chalcone derivatives (according to
Scheme 1)
[
5–9]
[9,10]
[11,12]
species,
T. cruzi,
and P. falciparum,
in addition to anti-
[16,17]
[
10,13]
[14,15]
oxidant, cytotoxic,
antiproliferative,
antitumoral,
Compound type
Substituent (C atom)
[
18]
[19]
antimicrobial,
antiulcer, and anti‐inflammatory activities,
I, Y = H II, Y = OH III, Y = NH
2
2
3
4
5
6
among others. Thus, a wide range of synthetic procedures had been
carried out to establish structure–activity relationships in these
compounds, but the structure requirements involved in these activ-
ities are still practically unknown. In this study, we report the in vitro
activity of 21 chalcone derivatives, with H‐bond formation and
electron‐donating substituents in the A and B rings, respectively,
against Leishmania braziliensis, T. cruzi, and P. falciparum.
1
2
3
4
5
6
7
8
15
16
17
18
19
20
21
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
9
OCH
OCH
H
3
OCH
3
OCH
OCH
H
3
10
11
12
13
14
3
H
3
NO
F
2
H
H
H
Cl
H
H
OCH
3
H
2
2
| RESULTS
.1 | Chemistry
mass spectroscopy (HRMS), with relatively good yields between 50%
and 96%. These compounds have already been described in the
literature; however, there are no reports of biological activity against
L. braziliensis, T. cruzi, and P. falciparum.
Through a Claisen–Schmidt condensation reaction (Scheme 1), three
types of chalcone derivatives were synthesized according to different
substituents in C‐2′ of ring A: H, OH, and NH
with electroattractant groups (NO ) and electron donors (OCH
and Cl) were placed in the B ring (Table 1).
2
. Also, substitutions
2
3
, F,
2.2 | Antiparasite activities and cytotoxicity
All compounds were isolated by acidification of the reaction
mixture, followed by dichloromethane extraction and chromato-
graphic separation. The compounds were identified by infrared (IR),
On the basis of a promising activity, with an EC50 value <20 µM
(equivalent to 4 µg/ml), it was observed that the synthesized com-
pounds were more active against L. braziliensis as compared with
1
13
H NMR (nuclear magnetic resonance), C NMR, and high‐resolution
SCHEME 1 The synthetic strategy for the
preparation of chalcones derivatives

GONZÁLEZ ET AL.
3 of 10
|
T. cruzi and P. falciparum (Table 1). In general, compounds of type I,
Y–H, were more active against intracellular amastigotes of L. brazi-
liensis, as 12 of the 21 synthesized compounds showed an EC50 va-
lue <20 µM. This concentration was lower with compounds 2, 3, 7,
effects of the molecules will focus on the results exhibited on
L. braziliensis and T. cruzi.
The effects of the compounds were analyzed according to the
structural parameters that guided their synthesis: the C‐2′ sub-
stitutions of ring A and ring B. Usually the biological effect of the
chalcones has been attributed to their ability to act as Michael ac-
ceptors of biological macromolecules, due to the α,β‐unsaturated
10, 15, 16, and 20, which exhibited an EC50 value <10 µM. Com-
pounds 11, 13, and 21 showed a moderate activity against L. brazi-
liensis, and the remaining compounds, 8, 14, and 19, exhibited weak
activity against the Leishmania parasite (Table 1). All compounds
were more active than AMB.
[
20,21]
carbonyl system.
The results of this study indicate that this
mechanism may be responsible for cytotoxicity, which was very high
for all compounds, and besides, for the activity and selectivity against
L. braziliensis. Thus, most of the compounds of the series I (Y = H, 1–7)
showed high leishmanicidal activity, with EC50 < 20.0 µM, and com-
Only compounds 8, 10, 13, 16, and 17 showed high activity
against T. cruzi, and the most powerful was 17, with EC50 = 8.1 ±
1
.1 µM, which was seven times more active than BNZ. A moderate
activity against T. cruzi was observed in compounds 4, 5, 6, 9, 12, 15,
8, 19, 20, and 21, whose activity was higher than 20 µM, but lower
2
pounds of the series III of the amine (Y = NH , 8–14) exhibited this
1
activity to a lesser extent. Type II compounds, which have a hydroxyl
group in C‐2′ (Y = OH, 15–21), exhibited a poor activity; this fact
could be explained on the basis of the reactivity of the conjugated
carbonyl system, as the formation of hydrogen bonds with the car-
bonyl decreases the reactivity and, therefore, the ability to form
Michael adducts with biological molecules (Figure 2). The failure to
establish an H‐bonding in the compounds of the series I maintains the
reactivity of carbonyl. As for the hydroxyl group, it forms a stronger
hydrogen bond, negatively affecting the reactivity of the system and,
therefore, its activity.
than 50 µM. For compounds 3 and 7, it was not possible to determine
the exact EC50 values against T. cruzi, given its toxicity to U‐937 host
cells, whereas the remaining compounds, 1, 2, 11, and 14, exhibited a
low activity against T. cruzi amastigotes (Table 2). However, in P.
falciparum, all compounds displayed a wide range of activity, from
marginal to a lack of activity, with EC50 values ranging from
59.2 ± 0.2 µM to 908.9 ± 53.0 µM.
Although all compounds were cytotoxic for U‐937 cells, with
LC50 values lower than 83 µM, when the activity and cytotoxicity
were correlated, compound 17 was identified as the most promising
compound, exhibiting an SI of 10.13 for T. cruzi and 3.6 for L. brazi-
liensis, followed by compounds 3, 10, 15, and 16, with SI values of 3.0,
[22]
In this regard, Bello et al.
synthesized several poly-
methoxylated chalcones substituted in the A ring, whereas ring B
possessed groups like ours in different positions. It was found that
substitution in ring A, especially trimethoxylation in ortho and meta
positions with respect to carbonyl positions of the unsaturated sys-
tem, significantly improved activity against L. braziliensis, whereas
ring B did not influence that activity. Although the authors make an
analysis of molecular orbitals to rationalize this activity, it is likely
that the inductive donor effect on carbonyl and, therefore, its
reactivity as part of the Michael adduct is the actual cause.
2.9, 2.8 and 2.6, respectively (Table 2).
3
| DISCUSSION
The search for drugs against neglected diseases is a persistent need
for millions of people, due to the reduced number of drugs available,
toxicological effects, and parasite resistance. Although natural pro-
ducts have been a good alternative as medicine sources, the drug
development process is affected by the low availability of natural
substances, as bioactive substances are usually found in low con-
centrations. For this reason, natural products are taken as templates,
whose structures are modified and optimized by organic synthesis.
Natural chalcones are relatively simple structures, with a very varied
spectrum of activities, including antiparasitic, and can be easily syn-
thesized with excellent yields.
Similarly, licochalcone
A
(3‐dimethylallyl‐4,4′‐dihydroxy‐6‐
methoxychalcone) that has hydroxyl groups in C‐4 and C‐4′ was
active in animal models of L. major and L. donovani, and also exhibited
[
23]
antimalarial activity against P. yoelii in mice,
but in our case, all the
evaluated compounds showed a low activity against P. falciparum,
with EC50 values >50 μM. In that case, the hydrogen bridge is not
involved, but there is possibly an electron‐donating effect of the OH
group on the conjugate system. However, the contribution of the B
ring is another factor that also seems to be involved in the activity.
In this study, we describe the synthesis of three types of chal-
3
Compounds 2, 9, and 16, which have a C‐4′–OCH group, are more
cone derivatives, which have the substituents H, OH, or NH
whereas, in ring B, they have groups with different electron‐donating
and electron‐withdrawing properties, such as OCH , NO , and ha-
2
in C‐2′,
active, as this group acts as an electron donor system to the carbonyl
system. Nevertheless, polymethoxylation at adjacent positions in that
carbon atom, as in 7, 14, and 21, does not improve activity, except in
the first compound, in which the lack of ability to form hydrogen
bonds seems to be essential, as it was mentioned already. The latter
is in agreement with the results observed for the other compounds
substituted in position C‐4′–OH. Thus, for example, compounds 5, 12,
and 19, which have a powerful electron‐attracting atom such as
fluorine, do not show significant activity against amastigotes of
L. braziliensis, as the Michel acceptor is destabilized. In the case of the
3
2
logens (Cl, F). In vitro effects of these substances on L. braziliensis,
T. cruzi, and P. falciparum were evaluated.
The results found showed that the synthesized molecules
have a high selectivity; thus, P. falciparum was insensitive to them,
unlike what was detected in T. cruzi, which exhibited a good
activity with some compounds. Greater sensitivity was observed
on the amastigotes of L. braziliensis. Hence, the discussion of the

4
of 10
GONZÁLEZ ET AL.
|
TABLE 2 In vitro cytotoxicity and antiparasite activity of chalcones
Antiparasitic activity (EC50: µM ± SEM)
a
SI
b
c
d
Compound
Cytotoxicity (LC50: µM ± SEM)
27.9 ± 0.5
18.3 ± 0.3
19.4 ± 0.1
22.5 ± 0.4
23.0 ± 0.1
21.5 ± 0.1
22.3 ± 0.0
37.9 ± 2.7
20.4 ± 1.0
19.4 ± 0.4
26.3 ± 1.9
24.0 ± 0.8
25.2 ± 1.2
30.7 ± 1.6
27.3 ± 1.3
17.6 ± 0.3
82.3 ± 1.4
23.1 ± 0.4
28.6 ± 2.0
24.1 ± 0.4
25.7 ± 1.2
41.6 ± 6.8
300.8 ± 10.1
>768.5
L. braziliensis
P. falciparum
768.9 ± 72.0
127.1 ± 15.0
T. cruzi
L. braziliensis
1.9
T. cruzi
0.3
P. falciparum
0.03
0.14
0.04
0.03
0.05
0.02
0.07
0.30
0.34
0.20
0.25
0.21
0.33
0.11
0.07
0.14
0.14
0.33
0.24
0.33
0.12
NA
e
1
2
3
4
5
6
7
8
9
15.0 ± 1.4
99.9 ± 39.0
77.8 ± 6.4
9.6 ± 1.0
7.4 ± 0.1
18.6 ± 2.0
22.6 ± 3.1
17.4 ± 2.9
9.7 ± 0.8
56.2 ± 4.9
15.0 ± 0.1
7.0 ± 1.4
33.8 ± 13.4
22.3 ± 9.0
29.5 ± 9.7
>30.7
1.9
0.2
f
485.6 ± 125.0
724.3 ± 68.0
477.3 ± 101.0
908.9 ± 53.0
310.4 ± 34.0
134.3 ± 23.2
59.2 ± 0.2
93.6 ± 3.5
105.2 ± 4.0
112.4 ± 4.5
76.3 ± 5.4
286.9 ± 38.2
135.8 ± 3.1
256.4 ± 48.0
590.6 ± 38.0
69.4 ± 6.7
119.5 ± 12.9
73.1 ± 3.1
220.5 ± 21.0
NA
>11.2
2.6
<1.7
0.6
35.2 ± 3.5
31.4 ± 3.9
29.3 ± 4.1
>12.6
1.2
1.0
0.7
1.2
0.7
2.3
<1.8
1.5
24.5 ± 0.4
45.5 ± 5.7
19.7 ± 3.2
85.7 ± 10.4
47.1 ± 11.9
24.0 ± 0.4
71.2 ± 70.4
41.2 ± 5.8
22.0 ± 2.6
8.1 ± 1.1
44.0 ± 3.4
45.6 ± 7.5
43.6 ± 8.2
26.9 ± 4.7
NA
0.7
1.4
0.4
10
11
12
13
14
15
16
17
18
19
20
21
2.8
1.0
0.8
0.3
1.1
0.5
0.9
1.1
<1.0
2.9
0.4
9.4 ± 3.1
5.7 ± 0.0
22.6 ± 0.2
15.7 ± 2.6
65.1 ± 31.9
8.2 ± 1.9
41.1 ± 16.7
0.3 ± 0.1
NA
0.8
3.1
0.5
3.6
10.2
0.6
1.5
0.4
0.6
2.9
0.6
0.6
1.0
AMB
CQ
138.7
NA
NA
NA
>16.8
NA
0.6 ± 0.0
NA
501.3
NA
BNZ
DOX
NA
NA
56.5 ± 1.5
NA
NA
0.6 ± 0.1
NA
NA
NA
NA
Note: The data show the average values ± standard deviation (SEM), in μM, of the cytotoxicity in U‐937 cells and the antiparasite activity for each
compound.
Abbreviations: AMB, amphotericin B for Leishmania braziliensis; BNZ, benznidazole; CQ, chloroquine; DOX, doxorubicin; EC50, Half maximal effective
concentration; LC50, cytotoxicity expressed as LC50, NA, not applicable; SI, selectivity index.
a
SI = LC50 U‐937/EC50
Leishmania braziliensis amastigotes.
.
b
c
Plasmodium falciparum.
d
Trypanosoma cruzi amastigotes.
e
In bold: Active compounds (EC50 < 20 µM; equivalent to 4 µg/ml).
f
Exact concentration value was not determined, because the toxic concentration was higher than the active concentration.
2
NO group, which has effects on meta positions, as in compounds 4,
11, and 18, the effect of the hydrogen bridge remains predominant.
Several substances exhibited moderate activity against T. cruzi,
and contrary to what was observed for L. braziliensis, the compounds
that can form hydrogen bonds with carbonyl, such as 10 and 17
2
(C‐2′–OH and C‐2′–NH , respectively), had the best trypanocidal
activity. The latter was especially interesting, as it showed an intense
FIGURE 2 The effect of H‐bonding on the reactivity of chalcones
like Michael acceptors
trypanocidal activity, with an EC50 value of 8.12 μM. Additionally, this

GONZÁLEZ ET AL.
5 of 10
|
compound has relatively good activity against amastigotes of L. bra-
ziliensis, with an EC50 value of 22.6 μM. Similarly, 2′‐hydroxy chal-
cones containing methoxy groups were very active against T. brucei
and T. cruzi. Against this parasite, there are very few drugs available
in the market, and it also exhibits high resistance, as evidenced in the
reduced effect of BNZ, which was used as a control.
2 4
spraying with vanillin (3% in H SO ) and heating at 110°C. The
chromatographic separations were performed using preparative
column chromatography with silica gel 60 (200–300 mesh; Merck,
Darmstadt, Germany). The melting points were determined using a
Mel‐Temp apparatus (Electrothermal, Staffordshire, UK). Fourier
transform infrared (FTIR) spectra were obtained on a Bruker Alpha
1
Therefore, it seems, that the mechanism of action of the chal-
cones is diverse because the formation of Michael adducts does not
explain the effects observed in this study, as the most active com-
pounds against L. braziliensis were 2–4, 7, 10, 15, 16, and 20. More-
over, this leishmanicidal effect is different from that observed in
T. cruzi, as, in the latter, the active compounds were 6, 8, 10, 13, 16,
and 21, and especially compound 17. This leishmanicidal effect
was depending on the type of substituent on C‐2′, as well as those on
the B ring. P. falciparum was virtually insensible to all synthesized
substances.
FTIR spectrometer (Bruker Optic GmbH, Ettlingen, Germany). H,
1
3
C, and two‐dimensional NMR spectra of the synthetic compounds
were recorded on a Bruker Fourier 300 spectrometer (Bruker Bio‐
1
Spin GmbH, Rheinstetten, Germany), operating at 300 MHz for
H
1
3
and 75 MHz for C NMR, using CDCl
3
(Sigma) as the solvent and
tetramethylsilane as an internal standard. Chemical shifts (δ) are
reported in ppm, and the coupling constants (J) are reported in Hz.
High‐resolution mass spectra were obtained using a UHR‐QqTOF
(Ultra‐High‐Resolution Qq‐Time‐Of‐Flight) mass spectrometer (Im-
pact II‐Bruker), with an electrospray ionization source in the positive
ion mode.
Fifteen of the 21 compounds synthesized in this study pre-
sented EC50 values lower than that shown by benznidazole, with
compound 17 standing out and being seven times more active
against T. cruzi. On the contrary, none of the compounds assessed
for L. braziliensis had an activity higher or similar to amphotericin B.
Nevertheless, it is important to highlight that the purpose of this
study is to identify compounds with a promising activity that allows
their selection as hit compounds and then to continue the studies in
animal models to validate these selected compounds as active
pharmaceutical ingredients.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
5.1.2 | General procedure for the preparation of
chalcone derivatives
The synthesis was conducted according to experimental procedures
[24–27]
described in the literature.
Briefly, a mixture of acetophenone
derivative (1 mmol) and benzaldehyde derivative (1.05 mmol) was dis-
solved in ethanol (20 ml) and kept at room temperature, with magnetic
stirring for 5 min. Then, a KOH/EtOH solution (1.1 mmol on 20 ml) was
added dropwise, and stirring was continued at 40°C for 12 hr. Further,
the reaction mixture was poured into ice water and acidified with 1 M
HCl. Then, compounds were extracted with dichloromethane, and
finally, compounds were purified using column chromatography with
silica gel 60, eluting with hexane/ethyl acetate 2:1.
4
| CONCLUSION
Some chalcones with selective activity against Leishmania and Try-
panosoma parasites were synthesized in this study. The role of sev-
eral substituents in the A and B ring in this biological activity was
demonstrated; these facts must be considered in the design of anti-
parasitic chalcones. Moreover, these compounds were obtained by a
simple route and with high yields, which would eventually allow them
in the amounts for confirmatory assays in animal models of leish-
maniasis and trypanosomiasis.
1,3‐Diphenyl‐2‐propen‐1‐one (1)
Yield 70%, m.p.: 55–56°C; IR (KBr) ʋ/cm: 3,059.61, 1,966.81,
1,905.40, 1,822.23, 1,661.41, 1,604.31, 1,574.43, 1,495.76, 1,446.77,
1,339.86, 1,311.40, 1,286.70, 1,217.96, 1,178.51, 1,153.83, 1,073.00,
1
and 977.14; H NMR (300 MHz, CDCl
3
) δ = 8.03 (d, J = 7.2 Hz, 2H),
5
5
5
| EXPERIMENTAL
.1 | Chemistry
7.82 (d, J = 15.7 Hz, 1H), 7.69–7.61 (m, 2H), 7.61–7.47 (m, 4H), and
1
3
7
3
.46–7.40 (m, 3H). C NMR (75 MHz, CDCl ) δ = 190.89, 145.18,
138.50, 135.18, 133.12, 130.88, 129.28, 128.95, 128.82, 128.78, and
+
1
22.37. HRMS (ESI) m/z, calculated for C15
H
12O [M+H] 209.0960;
.1.1 | General
found 209.0957.
All commercially available reagents and solvents were obtained from
commercial suppliers and used without further purification. Benzal-
dehydes and acetophenones (98–99%) were purchased from Sigma
Chemical Co. (St. Louis, MO). Thin‐layer chromatography (TLC), with
silica gel 60 F254‐impregnated aluminum sheets (0.25 mm; Merck,
Darmstadt, Germany), was used to check the progress or reactions,
and compounds were detected under UV light (254, 360 nm) after
1‐Phenyl‐3‐(3,4,5‐trimethoxyphenyl)prop‐2‐en‐1‐one (2)
Yield 88%, m.p.: 141–142°C; IR (KBr) ʋ/cm: 3,065.75, 2,996.02,
1,720.91, 1,660.78, 1,577.87, 1,503.92, 1,461.00, 1,420.16, 1,320.10,
1,280.13, 1,249.37, 1,215.71, 1,185.83, 1,128.96, 1,018.96, 1,081.91,
1
984.49, and 832.40;
H
NMR (300 MHz, CDCl
3
)
δ = 8.01
(d, J = 7.0 Hz, 2H), 7.72 (d, J = 15.6 Hz, 1H), 7.63–7.46 (m, 3H), 7.40
(d, J = 15.6 Hz, 1H), 6.86 (s, 2H), 3.92 (s, 6H), and 3.90 (s, 3H).

6
of 10
GONZÁLEZ ET AL.
|
1
3
C NMR (75 MHz, CDCl
3
) δ = 191.42, 153.76, 145.34, 140.68,
(d, J = 7.1 Hz, 2H), 7.79 (d, J = 15.6 Hz, 1H), 7.67–7.46 (m, 5H), 7.42
1
3
1
5
3
38.54, 131.99, 130.65, 128.91, 128.78, 121.73, 105.91, 61.29, and
(d, J = 15.6 Hz, 1H), and 6.94 (d, J = 8.7 Hz, 2H); C NMR (75 MHz,
CDCl ) δ = 191.01, 162.04, 145.10, 138.76, 132.93, 130.60, 128.92,
+
6.49. HRMS (ESI) m/z, calculated for
C H
18 18
4
O Na [M+Na]
3
21.1097; found 321.1035.
128.78, 127.96, 120.13, 114.77, and 55.77; HRMS (ESI) m/z, calcu-
+
lated for C16H
14
O
2
[M+H] 239.1065; found 239.1065.
3
‐(3,5‐Dimethoxyphenyl)‐1‐phenylprop‐2‐en‐1‐one (3)
Yield 80%, m.p.: 80–82°C; IR (KBr) ʋ/cm: 2,947.80, 2,835.42,
1‐(2‐Hydroxyphenyl)‐3‐phenylprop‐2‐en‐1‐one (8)
1
1
,665.84, 1,604.70, 1,460.84, 1,444.19, 1,428.42, 1,346.73, 1,323.47,
Yield 88%, m.p.: 89°C; IR (KBr) ʋ/cm 3,038.37, 1,689.91, 1,605.69,
,287.68, 1,249.68, 1,202.87, 1,153.53, 1,062.67, 1,017.55, 973.03,
1,577.23, 1,460.33, 1,405.64, 1,373.27, 1,342.48, 1,303.56, 1,227.87,
1
1
and 937.93; H NMR (300 MHz, CDCl
3
) δ = 8.01 (d, J = 7.5 Hz, 2H),
1,180.59, 985.49, and 957.38; H NMR (300 MHz, CDCl
3
) δ = 11.83
7
.72 (d, J 15.7 Hz, 1H), 7.63–7.42 (m, 4H), 6.78 (s, 2H), 6.53 (s, 1H),
(s, 1H), 7.00–6.86 (m, 2H), 6.73–6.62 (m, 3H), 6.54–6.38 (m, 4H), and
13
13
and 3.84 (s, 6H); C NMR (75 MHz, CDCl
3
) δ = 190.99, 161.41,
3
6.08–5.88 (m, 2H). C NMR (75 MHz, CDCl ) δ = 193.79, 163.63,
1
5
45.24, 138.50, 137.11, 133.19, 129.00, 122.97, 106.68, 103.10, and
145.54, 136.48, 134.62, 130.99, 129.71, 129.10, 128.72, 120.13,
+
5.84; HRMS (ESI) m/z, calculated for C17
16
H O
3
[M+H] 269.1172;
120.04, 118.92, and 118.69. HRMS (ESI) m/z, calculated for
+
found 269.1170.
C H
15 12
O
2
[M+H] 225.0910; found 225.0908.
3
‐(4‐Nitrophenyl)‐1‐phenylprop‐2‐en‐1‐one (4)
1‐(2‐Hydroxyphenyl)‐3‐(3,4,5‐trimethoxyphenyl)prop‐2‐en‐1‐one (9)
Yield 90%, m.p.: 158°C; IR (KBr) ʋ/cm: 2,942.71, 2,843.01, 1,685.83,
Yield 88%, m.p.: 138–140°C; IR (KBr) ʋ/cm: 2,930.33, 1,658.88,
1,608.64, 1,516.58, 1,446.66, 1,337.15, 1,219.91, 1,106.27, 1,016.02,
1,586.53, 1,461.40, 1,423.37, 1,391.45, 1,329.92, 1,206.04, 1,127.90,
1
1
and 983.59; H NMR (300 MHz, CDCl
3
) δ = 8.29 (d, J = 8.8 Hz, 2H),
and 992.47; H NMR (300 MHz, CDCl
3
) δ = 12.85 (s, 1H), 7.93
8
7
1
1
2
.04 (d, J = 7.5 Hz, 2H), 7.80 (d, J = 8.5 Hz, 3H), 7.69–7.59 (m, 2H), and
(d, J = 8.1 Hz, 1H), 7.85 (d, J = 15.4 Hz, 1H), 7.52 (dd, J = 15.7, 10.1 Hz,
1
3
3
.54 (t, J = 7.3 Hz, 2H); C NMR (75 MHz, CDCl ) δ = 190.02, 148.90,
2H), 7.03 (d, J = 8.3 Hz, 1H), 6.95 (t, J = 8.0 Hz, 1H), 6.88 (s, 2H), 3.92
1
3
41.88, 141.39, 137.86, 133.74, 129.29, 129.18, 128.95, 126.04, and
3
(s, 6H), and 3.71 (s, 3H); C NMR (75 MHz, CDCl ) δ = 193.85,
+
24.58. HRMS (ESI) m/z, calculated for C15
H11NO
3
Na [M+Na]
163.90, 153.84, 145.99, 141.09, 136.72, 130.38, 129.94, 120.32,
76.0631; found 276.0634.
119.56, 119.15, 118.98, 106.21, 61.37, and 56.58; HRMS (ESI) m/z,
+
calculated for C18
H O
18 5
[M+H] 315.1227, found 315.1228.
3
‐(4‐Fluorophenyl)‐1‐phenylprop‐2‐en‐1‐one (5)
Yield 75%, m.p.: 79–81°C; IR (KBr) ʋ/cm: 3,067.62, 3,037.36,
3‐(3,5‐Dimethoxyphenyl)‐1‐(2‐hydroxyphenyl)prop‐2‐en‐1‐one (10)
Yield 55%, m.p.: 102–103°C; IR (KBr) ʋ/cm: 3,010, 1,702.31,
1
1
,970.60, 1,907.19, 1,602.84, 1,574.84, 1,507.89, 1,446.07, 1,414.27,
,340.71, 1,317.28, 1,214.03, 1,159.36, 1,100.53, 1,031.12, 1,013.42,
40.75, 836.71, 820.28, 777.05, 754.72, 716.28, and 688.41;
1,604.87, 1,578.03, 1,476.68, 1,272.76, 1,201.18, 1,159.09, 1,072.45,
1
9
and 828.94; H NMR (300 MHz, CDCl
3
) δ = 12.81 (s, 1H), 7.89
1
H
NMR (300 MHz, CDCl3) δ = 8.02 (d, J = 7.2 Hz, 2H), 7.78
(d, J = 7.9 Hz, 1H), 7.80 (d, J = 15.4 Hz, 1H), 7.57 (d, J = 15.4 Hz, 1H),
(
(
(
d, J = 15.7 Hz, 1H), 7.70–7.56 (m, 3H), 7.55–7.41 (m, 3H), and 7.12
7.48 (t, J = 7.8 Hz, 1H), 7.01 (d, J = 8.9 Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H),
13
13
t, J = 8.6 Hz, 2H); C NMR (75 MHz, CDCl
3
) δ = 190.66, 164.37
3
6.77 (s, 2H), 6.52 (s, 1H), and 3.82 (s, 6H); C NMR (75 MHz, CDCl )
d, J = 251.8 Hz), 143.84, 138.42, 133.19, 131.44 (d, J = 3.2 Hz),
δ = 193.91, 163.84, 161.34, 145.71, 136.72, 129.97, 120.76, 120.22,
1
1
30.68 (d, J = 8.4 Hz), 128.80 (d, J = 13.4 Hz), 122.05 (d, J = 2.2 Hz),
119.14, 118.86, 107.36, 103.28, and 55.73; HRMS (ESI) m/z,
+
16.45, and 116.09. HRMS (ESI) m/z, calculated for C15
H11FO
calculated for C17
H O
17 4
[M+H] 285.1121; found 285.1120.
+
[
M+H] 227.0866; found 227.0800.
1‐(2‐Hydroxyphenyl)‐3‐(4‐nitrophenyl)prop‐2‐en‐1‐one (11)
3
‐(4‐Chlorophenyl)‐1‐phenylprop‐2‐en‐1‐one (6)
Yield 72%, m.p.: 141.8–142.1°C; IR (KBr) ʋ/cm: 3,363.81, 2,923.21,
1
Yield 80%, m.p.: 113–114°C; IR (KBr) ʋ/cm: 1,657.86, 1,602.92,
1,798.98, 1,640.58, 1,575.96, 1,272.99, and 1,179,85; H NMR
1,488.23, 1,446.13, 1,402.67, 1,333.54, 1,217.23, and 1,092.03;
3
(300 MHz, CDCl ) δ = 12.59 (s, 1H), 8.30 (d, J = 8.8 Hz, 2H), 7.99–7.88
1
H
NMR (300 MHz, CDCl
3
)
δ = 8.01 (d, J = 7.1 Hz, 2H), 7.74
(m, 2H), 7.88–7.76 (m, 3H), 7.60–7.51 (m, 1H), 7.06 (d, J = 8.4 Hz, 1H),
13
1
3
(
d, J = 15.7 Hz, 1H), 7.62–7.42 (m, 6H), and 7.37 (d, J = 8.5 Hz, 2H);
NMR (75 MHz, CDCl ) δ = 190.43, 143.54, 138.24, 136.66, 133.60,
33.22, 129.87, 129.49, 128.94, 128.77, and 122.64; HRMS (ESI) m/z,
C
and 6.98 (t, J = 7.6 Hz, 1H); C NMR (75 MHz, CDCl3) δ = 193.33,
3
164.09, 149.08, 142.46, 141.02, 137.40, 130.02, 129.49, 124.65,
1
124.44, 120.10, 119.47, and 119.20; HRMS (ESI) m/z, calculated for
+
+
calculated for C15
H
11ClO [M+H] 243.0571; found 243.0570.
C
15
H
11NO
4
[M+Na] 292.0580; found 292.0594.
3
‐(4‐Methoxyphenyl)‐1‐phenylprop‐2‐en‐1‐one (7)
3‐(4‐Fluorophenyl)‐1‐(2‐hydroxyphenyl)prop‐2‐en‐1‐one (12)
Yield 87%, m.p.: 80–82°C; IR (KBr) ʋ/cm: 3,013.96, 2,954.87,
Yield 56%, m.p.: 79–80°C; IR (KBr) ʋ/cm: 1,639.00, 1,579.40, 1,508.20,
1
2
1
9
,942.51, 2,902.20, 2,841.91, 1,657.84, 1,600.28, 1,511.88, 1,445.81,
1,341.87, 1,270.33, 1,208.23, 1,159.59, 1,024.16, and 829.07; H NMR
,419.03, 1,339.03, 1,304.35, 1,263.99, 1,213.97, 1,170.42, 1,017.57,
(300 MHz, CDCl
3
) δ = 12.80 (s, 1H), 7.97–7.81 (m, 2H), 7.73–7.59
1
84.70, and 824.98;
H
NMR (300 MHz, CDCl
3
)
δ = 8.01
(m, 3H), 7.52 (dd, J = 15.5, 7.0 Hz, 2H), 7.13 (t, J = 8.6 Hz, 2H), 7.03

GONZÁLEZ ET AL.
7 of 10
|
13
(
d, J = 8.4 Hz, 1H), and 6.95 (t, J = 7.6 Hz, 1H); C NMR (75 MHz,
7.30 (t, J = 7.7 Hz, 1H), 6.77 (d, J = 2.2 Hz, 2H), 6.74–6.67 (m, 2H),
13
3
CDCl ) δ = 193.86, 163.95, 164.61 (d, J = 252.6 Hz), 144.47, 136.82,
3
6.54–6.49 (m, 1H), and 3.84 (s, 6H); C NMR (75 MHz, CDCl )
131.18 (d, J = 11.8, 3.4 Hz), 130.96 (d, J 8.7, 0.0 Hz), 129.93, 120.25,
δ = 191.97, 161.33, 151.16, 143.28, 137.48, 134.71, 131.36, 123.92,
120.12, 119.22, 119.00, and 116.59 (d, J = 22.0 Hz); HRMS (ESI) m/z,
119.38, 117.72, 116.31, 106.49, 102.62, and 55.79; HRMS (ESI) m/z,
+
+
calculated for C15
H
11FO
2
[M+H] 243.0815; found 243.0815.
calculated for C17
H
18NO
3
[M+H] 284.1281; found 284.1282.
3
‐(4‐Chlorophenyl)‐1‐(2‐hydroxyphenyl)prop‐2‐en‐1‐one (13)
1‐(2‐Aminophenyl)‐3‐(4‐nitrophenyl)prop‐2‐en‐1‐one (18)
Yield 93%, m.p.: 149°C; IR (KBr) ʋ/cm: 2,958.90, 1,640.86, 1,563.97,
Yield 75%, m.p.: 145°C; IR (KBr) ʋ/cm: 3,460.29, 3,337.72, 1,646.71,
1
1
,487.07, 1,440.54, 1,406.14, 1,368.17, 1,341.36, 1,303.75, 1,263.06,
1,615.70, 1,578.40, 1,544.81, 1,411.48, 1,340.16, 1,207.79, 1,159.54,
1
1
,205.60, 1,159.31, 1,090.61, 1,022.97, 983.97, and 819.6; H NMR
1,109.31, 1,005.95, 986.22, 773.52, and 743.76; H NMR (300 MHz,
(
(
(
300 MHz, CDCl
3
) δ = 12.76 (s, 1H), 7.94–7.75 (m, 2H), 7.69–7.56
CDCl
(m, 4H), 7.31 (t, J = 7.7 Hz, 1H), and 6.76–6.67 (m, 2H); C NMR
(75 MHz, CDCl ) δ = 190.91, 151.66, 148.59, 141.84, 139.98, 135.20,
3
) δ = 8.25 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.3 Hz, 1H), 7.78–7.70
13
m, 3H), 7.51 (t, J = 7.8 Hz, 1H), 7.41 (d, J = 8.5 Hz, 2H), 7.04
1
3
d, J = 8.3 Hz, 1H), and 6.95 (t, J = 7.6 Hz, 1H); C NMR (75 MHz,
) δ = 193.80, 163.95, 144.28, 137.20, 136.91, 133.39, 130.13,
29.95, 129.68, 120.87, 120.24, 119.26, and 119.03. HRMS (ESI) m/z,
3
CDCl
3
131.29, 129.02, 127.34, 124.49, 118.75, 117.75, and 116.28. HRMS (ESI)
+
1
m/z, calculated for C15
H
12
N
2
O
3
[M+H] 269.0920; found 269.0920.
+
2
calculated for C15H11ClO [M+H] 259.0520; found 259.0549.
1
‐(2‐Aminophenyl)‐3‐(4‐fluorophenyl)prop‐2‐en‐1‐one (19)
1
1
‐(2‐Hydroxyphenyl)‐3‐(4‐methoxyphenyl)prop‐2‐en‐1‐one (14)
3
Yield 59%, m.p.: 90°C; H NMR (300 MHz, CDCl ) δ = 7.92–7.87
Yield 85%, m.p.: 91°C; IR (KBr) ʋ/cm: 2,969.64, 2,934.24, 1,639.09,
(m, 1H), 7.75 (d, J = 15.7 Hz, 1H), 7.70–7.64 (m, 2H), 7.59
1
9
7
7
,564.79, 1,512.46, 1,303.75, 1,260.68, 1,211.64, 1,162.63, 1,030.42,
(d, J = 15.6 Hz, 1H), 7.38–7.32 (m, 1H), 7.20–7.11 (m, 2H), 6.81–6.71
13
1
85.69, and 828.91; H NMR (300 MHz, CDCl
3
) δ = 12.95 (s, 1H),
3
(m, 2H), and 6.37 (s, 2H); C NMR (75 MHz, CDCl ) δ = 191.0, 164.0,
.98–7.85 (m, 2H), 7.64 (d, J = 8.6 Hz, 2H), 7.59–7.45 (m, 2H),
161.0, 151.0, 141.69, 136.0, 134.44, 131.01, 130.20, 126.0, 117.39,
1
3
.05–6.92 (m, 4H), and 3.87 (s, 3H); C NMR (75 MHz, CDCl
3
)
116.23, 116.16. and 115.0. HRMS (ESI) m/z, calculated for
+
δ = 194.05, 163.89, 162.38, 145.73, 136.52, 130.92, 129.89, 127.69,
C
15
H
12FNO [M+H] 242.0975; found 242.0974.
120.46, 119.12, 118.94, 117.92, 114.86, and 55.81. HRMS (ESI) m/z,
+
calculated for C16
H
14
O
3
Na [M+Na] 227.0835; found 227.0868.
1‐(2‐Aminophenyl)‐3‐(4‐chlorophenyl)prop‐2‐en‐1‐one (20)
Yield 85%, m.p.: 94°C; IR (KBr) ʋ/cm: 3,472.61, 3,325.33, 1,643.98,
1,612.83, 1,570.41, 1,492.35, 1,444.31, 1,404.72, 1,336.60, 1,292.96,
1
‐(2‐Aminophenyl)‐3‐phenylprop‐2‐en‐1‐one (15)
Yield 88%, m.p.: 50–54°C; IR (KBr) ʋ/cm: 3,442.86, 3,383.19, 3,078.44,
1,264.03, 1,210.78, 1,156.94, 1,088.42, 1,026.48, 1,008.26, 981.60,
1
1,643.38, 1,617.30, 1,576.07, 1,340.49, 1,209.27, 1,156.99, and 739.12;
and 842.88; H NMR (300 MHz, CDCl
3
) δ = 7.902 (d, J = 1.2 Hz, 1H),
1
H
NMR (300 MHz, CDCl
3
)
δ = 7.87 (d, J = 8.2 Hz, 1H), 7.75
7.874–7.588 (m, 4H), 7.438–7.304 (m, 3H), 6.766–6.719 (m, 2H), and
1
3
(d, J = 15.6 Hz, 1H), 7.68–7.58 (m, 3H), 7.41 (d, J = 5.2 Hz, 3H), 7.30
6.40 (s, 2H); C NMR (75 MHz, CDCl
3
) δ = 191.36, 151.15, 141.46,
13
(
t, J = 7.7 Hz, 1H), and 6.71 (dd, J = 7.7, 4.0 Hz, 2H); C NMR (75 MHz,
CDCl ) δ = 191.94, 151.25, 143.17, 135.50, 134.57, 131.27, 130.35,
29.14, 128.51, 123.36, 119.28, 117.56, and 116.11. HRMS (ESI) m/z,
135.96, 134.52, 133.79, 131.02, 129.45, 129.19, 123.57, 118.80,
3
117.40, and 115.96. HRMS (ESI) m/z, calculated for C15H12ClNO
+
1
[M+H] 258.0678; found 258.0678.
+
calculated for C15H13NO [M+H] 224.1069, found 224.1068.
1‐(2‐Aminophenyl)‐3‐(4‐methoxyphenyl)prop‐2‐en‐1‐one (21)
1
‐(2‐Aminophenyl)‐3‐(3,4,5‐trimethoxyphenyl)prop‐2‐en‐1‐one (16)
Yield 56%, m.p.: 67–68°C; IR (KBr) ʋ/cm: 3,455.33, 3,329.60, 3,076.14,
Yield 50%, m.p.: 128°C; IR (KBr) ʋ/cm: 3,468.67, 3,320.11, 2,991.19,
1,684.40, 1,644.34, 1,613.86, 1,572.25, 1,541.77, 1,510.32, 1,420.68,
2
1
8
,959.13, 2,938.87, 2,836.37, 1,644.19, 1,576.27, 1,542.03, 1,461.90,
1,353.03, 1,338.23, 1,305.89, 1,287.80, 1,247.72, 1,211.78, 1,158.14,
1
,357.04, 1,320.46, 1,286.48, 1,125.59, 1,012.88, 868.25, and
1,112.04, and 978.75; H NMR (300 MHz, CDCl
3
) δ = 7.86 (d, J = 8.3 Hz,
1
30.22; H NMR (300 MHz, CDCl
3
) δ = 7.89 (d, J = 8.3 Hz, 1H), 7.68
1H), 7.72 (d, J = 15.5 Hz, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.50 (d, J = 15.5 Hz,
(
d, J = 15.5 Hz, 1H), 7.52 (d, J = 15.5 Hz, 1H), 7.30 (t, J = 7.7 Hz, 1H),
1H), 7.32–7.23 (m, 1H), 6.93 (d, J = 8.7 Hz, 2H), 6.73–6.63 (m, 2H), 6.30
13
13
6
.87 (d, J = 2.0 Hz, 2H), 6.80–6.67 (m, 2H), and 3.93 (m, 9H); C NMR
3
(s, 2H), and 3.85 (s, 3H); C NMR (75 MHz, CDCl ) δ = 192.84, 161.36,
3
(75 MHz, CDCl ) δ = 191.72, 153.63, 151.24, 143.29, 140.19, 134.50,
150.90, 142.86, 134.13, 130.95, 130.01, 128.03, 120.82, 119.35,
1
5
31.17, 131.01, 122.60, 119.22, 117.56, 115.99, 105.63, 61.18, and
117.32, 115.89, 114.38, and 55.44. HRMS (ESI) m/z, calculated for
+
+
6.39; HRMS (ESI) m/z, calculated for C18
H
19NO
4
[M+H] 314.1386;
C
16
H
16NO
2
[M+H] 254.1175; found 254.1174.
found 314.1386.
1
‐(2‐Aminophenyl)‐3‐(3,5‐dimethoxyphenyl)prop‐2‐en‐1‐one (17)
Yield 63%, m.p.: 119–122°C; IR (KBr) ʋ/cm: 3,422.12, 3,301.63, 1,650.14,
,584.38, 1,451.88, 1,291.08, 1,248.01, 1,202.06, 1,164.68, and 1,070.73;
5.2 | Biological assays
1
The compounds were assayed in vitro for cytotoxicity on mammalian
cells and for leishmanicidal activity against intracellular amastigotes
1
3
H NMR (300 MHz, CDCl ) δ = 7.85 (d, J = 7.1 Hz, 1H), 7.70–7.52 (m, 2H),

8
of 10
GONZÁLEZ ET AL.
|
of L. braziliensis. The trypanocidal activity was carried out in in-
tracellular amastigotes of T. cruzi. Their antiplasmodial activity was
determined on nonsynchronized P. falciparum cultures. The anti-
protozoal activities were classified grouped according to EC50 values,
cells. Infected cells exposed to amphotericin B were used as con-
trols as a positive control (antileishmanial drug) in the assay,
whereas infected cells incubated in the absence of any compound or
[
29]
drug were used as controls for infection (negative control).
[
26]
based on the hit criteria proposed by Katsuno et al.
into high
(
EC50 < 10 μM), moderate (10 μM < EC50 < 50 μM), and low activity
EC50 > 50 μM).
(
5.2.3 | Antiplasmodial activity
The antiplasmodial activity was evaluated in vitro on asynchro-
5
.2.1 | Cytotoxic activity
nous cultures of P. falciparum (3D7 strain), maintained under
[
30]
standard culture conditions.
The activity of each compound
Cytotoxicity of the compounds was evaluated in the human monocyte
was assessed by measurement of the lactate dehydrogenase
cell line (U‐937 ATCC CRL‐1593.2) at the exponential growth phase,
(LDH) activity released from the cytosol of damaged cells into the
[31]
5
adjusted at 1 × 10 cells/ml in RPMI‐1640 medium (GIBCO Invitrogen,
supernatant, as previously described.
P. falciparum cultures
Basel, Switzerland) enriched with L‐glutamine (200 mM), 10% in-
activated fetal bovine serum (FBS), 1% penicillin (10,000 UI/ml) and
streptomycin (10,000 mg/ml). The compounds were added as a 100‐µl
solution in the same medium. For each compound, six serial 1:2 dilu-
tion concentrations, starting at 368 µM, were assayed. Doxorubicin
was assayed as a control drug under the same dilution pattern, starting
at 18 µM, respectively. Afterward, cells were incubated for 72 hr at
were adjusted to 0.5% parasitemia and 1% hematocrit in RPMI‐
1640 medium enriched with 1% Albumax II. Then, in each well of a
96‐well plate, 100 μl of parasite suspension was dispensed and
subsequently exposed against 100 μl of four serial dilutions of
compounds (100, 25, 3.125, and 0.78 μM), chloroquine (CQ) was
applied as a positive antiplasmodial drug control. Parasites un-
exposed to any compound were used as controls for both growth
and viability (negative control). Plates were incubated for 72 hr at
2
37°C and 5% CO , and cell viability was assayed by the MTT reduction
assay according to the optical density (OD) at 570 nm of the resulting
reduction of formazan in a Varioskan Flash Multimode Reader
2 2 2
37°C in an N (90%), CO (5%), and O (5%) atmosphere. After
incubation, the plates were harvested and parasites were sub-
jected to three 20‐min freeze–thaw cycles. Meanwhile, 100 μl of
Malstat reagent (400 μl Triton X‐100 in 80 ml deionized water, 4 g
L‐lactate, 1.32 g Tris‐buffer reagent, and 0.022 g acetylpyridine
adenine dinucleotide in 200 ml deionized water; pH 9.0) and 25 μl
of NBT/PES solution (16 g/l nitroblue tetrazolium salt and 0.8 g/l
phenazine methosulfate) were added to each well of a second flat‐
bottom 96‐well microtiter plate. Cultures in each well were lysed
by five freeze–thaw cycles, the lysate was homogenized by re-
petitive pipetting, and 15 μl from each well was transferred into a
replica plate containing Malstat and NBT/PES solution. After 1 hr
of incubation in the dark, color development of the LDH reaction
[17]
(Thermo Scientific, Waltham, MA).
Nonspecific absorbance was
corrected by subtracting the OD of the blank. Cells exposed to dox-
orubicin were used as positive controls, whereas unexposed cells were
[28]
used as negative controls.
5
.2.2 | Antileishmanial activity
The antileishmanial activity was evaluated in intracellular amasti-
gotes of L. braziliensis as follows. U‐937 human cells at a density of
5
3
× 10 cells/ml in Rosewell Park Memorial Institute (RPMI)‐1640
medium and 0.16 μM phorbol‐12‐myristate‐13‐acetate were dis-
pensed in a 24‐well microplate and then infected with stationary
L. braziliensis promastigotes transfected with the green fluorescent
protein gene (MHOM/CO/88/UA301‐EGFP), at a 15:1 parasite/cell
was monitored at 650 nm in a Varioskan, Thermo spectro-
fluorometer. Nonspecific absorbance was corrected by subtract-
ing the value of the blank.
2
ratio. Plates were incubated at 34°C and 5% CO for 3 hr, and cells
were then washed twice with phosphate‐buffered saline (PBS) to
eliminate noninternalized parasites. Fresh RPMI‐1640 medium was
added into each well (1 ml) and the plates were incubated again for
5.2.4 | Antitrypanosomal activity
T. cruzi (Tulahuen strain transfected with the β‐galactosidase gene)
metacyclic trypomastigotes were cultured at 26°C for 10 days in
modified NNN (Novy–McNeal–Nicolle) medium. U‐937 cells were
2
4 hr. Afterward, the RPMI‐1640 medium was replaced with com-
pound at four serial dilutions (250, 125, 62.5, and 31.25 μM) dis-
4
solved in a fresh culture medium, and plates were incubated at 34°C
seeded in 96‐well dishes at a density of 2.5 × 10 cells in 100 μl of
and 5% CO
2
for 72 hr. Then, cells were detached from the plate with
medium/well and exposed to PMA, as described above. After trans-
formation to macrophages, cells were infected using a ratio of five
parasites per cell and incubated in RPMI‐1640 medium with 10% FBS
1
00 μl of ethylenediaminetetraacetic acid/trypsin (0.25%) solution,
centrifuged (500g, 5 min, 4°C), and washed with 1 ml of cold PBS.
Cells were then suspended in 500 μl of 0.4% paraformaldehyde in
PBS and analyzed by flow cytometry (Cytomics™ FC 500; Coulter),
expressed as median fluorescence intensity. Nonspecific fluores-
cence was corrected by subtracting the FL1 channel in noninfected
2
for 24 hr at 37°C and 5% CO . Two washes with PBS removed
noninternalized parasites, and then fresh medium was added, con-
taining each of the six serial diluted concentrations of each testing
compound or benznidazole. After 72 hr of incubation under identical

GONZÁLEZ ET AL.
9 of 10
|
conditions, the viability of intracellular amastigotes was determined
by measuring the β‐galactosidase activity. For this, 100 μM of
chlorophenol red‐β‐D‐galactopyranoside and 0.1% Nonidet P‐40 were
added to each well and incubated for 4 hr at 37°C and at 24°C
protected from light. Then, measurement β‐galactosidase activity was
measured at 570 nm by a Varioskan, Thermo spectrophotometer.
Nonspecific absorbance (blank) was subtracted from the measure-
ment. Infected cells exposed to benznidazole (50, 12.5, and
[4] P. K. Pramanik, M. N. Alam, D. Roy Chowdhury, T. Chakraborti,
J. Glob. Antimicrob. Resist. 2019, 18, 1. https://doi.org/10.1016/j.jgar.
2
019.01.023
5] N. K. Sahu, S. S. Balbhadra, J. Choudhary, D. V. Kohli, Curr. Med. Chem.
012, 19, 209. https://doi.org/10.2174/092986712803414132
[
2
[6] M. V. Palmeria de Mello, B. de Azevedo Abrahim‐Vieira, T. F. Souza
Domingos, J. Barbosa de Jesus, A. C. Correa de Sousa, C. R.
Rodrigues, A. M. Teles de Souza, Eur. J. Med. Chem. 2018, 150, 920.
https://doi.org/10.1016/j.ejmech.2018.03.047
[
7] O. Kayser, A. F. Kiderlen, Phyther. Res. 2001, 15, 148.
3.125 μM) were used as controls for antitrypanosomal activity (po-
[8] N. Tajuddeen, M. B. Isah, M. A. Suleiman, F. R. van Heerden, M. A.
Ibrahim, Int. J. Antimicrob. Agents 2018, 51, 311. https://doi.org/10.
sitive control) and nontreated cells as controls for infection (negative
1
016/j.ijantimicag.2017.06.010
control). Determinations were done in triplicate with at least two
[
9] T. G. Passalacqua, L. A. Dutra, L. de Almeida, A. M. A. Velásquez, F. A.
[
32]
independent experiments.
Torres Esteves, P. R. Yamasaki, M. dos Santos Bastos, L. O. Regasini, P.
A. M. Michels, V. da Silva Bolzani, M. A. S. Graminha, Bioorg. Med. Chem.
Lett. 2015, 25, 3342. https://doi.org/10.1016/j.bmcl.2015.05.072
[
10] J. C. Aponte, M. Veras
Vaisberg, R. H. Gilman, G. B. Hammond, J. Med. Chem. 2008, 51,
230. https://doi.org/10.1021/jm800812k
tegui, E. Malaga, M. Zimic, M. Quiliano, A. J.
́ ́
5
.2.5 | Data analysis
6
All experiments were performed in triplicate. The cytotoxicity was
expressed as the Lethal Concentration 50 (LC50), whereas the anti-
protozoal activity was calculated as the median Effective Con-
centration (EC50). Both LC50 and EC50 values were calculated by the
Probit analysis. The LC50 values in U‐937 cells and the EC50 values in
P. falciparum were determined according to the percentage of
inhibition of cell or parasite viability, whereas the EC50 values in
L. braziliensis, T. cruzi amastigotes, and blood‐stage P. falciparum
parasites were determined according to the percentage of inhibition,
[
[
[
11] S. Kumar, A. Saini, J. Gut, P. J. Rosenthal, R. Raj, V. Kumar, Eur. J. Med.
Chem. 2017, 138, 993. https://doi.org/10.1016/j.ejmech.2017.06.009
12] L. C. Mishra, A. Bhattacharya, V. K. Bhasin, Acta Trop. 2009, 109, 194.
https://doi.org/10.1016/j.actatropica.2008.11.006
13] H. Yin, J. Dong, Y. Cai, X. Shi, H. Wang, G. Liu, Y. Tang, J. Liu, L. Ma,
Eur. J. Med. Chem. 2019, 180, 3550. https://doi.org/10.1016/j.ejmech.
2019.05.053
[
14] D. Quaglio, N. Zhdanovskaya, G. Tobajas, V. Cuartas, S. Balducci,
M. S. Christodoulou, G. Fabrizi, M. Gargantilla, E. M. Priego, A. C.
Pestana, D. Passarella, I. Screpanti, B. Botta, R. Palermo, M. Mori,
̃
F. Ghirga, M. J. P. Pérez, ACS Med. Chem. Lett. 2019, 10, 639. https://
[
28]
as described previously.
doi.org/10.1021/acsmedchemlett.8b00608
[
[
15] E. Corsini, G. Facchetti, S. Esposito, A. Maddalon, I. Rimoldi, M. S.
Christodoulou, Arch. Pharm. 2020, 353, e2000062. https://doi.org/
ACKNOWLEDGEMENTS
1
0.1002/ardp.202000062
L. A. Gonzalez is grateful to Minciencias for a scholarship in Doc-
torados Nacionales Program, 647‐2014. Financial support: Min-
ciencias and Universidad de Antioquia grant 111571249860,
contract 061‐2016.
16] A. Pesaran, F. Vafaei, M. Farokhpour, M. H. Nasr, A. R. Massah, A. R.
Iran, J. Pharm. Res. 2017, 16, 565.
[17] M. Das, K. Manna, J. Toxicol. 2016, 2016, 7651047. https://doi.org/
0.1155/2016/7651047
18] M. Xu, P. Wu, F. Shen, J. Ji, K. P. Rakesh, Bioorg. Chem. 2019, 91,
03133. https://doi.org/10.1016/j.bioorg.2019.103133
1
[
1
CONFLICTS OF INTEREST
[19] D. Biswaranjan, S. Subir, Int. J. Pharm. Res. 2015, 7, 1.
[20] P. Singh, A. Anand, V. Kumar, Eur. J. Med. Chem. 2014, 85, 758.
https://doi.org/10.1016/j.ejmech.2014.08.033
The authors declare that there are no conflicts of interest.
[
21] C. Borsari, N. Santarem, J. Torrado, A. I. Olías, M. J. Corral, C. Bap-
tista, S. Gul, M. Wolf, M. Kuzikov, B. Ellinger, G. Witt, P. Gribbon,
J. Reinshagen, P. Linciano, A. Tait, L. Costantino, L. H. Freitas‐Junior,
C. B. Moraes, P. Bruno dos Santos, L. M. Alcântara, C. H. Franco, C. D.
Bertolacini, V. Fontana, P. Tejera Nevado, J. Clos, J. M. Alunda,
A. Cordeiro‐da‐Silva, S. Ferrari, M. P. Costi, Eur. J. Med. Chem. 2017,
ORCID
Luis A. González
Yulieth A. Upegui
http://orcid.org/0000-0002-4935-3320
http://orcid.org/0000-0002-9348-6227
Luis Rivas
http://orcid.org/0000-0002-2958-3233
Fernando Echeverri
Gustavo Escobar
Sara M. Robledo
Wiston Quiñones
http://orcid.org/0000-0002-9530-8395
http://orcid.org/0000-0002-5387-4356
http://orcid.org/0000-0003-2752-4931
http://orcid.org/0000-0003-3088-1462
1
26, 1129. https://doi.org/10.1016/j.ejmech.2016.12.017
[
22] M. L. Bello, L. D. Chiaradia, L. R. S. Dias, L. K. Pacheco, T. R. Stumpf, A.
Mascarello, M. Steindel, R. A. Yunes, H. C. Castro, R. J. Nunes, C. R.
Rodrigues, Bioorg. Med. Chem. 2011, 19, 5046. https://doi.org/10.
1
016/j.bmc.2011.06.023
[
23] A. Foroumadi, S. Emami, M. Sorkhi, M. Nakhjiri, Z. Nazarian,
REFERENCES
S. Heydari, S. K. Ardestani, F. Poorrajab, A. Shafiee, Chem. Biol. Drug Des.
2010, 75, 590. https://doi.org/10.1111/j.1747-0285.2010.00959.x
[24] C. Zhuang, W. Zhang, C. Sheng, W. Zhang, C. Xing, Z. Miao, Chem.
Rev. 2017, 117, 7762. https://doi.org/10.1021/acs.chemrev.7b00020
[25] J. A. Donnelly, D. F. Farrell, J. Org. Chem. 1990, 55, 1757. https://doi.
org/10.1021/jo00293a017
[
[
[
1] World Health Organization: Tropical diseases. 2020. https://www.
who.int/topics/tropical_diseases/es/ (accessed: January 2020).
2] R. Laniado‐Laborín, M. N. Cabrales‐Vargas, Rev. Iberoam. Micol. 2009,
26, 223. https://doi.org/10.1016/j.riam.2009.06.003
3] G. Rojo, C. Castillo, J. Duaso, A. Liempi, D. Droguett, N. Galanti, J. D.
Maya, R. López‐Muñoz, U. Kemmerling, Acta Trop. 2014, 132, 112.
https://doi.org/10.1016/j.actatropica.2014.01.002
[26] K. Katsuno, J. N. Burrows, K. Duncan, R. H. van Huijsduijnen,
T. Kaneko, K. Kita, C. E. Mowbray, D. Schmatz, P. Warner, B. T.

10 of 10
GONZÁLEZ ET AL.
|
Slingsby, Nat. Rev. Drug Discov. 2015, 14, 751. https://doi.org/10.
038/nrd4683
[32] F. S. Buckner, C. L. Verlinde, A. C. La Flamme, W. C. van Voorhis,
1
Antimicrob. Agents Chemother. 1996, 40, 2592.
[
[
27] A. S. Bhambra, K. C. Ruparelia, H. L. Tan, D. Tasdemir, H. Burrell‐
Saward, V. Yardley, K. J. M. Beresford, R. R. J. Arroo, Eur. J. Med.
Chem. 2017, 128, 213. https://doi.org/10.1016/j.ejmech.2017.01.027
28] B. Insuasty, A. Montoya, D. Becerra, J. Quiroga, R. Abonia, S.
Robledo, I. D. Vélez, Y. Upegui, M. Nogueras, J. Cobo, Eur. J.
Med. Chem. 2013, 67, 252. https://doi.org/10.1016/j.ejmech.
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
2
013.06.049
[29] S. A. Pulido, D. L. Muñoz, A. M. Restrepo, C. V. Mesa, J. F. Alzate, I. D.
Vélez, S. M. Robledo, Acta Trop. 2012, 122, 36. https://doi.org/10.
How to cite this article: González LA, Upegui YA, Rivas L,
et al. Effect of substituents in the A and B rings of chalcones
on antiparasite activity. Arch Pharm. 2020;e2000157.
https://doi.org/10.1002/ardp.202000157
1
016/j.actatropica.2011.11.015
30] W. Trager, J. B. Jensen, J. Parasitol. 2005, 91, 484. https://doi.org/10.
645/0022-3395(2005)091[0484:HMPICC]2.0.CO;2
31] S. Nkhoma, M. Molyneux, S. Ward, Am. J. Trop. Med. Hyg. 2007, 76,
107.
[
[
1
1