Synthesis, in vitro antigiardial activity, SAR analysis and docking study of substituted chalcones

Article abstract of DOI:10.1007/s00044-019-02492-5

A series of 15 chalcones-bearing substituents at positions 2, 4, and 5 of rings A and B were synthesized using microwave-assisted Claissen–Smichdt condensation and evaluated for their activity against Giardia lamblia and Green monkey kidney cells. Five co

Full text of DOI:10.1007/s00044-019-02492-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02492-5
ORIGINAL RESEARCH
Synthesis, in vitro antigiardial activity, SAR analysis and docking
study of substituted chalcones
David Cáceres-Castillo1 Rubén M. Carballo1 Ramiro Quijano-Quiñones1 Gumersindo Mirón-López1
●
●
●
●
Manlio Graniel-Sabido1 Rosa E. Moo-Puc2 Gonzalo J. Mena-Rejón
1
●
●
Received: 2 October 2019 / Accepted: 11 December 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A series of 15 chalcones-bearing substituents at positions 2, 4, and 5 of rings A and B were synthesized using
microwave-assisted Claissen–Smichdt condensation and evaluated for their activity against Giardia lamblia and Green
monkey kidney cells. Five compounds exhibited activity against G. lamblia at IC50’s <5 μM. The chalcone 3m exhibited
the highest antigiardial activity (IC50 = 1.03 μM), even more than the positive control (metronidazole, IC50 = 1.4 μM),
and selectivity (SI = 38.9). A preliminary SAR study suggested that electrophylicity has not relationship with
antigiardiasic activity, and the docking study reveals that synthesized chalcones bind at zone 3 of colchicine site,
therefore presumably the action mechanism of the synthesized chalcones does not follow the Michael acceptor
mechanism.
●
●
●
●
Keywords Synthesis Substituted chalcones Antigiardial activity SAR analysis Docking study
Introduction
worldwide associated to G. lamblia infection were detected
applying the Child Health Epidemiology Reference Group
(CHERG) approach (Pires et al. 2015).
The flagellated and amitochondriate protozoan Giardia
lamblia (syn. G. intestinalis, G. duodenalis) is a human
enteric pathogen with the high morbidity worldwide. In
1998, WHO estimated that one billion people living in
developing countries was infected by this parasite, and ~280
million cases occurring annually. (Upcroft and Upcroft
2001). Two years later, giardiasis was referred as a ree-
merging infectious disease, (Thompson 2000), and in Sep-
tember 2004, it was included in the ‘WHO Neglected
Diseases Initiative’. (Savioli et al. 2006). More recently,
during 2010, 183 million cases of diarrheal disease
The infection by G. lamblia occurs when people ingest
water or food contaminated with cysts, or through person-
to-person contact, or by contact with contaminated fomites.
Signs of giardiasis vary between individuals and range from
acute or chronic diarrhea to total latency. Clinical symptoms
normally begin 1–3 weeks following transmission and may
include nausea, vomiting, weight loss, abdominal pain,
bloating, and diarrhea (Escobedo et al. 2010). The impact of
giardiasis is stronger on undernourished or immunodeficient
individuals and on young children (Ankarklev et al. 2010).
In 1–5 years old children the infection by Giardia can cause
failure to thrive syndrome, and delays in the physical
growth and cognitive-intellectual development (Halliez and
Buret 2013).
The most common drugs used in the treatment of giar-
diasis are 5-nitroimidazoles (metronidazole, tinidazole,
secnidazole, and ornidazole) of which metronidazole being
the first-choice option. Notwithstanding, it has been repor-
ted heavy side effects of metronidazole such as headache,
vertigo, nausea, allergic reactions, and in some cases neu-
rotoxicity, teratogenic, and mutagenic effects. Moreover,
resistance can occur in up to 20% of clinical cases
(Pasupuleti et al. 2014). This panorama makes necessary to
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02492-5) contains supplementary
material, which is available to authorized users.
* Gonzalo J. Mena-Rejón
mrejon@uady.mx
1
Facultad de Química, Universidad Autónoma de Yucatán, C. 43
No. 613, Col. Inalámbrica, Mérida 97069 Yucatán, Mexico
2
Unidad de Investigación Médica Yucatán, Unidad Médica de Alta
Especialidad, Centro Médico “Ignacio García Téllez”, IMSS, C.
41, No. 439, Col. Industrial, C.P., Mérida 97150 Yucatán, Mexico

Medicinal Chemistry Research
maintain a continuous searching for novel antigiardial drug
candidates, and natural product are promising scaffolds for
such compounds.
Materials and methods
Chemistry
The 1,3-diaryl-2-propen-1-one (chalcone) is a common
scaffold found in many natural products derived from plants
which possess a wide spectrum of interesting biological
activities. Also, chalcone derivatives exhibit high bioavail-
ability and tolerance in the organism of mammals (Gaonkar
and Vignesh 2017). Advantageously, the α, β-unsaturated
ketone moiety is easily synthesizable and chalcones are
generally prepared with good yields by condensation reac-
tions via base or acid catalysis (Claisen−Schmidt con-
densation). For these reasons, many chalcone derivatives
have been synthesized, and a high percentage of them have
resulted in biologically active compounds with clinical
potential against various diseases (Gomes et al. 2017).
Noteworthy, the importance of chalcone scaffold in med-
icinal chemistry has been increasing due to the new evi-
dence supporting it as a privileged structure (Zhuang et al.
2017).
Chalcone-based compounds have maintained the interest
of academia and industry across the 20th century due to
their wide-ranging biological activities, especially the anti-
infective ones (Sahu et al. 2012). Diverse chalcones deri-
vatives have been synthesized for 25 years to evaluate their
antimalarial and antileishmanial activities (Gomes et al.
2017; Zhuang et al. 2017). Besides, during the last decade, a
series of 25 variously substituted nonnatural chalcones were
synthesized to be tested for antigiardial activity. Although
only three compounds showed significant antigiardial
activity (Fig. 1), the obtained results revealed the impor-
tance of oxygenated and nonbulky substituents at para-
position in chalcone’s ring A with for such activity
(Montes-Avila et al. 2009).
All commercial reagents were obtained from Sigma-Aldrich
(Saint Louis, MO, USA) and used as received. Microwave
reactions were conducted in 25 mL open glass vessels using
a CEM Discover microwave reactor. Analytical and pre-
parative thin-layer chromatography (TLC) were performed
on 25 μm particle size silica gel GF-254 aluminium and
glass plates, respectively. Column chromatography was
undertaken with 0.063–0.037 mm particle size silica gel.
Melting points were determined on an Electrothermal
IA9100 apparatus in open capillaries. Fourier transform
infrared analysis was performed with
a
Nicolet
iS5 spectrometer in the range of 4000–600 cm−1 with
32 scans recorded at 4 cm−1 of resolution. H-NMR and
1
13C-NMR spectra were recorded in DMSO-d6 on Bruker
Avance 400 spectrometer at 400 and 100 MHz, respec-
tively, and the residual solvent peak was used as an internal
reference. The δ values are given in ppm. The low and high-
resolution mass spectra were recorded with a Jeol GCmate
II mass spectrometer by using electron impact mode at
70 eV.
General procedure for the preparation of chalcones
(3a–o)
An appropriate acetophenone (3.7 mmol) (1a–e) and the
corresponding benzaldehyde (3.7 mmol) (2a–c) were dis-
solved in EtOH (10 mL), and then 2.2 mL of a 50% NaOH
aqueous solution were added dropwise. The resulting
reaction mixture was subjected to MW irradiation at 80 °C
(70 W) from 2 to 4 h. After time completion, the reaction
mixture was poured on 20 mL of chilly water and acidified
with 10% HCl aqueous solution until pH 2. The crude
products were purified by crystallization or, in a few cases,
by silica gel chromatography.
As a part of our search into antiprotozoal compounds, we
synthesized a small library of chalcones derivatives and the
activity against G. lamblia of the resulting compounds was
evaluated together their calculated LogP and electrophilicity
indexes in a preliminary structure-antigiardiasic activity
(SAR) analysis. Finally, a docking study was performed
with the four most active chalcones.
(E)-3-(2-hydroxyphenyl)-1-(4-methylphenyl)-2-
propen-1-one (3a)
Pale-yellow powder, crystallized from EtOH-H2O (1:4),
Yield 49%, mp 148–149 °C. IR (KBr) νmax in cm−1: 3240
(OH), 1645 (C=O), 1604 (C=C). 1H NMR (400 MHz,
DMSO-d6): δ = 10.3 (1H, s, C-2′-OH), 8.05 (1H, d, J =
15.8 Hz, H-3), 8.01 (2H, d, J = 8.0 Hz, H-2′,H-6′), 7.86
(1H, m, H-3″), 7.85 (1H, d, J = 15.8 Hz, H-2), 7.36 (2H, d,
J = 8.4 Hz, H-3′, H-5′), 7.27 (1H, t, J = 8.4 Hz, H-5″), 6.95
(1H, d, J = 8.0 Hz, H-6″), 6.87 (1H, t, J = 7.6 Hz, H-4″),
2.38 (3H, s, C-4′-Me). 13C NMR (100 MHz, DMSO-d6):
δ = 188.9 (C, C-1), 157.2 (C, C-2″), 143.3 (C, C-4′), 139.2
(CH, C-3), 135.4 (C, C-1′), 132.0 (CH, C-5″), 129.4 (2CH,
O
R2
R3
R1
R1 R2 R3
C1 MeO F
C2 MeO H
C3
H
Cl
F
H
Me
Fig. 1 Reported antigiardiasic chalcones

Medicinal Chemistry Research
C-3′, C-5′), 128.7 (CH, C-3″), 128.5 (2CH, C-2′, C-6′),
121.5 (C, C-1″), 121.0 (CH, C-2), 119.5 (CH, C-4″), 116.3
(CH, C-6″), 21.2 (CH3, C-4′-Me). HRMS (EI+, 70 eV) m/z:
238.0990 for C16H14O2 (calcd. 238.0994).
6.94 (d, 1H, J = 8.2, H-6″), 6.87 (t, 1H, J = 7.5 Hz, H-4″),
3.85 (s, 3H, C-4′-OMe). 13C NMR (100 MHz, DMSO-d6):
δ = 188.1 (C, C-1), 163.5 (C, C-2″), 157.6 (C, C-4′), 139.1
(CH, C-3), 132.3 (C, C-1′), 131.2 (2CH, C-2′, C-6′), 131.1
(CH, C-5″), 129.0 (CH, C-3″), 122.0 (C, C-1″), 121.2 (CH,
C-2), 119.9 (CH, C-4″), 116.6 (CH, C-6″), 114.4 (2CH, C-3′),
55.9 (CH3, C-4′-OMe). HRMS (EI+, 70 eV) m/z: 254.0949
for C16H14O3 (calcd. 254.0949).
(E)-3-(4-methoxyphenyl)-1-(4-methylphenyl)-2-propen-1-
one (3b)
Pale-yellow powder, crystallized from EtOH-H2O (2:3),
Yield 71%, mp 103–104 °C. IR (KBr) νmax in cm−1: 2967
(CH3), 1656 (C=O), 1605 (C=C). 1H NMR (400 MHz,
DMSO-d6): δ = 8.04 (d, 2H, J = 8.0 Hz, H-2′, H-6′), 7.84 (d,
2H, J = 8.0 Hz, H-2″, H-6″), 7.78 (d, 1H, J = 16 Hz, H-3),
7.69 (d, 1H, J = 16 Hz, H-2), 7.36 (d, 2H, J = 8.0 Hz, H-3″,
H-5″), 7.01 (d, 2H, J = 8.0 Hz, H-3′, H-5′), 3.82 (s, 3H, C-
4″-OMe), 2.40 (s, 3H, C-4′-Me) ppm. 13C NMR (100 MHz,
DMSO-d6): δ 189.0 (C, C-1), 161.8 (C, C-4″), 144.1 (C, C-
4′), 143.8 (CH, C-3), 135.8 (C, C-1′), 131.2 (2CH, C-2″, C-
6″), 129.8 (C, C-1″), 129.0 (2CH, C-3′, C-5′), 127.8 (2CH,
C-2′, C-6′), 120.0 (CH, C-2), 114.9 (2CH, C-3″, C-5″), 55.8
(CH3, C-4″-OMe), 21.6 (CH3, C-4′-Me). HRMS (EI+,
70 eV) m/z: 252.3194 C17 H16 O2 (calcd. 252.3198).
(E)-1,3-bis-(4-methoxy-phenyl)-2-propen-1-one (3e)
Yellow powder, purified by column chromatography (silica
gel 74–37 μm, eluent hexane:ethyl acetate gradient), Yield
44%, mp 95–96 °C. IR (KBr) νmax in cm−1: 2962 (CH3),
1655 (C=O), 1601 (C=C). 1H NMR (400 MHz, DMSO-d6):
δ 8.15 (2H, d, J = 8.8 Hz, H-2′, H-6′), 7.85 (2H, d, J =
8.8 Hz, H-2″, H-6″), 7.8 (1H, d, J = 15.6 Hz, H-3), 7.68
(1H, d, J = 15.6 Hz, H-2), 7.08 (2H, d, J = 8.8 Hz, H-3′, H-
5′), 7.02 (2H, d, J = 8.8 Hz, H-3″, H-5″), 3.86 (3H, s, C-4′-
OMe), 3.82 (3H, s, C-4″-OMe). 13C NMR (100 MHz,
DMSO-d6): δ = 187.3 (C, C-1), 163.1 (C, C-4′), 161.2 (C,
C-4″), 143.2 (CH, C-2), 130.8 (2CH, C-2′, C-6′), 130.7
(2CH, C-2″, C-6″), 130.7 (C, C-1′), 127.5 (C, C-1″), 119.5
(CH, C-3), 114.4 (2CH, C-2″, C-6″), 114.0 (2CH, C-3′, C-
5′), 55.6 (CH3, C-4′-OMe), 55.4 (CH3, C-4″-OMe). HRMS
(EI+, 70 eV) m/z: 268.1104 for C17H16O3 (calcd. 268.1100).
(E)-3-(4-chloro-2-hydroxyphenyl)-1-(4-methylphenyl)-2-
propen-1- one (3c)
Orange powder, crystallized from EtOH-H2O (3:7), Yield
66%, mp 135–136 °C. IR (KBr) νmax in cm−1: 3226 (OH),
1651 (C=O), 1607 (C=C), 745 (C–Cl). 1H NMR
(400 MHz, DMSO-d6): δ = 10.6 (brs, 1H, C-2″-OH), 8.03
(m, 3H, H-2′, H-6′, H-6″), 7.98 (d, 1H, J = 16.0 Hz, H-3),
7.92 (d, 1H, J = 16 Hz, H-2), 7.35 (d, 2H, J = 7.8 Hz, H-
3′), 7.28 (dd, 1H, J = 6.8 and 1.8 Hz, H-4″), 6.94 (d, 1H,
J = 8.7, H-3″), 2.38 (s, 3H, C-4′-Me). 13C NMR (100 MHz,
DMSO-d6): δ = 188.8 (C, C-1), 159.0 (C, C-2), 143.7 (C,
C-4′), 137.4 (CH, C-3), 135.2 (CH, C-4″), 131.5 (C, C-1′),
129.5 (2CH, C-3′, C-5′), 128.9 (2CH, C-2′, C-6′), 127.5
(CH, C-6″), 123.4 (CH, C-2), 123.3 (C, C-5″), 122.1 (C, C-
1″), 118.0 (CH, C-3″), 21.3 (CH3, C-4′-Me). HRMS (EI+,
70 eV) m/z: 272.0606 for C16H13O2Cl (calcd. 272.0604);
274.0616 for C16H13O237Cl (calcd. 274.0575).
(E)-3-(5-Chloro-2-hydroxyphenyl)-1-(4-methoxyphenyl)-2-
propen-1-one (3f)
Yellow-orange powder, crystallized from EtOH-H2O (1:4),
Yield 61%, mp 150–151 °C. IR (KBr) νmax in cm−1: 3405
1
(OH), 1646 (C=O), 1601 (C=C), 756 (C-Cl). H NMR
(400 MHz, DMSO-d6): δ = 8.08 (d, 2H, J = 8 Hz, H-2′, H-
6′), 8.00 (d, 1H, J = 16 Hz, H-3), 7.90 (d, 1H, J = 16 Hz, H-
2), 7.62 (d, 1H, H-6″), 7.05 (d, 2H, J = 9.2 Hz, H-3′, H-5′),
7.02 (dd, 1H, J = 2.4 & 8.8 Hz, H-4″), 6.61 (d, 1H, J =
8 Hz, H-3″), 3.85 (s, 3H). 13C NMR (100 MHz, DMSO-d6):
δ = 188.4 (C, C-1), 164.9 (C, C-2″), 163.2 (C, C-4′), 141.1
(CH, C-3), 131.7 (C, C-1′), 131.6 (2CH, C-2′, C-6′), 131.0
(CH, C-4″), 128.6 (CH, C-6″), 123.8 (C, C-1″), 120.8 (C,
C-5″), 119.1 (CH, C-2), 117.9 (CH, C-3″), 114.3 (2CH, C-
3′, C-5′), 55.9 (CH3, C-4′-OMe). HRMS (EI+, 70 eV) m/z:
288.0562 for C16H13O3Cl (calcd. 288.0553); 290.0543 for
C16H13O337Cl (calcd. 290.0524).
(E)-3-(2-hydroxy-phenyl)-1-(4-methoxy-phenyl)-2-propen-1-
one (3d)
Yellow powder, purified by column chromatography (silica
gel 74–37 μm, eluent hexane:ethyl acetate gradient), Yield
43%, mp 146–147 °C. IR (KBr) νmax in cm−1: 3287 (OH),
2932 (CH3), 1645 (C=O), 1602 (C=C). 1H NMR (400 MHz,
DMSO-d6): δ = 10.3 (brs, 1H, C-2″-OH), 8.11 (d, 2H, J =
7.7 Hz, H-2′, H-6′), 8.04 (d, 1H, J = 15.7 Hz, H-3), 7.85 (d,
1H, J = 15.7 Hz, H-2), 7.84 (dd, 1H, J = 7.9 & 3.9 Hz, H-3″),
7.26 (t, 1H, J = 7.9 Hz, H-5″), 7.06 (d, 2H, J = 7.5 Hz, H-3′),
(E)-3-(2-hydroxy-phenyl)-1-(4-hydroxy-phenyl)-2-propen-1-
one (3g)
Yellow powder, crystallized from EtOH-H2O (2:3), Yield
45%, mp 147–148 °C. IR (KBr) νmax in cm−1: 3440 (OH),
1
1662 (C=O), 1625 (C=C). H NMR (400 MHz, DMSO-
d6): δ = 10.47 (brs, 1H, C-4′-OH), 10.29 (s, 1H, C-2″-OH),

Medicinal Chemistry Research
8.02 (m, 3H, H-2′, H-6′, H-3), 7.83 (d, 1H, J = 15.7 Hz, H-
2), 7.83 (d, 1H, J = 8.0 Hz H-3″), 7.25 (t, 1H, J = 7.6 Hz,
H-5″), 6.88 (m, 4H, H-3′, H-5′, H-4″, H-6″). 13C NMR
(100 MHz, DMSO-d6): δ = 187.7 (C, C-1), 162.2 (C, C-4′),
157.2 (C, C-2″), 138.4 (CH, C-3), 131.9 (CH, C-5″), 131.2
(2CH, C-2′, C-6′), 129.5 (CH, C-3″), 128.7 (C, C-1′), 121.7
(CH, C-2), 121.0 (CH, C-4″), 119.6 (CH, C-6″), 116.3 (C,
C-1″), 115.6 (2CH, C-3′, C-5′). HRMS (EI+, 70 eV) m/z:
240.0782 for C15H12O3 (calcd. 240.0787).
(OH), 1640 (C=O), 1615 (C=C). 1H NMR (400 MHz,
DMSO-d6): δ = 12.68 (1H, s, C-2′-OH), 10.42 (1H, s, C-2″-
OH), 8.19 (1H, d, J = 15.1 Hz, H-3), 8.19 (1H, m, H-6′),
7.98 (1H, d, J = 15.6 Hz, H-2), 7.91 (1H, d, J = 7.6 Hz, H-
3″), 7.55 (1H, t, J = 7.2 Hz, H-4′), 7.30 (1H, t, J = 7.2 Hz,
H-5″), 6.97 (3H, m, H-5′, H-3′, H-6″), 6.89 (1H, t, J =
7.2 Hz, H-4″). 13C NMR (100 MHz, DMSO-d6): δ = 193.9
(C, Bz-CO), 162.0 (C, C-2′), 157.6 (C, C-2″), 140.4 (CH,
C-3), 136.2 (CH, C-4′), 132.6 (CH, C-5″), 130.6 (CH, C-
6′), 129.0 (CH, C-3″), 121.0 (C, C-1′), 120.8 (C, C-1″),
120.3 (CH, C-2), 119.5 (CH, C-5′), 119.2 (CH, C-4″),
117.8 (CH, C-3′), 116.4 (CH, C-6″). HRMS (EI+, 70 eV)
m/z: 240.0778 for C15H12O3 (calcd. 240.0787).
(E)-1-(4-hydroxyphenyl)-3-(4-methoxyphenyl)-2-propen-1-
one (3h)
Yellow crystalline solid, crystallized from EtOH-H2O (2:3),
Yield 50%, mp 82–83 °C. IR (KBr) νmax in cm−1: 3225
(OH), 1650 (C=O), 1602 (C=C). 1H NMR (400 MHz,
DMSO-d6): δ = 10.51 (1H, brs, C-4′-OH), 8.07 (2H, d, J =
8.6 Hz, H-2′, H-6′), 7.81 (2H, d, J = 8.52 Hz, H-2″, H-6″)
7.77 (1H, d, J = 15.7 Hz, H-3), 7.67 (1H, d, J = 15.5 Hz, H-
2), 6.96 (2H, d, J = 8.6 Hz, H-3″, H-5″), 6.93 (2H, d, J =
8.6 Hz, H-3′, H-5′), 3.76 (3H, s, C-4″-OMe). 13C NMR
(100 MHz, DMSO-d6): δ = 187.4 (C, C-1), 162.3 (C, C-4′),
161.3 (C, C-4″), 143.0 (CH, C-2), 131.3 (2CH, C-2′, C-6′),
130.7 (2CH, C-2″, C-6″), 129.6 (C, C-1′), 127.7 (C, C-1″),
119.8 (CH, C-3), 115.6 (2CH, C-3′, C-5′), 114.5 (2CH, C-
3″ C-5″), 55.4 (CH3, C-4″-OMe). HRMS (EI+, 70 eV) m/z:
254.0947 for C16H14O3 (calcd. 254.0943).
(E)-1-(2-hydroxy-phenyl)-3-(4-methoxyphenyl)-2-propen-1-
one (3k)
Yellow powder, crystallized from EtOH-H2O (1:4); Yield:
77%; mp 85–87 °C. IR (KBr) νmax in cm−1: 2960 (CH3),
1
1636 (C=O), 1604 (C=C). H NMR (400 MHz, DMSO-
d6): δ = 12.78 (s, 1H, C-2′-OH), 8.25 (d, 1H, J = 8 Hz, H-
6′), 7.92 (d, 1H, J = 15.2 Hz, H-3), 7.88 (d, 2H, J = 8 Hz
H-2″, H-6″), 7.83 (d, 1H, J = 15.6 Hz, H-2), 7.55 (t, 1H,
J = 8 Hz, H-4′), 7.03 (m, 2H, H-4″, H-5″), 6.99 (m, 2H, H-
3′, H-5′), 3.82 (s, 3H, Bz-OCH3). 13C NMR (100 MHz,
DMSO-d6): δ = 193.6 (C, Bz-CO), 162.1 (C, C-2′), 161.8
(C, C-4″), 145.1 (CH, C-2), 136.2 (CH, C-4′), 131.6 (2CH,
C-2″, C-6″), 130.8 (CH, C-6′), 127.1 (C, C-1″), 120.6 (C,
C-1′0), 119.1 (CH, C-5′), 118.8 (CH, C-3), 117.8 (CH, C-
3′), 114.5 (2 CH, C-3″, C-4″), 55.4 (CH3, Bz-OMe). HRMS
(EI+, 70 eV) m/z: 254.0947 for C16H14O3 (calcd.
254.0943).
(E)-3-(5-chloro-2-hydroxyphenyl)-1-(4-hydroxyphenyl)-2-
propen-1-one (3i)
Yellow-orange powder, crystallized from EtOH-H2O (3:7),
Yield 69%, mp 177–178 °C. IR (KBr) νmax in cm−1: 3203
1
(OH), 1634 (C=O), 1603 (C=C), 757 (C-Cl). H NMR
(E)-3-(5-chloro-2-hydroxyphenyl)-1-(2-hydroxyphenyl)-2-
(400 MHz, DMSO-d6): δ = 10.52 (1H, s, C-4′-OH), 10.45
(1H, s, C-2″-OH), 8.07 (2H, d, J = 8.4 Hz, H-2′, H-6′), 8.00
(1H, d, J = 2.4 Hz, H-6″), 7.92 (2H, s, H-3, H-2), 7.27 (1H,
dd, J = 2.4, 8.8 Hz, H-4″), 6.93 (1H, d, J = 8.8 Hz, H-3″),
6.89 (2H, d, J = 8.5 Hz, H-3′, H-5′). 13C NMR (100 MHz,
DMSO-d6): δ = 187.3 (C, C-1), 162.2 (C, C-4′), 155.8 (C,
C-2″), 136.4 (CH, C-3), 131.3 (2CH, C-2′, C-6′), 131.1
(CH, C-4″), 129.3 (C, C-1′), 127.4 (CH, C-6″), 123.4 (C, C-
5″), 123.3 (C, C-1″), 122.2 (CH, C-2), 117.9 (CH, C-3″),
115.4 (2CH, C-3′, C-5′). HRMS (EI+, 70 eV) m/z:
274.0401 for C15H11O3Cl (calcd. 274.0397); 276.0388 for
C15H11O337Cl (calcd. 276.0367).
propen-1-one (3l)
Yellow solid; crystallized from EtOH-H2O (2:3); Yield
59%; mp 181–183 °C. IR (KBr) νmax in cm−1: 3331 (OH),
1645 (C=O), 1629 (C=C), 748 (C-Cl). 1H NMR
(400 MHz, DMSO-d6): δ = 12.64 (1H, s, C-2′-OH), 10.70
(1H, s, C-2″-OH), 8.27 (1H, d, J = 7.6 Hz, H-6′), 8.2 (1H,
d, J = 15.2 Hz, H-3), 8.1 (1H, m, H-3″), 8.0 (1H, d, J =
15.2 Hz, H-2), 7.56 (1H, brs, H-4′), 7.31 (1H, d, J = 7.2 Hz,
H-4″), 6.98 (3H, m, H-3′,H-5′, H-6″). 13C NMR (100 MHz,
DMSO-d6): δ = 193.8 (C, C-1), 162.1 (C, C-2′), 156.3 (C,
C-2″), 138.5 (CH, C-3), 136.4 (CH, C-4′), 131.9 (CH, C-
5″), 130.9 (CH,C-6′), 127.7 (CH, C-3″), 123.4 (C, C-4″),
122.9 (C, C-1′), 121.5 (CH, C-2), 120.7 (C, C-1″), 119.2
(CH, C-5′), 118.0 (CH, C-6″), 117.8 (CH, C-3′). HRMS
(EI+, 70 eV) m/z: 274.0390 for C15H11O3Cl (calcd.
274.0397); 276.0372 for C15H11O337Cl (calcd. 276.0367).
(E)-1,3-bis-(2-hydroxyphenyl)-2-propen-1-one (3j)
Yellow crystalline solid, crystallized from EtOH-H2O (2:3);
Yield: 78%; mp 155–156 °C. IR (KBr) νmax in cm−1: 3406

Medicinal Chemistry Research
(E)-1-(2-hydroxy-5-methylphenyl)-3-(2-hydroxyphenyl)-2-
propen-1-one (3m)
C-6′), 128.1 (C, C-5′), 127.6 (CH, C-6″), 123.5 (C, C-5″),
123.0 (C, C-1′), 121.5 (CH, C-2), 120.3 (C, C-1″), 118.1
(CH, C-3′), 117.7 (CH, C-3″), 20.1 (CH3, C-5′-Me). HRMS
(EI+, 70 eV) m/z: 288.0550 for C16H13O3Cl (calcd.
288.0553); 290.0525 for C16H13O337Cl (calcd. 290.0524).
Pale brown powder, crystallized from EtOH-H2O (2:3);
Yield 37%; mp 122–123 °C. IR (KBr) νmax in cm−1: 3333
1
(OH), 2924 (CH3), 1636 (C=O), 1604 (C=C). H NMR
(400 MHz, CDCl3): δ = 12.78 (s, 1H, C-2′-OH), 8.27 (1H,
d, J = 16 Hz, H-3), 8.27 (1H, d, J = 16 Hz, H-3), 7.84 (d,
1H, J = 16 Hz, H-2), 7.73 (s, 1H, H-3′), 7.65 (d, 1H, J =
8 Hz, H-5′), 7.32 (m, 2H, H-5″, H-6″), 7.0 (m, 2H, H-4″, H-
3″), 6.90 (d, 1H, J = 8 Hz, H-6′), 2.37 (s, 3H). 13C NMR
(100 MHz, DMSO-d6): δ = 194.6 (C, C-1), 161.4 (C, C-2′),
155.8 (C, C-2″), 141.0 (CH, C-3), 137.5 (CH, C-4′), 132.1
(CH, C-5″), 129.8 (CH, C-6′), 129.6 (CH, C-3″), 128.0 (C,
C-5′), 122.1 (C, C-1′), 121.1 (CH, C-2), 120.9 (C, C-1″),
119.8 (CH, C-4″), 118.3 (CH, C-3′), 116.6 (CH, C-6″), 20.6
(CH3, C-5′-Me). HRMS (EI+, 70 eV) m/z: 254.0953 for
C16H14O3 (calcd. 254.0943).
Antiprotozoal assay
G. lamblia strain IMSS:0696:1 was cultured in TYI-S-33
modified medium, supplemented with 10% calf serum
(Sigma-Aldrich Co.) and bovine bile (Sigma-Aldrich Co.).
For the bioassay (Cedillo-Rivera et al. 2002), the com-
pounds were dissolved in 1 mL of dimethylsulfoxide
(DMSO) and added to microtubes containing 1.5 mL of
medium to reach concentrations of 0.5, 1.0, 5.0 y
10.0 μg/mL. The solutions were inoculated with G. lamblia
trophozoites to achieve an inoculum of 4 × 104 trophozoites/
mL and then were incubated for 48 h at 37 °C. Each test
included metronidazole as positive control and trophozoites
incubated in culture medium with DMSO used in the
experiments as the negative control. The used DMSO
concentration was not higher than 0.05%. After the incu-
bation, trophozoites were washed and subcultured for
another 48 h in fresh medium alone. At the end of this
period, trophozoites were counted and the 50% inhibitory
concentration (IC50) was calculated by Probit analysis.
Experiments were carried out in triplicate and repeated at
least twice. The level of harmfulness on normal cells was
evaluated by determining the selectivity index (SI) of each
compound (Vonthron-Sénécheau et al. 2003), which is
calculated as the ratio of cytotoxicity on normal cells to
antigiardial activity (SI = IC50 Vero cells/IC50 G. lamblia).
(E)-1-(2-hydroxy-5-methyl-phenyl)-3-(4-methoxy-phenyl)-2-
propen-1-one (3n)
Yellow oil, purified by preparative TLC (eluent hexane:
diethyl ether 8:2), Yield 37%. IR (KBr) νmax in cm−1: 3400
1
(OH), 2919 (CH3), 1639 (C=O), 1604 (C=C). H NMR
(400 MHz, DMSO-d6): δ = 12.77 (1H, s, C-2′-OH), 7.90
(1H, d, J = 14.8 Hz, H-3), 7.66 (1H, s, H-6′), 7.65 (2H, d,
J = 8.76 Hz, H-2″, H-6″), 7.55 (1H, d, J = 15.2 Hz, H-2),
7.21 (1H, dd, J = 2, 8.4Hz, H-4′), 6.96 (2H, d, J = 8.72 Hz,
H-3″, H-5″), 6.93 (1H, d, J = 8.48 Hz, H-3′), 3.87 (3H, s,
C-4″-OCH3), 2.36 (3H, s, C-5′-CH3). 13C NMR (100 MHz,
DMSO-d6): δ = 193.8 (C, C-1), 162.1 (C, C-2′), 161.6 (C,
C-4″), 145.3 (CH, C-3), 137.34 (CH, C-4′), 130.7 (2CH, C-
2″, C-6″), 129.4 (CH, C-6′), 128.0 (C, C-5′), 127.6 (C, C-
1″), 119.9 (C, C-1′), 118.4 (CH, C-3′), 117.9 (CH, C-2),
114.7 (2CH, C-3″, C-5″), 55.6 (CH3, C-4″-OMe), 20.8
(CH3, C-5′-Me). HRMS (EI+, 70 eV) m/z: 268.1087 for
C17H16O3 (calcd. 268.1100).
Cytotoxicity assay
Cytotoxicity assay was performed according to the estab-
lished method of Rahman et al. (2001), where 1.5 × 104
viable cells, from the Green monkey kidney cell line (Vero
cells, ATCC-CCL-81) from the American Type Culture
Collection (ATCC), were seeded in a 96-well plate (Costar)
and incubated for 24–48 h. When cells reached >80%
confluence, the medium was replaced and cells were incu-
bated for 48 h with pure compounds (3a–o) at 6.25, 12.5,
25, and 50 μg/mL dissolved in DMSO at a maximum con-
centration of 0.05%. At the end of the exposure time, the
medium was removed, and the cells were fixed by adding
50 μL of 10% trichloroacetic acid solution to each well and
incubated at 4 °C for 30 min. After incubation, the tri-
chloroacetic acid was eliminated and 50 μL of sulforhoda-
mine B (0.1% sulforhodamine B in 1% acetic acid) were
added to each well and left in contact with the cells for
30 min, after which they were washed with 150 μL of 1%
acetic acid, and rinsed three times until only dye adhering to
(E)-3-(3-chloro-phenyl)-1-(2-hydroxy-5-methyl-phenyl)-2-
propen-1- one (3o)
Pale-yellow powder, crystallized from EtOH-H2O (1:1);
Yield 61%; mp 178–179 °C. IR (KBr) νmax in cm−1: 3292
1
(OH), 1637 (C=O), 1600 (C=C), 753 (C-Cl). H NMR
(400 MHz, DMSO-d6): δ = 12.45 (1H, s, C-2′-OH), 10.71
(1H, s, C-2″-OH), 8.09 (1H, d, J = 15.6 Hz, H-3), 8.05 (2H,
s, H-6′, H-6″), 8.01 (1H, d, J = 15.6 Hz, H-2), 7.37 (1H, d,
J = 8.4 Hz, H-4′), 7.32 (1H, d, J = 8.4 Hz, H-4″), 6.95 (1H,
d, J = 8.4 Hz, H-3′), 6.88 (1H, d, J = 8.4 Hz, H-3″), 2.31
(3H, s, C-5′-CH3). 13C NMR (100 MHz, DMSO-d6): δ =
193.8 (C, C-1), 160.2 (C, C-2′), 156.3 (C, C-2″), 138.4
(CH, C-3), 137.4 (CH, C-4′), 132.0 (CH, C-4″), 130.5 (CH,

Medicinal Chemistry Research
the cells was left. The plates were dried and 100 μL of
10 mM Tris base were added to each well to solubilize the
dye. The plates were shaken gently for 10 min and the
cellular proliferation was determined by measuring the
optical density (OD) at 540 nm using a bioassay reader
(BioRad, USA). Docetaxel (Taxotere®; Sigma-Aldrich Co.)
was used as positive control, whereas untreated cells were
used as a negative control. Each concentration was eval-
uated by triplicate in an assay by three independent
experiments. The cytotoxic activity was calculated as the
percentage of cells killed using the equation: growth inhi-
bition (%) = (ODcontrol − ODsample/ODcontrol) × 100.
The concentration of pure compounds that killed 50% of the
cells (CC50) was calculated by GraphPad Prim 4 software.
selected based on the lowest energy binding and the more
populated cluster. The visualization of the complex was
done using Maestro (Maestro 2019).
Results and discussion
A series of 15 chalcones, including a new one (3i), showing
substitution at rings A and B were synthesized by
Claisen–Schmidt condensation with the aid of microwave
irradiation (Scheme 1). The yields of the compounds
obtained by this procedure ranging from 37 to 78%. All the
1
compounds were characterized by mass spectrometry, H
NMR, 13C NMR, and melting points. The E configuration
of the double bond in the structure of the synthesized
1
Theoretical calculations
chalcones was confirmed by the presence in their H NMR
spectra of two doublets with high coupling constants (J =
15.1–15.8 Hz).
All synthesized chalcones were evaluated for in vitro
antigiardial activity as well as their cytotoxicity against
Green monkey kidney cells (Vero cells). The results of the
bioassay tests are summarized in Table 1.
The antiprotozoal activity (IC50) of five of the chalcones
synthesized in this work (3b, 3e, 3h, 3j, and 3k) have been
previously reported. Chalcone 3b exhibited an IC50 of
16.25 μM against G. lamblia, while it was reported to be
active against Entamoeba histolytica at 4.76 μM (Wani et al.
2012). The antiplasmodial activity of the compound 3j has
been investigated, finding a moderate IC50 of 14.7 μM
against P. falciparum (Cohen et al. 1998), but in this study,
it was resulting in the second more highly antigiardiasic
chalcone (IC50 = 2.29 μM) with a selectivity index of 20.83.
The molecular geometries of all compounds were fully
optimized using density functional theory with a 6–31G
(d, p) basis set. The exchange-correlation potential was
evaluated using the hybrid functional B3LYP (Lee et al.
1988). After optimization, a frequencies calculation was
performed to characterize all the stationary points at the
same computational level and no imaginary frequency was
observed. From this calculation, the electrophilicity indexes
(ω(eV)) were obtained. All the calculations were carried out
using the SPARTAN 16 program. The octanol-water par-
tition coefficients (CLogP) were calculated using the com-
puter program ChemDrawUltra ver. 12.0.2.1076. The
ClogP algorithm incorporated in the ChemDraw software is
licensed from BioByte Corporation.
The molecular docking studies were performed using the
software Autodock v 4.2 and the Autodock tools v 4.2
(ADT) graphical user interface was used to calculate the
Gasteiger–Marsili charges for the protein and to add polar
hydrogens. The homology model for β-tubulin for G.
intestinalis was obtained from Guzmán-Ocampo et al.
(2018). The polar hydrogen charges of Gasteiger-type were
assigned, and the nonpolar hydrogens were merged with the
carbon atoms. All the protein was considered as a rigid
body and the ligands being flexible. All the torsion and
rotatable bonds in the ligand were defined. The grid box for
chalcones derivatives were centered at the residue Pheβ200
in the Nocodazole (NZ) site reported in (Aguayo-Ortiz et al.
2013; Guzmán-Ocampo et al. 2018), with a dimension of
80 × 80 × 80 with a spacing grid of 0.375 Å. The search was
performed with the Lamarckian Genetic Algorithm as is
implemented in Autodock v 4.2 code. The population of
150 individuals was mutated with a mutation rate of 0.02
and envolved for 10 generations. The number of the
docking runs was 50. A cluster analysis was performed
based on a rms deviation values lower than 2.0 Å referenced
to the starting geometry. The best binding mode was
Compound 3k showed a significant weak activity (IC50
=
154.41 μM) against G. lamblia, in the same fashion as it has
been reported in the literature for its activity against Tri-
chomonas gallinae (IC50 = 393 μM) (Oyedapo et al. 2004).
Chalcones 3e and 3h have been previously reported as
nonactive compounds against G. lamblia (Montes-Avila
et al. 2009); similarly, in this study, the antigiardiasic
activity detected for these compounds (48.62 and 48.03 μM,
respectively) was considered weak.
Sixty percent of 15 synthesized chalcones were con-
sidered with significant activity against G. lamblia, ranging
from 1.3 to 9.35 μM (IC50). It is important remark that the
activity level of the most active compound against G.
lamblia (3m, IC50 1.03 μM) was slightly better than
metronidazole (1.4 μM); additionally, chalcone 3m resulted
in the most selective one (SI = 38.9).
The analysis of the results exhibited in Table 1 revealed
that compounds having no substitution at C4″ possess
antigiardial activity at IC’s50 below to 10 μM. The exception
to this pattern was chalcone 3f, which exhibited an IC50 of
12.86 μM. It was noteworthy that compounds belonging to

Medicinal Chemistry Research
O
O
O
Scheme 1 Synthetic scheme for
the synthesis of chalcones 3a–o
1) EtOH, 50% NaOH
MW, 80 °C, 2-4 h
R3
R2
5''
R6
R5
5'
R3
R2
R6
R5
CH3
H
1
3
2
A
B
+
2) 10% HCl
4'
2''
4''
R1
2'
R4
R1 R4
1a-e
2a-c
R4 R5 R6
2a OH
3a-o
R1 R2 R3
1a
H
H
H
Me
H
H
H
1b
1c
OMe H
2b
H
OMe H
Cl
OH
H
H
2c OH
H
1d OH
1e OH
H
H
Me
Table 1 Antigiardial activity,
selectivity index,
electrophilicity, and lipophilicity
of synthesized chalcones
Comp
Substituent position
G. lamblia
5″ IC50 μM SDa CC50 μM SD
6.48 1.55 25.83 1.29
MeO H 16.25 1.19 49.96 1.35
Vero Cells
SIb
CLogPc ω(eV)d
2′ 4′ 5′ 2″ 4″
3ª
H
H
H
H
H
H
H
H
H
Me
H
H
H
OH H
H
3.99 3.46
3.08 4.04
3.76 4.47
4.95 3.18
1.27 3.73
2.52 4.19
7.98 2.83
1.66 3.42
6.40 3.84
20.83 3.29
0.41 3.88
6.12 4.30
38.91 3.79
1.38 4.38
4.87 4.80
1.86
1.63
2.01
1.80
1.57
1.95
1.81
1.60
1.97
1.54
1.55
1.88
1.69
1.53
1.89
3b
3c
3d
3e
3f
Me
Me
H
OH H
Cl
H
5.98 1.11 22.52 1.29
9.35 1.41 46.26 1.02
MeO H OH H
MeO H
MeO H OH H
H
MeO H 48.62 1.22 61.57 1.20
Cl 12.86 1.03 32.36 1.20
5.34 1.37 42.63 1.33
MeO H 48.03 1.10 79.61 1.18
3g
3h
3i
OH
OH
OH
H
H
H
H
H
H
OH H
H
H
OH H
OH H
Cl
H
3.60 1.04 23.07 1.39
2.29 1.22 47.67 1.04
3j
HO H
HO H
HO H
HO H
HO H
HO H
3k
3l
H
MeO H 154.41 1.16 63.90 1.22
OH H
Cl
H
3.47 1.15 21.22 1.35
1.03 1.13 39.92 1.08
3m
3n
3o
Me OH H
Me H MeO H 40.93 1.45 56.40 1.19
Me OH H
Cl
2.20 1.24 10.73 1.36
Metronidazole
Docetaxel
1.40
1.30
aStandard deviation
bSelectivity index
cLogarithm of calculated octanol-water partition coefficient
dElectrophylicity index
this set having hydroxyl substituents at carbons 2′ and 2″, as
well as a methyl group at carbon 5′, exhibit the lowest
inhibitory concentrations against G. lamblia (3m and 3o).
Also, it was observed a slight decreasing in the bioactivity
of their analogs without substituents at C5′ (3j and 3l).
In addition, if the set of ten compounds above mentioned
is divided into two subgroups, based on the substituent
presents at C5″, it is possible to observe that the presence of
a chlorine atom, in general, provokes a decreasing of the
antigiardial activity. This effect was more pronounced over
the selectivity of the most active compounds, thereby
chalcone 3o was eightfold less selective than chalcone 3m,
and the selectivity of 3l decreased 3.4 times compared to
that of 3j.
It is interesting to note that the presence of a methoxyl
group at C4′ or C4″ together with the absence of sub-
stituents at C2″ cause a dramatic negative effect on the
activity against G. intestinalis. Thereby, chalcones 3e, 3h
and 3n were at least 40-fold less active than the most
active one while chalcone 3k was the less active compound
(IC50 = 154 μM).
The broad spectrum of biological activity of chalcones,
including antiprotozoal activity, has been attributed, mainly,
to their Michael acceptor features (Gaonkar and Vignesh
2017; Gomes et al. 2017; Zhuang et al. 2017); therefore, the
electrophilicity of the enone moiety may play an important
role in the mechanisms of action of this class of compounds.
In addition, the increase of lipophilicity has been also

Medicinal Chemistry Research
associated with an increase in the antigiardiasic activity of
some compounds (Mocelo-Castell et al. 2015).
It is known that the binding of microtubule target agents
at the colchicine site of tubulin can disrupt the formation of
microtubules. Colchicine site is a domain divided into three
zones: The zone 1 is found at the α unit of the interface,
zone 2 is located at β subunit, while zone 3 is buried deeper
in the β subunit (Li et al. 2017).
The affinity of chalcones to binding to the colchicine site
has been determined using a 5D-QSAR model for
combretastatin-like analogs (Ducki et al. 2005) due to
similarity between combretastatin A4 (CA-4) and this class
of compounds. CA-4 belongs to colchicine binding site
inhibitors (CBSIs) which have globular o butterfly shape
that allows them can accommodate into the binding pocket
at zones 1 and 2 (Pérez-Pérez et al. 2016).
Previous to molecular docking, the molecular geometries of
compounds to be docked were fully optimized. Surprisingly,
some low-energy conformers of each compound exhibited an
almost planar shape instead of the expected butterfly shape. It
is important to remark that this shape is similar to that
exhibited for benzimidazoles, one of the classes of the drugs
available for the treatment of giardiasis (Fennell et al. 2008),
when they are bonded to colchicine site (Li et al. 2017).
Benzimidazoles (nonclassical CBSIs) bind to the col-
chicine motif at zone 3 and also present a little overlapping
with zone 2 (Li et al. 2017). This feature, together with the
fact the homology model for Giardia β-tubulin only
describes the peptide sequence of zone 3 (Aguayo et al.
2013, 2), made it reasonable to perform docking of the
synthesized chalcones into zone 3 of the colchicine domain.
A molecular docking study of four most active chalcones
(3m, 3o, 3j, and 3l) was performed using a homology model
for Giardia β-tubulin as a target receptor, taken from
Guzmán-Ocampo et al. (2018). Remarkably, binding affi-
nity values of docked compounds ranged from −7.52 to
−6.88 kcal/mol, resulting very close to the docking score
obtained for the binding of Nocodonazole (NZ) with col-
chicine site (−8.05 kcal/mol). Noteworthy, it has been
reported that NZ is active against G. lamblia at an IC50 of
0.017 µM (Katiyar et al. 1984) and its binding energy is
−9.10 kcal/mol (Aguayo-Ortiz et al. 2013).
To explore the SAR relationships of synthesized chal-
cones, their octanol-water partition coefficients (CLogP)
and electrophilicity indexes were calculated (Table 1).
The CLogP’s ranging from 2.83 to 4.80. Noteworthy, the
calculated partition coefficients for the most active (3m) and
the less active (3k) chalcones (3.79 and 3.88, respectively)
exhibited a non-significant difference. Moreover, the anti-
giardiasic activity and CLogP (Table 1) of the four most
active compounds (3j, 3l, 3m, and 3o) exhibited a slight
inverse proportional relationship, thus the most active
chalcones 3m and 3j (IC50 = 1.03 and 2.20 μM, respec-
tively) were less lipophilic than their respective chlorine
analogs 3o and 3l (IC50 = 2.29 and 3.47 μM, respectively).
Contrary to our expectations, the relationship pattern
exhibited by the four most active chalcones was not
observed for the complete set of synthesized chalcones,
indeed it was not possible to find some correlation between
their ClogP’s and antiprotozoal activity. Then, the SAR
analysis was expanded adding the antigiardial activity
(IC50’s = 49.69, 55.29, and 63.79 µM) and CLogP’s (3.62,
4.02, and 4.23) of three previously reported chalcones C1,
C2 and C3, respectively (Fig. 1) (Montes-Avila et al. 2009).
Noteworthy, the slight inverse correlation was observed
again. It is important remark that chalcones C1, C2 and C3
present chlorine or fluorine atoms as substituents, thus the
weak inverse correlation could be associated with the pre-
sence of halogens in chalcones’ structure.
As it can see in Table 1, the electrophilicity indexes of
chalcones with significant activity present a modest inverse
correlation with the antiprotozoal activity. On the other
hand, the chalcones with values of IC50 higher than 10 µM
exhibited a slight direct relationship. Therefore, in the same
fashion of the calculated octanol-water partition coeffi-
cients, the electrophilicity indexes of the previously repor-
ted chalcones C1, C2 and C3 (1.97, 2.12, and 2.08,
respectively) were added to SAR analysis. Unfortunately,
the inclusion of the data mentioned above provokes the loss
of both observed trending. This contrasting behavior not
allowed to determine a clear correlation between anti-
protozoal activity and the electrophilicity of the studied
chalcones.
The binding site pocket for NZ in homology model for
Giardia β-tubulin consists of Tyr50, Gln134, Cys165,
Phe167, Glu198, Phe200, Thr237 Cys239, Leu240,
Leu250, Leu253, Leu257, and Phe266; among them, Glu98
has special importance due it plays a determinant role in the
stabilization of ligands through the formation of two
hydrogen bonds. After its formation, this interaction can be
reinforced by the establishment of interactions with Cys135
and Cys239 (Aguayo-Ortiz et al. 2013).
Surprisingly, in our docking study, it was found that the
four chalcones (3m, 3o, 3j, and 3l) established hydrogen
bonds between carbonyl group of enone system and Glu98
residue (Fig. 2), as it has been determined for NZ. The
Interestingly, although the Michael acceptor mechanism
is known as the representative mechanism of action of
chalcones, the obtained results suggest that antigiardial
activity exhibited for synthesized chalcones is not due to it.
Fortunately, tremendous efforts devoted to characterizing
the mechanisms of chalcones activity have resulted in the
identification of new therapeutic targets, like inhibition of
receptor tyrosine kinase, cyclooxygenase, and aldose
reductase activities, as well as the inhibition of microtubules
formation (Zhuang et al. 2017).

Medicinal Chemistry Research
Leu253
1.99
Glu198
Gln134
3.34
1.86
Glu198
1.83
2.03
Cys239
a
b
Glu198
Leu253
Glu198
2.21
2.01
1.74
1.94
Gln134
3.35
Cys236
1.99
c
d
Fig. 2 Docking images of four most active chalcones binding colchicine site of homology model for Giardia β-tubulin. a Compound 3m.
b Compound 3o. c Compound 3j. d Compound 3k
H-bond distances for chalcones ranged between 1.74 and
2.21 Å, while NZ exhibited distances of 1.71 and 2.35 Å.
Moreover, compound 3o was capable to interact with
Cys239 through another hydrogen bond. Thus, it could
support that the antigiardial activity exhibited by the syn-
thesized chalcones is due to their binding affinity toward the
zone 3 of the colchicine site of Giardia β-tubulin (Fig. 3).
Moreover, the hydrogen bond formed between the oxy-
gen atom of the enone carbonyl group and Glu198 residue
probably increases the electrophilicity of the enone β car-
bon. However, significantly, the docking study did not
show any interaction between the enone system carbons of
studied chalcones and the amino acid residues of zone 3 of
the colchicine site.
This fact, together with the unclear relationship between
the electrophilicity indexes of chalcones and their anti-
Fig. 3 Compound 3m docked at zone 3 of the colchicine site of the
protozoal activity, allows speculating that the action
mechanism of antigiardial activity may not follow the
Michael acceptor mechanism.
homology model of Giarda β-tubulin
by NMR and MS spectra, and tested against the pathogenic
intestinal protozoan Giardia lamblia, and Vero cells.
To the best of our knowledge, the (E)-3-(5-chloro-2-
hydroxyphenyl)-1-(4-hydroxyphenyl)-2-propen-1-one (3i,
IC50 = 3.60 μM) has not previous reports in the literature.
Conclusions
In summary, a total of 15 chalcones with different sub-
stituents at rings A and B were synthesized by microwave-
assisted Claisen–Schmidt condensation, fully characterized
Nine chalcones were active against G. lamblia at CI50
<
10 μM, among them, five exhibited CI50 values lower than

Medicinal Chemistry Research
5 μM. The chalcone 3m was the most active compound with
an IC50 of 1.03 μM and the highest selective one (SI =
38.9). It is important to note that, the antigiardial activity of
this compound was better than that showed by metronidazol
(CI50 = 1.4 μM). The structure relationship analysis sug-
gests that: (i) electro-donating groups at 2′, 2″ and 5′
positions enhanced the antigiardial activity, (ii) the presence
of chlorine at C5″ negatively affects the selectivity, (iii) the
electrophilicity of the most active chalcones presents a
slight inverse correlation with the antigiardial activity. The
docking study results allow supposing that antigiardial
chalcones bind to colchicine site at zone 3 and speculate
that their action mechanism does not follow the Michael
acceptor mechanism.
Fennell BJ, Naughton JA, Barlow J, Brennan G, Fairweather I, Hoey
E, McFerran N, Trudgett A, Bell A (2008) Microtubules as
antiparasitic drug targets. Expert Opin Drug Discov 3:501–518
Gaonkar SL, Vignesh UN (2017) Synthesis and pharmacological
properties of chalcones:
a review. Res Chem Intermed
43:6043–6077
Gomes M, Muratov E, Pereira M, Peixoto J, Rosseto L, Cravo P,
Andrade C, Neves B (2017) Chalcone derivatives: promising
starting points for drug design. Molecules 22:1210–1235
Guzmán-Ocampo DC, Aguayo-Ortiz R, Cano-González L, Castillo R,
Hernández-Campos A, Dominguez L (2018) Effects of the pro-
tonation state of titratable residues and the presence of water
molecules on nocodazole binding to β-tubulin. ChemMedChem
13:20–24
Halliez MC, Buret AG (2013) Extra-intestinal and long term con-
sequences of Giardia duodenalis infections. World J Gastro-
enterol 19:8974–8985
Katiyar SK, Gordon VR, Mclaughlin GL, Edlind TD (1984) Anti-
protozoal activities of benzimidazoles and correlations with
Finally, our findings encourage us to synthesize a series
of chalcones with electron-donating groups at 2′, 2″ and 5′
positions to perform a QSAR for their antigiardial activity.
β-tubulin
sequence.
38:2086–2090
Antimicrob
Agents
Chemother
Lee C, Yang W, Parr RG (1988) Development of the Colle-Salvetti
correlation-energy formula into a functional of the electron den-
sity. Phys Rev B 37:785–789
Li W, Sun H, Xu S, Zhu Z, Xu J (2017) Tubulin inhibitors targeting
the colchicine binding site: a perspective of privileged structures.
Future Med Chem 9:1765–1794
Acknowledgements Funding for this research was provided by
Facultad de Química, Universidad Autónoma de Yucatan. The dock-
ing analysis was performed as part of the research project 256657
granted by the Consejo Nacional de Ciencia y Tecnología - México
(CONACYT). In memory of Dr. José Antonio Manzanilla Cano
(1964-2015), for his invaluable support.
Maestro, Schrödinger, LLC, New York, NY, 2019
Mocelo-Castell R, Villanueva-Novelo C, Cáceres-Castillo D, Carballo
RM, Quijano-Quiñones RF, Quesadas-Rojas M, Cantillo-Ciau Z,
Cedillo-Rivera R, Moo-Puc RE, Moujir LM, Mena-Rejón GJ
(2015) 2-Amino-4-arylthiazole derivatives as anti-giardial agents:
synthesis, biological evaluation and QSAR studies. Open Chem
13:1127–1136
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Montes-Avila J, Díaz-Camacho SP, Sicairos-Félix J, Delgado-Vargas
F, Rivero IA (2009) Solution-phase parallel synthesis of sub-
stituted chalcones and their antiparasitary activity against Giardia
lamblia. Bioorg Med Chem 17:6780–6785
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Pasupuleti V, Escobedo AA, Deshpande A, Thota P, Roman Y,
Hernandez AV (2014) Efficacy of 5-nitroimidazoles for the
treatment of giardiasis: a systematic review of randomized con-
trolled trials. Plos Negl Trop Dis 8:e2733
Pérez-Pérez M-J, Priego E-M, Bueno O, Martins MS, Canela M-D,
Liekens S (2016) Blocking blood flow to solid tumors by
destabilizing tubulin: an approach to targeting tumor growth. J
Med Chem 59:8685–8711
Pires SM, Fischer-Walker CL, Lanata CF, Devleesschauwer B, Hall
AJ, Kirk MD, Duarte ASR, Black RE, Angulo FJ (2015)
Aetiology-specific estimates of the global and regional incidence
and mortality of diarrhoeal diseases commonly transmitted
through food. PLoS ONE 10:e0142927
References
Aguayo-Ortiz R, Méndez-Lucio O, Romo-Mancillas A, Castillo R,
Yépez-Mulia L, Medina-Franco JL, Hernández-Campos A (2013)
Molecular basis for benzimidazole resistance from a novel
β-tubulin binding site model. J Mol Graph Model 45:26–37
Ankarklev J, Jerlström-Hultqvist J, Ringqvist E, Troell K, Svärd SG
(2010) Behind the smile: cell biology and disease mechanisms of
Giardia species. Nat Rev Microbiol 8:413–422
Cedillo-Rivera R, Chavez B, Gonzalez-Robles A, Tapia A, Yepez-
Mulia L (2002) Physiology and ecology-in vitro effect of nita-
zoxanide against Entamoeba histolytica, Giardia intestinalis and
Trichomonas vaginalis trophozoites.
49:201–208
Cohen FE, McKerrow JH, Kenyon GL, Li Z, Chen X, Gong B, Li R
(1998) Inhibitors of metazoan parasite proteases. US Patent
1998-5739170, filled 30 Mar 1995, issued 14 Apr 1998
J
Eukaryot Microbiol
Oyedapo AO, Makanju VO, Adewunmi CO, Iwalewa EO, Adenowo
TK (2004) Antitrichomonal activity of 1,3-diaryl-2-propen-1-
ones on Trichomonas gallinae. Afr J Trad CAM 1:55–62
Rahman A, Choudhary MI, Thomsen WJ (2001) Bioassay techniques
for drug development. Harwood Academic Publishers,
Netherlands
Ducki S, Mackenzie G, Lawrence NJ, Snyder JP (2005) Quantitative
structure-activity
combretastatin-like analogues as inhibitors of tubulin assembly. J
Med Chem 48:457–465
relationship
(5D-QSAR)
study
of
Sahu NK, Balbhadra SS, Choudhary J, Kohli DV (2012) Exploring
pharmacological significance of chalcone scaffold: a review. Curr
Med Chem 19:209–225
Escobedo A, Almirall P, Robertson LJ, Franco RMB, Hanevik K,
Morch K, Cimerman S (2010) Giardiasis: The ever-present threat
of a neglected disease. Infect Disord Drug Targ 10:329–348
Savioli L, Smith H, Thompson A (2006) Giardia and Cryptosporidium
join the neglected diseases initiative’. Trends Parasitol
22:203–208

Medicinal Chemistry Research
Thompson RCA (2000) Giardiasis as a re-emerging infectious disease
and its zoonotic potential. Int J Parasitol 30:1259–1267
Wani MY, Bhat AR, Azam A, Lee DH, Choi I, Athar F (2012)
Synthesis and in vitro evaluation of novel tetrazole embed-
ded 1,3,5-trisubstituted pyrazoline derivatives as Enta-
moeba histolytica growth inhibitors. Eur J Med Chem
54:845–854
Zhuang C, Zhang W, Sheng C, Zhang W, Xing C, Miao Z (2017)
Chalcone: a privileged structure in medicinal chemistry. Chem
Rev 117:7762–7810
Upcroft P, Upcroft JA (2001) Drug targets and mechanisms of resis-
tance in the anaerobic protozoa. Clin Microbiol Rev 14:150–164
Vonthron-Sénécheau C, Bernard-Weniger B, Ouattara M, Tra-Bi F,
Kamenan A, Lobstein A, Brun R, Anton R (2003) In vitro anti-
plasmodial activity and cytotoxicity of ethnobotanically selected
Ivorian plants. J Ethnopharmacol 87:221–222