Antimicrobial, anti-inflammatory and antioxidant activities of polyoxygenated chalcones

Article abstract of DOI:10.21577/0103-5053.20180177

It was synthesized nine polyoxygenated chalcones with a potential and safe use as antioxidant, antimicrobial and anti-inflammatory therapies. Chalcones obtained by Claisen-Schmidt condensation were studied as antioxidant, inhibitors of human 5-lipoxygenas

Full text of DOI:10.21577/0103-5053.20180177

http://dx.doi.org/10.21577/0103-5053.20180177
J. Braz. Chem. Soc., Vol. 00, No. 00, 1-19, 2018
Printed in Brazil - ©2018 Sociedade Brasileira de Química
Article
Antimicrobial, Anti-Inflammatory and Antioxidant Activities of Polyoxygenated Chalcones
Yesseny A. Vásquez-Martínez,a Mauricio E. Osorio,*,b Diego A. San Martín,b
Marcela A. Carvajal,c Alejandra P. Vergara,c Elizabeth Sanchez,c Marcela Raimondi,d
Susana A. Zacchino,d Carolina Mascayano,e Claudia Torrent,e Francisco Cabezas,e
Sophia Mejias,f Margarita Montoyaf and Marcelo Cortez-San Martínf
aPrograma Centro de Investigaciones Biomédicas Aplicadas, Facultad de Ciencias Médicas,
Universidad de Santiago de Chile, 9170022 Santiago, Chile
bLaboratorio de Productos Naturales, Departamento de Química,
Universidad Técnica Federico Santa María, 2390123 Valparaíso, Chile
cCentro de Biotecnología CB-DAL, Universidad Técnica Federico Santa María,
2390136 Valparaíso, Chile
dFarmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas,
Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina
eDepartamento de Ciencias del Ambiente, Facultad de Química y Biología,
Universidad de Santiago de Chile, 9170022 Santiago, Chile
fDepartamento de Biología, Facultad de Química y Biología,
Universidad de Santiago de Chile, 9170022 Santiago, Chile
It was synthesized nine polyoxygenated chalcones with a potential and safe use as antioxidant,
antimicrobial and anti-inflammatory therapies. Chalcones obtained by Claisen-Schmidt
condensation were studied as antioxidant, inhibitors of human 5-lipoxygenase, antifungal,
antibacterial and antibiotic resistance modifiers. Two chalcones with catecholic moieties were
able to strongly decrease the minimum inhibitory concentration (MIC) of methicillin against
methicillin-resistant Staphylococcus aureus, increase the antiradical activity and significantly
inhibit the human 5-lipoxygenase. Only one of these chalcones was active synergistically with
methicillin. Chalcones with methoxyl substituents at different positions displayed the best activities
against Cryptococcus neoformans. Only one chalcone showed good activity against the plant
pathogenic bacteria Pseudomonas syringae whose half maximal inhibitory concentration (IC50)
value (2.5 µg mL-1) was similar to that observed with the antibiotic streptomycin (2.9 µg mL-1).
These simple chalcones have safe potential uses in antioxidant, antimicrobial and anti-inflammatory
therapies.
Keywords: antioxidant activity, polyoxygenated chalcones, human 5-lipoxygenase inhibitors,
antibacterial, antifungal
Introduction
condensation under homogeneous conditions in the
presence of acid or base being the most used. Traditionally,
strong alkaline media2-8 and the use of several Lewis acids9-15
have been employed for their synthesis. Some improved
strategies include solvent-free conditions,16 microwave17
and ultrasound irradiation18 and the grinding technique.19
The synthesis of chalcones in acid condition using SOCl2
in ethanol to generate HCl in situ have been used to prepare
chalcones with one free hydroxyl group,20 however, the
synthesis of chalcones with additional oxygenated groups
have not been proven using this method. Chalcones display
Chalcones (1,3-diphenyl-prop-2-en-1-ones) essentially
differ from other flavonoids by its open C-ring, thus
possessing a linear chain of three carbon atoms binding
A with B ring (Figure 1).1 Chalcones have been used
as intermediates for the preparation of compounds with
therapeutic value. A number of synthetic routes have been
reported for the synthesis of chalcones, the Claisen-Schmidt
*e-mail: mauricio.osorio@usm.cl, osomauricio@gmail.com

2
Antimicrobial, Anti-Inflammatory and Antioxidant Activities of Polyoxygenated Chalcones
J. Braz. Chem. Soc.
a large number of different biological activities, such as
antibacterial, antifungal, anti-cancer, anti-inflammatory,
analgesic, antiviral, anti-malarial, antipyretic and cytotoxic
activities.20,21
and different types of compounds have been used for their
control. However, these control efforts have not always
been successful and less toxic new products to replace the
existing ones are highly needed. To achieve this goal, the
worldwide trend is to explore new compounds against these
phytopathogens in order to minimize the risks associated
with the development of pathogen populations insensitive
to these chemical compounds.35 Also, many countries have
limited the use of some commercial antimicrobials based on
recommendations from the CodexAlimentarius36 according
to the maximum residue limits (MRLs) for residues of
pesticides or veterinary drugs in foods.
Figure 1. Representative structure of the chalcone backbone.
Multi-drug resistance is one of the major causes of
the alarming level of infectious diseases worldwide. The
discovery of new drugs with potent antimicrobial activity,
particularly against the resistant strains, is, therefore,
highly needed. In addition, since many of the currently
available antifungal drugs have undesirable side effects, are
ineffective against new or re-emerging fungi, or develop
a rapid resistance in the pathogen,22 there is an urgent
need of a next generation of new antifungal agents that
can overcome the above disadvantages. Several reports
highlighting the significance of chalcones as antimicrobial
and antiparasitic agents have been documented in the
literature.21 Chalcone derivatives with –OH and –OCH3
in the rings A and B have demonstrated potential anti-
leishmanial and anti-trypanosomal activities by acting on
a number of molecular targets.23
Fungal plant pathogens such as Botrytis cinerea
(B. cinerea),24 Gibberella fujikuroi (G. fujikuroi)25 and
Phytophthora cinnamomi (P. cinnamomi)26,27 attack a
wide range of agriculturally and ornamentally important
plants28 and significantly affect the economy of developing
countries causing large economic losses in agriculture.29
Numerous fungicides such as carboxin, captan, thiram,
tebuconazole and metalaxyl have been used to control
fungal phytopathogens, but all of them possess secondary
effects in other non-target microorganisms and living
beings.30 Natural and synthetic chalcones with –OH and
–OCH3 groups linked to different positions of the A and
B rings have shown bactericidal, antifungal, anthelmintic,
insecticidal, insect antifeedant, antiviral and phytotoxic
activities.31
Endogenous free-radical species like reactive oxygen
(ROS) and nitrogen species (RNS) and other reactive small
molecules have emerged as important regulators of many
physiological and pathological processes. ROS and RNS
are essential to maintain homeostasis and health of human
beings, but uncontrolled and excess ROS/RNS have been
implicated in the pathogenesis of various diseases including
cancer, cardiovascular and neurodegenerative diseases
as well as ageing. The human organism has developed
defense systems to neutralize excessive levels of ROS
and RNS and compensate for the oxidative stress.37 When
endogenous antioxidant protection is not able to maintain
the proper balance of free radicals due to their unlimited or
uncontrolled production, several health problems appear.
In such cases, additional external antioxidants are required
to restore the proper balance between free radicals and
antioxidants in the body.38 Chalcone derivatives with an
–OH substituent on ring A, and –SCH3 and –OCH3 groups
at the para position of ring B showed good anti-oxidant
activity.21
Different natural or synthetic chalcones has been
studied for their anticancer activities because they exert
cytotoxicity through multiple mechanisms, which include
cell cycle disruption as well as inhibition of angiogenesis,
tubulin polymerization, apoptosis and cell signaling.39 On
the other hand, high expression of 5-lipoxygenase (5-LOX)
has been described in cancer cells and its inhibition has
been widely characterized to control tumor cell growth.40,41
Moreover, ROS also have to be closely linked with tumor
initiation, development and progression, by promoting
cell survival, cell proliferation and migration.42 Thus,
inhibition of 5-LOX or the decrease in ROS levels can also
have impact on cancer cell survival and/or proliferation.
5-LOX belongs to a group of closely related non-heme
iron containing dioxygenases. These enzymes catalyze
the addition of molecular oxygen into poly-unsaturated
fatty acids (PUFAs) containing cis, cis-1,4-pentadiene
structures to give their hydroperoxy derivatives. LOXs are
further classified into 5-, 8-, 9-, 11-, 12-, and 15-LOXs
Plant pathogenic bacteria cause serious problems for
industrial agriculture because they affect crops of nutritional
and commercial interest. Agrobacterium tumefaciens
(A. tumefaciens), Pseudomonas syringae (P. syringae) and
Erwinia carotovora (E. carotovora) are Gram-negative
bacteria that are responsible for a number of economically
important diseases. These bacteria usually infect a wide
variety of fruits, vegetables, and ornamental plants,32-34

Vol. 00, No. 00, 2018
Vásquez-Martínez et al.
3
according to the positional specificity of arachidonate
oxygenation. In addition, the 5-LOX is the source of
potent pro-inflammatory mediators present in a variety of
inflammatory and allergic diseases.43 As 5-LOX have been
shown to have a major role in the pathogenesis of various
inflammatory disorders, including cancer, inhibitors of
5-LOX will have a wide range of therapeutic applications.44
Di-O-prenylated chalcone derivatives have shown to
possess good inhibitory potency of 5-LOX (half maximal
inhibitory concentration (IC50) = 4 µM), potent anti-
proliferative effects (50% growth inhibition (GI50) = 9 µM)
on the MCF-7 breast cancer cell line, and no appreciable
effects up to 100 µM on the immortalized nontumorigenic
human epidermal (HaCaT) cell line.43
In this paper, we report a facile synthesis of nine
chalcones to evaluate their effects against phytopathogens
and human pathogenic bacteria and fungi as possible
alternatives to antimicrobials currently in commercial
use. Additionally, as endogenous free radicals and human
5-lipoxygenase (5-hLOX) have been shown to have major
roles in the pathogenesis of various health problems
including cancer, cardiovascular and neurodegenerative
diseases and inflammatory disorders, the chalcones were
tested as 5-hLOX inhibitors and DPPH (2,2-diphenyl-
1-picrylhydrazyl) radical scavengers.
preferred when one or more free hydroxyl groups are
bound to the aromatic system of the chalcones. When
Claisen-Schmidt condensations using other 2’-hydroxy
or 2’-methoxy acetophenones were conducted in acidic
conditions, chalcones were not produced (12 and 19). This
was probably due to either the hydrogen-bond formed by
the OH or OMe groups on C-2’ with the carbonyl group
(that could hinder the formation of the enol intermediate)
or by problems of steric hindrance with the 2’-methoxyl
group.
The yields of all the synthesized chalcones were similar
or better than those reported in the literature and all were
obtained in only one step. Chalcone 12 is the only new
molecule in the series. The solid-state reactions (without
solvent) were not attempted.
The synthesis of chalcones with a hydroxyl group
in 4-position was incompatible with alkaline medium,
except for 12. Chalcones with 2’-OH group can be
synthesized in alkaline medium, but with low yield (11%
for 19). Protecting groups resistant to alkaline medium
(i.e., methoxymethyl (MOM), tetrahydropyranyl (THP))
should be considered to obtain chalcones in good yields
with two or more free hydroxyl groups. Chalcones with
3,4-diOH substituents (catechol group) on the B ring can
be synthesized in acid condition with moderate yields (47%
for 11 and 57% for 18).
Results and Discussion
In vitro antibacterial effects of chalcones 11-19 on bacteria
Synthesis
pathogenic to humans
Chalcones 11-19 were obtained in poor to good
yields (11-90%) via Claisen-Schmidt condensation of
acetophenones 1-6 with benzaldehydes 7-10 in acidic or
alkaline media (Scheme 1). Acidic medium was mostly
Determination of the minimum inhibitory concentration (MIC)
of the chalcones against bacteria
The result of the bacterial growth inhibition test
(Table 1) showed that chalcones 11 and 18 were active
Scheme 1. Preparation of chalcones 11-19 via Claisen-Schmidt condensation in acidic (i: SOCl2/EtOH) and alkaline (ii: KOH/MeOH/H2O) media.
Methodologies and yields of reaction are shown in brackets.

4
Antimicrobial, Anti-Inflammatory and Antioxidant Activities of Polyoxygenated Chalcones
J. Braz. Chem. Soc.
Table 1. Results of the in vitro antibacterial assay
Minimum inhibitory concentration (MIC) / (µg mL-1)
Compound
Structure
E. coli
E. coli
S. aureus
S. aureus
ATCC25922
33.1
NTCC8325-4
MRSA 97-7
11
> 100
> 100
> 100
> 100
70
100
12
> 100
> 100
13
14
15
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
16
17
18
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
50
> 100
> 100
50
19
> 100
> 100
> 100
> 100
Methicillin
Ciprofloxacin
Ampicillin
–
1
–
5
10
–
50
80
–
50
60
> 100
Antibacterial activity of chalcones in reference strain E. coli ATCC25922, multi-resistant E. coli 33.1, reference strain S. aureus NTCC8325-4 and
methicillin-resistant S. aureus MRSA 97-7 isolates.
against sensitive S. aureus strains with MICs of 50 and
70 µg mL-1, respectively. These compounds also inhibited
the growth of MRSA 97-7 strain (MICs of 50 and
90 µg mL-1, respectively).
When comparing the structural characteristics of 11 and
18, which are weakly active against S. aureus with the rest
of the chalcones, it can be observed that both compounds
possess a catechol moiety in their structures, which could
play a key role in the activity. Then, our results indicated
that the presence of catechol group at positions 3,4 on the B
ring is necessary to exhibit the anti-staphylococcal activity,
decreasing this activity when the hydroxyl at position 3

Vol. 00, No. 00, 2018
Vásquez-Martínez et al.
5
is methylated (chalcones 11, 13, 14, 16 and 17). These
results are similar to those obtained by Batovska et al.,45
who reported that the presence of hydroxyl group on ring
B alone is not enough for the chalcones to exhibit anti-
staphylococcal activity and the introduction of a methoxy
group next to o- and p-hydroxyl groups led to more inactive
chalcones, while the activity was kept when the methoxy
group was inserted next to the m-hydroxyl group. They also
showed that the lipophilicity of ring A is important for the
antibacterial activity against S. aureus.
In vitro antifungal effects on fungi pathogenic to humans
In the last decades, fungi have emerged as major
causes of human morbidity and mortality, mainly among
the immunocompromised and seriously-ill hospitalized
patients.50 Most of these mycoses-related deaths were
associated with Candida albicans (C. albicans) and
Cryptococcus neoformans (C. neoformans) and, although
there are several antifungal drugs in clinical use to treat
both opportunistic fungal species, fungal infections remain
very difficult to eradicate.
Since chalcones with different substituents than the
ones synthesized here have shown antifungal activities
in several previous papers,51-58 the antifungal properties
of these compounds could add new interesting data on
the antifungal behavior of chalcones. Thus, the nine
chalcones 11-19 were tested for antifungal properties
against the clinically important yeasts C. albicans
(ATCC 10231) and C. neoformans (ATCC 32264). To
assess antifungal activities, the broth microdilution method
M27-A3 for yeasts of the Clinical and Laboratory Standards
Institute (CLSI)59 was used.
Chalcones with hydroxyl and methylthio groups on
B ring at position 4 are being considered for a next work.
Evaluation of the ability of chalcones 11 and 18 to potentiate
the antibiotic activity of methicillin
The detection of what are known as resistance-
modifying agents (RMAs) represents an attractive strategy
to mitigate the spread of bacterial drug resistance, since
it could facilitate the recycling of well-established
antibiotics.46 Drug repositioning can accelerate the drug
development process as the already established compounds
are characterized by known safety profiles, pharmacology
and administration routes.47,48
The percentage of inhibition of each fungus was
determined for each compound at two-fold dilutions in the
range 250-3.9 µg mL-1 (Table 3). In the right column, IC50
represents the minimum concentration that inhibits fungal
growth by 50%. This value is accepted in the literature as
representative of the in vitro activity of target compounds.60
Although Table 3 shows that all compounds inhibit
both C. albicans and C. neoformans at different tested
concentrations, it is interesting to note that C. albicans is
more sensitive than C. neoformans. In Table 3, it can be
observed that IC50 of all compounds against C. albicans
ranged from 125 to > 250 µg mL-1, with five compounds
(11, 12, 14, 16 and 19) possessing an IC50 ≥ 250 µg mL-
1, one (13) with IC50 = 250 µg mL-1, two (15 and 18)
displaying IC50 = 125 µg mL-1 and one (17) with
IC50 = 50 µg mL-1. In the case of C. neoformans the IC50
ranged from 7.8 to > 250 µg mL-1 with two compounds
(15 and 17) displaying an IC50 between 7.8-15.6 µg mL-1,
Chalcones 11 and 18 showed an important capacity
to reduce the MIC of methicillin against MRSA 97-7,
re-sensitizing these resistant bacteria 2,500 times methicillin
and 2.5 times the synthetic chalcone 18. Compound 18
was found to act synergistically in conjunction with the
commercial antibiotic (Table 2), while compound 11
showed only partial synergy, although the dose was reduced
by the same amount as 18.
These findings add new data to the previously reported49
enhancing capacity of chalcones to antibiotics activity
showing that five chalcones possessing either 2-OH or
4-OH on the B ring enhanced the antibacterial activity of the
non β-lactam antibiotics doxycycline, ciprofloxacin and/or
gentamicin. In the present paper, the 3,4-diOH chalcones
either unsubstituted (18) or 3’,4’,5’-trimethoxylated (11)
on the A ring, showed potentiated activity of the β-lactam
antibiotic methicillin.
Table 2. Synergistic effects of chalcones 11 and 18 with methicillin in MRSA 97-7
MIC alone / MIC combination / FICs of each
Strain
Drug
DRI
FICI
0.40
Outcome
synergy
(µg mL-1)
(µg mL-1)
compound
18
50
50
20
0.40
2.5
2,500
2.0
methicillin
11
0.02
50
0.0004
0.5
MRSA 97-7
100
50
0.50
partial synergy
methicillin
0.02
0.0004
2,500
MIC: minimum inhibitory concentration; FIC: fractional inhibitory concentration; DRI: dose reduction index = MIC of each compound alone/MIC in
combination; FICI: FIC index.

6
Antimicrobial, Anti-Inflammatory and Antioxidant Activities of Polyoxygenated Chalcones
J. Braz. Chem. Soc.
Table 3. Percentages of inhibition of chalcones 11-19 against C. albicansATCC 10231 (Ca) and C. neoformansATCC 32264 (Cn) at different concentrations.
The compounds were ordered by structural similarity in order to facilitate the structure-activity analysis and not in numerical order
Concentration / (µg mL-1)
Compound
Structure
Fungi
a
250
125
62.5
31.2
15.6
7.8
3.9
IC50
Ca
Cn
66.3 1.9 40.6 0.0 24.4 0.1 17.8 0.2 11.1 1.9 8.1 0.7
3.6 0.2
0
125
18
24.4 2.1 10.9 2.0
0
0
0
0
> 250
Ca
Cn
100
100
100
100
100
100
45.9 1.5 18.2 0.6 8.8 0.0
100
5.2 0.5
50
17
11
93.5 0.1 15.4 0.1 8.6 0.4
15.6
Ca
Cn
25.0 2.8 18.4 1.6 12.6 0.8 8.4 0.4
45.9 0.2 34.7 2.7 9.1 0.2 7.6 0.5
5.1 1.0
3.3 0.5
3.4 0.2
2.8 0.1
2.3 0.9
1.7 0.5
250
250
Ca
Cn
74.6 3.3 27.51 1.35 10.4 1.0 6.7 0.3
60.6 1.6 46.6 0.4
3.8 0.7
0
2.4 1.2
0
1.8 0.1
0
250
125
13
15
0
0
Ca
Cn
84.0 2.7 54.5 0.0 31.0 2.3 17.0 2.0 8.0 0.2
100
3.7 0.0
0
0
125
7.8
95.6 0.8 95.5 2.9 86.3 0.5 75.3 1.5 61.5 1.0
Ca
Cn
27.4 8.3 15.1 2.1 13.5 0.3 9.4 1.1
86.0 0.0 80.9 1.9 5.5 0.6 0.00
4.9 0.9
0.00
3.4 0.3
0.00
2.3 0.3
0.00
> 250
125
12
Ca
Cn
42.3 2.1 30.7 1.9 19.6 1.5 13.5 1.2 8.5 1.2
6.0 0.9
0
3.6 0.0
0
> 250
125
14
16
85.6 2.1 66.5 1.3 27.4 0.0 3.2 0.1
0
Ca
Cn
27.1 1.3 15.3 0.9 12.1 0.6 8.2 0.7
6.2 0.1
2.5 0.8
0.2 0.5
> 250
31.25
100
85.8 0.0 77.1 1.3 45.5 0.1 32.0 0.5 19.9 0.0 14.8 0.2
Ca
Cn
18.4 1.6 11.0 0.8 5.9 0.2
5.1 0.2
0
2.6 0.0
0
1.8 0.3
0
1.6 0.1
0
> 250
> 250
19
23.7 0.5 7.1 3.7
0
Ca
Cn
100
100
100
100
100
100
100
100
100
100
100
100
100
100
1.5
1.0
Amphotericin B
aIC50 value represents the concentration of each compound that inhibits fungal growth by 50%.
one compound (16) with an IC50 = 31.25 µg mL-1, three
compounds with IC50 ≥ 125 µg mL-1 (12, 13 and 14) and
only two compounds (11 and 18) with IC50 > 250 µg mL-1.
The comparative behavior of all compounds against
C. albicans and C. neoformans can be clearly corroborated
by comparing the dose-responses curves in Figure 2.
Against C. albicans (Figure 2a), four (13, 15, 17 and 18)
out of the nine compounds produced an inhibition ≥ 50%
at 250 µg mL-1, one (15) produces ≥ 50% inhibition at
125 µg mL-1 and one (17) at 50 µg mL-1. While, against
C. neoformans, six compounds (12-17) produced an
inhibition ≥ 50% at 250 µg mL-1 and five of them (12,
14-17) produced the same inhibition at 125 µg mL-1
(Figure 2b).
To see the influence of the methylation of the 3-OH
group (on the B ring) on the growth of C. neoformans, the
anticryptococcal activity of 18 (with 3,4-diOH on the B ring)
was compared with 17 (with 3-OMe, 4-OH on the B ring),

Vol. 00, No. 00, 2018
Vásquez-Martínez et al.
7
Figure 2. Comparative antifungal activities of chalcones 11-19 against (a) C. albicans; (b) C. neoformans. The horizontal dotted line was drawn to clearly
see the number of compounds that displayed ≥ 50% inhibition of fungal growth. The vertical dotted lines were drawn to clearly see the effects of the
different compounds at 125 µg mL-1.
both with an un-substituted ringA (Figure 3a). With the same
purpose, the anticryptococcal activity of 11 (with 3,4-diOH
on the B ring) was compared with 13 (with 3-OMe, 4-OH
on the B ring), both with three methoxyl groups on the A
ring (Figure 3b). Results clearly show that the methylation
of the 3-OH increased the activity in different degrees on the
A ring unsubstituted and substituted chalcones. However, in
the compounds with an unsubstitutedA ring the difference in
inhibition between 17 and 18 (Figure 3a) was much greater
than that observed between 11 and 13 (Figure 3b), showing
that the substitution of ring A could play an important role
in the activity.
From Figure 4, it is clear that the chalcone with 3-OMe,
4-OH substituents and unsubstituted-ring A chalcone (17)
displayed the best activity reaching the highest inhibition
(100%) at 7.8 µg mL-1. The activity decreases in the following
order: 17 (unsubstituted-ring A) > 16 (3’,4’-(MeO)2
A ring) > 12 (2’5’-(MeO)2 A ring) > 14 (2’,4’- (MeO)2
A ring) > 13 (3’,4’,5’-(MeO)3 A ring).
In addition, the (MeO)2 derivatives possessing one of
the methoxyl groups at position 2’ (12 and 14) displayed
lower activities than 16 (3’,4’-(MeO)2 derivative), probably
due to steric hindrance of the OMe at position 2’.
Our results showed that these chalcones were more active
against C. neoformans than against C. albicans. Regarding
the relation between structure and anticryptococcal
activity, the methylation of the 3-OH of the B-ring led to
an improved activity. In turn, the pattern of substitution of
To see the influence of different substituents on ring A
on the anticryptococcal activity, all compounds possessing
3-OMe, 4-OH substituents on the B ring (12-14, 16, 17)
were compared in Figure 4.
Figure 3. Influence of the methylation of the 3-OH group on ring B on growth of C. neoformans, when ring A is (a) unsubstituted (chalcones 17 and 18)
or (b) trimethoxysubstituted (chalcones 11 and 13). Chalcones 18 and 13 possess 3,4-diOH as substituents on the B ring and chalcones 17 and 11 possess
3-OMe, 4-OH substituents on the B ring.

8
Antimicrobial, Anti-Inflammatory and Antioxidant Activities of Polyoxygenated Chalcones
J. Braz. Chem. Soc.
were less sensitive to the compounds. However, some
sensitivity was observed in the presence of compound 16
for A. tumefaciens, and in the presence of the compound 19
against E. carotovora and P. syringae.
Chalcones 19 and 12 showed an interesting effect on
P. syringae that is a Gram-negative bacterium, which is
classified as pathovars according to its host specificity.
Infection with this pathogen produces important economic
losses that may affect seriously the net production as well
as the quality of the product obtained.
In vitro fungicidal effects on fungi pathogenic to plants
The results of the antifungal activity assay using
phytopathogens (Table 5) indicate a high sensitivity to the
chalcones, with growth inhibition percentages higher than
51%, at all concentrations tested. Although the positive
controls, based on commercial antifungal agents (pure
active ingredients), showed strong inhibition (100%) at
all concentrations tested in the assays, some chalcones
showed inhibition values similar or slightly lower on the
growth of the oomycete P. cinnamomi (for the case of
compound 15) and G. fujikuroi (for the case of compounds
14, 15 and 17). This difference was statistically significant
with respect to the negative control. The foregoing indicates
that this type of compound may have great potential for
the treatment of these pathogens. Compound 15 was the
most active of this series inhibiting the growth of the
two fungi (B. cinerea and G. fujikuroi) and the oomycete
(P. cinnamomi). P. cinnamomi was the pathogen most
sensitive to 15, registering 100% growth inhibition with
the lowest concentration tested (250 µM), achieving effects
similar to those observed with metalaxyl.
Figure 4. Comparative anticryptococcal activity of 3-OMe, 4-OH
chalcones with different substituents on ring A [unsubstituted (17);
3’,4’,5’-(OMe)3 (13); 2’,4’-(OMe)2 (14), 2’,5’-(OMe)2 (12), 3’,4’-(OMe)2
(16)].
theA ring appears to play some role in the anticryptococcal
activity.
In vitro antibacterial effects on bacteria pathogenic to plants
Results of the antibacterial assays (Table 4) indicate
an outstanding antibacterial activity of compound 12 on
the growth of P. syringae with a low IC50 value (8 µM
or 2.5 µg mL-1). This effect is similar to that observed
with streptomycin whose IC50 value is close to 5 µM
(2.9 µg mL-1). Although the best inhibitory effect was
observed in the previous case, the remaining compounds of
the list do not show activity or show a moderate effect (i.e.,
19 shows an IC50 =191 µM and 17, IC50 = 343 µM) only
against P. syringae. The other two phytopathogenic bacteria
Additionally, an interesting sensitivity of the fungus
G. fujikuroi was observed compared to the complete series
of chalcones, with percentages of inhibition higher than
75.8%.
Table 4. IC50 values of chalcones towards phytopathogenic bacteria
IC50 / µM
Compound
A. tumefaciens E. carotovora
P. syringae
457
In vitro 5-hLOX enzyme assay
11
12
13
14
15
16
17
18
19
> 500
> 500
> 500
> 500
> 500
392
500
Three chalcones displayed an interesting activity as
human 5-hLOX inhibitors (Table 6). The analysis of the
relationship between structure and activity showed that the
most potent inhibitors 11, 18, and 19 possess free hydroxyl
groups on the A ring (19) or catechol groups on the B ring
(11 and 18) in the structure. The docking studies (Figure 5)
revealed that the catechol group present in chalcone 18 was
oriented towards the metal, which agrees with a competitive
mechanism found in a kinetics experiment (Figure 6).
Compound 11, which was the best inhibitor found, presented
a mixed mechanism and was unable to orient the catechol
> 500
> 500
> 500
> 500
> 500
> 500
> 500
442
8
> 500
> 500
> 500
494
> 500
> 500
500
500
343
191
Antibacterial activities are shown as IC50 values in µM concentrations;
IC50 values were calculated from the first kinetic assay.

Vol. 00, No. 00, 2018
Vásquez-Martínez et al.
9
Table 5. Percentage of growth inhibition of phytopathogenic fungi
Mycelial growth inhibition of fungal plant pathogens / %
Compound
P. cinnamomi
B. cinerea
G. fujikuroi
250 µM
500 µM
1000 µM
250 µM
500 µM
1000 µM
250 µM
500 µM
1000 µM
11
12
13
14
15
16
17
18
19
C+
C–
60.14 1.26 66.67 1.26 68.84 1.26 56.65 0.97 66.28 0.63 55.96 0.0 77.11 1.45 83.28 1.21 85.30 2.35
64.13 1.09 67.75 1.66 73.55 0.63 51.83 1.95 69.27 0.79 50.46 0.0 79.54 1.37 81.09 0.65 83.29 0.61
61.23 0.63 64.13 1.09 69.20 2.26 60.09 1.95 67.66 0.97 71.56 0.79 78.36 0.69 81.39 2.91 83.89 3.23
69.57 1.09 82.97 0.77
100 0.0 100 0.0
100 0.0
100 0.0
50.92 0.79 72.94 0.79
100 0.0
84.64 2.04 87.58 0.91 85.96 2.19
69.27 0.79 72.02 0.79 76.15 0.79 85.02 0.57 86.97 0.54 88.20 0.52
65.58 1.66 74.64 0.63 81.16 1.26 60.09 1.95 53.26 0.07 73.39 1.59 77.11 1.45 82.21 0.63 84.34 0.59
63.77 1.26 73.55 0.63 75.36 1.26 57.34 1.95 74.77 0.79 74.77 5.55 89.06 0.98 91.38 0.54 82.44 3.65
59.06 1.66 67.03 1.66 74.28 1.66 51.38 1.59 59.63 1.56 67.89 1.59 79.92 1.79 80.71 0.65 81.46 1.22
60.51 0.63 67.39 2.17 76.09 2.17 54.13 1.59 57.80 1.56 74,31 1.59 75.83 1.94 81.46 1.12 85.02 0,57
100 0.0a
100 0.0a
100 0.0a
100 0.0b
100 0.0b
100 0.0b
100 0.0b
100 0.0b
100 0.0b
0
0
0
0
0
0
0
0
0
The percentage of mycelial growth inhibition is based on mycelium area measurements after 72 h of incubation. Each point represents the mean of three
independent experiments. C+: positive control (ametalaxyl; bcaptan); C–: negative control (stock solution 1% ethanol/water).
group towards the iron, generating an interaction between
Asn425 and this group. Compound 19, that showed the worst
IC50 value and had a competitive mechanism, only oriented
a hydroxyl group towards the metal.
It is known that flavonoids containing ortho-diOH
groups have inhibitory activities against LOXs,61,62 although
it is not the main requirement. We show that there are
differences in the type and potency of the inhibition
between 11 and 18, so the presence of a catechol would
not be an absolute structural requirement for this activity.
Moreover, the most active compound (11) has three
additional methoxy groups in the 3’,4’,5’ positions.
compared with Trolox. Compounds 11 and 18 showed
more potent antioxidant activity than Trolox. The presence
of the catechol group resulted in a significant increase of
the antioxidant activity with respect to the compounds
without this group. Hydroxy groups in meta positions
(i.e., 19) and methoxy groups in meta and ortho positions
of the A ring (i.e., 14 and 16) and methoxy groups in
position 3 of the B ring (i.e., 13 and 17) decrease the
antioxidant activity. Hydroxy and methoxy groups on
the B and A rings, respectively, increase the antioxidant
activity.
In vitro cytotoxic activity of 18
DPPH radical scavenging activity
One of the most effective synthesized chalcone as
antioxidant and 5-hLOX inhibitory capacity was 18. Given
that both biological activities are related to proliferation
The results obtained from the antioxidant activity
assays are shown in Table 7. All the compounds were
Table 6. Results of 5-hLOX activity assays in presence of chalcones 11-19
Compound
Inhibitiona / %
W.A
IC50 / µM
N.D
Ki / µM
N.D
Type
N.D
16
17
W.A
N.D
N.D
N.D
18
88.36 2.18
58.60 5.42
96.30 1.47
W.A
0.023 0.004
0.707 0.037
0.011 0.004
N.D
0.01592
1.962
0.006326
N.D
competitive
competitive
mixed
N.D
19
11
12
13
W.A
N.D
N.D
N.D
14
41.35 4.33
11.67 13.13
93.42 5.87
N.D
N.D
N.D
15
N.D
N.D
N.D
NDGA
N.D
N.D
N.D
aChalcone at 10 µM; IC50: half maximal inhibitory concentration; Ki: inhibitory constant; W.A: without activity; N.D: not determined; NDGA:
nordihydroguaiaretic acid.

10
Antimicrobial, Anti-Inflammatory and Antioxidant Activities of Polyoxygenated Chalcones
J. Braz. Chem. Soc.
Figure 6. The Lineweaver-Burk plot of the inhibition of 5-hLOX by three
chalcones (11, 18 and 19).
Table 7. Screening results of DPPH radical scavenging activity of
chalcones 11-19
Compound
IC50 SD / µM
11.75 0.21
33.50 1.52
49.04 1.25
54.40 5.82
NA
11
Figure 5. Docking results for chalcones with 5-hLOX. (a) 19, with a
∆Gb = –3.68 kcal mol-1 and an experimental ki = 1.96 µM; (b) 18, with
a ∆Gb = –4.9 kcal mol-1 and an experimental ki = 0.016 µM; (c) 11, with
a ∆Gb = –4.25 kcal mol-1 and an experimental ki = 0.06 µM. The orange
ball represents the iron atom, located in the active site.
12
13
14
15
16
73.03 7.53
104.26 5.44
12.45 0.39
NA
17
and viability of cancer cells, we measured 18-induced
cytotoxic activity against MDA-MB-231 (human breast
adenocarcinoma cell line), B16-F10 (mouse melanoma
cells) and MEF (primary mouse embryonic fibroblast).
Our results in the Table 8 show that 18 exhibits a 4-fold
higher selectivity upon B16-F10 compared to the
effect on non-cancerous cells (MEF). 18 also exhibited
18
19
Trolox
22.54 0.61
Antioxidant activity is shown as IC50 (half maximal inhibitory
concentration) values in µM concentrations. NA: no activity. All
compounds were analyzed in triplicate and the results expressed as average
standard deviation (SD).

Vol. 00, No. 00, 2018
Vásquez-Martínez et al.
11
cytotoxic activity against MDA-MB-231, but with no
selectivity over MEF. When the cytotoxic effect of 18
against B16-F10 was compared with commonly used
chemotherapeutic drugs, it could be verified that 18
showed 29- and 8-fold lesser activity than taxol and
etoposide, respectively. Furthermore, 18 exhibited 300-
and 50-fold lesser cytotoxic activity against MDA-MB231
cell line. It is noteworthy that 18 exhibited antioxidant
activity and 5-LOX inhibition at lower concentrations
than the needed to exert cytotoxic activity.
Experimental
Chemistry
General data
All purchased chemical reagents (Merck or Sigma-
Aldrich) were of the highest available purity and were used
without previous purification. Melting points (mp in °C)
were measured on a Stuart-Scientific SMP3 melting point
apparatus (Stone, United Kingdom) and are uncorrected.
Infrared (IR) spectra were recorded as a thin film (14
and 15) or in KBr disks (11, 12, 13, 16, 17, 18 and 19)
in a Thermo Scientific Nicolet 6700 FT-IR spectrometer
(Massachusetts, USA), and wave numbers are reported in
cm-1. Low resolution mass spectra (MS) were recorded on a
Thermo Scientific, Trace GC Ultra, ISQ mass spectrometer
(Austin, USA) at 70 eV ionizing voltage and are given as
m/z (rel. int., in percentage). High-resolution mass spectra
(HRMS) were recorded on an ExactiveTM Plus Orbitrap
spectrometer (Thermo Scientific, Bremen, Germany) by
infusion, applying a voltage of 5 kV in the positive ionization
mode. The spectra were recorded using full scan mode,
covering a mass range from m/z 140 to 430. The resolution
was set to 140000, and maximum loading time for the ion
cyclotron resonance (ICR) cell was set to 200 ms. 1H and
13C nuclear magnetic resonance (NMR), 2D heteronuclear
single quantum correlation (HSQC) and 2D heteronuclear
multiple bond correlation (HMBC) spectra were recorded in
CDCl3 or DMSO-d6 solutions and referenced to the residual
Table 8. Cytotoxic activities of 18 against cancer and non-cancerous cells
IC50 SD / µM
Compound
MDA-MB-231
67.58 5.76
0.21 0.05
B16-F10
13.96 2.47
0.48 0.08
1.74 0.32
MEF
18
60.66 4.42
0.40 0.07
1.03 0.17
Taxol
Etoposide
1.33 0.28
SD: standard deviation; MDA-MB-231: human breast adenocarcinoma
cell line; B16-F10: mouse melanoma cells; MEF: primary mouse
embryonic fibroblast.
Conclusions
Apparently a 3,4-diOH substitution on the chalcone
skeleton plays a key role in the potentiation capacity of the
β-lactam antibiotic methicillin. For this reason, the chalcone
18 was subjected to cytotoxicity studies reinforcing a
possible use in the control of resistant pathogens in vivo.
These studies showed a low cytotoxicity against MEF cell
line. This clearly suggests that 18 could be useful for the
treatment against resistant bacteria, although further studies
are needed to confirm this possibility. Although chalcones
with 3,4-O-di-hydroxy groups are sometimes considered
interferers of all assays,63 it appears not to be applied here
because the most active chalcones against phytopathogens
and human pathogenic fungi (12, 15 and 17) do not have
this substitution pattern.
1
solvent peaks at d 7.26 and 2.50 ppm for H and d 77.0
and 39.5 ppm for 13C, respectively, on a Bruker Avance
400 NMR spectrometer (Rheinstetten, Germany) operating
at 400.1 MHz for H and 100.6 MHz for 13C. Chemical
1
shifts are reported as d (ppm), and coupling constants (J) are
given in Hz. Silica gel (200-400 mesh, Merck, Darmstadt,
Germany) was used for column chromatography and HF-254
silica gel plates for thin layer chromatography (TLC). TLC
spots were detected both under a UV lamp and by heating
after drenching in 10% H2SO4 in H2O. Antioxidant effects
were determined in a Thermo Scientific Multiskan GO
96-well plate photometer (Vantaa, Finland).
In both cases, bacteria and fungi, the sensitivity for
each molecule can vary, not only by cellular structural
characteristics such as wall and plasma membrane, but also
by differences in their mechanisms of tolerance to stress
and detoxification.64
General experimental procedure
Due to the versatility of biological properties
showed here to this kind of compounds, simple and safe
polyoxygenated chalcones not only have potential uses in
different biological areas, whose specificity or action target
depends largely on their functional groups, but also they
would help us to fight resistant pathogens to commercial
antibiotics through the specific re-sensitization.
General procedure in alkaline condition
To a solution of acetophenone (1 mol equiv.) and
appropriate benzaldehyde (1 mol equiv.) in methanol
(5 mL), KOH (4 mol equiv.) was added in one portion. The
mixture was warmed to 50 °C and stirred until completion
of reaction (monitored by TLC). Then the solvent was
removed under reduced pressure, poured into ice water and

12
Antimicrobial, Anti-Inflammatory and Antioxidant Activities of Polyoxygenated Chalcones
J. Braz. Chem. Soc.
neutralized with aqueous 10% HCl solution. The residue
was extracted with ethyl acetate (15 mL × 3 times). The
combined organic layer was washed with brine, dried
over anhydrous sodium sulfate and filtered; the solvent
was evaporated under reduced pressure. The mixture was
subjected to silica gel flash column chromatography (ethyl
acetate/hexanes as the mobile phase) to yield the products.
The fractions of the product were dried and recrystallized
from ethanol to obtain pure chalcones.
1-(2,5-Dimethoxyphenyl)-3-(4-hydroxy-3-methoxyphenyl)-
prop-2-en-1-one (12)
It was obtained from 1-(2,5-dimethoxyphenyl)
ethanone (2, 0.5 g, 2.78 mmol) and 4-hydroxy-
3-methoxybenzaldehyde (8, 0.426 g, 2.8 mmol) in 10 mL
of methanol and KOH (0.83 g, 14.8 mmol), stirring for
2 h as described above. mp 96-98 °C; IR (KBr) ν / cm-1
3391, 3067, 3002, 2941, 2836, 1650, 1577, 1513; MS
m/z (%): 314 (100), 299 (25), 283 (19), 177 (95), 137
(83); HRMS m/z, observed: 315.1227; C18H19O5 [M + H]+
1
General procedure in acid condition
requires: 315.1232; H NMR (400 MHz, CDCl3) d 7.55
To a stirred mixture of acetophenone (1 mol equiv.)
and benzaldehyde (1 mol equiv) in absolute ethanol
(5 mL), thionyl chloride (0.5 mL, 3 mol equiv.) was added
dropwise and the reaction was monitored by TLC. After
standing for 16 h, ice water (30 g approximately) was
added to the reaction mixture in order to decompose the
excess of SOCl2. Then, ethyl acetate (30 mL) was added
and the organic layer was separated, and a new extraction
with ethyl acetate was performed. The pooled organic
solutions were dried over anhydrous sodium sulfate
and filtered; the solvent was evaporated under reduced
pressure. The mixture was subjected to silica gel flash
column chromatography (ethyl acetate/hexanes as the
mobile phase) to yield the products. Solid chalcones were
recrystallized from ethanol.
(d, 1H, J 15.8 Hz, 2-CH=3-CH), 7.24 (d, 1H, J 15.8 Hz,
2-CH=3-CH), 7.18-7.16 (m, 2H, 1-ArH-6’, 3-ArH-6), 7.10
(d, 1H, J 1.6 Hz, 3-ArH-2), 7.04 (dd, 1H, J 8.9, 3.1 Hz,
1-ArH-4), 6.97-6.94 (m, 2H, 1-ArH-3, 3-ArH-5), 5.96
(s, 1H, 3-ArOH-4), 3.95 (s, 3H, ArOCH3), 3.87 (s, 3H,
ArOCH3), 3.83 (s, 3H, ArOCH3); 13C NMR (100 MHz,
CDCl3) d 55.8 (ArOCH3), 55.9 (ArOCH3), 56.6 (ArOCH3),
110.0 (3-ArCH-2), 113.4 (1-ArCH-3’), 114.4 (3-ArCH-6),
114.8 (3-ArCH-5), 118.6 (1-ArCH-4), 123.2 (1-ArCH-6),
124.7 (2-CH=3-CH), 127.6 (1-ArC-1), 130.0 (3-ArC-1),
144.1 (2-CH=3-CH), 146.7 (3-ArC-3), 148.1 (3-ArC-4),
152.3 (1-ArC-2), 153.6 (1-ArC-5), 192.8 (1-C=O).
1-(3,4,5-Trimethoxyphenyl)-3-(4-hydroxy-3-methoxy-
phenyl)-prop-2-en-1-one (13)
All structures were confirmed by IR and NMR spectra
as discussed below.
It was obtained from 1-(3’,4’,5’-trimethoxyphenyl)
ethanone (1, 0.5 g, 2.4 mmol) and 4-hydroxy-
3-methoxybenzaldehyde (8, 0.361 g, 2.4 mmol) in 5 mL
of ethanol and 0.5 mL of SOCl2 (0.82 g, 6.9 mmol), stirring
for 16 h as described above. mp 88-91 °C (lit.45 75-76 °C);
IR (KBr) ν / cm-1 3512, 3429, 3086, 2994, 2963, 2941,
2839, 1647, 1586, 1561, 1516; MS m/z (%): 344 (100), 329
(65), 313 (29), 145 (28); 1H NMR (400 MHz, CDCl3) d 7.75
(d, 1H, J 15.6 Hz, 2-CH=3-CH), 7.32 (d, 1H, J 15.6 Hz,
2-CH=3-CH), 7.30 (s, 2H, 1-ArH-2,6), 7.25 (dd, 1H,
J 9.6, 1.5 Hz, 3-ArH-6), 7.12 (d, 1H, J 1.5 Hz, 3-ArH-2),
6.97 (d, 1H, J 8.2 Hz, 3-ArH-5), 5.97 (s, 1H, 3-Ar-OH-4),
3.96 (s, 3H, 3-ArOCH3-3), 3.95 (s, 6H, 1-ArOCH3-3,5),
Physical data of chalcones
1-(3,4,5-Trimethoxyphenyl)-3-(3,4-dihydroxyphenyl)-prop-
2-en-1-one (11)
It was obtained from 1-(3’,4’,5’-trimethoxyphenyl)
ethanone(1, 0.5g, 2.4mmol)and3,4-dihydroxybenzaldehyde
(7, 0.327 g, 2.4 mmol) in 5 mL of ethanol and 0.5 mL of
SOCl2 (0.82 g, 6.9 mmol), stirring for 16 h as described above.
mp 151-153 °C (lit.65 158-159 °C); IR (KBr) ν / cm-1 3331,
2942, 1645, 1586, 1558; HRMS m/z, observed: 331.1178;
C18H19O6 [M + H]+ requires: 331.1182; 1H NMR (400 MHz,
CDCl3) d 7.75 (d, 1H, J 15.5 Hz, 2-CH=3-CH), 7.32 (d,
1H, J 15.5 Hz, 2-CH=3-CH), 7.27 (s, 1H, 3-ArH-2), 7.25
(s, 2H, 1-ArH-2,6), 7.14 (dd, 1H, J 8.2, 1.8 Hz, 3-ArH-6),
6.94 (d, 1H, J 8.2 Hz, 3-ArH-5), 6.81 (s, 1H, ArOH-3),
6.59 (s, 1H, ArOH-4), 3.96 (s, 3H, 1-ArOCH3-4), 3.94 (s,
13
3.94 (s, 3H, 1-ArOCH3-4); C NMR (100 MHz, CDCl3)
d 56.0 (ArOCH3), 56.4 (1-ArOCH3-3,5), 61.0 (ArOCH3),
106.1 (1-ArCH-2,6), 110.4 (3-ArCH-2), 114.9 (3-ArCH-5),
119.6 (2-CH=3-CH), 123.0 (3-ArCH-6), 127.5 (3-ArC-1),
133.8 (1-ArC-1), 142.3 (1-ArC-4), 145.2 (2-CH=3-CH),
146.8 (3-ArC-3), 148.3 (3-ArC-4), 153.1 (1-ArC-3,5),
189.5 (1-C=O).
13
6H, 1-ArOCH3-3,5); C NMR (100 MHz, CDCl3) d 56.4
(1-ArOCH3-3,5), 61.0 (1-ArOCH3-4), 106.1 (1-ArCH-2,6),
115.0 (3-ArC-2), 115.6 (3-ArCH-5), 119.3 (2-CH=3-CH),
122.8 (3-ArCH-6), 127.7 (3-ArC-1), 133.5 (1-ArC-1), 142.5
(1-ArC-4), 144.2 (3-ArC-3), 145.9 (2-CH=3-CH), 147.1
(3-ArC-4), 153.1 (1-ArC-3,5), 190.3 (1-C=O).
1-(2,4-Dimethoxyphenyl)-3-(4-hydroxy-3-methoxyphenyl)-
prop-2-en-1-one (14)
It was obtained from 1-(2,4-dimethoxyphenyl)ethanone
(3, 1.0 g, 5.6 mmol) and 4-hydroxy-3-methoxybenzaldehyde

Vol. 00, No. 00, 2018
Vásquez-Martínez et al.
13
(8, 0.93 g, 6.1 mmol) in 5 mL of ethanol and 0.81 mL of
SOCl2 (1.33 g, 11.1 mmol), stirring for 1 h as described
above. Pale yellow oil (lit.66 sticky-solid); IR (film) ν / cm-1
3370, 2940, 2840, 1648, 1601, 1512; MS m/z (%): 314
(400 MHz, CDCl3) d 7.73 (d, 1H, J 15.5 Hz, 2-CH=3-CH),
7.68 (dd, 1H, J 8.4, 1.8 Hz, 1-ArH-6), 7.62 (d, 1H,
J 1.7 Hz, 3-ArH-6), 7.40 (d, 1H, J 15.5 Hz, 2-CH=3-CH),
7.23 (dd, 1H, J 8.2, 1.6 Hz, 3-ArH-6), 7.12 (d, 1H, J 1.5 Hz,
3-ArH-2), 6.95-6.92 (m, 2H, 1-ArH-5, 3-ArH-5), 5.95
(s, 1H, 3-ArOH-4), 3.97 (m, 9H, ArOCH3); 13C NMR
(100 MHz, CDCl3) d 56.0-56.1 (ArOCH3), 109.9
(1-ArCH-5), 110.2 (3-ArCH-2), 110.8 (1-ArCH-2), 114.8
(3-ArCH-5), 119.4 (2-CH=3-CH), 122.8 (1-ArC-6), 123.0
(3-ArC-6), 127.6 (1-ArC-1), 131.5 (3-ArC-1), 144.3
(2-CH=3-CH), 146.8 (3-ArC-4), 148.1 (3-ArC-3), 149.2
(1-ArC-3), 153.1 (1-ArC-4), 188.7 (1-C=O).
1
(100), 299 (49), 178 (90), 165 (98), 137 (70); H NMR
(400 MHz, CDCl3) d 7.71 (d, 1H, J 8.7 Hz, 1-ArH-6), 7.60
(d, 1H, J 15.7 Hz, 2-CH=3-CH), 7.33 (d, 1H, J 15.7 Hz,
2-CH=3-CH), 7.15 (dd, 1H, J 8.2, 1.6 Hz, 3-ArH-6),
7.06 (d, 1H, J 1.6 Hz, 3-ArH-2), 6.92 (d, 1H, J 8.2 Hz,
3-ArH-5), 6.54 (dd, 1H, J 8.6, 2.1 Hz, 1-ArH-5), 6.48 (d,
1H, J 2.1 Hz, 1-ArH-3), 6.22 (s, 1H, 3-ArOH-4), 3.90 (s,
3H, 3-ArOCH3-3), 3.88 (s, 3H, 1-ArOCH3), 3.85 (s, 3H,
1-ArOCH3); 13C NMR (100 MHz, CDCl3) d 55.5 (ArOCH3),
55.7 (ArOCH3), 55.8 (ArOCH3), 98.6 (1-ArCH-3), 105.0
(1-ArCH-5), 110.2 (3-ArCH-2), 114.8 (3-ArCH-5), 122.2
(1-ArC-1), 122.7 (3-ArCH-6), 124.8 (2-CH=3-CH), 127.8
(3-ArC-1), 132.6 (1-ArCH-6), 142.8 (2-CH=3-CH), 146.7
(3-ArC-3), 147.8 (3-ArC-4), 160.1 (1-ArC-OCH3), 163.9
(1-ArC-OCH3), 190.9 (1-C=O).
1-Phenyl-3-(4-hydroxy-3-methoxyphenyl)-prop-2-en-1-one
(17)
It was obtained from acetophenone (5, 0.48 g, 4 mmol)
and 4-hydroxy-3-methoxybenzaldehyde (8, 0.502 g,
3.3 mmol) in 8 mL of ethanol and 0.24 mL of SOCl2 (0.39 g,
3.3 mmol), stirring for 6 h as described above. mp 91-92 °C
(lit.69 93 °C); IR (KBr) ν / cm-1 3322, 3001, 2938, 2835,
1655, 1583, 1564, 1525, 1454, 1420, 1369, 1248; MS m/z
(%): 254 (100), 237 (22), 223 (20), 165 (35), 145 (48), 124
(60); 1H NMR (400 MHz, CDCl3) d 8.01 (d, 2H, J 7.3 Hz,
1-ArH-2,6), 7.75 (d, 1H, J 15.6 Hz, 2-CH=3-CH), 7.60-7.56
(m, 1H, 1-ArCH-4), 7.52-7.48 (m, 2H, 1-ArH-3,5), 7.38
(d, 1H, J 15.6 Hz, 2-CH=3-CH), 7.22 (dd, 1H, J 8.2,
1.4 Hz, 3-ArH-6), 7.13 (d, 1H, J 1.4 Hz, 3-ArH-2), 6.96
(d, 1H, J 8.2 Hz, 3-ArH-5), 5.97 (s, 1H, 3-ArOH-4), 3.96
(s, 3H, 3-ArOCH3-3); 13C NMR (100 MHz, CDCl3) d 56.0
(3-ArOCH3-3), 110.0 (3-ArCH-2), 114.9 (3-ArCH-5), 119.8
(2-CH=3-CH), 123.4 (3-ArCH-6), 127.5 (3-ArC-1), 128.4
(1-ArCH-3,5), 128.6 (1-ArCH-2,6), 132.6 (1-ArCH-4),
138.5 (1-ArC-1), 145.2 (2-CH=3-CH), 146.8 (3-ArC-3),
148.3 (3-ArC-4), 190.7 (1-C=O).
1-(2,5-Dimethoxyphenyl)-3-phenyl-prop-2-en-1-one (15)
It was obtained from 1-(2’,5’-dimethoxyphenyl)
ethanone (2, 2.0 g, 11.1 mmol) and benzaldehyde (9,
1.3 g, 12.2 mmol) in 10 mL of methanol and KOH (2.5 g,
44.4 mmol), stirring for 2 h as described above.Yellow oil
(lit.67 yellow oil); IR (film) ν / cm-1 3060, 3001, 2942, 2911,
2835, 1658, 1594, 1575; MS m/z (%): 268 (100), 253 (15),
237 (25), 177 (60), 165 (70), 151 (61), 103 (65); 1H NMR
(400 MHz, CDCl3) d 7.65 (d, 1H, J 15.8 Hz, 2-CH=3-CH),
7.61-7.59 (m, 2H, 3-ArH-2,6), 7.42 (d, 1H, J 15.8 Hz,
2-CH=3-CH), 7.40-7.38 (m, 3H, 3-ArH-3,4,5), 7.19 (d, 1H,
J 3.1 Hz, 1-ArH-6), 7.03 (dd, 1H, J 9.0, 3.1 Hz, 1-ArH-4),
6.94 (d, 1H, J 9.0 Hz, 1-ArH-3), 3.86 (s, 3H, ArOCH3),
3.81 (s, 3H,ArOCH3); 13C NMR (100 MHz, CDCl3) d 55.8
(ArOCH3), 56.5 (ArOCH3), 113.4 (1-ArCH-6), 114.4
(1-ArC-3), 119.2 (2-CH=3-CH), 126.9 (1-ArCH-4), 128.4
(3-ArCH-2,6), 128.8 (3-ArCH-3,4,5), 129.6 (3-ArC-1),
130.2 (1-ArC-OCH3), 135.1 (1-ArC-OCH3), 143.2
(2-CH=3-CH), 152.6 (1-ArC-2), 153.6 (1-ArC-5), 192.4
(1-C=O).
1-Phenyl-3-(3,4-dihydroxyphenyl)-prop-2-en-1-one (18)
It was obtained from acetophenone (5, 0.5 g, 4.2 mmol)
and 3,4-dihydroxybenzaldehyde (7, 0.58 g, 4.2 mmol) in
5 mL of ethanol and 0.31 mL of SOCl2 (0.5 g, 4.2 mmol),
stirring for 16 h as described above. mp 198-200 °C dec.
(lit.70 200-201 °C); IR (KBr) ν / cm-1 3482, 3304, 1647,
1582, 1563, 1441, 1365, 1365, 1284; MS m/z (%): 240
(100), 223 (25), 163 (14), 110 (21), 77 (23); HRMS
m/z, observed: 241.0861; C15H13O3 [M + H]+ requires:
1-(3,4-Dimethoxyphenyl)-3-(4-hydroxy-3-methoxyphenyl)-
prop-2-en-1-one (16)
It was obtained from 1-(3’,4’-dimethoxyphenyl)
ethanone (4, 0.5 g, 2.8 mmol) and 4-hydroxy-
3-methoxybenzaldehyde (8, 0.42 g, 2.8 mmol) in 5 mL of
ethanol and 0.41 mL of SOCl2 (0.67 g, 5.6 mmol), stirring for
2 h as described above. mp 127-128 °C (lit.68 128-129 °C);
IR (KBr) ν / cm-1 3341, 3078, 2992, 2930, 2835, 1643,
1585, 1557, 1510, 1438, 1418, 1267; MS m/z (%): 314
1
241.0865; H NMR (400 MHz, DMSO-d6) d 9.42 (br
s, 2H, 3-ArOH-3,4), 8.09 (d, 2H, J 7.5 Hz, 1-ArH-3,5),
7.65-7.62 (m, 1H, 1-ArH-4), 7.60 (s, 2H, 2-CH=3-CH),
7.56-7.52 (m, 2H, 1-ArH-2,6), 7.25 (s, 1H, 3-ArH-2), 7.18
(d, 1H, J 8.1Hz, 3-ArH-6), 6.80 (d, 1H, J 8.1 Hz, 3-ArH-5);
13C NMR (100 MHz, DMSO-d6) d 115.5 (3-ArCH-2), 115.7
(3-ArCH-5), 118.4 (2-CH=3-CH), 122.2 (3-ArCH-6), 126.2
1
(100), 283 (65), 207 (90), 165 (58), 145 (39); H NMR

14
Antimicrobial, Anti-Inflammatory and Antioxidant Activities of Polyoxygenated Chalcones
J. Braz. Chem. Soc.
(3-ArC-1), 128.3 (1-ArCH-3,5), 128.7 (1-ArCH-2,6), 132.7
(1-ArCH-4), 138.0 (1-ArC-1), 145.0 (2-CH=3-CH), 145.6
(3-ArC-3), 148.8 (3-ArC-4), 189.0 (1-C=O).
DMSO (final concentration of ≤ 2.5%) served as negative
controls. The MIC was defined as the lowest concentration
of compounds resulting in the complete inhibition of visible
growth.
1-(2,4-Dihydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)-prop-
2-en-1-one (19)
Checkerboard dilution test
Itwasobtainedfrom1-(2’,4’-dimethoxyphenyl)ethanone
(3, 0.5 g, 3.3 mmol) and 3,4,5-trimethoxybenzaldehyde
(10, 0.65 g, 3.3 mmol) in 6 mL of methanol and KOH
(2 g, 35.6 mmol), stirring for 2 h as described above.
mp 181-183 °C (lit.71 188-190 °C); IR (KBr) ν / cm-1
3329, 3016, 2978, 2943, 2833, 1623, 1583, 1506, 1434,
1375, 1333, 1303; MS m/z (%): 330 (48), 194 (37), 181
The type of interaction of the combination of chalcones
11 and 18 with methicillin was investigated with the
checkerboard method.73 Briefly, the uppermost row (A)
of a 96-well microtiter plate contained substance X in
a concentration of about four times the expected MIC
of the microorganism examined. Each following row
(B-H) contained half the concentration of the previous
one. The same procedure was carried out along the
columns (1-12) with substanceY. So, each well contained
a unique combination of the two substances (X and Y).
At last 100 µL of Mueller-Hinton broth containing about
105 CFU mL-1 were added to the wells and incubated
at 37 °C for 24 h. The concentrations of the first wells
without visible growth along the stepwise boundary
between inhibition and growth were used to calculate the
fractional inhibitory concentration index (FICI) as follows:
FICI = fractional inhibitory concentration of A (FICA) +
fractional inhibitory concentration of B (FICB) = (MIC of
drug A in combination / MIC of drug A alone) + (MIC of
drug B in combination / MIC of drug B alone). FICI was
interpreted as follows: synergy, < 0.5; partial synergy,
0.5-0.75; additive effect, 0.76-1.0; indifference, 1.0-4.0;
and antagonism, > 4.0.74
1
(100); H NMR (400 MHz, DMSO-d6) d 13.51 (s, 1H,
1-ArOH-2), 10.75 (s, 1H, 1-ArOH-4), 8.23 (d, 1H, J 9.0 Hz,
1-ArH-6), 7.91 (d, 1H, J 15.4 Hz, 2-CH=3-CH), 7.75 (d,
1H, J 15.3 Hz, 2-CH=3-CH), 7.23 (s, 2H, 3-ArCH-2,6),
6.44 (dd, 1H, J 8.8, 2.3 Hz, 1-ArH-5), 6.29 (d, 1H,
J 2.3 Hz, 1-ArH-3), 3.86 (s, 6H, 3-ArOCH3-3,5), 3.71 (s,
3H, 3-ArOCH3-4); 13C NMR (100 MHz, DMSO-d6) d 56.1
(3-ArOCH3-3,5), 60.1 (3-ArOCH3-4), 102.6 (1-ArCH-3),
106.7 (3-ArCH-2,6), 108.2 (1-ArCH-5), 113.0 (1-ArC-1),
120.2 (2-CH=3-CH), 130.1 (3-ArC-1), 133.2 (1-ArCH-6),
139.8 (3-ArC-4), 144.2 (2-CH=3-CH), 153.1 (3-ArC-3,5),
165.2 (1-ArC-4), 165.9 (1-ArC-2), 191.5 (1-C=O).
Cytotoxicity assays
In vitro antibacterial activity assays, human pathogens
Minimum inhibitory concentration assay
In vitro antifungal activity assays against human pathogens
Two clinical isolates of methicillin resistant S. Aureus
(622-4 and 97-7), and one clinical isolate of E. coli 33.1,
kindly donated by Dr Marcela Wilkens from Universidad
de Santiago de Chile (Chile) were used, and as control
strains S. aureus (NCTC8325-4) and E. coli (ATCC25922)
were used. The antimicrobial activities of chalcones against
strains E. coli (ATCC25922), E. coli multi-resistant (33.1),
S. aureus (NCTC8325-4) and methicillin-resistant S. aureus
97-7, were determined using the microdilution method
established by CLSI.72 Briefly, stock solutions (5 mg mL-1)
of compounds in DMSO were diluted in Mueller-Hinton
broth (MHB) to the different two-fold assay concentrations.
The final concentration of DMSO was ≤ 2.5% and does not
affect the microbial growth. The obtained solution was then
added to MHB, and serially two-fold diluted (in a 96-well
microplate). 100 µL of inoculum 1.5 × 106 colony forming
unit (CFU) mL-1, prepared in MHB, was then added. The
plates were sealed with a tight-fitting plastic cover, then
incubated at 37 ºC for 18 h. The assay was repeated three
times. Wells containing MHB, 100 µL of inoculum and
Microorganisms and media
For the antifungal evaluation, standardized strains from
the American Type Culture Collection (ATCC, Rockville,
MD, USA), C. albicans ATCC 10231 and C. neoformans
ATCC 32264, were used. Strains were grown on
Sabouraud-chloramphenicol agar slants for 48 h at 30 °C
and sub-cultured every 15 days to prevent pleomorphic
transformations. Inocula were obtained according to
reported procedures59 and adjusted to 1-5 × 103 CFU mL-1.
Fungal growth inhibition
The fungal growth inhibition percentage was determined
with broth microdilution techniques performed in 96-well
microplates according to the CLSI reference method for
broth dilution antifungal susceptibility testing of yeasts,
approved standard M27-A3.59 For the assay, compound-
test wells (CTWs) were prepared with stock solutions of
each compound in DMSO (maximum concentration 1%),
diluted with Roswell Park Memorial Institute (RPMI)-1640

Vol. 00, No. 00, 2018
Vásquez-Martínez et al.
15
medium, to final concentrations range 250-3.9 µg mL-1.
An inoculum suspension (100 µL) was added to each well
(final volume in the well: 200 µL). A growth control well
(GCW) (containing medium, inoculum, and the same
amount of DMSO used in a CTW, but compound-free)
and a sterility control well (SCW) (sample, medium, and
sterile water instead of inoculum) were included for each
fungus tested. Microtiter trays were incubated in a moist,
dark chamber at 30 °C for 48 h for both yeasts. Microplates
were read in a VERSA Max microplate reader (Molecular
Devices, Sunnyvale, CA, USA). Amphotericin B (Sigma-
Aldrich, St. Louis, USA) was used as positive control.
Tests were performed in triplicate. Reduction of growth for
each compound concentration was calculated as follows:
inhibition(%) = 100 (OD405 CTW – OD405 SCW) / (OD405
GCW – OD405 SCW), where OD405 is the optical density
at 405 nm. The concentration of each compound that
produces 50% fungal growth reduction (IC50) was taken
as the MIC endpoint.
experiment (second kinetic assay) was conducted; this
experiment involved taking inoculum from the first kinetic
assay and adding it to MH in a new 96-well microplate,
which was then cultured for 7 h. The aim of this second
culture (second kinetic assay) was to observe and quantify
the ability of bacterial cultures to recover from the cytotoxic
effect caused by the compounds, which is evidenced by
the presence or absence of bacterial growth. This second
experiment allows a more accurate conclusion to be
reached: if the bacterial culture’s growth declines with
respect to the culture of the first kinetic assay, then it is
related to a bactericidal property. If, on the other hand, the
growth continues in line with that of the culture of first
kinetic assay, then it is related to a bacteriostatic property.
In vitro fungicidal activity assays, phytopathogens
A virulent isolate of B. cinerea obtained from
naturally infected grape berries79 was prepared and
maintained by placing it on potato dextrose agar (PDA)
at 5 °C. P. cinnamomi was kindly provided by the
National Institute of Agricultural Research (INIA, La
Platina, Santiago, Chile). The fungus G. fujikuroi was
kindly provided by Dr B. Fraga, Natural Products and
Agrobiology Institute, CSIC, Canary Islands, Spain. The
agar dilution technique was used to evaluate the effect
of the chalcones on the growth of P. cinnamomi,80,81
G. fujikuroi82 and B. cinerea.83,84 Fungicidal activity assays
of these compounds were performed in microcultures by
growing the fungi in sterile Petri dishes (35 mm) at a final
volume of 2 mL medium containing different compound
concentrations. Clarified V8 (Campbell Soup) medium
containing metalaxyl85 for P. cinnamomi or PDA medium
for B. cinerea and G. fujikuroi with captan86,87 was used
as the positive control (C+), and without fungicide as the
negative control (C−). The medium in each slot was then
inoculated with a small block (4 mm) of clarified V8 or
PDA medium containing fungal hyphae excised from the
edge of an actively growing culture. The fungal growth
was monitored at different times (24-72 h). To determine
the growth of mycelium in the agar plate, the surface of
the mycelium was measured using Sigma Scan Pro 5
software.88 The final results are expressed as percent growth
inhibition, calculated according to the control without
fungicide. Each treatment was independently performed
in triplicate.
In vitro antibacterial activity assays, phytopathogens
Liquid-dilution methods were used to evaluate the
effects of the compounds on the growth of E. carotovora
(NCPPB 312), A. tumefaciens (strain C58C1) and
P. syringae (NCPPB 281). Bacteria were grown in sterile
tubes with 10 mL of Mueller-Hinton (MH) medium and
incubated at 27 °C for 12 h with shaking, to produce an
initial culture. The antimicrobial activity was evaluated by
observing the growth response of both microorganisms in
samples with different concentrations of the compounds.75-77
All assays were performed on sterile 96-well microplates
with a final volume of 200 µL containing MH broth
inoculated with 1 µL aliquots of bacterial suspension
(105-106 CFU mL-1, initial culture) in the presence of
different concentrations of test compounds (3.9, 7.8, 15.6,
31.3, 62.5, 125 and 250 µM). MH was used as the negative
control (C–), and MH with streptomycin78 was used as the
positive control (C+). The plates were incubated for 7 h at
27 °C. Bacterial growth was monitored by measuring the
optical density at 595 nm every hour with a microplate
reader. All tests were performed in ten repetitions for each
microorganism evaluated. Bacterial growth was shown
as the arithmetic mean expressed in terms of the negative
control (100% growth). The lowest concentration of the
compound preventing the appearance of turbidity was
considered to be the MIC.
The first experiment (first kinetic assay) in which the
compound was exposed to the bacterial cultures in MH was
carried out over a period of 6 h. Subsequently, to determine
the minimal bactericidal concentration (MBC), a second
Statistics
A one-way analysis of variance (ANOVA) was
performed to identify significant differences among the

16
Antimicrobial, Anti-Inflammatory and Antioxidant Activities of Polyoxygenated Chalcones
J. Braz. Chem. Soc.
treatment and control groups. Tukey’s honest significant
difference test was applied to compare the means of every
treatment against the control and simultaneously establish
their significance (p < 0.05). All data are presented as
mean standard deviation (S.D.).
ranked cluster analysis, resulting in conformations with the
highest overall binding energy (most negative Gibbs free
energy binding value, –ΔG).
General procedure to determine the DPPH radical
scavenging activity
In vitro 5-LOX enzyme assay
The radical scavenging activity of the chalcones towards
the DPPH radical was measured as described,93 adapted to
a screen of 96-well plates. Briefly, stock solutions of each
compound were prepared in methanol at 1 mM concentration
(10 mL). Dilutions (1-200 µM) were prepared from the
stock solutions. Methanol (90 µL), each dilution (150 µL),
and DPPH (60 µL, Sigma-Aldrich, St. Louis, USA) in
methanol (0.5 mM), resulting in a final concentration of
0.1 mM DPPH, were added in a 96-well plate. Methanol
was used as the blank sample. The mixtures were left for
30 min at room temperature, and the absorbances were
then measured at 517 nm. Trolox was used as the standard
antioxidant. The radical scavenging activity was calculated
as follows: Inhibition(%) = [(blank absorbance – sample
absorbance) / blank absorbance] × 100. The mean of three
IC50 (concentration causing 50% inhibition) values for each
compound was determined graphically.
The commercially available enzyme 5-hLOX by
Cayman Chemicals Inc. (Ann Arbor, MI, USA), was
diluted (1:500) in the assay buffer (4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES) 50 mM,
ethylenediamine tetraacetic acid (EDTA) 2 mM, adenosine
triphosphate (ATP) 10 µM and CaCl2 10 µM at pH 7.5)
and mixed with 10 µM of H2DCFDA dye the reaction
mixture and incubated for 15 min in the assay plate. Later,
280 µL of buffer and 10 µM of inhibitor were added per
well, and finally incubated for 30 min at 37 °C. Finally,
the reaction was started by adding a suitable concentration
of arachidonic acid (0.5 µM) and the fluorescence was
read in a multimode detector Synergy™ HT Multi-Mode
Microplate Reader (Biotek, Bad Friedrichshall, Germany)
at 480 nm excitation/520 nm emission after an incubation
time of 1 h at room temperature. The IC50 values were
obtained for analysis of oxidation of H2DCFDA dye to
the highly fluorescent 2’,7’-dichloro-fluorescein (DCF)
product and obtained by using the non-linear curve-fitting
program. The kinetic assays to determine the mechanism of
inhibition were done with the best inhibitors, using different
concentrations of the inhibitor (0-1 µM) and substrate
(0.2-5 µM). Experimental conditions were carried out as
described previously. All data were collected in duplicate
and the assays were performed on different days to ensure
reproducibility of the method. The graphics for IC50 and
kinetics studies were performed by GraphPad Prism Demo
V.7.89
In vitro cytotoxic activity of 18
The cell lines used in this work included MDA-MB-231
human breast adenocarcinoma cells, B16-F10 mouse
metastatic melanoma cells and MEF primary mouse
embryonic fibroblast. Cells were maintained in DMEM
high glucose medium (Mediatech, Manassas, VA, USA)
supplemented with 10% (MDA-MB-231 and B16-F10) or
15% (MEF) heat-inactivated fetal bovine serum (HyClone
Laboratories, South Logan, USA), 100 IU mL-1 penicillin
and 100 µg mL-1 streptomycin and maintained at 37 °C
in a 5% CO2 humidified atmosphere. Cell viability was
measured using CyQuant Direct Cell ProliferationAssay
Kit (Life Technologies, Grand Island, NY, USA) following
the manufacturer’s instruction. Briefly, 5000 cells well-1
were seeded onto a flat-bottomed 96-well plate in 200 µL
final volume. Six hours after seeding, the culture medium
was replaced with the medium containing the tested
compounds at concentrations ranging from 0 up to 100 µM
dissolved in DMSO (0.1% final concentration) during
72 h. The concentrations used to calculate the IC50 values
were: 100.0, 30.0, 10.0, 3.0, 1.0, 0.3, 0.1, 0.01 and 0.0 µM.
Untreated cells (medium containing 0.1% DMSO) were
used as controls. At the end of the incubation, 100 µL of
culture medium was removed from each experimental well
and replaced by detection reagent solution provided by the
Docking of chalcones with human 5-LOX
The chalcone structures were built with the Molecular
Operating Environment software.90 ChelpG charges were
obtained at the B3LYP/6-31G** level theory, employing
the Gaussian 09 package.91 Docking of all inhibitors into
the active site of the crystal structure of 5-hLOX (PDB
code: 3O8Y, 2.39 Å resolution) was performed with the
AutoDock4 package,92 using a Lamarckian algorithm and
assuming total flexibility of the inhibitors. The grid maps
were made up to 60 × 60 × 60 points, with a grid-point
spacing of 0.375 Å and the Fe3+ as the center of the grid
map. The AutoTors option was used to define the ligand
torsions, and the docking results were then analyzed by a

Vol. 00, No. 00, 2018
Vásquez-Martínez et al.
17
kit (which is a 1:5 mix of the reagent CyQUANT® direct
nucleic acid stain and the reagent labeled as CyQUANT®
direct background suppressor I). Cells were incubated
for 1 h and fluorescence emission was measured at
535 nm with excitation at 480 nm in a microplate reader
(Infinite 200 PRO, Tecan, Grödig, Austria). At least
four independent experiments were performed for each
concentration. The results from each experiment were
transformed to percentage of controls and the IC50 values
were graphically obtained from the dose-response curves.
The IC50 value was obtained adjusting the dose-response
curve to sigmoidal curves (variable slope), generated using
GraphPad Prisma 6.0 software.94
10. Dong, X.; Zhang,Y.; He, J.-L.; Zhang, S.; Zeng, M.-M.; Chen,
J.; Zheng, Z.-P.; Food Chem. 2016, 197, 589.
11. Nam, N.-H.; Kim, Y.; You, Y.-J.; Hong, D.-H.; Kim, H.-M.;
Ahn, B.-Z.; Eur. J. Med. Chem. 2003, 38, 179.
12. Dong, F.; Jian, C.; Zhenghao, F.; Kai, G.; Zuliang, L.; Catal.
Commun. 2008, 9, 1924.
13. Narender, T.; Reddy, K. P.; Tetrahedron Lett. 2007, 48, 3177.
14. Calloway, N. O.; Green, L. D.; J. Am. Chem. Soc. 1937, 59,
809.
15. Iranpoor, N.; Kazemi, F.; Tetrahedron 1998, 54, 9475.
16. Ren, Y.-M.; Zhang, Z.; Jin, S.; Synth. Commun. 2016, 46, 528.
17. Kakati, D.; Sarma, R. K.; Saikia, R.; Barua, N. C.; Sarma, J.
C.; Steroids 2013, 78, 321.
18. Li, J.-T.;Yang, W.-Z.;Wang, S.-X.; Li, S.-H.; Li, T.-S.; Ultrason.
Sonochem. 2002, 9, 237.
Supplementary Information
19. Rateb, N. M.; Zohdi, H. F.; Synth. Commun. 2009, 39, 2789.
20. Petrov, O.; Ivanova, Y.; Gerova, M.; Catal. Commun. 2008, 9,
315.
Supplementary information is available free of charge
at http://jbcs.sbq.org.br as PDF file.
21. Singh, P.; Anand, A.; Kumar, V.; Eur. J. Med. Chem. 2014, 85,
758.
Acknowledgments
22. Vandeputte, P.; Ferrari, S.; Coste, A. T.; Int. J. Microbiol. 2012,
2012, article ID 713687.
This work was supported by FONDECYT (grant No.
1130924), Dirección General de Investigación, Innovación
y Postgrado (DGIIP) of the Universidad Técnica Federico
Santa María, DICYT-USACH (grant No. 021601VM) and
(grant No. 021641MC) of the Universidad de Santiago
de Chile. S. Z. and M. R. thank the Agencia Nacional de
Promoción Científica y Tecnológica (ANPCyT), grant
PICT 2014-1170.
23. Mahapatra, D. K.; Bharti, S. K.; Asati, V.; Eur. J. Med. Chem.
2015, 101, 496.
24. Williamson, B.; Tudzynski, B.; Tudzynski, P.; Van Kan, J. A.
L.; Mol. Plant Pathol. 2007, 8, 561.
25. Wulff, E. G.; Sørensen, J. L.; Lübeck, M.; Nielsen, K. F.; Thrane,
U.; Jan Torp, J.; Environ. Microbiol. 2010, 12, 649.
26. Sepúlveda-Chavera, G.; Salvatierra-Martínez, R.; Bilbao-Apata,
C.; Sepúlveda-Ramírez, P.; Allende-Castro, M.; Alache-
González, J.; Idesia 2013, 31, 41.
References
27. Gallo, L.; Siverio, F.; Rodríguez-Pérez,A.-M.; Ann. Appl. Biol.
2007, 150, 65.
1. Aksöz, B. E.; Ertan, R.; FABAD J. Pharm. Sci. 2011, 36, 223.
2. Sebti, S.; Solhy, A.; Tahir, R.; Abdelatif, S.; Boulaajaj, S.;
Mayoral, J.A.; García, J. I.; Fraile, J. M.; Kossir,A; Oumimoun,
H.; J. Catal. 2003, 213, 1.
28. Jiménez, E.; Dorta, F.; Medina, C.; Ramírez, A.; Ramírez, I.;
Peña-Cortés, H.; Mar. Drugs 2011, 9, 739.
29. El Khoury, W.; Makkouk, K.; J. Plant Pathol. 2010, 92, S4.35.
30. Yang, C.; Hamel, Ch.;Vujanovic,V.; Gan,Y.; ISRN Ecol. 2011,
2011, article ID 130289.
3. Kumar, S.; Lamba, M. S.; Makrandi, J. K.; Green Chem. Lett.
Rev. 2008, 1, 123.
4. Ndoile, M. M.; van Heerden, F. R.; Beilstein J. Org. Chem.
2013, 9, 1346.
31. Díaz-Tielas, C.; Graña, E.; Reigosa, M. J.; Sánchez-Moreiras,
A. M.; Planta Daninha 2016, 34, 607.
5. Sheng, R.; Lin, X.; Zhang, J.; Chol, K. S.; Huang, W.; Yang,
B.; He Q.; Hu, Y.; Bioorg. Med. Chem. 2009, 17, 6692.
6. Syam, S.; Abdelwahab, S. I.; Al-Mamary, M. A.; Mohan, S.;
Molecules 2012, 17, 6179.
32. Guo, M.; Block, A.; Bryan, C. D.; Becker, D. F.; Alfano, J. R.;
J. Bacteriol. 2012, 194, 5054.
33. Prajapat, R.; Marwal, A.; Jha, P. N.; Int. J. Curr. Microbiol.
Appl. Sci. 2013, 2, 83.
7. Nagaraju, M.; Deepthi, E. G.; Ashwini, C.; Vishnuvardhan, M.
V. P. S.; Nayak, V. L.; Chandra, R.; Ramakrishna, S.; Gawali,
B. B.; Bioorg. Med. Chem. Lett. 2012, 22, 4314.
8. Daskiewicz, J. B.; Comte, G.; Barron, D.; Di Pietro, A.;
Thomasson, F.; Tetrahedron Lett. 1999, 40, 7095.
9. Gall, E. L.; Texier-Boullet, F.; Hamelin, J.; Synth. Commun.
1999, 29, 3651.
34. Moore, L. W.; Pscheidt, J. W.; Pacific Northwest Plant Disease
Management Handbook; Pscheidt, J. W.; Ocamb, C. M., eds.;
Oregon State University: Corvallis, OR, USA, 2018. Available
at https://pnwhandbooks.org/node/408/print, accessed in
September 2018.
35. Lucera, A.; Costa, C.; Conte, A.; Del Nobile, M. A.; Front.
Microbiol. 2012, 3, article 287.

18
Antimicrobial, Anti-Inflammatory and Antioxidant Activities of Polyoxygenated Chalcones
J. Braz. Chem. Soc.
36. http://www.fao.org/fao-who-codexalimentarius/codex-texts/
Lobo, G.; Charris-Charris, J.; Cortés, J. C.; Ribas, J. C.; Devia,
C.; Rodríguez, A. M.; Enriz, R. D.; Bioorg. Med. Chem. 2001,
9, 1999.
maximum-residue-limits/en/, accessed on September 10, 2018.
37. Apak, R.; Ozyürek, M.; Güçlü, K.; Çapanoğlu, E.; J. Agric.
Food Chem. 2016, 64, 1046.
59. Clinical and Laboratory Standards Institute (CLSI); CLSI
Document M27-A3: Reference Method for Broth Dilution
Antifungal Susceptibility Testing ofYeasts; Approved Standard,
3rd ed.; CLSI: Wayne, PA, 2008.
38. Chand, K.; Rajeshwari; Hiremathad, A.; Singh, M.; Santos, M.
A.; Keri, R. S.; Pharmacol. Rep. 2017, 69, 281.
39. Karthikeyan, C.; Moorthy, N. S.; Ramasamy, S.; Vanam, U.;
Manivannan, E.; Karunagaran, D.; Trivedi, P.; Recent Pat. Anti-
Cancer Drug Discovery 2015, 10, 97.
60. Swinne, D.;Watelle, M.; Nolard, N.; Rev. Iberoam. Micol. 2005,
22, 24.
40. Ghosh, J.; Myers, C. E.; Proc. Natl. Acad. Sci. U. S. A. 1998,
95, 13182.
61. Mascayano, C.; Espinosa, V.; Sepúlveda-Boza, S.; Hoobler, E.
K.; Perry, S.; Chem. Biol. Drug Des. 2013, 82, 317.
62. Mascayano, C.; Espinosa, V.; Sepúlveda-Boza, S.; Hoobler, E.
K.; Perry, S.; Diaz, G.; Holman, T. R.; Chem. Biol. Drug Des.
2015, 86, 114.
41. Steinhilber, D.; Fischer, A. S.; Metzner, J.; Steinbrink, S. D.;
Roos, J.; Ruthardt, M.; Maier, T. J.; Front. Pharmacol. 2010,
1, article 143.
42. Chen,Y.; Zhang, H.; Zhou, H. J.; Ji, W.; Min, W.; Cancers 2016,
8, article 40.
63. Aldrich, C.; Bertozzi, C.; Georg, G. I.; Kiessling, L.; Lindsley,
C.; Liotta, D.; Merz Jr., K. M.; Schepartz, A.; Wang, S.; ACS
Cent. Sci. 2017, 3, 143.
43. Reddy, N. P.; Aparoy, P.; Reddy, T. C. M.; Achari, C.; Bioorg.
Med. Chem. 2010, 18, 5807.
64. Haruta, S.; Kanno, N.; Microbes Environ. 2015, 30, 123.
65. Rao, Y. K.; Fang, S.-H.; Tzeng, Y.-M.; Bioorg. Med. Chem.
2009, 17, 7909.
44. Ball, A. K.; Beilstein, K.; Wittmann, S.; Sürün, D.; Saul, M.
J.; Schnütgen, F.; Flamand, N.; Capelo, R.; Kahnt, A. S.; Frey,
H.; Schaefer, L.; Marschalek, R.; Häfner, A.-K.; Steinhilber,
D.; Biochim. Biophys. Acta 2017, 1862, 561.
66. Goswami, P.; Talukdar, M.; Bora, T. C.; Phukan, P.; Sarma, J.
C.; C. R. Chim. 2013, 16, 442.
45. Batovska, D.; Parushev, S.; Stamboliyska, B.; Tsvetkova, I.;
Ninova, M.; Najdenski, H.; Eur. J. Med. Chem. 2009, 44, 2211.
46. Abreu,A. C.; McBain,A. J.; Simões, M.; Nat. Prod. Rep. 2012,
29, 1007.
67. Kumar,V.; Kumar, S.; Hassan, M.;Wu, H.; Thimmulappa, R. K.;
Kumar, A.; Sharma, S. K.; Parmar, V. S.; Biswal, S.; Malhotra,
S. V.; J. Med. Chem. 2011, 54, 4147.
68. Pathak,V.;Ahmad, I.; Kahlon,A. K.; Hasanain, M.; Sharma, S.;
Srivastava, K. K.; Sarkar, J.; Shankar, K.; Sharma, A.; Gupta,
A.; RSC Adv. 2014, 4, 35171.
47. Butts, A.; Krysan, D. J.; PLoS Pathog. 2012, 8, e1002870.
48. Delattin, N.; De Brucker, K.; Vandamme, K.; Meert, E.;
Marchand, A.; Chaltin, P.; Cammue, B. P. A.; Thevissen, K.; J.
Antimicrob. Chemother. 2014, 69, 1035.
69. Mohamed, N.; Hamada, M.; Yousef, N.; Abdo, M.; Molecules
2015, 20, 10468.
49. Tran, T. D.; Do, T. H.; Tran, N. C.; Ngo, T. D.; Huynh, T. N. P.;
Tran, C. D.; Thai, K. M.; Bioorg. Med. Chem. Lett. 2012, 22,
4555.
70. Kakati, D.; Sarma, J. C.; Chem. Cent. J. 2011, 5, article 8.
71. Zhang, J.; Fu, X.-L.;Yang, N.; Wang, Q.-A.; Sci. World J. 2013,
article ID 649485.
50. Pfaller, M. A.; Diekema, D. J.; Clin. Microbiol. Rev. 2007, 20,
133.
72. Clinical and Laboratory Standards Institute (CLSI); CLSI
Document M26-A: Methods for Determining Bactericidal
Activity of Antimicrobial Agents; Approved Guideline; CLSI:
Wayne, PA, USA, 1999.
51. Sato, M.; Tsuchiya, H.; Akagiri, M.; Fujiwara, S.; Fujii, T.;
Takagi, N.; Matsuura, N.; Iinuma, M.; Lett. Appl. Microbiol.
1994, 18, 53.
73. Iten, F.; Saller, R.; Abel, G.; Reichling, J.; Planta Med. 2009,
75, 1231.
52. Batovska, D.; Parushev, St. P.; Slavova, A.; Bankova, V.;
Tsevetkova, I.; Ninova, M.; Najdenski, H.; Eur. J. Med. Chem.
2007, 42, 87.
74. Doern, C. D.; J. Clin. Microbiol. 2014, 52, 4124.
75. Cole, M. D.; Biochem. Syst. Ecol. 1994, 22, 837.
76. Zampini, I. C.;Vattuone, M.A.; Isla, M. I.; J. Ethnopharmacol.
2005, 102, 450.
53. Lathtchev, K. L.; Batovska, D.; Parushev, St. P.; Ubiyvovk, V.
M.; Sibirny, A. A.; Eur. J. Med. Chem. 2008, 43, 2220.
54. Gupta, D.; Jain, D.; J. Adv. Pharm. Technol. Res. 2015, 6,
114.
77. Cos, P.; Vlietinck, A. J.; Berghe, D. V.; Maes, L.; J.
Ethnopharmacol. 2006, 106, 290.
55. Mishra, P.; Sarma, B.; Singhai, P.; Singh, U.; Mycobiology 2007,
35, 72.
78. McManus, P. S.; Stockwell, V. O.; Sundin, G. W.; Jones, A. L.;
Ann. Rev. Phytopathol. 2002, 40, 443.
56. Jayasinghe, L.; Balasooriya, B. A.; Padmini, W. C.; Hara, N.;
Fujimoto, Y.; Phytochemistry 2004, 65, 1287.
57. Nowakowska, Z.; Eur. J. Med. Chem. 2007, 42, 125.
58. López, S. N.; Castelli, M.V.; Zacchino, S.A.; Domínguez, J. N.;
79. Riggoti, S.; Gindro, K.; Richter, H.;Viret, O.; FEMS Microbiol.
Lett. 2002, 209, 169.
80. Bekker, T.; Labuschagne, N.; Kaiser, C.; S. Afr. Avocado Grow.
Assoc. Yearb. 2005, 28, 60.

Vol. 00, No. 00, 2018
Vásquez-Martínez et al.
19
81. Horta, M.; Sousa, N.; Coelho, A. C.; Neves, D.; Cravador, A.;
Physiol. Mol. Plant Pathol. 2008, 73, 48.
Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg,
J. L.; Williams-Young, D.; Ding, F.; Lipparini, F.; Egidi, F.;
Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe,
D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang,
W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa,
J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.;
Vreven, T.; Throssell, K.; Montgomery Jr., J. A.; Peralta, J. E.;
Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K.
N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.;
Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.;
Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.;
Cammi, R.J.; Ochterski, W.; Martin, R. L.; Morokuma, K.;
Farkas, O.; Foresman, J. B.; Fox, D. J.; Gaussian 09, Revision
A.02; Gaussian, Inc., Wallingford, CT, 2016.
82. Azor, M.; Gené, J.; Cano, J.; Sutton, D. A.; Fothergill, A. W.;
Rinaldi, M. G.; Guarro, J.; Antimicrob. Agents Chemother. 2008,
52, 2228.
83. Salzman, R. A.; Tikhonova, I.; Bordelon, B. P.; Hasegawa, P.
M.; Bressan, R. A.; Plant Physiol. 1998, 117, 465.
84. Kirubakaran, S. I.; Begum, S. M.; Ulaganathan, K.; Sakthivel,
N.; Plant Physiol. Biochem. 2008, 46, 918.
85. Zhu, G.; Huang, F.; Feng, L.; Qin, B.; Yang, Y.; Hen, Y.; Lu,
X.; Agric. Sci. China 2008, 7, 831.
86. Meepagala, K. M.; Sturtz, G.; Wedge, D. E.; J. Agric. Food
Chem. 2002, 50, 6989.
87. Alizadeh, H. R.; Sharifi-Tehrani, A.; Hedjaroude, G. A.;
Commun. Agric. Appl. Biol. Sci. 2007, 72, 795.
92. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew,
R. K.; Goodsell, D. S.; Olson, A. J.; J. Comput. Chem. 2009,
16, 2785.
88. Sigma Scan Pro V. 5.0; Systat Software Inc., Chicago, Illinois,
USA, 2008.
89. GraphPad Prism Demo V.7.; GraphPad Software Inc., La Jolla
California, USA, 2017.
93. Osorio, M.; Quiroz, K.; Carvajal, M.;Vergara, A.; Sánchez, E.;
González, C.; Catalán, K.; J. Chil. Chem. Soc. 2016, 61, 3095.
94. GraphPad Prism, version 6.00 for Mac OS X; GraphPad
Software, La Jolla, California, USA, 2015.
90. Molecular Operating Environment (MOE), 2013.08; Chemical
Computing Group ULC, Montreal, QC, Canada, 2018.
91. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.;
Petersson, G.A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich,
A.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.;
Submitted: July 9, 2018
Published online: September 13, 2018
This is an open-access article distributed under the terms of the Creative Commons Attribution License.