Design, synthesis, biological activity and structure-activity relationship studies of chalcone derivatives as potential anti-Candida agents

Article abstract of DOI:10.1038/s41429-018-0048-9

Vulvovaginal candidiasis (VVC) affects millions of women around the world every year. Candida albicans is the most frequently isolated pathogen in women and its rapid ability to develop resistance to first and second line therapies has boosted the search

Full text of DOI:10.1038/s41429-018-0048-9

The Journal of Antibiotics
https://doi.org/10.1038/s41429-018-0048-9
ARTICLE
Design, synthesis, biological activity and structure-activity
relationship studies of chalcone derivatives as potential anti-
Candida agents
1,2
Jéssica T. Andrade1 Felipe R. S. Santos
William G. Lima1 Carla D. F. Sousa1 Lohanna S. F. M. Oliveira3
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Rosy I. M. A. Ribeiro3 Ana J. P. S. Gomes4 Marcelo G. F. Araújo5 José A. F. P. Villar2 Jaqueline M. S. Ferreira1
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Received: 9 November 2017 / Revised: 22 February 2018 / Accepted: 13 March 2018
© The Author(s) under exclusive licence to the Japan Antibiotics Research Association 2018
Abstract
Vulvovaginal candidiasis (VVC) affects millions of women around the world every year. Candida albicans is the most
frequently isolated pathogen in women and its rapid ability to develop resistance to first and second line therapies has
boosted the search for new and effective antifungal agents. In this study, we show the in vitro anti-Candida activity of fifteen
synthetic chalcone analogs and their antifungal potential in an in vivo model of VVC. Chalcone 12 showed potent antifungal
effects, being able to inhibit the growth of Candida spp. at a concentration of 15.6 μg mL−1. In addition, mechanism of
action studies have indicated the ergosterol fungal membrane as the target of this compound. Despite a considerable
antifungal activity, the chalcone 12 showed high cytotoxicity in kidney cells lineages. Moreover, this compound was able to
reduce Candida-associated virulence, impairing yeast–hyphal transition in C. albicans. An in vivo model of VVC showed
that chalcone 12 significantly reduces the fungal load. Taken together, these findings showed that the chalcone 12 is a potent
anti-Candida agent in vitro beyond of contribute to improve the fungal infection in a model of CVV. However, it showed
low selectivity and high toxicity, suggesting molecular modifications to minimize these proprieties.
Introduction
of vaginitis annually [3, 4]. Although most episodes of
symptomatic disease appear as sporadic attacks of acute
VVC, some women have chronic manifestations, and var-
ious patients have shown frequent infections with mani-
festations ranging from four or more episodes for year,
characterizing a board of VVC recurrent (VVCR) [5].
Candida albicans is the major etiological agent in VVC
and VVCR. However, studies have shown an increase in the
prevalence of non-albicans Candida which featured the
species C. glabrata, C. tropicalis, C. krusei, and C. dubli-
niensis [6]. Additionally, there are many problems related to
the currently available drugs and resistance to azoles and
echinocandins has been shown with frequency in C. albi-
cans and non-albicans [7]. In disparity to the steep rise in
Vulvovaginal Candidiasis (VVC) stands out among the
main causes of gynecological disorders worldwide [1]. This
infection is a significant problem, affecting 75% of all
women at least once during their lifetime [2]. In the USA,
almost 10 million women visit physicians for the treatment
These authors contributed equally: Jéssica T. Andrade, Felipe R. S.
Santos, William G. Lima.
Electronic supplementary material The online version of this article
(https://doi.org/10.1038/s41429-018-0048-9) contains supplementary
material, which is available to authorized users.
3
* Jaqueline M. S. Ferreira
Laboratório de Patologia Experimental, Universidade Federal de
jackmaria4@gmail.com
São João del-Rei (UFSJ)-Campus Centro Oeste Dona Lindu,
Divinópolis, Minas Gerais, Brazil
1
Laboratório de Microbiologia Médica, Universidade Federal de
4
Laboratório de Desenvolvimento Farmacotécnico, Universidade
Federal de São João del-Rei (UFSJ)-Campus Centro Oeste Dona
Lindu, Divinópolis, Minas Gerais, Brazil
São João del-Rei (UFSJ)-Campus Centro Oeste Dona Lindu,
Divinópolis, Minas Gerais, Brazil
2
Laboratório de Síntese Orgânica e Nanoestruturas, Universidade
5
Laboratório de Farmacologia, Universidade Federal de São João
del-Rei (UFSJ)-Campus Centro Oeste Dona Lindu,
Divinópolis, Minas Gerais, Brazil
Federal de São João del-Rei (UFSJ)-Campus Centro Oeste Dona
Lindu, Divinópolis, Minas Gerais, Brazil

J. T. Andrade et al.
number of VVC cases for this species, only a few drugs
have been developed over the past 5–6 decades [8].
Chemo-informatic study
In this context, chalcones have been highlighted as an
important class in the search for new antifungal agents.
Chemically known as 1,3-diaryl-2-propen-1-ones, the
chalcones consist of open-chain flavonoids in which the two
aromatic rings are joined by a three-carbon with an α,β-
unsaturated carbonyl system [9]. Naturally occurring chal-
cones and their synthetic analogs display a wide spectrum
of pharmacological activities, such as antibacterial [10] and
antileishmanial, [11], and some studies have described the
antifungal potential of this class [12–15]. However, limited
information is available regarding the antifungal action of
chalcones, and potential applications in in vivo infections
model, especially regarding candidiasis. Thus, the aims of
this paper were to evaluate the anti-Candida spectrum, the
mechanism of action, the effect on virulence factor
(yeast–hyphal transition) and the cytotoxicity in renal cells
lineages, of fifteen synthetic chalcones. Additionally, we
aimed to evaluate the potential utilization of the most active
chalcone in the treatment of VVC.
To calculate the physicochemical (LogP, LogS, Molecular
Weight, Numbers of acceptor and donors groups of
hydrogen bond) and pharmaceutical (Druglikeness) prop-
erties of compounds, chemo-informatics analyses were
employed. The two-dimensional chemical structures of the
chalcones were inserted into DataWarrior 4.2.2 software
and the parameters of interest were calculated [18]. After,
values of molecular weight, LogP, and number of donor and
acceptor groups of hydrogen bonds were compared with the
Lipinsk rules [19]. The descriptor 'respect all' was added to
the compounds that fit at all points. However, if any were
violated, the descriptor 'not respect all' was employed fol-
lowed by the description of the rule in non-compliance.
Cell culture and cytotoxicity assay
Cytotoxicity of active chalcones was evaluated using the
African green monkey kidney epithelium (Vero, ATCC
CCL-81) and Baby Hamster Kidney (BHK-21, ATCC CCL-
10) cells. These were cultured in Dulbecco’s Modified Eagle
supplemented with 10% fetal bovine serum (FBS) and 0.3%
of solution content penicillin, streptomycin, and amphoter-
icin B. Cell cultures were maintained at 35 2 °C in a
humidified atmosphere of 5% CO2. Cells lines were exposed
to the compounds for 48 h and the cell viability was pos-
teriorly quantified by the 3-(4.5-dimethylthiazol-2-yl)-2.5-
diphenyltetrazolium bromide (MTT) colorimetric assay.
Finally, the cytotoxic concentrations for 50% of the cells
(CC50) were calculated by linear regression analysis. [20]
Materials and methods
Synthesis and purification of chalcones
Reagents and solvents were purchased as reagent grade and
used without further purification. ¹H and ¹³C Nuclear
magnetic resonance (NMR) spectra for all chalcones were
acquired in a Bruker AVANCE DRX-400 or Bruker
Avance-600. Chemical shifts were reported as δ (ppm)
downfield from tetramethylsilane (TMS) and the J values
were reported in Hz. Spectra were recorded in chloroform-d
(CDCl3) or dimethylsulfoxide-d6 (DMSO-d6) solutions.
Mass spectra (MS) were recorded on a Bruker Daltonics
Amazon SL (Supplementary material). Preparative chro-
matography was performed using silica gel (230–400 mesh)
as described [16]. Thin layer chromatography (TLC) was
performed using silica gel GF254, with a thickness of 0.25
mm. TLC plates were visualized under ultraviolet light or
stained with iodine vapor or acidic vanillin. All procedures
and spectral data are presented.
Fifteen synthetic chalcones with different structural
modifications were synthesized, characterized and denomi-
nated from 1 to 15. Bromide C was obtained from com-
pound A by a previously described O-alkylation method
[17]. Compound D was synthesized by N-alkylation of
compound C with morpholine in the presence of triethyla-
mine and N, N-Dimethylformamide (DMF) at room tem-
perature for 3 h. Desired morpholinochalcone 15 was
synthesized by Claisen–Schmidt condensation of compound
D with benzaldehyde.
Antifungal activity
Fungal strains
Four fungal strains obtained from the American Type Culture
Collection (ATCC) were used: C. albicans ATCC 10231, C.
glabrata ATCC 2001, C. krusei ATCC 34135, and C. tro-
picalis ATCC 28707. In addition, eight clinical isolates were
provided by Marivalda A. R. Oliveira (Laboratory, Biocentro
Ltda, Divinopolis, Brazil) including three strains of C. albi-
cans and five of C. dubliniensis. According to ATCC speci-
fications, C. albicans ATCC 10231 is resistant to
anidulafungin, voriconazole, itraconazole, fluconazole, and
Ketoconazole. Moreover, C. glabrata ATCC 2001 showed
high value of MIC to azole antifungals [21].
Determination of minimum inhibitory concentrations (MIC)
and minimum fungicidal concentrations (MFC)
Minimum inhibitory concentration (MIC) was determined
by the microdilution method according to document M27-

Chalcones derivatives as anti-Candida agents
A3 of the Clinical and Laboratory Standards Institute [22],
with minor modifications [23]. The fifteen chalcones,
named from 1 to 15, were tested by employing concentra-
tions ranging from 3.9–1000 μg mL−1. Ketoconazole and
Nystatin were included as positive controls, and DMSO 2%
v/v was used as the solvent control. The minimal fungicidal
concentration (MFC), in turn, was determined as the
absence of visible colonies in Sabourad-Dextrose agar
(SDA), as previously described [24]. All tests were per-
formed in triplicate from at least three independent
experiments.
a magnification of ×400. Positive (ketoconazole) and sol-
vent (DMSO 2% v/v) controls were included [27].
Therapeutic effect in a VVC animal model
Preparation of vaginal cream
An oil/water emulsion was obtained according to Bancroft's
Rule, which contained (w/w) 5.0% nonionic emulsifying
wax (PolawaxTM), 3.0% mineral oil, 0.05% butylated
hydroxytoluene, 5.0% glycerol, 0.1% disodium ethylene-
diamine tetraacetic acid (EDTA), 2.0% propylene glycol,
0.2% parabens, and water q.s.p. 100% [28]. The vaginal
cream was prepared for the addition of 0.1, 0.5, and 1.0%
(w/w) of the most activity chalcone dispersed in isopropyl
myristate. Citric acid was added to the emulsion to a final
pH of 4.0–4.5 [29].
Mechanism of action studies
The probable mechanism of action was determined for the
highest activity chalcone using ergosterol exogenous bind-
ing and cytoprotection with sorbitol assays. The MIC of
compounds was determined as described above in the
absence and presence of ergosterol (200 μg mL−1) or sor-
bitol (0.8 M) [25]. Any increase in MIC after ergosterol
addition was considered to indicate that the compound
interferes with the fungal membrane. In the case of an
increase with the supplementation of sorbitol, the com-
pound was considered to be due to changes in the cell wall
[25]. Nystatin and Caspofungin were used as positive
controls in ergosterol and sorbitol assays, respectively.
VVC Model
Female Wistar rats (100–150 g) were maintained in accor-
dance with the institutional guidelines. The protocol was
approved by the Animal Experimentation Ethics Committee
of the Universidade Federal de São João Del-Rei (Protocol
002/2015). To establish a pseudo-estro state, the sub-
cutaneous administration of estradiol cypionate (Pfizer,
Brazil) at the dose of 0.2 mg mL−1 was performed for 24/24
h over 5 days before infection and for 48/48 h from of day
7. Rats were immunosuppressed by the administration of
cyclophosphamide (Sigma-Aldrich, Brazil, 50 mg kg-1 b.
w.). On the first day, 0.1 mL of a viable suspension of C.
albicans ATCC 10231 (5.0 × 107 CFU mL−1) was inocu-
lated into the rat vaginal cavity. Two days after infection
(day 0) the vaginal fungal load was evaluated through
lavage with 0.1 mL of saline, and determined in culture of
SDA supplemented with 0.05% chloramphenicol, with the
results expressed as CFU mL−1. The infection was con-
sidered sufficient if a count for the vaginal lavage cultures
from each rat was 102 CFU mL−1 [30].
Time-kill curve
The kinetics of anti-Candida activity was studied for the
most active chalcone, as previously described [26]. In brief,
tubes with 10 mL Sabouraud-Dextrose broth containing
C. albicans ATCC 10231 (106 CFU mL−1) and different
concentrations of the most active chalcone were incubated
at 35 2 °C. Then, 0.1 mL from these tubes were taken at
different time intervals (0, 2, 4, 6, 8, 10, 12, 24, 36, and 48
h) and inoculated on SDA plates. After, the plates were
incubated at 35 2 °C for 48 h for posterior colonies count,
with the results expressed in CFU mL−1. Ketoconazole and
DMSO (2% v/v) were included as positive and solvent
controls, respectively.
Treatment of animals
Inhibition of yeast-hyphae transition
Wistar females were randomized into seven equal groups
(n = 6 in each): non-infected (1); infected and untreated (2);
infected and treated with clotrimazole 1% cream vaginal
(Medley, Brazil) (3); infected and treated with vaginal
cream containing the most active chalcone at doses of 0.1%
(4), 0.5% (5), and 1% (6); and infected and treated with base
vaginal cream (7). Treatments were administered intravag-
inally once per day for 6 consecutive days. On days 0, 2, 4,
and 6 after infection, the vaginal fungal load was evaluated
to verify the effectiveness of the treatments [31].
The most active chalcone was studied regarding its ability
to inhibit yeast-hyphae transition in C. albicans ATCC
10231. Hyphal induction was conducted by the incubation
of C. albicans ATCC 10231 (103 CFU mL−1) in microplate
content FBS plus different concentrations of chalcone. The
microplates were incubated for 24, 48, and 72 h at 35 2 °C
and the hyphal formation in C. albicans was observed
through a light microscope (Nikon TE 2000-U Eclipse) with

J. T. Andrade et al.
Fig. 1 General route to the synthesis of chalcones 1–15. Reagents and
conditions: (i) PPTS, dichloromethane, DHP, r.t., 24 h; (ii) Aromatics
benzaldehyde, methanol, KOH, r.t., 24 h; (iii) Ethanol, HCl (4 mol L−1),
r.t., 4 h; (iv) 1,4-dibromobutane, K2CO3, acetonitrile, 45 °C, 5 h; (v)
Morpholine, DMF, triethylamine, r.t.,3 h; (vi) Benzaldehyde, metha-
nol, KOH, r.t., 24 h
Statistical analysis
by the nucleophilic substitution reaction of compound C
with morpholine in DMF. Morpholinochalcone (15) was
obtained by Claisen–Schmidt condensation with benzalde-
hyde, similarly to tetrahydropyranyl chalcones. The fifteen
novel chalcones were synthesized with yields ranging
between 70 and 86% (Fig. 1). All the compounds and their
intermediates were successfully characterized by ¹H and ¹³C
NMR and ESI-MS (Supplementary material).
All in vitro tests were performed in triplicate with at least
three independent experiments. For in vivo studies, the
normality was evaluated by Shapiro–Wilk test. An one-way
analysis of variance (ANOVA) was used to demonstrate the
differences between the groups. The Tukey test was used to
compare the results of the treatments and the Dunnett test to
compare the results of the treatment and control [32]. A p-
value less than 0.05 was considered statistically significant.
Fungistatic and fungicidal activity of chalcones
Chalcone 12 presents the best value of MIC and MFC (15.6
μg mL−1) against C. albicans. This activity pattern was also
observed against C. glabrata (MIC 15.6 μg mL−1 and MFC
125 μg mL−1), C. krusei (MIC and MFC 15.6 μg mL−1),
and C. tropicalis (MIC 15.6 μg mL−1 and MFC 31.25 μg
mL−1) (Table 1). In addition, chalcone 12 retained the
antifungal activity against clinical specimens obtained from
vaginal secretion. Three different clinical isolates of C.
albicans (MIC 31.25 μg mL−1), as well as five isolates of
non-albicans Candida (C. dubliniensis, MIC 31.25 μg mL−1)
were susceptible to chalcone 12. For the specimen of C.
albicans called CVV1, for example, the effect was observed
to the same level as ketoconazole (Table 2). Compound 15
was moderately active against C. krusei and C. tropicalis
(MIC 31.25 μg mL−1) (Table 1). The MIC and MFC results
for chalcones 1–7 were described in Tables S1 and S2 in
the supplementary material section.
Results
Synthesis of chalcones
As shown in Fig. 1, the treatment of resacetophenone (A)
with 3,4-Dihydro-2H-pyran (DHP) in dichloromethane and
catalytic amount of pyridinium-p-toluene sulfonate (PPTS)
gave the corresponding compound B. Tetrahydropyranyl
chalcones (1–7) were synthesized from compound B by
Claisen–Schmidt condensation with different aromatic
aldehydes. Dihydroxychalcones (8–14) were obtained by
the treatment of tetrahydropyranyl chalcones in ethanolic
acid media after 4 h at room temperature. Morpholi-
nochalcone (15) was synthesized from compound C, that
was prepared by selective O-alkylation of resacetophenone
with 1,4-dibromobutane. Then, compound D was prepared

Chalcones derivatives as anti-Candida agents
Table 1 Summary of fungistatic and fungicidal effects of eight chalcones evaluated against reference strains (ATCC) of different Candida species
Chalcones
C. albicans ATCC 10231
C. glabrata ATCC 2001
C. krusei ATCC 34135
C. tropicalis ATCC 28707
MICa
MFCa
MICa
MFCa
MICa
MFCa
MICa
MFCa
8
1000
1000
1000
>1000
15.6
1000
1000
250
>1000
>1000
>1000
ND
>1000
1000
>1000
>1000
15.6
1000
1000
125
ND
ND
ND
ND
125
ND
1000
500
>1000
4
1000
>1000
1000
>1000
15.6
ND
ND
>1000
ND
15.6
ND
ND
ND
7.8
>1000
>1000
>1000
>1000
15.6
ND
ND
ND
ND
31.25
ND
ND
500
62.5
2
9
10
11
12
13
14
15
KTZ
NYS
15.6
>1000
1000
>1000
>1000
4
>1000
>1000
31.25
7.8
1000
1000
31.25
31.25
2
125
125
4
4
8
8
All tests were performed in at least three independent experiments
MIC Minimum inhibitory concentration, MFC Minimum Fungicidal Concentration, KTZ Ketoconazole, NYS Nystatin, ND Not determine
a
Results expressed in μg mL−1
Cytotoxicity assay
Chemo-informatics analysis (Table 3) showed that
chalcone 12, which has fungistatic/fungicidal effect, pre-
sented physicochemical characteristics in accordance with
all of the Lipinski's rules (MW: 240.26 g mol−1; cLogP:
2.61; H-donors: 2; H-acceptors: 3), predicting good oral
bioavailability. Druglikeness, which suggests a significant
pharmaceutical potential when greater than 0, was mostly
unsatisfactory, ranging from −14.99 to 0.22. Chalcone 12
presented a considerable druglikeness (0.13); however, it
was considerable inferior to ketoconazole (8.14). Further-
more, all of the evaluated compounds (including the keto-
conazole) were predicted for reduced water solubility,
because cLogS ranged from −5.93 to −2.99.
The two active chalcones (12 and 15), showed cytotoxicity
in vitro against cells renal lineages. We found that the CC50
of chalcone 12 for BHK-21 and Vero cells was 8.67 4.16
μg mL−1 and 2.10 0.69 μg mL−1, respectively. In addition,
the CC50 of chalcone 15 showed values of 2.79 0.540 μg
mL−1 for BHK-21 and 5.21 0.0 μg mL−1 for Vero cells.
Ketoconazole was also considerably cytotoxic, showing a
CC50 of 42.48 1.16 μg mL−1 and 10.31 0.60 μg mL−1 for
Vero and BHK-21 cells, respectively.
Structure-activity relationship (SAR) and chemo-
informatics analysis
Mechanism of action of chalcone 12
The SAR studies showed that a lower biological activity is
associated with the presence of substituents in benzene ring
of dihydroxychalcones (8–14) (Fig. 2). In fact, the sub-
stitution with the OMe (8), OEt (9), OBu (10), OHex (11),
or Cl (13 and 14) groups into aromatic core decrease the
antifungal activity in these analogs. Furthermore, only the
unsubstituted chalcone (12) showed a potent antifungal
effect. Already the unsatisfactory activity of hydro-
xychalcones (1–7) can be justified by substitution of the
hydroxyl group by oxa-hexane (Fig. 2), which involves to
loss of one hydrogen bonding donor centers in relation the
dihydroxychalcones (Table 3). Chalcone 15, in turn, exhi-
bits a species-specific behavior, possessing a moderate
fungistatic effect against C. krusei and C. tropicalis, but
shows low activity against C. albicans, C. glabrata, and C.
dubliniensis. This effect is associated with the presence of
morpholine group, which is a pharmacophore exclusive this
compound.
To determine the target of action antifungal of chalcone 12,
the potential of this compound to bind to the ergosterol
membrane or to fungal cell walls was investigated
(Table 4). Chalcone 12 showed a two-fold increase of MIC
in the presence of exogenous ergosterol for C. albicans and
C. krusei (the test was performed in three independent
experiments using the same conditions). Thus, in these
species, it is suggest that the action of the compound
involve the bind with the sterol fungal. The MICs of nys-
tatin were increased in the presence of ergosterol, validating
the experimental conditions of this study.
In tests, after the addition of sorbitol, MICs remained
unchanged for all four species evaluated against chal-
cone 12, suggesting that this compound does not
interfere with the fungal cell wall. Caspofungin, as expec-
ted, showed an increase in the MICs after sorbitol
supplementation.

J. T. Andrade et al.
Table 2 Summary of fungistatic and fungicidal effects of eight chalcones evaluated against Candida species isolated of patients with vulvovaginal
candidiasis
Chalcones C. albicans
C. albicans
C. albicans
C. dubliniensis C. dubliniensis C. dubliniensis C. dubliniensis C.
CVV1
CVV2
CVV3
CVV1
CVV2
CVV3
CVV4
dubliniensis
CVV5
MICa MFCa MICa MFCa MICa MFCa MICa MFCa MICa MFCa MICa MFCa MICa MFCa MICa MFCa
8
1000 >1000 1000 >1000 1000 >1000 >1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
31.25 62.5
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
31.25 62.5
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
9
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
10
11
12
13
14
15
KTZ
NYS
31.25 31.25 31.25 31.25 31.25 31.25 31.25 125
31.25 31.25 31.25 250
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
>1000 ND
250
>1000 250
>1000 250
500 15.6
>1000 125
250 1.95
>1000 125
1.95 1.95
>1000 500
1.95 1.95
>1000 125
1.95 1.95
>1000 500
1.95 1.95
>1000
1.95
31.25 125
15.6
2
4
4
4
2
2
2
2
2
2
2
2
2
2
2
2
All tests were performed in at least three independent experiments
MIC Minimum inhibitory concentration, MFC Minimum Fungicidal Concentration, KTZ Ketoconazole, NYS Nystatin, ND Not determined
aResults expressed in μg mL−1
Time-kill curve study
chalcone 12 showed a cure rate of 60%. Clotrimazole, in
turn, showed a complete remission of the infection in all
animals on day 4, being statistically different from untreated
and chalcone-treated groups (p < 0.05).
The kill kinetics assay showed that after 4 h of exposure to
chalcone 12, a reduction of one log at the concentration of
2-fold MIC and two logs at the concentration of the MIC
was observed against C. albicans. Posteriorly, chalcone 12
showed a maximum fungicidal effect at 2-fold MIC and at
MIC with 6 and 10 h of incubation, respectively (Fig. 3).
Ketoconazole did not show a fungicidal effect, presenting
the same density of fungal cells during the entire period of
time considered in this experiment (48 h).
Discussion
In this study, we showed that chalcone 12 has considerable
antifungal activity in vitro and in vivo. The fungistatic
effect was equipotent to C. albicans, C. glabrata, C. krusei,
and C. tropicalis, revealing an extend-spectrum that covers
the most frequent species involved in candidiasis. Further-
more, the compound 15 showed promising antifungal
activity in vitro against C. krusei and C. tropicalis. In
contrast to the others compounds employed, this chalcone
was synthetized by the insertion of the morpholine group,
which was recently described as an important pharmaco-
phore in molecules with antifungal activity [33]. Similar to
these results, Kant and collaborators [14] showed the
potential antifungal activity of 25 chalcones against Can-
Inhibition of yeast-hyphae transition
The chalcone 12 was able to reduce yeast-hyphae transition
at all concentrations tested (7.8, 15.6, and 31.25 μg mL−1
)
in relation to untreated cells. This effect can be observed in
first 24 h of incubation, been remained for up to 72 h
(Fig. 4).
Therapeutic effect of chalcone 12 in a VVC animal
model
dida spp., revealing MICs in the range of 6.25–50 μg mL−1
.
Candida albicans was particularly sensitive to compounds
of the chalcone class, showing MICs that ranged from 1 to
64 μg mL−1 [34]. Thus, the results presented here corrobo-
rate those of previous works that point to chalcones as a
promising class in the development of prototypes targeted
to antifungal therapy.
Chalcone 12 presented fungicidal action against all
Candida species evaluated. Fungicidal activity is of para-
mount importance; because substances able to kill
Pharmaceutical preparations containing chalcone 12 were
employed to treat VVC in rats. Table 5 showed that in
relation to the infected group, the animals treated with
cream containing 0.5 and 1.0% of chalcone 12 showed a
significant reduction in fungal load (p < 0.05) after 6 days.
However, no interaction existed between the tested doses
(0.1, 0.5, and 1.0%) and the time evaluated (days 0, 2, 4,
and 6) (p > 0.05). On the last day, for example, all doses of

Chalcones derivatives as anti-Candida agents
Fig. 2 Representation of pharmacophoric scaffold detected in chalcones
Table 3 Chemical structures, physical-chemicals proprieties, and efficiency pharmacokinetica of chalcones derivatives
Chalcones
R
Total Mol weight (g mol-1)
cLogP
cLogS
H-Acceptors
H-Donors
Lipink's ruleb
Drug likeness
1
4-OMe
4-OEt
4-OBu
4-OHex
H
354.40
368.43
382.45
410.51
324.37
358.82
393.26
254.28
268.31
296.36
324.42
240.26
274.70
309.15
381.470
531.44
3.71
4.12
4.57
5.48
3.78
4.39
4.99
2.96
3.37
4.28
5.19
2.61
3.22
3.82
3.47
3.36
−4.48
−4.78
−5.05
−5.59
−4.46
−5.20
−5.93
−3.59
−3.75
−4.29
−4.83
−3.25
−3.99
−4.72
−3.59
−2.99
5
5
5
5
4
4
4
3
3
3
3
3
3
3
5
8
1
1
1
1
1
1
1
2
2
2
2
2
2
2
1
0
Respeite all
Respeite all
Respeite all
Non respeite allc
Respeite all
Respeite all
Respeite all
Respeite all
Respeite all
Respeite all
Non respeite alld
Respeite all
Respeite all
Respeite all
Respeite all
Non Respeite alld,e
−4.697
−6.1638
−4.961
−9.141
−4.8378
−4.7382
−4.7382
0.084809
−0.1375
−5.4354
−14.995
0.13358
0.22165
0.22165
−0.8394
8.14
2
3
4
5
6
4-Cl
7
2,3-Cl
4-Me
4-Et
8
9
10
11
12
13
14
15
KTZ
4-Bu
4-Hex
H
4-Cl
2,3-Cl
—
—
KTZ Ketoconazole
a
The physical-chemical proprieties and efficiency pharmacokinetic were calculated using DataWarrior software version 4.2.2
b Lipinski's rule follow the criteria: Molecular weight is less than 500; logP (the logarithm of the partition coefficient between water and 1-octanol)
is less than 5; Number of groups in the molecule that can donate hydrogen atoms to hydrogen is less than 5; Number of groups that can accept
hydrogen atoms to form hydrogen bonds is less than 10 [19].
c,d
LogP > 5
e
Molecular weight > 500 g mol−1

J. T. Andrade et al.
Table 4 Mechanism of action of chalcone 12 on the membrane and fungal cell wall for four Candida spp
MIC (μg mL−1 a
C. albicans ATCC 10231
)
C. glabrata ATCC 2001
C. krusei ATCC 34135
C. tropicalis ATCC 28707
MIC MIC with
Ergosterol
MIC with MIC MIC with MIC with MIC
MIC with
Ergosterol
MIC with MIC
Sorbitol
MIC with
Ergosterol
MIC with
Sorbitol
Sorbitol
Ergosterol
15.6 15.6
Sorbitol
12
15.6 31.25
15.6
ND
15.6
ND
1.0
15.6
7.8
31.25
31.25
15.6
ND
15.6
1.95
15.6
3.9
15.6
ND
NIS 3.9 31.25
CSP 3.9 ND
3.9 31.25
0.5 ND
15.6
0.0625 ND
0.25
0.0625 ND
0.25
NIS Nistatin, CSP Caspofungin, ND Not determined
a
Values are the average of three readings
was species-specific, as the compound was able to bind to
ergosterol only in C. albicans and C. krusei. These beha-
viors appoint that the binding of chalcone 12 to ergosterol is
not single action mechanism. In fact, chalcones are also
believed to inhibit the synthesis of 1,3-β-D-glucan [40], but
the action of chalcone 12 on this and others fungal cells
component remain to be determined in future studies.
Although chalcone 12 acts on targets conserved exclu-
sively in fungal cells (ergosterol), the cytotoxicity of this
compound was high against mammalian eukaryotic cells.
CC50 was considerably lower in Vero (CC50 2.10 0.69 μg
mL−1) and BHK-21 (CC50 8.67 4.16 μg mL–1) cells,
which was also observed in other studies that employed
chalcones as antimicrobial agents [41]. In contrast to the
several pharmacological proprieties attributed to chalcones
derivatives, it was also discovered that many of them have a
strong cytotoxic potential and might cause severe adverse
effects [42]. Synthetic and natural chalcones are known by
their myotoxicity, generally associated with the breakage
and collapse of myofibrils, reduction of cell numbers, and
disorganization of thick (myosin) and thin (actin) filaments
[43], as well as by their mitotoxicity, related with the ability
of chalcones in inhibit tubulin assembly and actuate on
other components of cell cycle [42]. Thus, chalcones deri-
vatives showed high toxicity on eukaryotic cells. Hence, as
both, fungal, and mammalian cells are eukaryotic, is not a
surprise that this compounds shown a no selective toxicity
[35, 44]. In other study, conducted by Zenger et al. (2015)
showed that cytotoxic effects are correlated with the pre-
sence and positions of the hydroxyl groups on the chalcone
scaffold [42]. In addition, the chemical characteristic of α,β-
double bond (Michael-system) is also a crucial element for
toxicity of chalcones derivatives [45]. Hence, it has been
shown that Michael-system is involved in the inhibition of
cell proliferation, reduction of mitochondrial mass, and
cytochrome c release from mammalian cells [42].
Fig. 3 Time-Kill curve test to chalcone 12 against Candida albicans
ATCC 10231. The compound was tested at concentrations of 1 × MIC
(15.6 μg mL−1) (●) and 2 × MIC (31.25 μg mL−1) (■). Ketoconazole
was employed as positive control in concentration of 1 × MIC (125 μg
mL−1) (▲) and fungal cells untreated as growth control (▼)
pathogens are strong candidates for clinical use [35]. The
time-kill curve study revealed that the fungicidal activity of
this compound was rapid, with a maximum effect after 6 h
at 2-fold MIC and 10 h at MIC. Thus, an efficient fungicidal
effect is attributed to chalcone 12, which is pharmacologi-
cally important because the rapid elimination of micro-
organisms guarantees therapeutic success to prevent the
spread of pathogens and the progression of disease to
boards of greater severity [36, 37].
One of the proposed mechanisms of actions for anti-
fungal agents is binding to membrane ergosterol, which
leads to fungal cell disruption and a loss of intracellular
content [38]. The exogenous ergosterol binding assay sug-
gests that chalcone 12 targets this sterol of the fungal
membrane. Corroborating with this result, the cell mem-
brane disrupting nature of Candida spp. by compounds of
the chalcones class was confirmed in other study, which
showed that these compounds increase the intracellular
levels of propidium iodide, a fluorescent intercalating agent
that only penetrates cells with damaged membranes [39].
However, in this study, the effect on membrane ergosterol
Candida albicans show several virulence factors such as
significant thermotolerance, dimorphism with production of
filamentous structures that aid tissue invasion, enzymatic
production, and the ability to form biofilms [32]. In this

Chalcones derivatives as anti-Candida agents
Fig. 4 Hyphal formation of
Candida albicans ATCC 10231.
Candida albicans were cultured
with different concentration of
chalcone 12 (7.8, 15.6, and
31.25 μg mL−1) during 24, 48,
and 72 h at 37 °C. Ketoconzole
was used as a positive control.
The experiments were
performed in duplicate and
repeated three times.
Representative
microphotographs are showed.
The white bar represents a
length of 50 μm (magnification
of ×400)
Table 5 Number of infected animals and quantification of fungal burden observed for animals treated with vaginal cream containing different
concentrations of chalcone 12 and their respective controls
Groups
Day 0
Day 2
Day 4
Day 6
Infected CFU mL−1
Infected CFU mL−1
Infected CFU mL−1
Infected
CFU mL−1
animals
(%)
animals
(%)
animals
(%)
animals (%)
Infected control
Positive control
5/5 (100) 2466.07 733.26 5/5 (100) 754.59 302.23 5/5 (100) 593.03 299.26 5/5 (100)
5/5 (100) 1551.35 735.79 3/5 (60) 10.20 7.11 0/0 (0) 1.00 0.00* 0/0 (0)
1674.19 505.59
1.00 0.00*
0.1% vaginal cream 5/5 (100) 273.61 156.25a 3/5 (60) 144.04 86.75a 2/5 (40) 222.60 108.88a 2/5 (40)
0.5% vaginal cream 5/5 (100) 466.39 201.93a 4/5 (80) 744.14 568.23a 4/5 (80) 74.36 18.8a
2/5 (40)
1.0% vaginal cream 5/5 (100) 308.36 182.01a 5/5 (100) 308.36 182.01a 4/5 (80) 158.13 30.47a 2/5 (40)
Vehicle 5/5 (100) 1906.37 877.11 4/5 (10) 653.06 299.74 4/5 (80) 356.97 101.95 4/5 (80)
Positive control Clotrimazole 1.0%, Vehicle vaginal cream
504.59 114.04a
30.47 21.83a,*
16.20 8.39a,*
583.67 249.47
a The same letter denotes non-significant differences between groups according to the parametric post hoc test (p < 0.05—Tukey test) *Significant
difference (p < 0.05) when compared with Infected control (Dunnett test with statistical difference at p < 0.05)

J. T. Andrade et al.
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