Antifungal activity of a novel quercetin derivative bearing a trifluoromethyl group on Candida albicans

Article abstract of DOI:10.1007/s00044-011-9750-x

In this study, a novel quercetin derivative bearing a trifluoromethyl group was synthesized by the nucleophilic aromatic substitution reaction between rutin and 2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene in basic medium. This synthetic quercetin showed antifungal activity against Candida albicans cultures. The results indicated a remarkable increase in the biological activity of this compound as compared to rutin.

Full text of DOI:10.1007/s00044-011-9750-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2217–2222
DOI 10.1007/s00044-011-9750-x
ORIGINAL RESEARCH
Antifungal activity of a novel quercetin derivative bearing
a trifluoromethyl group on Candida albicans
•
Tomas C. Tempesti M. Gabriela Alvarez Marcelo Francisco de Araujo
•
•
•
´
•
´
´
Francisco Eduardo Araga˜o Catunda Junior Mario Geraldo de Carvalho
Edgardo N. Durantini
Received: 8 May 2010 / Accepted: 8 July 2011 / Published online: 19 July 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In this study, a novel quercetin derivative
bearing a trifluoromethyl group was synthesized by the
nucleophilic aromatic substitution reaction between rutin
and 2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene in
basic medium. This synthetic quercetin showed antifungal
activity against Candida albicans cultures. The results
indicated a remarkable increase in the biological activity of
this compound as compared to rutin.
pathogen and has developed an extensive array of recognized
virulent mechanisms, which allows successful colonization
and infection of the host under suitable predisposing condi-
tions (White et al., 2002). In the past few years, resistance
of C. albicans is increasing against traditional antifungals,
such as fluconazole (Goldman et al., 2004; Briona et al.,
2007; Ribeiro and Rodrigues 2007).
Increasingly, flavonoids are becoming the subject of
medical research. They have been reported to possess many
useful properties, including anti-inflammatory, estrogenic,
antimicrobial, antiallergic, antioxidant, vascular, and
cytotoxic antitumor activities and enzyme inhibition
(Cushnie and Lamb 2005). Owing to the widespread ability
of flavonoids to inhibit spore germination of plant patho-
gens, they have been proposed for use against fungal
pathogens of human beings (Harborne and Williams 2000).
Extracts and natural products from plants containing
flavonoids offer an alternative for the treatment of oral
candidiasis. Extracts of the leaves and tips of the twigs of
Dodonaea viscosa var. angustifolia, an indigenous South
African plant, are traditionally used as a gargle for sore
throats and oral candidiasis (Patel and Coogan 2008).
Rutin (quercetin-3-rutinoside, Fig. 1) is a polyphenolic
flavonoid widely present in foods of plant origin, such as
buckwheat, parsley, and tomatoes (Guardia et al., 2001;
Erlund et al., 2000). Rutin is known as one of the common
and naturally occurring flavonoids with a variety of bio-
chemical and pharmacological activities. Recent reports
indicate that rutin scavenges free radicals (Duthie and
Dobson 1999), suppresses cellular immunity (Middleton
et al., 2000), and has anti-carcinogenic activity (Rotelli
et al., 2003) as well as anti-inflammatory effect (Guardia
et al., 2001). More recent reports show that rutin also has
antimicrobial activity (Pereira et al., 2007). A prenylated
flavanone isolated from the shrub Eysenhardtia texana,
Keywords Antifungal activity ꢀ Flavonoid ꢀ Rutin ꢀ
Quercetin ꢀ Candida albicans
Introduction
Resistance to antimicrobial agents has become an
increasingly important and pressing global problem. Anti-
microbial resistance complicates the treatment of important
nosocomial and community-acquired infections. Candidi-
asis is a significant infection in patients being treated with
chemotherapy and radiotherapy for cancer, and in patients
who are immunocompromised because of HIV infection
and AIDS. Candida albicans causes hematogeneous dis-
seminated candidiasis and local infections, such as thrush
and vaginitis. C. albicans is the most common fungal
T. C. Tempesti ꢀ M. G. Alvarez ꢀ E. N. Durantini (&)
´
´
Departamento de Quımica, Facultad de Ciencias Exactas Fısico-
´
´
Quımicas y Naturales, Universidad Nacional de Rıo Cuarto,
Agencia Postal Nro 3, X5804BYA Rio Cuarto, Argentina
e-mail: edurantini@exa.unrc.edu.ar
´
´
M. F. de Araujo ꢀ F. E. A. Catunda Junior ꢀ M. G. de Carvalho
´
Departamento de Quımica, Instituto de Ciencias Exatas,
Universidade Federal Rural do Rio de Janeiro, BR 465, Km 7,
ˆ
´
23890-000 Seropedica, RJ, Brazil
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Med Chem Res (2012) 21:2217–2222
OH
OH
OH
O2N
Cl
HO
O
O
HO
O
O
O2N
NO2
DMF
O
+
O
OH
HO
CF3
K2CO3
O
HO
OH
O
O2N
O
OH
80-90 ºC, 15 h
OH
CF3
OH
OH
OH
quercetin-CF3
rutin
Fig. 1 Synthesis of quercetin-CF3 derivative
identified as 40,5,7-trihydroxy-8-methyl-6-(3-methyl-[2-
butenyl])-(2S)-flavanone, has been shown to possess
activity against the opportunistic pathogen C. albicans
(Wachter et al., 1999). The flavonoid 7-hydroxy-30,40-
(methylenedioxy)flavan, isolated from Terminalia belleri-
ca fruit rind, was active against C. albicans (Valsaraj et al.,
1997). Recently, rutin was proposed to exert a therapeutic
effect on septic arthritis caused by C. albicans (Yongmoon
2009). However, a disadvantage of rutin is its poor solu-
bility in aqueous media. This is the reason for its poor
bioavailability. Therefore, it imposes some restraints to
further pharmaceutical use, especially for oral administra-
tion (Mauludin et al., 2009). Also, it is generally consid-
ered that when flavonols are supplied as glycosides in the
diet, they are first hydrolyzed by the digestive microflora
before they are absorbed (Manach et al., 1997).
spectra were captured using a Varian 1200L MS-APCI
interface. The HPLC experiments were performed in a
Waters 1525 liquid chromatograph equipped with a Varian
2550 UV–visible variable-wavelength detector and Phe-
nomenex Luna C18 (5 lm, 150 9 4.60 mm) column. The
separation was performed using a mobile phase composed
of 49% water/49% methanol/2% acetic acid (flow: 0.5 ml/
min). Semiempirical molecular orbital calculations (AM1)
were carried out using HyperChem software. All chemicals
from Aldrich were used without further purification. Silica
gel thin-layer chromatography (TLC) plates of 250
microns from Aldrich (Milwaukee, WI, USA) were used.
Synthesis of rutin derivative (quercetin-CF3)
A solution of 50 mg (0.08 mmol) of rutin and 11 mg
(0.04 mmol) of 2-chloro-5-(trifluoromethyl)-1,3-dinitro-
benzene in 5 ml the N,N-dimethylformamide (DMF) was
stirred with K2CO3 (100 mg) for 15 h at 80–90°C. Then,
the solution was extracted with ethyl acetate (3 9 15 ml)
and water (20 ml). The organic phase was separated, and
the water phase was washed three times with ethyl acetate.
Combined organic phase was evaporated in a rotary
evaporator, and the solid was dried under reduced pressure.
The product (quercetin-CF3) was obtained with 90% yield.
M.p. 143–145°C. 1H-NMR (DMSO-d6, TMS) d [ppm] 6.17
(d, 1H, J = 2.0 Hz, C-6), 6.39 (d, 1H, J = 2.0 Hz, C-8),
6.87 (d, 1H, J = 8.3 Hz, C-50), 7.53 (dd, 1H, J = 2.0,
8.3 Hz, C-60), 7.66 (d, 1H, J = 2.0 Hz, C-20), 8.1 (s, 2H,
C-300 and C-500). 13C-NMR (DMSO-d6, TMS) 93.8 (C-8),
98.6 (C-6), 103.5 (C-10), 115.5 (C-20), 116.1 (C-50), 120.4
(C-60), 122.1 (CF3), 122.4 (C-10), 126.8 (C-400), 126.9 (C-300
and C-500), 136.2 (C-3), 143.1 (C-100), 145.5 (C-30), 147.3
(C-40), 148.2 (C-200 and C-600), 156.6 (C-9), 157.1 (C-2),
161.2 (C-5), 164.3 (C-7), 176.3 (C-4). APSI-MS [m/z] 575
(M ? K)? (575.00 calculated for C22H11F3N2O11K).
On the other hand, novel quercetin-3-O-amino acid-esters
were discovered as a new class of Src tyrosine kinase
inhibitors (Huang et al., 2009). Quercetin disappears
immediately from the plasma when administered intrave-
nously to rodents. This suggests that quercetin is metabolized
rapidly and excreted into the urine with no accumulation in
tissues and biological fluids (Murakami et al., 2008).
In order to discover a new rutin prodrug with improved
bioavailability, in the present investigation, a novel deriv-
ative bearing a trifluoromethyl group was synthesized
(quercetin-CF3, Fig. 1). The influence of the trifluoro-
methyl group in biologically active molecules is often
associated with the increased lipophilicity that this sub-
stituent imparts (McClinton and McClinton 1992; Ando
and Kumadaki 1999). The antifungal activity of this syn-
thetic compound was compared to that of rutin on C.
albicans cultures. The results showed a remarkable
increase in the biological activity of this compound.
Materials and methods
General
Solutions
UV–visible spectra were recorded at 25.0 0.5°C using
1-cm path length quartz cells on a Shimadzu UV-2401PC
spectrometer. NMR spectra were recorded on a FT-NMR
Bruker Advance 400 and 200 MHz spectrometer. Mass
Stock solutions of rutin or quercetin-CF3 (10 mg/ml) were
prepared by dissolution in 5 ml of water. Cloves of garlic
were peeled and pressed through a garlic presser into eight
layers of cheesecloth, and then squeezed to extract garlic
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Med Chem Res (2012) 21:2217–2222
2219
juice. The liquid was then centrifuged with a filter at
13000 rpm for 5 min. The liquid was then weighed, and a
stock concentration of 10 mg/ml was prepared in water.
Stock solution of fluconazole (500 lg/ml) was prepared by
dissolution in 2 ml of water.
experiments were carried out under the same conditions in
the absence of extracts. Each experiment was repeated
separately three times.
Effect on growth of C. albicans
Partition coefficient measurements
Cultures of C. albicans cells were grown overnight as
described above. A portion (1 ml) of this culture was
transferred to 20 ml of fresh Sabouraud broth medium. The
suspension was homogenized, and aliquots of 2 ml were
incubated with different concentrations of the flavonoid
derivatives at 37°C. The culture grown was measured by
turbidity at 660 nm using a Tuner SP-830 spectropho-
tometer. In all the cases, control experiments were carried
out in the absence of the extracts. Each experiment was
repeated separately three times.
1-Octanol/water partition coefficients (P) were determined
at 25°C using equal volumes of water (2 ml) and 1-octanol
(2 ml). Typically, a solution of each flavonoid (*100 lM)
was stirred in a thermostat after equilibrium had been
reached (8 h). An aliquot (200 ll) of aqueous and organic
phases were dissolved in 2 ml of methanol and the final
flavonoid concentration determined by absorption spec-
troscopy (Scalise and Durantini 2004).
Microorganism and growth conditions
Statistical analysis
Candida albicans strain PC31, recovered from human skin
lesion, was previously characterized and identified
according to conventional procedures (Cormick et al.,
2008, 2009). Primary classification of colonies from plates
was based on colony characteristics (pigmentation and
shape), mode of vegetative reproduction, formation of
pseudohyphae, and ascopore production. Identification of
All the data were presented as the mean standard devi-
ation of each group. Variation between groups was evalu-
ated using the Students t-test, with a confidence level of
95% (P \ 0.05) considered statistically significant.
Results and discussion
the yeast isolates to species level was done using the API
0
´
20C AUX (BioMerieux, Marcy l Etoile, France) system of
Synthesis
carbohydrate assimilation profiles. The strain of C. albi-
cans was grown aerobically overnight in Sabouraud (Bri-
tania, Buenos Aires, Argentina) broth (4 ml) at 37°C to
stationary phase. Viable C. albicans cells were monitored,
and the number of colony-forming units (CFU) was
determined on Sabouraud agar plates after *48-h incu-
bation at 37°C. This procedure produces *107 CFU/ml
after an overnight incubation.
Coupling of rutin and 2-chloro-5-(trifluoromethyl)-1,3-
dinitrobenzene by an ether bond allows for the formation of
the new flavonoid derivate quercetin-CF3, as shown in
Fig. 1. To synthesize this compound, the nucleophilic
aromatic substitution reaction was performed in DMF at
80–90°C for 15 h. The basic medium was employed to
activate the –OH group to nucleophilic reaction with the
chlorine aromatic substrate. This procedure was also
accompanied by the hydrolysis of the sugar group. The
quercetin-CF3 product was isolated with 90% yield.
The quercetin-CF3 bears a highly lipophilic trifluoro-
methyl group, which increases the amphiphilic character of
the structure. The influence of the trifluoromethyl group in
biologically active molecules is often associated with
the increased lipophilicity that this substituent imparts
(McClinton and McClinton 1992; Ando and Kumadaki
1999).
Antifungal activity
After overnight incubation, the cells were appropriately
diluted to obtain *104 CFU/ml in Sabouraud broth. In all
the experiments, 2 ml of the cell suspensions in Pirex brand
culture tubes (13 9 100 mm) was used, and the flavonoid
derivatives were added from a stock solution *10 mg/ml
in DMF:water (1:1). The minimal inhibitory concentration
(MIC) values were defined as the lowest concentration of
flavonoid derivatives, which inhibits the visible growth
of C. albicans after 48-h incubation at 37°C (range
0.06–2.50 mg/ml). Thus, no turbidity after incubation was
indicative of growth inhibition. Before the assays, it was
verified that the loading solvent, N,N-dimethylformam-
ide:water (1:1), was completely inactive against the test
organisms under the assay conditions. Also, control
The product was purified for HPLC. As expected for its
lipophilicity, the retention time (tR) of the compound
quercetin-CF3 (tR = 18 min) was longer than that of rutin
(tR = 5 min). NMR spectroscopic studies confirm the
1
suggested structure (Fig. 2). The comparison of H-NMR
spectra (in DMSO-d6) of rutin and quercetin-CF3 shows
that the signals in the region of 7.5–7.6 ppm of rutin,
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Med Chem Res (2012) 21:2217–2222
1.2
1.0
0.8
0.6
0.4
0.2
0.0
5'
2'
OH
O2N
6'
1'
4'
3'
8
HO
O
4
7
2
3
3''
1''
O
250
300
350
400
450
500
550
6
CF3
OH
5
Wavelength (nm)
O2N
5''
OH
O
Fig. 3 Absorption spectra of quercetin-CF3 (solid line) and rutin
(dashed line) in methanol
Fig. 2 Structure of quercetin-CF3
Maximum wavelength of band I of quercetin-CF3 deriva-
tive presents a bathochromic shift of *12 nm respect to
rutin. Similar behavior has previously been observed for
quercetin in different media (Le Nest et al., 2004).
The n-octanol/water partition coefficients (P) of flavo-
noids were evaluated at 25°C (P = [porphyrin]o/[porphy-
rin]w). As can be observed in Table 1, results indicate that
the lipophilic character increased in the quercetin-CF3
structure with respect to rutin because of the presence of a
trifluoromethyl group.
corresponding to the ortho protons and the hydroxy group
in the benzene ring, suffer a splitting in the compound
quercetin-CF3 as defined by two signals, one at 7.5 ppm
and the other at 7.7 ppm, being the effect of the substituent.
The COSY and HMQC spectra were recorded to establish
the specific assignment of protons. It also notes the dis-
appearance of signals corresponding to the sugars of the
routine and the emergence of a new signal at 8.1 ppm
which is correlated with the protons of the substrate con-
taining the –CF3 group.
To evaluate the effect produced by the distribution of
different polarity groups upon the intramolecular polarity,
the dipole moments of the porphyrins were estimated. The
semi-empirical method for molecular modeling (AM1) was
used in structure geometry optimization calculations
(Fig. 2). A value of 5.2 D was found for quercetin-CF3.
This value is higher than that found for the corresponding
rutin derivative without substitution by amino groups
(3.8 D). As expected, the presence of a lipophilic group in
the periphery of quercetin structure enhances the dipole
moment. The combination of hydrophobic and hydrophilic
substituents in the molecular structure results in an intra-
molecular polarity axis, which can facilitate membrane
penetration and produces a better accumulation in subcel-
lular compartments, enhancing the effective antifungal
agent.
Antimicrobial activity of flavonoid derivatives
on C. albicans
The biological activity of flavonoid derivatives was inves-
tigated on C. albicans cellular suspension in Sabouraud
medium. The extracts were evaluated in a range of
0.060–2.5 mg/ml. The antimicrobial activities on C. albi-
cans are summarized in Table 2. From these results, MIC
values of 2.20 0.10 and 0.60 0.10 mg/ml were cal-
culated for rutin and quercetin-CF3, respectively. As can be
observed, quercetin-CF3 showed about fourfold higher
antifungal activity than rutin. On the other hand, the sus-
ceptibility of this yeast was evaluated using fluconazole and
garlic extract. Azole derivatives are standard active anti-
fungals established to eradicate C. albicans (Soysa et al.,
Table 1 UV-visible absorption characteristics in methanol and
1-octanol/water partition coefficients (P) of rutin and quercetin-CF3
UV–visible absorption studies and lipophilic properties
Compound
k (nm)
k (nm)
Log P
(e M-1 cm-1
)
(e M-1 cm-1
)
The UV–visible absorption spectra of rutin and quercetin-
CF3 in methanol are compared in Fig. 3. The flavonoid
derivative shows the typical band I around 370 nm and
band II at 260 nm, according to the molecular structure.
Rutin
256 (14100)
254 (18800)
360 (12200)
372 (17100)
0.31
0.62
Quercetin-CF3
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Med Chem Res (2012) 21:2217–2222
2221
0.06
Table 2 Antifungal activity of flavonoid derivatives on C. albicans
Compound
Concentration (mg/ml)
2.50
2.20
2.10
1.90
1.60
1.30
1.25
0.60
0.50
0.40
0.30
0.15
Rutin
?
?
?
?
-
?
-
?
-
?
-
?
-
?
-
?
-
-
-
-
-
-
-
-
-
-
Quercetin-CF3
? inhibition of visible growth, - no inhibition
2004). Also, garlic oil has shown to have anti-Candida
activity at higher concentrations, indicating that they may
be useful in the topical treatment of superficial Candida
infections (Iwalokun et al., 2004). The MIC values obtained
were 4 lg/ml and 2.0 mg/ml for fluconazole and garlic
extract, respectively. Thus, natural antimycotic remedies,
such as rutin and garlic extract, are likely not to be as
effective as azole antifungal agents, and may require higher
doses to produce similar results. However, chemical mod-
ification of rutin to produce quercetin-CF3 was accompa-
nied by a considerable increase in the antifungal activity.
Taking into account these results, growth delay of
C. albicans cultures produced by flavonoids was carried
out in Sabouraud medium. Thus, different amounts of
flavonoids were added to fresh cultures of C. albicans
reaching the log phase, and the flasks were incubated at
37°C. Three concentrations of rutin and quercetin-CF3,
ranging above and below the MIC values, were analyzed
under these conditions. As can be observed in Fig. 4,
growth was suppressed when C. albicans cultures were
treated with both extracts using concentration identical to or
higher than MIC. After 30 min of incubation in the presence
of 2.2 or 2.5 mg/ml of rutin, the cells no longer appeared to
be growing as measured by turbidity at 660 nm. Under
these conditions, the effect of quercetin-CF3 was more
efficient than that of rutin, because it was effective between
0.6 and 0.7 mg/ml. On the other hand, C. albicans cells
incubated with a lower concentration of flavonoid (less than
MIC value) showed only a small growth delay compared
with controls. Under this condition, Lag phase was
increased with respect to the control. Therefore, with the
greater than MIC values , the growth did not occur, and at
concentrations less than the MIC values, growth occurred
but with a prolonged lag phase. These data illustrate that the
observed growth delay is due to the antimicrobial effect of
the quercetin-CF3 on the C. albicans cells.
The results obtained in the present study indicate that a
stronger antifungal activity is produced by quercetin-CF3 in
comparison with that exerted by rutin. Also, compound
quercetin-CF3 has a higher log P value. In previous studies,
it had been observed that glycosylation of quercetin
reduced the vasodilator activity (Chen et al., 2004). In
general, in this kind of compounds, a stronger biological
activity is accompanied by an increase of log P values.
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Control
Rutin 1.9 mg/mL
Rutin 2.2 mg/mL
Rutin 2.5 mg/mL
Rutin-CF3 0.5 mg/mL
Rutin-CF3 0.6 mg/mL
Rutin-CF3 0.7 mg/mL
Conclusions
A new rutin derivative was synthesized by the nucleophilic
aromatic substitution reaction between rutin and 2-chloro-
5-(trifluoromethyl)-1,3-dinitrobenzeneound in basic med-
ium. The reaction conditions also produced the loss of
sugar structure from the flavonoid, to obtain quercetin-CF3
with a high yield. The potential antifungal activities of
rutin and quercetin-CF3 were compared against the strains
of C. albicans. Quercetin-CF3 was more effective than
rutin against C. albicans. Therefore, this study indicates
that quercetin-CF3 is an interesting antifungal agent with
considerably higher activity than rutin.
0
100 200 300 400 500 600 700 800
Time (min)
Fig. 4 Growth delay curves of C. albicans cells incubated with
different concentrations of rutin [(open down triangle) 1.9 mg/ml,
(open square) 2.2 mg/ml, (open up triangle) 2.5 mg/ml] and quer-
cetin-CF3 [(filled down triangle) 0.5 mg/ml, (filled square) 0.6 mg/ml
and (filled up triangle) 0.7 mg/ml] in Sabouraud broth at 37°C.
Control cultures: cells without addition of flavonoid derivatives (open
circle). Values represent mean standard deviation of three separate
experiments
Acknowledgments The authors are grateful to the Consejo Nac-
´
´
ional de Investigaciones Cientıficas y Tecnicas (CONICET) of
Argentina for financial support. M.G.A. and E.N.D. are Scientific
Members of CONICET. T.C.T. thanks CONICET for a research
fellowship.
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catechin, and derivatives: I optical and NMR studies. Inorg Chim
Acta 357:775–784. doi:10.1016/j.ica.2003.09.014
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