Antifungal activity of chalcones: A mechanistic study using various yeast strains

Article abstract of DOI:10.1016/j.ejmech.2007.12.027

We reported the synthesis, antifungal evaluation and study on substituent effects of 21 chalcones. A lot of genetically defined strains belonging to different yeast genera and species, namely Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis, were used as test organisms. Concerning the mode of the antifungal action of chalcones it was shown that DNA was probably not the main target for the chalcones. It was revealed that the yeast's intracellular glutathione and cysteine molecules play significant role as defence barrier against the chalcone action. It was also shown that chalcones may react with some proteins involved in cell separation.

Full text of DOI:10.1016/j.ejmech.2007.12.027

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2220e2228
http://www.elsevier.com/locate/ejmech
Original article
Antifungal activity of chalcones: A mechanistic study
using various yeast strains
b
c
c
K.L. Lahtchev a, D.I. Batovska b, , St.P. Parushev , V.M. Ubiyvovk , A.A. Sibirny
*
a The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Street,
Block 26, Sofia 1113, Bulgaria
b Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, Block 9,
Sofia 1113, Bulgaria
c Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine
Received 30 May 2007; received in revised form 26 December 2007; accepted 26 December 2007
Available online 12 January 2008
Abstract
We reported the synthesis, antifungal evaluation and study on substituent effects of 21 chalcones. A lot of genetically defined strains belong-
ing to different yeast genera and species, namely Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis, were used as test
organisms. Concerning the mode of the antifungal action of chalcones it was shown that DNA was probably not the main target for the chal-
cones. It was revealed that the yeast’s intracellular glutathione and cysteine molecules play significant role as defence barrier against the chal-
cone action. It was also shown that chalcones may react with some proteins involved in cell separation.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Chalcones; Yeasts; Antifungal activity; Substituent effects
1. Introduction
introduction of p-EW but not p-ED substituents is effective
for the synthesis of chalcones with antifungal properties [1,3].
Some exceptions of this rule are known. For example, p-nitro-
chalcone (strong p-EW group) showed the same or weaker an-
tifungal activity if compared to p-chlorochalcone (poor EW
group) and p-methylchalcone (ED group) [1]. Besides, the
ED hydroxyl groups are considered to play a significant role,
though it is not clear if and how exactly the number and precise
locations of the phenolic groups correlate with the antifungal
activity of chalcones [1,5,8e11].
Previously we reported the effects of various substitutions
of a series of synthetic chalcones against the pathogenic yeast
Candida albicans [12]. It was shown that the potency of the
chalcones against C. albicans to a large extent depended on
their ability to interact with sulfhydryl groups. However, addi-
tional studies are needed to determine the discrete cellular tar-
gets for the chalcone action.
Chalcones (1,3-diaryl-2-propen-1-one) are natural or syn-
thetic flavonoids displaying an impressive array of biological
properties [1e6]. Their antimicrobial activity and particularly
the antifungal action have been largely attributed to the reactive
enone moiety [1,3]. As a Michael reaction acceptor the enone
unit binds thiol groups of certain proteins. Probably in that man-
ner, most chalcones inhibit biosynthesis of yeast cell wall and
thus unfold their antifungal potential [7]. The Michael reactions
of chalcones are facilitated by electron withdrawing (EW)
groups at p-position in ring B. Such substituents increase the
electron deficiency at C-b transforming it into an attractive
electrophilic centre for the thiol attack. The alternative p-elec-
tron donating ( p-ED) groups hamper this reaction. Therefore,
For deeper insight of the antifungal mechanisms of chal-
cones, it is necessary to employ suitable organisms. Yeasts
* Corresponding author. Tel.: þ359 2 9606 184; fax: þ359 2 8700 225.
E-mail address: danibat@orgchm.bas.bg (D.I. Batovska).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.12.027

K.L. Lahtchev et al. / European Journal of Medicinal Chemistry 43 (2008) 2220e2228
2221
are appropriate and useful eukaryotic models for such studies.
They allow for rapid and low-cost experiments and avoid the
ethical questions inherent in the use of animals. To date,
chalcones have been tested mainly against clinical isolates
of various medically important yeasts such as Cryptococcus
neoformans [1,3,11], Candida [1,8,9,12,13], Microsporum
[1,3], Trichophyton [1,3] and Penicillium species [1,13]. How-
ever, these strains have been with unknown ploidy and carried
many unknown natural determinants giving rise to resistance
against chemicals. The employment of such strains allows de-
termination of compounds with very strong activity only and
hampers elucidation of mechanisms of antifungal action of
chalcones. These problems can be avoided by employment
of genetically defined strains belonging to well-investigated
yeast species.
or cinnamaldehyde (Scheme 1). The desired products were ob-
tained in average yield of 80% after purification. Their struc-
tures were established with UV, IR, NMR, mass spectrometry
1
and elemental analysis. H NMR spectra showed that only E-
chalcones were obtained. The spectral data of the recently syn-
thesized compounds are presented in Sections 4.1.1.1 and
4.1.1.2.
2.2. Microbiology
The chalcones were tested for their growth inhibitory ac-
tivity against 33 strains belonging to S. cerevisiae (13
strains), H. polymorpha (17 strains) and K. lactis (3 strains).
Their MICs were determined by the agar dilution method.
The most active chalcones were 1, 2, 5, 7 and 8 (Tables
2e4). The rest of the compounds exhibited antifungal activ-
ity at very high concentrations (>40 mg/ml) and were taken
into consideration only for the analysis of the substituent
effects.
K. lactis appeared to be the most sensitive species among
the tested yeasts (Table 4). On the other side, S. cerevisiae
was more resistant to the parent chalcone 1 than H. polymor-
pha and the opposite was true for compound 7 bearing a
m-hydroxyl group in ring B (Tables 2 and 3).
In this study a series of 21 chalcones with various substit-
uents were synthesized and examined in vitro against 33 yeast
strains of Saccharomyces cerevisiae, Hansenula polymorpha
and Kluyveromyces lactis. All the strains employed were
with well-defined ploidy and carrying specific mutations in ei-
ther the biosynthetic pathways or defence systems.
2. Results and discussion
2.1. Chemistry
2.3. DNA is not the main target for the antifungal action
of chalcones
A series of 7 recently synthesized and 14 known chalcones
(Table 1) were prepared by a cold or hot variant of the Clai-
seneSchmidt condensation between acetophenones (acetophe-
none or p-chloroacetophenone) and appropriate aryl aldehydes
The antifungal activity of chalcones was examined against
9 laboratory employed haploid strains of S. cerevisiae and 4
artificially constructed or naturally occurring diploids. No
clear relationship was observed between the ploidy level and
the strain sensitivity (Table 2). Similar results were found
for the 16 H. polymorpha haploids and a diploid strain desig-
nated as D22 (Table 3). These results show that the ploidy has
no significant influence on the observed antifungal effects and
that probably DNA is not the main target for the chalcone ac-
tion. To verify the last statement chalcones were examined for
their ability to induce nuclear and mitochondrial mutations.
For this purpose, two diploid strains of S. cerevisiae were
used: PV-2 [MATa/MATa his7-1/his7-1 ade2-475/ade2-475
lys2-67/lys2-68 rad1-5/RAD1] and its ultraviolet-light-sensi-
tive (homozygous for the rad1-5 mutation) derivate designated
as PV-3 [14]. These strains give a possibility for detection of
various genetic events such as reversion of ochre mutation
his7-1 [his7-1 / Hisþ], reversion of frame-shift mutation
ade2-475 [ade2-475 / Adeþ], mitotic gene conversion at
LYS2 gene [lys2-67/lys2-68 / Lysþ], illegitimate mating,
etc. Chalcones 1, 6, 7 and 13 were tested without metabolic
activation by the method of Fink and Lowenstein [25]. No
cases of enhanced appearance of Adeþ, Hisþ and Lysþ col-
onies were detected suggesting that these substances cannot
act as strong mutagens or recombinogens at the used condi-
tions. In separate experiments, chalcones 1 and 7 were tested
for their ability to induce loss of mitochondrial DNA. In
S. cerevisiae such alternations cause appearance of respiratory
deficient ‘petite’ mutants, which form invariable small sized
Table 1
Substitution patterns of the chalcones studied for the antifungal effects
R2
O
R3
A
B
n
R'4 p'
R4
p
R5
4
Compound
n
R0
R2
R3
R4
R5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
2
H
OH
H
H
H
3
H
OH
OH
OCH3
H
H
4
H
OH
OH
H
H
5
H
H
6
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
7
OH
H
H
H
8
OH
OH
OCH3
OH
OH
H
H
9
OH
OH
H
10
11
12
13
14
15
16
17
18
19
20
21
H
OCH3
OCH3
OCH3
OCH3
H
H
OCH3
H
H
Cl
H
H
H
CN
NO2
NHCOCH3
CH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H

2222
K.L. Lahtchev et al. / European Journal of Medicinal Chemistry 43 (2008) 2220e2228
R2
O
R2
R5
O
O
R3
R4
R3
R4
a
b
H
CH3
+
R'4
R'4
R5
Scheme 1. General routes used for the chalcone synthesis. Reagents and conditions: (a) 6 M NaOH, MeOH, keeping in refrigerator overnight; (b) KOH, MeOH,
reflux for 1.5 h.
colonies. The strains PV-2 and PV-3 were grown on liquid
YPD supplemented with 20 mg/ml of the chalcones 1 and 7.
After 2 days of incubation yeast cells were washed, serially di-
luted and plated onto YPD plates. The lack of ‘petite’ colonies
in the experimental plates compared to the controls indicated
that the chalcones did not react with mitochondrial DNA.
compounds 2 and 7 acted on some proteins involved in the
cell division cycle. The strains MC1, 2a, AS1 and AS2
were very sensitive to most of the chalcones regardless of
their cell density and the chalcone concentration (Figs. 1
and 2). Their increased susceptibility was due to a diminished
pool of thiol compounds as this can be judged by the spec-
ificity of the carried mutations. MC1 and 2a possess cys1-1
allele, which strongly requires cysteine and encodes the en-
zyme O-acetyl homoserine sulfhydrylase. The strain AS1 is
impaired in biosynthesis of S-adenosyl-L-methionine uropor-
phyrogen III methyltransferase. This enzyme is responsible
for sirohem biosynthesis and it is required for the sulfite/sul-
fide reduction due to the mutation gsh1 [20]. The most sen-
sitive strain AS2 is impaired in the gene GSH2 encoding g-
glutamylcysteine synthetase, the enzyme controlling the first
step of glutathione biosynthetic pathway. An increased mor-
tality of AS2 cells was observed upon their treatment with
various concentrations of chalcone 1. This was due to the
gsh2 mutation (Fig. 1). The obtained results show that at
least one of the steps involved in chalcone detoxification is
based on the attack from the available thiol molecules. The
higher sensitivity of gsh2 deletion mutant AS2 in comparison
with the strains carrying cys1 and gsh1 mutations pointed at
the cellular glutathione as the major yeast barrier towards
chalcones.
2.4. Role of chromosomal mutations
For better evaluation of the antifungal activity of chalcones
we used strains carrying many chromosomal mutations con-
trolling specific metabolic pathways. Those with mutations
impaired in purine, pyrimidine and some amino acid biosyn-
thetic pathways did not show high sensitivity against the
chalcones.
Some of the H. polymorpha strains revealed great differ-
ences in their sensitivities depending on the cellular geno-
type. The strain CTA1 carrying mutation cat1 (lack of
catalase activity as a result of disruption of the structural
gene for catalase) showed increased resistance against the
chalcones. This might be due to accumulation of hydrogen
peroxide because of the lack of catalase activity. Hydrogen
peroxide is highly reactive and may transform chalcones
into less effective epoxides [5]. The mutants R3 and M4
are with altered morphology of colonies and cells. They
were very sensitive to chalcones 2 and 7. The phenotype
of R3 (chains of undivided cells) is caused by mutation
rgh3, whereas M4 carries mutation rpm2 leading to pseudo-
hyphal and invasive growth [21]. Both mutations are involved
in the process of cell separation, suggesting that the
2.5. Effect of the chalcone concentration and
strain cell density
The yeast survival depended on the chalcone concentration
(Fig. 1) and the strain cell density. At high chalcone concen-
tration (>40 mg/ml) and low cell density (103 cells/ml),
a strong killing effect was observed. At concentration of
40 mg/ml, growth was observed only for strains with high
cell density (109 cells/ml). Moderate concentrations of the
chalcones (20e30 mg/ml) caused a clear growth inhibition af-
ter 1 day of incubation and restoration of the normal growth
after 2 days of incubation. Such a stasis effect was observed
regardless of the manner of the cell plating (spotted, Fig. 2 or
streaked, Fig. 3) and the nature of the cultures (solid, Figs. 2
and 3 or liquid, Fig. 4). The observed effect may be due to
structural alterations of the chalcones during the experiments
and/or development of yeast cellular resistance. These possi-
bilities were verified as follows. The stability of chalcones 1
and 5 was examined via UV spectrophotometrically determi-
nation of their concentrations in the culture media (liquid
YPD) at 37 ꢀC. No change in their quantities was registered
after 24 h incubation. After 48 h the quantity of compound
Table 2
MIC values (mg/ml) of chalcones acting against S. cerevisiae strains
Strains
Ploidy level
Source
Chalcones
1
2
5
7
8
1S
PV-2
Haploid
Diploid
Diploid
Haploid
Haploid
Diploid
Diploid
Haploid
Haploid
Haploid
Haploid
Haploid
Haploid
[14]
[14]
[14]
[15]
[15]
[16]
[16]
[15]
[15]
[15]
[17]
[17]
[17]
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
25
25
25
30
25
25
25
25
25
25
25
25
20
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
35
20
18
15
20
16
20
25
15
20
18
15
15
18
>40
35
PV-3
1169
35
>40
>40
35
2180
1a
29a
>40
>40
>40
>40
>40
>40
>40
S1278b
D3C
FY4
10560-6B
YSH1170
YSH1171
>40

K.L. Lahtchev et al. / European Journal of Medicinal Chemistry 43 (2008) 2220e2228
2223
Table 3
MIC values (mg/ml) of chalcones acting against H. polymorpha strains
Strains
Genotype
Source
Chalcones
1
2
5
7
8
WT1
WT2
CD1
CD2
CD3
WA1a
2aa
ade1 ura3
ade1 met6
[18]
[18]
[19]
[19]
[19]
[20]
[21]
[21]
[19]
30
30
35
25
25
25
25
>40
>40
>40
>40
>40
30
25
>40
>40
>40
>40
>40
30
<20
<20
<20
<20
<20
<20
25
ade1 leu2 cds1
ade1 leu2 cds2
ade1 leu2 cds3
ade1 leu2 gsh1
arg1 leu2 ura3 cys1
arg1 ade1 leu2
arg1 ade1 leu2 URA3 CYS1
arg1 ADE1 leu2 ura3 cys1
met2
30
30
30
<20
25
<20
25
30
35
2b
D22
30
30
25
25
>40
>40
>40
>40
<20
M2
[21]
[21]
[20]
[21]
[21]
[21]
[21]
[19]
30
20
25
20
20
30
35
35
20
20
>40
30
25
<20
<20
25
>40
30
MC1a
AS1a
M4
met2 cys1
arg1 gsh2D
20
30
30
met4
ade3 ura3
<20
<20
30
>40
>40
>40
>40
>40
>40
>40
>40
w40
w40
AU05
CTA1
R3a
25
25
leu2 cat1
rgh3
leu2 CDR2
>40
>40
<20
<20
CRD2
a
Strain is very sensitive to the chalcone action when plated at low cell density.
1 decreased to 7.1 ꢁ 0.3 mg/ml while that of the compound 5
retained. When the YPD media were inoculated with cells of
H. polymorpha strain (WT1) the concentration of the chal-
cone 1 dropped to 1.4 ꢁ 0.2 mg/ml and was not detected after
48 h. The quantity of chalcone 5 was 17.5 ꢁ 0.1 mg/ml after
24 h and decreased to 0.41 ꢁ 0.01 mg/ml after 48 h. These re-
sults indicate that both compounds interact with yeast cells
but in different ways. The development of cellular resistance
against chalcones’ action was evident from the second group
of experiments. Yeast cells were pre-incubated on YPD plates
supplemented with 35 mg/ml of chalcone 1 for 3 days and
then streaked onto plates containing a freshly prepared solu-
tion of 1. The pre-incubated strains were found to be more
resistant than the control cells. Taken collectively these ex-
periments indicated that the observed stasis effect was due
to depletion of chalcone molecules during the incubation,
which tolerated the growth restoration of surviving yeast cells
and development of cellular resistance.
1. Introduction of EW substituents (Cl, CN and NO2 groups)
in p-position in ring B yielded less active chalcones (com-
pounds 15e17) than the parent chalcone 1.
2. Introduction of ED substituents (OH, CH3 and OCH3
groups) in p-position in ring B produced inactive chal-
cones (compounds 3, 19 and 20).
3. Presence of a single hydroxyl group was effective at m-
position in ring B. Introduction of a single methoxy group
at m-position in ring B led to inactive compound.
4. The combination of m-hydroxyl and p-methoxy groups in
ring B was effective (compound 5). Loss of activity was
observed with the interchange of the positions of the hy-
droxyl and methoxy groups (compound 11) and when
the hydroxyl group was placed in o-position and the me-
thoxy group was in m-position (compound 14).
5. Introduction of p0-chloro atom in ring A was beneficial
only for the chalcones with a single hydroxyl group at
m- and p-positions (compounds 7 and 8). The m-position
was more favourable than the p-position. Presence of m-
and p-hydroxyl groups together led to the inactive chal-
cone 9.
2.6. Substituent effects
6. Elongation of the conjugated system by introduction of
one additional double bond between the ketovinyl moiety
and the ring B did not produce an active compound (com-
pound 21).
The antifungal effects of the substituted chalcones were
compared with those of the parent chalcone 1. The following
correlations were observed.
Based on these observations it could be concluded that
the electronic effects of the p-substituents in ring B of chal-
cones are not crucial for displaying antifungal activity to-
wards the tested fungi. This is contradictory to the
antifungal effects, which chalcones with EW and ED sub-
stituents in ring B have shown against several dermato-
phytes and the yeast C. albicans [1,3,12]. Besides, in this
study the position of the hydroxyl group in ring B was
Table 4
MIC values (mg/ml) of chalcones acting against K. lactis strains
Strains Genotype
Source Chalcones
1
2
5
7
8
2-18B MATa metA1 ura3 leu2 [22]
<20 <20 25 <20
<20 <20 25 <20 >40
25 <20 25 <20 35
35
1-6A
MATa uraA lys arg
[23]
[24]
2-19B MATa ura lys arg

2224
K.L. Lahtchev et al. / European Journal of Medicinal Chemistry 43 (2008) 2220e2228
activity of hydroxyl-chalcones against C. albicans [12].
The reason for these effects is unknown.
Concentrations of chalcone 1 [µg/ml]
0
10
20
30
40
100
10
1
3. Conclusion
We report a study on the antifungal mechanism of 21 chal-
cones with diverse substituents in their aryl ring B. A large
number of genetically defined strains of various yeast genera
and species were used as test organisms. In general, the anti-
fungal activity of chalcones depended on their substitution
pattern as well as on the yeast genotype and strain cell
density.
The data obtained widen the knowledge about the mode of
antifungal action of chalcone. The role of the ploidy level was
clarified by utilization of haploid and diploid strains and ac-
cording to the results DNA is probably not the main target
for the chalcones. At least one of the steps involved in chal-
cone detoxification is based on thiol alkylation. This was
proved by the increased sensitivities of strains carrying muta-
tions in genes impaired in sulphur (GSH1), cysteine (CYS1)
and glutathione (GSH2) metabolism. For the first time a precise
link of chalcone action to particular protein targets encoded by
the genes GSH1, GSH2 and CYS1 was experimentally demon-
strated. Our data also showed that chalcones may react with
proteins involved in cell separation as estimated by the in-
creased sensitivity of strains with mutation rgh3 to the lethal
action of chalcones 2 and 7.
0,1
Fig. 1. Survival rates of H. polymorpha strains under the action of various concen-
trations of chalcone 1: ( ) WT1; (-) cys1(2a);( ) gsh1(AS1);( ) gsh2(AS2).
A
:
6
found important for the chalcone activity as opposed to
some other antifungal studies [5,11]. Interestingly, the fav-
oured location for the hydroxyl group was the m-position
in ring B. In contrast, in our previous study we showed
that o-position was the most favourable for the antifungal
Cell
number
(cells/ml)
103 105 107 109
103 105 107 109
103 105 107 109
WT
gsh1
gsh2
cys1
A
B
C
WT
gsh1
gsh2
cys1
D
E
F
Fig. 2. Growth of wild type and mutant strains of H. polymorpha on rich media supplemented with 15 (B and E) and 20 mg/ml of chalcone 1 (C and F). The strains
WT1 (wild type), AS1 ( gsh1), AS2 ( gsh2) and 2a (cys1) were used; yeast cells were plated at 100-fold dilutions indicated on the top. Results were scored after
48 h (AeC) and 72 h (DeF) incubations at 37 ꢀC.

K.L. Lahtchev et al. / European Journal of Medicinal Chemistry 43 (2008) 2220e2228
2225
Fig. 3. Growth of wild type and mutant strains of H. polymorpha on rich media supplemented with 15 (B and F), 20 (C and G) and 30 mg/ml of chalcone 1 (D and
H). 1 ¼ WT1 (wild type), 2 ¼ AS1 ( gsh1), 3 ¼ AS2 ( gsh2) and 4 ¼ 2a (cys1).
The approach used is suitable for studying the interaction of
chalcones with living cells and finding new chalcone-like
compounds with pharmacological activities.
[26], 40-chloro-4-hydroxychalcone (8) [26], 40-chloro-3,4-
dihydroxychalcone (9) [29], 40-chloro-3-methoxy-4-hydro-
xychalcone (11) [30], 40,4-dichlorochalcone (15) [26],
40-chloro-4-nitrochalcone (17) [31], 40-chloro-4-methylchalcone
(19) [12], 40-chloro-4-methoxychalcone (20) [12] and 1-(4-
chloro-phenyl)-5-phenyl-penta-2,4-dien-1-one (21) [32].
4. Experimental protocols
Infrared and UV spectra were measured on Bruker IFS
113V and Helios gamma UVevisible spectrophotometers, re-
spectively. 1H and 13C NMR spectra were recorded on Bruker
AM 250 spectrometers with tetramethylsilane as internal refer-
ence. Chemical shifts are given in parts per million (d-scale);
coupling constants (J ) are in hertz. Splitting patterns are de-
scribed as singlet (s), doublet (d), triplet (t) and multiplet
(m). Mass spectral analyses were accomplished on a Hew-
lettePackard 5972 using Mass Selective Detector with EI
(70 eV). The melting points were obtained using Mel-Temp
1102D-230 VAC and were uncorrected. The reactions were
monitored on silica gel 60 F254 using PE:acetone 7:3 or tolue-
ne:Et2O 4:1. Flash chromatography was performed for purifi-
cation of the chalcones on silica gel 60 (230e400 mesh
ASTM) using eluent PE:acetone 7:3.
1E+09
1E+08
1E+07
1E+06
1E+05
4.1. Chemistry
0
1
2
3
Days of incubations
4
4.1.1. Synthesis of chalcones
Preparation of the following chalcones was reported pre-
viously: chalcone (1) [3,26], 3-hydroxychalcone (2) [26], 4-
hydroxychalcone (3) [26], 3,4-dihydroxychalcone (4) [27],
40-chlorochalcone (6) [28], 40-chloro-3-hydroxychalcone (7)
Fig. 4. Growth of wild type and mutant H. polymorpha strains in liquid YPD
media supplemented with 20 mg/ml of chalcone 1. ( ) WT1; (-) cys1(2a);
:
( ) gsh1(AS1) and gsh2(AS2).
A

2226
K.L. Lahtchev et al. / European Journal of Medicinal Chemistry 43 (2008) 2220e2228
Chalcones 4, 9, 12 and 14 were prepared applying a hot var-
(s, 3H, OCH3). 13C NMR (CDCl3) d: 189.1 (C]O), 159.9
(C-3), 145.2 (C-b), 139.2 (C-40), 136.4 (C-1), 136.0 (C-10),
130.0 (C-20, C-60), 129.9 (C-5), 128.9 (C-30, C-50), 121.8 (C-
a), 121.1 (C-6), 116.4 (C-4), 113.5 (C-2), 55.3 (OCH3).
Mass (m/z, %) 272 (Mþ, 100), 257 (14), 241 (98), 237 (56),
161 (28), 139 (55), 111 (39). Anal. Calc. for C16H13ClO2
(%): C, 70.46; H, 4.80; Cl, 13.00. Found: C, 70.79; H, 4.90;
Cl, 13.22.
iant of the ClaiseneSchmidt condensation, whereas the rest of
the compounds were obtained by a cold variant of the same
method.
4.1.1.1. A cold variant of the ClaiseneSchmidt condensation.
Acetophenone (97 ml, 0.8 mmol) or p-chloroacetophenone
(84 ml, 0.7 mmol) was added to equimolar quantities of appro-
priate aryl aldehydes or cinnamaldehyde, dissolved in MeOH
(0.8 ml). To this solution 6 M NaOH (0.4 ml) was added drop-
wise and the reaction mixture was stirred for 40 min and then
kept in refrigerator overnight. The product crystals were fil-
trated and washed carefully with ice water and cold MeOH
to neutral reaction. The resulting chalcones were purified by
recrystallisation from MeOH. The reaction mixtures contain-
ing chalcones soluble in MeOH were evaporated and frac-
tioned between EtOAc and water. The organic layer was
evaporated and submitted to flash chromatography.
4.1.1.1.4. 40-Chloro-4-cyanochalcone (16). Yield 71%, yel-
low crystals, m.p. 139e141 ꢀC (MeOH). UV (MeOH) l/nm:
228, 310. IR (KBr) n/cmꢂ1: 2210 (C^N), 1656 (C]O),
1
1583 (C]C). H NMR (DMSO-d6) d: 7.96 (d, 2H, H-20, H-
60, J ¼ 8.8 Hz), 7.78 (d, 1H, H-b, J ¼ 15.8 Hz), 7.71 (s, 4H,
H-2, H-3, H-5, H-6), 7.55 (d, 1H, H-a, J ¼ 15.8 Hz), 7.49
(d, 2H, H-30, H-50, J ¼ 8.8 Hz). 13C NMR (DMSO-d6) d:
188.4 (C]O), 142.5 (C-b), 139.8 (C-40), 138.9 (C-1), 135.9
(C-10), 132.7 (C-3, C-5), 129.9, 129.1 (C-20, C-30, C-50, C-
60), 128.7 (C-2, C-6), 124.4 (C-a), 118.3 (C^N), 113.6 (C-
4). Mass (m/z, %) 267 (Mþ, 68), 232 (100), 156 (37), 139
(53), 128 (32), 111 (42). Anal. Calc. for C16H10ClNO (%):
C, 71.78; H, 3.77; Cl, 13.24; N, 5.23. Found: C, 72.15; H,
3.92; Cl, 13.02; N, 5.38.
4.1.1.1.1. 3-Hydroxy-4-methoxychalcone (5). Yield 96%,
yellow crystals, m.p. 92e94 ꢀC (MeOH). UV (MeOH) l/nm:
265, 360. IR (KBr) n/cmꢂ1: 3260 (OH), 1646 (C]O), 1571
1
(C]C), 1260 (CeO). H NMR (CDCl3) d: 7.99e8.03 (m,
2H, H-20, H-60), 7.74 (d, 1H, H-b, J ¼ 15.5 Hz), 7.44e7.58
(m, 3H, H-30, H-40, H-50), 7.40 (d, 1H, H-a, J ¼ 15.5 Hz),
7.28 (d, 1H, H-2, J ¼ 2.3 Hz), 7.13 (dd, 1H, H-6, J ¼ 8.2,
2.2 Hz), 6.87 (d, 1H, H-5, J ¼ 8.5 Hz), 5.77 (s, 1H, OH),
3.93 (s, 3H, OCH3). 13C NMR (CDCl3) d: 190.5 (C]O),
148.8 (C-4), 145.9 (C-3), 144.8 (C-b), 138.4 (C-10), 132.6
(C-40), 128.6, 128.4 (C-1, C-20, C-30, C-50, C-60), 122.7 (C-
a), 120.3 (C-6), 113.0 (C-5), 110.6 (C-2), 56.0 (OCH3).
Mass (m/z, %): 254 (Mþ, 100), 239 (55), 223 (14), 177 (36),
105 (33), 77 (47). Anal. Calc. for C16H14O3 (%): C, 75.57;
H, 5.55. Found C, 75.68; H, 5.82.
4.1.1.1.5. 40-Chloro-4-acetamidochalcone (18). Yield 84%,
yellow crystals, m.p. 191e194 ꢀC (96% EtOH). UV (MeOH)
l/nm: 260, 345. IR (KBr) n/cmꢂ1: 3321 (NH), 1671
(NHeC]O, amide-I), 1639 (C]O), 1582 (C]C), 1504
(CeNeH, amide-II), 1307 (CH3), 814 (CeCl). 1H NMR
(DMSO-d6) d: 10.20 (s, 1H, NH), 8.14 (d, 2H, H-20, H-60,
J ¼ 8.8 Hz), 7.82 (d, 2H, H-3, H-5, J ¼ 8.8 Hz), 7.78 (s, 1H,
H-b), 7.71 (s, 1H, H-a), 7.65 (d, 2H, H-30, H-50,
J ¼ 8.5 Hz), 7.61 (d, 2H, H-2, H-6, J ¼ 8.8 Hz), 2.06 (s, 3H,
CH3). 13C NMR (DMSO-d6) d: 187.9 (C]O), 168.8
(CONH), 144.4 (C-b), 141.7 (C-4), 138.0 (C-40), 136.4 (C-
10), 130.4 (C-20, C-60), 130.0 (C-30, C-50), 129.2 (C-1), 128.9
(C-2, C-6), 119.8 (C-a), 118.8 (C-3, C-5), 24.2 (CH3). Mass
(m/z, %) 299 (Mþ, 100), 264 (20), 256 (53), 241 (26), 139
(22), 111 (27). Anal. Calc. for C17H14ClNO2 (%): C, 68.12;
H, 4.71; Cl, 11.83; N, 4.67. Found: C, 68.22; H, 5.00; Cl,
11.23; N, 4.41.
4.1.1.1.2. 40-Chloro-3-hydroxy-4-methoxychalcone (10). Yield
97%, yellow crystals, m.p. 195e196 ꢀC (96% EtOH). UV
(MeOH) l/nm: 265, 360. IR (KBr) n/cmꢂ1: 3370 (OH),
1620 (C]O), 1563 (C]C), 1483, 1250 (CeO), 800 (Ce
1
Cl). H NMR (CD3OD) d: 8.04 (d, 2H, H-20, H-60,
J ¼ 8.5 Hz), 7.70 (d, 1H, H-b, J ¼ 15.5 Hz), 7.69 (s, 1H,
OH), 7.52 (d, 2H, H-30, H-50, J ¼ 8.8 Hz), 7.45 (d, 1H, H-a,
J ¼ 15.5 Hz), 7.09 (s, 1H, H-2), 6.77 (s, 2H, H-5, H-6), 3.82
(s, 3H, OCH3). 13C NMR (CD3OD) d: 191.4 (C]O), 158.1
(C-4), 156.7 (C-3), 149.8 (C-b), 139.9 (C-40), 138.5 (C-10),
131.1 (C-20, C-60), 129.9 (C-30, C-50), 129.3 (C-1), 119.8 (C-
a), 117.9 (C-6), 116.8 (C-5), 111.2 (C-2), 55.7 (OCH3).
Mass (m/z, %): 288 (Mþ, 100), 273 (50), 271 (36), 257 (13),
253 (49), 177 (26), 139 (27), 111 (32). Anal. Calc. for
C16H13ClO3 (%): C, 66.56; H, 4.54; Cl, 12.28. Found: C,
66.80; H, 4.64; Cl, 12.16.
4.1.1.2. A hot variant of the ClaiseneSchmidt condensation
[33]. Solid KOH (4.2 g, 75.0 mmol) was added to a mixture
of p-chloroacetophenone (274 ml, 2.1 mmol) and aryl aldehyde
(2.1 mmol) in MeOH (4.2 ml) and H2O (2.1 ml). The resulting
solution was refluxed for 1.5 h, then cooled in an ice-water
bath and acidified with 8.4 ml conc. HCl. The solution was di-
luted with water (20 ml) and stored in a refrigerator overnight.
The formed crystals were filtrated, washed with water, dried
out and recrystallised.
4.1.1.1.3. 40-Chloro-3-methoxychalcone (13). Yield 94%,
white crystals, m.p. 65e66 ꢀC (MeOH). UV (MeOH) l/nm:
310. IR (KBr) n/cmꢂ1: 1650 (C]O), 1576 (C]C), 1247
4.1.1.2.1. 40-Chloro-4-hydroxy-3,5-dimethoxychalcone (12).
Yield 83%, yellow crystals, m.p. 126e128 ꢀC (MeOH). UV
(MeOH) l/nm: 213.5, 261.0, 371.5. IR (KBr) n/cmꢂ1: 3432
(OH), 1643 (C]O), 1590 (C]C), 1287 (CeOeC), 821
1
(CeO), 777 (CeCl). H NMR (CDCl3) d: 7.96 (d, 2H, H-20,
1
H-60, J ¼ 8.5 Hz), 7.77 (d, 1H, H-b, J ¼ 15.5 Hz), 7.45 (d,
1H, H-a, J ¼ 15.5 Hz), 7.44 (d, 2H, H-30, H-50, J ¼ 8.5 Hz),
7.34 (t, 1H, H-5, J ¼ 7.8 Hz), 7.22 (d, 1H, H-6, J ¼ 7.8 Hz),
7.14 (s, 1H, H-2), 6.97 (dd, 1H, H-4, J ¼ 8.0, 1.8 Hz), 3.85
(CeCl). H NMR (DMSO-d6) d: 8.15 (d, 2H, H-20, H-60,
J ¼ 8.8 Hz), 7.77 (d, 1H, H-b, J ¼ 15.5 Hz), 7.67 (d, 1H,
H-a, J ¼ 15.5 Hz), 7.61 (d, 2H, H-30, H-50, J ¼ 8.8 Hz), 7.19
(s, 2H, H-2, H-6), 3.82 (s, 6H, 2 ꢃ OCH3). 13C NMR

K.L. Lahtchev et al. / European Journal of Medicinal Chemistry 43 (2008) 2220e2228
2227
(DMSO-d6) d: 187.9 (C]O), 148.1 (C-3, C-5), 146.0 (C-b),
139.1 (C-40), 137.9 (C-10), 136.6 (C-4), 130.4 (C-20, C-60),
128.9 (C-30, C-50), 125.0 (C-1), 118.7 (C-a), 107.1 (C-2, C-
6), 56.2 (2 ꢃ OCH3). Mass (m/z, %): 318 (Mþ, 100), 303
(9), 287 (74), 207 (11), 139 (27), 111 (25). Anal. Calc. for
C17H15ClO4 (%): C, 64.06; H, 4.74; Cl, 11.12. Found: C,
64.20; H, 4.86; Cl, 10.85.
4.1.1.2.2. 40-Chloro-2-hydroxy-3-methoxychalcone (14).
Yield 80%, yellow crystals, m.p. 129e131 ꢀC (MeOH). UV
(MeOH) l/nm: 215, 321. IR (KBr) n/cmꢂ1: 3279 (OH),
1655 (C]O), 1595 (C]C), 1210 (CeOeC), 834 (CeCl).
1H NMR (CDCl3) d: 8.03 (d, 2H, H-b, J ¼ 15.8 Hz), 7.97
(d, 2H, H-20, H-60, J ¼ 8.8 Hz), 7.71 (d, 1H, H-a,
J ¼ 15.8 Hz), 7.46 (d, 2H, H-30, H-50, J ¼ 8.8 Hz), 7.14e
7.19 (m, 1H, H-5), 6.83e6.91 (m, 2H, H-4, H-6), 6.36 (s,
1H, OH), 3.92 (s, 3H, OCH3). 13C NMR (CDCl3) d: 189.85
(C]O), 146.8 (C-3), 145.9 (C-2), 140.7 (C-b), 138.9 (C-40),
136.7 (C-10), 129.9 (C-20, C-60), 128.8 (C-30, C-50), 122.9
(C-a), 121.8 (C-5), 121.0 (C-1), 119.7 (C-6), 112.0 (C-4),
56.2 (OCH3). Mass (m/z, %): 288 (Mþ, 100), 271 (75), 257
(28), 177 (32), 149 (56), 139 (66). Anal. Calc. for
C16H13ClO3 (%): C, 66.56; H, 4.54; Cl, 12.28. Found: C,
66.66; H, 4.75; Cl, 12.56.
supplemented with 50 mg/ml adenine, histidine or lysine but
lacked one of the listed compounds.
4.2.3. Antifungal assays
The antifungal activity of chalcones was evaluated by the
agar dilution method using rich YPD medium for all yeast
strains. Stock solutions of compounds prepared in 96%
EtOH were added to each medium resulting in concentrations
ranging from 20 to 50 mg/ml. Yeast cells were suspended into
water and serially diluted to cell densities of 109, 107, 105 and
103 cells/ml. These cell suspensions were filled into wells of
metal replicator and plated onto agar surfaces. The MIC for
each compound was defined as the lowest concentration,
which completely inhibited the yeast growth after incubation
time. Drug-free solution was used as a blank control and keto-
konazol was used as a positive control.
4.2.4. Mutagenicity assays
The PV-2 and PV-3 strain cells were grown on liquid YPD
medium and spread onto YPD plates. In the centre of each
plate was placed filter paper and solutions containing 30 and
40 mg/ml of compounds tested were poured. These plates
were grown at 30 ꢀC for 2 days and replica-plated onto omis-
sion media lacking adenine, histidine and lysine. After another
2 days of incubation, plates were inspected for the appearance
of ring of prototrophic colonies around the filter paper.
4.2. Microbiology
4.2.1. Strains
The number, genotypes and sources of yeast strains used are
listed in Tables 2e4. They included 16 haploids and 1 diploid of
H. polymorpha, three haploids of K. lactis as well as 9 haploid
and 4 diploid strains belonging to S. cerevisiae. Diploid S. cer-
evisiae strain PV-3 was used in experiments for testing of
some chalcones for their mutagenic and recombinogenic activ-
ities. All the S. cerevisiae and K. lactis strains were grown at
30 ꢀC and those belonging to H. polymorpha at 37 ꢀC. The
isolation and genetic properties of strains belonging to H. poly-
morpha genetic breading stock were described previously [21].
Auxotrophic mutations arg1, ade1, ade3, leu2, ura3, met2, met4
and cys1 require arginine, adenine, leucine, uracil, methionine
and cysteine for growth, respectively. Mutations cds1, cds2
and cds3 are recessive alleles conferring sensitivity to cadmium
ions, whereas CDR1 is a dominant mutation conferring in-
creased resistance to cadmium ions. Mutation cat1 abolishes
the function of catalase enzyme and mutation rgh3 affects cell
separation. Mutation gsh1 is involved in glutathione biosynthe-
sis [20]. With MATa and MATa the alleles of mating type locus
for K. lactis and S. cerevisiae are designated.
4.2.5. Determination of the concentrations of the chalcones
1 and 5 in the liquid YPD media
Stock solutions of chalcones 1 and 5 prepared in 96% EtOH
were added to liquid YPD medium resulting in concentrations
of 30 mg/ml. The media were inoculated with 105 cells/ml of
H. polymorpha strain (WT1). The obtained solutions were in-
cubated at 37 ꢀC. Samples were taken after 24 and 48 h incu-
bation and centrifuged. The concentrations of the chalcones in
the culture supernatants were measured at 308 nm (for com-
pound 1) and at 361.5 nm (for compound 5). Solutions of chal-
cones 1 and 5 in YPD media (30, 25, 15, 10 and 5 mg/ml) were
used as references. Blank samples were YPD media. The con-
trol experiments were conducted in YPD media without yeast
cells. Every assay was carried out in triplicates.
Acknowledgements
4.2.2. Media
This work was supported in part by the Bulgarian National
Science Fund (Contract X-1514). The authors are very much
thankful to Dr. D. Staneva, Institute of Molecular Biology,
Bulgarian Academy of Sciences for providing with K. lactis
strains, to Dr. P. Denkova, Laboratory of Nuclear Magnetic
Resonance, Institute of Organic Chemistry with Centre of
Phytochemistry for the help with interpreting the NMR spectra
and to Prof. Dr. V. Bankova, Institute of Organic Chemistry
with Centre of Phytochemistry for the creative support.
All the strains were maintained and propagated onto solid
YPD media (0.5% yeast extract, 0.5% bacto peptone, 0.5%
glucose). The chalcones were dissolved into 96% ethanol
and incorporated into YPD medium by the addition of appro-
priate amounts into hot and sterile media. In experiments with
detection for mutagenic and recombinogenic action three
kinds of omission media containing 0.7% yeast nitrogen
base and 1% glucose were used. These media were

2228
K.L. Lahtchev et al. / European Journal of Medicinal Chemistry 43 (2008) 2220e2228
[15] R.K. Mortimer, J.R. Johnson, Genetics 113 (1986) 35e43.
References
[16] K.L. Lahtchev, M. Pesheva, O. Tzvetanov, J. Wine Res. 2 (1991) 191e
201.
[1] P. Boeck, P.C. Leal, R.A. Yunes, V.C. Fiiho, S. Lopez, M. Sortino,
A. Escalante, R.L.E. Furlan, S. Zacchino, Arch. Pharm. Chem. Life
Sci. 338 (2005) 87e95.
[2] L. Ni, C.Q. Meng, J.A. Sikorski, Expert Opin. Ther. Pat. 14 (2004)
1669e1691.
[17] N. Pettersson, C. Filipsson, E. Becit, L. Brive, S. Hohmann, Biol. Cell 97
(2005) 487e500.
[18] H.R. Waterham, V.I. Titorenko, I.-J. van der Klei, W. Harder,
M. Veenhuis, Yeast 8 (1992) 961e972.
[19] This study.
[3] S.N. Lopez, M.V. Castelli, S.A. Zacchino, J.N. Dominguez, C. Lobo,
J. Charris-Charris, J.C.G. Cortes, J.C. Ribas, C. Devia, A.M. Rodriguez,
R.D. Entriz, Bioorg. Med. Chem. 9 (2001) 1999e2013.
[4] V. Opletalova, P. Ricicarova, D. Sedivy, D. Meltrova, J. Krivakova, Folia
Pharm. Univ. Carol. XXV (2000) 21e33.
[20] V.M. Ubiivovk, T.Y. Nazarko, E.G. Stasyk, A.A. Sibirnyi, Microbiolo-
giya 71 (2002) 829e835.
[21] K.L. Lahtchev, V.D. Semenova, I.I. Tolstorukov, I.-J. van der Klei,
M. Veenhuis, Arch. Microbiol. 177 (2002) 150e158.
[22] D. Staneva, D. Uccelletti, F. Farina, P. Venkov, C. Palleschi, Yeast 21
(2004) 41e51.
[5] J.R. Dimmock, D.W. Elias, M.A. Beazely, N.M. Kandepu, Curr. Med.
Chem. 6 (1999) 1125e1149.
[23] D. Staneva, D. Uccelletti, N. Petrova, P. Venkov, C. Palleschi, C. R.
Acad. Bulg. Sci. 58 (2005) 233e238.
[24] D. Uccelletti, D. Staneva, S. Rufini, P. Venkov, C. Palleschi, FEMS Yeast
Res. 5 (2005) 735e746.
[6] V. Opletalova, D. Sedivy, Ceska Slov. Farm. 48 (1999) 252e255.
[7] K. Bowden, A. Dal Pozzo, C.K. Duah, J. Chem. Res., Synop. 12 (1990)
2801e2830.
[8] M. Sato, H. Tsuchiya, M. Akagiri, S. Fujiwara, T. Fujii, N. Takagi,
N. Matsuura, M. Iinuma, Lett. Appl. Microbiol. 18 (1994) 53e55.
[9] H. Tsuchiya, M. Sato, M. Akagiri, N. Takagi, T. Tanaka, M. Iinuma,
Pharmazie 49 (1994) 756e758.
[25] G.R. Fink, R. Lowenstein, J. Bacteriol. 100 (1969) 1126e1127.
[26] J.F. Miquel, Memoires Presentes a la Societe Chimique 7e12 (1961)
1369e1376.
[27] C.-N. Lin, J. Pharm. Pharmacol. 49 (1997) 530e536.
[28] P. Wang, S.J. Wu, Photochem. Photobiol. A: Chem. 77 (1994) 127e
131.
[10] M. Sato, H. Tsuchiya, M. Akagiri, N. Takagi, M. Iinuma, Aust. Dent. J.
42 (1997) 343e346.
[11] A.A. Bilgin, N. Yulug, S. Sarac, A. Tayhan, E.D. Berkman, Hacettepe
Ecz. Fak. Der. 7 (1987) 39e50.Chem. Abstr. 109, 89593k.
[12] D. Batovska, St. Parushev, A. Slavova, V. Bankova, I. Tsvetkova,
M. Ninova, H. Najdenski, Eur. J. Med. Chem. 42 (2007) 87e92.
[13] S. Alam, J. Chem. Sci. 116 (2004) 325e331.
[29] G.J. Martin, J.M. Beiler, S. Avakian, J.N. Moss, U.S. Patent 2,647,148,
1953; Chem. Abstr.
[30] I.S. Arty, H. Timmerman, M. Samhoedi, S. Sastrohamidjojo, H. Van der
Goot, Eur. J. Med. Chem. 35 (2000) 449e457.
[31] W. Davey, J.R. Gwilt, J. Chem. Soc. (1957) 1008e1014.
[32] J.-T. Li, W.-Z. Yang, S.-X. Wang, S.-H. Li, T.-S. Li, Ultrason. Sonochem.
9 (2002) 237e239.
[14] S.G. Inge-Vechtomov, Y.I. Pavlov, V.N. Noskov, M.V. Repnevskaya,
T.S. Karpova, N.N. Khromov-Borisov, L. Chekuolene, D. Chitavichus,
in: D.L. Ashby, D.L. de Serres (Eds.), Progress in Mutation Research,
Elsevier, Amsterdam, 1985, pp. 243e255.
[33] F.A. Davis, B.-C. Chen, J. Org. Chem. 58 (1993) 1751e1753.