Study on the substituents' effects of a series of synthetic chalcones against the yeast Candida albicans

Article abstract of DOI:10.1016/j.ejmech.2006.08.012

A large series of chalcones were synthesized and studied for activity against Candida albicans. The SAR analysis showed that the antifungal activity was highly dependent on the substitution pattern of the aryl rings and correlated to a large extent with the ability of compounds to interact with sulfhydryl groups. The most active were the hydroxylated chalcones as their activity related to the location of the phenolic group in the aryl ring B as follows: o-OH > p-OH ～ 3,4-di-OH > m-OH. These and other correlations obtained strongly contribute to the knowledge for design of anticandidal chalcones.

Full text of DOI:10.1016/j.ejmech.2006.08.012

European Journal of Medicinal Chemistry 42 (2007) 87e92
http://www.elsevier.com/locate/ejmech
Short communication
Study on the substituents’ effects of a series of synthetic
chalcones against the yeast Candida albicans
a
a
a
b
D. Batovska a, , St. Parushev , A. Slavova , V. Bankova , I. Tsvetkova ,
*
M. Ninova b, H. Najdenski b
a Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Street,
Building 9, Sofia 1113, Bulgaria
b The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, Building 26, Sofia 1113, Bulgaria
Received 31 July 2006; received in revised form 16 August 2006; accepted 16 August 2006
Available online 26 September 2006
Abstract
A large series of chalcones were synthesized and studied for activity against Candida albicans. The SAR analysis showed that the antifungal
activity was highly dependent on the substitution pattern of the aryl rings and correlated to a large extent with the ability of compounds to in-
teract with sulfhydryl groups. The most active were the hydroxylated chalcones as their activity related to the location of the phenolic group in
the aryl ring B as follows: o-OH > p-OH w 3,4-di-OH > m-OH. These and other correlations obtained strongly contribute to the knowledge for
design of anticandidal chalcones.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Chalcones; Antifungal activity; Candida albicans; SAR
1. Introduction
in chalcone detoxification in yeasts is based on thiol alkylation
[5]. From a therapeutic standpoint it is also interesting that
chalcones can inhibit glutathione-S-transferases (GSTs), en-
zymes that appear to be involved in drug resistance [3]. To
be of help for developing anticandidal chalcones these mech-
anistic data require extensive study of the structureeactivity
relationships (SARs).
Up-to-now the SAR analysis has shown that the anticandidal
activity of chalcones was lost by their cyclization to correspond-
ing flavanones or by reduction to dihydrochalcones [6]. These
results indicate that the chalcone skeleton is fundamental for
the growth inhibitory property against Candida spp. In addition,
some contradictory data exist about the anticandidal activity of
hydroxyl-chalcones. According to Sato et al. the chalcones lack-
ing phenolic groups were either inactive or having low potency
[7]. At the same time some hydroxyl-chalcones derived from
plants did not show marked activity towards C. albicans [8,9].
Most of the naturally occurring chalcones are hydroxylated in
their aryl ring A and therefore such compounds have mainly
been object of antifungal studies [4]. Little is known about the
position effect of a phenolic group located in the aryl ring B.
Candida albicans is the major human pathogen giving rise
to opportunistic oral and vaginal infections. It is a leading
cause of invasive fungal disease in premature infants, dia-
betics, surgical, AIDS and other immunocompromised pa-
tients. In these cases the intensive prophylactic use of
antifungal drugs leads to emergence of resistant strains of
C. albicans. This causes a great concern for finding suitable
new therapeutics [1].
Chalcones (1,3-diaryl-2-propen-1-ones) seem to be promis-
ing antifungal drug candidates. They have been shown to in-
hibit the growth of various fungi and yeasts, including
representatives of the Candida species [2,3]. It is known that
the mode of the antifungal action of chalcones relates to inhi-
bition of the fungal cell wall [4]. Recently, we have widened
this knowledge showing that at least one of the steps involved
* Corresponding author. Fax: þ359 2 87 00 225.
E-mail address: danibat@orgchm.bas.bg (D. Batovska).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.08.012

88
D. Batovska et al. / European Journal of Medicinal Chemistry 42 (2007) 87e92
Besides this uncertain point, the question if and how the elec-
tronic effects of the aryl substituents correlate with the antican-
didal activity of chalcones still remains unclear. Obviously,
additional studies are necessary to verify the substituents’
effects in the aromatic rings.
In this study, we synthesized a large series of chalcones
with diverse substituents in the aromatic rings, mainly in
ring B or with a modified enone moiety and probed the struc-
tureeactivity correlations towards the yeast C. albicans. Our
objective was to find important SARs for developing new anti-
candidal agents based on the chalcone skeleton.
To verify the hypothesis that the anticandidal activity of
chalcones might be based on the better acceptance of Michael
reaction we studied variously substituted chalcones. Twenty-
two of the compounds had p-EW or p-ED group in ring B
and were differently substituted in ring A (Table 2). As ex-
pected, the chalcones with p-EW group were much more ac-
tive than those having p-ED substituent. However, either
type of substituted chalcones was less effective than the un-
substituted chalcone 1. Its higher activity may be due to inhi-
bition on the enzymes involved in chalcone detoxification,
such as the GSTs. GSTs contribute to phase 2 cell biotransfor-
mation of endo- and xenobiotics by conjugating these com-
pounds with the reduced glutathione. Inhibition of GSTs by
xenobiotics makes the yeast cell more susceptible for alkylat-
ing agents and can thus have toxic consequences [12]. It is
known that within a series of chalcones with various p- or
p0-groups the unsubstituted chalcone has shown the highest in-
hibitory activity against GSTs [13,14].
As it can be seen from Table 2 the chalcones with p-chloro
group were as active as the chalcones with p-ED group prob-
ably because of the poor electron withdrawing ability of the
halogen substituent. The most active ones were the chalcones
with p-hydroxyl group. Obviously, their activity cannot be as-
cribed to increased electrophilicity of the C-b atom and must
depend on some other properties of their molecules.
As expected, the electronic effects of substituents in the
aryl ring A were not significant. The p0-chloro group did not
affect the antifungal activity of the chalcones with p-EW or
p-OH group in the ring B and depleted the activity of the other
compounds. The chalcones with methoxylated ring A were in-
active, which might be due to sterical reasons.
2. Results and discussion
2.1. Chemistry
A series of 44 chalcones and three diphenyl alkenones were
easily prepared by the ClaiseneSchmidt condensation between
acetophenone derivatives or acetone and appropriate aryl alde-
hydes (Table 1, Fig. 1). Five new compounds were synthe-
sized. The desired products were obtained on an average
yield of 80% after purification. Their structures were estab-
lished with UV, IR, NMR, mass spectrometry and elemental
1
analysis. H NMR spectra showed that typically for this reac-
tion the E-isomers were specifically generated. The spectral
data of the novel compounds are presented in Section 4.
2.2. Microbiology
Antifungal activity was checked by the agar cup method of
Spooner and Sykes with C. albicans grown on Sabouraud agar
[10]. Chalcones giving an inhibitory zone with a diameter (d )
of at least 15 mm were initially considered as active. The min-
imal inhibitory concentration (MIC) of all compounds was de-
termined by the method of serial dilutions as described by
Hindler [11]. The hydroxylated chalcones gave relatively
small inhibitory zones with diameters between 14 and
21 mm, which might be due to their low diffusion potential
into the agar media. All these compounds were highly active
when tested in liquid media (meat-pepton broth) yielding
MICs around 62.5 mg/ml.
2.3.2. Effect of the position of hydroxyl groups
Several studies including this one reveal the importance of
phenolic groups for the antifungal activity of chalcones against
various Candida spp. [3]. According to some authors antican-
didal chalcones must possess 2-hydroxyl function [6,7] while
Bilgin et al. have demonstrated that the precise location of the
hydroxyl group was not important [15].
To clarify this matter a number of chalcones with hydroxyl
groups in different positions in ring B were examined for ac-
tivity against C. albicans. Regarding to the position of the phe-
nolic group the antifungal activity decreased in the following
order: o-OH (8) > p-OH (10) w 3,4-di-OH (11) > m-OH (9)
(Table 3). Interestingly, this result correlated with the sugges-
tion of Dinkova-Kostova et al. that the presence of o-, but not
m- or p-hydroxyl group had a powerful accelerating effect on
the reactivity of chalcones with sulfhydryl groups [16]. These
authors have proposed a mechanism by which the o-hydroxyl
groups are involved in extensive intermolecular hydrogen
bond formation facilitating the addition of thiols. However,
it is very likely that the antifungal action of o-hydroxyl-chal-
cones works in a multiple mechanism including not only thiol
alkylation. This view is supported by the fact that these com-
pounds are also known as strong inhibitors of GSTs and are
supposed to chelate the metals in metalloproteases [3].
2.3. Structureeactivity relationships
2.3.1. Electronic effects of the p-substituents in B ring
Our study of the electronic effects of the aryl substituents of
chalcones was based on the knowledge that the antifungal
mechanism of these compounds is partially by thiol alkylation
[5]. Such reactions may occur between chalcones and the sulf-
hydryl groups of intracellular thiols like cysteine, homocys-
teine and glutathione, representing a prime line of cell
defence. Thiol alkylation of chalcones is a Michael addition
at their electrophilic centre C-b resulting in adducts’ forma-
tion. It may be facilitated by EW groups located in p-position
in the aryl ring B and hampered by p-electron donating (ED)
groups in the same ring. Meanwhile, the electronic effects of
the substituents in ring A if there are any should be negligible.

D. Batovska et al. / European Journal of Medicinal Chemistry 42 (2007) 87e92
89
Table 1
Substitution pattern of the chalcones studied against C. albicans
R2
R2
R2
R5
O
O
O
R3
R4
R3
R4
H3CO
H3CO
R3
R4
Cl
R5
R5
OCH3
X
Y
Z
Chalcone
Type
R2
R3
R4
R5
1
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Z
H
H
H
H
H
H
H
OH
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2
3
eOCH2Oe
H
4
N(CH3)2
NHCOCH3
NO2
CN
5
H
6
H
7
H
8
H
OH
H
9
H
OH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
H
OH
OH
H
OCH3
OH
OCH3
OCH3
CH3
H
H
H
H
H
eOCH2Oe
Cl
H
N(CH3)2
NHCOCH3
NO2
CN
H
H
H
H
OH
H
H
OH
H
OH
OH
H
OCH3
OH
OCH3
OH
OH
OCH3
OCH3
OCH3
OCH3
H
H
H
OCH3
OCH3
OCH3
OCH3
CH3
H
H
H
OCH3
H
OCH3
H
H
H
Z
H
eOCH2Oe
H
Cl
H
Z
H
Z
N(CH3)2
NHCOCH3
CN
H
Z
H
H
Z
H
H
Z
OCH3
H
H
H
Z
OCH3
CH3
H
Z
H
H
This table represents the structures of all the chalcones synthesized.
In contrast, in our previous study we have shown that m-OH
group in ring B was the most favourable for the antifungal ac-
tivity of hydroxyl-chalcones against a large number of yeast
strains belonging to species other than Candida [5]. This dif-
ference underlines the importance of the yeast genotype along
with the necessity of SAR studies.
The introduction of p0-Cl group in the aryl ring A almost
equalized the MIC values: o-OH (23) w p-OH (25) w 3,4,-di-
OH (26) > m-OH (24). Presence of the halogen was
advantageous only when it was inserted into the chalcone with
m-OH group (9) as this led to a two-fold increase of its activity.
2.3.3. Effect of the combination of hydroxyl and methoxy
groups in ring B
Table 3 shows the potency of chalcones having one hy-
droxyl and one methoxy group in vicinal positions. For the
hydroxyl group it was observed that the location at C-2 was
the most favourable, followed by positions C-4 and C-3, of

90
D. Batovska et al. / European Journal of Medicinal Chemistry 42 (2007) 87e92
O
decided to check if elongation of the conjugated linker be-
tween the aromatic rings of chalcones would increase their ac-
tivity. We synthesized the diphenyl alkenones 45, 46 and 47
(Fig. 1) and examined their activity against C. albicans. Sur-
prisingly, none of these compounds showed any activity
when tested in high concentration (2000 mg/ml). This result in-
dicates that insertion of one or more double bonds from either
side of the carbonyl group is unfavourable and supports the
fact that the diphenyl-2-propen-1-one skeleton is fundamental
for the antifungal activity of chalcones.
m
n
45, n=0; m=2; 46, n=m=1; 47, n=m=2
Fig. 1. Chalcones with elongated conjugated linker between the aromatic
rings. It presents the formulas of three diphenyl alkenones synthesized and
studied against Candida albicans.
which the substitution at C-4 led to much more active com-
pounds. It turned out that the best surrounding for the p-hy-
droxyl group was two vicinal methoxy groups.
Introduction of p0-Cl group led to an eight-fold decrease in
the activity of compound 12 having o-OH and m-OCH3 groups
and did not affect the activity of the chalcone 13 with m-OH
and p-OCH3 functions.
3. Conclusion
This is the first SAR study to show that the potency of the
chalcones against C. albicans to a large extent depends on
their ability to interact with sulfhydryl groups. This conclusion
was based on how the electronic effects of the aryl substituents
influenced the antifungal activity. The hydroxyl-chalcones
were the most active compounds. Their antifungal activity
strongly correlated with the location of the hydroxyl group
in ring B. The o-position was most favourable, which sup-
ported the presumption for a thiol alkylation of these chal-
cones. Insertion of one and more double bonds between the
phenyl rings did not produce active compounds. Although
our study shows that chalcones most probably interact with
thiol cellular components of C. albicans we consider that the
mechanism of their action is multiple and complex and
deserves further investigation.
The introduction of one, two or three methoxy groups or
a 3,4-methylenedioxy group in ring B of the non-hydroxylated
chalcones produced inactive compounds, which was in consis-
tence with the electronic effects of these substituents.
2.3.4. Influence of the position of the keto-group and length
of the conjugated linker between both aromatic rings
From a structural point of view chalcones could be also
considered as aryl styryl ketones. Some non-aryl, but conju-
gated styryl ketones are known to display activity against
C. albicans as they inhibit one or more of the enzymes in
the glutathione metabolic pathway [17,18]. Therefore we
Table 2
Electronic effects of p-substituents in ring B on the activity of the chalcones against C. albicans
O
2'
A
5'
2
1'
1
3
4
3'
4'
B
5
(p-position)
6'
6
Ring Aa
Ring B
Unsubstituted ring B
eH
Electron withdrawing groups in p-position in ring B
eNO2
eCN
eCl
dinh (mm)
MIC (mg/ml)
dinh (mm)
MIC (mg/ml)
dinh (mm)
MIC (mg/ml)
dinh (mm)
MIC (mg/ml)
40-H
30 ꢀ 0
125
500
500
17 ꢀ 1
15 ꢀ 1
n.d.
250
250
n.d.
17 ꢀ 1
23 ꢀ 1
0
250
250
500
0
0
0
500
500
500
40-Cl
0
0
30,40,50-Trimethoxy-
Electron donating groups in p-position in ring B
eOH
eN(CH3)2/eOCH3
dinh (mm)
eCH3/eNH(CO)CH3
dinh (mm)
dinh (mm)
MIC (mg/ml)
MIC (mg/ml)
MIC (mg/ml)
40-H
15 ꢀ 1
21 ꢀ 1
n.d.
62.5
62.5
n.d.
0
0
0
500
500
500
0
0
0
500
500
500
40-Cl
30,40,50-Trimethoxy-
The anticandidal activities of the chalcones having p-electron withdrawing or p-electron donating group in the aryl ring B are compared. The aryl ring A of the
same compounds is unsubstituted or having 40-chloro or 30,40,50-trimethoxy-groups.
a
The substitution patterns of the aryl ring A respond to the types X, Y and Z from Table 1.

D. Batovska et al. / European Journal of Medicinal Chemistry 42 (2007) 87e92
91
Table 3
Effect of the position of the phenolic group and its surrounding in ring B on
the chalcone activity against Candida albicans
then kept in refrigerator overnight. The product crystals
were filtrated and washed carefully with ice water and cold
MeOH to neutral reaction. The resulting chalcones were puri-
fied by recrystallization except for the compounds 9, 19 and
20, which were subjected to flash chromatography over silica
gel using toluene, toluene/Et2O 8:1 or PE/acetone 3:1 as the
eluent [5].
R2
R2
O
O
R3
R4
R3
R4
Cl
R4
R4
X
Y
4.1.2. Synthesis of diphenyl alkenones 45, 46 and 47
Compound Type R2
R3
R4
R5
dinh
(mm)
MIC
(mg/ml)
Benzaldehyde or cinnamaldehyde (1.46 mmol) was dis-
solved in 1.0 ml MeOH and acetone (0.73 mmol), and
1.4 ml 6 M NaOH was added. In a few minutes solids formed.
The products were isolated after filtration, washing the crystals
with cold methanol and recrystallization from MeOH.
8
X
X
X
X
X
X
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
21 ꢀ 1
31.25
9
OH
H
H
17 ꢀ 1 250
10
11
12
13
23
24
25
26
27
28
29
30
31
33
18
34
H
OH
OH
H
15 ꢀ 1
15 ꢀ 1
12 ꢀ 0
0
62.5
H OH
OH OCH3
62.5
62.5
H
OH
H
OCH3
H
500
62.5
4.1.3. Structural data
OH
H
16 ꢀ 0
OH
H
H
20 ꢀ 0 125
4.1.3.1. p-Cyanochalcone (7). Yield 98%, white crystals, m.p.
140e141 ꢁC (MeOH). UV (MeOH): 225, 230, 305 nm. IR
(KBr): 2210 (C^N), 1650 (C]O), 1592 (C]C) cmꢂ1. MS
H
OH
OH
H
21 ꢀ 1
62.5
62.5
500
500
H
OH
OH OCH3
14 ꢀ 0
0
0
ꢃ
m/z [Mþ ] 233 (100), [M ꢂ 77]þ 156 (39), [M ꢂ 105]þ 128
H
H
H
H
H
H
H
OH
OCH3
OH
OH
H
OCH3
OCH3
OCH3
OCH3
eOCH2Oe
OCH3
19 ꢀ 1 250
(33), [M ꢂ 128]þ 105 (53), [M ꢂ 156]þ 77 (59). 1H NMR
(CDCl3, 250 MHz): d 8.00e8.05 (m, 2H, H-20, H-60), 7.77
(d, J ¼ 15.8 Hz, 1H, H-b), 7.67e7.75 (m, 3H, H-2, H-6, H-
40), 7.60 (d, J ¼ 15.8 Hz, 1H, H-a), 7.44e7.65 (m, 4H, H-30,
H-50, H-3, H-5). 13C NMR (CDCl3, 250 MHz): d 189.7
(C]O), 142.0 (C-b), 139.2, 137.6 (C-10, C-1), 133.3 (C-40),
132.6 (C-3, C-5), 128.8, 128.7, 128.5 (C-20, C-30, C-50, C-60,
C-2, C-6), 125.1 (C-a), 118.3 (C^N), 113.5 (C-4). Anal. for
C16H11NO. Calc. (%): C, 82.38; H, 4.75; N, 6.00. Found
(%): C, 82.49; H, 4.96; N, 6.15.
OCH3 15 ꢀ 1 125
H
H
H
15 ꢀ 1 500
OCH3
0
0
0
500
500
500
OCH3 OCH3
The table represents data about the anticandidal activity of the chalcones
having hydroxyl groups or a combination of hydroxyl and methoxyl groups
in ring B.
4. Experimental protocols
Infrared and UV spectra were recorded on Bruker IFS 113V
and Helios gamma UVevis spectrophotometers, respectively.
1H and 13C NMR spectra were obtained with Bruker AM 250
spectrometer with tetramethylsilane as internal reference.
Chemical shifts are given in ppm (d-scale); coupling constants
(J ) are in Hz. Splitting patterns are described as singlet (s),
doublet (d), triplet (t) and multiplet (m). Mass spectral analy-
ses were accomplished on a HewlettePackard 5972 using
Mass Selective Detector with EI (70 eV). The melting points
were obtained using Mel-Temp 1102D-230 VAC and were un-
corrected. The reactions were monitored on silica gel 60 F254
using PE/acetone 7:3 or toluene/Et2O 4:1. Flash chromatogra-
phy was performed for purification of the chalcones on silica
gel 60 (230e400 mesh ASTM) using eluent PE/acetone 7:3.
4.1.3.2. 4-Acetamido-30,40,50-trimethoxychalcone (40). Yield
85%, yellow crystals, m.p. 186e188 ꢁC (MeOH). UV
(MeOH): 345 nm. IR (KBr): 3350 (NH), 1700 (NHeC]O,
amide-I), 1643 (C]O), 1550 (C]C), 1496 (CeNeH, am-
ꢃ
ide-II), 1300 (CH3) cmꢂ1. MS m/z [Mþ ] 355 (100),
[M ꢂ 15]þ 340 (50), [M ꢂ 31]þ 324 (22), [M ꢂ 43]þ 312
(19), [M ꢂ 57]þ 298 (24), [M ꢂ 160]þ 195 (15), [M ꢂ 167]þ
1
188 (6), [M ꢂ 188]þ 167 (5). H NMR (CDCl3, 250 MHz):
d 7.87 (bs, 1H, NH), 7.76 (d, J ¼ 15.5 Hz, 1H, H-b), 7.60 (s,
4H, H-2, H-3, H-5, H-6), 7.40 (d, J ¼ 15.5 Hz, 1H, H-a),
7.26 (s, 2H, H-20, H-60), 3.92 (s, 9H, 3 ꢄ CH3O), 2.19 (s,
3H, CH3). 13C NMR (CDCl3, 250 MHz): d 189.3 (C]O),
168.6 (CONH), 153.1 (C-30, C-50), 144.2 (C-b), 142.4 (C-4),
140.2 (C-40), 133.6 (C-10), 130.6 (C-1), 129.4 (C-2, C-6),
120.6 (C-a), 119.7 (C-3, C-5), 106.1 (C-20, C-60), 60.9
(CH3O), 56.4 (2 ꢄ CH3O), 24.6 (CH3). Anal. for
C20H21NO5. Calc. (%): C, 67.59; H, 5.96; N, 3.94. Found
(%): C, 67.86; H, 6.14; N, 3.65.
4.1. Chemistry
4.1.1. General procedure for synthesis of chalcones
Acetophenone (97 ml, 0.83 mmol), p-chloroacetophenone
4.1.3.3. 4-Cyano-30,40,50-trimethoxychalcone (41). Yield 91%,
pale yellow crystals, m.p. 171e173 ꢁC (MeOH). UV (MeOH):
308 nm. IR (KBr): 2214 (C^N), 1657 (C]O), 1575 (C]C),
(84 ml,
0.65 mmol)
or
3,4,5-trimethoxyacetophenone
(150 mg, 0.71 mm) was added to equimolar quantities of ap-
propriate aryl aldehydes or cinnamaldehyde and dissolved in
MeOH (0.8 ml). To this solution 6 M NaOH (0.4 ml) was
added and the reaction mixture was stirred for 40 min and
ꢃ
1336 (CeOeAr) cmꢂ1. MS m/z [Mþ ] 323 (100), [M ꢂ 15]þ
308 (32), [M ꢂ 31]þ 292 (15), [M ꢂ 43]þ 280 (35),
[M ꢂ 128]þ 195 (29), [M ꢂ 167]þ 156 (20), [M ꢂ 195]þ 128

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D. Batovska et al. / European Journal of Medicinal Chemistry 42 (2007) 87e92
1
(22). H NMR (DMSO-d6, 250 MHz): d 7.77 (d, J ¼ 15.5 Hz,
1H, H-b), 7.72 (s, 4H, H-2, H-3, H-5, H-6), 7.54 (d,
J ¼ 15.5 Hz, 1H, H-a), 7.27 (s, 2H, H-20, H-60), 3.95 (s, 6H,
CH3O), 3.94 (s, 3H, CH3O). 13C NMR (DMSO-d6,
250 MHz): d 188.4 (C]O), 153.2 (C-30, C-50), 143.0 (C-1),
141.9 (C-b), 139.2 (C-40), 132.8 (C-10), 132.7 (C-3, C-5),
129.6 (C-2, C-6), 124.8 (C-a), 118.3 (C^N), 113.5 (C-4),
106.3 (C-20, C-60), 61.0 (CH3O), 56.4 (2 ꢄ CH3O). Anal. for
C19H17NO4. Calc. (%): C, 70.58; H, 5.30; N, 4.33. Found
(%): C, 70.56; H, 5.51; N, 4.04.
diameter. One hundred microliter of each sample, dissolved
in 96% EtOH (5000 mg/ml) was added to the appropriate
well. For pre-diffusion the Petri dishes were placed at 4 ꢁC
for 2 h. The antifungal activity was estimated by the diameter
of inhibitory zones in the agar layer after incubation at 37 ꢁC
for 48 h. Control experiments were carried out with the pure
solvent.
4.2.2. Measurement of MIC
MIC was determined by serial dilution of each chalcone
(0.0e2000 mg/ml) in test tubes using meat-pepton broth.
Each test tube was inoculated with fungal suspension contain-
ing 105 cells/ml and incubated at 37 ꢁC for 24 h. The lowest
dilution that visibly showed no growth compared to drug-
free broth inoculated with microbial suspension was consid-
ered the MIC. For more precise detection, tubes that showed
no visible growth were streaked on fresh meat-pepton agar
plates, incubated at 37 ꢁC for 24 h and checked for growth.
4.1.3.4. 3,30,40,50-Tetramethoxychalcone (42). Yield 78%, yel-
low crystals, m.p. 65e67 ꢁC (MeOH). UV (MeOH): 320 nm.
IR (KBr): 1650 (C]O), 1571 (C]C), 1329 (CeOeAr),
1214 (CeO) cmꢂ1. MS m/z [Mþ] 328 (100), [M ꢂ 15]þ 313
(41), [M ꢂ 31]þ 297 (46), [M ꢂ 43]þ 285 (22), [M ꢂ 133]þ
195 (27), [M ꢂ 167]þ 161 (16), [M ꢂ 195]þ 133 (10). 1H
NMR (CDCl3, 250 MHz): d 7.78 (d, J ¼ 15.5 Hz, 1H, H-b),
7.46 (d, J ¼ 15.5 Hz, 1H, H-a), 7.35 (t, J ¼ 8.0 Hz, 1H, H-
5), 7.22e7.27 (m, 3H, H-20, H-60, H-6), 7.17 (t, J ¼ 2.0 Hz,
1H, H-2), 6.97 (dd, 1 H, J1 ¼ 2.8 Hz, J2 ¼ 8.0 Hz, H-4), 3.95
(s, 6H, 2 ꢄ CH3O), 3.94 (s, 3H, CH3O), 3.83 (s, 3H, CH3O).
13C NMR (CDCl3, 250 MHz): d 189.2 (C]O), 159.9 (C-3),
153.1 (C-30, C-50), 144.6 (C-b), 142.4 (C-40), 136.2 (C-1),
133.4 (C-10), 129.9 (C-4), 122.1 (C-a), 121.0 (C-6), 116.0
(C-4), 113.7 (C-2), 106.0 (C-20, C-60), 60.9 (CH3O), 56.4
(2 ꢄ CH3O), 55.3 (CH3O). Anal. for C19H20O5. Calc. (%):
C, 69.50; H, 6.14. Found (%): C, 69.61; H, 6.40.
Acknowledgments
This work was supported by the Bulgarian National Foun-
dation for Scientific Research (Contract X-1514). We are very
much thankful to our colleague Dr. I. Timcheva for the fruitful
discussions.
References
4.1.3.5. 30,40,50-Trimethoxy-4-methylchalcone (44). Yield 96%,
white crystals, m.p. 120e123 ꢁC (MeOH). UV (MeOH):
325 nm. IR (KBr): 1650 (C]O), 1575 (C]C) cmꢂ1. MS m/
z [Mþ] 312 (100), [M ꢂ 15]þ 297 (72), [M ꢂ 31]þ 281 (25),
[M ꢂ 43]þ 269 (29), [M ꢂ 117]þ 195 (26), [M ꢂ 167]þ 145
(19), [M ꢂ 195]þ 117 (13). 1H NMR (CDCl3, 250 MHz):
d 7.79 (d, J ¼ 15.5 Hz, 1H, H-b), 7.54 (d, J ¼ 8.0 Hz, 2H,
H-2, H-6), 7.43 (d, J ¼ 15.5 Hz, 1H, H-a), 7.27 (s, 2H, H-20,
H-60), 7.22 (d, J ¼ 8.3 Hz, 1H, H-3, H-5), 3.94 (s, 6H,
2 ꢄ CH3O), 3.93 (s, 3H, CH3O), 2.39 (s, 3H, CH3). 13C
NMR (CDCl3, 250 MHz): d 189.3 (C]O), 153.1 (C-30, C-
50), 144.8 (C-b), 142.4 (C-40), 141.1 (C-4), 133.6 (C-1),
132.1 (C-10), 129.7 (C-3, C-5), 128.4 (C-2, C-6), 120.8 (C-
a), 106.1 (C-20, C-60), 60.9 (CH3O), 56.4 (2 ꢄ CH3O), 21.5
(CH3). Anal. for C19H20O4. Calc. (%): C, 73.06; H, 6.45.
Found (%): C, 73.10; H, 6.49.
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