Examination of growth inhibitory properties of synthetic chalcones for which antibacterial activity was predicted

Article abstract of DOI:10.1016/j.ejmech.2008.05.010

A large series of chalcones were synthesized and studied against Staphylococcus aureus and Escherichia coli. Chalcones were either unsubstituted in ring A or possessed 4′-chloro or 3′,4′,5′-trimethoxy groups. Their other ring B was variously substituted. It was found that the anti-staphylococcal activity of chalcones was related to the energy difference between the two highest occupied molecular orbitals (HOMO and HOMO-1). Presence of hydroxyl group in ring B was not a determinant factor for the anti-staphylococcal activity, but the lipophilicity of ring A of the hydroxyl chalcones was of importance.

Full text of DOI:10.1016/j.ejmech.2008.05.010

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European Journal of Medicinal Chemistry 44 (2009) 2211e2218
http://www.elsevier.com/locate/ejmech
Short communication
Examination of growth inhibitory properties of synthetic chalcones for
which antibacterial activity was predicted
a
a
b
Daniela Batovska a, , Stoyan Parushev , Bistra Stamboliyska , Iva Tsvetkova ,
*
Mariana Ninova b, Hristo Najdenski b
a Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, Bl. 9, Sofia 1113, Bulgaria
b The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, Bl. 26, Sofia 1113, Bulgaria
Received 26 March 2008; received in revised form 17 May 2008; accepted 17 May 2008
Available online 23 May 2008
Abstract
A large series of chalcones were synthesized and studied against Staphylococcus aureus and Escherichia coli. Chalcones were either unsub-
stituted in ring A or possessed 40-chloro or 30,40,50-trimethoxy groups. Their other ring B was variously substituted. It was found that the anti-
staphylococcal activity of chalcones was related to the energy difference between the two highest occupied molecular orbitals (HOMO and
HOMO-1). Presence of hydroxyl group in ring B was not a determinant factor for the anti-staphylococcal activity, but the lipophilicity of
ring A of the hydroxyl chalcones was of importance.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Chalcones; Antibacterial activity; Staphylococcus aureus; Escherichia coli; SAR
1. Introduction
found that some of these compounds had been included in in-
dexed sub-libraries subjected to antibacterial studies against
several bacteria including Staphylococcus aureus and Escher-
ichia coli [4]. The sub-libraries of chalcones with unsubsti-
tuted and methoxylated ring A showed one of the best
inhibitory activities as was estimated by agar-well diffusion
method. The same method was used for the determination of
the minimal inhibitory concentration (MIC) of some of the
individual chalcones. As such only one of the 30,40,50-tri-
methoxychalcone series was synthesized and examined. To
our knowledge, the examination of chalcones by only agar
diffusion techniques may not be reliable enough, particularly
for giving the quantitative measure of their antibacterial
activity. The reason is that some of these compounds have
a low rate of diffusion into the agar media [5]. This problem
can be eliminated by using liquid media for the determination
of the MICs of chalcones.
Chalcones are open-chain flavonoids with common skele-
ton of 1,3-diaryl-2-propen-1-one. Their wide-range biological
properties, including antimicrobial activities, are largely attrib-
uted to the a,b-unsaturated ketone moiety. Introduction of
various substituents into the two aryl rings is also a subject of
interest because it leads to useful SAR conclusions and thus
helps to synthesize pharmacologically active chalcones [1,2].
Recently, we prepared a large series of chalcones and
showed that their potency against various yeast strains de-
pended to a large extent on their ability to interact with yeast’s
intracellular thiols, such as glutathione and cysteine [3]. The
studied chalcones had various functional groups in their ring
B and belonged to three structural types in respect of their
other ring A, namely chalcones with either no substitutions
or possessing 40-chloro or 30,40,50-trimethoxy groups. We
In this study, we enlarged the series of chalcones previously
synthesized by us, preparing some novel hydroxylated com-
pounds and chalcone derivatives with altered linker between
the two aryl rings. Antibacterial effects of all the compounds
Abbreviations: ED, electron donating; EW, electron withdrawing.
* Corresponding author. Tel.: þ359 2 9606 184; fax: þ359 2 87 00 225.
E-mail address: danibat@orgchm.bas.bg (D. Batovska).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.05.010

2212
D. Batovska et al. / European Journal of Medicinal Chemistry 44 (2009) 2211e2218
were investigated towards the most widely distributed patho-
gens in humans e S. aureus and E. coli. The MIC of the com-
pounds was determined by the method of serial dilution
in meat-peptone broth. Based on the results some structuree
activity relationships were found and discussed in the light
of existing hypotheses.
4. Results, discussion and conclusion
Chalcones were not active towards E. coli in concentrations
under 500 mg/ml. Interesting relationships were observed
between the anti-staphylococcal activity of chalcones and their
type of substitution. Presence of certain functional groups in
the two aryl rings A and B was found to be of importance.
2. Chemistry
4.1. Electronic effects of the p-substituent in ring B
We prepared 62 chalcones and their derivatives by a
ClaiseneSchmidt condensation between acetophenones
(acetophenone, p-chloroacetophenone and 3,4,5-trimethoxy-
acetophenone) or acetone and appropriate aryl aldehydes
(Table 1). The desired products were obtained in yields be-
tween 50% and 95% after purification, following procedures
described previously [3,6]. The structures were established
with UV, IR, NMR, mass spectrometry and elemental analy-
Mode of the antibacterial action of chalcones has long been
believed to be due to their reaction with important thiol groups
of essential enzymes via Michael addition at the ketovinyl dou-
ble bond [9]. If this is the case, electronic effect of the p-substit-
uent in ring B is of great importance. p-Electron withdrawing
(EW) group is favourable because it will increase the electro-
philicity of C-b and thus facilitate the nucleophilic attack of
the cellular thiol groups. The opposite is true for the p-ED
groups. To clarify if there is a relationship between the elec-
tronic effect of the p-substituent in ring B and the antibacterial
activity of chalcones we studied 27 chalcones with p-EW or
p-electron donating (ED) group as a single substituent in ring
B. The results obtained for these compounds were compared
to those obtained for chalcones with unsubstituted ring B.
Also, three types of substitutions in ring A e X, Y and Z, were
scrutinized. All the chalcones with p-ED groups in ring B
were inactive under concentration of 500 mg/ml except for com-
pounds 10 and 27 possessing p-OH group. Meanwhile, it was
found that the presence of p-EW groups enhanced the activity
only of chalcones with methoxylated ring A (Z-type) (Table 2).
In order to understand the experimental results a detailed
quantum chemical study was made on all 27 chalcones. Mul-
liken atomic charges of the C-b were calculated at B3LYP/6-
31G* level of theory (Table 2). In most cases the presence of
donor and acceptor groups in ring B had a direct influence on
the charge of this C atom. However, this appeared not to be
a determinant factor for the antibacterial activity of chalcones.
The presence of three methoxy groups in ring A, which we
found to be of great importance for the anti-staphylococcal ac-
tivity, did not contribute to the changes in electrophilicity of
C-b. In other words this electron parameter is not linked to
the antibacterial activity of studied compounds.
1
sis. H NMR spectra showed that only trans-chalcones were
obtained. Most of the chalcones were synthesized at 0 ꢀC
using sodium hydroxide as a catalyst. In this way we ob-
tained 3-hydroxychalcone 9 with a yield of 92% in 24 h.
Our attempt to shorten the reaction time by refluxing a reac-
tion mixture of acetophenone and m-hydroxybenzaldehyde
for 15 min resulted in only one product different from the ex-
1
pected chalcone. Its H NMR spectrum showed no character-
istic signals of the a,b-olefinic protons and 14 aromatic
protons instead of the nine expected ones. In addition, two
more signals were observed e two doublets of doublets for
four protons at d 3.40 and a pentet for one proton at d 4.0.
This information showed that at heating the reaction runs
fast to receive the chalcone, which was further attacked by
acetophenone to give the Michael adduct 56 (Fig. 1). Its
structure was additionally confirmed by the 13C NMR and
mass spectral analyses. To our knowledge this compound
has not been described in the literature up till now. The
same applies to eight other compounds synthesized in this
study (33, 44, 50e54 and 61). Their spectral data are pre-
sented in Section 5.
3. Microbiology
Antibacterial activity was checked by the agar-well diffu-
sion method of Spooner and Sykes with S. aureus and E.
coli grown on meat-peptone agar [7]. Chalcones and their
derivatives giving an inhibitory zone with a diameter (d ) of
at least 15 mm were initially considered active. The minimal
inhibitory concentration (MIC) of all compounds was deter-
mined by the method of serial dilutions as described by
Hindler [8]. None of the compounds tested showed activity
towards the Gram-negative bacterium E. coli while most of
the chalcones inhibited the growth of the Gram-positive bacte-
rium S. aureus. Four of the chalcones with anti-staphylococcal
activity (compounds 30, 40, 44 and 45) gave no inhibitory
zones probably due to their low diffusion potential into the
agar media. When tested in liquid media (meat-peptone broth)
these compounds yielded MICs around 62.5 mg/ml.
We tried to correlate the MIC results of chalcones with
other descriptors. Parameters such as refractivity, polarizabil-
ity, mass, the coefficient of molecular partition octanolewater
(log P), dipole moments, HOMO and LUMO energies, were
disregarded due to their inability to differentiate the active
compounds (data not given). A good correlation was observed
with D, the energy difference between the two highest occu-
pied molecular orbitals (HOMO and HOMO-1) (Table 2).
This descriptor clearly discriminates between the active
(MIC ꢁ 250 mg/ml) and inactive (MIC ¼ 500 mg/ml) chal-
cones (Fig. 2). The horizontal lines on the dendrogram
(Fig. 2) represent the chalcones while the vertical ones show
the similarity between pairs of them based on their MIC and
D values. The parameter D has so far been successfully used
for the prediction of diversified biological activities of

D. Batovska et al. / European Journal of Medicinal Chemistry 44 (2009) 2211e2218
2213
Table 1
Substitution pattern of the chalcones studied against S. aureus
R2
R2
R2
O
O
O
R3
H3CO
H3CO
R3
R4
R3
R4
R4
Cl
R5
OCH3
R5
R5
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
X
X
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Z
H
H
H
Cl
H
2
H
H
eOCH2Oe
H
3
H
H
4
H
H
N(CH3)2
NHCOCH3
NO2
CN
H
5
H
H
H
6
H
H
H
7
H
H
H
8
OH
H
H
OH
H
H
9
H
OH
H
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
45
46
47
48
49
50
51
52
53
54
55
H
H
OH
H
H
OH
H
H
OH
H
OCH3
OH
H
OCH3
OH
H
H
OCH3
OCH3
H
H
H
H
H
H
OCH3
CH3
H
H
H
H
H
H
H
H
H
H
eOCH2Oe
Cl
H
H
H
H
H
N(CH3)2
NHCOCH3
NO2
CN
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
OH
H
H
OH
H
H
H
H
OH
OCH3
OH
OCH3
OCH3
H
OH
H
H
OH
H
H
OCH3
OH
OH
H
H
H
H
OCH3
NO2
H
OH
H
H
OCH3
H
H
H
OCH3
OCH3
OCH3
CH3
H
H
H
OCH3
OCH3
H
H
H
OCH3
H
H
H
H
H
Z
H
H
Cl
H
Z
H
eOCH2Oe
H
H
Z
H
N(CH3)2
NHCOCH3
NO2
H
Z
H
H
H
Z
H
H
H
Z
H
H
CN
H
H
Z
H
OCH3
H
H
Z
H
OCH3
OCH3
OCH3
H
H
Z
H
OCH3
OCH3
H
H
Z
H
OCH3
H
Z
OH
H
Z
OH
H
H
OH
H
Z
H
H
Z
H
OH
OCH3
H
OCH3
OH
CH3
H
Z
H
H
Z
H
H
This table represents the structures of all the chalcones synthesized.

2214
D. Batovska et al. / European Journal of Medicinal Chemistry 44 (2009) 2211e2218
4.2. Presence of hydroxyl group in ring B
Bowden et al. [11] supposed that chalcones inhibit the
growth of S. aureus by interaction with an enzyme receptor
consisting of two areas e a lipophilic region, associated
with ring A and a hydrophilic region, which reacts with a hy-
droxyl group on ring B presumably by hydrogen bond forma-
tion. We studied 21 chalcones having hydroxylated ring B. All
they possessed a lipophilic ring A, which was of structural
type X, Y or Z (Table 3). Our results showed that the presence
of hydroxyl group in ring B alone is not enough for the chal-
cones to exhibit anti-staphylococcal activity. Furthermore, the
activity of chalcones was retained when an additional
hydroxyl group was introduced (compounds 11 and 28). How-
ever, the lipophilicity of ring A was found to be of importance.
The hydroxyl chalcones of the less lipophilic Z-type were all
inactive while the hydroxylated compounds of the more lipo-
philic X- and Y-types showed activity between 62.5 and
O
O
OH
Compound 56
Fig. 1. The Michael adduct (compound 56) obtained from the Claisene
Schmidt condensation between acetophenone and 3-hydroxybenzaldehyde un-
der reflux.
thousands of compounds. It is supposed to be an indirect mea-
sure of the chemical reactivity and lifetime of excited states in
the process of charge-transfer and/or covalent bond formation
to cellular components [10].
Table 2
EIM electronic variables
O
O
O
H3CO
R4
H3CO
R4
R4
Cl
OCH3
X
Y
Z
Compound
Type
R4
Charge on C-b
H (eV)
H-1 (eV)
D (eV)
dinh (mm)
MIC (mg/ml)
1
X
X
X
X
X
X
X
X
X
Y
Y
Y
Y
Y
Y
Y
Y
Y
Z
H
NO2
ꢂ0.127
ꢂ0.138
ꢂ0.136
ꢂ0.128
ꢂ0.119
ꢂ0.123
ꢂ0.118
ꢂ0.114
ꢂ0.125
ꢂ0.124
ꢂ0.155
ꢂ0.133
ꢂ0.125
ꢂ0.116
ꢂ0.121
ꢂ0.118
ꢂ0.114
ꢂ0.125
ꢂ0.128
ꢂ0.139
ꢂ0.138
ꢂ0.128
ꢂ0.120
ꢂ0.124
ꢂ0.120
ꢂ0.121
ꢂ0.128
ꢂ8.424
ꢂ9.294
ꢂ8.991
ꢂ8.559
ꢂ8.019
ꢂ8.208
ꢂ7.943
ꢂ7.322
ꢂ8.565
ꢂ8.559
ꢂ7.074
ꢂ9.126
ꢂ8.694
ꢂ8.127
ꢂ8.316
ꢂ8.058
ꢂ7.416
ꢂ8.692
ꢂ9.113
ꢂ8.586
ꢂ8.598
ꢂ8.424
ꢂ8.046
ꢂ8.176
ꢂ8.204
ꢂ7.644
ꢂ8.131
ꢂ9.099
ꢂ9.396
ꢂ9.342
ꢂ9.207
ꢂ9.072
ꢂ9.072
ꢂ9.057
ꢂ8.978
ꢂ8.982
ꢂ9.207
ꢂ7.209
ꢂ9.430
ꢂ9.585
ꢂ9.180
ꢂ9.180
ꢂ9.163
ꢂ9.080
ꢂ9.051
ꢂ9.261
ꢂ8.667
ꢂ8.679
ꢂ8.505
ꢂ8.316
ꢂ8.494
ꢂ8.519
ꢂ8.409
ꢂ8.847
0.675
0.102
0.351
0.648
1.053
0.864
1.114
1.656
0.417
0.648
0.135
0.304
0.891
1.053
0.864
1.105
1.665
0.359
0.148
0.081
0.081
0.081
0.270
0.318
0.314
0.765
0.716
29 ꢃ 1
125
250
500
500
125
500
500
500
500
500
250
250
500
125
500
500
500
500
250
62.5
62.5
62.5
500
500
500
500
500
6
17 ꢃ 1
7
CN
Cl
16 ꢃ 0
2
0
10
17
16
4
OH
CH3
19 ꢃ 1
0
OCH3
N(CH3)2
NHCOCH3
H
0
0
5
0
18
23
24
19
27
38
35
21
22
39
44
45
40
52
55
47
42
43
0
NO2
CN
17 ꢃ 1
21 ꢃ 1
Cl
OH
0
31 ꢃ 1
CH3
0
0
0
0
0
0
0
0
0
0
0
0
0
OCH3
N(CH3)2
NHCOCH3
H
Z
NO2
CN
Z
Z
Cl
OH
Z
Z
CH3
Z
OCH3
N(CH3)2
NHCOCH3
Z
Z
This table presents data for some EIM variables including energy difference HOMO ꢂ (HOMO-1), where HOMO refers to the highest occupied molecular orbital
as well as data for the anti-staphylococcal activity of chalcones.
Mulliken atomic charges of the C-b; HOMO (H ) and HOMO-1 (H-1) energies and contribution of their difference (D) for the antibacterial activity of chalcones of
X, Y and Z-types having a single substituent at p-position in ring B.

D. Batovska et al. / European Journal of Medicinal Chemistry 44 (2009) 2211e2218
2215
Chalcone 1
Chalcone 10
Chalcone 27
Chalcone 6
Chalcone 23
Chalcone 39
Chalcone 24
Chalcone 44
Chalcone 40
Chalcone 45
Chalcone 2
Chalcone 18
Chalcone 42
Chalcone 43
Chalcone 17
Chalcone 38
Chalcone 19
Chalcone 16
Chalcone 35
Chalcone 4
Chalcone 21
Chalcone 7
Chalcone 22
Chalcone 5
Chalcone 52
Chalcone 55
Chalcone 47
Active
Inactive
0
20
40
60
80
100
120
(Dlink/Dmax)*100
Fig. 2. Hierarchical dendrogram obtained with the Ward’s method of linkage. It shows the distribution of active and inactive chalcones having one substituent at
p-position in ring B, in respect of their D values.
Table 3
Effect of the position of the phenolic group and its surrounding in ring B on the chalcone activity against S. aureus
R2
R2
R2
O
O
O
R3
R4
H3CO
H3CO
R3
R4
R3
R4
Cl
R5
R5
OCH3
R5
X
Y
Z
Compound
Type
R2
R3
R4
R5
dinh (mm)
MIC (mg/ml)
8
X
X
X
X
X
X
X
Y
Y
Y
Y
Y
Y
Y
Y
Y
Z
OH
H
H
H
H
H
22 ꢃ 1
62.5
250
125
125
125
250
500
125
125
125
125
250
62.5
250
500
500
500
500
500
500
500
9
OH
H
H
20 ꢃ 0
10
11
12
13
14
25
26
27
28
29
30
31
32
33
50
51
52
53
54
H
OH
OH
H
H
19 ꢃ 1
H
OH
OCH3
OH
OCH3
H
H
21 ꢃ 1
OH
H
H
19 ꢃ 1
OCH3
OH
H
H
0
H
H
0
OH
H
H
20 ꢃ 0
OH
H
H
H
25 ꢃ 0
H
OH
OH
H
H
31 ꢃ 1
H
OH
OCH3
OH
OCH3
OCH3
H
H
19 ꢃ 0
OH
H
H
0
OCH3
OH
OH
H
H
0
H
H
21 ꢃ 1
H
OCH3
NO2
H
19 ꢃ 1
OH
OH
H
0
0
0
0
0
0
H
H
Z
OH
H
H
H
Z
H
OH
OCH3
OH
H
Z
H
OH
OCH3
H
Z
H
H
This table presents data about the anti-staphylococcal activity of the chalcones having hydroxyl groups or a combination of hydroxyl and methoxyl groups in ring B.

2216
D. Batovska et al. / European Journal of Medicinal Chemistry 44 (2009) 2211e2218
250 mg/ml. The position of hydroxyl group mattered only for
the chalcones with unsubstituted ring A (X-type) as the o-po-
sition was most favourable, followed by p ¼ 3,4 position and
m-position. Introduction of a methoxy group next to o- and
p-hydroxyl groups led to more inactive chalcones while the
activity was kept when the methoxy group was inserted next
to the m-hydroxyl group. Transformation of the m-hydroxy-
chalcone to the Michael adduct (compound 56) resulted in
the loss of activity probably because in this way no Michael
addition of thiol groups was possible.
In conclusion, the energy difference between the two high-
est occupied molecular orbitals (HOMO and HOMO-1) of
chalcones with a single substituent at p-position in ring B dif-
ferentiated the active compounds. Presence of hydroxyl group
in ring B was not a determinant factor for the anti-staphylo-
coccal activity, but the lipophilicity of ring A of the hydroxyl
chalcones was of importance.
5. Experimental and computational details
Since previous papers highlighted the importance of the
bulky substituents at C-5 in ring B for the antibacterial activity
of chalcones we decided to introduce a nitro group at C-5 of
the 40-chloro-2-hydroxychalcone [12]. However, the synthe-
sized chalcone 33 was not active.
Infrared and UV spectra were recorded on Bruker IFS
113 Vand Helios gamma UVevis spectrophotometers, respec-
1
tively. H and 13C NMR spectra were obtained with Bruker
AM 250 spectrometer with tetramethylsilane as internal refer-
ence. Chemical shifts are given in ppm (d-scale); coupling
constants (J ) are in Hz. Splitting patterns are described as sin-
glet (s), doublet (d), triplet (t) and multiplet (m). Mass spectral
analyses 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 uncorrected. The reactions were monitored on silica
gel 60 F254 using petroleum ether/acetone 7:3 or toluene/
Et2O 4:1.
The quantum chemical calculations were performed using
the GAUSSIAN-98 program package [13]. The geometries
of all possible conformational isomers of studied chalcones
were fully optimized at B3LYP/6-31G* level of theory without
constraints. We used just the results for the most stable
conformers according to the calculation in the analysis of
structureeactivity relationship.
4.3. Influence of the position of the keto-group and length
of the conjugated linker between both aromatic rings
In some cases, structural alteration of chalcones, such as
replacement of ring A or ring B by phenanthryl, naphthyl or
pyridinium functions has resulted in a series of compounds
with growth inhibitory properties to S. aureus and E. coli
[1]. We decided to check if elongation of the conjugated linker
between the aromatic rings of chalcones would affect their
antibacterial activity. Thus, we synthesized six diphenyl alke-
nones (57e62) and examined their activity against S. aureus.
These compounds can be regarded as chalcones in the mole-
cules of which one or two double bonds were introduced either
between ring A and the carbonyl function or between the
carbonyl function and the double bond. It was observed that
the insertion of one double bond between ring A and the
carbonyl group led to compounds 60 and 61, which were
two-fold more active than the correspondent chalcones 19
and 49 (Table 4). The compounds with two double bonds
from either one or both sides of the carbonyl group were inac-
tive (MIC ¼ 500 mg/ml).
5.1. Chemistry
5.1.1. General procedure for synthesis of chalcones
Acetophenone (97 ml, 0.8 mmol), p-chloroacetophenone
(84 ml, 0.7 mmol) or 3,4,5-trimethoxyacetophenone (150 mg,
0.7 mmol) was added to equimolar quantities of appropriate
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 then kept in
refrigerator overnight. The product crystals were filtered and
washed carefully with ice-water and cold MeOH to neutral
reaction. The chalcones were purified by recrystallization.
Table 4
Effect of the change of the linker between the two aromatic rings on the acti-
vity against S. aureus
O
3
R3
R4
'
R
R
5.1.1.1. 40-Chloro-2-hydroxy-5-nitrochalcone (33). Yield
56%, orange crystals, m.p. 292e293 ꢀC (MeOH). UV
(MeOH): 287, 456 nm. IR (KBr): 3280 (OH), 1655 (C]O),
n
m
4
'
5
R5
ꢄ
1595 (C]C), 836 (CeCl) cmꢂ1. MS m/z: [Mþ ] 303 (100),
'
R
a
0
0
[M ꢂ 17]þ 286 (65), [M ꢂ 35]þ 268 (20), [M ꢂ 111]þ 192
Entry
n
m
R3
0
R4
R5
R3
R4
R5
dinh
MICa
1
57
58
59
60
61
62
0
0
0
1
1
2
2
2
2
1
1
2
H
H
H
Cl
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
0
0
500
500
(57), [M ꢂ 164]þ 139 (60), [M ꢂ 139]þ 164 (45). H NMR
(DMSO-d6, 250 MHz): d 8.19 (d, J ¼ 15.3 Hz, 1H, H-b),
8.02e8.07 (m, 2H, H-20, H-60), 7.94 (d, J ¼ 15.3 Hz, 1H, H-
a), 7.77 (dd, J1 ¼ 8.3 Hz, J2 ¼ 2.0 Hz, 1H, H-4), 7.64 (d,
J ¼ 2.0 Hz, 1H, H-6), 7.57e7.62 (m, 2H, H-30, H-50), 6.07 (t,
J ¼ 7.4 Hz, 1H, H-3). 13C NMR (DMSO-d6, 250 MHz):
d 189.0 (C]O), 174.4 (C-2), 144.2 (C-b), 138.9 (C-5), 137.5
(C-4), 137.2 (C-10), 130.0 (C-20, C-60), 129.8 (C-a), 129.5
OCH3 OCH3 OCH3
Cl
19 ꢃ 1 500
H
H
0
0
0
250
250
500
OCH3 OCH3 OCH3 OCH3 OCH3 OCH3
H
H
H
H
H
H
This table presents data about the anti-staphylococcal activity of the chalcones
with altered linker between their aromatic rings.
a
The diameter of inhibitory zone dinh is given in mm; the MIC unit is mg/ml.

D. Batovska et al. / European Journal of Medicinal Chemistry 44 (2009) 2211e2218
2217
(C-30, C-50), 121.5 (C-6), 117.5 (C-1), 107.1 (C-3). Anal. for
C15H10ClNO4. Calcd (%): C, 59.32; H, 3.32; Cl, 11.67; N,
4.61. Found (%): C, 59.35; H, 3.37; Cl, 11.70; N, 4.65.
(MeOH): 260, 365 nm. IR (KBr): 3260 (OH), 1636 (C]O),
ꢄ
1561 (C]C), 1328 (CeOeAr) cmꢂ1. MS m/z: [Mþ ] 344
(31), [M ꢂ 15]þ 329 (18), [M ꢂ 31]þ 313 (9), [M ꢂ 149]þ
195 (100). 1H NMR (CD2Cl2, 250 MHz): d 7.69 (d,
J ¼ 15.5 Hz, 1H, H-b), 7.37 (d, J ¼ 15.5 Hz, 1H, H-a), 7.20
(s, 2H, H-20, H-60), 7.15 (dd, J ¼ 8.1, 2.0 Hz, 1H, H-6), 7.09
(d, J ¼ 2.0 Hz, 1H, H-2), 6.77 (d, J ¼ 8.1 Hz, 1H, H-5), 3.89
(s, 3H, CH3O), 3.86 (s, 6H, 2 ꢅ CH3O), 3.81 (s, 3H, CH3O).
13C NMR (CDCl3, 250 MHz): d 191.7 (C]O), 158.8 (C-4),
156.8 (C-30, C-50), 154.5 (C-40), 149.4 (C-b, C-3), 135.6 (C-
1, C-10), 129.5 (C-a), 119.1 (C-6), 117.1 (C-5), 111.2 (C-2),
107.3 (C-20, C-60), 61.3 (CH3O), 56.9 (2 ꢅ CH3O), 55.8
(CH3O). Anal. for C19H20O6. Calcd (%): C, 66.27; H, 5.85.
Found (%): C, 66.35; H, 5.92.
5.1.1.2. 4-Nitro-30,40,50-trimethoxychalcone (44). Yield 83%,
pale yellow crystals, m.p. 169e170 ꢀC (MeOH). UV
(MeOH): 278 nm. IR (KBr): 1650 (C]O), 1586 (C]C),
ꢄ
1325 (CeOeAr) cmꢂ1
.
MS m/z: [Mþ ] 343 (100),
[M ꢂ 15]þ 328 (45), [M ꢂ 31]þ 312 (20), [M ꢂ 43]þ 300
(30), [M ꢂ 148]þ 195 (32), [M ꢂ 167]þ 176 (25),
[M ꢂ 215]þ 148 (28). 1H NMR (DMSO-d6, 250 MHz):
d 8.23e8.28 (m, 2H, H-3, H-5), 7.77 (d, J ¼ 15.8 Hz, 1H,
H-b), 7.63 (d, J ¼ 8.5 Hz, 2H, H-2, H-6), 7.52 (d,
J ¼ 15.8 Hz, 1H, H-a), 7.18 (s, 2H, H-20, H-60), 3.93 (s, 3H,
CH3O), 3.91 (s, 6H, 2 ꢅ CH3O). 13C NMR (DMSO-d6,
250 MHz): d 189.3 (C]O), 154.3 (C-30, C-50), 148.1 (C-4),
145.6 (C-b), 145.2 (C-40), 139.8 (C-1), 133.1 (C-10), 128.2
(C-2, C-6), 122.4 (C-3, C-5), 121.0 (C-a), 107.8 (C-20, C-
60), 56.4 (CH3O), 56.0 (2 ꢅ CH3O). Anal. for C18H17NO6.
Calcd (%): C, 62.97; H, 4.99; N, 4.08. Found (%): C, 63.01;
H, 5.04; N, 4.15.
5.1.1.6. 4-Hydroxy-3,30,40,50-tetramethoxychalcone (54). Yield
63%, white crystals, m.p. 75e76 ꢀC (MeOH). UV (MeOH):
280 nm. IR (KBr): 3258 (OH), 1654 (C]O), 1585 (C]C),
ꢄ
1279 (CeOeAr) cmꢂ1. MS m/z: [Mþ ] 344 (35), [M ꢂ 15]þ
329 (20), [M ꢂ 31]þ 313 (12), [M ꢂ 149]þ 195 (100). 1H
NMR (CD2Cl2, 250 MHz): d 7.66 (d, J ¼ 15.5 Hz, 1H, H-b),
7.30 (d, J ¼ 15.5 Hz, 1H, H-a), 7.19 (s, 2H, H-20, H-60),
7.17 (dd, J1 ¼ 8.1 Hz, J2 ¼ 2.0 Hz, 1H, H-6), 7.11 (d,
J ¼ 2.0 Hz, 1H, H-2), 6.87 (d, J ¼ 8.1 Hz, 1H, H-5), 3.88 (s,
3H, CH3O), 3.85 (s, 6H, 2 ꢅ CH3O), 3.78 (s, 3H, CH3O).
13C NMR (CD2Cl2, 250 MHz): d 189.1 (C]O), 153.6 (C-30,
C-50, C-3), 148.7 (C-40), 147.3 (C-4), 144.9 (C-b), 134.2 (C-
10), 127.9 (C-1), 123.4 (C-a), 119.8 (C-6), 115.0 (C-5),
110.6 (C-2), 106.3 (C-20, C-60), 60.9 (CH3O), 56.7
(2 ꢅ CH3O), 56.4 (CH3O). Anal. for C19H20O6. Calcd (%):
C, 66.27; H, 5.85. Found (%): C, 66.52; H, 5.91.
5.1.1.3. 3-Hydroxy-30,40,50-trimethoxychalcone (51). Yield
82%, red crystals, m.p. 280e281 ꢀC (MeOH). UV (MeOH):
315 nm. IR (KBr): 3425 (OH), 1639 (C]O), 1560 (C]C),
ꢄ
1375 (CeOeAr) cmꢂ1. MS m/z: [Mþ ] 314 (37), [M ꢂ 15]þ
299 (22), [M ꢂ 17]þ 297 (86), [M ꢂ 31]þ 283 (15),
[M ꢂ 119]þ 195 (100). 1H NMR (DMSO-d6, 250 MHz):
d 7.70 (d, J ¼ 15.8 Hz, 1H, H-b), 7.59 (d, J ¼ 15.8 Hz, 1H,
H-a), 7.30 (t, J ¼ 8.2 Hz, 1H, H-5), 7.20e7.26 (m, 3H, H-20,
H-60, H-6), 7.10 (t, J ¼ 2.0 Hz, 1H, H-2), 6.90 (dd,
J1 ¼ 2.0 Hz, J2 ¼ 8.2 Hz, 1H, H-4), 3.95 (s, 3H, CH3O), 3.93
(s, 6H, 2 ꢅ CH3O). 13C NMR (DMSO-d6, 250 MHz):
d 189.3 (C]O), 159.0 (C-3), 153.1 (C-30, C-50), 145.6 (C-
b), 143.2 (C-40), 136.2 (C-1), 133.6(C-10), 128.7 (C-5),
122.3 (C-a), 115.9 (C-4), 112.6 (C-2), 107.0 (C-20, C-60),
60.9 (CH3O), 56.4 (2 ꢅ CH3O). Anal. for C18H18O5. Calcd
(%): C, 68.78; H, 5.77. Found (%): C, 68.84; H, 5.82.
5.1.2. Preparation of 2-hydroxy-30,40,50
-trimethoxychalcone (50)
Solid KOH (2.1 g, 37.5 mmol) was added to a mixture of
3,4,5-trimethoxyacetophenone (200 mg, 0.9 mmol) and salicyl
aldehydes (101 ml, 0.9 mmol) in MeOH (2.5 ml) and H2O
(1.3 ml). The resulting solution was refluxed for 2 h, then
cooled in an ice-water bath and acidified with 4.2 ml conc.
HCl. The solution was diluted with distilled water (10 ml)
and stored in a refrigerator overnight. The formed crystals
were filtered, washed with water, dried out, purified by prepar-
ative thin-layer chromatography (petroleum ether:acetone 7:4)
and recrystallized. Yield 50%, cream-colored crystals, m.p.
211e212 ꢀC (MeOH). UV (MeOH): 280 nm. IR (KBr):
3490 (OH), 1650 (C]O), 1575 (C]C), 1375 (CeOe
5.1.1.4. 4-Hydroxy-30,40,50-trimethoxychalcone (52). Yield
62%, yellow crystals, m.p. 179e180 ꢀC (MeOH). UV
(MeOH): 350 nm. IR (KBr): 3430 (OH), 1650 (C]O), 1565
ꢄ
(C]C), 1380 (CeOeAr) cmꢂ1. MS m/z: [Mþ ] 314 (29),
[M ꢂ 15]þ 299 (18), [M ꢂ 17]þ 297 (75), [M ꢂ 31]þ 283
(12), [M ꢂ 119]þ 195 (100). 1H NMR (DMSO-d6,
250 MHz): d 7.78 (d, J ¼ 15.5 Hz, 1H, H-b), 7.68 (s, 4H, H-
2, H-3, H-5, H-6), 7.40 (d, J ¼ 15.5 Hz, 1H, H-a), 7.20 (s,
2H, H-20, H-60), 3.93 (s, 3H, CH3O), 3.90 (s, 6H,
2 ꢅ CH3O). 13C NMR (DMSO-d6, 250 MHz): d 189.6
(C]O), 165.3 (C-4), 153.3 (C-30, C-50), 145.0 (C-b), 144.5
(C-40), 133.4 (C-10), 130.5 (C-1), 129.5 (C-2, C-6), 120.7
(C-a), 117.6 (C-3, C-5), 107.0 (C-20, C-60), 60.8 (CH3O),
56.4 (2 ꢅ CH3O). Anal. for C18H18O5. Calcd (%): C, 68.78;
H, 5.77. Found (%): C, 68.82; H, 5.84.
ꢄ
Ar) cmꢂ1. MS m/z: [Mþ ] 314 (34), [M ꢂ 15]þ 299 (18),
[M ꢂ 17]þ 297 (80), [M ꢂ 31]þ 283 (17), [M ꢂ 119]þ 195
(100). 1H NMR (DMSO-d6, 250 MHz):
d
7.90 (d,
J ¼ 15.8 Hz, 1H, H-b), 7.63 (d, J ¼ 15.8 Hz, 1H, H-a),
7.23e7.35 (m, 3H, H-4, H-5, H-6), 6.89 (s, 2H, H-20, H-60),
6.69 (d, J ¼ 8.0 Hz, 1H, H-3), 3.96 (s, 3H, CH3O), 3.93 (s,
6H, 2 ꢅ CH3O). 13C NMR (DMSO-d6, 250 MHz): d 189.6
(C]O), 158.9 (C-2), 153.4 (C-30, C-50), 145.0 (C-b), 144.1
(C-40), 133.2 (C-10), 130.0 (C-4), 128.7 (C-6), 122.5 (C-a),
120.7 (C-5), 116.9 (C-1), 107.3 (C-20, C-60), 60.9 (CH3O),
5.1.1.5. 3-Hydroxy-30,4,40,50-tetramethoxychalcone (53). Yield
95%, brown crystals, m.p. 250e252 ꢀC (MeOH). UV

2218
D. Batovska et al. / European Journal of Medicinal Chemistry 44 (2009) 2211e2218
56.4 (2 ꢅ CH3O). Anal. for C18H18O5. Calcd (%): C, 68.78; H,
sample, dissolved in 96% EtOH (5000 mg/ml) was added to
appropriate wells. For pre-diffusion the Petri dishes were
placed at 4 ꢀC for 2 h. The antibacterial activity was estimated
by the diameter of inhibitory zones formed in the agar layer
after incubation at 37 ꢀC for 48 h. Compounds giving an inhib-
itory zone with a diameter of at least 15 mm were considered
active. Control experiments were carried out with the pure
solvent.
5.77. Found (%): C, 68.83; H, 5.80.
5.1.3. Preparation of 3-(3-hydroxyphenyl)-1,5-
diphenylpentane-1,5-dione (56)
m-Hydroxybenzaldehyde (98 mg, 0.8 mmol) and acetophe-
none (93 ml, 0.8 mmol) were dissolved in 0.8 ml MeOH and to
this solution 0.4 ml of 6 M NaOH was added. The reaction
mixture was then refluxed for 10 min. The product obtained
was purified with column chromatography over silica using
petroleum ether:acetone 7:1 to yield amorphous powder
5.2.2. Measurement of MIC
MIC was determined by serial dilution of each chalcone to
0.0e2000 mg/ml in test tubes using meat-peptone broth. Each
test tube was inoculated with bacterial suspension containing
105 cells/ml and incubated at 37 ꢀC for 24 h. The lowest dilu-
tion that visibly showed no growth compared to drug-free
broth inoculated with microbial suspension was considered
the MIC. For more precise detection, tubes that showed no vis-
ible growth were streaked on fresh meat-peptone agar plates,
incubated at 37 ꢀC for 24 h, and checked for growth.
(56), yield 67%. UV (MeOH): 245, 280 nm. IR (KBr): 3375
ꢄ
(OH), 1657 (C]O) cmꢂ1
.
MS m/z: [Mþ ] 344 (7),
[M ꢂ 18]þ 326 (8), [M ꢂ 119]þ 225 (100), [M ꢂ 239]þ 105
(95), [M ꢂ 267]þ 77 (59). 1H NMR (CDCl3, 250 MHz):
d 7.92e7.97 (m, 4H, Ar-H), 7.50e7.57 (m, 2H, Ar-H),
7.39e7.46 (m, 4H), 7.10 (t, J ¼ 8.01 Hz, 1H, Ar-H), 6.78e
6.81 (m, 2H, Ar-H), 6.63e6.67 (m, 1H, Ar-H), 6.14 (s, 1H,
OH), 4.00 (p, J ¼ 7.01 Hz, 1H, CH), 3.32, 3.47 (two dd,
J1 ¼ 16.5 Hz, J2 ¼ 7.0 Hz, 4H, 2 ꢅ CH2). 13C NMR (CDCl3,
250 MHz): d 199.1 (2 ꢅ C]O), 156.1, 145.4, 136.7, 133.2,
129.8, 128.6, 128.3, 128.2, 128.0, 119.3, 114.6, 113.9, 44.8,
37.1, 30.9. Anal. for C23H20O3. Calcd (%): C, 80.21; H,
5.85. Found (%): C, 80.02; H, 5.94.
Acknowledgments
This work was supported by the Bulgarian National
Science Fund (contract X-1514).
5.1.4. Synthesis of 1,5-bis-(3,4,5-trimethoxy-phenyl)-
penta-1,4-dien-3-one (61)
References
3,4,5-Trimethoxybenzaldehyde (294 mg, 1.5 mmol) was
dissolved in 1.0 ml MeOH and acetone (53 ml, 0.73 mmol),
and 1.4 ml of 6 M NaOH was added. In a few minutes solid
formed. The product (61) was isolated after filtration, washing
of the crystals with cold methanol and recrystallization. Yield
90%, green crystals, m.p. 108e110 ꢀC (MeOH). UV (MeOH):
250, 365 nm. IR (KBr): 1610 (þC]O), 1570 (C]C)þcmꢂ1. MS
m/z: [Mþꢄ] 414 (41), [M ꢂ 15] 399 (22), [M ꢂ 31] 383 (20),
[M ꢂ 33]þ 181 (100). 1H NMR (CDCl3, 250 MHz): d 7.66 (d,
J ¼ 15.8 Hz, 2H, 2 ꢅ H-b), 6.97 (d, J ¼ 15.8 Hz, 2H, 2 ꢅ H-
a), 6.84 (s, 4H, Ar-H), 3.910 (s, 12H, 4 ꢅ CH3O), 3.89 (s,
6H, 2 ꢅ CH3O). 13C NMR (CDCl3, 250 MHz): d 188.5,
153.5, 143.3, 140.4, 130.2, 124.8, 105.6, 61.0 (2 ꢅ CH3O),
56.2 (4 ꢅ CH3O). Anal. for C23H26O7. Calcd (%): C, 66.65;
H, 6.32. Found (%): C, 66.85; H, 6.65.
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5.2. Microbiology
The strains of S. aureus (Rosenbach 209) and E. coli (Cas-
tellani and Chalmers) WFþ were obtained from the collection
of the Stefan Angeloff Institute of Microbiology, Bulgarian
Academy of Sciences and the Institute for State Control of
Drugs, Sofia, Bulgaria.
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5.2.1. Agar-well diffusion method
Two hundred-microliter suspension of the bacteria
(105 cells/ml) was plated on the agar layer in Petri dishes
(10 cm in diameter). Five wells per dish were prepared,
each 10 mm in diameter. One hundred microliters of each