A novel one-pot synthesis and preliminary biological activity evaluation of cis-restricted polyhydroxy stilbenes incorporating protocatechuic acid and cinnamic acid fragments

Article abstract of DOI:10.1016/j.ejmech.2013.05.040

A series of new stilbenes 4a -e, 5 were synthesized through a novel one-pot Perkin-like reaction between 6,7-dimethoxyhomophthalic anhydride and aromatic aldehydes, followed by treatment with BBr₃. This synthesis is straightforward and allows polyhydroxylated cis-stilbenes gathering two well-known pharmacophoric fragments to be obtained in good yields and for short reaction times. The structure of the newly synthesized compounds was established by spectroscopic methods (¹H NMR, ¹³C NMR, IR and HRMS) and the double bond configuration was unequivocally elucidated by means of gated decoupling ¹³C NMR spectra and 2D NOESY experiments. Preliminary differentiating screening of their radical scavenging, antibacterial, anti-fungal and tyrosinase inhibitory activity was further performed. The results obtained suggest that the tested compounds possess a triple biological action as potent radical scavengers, antifungal agents and tyrosinase inhibitors in micromolar concentration. The most promising bioactive compound amongst the others was 4a, acting as excellent radical scavenger against DPPH? radical (IC50 ≤10 μM), antifungal agent suppressing the growth of Fusarium graminearum (89% inhibition at 0.17 μmol/mL), and tyrosinase inhibitor showing higher activity than hydroquinone at 23 μM.

Full text of DOI:10.1016/j.ejmech.2013.05.040

Accepted Manuscript
A Novel One-pot Synthesis and Preliminary Biological Activity Evaluation of cis-
Restricted Polyhydroxy Stilbenes Incorporating Protocatechuic acid and Cinnamic
Acid Fragments
Mitko Miliovsky, Ivan Svinyarov, Yavor Mitrev, Yana Evstatieva, Dilyana Nikolova,
Maya Chochkova, Milen G. Bogdanov
PII:
S0223-5234(13)00358-9
10.1016/j.ejmech.2013.05.040
EJMECH 6218
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 March 2013
Revised Date: 20 May 2013
Accepted Date: 25 May 2013
Please cite this article as: M. Miliovsky, I. Svinyarov, Y. Mitrev, Y. Evstatieva, D. Nikolova, M.
Chochkova, M.G. Bogdanov, A Novel One-pot Synthesis and Preliminary Biological Activity Evaluation
of cis-Restricted Polyhydroxy Stilbenes Incorporating Protocatechuic acid and Cinnamic Acid
Fragments, European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.05.040.
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ACCEPTED MANUSCRIPT
Research Highlights:
ꢀ Polyhydroxylated cis-stilbenes are readily available via a novel one-pot
synthetic procedure
ꢀ trans-Cinnamic acid fragment ensures cis-configuration of the stilbene
backbone
ꢀ cis-Stilbenes possess a triple biological action
ꢀ Combination of two pharmacophoric fragments in one molecule evoke
synergistic effect
ꢀ Dicarboxylic acid fragment might be important for tyrosinase inhibition

ACCEPTED MANUSCRIPT
A Novel One-pot Synthesis and Preliminary Biological Activity Evaluation of cis-Restricted
Polyhydroxy Stilbenes Incorporating Protocatechuic acid and Cinnamic Acid Fragments
Mitko Miliovsky,a Ivan Svinyarov,a Yavor Mitrev,b Yana Evstatieva,c Dilyana Nikolova,c Maya
Chochkova,d and Milen G. Bogdanov*a
a Faculty of Chemistry and Pharmacy, University of Sofia “St. Kl. Ohridski”, 1, James Bourchier
Blvd., 1164 Sofia, Bulgaria
b Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences,
Acad. G. Bonchev Str. Bl. 9, Sofia 1113, Bulgaria
c
Faculty of Biology, University of Sofia “St. Kl. Ohridski”, 8, Dragan Tsankov Blvd., 1164
Sofia, Bulgaria
d Faculty of Mathematics and Natural Sciences, South-West University ‘‘Neofit Rilski’’,66, Ivan
Mihailov Str., 2700 Blagoevgrad, Bulgaria
*Corresponding author:
Milen G. Bogdanov
Faculty of Chemistry and Pharmacy
Sofia University “St. Kl. Ohridski”
1 J. Bourchier blvd.
1164 Sofia
Bulgaria
E-mail: mbogdanov@chem.uni-sofia.bg
Tel:
Fax:
(+359 2) 81 61 228
(+359 2) 962 54 38

ACCEPTED MANUSCRIPT
Abstract:
A series of new stilbenes 4a-e, 5 were synthesized through a novel one-pot Perkin-like reaction
between 6,7-dimethoxyhomophthalic anhydride and aromatic aldehydes, followed by treatment
with BBr3. This synthesis is straightforward and allows polyhydroxylated cis-stilbenes gathering
two well-known pharmacophoric fragments to be obtained in good yields and for short reaction
times. The structure of the newly synthesized compounds was established by spectroscopic
methods (1H NMR, 13C NMR, IR and HRMS) and the double bond configuration was
unequivocally elucidated by means of gated decoupling 13C NMR spectra and 2D NOESY
experiments. Preliminary differentiating screening of their radical scavenging, antibacterial, anti-
fungal and tyrosinase inhibitory activity was further performed. The results obtained suggest that
the tested compounds possess a triple biological action as potent radical scavengers, antifungal
agents and tyrosinase inhibitors in micromolar concentration. The most promising bioactive
compound amongst the others was 4a, acting as excellent radical scavenger against DPPH●
radical (IC50 ≤ 10 µM), antifungal agent suppressing the growth of F. graminearum (89%
inhibition at 0.17 µmol/mL), and tyrosinase inhibitor showing higher activity than hydroquinone
at 23 µM.
Keywords: homophthalic anhydride, polyhydroxy stilbenes, antioxidant activity, antifungal
activity, tyrosinase inhibitors

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1. Introduction
Stilbenoids are a class of natural compounds, formed by a particular branch of the flavonoid
biosynthetic pathway. Being first isolated from plants in 1899, they attracted the scientific
community attention due to their chemical diversity and wide range of biological effects [1].
Nowadays it is widely accepted, that stilbenoids play an important role as phytoalexins in plant
resistance to fungal pathogens [2]. Recently, natural hydroxy stilbenes (a particular group of
stilbenoids) such as resveratrol and combretastatin, as well as their synthetic analogues, become
of a great interest to the medicinal chemists. These compounds have been proved to be
antioxidant reagents, that inhibit cell apoptosis, suppress growing cancer cells, influence the
activity of specific enzymes, effect the animal ageing and metabolisms of estrogens [3-12], etc.
Although the hydroxy stilbenoids possess remarkable medical benefits, it is noteworthy that their
bioavailability is very narrow. In fact they are only produced in nanogram scale in plants, in
response to stress situations such as fungal infection or injury, and thus, it is hardly to be
obtained in large quantities from their natural sources. To overcome this, various synthetic
approaches for the synthesis of stilbenes have been developed including Colvin rearrangement to
an alkyne, followed by selective semi-reduction [13], Suzuki cross-coupling [14,15], Heck [16],
Wittig [17-18] and Perkin reactions [19-22], to name just a few. It is however rather important to
be mentioned, that the formation of the carbon–carbon double bond between the two aromatic
rings is the key step in all the synthetic approaches developed, but in most of the cases a mixture
of cis- and trans- isomers is produced. Thus, additional work is required in order these isomeric
mixtures to be separated, which lowers the products yields and limits the benefits of these
approaches from an applied standpoint. Moreover, the classical methods such as Suzuki and
Heck reactions usually require relatively long synthetic sequences and use of expensive catalyst
and reagents. Therefore, the need from fast and highly efficient stereo specific synthetic methods
for large-scale preparation of such compounds can be put forward.

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HOOC
HO
MeO
OH
OMe
MeO
OMe
OH
Protocatechuic acid
Combretastatin A-4
HOOC
HOOC
HOOC
R2
R
R1
HO
OH
R
R
cinnamic acid: R1 = R2 = H
p-coumaric acid: R1 = OH; R2 = H
caffeic acid: R1 = R2 = OH
Target compounds
in this study
R = H or OH or t-Bu
In this context, herein we report a novel highly diastereoselective synthesis of polyhydroxy cis-
stilbenes by means of one-pot procedure, including initial Perkin-like reaction between
commercially available homophthalic anhydrides and aromatic aldehydes, followed by treatment
with BBr3. This straightforward procedure allows the synthesis of polyhydroxylated stilbenes in
high yields and as single cis-isomers, which are structurally based on two independent
pharmacophoric fragments, such as protocatechuic acid and cinnamic acid. It is noteworthy, that
the incorporated trans-cinnamic acid fragment into the target compounds structure ensures cis-
configuration of the stilbene backbone and prevents further isomerization along the carbon–
carbon double bond. Furthermore, the combination of two different and independently acting
fragments of well-known pharmacological profiles [23-29] into one covalently bonded hybrid
molecule can evoke synergistic effect, which is a precondition of an enhanced biological activity
to be expected. Therefore, in order the synergistic effect of this combination of pharmacophoric
moieties to be assessed, the synthesized compounds were subjected to a preliminary in vitro
study of their radical scavenging, antibacterial, antifungal and tyrosinase inhibitory activities.
2. Results and discussion
2.1. Chemistry
Amongst the above mentioned synthetic approaches for the formation of carbon-carbon double
bond, the Perkin reaction between benzaldehydes and phenylacetic acids in the presence of acetic

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anhydride and a base proved to be useful in the synthesis of stilbenes [19-22]. Despite its utility,
however, the classical Perkin reaction requires high temperature and prolonged reaction time.
Moreover, in order polyhydroxylated stilbenes to be synthesized, one should follow multistep
approach involving sequential protection, condensation, decarboxylation and deprotection steps.
This sequence is necessary due to the extreme susceptibility of the hydroxyl function towards
oxidation and polymerization, but results in a low overall product yields. To overcome these
limitations, we developed a novel one-pot procedure for the synthesis of polyhydroxy stilbenes,
including initial Perkin-like reaction between commercially available homophthalic anhydrides
and aromatic aldehydes, followed by treatment with BBr3. The synthetic strategy towards the
target compounds
4 and their substitution pattern are presented in Scheme 1. The idea behind is
coming from recent studies of ours [30-32], where we have shown, that homophthalic anhydride
reacts with substituted benzaldehydes in the presence of 4-dimethylaminopyridine (DMAP) at
room temperature to produce diastereomeric mixture of cis- and trans-3-aryl-3,4-
dihydroisocoumarin-4-carboxylic acids (
undergo rapid lactone ring opening reaction to form an equilibrium mixture of
corresponding dicarboxylic acid of type [32]. It is noteworthy, that were obtained as single
3
) [30] and that in presence of a base these acids
3
and the
4
4
diastereomers with cis-stilbene backbone, regardless the relative configuration of the starting
compounds. Based on the above, we were initially interested in evaluating of the behavior of
acids
under demethylation reaction conditions. So, we found that acid trans-3a (used as a model
compound) undergo quantitative conversion to the hydroxystilbene acid 4a. Considering as
3 in the presence of BBr3 (CH2Cl2 solution) and to check the stability of the lactone ring
3
intermediates in the reaction scheme towards the target compounds, we further assumed possible
synthesis shortening by addition of BBr3 solution to the anhydride-aldehyde reaction mixture,
immediately after the corresponding acids
Indeed, this approach proved successful, showing the possible formation of
of any intermediates. Following that procedure, aromatic aldehydes of different substitution
pattern (2а-е) were initially reacted with 6,7-dimethoxyhomophthalic anhydride ( ). On the one
3 are formed.
4
without isolation
1
hand, this provides the protocatechuic acid pharmacophoric fragment as ring A at the end of the
synthetic sequence, and on the other, it allows the influence of the substituents in ring B on the
biological activity of compounds
4 to be studied. The diastereomeric mixtures of cis- and trans-
3
, produced from the above reaction in presence of DMAP/CH2Cl2, were further successfully

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converted into
4
by direct addition of BBr3/CH2Cl2 solution at room temperature. The liquid
chromatography analysis of the reaction mixtures showed, that
3 were formed in 10-15 min in all
cases, whereas the time needed for the formation of depends on the number of groups, which
4
have to be demethylated. To study this, individual reactions were performed for each compound,
and the products yields were estimated after working-up the reaction mixtures on every 10 min.
The results obtained showed that the least substituted 4a (two hydroxyl groups) and the highly
substituted 4d (5 hydroxyl groups) were obtained in 10 and 60 min, respectively, i.e. the higher
substituted nuclei, lead to an increase in reaction time. Summarizing, if one follows that one-pot
procedure, polyhydroxylated cis-stilbenes are easily available from commercial starting materials
in less than 90 min. The compounds synthesized in this way were purified by column
13
chromatography and their structures were characterized by spectral methods (1H and C NMR,
IR, HRMS). The configuration of the stilbene double bond was estimated as describe in Section
2.2.
It is noteworthy, that when
1
reacts with 3,5-di-tert-butyl-4-hydroxybenazaldehyde (2e), cis-
. The latter can
stilbene was formed instead the expected 3,4-dihydroisocoumarin acid of type
5
3
be attributed to the positive mesomeric effect of the hydroxyl group, which induces a lactone
ring opening reaction. This chance, however, allowed the influence of the catechol moiety in the
protocatechuic acid fragment on the biological effects to be assessed. On the other hand, it
showed the possibility for diversification of the developed synthetic procedure, and now
experiments on the reactions of homophthalic anhydrides with p-hydroxybenzaldehyde
derivatives are in progress in our laboratory.
2.2. Structure elucidation
It is noteworthy, that the unique substitution pattern of the polyhydroxystilbene acids 4a-e
poses difficulties in their structural elucidation. In most of the cases, their proton NMR spectra
consist mainly of singlets, and thus, the lack of proton-proton coupling constants data yields
practically no information in respect to the double bond configuration. Very recently Zou and
coworkers [21] found, that the vinyl protons in E-isomers of a series of E-2,3-diarylacrilic acids
appear at lower field compared to the same protons in the Z-isomers, which was attributed to the
field effect of the carboxylic group. Nevertheless, the lack of spectroscopic data for the opposite
diastereomers suggests limited applicability of this approach for determination of the double

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bond configuration of such systems, since the chemical shifts for the vinyl proton (H8) seem to
be dependent on the substituents electronic effects (see Table 1). Moreover, the possible signal
overlapping in some cases could lead to wrong data interpretation. To overcome this problem, an
approach based on the heteronuclear coupling constants could be applied, since it is known, that
the vicinal heteronuclear coupling constants exhibit Karplus type relationship between its value
and the dihedral angle between the interacting nuclei [33]. This approach has already proved
successful and accurate for configuration studies of similar compounds, even when the
diastereomers with the opposite configuration are not available [32]. In order to elucidate the
configuration of the herein synthesized compounds and to evaluate the substituents effect on
their spectral properties, acids 4a
,
4b and 4d containing no, one and three hydroxylic groups into
13
ring B, respectively, were chosen as model compounds. Gated decopuling C NMR spectra for
those compounds were recorded and the corresponding H8-C7 and H2-C1a coupling constants
were extracted. The assignment of the two carbonyl signals was made using HSQC and HMBC
spectra. The data obtained is summarized in Table 1.
The most important information gained from the above mentioned experiments is the
observed low values of the coupling constants for H2-C1a and H8-C7, which were found in all
cases in the range about 4 Hz and 7 Hz for, respectively. These results are in excellent agreement
with the literature data for cis-orientation along the double bond of such a spin system [32,33]
and allow cis-configuration of the stilbene moiety in compounds
4 to be assigned. It should be
noted, that the opposite trans-orientation results in higher (14-16 Hz) heteronuclear coupling
constants [33], which is obviously not the case here. Nevertheless, the assigned configuration for
4
was further independently estimated by means of 2D NOESY experiments. Using this NMR
technique the spatial proximity between particular nuclei could be established. The performed
analysis showed no cross-peaks between H8 and H5 which allowed the trans-orientation of H8
and aromatic ring A to be proved.
Regarding the relationship chemical shifts–substituents effect, it is noteworthy, that the
presence of hydroxyl groups in ring B does not influence significantly the chemical shifts of the
two carboxylic groups and the protons in ring A, thus making them useful probe for the
configuration assignment of structural analogues. In all cases, the signal corresponding to H2
resonates at 7.45 ppm and that for H5 is around 6.35-6.40, which can be explained by the
shielding effect of ring B, and the C-7 carbonyl has chemical shift in the interval between

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168.25-168.74 ppm. This allowed us to attribute cis-configuration of the stilbene moiety in acids
4c,
4e and
5 just by comparing the chemical shifts for the selected signals.
In summary, although it was possible to prove the double bond configuration by NMR in this
particular study, the full characterization of those systems is far from trivial and requires more
sophisticated experiments, along with meticulous extraction and interpretation of all
heteronuclear coupling constants.
2.3. Biological activity
2.3.1. DPPH● scavenging activity
The DPPH● radical is one of the commercially available stable organic nitrogen radicals, which
does not have to be generated prior to analysis. It is easy to perform, highly reproducible and
comparable with other methods such as ABTS assay, reduction of superoxide anion and
inhibition of lipid peroxidation [34], and has been successfully applied for evaluation of the
antioxidant activity of both synthetic and natural antioxidants. Therefore, we applied this method
to assess the radical scavenging activity of the synthesized polyhydroxy cis-stilbenes. The
preliminary assay was performed at 3.3 µM concentration of each of the tested compounds, and
the radical scavenging activity was evaluated by measuring in vitro the residual DPPH●. For
comparison, caffeic acid (CA), protocatechuic acid (PCA) and their equimolar mixture
(CA+PCA) were used as positive controls. The results obtained are presented in Fig. 1.
In general, the group of compounds shows higher DPPH● radical scavenging activity than the
standards used. The activity increases as follows: CA < 5 ≤ PCA ≈ 4a ≈ (CA + PCA) ≤ 4b
< 4e <
4c 4d. As can be seen,
<
5
is the least active compound scavenging 18% of DPPH● radicals, and
this can be attributed to the blocked (methylated) catechol moiety into the protocatechuic acid
fragment (ring A) and presence of not only one, but sterically hindered hydroxyl group into ring
B. Considering compounds 4a-4d, it can be seen that the presence of a single hydroxyl group at
4th position in ring B (4b) did not affect significantly the activity of 4a, but the addition of
second or third did it in a great value, and thus compounds 4c and 4d are respectively two- and
four-folds of higher activity than the former. This suggests that in addition to the number of the
hydroxyl groups, their distribution into the basic stilbene backbone is of a great importance for
the hydrogen transfer ability and for the resulting radical stabilization. Interestingly, in case of 4c
a synergistic effect from the combination of two active fragments into one hybrid molecule could

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be established. As it can be seen 4c shows higher activity than CA, PCA and their equimolar
mixture, and this could be attributed to the formation of intramolecular intermediates capable to
scavenge DPPH● radicals. It is noteworthy that the expected IC50 (concentration needed to
scavenge 50% of DPPH●) for some of the compounds studied should be lower than 3.3
µM, and
for the rest lower than 10 M. This makes them attractive targets for further detailed studies of
µ
their antioxidant activity, since it is well-known that one of the main characteristics responsible
for the antioxidant activity of phenolic compounds is their ability to scavenge free radicals. The
work in this direction is in progress and the results will be published in due course.
2.3.2. Antimicrobial activity
The antimicrobial activity of 4a-e, 5 was screened against four bacterial strains – Gram-
positive Bacillus subtilis (ATCC 6633), Staphilococcus aureus (ATCC 25923), Gram-negative
Escherichia coli (ATCC 25922) and Pseudomonas aeroginosa (ATCC 10145), one yeast strain –
Candida albicans (ATCC 10231) and three fungal strains – Aspergillus niger (A3), Aspergillus
flavus (IMI 052104) and Fusarium graminearum (ATCC 24373). The analyses were performed
as described in Sections 4.3.2 and 4.3.3, and the results obtained for the antifungal activity are
presented in Table 2.
Regarding the antibacterial activity, compounds 4a-e can be considered not to be active
against the four bacterial strains in concentration 50 µg/mL. Exception is compound 5, which
shows 44% of the inhibitory effect of the standard Amikacine against B. Subtilis at the same
concentration. By contrary, the whole group of compounds can be considered as potent
antifungal agents. Their activity against the yeast strain C. albicans is 34-48% of the inhibitory
effect of the standard Nystatin in concentration 50 µg/mL and is in the same range for A. niger
and A. flavus. Considering that narrow range, the apparent antifungal activity can be attributed to
some specific interactions between the cis-stilbenes and the fungal cells, since it does obviously
not depend on the number of the hydroxyl groups. However, the presence of t-Bu substituents
slightly increases the activity, and thus 4e and
5 can be considered as the most active amongst
the others in the series. It is noteworthy the higher antifungal activity of the tested compounds
against F. graminearum, and in particular, that of 4a which inhibit significantly its growth at
concentration 0.17
µmol/mL. This is of a great importance from an industrial standpoint since F.
graminearum is known as a plant pathogen [35] that causes various diseases, such as gibberella

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ear rot in corn and fusarium head blight or scab in wheat and other grains. Moreover, the major
mycotoxins produced by F. graminearum – deoxynivalenol and zearalenone – have been found
to be toxigenic in humans and animals [36,37]. Consequently, the investigation of the antifungal
activity of other derivatives of
4 against that microorganism should be further considered.
2.3.3. Tyrosinase inhibitory activity
Phenolic compounds, such as hydroxycinnamic acids and their derivatives, have been reported
to act as promising tyrosinase inhibitors [38-43]. Considering the synthesized cis-stilbenes as
cinnamic acid analogues, their tyrosinase inhibitory activities were examined using mushroom
tyrosinase and L-tyrosine as a substrate. This preliminary assay was performed at 23
µM
concentration of each of the tested compounds and the monophenolase inhibitory activity was
evaluated by measuring the dopachrome product in vitro as reported previously by one of us
[42]. For comparison, hydroquinone (known as strong, but toxic tyrosinase inhibitor), caffeic
acid and protocatechuic acid were used as positive controls. The results obtained are presented in
Fig. 2.
Compared to standards, the inhibitory activities increase in the following order: CA ≤ 4d ≤ 5 ≈
4e
4e and
4a 4b and 4c are superior to the rest in the inhibition of tyrosinase and that the number and the
<
4c ≤ HQ
≤
PCA ≈ 4b ≤ 4a, and thus the compounds tested can be considered as weak (4d
,
5
) or strong (4a
,
4b and 4c) mushroom tyrosinase inhibitors. These results suggest that
,
position of substituents into the cinnamic acid moiety (ring B) seem to play an important role in
triggering the inhibitory effects. Considering the former compounds, it can be seen that the
introduction of an additional hydroxyl group slightly decreases the enzyme inhibition, and thus
to be concluded that the presence of up to two hydroxyl groups into ring B is essential for potent
inhibitory activity. Furthermore, the insertion of third hydroxyl group into position 5 in ring B
diminished dramatically the tyrosinase inhibitory effect, and thus 4d is the least active compound
in the series. It is worthy to note, that the same effect was observed for the two butylated cis-
stilbenes 4e and 5, suggesting that the steric effects of the substituents play an important role,
when the solute is approaching to the active site of the enzyme. Based on the above arguments,
the compounds studied can be considered as competitive tyrosinase inhibitors due to their
structure similarity to the natural substrates L-tyrosine and L-DOPA (Fig. 3). However,
considering the results obtained for CA and PCA, it could be deduced that the protocatechuic

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acid fragment contributes in a great value to the inhibitory effect since it ensures a catechol
group capable to chelate copper in the enzyme. On the other hand, it may not be illogical to be
assumed that the homophthalic acid fragment may be also in charge of the apparent activity, and
thus the presence of the two carboxylic groups to be taken into account. This assumption can be
supported by recent studies [46-48], reporting that homophthalic acid may play a role as a linker
in the formation of metal complexes (including Cu(II)) with rigid nitrogen containing ligands.
Based on the above, compounds
4 can be also considered as non-competitive or mixed tyrosinase
inhibitors, which binds equally well to the enzyme whether or not it has already bound the
substrate. However, in order the role of each fragment incorporated into the compounds structure
to be clarified and the above speculations to be converted to a conclusive interpretation,
additional work is needed.
3. Conclusions
A series of polyhydroxy cis-stilbenes, structurally based on two independent pharmacophoric
fragments such as protocatechuic acid and cinnamic acid, were synthesized by means of a novel
one-pot procedure employing commercially available starting materials. The latter allows the
room temperature synthesis of stilbenes gathering two well-known pharmacophoric fragments, in
good yields and for short reaction times. The incorporated trans-cinnamic acid fragment into the
target compounds structure was shown to ensure cis-orientation of the stilbene backbone and to
prevent further isomerization along the carbon–carbon double bond, the configuration of which
13
was unequivocally elucidated by means of gated decopuling C NMR spectra and 2D NOESY
experiments.
The conducted preliminary biological screening showed that the tested compounds possess a
triple biological action as potent radical scavengers, antifungal agents and tyrosinase inhibitors in
micromolar concentrations. The most promising bioactive compound amongst the others was 4a
acting as excellent radical scavenger against DPPH● radical (IC50
10 M), antifungal agent
suppressing the growth of F. graminearum (89% at 0.17 mol/mL) and better than hydroquinone
tyrosinase inhibitor at 23 M. Moreover, it was shown that the combination of two different and
,
≤
µ
µ
µ
independently acting fragments of well-known pharmacological profiles into one covalently
bonded hybrid molecule can evoke synergistic effect resulting in higher than the expected
activity.

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The biological potential of the synthesized compounds 4a-e and
5 found in this preliminary
study suggest that further work is worth performing. In this regard, in order the role of each
fragment incorporated into the compounds structure to be clarified, experiments on the synthesis
of more complete series of structural analogues are in progress in our laboratory and the results
will be published in due course.
4. Experimental section
4.1. General remarks
Most chemicals and mushroom tyrosinase (EC 1.14.18.1) used in this study were purchased
from Sigma–Aldrich (FOT, Bulgaria). Hydroquinone was obtained from Ferak (Germany). The
organic solvents were of analytical grade. The test microorganism strains used for the
microbiological assays were taken from National Bank for Industrial Microorganisms and Cell
Cultures (NBIMCC), Bulgaria, except for Aspergillus niger (A3), which belongs to the
Department of Biotechnology, Biological Faculty, Sofia University, Bulgaria. Melting points
were determined on a Kofler microscope Boetius PHMK 0.5. All the reactions were monitored
by thin layer chromatography (TLC) on pre-coated polyesters sheets POLIGRAM® SIL G/UV254;
spots were visualized with UV light. MASCHEREY-NAGEL Silica gel 60 (230-400 mesh) was
used for column chromatography. The IR spectra were acquired in Nujol on a Specord 75 and
are reported in reciprocal centimeters. NMR spectra were recorded on a Bruker Avance (250.13
1
13
MHz and 62.90 MHz for H and C, respectively) using DMSO-d6 as
internal standard. The chemicals shifts ( ) are given in ppm and J values are reported in Hz. The
exact mass of compounds 4a-e was determinate by HRMS analyses on DFS High Resolution
а solvent and TMS as an
δ
,
5
GC/MS (Thermo), after derivatization with (CH3)3SiCl. The DPPH● radical scavenging activity
assay was performed on Shimadzu UV/VIS CECIL Aurius 3021 spectrophotometer and the
tyrosinase inhibitory activity on Agilent 8453 UV–Vis spectrophotometer.
4.2. Chemistry
General procedure for one-pot synthesis of polyhydroxy (E)-2-(1-carboxy-2-phenylvinyl)benzoic
acids (4a-е
An equimolar mixture of 6,7-dimetoxihomophthalic anhydride (
in dry dichloromethane (10 mL) was stirred at room temperature and the consumption of the
)
1
), aldehyde 2a-e and DMAP

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reagents was monitored by TLC. At the end of the reaction (10-15 min), boron tribromide (1.6 M
solution in dichloromethane) was slowly added (one equivalent per heteroatom) and the reaction
mixture was stirred additionally for 10-60 min. At the end of the reaction, the mixture was
treated with 50% NaOH (pH = 12), stirred for 10 min and acidified with conc. HCl (pH = 1). The
water layer was then saturated with NaCl and the products were extracted with ethyl acetate. The
organic phase was washed with brine to a constant pH, dried over anhydrous Na2SO4 and the
solvent was evaporated under reduced pressure. The residue was further purified by column
chromatography (Mobile phase: acetone/petroleum ether/formic acid – 1/1/0.01) and the
products 4a-e were isolated after crystallization from appropriate mixture of solvents.
4.2.1. (E)-2-(1-carboxy-2-phenylvinyl)-4,5-dihydroxybenzoic acid (4a
)
0.986 g oil (99%) from reaction of 6,7-dimetoxihomophthalic anhydride (
1
) (0.74 g, 3.33
mmol), benzaldehyde (2a) (0.34 mL, 3.33 mmol), 4-dimethylaminopyridine (0.40 g, 3.33 mmol)
and boron tribromide (18.7 mL, 29.97 mmol) in 10 mL dichloromethane. White crystals (0.51 g,
51%) after column chromatography and crystallization form methanol/dichloromethane – 1/5.
M.p. = 229-231 oC; IR (Nujol)
6.36 (s, 1H), 6.97-7.09 (m, 2H, =CH–), 7.15-7.26 (m, 3H, =CH–), 7.46 (s, 1H, =CH–), 7.49 (s,
1H, =CH–), 9.38 (bs, 1H, OH), 9.63 (bs, 1H, OH), 12.23 (bs, 2H, 2 x COOH); 13C NMR
(DMSO-d6, 62.5 MHz) 117.6 (=CH–), 118.2 (=CH–), 121.5 (C), 128.4 (2 x =CH–), 128.5
ν
(cm-1): 1680 (C=O, COOH); 1H NMR (DMSO-d6, 250.13 MHz)
δ
δ
(=CH–), 130.0 (2 x =CH–), 130.8 (C), 135.1 (=CH–), 135.2 (C), 144.5 (C), 149.4 (C), 167.2
(COOH), 168.3 (COOH); HRMS: m/z calculate: 588.2215, found: 588.2199 for C28H44O6Si4.
4.2.2. (E)-2-(1-carboxy-2-(4-hydroxyphenyl)vinyl)-4,5-dihydroxybenzoic acid (4b
0.95 g oil (95%) from reaction of 6,7-dimetoxihomophthalic anhydride (
)
1
) (0.70 g, 3.16
mmol), 4-methoxybenzaldehyde (2b) (0.38 mL, 3.162 mmol), 4-dimethylaminopyridine (0.39 g,
3.16 mmol) and boron tribromide (19.8 mL, 31.62 mmol) in 10 mL dichloromethane. Yellow
crystals (0.47 g, 47%) after column chromatography and crystallization from methanol/ethyl
acetate – 1/5. M.p. = 186-188 oC; IR (Nujol)
250.13 MHz) 6.38 (s, 1H), 6.58 (d, 2H, =CH–, J = 8.5), 6.85 (d, 2H, =CH–, J = 8.5), 7.41 (s,
1H, =CH–), 7.45 (s, 1H, =CH–), 9.31 (bs, 1H, OH), 9.60 (bs, 1H, OH), 9.74 (bs, 1H, OH), 12.01
(bs, 2H, 2 x COOH); 13C NMR (DMSO-d6, 62.5 MHz)
115.2 (2 x =CH–), 117.7 (=CH–), 118.2
ν
(cm-1): 1660 (C=O, COOH); 1H NMR (DMSO-d6,
δ
δ

ACCEPTED MANUSCRIPT
(=CH–), 121.4 (C), 126.0 (C), 131.2 (C), 131.4 (C), 131.9 (2 x =CH–), 135.6 (=CH–), 144.3 (C),
149.4 (C), 158.0 (C), 167.2 (COOH), 168.6 (COOH); HRMS: m/z calculate: 676.25594, found:
676.25572 for C31H52O7Si5.
4.2.3. (E)-2-(1-carboxy-2-(3,4-dihydroxyphenyl)vinyl)-4,5-dihydroxybenzoic acid (4c
)
0.92 g oil (92%) from reaction of 6,7-dimetoxihomophthalic anhydride ( ) (0.70 g, 3.01
1
mmol), 3,4-dimethoxybenzaldehyde (2c) (0.50 g, 3.01 mmol), 4-dimethylaminopyridine (0.37g,
3.01 mmol) and boron tribromide (20.7 mL, 33.11 mmol) in 10 mL dichloromethane. Solid
1
foam (0.43 g, 43%) after column chromatography. IR (Nujol)
NMR (DMSO-d6, 250.13 MHz)
ν
(cm-1): 1675 (C=O, COOH); H
δ
6.29-6.45 (m, 3H, =CH–), 6.56 (d, 1H, =CH–, J = 8.1), 7.32
(s, 1H, =CH–), 7.45 (s, 1H, =CH–), 8.86 (bs, 1H, OH), 9.28 (bs, 2H, 2 x OH), 9.56 (bs, 1H, OH),
13
12.00 (bs, 2H, 2 x COOH); C NMR (DMSO-d6, 62.5 MHz)
δ 115.3 (=CH–), 117.5 (=CH–),
117.8 (=CH–), 118.2 (=CH–), 121.3 (C), 122.8 (=CH–), 126.5 (C), 131.2 (C), 131.3 (C), 136.0
(=CH–), 144.2 (C), 144.8 (C), 146.5 (C), 149.3 (C), 167.2 (COOH), 168.7 (COOH); HRMS: m/z
calculate: 764.29038, found: 764.28984 for C34H60O8Si6.
4.2.4. (E)-2-(1-carboxy-2-(3,4,5-trihydroxyphenyl)vinyl)-4,5-dihydroxybenzoic acid (4d
)
0.95 g (95%) oil from reaction of 6,7-dimetoxihomophthalic anhydride ( ) (0.64 g, 2.87
1
mmol), 3,4,5-trimethoxybenzaldehyde (2d) (0.56 g, 2.87 mmol), 4-dimethylaminopyridine (0.35
g, 2.87 mmol) and boron tribromide (21.5 mL, 34.46 mmol) in 10 mL dichloromethane. Yellow
crystals (0.42 g, 42%) after column chromatography and crystallization form
o
dichloromethane/ethyl acetate – 1/5. M.p. = 178-179 C; IR (Nujol)
ν
(cm-1): 1680 (C=O,
1
COOH); H NMR (DMSO-d6, 250.13 MHz)
δ 6.02 (s, 2H, =CH–), 6.36 (s, 1H, =CH–), 7.22 (s,
1H, =CH–), 7.44 (s, 1H, =CH–), 8.77 (bs, 3H, 3 x OH), 9.23 (bs, 1H, OH), 9.56 (bs, 1H, OH),
12.01 (bs, 2H, 2 x COOH); 13C NMR (DMSO-d6, 62.5 MHz)
110.0 (2 x =CH–), 117.8 (=CH–),
δ
118.2 (=CH–), 121.3 (C), 125.4 (C), 131.2 (C), 131.5 (C), 134.6 (C), 136.3 (=CH–), 144.2 (C),
145.4 (2 x C), 149.2 (C), 167.3 (COOH), 168.8 (COOH) HRMS: m/z calculate: 852.32482,
found: 852.32428 for C37H68O9Si7.
4.2.5. (E)-2-(1-carboxy-2-(3,5-di-tert-butyl-4-hydroxyphenyl)vinyl)-4,5-dihydroxybenzoic acid
(4e)

ACCEPTED MANUSCRIPT
0.96 g (96%) oil from reaction of 6,7-dimetoxihomophthalic anhydride (1) (0.52 g, 2.33
mmol), 3,5-di-tert-butyl-4-hydroxybenzaldehyde
(
2e
)
(0.58 g, 2.33 mmol), 4-
dimethylaminopyridine (0.29 g, 2.33 mmol) and boron tribromide (14.6 mL, 23.34 mmol) in 10
mL dichloromethane. Yellow crystals (0.50 g, 50%) after column chromatography and
crystallization form dichloromethane/ethyl acetate – 1/5. M.p. = 236-238 oC; IR (Nujol)
1725 (COOH), 1680 (COOR); 1H NMR (DMSO-d6, 250.13 MHz)
1.20 (s, 18H 2 x t-Bu), 6.41
(s, 1H, =CH–), 6.89 (s, 2H, =CH–), 7.18 (s, 1H), 7.43 (s, 1H), 7.46 (s, 1H), 9.23 (bs, 1H, OH),
9.58 (bs, 1H, OH), 12.01 (bs, 2H, 2 x COOH); 13C NMR (DMSO-d6, 62.5 MHz)
30.1 (6 x
ν
(cm-1):
δ
δ
CH3), 34.3 (2 x C), 117.6 (=CH–), 118.1 (=CH–), 121.2 (C), 126.1 (C), 127.6 (2 x =CH–), 131.4
(C), 131.5 (C), 136.6 (=CH–), 138.3 (2 x C), 144.3 (C), 149.6 (C), 154.7 (C), 167.1 (COOH),
168.6 (COOH); HRMS: m/z calculate: 716.34161, found: 716.34107 for C36H60O7Si4.
4.2.6.
dimethoxybenzoic acid (
To a mixture 6,7-dimethoxyhomophthalic anhydride (
Synthesis
of
(E)-2-(1-carboxy-2-(3,5-di-tert-butyl-4-hydroxyphenyl)vinyl)-4,5-
5
)
1
) (0.49 g, 2.19 mmol) and 1.1 equiv. of
3,5-di-tert-butyl-4-hydroxybenzaldehyde (2e) (0.57 g, 2.41 mmol) in 5 mL dry dichloromethane,
1 equiv. DMAP (0.27 g, 2.19 mmol) was added. The reaction mixture was stirred for 15 min at
room temperature. At the end of the reaction (TLC), the reaction mixture was acidified (pH = 3)
with 15% HCl, diluted with ethyl acetate and the obtained carboxylic acids were extracted with
10% NaHCO3. The aqueous layer was acidified (pH = 3) with 15% HCl and extracted with ethyl
acetate. The organic layer was dried with Na2SO4, filtered and the solvent was evaporated under
reduced pressure. The residual oil crystallized from ethyl acetate/petroleum ether – 1/1. Yellow
o
1
crystals (0.53 g, 53%). M.p. = 214-216 C; IR (Nujol)
(DMSO-d6, 250.13 MHz) 1.18 (s, 18H, 2 x t-Bu), 3.65 (s, 3H, O–CH3), 3.82 (s, 3H, O–CH3),
6.63 (s, 1H), 6.85 (s, 2H), 7.23 (s, 1H), 7.51 (s, 1H), 7.56 (s, 1H), 12.33 (bs, 2 x COOH); 13C
NMR (DMSO-d6, 62.5 MHz) 30.0 (6 x CH3), 34.7 (2 x C), 55.8 (2 x O–CH3), 113.9 (2 x =CH–
ν
(cm-1): 1680 (C=O, COOH); H NMR
δ
δ
), 122.7 (C), 126.0 (C), 127.6 (2 x =CH–), 130.8 (C), 133.1 (C), 137.2 (=CH–), 138.4 (2 x C),
147.7 (C), 152.3 (C), 154.9 (C), 167.0 (COOH), 168.4 (COOH); HRMS: m/z calculate:
600.29386, found: 600.29346 for C32H48O7Si2.

ACCEPTED MANUSCRIPT
4.3. Biological activity
4.3.1. DPPH● radical assay
For estimation of the DPPH● radical scavenging activity, the analyses were performed at 3.3
µM of compounds 4a-e, 5. In general, 0.5 mL methanol solution with exact concentration of the
tested compounds was added to 0.5 mL of DPPH● stock solution (100
ꢀM) and the absorbance
of the DPPH● at 518 nm was recorded at every 10 min until the reaction reached equilibrium.
The experiments were performed in an excess of DPPH● radical (molar ratio DPPH●/substrate of
15) in order to exhaust the H-donating capacity of polyphenols. Methanol was used as blank
sample and caffeic acid (CA) was used as a reference compound. The scavenging activity was
calculated by means of Eq. 1.
DPPH reduction, % = (A0-Af)/A0 × 100
(1),
where A0 and Af correspond to the absorbencies of DPPH● radical at the beginning and at the end
of the reaction, respectively.
4.3.2. Antibacterial and Anti-yeast Activity (In Vitro)
The synthesized compounds were screened for antibacterial activity against four bacterial
strains. i.e., Bacillus subtilis (ATCC 6633), Staphilococcus aureus (ATCC 25923), Escherichia
coli (ATCC 25922), Pseudomonas aeroginosa (ATCC 10145) and one yeast strain Candida
albicans (ATCC 10231) using agar well diffusion method. Overlays of each test strain (106
CFU/mL) were prepared on agar plates and allowed to dry. Wells (7 mm) were made in the agar
plates and 100 µL of the test sample (0.5 mg/mL in DMSO) was introduced into respective
wells. Other wells were supplemented with DMSO and reference antimicrobial drugs (amikacin
30 µg dick for bacterial test strains and 100 µL nystatine 10000 IU/mL for C. albicans) serving
as negative and positive controls, respectively. The plates were allowed to diffuse through the
o
o
agar for 60 min at 4 C prior to incubation for 24 h at 30 or 37 C, after which inhibitory zones
were measured (in mm). All tests were done in triplicate. Growth inhibition was calculated with
reference to positive control.

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4.3.3. Antifungal Activity (In Vitro)
The synthesized compounds were tested for antifungal activity against three fungal strains
Aspergillus niger (A3), Aspergillus flavus (IMI 052104) and Fusarium graminearum (ATCC
24373). Stock solutions of pure compounds (1 mg/mL) were prepared in sterile DMSO. Test
o
compounds (0.5 mL) were mixed with 20 mL temperate PDA (Oxoid) agar at 45 C into plates.
After drying of the plates, 3
µL of each molds spore suspension (105 spores/mL) were dropped
o
on the agar, and the plates were incubated for 7 days at 30 C. The diameter of the mould
colonies was measured and compared with the controls on media with 0.5 mL DMSO and pure
PDA. All tests were done in triplicate. The inhibition effect was calculated as:
Ihibition effect, % = 100-(d[colony]/d[control colony]*100)
4.3.4. Mushroom Tyrosinase Assay
Tyrosinase activity was determined spectrophotometrically by the modified dopachrome
method, using L-tyrosine as a substrate. The reaction media (3 mL) contained: phosphate buffer
(1.0 mL, 0.1 M, pH 6.8); tyrosine (1.0 mL, 1.5 mM) dissolved in deionized water; inhibitor (0.35
mL, 0.2 mM) dissolved in DMSO; deionized water (0.35 mL); mushroom tyrosinase aqueous
solution (0.30 ml, 192 U/mL). The reference solution was prepared with 0.35 mL of DMSO
instead of inhibitor. After adding of mushroom tyrosinase solution, the reaction mixture was
incubated at 37 oC for 30 min. The UV absorbance of the reaction mixture was measured at 475
nm. The percentage of mushroom tyrosinase inhibitory activity was calculated by following
equation:
Mushroom tyrosinase activity, % = B/A x 100
where A is the absorbance of the reference solution, and B is the absorbance of the test sample
solution. Each experiment was performed in triplicate and averaged. Hydroquinone was used as
positive controls.

ACCEPTED MANUSCRIPT
Acknowledgements
The financial support of the National Science Fund of Bulgaria at the Ministry of Education,
Youth and Science (projects DMU-03-10/2011 and DRNF-02-13/2009) and Sofia University
Fund (project 029/2013) is greatly acknowledged by the authors.
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Table, Scheme and Figure Captions
Table 1. Structure and atom numbering of 4a-e. Chemical shifts (δ, ppm) and heteronuclear
coupling constants (in brackets, J, Hz) for selected representative nuclei.
Table 2. In vitro anti-yeast and antifungal activity of 4a-e and 5.
Scheme 1. One-pot synthesis of cis-restricted polyhydroxy stilbenes 4a-e. Reagents and
conditions: (i) DMAP/CH2Cl2, 10-15 min, rt, then (ii) BBr3/CH2Cl2, rt, 10-60 min.
Figure 1. DPPH● scavenging activity of 4a-e, 5 at 3.3
µM. The analyses were performed in a
molar ratio DPPH●/substrate of 15. Caffeic acid (CA), protocatechuic acid (PCA) and their
equimolar mixture were used as reference compounds.
Figure 2. Mushroom tyrosinase inhibitory activity of 4a-e, 5 at 23 µM. The analyses were
performed in a mole ratio tyrosine/substrate of 22. Hydroquinone (HQ), caffeic acid (CA) and
protocatechuic acid (PCA) were used as reference compounds.
Figure 3. Possible tyrosinase inhibition mechanism. Given structural fragments suggest
4 as both
competitive and non-competitive tyrosinase inhibitors.

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