Resveratrol-related dehydrodimers: Laccase-mediated biomimetic synthesis and antiproliferative activity

Article abstract of DOI:10.1002/ejoc.201200664

Seven resveratrol-related monomeric stilbenoids were submitted to biomimetic oxidative coupling in the presence of laccase from Trametes versicolor (TvL), and gave racemic dihydrobenzofuran dehydrodimers (±)-15 to (±)-21. These products, after spectral characterization, were submitted to an antiproliferative activity bioassay against SW480 human colon cancer cells. Five racemates were found to be active, and were resolved by chiral HPLC. The pure enantiomers were subjected to circular dichroism measurements to establish their absolute configurations at C-7 and C-8. These enantiomerically pure compounds were submitted to the antiproliferative activity assay towards SW480 cells, and were all shown to be active with IC₅₀ values in the approximate range 20-90 μM. In some cases, a significant difference between the activity of the 7R,8R and 7S,8S enantiomers was observed, but a defined configuration of the stereogenic centers does not appear to be a structural requirement for the activity. The comparison between the most active compounds and the inactive ones strongly suggests that the presence of a methoxy group in the position ortho to the C-4 hydroxy group is highly detrimental to the activity. Copyright

Full text of DOI:10.1002/ejoc.201200664

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FULL PAPER
DOI: 10.1002/ejoc.201200664
Resveratrol-Related Dehydrodimers: Laccase-Mediated Biomimetic Synthesis
and Antiproliferative Activity
Vedamurthy M. Bhusainahalli,[a] Carmela Spatafora,[a] Malik Chalal,[b]
Dominique Vervandier-Fasseur,[b] Philippe Meunier,[b] Norbert Latruffe,[c] and
Corrado Tringali*[a]
Dedicated to the memory of Ernesto Fattorusso
Keywords: Medicinal chemistry / Biomimetic synthesis / Enzyme catalysis / Antiproliferation / Antitumor agents /
Structure-activity relationships
Seven resveratrol-related monomeric stilbenoids were sub-
mitted to biomimetic oxidative coupling in the presence of
laccase from Trametes versicolor (TvL), and gave racemic di-
hydrobenzofuran dehydrodimers (Ϯ)-15 to (Ϯ)-21. These
products, after spectral characterization, were submitted to
an antiproliferative activity bioassay against SW480 human
colon cancer cells. Five racemates were found to be active,
and were resolved by chiral HPLC. The pure enantiomers
were subjected to circular dichroism measurements to estab-
lish their absolute configurations at C-7 and C-8. These en-
antiomerically pure compounds were submitted to the anti-
proliferative activity assay towards SW480 cells, and were all
shown to be active with IC50 values in the approximate range
20–90 μM. In some cases, a significant difference between the
activity of the 7R,8R and 7S,8S enantiomers was observed,
but a defined configuration of the stereogenic centers does
not appear to be a structural requirement for the activity. The
comparison between the most active compounds and the in-
active ones strongly suggests that the presence of a methoxy
group in the position ortho to the C-4 hydroxy group is highly
detrimental to the activity.
studies, including in vivo studies, which have been summa-
rized by recent books and reviews.[4] Less attention has been
dedicated to the study of natural or synthetic oligomers of
resveratrol, even though this group of compounds of natu-
ral origin includes many biologically active members. Ex-
amples of dimers of this class are: ε-viniferin (2), a resvera-
trol dehydrodimer found in Vitis vinifera and other natural
sources,[5] which is known for its antifungal,[6] antimicro-
bial,[7] antiinflammatory,[8] and antiproliferative activities;[9]
its glucoside (3), and the related compound scirpusin A (4),
both of which have recently been reported to show ex-
tremely efficient inhibition of β-amyloid peptide aggrega-
tion, a phenomenon that is observed in the progression of
Alzheimer’s disease;[10] the dehydrodimer δ-viniferin (5),
identified as a metabolite of Vitis vinifera cultures[11] and as
a product of resveratrol biotransformation by Botritis ci-
nerea fungal cultures,[12] which has been shown to be mod-
erately cytotoxic against CEM (human lymphoblastoid)
cells;[12] pallidol (6), originally isolated from Cissus
pallida,[13] which has also been reported to be active against
CEM and, more recently, to be an inhibitor of tumorigenic
colon cell growth;[14] and its hexaacetate (7), which has also
shown significant cytotoxicity against KB cells.[15]
Introduction
Resveratrol – (E)-3,5,4Ј-trihydroxystilbene (1) – a stilb-
enoid originally isolated from Veratrum grandiflorum,[1]
and later obtained from the roots of the Asian medicinal
plant Polygonum cuspidatum,[2] has, in recent times, become
one of the most cited natural products. This impressive
growth of popularity started with the possible correlation
between its presence in grapes and the so-called “French
paradox”, which refers to the inverse correlation between a
high-fat diet and low mortality risk from heart disease ob-
served in some southern regions of France, and attributed
to red wine consumption.[3] The subsequent evidence in
favor of its possible role in the prevention of cancer and
other degenerative diseases has stimulated a wide variety of
[a] Dipartimento di Scienze Chimiche, Università di Catania,
Viale A. Doria 6, 95125 Catania, Italy
Homepage: http://www.dipchi.unict.it/cat/docenti/
tringali-corrado/
[b] ICMUB, UMR 6302, Université de Bourgogne,
21000 Dijon, France
[c] LBMN, INSERM U866, Université de Bourgogne,
21000 Dijon, France
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200664.
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These stilbenoids were used as substrates for oxidative
coupling according to the general Scheme 1, and gave de-
hydrodimers (Ϯ)-15 to (Ϯ)-21, incorporating a dihydro-
benzofuran moiety, as major products. It is worth emphas-
izing that the stilbenoid dimerization reactions, even the en-
zyme-mediated reactions, occured with regio- and dia-
stereoselectivity, but not enantioselectivity,[16e,19] and conse-
quently gave racemic mixtures.
The mechanism of formation of resveratrol oligomers by
oxidative coupling has been investigated by several authors,
and the involvement of radical species is widely accepted.
The hypothesis of a radical attack on the unactivated start-
ing material has been considered as an alternative to a radi-
cal–radical coupling mechanism.[16d,16f,19,20] However, the
involvement of a quinone–methide radical is frequently
claimed, and the mechanism illustrated in Scheme 2, sup-
ported by experimental evidence,[16e] seems reasonable for
the laccase-mediated formation of resveratrol dehydrodi-
mers with the δ-viniferin skeleton. The observed regioselec-
tivity of these reactions is in agreement with this mecha-
nism. A plausible explanation of the diastereoselectivity in
favor of the 7,8-trans-substituted diastereomers, which has
also been observed in all previous syntheses of dihydroben-
zofuran-based stilbene dimers,[21] is that the less hindered
transition state (TS) leading to the trans product is largely
favoured. Moreover, recently reported AM1 calculations on
compound 19[16f] indicated that the trans diastereomer is
more stable than its cis isomer. This was supported by our
recently published data on the oxidative coupling of caffeic
acid esters to give benzoxanthene and trans-aryldihydro-
naphthalene lignans.[22] The calculated ΔHf values for the
possible TS originating from a quinone–methide intermedi-
ate clearly indicated that the TS leading to the trans aryldi-
hydronaphthalene racemate is by far the most stable. With
regard to the lack of enantioselectivity in the enzyme-medi-
ated oxidative coupling, it is worth noting that in the bio-
synthesis of lignans, where the proposed mechanism is the
oxidative coupling of two phenylpropanoid units followed
by quinone–methide cyclization, it has been proved that the
The interesting properties of the natural resveratrol di-
mers have stimulated the preparation of synthetic analogues
by both chemical and enzymatic methods.[16] Nevertheless,
to the best of our knowledge, none of these synthetic efforts
has been accompanied by a biological evaluation of the
products. Thus, as a continuation of our studies on antitu-
mor resveratrol analogues,[17] we have now synthesized a
small library of resveratrol-related dimers by a biomimetic
route based on enzyme-mediated oxidative coupling using
the laccase from Trametes versicolor. The products were
purified and evaluated for antiproliferative activity against
SW480 colon cancer cells. The results and some structure–
activity relationship considerations are presented in this pa-
per.
Results and Discussion
Monomeric resveratrol analogues 8–14 were obtained by
Wittig and/or Heck reactions, and their synthesis has pre-
viously been reported in detail by some of us.[18]
Scheme 1. Oxidative coupling of compounds 8–14.
2
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Resveratrol-Related Dehydrodimers
Scheme 2. Mechanism of formation of compounds (Ϯ)15–(Ϯ)-21.
oxidase enzyme only generates the radical species, and that subjected to an analogous reaction in the presence of TvL,
the stereochemistry is controlled by a “dirigent protein”.[21] and the major product was isolated by flash chromatog-
In the absence of this protein, racemic mixtures are ob- raphy. The ESI-MS spectrum of this product showed a peak
tained.[23]
at m/z = 465.1 [M + Na]+, consistent with the expected
We used a biomimetic oxidative coupling methodology molecular weight for (Ϯ)-16, a previously unreported dimer.
that mimics the biosynthesis of natural dihydrobenzofuran For this compound, we carried out an extensive 1H and 13
C
dimers,[21] as outlined in the literature.[6,16d,21] Although NMR spectroscopic analysis, including two-dimensional
various synthetic methodologies have been used for the syn- methods (COSY, HSQC and HMBC, see Supporting Infor-
thesis of stilbenoid oligomers, the biomimetic approach has mation and Figure 1), in order to unambiguously establish
1
the advantage of simplicity and brevity. An enzyme-medi- the structure and assign all H and 13C NMR signals, as
ated reaction is also a sustainable chemical process. Reac- reported in the Experimental section. Stilbenoid 10, bearing
tion conditions were evaluated in preliminary experiments a methoxy group at C-4Ј, was treated as described above,
on an analytical scale, using both chemical and enzymatic and one major product was obtained. The product was
methods.
characterized as dimer (Ϯ)-17 on the basis of the analysis
The reactions were carried out on three resveratrol ana- of its ESI-MS and NMR spectroscopic data and compari-
logues, 9, 10 and 14, using either Mn(OAc)3 or laccase from son with literature data.[16e] In a similar way, substrate 11,
the fungus Trametes versicolor (TvL). In the enzymatic reac- with three oxygenated functionalities (one hydroxy group at
tions, a shaken biphasic system with an acetate buffer and C-4 and two methoxy groups at C-3 and C-4Ј), was treated
a co-solvent was used at room temperature. In the Mn-me- with TvL in the biphasic system, and one major product
1
diated reactions, dichloromethane and chloroform gave bet- was obtained. Analysis of the ESI-MS, H and 13C NMR
ter results than ethyl acetate. In the TvL-mediated reac- spectra of the dimer led to structure (Ϯ)-18, in agreement
tions, poor conversions were observed when dichlorometh- with previously reported data.[16e] When this method was
ane and chloroform were used as co-solvents, whereas ethyl applied to stilbenoid 12, bearing two methoxy groups at C-
acetate gave good conversion rates. In a further preliminary 3Ј and C-5Ј, it led to the formation of racemate (Ϯ)-19 as
evaluation of the metal-mediated methodology, the sub- the major product, whose ESI-MS and NMR spectroscopic
strate/Mn(OAc)3 ratio was evaluated, and a 1:4 ratio gave data were in perfect agreement with those previously re-
the best results. In summary, the TvL-mediated conditions ported.[16e,16f] The pentasubstituted substrate 13 (bearing
appeared to be the most promising, both in terms of the four methoxy groups at C-3, C-3Ј, C-4Ј and C-5Ј, in ad-
higher conversion of the substrate, and for giving essentially dition to the C-4 hydroxy group), treated as described
one major product. Thus, the enzymatic methodology was above, gave a major product that had not been reported
1
selected for the preparative reactions, also in view of its sim- previously. Analysis of its H and 13C NMR spectra was
ple work-up and of its “eco-friendly” and heavy-metal-free facilitated by using two-dimensional methods (COSY,
features.
NOESY, HSQC and HMBC, see Supporting Information
For the preparation of dimer 15, monohydroxylated stil- and Figure 1), which allowed a complete assignment of the
bene 8 was treated with TvL in ethyl acetate/acetate buffer. NMR signals and confirmed the expected structure [i.e.,
The major product was isolated by flash chromatography. (Ϯ)-20]. In particular, due to the presence of eight methoxy
1
Analysis of the ESI-MS, H and 13C NMR spectra of the groups in this compound, the NOESY spectrum was of crit-
1
product allowed its identification as the E-dihydrobenzofur- ical importance for the assignment of the six pertinent H
an dimer (Ϯ)-15, based also on comparison with spectro- signals (See Experimental section). Finally, brominated stilb-
scopic data previously reported in the literature.[16e] The J- ene 14 was submitted to the dimerization reaction condi-
value of the 7Ј-H,8Ј-H coupling (J = 16.3 Hz) established tions, and the major product gave a peak at m/z = 546.9
the E configuration of the double bond, and that of the 7- [M – H]–, which was consistent with the expected molecular
H,8-H coupling (J = 8.5 Hz), confirmed the trans relative weight of (Ϯ)-21. The structural determination of this un-
configuration of the C-7 and C-8 substituents. Monohy- precedented dimer was also supplemented by two-dimen-
droxylated stilbenoid 9, bearing a vinyl group at C-4Ј, was sional NMR spectroscopic methods (COSY, HSQC and
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Figure 1. Selected 2D-NMR correlations for (Ϯ)-16, (Ϯ)-20 and (Ϯ)-21.
HMBC, see Supporting Information and Figure 1), to allow fied enantiomers were subjected to Circular Dichroism
the complete assignment of the NMR signals (See Experi- (CD) measurements to establish their absolute configura-
mental section).
tion at the stereogenic centers C-7 and C-8. The compari-
After their structural characterization, the racemic dihy- son of CD spectra with that of a closely related compound
drobenzofuran dimers [i.e., (Ϯ)-15–(Ϯ)-21] were submitted is one of the most useful methods for determining the abso-
to an antiproliferative activity bioassay against SW480 hu- lute configuration of natural products or their synthetic an-
man colon cancer cells.
alogues,[24] and it has been applied to similar cases of dihy-
Resveratrol (1) was used as a reference compound, taking drobenzofuran dimers.[25] For the sake of brevity, only the
into account its structural relationship with the dimers and CD spectra of the enantiomers 17a and 17b are reported
the antitumor properties of this well-known stilbenoid. The here (Figure 2). The CD spectra of all of the enantiomeric
results are reported in Table 1.
pairs are reported in the Supporting Information. The abso-
lute configuration of 17a and 17b was determined by com-
parison of their CD spectra with that of (–)-ε-viniferin
(2),[25d] whose absolute configuration has previously been
established as 7R,8R.[25b] Both 17a and 7R,8R-viniferin (2)
show a negative Cotton effect in the range 230–240 nm, and
consequently, the 7R,8R configuration was assigned to 17a,
and the 7S,8S configuration was assigned to 17b.
Table 1. Antiproliferative activity towards SW480 cells of com-
pounds (Ϯ)-15–(Ϯ)-21.
Compound
IC50 [μm][a]
1
68.1Ϯ5.5
22.5Ϯ2.0
22.3Ϯ2.7
33.4Ϯ2.9
Ͼ 100
27.8Ϯ3.1
Ͼ 100
(Ϯ)-15
(Ϯ)-16
(Ϯ)-17
(Ϯ)-18
(Ϯ)-19
(Ϯ)-20
(Ϯ)-21
30.7Ϯ2.8
[a] IC50 and standard deviation determined after 48 h of treatment;
values are the mean of three experiments.
A first obvious structure–activity relationship is that two
dimers (Ϯ)-18 and (Ϯ)-20, the only ones bearing a methoxy
group in the position ortho to the C-4 hydroxy group, are
essentially inactive (IC50 Ͼ 100 μm), whereas all the other
racemic dimers show antiproliferative activity with IC50 val-
ues in the approximate range 22–33 μm, i.e., significantly
higher than that of resveratrol. It is particularly interesting
that the most active dimers, (Ϯ)-16 and (Ϯ)-15, are those
with fewest substituents. Of course, these results do not al-
Figure 2. CD spectra of 17a and 17b.
The absolute configurations of the other enantiomeric
low the assessment of the possible consequences of different pairs were determined on the basis of an analogous com-
configurations at the stereogenic centers at C-7 and C-8. parison. Thus, the ten enantiomerically pure compounds
Thus, we carried out the chiral resolution of the five active were established as follows: 7R,8R-15a; 7S,8S-15b; 7R,8R-
racemates to isolate the pure enantiomers, in order to estab- 16a; 7S,8S-16b; 7R,8R-17a; 7S,8S-17b; 7R,8R-19a; 7S,8S-
lish their absolute configuration and evaluate their specific 19b; 7R,8R-21a; 7S,8S-21b. All of the enantiomers that
antiproliferative activity. Each racemic mixture was sub- eluted first from the Chiralpack IA column had the 7R,8R
jected to chiral HPLC separation on a Chiralpack IA col- configuration.
umn (see Experimental section for details). From here on,
The purified enantiomeric forms of 15, 16, 17, 19 and 21
the enantiomer with the shorter retention time of each pair were submitted to the antiproliferative activity assay
is indicated as “a” to distinguish it from the enantiomer towards SW480 colon cancer cells. The results are summa-
with the longer retention time, indicated as “b”. The puri- rized in Table 2.
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Resveratrol-Related Dehydrodimers
Table 2. Antiproliferative activity towards SW480 cells of the enan-
tiomeric forms of compounds 15, 16, 17, 19, 21.
The racemates were submitted to an antiproliferative ac-
tivity bioassay towards SW480 human colon cancer cell cul-
tures, and from this assay, five dimers showed significant
activity, in the order (Ϯ)-16 Ͼ (Ϯ)-15 Ͼ (Ϯ)-19 Ͼ (Ϯ)-
21 Ͼ (Ϯ)-17. The racemic mixtures were resolved by chiral
HPLC, and the pure enantiomers were submitted to CD
measurements in order to establish their absolute configura-
tion. The enantiomerically pure compounds were also
evaluated for their antiproliferative activity. The results of
the bioassays are not simple to interpret, but the following
observations on possible structure–activity relationships
can be made: (a) A comparison between the most active
compound (i.e., 17a) and inactive (Ϯ)-18 strongly suggests
that the methoxy group ortho to the C-4 hydroxy group is
highly detrimental to the activity. The data also suggest a
critical role for the C-4 hydroxy group, for instance in estab-
lishing hydrogen bonds with a biological target;
(b) Although the three most active compounds (i.e., 17a,
16a and 21a) have a 7R,8R configuration, the configuration
of the stereogenic centers does not appear to be a vital
structural determinant for activity. However, it could be-
come important when other factors have a minor role, as
suggested by the marked difference in activity between
some pairs of enantiomers. For instance, in dimers without
hindering substituents close to the C-4 hydroxy group, fur-
ther interactions involving the stereogenic centers could be
established with the biological target.
Compound
IC50 [μm][a]
7R,8R-15a
7S,8S-15b
7R,8R-16a
7S,8S-16b
7R,8R-17a
7S,8S-17b
7R,8R-19a
7S,8S-19b
7R,8R-21a
7S,8S-21b
88.1Ϯ2.1
22.5Ϯ0.8
20.0Ϯ0.4
22.5Ϯ0.7
19.9Ϯ0.6
73.4Ϯ2.3
43.8Ϯ1.5
24.9Ϯ0.9
21.7Ϯ0.4
35.9Ϯ0.8
[a] IC50 and standard deviation determined after 48 h of treatment;
values are the mean of three experiments.
From these results on the inhibition of SW480 colon can-
cer cell growth, it is clear that all of the individual enantio-
mers are active, with IC50 values in the approximate range
20–90 μm. Although the highest activity was shown by
7R,8R-17a, the most active enantiomers of each pair were
equally distributed between the 7R,8R and 7S,8S configura-
tions, in the order: 7R,8R-17a Ͼ 7R,8R-16a Ͼ 7R,8R-21a
Ͼ 7S,8S-15b = 7S,8S-16b Ͼ 7S,8S-19b. The most potent
compound 17a (IC50 = 19.9 μm) also showed the highest
difference in activity with respect to its enantiomer 17b
(IC50 = 73.4 μm). Compound 15b (IC50 = 22.5 μm) was four
times more active than its enantiomer 15a (IC50 = 88.1 μm),
but a less marked difference was observed between 21a and
21b and between 19b and 19a, whereas 16a and 16b had
comparable activities. On the basis of these data, a defined
configuration of the stereogenic centers does not appear to
be a structural determinant for the activity, although it
could have a role in some cases. All of the most active de-
hydrodimers 17a, 16a and 21a, which showed comparable
IC50 values, bear only one substituent at C-4Ј position, and
their activity was close to that of 15b, which has no substit-
uent on the second (non-hydroxylated) ring. This corrobo-
rates the observation reported above that the compounds
bearing fewer substituents show higher activity. However,
other factors, such as diffusion, membrane permeability,
metabolic stability, and others, may affect the activity in
bioassays on cell cultures.
In conclusion, although only a small library of com-
pounds was evaluated, and a more detailed definition of the
structural determinants for resveratrol-related dihydroben-
zofuran dimers will require further structure–activity rela-
tionship studies, the results discussed here offer first indica-
tions for further optimization of these promising lead com-
pounds.
Experimental Section
General: NMR spectra were obtained with a Varian Unity Inova
spectrometer operating at 499.86 (1H) and 125.70 MHz (13C),
equipped with gradient-enhanced, reverse-detection probe. Chemi-
cal shifts (δ) are indirectly referenced to TMS using solvent signals.
All NMR experiments, including two-dimensional spectra, were
performed using software supplied by the manufacturers and ac-
quired at constant temperature (298 K) using CDCl3 as solvent.
NMR spectroscopic data of the previously unreported compounds
(16, 20 and 21) are reported below, copies of the pertinent spectra
are included in the Supporting Information.
Conclusions
In this work, we have synthesized seven racemic resvera-
trol-related dehydrodimers incorporating a dihydrobenzo-
furan moiety (i.e., 15–21), using an eco-friendly, biomimetic
methodology based on laccase-mediated oxidative coupling
of synthetic stilbenoids 8–14. Three previously unreported
dimers (i.e., 16, 20, and 21) were obtained; the other prod-
ucts (i.e., 15, 17, 18, and 19) have been reported previously,
but using the laccase from Trametes versicolor, we obtained
both higher yields and shorter reaction times (for com-
pounds 15, 17 and 19), or at least a shorter reaction time
(for compound 18), than had previously been obtained
using the laccase from Trametes pubescens.[16e]
Electrospray ionization mass spectra (ESI-MS) were recorded with
a
Thermo-Finnigan LCQ-DECA ion-trap mass spectrometer
equipped with an ESI ion source, operating in MS negative- or
positive-ion mode under the following conditions: capillary tem-
perature, 180 °C; sheath gas 20 a.u.; spray voltage 4 kV. Elemental
analyses were performed with a Perkin–Elmer 240B microanalyzer.
High-performance liquid chromatography (HPLC) was carried out
using an Agilent Series G1354A pump and a photodiode array
detector Agilent Series 1200 G1315C/D. An auto-sampler Agilent
Series 1100 G1313A was used for sample injection. The chiral
HPLC-UV of racemates was carried out on a Chiralpak IA (5 μm;
4.6ϫ250 mm) column; details of elution and flow rate are reported
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below for each separation. The separation protocol was repeated a
sufficient number of times to achieve the amount (3–5 mg) required
for CD measurements and bioassays.
extraction of the water solution. The dried organic extracts were
evaporated, and the crude residue was purified by flash chromatog-
raphy (petroleum ether/dichloromethane, 6:4) to give (Ϯ)-16 (9 mg,
24%) as an amorphous white powder. Rf (TLC) = 0.41 (25%
EtOAc/n-hexane). 1H NMR (500 MHz, CDCl3, 298 K): δ = 7.41
CD spectra were run in methanol on a Jasco J610 spectrometer.
The CD spectra of pure enantiomers are included in the Support-
ing Information.
3
(d, JH,H = 8.5 Hz, 4 H, 11-H, 13-H, 11Ј-H, and 13Ј-H), 7.38* (d,
3
3JH,H = 8.5 Hz, 1 H, 2Ј-H), 7.37* (d, JH,H = 8.5 Hz, 2 H, 10Ј-H
3
and 14Ј-H), 7.23 (d, JH,H = 8.5 Hz, 1 H, 2-H, 6-H), 7.19# (br. s, 1
Flash chromatography was performed on Silica gel 60 (Merck) and
DIOL Silica gel (Merck) using different solvent systems, as re-
ported for each compound. TLC was carried out using pre-coated
silica gel F254 plates (Merck); cerium sulfate and phosphomolybdic
acid were used as chromogenic spray reagents.
3
H, 6Ј-H), 7.18# (d, JH,H = 8.5 Hz, 4 H, 10-H and 14-H), 7.04 (d,
3
3JH,H = 16.2 Hz, 1 H, 7Ј-H), 6.94 (d, JH,H = 8.5 Hz, 1 H, 3Ј-H),
3
3
6.90 (d, JH,H = 16.2 Hz, 1 H, 8Ј-H), 6.82 (d, JH,H = 8.5 Hz, 2 H,
3
3-H and 5-H), 6.74 (dd, JH,H = 16.2, 10.5 Hz, 1 H, 15-H), 6.70
3
3
(dd, JH,H = 16.2, 10.5 Hz, 1 H, 15Ј-H), 5.78 (d, JH,H = 16.2 Hz,
Materials and Methods: All chemicals were of reagent grade and
were used without further purification. Laccase from Trametes ver-
sicolor (TvL) and manganese acetate were purchased from Sigma
(Milan, Italy).
1 H, 16b-H), 5.75 (d, 3JH,H = 16.2 Hz, 1 H, 16bЈ-H), 5.49 (d, 3JH,H
3
= 8.7 Hz, 1 H, 7-H), 5.28 (d, JH,H = 10.5 Hz, 1 H, 16a-H), 5.24
(d, JH,H = 10.5 Hz, 1 H, 16aЈ-H), 4.90 (br. s, 1 H, OH), 4.56 (d,
3
3JH,H = 8.7 Hz, 1 H, 8-H) ppm. The signals with identical super-
scripts (* or #) were partially overlapping. 13C NMR (125 MHz,
CDCl3, 298 K): δ = 159.6 (C-4Ј), 155.6 (C-4), 140.9 (C-9), 137.2
(C-5Ј), 136.7 (C-12Ј), 136.5 (C-15), 136.4 (C-15Ј), 132.5 (C-1), 131.0
(C-1Ј), 130.9 (C-9Ј), 128.6 (C-7Ј), 128.3 (C-6Ј), 127.9 (C-2Ј), 127.6
(C-2 and C-6), 126.7 (C-10Ј and C-14Ј), 126.5 (C-11, C-13, C-11Ј
and C-13Ј), 126.3 (C-12), 125.9 (C-8Ј), 122.8 (C-10 and C-14),
115.5 (C-3 and C-5), 113.9 (C-16), 113.4 (C-16Ј), 109.7 (C-3Ј), 93.2
(C-7), 57.4 (C-8) ppm. For selected COSY and HMBC correlations,
see Figure 1. ESI-MS: m/z = 465.1 [M + Na]+. C32H26O2 (442.5):
calcd. C 86.85, H 5.92; found C 86.91, H 5.86. The chiral HPLC
of racemic (Ϯ)-16 was carried out by HPLC-UV using a Chiralpak
IA (5 μm; 4.6ϫ250 mm) column and an isocratic elution (2-prop-
anol/n-hexane, 6:4, flow rate 0.6 mL/min). Pure enantiomer 7R,8R-
16a was isolated with a retention time of 12.0 min, and 7S,8S-16b
was isolated with a retention time of 14.0 min. 7R,8R-16a: CD (c
= 1.8ϫ10–4 m, MeOH) nm (Δε): 235 (–0.508); 7S,8S-16b: CD (c =
2.2ϫ10–4 m, MeOH) nm (Δε): 235 (0.417).
Synthesis of the Stilbenoids 8–14: The monomeric resveratrol ana-
logues 8–14 were synthesized by Wittig and/or Heck reactions ac-
cording to the procedures previously reported by some of us.[18]
Biomimetic Synthesis of Compounds (؎)-15 to (؎)- 21
Preliminary Experiments. Metal-Mediated Reactions: Each sub-
strate (9, 10, and 14; 2 mg) was dissolved in three different solvents,
namely dichloromethane, chloroform and ethyl acetate (2 mL).
Subsequently, Mn(OAc)3 (1 equiv.) was added to the solutions. The
reaction mixture was stirred at room temperature and monitored
by TLC. After 24 h, the reaction was quenched with a solution of
ascorbic acid in water, and a further amount (3ϫ 2 mL) of the
pertinent organic solvent (dichloromethane, chloroform or ethyl
acetate) was added. After extraction, the dried organic phase was
evaporated to dryness under vacuum, and the residue was analyzed
by TLC. Two further substrate/oxidant ratios (1:2 and 1:4) were
also evaluated in chloroform and dichloromethane.
Enzyme-Mediated Reactions: Each substrate (9, 10, and 14; 2 mg)
was dissolved in three different solvents, namely dichloromethane,
chloroform and ethyl acetate (1 mL). A solution of TvL (2 mg) in
acetate buffer (1 mL, 0.1 m, pH = 4.7) was added, the mixture was
stirred at room temperature, and the reaction was monitored by
TLC. After 24 h, the product was obtained by phase-separation.
Synthesis of (؎)-17: Substrate 10 (100 mg, 0.44 mmol) was dis-
solved in ethyl acetate (40 mL), and TvL (10 U, 15 mg) was dis-
solved in acetate buffer, (pH 4.7; 15 mL). The biphasic system was
stirred at room temperature for 20 h and monitored by TLC. The
reaction was quenched by phase separation, and this was followed
by EtOAc extraction of the water solution. The dried organic ex-
tracts were evaporated. The crude residue was purified by flash
chromatography using petroleum ether/ethyl acetate (8:2) as eluent
to give (Ϯ)-17 (65 mg, 65%). 1H and 13C NMR spectroscopic data
were in agreement with previous data reported in the literature.[16e]
ESI-MS: m/z = 449.3 [M – H]–. The chiral HPLC of racemic (Ϯ)-
17 was carried out by HPLC-UV using a column Chiralpak IA
(5 μm; 4.6ϫ250 mm) and an isocratic elution with (2-propanol/n-
hexane, 3:7, flow rate 0.5 mL/min). Pure enantiomer 7R,8R-17a
was isolated with a retention time of 21.0 min, and 7S,8S-17b was
isolated with a retention time of 25.1 min. 7R,8R-17a: CD (c =
1.1ϫ10–4 m, MeOH) nm (Δε): 235 (–0.508); 7S,8S-17b: CD (c =
1.2ϫ10–4 m, MeOH) nm (Δε): 235 (0.417).
Synthesis of (؎)-15: Substrate 8 (140 mg, 0.71 mmol) was dissolved
in ethyl acetate (35 mL), and TvL (10 U, 30 mg) was dissolved in
acetate buffer, (pH 4.7; 15 mL). The biphasic system was stirred at
room temperature for 10 h and monitored by TLC. The reaction
was quenched by phase separation, and this was followed by EtOAc
extraction of the water solution. The dried organic extracts were
evaporated, and the crude residue was purified by flash chromatog-
raphy using petroleum ether/dichloromethane (6:4) as the eluent to
give (Ϯ)-15 (38 mg, 28%). 1H and 13C NMR spectroscopic data
were in agreement with previous data reported in the literature.[16e]
ESI-MS: m/z = 413.1 [M + Na]+. The resolution of racemic (Ϯ)-
15 was carried out by chiral HPLC-UV using a Chiralpak IA col-
umn (5 μm; 4.6ϫ250 mm), with an isocratic elution (2-propanol/
n-hexane, 8:2, flow rate 0.4 mL/min). Pure enantiomer 7R,8R-15a
was isolated with a retention time of 14.3 min, and 7S,8S-15b was
isolated with a retention time of 18.1 min. 7R,8R-15a: CD (c =
2.5ϫ10–4 m, MeOH) nm (Δε): 230 (–0.334); 7S,8S-15b: CD (c =
2.7ϫ10–4 m, MeOH) nm (Δε): 230 (0.421).
Synthesis of (؎)-18: Substrate 11 (30 mg, 0.11 mmol) was dissolved
in EtOAc (15 mL), and TvL (10 U, 10 mg) was dissolved in of acet-
ate buffer (pH 4.7; 10 mL). The biphasic system was stirred at room
temperature for 24 h and monitored by TLC. The reaction was
quenched by phase separation, and this was followed by EtOAc
extraction of the water solution. The dried organic extracts were
evaporated. The crude residue was purified by flash chromatog-
raphy using petroleum ether/ethyl acetate (8:2) as eluent to give
(Ϯ)-18 (19 mg, 67%). 1H and 13C NMR spectroscopic data were
in agreement with previous data reported in the literature.[16e] ESI-
MS: m/z = 509 [M – H]–.
Synthesis of (؎)-16: Substrate 9 (30 mg, 0.13 mmol) was dissolved
in ethyl acetate (20 mL), and TvL (10 U, 15 mg) was dissolved in
acetate buffer, (pH 4.7; 15 mL). The biphasic system was stirred at
room temperature for 24 h and monitored by TLC. The reaction
was quenched by phase separation, and this was followed by EtOAc
6
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20664
/KAP1
Date: 25-07-12 15:51:20
Pages: 9
Resveratrol-Related Dehydrodimers
3
Synthesis of (؎)-19: Substrate 12 (80 mg, 0.31 mmol) was dissolved
in EtOAc (35 mL), and TvL (10 U, 15 mg) was dissolved in acetate
buffer (pH 4.7; 15 mL). The biphasic system was stirred at room
temperature for 10 h and monitored by TLC. The reaction was
quenched by phase separation, and this was followed by EtOAc
extraction of the water solution. The dried organic extracts were
evaporated. The residue was subjected to column chromatography
using DIOL silica gel and petroleum ether/ethyl acetate (8:2) as
eluent to give (Ϯ)-19 (12 mg, 25%). 1H NMR spectroscopic data
were in perfect agreement with those previously reported.[16f] ESI-
MS: m/z = 509 [M – H]–. The chiral HPLC of racemic (Ϯ)-19 was
carried out by HPLC-UV using a column Chiralpak IA (5 μm;
4.6ϫ250 mm) and an isocratic elution with (2-propanol/n-hexane,
6:4, flow rate 0.6 mL/min). Pure enantiomer 7R,8R-19a was iso-
lated with a retention time of 13.4 min, and 7S,8S-19b was isolated
2 H, 3-H and 5-H), 6.834* (d, JH,H = 16.3 Hz, 1 H, 8Ј-H), 5.44
3
3
(d, JH,H = 8.7 Hz, 1 H, 7-H), 4.99 (s, 1 H, 4-OH), 4.53 (d, JH,H
= 8.7 Hz, 8-H) ppm. The signals with identical superscript (*) were
partially overlapping. 13C NMR (125 MHz, CDCl3, 298 K): δ =
159.8 (C-4Ј), 155.8 (C-4), 140.3 (C-9), 136.5 (C-9Ј), 132.0 (C-11Ј
and C-13Ј), 131.7 (C-11 and C-13), 130.8 (C-1), 130.6 (C-5Ј), 130.1
(C-10, C-14), 129.0 (C-7Ј), 128.24 (C-1Ј), 128.23 (C-2Ј), 127.6 (C-2
and C-6), 127.6 (C-10Ј and C-14Ј), 125.2 (C-8Ј), 122.8 (C-6Ј), 121.3
(C-12), 120.8 (C-12Ј), 115.5 (C-3 and, C-5), 109.9 (C-3Ј), 93.3 (C-
7), 57.1 (C-8) ppm. For selected COSY and HMBC correlations,
see Figure 1. ESI-MS: m/z = 546.9 [M – H]–. C28H20Br2O2 (548.3):
calcd. C 61.34, H 3.68, Br 29.15; found C 61.37, H 3.58, Br 29.22.
The chiral HPLC of the racemate was carried out by HPLC-UV
using a column Chiralpak IA (5 μm; 4.6ϫ250 mm) and an iso-
cratic elution (2-propanol/n-hexane, 5:5; flow rate 0.8 mL/min).
with a retention time of 16.0 min. 7R,8R-19a: CD (c = 2.7ϫ10–4 m, Pure enantiomer 7R,8R-21a was isolated with a retention time of
MeOH) nm (Δε): 234 (–0.378); 7S,8S-19b: CD (c = 3.3ϫ10–4 m,
MeOH) nm (Δε): 234 (0.392).
10.4 min, and enantiomer 7S,8S-21b was isolated with a retention
time of 12.5 min. 7R,8R-21a: CD (c = 1.8ϫ10–4 m, MeOH) nm
(Δε): 235 (–1.853); 7S,8S-21b: CD (c = 2.4ϫ10–4 m, MeOH) nm
(Δε): 235 (1.515).
Synthesis of (؎)-20: Substrate 13 (100 mg, 0.32 mmol) was dis-
solved in EtOAc (25 mL), and TvL (10 U, 15 mg) was dissolved in
acetate buffer (pH 4.7; 15 mL). The biphasic system was stirred at
room temperature for 8 h and monitored by TLC. The reaction was
quenched by phase separation, and this was followed by EtOAc
extraction of the water solution. The organic solvents were evapo-
rated. The crude residue was purified by flash chromatography
using petroleum ether/ethyl acetate (6:4) as eluent to give (Ϯ)-20
(30 mg, 30%) as an amorphous white powder. Rf (TLC) = 0.58
Biological Methods
Cell Culture: The human colon carcinoma cell line SW480 was ob-
tained from ATCC (American Type Culture Collection). The cells
were cultured in RPMI-Medium, supplemented with 10% (v/v)
heat-inactivated fetal bovin serum and 1% antibiotics (penicillin,
streptomycin); the cell cultures were maintained at 37 °C in a hu-
midified CO2 (5%) incubator.
1
(40% EtOAc/n-hexane). H NMR (500 MHz, CDCl3, 298 K): δ =
3
Proliferation Inhibition Assays: Proliferation inhibition assays were
performed in 96-well plates in triplicate. Each well was seeded with
105 cells, and after 24 h, the cells were incubated with the com-
pounds under study dissolved in 0.1% dimethyl sulfoxide (DMSO),
at different concentrations (0.75–100 μm). After 48 h incubation at
37 °C, the medium was carefully removed from the wells, and the
PBS was warmed at room temperature before washing the plates.
Then the plates were washed several times with tap water to remove
the excess stain. The remaining crystal violet incorporated into the
nucleus was solubilized in sodium citrate solution. Plates were agi-
tated on an orbital shaker until the color was uniform, with no
areas of dense coloration in the bottom of the wells. The ab-
sorbance was read on each plate at 540 nm with a spectrophotome-
ter (Dynex MRX-TC Revelation). The absorbance is proportional
to the density of cells adhering to the multi-well dishes relative to
the absorbance of the control well-plate (5% DMSO).
7.03 (s, 1 H, 2Ј-H), 6.95 (d, JH,H = 16.0 Hz, 1 H, 7Ј-H), 6.90 (d,
3JH,H = 2.0 Hz, 1 H, 6Ј-H), 6.88 (d, 3JH,H = 8.0 Hz, 1 H, 5-H), 6.86
3
3
4
(d, JH,H = 16.0 Hz, 1 H, 8Ј-H), 6.82 (dd, JH,H = 8.0, JH,H
=
1.5 Hz, 1 H, 6-H), 6.80 (d, 3JH,H = 1.5 Hz, 1 H, 2-H), 6.70 (s, 2 H,
10Ј-H and 14Ј-H), 6.38 (s, 2 H, 10-H and 14-H), 5.52 (d, JH,H
3
=
9.2 Hz, 1 H, 7-H), 4.52 (d, 3JH,H = 9.2 Hz, 1 H, 8-H), 4.01 (s, 3 H,
3Ј-OCH3), 3.90 (s, 6 H, 11Ј-OCH3 and 13Ј-OCH3), 3.874 (s, 3 H,
3-OCH3), 3.872 (s, 3 H, 12Ј-OCH3), 3.86 (s, 3 H, 12-OCH3), 3.78 (s,
6 H, 11-OCH3 and 13-OCH3) ppm. 13C NMR (125 MHz, CDCl3,
298 K): δ = 153.5 (C-11, C-13), 153.4 (C-11Ј and C-13Ј), 148.1 (C-
3), 146.6 (C-4Ј), 145.8 (C-4), 144.5 (C-3Ј), 137.7 (C-12Ј), 137.2 (C-
12), 136.8 (C-5Ј), 133.2 (C-1Ј), 131.9 (C-1), 131.7 (C-9), 131.5 (C-
9Ј), 128.2 (C-7Ј), 126.6 (C-8Ј), 119.6 (C-2), 115.6 (C-6), 114.2 (C-
5), 109.9 (C-2Ј), 108.7 (C-6Ј), 105.4 (C-10 and C-14), 103.3 (C-10Ј
and C-14Ј), 94.2 (C-7), 60.9 (12-OCH3), 60.8 (12Ј-OCH3), 58.5 (C-
8), 56.2 (3Ј-OCH3), 56.1 (11-OCH3, 13-OCH3), 56.05 (11Ј-OCH3,
13Ј-OCH3), 56.01 (3-OCH3) ppm. For selected COSY, NOESY and
HMBC correlations, see Figure 1. ESI-MS: m/z = 629.2 [M – H]–.
C36H38O10 (630.7): cald. C 68.56, H 6.07; found C 68.34, H 6.21.
Supporting Information (see footnote on the first page of this arti-
cle): ESI mass, NMR and CD spectra.
Synthesis of (؎)-21: Substrate 14 (50 mg, 0.2 mmol) was dissolved
in EtOAc (20 mL), and TvL (10 U, 15 mg) was dissolved in acetate
buffer (pH 4.7; 10 mL). The biphasic system was stirred at room
temperature for 5 d and monitored by TLC. The reaction was
quenched by phase separation, and this was followed by EtOAc
extraction of the water solution. The dried organic extracts were
evaporated. The crude residue was purified by flash chromatog-
raphy using petroleum ether/ethyl acetate (94:6) as eluent to give
(Ϯ)-21 (19 mg, 20%) as an amorphous white powder. Rf (TLC) =
Acknowledgments
This research was supported by a grant of Ministero dell’Università
e della Ricerca (MIUR) (PRIN 2009, Rome, Italy) and by the Uni-
versità degli Studi di Catania (Progetti di Ricerca di Ateneo, Cat-
ania, Italy). The authors acknowledge Prof. R. Purrello and Dr. A.
D’Urso (Università degli Studi di Catania) for the Circular Dichro-
ism facility. Dr. Malik Chalal benefited of a PhD grant from the
Algerian government (Ministère de la Recherche et de l’Enseigne-
ment), which is sincerely acknowledged.
1
0.29 (20% EtOAc/n-hexane). H NMR (500 MHz, CDCl3, 298 K):
3
3
δ = 7.49 (d, JH,H = 8.5 Hz, 2 H, 11-H and 13-H), 7.44 (d, JH,H
3
= 8.0 Hz, 2 H, 11Ј-H and 13Ј-H), 7.38 (d, JH,H = 8.5 Hz, 1 H, 2Ј-
3
3
H), 7.31 (d, JH,H = 8.0 Hz, 2 H, 10Ј-H and 14Ј), 7.21 (d, JH,H
=
[1] M. J. Takaoka, Journal of the Faculty of Science Hokkaido Im-
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8.0 Hz, 2 H, 2-H and 6-H), 7.14 (br. s, 1 H, 6Ј-H), 7.08 (d, JH,H
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= 8.5 Hz, 2 H, 10-H and 14-H), 7.02 (d, JH,H = 16.3 Hz, 1 H, 7Ј-
3
3
H), 6.94 (d, JH,H = 8.0 Hz, 1 H, 3Ј-H), 6.836* (d, JH,H = 8.0 Hz,
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7

Job/Unit: O20664
/KAP1
Date: 25-07-12 15:51:20
Pages: 9
C. Tringali et al.
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Received: May 16, 2012
Published Online: ᭿
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Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20664
/KAP1
Date: 25-07-12 15:51:20
Pages: 9
Resveratrol-Related Dehydrodimers
Medicinal Chemistry
Seven resveratrol-related dehydrodimers
were obtained by biomimetic oxidative
coupling in the presence of laccase from
Trametes versicolor. Some of these products
were found to be active in an antiproliferat-
ive bioassay against SW480 human colon
cancer cells.
V. M. Bhusainahalli, C. Spatafora,
M. Chalal, D. Vervandier-Fasseur,
P. Meunier, N. Latruffe,
C. Tringali* ....................................... 1–9
Resveratrol-Related Dehydrodimers: Lac-
case-Mediated Biomimetic Synthesis and
Antiproliferative Activity
Keywords: Medicinal chemistry / Biomi-
metic synthesis / Enzyme catalysis / Anti-
proliferation / Antitumor agents /
Structure-activity relationships
Eur. J. Org. Chem. 0000, 0–0
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