A novel and efficient synthesis of highly oxidized lignans by a methyltrioxorhenium/hydrogen peroxide catalytic system. Studies on their apoptogenic and antioxidant activity

Article abstract of DOI:10.1016/j.bmc.2009.06.010

Highly oxidized lignans produced during the cytochrome P-450 metabolism in the cells show biological activities significantly different from those of their parent natural compounds. Lignans precursors of mammalian enterolignans were treated with a methylt

Full text of DOI:10.1016/j.bmc.2009.06.010

Bioorganic & Medicinal Chemistry 17 (2009) 5676–5682
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A novel and efficient synthesis of highly oxidized lignans
by a methyltrioxorhenium/hydrogen peroxide catalytic system.
Studies on their apoptogenic and antioxidant activity
a
b
a
a
Roberta Bernini a, , Giampiero Gualandi , Claudia Crestini , Maurizio Barontini , Maria Cristina Belfiore ,
*
Stefan Willför c, Patrik Eklund c, Raffaele Saladino a,
*
a Dipartimento di Agrobiologia e Agrochimica, Università degli Studi della Tuscia, Via S. Camillo De Lellis snc, 01100 Viterbo, Italy
b Dipartimento di Scienze e Tecnologie Chimiche, Università Tor Vergata, Via della Ricerca Scientifica, 00133 Roma, Italy
c Åbo Akademi Process Chemistry Centre, Åbo Akademi University, Porthansgatan 3, FI-20500 Åbo, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 January 2009
Revised 3 June 2009
Accepted 6 June 2009
Available online 13 June 2009
Highly oxidized lignans produced during the cytochrome P-450 metabolism in the cells show biological
activities significantly different from those of their parent natural compounds. Lignans precursors of
mammalian enterolignans were treated with a methyltrioxorhenium/hydrogen peroxide catalytic system
to afford new compounds oxidized at benzylic as well as in arylic positions. The evaluation of the anti-
oxidant and apoptogenic activity by in vivo protocols of these compounds showed some interesting
structure–activity relationships related to the oxidation degree of the molecules.
Keywords:
Lignans
Ó 2009 Elsevier Ltd. All rights reserved.
Oxidation
Methyltrioxorhenium
Antioxidant activity
Apoptogenic activity
1. Introduction
the activity, the presence of a keto group in this position being
more inhibitory than that of the hydroxyl moiety. It is worth to
Lignans are a large group of naturally occurring phenols widely
spread within the plant kingdom, that are derived from the shiki-
mic acid biosynthetic pathway. These compounds show dimeric
structures formed by a b,b0-linkage between two phenyl propane
units with a different degree of oxidation in the side-chain and
by a different substitution pattern in the aromatic moieties.1 Be-
cause of their biological activities, such as antioxidant,2 antimicro-
bial,3 antitumour,4 anti-inflammatory5 and antiviral properties,6
lignans have been used for a long time both in the ethnical and
conventional medicine.7 To date, most attention has been devoted
to the antioxidant activity, mainly because due to the radical scav-
enging properties of these compounds. Some activity–structure
relationship studies have been made to correlate the antioxidant
activity to the degree of oxidation of the molecule.2a As a general
reaction pattern, the presence of a catechol moiety increases the
antioxidant activity, while oxidation at the benzylic position (the
C-1 position in the side chain of the phenyl propane unit) decreases
note that these studies are usually performed under in vitro condi-
tions by monitoring the conversion of a sacrificial substrate in the
presence of active oxygen species and lignans. On the other hand,
lignans that are characterized by a high value of antioxidant activ-
ity under in vitro conditions can modify their pharmacological pro-
file in vivo, due to the metabolism in the cell. In this general
context, lignans are subjected to oxidative metabolic transforma-
tions by enzymes of the cytochrome P-450 family.8 During these
transformations, lignans are oxidized both in the benzylic position
by oxygen atom insertion on the activated sigma C–H bond, and on
the arene moieties. In the latter case, the reaction proceeds
through an initial epoxidation process, with or without de-O-
methylation of the guaiacyl group, and successive oxidation steps
to afford the corresponding hydroquinone and catechol or quinone
derivatives. It is well known from the literature that these metab-
olites can tune the redox potential of the cell and, in particular qui-
nones, can act as electrophilic species in alkylation of nucleophilic
sites in proteins and nucleic acids.9 Thus, the biological activity of
lignans should be compared to that of the corresponding highly
oxidized metabolites, to account for the potential role of metabo-
lism in their biological activity. Unfortunately, only few data about
the biological activity of oxidized lignans are available.10 This is
* Corresponding authors. Tel.: +39 0761 357452; fax: +39 0761 357242 (R.B.);
tel.: +39 0761 357314; fax: +39 0761 357242 (R.S.).
E-mail addresses: berninir@unitus.it (R. Bernini), saladino@unitus.it (R. Saladi-
no).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.010

R. Bernini et al. / Bioorg. Med. Chem. 17 (2009) 5676–5682
5677
mainly due to the small amount of lignan metabolites recoverable
from physiological fluids during in vivo treatments (less than ppm
amounts of lignans metabolites are detectable by GC–MS and LC–
MS analyses of organic fluid samples), associated with the diffi-
culty to design efficient chemical strategies for the selective trans-
formation of lignans stable, and some time recalcitrant, to
oxidation.
However, little is known about the mechanistic basis for its anti-
cancer activity. In fact, these compounds can have strong apopto-
genic activity on tumour cells by various mechanisms of action.
In this paper, novel lignans have been evaluated for their cytotox-
icity by an apoptosis assay and in vivo antioxidant activity to de-
fine qualitative structure–activity relationships with respect to
parent compounds.
In the last few years we have been involved in the design and
development of novel stoichiometric and catalytic procedures for
the oxidation of biologically active natural substances, including
products from the primary (nucleic acids components, amino acids
and peptides) and secondary (phenols and terpenes) metabolic
pathways.11 During these studies we observed that methyltrioxo-
rhenium (MeReO3, MTO), in combination with hydrogen peroxide
(H2O2) as the primary oxidant, was a selective and efficient organo-
metallic catalyst for the oxidation of several phenols,12 flavo-
noids,13 lignans, and lignin derivatives.14 These oxidations, which
proceed through two reactive intermediates, the monoperoxo
2. Chemical part
2.1. Catalytic oxidation with H2O2/MTO
For the oxidation of lignans 1–4 with MTO, the appropriate sub-
strate (1.0 mmol) was added to a solution of MTO (5.0% w/w of the
substrate) in CH2Cl2/CH3CN (1:1 ratio; 12.0 mL), and a small excess
of the primary oxidant H2O2 (1.5 equiv; 35% water solution) was
added to the reaction mixture in several batches at 0 °C. In the
absence of MTO, less than 2% of conversion of the substrate was
observed. Treatment of 1 with the MTO/ H2O2 system gave
3,30-dimethoxy-4,40,7,9-tetrahydroxy-7,90-epoxylignan 5, a prod-
uct of oxygen atom insertion at the C-7 benzylic ethereal position
(Scheme 1) as the only recovered product at an acceptable conver-
sion of substrate and high yield, besides the unreacted substrate
(Table 1, entry 1). The absence of oxidation products characteristic
of benzylic C-70 position suggests that the electronic effect of the
oxygen atom on the furan scaffold was a crucial factor for the reg-
ioselectivity of the reaction. Moreover, other possible products of
the oxidation at the C-9 position (primary alcohol) and of the aro-
matic moieties were not detected in the reaction mixture by gas
chromatography–mass spectrometry (GC–MS). These results are
in accordance with previously reported data on the oxidation of
the tetrahydrofuran lignan asarinin (not shown) with MTO, in
which case the benzylic ethereal position was selectively oxidized.
The configuration of the C-7 carbon in 5 was confirmed by NOE be-
tween H-8 and H-2(H-6). Unfortunately, compound 5 showed a
low stability in solution even when stored at low temperature
probably due to the ring-opening of the hemiacetalic moiety. The
oxidation of isolariciresinol 2 afforded isoshonanin 6 and the cate-
chol derivative 7 as the only recovered products in acceptable
yields besides the presence of unreacted substrate (Scheme 1, Ta-
ble 1, entry 2). No products of the benzylic oxidation of the C-7
and C-70 positions were observed. In this latter case, MTO showed
some multifunctional catalytic properties, acting contemporary as
an acidic catalyst during the ring-closure of the new furan ring by
dehydration of the C-9 and C-90 primary alcohols, and as oxidative
catalyst in the de-O-methylation of the guaiacyl moiety. The de-O-
methylation process was regioselective at the guaiacyl moiety on
the C-ring as suggested by the disappearance of the down-shielded
C(30)-OMe (dH 3.80 ppm) characteristic of the C-ring in both com-
pounds 2 and 6 in 1H NMR, and by the presence of the character-
[MeRe(O)2(O2)] and bis-peroxo [MeRe(O)(O2)2]
g
2-rhenium com-
plexes,15 showed a selectivity that strictly depends on the stereo-
electronic properties and substituents pattern of the substrates.
As a general pathway, the oxidation of arene derivatives proceeds
by a concerted oxygen atom transfer by a butterfly-like transition
state, with formation of epoxide intermediates that are further
rearranged and oxidized to corresponding quinones and hydroqui-
none or catechol derivatives. No radical pathways have been ob-
served suggesting that the homolytic activation of H2O2 is not an
operative process under these experimental conditions.
In the case of the oxidation of lignans of the aryltetralin family,
we described that the oxidation of podophyllotoxin, isolated from
different plants of the genus Podophyllum, with the MTO/H2O2 sys-
tem, afforded quinone and isopodophyllotoxone derivatives, which
have never been previously reported.14a The evaluation of the tox-
icity of these compounds showed an unprecedented shift of the
biological activity from the inhibition of cell division at the level
of the microtubule assembly by freezing the polymerization of
tubulin at the colchicine site, which is characteristic of the parent
podophyllotoxin, to the inhibition of topoisomerase II, a more
interesting goal from a therapeutic point of view. More exciting
was that the biological activity changed gradually so that some
oxidation products showed both properties, inhibition of tubulin
and activity against topoisomerase II. In particular, we observed
a decreased inhibitory activity of polymerization of tubulin in the
case of 20,30-ortho-benzoquinone derivatives versus a selective
activity against topoisomerase II, when the oxidation of the ben-
zylic C-7 position, affording isopodophyllotoxone, was accompa-
nied by the ring-opening of the D
-lactone moiety.14a
A similar structure activity relationships was also observed in
the evaluation of the apoptogenic activity of products of the
MTO/H2O2 oxidation of galbulin, an aryltetraline lignan isolated
from Galbulimima belgraveana, against both human lymphoma
cells lines BL41 (EBV-) prone to apoptosis and E2R (EBV+), which
is strongly resistant to chemical treatment.14a Moreover, an
unprecedented diastereoselective dearylation process, and produc-
tion of rare and enantiomerically pure acuminatolide, was ob-
served by treatment of the furfuran lignan asarinin with the
MTO/H2O2 system.14e
istic fragment m/z 266 which indicated the presence of
a
catechol moiety on the C-ring in GC–MS analysis.2a Probably, the
reaction proceeded by initial formation of 6 and successive de-O-
methylation to 7. This hypothesis was confirmed by the quantita-
tive oxidation of isolated 6 to 7 under identical experimental
conditions.
Next we studied the oxidation of the butyrolactone lignans,
matairesinol 3 and 7-hydroxymatairesinol 4. The oxidation of 3
afforded a mixture of three reaction products in similar yield, 3-
As a continuation of this study and with the aim to further eval-
uate the biological activity of highly oxidized lignans, we focused
here the attention on the oxidation of lariciresinol 1, isolariciresi-
nol 2, matairesinol 3 and 7-hydroxymatairesinol 4, which are all
precursors of mammalian enterolignans, with the MTO/H2O2 sys-
tem. Enterolignans, that are enterolactones and enterodiols, are
phytoestrogens that are formed by the human colonic microflora
from plant lignans. Enterolactones have been suggested to inhibit
the growth and development of breast and prostate cancer.16,17
methoxy-30,4,40-trihydroxylignano-9,90-butyrolactone
8,
9 and the c-hydroxyacid derivative 10
70-
hydroxymatairesinol
(Scheme 2, Table 1, entry 3). It is interesting to note that, in accor-
dance with data previously reported for the oxidation of 2, MTO
again catalyzed the regioselective de-O-methylation of only one
of the guaiacyl moieties in 3, to give 8 that was identical to an

5678
R. Bernini et al. / Bioorg. Med. Chem. 17 (2009) 5676–5682
OH
OH
MeO
MeO
3'
H
1'
7'
H
9'
MTO/H2O2
O
8'
O
1
CH2Cl2/MeCN
OH
8
7
HO
H
HO
2
3
H
9
6
OMe
OMe
4
OH
5
OH
1
5
7
9
1
MeO
HO
MeO
HO
MeO
OH
OH
MTO/H2O2
O
O
7'
2
2'
HO
CH2Cl2/MeCN
9'
1'
MeO
HO
MeO
OH
OH
OH
6
7
2
Scheme 1. Oxidation of lariciresinol 1 and isolariciresinol 2 with the hydrogen peroxide/methyltrioxorhenium catalytic system.
Table 1
benzylic C-7 position to afford 12, followed by successive oxygen
atom insertion into the C-7 benzylic position (as in the case of
11) or a dehydration process to give isomers 13 and 14. This reac-
tion pattern is similar to that previously reported for the oxidation
of 4 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in
dioxane.19 In this latter case, the initial formation of benzylic cat-
ions by abstraction of hydride ion was a crucial step. Instead, on
the basis of the known property of activated MTO complexes, the
transfer of the oxygen atom to the substrate in our case required
a concerted mechanism.15
Experimental data of oxidation as depicted in Schemes 1 and 2a
Entry
Substrate
Conv. (%)
Yieldb (%)
1
2
1
2
60
70
5: 98
6: 30
7: 57
8: 30
3
4
3
4
55
82
9: 30
10: 40
11: 35
12: 37
13: 16
14: 8
3. Biological part
a
In a typical experiment, hydrogen peroxide (1.5 equiv, 35% water solution) and
the substrate (1.0 mmol) to be oxidized were added to a solution of MTO (5% w/w)
in CH2Cl2/CH3CN at T = 0 °C.
3.1. Apoptosis evaluation
b
Calculated on converted substrate.
The cytotoxicity of selected lignans 6–7, 8–9 and 11–13 and
their parent compounds 2, 3, and 4 have been evaluated by apop-
authentic sample (see for example [
a
a
]
of 8 ꢀ21.3 c 0.114 MeOH
ꢀ22.8 c 0.114 MeOH).18
tosis induction (Figs. 1 and 2). Lignan 1 and a-conidendrin 15 (not
D
versus [
a
]
D
ꢀ20° c 0.114 MeOH or [
]
shown) were also evaluated as references. Lignan 5 has not been
included in our study since it was not stable enough in solution
to perform biological tests. Apoptosis induction has been tested
on prone human B cell lymphoma line (BL41) and on the resistant
isogenic derivative (E2R).20 The apoptosis assay was done in paral-
lel with both cell lines. In addition, E2R cell line (apoptosis resis-
tant) was employed in an acute in vivo test to evaluate the
ability of the lignans to modulate the oxidative status of the cells.
The parent lignans lariciresinol 1, isolariciresinol 2, hydroxyma-
D
These data suggest that the reactivity of the guaiacyl moiety to-
wards electrophilic oxidants like activated MTO complexes strictly
depends on its position on the lignan scaffold probably due to ste-
reoelectronic effects exerted by the other substituents on the arene
moieties. A significative regioselectivity was also observed in the
formation of 9 in which case the oxygen atom insertion at the C-
70 benzylic position was the only oxidative process operative in
the side-chain of the molecule. In this latter case, stereoelectronic
effects occurred even if a possible orientating effect of the carbonyl
group on the approach of the catalytic species of the substrate can
not be completely ruled out. Finally, compound 10 was simply ob-
tained by hydrolytic ring-opening of the lactone moiety in 3.
During the oxidation of 4, a more complex mixture of reaction
products was observed, including 70-hydroxy-7-oxomatairesinol
11, 7-oxo-matairesinol 12 and isomeric 70,80-dehydro-7-hydrox-
ymatairesinol (Z) 13 and 70,80-dehydro-7-hydroxymatairesinol (E)
14, compounds 11 and 12 being synthesized in highest yield (Table
1, entry 4). The structures of all products were confirmed by com-
parison with authentic samples.19 The products formed in the reac-
tion above support the presence of an initial oxidation of the
tairesinol 4, and a-conidendrin were scarcely apoptogenic in both
cell lines BL41 and E2R, with the exception of matairesinol 3 which
showed an appreciable activity. In the case of isolariciresinol-type
lignans 6 and 7, compound 6 resulting from the ring-closure of the
dihydroxy moiety present in parent lignan, and resembling to a
deoxo-a-conidendrin derivative, showed the highest apoptogenic
activity for both cell lines. Thus, the process of ring-closure of
the dihydroxy moiety increases the cytotoxicity. On the contrary,
the de-O-methylation of the guaiacyl moiety in parent compound,
decreases the cytotoxicity as shown by the comparison between
derivatives 6 and 7, the apoptogenic activity of 7 being comparable
to that of the low-toxic lignan 2 (Fig. 1, panels A and B).

R. Bernini et al. / Bioorg. Med. Chem. 17 (2009) 5676–5682
5679
O
MeO
O
HO
OH
O
H
H
O
MeO
HO
H
OH
7'
9'
3'
1'
MeO
HO
O
8'
O
9
OH
8
MTO/H2O2
7
1
8
H
4
CH2Cl2/MeCN
2
6
OMe
O
3
H
OMe
MeO
HO
5
OH
OH
OH
OH
9
H
3
OMe
OH
10
OH
O
O
MeO
HO
MeO
HO
O
O
O
O
OMe
OMe
O
7'
7
OH
12
OH
1'
3'
8'
MeO
HO
9'
O
11
8
MTO/H2O2
HO
6
9
2
3
1
HO
CH2Cl2/MeCN
OMe
OMe
O
5
4
MeO
HO
O
O
OH
O
4
O
O
OMe
OMe
OH
14
OH
13
Scheme 2. Catalytic oxidation of matairesinol 3 and 7-hydroxymatairesinol 4.
In the family of butyrolactone lignans, the oxidation of the C-7
position (compound 4) or C-70 position (compound 9) decreases
the apoptogenic activity against both cell lines with respect to 3,
in which case there are not hydroxyl moieties in the side-chain
(Fig. 2, panels A and B). On the other hand, when both benzylic
C-7 and C-70 positions are contemporary oxidized, one of which
to carbonyl moiety (C-7 position), as in the case of 11, the most
intensive apoptogenic activity was obtained for the resistant E2R
cell line. A similar behaviour was observed in the case of side-chain
dehydrated 13. In accordance with data previously shown for com-
pound 7, the presence of the cathecol moiety in 8 slightly decreases
the apoptogenic activity with respect to parent matairesinol 3.
selective probe versus oxygen and nitrogen peroxides,20 in flow
cytometric analysis. Cells were exposed to the same doses of lignan
samples employed for the apoptosis evaluation, but for a short
exposition time to avoid interference of possible degenerative pro-
cesses (apoptosis, necrosis, etc.) on the measure of oxidative status
of the cells. The flow cytometric analysis was both qualitative,
allowing to evaluate the oxidation at single cell level and providing
information on the overall status of cell population in the culture,
and quantitative in that the results can be treated by integrating
the values of population/subpopulations from the pool of single
measurements. As shown in Figure 3 none of the parent lignans
showed a pro-oxidant activity. Matairesinol 3 and
15 were the only compounds among the parent lignans that dis-
played significant antioxidant activity. Since compound
showed a significant high toxicity compared to -conidendrin
a-conidendrin
3.2. Measuring the oxidative intracellular status
a
3
a
Next, we examined the antioxidant or pro-oxidant effect of the
lignans on the intracellular redox level. This in vivo assay was per-
formed by using the resistant E2R cell line in the presence of the
probe dichlorodehydrofluoresceine diacetate (DCFH-DA) that is a
(see above), the toxicity of these compounds can not be directly
correlated to their in vivo antioxidant activity. Lignans 6 and 7
did not show a significant antioxidant activity (Fig. 3, panel A). In
the family of butyrolactone lignans, 3 and 8 were more active than

5680
R. Bernini et al. / Bioorg. Med. Chem. 17 (2009) 5676–5682
Panel A
Panel A
100
80
60
40
20
0
1.4
1.2
1
0.8
0.6
0.4
0.2
0
1
2
6
7
15
1
2
6
7
15
-20
0
25
50
75
100
0
25
50
75
100
Dose ( M)
µ
Dose ( M)
µ
Panel B
Panel B
100
80
60
40
20
0
1.4
1.2
1
0.8
0.6
0.4
0.2
0
1
2
6
7
3
4
8
9
11
13
15
-20
0
25
50
Dose ( M)
75
100
0
25
50
Dose ( M)
75
100
µ
µ
Figure 1. Apoptosis evaluation of compounds 1, 2, 6, 7 and 15. Panel A: prone
human B cell lymphoma line (BL41). Panel B: resistant isogenic derivative (E2R).
Figure 3. Antioxidant or pro-oxidant effect of the lignans on the intracellular redox
level.
Panel A
presence of a cathecol moiety or the mono-oxidation of the ben-
zylic C-7 and C-70positions decreases the cytotoxicity, while the
ring-closure process to a novel tetrahydro furane ring, as well as,
the contemporary oxidation of both benzylic positions increases
the toxicity. Finally, the presence of the lactone moiety do not
seems to be relevant per se for the apoptogenic activity. About
the antioxidant activity, all lignans that are characterized by a rel-
evant antioxidant activity contain the lactone moiety. In this latter
case, the oxidation of the benzylic positions always decreases the
antioxidant activity.
100
80
60
40
20
0
3
4
8
9
11
13
-20
0
25
50
75
100
Dose ( M)
µ
5. Experimental
Panel B
100
80
60
40
20
0
All solvents and reagents used were of analytical grade and
were purchased from Aldrich Chemical Co. Silica Gel 60 F254 plates
and Silica Gel 60 were furnished from Merck. Lignans 1–4 were
purchased from Oy ArboNova Ab. The nomenclature of lignans
and neolignans has been attributed according to IUPAC-IUB Joint
Commission on Biochemical Nomenclature (JCBN), Recommenda-
tions 2000.
3
4
8
9
11
13
-20
0
25
50
Dose ( M)
75
100
5.1. Oxidation with MTO/H2O2. General procedure
µ
In a typical experiment, hydrogen peroxide (1.5 equiv, 35%
water solution) and the substrate (1.0 mmol) to be oxidized were
added to a solution of the methyltrioxorhenium (5% w/w) in
CH2Cl2/CH3CN, 1:1 (12 mL) and the mixture was stirred at
T = 0 °C. Oxidations were monitored by TLC using the mixture
CH2Cl2/CH3OH (9.2/0.8 v/v) as eluent. At the end of the reaction,
MnO2 was added to the solution to degrade the excess of hydrogen
peroxide and the reaction mixture was vigorously stirred for
15 min. After filtration, the solvent mixture was evaporated under
vacuum. The reaction products were extracted with ethyl acetate
Figure 2. Apoptosis evaluation of compounds 3, 4, 8, 9, 11 and 13. Panel A: prone
human B cell lymphoma line (BL41). Panel B: resistant isogenic derivative (E2R).
4, 9, 11 and 13 (Fig. 3, panel B). It is worth to note that the catechol
moiety has a significant role in the antioxidant activity while the
oxidation of the benzylic C-7 and C-70 positions did not influence
or even decreases the antioxidant activity (compare activity of
compounds 8 and 9).
4. Conclusions
(3 ꢁ 20 mL) and washed with
a
NaCl saturated solution
(2 ꢁ 20 mL). The organic layers were dried over anhydrous sodium
sulfate. The crude product was purified by preparative TLC using
dichloromethane/methanol (9.5/0.5 v/v) mixtures as eluents. All
On the basis of the data previously reported on the apoptogenic
activity of lignan derivatives it is reasonable to suggest that the

R. Bernini et al. / Bioorg. Med. Chem. 17 (2009) 5676–5682
5681
the products were identified by 1H NMR, 13C NMR and GC–MS
spectra. 1H NMR and 13C NMR were recorded on a Bruker
200 MHz spectrometer using CDCl3 (99.8% in deuterium), CD3OD
(99.8% in deuterium) or CD3COCD3 (99.8% in deuterium) as sol-
vents. All chemical shift are expressed in parts per million (d scale)
and coupling constants in Hertz (Hz).
40), 147.0 (C-30), 147.3 (C-4), 148.9 (C-3), 177.7 (C-9). MS (EI): m/z
392 (M+).
5.1.7. 70-Hydroxy-7-oxomatairesinol (11)
Oil.19 dH (200 MHz, CDCl3, (CD3)2CO): 3.38 (1H, m, H-80), 3.47
(3H, s, OMe), 3.73 (3H, s, OMe), 3.90–4.10 (3H, m, H-9 and H-8),
5.11–5.15 (1H, m, H-70), 6.57–6.77 (6H, m, Ar-H); dC (200 MHz,
CDCl3, (CD3)2CO): 45.6 (C-8), 50.0 (C-80), 56.0 (2 ꢁ OMe), 67.2 (C-
9), 73.6 (C-70), 109.2 (C-2), 110.9 (C-20), 114.3 (C-5), 117.0 (C-50),
120.2 (C-6), 124.6 (C-60), 129.7 (C-1), 133.2 (C-1), 147.0 (C-3),
149.7 (C-30), 151.6 (C-4), 176.3 (C-90),194.7 (C-7). MS (EI): m/z
388 (M+).
5.1.1. 3,30-Dimethoxy-4,40,7,9-tetrahydroxy-7,90-epoxylignan (5)
Oil. dH (200 MHz, CDCl3, (CD3)2CO): 2.65 (1H, m, H-80), 2.7 (1H,
m, H-8), 3.0 (2H, m, H-6), 4.04–4.14 (4H, m, H-9 and H-90), 4.22
(3H, s, OMe), 4.26 (3H, s, OMe), 7.02–7.20 (6H, m, Ar-H); dC
(200 MHz, CDCl3, (CD3)2CO): 36.3 (C-70), 40.2 (C-80), 55.4
(2 ꢁ OMe), 59.8 (C-9), 61.5 (C-8), 72.4 (C-90), 109.5 (C-7), 112.1
(C-20and C-2), 114.4 (C-50), 114.5 (C-5), 119.7 (C-60), 121.8 (C-6),
132.5 (C-60), 132.5 (C-10), 136.1 (C-1), 144.5 (C-40), 145.0 (C-4),
147.1 (C-30),147.2 (C-3). MS (EI): m/z 376 (M+).
5.1.8. 7-Oxo-matairesinol (12)
Oil.19 dH (200 MHz, CDCl3, (CD3)2CO) 2.90–2.96 (2H, m, H-70),
3.44 (1H, m, H-80), 3.67 (3H, s, OMe), 3.86 (3H, s, O-Me), 4.0–4.30
(3H, m, H-8 and H-9), 6.48 (1H, m, H-60), 6.54 (1H, m, H-20), 6.55
(1H, m, H-50), 6.81 (1H, m, H-5), 7.13 (1H, m, H-6), 7.27 (1H, m,
H-2); dC (200 MHz, CDCl3, (CD3)2CO): 34.5 (C-70), 44.9 (C-80), 46.5
(C-8), 55.8 (OMe), 56.2 (OMe), 69.4 (C-9), 110.1 (C-2), 111.8 (C-
20), 113.9 (C-5), 114.4 (C-50), 122.37 (C-60), 123.6 (C-6), 128.7 (C-
1), 128.9 (C-10), 144.7 (C-40), 146.6 (C-30), 147.1 (C-3), 151.4 (C-
4),177.3 (C-90), 195.0 (C-7). MS (EI): m/z 372 (M+).
5.1.2. Isoshonanin (6)
Oil.21 dH (200 MHz, CDCl3): 2.24 (2H, m, H-8 and H-80), 2.70 (1H,
m, H-7), 2.96 (1H, m, H-7), 3.50–3.82 (4H, m, H-9 and H-70 and H-
90), 3.80 (3H, s, OMe), 3.85 (3H, s, OMe), 4.2 (1H, m, H-9), 6.37 (1H,
s, H-3), 6.54–6.82 (4H, m, H-20, H-6, H-60 and H-50); dC (200 MHz,
CDCl3): 32.5 (C-7), 42.4 (C-80), 49.6 (C-70), 50.6 (C-8), 55.8 (OMe),
55.9 (OMe), 72.4 (C-90), 73.2 (C-9), 110.4 (C-6), 111.0 (C-20),
114.3 (C-50), 115.2 (C-3), 121.5 (C-60), 127.6 (C-1), 133.0 (C-2),
136.5 (C-10), 143.8 (C-40), 144.4 (C-4), 145.0 (C-30), 146.7 (C-5).
MS (EI): m/z 342 (M+).
5.1.9. 70,80-Dehydro-7-hydroxymatairesinol (Z) (13)
Oil.19 dH (200 MHz, CDCl3, (CD3)2CO): 3.36 (1H, m, H-8), 3.81
(3H, s, OMe), 3.85 (3H, s, OMe), 4.29 (1H, m, H-9), 4.52 (1H, m,
H-9), 4.80 (1H, m, H-7), 6.50 (1H, m, H-70), 6.78-7.10 (5H, m, H-2,
H-5, H-50, H-6, H-60), 8.11 (1H, d, J = 2.0 Hz, H-20); dC (200 MHz,
CDCl3, (CD3)2CO): 50.9 (C-8), 56.0 (OMe), 56.4 (OMe), 67.3 (C-9),
75.9 (C-7), 111.0 (C-2), 115.0 (C-20), 115.1 (C-50), 115.3 (C-5),
120.0 (C-6), 123.9 (C-80), 126.9 (C-60), 127.2 (C-10), 135.5 (C-1),
142.3 (C-70), 146.6 (C-4), 147.5 (C-3), 149.0 (C-40), 170.8 (C-90).
MS (EI): m/z 372 (M+).
5.1.3. 30,40-Dihydroxy-5-methoxy-2,70-cyclolignan-9,90-furan (7)
Oil. dH (200 MHz, CDCl3): 1.94–2.22 (2H, m, H-80 and H-8), 2.51–
2.92 (2H, m, H-7), 3.34–3.82 (3H, m, H-70 and H-9), 3.84 (3H, s,
OMe), 4.27–4.44 (2H, m, H-90), 6.48 (1H, s, H-6), 6.59 (1H, s, H-
60),6.67–7.12 (3H, m, H-3, H-20and H-50); dC (200 MHz, CDCl3):
37.7 (C-7), 38.3 (C-80), 49.6 (C-8), 53.6 (C-70), 55.8 (OMe), 72.2
(C-9), 78.7 (C-90), 112.9 (C-6), 114.2 (C-20), 114.3 (C-50), 117.9 (C-
3), 121.2 (C-60), 128.8 (C-1), 133.6 (C-10), 138.4 (C-2), 143.4 (C-
40), 144.2 (C-30), 144.9 (C-4), 145.5 (C-5). MS (EI): m/z 328 (M+).
5.1.10. 70,80-Dehydro-7-hydroxymatairesinol (E) (14)
Oil.19 dH (200 MHz, CDCl3, (CD3)2CO) 3.86 (3H, s, OMe), 3.92 (3H,
s, OMe), 4.10 (1H, m, H-8), 4.14 (1H, m, H-9), 4.51 (1H, m, H-9),
5.11 (1H, m, H-7), 6.80(1H, m, H-5), 6.80–7.29(5H, m, H-2, H-20,
H-50, H-6, H-60), 7.45 (1H, d, J = 2.1 Hz, H-70); dC (200 MHz, CDCl3,
(CD3)2CO): 45.7 (C-8), 56.1 (OMe), 56.4 (OMe), 66.2 (C-9), 71.6
(C-7), 110.0 (C-2), 114.2 (C-20), 114.9 (C-50), 116.0 (C-5), 118.9
(C-6), 121.7 (C-80), 124.8 (C-60), 127.1 (C-10), 134.6 (C-1), 137.3
(C-70), 146.3 (C-4), 148.2 (C-3),149.1 (C-40), 173.0 (C-90). MS (EI):
m/z 372 (M+).
5.1.4. 3-Methoxy-30,4,40-hydroxylignan-9,90-butyrolactone (8)
Amorphorus powder.18b dH (200 MHz, CDCl3, (CD3)2CO): 2.45–
2.64 (4H, m, H-7, H-8 and H-80), 2.85 (2H, m, H-70), 3.83 (3H, s,
OMe), 3.88 (1H, m, H-9), 4.12 (1H, m, H-9), 6.45–6.80 (6H, m, Ar-
H); dC (200 MHz, CDCl3, (CD3)2CO): 35.2 (C-70), 38.7 (C-7), 42.4
(C-8), 47.9 (C-80), 56.3 (OMe), 72.8 (C-9), 113.8 (C-20), 116.0 (C-
50), 116.3 (C-5), 116.7 (C-2), 120.9 (C-6), 130.5 (C-10), 131.3 (C-1),
122.9 (C-60), 144.8 (C-4), 146.2 (2ꢁ, C-3 and C-40), 148.1 (C-30),
181.4 (C-90). MS (EI): m/z 344 (M+).
5.2. Cell culture
5.1.5. 70-Hydroxymatairesinol (9)
BL 41 is an EBV negative cell line derived from a Burkitt Lym-
phoma while E2R is the isogenic counterpart, converted to EBV po-
sitive, and apoptosis resistant.20 Both cell lines were grown in RPMI
medium with 4 mM L-glutamine and 10% FCS. All incubations were
at 37 °C in a 5% CO2 atmosphere.
Oil. dH (200 MHz, CDCl3): 2.15–2.55 (3H, m, H-80 and H-70), 2.93
(1H, m, H-8), 3.62 (3H, s, OMe), 3.79 (3H, s, OMe), 3.92 (2H, m, H-
90), 5.35 (1H, s, H-7), 6.41–6.82 (6H, m, H-2, H-20, H-6, H-60, H-5
and H-50); dC (200 MHz, CDCl3): 35.6 (C-70), 42.8 (C-80), 55.5 (C-
8), 55.8 (OMe), 55.9 (OMe), 72.0 (C-90), 74.5 (C-7), 110.9 (C-2),
111.2 (C-20), 114.3 (C-50), 115.9 (C-5), 120.2 (C-6), 120.8 (C-60),
132.6 (C-10), 133.2 (C-1), 145.2 (C-40), 146.3 (C-30), 147.0 (C-4),
148.7 (C-3), 178.1 (C-9). MS (EI): m/z 374 (M+).
5.3. Apoptosis evaluation
Both BL41 and E2R cell lines were seeded at concentration of
3 ꢁ 105 cells/mL and the compounds were added for 18 h of incu-
bation. Compounds were dissolved in DMSO which never exceeded
0.5% v/v. For morphological analysis of apoptogenic activity cell
were fixed in 4% (v/v) p-formaldehyde and stained with a solution
5.1.6.
c-Hydroxyacid derivative (10)
Oil. dH (200 MHz, CDCl3): 2.08–2.61 (2H, m, H-80 and H-8), 3.46
(2H, m, H-90), 3.76 (3H, s, OMe), 3.79 (3H, s, OMe), 5.13 (1H, s, H-7),
6.31–6.57 (6H, m, H-2, H-20, H-6, H-60, H-5 and H-50); dC (200 MHz,
CDCl3): 36.6 (C-70), 39.1 (C-80), 56.5 (C-8), 55.7 (OMe), 55.8 (OMe),
65.3 (C-90), 76.4 (C-7), 110.7 (C-2), 112.5 (C-20), 115.7 (C-50), 116.1
(C-5), 119.9 (C-6), 122.8 (C-60), 131.8 (C-1), 132.2 (C-10), 145.6 (C-
(0.2 lg/mL) of DAPI. Apoptosis was quantified by scoring cells con-
densed and fragmented nuclei according to the reported proce-
dure.22 At least 500 cells, in random fields, were scored using an
epifluorescent microscopy.

5682
R. Bernini et al. / Bioorg. Med. Chem. 17 (2009) 5676–5682
10. For the effect of a tertiary hydroxyl group on the main lignin structure see: (a)
5.4. Flow cytometric analysis of ROS
Yamauchi, S.; Ina, T.; Kirikishira, T.; Masuda, T. Biosci. Biotechnol. Biochem. 2004,
68, 183; (b) Yamauchi, S.; Hayashy, Y.; Kirikihira, T.; Masuda, T. Biotechnol.
Biochem. 2005, 69, 581; For the effect of benzylic oxygen on antioxidant
activity see: Yamauchi, S.; Hayashi, H.; Nakashima, Y.; Kirikihira, Y.; Yamada,
K.; Masuda, T. J. Nat. Prod. 2005, 68, 1459; For the effect of benzylic oxygen on
antimicrobial activity see: Akiyama, K.; Maruyama, M.; Yamauchi, S.;
Nakashima, Y.; Nakato, T.; Tago, R.; Sugahara, T.; Kishida, T.; Koba, Y. Biosci.
Biotechnol. Biochem. 2006, 71, 1745.
Intracellular peroxide levels were assessed using an oxidation-
sensitive fluorescent probe DCFH-DA (Sigma–Aldrich). In presence
of a variety of intracellular peroxides, DCFH-DA is oxidized to 20,70-
dichlorofluorescein. Cells after treatments of 1 h with the tested
compounds (dissolved in DMSO 0.2%.) in RPMI medium were incu-
bated in the same medium with 10 l
M DCFH-DA for 200 and at
11. (a) Saladino, R.; Crestini, C.; Bernini, R.; Mincione, E.; Ciafrino, R. Tetrahedron
Lett. 1995, 36, 2665; (b) Saladino, R.; Bernini, R.; Crestini, C.; Mincione, E.;
Bergamini, A.; Marini, S.; Palamara, A. T. Tetrahedron 1995, 51, 7561; (c)
Saladino, R.; Bernini, R.; Mincione, E.; Tagliatesta, P.; Boschi, T. Tetrahedron Lett.
1996, 37, 2647; Bernini, R.; Mincione, E.; Sanetti, A.; Bovicelli, P.; Lupattelli, P.
Tetrahedron Lett. 1997, 38, 4651; (d) Mincione, E.; Sanetti, A.; Bernini, R.; Felici,
M.; Bovicelli, P. Tetrahedron Lett. 1998, 39, 8699; (e) Tagliatesta, P.; Bernini, R.;
Crestini, C.; Monti, D.; Boschi, T.; Mincione, E.; Saladino, R. J. Org. Chem. 1999,
64, 5361.
12. (a) Saladino, R.; Neri, V.; Mincione, E.; Marini, S.; Coletta, S.; Fiorucci, C.;
Filippone, P. J. chem. Soc. Perkin Trans 1 2000, 4, 581; (b) Saladino, R.; Neri, V.;
Mincione, E.; Filippone, P. Tetrahedron 2002, 58, 8493; (c) Saladino, R.;
Mincione, E.; Attanasi, O. A.; Filippone, P. Pure Appl. Chem. 2003, 75, 265; (d)
Bernini, R.; Mincione, E.; Barontini, M.; Crisante, F.; Gambacorta, A. Tetrahedron
2007, 63, 6895.
13. (a) Bernini, R.; Mincione, E.; Cortese, M.; Aliotta, G.; Oliva, A.; Saladino, R.
Tetrahedron Lett. 2001, 42, 5401; (b) Bernini, R.; Mincione, E.; Cortese, M.;
Saladino, R.; Gualandi, G.; Belfiore, M. C. Tetrahedron Lett. 2003, 44, 4823; (c)
Bernini, R.; Coratti, A.; Fabrizi, G.; Goggiamani, A. Tetrahedron Lett. 2003, 44,
8991; (d) Bernini, R.; Mincione, E.; Provenzano, P.; Fabrizi, G. Tetrahedron Lett.
2005, 46, 2993; (e) Bernini, R.; Mincione, E.; Barontini, M.; Fabrizi, G.;
Pasqualetti, M.; Tempesta, S. Tetrahedron 2006, 62, 7733; (f) Bernini, R.;
Mincione, E.; Provenzano, G.; Fabrizi, G.; Tempesta, S.; Pasqualetti, M.
Tetrahedron 2008, 64, 7561.
14. (a) Crestini, C.; Caponi, M. C.; Argyropoulous, D. S.; Saladino, R. Bioorg. Med.
Chem. 2006, 14, 5292; (b) Crestini, C.; Pro, P.; Neri, V.; Saladino, R. Bioorg.
Med. Chem. 2005, 13, 2569; (c) Saladino, R.; Fiani, C.; Crestini, C.;
Argyropoulous, D. S.; Marini, S.; Coletta, M. J. Nat. Prod. 2007, 70, 39; (d)
Saladino, R.; Fiani, C.; Belfiore, M. C.; Gualandi, G.; Penna, S.; Mosesso, P.
Bioorg. Med. Chem. 2005, 13, 5949; (e) Saladino, R.; Fiani, C.; Crestini, C.;
Argyropoulos, D. S.; Marini, S.; Coletta, M. J. Nat. Prod. 2007, 70, 39; (f)
Saladino, R.; Gualandi, G.; Farina, A.; Crestini, C.; Nencioni, L.; Palamara, A. T.
Curr. Med. Chem. 2008, 15, 1500.
37 °C in CO2 incubator. The analysis was performed using FACScal-
ibur cytometer (Becton & Dickinson) with excitation at k = 488 nm,
emission at k = 533 nm recorded in FL-1 and 10,000 events were
recorded for each sample.
Acknowledgments
The authors like to thank Interuniversity Consortium ‘Chemis-
try for the Environment’ (INCA); PNR-FIRB; ASI and EU COST
CM07035 System, as well as the Academy of Finland for financial
support.
References and notes
1. Ayres, D. C.; Loike, J. D. In Lignans: Chemical, Biological and Clinical Properties;
Cambridge University Press, 1990.
2. (a) Eklund, P. C.; Långvik, O. K.; Wärnå, J. P.; Salmi, T. O.; Willför, S. M.; Sjöholm,
R. E. Org. Biomol. Chem. 2005, 3, 3336; (b) Yamauchi, S.; Hayashi, Y.; Nakashima,
Y.; Kirikihira, T.; Yamada, K.; Masuda, T. J. Nat. Prod. 2005, 68, 1459.
3. (a) Akiyama, K.; Maruyama, M.; Yamauchi, S. I.; Nakashima, Y.; Nakato, T.;
Tago, R.; Sugahara, T.; Kishida, T.; Koba, Y. Biosci. Biotech. Biochem. 2007, 71,
1745; (b) Rahman, M. M.; Gray, A. I.; Khondkar, P.; Islam, M. A. Nat. Prod.
Commun. 2008, 3, 45.
4. (a) Awad, K. S. Cancer cells 2007, 156; (b) Mulabagal, V.; Subbaraju, G. V.;
Ramani, M. V.; DeWitt, D. L.; Nair, M. G. Nat. Prod. Commun. 2008, 3, 1793.
5. Jeng, K. C. G.; Hou, R. C. W. Curr. Enzyme Inhib. 2005, 1, 11.
6. (a) Charlton, J. L. J. Nat. Prod. 1998, 61, 1447; (b) Li, S.-Y.; Wu, M.-D.; Wang, C.-
W.; Kuo, Y.-H.; Huang, R.-L.; Lee, K.-H. Chem. Pharm. Bull. 2000, 48, 1992.
7. See as a general review: Osawa, T. In Phenolic Compounds in Food and their Effect
on Health; Huang, M. T., Ho, C. T., Lee, C. Y., Eds.; American Chemical Society:
Washington, D.C., 1992; p 135.
15. Herrmann, W. A.; Fischer, R. W.; Scherer, W.; Rouch, M. V. Angew. Chem., Int. Ed.
Engl. 1993, 32, 1157.
16. Li-Hua, C.; Jing, F.; Huaixing, L.; Wendy, D.-W.; Xu, L. Mol. Cancer Ther. 2007, 6,
2581.
8. For some examples of the effect of cytochrome P450-dependent metabolic
process on lignans see: (a) Sinha, B. K.; Eliot, H. M.; Kalayanaraman, B. FEBS Lett.
1988, 227, 240; (b) Niemeyer, H. B.; Metzler, M. Anal. Technol. Life Sci. 2002, 777,
321; (c) Jancova, P.; Anzenbacherova, E.; Papouskova, B.; Lemr, K.; Luzna, P.;
Veinlichova, A.; Anzenbacher, P.; Vilim, S. Drug Met. Disp. 2007, 35, 2035.
9. (a) Cavalieri, E. L.; Li, K. M.; Balu, N.; Saeed, M.; Decanesan, P.; Higginbotham,
S.; Zhao, J.; Gross, M. L.; Rogan, F. G. Carcinogenesis 2002, 23, 1071; (b) Wolf, K.
K.; Wood, S. G.; Allard, J. L.; Hunt, J. A.; Gorman, N.; Walton-Strong, B. W.;
Szakacs, J. G.; Duan, S. X.; Hao, Q.; Court, M. H.; Von Moltke, L. L.; Greenblatt, D.
J.; Kostrubsky, V.; Jeffery, E. H.; Wrighton, S. A.; Gonzalez, F. J.; Sinclair, P. R.;
Sinclair, J. F. Drug Metab. Dispos. 2007, 35, 1223.
17. Miura, D.; Saarinen, N. M.; Miura, Y.; Santti, R.; Yagasaki, K. Nutr. Cancer 2007,
58, 49.
18. (a) Xie, L.-H.; Akao, T.; Hamasaki, K.; Deyama, T.; Hattori, M. Chem. Pharm. Bull.
2003, 51, 508; (b) Jing, J.-S.; Zhao, Y.-F.; Nakamura, N.; Akao, T.; Kakiuchi, N.;
Hattori, M. Biol. Pharm. Bull. 2007, 30, 904.
19. Eklund, P. C.; Sjöholm, R. E. Tetrahedron 2003, 59, 4515.
20. Belfiore, M. C.; Natoni, A.; Barzellotti, R.; Merendino, N.; Pessina, G.; Ghibelli, L.;
Gualandi, G. Cancer Lett. 2007, 25, 236.
21. Nose, M.; Bernards, M. A.; Furlan, M.; Zajicek, J.; Eberhardt, J.; Lewis, N. G.
Phytochemistry 1995, 39, 71.
22. Ghibelli, L.; Maresca, V.; Coppola, S.; Gualandi, G. FEBS Lett. 1995, 377, 9.