A selective de-O-methylation of guaiacyl lignans to corresponding catechol derivatives by 2-iodoxybenzoic acid (IBX). the role of the catechol moiety on the toxicity of lignans

Article abstract of DOI:10.1039/b822661j

We report here the first selective de-O-methylation of a large panel of guaiacyl lignans to the corresponding catechol derivatives by using IBX as primary oxidant under green conditions (dimethyl carbonate-H₂O solvent) through an in situ reduction procedure. The influence of the catechol moiety on the cytotoxicity and genotoxicity of new lignan derivatives has been investigated. The results obtained indicated that the presence of the catechol moiety sharply enhances the clastogenic potential (e.g. induction of chromosomal aberrations), the cytotoxicity and the modulation of cell cycle progression with respect to the parent compounds. Thus, despite the in vitro antioxidant activity usually described for catechol derivatives, our results show for the first time the generation of a clastogenic potential, highly indicative of a long-term genetic and cancer risk. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b822661j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A selective de-O-methylation of guaiacyl lignans to corresponding catechol
derivatives by 2-iodoxybenzoic acid (IBX). The role of the catechol moiety
on the toxicity of lignans
Roberta Bernini,*a Maurizio Barontini,a Pasquale Mosesso,a Gaetano Pepe,a Stefan M. Willfo¨r,b
Rainer E. Sjo¨holm,b Patrik C. Eklundb and Raffaele Saladino*a
Received 17th December 2008, Accepted 5th March 2009
First published as an Advance Article on the web 3rd April 2009
DOI: 10.1039/b822661j
We report here the first selective de-O-methylation of a large panel of guaiacyl lignans to the
corresponding catechol derivatives by using IBX as primary oxidant under green conditions (dimethyl
carbonate–H2O solvent) through an in situ reduction procedure. The influence of the catechol moiety
on the cytotoxicity and genotoxicity of new lignan derivatives has been investigated. The results
obtained indicated that the presence of the catechol moiety sharply enhances the clastogenic potential
(e.g. induction of chromosomal aberrations), the cytotoxicity and the modulation of cell cycle
progression with respect to the parent compounds. Thus, despite the in vitro antioxidant activity usually
described for catechol derivatives, our results show for the first time the generation of a clastogenic
potential, highly indicative of a long-term genetic and cancer risk.
concomitant decrease of GH and protein-SH, which are sacrificed
Introduction
to protect nucleic acids from radical damage.10
Lignans are a large family of natural phenols, widely diffused on
More generally, the biological effect of highly oxidized phenols
the plant kingdom, and characterized by the b–b¢-linkage between
two phenylpropane units or between their biogenetic equivalents.1
These compounds, having different degrees of oxidation of the
side chain or differences in the aromatic substitution, show
several biological activities,2 including antiviral,3 antitumoral,4
antiinflammatory5 and antioxidant properties.6
is further complicated by considering their specific electronic
properties. In fact, quinones are highly electrophilic species that
can easily react with the nucleophilic sites of biomolecules,
the reaction proceeding by a Michael-like addition at the
a,b-unsaturated carbonyl moiety present in their structure. This
property potentially confers to quinones high genotoxicity and
cytotoxicity. On the other hand, hydroquinone and catechol
derivatives – the reduced form of quinones – protect nucleic
acids by radical damage, entrapping the ROS via a H-abstraction
process with generation of less reactive radicals usually stabilized
through mesomeric effects.11 The oxidation of the reactive benzylic
positions in the side-chain can also affect the biological activity.
On the basis of this scenario, it is expected that highly oxidized
lignan derivatives should possess biological activities different
from that of the parent lignans. Thus, in a systematic approach,
the biological activity of lignans should be compared to that
of the corresponding oxidized derivatives, to account for their
potential metabolic role. Unfortunately, little data is available
about the biological activity of oxidized lignans.12 This is mainly
due to the low quantities of lignan metabolites recoverable from
physiological fluids during in vivo treatments (less than ppm of
lignan metabolites are detectable by GC-MS and LC-MS analyses
of organic samples), associated with the difficulty of designing
efficient chemical strategies for the selective oxidation of lignans.
In recent years, we have been involved in the design and
development of novel stoichiometric and catalytic procedures for
the oxidation of natural phenols, including lignin and lignan
derivatives.13 For example, the oxidation of podophyllotoxin
(a lignan of the aryltetralin family isolated from various plants
of the genus Podophyllum) with methyltrioxorhenium (MeReO3,
MTO), in combination with H2O2 as primary oxidant, afforded
various quinone and isopodophyllotoxone derivatives, some of
Once introduced into an organism, lignans are metabolized by
the action of oxidative enzymes containing the cytochrome P-450
molecule as a cofactor at the reactive site.7 During these metabolic
pathways, lignans can be de-O-methylated at the guaiacyl or
syringyl groups in the aromatic moieties, oxidized at reactive
benzylic positions by oxygen atom insertion into the C–H bond,
or transformed to corresponding hydroquinones, catechols and
ortho- or para-benzoquinone derivatives. It is worth noting that
highly oxidized forms of phenol derivatives, such as quinones and
hydroquinones, can modify the value of the redox-potential in
the cell. This process, which has consequences for the lifetime of
the cell, is performed by various reaction processes. In the first
case, highly oxidized phenols react with the components of the
cellular machinery devoted to the control of the redox-potential,
such as glutathione (GH) and mercapto-group containing proteins
(protein-SH).8 According to their mode of action, they result in
depletion of the molecular redox controllers, mainly by alkylation
of the nucleophilic mercapto (SH) group.9 Alternatively, highly
oxidized phenols can generate reactive oxygen species (ROSs),
such as hydroxyl radicals (HO∑) or peroxyl radicals (HOO∑), with a
aDepartment of Agrobiology and Agrochemistry, University of Tuscia, 01100,
Viterbo, Italy. E-mail: berninir@unitus.it, saladino@unitus.it; Fax: +39
0761 357242; Tel: +39 0761 357452-357284
b
˚
˚
Abo Akademi Process Chemistry Centre, Abo Akademi University,
˚
Porthansgatan 3, FI-20500, Abo, Finland
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which were not previously reported.14 These compounds showed
various mechanisms of action, from the inhibition of cell division
at the level of the microtubule assembly by freezing polymerization
of tubulin (a characteristic of the parent podophyllotoxin), to
the inhibition of topoisomerase II, which is a more interesting
therapeutic target.
nans and neolignans to the corresponding catechol derivatives
by using IBX as the primary oxidant under green conditions
(dimethyl carbonate–H2O solvent) through the in situ reduction
procedure.21,22 The role of the catechol moiety on the cytotoxicity
and genotoxicity of new lignan derivatives has been investigated.
More exciting was that the biological activity is correlated
to the oxidation state of the molecule, from (a) the relatively
low inhibitory activity towards tubulin polymerization of in
2¢,3¢-ortho-benzoquinone derivatives, to (b) an enhanced in-
hibitory activity towards tubulin polymerization and concurrent
emergence of antitopoisomerase II activity by oxidation of the C-7
position of the C-ring in isopodophyllotoxone, to (c) a selective
activity against topoisomerase II when the oxidation of the C-7
position was accompanied by the ring-opening of the D-lactone
moiety. A similar structure–activity relationship was observed in
the apoptogenic activity of products of oxidation of galbulin, an-
other aryltetraline lignan isolated from Galbulimima belgraveana,
against both human lymphoma cell line BL41 (EBV-), prone
to apoptosis, and E2R (EBV+), which is strongly resistant to
chemical treatment.14
Results and discussion
Initially, we studied the de-O-methylation procedure with IBX
for three representative guaiacyl derivatives, two monolig-
nols (vanillic acid 1 and ferulic acid 2) and two dimeric
derivatives, (E)-4-(4-hydroxy-3-methoxyphenyl)-2-(4-hydroxy-3-
methoxyphenylmethyl)but-3-enoic acid 3, which is formed in the
treatment of the natural lignan hydroxymatairesinol in strongly
basic conditions,23 and 5,5¢-dihydroxy-4,4¢-dimethoxybiphenyl-
2,2¢-dicarbaldehyde 4 (Fig. 1). Since a free carboxylic moiety
decreases the efficiency of product extraction in the IBX procedure,
acids 1–3 were converted also into the corresponding methyl
esters 5–7 by reported protocols before the oxidative treatment
(Scheme 1).23–26
As a continuation of this study, with the aim to further elucidate
the biological activity of highly oxidized lignan derivatives, we
focused our attention on the de-O-methylation of the guaiacyl
(4-hydroxy-3-methoxyphenyl) moiety to the corresponding cat-
echol (3,4-dihydroxyphenyl) group. In fact, despite the higher
radical scavenging capacity reported for lignans with catechol
(3,4-dihydroxyphenyl) moieties compared to guaiacyl (3-methoxy-
4-hydroxyphenyl) lignans,6a no general chemical procedures are
available to mimic the metabolic activity of cytochrome P-450
enzymes in the de-O-methylation of guaiacyl lignans.15 Moreover,
even though some cytotoxicity data are available for guaiacyl
lignans (mainly matairesinol derivatives),16 no cytotoxicity or
genotoxicity data are available for de-O-methylated guaiacyl
derivatives.
Fig. 1 Chemical structure of monolignols 1 and 2, and dimeric derivatives
3 and 4.
An efficient reagent useful for obtaining the catechol
5
moiety from guaiacyl derivatives is 1-hydroxy-1-oxo-1H-1l -
benz[d][1,2]iodoxol-3-one (2-iodoxybenzoic acid, IBX), first pre-
pared by Hartmann and Mayer by oxidizing 2-iodobenzoic
acid (IBA) with potassium bromate in aqueous solution.17
A
convenient and safe procedure for its preparation, involving Oxone
as oxidant, was reported by Santagostino and coworkers.18 When
phenolic methyl ethers are oxidized by IBX, the corresponding
ortho-quinone derivatives are obtained.19 If this oxidative step is
followed by in situ reduction, the catechol moiety is produced, with
a selectivity similar to that of a demethylating enzyme. Despite this
remarkable capacity, only some examples of catecholic compounds
have been prepared using this demethylating-IBX methodology.20
Recently, we have been involved in the optimization of this method
and its application to a wide variety of natural organic compounds
to obtain the corresponding bioactive catechol derivatives. For
example, 2-(4-hydroxy-3-methoxyphenyl)ethanol (homovanillyl
alcohol), a natural guaiacyl derivative, has been utilized as starting
material for the synthesis of 2-(3¢,4¢-dihydroxyphenyl)ethanol
(hydroxytyrosol) and its lipophilic derivatives, useful molecules
for cosmetic and pharmaceutical applications.21 Two patents
regarding these results have been deposited.22
Scheme 1 Methylation of carboxylic acids 1, 2 and 3 with methanol/
sulfuric acid.
As a general procedure, the acids 1–3, methyl esters 5–7, and
vanillin derivative 4 (1.0 mmol) were treated with a small excess
of IBX (1.2 equivalents) in H2O/DMC (1.0 : 9.0) solution at
room temperature for 3–5 hrs. The quenching of the reaction
was performed by adding water (1.0 ml) and sodium dithionite
(Na2S2O4, 2.4 equivalents) over several minutes, with a colour
change in the solution being observed. Under these experimental
We report here the first selective de-O-methylation of a large
panel of representative guaiacyl monolignols and guaiacyl lig-
2368 | Org. Biomol. Chem., 2009, 7, 2367–2377
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Table 1 Oxidation of lignans and neolignans with IBX
Entry
Substrate
Product
Conv. (%)
Yield (%)a
1
2
3
4
5
6
7
8
5
6
8
9
>98
60
>98
>98
>98
>98
50
73
82
25b
55
70
40
85
70b
7
10
14
15
16
21
22
11
12
13
19
20
65
a Yield based on conversion of substrate. b Total yield after acetylation.
Scheme 2 Oxidation of monolignol derivatives 5–7 by IBX. Reagents and
conditions: a) IBX, DMC, H2O; b) Na2S2O4, H2O; c) Ac2O, py.
conditions, the oxidations of acids 1–3 and vanillin derivative
4 failed. In these cases, the low efficiency of the oxidation is
in accordance with previously reported data for the reactivity
of phenols containing electron-withdrawing groups (EWGs, e.g.
C(O)R and NO2 groups) with IBX.19,20 On the other hand, the
methyl esters of catechol monolignol derivatives such as methyl
3,4-dihydroxybenzoate 8 (methyl protocatechuate) and methyl
3,4-dihydroxycinnamate 9 (methyl caffeate) were obtained in
acceptable to high yield (Scheme 2, Table 1, entries 1 and 2).
To the best of our knowledge, the de-O-methylation of vanillic
acid and ferulic acid has only previously been reported using
enzymatic systems.26 In the case of 7 we were able to isolate
the reaction product, methyl (3E)-2-(3,4-diacetoxybenzyl)-4-(3,4-
diacetoxyphenylmethyl)but-3-enoate 10, only after acetylation
under usual experimental conditions (Scheme 2, Table 1, entry 3).
In accordance with previously reported data,20 the IBX-de-
O-methylation proceeded by a ionic mechanism during which
guaiacyl derivative adds to the iodine(V) center of IBX to give a
2-iodobenzoic acid (IBA), the only by-product of the reaction.
Catechols were then obtained during the final reductive step.
Next, we applied the oxidation procedure with IBX to three
selected guaiacyl lignan derivatives, 7-hydroxymatairesinol 11
(butyrolactone lignan), a-conidendrin 12 (aryltetralin lignan) and
lariciresinol 13 (tetrahydrofuran lignan), extracted from knots
of Norway Spruce (Picea abies).27 The oxidation of lignans
11–13 afforded new catechols 14–16 in acceptable yields, as well as
unreacted substrate and some over-oxidation or oligomerization
products28 that we were not able to recover from the reaction
mixture, probably because of their high polarity (Scheme 3, Table
1, entries 4–6). In these reactions, a-conidendrin 12 was the most
reactive lignan, affording the catechol derivative 15 in quantitative
conversion and a yield (70%) higher than the other lignans. The
catechol derivatives 15–16 were also acetylated to compounds
17–18, as selected examples to compare the biological activity with
parent guaiacyl and catechol compounds (vide infra). To the best
of our knowledge, this is the first example of synthesis of catechol
lignan derivatives through a chemical procedure that mimics the
reaction products of cytochrome P-450 activity in the cell.
5
l -iodanyl intermediate. The intramolecular (and regioselective)
5
delivery of an oxygen atom from the l -iodanyl intermediate
3
leads to a more stable l -iodanyl intermediate that is then
hydrolyzed into the corresponding ortho-quinone derivatives and
Scheme 3 Oxidation of lignans 11–13 by IBX. Reagents and conditions: a) IBX, DMC, H2O; b) Na2S2O4, H2O; c) Ac2O, py.
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To further evaluate the generality of this procedure, we extended
the protocol of IBX oxidation to two neolignan derivatives, 19
and 20, in which the phenylpropane units are not bonded into
the b and b¢ positions. Neolignans, which are the most abundant
sub-structures in lignin,29 are recalcitrant to oxidation processes
and usually furnish very complex reaction mixtures under forcing
experimental conditions.13a When freshly prepared 19 was treated
with IBX under previously reported experimental conditions,
the catechol derivative 21 was obtained as the only recovered
product in acceptable yield in addition to unreacted substrate
(Scheme 4, Table 1, entry 7). We were not able to recover the
catechol derivative of 20, which was obtained from the reaction
mixture only as a fully acetylated derivative 22 (Scheme 4, Table 1,
entry 8).
The catechol derivatives 8, 9, 14, 15, 16, 21 and the acetylated
derivatives 10, 17, 18, and 22 were evaluated for their toxicity
relative to their parent natural compounds 5, 6, 11, 12, 13 and 19
to better define the role of the catechol moiety on structure–activity
relationships. In particular, we assessed potential genotoxic effects
in mammalian cells in vitro by analyzing the induction of
chromosomal aberrations, which are highly predictive of long
term genetic and cancer risk.29,30 Analyses of the mitotic index
(MI), an indirect parameter used to evaluate cytotoxic effects, and
proliferative replication index (PRI), used to assess cell division
kinetics and interference with cellular check-points, were also
determined.
Scheme 4 Oxidation of neolignans 19 and 20 by IBX. Reagents and
conditions: a) IBX, DMC, H2O; b) Na2S2O4, H2O; c) Ac2O, py.
Among the different parent compounds and corresponding
catechols and acetylated catechol derivatives, the monolignols
methyl vanillate 5 and methyl ferulate 6 did not induce marked
cytotoxicity at any dose-level employed and were not clastogenic.
MI and PRI values were only slightly lower (250 and 500 mM) in
the cultures treated with methyl ferulate 6 (Tables 2 and 3). No
statistically significant increases in the incidence of chromosomal
Table 2 MI values observed in V79 cells for compounds 5 and 8
Frequency of
Compound
Dose level (mM)
Mean MI (%)
Relative MI
M1
M2
M3
PRI
Relative PRI
None (control, DMSO)
1%
25
50
100
250
500
1%
25
5.2
4.6
5.1
5.5
5.1
4.0
4.0
3.1
2.9
2.4
1.9
1.7
100
88
98
105
98
78
100
78
71
60
47
3
9
7
7
9
96
91
93
93
91
91
75
56
70
63
52
5
1
0
0
0
0
0
0
0
0
0
0
0
1.98
1.91
1.93
1.93
1.91
1.91
1.75
1.72
1.70
1.63
1.42
1.05
100
96
97
97
96
96
100
98
97
93
81
60
5
5
5
5
5
9
None (control, DMSO)
25
44
30
37
48
95
8
8
8
8
8
50
100
250
500
43
Table 3 MI values observed in V79 cells for compounds 6 and 9
Frequency of
Compound
Dose level (mM)
Mean MI (%)
Relative MI
M1
M2
M3
PRI
Relative PRI
None (control, DMSO)
1%
25
50
100
250
500
1%
25
4.1
3.4
3.1
3.1
2.9
2.7
4.0
3.3
2.2
1.9
0.7
0.0
100
83
76
76
72
67
100
81
53
47
17
0
12
42
40
37
40
37
25
60
62
92
100
0
88
58
60
63
60
63
75
40
38
8
0
0
0
0
0
0
0
0
0
0
0
0
1.88
1.78
1.68
1.63
1.60
1.68
1.75
1.40
1.38
1.08
1.00
1.00
100
95
89
87
85
89
100
80
79
62
57
57
6
6
6
6
6
None (control, DMSO)
9
9
9
9
9
50
100
250
500
0
0
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aberrations were observed at any dose-level employed (Fig. 2
and 3). In contrast, their corresponding catechol derivatives,
methyl protocatechuate 8 and methyl caffeate 9, showed marked
cytotoxicity and dose-related, statistically significant, increases of
chromosomal aberrations (Figs. 2 and 3). MI and PRI values were
reduced up to 43 and 60% respectively by methyl protocatechuate 8
at 500 mM, and up to 17 and 57% respectively by methyl caffeate 9
at 250 mM compared to the relevant solvent control (Tables 2
and 3). At higher dose-levels, methyl caffeate 9 proved to be
severely toxic, and no cytogenetic analysis was performed since
no metaphases were recovered (Table 3).
Among the group of lignans assessed, 7-hydroxymatairesinol 11
and lariciresinol 13, similarly to the monolignols methyl vanillate
5 and methylferulate 6, did not show marked cytotoxicity and
did not induce chromosomal aberrations at any of the dose-
levels employed (Figs. 4 and 5). MI values were only reduced in
11-treated cultures up to 67% of the relevant solvent control value
at 500 mM, while PRI values were not affected in either compound
(Tables 4 and 5).
Fig. 2 Frequency of aberration-bearing cells induced by the monolignol
methyl vanillate 5 and its catechol derivative methyl protocatechuate 8 in
V79 cells in vitro. * and ***: Statistically significant at P < 0.05 and 0.001
respectively by Fisher’s Exact test.
Fig. 4 Frequency of aberration-bearing cells induced by the lignan
7-hydroxymatairesinol 11 and its catechol derivative 14 in V79 cells in vitro.
*: Statistically significant at P < 0.05 by Fisher’s Exact test.
Alternatively, the remaining lignan a-conidendrin 12 proved to
be a strong clastogenic compound without marked cytotoxicity. It
induced statistically significant increases in chromosomal aberra-
tions at 100 and 250 mM (Fig. 6) without significant cytotoxicity,
since the MI and PRI were not different from the relevant solvent
control values (Table 6). However, all the corresponding cate-
chol derivatives in this group showed pronounced clastogenicity
(Figs. 4–6) and selective blocking of cell kinetics at M1 metaphases
(Tables 4–6). Cytotoxicity was also markedly enhanced, but only
in 14 and 16 were MI values were significantly reduced at higher
dose-levels, dropping to zero in the case of catechol derivative 14
at 500 mM (Table 4).
Fig. 3 Frequency of aberration-bearing cells induced by the monolignol
methyl ferulate 6 and its catechol derivative methyl caffeate 9 in V79 cells
in vitro. *, ** and ***: Statistically significant at P < 0.05, 0.01 and 0.001
respectively by Fisher’s Exact test.
Table 4 MI values observed in V79 cells for compounds 11 and 14
Frequency of
Compound
Dose level (mM)
Mean MI (%)
Relative MI
M1
M2
M3
PRI
Relative PRI
None (control, DMSO)
1%
25
50
100
250
500
1%
25
4.9
4.0
4.5
4.1
4.3
3.3
4.1
3.4
2.6
1.4
0.9
0.0
100
82
92
84
88
67
100
83
62
35
3
8
6
96
92
94
92
90
84
82
87
75
70
0
1
0
0
0
0
0
0
0
0
0
0
0
1.98
1.92
1.94
1.92
1.90
1.92
1.88
1.87
1.75
1.70
1.00
0.00
100
97
98
97
99
97
100
99
93
90
53
0
11
11
11
8
11
10
16
12
13
25
30
100
0
11
None (control. DMSO)
14
14
14
14
14
50
100
250
500
22
0.0
0
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Table 5 MI values observed in V79 cells
Frequency of
Compound
Dose level (mM)
Mean MI (%)
Relative MI
M1
M2
M3
PRI
Relative PRI
None (control, DMSO)
1%
25
50
100
250
500
1%
25
50
100
250
500
1%
25
4.9
4.0
4.7
4.9
6.4
6.0
4.13
3.48
3.31
2.24
2.12
0.50
4.13
3.62
2.55
2.50
2.39
2.10
100
82
95
3
6
3
3
5
96
94
97
97
95
93
88
88
92
86
64
0
88
90
88
85
63
15
1
0
0
0
0
0
1.98
1.93
1.95
1.97
1.97
1.94
1.98
1.88
1.92
1.86
1.64
1.00
1.98
1.90
1.88
1.85
1.63
1.15
100
82
95
13
13
13
99
99
13
131
122
100
84
80
54
51
12
100
88
62
131
122
100
95
97
94
88
61
100
96
95
13
7
None (control, DMSO)
12
12
8
16
0
0
0
0
0
0
0
0
0
0
0
16
16
14
36
100
12
10
12
15
37
85
16
16
None (control, DMSO)
18
18
18
18
18
50
100
250
500
61
58
51
93
88
71
cytotoxic since the MI values were not lower than the relevant
solvent control values. This interesting finding indicates that 15
could trigger specific mitotic check-points located between the
onset of mitosis and the activation of the anaphase-promoting
complex (APC). This aspect is supported by the significant
induction of endoreduplicated cells (9%) at 500 mM (data not
shown). Endoreduplication consists of two successive DNA
synthetic rounds without an intervening mitosis stage, leading
to the formation of diplochromosomes in the following mitotic
metaphases, and thus generating tetraploid and polyploid cells.
Polyploid cells are genetically unstable and lose chromosome
randomly to give aneuploidy (Fig. 7). As far as the acetylated
catechol derivatives were concerned, compound 17 did not prove
to be clastogenic but showed a marked cytotoxicity. MI and PRI
values were reduced up to 28 and 53% of solvent control values
at 250 mM respectively. At 500 mM it was extremely cytotoxic and
no metaphases were recovered. Compound 18 induced a single
positive result in terms of induction of chromosomal aberrations
at 500 mM accompanied by a mild cytotoxicity. MI and PRI values
were reduced up to 51 and 71% of solvent control values at 500 mM
Fig. 5 Frequency of aberration-bearing cells induced by the lignan
lariciresinolo 13 and their catechol and acetylated catehcol derivatives 16
and 18 respectively in V79 cells in vitro. * and **: Statistically significant
at P < 0.05 and 0.01 respectively by Fisher’s Exact test.
Fig. 6 Frequency of aberration-bearing cells induced by the lignan
a-conidendrin 12, its catechol derivative 15 and its acetylated catechol
derivative 17 in V79 cells in vitro. *, ** and ***: Statistically significant at
P < 0.05, 0.01 and 0.001 respectively by Fisher’s Exact test.
In contrast, compound 15, though it induced selective block
of cell cycle kinetic similar to 14 and 16, did not prove to be
Fig. 7 Endoreduplicated cell from a V79 cell line treated with the catechol
derivative 15 of lignan a-conidendrin at 500 mM.
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Table 6 MI values observed in V79 cells for compounds 12, 15 and 17
Frequency of
Compound
Dose level (mM)
Mean MI (%)
Relative MI
M1
M2
M3
PRI
Relative PRI
None (control, DMSO)
1%
25
50
100
250
500
1%
25
5.2
5.0
4.5
4.7
5.8
5.6
4.0
2.0
1.8
1.7
3.4
3.6
4.1
3.4
2.8
2.3
1.2
0.0
100
95
87
3
6
4
6
2
96
94
96
94
98
93
88
68
65
55
19
5
90
13
20
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1.98
1.94
1.96
1.94
1.98
1.93
1.88
1.68
1.65
1.55
1.19
1.05
1.9
100
98
99
98
100
97
100
89
88
82
63
56
100
64
63
53
53
0
12
12
12
89
12
112
107
100
50
44
42
84
89
100
82
68
12
7
None (control, DMSO)
12
32
35
45
81
95
10
87
80
99
100
0
15
15
50
15
100
250
500
1%
25
15
15
None (control, DMSO)
17
17
17
17
17
1.21
1.20
1.01
1.00
0.0
50
100
250
500
54
28
0
Table 7 MI values observed in V79 cells
Frequency of
Compound
Dose level (mM)
Mean MI (%)
Relative MI
M1
M2
M3
PRI
Relative PRI
None (control, DMSO)
1%
25
50
100
250
500
1%
25
50
4.9
5.1
4.1
4.2
4.7
4.3
4.0
1.7
1.7
1.4
1.2
0.4
100
104
84
85
96
88
100
42
43
34
3
4
3
4
4
10
25
45
60
90
100
100
96
96
97
96
96
90
75
55
40
10
0
1
0
0
0
0
0
0
0
0
0
0
0
2.0
1.9
2.0
2.0
2.0
1.9
1.8
1.6
1.4
1.1
1.0
1.0
100
97
98
99
99
97
100
89
80
63
57
57
19
19
19
19
19
None (control, DMSO)
21
21
21
21
21
100
250
500
29
10
0
respectively (Table 6). Furthermore, the catechol derivative 21 of
neolignan 19 showed a similar pattern of results observed for
the corresponding catechol of monolignols 5 and 6 and lignans
11, 12 and 13 (Table 7). It induced dose-related and statistically
significant increases in chromosomal aberrations at all dose-levels
assayed (Fig. 8) accompanied by marked cytotoxicity. MI values
were reduced up to 10% of the relevant solvent control value at
500 mM while the PRI was reduced to 63% at 100 mM, and up
to 57% at 250 and 500 mM, where only metaphase 1 (M1) was
observed.
Experimental
Fig. 8 Frequency of aberration-bearing cells induced by the neolignan 19
and its catechol derivative 21 in V79 cells in vitro. *, ** and ***: Statistically
significant at P < 0.05, 0.01 and 0.001 respectively by Fisher’s Exact test.
Chemical section
Reagents and lignans. 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. IBX was prepared in laboratory as described in the
literature.18 Guaiacyl derivative 3 and lignan derivatives 11, 12
and 13 were purchased from Oy ArboNova Ab. Neolignans 19
and 20 were synthesized according to the reported literature.31
Methyl esters 5, 6, and 7 were prepared as described in the
literature.23–25 The nomenclature of lignans and neolignans has
been assigned according to the IUPAC-IUB Joint Commission
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on Biochemical Nomenclature (JCBN), Recommendations
(2000).
Methyl 4-hydroxy-3-methoxycinnamate (Methyl ferulate, 6).
Solid. Yield 70%. Mp 66 ◦C.25
Oxidation with IBX. Preparation of catecholic compounds.
Substrate (1.0 mmol) was dissolved in dimethyl carbonate–water
9:1 (10 ml) and then IBX (1.2 eq.) was added, and the mixture
stirred at room temperature for 3–5 h, depending on the substrate.
At the end, water (10 ml) and Na2S2O4 (2.4 eq.) were added and
the solution was stirred for 5–10 minutes. After evaporation of
the solvent under vacuum, the residue was solubilized with ethyl
acetate (20 ml) and treated with a saturated solution of NaHCO3
(10 ml). The aqueous phase was extracted with ethyl acetate
(3 ¥ 10 ml). The organic phases were washed with a saturated
solution of NaCl (20 ml) and dried over Na2SO4. After evaporation
of the solvent, catecholic lignan derivatives 8, 9, 14, 15, 16, and
21 were isolated by chromatographic purification on silica gel by
using ethyl acetate–hexane (ratio 1:2, 2:1, 3:1 depending on the
compound) as eluent.
Methyl (3E)-4-(4-hydroxy-3-methoxyphenyl)-2-(4-hydroxy-3-
methoxyphenylmethyl)but-3-enoate (7). Solid. Yield 88%.23
Methyl 3,4-dihydroxybenzoate (Methyl protocatechuate 8).
Solid. Yield 73%. Mp 135 ◦C.25
Methyl (2E)-3-(3,4-dihydroxyphenyl)prop-2-enoate (Methyl caf-
feate 9). Solid. Yield 82%. Mp 159 ◦C.25
Methyl (3E)-2-(3,4-diacetoxybenzyl)-4-(3,4-diacetoxyphenyl)-
but-3-enoate (10). Solid. Yield 25%. Mp 144 ◦C.23 dH (200 MHz,
CDCl3) d: 2.25 (6H, s, 2xMe), 2.26 (3H, s, Me), 2.27 (3H, s, Me),
2.81–2.92 (1H, dd, J1= 13.7 Hz, J2= 6.9 Hz, H-7a¢¢), 3.07–3.18
(1H, dd, J1= 14.0 Hz, J2= 8.0 Hz, H-7b¢¢), 3.36–3.47 (1H, m,
H-2), 3.64 (3H, s, OMe), 6.06–6.18 (1H, dd, J1= 15.9 Hz, J2=
8.4 Hz, H-3), 6.31–6.38 (1H, d, J= 15.7 Hz, H-4), 7.04–7.19 (6H,
m, H-2¢,2¢¢,5¢,5¢¢,6¢,6¢¢); dC (50 MHz, CDCl3) 20.2 (2 ¥ OAc), 20.6
(2 ¥ OAc), 38.1 (C-1), 50.8 (OMe), 52.0 (C-2), 121.0 (C-2¢), 123.2
(C-2¢¢),123.4 (C-3), 124.0 (C-5¢),/124.7 (C-5¢¢), 127.2 (C-6¢), 127.6
(C-6¢¢), 131.4 (C-4), 135.5 (C-1¢¢), 137.2 (C-1¢), 140.6 (C-3¢), 141.4
(C-3¢¢), 141.8 (C-4¢), 142.1 (C-4¢¢), 168.2 (4 ¥ OCOMe), 173.2
(COOMe). HR-MS m/z: 498.47864 (M+).
Preparation of methyl (3E)-2-(3,4-diacetoxybenzyl)-4-(3,4-
diacetoxyphenyl)but-3-enoate (10). The substrate (1.0 mmol) was
dissolved in dimethyl carbonate–water 9:1 (10 ml) and then IBX
(1.2 eq.) was added at room temperature for 3–5 h depending on
the substrate. At the end, water (10 ml) and Na2S2O4 (2.4 eq.)
were added and the solution was stirred for 5–10 minutes.
After evaporation of the solvent under vacuum, the residue was
solubilized with ethyl acetate (20 ml) and treated with 1 M HCl
(10 ml). The aqueous phase was extracted with ethyl acetate (3 ¥
10 ml). The organic phases were washed with a saturated solution
of NaCl (10 ml) and dried over Na2SO4. After evaporation of the
solvent, the mixture reaction was solubilized in pyridine (10 ml),
acetic anhydride (10 ml) was added, and the mixture stirred
overnight. At the end of the reaction, a 1 M solution of HCl
(10 ml) was added and the final products were extracted with
ethyl acetate (3 ¥ 10 ml). The organic phases were washed with a
saturated solution of HCl (20 ml) and with a saturated solution of
NaCl (20 ml), then dried over Na2SO4. After evaporation of the
solvent, and chromatographic purification on silica gel, acetylated
compound 10 was isolated.
3,3¢,4,4¢,7¢-Pentahydroxylign-9,9¢-lactone (14). Oil. Yield 55%.
[a]20D -4.5◦ (c 0.5, acetone). dH (200 MHz, CDCl3) 2.43–3.11 (4H,
m, H-7,8,8¢), 3.88–3.98 (3H, m, H-7¢,9a¢,9b¢), 6.47–6.59 (2H, m,
H-5¢,6¢), 6.64 (1H, d, J= 2.1, H-6) 6.70–6.78 (3H, m, H-2,2¢,5); dC
(50 MHz, CDCl3) 31.7 (C-7), 41.2 (C-8), 43.9 (C-8¢), 70.5 (C-9¢),
76.6 (C-7¢), 115.7 (C-2¢), 115.9 (C-5¢), 116.4 (C-2), 117.8 (C-6¢),
120.6 (C-5), 122.5 (C-6), 127.9 (C-1), 131.4 (C-1¢), 144.1 (C-4),
144.7 (C-3¢), 145.5 (C-3), 145.7 (C-4¢), 178.3 (C-9). HR-MS m/z:
346.33737 (M+).
3¢,4,4¢,5-Tetrahydroxy-2,7¢-cyclolignan-9,9¢-lactone (15). Oil.
Yield 70%.32 [a]20 -75.0◦ (c 0.5, acetone). dH (200 MHz, CDCl3)
D
2.56–3.02 (4H, m, H-7a,7b,8,8¢), 3.87–4.06 (3H, m, H-7¢,9a¢,9b¢),
6.23 (1H, s, H-6), 6.54–6.66 (3H, m, H-2¢,3,6¢), 6.79 (1H, d, J=
7.9 Hz, H-5¢); dC (50 MHz, CDCl3) 25.1 (C-7), 46.1 (C-8¢), 52.0 (C-
8), 54.2 (C-7¢), 76.4 (C-9¢), 120.4 (C-2¢), 120.8 (C-6), 121.0 (C-3),
124.2 (C-5¢), 125.2 (C-6¢), 131.6 (C-1), 136.2 (C-1¢), 140.2 (C-2),
148.5 (C-5), 148.9 (C-4¢), 149.2 (C-3¢), 150.4 (C-4), 181.8 (C-9).
HR-MS m/z: 328.32211 (M+).
Acetylation of catecholic compounds. Substrate (1.0 mmol)
was solubilized in pyridine (10 ml), acetic anhydride (10 ml)
was added, and the mixture stirred overnight. At the end of the
reaction, a 1 M solution of HCl (10 ml) was added and the final
products were extracted with ethyl acetate (3 ¥ 10 ml). The organic
phases were washed with a saturated solution of HCl (20 ml)
and with a saturated solution of NaCl (20 ml), then dried over
Na2SO4. After evaporation of the solvent, and chromatographic
purification on silica gel, acetylated compounds 17, 18 and 22 were
isolated.
3,3¢,4,4¢,9-Pentahydroxy-7,9¢-epoxylignan (16). Oil. Yield
40%. [a]20 +46.7◦ (c 0.1, acetone). dH (200 MHz, CDCl3)
D
2.25–2.81 (4H, m, H-7a¢,7b¢,8,8¢), 3.59–3.67 (2H, m, H-9a,9b),
3.78–3.91 (2H, m, H-9a¢,9b¢), 4.74 (1H, d, J= 5.7 Hz, H-7),
6.49–6.83 (6H, m, H-2,2¢,5,5¢,6,6¢); dC (50 MHz, CDCl3) 36.2
(C-7¢), 46.3 (C-8¢), 57.3 (C-8), 63.4 (C-9), 76.1 (C-9¢), 86.1 (C-7),
116.8 (C-5), 118.7 (C-2), 119.1 (C-2¢), 119.5 (C-5¢), 121.0 (C-6),
123.7 (C-6¢), 136.7 (C-1), 140.2 (C-1¢), 147.1 (C-4¢), 147.8 (C-3),
148.7 (C-4), 148.8 (C-3¢). HR-MS m/z: 332.12599 (M+).
Identification and characterization of oxidation products. 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 solvents.
All chemical shifts are expressed in parts per million (d scale) and
coupling constants in Hertz (Hz). HR-MS were recorded with a
Micromass Q-TOF micro mass spectrometer (Waters).
2,7¢-Cyclolignan-9,9¢-lactone-4,5,3¢,4¢-tetrayl-tetraacetate (17).
Oil. Yield 70%. [a]20D -85.0◦ (c 0.5, acetone). dH (200 MHz, CDCl3)
2.19 (3H, s, OAc), 2.25 (6H, s, OAc), 2.26 (3H, s, OAc), 2.48–2.61
(2H, m, H-8,8¢), 2.92–3.05 (1H, m, H-7a), 3.22–3.32 (1H, m, H-
7b), 3.96–4.30 (3H, m, H-7¢,9¢,9b¢), 6.71 (1H, s, H-6), 6.95–7.05
(3H, m, H-3,5¢,6¢), 7.15 (1H, d, J= 8.2 Hz, H-2¢); dC (50 MHz,
Methyl 4-hydroxy-3-methoxybenzoate (Methyl vanillate, 5).
Solid. Yield 93%. Mp 70 ◦C.24
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CDCl3) 20.5 (OAc), 20.6 (OAc), 20.7 (OAc), 29.2 (C-7), 41.4 (C-
8¢), 47.2 (C-8), 49.7 (C-7¢), 71.4 (C-9¢), 123.3 (C-2¢), 124.2 (C-6),
124.4 (C-5¢), 126.2 (C-3), 133.8 (C-6¢), 136.3 (C-1), 140.0 (C-2),
140.7 (C-4), 141.0 (C-1¢), 141.5 (C-4¢), 141.5 (C-5), 142.5 (C-3¢),
167.9 (OCOMe), 168.1 (OCOMe), 168.3 (OCOMe), 176.0 (C-9).
HR-MS m/z: 496.47143 (M+).
cells from the same frozen stocks but thawed on different days.
Mitotic indices observed in the vehicle controls (0 mM) were in the
range 4.0–5.2. This slight variation is considered “physiological”
and is compatible with our historical control range values. Cultures
of the cells are grown in Eagle minimal essential medium (EMEM)
supplemented with 10% Foetal Calf Serum, 2 mM L-Glutamine
and antibiotics (1% w/v Penicillin and 86 mM Streptomycin). All
incubations were at 37 ◦C in a 5% carbon dioxide atmosphere and
100% nominal humidity.
(7,9¢-Epoxylignan-3,3¢,4,4¢,9-pentayl)pentaacetate (18). Oil.
Yield 85%. [a]20 +12.5◦ (c 0.4, acetone). dH (200 MHz, CDCl3)
D
1.54 (3H, s, OAc), 2.26 (12H, s, 3xOAc), 2.43–2.86 (4H, m,
H-7a¢,7b¢,8,8¢), 4.03–4.31 (4H, m, H-9a¢,9b¢), 4.85 (1H, d, J =
5.4 Hz, H-7), 6.73–7.15 (6H, m, H-2,2¢,5,5¢,6,6¢); dC (50 MHz,
CDCl3) 20.6 (OAc), 20.8 (OAc), 32.8 (C-7¢), 41.7 (C-8¢), 49.0
(C-8), 62.4 (C-9), 72.6 (C-9¢), 82.2 (C-7), 120.6 (C-2¢), 123.3
(C-2), 123.4 (C-5¢), 123.5 (C-5), 123.6 (C-6), 126.7 (C-6¢), 138.6
(C-1), 138.6 (C-1¢), 141.6 (C-3), 141.6 (C-3¢), 142.0 (C-4), 142.0
(C-4¢), 168.1 (OCOMe), 168.2 (OCOMe), 168.3 (OCOMe), 170.9
(OCOMe). HR-MS m/z: 542.54067 (M+).
Chromosomal aberration assays. Test compound treatments
of Chinese hamster V79 cells were performed in the absence of
rat liver metabolism. The study was designed to comply with the
experimental methods indicated in the OECD Guidelines for the
Testing of Chemicals No. 487 (Draft June 2004).
Following dose-range finding experiments, the assay was per-
formed using dose levels of 500, 250, 100, 50, and 25 mM
and a three hour treatment time. Solvent-treated cells served as
negative control. At the end of treatment (3 hours), cultures
were washed twice with a PBS solution and re-incubated at
37 ◦C in fresh complete culture medium for further 18 hours
(approximately 1.5 cell cycle). Cultures set up for analysis of
proliferative replication index (PRI) received also BrdU at 9.8 mM
to differentiate sister chromatids. Colcemid at 0.27 mM was added
during the last 3 hours of culture to accumulate cells in metaphase.
Hypotonic shock was induced by 1% trisodium citrate solution for
10 minutes. Cell suspension was fixed in a mixture of methanol and
glacial acetic acid (v/v 3:1) followed by three washes. Cytogenetic
preparations for analyses of chromosomal aberrations and mitotic
indices were stained with an aqueous solution of Giemsa 1%.
The fluorescence-plus-Giemsa (FPG) technique33 was used for
sister chromatid differentiation (SCD) staining. Slides were stained
for 20 min with Hoechst 33258 (5 mg/ml), mounted in saline
sodium citrate buffer (SSC) 2X powder (A◦nidra Company), and
exposed to “black light” for 20 min at 50 C. Finally, cells were
stained with Giemsa and air-dried for evaluation of the frequency
of cells in their first (M1), second (M2) and third (M3) cell
division.
2-(2¢-Methoxyphenoxy)-1-(3¢¢,4¢¢-dihydroxyphenyl)ethanol (21).
Oil. Yield 85%. dH (200 MHz, CDCl3) 3.80 (3H, s, OMe), 3.89–4.06
(2H, m, H-2a,2b), 4.86–4.93 (1H, m, H-1), 6.79–7.01 (7H, m, H-
3¢,4¢,5¢,6¢, 2¢¢,5¢¢,6¢¢); dC (50 MHz, CDCl3) 56.1 (OMe), 72.4 (C-2),
76.2 (C-1), 113.4 (C-2¢¢), 114.3 (C-5¢¢), 115.6 (C-3¢), 115.8 (C-6¢),
118.6 (C-6¢¢), 121.7 (C-5¢), 122.3 (C-4¢), 134.3 (C-1¢¢), 145.1 (C-3¢¢),
145.5 (C-4¢¢), 149.6 (C-2¢), 150.9 (C-1¢). HR-MS m/z: 276.28969
(M+).
2-(2¢-Methoxyphenoxy)-1-(3¢¢,4¢¢-diacetoxyphenyl)propan-1,3-
diacetate (22). Oil. Yield 70%. dH (200 MHz, CDCl3) 1.98 (3H,
s, OAc), 2.18 (6H, s, OAc), 2.21 (3H, s, OAc), 3.79 (3H, s, OMe),
4.04–4.28 (2H, m, H-3a,3b), 4.37–4.46 (1H, m, H-1), 6.06–6.26
(1H, m, H-3¢), 6.83–7.26 (6H, m, H-2¢¢,4¢,5¢,5¢¢6¢,6¢¢); dC (50 MHz,
CDCl3) 20.4 (OAc), 20.6 (OAc), 20.9 (OAc), 55.7 (OMe), 62.2
(C-3), 73.8 (C-1), 80.1 (C-2), 112.0 (C-3¢), 118.7 (C-6¢), 120.0 (C-
5¢), 120.8 (C-4¢), 120.9 (C-2¢¢), 122.4 (C-5¢¢), 123.8 (C-6¢¢), 135.2
(C-1¢¢), 141.9 (C-4¢¢), 142.0 (C-3¢¢), 147.7 (C-1¢), 151.2 (C-2¢),
167.8 (OCOMe), 169.1 (OCOMe), 170.5 (OCOMe). HR-MS m/z:
474.46531 (M+).
Cell division kinetics was determined by the proliferative
replication index (PRI) according to the formula:34
Biological section
(1 ¥ M1) + (2 ¥ M2) + (3 ¥ M3)
Test compounds. All lignans and derivatives were prepared
immediately before treatment in dimethyl sulfoxide (DMSO)
obtained from Sigma-Aldrich and added to the culture medium
such that the final concentration of solvent did not exceed 1%.
5-Bromo-2¢-deoxyuridine (BrdU) used to evaluate the frequency
of cells in first (M1), second (M2) and third (M3) cell division, and
Colcemid to accumulate cells in metaphase, were also purchased
from Sigma-Aldrich.
PRI =
100
where M1, M2, and M3 are the proportions of first, second and
third generations of mitotic cells respectively.
To evaluate the mitotic index (MI), cytogenetic preparations
stained by Giemsa were analyzed in a light microscope at 400¥
magnification. MI was expressed as number of metaphases per
1000 nuclei analyzed.
Test systems and culture conditions. Chinese hamster V79 cells
were originally obtained from Dr. J. Williamson (British American
Tobacco, UK). The karyotype, generation time, plating efficiency,
and absence of mycoplasmal contamination were checked at
regular intervals. Permanent stocks of V-79 cells were stored in
liquid nitrogen (-170 ◦C) and subcultures are prepared from
these stocks for experimental use. At the end of assay the cells
were discarded and a new ampoule (containing cells from the
stock under liquid nitrogen) was used. In our case experiments on
different compounds have been conducted on different days using
Scoring for chromosomal damage was undertaken blind with
coded slides. A minimum of 100 metaphases per culture were
scored for chromosomal aberrations. Chromosomal aberrations
were classified as chromatid-type gaps, chromatid-type breaks,
chromatid-type exchanges, chromosome-type gaps, chromosome-
type breaks, chromosome-type exchanges and isolocus events
(which include isochromatid and isolocus breaks when these
cannot be distinguished), as described by Savage35.
For the chromosome aberration assay the number of aberration-
bearing cells (excluding gaps) was utilized for statistical analyses.
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To determine the statistical significance Fisher’s exact test was
used. The test substance is considered positive when statistically
significant increases in aberration-bearing cells are observed at two
consecutive dose-levels or at the higher dose-level and exceed the
historical control mean values.
7 For some examples of the effect of cytochrome P450-deppendent
metabolic process on lignans, see: (a) B. K. Sinha, H. M. Eliot and
B. Kalayanaraman, FEBS Lett., 1988, 227, 240; (b) H. B. Niemeyer
and M. Metzler, Anal. Technol. Life Sci., 2002, 777, 321–327.
8 K. K. Wolf, S. G. Wood, J. L. Allard, J. A. Hunt, N. Gorman, B.
W. Walton-Strong, J. G. Szakacs, S. X. Duan, Q. Hao, M. H. Court,
L. L. Von Moltke, D. J. Greenblatt, V. Kostrubsky, E. H. Jeffery, S. A.
Wrighton, F. J. Gonzalez, P. R. Sinclair and J. F. Sinclair, Drug Metab.
Dispos., 2007, 35, 1223–1231.
Conclusions
9 E. L. Cavalieri, K. M. Li, N. Balu, M. Saeed, P. Devanesan, S.
Higginbotham, J. Zhao, M. L. Gross and F. G. Rogan, Carcinogenesis,
2002, 23, 1071–1077.
In this paper we have described a general and selective procedure
for the de-O-methylation of guaiacyl monolignols, lignans and
neolignans to the corresponding catechol derivatives by a modifi-
cation of the Quideau demethylation reaction20 here performed in
an aqueous (green) solvent system. To the best of our knowledge,
this is the first example of an oxidative system able to mimic one of
the activities of cytochrome P-450 enzymes in the metabolic path-
way of the guaiacyl moiety in the cell. Reactions proceeded with
satisfactory conversions of substrate and yield of products, even
for such recalcitrant substrates as neolignans. In some selected
examples, the novel catechol derivatives were also acetylated to
afford the corresponding esters as useful references to compare the
biological activity with parent compounds. The catechols and the
acetylated derivatives were evaluated for the toxicity in comparison
to the parent natural compounds. The results obtained indicate
that the presence of the catechol moiety sharply enhances the clas-
togenic potential, the cytotoxicity and the modulation of cell cycle
progression. In contrast, acetylated derivatives showed a markedly
reduced clastogenic activity, but preserving the cytotoxicity and
the modulation of cell cycle progression. The only exception to
this general trend was the catechol derivative of a-conidendrin,
for which cytotoxicity was not observed, thus conferring the
property to induce a new effect, the enderoduplication process.
Thus, despite the in vitro antioxidant activity usually described for
catechol derivatives, our results show for the first time the gen-
eration of a clastogenic potential (e.g. induction of chromosomal
aberrations), highly indicative of a long-term genetic and cancer
risk.
10 (a) P. Arivazhagan, K. Ramanathan and C. Panneerselvam, Experi-
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