Methyltrioxorhenium catalysed synthesis of highly oxidised aryltetralin lignans with anti-topoisomerase II and apoptogenic activities

Article abstract of DOI:10.1016/j.bmc.2005.07.017

A novel and efficient procedure to prepare highly oxidised aryltetralin lignans, such as isopodophyllotoxone and (-)-aristologone derivatives, by oxidation of podophyllotoxin and galbulin with methylrhenium trioxide (MTO) and novel MTO heterogeneous catal

Full text of DOI:10.1016/j.bmc.2005.07.017

Bioorganic & Medicinal Chemistry 13 (2005) 5949–5960
Methyltrioxorhenium catalysed synthesis of highly oxidised
aryltetralin lignans with anti-topoisomerase II
and apoptogenic activities
b
b
b
Raffaele Saladino,a,b, Cinzia Fiani, Maria Cristina Belfiore, Giampiero Gualandi,
*
Sabrina Pennab and Pasquale Mosessob
aINFM, della Tuscia, via S.Camillo De Lellis, I-01100 Viterbo, Italy
`
Dipartimento A.B.A.C., Universita della Tuscia, via S.Camillo De Lellis, I-01100 Viterbo, Italy
b
Received 10 March 2005; revised 1 July 2005; accepted 8 July 2005
Available online 19 August 2005
Abstract—A novel and efficient procedure to prepare highly oxidised aryltetralin lignans, such as isopodophyllotoxone and (ꢀ)-aris-
tologone derivatives, by oxidation of podophyllotoxin and galbulin with methylrhenium trioxide (MTO) and novel MTO hetero-
geneous catalysts is reported. It is noteworthy that in the case of isopodophyllotoxone derivatives the functionalisation of the
C-4 position of the C-ring and the ring-opening of the D-lactone moiety increased the activity against topoisomerase II while caus-
ing the undesired inhibition of tubulin polymerisation to disappear. The novel (ꢀ)-aristologone derivatives showed apoptogenic
activity against resistant human lymphoma cell lines.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
odophyllotoxin), which have been widely used for the
treatment of small-cell lung cancer, testicular cancer,
lymphoma and acute lymphocytic leukaemia.5 Etopo-
side and teniposide show different side effect profiles
compared to podophillotoxin due to their action as
selective inhibitors of DNA topoisomerase II, a key
enzyme involved in DNA transcription, replication,
recombination and possibly DNA repair.6 In recent
years, several syntheses of podophyllotoxin and
aryltetralin lignan derivatives have been reported in
the literature which mainly focused on structural mod-
ifications of leader molecules to obtain less toxic ana-
logues with high biological activities. On the other
hand, a few data are available on oxidative functionali-
sation,7 a process that plays a relevant role in their
biological mechanism of action. For example, it is
known that etoposide undergoes oxidative ortho-
demethylation by a cytochrome P450-dependent meta-
bolic process to a 30,40-dihydroxy derivative.8 This
derivative is further oxidised to the corresponding
ortho-benzoquinone, a highly reactive intermediate
responsible for the irreversible binding to proteins
and DNA.9 In a similar way, a semibenzoquinone free
radical intermediate of etoposide is responsible for
DNA strand breakage.10 Since general and selective
methods for the oxidation of aryltetralin lignans are
still lacking,11 novel synthetic strategies are needed to
Lignans are a family of natural products with a broad
variety of biological and pharmacological activities.1
Among them, aryltetralin derivatives are of special
interest owing to their powerful antitumoral, antimitot-
ic, antiviral, cardiovascular and immunosuppressive
activity.2 Aryltetralin derivatives show also a selective
nonredox inhibition of 5-lipoxygenase by interaction
with the arachidonic acid binding site and could form
a new class of therapeutic agents for the treatment of
asthma and rheumatoid arthritis.3 Podophyllotoxin 1,
isolated from different plants of the genus Podophyl-
lum, is the most investigated aryltetralin derivative.
It is a well-established inhibitor of cell division at
the level of the microtubule assembly by freezing poly-
merisation of tubulin at the colchicine site.4 This activ-
ity has led to the design of semi-synthetic derivatives,
such as etoposide (40-demethyl-7-[4,6-O-ethylidene-b-D-
gluco-pyranosyl epipodophyllotoxin) and teniposide
(40-demethyl-7-[4,6-O-thenilidene-b-D-glucopyranosyl epip-
Keywords: Lignans; Oxidative functionalisation; Methyltrioxorheni-
um; Topoisomerase II; Apoptogenic activity.
*
Corresponding author. Tel.: + 39 076 13 572 84; fax: + 39 076 13 57
242; e-mail: saladino@unitus.it
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.017

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R. Saladino et al. / Bioorg. Med. Chem. 13 (2005) 5949–5960
prepare highly oxidised derivatives for the evaluation
of their biological activity. A novel catalyst useful for
this purpose is methyltrioxorhenium (MeReO3,
MTO).12 In the last decade MTO, in combination with
H2O2, has been used in several organic trasformations.13
The reactive intermediates for these oxidations are mo-
noperoxo [MeRe(O)2O2] and bis-peroxo [MeReO-
(O)2]g2-rhenium complexes.14 Accordingly, with this
high reactivity, MTO has been used for the oxidation
of phenols and methoxy benzene derivatives.15 In this
latter case, the reaction proceeds through the formation
of epoxide intermediates that are further rearranged and
oxidised to corresponding benzoquinones.16 The efficient
oxidation of several aromatic derivatives to ortho- and
para-benzoquinones has also been reported by use of
novel heterogeneous rhenium compounds based on the
heterogenation of MTO on easily available and low-cost
poly(4-vinylpyridine) 2% cross-linked with divinyl-
benzene (PVP-2/MTO, I) and microencapsulation on
polystyrene (PS-2/MTO, II).13,17
bly, the oxidation of the C-4 position of the C-ring and
the concomitant ring-opening of the D-lactone moiety
enhances the activity against topoisomerase II, while
causing the undesired inhibition of tubulin polymerisa-
tion to disappear. The generality of this procedure was
further shown by the unprecedented synthesis of benzo-
quinone derivatives of galbulin, an aryltetralin lignan
isolated from Galbulimima belgraveana.18 Benzoquinone
derivatives of galbulin were evaluated for apoptogenic
activity against human lymphoma cell lines BL41
(EBVꢀ) prone to apoptosis and E2R (EBV+) which is
strongly resistant to chemical treatment.
2. Chemistry
Initially, the oxidation of aryltetralin derivatives was
investigated using MTO under homogeneous condi-
tions. As a general procedure, podophyllotoxin 1
(1.0 mmol) was added to
a solution of MTO
(0.05 mmol) in the appropriate solvent (vide infra).
Hydrogen peroxide (H2O2, 4.0 mmol, 30% aqueous
solution) was added to the reaction mixture repeatedly
over 24 h period. The results of the oxidations are sum-
marised in Table 1 and Scheme 1.
Here, we report that MTO and MTO heterogeneous cat-
alysts I and II, whose structures are reported in Figure 1,
can be used for the unprecedented one-pot preparation
of highly oxidised isopodophyllotoxone derivatives from
podophyllotoxin using H2O2 as primary oxidant. Nota-
In the absence of the catalyst, less than 2% conversion of
substrate took place under otherwise identical condi-
tions. The reaction performed in acetic acid (AcOH,
5 mL) at 25 ꢁC gave the ortho-benzoquinone of isopodo-
phyllotoxone, compound 2, as the only recovered prod-
uct in 35% yield and quantitative conversion of
substrate (Table 1, entry 1).
MTO
N
MTO
N
In accordance with the high reactivity reported for
MTO in the presence of AcOH, the low-mass balance
with respect to isolated product might be due to the for-
mation of polar hydrophilic over-oxidation products not
recovered by usual work-up procedures.19 Probably, the
reaction involves the formation of highly reactive arene
oxide intermediates that can be further rearranged and
oxidised at both the C- and E-rings.15a ortho-Benzoqui-
none analogues of podophyllotoxin and 40-de-O-meth-
ylpodophyllotoxin (not shown) bearing the quinone
MTO
n
PVP-2/MTO (I)
Figure 1.
PS/MTO (II)
Table 1. Oxidative functionalisation of podophyllotoxin 1 and galbulin 5 with MTO and MTO heterogeneous catalysts I and II
Entry
Solvent
Substrate
T (ꢁC)
t (h)
Catalyst
Conversion (%)
Product(s)
Yield (%)
1
2
CH3COOH
1
1
1
1
1
1
1
5
5
5
20
50
24
24
24
24
66
66
66
24
66
7
MTOa
>96
80
2
35
60
CH2Cl2/CH3CN
CH2Cl2/EtOH
CH2Cl2/EtOH
CH3COOH
MTOa
2
3
20
MTOa
MTOa
>99
>99
>99
>99
>99
44
2
15
71
4
ꢀ10
40
2
5
PVP-2/MTO(I)b,c
PS/MTO(II)b,c
PVP-2/MTO(I)b,c
MTO
2
69
13(4)[62]
65
6
CH3COOH
20
20
2(3)[4]
2
7
CH2Cl2/EtOH
CH2Cl2/CH3CN
CH2Cl2/CH3CN
CH2Cl2/CH3CN
8
20
20
6(7)[8]{9}
6(7)[8]{9}
7(8)[9]
25(17)[21]{28}
36(6)[10]{9}
9(12)[40]
9
10
PVP-2/MTO(I)b,c
PS/MTO(II)b,c
40
65
20
a All the reactions were performed with H2O2 (35% aqueous solution) using 0.05 mmol catalyst and 1.0 mmol substrate.
b Catalysts I and II were prepared adding MTO (1.0 mmol) to a suspension of the resin [poly(4-vinylpyridine) or polystyrene 2% cross-linked with
divinyl benzene; 1.0 g] in ethanol (5 mL) at room temperature.
c All the reactions were performed treating podophyllotoxin 1 and galbulin 5 (1.0 mmol), dissolved in the appropriate solvent, with catalysts I and II
(100 mg, loading factor 1.0) and H2O2 (4.0 mmol).

R. Saladino et al. / Bioorg. Med. Chem. 13 (2005) 5949–5960
5951
O
O
O
O
O
O
O
O
O
O
O
O
OCH3
H3CO
OCH3
OH
7
OCH3
OCH3
5
B
2
9
6
1
4
3
8
O
A
O
3
2
C
D O
9'
catalyst, H2O2 (35%)
solvent
8'
7'
1'
O
2'
3'
6'
O
O
E
5'
O
O
H3CO
OCH3
4'
O
CH3
OCH3
OH
1
O
H3CO
OCH3
OCH3
4
Scheme 1.
moiety on the 30- and 40-positions of the E-ring have
been previously synthesized by oxidative demethylation
with sodium metaperiodate and nitric acid.20 In this
latter case, the presence of a substituent on the C-20 po-
sition of the E-ring was found to stabilise the stereo-
chemistry of products against epimerisation.21 To the
best of our knowledge, this is the first example of synthe-
sis of ortho-benzoquinone derivative of podophyllotoxin
showing the quinone moiety on the 20-, 30- positions of
the E-ring. When the reaction was performed in
CH2Cl2/CH3CN (5 mL, 1:1 v/v) at 50 ꢁC compound 2
was isolated as the only recovered product in 60% yield
and 80% conversion of substrate (Table 1, entry 2). A
better result was observed performing the reaction in
CH2Cl2/EtOH (5 mL, 1:1 v/v) at ꢀ10 ꢁC in which case
compound 2 was recovered in 71% yield (Table 1, entry
4). In contrast, a low selectivity was obtained in CH2Cl2/
EtOH (5 mL, 1:1 v/v) at 25 ꢁC (Table 1, entry 3). Next,
we studied the oxidation of podophyllotoxin 1 with het-
erogeneous catalysts I and II. The reactivity and selec-
tivity of MTO in these catalysts can be tuned by the
chemical–physical properties of the resin used as a sup-
port of the active species.22 Briefly, MTO (1.0 mmol)
was added to the suspension of the appropriate resin
poly(4-vinylpyridine) (PVP-2) or polystyrene (PS) 2%
cross-linked with divinylbenzene, in ethanol (5 mL) at
25 ꢁC. After 1 h, the solvent was removed by filtration
and the catalysts were used without further purification.
Podophyllotoxin 1 dissolved in the appropriate solvent
(vide infra) was added portion wise to catalysts I and
II (100 mg, loading factor 1. The loading factor is re-
ferred as mmol of active species for gram of resin) and
H2O2 (4.0 mmol) at 20–40 ꢁC. At the end of the reaction
catalysts were recovered by filtration. As reported in
Scheme 1, the performance of poly(4-vinylpyridine)/
MTO catalyst I in AcOH at 40 ꢁC was similar to that
observed for MTO in mixed solvents affording com-
pound 2 as the only recovered product in quantitative
conversion of substrate and 69% yield (Table, entry 5
vs entries 2 and 4). Similar results were obtained in
CH2Cl2/EtOH mixture (1:1, v/v) (Table 1, entry 6). Not-
ably, a different reaction pathway was observed with
microencapsulated MTO catalyst II. In this latter case,
the oxidation in AcOH at 25 ꢁC gave the novel acetylat-
ed D-ring opened isopodophyllotoxone derivative 4 as
the main product (62%) beside compound 2 (13%) and
low amount of isopodophyllotoxone 3 (4%) (Table 1,
entry 7).23 Modification of the D-ring of podophyllotox-
in including the ring-opening of the lactone moiety has
been reported in the literature to give products with dif-
ferent degrees of oxidation at positions C-9 and C-90.24
These modifications include the synthesis of the hydra-
zine derivative, GP-11, which is almost equipotent than
etoposide.25 A different selectivity depending on the nat-
ure of the support used for the heterogenisation process
of MTO has been previously observed in the oxidation
of N,N0-disubstituted hydroxylamines to nitrones, and
during the preparation of benzoquinone and c-lactone
derivatives from natural phenols. For example, higher
conversions and yields of benzoquinones were obtained
during the oxidation of substituted hydrogenated carda-
nols (3-n-pentadecylphenols) with catalyst I with respect
to MTO and catalyst II because of a support mediated
molecular recognition process based on hydrogen bond-
ing interactions between the pyridinyl moiety and the
phenolic group of substrate.26 Irrespective of the cata-
lyst used for the oxidation, the lower reactivity of heter-
ogeneous catalysts I and II with respect to MTO (see for
example reaction times in Table 1: entries 5 and 6 vs 1)
was probably due to the presence of a kinetic barrier to
the approach of compound 1 to rhenium polymeric
compounds.
To evaluate the generality of this procedure, galbulin
5 was oxidised with MTO under similar experimental
conditions. Treatment of 5 (1.0 mmol) with MTO

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R. Saladino et al. / Bioorg. Med. Chem. 13 (2005) 5949–5960
(0.05 mmol) and H2O2 (4.0 mmol, 30% aqueous solu-
tion) in CH2Cl2/CH3CN (1:1 v/v, 5 mL) at 25 ꢁC gave
the para-benzoquinone derivatives of (ꢀ)-aristoligone
[(70R,8S,80R)-8,80-dimethyl-30,40,4,5-tetramethoxy-2,70-
cyclolignan-7-one], compounds 6–9, in similar yields
and acceptable conversion of substrate (Table 1, entry
8, Scheme 2). A better result was obtained performing
the reaction with catalyst I in which case para-benzoqui-
none 6 was recovered as the main reaction product in
36% yield (Table 1, entry 9, Scheme 2). Again, a different
selectivity was observed with catalyst II. In this latter
case, the reaction performed under similar experimental
conditions afforded para-benzoquinone 9 as the main
reaction product in 40% yield beside low amount of
compounds 7 and 8 (Scheme 2, Table 1, entry 10).
in metaphase by inhibiting the depolymerisation of
tubulin was also included at 0.2 lg/mL, a very low con-
centration routinely employed in cytogenetic assays to
accumulate cells in a metaphase-like stage. Evaluation
of mitotic indices (number of metaphases out of 1000
cells scored expressed as percentage) was used to assess
the interference of compounds 2–4 with polymerisation
of tubulin during assembly of mitotic spindle apparatus
while analyses of chromosomal aberrations in the G2
phase of cell cycle were used to assess the inhibition of
DNA topoisomerase II. This indirect evaluation of
activity against topoisomerase II is based on the fact
that non catalytic inhibitors of both DNA topoisome-
rases I and II act by stabilising a ternary complex known
as the Ôcleavable complexÕ in which the enzyme is cova-
lently linked to DNA. In the topoisomerase II reaction
both single (SSBs) and double DNA strand breaks
(DSBs) are formed while in the topoisomerase I reaction
only DNA SSBs are generated. These DNA breaks are
therefore produced by an enzyme-mediated process
and being Ôprotein concealed,Õ they can be detected by
DNA filter elution methodology only if the cell lysate
is digested with a proteinase before elution.28 Inhibitors
of DNA topoisomerase II, which give rise to DSBs, the
ultimate lesion responsible for the production of chro-
mosomal aberrations, are able to induce chromosomal
aberrations in all phases of the cell cycle, acting similar
to ionising radiation by a typical ÔS-independentÕ mech-
anism. Furthermore, unlike the majority of ÔS-indepen-
dentÕ agents, they are also able to induce sister
chromatid exchanges (SCEÕs) when the treatment is per-
formed in the S phase of the cell cycle.28b
Low amounts of (ꢀ)-aristologone have been detected in
the roots of Holostylis reniformis and in Aristolochia
species used in Brazilian traditional medicine.27 To the
best of our knowledge there are no data available in
the literature on the synthesis and biological activity of
benzoquinone derivatives of (ꢀ)-aristoligone.
3. Biology
3.1. Activity of isopodophyllotoxone derivatives 2–4
against topoisomerase II
To evaluate the activity and mechanism of action of
isopodophyllotoxone derivatives 2–4, we analysed the
mitotic indices (MI) and the frequencies of chromos-
omal aberrations following a 3 h treatment with four
different dose levels of each compound (selected in pre-
liminary dose-range finder experiments) in the G2 phase
of cell cycle in a Chinese hamster ovary (CHO) cell line.
This cell line is routinely used in mutagenicity testing as
indicated in the guidelines of EEC Council 79/831 and
OECD for the test of chemical No. 471. The positive
control Colcemidꢂ which causes accumulation of cells
MI for the podophyllotoxin and isopodophyllotoxone
derivatives 2–4 are displayed in Figure 2, A–D,
respectively. Tables 2–5 show the incidence of chromo-
somal aberrations induced by phodophillotoxin and
compounds 2–4, respectively. In these latter tables, at
each dose level, the total number of cell scored, the
frequency of chromatid and chromosome deletions,
OH
H3CO
CH3
H3CO
H3CO
CH3
H3CO
CH3
O
CH3
O
O
OCH3
O
OCH3
9
6
A
3
7
5
4
8
1
2
H3CO
H3CO
CH3
OCH3
OCH3
catalyst, H2O2 (35%)
solvent
B
7'
7
6
CH3
9'
8'
1'
O
2'
6'
C
3'
H3CO
H3CO
CH3
H3CO
H3CO
CH3
5'
OCH3
4'
CH3
O
OCH3
CH3
O
HO
5
O
OCH3
O
OCH3
OCH3
OCH3
8
9
Scheme 2.

R. Saladino et al. / Bioorg. Med. Chem. 13 (2005) 5949–5960
5953
A
12
10
8
6
4
2
0
Untreated Colcemid
0.05
0.10
0.25
0.50
mM
B
12
10
8
6
4
2
Cytotoxic
0
Untreated Colcemid
0.05
0.10
0.25
0.50
mM
C
12
10
8
6
4
2
0
Untreated Colcemid
0.05
0.10
0.25
0.50
mM
D
12
10
8
6
4
2
0
Untreated Colcemid
0.05
0.10
0.25
0.50
mM
Figure 2. Mitotic index values (MI) of podophyllotoxin 1 (A), benzoquinone derivative 2 (B), isopodophyllotoxone 3 (C), and isopodophyllotoxone
derivative 4 (D) in a Chinese hamster ovary (CHO) cell line.
exchanges and the percentage of cells bearing aberra-
tions excluding gaps are reported. Statistical significance
calculated on the number of cells bearing aberrations is
also shown.
Podophyllotoxin 1 (Fig. 2A, Table 2) induced marked
and dose-related increases in the MI, compared to the
untreated and positive control (colcemid)-treated cul-
tures. At the high-dose level this increase was 1.74 times

Table 2. Incidence of chromosomal aberrations in CHO cells by podophyllotoxin 1
Treatment
Dose level (mM)
N
Total number of aberration (%)
Chromatid Chromosome
Breaks Exch. Breaks Exch.
F
Aberrant cells (%)
Stat. sig.
Polyploid cells (%)
Relative MI (%)
Untreaded positive control
Colcemid
0.00
0.20
100
100
2
2
0
0
0
0
0
0
0
0
2.0
0.0
NS
—
0
26
2.0
100
Compound 1
Compound 1
Compound 1
Compound 1
0.05
0.10
0.25
0.50
100
100
100
100
1
4
2
5
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
2.0
4.0
2.0
6.0
NS
NS
NS
NS
1.0
2.0
4.0
3.0
105
115
130
173
N: total number of metaphases scored (100 metaphases/culture). F: isolocus events which include isochromatid and isolocus breaks when these cannot be distinguished.
Aberrant cells (%): percentage of cells bearing aberrations (excluding gaps).
Relative MI: mitotic index relative to colcemid-treated culture (percent).
Stat. sig.: statistical significance (test compound-treated culture vs colcemid treated culture). NS: not significant.
Table 3. Incidence of chromosomal aberrations in CHO cells by benzoquinone 2
Treatment
Dose level (mM)
N
Total number of aberration (%)
Chromatid Chromosome
Breaks Exch. Breaks Exch.
F
Aberrant cells (%)
Stat. sig.
Polyploid cells (%)
Relative MI (%)
Untreaded positive control
Colcemid
0.00
0.20
100
100
2
2
0
0
0
0
0
0
0
0
2.0
2.0
NS
NS
0
26
2.0
100
Compound 2
Compound 2
Compound 2
Compound 2
0.05
0.10
0.25
0.50
100
100
0
5
0
0
0
1
0
0
0
0
0.0
6.0
NS
NS
1.0
10.0
134
104
No metaphases found (cytotoxic)
No metaphases found (cytotoxic)
N: total number of metaphases scored (100 metaphases/culture). F: isolocus events which include isochromatid and isolocus breaks when these cannot be distinguished.
Aberrant cells (%): percentage of cells bearing aberrations (excluding gaps).
Relative MI: mitotic index relative to colcemid-treated culture (percent).
Stat. sig.: statistical significance (test compound-treated culture vs colcemid treated culture). NS: not significant.

Table 4. Incidence of chromosomal aberrations in CHO cells by isopodophyllotoxone 3
Treatment
Dose level (mM)
N
Total number of aberration (%)
Chromatid Chromosome
Breaks Exch. Breaks Exch.
F
Aberrant cells (%)
Stat. sig.
Polyploid cells (%)
Relative MI (%)
Untreaded positive control
Colcemid
0.00
0.20
100
100
2
2
0
0
0
0
0
0
0
0
2.0
0.0
NS
—
0
26
2.0
100
Compound 3
Compound 3
Compound 3
Compound 3
0.05
0.10
0.25
0.50
100
100
100
100
0
5
0
0
0
0
0
0
0
0
0
1
1
0
0
3
0
0
0.0
9.0
NS
**
1.0
0.0
4.0
1.0
196
151
191
160
1
10
2.0
10.0
NS
***
N: total number of metaphases scored (100 metaphases/culture). F: isolocus events which include isochromatid and isolocus breaks when these cannot be distinguished.
Aberrant cells (%): percentage of cells bearing aberrations (excluding gaps).
Relative MI: mitotic index relative to colcemid-treated culture (percent).
Stat. sig.: statistically significance (test compound-treated culture vs colcemid treated culture). NS: not significant.
** Statistically at P < 0.05 with FisherÕs exact test.
*** Statistically at P < 0.01 with FisherÕs exact test.
Table 5. Incidence of chromosomal aberrations in CHO cells by isopodophyllotoxone derivative 4
Treatment
Dose level (mM)
N
Total number of aberration (%)
Chromatid Chromosome
Breaks Exch. Breaks Exch.
F
Aberrant cells (%)
Stat. sig.
Polyploid cells (%)
Relative MI (%)
Untreaded positive control
Colcemid
0.00
0.20
100
100
2
2
0
0
0
0
0
0
0
0
2.0
0.0
NS
—
0
26
2.0
100
Compound 4
Compound 4
Compound 4
Compound 4
0.05
0.10
0.25
0.50
100
100
3
20
0
0
0
0
3
0
0
0
6.0
20.0
NS
***
3.0
4.0
106
70
Very few metaphases (cytotoxic)
Very few metaphases (cytotoxic)
25
15
N: total number of metaphases scored (100 metaphases/culture). F: isolocus events which include isochromatid and isolocus breaks when these cannot be distinguished.
Aberrant cells (%): percentage of cells bearing aberrations (excluding gaps).
Relative MI: mitotic index relative to colcemid-treated culture (percent).
Stat. sig.: statistical significance (test compound-treated culture vs colcemid treated culture). NS: not significant.
*** Statistically at P < 0.01 with FisherÕs exact test.

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R. Saladino et al. / Bioorg. Med. Chem. 13 (2005) 5949–5960
the value of colcemid-treated cultures. Alternatively,
podophyllotoxin did not induce significant increases in
the frequency of chromosomal aberrations compared
to control values. Polyploid cells were also observed.
(i) compound 2, characterised by the presence of the
20,30-ortho-benzoquinone moiety on the E-ring and by
the contemporary oxidation of the C-7 benzylic position
on the C-ring, shows a decreased inhibitory activity
polymerisation of tubulin accompanied by a global in-
crease of cytotoxicity. (ii) Isopodophyllotoxone 3, in
which the oxidation of the C-7 benzylic position on
the C-ring is the only oxidative functionalisation, shows
an enhanced inhibitory activity of polymerisation of
tubulin and concurrent generation of anti-topoisomer-
ase II activity. (iii) Finally, the oxidation of the C-7 ben-
zylic position, the concomitant ring-opening of the
D-lactone moiety and the acetylation of the newly
formed hydroxyl group further enhance the activity of
compound 4 against topoisomerase II causing at the
same time a significant decrease in the inhibitory activity
against tubulin polymerisation. To the best of our
knowledge, this is the first example reported in the liter-
ature of a isopodophyllotoxone derivative characterised
by a significant anti-topoisomerase II activity.
Compound 2 showed a cytotoxic effect at the two high-
er-dose levels as indicated by the absence of mitotic
activity. At lower-dose levels an increase in the mitotic
index values similar to podophyllotoxin was observed
(Fig. 2B, Table 3).
On the contrary, treatments with compound 3 induced
significant increases in the frequency of chromosomal
aberrations compared to control cultures and elevated
values of mitotic indices not related to the dose levels
which in some cases were even higher than podophyllo-
toxin ones (Fig. 2C, Table 4).
Notably, compound 4 induced marked and dose-related
decreases of mitotic indices compared to control values.
At the two higher-dose levels employed, mitotic indices
reached 25% and 47% of control values indicating a
clear cytotoxic effect. At immediate lower-dose level,
mitotic index value reached 68% of control one and sig-
nificant increases in chromosomal aberrations (p < 0.01)
were observed at this dose level. Such a result indicates
that the mechanism of action of compound 4 is different
compared to the podophyllotoxin . In fact, in this latter
case the polymerisation of tubulin into microtubule ap-
pears not be influenced as evinced by decreased mitotic
index values. Alternatively, a new plausible antitopois-
merase II activity appears to be generated as evinced
by induction of chromosomal aberrations (Fig. 2D, Ta-
ble 5). The profile of results on MI and chromosomal
aberrations for compound 4 is influenced by general
cytotoxic effects. A possible buffering effect on tubulin
can be excluded by the observation of very few metapha-
ses (cytotoxic effects) at dose levels higher than 0.1 mM
(Table 5).
4. Apoptogenic activity of (ꢀ)-aristologone derivatives
6–9 against human lymphoma cells lines, BL41 (EBVꢀ)
and E2R (EBV+)
Lignans are a promising class of apoptogenic com-
pounds. For example, NDGA, epiashantin and arctige-
nin causes mitochondrial dependent apoptosis in
colorectal tumor cells.29 Lignans are inhibitors of 50-
lipooxygenase LOX (e.g., NDGA) and generally of
arachidonic acid metabolism whose modulation or
inhibition has dramatic effect on survival/apoptosis of
cancer cells. The choice of the cell line utilised to assay
the toxicity of these compounds seems to be of great rel-
evance. In fact, the extent of apoptosis resistance, which
largely differs among the cell lines, may lead to mis-in-
terpretation of the results. So the utilisation of a couple
of isogenic cell lines, which differ for apoptosis resis-
tance, may give a more complete information about tox-
icity and may be of great interest to identify new
compounds capable of inducing apoptosis in resistant
On the basis of these results it is possible to define the
following qualitative structure–activity relationships:
BL41
E2R
A
B
70
60
50
40
30
20
10
0
70
5
6
7
8
9
5
6
7
8
9
60
50
40
30
20
10
0
0
20
40
60
80
100
120
0
20
40
60
80
100
120
µM
µM
Figure 3. Apoptogenic activity of galbulin 5 and (ꢀ)-aristologone derivatives 6–9 against human lymphoma cell line BL41(EBVꢀ) (A) prone to
apoptosis and strongly resistant cells E2R (EBV+) (B).

R. Saladino et al. / Bioorg. Med. Chem. 13 (2005) 5949–5960
5957
cell lines. In this light,30 galbulin 5, and (ꢀ)-aristologone
derivatives 6–9 were tested on human lymphoma cell
line BL41(EBVꢀ) prone to apoptosis and on the con-
verted ones E2R (EBV+) which is strongly resistant.31
Galbulin 5, and compounds 6 and 8 resulted to be weak
inducers of apoptosis only in the sensitive cell line
(Fig. 3A and B). Notably, compounds 9 and 7 resulted
to be strongly apoptogenic both on sensitive and apop-
tosis resistant cell lines, being 7 more effective on E2R.
Thus, the highest apoptogenic activity was observed in
(ꢀ)-aristologone derivatives 9 and 7 characterised by
both the para-benzoquinone moieties on the C-ring
and by the oxidation of the benzylic C-7 position of
the B-ring to ketone or hydroxyl moiety. On the con-
trary, the oxidation of the benzylic C-1 position of the
B-ring was not important for the apoptogenic activity.
genic activity. The highest activity was observed when
the benzylic C-7 position on the B-ring was also oxidised
as, for example, in the case of compound 7. Noteably,
the oxidation of the benzylic C-70 position in 8 does
not enhance the biological activity, showing that the site
of the oxy-functionalisation on the B-ring is of relevant
importance for the apoptogenic properties of these
derivatives.
6. Experimental
1H NMR and 13C NMR spectra were recorded on a
Bruker AM 400, or Bruker 200 spectrometers. Mass
spectroscopy (MS) was performed with a GC Shimadzu
GC-17A and a mass-selective detector QP 6000. All sol-
vents were of ACS reagent grade, and were redistilled
and dried according to the standard procedures. Chro-
matographic purifications were performed on columns
packed with Merck silica gel 60, 230–400 mesh for flash
technique. Thin layer chromatography was carried out
using Merck platten Kieselgel 60 F254.
The compounds, at the tested doses, did not show the
capability to producing necrosis, using the trypan blue
test (data not shown) hence stressing the notion that
the strong apoptogenic activities on human lymphoma
cell lines are specific.
6.1. Starting materials
5. Conclusions
Podophyllotoxin 1 and MTO were commercial availa-
bles (Aldrich). Galbulin 5 was prepared as previously
reported.33 Dimethylsulfoxide (DMSO) of spectroscopic
grade was obtained from Fluka AG (Switzerland). Col-
cemid (Gibco BRL) was used at a final concentration of
0.2 lg/mL. Poly(4-vinyl pyridine)/MTO I and polysty-
rene/MTO II catalysts were prepared as previously
reported.17 In summary, to a suspension of 600 mg of
the appropriate resin in 4 mL of ethanol (tetrahydrofu-
ran in the case of polystyrene) 77 mg (0.3 mmol)
MTO, was added and the mixture was stirred for 1 h
using a magnetic stirrer. Coocervates were found to
envelop the solid core dispersed in the medium and
5.0 mL hexane added to harden the capsule walls. The
solvent was removed by filtration, and the solid residue
was washed with ethyl acetate and finally dried under
high vacuum. In each case, MTO had completely be-
come bound to the polymer. This result was confirmed
by spectroscopic analysis of the residue obtained after
evaporation of the organic layers. Catalysts were used
without any further purification.
MTO, poly(4-vinylpyridine)/MTO I and microencapsu-
lated polystyrene/MTO II were efficient catalysts for
the functionalisation of podophyllotoxin 1 and galbulin
5 with H2O2 as primary oxidant. Irrespective of experi-
mental conditions used for the transformations, both
aryltetralin derivatives were oxidised at the correspond-
ing benzylic positions and on the aromatic moieties. In
the oxidation of 1, the ortho-benzoquinone 2 produced
by de-alkylation of one methoxy substituent and con-
temporary oxygen atom insertion on the benzylic C-7
position was obtained as the only recovered product
with MTO and catalyst I. The products of C-7 benzylic
oxidation and ring-opening of the lactone on the D-ring,
compounds 3 and 4, were isolated in the presence of cat-
alyst II. para-Benzoquinones 6–9, with a different degree
of oxidation at the benzylic positions, were synthesized
by oxidation of 5 both with MTO and I.
From the biological point of view, the presence of the
ortho-benzoquinone moiety in 2 does not increase the
activity against topoisomerase II. On the contrary, the
D-ring opened derivative 4 showed the highest biologi-
cal activity as evaluated by mitotic index and aberration
studies, with isopodophyllotoxone 3 possessing an inter-
mediate behaviour. The anti-topoisomerase II activity of
4 is noteworthy. It is known from the literature that
some cyclolignan derivatives lacking the lactone moiety
are potent inhibitors of topoisomerase II.24c In accor-
dance with the hypotheses suggested to rationalise this
behaviour,32 the significant activity of 4 could be
accounted for in terms of a spontaneous or enzyme-
mediated transformation of the acetylated alcoholic
moiety into a more reactive electrophilic intermediate
that was able to react with nucleophiles of biomolecules.
In the case of (ꢀ)-aristologone derivatives 6–9, the pres-
ence of the para-benzoquinone moiety on the C-ring was
an important structural property to increase the apopto-
6.2. Oxidation of podophyllotoxin 1 and galbulin 5
6.2.1. Homogeneous oxidation. General procedure. A
10 mL reaction flask was charged sequentially with sub-
strate (1.0 mmol), the appropriate solvent (5 mL; AcOH,
CH2Cl2/CH3CN 1:1 v/v, CH2Cl2/EtOH 1:1 v/v), MTO
(0.05 mmol) and H2O2 (35% aqueous solution,
4.0 mmol). The stirred solution became yellow due to
formation of peroxo species. The mixture was stirred
at the appropriate temperature (see Table 1) until no
more starting material could be detected on TLC. The
mixture was extracted with CH2Cl2 (3· 50 mL), dried
over Na2SO4, and then concentrated. The crude was
purified by preparative TLC (CH2Cl2/MeOH 9.7:0.3)
and by flash chromatography. Products were identified

5958
R. Saladino et al. / Bioorg. Med. Chem. 13 (2005) 5949–5960
1
by their H NMR, 13C NMR, MS and elemental analy-
ses, and by comparison with authentic sample (in the
case of isopodophyllotoxone).
113.39 (C), 112.23 (PhH), 61.01 (CH3O), 60.82
(CH3O), 55.89 (CH3O), 55.7 (CH3O), 44.97 (CH),
37.16 (CH), 35.41 (CH), 18.57(CH3), 14.42(CH3); m/z
(EI) 386. (M+). Anal. Calcd for C22H26O6: C, 68.38;
H, 6.78. Found: C, 68.28; H, 6.75.
6.2.2. Heterogeneous oxidation. General procedure. To
the solution of the substrate (1.0 mmol) in AcOH or
CH2Cl2 /EtOH 1:1 v/v (5.0 mL) were added catalysts I
and II (100 mg, loading factor 1) and H2O2 (4.0 mmol)
at 25–50 ꢁC. The mixture was stirred until no more start-
ing material could be detected on TLC. The suspension
was filtered off, and the recovered catalyst was washed
with CH2Cl2. The crude was purified by preparative
TLC (CH2Cl2/MeOH 9.7:0.3) and by flash chromatog-
raphy. Products were identified by 1H NMR, 13C
NMR, MS and elemental analyses, and by comparison
with authentic sample (in the case of isopodo-
phyllotoxone).
6.2.7. Benzoquinone 7. dH (CDCl3): 6.98 (1H, s, PhH),
6.92 (1H, s, PhH), 5.18 (1H, s, PhH), 4.51 (1H, br s,
OH), 3.84 (3H, s, OCH3), 3.81 (3H, s, OCH3), 3.66
(3H, s, OCH3), 3.62 (1H, m, CH), 3.39 (3H, s, OCH3),
2.8 (1H, m, CH), 1.84–1.4 (2H, m, CH2), 1.2 (3H, d,
J = 6.55 Hz, CH3), 0.85 (3H, d, J = 6.42 Hz, CH3). dC
(CDCl3): 183.46 (C@O), 177.41 (C@O), 149.94 (C),
147.98 (C), 147.78 (C), 145.23 (C), 144.21 (C), 129.88
(C), 129.36 (C), 129.18 (PhH), 113.66 (PhH), 112.01
(PhH), 71.58 (CH), 61.01 (CH3O), 60.82 (CH3O), 55.9
(CH3O), 44.84 (CH), 43.67 (CH), 34.99 (CH), 14.82
(CH3), 14.29 (CH3); m/z (EI) 402. (M+). Anal. Calcd
for C22H26O7: C, 65.66; H, 6.51. Found: C, 65.59; H,
6.50.
6.2.3. Benzoquinone 2. dH (CDCl3): 7 (1H, s, PhH), 6.25
(1H, s, PhH), 6.13 (1H, s, PhH), 6 (2H, m, CH2), 4.9
(1H, d, J = 6.4 Hz, CH), 4.8–4.5 (2H, m, CH2), 4.0
(6H, s, OCH3), 3–2.6 (2H, m, CH). dC (CDCl3): 207
(C@O), 183.95 (C@O), 182.61 (C@O), 174.24 (C),
149.44 (C), 147.47 (C), 145.37 (C), 144.07 (C), 134.44
(C), 132.65 (C), 128.58 (PhH), 128.25 (C), 108.77
(PhH), 106.04 (PhH), 101.62 (CH2), 71.63 (CH2), 61.28
(CH2), 60.97 (OCH3), 43.81 (CH), 41.70 (CH), 33.57
(CH); m/z (EI) 412 (M+). Anal. Calcd for C21H16O9:
C, 61.17; H, 3.91. Found: C, 61; H, 3.89.
6.2.8. Benzoquinone 8. dH (CDCl3): 6.96 (1H, s, PhH),
6.68 (1H, s, PhH), 5.24 (1H, s, PhH), 4.38 (1H, br s,
OH), 3.87 (3H, s, OCH3), 3.83 (3H, s, OCH3), 3.64
(3H, s, OCH3), 3.39 (3H, s, OCH3), 2.92–2.5 (2H, m,
CH2), 1.5 (2H, m, CH), 1.23–1.20 (3H, d, J = 6.2 Hz,
CH3), 1.09–1.05 (3H, d, J = 6.0 Hz, CH3). dC (CDCl3):
193.52 (C@O), 192.9 (C@O), 155.35 (C), 147.9 (C),
147.62 (C), 138 (C), 134.15 (PhH), 127.95 (C), 113.68
(PhH), 109 (PhH), 79.92 (C), 73.78 (CH), 73.56 (CH),
57.41 (CH3O), 55.98 (CH3O), 55.73 (CH3O), 45.27
(CH), 36.75 (CH2), 34.38 (CH), 19 (CH3), 12.46
(CH3); m/z (EI) 404. (M+). Anal. Calcd for C22H28O7:
C, 65.33; H, 6.98. Found: C, 65.28; H, 6.95.
6.2.4. Isopodophyllotoxone 3. dH (CDCl3): 7.095 (1H, s,
PhH), 6.49 (1H, s, PhH), 6.35 (2H, s, PhH), 5.95 (2H,
m, CH2), 4.59 (2H, m, CH2), 4.08 (1H, m, CH), 3.79
(9H, s, OCH3), 2.81–2.75 (2H, m, CH). dC (CDCl3):
188 (C@O), 173 (C@O), 153 (C), 152.7 (C), 152.2(C),
147.8 (C), 140.36 (C), 137.55 (C), 133.9 (C), 128 (C),
110.2 (PhH), 107.5 (PhH), 105.5 (PhH), 102.2 (CH2),
66.8 (CH2), 60.6 (CH3O), 56.1 (CH3O), 46.5 (CH),
44.5 (CH), 43.3 (CH); m/z (EI) 412 (M+). Anal. Calcd
for C22H20O8: C, 64.07; H, 4.89. Found: C, 63.98; H,
4.79.
6.2.9. Benzoquinone 9. dH (CDCl3): 6.71 (1H, s, PhH),
6.62 (1H, s, PhH), 5.63 (1H, s, PhH), 3.86 (3H, s,
OCH3), 3.84 (3H, s, OCH3), 3.82 (3H, s, OCH3), 3.67
(3H, s, OCH3), 3.55 (1H, m, CH), 2–1.9 (2H, m, CH),
1.0 (3H, d, J = 6.45 Hz, CH3), 0.89 (3H, d, J = 6.33
HZ, CH3). dC (CDCl3): 201.49 (C@O), 183.46 (C@O),
177.41 (C@O), 149.94 (C), 149.02 (C), 145.23 (C),
144.21 (C), 133.6(C), 129.8 (PhH), 129.26 (C), 113.9
(CH), 110.77 (PhH), 61.01 (CH3O), 60.82 (CH3O),
55.89 (CH3O), 55.65 (CH3O), 44.2 (CH), 42.67 (CH),
35.55 (CH), 14.68 (CH3), 14.36 (CH3); m/z (EI) 400.
(M+). Anal. Calcd for C22H24O7: C, 65.99, H, 6.04.
Found: C, 65.87; H, 6.0.
6.2.5. Isopodophyllotoxone derivative 4. dH (CDCl3): 6.85
(1H, s, PhH), 6.53 (1H, s, PhH), 6.24 (2H, s, PhH), 5.96
(2 H, m, CH2), 4.65 (1 H, m, CH2), 4.33 (1H, m, CH2), 4
(1H, m, CH), 3.83 (9H, m, OCH3), 3–2.80 (2H, m, CH),
2.10 (3H, s, COOCH3). dC (CDCl3): 197 (C@O), 174.12
(COOH), 170.55 (C), 152.67 (OCH3), 147.43 (C), 134.51
(C), 132.88 (C), 127.79 (C), 110.19 (PhH), 109.05 (PhH),
108.12 (PhH), 101.65 (PhH), 68.08 (C@O), 67.42 (CH2),
60.71 (CH3O), 60.35 (CH3O), 56.23 (CH3O), 43.79
(CH), 41.47 (CH), 36.71 (CH); m/z (EI) 472 (M+). Anal.
Calcd for C24H24O10: C, 61.01; H, 5.12. Found: C,
59.98; H, 5.12.
6.3. Biological assay
6.3.1. Evaluation of mitotic index and anti-topoisomerase
II activity. The parent compound podophyllotoxin 1
and isopodophyllotoxone derivatives 2–4 were dissolved
in dimethylsulfoxide (DMSO). Stock solutions were pre-
pared at 5.0 mM and kept frozen at ꢀ20 ꢁC. For treat-
ment of cultures, 50 lL of appropriately diluted
solutions were added to 5 mL culture such that the final
concentration of solvent did not exceed 1%.
6.2.6. Benzoquinone 6. dH (CDCl3): 6.95 (1H, s, CH), 6.7
(1H, s, CH), 5.24 (1H, s, CH), 3.87 (3H, s, CH3), 3.83
(3H, s, CH3), 3.62 (3H, s, CH3), 3.52 (3H, s, CH3),
3.15 (1H, m, CH), 2.65–2.1 (2H, m, CH2), 1.8–1.5
(2H, m, CH), 1.1 (3H, d, J = 7.6 Hz, CH3), 0.85 (3H,
d, J = 6.2 Hz, CH3). dC (CDCl3): 183.46 (C@O),
177.41 (C@O), 149.94 (C), 147.36 (C), 147 (C), 145.23
(C), 144.21 (C), 130.59 (C), 129.18 (PhH), 129.12 (C),
6.3.1.1. Cell cultures and media. Chinese hamster ova-
ry (CHO) cells used in this work were obtained from
Prof. A.T. Natarajan (State University of Leiden, The

R. Saladino et al. / Bioorg. Med. Chem. 13 (2005) 5949–5960
5959
Netherland). This cell line derives from the CHO
isolated from an explant of the ovary of the Chinese
hamster (Cricetulus griseus, 2n = 22) originally described
by Kao and Puck.34 The Chinese hamster ovary (CHO)
cell line is particularly useful for this kind of studies be-
cause of its stable karyotype (modal number is 21 chro-
mosomes), short cell cycle (12–14 h) and its high plating
efficiency.
For morphological analysis of apoptogenic activity,
2–6 · 105 cells were fixed in 4 % (v/v) paraformeldehyde
and stained with a solution (0.2 lg/mL) of 4,6-diamino-
2-phenyl-indole (DAPI). Apoptosis was quantified by
scoring cells with condensed and fragmented nuclei,
according to Ghibelli et al.36 major than 500 cells in ran-
dom fields were scored using fluorescent microscopy.
Permanent stocks of CHO cells are stored at ꢀ163 ꢁC
under liquid nitrogen and subcultures are prepared from
these stocks for experimental use. Cultures are grown as
monolayer cultures in HamÕs F-10 medium (Gibco
BRL) supplemented with 15% foetal bovine serum,
4mM L-glutamine, and the antibiotics penicillin
(50 IU/mL) and streptomycin (50 lg/mL). All incuba-
tions are at 37 ꢁC in a 5% carbon dioxide (CO2) atmo-
sphere (100% humidity nominal).
Acknowledgment
Italian Murst, cofin 2003 ÔLa catalisi dei metalli di
transizione nello sviluppo di strategie sintetiche inno-
vative di eterocicliÕ is acknowledged for financial
support.
References and notes
6.3.1.2. G2 treatment of cells. Approximately, 24–30 h
before treatment exponentially growing cells were de-
tached by trypsin action and an appropriate number
of 25 sq cm plastic cell culture flasks containing 5 mL
complete culture medium was individually inoculated
with 3.0 · 105 cells.
1. (a) For example of recent reviews on lignans, neolignans
and related compounds see: Ward, R. S. Nat. Prod. Rep.
1999, 16, 75; (b) Ayres, D. C.; Loike, J. D. Lignans.
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Nat. Prod. Rep. 1997, 104, 43.
2. (a) Bohlin, L.; Rosen, B. Drug Discovery Today 1996, 1,
343; (b) Buss, A. D.; Waigh, R. D. In Natural Product as
leads for New Pharmaceuticals; Wolff, M. E., Ed.; John
Wiley & Sons: New York, 1995.
Cultures were treated according to the following experi-
mental schedule: (i) Three hours incubation with podo-
phyllotoxin
1 or individually isopodophyllotoxone
3. Delorme, D.; Ducharme, C.; Brideau, C.; Chan, C.-C.;
`
derivatives 2–4 at 0.05, 0.1, 0.25 and 0.50 mM. (ii) Three
hours pre-treatment with colcemid at 0.2 lg/mL which
served as positive control for inhibition of cell division
interfering with the microtubule assembly by freezing
polymerisation of tubulin. Untreated and solvent treated
controls were also included. At the end of treatment cul-
tures were trypsinised, and hypotonic treatment (KCl
0.075 M) and fixation were carried out according to stan-
dard procedures. Air-dried preparations were stained
with Giemsa (3%) for 5 min. For each experimental point
100 metaphases were scored for chromosomal aberra-
tions and were classified according to the description of
Savage.35 The mitotic index was expressed in percentage
based on the number of metaphases present after a total
of 1000 cells scored (interphases and metaphases).
Chareut, N.; Desmarais, S.; Dube, D.; Falgueyret, P.;
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6.3.1.3. Evaluation of results. For the chromosome
aberration assay the number of aberration-bearing cells
(excluding gaps) was utilised for statistical analyses. To
determine the statistical significance FisherÕs exact test
was used. The test compounds were considered positive
when statistically significant increases in aberration-
bearing cells compared with the concurrent solvent con-
trols were observed at two consecutive dose levels or the
higher-dose level end exceeded the historical control
mean values.
6.3.2. Evaluation of apoptogenic activity of galbulin 5 and
(ꢀ)-aristologone derivatives 6–9. Human lymphoma cell
lines BL41(EBVꢀ) and E2R (EBV+) were seeded at
conc 3 · 105cells/mL and the compounds 5–9 were add-
ed during 18 h. Dimethylsulphoxide (DMSO) used
to solubilise the compounds never excedeed 0.5% v/v.
9. Kalayanaraman, B.; Nemec, J.; Sinha, B. K. Biochemistry
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