Synthesis and cytotoxicity of hydrophobic esters of podophyllotoxins

Article abstract of DOI:10.1016/j.bmcl.2003.12.039

Diverse norbornenecarboxylate esters of podophyllotoxin and its epimers and diastereoisomers have been prepared through Diels-Alder cycloaddition by treating the dienophilic acrylates of cyclolignans with cyclopentadiene. Their cytotoxicities against seve

Full text of DOI:10.1016/j.bmcl.2003.12.039

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1283–1286
Synthesis and cytotoxicity of hydrophobic esters of
podophyllotoxins
J. L. Lopez-Perez,a,* E. del Olmo,a B. de Pascual-Teresa,b A. Abadc
and A. San Felicianoa
a
´
Dpto. de Quımica Farmaceutica, Universidad de Salamanca, 37007 Salamanca, Spain
´
Dpto. de Quımica Organica y Farmaceutica, Universidad San Pablo, Madrid, Spain
b
´
´
Dpto. de Quımica, Facultad de Ciencias, Universidad de Los Andes, Merida, Venezuela
c
´
´
Received 30 July 2003; revised 30 October 2003; accepted 9 December 2003
Abstract—Diverse norbornenecarboxylate esters of podophyllotoxin and its epimers and diastereoisomers have been prepared
through Diels–Alder cycloaddition by treating the dienophilic acrylates of cyclolignans with cyclopentadiene. Their cytotoxicities
against several cancer cell lines have been evaluated and the results compared with those found for other lignan esters. Podo-
phyllotoxin adducts showed a one-fold increase in activity when compared to the natural product. The preparation of more
hydrophobic esters, which showed less cytotoxicity, demonstrated that this activity is not primarily due to the lipophilic factor, but
mainly to the spatial arrangement of the bulky moiety, which could contribute to increase the binding to the target site.
# 2003 Elsevier Ltd. All rights reserved.
Currently, etoposide (1a) is one of the most prescribed
anticancer drugs, with good clinical perspectives against
several types of cancer.1 Etopophos (1b) is a new eto-
poside phosphate designed to overcome the limitations
associated with the poor solubility of etoposide.2 The
introduction of these compounds into the market, is a
good example of the development of therapeutically
useful drugs derived from a natural product, such as
podophyllotoxin (2a) in this case. NK6113 (1c) and
GL331 (1d) are two related derivatives which are pre-
sently under clinical trials.4 GL331 has a promising
potential in the treatment of gastric carcinoma, colon
cancer and non-small-cell lung cancer.5 1a and 1b, two
40-demethylepipodophyllotoxin derivatives, are potent
inhibitors of DNA topoisomerase II, while podophyllo-
toxin acts by a different cytotoxic mechanism, primarily
inhibiting tubulin polymerization through its interaction
at the colchicine binding site (Fig. 1).
In addition, podophyllotoxin and all of its related dis-
play the typical adverse effects common to most anti-
neoplastics, inducing anemia, hair loss and severe
gastrointestinal disturbances. Consequently, rather than
more potent antimitotics of this type, it would be most
interesting to discover more selective drugs with less
adverse effects and, if possible, displaying an even better
affinity for the colchicine binding site. These substances
could lead to therapeutical agents that could work by
tubulin polymerization inhibition or, much better, by
inhibition of both tubulin and DNA–topoisomerase II.
With this objective in mind, we have prepared several
bulky esters of podophyllotoxin and epipodophyllo-
toxin with the aim of adding an extra binding group
that could improve the affinity for the target.
It is widely accepted from structure–activity studies in
the field of podolignans that the trans-lactones are more
potent antineoplastics than the cis-lactones.7 Never-
theless, we have prepared not only trans-lactones, but
also several cis-lactone derivatives aiming to study the
efficacy of the different epimers and diastereoisomers as
chiral auxiliaries of dienophiles in the control of the
stereochemistry during the Diels–Alder reaction
(Scheme 1) used for the synthesis of these derivatives.
The results of these chemical studies will be published in
the near future.
Despite intensive efforts, which include research for
better podophyllotoxin derivatives, no substance has
been found that can outperform the natural product in
its efficacy for the inhibition of microtubule assembly.6
Keywords: Podophyllotoxin; Lignans; Norbornenecarboxylate esters.
* Corresponding author. Tel.:+34-923-294528; fax:+34-923-294515;
e-mail: lopez@usal.es
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.039

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J. L. Lopez-Perez et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1283–1286
1284
Figure 1. Lignans in the market or under clinical trials.
Scheme 1. Synthesis of acrylate and norbornenecarboxylate esters of podolignans.
In accordance with previously published results,7 all cis-
lactone derivatives described in this paper displayed less
activity than the corresponding trans-lactones. The
esters of podophyllotoxin8 and epipodophyllotoxin9 at
the C-7 or C-40 (previously demethylated) hydroxyl
groups reported in the literature, are less potent than
the parent lignan alcohols. Similar results were obtained
by us when a number of similar derivatives were pre-
pared (Scheme 2). When norbornenecarboxylates were
obtained from the acrylate trans-lactone dienophiles,
however, there was a one-fold increase in the cytotoxic
potency with respect to the parent alcohols.
resin. The other three stereoisomers (2b, c and d) were
obtained from podophyllotoxin by simple transform-
ations: picropodophyllin (2b) by treatment of 2a with
methanolic KOH; epipodophyllotoxin (2c) by treatment
with acid10 and epipicropodophyllotoxin (2d) by treat-
ment with acid followed by methanolic KOH. The
acrylic esters (3a, b, c and d) were prepared by treatment
of the corresponding alcohols with acryloyl chloride in
the presence of triethylamine. The esters 3, were then
treated with cyclopentadiene affording the adducts 4
and 5. Cyclopentadiene was prepared by cracking of
dicyclopentadiene and used immediately.11 It has been
mentioned that the use of a Lewis acid catalyst in the
Diels–Alder cycloaddition usually increases the rate of
the endo/exo selectivity by controling the conformation
of the transition state.12 With this idea in mind, several
Lewis acids were used, but no satisfactory results were
obtained in any of the cases. When a recently distilled
The synthesis of the proposed bulky lipophilic esters has
been achieved through Diels–Alder cycloaddition of
cyclopentadiene to several podophyllotoxin related
acrylates as it is summarized in Scheme 1. The starting
material 2a was isolated from commercial podophyllin
Scheme 2. Acylation of podophyllotoxin and epipodophyllotoxin.

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1285
solution of BF3OEt2 was carefully added at ꢀ78 ꢁC in an
inert atmosphere, no reaction was observed, whereas if the
temperature was slowly increased, an intense coloration
appeared, giving place to a mixture of products that could
not be resolved. When other catalysts such as SnCl4, TiCl4,
AlCl3 or ZnCl3 were added at ꢀ78 ꢁC, the reaction did not
take place and it was necessary to increase the temper-
ature up to 0 ꢁC to obtain the desired adducts. In this
case, however, no increase in the rate of the endo/exo
selectivity was observed with respect to the reaction in
thermal conditions. For this reason, all the cycloaddi-
tion reactions described in this work, were finally per-
formed at ꢀ10 ꢁC, in absence of catalyst.
Table 1. Cytotoxicity of lignans esters against four neoplastic cell
lines. (Etoposide and podophyllotoxin as reference compounds. Data
are expressed as IC50 nM values)
Compd
P-388
A-549
HT-29
MEL-28
1a*
2a*
3a
4a
5a
170
12
21
5
170
12
21
4
1700
24
21
4
850
—
21
4
5
4
4
4
6a
7a
190
24
190
48
190
48
190
48
8a
9a
10a
2b
47
94
94
94
184
454
60
184
454
60
184
454
60
184
454
—
3b
55
19
53
19
55
19
53
19
1930
188
9182
The determination of the diastereomeric excess of the
four Diels–Alder reactions was performed by HPLC
analysis. The complete chemistry results will be published
elsewhere. In the four cases, the two endo compounds
were the major products while the two exo compounds
appeared in proportion less than 0.5% of the crude
reaction products. NMR studies of the diastereoisomeric
pairs obtained from the four Diels–Alder reactions,
showed that the major compounds were the endo
adducts, in agreement with theoretical predictions. The
mixture of the endo stereoisomers 4a and 5a was
resolved by flash chromatography. The identification of
every compound of each diastereoisomeric pair was
achieved on the basis of specific rotation studies.13
4b+5b
7b
8b
9b
2c
1930
188
4591
6000
213
1122
2900
213
468
1930
188
9182
6000
213
1122
2900
213
468
1930
188
9182
6000
213
1122
2900
213
468
3c
213
1122
—
213
468
4c+5c
2d
3d
4d+5d
is quite obvious and can be justified by their close simi-
larity or identity in lipophilicity, configuration at C-7,
global conformation and spatial distribution, in spite of
the different configuration at position C-5 of the nor-
bornene moiety, with the additional assumption of free
rotation around the CO–norbornene bond.
The preparation of podophyllotoxin esters 6a–10a was
carried out with good yield, by treatment of podophyllo-
toxin, suspended in methylene dichloride, with the corres-
ponding acid chloride (heptanoic, benzoic, phenylacetic,
trans-cinnamic and norbornylacetic acids) in the presence
of pyridine. Nevertheless, in the case of the preparation
of epipodophyllotoxin esters (6b–10b), due to the axial
disposition of the hydroxyl group, better results were
achieved when using the Hassner14 procedure, in which,
epipodophyllotoxin is treated with the corresponding
free acid (heptanoic, phenylacetic, trans-cinnamic and
norbornylacetic) in presence of 1,3-dicyclohexylcarbodi-
imide (DCC) and 4-dimethylaminopyridine (4-DMAP).
In conclusion, the introduction of linear or aromatic
acyl moieties at the C-7-OH of podolignans, generally
induces either a loss of, or no effect upon, the cytotoxic
activity of the parent hydroxylic derivatives. Never-
theless, when the acyl residue contains a norbornene
fragment, almost a one-fold increase of activity is
observed in the case of trans-lactones. This improve-
ment of activity is more significant in the case of podo-
phyllotoxin norbornene derivatives. The introduction of
one additional methylene unit between the bicylic sys-
tem and the carboxyl group (2-norborneneacetate of
cyclolignan, 10a) that would increase the lipophicity
and the volume virtually occuped by the substituent, led
to a diminished activity. These findings indicate that the
main responsibility for the cytotoxicity change is not
due to the lipophilic factor, but to the precise spatial
arrangement of the bulky moiety, which may also con-
tribute with additional non-polar interactions to
enhance the binding to the target site.
Cytotoxicities of all of the compounds prepared were
tested in vitro following an adapted procedure of the
method described by Bergeron15 against different neo-
plastic cell lines: P-388 (suspension culture of a lymphoid
neoplasm from DBA/2 mouse), A-549 (monolayer culture
of human lung carcinoma), HT-29 (monolayer culture
of human colon carcinoma) and MEL-28 (monolayer
culture of human malign melanoma). The results of
these assays were used to obtain the corresponding
dose–response curves, from which IC50 (nM) values
were calculated.
Other compounds, mainly 40-demethylderivatives will be
prepared in order to clarify the structure–activity rela-
tionship for this family of compounds in relation with
the DNA-topoisomerase II inhibition.
The results obtained are shown in Table 1. As it can be
seen, the IC50 values of the majority of esters were either
similar or better than those of the parent alcohols.
Nevertheless, both podophyllotoxin norbornene-
carboxylates 4a and 5a showed IC50 values about 4 nM,
that represented an improvement in their potency with
respect to the podophyllotoxin 2a. The equipotency
shown by compounds 4a and 5a against neoplastic cells
Acknowledgements
Financial support came from Spanish MEC (AGL2000-
0039-P4-02). This research has been performed under
the auspices of CYTED Program. Subprogram X.

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1286
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