Synthesis of a resveratrol analogue with high ceramide-mediated proapoptotic activity on human breast cancer cells

Article abstract of DOI:10.1021/jm050528k

Resveratrol, a natural product with a stilbene structure, exerts profound proapoptotic activity in human cancer cells, by triggering the accumulation of ceramide, a bioactive sphingolipid. We studied the biological effects of seven methoxylated and/or nap

Full text of DOI:10.1021/jm050528k

J. Med. Chem. 2005, 48, 6783-6786
6783
Scheme 1a
Synthesis of a Resveratrol Analogue with
High Ceramide-Mediated Proapoptotic
Activity on Human Breast Cancer Cells
Filippo Minutolo,†,| Giusy Sala,‡,|
Annalisa Bagnacani,‡ Simone Bertini,†
Isabella Carboni,† Giorgio Placanica,†
Giovanni Prota,† Simona Rapposelli,†
Nicoletta Sacchi,§ Marco Macchia,*,† and
Riccardo Ghidoni*,‡
a Key: (a) Pd(PPh3)4, K3PO4 (aq), dioxane, 80 °C, 16 h. (b) BBr3,
CH2Cl2, -78 °C, 3 h.
Scheme 2a
Department of Pharmaceutical Sciences, University of Pisa,
via Bonanno 6, 56126 Pisa, Italy, Laboratory
of Biochemistry and Molecular Biology, San Paolo
University Hospital, Medical School, University of Milan,
20142 Milan, Italy, and Department of Cancer Genetics,
Roswell Park Cancer Institute, Buffalo, New York 14263
Received June 6, 2005
a Key: (a) (1) Pd(PPh3)4, K3PO4 (aq), dioxane, 80 °C, 16 h. (2)
HCl (aq).
Abstract: Resveratrol, a natural product with a stilbene
structure, exerts profound proapoptotic activity in human
cancer cells, by triggering the accumulation of ceramide, a
bioactive sphingolipid. We studied the biological effects of
seven methoxylated and/or naphthalene-based resveratrol
analogues and compared these compounds with resveratrol
with the objective to identify an analogue with higher cera-
mide-mediated proapoptotic activity relative to resveratrol.
Here we show that the compound with three hydroxyls and a
naphthalene ring is the most effective in triggering apoptosis
coupled to the induction of endogenous ceramide in human
cancer cells.
Because methoxylated resveratrol analogues have
been identified in plants,9-12 we synthesized three
analogues (compounds 1-3), by replacing the hydroxyl
groups with methoxyl groups. In addition we tried to
overcome both the chemical and metabolic instability
of resveratrol by substituting the stilbene double bond
present in resveratrol with a naphthalene ring. To this
end, we synthesized four naphthalene-based resveratrol
analogues that do not contain the unstable double bond
within their structure, either with or without methoxyl
groups (compounds 4-7). This simple, yet unexplored,
change enables preservation of the original geometrical
orientation of the pharmacophoric groups of resveratrol,
while adding more conformational rigidity to the whole
structure.
When we tested these seven analogues for their
ceramide-mediated proapoptotic activity, we found that
the analogue with three hydroxyls and a naphthalene
ring was the most effective in triggering apoptosis
coupled to the induction of endogenous ceramide in the
human breast cancer cell line.
Synthesis of Resveratrol Analogues. Compounds
1-3 were synthesized as outlined in the Supporting
Information. By using the strategy outlined in Scheme
1, we synthesized compounds 4, 6, and 7. A Suzuki
Pd-catalyzed cross-coupling reaction between 3,5-
dimethoxyphenylboronic acid (10) and 6-bromo-2-naph-
thol (8) or 6-methoxy-2-bromo-naphthalene (9) was used
to synthesize compounds 6 and 7, respectively. The
completely demethylated compound 4 was obtained by
treating 6 with boron tribromide at -78 °C.
The synthesis of monomethylated compound 5 was
achieved as shown in Scheme 2. In this case 6-meth-
oxynaphthylboronic acid (11)13 was again submitted to
a Suzuki reaction with 3,5-(di-tert-butyldimethylsilyl-
oxy)-1-bromobenzene (12).14 Acidic aqueous workup
caused complete desilylation of the protected phenolic
groups, leading to the synthesis of pure compound 5.
Resveratrol, a naturally occurring phytoalexin (3,5,4′-
trans-trihydroxystilbene) present in medicinal plants,
grape skin, peanuts, and red wine,1 has been reported
to have chemopreventive activity,2 by exerting antipro-
liferative and proapoptotic effects in human cancer
cells.3 We recently showed that resveratrol can affect
cancer cell growth and induce apoptosis by triggering
the synthesis of endogenous ceramide,4.5 a bioactive
sphingolipid.6,7 Specifically, we observed that resveratrol
leads to the accumulation of ceramide in association
with apoptosis both in metastatic breast4 and prostate5
cancer cells. Ceramide is a promising pharmacological
target when either major apoptotic pathways are dis-
rupted or resistance to DNA damage is elevated. More-
over, drugs that trigger ceramide, while highly effective
in malignant cells, are less toxic to normal cells and
tissues.8
For this reason, objective of the present study was to
design and synthesize resveratrol analogues more ef-
fective than resveratrol at inducing ceramide-mediated
apoptosis in the metastatic breast cancer cell line, MDA-
MB-231. In these cells we found that resveratrol induced
ceramide by activation of its de novo synthesis.4
* Corresponding authors. R.G.: Phone: +39 02 50323250. E-mail:
riccardo.ghidoni@unimi.it. M.M.: Dipartimento di Scienze Farmaceu-
tiche, Universita` di Pisa, Via Bonanno, 6-56126 Pisa, Italy. Phone: +39
050 2219553. Fax: +39 050 2219553. E-mail: mmacchia@farm.unipi.it.
† University of Pisa.
‡ University of Milan.
All Resveratrol Analogues Display Antiprolif-
erative Effect in MDA-MB-231 Cancer Cells. The
§ Roswell Park Cancer Institute.
| These authors equally contributed to the present work.
10.1021/jm050528k CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/04/2005

6784 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Letters
6 days (Figure 2). The sulforhodamine B (SRB) cell
proliferation assay was used. The proliferation index at
the beginning of the experiment (time 0) was set at 1.
The IC50 for the seven resveratrol analogues are re-
ported in Table 1, together with the IC50 of resveratrol.
Compounds 2, 3, 4, and 6 had a stronger antiprolifera-
tive effect than resveratrol.
Compound 3 showed the highest antiproliferative
Figure 1. Structures of stilbene- and naphthalene-type
effect (IC50 ) 1.2 µM), followed by compounds 6 (IC50
)
resveratrol analogues.
7.2 µM), 2 (IC50 ) 10.0 µM), and 4 (IC50 ) 12.7 µM).
The antiproliferative effect of compounds 1, 5, and 7
were by far lower (IC50 ) >50, 28.3, and 45.1 µM,
respectively). Some compounds tested were able to
impair cell growth at a concentration as low as 1 µM
(Figure 2).
seven resveratrol analogues were compared for their
growth inhibitory effect on MDA-MB-231 breast cancer
cells. Cells were treated with compounds 1-7 at differ-
ent concentrations, ranging from 0.25 µM to 64 µM for
Figure 2. Antiproliferative effect of resveratrol and synthetic resveratrol analogues. Proliferation index was evaluated by the
SRB assay in MDA-MB-231 cells treated with 0.25-64 µM resveratrol or synthetic analogues for up to 6 days. The values are the
mean ( SD of three independent experiments. The symbols and lines represent different drug concentrations, as shown in the
figure insets.

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6785
Table 1. IC50 Values for Resveratrol and Resveratrol
more than 5-fold by compound 4 than by resveratrol. A
modest, but not significant ceramide accumulation was
induced also by compound 5, a monomethylated, naph-
thalene ring resveratrol analogue.
Altogether, our data show that the introduction of a
naphthalene ring on the intact resveratrol molecule
confers an improved ceramide-mediated proapoptotic
activity.
In this study we report that there is a relationship
between the structure of resveratrol analogues and the
ceramide-mediated proapoptotic activity in human can-
cer cells.
Resveratrol biological activity in cancer cells is
limited by its photosensitivity and metabolic instability.
Under UV irradiation, resveratrol converts to the (Z)-
isomer15a,16a and this was claimed as one of the reason
for the presence of small amounts of (Z)-resveratrol in
wines. The conversion from an (E)- to a (Z)-configuration
makes resveratrol less stable16b,c and consequently
diminishes its biological activity. Moreover, stilbene
double bonds are readily oxidized by cytochrome P450
monooxygenases into highly reactive epoxides, that may
act as carcinogenic metabolites.15b-d We then synthe-
sized novel resveratrol analogues with a naphthalene
ring instead of a stilbene double bond and tested
whether they were able to induce ceramide-mediated
apoptosis.
Analoguesa
compound
IC50 (µM)
20.5 ( 2.6
RES
1
2
3
4
5
6
7
>50
10.0 ( 1.8
1.2 ( 0,2
12.7 ( 1.3
28.3 ( 3.8
7.2 ( 1.0
45.1 ( 6.8
a IC50 (µM) was calculated as the mean ( SD of the proliferation
values obtained at 2, 4, and 6 days of treatment with each
compound used at different concentrations in three independent
experiments.
First, we observed that the stabilization of the double
bond of resveratrol, accompanied by an increase in
molecular rigidity which is operated by the addition of
a fused benzene ring as in compound 4, not only
preserves, but considerably improves the biological
activity of natural resveratrol (IC50 ) 12.7 µM of
compound 4 versus IC50 ) 20.5 µM of resveratrol, and
IC50 ) 28.3 µM of compound 5 versus IC50 > 50 µM of
compound 1). Like resveratrol, yet at higher efficiency,
compound 4 exerts an antiproliferative effect via a
ceramide-mediated proapoptotic mechanism, as shown
by an increase of endogenous ceramide coupled to
cleavage of PARP. All the other six compounds tested
exerted at the IC50 variable antiproliferative effects
without significant induction of either ceramide or
apoptosis. Thus, both resveratrol and compound 4 with
three intact hydroxyl groups seem to affect proliferation
with a ceramide-mediated apoptotic mechanism.
Second, we confirmed that ceramide-independent
antiproliferative activity of resveratrol can be increased
by replacing the 3,5-hydroxy groups with methoxy
functions (compounds 2 and 3), regardless of the pres-
ence of two or three aromatic rings. A resveratrol
analogue (3,5-di-O-methyl-3′-hydroxy-trans-stilbene), very
similar to compound 2, has been recently reported to
be the most potent antiproliferative analogue of a group
of methylated analogues of resveratrol17 toward the
breast cancer cell line MDA-MB-468. The addition of a
third methoxy function in the 4′-position of resveratrol
(compound 3) resulted in a dramatic antiproliferative
and cytotoxic effect, not associated with ceramide ac-
cumulation. In other cell lines18,19 trimethoxystilbene
was found to be highly cytotoxic, and this is due to
interference with the mitotic process by microtubule
disassembly, impairment of tubulin polymerization, and
depletion of polyamine intracellular pool. Interestingly,
the presence of an additional aromatic ring to methoxy
Figure 3. Proapoptotic effect and ceramide accumulation by
resveratrol and synthetic analogues. MDA-MB-231 cells were
treated for 4 days with resveratrol and resveratrol analogues
at the respective IC50. (A) The proapoptotic effect was deter-
mined by the PARP cleavage assay, evaluated as appearance
of the 86 kDa band. (B) Ceramide accumulation determined
by the DGK assay. Ceramide spots of a representative experi-
ment and ceramide quantification, as the mean ( SD of three
independent experiments. Ceramide content of treated cells
is expressed as fold-increase (** ) p < 0.01) respect to
untreated cells.
Compound 4 Exerts an Antiproliferative Effect
through an Apoptotic Mechanism. To assess whether
the antiproliferative effect could be traced to an apop-
totic mechanism, we tested the induction of poly-(ADP-
ribose)-polymerase (PARP) cleavage by the seven ana-
logues after treating MDA-MB-231 cells with the IC50
(see Table 1) for 4 days (Figure 3A). The 86 kDa band,
which is an evidence of PARP cleavage, was detected
only with compound 4. Thus, only the resveratrol
analogue with intact hydroxyls and an additional naph-
thalene ring exerts an antiproliferative effect via an
apoptotic mechanism. Since PARP is a substrate for
caspase-3, the proapoptotic effect of compound 4 might
be caspase-mediated.
Compound 4 Induces a Level of Endogenous
Ceramide Higher Than the Ceramide Level In-
duced by Resveratrol. Next, we tested whether the
antiproliferative effect of the seven analogues involved
ceramide accumulation. By using the diacylglycerol-
kinase (DGK) assay, we found that MDA-MB-231 cells
treated for 4 days with the seven resveratrol analogues
at the corresponding IC50 (see Table 1) displayed a level
of ceramide significantly higher (p < 0.01) than the level
induced by resveratrol only when treated with com-
pound 4 (Figure 3B). Specifically ceramide was induced

6786 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
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JM050528K