Stilbene-based anticancer agents: Resveratrol analogues active toward HL60 leukemic cells with a non-specific phase mechanism

Article abstract of DOI:10.1016/j.bmcl.2006.03.028

Several stilbenes, related to known resveratrol, have been synthesized and tested for their anticancer effect on HL60 leukemia cell line, taking particular care of the cell cycle analysis. The most potent compound was the known (Z)-3,4′,5-trimethoxystilbe

Full text of DOI:10.1016/j.bmcl.2006.03.028

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3245–3248
Stilbene-based anticancer agents: Resveratrol analogues active
toward HL60 leukemic cells with a non-specific phase mechanism
a,
b
c
c
*
Daniele Simoni, Marinella Roberti, Francesco Paolo Invidiata, Enrico Aiello,
c
a
a
a
Stefania Aiello, Paolo Marchetti, Riccardo Baruchello, Marco Eleopra,
d
d
e
Antonietta Di Cristina, Stefania Grimaudo, Nicola Gebbia,
f
g
d
Lucia Crosta, Francesco Dieli and Manlio Tolomeo
a
Dipartimento di Scienze Farmaceutiche, Via Fossato di Mortara 17-19, Universit a` di Ferrara, 44100 Ferrara, Italy
b
Dipartimento di Scienze Farmaceutiche, Universit a` di Bologna, Italy
Dipartimento Farmacochimico Tossicologico e Biologico, Universit a` di Palermo, Palermo, Italy
c
d
Divisione di Ematologia e Servizio AIDS, Policlinico, Universit a` di Palermo, Palermo, Italy
Dipartimento di Oncologia, Unit a` Operativa Oncologia Medica, Policlinico, Universit a´ di Palermo, Palermo, Italy
e
f
Consorzio di Ricerca sul Rischio Biologico in Agricoltura (Co.Ri.Bi.A), Palermo, Italy
Dipartimento di Biopatologia e Metodologie Biomediche, Universit a` di Palermo, Palermo, Italy
g
Received 14 February 2006; revised 13 March 2006; accepted 13 March 2006
Available online 31 March 2006
Abstract—Several stilbenes, related to known resveratrol, have been synthesized and tested for their anticancer effect on HL60 leu-
0
kemia cell line, taking particular care of the cell cycle analysis. The most potent compound was the known (Z)-3,4 ,5-trimethoxy-
stilbene (6b) which was active as apoptotic agent at 0.24 lM. Differently from other stilbenes (including resveratrol) that induced a
prevalent recruitment of cells in S phase of cell cycle, we found a peculiar behavior of 6b that caused a decrease of cells in all phases
0 1 2
of cell cycle (G –G , S, and G –M) and a proportional increase of apoptotic cells. The potent pro-apoptotic activity shown by com-
pound 6b and its effects on cell cycle make this compound of great interest for further investigations.
Ó 2006 Elsevier Ltd. All rights reserved.
Resveratrol (1), a phytoalexin present in grapes and
OH
OCH3
1
,2
other food products, has recently been suggested as
a potential cancer chemopreventive agent based on its
striking inhibitory effects on cellular events associated
B
HO
H3CO
H3CO
A
3
with cancer initiation, promotion, and progression.
This triphenolic stilbene has also been shown to induce
apoptosis (programmed cell death) in different cell
OH
OCH3
Resveratrol (1)
2
4
–6
lines. Since reduced apoptosis has been implicated
in the development and progression of malignant
Figure 1. Resveratrol and a known methoxylate analogue with potent
cancer growth inhibitory activity.
7
,8
tumors
phenotypes,
and in the occurrence of chemoresistant
9
–12
resveratrol-induced apoptosis might
therefore contribute to its antitumor activity (Fig. 1).
other research fields of medicinal chemistry. The sim-
plicity of resveratrol, associated with its interesting anti-
cancer activity, offers promises for the rational design of
new chemotherapeutic agents, and efforts have recently
been devoted regarding a detail study on the struc-
ture–activity relationship (SAR) of this type of substi-
The complex variety of biological activities shown by
resveratrol and other natural polyphenolic derivatives
is a feature shared with several results observed from
1
3–16
tuted stilbene derivatives.
Keywords: Resveratrol analogues; Anticancer agents; Cell cycle
analysis.
*
Corresponding author. Tel.: +39 0532 291291; fax: +39 0532
91296; e-mail: smd@unife.it
In previous works, we described the synthesis of a series
of cis- and trans-stilbene-based resveratrols with the aim
2
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.028

3246
D. Simoni et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3245–3248
0
of discovering new lead compounds with clinical poten-
1
trans compounds 5e,f, (3,4-dimethyl- or 4-nitro-4 -
7,18
tial.
dimethoxy analogues of rhapontigenin and its 3 -amino-
The best results were obtained with cis-3,5-
0
hydroxystilbene) through Horner–Emmons–Wadsworth
reaction with (4-benzyloxybenzyl)-phosphonic acid
diethyl ester (3b), NaH, and 3,4-dimethyl benzaldehyde
(4e) or 4-nitrobenzaldehyde (4a), respectively, in THF
(40–50% yield), and subsequent debenzylation with
0
and 3 -hydroxy derivatives, which showed apoptotic
7
1
activity at nanomolar concentration. We have also
0
demonstrated that 3 -hydroxy stilbenes possess interest-
ing antileukemic properties and they may constitute
effective and powerful drugs in MDR and apoptosis-re-
N,N-dimethylaniline (3 equiv), AlCl (4 equiv) in dichlo-
3
romethane (60% yield). Meanwhile, procedures were
attempted also with employment of resin-bound triphe-
nyl phosphine and 3,5-dimethoxybenzyl bromide (for
compounds 5a–d and 6a–d) or Wang-bromo resin as
analogue of resin-bound 4-hydroxybenzyl bromide for
compounds 5e,f, but we did not find improvements in
synthetic yields.
1
8
sistant hematological malignancies.
intriguing results were recently described regarding the
Of interest,
0
3
,4,5,4 -tetramethoxystilbene (2), a methoxylated ana-
logue of resveratrol, which was found to potently inhibit
the growth of cancer cell lines, but with almost no inhib-
1
9
itory effect on the growth of normal cells. Thus, on the
basis of our previous observations regarding the impor-
tance of the 3,5-dimethoxy motif in conferring pro-
apoptotic activity, together with the interesting selective
tumor growth inhibitory activity of 2, we further
explored other modified resveratrols and, therefore, we
planned the preparation of a small library mainly based
on methoxylated analogues with the aim to obtain
potent and selective proapoptotic agents.
In this work, the effects of 10 different stilbenes on the
myeloblastic leukemia cell line HL60 cells (myeloblastic
acute leukemia) are described. Antiproliferative (IC )
5
0
and apoptosis-inducing (AC ) activities are reported
5
0
in Table 1. Apoptosis was detected by morphological
examination and confirmed by Annexin V test. The
most active compounds resulted in the stilbenes
1
3,14,16,20
21
22
13,16,23
24
25
5
b,
e, f, 6b,
c, d. Compounds
In this letter, we observe that some of the synthesized
stilbenes are potent inducers of apoptosis but, for the
first time, we demonstrate that the 3,4 ,5-trimethoxystil-
5b,e,f and 6d showed a similar antiproliferative and
apoptotic-inducing activity, a little less than that of
compound 6c. Compound 5b showed an apoptotic
activity about 10–12 times higher than the natural ana-
logues resveratrol and piceatannol, and 19 times higher
0
bene 6b causes a not phase-specific block of cell cycle,
suggesting that its mechanism of cytotoxicity on neo-
plastic cells could be different from those of other stilb-
enes. Our data suggest that 6b could be a compound
effective in cancers with different kinetics and it should
be useful alone or in combination with other anticancer
agents to decrease the percentage of minimal residual
disease caused by kinetic factors.
1
8
than that of pterostilbene. The most active compound
of this series, however, was 6b, which was about 10 and
17 times more active than compounds 6c and 5b. From a
structure–activity relationship point of view, remarkable
results were obtained when the hydroxyl group of ptero-
stilbene was changed to the methoxy derivative 5b, hav-
1
8
ing the latter about 14 times smaller IC . Analogous
5
0
Stilbenes were prepared through classic Wittig couplings
from ylide 3a and aldehydes 4a–d (Scheme 1) or, for
results were also obtained with the cis-methoxy deriva-
tive 6b that is about 13 times more active than the cor-
R1
R2
MeO
+
_
PPh3Br
MeO
R3
1
3
2
4
4
4
a, 5a, 6a: R =R =H, R =NO
2
MeO
(i)
1
3
2
b, 5b, 6b: R =R =H, R =OCH
3
3
a
OMe
1
2
3
c, 5c, 6c: R =R =R =OCH
3
5
OMe
a-d
R1
1
2
3
+
R1
4d, 5d, 6d: R =OCH3, R =R =H
R2
R2
CHO
R3
R3
MeO
4
a-d
6a-d
R1
BzO
CH2PO3Et2
R2
3
b
(ii, iii)
R2
CHO
1
2
HO
4e, 5e: R =R =CH3
1
2
5
f: R =H, R =NO
R1
2
4a,e
Scheme 1. Reagents and conditions: (i) 3a (1.1 equiv), 4a–d (1 equiv), NaH (1.2 equiv), THF, 4–16 h, room or reflux temperature, 30–50% yield, 1:1
to 1:3 Z/E isomer ratio; (ii) same conditions as (i), but only E-isomers were recovered; (iii) AlCl , N,N-dimethylaniline, CH Cl , rt, 60% yield.
3
2
2

D. Simoni et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3245–3248
3247
Table 1. Antiproliferative effects (expressed as IC50) and apoptosis-
inducing effects (expressed as AC50) of stilbene derivatives on HL60
cells
The effects on cell cycle of the most active cis (6b,c) and
trans (5b,f) were examined by flow cytometry after stain-
ing of cells with propidium iodide. HL60 cells were
exposed to each compound at the concentrations
reported in Figure 2. In previous studies, we observed
that most stilbene compounds cause a block of cells in
a specific phase of cell cycle, acting as phase-specific
drugs; in particular many trans-stilbenes (trans-resvera-
trol and natural resveratrol analogues such as piceatan-
nol) induced a prevalent block in S phase, while
Compound
IC50 (lM)
AC50 (lM)
a
1
5 (±2)
35 (±3)
50 (±6)
68 (±5.6)
4 (±2.1)
5
5
5
5
5
5
6
6
6
6
a
b
c
d
e
f
2.5 (±0.6)
22 (±3.7)
37 (±2.9)
3.5 (±0.2)
2.5 (±0.3)
10 (±1.8)
0.15 (±0.01)
1.8 (±0.22)
2.8 (±0.4)
42 (±6.2)
70 (±10)
6 (±0.7)
4.8 (±0.6)
28 (±2.1)
0.24 (±0.017)
2.6 (±0.3)
5.4 (±0.4)
cis-stilbenes (such as combretastatin analogues) in
26,27
a
b
c
d
G –M phase,
2
although some trans-stilbenes can still
1
6
cause a block in G –M. Similar to resveratrol, com-
2
pounds 5b,f and 6c induced a partial block of cells in S
phase and an apoptotic sub-G –G peak corresponding
0
1
a
Ref. 18.
of about 20%, suggesting that these compounds act on
HL60 cells as phase-specific cytotoxic agents. In contrast,
compound 6b caused an evident sub-G –G peak increase
0
1
0
17
but no modification in cell cycle distribution (phases G0–
responding 4 -hydroxy derivative. Of note, by locating
0
G , S, and G –M) respect to the control.
1
the methoxy substituent at the 4 -position, as in 5b and
2
6
activity than the corresponding 3 -methoxy derivatives
b, we obtained compounds with significantly better
0
To better understand the effects of 6b on cell cycle, we
exposed HL60 cells to different concentration of 6b
5
d and 6d.
Figure 2. Flow cytometry analysis of cell cycle. HL60 cells were exposed 24 h to 3.5 lM 5b (b), 2.5 lM 6c (c), 4 lM 5f (d) and 0.2 lM 6b (e). (a)
Control; A, sub-G –G peak.
0
1
Figure 3. Flow cytometry analysis of cell cycle in HL60 cells exposed 24 and 48 h to different concentrations of compound 6b.

3248
D. Simoni et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3245–3248
for 24 and 48 h. As shown in Figure 3, 6b caused a
decrease of G –G , S, and G –M peaks, and a pro-
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0
1
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0
1
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8 h.) and to the concentration used. These data indi-
1
1
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ticularly interesting, because it could be useful alone
or in combination with other anticancer agents to de-
crease the percentage of minimal residual disease
caused by kinetic factors.
1
1
1
1
1
In conclusion, our results indicate that introduction of
methoxy groups at the stilbene motif of resveratrol is
important to confer cytotoxic and apoptotic activity to
this class of compounds and, in some cases, the methoxy
derivatives were more active than the corresponding
phenols. The potent apoptosis-inducing activity and
the ability of 6b to decrease the number of neoplastic
cells in all phases of cell cycle make this compound of
great interest for further investigations.
1
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Acknowledgment
2
0. Murias, M.; Handler, N.; Erker, T.; Pleban, K.; Ecker, G.;
Saiko, P.; Szekeres, T.; J a¨ ger, W. Bioorg. Med. Chem.
This work has been supported in part by Ministero
dell’Universit a` e della Ricerca Scientifica e Tecnologica
2
004, 12, 5571.
21. Colourless solid. Mp = 150–153 °C. H NMR: d 2.27 (s,
H), 2.29 (s, 3H), 4.76 (s, 1H), 6.82 (d, J = 8.6, 2H), 6.92
d, J = 16.4, 1H), 7.00 (d, J = 16.4, 1H), 7.10 (d, J = 8,
1
(
MURST), Rome, Italy.
3
(
1
3
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1
1
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3
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24. Yellow oil. H NMR: d 3.67 (s, 12H), 3.81 (s, 3H), 6.31 (t,
1
3
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