Design, synthesis and anticancer activities of stilbene-coumarin hybrid compounds: Identification of novel proapoptotic agents

Article abstract of DOI:10.1016/j.bmc.2010.03.069

The naturally occurring coumarins and resveratrol, attract great attention due to their wide range of biological properties, including anticancer, antileukemic, antibacterial and anti-inflammatory activities; moreover, their cancer chemopreventive property have been recently emphasized. A novel class of hybrid compounds, obtained by introducing a substituted trans-vinylbenzene moiety on a coumarin backbone, was synthesized and evaluated for the antitumor profile. A number of derivatives showed a good antiproliferative activity, in some cases higher to that of the reference compound resveratrol. The most promising compounds in this series were 14 and 17, endowed with excellent antiproliferative and proapoptotic activities. The present study suggests that the 7-methoxycoumarin nucleus, together with the 3,5-disubstitution pattern of the trans-vinylbenzene moiety, are likely promising structural features to obtain excellent antitumor compounds endowed with a apoptosis-inducing capability.

Full text of DOI:10.1016/j.bmc.2010.03.069

Bioorganic & Medicinal Chemistry 18 (2010) 3543–3550
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and anticancer activities of stilbene-coumarin hybrid
compounds: Identification of novel proapoptotic agents
b
c
c
c
c
Federica Belluti a, , Gabriele Fontana , Laura Dal Bo , Nives Carenini , Chiara Giommarelli , Franco Zunino
*
a Dipartimento di Scienze Farmaceutiche, Via Belmeloro, 6, I-40126 Bologna, Italy
b Indena S.p.A., Viale Ortles, Milan, Italy
c Fondazione IRCCS Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The naturally occurring coumarins and resveratrol, attract great attention due to their wide range of bio-
logical properties, including anticancer, antileukemic, antibacterial and anti-inflammatory activities;
moreover, their cancer chemopreventive property have been recently emphasized. A novel class of hybrid
compounds, obtained by introducing a substituted trans-vinylbenzene moiety on a coumarin backbone,
was synthesized and evaluated for the antitumor profile. A number of derivatives showed a good antipro-
liferative activity, in some cases higher to that of the reference compound resveratrol. The most promis-
ing compounds in this series were 14 and 17, endowed with excellent antiproliferative and proapoptotic
activities. The present study suggests that the 7-methoxycoumarin nucleus, together with the 3,5-disub-
stitution pattern of the trans-vinylbenzene moiety, are likely promising structural features to obtain
excellent antitumor compounds endowed with a apoptosis-inducing capability.
Received 11 November 2009
Revised 19 March 2010
Accepted 25 March 2010
Available online 29 March 2010
Keywords:
Coumarins
Resveratrol
Apoptosis
Antitumor
Ó 2010 Elsevier Ltd. All rights reserved.
Hybrid compound
1. Introduction
lar, coumarin itself (1a) and its metabolite 7-hydroxycoumarin (1b,
Fig. 1) have been investigated as potential candidates for cancer
Cancer is at present one of the most leading causes of death in
the United States and in the developed countries,1 and many ef-
forts have been made to discovery agents endowed with cytostatic
action.2 For several chemotherapeutic agents, a direct correlation
between the antitumor efficacy and the ability to induce apoptosis
was established, and the development of new anticancer ap-
proaches, aimed at promoting apoptosis in cancer cells, gained a
paramount importance.3
Natural products, with their ability to interact with more to one
target, represent, in medicinal chemistry, a significant source of
inspiration for the design of structural analogues with improved
pharmacological profile.
Coumarins are a wide group of naturally occurring compounds
that, due to their remarkable array of biological activities, usually
associated to low toxicity, form an elite class of compounds which
occupy a special role in nature. Indeed, a large number of couma-
rins have been isolated from a variety of plant sources and under-
went extensive investigations aimed to assess their potential
therapeuticapplications:4 somerelevant actions were reported such
as: anticancer,5,6 anti-HIV,7,8 anticoagulant,9 antimicrobial,10,11
antioxidant and anti-inflammatory.12 The antitumor effects, which
are related to the inhibition of the cellular proliferation, through a
variety of mechanisms, were extensively examined.13–17 In particu-
therapy,18,17,19 scopoletin (6-methoxy-7-hydroxycoumarin, 1c)
and esculetin (6,7-dihydroxycoumarin, 1d) have been shown to
exhibit an antiproliferative effect in leukaemic cells by inducing
apoptosis.20,21
This inherent biological relevance has attracted much interest
in the drug discovery field, and the benzopyran-2-one nucleus
emerged as a valuable molecular template for the design of a vari-
ety of analogues. A lot of efforts aimed at the design of properly
functionalized synthetic coumarins as effective agents able to
potently and selectively affect some cellular functions, have been
performed.22–31
Recently, considerable attention has been also focused on resve-
ratrol (3,5,40-trans-trihydroxystilbene, 2, Fig. 1), a well-known nat-
ural polyphenol found in large amount in grapes,32 that has been
reported to exert multiple biological activities33 including anti-
inflammatory,34 anti-oxidant,35 inhibition of platelet aggrega-
tion,36 antitumor,37 and induction of apoptosis.38 Remarkably, the
cancer chemopreventive activity39,40 of 2 represents an important
add value and it seems to be strictly connected to the antitumor,
and the proapoptotic effects.41 Structure–activity relationship
(SAR) studies, performed on a number of resveratrol analogues, al-
lowed to establish the chemical features responsible for the antitu-
mor and the proapoptotic activities; for example the 3,5-
dimethoxyphenyl moiety was identified to play a pivotal role in
conferring both the antitumor and the proapoptotic activities.42–44
Generally, the presence of a number of methoxy groups seems to
* Corresponding author. Fax: +39 051 2099734.
E-mail address: federica.belluti@unibo.it (F. Belluti).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.069

3544
F. Belluti et al. / Bioorg. Med. Chem. 18 (2010) 3543–3550
H3C
OH
O
O
O
CH3
CH3
R'
OH
H3C
R
O
O
HO
O
1a: R, R' = H
3 (DMU-212)
1b: R = OH, R' = H
1c: R = OH, R' = OCH3
1d: R, R' = OH
2 (resveratrol)
insertion
R
compd
R1
R2
R3
R4
position
R2
12
13
14
15
16
17
18
19
20
21
4
4
4
4
4
4
4
4
3
4
H
H
H
H
H
OCH3
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
CH3
H
H
H
OCH3
OCH3
H
OC5H9
H
OCH3
OCH3
OCH3
OCH3
H
CH3
H
H
R1
R3
R4
6-OCH3
7-OCH3
7-OCH3
7-OCH3
7-OCH3
7-OCH3
7-OCH3
7-OCH3
7-OH
R
H
O
O
OCH3
OCH3
OH
H
H
OCH3
OH
12-21
Figure 1. Chemical structures of coumarins and stilbene analogues mentioned in this study and general structure of the synthesized compounds (12–21).
be a fundamental requirement to obtain potent cytotoxic agents,
and the 3,4,5,40-tetramethoxystilbene (DMU-212, 3, Fig. 1) proved
to be more potent and selective than the parent compound 2 in
the inhibition of the cancer cell growth.45,46
2. Chemistry
Preparation of the derivatives 12–21 was accomplished by
means of a common synthetic procedure shown in Scheme 1.
Bromination of the 3 or 4-methylcoumarin derivatives with N-bro-
mosuccinimide (NBS) gave the 3 or 4-bromomethyl intermediates
4–7, that were reacted with triethylphosphate, to obtain the corre-
sponding phosphonic acid diethyl ester derivatives 8–11. Horner–
Emmons–Wadsworth reaction of the above obtained compounds
with the selected aldehydes in the presence of sodium methoxide
as base, afforded the stilbene derivatives 12–20 in a 9:1 mixture of
E/Z isomers. The desired E-isomer (trans) was achieved by means of
the fractionated crystallization from toluene. The formation of the
E isomers was confirmed by the characteristic 1H NMR coupling
constants (J) of 15–18 Hz observed for the olefinic protons.51 Cleav-
age of the methoxy functions of compound 14, with BBr3 at low
temperature, afforded compound 21.
Over the last few years, with the progress of the medicinal
chemistry, the hybrid approach has received significant attention
since it allowed obtaining molecules with improved biological
activity with respect to the corresponding lead compounds. Thus,
the design and synthesis of hybrid molecules encompassing two
pharmacophores in one molecular scaffold is a well established ap-
proach to the synthesis of more potent drugs.47 Using this ap-
proach, several research groups have recently designed and
synthesized hybrid molecules by coupling coumarins with a num-
ber of bioactive molecules such as: resveratrol, maleimide and al-
pha-lipoic acid; these efforts resulted in new molecules endowed
with vasorelaxant and platelet antiaggregating, imaging, and anti-
oxidant and anti-inflammatory properties, respectively.48–50
Objective of the present study was to synthesize a novel class of
conjugates (compounds 12–21), which general formula was de-
picted Figure 1, obtained by the insertion of a properly substituted
trans-vinylbenzene moiety on a coumarin backbone. As first line of
search, the trans-3,5-dimethoxyphenylvinyl function was intro-
duced on a number of coumarins (compounds 12–14 and 20)
and the 7-methoxycoumarin bearing the trans-vinylbenzene moi-
ety in the 4 position (14) proved to be the derivative endowed with
the higher antiproliferative activity. These results prompted to fur-
ther explore this framework and some structural analogues of 14,
bearing different substitution patterns on the vinylbenzene por-
tion (compounds 15–19 and 21) were prepared. The new couma-
rin-resveratrol hybrids were tested for their antiproliferative
activity against a human lung carcinoma cell line (H460). The
two most promising compounds (14 and 17) underwent addi-
tional biological investigations regarding: the inhibitory effects
against proliferation of squamous cell carcinoma (A431) and mel-
anoma (JR8); the ability to induce apoptosis, together with the
cellular basis and the molecular events responsible for their anti-
cancer profile.
3. Biological results and discussion
The synthesized analogues 12–21 were initially tested for their
antiproliferative activity in H460 cells and the results were re-
ported in Table 1. The analogues 12–14, obtained by inserting
the trans-3,5-dimethoxyphenylvinyl moiety in the positions 4 of
coumarin, 6-methoxy and 7-methoxycoumarin scaffolds, respec-
tively, showed quite different antiproliferative potencies: com-
pound 12, with IC50 value of 2.6
active than derivatives 13 and 2; compound 14, which showed a
IC50 value of 0.45 M, was 6- and 28-fold more active than deriv-
atives 12 and 2, respectively, and undoubtedly emerged as one of
the most active compounds within this subset, thus offering a
proof about the importance of the 7-methoxycoumarin nucleus
as main backbone. Accordingly, shifting the trans-3,5-dimethoxy-
phenylvinyl moiety of derivative 14 from the C-4 to the C-3 posi-
tion, to yield compound 20, a dramatic drop of potency was
observed, suggesting that also the C-4 insertion position plays a
lM, was about fivefold more
l

F. Belluti et al. / Bioorg. Med. Chem. 18 (2010) 3543–3550
3545
O
OEt
OEt
CH
P
3
Br ii
i
R
R
R
O
O
O
O
O
O
8-11
4-7
2
R
Insertion position 4:
4, 8: R = H
1
3
R
R
5, 9: R = 6-OCH
6, 10: R = 7-OCH
3
4
R
iii
3
Insertion position 3:
7, 11: R = 7-OCH
R
3
O
O
Insertion position 4:
12-20
1
3
2
4
12: R=H, R , R =H, R ,R =OCH
3
1
3
2
4
13: R=6-OCH , R ,R =H, R ,R =OCH
iv
3
3
3
3
for 14
1
3
2
4
14: R=7-OCH R ,R =H, R ,R =OCH
3,
1
2
3
4
HO
OH
15: R=7-OCH R =H, R ,R ,R =OCH
3,
1
2
3
4
16: R=7-OCH , R ,R ,R =OCH , R =H
3
3
1
3
2
4
17: R=7-OCH , R ,R =H, R ,R =CH
3
3
1
2
4
3
18: R=7-OCH , R ,R ,R =H, R =OC H
5 9
3
1
3
4
2
19: R=7-OCH , R , R ,R =H, R =OCH
3
3
Insertion position 3:
HO
O
O
1
3
2
4
20: R=7-OCH R ,R =H, R ,R =OCH
3,
3
21
Scheme 1. Reagents and conditions: (i) NBS, (PhCOO)2O, CCl4, reflux; (ii) PO(OEt)3, 150 °C; (iii) Ar-CHO, NaOCH3, DMF, 0–100 °C; (iv) BBr3, DCM, 0 °C, rt.
Table 1
to be slightly more potent than the corresponding lead molecule
14.
Antiproliferative activity of synthetic coumarins 12–21 in human tumour cells from
different tumour types (lung carcinoma H460, squamous cell carcinoma A431,
melanoma JR8)a Figure 4
The two most effective compounds of the series (14 and 17)
underwent additional biological investigations such as: their po-
tency against A431 and JR8 cells was evaluated; in both these cell
systems a comparable activity (at micromolar level) was observed.
Furthermore, in an attempt to better characterize the cellular
basis of their antiproliferative effects, the ability of these selected
compounds to induce apoptosis in the most sensitive cell line
(i.e., H460) was investigated. At concentrations causing compara-
ble antiproliferative effects (IC80), 14 and 17 produced a level of
apoptosis similar to that of cisplatin and substantially higher to
that of 2, which was a poor inducer of apoptosis in this cell system
(Fig. 2A). In particular, the proapoptotic effect of 14 was confirmed
by analysis of cleavage of caspase 3 and PARP. As shown in Figure
2B, activation of caspase 3 and cleavage of PARP was already
detectable after 24 h exposure.
Compd
IC50 (lM)
H460
A431
JR8
Resveratrol
12.9 3.1
2.6 0.2
11.0
0.45 0.09
>100
>50
0.29 0.08
>50
12.9 1.3
>10
n.t.
n.t.
n.t.
3.44
n.t.
n.t.
3.5
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
3.2
n.t.
n.t.
3.5
n.t.
n.t.
n.t.
n.t.
12
13
14
15
16
17
18
19
20
21
1
27.0 3.0
Chemical structures of coumarins and stilbene mentioned in this study and general
structure of the synthesized compounds.
Besides, to gain insights into the cellular basis of the peculiar
behaviour of these selected compounds some aspects of cellular re-
sponse in H460 cells have been investigated (Fig. 3): when the cells
were exposed to antiproliferative concentrations (IC50) for 24 h,
cell cycle analysis showed a moderate arrest at the G2/M phase,
this effect was dose-dependent because the cell accumulation in
G2/M phase was increased at more toxic concentrations. This re-
sponse, in terms of perturbation of cell cycle, was somewhat sim-
ilar to that produced by resveratrol at substantially higher
n.t.: not tested.
a
Drug effects were determined after 72 h exposure.
pivotal role. A detrimental effect was also observed with the
replacement of the methoxy groups of 14 with hydroxyls: the
derivative 21 was 60-fold less active than 14 and only twofold less
active than 2. Taken these data into account, further structural
modifications of 14, purposely focused on the vinybenzene portion
of the molecule, were performed; a noteworthy reduction of the
antiproliferative activity was observed for compounds 15 and 16,
bearing a supplementary methoxy group, and for derivative 18,
with a p-3,3-dimethylalliloxy side chain. Moreover, the removal
of one methoxy group of 14 caused a loss of potency: compound
19 was equipotent to 2. These findings outlined as the 3,5-dime-
thoxy groups on the trans-vinylbenzene moiety play a pivotal role;
their replacement with methyls lead to compound 17, that proved
concentrations (20 lM).
Moreover, the molecular events involved in cellular response to
the effective compounds, were investigated and to this end, the
levels of regulatory proteins implicated in G2 arrest, including
p21, cyclin B1, p34cdc2 were examined (Fig. 4A). Protein p21 plays
a crucial role in G2 arrest trough accumulation of inactive cyclin
B1/p34cdc2 complex. In fact, under the same conditions used for
analysis of cell cycle perturbation (24 h exposure to 1.5 lM for

3546
F. Belluti et al. / Bioorg. Med. Chem. 18 (2010) 3543–3550
1µM
35
30
25
20
15
10
5
1.5µM
3µM
c
14
pro-caspase 3
CPP32
cleaved PARP
0.5 µM
0.3 µM
20 µM
10µM
0
c
cisPt
Resv
17
14
Figure 2. Induction of apoptotic cell death in H460 cells by compounds 14 and 17 and effects of 14 on apoptosis-related proteins. (A) Comparison of apoptosis induced by
cisplatin (cisPt) resveratrol (Resv), compounds 14 and 17. After 72 h exposure to equitoxic concentrations of cisPt (1 M), resveratrol (10 and 20 M), 17 (0.3 and 3 M) and
14 (0.5 and 1.5 M), apoptosis was detected by TUNEL assay. Drug effects are reported as percent of TUNEL-positive cells. (c) control untreated. (B) Biochemical analysis of
l
l
l
l
caspase 3 and PARP cleavage. Total cellular extracts were obtained after 24 h exposure to 14 (1.5 lM). Western blot analyses were then performed to assess protein
expression. Actin is shown as a control for protein loading.
c
14
G1
S
G2/M
c
17
17 (3 µM)
p21
17 (0.3 µM)
cyclin B1
p34 CdC2
actin
14 (1.5 µM)
14 (0.5 µM)
Resv(20 µM)
Resv(10 µM)
c
14
c
0
10
20
30
40
50
60
IP: p34 CdC2, WB: p34 CdC2
IP: p34 CdC2, WB: p21
Figure 3. Cell cycle analysis. H460 cells were treated for 24 h with equitoxic
concentrations of resveratrol (Resv) (10 and 20 M) or 14 (0.5 and 1.5 M) or 17
(0.3 and 3 M).
l
l
l
14 and 3 lM for 17, respectively), there was a marked increase of
Figure 4. Modulation of cell cycle regulatory proteins in H460 cells treated with 14
or 17. (A) Cyclin B1, p34cdc-2 and p21 protein expression. Total cellular extracts
p21 expression concomitant with upregulation of cyclin B1 and
p34cdc2 (Fig. 4A). All together these events are consistent with a
critical role of p21 in G2 arrest as a consequence of its association
with p34cdc2 and inactivation of cyclin B1–p34cdc2 complex. In-
deed, Western blot analysis of immunoprecipitates with p34cdc2
antibody revealed an accumulation of p21–p34cdc2 complexes
(Fig. 4B).
were obtained after 24 h exposure to 14 (1.5
Western blotting. Actin is shown as control for protein loading. (B) Co-
immunoprecipitation analysis of p34cdc-2/p21 in H460 cells after 24 h exposure to
14 (1.5 M). After treatment cell lysates were harvested and immunoprecipitated
lM) or 17 (3 lM) and analyzed by
a
l
with anti-p34cdc-2 rabbit polyclonal antibody. Immunoprecipitates were immuno-
blotted for p21. Blots were stripped and probed for p34cdc-2
.
Thus, a tentative explanation of the proapoptotic activity of 14
and 17 could be related to activation of apoptotic signals, as a con-
sequence of arrest in G2 phase, and inhibition of G2?M transition,
which favours apoptosis activation.
On the basis of the promising features at cellular level, com-
pound 14 was also tested in the human lung carcinoma H460
xenograft model. In this preliminary experiment, when adminis-
tered orally at 10–20 mg/kg with a daily treatment schedule, the
derivative produced an appreciable tumour growth inhibition (by
ꢀ50%) without evidence of toxic effects. The optimal treatment
conditions remain to be defined.
4. Conclusions
A series of hybrid molecules (derivatives 12–21), composed by a
coumarin scaffold and a properly substituted trans-vinylbenzene
moiety, were synthesized and tested to assess their antiprolifera-
tive activity on H460 lung carcinoma cells. For the most potent
compounds the proapoptotic effects were also evaluated. The
derivatives 15, 16 and 18, in which the para position of the vinyl-
benzene moiety was occupied by a substituent, proved to be the
less active of the series; in particular, 18, with an hindering substi-

F. Belluti et al. / Bioorg. Med. Chem. 18 (2010) 3543–3550
3547
tuent in this position was designed as the negative control com-
pound. An increment of potency was observed for compound 19,
bearing a meta methoxyl, that showed the same potency of the ref-
erence compound 2. The presence of the methoxyls at specific
positions, in both fragments of the hydrid molecules, demon-
strated to be critical in conferring the antiproliferative activity:
the subset of compounds 12–14 and 20, characterized by the pres-
ence of a 3,5-dimethoxyphenylvinyl moiety, demonstrated that the
substitution pattern on the coumarin scaffold significantly affected
potency. Specifically, 12, without substituent on the coumarin nu-
cleus, was fivefold more active than 2, 13, bearing a methoxy group
in the 6 position of the coumarin scaffold, demonstrated the same
potency of 2, and 14, the C-7 methoxylated analogue, showed a
substantial enhancement of activity, proving to be a good antipro-
obtained for those elements are within 0.4% of the theoreticalvalues.
Column chromatography was carried out with silica gel (Kiesel gel
40, 0.040–0.063 mm; Merck) using the flash technique. Yields were
reported after crystallization or chromatographic purification. Com-
pounds were named following IUPAC rules as applied by Beilstein-
Institut AutoNom (version 2.1), a PC integrated software package
for systematic names in organic chemistry.
5.2. General procedure for the preparation of bromomethyl-2H-
chromen-2-one derivatives (4–7)
To
a suspension of a suitable methylcoumarin derivative
(10 mmol) and NBS (11 mmol) in CCl4 (100 mL), a catalytic amount
of benzoyl peroxide was added. The reaction mixture was refluxed
for 8 h, the succinimide produced during the reaction was filtered
off, and the solvent was washed with H2O, dried and removed under
reduced pressure. The crude was crystallized from ligroin to afford
the desired bromomethyl derivative as a pure crystalline compound.
liferative agent with IC50 values of 0.45 lM. A decrease of activity
was observed by shifting the 3,5-dimethoxy trans-vinylbenzene
function of 14 from C-4 to the C-3 position (20). Replacement of
both the methoxyls of 14 with the hydroxyls, to obtain 21, resulted
in about a twofold reduction in activity with respect to the strictly
related molecule 2; on the contrary, promising results were ob-
tained by replacing the methoxyls at the 3 and 5 positions of the
vinylbenzene moiety with methyls, as documented by the effects
of 17, characterized by submicromolar IC50 value. Derivatives 14
and 17 exhibited pharmacologically relevant antiproliferative
activity and underwent deep biological investigations, thus reveal-
ing their ability to induce an appreciable level of apoptosis (ꢀ30%
of TUNEL-positive cells), comparable to that of cisplatin, and to a
great extent superior to that of 2. This effect was associated with
a partial cell accumulation in G2/M phase of cell cycle. Indeed,
the presence of particular substituents in specific positions of both
fragments of the hydrid molecule, demonstrated to be critical in
conferring the antiproliferative activity; the 7, 30 and 50 substitu-
tion pattern demonstrated to be a favourable feature for both the
antitumor and the proapoptotic activities.
5.2.1. 4-Bromomethyl-2H-chromen-2-one (4)
Starting from 4-methyl-2H-chromen-2-one52 (1.6 g, 10 mmol),
the desired derivative 4 was obtained (2.0 g, 85% yield); mp 175–
177 °C. 1H NMR (CDCl3): d 4.58 (s, 2H), 6.35 (s, 1H), 7.25–7.42
(m, 2H), 7.51 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H).
5.2.2. 4-Bromomethyl-6-methoxy-2H-chromen-2-one (5)
Starting from 6-methoxy-4-methyl-2H-chromen-2-one52 (1.9 g,
10 mmol), 5 was obtained (2.1 g, 75% yield); mp 198–200 °C. 1H
NMR (CDCl3): d 3.84 (s, 3H), 4.5 (s, 2H), 6.33 (s, 1H), 6.95 (s,
J = 2.0 Hz, 1H), 7.05 (dd, J = 2.0 and 8.4 Hz, 1H), 7.42 (d, J = 8.4 Hz,
1H).
5.2.3. 4-Bromomethyl-7-methoxy-2H-chromen-2-one (6)
Staring from 7-methoxy-4-methyl-2H-chromen-2-one53 (1.9 g,
10 mmol), 6 was obtained (1.95 g, 73% yield); mp 214–215 °C. 1H
NMR (CDCl3): d 3.90 (s, 3H), 4.48 (s, 2H), 6.39 (s, 1H), 6.88 (d,
J = 2.2 Hz, 1H), 6.92 (dd, J = 2 and 8.8 Hz, 1Y, 7.64 (d, J = 8.5 Hz, 1H).
In conclusion, in this study, among the newly synthesized hy-
brid molecules, compounds 14 and 17 were identified to be very
promising candidates; their excellent antiproliferative potency, to-
gether with their remarkable apoptosis-inducing activity, make
them leads of great interest for further studies.
5.2.4. 3-Bromomethyl-7-methoxy-2H-chromen-2-one (7)
Starting from 7-methoxy-3-methylchromen-2-one28 (1.9 g,
10 mmol), 7 was obtained (1.9 g, 71% yield); mp 131–132 °C. 1H
NMR (CDCl3): d 3.89 (s, 3H), 4.43 (s, 2H), 6.80–6.95 (m, 2H), 7.40
(d, J = 8.8 Hz, 1H), 7.80 (s, 1H).
5. Experimental section
5.1. Chemistry
5.1.1. Materials and methods
5.3. General procedure for the preparation of the phosphonic
acid diethyl ester derivatives (8–11)
Chemicals and reagents were obtained from commercial
sources and used without further purification. All melting points
were determined in open glass capillaries using a Büchi apparatus
and are uncorrected. Product mixtures were monitored by thin-
layer chromatography (TLC) on Merk precoated Silica Gel F254
plates. Nuclear magnetic resonance (1H NMR) spectra were re-
corded with a Varian VXR 200 and 300 MHz spectrometers, peaks
positions are given in parts per million downfield from tetrameth-
ylsilane (TMS) as the internal standard and spin multiplicities are
given as s (singlet), d (doublet), t (triplet), q (quartet), dd (double
doublet), dt (double triplet), m (multiplet) or br (broad). Coupling
constants (J) are reported in hertz (Hz). OH protons were detected
upon proton exchange with D2O. Mass spectra were obtained with
Waters Micromass ZQ 4000 (ES-MS spectra) or V. G. 7070 E (EI-MS
spectra) apparatuses. The purity of the tested compounds was deter-
mined by HPLC analysis, performed on a Jasco LC 1500 PU-1587; the
A mixture of bromomethyl derivative 4–7 (10 mmol) and tri-
ethylphosphate (10 mL) was heated at 130 °C for 2 h, then cooled
at room temperature and left to stand overnight. The desired com-
pound precipitates as a white solid that was collected by filtration,
washed with diethyl ether and dried.
5.3.1. (2-Oxo-2H-chromen-4-ylmethyl)phosphonic acid diethyl
ester (8)
Starting from 4 (2.39 g, 10 mmol) 8 was obtained (1.4 g, 60%
yield), mp 195–197 °C. 1H NMR (CDCl3): d 1.27 (t, J = 6.9 Hz, 6H),
3.36 (d, J = 22.8 Hz, 2H), 4.16 (q, J = 7.2 Hz, 4H), 6.30 (s, 1H),
7.01–7.35 (m, 2H), 7.49–7.58 (m, 1H), 7.76 (d, J = 8.2 Hz, 1H). ES-
MS m/z: 319 (M+23). Anal. Calcd for C14H17O5P: C, 56.76; H, 5.78.
Found: C, 56.81; H, 5.72.
column used was a Phenomenex Luna C18(2) 5
elution conditions: mobile phase 70:30 CH3CN/H2O; the flow-rate
was 0.8 ml/min and the injection volume was 5 l; peaks were de-
l
m 4.60 ꢁ 150 mm;
5.3.2. (6-Methoxy-2-oxo-2H-chromen-4-ylmethyl)phosphonic
acid diethyl ester (9)
l
tected at 250 nm and results were >95% purity.Wherever analyses
are only indicated with elements symbols, analytical results
Starting from 5 (2.7 g, 10 mmol), 9 was obtained (2.6 g, 81%
yield); mp 265–268 °C. 1H NMR (CDCl3): d 1.27 (t, J = 6.9 Hz, 6H),

3548
F. Belluti et al. / Bioorg. Med. Chem. 18 (2010) 3543–3550
3.28 (d, J = 22.8 Hz, 2H), 3.88 (s, 3H), 4.11 (q, J = 7.2 Hz, 4H), 6.30 (s,
1H), 7.02 (d, J = 2.0 Hz, 1H), 7.08 (dd, J = 2.0 and 8.4 Hz, 1H), 7.25 (d,
J = 8.4 Hz, 1H). ES-MS m/z: 327 (M+1). Anal. Calcd for C15H19O6P: C,
55.22; H, 5.87. Found: C, 55.29; H, 5.82.
m/z: 361 (M+23). EI-MS m/z: 338.12 (100%), 339.12 (23%). Anal.
Calcd for C20H18O5: C, 70.99; H, 5.36. Found: C, 70.95; H, 5.40.
5.4.4. 7-Methoxy-4-[(E)-2-(3,4,5-trimethoxyphenyl)vinyl]-2H-
chromen-2-one (15)
5.3.3. (7-Methoxy-2-oxo-2H-chromen-4-ylmethyl)phosphonic
acid diethyl ester (10)
Starting from 10 (0.9 g, 3.0 mmol) and 3,4,5-trimethoxybenzal-
dehyde (0.57 g, 3.0 mmol), 15 was obtained (0.80 g, 72% yield); mp
181–182 °C. 1H NMR (CDCl3): d 3.90 (s, 3H), 3.91 (s, 3H), 3.94 (s,
3H), 3.95 (s, 3H), 6.44 (s, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.88 (d,
J = 2.2 Hz, 1H), 6.91 (dd, J = 2.2 and 8.8 Hz, 1H), 7.29 (d,
J = 15.9 Hz, 1H), 7.33 (d, J = 8.6 Hz, 1H), 7.50 (d, J = 15.9 Hz, 1H),
7.71 (d, J = 8.8 Hz, 1H). EI-MS m/z: 368 (100%), 369 (24%). Anal.
Calcd for C21H20O6: C, 68.47; H, 5.47. Found: C, 68.44; H, 5.38.
Staring from 6 (2.7 g, 10 mmol), 10 was prepared (2.3 g, 71%
yield); mp 238–241 °C. 1H NMR (CDCl3): d 1.31 (t, J = 6.9 Hz, 6H),
3.90 (s, 3H), 3.25 (d, J = 22.7 Hz, 2H), 4.07 (q, J = 7.2 Hz, 4H), 6.39
(s, 1H), 6.88 (d, J = 2.2 Hz, 1H), 6.92 (dd, J = 2 and 8.8 Hz, 1Y, 7.64
(d, J = 8.5 Hz, 1H). ES-MS m/z: 349 (M+23). Anal. Calcd for
C15H19O6P: C, 55.22; H, 5.87. Found: C, 55.25; H, 5.83.
5.3.4. (7-Methoxy-2-oxo-2H-chromen-3-ylmethyl) phosphonic
acid diethyl ester (11)
5.4.5. 7-Methoxy-4-[(E)-2-(2,3,4-trimethoxyphenyl)vinyl]-2H-
chromen-2-one (16)
Starting from 7 (2.7 g, 10 mmol), 11 was prepared (2.1 g, 65%
yield); mp 225–228 °C. 1H NMR (CDCl3): d 1.31 (t, J = 7.4 Hz, 6H),
3.15 (d, J = 21.6 Hz, 2H), 3.87 (s, 3H), 4.13 (q, J = 7.4 Hz, 4H),
6.80–6.87 (m, 2H), 7.37 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 4.0 Hz, 1H).
ES-MS m/z: 327 (M+1). Anal. Calcd for C15H19O6P: C, 55.22; H,
5.87. Found: C, 55.17; H, 5.88.
Starting from 10 (0.9 g, 3.0 mmol) and 2,3,4-trimethoxybenzal-
dehyde (0.57 g, 3.0 mmol), 16 was obtained (0.73 g, 66% yield); mp
137–139 °C. 1H NMR (CDCl3): d 3.89 (s, 3H), 3.90 (s, 3H), 3.92 (s,
3H), 3.95 (s, 3H), 6.41 (s, 1H), 6.71 (d, J = 8.8 Hz, 1H), 6.77 (d,
J = 2.2 Hz, 1H), 6.83 (dd, J = 2.2 and 8.8 Hz, 1H), 7.25 (d,
J = 15.9 Hz, 1H), 7.32 (d, J = 8.6 Hz, 1H), 7.50 (d, J = 15.9 Hz, 1H),
7.65 (d, J = 8.8 Hz, 1H). EI-MS m/z: 368 (100%), 369 (24%). Anal.
Calcd for C21H20O6: C, 68.47; H, 5.47. Found: C, 68.49; H, 5.55.
5.4. General procedure for the synthesis of stilbene derivatives
(12–20)
5.4.6. 4-[(E)-2-(3,5-Dimethylphenyl)ethyl]-7-methoxy-2H-chro-
men-2-one (17)
To a solution of a suitable phosphonic acid diethyl ester (8–11)
(3.0 mmol) in DMF (1.5 mL) at 0 °C, NaOCH3 (4.5 mmol) was care-
fully added. The mixture was stirred at the same temperature for
1 h, then the selected aldehyde (3.0 mmol) in DMF (2.5 mL) was
added dropwise and the reaction mixture was stirred at room tem-
perature for 1.5 h and heated at 95 °C for 1 h. After cooling, the
mixture was poured in ice water and left to stand overnight. The
solid formed was collected by filtration, washed with water and
dried. Crystallization from toluene gave the desired stilbene deriv-
ative as a pure E-isomer.
Starting from 10 (0.9 g, 3.0 mmol) and 3,5-dimethylbenzalde-
hyde (0.40 g, 3.0 mmol), 17 was obtained (0.5 g, 66% yield); mp
146–148 °C. 1H NMR (CDCl3):d 2.37 (s, 6H), 3.89 (s, 3H), 6.43 (s,
1H), 6.86 (d, J = 1.8 Hz, 1H), 6.88 (dd, J = 1.8 and 8.7 Hz, 1H), 7.02
(s, 1H), 7.20 (s, 2H), 7.24 (d, J = 16.2 Hz, 1H), 7.30 (d, J = 16.2 Hz,
1H), 7.70 (d, J = 8.4 Hz, 1H). ES-MS m/z: 329 (M+23). Anal. Calcd
for C20H18O3: C, 78.41; H, 5.92. Found: C, 78.31; H, 6.00.
5.4.7. 7-Methoxy-4-[(E)-{2-[4-(3-methylbut-2-enyloxy)phenyl]-
vinyl}-2H-chromen-2-one (18)
5.4.1. 4-(E)-[2-(3,5-Dimethoxyphenyl)vinyl]-2H-chromen-2-one
(12)
Starting from 10 (0.9 g, 3.0 mmol) and 4-(3-methylbut-2-enyl-
oxy)benzaldehyde36 (0.57 g, 3.0 mmol), 18 was obtained (0.77 g,
71% yield); mp 134–136 °C. 1H NMR (CDCl3): d 1.75 (s, 3H), 1.85
(s, 3H), 3.95 (s, 3H), 4.60 (d, J = 6.9 Hz, 2H), 5.48–5.58 (m, 1H),
6.44 (s, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 2.5 Hz, 1H), 6.97
(d, J = 8.8 Hz, 2Y, 7.18 (d, J = 16.0 Hz, 1H), 7.28 (d, J = 16.0 Hz, 1H),
7.53 (d, J = 8.6 Hz, 2H), 7.72 (d, J = 8.5 Hz, 1H). ES-MS m/z: 385
(M+23). Anal. Calcd for C23H22O4: C, 76.22; H, 6.12. Found: C,
76.21; H, 6.05.
Starting from 8 (0.9 g, 3.0 mmol) and 3,5-dimethoxybenzalde-
hyde (0.49 g, 3.0 mmol), 12 was obtained (0.6 g, 65% yield); mp
143–145 °C. 1H NMR (CDCl3): d 3.85 (s, 6H), 6.53 (t, J = 2.2 Hz,
1H), 6.62 (s, 1H), 6.75 (d, J = 2.2 Hz, 2H), 7.25 (d, J = 16.0 Hz, 1H),
7.30 (d, J = 8.6 Hz, 1H), 7.37 (d, J = 16.0 Hz, 1H), 7.35–7.45 (m,
1H), 7.55–7.65 (m, 1H), 7.82 d, J = 8.2 Hz, 1Y. ES-MS m/z: 331
(M+23). Anal. Calcd for C19H16O4: C, 74.01; H, 5.23. Found: C,
73.95; H, 5.16.
5.4.8. 7-Methoxy-4-[(E)-2-(3-methoxyphenyl)vinyl]-2H-chromen-
2-one (19)
5.4.2. 4-[(E)-2-(3,5-Dimethoxyphenyl)vinyl]-6-methoxy-2H-chro-
men-2-one (13)
Starting from 10 (0.9 g, 3.0 mmol) and 3-methoxylbenzalde-
hyde (0.41 g, 3.0 mmol), 19 was obtained (0.49 g, 54% yield), mp
137–139 °C. 1H NMR (CDCl3): d 3.78 (s, 3H), 3.81 (s, 3H), 6.46 (s,
1H), 6.85–6.97 (m, 3H), 7.08 (s, 1H), 7.18 (d, J = 7.6 Hz, 1H);
7.22–7.38 (m, 3H), 7.69 (d, J = 8.1 Hz, 1H). ES-MS m/z: 331
(M+23). Anal. Calcd for C19H16O4: C, 74.01; H, 5.23. Found: C,
73.95; H, 5.19.
Starting from 9 (0.9 g, 3.0 mmol) and 3,5-dimethoxybenzalde-
hyde (0.49 g, 3.0 mmol), 13 was obtained (0.51 g. 51% yield); mp
191–193 °C. 1H NMR (CDCl3): d 3.85 (s, 6H), 3.88 (s, 3H), 6.51 (t,
J = 2.2 Hz, 1H), 6.59 (s, 1H), 6.71 (d, J = 2.2 Hz, 2H), 6.15–6.25 m,
3H), 7.27 (d, J = 8.8 Hz, 1H), 7.29 (d, J = 17.6 Hz, 1H). ES-MS m/z:
338 (M-1) and 361 (M+23). Anal. Calcd for C20H18O5: C, 70.99; H,
5.36. Found: C, 70.90; H, 5.42.
5.4.9. 3-[(E)-2-(3,5-Dimethoxyphenyl)vinyl]-7-methoxy-2H-chro-
men-2-one (20)
5.4.3. 7-Methoxy-4-[(E)-2-(3,5-dimethoxyphenyl)vinyl]-2H-chro-
men-2-one (14)
Starting from 11 (0.9 g, 3.0 mmol) and 3,5-dimethoxybenzalde-
hyde (0.49 g, 3 mmol), 20 was obtained (0.56 g, 55% yield); mp
198–200 °C. 1H NMR (CDCl3) : d 3.82 (s, 6H), 3.88 (s, 3H), 6.41 (t,
J = 2.2 Hz, 1H), 6.69 (d, J = 2.2 Hz, 2H), 6.82 (d, J = 2.2 Hz, 1H),
6.86 (dd, J = 2.2 and 8.8 Hz, 1H), 7.07 (d, J = 16.2 Hz, 1H), 7.42 (d,
J = 8.8 Hz, 1H), 7.48 (d, J = 16.4 Hz, 1H), 7.75 (s, 1H). ES-MS m/z:
Starting from 10 (0.9 g, 3.0 mmol) and 3,5-dimethoxybenzalde-
hyde (0.49 g, 3 mmol), 14 was obtained (0.69 g, 68% yield); mp
176–178 °C. 1H NMR (CDCl3): d 3.85 (s, 6H), 3.89 (s, 3H), 6.43 (s,
1H), 6.49–6.51 (m, 1H), 6.71 (d, J = 2 Hz, 2H), 6.86 (d, J = 2 Hz,
1H), 6.91 (dd, J = 2.2 and 8.8 Hz, 1H), 7.21 (d, J = 15.6 Hz, 1H),
7.27 (d, J = 15.6 Hz, 1H), 7.68 (dd, J = 2.4 and 8.8 Hz, 1H). ES-MS

F. Belluti et al. / Bioorg. Med. Chem. 18 (2010) 3543–3550
3549
361 (M+23). Anal. Calcd for C20H18O5: C, 70.99; H, 5.36. Found: C,
71.05; H, 5.29.
pase 3 (BD Transduction Laboratories, Lexington, USA), anti-
cleaved CCP32 (Asp175) (Cell Signalling Technology, USA), anti-
PARP-1 (Calbiochem, USA), anti-actin (Sigma).
5.4.10. 4-[(E)-2-(3,5-Dihydroxyphenyl)vinyl]-7-hydroxychromen-
2H-2-one (21)
5.9. Co-immunoprecipitation and immunoblot analysis
BBr3 (1 M solution in CH2Cl2, 1.35 mL, 1.35 mmol) was added
dropwise to a solution of 14 (0.1 g, 0.3 mmol) in CH2Cl2 (5 mL) at
0 °C. The solution was warmed to room temperature and stirred
at the same temperature for 4 h. The solid formed was collected
by filtration and purified by flash chromatography on silica gel
(CH2Cl2/methyl alcohol: 9.5/0.5) to obtain 21 (0.028 g, 31% yield);
mp 280–282 °C (dec). 1H NMR (DMSO-d6): d 2.86 (br, 3H), 6.41–
6.43 (m, 2H), 6.74 (d, J = 2.2 Hz, 1H), 6.78 (d, J = 2.4 Hz, 1H), 6.86
(d, J = 2 Hz, 1Y, 6.88 dd, J = 2.4 and 8.8 Hz, 1H), 7.36 (d,
J = 16.0 Hz, 1H), 7.49 (d, J = 15.6 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H).
ES-MS m/z: 297 (M+1). Anal. Calcd for C17H12O5: C, 68.92; H,
4.08. Found: C, 69.05; H, 4.02.
Following the designated treatments, cells were lysed in NET
buffer [50 mmol/L Tris (pH 7.4), 150 mmol/L sodium chloride,
0.1% NP40,1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L
EDTA, 2.5 lg/mL leupeptin, 5 lg/mL aprotinin] first for 30 min on
ice and then for 30 min at 4 °C in rotation. The nuclear and cellular
debris were cleared by centrifugation (10,000g, 10 min, 4 °C). Total
cellular proteins were then quantified using the BCA protein assay.
Cell lysates (500
clonal antibody for 2 h at 4 °C. Protein A-sepharose (Sigma–Al-
drich, Germany) (90 l), previously prepared in TNT solution
lg) were incubated with the cdc-2-specific mono-
l
[20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton X-100] was
added to the lysates and incubated overnight at 4 °C. The immuno-
precipitates (separated by centrifugation 15,000g, 2 min, 4 °C)
were washed twice in NET buffer and twice in PBS with 1% aproti-
nin and 1 mmol/L PMSF. Proteins were eluted with the SDS sample
loading buffer before the immunoblot analyses with specific anti-
bodies against p34cdc2 or p21 (Santa Cruz).
5.5. Cellular sensitivity to drugs
Cellular sensitivity to drugs was evaluated by growth-inhibition
assay 72 h after drug exposure. Tumour cells, in the logarithmic
phase of growth, were seeded in duplicate into 6-well plates.
Twenty-four hours after seeding, the drug was added to the med-
ium and 72 h after drug exposure cells were harvested and counted
with a cell counter. IC50 is defined as the drug concentration caus-
ing a 50% reduction of cell number compared with that of un-
treated control.
Acknowledgements
This work was partially supported by the Associazione Italiana
per la Ricerca sul Cancro, Milan and by the Fondazione CARIPLO,
Milan, Italy.
5.6. Determination of apoptosis
References and notes
Apoptosis was determined in H460 lung carcinoma cells by
TUNEL assay following a 72 h exposure to the drug. Floating and
adherent cells were fixed in 4% paraformaldehyde, for 60 min, at
room temperature, washed and resuspended in ice-cold PBS. The
in situ cell death detection kit fluorescein (Roche, Mannheim, Ger-
many) was used according to the manufacturers’ instructions and
the samples were analyzed by flow cytometry (Becton Dickinson).
1. Sloane, D. Methods Mol. Biol. 2009, 471, 65.
2. Hotta, K.; Ueoka, H. Crit. Rev. Oncol. Hematol. 2005, 55, 45.
3. Fesik, S. W. Nat. Rev. Cancer 2005, 5, 876.
4. Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte, E. Curr. Med. Chem.
2005, 12, 887.
5. Riveiro, M. E.; Moglioni, A.; Vazquez, R.; Gomez, N.; Facorro, G.; Piehl, L.; de
Celis, E. R.; Shayo, C.; Davio, C. Bioorg. Med. Chem. 2008, 16, 2665.
6. Feuer, G.; Kellen, J. A.; Kovacs, K. Oncology 1976, 33, 35.
7. Kashman, Y.; Gustafson, K. R.; Fuller, R. W.; Cardellina, J. H.; McMahon, J. B.;
Currens, M. J.; Buckheit, R. W.; Hughes, S. H.; Cragg, G. M.; Boyd, M. R. J. Med.
Chem. 1993, 36, 1110.
5.7. Cell cycle analysis
Early events associated with drug effects (cell cycle perturba-
tion and biochemical modulation) were examined within 24 h of
drug exposure to avoid any influence of indirect effects of cellular
response. Cell cycle analyses were performed in H460 cells 24 h
after drug treatments. Briefly, cells were washed, fixed in ice-cold
70% ethanol, and stored at ꢂ20 °C. Subsequently, samples were
8. Shikishima, Y.; Takaishi, Y.; Honda, G.; Ito, M.; Takfda, Y.; Kodzhimatov, O. K.;
Ashurmetov, O.; Lee, K. H. Chem. Pharm. Bull. 2001, 49, 877.
9. Gage, B. F. Hematol. Am. Soc. Hematol. Educ. Program. 2006, 467.
10. Ostrov, D. A.; Hernández Prada, J. A.; Corsino, P. E.; Finton, K. A.; Le, N.; Rowe, T.
C. Antimicrob. Agents Chemother. 2007, 51, 3688.
11. Gormley, N. A.; Orphanides, G.; Meyer, A.; Cullis, P. M.; Maxwell, A.
Biochemistry 1996, 35, 5083.
12. Fylaktakidou, K. C.; Hadjipavlou-Litina, D. J.; Litinas, K. E.; Nicolaides, D. N. Curr.
Pharm. Des. 2004, 10, 3813.
rehydratated with PBS and cellular DNA was stained with 10 lg/
13. Whang, W. K.; Park, H. S.; Ham, I.; Oh, M.; Namkoong, H.; Kim, H. K.; Hwang, D.
W.; Hur, S. Y.; Kim, T. E.; Park, Y. G.; Kim, J. R.; Kim, J. W. Exp. Mol. Med. 2005,
37, 436.
14. Thornes, D.; Daly, L.; Lynch, G.; Browne, H.; Tanner, A.; Keane, F.; O’Loughlin, S.;
Corrigan, T.; Daly, P.; Edwards, G.; Breslin, B.; Browne, H.; Shine, M.; Lennon, F.;
Hanley, J.; McMurray, N.; Gaffney, E. Eur. J. Surg. Oncol. 1989, 15, 431.
15. Riveiro, M. E.; Vazquez, R.; Moglioni, A.; Gomez, N.; Baldi, A.; Davio, C.; Shayo,
C. Biochem. Pharmacol. 2008, 75, 725.
ml propidium iodide in PBS, containing RNase A (66 U/ml). Cell cy-
cle distribution was determined by flow cytometry, and data were
analyzed by Cell QuestÒ software; for each sample 40,000 events
were collected.
5.8. Protein expression analysis
16. Alvarez-Delgado, C.; Reyes-Chilpa, R.; Estrada-Muniz, E.; Mendoza-Rodriguez,
C. A.; Quintero-Ruiz, A.; Solano, J.; Cerbon, M. A. J. Biochem. Mol. Toxicol. 2009,
23, 263.
17. Omarbasha, B.; Fair, W. R.; Heston, W. D. W. Cancer Res. 1989, 49, 3045.
18. Weber, U. S.; Steffen, B.; Siegers, C. P. Res. Commun. Mol. Pathol. Pharmacol.
1998, 99, 193.
19. Marshall, M. E.; Kervin, K.; Benefield, C.; Umerani, A.; Albainy-Jenei, S.; Zhao,
Q.; Khazaeli, M. B. J. Cancer Res. Clin. Oncol. 1994, 120.
20. Kim, E. K.; Kwon, K. B.; Shin, B. C.; Seo, E. A.; Lee, Y. R.; Kim, J. S.; Park, J. W.;
Park, B. H.; Ryu, D. G. Life Sci. 2005, 77, 824.
Total cell lysates were prepared rinsing cells twice with ice-cold
PBS supplemented with 0.1 mM sodium orthovanadate, and then
lysing them in hot sample buffer.54 After determination of protein
concentration, whole-cell extracts were separated by SDS poly-
acrylamide gel electrophoresis (PAGE) and transferred onto nitro-
cellulose membranes. Detection of proteins was accomplished
using horseradish peroxidase-conjugated secondary antibodies
and enhanced chemiluminescence purchased from Amersham Bio-
sciences, Rockford, IL. Primary antibodies used in this study are:
anti-p21 (NeoMarker, USA), anti-p34cdc2 (GIBCO, Invitrogen, NY,
USA), anti-cyclin B1 (Santa Cruz Biotechnology, USA), anti-pro-cas-
21. Chu, C. Y.; Tsai, Y. Y.; Wang, C. J.; Lin, W. L.; Tseng, T. H. Eur. J. Pharmacol. 2001,
416, 25.
22. Chimichi, S.; Boccalini, M.; Salvador, A.; Dall’Acqua, F.; Basso, G.; Viola, G.
ChemMedChem 2009, 4, 769.
´
ˇ
23. Cavar, S.; Kovac, F.; Maksimovic, M. Food Chem. 2009, 117, 135.

3550
F. Belluti et al. / Bioorg. Med. Chem. 18 (2010) 3543–3550
24. Ghate, M.; Kusanur, R. A.; Kulkarni, M. V. Eur. J. Med. Chem. 2005, 40, 882.
25. Khan, K. M.; Saify, Z. S.; Khan, M. Z.; Zia, U.; Choudhary, I. M.; Atta Ur, R.;
Perveen, S.; Chohan, Z. H.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2004, 19,
373.
26. Kirkiacharian, S.; Thuy, D. T.; Sicsic, S.; Bakhchinian, R.; Kurkjian, R.; Tonnaire,
T. Farmaco 2002, 57, 703.
27. Kontogiorgis, C. A.; Hadjipavlou-Litina, D. J. J. Med. Chem. 2005, 48, 6400.
28. Leonetti, F.; Favia, A.; Rao, A.; Aliano, R.; Paluszcak, A.; Hartmann, R. W.;
Carotti, A. J. Med. Chem. 2004, 47, 6792.
29. Raad, I.; Terreux, R.; Richomme, P.; Matera, E. L.; Dumontet, C.; Raynaud, J.;
Guilet, D. Bioorg. Med. Chem. 2006, 14, 6979.
30. Riveiro, M. E.; Maes, D.; VÃÀzquez, R.; Vermeulen, M.; Mangelinckx, S.; Jacobs,
J.; Debenedetti, S.; Shayo, C.; De Kimpe, N.; Davio, C. Bioorg. Med. Chem. 2009,
17, 6547.
31. Valenti, P.; Fabbri, G.; Rampa, A.; Bisi, A.; Gobbi, S.; Da Re, P.; Carrara, M.;
Sgevano, A.; Cima, L. Anticancer Drug Des. 1996, 11, 243.
32. Burns, J.; Yokota, T.; Ashihara, H.; Lean, M. E. J.; Crozier, A. J. Agric. Food Chem.
2002, 50, 3337.
40. Jang, M.; Cai, L.; Udeani, G. O.; Slowing, K. V.; Thomas, C. F.; Beecher, C. W. W.;
Fong, H. H. S.; Farnsworth, N. R.; Kinghorn, A. D.; Mehta, R. G.; Moon, R. C.;
Pezzuto, J. M. Science 1997, 275, 218.
41. Sun, W.; Wang, W.; Kim, J.; Keng, P.; Yang, S.; Zhang, H.; Liu, C.; Okunieff, P.;
Zhang, L. Adv. Exp. Med. Biol. 2008, 179.
42. Simoni, D.; Roberti, M.; Invidiata, F. P.; Aiello, E.; Aiello, S.; Marchetti, P.;
Baruchello, R.; Eleopra, M.; Di Cristina, A.; Grimaudo, S.; Gebbia, N.; Crosta, L.;
Dieli, F.; Tolomeo, M. Bioorg. Med. Chem. Lett. 2006, 16, 3245.
43. Pettit, G. R.; Grealish, M. P.; Jung, M. K.; Hamel, E.; Pettit, R. K.; Chapuis, J. C.;
Schmidt, J. M. J. Med. Chem. 2002, 45, 2534.
44. Roberti, M.; Pizzirani, D.; Simoni, D.; Rondanin, R.; Baruchello, R.; Bonora, C.;
Buscemi, F.; Grimaudo, S.; Tolomeo, M. J. Med. Chem. 2003, 46, 3546.
45. Sale, S.; Verschoyle, R. D.; Boocock, D.; Jones, D. J. L.; Wilsher, N.; Ruparelia, K.
C.; Potter, G. A.; Farmer, P. B.; Steward, W. P.; Gescher, A. J. Br. J. Cancer 2004,
90, 736.
46. Sale, S.; Tunstall, R. G.; Ruparelia, K. C.; Potter, G. A.; Steward, W. P.; Gescher, A.
J. Int. J. Cancer 2005, 115, 194.
47. Meunier, B. Acc. Chem. Res. 2007, 41, 69.
33. Fre’mont, L. Life Sci. 2000, 66, 663.
34. Udenigwe, C. C.; Ramprasath, V. R.; Aluko, R. E.; Jones, P. J. H. Nutr. Rev. 2008,
66, 445.
35. Burns, J.; Gardner, P. T.; O’Neil, J.; Crawford, S.; Morecroft, I.; McPhail, D. B.;
Lister, C.; Matthews, D.; MacLean, M. R.; Lean, M. E. J.; Duthie, G. G.; Crozier, A.
J. Agric. Food Chem. 2000, 48, 220.
36. Pace-Asciak, C. R.; Hahn, S.; Diamandis, E. P.; Soleas, G.; Goldberg, D. M. Clin.
Chim. Acta 1995, 235, 207.
37. Savouret, J. F.; Quesne, M. Biomed. Pharmacother. 2002, 56, 84.
38. Dorrie, J.; Gerauer, H.; Wachter, Y.; Zunino, S. J. Cancer Res. 2001, 61, 4731.
39. Aggarwal, B. B.; Bhardwaj, A.; Aggarwal, R. S.; Seeram, N. P.; Shishodia, S.;
Takada, Y. Anticancer Res. 2004, 24, 2783.
48. Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Yánez, M.; Fraiz, N.; Alcaide, C.;
Cano, E.; Orallo, F. Bioorg. Med. Chem. Lett. 2006, 16, 257.
49. Song, H. Y.; Ngai, M. H.; Song, Z. Y.; MacAry, P. A.; Hobley, J.; Lear, M. J. Org.
Biomol. Chem. 2009, 7, 3400.
50. Melagraki, G.; Afantitis, A.; Igglessi-Markopoulou, O.; Detsi, A.; Koufaki, M.;
Kontogiorgis, C.; Hadjipavlou-Litina, D. J. Eur. J. Med. Chem. 2009, 44, 3020.
51. Silverstein, E. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of
Organic Compounds; John Wiley and Sons: New York, 1981. pp 264–265.
52. Mali, R. S.; Yadav, V. J. Synthesis 1997, 1977, 464.
53. von Pechmann, H.; Duisberg, C. U. Chem. Ber. 1883, 16, 2119.
54. Zuco, V.; Zanchi, C.; Cassinelli, G.; Lanzi, C.; Supino, R.; Pisano, C.; Zanier, R.;
Giordano, V.; Garattini, E.; Zunino, F. Cell Death Differ. 2004, 11, 280.