Synthesis and biological evaluation of resveratrol and analogues as apoptosis-inducing agents

Article abstract of DOI:10.1021/jm030785u

Resveratrol 1 (3,4′,5-trihydroxy-trans-stilbene), a phytoalexin present in grapes and other food products, has recently been suggested as a potential cancer chemopreventive agent based on its striking inhibitory effects on cellular events associated with cancer initiation, promotion, and progression. This triphenolic stilbene has also displayed in vitro growth inhibition in a number of human cancer cell lines. In this context, a series of cis- and trans-stilbene-based resveratrols were prepared with the aim of discovering new lead compounds with clinical potential. All the synthesized compounds were tested in vitro for cell growth inhibition and the ability to induce apoptosis in HL60 promyelocytic leukemia cells. The tested trans-stilbene derivatives were less potent than their corresponding cis isomers, except for trans-resveratrol, whose cis isomer was less active. The best results were obtained with compounds 11b and 7b, the cis-3,5-dimethoxy derivatives of rhapontigenin 10a (3,5,3′-trihydroxy-4′methoxy-transstilbene) and its 3′-amino derivative 10b, respectively, which showed apoptotic activity at nanomolar concentrations. The corresponding trans isomers 12b and 8b were less active both as antiproliferative and as apoptosis-inducing agents. Of interest, 11b and 7b were active toward resistant HL60R cells and their activity was higher than that of several classic chemotherapeutic agents. The flow cytometry assay showed that at 50 nM compounds 7b or 11b were able to recruit almost all cells in the apoptotic sub-G₀-G₁ peek, thus suggesting that the main mechanism of cytotoxicity of these compounds could be the activation of apoptosis. These data indicate unambiguously that structural alteration of the stilbene motif of resveratrol can be extremely effective in producing potent apoptosis-inducing agents.

Full text of DOI:10.1021/jm030785u

3546
J . Med. Chem. 2003, 46, 3546-3554
Syn th esis a n d Biologica l Eva lu a tion of Resver a tr ol a n d An a logu es a s
Ap op tosis-In d u cin g Agen ts
Marinella Roberti,† Daniela Pizzirani,† Daniele Simoni,*,# Riccardo Rondanin,# Riccardo Baruchello,#
Caterina Bonora,# Filippo Buscemi,‡ Stefania Grimaudo,‡ and Manlio Tolomeo‡
Dipartimento di Scienze Farmaceutiche, Universita` di Bologna, Italy, Divisione di Ematologia e Servizio AIDS,
Policlinico di Palermo, Palermo, Italy, and Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara,
Via Fossato di Mortara 17-19, 44100 Ferrara, Italy
Received J anuary 23, 2003
Resveratrol 1 (3,4′,5-trihydroxy-trans-stilbene), a phytoalexin present in grapes and other food
products, has recently been suggested as a potential cancer chemopreventive agent based on
its striking inhibitory effects on cellular events associated with cancer initiation, promotion,
and progression. This triphenolic stilbene has also displayed in vitro growth inhibition in a
number of human cancer cell lines. In this context, a series of cis- and trans-stilbene-based
resveratrols were prepared with the aim of discovering new lead compounds with clinical
potential. All the synthesized compounds were tested in vitro for cell growth inhibition and
the ability to induce apoptosis in HL60 promyelocytic leukemia cells. The tested trans-stilbene
derivatives were less potent than their corresponding cis isomers, except for trans-resveratrol,
whose cis isomer was less active. The best results were obtained with compounds 11b and 7b,
the cis-3,5-dimethoxy derivatives of rhapontigenin 10a (3,5,3′-trihydroxy-4′methoxy-trans-
stilbene) and its 3′-amino derivative 10b, respectively, which showed apoptotic activity at
nanomolar concentrations. The corresponding trans isomers 12b and 8b were less active both
as antiproliferative and as apoptosis-inducing agents. Of interest, 11b and 7b were active
toward resistant HL60R cells and their activity was higher than that of several classic
chemotherapeutic agents. The flow cytometry assay showed that at 50 nM compounds 7b or
11b were able to recruit almost all cells in the apoptotic sub-G0-G1 peek, thus suggesting that
the main mechanism of cytotoxicity of these compounds could be the activation of apoptosis.
These data indicate unambiguously that structural alteration of the stilbene motif of resveratrol
can be extremely effective in producing potent apoptosis-inducing agents.
In tr od u ction
Stilbene-based compounds are widely represented in
nature and have become of particular interest to chem-
ists and biologists because of their wide range of
biological activities.1,2 Stilbene itself does not occur in
nature, but hydroxylated stilbenes have been found in
a multitude of medicinal plants. Resveratrol 1 (3,4′,5-
F igu r e 1. Resveratrol (1) and stilbene-related proapoptotic
trihydroxy-trans-stilbene, Figure 1), a phytoalexin present
compounds.
in grapes and other food products,2-4 has been reported
to play a role in the prevention of heart diseases
The simplicity of resveratrol, associated with its
associated with red wine consumption because it inhib-
interesting anticancer activity, offers promises for the
its platelet aggregation,5 alters eicosanoid synthesis,5,6
rational design of new chemotherapeutic agents, and in
and modulates lipid and lipoprotein metabolism.7,8
this context, efforts have recently been devoted to the
Resveratrol has recently been suggested as a potential
detailed study of structure-activity relationships (SAR)
cancer chemopreventive agent based on its striking
of this type of substituted stilbene derivative.12-15
inhibitory effects on cellular events associated with
Resveratrol has also been shown to induce apoptosis in
cancer initiation, promotion, and progression.9 This
different cancer cell lines.16-18 Since reduced apoptosis
triphenolic stilbene possesses strong antioxidative10 and
(programmed cell death) has been implicated in the
antiinflammatory6 activities that may contribute to its
development and progression of malignant tumors19,20
chemopreventive/chemoprotective properties. In addi-
and in the occurrence of chemoresistant phenotypes,21-24
tion to the anticancer-promoting activity, 1 has dis-
resveratrol-induced apoptosis might therefore contribute
played in vitro growth inhibition in a number of human
to its antitumor activity.
cancer cell lines.11
Because we recently started a study aimed at evalu-
ating the apoptotic activity of a novel class of stilbene
compounds structurally related to vitamin A25,26 and
because some derivatives (2a ,b; Figure 1) were found
to be endowed with potent apoptotic activity in both
* To whom correspondence should be addressed. Phone: +39-0532-
291291. Fax: +39-0532-291296. E-mail: smd@dns.unife.it.
† Universita` di Bologna.
#
Universita` di Ferrara.
‡ Policlinico di Palermo.
10.1021/jm030785u CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/01/2003

Resveratrol and Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3547
Sch em e 1a
a
Reagents and conditons: (a) n-BuLi, THF, -78 °C.
Sch em e 2a
normal and MDR cell lines,27,28 we believed that it would
be further informative to deeply explore other classes
of related stilbenes as a logical starting point in the
quest of novel anticancer chemotherapeutics. Therefore,
on the bases of these premises, we prepared various
derivatives in order to explore new areas of structural
alteration of resveratrol. A variety of substituents were
introduced at positions 3′ and 4′ (OH, NH2, OCH3), and
the replacement of the 3,5-hydroxy groups with methoxy
functions was also investigated in this series, together
with the determination of the effects of double bond
isomerization (E and Z geometries).
All the synthesized compounds were tested in vitro
for cell growth inhibition and the ability to induce
apoptosis in HL60 premyelocytic leukemia cells. Two
of them (7b and 11b) showed an activity comparable to
that of daunorubicin and proved to be potent apoptosis-
inducing agents also active in multidrug-resistant (MDR)
cell lines, in particular resistant to the apoptotic effects
of several chemoterapeutic drugs such as daunorubicin,
etoposide, and citarabine.
a
Reagents and conditons: (a) Na2S2O4, acetone/H2O, 50 °C.
higher field compared with the E isomers. The coupling
constants of the vinylic protons of the E isomers were
about 16 Hz, whereas the Z isomers showed coupling
constants of 12 Hz. Nitro derivatives 5c,d ,i and the
corresponding E isomers 6c,d ,i were then reduced with
sodium dithionite to give the respective amino deriva-
tives 7a -c and 8a -c in satisfactory yields (Scheme 2).
Finally, deprotection of the benzyloxy derivatives
5a ,b, 7a and 6a ,b, 8a performed with a complex
prepared from aluminum chloride and N,N-dimethyla-
niline gave the desired compounds 9a -c and 1, 10a ,b
(Scheme 3), whereas removal of the tert-butyldimeth-
ylsilyl (TBDMS) group from stilbenes 5e-h and 6e-h
using tetrabutylammonium fluoride afforded compounds
11a -d and 12a -d in appreciable yields (Scheme 4).
Ch em istr y
Preparation of the stilbene derivatives 5a -i and 6a -i
was accomplished by means of the Wittig reaction
between the appropriate aromatic aldehydes 4a -h and
the suitable aromatic ylides, in turn obtained from the
phosphonium salts 3a ,b (Scheme 1). The reaction
produced a mixture of E and Z isomers (7:3) purified
and separated by flash chromatography. Spectral data
of known and new compounds were consistent with
those of stilbene derivatives previously described in the
literature. Bond geometry of compounds was established
1
by comparison of the H NMR of the isomeric pairs. Z
isomers showed the olefinic protons at 0.3-0.4 ppm

3548 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Roberti et al.
Sch em e 3a
cis-resveratrol 9c was scarcely active, being its IC50 was
10 times higher than that of trans-resveratrol and
having its AC50 higher than 200 µM. This was somewhat
surprising to us because the potent apoptotic activity
of other cis-stilbenoids is well documented.27,28 The
natural rhapontigenin 10a and its 3′-amino derivative
10b had low cytotoxic activity and the respective cis
isomers 9a and 9b were only slightly more active.
Pterostilbene 12a , the natural 3,5-dimethoxy analogue
of resveratrol, was less active as antiproliferative and
apoptosis-inducing agent than the parent compound.
However, unlike the cis derivatives 9a -c described
above, the cis isomer 11a showed interesting cytotoxicity
associated with potent apoptotic activity (IC50 ) 2 µM;
AC50 ) 5 µM). Indeed, although this compound had an
antiproliferative activity of only 2.5-fold higher than
trans-resveratrol, its ability to induce programmed cell
death was 10-fold higher.
On the basis of these results, it appeared convenient
to investigate the synthesis of new resveratrol analogues
bearing the 3,5-dimethoxy motif at the A phenyl ring;
moreover, amino, methoxy, and hydroxy moieties were
considered at the 3′- and 4′-positions. The cytotoxic and
the apoptotic activities were both diminished when the
cis- and trans-pterostilbene analogues were tested as
4′-O-TBDMS (compounds 5e and 6e). When the 4′-OH
group was replaced by the amino function, the activity
of the trans isomer 8c was higher than that of the
parent compound 12a , whereas the cis derivative 7c
showed an activity comparable to the cis-pterostilbene
(11a ). Additionally, it is worth noting that in switching
the methoxy and hydroxy moieties as in compounds 11b
and 12b vs 11c and 12c, we may greatly influence the
activity, given that derivatives 3′-hydroxy-4′-methoxy
(11b and 12b) appear more active than the 3′-methoxy-
4′-hydroxy compounds 11c and 12c. A positive effect if
compared with pterostilbene 12a was obtained by the
introduction of a second hydroxy function at the 3′-
position (compounds 11d and 12d ), which resulted in
about a 40- to 70-fold increase in both antiproliferative
and apoptotic activity. The best results in this series
were obtained with compounds 11b and 7b, the cis-3,5-
dimethoxy derivatives of rhapontigenin 10a , and its 3′-
amino derivative 10b. They were active at nanomolar
concentrations (IC50 and AC50 of about 35 nM), whereas
the corresponding trans isomers 12b and 8b were less
active as antiproliferative and apoptosis-inducing agents.
a
Reagents and conditons: (a) AlCl3, (CH3)2NC6H5, CH2Cl2, 0
°C.
Sch em e 4a
a
Reagents and conditons: (a) Bu4NF, THF, room temp.
Resu lts a n d Discu ssion
The interesting anticancer properties of resveratrol
combined with our previous finding concerning the
remarkable apoptotic activity associated with the stil-
bene motif of arotinoids25-28 prompted us to investigate
a series of resveratrol structural modifications as an
extension of our quest for novel stilbene-based anti-
cancer chemotherapeutics. The new resveratrol-based
analogues were prepared with the aim of discovering
new lead compounds with clinical potential. Particular
attention was focused on the identification of new
structural features for apoptotic activity with the main
objective of investigating the importance of resveratrol’s
phenolic moieties in conferring cell death induction
activity.
All the new synthesized compounds were assayed in
vitro for cell growth inhibition and for their ability to
induce apoptosis in HL60 cells. As shown in Table 1,
trans-resveratrol 1 was effective as an antiproliferative
agent in HL60 cells with an IC50 of 5 µM. In contrast,
it was less effective as an apoptosis-inducing agent,
showing an AC50 of only 50 µM. Quite surprisingly, the
As shown in Table 1, compounds 7b and 11b are the
only compounds having similar IC50 and AC50. This
suggests that the main mechanism of cytotoxicity of
these compounds could be the activation of apoptosis.
The flow cytometry assay conducted in HL60 cells after
staining with propidium iodide showed that at 50 nM
compounds 7b and 11b were able to recruit almost all
cells in the apoptotic sub-G0-G1 peak (Figure 2). The
activity of these compounds was higher than several
classic chemotherapeutic agents commonly used in
clinical practice, such as etoposide, citarabine, 5-fluo-
ruracil, and cis-platinum, but lower than that shown
by daunorubicin (Table 2). However, unlike daunoru-
bicin, etoposide, and citarabine, compounds 7b and 11b
were also active in HL60R cells, a cell line derived from
HL60 and expressing the multidrug-resistance pheno-
type (Table 3). It is worth noting that the classical MDR

Resveratrol and Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3549
F igu r e 2. Flow cytometry determination of apoptosis. HL60 cells were exposed to 50 nM of 7b (b) or 11b (c). After 48 h, the cells
were collected and stained with PI. Apoptosis was determined by FACscan flow cytometry assay using the CellQuest software.
The hypodiploid sub-G0-G1 peak, designed as AP, represents cells undergoing apoptosis. The data for the control are shown in (a).
Ta ble 1. Antiproliferative (IC50) and Apoptotic-Inducing Activity (AC50) of trans-Resveratrol 1 and Its Derivatives in the HL60 Cell
Line: Comparison of Cis and Trans Compounds
reversing agent verapamil was unable to completely
reverse the resistance of HL60R cells toward drugs such
as daunorubicin and etoposide (Table 4). On the con-
trary, the IC50 and AC50 of compounds 7b and 11b
evaluated in resistant HL60R cells were almost similar
to those obtained in sensitive parental cells, also without
use of verapamil. Resistance to chemotherapeutic agents
is a major problem in cancer therapy. This phenomenon

3550 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Roberti et al.
Ta ble 2. IC50 and AC50 of Daunorubicin, Etoposide,
Citarabine, 5-Fluorouracil, and cis-Platinum in the HL60 Cell
Line: Comparison with 7b and 11b
dimethoxy motif may be required for good proapoptotic
action. Moreover, the greater activity of 3′,4′-dihydroxy
analogue 11d with respect to the monohydroxylated
derivative 11a pointed out the importance of a hydroxy
function at the C-3′ position. This finding was also
supported by comparing the activity of compound 11b
(AC50 ) 0.04 µM) and its isomer 11c (AC50 ) 38 µM) in
which the 3′-hydroxy function was switched with the
methoxy group at C-4′. Finally, replacement of a 3′-
hydroxy group with the amino function did not affect
the proapoptotic activity. Thus, the introduction of
either a hydroxy or an amino group at the C-3′ position
and a methoxy group at C-3, C-4′, and C-5 produces the
greatest proapoptotic effects as seen in compounds 7b
and 11b.
compd
IC50 (µM)
AC50 (µM)
daunorubicin
etoposide
citarabine
5-fluorouracil
cis-platinum
7b
0.005 ( 0.0017
0.123 ( 0.07
0.08 ( 0.01
1.5 ( 0.3
0.04 ( 0.006
0.45 ( 0.08
0.15 ( 0.05
6.5 ( 0.8
0.6 ( 0.08
1.2 ( 0.1
0.03 ( 0.005
0.03 ( 0.0012
0.04 ( 0.0012
0.04 ( 0.0045
11b
Ta ble 3. IC50 and AC50 of 7b and 11b in the HL60R Cell
Line: Comparison with Daunorubicin, Etoposide, and
Citarabine
compd
IC50 (µM)
AC50 (µM)
In summary, structural alteration of the natural
trans-resveratrol offered the discovery of two potent
apoptosis-inducing agents. The activity of these com-
pounds was higher than that of several classic chemo-
therapeutic agents, and they could be useful for treat-
ment of malignancies expressing the MDR phenotype.
The cis stereochemistry of the novel stilbenoids seems
to be associated with their potent apoptotic activity.
Consequently, because the cis-resveratrol does not pos-
sess activity, the interpretation of these data does not
appear straightforward. One possibility is that the
activity of 7b and 11b could be related, in some ways,
to their structural similarity to other known natural
stilbenes such as combretastatine A4 and its ana-
logues.36 However, of note, our data suggest unambigu-
ously that structural alteration of the stilbene motif of
resveratrol can be extremely effective in producing
potent apoptosis-inducing agents, and our work is
continuing in the exploration of structurally restricted
derivatives.
7b
11b
daunorubicin
etoposide
citarabine
0.035 ( 0.002
0.025 ( 0.003
1.5 ( 0.2
0.05 ( 0.0012
0.03 ( 0.0045
10 ( 8
6.5 ( 0.9
15 ( 3.2
0.35 ( 0.04
8 ( 12
Ta ble 4. IC50 of 7b, 11b, Daunorubicin, and Etoposide in
Combination with 5 µM Verapamil in the HL60R Cell Line
compd
IC50 (µM)
7b
0.032 ( 0.004
0.02 ( 0.003
0.04 ( 0.003
0.5 ( 0.008
11b
daunorubicin
etoposide
has often been ascribed to the overexpression of multi-
drug transporters on the surface membrane of cancer
cells such as P-glycoprotein (which confer the classic
MDR phenotype) or to qualitative and quantitative
alterations in anticancer drug targets such as topo-
isomerases and thimidylate synthase.29-31
Several MDR-reversing agents are known, and they
are currently investigated in various stages of clinical
development. However, despite the interesting results
obtained in vitro with MDR modulators such as calcium
channel blockers, antimalarials, antibiotics, cyclospor-
ins, hormones, and others and despite occasional re-
sponses in patients with hematologic malignancies or
non-small-cell lung cancer,32-34 several limitations to
the use of these modulators are well-known, including
multiple cellular mechanisms of resistance, alterations
in pharmacokinetics of cytotoxic agents, and clinical
toxicities.35 Therefore, the development of compounds
active toward sensitive and MDR cells such as 7b and
11b could be a useful tool for the treatment of malig-
nancies expressing the MDR phenotype.
Additionally, a general conclusion arising from the
SAR study of resveratrol analogues based on their
apoptotic activity on the HL60 cell line can be offered.
All the tested trans-stilbene analogues were less potent
than their corresponding cis isomers excepting trans-
resveratrol, whose cis counterpart was inactive. There-
fore, although we recently hypothesized that apoptotic
activity may be associated with the cis stereochemistry
of stilbenoids,25,27 this cannot be considered an absolute
prerequisite. However, the cis configuration of the
stilbene architecture seems to be at least an important
feature in conferring apoptotic activity. The low activity
of compounds 9a -c, compared with the good activity
shown by the corresponding 3,5-dimethoxy derivatives
11a ,b and 7b, suggests that introduction of the 3,5-
Ma ter ia ls a n d Meth od s
All solvents were redistilled prior to use. All reactions were
carried out under an inert atmosphere. Solvent extracts of
aqueous solutions were dried over anhydrous sodium sulfate.
Reactions were monitored by thin-layer chromatography (TLC)
on precoated silica gel plates (Kieselgel 60 F254, Merck). Flash
column chromatographies were performed on silica gel (par-
ticle size of 40-63 µM, Merck). Melting points were determined
on a Gallenkamp melting point apparatus and are uncorrected.
NMR spectra were recorded on a Varian VXR 300 instruments
with CDCl3 (TMS as internal reference) as solvent unless
otherwise noted. IR spectra were obtained on a Nicolet Avatar
320 E.S.P. instrument; νmax is expressed in cm-1. The elemen-
tal composition of compounds agreed to within (0.4% of the
calculated value. When the elemental analysis is not included,
crude compounds were used in the next step without further
purification.
Compounds 4a and 4b were obtained by benzylation of
commercially available 4-hydroxybenzaldehyde and 3-hydroxy-
4-methoxybenzaldehyde, respectively. Compounds 4d ,12 4e,37
4f,38 and 4g38 were obtained from the commercially available
4-hydroxybenzaldehyde, 3-hydroxy-4-methoxybenzaldehyde,
4-hydroxy-3-methoxybenzaldehyde, and 3,4-dihydroxybenzal-
dehyde, respectively, by protection of phenols with the tert-
butyldimethylsilyl group (TBDMS) following standard proce-
dure.
Gen er a l P r oced u r e for P r ep a r a tion of Stilben es 5a -i
a n d 6a -i. To the phosphonium bromide salt 3a ,b (1.0 equiv)
in anhydrous tetrahydrofuran at -78 °C was added n-
butyllithium (2 M in hexanes, 1.0 equiv), and the resulting
red solution was stirred under nitrogen for 2 h. A solution of
aldehydes 4a -h (1.0 equiv) in tetrahydrofuran was added
dropwise over 30 min, and the mixture was stirred for 2-6 h

Resveratrol and Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3551
at room temperature. The resulting suspension was poured
into water and extracted with dichloromethane. The organic
phase was washed with brine, and removal of the solvent in
vacuo afforded a mixture of the cis/ trans-stilbenes 5a -i and
6a -i that were separated by flash chromatography (9.7:0.3
petroleum ether/ethyl acetate). The cis-stilbenes were eluted
first, followed by the trans isomers.
106.6, 111.6, 121.3, 122.8, 128.7, 129.9, 130.2, 139.5, 144.4,
150.2, 160.5. 6f: 0.24 g (18% yield); yellow powder; mp 55 °C;
1H NMR δ 0.25 (s, 6H), 1.10 (s, 9H), 6.43 (t, 1H, J ) 2.2 Hz),
6.71-6.72 (m, 1H), 6.87 (m, 1H), 6.92 (d, 1H, J ) 16.6 Hz),
6.95 (m, 2H), 7.08-7.13 (m, 2H); 13C NMR δ -4.7, 18.4, 25.7,
29.6, 55.1, 99.5, 104.1, 111.8, 118.5, 120.4, 126.4, 128.6, 130.1,
139.4, 144.9, 150.7, 160.7. Anal. (C23H32SiO4) C, H.
3,3′,5-Tr im e t h oxy-4′-t er t -b u t yld im e t h ylsilyloxyst il-
ben es (5g, 6g). Reaction of 3,5-dimethoxybenzyltriphenylphos-
phonium bromide 3b and 4-(tert-butyldimethylsilyloxy)-3-
methoxybenzaldehyde 4f (0.74 g, 2.77 mmol) gave a mixture
of cis-stilbene 5g and the trans-isomer 6g. 5g: 0.46 g (41%
yield); yellow oil; 1H NMR δ 0.13 (s, 6H), 0.98 (s, 9H), 3.59 (s,
3,4′,5-Tr i(ben zyloxy)stilben es (5a , 6a ). Reaction of 3,5-
di(benzyloxy)benzyl]triphenylphosphonium bromide 3a 39 and
4-benzyloxybenzaldehyde 4a (1.2 g, 5.65 mmol) gave a mixture
of cis-stilbene 5a and the trans-isomer 6a . 5a : 0.72 g (15%
yield); light-yellow oil; 1H NMR δ 4.96 (s, 4H), 5.10 (s, 2H),
6.62 (m, 5H), 6.94 (m, 2H), 7.27-7.46 (m, 17H). 6a : 0.43 g
3H), 3.66(s, 6H), 6.31 (m, 2H), 6.46 (m, 3H), 6.75 (m, 3H); 13
C
1
(26% yield); light-yellow oil; H NMR δ 5.07 (s, 2H), 5.15 (s,
NMR δ -4.6, 18.5, 25.7, 55.2, 99.7, 106.5, 107.0, 112.6, 120.5,
4H), 6.30-6.40 (m, 2H), 6.50 (s, 1H), 6.65 (s, 1H), 6.85 (m, 1H),
122.0, 128.7, 130.4, 130.6, 139.4, 144.2, 150.3, 160.4. 6g: 0.39
6.95-7.13 (m, 4H), 7.44-7.53 (m, 15H). Anal. (C35H30O3) C,
1
g (35% yield); yellow oil; H NMR δ 0.21 (s, 6H), 1.04 (s, 9H),
H.
3.85 (s, 6H), 3.89 (s, 3H), 6.40 (t, 1H, J ) 2.2 Hz), 6.67 (m,
2H), 6.87 (m, 2H), 7.04 (m, 3H); 13C NMR δ -4.7, 18.5, 25.6,
55.2, 55.3, 99.4, 104.1, 109.6, 119.7, 120.8, 126.5, 129.0, 130.8,
139.4, 144.9, 150.8, 160.7. Anal. (C23H32SiO4) C, H.
3,3′,5-Tr i(ben zyloxy)-4′-m eth oxystilben es (5b, 6b). Re-
action of [3,5-di(benzyloxy)benzyl]triphenylphosphonium bro-
mide 3a and 3-benzyloxy-4-methoxybenzaldehyde 4b (1.2 g,
4.95 mmol) gave a mixture of cis-stilbene 5b and the trans-
isomer 6b. 5b: 0.50 g (19% yield); white powder; mp 134 °C;
1H NMR δ 3.89 (s, 3H), 4.95 (s, 6H), 6.45-6.65 (m, 5H), 6.8-7
(m, 3H), 7.32-7.38 (m, 15H). 6b: 1.41 g (54% yield); white
powder; mp 49-52 °C; 1H NMR δ 3.93 (s, 3H), 5.1 (s, 4H), 5.22
(s, 2H), 6.56 (t, 1H, J ) 2.2 Hz), 6.76 (m, 1H), 6.82 (d, 1H, J
) 16.6 Hz), 6.91 (m, 2H), 6.99 (d, 1H, J ) 16.2 Hz), 7.06-7.10
(m, 2H), 7.33-7.52 (m, 15H). Anal. (C36H32O4) C, H.
3,5-Di(ben zyloxy)-3′-n itr o-4′-m eth oxystilben es (5c, 6c).
Reaction of [3,5-di(benzyloxy)benzyl]triphenylphosphonium
bromide 3a and the commercially available 3-nitro-4-meth-
oxybenzaldehyde 4c (1 g, 5.52 mmol) gave a mixture of cis-
stilbene 5c and the trans-isomer 6c. 5c: 0.42 g (16% yield);
yellow oil; 1H NMR δ 3.94 (s, 3H), 4.98 (s, 4H), 6.51-6.60 (m,
5H), 6.88-6.92 (m, 2H), 7.37-7.44 (m, 10 H), 7.80-7.81 (m,
1H). 6c: 0.68 g (26% yield); yellow powder; mp 111 °C; 1H
NMR δ 4.01 (s, 3H), 5.10 (s, 4H), 6.78 (m, 2H), 7.00 (m, 2H),
7.28-7.46 (m, 13H), 8.02 (m, 1H). Anal. (C29H25NO5) C, H, N.
3,4′,5-Tr im eth oxy-3′-n itr ostilben es (5d , 6d ). Reaction of
3,5-dimethoxybenzyltriphenylphosphonium bromide 3b12 and
3-nitro-4-methoxybenzaldehyde 4c (0.7 g, 3.86 mmol) gave a
mixture of cis-stilbene 5d and the trans-isomer 6d . 5d : 0.33 g
(27% yield); yellow crystals; mp 49-52 °C; 1H NMR δ 3.67 (s,
6H), 3.90 (s, 3H), 6.34 (m, 3H), 6.44 (d, 1H, J ) 12.3 Hz), 6.57
(d, 1H, J ) 12 Hz), 6.89 (m, 1H), 7.38 (m, 1H), 7.74 (m, 1H).
6d : 0.32 g (32% yield); yellow crystals; mp 107-110 °C; 1H
NMR δ 3.83 (s, 6H), 3.98 (s, 3H), 6.41 (t, 1H, J ) 2.4 Hz), 6.65
(m, 2H), 6.98 (m, 2H), 7.07 (m, 1H), 7.65 (m, 1H), 7.99 (m,
1H). Anal. (C17H17NO5) C, H, N.
3,5-Dim eth oxy-3′,4′-d i-(ter t-bu tyld im eth ylsilyloxy)stil-
ben es (5h , 6h ). Reaction of 3,5-dimethoxybenzyltriphenylphos-
phonium bromide 3b and 3,4-di(tert-butyldimethylsilyloxy)-
benzaldehyde 4g (1.35 g, 3.68 mmol) gave a mixture of cis-
stilbene 5h and the trans-isomer 6h . 5h : 0.46 g (25% yield);
colorless oil; 1H NMR δ 0.10 (s, 6H), 0.22 (s, 6H), 0.96 (s, 9H),
1.01 (s, 9H), 3.71 (s, 6H), 6.35 (t, 1H, J ) 2.2 Hz), 6.47 (m,
4H), 6.77 (m, 3H). 13C NMR δ -4.0, 4.3, 18.4, 18.5, 25.9, 26.0,
55.1, 99.6, 106.5, 120.5, 121.3, 122.5, 128.6, 130.2, 130.4, 139.5,
146.1, 146.3, 160.5. 6h : 0.37 g (20% yield); oil; 1H NMR δ 0.25
(s, 6H), 0.26 (s, 6H), 1.02 (s, 9H), 1.04 (s, 9H), 3.80 (s, 3H),
3.86 (s, 3H), 6.38 (m, 2H), 6.67 (m, 2H), 6.86 (m, 1H), 7.01 (m,
2H), 7.28 (m, 1H). 13C NMR δ -4.0, 18.5, 21.9, 26.0, 55.2, 55.4,
97.4, 99.6, 104.3, 107.0, 119.2, 120.0, 121.1, 126.6, 128.9, 130.7,
139.6, 146.9, 160.8. Anal. (C28H44SiO4) C, H.
3,5-Dim eth oxy-4′-n itr ostilben es (5i, 6i). Reaction of 3,5-
dimethoxybenzyltriphenylphosphonium bromide 3b and the
commercially available 4-nitrobenzaldehyde 4h (0.4 g, 2.65
mmol) gave a mixture of cis-stilbene 5i and the trans-isomer
6i.35 5i: 0.25 g (33% yield); yellow solid; mp 72 °C; 1H NMR δ
3.66 (s, 3H), 6.33 (m, 3H), 6.58 (d, 1H, J ) 12 Hz), 6.73 (d, 1H,
J ) 12.3), 7.38 (d, 2H, J ) 9 Hz), 8.07 (d, 2H, J ) 8.7). 6i:
1
0.35 g (46% yield); yellow solid; mp 134-136 °C; H NMR δ
3.83 (s, 6H), 6.44 (t, 1H, J ) 2.1), 6.68 (d, 2H, J ) 1.8 Hz),
7.09 (d, 1H, J ) 16.2 Hz), 7.19 (d, 1H, J ) 16.2 Hz), 7.61 (d,
2H, J ) 8.7), 8.20 (d, 2H, J ) 9 Hz). Anal. (C16H15NO4) C, H,
N.
Gen er a l P r oced u r e for th e Red u ction of Nitr o Der iva -
tives 5c,d ,i a n d 6c,d ,i. The nitrostilbene derivatives 5c,d ,i
or 6c,d ,i (1.0 equiv) was dissolved in acetone/water (10:5 mL),
and the mixture was heated to 50 °C. After 30 min, sodium
dithionite (25.0 equiv) was slowly added, and the mixture was
heated to reflux (1-4 h) and then cooled to room temperature.
Water was added, and the product was isolated by extraction
with ethyl acetate. The organic phase was washed with brine,
and the solvent was removed in vacuo. The crude products
were used in a further reaction without purification.
cis-3,5-Di(ben zyloxy)-3′-a m in o-4′-m eth oxystilben e (7a ).
Nitro derivative 5c (0.37 g, 0.8 mmol) led to 7a as a yellow oil
3,5-D im e t h o x y -4′-t er t -b u t y ld im e t h y ls ily lo x y s t il-
ben es (5e, 6e). Reaction of 3,5-dimethoxybenzyltriphenylphos-
phonium bromide 3b and 4-(tert-butyldimethylsilyloxy)ben-
zaldehyde 4d (1.3 g, 5.7 mmol) gave a mixture of cis-stilbene
5e and the trans-isomer 6e. 5e: 0.54 g (25% yield); yellow oil;
IR νmax (Nujol) cm-1 3380, 1593, 1506, 1454, 1255, 1148, 1065,
1
907, 835, 784, 682; H NMR δ 0.22 (s, 6H), 1.02 (s, 9H), 3.70
(s, 6H), 6.36 (t, 1H, J ) 2.4 Hz), 6.50 (m, 4H), 6.74-6.77 (m,
2H), 7.19-7.21 (m, 2H); 13C NMR δ -4.4, 18.3, 25.7, 55.2, 99.8,
106.5, 119.7, 128.7, 130.1, 139.3, 154.8, 160.4. 6e: 0.32 g (15%
yield); yellow oil; IR νmax (Nujol) cm-1 3385, 3298, 3180, 1670,
1588, 1506, 1265, 1204, 1147, 1060, 958, 912, 840; 1H NMR δ
0.26 (s, 6H), 1.04 (s, 9H), 3.87 (s, 6H), 6.43 (t, 1H, J ) 2.4 Hz),
6.69-6.70 (m, 2H), 6.87-6.89 (m, 2H), 6.94 (d, 1H, J ) 16.2),
7.08 (d, 1H, J ) 16.2), 7.42-7.45 (m, 2H); 13C NMR δ -4.3,
18.3, 25.7, 55.3, 99.5, 104.3, 107.0, 120.2, 126.6, 127.6, 128.7,
130.4, 139.6, 155.4, 160.8. Anal. (C22H30SiO3) C, H.
1
(0.32 g, 92% yield); H NMR δ 3.85 (s, 3H), 4.94 (s, 4H), 6.42
(d, 1H, J ) 12.2 Hz), 6.52 (t, 1H, J ) 2.6 Hz), 6.51 (d, 1H, J )
12.2 Hz), 6.62 (m, 2H), 6.70 (m, 3H), 7.39 (m, 10 H).
tr a n s-3,5-Di(b en zyloxy)-3′-a m in o-4′-m et h oxyst ilb en e
(8a ). Nitro derivative 6c (0.53 g, 1.1 mmol) led to 8a as a
yellow oil at room temperature (0.29 g, 42% yield); 1H NMR δ
3.89 (s, 3H), 5.07-5.11 (m, 4H), 6.58-6.99 (m, 8H), 7.40-7.49
(m, 10H).
3,4′,5-Tr im e t h oxy-3′-t er t -b u t yld im e t h ylsilyloxyst il-
ben es (5f, 6f). Reaction of 3,5-dimethoxybenzyltriphenylphos-
phonium bromide 3b and 3-(tert-butyldimethylsilyloxy)-4-
methoxybenzaldehyde 4e (0.92 g, 3.46 mmol) gave a mixture
of cis-stilbene 5f and the trans-isomer 6f. 5f: 0.33 g (24%
yield); yellow oil; 1H NMR δ 0.10 (s, 6H), 0.97 (s, 9H), 3.71 (s,
6H), 3.81 (s, 3H), 6.35 (t, 1H, J ) 2.6 Hz), 6.46-6.50 (m, 4H),
6.73-6.90 (m, 3H); 13C NMR δ -4.8, 18.3, 25.7, 55.1, 55.4, 99.5,
cis-3,4′,5-Tr im eth oxy-3′-a m in ostilben e (7b). Nitro de-
rivative 5d (0.13 g, 0.4 mmol) led to 7b as a yellow oil (0.04 g,
1
32% yield); H NMR δ 3.70 (s, 6H), 3.85, (s, 3H), 6.35 (t, 1H,
J ) 2.2), 6.45-6.54 (m, 4H), 6.70 (m, 3H); 13C NMR δ 55.2,
55.5, 99.6, 106.6, 109.9, 115.3, 119.5, 128.3, 129.8, 130.6, 135.6,
139.5, 146.6, 160.3. Anal. (C17H19NO3) C, H, N.

3552 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Roberti et al.
tr a n s-3,4′,5-Tr im eth oxy-3′-a m in ostilben e (8b). Nitro de-
rivative 6d (0.2 g, 0.6 mmol) led to 8b as a yellow oil (0.09 g,
50% yield); 1H NMR δ 3.85 (s, 6H), 3.90 (s, 3H), 6.41 (t, 1H, J
) 2.2 Hz), 6.68 (m, 2H), 6.89 (d, 1H, J ) 16.0 Hz), 6.90-6.95
(m, 3H), 7.02 (d, 1H, J ) 16.2); 13C NMR δ 55.3, 55.5, 99.5,
104.3, 110.3, 112.3, 117.8, 126.2, 129.2, 130.2, 136.2, 139.7,
147.4, 160.8. Anal. (C17H19NO3) C, H, N.
cis-3,5-Dim eth oxy-4′-a m in ostilben e (7c). Nitro deriva-
tive 5i (0.14 g, 0.5 mmol) led to 7c as a yellow powder (0.06 g,
48% yield); mp 44-47 °C; 1H NMR δ 3.57 (s, 6H), 6.20 (t, 1H,
J ) 2.4 Hz), 6.25 (m, 1H), 6.34-6.43 (m, 5H), 6.99 (m, 2H);
13C NMR δ 55.2, 99.5, 106.6, 114.6, 115.1, 127.4, 130.2, 130.6,
139.8, 145.5, 160.4. Anal. (C16H17NO2) C, H, N.
tr a n s-3,5-Dim eth oxy-4′-a m in ostilben e (8c).40 Nitro de-
rivative 6i (0.15 g, 0.5 mmol) led to 8c as yellow solid (0.05 g,
37% yield); mp 86-90 °C; 1H NMR δ 3.85 (s, 6H), 6.38 (m,
1H), 6.66 (m, 4H), 6.86 (d, 1H, J ) 16.2 Hz), 7.03 (d, 1H, J )
16.2 Hz), 7.33 (m, 2H); 13C NMR δ 54.3, 98.1, 102.9, 113.9,
123.0, 126.7, 128.3, 138.8, 146.0, 159.7. Anal. (C16H17NO2) C,
H, N.
102.5, 105.5, 111.1, 112.3, 117.5, 126.8, 129.6, 131.2, 138.3,
140.8, 148.0, 159.4. Anal. (C15H15NO3) C, H, N.
Gen er a l P r oced u r e for th e Silyloxy Dep r otection of
Com p ou n d s 5e-h a n d 6e-h . To a solution of the silyloxy-
protected stilbene 5e-h or 6e-h (1 equiv) in anhydrous
tetrahydrofuran (10 mL) tetrabutylammonium fluoride (1 M
in THF, 3 equiv) was added. The pale-yellow solution was
stirred for 45 min, poured into water, and extracted with
dichloromethane. Removal of solvent in vacuo from the organic
phase yielded the crude hydroxystilbenes 11a -d and 12a -d ,
which were purified by flash chromatography.
cis-3,5-Dim eth oxy-4′-h yd r oxystilben e (11a ). 11a was
obtained from 5e (0.43 g, 1.2 mmol). Flash chromatography
of the final reaction mixture (petroleum ether/ethyl acetate,
7.5:2.5) gave 11a (0.07 g, 23% yield) as a light-yellow oil.12
Anal. (C16H16O3) C, H.
tr a n s-3,5-Dim et h oxy-4′-h yd r oxyst ilb en e (P t er ost il-
ben e) (12a ). 12a was obtained from 6e (0.24 g, 0.7 mmol).
Flash chromatography of the final reaction mixture (petroleum
ether/ethyl acetate, 8:2) gave 12a (0.04 g, 24% yield) as a white
powder: mp 88°C (lit.43 mp 87-88 °C). Anal. (C16H16O3) C, H.
cis-3,4′,5-Tr im eth oxy-3′-h yd r oxystilben e (11b). 11b was
obtained from 5f (0.33 g, 0.8 mmol). Flash chromatography of
the final reaction mixture (petroleum ether/ethyl acetate, 9:1)
gave 11b (0.21 g, 89% yield) as a yellow oil: 1H NMR δ 3.70 (s,
6H), 3.85 (s, 3H), 6.36 (t, 1H, J ) 2.2 Hz), 6.50 (m, 4H), 6.71-
6.80 (m, 2H), 6.92 (m, 1H); 13C NMR δ 55.1, 55.8, 99.6, 106.6,
110.2, 115.0, 121.0, 128.8, 130.0, 130.3, 139.1, 145.0, 145.7,
160.3. Anal. (C17H18O4) C, H.
tr a n s-3,4′,5-Tr im eth oxy-3′-h yd r oxystilben e (12b). 12b
was obtained from 6f (0.24 g, 0.6 mmol). Flash chromatography
of the final reaction mixture (petroleum ether/ethyl acetate,
9:1) gave 12b (0.12 g, 70% yield) as a white powder: mp 90-
91 °C (lit.44 mp 89-90 °C). Anal. (C17H18O4) C, H.
cis-3,3′,5-Tr im eth oxy-4′-h yd r oxystilben e (11c). 11c was
obtained from 5g (0.37 g, 0.9 mmol). Flash chromatography
of the final reaction mixture (petroleum ether/ethyl acetate,
9:1) gave 11c (0.23 g, 87% yield) as a yellow oil: 1H NMR δ
3.69 (s, 3H), 3.71 (s, 6H), 6.36 (t, 1H, J ) 2.2), 6.51 (m, 4H),
6.84 (m, 3H); 13C NMR δ 55.1, 55.6, 99.4, 106.5, 111.2, 113.9,
122.5, 128.3, 128.9, 130.2, 139.4, 144.8, 145.8, 160.4. Anal.
(C17H18O4) C, H.
Gen er a l P r oced u r e for th e Clea va ge of th e Ben zyloxy
Gr ou p of Com p ou n d s 5a ,b, 7a a n d 6a ,b, 8a . Freshly
distilled N,N-dimethylaniline (3.0 equiv) was added to a well-
stirred solution of the benzyloxy derivative 5a ,b, 7a or 6a ,b,
8a (1.0 equiv) in dry methylene chloride (10 mL) under a
nitrogen atmosphere at 0 °C. After 5 min, anhydrous AlCl3
(4.0 equiv) was added in one portion. After 2-8 h, the reaction
mixture was cooled and 1.0 M HCl (10 mL) was added. The
resulting mixture was extracted with ethyl acetate, and the
combined extracts were washed with brine. Removal of solvent
in vacuo from the organic phase yielded the crude hydroxy-
stilbene, which was purified by flash chromatography.
cis-3,4′,5-Tr ih yd r oxystilben e (9c). 9c was prepared from
5a (0.56 g, 1.1 mmol). Flash chromatography of the final
reaction mixture (petroleum ether/ethyl acetate, 6:4) gave 9c
as a white powder (0.062 g, 24% yield): mp 169-170 °C (lit.41
mp 170-174 °C). Anal. (C14H12O3) C, H.
tr a n s-3,4′,5-Tr ih yd r oxystilben e (Resver a tr ol) (1). 1 was
prepared from 6a (0.42 g, 0.9 mmol). Flash chromatography
of the final reaction mixture (petroleum ether/ethyl acetate,
7:3) gave 1 as a white powder (0.07 g, 36% yield): mp 258 °C
(lit.41 mp 260 °C). Anal. (C14H12O3) C, H.
tr a n s-3,3′,5-Tr im eth oxy-4′-h yd r oxystilben e (12c). 12c
was obtained from 6g (0.30 g, 0.75 mmol). Flash chromatog-
raphy of the final reaction mixture (petroleum ether/ethyl
acetate, 9:1) gave 12c (0.19 g, 88% yield) as a white solid: mp
82-84 °C (lit.44 mp 85-86 °C). Anal. (C17H18O4) C, H.
cis-3,5-Dim eth oxy-3′,4′-d ih yd r oxystilben e (11d ). 11d
was obtained from 5h (0.37 g, 0.7 mmol). Flash chromatogra-
phy of the final reaction mixture (petroleum ether/ethyl
acetate, 8:2) gave 11d (0.04 g, 20% yield) as a yellow oil (lit.
data in ref 45): 1H NMR δ 3.68 (s, 6H), 5.28 (br, 2H), 6.33 (t,
1H, J ) 2.2 Hz), 6.46 (m, 4H), 6.78 (m, 3H); 13C NMR δ 55.3,
99.7, 106.8, 115.0, 115.7, 122.3, 128.6, 129.9, 130.1, 139.4,
143.0, 143.1, 160.3. Anal. (C16H16O4) C, H.
tr a n s-3,5-Dim eth oxy-3′,4′-d ih yd r oxystilben e (12d ). 12d
was obtained from 6h (0.25 g, 0.5 mmol). Flash chromatogra-
phy of the final reaction mixture (petroleum ether/ethyl
acetate, 9:1) gave 12d (0.04 g, 29% yield) as a white solid: mp
112-115 °C (lit.46 mp 72-74 °C); 1H NMR δ 3.85 (s, 6H), 5.70
(br, 2H), 6.42 (m, 1H), 6.66 (m, 2H), 6.90 (m, 4H), 7.08 (m,
1H). Anal. (C16H16O4) C, H.
Biology. Cell Cu ltu r e. Cells were grown in RPMI 1640
(Gibco Grand Island, NY) containing 10% FCS (Gibco), 100
U/mL penicillin (Gibco), 100 µg/mL streptomycin (Gibco), and
2 mM l-glutamine (Sigma Chemical Co., St. Louis, MO) in a
5% CO2 atmosphere at 37 °C.
Sa m p le P r ep a r a tion . Each compound was dissolved in
DMSO or PBS. They were diluted to the appropriate experi-
mental concentrations in tissue medium and protected from
light. In each experiment, DMSO never exceeded 0.1%, and
this percentage did not interfere with cell growth.
cis-3,3′,5-Tr ih yd r oxy-4′-m eth oxystilben e (9a ). 9a was
prepared from 5b (0.50 g, 0.95 mmol). Flash chromatography
of the final reaction mixture (petroleum ether/ethyl acetate,
8:2) gave 9a as a light-yellow oil (0.038 g, 15% yield): 1H NMR
(CD3COCD3) δ 3.84 (s, 3H), 6.24 (t, 1H, J ) 2.2 Hz), 6.31-
6.41 (m, 4H), 6.77-6.85 (m, 3H), 7.3 (br, 1H), 8.16 (s, 2H); 13
C
NMR (CD3COCD3) δ 56.1, 102.3, 107.8, 111.9, 116.2, 121.5,
129.5, 130.4, 131.0, 140.3, 146.8, 147.5, 159.1. Anal. (C15H14O4)
C, H.
tr a n s-3,3′,5′-Tr ih yd r oxy-4-m eth oxystilben e (Rh a p on -
tigen in ) (10a ). 10a was prepared from 6b (0.50 g, 0.90 mmol).
Flash chromatography of the final reaction mixture (petroleum
ether/ethyl acetate, 7:3) gave 10a as a white powder (0.040 g,
16% yield): mp 195°C (lit.42 mp 195 °C). Anal. (C15H14O4) C,
H.
cis-3,5-Dih yd r oxy-3′-a m in o-4′-m eth oxystilben e (9b). 9b
was prepared from 7a (0.32 g, 0.7 mmol). Flash chromatog-
raphy of the final reaction mixture (petroleum ether/ethyl
acetate, 7:3) gave 9b as a yellow powder (0.11 g, 58% yield):
mp 65 °C; 1H NMR (CD3COCD3) δ 3.15 (br, 1H), 3.82 (s, 3H),
4.30 (br, 1H), 6.23 (t, 1H, J ) 2.2 Hz), 6.32 (d, 1H, J ) 12.3
Hz), 6.35 (m, 2H), 6.41 (d, 1H, J ) 12.3 Hz), 6.58-6.61 (m,
1H), 6.70-6.73 (m, 2H), 8.15 (br, 2H); 13C NMR (CD3COCD3)
δ 55.7, 102.3, 108.0, 110.7, 115.5, 118.9, 129, 130.8, 131.1,
137.8, 140.5, 147.1, 159.1. Anal. (C15H15NO3) C, H, N.
tr a n s-3,5-Dih ydr oxy-3′-am in o-4′-m eth oxystilben e (10b).
10b was prepared from 8a (0.37 g, 0.8 mmol). Flash chroma-
tography of the final reaction mixture (petroleum ether/ethyl
acetate, 7.5:2.5) gave 10b as a red powder (0.09 g, 42% yield):
1
mp 163 °C; H NMR (CD3COCD3) δ 3.86 (s, 3H), 6.28 (t, 1H,
J ) 2.2 Hz), 6.54-6.55 (m, 2H), 6.81 (s, 1H), 6.80-6.92 (m,
Cytotoxicity Assa ys. To evaluate the number of live and
dead cells, cells were stained with trypan blue and counted
3H), 7.00 (m, 2H), 8.20 (br, 2H); 13C NMR (CD3COCD3) δ 55.8,

Resveratrol and Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3553
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treated with different concentrations of each drug. After 48 h
of incubation, the number of viable cells was determined and
expressed as a percent of control proliferation.
Eva lu a tion of Ap op tosis by An n exin e V. Apoptosis was
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staining kit (Roche Molecular Biochemicals, Mannheim, Ger-
many). Cells (106) were washed with PBS and centrifuged at
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staining solution containing annexine-V-fluorescein labeling
reagent and propidium iodide and was incubated for 15 min
at 20 °C. Annexine V propidium iodide negative cells were
evaluated by flow cytometry (Becton Dickinson) and fluores-
cence microscopy.
F low Cytom etr ic Deter m in a tion of Ap op tosis. Cell
cycle progression and apoptosis were analyzed by quantitating
DNA content after staining with propidium iodide (PI).
Untreated or drug-treated cells were collected by centrifuga-
tion, suspended in PBS, and fixed in FACS permeabilizing
solution 2 (Becton-Dickinson) (ratio 1:10) for 10 min at 20 °C.
After centrifugation at 500g for 5 min, cells were suspended
in 1 mL of PBS containing PI (50 mg/mL) and RNAse (0.2 mg/
mL) in polypropylene tubes and incubated at room tempera-
ture for 30 min. The tubes were then placed at 4 °C in the
dark until use in flow cytometry analysis using an FACscan
flow cytometer (Becton-Dickinson) with CellQuest software.
The data were registered on a linear scale. Apoptotic cells were
evaluated as a sub-G0-G1 hypodiploid peak.
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Ack n ow led gm en t. This work was supported in part
by the Ministero dell’Universita` e della Ricerca Scien-
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