Design, synthesis and structure-activity relationships of some novel, highly potent anti-invasive (E)- and (Z)-stilbenes

Article abstract of DOI:10.1016/j.bmc.2013.06.048

In our ongoing exploration of the structure-activity landscape of anti-invasive chalcones, we have prepared and evaluated a number of structurally related (E)- and (Z)-stilbenes. These molecules exhibited an extraordinary high in vitro potency in the chick heart invasion assay, being active up to 10 nmol L^-1, a concentration level a 100-fold lower than the lowest effective doses that have been reported for natural analogues. Furthermore, they possess an interesting pharmacological profile in silico.

Full text of DOI:10.1016/j.bmc.2013.06.048

Bioorganic & Medicinal Chemistry 21 (2013) 5054–5063
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and structure–activity relationships of some novel,
highly potent anti-invasive (E)- and (Z)-stilbenes
Bart I. Roman a,1, Laurens M. De Coen a, Séverine Thérèse F.C. Mortier a, Tine De Ryck b,
Barbara W. A. Vanhoecke b, Alan R. Katritzky c,d, Marc E. Bracke b, Christian V. Stevens a,
⇑
a Research Group SynBioC, Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Coupure Links 653, B-9000 Ghent, Belgium
b Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, De Pintelaan 185, B-9000
Ghent, Belgium
c Center for Heterocyclic Compounds, Department of Chemistry, 127 Chemistry Research Building, University of Florida, Gainesville, FL 32611-7200, USA
d Department of Chemistry, King Abdulaziz University, Jeddah 21589, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
In our ongoing exploration of the structure–activity landscape of anti-invasive chalcones, we have pre-
pared and evaluated a number of structurally related (E)- and (Z)-stilbenes. These molecules exhibited
Received 16 May 2013
Revised 17 June 2013
Accepted 19 June 2013
Available online 27 June 2013
an extraordinary high in vitro potency in the chick heart invasion assay, being active up to 10 nmol Lꢀ1
,
a concentration level a 100-fold lower than the lowest effective doses that have been reported for natural
analogues. Furthermore, they possess an interesting pharmacological profile in silico.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Invasion
Cancer
Stilbene
SAR
Chalcone
1. Introduction
cells. Our attention was primarily drawn towards chalcones 1,4
which emerged as promising leads from the Indo-Belgian anti-
A cardinal challenge tackled by contemporary cancer research is
the prevention or suppression of metastasis, a process which ac-
counts for 90% of human cancer fatalities.1,2 The development of
novel, efficient anti-metastatic drugs is thus quite literally of vital
importance and would represent a giant leap forward in the man-
agement of malignant neoplasms.3
A first, fundamental step during metastases development is
invasion, the process by which cancer cells migrate from the pri-
mary tumor into the surrounding tissue. Although the mechanisms
of invasion remain somewhat enigmatic, pathways involved in tu-
mor–stromal interactions, anoikis and cell motility have been for-
warded as possible sites of therapeutic intervention. Nonetheless,
these targets have only been poorly addressed to date. As a conse-
quence, no anti-invasive drugs are available in the clinic yet.
Recently, we embarked on a quest for novel small molecules
that can effectively suppress the invasive tendency of malignant
invasive screening program.5,6 As the molecular target of these
substances has not yet been revealed, we have developed and val-
idated a predictive quantitative structure–activity relationship
(QSAR) model for chalcones as a rationale behind our work.7 Fur-
thermore, we have assessed the anti-invasive potential of their
conformationally restricted heterocyclic analogues 2–3.8 During
these investigations, methoxy- and fluoro chalcones surfaced as
remarkably interesting molecules, with (E)-3-(4-fluoro)-1-(3,4,5-
trimethoxy)propenone 1 as an example. An obvious next step in
the elaboration of this SAR study on potent chalcones was an eval-
uation of the neighboring 1,2-diarylethene congeners 4–5, better
known as stilbenes.
Stilbenes are naturally occurring, widely distributed plant sec-
ondary metabolites, of which the most abundant are resveratrol,
pterostilbene, and the combretastatins.9 The antimalarial,10 anti-
inflammatory and anti-asthmatic11 potency of stilbenes has been
confirmed in vivo, and some derivatives have been included in
clinical trials as potential orally available anti-cancer drugs.12,13
Of these, fosbretabulin, the phosphorylated prodrug of combretast-
atin-A4 (Zybrestat™, OXiGENE), is likely the best-known example.
⇑
Corresponding author. Tel.: +32 9 264 5957; fax: +32 9 264 6221.
E-mail address: chris.stevens@ugent.be (C.V. Stevens).
URL: http://www.ugent.be/bw/doct/en (Christian V. Stevens).
This molecule,
a dual antimitotic and vascular disrupting
1
B.I.R. is
a postdoctoral fellow of the Research Foundation—Flanders
agent, has entered phase II/III trials for the treatment of several
(FWO—Vlaanderen).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.048

B. I. Roman et al. / Bioorg. Med. Chem. 21 (2013) 5054–5063
5055
malignancies.14 Nevertheless, only few reports on the anti-invasive
O
Br
a
effects of stilbenes in malignant cells have appeared to date,15–24
thereby leaving ample room for additional structure–activity rela-
tionship studies (SAR) in this area.
b
H
R
PPh3
Br
R
6
7
9-13
8
1,2-Diarylethenes bear high structural resemblance to chal-
cones, as only the carbonyl group is omitted from the linker be-
tween the two aromatic moieties. Hence, the biological
evaluation of stilbenes bearing the same decoration pattern as po-
tent chalcones could provide valuable information on the active
conformation of the latter.8 Besides, the existence and significance
of a presumed interaction between the carbonyl group of chal-
cones and the molecular target may be clarified in this way.7
Additionally, the possibility to discern between the (E)- and (Z)-
isomers of stilbenes may lead to insight in the conformational de-
mands for an active ligand. Indeed, a striking difference in potency
between stilbene stereoisomers has been noted in several in-
stances, for example in the case of antitumoral combretastatin ana-
logues25 and in the antiproliferative activity of resveratrol
analogues.26 Such acceptor discrimination was also observed for
1,3-dimethoxy-5-(4-methoxystyryl)benzene, for which the (Z)-iso-
mer was more potent against HT-29 cells in vitro. Intriguingly,
however, due to low bioavailability of this isomer in vivo, only
the (E)-stilbene successfully inhibited tumor growth in SCID
mice.27
Scheme 2. Reagents and conditions: (a) 1 equiv PPh3, dry Et2O, N2,
D
D
, 12 h; (b)
, for details
2 equiv KOtBu, dry Et2O, N2,
D
, 1 h; then 1 equiv aldehyde 8, 0 °C to
see Table 1.
Table 1
Attempts at the synthesis of substituted (E)-stilbenes via a classic Wittig-approach
(see Scheme 2)
Product
R
Time (h)
E/Z-ratioa
Isolated yieldb (%)
E-Isomer
Z-Isomer
9
H
3-F
5
>99:1
>99:1
60:40
70:30
50:50
70
47
—
—
23
10
11
12
13
16
5
4-OMe
4-F
29
5
5
N.d.c
N.d.c
N.d.c
N.d.c
4-Cl
Determined by 1H NMR analysis of the crude.
Yield over two steps, from 6.
Not determined: isomers were not separated.
a
b
c
with triphenylphosphine in dry diethyl ether under reflux condi-
tions, which quantitatively furnished the desired benzyltriphenyl-
phosphonium salt 7. To the same pot, 2 equiv of KOtBu were added
in order to generate the phosphonium ylide. Reaction of this spe-
cies with benzaldehyde at room temperature proved sluggish. A
fair yield of non-substituted stilbene 9, however, was obtained
when the mixture was heated to reflux temperature for several
hours. Furthermore, the transformation proceeded with excellent
stereoselectivity, furnishing the (E)-stilbene 9 in an (E)/(Z)-ratio
exceeding 99:1. Unreacted phosphonium salt was removed via
extraction employing glacial acetic acid and hexanes.
Under identical conditions, the (E)-isomer of 3-fluorostilbene
10 was stereoselectively obtained, though in a mere 47% yield,
even upon prolonged heating. No pronounced stereoselectivity
was observed in the formation of 4-methoxystilbene 11. However,
its (E)- and (Z)-isomers were successfully isolated via preparative
thin layer chromatography (TLC) using an eluent mixture of petro-
leum ether and diethyl ether (4:1), albeit in disappointing yields.
To our discontent, the formation of stilbenes 12 and 13 also lacked
selectivity. Further trials employing LiOHꢁH2O as a base, following
the findings of Antonioletti et al. also gave complex reaction mix-
tures.28 Clearly, the Wittig approach deemed unsatisfactory and
was hence abandoned.
In summary, the present report provides an overview of our at-
tempts to synthesize stilbenoid analogues of the most potent chal-
cones reported in our previous communications,7,8 and discloses
their in vitro anti-invasive activity profile. An initial survey on their
druglikeness is also presented.
2. Results and discussion
Given the extraordinary in vitro anti-invasive activity of 1,7 we
were intrigued to assess if its stilbenoid analogues are endowed
with such high potency as well. Hence, we focused our synthetic
efforts on (tri)methoxylated and fluorinated stilbenes, both in the
(E)- and the (Z)-configuration (see Scheme 1).
2.1. Synthesis
2.1.1. Synthesis of (E)-stilbenes
In a first attempt to stereoselectively synthesize the (E)-stilb-
enes of interest, we evaluated a fairly standard Wittig approach
(Scheme 2, Table 1). Thus, benzyl bromide 6 was reacted overnight
O
In aspiration of higher yields and better (E)-selectivity, we
quickly turned our attention towards the Horner–Wadsworth–Em-
mons (HWE) reaction (Scheme 3). In a first step, the required phos-
phonates 15 were prepared from the corresponding benzyl
bromides 14 via a classical solvent-free Arbusov reaction. Excess
triethyl phosphite was readily distilled off under vacuum, which
afforded the pure phosphonate.
O
MeO
MeO
MeO
MeO
-1
F
OMe
OMe
F
active up to 0.01 µmol.L
1a
1b
s-cis conformer
s-trans conformer
N
O
O
N
F
MeO
Next, the HWE reaction was evaluated.
A first attempt,
MeO
MeO
employing the conditions of Smith et al. (KOtBu/THF), yielded
MeO
OMe
OMe
F
2
3
previously evaluated
O
8
-1
-1
active up to 10 µmol.L
active up to 1 µmol.L
a
1
O
1
R
H
R
2
Br
R
P(OEt)2
1
1
R
R
1
14
15
R
b
2
R
2
R
10-13, 16-22
2
R
4
5
targets of the present study
Scheme 3. Reagents and conditions: (a) Excess P(OEt)3, neat, 130 °C, 5 h; (b)
Scheme 1. SAR studies on anti-invasive chalcones and analogues. Lowest active
1.1 equiv NaOMe, 0.67 equiv 8, dry DMF, 0 °C to rt, 1 h, then D, 1 h, then left
concentrations in the chick heart invasion assay are indicated.
standing at rt, overnight; see Table 2.

5056
B. I. Roman et al. / Bioorg. Med. Chem. 21 (2013) 5054–5063
mixtures of (E)- and (Z)-isomers.29 However, using NaOMe as a
base and DMF as a solvent, we were able to obtain the desired
(E)-stilbenes 10–13 and 16–22 exclusively, in fair to excellent
a
b or c
I
1
1
2
1
2
2
R
R
R
R
R
R
23
25-30
9, 11, 12, 31-33
24
yields (Table 2). Moreover, these conditions allowed for
a
straightforward work-up, since addition of a 1:1-mixture of water
and methanol upon reaction completion provided the pure prod-
uct as a precipitate. Only in the case of stilbenes (E)-20 and (E)-17
(Table 2, entry k, as compared to entry f), no precipitation oc-
curred, since conversion grades were rather low. Attempts to iso-
late these products via extraction with diethyl ether failed.
Apparently, in these runs, the combination of the deactivated
benzaldehyde and the stabilized ylide was too unfavorable to al-
low the reaction to proceed at a reasonable rate. However, prod-
uct (E)-17 was obtained employing reagents with an inverse
substitution pattern (entry f).
Scheme 4. Reagents and conditions: (a) 0.77 equiv aryl iodide 24, 0.02 equiv
PdCl2(PPh3)2, H2O/sec-BuNH2 (1:1), Ar, rt, see Table 3; (b) 1.5 equiv EtOMe2SiH,
7 mol % PtO2, N2, 60 °C, 15 h, then 3 equiv TBAF, THF, N2, 0 °C to rt, 15 h, see Table 4;
(c) 30 mol % Lindlar’s catalyst, 1 mol % additive, H2 (1 atm), rt, see Table 5 for
additional details.
Table 3
Synthesis of diarylalkynes 25–30
Product
R1
R2
Time (h)
Yield (%)
25
26
27
28a
29
30
H
H
H
H
4-F
4-OMe
H
2
92
96
95
99
98
99
2.5
3.5
1.5
2.5
2.5
4-I
2.1.2. Synthesis of (Z)-stilbenes
H
3,4,5-triOMe
3,4,5-triOMe
In our view, a strategy involving the stereoselective syn-hydro-
genation of diarylalkynes represented the most attractive ap-
proach. Indeed, the intermediate acetylenes are readily
obtainable and various approaches for their conversion into (Z)-
stilbenes have been reported.9
The most straightforward way to prepare a diarylalkyne is
through Sonogashira coupling of an aryl halide and a phenylacety-
lene, for which a myriad of protocols are available (Scheme 4).30
We considered the copper-free approach of Komáromi to be one
of the most appealing.31 Although the original report recommends
a reaction time of 90 min, we observed precipitation of the target
substances from the reaction mixture after a longer period of time.
This allowed for a straightforward isolation of the pure diarylalky-
nes 25–30 in excellent yields by filtration (Table 3).
4-F
a
1,4-bis(phenylethynyl)benzene, produced with 2 equiv of acetylene 23.
Table 4
Results for the hydrosilylation-protodesilylation of diarylalkynes towards stilbenes
Product
R1
R2
Z/E-Ratioa
Isolated yield (%)
(Z)-Stilbene (E)-Stilbene
9
H
H
H
H
H
60/40
>99:1
>99:1
N.d.b
80/20
50/50
6c
—
—
—
—
5c
3c
11
12
31
32
33
4-OMe
4-F
4-I
3,4,5-triOMe
3,4,5-triOMe
87
51
—
H
13c
—
4-F
Stereoselective conversion of the thus obtained alkynes into (Z)-
stilbenes was firstly attempted via a hydrosilylation–protodesilyla-
tion sequence. Conventional catalysts for the hydrosilylation of al-
kynes are platinum species; this transformation is generally
assumed to proceed according to the Chalk–Harrod mechanism,
through hydrometalation of the alkyne.32 Recently, heterogeneous
PtO2 was proposed as a highly efficient catalyst for this first stage
of the (Z)-selective semi-reduction. Subsequent TBAF-mediated
transformation of the resulting vinylsilanes into the desired (Z)-
stilbenes was easily achieved in the same pot.33
Based on 1H NMR analysis.
Not determined: complex reaction mixture.
After purification via preparative TLC.
a
b
c
In aspiration of superior stereoselectivities, we turned our
attention towards alternatives for the hydrosilylation–protodesi-
lylation strategy. An obvious choice was the well-established
Lindlar-catalyzed (Z)-selective semi-hydrogenation of alkynes to
alkenes. In a first attempt (Table 5, entry a), a DMF-solution of
Regrettably, in our hands, only stilbenes 11 and 12 were formed
with excellent (Z)-selectivity (Table 4). All other alkenes were ob-
tained as mixtures of the two stereoisomers. The ensuing rather
strenuous isolation of these virtually equipolar isomers via pre-
parative TLC engendered significant product losses, resulting in
poor yields recorded for stilbenes 9, 32 and 33. Nevertheless, these
substances were obtained in a high degree of purity, suitable for
biological screening. For 1,4-distyrylbenzene 31, on the contrary,
product isolation failed entirely.
1,2-diphenylethyne was kept at room temperature under
hydrogen atmosphere (2 atm) for 4 h, in the presence of ethy-
lenediamine (EDA) as poison of Lindlar’s catalyst (Pd/Pb/
a
a
CaCO3).34 Unfortunately, the starting material was recovered
completely. Increasing the pressure to 3.5 atm gave an identical
outcome. Next, we switched the solvent to benzene and the cat-
alyst poison to quinoline, concurrently increasing the amount of
catalyst to 30 wt.% while reducing the pressure to 1 atm (run
b). To our satisfaction, after three hours, complete conversion
to the stilbene in an excellent stereochemical outcome was de-
tected. Usage of toluene as a less toxic alternative to benzene
proved unfeasible (entry c).
Table 2
Synthesis of (E)-stilbenes
R1
R2
Yielda (%)
Submission of 1,4–bis(diphenylethyny)benzene 28 to the opti-
mized conditions (run d) gave excellent conversion of the starting
material, but an inseparable mixture of stereoisomers was obtained.
(Z)-40-fluoro-3,4,5-trimethoxybenzene 33 was synthesized employ-
ing 100 wt.% of catalyst and 5.6 equiv of quinoline, albeit in very
moderate yield (43%). The sluggishness of the reaction may be as-
cribed to an increased steric hindrance, caused by the three methoxy
moieties, when approaching the metal surface.
Entry
Product
a
b
c
d
e
f
g
h
i
(E)-10
(E)-11
(E)-12
(E)-13
(E)-16
(E)-17
(E)-18
(E)-19
(E)-20
(E)-21
(E)-17
(E)-22
3-F
4-OMe
4-F
4-Cl
3-F
4-OMe
4-F
4-Cl
3-F
4-OMe
4-F
H
H
H
H
4-F
4-F
4-F
4-F
4-OMe
4-OMe
4-OMe
3-OMe
79
84
51
74
99
50
48
81
—
j
k
l
73
—
95
2.2. Biological activity and SAR
4-Cl
The goal of the present research project is to develop anti-inva-
sive agents with in vivo efficacy. Contrary to traditional antiprolif-
a
Combined yield over two steps, starting from 14.

B. I. Roman et al. / Bioorg. Med. Chem. 21 (2013) 5054–5063
5057
Table 5
Attempts at (Z)-selective partial hydrogenation of diarylalkynes towards stilbenes employing Lindlar’s catalyst
Entry
Solvent
Equiv Lindlar’s catalyst (wt.%)
Additive
p (atm)
Time (h)
Conversion (%) and Z/E-ratio
Yield (%)
Synthesis of (Z)-9: R1 = H, R2 = H
a
b
c
DMF
Benzene
Toluene
10
30
30
1.2 equiv EDA
2.3 equiv quinoline
2.3 equiv quinoline
2
1
1
4
3
4
0
—
83
—
100 (>99:1)
0
Synthesis of (Z)-1,4-distyrylbenzene 31
Benzene 30
d
2.5 equiv quinoline
1
7
95 (N.d.)
—
Synthesis of (Z)-33: R1 = 4-F, R2 = 3,4,5-triOMe
e
f
g
Benzene
Benzene
Benzene
30
100
30
1.7 equiv quinoline
5.6 equiv quinoline
10 equiv EDA
1
1
1
6
5
6
0
—
43
—
N.d. (95:5)
0
N.d.: not determined; ⁄1,4-bis(phenylethynyl)benzene was used as a substrate.
C
Grade Observation
A
0
Only PHF is present; no confronting cells are observed.
Confronting cells are attached to the PHF, but do not occupy
the heart tissue, even not the outermost fibroblastic cell layers
I
II
Occupation is limited to the outer fibroblast-like and myoblast
cell layers; confronting cells may have surrounded the cell
aggregate.
B
D
III
Confronting cells have occupied less than half of the original
amount of heart tissue.
IV
Confronting cells have occupied more than half of the original
volume of the PHF.
Figure 1. This illustration of the chick heart invasion assay displays 7 lm thick sections from confronting cultures between precultured heart fragments (PHF) and invasive
human mammary carcinoma cells (MCF-7/6). Panel A: scoring criteria. Panel B results from an untreated culture, in which the confronting cells have occupied the PHF, and
was attributed an invasion grade of III. In panel C, addition of an anti-invasive agent resulted in a clear histological separation between the confronting partners; the invasion
grade is denoted as I/II. Panel D displays the outcome for a product with selective toxicity for MCF-7/6 cells with respect to the PHF. In this case, no judgment of the anti-
invasive potency can be made; this pattern is coined grade 0.
erative chemotherapeutics, such molecules need not per sé be
toxic towards neoplastic cells: their main task is to suppress
metastasis, not primary tumor growth. Also, when developing mol-
ecules with such a profile, the involvement of the host tissue in the
micro-ecosystem that governs tumor behavior should not be
neglected.5
An optimal screening strategy for anti-invasive molecules
therefore has to discern (i) effects on tumor cells from effects on
normal host tissue, and (ii) effects on invasiveness from mere tox-
icity. In such a case, a phenotypic, cell-based assay provides the
most valuable information. The chick heart invasion model, devel-
oped at the Bracke lab (University Hospital, Ghent, Belgium), pos-
sesses all of these properties, and was accordingly used to evaluate
the anti-invasive potency of ten of our stilbenes.35 These molecules
were selected based on their structural resemblance with previ-
ously assessed interesting chalcones,7 and with the intention of
creating a dataset suitable for SAR analysis. Although highly rele-
vant, the CHI assay is time- and labor-intensive; therefore, evalua-
tion of each of the 18 stilbenes prepared in this work was
infeasible.
agents (e.g., vinblastine, vincristine, taxol, nocodazole, podophyllo-
toxin) in this model is well-documented.36
For each of the ten selected stilbenes, the invasion grade in the
CHI assay was scored at four different concentrations (10, 1, 0.1
and 0.01
l
mol Lꢀ1, Table 6). In order to translate the assay data
into one anti-invasive value per compound, we introduced ‘log
cmin’, the logarithm of the ‘lowest active concentration cmin’ for a
compound, being the lowest concentration at which a substance
exhibits an anti-invasive behavior (invasion grades I or II, Eq. (1)):
anti-invasive activity class ¼ log cmin
ð1Þ
The lowest anti-invasive concentration was thus defined as the
lowest concentration at which both repeats could be attributed an
invasion grade I or II. If inconclusive, further repeats at that con-
centration were conducted. Conversely, if the first of two repeats
at a certain concentration yielded a grade I or II, and all repeats
at a 10-fold higher concentration did so as well, then the single re-
sult at the former, lower concentration was considered satisfac-
tory. Besides, runs at 10 l
mol Lꢀ1 were often not scored in duplo,
given the lesser therapeutic relevance of anti-invasive effects at
this concentration level; the assessment of toxic effects was rather
In the CHI in vitro screening technique, precultured heart tissue
fragments (PHFs), dissected from 9-day-old chick embryos, are
confronted with aggregates of human invasive MCF-7/6 mammary
carcinoma cells in the presence of a certain concentration of the
test compound. After eight days of incubation at 37 °C, the interac-
tion between the cancer cells and the PHF is evaluated histologi-
cally and is classified along a 5-grades scale (Fig. 1). Compounds
are coined ‘active’ or ‘anti-invasive’ when the invasion grade is I
or II, and ‘inactive’ when the grade is III or IV. For substances that
exhibit selective toxicity versus MCF-7/6 cells, denoted as invasion
grade 0, a judgment on their anti-invasive potency cannot be
made. Importantly, the behavior of numerous antiproliferative
the goal at this high concentration level
.
According to the criteria set out above, six activity levels were
defined, ranging from class-2 for the most active compounds (inva-
sion grade I or II at the 0.01
with no apparent effect at a concentration of 100
l
mol Lꢀ1 level) to class 3 (compounds
l
mol Lꢀ1).
To our satisfaction, four out of the ten evaluated molecules
exhibited a clear anti-invasive effect at concentrations as low as
Cultures of conditions not scored in duplo were archived embedded in paraffin,
and are available for consultation whenever necessary.

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B. I. Roman et al. / Bioorg. Med. Chem. 21 (2013) 5054–5063
Table 6
In vitro anti-invasive activity data against MCF-7/6 cells, obtained for 10 stilbenes in the CHI-assay (0 = toxic for MCF7/6; I and II = no invasion, the compound is coined active; III
and IV: invasion, the compound is inactive)
Structure
Invasion grade at different concentrations (
l
mol Lꢀ1
)
Anti-invasive activity classa
10
III
1
0.1
0.01
(E)-9
II/I
II
II/III/III
ꢀ1
ꢀ1
(E)-11
IV
II/II/III
II/II/Ib
II/III
MeO
F
(E)-12
(Z)-12
IV/II/II/IV/III
II
III
III/IIc
Inconclusive
II/III
III/II/II
IId/III/II/II
IId/II
ꢀ2
F
F
(E)-17
(E)-18
II
II
III/III/II/III
IV/II/IId
II/II
II/II
II
ꢀ2
ꢀ2
MeO
F
II/II
F
MeO
MeO
(E)-32
(Z)-32
II
II
III/IIc/III
II/III/II
III/II/III/II
ꢀ1
ꢀ2
OMe
MeO
II/I
II
II/II
MeO
OMe
F
MeO
(E)-33
II
III/II/II
III/III
III/III
III/II
0
1
MeO
OMe
MeO
(Z)-33
Ib
III/III
IV/II/III
MeO
OMe
F
a
b
c
Logcmin
.
few MCF-7/6 cells left.
Toxic for PHF.
PHF dedifferentiated.
d
0.01
l
mol Lꢀ1 (class-2, compounds (Z)-12, (E)-17, (E)-18 and (Z)-
of a methoxy and a fluorine substituent (17), or of two fluorine
substituents (18), enhances potency. Intriguingly, stilbene 33,
bearing a 40-fluoro-3,4,5-trimethoxy substituent pattern, is much
less active. Besides, (E)-11 exhibited a selective detrimental effect
32). In our preceding research on novel anti-invasive chalcones,
only two out of the 33 newly synthesized substances were active
at this level.7 Moreover, three other stilbenes proved potent up
to 0.1
l
mol Lꢀ1. The extraordinary activity profile of some of these
versus MCF-7/6 cells at 0.1 l .
mol Lꢀ1
stilbenes is even more perceivable when contrasted with the re-
sults from the original library of mostly natural chalcones: for
these substances, no anti-invasive tendency at concentrations be-
A similar observation can be made when examining the scores
of the (Z)-stilbenes: whereas fluorinated or trimethoxylated deriv-
atives 12 and 32 emerge as extraordinarily potent, analogue 33,
combining both decoration patterns, displays only very weak po-
tency. The somewhat capricious behavior of (Z)- and (E)-stilbenes
33 certainly warrants further investigation. Unfortunately, since
the molecular target of our stilbenes has not yet been identified,
any attempt at providing an explanation at this stage would be
speculative.
low 1 l
mol Lꢀ1 has ever been reported.6 Importantly, not all stilb-
enes proved active in the assay, which contributes to its credibility
as an appropriate model.
Interestingly, parent (E)-stilbene scaffold 9 is remarkably active.
The introduction of a 4-methoxy or a 3,4,5-trimethoxy substituent
pattern does not alter activity (11 and 32), while the combination

B. I. Roman et al. / Bioorg. Med. Chem. 21 (2013) 5054–5063
5059
When comparing the results of both isomers of stilbenes 32 or
33, no large stereochemical discrimination is tangible. Even more
so, some toxicity versus the PHF was noted for both 4-fluorostil-
bene isomers. Besides the absence of discrimination between both
isomers by the involved receptor(s), this observation may also
point to a certain degree of isomerization of the (Z)-stilbenes to
their thermodynamically more stable (E)-counterparts under the
assay conditions. Indeed, the conversion of (Z)- to (E)-diaryleth-
enes has been of concern in biological studies, both in vitro and
in vivo.27 Moreover, the presence of a para-methoxy group, as in
stilbenes 32 and 33, has been shown to facilitate this
transformation.37,38
In all, the obtained results demonstrate that two-atom spacers
between the aromatic moieties are well-tolerated, and that the
stilbene scaffold accordingly constitutes a highly suitable frame-
work for the design of in vitro anti-invasive molecules. At first
glance, the high potency of the stilbenes further seems to indicate
that the presence of a carbonyl group in the linker (as in chalcones)
is not an absolute requisite for activity (Fig. 2). However, stilbenes
33, formally obtained by the removal of the carbonyl group in
30,40,50-trimethoxy-4-fluorochalcone 1, prove a factor 100 or 1000
less potent than the latter chalcone. These results corroborate
our prior observations for the conformationally analogous 4,5-
diarylisoxazole 3. Hence, introduction of the decoration pattern
of a potent chalcone on a 1,2-diarylethene framework will not nec-
essarily yield an equally active stilbene.
their cLogP value. In this light, (E)- and (Z)-stilbenes 32 are to be
considered the most interesting members of the current library.
Attention should also be paid to the polar surface area (PSA), as this
parameter serves as a good indicator of intestinal absorption. In-
deed, in a study by Palm et al. 90% of all evaluated highly absorbed
drugs had a PSA of less than 60 Å2.40 According to this benchmark,
all compounds in Table 7 may exhibit good intestinal uptake.
Besides, (E)- and (Z)-stilbenes 32 attain a very interesting drug
score in the Osiris Property Explorer (OPE).41 This tool calculates a
number of drug-relevant properties, and compiles these in a single
‘drug score’ value on a scale between 0 (extremely poor) and 1
(highly druglike). It also recognizes structural fragments that pose
elevated risks regarding mutagenicity, tumorigenicity, irritation
and reproductive effects. In this light, elaboration of the less substi-
tuted stilbenoids is undesirable since they may exhibit mutagenic
behavior.
Taken together, trimethoxy-substituted stilbenes 32 exhibit the
most propitious druggability profile from a theoretical point of
view. In pursuit of further evidence for this assumption, we turned
our attention to the literature on the pharmacokinetics of stilbenes
in vivo.
A number of studies in mice, rats, dogs and humans indicate
that natural stilbenes such as resveratrol suffer from poor systemic
bioavailability, much like other putative antineoplastic polyphe-
nols.44–47 Due to the presence of hydroxyl groups, these molecules
undergo extensive phase II metabolism, i.e. glucoronidation or sul-
fatation. As a consequence, systemic levels of resveratrol are rarely
commensurate with anticancer efficacy, while its conjugate metab-
olites suffer from short elimination half-life.48 Therefore, further
investigations on the utility of such polyphenols may better be
limited to the targeted treatment of gastrointestinal diseases.49
Still, ample evidence indicates that (per)methylation protects
hydroxystilbenes against conjugation, thereby greatly enhancing
their biostability and pharmacokinetic characteristics. Combined
with a relatively efficient uptake, these substances exhibit good
oral bioavailability. Care should be taken while exploiting this ap-
proach, since methylation can also give rise to methyl group-spe-
cific target site interactions.50 Nonetheless, Lin et al. pointed out
that (E)-3,40,5-trimethoxystilbene, the permethylation product of
resveratrol, has greater plasma exposure, longer half-life and lower
clearance compared to its trihydroxy congener.51 Another trimeth-
oxylated analogue, (Z)-3,40,5-trimethoxy-30-aminostilbene, also
showed a satisfactory pharmacokinetic profile in a xenograft mod-
el in nude mice, with preferential accumulation in tumor, brain, li-
ver and kidneys, and persistently high levels of the compound in
the tumor xenograft.52
Further, since (E)- as well as (Z)-stilbene geometries rather
resemble an s-trans- than an s-cis chalcone conformation, the assay
results seem to indicate that the active conformer of chalcone 1
may be more s-trans- than s-cis-like, which is in agreement with
our earlier report on conformationally restrained chalcone
analogues.8
Further investigation into the binding modes of these molecules
is, however, needed to confirm these hypotheses. Hence, target
identification efforts are currently being undertaken for the most
active compounds.
3. Druglikeness
Given the extraordinary anti-invasive potency of some of the
presented stilbenes, we decided to take a closer look at their phys-
icochemical properties. An array of calculated properties that can
provide initial clues of which molecules possess the most promis-
ing profile is presented in Table 7. All compounds adhere to Lipin-
ski’s rule of five (ROF), which serves as a basic touchstone of their
oral bioavailability and distribution profile.39
The beneficial effect of methylation was also observed in the
case of piceatannol, a tetrahydroxystilbene.53 Intriguingly, upon
p.o. administration, the isomeric 3,4,5,40-tetramethoxystilbene is
more readily available than resveratrol in intestinal and colonic
Although the cLogP-values of the presently evaluated stilbenes
are still acceptable (ꢂ3–4.5), these substances are quite lipophilic.
From a pharmacological point of view, care should thus be taken
during ongoing development steps to avoid a further increase of
F
F
MeO
MeO
MeO
MeO
OMe
F
MeO
OMe
(E)-9
(E)-17
(E)-33
(Z)-33
class -1
class -2
class 0
class 1
O
O
O
MeO
MeO
F
MeO
F
OMe
34
35
1
class 0
class 0
class -2
Figure 2. Comparison of the activity of identically decorated stilbenes and chalcones in the chick heart invasion assay.

5060
B. I. Roman et al. / Bioorg. Med. Chem. 21 (2013) 5054–5063
Table 7
Activity and drug-relevant properties for the ten in vitro evaluated stilbenes
Anti-invasive activity class
Lipinski-related properties
cLogSe
Drug scoref
PSAg (Å2)
cLogPa
ACD/Labs
MW (g molꢀ1
)
HBDb
HBAc
FRBd
OPE
(E)-9
ꢀ1
ꢀ1
inc.h
ꢀ2
ꢀ2
ꢀ2
ꢀ1
ꢀ2
0
4.02
3.91
4.08
4.08
3.97
4.13
3.70
3.70
3.76
3.76
3.89 0.19
3.94 0.22
4.38 0.38
4.38 0.38
4.43 0.40
4.87 0.53
3.35 0.33
3.35 0.33
4.43 0.44
4.43 0.44
180.25
210.27
198.24
198.24
228.26
216.23
270.32
270.32
288.31
288.31
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
3
3
3
3
2
3
2
2
3
2
5
5
5
5
ꢀ3.75
ꢀ3.77
ꢀ4.07
ꢀ4.07
ꢀ4.08
ꢀ4.38
ꢀ3.81
ꢀ3.81
ꢀ4.12
ꢀ4.12
0.09
0.09
0.08
0.08
0.15
0.14
0.73
0.73
0.56
0.56
0
9.23
0
0
9.23
0
27.7
27.7
27.7
27.7
(E)-11
(E)-12
(Z)-12
(E)-17
(E)-18
(E)-32
(Z)-32
(E)-33
(Z)-33
1
a
b
c
41
Calculated LogP values, obtained from Osiris Property Explorer (OPE
HBD: number of hydrogen bond donors.
HBA: number of hydrogen bond acceptors.
)
or Advanced Chemistry Development (ACD/Labs) Software V11.02.42
d
e
f
FRB: number of freely rotatable bonds.
estimation of LogS by OPE, the unit stripped logarithm of the aqueous solubility in mol Lꢀ1
.
Drug Score, general estimation of druglikeness (on a scale from 0 to 1) calculated by OPE
.
g
h
PSA: polar surface area as predicted by the tool ‘interactive calculation of molecular PSA’ provided by Novartis.43
Inconclusive.
mucosae and in the brain, but not in the plasma, liver or heart.54,55
One explanation may be that the introduction of four methoxy
moieties inflicts too high a lipophilicity on the stilbene. Besides,
this compound proved prone to hepatic metabolic demethylation
and hydroxylation.
From the above in vivo literature data, it seems that trimethoxy
stilbenes possess a good balance between metabolic stability and
lipophilicity.2 These observations are consonant with the interest-
ing in silico druggability profile of stilbenes 32. Combined with an
extraordinary intrinsic potency in vitro, we therefore consider the
(Z)- and (E)-isomers of stilbene 32 as interesting hits in the search
for potent anti-invasive drug candidates.
High resolution 1H (300 MHz), 13C (75 MHz) and 19F (282 MHz)
magnetic resonance (NMR) spectra were recorded on a Jeol
Eclipse+300 FT NMR spectrometer in deuterated solvents. Chemi-
cal shifts are reported using TMS and/or CFCl3 as an internal ref-
erence, unless otherwise indicated. Peak assignments were
obtained with the aid of DEPT, HSQC, HMBC and COSY spectra.
Attenuated total reflection (ATR) IR spectra were recorded on a
Perkin Elmer Spectrum BX spectrometer, equipped with a ZnSe-
crystal, at room temperature (neat). Low-resolution mass spectra
were recorded on an Agilent Technologies 1100 series VL mass
spectrometer (ESI, 70 eV). HRMS analyses were conducted using
an HPLC coupled to an Agilent Technologies 6210 series time-
of-flight mass spectrometer equipped with an ESI/APCI-multi-
mode source. Melting points were measured with a Büchi B-540
apparatus and are uncorrected.
4. Conclusion
The present study sheds light on the hitherto scarcely explored
anti-invasive potency of stilbenes. A total of 13 (E)- and 5-(Z)-stil-
benes were prepared in high purity following various synthetic
strategies. Ten of these molecules were evaluated for their anti-
invasive activity in the in vitro chick heart invasion assay. Four
stilbenes proved efficacious down to 10 nmol Lꢀ1, an activity level
100 times lower than that of the natural chalcones which served as
hit compounds in our quest for anti-invasive therapeutics. A preli-
minary druggability survey points towards trimethoxy stilbenes as
the most suitable subjects of further investigation. Hence, we will
further elaborate the in vitro and in vivo pharmacology of these
stilbenes and related compounds, in the pursuit of highly
promising anti-invasive drug candidates.
5.1.1. Preparation of (E)-stilbenes 9-11 via a Wittig reaction
Benzyl bromide (1 mmol) and triphenylphosphine (0.26 g,
1 equiv) were added to a flame-dried, round-bottomed flask filled
with 5 mL of dry diethyl ether. The resulting suspension was
heated to reflux under a nitrogen atmosphere for 12 h, where-
upon the precipitated phosphonium salt was isolated by
filtration.
The resulting suspension could also be used as such in the
Wittig reaction. In this case, 1 equiv of KOtBu was added, after
which the mixture was heated to reflux, still under a nitrogen
atmosphere, for a further hour. Next, the contents of the flask were
cooled to 0 °C and the aldehyde (0.5 equiv) was added dropwise.
The mixture was subsequently heated to reflux temperature for
several hours, while reaction progress was monitored by means
of TLC.
Upon reaction completion, the suspension was quenched by
addition of an equal volume of water, and the emulsion was ex-
tracted with diethyl ether (2ꢃ). The combined organic layers were
dried over MgSO4, and the solvent was evaporated in vacuo. The
crude material was taken up with hexane; impurities were
removed by filtration and the hexane phase was concentrated in
vacuo, furnishing the desired stilbene. If a mixture of (E)- and
(Z)-isomers was formed, separation was conducted by means of
preparative TLC, employing an eluent mixture of petroleum ether
and diethyl ether (4:1).
5. Experimental part
5.1. Synthesis
All reagents were purchased from commercial sources (Aldrich,
Acros) and used without further purification. Solvents were pur-
chased from commercial sources (Aldrich) and employed as is. The
petroleum ether, employed during product purification steps, had
a boiling range 40–60 °C. Microwave reactions were carried out in
a CEM Benchmate apparatus (maximum power: 300 W). Crude reac-
tion mixtures were analyzed on LC/MS/UV. Thin layer chromatogra-
phy was carried out on silica gel 60F254 plates (Merck).

B. I. Roman et al. / Bioorg. Med. Chem. 21 (2013) 5054–5063
5061
5.1.2. Preparation of (E)-stilbenes 10–13 and 16–22 via Horner–
Wadsworth–Emmons reaction
5.1.7. Synthesis of 1,2-diarylacetylenes 25–30 via Sonogashira-
coupling
A mixture of a suitable benzyl bromide (1 mmol) and excess tri-
ethyl phosphite were stirred at 130 °C for 20 h. Removal of the
remaining phosphite by vacuum distillation furnished the desired
phosphonate as the residue. This intermediate was dissolved in
2 mL of dry DMF, contained in a flame-dried round-bottomed flask
A round-bottomed flask was loaded with PdCl2(PPh3)2
(0.02 mmol, 2 mol %) and thoroughly flushed with argon. sec-
Butylamine (0.5 mL), water (0.5 mL), the aryl iodide (1 mmol)
and the acetylene (1.3 mmol, 1.3 equiv) were added respectively,
after which the headspace was flushed with argon once again.
The resulting mixture was stirred at 25 °C, under an inert atmo-
sphere, for a suitable time. The desired acetylene was obtained
as a precipitate, which was isolated via filtration and air-dried.
under
a nitrogen atmosphere. Next, a solution of NaOMe
(0.73 equiv) in methanol was added dropwise. The contents of
the flask were cooled to 0 °C, upon which the aldehyde (0.67 equiv)
was added. The resulting mixture was then successively stirred at
room temperature for 1 h, heated to reflux temperature for another
hour and left standing overnight. Afterward, a mixture of water
and methanol (1:1) was added, which provided the desired stil-
bene as a precipitate. A second crop of crystals was harvested from
the mother liquor.
5.1.8. 1,2,3-Trimethoxy-5-(phenylethynyl)benzene 29
Yield: 98%, as a brown solid; mp = 20–21 °C; Rf: 0.68 (petroleum
ether/EtOAc 1:1); IR (ATR, cmꢀ1):
mmax: 1574, 1503, 1462, 1409,
1355, 1256, 1233; 1H NMR (CDCl3, 300 MHz, ppm): d 3.88 (s, 3H,
C2OMe), 3.89 (s, 6H, C1OMe, C3OMe), 6.78 (s, 2H, H4, H6), 7.30–
0
0
0
0
0
7.40 (m, 3H, H3 , H4 , H5 ), 7.50–7.56 (m, 2H, H2 , H6 ); 13C NMR (ace-
5.1.3. (E)-3-Fluorostilbene 10
tone-d6, 75 MHz, ppm): d 56.5 (C1OMe, C3OMe), 60.7 (C2OMe),
0
Yield: 79%, as white crystals; mp: 75–76 °C; IR (ATR, cmꢀ1):
88.8, 90.4 (2ꢃ Cq), 109.8 (C4, C6), 118.9 (C5), 124.1 (C1 ), 129.3
0
0
0
0
0
m
max: 1606, 1580, 1495, 1484, 1466, 1447, 1266; 1H NMR (CDCl3,
(C4 ), 129.5 (C3 , C5 ), 132.2 (C2 , C6 ), 140.2 (C2), 154.4 (C1, C3);
300°MHz, ppm): d 6.95 (ddd, 1H, Jvic = 8.3 Hz, JHF = 8.3 Hz, Jallyl
=
=
ESI-MS (+) m/z: 269.2 ([M+H]+, 100).
1.7 Hz, H4), 7.05 (d, 1H, Jtrans = 16.0 Hz, CH), 7.12 (d, 1H, Jtrans
16.0 Hz, CH), 7.22 (dt, 1H, JHF = 10.0 Hz, Jallyl = 1.6 Hz, H2), 7.26–
5.1.9. 1,2,3-Trimethoxy-5-(40-fluorophenylethynyl)benzene 30
0
7.33 (m, 3H, H5, H6, H4 ), 7.37 (dt, 2H, Jvic = 7.4 Hz, Jallyl = 1.7 Hz,
Yield: 99%, as brown crystals; mp = 92–93 °C; IR (ATR, cmꢀ1):
0
0
H3’, H5’), 7.51 (dd, 2H, Jvic = 7.4 Hz, Jallyl = 1.7 Hz, H2 , H6 ); 13C
NMR (CDCl3, 75 MHz, ppm): d 112.8 (d, JCF = 21.9 Hz, C2), 114.4
m
max: 1574 1506, 1463, 1448, 1356, 1237; 1H NMR (CDCl3, 300
MHz, ppm): d 3.88 (s, 3H, C2OMe), 3.89 (s, 6H, C1OMe, C3OMe),
0
0
0
(d, JCF = 21.9 Hz, C4), 122.5 (d, JCF = 2.3 Hz, C6), 126.7 (C2 , C6 ),
6.76 (s, 2H, H4, H6), 7.05 (dd, 2H, JHF = 8.8 Hz, JHvic = 8.8 Hz, H3 ,
0
0
0
0
0
a
127.5 (d, JCF = 2.3 Hz, C ), 128.0 (Cb), 128.7 (C3 , C5 ), 130.0, 130.2
H5 ), 7.51 ppm (dd, 2H, JHvic = 8.8 Hz, JHF = 5.5 Hz, H2 , H6 ); 13C
NMR (acetone-d6, 75 MHz, ppm): d 56,5 (C1OMe, C3OMe), 60.7
(C2OMe), 87.7 (Cq), 90.1 (Cq), 109.8 (C4, C6), 116.6 (d, JCF = 21,9 Hz,
0
0
(C4 , C5), 136.8 (C1 ), 139.7 (d, JCF = 6.9 Hz, C1), 163.2 (d, JCF = 245.8 Hz,
C3); ESI-MS (+) m/z: 198 ([M+H]+, 100).
0
0
C3 , C5 ), 118.7 (C5), 120.5 (d, JCF = 3.5 Hz, C1), 134.4 (d, JCF = 8.1 Hz,
0
0
0
5.1.4. (E)-3,40-Difluorostilbene 16
C2 , C6 ), 140.3 (C2), 154.4 (C1, C3), 163.4 ppm (d, JCF = 246.9 Hz, C4 );
ESI-MS (+) m/z: 287.3 ([M+H]+, 100).
Yield: 99%, as white crystals; mp: 92–93 °C; IR (ATR, cmꢀ1):
m
max: 1597, 1578, 1507, 1482, 1444, 1266, 1228, 1217; 1H NMR
(acetone-d6, 300 MHz, ppm): d 6.98–7.09 (m, 1H, H3), 7.17 (dd,
5.1.10. Synthesis of (Z)-stilbenes 9, 11, and 32 via a
hydrosilylation–protodesilylation sequence. Isolation of (E)-32
and (E)-33
0
0
4H, JHF = 8.8 Hz, JHvic = 8.8 Hz, H3 , H5 ), 7.22 (d, 1H, Jtrans
=
16.2 Hz, CH), 7.34 (d, 1H, Jtrans = 16.2 Hz, CH), 7.37–7.42 (m, 3H,
0
0
H2, H5, H6), 7.68 (dd, 2H, JHF = 5.5 Hz, JHvic = 8.8 Hz, H2 , H6 ); 13C
NMR (CDCl3, 75 MHz, ppm): d 112.7 (d, JCF = 21.9 Hz, C2), 114.4
A flame-dried, round-bottomed flask was loaded, in turn, with
the diaryl acetylene (1 equiv), EtOMe2SiH (1.5 equiv) and PtO2
(7 mol %). The resultant mixture was kept overnight at 60 °C, under
a nitrogen atmosphere. Next, the excess EtOMe2SiH was evapo-
rated under a high vacuum atmosphere, upon which the residue
was dissolved in dry THF. The contents of the flask were cooled
to 0 °C, and a solution of tetra-n-butylammonium fluoride (TBAF)
in THF was added dropwise (3 equiv). The mixture was stirred
overnight under a nitrogen atmosphere, while the temperature
was allowed to rise to 20 °C. The contents of the flask were filtered
over Celite; the filtrate was concentrated in vacuo, taken up in
diethyl ether and washed with brine (2ꢃ). The organic phase was
subsequently dried over MgSO4 and concentrated by rotary evapo-
ration, furnishing the desired stilbene as a mixture of stereoiso-
mers. The (E)- and (Z)-isomers were isolated by means of
preparative TLC, employing an eluent mixture of petroleum ether
and diethyl ether (4:1).
0
0
(d, JCF = 21.9 Hz, C4), 115.7 (d, JCF = 21.9 Hz, C3 , C5 ), 122.4 (d,
0
0
JCF = 2.3 Hz, C6), 127.3 (@CH), 128.2 (d, JCF = 8.1 Hz, C2 , C6 ), 128.8
0
(@CH), 130.1 (d, JCF = 8.1 Hz, C5), 133.0 (d, JCF = 3.5 Hz, C1 ), 139.5
0
(d, JCF = 8.1 Hz, C1), 162.6 (d, JCF = 244.6 Hz, C4 ), 163.2 ppm (d,
JCF = 248.1 Hz, C3); ESI-MS (+) m/z: 216 ([M+H]+, 100).
5.1.5. (E)-4,40-Difluorostilbene 18
Yield: 48%, as yellow–brown crystals; mp: 116-117 °C; IR (ATR,
cmꢀ1):
m
max: 1599, 1506, 1255, 1230, 1205; 1H NMR (CDCl3, 300
a
MHz, ppm): d 6.98 (s, 2H, H , Hb), 7.05 (dd, 4H, JHF = 8.8 Hz, JHvic
0
0
= 8.8 Hz, H3, H5, H3 , H5 ), 7,46 (dd, 4H, JHF = 5.5 Hz, JHvic = 8.8 Hz,
0
0
H2, H6, H2 , H6 ); 13C NMR (CDCl3, 75 MHz, ppm): d 116.3 (d,
0
0
JCF = 21.9 Hz, C3, C5, C3 , C5 ), 128.2 (C , Cb), 129.2 (d, JCF = 8.1 Hz,
a
0
0
0
C2, C6, C2 , C6 ), 134.8 (d, JCF = 3.5 Hz, C1, C1 ), 163.2 (d, JCF = 244.6 Hz,
C4); ESI-MS (+) m/z: 216 ([M+H]+, 100).
5.1.6. (E)-40-Chloro-3-methoxystilbene 22
Yield: 95%, as white crystals; mp: 71–72 °C; IR (ATR, cmꢀ1):
5.1.11. (Z)-3,4,5-Trimethoxystilbene 32
max: 1604, 1575, 1444, 1431, 1269, 1251; 1H NMR (CDCl3,
Yield: 13%, as a yellow oil; Rf: 0.72 (petroleum ether/EtOAc 1:1);
m
IR (ATR, cmꢀ1):
mmax: 1579, 1505, 1462, 1451, 1420, 1403, 1327,
300 MHz, ppm): d 3.83 (s, 3H, OMe), 6.82 (dd, 1H, Jvic = 7.7 Hz,
1236, 1123; 1H NMR (CDCl3, 300 MHz, ppm): d 3.64 (s, 6H,
C3OMe, C5OMe), 3.83 (s, 3H, C4OMe), 6.46 (s, 2H, H2, H6), 6.49 (d,
1H, Jcis = 12.1 Hz, CH), 6.59 (d, 1H, Jcis = 12.1 Hz, CH), 7.14–7.38
Jallyl = 2.6 Hz, H4), 6.97–7.09 (m, 3H, H2, H , Hb), 7.09 (d, 1H,
a
Jvic = 7.7 Hz, H6), 7.27 (t, 1H, Jvic = 7.7 Hz, H5), 7.31 (d, 2H, Jvic = 8.5 -
0
0
0
0
Hz, H3 , H5 ), 7.42 (d, 2H, Jvic = 8.5 Hz, H2 , H6 ); 13C NMR (CDCl3,
75 MHz, ppm): d 55.4 (OMe), 111.9 (C2), 113.6 (C4), 119.4 (C6),
0
0
0
0
0
(m, 5H, H2 , H3 , H4 , H5 , H6 ); 13C NMR (CDCl3, 75 MHz, ppm): d
0
0
0
0
40
55.8 (C3OMe, C5OMe), 60.9 (C4OMe), 106.0 (C2, C6), 127.1 (C ),
127.8 (br.s, C2 , C6 , CH), 129.0 (br. s, C3 , C5 ), 129.3 (CH), 129.8
0
0
0
20
60
50
128.2 (C , C ), 128.9 (C3 , C ), 130.0 (CH), 130.1 (CH), 132.4 (C1),
(br. s, C5), 133.3, 135.8, 138.5 (C1, C1 , C4 ), 160.0 (C3); ESI-MS (+)
0
137.1 (C4), 137.4 (C1 ), 152.8 ppm (C3, C5); ESI-MS (+) m/z: 271.3
m/z: 245.2 ([M+H]+, 100), 247.2 ([M+2+H]+, 35).

5062
B. I. Roman et al. / Bioorg. Med. Chem. 21 (2013) 5054–5063
+
([M+H]+, 100); ESI-HRMS (+) m/z: Anal. Calcd for C17H19O3
hematoxylin and eosin. In alternating sections, cancer cells were
stained with the 5D10 monoclonal antibody, so as to evaluate
the interaction between the confrontation partners.
(M+H)+: 271.1329. Found: 271.1335.
5.1.12. (E)-3,4,5-Trimethoxystilbene 32
Spectral data were in full accordance with those reported by
Borate et al.56
All compounds were tested in duplo at 10, 1, 0.1 and 0.01
l
mol Lꢀ1 and added as DMSO solutions. Vehicle cultures (negative
control, DMSO) yielded a grade III or IV result in all cases. Further
information on this assay is included in the relevant parts of this
manuscript.
5.1.13. (E)-40-Fluoro-3,4,5-trimethoxystilbene 33
Yield: 3%, as a yellow oil; Rf: 0.69 (petroleum ether/EtOAc 1:1);
IR (ATR, cmꢀ1):
mmax: 1582, 1521, 1508, 1449, 1334, 1239, 1225,
1152, 1128; 1H NMR (CDCl3, 300 MHz, ppm): d 3.87 (s, 3H,
C4OMe), 3.92 (s, 6H, C3OMe, C5OMe), 6.72 (s, 2H, H2, H6), 6.93 (d,
1H, Jtrans = 16.5 Hz, @CH), 6.99 (d, 1H, Jtrans = 16.5 Hz, @CH), 7.05
Acknowledgements
The authors thank the Research Foundation – Flanders (Fonds
voor Wetenschappelijk Onderzoek – Vlaanderen) for financial sup-
port for this research, and would like to express their sincere
appreciation to Marleen De Meulemeester for meticulously con-
ducting the CHI-assay on all compounds investigated in this study.
0
0
(dd, 2H, JHF = 8.8 Hz, JHvic = 8.8 Hz, H3 , H5 ), 7.47 (dd, 2H, JHvic = 8.9 -
0
0
Hz, JHF = 5.0 Hz, H2 , H6 ); 13C NMR (CDCl3, 75 MHz, ppm): d 56.1
(C3OMe, C5OMe), 61.0 (C4OMe), 103.5 (C2, C6), 115.7 (d, JCF = 21.9 -
0
0
0
0
Hz, C3 , C5 ), 127.0 (@CH), 127.9 (d, JCF = 8.1 Hz, C2 , C6 ), 128.4
0
(@CH), 132.9 (C1), 133.4 (d, JCF = 3.5 Hz, C1 ), 137.9 (C4), 153.4 (C3,
0
C5), 162.3 (d, JCF = 246.9 Hz, C4 ); ESI-MS (+) m/z: 289.2 ([M+H]+,
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