Inhibitory effect of cytotoxic stilbenes related to resveratrol on the expression of the VEGF, hTERT and c-Myc genes

Article abstract of DOI:10.1016/j.ejmech.2015.09.014

A group of thirty-nine stilbene derivatives, prepared by means of Heck coupling reactions, has been investigated for their cytotoxicity, as well as for their ability to inhibit the production of the vascular endothelial growth factor (VEGF) and the activation of telomerase. The ability of these compounds to inhibit proliferation of two tumoral cell lines (HT-29 and MCF-7) and one non tumoral cell line (HEK-293) was first determined. Subsequently, we determined the capacity of the compounds to inhibit the secretion of VEGF in the aforementioned cell lines and to downregulate the expression of the VEGF, hTERT and c-Myc genes, the two latter involved in the control of the activation of telomerase. One of the synthetic stilbenes, (E)-4-(4-methoxystyryl)aniline, showed strong cytotoxicity and proved able to cause a marked decrease both in the secretion of VEGF and in the expression of the hTERT and c-Myc genes, in all cases at concentrations in the low nanomolar range.

Full text of DOI:10.1016/j.ejmech.2015.09.014

Accepted Manuscript
Inhibitory effect of cytotoxic stilbenes related to resveratrol on the expression of the
VEGF, hTERT and c-Myc genes
Rosa Martí-Centelles, Eva Falomir, Juan Murga, Miguel Carda, J. Alberto Marco
PII:
S0223-5234(15)30258-0
10.1016/j.ejmech.2015.09.014
EJMECH 8111
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 March 2015
Revised Date: 8 September 2015
Accepted Date: 9 September 2015
Please cite this article as: R. Martí-Centelles, E. Falomir, J. Murga, M. Carda, J.A. Marco, Inhibitory
effect of cytotoxic stilbenes related to resveratrol on the expression of the VEGF, hTERT and c-Myc
genes, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.09.014.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Inhibitory effect of cytotoxic stilbenes related to resveratrol on the expression of
the VEGF, hTERT and c-Myc genes
*
*
Rosa Martí-Centelles, Eva Falomir Juan Murga, Miguel Carda and J. Alberto Marco
Graphical Abstract
Thirty-nine stilbenes have been prepared. Their cytotoxicities and ability to inhibit the expression of
VEGF, h-TERT and c-Myc genes have been measured. One compound is very active in these
properties.
2

ACCEPTED MANUSCRIPT
Inhibitory effect of cytotoxic stilbenes related to resveratrol on the expression of
the VEGF, hTERT and c-Myc genes
*
*
Rosa Martí-Centelles, Eva Falomir, Juan Murga, Miguel Carda and J. Alberto Marco
Highlights
•
Thirty-nine stilbene derivatives related to resveratrol were prepared through Heck type
coupling reactions
•
•
•
•
The molecules were tested for cytotoxicity on a normal and two tumoral cell lines
The molecules were tested as regards inhibition of secretion of VEGF protein
The molecules were tested as regards downregulation of VEGF, h-TERT and c-Myc genes
(E)-4-(4-methoxystyryl)aniline showed cytotoxicity at the low nanomolar level and was
able to significantly inhibit secretion of VEGF protein and the expression of the VEGF, h-
TERT and c-Myc genes at similarly low concentrations
3

ACCEPTED MANUSCRIPT
Inhibitory effect of cytotoxic stilbenes related to resveratrol
on the expression of the VEGF, hTERT and c-Myc genes
a
a
a
a
b
Rosa Martí-Centelles, Eva Falomir, * Juan Murga, Miguel Carda, * and J. Alberto Marco
a
Depart. de Q. Inorgánica y Orgánica, Univ. Jaume I, E-12071 Castellón, Spain
b
Depart. de Q. Orgánica, Univ. de Valencia, E-46100 Burjassot, Valencia, Spain
*
Authors to whom correspondence should be addressed. E-Mail addresses: efalomir@qio.uji.es,
mcarda@qio.uji.es
ABSTRACT
A group of thirty-nine stilbene derivatives, prepared by means of Heck coupling reactions, has been
investigated for their cytotoxicity, as well as for their ability to inhibit the production of the vascular
endothelial growth factor (VEGF) and the activation of telomerase. The ability of these compounds to
inhibit proliferation of two tumoral cell lines (HT-29 and MCF-7) and one non tumoral cell line (HEK-
2
93) was first determined. Subsequently, we determined the capacity of the compounds to inhibit the
secretion of VEGF in the aforementioned cell lines and to downregulate the expression of the VEGF, h-
TERT and c-Myc genes, the two latter involved in the control of the activation of telomerase. One of the
synthetic stilbenes, (E)-4-(4-methoxystyryl)aniline, showed strong cytotoxicity and proved able to
cause a marked decrease both in the secretion of VEGF and in the expression of the h-TERT and c-Myc
genes, in all cases at concentrations in the low nanomolar range.
KEYWORDS
resveratrol, stilbenes, VEGF, gene regulation, telomerase, hTERT, c-Myc.
4

ACCEPTED MANUSCRIPT
1
. Introduction
Resveratrol, a natural compound belonging to the class of stilbenes (Fig. 1) is a polyphenolic
compound which occurs in grapes, red wine, chocolate, and certain berries and roots. It became popular
since the 1993 paper on its inhibitory effects against the oxidation of human low-density lipoproteins, a
chemical event at the onset of the pathogenesis of atherosclerosis [1]. In later years, further important
biological features were reported for the compound, most particularly antioxidant, anticarcinogenic,
and anti-inflammatory properties [2]. These properties were related to the ability of the compound to
retard the onset of age-related diseases [3] and to its beneficial effect on other ailments such as viral
attacks [4], diabetes [5], inflammation [6] and neurodegenerative diseases [7]. In addition, resveratrol
has been shown to display, among other features: a) cancer chemopreventive effects by means of
inhibiting the initiation step of the carcinogenesis process [8]; b) anticancer activity through inhibition
of cell cycle progression and induction of apoptotic cell death [9]; and c) antiangiogenic activity with
sufficient potency to suppress FGF-2 and VEGF-induced neovascularisation in vivo [10] and to inhibit
bovine aorta endothelial cell proliferation, migration and tube formation in vitro [11].
Figure 1. Structure of resveratrol
Tumor angiogenesis is a very complex process and involves the tight interplay of many factors [12].
Vascular endothelial growth factor (VEGF) is an inducer of angiogenesis and promotes endothelial cell
survival, proliferation and migration while increasing vascular permeability [13]. In fact,
overexpression in the production of VEGF has been reported to occur in various types of tumors [14].
Not unexpectedly, VEGF has become one further key target molecule in cancer therapy [15,16].
Cancer cells are able to evolve the ability to overcome senescence upon telomerase reactivation
[
17]. This enzyme has been detected in about 90% of all malignant tumors [18], so that drugs with
ability to inhibit telomerase activity are potentially useful weapons in the fight against cancer, aging
and other diseases related to premature telomere shortening [19]. Recently, it has been reported that
resveratrol downregulates the telomerase activity of MCF-7 cells and the nuclear levels of hTERT, the
reverse transcriptase subunit of the telomerase complex [20].
5

ACCEPTED MANUSCRIPT
2
. Research purpose
When aiming at a more efficient anticancer therapy, compounds exhibiting not only high cytotoxic
activity but also the ability to suppress tumoral angiogenesis and the expression of genes related to
telomerase activation would be highly desirable [21]. On our ongoing research on anticancer properties
of synthetic analogues derived from natural products, we have recently reported on the inhibition of
VEGF formation in cancer cells and on endothelial cell differentiation promoted by synthetic stilbene
derivatives [22]. Our study has established that resveratrol and several stilbene derivatives lead to a
decrease in the production of VEGF by HT-29 cells. In view of these precedents we decided to evaluate
the influence of new synthetic stilbene analogues not only on VEGF production, but also on the
expression of the hTERT gene, involved in the process of telomerase activation. In addition, we further
decided to measure the inhibition of expression of c-Myc, an oncogene which encodes the c-Myc
protein that acts as a transcription factor for hTERT expression.
The structures of the synthetic resveratrol analogues which are the subject of this work are depicted
in Figure 2. These compounds belong to two types: one corresponds to stilbenes carrying hydroxy
and/or methoxy groups (a-series in Figure 2), whereas the other (b-series) includes stilbenes carrying
amine or amide functions in one of the rings, as well as hydroxyl or methoxy groups in the other.
1
2
1
2
1
2
1
2
3
4
5
a R = 2-OH R = 2´-OH
6a R = 4-OH R = 4´-OH
11a R = 3-OMe R = 3´-OH
1
2
1
2
1
2
a R = 3-OH R = 2´-OH
7a R = 2-OMe R = 2´-OH
12a R = 4-OMe R = 3´-OH
1
2
1
2
1
2
a R = 4-OH R = 2´-OH
8a R = 3-OMe R = 2´-OH
13a R = 2-OMe R = 4´-OH
1
2
1
2
1
2
a R = 3-OH R = 3´-OH
9a R = 4-OMe R = 2´-OH
14a R = 3-OMe R = 4´-OH
1
2
1
2
1
2
a R = 4-OH R = 3´-OH 10a R = 2-OMe R = 3´-OH
15a R = 4-OMe R = 4´-OH
1
2
1
2
1
2
1
2
3
4
5
6
7
8
b R = 2-NH R = H
9b R = 3-NH R = 4´-OH
17b R = 3-NH R = 3´-OMe
2
2
2
1
2
1
2
1
2
b R = 3-NH
2
2
2
R = H
10b R = 4-NH
2
2
2
R = 2´-OH
18b R = 3-NH
2
2
2
R = 4´-OMe
1
2
1
2
1
2
b R = 4-NH
R = H
11b R = 4-NH
R = 3´-OH
19b R = 4-NH
R = 2´-OMe
1
2
1
2
1
2
b R = 2-NH
R = 2´-OH 12b R = 4-NH
R = 4´-OH
20b R = 4-NH
R = 3´-OMe
1
2
1
2
1
2
b R = 2-NH R = 3´-OH 13b R = 2-NH R = 2´-OMe 21b R = 4-NH R = 4´-OMe
2
2
2
1
2
1
2
1
2
b R = 2-NH R = 4´-OH 14b R = 2-NH R = 3´-OMe 22b R = 2-NHCO(CH ) Me R = H
2
2
2 10
1
2
1
2
1
2
b R = 3-NH R = 2´-OH 15b R = 2-NH R = 4´-OMe 23b R = 3-NHCO(CH ) Me R = H
2
2
2 10
1
2
1
2
1
2
b R = 3-NH R = 3´-OH 16b R = 3-NH2 R = 2´-OMe 24b R = 4-NHCO(CH ) Me R = H
2
2 10
Figure 2. Structures of synthetic resveratrol analogues.
6

ACCEPTED MANUSCRIPT
3
. Synthetic work
Stilbene derivatives 1a-15a were prepared by means of palladium-catalyzed Heck-type cross
coupling reactions [23]. Monohydroxylated styrenes were used as the nucleophilic partners whereas
hydroxy or methoxy bromobenzenes played the role of the electrophilic components, with Pd(PPh3)4
being the catalyst [24] (conditions A in Scheme 1). These reactions were performed under microwave
irradiation, as these conditions have often been found to accelerate this kind of coupling reactions [25]
(
for details and yields, see Experimental section).
Scheme 1. Synthesis of stilbene derivatives by means of palladium-catalyzed Heck couplings (for more
detailed descriptions of the reaction conditions and yields, see Supporting Information).
Preparation of compounds belonging to the b-series was also carried out with Heck-type cross
coupling reactions. The same type of nucleophilic partners as above were used, with the electrophilic
components being now bromoanilines. The aforementioned reaction conditions A, however, used in the
synthesis of compounds of the a-series, were successful in the preparation of some compounds (e. g.
4
b, 5b, 7b, 9b), but proved inefficient in other cases or else failed completely. In order to obtain the
remaining amino stilbenes without protection of the amino groups, we undertook a survey of catalysts
and conditions which resulted in three new reactions conditions, B, C and D (Scheme 1). In this case,
3 2 2
Pd(NH ) Cl was the catalyst [26], the differences residing in the reaction conditions. All Heck
reactions were highly stereoselective and afforded, after chromatographic purification, the
corresponding stilbenes of E-configuration with no traces of Z-isomers. Yields ranged from 50% to
8
8% and, most importantly, no protection/deprotection steps were needed.
Amides 22b-24b were prepared with the aim at observing whether the introduction of a lipophilic
side chain would cause visible changes in the biological properties. Amide 23b was synthesized upon
7

ACCEPTED MANUSCRIPT
coupling compound 2b with dodecanoic acid in the presence of chlorosulfonyl isocyanate [27].
Attempts at preparing 22b and 24b in the same way by means of acylation of 1b and 3b, respectively,
were unsatisfactory. Thus, 22b and 24b were obtained by first acylating the corresponding
bromoaniline followed by Heck coupling of the resulting amide with styrene under conditions C.
4
. Biological work
4
.1. Inhibition of cell proliferation
We carried out a measurement of the cytotoxic activity of our synthetic stilbenes using two tumoral
cells, the human colon HT-29 and the breast adenocarcinoma MCF-7 cell lines, as well as one non
tumoral cell line, the human embryonic kidney cell line, HEK-293 [28]. Table 1 shows cytotoxicity
values for stilbenes of the a-series, expressed as the compound concentration (µg/mL) that causes 50%
inhibition of cell growth (IC ). Table 1 further shows the α and β coefficients, obtained by dividing
5
0
the IC values of the normal cell line by those of either the HT-29 or the MCF-7 cell line, respectively
5
0
(
see footnote in Table 1). The higher the value of either coefficient, the higher the therapeutic safety
margin of the compound in the corresponding cell line.
Table 1. IC values (µg/mL) and selectivity coefficients for compounds of the a-series.
5
0
b
c
Compound
Resveratrol
HT-29
MCF-7
HEK-293
α
β
34.1 ± 1.4
25± 7
16.1 ± 1.1
23.0 ± 1.3
20.1 ± 2.1
21 ± 3
7.1 ± 1.0
24.3 ± 2.3
18 ± 8
0.2
0.9
0.7
0.8
0.8
0.2
< 0.1
0.7
0.8
2.1
1.4
1.6
1.1
1.7
0.9
0.4
1.1
0.9
0.8
1
1a
2a
3a
4a
5a
6a
7a
8a
9a
27 ± 7
22 ± 9
17.5 ± 1.5
21 ± 5
25.9 ± 1.0
6.9 ± 1.9
>100
21 ± 6
23 ± 3
1.1 ± 0.7
1.2 ± 0.1
15.0 ± 2.5
20 ± 4
< 0.1
0.2
0.8
1.1
1.2
1
6.8 ± 0.2
17.9 ± 2.4
18 ± 4
23 ± 6
26 ± 3
10 ± 4
17 ± 6
21 ± 4
1
1
1
1
1
0a
1a
2a
3a
4a
17 ± 4
24.3 ± 1.0
13 ± 3
24 ± 3
11.5 ± 3
22 ± 4
19 ± 7
1.5
1.1
2.2
1.1
21 ± 5
24 ± 3
24 ± 3
19 ± 4
41 ± 3
26 ± 4
21.0 ± 1.5
24 ± 3
8

ACCEPTED MANUSCRIPT
1
5a
17.4 ± 0.2
11.3 ± 2.3
3.9 ± 0.5
0.2
0.3
a
Values are the average (± s.d.) of three different measurements performed as described in the Material
b
c
and Methods section. α = IC50 (HEK-293) / IC50 (HT-29). β = IC50 (HEK-293) / IC50 (MCF-7). Values
of α and β have been rounded off to a decimal figure.
The observed values for IC are in the low to medium micromolar range, with compound 5a
50
showing the lowest values in the HT-29 cell line and compound 6a showing the lowest values in the
MCF-7 cell line. In the case of HT-29 cell lines, all synthetic compounds exhibit better IC values than
5
0
resveratrol. As regards the α and β coefficients, the calculated values are is most cases close to, or even
lower than 1. This means that the cytotoxic activity of these compounds is similar in both the non
tumoral and the tumoral line.
Table 2. IC values (µg/mL) and selectivity coefficients for compounds of the b-series.
5
0
b
c
Compound
Resveratrol
HT-29
MCF-7
16.1 ± 1.1
1.4 ± 0.1
14 ± 4
HEK-293
7.1 ± 1.0
1.3 ± 0.2
84 ± 11
11.4 ± 0.4
27 ± 7
α
β
34.1 ± 1.4
17 ± 4
0.2
0.1
0.4
0.9
6
1
2
3
4
5
6
7
8
9
b
b
b
b
b
b
b
b
b
12.9 ± 0.9
4.4 ± 1.1
36 ± 5
6.5
16 ± 0.1
39 ± 3
2.6
0.7
0.7
1.2
< 0.1
3.9
1.8
1.2
1
0.7
30 ± 5
17 ± 3
20 ± 7
0.7
>100
79 ± 3
7 ± 3
< 0.1
3.2
30.4 ± 1.8
43 ± 16
24.7 ± 0.2
54 ± 7
96 ± 15
>100
2.3
51 ± 16
6 ± 3
7 ± 3
0.1
1
1
1
1
1
1
1
1
1
1
2
2
2
0b
1b
2b
3b
4b
5b
6b
7b
8b
9b
0b
1b
2b
37 ± 5
23 ± 4
21.4 ± 1.4
25 ± 8
0.6
42 ± 6
24.8 ± 0.4
2.2 ± 0.4
>100
0.6
1
30.6 ± 2.4
>100
0.8 ± 0.1
2.1 ± 0.7
0.5 ± 0.1
0.6 ± 0.1
45 ± 0.3
16 ± 5
0.03
< 0.1
< 0.1
< 0.1
1.8
0.4
< 0.1
< 0.1
1.4
1.2
0.5
0.5
0.3
0.3
5.7
1.3
21.2 ± 1.5
73 ± 5
6.1 ± 1.8
0.4 ± 0.1
38.7 ± 0.6
34 ± 5
25.3 ± 0.1
19.6 ± 2.0
16.3 ± 0.4
22 ± 4
0.8
23 ± 4
11 ± 0.3
1 ± 0.3
0.7
3.2 ± 1.7
52 ± 6
< 0.1
1.1
16.1 ± 0.1
0.0036 ± 0.0015
2.5 ± 0.7
18 ± 4
0.0021 ± 0.0005
12 ± 0.9
0.012 ± 0.004
16 ± 9
3.4
6.4
9

ACCEPTED MANUSCRIPT
2
2
3b
4b
>100
>100
3.5 ±0.5
5.8 ± 0.1
11.6 ± 0.6
10 ± 4
< 0.1
< 0.1
3.3
1.7
a
Values are the average (± s.d.) of three different measurements performed as described in the Material
b
c
and Methods section. α = IC50 (HEK-293) / IC50 (HT-29). β = IC50 (HEK-293) / IC50 (MCF-7). Values
of α and β have been rounded off to a decimal figure.
Cytotoxicity values for stilbenes of the b-series are shown in Table 2, with most compounds
showing cytotoxicities of the same order of magnitude as those of Table 1. It can be further observed
that the introduction of the aliphatic side chains (compounds 22b-24b) did not cause improvements in
the cytotoxic activities. However, one of the stilbene derivatives calls for attention: compound 21b
showed cytotoxic values in the low nanomolar range for both cancer cell lines. The α and β
coefficients, clearly above 1, further show that the compound is noticeably more cytotoxic for tumoral
cell lines than for normal ones, a desirable feature.
4
.2. Effect of stilbene derivatives on VEGF protein secretion and VEGF gene inhibition
The action of stilbene derivatives on VEGF secretion and VEGF gene inhibition was conducted on
the HT-29 tumoral cell line. For these measurements compounds 9a-13a, 2b, 3b, 7b, 8b, 16b and 20b-
2
1
2b were selected because they had α-coefficients above 1. In addition, compounds 1b, 14b, 17b and
8b were further selected because they had lower IC50 values than resveratrol.
The secretion of the vascular endothelial growth factor (VEGF) was first determined by means of
the ELISA procedure [29], as described in the Experimental Section. Results for all the selected
compounds (except 21b, which will be discussed later) are depicted in Figure 3, which shows the
percentage of VEGF secreted to the culture medium after 72 h of incubation in the presence of DMSO
(
control experiment) and in the presence of each of the compounds investigated at a concentration of 10
µg/mL (lower than their IC50 values).
1
0

ACCEPTED MANUSCRIPT
Figure 3. VEGF protein secretion from HT-29 cells determined by means of the ELISA procedure. At
least three measurements were performed in each case. Concentration of all compounds was 10 µg/mL.
Bars represent mean values of VEGF secretion (percentage values related to control) and error bars
indicate standard errors of the mean. The statistical significance was evaluated using one-sample t-tests
(
P < 0.001).
While the majority of the compounds showed an inhibitory effect on VEGF production, the values
were very similar to that of resveratrol, which reduced VEGF secretion to 78% of the control value.
The most active is compound 14b, which causes a decrease to 51%.
The high cytotoxicity of compound 21b, as compared with the remaining stilbenes, justified a
separate study. It was evaluated as regards inhibition of VEGF production on both the HT-29 and the
MCF-7 cell line (Figure 4). When a concentration of 1 ng/mL was used, no noticeable effect on VEGF
production was detected in either of the two cell lines. However, at 2 ng/mL concentration, compound
2
1b inhibited VEGF secretion to 84% of the control value in the case of HT-29 cells and to 75% in the
case of MCF-7. When HT-29 cells were cultivated in the presence of 3 ng/mL of compound 21b,
VEGF protein secretion further dropped to 59%. These values are similar to that achieved by
resveratrol at a concentration of 10
µg/mL. Compound 21b therefore is three orders of magnitude
more active than resveratrol in inhibiting VEGF production.
1
1

ACCEPTED MANUSCRIPT
Figure 4. VEGF protein secretion from HT-29 and MCF-7 cells with compound 21b as determined by
means of the RT-qPCR methodology. At least three measurements were performed in each case.
Concentration of resveratrol was 10 µg/mL. Bars represent mean values of VEGF secretion (percentage
values related to control) and error bars indicate standard errors of the mean. The statistical significance
was evaluated using one-sample t-tests (P < 0.001).
While the results depicted in Figures 3 and 4 point out that the investigated compounds cause
inhibition of the VEGF production, they do not permit conclusions about the precise phase of the
VEGF generation process with which they interfere. In order to further investigate the mechanism of
action of the compounds under study, we proceeded to determine whether they were able to control
protein production at the transcriptional level. The VEGF production we have determined with the
ELISA procedure is that secreted to the culture medium and corresponds mainly to the lighter VEGFA-
1
65 isoform [30]. Therefore, in order to evaluate the ability of our compounds to inhibit the expression
of the VEGF gene, we incubated HT-29 cells for 48 h with several selected stilbene derivatives at a
concentration of 10 µg/mL, again with DMSO as the control test. For that purpose, the reverse
transcription quantitative PCR (RT-qPCR) methodology [31] was used as described in the
Experimental Section. Results for all the selected compounds (except 21b) are shown in Figure 5 as
percentage of the VEGF gene expression. All values were standardized (100%) to control (DMSO) and
to β-actine.
1
2

ACCEPTED MANUSCRIPT
Figure 5. Expression percentage of the VEGF gene after 48 h of incubation of HT-29 cells determined
by means of the RT-qPCR methodology. At least three measurements were performed in each case.
Concentration of all compounds was 10 µg/mL. Error bars indicate standard errors of the mean. The
statistical significance was evaluated using one-sample t-tests (P < 0.001).
Comparison of Fig. 5 with Fig. 4 reveals much more meaningful differences in activity between the
compounds investigated. Thus, several compounds showed a visibly stronger inhibitory effect on the
transcription of VEGF mRNA than resveratrol, which lowered VEGF gene expression to 65% of the
control value. The reduction degrees were particularly strong for compounds 3b and 17b, which caused
inhibition of the gene expression to 24% and 23%, respectively, of the control value.
The results displayed in Figs. 4 and 5 do not show correlation between the VEGF amount excreted
to the medium and the degree of gene expression. Compounds 3b and 17b, for instance, proved highly
effective in the inhibition of the VEGF gene expression but much less so in decreasing the amount of
VEGF secreted to the medium. This suggests that these compounds exert the control of VEGF
production at a phase different from that of gene transcription, perhaps during the post-translational
stage, during which the lighter isoforms such as VEGFA-165 are formed and secreted to the medium
[
30].
As in the previous case, a separate study was performed for compound 21b. The inhibition of the
VEGF gene expression was measured in this case on both HT-29 and MCF-7 cancer cell lines, the
results being depicted in Figure 6. When HT-29 cells were incubated in the presence of 1, 2 or 3 ng/mL
of compound 21b, the level of VEGF gene expression was similar to that achieved with resveratrol at a
1
3

ACCEPTED MANUSCRIPT
concentration of 10 µg/mL In other words, the potency of compound 21b in downregulating VEGF
gene expression is three orders of magnitude higher than that of resveratrol. In addition, when MCF-7
cells were treated with 2 ng/mL of compound 21b, only 36 % of VEGF gene expression was detected.
Thus, compound 21b is about 5000 times more active than resveratrol.
Figure 6. Expression percentage of the VEGF gene after 48 h of incubation of HT-29 and MCF-7
cells with compound 21b as determined by means of the RT-qPCR methodology. At least three
measurements were performed in each case. Concentration of resveratrol was 10 µg/mL. Error bars
indicate standard errors of the mean. The statistical significance was evaluated using one-sample t-
tests (P < 0.001).
4
.3. Effect of stilbene derivatives on the inhibition of the hTERT and c-Myc genes
Human telomerase contains an RNA component (hTERC) that serves as a template for the addition
of the repeat nucleotide sequences and also a reverse transcriptase subunit (hTERT) which catalyzes
the nucleotide polymerization process. In addition, there are other associated protein factors, the role of
which has not yet been completely elucidated. Human telomerase is regulated during development and
differentiation, mainly through transcriptional control of the hTERT gene, the expression of which is
restricted to cells that exhibit telomerase activity. This indicates that the hTERT subunit is the rate
limiting factor of the enzyme complex. For the expression of the hTERT gene, several transcriptional
factors such as c-Myc, a proto-oncoprotein [32], have been found to play an important role through
1
4

ACCEPTED MANUSCRIPT
upregulation of the mRNA encoding the hTERT protein subunit [33]. Thus, and as an initial study of
the potential activity of these stilbene derivatives as inhibitors of telomerase activation, we have
investigated their ability to inhibit the expression of the hTERT and c-Myc genes, which are responsible
for the production of the corresponding proteins.
In order to determine whether the studied compounds were able to regulate the expression of the two
aforementioned genes, we have performed a RT-qPCR analysis using again HT-29 tumoral cells. The
cells were incubated for 48 h in the presence of DMSO (control) and 10 µg/mL of each of the studied
compounds (lower than the IC50 values). Results are depicted in Fig. 7 (hTERT gene expression) and
Fig. 8 (c-Myc gene expression). As in previous experiments, the highly cytotoxic compound 21b was
the subject of a separate study, which will be described below.
Figure 7. Expression percentage of the hTERT gene after 48 h of incubation with HT-29 cells as
determined by means of the RT-qPCR methodology. At least three measurements were performed in
each case. Concentration of all compounds was 10 µg/mL. Bars represent mean values of VEGF
secretion (percentage values related to control) and error bars indicate standard errors of the mean. The
statistical significance was evaluated using one-sample t-tests (P < 0.001).
1
5

ACCEPTED MANUSCRIPT
Figure 8. Expression percentage of the c-Myc gene after 48 h of incubation with HT-29 cells as
determined by means of the RT-qPCR methodology. At least three measurements were performed in
each case. Concentration of all compounds was 10 µg/mL. Bars represent mean values of VEGF
secretion (percentage values related to control) and error bars indicate standard errors of the mean. The
statistical significance was evaluated using one-sample t-tests (P < 0.001).
As shown in Fig. 7, compounds 10a-12a, 3b, 7b, 14b, 16b and 22b showed the highest ability to
downregulate the expression of the hTERT gene, their activities being somewhat higher than that of
resveratrol. As regards the expression of the c-Myc gene (Fig. 8), more or less the same compounds
showed the highest inhibitory activity. In any case, however, differences in activity with resveratrol
were perceptible but not particularly worth noting.
Inhibition of h-TERT and c-Myc gene expression for compound 21b was measured on both HT-29
and MCF-7 cancer cell lines. Results are shown in Figs. 9 and 10, which show that compound 21b was
able to downregulate the expression of the h-TERT and c-Myc genes. The expression of the h-TERT
gene was decreased to 48% of the control value when 3 ng/mL concentration was used on HT-29 cells,
and to 51% when 2 ng/mL concentration was used on MCF-7 cells. Again, the potency of compound
2
1b in downregulating the h-TERT and c-Myc genes is 3000-5000 times greater than that of resveratrol.
1
6

ACCEPTED MANUSCRIPT
Figure 9. Expression percentage of the hTERT gene after 48 h of incubation with compound 21b as
determined by means of the RT-qPCR methodology. At least three measurements were performed in
each case. Concentration of resveratrol was 10 µg/mL. Bars represent mean values of VEGF secretion
(
percentage values related to control) and error bars indicate standard errors of the mean. The statistical
significance was evaluated using one-sample t-tests (P < 0.001).
Figure 10. Expression percentage of the c-Myc gene after 48 h of incubation of HT-29 and MCF-7
cells with compound 21b as determined by means of the RT-qPCR methodology. At least three
measurements were performed in each case. Concentration of resveratrol was 10 µg/mL. Bars represent
1
7

ACCEPTED MANUSCRIPT
mean values of VEGF secretion (percentage values related to control) and error bars indicate standard
errors of the mean. The statistical significance was evaluated using one-sample t-tests (P < 0.001).
Table 3 summarizes the results observed for the synthetic compounds 12a, 3b, 7b, 14b, 16b, 17b,
2
1b and 22b, as well as for resveratrol. As regards inhibition of the expression of the VEGF, hTERT
and c-Myc genes, stilbene derivatives bearing amino groups (b-series) feature much better than those
lacking them (a-series). Within stilbenes carrying an amide function, the most active is compound 22b
whereas within amino stilbenes, those having a methoxy group (14b, 16b, 17b and 21b) perform better
than those having a hydroxy group. A very particular case is compound 21b, which is able to
downregulate the secretion of VEGF protein and the expression of VEGF, h-TERT and c-Myc genes at
concentrations in the low nanomolar level.
Table 3. Compared percentages of excretion of the VEGF protein and of the expression of the VEGF,
a
hTERT and c-Myc genes.
Cytotoxicity
% Secretion of % Expression
VEGF protein of VEGF gene of hTERT gene of c-Myc gene
% Expression % Expression
Compd
(
IC50, µg/mL)
34.1 ± 1.4
22 ± 4
Resv.
78
73
78
76
51
75
73
59
79
65
38
24
38
100
67
23
67
37
57
48
65
52
35
52
100
48
53
51
46
46
37
79
45
34
69
45
1
2a
3
b
b
4.4 ± 1.1
7
30.4 ± 1.8
21.2 ± 1.5
25.3 ± 1
14b
16b
17b
19.6 ± 2
b
2
1b
0.0036 ± 0.001
2.5 ± 0.7
22b
a
All values refer to HT-29 cells. The values observed for the control (DMSO without added
drugs) are taken as the 100% reference of protein excretion or gene expression. Therefore, the
lower the value, the higher the activity of the drug in the corresponding activity. Except for 21b,
measurements were performed at concentration values of 10 µg/mL.
b
At a concentration of 3 ng/mL.
The biological activities of resveratrol are often ascribed, at least in part, to its antioxidant activity.
The various biological activities of resveratrol analogues depend upon the number and location of the
1
8

ACCEPTED MANUSCRIPT
hydroxy groups [34], minor structural changes in such stilbenes having major effects on their biological
activity [35]. In the present study, a series of 39 stilbenes carrying hydroxy, methoxy and amino groups
were compared with resveratrol for their cytotoxicity and for their ability to inhibit VEGF secretion and
the expression of the VEGF, h-TERT and c-Myc genes. Several compounds were found to be more
active than resveratrol, the case of (E)-4-(4-methoxystyryl)aniline (21b) being particularly interesting
because of its high cytotoxicity. In order to find some relationships between chemical structure and
biological activity, we have graphically represented α coefficients vs. β coefficients of the studied
compounds (Fig. 11). Figs. 11a to 11c display the α and β values for stilbenes carrying an ortho-, meta-
and para-hydroxy group, respectively. Figure 11d shows values for the remaining compounds.
Figure 11. α coefficients vs. β coefficients of the synthetic stilbenes.
1
9

ACCEPTED MANUSCRIPT
Figure 11 (cont.). α coefficients vs. β coefficients of the synthetic stilbenes.
As mentioned above, the higher the coefficient values, the better the safety margin for the
corresponding compound from the therapeutic point of view. Aside from compound 21b, which is three
orders of magnitude more cytotoxic than the others, the best safety margins are achieved with
compounds 9a (2’-OH, 4-OMe), 13a (4’-OH, 2-OMe), 7b (2’-OH, 3-NH ), 8b (3’-OH, 3-NH ) and
2
2
1
2
6b (2’-OMe, 3-NH ). These three amino stilbenes share in common an amino group in meta position,
regardless of the position occupied by the hydroxy or methoxy group. Compound 21b, however, which
is cytotoxic at nanomolar level and shows the highest safety margins, has its amino group in the para
position. This may possibly indicate that, in comparison with the rest of compounds, 21b shows a
different mode of action.
2
0

ACCEPTED MANUSCRIPT
In Figure 12, the percentage of VEFG protein secretion is graphically represented vs. the percentage
of h-TERT gene expression. Compounds 14b and 21b are located in the lower left quadrant, where high
inhibition of VEGF secretion and high inhibition of h-TERT gene expression are combined. It is worth
noting that this desirable dual mode of action is exhibited by compound 21b at low nanomolar
concentrations, whereas compound 14b achieves it only at the micromolar level.
Figure 12. Percentage of VEFG protein secretion vs. percentage of h-TERT gene expression.
5
. Summary and conclusions
In conclusion, 39 stilbene derivatives have been synthesized and evaluated for their biological
activity. One of them, (E)-4-(4-methoxystyryl)aniline (compound 21b) has proven particularly
interesting. Not only has this compound a strong cytotoxicity at the low nanomolar level and a good
safety margin, it further combines these features with an ability to inhibit VEFG protein secretion and
the expression of the telomerase related h-TERT and c-Myc genes at similarly low concentrations.
Compound 21b therefore may possibly display anticancer activity through a multiple mode of action. It
is worth noting that 21b shows an extremely high difference in activity with all other compounds,
including its positional isomers 19b and 20b, and its demethyl derivative 12b. This may possibly
suggest that its interactions with its (still unknown) biomolecular targets, and perhaps also the targets
themselves, are of a different nature. More studies will be required to acquire a knowledge about its
mechanism of action.
2
1

ACCEPTED MANUSCRIPT
6
. Experimental
6
.1. Chemistry. General procedures
The general reaction conditions and the physical and spectral data of all synthetic intermediates and
final compounds are described in detail in the Supporting Information. The samples of compounds used
for the biological studies were purified to > 95% by means of preparative HPLC.
6
.2. Biological studies. Materials and methods
.2.1. Reagents and cell culture
Cell culture media were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS)
6
was a product of Harlan-Seralab (Belton, U.K.). Supplements and other chemicals not listed in this
section were obtained from Sigma Chemicals Co. (St. Louis, Mo., USA). Plastics for cell culture were
TM
supplied by Thermo Scientific BioLite. All tested compounds were dissolved in DMSO at a
concentration of 10 µg/mL and stored at –20°C until use.
Cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing glucose (1
g/L), glutamine (2 mM), penicillin (50 IU/mL), streptomycin (50 µg/mL) and amphoterycin (1.25
µg/mL), supplemented with 10% FBS.
6
.2.2. Cytotoxicity assays
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., St.
Louis, MO) dye reduction assay in 96-well microplates was used, as previously described [36]. Some 5
3
x 10 cells of HT-29, MCF-7 or HEK-293 cells in a total volume of 100 µL of their respective growth
media were incubated with serial dilutions of the tested compounds. After 3 days of incubation (37 °C,
5
% CO in a humid atmosphere), 10 µl of MTT (5 mg/ml in PBS) were added to each well and the
2
plate was incubated for further 4 h (37 °C). The resulting formazan was dissolved in 150 µL of 0.04 N
HCl/2-propanol and read at 550 nm. All determinations were carried out in triplicate.
6
.2.3. ELISA analysis
HT-29 cells at 70–80% confluence were collected after serum starvation for 24 h. Cells were
incubated with the selected stilbene derivatives in DMSO for 72 h (Fig. 3). Culture supernatants were
collected and VEGF secreted by HT-29 cells was determined using Invitrogen Human Vascular
Endothelial Growth Factor ELISA Kit according to the manufacturer’s instructions.
2
2

ACCEPTED MANUSCRIPT
6
.2.4. RT-qPCR analysis
HT-29 cells at 70–80% confluence were collected after serum starvation for 24 h. Cells were
incubated with 10 µg/mL of the corresponding drugs in DMSO for 48 h. Cells were collected and the
total cellular RNA from HT-29 cells was isolated using Ambion RNA extraction Kit according to the
manufacturer’s instructions. The cDNA was synthesized by MMLV-RT with 1-21 µg of extracted
RNA and oligo(dT)15 according to the manufacturer’s instructions.
Amplification of the genes was performed by use of a StepOnePlus™ thermalcycler. Fast TaqMan
Gene Expression Master Mix containing the appropriate buffer for the amplification conditions,
dNTPs, thermostable DNA polymerase enzyme and a passive reference probe was used. Each of the
genes were amplified using predesigned primers by Life Technologies TaqMan® Gene Expression
Assays, Hs99999903-m1 (β-actin), Hs00900055-m1 (VEGF), Hs00972646-m1 (hTERT) and
Hs00153408-m1 (c-Myc).
6
.2.5. Statistical analysis
Data are expressed as the mean ± SEM. Statistical analyses were done using Microsoft Excel and
®
GraphPad Prism . Differences between means were determined using Student’s t-Test or one-way
ANOVA with Dunnett's Multiple Comparison Test, and considered to be statistically significant at
p≤0.001.
Acknowledgments
Financial support has been granted by the Spanish Government (MINECO, Ministerio de Economía y
Competitividad, project CTQ2011-27560), by the Consellería d’Empresa, Universitat i Ciencia de la
Generalitat Valenciana (projects PROMETEO/2013/027 and ACOMP/2014/272) and by the
Universitat Jaume I (PI-1B-2011-37). R. M.-C. thanks the University Jaume I for a predoctoral
fellowship.
Supplementary Information
Description of general features and reaction conditions. Spectral data and graphical NMR spectra of
all new compounds.
2
3

ACCEPTED MANUSCRIPT
References
[
1] E. N. Frankel, A. L. Waterhouse, J. E. Kinsella, Inhibition of human LDL oxidation by
resveratrol, Lancet 341 (1993) 1103-1104.
[
2] (a) J. Marchal, F. Pifferi, F. Aujard, Resveratrol in mammals: effects on aging biomarkers, age-
related diseases, and life span, Ann. N. Y. Acad. Sci. 1290 (2013) 67-73.
(b) R. S. Yao, X. Q. Lu, Q. X. Guan, L. Zheng, X. Lu, B. F. Ruan, Synthesis and biological
evaluation of some novel resveratrol amide derivatives as potential anti-tumor agents, Eur. J. Med.
Chem. 62 (2013) 222-231.
(c) M. Miliovsky, I. Svinyarov, Y. Mitrev, Y. Evstatieva, D. Nikolova, M. Chochkova, M. G.
Bogdanov, A novel one-pot synthesis and preliminary biological activity evaluation of cis-
restricted polyhydroxy stilbenes incorporating protocatechuic acid and cinnamic acid fragments,
Eur. J. Med. Chem. 66 (2013) 185-192.
(
d) A. Csiszar, Anti-inflammatory effects of resveratrol: possible role in prevention of age-related
cardiovascular disease, Ann. N. Y. Acad. Sci. 1215 (2011) 117-122.
[
[
3] S. Quideau, D. Deffieux, L. Pouységu, Resveratrol Still Has Something To Say about Aging!,
Angew. Chem. Int. Ed. 51 (2012) 6824-6826.
4] V. Krishnan, S. L. Zeichner, Host cell gene expression during human immunodeficiency virus
type I latency and reactivation and effects of targeting genes that are differentially expressed in
viral latency, J. Virol. 78 (2004) 9458-9473.
[
[
[
[
5] P. J. Elliott, S. M. Walpole, L. Morelli, P. D. Lambert, W. Lunsmann, C. H. Westphal, S. Lavu,
Resveratrol/SRT501 Sirtuin SIRT1 Activator Treatment of Type 2 Diabetes, Drugs Fut. 34 (2009)
291-295.
6] C. R. Pace-Asciak, S. Hahn, E. P. Diamandis, G. Soleas, D. M. Goldberg, The red wine phenolics
trans-resveratrol and quercetin block human platelet-aggregation and eicosanoid synthesis -
implications for protection against coronary heart-disease, Clin. Chim. Acta 235 (1995) 207-219.
7] J. A. Parker, M. Arango, S. Abderrahmane, E. Lambert, C. Tourette, H. Catoire, C. Neri,
Resveratrol rescues mutant polyglutamine cytotoxicity in nematode and mammalian neurons,
Nat. Genet. 37 (2005) 349-350.
8] (a) M. S. Jang, E. N. Cai, G. O. Udeani, K. V. Slowing, C. F. Thomas, C. W. W. Beecher, H. H.
S. Fong, N. R. Farnsworth, A. D. Kinghorn, R. G. Mehta, R. C. Moon, J. M. Pezzuto, Cancer
chemopreventive activity of resveratrol, a natural product derived from grapes, Science 275
(1997) 218-220.
2
4

ACCEPTED MANUSCRIPT
(b) T. P. Kondratyuk, E.-J. Park, L. E. Marler, S. Y. Ahn, Y. Yuan, Y. S. Choi, R. Yu, R. B. van
Breemen, B. Sun, J. Hoshino, M. Cushman, K. C. Jermihov, A. D. Mesecar, C. J. Grubbs, J. M.
Pezzuto, Resveratrol derivatives as promising chemopreventive agents with improved potency
and selectivity, Mol. Nutr. Food Res. 55 (2011) 1249-1265.
[
[
9] D. Delmas, E. Solary, N. Latruffe, Resveratrol, a Phytochemical Inducer of Multiple Cell Death
Pathways: Apoptosis, Autophagy and Mitotic Catastrophe, Curr. Med. Chem 18 (2011) 1100-
1
121.
10] E. Brakenhielm, R. H. Cao, Y. H. Cao, Suppression of angiogenesis, tumor growth, and wound
healing by resveratrol, a natural compound in red wine and grapes, FASEB J. 15 (2001) 1798-
1
800.
[
[
11] K. Igura, T. Ohta, Y. Kuroda, K. Kaji, Resveratrol and quercetin inhibit angiogenesis in vitro,
Cancer Lett. 171 (2001) 11-16.
12] (a) N. Ferrara, VEGF: an update on biological and therapeutic aspects, Curr. Opin. Biotechnol.
1
1 (2000) 617-624.
(b) P. Carmeliet, R. K. Jain, Molecular mechanisms and clinical applications of angiogenesis,
Nature 473 (2011) 298-307.
[
13] S. M. Moghaddam, A. Amini, D. L. Morris, M. H. Pourgholami, Significance of vascular
endothelial growth factor in growth and peritoneal dissemination of ovarian cancer, Cancer
Metastasis Rev. 31 (2012) 143-162.
[
14] (a) Z. P. Zhu, L. Witte, Inhibition of tumor growth and metastasis by targeting tumor-associated
angiogenesis with antagonists to the receptors of vascular endothelial growth factor, Invest. New
Drugs 17 (1999) 195-212.
(b) R. B. Caldwell, M. Bartoli, M. A. Behzadian, A. E. B. El-Remessy, M. Al-Shabrawey, D. H.
Platt, G. I. Liou, R. W. Caldwell, Vascular endothelial growth factor and diabetic retinopathy:
Role of oxidative stress, Curr. Drug Targets 6 (2005) 511-524.
(c) A. F. C. Okines, A. R. Reynolds, D. Cunningham, Targeting angiogenesis in esophagogastric
adenocarcinoma, Oncologist 16 (2011) 844-858.
15] For some relevant references, see, for example:
[
(
a) M. Potente, H. Gerhardt, P. Carmeliet, Basic and therapeutic aspects of angiogenesis , Cell
46 (2011) 873-887.
b) G. Korpanty, E. Smyth, Anti-VEGF Strategies - from antibodies to tyrosine kinase inhibitors:
1
(
background and clinical development in human cancer, Curr. Pharm. Des. 18 (2012) 2680-2701.
2
5

ACCEPTED MANUSCRIPT
(c) A. G. Linkous, E. M. Yazlovitskaya, Novel therapeutic approaches for targeting tumor
angiogenesis, Anticancer Res. 32 (2012) 1-12.
[
16] Antiangiogenic therapies are not completely devoid of problems. See, for example:
(
a) A. R. Quesada, M. A. Medina, R. Muñoz-Chapuli, A. L. G. Ponce, Do not say ever never
more: the ins and outs of antiangiogenic therapies, Curr. Pharm. Des. 16 (2010) 3932-3957.
b) K. De Bock, M. Mazzone, P. Carmeliet, Antiangiogenic therapy, hypoxia, and metastasis:
risky liaisons, or not?, Nature Rev. Clin. Oncol. 8 (2011) 393-404.
c) L. Moserle, G. Jiménez-Valerio, O. Casanovas, Antiangiogenic therapies: going beyond their
(
(
limits, Cancer Discov. 4 (2014) 31-41.
[
17] (a) N. W. Kim, M. A. Piatyszek, K. R. Prowse, C. B. Harley, M. D. West, P. L. C. Ho, G. M.
Coviello, W. E. Wright, S. L. Weinrich, J. W. Shay, Specific association of human telomerase
activity with immortal cells and cancer, Science 266 (1994) 2011-2015.
(b) B.-S. Herbert, A. E. Pitts, S. I. Baker, S. E. Hamilton, W. E. Wright, J. W. Shay, D. R. Corey,
Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening
and cell death, Proc. Natl. Acad. Sci. 96 (1999) 14276-14281.
[
[
18] (a) J. W. Shay, W. E. Wright, Role of telomeres and telomerase in cancer, Semin. Cancer Biol.
2
1 (2011) 349-353.
b) V. A. Zakian, Telomeres: the beginnings and ends of eukaryotic chromosomes, Exp. Cell Res.
18 (2012) 1456-1460.
(
3
19] (a) K. A. Olaussen, K. Dubrana, J. Domont, J.-P. Spano, L. Sabatier, J.-C. Soria, Telomeres and
telomerase as targets for anticancer drug development, Crit. Rev. Oncol. Hematol. 57 (2006) 191-
2
14.
(
(
(
b) D. R. Corey, Telomeres and telomerase: from discovery to clinical trials, Chem. Biol. 16
2009) 1219-1223.
c) C. M. Buseman, W. E. Wright, J. W Shay, Is telomerase a viable target in cancer?, Mut. Res.
7
30 (2012) 90-97.
[
[
20] G. Lanzilli, M. P. Fuggetta, M. Tricarico, A. Cottarelli, A, Serafino, R. Falchetti, G. Ravagnan,
M. Turriziani, R. Adamo, O. Franzese, E. Bonmassar, Resveratrol down-regulates the growth and
telomerase activity of breast cancer cells in vitro, Int. J. Oncol. 28 (2006) 641-648.
21] See, for example: C.-T. Chen, M.-C. Hung, Beyond anti-VEGF: dual-targeting antiangiogenic
and antiproliferative therapy, Am. J. Transl. Res. 5 (2013) 393-403.
2
6

ACCEPTED MANUSCRIPT
[
[
22] R. Martí-Centelles, R. Cejudo-Marín, E. Falomir, J. Murga, M. Carda, J. A. Marco, Inhibition of
VEGF expression in cancer cells and endothelial cell differentiation by synthetic stilbene
derivatives, Bioorg. Med. Chem. 21 (2013) 3010-3015.
23] (a) R. Heck, Palladium-catalyzed reactions of organic halides with olefins, Acc. Chem. Res. 12
(1979) 146-151.
(b) I. P. Beletskaya, A. V. Cheprakov, The Heck reaction as a sharpening stone of palladium
catalysis, Chem. Rev. 100 (2000) 3009-3066.
c) A. F. Schmidt, A. Al Halaiqa, V. V. Smirnov, Interplays between reactions within and without
(
the catalytic cycle of the Heck reaction as a clue to the optimization of the synthetic protocol,
Synlett (2006) 2861-2878.
[
24] N. Sharma, D. Mohanakrishnan, A. Shard, A. Sharma, Saima, A. K. Sinha, D. Sahal, Stilbene-
chalcone hybrids: design, synthesis, and evaluation as a new class of antimalarial scaffolds that
trigger cell death through stage specific apoptosis, J. Med. Chem. 55 (2012) 297-311.
[
[
25] V. P. Mehta, E. V. Van der Eycken, Microwave-assisted C-C bond forming cross-coupling
reactions: an overview, Chem. Soc. Rev. 40 (2011) 4925-4936.
26] S.-H. Huang, J.-R. Chen, F.-Y. Tsai, Palladium(II)/cationic 2,2 '-bipyridyl system as a highly
efficient and reusable catalyst for the Mizoroki-Heck reaction in water, Molecules 15 (2010) 315-
3
30.
[
[
[
27] K. S. Keshavamurthy, Y. D. Vankar, D. N. Dhar, Preparation of acid anhydrides, amides, and
esters using chlorosulfonyl isocyanate as a dehydrating agent, Synthesis (1982) 506-508.
28] N. Arden, M. J. Betenbaugh, Life and death in mammalian cell culture: strategies for apoptosis
inhibition, Trends Biotechnol. 22 (2004) 174-180.
29] (a) B. K. Van Weemen, A. H. W. M. Schuurs, Immunoassay using antigen-enzyme conjugate,
FEBS Lett. 15 (1971) 232-236.
(
b) E. Engvall, P. Perlmann, Enzyme-linked immunosorbent assay, ELISA .3. quantitation of
specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes, J. Immunol.
09 (1972) 129-135.
c) A. J. O’Beirne, H. R. Cooper, Heterogeneous enzyme immunoassay, J. Hystochem.
Cytochem. 27 (1979) 1148-1162.
1
(
2
7

ACCEPTED MANUSCRIPT
[
[
30] M. Nakamura, Y. Abe, T. Tokunaga, Pathological significance of vascular endothelial growth
factor A isoform expression in human câncer, Pathol. Int. 52 (2002) 331-339.
31] S. A. Bustin, V. Benes, J. A. Garson, J. Hellemans, J. Huggett, M. Kubista, R. Mueller, T. Nolan,
M. W. Pfaffl, G. L. Shipley, J. Vandesompele, C. T. Wittwer, The MIQE guidelines: minimum
information for publication of quantitative Real-Time PCR Experiments, Clin. Chem. 55 (2009)
6
11-622.
[
[
32] The Myc family of genes and proteins, involved in many aspects of cell metabolism, is subjected
to a tight control in normal cells but becomes deregulated in most tumor cells. For a review, see:
A. Albihn, J. I. Johnsen, M. A. Henriksson, MYC in oncogenesis and as a target for cancer
therapies, Adv. Cancer Res. 107 (2010) 163-224.
33] (a) J. Dwyer, H. Li, D. Xu, J.-P. Liu, Transcriptional regulation of telomerase activity - Roles of
the the Ets transcription factor family, Ann. N. Y. Acad. Sci. 1114 (2007) 36-47.
(
b) S. Kyo, M. Takakura, T. Fujiwara, M. Inoue, Understanding and exploiting hTERT promoter
regulation for diagnosis and treatment of human cancers, Cancer Sci. 99 (2008) 1528-1538.
c) M. Daniel, G. W. Peek, T. O. Tollefsbol, Regulation of the human catalytic subunit of
(
telomerase (hTERT), Gene 498 (2012) 135-146.
[
[
[
34] J. J. Heynekamp, W. M. Weber, L. A. Hunsaker, A. M. Gonzales, R. A. Orlando, L. M. Deck, D.
L. V. Jagt, Substituted trans-stilbenes, including analogues of the natural product resveratrol,
inhibit the human tumor necrosis factor alpha-induced activation of transcription factor nuclear
factor KappaB, J. Med. Chem. 49 (2006) 7182-7189.
35] L. A. Stivala, M. Savio, F. Carafoli, P. Perucca, L. Bianchi, G. Maga, L. Forti, U. M. Pagnoni, A.
Albini, E. Prosperi, V. Vannini, Specific structural determinants are responsible for the
antioxidant activity and the cell cycle effects of resveratrol, J. Biol. Chem. 276 (2001) 22586-
2
2594.
36] S. Rodríguez-Nieto, M. A. Medina, A. R. Quesada, A re-evaluation of fumagillin selectivity
towards endothelial cells, Anticancer Res. 21 (2001) 3457-3460.
2
8

ACCEPTED MANUSCRIPT
List of captions
Figure 1. Structure of resveratrol
Figure 2. Structures of synthetic resveratrol analogues.
Scheme 1. Synthesis of stilbene derivatives by means of palladium-catalyzed Heck couplings (for more
detailed descriptions of the reaction conditions and yields, see Supporting Information).
Table 1. IC values (µg/mL) and selectivity coefficients for compounds of the a-series.
5
0
Table 2. IC50 values (µg/mL) and selectivity coefficients for compounds of the b-series.
Figure 3. VEGF protein secretion from HT-29 cells determined by means of the ELISA procedure. At
least three measurements were performed in each case. Concentration of all compounds was 10 µg/mL.
Bars represent mean values of VEGF secretion (percentage values related to control) and error bars
indicate standard errors of the mean. The statistical significance was evaluated using one-sample t-tests
(
P < 0.001).
Figure 4. VEGF protein secretion from HT-29 and MCF-7 cells with compound 21b as determined by
means of the RT-qPCR methodology. At least three measurements were performed in each case.
Concentration of resveratrol was 10 µg/mL. Bars represent mean values of VEGF secretion (percentage
values related to control) and error bars indicate standard errors of the mean. The statistical significance
was evaluated using one-sample t-tests (P < 0.001).
Figure 5. Expression percentage of the VEGF gene after 48 h of incubation of HT-29 cells determined
by means of the RT-qPCR methodology. At least three measurements were performed in each case.
Concentration of all compounds was 10 µg/mL. Error bars indicate standard errors of the mean. The
statistical significance was evaluated using one-sample t-tests (P < 0.001).
Figure 6. Expression percentage of the VEGF gene after 48 h of incubation of HT-29 and MCF-7 cells
with compound 21b as determined by means of the RT-qPCR methodology. At least three
measurements were performed in each case. Concentration of resveratrol was 10 µg/mL. Error bars
indicate standard errors of the mean. The statistical significance was evaluated using one-sample t-tests
(
P < 0.001).
Figure 7. Expression percentage of the hTERT gene after 48 h of incubation with HT-29 cells as
determined by means of the RT-qPCR methodology. At least three measurements were performed in
each case. Concentration of all compounds was 10 µg/mL. Bars represent mean values of VEGF
secretion (percentage values related to control) and error bars indicate standard errors of the mean. The
statistical significance was evaluated using one-sample t-tests (P < 0.001).
Figure 8. Expression percentage of the c-Myc gene after 48 h of incubation with HT-29 cells as
determined by means of the RT-qPCR methodology. At least three measurements were performed in
each case. Concentration of all compounds was 10 µg/mL. Bars represent mean values of VEGF
secretion (percentage values related to control) and error bars indicate standard errors of the mean. The
statistical significance was evaluated using one-sample t-tests (P < 0.001).
2
9

ACCEPTED MANUSCRIPT
Figure 9. Expression percentage of the hTERT gene after 48 h of incubation with compound 21b as
determined by means of the RT-qPCR methodology. At least three measurements were performed in
each case. Concentration of resveratrol was 10 µg/mL. Bars represent mean values of VEGF secretion
(
percentage values related to control) and error bars indicate standard errors of the mean. The statistical
significance was evaluated using one-sample t-tests (P < 0.001).
Figure 10. Expression percentage of the c-Myc gene after 48 h of incubation of HT-29 and MCF-7
cells with compound 21b as determined by means of the RT-qPCR methodology. At least three
measurements were performed in each case. Concentration of resveratrol was 10 µg/mL. Bars represent
mean values of VEGF secretion (percentage values related to control) and error bars indicate standard
errors of the mean. The statistical significance was evaluated using one-sample t-tests (P < 0.001).
Table 3. Compared percentages of excretion of the VEGF protein and of the expression of the VEGF,
a
hTERT and c-Myc genes.
Figure 11. α coefficients vs. β coefficients of the synthetic stilbenes.
Figure 11 (cont.). α coefficients vs. β coefficients of the synthetic stilbenes.
Figure 12. Percentage of VEFG protein secretion vs. percentage of h-TERT gene expression.
3
0