Cytotoxic, Antiangiogenic and Antitelomerase Activity of Glucosyl- and Acyl- Resveratrol Prodrugs and Resveratrol Sulfate Metabolites

Article abstract of DOI:10.1002/cbic.201600084

Resveratrol (RES) is a natural polyphenol with relevant and varied biological activity. However, its low bioavailability and rapid metabolism to its glucuronate and sulfate conjugates has opened a debate on the mechanisms underlying its bioactivity. RES prodrugs are being developed to overcome these problems. We have synthesized a series of RES prodrugs and RES sulfate metabolites (RES-S) and evaluated their biological activities. RES glucosylated prodrugs (RES-Glc) were more cytotoxic in HT-29 and MCF-7 cells than RES itself whereas RES-S showed similar or higher cytotoxicity than RES. VEGF production was decreased by RES-Glc, and RES-disulfate (RES-diS) diminished it even more than RES. Finally, RES-Glc and RES-diS inhibited hTERT gene expression to a higher extent than RES. In conclusion, resveratrol prodrugs are promising candidates as anticancer drugs. In addition, RES-S showed distinct biological activity, thus indicating they are not simply RES reservoirs.

Full text of DOI:10.1002/cbic.201600084

DOI: 10.1002/cbic.201600084
Full Papers
Cytotoxic, Antiangiogenic and Antitelomerase Activity of
Glucosyl- and Acyl- Resveratrol Prodrugs and Resveratrol
Sulfate Metabolites
Eva Falomir,[a] Ricardo Lucas,[b] Pablo PeÇalver,[c] Rosa Martꢀ-Centelles,[a] Alexia Dupont,[b]
Alberto Zafra-Gꢁmez,[d] Miguel Carda,*[a] and Juan C. Morales*[b, c]
Resveratrol (RES) is a natural polyphenol with relevant and
varied biological activity. However, its low bioavailability and
rapid metabolism to its glucuronate and sulfate conjugates
has opened a debate on the mechanisms underlying its bio-
activity. RES prodrugs are being developed to overcome these
problems. We have synthesized a series of RES prodrugs and
RES sulfate metabolites (RES-S) and evaluated their biological
activities. RES glucosylated prodrugs (RES-Glc) were more cyto-
toxic in HT-29 and MCF-7 cells than RES itself whereas RES-S
showed similar or higher cytotoxicity than RES. VEGF produc-
tion was decreased by RES-Glc, and RES-disulfate (RES-diS) di-
minished it even more than RES. Finally, RES-Glc and RES-diS
inhibited hTERT gene expression to a higher extent than RES.
In conclusion, resveratrol prodrugs are promising candidates as
anticancer drugs. In addition, RES-S showed distinct biological
activity, thus indicating they are not simply RES reservoirs.
Introduction
Resveratrol (trans-3,5,4’-trihydroxystilbene, RES, 1; Scheme 1) is
a phytoalexin generated in response to environmental stress
or pathogenic attack in grapes, blueberries, peanuts, cocoa,
and plants such as the Japanese knotweed Polygonum cuspida-
tum. RES has attracted great attention because of its relevant
and varied biological activities, such as its cardiovascular pro-
tective properties,[1] its anti-inflammatory activity,[2] and its ca-
pacity to extend lifespan in various species.[3] The cancer che-
mopreventive and chemotherapeutic potential of RES has
been demonstrated in different models of carcinogenesis, both
in vitro and in vivo. It inhibits proliferation in various cancer
cell lines,[4] and in animal studies RES was able to interfere with
the formation of azoxymethane-induced aberrant crypt foci in
rat colon,[5] reduce mammary tumor formation in N-methyl-N-
nitrosourea-(NMU)-treated rats,[6] and suppress prostate cancer
in SV-40 tag rats.[7] Extensive in vitro studies have revealed
multiple intracellular targets of resveratrol; this affects cell
growth, inflammation, apoptosis, and metastasis.[8]
Tumor angiogenesis plays a critical role in the development
of cancer. RES and piceid (resveratrol-3-b-glucoside, 2) have
been shown to inhibit the formation of capillary-like tube for-
mation from human umbilical vein endothelial cells.[9] Vascular
endothelial growth factor (VEGF) is also crucial for angiogene-
sis and tumor growth. In breast cancer cells, a notable de-
crease in extracellular VEGF has been associated with apopto-
sis after resveratrol treatment.[10] A relevant observation in 90%
of malignant tumors is the maintenance of telomere length
(cellular DNA) through the expression of the human telomer-
ase reverse transcriptase (hTERT). This process allows cancer
cells to evade the progressive shortening of telomeres and
thus avoid apoptosis. Resveratrol showed an inhibitory effect
on MCF-7 tumor cells mainly by the induction of S-phase
arrest and apoptosis, with reduced expression of hTERT.[11]
However, as is the case with many polyphenols with puta-
tive anticancer properties, the bioavailability of resveratrol is
very low.[12] RES is well absorbed but avidly glucuronidated and
sulfated, both in the liver and in intestinal epithelial cells.[13] In
order to counteract this, high doses of resveratrol have been
used in cellular, animal, and clinical studies. This could be the
cause of the toxicity observed in certain cases, such as a multi-
ple myeloma clinical trial with 5 g a day of a formulation of re-
sveratrol.[14]
[a] Dr. E. Falomir, Dr. R. Martꢀ-Centelles, Dr. M. Carda
Department of Inorganic and Organic Chemistry, University Jaume I
Avda Sos Baynat, sn 12071 Castellꢁn (Spain)
E-mail: mcarda@qio.uji.es
[b] Dr. R. Lucas, A. Dupont, Dr. J. C. Morales
Department of Bioorganic Chemistry
Instituto de Investigaciones Quꢀmicas (IIQ)
CSIC-Universidad de Sevilla
Avda Americo Vespucio, 4941092 Sevilla (Spain)
[c] Dr. P. PeÇalver, Dr. J. C. Morales
Instituto de Parasitologꢀa y Biomedicina “Lꢁpez Neyra”, CSIC
Parque Tecnolꢁgico Ciencias de la Salud
Avenida del Conocimiento, 17, 18016 Armilla, Granada (Spain)
E-mail: jcmorales@ipb.csic.es
The low systemic levels of resveratrol have brought about a
debate on the possible contribution of the RES metabolites to
the in vivo biological activities attributed to RES.[12,15] It has
been proposed that resveratrol metabolites could contribute
as a RES reservoir through their deconjugation, or that RES me-
tabolites could enter the cell via ABC transporters and interact
directly with different intracellular targets.[16]
[d] Dr. A. Zafra-Gꢁmez
Department of Analytical Chemistry
Faculty of Sciences, University of Granada
C/ Severo Ochoa, s/n, 18001 Granada (Spain)
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cbic.201600084.
ChemBioChem 2016, 17, 1 – 7
1
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
Scheme 1. Resveratrol (RES, 1), piceid (2), RES derivatives (3–7) and RES metabolites (8–10) prepared and assayed in this study.
RES prodrugs have been investigated as an alternative to in-
crease resveratrol bioavailability.[17] We have prepared a family
of RES prodrugs (and pro-prodrugs) by attaching glucosyl or
acyl groups to 1 or 2 and examined their anti-inflammatory ef-
fects in a mouse model of inflammatory bowel disease (IBD).[18]
Oral administration of trans-resveratrol-3-O-(6’-O-butanoyl)-b-d-
glucopyranoside (6) and trans-resveratrol-3-O-(6’-O- octanoyl)-
b-d-glucopyranoside (7) resulted in increased delivery of RES
to the colon, prevented the rapid metabolism of RES, and re-
duced inflammation in a murine dextran sodium sulfate (DSS)
model of inflammatory bowel disease.
tected resveratrol compounds obtained by random silylation
of 1 were glycosylated by using 2,3,4,6-tetra-O-benzoyl-d-glu-
copyranosyl trichloroacetimidate as the glycosyl donor. One-
step deprotection under aqueous strong basic conditions
yielded the desired products 3 and 4. Piceid acyl derivatives 6
and 7 were synthesized by enzymatic acylation, by using Ther-
momyces lanuginosus lipase immobilized on granulated silica
(Lipozyme TL IM) as reported previously.[18]
RES sulfate metabolites 8–10 were synthesized by following
the strategy of Hoshino et al.[19] TBDMS-protected resveratrol
derivatives 11–15 were obtained from RES, and each com-
pound was separated by flash column chromatography. Subse-
Here, we report cytotoxicity, VEGF expression, and telomer-
ase inhibition by five RES prodrugs (3–7) modified with gluco-
syl or acyl groups. Our intention was to determine their poten-
tial as cancer chemopreventive or chemotherapeutic agents
and if they act directly or as a RES reservoir. In addition, we
synthesized three RES sulfate metabolites (8–10) by an im-
proved synthetic route. We tested their biological activities
and compared them with RES in order to shed light on the
controversy concerning the biological activity of RES metabo-
lites.
.
quent sulfation of each RES derivative by using SO3 NMe3 as
the sulfating reagent in acetonitrile under reflux followed by
silyl deprotection resulted in very low yields (10–26%). The dif-
ficulties might have been due to incomplete sulfation and the
complicated purification of these highly polar products. Thus,
instead, we used microwave-assisted synthesis as reported by
Desai and co-workers for highly sulfated organic scaffolds.[20]
.
TBDMS-protected 11–13 were treated with SO3 NMe3 and NEt3
in acetonitrile and microwave irradiated at 1008C for 20–
40 min (Scheme 2). The crude product was purified by LH-20
chromatography, and desilylation was carried out with KF in
MeOH for 18 h. Final crude purification was carried out by re-
versed-phase chromatography to obtain 8–10 in good yields
(73–89%, calculated from the TBDMS-protected RES deriva-
tives). Mattarei et al. recently reported the synthesis of resvera-
trol sulfates by a similar approach: selective protection by silyl
ether and sulfation with chlorosulfonic acid.[21]
Results and Discussion
Synthesis of RES prodrugs and RES sulfate metabolites
Diglucosylated RES derivatives, trans-resveratrol-3,5-di-O-b-d-
glucopyranoside (3) and trans-resveratrol-3,4’-di-O-b-d-gluco-
pyranoside (4) were synthesized from 1 as previously de-
scribed.[18] Briefly, mono-TBDMS (tert-butyldimethylsilyl)-pro-
&
ChemBioChem 2016, 17, 1 – 7
www.chembiochem.org
2
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
reported values, and lower than for 1 in both cell lines (HT-29
and MCF-7). The differences in IC50 values for 2 might arise
from the different methods used to measure cell viability or
from differences in purity.
It is important to note that 2–4 were more toxic with tumor
cells than nontumor ones (an obviously desirable feature). This
can be better appreciated by examining the a and b coeffi-
cients, which are obtained by dividing the IC50 values for the
noncancerous cell line (HEK-293) by those for a tumor cell line
(see footnote to Table 1). In general, a higher value reflects
a higher therapeutic safety margin in the corresponding cell
line. Thus, 1 was relatively more cytotoxic for the noncancer-
ous cell line than for both cancer cell lines. In contrast, 2 and 3
showed good selectivity for HT-29 (a>5.6 and 4.2, respective-
ly). Zhang et al.[23a] also showed that 2 is more potent with
cancer cells than non-cancer cells for a range of cell lines.
Storniolo et al. reported recently that 2 inhibited Caco-2 cell
growth (IC50 =25 mm).[25] Because the authors did not observe
b-glucosidase activity, they proposed that 2 exhibits antiproli-
ferative effects on intestinal epithelial cells directly and not as
a resveratrol reservoir. This could also be the case in our assays
with 2 and glycosylated RES derivatives 3 and 4. However, we
previously observed that the glucosyl or acyl modifications in
prodrugs 2, 3, 4, 6, and 7 retarded their metabolism in Caco- 2
cells, although they were metabolized to RES and its conju-
gates (glucuronates, sulfates, etc.) after 6–24 h incubation.[18]
The chemical stability and metabolism of 1, 2, and 7 were
evaluated after incubation with MCF-7, HT-29, and HEK-293
cells for different times (1, 12, 24, and 72 h; see the Supporting
Information). We observed low amounts of 1 (<3%) after
short times in the three cell lines, either as a consequence of
fast metabolism or, more probably, binding to serum proteins
such as albumin.[26] Increasing amounts of resveratrol-3-sulfate
(8) and resveratrol glucuronide were observed, with 8 as the
major metabolite. Resveratrol-4’-sulfate (9) was also observed,
but its concentration diminished after 1 h of incubation. When
2 and 7 were incubated, we observed similar behaviors in the
three cell lines. Their concentrations diminished over time and
yielded increasing amounts of 8, with 9 as the main metabolite
at 1 h (decreasing over time). Neither 1 nor resveratrol glucuro-
nide were observed. Therefore, 2 and 7 seem to act as pro-
drugs thus delivering RES (1)which is mainly transformed into
RES sulfated metabolites in the cells.
Scheme 2. a) TBDMSOTf, DIPEA, CH2Cl2; b) SO3·NMe3, TEA, CH3CN, 1008C,
20 min; c) KF, MeOH.
Cytotoxicity
The cytotoxicities of 2–10 were evaluated in vitro against two
cancer cell lines: human colon adenocarcinoma HT-29 and
breast adenocarcinoma MCF-7. In addition, one noncancerous
cell line, human embryonic kidney cell line (HEK-293), was em-
ployed for comparison.[22] RES was used as the positive control.
A standard MTT assay was used (see the Experimental Section),
and cytotoxicity values (IC50) are shown in Table 1. In most
cases, the values were in the low-to-medium micromolar
range. Among the RES derivatives, glucosylated compounds (2,
3, and 4) and piceid octanoate 7 showed the highest cytotox-
icity for HT-29, with IC50 values lower than for RES. A similar
trend was observed for MCF-7, with the lowest IC50 values for
2 and 7.
Reported cytotoxicity for 2 in several cancer cell lines has a
wide IC50 range, for example 1.5 to 300 mm for MCF-7 cells.[23]
IC50 values for 2 were in the same range as or higher than for
1.[23b,24] In our case, IC50 values for 2 were within previously
Table 1. Cytotoxicity of resveratrol and 2–10
IC50 [mm][a]
a[b]
b[c]
Among sulfated metabolites 8–10, we found low IC50 values
for 8 and 10 with MCF-7 cells (17 and 26 mm, respectively;
more toxic than 1 (127 mm)). With HT-29 cells, only 10 was
slightly more toxic than 1. It is noteworthy that 8–10 had low
toxicity on HEK-293, so the high safety margins for 8 and 10 in
the MCF-7 line (b>5.9 and 3.8, respectively) are especially in-
teresting.
HT-29
MCF-7
HEK-293
RES 1
PIC 2
3
4
5
6
7
8
9
70Æ3
127Æ22
38Æ18
72Æ18
89Æ16
115Æ12
110Æ7
52Æ2
12Æ3.5
0.2
0.1
18Æ8
24Æ0.5
45Æ9
>100
>100
>100
>100
>100
>5.6
>4.2
>2.2
>1.0
>0.7
0.4
>0.9
>1.0
>1.8
>2.6
>1.4
>1.1
>0.9
>0.9
0.3
>5.9
>0.7
>3.8
99Æ24
137Æ20
39Æ2
15Æ4
Several studies have found low (or no) cytotoxicity (>
100 mm) for RES sulfated metabolites in various cancer cell
lines (MDA-MB-231, ZR-55-1, KB, and neuroblastoma).[19,27]
More recent studies have described higher cytotoxicity for 8
(11–35 mm) in other cancer cell lines (Caco-2, CCL-228, HCT-116,
SW620, and SW480);[28] in these studies, resveratrol 3-O-d-glu-
curonide and resveratrol 4’-O-d-glucuronide (common resvera-
115 Æ17
17Æ1
>100
>100
>100
>100
144Æ32
26Æ4
10
56Æ4
[a] Concentration that causes 50% inhibition in an MTT assay. The values
are the average (ÆSD) of three different measurements. [b] a=IC50 (HEK-
293)/IC50 (HT-29). [c] b=IC50 (HEK-293)/IC50 (MCF-7). Values of a and b are
rounded to one decimal place.
ChemBioChem 2016, 17, 1 – 7
www.chembiochem.org
3
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
trol metabolites) also proved cytotoxic (10-35 mm), except on
SW620 and SW480 cell lines. For MCF-7 cells, Hoshino et al. re-
ported IC50 >50 mm for 8.[19] We measured slightly higher toxici-
ty for 8 (similar to 10). Polycarpou et al. suggested that the dif-
ferences in reported cytotoxicity for resveratrol metabolites
could be due to the use of different assays to measure cell via-
bility.[28c]
Telomerase production
Human telomerase contains an RNA component (hTER) that
serves as a template for the addition of the repeated nucleo-
tide sequences and a protein subunit (hTERT) that catalyzes
nucleotide polymerization. Human telomerase is regulated
during development and differentiation, mainly through tran-
scriptional control of hTERT, the expression of which is restrict-
ed to cells that exhibit telomerase activity. This indicates that
hTERT is the rate-limiting factor in the enzyme complex.[32]
Transcriptional factors c-Myc and Sp1 (among others) are impli-
cated in the expression of hTERT, by upregulating hTERT
mRNA.[33] Thus, for a preliminary study of the potential anti-
telomerase activity of RES derivatives, we investigated their
ability to inhibit the expression of hTERT and c-Myc mRNA. We
selected the same group of RES prodrugs (2, 3, and 7) and me-
tabolites (8, 9, and 10) as for VEGF inhibition. RES was used
again as the positive control.
RES sulfate metabolites have been suggested to provide an
intracellular pool for resveratrol generation.[29] Our results show
that RES sulfate metabolites are equally or more active than
RES as antiproliferative agents; thus they probably contribute
to the in vivo biological activity observed for RES.
VEGF production in HT-29 cells
One of the key factors in angiogenesis is the release of VEGF
from cells. We decided to examine whether our RES derivatives
and metabolites were able to inhibit or at least decrease the
activation of VEGF genes in HT-29 tumor cells. We selected glu-
cosylated RES compounds 2 and 3, piceid octanoate 7, and
RES sulfate metabolites 8, 9, and 10. Again, RES 1 was used as
the positive control. We determined VEGF protein production
by ELISA in culture supernatants. Figure 1 shows the results
obtained in ELISA measurements after treatment of HT-29 cells
with RES derivatives in DMSO: 2, 3, 7, and 10 decreased VEGF
secretion in comparison with untreated cells; 3 and 7 were
more effective than RES itself.
Treatment of HT-29 cells with RES and the derivatives led to
various degrees of reduction in the transcription of hTERT and
c-Myc mRNA (Figures 2 and 3). The most active compounds for
the inhibition of hTERT were 3 and 10 (both more potent than
RES). Particularly interesting is 10, which reduced the expres-
sion to 39% (RES: 61%).
Figure 2. Expression of hTERT mRNA is normalized to that of the housekeep-
ing gene b-actin. Data are meanÆSEM (n>2) relative to control. Statistical
significance was evaluated using one-sample t-tests (***p<0.001).
Figure 1. Percentage VEGF from HT-29 cells treated with DMSO, 1, 2, 3, 7, 8,
9, and 10 (20 mgmLÀ1 for 1 and 10 mgmLÀ1 others) related to not treated
cells. Data are meanÆSEM (n>2) relative to control. Statistical significance
was evaluated using one-sample t-tests (***p<0.001).
Piceid 2 had been shown previously[9] to inhibit angiogene-
sis on human umbilical vein endothelial cells at concentrations
of 100 to 1000 mm, in accordance with our findings. Two per-
methylated stilbene derivatives[30] and a family of RES dimers[31]
have also been reported to inhibit VEGF production. In the
case of the sulfate RES derivatives, it is surprising that disulfate
10 reduced VEGF secretion whereas monosulfates 8 and 9 did
not. It is difficult to imagine that 10 could be a resveratrol re-
servoir, as neither 8 nor 9 displayed antiangiogenic activity.
Figure 3. Expression of c-Myc mRNA is normalized to that of b-actin. At least
three measurements were performed in each case. Data are meanÆSEM
(n>2) relative to control. The statistical significance was evaluated using
one-sample t-tests (***p<0.001).
&
ChemBioChem 2016, 17, 1 – 7
www.chembiochem.org
4
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
Resveratrol sulfate metabolites: General procedure for micro-
wave-assisted O-sulfonation and KF-mediated desilylation: Mi-
crowave-based sulfonation reactions were performed by using a mi-
crowave synthesizer (BIOTAGE Initiator+) in sealed reaction vessels.
Sulfur trioxide–trimethylamine complex was washed with H2O,
MeOH, and CH2Cl2 and dried under high vacuum. TBDMS-protected
resveratrol derivative (1.0 equiv), sulfur trioxide–trimethylamine
complex (5 equiv per OH) and a magnetic stirrer bar were placed
in a 2–5 mL microwave reaction vial and fitted with a septum,
which was then pierced with a needle. The closed vial was then
evacuated under a vacuum for 2 h. The mixture was dissolved in
dry CH3CN (2.0 mL), then NEt3 (0.3–1.0 mL) was added. The mixture
was subjected to microwave radiation (20–40 min, 1008C, 50–60W
average power). MeOH (1 mL) and CH2Cl2 (1 mL) were added, and
the solution was layered on a Sephadex LH-20 column and with
CH2Cl2/MeOH (1:1) to obtain the corresponding triethylammonium
salt. The product and KF (2.0 equiv) were dissolved in MeOH
(5 mL). The mixture was stirred at room temperature for 18 h, then
the solvent was removed under vacuum. The crude product was
purified on an RP-C18 column with elution in H2O/CH3OH (100:0 to
70:30). Fractions containing the desired product were concentrated
and freeze-dried to afford the resveratrol sulfated compound
(yields: 8 (73%), 9 (82%), 10 (93%)). Compound characterization
(1H NMR, 13C NMR and MS) agreed with previously reported data.[18]
For c-Myc inhibition, RES and 3 were the most active (48%
and 66% respectively; Figure 3); 2, 8, 9, and 10 proved to be
inactive. RES and 3 show some correlation between inhibition
of c-Myc and hTERT gene expression (61 and 52%, Figure 3).
This indicates that RES and 3 might downregulate the expres-
sion of hTERT by reducing the transcription of c-Myc. In con-
trast, 10 showed the highest inhibition of hTERT in this series
but did not downregulate c-Myc. A different mechanism seems
to be used by 10, thus pointing out again that this RES metab-
olite is not just a RES reservoir but exerts its own biological re-
sponses.
Conclusion
RES derivatives 2, 3, 4, and 7 displayed higher antiproliferative
activity than RES, and showed less toxicity with HEK-293 cells.
Moreover, they showed a notable decrease in the production
of VEGF. These derivatives act as RES prodrugs, thus delivering
RES more slowly to cancer cells and therefore increasing the
biological effect. Therefore, RES prodrugs such as resveratrol-
3,5-diglucoside 3 might be promising anticancer drugs. The
RES sulfate metabolites (8–10) showed equal or higher cyto-
toxicity than RES for HT-29 and MCF-7 cells together with
lower toxicity towards noncancerous cells. The fact that RES di-
sulfate (10) decreased VEGF secretion whereas no activity was
observed for the monosulfates (8 and 9) seems to indicate
a distinct activity of 10 rather than just as a RES reservoir. Final-
ly, 10 seemed to decrease hTERT expression by a mechanism
different to that of resveratrol, as it did not affect c-Myc ex-
pression. These results indicate that RES metabolites contribute
to the in vivo biological activity observed for RES and do not
act only as a RES reservoir.
Cell culture: Cell culture media were purchased from Gibco (Grand
Island, NY). Fetal bovine serum (FBS) was from Seralab (Belton, UK).
Supplements were obtained from Sigma–Aldrich. BioLite plastics
for cell culture were supplied by Thermo Fisher Scientific. All
tested compounds were dissolved (10 mgmLÀ1) in DMSO and
stored at À208C. Cell lines were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) containing glucose (1 gLÀ1), glutamine
(2 mm), penicillin (50 UmLÀ1), streptomycin (50 mgmLÀ1) and am-
photerycin (1.25 mgmLÀ1), supplemented with FBS (10%).
Cytotoxicity assays: An MTT assay was used with 96-well micro-
plates, as previously described.[35] HT-29, MCF-7, or HEK-293 cells (~
5ꢃ103 in growth medium (100 mL)) were incubated (378C, CO2
(5%), humid atmosphere) with serial dilutions of the tested com-
pound. After three days, MTT (10 mL, 5 mgmLÀ1 in PBS; Sigma–Al-
drich) was added and the cells were incubated for a further 4 h
(378C). The resulting formazan was dissolved in HCl (150 mL, 0.04 n
in 2-propanol) and read at 550 nm. Each determination was carried
out in triplicate and all experiments were repeated at least three
times.
Experimental Section
General experimental methods: All commercial chemicals were
used without further purification, unless otherwise noted. All re-
actions were monitored by TLC on precoated Silica-Gel 60 F254
plates, and detected by heating with Mostain (H2SO4 (10%,
500 mL), (NH4)6Mo7O24·4H2O (25 g), Ce(SO4)2·4H2O (1 g)). Products
were purified by flash chromatography with Merck Silica gel 60
(200–400 mesh). MS analyses were run in the electrospray mode
(ESI-MS). NMR spectra were recorded on 300 or 400 MHz spectrom-
eters, at room temperature for solutions in CDCl3 or D2O. Chemical
shifts are referred to the solvent signal. Metabolites were purified
by Sephadex LH-20 and RP-C18 chromatography.
ELISA analysis: HT-29 cells (~150000) were placed on six-well
dishes, and resveratrol derivatives were added after 24 h. The cul-
ture supernatants were collected after 72 h, and secreted VEGF
was determined with a Human VEGF ELISA Kit (Invitrogen/Thermo
Fisher Scientific).
RT-qPCR analysis: HT-29 cells were incubated with resveratrol de-
rivative for 48 h. Cells were collected, and total RNA was isolated
with an Ambion RNA extraction Kit (Thermo Fisher Scientific). The
cDNA was synthesized by MMLV-RT with 1–21 mg of extracted RNA
and oligo(dT)15 according to the Maxima cDNA synthesis kit from
Thermoscientific. Genes were amplified by use of a thermal cycler
and StepOnePlus TaqMan probes. TaqMan Gene Expression Master
Mix Fast containing the appropriate buffer for the amplification
conditions, dNTPs, thermostable DNA polymerase enzyme and
a passive reference probe was used. For amplification of each gene
primers from Life Technologies were used: Hs99999903_m1 (b-
actin), Hs00900055_m1 (VEGFA), Hs00972646_m1 (TERT), and
Hs00153408_m1 (MYC).
Synthetic procedures
Glucosylated resveratrol derivatives: Resveratrol (1, RES) and
piceid (trans-resveratrol-3-O-b-d-glucopyranoside, 2, PIC) are com-
mercially available (Sigma Aldrich). Diglucosylated resveratrol deriv-
atives 3 and 4 were prepared as reported previously.[18]
Acylated resveratrol derivatives: 3-O-stearoyl-trans-resveratrol (5)
was kindly provided by Dr. Francisco J. Plou (Institute of Catalysis
and Petrochemistry, CSIC). Briefly, the preparation of 5 involves en-
zymatic acylation of RES with immobilized lipase from Alcaligenes
sp. (lipase QLG) as reported previously.[34] Piceid acyl derivatives 6
and 7 were synthesized by enzymatic acylation as previously de-
scribed.[18]
ChemBioChem 2016, 17, 1 – 7
www.chembiochem.org
5
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
[18] M. Larrosa, J. Tomꢆ-Carneiro, M. J. YꢅÇez-Gascꢁn, D. Alcꢅntara, M. V.
Selma, D. Beltrꢅn, M. T. Garcia-Conesa, C. Urbꢅn, R. Lucas, F. Tomꢅs-Bar-
berꢅn, J. C. Morales, J. C. Espin, J. Med. Chem. 2010, 53, 7365–7376.
[19] J. Hoshino, E.-J. Park, T. P. Kondratyuk, L. Marler, J. M. Pezzuto, R. B. van
Breemen, S. Mo, Y. Li, M. Cushman, J. Med. Chem. 2010, 53, 5033–5043.
[20] a) A. Raghuraman, M. Riaz, M. Hindle, U. R. Desai, Tetrahedron Lett. 2007,
48, 6754–6758; b) R. A. Al-Horani, U. R. Desai, Tetrahedron 2010, 66,
2907–2918.
Acknowledgements
Financial support has been granted by the Spanish Government
(Ministerio de Economꢀa y Competitividad, projects CTQ2011–
27560 and CTQ2012–35360), by the Consellerꢀa d’Empresa, Uni-
versitat i Ciencia de la Generalitat Valenciana (projects PROME-
TEO/2013/027) and by the Universitat Jaume I (project PI-1B-
2011–37).
[21] A. Mattarei, L. Biasutto, M. Romio, M. Zoratti, C. Paradisi, Tetrahedron
2015, 71, 3100–3106.
[22] N. Arden, M. J. Betenbaugh, Trends Biotechnol. 2004, 22, 174–180.
[23] a) Y. Zhang, Z. Zhuang, Q. Meng, Y. Jiao, J. Xu, S. Fan, Oncol. Lett. 2014,
7, 295–301; b) D. Su, Y. Cheng, M. Liu, D. Liu, H. Cui, B. Zhang, S. Zhou,
T. Yang, Q. Mei, PLoS One 2013, 8, e54505; c) H. Liu, S. Zhao, Y. Zhang, J.
Wu, H. Peng, J. Fan, J. Liao, J. Cell. Biochem. 2011, 112, 3695–3703.
[24] T. H. Do, Q. C. Chen, T. M. Hung, U. J. Youn, T. M. Ngoc, P. T. Thuong, H. J.
Kim, Y. H. Seong, B. S. Min, K. Bae, Arch. Pharmacal Res. 2009, 32, 177–
183.
Keywords: angiogenesis · antiproliferation · cytotoxicity ·
metabolite · prodrug · resveratrol
[1] E. N. Frankel, A. L. Waterhouse, J. E. Kinsella, Lancet 1993, 341, 1103–
1104.
[2] M. Jang, L. Cai, G. O. Udeani, K. V. Slowing, C. F. Thomas, C. W. W. Beech-
er, H. H. S. Fong, N. R. Farnsworth, A. D. Kinghorn, R. G. Mehta, R. C.
Moon, J. M. Pezzuto, Science 1997, 275, 218–220.
[25] C. E. Storniolo, P. Quifer-Rada, R. M. Lamuela-Raventos, J. J. Moreno,
Food Funct. 2014, 5, 2137–2144.
[26] a) B. Jannin, M. Menzel, J.-P. Berlot, D. Delmas, A. LanÅon, N. Latruffe,
Biochem. Pharmacol. 2004, 68, 1113–1118; b) C. N. N’soukpoꢆ-Kossi, C.
St. -Louis, M. Beauregard, M. Subirade, R. Carpentier, S. Hotchandani,
H. A. Tajmir-Riahi, J. Biomol. Struct. Dyn. 2006, 24, 277–283; c) Z. Lu, Y.
Zhang, H. Liu, J. Yuan, Z. Zheng, G. Zou, J. Fluoresc. 2007, 17, 580–587;
d) N. Latruffe, M. Menzel, D. Delmas, R. Buchet, A. LanÅon, Molecules
2014, 19, 17066–17077.
[3] J. S. Allard, E. Perez, S. Zou, R. de Cabo, Mol. Cell. Endocrinol. 2009, 299,
58–63.
[4] J. Gusman, H. Malonne, G. Atassi, Carcinogenesis 2001, 22, 1111 – 1117.
[5] L. Tessitore, A. Davit, I. Sarotto, G. Caderni, Carcinogenesis 2000, 21,
1619–1622.
[6] K. P. L. Bhat, D. Lantvit, K. Christov, R. G. Mehta, R. C. Moon, J. M. Pezzu-
to, Cancer Res. 2001, 61, 7456–7463.
[27] a) M. Miksits, K. Wlcek, M. Svoboda, O. Kunert, E. Haslinger, T. Thalham-
mer, T. Szekeres, W. Jꢇger, Planta Med. 2009, 75, 1227–1230; b) J. D. Ke-
nealey, L. Subramanian, P. R. Van Ginkel, S. Darjatmoko, M. J. Lindstrom,
V. Somoza, S. K. Ghosh, Z. Song, R. P. Hsung, G. S. Kwon, K. W. Eliceiri,
D. M. Albert, A. S. Polans, J. Agric. Food Chem. 2011, 59, 4979–4986.
[28] a) C. E. Storniolo, J. J. Moreno, Food Chem. 2012, 134, 1385–1391; b) V.
Aires, E. Limagne, A. K. Cotte, N. Latruffe, F. Ghiringhelli, D. Delmas, Mol.
Nutr. Food Res. 2013, 57, 1170–1181; c) E. Polycarpou, L. B. Meira, S. Car-
rington, E. Tyrrell, H. Modjtahedi, M. A. Carew, Mol. Nutr. Food Res. 2013,
57, 1708–1717.
[29] K. R. Patel, C. Andreadi, R. G. Britton, E. Horner-Glister, A. Karmokar, S.
Sale, V. A. Brown, D. E. Brenner, R. Singh, W. P. Steward, A. J. Gescher, K.
Brown, Sci. Transl. Med 2013, 5, 205ra133.
[30] a) M. Belleri, D. Ribatti, S. Nicoli, F. Cotelli, L. Forti, V. Vannini, L. A. Stivala,
M. Presta, Mol. Pharmacol. 2005, 67, 1451–1459; b) D. Alex, E. C. Leong,
Z.-J. Zhang, G. T. H. Yan, S.-H. Cheng, C.-W. Leong, Z.-H. Li, K.-H. Lam, S.-
W. Chan, S. M.-Y. Lee, J. Cell. Biochem. 2010, 109, 339–346; c) L. Zhang,
H. Jing, L. Cui, H. Li, B. Zhou, G. Zhou, F. Dai, J. Cell. Biochem. 2013, 114,
697–707.
[7] C. E. Harper, L. M. Cook, B. B. Patel, J. Wang, I. A. Eltoum, A. Arabshahi, T.
Shirai, C. A. Lamartiniere, Prostate 2009, 69, 1668–1682.
[8] M. Athar, J. H. Back, L. Kopelovich, D. R. Bickers, A. L. Kim, Arch. Biochem.
Biophys. 2009, 486, 95–102.
[9] a) Y. Kimura, H. Okuda, J. Pharm. Pharmacol. 2000, 52, 1287–1295; b) Y.
Kimura, H. Okuda, J. Nutr. 2001, 131, 1844–1849.
[10] S. Garvin, K. ꢄllinger, C. Dabrosin, Cancer Lett. 2006, 231, 113–122.
[11] G. Lanzilli, M. P. Fuggetta, M. Tricarico, A. Cottarelli, A. Serafino, R. Fal-
chetti, G. Ravagnan, M. Turriziani, R. Adamo, O. Franzese, E. Bonmassar,
Int. J. Oncol. 2006, 28, 641–648.
[12] C.-H. Cottart, V. Nivet-Antoine, C. Laguillier-Morizot, J.-L. Beaudeux, Mol.
Nutr. Food Res. 2010, 54, 7–16.
[13] a) J.-F. Marier, P. Vachon, A. Gritsas, J. Zhang, J.-P. Moreau, M. P. Duch-
arme, J. Pharmacol. Exp. Ther. 2002, 302, 369–373; b) T. Walle, F. Hsieh,
M. H. DeLegge, J. E. Oatis, Jr., U. K. Walle, Drug Metab. Dispos. 2004, 32,
1377–1382; c) A. Burkon, V. Somoza, Mol. Nutr. Food Res. 2008, 52,
549–557; d) E. Wenzel, V. Somoza, Mol. Nutr. Food Res. 2005, 49, 472–
481.
[14] GlaxoSmithKline halted all further development of resveratrol drug
SRT501. The Myeloma Beacon 2010 Nov 30 [Internet]. Available at
http://www.myelomabeacon.com/news/2010/11/30/glaxosmithkline-
halts-all-further-development-of-resveratrol-drug-srt501, last revised No-
vember 30, 2010.
[31] K. Kunimasa, T. Ohta, H. Tani, E. Kato, R. Eguchi, K. Kaji, K. Ikeda, H. Mori,
M. Mori, T. Tatefuji, Y. Yamori, Mol. Nutr. Food Res. 2011, 55, 1730–1734.
[32] K. A. Olaussen, K. Dubrana, J. Domont, J.-P. Spano, L. Sabatier, J.-C.
Soria, Crit. Rev. Oncol. Hematol. 2006, 57, 191–214.
[33] a) J. Dwyer, H. Li, D. Xu, J.-P. Liu, Ann. NY Acad. Sci. 2007, 1114, 36–47;
b) S. Kyo, M. Takakura, T. Fujiwara, M. Inoue, Cancer Sci. 2008, 99, 1528–
1538; c) M. Daniel, G. W. Peek, T. O. Tollefsbol, Gene 2012, 498, 135–146.
[34] P. Torres, A. Poveda, J. Jimenez-Barbero, A. Ballesteros, F. J. Plou, J. Agric.
Food Chem. 2010, 58, 807–813.
[15] J. A. Baur, D. A. Sinclair, Nat. Rev. Drug Discovery 2006, 5, 493–506.
[16] A. Maier-Salamon, M. Bçhmdorfer, J. Riha, T. Thalhammer, T. Szekeres,
W. Jaeger, Ann. NY Acad. Sci. 2013, 1290, 98–106.
[17] a) L. Biasutto, E. Marotta, A. Bradaschia, M. Fallica, A. Mattarei, S. Garbi-
sa, M. Zoratti, C. Paradisi, Bioorg. Med. Chem. Lett. 2009, 19, 6721–6724;
b) A. Mattarei, M. Azzolini, M. Carraro, N. Sassi, M. Zoratti, C. Paradisi, L.
Biasutto, Mol. Pharm. 2013, 10, 2781–2792; c) L. Ma, Y. Zhao, B. Li, Q.
Wang, X. Liu, X. Chen, Y. Nan, L. Liang, R. Chang, L. Liang, P. Li, F. Jin,
Respir. Physiol. Neurobiol. 2013, 185, 608–614; d) A. Madrona, G. Per-
eira-Caro, R. Mateos, G. Rodriguez, M. Trujillo, J. Fernꢅndez-BolaÇos, J. L.
Espartero, Molecules 2009, 14, 1762–1772; e) L. Biasutto, M. Zoratti,
Curr. Drug Metab. 2014, 15, 77–95.
[35] S. Rodriguez-Nieto, M. A. Medina, A. R. Quesada, Anticancer Res. 2001,
21, 3457–3460.
Manuscript received: February 11, 2016
Accepted article published: May 4, 2016
Final article published: && &&, 0000
&
ChemBioChem 2016, 17, 1 – 7
www.chembiochem.org
6
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

FULL PAPERS
E. Falomir, R. Lucas, P. PeÇalver,
R. Martꢀ-Centelles, A. Dupont,
A. Zafra-Gꢁmez, M. Carda,* J. C. Morales*
&& – &&
Cytotoxic, Antiangiogenic and
Improved resveratrol activity: Glucosy-
lated resveratrol prodrugs and resvera-
trol sulfate metabolites show improved
anti-proliferative and anti-angiogenic
activity in comparison to resveratrol
itself. These results suggest resveratrol
derivatives as potential drugs and con-
firm that resveratrol sulfate metabolites
contribute to the in vivo biological ac-
tivity observed for the parent com-
pound.
Antitelomerase Activity of Glucosyl-
and Acyl- Resveratrol Prodrugs and
Resveratrol Sulfate Metabolites
ChemBioChem 2016, 17, 1 – 7
www.chembiochem.org
7
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ