Alkylated resveratrol prodrugs and metabolites as potential therapeutics for neurodegenerative diseases

Article abstract of DOI:10.1016/j.ejmech.2018.01.037

Resveratrol is a naturally occurring stilbene which has shown promising results as treatment for several neurodegenerative diseases. However, its application is limited due to its low efficacy and bioavailability. Here, we have designed and synthesized alkylated resveratrol prodrugs combining structural modification to improve antioxidant and anti-inflammatory properties and the preparation of prodrugs to extend drug bioavailability. For comparison we also studied resveratrol prodrugs and alkylated resveratrol derivatives. Methylated and butylated resveratrol derivatives showed the best in vitro neuroprotective and anti-inflammatory activity. The glucosyl- and glucosyl-acyl- prodrugs of these derivatives showed lower toxicity on zebra fish embryo. When neuroprotection was examined on pentylenetetrazole challenged zebra fish, they were capable of reverting neuronal damage but to a lower extent than resveratrol. Nevertheless, 3-O-(6′-O-octanoyl)-β-D-glucopyranoside resveratrol (compound 8) recovered AChE activity over 100% whereas resveratrol only up to 92%. In a 3-nitropropionic acid mice model of Huntington's disease, resveratrol derivative 8 delayed the onset and reduced the severity of HD-like symptoms, by improving locomotor activity and protecting against weight loss. Its effects involved an equal antioxidant but better anti-inflammatory profile than resveratrol as shown by SOD2 expression in brain tissue and circulating levels of IL-6 (11 vs 18 pg/mL), respectively. Finally, the octanoyl chain in compound 8 could be playing a role in inflammation and neuronal development indicating it could be acting as a double-drug, instead of as a prodrug.

Full text of DOI:10.1016/j.ejmech.2018.01.037

Accepted Manuscript
Alkylated resveratrol prodrugs and metabolites as potential therapeutics for
neurodegenerative diseases
Pablo Peñalver, Efres Belmonte-Reche, Norma Adán, Marta Caro, María Luisa
Mateos-Martín, Mario Delgado, Elena González-Rey, Juan Carlos Morales
PII:
S0223-5234(18)30050-3
10.1016/j.ejmech.2018.01.037
EJMECH 10114
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 December 2017
Revised Date: 9 January 2018
Accepted Date: 11 January 2018
Please cite this article as: P. Peñalver, E. Belmonte-Reche, N. Adán, M. Caro, Marí.Luisa. Mateos-
Martín, M. Delgado, E. González-Rey, J.C. Morales, Alkylated resveratrol prodrugs and metabolites as
potential therapeutics for neurodegenerative diseases, European Journal of Medicinal Chemistry (2018),
doi: 10.1016/j.ejmech.2018.01.037.
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ACCEPTED MANUSCRIPT
Alkylated resveratrol prodrugs and metabolites as potential therapeutics for
neurodegenerative diseases
Pablo Peñalver,# Efres Belmonte-Reche,# Norma Adán, Marta Caro, María Luisa Mateos-Martín, Mario
Delgado, Elena González-Rey* and Juan Carlos Morales*
Affiliations
Department of Biochemistry and Molecular Pharmacology and Department of Cellular Biology and
Immunology, Instituto de Parasitología y Biomedicina López Neyra, CSIC, PTS Granada, Avda. del
Conocimiento, 17, 18016 Armilla, Granada, Spain.
Author Contributions
# P.P. and E.B-R. contributed equally.
Corresponding Authors
*E.G.-R.: phone, +34-958181670; e-mail: elenag@ipb.csic.es
*J.C.M: phone, +34-958181644; e-mail: jcmorales@ipb.csic.es
KEYWORDS
Resveratrol, neuroprotection, inflammation, prodrug, zebra fish, neurodegenerative diseases

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ABSTRACT
Resveratrol is a naturally occurring stilbene which has shown promising results as treatment for several
neurodegenerative diseases. However, its application is limited due to its low efficacy and bioavailability.
Here, we have designed and synthesized alkylated resveratrol prodrugs combining structural modification
to improve antioxidant and anti-inflammatory properties and the preparation of prodrugs to extend drug
bioavailability. For comparison we also studied resveratrol prodrugs and alkylated resveratrol derivatives.
Methylated and butylated resveratrol derivatives showed the best in vitro neuroprotective and anti-
inflammatory activity. The glucosyl- and glucosyl-acyl- prodrugs of these derivatives showed lower
toxicity on zebra fish embryo. When neuroprotection was examined on pentylenetetrazole challenged
zebra fish, they were capable of reverting neuronal damage but to a lower extent than resveratrol.
Nevertheless, 3-O-(6’-O-octanoyl)-β-D-glucopyranoside resveratrol (compound 8) recovered AChE
activity over 100% whereas resveratrol only up to 92%. In a 3-nitropropionic acid mice model of
Huntington’s disease, resveratrol derivative 8 delayed the onset and reduced the severity of HD-like
symptoms, by improving locomotor activity and protecting against weight loss. Its effects involved an
equal antioxidant but better anti-inflammatory profile than resveratrol as shown by SOD2 expression in
brain tissue and circulating levels of IL-6 (11 vs 18 pg/mL), respectively. Finally, the octanoyl chain in
compound 8 could be playing a role in inflammation and neuronal development indicating it could be
acting as a double-drug, instead of as a prodrug.
HIGHLIGHTS
• A series of alkylated resveratrol (RES) prodrugs has been synthesized
• Cytotoxicity, neuroprotective and anti-inflammatory activity were measured
• 3-O-(6’-O-octanoyl)-β-D-glucopyranoside resveratrol (8) was better neuroprotector than RES
• Compound 8 reduced the severity in a Huntington’s disease mice model
• Compound 8 could be a double-drug due to the role of octanoic acid in its structure

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1. Introduction
Neurodegenerative diseases are the result of progressive loss of neurons and neuronal connections in the
central nervous system (CNS) which normally lead to cognition and motor dysfunction. Despite the
differences in clinical manifestations of Alzheimer’s disease (AD), Parkinson’s disease (PD),
Huntington’s disease (HD), multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS), among
others, the pathological processes appear similar, suggesting common neurodegenerative pathways [1].
Mitochondrial malfunction and oxidative stress [2-4] together with the activation of innate immune
responses leading to inflammation [5] are known to play a major role in the pathophysiology of
neurodegenerative diseases.
A huge effort is being made to obtain drugs to prevent, treat or cure neurodegenerative diseases. The
majority of drug discovery programs are based on a ‘one-target-one-disease’ approach that has not been
successful up to now. Thus, alternative approaches such as ‘multi-target’ strategies have been proposed
[6-9]. The main aim is to modulate simultaneously different targets involved in the disease with just a
single drug. Natural products have been proposed as sources of multi-targeted lead compounds with
proven biological efficacy and safety [8, 10]. In fact, they can be considered as pre-optimized leads for
additional structural optimization by improving their bioactivity and/or their pharmacokinetic properties.
Resveratrol 1 (Figure 1) is a natural product with a stilbene scaffold that has shown antiplatelet [11],
antitumor [12, 13], anti-inflammatory [14] and neuroprotective activities [15-17]. Its bioactivity comes
from direct interactions of resveratrol with multiple molecular targets involved on inflammation, cell cycle
arrest, cell signaling, metabolism and posttranslational modification [18, 19]. Resveratrol has shown
promising results as treatment of neurodegenerative diseases and thus, has reached clinical trials for AD
[20, 21], PD and recently for HD (clinicaltrials.gov). When patients with mild-moderate Alzheimer’s
disease were treated with resveratrol (1 g by mouth twice daily), declines in cerebrospinal fluid A
β40
levels and activities of daily living scores were attenuated. However, high amounts of resveratrol need to

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be administered due to its low bioavailability. These results indicate that resveratrol could be considered a
lead compound that needs optimization.
Fig. 1. Resveratrol and related natural products
Two simple natural resveratrol derivatives pinostilbene 2 and pterostilbene 3 (Figure 1) have shown
improved neuroprotective capacity with respect to resveratrol. Pinostilbene 2 was reported to exert a
potent neuroprotective effect in SH-SY5Y neuroblastoma cells with a wider effective concentration than
resveratrol [22]. Low doses of pterostilbene 3 improved significantly radial arm water maze function in
SAMP8 mice, a model of AD, together with positive modulation of inflammation and cellular stress
whereas resveratrol showed no effect [23]. These results have inspired several groups to prepare O-
alkylated and C- alkylated resveratrol derivatives and to examine their in vitro neuroprotective potential.
Villalonga-Barber et al. [24] reported that 3’,5’-C-alkylated-resveratrol derivatives were 100-fold more
potent than resveratrol modulating oxidative stress on glutamate-challenged HT22 hippocampal neurons.
Puksasook et al. [25] found that 4-prenylresveratrol exhibited better anti-Aβ aggregation activity than
resveratrol and similar anti-BACE inhibitory activity. Other modifications on resveratrol such as imine
resveratrol derivatives, [26] resveratrol tacrine hybrids [27] and resveratrol clioquinol hybrids [28] have
also been explored as neuroprotective agents.
Due to the low bioavailability, rapid metabolism and low water solubility of resveratrol, several
resveratrol prodrugs have been prepared [29, 30]. Our group previously reported a series of resveratrol

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glucosylated and acylated prodrugs (Figure 2) and evaluated their anti-inflammatory activity. Compounds
7 and 8 were capable of decreasing colon inflammation in a mouse dextran sulfate sodium model to a
much higher extent than resveratrol [31]. Surprisingly, piceid 4 (Figure 1) and resveratrol lipoconjugates
[32] are the only resveratrol prodrugs that have been examined as neuroprotective drugs. Piceid, was able
to protect the brain on an ischemic stroke animal model, possibly by up-regulating the expression of Gli1,
Ptch1 and SOD1 and down-regulating the expression of NF-κB [33].
Fig. 2. Resveratrol prodrugs prepared previously.
OH
HO
HO
O
O
O
OH
HO
OH
R
O
O
O
HO
HO
HO
HO
O
OH
O
5
HO
OH
OH
OH
OH
7
8
R=C3H7
R=C7H15
O
O
HO
OH
HO
HO
O
O
HO
OH
6
OH
In this work, our aim was to combine two strategies in the same molecule to optimize resveratrol, i.e.
structural optimization through the synthesis of alkylated resveratrol derivatives to increase efficacy and
the improvement of bioavailability through the preparation of their prodrugs (Figure 3, Table 1). We have
also added resveratrol sulfate metabolites to our approach since metabolites of several drugs have shown
to be more active than the drugs themselves, as in the case of morphine-6-glucuronide [34]. Cytotoxicity,
neuroprotective capacity and anti-inflammatory activity of alkylated resveratrol derivatives 11-25 were
first evaluated in vitro in order to optimize the resveratrol scaffold. Then, prodrugs and metabolites of the
best alkylated derivatives were synthesized and their toxicity and neuroprotection capacity was evaluated
on zebrafish. Finally, the best resveratrol derivative, compound 8, was investigated as potential treatment

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for neurodegeneration using a preclinical model for Huntington’s disease. We have chosen this disease
because it is a hereditary and degenerative brain disorder (characterized by behavioural, cognitive and
motor dysfunctions), for which there is no cure or treatments which can slow down its progression. New
approaches in order to develop new therapeutics are strongly needed.
Fig. 3. Strategy for the design of novel resveratrol derivatives, metabolites and prodrugs.

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Table 1. Resveratrol derivatives examined in this work.
Comp.
RES prodrugs
R1
R2
R3
R4
Comp.
24
25
R1
iPr
H
R2
H
H
R3
H
iPr
R4
-
-
Glc
Glc
H
H
H
Glc
H
H
H
5
6
7
8
Glc
Glc
Glc
Butyl RES prodrugs
COC3H7
COC7H15
But
But
Glc
Glc
Glc
Glc
But
Glc
But
H
H
H
26
27
28
H
H
RES metabolites
-
9
10
SO3-
H
H
H
SO3
-
-
Butyl RES metabolites
-
-
SO3
But
But
SO3
SO3-
SO3-
SO3
But
SO3
But
-
-
-
29
30
31
-
Alkylated RES
-
Me
Me
H
Et
Et
Me
H
H
Et
H
H
H
But
H
H
H
Me
Me
Me
Et
Et
H
-
-
-
-
-
-
-
-
-
-
-
-
-
11
12
13
14
15
16
17
18
19
20
21
22
23
Methyl RES prodrugs
Me
Glc
Me
Me
Glc
Me
Me
Me
Me
Me
Glc
Glc
Me
Glc
Glc
Me
Glc
Me
Me
Me
Me
Me
Glc
Me
Me
Glc
Me
Glc
Malt
Malt
H
H
H
32
33
34
35
36
37
38
39
40
41
Et
H
COC7H15
COC7H15
COC7H15
COC3H7
COC3H7
H
Et
But
But
But
H
iPr
iPr
But
But
H
But
iPr
iPr
iPr
H
COC7H15
2. Chemistry
Resveratrol glucosylated prodrugs 5 and 6 were synthesized by chemical glycosylation of TBDMS-
protected resveratrol derivatives using a peracylated trichloroacetimidate donor, and posterior one-step
deprotection with NaOH in MeOH/THF, as previously reported [31]. Piceid acylated prodrugs 7 and 8
were prepared by enzymatic acylation of piceid 4 using Thermomyces lanuginosus lipase immobilized on

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granulated silica (Lipozyme TL IM). The reactions were carried out in tert-butyl alcohol with the
corresponding fatty acid vinyl esters to obtain high yields of the acylated compounds (92-97%) [31].
Resveratrol sulfate metabolites 9 and 10 were prepared following the procedure described by Hoshino et
al. [35] with an improved purification methodology reported by our group.[36] Briefly, TBDMS-protected
resveratrol derivatives were treated with SO3•NMe3 and NEt3 in acetonitrile under microwave irradiation.
The crude was first purified by LH-20 chromatography, then desilylated with KF in MeOH and finally
purified by reversed-phase chromatography.
Methyl, ethyl, isopropyl and butyl resveratrol derivatives were synthesized by random alkylation in
DMF using potassium carbonate and the corresponding 1-iodoalkane (Scheme 1) [25, 37]. Subsequent
chromatographic separation afforded mono-, di- and trialkyl resveratrol derivatives 11-25 although several
derivatives such as pterostilbene (3) could not be isolated under the chromatographic conditions used.
Compound characterization was easily carried out using NMR spectroscopy due to the differences in
symmetry between the mono-substituted regioisomers and the same applies to the di-substituted
regioisomers.
Scheme 1. Synthesis of alkylated resveratrol derivatives 11-25. Reagents and conditions: (a) MeI or EtI or
BuI or iPrI, K2CO3, DMF.
Since butyl resveratrol derivatives 18-21 showed the best overall in vitro neuroprotective activity and
lowest cytotoxicity among the alkylated derivatives examined (see below), we decided to prepare their
glucosylated prodrugs 26-28 and sulfate metabolites 29-31 for further investigation. Reaction of a

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peracylated glucosyl trichloroacetimidate donor with the corresponding butyl resveratrol derivatives and
posterior basic deprotection with NaOMe in MeOH (Scheme 2) resulted on compounds 26-28. Sulfation
of butyl resveratrol derivatives was carried out using the same conditions described above for the silyl
resveratrol derivatives and compounds 29-31 were obtained (Scheme 2).
Scheme 2. Synthesis of butylated resveratrol prodrugs and metabolites 26-31. Reagents and conditions:
(a) BF3·Et2O, CH2Cl2; (b) NaOMe/MeOH; (c) SO3·NMe3, TEA, CH3CN, 100ºC, 20 min, MW.
Since pterostilbene (3,5-dimethyl resveratrol) 3 has been reported to exert better neuroprotective and
anti-inflammatory activity than resveratrol [23, 38], and the methyl resveratrol derivatives 11-13 were the
second best family of alkylated derivatives on in vitro neuroprotection studies (see below), we decided to
prepare several methyl resveratrol prodrugs 32-41. Methyl resveratrol glucosylated derivatives 32-34 and
40 were prepared from the corresponding methyl resveratrol derivatives by reaction with peracylated
glucosyl or maltotriosyl trichloroacetimidate donors and posterior basic deprotection with NaOMe in
MeOH (Scheme 3). Since resveratrol sulfate metabolites showed high toxicity on the zebra fish embryo

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acute toxicity assay (see below), we decided to prepare acylated derivatives of methyl glucosyl resveratrol
derivatives instead. Moreover, the butanoyl or octanoyl fatty acids couldbe adding an intrinsic anti-
inflammatory effect (see below). Thus, enzymatic acylation of compounds 32-34 and 40 was carried out
with Lipozyme TL IM and butyric or octanoic acid vinyl esters to obtain compounds 35-39 and 41
(Scheme 3).
Scheme 3. Synthesis of methylated resveratrol prodrugs 32-41. Reagents and conditions: (a) BF3·Et2O,
CH2Cl2; (b) NaOMe/MeOH; (c) Lipozyme TL IM, vinyl fatty acid ester, 55 ºC, 18h.

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3. Results and discussion
3.1. Screening for neuroprotection against oxidative stress
To test if the alkylated modifications on resveratrol had any effect on its reported neuroprotective capacity
[39], we evaluated viability of SH-SY5Y neuroblastoma cells after oxidative stress challenge with
hydrogen peroxide in the presence or absence of the prepared alkylated resveratrol derivatives 11-25 [40].
We previously determined their cytotoxic effects at 10 µM concentration on SH-SY5Y cells (Fig. S1) and
found none of the compounds were toxic at the examined concentration. All alkylated resveratrol
compounds 11-25 investigated counteracted to some extent oxidative damage on neuroblastoma cells
produced by hydrogen peroxide at 1 and 10 µM concentration. Methylated and butylated resveratrol
derivatives, 11-13 and 18-21 respectively, showed a recovery up to 80-100% cell viability whereas
resveratrol 1 only recovered up to 55% cell viability. These results are in accordance with previously
reported results on the capacity to reduce oxidative stress of methylated resveratrol derivatives [22] and
other alkylated derivatives [25] on similar cellular systems. At 100 µM concentration several alkylated
derivatives (12, 16, 17, 19-21 and 23-25) showed no neuroprotection possibly due to their toxicity at
higher concentrations as it could be deduced from toxicity data in zebra fish embryo (see below).

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Fig. 4. Neuroprotective activity of alkylated resveratrol derivatives 11-25 on hydrogen peroxide-treated
SH-SY5Y cells. Cell viability was evaluated in SH-SY5Y cells after 24h of hydrogen peroxide incubation
(100 µM) in combination with the compounds at 1, 10, and 100 µM. Dashed line: reference value for
viability found on the H2O2 control without compounds; black bar: control SH-SY5Y cell viability; white
bar: SH-SY5Y cell viability after hydrogen peroxide incubation. Each bar represents mean ± standard
deviation of at least two different experiments run in quadruplicate.
3.2. Screening for inhibitory activity against LPS-induced TNF-a and IL-6 release
Resveratrol has been reported to decrease inflammation by inhibiting the induced production of pro-
inflammatory cytokines, such as TNF-α, IL-1β, IL-6, IL-8 and matrix metalloproteinases MMP-2, MMP-
3, MMP-9 and MMP-13, both on in vitro and on in vivo models [41-43]. We measured two inflammatory
markers (TNF-α and IL-6) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells in the
presence or absence of the alkylated resveratrol derivatives prepared. Peralkylated resveratrol derivatives
were discarded due to their high hydrophobicity and no possibility of further modification. We found that
the selected alkylated resveratrol compounds showed a higher inhibition of TNF-α production than

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resveratrol, except for 4’-ethylresveratrol 17 and 4’-isopropylresveratrol 25 (Figure 5A). The levels of IL-
6 produced were similar for resveratrol and all the investigated alkylated derivatives (Figure 5B).
Fig. 5. Anti-inflammatory profile of alkylated resveratrol derivatives in RAW macrophages. TNF-α (A)
and IL-6 (B) production was determined in RAW cells after 24h of LPS incubation (100 ng/ml) in
combination with the compounds at 10 µM concentration. Data are shown as percentage of cytokine
inhibition and they were normalized using the values corresponding to LPS and basal- production in RAW
cells as reference values for maximal and minimum cytokine production, respectively. Dashed line:
reference values for anti-inflammatory activity exerted by unmodified resveratrol 1. Each bar represents
mean ± standard deviation of at least two different experiments run in quadruplicate.
3.3. Assessment of acute toxicity on zebra fish
To screen compound toxicity on zebra fish is a cost-effective model due to the high fecundity, rapid
embryonic development and high homology to mammalian species of zebrafish [44]. Moreover, the
embryo is preferred to adult fish because it is predicted that early life stages feel less pain and distress than
adult fish. Fast acute toxicity was measured by incubation of zebrafish embryos with increasing
concentration of each compound and cumulative mortality/toxicity was observed after 96 hpf (hours post

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fertilization) [45]. It is important to note that cumulative mortality/toxicity is due to both developmental
impact and organotoxicity.
In vivo toxicity on zebrafish embryo was measured for representative compounds of each family of
resveratrol derivatives under study, alkylated resveratrol derivatives 11-25 and their corresponding
synthesized prodrugs and metabolites 26-41. We also measured toxicity for several resveratrol prodrugs 5-
8, metabolites 9-10 and resveratrol 1 itself. Table 2 and Figure S3 show NOEC, LOEC and LC50 values
for acute toxicity on zebrafish embryos. We observed that alkylated resveratrol derivatives 16, 20 and 24
were quite toxic with LC50 values from 11 to 13 µM whereas methylated resveratrol derivative 13 and
pterostilbene 3 were slightly less toxic with LC50 values of 32 and 36 µM, respectively. Addition of a
sulfate group to the butyl resveratrol structure as in compound 30 did not improve toxicity (10 µM). In
contrast, adding a glucose unit to the butyl resveratrol structure as in compounds 27 and 28 resulted in less
toxic derivatives.
In the case of the methylated resveratrol derivatives, their modification with glucosyl or glucosyl-acyl
groups (compounds 34-41) resulted in similar toxicity to the parent compounds with LC50 values from 29-
45 µM. The exceptions were compounds 36 (8 µM) and 40 (379 µM). The least toxic compounds of the
entire series were resveratrol 1 itself together with resveratrol prodrugs piceid 4, diglucosyl resveratrol
derivatives 5 and 6, piceid butyrate 7, piceid octanoate 8 and 4’-O-maltotriosyl pterostilbene 40.

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Table 2. NOEC, LOEC and LC50 values for acute toxicity on zebra fish embryos for resveratrol prodrugs,
metabolites and derivatives.
Compound
NOEC (µM)
LOEC (µM)
LC50 (µM)
1000
10
>1000
100
>1000
36
1
3
100
1000
114
4
RES prodrugs
1000
1000
100
1
>1000
>1000
1000
10
>1000
>1000
315
5
6
7
8
138
RES metabolites
10
100
1
29
17
9
0.1
10
Alkylated RES
10
1
100
10
1
32
13
11
11
13
16
20
24
0.1
0.1
1
Butylated RES prodrugs
10
10
100
100
35
21
27
28
Butylated RES metabolites
0.1
1
10
30
Methylated RES prodrugs
10
10
1
100
100
10
29
32
8
34
35
36
37
38
39
40
41
10
10
10
10
10
100
100
100
100
100
45
32
44
379
38
LC50 (median lethal dose), calculated by fitting sigmoidal curve to mortality data (y
= Bot + (Top-Bot) / (1 + 10 ^ (k*(x0 –Log(C)))). Bot, minimum mortality; Top,
maximum mortality; k, curve slope; x0, LC50 estimated. NOEC (No observed effect
concentration, with mortality score > 20% assumed as the effect). LOEC (Lowest
observed effect concentration, with mortality score > 20% assumed as the effect).
Negative control: 0.1% DMSO, in three replicates. Positive controls: 4-
Diethylaminobenzaldehyde (DEAB) at 5 different concentrations (0.1µM, 1µM,
10µM, 100µM, 1mM). DEAB is
a competitive inhibitor of aldehyde
dehydrogenases known to generate toxic and teratogenic effects.

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3.4. Assessment of neuroprotective activity on zebra fish
Pentylentetrazole (PTZ) is a competitive GABA antagonist which acts blocking Cl- anion conductance and
the formation of inhibitory postsynaptic potentials [46]. In zebra fish, PTZ acts as a pro-convulsant agent
that blocks the GABAergic inhibitory synaptic transmission [47]. PTZ has been used previously in rats to
induce neurodegeneration associated to an increase of damaged neurons, oxidative stress and
neuroinflammation [48-51]. We measured acetylcholinesterase (AChE) activity on PTZ challenged zebra
fish embryo since acetylcholine is a neurotransmitter involved in movement control and an important
modulator of cognitive functions such as learning and memory [52]. Therefore, appropriate levels of
AChE reflect a healthy neuronal state. Five days post fertilization (dpf) zebra fish larvae were pre-
incubated with each of the resveratrol derivatives for 1h. Then, media was changed and larvae were co-
incubated with PTZ and each of the resveratrol derivatives for 6h. Finally, larvae were processed to
measure AChE activity under each condition. A group treated with physostigmine (Phys), a commercial
inhibitor of the enzyme AChE, was included as positive control.
In a first assay we examined two prodrugs, 3,5-diglucosyl-resveratrol 5 and piceid octanoate 8 and
the three butylated resveratrol glucosylated prodrugs 26-28 together with control resveratrol 1. Butylated
resveratrol sulfate metabolites were not included due to their toxicity on zebrafish embryo. We found that
AChE activity of resveratrol-3-O-(6’-O-octanoyl)-β-D-glucopyranoside 8 group was fully recovered even
over 100% (Figure 6A). In fact, its AChE activity was higher than the one found for resveratrol (92%) and
for the other resveratrol prodrugs in this series. In a second assay we evaluated a series of methylated
resveratrol prodrugs (35, 37, 39 and 41) to check the possible relevance of the methyl group position, the
number of glucosyl groups and the optimum fatty acid group (Figure 6B). In this second assay all the
compounds examined possessed a fatty acid chain because the results on the first assay pointed to their
possible relevant role in neuroprotection. We found the methyl resveratrol prodrugs examined were
capable of partially recovering from PTZ damage but none of them showed higher AChE activity than
resveratrol.

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Fig. 6. Acetylcholinesterase (AChE) activity of resveratrol derivatives. The percentage of AChE (mU)
enzyme activity normalized to total protein (µg) vs control (considered as 100%) is shown. A) Series of
resveratrol glucosylated prodrugs and the butyl resveratrol prodrugs examined: B) Series of methyl
resveratrol prodrugs examined. Resveratrol was used as internal control. PTZ is pentylenetetrazole and
Phys is physostigmine. Two independent experiments were carried out with ten replicates of each
experimental condition. Each bar represents mean ± standard error of the mean (SEM). ANOVA statistics
were carried out followed by Dunnett's test with multiple comparisons. It is considered significant when
#P<0.05, ##P<0.01 respect to the control; **P<0.01 respect to the Control + PTZ.

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We observed in our neuroprotective assay in zebra fish that 3,5-O-di-β-D-glucopyranosyl resveratrol
5, a similar prodrug to resveratrol-3-O-(6’-O-octanoyl)-β-D-glucopyranoside 8 failed to prevent AChE
activity loss. This result indicates that the octanoyl chain could be playing a key active role. Short and
medium chain fatty acids have been reported to possess anti-inflammatory activity[53] and octanoic acid
was capable of partly restoring mitochondrial respiration on starved human endothelial cells and
monocytes under inflammatory conditions [54]. Octanoic acid is the main component of medium chain
triglycerides (MCT), and diets with a carbohydrate-reduction and an increase in MCT intake, constitute an
alternative for the treatment of drug-resistant epilepsy in children [55, 56]. Moreover, octanoic acid
induces neurite outgrowth in PC12 neuronal cells and shows a maximum effect in comparison with shorter
or longer fatty acids [57]. Growth of neurite processes from the cell body is a critical step in neuronal
development. Kamata et al. [57] suggested that the mechanism responsible is related with the activation of
p38 mitogen-activated protein kinase (MAPK) and the extracellular signal-regulated kinase (ERK). This
scenario, where the medium chain fatty acid and resveratrol would be playing a role side-by-side,
indicates that compound 8 could indeed be a double-drug. Other double-drugs have been reported earlier
as antimalarial [58], anti-HIV [59] or as antiproliferative agents [60].
Taking together, these results indicate that piceid octanoate 8 appeared to be one of the most effective
resveratrol derivatives on neuroprotection. Therefore, we decided to evaluate its protective effect in a
preclinical model of neurodegeneration.
3.5. Neuroprotective activity on a 3-nitropropionic acid mice model
3-Nitropropionic acid (3-NP) is an irreversible inactivator of the succinate dehydrogenase and an inducer
of mitochondrial dysfunction, which leads to an energy deprivation and oxidative stress exacerbation that
promotes neuronal damage. 3-NP-induced neurodegeneration results in a striking preferential damage of
striatum [61-63], the mainly anatomical region affected in Huntington’s disease (HD). HD is a mortal
progressive disorder caused by a dominant inherited expansion of CAG repeats in the Huntington gene.

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HD is characterized by neurological and systemic manifestations including cognitive, psychiatric and
motor defects which are accompanied and even preceded in time by an increment of circulating pro-
inflammatory cytokines (i.e. IL-6, TNF-α) [64, 65]. A plethora of evidences shows that 3-NP chronically
administrated in rodents and primates can replicate key abnormalities underlying HD pathogenesis,
including molecular alterations dependent of mitochondrial dysfunction (energetic deficit, oxidant species
accumulation) as well as clinical (motor and cognitive) signs [61, 63, 66, 67] supporting the potential
utility of the 3-NP intoxication model in HD studies [68, 69].
To analyze the neuroprotective and immune actions exerted by compound 8, we used the 3-NP
intoxication model (as described in the experimental section). Mice were given increasing amounts of the
toxin accompanied by the administration of compound 8 or resveratrol 1 which was used as reference.

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Fig. 7. Evaluation of the activity of compound 8 in a 3-NP-induced neurodegeneration animal model.
Mice were treated with vehicle (Veh), 3-NP, 3-NP plus resveratrol (1) or 3-NP plus compound 8, and
semi-quantitative motor-deficits (score from 0 to 8) (A) and body weight changes (relative to initial
&
#
weight) (B) were evaluated at different time points (n = 7 mice). P < 0.05 3-NP vs. veh, P < 0.05 3-NP
a
vs. 1+3-NP, P < 0.05 3-NP vs. 8+3-NP. At the end of experiment, (C) SOD2 and p53 mRNA levels in
whole brain-derived samples were determined by qRT-PCR (n = 4-7 mice) and (D) serum IL-6 levels
detected were assayed by ELISA (n = 6-7 mice). Results represent mean ± SEM. *P < 0.05, ***P < 0.005,
n.s.: non-significant.
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
105
100
95
Veh
3-NP
1+3-NP
8+3-NP
A)
B)
a
#
&
#
&
&
a
#
&
a
#
&
#
&
90
#
&
a
#
&
Veh
3-NP
1+3-NP
8+3-NP
85
&
a
#
#
&
80
75
Cumulated 3-NP [mg/kg]
Cumulated 3-NP [mg/kg]
p53
SOD2
6.0
5.0
4.0
3.0
2.0
1.0
0.0
3.0
2.5
2.0
1.5
1.0
0.5
0.0
40
D)
n.s.
C)
n.*s.**
***
35
*
30
*
25
20
15
10
5
n.s.
0

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We found an increment of the motor deficit-score scale and the weight loss in response to increasing
doses of 3-NP (Figure 7A-B). Furthermore, administration of compound 8 had a protective effect similar
to resveratrol on ameliorating loss of both motor coordination (Figure 7A) and weight induced by toxin
(Figure 7B). To evaluate the mechanisms of action of compound 8 on molecular hallmarks of HD such as
oxidative stress, neuronal death, and pro-inflammatory cytokines overproduction, we measured the
expression of SOD2 and p53, as well as the serum circulating level of IL-6 (Figure 7C-D). 3-NP increased
the expression of the proapoptotic p53 but neither resveratrol nor compound 8 counteracted its effect.
However, we found that although 3-NP did not appears to modify the basal level of SOD2, both
resveratrol and compound 8 induced in a similar way the overexpression of SOD2, an indicative result of
their antioxidant capacity (Figure 7C). In addition, 3-NP administration replicated the pro-inflammatory
phenotype of HD leading an increase of circulating IL-6 production. Interestingly, IL-6 stimulation was
efficiently counteracted by compound 8, while resveratrol produced a slight but not significant
amelioration (Figure 7D). All together these results show that compound 8 could exert neuroprotective
actions accompanied by antioxidant and anti-inflammatory effects in vivo as good as, or even better than
resveratrol itself, making of compound 8 a promising candidate for the therapeutic treatment of
neurodegenerative diseases such as HD.
4. Conclusions
In this study, we have combined two approaches for lead optimization of resveratrol for neurodegenerative
diseases, structural optimization through the synthesis of resveratrol derivatives to improve its efficacy
and an improvement in bioavailability through the preparation of their glucosylated and acylated prodrugs.
We have designed and prepared resveratrol prodrugs, alkylated resveratrol derivatives and alkylated
resveratrol prodrugs, together with sulfate metabolites of some of these resveratrol derivatives. Methylated
and butylated resveratrol derivatives showed better neuroprotective and anti-inflammatory activity than
resveratrol in the initial in vitro drug screening. When we examined toxicity in animals using a zebra fish

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embryo model, we observed that alkylated resveratrol derivatives were highly toxic whereas their
glycosylated prodrugs showed lower toxicity. When we investigated neuroprotection activity on zebra fish
on a pentylenetetrazole challenged zebra fish, resveratrol-3-O-(6’-O-octanoyl)-β-D-glucopyranoside 8
displayed better neuroprotective activity than resveratrol. Methylated and butylated resveratrol prodrugs
also could partially recover zebra fish embryo from neuronal damage although to a lower extent than
resveratrol. We cannot rule out whether the intrinsic toxicity of the alkylated resveratrol derivatives are
preventing a better recovery from the PTZ damage. We also observed that resveratrol-3-O-(6’-O-
octanoyl)-β-D-glucopyranoside 8 was capable of ameliorating the onset and reducing the severity of HD-
like symptoms induced by 3-nitropropionic acid in mice, used as a model of HD. Compound 8 improved
mice locomotor activity and prevented weight loss in a similar way to resveratrol. Whereas their
antioxidant effect seems to be comparable as revealed by SOD2 expression levels in brain tissue, the anti-
inflammatory activity of compound 8 appears to be better than that of resveratrol as indicated by the
circulating levels of IL-6. Finally, the octanoic acid chain within the structure of compound 8 could be
playing an active role on inflammation and neuronal development what suggests 8 could be acting as a
double-drug.
5. Experimental section
5.1. Chemistry
All solvents and chemicals were used as purchased without further purification. All reactions were
monitored by TLC on precoated silica gel 60 plates F254 (Merck) and detected by heating after staining
with H2SO4:EtOH (1:9, v/v), anisaldehyde (450 ml ethanol, 25 ml anisaldehyde, 25 ml H2SO4 and 1 ml
AcOH) or Mostain (500 ml of 10% H2SO4, 25g of (NH4)6Mo7O24•4H2O, 1g Ce(SO4)2•4H2O). Products
were purified by flash chromatography with silica gel 60 (200-400 mesh). Eluents are indicated for each

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particular case. NMR spectra were recorded on Bruker Advance 300, 400 or 500 MHz [300, 400 or 500
MHz (1H), 75, 101 or 126 (13C)] NMR spectrometers, at room temperature for solutions in CDCl3, or
CD3OD. Chemical shifts are referred to the solvent signal. 2D experiments (COSY, TOCSY, ROESY, and
HMQC) were done when necessary to assign the new compounds. Chemical shifts are in ppm. Low
resolution mass spectra were obtained on an ESI/ion trap mass spectrometer. High resolution mass spectra
(HRMS) were obtained on an ESI/quadrupole mass spectrometer (WATERS, ACQUITY H CLASS). If
necessary, the purity was determined by high performance liquid chromatography (HPLC). Purity of all
final compounds was 95% or higher. The instrument used for chromatographic separation was a Waters
Acquity UPLCTM H-class system (Waters, Manchester, UK). The column was an Acquity UPLCR BEH
C18 (2.1 x 100 mm, 1.7 µM). A QDA single quadrupole mass spectrometer (Waters) equipped with an
orthogonal Z-sprayTM electrospray ionization (ESI) source was used for metabolites detection. Empower 3
software was used for instrument control, peak detection and integration.
5.1.1. General procedure for etherification. Resveratrol (1 eq.) and potassium carbonate (2 eq.) were
added to DMF (2.85 ml/mmol of resveratrol) under agitation in a round-bottomed flask. 1-Iodoalkane (1-
1.5 eq) was added dropwise and reaction was stirred for 6 h at room temperature. The reaction mixture
was filtered, diluted with water and extracted with ethyl acetate (3 x 20 mL). The combined organic
layers were dried with MgSO4 and the mixture filtered, concentrated. The crude was purified by flash
column chromatography using different hexane/ethyl acetate mixtures.
5.1.1.1. Preparation of butylated resveratrol derivatives (18-21). Following the general procedure and
starting from resveratrol (513 mg, 2.25 mmol) and 1-iodobutane (0.38 mL, 3.37 mmol) the reaction
yielded 18-21 after purification with flash chromatography whilst eluting with a gradient concentration of
hexane:ethyl acetate (8:1 to 1:1).
5.1.1.1.1. (E)-1-(3,5-dibutoxyphenyl)-2-(4-butoxyphenyl)ethene (18). Yield=16%; Rf = 0.9 (Hexane/ethyl
acetate, 2:1). Compound characterization in accordance to literature [70].

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5.1.1.1.2. (E)-1-(3-butoxy-5-hydroxyphenyl)-2-(4-butoxyphenyl)ethene (19). Yield=31%; Rf = 0.6
1
(Hexane/ ethyl acetate, 2:1). H NMR (400 MHz, CDCl3) δ = 7.44 (d, J = 8.7 Hz, 2H), 7.02 (d, J = 16.3
Hz, 1H), 6.97 – 6.82 (m, 3H), 6.74 – 6.63 (m, 1H), 6.63 – 6.54 (m, 1H), 6.35 (t, J = 2.2 Hz, 1H), 4.04 –
3.94 (m, 4H), 1.89 – 1.76 (m, 4H), 1.60 – 1.43 (m, 4H), 1.08 – 0.94 (m, 6H). 13C NMR (101 MHz,
CDCl3) δ = 160.64, 158.97, 156.84, 140.02, 129.76, 128.90, 127.83, 126.17, 115.70, 114.77, 105.61,
105.38, 105.00, 101.12, 67.86, 31.33, 19.28, 13.89; ESI-HRMS [M + H] calcd for C22H28O3 341.2111,
found 341.2104.
5.1.1.1.3. (E)-1-(3-butoxy-5-hydroxyphenyl)-2-(4-hydroxyphenyl)ethene (20). Yield=13%; Rf = 0.25
1
(Hexane/ ethyl acetate, 2:1). H NMR (400 MHz, CD3OD) δ = 7.38 (d, J = 8.3 Hz, 2H), 7.01 (d, J = 16.3
Hz, 1H), 6.86 (d, J = 16.3 Hz, 1H), 6.79 (d, J = 8.3 Hz, 2H), 6.57 (s, 2H), 6.27 (s, 1H), 3.95 (t, J = 6.4 Hz,
2H), 1.81 – 1.69 (m, 2H), 1.51 (dd, J = 14.9, 7.4 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz,
CD3OD) δ = 160.58, 158.22, 157.00, 139.91, 129.02, 128.27, 127.51, 125.62, 115.12, 105.16, 103.76,
100.57, 67.30, 31.20, 18.96, 12.87; ESI-HRMS [M + H] calcd for C18H20O3 285.1485, found 285.1480.
5.1.1.1.4. (E)-1-(3,5-dihydroxyphenyl)-2-(4-butoxyphenyl)ethene (21). Yield=35%; Rf
=
0.3
1
(Hexane/ethyl acetate, 2:1). H NMR (400 MHz, CD3OD) δ = 7.40 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 16.3
Hz, 1H), 6.90 – 6.81 (m, 3H), 6.51 (s, 2H), 6.23 (s, 1H), 3.92 (t, J = 6.4 Hz, 2H), 1.77 – 1.65 (m, 2H), 1.48
(dd, J = 15.0, 7.5 Hz, 2H), 0.97 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ = 158.90, 158.28,
139.87, 129.91, 127.85, 127.36, 126.24, 114.33, 104.56, 101.43, 67.39, 31.14, 18.93, 12.87; ESI-HRMS
[M + H] calcd for C18H20O3 285.1485, found 285.1484.
5.1.1.2. Preparation of isopropylated resveratrol derivatives (22-25). Following the general procedure and
starting from resveratrol (529 mg, 2.32 mmol) and 2-iodopropane (0.23 mL, 2.32 mmol) the reaction
yielded 22-25 after purification with flash chromatography whilst eluting with a gradient concentration of
hexane:ethyl acetate (8:1 to 2:1).
5.1.1.2.1. (E)-1-(3,5-di-isopropoxyphenyl)-2-(4-isopropoxyphenyl)ethene (22). Yield=2%; Rf = 0.8
(Hexane/ ethyl acetate, 2:1). Compound characterization in accordance to literature [71].

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5.1.1.2.2. (E)-1-(3-isopropoxy-5-hydroxyphenyl)-2-(4-isopropoxyphenyl)ethene (23). Yield=15%; Rf =
1
0.5 (Hexane/ethyl acetate, 2:1). H NMR (400 MHz, CDCl3) δ = 7.39 (d, J = 8.4 Hz, 2H), 7.00 (d, J =
16.3 Hz, 1H), 6.91 – 6.77 (m, 3H,), 6.58 (s, 1H), 6.56 (s, 1H), 6.28 (s, 1H), 4.62 – 4.41 (m, 2H), 1.28 (dd,
J = 8.5, 6.4 Hz, 12H). 13C NMR (101 MHz, CDCl3) δ = 159.31, 157.64, 156.89, 140.07, 129.82, 128.86,
127.89, 126.23, 116.16, 106.91, 105.66, 102.37, 70.21, 22.11, 22.07; ESI-HRMS [M - H] calcd for
C20H24O3 311.1647, found 311.1633.
5.1.1.2.3. (E)-1-(3-isopropoxy-5-hydroxyphenyl)-2-(4-hydroxyphenyl)ethene (24). Yield=12%; Rf = 0.15
1
(Hexane/ethyl acetate, 2:1). H NMR (400 MHz, CD3OD) δ = 7.36 (d, J = 8.5 Hz, 2H), 6.99 (d, J = 16.2
Hz, 1H), 6.89 – 6.75 (m, 3H), 6.56 (s, 2H), 6.56 (s, 1H), 6.29 (s, 1H), 4.46 (dt, J = 12.0, 6.0 Hz, 1H), 1.24
(d, J = 6.1 Hz, 6H). 13C NMR (101 MHz, CD3OD) δ = 158.26, 157.55, 139.99, 129.89, 128.01, 127.50,
126.27, 115.79, 104.77, 101.56, 69.75, 21; ESI-HRMS [M + H] calcd for C17H18O3 09271.1329, found
271.1323.
5.1.1.2.4. (E)-1-(3,5-dihydroxyphenyl)-2-(4-isopropoxyphenyl)ethene (25). Yield=33%; Rf = 0.4
(Hexane/ethyl acetate, 1:1). 1H NMR NMR (400 MHz, CD3OD) δ = 7.38 (d, J = 8.4 Hz, 2H), 7.00 (d, J =
16.3 Hz, 1H), 6.85 (d, J = 16.3 Hz, 1H), 6.80 (d, J = 8.4 Hz, 2H), 6.58 (s, 1H), 6.56 (s, 1H), 6.28 (s, 1H),
4.56 (dt, J = 12.0, 6.0 Hz, 1H), 1.31 (d, J = 6.0 Hz, 6H). 13C NMR (101 MHz, CD3OD) δ = 159.22,
158.25, 156.98, 139.98, 129.05, 128.29, 127.54, 125.65, 115.16, 105.40, 105.23, 102.03, 69.64, 21.10;
ESI-HRMS [M + H] calcd for C17H18O3 271.1329, found 271.1331.
5.1.2.1. General Procedure for glycosylation. 2,3,4,6-tetra-O-benzoyl-α/β-D-glucopyranosyl trichloro
acetimidate 4253 (1 mmol) was dissolved in anhydrous CH2Cl2 (15 mL) in a 50 mL round-bottomed flask
under argon atmosphere. Butylated resveratrol derivative (0.33 mmol) and boron trifluoride diethyl
etherate (0.033 mmol) were added to the stirring solution and after 30 min, the reaction was stopped with
triethylamine (5 mL) and concentrated. The crude product was purified by flash column chromatography
using different hexane/ethyl acetate mixtures with 1% TEA.

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5.1.2.1. (E)-1-(3-butoxy-5-(2,3,4,6-tetra-O-benzoyl-
β-D-glucopyranosyloxy)phenyl)-2-(4-butoxyphenyl)
ethene (43). Following the general procedure and starting from 3,4’-di-O-butyl-resveratrol 19, the reaction
yielded 43 after purification with flash chromatography whilst eluting with a gradient concentration of
hexane:ethyl acetate (6:1). Yield=95%; Rf = 0.6 (Hexane/ethyl acetate, 2:1). 1H NMR (300 MHz,
CDCl3) δ = 7.93 (d, J = 7.4 Hz, 1H), 7.90 – 7.77 (m, 5H), 7.75 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 7.4 Hz,
1H), 7.32 – 7.24 (m, 3H), 7.17 (dd, J = 16.9, 8.6 Hz, 8H), 7.09 (d, J = 3.7 Hz, 2H), 6.83 (d, J = 16.2 Hz,
1H), 6.76 – 6.57 (m, 3H, CH), 6.51 (s, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 6.05 – 5.85 (m, 1H), 5.79 – 5.59
(m, 2H), 5.50 – 5.37 (m, 1H), 4.66 – 4.47 (m, 1H), 4.47 – 4.33 (m, 1H), 4.33 – 4.20 (m, 1H), 3.79 (t, J =
6.5 Hz, 2H), 3.68 (t, J = 5.5 Hz, 2H), 1.66 – 1.54 (m, 2H), 1.49 (dd, J = 14.4, 6.6 Hz, 2H), 1.40 – 1.15 (m,
4H), 0.88 – 0.67 (m, 6H); 13C NMR (75 MHz, CDCl3) δ = 166.49, 166.36, 166.12, 165.99, 165.91,
165.60, 165.51, 163.92, 160.73, 159.38, 158.55, 140.29, 133.87, 133.68, 133.56, 133.45, 130.28, 130.11,
130.00, 129.80, 129.76, 129.54, 129.37, 129.06, 129.03, 129.00, 128.78, 128.71, 128.67, 128.21, 126.16,
117.69, 115.00, 107.92, 107.46, 103.30, 99.90, 92.47, 89.13, 73.21, 72.90, 72.17, 70.74, 69.99, 68.64,
68.02, 63.70, 62.55, 31.60, 31.50, 31.28, 30.02, 19.55, 19.50, 14.19, 14.17; ESI-HRMS [M + H] calcd for
C56H54O12 919.3694, found 919.3720.
5.1.2.2. (E)-1-(3-butoxy-5-(2,3,4,6-tetra-O-benzoyl-
β-D-glucopyranosyloxy)phenyl)-2-(4-(2,3,4,6-tetra-O-
benzoyl- -D-glucopyranosyloxy)phenyl)ethene (44). Following the general procedure and starting from 3-
β
O-butyl-resveratrol 20 (0.33 mmol), the reaction yielded 44 after purification with flash chromatography
whilst eluting with a gradient concentration of hexane:ethyl acetate (4:1). Yield = 88%; Rf = 0.3
1
(Hexane/ethyl acetate, 2:1). H NMR (300 MHz, CDCl3) δ =7.92 (d, J = 7.7 Hz, 2H), 7.85 (t, J = 8.6 Hz,
10H), 7.76 (d, J = 7.6 Hz, 4H), 7.39 – 7.12 (m, 24H), 7.07 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H),
6.77 (d, J = 16.2 Hz, 1H), 6.67 – 6.54 (m, 3H, CH), 6.38 (s, 1H), 5.95 (t, J = 9.5 Hz, 2H), 5.75 (dd, J =
13.3, 5.5 Hz, 2H), 5.66 (dd, J = 12.4, 6.4 Hz, 2H), 5.43 (d, J = 7.6 Hz, 1H), 5.37 (d, J = 7.7 Hz, 1H), 5.12
(s, 1H), 4.62 (t, J = 9.1 Hz, 2H), 4.54 – 4.38 (m, 3H), 4.34 – 4.22 (m, 2H), 3.73 (t, J = 5.7 Hz, 2H), 1.61 –
1.49 (m, 2H), 1.30 (dd, J = 14.9, 7.4 Hz, 2H), 0.81 (t, J = 7.4 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ =
171.40, 166.42, 166.28, 166.05, 165.55, 160.70, 158.50, 156.80, 139.84, 133.83, 133.65, 130.05, 129.93,

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129.35, 128.96, 128.70, 128.63, 128.03, 117.62, 108.06, 107.61, 99.78, 73.08, 72.94, 72.02, 69.89, 68.09,
63.49, 60.65, 53.75, 31.46, 30.19, 29.91, 21.28, 19.45, 14.46, 14.11; ESI-HRMS [M + Na] calcd for
C86H72O21 1463.4464, found 1463.4453.
5.1.2.3.
(E)-1-(3,5-di-(2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyloxy)phenyl)-2-(4-butoxy)phenyl)
ethene (45). Following the general procedure and starting from 4’-O-butyl-resveratrol 21 (0.33 mmol), the
reaction yielded 45 after purification with flash chromatography whilst eluting with a gradient
1
concentration of hexane:ethyl acetate (4:1). Yield = 87%; Rf = 0.3 (Hexane/ethyl acetate, 2:1). H NMR
(300 MHz, CDCl3) δ = 7.84 (dd, J = 13.3, 7.5 Hz, 12H), 7.74 (d, J = 7.5 Hz, 4H), 7.40 – 7.08 (m, 27H),
6.74 (dd, J = 17.5, 11.8 Hz, 4H), 6.60 – 6.42 (m, 2H), 5.90 (t, J = 9.5 Hz, 2H), 5.65 (dd, J = 19.6, 9.6 Hz,
4H), 5.29 (s, 1H), 5.26 (s, 1H), 4.56 (d, J = 9.7 Hz, 2H), 4.41 (dd, J = 12.0, 5.7 Hz, 2H), 4.21 – 4.05 (m,
2H), 3.82 (t, J = 6.5 Hz, 2H), 1.60 (dd, J = 14.4, 6.5 Hz, 2H), 1.34 (dd, J = 14.9, 7.4 Hz, 2H), 0.82 (t, J =
7.4 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ = 171.40, 166.45, 166.06, 165.54, 165.29, 159.47, 158.33,
140.81, 133.82, 133.64, 133.46, 130.13, 130.02, 129.66, 129.50, 129.38, 129.00, 128.76, 128.72, 128.64,
128.29, 125.34, 114.92, 110.33, 99.68, 73.02, 72.71, 72.05, 70.01, 68.01, 63.56, 60.66, 31.56, 29.98,
21.29, 19.51, 14.47, 14.15; ESI-HRMS [M + Na] calcd for C86H72O21 1463.4464, found 1463.4445.
5.1.3. General Procedure for benzoyl deprotection. Butyl perbenzoylated glucopyranosyl resveratrol
derivative (0.35 mmol) was dissolved in CH2Cl2 (5 mL) in a 25 mL round-bottomed flask to give a yellow
solution. A solution of 25% sodium methoxide in methanol (5 mL) was added and left under stirring for 5
h. CH2Cl2 (20 mL) was then added and extracted with water (10 mL x 3). The aqueous phase was
neutralized with IR-120 H+ and concentrated. In the case of derivative 45, the organic phase was
concentrated and purified by flash column gel cromatography eluting with Hexane:EtOAc (1:3).
5.1.3.1. (E)-1-(3-butoxy-5-(β-D-glucopyranosyloxy)phenyl)-2-(4-butoxyphenyl)ethene (26). Yield = 63%,
white solid; 1H NMR (300 MHz, CDCl3) δ = 7.44 (d, J = 8.7 Hz, 2H), 7.07 (d, J = 16.4 Hz, 1H), 6.92 (d, J
= 16.4 Hz, 1H), 6.91 – 6.85 (m, 3H), 6.72 (s, 1H), 6.58 (s, 1H), 4.91 (d, J = 7.2 Hz, 1H), 3.97 (q, J = 6.5
Hz, 4H), 3.93 (dd, J = 12.2, 1.8 Hz, 2H), 3.71 (dd, J = 12.0, 6.1 Hz), 3.54 – 3.44 (m, 2H), 3.39 (d, J = 9.0

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Hz, 1H), 1.81 – 1.70 (m, 4H), 1.56 – 1.45 (m, 4H), 0.99 (td, J = 7.4, 3.2 Hz, 6H); 13C NMR (75 MHz,
CDCl3) δ = 160.34, 159.05, 159.00, 139.83, 129.82, 128.59, 127.47, 127.31, 125.83, 116.52, 114.28,
106.56, 106.48, 102.18, 101.18, 76.90, 76.63, 73.58, 70.16, 67.45, 67.35, 61.23, 31.14, 31.12, 18.93,
18.90, 12.83, 12.81; ESI-HRMS [M + H] calcd for C28H38O8 503.2632, found 503.2645.
5.1.3.2 (E)-1-(3-butoxy-5-(β-D-glucopyranosyloxy)phenyl)-2-(β-D-glucopyranosyloxy)phenyl) ethene
(27). Yield = 62%, white solid; 1H NMR (300 MHz, CDCl3) δ = 8.01 (d, J = 7.4 Hz, 1H), 7.47 (d, J = 7.4
Hz, 1H), 7.45 (d, J = 7.3 Hz, 1H), 7.10 (d, J = 16.3 Hz, 1H), 6.95 (s, 1H), 6.88 (d, J = 8.3 Hz, 2H), 6.76 (s,
1H), 6.66 (s, 1H), 4.94 (d, J = 6.3 Hz, 2H), 4.01 – 3.94 (m, 2H), 3.94 – 3.70 (m, 4H), 3.70 – 3.60 (m, 2H),
3.59 – 3.51 (m, 2H), 3.50 – 3.44 (m, 2H), 3.37 (d, J = 9.4 Hz, 2H), 1.80 – 1.68 (m, 2H), 1.57 – 1.42 (m,
2H), 0.98 (t, J = 7.3 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ = 168.54, 159.17, 158.87, 140.01, 132.74,
129.40, 129.01, 128.16, 127.62, 125.6, 114.40, 108.50, 103.70, 100.94, 76.95, 76.71, 73.63, 70.35, 67.47,
61.44, 61.33, 31.22, 19.01, 12.90; ESI-HRMS [M + Na] calcd for C30H40O13 631.2367, found 631.2421.
5.1.3.3. (E)-1-(3,5-di-(β-D-glucopyranosyloxy)phenyl)-2-(4-butoxy)phenyl)ethene (28). Yield = 71%,
white solid; 1H NMR (300 MHz, CDCl3) δ = δ = 8.03 – 8.02 (m, 1H), 8.01 (d, J = 1.3 Hz, 1H), 7.58 (ddd,
J = 7.0, 2.5, 1.3 Hz, 1H), 7.47 – 7.43 (m, 2H), 7.11 – 7.07 (m, 1H), 6.98 – 6.91 (m, 1H, m, 1H), 6.74 (s,
1H), 6.59 (s, 1H), 4.94 (s, 2H), 4.01 – 3.88 (m, 4H), 3.94 – 3.88 (m, 2H), 3.75 – 3.70 (m, 2H), 3.56 – 3.39
(m, 6H), 1.73 (tt, J = 13.5, 6.8 Hz, 2H), 1.50 (td, J = 14.9, 7.5 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H); 13C NMR
(75 MHz, CDCl3) δ = 168.54, 160.34, 158.99, 157.32, 132.70, 130.43, 129.32, 128.08, 127.41, 116.53,
114.36, 101.09, 100.74, 76.83, 76.68, 76.50, 73.57, 73.50, 70.16, 69.98, 67.51, 61.21, 61.10, 31.11,
18.93, 12.87; ESI-HRMS [M + Na] calcd for C30H40O13 631.2367, found 631.2366.
5.1.4. General Procedure for sulfation. Butylated resveratrol derivative (1.4 mmol) and sulfur trioxide
trimethylamine complex (14 mmol) were dissolved in anhydrous acetonitrile (10 mL) in a 25 mL round-
bottomed flask. Triethylamine (28 mmol) was then added. The reaction was carried out under agitation in
a microwave reactor (150 W, 60 ºC, 1 h). After completion the mixture was filtered and concentrated. The

ACCEPTED MANUSCRIPT
product was resuspended in 2-propanol, filtered and purified by flash column gel cromatography eluting
with Hexane:EtOAc (1:1).
5.1.4.1 Potassium (E)-3-(4-butoxystyryl)-5-butoxyphenyl sulfate (29). Yield=66%, dark yellow oil,
1
Eluents = Hexane:AcOEt (1:1), H NMR (300 MHz, CD3OD) δ = 7.27 (d, J = 8.7 Hz, 2H), 6.96 – 6.85
(m, 2H), 6.76 (d, J = 16.4 Hz, 1H), 6.69 (d, J = 8.4 Hz, 3H), 6.61 (d, J = 1.9 Hz, 1H), 3.83 – 3.72 (m, 4H),
1.61 – 1.47 (m, 4H), 1.30 (dt, J = 14.6, 7.3 Hz, 4H), 0.79 (td, J = 7.3, 5.3 Hz, 6H); 13C NMR (75 MHz,
CD3OD) δ = 187.82, 160.35, 159.40, 154.06, 139.91, 129.83, 129.23, 127.94, 125.70, 114.70, 111.13,
108.94, 106.78, 67.88, 67.68, 31.41, 19.25, 19.23, 13.25; ESI-HRMS [M + Na] calcd for C22H27O6
419.1521, found 419.1528.
5.1.4.2. Dipotassium (E)-5-(4-butoxystyryl)phenyl-1,3-disulfate (30). Yield=24%, yellow oil; Rf = 0.6
(Hexane:AcOEt - 1:1) , Column eluents = Hexane:AcOEt (1:1); 1H NMR (300 MHz, CDCl3) δ = 7.31 (d,
J = 8.6 Hz, 2H), 6.96 (d, J = 16.5 Hz, 1H), 6.80 (d, J = 16.5 Hz, 1H), 6.76 (d, J = 8.6 Hz, 2H), 6.56 (s,
1H), 6.52 (s, 1H), 6.25 (s, 1H), 3.86 (t, J = 6.4 Hz, 2H), 1.73 – 1.58 (m, 2H), 1.48 – 1.36 (m, 2H), 0.91 (t,
J = 7.3 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ = 188.77, 172.99, 161.76, 159.33, 158.10, 141.11, 130.25,
129.52, 128.81, 126.89, 116.39, 106.43, 105.11, 101.88, 68.54, 32.41, 20.20, 14.21; ESI-HRMS [M + H]
calcd for C18H18O9 S2 443.0470, found 443.0461.
5.1.4.3. Dipotassium (E)-3-(4-sulphate-styryl)-5-butoxyphenyl sulfate (31). Yield=32%, yellow oil; Rf =
0.65 (Hexane:AcOEt - 1:1), Eluents = Hexane:AcOEt (1:1); 1H NMR (300 MHz, CD3OD) δ = 7.31 (d, J =
8.3 Hz, 2H), 6.96 (d, J = 16.2 Hz, 1H), 6.80 (d, J = 16.2 Hz, 1H), 6.76 (d, J = 8.0 Hz, 2H), 6.56 (s, 1H),
6.52 (s, 1H), 6.25 (s, 1H), 3.85 (t, J = 5.7 Hz, 2H), 1.68-1.61 (m, 2H), 1.52 – 1.33 (m, 2H), 0.91 (t, J = 7.2
Hz, 3H). 13C NMR (75 MHz, CD3OD) δ = 187.82, 160.85, 158.58, 157.39, 140.05, 129.15, 128.60,
127.90, 125.79, 115.50, 105.42, 100.91, 100.88, 67.60, 31.46, 19.25, 13.21; ESI-HRMS [M + H] calcd for
C18H18O9 S2 443.0470, found 443.0476.
5.1.5. Preparation of methylated resveratrol prodrugs (32-41). General Procedure for glycosylation.
2,3,4,6-tetra-O-acetyl-α/β-D-glucopyranosyl trichloroacetimidate 46 [72] or per-O-acetyl-α/β-D-