Silyl resveratrol derivatives as potential therapeutic agents for neurodegenerative and neurological diseases

Article abstract of DOI:10.1016/j.ejmech.2021.113655

Natural phenolic compounds found in food have demonstrated interesting preventive and therapeutic effects on a large variety of pathologies. Indeed, some of them, such as resveratrol (RES), have been examined in clinical trials. Nevertheless, their success has been scarce mainly due to their low bioavailability. In this study, we found serendipitously that O-silyl RES derivatives exerted a better neuroprotective activity than resveratrol itself and decided to explore them as potential drugs for neurodegenerative and neurological diseases. We have also designed and prepared a series of O-silyl RES prodrugs to improve their bioavailability. We found that di-triethylsilyl and di-triisopropylsilyl RES derivatives were better in vitro neuroprotective and anti-inflammatory agents than RES. Among these derivatives and their corresponding acyl-, glycosyl- and carbamoyl-prodrugs, 3,5-triethylsilyl-4’-(6″-octanoylglucopyranosyl) resveratrol 26 showed the best profile on toxicity and neuroprotective activity in zebra fish embryo. Compound 26 was also capable of reducing the loss of motor coordination in a 3-nitropropionic acid mice model of Huntington's disease, in a similar way to RES. However, 26 diminished pro-inflammatory cytokine IL-6 to a higher extent than RES and improved the latency to fall in the rotarod test by 10% with respect to RES. Finally, we investigated 26 and RES as potential treatments on an experimental autoimmune encephalomyelitis (EAE) multiple sclerosis mice model. We observed that, in a therapeutic regimen, 26 significantly diminished the progression of EAE severity and reduced the percentage of animals with moderate to severe clinical score, whereas RES showed no improvement.

Full text of DOI:10.1016/j.ejmech.2021.113655

European Journal of Medicinal Chemistry 223 (2021) 113655
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Silyl resveratrol derivatives as potential therapeutic agents for
neurodegenerative and neurological diseases
1
1
~
Efres Belmonte-Reche , Pablo Penalver , Marta Caro-Moreno,
**
ꢀ
ꢀ
María Luisa Mateos-Martín, Norma Adan, Mario Delgado, Elena Gonzalez-Rey ,
Juan Carlos Morales*
Department of Biochemistry and Molecular Pharmacology and Department of Cellular Biology and Immunology, Instituto de Parasitología y Biomedicina
ꢀ
Lopez Neyra, CSIC, PTS Granada, Avda. del Conocimiento, 17, 18016, Armilla, Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Natural phenolic compounds found in food have demonstrated interesting preventive and therapeutic
effects on a large variety of pathologies. Indeed, some of them, such as resveratrol (RES), have been
examined in clinical trials. Nevertheless, their success has been scarce mainly due to their low
bioavailability. In this study, we found serendipitously that O-silyl RES derivatives exerted a better
neuroprotective activity than resveratrol itself and decided to explore them as potential drugs for
neurodegenerative and neurological diseases. We have also designed and prepared a series of O-silyl RES
prodrugs to improve their bioavailability. We found that di-triethylsilyl and di-triisopropylsilyl RES de-
rivatives were better in vitro neuroprotective and anti-inflammatory agents than RES. Among these
derivatives and their corresponding acyl-, glycosyl- and carbamoyl-prodrugs, 3,5-triethylsilyl-4’-(600-
octanoylglucopyranosyl) resveratrol 26 showed the best profile on toxicity and neuroprotective activity
in zebra fish embryo. Compound 26 was also capable of reducing the loss of motor coordination in a 3-
nitropropionic acid mice model of Huntington's disease, in a similar way to RES. However, 26 diminished
pro-inflammatory cytokine IL-6 to a higher extent than RES and improved the latency to fall in the
rotarod test by 10% with respect to RES. Finally, we investigated 26 and RES as potential treatments on an
experimental autoimmune encephalomyelitis (EAE) multiple sclerosis mice model. We observed that, in
a therapeutic regimen, 26 significantly diminished the progression of EAE severity and reduced the
percentage of animals with moderate to severe clinical score, whereas RES showed no improvement.
© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-
NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Received 17 February 2021
Received in revised form
28 May 2021
Accepted 14 June 2021
Available online 18 June 2021
Keywords:
Resveratrol
Silyl group
Prodrug
Neurodegenerative diseases
Huntington disease
Multiple sclerosis
1. Introduction
RES has shown beneficial effects in animal models of a wide
range of pathologies including cancer [3e5], cardiovascular dis-
Natural products have been one of the most successful sources
for novel drug candidates. In the last decades, some of these
compounds have been found as minor components of food. One
example is resveratrol (RES, 1) (Fig. 1), a phenolic derivative present
in red grapes, mulberries, peanuts, wine and tea. RES is a herbal
biosynthetic defense mechanism plants use against pathogenic
attacks and adverse environmental conditions, and is produced
from the conversion of p-coumaric acid through the phenyl-
propanoid pathway [1,2].
eases
[6e8],
antiplatelet
blood-related
disorders
[9],
inflammatory-related diseases and neurodegenerative diseases
such as Alzheimer disease (AD) [10e13], Huntington's disease
(HD) [14,15], Parkinson's disease (PD) [16,17] and amyotrophic
lateral sclerosis (ALS) [18]. Although with some controversy, RES
has also shown promising results on multiple sclerosis, an
immune-related neurological disease in which an abnormal
response causes inflammation and oxidative stress in neurons
[19e21].
Even though the mode of action of RES is not fully understood, it
modulates a wide range of signaling transduction pathways,
affecting cell growth and differentiation, apoptosis, angiogenesis
and metastasis [22] [e] [24]. RES also activates SIRT-1 and hence,
modulates essential metabolic regulatory transcription factors and
inflammatory responses. Additionally, it decreases oxidative stress
by reducing the toxicity of glutamate and calcium over exposure,
* Corresponding author.
** Corresponding author.
ꢀ
E-mail addresses: elenag@ipb.csic.es (E. Gonzalez-Rey), jcmorales@ipb.csic.es
(J.C. Morales).
1
These authors equally contributed to the paper.
https://doi.org/10.1016/j.ejmech.2021.113655
0223-5234/© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).

~
E. Belmonte-Reche, P. Penalver, M. Caro-Moreno et al.
European Journal of Medicinal Chemistry 223 (2021) 113655
Fig. 1. Schematic representation of resveratrol and derivatives: a) resveratrol (RES, 1) and resveratrol derivative 2 prepared previously; b) strategy used for the preparation of the
silyl resveratrol prodrugs.
and it modulates glucose metabolism and distribution via vaso-
relaxation, all of which improves neuronal functioning.
investigated in the preclinical models of Huntington's disease and
multiple sclerosis as a potential treatment.
The main drawbacks of using RES as a drug are its low solubility
and its very low bioavailability due to rapid metabolism and
elimination [25,26]. One of the approaches to avoid these problems
has been to increase the dosage. However, mild to severe side ef-
fects appear especially when very high doses are administered to
humans (2e5 g/day) [27]. From a medicinal chemistry perspective,
RES can be considered a Nature optimized hit compound that can
be further optimized to a lead compound. In fact, several research
groups have designed and prepared RES analogues and derivatives
trying to improve the bioavailability and the pharmacological ac-
tivity of RES, mostly as anticancer drugs [28e30].
2. Chemistry
O-Silyl resveratrol derivatives were synthesized by random
silylation of RES in DMF using imidazole and the corresponding silyl
chloride (Scheme 1) as previously reported for TBDMS derivatives
[34,40]. Subsequent chromatographic separation afforded mono-,
di- and trisilyl resveratrol derivatives 3e17 (Scheme 1). The trisilyl
resveratrol derivatives 3, 8 and 13 were not examined due to their
high lipophilicity and lack of available phenolic OH groups for
further modification.
RES prodrugs have also been synthesized and examined for the
treatment of a variety of pathologies [31e33]. Our group prepared
acyl- and glucosyl- RES derivatives as prodrugs and evaluated them
as anti-inflammatory [34] and anticancer drugs [35]. More recently,
we have combined two concepts, the preparation of O-alkyl RES
derivatives to increase RES efficacy and the synthesis of their acyl-
and glucosyl-prodrugs [36]. These new compounds were evaluated
as potential drugs for the treatment of neurodegenerative diseases
Due to the cytotoxicity observed for all the monosilyl resveratrol
derivatives except 40-triethylsilyl resveratrol (12) on SH-SY5Y
neuroblastoma and RAW macrophage cells (see Figs. S1 and S2),
we decided to prepare prodrug derivatives of the disilyl resveratrol
derivatives. We finally focused only on derivatives containing
triethylsilyl (TES) and triisopropylsilyl (TIPS) groups since 3,5-tert-
butyldimethylsilyl (TBDMS) resveratrol (5) showed some toxicity
on RAW macrophages at 10 mM concentration and also lower cell
viability when challenged with LPS (see Figs. S2 and S3).
and
resveratrol-3-O-(60-O-octanoyl)-
b
-D-glucopyranoside
(2)
(Fig. 1) was capable of reducing the severity of Huntington disease-
like symptoms induced by 3-nitropropionic acid in mice, improving
mice locomotor activity and diminishing inflammatory markers
such as IL-6 in plasma.
In the case of di-TES resveratrol derivatives, we modified the
remaining OH in the structure by enzymatic acylation. Previously,
this type of modification has improved the efficacy of piceid (3-
glucosyl resveratrol), such as for compound 2, on decreasing co-
lon inflammation in mice models of inflammatory-bowel diseases
[34] and improving locomotor activity in a mice model of Hun-
tington disease [36]. We selected 3,5-ditriethylsilyl resveratrol (10)
as starting material based on its toxicity, antioxidant and anti-
inflammatory activity (see Figs. 2e3 and S2-S4). Enzymatic acyla-
tion of 10 was carried out using Novozym® 435 lipase. The re-
actions were carried out in tert-butyl alcohol with the
corresponding fatty acid vinyl esters by reaction at 55 ꢀC for 72 h to
obtain high yields of the acylated compounds 18e22 (34e90%)
(Scheme 2). Other chemical modifications on compound 10 to
prepare prodrugs are not simple due to the chemical lability of the
O-silyl bond of TES to weak acid and basic conditions.
During the course of preliminary in vitro screening experiments
on neuroprotective activity of resveratrol derivatives, we discov-
ered the high efficacy of O-silyl resveratrol derivatives reducing
oxidative stress in SH-SY5Y neuroblastoma cells in comparison to
that of RES. In fact, silicon can be considered a bioisostere of carbon
and has been used previously in drug discovery [37e39]. Thus, we
decided to explore this type of compounds as potential drugs for
neurodegenerative diseases. In this work, we present the synthesis
of O-silyl resveratrol derivatives and, similarly to our previous work
with O-alkyl RES derivatives, we have also prepared the corre-
sponding prodrugs by modifications with acyl-, glucosyl- and
carbamoyl-groups (Fig. 1b and Table 1). Cytotoxicity, neuro-
protective capacity and anti-inflammatory activity of the new silyl
resveratrol derivatives 3e17 were first examined in vitro in order to
select the best silyl group and substitution pattern. Then, prodrugs
of the best silyl derivatives were synthesized and their toxicity and
neuroprotective capacity was evaluated on zebrafish embryo.
Finally, the best resveratrol derivative, compound 26, was
In the case of di-TIPS resveratrol derivatives 14 and 15, we first
modified the remaining OH by glycosylation. Reaction of peracy-
lated glucosyl trichloroacetimidate donor 29 with the corre-
sponding di-TIPS resveratrol derivative and posterior basic
deprotection with NaOMe in methanol (Scheme 3) lead to com-
pounds 23 and 24. Next, we acylated these derivatives with
2

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E. Belmonte-Reche, P. Penalver, M. Caro-Moreno et al.
European Journal of Medicinal Chemistry 223 (2021) 113655
Table 1
Resveratrol derivatives examined in this work.
Comp.
R1
R2
R3
Comp.
R1
R2
R3
R4
Silyl RES
TES RES prodrugs
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
TBDMS
TBDMS
TBDMS
TBDMS
H
TES
TES
TES
TES
TBDMS
H
TBDMS
H
H
TES
H
TES
H
H
TIPS
H
TBDMS
TBDMS
H
18
19
20
21
TES
TES
TES
TES
TES
TES
TES
TES
TES
TES
COC2H5
COC3H7
COC5H11
COC7H15
COC9H19
-
-
-
-
-
H
TBDMS
TES
TES
H
22
TIPS RES prodrugs
23
24
25
26
27
28
TIPS
TIPS
TIPS
TIPS
TIPS
TIPS
Glc
TIPS
Glc
TIPS
NHCOC2H5
TIPS
TIPS
Glc
TIPS
Glc
TIPS
NHCOC2H5
H
H
H
COC7H15
COC7H15
-
-
H
TES
TIPS
TIPS
H
H
TIPS
TIPS
TIPS
TIPS
TIPS
H
TIPS
H
H
Scheme 1. Synthesis of silyl RES derivatives 3e17.
Scheme 2. Synthesis of di-TES resveratrol prodrugs 18e22.
octanoic acid mimicking the modifications made in our previous
work to obtain compound 2. Again, we used Novozym® 435 lipase
as the acylating agent for the reaction between glucosyl silyl
resveratrol derivatives and vinyl octanoate in tert-butyl alcohol.
Compounds 25 and 26 were obtained in 35 and 40%, respectively.
Finally, we synthesized carbamoyl prodrugs of di-TIPS resveratrol
derivatives. Compounds 14 and 15 were reacted with ethyl isocy-
anate in dichloromethane to obtain prodrugs 27 and 28 (68 and 79%
yield, respectively).
Aqueous solubility of these new potential drugs is an important
aspect since resveratrol has a low aqueous solubility (0.05 g/L,
2.19 ꢁ 10ꢂ4 M) [41]. Thus, adding silyl groups makes the new RES
derivatives even less soluble in water. We have calculated compu-
tationally the solubility of several compounds of the TIPS RES
family and TIPS RES prodrugs (Table S1, Supporting information),
and their values range from 1.03 ꢁ 10ꢂ7 to 6.31 ꢁ 10ꢂ13 M. In order
to evaluate compound 26 in animal models we have used a
formulation containing hydroxypropyl-b-cyclodextrin and tylox-
apol to increase its aqueous concentration up to 1.0 ꢁ 10ꢂ2 M.
The hydrolytic stability of 26, the compound selected for in vivo
studies and representative members of the TIPS family (compounds
15 and 23) was measured in PBS buffer 1x (pH ¼ 7.4) and in the
culture media used for SH-SY5Y neuroblastoma cells after 72 h
incubation. Resveratrol was also included as a control. All the tested
compounds except resveratrol were quite stable both in PBS and
cell culture media (more than 80% of compound remaining after
72 h) (Fig. S1, Supporting information). At the same time, hydrolytic
stability of 26 was also measured in mice serum at 1 h and 24 h. We
3

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E. Belmonte-Reche, P. Penalver, M. Caro-Moreno et al.
European Journal of Medicinal Chemistry 223 (2021) 113655
Scheme 3. Synthesis of di-TIPS resveratrol prodrugs 23e28.
observed 26 was stable at short times (at 1 h, 75% of compound
remained), but less stable at long times (at 24 h, 15% of compound
remained). In the case of the 40-acylated TES RES derivatives,
compounds 18e22, we observed the triethylsilyl groups are quite
labile to mild acid conditions what made difficult their character-
ization and further biological examination. Thus, only compound
19 was evaluated in the zebra fish assays.
inflammatory cytokines, such as TNF-a, IL-1 b, IL-6 or IL-8 and ma-
trix metallopeptidases MMP-2, MMP-3, MMP-9 and MMP-13 [42]. In
orderto assess the anti-inflammatorycapacityof the silylresveratrol
derivatives, we measured three inflammatory markers (TNF-a,
interleukin-6 and NO) on lipopolysaccharide (LPS)-stimulated RAW
264.7 macrophage cells. These experiments were carried out in the
presence or absence of the silyl resveratrol derivatives prepared, and
RES and piceid octanoate 2 were used as controls.
We selected to evaluate the disilyl resveratrol derivatives
together with mono-TES resveratrol derivatives 11 and 12 for this
experiment based on their low toxicity on SH-SY5Y neuroblastoma
cells and on RAW 264.7 macrophage cells (see Figs. S1 and S2). We
observed that di-TBDMS resveratrol derivatives 4 and 5, and 3,40-
3. Results and discussion
3.1. Screening for neuroprotection against oxidative stress
First, toxicity of the O-silyl resveratrol derivatives was examined
di-triisopropylsilyl resveratrol 14 showed lower TNF-a values than
on SH-SY5Y neuroblastoma cells at 10 mM concentration. We found
RES, whereas values for 4 and 5 were in the same range than
control compound 2 (Fig. 3A). At the same time, di-TES resveratrol
derivatives 9 and 10, mono-TES resveratrol derivatives 11 and 12,
several mono-silyl derivatives, compounds 6, 16 and 17, that pro-
duced more than 50% decrease on cell viability in comparison to the
control (Fig. S1). Compound 7 was not assayed due to solubility
problems. Next, neuroprotective capacity was investigated
measuring viability of SH-SY5Y cells after oxidative stress challenge
with hydrogen peroxide in the presence or absence of the silyl
resveratrol derivatives. The compounds were investigated at 1, 10
and 3,5-di-triisopropylsilyl resveratrol 15 showed similar TNF-
a
values than RES. In the case of IL-6, all silyl resveratrol derivatives
inhibited this cytokine in the same range as RES, except compounds
14 and 15 that showed slight less inhibition (Fig. 3B). Finally, in-
hibition of NO was only observed for 3,5-triethylsilyl resveratrol 10
(Fig. S4).
and 100
All Resveratrol prodrugs showed better neuroprotection than
resveratrol when examined at 1 M concentration, except 3-
triethylsilyl resveratrol 11. However, at 10 M concentration only
mM concentration and RES was used as control (Fig. 2).
m
m
3.3. Acute toxicity on zebrafish
disilyl resveratrol derivatives 4, 5, 9, 14 and 15 maintained cell
viability as good as or even better than RES, possibly due to the
toxicity already detected for monosilyl resveratrol derivatives 6, 16
Zebrafish is a very convenient animal model to measure in vivo
toxicity [43,44] and also a great model of a variety of pathologies.
This is due to their high fecundity, rapid embryonic development
and high homology to mammalian species. We selected several
disilyl resveratrol derivatives and some of their prepared prodrugs
to measure their acute toxicity. Since most monosilyl resveratrol
derivatives showed high in vitro toxicity we decided to discard
them. Zebrafish embryos were incubated with increasing concen-
tration of each compound and cumulative mortality/toxicity was
observed after 96 hpf (hours post fertilization). It is important to
remark that cumulative mortality/toxicity can be due to both
developmental impact and organotoxicity.
and 17 at this concentration (Fig. S1). Interestingly, at 100
mM
concentration 3,5-di-triisopropylsilyl resveratrol 15 was the only
compound retaining 100% cell viability and 3,40-di-triisopropylsilyl
resveratrol 14 still protected neuroblastoma cells for oxidative
stress better than RES.
3.2. In vitro anti-inflammatory activity
The anti-inflammatory activity of RES is well known and is
mainly due to the inhibition in the production of relevant pro-
4

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E. Belmonte-Reche, P. Penalver, M. Caro-Moreno et al.
European Journal of Medicinal Chemistry 223 (2021) 113655
Fig. 2. Neuroprotective profile for mono- and disilyl RES derivatives on hydrogen peroxide-incubated SH-SY5Y cells. Cell viability was evaluated after 24 h of treatment with
hydrogen peroxide (100 nM) in combination with the compounds at 1, 10 and 100 M. Dashed line: reference value for cell viability after H2O2 incubation in the absence of
m
compounds (represented by red bar); black bar: control cell viability without H2O2; Values are expressed as the mean SD (n ¼ 4). (*) Significant difference (p < 0.05) compared to
control; (#) Significant difference (p < 0.01) compared to control (red dashed line).
NOEC, LOEC and LC50 values for acute toxicity on zebrafish
embryos were calculated and are represented on Table 2. The values
obtained indicated low toxicity of all measured compounds, with
LC50 values between those of RES and piceid octanoate 2 (see
Table 2 and Fig. S5).
protecting the zebrafish embryo from the PTZ challenge and
showed similar AChE activity as the control group. Moreover, 3,5-
triethylsilyl-4’-(600-octanoylglucopyranosyl) resveratrol 26 was the
best compound of the series showing the highest AChE activity
under these experimental conditions.
3.4. Neuroprotection activity on zebrafish
3.5. Neuroprotective activity on a 3-nitropropionic acid mice model
As mentioned previously, zebrafish is becoming an animal
model increasingly accepted to study a large variety of diseases
such as cancer [45], cardiovascular diseases [46] or bone diseases
[47]. Recent advances in zebrafish-based CNS disease modeling
have also significantly improved translational neuroscience and
experimental neurology research and drug discovery [48,49]. In
order to assess the neuroprotective activity of the silyl resveratrol
derivatives and their prodrugs, we challenged zebrafish embryo
with pentylenetetrazole (PTZ) which produces disorders on
neuronal development and neuronal death [50]. In fact, PTZ is a
pro-convulsant compound that blocks the GABAergic inhibitory
synaptic transmission. In this assay, acetylcholinesterase (AChE)
activity was measured on PTZ challenged zebra fish embryo since
acetylcholine is a neurotransmitter participating in movement
control and a relevant modulator of cognitive functions such as
learning and memory. Thus, the levels of acetylcholinesterase
indicate us if the neuronal state is optimum [36]. The experiment
was run on 5 days post fertilization (dpf) zebra fish larvae who were
pre-incubated with each of the resveratrol derivatives for 1 h. Then,
after changing media, larvae were co-incubated with PTZ and each
of the resveratrol derivatives for 6 h. Finally, AChE activity was
measured after the larvae were processed. A group treated with
physostigmine (Phys), an AChE inhibitor was included as control.
We investigated three silyl resveratrol derivatives with TES and
TIPS groups since derivatives with TBDMS groups have shown
some toxicity on RAW macrophages (Fig. S2). We observed that 3,5-
triethylsilyl resveratrol 10 showed similar AChE activity than RES
(1) whereas 3,40-triethylsilyl resveratrol 9 and 3,5-triisopropylsilyl
resveratrol 15 showed less AChE activity (Fig. 4). In the case of
the silyl resveratrol prodrugs examined, all of them were capable of
Systemic administration of 3-nitropropionic acid (3-NP), an in-
hibitor of the mitochondrial citric acid cycle, results in a progressive
locomotor deterioration resembling that of Huntington's disease
(HD). It is known that HD is caused by a dominant inherited
expansion of CAG repeats in the Huntington gene, and therefore
several aspects of HD cannot be mimicked by 3-NP administration.
However, recent research shows that oxidative stress and mito-
chondrial dysfunction may play a key role in HD pathogenesis,
further supporting the potential utility of the 3-NP model of striatal
degeneration in preclinical studies [51,52].
We selected compound 26 to examine its potential as treatment
for HD in a 3-NP intoxication model. Increasing amounts of the
neurotoxin were administered to mice together with the silyl
resveratrol derivative 26 or RES 1 which was used for comparison
purposes (Fig. S6). Both drugs were administered to the animals
using intraperitoneal injection in contrast to oral administration
which is normally used for resveratrol. The purpose is to reduce the
rapid metabolism of the phenolic drugs thus potentially increasing
the efficacy against the pathology under study.
Increasing doses of 3-NP resulted on an increment of the motor
deficit-score scale and on the weight loss in all mice (Fig. 5A and B).
Both, compound 26 and RES were able to decrease the loss of motor
coordination in a similar way, although treatment with compound
26 showed a better reduction in some motor deficits such as truncal
dystonia (Fig. S7). In the case of 26, severity-score diminished 26.5%
with respect to the 3-NP group at day 5 (end of treatment). Simi-
larly, 26 and RES partially compensate the weight loss of mice
(Fig. 5B), by 40% in the case of 26 at day 5 (94.8% weight versus
91.4% in the 3-NP mice group). Moreover, compound 26 improved
latency to fall in the rotarod test by 10% with respect to RES and the
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European Journal of Medicinal Chemistry 223 (2021) 113655
Fig. 3. Anti-inflammatory activity of mono- and disilyl RES derivatives in stimulated macrophages. TNF-
a
(A) and Interleukin-6 (B) production was determined in LPS-stimulated
RAW cells (100 ng/mL) co-incubated with 10
of maximal cytokine production. (*) Significant difference (p < 0.05) compared to control; (**) Significant difference (p < 0.01) compared to control.
m
M of each silyl RES derivatives. Values (in ng of enzyme per ml) are expressed as the mean SD (n ¼ 4). Dashed line: reference value
3-NP mice group, although it was not statistically significant
(Fig. 5C). Several biomolecular markers such as the expression of
SOD2 and the serum circulating level of IL-6 were measured in
order to better understand the mode of action of compound 26
(Fig. 5D and E). We observed that the increase on pro-inflammatory
cytokine IL-6 due to the 3-NP treatment was partially neutralized
by RES and 26, with the silyl resveratrol prodrug being more
effective (Fig. 5D). Although 3-NP administration usually can orig-
inate some reduction in antioxidant enzymes (such as catalase or
SOD2), we could not appreciate that it modified the basal mRNA
expression level of the antioxidant protein SOD2 in our model.
However, both RES and compound 26 administered simultaneously
to the neurotoxin, induced a similar overexpression of SOD2, sug-
gesting a potential antioxidant capacity against the 3-NP oxidative
stress-induced effect. Together, these results suggest a neuro-
protective role for the compound 26 based on anti-inflammatory
and antioxidant properties and indicate that 26 could be a prom-
ising treatment of neurodegenerative diseases.
3.6. Treatment with compound 26 reduces EAE severity
Next, we investigated the effect of the systemic treatment with
silyl resveratrol prodrug 26 in a preclinical model of chronic pro-
gressive EAE [53]. This model, induced by injection of myelin
oligodendrocyte protein MOG35-55 in C7BL/6 mice, mimics 20% of
clinical multiple sclerosis and is frequently used for investigating
novel therapeutic approaches that avoid progressive clinical
disability. As showed in Fig. 6, vehicle-treated mice developed
moderate clinical symptoms and never recovered from the disease.
In this study, we evaluated two different therapeutic regimens: a
preventive treatment with compound 26 or RES, that were daily
administered from day 5 post-immunization during 3 weeks
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European Journal of Medicinal Chemistry 223 (2021) 113655
Table 2
mice developed moderate to severe clinical signs (Fig. 6E). These
results suggest that a short treatment with compound 26 was
enough to generate a long lasting protective effect. RES treatment
was completely inefficient in a therapeutic regimen. It is worth
noting that, in both regimens, we administered much less amount
of compound 26 than RES as we use the same dose for both com-
pounds (20 mg/kg) although the molecular weight of 26 is three
times that of RES. It is also important to note that the solution
NOEC, LOEC and LC50 values for acute toxicity on zebrafish embryos for resveratrol
prodrugs, metabolites and derivatives.
Compound
NOEC (
m
M)
LOEC (
m
M)
LC50 (mM)
1
2
1000
1
>1000
10
>1000
138
Silyl RES
9
10
14
100
100
>1000
1000
1000
1000
380
475
>1000
formulation for compound 26 contained HP-b-cyclodextrin (13%
conc.) and tween 80 (2% conc.) and also for the corresponding
vehicle solution, whereas RES did not contain the tween-80
emulgent and neither its corresponding vehicle. This could be the
reason for the differences observed in the clinical score of mice
treated with vehicle on each experimental setup.
TES RES prodrugs
19
TIPS RES prodrugs
23
24
25
26
28
100
1000
407
1000
1000
100
100
1000
1000
1000
1000
1000
1000
>1000
>1000
407
407
563
4. Conclusions
LC50 (median lethal dose), calculated by fitting sigmoidal curve to mortality data
(y ¼ Bot þ (Top-Bot)/(1 þ 10 ^ (k*(x0 eLog(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-
In this study, we have prepared silyl resveratrol derivatives and
examined them as potential therapeutic agents for neurodegener-
ative diseases. We have also synthesized and investigated several
prodrugs of these compounds with modifications such as acyl,
glucosyl and carbamoyl, or combinations of them. The idea on both
cases was to try improving resveratrol efficacy and bioavailability.
Cytotoxicity studies on SH-SY5Y neuroblastoma cells and on RAW
macrophages indicated that monosilyl resveratrol derivatives were
quite toxic whereas disilyl derivatives did not show toxicity at
Diethylaminobenzaldehyde (DEAB) at 5 different concentrations (0.1
10 M, 100 M, 1 mM). DEAB is a competitive inhibitor of aldehyde dehydrogenases
known to generate toxic and teratogenic effects.
mM, 1 mM,
m
m
(excluding weekends) (Fig. 6A and B); and a therapeutic treatment,
in which both compounds were daily administered at disease onset
during 2 weeks (Fig. 6C and D). Although the preventive treatment
with RES showed a beneficial effect only during the first 2 weeks
after which the disease showed again a clinical progression, pre-
ventive use of compound 26 was ineffective (Fig. 6A and B). How-
ever, therapeutic administration of compound 26 reduced
incidence and severity and avoid clinical progression even 20 days
after finishing treatment (Fig. 6C and D). In fact, at the end of the
clinical evaluation most of the mice treated with compound 26
showed only mild symptoms while almost 80% of vehicle-treated
10
mM concentration. In addition, di-triethylsilyl and di-
triisopropylsilyl resveratrol derivatives showed better neuro-
protective capacity and anti-inflammatory activity than resveratrol
itself and thus, were the best candidates to prepare prodrugs.
Among the acyl, glucosyl and carbamoyl disilyl resveratrol com-
pounds investigated on zebrafish for toxicity and neuroprotective
activity, 3,5-triethylsilyl-4’-(600-octanoylglucopyranosyl) resvera-
trol 26 showed the best results. When 26 was examined on the 3-
nitropropionic acid mice model of Huntington disease it was
capable of ameliorating the severity of motor deficits and of
Fig. 4. Acetylcholinesterase (AChE) activity of resveratrol derivatives with respect to the control. The percentage of AChE (mU) enzyme activity normalized to total protein (mg) vs
control (considered as 100%) is shown. PTZ: pentylenetetrazole; Phys: physostigmine. ANOVA statistics were carried our followed by Dunnett's test with multiple comparisons. It is
considered significant when #P < 0.05 respect to Control; *P < 0.05, **P < 0.01 respect to Control þ PTZ.
7

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E. Belmonte-Reche, P. Penalver, M. Caro-Moreno et al.
European Journal of Medicinal Chemistry 223 (2021) 113655
Fig. 5. Analysis of the functional activity of resveratrol (RES, 1) and compound 26 in a 3-NP-induced neurotoxic model. Mice were treated with vehicle (Veh), 3-NP, 3-NP plus
resveratrol (1þ3-NP) or 3-NP plus compound 26 (26þ3-NP). A) Semi-quantitative motor symptoms evaluation (score severity from 0 to 8); B) Body weight analysis (relative to
initial weight for each mice); C) Rotarod test, latency to fall (in seconds) after 360 mg/kg accumulated administration of 3NP; D) IL-6 levels detected in serum samples; E) SOD2,
relative mRNA expression in whole brain-derived samples. 2-ways ANOVA test was carried out in Fig. 5A) and B) to compare 3-NP data vs 3-NP þ resveratrol (#) or 3-
NP þ compound 26 ($). Results with a P < 0.05 are considered statistically significant (# or $ P < 0.05; ## or $$ P < 0.01; ### or $$$ P < 0.001). Mann Whitney test with
multiple comparisons was applied in Fig. 5C)e5E) to compare vehicle (5C and 5D) or 3-NP (5 E) data vs 3-NP þ resveratrol or 3-NP þ compound 26. Results with a P < 0.05 are
considered statistically significant (*P < 0.05; **P < 0.01).
reducing the weight loss to a similar extent than RES. But, 26
showed better results in the rotarod test with respect to RES and
the 3-NP mice group, although not statistically significant. In
addition, compound 26 showed a relevant anti-inflammatory and
antioxidant activities. Moreover, silyl resveratrol prodrug 26
showed a therapeutic beneficial effect for chronic EAE while no
effect was observed for RES. These results indicate that silyl mod-
ifications on resveratrol together with a prodrug strategy can yield
compounds such as 26 that improve the efficacy of RES on in vivo
neurodegenerative and neuroinflammatory disease models.
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).
5.1.1. General procedure for silylation
In a round-bottom flask under agitation was resveratrol (1 eq.)
and imidazole (2.5 eq.) suspended in DMF (3 mL/mmol of resver-
atrol) and cooled to 0 ꢀC. The corresponding silyl-chloride (1.4e1.55
eq.) was added dropwise in a two-step process whilst stirring, half
at t ¼ 0 h and the other at t ¼ 3 h. The reaction was stirred for
another 6 h at r. t. The reaction mixture was filtered, diluted with
water and extracted with ethyl acetate (3 ꢁ 50 mL). The combined
organic layers were dried with MgSO4 and then filtered, concen-
trated and added to a silica gel column eluting with gradient con-
centrations of hexane/ethyl acetate.
5. Experimental section
5.1. Chemistry
All solvents and chemicals were used as purchased without
further purification. All reactions were monitored by TLC on pre-
coated 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, 25 g of (NH4)6Mo7O24ꢃ4H2O, 1 g
Ce(SO4)2ꢃ4H2O). Products were purified by flash chromatography
with silica gel 60 (200e400 mesh). Eluents are indicated for each
particular case. NMR spectra were recorded on Bruker Advance
300, 400 or 500 MHz [300, 400 or 500 MHz (1H), 75, 101 or 126
5.1.2. Preparation of tert-butyldimethylsilyl resveratrol derivatives
(3e7)
Following the general procedure and starting from resveratrol
(830 mg, 3.64 mmol) and terbutyldimethylsilyl chloride
(849.55 mg, 5.64 mmol) yielded 3e7 after purification with flash
chromatography whilst eluting with a gradient concentration of
hexane: ethyl acetate e (8:1 to 2:1). Compounds were character-
ized according to literature [40].
(
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
8

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E. Belmonte-Reche, P. Penalver, M. Caro-Moreno et al.
European Journal of Medicinal Chemistry 223 (2021) 113655
Fig. 6. Beneficial effect of treatment with compound 26 in chronic EAE. Chronic progressive EAE was induced in C57BL/6 mice by immunization with MOG35e55. Mice were treated i.
p. with vehicle (EAE control, A, B, black circles), with vehicle with Tween-80 (EAE control-T, C, D, black circles), RES (A, B, red squares) or compound 26 (C, D, blue triangles) (20 mg/
kg for both compounds). Preventive treatment is represented on A and C, and consisted on treatment for 3 weeks (avoiding weekends) starting 5 days post-immunization. Ther-
apeutic treatment is represented on B and D, and consisted on treatment for 2 weeks (avoiding weekends) after disease onset (day 12 post-immunization). E) Disease incidence
(expressed as percentage) at the end of the experiment was quantified by evaluating mice with none to mild symptoms (clinical score ꢄ 1.5) or with moderate to severe signs
(clinical score > 1.5).
5.1.2.1. 3,4’,5-O,O,O-tri-tert-butyldimethylsilyl
Yield ¼ 6%; Rf ¼ 0.9 (Hexane: AcOEt, 7:3).
resveratrol,
resveratrol,
resveratrol,
3.
4.
2.8 Hz, 27H), 0.78 (q, J ¼ 7.9 Hz, 18H). 13C NMR (101 MHz, CDCl3)
d
¼ 156.61, 155.45, 139.48, 130.60, 128.44, 127.73, 126.76, 120.17,
111.41, 110.93, 6.67, 5.05; TOF MS ES þ Calculated mass for
5.1.2.2. 3,40-O,O-di-tert-butyldimethylsilyl
Yield ¼ 22%; Rf ¼ 0.6 (Hexane: AcOEt, 7:3).
C
32H54O3Si3 ¼ 570.34, Found mass [M þ H] ¼ 571.5.
5.1.3.2. 3,40-O,O- di-triethylsilyl resveratrol, 9. Yield ¼ 15%; Rf ¼ 0.6
5.1.2.3. 3,5-O,O-di-tert-butyldimethylsilyl
Yield ¼ 6%; Rf ¼ 0.5 (Hexane: AcOEt, 7:3).
5.
(Hexane: Ethyl Acetate e 3:1). 1H NMR (400 MHz, CD3OD)
d
¼ 7.35
(d, J ¼ 8.6 Hz, 2H), 6.97 (d, J ¼ 16.2 Hz, 1H), 6.88e6.78 (m, 3H), 6.66
(s, 1H), 6.53 (s, 1H), 6.30 (s, 1H), 1.04e0.96 (m, 18H), 0.79e0.69 (m,
12H). 13C NMR (101 MHz, CD3OD)
d
¼ 158.19, 156.71, 155.24, 139.74,
5.1.2.4. 3-O-tert-butyldimethylsilyl resveratrol, 6. Yield
Rf ¼ 0.2 (Hexane: AcOEt, 7:3).
¼
¼
23%;
23%;
130.77, 128.02, 127.48, 126.55, 119.78, 109.46, 106.28, 106.08, 5.85,
4.68; TOF MS ES þ Calculated mass for C26H40O3Si2 ¼ 456.2, Found
mass [M þ H] ¼ 457.4.
5.1.2.5. 40-O-tert-butyldimethylsilyl resveratrol, 7. Yield
Rf ¼ 0.15 (Hexane: AcOEt, 7:3).
5.1.3.3. 3,5 eO,O- di-triethylsilyl resveratrol, 10. Yield ¼ 7%; Rf ¼ 0.5
(Hexane: Ethyl Acetate e 3:1). 1H NMR (400 MHz, CD3OD)
d
¼ 7.36
5.1.3. Preparation of triethylsilyl resveratrol derivatives (8e12)
(d, J ¼ 8.3 Hz, 2H), 6.96 (d, J ¼ 16.2 Hz, 1H), 6.81 (t, J ¼ 12.6 Hz, 3H),
Following the general procedure and starting from resveratrol
(809 mg, 3.54 mmol) and triethylsilyl chloride (0.92 mL,
5.49 mmol) yielded compounds 8e12 after purification with flash
chromatography whilst eluting with a gradient concentration of
hexane: ethyl acetate (10:1 to 1:1).
6.63 (s, 2H), 6.25 (s, 1H), 1.02 (t, J ¼ 7.9 Hz, 18H), 0.76 (q, J ¼ 7.9 Hz,
12H). 13C NMR (101 MHz, CD3OD)
d
¼ 157.10, 156.53, 140.03, 128.82,
128.65, 127.63, 127.53, 125.15, 115.18, 110.98, 110.07, 5.78, 4.66; TOF
MS ES þ Calculated mass for C26H40O3Si2 ¼ 456.2, Found mass [M þ
H] ¼ 457.4.
5.1.3.1. 3,4’,5 -O,O,O- tri-triethylsilyl resveratrol, 8. Yield ¼ 4%;
Rf ¼ 0.95 (Hexane: Ethyl Acetate e 3:1). 1H NMR (400 MHz, CDCl3)
5.1.3.4. 3-O- triethylsilyl resveratrol, 11. Yield ¼ 9%; Rf ¼ 0.17
d
¼ 7.40 (d, J ¼ 8.5 Hz, 2H), 6.98 (d, J ¼ 16.2 Hz, 1H), 6.90e6.82 (m,
(Hexane: Ethyl Acetate e 3:1). 1H NMR (400 MHz, CD3OD)
d
¼ 7.37
3H), 6.64 (d, J ¼ 2.0 Hz, 2H), 6.31 (t, J ¼ 2.0 Hz, 1H), 1.04 (td, J ¼ 7.8,
(d, J ¼ 8.3 Hz, 2H), 6.98 (d, J ¼ 16.2 Hz, 1H), 6.83 (t, J ¼ 12.0 Hz, 3H),
9

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European Journal of Medicinal Chemistry 223 (2021) 113655
6.65 (s, 1H), 6.53 (s, 1H), 6.28 (s, 1H), 1.02 (t, J ¼ 7.9 Hz, 9H), 0.75 (q,
ES þ Calculated mass [M þ H] ¼ 385.2193, Found mass for
J ¼ 7.9 Hz, 6H). 13C NMR (101 MHz, CD3OD)
d
¼ 158.08, 156.90,
C
23H32O3Si [M þ H] ¼ 385.2181.
156.72, 139.99, 129.04, 128.38, 127.62, 125.51, 115.21, 109.43, 106.11,
105.87, 5.82, 4.66; TOF MS ES- Calculated mass for
5.1.5. General procedure for glycosylation
2,3,4,6-tetra-O-acetylyl- -glucopyranosyl trichloro aceti-
C
20H26O3Si ¼ 342.1, Found mass [M ꢂ H] ¼ 341.1.
a/b-D
midate 29 (1 mmol) was dissolved in anhydrous CH2Cl2 (15 mL) in a
50 mL round-bottomed flask under argon atmosphere. Silylated
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% triethylamine.
5.1.3.5. 40-O- triethylsilyl resveratrol, 12. Yield ¼ 16%; Rf ¼ 0.14
(Hexane: Ethyl Acetate e 3:1). 1H NMR (400 MHz, CD3OD)
d
¼ 7.36
(d, J ¼ 8.3 Hz, 2H), 7.00 (d, J ¼ 16.2 Hz,1H), 6.84 (m, 3H), 6.55 (s, 2H),
6.27 (s, 1H), 0.98 (t, J ¼ 8.0 Hz, 9H), 0.72 (q, J ¼ 7.9 Hz, 6H). 13C NMR
(101 MHz, CD3OD)
d 158.19, 155.20, 139.83, 130.86, 127.84, 127.43,
126.68, 119.79, 104.77, 101.59, 5.76, 4.60; TOF MS ES þ Calculated
mass for C20H26O3Si ¼ 342.1, Found mass [M ꢂ H] ¼ 343.2.
5.1.4. Preparation of triisopropylsilyl resveratrol derivatives
(13e17)
5.1.6. General procedure for acetyl deprotection
Silyl peracetylated glucopyranosyl resveratrol derivative
(0.75 mmol) was dissolved in methanol (10 mL) in a 25 mL round-
bottomed flask. A solution of 25% sodium methoxide in methanol
(0.5 mL) was added slowly and left under stirring at room tem-
perature for 1 h. The solution was filtered first through IR-120Hþ to
neutralize it, filtered through Celite to remove salt excess and
finally concentrated.
Following the general procedure and starting from resveratrol
(816 mg, 3.57 mmol) and triisopropylsilyl chloride (1.19 mL,
5.54 mmol) yielded 13e17 after purification with flash chroma-
tography whilst eluting with a gradient concentration of hexane:
ethyl acetate (10:1 to 1:1).
5.1.4.1. 3,4’,5
eO,O,O-
tri-triisopropylsilyl
resveratrol,
13.
Yield ¼ 19%; Rf ¼ 0.9 (Hexane: Ethyl Acetate e 3:1). 1H NMR
5.1.7. General procedure for esterification
(400 MHz, CDCl3)
d
¼ 7.44 (d, J ¼ 8.3 Hz, 2H), 7.03 (d, J ¼ 16.2 Hz,
Starting material (1 eq.) and vinyl ester (8 eq.) were dissolved in
t-BuOH (10 mL) in a round-bottomed flask. Novozyme® 435 was
then added to the solution (same mass of enzyme than that of
starting material) and reaction was stirred overnight at 55 ꢀC in an
orbital shaker. The reaction mixture was filtered to remove the
enzyme and the crude was purified by flash column chromatog-
raphy using different hexane/ethyl acetate/methanol mixtures.
1H), 6.93 (dd, J ¼ 12.4, 6.4 Hz, 3H), 6.74 (s, 2H), 6.44 (s, 1H),
1.37e1.30 (m, 9H, CHeSi), 1.25e1.13 (m, 54H, CH3). 13C NMR
(101 MHz, CDCl3)
d
¼ 157.08, 155.95, 139.43, 130.44, 128.38, 127.72,
126.82, 120.16, 111.25, 110.93, 18.02, 12.79; TOF MS ES þ Calculated
mass for C41H72O3Si3 ¼ 696.5, Found mass [M þ H] ¼ 697.6.
5.1.4.2. 3,4’
eO,O-
di-triisopropylsilyl
resveratrol,
14.
Yield ¼ 26%; Rf ¼ 0.65 (Hexane: Ethyl Acetate e 3:1). 1H NMR
5.1.8. General procedure for carbamate derivatives synthesis
In a round flask underargon atmosphere, starting material (1 eq.)
was dissolved in anhydrous DCM (10 mL). Triethylamine (2 eq.) and
ethyl isocyanate (1.5 eq.) were added slowly whilst the reaction was
stirred. After TLC verification of the complete reaction of the initial
product, an aqueous solution of NaHCO3 (10% - 50 mL) was added,
and extracted several times with DCM (3 ꢁ 20 mL). The combined
organic phases were concentrated and added to a flash chromato-
graphic column eluting with Hexane: Ethyl Acetate e 10:1.
(400 MHz, CD3OD)
d
¼ 7.34 (d, J ¼ 8.2 Hz, 2H), 6.96 (d, J ¼ 16.2 Hz,
1H), 6.82 (t, J ¼ 12.0 Hz, 3H), 6.64 (s, 1H), 6.54 (s, 1H), 6.33 (s, 1H),
1.28e1.20 (m, 6H), 1.11 (dd, J ¼ 16.0, 7.4 Hz, 36H). 13C NMR
(101 MHz, CD3OD)
d
¼ 158.13, 157.11, 155.67, 139.62, 130.55, 127.97,
127.46,126.56,119.76,109.57,106.15,106.05,17.41,17.37,17.10,12.62,
12.59, 12.33; TOF MS ES þ Calculated mass for C32H52O3Si2 ¼ 540.9,
Found mass [M þ H] ¼ 541.5.
5.1.4.3. 3,5
eO,O-
di-triisopropylsilyl
resveratrol,
15.
Yield ¼ 9%; Rf ¼ 0.5 (Hexane: Ethyl Acetate e 3:1). 1H NMR
5.1.9. Preparation of TES resveratrol prodrugs derivatives (18e22)
TES resveratrol prodrugs derivatives were synthesized following
the general procedure for esterification, using compound 10 and
vinyl esters of different chain length as starting materials.
(400 MHz, CD3OD)
d
¼ 7.37 (d, J ¼ 8.4 Hz, 2H), 6.95 (d, J ¼ 16.2 Hz,
1H), 6.81 (t, J ¼ 12.8 Hz, 3H), 6.64 (d, J ¼ 1.0 Hz, 2H), 6.31 (s, 1H),
1.29e1.22 (m, J ¼ 14.7, 7.1 Hz, 6H), 1.12 (d, J ¼ 7.5 Hz, 36H). 13C NMR
(101 MHz, CD3OD)
d
¼ 157.11, 156.92, 139.86, 128.80, 128.57, 127.63,
125.26, 115.18, 110.85, 110.14, 17.25, 12.58; TOF MS ES- Calculated
5.1.9.1. 3,5
-O,O-ditriethylsilyl-40-propionate
resveratrol,
18.
mass for C32H52O3Si2 ¼ 540.3, Found mass [M ꢂ H] ¼ 539.4.
Yield ¼ 34%; Rf ¼ 0.37 (Hexane:AcOEt 8:1); 1H NMR (400 MHz,
CDCl3)
d
¼ 7.39 (d, J ¼ 8.4 Hz, 2H), 6.95 (d, J ¼ 16.2 Hz, 1H),
5.1.4.4. 3 -O- triisopropylsilyl resveratrol, 16. Yield ¼ 15%; Rf ¼ 0.2
6.85e6.79 (m, 3H), 6.62 (d, J ¼ 1.7 Hz, 2H), 6.29 (s, 1H), 1.02 (t,
(Hexane: Ethyl Acetate e 3:1). 1H NMR (400 MHz, CD3OD)
d
¼ 7.36
J ¼ 7.9 Hz, 18H), 0.94 (t, J ¼ 7.9 Hz, 3H), 0.76 (q, J ¼ 7.9 Hz, 12H), 0.66
(d, J ¼ 8.3 Hz, 2H), 6.97 (d, J ¼ 16.2 Hz, 1H), 6.82 (t, J ¼ 12.7 Hz, 3H),
(td, J ¼ 16.2, 8.2 Hz, 2H). 13C NMR (101 MHz, CDCl3)
d
¼ 156.57,
6.62 (s, 1H), 6.54 (s, 1H), 6.30 (s, 1H), 1.25 (m, 3H), 1.12 (d, J ¼ 7.4 Hz,
155.28, 139.41, 130.21, 128.25, 127.93, 126.62, 115.57, 111.40, 110.94,
29.68, 6.60, 6.54, 5.00, 4.88;
18H); 13C NMR (101 MHz, CD3OD)
d
¼ 158.06, 157.15, 156.92, 139.86,
129.01, 128.30, 127.58, 125.54, 115.19, 109.42, 105.89, 105.83, 17.21,
12.57. TOF MS ES Calculated mass for
23H32O3Si [M þ
H] ¼ 385.2193, Found mass [M þ H] ¼ 385.2182.
þ
C
5.1.9.2. 3,5
-O,O-ditriethylsilyl-40-butanoate
resveratrol,
19.
Yield ¼ 39%; Rf ¼ 0.29 (Hexane:AcOEt 8:1); 1H NMR (400 MHz,
CDCl3)
d
¼ 7.39 (d, J ¼ 8.3 Hz, 2H), 6.95 (d, J ¼ 16.2 Hz, 1H),
5.1.4.5. 4’ -O- triisopropylsilyl resveratrol, 17. Yield ¼ 18%; Rf ¼ 0.1
(Hexane: Ethyl Acetate e 3:1). 1H NMR (400 MHz, CD3OD)
6.85e6.80 (m, 3H), 6.62 (d, J ¼ 1.8 Hz, 2H), 6.29 (s, 1H), 1.72e1.63
(m, 2H), 1.07e0.96 (m, 18H), 0.92e0.84 (m, 3H), 0.77 (q, J ¼ 7.9 Hz,
12H), 0.65 (dt, J ¼ 23.8, 7.9 Hz, 2H). 13C NMR (101 MHz, CDCl3)
d
¼ 7.34
(d, J ¼ 7.9 Hz, 2H), 6.99 (d, J ¼ 16.2 Hz, 1H), 6.87e6.76 (m, 3H), 6.56
(s, 2H), 6.29 (s, 1H), 1.20 (dd, J ¼ 14.6, 7.1 Hz, 3H), 1.07 (d, J ¼ 7.5 Hz,
d
¼ 156.57, 155.33, 139.41, 130.17, 130.14, 128.44, 128.27, 127.92,
18H). 13C NMR (101 MHz, CD3OD)
127.91, 127.45, 126.59, 119.80, 104.86, 101.62, 17.31, 12.56. TOF MS
d
¼ 158.16, 155.66, 139.88, 130.59,
126.58, 115.58, 111.39, 110.93, 29.67, 22.66, 14.08, 6.60, 6.53, 4.99,
4.88.
10

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European Journal of Medicinal Chemistry 223 (2021) 113655
5.1.9.3. 3,5
-O,O-ditriethylsilyl-40-hexanoate
resveratrol,
20.
5.1.10.3. 3,40-O,O- di-triisopropylsilyl-5-glucosyl resveratrol, 23.
Yield ¼ 69%; Rf ¼ 0.4 (DCM). Purification eluent ¼ Hexane: Ethyl
Yield ¼ 66%; Rf ¼ 0.29 (Hexane:AcOEt 8:1); 1H NMR (400 MHz,
CDCl3)
d
¼ 7.39e7.36 (m, 2H), 6.92 (d, J ¼ 16.2 Hz, 1H), 6.84e6.80
Acetate e 1:1 to 1:3.1HNMR (500 MHz, CD3OD)
d
¼ 7.43 (d, J ¼ 8.6 Hz,
(m, 3H), 6.61 (d, J ¼ 2.2 Hz, 2H), 6.28 (t, J ¼ 2.1 Hz, 1H), 2.34 (t,
J ¼ 7.5 Hz, 9H), 1.68e1.60 (m, 9H), 1.02 (t, J ¼ 7.9 Hz, 12H), 0.92e0.86
(m, 15H), 0.77 (q, J ¼ 7.9 Hz, 9H). 13C NMR (101 MHz, CDCl3)
2H), 7.05 (d, J ¼ 16.3 Hz, 1H), 6.98e6.89 (m, 2H), 6.87 (d, J ¼ 8.6 Hz,
2H), 6.70 (s,1H), 6.55 (t, J ¼ 2.0 Hz,1H), 4.92e4.88 (m,1H), 3.94e3.88
(m, 1H), 3.75 (dd, J ¼ 11.9, 4.8 Hz, 1H), 3.52e3.38 (m, 4H), 1.36e1.23
d
¼ 180.26, 156.54, 155.39, 139.41, 130.29, 130.06, 128.27, 127.87,
(m, 6H), 1.18e1.10 (m, 36H). 13C NMR (126 MHz, CD3OD)
d
¼ 158.94,
126.49, 115.56, 115.03, 113.64, 111.35, 110.88, 34.00, 31.15, 29.64,
24.31, 22.23, 13.79, 6.56, 6.49, 4.97, 4.85.
156.93, 155.80, 139.83, 130.51, 128.55, 127.51, 126.04, 119.69, 111.96,
107.30, 107.07, 101.13, 76.86, 76.59, 73.48, 69.90, 61.01, 17.05, 16.99,
12.53, 12.51. TOF MS ES þ Calculated mass for C38H62O8Si2 [M þ
Na] ¼ 725.3881, Found mass [M þ Na] ¼ 725.3862.
5.1.9.4. 3,5
-O,O-ditriethylsilyl-40-octanoate
resveratrol,
21.
Yield ¼ 63%; Rf ¼ 0.26 (Hexane:AcOEt 8:1); 1H NMR (400 MHz,
5.1.10.4. 3,5eO,O-di-triisopropylsilyl-40-glucosyl resveratrol, 24.
Yield ¼ 67%; Rf ¼ 0.1 (Hexane: Ethyl Acetate e 1:4). Purification
eluent ¼ Hexane: Ethyl Acetate e 1:1 to 1:3.1H NMR (500 MHz,
CDCl3)
d
¼ 7.38 (d, J ¼ 8.5 Hz, 2H), 6.94 (d, J ¼ 16.2 Hz, 1H), 6.82 (dd,
J ¼ 12.2, 3.7 Hz, 3H), 6.60 (d, J ¼ 1.9 Hz, 2H), 6.28 (d, J ¼ 1.8 Hz, 1H),
2.35 (t, J ¼ 7.5 Hz, 15H), 1.67e1.60 (m, 15H), 1.01 (t, J ¼ 7.9 Hz, 12H),
0.88 (t, J ¼ 6.6 Hz, 20H), 0.75 (q, J ¼ 7.9 Hz, 8H). 13C NMR (101 MHz,
CD3OD)
d
¼ 7.49 (d, J ¼ 8.7 Hz, 2H), 7.11 (d, J ¼ 8.7 Hz, 1H), 6.97 (q,
J ¼ 16.3 Hz, 3H), 6.68 (d, J ¼ 2.0 Hz, 2H), 6.33 (t, J ¼ 2.1 Hz, 1H),
4.96e4.92 (m, 1H), 3.92 (dd, J ¼ 12.1, 1.9 Hz, 1H), 3.74 (dd, J ¼ 12.1,
5.3 Hz, 1H), 3.51e3.47 (m, 2H), 3.45 (dd, J ¼ 5.0, 1.7 Hz, 1H),
CDCl3)
d
¼ 180.40, 156.56, 155.45, 139.42, 130.30, 130.06, 128.29,
127.88, 126.49, 115.57, 111.35, 110.88, 34.07, 31.59, 28.98, 28.85,
24.64, 22.55, 13.98, 6.58, 4.98.
3.44e3.38 (m, 1H), 1.31e1.23 (m, 6H), 1.14 (d, J ¼ 7.3 Hz, 36H). 13
C
NMR (126 MHz, CD3OD)
d
¼ 157.41, 156.96, 139.63, 131.54, 128.05,
5.1.9.5. 3,5
-O,O-ditriethylsilyl-40-decanoate
resveratrol,
22.
127.37, 126.65, 116.54, 110.93, 110.28, 100.83, 76.75, 76.57, 73.49,
Yield ¼ 90%; Rf ¼ 0.26 (Hexane:AcOEt 8:1); 1H NMR (400 MHz,
69.94, 61.10, 17.05, 12.55. TOF MS ES þ Calculated mass for
CDCl3)
d
¼ 7.37 (d, J ¼ 8.6 Hz, 2H), 6.94 (d, J ¼ 16.2 Hz, 1H), 6.81 (dd,
C
38H62O8Si2 [M
þ
Na]
¼
725.3881, Found mass [M
þ
J ¼ 12.3, 3.8 Hz, 3H), 6.61 (d, J ¼ 2.2 Hz, 2H), 6.28 (t, J ¼ 2.1 Hz, 1H),
Na] ¼ 725.3907.
2.34 (t, J ¼ 7.5 Hz, 14H), 1.63 (dt, J ¼ 14.9, 7.5 Hz, 14H), 1.02 (t,
J ¼ 7.8 Hz, 12H), 0.88 (t, J ¼ 6.9 Hz, 18H), 0.74 (q, J ¼ 7.9 Hz, 9H). 13
C
5.1.10.5. 3,40-O,O-di-triisopropylsilyl-5-(600-octanoyl)glucosyl resver-
atrol, 25. Yield ¼ 35%; Rf ¼ 0.8 (Hexane: Ethyl Acetate e 1:5), pu-
rification eluents ¼ Hexane: Ethyl Acetate ꢂ1:1 to 0:1.1H NMR
NMR (101 MHz, CDCl3)
d
¼ 180.45, 156.55, 155.50, 139.44, 130.01,
128.31, 127.88, 126.45, 115.58, 111.36, 110.87, 34.09, 31.82, 29.35,
29.21, 29.20, 29.02, 24.64, 22.62, 14.03, 6.57, 4.99.
(400 MHz, CDCl3)
d
¼ 7.32 (d, J ¼ 8.5 Hz, 2H), 6.94 (d, J ¼ 16.2 Hz,1H),
6.86e6.79 (m, 3H), 6.77 (d, J ¼ 1.7 Hz, 1H), 6.66 (s, 1H), 6.46 (d,
J ¼ 1.8 Hz,1H), 4.91e4.87 (m, 1H), 4.36 (d, J ¼ 3.9 Hz, 2H), 3.68e3.64
(m, 2H), 3.63e3.60 (m, 1H), 3.49 (dd, J ¼ 11.4, 6.8 Hz, 1H), 2.28 (t,
J ¼ 7.6 Hz, 6H),1.66e1.54 (m, 4H),1.27e1.20 (m, 24H),1.08 (dd, J ¼ 7.2,
2.6 Hz, 20H), 0.85 (t, J ¼ 6.7 Hz, 3H). 13C NMR (101 MHz, CDCl3)
5.1.10. Preparation of TIPS resveratrol prodrugs derivatives (23e28)
TIPS resveratrol prodrugs derivatives 23e26 were synthesized
following the general procedure for glycosylation, acetyl depro-
tection and esterification, using compounds 14 and 15 as starting
materials. Compounds 27 and 28 were obtained following the
general procedure for carbamate preparation.
d
¼ 179.50, 174.30, 158.23, 157.02, 155.96, 139.58, 130.07, 128.83,
127.66, 126.28, 120.06, 112.61, 107.94, 107.40, 100.73, 76.51, 74.07,
73.34, 70.11, 63.35, 45.13, 34.72, 34.01, 31.63, 31.58, 29.65, 29.11,
28.95, 28.91, 28.85, 25.01, 24.77, 22.55, 22.53, 17.90, 17.84, 13.99,
5.1.10.1. 3,40-O,O-di-triisopropylsilyl-5-(2,3,4,6-tetracetylglucosyl)
12.63, 8.34. TOF MS AP- Calculated mass for
C46H76O9Si2
resveratrol, 30. Yield ¼ 68%; Rf ¼ 0.2 (Hexane: Ethyl Acetate e 3:1);
[M ꢂ H] ¼ 827.4950, Found mass [M ꢂ H] ¼ 827.4922.
Purification eluent: DCM. 1H NMR (300 MHz, CDCl3)
d
¼ 7.39 (d,
J ¼ 8.5 Hz, 2H), 7.00 (d, J ¼ 16.2 Hz, 1H), 6.90e6.82 (m, 3H), 6.75 (s,
2H), 6.44 (s, 1H), 5.33 (dd, J ¼ 16.4, 8.2 Hz, 2H), 5.22 (dd, J ¼ 17.9,
8.3 Hz, 1H), 5.11 (d, J ¼ 7.4 Hz, 1H), 4.35 (dd, J ¼ 12.3, 5.2 Hz, 1H),
4.24e4.10 (m, 1H), 3.97e3.84 (m, 1H), 2.08 (t, J ¼ 4.9 Hz, 12H),
1.37e1.22 (m, 6H), 1.14 (dd, J ¼ 6.9, 2.6 Hz, 36H). 13C NMR (75 MHz,
5.1.10.6. 3,5-O,O-di-triisopropylsilyl-4’-(600-octanoyl)glucosyl resver-
atrol, 26. Yield ¼ 40%; Rf ¼ 0.8 (Hexane: Ethyl Acetate e 1:5), pu-
rification eluents ¼ Hexane: Ethyl Acetate ꢂ1:1 to 0:1.1H NMR
(300 MHz, CDCl3)
d
¼ 7.41 (d, J ¼ 8.5 Hz, 2H), 7.04 (d, J ¼ 8.4 Hz, 2H),
6.90 (q, J ¼ 16.2 Hz, 2H), 6.63 (s, 2H), 6.34 (s, 1H), 4.93 (d, J ¼ 6.0 Hz,
1H), 4.38 (q, J ¼ 12.1 Hz, 2H), 3.73 (d, J ¼ 6.6 Hz, 2H), 3.66 (dd,
J ¼ 15.1, 10.1 Hz, 1H), 3.52 (m, 1H), 2.33 (t, J ¼ 7.5 Hz, 3H), 1.28 (dd,
J ¼ 14.4, 6.8 Hz, 18H), 1.14 (d, J ¼ 7.0 Hz, 36H), 0.88 (dd, J ¼ 15.1,
CDCl3)
d
¼ 170.91, 170.52, 169.65, 169.60, 158.25, 157.44, 156.38,
140.13, 130.20, 129.46, 128.00, 126.29, 120.43, 113.33, 107.74, 99.46,
72.99, 72.31, 71.41, 68.52, 62.21, 20.90, 20.85, 18.19, 18.16, 12.92. TOF
MS ES þ Calculated mass for C46H70O12Si2 [M þ H] ¼ 871.4484,
Found mass [M þ H] ¼ 871.4449.
6.8 Hz, 5H). 13C NMR (75 MHz, CDCl3)
d
¼ 180.33, 174.74, 174.12,
157.31, 156.87, 139.28, 132.64, 127.88, 117.16, 111.51, 99.28, 75.56,
74.27, 73.62, 70.87, 63.38, 34.61, 34.35, 31.89, 29.36, 29.28, 29.15,
25.28, 25.13, 24.94, 22.84, 18.17, 14.28, 12.94. TOF MS
ES þ Calculated mass for C46H76O9Si2 [M þ Na] ¼ 851.4926, Found
mass [M þ Na] ¼ 851.4966.
5.1.10.2. 3,5-O,O-di-triisopropylsilyl-4’-(2,3,4,6-tetracetylglucosyl)
resveratrol, 31. Yield ¼ 65%; Rf ¼ 0.3 (Hexane: Ethyl Acetate e 3:1);
Purification eluant: DCM. 1H NMR (300 MHz, CDCl3)
d
¼ 7.47 (d,
J ¼ 8.5 Hz, 1H), 7.04e6.83 (m, 4H), 6.66 (s, 2H), 6.38 (s, 1H),
5.40e5.25 (m, 2H), 5.25e5.09 (m, 2H), 4.34 (dd, J ¼ 12.2, 5.1 Hz,1H),
4.21 (d, J ¼ 12.2 Hz, 1H), 3.96e3.86 (m, 1H), 2.19e2.03 (m, 12H),
1.36e1.22 (m, 6H), 1.15 (d, J ¼ 7.2 Hz, 36H). 13C NMR (101 MHz,
5.1.10.7. 3,40-O,O-di-triisopropylsilyl-5-ethylcarbamoyl resveratrol,
27. Yield ¼ 68%; Rf ¼ 0.4 (Hexane: Ethyl Acetate e 6:1). 1H NMR
(300 MHz, CDCl3)
d
¼ 7.36 (d, J ¼ 8.5 Hz, 2H), 6.99 (d, J ¼ 16.2 Hz,
CD3OD)
d
¼ 170.88, 170.51, 169.70, 169.61, 157.31, 156.55, 139.18,
1H), 6.93e6.80 (m, 5H), 6.57 (s, 1H), 3.40e3.20 (m, 2H), 1.27 (ddd,
133.01, 128.35, 127.96, 127.87, 117.40, 111.53, 111.37, 99.30, 72.98,
72.30, 71.45, 68.55, 20.97, 20.89, 20.86, 20.83, 18.19, 12.93. TOF MS
ES þ Calculated mass for C46H70O12Si2 [M þ H] ¼ 871.4484, Found
mass [M þ H] ¼ 871.4443.
J ¼ 10.6, 7.4, 3.7 Hz, 9H), 1.12 (dd, J ¼ 7.0, 3.1 Hz, 36H). 13C NMR
(75 MHz, CDCl3)
d
¼ 157.05, 156.30, 152.32, 139.77, 130.40, 129.39,
128.02,126.26,120.40, 115.13,112.32, 36.38,18.21,18.19,17.97,15.40,
12.95, 12.93, 12.57.
11

~
E. Belmonte-Reche, P. Penalver, M. Caro-Moreno et al.
European Journal of Medicinal Chemistry 223 (2021) 113655
5.1.10.8. 3,5-O,O-di-triisopropylsilyl-40-ethylcarbamoyl resveratrol,
using the Griess reagent [55]. A minimum of two independent sets
of experiments and three replicates per experiment were carried
out. All data are expressed as mean SEM.
28. Yield ¼ 79%; Rf ¼ 0.3 (Hexane: Ethyl Acetate e 6:1). 1H NMR
(300 MHz, CDCl3)
d
¼ 7.51 (d, J ¼ 8.0 Hz, 2H), 7.15 (d, J ¼ 8.0 Hz, 2H),
7.01 (d, J ¼ 16.4 Hz, 1H), 6.93 (d, J ¼ 16.1 Hz, 1H), 6.67 (s, 2H), 6.38 (s,
1H), 3.41e3.28 (m, 2H), 1.32e1.22 (m, 9H), 1.15 (d, J ¼ 7.2 Hz, 36H).
5.2.6. Zebrafish toxicity assay
13C NMR (75 MHz, CDCl3)
d
¼ 157.31, 157.25, 139.15, 128.14, 128.00,
Fertilized embryos of zebrafish (Danio rerio e strain AB) were
harvested 3 h post fertilization and grouped into wells (20 embryos
per well) in E3 medium with the desired compounds in gradient
concentrations. They were further incubated at 28.5 ꢀC for 93 more
hours and then mortality, median lethal dose, no observed effect
concentration (NOEC) and lowest observed effect concentration
(LOEC) were calculated whilst using as negative controls 0.1% of
DMSO and as positive control 4-diethylaminobenzaldehyde (DEAB)
127.59, 121.99, 116.20, 116.04, 112.17, 111.61, 111.38, 45.95, 36.41,
18.20, 12.95. TOF MS ES þ Calculated mass for C35H57NO4Si2 [M þ
H] ¼ 612.3904, Found mass [M þ H] ¼ 612.3900.
5.2. Biological evaluation
5.2.1. Cell cultures
SH-S5Y5 neurons were cultured in collagen-pretreated petri-
dishes with DMEM-F12 medium supplemented with Penicillin/
Streptomycin and 10% inactivated fetal bovine serum (iFBS). RAW
264.7 macrophages were cultured in DMEM high glucose medium
supplemented with Penicillin/Streptomycin and 10% iFBS.
at 5 different concentrations (from 0.1 mM to 1 mM).
5.2.7. PTZ neuroprotection assay
Fertilized embryos of zebrafish (Danio rerio e strain AB) were
seeded into a Petri dish with 50 mL of dilution water until they
reached larva state (120 h post fertilization). After verification of no
abnormalities, five larvae per well were transferred into a 24 well
plate and ten replicates of each experimental condition were car-
ried out. Two independent experiments were performed. Pre-
treatment: larvae were incubated for 1 h at 26 ꢀC in a volume of
2 mL of dilution water containing 0.1% DMSO (control and
control þ PTZ groups), the positive control group (Phys) included
5.2.2. Cell viability assays
Neuron assays were done in collagen-pretreated 96 well plates
by seeding 2 ꢁ 104 neurons per well in a 100
mL volume and with
24 h of incubation time before compound addition. Macrophage
assays were done in 96 well plates by seeding 2.5 ꢁ 104 macro-
phages per well in a 100 mL volume with 4 h of incubation time
before compound addition. Tested compounds dissolved in DMSO
were then added at different concentrations (1, 10 and 100 M) to
physostigmine (20
examined included 10
ment, media was changed and larvae were incubated for 6 h at
26 ꢀC with Phys (20
M) or each compound (10 M) in combination
mM) and the groups with each compound to be
m
mM of the compound. After this pretreat-
determine compound toxicity. Final DMSO percentage in each cell
was adjusted to 1% DMSO. Cell viability was evaluated 24 h after
compound addition by mitochondrial MTT assay, according to
manufacturer. Averages and standard deviations of at least eight
different readings from various experiments were calculated.
m
m
with a final concentration of 5 mM PTZ. Then, larvae were exam-
ined and their overall status was normal, without visible abnor-
malities and with normal behavior. Finally, in order to determine
the levels of AChE activity, the larvae were mechanically homoge-
nized and the samples centrifuged to obtain the supernatant, which
was then directly tested. AChE activity was measured following
methodology by Ellman et al. [56]. Total protein was quantified
using BCA methodology in order to normalize the obtained enzy-
matic values. Lastly, the negative control values were considered to
be the 100% values. A statistical analysis was carried out using the
Dunnett multiple comparison test (One-way ANOVA) with the
GraphPad Prism program.
5.2.3. Neuroprotective assay
Neurons were cultured and plated as described in the cell
viability assay. Tested compounds dissolved in DMSO were added
at different concentrations (1, 10 and 100
mM) and after 10 min
incubation 100 M of hydrogen peroxide was added. Final DMSO
m
percentage in each cell was adjusted to 1% DMSO. Cell viability was
evaluated 24 h after compound addition by mitochondrial MTT
assay, according to manufacturer. Averages and standard deviations
of at least eight different readings from various experiments were
calculated. Neuron recovery was calculated by normalizing the
results from H2O2-neuron viability to the H2O2 positive control.
5.2.8. Induction of neurodegenerative preclinical models
In order to assay potential neuroprotective effect of resveratrol
derivatives, we performed two in vivo mice models mimicking
neurodegenerative diseases, such as Huntington's disease and
multiple sclerosis.
C57BL/6 mice 12 weeks old were obtained from Charles River
and housed under standard conditions (12 h light/dark cycle) with
access to food and water ad libitum. Animals were handled and
habituated to the experimenters one week before any behavioral
assessment. All experiments with animals were performed in
accordance with the European ethical guidelines and approved by
the Animal Care Unit Committee from the Institute of Parasitology
5.2.4. LPS damage mitigation assay
Macrophages were cultured and plated as described above
Tested compounds dissolved in DMSO were added at different
concentrations (1, 10 and 100
mM) and after 10 min incubation,
100 ng/mL of LPS was added. Final DMSO percentage in each cell
was adjusted to 1% DMSO. Cell viability was evaluated 24 h after
compound addition by mitochondrial MTT assay, according to
manufacturer. Averages and standard deviations of at least eight
different readings from various experiments were calculated.
ꢀ
and Biomedicine Lopez-Neyra - Consejo Superior Investigaciones
5.2.5. Cytokine production studies
Científicas. Procedures were designed to minimize the number of
animals used and their suffering.
To determine cytokine production, 5 ꢁ 105 RAW 264.7 macro-
phages were seeded in 24-well plates (in 0.5 mL). Compounds
(10
or not by adding LPS (1
at the well, 100 ng/mL LPS). After 24 h, levels of IL-6 and TNF-
m
M) were then added and macrophages were either stimulated
g/mL) to the medium (final concentration
in
5.2.8.1. 3-Nitropropionic
3-nitropropionic acid (3-NP, Sigma) was prepared and adminis-
tered as previously indicated with minor modifications [57,58]. 3-
acid
intoxication
model.
m
a
the supernatants were determined by ELISA using capture/bio-
tinylated detection antibodies from BD PharMingen and PrepoTech
[54]. The levels of NO in culture supernatants (at 24 h) were
determined indirectly by measuring the concentration of nitrite
NP was dissolved in distilled water final pH 7.4, filtered (0.22 mm,
Millipore), protected from light and kept at 4 ꢀC until use. Resver-
atrol 1 and compound 26 were dissolved in an aqueous solution
containing
13%
(2-hydroxypropyl)-
b
-cyclodextrin
(average
12

~
E. Belmonte-Reche, P. Penalver, M. Caro-Moreno et al.
European Journal of Medicinal Chemistry 223 (2021) 113655
MW z 1380) and 2% Tween-80.28 animals were grouped as follow
for treatment administration: vehicle (Veh; n ¼ 7), 3-NP (n ¼ 7), 3-
NP þ 1 (n ¼ 7), and 3-NP þ 26 (n ¼ 7). For 3-NP intoxication, an-
imals received twice daily i. p. injections (8:00 a.m.: 6:00 p.m.) 10 h
apart with the next regimen 4 ꢁ 20 mg/kg, 4 ꢁ 40 mg/kg,
2 ꢁ 60 mg/kg (cumulated dose: 360 mg/kg in 5 days) (Fig. S6).
Resveratrol 1 (5.5 mg/kg [5 mM solution]) and compound 26
(20 mg/kg [5 mM solution]) were i. p. injected only and immedi-
ately before the morning dose of 3-NP. All treatments were
phosphate buffer pH 9.0. The plate was washed and blocked with
0.1 M PBS containing 10% fetal bovine serum at room temperature
for 3 h. Then, serum samples or different concentrations of the
recombinant IL-6 were added and incubated overnight at 4 ꢀC,
followed by a 2 h period incubation with biotynilated secondary
antibody at RT. Finally, avidin peroxidase (Sigma) was added fol-
lowed by the ABTS substrate addition at RT, and plate was protected
from light until color development. Three washing buffer (0.1 M
PBS plus 0.05% Tween-20) washes were applied between steps.
Absorbance was obtained in a reader plate at 405 nM, and amount
of IL-6 was calculated from the standard curve.
administered in a volume of z100 mL and always after behavioral
evaluations. For this, mice were weighed, and then a semi-
quantitative motor symptoms evaluation was performed. Behav-
ioral assessments were always carried out just before injections,
except the last one, performed 3 h after the final 3-NP injection
(60 mg/kg). For clinical semi-quantitative motor symptoms
assessment, we used a previously reported motor scale with four
items: global locomotor activity, hindlimb clapsing, hindlimb dys-
tonia and truncal dystonia [57,58]. Each was rated on a three levels
scale of severity (0-absent, 1-slight to moderate and 2-severe)
resulting in a total score ranging from 0 to 8.
5.2.11. qRT-PCR
RNA was isolated from brain tissues from controls and 3-NP
treated mice, using Tripure (Roche) accordingly the manufac-
turer's indications. Total RNA (1
20 L volume using the reagents supplied in the synthesis cDNA kit
(Thermo Scientific RevertAid First Strand K1622, Vilnius, Lithuania).
20 ng cDNA (2 L) were amplified by real-time PCR using SensyFast
mg) was reverse transcribed in a
m
m
and sequences of specific primers for SOD2and GAPDH (Glyceral-
dehyde 3-phosphate dehydrogenase) as housekeeping [see
Table S2]. All PCR reaction were carried out in a Bio-Rad CFX
equipment (Bio-Rad). Thermal cycling profile consisted of a pre-
incubation step at 94 ꢀC for 5 min, followed by 40 cycles of dena-
turation (94 ꢀC, 30 s), annealing (temperature adjusted for each
gene (Table S2), 30 s), and extension (72 ꢀC, 30 s).
5.2.8.2. Induction and treatment of experimental autoimmune
encephalomyelitis (EAE). To induce chronic EAE, female C57BL/6
mice (8 weeks old, Charles River) were immunized subcutaneously
(s.c). with 200
MEVGWYRSPFSRVVHLYRNGK, GeneScript) emulsified in complete
Freund' s adjuvant (CFA) containing 400 g of Mycobacterium
mg of myelin oligodendrocyte protein (MOG35e55,
m
tuberculosis H37 RA (Difco). Mice also received i. p. injections of
200 ng of pertussis toxin (Sigma) on days 0 and 2. Treatment
consisted in the i. p. injection of, vehicle (aqueous solution con-
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
taining
13%
(2-hydroxypropyl)-
b
-cyclodextrin
(average
MW z 1380) with/without 2% Tween-80, EAE controls), Resvera-
trol 1 (20 mg/kg) dissolved in vehicle without tween-80 or com-
pound 26 (20 mg/kg) dissolved in vehicle with 2% Tween-80.
Solutions were prepared fresh daily with sonication and vortex
for 30 min, and kept at 4 ꢀC until used. We distributed 10 animals by
treatment: vehicle (EAE control), vehicle þ Tween-80 (EAE control-
Acknowkedgments
This work was supported by the Junta de Andalucía (FQM-7316)
and Spanish Ministerio de Economía y Competitividad (SAF2017-
85602-R). NA's work was supported by a Juan de la Cierva
Fellowship from the Spanish Ministerio de Economia
Competitividad.
T), resveratrol (EAE
þ
compound 1), compound 26
(EAE þ compound 26), and performed two different regimens of
administration: i) preventive treatment, 15 i. p. injections of vehicle,
RES or compound 26 starting 5 days post-immunization; ii) ther-
apeutic treatment, 10 i. p. injections of vehicle, RES or compound 26
starting at disease onset (12 days post-immunization). Mice were
scored daily for signs of EAE according to the following clinical
scoring system [53]: 0, no clinical signs; 0.5, partial loss of tail
tonicity; 1, complete loss of tail tonicity; 2, flaccid tail and abnormal
gait; 3, hind leg paralysis; 4, hind leg paralysis with hind body
paresis; 5, hind and fore leg paralysis; and 6, death. Disease inci-
dence at the end of the experiment (49 days post-immunization)
was graded as none (no clinical signs), mild (clinical score 0.5e1),
moderate (clinical score 1.5e3), and severe (clinical score 3.5e6).
y
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113655.
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