TRPA1 channels as targets for resveratrol and related stilbenoids

Article abstract of DOI:10.1016/j.bmcl.2015.12.065

A series of twenty resveratrol analogues was synthesized and tested on TRPA1 and TRPV1 channels. None was able to significantly modulate TRPV1 channels. Conversely, most of them exhibited remarkably higher TRPA1 modulating activity than resveratrol. Optimal potency was observed with ortho monoxygenated stilbenes 6 and 17.

Full text of DOI:10.1016/j.bmcl.2015.12.065

Accepted Manuscript
TRPA1 channels as targets for resveratrol and related stilbenoids
Marianna Nalli, Giorgio Ortar, Aniello Schiano Moriello, Enrico Morera,
Vincenzo Di Marzo, Luciano De Petrocellis
PII:
S0960-894X(15)30378-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.12.065
BMCL 23426
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 November 2015
14 December 2015
19 December 2015
Please cite this article as: Nalli, M., Ortar, G., Moriello, A.S., Morera, E., Marzo, V.D., Petrocellis, L.D., TRPA1
channels as targets for resveratrol and related stilbenoids, Bioorganic & Medicinal Chemistry Letters (2015), doi:
http://dx.doi.org/10.1016/j.bmcl.2015.12.065
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Graphical Abstract
TRPA1 channels as targets for resveratrol
and related stilbenoids
Leave this area blank for abstract info.
Marianna Nalli,* Giorgio Ortar, Aniello Schiano Moriello, Enrico Morera, Vincenzo Di Marzo, Luciano De
Petrocellis*

Bioorganic & Medicinal Chemistry Letters
TRPA1 channels as targets for resveratrol and related stilbenoids
Marianna Nallia,*, Giorgio Ortara, Aniello Schiano Moriellob, Enrico Moreraa, Vincenzo Di Marzob,
Luciano De Petrocellisb,*
aDipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, piazzale Aldo Moro 5, 00185 Roma, Italy
bEndocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, via dei Campi Flegrei 34, 80078, Pozzuoli (Napoli),
Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
A series of twenty resveratrol analogues was synthesized and tested on TRPA1 and TRPV1
channels. None was able to significantly modulate TRPV1 channels. Conversely, most of them
exhibited remarkably higher TRPA1 modulating activity than resveratrol. Optimal potency was
observed with ortho monoxygenated stilbenes 6 and 17.
Accepted
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
TRP channels
TRPA1
TRPV1
Resveratrol
Stilbenoids
The polyphenol resveratrol [(E)-3,4',5-trihydroxystilbene]
(Figure 1) is a phytoalexin produced in a wide range of plants in
response to environmental stress or pathogenic attack and has
been detected in various food sources such as grapes, berries,
chocolate, and peanuts.1 Over the last decade, about 3000 articles
(PubMed, November 2015) concerning health benefits in humans
of resveratrol and related stilbenoids (in particular, pinostilbene
and pterostilbene) have been published. The chemopreventive
properties of resveratrol include the cardiovascular system,
inflammation, cancer development, aging processes, diabetes, and
cerebral functions2 and are ascribed to its antioxidant activity and
ability to interfere with multiple signalling pathways
(inflammatory mediators, transcription factors, cell cycle
regulatory genes, angiogenic and apoptotic genes, antioxidant
enzymes, protein kinases).3 The detailed mechanisms by which
resveratrol exerts its effects are however still unclear.
The transient receptor potential (TRP) channels ankyrin type-1
(TRPA1) and vanilloid type-1 (TRPV1) are two prominent
members of the TRP family of structurally related non-selective
cation channels4 that play numerous roles in many physiological
and pathophysiological processes.5 In particular, TRPA1 and
TRPV1 are involved in the perception of nociceptive and
inflammatory pain via sensory nerve activation.5c,d,k Accordingly,
the possibility that antinociceptive, antioxidant, and anti-
inflammatory properties of resveratrol could derive, at least in
part, from a TRPA1 and/or TRPV1 inhibitory activity has been
_____
Figure 1. Structures of resveratrol and some related stilbenoids.
recently investigated by Yu et al.6 Resveratrol was found indeed
to suppress the allyl isothiocyanate (AITC)-induced currents in
mTRPA1-transfected HEK293 cells with an IC50 value of
approximately 0.75 M. AITC, the pungent component of
wasabi, horseradish, and mustard oil, is probably the best
recognized TRPA1 agonist.5a-c,h It covalently binds to cysteine
residues in the cytoplasmatic N-terminal domain of TRPA1 and
opens the channel, increasing intracellular Ca2+ concentrations
and ultimately leading to a noxious or nociceptive response. In
contrast to TRPA1, the TRPV1 channel was not inhibited
significantly by resveratrol. Interestingly, and rather surprisingly,
an opposite behaviour was exhibited by pinosylvin methyl ether.
Since previous SAR studies have shown that some resveratrol
analogues exhibited enhanced antimicrobial, antioxidant, and
cytotoxic activities,7 we have now prepared 20 resveratrol
analogues (compounds 1-20) and examined their functional
activity at TRPA1 and TRPV1 channels, with the aim of
identifying more potent TRPA1 and/or TRPV1 modulators based on
the resveratrol structure. The compounds differed by the number
* Corresponding authors. Tel.: +39 6 49913647; fax: +39 6
49913133 (M.N.); tel.: +39 081 8675173 (L.D.P.).
E-mail addresses: : marianna.nalli@uniroma1.it (M. Nalli),
l.depetrocellis@icb.cnr.it (L. De Petrocellis).

and position of hydroxy and/or methoxy or acetyloxy groups and
were synthesized using standard chemical methodologies7b,8,9
(resveratrol was obtained from a commercial supplier and was of
analytical grade). In detail, stilbenoids 4, 6, 7, 9-17 were
synthesized by a Mizoroki-Heck coupling reaction between
appropriate iodoarenes 21 and styrenes 22 (Scheme 1).7c,8c Non-
commercially available 3,5-dimethoxystyrene was obtained by a
Wittig
reaction
of
3,5-dimethoxybenzaldehyde
and
triphenylphosphonium iodide and t-BuOK.9 Preparation of
hydroxystilbenes 1, 2, 3, 5, 8 was accomplished by BBr3-
promoted demethylation of the corresponding methoxystilbenes
12, 9, 14, 16, 12, respectively (Scheme 2).8e Finally, acetylation
of resveratrol and of hydroxystilbenes 4, 6 with acetyl chloride
afforded acetyloxystilbenes 18, 19, 20 (Scheme 3).8a,b,d IR and
NMR data of compounds 1-20 were consistent with those
reported in the literature.10
Scheme 1. Synthesis of compounds 4, 6, 7, 9-17. Reagents and conditions:
(a) Pd(OAc)2, N(CH2CH2OH)3, DMF, 100 °C, 1 h (42-67%).
Resveratrol and the 20 stilbenoids synthesized here were
tested for their ability to induce intracellular Ca2+ elevation in
HEK293 cells stably transfected with the rat TRPA1, or the
human TRPV1 cDNAs.11 The antagonist or desensitizing activity
was assessed by adding the test compounds 5 min before
stimulation of cells with reference agonists AITC and capsaicin.11
As concerns TRP assays on TRPV1-HEK293 cells, neither
resveratrol nor any of 20 analogues 1-20 were able to
significantly modulate this channel (efficacies and IC50 values
were invariably <10% and >10 M, respectively). Pinosylvin
methyl ether (8) was reported by Yu et al.6 to significantly
suppress TRPV1 activity both in heterologous HEK293 cells and
DRG neurons. Actually, however, an IC50 in the range 15 and 30
M could be inferred from their bars graph showing the
suppressive effect of pinosylvin methyl ether on 20 nM
capsaicin-induced currents in TRPV1-HEK293 cells, a value
comparable with that observed by us (23.7 M, Figure 2).
On the contrary, rat TRPA1-HEK293 cells exhibited a sharp
Scheme 2. Synthesis of compounds 1, 2, 3, 5, 8. Reagents and conditions: (a)
BBr3, CH2Cl2, 0 °C, 1 h (26-37%).
Scheme 3. Synthesis of compounds 18-20. Reagents and conditions: (a)
AcCl, pyridine, CH2Cl2, rt, 1 h (64-73%).
Table 1. Results of the TRPA1 assays of resveratrol and related stilbenoids 1-20a
TRPA1
TRPA1
TRPA1
TRPA1
TRPA1
TRPA1
Compound
resveratrol
Compound
(efficacy)b (EC50, M) (IC50, M)c
(efficacy)b (EC50, M) (IC50, M)c
<10
<10
73.4
40.0
61.9
77.4
88.4
71.1
71.4
41.8
72.1
ND
ND
13.5
11.0
8.4
19.9
12.1
16.5
25.2
5.5
72.5
84.1
1.7
1.7
3.0
3.4
11
12
13
14
15
16
17
18
19
20
1
(pinosylvin)
75.0
1.5
2.9
2
3
4
5
6
86.7
4.2
10.6
17.8
2.3
49.0
2.4
1.8
3.4
101.2
83.4
1.7
0.58
3.6
2.2
0.88
7.5
1.7
7
7.5
127.1
89.7
11.8
29.4
8.6
(pterostilbene)
8
(pinosylvin
methyl ether)
3.5
6.9
16.3
7.2
6.6
10.1
10.0
135.1
9
10.1
10
aData are means of at least 3 separate determinations. Standard errors are not shown for the sake of clarity and were never higher than 10% of the means. bAs percent
of allyl isothiocyanate (100 M). cDetermined against the effect of allyl isothiocyanate (100 M).

desensitization), while 15 behaved as a weakly desensitizing
agonist. During preparation of this manuscript, short
a
communication highlighting the ability of pinosylvin to inhibit
TRPA1-induced calcium influx in hTRPA1-HEK293 cells with
an IC50 value slightly higher than that presented here (26.5 vs
12.1 M) appeared online.12
As mentioned initially, the inhibition of AITC-induced
currents by resveratrol was reported by Yu et al. to appear from
low concentration with an IC50 value of approximately 0.75 M,
a distinctly lower value than ours (19.9 M, Figure 3). Yu et al.
measured however an EC50 of 61.2 and 61.7 M for AITC and
AITC with resveratrol, respectively, while the corresponding
values recorded by us were 1.40 and 2.74 M, respectively
(Figure 4). Clearly, under experimental conditions in which
agonists exhibit lower potency, stronger inhibitory effects with
antagonists and/or desensitizing agents will be observed.
The TRPA1 modulating activity of resveratrol and
hydroxystilbenes 1-6 exhibited an inverse relationship with the
number of hydroxyl groups, being the monohydroxystilbenes 4-6
the most active ones. The position of the OH group was also
important for activity, with the ortho isomer 6 endowed with a
submicromolar activating potency. By contrast, the number of
MeO groups did not influence appreciably the activity of
methoxystilbenes 11-17, but, again, the ortho isomer (compound
17) was the most active member of the series.
Figure 2. Dose-response curve showing inhibition of capsaicin-induced
hTRPV1 intracellular Ca2+ elevation by pinosylvin methyl ether (PME).
Overall, methoxystilbenes exhibited
a superiority over
hydroxystilbenes and stilbenes containing both hydroxy and
methoxy groups (compare resveratrol with compounds 7 and 11,
1 with 8 and 12, 2 with 9 and 13, 3 with 10 and 14).
Monohydroxystilbenes were however roughly equipotent with
the corresponding monomethoxystilbenes. It has to be noted that,
in contrast to the study by Yu et al.,6 pinosylvin methyl ether (8)
showed here a significant effect on TRPA1 channels, a difference
that might be attributed to the use of different cell types (TRPA1-
HEK293 cells vs DRG neurons). While DRG neurons provide
one of the best 'in vitro' model systems for the study of
nociceptors, heterologous cell lines such as HEK293 cells
represent an additional and widespread model. Often, the two
systems are found to exhibit response profiles for TRPA1 very
similar,13 but important differences between them have been also
observed14 as a consequence of the overlapping expression of
TRPA1 and TRPV1 in DRG neurons, with a marked dependence
of the relative expression pattern upon the target tissue.15 This is
why we preferred to investigate the effects of our compounds on
TRPA1-HEK293 cells and not on DRG neurons that
heterogeneously express different TRP channels, which may
even cross-desensitize each other. This is, of course, a critical
point, as the identification of a suitable 'in vitro' screening will
have far-reaching effects on the future preclinical development.
Derivatization of the hydroxystilbenes with acetyl groups
proved to be detrimental to the TRPA1 modulating activity, as
the acetyloxystilbenes 18-20 were generally less potent than the
corresponding hydroxy- and methoxystilbenes.
As concerns the mechanism of action of compounds 1-20, Yu
et al.6 suggested that a covalent modification of cysteine residues
was not involved in the resveratrol-induced inhibition of
TRPA1.5h Given their non-electrophilic nature and on the basis of
SAR analysis, the binding to a hydrophobic pocket of TRPA1
channel via a non-covalent mechanism has been proposed for
other phenol-derived compounds (eugenol, gingerol, some alkyl-
substituted phenols) as well.5h When one of our two most potent
TRPA1 modulators (compound 6) was treated with the
biologically relevant model thiol cysteamine16 in DMSO, no
reaction was observed within 72 h.17 We reasonably assume
therefore that the activities observed for all resveratrol analogues
presented in Table 1 might result from a mechanism of action
Figure 3. Dose-response curve showing inhibition of AITC-induced rTRPA1
intracellular Ca2+ elevation by resveratrol.
Figure 4. Dose-response curves of AITC in the absence (triangles) and
presence (squares) of resveratrol.
increase in intracellular Ca2+ upon application of 19 out of the 21
compounds examined (Table 1). Only resveratrol and pinosylvin
(compound 1) were devoid of rat TRPA1 activating ability. Seven
of the stilbenoids 2-20, that is, 5, 6, 11-13, 16, 17 produced a
robust activation of TRPA1 in transfected cells with EC50 values
< 2 M.
Five min preincubation of TRPA1-HEK293 cells with
resveratrol and 20 analogues, and then continued incubation with
AITC caused inhibition of TRPA1 response to this agonist with
IC50 values between 1.7 M and 29.4 M (Table 1). The EC50
and IC50 values were, with the exception of resveratrol,
pinosylvin (1) and, partially, 15, comparable and thus, only
resveratrol and pinosylvin acted as 'true' antagonists (that is,
inhibition without agonism per se, and hence not due to

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similar to that hypothesized for resveratrol and other phenol-
derived compounds.
TRPA1 channels have been validated as a promising target for
various potential therapeutic applications including neuropathic
and inflammatory pain and airway disorders.5c,k Pre-
administration of resveratrol or of pinosylvin has been reported
by Yu et al.6 and by Moilanen et al.12 to suppress intraplantar
injections of AITC-evoked nocifensive behaviours in rats or
mice, and a mixture of TRPA1 modulators including resveratrol
was found to prevent, improve, or treat respiratory failure
induced by defect in oxygen sensing mechanism.18 The
identification of resveratrol analogues 5, 6, 11, 12, 13, 16, and 17
as more potent TRPA1 modulators than resveratrol itself should
therefore designate them as useful pharmacological tools in
preclinical rodent models for the evaluation of TRPA1 as a target
for the treatment of inflammatory and neuropathic pain.
8.
9.
10. General procedure for the synthesis of resveratrol analogues 4, 6,
7, 9-17. A mixture of the appropriate iodoarene 21 (1 mmol), the
appropriate styrene 22 (1 mmol), triethanolamine (0.13 mL, 1
mmol), and Pd(OAc)2 (9 mg, 0.04 mmol) was stirred at 100 °C for
1 h under nitrogen. The reaction mixture was then cooled to room
temperature, diluted with 2 N HCl and extracted with AcOEt. The
organic phase was washed with saturated NaHCO3 and brine,
dried (Na2SO4), and evaporated under vacuum. The residue was
purified by column chromatography (silica gel, petroleum
ether/AcOEt mixtures). General procedure for the synthesis of
resveratrol analogues 1-3, 5, 8. A 1 M solution of BBr3 in CH2Cl2
Acknowledgements
This research was supported by Sapienza Università di Roma
(grant n. C26A14TLFT).
(3-6 mmol) was added dropwise to
a stirred solution of
methoxystilbene 12 (or 9, 14, 16) (1 mmol) in CH2Cl2 (10 mL) at
0 °C. The mixture allowed to warm at room temperature and
stirred at room temperature for 30 min. The mixture was diluted
with ice-water and extracted with AcOEt. The organic phase was
washed with brine, dried (Na2SO4), and evaporated under vacuum.
In the case of 12, a 1:3 mixture of 1 and 8 was obtained. The
residue was purified by column chromatography (silica gel,
petroleum ether/AcOEt mixtures). General procedure for the
synthesis of resveratrol analogues 18-20. AcCl (4 mmol) was
added dropwise to a stirred solution of resveratrol (or 4, 6) (1
mmol) and pyridine (0.32 mL, 4 mmol) in CH2Cl2 (10 mL) at
room temperature. The mixture was stirred at room temperature
for 1 h. The mixture was diluted with ice-water and extracted with
AcOEt. The organic phase was washed with 2N HCl, saturated
NaHCO3, and brine, dried (Na2SO4), and evaporated under
vacuum. The residue was purified by column chromatography
(silica gel, petroleum ether/AcOEt mixtures).
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11. TRPA1 and TRPV1 channel assays. HEK293 (human embryonic
kidney) cells stably over-expressing recombinant rat TRPA1 or
human TRPV1 were grown on 100 mm diameter Petri dishes as
mono-layers in minimum essential medium (EMEM)
supplemented with non-essential amino acids, 10% foetal bovine
serum, and 2 mM glutamine, and maintained at 5% CO2 at 37 °C.
Stable expression of each channel was checked by quantitative
PCR (data not shown). The effect of the substances on
intracellular Ca2+ concentration ([Ca2+]i) was determined by using
Fluo-4, a selective intracellular fluorescent probe for Ca2+. On the
day of the experiment, cells were loaded for 1 h at room
temperature with the methyl ester Fluo-4-AM (4 M in dimethyl
sulfoxide containing 0.02% Pluronic F-127, Invitrogen) in EMEM
without foetal bovine serum, then were washed twice in Tyrode’s
buffer (145 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl2, 1.2 mM
MgCl2, 10 mM D-glucose, and 10 mM HEPES, pH 7.4),
resuspended in the same buffer, and transferred (about 100000
cells) to the quartz cuvette of the spectrofluorimeter (Perkin-Elmer
LS50B equipped with PTP-1 Fluorescence Peltier System;
PerkinElmer Life and Analytical Sciences, Waltham, MA, USA)
under continuous stirring. The changes in [Ca2+]i were determined
before and after the addition of various concentrations of test
6.
7.
Yu, L.; Wang, S.; Kogure, Y.; Yamamoto, S.; Noguchi, K.; Dai,
Y. Molecular Pain 2013, 9, 3. An inhibitory action of resveratrol
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compounds by measuring cell fluorescence (EX = 488 nm, EM
=
516 nm) at 25 °C. Curve fitting (sigmoidal dose-response variable
slope) and parameter estimation were performed with GraphPad
Prism® (GraphPad Software Inc., San Diego, CA). Potency was
expressed as the concentration of test substances exerting a half-
maximal agonist effect (i.e. half-maximal increases in [Ca]i)
(EC50). The effects of TRPA1 agonists are expressed as a
percentage of the effect obtained with 100 M allyl isothiocyanate
(AITC). In the case of TRPV1 assays, the efficacy of the agonists
was first determined by normalizing their effect to the maximum
Ca2+ influx effect on [Ca2+]i observed with application of 4 μM

ionomycin (Alexis). When significant, the values of the effect on
[Ca2+
in wild-type (i.e. not transfected with any construct)
]
i
HEK293 cells were taken as baseline and subtracted from the
values obtained from transfected cells. Antagonist/desensitizing
behaviour was evaluated against AITC (100 μM) for TRPA1, and
capsaicin (0.1 M) for TRPV1, by adding the test compounds in
the quartz cuvette 5 min before stimulation of cells with agonists.
Data are expressed as the concentration exerting a half-maximal
inhibition of agonist-induced [Ca2+]i elevation (IC50), which was
calculated again using GraphPad Prism® software. The effect on
[Ca2+
] exerted by agonist alone was taken as 100%. Dose
i
response curves were fitted by a sigmoidal regression with
variable slope. All determinations were performed at least in
triplicate. Statistical analysis of the data was performed by
analysis of variance at each point using ANOVA followed by the
Bonferroni’s test.
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was monitored periodically by 1H NMR.
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