Synthesis of resveratrol derivatives as new analgesic drugs through desensitization of the TRPA1 receptor

Article abstract of DOI:10.1016/j.bmcl.2017.05.025

A series of 31 resveratrol derivatives was designed, synthesized and evaluated for activation and inhibition of the TRPA1 channel. Most acted as activators and desensitizers of TRPA1 channels like resveratrol or allyl isothiocyanate (AITC). Compound 4z (HUHS029) exhibited higher inhibitory activity than resveratrol with an IC₅₀ value of 16.1?μM. The activity of 4z on TRPA1 was confirmed in TRPA1-expressing HEK293 cells, as well as in rat dorsal root ganglia neurons by a whole cell patch clamp recording. Furthermore, pretreatment with 4z exhibited an analgesic effect on AITC-evoked TRPA1-related pain behavior in vivo.

Full text of DOI:10.1016/j.bmcl.2017.05.025

Accepted Manuscript
Synthesis of Resveratrol Derivatives as New Analgesic Drugs Through Desen-
sitization of the TRPA1 receptor
Syuhei Nakao, Miyuki Mabuchi, Shenglan Wang, Yoko Kogure, Tadashi
Shimizu, Koichi Noguchi, Akito Tanaka, Yi Dai
PII:
S0960-894X(17)30506-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.05.025
BMCL 24970
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 March 2017
2 May 2017
8 May 2017
Please cite this article as: Nakao, S., Mabuchi, M., Wang, S., Kogure, Y., Shimizu, T., Noguchi, K., Tanaka, A.,
Dai, Y., Synthesis of Resveratrol Derivatives as New Analgesic Drugs Through Desensitization of the TRPA1
receptor, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.05.025
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Synthesis of Resveratrol Derivatives as New
Analgesic Drugs Through Desensitization of
the TRPA1 receptor
Syuhei Nakao, Miyuki Mabuchi, Shenglan Wang, Yoko Kogure, Tadashi Shimizu, Koichi Noguchi, Akito
Tanaka, and Yi Dai
Pretreatment of HUHS029
OH
Ring A
HO
suppresses AITC-induced
nocifensive pain behavior
in rats.
OH
Ring B
Br
OH
HO
Novel TRPA1 desesitizer
HUHS029 (4z)
Resveratrol (1)
Br
HUHS029 (4z)
allyl isothiocyanate (AITC)

Bioorganic & Medicinal Chemistry Letters
Synthesis of Resveratrol Derivatives as New Analgesic Drugs Through
Desensitization of the TRPA1 receptor
Syuhei Nakaoa,b, Miyuki Mabuchia, Shenglan Wangb,c, Yoko Kogureb, Tadashi Shimizua,b, Koichi
Noguchid, Akito Tanakaa,b *, and Yi Daib,c*
a Advanced medical research center, Hyogo University of Health Sciences, 1-3-6 Minatojima, KOBE 650-8530, JAPAN.
b School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, KOBE 650-8530, JAPAN.
c Traditional Medicine Research Center, Chinese Medicine Confucius Institute at Hyogo College of Medicine, KOBE 650-8530, JAPAN.
d Department of Anatomy and Neuroscience, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, JAPAN.
*Corresponding authors, Authors A.T. and Y. D. contributed equivalently to this work.
E-mail: tanaa-a@huhs.ac.jp, tel: +81-78-304-3025, fax: +81-78-304-2767.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of 31 resveratrol derivatives was designed, synthesized and evaluated for activation and
inhibition of the TRPA1 channel. Most acted as activators and desensitizers of TRPA1 channels
like resveratrol or allyl isothiocyanate (AITC). Compound 4z (HUHS029) exhibited higher
inhibitory activity than resveratrol with an IC50 value of 16.1 M. The activity of 4z on TRPA1
was confirmed in TRPA1-expressing HEK293 cells, as well as in rat dorsal root ganglia neurons
by a whole cell patch clamp assay. Furthermore, pretreatment with 4z exhibited an analgesic
effect on AITC-evoked TRPA1-related pain behavior in vivo.
Keywords:
TRPA1
Resveratrol
Analgesic Drug
HUHS029
2009 Elsevier Ltd. All rights reserved.
Resveratrol (1) is a natural phenol and phytoalexin that
consists of two aromatic rings (A and B rings) attached by an
ethenyl moiety (Figure 1). Resveratrol was first isolated from the
roots of Polygonum cuspidatum, a plant used in traditional
Chinese medicine1, and has been reported to have beneficial
effects on human health such as anti-inflammatory, antioxidant,
TRPA1 is a critical molecule for the detection and modulation of
pain sensations.
In our previous report, resveratrol concentration-dependently
suppressed the AITC-induced currents (IAITC) in HEK293 cells
that express TRPA1 with an IC50 value of approximately 0.75
M9. Nalli et al. have reported simple modifications of
resveratrol and the activities of the resulting analogues against
TRPA110. To further explore resveratrol analogues with better
bioactivity against TRPA1, we designed, synthesized, and
evaluated in detail 31 resveratrol derivatives for their modulatory
effect on the TRPA1 channel.
anti-tumor,
anti-aging,
cardiovascular
protective
and
chemoprotective bioactivity2. In addition, analgesic properties of
resveratrol against pain triggered by nocifensive stimuli,
inflammation, or nerve injury have been demonstrated3. The
molecular target of resveratrol had been elusive until our group
discovered that resveratrol modulates transient receptor potential
A1 (TRPA1) channels4.
OH
OH
Ring A
HO
Br
TRPA1 is expressed by a subset of small-sized dorsal root
ganglia (DRG) or trigeminal ganglia neurons5. In addition to
cold, many noxious compounds including allyl isothiocyanate
(AITC), cinnamaldehyde, cannabinoids, and allicin activate
TRPA16. TRPA1 is also activated by endogenous molecules
such as bradykinin, intracellular alkalization, and 4-
hydroxynonenal7. Pharmacological or genetic block of the
channels has demonstrated that TRPA1 is an important
component of the transduction machinery that elicits
inflammatory and neuropathic pain8. This evidence indicates that
HO
Ring B
HUHS029 (4z)
Br
OH
TRPA1 desensitizer
Resveratrol (1)
N
C
S
OR
Previously reported 1 derivatives with
inhibitory activiy against TRPA110)
Known TRPA1 activator
AITC (Allyl isothiocyanate) (2)
R=H (3a), Me (3b) :
IC50(TRPA1) = 1.7 - 2.2 M
Figure 1. Structures of resveratrol (1), AITC (2), and previously reported
TRPA1 inhibitors (3).

Resveratrol derivatives (4a-4ee) were synthesized according
to methods described in previous papers11 (Scheme 1 - 4).
Compounds 4a-4f were directly synthesized from resveratrol (1),
and amide derivatives (4g-4h) were obtained by amide coupling
reactions of the corresponding amines and carboxylic acids
HO
R''
AcO
COCl
a
+
R''
OH
OAc
4ff: R''=tBu
4gg: R''=cHex
9a: R''=tBu for 4ff
9b: R''=cHex for 4gg
7b
OH
(Scheme 1).
Compounds 4i-4n were obtained from the
Br
MeO
B(OH)2
b
HO
corresponding Wittig reagents (5) and benzaldehydes (6)
(Scheme 2). Compound 4n was similarly prepared by a Wittig-
Horner reaction. Synthesis of compounds 3b and 4o-4ee is
shown in Scheme 3. Coupling of acid chlorides (7) with
substituted styrenes (8) was carried out using a palladium-
catalyzed decarbonylative Heck reaction with continuous
demethylation with BBr3 or deacetylation with aqueous solution
of NaOH to give 4o-4ee. Compounds 4ff-4gg were also prepared
by the palladium-catalyzed decarbonylative Heck coupling, and
biphenyl derivative 4hh was obtained by a palladium-catalyzed
Suzuki coupling (Scheme 4).
+
4hh
OMe
OMe
OH
Scheme 4. Synthesis of compounds 4ff-4hh.
Reagents and conditions: (a) (i) Pd(OAc)2, N-ethylmorpholine, (ii) 1N
NaOH, (b) (i) Pd(PPh3)4, Na2CO3, (ii) BBr3
We evaluated compounds for their inhibitory activity of the
TRPA1 receptor by the increase in concentration of free Ca2+ in
HEK293 cells expressing the hTRPA1 receptor after stimulation
with AITC using the Calcium KitⅡ- Fluo4 (Table 1).
Desensitization of receptors is a fundamental approach for
reducing TRPA1 channel activation. Therefore, the effects of
pretreatment with the compounds in TRPA1-expressing HEK293
cells on calcium influx, an agonistic effect, were also examined
(Table 1). As shown in Table 1, all compounds almost equally
stimulated TRPA1 channel (relative activity: 0.3-0.7) and among
of those that had inhibitory activity, they might be desensitizers
such as resveratrol against AITC. In this study, all compounds,
including resveratrol (1), the previously reported resveratrol
derivative (3a), and 4a-4ii, were assessed first at 30 M (Table 1
and 2). Compound 3a10 showed 1.1 times more potent inhibitory
activity than resveratrol. Inhibitory activities were reduced by
acetylation or alkylations of the phenolic hydroxyl groups (4a-
4e). Modification of the hydroxyl group of ring B of 1 (4p-4o,
4m-4q, 4w-4bb) also decreased the activities. Among them,
compound 4x, bearing a hydroxyl group at the 3 position, showed
R'
a
R
1(Resveratrol)
4a: R=3,5-di-AcO R'=4-AcO
4b: R=3,5-di-MeO, R'=4-MeO
4c: R=3,5-di-EtO, R'=4-EtO
4d: R=3,5-di-nPrO, R'=4-nPrO
4e: R=3,5-di-MOMO, R'=4-MOMO
b
OH
HO
4f
OH
R''
O
MOMO
COOH
+
c
HO
X
N
H
H2
N
OMOM
4g: R''=OH
4h: R''=OMe
OH
X=OMOM for 4g
=OMe for 4h
Scheme 1. Synthesis of compounds 4a - 4h.
Reagents and conditions: (a) Ac2O, Py for 4a, MeI, K2CO3 for 4b, EtBr,
KI, K2CO3 for 4c, nPrI, K2CO3 for 4d, and MeOCH2-Cl (MOM-Cl), DIPEA
for 4e, (b) H2/Pd-C, (c) (i) EDC hydrochloride, DMAP, (ii) 4 N HCl/AcOEt.
relatively potent inhibitory activity.
Replacement of the
X-
hydroxyl group of 1 on ring B with a chlorine atom maintained
activity (4q), which indicated the hydroxyl group was not vital
for the inhibition. Replacement of the hydroxyl groups with
chlorine atoms was therefore carried out on ring A as well (4i-
4k). Compounds 4i and 4j, bearing 3,5-dichloro atoms on ring
A, exhibited relatively potent inhibitory activity as expected.
Addition of a hydroxyl group on ring B (4ee), methylation of the
hydroxyl group of 3a, and removal of a hydroxyl group of ring A
of 1 decreased inhibitory activities. The compound bearing 3,4-
dihydroxy groups on ring A (4y) had diminished inhibitory
activity. Replacement of the 3,5-dihydroxyphenyl of 1 with 3,5,-
dibromo-4-hydroxyphenyl (4z) resulted in 1.4 times increased the
inhibitory activity, while replacement of the 4-hydroxyphenyl
with a 3,5-dibromophenyl moiety (4aa) did not affect the
activity. Replacement of the 2-hydroxy group of 3a with a 4-
hydroxy
R'
a
PPh3
R
R
CHO
5
4i: R=3,5-di-Cl, R'=4-Cl
4j: R=3,5-di-Cl, R'=H
4k: R=H, R'=4-Cl
6
R'
4l: R=H, R'=3,5-di-MeOH
Br-
b
c
MOMO
HO
PPh3
CHO
4m
OMe
OMe
MeO
OMOM
b
PO(OEt)2
MOMO
HO
CHO
OH
4n
OMOM
Scheme 2. Synthesis of compounds 4i - 4n..
Reagents and conditions: (a) n-BuLi (X=Br) for 4i, n-BuLi (X=Cl) for 4j,
n-BuLi (X=Br) for 4k, n-BuLi (X=Cl) for 4l, (b) n-BuLi, (c) 4 N HCl/AcOEt.
COCl
R'
a
R
+
R'
R
3b: R=H, R'=2-MeO
4o: R=3,5-di-HO, R'=4-MeO
4p: R=3,5-di-HO, R'=H
4q: R=3,5-di-HO, R'=4-Cl
4r: R=3,5-di-HO, R'=4-EtO
4s: R=3,5-di-HO, R'=4-nPrO
4t: R=3,5-di-HO, R'=4-nHexO
4u: R=3,5-di-HO, R'=4-nHexCH2O
4v: R=3,5-di-HO, R'=4-BzlO
4w: R=3,5-di-HO, R'=2-HO
4x: R=3,5-di-HO, R'=3-HO
7a: R=H for 3b
8a: R'=4-MeO for 4o
7b: R=3,5-di-AcO for 4o-x, 4bb-4ee
8b: R'=H for 4p
7c: R=3,4-di-MeO for 4y
8c: R'=4-Cl for 4q
7d: R'=3,5-di-Br-4-MeO for 4z, 4aa
8d: R'=4-EtO for 4r
8e: R'=4-nPrO for 4s
8f: R'=4-nHexO for 4t
8g: R'=4-cHexCH2O for 4u
8h: R'=4-BzlO for 4v
8i: R'=2-MeO for 4w, 3b
8j: R'=3-MeO for 4x
8k: R'=4-AcO for 4y, 4z
8l: R'=3,5-di-MeO for 4aa
8m: R'=3,4-OCH2O- for 4bb
8n: R'=3,4-di-MeO for 4cc, 4dd
8o: R'=3,4,5-tri-MeO for 4ee
4y: R=3,4-di-HO, R'=4-HO
4z: R=3,5-di-Br, 4-HO, R'=4-HO
4aa: R=3,5-di-Br, 4-HO, R'=3,5-di-HO
4bb: R=3,5-di-HO, R'=3,4-OCH2O-
4cc: R=3,5-di-HO, R'=3,4-di-MeO
4dd: R=3,5-di-HO, R'=3,4-di-HO
4ee: R=3,5-di-HO, R'=3,4,5-tri-HO
Scheme 3. Synthesis of compounds 4o - 4ee.
Reagents and conditions: (a) (i) Pd(OAc)2, N-ethylmorpholine, (ii) 1N
NaOH for 4o-4v and 4bb-4cc, BBr3 for 4w-4aa, and 4dd-4ee.

Table 1. Structures of resveratrol derivatives with modifications of
Table 3. Concentration-dependent activities of selected compounds in
hydroxy groups and TRPA1 inhibitory
TRPA1 inhibition
7
R
6
R
Inhibitory value (%)
B ring
1
EC50 M)**)
IC50 M)*)
R
5
R
A ring
4
R
2
10 M
3 M
30 M
R
3
R
>30
1
-3.8
2.5
37.5
>30
(Resveratrol)
Relative
agonistic
activity
Relative
inhibitory
activity
B ring
A ring
>10
>30
>30
>30
>30
>10
3a
4x
4q
4i
-1.9
-19.7
6.1
3.0
-16.3
14.0
-5.0
-17.8
5.6
7.2
6.2
-9.5
1.4
18.6
20.6
8.4
>30
>30
>30
>30
>30
16.1
R1
R2
H
R3
R4
H
R5
H
R6
R7
H
*)
**)
at 30 M
at 30 M
1
OH
OH
OH
=1.0
0.4
4j
(Resveratorol)
4z
37.3
66.7
3a
3b
4a
4b
4c
4d
4e
4p
4x
4o
4l
4m
4r
4s
4t
4u
4v
4q
4i
4j
4k
4w
4dd
4cc
4bb
4ee
4ii
H
H
OAc
OMe
OEt
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
H
H
H
OH
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
OAc
OMe
OEt
OnPr
OMOM
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
Br
1.1
0.3
0.5
0.3
0.4
-0.1
0.4
-0.1
0.8
0.3
0.5
0.6
-0.2
0.1
0.1
-0.1
0.7
0.5
0.8
0.7
-0.2
0.6
0.0
0.4
0.6
0.4
0.1
-0.3
1.4
0.8
0.6
0.4
0.5
0.3
0.4
0.3
0.5
0.4
0.4
0.3
0.6
0.7
0.3
0.3
0.5
0.4
0.3
0.3
0.3
0.3
0.3
0.4
0.3
0.3
0.3
0.3
0.4
0.4
0.5
0.3
(HUHS029)
>30
>30
4f
4n
-12.7
-5.9
1.2
-8.2
-10.2
11.8
>30
>30
OAc
OMe
OEt
O-nPr
OMOM
OH
OH
OH
OMe
OMe
OH
OH
OH
OH
OH
OH
Cl
*)
Inhibitory activity in the TRPA1 assy against AITC (30 M) stimulation.
O-nPr
OMOM
OH
OH
OH
OMe
OH
OH
OH
OH
OH
OH
OH
Cl
**)
Agonistic activity in the TRPA1 assy.
H
OMe
H
Using
a
cell-based calcium assay, we obtained
a
H
concentration-response curve for 4z and AITC-induced calcium
influx in TRPA1-transfected-HEK cells (Figure 2). The EC50 of
4z on TRPA1 was 29.9 M, which was 4-fold higher than that of
AITC. The maximum responses of 4z and AITC were almost
equal (Figure 2), indicating that 4z was a full agonist for TRPA1.
To further determine the agonistic action of 4z on TRPA1
channels, activated currents in TRPA1-transfected HEK293 cells
elicited by this compound were examined (Figure 3). HUHS029
(4z) did not induce any current in untransfected HEK293 cells as
measured by voltage clamp recordings (Figure 3A). In contrast,
4z induced significant inward currents in TRPA1-transfected
HEK293 cells in a concentration-dependent manner (Figure 3B,
C). The activated currents of 4z were blocked completely and
reversibly by HC-030031, a TRPA1-selective antagonist (Figure
3B).
OEt
OnPr
OnHex
OCH2cHex
OBzl
Cl
Cl
H
Cl
H
OH
OMe
H
H
H
H
Cl
H
Cl
H
OH
OH
OH
OH
OH
H
OH
Br
OH
OH
OH
OH
OH
OH
H
H
Br
OH
OH
OMe
-OCH2O-
OH
H
H
H
Br
OH
OH
OH
OH
OH
4y
4z
4aa
**)
.
*)
Effective activity of AITC =1.0
Inhibitory activity of resveratrol=1.0.
group (4ii) decreased the relative inhibitory activity while the
relative effective activity was almost the same. 5-Substituted
resorcinol derivatives were also synthesized (Table 2).
Reduction of double bond of 1 increased the inhibitory activity
(4f). Replacement of the double bond of 1 with an amide bond
(4g-4h) and removal of that group (4hh) abolished the inhibitory
activity. Replacement of the 4-hydroxyphenyl with a 1-naphthyl
moiety (4n) gave 1.1 times increased the activity while those
with aliphatic moieties (4gg-4ff) had reduced activities. Among
these compounds, compound 4z (HUHS029) exhibited the more
potent inhibitory activity than resveratrol (1) and previously
reported compounds (3a - 3b).
Pharmacological desensitization of receptors is a fundamental
approach for reducing channel activation. The effects of
pretreatment of 4z on the AITC-induced currents in TRPA1-
expressing HEK293 cells in a Ca2+-containing extracellular
solution were examined. A single application of 300 μM AITC
(without 4z pretreatment) induced robust inward currents with a
current density of -121.3 ± 42.2 pA/pF (Figure 3D), which were
attenuated significantly by pretreatment of 4z in a concentration-
dependent manner (Figure 3E, F). These data indicated that 4z
can inhibit AITC-activated TRPA1 channel activation through
channel desensitization, which was supported by the above
findings in the calcium assay (Table 3). In the series of the patch
clamp experiments, we found that 4z at 5 M induced smaller
inward currents by itself but caused a larger inhibition on AITC-
induced current (Figure 3C and 3F), suggesting that appropriate
dosage of 4z might alleviate TRPA1-related pain without striking
nociceptive effects.
We next measured concentration-dependent activities of
compounds 1, 3a, 4x, 4q, 4i, 4j, 4z, 4f, and 4n (Table 3). Among
them, compound 4z exhibited the most potent inhibitory activity
with an IC50 value of 16.1 M, while reported TRPA1 inhibitors
such as 1 and 3a had IC50 values of more than 30 M in our
assay. Therefore, we selected 4z (HUHS029) for further study
and analyzed its effects on the electrophysiological properties of
the TRPA1 channel and AITC-evoked pain behavior (Figure 3-
5).
AITC EC50: 7.3 
4z
EC50: 29.9 M
(HUHS029)
Table 2. Structures of 3,5-dihdroxyphenyl-5-substituted derivatives and
TRPA1 inhibitory activities
Relative
agonisitic
activity
Relative
Inhibitory
activity
HO
R
**)
*)
OH
at 30 M
at 30 M
-CH2CH2-(4-HO)Ph
-CONH-(4-HO)Ph
-CONH-(4-MeO)Ph
(E) -CH=CH--Naphtyl
(E) -CH=CH-cHex
(E) -CH=CH-tert Butyl
-(4-HO)Ph
1.2
-0.6
-0.4
1.1
0.5
0.8
0.4
0.3
0.3
0.3
0.3
0.3
0.5
4f
4g
4h
4n
4gg
4ff
Concentration of compound
Figure 2. Concentration−response curve for AITC (open circles) and
compound 4z (filled squares)-induced calcium influx assays in TRPA1-
expressing HEK293 cells.
0.8
4hh
*) **)
See footnotes of Table 1.

HUHS029 (4z) was demonstrated to activate TRPA1-
expressing HEK293 cells in the present study. Next, we raised
the question as to whether 4z could activate sensory neurons.
Cultured DRG neurons from Sprague-Dawley rats were used to
answer the question. TRPA1 is thought to be co-expressed with
TRPV1 in DRG neurons13. AITC application followed by a
capsaicin application, which can activate TRPV1, to the DRG
neurons has often been used to detect TRPA1 currents9,14. Our
data showed that 4z (30 μM) evoked currents in rat DRG
neurons, which were also activated by AITC (300 μM) and
capsaicin (1 μM) (Figure 4).
an irritant effect of 4z may be due to its low potency and efficacy
for activating TRPA1. As expected, the pretreatment with 4z (5
min before AITC injection) resulted in a significant decrease in
the licking time of the hind paw in the initial 5 min after the
intraplantar AITC injection (Figure 5).
Figure 4. HUHS029 (4z)-induced TRPA1 activation in DRG neurons.
Representative trace showing 4z (30 μM, 30 sec)-, AITC (300 μM, 30 sec) and
capsaicin (CAP, 1 μM for 30 sec)-induced currents in one DRG neuron.
A
B
60
50
40
30
20
10
50
40
30
20
10
Figure 3. Compound 4z-induced activation and desensitization of TRPA1
in heterologous TRPA1-expressing HEK293 cells. (A) Representative traces
for 4z (10 μM)-induced activation in native HEK293 cells. (B) Representative
traces for 4z (10 μM)-induced activation in TRPA1-expressing HEK293
cells. HC-030031 (10 μM), a TRPA1 selective antagonist, inhibited the 4z-
induced TRPA1 current. (C) Concentration -dependency of 4z-induced
TRPA1 activation. (D) Representative traces for the AITC (30 μM, 1 min)-
induced TRPA1 currents in the absence of 4z, (E) in the presence of 4z (5
0
4z
0
4z
vehci el
4Z
4z+ATI
C
A
TI
C
vehci el
4Z
A
TI
C
4z+ATI C
( - )
2 mM
( - )
2 mM
5 mM
( - )
2 mM
( - )
2 mM
5 mM
AITC
( - )
( - )
5 mM
AITC
( - )
( - )
5 mM
Figure 5. Pretreatment with 4z suppresses AITC-induced nocifensive
pain behavior in rats. Five min before the AITC (5 mM, 30 uL) injection, 4z
(2 mM, 30 uL) or vehicle (10% DMSO in paraffin) were intraplantarly
injected into the right hind paw (see method). Bar graphs showing the number
of AITC-induced paw lifts (A) and the time of AITC-induced paw licking
during the initial 5 min after AITC injection.
μM,
1 min) pre-application. (F) Concentration-dependency of current
densities of AITC (30 μM) in the absence or presence of 4z (3 μM,5 μM, 10
μM or 30 μM) pre-application. *, p<0.05, vs. AITC without 4z pre-
application. The results are shown as the means ± SEM, Numbers of the
tested cells are shown in parenthesis.
In conclusion, we designed and synthesized a series of
resveratrol derivatives and tested their modulatory effect on the
TRPA1 channel. Among the synthesized compounds, HUHS029
(4z) showed agonism and antagonism on the TRPA1 channel.
Further electrophysiological and behavioral analysis indicated
that 4z can activate TRPA1 and subsequently desensitize the
channel, and it therefore may serve as a candidate for novel
analgesic targeting TRPA1 since HUHS029 (4z) is a derivative
from the widely used natural product, resveratrol (1), and
expected to be safety while other synthetic inhibitors arose from
random screenings have been terminated by mainly side effects
such as burning pain.
Activation of TRPA1 by pungent natural products suggests a
nociceptive role for TRPA1. Desensitization of TRPA1 channels
was expected to be a therapeutic approach in pain relief. Due to
its agonism, 4z activated and then desensitized TRPA1 in the in
vitro experiments. To further examine if 4z could also suppress
the TRPA1-mediated pain behavior in vivo, we administered
intraplantar injections of 4z followed by AITC injections to
Sprague-Dawley rats and recorded the AITC-induced nocifensive
behaviors.
injection without 4z pretreatment induced
Consistent with our previous studies15, AITC
significant
a
nocifensive behavior represented as increases in paw lifting and
licking at the site of injection. Interestingly, injection of 4z (2
mM, 30 µL in 10% DMSO in paraffin) itself did not cause acute
nocifensive behavior such as paw lifting or licking. Paw-lifting
is a reflex response occurs in the spinal level, while paw-licking
is a conscious behavior which follows orders from the cerebral
cortex. Therefore, licking time is thought to indicate pains of rats
because they lift and lick paw when they feel pain. This lack of
.
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Acknowledgments
This research was supported by a CMCIHCM Collaborative
Research Grants for CMCIHCM Collaborative Research
Grants for Yong Scientists.

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12. Typical procedure for the synthesis of resveratrol derivatives in
this study (preparation of HUHS029 (4z)). To a solution of 7d
(400 mg, 1.3 mmol) in p-xylene (4 mL) was added 8k (244 mg 1.5
mmol), N-ethylmorpholine (147 mg, 1.3 mmol) and then acetic
acid palladium(II) salt (2.9 mg, 0.013 mmol) under nitrogen,
which was stirred at 100 °C for 5 h. The reaction mixture was
diluted with ethyl acetate and filtered with celite. The filtrate
concentrated under reduced pressure. The resulting residue was
purified using silica gel column chromatography (1% ethyl acetate
in n-hexane) to give acetic acid 4-[2-(3,5-dibromo-4-methoxy-
phenyl)-vinyl]-phenyl ester as a white powder (76 mg, 14%), was
used for the next step without further purification. The
intermediate (33 mg, 0.077 mmol) was diluted in dichloromethane
(1.0 mL) and added 1.0 M dichloromethane solution of boron
tribromide (0.23 mL, 0.23 mmol) under nitrogen and ice cooling.
The mixture was stirred at room temperature for 2.5 h. The
reaction mixture was added to cooled water under ice cooling and
extracted with ethyl acetate. The combined organic layer was
washed with water and brine, dried over anhydrous MgSO4,
filtered and then concentrated under reduced pressure. The
resulting residue was purified by silica gel column
chromatography (20% ethyl acetate in n-hexane) to give 4z as a
white powder (26 mg, 91%).
hTRPA1 screening assay was
carried out as below: Calcium influx to the cells after stimulation
of the receptor were examined using Calcium KitII-Fluo4
(DOJINDO, #CS32). The loading buffer of the kit was loaded to
the hTRPA1-transfected HEK293 cells or untransfected cells in a
96-well plate, after 1 h, Ca2+-dependent fluorescence intensity of
each wells were previously measured for 30 sec with an excitation
wavelength of 485 nm and an emission wavelength of 525 nm on
FlexStation3 (Molecular Devices). Then, compounds or 0.1%
DMSO in the culture medium were added and measured for 6 min
at room temperature; after that, AITC or 0.1% DMSO was added,
and immediately, the fluorescence intensity was measured for 1
min. The change value of fluorescence was calculated as the rate
of the average fluorescence value, after compound administration /
initial fluorescence average value for agonistic effect, after AITC
administration / initial fluorescence average value for antagonistic
effect, furthermore, inhibition (%) of compounds for AITC
activation of hTRPA1 was also calculated about each compound
as 0% inhibition at 0.1% DMSO control. To compare the
inhibitory activity of the compounds, relative inhibitory activity of
Resveratrol was calculated.