Synthesis and evaluation of new alkylamides derived from α-hydroxysanshool, the pungent molecule in szechuan pepper

Article abstract of DOI:10.1021/jf803067r

Szechuan pepper is widely used in Asia as a spice for its pleasant pungent and tingling sensations, produced by natural alkylamides called sanshools. R-Hydroxysanshool, the main alkylamide found in the pericarp of the fruit, stimulates sensory neurons innervating the mouth by targeting two chemosensitive members of the transient receptor potential (TRP) channels, TRPV1 and TRPA1. As it was previously found that configuration of the unsaturations in the R-hydroxysanshool alkyl chain is required for TRPA1 but not TRPV1 selectivity, this study aimed at obtaining more potent and selective TRPA1 agonists using R-hydroxysanshool as a starting material. This paper reports the preparation of new alkylamides derived from sanshool and their efficacy in stimulating TRPA1 and TRPV1 receptors. The data provide knowledge of the main sanshool chemical functionalities required for TRP channel activation, but they also evidence new selective and potent TRPA1 agonists based on α-hydroxysanshool.

Full text of DOI:10.1021/jf803067r

1982 J. Agric. Food Chem. 2009, 57, 1982–1989
Synthesis and Evaluation of New Alkylamides
Derived from r-Hydroxysanshool, the Pungent
Molecule in Szechuan Pepper
†
†
CANDICE MENOZZI-SMARRITO, CELINE E. RIERA, CAROLINE MUNARI,
JOHANNES LE COUTRE, AND FABIEN ROBERT*
Nestl e´ Research Center, Vers-Chez-Les-Blanc, 1000 Lausanne 26, Switzerland
Szechuan pepper is widely used in Asia as a spice for its pleasant pungent and tingling sensations,
produced by natural alkylamides called sanshools. R-Hydroxysanshool, the main alkylamide found
in the pericarp of the fruit, stimulates sensory neurons innervating the mouth by targeting two
chemosensitive members of the transient receptor potential (TRP) channels, TRPV1 and TRPA1.
As it was previously found that configuration of the unsaturations in the R-hydroxysanshool alkyl
chain is required for TRPA1 but not TRPV1 selectivity, this study aimed at obtaining more potent
and selective TRPA1 agonists using R-hydroxysanshool as a starting material. This paper reports
the preparation of new alkylamides derived from sanshool and their efficacy in stimulating TRPA1
and TRPV1 receptors. The data provide knowledge of the main sanshool chemical functionalities
required for TRP channel activation, but they also evidence new selective and potent TRPA1 agonists
based on R-hydroxysanshool.
KEYWORDS: TRPA1 and TRPV1 agonists; r-hydroxysanshool; synthesis; chemoreceptors
INTRODUCTION
from the configuration of one double bond (R- and ꢀ-sanshools,
for instance) and the length of the polyenic system (12 carbons
for R- and ꢀ-sanshools versus 14 carbons for γ- and δ-san-
shools). Interestingly, sensory evaluations brought to the fore
that R-hydroxysanshool produced a tingling sensation, whereas
δ-, γ-, and R-sanshools were perceived as burning and the
Szechuan pepper (Zanthoxylum piperitum), which is com-
monly used in Asia as a spice and in traditional medicine, is
particularly appreciated for its pleasant pungent and tingling
sensations. Recent studies showed that R-hydroxysanshool, the
major alkylamide in the pepper, is responsible for these
sensations and activates TRPV1 and, to a lower extent, TRPA1
channels (1-3). TRPV1 receptors are involved in sensing a
multitude of noxious stimuli, the most notable being capsaicin,
the burning molecule in red hot chili pepper (4) and also
common vanilloids such as zingerone and gingerol from ginger
ꢀ
-sanshools as rather numbing (1, 21). One common charac-
teristic of all these natural compounds is their agonistic activity
on TRPV1, consistent with their burning properties (1). More-
over, the sanshool pungent “sharp” and “biting” sensations may
originate from TRPV1 and TRPA1 stimulation, but their
contribution to its numbing effects has been controversial. Very
recently, two-pore potassium channels sensing volatile anesthe-
(
5), eugenol from clove (6), or piperine contained in black and
white pepper (7) (Figure 1). TRPA1 receptors are found in a
subset of nociceptive sensory neurons, where they are coex-
pressed with TRPV1 (8) and respond specifically to noxious
cold and pungent compounds such as isothiocyanate (mustard
oil, wasabi, and horseradish) and cinnamaldehyde from
cinnamon (9-12). Previous studies have highlighted the structure-
activity relationship (SAR) between the agonist and TRP
channels where chemical modification of the ligand modulates
the receptor responsiveness, as observed in the capsaicin
hydrophobic region on TRPV1 (13-15).
The main sanshools found in Szechuan pepper are R-, ꢀ-,
γ-, and δ-sanshools and their analogues possessing one hydroxyl
group on the amide moiety (Figure 2) (16-20). They differ
*
Corresponding author [telephone (0041) 21 785-9372; fax (0041)
2
1 785-8554; e-mail fabien.robert@rdls.nestle.com].
†
These authors contributed equally to this work.
Figure 1. Examples of natural products activating TRPV1.
1
0.1021/jf803067r CCC: $40.75  2009 American Chemical Society
Published on Web 02/11/2009

Chemosensory Attributes of Alkylamides
J. Agric. Food Chem., Vol. 57, No. 5, 2009 1983
Figure 2. Structures of the main sanshools found in Szechuan pepper.
tics (22, 23) were found to be stimulated by applications of
R-hydroxysanshool, rationalizing some of the complex numbing
and long-lasting perceptions associated with the spice (3). In
our previous work (24), we observed that R-hydroxysanshool
is likely to gate TRPA1 via covalent interaction on its reactive
cysteines, whereas this mechanism is unlikely to occur for
TRPV1, highlighting that the two channels behave differently
in their responses. These results may account for dissociated
sensory roles of the channels in their response to this compound.
If TRPV1’s crucial role in pungency mediated by burning
molecules is well established (4, 25), the physiological implica-
tion of TRPA1 in sensory perception remains so far partially
elucidated (26). Therefore, to better understand the differences
between TRPV1 and TRPA1 in sensing these compounds, we
aimed at developing new synthetic molecules based on R-hy-
droxysanshool structure, which would possess a TRPA1 rather
than a TRPV1 selectivity.
pump SL, high-performance autosampler, diode array detector SL and
thermostated column compartment SL with an Agilent 6210 time-of-
flight mass spectrometer.
Purification of Green Szechuan Extract. 800 mg of Green
Szechuan extract was purified by chromatography column on silica gel
using a gradient of ethyl acetate in petroleum ether. Column FLASH
4
0+M was used on a Biotage HPFC purification system with a flow
rate of 40 mL/min. The hydroxysanshools were detected at 275 nm
and collected in 21 mL fractions. Fractions were concentrated under
reduced pressure to give finally 200 mg of hydroxysanshools as a
mixture of three isomers that are not separable by this technique of
purification (25% yield by weight). The ratio after purification was
established by HPLC and stayed unchanged (m/R/ꢀ 3.7:75.4:20.8); R
0.35 (ethyl acetate).
f
)
1
3
R-Hydroxysanshool: H NMR (360 MHz, CDCl , TMS as reference)
δ 6.88 (dt, J ) 15.3, 6.7 Hz, 1H), 6.36-5.92 (m, 5H), 5.84 (dt, J )
1
6
4
5.3, 1.3 Hz, 1H), 5.74 (dt, J ) 13.8, 6.5 Hz, 1H), 5.37 (dt, J ) 10.8,
.7 Hz, 1H), 3.32 (d, J ) 6.1 Hz, 2H), 2.54 (s, 1H), 2.36-2.27 (m,
13
3
H), 1.78 (d, J ) 6.7 Hz, 3H), 1.23 (s, 6H); C NMR (90 MHz, CDCl ,
Here we report the elaboration of new alkylamides that are
able to activate selectively TRPA1 receptors expressed in HEK
TMS as reference) δ 166.95, 144.47, 133.51, 131.77, 130.2, 129.64,
29.53, 125.44, 123.70, 71.06, 50.41, 32.07, 27.34, 26.46, 18.34.
-Hydroxysanshool: 13C NMR (90 MHz, CDCl
, TMS as reference)
1
2
93 cells. We first focused on sanshool derivatives having
ꢀ
3
variations in their alkyl chains. These analogues would be very
useful to identify main chemical functionalities for the TRP
channel activation. The second point was to evaluate the
influence of the amide part, replacing the 2-methylpropan-2-ol
moiety by various amino acids having different chemical
properties. This study finally led to potentially new selective
TRPA1 agonists easier to elaborate and more potent than the
natural R-hydroxysanshool.
δ 166.93, 144.35, 132.03, 131.63, 131.57, 131.44, 130.07, 129.39,
123.71, 71.06, 50.42, 31.90, 31.37, 27.34, 18.29.
Ethyl (2E,6Z)-dodeca-2,6-dienoate 2. To a solution of (ethoxycar-
bonylmethyl) triphenylphosphorane (9.40 g, 27 mmol, 2 equiv) in
anhydrous THF (40 mL) was added dropwise 4-(Z)-decenal 1 (2.46
mL, 13.5 mmol, 1 equiv), and the mixture was stirred overnight at
room temperature. The solution was then concentrated to 25 mL, and
petroleum ether was added to precipitate the phosphonium salt. The
white solid was filtered, and the filtrate was concentrated under reduced
pressure. The resulting residue was purified by flash chromatography
column on silica gel using a gradient of ethyl acetate in petroleum
ether (boiling range, 40-60 °C). Ethyl (2E,6Z)-dodeca-2,6-dienoate 2
MATERIALS AND METHODS
Chemistry. All commercially available reagents were obtained from
Sigma Aldrich (Buchs, Switzerland) and used as received. Water- and
air-sensitive reactions were carried out under argon atmosphere.
Analytical thin layer chromatography (TLC) was carried out on silica
was isolated as a colorless oil (1.65 g, 7.35 mmol, 54%); R
(petroleum ether/ethyl acetate 95:5); H NMR (360 MHz, CDCl , TMS
f
) 0.62
3
1
as internal reference) δ 6.96 (dt, J ) 15.6, 6.6 Hz, 1H), 5.83 (dt, J )
15.6, 1.5 Hz, 1H), 5.45-5.30 (m, 2H), 4.18 (q, J ) 7.0 Hz, 2H),
2.25-2.18 (m, 4H), 2.04-1.98 (m, 2H), 1.36-1.26 (m, 6H), 1.28 (t,
6
0 F254 (Merck) and RP-18 F254s (Merck) plates. The TLC plates
were visualized by shortwave UV light with ceric ammonium molybdate
stain. Flash chromatography was performed using a Biotage SP1 HPFC
1
3
3
J ) 6.7 Hz, 3H), 0.88 (t, J ) 6.7 Hz, 3H); C NMR (90 MHz, CDCl ,
1
system and a FLASH cartridge (25+M and 40+M KP-SIL). H NMR
TMS as internal reference) δ 166.67, 148.64, 131.64, 127.74, 121.57,
60.15, 32.30, 31.49, 29.27, 27.23, 25.75, 22.56, 14.28, 14.06.
1
3
(
360.13 MHz) and C NMR (90.56 MHz) spectra were recorded on a
Bruker DPX-360 spectrometer equipped with a broadband multinuclear
z-gradient probehead. The chemical shifts (in ppm) were expressed with
respect to an internal reference (TMS or TSP). Multiplicities are
reported as follows: s ) singlet, d ) doublet, t ) triplet, q ) quadruplet,
m ) multiplet, bs ) broad singlet. Melting points were recorded on
Buchi B-545 melting point apparatus and are uncorrected. Optical
rotations were measured with a JASCO P2000 polarimeter. Elementary
analyses were performed at the University of Geneva (Service de
microanalyse). HPLC of purified green Szechuan extract was carried
out with a N-Nucleosil 100 column (Macherey-Nagel, 5 µm, C-18,
(2E,6Z)-Dodeca-2,6-dienoic acid 3d. To a solution of ethyl (2E,6Z)-
dodeca-2,6-dienoate 2 (800 mg, 3.56 mmol, 1 equiv) in THF/water
(v/v 1/:1, 40 mL) was added LiOH (512 mg, 21.4 mmol, 6 equiv), and
the mixture was stirred for 12 h at room temperature and for 4 h at 80
°C. The medium was then acidified with 1N HCl to pH 5. The organic
was dried over sodium sulfate, filtered, and concentrated under reduced
pressure. The resulting oil was purified by flash chromatography column
on silica gel using a gradient of ethyl acetate in petroleum ether. 420
mg of (2E,6Z)-dodeca-2,6-dienoic acid 3d was isolated as a colorless
oil (420 mg, 2.13 mmol, 60%); R
2:1); H NMR (360 MHz, CDCl , TMS as internal reference) δ 7.07
f
) 0.35 (petroleum ether/ethyl acetate
3
1
2
50 × 4 mm), with UV detection at 254 nm and a flow rate of 1 mL/
min. Solvents used are A ) 0.1% (g/vol) TFA in H O and B )
2
(dt, J ) 15.7, 6.4 Hz, 1H), 5.84 (dt, J ) 15.6, 1.6 Hz, 1H), 5.46-5.29
(m, 2H), 2.31-2.20 (m, 4H), 2.04-1.96 (m, 2H), 1.38-1.21 (m, 6H),
Acetonitrile. HPLC/DAD/ESI-TOF-MS analyses were performed on
an Agilent-1200 series Rapid Resolution LC System including binary
1
3
3
0.88 (t, J ) 7.0 Hz, 3H); C NMR (90 MHz, CDCl , TMS as internal

1984 J. Agric. Food Chem., Vol. 57, No. 5, 2009
Menozzi-Smarrito et al.
,
a b
Table 1
(5Z)-N-(2-Hydroxy-2-methylpropyl)-dodec-5-enamide 5c: 57% yield;
1
pale orange oil; R
f
) 0.60 (ethyl acetate); H NMR (360 MHz, CDCl
3
,
TMS as internal reference) δ 5.87 (s, 1H), 5.37-5.22 (m, 2H), 3.19
d, J ) 6.0 Hz, 2H), 2.16 (t, J ) 7.4 Hz, 2H), 2.01 (q, J ) 6.9 Hz,
H), 1.93 (q, J ) 6.4 Hz, 2H), 1.64 (q, J ) 7.5 Hz, 2H), 1.27-1.15
(
2
1
3
(
m, 8H), 1.15 (s, 6H), 0.81 (t, J ) 7.0 Hz, 3H); C NMR (90 MHz,
CDCl , TMS as internal reference) δ 176.95, 133.96, 131.21, 73.73,
3.13, 39.94, 34.57, 32.48, 31.80, 30.10, 30.09, 29.48, 28.56, 25.45,
3
5
1
-
6.91; LC/ESI-TOF-MS, obsd m/z [M - 1] 268.23. Anal. Calcd for
+ 0.2H O: C, 70.39%; H, 11.59%; N, 5.13%. Found: C,
0.58%; H, 11.56%; N, 5.16%.
2E,6Z)-N-(2-Hydroxy-2-methylpropyl)-dodeca-2,6-dienamide 5d:
7% yield; [ratio (6Z,2E)/(6E,2E) 87.5/12.4, 77%]; colorless oil; R
C
7
16
H
31NO
2
2
(
7
0
f
)
1
3
.51 (ethyl acetate); H NMR (360 MHz, CDCl , TMS as internal
reference) δ 6.87 (dt, J ) 15.3, 6.7 Hz, 1H), 5.95 (s, 1H), 5.83 (d, J )
1
1
5.4 Hz, 1H), 5.44-5.30 (m, 2H), 3.33 (d, J ) 5.9 Hz, 2H), 2.59 (s,
H), 2.27-2.15 (m, 4H), 2.04-1.97 (m, 2H), 1.36-1.21 (m, 6H), 1.23
1
3
a
(s, 6H), 0.88 (t, J ) 6.5 Hz, 3H); C NMR (90 MHz, CDCl
internal reference) δ 166.99, 144.81, 131.21, 127.89, 123.34, 71.04,
0.41, 32.19, 31.47, 29.27, 27.31, 27.22, 25.91, 22.54, 14.05; LC/ESI-
3
, TMS as
Reagents and conditions: (a) (benzyltriazol-1-yloxy) tripyrrolidinophosphonium
b
2
hexafluorophosphate, iPr EtN, DMF, rt, 12 h. Isolated yields.
5
-
2
TOF-MS, obsd m/z [M - 1] 266.18. Anal. Calcd for C16H29NO +
0
1
reference) δ 171.55, 151.75, 131.56, 127.49, 120.83, 32.49, 31.49,
9.26, 27.24, 25.60, 22.56, 14.20.
-Amino-2-methylpropan-2-ol 4. To a solution of 1,1-dimethylep-
.3H
0.97%; N, 5.10%.
Typical Procedure for the Preparation of Compounds 7a-d and
2
O: C, 70.44%; H, 10.94%; N, 5.13%. Found: C, 70.53%; H,
2
1
oxide (4 mL, 44.98 mmol, 1 equiv) and benzylamine (5.39 mL, 49.42
mmol, 1.1 equiv) in 250 mL of water was added dropwise triethylamine
8a-c (Tables 2 and 3). To a solution of carboxylic acid 3b-f (1 equiv),
amino acid methyl ester hydrochloride 6a-d (1 equiv), and di-
isopropylethylamine (2 equiv) in DMF (0.084 M) was added the
(benzyltriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate
(1 equiv) at 0 °C. The solution was stirred for 12 h at room temperature
and then diluted with water and ethyl acetate. The organic layer was
washed with water, dried over sodium sulfate, filtered, and concentrated
under reduced pressure. The resulting oil was purified by flash
chromatography column on silica gel using a gradient of ethyl acetate
in petroleum ether. The resulting methyl ester (1 equiv) was dissolved
(0.062 mL, 0.449 mmol, 0.01 equiv) at room temperature. The mixture
was stirred overnight at the same temperature and then concentrated
under reduced pressure. The resulting oil was purified by flash
chromatography column on silica gel using a gradient of methanol in
dichloromethane. A colorless oil (7.95 g) was isolated as a mixture of
1
-(benzylamino)-2-methylpropan-2-ol and benzylamine. The oil was
dissolved in methanol (130 mL) and Pd/C (5%, 3.00 g) was added.
The mixture was successively degassed and submitted to hydrogen
atmosphere. The same operation was performed three times, and then
the mixture was stirred for 1.5 h at room temperature under hydrogen
atmosphere. The suspension was then filtered over Celite and concen-
trated under reduced pressure to give the 1-(amino)-2-methylpropan-
2
in THF/H O (0.084 M, v/v, 3:1), and LiOH (3 equiv) was added at
room temperature. The mixture was stirred for 2 h and then acidified
with 0.5 N HCl to pH 6. Ethyl acetate was added, and the aqueous
phase was concentrated under reduced pressure, purified by flash
chromatography column on reverse phase using a gradient of methanol
in water, and finally freeze-dried to give a white foam.
2
1
-ol 4 as a colorless oil (2.54 g, 28.49 mmol, 62% from the
1
3
,1-dimethylepoxide): H NMR (360 MHz, CDCl , TMS as internal
1
3
reference) δ 2.60 (s, 2H), 2.04 (s, 2H), 1.16 (s, 6H); C NMR (90
MHz, CDCl , TMS as internal reference) δ 69.76, 52.37, 26.74.
3
(S,Z)-2-Dodec-5-enamido-3-hydroxypropanoic acid 7a: 78% yield;
2
4
General Procedure for the Preparation of Sanshool Analogues
white foam; R
f
) 0.55 (methanol/water/AcOH 80:20:1); [R]
D
+24.07°
1
5
a-d (Table 1). To a solution of acid 3a-d (1 equiv), 1-amino-2-
(c 1.08 mg/mL, MeOH); H NMR (360 MHz, MeOD-d
4
, TMS as
methylpropan-2-ol 4 (1 equiv), and di-isopropylethylamine (2 equiv)
in anhydrous DMF (0.084 M) was added the (benzyltriazol-1-yloxy)
tripyrrolidinophosphonium hexafluorophosphate (1 equiv) at 0 °C. The
solution was stirred for 12 h at room temperature and then diluted with
water and ethyl acetate. The organic layer was washed with water, dried
over sodium sulfate, filtered, and concentrated under reduced pressure.
The resulting oil was purified by flash chromatography column on silica
gel using a gradient of ethyl acetate in petroleum ether.
internal reference) δ 5.45-5.34 (m, 2H), 4.31 (t, J ) 4.8 Hz, 1H),
3.81 (d, J ) 4.8 Hz, 2H), 2.29 (t, J ) 7.1 Hz, 2H), 2.11 (q, J ) 6.9
Hz, 2H), 2.06 (q, J ) 6.3 Hz, 2H), 1.71 (dq, J ) 7.9, 7.9 Hz, 2H),
1
3
1.36-1.30 (m, 8H), 0.91 (t, J ) 6.3 Hz, 3H); C NMR (90 MHz,
4
MeOD-d , TMS as internal reference) δ 178.16, 176.96, 133.11, 131.27,
65.75, 59.43, 38.28, 34.34, 32.24, 31.48, 29.60, 29.19, 28.34, 25.11,
-
15.83; LC/ESI-TOF-MS, obsd m/z [M - 1] 284.16 (M - H). Anal.
Calcd for C14
C, 60.22; H, 8.83; N, 4.62.
(S,Z)-2-Dodec-5-enamidopentanedioic acid 7b: 69% yield; white
H
26NO
4
2
Li + 0.4H O: C, 60.35; H, 9.05; N, 4.69. Found:
N-(2-Hydroxy-2-methylpropyl)-dodecanamide 5a: 57% yield, white
1
solid; R
f
) 0.51 (ethyl acetate); H NMR (360 MHz, CDCl
3
, TMS as
2
4
internal reference) δ 6.48 (s, 1H), 3.28 (d, J ) 5.7 Hz, 2H), 3.17 (s,
solid; R
f
) 0.50 (methanol/water/AcOH 60:40:1); [R]
D
-4.90° (c 1.02
1
4
1
1
H), 2.28 (t, J ) 7.5 Hz, 2H), 1.67-1.61 (m, 2H), 1.34-1.23 (m, 16H),
mg/mL, MeOH); H NMR (360 MHz, MeOD-d , TMS as internal
reference) δ 5.45-5.33 (m, 2H), 4.43 (dd, J ) 8.9, 5.0 Hz, 1H), 2.40
(td, J ) 7.9, 1.2 Hz, 2H), 2.27 (t, J ) 7.4 Hz, 2H), 2.17-1.89 (m,
1
3
3
.23 (s, 6H), 0.87 (t, J ) 6.6 Hz, 3H); C NMR (90 MHz, CDCl ,
TMS as internal reference) δ 177.83, 73.71, 53.41, 39.28, 34.71, 32.41,
2.29, 32.14, 32.10, 30.05, 28.74, 25.49, 16.92; LC/ESI-TOF-MS, obsd
3
6H), 1.68 (dq, J ) 7.3, 7.3 Hz, 2H), 1.35-1.30 (m, 8H), 0.91 (t, J )
-
13
m/z [M - 1] 270.24. Anal. Calcd for C16
2.29%; N, 5.12%. Found: C, 70.83%; H, 12.29%; N, 5.12%.
2E)-N-(2-Hydroxy-2-methylpropyl)-dodec-2-enamide 5b: 66% yield;
H
33NO
2
: C, 70.80%; H,
6.3 Hz, 3H); C NMR (90 MHz, MeOD-d
4
, TMS as internal reference)
1
δ 177.88, 177.56, 176.74, 133.18, 131.20, 54.66, 37.79, 34.34, 32.79,
(
32.22, 31.46, 29.59, 29.46, 29.10, 28.38, 25.11, 15.83; LC/ESI-TOF-
1
+
pale orange oil; R
f
) 0.60 (ethyl acetate); H NMR (360 MHz, CDCl
3
,
MS, obsd m/z [M + 1] 345 (M + H
2
O). Anal. Calcd for C17
H
29NO
5
TMS as internal reference) δ 6.79 (dt, J ) 15.3, 7.0 Hz, 1H, H-10),
2
+ 0.3H O: C, 61.35; H, 8.96; N, 4.21. Found: C, 61.47; H, 8.81; N,
6
2
1
.03 (s, 1H), 5.75 (dt, J ) 15.2, 1.5 Hz, 1H), 3.26 (d, J ) 6.0 Hz, 2H),
4.18.
.06 (qd, J ) 7.7, 1.4 Hz, 2H), 1.40-1.34 (m, 2H), 1.24-1.17 (m,
(Z)-2-Dodec-5-enamidoacetic acid 7c: 64% yield; white foam; R
f
13
1
2H), 1.16 (s, 6H), 0.81 (t, J ) 6.7 Hz, 3H); C NMR (90 MHz, CDCl
3
,
) 0.42 (methanol/water/AcOH 80:20:1); H NMR (360 MHz,
4
TMS as internal reference) δ 167.35, 145.79, 123.23, 71.14, 50.57,
2.21, 32.00, 29.93, 29.55, 29.42, 29.33, 28.36, 27.42, 22.79, 14.23;
MeOD-d , TMS as internal reference) δ 5.44-5.33 (m, 2H), 3.37
3
(s, 2H), 2.26 (t, J ) 7.8 Hz, 2H), 2.10 (q, J ) 7.0 Hz, 2H), 2.06 (q,
J ) 6.3 Hz, 2H), 1.64 (qd, J ) 7.5, 7.5 Hz, 2H), 1.38-1.30 (m,
-
LC/ESI-TOF-MS, obsd m/z [M - 1] 268.23. Anal. Calcd for
+ 0.2H O: C, 70.39%; H, 11.59%; N, 5.13%. Found: C,
0.36%; H, 11.47%; N, 5.08%.
1
3
C
7
16
H
31NO
2
2
8H), 0.91 (t, J ) 6.3 Hz, 3H); C NMR (90 MHz, MeOD-d
4
, TMS
as internal reference) δ 177.86, 177.03, 133.11, 131.26, 45.92, 38.06,

Chemosensory Attributes of Alkylamides
J. Agric. Food Chem., Vol. 57, No. 5, 2009 1985
,
a b
Table 2
a
b
O, rt, 2 h. Isolated
Reagents and conditions: (a) (benzyltriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate, iPr
2
EtN, DMF, rt, 12 h; (b) LiOH, THF/H
2
yields.
,
a b
Table 3
a
2
Reagents and conditions: (a) alanine chloride methyl ester 6d, (benzyltriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate, iPr EtN, DMF, rt, 12 h; (b) LiOH,
b
2
THF/H O, rt, 2 h. Isolated yields.
3
4.34, 32.23, 31.48, 29.60, 29.17, 28.32, 25.11, 15.84; LC/ESI-TOF-
32.94, 30.83, 30.08, 28.20, 27.73, 27.01, 23.71, 19.45, 15.84; LC/ESI-
TOF-MS, obsd m/z [M - 1] 268.20 (M - H). Anal. Calcd for
-
-
MS, obsd m/z [M - 1] 254.20 (M - H). Anal. Calcd for
Li + 0.35H O: C, 62.42; H, 9.32; N, 5.20. Found: C,
2.28; H, 9.27; N, 5.19.
S,Z)-2-Dodec-5-enamidopropanoic acid 7d: 56% yield; white
C
6
14
H
24NO
3
2
C
15
H
26NO
3
Li + 0.35H
H, 9.48; N, 4.94.
(S,E,Z)-2-[Dodeca-2,6-dienamido]propanoic acid 8a: 73% yield;
2
O: C, 63.97; H, 9.56; N, 4.97. Found: C, 64.14;
(
2
4
25
foam; R
.20 mg/mL, MeOH); H NMR (360 MHz, MeOD-d , TMS as internal
reference) δ 5.41-5.33 (m, 2H), 4.21 (q, J ) 7.1 Hz, 1H), 2.22 (t, J
7.5 Hz, 2H), 2.06 (q, J ) 6.8 Hz, 2H), 2.02 (q, J ) 6.3 Hz, 2H),
.70-1.61 (m, 2H), 1.36-1.28 (m, 8H), 1.32 (d, J ) 7.1 Hz, 3H),
f
) 0.39 (methanol/water/AcOH 80:20:1); [R]
D
+ 14.17° (c
white solid, R
f
) 0.71 (methanol/water 80:20); mp ) 95-97 °C; [R]
D
1
4
1
4
1
-7.84° (1.02 mg/mL, MeOH); H NMR (360 MHz, MeOD-d , TMS
as reference) δ 6.78 (dt, J ) 15.4, 6.5 Hz, 1H), 5.95 (d, J ) 15.3 Hz,
1H), 5.44-5.34 (m, 2H), 4.40 (qd, J ) 7.2, 7.2 Hz, 1H), 2.26-2.15
)
1
(m, 4H), 2.03 (q, J ) 6.9 Hz, 2H), 1.39 (d, J ) 7.2 Hz, 3H),1.39-1.30
1
3
13
0
.91 (t, J ) 6.3 Hz, 3H); C NMR (90 MHz, MeOD-d
4
, TMS as
(m, 6H), 0.88 (t, J ) 6.5 Hz, 3H); C NMR (90 MHz, MeOD-d
4
,
internal reference) δ 179.87, 174.91, 131.71, 129.86, 51.83, 36.82,
TMS as reference) δ 177.03, 168.04, 145.40, 132.01, 129.22, 124.85,

1986 J. Agric. Food Chem., Vol. 57, No. 5, 2009
Menozzi-Smarrito et al.
a
4
9.97, 33.26, 32.66, 29.27, 28.19, 27.13, 23.64, 18.23, 14.43; LC/ESI-
Scheme 1
-
TOF-MS, obsd m/z [M - 1] 266.19. Anal. Calcd for C15
H
24LiNO
3
+
0
4
.5H
.81.
Z,Z,Z)-2-[Octadeca-9,12,15-trienamido]propanoic acid 8b: 33%
yield; white solid, R ) 0.24 (methanol/water 80:20); mp ) 100-102
2
O: C, 63.82; H, 8.93; N, 4.96. Found: C, 63.93; H, 8.97; N,
(
f
25
1
°
C; [R]
d
3
D
+5.32° (0.94 mg/mL, MeOH); H NMR (360 MHz, MeOD-
, TMS as reference) δ 7.27 (d, J ) 6.3 Hz, 1H), 5.39-5.26 (m, 6H),
.70 (qd, J ) 6.8, 6.8 Hz, 1H), 2.77 (t, J ) 5.8 Hz, 4H), 2.06-1.99
a
4
3
Reagents and conditions: (a) Ph PCHCOOEt, THF, rt, 12 h, 54%; (b)
LiOH, THF/H
2
O, rt, 12 h, and then 80 °C, 4 h, 60%.
(
m, 6H), 1.45 (qd, J ) 7.3, 7.3 Hz, 2H), 1.32-1.20 (m, 8H), 1.12 (d,
13
J ) 6.9 Hz, 3H), 0.92 (t, J ) 7.5 Hz, 3H); C NMR (90 MHz, MeOD-
olefination, using the (ethoxycarbonylmethyl) triphenylphos-
phorane in anhydrous THF, provided the diene 2 in 12 h at
room temperature (54% yield). The ester function in 2 was then
hydrolyzed under standard saponification conditions (LiOH,
d
1
2
1
4
, TMS as reference) δ 178.10, 174.64, 135.57, 133.99, 132.02, 131.98,
31.61, 131.06, 54.01, 36.48, 29.76, 29.46, 29.43, 29.32, 27.36, 26.12,
5.91, 25.82, 20.76, 20.17, 14.85; LC/ESI-TOF-MS, obsd m/z [M -
]
-
348.28. Anal. Calcd for C21
H
34NO
3
2
+ 0.6H O: C, 68.87; H, 9.69;
THF/H
coupling reaction of carboxylic acids 3a-d and the 1-amino-
-methylpropan-2-ol 4, activated by the (benzyltriazol-1-yloxy)
2
O) to afford the carboxylic acid 3d in 60% yield. The
N, 3.82. Found: C, 69.02; H, 9.54; N, 3.74.
(
Z)-2-[Hexadeca-9-enamido]propanoic acid 8c: 76% yield; white
2
2
5
solid, R
f
) 0.24 (methanol/eater 80:20); mp ) 56-58 °C; [R]
D
1
tripyrrolidinophosphonium hexafluorophosphate, led after 12 h
at room temperature to the R-hydroxysanshool derivatives 5a-d,
respectively, in 57, 66, 57, and 77% yields (Table 1) (28). The
alkylamide 5d was isolated as a mixture of (6Z,2E) and (6E,2E)
diastereoisomers due to the commercial 4-(Z)-decenal 1, which
contains 10% of (E)-isomer. The ratio (6Z,2E)/(6E,2E) was 88:
2 and was established by HPLC.
The synthesis of alkylamides 7a-d was achieved from the
commercially available 5-(Z)-dodecenoic acid 3b and various
amino acid methyl esters 6a-d. Polar (glycine, serine) and
nonpolar (alanine) as well as acidic amino acids (glutamic acid)
were used to prepare new components 7a-d. The previous
coupling reaction [(benzyltriazol-1-yloxy) tripyrrolidinophos-
phonium hexafluorophosphate, iPr EtN, DMF] was followed by
2
a saponification step under standard conditions (LiOH, THF/
H O). Alkylamides 7a-d were finally isolated, respectively, in
78, 69, 64, and 56% yields over the two steps (Table 2).
Moreover, various carboxylic acids containing at least one
Z-insaturation in the alkyl chain were coupled with alanine
hydrochloride methyl ester 6d using the same procedure as
previously. The amidation reaction on (2E,6Z)-dodecadienoic
-
14.42° (1.04 mg/mL, MeOH); H NMR (360 MHz, DMSO-d
as reference) δ 8.04 (d, J ) 7.2 Hz, 1H), 5.32 (t, J ) 5.3 Hz, 2H),
.17 (qd, J ) 7.3, 7.3 Hz, 1H), 2.07 (t, J ) 7.3 Hz, 2H), 1.98-1.96
m, 4H), 1.48-1.45 (m, 2H), 1.39-1.21 (m, 19H), 0.85 (t, J ) 6.2
6
, TMS
4
(
1
3
Hz, 3H); C NMR (90 MHz, DMSO-d
6
, TMS as reference) δ 174.52,
72.12, 129.82, 47.85, 35.17, 31.33, 29.30, 29.28, 28.88, 28.79, 28.74,
8.47, 26.79, 25.37, 22.28, 17.42, 14.12; LC/ESI-TOF-MS, obsd m/z
1
2
1
-
[
1
M - 1] 324.29. Anal. Calcd for C19
H
35NO
3
+ 0.3H
0.84; N, 4.23. Found: C, 68.96; H, 10.74; N, 3.99.
Molecular Biology. Cloning and Expression of Human TRPV1 and
2
O: C, 68.97; H,
TRPA1 Receptors in HEK 293 Cells. Cloning and expression of these
receptors was performed following previously published protocols (27).
Briefly, cloned human TRPV1 cDNA was obtained from RZPD
(Germany) and hTRPA1 cDNA from OriGene (Rockville, MD). Genes
were subcloned into pcDNA5/FRT (Invitrogen, Carlsbad, CA) to
generate stable cell lines using the Flp-In system (Invitrogen) after
sequencing verification.
2
Measurement of Intracellular Calcium LeVels [Ca2+
i
] and Membrane
Potential Variation in HEK 293 Cells Using a Fluorescent Plate
Reader. Cell lines stably expressing TRP channels were seeded into
9
6-well plates previously coated with poly-D-lysine. Cells were
incubated in Hank’s Balanced Salt Solution (HBSS) supplemented with
mM CaCl and 20 mM HEPES (pH 7.4), containing the cytoplasmic
2
2
3
d and linolenic and palmitoleic acids 3e-f followed by the
calcium indicator Fura-2/AM at 2 µM (Molecular Devices, Sunnyvale,
CA) as previously described (27). Experiments were conducted at room
temperature. [Ca
hydrolysis of the methyl ester under basic conditions afforded
the carboxylic acids 8a-c, respectively, in 73, 33, and 76%
yields over the two steps (Table 3).
2
+
i
] fluxes from a homogeneous cell population
(approximately 100000 cells) were measured as changes in fluorescence
intensity when stimulated with agonists using a FLEXstation (Molecular
Devices). Cells were then challenged with the different sanshool
derivatives. Mock cells transfected with pcDNA5/FRT were used as
negative controls. Cell viability was checked after alkylamide stimula-
tion using 100 µM ATP.
Data Analysis. Responses of molecules in HEK 293 cells were
expressed as a percentage of maximum responses evoked by 150 µM
cinnamaldehyde for TRPA1 and 1 µM capsaicin for TRPV1 (these
concentrations were assessed independently to be saturating under these
conditions). For all experiments, calcium fluxes were measured as
changes in fluorescence intensity, before and after the addition of
agonists. The peak response was taken to be the characteristic value
and was obtained by subtracting the peak value from the baseline (value
before injection). Dose-response curves were fitted using the Hill
equation (GraphPad Prism Software, San Diego, CA) to obtain EC50
values. Data obtained from this study were expressed as mean ( SEM.
Statistical analysis was performed using the unpaired Student t test.
In Vitro Pharmacology and SAR. The natural compound
R-hydroxysanshool provided the framework for establishing
more potent and selective TRPA1 agonists. Despite the fact that
the importance of the polyenic chain and particularly the olefin
configuration in the activation of TRP channels has been already
reported (2), the role of each specific double bond had never
been clarified. Hence, the activation of the channels TRPV1
and TRPA1 by the four sanshool analogues 5a-d, which
possess variations in the R-hydroxysanshool alkyl chain (Table
1), was first evaluated (Figure 3). To assess TRPV1- and
TRPA1-induced activities by our compounds, intracellular
2
+
calcium [Ca ] increases in HEK 293 cells were monitored
i
2+
i
using Fura-2-based calcium imaging. No [Ca ] increase was
monitored in mock-transfected cells when stimulated with the
different compounds for the concentration range tested. Re-
sponses were expressed relative to the maximum responses
evoked by 150 µM cinnamaldehyde for TRPA1 and 1 µM
capsaicin for TRPV1 receptors (assessed independently to be
saturating concentrations). Normalized maximal induced re-
sponses of compounds 5a-d were compared with that of a
mixture of natural hydroxysanshools SOH extracts. The latter
was obtained by purification of Green Szechuan oil as described
under Materials and Methods. The resulting purified extract
contained R-,ꢀ-hydroxysanshool and a nonidentified isomer (m)
RESULTS AND DISCUSSION
The syntheses of four R-hydroxysanshool derivatives 5a-d
with various alkyl chains were achieved starting from the
corresponding carboxylic acids 3a-d and the 1-amino-2-
methylpropan-2-ol 4. Carboxylic acids 3a-c were commercially
available, whereas the polyene 3d was synthesized in a two-
step sequence from the 4-(Z)-decenal 1 (Scheme 1). Wittig

Chemosensory Attributes of Alkylamides
J. Agric. Food Chem., Vol. 57, No. 5, 2009 1987
Figure 4. Normalized maximum induced responses of alkylamides 5c
and 7a-d. Values are presented as means ( SEM, n ) 4.
Figure 3. Normalized maximum induced responses of four sanshool
analogues 5a-d. Values are presented as means ( SEM, n ) 4.
changing the 2-methylpropan-2-ol part of 5c by serine or
glutamic acid moieties (compounds 7a and 7b) resulted in a
significant decrease of TRPA1 activation (p < 0.0001). Interac-
tions between these molecules and the TRPA1 receptors could
be disfavored either by steric hindrance or by an electronic
repulsion due to the lateral chain present in serine (hy-
droxymethanyl) and glutamic acid (propanoic acid group).
Maximal responses of expressed TRPV1 receptors induced by
500 µM monoacid 7a, diacid 7b, and glycinyl containing 7c
were slightly lower compared with that evoked by a 500 µM
concentration of the alkylamide 5c. However, a drastic change
superior to 100-fold is observed in the transition of EC50 values
from 5c to 7a-c, as a consequence of hampered hydrophobicity
of the amide moiety. With regard to TRPA1 activation,
component 7c was a more potent agonist than 7a and 7b on
TRPA1 channels but was still a partial agonist inducing ≈60%
of cinnamaldehyde response. In addition, acid 7c was not able
to discriminate significantly the TRPA1 and TRPV1 receptors
(p>0.05). Interestingly, when glycine was replaced with alanine,
alkylamide 7d exhibited a stronger TRPA1 activation than that
caused by sanshool analogue 5c and SOH. Furthermore,
component 7d exhibited specificity toward TRPA1 as it was a
full agonist at TRPA1 receptors and had only a low effect on
TRPV1 channels. However, its activity at TRPA1 was monitored
in the high micromolar range (Table 4).
Having identified alanine as an optimal amide substituent,
an additional SAR study was carried out with alkylamides 8a-c
containing at least one Z-insaturation and various lengths of
alkyl chain. As shown in Figure 5A, TRPA1-expressing cells
responded to all four alkylamides 7d and 8a-c in a dose-
dependent manner. Interestingly, the gain in activity and
specificity obtained from 5c to 7d, when the 2-methylpropan-
2-ol was replaced by the alanine, was not observed in the
alkylamides 5d to 8a. Therefore, the presence of the R,ꢀ-
unsaturation does not significantly modify the TRP activity
induced by the R-hydroxysanshool analogue 5d compared to
that obtained with 5c (Figure 3). However, this unsaturation
negatively affects the transition from the alanine conjugate 7d
(Figure 4) and 8a (Figure 5A). These effects underlie the fact
that both the alkyl chain tail and the polar amide head of the
components play a role in TRPA1 activation. The best synergy
between the head and the tail of the molecule to produce
maximal responses at TRPA1 was obtained with 7d. From these
results, it is difficult to weigh for the TRPA1 activation, first,
the importance of each part of the molecule (tail versus head)
and, second, the importance of the intrinsic molecular shape
versus the self-aggregation of these components in solution. On
the basis of these findings, we selected mono- and pluri-Z
Table 4. EC50 Values of Selected TRPV1 and TRPA1 Ligands
EC50 (µM)
TRPA1
69.4
TRPV1
SOH
1.1
7.0
10.2
3.5
5a
5b
5c
5d
7a
7b
7c
7d
8a
8b
8c
100.1
125.2
271
539.6
259.2
263.1
66.8
20.3
25
5.0
250.4
350.9
283.7
-
27.5
-
115.1
with a R/ꢀ/m ratio of 76:21:3 (24). Because previous studies
demonstrated that only R-hydroxysanshool was able to excite
sensory neurons (2), the response elicited from the mixture
containing predominately the R-hydroxysanshool was assimi-
lated to that induced by the R-hydroxysanshool itself. As shown
in Figure 3 and Table 4, maximal activation of TRPV1 channels
by all four sanshool analogues 5a-d and half-maximum
activation concentrations (EC50) were quite similar to those
induced by natural SOH. These alkylamides elicited saturating
responses on TRPV1 of about 80% of capsaicin response and
EC50 values of e10 µM. Consequently, modification of the alkyl
system seemed to have very low influence on the TRPV1
activation, meaning that this channel does not exhibit selectivity
relative to the sanshool unsaturations. On the contrary, TRPA1
activation by 500 µM sanshool analogues 5a-d was dependent
on the alkyl moiety (Figure 3). We observed weak responses
induced by the fully saturated compound 5a and the R,ꢀ-
unsaturated component 5b, whereas analogues 5c,d, possessing
the Z-olefin, were active on TRPA1 and evoked increases of
the intracellular calcium in HEK 293 cells similar to those
of the SOH with EC50 values of ≈100 µM and maximal
responses reaching 60% of cinnamaldehyde response. These
results point toward the crucial role of olefins in the alkyl chain
and particularly that of the Z-olefin in the activation of expressed
TRPA1 receptors.
With the alkyl moiety fixed as being 5-(Z)-undecenyl, a
similar SAR study was initiated by modifying the amide part.
Thus, the 2-methylpropan-2-ol was replaced by various amino
acids to develop more selective and potent TRPA1 agonists.
Normalized maximal activation of TRPA1 and TRPV1 channels
by new alkylamides 7a-d was evaluated and compared with
activation induced by compound 5c (Figure 4). It is seen that

1988 J. Agric. Food Chem., Vol. 57, No. 5, 2009
Menozzi-Smarrito et al.
to the elaboration of new selective and more potent TRPA1
agonists 7d and 8b. In particular, it has been shown that
unsaturations in the alkyl chain and the amide moiety modulate
differently TRPA1 and TRPV1 channels. In the future, the
alkylamide 8b, which is easily accessible from the natural
linolenic acid, would be prepared in food grade quality to
evaluate its sensory characteristics and correlate them with its
TRPA1 activity.
LITERATURE CITED
(
(
(
1) Sugai, E.; Morimitsu, Y.; Iwasaki, Y.; Morita, A.; Watanabe, T.;
Kubota, K. Pungent qualities of sanshool-related compounds
evaluated by a sensory test and activation of rat TRPV1. Biosci.,
Biotechnol., Biochem. 2005, 69, 1951–1957.
2) Koo, J. Y.; Jang, Y.; Cho, H.; Lee, C. H.; Jang, K. H.; Chang,
Y. H.; Shin, J.; Oh, U. Hydroxy-R-sanshool activates TRPV1 and
TRPA1 in sensory neurons. Eur. J. Neurosci. 2007, 26, 1139–
1
147.
3) Bautista, D. M.; Sigal, Y. M.; Milstein, A. D.; Garrison, J. L.;
Zorn, J. A.; Tsuruda, P. R.; Nicoll, R. A.; Julius, D. Pungent agents
from Szechuan peppers excite sensory neurons by inhibiting two-
pore potassium channels. Nat. Neurosci. 2008, 11, 772–779.
4) Caterina, M. J.; Schumacher, M. A.; Tominaga, M.; Rosen, T. A.;
Levine, J. D.; Julius, D. The capsaicin receptor: a heat-activated
ion channel in the pain pathway. Nature 1997, 389, 816–824.
5) Dedov, V. N.; Tran, V. H.; Duke, C. C.; Connor, M.; Christie,
M. J.; Mandadi, S.; Roufogalis, B. D. Gingerols: a novel class of
vanilloid receptor (VR1) agonists. Br. J. Pharmacol. 2002, 137,
(
(
7
93–798.
(
6) Yang, B. H.; Piao, Z. G.; Kim, Y. B.; Lee, C. H.; Lee, J. K.;
Park, K.; Kim, J. S.; Oh, S. B. Activation of vanilloid receptor 1
(
VR1) by eugenol. J. Dent. Res. 2003, 82, 781–785.
(
7) McNamara, F. N.; Randall, A.; Gunthorpe, M. J. Effects of
piperine, the pungent component of black pepper, at the human
vanilloid receptor (TRPV1). Br. J. Pharmacol. 2005, 144, 781–
Figure 5. (A) Alkylamides 7d and 8a-c induced dose-dependent
2+
increases in [Ca ] rise in TRPA1 expression in HEK 293 cells.
i
7
90.
Dose-responses were monitored until saturation concentrations were
reached and were as follows: 7d, 8a (0.001-1 mM), 8b (1-250 µM),
(
(
8) Story, G. M.; Peier, A. M.; Reeve, A. J.; Eid, S. R.; Mosbacher,
J.; Hricik, T. R.; Earley, T. J.; Hergarden, A. C.; Andersson, D. A.;
Hwang, S. W.; McIntyre, P.; Jegla, T.; Bevan, S.; Patapoutian,
A. ANKTM1, a TRP-like channel expressed in nociceptive
neurons, is activated by cold temperatures. Cell 2003, 112, 819–
8c (1-500 µM). (B) Normalized maximum induced responses of
alkylamides 5c, 5d, 7d, and 8a-c. Values are presented as means (
SEM, n ) 4.
8
29.
unsaturated moieties to be coupled to alanine being compounds
9) Bandell, M.; Story, G. M.; Hwang, S. W.; Viswanath, V.; Eid,
S. R.; Petrus, M. J.; Earley, T. J.; Patapoutian, A. Noxious cold
ion channel TRPA1 is activated by pungent compounds and
bradykinin. Neuron 2004, 41, 849–857.
(10) Jordt, S. E.; Bautista, D. M.; Chuang, H. H.; McKemy, D. D.;
Zygmunt, P. M.; Hogestatt, E. D.; Meng, I. D.; Julius, D. Mustard
oils and cannabinoids excite sensory nerve fibres through the TRP
channel ANKTM1. Nature 2004, 427, 260–265.
11) Macpherson, L. J.; Geierstanger, B. H.; Viswanath, V.; Bandell,
M.; Eid, S. R.; Hwang, S.; Patapoutian, A. The pungency of garlic:
activation of TRPA1 and TRPV1 in response to allicin. Curr.
Biol. 2005, 15, 929–934.
12) Bautista, D. M.; Movahed, P.; Hinman, A.; Axelsson, H. E.;
Sterner, O.; Hogestatt, E. D.; Julius, D.; Jordt, S. E.; Zygmunt,
P. M. Pungent products from garlic activate the sensory ion
channel TRPA1. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12248–
12252S.
13) Walpole, C. S.; Wrigglesworth, R.; Bevan, S.; Campbell, E. A.;
Dray, A.; James, I. F.; Perkins, M. N.; Reid, D. J.; Winter, J.
Analogues of capsaicin with agonist activity as novel analgesic
agents; structure-activity studies. 1. The aromatic “A-region”.
J. Med. Chem. 1993, 36, 2362–2372.
14) Walpole, C. S.; Wrigglesworth, R.; Bevan, S.; Campbell, E. A.;
Dray, A.; James, I. F.; Masdin, K. J.; Perkins, M. N.; Winter, J.
Analogues of capsaicin with agonist activity as novel analgesic
agents; structure-activity studies. 2. The amide bond “B-region”.
J. Med. Chem. 1993, 36, 2373–2380.
8
b derived from linolenic acid and 8c derived from palmitoleic
acid. Consistent with the results obtained with 7d, we observed
that the monounsaturated acid 8c induced a relatively high
TRPA1 activation but still exhibited an important TRPV1
response (Figure 5). In addition, the obtained half-maximal
activation concentration EC50 (25 µM) was greatly superior to
that obtained with 7d (263 µM). Finally, the best results were
observed with the alkylamide 8b, which contained three
Z-olefins in its C18 carbon chain. Actually, 100 µM 8b induced
potent and selective TRPA1 activation, meaning its effect on
TRPV1 was relatively low. From all tested molecules, com-
pound 8b is the most potent and selective TRPA1 agonist.
Normalized maximum value was similar to that evoked by 500
µM compound 7d, but its EC50 was smaller than 8a and 7d,
with only 20.3 versus 25 and 263 µM for 8a and 7d,
respectively. Because components 8a and 8b, which differ only
in the number of insaturations in their alkyl system (1Z for 8a
vs 3Z olefins in 8b), had different reactivities toward TRPA1
channels, this result reinforced the importance of Z-olefins to
interact strongly with TRPA1 receptors.
(
(
(
(
New alkylamides were designed from R-hydroxysanshool,
the molecule responsible for the burning and tingling perceptions
of Szechuan pepper. Their evaluation on TRPA1 and TRPV1
channels, two TRPs involved in the trigeminal perception, led

Chemosensory Attributes of Alkylamides
J. Agric. Food Chem., Vol. 57, No. 5, 2009 1989
(
15) Walpole, C. S.; Wrigglesworth, R.; Bevan, S.; Campbell, E. A.;
Dray, A.; James, I. F.; Masdin, K. J.; Perkins, M. N.; Winter, J.
Analogues of capsaicin with agonist activity as novel analgesic
agents; structure-activity studies. 3. The hydrophobic side-chain
neurotransmitters share a molecular site of action. J. Biol. Chem.
2002, 277, 17733–17742.
(24) Riera, C. E.; Menozzi-Smarrito, C.; Affolter, M.; Michlig, S.;
Munari, C.; Robert F.; Vogel, H.; S.A. Simon; le. Coutre, J.
Covalent and non-covalent ligand interactions in TRPA1 and
TRPV1 channels with spicy molecules from Sichuan and Me-
legueta peppers. Br. J. Pharmacol., submitted.
25) Caterina, M. J.; Leffler, A.; Malmberg, A. B.; Martin, W. J.;
Trafton, J.; Petersen-Zeitz, K. R.; Koltzenburg, M.; Basbaum,
A. I.; Julius, D. Impaired nociception and pain sensation in mice
lacking the capsaicin receptor. Science 2000, 288, 306–313.
26) Kwan, K. Y.; Allchorne, A. J.; Vollrath, M. A.; Christensen, A. P.;
Zhang, D. S.; Woolf, C. J.; Corey, D. P. TRPA1 contributes to
cold, mechanical, and chemical nociception but is not essential
for hair-cell transduction. Neuron 2006, 50, 277–289.
“
C-region”. J. Med. Chem. 1993, 36, 2381–2389.
(
16) Yasuda, I.; Takeya, K.; Itokawa, H. Distribution of unsaturated
aliphatic acid amides in japanese Zanthoxylum species. Phy-
tochemistry 1982, 21, 1295–1298.
(
(
(
(
17) Marcos, M.; Jimenez, C.; Villaverde, M. C.; Riguera, R.; Castedo,
L.; Stermitz, F. Lignans and other constituents from South and
Central American Zanthoxylum species. Planta Med. 1990, 56,
8
9–91.
(
(
(
(
(
(
18) Kashiwada, Y.; Ito, C.; Katagiri, H.; Mase, I.; Komatsu, K.;
Namba, T.; Ikeshiro, Y. Amides of the fruit of Zanthoxylum spp.
Phytochemistry 1997, 44, 1125–1127.
19) Xiong, Q.; Shi, D.; Yamamoto, H.; Mizuno, M. Alkylamides from
27) Riera, C. E.; Vogel, H.; Simon, S. A.; le Coutre, J. Artificial
sweeteners and salts producing a metallic taste sensation activate
TRPV1 receptors. Am. J. Physiol. Regul. Integr. Comp. Physiol.
pericarps of Zanthoxylum bungeanum. Phytochemistry 1997, 46,
1
123–1126.
20) Chen, I. S.; Chen, T. L.; Lin, W. Y.; Tsai, I. L.; Chen, Y. C.
Isobutylamides from the fruit of Zanthoxylum integrifoliolum.
Phytochemistry 1999, 52, 357–360.
21) Bryant, B. P.; Mezine, I. Alkylamides that produce tingling
paresthesia activate tactile and thermal trigeminal neurons. Brain
Res. 1999, 842, 452–460.
2
007, 293, R626–R634.
(28) Esteves, A. P.; Rodrigues, L. M.; Silva, M. E.; Gupta, S.; Oliveira-
Campos, A. M. F.; Machalicky, O.; Mendonca, A. J. Synthesis
and characterization of novel fluorescent N-glycoconjugates.
Tetrahedron 2005, 36, 8625–8632.
22) Patel, A. J.; Honore, E.; Lesage, F.; Fink, M.; Romey, G.;
Lazdunski, M. Inhalational anesthetics activate two-pore-domain
background K+ channels. Nat. Neurosci. 1999, 2, 422–426.
23) Talley, E. M.; Bayliss, D. A. Modulation of TASK-1 (Kcnk3)
and TASK-3 (Kcnk9) potassium channels: volatile anesthetics and
Received for review October 1, 2008. Revised manuscript received
January 4, 2009. Accepted January 10, 2009.
JF803067R