Modulation of thermo-transient receptor potential (thermo-TRP) channels by thymol-based compounds

Article abstract of DOI:10.1016/j.bmcl.2012.03.055

A series of thirty-three thymol, p-cymene-3-carboxylic acid, and 3-amino-p-cymene derivatives was synthesized and tested on TRPA1, TRPM8, and TRPV3 channels. Most of them acted as strong modulators of TRPA1, TRPM8, and TRPV3 channels with EC₅₀ and/or IC₅₀ values distinctly lower than those of thymol and related monoterpenoids. Some of the compounds examined, that is, 3c, 4e, f, 6b, and 8b exhibited an appreciable subtype-selectivity.

Full text of DOI:10.1016/j.bmcl.2012.03.055

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3535–3539
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Modulation of thermo-transient receptor potential (thermo-TRP) channels
by thymol-based compounds
Giorgio Ortar a, , Ludovica Morera a, Aniello Schiano Moriello b, Enrico Morera a, Marianna Nalli a,
⇑
Vincenzo Di Marzo c, Luciano De Petrocellis b
a Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, piazzale Aldo Moro 5, 00185 Roma, Italy
b Endocannabinoid Research Group, Istituto di Cibernetica, Consiglio Nazionale delle Ricerche, via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
c Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thirty-three thymol, p-cymene-3-carboxylic acid, and 3-amino-p-cymene derivatives was syn-
thesized and tested on TRPA1, TRPM8, and TRPV3 channels. Most of them acted as strong modulators of
TRPA1, TRPM8, and TRPV3 channels with EC50 and/or IC50 values distinctly lower than those of thymol
and related monoterpenoids. Some of the compounds examined, that is, 3c, 4e, f, 6b, and 8b exhibited
an appreciable subtype-selectivity.
Received 11 November 2011
Revised 12 March 2012
Accepted 14 March 2012
Available online 20 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Thermo-TRP channels
TRPA1
TRPM8
TRPV3
Tymol derivatives
p-Cymene-3-carboxylic acid derivatives
3-Amino-p-cymene derivatives
The transient receptor potential (TRP) family of ion channels are
cation-permeable ion channels divided into six mammalian sub-
families based on sequence homology and denoted TRPA (Ankyrin),
TRPC (Canonical), TRPM (Melastatin), TRPML (Mucolipin), TRPP
(Polycystin), and TRPV (Vanilloid).1 They are involved in a wide ar-
ray of physiological functions including vision, taste, olfaction,
hearing, touch, and thermo- and osmosensation and have emerged
as important drug targets in pathological conditions such as pain,
cancer, and bone development, kidney, and respiratory disorders.2
Several TRP channels act as polymodal sensors that are activated
by physical stimuli such as temperature as well as chemical stim-
uli, a property that is exploited by several natural product derived
ligands.3 Indeed, naturally occurring modulators, in particular
monoterpenoids, have now been reported for most thermo-TRP
channels, that is, TRP channels activated by distinct temperature
thresholds,2c,4 specifically TRPA1, TRPM8, and TRPV1-4 (Fig. 1).
Thermo-TRPs are however generally not highly specific for one gi-
ven natural ligand, but are activated by a number of chemically re-
lated monoterpenoid agonists and overlapping features have been
identified in agonist profiles of thermo-TRPs, such as TRPA1,
TRPM8, TRPV1, and TRPV3.
OH
OH
OH
O
(+)-Borneol
Camphor
Carvacrol
(-)-Carveol
OH
OH
O
Eucalyptol
Geraniol
( )-Linalool
OH
OH
OH
(-)-Isopulegol
(-)-Menthol
Thymol (1)
Figure 1. Structures of some monoterpenoid modulators of thermo-TRPs.
In this respect, thymol (1), an important phenolic monoterpene
component of oils derived from thyme (Thymus vulgaris) and oreg-
⇑
Corresponding author. Tel.: +39 6 49913612; fax: +39 6 49693268.
E-mail address: giorgio.ortar@uniroma1.it (G. Ortar).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.055

3536
G. Ortar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3535–3539
Table 1
Results of TRPA1, TRPM8, and TRPV3 assays of thymol-based compounds 2–8, thymol (1), and 3-amino-p-cymene hydrochloride (10)a
O
O
O
O
O
H
N
O
O
R
R
O
NHR
R
N
H
R
N
H
OR
N
H
NHR
O
O
2
3
4
5
7
8
6
Compd
R
TRPA1
(efficacy)b
TRPA1
(EC50
TRPA1
M) (IC50
TRPM8
TRPM8
TRPM8
M) (IC50
TRPM8
M)e (IC50
TRPV3
TRPV3
TRPV3
M) (IC50, l
M)g
,
l
,
l
M)c (efficacy)d (EC50
,
l
,
l
,
l
M)f (efficacy)d (EC50
,
l
2a
2b
2c
2d
2e
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
Ph-4-Me
Ph-4-t-Bu
Ph-4-Ph
Ph-4-OMe
CH2Ph-4-Ph
Ph-4-t-Bu
Ph-4-Ph
205.7 17.9 3.1 1.3
194.1 3.9 12.7 1.3
204.4 10.7 3.3 0.7
149.3 1.1 1.0 0.2
225.7 23.8 1.8 1.0
8.1 1.0
11.9 1.1
5.5 0.3
2.7 0.3
20.9 6.0
<10
<10
<10
<10
<10
<10
<10
<10
<10
ND
ND
ND
ND
ND
ND
ND
ND
ND
5.2 0.7
42.7 4.3
21.2 1.0
6.5 0.3
>100
>100
>100
>100
>100
3.6 0.4
57.8 0.6
18.6 0.7
5.0 0.3
>100
>100
>100
>100
77.4 1.6
1.2 0.1
4.4 0.1
3.4 0.3
5.0 0.15
2.8 0.4
55.5 1.2 5.7 0.6
6.9 2.2
27.8 2.2
6.5 0.8
9.7 1.5
1.5 0.3
>100
2.9 0.5
16.6 4.3
5.0 1.2
1.1 0.2
17.7 0.3 21.4 0.9
33.8 2.6 7.7 1.5
23.1 0.4 4.9 0.3
52.7 3.7 3.8 1.3
25.3 0.4 12.2 0.6
64.3 6.4 14.5 5.9
42.3 0.5 20.9 0.8
27.1 1.2 4.0 1.0
36.5 3.6 1.2 0.4
244.7 42.6 14.1 10.4 >100
195.2 27.2 7.4 2.8
259.6 46.1 4.4 3.1
194.2 7.4 3.6 0.5
12.7 0.7
9.2 4.2
3.6 1.2
0.84 0.07 40.0 2.5
3.6 0.8
3.4 0.8
26.15 4.0 43.2 1.0
60.0 0.5
>100
Ph-4-Cl
Ph-4-OMe
Ph-4-t-Bu
Ph-4-CF3
Ph-4-Ph
Ph-4-Cl
Ph-4-OMe
CH2CH2Ph
97.9 5.4
65.4 8.7
0.6 0.1
3.9 2.4
0.6 0.3
45.8 23.4 3.2 0.1
ND
0.85 0.2
1.7 0.2
1.9 0.2
13.9 1.3
<10
22.1 0.6 59.7 10.9 75.1 4.4
40.9 1.0 3.6 0.4
43.4 2.2 11.8 2.8
194.1 3.9 11.1 0.9
12.3 1.4
4.8 0.4
3.4 0.2
4.0 0.8
4.9 2.2
59.5 1.1
75.4 1.6
<10
ND
35.3 0.1
<10
12.4 0.1
ND
65.3 1.5
64.1 0.5
<10
ND
ND
0.27 0.01 0.6 0.01
0.32 0.01 <10
OBz
4g
241.1 5.3 25.9 3.2
204.4 10.7 6.4 1.6
70.2 5.6
<10
ND
69.4 1.9
61.4 0.1
<10
ND
41.9 6.5
N
H
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
7a
7b
7c
8a
8b
8c
1
Ph-4-t-Bu
Ph-4-Ph
8.8 0.6
17.9 5.9
>100
24.5 3.1
<10
58.1 1.2
<10
<10
<10
0.44 0.14 1.0 0.1
ND
4.8 0.4
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
5.1 0.5
ND
ND
ND
52.2
43.4 6.6
1.0 0.1
4.1 0.8
15.2 0.4
78.3 8.4
14.1 1.9
0.9 0.3
34.8 1.8 10.6 2.8
43.2 2.6 0.8 0.3
<10
39.3 0.1 6.3 0.1
15.0 0.1 15.9 0.1
<10
6.2 0.7
2.5 0.6
>100
59.2 6.3
11.3 4.7
0.9 0.05
14.7 0.8
31.2 4.0
11.6 1.4
0.3 0.03
0.7 0.1
23.1 4.0
5.8 1.0
6.0 0.9
9.8 0.2
11.8 1.8
31.7 2.7
2.5 0.4
>100
Ph-4-OMe
CH2Ph-4-Ph
CH2Ph-3-I
(CH2)7Ph
Ph-4-t-Bu
Ph-4-CF3
Ph-3-Ph
149.3 1.1 45.9 0.6
212.3 4.5 6.5 0.5
154.3 29.8 20.5 11.3 43.2 4.0
93.4 7.3 0.8 0.3 1.4 0.3
132.1 3.1 1.3 0.2
178.3 17.7 41 16
162.8 2.7 4.7 0.4
175.9 4.6 12.7 1.2
159.5 5.3 34.5 4.2
145.2 12.9 4.1 1.8
133.6 13.1 4.8 2.0
263.6 12.2 7.4 1.5
234.7 18.6 4.3 2.1
ND
3.9 0.3
29.7 3.9
20.4 1.7
39.5 11.2
1.9 0.3
50.3 4.2
40.9 0.6
9.6 0.8
59.7 8.7
2.8 0.8
92.5 30.9
64.3 14.3
3.3 0.9
12.8 2.4
85.4 8.2
—
ND
0.78 0.04 <10
0.85 0.04 62.5 1.0 1.4 0.2
4.1 1.3
7.2 0.6
4.3 0.3
25.7 0.2
24.4 3.8
16.8 2.2
4.5 0.2
25.0 5.8
<10
<10
<10
<10
<10
<10
30.3 0.2
<10
<10
73.7 6.4
13.9 1.6
16.2 0.9
10.4 0.4
6.1 0.2
11.7 3.1
5.2 0.3
26.7 1.3
25.4 1.3
77.5 1.8
<10
ND
32.2 1.3 34.3 4.9
64.8 0.2 5.2 0.1
<10
53.4 0.9 2.5 0.3
27.6 1.5 15.1 2.6
<10
31.6 5.0 7.6 5.2
16.9 0.1 5.9 0.1
<10
Ph-4-Ph
Ph-4-OMe
Ph-4-t-Bu
Ph-4-Ph
Ph-4-OMe
Ph-4-t-Bu
Ph-4-Ph
ND
ND
223.9 54.2 32.2 26.3 27.9 5.1
114.1 2.3 14.1 0.9 34.4 1.6
118.7 9.6 286.6 86.3 125.4 0.5 31.15
57.4 0.6 64.2 1.4 212.3 8.0 33.8 2.3
37.4 0.4
38.5 3.4
Ph-4-OMe
<10
ND
314.6 8.9 169.1 12.5 34.7 0.2 84.1 1.6
238.0 1.3 113.4 2.8 <10 ND
10
>100
a
b
c
d
e
f
Data are means SEM of N = 3 determinations.
As percent of allyl isothiocyanate (100 M).
Determined against the effect of allyl isothiocyanate (100
As percent of ionomycin (4 M).
Determined against the effect of icilin (0.25
l
lM).
l
lM).
Determined against the effect of menthol (20
Determined against the effect of thymol (100
l
l
M).
M). ND, not determined when efficacy is lower than 10%.
g
ano (Origanum vulgare), behaves as a promiscuous TRP agonist,
activating TRPA1, TRPM8, and TRPV3 channels.2c,5 The evaluation
of the ability of eight thymol-related alkyl-substituted phenols to
activate hTRPA1 revealed that the potency of these compounds
generally diminished with decreasing logP values, while the
presence of sterically hindered and branched alkyl groups at the
ortho-position of the seemingly essential hydroxyl group increased
potency.5a Taken together, the SAR data suggest that thymol and
related alkylphenols might bind in a hydrophobic pocket to acti-
vate TRPA1 via a non covalent mechanism.
In a recent work we have disclosed a series of (ꢀ)-menthyl-
amine derivatives acting as potent TRPM8 antagonists with IC50
values similar or lower than those of previously reported unselec-
tive antagonists.6 Since identification of monoterpenoid pharmaco-
phore through systematic SAR approaches can be expected to lead
to the individuation of TRP subtype-selective modulators and, per-
haps, of novel potential treatment options for TRP ‘channelopa-
thies’, we aimed at further investigating the pharmacological
profile of some thermo-TRPs by synthesizing and testing on TRPA1,
TRPM8, and TRPV3 channels a series of compounds based on the

G. Ortar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3535–3539
3537
thymol structure (Table 1). In these compounds, the hydroxyl
group of thymol has been replaced by bulky groups connected to
the p-cymene moiety through ester, amide, carbamate, and urea
functionalities in the hope that these modifications would enhance
efficacy/potency and/or selectivity, in analogy to previous results
with some menthol-based compounds.6,7 Thus, for example, some
N-menthyl arylcarboxamides and arylcarbamates with substitu-
ents like t-Bu, CF3, Ph, Cl on the aryl moiety potently antagonized
TRPM8,6 while some N-aryl-p-menthane-3-carboxamides like
WS-12, WS-5, and CPS-125 were reported to robustly and selec-
tively activate TRPM8 with EC50 values up to 16-fold lower than
menthol.7a The choice of substituents was also partly inspired by
the lipophilic nature common to many TRPA1 modulators.2c
The synthesis of esters 2 and 3 was carried out by condensation
of 1 with the appropriate carboxylic acids, or of p-cymene-3-car-
boxylic acid (9) with the appropriate phenols, respectively, using
N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC) as the carboxylate activator and 4-dimethylaminopyridine
(DMAP) as the nucleophilic catalyst (Schemes 1 and 2). Amides 4
and 5 were prepared by condensation of 9 with the appropriate
amines or of 3-amino-p-cymene hydrochloride (10) with the
appropriate carboxylic acids, respectively, using 1-hydroxybenzo-
triazole (HOBt)/EDC as the carboxylate activator (Schemes 2
O
a
.
NH2 HCl
N
H
R
10
5a-f
Scheme 3. Synthesis of compounds 5a–f. Reagents and conditions: (a) RCO2H,
HOBt/EDC, rt, 1 h, then 10, Et3N, DMF, rt, 16 h.
the rat TRPM8, or the rat TRPV3 cDNAs.12 Rat TRPA1-HEK293 cells
exhibited a sharp increase in intracellular Ca2+ upon application of
most of the 33 compounds. With the exception of 4d, f, all com-
pounds were more potent than 1 and 10 at activating rat TRPA1
channels. Thymol proved indeed in our hands to be a weak rat
TRPA1 activator (EC50 = ꢁ287
l
M), while EC50 values ranging from
M have been reported by using different techniques
6 to 127
l
when thymol was tested on human TRPA1.5a This result confirms
interspecies differences observed in the profile of some TRPA1 li-
gands, including thymol.2c Eighteen of the compounds examined
here, that is, 2a, c–e, 3b–d, 4a, b, 5a, d, f, 6a, c, 7a–c, and 8a pro-
duced a robust activation of TRPA1 in transfected cells with EC50
and 3). Acid
9
was prepared by
a
palladium-catalyzed
values between 0.6 and 7.4 lM without any theoretical ability, at
hydroxycarbonylation reaction8 on thymyl triflate (11)9
(Scheme 4). 3-Amino-p-cymene hydrochloride (10) was obtained
by a palladium-catalyzed coupling between thymyl triflate (11)
and benzophenone imine, followed by acid hydrolysis of the
resulting imine 12 (Scheme 4).10 Carbamates 6 and ureas 8 were
synthesized by condensation of 10 with the appropriate aryl
chloroformates or isocyanates (Scheme 5), while the preparation
of carbamates 7 was accomplished by condensation of 1 with the
appropriate isocyanates (Scheme 6).11
least in the case of compounds of the 2–5 and 8 series, to form
covalent adducts with critical cysteine and lysine residues of the
channel.2c Given their electrophilic nature, the possibility that car-
bamates of the 6 and 7 series might act via covalent interactions
cannot in principle be ruled out. However, the finding that the car-
bamate URB597, a well known covalent fatty acid amide hydrolase
(FAAH) inhibitor,13 activates TRPA1 channels, whereas the closely
related carbamate URB523 had no effect on the channel,14 would
not suggest a covalent binding mechanism for this class of com-
pounds. Overall, esters 2 and 3 and carbamates 7 exhibited a slight
superiority over amides 4 and 5, carbamates 6, and ureas 8,
although the two most potent TRPA1 activators proved to be
amides 4a and 5f. None of above eighteen compounds, with the
partial exception of 3c, was selective versus TRPM8 and/or TRPV3
activation. Five-min preincubation of TRPA1-HEK293 cells with
some of compounds 2–7, that is, 2a, c, d 3c, d 4a–c, 5a, d, f, 6a–
d, and 7c and then continued incubation with allyl isothiocyanate
caused inhibition of TRPA1 response to this agonist with IC50 val-
The 33 derivatives synthesized here and their precursors 1 and
10 were tested for their ability to induce intracellular Ca2+ eleva-
tion in HEK293 cells stably transfected with either the rat TRPA1,
a
O
OH
O
R
ues <10 lM. The carbamate 6 moiety appeared to serve as the best
functionality for TRPA1 desensitization, followed by esters and
amides, while carbamates 7 and ureas 8 were generally poor
TRPA1 desensitizers. In particular, compound 6b behaved as a
rather selective full TRPA1 antagonist.
As concerns TRP assays on TRPM8-HEK293 cells, most of amides
4 were able to both activate/desensitize this channel, with EC50 and
1
2a-e
Scheme 1. Synthesis of compounds 2a–e. Reagents and conditions: (a) RCO2H,
EDC/DMAP, CH2Cl2, rt, 16 h.
IC50 values between 0.27 and 1.7
lM and between 0.32 and
5.0 M, respectively. The N-phenethyl substituent appeared to be
l
particularly effective at increasing both activation and desensitiza-
tion potencies (compound 4f). Two of amides 4, that is, 4e, f were
also selective versus TRPA1 and TRPV3 channels. In contrast, none
of the other functionalities, with the exception of 5a, c and 7c acti-
vated significantly TRPM8. Interestingly, a ‘true’ TRPM8 antago-
nism (that is, inhibition without agonism per se, and hence not
a
O
R
O
CO2H
3a-d
due to desensitization) with IC50 values <10 lM could be observed
b
9
with esters 2a, d, amides 5b, f and carbamate 6a. However, these
compounds were not selective versus the other channels. Neither
esters 3 nor ureas 8 had any effect on TRPM8 in terms of activation
or inhibition. In agreement with literature data,5b thymol did not
activate/desensitize TRPM8 to a major extent.
Fourteen compounds belonging to all seven structural classes
explored, that is, 2a, c–e, 3d, 4a, c, 5b,d, 6a, d, 7a, and 8a, b ele-
vated intracellular Ca2+ in rat TRPV3-HEK293 cells with EC50 values
H
N
R
O
4a-g
Scheme 2. Synthesis of compounds 3a–d or 4a–g. Reagents and conditions: (a)
ROH, EDC/DMAP, CH2Cl2, rt, 16 h. (b) HOBt/EDC, rt, 1 h, then RNH2, DMF, rt, 16 h.

3538
G. Ortar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3535–3539
a
CO2H
OSO2CF3
9
b
c
11
Ph
Ph
.
N
NH2 HCl
12
10
Scheme 4. Synthesis of compounds 9 and 10. Reagents and conditions: (a) Pd(OAc)2, dppf, CO, AcOK, DMSO, 60 °C, 3 h. (b) Pd(OAc)2, ( )-BINAP, Cs2CO3, HN = C(Ph)2, dioxane,
80 °C, 16 h. (c) 2 N HCl, THF, rt, 16 h.
Supplementary data
O
O
a
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
03.055.
or
R
.
R
NH2 HCl
N
H
O
N
H
N
H
10
6a-e
8a-c
References and notes
Scheme 5. Synthesis of compounds 6a–e and 8a–c. Reagents and conditions: (a)
ROCOCl or RNCO, Et3N, DMF, rt, 16 h.
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O
a
R
OH
O
N
H
1
7a-c
Scheme 6. Synthesis of compounds 7a–c. Reagents and conditions: (a) RNCO, Et3N,
DMF, rt, 16 h.
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7. (a) Sherkheli, M. A.; Vogt-Eisele, A. K.; Bura, D.; Beltrán Márques, L. R.;
Gisselmann, G.; Hatt, H. J. Pharm. Pharm. Sci. 2010, 13, 242; (b) Kühn, F. J. P.;
Kühn, C.; Lückhoff, A. J. Biol. Chem. 2009, 284, 4102.
8. Cacchi, S.; Lupi, A. Tetrahedron Lett. 1992, 33, 3939.
9. Radivoy, G.; Alonso, F.; Yus, M. Tetrahedron 1999, 55, 14479.
10. Wolfe, J. P.; Ahman, J.; Sadighi, J. P.; Singer, R. A.; Buchwald, S. L. Tetrahedron
Lett. 1997, 38, 6367.
more than 11-fold lower than thymol (0.8–7.6 vs 84.1 lM), a result
in contrast with data by Vogt-Eisele et al.5b who suggested that the
presence of a hydroxyl group in a series of monoterpenoids includ-
ing thymol is critical for TRPV3 activation and that its derivatiza-
tion abolishes the activity. Five-min exposure of rTRPV3-HEK293
cells to compounds 2a,c–e, 3b, d, 4a, c, d, 5a, b, 6a, d, 7a, and 8a
before stimulation with thymol induced desensitization of this
channel with IC50 values between 1.1 and 9.6 lM. Esters 2 ap-
peared to be superior to the other derivatives for both activation
11. General procedure for the synthesis of compounds 2 and 3. A solution of the
appropriate carboxylic acid or 9 (0.33 mmol), 1 or the appropriate phenol
(0.30 mmol), EDC (0.55 mmol), and DMAP (0.5 mmol) in dry CH2Cl2 (2 mL) was
stirred at room temperature overnight. The mixture was 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. General procedure for the synthesis
of compounds 4 and 5. To a stirred solution of 9 or the appropriate carboxylic
acid (0.34 mmol) in DMF (3 mL) HOBt (0.35 mmol) and EDC (0.35 mmol) were
added at 0 °C. The mixture was stirred at 0 °C for 15 min and at room
temperature for 1 h. Then, the appropriate amine or 10 (0.40 mmol) and Et3N
(in the case of 10, 0.40 mmol) were added, and the mixture was stirred at room
temperature overnight. The mixture was diluted with brine and extracted with
AcOEt. The organic phase was washed with 2 N HCl solution, saturated
NaHCO3, and brine, dried (Na2SO4), and evaporated under vacuum. The residue
was purified by column chromatography. General procedure for the synthesis
of compounds 6. To a stirred 20% phosgene solution in toluene (0.72 mL,
and desensitization properties.
Finally, as concerns the influence of aromatic substitution pat-
terns, a comparison between the three R-groups that are the same
across all seven functionalities, that is, Ph-4-t-Bu, Ph-4-Ph, and Ph-
4-OMe, revealed that their order of TRP modulating ability (EC50
and IC50 values <10 lM were taken into account) was as follows:
TRPA1, Ph-4-t-Bu > Ph-4-OMe > Ph-4-Ph; TRPM8, Ph-4-OMe > Ph-
4-t-Bu > Ph-4-Ph; TRPV3, Ph-4-Ph > Ph-4-t-Bu > Ph-4-OMe. The
differences were not large however and none of substituents ap-
peared in general to be critical per se for selectivity.
In conclusion, in this Letter we have presented a series of deriv-
atives of thymol, p-cymene-3-carboxylic acid, and 3-amino-p-cym-
ene that act as strong modulators of TRPA1, TRPM8, and TRPV3
channels with EC50 and/or IC50 values distinctly lower than those
of thymol and related monoterpenoids. The subtype-selectivity of
some of the compounds examined, that is, 3c, 4e, f, 6b, and 8b
may stimulate their use as pharmacological tools and contribute
to the basic knowledge of thermo-TRP channels.
1.37 mmol)
a solution of the appropriate phenol (0.34 mmol) and Et3N
(0.40 mmol) in dry toluene (3.4 mL) was added dropwise at 0 °C. The
reaction mixture was stirred at room temperature for 3 h and evaporated
under vacuum. The residue of the crude chloroformate was dissolved in dry
CH2Cl2 (1.4 mL) and a solution of 10 (0.29 mmol) and Et3N (0.57 mmol) in dry
DMF (1.23 mL) was added dropwise at room temperature with stirring. The
reaction mixture was stirred at room temperature overnight, diluted with

G. Ortar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3535–3539
3539
water, and extracted with AcOEt. The organic phase was washed twice with
brine, dried (Na2SO4), and evaporated under vacuum. The residue was purified
by column chromatography. General procedure for the synthesis of
compounds 7 and 8. A solution of the appropriate isocyanate (0.48 mmol), 1
or 10 (0.48 mmol), and Et3N (0.72 mmol) in dry DMF (2 mL) was stirred at
room temperature overnight. The mixture was diluted with brine and
extracted with AcOEt. The organic phase was washed twice with brine, dried
(Na2SO4), and evaporated under vacuum. The residue was purified by column
chromatography.
12. See Supplementary Material for details of biochemical assays.
13. (a) Otrubova, K.; Ezzili, C.; Boger, D. L. Bioorg. Med. Chem. Lett. 2011, 21, 4674;
(b) Petrosino, S.; Di Marzo, V. Curr. Opin. Invest. Drugs 2010, 11, 51.
14. Niforatos, W.; Zhang, X.-F.; Lake, M. R.; Walter, K. A.; Neelands, T.; Holzman, T.
F.; Scott, V. E.; Faltynek, C. R.; Moreland, R. B. Mol. Pharmacol. 2007, 71, 1209.