Effect of acyclic monoterpene alcohols and their derivatives on TRP channels

Article abstract of DOI:10.1016/j.bmcl.2014.10.012

A series of thirty-six geraniol, nerol, citronellol, geranylamine, and nerylamine derivatives was synthesized and tested on TRPA1, TRPM8, and TRPV1 channels. Most of them acted as strong modulators of TRPA1 channels with EC₅₀ and/or IC₅₀ values <1 μM. None was able to significantly activate TRPM8 channels, while thirteen of them behaved as 'true' TRPM8 antagonists. Little or no effect was generally observed on TRPV1 channels. Some of the compounds examined, that is, compounds 1d,g,n, 2c,d,h,i,o, 3b,e exhibited an appreciable selectivity for TRPA1 subtype.

Full text of DOI:10.1016/j.bmcl.2014.10.012

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5507–5511
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Effect of acyclic monoterpene alcohols and their derivatives on TRP
channels
b
a
a
b
Giorgio Ortar a, , Aniello Schiano Moriello , Enrico Morera , Marianna Nalli , Vincenzo Di Marzo ,
⇑
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 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-six geraniol, nerol, citronellol, geranylamine, and nerylamine derivatives was synthe-
sized and tested on TRPA1, TRPM8, and TRPV1 channels. Most of them acted as strong modulators of
TRPA1 channels with EC50 and/or IC50 values <1 lM. None was able to significantly activate TRPM8 chan-
nels, while thirteen of them behaved as ‘true’ TRPM8 antagonists. Little or no effect was generally
observed on TRPV1 channels. Some of the compounds examined, that is, compounds 1d,g,n, 2c,d,h,i,o,
3b,e exhibited an appreciable selectivity for TRPA1 subtype.
Received 4 August 2014
Revised 30 September 2014
Accepted 1 October 2014
Available online 12 October 2014
Keywords:
Transient receptor potential channels
Ó 2014 Elsevier Ltd. All rights reserved.
TRPA1
TRPM8
TRPV1
Acyclic monoterpene alcohols
Mammalian transient receptor potential (TRP) channels consti-
tute a large family of non-selective cation-permeable channels
subdivided into six subfamilies on the basis of their amino acid
sequence homology: TRPA (ankyrin), TRPC (canonical), TRPM
(melastatin), TRPML (mucolipin), TRPP (polycystin), and TRPV
(vanilloid).1 They are cellular sensors involved in a host of physio-
logical functions (such as nociception, taste perception, vision,
mechano-, osmo-, and thermosensation). Dysfunctions of TRP
channels have been implicated in various disease states (such as
chronic pain, overactive bladder, obesity, diabetes, chronic cough,
chronic obstructive pulmonary disease, dermatological disorders,
cancer).2 Therefore, the therapeutic potential of targeting TRP
channels by agonists or antagonists has been extensively and
increasingly explored over the past 15 years. Thermo-TRPs, that
is, TRP channels activated by distinct temperature thresholds
(TRPA1, TRPM8, TRPV1-4),2g appear to be preferred targets for
numerous plant-derived chemicals.3 Thus, for example, TRPA1
responds to cinnamaldehyde, curcumin, thymol, gingerols, euge-
nol, TRPM8 is the ‘menthol receptor’, and TRPV1 is activated by
capsaicin, resiniferatoxin, piperine, and camphor. Many naturally
occurring ligands exhibit however relatively low potencies and
poor specificity for individual members of the TRP family.
Among promiscuous TRP modulators are monoterpenic alco-
hols geraniol, nerol, and citronellol (Fig. 1). In detail, geraniol and
nerol were described as weak activators of TRPA1, TRPM8, and
TRPV1,4a,c while, at higher concentrations, they also inhibited
currents thereby induced. The dose-response curves for TRPV1
activation by citronellol and geraniol revealed a rank order potency
of citronellol (43 lM) > geraniol (102 l
M),4b while geranyl acetate,
the main constituent of thyme geraniol essential oil, did not affect
TRPV1 activity. Geraniol proved to be an extremely weak TRPM8
agonist with a rather low efficacy (28%) and potency (5.9 mM).4d
TRPM8 and TRPV1, but not TRPA1, were found to be involved in
the antinociceptive effects of citronellyl acetate.5 N-Geranyl
cyclopropylcarboxamide enhanced Ca2+ influx in hTRPV1- and
hTRPA1-expressing cells with EC50 values of 115
lM and
83.6
l
M, respectively.6 A number of terpenoid analogues, includ-
ing geraniol and geranylamine derivatives, designed to be used
for treating disorders of nerve transmission, was claimed in a pat-
ent application to exhibit various degrees of agonist and antagonist
activity at TRPV1 channels.7 Eventually, citronellyl acetate has
been included in a list of TRPA1 antagonists intended to reduce
the burn associated with hydrogen peroxide in oral care composi-
⇑
Corresponding authors. Tel.: +39 6 49913612; fax: +39 6 49913133 (G.O.); tel.:
+39 081 8675173 (L.D.P.).
tions (70% reduction of H2O2 TRPA1 activation at 400 l
M).8
Our previous structure–activity studies on natural product
E-mail addresses: giorgio.ortar@uniroma1.it (G. Ortar), l.depetrocellis@icb.cnr.it
ligands of TRP channels have resulted in the identification of some
(L. De Petrocellis).
http://dx.doi.org/10.1016/j.bmcl.2014.10.012
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

5508
G. Ortar et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5507–5511
OH
examined, that is, 1b,d,f–i,l,n,o, 2b–j,m,o,p, 3b–e produced a
robust activation of TRPA1 in transfected cells with EC50 values
<1 M. Most of these compounds are non-electrophilic and it is
OH
OH
l
therefore unlikely that they act on TRPA1forming covalent adducts
with critical cysteine residues of the channel.2e On the other hand,
given their electrophilic nature, the possibility that carbamates
1n,o, 2o,p might act via covalent interactions cannot in principle
be ruled out. However, the structurally related carbamate
URB597, a known potent FAAH covalent inhibitor,17 was reported
to activate TRPA1 channels probably through a non-covalent,
direct gating mechanism.18 Furthermore, our most potent carba-
mate 2o gave a very sluggish reaction (43% conversion after
24 h) with the biologically relevant model thiol cysteamine19 and
the N-carbamylated cysteamine 6 was isolated as the main product
(Scheme 6).20 These data argue against a covalent binding mecha-
nism with thiol groups of TRPA1 channel for carbamates as well.
Overall, esters and carbamates exhibited a clear superiority
over amides and ureas. Nerol derivatives were better TRPA1
activators than derivatives of the other two alcohols (compare
compounds 2b with 1b and 3b, 2e with 1f and 3c, 2m with 1l,
and 2o with 1n). A number of the aforementioned twenty-five
compounds was selective (P100 fold) versus TRPM8 and TRPV1
activation (compounds 1d,g,n, 2c,d,h,i,o, 3b,e). Five-min preincu-
bation of TRPA1-HEK293 cells with each of the twenty-five
compounds, and then continued incubation with allyl isothiocya-
nate caused inhibition of TRPA1 response to this agonist with
Geraniol
Nerol
(
)-Citronellol
NH2
NH2
Geranylamine
Nerylamine
Figure 1. Structures of the acyclic monoterpene alcohols and amines derivatized in
this study.
(ꢀ)-menthylamine derivatives as potent and selective TRPM8
antagonists,9 the elucidation of structural determinants for the
TRPA1 and TRPV1 agonist properties of gingerols,10 the identifica-
tion of thymol-based compounds as modulators of some thermo-
TRP channels,11 and the report of 3-ylidenephthalides as dual
modulators of TRPA1 and TRPM8 channels.12
In continuation of these studies and with the aim of better
defining the TRP modulating properties of geraniol, nerol, citronel-
lol, geranylamine, and nerylamine (Fig. 1) and identifying more
potent TRP modulators based on the structure of these monoterp-
enoids, we have now prepared 36 derivatives and examined their
functional activity at TRPA1, TRPM8, and TRPV1 channels
(Table 1).
IC50 values 61 lM. Carbamates 1n, 2o and 4-chlorobenzyl esters
The hydroxyl/amino group of the reference compounds has
been replaced by lipophilic groups connected to the monoterpene
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- and thymol-based compounds.9,11 It is well known
that the TRP domain of the different transient potential receptor
channels contains various sets of hydrophobic residues, involved
in channel assembly and activation.13
Esters 1a–i, 2a–j, 3a–e were prepared by acylation of the corre-
sponding alcohols with either acetic anhydride (esters 1a, 2a, 3a)
or the appropriate carboxylic acids, using N-ethyl-N0-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (EDC) as the carboxylate
activator and 4-dimethylaminopyridine (DMAP) as the nucleo-
philic catalyst (esters 1b–i, 2b–j, 3b–e) (Scheme 1). Amides 1j,k
and 2k,l were prepared by condensation of geranylamine (4a)
and nerylamine (4b), respectively, with the appropriate carboxylic
acids, using 1-hydroxybenzotriazole (HOBt)/EDC as the carboxyl-
ate activator (Scheme 2). Ureas 1l,m and 2m,n were synthesized
by condensation of amines 4a,b with the appropriate isocyanates
(Scheme 3), while the preparation of carbamates 1n,2o and 1o,2p
was accomplished by condensation of 4a,b with 4-tert-butylphenyl
chloroformate or of 4-tert-butylaniline with geranyl/neryl chloro-
formate, respectively (Scheme 4). Amines 4a,b were prepared from
the corresponding alcohols by a Mitsunobu reaction, using potas-
sium phthalimide as the nucleophile, followed by hydrazinolysis
of the phthalimide intermediates 5a,b (Scheme 5).14,15
of nerol and citronellol (compounds 2f, 3d) appeared to serve as
the best functionality for TRPA1 desensitization, while reverse
carbamates 1o, 2p were ꢁ10 times less potent and amides
were generally poor TRPA1 desensitizers. The EC50 and IC50 values
were, with the partial exception of 1o, 2h,p, comparable and
thus, none of compounds acted as ‘true’ antagonist, in a fashion
similar to the effect of capsaicin on the TRPV1 channel.21
Consistent with their interaction with a series of environmental
irritants and endogenous mediators of inflammation and pain,
TRPA1 channels have been validated as a promising target for var-
ious potential therapeutic applications including neuropathic and
inflammatory pain and airway disorders.22 Although a large num-
ber of TRPA1 modulators has been already reported, many of them
tend to be either reactive or of low potency and/or selectivity and
therefore they are not optimal tools for pharmacological studies.
The identification of easily synthesized, non-reactive, non-volatile,
stable compounds 1d,g,n, 2c,d,h,i,o, 3b,e, as potent and selective
TRPA1 modulators should enable further assessment of the
TRPA1 pharmacology and designate them as candidates for future
evaluation of in vivo efficacy in rodent models of inflammatory and
neuropathic pain.
As concerns TRP assays on TRPM8-HEK293 cells, none of thirty-
six derivatives was able to significantly activate this channel, while
thirteen of them behaved as ‘true’ antagonists (that is, inhibition
without agonism per se, and hence not due to desensitization) with
IC50 values <1
lM (compounds 2e,f,j,m, 3d) or between 1 lM and
10 M (compounds 1f,j,k, 2b,g,k,l, 3c). The nerol chain appeared to
l
The 36 derivatives synthesized here and the reference alcohols
were tested for their ability to induce intracellular Ca2+ elevation in
HEK293 cells stably transfected with either the rat TRPA1, the rat
TRPM8, or the human TRPV1 cDNAs (Table 1). The antagonist or
desensitizing activity was assessed by adding the test compounds
5 min before stimulation of cells with reference agonists.16
Rat TRPA1-HEK293 cells exhibited a sharp increase in intracel-
lular Ca2+ upon application of most of the 36 compounds which
were 1–3 orders of magnitude more potent than the corresponding
alcohols as rTRPA1 activators. Geraniol, nerol, and citronellol
proved indeed to be weak rat TRPA1 activators with efficacies
<10, 14.1, and 26.1, respectively. Twenty-five of the compounds
be particularly effective in this respect, as eight of the thirteen
TRPM8 antagonists belong to this series. It is worth noting the
marked difference between compounds 2b,f,j,m and the corre-
sponding geraniol derivatives 1b,g,i,l which are completely
inactive.
Together with TRPA1, TRPM8 is involved in the development
and maintenance of cold allodynia arising from traumatic neurop-
athy, as well as in bladder pain, and TRPM8 antagonists comprising
a variety of chemotypes with a potential role in these chronic
pain conditions has been recently disclosed.23 In addition, TRPM8
represents a prostate tumor marker with a potential use in the
diagnosis as well as in the therapy of this type of cancer. Indeed,

G. Ortar et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5507–5511
5509
Table 1
Results of the TRPA1, TRPM8, and TRPV1 assays of monoterpenic alcohols geraniol, nerol, and citronellol and their derivatives/analogues 1–3a
X
R
X
R
X
R
O
O
O
1
2
3
Compound
X
R
TRPA1
TRPA1
TRPA1
(IC50
TRPM8
TRPM8
TRPM8
(IC50
TRPV1
TRPV1
TRPV1
(IC50, l
M)f
(efficacy)b
(EC50
,
l
M)
,
l
M)c
(efficacy)d
(EC50
,
l
M)
,
l
M)e
(efficacy)d
(EC50
,
l
M)
Geraniol
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
Nerol
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
<10
ND
>100
21.2
1.0
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
>100
>100
>100
>100
>100
>100
5.8
>100
>100
>100
3.8
<10
<10
17.6
<10
<10
<10
<10
<10
13.2
27.7
57.5
10.5
21.2
36.0
22.4
21.9
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
57.9
<10
20.1
50.1
15.8
24.0
<10
<10
<10
<10
<10
<10
ND
ND
6.0
ND
ND
ND
ND
ND
8.0
4.0
1.1
16.2
2.5
4.8
10.7
8.7
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.43
ND
1.7
8.9
6.5
7.8
ND
ND
ND
ND
ND
ND
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.89
>100
0.71
9.9
92.3
76.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.53
>100
0.38
15.7
>100
74.6
O
O
O
O
O
O
O
O
R = Me
R = Ph-4-Me
R = Ph-3-Me
R = Ph-4-CN
83.9
75.2
69.8
72.6
18.8
85.5
86.1
88.8
85.6
89.1
97.1
123.6
74.9
107.7
72.7
14.1
58.5
82.0
78.0
78.3
73.2
81.8
88.0
61.6
73.4
93.5
99.7
72.7
104.3
79.4
100.3
73.6
26.1
32.2
55.2
88.9
88.0
77.1
25.8
0.33
2.4
0.48
11.9
0.36
0.12
0.51
0.39
1.2
1.4
0.73
>100
0.32
0.13
0.38
0.17
1.4
R = Ph-3,5-CF3
R = CH2Ph-4-Me
R = CH2Ph-4-Cl
R = CH2Ph-4-OMe
R = CH2CH2Ph
R = CH2Ph-4-Cl
R = CH2CH2Ph
R = NHPh-3-Cl
R = NHcC6H11
R = OPh-4-t-Bu
R = NHPh-4-t-Bu
O
NH
NH
NH
NH
NH
O
7.5
0.61
4.9
7.4
5.5
0.48
11.8
0.038
0.57
>100
20.7
0.11
0.14
0.34
0.041
0.013
0.26
0.94
0.18
0.11
0.94
13.9
0.41
4.4
0.029
0.29
>100
>100
0.87
0.12
0.048
0.34
>100
>100
86.5
>100
>100
>100
1.2
30.8
>100
0.28
0.75
9.7
0.045
0.092
11.7
13.3
0.093
0.14
0.26
0.043
0.016
0.14
0.10
O
O
O
O
O
O
O
O
R = Me
R = Ph-4-Me
R = Ph-3-Me
R = Ph-4-CN
R = CH2Ph-4-Me
R = CH2Ph-4-Cl
R = CH2Ph-4-OMe
R = CH2Ph-3-I
R = CH2-2-Naft
R = CH2CH2Ph
R = CH2Ph-4-Cl
R = CH2CH2Ph
R = NHPh-3-Cl
R = NHcC6H11
R = OPh-4-t-Bu
R = NHPh-4-t-Bu
>100
>100
0.29
2.2
O
O
0.081
0.11
1.0
NH
NH
NH
NH
NH
O
6.6
2.2
0.43
2.65
0.018
0.041
>100
12.2
0.28
0.090
0.053
0.16
0.71
30.0
>100
>100
>100
>100
>100
2.4
2p
Citronellol
3a
3b
3c
3d
>100
>100
>100
>100
>100
>100
O
O
O
O
O
R = Me
R = Ph-4-Me
R = CH2Ph-4-Me
R = CH2Ph-4-Cl
R = CH2CH2Ph
0.83
>100
3e
a
b
c
d
e
f
Data 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.
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
Determined against the effect of capsaicin (0.1
l
l
M).
l
l
M).
l
M). ND, not determined when efficacy is lower than 10%.
both activation by monoterpenoid menthol24 or blockade by a
menthylamine derivative9 of TRPM8 channels induce apoptosis of
TRPM8-expressing LNCaP prostate carcinoma cells.
specific and not to represent a general property of the class, as just
a few members were able to effectively modulate these channels,
while the majority of the compounds were significantly less potent
or completely inactive.
Eleven compounds belonging to the geraniol and nerol series,
that is, 1b,h–j,l,m,o, 2m–p, modestly elevated intracellular Ca2+ in
In conclusion, we have disclosed here a series of derivatives of
the acyclic monoterpene alcohols geraniol, nerol, and citronellol
and of the related amines geranylamine and nerylamine that act
as potent and selective TRPA1 channel modulators with EC50 and
IC50 values distinctly lower than those of reference alcohols, com-
pare favourably in terms of potency and/or selectivity with most of
other chemotypes presented as TRPA1 modulators and published
recently,22,25 and qualify therefore as useful pharmacological tools
in studies on TRP channel modulation using natural products as
lead compounds.
hTRPV1-HEK293 cells with EC50 values ranging from 1 to 10 lM.
The sole TRPV1 activator endowed with submicromolar potency
was the amide 2k. Five-min exposure of hTRPV1-HEK293 cells to
compounds 1j,l,m, 2k,m before stimulation with capsaicin induced
desensitization of this channel with IC50 values between 0.38
and 9.9 M. Amides and ureas appeared to be superior to the other
derivatives as far as TRPV1 desensitization was concerned.
lM
l
Contrary to what observed on TRPA1 channels, the effects of the
thirty-six derivatives on TRPM8 and TRPV1 channels appear to be

5510
G. Ortar et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5507–5511
OCOCH3
H
N
H
N
H
N
O
Ph-4-t-Bu
SH
a
O
O
a
b
OH
1a, 2a, 3a
2o
6
O
R
Scheme 6. Reaction of 2o with cysteamine. Reagents and conditions: (a) cyste-
amine, DMSO, rt, 72 h.
O
geraniol or nerol or citronellol
1b-i, 2b-j, 3b-e
References and notes
Scheme 1. Synthesis of compounds 1a–i, 2a–j, 3a–e. Reagents and conditions: (a)
Ac2O, Et3N, CH2Cl2, rt, 16 h. (b) RCO2H, EDC/DMAP, CH2Cl2, rt, 16 h.
1. (a) Li, M.; Yu, Y.; Yang, J. Adv. Exp. Med. Biol. 2011, 704, 1; (b) Liu, Y.; Qin, N. Adv.
Exp. Med. Biol. 2011, 704, 185.
2. (a) Kaneko, Y.; Szallasi, A. Br. J. Pharmacol. 2014, 171, 2474; (b) Bishnoi, M.;
Premkumar, L. S. Open Pain J. 2013, 6, 10; (c) Szallasi, A.; Sheta, M. Expert Opin.
Investig. Drugs 2012, 21, 1351; (d) Moran, M. M.; McAlexander, M. A.; Bíró, T.;
Szallasi, A. Nat. Rev. Drug Disc. 2011, 10, 601; (e) Baraldi, P. G.; Preti, D.;
Materazzi, S.; Geppetti, P. J. Med. Chem. 2010, 53, 5085; (f) Di Marzo, V.; De
Petrocellis, L. Curr. Med. Chem. 2010, 17, 1430; (g) Vay, L.; Gu, C.; McNaughton,
P. A. Expert Rev. Clin. Pharmacol. 2010, 3, 687; (h) Trevisani, M.; Szallasi, A. Open
Drug Discov. J. 2010, 2, 37; (i) Cortright, D. N.; Szallasi, A. Curr. Pharm. Des. 2009,
15, 1736; (j) Patapoutian, A.; Tate, S.; Woolf, C. J. Nat. Rev. Drug Disc. 2009, 8, 55;
(k) Gunthorpe, M. J.; Szallasi, A. Curr. Pharm. Des. 2008, 14, 32; (l) Vennekens,
R.; Owsianik, G.; Nilius, B. Curr. Pharm. Des. 2008, 14, 18; (m) Westaway, S. M. J.
Med. Chem. 2007, 50, 2589; (n) Gharat, L.; Szallasi, A. Drug Dev. Res. 2007, 68,
477; (o) Venkatachalam, K.; Montell, C. Annu. Rev. Biochem. 2007, 76, 387.
3. (a) Premkumar, L. S. ACS Chem. Neurosci. 2014. article ASAP; (b) Vetter, I.; Lewis,
R. J. Adv. Exp. Med. Biol. 2011, 704, 41; (c) Appendino, G.; Minassi, A.; Pagani, A.;
Ech-Chahad, A. Curr. Pharm. Des. 2008, 14, 2.
H
NH2.HCl
N
R
O
a
4a b
,
1j,k, 2k,l
Scheme 2. Synthesis of compounds 1j,k, 2k,l. Reagents and conditions: (a) RCO2H,
HOBt/EDC, rt, 1 h, then 4a or 4b, Et3N, DMF, rt, 16 h.
4. (a) Lübbert, M.; Kyereme, J.; Schöbel, N.; Beltrán, L.; Horst Wetzel, C.; Hatt, H.
PLoS ONE 2013, 8, e77998; (b) Ohkawara, S.; Tanaka-Kagawa, T.; Furukawa, Y.;
Nishimura, T.; Jinno, H. Biol. Pharm. Bull. 2010, 33, 1434; (c) Stotz, S. C.; Vriens,
J.; Martyn, D.; Clardy, J.; Clapham, D. E. PLoS ONE 2008, 3, e2082; (d) Behrendt,
H.-J.; Germann, T.; Gillen, C.; Hatt, H.; Jostock, R. Br. J. Pharmacol. 2004, 141,
737.
5. Rios, E. R.; Rocha, N. F.; Carvalho, A. M.; Vasconcelos, L. F.; Dias, M. L.; de Sousa,
D. P.; de Sousa, F. C.; Fonteles, M. M. Chem.-Biol. Interact. 2013, 203, 573.
6. Kim, M. J.; Son, H. J.; Kim, Y.; Kweon, H.-J.; Suh, B.-C.; Lyall, V.; Rhyu, M.-R. PLoS
ONE 2014, 9, e89062.
H
N
H
N
NH2.HCl
R
O
a
4a b
,
1l,m, 2m,n
Scheme 3. Synthesis of compounds 1l,m, 2m,n. Reagents and conditions: (a) RNCO,
Et3N, DMF, rt, 16 h.
7. Reed, M. A.; Weaver, D.; Sun, S., McLellan, A.; Lu, E. PCT Int. App. WO034232,
2012.
8. Haught, J. C.; Sreekrishna, K. T.; Das, S.; Hoke, S. H. H.; Coffindaffer, T. W.; Bakes,
K. A.; Glandorf, W. M. PCT Int. App. WO176897, 2013.
9. Ortar, G.; De Petrocellis, L.; Morera, L.; Schiano Moriello, A.; Orlando, P.;
Morera, E.; Nalli, M.; Di Marzo, V. Bioorg. Med. Chem. Lett. 2010, 20, 2729.
10. Morera, E.; De Petrocellis, L.; Morera, L.; Schiano Moriello, A.; Nalli, M.; Di
Marzo, V.; Ortar, G. Bioorg. Med. Chem. Lett. 2012, 22, 1674.
H
NH2.HCl
N
O
Ph-4-t-Bu
O
a
11. Ortar, G.; Morera, L.; Schiano Moriello, A.; Morera, E.; Nalli, M.; Di Marzo, V.; De
Petrocellis, L. Bioorg. Med. Chem. Lett. 2012, 22, 3535.
12. Ortar, G.; Schiano Moriello, A.; Morera, E.; Nalli, M.; Di Marzo, V.; De
Petrocellis, L. Bioorg. Med. Chem. Lett. 2013, 23, 561.
4a b
,
1n, 2o
13. Bonache, M. Á.; Balsera, B.; López-Mendéz, B.; Millet, O.; Brancaccio, D.;
Gómez-Monterrey, I.; Carotenuto, A.; Pavone, L. M.; Reille-Seroussi, M.; Gagey-
Eilstein, N.; Vidal, M.; de la Torre-Martínez, R.; Fernández-Carvajal, A.; Ferrer-
Montiel, A.; García-López, M. T.; Martín-Martínez, M.; Pérez de Vega, M. J.;
González-Muñiz, R. ACS Comb. Sci. 2014, 16, 250.
H
N
O
Cl
O
Ph-4-t-Bu
O
O
b
14. Wie˛cek, M.; Kottke, T.; Ligneau, X.; Schunack, W.; Seifert, R.; Stark, H.;
´
Handzlik, J.; Kiec-Kononowicz, K. Bioorg. Med. Chem. 2011, 19, 2850.
1o, 2p
15. General procedure for the synthesis of esters 1b–i, 2b–j, 3b–e. A solution of the
appropriate carboxylic acid (0.55 mmol), geraniol or nerol or citronellol
(0.50 mmol), EDC (0.90 mmol), and DMAP (0.82 mmol) in dry CH2Cl2 (3 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
amides 1j, k, 2k,l. To a stirred solution of the appropriate carboxylic acid
(0.50 mmol) in DMF (4 mL), HOBt (0.53 mmol) and EDC (0.53 mmol) were
added at 0 °C. The mixture was stirred at 0 °C for 15 min and at room
temperature for 1 h. Then, 4a or 4b (0.60 mmol) and Et3N (0.75 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 ureas 1l,m, 2m,n. A
solution of the appropriate isocyanate (0.48 mmol), 4a or 4b (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. General procedure for the synthesis of carbamates 1n,o, 2o, p.
To a stirred 20% phosgene solution in toluene (1.20 mL, 3.30 mmol) a solution
of 4-tert-butylphenol or of geraniol or nerol (0.58 mmol) and Et3N (0.72 mmol)
in dry toluene (5.8 mL) was added dropwise at 0 °C. The reaction mixture was
Scheme 4. Synthesis of compounds 1n, 2o, 1o, 2p. Reagents and conditions: (a) 4-t-
BuPhOCOCl, Et3N, DMF, rt, 16 h. (b) 4-t-BuPhNH2, Et3N, DMF, rt, 16 h.
O
OH
N
a
O
geraniol or nerol
5a b
,
NH2.HCl
b,c
4a b
,
Scheme 5. Synthesis of compounds 4a,b. Reagents and conditions: (a) potassium
phthalimide, PPh3, DIAD, THF, rt, 16 h. (b) NH2NH2ꢂH2O, MeOH, reflux, 2.5 h. (c) HCl,
Et2O, 0 °C.

G. Ortar et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5507–5511
5511
stirred at room temperature for 3 h and evaporated under vacuum. The residue
of the crude chloroformate was dissolved in dry CH2Cl2 (4 mL) and a solution of
4a or 4b or of 4-tert-butylaniline (0.48 mmol) and Et3N (0.96 mmol) in dry
DMF (2.1 mL) was added dropwise at room temperature with stirring. The
reaction mixture was stirred at room temperature overnight, diluted with
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. Data for selected compounds: compound 1g: yield
isothiocyanate (AITC). In the case of TRPM8 assays, the efficacy of the
agonists was first determined by normalizing their effect to the maximum
Ca2+ influx effect on [Ca2+
] observed with application of 4 lM ionomycin
i
(Alexis). 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
l
M ionomycin (Alexis). When significant,
] in wild-type (i.e., not transfected with any
i
the values of the effect on [Ca2+
construct) HEK293 cells were taken as baseline and subtracted from the values
obtained from transfected cells. Antagonist/desensitizing behaviour was
78%; oil; IR (ATR) 2918, 1733, 1492, 1242, 1150, 970 cmꢀ1 1H NMR (300 MHz,
;
CDCl3) d 1.60 (3H, s), 1.68 (6H, s), 2.01–2.10 (4H, m), 3.59 (2H, s), 4.61 (2H, d,
J = 7.2 Hz), 5.07 (1H, m), 5.32 (1H, t, J = 7.2 Hz), 7.21 (2H, d, J = 8.1 Hz), 7.29 (2H,
d, J = 8.1 Hz). 13C NMR (75 MHz, CDCl3) d 16.48, 17.69, 25.69, 26.28, 39.51,
40.66, 61.95, 118.00, 123.68, 128.66, 130.64, 131.85, 132.56, 133.00, 142.65,
171.17. Compound 1n: yield 46%; oil; IR (ATR) 3327, 2964, 1714, 1504, 1209,
evaluated against AITC (100
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
lM) for TRPA1, icilin (0.25 lM) for TRPM8, and
l
the concentration exerting a half-maximal inhibition of agonist-induced [Ca2+
]
i
elevation (IC50), which was calculated again using GraphPad PrismÒ software.
1175 cmꢀ1 1H NMR (300 MHz, CDCl3) d 1.30 (9H, s), 1.61 (3H, s), 1.69 (6H, s),
;
The effect on [Ca2+]i exerted by agonist alone was taken as 100%. Dose response
2.03–2.14 (4H, m), 3.86 (2H, t, J = 6.0 Hz), 4.96 (1H, br s), 5.09 (1H, m), 5.26 (1H,
t, J = 6.9 Hz), 7.04 (2H, d, J = 8.7 Hz), 7.35 (2H, d, J = 8.7 Hz); 13C NMR (75 MHz,
CDCl3) d 16.28, 17.70, 25.68, 26.44, 31.45, 34.42, 39.10, 39.51, 119.98, 120.96,
123.86, 126.14, 131.78, 140.02, 147.98, 148.82, 154.73. Compound 2e: yield
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.
81%.; oil; IR (ATR) 2922, 1733, 1445, 1244, 1144, 972 cmꢀ1
;
1H NMR (300 MHz,
17. (a) Otrubova, K.; Ezzili, C.; Boger, D. L. Bioorg. Med. Chem. Lett. 2011, 21, 4674;
(b) Seierstad, M.; Breitenbucher, J. G. J. Med. Chem. 2008, 51, 7327.
18. 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.;Chen, J. Mol. Pharmacol. 2007, 71, 1209.
19. Avonto, C.; Tagliatela-Scafati, O.; Pollastro, F.; Minassi, A.; Di Marzo, V.; De
Petrocellis, L.; Appendino, G. Angew. Chem., Int. Ed. 2011, 50, 467.
CDCl3) d 1.59 (3H, s), 1.67 (3H, s), 1.75 (3H, s), 2.02–2.13 (4H, m), 2.32 (3H, s),
3.57 (2H, s), 4.57 (2H, d, J = 7.2 Hz), 5.09 (1H, m), 5.35 (1H, t, J = 7.2 Hz), 7.12
(2H, d, J = 8.4 Hz), 7.17 (2H, d, J = 8.4 Hz); 13C NMR (75 MHz, CDCl3) d 17.65,
21.06, 23.51, 25.69, 26.65, 32.19, 40.96, 61.50, 119.13, 123.59, 129.12, 129.22,
131.06, 132.12, 136.59, 142.60, 171.79. Compound 2f: yield 62%; oil; IR (ATR)
2916, 1733, 1493, 1244, 1151, 971 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d 1.59 (3H,
20. The reaction between carbamate 2o (49 mg, 0.15 mmol) and cysteamine
s), 1.67 (3H, s), 1.76 (3H, s), 2.04–2.09 (4H, m), 3.59 (2H, s), 4.58 (2H, d,
J = 7.2 Hz), 5.07 (1H, m), 5.34 (1H, t, J = 7.2 Hz), 7.21 (2H, d, J = 8.4 Hz), 7.29 (2H,
d, J = 8.4 Hz); 13C NMR (75 MHz, CDCl3) d 17.65, 23.52, 25.69, 26.66, 32.20,
40.68, 61.70, 118.90, 123.54, 128.67, 130.65, 130.71, 132.20, 132.55, 142.96,
171.16. Compound 2o: yield 57%; oil; IR (ATR) 3329, 2964, 1715, 1504, 1210,
(19 mg, 0.25 mmol) in DMSO-d6 (0.75 mL) in a standard NMR tube was
monitored by 1H NMR. After completion of the reaction (ꢁ72 h), the solution
was diluted with water, and extracted with AcOEt. The organic phase was
washed twice with water, dried (Na2SO4), and evaporated under vacuum. The
residue was purified by preparative layer chromatography (silica gel, 0.5 cm
thick) and CH2Cl2/AcOEt = 7/3 as eluent to afford 18 mg (47%) of 6: wax; IR
1175 cmꢀ1 1H NMR (300 MHz, CDCl3) d 1.30 (9H, s), 1.61 (3H, s), 1.70 (3H, s),
;
1.74 (3H, s), 2.04–2.15 (4H, m), 3.83 (2H, t, J = 6.3 Hz), 4.91 (1H, br s), 5.11 (1H,
m), 5.28 (1H, t, J = 7.2 Hz), 7.03 (2H, d, J = 8.4 Hz), 7.35 (2H, d, J = 8.4 Hz); 13C
NMR (75 MHz, CDCl3) d 17.68, 23.32, 25.70, 26.43, 31.45, 31.96, 34.41, 38.79,
120.85, 120.96, 123.75, 126.13, 132.35, 140.20, 147.96, 148.83, 154.67.
Compound 3d: yield 82%; oil; IR (ATR) 2914, 1735, 1492, 1250, 1156, 1091,
(ATR) 3347, 2925, 1630, 1570, 1439, 1253 cmꢀ1 1H NMR (300 MHz, CDCl3) d
;
1.60 (3H, s), 1.65 (1H, s), 1.68 (3H, s), 1.70 (3H, s), 2.01–2.08 (4H, m), 2.77 (2H,
t, J = 6.6 Hz), 3.52 (2H, q, J = 6.6 Hz), 3.73 (2H, t, J = 6.0 Hz),), 5.07 (2H, m), 5.21
(1H, t, J = 6.9 Hz), 5.59 (1H, m). 13C NMR (75 MHz, CDCl3) d 17.68, 23.37, 25.71,
26.64, 32.06, 38.13, 38.89, 121.96, 123.82, 132.07, 139.19, 158.50. By
comparison, no reaction occurred under the same conditions with the most
potent ester 2f.
806 cmꢀ1 1H NMR (300 MHz, CDCl3) d 0.88 (3H, d, J = 6.3 Hz), 1.09–1.53 (4H,
;
m), 1.60 (3H, s), 1.61–1.71 (1H, m), 1.68 (3H, s), 1.88–2.10 (2H, m), 3.57 (2H, s),
4.12 (2H, m), 5.07 (1H, t, J = 7.2 Hz), 7.21 (2H, d, J = 8.4 Hz), 7.29 (2H, d,
J = 8.4 Hz); 13C NMR (75 MHz, CDCl3) d 17.66, 19.36, 25.39, 25.73, 29.46, 35.37,
36.93, 40.77, 63.60, 124.50, 128.66, 130.62, 131.36, 132.57, 133.00, 171.21.
16. TRPA1, TRPM8, and TRPV1 channel assays. HEK293 (human embryonic kidney)
cells stably over-expressing recombinant rat TRPA1, rat TRPM8 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+
21. Ruparel, N. B.; Patwardhan, A. M.; Akopian, A. N.; Hargreaves, K. M. Pain 2008,
135, 271.
22. Copeland, K. W.; Boezio, A. A.; Cheung, E.; Lee, J.; Olivieri, P.; Schenkel, L. B.;
Wan, Q.; Wang, W.; Wells, M. C.; Youngblood, B.; Gavva, N. R.; Lehto, S. G.;
Geuns-Meyer, S. Bioorg. Med. Chem. Lett. 2014, 24, 3464. and references therein.
23. Horne, D. B.; Tamayo, N. A.; Bartberger, M. D.; Bo, Y.; Clarine, J.; Davis, C. D.;
Gore, V. K.; Kaller, M. R.; Lehto, S. G.; Ma, V. V.; Nishimura, N.; Nguyen, T. T.;
Tang, P.; Wang, W.; Youngblood, B. D.; Zhang, M.; Gavva, N. R.; Monenschein,
H.; Norman, M. H. J. Med. Chem. 2014, 57, 2989. and references therein.
24. Zhang, L.; Barritt, G. J. Cancer Res. 2004, 64, 8365.
25. (a) Rooney, L.; Vidal, A.; D’Souza, A.-M.; Devereux, N.; Masick, B.; Boissel, V.;
West, R.; Head, V.; Stringer, R.; Lao, J.; Petrus, M. J.; Patapoutian, A.; Nash, M.;
Stoakley, N.; Panesar, M.; Verkuyl, J. M.; Schumacher, A. M.; Petrassi, H. M.;
Tully, D. C. J. Med. Chem. 2014, 57, 5129; (b) Laliberté, S.; Vallée, F.; Fournier, P.-
A.; Bedard, L.; Labrecque, J.; Albert, J. S. Bioorg. Med. Chem. Lett. 2014, 24, 3204;
(c) Hu, Y.-J.; St.-Onge, M.; Laliberté, S.; Vallée, F.; Jin, S.; Bedard, L.; Labrecque,
J.; Albert, J. S. Bioorg. Med. Chem. Lett. 2014, 24, 3199; (d) Bassoli, A.; Borgonovo,
G.; Morini, G.; De Petrocelllis, L.; Schiano Moriello, A.; Di Marzo, V. Food Chem.
2013, 141, 2044; (e) Vallin, K. S. A.; Sterky, K. J.; Nyman, E.; Bernström, J.; From,
R.; Linde, C.; Minidis, A. B. E.; Nolting, A.; Närhi, K.; Santangelo, E. M.;
Sehgelmeble, F. W.; Sohn, D.; Strindlund, J.; Weigelt, D. Bioorg. Med. Chem. Lett.
2012, 22, 5485; (f) Gijsen, H. J. M.; Berthelot, D.; De Cleyn, M. A. J.; Geuens, I.;
Brône, B.; Mercken, M. Bioorg. Med. Chem. Lett. 2012, 22, 797; (g) Baraldi, P. G.;
Romagnoli, R.; Saponaro, G.; Tabrizi, M. A.; Baraldi, S.; Pedretti, P.; Fusi, C.;
Nassini, R.; Materazzi, S.; Geppetti, P.; Preti, D. Bioorg. Med. Chem. 2012, 20,
1690; (h) Ryckmans, T.; Aubdool, A. A.; Bodkin, J. V.; Cox, P.; Brain, S. D.;
Dupont, T.; Fairman, E.; Hashizume, Y.; Ishii, N.; Kato, T.; Kitching, L.; Newman,
J.; Omoto, K.; Rawson, D.; Strover, J. Bioorg. Med. Chem. Lett. 2011, 21, 4857.
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
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 -glucose,
lM in dimethyl sulfoxide containing 0.02% Pluronic F-127, invitrogen) in
D
and 10 mM HEPES, pH 7.4), resuspended in the same buffer, and transferred
(about 100,000 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+
] were determined before and after the addition of various
i
concentrations of test compounds by measuring cell fluorescence
(kEX = 488 nm, kEM = 516 nm) at 25 °C. Curve fitting (sigmoidal dose–response
variable slope) and parameter estimation were performed with GraphPad
PrismÒ (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 [Ca2+]i) (EC50). The effects of TRPA1 agonists are
expressed as
a percentage of the effect obtained with 100 lM allyl