(-)-Menthylamine derivatives as potent and selective antagonists of transient receptor potential melastatin type-8 (TRPM8) channels

Article abstract of DOI:10.1016/j.bmcl.2010.03.076

A series of twenty-two (-)-menthylamine derivatives was synthesized and tested on TRPM8, TRPV1, and TRPA1 channels. Five of the novel compounds, that is, 1d, 1f, 2b, 2c, and 2e behaved as potent TRPM8 antagonists with IC₅₀ values versus icilin and (-)-menthol between 20 nM and 0.7 μM, and were between 4- and ～150-fold selective versus TRPV1 and TRPA1 activation. Compound 1d also induced caspase 3/7 release in TRPM8-expressing LNCaP prostate carcinoma cells, but not in non-TRPM8 expressing DU-145 cells. Five other derivatives, that is, 1a, 1g, 1h, 2f, and 2h were slightly less potent than previous compounds but still relatively selective versus TRPV1 and TRPA1.

Full text of DOI:10.1016/j.bmcl.2010.03.076

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2729–2732
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
(ꢀ)-Menthylamine derivatives as potent and selective antagonists of
transient receptor potential melastatin type-8 (TRPM8) channels
b,
a
b
Giorgio Ortar a, , Luciano De Petrocellis , Ludovica Morera , Aniello Schiano Moriello ,
*
*
Pierangelo Orlando c, Enrico Morera a, Marianna Nalli a, Vincenzo Di Marzo d
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 Campi Flegrei 34, 80078, Pozzuoli (Napoli), Italy
c Endocannabinoid Research Group, Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, via P. Castellino 111, 80131 Napoli, Italy
d 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 twenty-two (ꢀ)-menthylamine derivatives was synthesized and tested on TRPM8, TRPV1, and
TRPA1 channels. Five of the novel compounds, that is, 1d, 1f, 2b, 2c, and 2e behaved as potent TRPM8
Received 4 February 2010
Revised 17 March 2010
Accepted 19 March 2010
Available online 25 March 2010
antagonists with IC50 values versus icilin and (ꢀ)-menthol between 20 nM and 0.7
lM, and were between
4- and ꢁ150-fold selective versus TRPV1 and TRPA1 activation. Compound 1d also induced caspase 3/7
release in TRPM8-expressing LNCaP prostate carcinoma cells, but not in non-TRPM8 expressing DU-145
cells. Five other derivatives, that is, 1a, 1g, 1h, 2f, and 2h were slightly less potent than previous com-
pounds but still relatively selective versus TRPV1 and TRPA1.
Keywords:
Transient receptor potential channels
TRPA1
Ó 2010 Elsevier Ltd. All rights reserved.
TRPV1
TRPM8
(ꢀ)-Menthylamine derivatives
Ion channels belonging to the transient receptor potential (TRP)
superfamily that are activated by distinct temperature thresholds
are referred to as ‘thermo-TRPs’1 and their targeting represents a
novel and promising strategy in pain relief.2 Four of these channels
(TRPV1–TRPV4) respond to heat and two others (TRPA1 and
TRPM8) are sensitive to cold. One of the best-investigated ther-
mo-TRPs is the transient receptor potential channel of melastatin
type-8 (TRPM8), which is activated by moderately cool tempera-
tures (<23–28 °C) and by compounds that evoke a sensation of
coolness, such as (ꢀ)-menthol, the homomenthylamide WS-12
and icilin (Fig. 1).3 Interestingly, both (ꢀ)-menthol and icilin stim-
ulate human TRPA1 channels as well,4 and a number of TRPV1
antagonists such as capsazepine, BCTC, and SB-452533 and the
non-specific blocker of various calcium channels SKF96365 have
been reported to be also potent TRPM8 antagonists,5 indicating a
significant degree of pharmacological overlap between TRPA1,
TRPV1 and TRPM8 channels. In contrast, WS-12 elicits a robust re-
sponse in TRPM8 expressing HEK cells (EC50 = 306 and 193 nM3)
thermo-TRPs7 are activated at a concentration optimally effective
for TRPM8 responses. The localization of TRPM8 in both Ad and
C-fibers may account for abnormal cold sensitivity in some patho-
logic states, thus providing a rationale for the design of TRPM8
modulators as novel antihyperalgesic or antiallodynic agents,8
although the role of TRPM8 in cold allodynia in patients with cold
injury was recently questioned.9
The expression of TRPM8 in tissues not subjected to tempera-
ture changes suggests, however, other important functions for this
ion channel and indeed a series of benzyloxyphenylamides and
carbamates,10 and of phosphorus-containing benzothiophene and
benzofuran derivatives11 has been disclosed in the patent litera-
ture as TRPM8 antagonists for the treatment of urological and
respiratory disorders. Last but not least, TRPM8 is overexpressed
in a range of cancers including prostate, breast, lung, and colon,
while, within normal tissues, it is predominantly expressed in
the human prostate.6,12 Its knock-down in human prostate carci-
noma cells using mRNA silencing techniques was reported to inhi-
bit cell proliferation.12 Thus, TRPM8 is a tumor marker with
potential use in cancer diagnosis as well as therapy.
and in Xenopus oocytes (EC50 = 12 l
M),7 while none of related
TRP channels like TRPM3 and TRPV63 and of the other
With this background, TRPM8 agonists and antagonists based
on the 3-substituted-p-menthane structure have been reported in
a patent application to be effective at inhibiting growth of cells
expressing TRPM8 and/or inducing their apoptosis and/or necrosis,
but no evidence for their capability of modulating the functional
* Corresponding authors. Tel.: +39 6 49913612; fax: +39 6 49693268 (G.O.); tel.:
+39 081 8675173 (L.D.P.).
E-mail addresses: giorgio.ortar@uniroma1.it (G. Ortar), l.depetrocellis@cib.na.cn-
r.it (L.D. Petrocellis).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.076

2730
G. Ortar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2729–2732
Cl
H
S
N
O
O2N
OH
H
N
HO
HO
N
N
H
N
OH
O
OMe
(-)-Menthol
WS-12
Icilin
Capsazepine
N
N
OMe
N
O
N
O
O
N
Cl
N
N
N
H
N
H
N
H
Cl
Br
N
OMe
BCTC
SB-452533
SKF96365
Clotrimazole
Figure 1. Structures of some TRPM8 activators and antagonists.
activity of recombinant TRPM8 selectively over other TRP channels
was provided.13 Recently, clotrimazole, an antimycotic drug widely
used for the topical treatment of candidiasis and ringworm infec-
channels, that is, menthol or icilin, capsaicin, and mustard oil iso-
thiocyanate, respectively, to check whether or not they exhibited
any functional antagonism. Most compounds were found to effi-
ciently antagonize the agonist effect of either menthol or icilin
on TRPM8-mediated intracellular Ca2+ elevation in HEK-293 cells
overexpressing rat TRPM8 channels, and, usually only at much
higher concentrations, to stimulate intracellular Ca2+ elevation in
HEK-293 cells overexpressing rat TRPA1 or human TRPV1 chan-
nels, but not in wild-type HEK-293 cells.
tions, was described as
a potent TRPM8 antagonist (IC50
ꢁ200 nM for the inhibition of inward TRPM8 currents) and a useful
tool to discriminate between TRPM8- and TRPA1-mediated re-
sponses, although this compound also activated TRPV1 channels.14
Thus, lack of selectivity represents the most important problem
that prevents the use of known TRPM8 antagonists, as it compli-
cates the interpretation of their effects in vitro and in vivo. To
the best of our knowledge, no truly selective TRPM8 antagonist
has been reported to date.
With the aim of gaining further insight into the functional and
pharmacological properties of thermo-TRPs and in an attempt to
identify compounds with the ability to discriminate between
TRPM8, TRPV1 and TRPA1, we prepared 22 new derivatives of
(ꢀ)-menthylamine and examined their functional activity at these
three channels (Table 1).
The synthesis of amides 1a–i was carried out by condensation
of (ꢀ)-menthylamine hydrochloride (4)15 and the appropriate car-
boxylic acids 5a–i using 1-hydroxybenzotriazole (HOBt)/N-ethyl-
N0-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as
the carboxylate activator (Scheme 1).16 Carbamates 2a–j and ureas
3a–c were synthesized by condensation of (ꢀ)-menthylisocyanate
(6)15 with the appropriate phenols 7a–j or amines 8a–c (Scheme
2).16
The choice of analogues was based on substituents found in
other TRP modulators, with the reasoning that their presence on
the (ꢀ)-menthylamine scaffold could boost their potency or selec-
tivity toward TRPM8. Thus, for instance, the t-Bu-Ph, oleyl, Cl-Ph,
CF3-Ph, serotonin and vanillamine groups of 1b, 1c, 1g, 1h, 2b,
2c, 2d, 2e, 2h, 2i, 3b, and 3c could mimic the same groups of TRPV1
ligands BCTC, capsazepine, N-oleoylethanolamine, N-arachidono-
ylserotonin (AA-5-HT), capsaicin, and of piperazinyl carbamates
and ureas recently found to act as fatty acid amide hydrolase
(FAAH) and TRP ligands.17
The results of the pharmacological assays are shown in Table 1
and the salient aspects of the structure–activity relationships
(SARs) can be summarized as follows. Five of the novel compounds
examined, that is, 1d, 1f, 2b, 2c, and 2e behaved as potent TRPM8
antagonists with IC50 values versus icilin and (ꢀ)-menthol between
20 nM and 0.7
lM, and were between 4- and ꢁ150-fold selective
versus TRPV1 and TRPA1 activation. Five other derivatives, that
is, 1a, 1g, 1h, 2f, and 2h, although slightly less potent than the
above mentioned compounds, were still relatively selective versus
TRPV1 and TRPA1. Three other compounds, that is, 1e, 2a, and 2j
elicited a potent TRPM8 antagonist activity but were not selective
versus TRPA1 activation. Finally, three compounds, that is, 1b, 3a,
and 3c exhibited a significant and rather selective TRPV1 agonist
activity. With the exception of 1b, 1c, and 1i, all N-menthyl amides
1 and N-menthyl carbamates 2 showed potent TRPM8 antagonism
with no clear-cut superiority of one type of functionality over the
other. The inactivity of compound 1b was therefore rather unex-
pected in view of the good antagonist activity of the carbamate
congener 2e. Thus, reversal of the amide linkage shifted the agon-
ism of p-menthane-based TRPM8 ligands toward antagonism,
whereas that of the carbamate function retained full antagonist
activity.13 The reverse amide 1i, which directly compares with
WS-12, did not exhibit however a particularly good TRPM8 antag-
onist potency. The selectivity towards TRPV1 and TRPA1 of com-
pounds of the 1 and 2 series was appreciably influenced by the
nature and position of the substituent on the aromatic moiety
and by the length of the alkyl chain in the case of
x-phenylalkyla-
The novel 22 compounds synthesized here were tested for their
ability to induce intracellular Ca2+ elevation in HEK-293 cells stably
transfected with either the human TRPV1, the rat TRPA1, or the rat
TRPM8 cDNAs. Control experiments were carried out using non-
transfected HEK-293 cells. The compounds that were found to be
inactive at elevating intracellular Ca2+ were then given to cells
5 min before known agonists of TRPM8, TRPV1, and TRPA1
mides. In this respect, compounds with meta substituted aromatic
rings were generally more selective than the para substituted ones,
irrespective of the nature of the substituent (see compounds 2b, 2f,
2h and 2c, 2g, 2i). Finally, the three urea derivatives 3 were uni-
formly inactive as TRPM8 antagonists.
The most potent and highly selective (>80-fold) TRPM8 func-
tional antagonist synthesized here, compound 1d, was also tested

G. Ortar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2729–2732
2731
Table 1
Results of TRPM8, TRPV1, and TRPA1 assays of (ꢀ)-menthylamine derivatives 1–3a
O
N
H
R
Compound
R
TRPM8b (IC50
,
l
M) TRPM8c (IC50
,
l
M) TRPV1
TRPV1 (EC50
,
l
M) TRPA1
(efficacy)e
TRPA1 (EC50, lM)
(efficacy)d
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
2d
2e
2f
2g
2h
2i
Ph-4-Me
Ph-4-t-Bu
Oleyl
2.5 0.1
60.8 9.3
>100
2.2 0.1
5.4 2.1
27.4 1.9
4.4 0.4
NM
0.25 0.10
NM
NM
NM
NM
137.6 10.1
NM
34.7 0.01
42.8 1.9
67.0 2.2
NM
222.6 20.7
NM
73.9 2.4
220.2 20.7
88.9 9.9
272.8 35.2
76.0 2.3
282.5 16.1
87.2 6.9
309.0 52.1
181.2 25.4
134.7 30.3
NM
4.9 1.7
NM
4.1 0.01
0.4 0.1
26.7 1.3
NM
40.2 12.8
NM
0.7 0.1
13.8 7.8
3.0 1.4
36.7 17.7
11.7 1.4
31.0 5.2
6.7 2.6
30.0 17.5
7.1 3.7
3.2 2.0
97.6 11.9
60.6 4.4
0.02 0.002
0.4 0.03
0.4 0.04
2.8 0.2
0.7 0.02
12.8 0.6
1.1 0.04
0.3 0.04
0.2 0.01
5.9 1.1
0.1 0.02
0.05 0.002
0.7 0.06
1.5 0.2
0.8 0.03
2.2 0.2
Ph-4-Ph
0.05 0.01
0.4 0.01
0.5 0.1
2.1 0.1
1.6 0.1
9.6 3.5
0.7 0.1
0.6 0.03
0.7 0.06
3.3 0.6
0.08 0.01
1.5 0.4
7.4 0.4
0.5 0.04
4.1 0.4
2.6 0.2
7.8 0.3
(CH2)6Ph
(CH2)7Ph
Ph-3-Cl
8.0 0.01
5.5 0.01
41.9 1.8
53.8 0.8
9.6 0.5
NM
7.6 0.6
4.1 0.2
NM
NM
NM
Ph-4-Cl
Ph-4-OMe
OPh-4-Me
OPh-3-CF3
OPh-4-CF3
OPh-3-t-Bu
OPh-4-t-Bu
OPh-3-Ph
OPh-4-Ph
OPh-3-Cl
OPh-4-Cl
OPh-4-OMe
3.4 1.0
7.4 1.5
3.3 0.01
16.3 0.6
23.8 1.1
50.9 1.0
4.4 1.8
22.6 3.1
15.1 3.8
46.6 3.0
NM
5.8 1.0
18.3 2.4
7.1 0.6
NM
19.2 8.3
42.8 23.8
53.8 3.6
2j
3a
3b
3c
>100
>100
24.4 0.1
12.7 0.01
63.2 0.8
1.0 0.06
52.2 0.03
0.23 0.02
158.8 12.4
11.2 3.4
N
H
OH
HN
31.0 0.7
19.8 0.6
15.8 0.8
30.5 2.9
NM
NM
N
H
OMe
OH
N
H
NM
NM
NM, not measurable when efficacy is lower than 10%.
a
Data are means SEM of N = 3 determinations.
b
Determined against the effect of icilin (0.25
lM).
c
d
e
Determined against the effect of menthol (20
As percent of ionomycin (4 M).
As percent of allyl isothiocyanate (100
l
M).
l
lM).
on the human androgen-responsive prostate carcinoma cell line,
LNCaP, which expresses high levels of TRPM8, and as a negative
control, on the DU-145 cell line, which, like other androgen-unre-
sponsive prostate carcinoma cell lines, does not express apprecia-
ble levels of TRPM8 channels.18 As expected, and as previously
5
RCO2H ( )
O
R
.
NH2 HCl
N
H
shown for other TRPM8 antagonists,13 compound 1d (1
lM) in-
1a-i
4
duced the apoptosis of LNCaP, but not DU-145, cells, as measured
by the release of caspase 3/7 (from 16521 3779 to
58576 29921, in LNCaP cells, P <0.01; and from 9281 247 to
9768 1496, not significant, in DU-145 cells; arbitrary units,
Scheme 1. Synthesis of compounds 1a–i. Reagents and conditions: 5, HOBt/EDC, rt,
1 h, then 4, Et3N, DMF, rt, 16 h.
7a-j) or
RNH2 HCl (
ArOH (
O
O
.
8a-c
)
or
R
R
N
H
O
N
C
O
N
H
N
H
6
2a-j
3a-c
Scheme 2. Synthesis of compounds 2a–j and 3a–c. Reagents and conditions: Et3N, DMF or AcOEt, rt, 16 h.

2732
G. Ortar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2729–2732
8. (a) Fleetwood-Walker, S.; Mitchell, R.; Proudfoot, C. W. J. PCT Int. Appl.
WO2008015403, 2008.; (b) Xing, H.; Chen, M.; Ling, J.; Tan, W.; Gu, J. G. J.
Neurosci. 2007, 27, 13680; (c) Proudfoot, C. J.; Garry, E. M.; Cottrell, D. F.; Rosie,
R.; Anderson, H.; Robertson, D. C.; Fleetwood-Walker, S. M.; Mitchell, R. Curr.
Biol. 2006, 16, 1591.
means SD of N = 3 experiments). In LNCaP cells, the compound
was as efficacious as the standard pro-apoptotic mixture of anti-
FAS antibody and camptothecin19 (from 16521 3779 to
58954 5130 in LNCaP cells, P <0.01; and from 9281 247 to
165595 15965, in DU-145 cells; arbitrary units, means SD of
N = 3 experiments).
In conclusion, in the present work we have disclosed a series of
derivatives of (ꢀ)-menthylamine that act as potent TRPM8 antag-
onists with IC50 values similar or lower than those of previously re-
ported unselective antagonists. Special attention should be paid to
compounds 1d, 1f, 2b, 2c, and 2e, the excellent selectivity of which
may allow for their use as pharmacological tools, thus aiding future
biological studies aimed at deciphering the multiple roles of
TRPM8 in mammalian species. The pro-apoptotic effect of 1d also
warrants further studies aiming at investigating its potential ther-
apeutic use against prostate carcinoma.
9. Namer, B.; Kleggetveit, I. P.; Handwerker, H.; Schmelz, M.; Jorum, E. Pain 2008,
139, 63.
10. (a) Lampe, T.; Alonso-Alija, C.; Beck, H.; Rosentreter, U.; Sandner, P.; Stahl, E.;
Stelte-Ludwig, B. PCT Int. Appl. WO2007017094, 2007.; (b) Lampe, T.; Alonso-
Alija, C.; Beck, H.; Rosentreter, U.; Sandner, P.; Stahl, E.; Stelte-Ludwig, B. PCT
Int. Appl. WO2007017093, 2007.; (c) Lampe, T.; Alonso-Alija, C.; Bauser, M.;
Beck, H.; Rosentreter, U.; Sandner, P.; Stahl, E.; Stelte-Ludwig, B. PCT Int. Appl.
WO2007017092, 2007.; (d) Lampe, T.; Alonso-Alija, C.; Stelte-Ludwig, B.;
Sandner, P.; Bauser, M.; Beck, H.; Lustig, K.; Rosentreter, U.; Stahl, E.; Takagi, H.
PCT Int. Appl. WO2006040136, 2006.
11. Colburn, R. W.; Dax, S. L.; Flores, C.; Matthews, J.; Qin, N.; Youngman, M. A.;
Teleha, C.; Reany, L. PCT Int. Appl. WO2007134107, 2007.
12. (a) Kim, S.-H.; Nam, J.-H.; Park, E.-J.; Kim, B.-J.; Kim, S.-J.; So, I.; Jeon, J.-H.
Biochim. Biophys. Acta, Mol. Bas. Dis. 2009, 1792, 33; (b) Prevarskaya, N.; Zhang,
L.; Barritt, G. Biochim. Biophys. Acta, Mol. Bas. Dis. 2007, 1772, 937; (c) Flockerzi,
V.; Wissenbach, U. BIOspektrum 2005, 11, 620.
13. Natarajan, S. K.; Moreno, O.; Graddis, T. J.; Duncan, D.; Laus, R.; Chen, F. PCT Int.
Appl. WO2005020897, 2005.
14. Meseguer, V.; Karashima, Y.; Talavera, K.; D’Hoedt, D.; Donovan-Rodriguez, T.;
Viana, F.; Nilius, B.; Voets, T. J. Neurosci. 2008, 28, 576.
Acknowledgment
15. Schopohl, M. C.; Bergander, K.; Kataeva, O.; Fröhlich, R.; Waldvogel, S. R.
Synthesis 2003, 2689.
The authors are grateful to Roberta Verde for valuable technical
help.
16. General procedure for the synthesis of compounds 1a–i. To a stirred solution of
5a–i (0.25 mmol) in DMF (1 mL) were added at 0 °C HOBt (0.26 mmol) and EDC
(0.26 mmol). The mixture was stirred for 15 min at 0 °C and for 1 h at room
temperature. Then (ꢀ)-menthylamine hydrochloride (4) (0.30 mmol) and Et3N
(0.30 mmol) were added, and the mixture was stirred overnight at room
temperature. 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 or crystallized from CH2Cl2/hexane. General
procedure for the synthesis of compounds 2a–j and 3a–c. A solution of (ꢀ)-
menthylisocyanate (6) (0.47 mmol), 7a–j or 8a–c (0.47 mmol), and Et3N
(0.56 mmol) in dry DMF or AcOEt (1 mL) was stirred overnight at room
temperature. 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 or crystallized from MeOH.
Supplementary data
Supplementary data (detailed experimental procedures and
characterization data for all products and biochemical assays)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.03.076.
References and notes
1. Venkatachalam, K.; Montell, C. Annu. Rev. Biochem. 2007, 76, 387.
2. Cortright, D. N.; Szallasi, A. Curr. Pharm. Des. 2009, 15, 1736.
3. Bödding, M.; Wissenbach, U.; Flockerzi, V. Cell Calcium 2007, 42, 618.
4. Kühn, F. J. P.; Kühn, C.; Lückhoff, A. J. Biol. Chem. 2009, 284, 4102.
5. Wiel, A.; Moore, S. E.; Waite, N. J.; Randall, A.; Gunthorpe, M. J. Mol. Pharmacol.
2005, 68, 518.
6. Beck, B.; Bidaux, G.; Bavencoffe, A.; Lemonnier, L.; Thebault, S.; Shuba, Y.;
Barritt, G.; Skryma, R.; Prevarskaya, N. Cell Calcium 2007, 41, 285.
7. Sherkheli, M. A.; Gisselmann, G.; Vogt-Eisele, A. K.; Doerner, J. F.; Hatt, H. Pak. J.
Pharm. Sci. 2008, 21, 370.
17. Morera, E.; De Petrocellis, L.; Morera, L.; Schiano Moriello, A.; Ligresti, A.; Nalli,
M.; Woodward, D. F.; Di Marzo, V.; Ortar, G. Bioorg. Med. Chem. Lett. 2009, 19,
6806.
18. Prevarskaya, N.; Flourakis, M.; Bidaux, G.; Thebault, S.; Skryma, R. Biochem. Soc.
Trans. 2007, 35, 133.
19. Costa-Pereira, A. P.; Cotter, T. G. Br. J. Cancer 1999, 80, 371.