The design and synthesis of novel, phosphonate-containing transient receptor potential melastatin 8 (TRPM8) antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.02.060

A series of benzothiophene-based phosphonates was synthesized and many analogs within the series were shown to be potent antagonists of the TRPM8 channel. The compounds were obtained as a racemic mixture in 5 synthetic steps, and were tested for TRPM8 antagonist activity in a recombinant, canine TRPM8-expressing cell line using a fluorometric imaging plate reader (FLIPR) assay. Structure-activity relationships were developed initially by modification of the core structure and subsequently by variation of the aromatic substituents and the phosphonate ester. Compound 9l was administered intraperitoneally to rats and demonstrated engagement of the TRPM8 target in both prevention and reversal-modes in an icilin-induced 'wet-dog' shake model.

Full text of DOI:10.1016/j.bmcl.2012.02.060

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2922–2926
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The design and synthesis of novel, phosphonate-containing transient
receptor potential melastatin 8 (TRPM8) antagonists
Jay M. Matthews a, , Ning Qin b, Raymond W. Colburn c, Scott L. Dax d, Mike Hawkins e,
⇑
James J. McNally a, Laura Reany c, Mark A. Youngman f, Judith Baker c, Tasha Hutchinson g, Yi Liu a,
Mary Lou Lubin h, Michael Neeper a, Michael R. Brandt c, Dennis J. Stone c, Christopher M. Flores a
a Janssen Pharmaceuticals, Welsh & McKean Roads, Spring House, PA 19477-0776, United States
b Roche R&D, Ltd, China
c Formerly of Johnson & Johnson Pharmaceutical Research & Development, L.L.C., United States
d Galleon Pharmaceuticals, 213 Witmer Rd., Horsham, PA 19044, United States
e Trevena, Inc., 1018 W 8th Ave. #A, King of Prussia, PA 19406, United States
f Purdue Pharma, 6 Cedarbrook Dr., Cranbury Township, NJ 08512, United States
g PhaseBio Pharmaceuticals, Inc., 1 Great Valley Parkway, Suite 30, Malvern, PA 19355, United States
h Merck & Co., 770 Sumneytown Pike, West Point, PA 19486, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzothiophene-based phosphonates was synthesized and many analogs within the series
were shown to be potent antagonists of the TRPM8 channel. The compounds were obtained as a racemic
mixture in 5 synthetic steps, and were tested for TRPM8 antagonist activity in a recombinant, canine
TRPM8-expressing cell line using a fluorometric imaging plate reader (FLIPR) assay. Structure–activity
relationships were developed initially by modification of the core structure and subsequently by
variation of the aromatic substituents and the phosphonate ester. Compound 9l was administered intra-
peritoneally to rats and demonstrated engagement of the TRPM8 target in both prevention and reversal-
modes in an icilin-induced ‘wet-dog’ shake model.
Received 5 December 2011
Revised 16 February 2012
Accepted 17 February 2012
Available online 27 February 2012
Keywords:
Transient receptor potential
Melostatin 8
Cold menthol receptor (CMR1)
TRPM8
Ó 2012 Published by Elsevier Ltd.
Cold allodynia
Transient receptor potential (TRP) channels are non-selective
cation channels that are activated by a variety of stimuli. Numer-
ous members of this ion channel family have been identified to
date, a number of which are thermosensitive. Of particular interest
to us is the melastatin family, type 8 channel (TRPM8), also called
the cold-menthol receptor 1 (CMR1).1 TRPM8 is stimulated by cool
to cold temperatures as well as by the chemical agonists menthol
and icilin,2,3 which likely accounts for the cooling sensation that
these agents provoke. TRPM8 is located on nociceptive A-delta
and C-fiber neurons and is also modulated by inflammation-med-
iated second messenger signals,4,5 thereby potentially providing a
molecular basis for abnormal cold sensitivity in pathologic condi-
tions wherein pain, often of a burning nature, is a hallmark.6–8
Hypersensitivity to cold stimuli is a well-documented symptom
of inflammatory and neuropathic pain. Recent studies using
TRPM8 knock-out mice demonstrate a critical role for TRPM8 in
cold sensation.9–11 TRPM8 expression on nociceptive neurons is
up-regulated under pathological conditions, resulting in an
enhanced sensitivity to innocuous cold (i.e., cold allodynia),12,13
whereas cold allodynia seen following nerve ligation14 is attenu-
ated in TRPM8 null mice.9 The effective treatment of chronic pain,
especially following nerve injury, remains an elusive goal and still
represents a large unmet clinical need. Because cold intolerance
and paradoxical burning sensations induced by chemical or ther-
mal cooling closely parallel symptoms seen in a wide range of clin-
ical disorders, there is a strong rationale for the development of
TRPM8 modulators as novel therapeutics.15
Recently, potent, small molecule TRPM8 antagonists have been
reported.16,17 This communication describes the discovery and
optimization of a series of potent and selective TRPM8 antagonists
that are based on a benzothiophene-phosphonate template. This
structural motif afforded compounds that antagonize TRPM8-med-
iated behaviors in rats and offer the potential for treatment of cold
allodynia and other aberrations in cold sensing.
In the present study, high-throughput screening in a TRPM8 ca-
nine FLIPR18 assay at a concentration of 4
l
M led to the identifica-
tion and confirmation of a diaryl phosphonate lead, 1, which
demonstrated modest potency (IC50 = 3.6 M). The low molecular
weight and multiple points for chemical diversification were
l
⇑
Corresponding author. Tel.: +1 215 628 5214; fax: +1 215 540 4612.
E-mail address: jmatthew@its.jnj.com (J.M. Matthews).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2012.02.060

J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2922–2926
2923
O
P
OEt
attractive features of this phosphonate hit. As shown in Figure 1,
initial modification of 1 via insertion of a methylene group afforded
homolog 2, which demonstrated a four-fold improvement in po-
O
P
OEt
a
OEt
OEt
Ar
Ar
tency (IC50 = 0.95 lM).
3
Investigation of alternate scaffolds related to compound 2, using
chemistry outlined in Scheme 1, resulted in the synthesis of com-
pounds 4,19,20 as shown in Table 1. Though several of the com-
pounds had similar potency to compound 2, analogs 2-naphthyl
4a-g
F
Scheme 1. Reagents and conditions: (a) THF, n-BuLi; 4-fluorobenzyl bromide,
À70 °C (35–60%).
4c (IC50 = 0.12
lM), 2-benzothienyl 4e (IC50 = 0.25
lM), and 3-
M) demonstrated a 4- to
methyl-2-benzothienyl 4f (IC50 = 0.184
l
8-fold improvement in potency. Additionally, compound 4f repre-
sented a novel scaffold for further studies of structure–activity
relationships.
Table 1
In vitro TRPM8 IC50 data for analogs, 4a–g
OEt
O
Analogs of compound 4f were prepared utilizing the synthetic
approach outlined in Scheme 2. 3-Substituted benzothiophenes 5
were treated with n-butyllithium at reduced temperatures, fol-
lowed by treatment of the resulting anion with DMF to give 2-
formylbenzothiophenes 6. Reduction of the aldehyde with sodium
borohydride, followed by conversion of the resulting alcohol to the
bromide with phosphorous tribromide afforded bromomethyl-
benzothiophenes 7. Arbuzov reaction with a trialkylphosphite gave
phosphonate intermediates 8, which were deprotonated with n-
butyl lithium and alkylated with an appropriately substituted ben-
zyl bromide to afford compounds 9.
In cases where the requisite 3-substituted benzothiophenes
were not commercially available, Scheme 3 was utilized to synthe-
size the starting materials. For example, the 2-fluoro-aryl ketones
10 were treated with ethyl thioglycolate, in the presence of base
and heat, to afford benzothiophene carboxylate esters 11. Subse-
quent reduction of the ethyl ester gave alcohols 12, which, using
the steps outlined in Scheme 2(b–d), were converted to target
compounds 13.
P
OEt
Ar
4
F
Compda
Ar
IC50 (lM)
4a
4b
4c
4d
4e
4f
5-Chloro-1-phenyl-3-indolyl
5-Fluoro-3-benzothienyl
2-Naphthyl
1-Naphthyl
2-Benzothienyl
1.40
0.70
0.12
0.50
0.25
0.18
0.50
3-Methyl-2-benzothienyl
2-Benzofuranyl
4g
a
Target compounds were purified by reverse-phase semi-prep HPLC and purities
were judged by reverse-phase HPLC/MS with UV detection at 215 and 254 nm (YMC
J’Sphere C-18 column, 0.4 Â 5 cm; mobile phase: MeCN–H2O (0.1% TFA)). All new
compounds were characterized by ESI-MS and 400-MHz 1H NMR. Compounds were
isolated as racemic mixtures.
The 3-isopropyl benzothiophene analogs were synthesized
according to Scheme 4. Thiophenol 14 was treated with 1-bro-
mo-3-methyl-butan-2-none21 and sodium hydride, to afford 3-
methyl-1-phenylsulfanyl-butan-2-one 16, which was cyclized
using polyphosphoric acid (PPA) in the presence of heat to give
benzothiophene 17. Compound 17 was converted to compound
18 using analogous chemistry to that depicted in Scheme 2 (steps
a–d).
a
b
Br
CHO
S
S
S
5
6
7
Compound 9l was monodealkylated using DMF and sodium
azide to afford the acid analog 19, as shown in Scheme 5.
In addition, phosphine oxide analogs were prepared as shown
in Scheme 6 to study the importance of the phosphonate ester moi-
ety for TRPM8 antagonist activity. Diethyl phosphate 20 was trea-
ted with an n-propyl Grignard reagent to afford intermediate 21,22
which was subsequently treated with base and compound 7 to af-
ford dialkyl-phosphine oxide 22. Treatment of compound 22 with
strong base and 3,4-difluorobenzyl bromide gave compound 23.
The functional activity of compounds 4, 9, 13, 18, 19 and 23–26
was determined in HEK293 cells stably expressing canine TRPM8
by measuring changes in intracellular calcium concentration with
a Ca2+-sensitive fluorescent dye upon activation with icilin.18,23,24
All analogs were similarly evaluated for potential cross-reactivity
with the genetically related TRPV1 channel, another thermoTRP
O
P(OR1)2
O
P(OR1)2
d
c
S
S
8
9
R2
Scheme 2. Reagents and conditions: (a) (1) THF, n-BuLi; (2) DMF; (3) 1 N HCl,
À70 °C (99%); (b) (1) THF, MeOH, NaBH4 (84%); (2) CH2Cl2, PBr3 (98%); (c) Toluene,
P(OR1)3 (85–98%); (d) THF, n-BuLi, R2 benzylbromide (23–66%).
O
R
R
a
b
OH
R
CO2Et
F
S
S
11
12
10
OEt
O
R
O
P
OEt
OEt
OEt
O
P(OR1)2
P
S
R2
1
Br
2
13
Br
Scheme 3. Reagents and conditions: (a) DMF, NaH, ethyl thioglycolate, 90 °C (39–
Figure 1. Structure of HTS lead 1 and homolog 2.
81%); (b) (1) THF, LiAlH4 (53–100%).

2924
J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2922–2926
Table 3
channel that is activated by, inter alia, noxious heat, protons and
capsaicin.25
In vitro TRPM8 IC50 data for compounds 13a–13f and 18
R
O
P
Initially, the focus was on exploring the substituents on the
benzyl moiety using chemistry depicted in Scheme 1. As illustrated
in Table 2, replacement of the 4-fluoro group with hydrogen gave
an analog (9a) that was slightly less potent than compound 4f in
the functional assay. However, analogs with small, lipophilic
groups in the 3-position of the phenyl ring (cf. 9b, 9d, and 9f), were
more potent than lead compound 4f. Disubstitution of the 3,4-and
3,5-positions with fluoro groups was also well tolerated (cf. 9k and
OEt
OEt
S
F
F
Compda
R
IC50
(lM)
b
9l). All analogs were inactive against TRPV1 at 10 lM.
13a
13b
13c
13d
13e
13f
18
H
0.390
0.116
0.146
0.026
0.350
0.160
0.012
-c-C3H5
-c-C4H7
-c-C5H9
-c-C6H11
–CH2C(CH3)3
–CH(CH3)2
As it was assumed that the 3,4-difluorobenzyl substitution of
compound 9l would prevent extensive metabolism of the benzyl
ring, we next altered the 3-position of the benzothiophene ring
(Table 3) and the R1 phosphonate ester moiety (Table 4), while
holding the benzyl moiety constant.
Potency in the functional assay was somewhat sensitive to the
steric bulk of the 3-position substituent, as cyclopropyl-(13b),
cyclobutyl-(13c) and cyclohexyl-(13e) analogs exhibited higher
IC50 values than compound 9l. Nevertheless, potency could be
maintained or further improved through the judicious choice of a
substituent with the appropriate steric and electronic properties.
For instance the cyclopentyl analog 13d, with an IC50 value of
a
Target compounds were purified by reverse-phase semi-prep HPLC and purities
were judged by reverse-phase HPLC/MS with UV detection at 215 and 254 nm (YMC
J’Sphere C-18 column, 0.4 Â 5 cm; mobile phase: MeCN–H2O (0.1% TFA). All new
compounds were characterized by ESI-MS and 400-MHz 1H NMR. Compounds were
isolated as racemic mixtures.
b
Inhibition of canine TRPM8 is reported as mean IC50 values. All analogs were
inactive when counter-screened against the human TRPV1 cell line at 10 lM.
0.026
18 demonstrated a marked improvement in functional activity
(IC50 = 0.012 M) when compared to compound 9l.
lM, demonstrated similar potency to 9l. Isopropyl analog
moieties of the phosphonate ester with the alkyl chains of a phos-
phine oxide (e.g., 23 and 25) using the chemistry shown in Scheme
6 resulted in considerably less potent analogs, with IC50 values of
l
The effect of the ester functionality of compound 9l on TRPM8
antagonist potency was investigated by either altering the size of
the ester moiety or by removal of one of the ester groups. As illus-
trated in Table 4, conversion to the mono-acid/mono-ester 19 as
per the chemistry in Scheme 5, resulted in complete loss of TRPM8
functional activity, demonstrating the vital importance of a hydro-
phobic moiety in this portion of the molecule. We next explored
the size requirement of the ester, and showed that optimal potency
was realized when the ethyl esters were replaced by isopropyl es-
0.165 and 0.123 lM, respectively.
A tool compound was required to establish proof of pharmaco-
dynamic activity in vivo for a TRPM8 antagonist. Racemates 9l and
24 were identified as phosphonate analogs of interest in light of
their potency in the calcium mobilization assay (TRPM8 IC50 = 64
and 24 nM, respectively). Both compounds were assessed for their
metabolic stability in rat and human liver microsomes and for their
absorption potential in Caco-2 cells. Compounds 9l and 24 exhib-
ited similar microsomal stability, with 53% and 63% remaining,
respectively, after incubation in rat liver microsomes for 10 min,
and 60% and 39% remaining, respectively, after incubation in hu-
man liver microsomes for 10 min.26 However, compound 9l dem-
onstrated a much higher absorption potential in Caco-2 cells,
with a Papp A to B of 3.99 Â 10À6 cm/s, compared to compound
24, with a Papp A to B of 0.075 Â 10À6 cm/s. Despite the modest
ters (e.g., 24: IC50 = 0.024 lM). However, replacement of the alkoxy
Table 2
In vitro TRPM8 IC50 data for compounds 9a–9o
O
P
OEt
OEt
S
9
Table 4
R2
In vitro TRPM8 IC50 data for compounds 19, 23–26
Compda
R2
IC50 (lM)
R1
R3
O
P
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
H
0.360
0.031
0.114
0.022
0.145
0.065
0.138
0.121
0.365
0.240
0.058
0.064
0.300
0.525
0.120
3-Fluoro
2-Fluoro
3-Chloro
3-Bromo
3-Methyl
3-Trifluoromethyl
3-Methoxy
4-Trifluoromethyl
4-Trifluoromethoxy
3,5-Difluoro
3,4-Difluoro
3,4-Dichloro
4-Chlroro-3-trifluoromethyl
4-Fluoro-3-trifluoromethyl
S
F
F
R3
Compda
R1
IC50
NA
(lM)
b
19
23
24
25
26
–OEt
–C3H7
–O–CH(CH3)2
–CH2CH(CH3)2
–O-c-C6H13
–OH
–C3H7
–O–CH(CH3)2
–CH2CH(CH3)2
–O-c-C6H13
0.165
0.024
0.123
0.750
a
Target compounds were purified by reverse-phase semi-prep HPLC and purities
were judged by reverse-phase HPLC/MS with UV detection at 215 and 254 nm (YMC
J’Sphere C-18 column, 0.4 Â 5 cm; mobile phase: MeCN–H2O (0.1% TFA). New
compounds were characterized by ESI-MS and 400-MHz 1H NMR. Compounds were
isolated as racemic mixtures.
a
Target compounds were purified by reverse-phase semi-prep HPLC and purities
were judged by reverse-phase HPLC/MS with UV detection at 215 and 254 nm (YMC
J’Sphere C-18 column, 0.4 Â 5 cm; mobile phase: MeCN–H2O (0.1% TFA)). All new
compounds were characterized by ESI-MS and 400-MHz 1H NMR. Compounds were
isolated as racemic mixtures. All analogs were inactive when counter-screened
b
Inhibition of canine TRPM8 is reported as mean IC50 values; NA, <20% inhibition
at 5
lM. All analogs were inactive when counter-screened against the human
against the human TRPV1 cell line at 10 lM.
TRPV1 cell line at 10
l
M.

J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2922–2926
2925
Figure 2. Time course of icilin-induced ‘wet-dog’ shakes in rats after ip administration of vehicle or Cpd 9l.
a
b
O
S
SH
O
Br
S
16
14
17
15
O
P
OEt
OEt
S
F
Figure 3. Cumulative reduction of subsequent icilin-induced ‘wet-dog’ shakes
behavior in rats after ip administration of vehicle or Compd 9l.
18
F
Scheme 4. Reagents and conditions: (a) DMF, NaH, 15,21 90 °C (69%); (b) PPA,
140 °C (82%).
O
a
P(OEt)(OH)
9l
S
F
19
F
Figure 4. Reversal of icilin-induced ‘wet-dog’ shakes in rats after oral administra-
tion of vehicle or Compd 9l.
Scheme 5. Reagents and conditions: (a) DMF, NaN3 (82%).
metabolic stability in the rat and human liver microsomes, com-
pound 9l was chosen for further in vivo evaluation. To overcome
high metabolism and consequent low exposure (Cmax = 41 ng/mL)
when administered orally,27 9l was administered intraperitoneally
at 30 mg/kg in a rat pharmacodynamic ‘wet-dog’ shaking (WDS)
assay. Icilin (AG 3–5) is a super cooling agent that activates the
TRPM8 channel, and causes WDS behavior in rats after intraperito-
neal administration at 1 mg/kg.1,28–30 A study in TPRM8 knockout
mice further demonstrated that icilin-induced WDS behavior was
mediated by TRPM8.9 To verify that the phosphonate series was
acting at the TRPM8 channel, compound 9l was initially tested in
a preventive mode.31 Specifically, 9l was administered to rats
(30 mg/kg, ip) 15 min prior to treatment with icilin (1 mg/kg, ip),
the number of WDS behaviors was measured for 30 min in two-
min intervals, and the results were compared to vehicle pretreat-
ment. As shown in Figure 2, 9l antagonized the effects of icilin
throughout the 30-min time course of the experiment, cumula-
tively reducing WDS behaviors by 80% (Fig. 3).
O
O
P
OEt
20
a
O
P
b
P(n-Pr)2
H
OEt
H
S
21
22
O
P(n-Pr)2
c
S
23
F
F
Scheme 6. Reagents and conditions: (a) n-PrMgCl, H2O, K2CO3,22 (b) Toluene, NaH
(60%), 7 (42%); (c) THF, n-BuLi, 3,4-difluorobenzyl bromide (73%).
To further characterize 9l in vivo, the antagonism of the TRPM8
channel was also measured in the reversal mode of the rat icilin-in-
duced WDS model. In this mode, WDS behaviors were measured
between 10 and 14 min after administration of icilin (3 mg/kg,
po), which served as baseline. Compound 9l (30 mg/kg, ip) was
administered at 14 min, the number of shakes was counted in
4-min bins for an additional 24 min, and the results were
compared to vehicle control ( Fig. 4, depicts final 4 min 4 bin).
Compound 9l is effective in both preventing—as seen in the previ-
ous experiment—and reversing existing icilin-induced behaviors.
In conclusion, members of a novel series of benzothiophene
derived phosphonate esters have been shown to be potent

2926
J. M. Matthews et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2922–2926
14. Caspani, O.; Zurborg, S.; Labuz, D.; Heppenstall, P. A. PLoS One 2009, 4, 7383.
15. McKemy, D. D. The Open Drug Discovery J. 2010, 2, 81.
16. DeFalco, J.; Steiger, D.; Dourado, M.; Emerling, D.; Duncton, M. Bioorg. Med.
Chem. Lett. 2010, 20, 7076.
17. Parks, D. J.; Parsons, W. H.; Colburn, R. W.; Meegalla, S. K.; Ballentine, S. K.; Illig,
C. R.; Qin, N.; Liu, Y.; Hutchinson, T. L.; Lubin, M. L.; Stone, D. J.; Baker, J. F.;
Schneider, C. R.; Ma, J.; Damiano, B. P.; Flores, C. M.; Player, M. R. J. Med. Chem.
2011, 54, 233.
18. The functional activity of the analogs was determined by measuring changes in
intracellular calcium concentration using a Ca2+-sensitive fluorescent dye and
FLIPRTM technology. Increases in intracellular Ca2+ concentration were readily
detected upon activation with the TRPM8 agonist icilin. HEK293 cells stably
expressing canine TRPM8 were grown on poly-D-lysine coated 384-well black-
walled plates and loaded with Calcium 3 Dye (Molecular Devices) for 35 min at
37 °C, under 5% CO2 and then for 25 min at room temperature and atmosphere.
Cells were challenged with test compounds (at varying concentrations) and
intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all
wells to achieve a final concentration of 60 nM which produces approximately
an 80% maximal response. The IC50 value for each analog was determined from
eight-point dose–response studies. Curves were generated using the average of
quadruplicate wells for each data point.
antagonists of the TRPM8 channel. Compound 9l was identified as
a highly selective and robust TRPM8 antagonist in vitro that inhib-
ited icilin-induced behaviors in a rat WDS model. On the basis of
these findings, the synthesis and identification of potent, selective,
and orally bioavailable benzothiophene-derived phosphonate es-
ters are being pursued. Ultimately, this work aspires to generate
a safe and effective TRPM8 antagonist therapeutic that may be
used to treat pain and other disorders in which patients suffer from
a pathophysiologic hypersensitivity to cold.
Acknowledgments
The authors thank Gary Caldwell, John Masucci, and Andrew
Mahan for their assistance with LC–MS and NMR support, and
Norman Huebert, Risa Batta, Deping Cheng and Karen Diloretto
for their pharmacokinetic support.
19. Greco, M. N.; Hawkins, M. J.; Powell, E. T.; Almond, H. R.; de Garavilla, L.; Hall,
J.; Minor, L. K.; Wang, L.; Corcoran, T. W.; Di Cera, E.; Cantwell, A. M.; Savvides,
S. N.; Damiano, B. P.; Maryanoff, B. E. J. Med. Chem. 2007, 50, 1727.
20. Hawkins, M. J.; Greco, M. N.; Powell, E.; de Garavilla, L.; Maryanoff, B. E. WO
2005073214, 2005.
21. Kumar, R. N.; Selvi, S. T.; Suresh, T. M.; Palathurai, S. Heterocycles 2002, 57, 357.
22. (a) Hays, H. R. J. Org. Chem. 1968, 33, 3690; (b) Aksnes, G.; Majewski, P.
Phosphorus Sulfur 1986, 26, 261.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.02.060.
23. Flores, C. M.; Liu, Y.; Lubin, M. L.; Qin, N. WO 2005100386, 2005.
24. Liu, Y.; Lubin, M. L.; Reitz, T. L.; Wang, Y.; Colburn, R. W.; Flores, C. M.; Qin, N.
Eur. J. Pharmacol. 2006, 530, 23.
25. The TRPV1 functional assay is very similar to the TRPM8 functional assay with
exceptions. Increases in Ca2+ concentration were readily detected upon
challenge with the TRPV1 agonist capsaicin. HEK293 cells were expressed
with human VR1 and intracellular Ca2+ was measured for 5 min prior to the
addition of capsaicin to all wells.
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Labs, n = 4–6/treatment) in PEG-400 at
a concentration 1.0 mg/kg, ip
Spontaneous WDS behaviors were counted in 2 min bins over 30 min post-
icilin. Compound 9l or vehicle was administered ip 15 min before icilin to
measure its ability to inhibit this spontaneous WDS behavior.