Arylglycine derivatives as potent transient receptor potential melastatin 8 (TRPM8) antagonists

Article abstract of DOI:10.1016/j.bmcl.2013.01.062

A series of arylglycine-based analogs was synthesized and tested for TRPM8 antagonism in a cell-based functional assay. Following structure-activity relationship studies in vitro, a number of compounds were identified as potent TRPM8 antagonists and were subsequently evaluated in an in vivo pharmacodynamic assay of icilin-induced 'wet-dog' shaking in which compound 12 was fully effective. TRPM8 antagonists of the type described here may be useful in treating pain conditions wherein cold hypersensitivity is a dominant feature.

Full text of DOI:10.1016/j.bmcl.2013.01.062

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2234–2237
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Arylglycine derivatives as potent transient receptor potential
melastatin 8 (TRPM8) antagonists q
⇑
Bin Zhu , Mingde Xia, Xiaoqing Xu, Donald W. Ludovici, Manomi Tennakoon, Mark A. Youngman,
Jay M. Matthews, Scott L. Dax, Raymond W. Colburn, Ning Qin, Tasha L. Hutchinson, Mary Lou Lubin,
Michael R. Brandt, Dennis J. Stone, Christopher M. Flores, Mark J. Macielag
Janssen Research & Development, L.L.C., Welsh & McKean Roads, Spring House, PA 19477, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of arylglycine-based analogs was synthesized and tested for TRPM8 antagonism in a cell-based
functional assay. Following structure–activity relationship studies in vitro, a number of compounds were
identified as potent TRPM8 antagonists and were subsequently evaluated in an in vivo pharmacodynamic
assay of icilin-induced ‘wet-dog’ shaking in which compound 12 was fully effective. TRPM8 antagonists
of the type described here may be useful in treating pain conditions wherein cold hypersensitivity is a
dominant feature.
Received 21 September 2012
Revised 10 January 2013
Accepted 16 January 2013
Available online 4 February 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Transient receptor potential melastatin 8
TRPM8 antagonist
Arylglycine
Cold allodynia
Pain
Transient receptor potential melastatin 8 (TRPM8) is a member
of the transient receptor potential (TRP) superfamily, comprising a
diverse group of non-selective cation channels that are activated
by a variety of physical and chemical stimuli. First described in
2002, TRPM8 was shown to be activated by innocuous cool to nox-
ious cold temperatures as well as by chemical agonists, such as
menthol and icilin.1 TRPM8 is expressed on primary nociceptive
Ad and C fibers,1,2 through which cold responses are transmitted.
Studies on TRPM8 knock-out mice have demonstrated decreased
sensitivity to cold temperature as well as decreased hypersensitiv-
ity to cold after nerve injury or inflammation.3 Thus, antagonism of
TRPM8 provides an attractive approach to the treatment of cold-re-
lated painful conditions, such as cold hyperalgesia and cold allo-
dynia, which are commonly associated with certain types of
neuropathic and inflammatory pain.4 A number of small molecule
TRPM8 antagonists have been reported in the literature as poten-
tial pain therapeutics.5 Herein, we describe a series of arylgly-
cine-based analogs that are potent TRPM8 antagonists (Fig. 1).
Several synthetic routes have been utilized to prepare the de-
signed analogs. The synthesis of compounds 7 is shown in Scheme
1. Substituted phenylacetic acid 1 was treated with concd HCl in
methanol to give its corresponding methyl ester, which was then
reacted with N-bromosuccinimide (NBS) in the presence of a
catalytic amount of aqueous 48% HBr in refluxing CCl4 to give the
R
NH
N
Ar1
Ar2
O
Figure 1. Arylglycine-based TRPM8 antagonists.
R
H2N
Br
NH
R
OH
Ar1
a,b
OCH3
3
Ar1
OCH3
Ar1
c
O
O
O
2
1
4
R
R
Ar2
N
d
NH
NH
H
6
e
N
OH
Ar1
Ar1
Ar2
O
O
5
7
q
All authors were employed by Janssen Research & Development, L.L.C. at the
Scheme 1. Reagents and conditions: (a) concd HCl, MeOH, rt, 16 h; (b) NBS, cat. aq
48% HBr, CCl4, reflux, 2 h; (c) CH3CN, reflux, 16 h; (d) LiOH, H2O, THF, rt, 3 h; (e) (i)
HATU, Et3N, CH2Cl2, rt, 16 h, only for compounds 7q, 7r, and 7x: (ii) LiOH, THF,
MeOH, H2O, rt, 16 h.
time the work reported herein was conducted.
⇑
Corresponding author.
E-mail address: bzhu@its.jnj.com (B. Zhu).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.062

B. Zhu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2234–2237
2235
a
-bromo compound 2. Replacement of the
a
-bromo group with
benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophos-
phate (HATU), triethylamine, CH2Cl2] to give the final products 7.
Scheme 2 depicts a more efficient route to the designed com-
pounds via a boronic acid Mannich reaction.6 The three-compo-
nent condensation of boronic acid 8, substituted aniline 3 and
glyoxylic acid in refluxing acetonitrile led to carboxylic acid 9. Cou-
pling of 9 with aryl-substituted pyrrolidine 6 gave the desired final
products 10. Further functionalization at the Ar1 group was
achieved through palladium-catalyzed coupling reactions between
4-bromo-phenyl analog 7y and aryl/heteroaryl boronic acids
(Scheme 3).
The functional activity of the prepared analogs was determined
in the canine TRPM8 Ca2+ flux assay, in which icilin-induced
changes of intracellular calcium concentration in HEK293 cells sta-
bly expressing canine TRPM8 channels were measured using a
Ca2+-sensitive fluorescent dye.7,8 Initially, the Ar1 region was kept
constant as a para-CF3-phenyl group to expedite structure–activity
relationship (SAR) studies of the R and Ar2 groups. As illustrated in
Table 1, analogs containing a 2-F-aniline (R = 2-F, 7f, 7g, 7m) are
more potent TRPM8 antagonists than those with an unsubstituted
aniline (R = H, 7a–c). The position of the fluorine group on the ani-
line is critical, as 3- or 4-substitution (R = 3-F or R = 4-F) led to
much less potent compounds (7j vs 7k, 7l). Exploring SAR of the
Ar2 group indicated that both phenyl (7f) and pyridyl groups
(7n–7p) afforded relatively high potency. Less polar substituents,
such as halogen (7g–7j) and methoxy (7m), were tolerated on
the phenyl group, whereas more polar groups, such as carboxylic
acid (7q, 7r), led to slightly reduced potency. The stereochemistry
substituted aniline 3 was achieved by using an excess amount of
3 at elevated temperature. The ester group of compound 4 was
hydrolyzed under basic conditions (LiOH, H2O/THF), and the result-
ing carboxylic acid 5 was coupled with aryl-substituted pyrrolidine
6 under standard amide bond formation conditions [O-(7-aza-
R
O
OH
a
NH
OH
+
H2N
+
B
H
Ar1
OH
R
OH
Ar1
O
O
8
3
9
R
Ar2
N
NH
H
6
b
N
Ar1
Ar2
O
10
Scheme 2. Reagents and conditions: (a) CH3CN, reflux, 3 h; (b) (i) HATU, Et3N,
CH2Cl2, rt, 16 h, only for compounds 10d and 10e: (ii) LiOH, THF, MeOH, H2O, rt.
F
F
Ar3-B(OH)2
a
NH
NH
N
N
Ar2
Ar2
O
O
Ar3
Br
Table 2
11
7y
Effect of substituents Ar1 and Ar2 on icilin-induced in vitro canine TRPM8 functional
activity
Scheme 3. Reagents and conditions: (a) (i) Pd(dppf)Cl2, K2CO3, EtOH, H2O,
microwave, 130 °C, only for compound 11d: (ii) LiOH, THF, MeOH, H2O, rt.
S
Table 1
Compd
Ar1
Ar2
IC50 (lM)
Effect of substituents R and Ar2 on icilin-induced in vitro canine TRPM8 functional
activity
10a
(S)-(2-F-phenyl)
0.003
0.008
0.025
0.078
R
S
S
S
S
10b
10c
10d
2-Pyridyl
NH
N
Ar2
3-Pyridyl
O
F3C
Compd
R
Ar2
IC50 (lM)
3-CO2H-phenyl
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
H
H
H
H
Phenyl
2-F-phenyl
2-OMe-phenyl
(S)-(2-Cl-phenyl)
(R)-(2-Cl-phenyl)
Phenyl
2-F-phenyl
3-F-phenyl
4-F-phenyl
2-Cl-phenyl
2-Cl-phenyl
2-Cl-phenyl
2-OMe-phenyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
3-CO2H-phenyl
4-CO2H-phenyl
(S)-Phenyl
0.060
0.030
0.033
0.041
0.168
0.005
0.007
0.009
0.037
0.011
0.138
0.073
0.012
0.025
0.007
0.019
0.040
0.066
0.005
0.043
0.008
0.044
0.017
0.033
10e
10f
7y
4-CO2H-phenyl
(S)-(2-F-phenyl)
(S)-(2-F-phenyl)
(S)-(2-F-phenyl)
0.041
0.014
0.009
0.007
O
H
Br
2-F
2-F
2-F
2-F
2-F
3-F
4-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
N
11a
N
N
11b
11c
2-F-phenyl
2-F-phenyl
0.025
0.010
N
HO2C
F
7s
7t
7u
7v
7w
7x
(R)-Phenyl
(S)-(2-F-phenyl)
(R)-(2-F-phenyl)
(S)-(2-Cl-phenyl)
(S)-(3-CO2H-phenyl)
NH
11d
(S)-(2-F-phenyl)
0.056
N
Ar1
Ar2
O

2236
B. Zhu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2234–2237
F
F
F
NH
NH
NH
N
+
N
S
N
S
R
F
S
S
F
RS
F
O
O
O
F3C
F3C
F3C
13
IC50 = 0.045 µM
12
7u
IC50 = 0.006 µM
Scheme 4. Separation of enantiomers 12 and 13.
kg. Ongoing WDS was counted again for 10 min at 60–70 min
post-drug to assess treatment effects. The results are presented
in Table 3 as a percent inhibition of WDS (average standard er-
ror), which was calculated as [1 ꢀ (test compound WDS count/
vehicle WDS count)] ꢁ 100%.
Table 3
Inhibition of icilin-induced WDS in rats
Compound
% Inhibition
7s
56 19
63 15
79 13
7u
7w
11a
12
As indicated in Table 3, compounds 7s, 7u, 7w and 12 exhibited
moderate to full efficacy in the reversal of icilin-induced WDS, with
the enantiomerically pure compound 12 being the most effective
at the dose tested. On the other hand, the pyridyl-substituted ana-
log 11a showed no efficacy in this model, possibly due to low plas-
ma levels upon oral dosing.
ꢀ13 31
99
1
of the pyrrolidine carbon to which Ar2 is attached plays an impor-
tant role. The S configuration is preferred over the R configuration
(7d vs 7e, 7s vs 7t, and 7u vs 7v).
In summary, a series of arylglycine-based analogs have been
prepared and evaluated as TRPM8 antagonists. SAR studies led to
a number of compounds with potent in vitro activity. Selected
compounds also demonstrated robust in vivo efficacy in the ici-
lin-induced WDS assay. In particular, compound 12 exhibited an
excellent in vitro and in vivo profile and emerged as a strong can-
didate for further investigation. Ultimately, it is the goal of this re-
search to identify TRPM8 antagonists that may be useful in treating
pain and/or other conditions wherein cold hypersensitivity is a
dominant feature.
Once the optimal R and Ar2 groups had been identified through
the initial screening, the scope of the Ar1 substituent was expanded
to include a variety of substituted phenyl groups and heteroaro-
matics (Table 2). The goal was to reduce lipophilicity and improve
physiochemical properties of the resulting analogs. Fused bicyclic
Ar1 groups, such as benzothiophene (10a–10e) and 2,3-dihydro-
benzofuran (10f), were investigated. Benzothiophene shared simi-
lar SAR with para-CF3-phenyl as the Ar1 group. In particular,
benzothiophene analogs exhibited relatively high potency when
Ar2 was a 2-F-phenyl (10a) or a pyridyl group (10b, 10c), whereas
reduced potency was observed when the Ar2 group was carboxy-
phenyl (10d, 10e). For biaryl/heteroaryl Ar1 groups, substitution
of the phenyl ring with a pyridyl or pyrimidyl group was well tol-
erated, and the corresponding analogs (11a–11c) achieved potency
equivalent to that of the para-CF3-phenyl analogs. On the other
hand, introduction of a carboxyphenyl substituent led to a slightly
less potent analog 11d.
References and notes
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Because stereoselective synthetic methods were not utilized, the
final products 7, 10 and 11 were all obtained as mixtures of (R) and
(S) isomers at the carbon to which the aniline is attached. To identify
the optimal stereochemistry at this position, the two diastereomers
comprising compound 7u were separated as the (S,S)-isomer 12 and
the (R,S)-isomer 13.9 Compound 12 (IC50 = 0.006
lM), having the
(S)-configuration at the asymmetric center, was more potent than
compound 13 (IC50 = 0.045 lM), having the (R)-configuration
(Scheme 4).
Several selected compounds were assessed in a rat ‘wet-dog’
shaking (WDS) assay. Administration of icilin, a potent TRPM8 ago-
nist, causes WDS in rats, mice and other animals.1a,10 This effect is
mediated by TRPM8, because such behaviors are not manifested in
TRPM8 knock-out mice.3b Treating rats with a TRPM8 antagonist
reverses icilin-induced WDS, thereby providing a convenient phar-
macodynamic assay to evaluate the test compounds in vivo. Icilin
was administered at 3 mg/kg (ip) in 10% solutol/H2O, and instances
of spontaneous WDS were counted over a 10-min interval at 10–
20 min post-icilin. Animals that exhibited 10 or more instances
of WDS within this 10-min period were randomized into treatment
groups and orally administered the test compounds at a dose of
30 mg/kg in 10% solutol/H2O or the vehicle at a volume of 5 mL/
6. Petasis, N. S.; Goodman, A.; Zavialov, I. A. Tetrahedron 1997, 53, 16463.
7. Liu, Y.; Lubin, M. L.; Reitz, T.; Wang, Y.; Colburn, R. W.; Flores, C. M.; Qin, N. Eur.
J. Pharmacol. 2006, 530, 23.
8. TRPM8 functional activity was determined by measuring changes in
intracellular calcium concentration using a Ca2+ sensitive fluorescent dye.
The changes in fluorescent signal were monitored by a fluorescence plate
reader, either FLIPR (manufactured by Molecular Devices) or FDSS

B. Zhu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2234–2237
2237
(manufactured by Hamamatsu). Increases in intracellular Ca2+ concentration
were readily detected upon activation with icilin. At 24 h prior to the assay,
HEK293 cells stably expressing canine TRPM8 were seeded in culture medium
the addition of icilin to all wells to achieve a final concentration that produces
approximately an 80% maximal response. The IC50 values were determined
from eight-point concentration–response studies. Curves were generated using
the average of quadruplicate wells for each data point.
in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences,
NJ, USA) and grown overnight in 5% CO2 at 37 °C. On assay day, the growth
media was removed and cells were loaded with Calcium 3 Dye (Molecular
Devices) for 35 min at 37 °C under 5% CO2 and then for 25 min at rt.
Subsequently, cells were tested for agonist induced increases in intracellular
Ca2+ levels using FLIPR or FDSS. Cells were then exposed to test compound (at
varying concentrations), and intracellular Ca2+ was measured for 5 min prior to
9. The absolute configurations of compounds 12 and 13 were determined by
Vibrational Circular Dichroism (VCD).
10. (a) Behrendt, H. J.; Germann, T.; Gillen, C.; Hatt, H.; Jostock, R. Br. J. Pharmacol.
2004, 141, 737; (b) Werkheiser, J. L.; Rawls, S. M.; Cowan, A. Eur. J. Pharmacol.
2006, 547, 101; (c) Werkheiser, J. L.; Rawls, S. M.; Cowan, A. Amino Acids 2006,
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