Discovery of vinylcycloalkyl-substituted benzimidazole TRPM8 antagonists effective in the treatment of cold allodynia

Article abstract of DOI:10.1016/j.bmcl.2012.01.060

Thermosensitive transient receptor potential melastatin 8 (TRPM8) antagonists are considered to be potential therapeutic agents for the treatment of cold hypersensitivity. The discovery of a new class of TRPM8 antagonists that shows in vivo efficacy in the rat chronic constriction injury (CCI)-induced model of neuropathic pain is described.

Full text of DOI:10.1016/j.bmcl.2012.01.060

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1903–1907
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of vinylcycloalkyl-substituted benzimidazole TRPM8 antagonists
effective in the treatment of cold allodynia
Raul R. Calvo, Sanath K. Meegalla, Daniel J. Parks, William H. Parsons, Shelley K. Ballentine,
Mary Lou Lubin, Craig Schneider, Raymond W. Colburn, Christopher M. Flores, Mark R. Player
⇑
Janssen Research & Development, Welsh and McKean Roads, Spring House, PA 19477-0776, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Thermosensitive transient receptor potential melastatin 8 (TRPM8) antagonists are considered to be
potential therapeutic agents for the treatment of cold hypersensitivity. The discovery of a new class of
TRPM8 antagonists that shows in vivo efficacy in the rat chronic constriction injury (CCI)-induced model
of neuropathic pain is described.
Received 20 December 2011
Revised 13 January 2012
Accepted 17 January 2012
Available online 28 January 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
TRPM8
Transient receptor potential melastatin
Ion channel
Pain
Cold allodynia
Cold hypersensitivity
Benzimidazole
Transient receptor potential, melastatin sub-family, type 8
(TRPM8) is a member of a larger family of non-selective cation
channels that are activated by a variety of endogenous and envi-
ronmental stimuli. A subset of these TRP channels, including
TRPM8, is thermosensitive and connotes sensory responsiveness
to the temperature spectrum, selectively responding to thermal
stimuli, as well as to certain chemicals that mimic these sensa-
tions. Specifically, TRPM8 is stimulated by normally innocuous
cool to noxious cold temperatures as well as by the chemical ago-
nists menthol and icilin, which produce a cooling sensation.1
TRPM8 is located on primary nociceptive neurons (Ad and C fi-
bers) and becomes sensitized by second messenger signals induced
by tissue injury. This localization and sensitization profile may pro-
vide a basis for abnormal cold hypersensitivity in pathologic condi-
tions, such as neuropathic cold allodynia, which is often sensed as
burning pain.2–4
provide evidence of a critical role for TRPM8 in the development
of cold hypersensitivity.5
Recent literature, both patent applications and journal articles,
have disclosed a variety of TRPM8 antagonist chemotypes for the
treatment of various disease states, demonstrating the current
interest in TRPM8 as a therapeutic target.6
A focused high-throughput screen of in-house chemical li-
braries, followed by further structural elaborations, identified pro-
totypical 2-vinyl cycloalkyl-substituted benzimidazoles (1) as
moderately potent TRPM8 antagonists. Herein is reported a de-
tailed account of structure–activity relationships and optimization
of this series of compounds (Fig. 1).
Synthetic routes for the preparation of the target compounds
are shown in Scheme 1. The key step in these syntheses is the
acid-catalyzed intramolecular cyclization of the amino-amide
intermediate 3 to construct the benzimidazole core. Intermediate
It has been shown that TRPM8 null mice demonstrate normal
survival during postnatal development, are fertile and exhibit a
normal lifespan. However, in contrast to wild-type mice, these
TRPM8 knockout mice exhibit a significantly decreased aversion
to cold, are essentially unresponsive to the behavioral effects of
the TRPM8 agonist icilin (shaking/jumping) and fail to develop cold
allodynia following peripheral nerve injury. These observations
3
5
R
N
N
H
1
7
R = Cycloalkyl
1
⇑
Corresponding author. Tel.: +1 215 628 7860.
E-mail address: mplayer@its.jnj.com (M.R. Player).
Figure 1. Generic structure of 2,5-disubstituted benzimidazoles.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.060

1904
R. R. Calvo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1903–1907
Route A
Ar
N
R
Br
NH2
NH2
NH2
Br
c
N
R
b
a
Br
N
H
N
H
NHCOCH=CHR
2
3
4
5
Route B
O
B
Ar
Ar
NO2
NHCOCH=CHR
N
R
NO2
NH2
Ar
NO2
O
f
d
e
N
H
NH2
6
7
8
5
Scheme 1. General synthetic route for the preparation of compounds of structure 1. Reagents and conditions: (a) RCH@CHCOCl, toluene, Et3N; (b) p-TSA, toluene, 110 °C; (c)
Ar-B(OH)2, PdCl2 dppf, 2 M Na2CO3, DME, 95 °C; (d) ArX, PdCl2 dppf, 2 M Na2CO3, DME, 95 °C (e) (i) NaH/THF/0 °C, (ii) RCH@CHCOCl; (f) Fe/HOAc/70 °C.
CO2H
d
COCl
H
OMe
O
Table 1
O
X
Structure–activity relationships of the C-5 aryl substitution
a
b
c
3
5
Ar
N
X
X
X
X
6
9
10
11
12
13
N
H
1
X = CH2, O, S, N-BOC
COCl
CO2H
d
CO2Et
CO2Et
CHO
Compound Ar
cTRPM8 IC50 (nM)a Stabilityb HLM/RLM/DLM
e
c
f
19
110
ND
Y
Y
Y
Y
Y
Y = O, -OCH2CH2O-
14 15
F
18
16
17
20
21.0
98/34/75
Scheme 2. General synthetic route for the preparation of acryloyl chlorides.
Reagents and conditions: (a) Ph3PCH2OMeCl, LiHMDS, THF, ꢀ10 °C; (b) H+, heat; (c)
Malonic acid, piperidine, pyridine, 70 °C; (d) (COCl)2, cat. DMF; (e) tert BuOK, MeI,
THF/ꢀ10 °C; (f) (i) LAH/THF/0 °C; (ii) PDC/DCM.
Cl
21
22
23
8.9
7.1
8.9
71/39/ND
77/15/13
62/10/ND
OMe
3 was synthesized via two general methods. In the first method
(Scheme 1, Route A) the diaminobenzene 2 was acylated with an
acryloyl chloride RCH@CHCOCl to obtain a mixture of the desired
mono-acylated product 3, which was contaminated with the corre-
sponding bis-acylated byproduct, necessitating chromatographic
removal prior to the cyclization. Amino-amide 3 was cyclized un-
der acidic conditions in refluxing toluene to obtain 4.
OCF3
CF3
24
25
14
71/91/ND
85/58/20
11
In the second synthetic route (Scheme 1, Route B), 2-nitroani-
line 6 was used in place of diaminobenzene 2 to circumvent the
bis-acylation contamination issue in Route A. The sodium salt of
the nitro-aniline 7 was first reacted with the acrolyl chlorides to af-
ford 2-nitrobenzamide 8. Treatment of 8 with Fe/HOAc at elevated
temperatures affected both the reduction of the nitro moiety as
well as the subsequent cyclization of the resulting amino-amide
intermediate in one pot to yield benzimidazoles 5. Conversely,
when the 2-nitrobenzamide 8 was treated with Fe/NH4Cl, the cor-
responding amino-amide was obtained without cyclization but
could then be cyclized under acidic conditions (e.g., p-TsOH in tol-
uene) at higher temperatures to obtain benzimidazoles 5.
CF3
26
12.3
38.0
8.6
56/44/61
90/72/42
67/66/42
CF3
CF3
CF3
27
28
N
F
The introduction of the C-5 aryl substituent was effected with a
Suzuki coupling protocol in both synthetic routes. With non-hin-
dered arylboronic acids, the Suzuki coupling reactions were carried
out as the last step of the synthesis with the penultimate interme-
ND-not determined.
a
IC50 values or % inhibition averaged from 4 determinations (n = 4) by FDSSÒ
.
b
% Remaining after 10 min incubations in human (H), rat (R), and dog (D) liver
microsomal preparations.

R. R. Calvo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1903–1907
1905
Table 2
Structure–activity relationships of the vinyl cycloalkyl substituent
R1
N
Cy
N
H
Compound
R1
Cy
cTRPM8 IC50 (nM)a
110
Stabilityb HLM/RLM/DLM
29
CF3O–
84/71/ND
47/100/39
F
30
CF3–
21.0
F
Me
Me
F
31
32
CF3–
CF3–
8.9
7.1
97/84/28
93/79/51
F
O
O
33
34
35
36
37
38
39
CF3–
CF3–
CF3–
CF3–
CF3–
CF3–
CF3–
8.9
31
62/20/35
67/6/8
S
SO2
NH
NAc
63%
16%
1%
91/74/60
99/86/98
100/85/100
ND/ND/75
ND
NSO2Me
NMe
14%
1%
ND-not determined.
a
IC50 values or % inhibition at a test concentration of 0.2 l .
M averaged from four determinations (n = 4) by FDSSÒ
% Remaining after 10 min incubations in human (H), rat (R), and dog (D) liver microsomal preparations.
b
diate 4. Since the hindered boronic acids or esters, such as the 2,6-
disubstituted variety, produced poor yields in this reaction, they
were introduced in the very first step in the synthetic sequence
(Route B). In these instances, 2,6-disubstituted phenyl bromides
or iodides were coupled with the commercially available 3-nitro-
4-amino-phenylboronic acid pinacol ester 6 to obtain intermedi-
ates 7, which were subsequently employed in the benzimidazole
ring construction procedure.
Scheme 2 illustrates the general synthetic routes for the prepa-
ration of the required acryloyl chlorides. The reaction of aldehyde
11 with malonic acid in the presence of an organic base, such as
piperidine, under thermal conditions was utilized to prepare the
acrylic acid 12 in one step. When the desired aldehydes 11 were
not commercially available, they were prepared from ketone 9
using a Wittig homologation with methoxymethyltriphenyl-phos-
phonium chloride, followed by the acid-catalyzed hydrolysis of the
resulting enol ether 10.
For the introduction of the substituent at the ring attachment
point, ester 14 was treated with potassium t-butoxide and methyl
iodide to obtain 15. A two-step reduction/oxidation sequence
afforded the desired aldehyde 16. The desired acryloyl chloride
18 was obtained from aldehyde 16 following the previously de-
scribed sequence (Scheme 2).
The gem difluoro compound 31 was prepared by the action of
DAST on the corresponding ketone which in turn was prepared
by the deprotection of the corresponding ketal. The ketal was ob-
tained following the chemistry described in Scheme 1 and 2.
Table 3
Profile of compound 25
In vitro
profile
cTRPM8 (IC50, nM) = 11
hTRPM8 (IC50, nM) = 15
rTRPM8 (IC50, nM) = 20
inhibition
Recombinant CYPs (IC50
1A2 > 10.
CYP
,
l
M): 3A4 = 3.5, 2C9 = 3.5, 2C19, 2D6,
CYPs in HLM (IC50
2C9 = 62% at 3.3
, lM): 3A4, 2C19 > 30,
l
M
Rat PK
F = 14%, CL = 22 mL/min/kg, T1/2 = 2.9 h, Cmax = 225 ng/mL,
Vss = 9.5 L/kg
Initial evaluation of new analogs were carried out in the canine
TRPM8 (cTRPM8) Ca2+-flux assay utilizing FDSSÒ technology,
which measures the intracellular calcium concentration using
Ca2+-sensitive dyes in HEK293 cells stably expressing the cTRPM8
channel.7 Icilin was employed as the agonist in these assays.
Metabolic stabilities of the compounds were evaluated using
10 min incubations of the test compound with preparations of
rat, dog or human liver microsomes (RLM, DLM and HLM, respec-
tively) and are reported as percent remaining at the end of the
incubation period.
The cTRPM8 IC50 values and metabolic stability data of the C-5
substituted variants are shown in Table 1. The presence of

1906
R. R. Calvo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1903–1907
Compound 25, 10 mpk
100
90
80
70
60
50
40
30
20
10
0
**
20% HPβCD
Compound 25, 30 mpk
**
20% HPβCD
***
***
***
***
***
100
80
60
40
20
*
***
**
VEH
3
10
30
0
Compound 25 (mg/ kg, p.o.)
0
1
2
3
4
Hours after oral dose
Figure 2. Inhibition of icilin-induced (3 mg/kg, ip) WDS in rats following 60 min
pretreatment with compound 25 (⁄⁄p < 0.01, one-way ANOVA; Dunnett’s post hoc
test).
Figure 3. Time-course response in the rat CCI cold allodynia model following oral
dosing of 10 mg/kg of compound 25. (⁄p < 0.05; ⁄⁄⁄p < 0.001; two-way ANOVA for
repeated measure; Bonferroni post hoc test).
zymes, whereas inhibition of the other CYP isoforms tested was
insignificant. Further evaluation in HLM revealed that 25 showed
no appreciable 3A4 inhibitory activity, but inhibition of 2C9 re-
mained. The propensity for 25 to react with native substrates with
or without reactive intermediate formation was also evaluated in
an in vitro glutathione conjugation assay,9 and no adduct forma-
tion was observed.
The rat pharmacokinetic study at 10 mg/kg (Table 3) provided
evidence for extensive tissue distribution of 25, possessing a
high volume of distribution (9.5 L/kg) compared to total body
water (0.688 L/kg). The compound had a modest half-life (ca.
3 h) with a high clearance rate, a low Cmax of 225 ng/mL peaking
at 1.6 h, and only 14% oral bioavailability. Nevertheless, the over-
all favorable profile of this compound warranted further
evaluation.
The efficacy of 25 was first evaluated in the icilin-induced ‘wet
dog shakes’ (WDS) pharmacodynamic model.8 As shown in Figure
2, the compound inhibited icilin-induced WDS in a dose-depen-
dent manner at 3, 10 and 30 mg/kg, po when given 1 h prior to
icilin administration (3 mg/kg, ip), corresponding to an oral ED50
of 6.4 mg/kg. Plasma concentrations of 25 demonstrated good
dose linearity over the range studied. The plasma concentration
at the ED50 dose was 186 ng/mL.
substituents in the ortho position of the C-5 aryl moiety on the
benzimidazole core structure uniformly enhanced the potency of
the compounds (19 vs 21), suggesting that the 5-aryl group needs
to be non-planar with the benzimidazole core for optimum binding
in the TRPM8 binding site. However, introduction of a second sub-
stituent in the 6-position of the aryl ring did not have a significant
effect on the potency of the compounds but reduced metabolic sta-
bility (25 vs 26). Although the electron donating or withdrawing
ability of the substituent on the C-5 aryl ring did not have a signif-
icant impact on the potency of the compounds (20–25), the
replacement of the C-5 aryl ring with a heteroaryl ring caused a
three- to four-fold decrease in the TRPM8 potency (25 vs 27).
Preliminary metabolite identification studies carried out with
25 after RLM incubation showed that this substrate was exten-
sively metabolized. The analysis of the major metabolites re-
vealed that the vinyl cyclohexyl ring is the major metabolic ‘hot
spot’. Both mono- and bis-hydroxylations of the cyclohexyl ring
were detected as metabolites, however the positions of the
hydroxylations are unknown. Thus, limited work was carried
out to evaluate the feasibility of altering the metabolic vulnerabil-
ity of the cyclohexyl ring (Table 2). The replacement of the cyclo-
hexyl ring with the smaller cyclopentyl ring resulted in a three-
fold decrease in TRPM8 potency (24 vs 29). Difluoro substitution
of the 4-position of the cyclohexyl ring (30) did not improve met-
abolic stability, although the compound remained active in the
in vitro TRPM8 assay. Methylation of the allylic 10 position of
40,40-difluorocyclohexane 31 neither altered the TRPM8 activity
nor improved the DLM stability.
The replacement of the cyclohexyl moiety with tetrahydrothio-
pyranyl (34) worsened the observed metabolic stabilities in both
RLM and DLM while also reducing cTRPM8 potency by threefold
versus 25. In contrast, the corresponding tetrahydropyranyl analog
(32) was equipotent to 25 and displayed marginally improved met-
abolic stability. The introduction of polar functionalities at the 4-
position of the cyclohexyl moiety (35–39) significantly diminished
cTRPM8 antagonist activity.
Although compound 25 did not have robust stability against
DLM preparations, it displayed adequate stability in both HLM
and RLM incubations. Thus compound 25 was chosen as a suitable
candidate for further profiling and initial efficacy studies. It was
equipotent in TRPM8 FDSSÒ in all three species (dog, human and
rat) evaluated. In general the tested compounds exhibited compa-
rable in vitro activities across rat, human, and dog.
Compound 25 was further evaluated for its ability to reverse
cold hypersensitivity in the chronic constriction injury (CCI)
Compound 25, 2h
Compound 25, 4h
20% HPβCD, 2h
20% HPβCD, 4h
100
80
60
40
20
0
***
*
1
Veh
3
10
Oral Dose (mg/kg, p.o.)
Figure 4. Dose response in rat CCI cold allodynia model at 2 and 4 h post dose
following administration of compound 25 at 1, 3 and 10 mg/kg, po. (⁄p < 0.05;
⁄⁄⁄p < 0.001; two-way ANOVA for repeated measure; Bonferroni post hoc test).
In the recombinant cytochrome P450 (CYP) inhibition assay, 25
showed 3.5 and 5.5 lM inhibitory activity against 3A4 and 2C9 iso-

R. R. Calvo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1903–1907
1907
model of neuropathic pain.10 In a time course study, doses of 10
and 30 mg/kg showed an approximately 90% peak inhibition of
response to cold stimulation at 2–3 h post-dose (Fig. 3). In a sub-
sequent dose–response study (1, 3 and 10 mg/kg, po) in the CCI
model (Fig. 4), 25 showed statistically significant effects at both
2 and 4 h time points at 10 mg/kg as compared to the vehicle
control group (p < 0.05 and p < 0.001, respectively).
In conclusion, a novel series of high affinity vinylcycloalkyl-
substituted benzimidazole TRPM8 antagonists, exhibiting robust
in vitro potency and in vivo efficacy in a model of neuropathic pain,
has been identified. These antagonists provide further evidence
that in vivo antagonism of TRPM8 may provide a strategy for the
alleviation of disorders in which cold hypersensitivity is a promi-
nent feature.
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