Synthesis of benzamide derivatives as TRPV1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2007.12.007

From hit compounds identified by high throughput screening (HTS), we have found compound 1 as a lead TRPV1 antagonist and confirmed its potential as a treatment for pain. Compound 1 has led to potent TRPV1 antagonistic benzamide derivatives ((±)-2: human IC₅₀ = 23 nM, (±)-3: human IC₅₀ = 14 nM in the capsaicin-induced calcium influx assay) containing indole and naphthyl moieties, obtained by elaboration of the tryptamine scaffold or via bioisosteric replacements.

Full text of DOI:10.1016/j.bmcl.2007.12.007

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1072–1078
Synthesis of benzamide derivatives as TRPV1 antagonists
Yuji Shishido,* Madoka Jinno, Takafumi Ikeda, Fumitaka Ito, Masaki Sudo,
Naoya Makita, Atsuko Ohta, Ayako Iki-Taki, Takashi Ohmi, Yoshihito Kanai,
Tetsuya Tamura and Masato Shimojo
Pfizer Global Research & Development, Nagoya Laboratories, 5-2 Taketoyo, Aichi 470-2394, Japan
Received 19 September 2007; revised 13 November 2007; accepted 6 December 2007
Available online 26 December 2007
Abstract—From hit compounds identified by high throughput screening (HTS), we have found compound 1 as a lead TRPV1 antag-
onist and confirmed its potential as a treatment for pain. Compound 1 has led to potent TRPV1 antagonistic benzamide derivatives
(( )-2: human IC50 = 23 nM, ( )-3: human IC50 = 14 nM in the capsaicin-induced calcium influx assay) containing indole and naph-
thyl moieties, obtained by elaboration of the tryptamine scaffold or via bioisosteric replacements.
Ó 2008 Elsevier Ltd. All rights reserved.
TRPV1 (transient receptor potential vanilloid subfamily
member 1,1 also known as VR1) is a non-selective cation
channel, predominantly expressed by neurons of the
peripheral and central nervous system,2 which plays a
key role in the detection of noxious painful stimuli such
as acid (low pH),3 noxious heat, and vanilloids such as
capsaicin4 (the pungent component of hot chili peppers).
TRPV1 has been associated with various biological dis-
orders such as hyperalgesia, neuropathic pain, migraine,
asthma, visceral pain, chronic obstructive pulmonary
disease (COPD), inflammatory bowl disease (IBD), irri-
table bowel syndrome (IBS), interstitial cystitis/inconti-
nence, and osteoarthritis. Disruption of the TRPV1
gene in mice causes reduction in both thermal nocicep-
tion and inflammation-induced hyperalgesia.5 Recently,
substantial advances by several pharmaceutical compa-
nies have resulted in the progress to clinical trials of
TRPV1 antagonist clinical candidates such as SB-
705498 (GSK, phase II for migraine and IBS),
GRC6211 (Glenmark/Eli lilly, Phase II for acute pain),
and NGD-8243/MK-2295 (Neurogen/Merck, phase II
for acute pain).
development stage of potent TRPV1 antagonistic benz-
amide compounds ( )-2 and ( )-3, containing indole
and naphthyl moieties, respectively. This report summa-
rizes our SAR pursuit from the original hit compound 4
to the more advanced compounds ( )-2 and ( )-3 focus-
ing on the structural novelty, the avoidance of an aniline
moiety (Tables 2–5), and the bioisosteric replacement of
the obtained tryptamine scaffold (Fig. 5).6
The TRPV1 program at Pfizer was initiated with two hit
compounds (4 and 5) discovered through a HTS cam-
paign in both the capsaicin(CAP)-induced calcium in-
flux and the carbachol-induced calcium influx7 on
HEK-293 cells as shown in Table 1. The TRPV1-selec-
tive small molecule 4 was selected and modified, focus-
ing on inhibitory activity in the capsaicin-induced
calcium influx assay. At first, modifications of the triflu-
oroacetyl moiety were investigated, followed by replace-
ments of the iodide. The trifluoroacetyl group could be
converted to trifluoromethyl, trifluoromethoxy, and
2,2,1,1-tetrafluoroethoxy groups as shown in com-
pounds 6c, 6d, and 1. Having set R1 as trifluoromethoxy
or 2,2,1,1-tetrafluoroethoxy, the iodide was replaced by
a tert-butyl group, thus lowering the molecular weight
as shown in compounds 7a and 1. In the preliminary
SAR around HTS hit compound 4, compound 1 dem-
onstrated a human IC50 value of 70 nM, an increase
of antagonistic activity of more than 10-fold over that
of 4.
Having identified HTS hit compounds by high through-
put screening (HTS), our group has profiled compound
1 as a TRPV1 antagonist lead and confirmed its poten-
tial for the treatment of pain (Fig. 1). Elaboration of
compound 1 has led to the progression to the early
Keywords: TRPV1; Antagonist; Vanilloid; Bioisostere; Tetrahydrocarbazole.
*
Around the same time that the first potent TRPV1
antagonist was reported,8 the newly discovered
Corresponding author. E-mail: Yuji.Shishido@pfizer.com
Present address: Takeda Pharmaceutical Company Limited.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.007

Y. Shishido et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1072–1078
1073
Figure 1. Advanced lead compound and potent TRPV1 antagonistic benzamide derivatives.
Table 1. Preliminary SAR from HTS Hit Compound 4
Compound
R1
IC50 (nM)a
Compound
R1
R2
IC50 (nM)a
6a
6b
6c
6d
6e
6f
4-COCF3
4-COMe
4-OCF3
4-CF3
790
7a
7b
7c
7d
7e
7f
7g
7h
7i
4-OCF3
4-CF3
4-t-Bu
4-t-Bu
120
220
>10,000
280
330
4-OCH2CF3
4-OCF3
4-OCF3
4-t-Bu
4-CF3
>10,000
220
210
3-CF3
2-CF3
>10,000
>10,000
>10,000
N.T.
3, 4-cyclopentyl
3, 5-di-t-Bu
4-cyclohexyl
4-phenyl
4-t-Bu
4-OCF3
4-OCF3
>10,000
>10,000
>10,000
253
6g
6h
4-SO2Me
4-OCF2CF2H
4-OCF3
4-SCF3
4-OCF2CF2H
1
4-t-Bu
70
N.T., not tested.
a The value of IC50 means 50% inhibitory concentration in CAP-induced Ca2+ influx on human HEK-293 cells.
compound 1 was evaluated in various models and dem-
onstrated inhibitory activities9 in both CCI (MED: 59%
inhibition, @100 mg/kg) and carragenan-induced ther-
mal hyperalgesia (52% inhibition, @30 mg/kg) by sys-
temic administration (ip) as shown in Figure 2. Thus,
the benzanilide derivative 1 proved to be a useful probe
for evaluating the treatment of painful diseases by sys-
temic administration, and a key scaffold for further
modification.
In further SAR studies based on the benzanilide scaf-
fold, our efforts focused on increasing the structural
novelty and on avoiding the aniline substructure by
the modification of the linker moiety or cyclization to
benzimidazole and indole derivatives,10 as shown in
Figure 3.
Replacements of the anilide with various amide-equiv-
alent linkers caused a significant decrease of activity

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Y. Shishido et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1072–1078
Figure 2. In vitro, in vivo, and PK data of compound 1.
Figure 3. SAR strategies to avoid the anilide moiety.
Table 2. Linkage modifications based on the benzanilide scaffold
and only derivative 8g, a compound containing an
imidazole linker and a free NH, showed weak activity
with a human IC50 value of 6000 nM in the in vitro
assay, as shown in Table 2. In subsequent reports,11
it was found that the NH group of the linker plays
an important role in the inhibitory activity against
TRPV1 receptor. Furthermore, the fused benzimid-
azole derivative 10, also equipped with the free NH,
was 15-fold more potent than 8g with a human IC50
value of 380 nM, a comparable value to the IC50 of
the positive control capsazepin (Table 3).12 However,
the urea and reversed amide derivatives (8a and 8f)
demonstrated a drastic decrease of activity despite
the presence of the NH moiety.
a
IC50 (nM)
Compound
R1
R1-linker-R2
R2
I
O
8a
CF3
>10,000
N
H
N
H
O
8b
CF3
t-Bu
>10,000
O
8c
8d
8e
OCF3
CF3
t-Bu
t-Bu
t-Bu
>10,000
>10,000
>10,000
The compounds of Table 4 were prepared by an efficient
parallel synthesis protocol using 4-trifluoromethyl- or 4-
trifluoromethoxybenzoyl chloride. As a result of screen-
ing non-aniline amine monomers, substituted trypt-
amine and phenethylamine monomers were found to
lead to compounds in the desired activity range with hu-
man IC50s of 200–2000 nM such as compounds 14e–h
and 14l–n. The phenethylamine SAR revealed that the
2,6-dichloro derivative 14l had comparable activity to
capsazepine (human IC50 of 250–300 nM). However,
after extensive screening of liquid samples made by par-
allel synthesis, as well as powder samples obtained by
conventional methods, no further improvement over
the potency of 14l could be achieved. While the trypt-
amine derivatives showed antagonistic activity at the
sub-micromolar level, our focus on the structural nov-
elty led us to investigate substituents at the 5-position
of the indole ring. Thus we soon discovered that modi-
fications at the 5-position are important for the mainte-
NH2
CN
O
OCF3
N
Me
O
8f
CF3
I
>10,000
N
H
N
8g
8h
CF3
CF3
Br
6000
N
H
N
N
t-Bu
>10,000
N
N
a The value of IC50 means 50% inhibitory concentration in CAP-
induced Ca2+ influx assay on human HEK-293 cells.

Y. Shishido et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1072–1078
1075
Table 3. Conversion of benzanilide derivatives to benzimidazole
derivatives
In addition to amine screening, variations of the benzoyl
substituent R1 were also investigated when coupled with
5-methoxytryptamine, as shown in Table 5. While the
phenyl derivative 15b showed a 3-fold improvement
over the trifluoromethoxy group (15a), this result was
assumed to be due to the increase in lipophilicity (and
decrease in solubility) in the narrow benzamide SAR.
In order to avoid increasing the lipophilicity, the pyri-
dine derivatives (15h–j), as well as derivatives containing
a basic R1 group (15f and g), were synthesized, but the
results showed a 3-fold decrease in potency for 15h
and 15i compared to 15a, and a drastic decrease of activ-
ity for 15f, 15j, and 15g.
a
Compound Cyclic compound
IC50
(nM)
9
>10,000
The follow-up SAR on tryptamine derivatives is summa-
rized in Figure 4. The introduction of a methoxy group at
the 5-position (14e) followed by mono-methylation at the
benzylic position gave a compound (16) with comparable
antagonistic activity to capsazepine. In the next step, the
mono-benzyl methyl group was linked to the 2-position
of the indole ring to give tetrahydrocarbazole derivative
(20). Removal of the O-methyl group of 20 resulted in
compound ( )-2 (human IC50 = 23 nM) with 60-fold
improvement in inhibitory activity over the open-chain
compound 14e. It is clear that the conformational restric-
tion and presence of the free hydroxyl group at the appro-
10
11
380
477
12
13
1420
4970
priate position result in
a dramatic increase of
antagonistic activity. Compound ( )-16 was determined
to be TRPV1-selective and a competitive, full antagonist
(pA2 = 6.84; 143 nM) against capsaicin, after confirma-
tion of other signals with various kinase, GPCR/Channel
receptor, and enzyme assays.
a The value of IC50 means 50% inhibitory concentration in CAP-
induced Ca2+ influx assay on human HEK-293 cells.
nance of activity and that 5-methoxy and 5-hydroxy
compounds (14e and h) showed a 5-fold boost in activity
compared with the parent 14a.
Because these benzamide derivatives based on the trypt-
amine scaffold are similar to melatonin derivatives con-
Table 4. Amidation of tryptamine and phenethylamine monomers with 4-substituted benzoyl chloride
R1
R2
R3
a
IC50 (nM)
Arylamines
14a–r
a
b
c
d
e
f
OCF3
H
H
H
H
Me
H
H
H
H
H
H
H
7350
N.T.
OCF2CF2H
CF3
CF3
H
H
4230
H
>10,000
1310
2090
OCF3
CF3
5-Methoxy
5-Methoxy
5-Methoxy
5-OH
5-Cl
g
h
i
OCF2CF2H
OCF3
OCF3
1640
1730
3720
>10,000
2620
j
OCF3
OCF3
5-t-Bu
5-F
k
l
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
2,6-Cl
2-Cl
3-Cl
205
776
m
n
o
p
q
r
1930
2560
3700
>10,000
3490
2,4-Cl
3,4-Cl
3-Cl
2-F
a The value of IC50 means 50% inhibitory concentration in CAP-induced Ca2+ influx assay on human HEK-293 cells.

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Y. Shishido et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1072–1078
Table 5. Screening of benzoic moiety based on 5-methoxytryptamine
a
IC50 (nM)
Compound
R1
X
15a
15b
15c
15d
15e
15f
15g
15h
15i
Trifluoromethoxy
Phenyl
Phenoxy
C
C
C
C
C
C
C
N
N
N
1310
370
1890
1160
Cyclohexyl
Isobutyl
3430
4-Pyridyl
N,N-Dimethylaminoethoxy
Phenyl
>10,000
>10,000
3940
2,2,2-Trifluoroethoxy
Trifluoromethyl
3400
>10,000
15j
a The value of IC50 means 50% inhibitory concentration in CAP-induced Ca2+ influx assay on human HEK-293 cells.
Figure 4. Conformational approach based on tryptamine derivative (14e).
taining the same acetamide moiety, we hypothesized
that the bioisosteric replacements13 which were success-
ful for the melatonin receptor SAR would also be use-
ful in the TRPV1 antagonists program as shown in
Figure 5. In fact, the tetrahydrocarbazole moiety14 of
( )-2, 20, and 21 was known to be a bioisostere of the
tryptamine skeleton. As an example of conversion to
another known bioisostere, the non-substituted naph-
thyl derivative (22a) showed moderate activity with an
IC50 value of 2600 nM, as shown in Figure 5. In most
cases, the introduction of a hydroxyl group at the 7-po-
sition of the bicyclic system increased the antagonistic
activity over the unsubstituted compounds (22a–g). In
particular, the naphthol derivative (24a) demonstrated
a human IC50 of 150 nM, a 9-fold improvement in
antagonistic activity compared to the human
IC50 = 1310 nM for tryptamine derivative (14e). Fur-
thermore, the introduction of a methyl group at the ben-
zylic position also caused a 2- to 5-fold improvement in
potency, compared to the non-methyl analogs (24a–c).
As a result, unsubstituted naphthyl derivative (22a)
could be transformed into the optimized hydroxyl-
substituted compound (( )-3) with human and rat IC50
values of 14 nM and 44 nM, respectively.

Y. Shishido et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1072–1078
1077
Figure 5. Approach of tryptamine moiety to bioisosteric replacements.
Table 6. Bioavailability of compound ( )-2 using various vehicles
iv Administration (rat)
po Administration (rat)
po Administration (rat)
AUC (0–tmax) 662
T1/2: 2.1 h
F = 0.7 0.4%
T1/2: 1.2 h
F = 30.2 10.9%
T1/2: 2.1 h
Vehicle: 5% DMSO/PEG400
Vehicle: 0.1% MC solution
Vehicle: PEG400

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Y. Shishido et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1072–1078
4. Caterina, M. J.; Schumacher, M. A.; Tominaga, M.; Rosen,
T. A.; Levine, J. D.; Julius, D. Nature 1997, 389, 816.
5. (a) Caterina, M. J.; Leffler, A.; Malmberg, A. B.; Martin,
W. J.; Trafton, J.; Petesen-Zeiet, K. R.; Basbaum, A. I.;
Julius, D. Science 2000, 288, 306; (b) Davis, J. B.; Gray, J.;
Gunthope, M. J.; Hatcher, J. P.; Davey, P. T.; Overend,
P.; Harries, M. H.; Latcham, J.; Claphams, C.; Atkinson,
K.; Hughes, S. A.; Rance, K.; Grau, E.; Harper, A. J.;
Pugh, P. L.; Rogers, D. C.; Bingham, S.; Randall, A.;
Sheardown, S. A. Nature 2000, 405, 183.
In summary, structure–activity relationship studies start-
ing from hit compound 4 have led to advanced compound
( )-2 with human and rat IC50 values of 23 nM and
410 nM, respectively, in the capsaicin-induced calcium in-
flux assay. Moreover, the naphthyl derivative ( )-3 was
obtained by bioisosteric replacement of the tryptamine
scaffold, and showed solid activity with human and rat
IC50 values of 14 nM and 44 nM, respectively. However,
the major issue with compound ( )-2 was its low bioavail-
ability (Foral = 0.7%, vehicle: 0.1% MC solution), possibly
due to low solubility. As shown in Table 6, the PK study of
compound ( )-2 using PEG400 showed moderate bio-
availability (Foral = 30.2 10.2%) and this compound is
expected to have improved bioavailability by spray dryer
dispersion (SDD) (or solid dispersion) formulation on the
free base.
6. Nakao, K.; Nagayama, S.; Shishido, Y.; Hirano, M.;
Sudo, M.; Takada, J.; Kanai, Y. Abstracts of Papers,
232th ACS National Meeting, San Francisco, CA, United
States, September 10–14, 2006.
7. This assay determines the influence of Ca2+ entry followed
by the activation of intrinsic muscarinic receptors
expressed on HEK-293 cells.
8. N-(4-tert-Butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydro-
pyrazine-1(2H)-carboxamide (BCTC) (a) Valenzano, K.
J.; Grant, E. R.; Wu, G.; Hachicha, M.; Schmid, L.;
Tafesse, L.; Sun, Q.; Rotshteyn, Y.; Francis, J.; Limberis,
J.; Malik, S.; Whittemore, E. R.; Hodges, D. J. Pharmacol.
Exp. Ther. 2003, 306, 377; (b) Sun, Q.; Tafesse, L.; Islam,
K.; Zhou, X.; Victory, S. F.; Zhang, C.; Hachicha, M.;
Schmid, L.; Patel, A.; Rotshteyn, Y.; Valenzano, K. J.;
Kyle, D. J. Bioorg. Med. Chem. Lett. 2003, 13, 3611.
9. In this assay, we confirmed that compound 4 did not convert
to TRPV1 agonist metabolites after systemic administra-
tion by monitoring the rectal temperature of the mice/rats.
10. (a) Shao, B.; Huang, J.; Sun, Q.; Valenzano, K. J.;
Schmid, L.; Nolan, S. Bioorg. Med. Chem. Lett. 2005, 15,
719; (b) Fletcher, S. R.; McIver, E.; Lewis, S.; Burkamp,
F.; Leech, C.; Mason, G.; Boyce, S.; Morrison, D.;
Richards, G.; Sutton, K.; Jones, A. B. Bioorg. Med. Chem.
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Acknowledgments
The authors thank Mr. Kazunari Nakao for providing
helpful advice and Dr. Bernard Hulin of ChemWrite
for the editing of the manuscript.
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