Discovery of potent transient receptor potential vanilloid 1 antagonists: Design and synthesis of phenoxyacetamide derivatives

Article abstract of DOI:10.1016/j.bmcl.2013.04.016

We aimed to discover a novel type of transient receptor potential vanilloid 1 (TRPV1) antagonist because such antagonists are possible drug candidates for treating various disorders. We modified the structure of hit compound 7 (human TRPV1 IC₅₀ = 411 nM) and converted its pyrrolidino group to a (hydroxyethyl)methylamino group, which substantially improved inhibitory activity (15d; human TRPV1 IC₅₀ = 33 nM). In addition, 15d ameliorated bladder overactivity in rats in vivo.

Full text of DOI:10.1016/j.bmcl.2013.04.016

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3154–3156
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent transient receptor potential vanilloid 1
antagonists: Design and synthesis of phenoxyacetamide derivatives
⇑
Eiki Takahashi , Noriyuki Hirano, Takashi Nagahara, Satoru Yoshikawa, Shinobu Momen,
Hiroshi Yokokawa, Ryoji Hayashi
Pharmaceutical Research Laboratories, Toray Industries, Inc., 10-1-6 Tebiro, Kamakura, Kanagawa 248-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 December 2012
Revised 1 April 2013
Accepted 5 April 2013
Available online 13 April 2013
We aimed to discover a novel type of transient receptor potential vanilloid 1 (TRPV1) antagonist because
such antagonists are possible drug candidates for treating various disorders. We modified the structure of
hit compound 7 (human TRPV1 IC50 = 411 nM) and converted its pyrrolidino group to a (hydroxy-
ethyl)methylamino group, which substantially improved inhibitory activity (15d; human TRPV1
IC50 = 33 nM). In addition, 15d ameliorated bladder overactivity in rats in vivo.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
TRPV1
Transient receptor potential vanilloid 1
VR1
Antagonist
Me
O
Transient receptor potential vanilloid 1 (TRPV1) is a Ca2+-per-
meable non-selective cation channel composed of six transmem-
brane domains. The channel is activated by stimuli including
heat, acids, and endogenous substances such as anandamide and
lipoxygenase products, as well as by chemical compounds such
as capsaicin (1) and resiniferatoxin (2) (Fig. 1).1 TRPV1 antagonists
are expected to have therapeutic potential for various disorders
such as hyperalgesia, neuropathic pain, migraine, irritable bowel
syndrome, osteoarthritis, and overactive bladder.2 We therefore
initiated a research program to discover a novel type of TRPV1
antagonist.
Capsazepine (3) was the first competitive TRPV1 antagonist to
be discovered and exhibits antihyperalgesic activity in rats.3 Sub-
sequently, various structural classes of TRPV1 antagonists have
been reported, including SB-704598 (4), ABT-102 (5), and AMG-
517 (6) (Fig. 2).4
Me
H
O
O
Me
H
H
O
MeO
HO
Me
Me
O
OMe
OH
N
H
HO
O
O
1
2
Figure 1. Representative TRPV1 agonists.
ovary K1 cells.5 We found a phenoxyacetamide derivative (7;
Fig. 3) with moderate inhibitory activity against human TRPV1
(hTRPV1 IC50 = 411 nM). Aiming to improve TRPV1 inhibition, we
explored structural modifications of 7.
Phenoxyacetamide derivatives were efficiently synthesized as
shown in Scheme 1. Coupling between amine 8 and nitropyridine
9, followed by reduction of the nitro group provided aminopyridine
11. Phenoxyacetic acid 14 was prepared by O-alkylation of phenol
12, followed by hydrolysis of tert-butyl phenoxyacetate 13. Pheno-
xyacetamide derivatives 15a–n were obtained by using a coupling
reagent for the condensation reaction between aminopyridine 11
and phenoxyacetic acid 14. The obtained compounds were con-
verted into corresponding salts for pharmacological tests as appro-
priate and were evaluated for antagonistic activity toward hTRPV1.
We evaluated the impact of changes of the left-part pyrrolidine
moiety at the 2-position of the pyridine ring (Table 1). Introduction
of a hydroxyl group at the 3-position of pyrrolidine did not mark-
edly affect antagonistic activity toward hTRPV1 (15a). Conversion
Here we report the design, synthesis, and biological evaluation
of novel TRPV1 antagonists that, compared with SB-705498 (4), are
more potent TRPV1 inhibitors.
First, we screened a chemical library in order to find antagonists
to human TRPV1 recombinantly expressed in Chinese hamster
Abbreviations: CYP, cyclophosphamide; HBSS, Hank’s balanced salt solution;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; hTRPV1, human tran-
sient receptor potential vanilloid 1; SEM, standard error of mean; TRPV1, transient
receptor potential vanilloid 1.
⇑
Corresponding author. Tel.: +81 467 32 9643; fax: +81 467 32 9931.
E-mail address: eiki_takahashi@nts.toray.co.jp (E. Takahashi).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.016

E. Takahashi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3154–3156
3155
Cl
N
N
O
O
O
S
N
CF3
N
S
N
H
N
H
N
H
N
H
N
F3C
HN
N
H
HO
HO
N
Br
O
N
N
H
3
4
5
6
Figure 2. Representative TRPV1 antagonists.
left-part
linker
right-part
Table 1
Antagonist activity of phenoxyacetamide derivatives toward hTRPV1
H
R5
N
N
N
O
H
N
O
R4
N
O
R1
O
N
R2 R3
7
(hTRPV1 IC50 = 411 nM)
Compound R1
R2
R3
R4
R5
htrpv1 IC50
(nM)a
Figure 3. Compound discovered by a screening campaign.
4 (SB-
704598)
120
NH2
7
–(CH2)4–
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-tert-
Butyl
4-tert-
Butyl
4-tert-
Butyl
4-tert-
Butyl
4-tert-
Butyl
4-tert-
Butyl
4-tert-
Butyl
3-tert-
Butyl
2-tert-
Butyl
H
4-i-propyl
4-CF3
4-
Admanthyl
4-tert-
Butyl
411
516
59
NO2
NO2
b
R1
N
a
N
N
R1
NH
R2
+
R1
N
N
X
15a
15b
15c
15d
15e
15f
15g
15h
–(CH2)2–
CH2OHCH2–
Me
R2 R3
R2 R3
R3
9
8
10
11
Me
Me
Et
583
33
R4
R4
R5
R4
R5
c
d
(CH2)2OH Me
HO
t-BuO
R5
HO
O
O
O
O
13
(CH2)2OH Me Me
(CH2)2OH Me Cl
130
44
12
14
R4
R5
H
N
(CH2)2OH Me
(CH2)2OH Me
H
H
2620
796
e
N
O
R1
O
N
R2 R3
15
15i
15j
15k
15l
(CH2)2OH Me
(CH2)2OH Me
(CH2)2OH Me
(CH2)2OH Me
H
H
H
H
H
H
H
H
>10
110
>10
>10
l
M
(R1, R2) = (Me, Me), (Me, Et), (Me, CH2CH2OH), (CH2CH2CH2CH2), (CH2CH2CH2OHCH2),
3 = H, Me, Cl
lM
M
R
(R4, R5) = (H, 4-tert-butyl), (H, H),(H, 4-i-Pr), (H, 4-CF3), (H, 4-adamantyl),
(H, 2-tert-Butyl), (H, 3-tert-butyl), (2,4-di-tert-butyl),
(2-Br, 4-di-tert-butyl), (2-piperidinyl, 4-di-tert-butyl)
X = Cl, Br
l
15m
15n
15o
(CH2)2OH Me
(CH2)2OH Me
(CH2)2OH Me
H
H
H
2-tert-
Butyl
2-Br
107
86
4-tert-
Butyl
4-tert-
Scheme 1. Reagents and conditions: (a) K2CO3, dimethylformamide, rt, 72–99%; (b)
Pd/C, H2, MeOH, rt or Fe, NH4Cl, tetrahydrofuran, EtOH, H2O, 70 °C, 77–99%; (c) tert-
butyl bromoacetate, K2CO3, acetone, 50 °C, 58–99%; (d) trifluoroacetic acid, CH2Cl2,
40 °C, 58–99%, (e) 11, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N-diiso-
propylethylamine, CH2Cl2, rt, 10–99%.
2-
941
Piperidinyl Butyl
a
Inhibition assays were carried out in hTRPV1-transfected Chinese hamster
ovary K1 cells. IC50 were determined from data at various concentrations of
derivatives in triplicate.
of a pyrrolidino group to dimethyamino group considerably im-
proved activity (15b), while the activity of ethylmethylamino com-
pound 15c was comparable with that of 7. To our surprise,
(hydroxyethyl)methylamino compound 15d showed the most po-
tent activity with an IC50 = 33 nM. The effects of the hydroxyethyl
group are not yet fully understood. The chlorine substituent at the
3-position of the pyridine ring of 15d had a similar effect, but a
methyl group was not favorable (15e and 15f).
We next turned our attention to modifying the right part (Table
1; 15g–15o). To clarify the substituent effects, we took 15d as a
starting point and modified the substituents of the right-part phe-
nyl ring. Moving the tert-butyl group from the 4-position to the 2-
or 3-position of the phenyl ring decreased antagonistic activity
(15g and 15h). Furthermore, we introduced hydrogen or substitu-
ents such as trifluoromethyl, isopropyl, and adamantly groups at
the 4-position. However, these substituents were not as effective
as the tert-butyl group. Adding a substituent at the 2-position of
right-part phenyl ring in 15d caused a decrease in antagonistic
activity (15m, 15n, and 15o).
We next evaluated the pharmacological effects of 15d in vivo in
an overactive bladder rat model, because 15d was a potent TRPV1
inhibitor in vitro. Specifically, we evaluated the effects of 15d on
capsaicin-induced bladder contraction in rats (mechanistic mod-
el)6 and on cyclophosphamide (CYP)-induced cystitis in rats (dis-
ease model)7 ( Figs. 4 and 5). At a dose of 3 mg/kg, iv, 15d
suppressed capsaicin (30 lg/kg, iv)-induced bladder contraction
in rats. Moreover, at a dose of 3 mg/kg (iv), 15d reduced the void-
ing frequency in rats with CYP-induced cystitis and pollakiura.

3156
E. Takahashi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3154–3156
References and notes
1. (a) Caterina, M. J.; Schumacher, M. A.; Tominaga, M.; Rosen, T. A.; Levine, J. D.;
Julius, D. Nature 1997, 389, 816; (b) Tominaga, M.; Caterina, M. J.; Malmberg, A.
B.; Rosen, T. A.; Gilbert, H.; Skinner, K.; Raumann, B. E.; Basbaum, A. I.; Julius, D.
Neuron 1998, 21, 531; (c) Gunthorpe, M. J.; Harries, M. H.; Prinjha, R. K.; Davis, J.
B.; Randall, A. J. Physiol. 2000, 3, 747; (d) Piper, A. S.; Yeats, J. C.; Bevan, S.;
Docherty, R. J. J. Physiol. 1999, 518, 721; (e) Zygmunt, P. M.; Petersson, J.;
Andersson, D. A.; Chuang, H.-H.; Sorgard, M.; Di Marzo, V.; Julius, D.; Hogestatt,
E. D. Nature 1999, 400, 452; (f) Hwang, S. W.; Cho, H.; Kwak, J.; Lee, S. Y.; Kang, C.
J.; Jung, J.; Cho, S.; Min, K. H.; Suh, Y. G.; Kim, D.; Oh, U. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 6155.
2. (a) Szallasi, A.; Cortright, D. N.; Blum, C. A.; Eis, S. R. Nat. Rev. Drug Disc. 2007, 6,
357; (b) Gunthorpe, M. J.; Chizh, B. A. Drug Discovery Today 2009, 14, 56; (c)
Khairatkar-Joshi, N.; Szallasi, A. Trends Mol. Med. 2009, 15, 14; (d) Charrua, A.;
Cruz, C. D.; Narayanan, S.; Gharat, L.; Gullapalli, S.; Cruz, F.; Avelino, A. J. Urology
2009, 181, 379; (e) Urbahns, K.; Yura, T.; Mogi, M.; Tajimi, M.; Fujishima, H.;
Masuda, T.; Yoshida, N.; Moriwaki, T.; Madge, D.; Chan, F.; Gupta, J. B. Bioorg.
Med. Chem. Lett. 2011, 21, 3354.
3. (a) Walpole, C. S.; Bevan, S.; Boverman, G.; Boelsterli, J. J.; Breckenridge, R.;
Davies, J. W.; Hughes, G. A.; James, I.; Oberer, L. J. Med. Chem. 1942, 1994, 37; (b)
Walker, K. M.; Urban, L.; Medhurst, S. J.; Patel, S.; Panesar, M.; Fox, A. J.;
Mcintyre, P. J. Pharmacol. Exp. Ther. 2003, 304, 56.
4. (a) Rami, H. K.; Thompson, M.; Stemp, G.; Fell, S.; Jerman, J. C.; Stevens, A. J.;
Smart, D.; Sargent, B.; Sanderson, D.; Randall, A. D.; Gunthorpe, M. J.; Davis, J. B.
Bioorg. Med. Chem. Lett. 2006, 16, 3287; (b) Gomtsyan, A.; Bayburt, E. K.;
Schmidt, R. G.; Surowy, C. S.; Honore, P.; Marsh, K. C.; Hannick, S. M.; McDonald,
H. A.; Wetter, J. M.; Sullivan, J. P.; Jarvis, M. F.; Faltynek, C. R.; Lee, C.-H. J. Med.
Chem. 2008, 51, 392; (c) Doherty, E. M.; Fotsch, C.; Bannon, A. W.; Bo, Y.; Chen,
N.; Dominguez, C.; Falsey, J.; Gavva, N. R.; Katon, J.; Nixey, T.; Ognyanov, V. I.;
Pettus, L.; Rzasa, R. M.; Stec, M.; Surapaneni, S.; Tamir, R.; Zhu, J.; Treanor, J. J. S.;
Norman, M. H. J. Med. Chem. 2007, 50, 3515.
Figure 4. Effect of 15d on capsaicin-induced bladder contraction in rats. Compound
15d dose-dependently suppressed capsaicin-induced bladder contraction in rats.
Data are shown as the mean SEM of 4 animals. ⁄⁄P <0.01 compared with the
vehicle group.
5. Calcium mobilization assays: hTRPV1-expressing Chinese hamster ovary K1 cells
were seeded into 96-well plates and grown to 90% confluency. The cells were
then incubated at rt for 90 min in the Hank’s balanced salt solution (HBSS)/4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (20 mM HEPES,
HBSS; pH 7.4) supplemented with calcium-sensitive fluorescent dye (2 lM,
Fluo4-AM, Dojindo). The wells were then washed three times with HBSS–HEPES
buffer. Addition of 100 nM capsaicin to the wells resulted in concentration-
dependent elevation of intracellular calcium levels and subsequent activation of
Fluo4 fluorescence. Fluorescence imaging plate readers (FLIPR, Molecular
Devices) were used to monitor changes in the fluorescence and to determine
the maximum fluorescence signal during 120 s after the application of capsaicin
by statistics (FLIPR Software). The derivatives at various concentrations were
added to the wells together with capsaicin. The maximum signals by capsaicin
in the presence or absence of the derivatives at various concentrations were
averaged in triplicate and then, IC50 values (the concentration required for 50%
inhibition of the control reaction) were determined in GraphPad Prism, version
4 (GraphPad Software).
Figure 5. Effect of 15d on pollakiuria in rats with CYP-induced cystitis. Compound
15d dose-dependently reduced voiding time in rats with CYP-induced cystitis with
pollakiuria. Data are shown as the mean SEM of 4 animals. ⁄P <0.05 compared
with the vehicle group.
6. Capsaicin-induced bladder contraction in rats: Female Sprague–Dawley rats were
anesthetized with urethane (1.0 g/kg, ip), and then a catheter was inserted into
the bladder through the external urethral orifice. Physiological saline at rt was
infused into the bladder (3.0 mL/h, 5 min; 0.25
lL) to obtain the basal
intravesical pressure. Capsaicin (30 g/kg, iv) was injected at 10 min intervals
l
to induce bladder contraction. Stable bladder contractions (control) were
obtained, 15d or the vehicle (20% cremophor EL) was administered (iv) which
was followed by additional application of capsaicin. A percent change in the
capsaicin-induced bladder contraction was calculated. Statistical significance
was evaluated using Bartlett’s test followed by Williams’ tests compared to
vehicle group. P values <0.05 were considered significant.
In conclusion, we identified hit compound 7, which has a phe-
noxyacetamide structure, from our chemical library. The pyrrolidi-
no group of 7 was converted to other substituents. As a result, we
found that 15d bearing a (hydroxyethyl)methylamino group has
potent antagonistic activity toward hTRPV1. Moreover, we demon-
strated that 15d, which showed the most potent antagonistic activ-
ity in vitro, ameliorated bladder overactivity in rats in vivo. These
data suggest that 15d has potential as a drug for treating overac-
tive bladder. To broaden the therapeutic potential, we plan to eval-
uate the discovered TRPV1 antagonists in other disease models.
7. Cystometry in pollakiuria rats with CYP-induced cystitis: Female Sprague–Dawley
rats were used. Cystitis was induced by administering cyclophosphamide
(150 mg/kg, ip) to rats. At 4 h after application, an intravesical catheter was
implanted and then physiological saline at rt was infused into the bladder
(3.6 mL/h) to obtain continuous cystometrograms. After
a stable voiding
frequency was obtained, 15d or the vehicle (20% cremophor EL) was
administered (iv). Voiding times for 20 min before and after the
administration was recorded; the percent change in voiding frequency was
calculated for each group. Statistical significance was evaluated by Bartlett’s test
followed by Williams’ test for comparison with the vehicle group. P values <0.05
were considered significant.
Acknowledgments
The authors gratefully thank Mr. Yuji Sugawara (Toray Indus-
tries, Inc.) for the editing of the manuscript.