Discovery of 1-(1H-indazol-4-yl)-3-((1-phenyl-1H-pyrazol-5-yl)methyl) ureas as potent and thermoneutral TRPV1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2020.127548

A series of 1-indazol-3-(1-phenylpyrazol-5-yl)methyl ureas were investigated as hTRPV1 antagonists. The structure–activity relationship study was conducted systematically for both the indazole A-region and the 3-trifluoromethyl/t-butyl pyrazole C-region to optimize the antagonism toward the activation by capsaicin. Among them, the antagonists 26, 50 and 51 displayed highly potent antagonism with K_i(CAP) = 0.4–0.5 nM. Further, in vivo studies in mice indicated that these derivatives both antagonized capsaicin induced hypothermia, consistent with their in vitro activity, and themselves did not induce hyperthermia. In the formalin model, 51 showed anti-nociceptive activity in a dose-dependent manner.

Full text of DOI:10.1016/j.bmcl.2020.127548

Bioorganic&MedicinalChemistryLetters30(2020)127548
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 1-(1H-indazol-4-yl)-3-((1-phenyl-1H-pyrazol-5-yl)methyl)
ureas as potent and thermoneutral TRPV1 antagonists
Jin Mi Kanga, Sun Ok Kwona, Jihyae Anna, Peter M. Blumberga, Heejin Hab, Young Dong Yoob,
Robert Frank-Foltync, Bernhard Leschc, Gregor Bahrenbergc, Hannelore Stockhausenc,
⁎
Thomas Christophc, Jeewoo Leea,
a Laboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
b Medifron DBT, Seoul 08502, Republic of Korea
c Grünenthal Innovation, Grünenthal GmbH, D-52078 Aachen, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of 1-indazol-3-(1-phenylpyrazol-5-yl)methyl ureas were investigated as hTRPV1 antagonists. The
structure–activity relationship study was conducted systematically for both the indazole A-region and the 3-
trifluoromethyl/t-butyl pyrazole C-region to optimize the antagonism toward the activation by capsaicin. Among
them, the antagonists 26, 50 and 51 displayed highly potent antagonism with Ki(CAP) = 0.4–0.5 nM. Further, in
vivo studies in mice indicated that these derivatives both antagonized capsaicin induced hypothermia, consistent
with their in vitro activity, and themselves did not induce hyperthermia. In the formalin model, 51 showed anti-
nociceptive activity in a dose-dependent manner.
Vanilloid Receptor 1
TRPV1 Antagonist
Analgesic
The transient receptor potential vanilloid 1 (TRPV1) is a non-
selective cation channel expressed in primary sensory neurons, func-
tioning as a molecular integrator of nociceptive stimuli. The blocking of
TRPV1 activation is regarded as one of the more promising therapeutic
strategies for the treatment of neuropathic pain.1–3 Extensive efforts
have been devoted to the development of clinical TRPV1 antagonists as
novel non-opioid analgesics.4 However, so far almost all of the an-
tagonists have failed at the clinical stage due to mechanism-associated
side effects such as hyperthermia or loss of sensitivity to thermal pain.
TRPV1 is activated by multiple stimuli, of which low pH
(pH < 6), elevated temperature (> 42 °C), and ligands such as cap-
saicin and endogenous endovanilloids are the most prominent.5–7
Reflecting the complexity of the interaction of antagonists with
TRPV1, it is now apparent that some antagonists are not equally ef-
fective at blocking these various activation modes.8,9 Likewise, not all
antagonists show an identical pattern of response at the whole animal
level. Importantly, a mode-selective TRPV1 antagonist was reported
that potently blocked channel activation by capsaicin but did not itself
cause hyperthermia.9 This complexity of response of TRPV1 to an-
tagonists fuels the current objective in the development of second-
generation vanilloid antagonists, that is to identify potent, novel
TRPV1 antagonists that themselves do not induce hyperthermia in vivo
or other side effects.10,11
ABT-102 (1) is a potent and selective TRPV1 antagonist that dis-
played promising analgesic efficacy in inflammatory, post-operative,
osteoarthritic and bone cancer pain models and drug-like properties.12
While ABT-102 induced hyperthermia in preclinical models, the hy-
pothermia was found to be attenuated upon repeated administration,
making it a promising lead compound for further SAR exploration.13,14
Structurally, ABT-102 contains an indazole A-region, a urea B-region,
and a t-butyldihydroindenyl C-region corresponding to the three
pharmacophoric regions previously designated for capsaicin.15
As part of our continuing effort to discover TRPV1 antagonists as
clinical candidates for neuropathic pain, we have investigated a new
scaffold (2) with an indazole A-region in which urea was employed as
the B-region and a variety of 1,3-disubstituted pyrazole derivatives
previously studied16 were explored as the C-region (Fig. 1). In this
study, we describe the syntheses of a series of 1-indazol-3-(1-phe-
nylpyrazol-5-yl)methyl urea analogues and characterize their antag-
onism toward activation of hTRPV1 by capsaicin. With the selected
potent antagonists in the series, we further characterize in detail the
in vivo mechanism and effect on body temperature. Finally, we ex-
amined antinociceptive activity in the formalin pain model. While we
did not determine whether these derivatives were activation-mode
selective, we did show that they did not induce hyperthermia by
themselves.
⁎ Corresponding author.
E-mail address: jeewoo@snu.ac.kr (J. Lee).
https://doi.org/10.1016/j.bmcl.2020.127548
Received 21 July 2020; Received in revised form 3 September 2020; Accepted 6 September 2020
Availableonline12September2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

J.M. Kang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127548
Scheme 3. Synthesis of the 3-trifluoromethyl pyrazole C-region. Reagents and
conditions: (a) LiAlH4, Et2O, −78 °C, 2 h; (b) R-NHNH2, EtOH, reflux, 5 h; (c)
NCS, DMF, r.t.; (d) 2-chloroacrylonitrile, TEA, toluene, 80 °C, 20 h; (e) LiAlH4,
THF, 0 °C to r.t., 3 h.
Fig. 1. Design of the indazole scaffold analogues.
For the synthesis of the C-region, (1-phenyl-3-trifluoromethyl-1H-
pyrazol-5-yl)methylamines were synthesized from ethyl tri-
fluoroacetate in 5 steps using different substituted phenyl hydrazines
(Scheme 3). The starting ester was reduced to the corresponding alde-
hyde and then was condensed with the phenyl hydrazines to afford the
corresponding phenyl hydrazones. The chlorination of hydrazone fol-
lowed by the reaction with 2-chloroacrylonitrile provided 4-cyanopyr-
azole which was reduced to give the 3-trifluoropyrazole C-region amine
(11).
Scheme 1. Synthesis of the 5- and 6-amino indazole A-region. Reagents and
conditions: (a) H2, Pd-C, MeOH, r.t.; (b) phenylchloroformate, pyridine, THF-
MeCN, r.t.; (c) NaH, MeI, DMF, 0 °C-r.t.
Similarly, (1-phenyl-3-t-butyl-1H-pyrazol-5-yl)methylamines were
prepared from 4,4-dimethyl-3-oxopentanenitrile in 4 steps using dif-
ferent substituted phenyl hydrazines (Scheme 4). The starting nitrile
was condensed with phenyl hydrazines to provide 1-phenyl 3-t-butyl-5-
aminopyrazoles whose amino group was converted to the corre-
sponding nitrile group and then reduced to afford the 3-t-butylpyrazole
C-region amine (12).
The syntheses of a series of indazole analogues were described in
Schemes 1–5. For the synthesis of 5- and 6-amino indazole A-region
intermediates (Scheme 1), commercially available
5 (or 6)-ni-
troindazole was reduced to the respective aminoindazole, which was
converted to the corresponding phenyl carbamate (3, 4) using phe-
nylchloroformate. The methylation of the nitroindazoles followed by
the same protocol above provided the N-methyl indazole A-region
carbamates (5, 6).
Scheme 4. Synthesis of the 3-t-butyl pyrazole C-region. Reagents and condi-
tions: (a) R-NHNH2·HCl, EtOH:H2O (1:1), reflux, overnight; (b) t-BuONO, CuI,
CH3CN, 0 °C to 65 °C; (c) Zn(CN)2, Pd(PPh3)4, DMF, reflux, overnight; (d)
LiAlH4, THF, 0 °C to r.t., 1 h.
The prepared indazole phenyl carbamates (A-region) were coupled
with the 3-trifluoromethyl or 3-t-butyl pyrazole amines (C-region) to
provide the final urea compounds, respectively (Scheme 5).19
Scheme 2. Synthesis of the 4-amino indazole A-region. Reagents and condi-
tions: (a) NaNO2, H2O, AcOH, r.t.; (b) H2, Pd-C, EtOH-THF (1:1), r.t. ; (c)
phenylchloroformate, pyridine, THF-MeCN, r.t.; (d) NaH, MeI, THF, r.t.; (e) i)
AcOK, Ac2O, toluene, r.t. ii) isoamyl nitrite, reflux.
Scheme 5. Synthesis of final urea compounds. Reagents and conditions: (a)
DMAP, DMF, 50 °C.
For the synthesis of the 4-amino indazole A-region intermediate
(Scheme 2), commercially available 2-methyl-3-nitroaniline was cy-
clized into 4-nitroindazole employing the method of Ruchardt and
Hassmann.17 The methylation of 4-nitroindazole yielded a mixture of
its N1- and N2-methyl isomers, respectively. On the other hand, 2-
methyl-3-nitroaniline was also reacted with potassium acetate and
acetic anhydride followed by isoamylnitrite to provide the N-acetyl 4-
nitro indazole.18 The prepared 4-nitroindazoles were converted to the
corresponding phenylcarbamates (7–10), respectively.
The in vitro assay for TRPV1 antagonism was performed using a
fluorometric imaging plate reader (FLIPR) with hTRPV1 heterologously
expressed in Chinese hamster ovary (CHO) cells.20 The activity of the
synthesized compounds was measured by inhibition of TRPV1 activa-
tion by capsaicin (100 nM) and expressed as binding affinity (Ki(CAP)).
The results are summarized in Tables 1–5.
First, we investigated the antagonistic activities of the positional
isomers of the indazole A-region. For comparison, the indazole ureas
were fixed with the same prototype C-region, the 1-(3-chlorophenyl)-3-
trifluoromethyl-pyrazole
or
1-(3-chlorophenyl)-3-t-butyl-pyrazole
groups, respectively (Table 1). Among the 16 representatives studied
2

J.M. Kang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127548
(13–28), only the 4-amino 1H-indazole and the 1-methyl-indazole
analogues (17, 18, 25, 26) exhibited full and potent antagonism with a
range of Ki(CAP) = 0.4–2.3 nM, respectively. The other isomers were
found to be agonists (19, 21, 22, 24, 27), partial agonists (16, 23) or
weak/moderate antagonists (14, 15, 20, 28).
Table 1 (continued)
25
0.6
0.4
(
(
0.2)
0.1)
26
27
The SAR analysis of the indazole A-region indicated that (1) the
position of the indazole in the A-region is critical for a proper inter-
action with the receptor in which the 4-aminoindazole isomer showed
the most promising antagonism. (2) The NH of the indazole appears not
to be involved in hydrogen bonding with the receptor for activity be-
cause both 1-H and 1-methyl indazole exhibited similar and potent
antagonism (17 vs 18, 25 vs 26).
AG
28
29.5
(
5.8)
Table 1
In vitro hTRPV1 antagonistic activities for indazole derivatives (Ki[CAP] (nM)a).
a
Values from triplicate experiments (AG: agonist, pAG: partial agonist, NT:
not tested).
In order to further optimize the antagonistic activities of the potent
4-amino 1H-indazole and 1-methyl-indazole analogues (17, 18, 25,
26), the SAR of the N-1 phenyl group in the southern part of the pyr-
azole C-region were investigated by incorporating various substituents
at the 3 or/and 4 position.
R
Ki[CAP] (nM)a
1
0.3
NT
For the analogues of the 3-trifluoromethylpyrazole indazole
(Table 2), compared to 17, whereas the 3-CF3 (29) derivative retained
activity, the 4-substituted derivatives showed slightly lower activity. In
contrast, addition to 17 of the small fluorine group at the 4-position
provided a more potent analogue (33).
13
14
61
(
8.2)
For the analogues of the 3-trifluoromethylpyrazole 1-methyl in-
dazole (Table 3), compared to 18, whereas the 3-CF3 (36) and 3-chloro-
4-fluoro (42) derivatives showed improved activities, the 4-substituted
derivatives displayed lower activities as had been seen for the previous
series above.
15
16
17
WE
pAG
(
For the analogues of 3-t-butylpyrazole indazole (Table 4), compared
to 25, whereas the 3-trifluoromethylphenyl (45), 3,4-difluorophenyl
(50) and 3-chloro-4-fluorophenyl (51) derivatives exhibited enhanced
activities, the 4-substituted phenyl derivatives once again showed lower
activities.
0.9
2.3
0.3)
0.9)
18
19
(
For the analogues of 3-t-butylpyrazole 1-methyl indazole (Table 5),
the SAR pattern was similar to those above in which the 3-trifluoro and
3-chloro-4-fluoro derivatives exhibited comparable activity to that of
26.
AG
The SAR analysis of the N1-position in the pyrazole C-region in-
dicated that the 3-trifluoro and 3-t-butylpyrazole analogues showed
similar SAR patterns in which (1) a lipophilic and electron withdrawing
group such as chloro or trifluoromethyl as a 3-substituent is optimal for
antagonism. (2) The 3-substituted phenyl derivatives displayed better
antagonism than did the corresponding 4-substituted phenyl deriva-
tives. (3) The addition of 4-F to the 3-chloro substituted derivative
further improved the antagonism.
20
17.4
(
4.8)
R
Ki[CAP] (nM)a
AG
21
22
AG
23
24
pAG
AG
3

J.M. Kang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127548
Table 2
Table 4
In vitro hTRPV1 antagonistic activities for 4-amino indazole derivatives.
In vitro hTRPV1 antagonistic activities for 4-amino indazole derivatives (Ki[CAP]
(nM)a).
R
Ki[CAP] (nM)a
(
R
Ki[CAP] (nM)a
(
17
29
0.9
1.3
0.4)
0.5)
25
44
45
0.6
1.3
0.3
0.2)
0.4)
0.1)
(
(
(
30
31
32
33
2.5
3.8
4.1
0.6
(
(
(
(
0.6)
1.2)
0.8)
0.2)
46
47
48
49
50
2.2
19.6
62.9
40.0
0.5
(
(
0.8)
7.2)
(
(
18.9)
11.5)
0.2)
34
2.8
(
0.9)
a
(
Values from triplicate experiments.
51
52
0.5
(
0.1)
Table 3
In vitro hTRPV1 antagonistic activities for 4-amino indazole derivatives.
pAG
a
Values from triplicate experiment.
Table 5
In vitro hTRPV1 antagonistic activities for 4-amino indazole derivatives (Ki[CAP]
R
Ki[CAP] (nM)a
(
(nM)a).
18
35
36
2.3
7.0
1.4
0.7)
2.7)
0.5)
(
(
R
Ki[CAP] (nM)a
(
37
38
39
40
41
2.7
8.4
(
(
(
(
(
0.6)
2.8)
0.6)
3.6)
0.8)
26
53
0.4
0.6
0.2)
0.3)
(
3.9
13.2
4.9
54
55
56
57
58
9.5
4.5
(
(
2.2)
1.6)
3.4)
10.2)
0.4)
12.4
46.3
1.0
(
42
43
1.9
3.5
(
(
0.7)
0.9)
(
(
a
59
0.6
(
0.2)
Values from triplicate experiment.
a
Values from triplicate experiments.
4

J.M. Kang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127548
For evaluating in vivo activity and the thermal effect of selected
Declaration of Competing Interest
antagonists (26, 50, 51), we measured the change of body temperature
after administration to mice of the antagonists alone or in combination
with the TRPV1 agonist capsaicin. Consistent with its in vitro me-
chanism of action, the antagonists were able to block in vivo the acute
hypothermic response to capsaicin. We measured the inhibition of the
hypothermia induced by capsaicin (3 mg/kg) injected intraperitoneally
(ip) after the oral administration (po) of 10 mg/kg of each compound
(Table 6). The level of inhibition fell within the range of 14–25% under
these conditions. We also determined the effect of the antagonists by
themselves on thermoregulation, measuring the body temperature after
the oral administration (po) of 10 mg/kg of each compound. None
caused hyperthermia, with one inducing weak hypothermia.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This work was supported by the Midcareer Researcher Program
(NRF-2019R1A2C2006837) funded by the National Research
Foundation of Korea (NRF).
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