Pharmacological evaluation of novel (6-aminopyridin-3-yl)(4-(pyridin-2-yl)piperazin-1-yl) methanone derivatives as TRPV4 antagonists for the treatment of pain

Article abstract of DOI:10.1016/j.bmc.2017.02.047

A novel series of (6-aminopyridin-3-yl)(4-(pyridin-2-yl)piperazin-1-yl) methanone derivatives were identified as selective transient receptor potential vanilloid 4 (TRPV4) channel antagonist and showed analgesic effect in Freund's Complete Adjuvant (FCA) induced mechanical hyperalgesia model in guinea pig and rat. Modification of right part based on the compound 16d which was disclosed in our previous communication led to the identification of compound 26i as a flagship compound. In this paper, we described the details about design, synthesis and structure-activity relationship (SAR) analysis at right and left part of these derivatives (Fig. 1).

Full text of DOI:10.1016/j.bmc.2017.02.047

Accepted Manuscript
Pharmacological evaluation of novel (6-aminopyridin-3-yl)(4-(pyridin-2-yl)pi-
perazin-1-yl) methanone derivatives as TRPV4 antagonists for the treatment of
pain
Naoki Tsuno, Akira Yukimasa, Osamu Yoshida, Shinji Suzuki, Hiromi Nakai,
Tomoyuki Ogawa, Motohiro Fujiu, Kenji Takaya, Azusa Nozu, Hiroki
Yamaguchi, Hidetoshi Matsuda, Satoko Funaki, Natsue Yamanada, Miki
Tanimura, Daiki Nagamatsu, Toshiyuki Asaki, Narumi Horita, Miyuki
Yamamoto, Mikie Hinata, Masahiko Soga, Masayuki Imai, Yasuhide Morioka,
Toshiyuki Kanemasa, Gaku Sakaguchi, Yasuyoshi Iso
PII:
S0968-0896(16)31003-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.047
BMC 13577
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
6 December 2016
13 February 2017
15 February 2017
Please cite this article as: Tsuno, N., Yukimasa, A., Yoshida, O., Suzuki, S., Nakai, H., Ogawa, T., Fujiu, M., Takaya,
K., Nozu, A., Yamaguchi, H., Matsuda, H., Funaki, S., Yamanada, N., Tanimura, M., Nagamatsu, D., Asaki, T.,
Horita, N., Yamamoto, M., Hinata, M., Soga, M., Imai, M., Morioka, Y., Kanemasa, T., Sakaguchi, G., Iso, Y.,
Pharmacological evaluation of novel (6-aminopyridin-3-yl)(4-(pyridin-2-yl)piperazin-1-yl) methanone derivatives
as TRPV4 antagonists for the treatment of pain, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
1
0.1016/j.bmc.2017.02.047
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Pharmacological evaluation of novel (6-
aminopyridin-3-yl)(4-(pyridin-2-yl)piperazin-1-
yl) methanone derivatives as TRPV4
antagonists for the treatment of pain
a
a
a
a
a
Naoki Tsuno , Akira Yukimasa , Osamu Yoshida , Shinji Suzuki , Hiromi Nakai ,
a
a
a
a
a
Tomoyuki Ogawa , Motohiro Fujiu , Kenji Takaya , Azusa Nozu , Hiroki Yamaguchi ,
b
d
c
c
Hidetoshi Matsuda , Satoko Funaki , Natsue Yamanada , Miki Tanimura , Daiki
d
c
b
c
c
Nagamatsu , Toshiyuki Asaki , Narumi Horita , Miyuki Yamamoto , Mikie Hinata ,
c
e
c
c
Masahiko Soga , Masayuki Imai , Yasuhide Morioka , Toshiyuki Kanemasa , Gaku
e
f
Sakaguchi and Yasuyoshi Iso
a
Medicinal Chemistry Research Laboratry, Shionogi & Co., Ltd.
b
Shionogi Techno Advance Research Co., Ltd.
c
Drug Discovery & Disease Research Laboratory, Shionogi & Co., Ltd.
d
e
Research Laboratory for Development, Shionogi & Co., Ltd.
Global Innovation Office, Shionogi & Co., Ltd.
f
IMP Manufacturing Center, CMC R&D Division, Shionogi & Co., Ltd.

Key words: Transient Receptor Potential Vanilloid 4, TRPV4 antagonist, ion channel,
vanilloid receptor, pain
Author information:
Corresponding author
*
Phone: +81 (6) 6331 6173. Fax: +81 (6) 6332 6385. E-mail:
naoki.tsuno@shionogi.co.jp
ABSTRACT
A novel series of (6-aminopyridin-3-yl)(4-(pyridin-2-yl)piperazin-1-yl) methanone
derivatives were identified as selective transient receptor potential vanilloid 4 (TRPV4)
channel antagonist and showed analgesic effect in Freund’s Complete Adjuvant (FCA)
induced mechanical hyperalgesia model in guinea pig and rat. Modification of right part
based on the compound 16d which was disclosed in our previous communication led to
the identification of compound 26i as a flagship compound. In this paper, we described
the details about design, synthesis and structure-activity relationship (SAR) analysis at
right and left part of these derivatives (Figure 1).
1
.
Introduction

TRPV4 is a cation channel and a member of the TRPV family composed of TRPV1/2,
1
-6
TRPV3, TRPV4 and TRPV5/6 . TRPV4 was first disclosed as an osmotic-sensitivity
7,8
receptor activated by osmotic cell swelling and mechanical stimuli . Moreover, it was
activated by several stimulus including low pH, warm temperature (~35°C), mechano-
stimuli, endogenous ligand and some chemical stimulus such as 4-phorbol 12,13-
8
-14
didecanoate (4αPDD) . The compound which exhibits high affinity to TRPV4 has
been expected a high potential as useful medicine in the therapy and/or prevention of
TRPV4 receptor mediated disorder such as heart failure, pulmonary edema, pain,
1
5-23
osteoporosis and bladder function
. Because, TRPV4 is widely expressed such as in
epithelial cells of urinary system, airway epithelia of the trachea and the lungs, in the
cilia of bronchial epithelium, in the ciliated epithelia of the bile ducts, in the epidermal
5
keratinocytes, in the vascular endothelium, in the inner ear, in osteoblasts of born .
Furthermore, there are a lot of reports which suggests the relationship between TRPV4
2
4-30
and pain
. The activation of the C-fiber by hypotonic stimulation under the
inflammatory environment induced by inflammatory mediators is also reported that
TRPV4 relates to this activation. Moreover, it is also revealed that TRPV4 is activated
by fluid pressure and mechanical stimuli, and TRPV4 relates to hyperalgesia caused by
3
1
mechanical stimuli, relates to paclitaxel-induced pain . According to these information,

we focused on the TRPV4 as the target of pain and conducted some in vivo validation
as same as HTS to afford some hit compounds including bis-thiazole amine. Recently,
we reported that novel thiazole derivatives showed more potent antagonism of TRPV4
and showed analgesic effect in FCA induced mechanical hyperalgesia model in
3
2
previous communication . The further optimization of left and right part around the
compound 16d, the advanced compound 26i which improved the rat TRPV4 potency
was found and moved on the in vivo and selectivity study while maintaining the species
differences between human and rat TRPV4 potency. As the result of these studies
resulted in identification of 26i as a potent and highly selective TRPV4 antagonist
which shows analgesic effect in FCA induced mechanical hyperalgesia model in guinea
pig and rat. In this paper, we report the details of SAR study based on the compound
1
6d and their synthesis.

Figure 1. General structure of the SAR compound.

2
2
.
Result and discussion
.1. Chemistry. The key intermediates of carboxylic acid 4a-d were prepared as
described in Scheme 1. After bromination with NBS, the resulting α-bromo ketones
were reacted with thiourea to afford the cyclized compounds 2a-d, respectively. The
amines were coupled with ethyl-6-chloronicotinate under Buchwald-Hartwig condition
to afford ester 3a-d, which was then hydrolyzed with sodium hydroxide to afford
corresponding carboxylic acid. The synthesis of carboxylic acid 11a and 11b was
described in Scheme 2. The amination of chloride on thiadiazole with ammonia aqueous
solution was given amine 6 followed by protection with Boc group to provide 7, which
was coupled with boronic ester followed by deprotection of Boc group to afford
compound 9a and 9b, respectively. The next coupling reaction with ethyl-6-
chloronicotinate and hydrolysis step was almost same manner with synthesis method
from 2a-d to 4a-d. The right part including bicyclic amines was synthesized according
to Scheme 3. The commercially available Boc protected diazabicyclo[3.2.1]octane was
coupled with allyl bromide under Buchwald-Hartwig condition to afford 13a and 13b,
followed by deprotection of Boc group to provide 14a and 14b, respectively. In the case
of 2-chloropyrimidine as other end with coupling reaction, the usual alkylation was
given 13c and 13d which were treated with hydrochloric acid to afford 14c and 14d

respectively. The resulting carboxylic acids 4a-d, 11a, 11b and amines 14a-d,
commercially available 15 were treated with COMU under basic condition to provide
corresponding target compound 16a-d, 18-22 and intermediate 17, respectively
(Scheme 4). However, intermediate 17d was not obtained by amidation with
corresponding carboxylic acid due to low reactivity of trifluoromethyl piperazine. After
acylation with piperazine using acid chloride, the resulting compound 24 was coupled
with 2a to provide 17d (Scheme 5). After deprotection of Boc group in 17a-j, the usual
alkylation with aryl chloride was provided target compound 26a-i and 27-35,
respectively (Scheme 6). Conversion of the ester to alcohol 36, carboxylic acid 37 and
amide 38 was shown in Scheme 7, which was prepared by usual and general condition
to afford target compounds.

Scheme 1. Synthesis of the carboxylic acid 4a-d. Reagents and conditions: (a)
TMSOTf, Et N, THF, 0°C, 2h; (b) NBS, THF, 0°C, 0.5h; (c) thiourea, EtOH, reflux, 1h;
3
(
d) ethyl-6-chloronicotinate, sodium carbonate, Pd(dba) , xantphos, 1,4-dioxane, reflux,
2
1
6h; (e) 2N NaOH, EtOH, 80°C, 5h.

Scheme 2. Synthesis of the the carboxylic acid 11a and 11b. Reagents and conditions:
a) 28% ammonia aqueous, EtOH, 70°C, 3h; (b) (Boc) O, DMAP, THF, 50°C, 1h; (c)
(
2
2
4
9
,4-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiazole or 1,3-dimethyl-
-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, CsF, 1,4-dioxzne, H O,
2
0°C, overnight; (d) 4N HCl in EtOAc, r.t., overnight; (e) ethyl-6-chloronicotinate,
potassium phosphate, Pd (dba) , xantphos, 1,4-dioxane, 130°C, 2h, microwave
2
3
irradiation; (f) 2N NaOH, EtOH, 70°C, 8h.

Scheme 3. Synthesis of the intermediate 14a-d. Reagents and conditions: (a) ArBr,
Pd(dba) , RuPhos, NaOt-Pen, 1,4-dioxane, 100°C, 2h, microwave irradiation. For 13a
2
and 13b. (b) 2-chloro-pyrimidine, diisopropyl ethylamine, NMP, 80°C, 5h. For 13c and
1
3d; (c) 4N HCl in 1,4-dioxane, r.t., overnight.

Scheme 4. Synthesis of the compound 16-22. Reagents and conditions: (a) COMU,
Et N, DMF, r.t., 1.5h.
3

Scheme 5. Synthesis of the intermediate 17d. Reagents and conditions: (a) 6-
chloronicotinoyl chloride , diisopropyl ethylamine, THF/CH Cl =1/1, 0°C~r.t., 0.5h; (b)
2
2
2
a, sodium carbonate, Pd(dba) , xantphos, 1,4-dioxane, reflux, 16h.
2

Scheme 6. Synthesis of the compound 26a-i and 27-35. Reagents and conditions: (a)
4
2
N HCl in 1,4-dioxane, 0°C~r.t., 2h; (b) 2-chloro-pyrimidine or 3-chloro-pyridazine or
-chloro-pyridine, K CO , NMP, 90°C, 3h.
2
3

Scheme 7. Synthesis of the compound 36-38. Reagents and conditions: (a) LiBH4,
THF, MeOH, r.t., 2.5h; (b) 2N NaOH, THF, EtOH, r.t.~60°C, 4h; (c) COMU, NH Cl,
4
Et N, DMF, r.t., 24h.
3

2
.2. In vitro pharmacology. The synthesized TRPV4 ligands were evaluated in
vitro for antagonism as measured by inhibition of activation by 4αPDD or osmotic
stimuli. The assays were conducted using a fluorometric imaging plate reader (FLIPR)
with human, guinea pig or rat TRPV4 stably expressed in recombinant Chinese hamster
ovary (CHO) K1 cells. The results were summarized in Table 1-4 as following, which
included some previously reported antagonist values for comparing the results. Table 1
showed human TRPV4 antagonist potencies against 4αPDD as TRPV4 selective agonist
and human hypotonicity IC50 values as using osmotic stimuli. The human TRPV4
antagonist potency against 4αPDD of compound 27 resulted in nearly 2-fold
improvement compared with compound 16a and 3,5-pyrimidine analog 16b resulted in
nearly 3-fold loss of potency compared with 27, which indicated the improvement of
the potency to add the nitrogen at 2-position on phenyl ring. The pyridine 27, pyridazine
2
8 and pyrazine 29 showed almost same TRPV4 antagonist potency against 4αPDD,
which indicated no influence of the improvement of the potency based on the nitrogen
position of 27. Additional 6-position nitrogen substituted on 2-pyridine ring as in the
case of 16d resulted in a significant improvement of human TRPV4 antagonist potency
against 4αPDD. Moreover, the hypotonic potency showed almost same trend of 4αPDD
potency except for 29. Most potent analog 16d against 4αPDD showed best TRPV4

hypotonic potency and 16b which indicated weaker potency against 4αPDD than 16d
showed slightly loss in hypotonic potency. There are some exceptions in the correlation
between 4αPDD and hypotonicity IC values, but it could be predicted by the result of
5
0
the 4αPDD as using the activation in human TRPV4 antagonist potency. The influence
of the substitution at 4-position on 2,6-pyrimidine ring was given in Table 2. The
compounds which have halogen, methanesulfonyl, cyano and trifluoromethyl group
showed more potent for human TRPV4 and resulted in a slightly improvement of non-
substituted analog 30. The alcohol analog 36 retained good potency, but amide analog
3
8 resulted in 8-fold loss in potency. Furthermore, the carboxylic acid analog 37 showed
significant loss in potency. These result suggested that the region of near the 4-position
is not to be tolerated in improvement of the human TRPV4 potency. Next, the SAR and
result of human, guinea pig and rat of the piperazine ring was shown in Table 3. The
(
S)-methyl isomer at R position 26b resulted in 10-fold improvement in human TRPV4
1
potency compared with non-substituted piperazine analog 26a. On the other hand, the
R)-methyl isomer at R position 26c showed slightly improvement compared with 26a,
(
1
which suggested that the potency could be depended on the conformation on this region.
The compound 26d having enantio-mixture trifluoromethyl group at R position
1
resulted in nearly 20-fold improvement in human TRPV4 potency. Comparison with the

result of the 26b suggested that it could be important for improvement of the TRPV4
potency to increase the lipophilicity. There is a support information that the compound
2
6e including alcohol, hydrophilic group at R position showed 10-fold loss in human
1
TRPV4 potency. The regio-substitution of methyl group at R position showed slightly
3
differences their potencies, as in 26f and 26g. The compounds including bridge structure
at R and R showed more potent than compound 26h including bridge structure at R
1
2
3
and R position, which indicated that the steric hindrance near the R and R region
4
1
2
could be important to improve the human TRPV4 potency. There is no support data
which influence is important or not, but these data suggested that it was very important
for keeping the good potency to maintain high lipophilicity and/or steric hindrance at R1
and R region. The guinea pig TRPV4 antagonist potencies showed almost same result
2
with human, which indicated that there are about 1-4 fold species differences between
them. On the other hand, there are big species differences between human and rat,
which indicated the chemo type dependence in Table 3. Fortunately, more human
TRPV4 potent compound 26d, 26i and 16d showed single nano-molar IC values for
50
rat TRPV4 in spite of about 20-30 fold species differences. The left part SAR was
shown in Table 4. The 1,3-dimethyl-5-substituted pyrazole analog 18 and the compound
including alcohol group at 2-position at thiazole ring, as in 19, showed 6-fold loss in

potency compared with 16d. The potency of 1,3-dimethyl-4-substituted pyrazole analog
0 showed as same TRPV4 IC value as 16d. On the other hand, 3-substituted-1,2,4-
2
5
0
thiadiazol-5-amine analogs, as in 21 and 22, resulted in a significant loss in TRPV4
potency compared with corresponding thiazole analogs 20 and 16d, respectively. The
degree of the species differences between human and rat showed almost same result in
Table 3.

Table 1. Relationship between human 4αPDD and hypotonicity as agonist with
trifluoromethyl benzene and hetero aromatic ring on the right part.
b,c
a,c
h_hypotonicity
h_4αPDD
compd.
R
IC (nM)
5
0
IC (nM)
5
0
16a
3.9
-
2
7
8
9
2.2
2.2
2.6
6.3
1.4
1.3
2
2
0.71
23
1
1
6b
6d
0.22
0.4
a
Concentration of 4αPDD was 1000nM, determined as ED for human TRPV4.
7
0
b
c
Hypotonic solution(218 mOsm), equivalent to ED of 4αPDD. The IC value of HC-
7
0
50
0
2
67047 with using the 4αPDD and /or hypotonicity as the agonist was 57nM and /or
9nM for human TRPV4 as a control of the assay respectively.

Table 2. SAR with human 4αPDD and microsomal stability of pyrimidine at 4-position
a
h_4αPDD
h_microsomal
stability (%)
c
compd.
R
LogD
b
IC (nM)
5
0
3
3
3
3
3
3
3
3
3
0
1
2
3
4
5
6
7
8
H
F
0.5
8.5
3.30
3.51
3.97
4.05
2.83
3.18
2.70
1.46
2.31
4.25
0.31
0.3
38.4
76.4
72.8
52.7
37.7
61.4
86.2
51.8
94.8
Cl
Br
0.21
0.39
0.18
0.65
170
4.2
SO Me
2
CN
CH OH
2
COOH
CONH
CF
2
16d
3
0.22
a
Concentration of 4αPDD was 1000nM, determined as ED for human TRPV4.
7
0
b
percent of compound remaining after 30 min incubation with human liver microsomes
c
(
0.5 mg protein/mL) at 37°C with or without NADPH generating system. LogD was
calculated value by Pipeline pilot version 9.5.0.831.

Table 3. SAR of the piperidine ring and each species TRPV4 antagonist potencies
against 4αPDD as agonist.
a, b
4αPDD
h_microsomal
stability (%)
d
compd.
R , R2
R , R4
LogD
1
3
c
h_IC50
g_IC50
r_IC50
(nM)
(
nM)
6.8
(nM)
2
6a
6b
6c
6d
6e
6f
6g
6h
H, H
H, H
H, H
-
1.3
2.1
1.1
-
-
77.5
69.5
76.3
88.4
82.1
75.1
64.1
57.4
79.8
94.8
3.71
4.09
4.09
4.75
3.20
3.69
3.69
5.01
3.47
4.25
2
(S)Me, H
(R)Me, H
0.62
2.4
22
62
8.2
-
2
H, H
2
CF
3
H, H
0.29
64
2
CH OH, H
2
H, H
2
H, H
H, H
H, H
(R)Me, H
(S)Me, H
0.99
2
-
-
2
1.8
1.2
0.73
0.9
47
50
4.4
8.6
2
1
0.97
0.19
0.22
26i
H, H
H, H
6d
a
Concentrations of 4αPDD were 1000nM, 250nM, 300nM for humans, guinea pigs, and
b
rats, respectively, determined as ED for each TRPV4. The IC value of HC-067047
7
0
50
c
was 180nM for rat TRPV4 as a control of the assay. percent of compound remaining
after 30 min incubation with human liver microsomes (0.5 mg protein/mL) at 37°C with
d
or without NADPH generating system. LogD was calculated value by Pipeline pilot
version 9.5.0.831.

Table 4. SAR of the azole on the left part and each species TRPV4 antagonist potencies
against 4αPDD as agonist.
a
4αPDD
h_microsomal
stability (%)
c
compd.
R
X
C
C
LogD
b
h_IC50
g_IC50
r_IC50
(nM)
(
nM)
(nM)
1
1
8
9
1.3
1.3
1.4
39
38
99.7
79.9
4.16
1.8
4.06
2
2
2
0
1
2
C
N
N
C
0.87
6.2
0.93
4
-
180
-
89
3.87
3.43
3.80
4.25
96.7
85.2
94.8
3.7
2.6
0.9
16d
0.22
8.6
a
Concentrations of 4αPDD were 1000nM, 250nM, 300nM for humans, guinea pigs, and
b
rats, respectively, determined as ED for each TRPV4. percent of compound
7
0
remaining after 30 min incubation with human liver microsomes (0.5 mg protein/mL) at
c
3
7°C with or without NADPH generating. LogD was calculated value by Pipeline pilot
version 9.5.0.831.

2
.3. Human microsomal stability. According to our expectation of future oral drug,
the human microsomal stability data and corresponding calculated LogD as the
reference were put on the table 2-4. This chemo type showed good human microsomal
stability in spite of high lipophilic derivatives, but it was depended on the 4-position
substitutions on pyridine or substitutions on piperazine ring. In Table 2, non-substituted
pyrimidine analog 30 showed poor microsomal stability due to the position to be easily
metabolize at 4-position on pyrimidine ring, generally. The florine substituted at 4-
position analog 31 showed better microsomal stability than 30, which indicated the
support data. The chlorine, bromine and trifluoromethyl substituted analogs 32, 33 and
1
6d showed more stable respectively. Contrary to our expectations, the methane
sulfonyl, cyano and amide substituted analog, as in 34, 35 and 38, resulted in poor
metabolic stability in spite of lower calculated LogD. The compounds including
hydrophilic group at 4-position on pyrimidine showed poor or moderate microsomal
stability. In Table 3, there seems to be no influence the substitutions at R and R on
1
2
piperazine ring. On the other hand, the R and R substituted compound showed slightly
3
4
decreased in human microsomal stability, as in 26h, compared with corresponding
analogs, as in 16d, which indicated one possibility that higher LogD showed lower
human microsomal stability. However, the compound including alcohol group at 2-

position at thiazole ring, as in 19, decreased the human microsomal stability compared
with 16d in spite of lower lipophilicity than 16d. As the result, microsomal stability of
these derivatives did not depend on the lipophilicity as general theory.

2
.4. In vivo pharmacology and selectivity for TRPV1. The analgesic effect of
compound 16d and 26i was evaluated via subcutaneous administration in FCA induced
mechanical hyperalgesia model in guinea pig and its effect was compared with
ibuprofen as the reference compound in Table 5. The analgesic effect of compound 16d
has already been confirmed via oral administration and its result was reported in our
3
2
previous communication . In this paper, we put the analgesic effect of compound 16d
as the comparison data with 26i via subcutaneous administration. Both compounds 16d
and 26i showed dose-dependent reversal of mechanical hyperalgesia with 3mg/kg,
1
1
0mg/kg and 30mg/kg and its effect showed significant effect compared with ibuprofen
0mg/kg and 30mg/kg. Furthermore, the 3mg/kg dose of compound 26i showed better
efficacy than corresponding dose of 16d, which might be possible to give a target
efficacy with lower plasma exposure level than 16d. In Table 6, the analgesic effect via
subcutaneous administration in FCA induced mechanical hyperalgesia model in rat of
compound 26i was illustrated, which was plateaued in a dose of 10mg/kg and 100mg/kg
due to the solubility issue or ceiling effect. However, improvement of rat potency has
led us to confirm the analgesic effect in rat FCA induced mechanical hyperalgesia
model in spite of species differences between human, guinea pig and rat. In Table 7, the
selectivity data for human TRPV1 of compound 16d and 26i was indicated and its

potency was compared to those of reference BCTC as known TRPV1 selective
antagonist. As the result, both compounds did not show the human TRPV1 antagonist
potency.

Table 5. In vivo efficacy comparison data of ibuprofen, 16d and 26i in guinea pig FCA
induced mechanical hyperalgesia model at 3h.
analgesic effect (%reversal)
dose (s.c.)
a
ibuprofen
-2.7±10.1
-
16d
26i
vehicle (DMAA+PG 1:1)
mg/kg
-2.4±2.9
4.0±2.4
20.5±2.7
30.9±2.6
-1.7±1.2
21.4±4.8
29.0±2.6
34.7±3.6
3
1
3
0 mg/kg
0 mg/kg
10.1±5.4
13.6±4.1
a
comparison.

Table 6. In vivo efficacy of 26i in rat FCA induced mechanical hyperalgesia model.
analgesic effect (%reversal)
dose (s.c.)
1
h
3h
vehicle (0.5%MC)
0 mg/kg
00 mg/kg
0±28.9
0±16.9
1
31.6±14.0
39.7±11.8
45.6±13.7
47.3±7.4
1

Table 7. Comarison data of 16d and 26i about hTRPV4 antagonist potency against
αPDD as agonist and selectivity for hTRPV1 antagonist potency of activation by
capsaicin.
4
h_TRPV4
h_TRPV1
compd.
c
4
αPDD IC (nM)
CAP IC (nM)
5
0
50
1
6d
6i
0.22
>10000
>10000
1.83
2
0.19
a
b
BCTC
n.t.
a
N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2H)-
b
c
carboxyamide as selective TRPV1 ligand. no try. The IC values of TRPV1 was
5
0
determined against 100nM capsaicin in human TRPV1 CHO cells.