Discovery of novel somatostatin receptor subtype 5 (SSTR5) antagonists: Pharmacological studies and design to improve pharmacokinetic profiles and human Ether-a-go-go-related gene (hERG) inhibition

Article abstract of DOI:10.1016/j.bmc.2017.06.003

Somatostatin (SST) is a peptide hormone comprising 14 or 28 amino acids that inhibits endocrine and exocrine secretion via five distinct G-protein-coupled receptors (SSTR1–5). SSTR5 has an important role in inhibiting the secretion of pancreatic and gastrointestinal hormones (e.g., insulin, GLP-1, PYY) through the binding of SSTs; hence, SSTR5 antagonists are expected to be novel anti-diabetic drugs. In the course of our lead generation program of SSTR5 antagonists, we have discovered a novel spiroazetidine derivative 3a. However, pharmacological evaluation of 3a revealed that it had to be administered at a high dose (100?mg/kg) to show a persistent glucose-lowering effect in an oral glucose tolerance test (OGTT). We therefore initiated an optimization study based on 3a aimed at improving the antagonistic activity and mean residence time (MRT), resulting in the identification of 2-cyclopropyl-5-methoxybiphenyl derivative 3k. However, 3k did not show a sufficient persistent glucose-lowering effect in an OGTT; moreover, hERG inhibition was observed. Hence, further optimization study of the biphenyl moiety of compound 3k, focused on improving the pharmacokinetic (PK) profile and hERG inhibition, was conducted. Consequently, the introduction of a chlorine atom at the 6-position on the biphenyl moiety addressed a putative metabolic soft spot and increased the dihedral angle of the biphenyl moiety, leading to the discovery of 3p with an improved PK profile and hERG inhibition. Furthermore, 3p successfully exhibited a persistent glucose-lowering effect in an OGTT at a dose of 3?mg/kg.

Full text of DOI:10.1016/j.bmc.2017.06.003

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel somatostatin receptor subtype 5 (SSTR5) antagonists:
Pharmacological studies and design to improve pharmacokinetic profiles
and human Ether-a-go-go-related gene (hERG) inhibition
a,
⇑
a,c
a
a
a
Takeshi Yamasaki , Hideki Hirose , Tohru Yamashita , Nobuyuki Takakura , Sachie Morimoto ,
a
a
a,b
a
a,c
Takashi Nakahata , Asato Kina , Yoshihide Nakano , Yumiko Okano Tamura , Jun Sugama
,
a
a
a
a,c
a,c
Tomoyuki Odani , Yuji Shimizu , Shinji Iwasaki , Masanori Watanabe , Tsuyoshi Maekawa
,
a,c
Shizuo Kasai
a
Research Division, Takeda Pharmaceutical Co., Ltd., 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
Global Procurement, Takeda Pharmaceutical Co., Ltd., 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Somatostatin (SST) is a peptide hormone comprising 14 or 28 amino acids that inhibits endocrine and
exocrine secretion via five distinct G-protein-coupled receptors (SSTR1–5). SSTR5 has an important role
in inhibiting the secretion of pancreatic and gastrointestinal hormones (e.g., insulin, GLP-1, PYY) through
the binding of SSTs; hence, SSTR5 antagonists are expected to be novel anti-diabetic drugs. In the course
of our lead generation program of SSTR5 antagonists, we have discovered a novel spiroazetidine deriva-
tive 3a. However, pharmacological evaluation of 3a revealed that it had to be administered at a high dose
Received 9 May 2017
Revised 2 June 2017
Accepted 3 June 2017
Available online xxxx
Keywords:
Somatostatin
(100 mg/kg) to show a persistent glucose-lowering effect in an oral glucose tolerance test (OGTT). We
therefore initiated an optimization study based on 3a aimed at improving the antagonistic activity and
mean residence time (MRT), resulting in the identification of 2-cyclopropyl-5-methoxybiphenyl deriva-
tive 3k. However, 3k did not show a sufficient persistent glucose-lowering effect in an OGTT; moreover,
hERG inhibition was observed. Hence, further optimization study of the biphenyl moiety of compound
3k, focused on improving the pharmacokinetic (PK) profile and hERG inhibition, was conducted.
Consequently, the introduction of a chlorine atom at the 6-position on the biphenyl moiety addressed
a putative metabolic soft spot and increased the dihedral angle of the biphenyl moiety, leading to the dis-
covery of 3p with an improved PK profile and hERG inhibition. Furthermore, 3p successfully exhibited a
persistent glucose-lowering effect in an OGTT at a dose of 3 mg/kg.
Somatostatin receptor subtype 5 (SSTR5)
antagonist
Anti-diabetic drug
hERG
Oral glucose tolerance test (OGTT)
Pharmacokinetic
Ó 2017 Elsevier Ltd. All rights reserved.
1
. Introduction
Somatostatin (SST) is a mammalian peptide hormone compris-
hormone, insulin, glucagon, gastrin, secretin, peptide YY (PYY), gas-
tric inhibitory polypeptide (GIP), and glucagon-like peptide-1
2
(GLP-1). SSTs show multiple biological functions including
3
ing 14 or 28 amino acids that is widely distributed throughout the
antiproliferative, hormonal, and neurotransmitter activity as well
body1 and suppresses the secretion of growth hormone, thyroid
,2
as the inhibition of endocrine and exocrine secretion. SSTs act via
five distinct G-protein-coupled receptors (SSTR1–5).1 SSTR5 in
particular has an important role in controlling the secretion of pan-
creatic and gastrointestinal hormones (e.g., insulin, GLP-1, PYY)
–4
Abbreviations: Boc, tert-butoxycarbonyl; Pd(OAc)
2 3
, palladium acetate; PBu ,
tributylphosphine; DIBAL-H, diisobutylaluminum hydride; S-phos, 2-dicyclo-
hexylphosphino-2 ,6 -dimethoxybiphenyl; Pd
dipalladium; NaH, sodium hydride; LiAlH
glucose; HFD, high-fat diet.
⇑
5
–8
0
0
through the binding of SSTs.
an anti-diabetic phenotype.
In addition, SSTR5 KO mice express
Therefore, SSTR5 antagonists are
2
(dba)
3
, tris(dibenzylideneacetone)
9
–11
4
, lithium aluminum hydride; BG, blood
expected to be novel anti-diabetic drugs and several SSTR5 antag-
Corresponding author.
12–17
onists have been reported.
E-mail address: takeshi.yamasaki@takeda.com (T. Yamasaki).
Present address: Research Division Medicinal Chemistry Laboratory, SCOHIA
In the course of our lead generation study for novel SSTR5
antagonists, we discovered spiroazetidine derivative 3a (Fig. 1) as
a promising lead compound, which showed a glucose-lowering
c
PHARMA, Inc., 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555,
Japan.
http://dx.doi.org/10.1016/j.bmc.2017.06.003
0
968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yamasaki T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.003

2
T. Yamasaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
ester 8. Suzuki-coupling reaction of 8 with cyclopropylboronic acid
OEt
N
4
yielded 9, which was converted to aldehyde 10e over two steps.
Synthesis of 2,3,4,5-substituted benzaldehyde 10m is outlined
in Scheme 3, and the other 2,3,4,5-substituted aldehydes and
HO C
2
N
O
N
OEt
IC50 (nM) : 9.6 / 57 (human / mouse)
Brief SAR of spiroazetidine derivatives
3
a
F
2
,4,5-substituted aldehydes were synthesized in a similar manner.
Suzuki-coupling reaction of methyl 4-iodo salicylate 11 with 4-flu-
orophenylboronic acid gave 12 in a quantitative yield. Regioselec-
tive bromination of 12 with bromine was carried out to afford
desired compound 13. Suzuki-coupling reaction of 13 with cyclo-
propylboronic acid afforded 14 in a quantitative yield. Introduction
of a chlorine atom at the 6-position was achieved by using N-
chlorosuccinimide (NCS) as a chlorine source to provide 15 at a
good yield. Alkylation of the hydroxyl group of 15 afforded 16,
which was converted into a benzaldehyde 10m via reduction and
oxidation.
Following substructures are important for antagonistic activity
•
•
Benzyl amine structure (red)
Isonipecotic acid (green)
Optimization strategies
•
•
Introduction of a substituent at the 4-position of piperidine part
Chemical modifications of biphenyl moiety (blue)
Fig. 1. Brief SAR of spiroazetidine derivative 3a and optimization strategies.
effect in an oral glucose tolerance test (OGTT) conducted 1 h after
oral administration (1 h-OGTT) using HFD-fed C57BL/6 J mice,
18
from 1 mg/kg in a dose-dependent manner. However, compound
a had to be administered at a dose of 100 mg/kg to exhibit a per-
3. Results and discussion
3
sistent glucose-lowering effect in an OGTT conducted 8 h after oral
administration (8 h-OGTT). As a rule, a persistent glucose-lowering
effect is very important for the treatment of diabetes, so we initi-
ated a lead optimization study based on 3a aimed at identifying
a novel clinical candidate of an SSTR5 antagonist, which shows a
sufficient persistent glucose-lowering effect.
The newly synthesized compounds were tested for functional
SSTR5 antagonism using a cAMP reporter assay in CHO cells stably
expressing human and mouse SSTR5.
Table 1 shows the results of initial SAR study with the aim of
improving SSTR5 antagonistic activity and MRTiv. We first intro-
duced a substituent at the 4-position of the piperidine unit of 3a.
Introduction of a fluorine atom (3b), which is well known as a bioi-
sostere of hydrogen, shortened MRTiv without improving the
antagonistic activity (h/m IC50: 58/78 nM, MRTiv 0.29 h). On the
other hand, introduction of a small alkyl group such as methyl
(3c) or ethyl (3d) enhanced mouse SSTR5 antagonistic activity with
the retention of human SSTR5 antagonistic activity. These results
indicated that the 4-position of the piperidine unit is directed
toward a lipophilic space in mSSTR5, wherein an alkyl substituent
was preferred to enhance mSSTR5 antagonistic activity. We subse-
Fig. 1 displays an overview of the structure-activity relationship
(
SAR) of spiroazetidine derivatives and our optimization strategies.
1
8
Lead generation study of the spiroazetidine derivatives revealed
that the benzyl amine structure is crucial for showing antagonistic
activity and that isonipecotic acid is also a favorable substructure
to exhibit such activity. However, chemical modifications of biphe-
nyl moieties have not been studied in detail. Hence, our efforts at
synthesis were focused on the optimization of isonipecotic acid
and biphenyl moieties, with the aim of improving the antagonistic
activity and PK profile, especially the mean residence time (MRT).
Additionally, we addressed human-Ether-a-go-go-related gene
quently investigated the SAR of the biphenyl moiety based on 3c
,
which showed potent activity with elongated MRTiv (h/m IC50
14/16 nM, MRTiv 1.98 h).
:
2
6,27
(
hERG)
inhibition issues, which emerged during the optimiza-
tion study. Here we report our results on the optimization of 3a
and the anti-diabetic effect of the newly discovered 2,5,6-substi-
tuted-biphenyl derivative 3p.
The chemical modification was initiated from the 2-position
(R ) of the biphenyl moiety (3e–h). Methoxy derivative 3e
decreased the in vitro activity (h/m IC50 3c: ethoxy (14/16 nM)
4
>3e: methoxy (18/62 nM)); however, replacement with a small
alkyl group such as methyl (3f) or cyclopropyl (3h) showed sin-
gle-digit nM IC50 values in both human and mouse (3f:
2
. Chemistry
6
.1/7.3 nM, 3h: 5.7/7.8 nM).
The 5-oxa-2,6-diazaspiro[3,4]oct-6-ene derivatives 3a–p
We subsequently investigated the effect of the substituent posi-
were synthesized in accordance with
a
previously reported
Ar reaction of tert-butyl
-bromo-5-oxa-2,6-diazaspiro[3,4]oct-6-ene-2-carboxylate 1 with
tion. Transposition of the ethoxy group from the 6-position to the
5-position (3i) resulted in an approximately 4–10-fold enhance-
ment of antagonistic activity (h/m IC50: 2.6/4.3 nM) and extended
MRTiv (2.24 h) relative to compound 3a. On the other hand, dis-
placement of the cyclopropyl group from the 2-position to the 3-
position (3j) showed a 10-fold reduction against mouse SSTR5
(3j: 85 nM vs. 3h: 7.8 nM). These findings suggested that a 2,5-sub-
stituted analog is favorable for antagonistic activity and the pro-
longation of MRTiv; therefore, the introduction of an alkoxy/alkyl
group such as methoxy (3k), isopropoxy (3l), or ethyl (3m) at the
method,1 as shown in Scheme 1. The S
8
N
7
commercially available 4-substituted ethyl piperidine carboxylate
derivatives afforded corresponding N-Boc-spiroisoxazoline analogs
2
a–c. After deprotection of the Boc group of 2a–c with formic acid,
reductive amination with corresponding benzaldehyde derivatives
0a–m using NaBH(OAc) followed by hydrolysis gave the
1
3
proposed carboxylic acids 3a–p. Compound 10a was prepared by
a previously reported method, the synthesis of benzaldehydes
2
1
0e and 10m is described in Schemes 2 and 3, and that of the
5-position (R ) with a fixed cyclopropyl group at the 2-position
others is described in the Supporting information.
was attempted. Compared with the alkoxy derivatives 3k and 3l,
methoxy derivative 3k showed a slight decrease in antagonistic
activity (h/m IC50: 3.9/7.6 nM) with extended MRTiv (2.77 h), and
isopropoxy derivative 3l exhibited improvement of both antago-
nistic activity (h/m IC50: 2.1/3.9 nM) and MRTiv (2.57 h). In con-
trast, ethyl derivative 3m showed a 4-fold decline in mouse
SSTR5 antagonistic activity compared with 3k. These results sug-
gested that the 5-position of the biphenyl moiety could be located
in a hydrophobic site of SSTR5 during interaction; the antagonistic
activity was correlated with the alkoxy group size at the 5-position
To access various benzaldehyde derivatives, we employed
several synthetic methods. Synthesis of the representative 3,4,5-
substituted benzaldehyde 10e is shown in Scheme 2, and the other
3
,4,5-substituted aldehydes (10b–d) were prepared in a similar
manner. Starting with commercially available benzoic acid 4,
esterification followed by alkylation gave an ethyl 4-bromoben-
zoate derivative 6, which was converted into 7 by the Suzuki-
coupling reaction. The hydroxyl group of 7 was reacted with triflu-
oromethanesulfonyl anhydride to afford trifluoromethanesulfonyl
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T. Yamasaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
R2
R4
R1
O
NBoc
a)
b), c), d), e)
O
R3
NBoc
EtO
N
N
Br
HO
O
HN
N
O
O
R
2
R1
N
N
R5
R1
OEt
R3
O
N
2a-c
H
1
F
3
a-p
O
R5
R4
0a-m
F
1
Scheme 1. Synthesis of 5-oxa-2,6-diazaspiro[3,4]oct-6-ene derivatives 3a–p. Reagents and conditions: (a) Na
2
CO
, piperidine derivatives (R1 = Me, F, Et), DMF, 130 °C, 35%–
3
8
6%; (b) HCO
2 3
H, 60 °C; (c) 10a–m, NaBH(OAc) , THF, r.t.; (d) NaOH(aq.), EtOH, r.t.; (e) recrystallization (EtOH–water), 27–94% over four steps for 3a–p.
O
O
O
O
O
OH
OH
Br
OEt
Br
OEt
OEt
HO
a
EtO
b
EtO
c
EtO
d
EtO
Br
OH
OH
OH
OH
OTf
F
F
4
5
6
7
8
O
O
OEt
OEt
f,g
e
H
EtO
F
F
9
10e
Scheme 2. Synthesis of 3,4,5-substituted benzaldehyde derivative 10e. Reagents and conditions: (a) concd H
fluorophenylboronic acid, Pd(OAc) , PBu , K PO , toluene, water, 90 °C, under Ar, 100%; (d) Tf O, pyridine, 0 °C, 91%; (e) cyclopropylboronic acid, Pd
aq., toluene, 100 °C under Ar, 98%; (f) LiAlH , THF, r.t., 80% over two steps.
2 4
CO , EtOH, reflux, 94%; (b) NaH, EtI, DMF, 0 °C, 65%; (c) 4-
2
3
3
4
2
2
(dba)
3 2 3
, S-phos, Na CO
4
, THF, 0 °C; (g) MnO
2
O
OH
O
OH
O
OH
Br
O
OH
O
OH
Cl
a
b
c
d
F
MeO
MeO
MeO
MeO
MeO
I
F
F
F
11
12
13
14
15
O
OMe
O
OMe
e
Cl
f,g
Cl
MeO
H
F
F
16
10m
Scheme 3. Synthesis of 2,3,4,5-substituted benzaldehyde derivative 10m. Reagents and conditions: (a) 4-fluorophenylboronic acid, Pd(OAc)
2 3 3 4
, PBu , K PO , toluene, water,
9
0 °C, under Ar, 100%; (b) bromine, AcOEt, 50 °C, 74%; (c) cyclopropylboronic acid, Pd (dba) , S-phos, Na CO aq. toluene, 100 °C, under Ar, 100%; (d) N-chlorosuccinimide,
2
3
2
3
DMF, r.t., 80%; (e) MeI, K
2
CO
3
, DMF, 60 °C, 83%; (f) DIBAL-H, THF, 0 °C; (g) MnO
2
, THF, 60 °C, 69% over two steps.
(
3
R2) and the lipophilicity of compounds (logD; 3l: 4.24, 3i: 3.98, 3k:
.53). We also considered that an oxygen atom of the alkoxy group
the aim of improving the PK profile, MRTiv, and AUC, and hERG
inhibition based on 3k.
at the 5-position would form a hydrogen bonding interaction with
SSTR5 and be indispensable to obtain decent antagonistic activity
To improve the PK profile, we guessed the identity of a metabol-
ically vulnerable moiety of compound 3k. In general, compounds
bearing an alkoxy group are metabolized into dealkylation
(3k vs. 3m).
2
9a-b
Among the 2,5-substituted derivatives 3i, 3k, and 3l with
metabolites by CYP1A2
. Based on this general term, we
improved antagonistic activity and MRTiv, we selected 3k for eval-
uation of the glucose-lowering effect in an 8 h-OGTT owing to its
longer duration (MRTiv = 2.77 h) and lower lipophilicity
speculated that 3k was also metabolized by CYP1A2 and that the
introduction of a substituent adjacent to the alkoxy group would
3
0
inhibit the dealkylation by CYP1A2.
(
logD = 3.53). Unfortunately, 3k did not show a significant glu-
In terms of addressing hERG issues, our investigation of the
relationship between hERG inhibition and our synthetic com-
pounds revealed that 2,6-substituted derivatives (3e–h) displayed
weaker hERG inhibition than 2,5-substituted derivatives (3i–l)
(Table 2).
This finding indicated that the biphenyl moiety is the structure
responsible for hERG inhibition and that optimizing the biphenyl
moiety of 2,5-substituted derivatives would avoid hERG inhibition.
cose-lowering effect in this test at a dose of 30 mg/kg (data not
shown). We considered that the blood concentration of 3k after
8
h was not sufficient to show glucose-lowering effect. Moreover,
it was revealed that 3k and other 2,5-substituted derivatives such
as 3i and 3l displayed hERG inhibition (3k: 66% inhibition at 30
lM
compound concentration). Therefore, we focused our efforts at
synthesis on the further optimization of the biphenyl moiety with
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4
T. Yamasaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 1
Effects of substituent at the 4-position of the piperidine unit.
R2
O
R3
5
6
N
HO
3
R1
N
R5
O
2
N
R4
F
Compound
R1
R2
R3
R4
R5
IC50 (nM)a hb/mc
Mouse cassetted MRTive (h)
logDf
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
g
h
i
H
F
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
H
OEt
OEt
OEt
OEt
OMe
Me
Et
cPr
cPr
H
H
H
H
H
H
H
H
H
H
9.6/57
58/78
14/16
14/19
18/62
6.1/7.3
6.1/23
5.7/7.8
2.6/4.3
4.6/85
3.9/7.6
2.1/3.9
5.1/29
1.73
0.29
1.98
1.72
2.99
2.94
3.33
3.58
2.84
3.33
3.65
3.75
3.98
g
NT
0.88
0.67
1.44
2.24
2.40
2.77
2.57
3.50
OEt
H
g
j
k
l
OEt
H
H
cPr
H
H
NT
OMe
cPr
cPr
cPr
3.53
4.24
4.37
i
O Pr
m
Et
H
H
a
b
c
d
e
f
IC50 values were calculated using an experiment performed in duplicate.
Antagonistic activity against human SSTR5.
Antagonistic activity against mouse SSTR5.
n = 3; ICR mouse (male, 8 weeks old), iv: 0.1 mg/kg, po: 1 mg/kg.
Mean residence time.
logD value at pH 7.4.
Not tested.
g
Table 2
hERG inhibition of our synthetic compounds (3e–h: 2,6-substituted, 3i–l: 2,5-substituted).
Compound
hERG%a
3e
3f
3g
3h
3i
3k
3l
11
11
28
31
79
66
109
a
hERG inhibitory activity at 30
lM compound concentration.
In the comparison of 2,6-substituted derivatives with 2,5-substi-
tuted derivatives, we focused on the dihedral angle in the biphenyl
moiety of these analogs. Fig. 2 shows dihedral angles of 2,6-substi-
tuted derivative 3h and 2,5-substituted derivative 3i calculated by
Molecular Operating Environment software (MOE).28 This calcula-
tion indicated that 3h had a slightly larger dihedral angle than 3i
inhibit the dealkylation by CYP1A2, leading to improvement of
the PK profile. The dihedral angle in the biphenyl moiety of 3n
was larger than that of compound 3h (3n: 71.6° vs. 3h: 64.8°),
whereas the hERG inhibition of compound 3n was not declined
(Fig. 4).
In general, reduction of the electron density of compounds to
(
3
Fig. 2, 3h: 64.8°, 3i: 59.5°). Based on this result, we considered that
h with its larger dihedral angle in the biphenyl moiety decreased
reduce electrostatic (
be effective to disrupt the interaction with the hERG channel.
p–p/CH–p) interactions is also considered to
24,25
p
-related interaction with the hERG channel. Indeed, several
Therefore, we hypothesized that the combination of a large
dihedral angle and an electron-withdrawing group on the biphenyl
moiety would additively decline hERG inhibition. Based on this
hypothesis, a chlorine atom was selected as a substituent at the
6-position, which is an electron-withdrawing group with steric
bulkiness to twist the biphenyl moiety sufficiently. Indeed, the
calculation by MOE suggested that the introduction of a chlorine
atom at the 6-position sufficiently twisted the biphenyl moiety
(3o: 70.8°, 3p: 71.7°), resulting in 6-chloro derivatives 3o and 3p
successfully declining hERG inhibition (3o: 4%, 3p: 24%) and
improving the PK profile (MRTiv/AUCpo: 3o: 2.88 h/2510 ngꢀh/
mL, 3p: 3.40 h/1480 ngꢀh/mL) (Fig. 5).
1
9a,b,20–23
reports
have indicated that some aromatic amino acid
residues of the hERG channel, such as Tyr652 and Phe656, play a
key role in creating -related interactions and one of the most
effective methods for declining hERG inhibition is to modify the
structural conformation of compounds to reduce the
interactions.
Taking account of our considerations for improving both the PK
profile and hERG inhibition, we attempted to introduce a sub-
stituent at the 6-position (R ) for inhibiting the dealkylation of
p
p-related
3
the alkoxy group by CYP1A2 and twisting the biphenyl moiety to
the same level as in 2,6-substituted derivative 3h (Fig. 3).
Table 3 summarizes the effect of the substituent at the
Given that these two compounds, 3o and 3p, were endowed
with improved PK profiles and hERG inhibition, we next examined
their glucose-lowering effects in an 8 h-OGTT in HFD-fed C57BL6
mice. Of these two compounds, we selected 3p for in vivo evalua-
tion because it showed longer duration (MRTiv; 3o: 2.88 h vs. 3p:
3.40 h) and good bioavailability (F = 85%) (Table 4)
3
6
-position (R ) on PK profiles and hERG inhibition. We first intro-
duced a methyl group to yield 3n. Compound 3n slightly decreased
mouse SSTR5 antagonistic activity, although it markedly improved
AUCpo (3n: 1830 ngꢀh/mL). This indicated that the introduction of
a substituent adjacent to the alkoxy group would successfully
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T. Yamasaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Fig. 2. Dihedral angle of biphenyl moiety in compounds 3h (64.8°) and 3i (59.5°).
Introduction of substituent at the 6-position
• To inhibit metabolism of alkoxy group
• To twist biphenyl moiety
OMe
5
Me
HO
N
6
N
O
2
N
O
F
Fig. 3. Our synthetic strategy to improve PK profiles and hERG inhibition.
Table 3
3
Effect of substituent at the 6-position (R ) on PK profiles and hERG inhibition.
R2
O
R3
N
HO
Me
N
O
N
F
R2
R3
IC50 (nM)a hb/mc
logDd
Mouse cassettee
hERG inhibition%
i
Dihedral angle
Compound
f
g
MRTiv (h)
AUCpo (ngꢀh/mL)
3
3
3
3
i
OEt
OEt
OEt
OMe
H
Me
Cl
2.6/4.3
6.7/37
7.3/25
6.2/25
3.98
4.11
4.18
3.74
2.24
2.69
2.88
3.40
566
79
76
4
59.5°
71.6°
70.8°
71.7°
n
o
p
1830
2510
1479
Cl
24
a–f
See corresponding footnotes of Table 3.
Area under the plasma concentration-time curve (0–8 h).
hERG inhibitory activity at 30 lM compound concentration.
g
i
Compounds 3a, 3p, and vehicle were administered orally in
optimize spiroazetidine derivative 3a. We first attempted to mod-
ify isonipecotic acid and biphenyl moiety to improve the antago-
nistic activity and MRTiv. Introduction of a methyl group at the
4-position of the piperidine unit and exploration of substituents
and their position on the biphenyl moiety adequately improved
antagonistic activity and MRTiv, leading to the identification of
2-cyclopropyl-5-alkoxybiphenyl derivative 3k. Compound 3k
exhibited more potent antagonistic activity and longer MRTiv than
lead compound 3a, but it did not show a sufficient persistent glu-
cose-lowering effect in an 8 h-OGTT. Additionally, compound 3k
and other 2-cyclopropyl-5-alkoxybiphenyl analogs were found to
show hERG inhibition. Therefore, our subsequent efforts at
synthesis focused on structural modification of the central benzene
ring of compound 3k with the aim of further improving the PK
mice 8 h before glucose loading, and blood glucose levels were
monitored for 2 h. Compound 3a (100 mg/kg), which exhibited
the maximum effect in this test, was used as a positive control.
As shown in Fig. 6, compound 3p exhibited a persistent glucose-
lowering effect comparable to that of 3a (100 mg/kg) at a lower
dose (3 mg/kg, plasma concentration at 8 h after dosing:
0
.484
lg/mL), indicating that 3p exerts a robust and long-lasting
anti-diabetic effect in mice.
4
. Conclusion
To develop a novel SSTR5 antagonist showing a persistent
glucose-lowering effect at a lower dose, we initiated a study to
Please cite this article in press as: Yamasaki T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.003

6
T. Yamasaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
cial suppliers and used without further purification. Thin-layer
chromatography (TLC) was performed on Merck silica gel plates
0F254. Column chromatography was performed on silica gel 60
6
(
(
0.063–0.200 or 0.040–0.063 mm, E. Merck), basic silica gel
Chromatorex NH, 100–200 mesh, Fuji Silysia Chemical Ltd.), or
Purif-Pack (Si or NH, Moritex Corporation). LC–MS analysis was
performed on a Waters, Agilent, or Shimadzu Liquid Chromatogra-
phy–Mass Spectrometer System, operating in APCI (+ or ꢁ) or ESI
(
+ or ꢁ) ionization mode. Analytes were eluted using a linear gra-
dient of 0.05% TFA containing water/acetonitrile or 5 mM ammo-
nium acetate containing water/acetonitrile mobile phase.
Determination of chemical purity by HPLC (detection at 220 nm)
was conducted using a Shimadzu Liquid Chromatography System
with 0.05% TFA containing water/acetonitrile mobile phase.
Elemental analyses and high-resolution mass spectroscopy (HRMS)
measurements (ESI-MS) were performed by Takeda Analytical
Research Laboratories, Ltd. Yields are not optimized.
Fig. 4. Dihedral angle of 3n (71.6°).
profile and hERG inhibition. As a result, the introduction of a
chlorine atom at the 6-position on the biphenyl moiety protected
the presumed metabolically vulnerable moiety and increased the
5
.1.1. tert-Butyl 7-(4-(ethoxycarbonyl)-4-methylpiperidin-1-yl)-5-
oxa-2,6-diazaspiro[3.4]oct-6-ene-2-carboxylate (2b)
A mixture of 1 (23 g, 79.0 mmol), ethyl-4-methylpiperidine-4-
dihedral angle of the biphenyl moiety to decrease the
p-related
2 3
carboxylate (18.9 g, 111 mmol) and Na CO (25.1 g, 237 mmol) in
interaction with the hERG channel, resulting in the discovery of
DMF (100 ml) was stirred at 130 °C overnight. The mixture was
poured into water, brine and extracted with EtOAc twice. The
organic layer was separated, washed with brine twice, dried over
0
1
-(2-((2-chloro-6-cyclopropyl-4 -fluoro-3-methoxybiphenyl-4-yl)
methyl)-5-oxa-2,6-diazaspiro[3,4]oct-6-en-7-yl)-4-methylpiperi-
dine-4-carboxylic acid 3p with an improved PK profile
MgSO
4
and concentrated in vacuo. The residue was crystallized
(
MRTiv = 3.40 h, AUCpo = 1480 ngꢀh/mL) and hERG inhibition. Fur-
from EtOAc–hexane (10 ml–100 ml) to give the title compound
thermore, 3p successfully exhibited a persistent glucose-lowering
effect in an 8 h-OGTT at a dose of 3 mg/kg. These results suggested
that the newly discovered SSTR5 antagonist 3p is promising for
anti-diabetic treatment, and further evaluations of its pharmaco-
logical effects are underway.
1
(
26.0 g, 86%) as off-white crystals. H NMR (400 MHz, DMSO-d
6
)
d 1.11–1.23 (6H, m), 1.35–1.46 (11H, m), 1.93 (2H, d, J = 13.8 Hz),
2
4
.82–2.91 (2H, m), 3.25–3.34 (4H, m), 3.88 (2H, d, J = 9.3 Hz),
.01 (2H, d, J = 9.3 Hz), 4.11 (2H, q, J = 7.0 Hz).
5.1.2. tert-Butyl 7-(4-(ethoxycarbonyl)-4-fluoropiperidin-1-yl)-5-oxa-
2
,6-diazaspiro[3.4]oct-6-ene-2-carboxylate (2a)
Compound 2a was prepared from 1 and 4-fluoro-piperidine-4-
5
. Experimental section
carboxylicacid ethyl ester in a manner similar to that described
for compound 2b. H NMR (400 MHz, CDCl ) d 1.32 (3H, t,
3
5.1. Chemistry
1
J = 7.1 Hz), 1.44 (9H, s), 1.93–2.20 (4H, m), 3.16–3.27 (4H, m),
3.52 (2H, d, J = 12.9 Hz), 4.00 (2H, d, J = 9.7 Hz), 4.18–4.30 (4H, m).
Melting points were determined in open capillary tubes on a
1
Büchi melting point apparatus B545 and are uncorrected.
H
NMR spectra were recorded on a Bruker AVANCE III (300 MHz),
Bruker AVANCE 300 (300 MHz), or Bruker Advance III plus
(
5.1.3. tert-Butyl 7-(4-(ethoxycarbonyl)-4-ethylpiperidin-1-yl)-5-oxa-
2,6-diazaspiro[3.4]oct-6-ene-2-carboxy late (2c)
400 MHz) spectrometer. Chemical shifts for 1H NMR are given in
parts per million (ppm) downfield from tetramethylsilane (d) as
Compound 2c was prepared from 1 and ethyl-4-ethylpiperi-
an internal standard in deuterated solvent and coupling constants
dine-4-carboxylate in a manner similar to that described for com-
1
(J) are in Hertz (Hz). Data are reported as follows: chemical shift,
pound 2b. H NMR (300 MHz, DMSO-d
6
) d 0.75 (3H, t, J = 7.5 Hz),
integration, multiplicity (s = singlet, d = doublet, t = triplet,
q = quartet, m = multiplet, dd = doublet of doublet, td = triplet of
doublet, qd = quartet of doublet, brs = broad singlet), and coupling
constants. All solvents and reagents were obtained from commer-
1.19 (3H, t, J = 7.1 Hz), 1.30–1.44 (11H, m), 1.45–1.57 (2H, m),
1.97 (2H, d, J = 13.5 Hz), 2.78 (2H, t, J = 11.3 Hz), 3.25–3.41 (4H,
m), 3.87 (2H, d, J = 9.2 Hz), 4.02 (2H, d, J = 9.4 Hz), 4.12 (2H, q,
J = 7.1 Hz).
Fig. 5. Dihedral angles of 3o (70.8°) and 3p (71.7°).
Please cite this article in press as: Yamasaki T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.003

T. Yamasaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
Table 4
a
Mouse cassette dosing data of 3p.
iv: 0.1 mg/kg
po: 1.0 mg/kg
MRTivb hour
3.40
VDssc mL/kg
2013
Cltotald mL/h/kg
591
Cmaxe ng/mL
Tmax hour
2.70
f
AUCpog ngh/mL
1479
MRTpob hour
3.90
Fh
%
C5 min ng/mL
3.6
AUCiv ngh/mL
173.8
4
260
85
a
b
c
iv: 0.1 mg/kg, po: 1 mg/kg, n = 3; ICR mouse (male, 8 weeks old).
Mean residence time.
Volume of distribution at a steady state.
Total clearance.
d
e
f
Maximal plasma concentration.
Time of maximal concentration.
Area under the plasma concentration-time curve (0–8 h).
Bioavailability.
g
h
Fig. 6. Oral glucose tolerance test in HFD-fed C57BL6 mice: Male C57BL/6J Jcl mice (9 weeks of age, n = 6) were fasted at 7:00 a.m. and orally administered vehicle (0.5%
methylcellulose) or compounds after an hour. Eight hours later, all animals underwent oral glucose loading (5 g/kg). Blood samples were collected from the tail vein before
compound administration (pre), just before glucose loading (time 0), and 10, 30, 60, and 120 min thereafter. Blood glucose was measured by ACCU–CHECK Aviva. Statistical
*
**
***
analysis was conducted using Student’s t-test, Welch’s test, Williams’ test, or Shirley Williams’ test. p < 0.05 versus vehicle, p < 0.05 versus vehicle, p < 0.05 versus vehicle
(
Student’s t-test), ##p < 0.01 versus vehicle, ###p < 0.05 versus vehicle (Williams’ test).
0
5
.1.4. 1-(2-((2,6-Diethoxy-4 -fluorobiphenyl-4-yl)methyl)-5-oxa-2,6-
J = 10.4 Hz), 3.16 (2H, d, J = 8.2 Hz), 3.23–3.38 (4H, m), 3.46 (2H,
d, J = 7.9 Hz), 3.57 (2H, s), 3.95 (4H, q, J = 7.0 Hz), 6.61 (2H, s),
7.10–7.19 (2H, m), 7.22–7.31 (2H, m). Anal. Calcd for C29H N O -
36 3 5
F: C, 66.27; H, 6.90; N, 7.99; F, 3.61. Found: C, 66.18; H, 7.01; N,
7.88.
diazaspiro[3.4]oct-6-en-7-yl)-4-methylpiperidine-4-carboxylic acid
3c)
A solution of 2b (608 mg, 1.59 mmol) in formic acid (3 ml) was
(
stirred at 60 °C for 1 h. The mixture was concentrated in vacuo to
give an pale yellow oil, which was diluted with THF (10 ml). Com-
pound 10a (460 mg, 1.59 mmol) and NaBH(OAc)
.39 mmol) was added to the solution at room temperature. The
mixture was stirred at room temperature for 1 h. The mixture
was quenched with sat. NaHCO aq. at room temperature and
extracted with EtOAc. The organic layer was separated, washed
with brine, dried over MgSO and concentrated in vacuo. The resi-
3
(507 mg,
0
5
.1.5. 1-(2-((2,6-Diethoxy-4 -fluorobiphenyl-4-yl)methyl)-5-oxa-2,6-
2
diazaspiro[3.4]oct-6-en-7-yl)-4-fluoropiperidine-4-carboxylic acid
3b)
Compound 3b was prepared by using procedure described for
the synthesis of 3c except that 2b was replaced with 2a. White
(
3
+
1
4
solid. Yield. 81% mp 140–141 °C, MS (ESI) m/z 530.5[M+H] .
NMR (400 MHz, DMSO-d ) d 1.15 (6H, t, J = 7.0 Hz), 1.78–2.10
4H, m), 3.02 (2H, t, J = 11.5 Hz), 3.25 (2H, d, J = 8.0 Hz), 3.31–
H
due was purified by column chromatography (NH silica gel, eluted
with 10–50% EtOAc in hexane) to give a precursor of 3c as white
crystals. 2M NaOH (3.5 ml, 7.00 mmol) was added to a solution
of the above precursor (658 mg, 1.19 mmol) in EtOH (6 ml) at room
temperature. The mixture was stirred at 60 °C for 1 h and neutral-
ized with 2M HCl (3.5 ml). After being stirred for 30 min, the crys-
tals were filtered, washed with water and dried to give crude of 3c,
6
(
3
3
.38 (2H, m), 3.45 (2H, d, J = 12.5 Hz), 3.54 (2H, d, J = 7.9 Hz),
.63 (2H, s), 3.95 (4H, q, J = 6.9 Hz), 6.63 (2H, s), 7.09–7.19 (2H,
m), 7.23–7.31 (2H, m). C NMR (75MHz, DMSO-d ) d 172.3 (d,
6
J = 24.2 Hz), 160.5, 160.8 (d, J = 242.6 Hz), 156.2, 138.2, 132.8 (d,
J = 8.3 Hz), 130.1 (d, J = 3.3 Hz), 117.0, 114.0 (d, J = 20.9 Hz),
05.7, 91.7 (d, J = 183.2 Hz), 79.3, 66.0, 63.7, 62.0, 42.3, 42.2, 30.9
1
3
2
which was recrystallized from EtOH–H O (10 ml–3 ml) to give the
1
(
1
1
1
3
title compound (566 mg, 74% from 2b). mp 215–216 °C, MS (ESI)
d, J = 22.6 Hz), 14.5. C NMR (75 MHz, DMSO-d
6
) d 177.6, 160.8,
60.8 (d, J = 243.2 Hz), 156.1, 139.5, 132.8 (d, J = 8.3 Hz, 1C),
30.2, 116.6, 114.0 (d, J = 20.9 Hz), 105.4, 79.2, 66.4, 63.7, 62.9,
+
1
m/z 526.6[M+H] . H NMR (400 MHz, DMSO-d
6
) d 1.09–1.19 (9H,
m), 1.37 (2H, t, J = 9.8 Hz), 1.91 (2H, d, J = 13.3 Hz), 2.88 (2H, t,
Please cite this article in press as: Yamasaki T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.003

8
T. Yamasaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4
6
4.0, 42.6, 40.4, 33.3, 25.2. Anal. Calcd for C28
1.41; H, 6.44; N, 7.67; F, 3.61. Found: C, 61.29; H, 6.51; N, 7.58.
H
33
N
3
O
5
F
2
ꢀ1H
2
O: C,
25.9, 25.2, 15.4, 14.5. Anal. Calcd for C29
H, 7.19; N, 8.10. Found: C, 67.04; H, 7.04; N, 8.07.
H
36
N
3
O
4
2
Fꢀ1H O: C, 67.16;
0
5
.1.6. 1-(2-((2,6-Diethoxy-4 -fluorobiphenyl-4-yl)methyl)-5-oxa-2,6-
0
5
.1.10. 1-(2-((2-Cyclopropyl-6-ethoxy-4 -fluorobiphenyl-4-yl)
diazaspiro[3.4]oct-6-en-7-yl)-4-ethylpiperidine-4-carboxylic acid (3d)
Compound 3d was prepared by using procedure described for
the synthesis of 3c except that 2b was replaced with 2c. White
methyl)-5-oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)-4-methylpiperidine-
4-carboxylic acid (3h)
White solid. Yield. 81% mp 211–212 °C, MS (ESI) m/z 522.5 [M
H] . H NMR (400 MHz, DMSO-d ) d 0.57 (2H, d, J = 4.0 Hz), 0.71
6
2H, d, J = 7.5 Hz), 1.02–1.20 (6H, m), 1.37 (2H, t, J = 10.0 Hz),
.43–1.53 (1H, m), 1.91 (2H, d, J = 13.3 Hz), 2.88 (2H, t,
J = 10.7 Hz), 3.14 (2H, d, J = 7.5 Hz), 3.20–3.33 (4H, m), 3.43 (2H,
d, J = 7.5 Hz), 3.54 (2H, s), 3.92 (2H, q, J = 6.8 Hz), 6.42 (1H, s),
6
.76 (1H, s), 7.14–7.32 (4H, m). C NMR (75MHz, DMSO-d ) d
77.6, 160.8, 160.9 (d, J = 242.1 Hz), 155.4, 142.0, 138.7, 133.2 (d,
J = 3.3 Hz), 132.3 (d, J = 8.3 Hz), 128.5, 115.5, 114.5 (d,
J = 20.9 Hz), 109.6, 79.2, 66.3, 63.5, 62.7, 44.0, 42.6, 40.4, 33.3,
+
1
solid. Yield. 94% mp 212–213 °C, MS (ESI) m/z 540.6[M+H] .
H
+ 1
+
(
1
NMR (400 MHz, DMSO–d ) d 0.78 (3H, t, J = 7.3 Hz), 1.15 (6H, t,
6
J = 6.8 Hz), 1.34 (2H, t, J = 10.4 Hz), 1.44–1.54 (2H, m), 1.93 (2H,
d, J = 12.8 Hz), 2.81 (2H, t, J = 11.2 Hz), 3.16 (2H, d, J = 7.3 Hz),
.21–3.39 (4H, m), 3.46 (2H, d, J = 7.5 Hz), 3.57 (2H, s), 3.90–3.99
3
(
4H, m), 6.61 (2H, s), 7.14 (2H, t, J = 8.5 Hz), 7.22–7.32 (2H, m).
13
6
1
1
3
C
NMR (75MHz, DMSO-d
J = 242.1 Hz), 156.1, 139.5, 132.8 (d, J = 7.7 Hz), 130.2 (d,
J = 3.3 Hz), 116.6, 114.0 (d, J = 21.5 Hz), 105.4, 79.2, 66.4, 63.7,
2.9, 44.7, 44.2, 42.6, 31.7, 14.6, 8.3, 1C was hidden. Anal. Calcd
for C30 F: C, 66.77; H, 7.10; N, 7.79. Found: C, 66.61; H,
.22; N, 7.57.
Compound 3e–p was prepared by using procedure described for
the synthesis of 3c except that 10a was replaced with correspond-
ing benzaldehyde 10b–m.
6
)
d
176.5, 160.9, 160.8 (d,
6
2
6
36 3 4
5.2, 14.6, 13.4, 9.2. Anal. Calcd for C30H N O F: C, 69.08; H,
.96; N, 8.06. Found: C, 68.96; H, 7.02; N, 7.95.
38 3 5
H N O
7
0
5
.1.11. 1-(2-((2-Cyclopropyl-5-ethoxy-4 -fluorobiphenyl-4-yl)
methyl)-5-oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)-4-methylpiperidine-
4-carboxylic acid (3i)
White solid. Yield. 83% mp 184–185 °C, MS (ESI) m/z 522.4 [M
H] . H NMR (400 MHz, DMSO-d ) d 0.49–0.56 (2H, m), 0.75
6
2H, d, J = 8.4 Hz), 1.14 (3H, s), 1.28–1.42 (5H, m), 1.69–1.78 (1H,
m), 1.91 (2H, d, J = 13.7 Hz), 2.89 (2H, t, J = 10.2 Hz), 3.16 (2H,
brs), 3.31 (4H, brs), 3.41–3.60 (4H, m), 4.02 (2H, q, J = 6.9 Hz),
0
5
.1.7. 1-(2-((2-Ethoxy-4 -fluoro-6-methoxybiphenyl-4-yl)methyl)-5-
+
1
+
(
oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)-4-methylpiperidine-4-
carboxylic acid (3e)
+
White solid. Yield. 81% mp 227–228 °C, (ESI) m/z 512.5[M+H] .
H NMR (300 MHz, DMSO-d ) d 1.05–1.22 (6H, m), 1.37 (2H, ddd,
J = 13.57, 10.03, 3.97 Hz), 1.91 (2H, d, J = 13.69 Hz), 2.82–2.97
1
6
6
.73 (1H, s), 6.84 (1H, s), 7.26 (2H, t, J = 8.7 Hz), 7.47 (2H, dd,
1
3
J = 8.3, 5.8 Hz), 12.37 (1H, brs). C NMR (75MHz, DMSO-d
77.6, 160.9, 161.2 (d, J = 242.6 Hz), 153.9, 140.0, 137.5 (d,
J = 3.3 Hz), 131.4, 131.2 (d, J = 8.3 Hz), 125.5 (d, J = 5.5 Hz), 115.0,
6
) d
(
(
7
2H, m), 3.17 (2H, d, J = 8.50 Hz), 3.23–3.35 (4H, m), 3.41–3.52
1
2H, m), 3.59 (2H, s), 3.66 (3H, s), 3.88–4.03 (2H, m), 6.63 (2H, s),
.05–7.32 (4H, m), 12.42 (1H, br. s.). 13C NMR (75MHz, DMSO-d
)
6
1
1
7
14.7, 112.8, 79.2, 66.5, 63.4, 56.1, 44.0, 42.5, 40.4, 33.3, 25.2,
4.8, 12.9, 8.9. Anal. Calcd for C30 O: C, 65.67; H,
d 177.6, 160.8, 160.8 (d, J = 242.6 Hz), 156.9, 156.1, 139.6, 132.7
d, J = 7.7 Hz), 130.3, 116.4, 114.2 (d, J = 20.9 Hz), 105.4, 104.1,
9.2, 66.4, 63.7, 62.9, 55.6, 44.0, 42.6, 33.3, 25.23, 14.55, 1C was
hidden. Anal. Calcd for: C28 F: C, 65.74; H, 6.70; N, 8.21.
H
36
N
3
O
4
Fꢀ1H
2
(
7
.16; N, 7.66. Found: C, 65.41; H, 6.96; N, 7.55.
34 3 5
H N O
0
5
.1.12. 1-(2-((5-cyclopropyl-2-ethoxy-4 -fluorobiphenyl-4-yl)
Found: C, 65.67; H, 6.73; N, 8.15.
methyl)-5-oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)-4-methylpiperidine-
4-carboxylic acid (3j)
0
5
.1.8. 1-(2-((2-Ethoxy-4 -fluoro-6-methylbiphenyl-4-yl)methyl)-5-
White solid. Yield. 89% mp 204–205 °C, MS (ESI) m/z 522.5 [M
oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)-4-methylpiperidine-4-
carboxylic acid (3f)
White solid. Yield. 39% mp 204–205 °C, MS (ESI) m/z 496.3[M
6
H] . H NMR (300 MHz, DMSO-d ) d 1.04–1.18 (6H, m), 1.37 (2H,
ddd, J = 13.50, 9.91, 3.97 Hz), 1.91 (2H, d, J = 13.69 Hz), 1.98 (3H,
s), 2.79–2.99 (2H, m), 3.14 (2H, d, J = 8.50 Hz), 3.21–3.37 (4H, m),
.45 (2H, d, J = 8.50 Hz), 3.55 (2H, s), 3.92 (2H, q, J = 6.99 Hz),
+
1
+
0
H] . H NMR (400 MHz, DMSO-d
6
) d 0.55–0.63 (2H, m), 0.82–
.89 (2H, m), 1.14 (3H, s), 1.25 (3H, t, J = 6.9 Hz), 1.32–1.42 (2H,
m), 1.86–2.00 (3H, m), 2.89 (2H, t, J = 10.3 Hz), 3.21 (2H, d,
J = 8.0 Hz), 3.24–3.38 (4H, m), 3.51 (2H, d, J = 8.0 Hz), 3.77 (2H, s),
+
1
+
4
.01 (2H, q, J = 7.0 Hz), 6.84 (1H, s), 7.00 (1H, s), 7.19 (2H, t,
13
J = 8.9 Hz), 7.51 (2H, dd, J = 8.5, 5.7 Hz), 12.43 (1H, brs). C NMR
(75MHz, DMSO-d ) d 177.6, 160.8, 161.1 (d, J = 243.2 Hz), 153.1,
3
6
1
3
.79 (2H, d, J = 3.78 Hz), 7.13–7.25 (4H, m), 12.41 (1H, brs).
) d 177.6, 160.8, 161.0 (d, J = 242.6 Hz),
55.7, 138.4, 136.5, 133.4 (d, J = 3.3 Hz), 131.9 (d, J = 7.7 Hz),
28.1, 122.1, 114.7 (d, J = 20.9H), 109.9, 79.2, 66.4, 63.5, 62.6,
4.0, 42.6, 40.4, 33.3, 25.2, 20.3, 14.5. Anal. Calcd for C28 F:
C
6
1
1
4
38.3, 134.5 (d, J = 3.3 Hz), 132.9, 131.0 (d, J = 7.7 Hz), 127.5,
26.8, 114.6 (d, J = 20.9 Hz), 112.8, 79.3, 66.7, 63.7, 59.9, 44.0,
NMR (75MHz, DMSO-d
6
1
1
4
2.6, 40.4, 33.3, 25.2, 14.6, 11.7, 6.8. Anal. Calcd for C30
36 3 3
H N O -
Fꢀ0.2H
2
O: C, 70.76; H, 7.21; N, 8.25. Found: C, 70.86; H, 7.25; N,
34 3 4
H N O
8
.13.
C, 67.86; H, 6.92; N, 8.48. Found: C, 67.79; H, 6.92; N, 8.45.
0
0
5
2
.1.9. 1-(2-((2-Ethoxy-6-ethyl-4 -fluorobiphenyl-4-yl)methyl)-5-oxa-
,6-diazaspiro[3.4]oct-6-en-7-yl)-4-methylpiperidine-4-carboxylic
5.1.13. 1-(2-((2-Cyclopropyl-4 -fluoro-5-methoxybiphenyl-4-yl)
methyl)-5-oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)-4-methylpiperidine-
4-carboxylic acid (3k)
acid (3g)
White solid. Yield. 82% mp 207–208 °C, MS (ESI) m/z 510.5 [M
H] . H NMR (400 MHz, DMSO-d
White solid. Yield. 76% mp 213–214 °C, MS (ESI) m/z 508.4 [M
+
1
+H]+. H NMR (400 MHz, DMSO-d
1
+
6
) d 0.93 (3H, t, J = 7.4 Hz),
6
) d 0.53 (2H, q, J = 4.9 Hz),
1
.03–1.18 (6H, m), 1.36 (2H, t, J = 10.2 Hz), 1.91 (2H, d,
0.72–0.80 (2H, m), 1.14 (3H, s), 1.32–1.42 (2H, m), 1.70–1.79 (1H,
m), 1.90 (2H, d, J = 13.8 Hz), 2.83–2.93 (2H, m), 3.12–3.20 (2H,
m), 3.24–3.38 (4H, m), 3.41–3.48 (2H, m), 3.55 (2H, brs), 3.76
J = 13.1 Hz), 2.30 (2H, q, J = 7.2 Hz), 2.89 (2H, t, J = 10.8 Hz), 3.15
(
(
(
2H, d, J = 7.7 Hz), 3.20–3.37 (4H, m), 3.45 (2H, d, J = 7.4 Hz), 3.57
2H, s), 3.91 (2H, q, J = 6.7 Hz), 6.79 (2H, d, J = 4.6 Hz), 7.05–7.35
(3H, s), 6.75 (1H, s), 6.84 (1H, s), 7.26 (2H, t, J = 8.8 Hz), 7.48 (2H,
4H, m), 12.40 (1H, brs). 13C NMR (75MHz, DMSO-d
) d 177.6,
60.8, 161.0 (d, J = 242.6 Hz), 155.7, 142.7, 138.7, 133.3 (d,
13
6
6
dd, J = 8.3, 5.7 Hz). C NMR (75 MHz, DMSO-d ) d 177.6, 160.9,
1
161.3 (d, J = 243.7 Hz), 154.6, 140.0, 137.5 (d, J = 3.3 Hz), 131.4,
131.2 (d, J = 8.3 Hz), 125.4, 125.3, 114.9 (d, J = 21.5 Hz), 111.9,
79.2, 66.4, 56.3, 55.4, 44.0, 42.5, 40.4, 33.3, 25.2, 12.8, 8.9. Anal.
J = 3.9 Hz), 131.9 (d, J = 8.3 Hz), 127.7, 120.4, 114.6 (d,
J = 21.5 Hz), 109.8, 79.2, 66.4, 63.5, 62.7, 44.0, 42.6, 40.4, 33.3,
Please cite this article in press as: Yamasaki T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.003

T. Yamasaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
0
Calcd for C29
H
34
N
3
O
4
F: C, 68.62; H, 6.75; N, 8.28; F, 3.74. Found: C,
5.1.18. 1-(2-((2-Chloro-6-cyclopropyl-4 -fluoro-3-methoxybiphenyl-
4-yl)methyl)-5-oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)-4-
methylpiperidine-4-carboxylic acid (3p)
6
8.44; H, 6.69; N, 8.26.
0
White solid. Yield. 73% mp 197–198 °C, MS (ESI) m/z 542.4 [M
5
.1.14. 1-(2-((2-Cyclopropyl-4 -fluoro-5-isopropoxybiphenyl-4-yl)
+
1
+
0
6
H] . H NMR (300 MHz, DMSO-d ) d 0.54–0.62 (2H, m), 0.66–
methyl)-5-oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)-4-methylpiperidine-
.74 (2H, m), 1.14 (3H, s), 1.31–1.48 (3H, m), 1.91 (2H, d,
4-carboxylic acid (3l)
J = 13.5 Hz), 2.82–2.95 (2H, m), 3.19 (2H, d, J = 8.2 Hz), 3.25–3.35
(4H, m), 3.47 (2H, d, J = 8.2 Hz), 3.63 (2H, s), 3.76 (3H, s), 6.87
White solid. Yield. 84% mp 165–166°C, MS (ESI) m/z 536.2 [M
H] . H NMR (300 MHz, DMSO-d
.80 (2H, m), 1.13 (3H, s), 1.25 (6H, d, J = 5.9 Hz), 1.30–1.43 (2H,
+
1
+
0
6
) d 0.48–0.56 (2H, m), 0.70–
1
3
(
1
1H, s), 7.25–7.32 (4H, m). C NMR (75MHz, DMSO-d
6
) d 177.6,
60.9, 161.4 (d, J = 243.7 Hz), 151.4, 139.5, 138.4, 134.1 (d,
J = 3.3 Hz), 133.0, 131.7 (d, J = 8.3 Hz), 126.5, 123.6, 115.2 (d,
J = 20.9 Hz), 79.1, 66.5, 60.7, 56.6, 44.0, 42.5, 40.4, 33.3, 25.2,
13.8, 9.0. Anal. Calcd for C29H33N O ClF: C, 64.26; H, 6.14; N,
3 4
.75; Cl, 6.54; F, 3.50. Found: C, 64.08; H, 6.22; N, 7.76.
m), 1.66–1.79 (1H, m), 1.91 (2H, d, J = 13.6 Hz), 2.88 (2H, t,
J = 10.3 Hz), 3.14 (2H, d, J = 8.1 Hz), 3.21–3.38 (4H, m), 3.44 (2H,
d, J = 8.2 Hz), 3.53 (2H, s), 4.50–4.63 (1H, m), 6.74 (1H, s), 6.84
1H, s), 7.20–7.31 (2H, m), 7.46 (2H, dd, J = 8.4, 5.8 Hz). C NMR
75MHz, DMSO-d ) d 177.6, 160.8, 161.2 (d, J = 243.2 Hz), 152.8,
39.9, 137.5 (d, J = 3.3 Hz), 131.4, 131.2 (d, J = 8.3 Hz), 126.5,
25.6, 114.8 (d, J = 21.5 Hz), 114.4, 79.2, 69.8, 66.5, 56.2, 44.0,
2.5, 40.4, 33.3, 25.2, 22.0, 12.9, 8.9. Anal. Calcd for C31
O: C, 67.25; H, 7.28; N, 7.59. Found: C, 67.10; H, 7.25; N, 7.54.
1
3
(
(
7
6
1
1
4
Fꢀ1H
5.2. Estimation of LogD at pH 7.4
38 3 4
H N O -
2
LogD7.4, which is a partition coefficient between 1–octanol and
aqueous buffer pH 7.4, of the compounds was measured on the
chromatographic procedure whose condition was developed based
on a published method. The instruments were Waters Alliance
0
5
5
.1.15. 1-(2-((2-Cyclopropyl-5-ethyl-4 -fluorobiphenyl-4-yl)methyl)-
-oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)-4-methylpiperidine-4-
2
795 HPLC system with 2996 UV–vis detector (Milford, MA, USA).
carboxylic acid (3m)
White solid. Yield. 63% mp 225–226 °C, MS (ESI) m/z 506.2 [M
+
1
+
H] . H NMR (300 MHz, DMSO-d
6
) d 0.55–0.65 (2H, m), 0.74–
5.3. Calculation of pharmacokinetics parameters
0
.84 (2H, m), 1.09–1.18 (6H, m), 1.30–1.43 (2H, m), 1.69–1.81
(
1H, m), 1.90 (2H, d, J = 13.5 Hz), 2.62 (2H, q, J = 7.5 Hz), 2.88 (2H,
Pharmacokinetic parameters were calculated by the non-com-
partmental analysis. The area under the concentration-time curve
(AUC) and the area under the first moment curve (AUMC) were cal-
culated using the linear trapezoidal method. The mean residence
time (MRT) was calculated as AUMC/AUC. The total clearance
(CLtotal) was calculated as doseiv/AUCiv. The volume of distribution
(VDss) was calculated as CLtotal*MRTiv. Oral bioavailability (F) was
calculated as (AUCpo/dosepo)/(AUCiv/doseiv)*100. All of the pharma-
cokinetic calculations were performed with Microsoft Excel
(Microsoft Corporation, Redmond, WA).
t, J = 10.2 Hz), 3.14 (2H, d, J = 8.2 Hz), 3.22–3.37 (4H, m), 3.42
(
2H, d, J = 8.0 Hz), 3.58 (2H, s), 6.85 (1H, s), 6.96 (1H, s), 7.20–
1
3
7
.30 (2H, m), 7.44 (2H, dd, J = 8.6, 5.7 Hz), 12.45 (1H, brs).
) d 177.6, 160.8, 161.2 (d, J = 243.2 Hz),
39.2, 139.1, 137.5 (d, J = 3.3 Hz), 137.1, 135.1, 131.1 (d,
J = 8.3 Hz), 129.4, 124.4, 114.8 (d, J = 20.9 Hz), 79.2, 66.5, 56.0,
4.0, 42.5, 40.4, 33.3, 25.2, 24.2, 15.1, 13.0, 9.2. Anal. Calcd for C30
Fꢀ0.2H O: C, 68.60; H, 6.99; N, 8.00. Found: C, 68.59; H,
.97; N, 7.95.
C
NMR (75MHz, DMSO-d
6
1
4
H
6
-
36
N
3
O
4
2
0
5
.1.16. 1-(2-((6-Cyclopropyl-3-ethoxy-4 -fluoro-2-methylbiphenyl-4-
5.4. In vitro SSTR5 antagonistic activity
yl)methyl)-5-oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)-4-
methylpiperidine-4-carboxylic acid (3n)
Chinese hamster ovary (CHO) cells stably expressing human
SSTR5 or mouse SSTR5 were suspended in assay buffer (Hank’s bal-
anced salt solution supplemented with 5 mM HEPES (pH7.5), 0.1%
fatty acid–free BSA and 500 lM IBMX). The cells were incubated
with various concentrations of test compounds for 15 min at room
White solid. Yield. 47% mp 206–207 °C, MS (ESI) m/z 536.1 [M
+
1
+
0
2
6
H] . H NMR (300 MHz, DMSO-d ) d 0.47–0.55 (2H, m), 0.59–
.68 (2H, m), 1.13 (3H, s), 1.28–1.43 (6H, m), 1.82–1.96 (5H, m),
.88 (2H, t, J = 10.2 Hz), 3.15 (2H, d, J = 8.2 Hz), 3.24–3.37 (4H,
m), 3.44 (2H, d, J = 8.3 Hz), 3.58 (2H, s), 3.79 (2H, q, J = 6.8 Hz),
temperature. Then, the cells were stimulated with 1 or 3 nM (for
human SSTR5 or mouse SSTR5, respectively) SST14 and 0.3 lM fol-
skolin for 30 min at room temperature. Intracellular cAMP level
was determined using HTRF cAMP dynamic 2 kit (Cisbio Bioassays)
by following the manufactures’ instructions. HTRF signal was mea-
sured using Envision (PerkinElmer).
6
1
.73 (1H, s), 7.18–7.32 (4H, m). 13C NMR (75MHz, DMSO-d
77.6, 160.8, 161.0 (d, J = 242.6 Hz), 153.1, 140.9, 136.3 (d,
6
) d
J = 3.3 Hz), 135.9, 131.3 (d, J = 7.7 Hz), 130.2, 128.5, 122.0, 115.1
d, J = 21.5 Hz), 79.2, 68.4, 66.6, 57.1, 44.0, 42.5, 40.4, 33.3, 25.2,
(
1
7
38 3 4
5.6, 13.9, 13.5, 8.6. Anal. Calcd for C31H N O F: C, 69.51; H,
.15; N, 7.84. Found: C, 69.28; H, 7.10; N, 7.75.
5.5. Oral glucose tolerance test
0
5
.1.17. 1-(2-((2-Chloro-6-cyclopropyl-3-ethoxy-4 -fluorobiphenyl-4-
yl)methyl)-5-oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)-4-
methylpiperidine-4-carboxylic acid (3o)
The care and use of the animals and the experimental protocols
used in this research were approved by the Experimental Animal
Care and Use Committee of Takeda Pharmaceutical Company
Limited. Male C57BL/6J Jcl mice were obtained from CLEA Japan.
Inc (Japan). They were fed a high fat diet (D12492, Research Diets
Inc.) and tap water ad libitum. Male C57BL/6J Jcl mice (9 weeks of
age) were fasted at 7:00 am. and orally given vehicle (0.5%
methylcellulose) or compounds after an hour. Eight hours later,
all animals were received an oral glucose load (5 g/kg). Blood sam-
ples were collected from tail vein before drug administration (pre),
and just before glucose load (time 0), and 10, 30, 60 and 120 min
after glucose load. Blood glucose was measured by ACCU–CHECK
Aviva (Roche Diagnostics, Japan). Statistical analysis was
White solid. Yield. 77% mp 217–218 °C, MS (ESI) m/z 556.4 [M
+
1
+
H] . H NMR (300 MHz, DMSO-d
6
) d 0.53–0.62 (2H, m), 0.65–
0
.75 (2H, m), 1.14 (3H, s), 1.30–1.47 (6H, m), 1.90 (2H, d,
J = 13.8 Hz), 2.88 (2H, t, J = 10.2 Hz), 3.19 (2H, d, J = 8.1 Hz), 3.23–
.40 (4H, m), 3.46 (2H, d, J = 8.2 Hz), 3.62 (2H, s), 3.95 (2H, q,
3
13
J = 6.8 Hz), 6.86 (1H, s), 7.24–7.33 (4H, m). C NMR (75MHz,
DMSO-d ) d 177.6, 160.8, 161.4 (d, J = 244.3 Hz), 150.6, 139.3,
38.1, 134.3 (d, J = 3.3 Hz), 132.6, 131.7 (d, J = 7.7 Hz), 126.6,
23.3, 115.2 (d, J = 21.5 Hz), 79.2, 69.0, 66.6, 56.9, 44.0, 42.5,
0.4, 33.3, 25.2, 15.4, 13.3, 9.0. Anal. Calcd for C30 FCl: C,
4.80; H, 6.34; N, 7.56. Found: C, 64.67; H, 6.41; N, 7.56.
6
1
1
4
6
35 3 4
H N O
Please cite this article in press as: Yamasaki T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.003

1
0
T. Yamasaki et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
conducted by using Student’s t–test, Welch’s test, Williams’s test
or Shirley Williams’s test.
15. Martin RE, Mohr P, Maerki HP, et al. Bioorg Med Chem Lett. 2009;19:6106–6113.
1
1
6. Alker A, Binggeli A, Christ AD, et al. Bioorg Med Chem Lett. 2010;20:4521–4525.
7. (a) Binggeli A, Christ AD, Green LGG, et al., PCT Int Appl WO2006/094682 A1,
2006.(b) Binggeli A, Christ AD, Green LGG, et al., PCT Int Appl WO2006/128803
Acknowledgements
A1, 2006.(c) Binggeli A, Christ AD, Green L, et al., PCT Int Appl WO2007/025897
A2, 2007.(d) Binggeli A, Christ AD, Maerki HP, Martin RE. PCT Int Appl WO2007/
1
2
10340 A2, 2007.(e) Christ AD, Martin RE. PCT Int Appl WO2008/000692 A2,
007.(f) Binggeli A, Christ AD, Maerki HP, Martin RE. PCT Int Appl WO2008/
We thank Mr. Masaki Ogino and Dr. Ryo Mizojiri for helpful
discussions and identification of the key compound 3a. We also
thank Dr. Masaaki Funata and Mr. Akito Hata for establishing the
cAMP reporter assay system for evaluating the compounds, and
Mr. Hiroaki Yashiro and Mr. You Muraki for fruitful discussions
on pharmacological aspects of this work.
019967 A2, 2007.(g) Christ AD, Martin RE, Mohr P. PCT Int Appl WO2008/
3173538 A1, 2008.(h) Bleicher K, Christ AD, Martin RE, Mohr P.CT Int Appl
0
WO2008/122510 A1, 2008.(i) Christ AD, Martin RE, Mohr P. PCT Int Appl
WO2008/145524 A2, 2008.(j) Christ AD, Martin RE, Mohr P. PCT Int Appl
WO2008/148710 A2, 2008.(k) He J, Bawiec J, Liu W, Liang GB, Yang L, PCT Int
Appl WO2010/056717 A1, 2010.(l) Aster SD, Duffy JL, Liang GB, Shao P, Ye F.
PCT Int Appl WO2010/129729 A1, 2010.(m) Duffy JL, Bao J, Ondeyka DL, et al.,
PCT Int Appl WO2011/146324 A1 2011.(n) Duffy JL, Bao J, Ondeyka DL, et al.,
PCT Int Appl WO2012/024183 A1, 2012.
8. Hideki Hirose et al. To be prepared.
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A. Supplementary data
1
1
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.06.003.
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Please cite this article in press as: Yamasaki T., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.003