Synthesis and biological evaluation of novel chiral diazepine derivatives as bombesin receptor subtype-3 (BRS-3) agonists incorporating an antedrug approach

Article abstract of DOI:10.1016/j.bmc.2014.11.018

Novel compounds based on the lead BRS-3 agonists from our HTS compounds 2a and 2b have been synthesized with the focus on obtaining peripheral BRS-3 agonists. To identify potent anti-obesity compounds without adverse effects on the central nerve system, a

Full text of DOI:10.1016/j.bmc.2014.11.018

Bioorganic & Medicinal Chemistry 23 (2015) 89–104
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel chiral diazepine
derivatives as bombesin receptor subtype-3 (BRS-3) agonists
incorporating an antedrug approach
a,
⇑
a
a
a
a
Tetsuyoshi Matsufuji , Kousei Shimada , Shozo Kobayashi , Masanori Ichikawa , Asuka Kawamura ,
a
a
b
b
b
b
Teppei Fujimoto , Tsuyoshi Arita , Takashi Hara , Masahiro Konishi , Rie Abe-Ohya , Masanori Izumi ,
Yoshitaka Sogawa , Yoko Nagai , Kazuhiro Yoshida , Yasuyuki Abe , Takako Kimura ,
b
c
c
d
e
a
Hisashi Takahashi
a
Medicinal Chemistry Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
Cardiovascular Metabolics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
b
c
d
e
Drug Discovery and Biomedical Technology Unit, Daiichi Sankyo RD NOVARE Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel compounds based on the lead BRS-3 agonists from our HTS compounds 2a and 2b have been syn-
thesized with the focus on obtaining peripheral BRS-3 agonists. To identify potent anti-obesity com-
pounds without adverse effects on the central nerve system, a labile carboxylic ester with an antedrug
functionality was introduced onto the terminal position. Through the extensive synthetic exploration
and the pharmacokinetic studies of oral administration in mice, the phenol ester 17c was selected due
to the most suitable pharmacological profile. In the evaluation of food intake suppression in B6 mice,
Received 9 September 2014
Revised 13 November 2014
Accepted 14 November 2014
Available online 21 November 2014
Keywords:
Anti-obesity
Antedrug
Bombesin receptor subtype-3 (BRS-3)
Brain penetration
Diazepine
17c showed significant in vivo efficacy and no clear adverse effect on heart rate and blood pressure
change in dog iv infusion. Our study paved the way for development of anti-diabetes and obesity drugs
with a safer profile.
Ó 2014 Elsevier Ltd. All rights reserved.
1
. Introduction
Obesity is a highly prevalent major public health problem, and
and undesirable adverse effects mainly on the central nervous sys-
tem (CNS). Therefore, there are significant unmet medical needs for
5
the treatment of obesity with safer profiles.
has been known as a chronic medical condition internationally
since the World Health Organization officially defined obesity a
disease in 1998. Considering that related disorders such as type
The bombesin receptor subtype-3 (BRS-3), which is known as
an orphan G-protein coupled receptor, is expressed not only in
the brain, but also in peripheral organs such as the intestine, liver,
1
6
2
diabetes, cardiovascular diseases and hypertension could exacer-
lung, testes and pancreas. Since BRS-3 deficient mice develop
2
bate the condition, obesity is no longer a disorder that should be
resolved by self-treatment, but rather a malady that demands
medical therapy. There are presently several FDA-approved drugs
for chronic treatment of obesity: the lipase inhibitor, orlistat; a
selective serotonin 5-HT2C receptor agonist, lorcaserin; and the
obesity and impaired glycemic regulation, finding ligands that
modulate BRS-3 signaling is useful for developing a novel treat-
7
ment for diabetes and obesity. Although the exact physiological
effects by the activation of BRS-3 are unknown since the natural
ligand for BRS-3 has yet to be identified, BRS-3 not only in the
CNS, but also in peripheral organs such as the intestine can be a
promising drug target with an important function in regulating
endocrine and metabolic processes via a vagus nerve.8
3
combination of phentermine and topiramate, Qsymia. However,
more efficacious and safer treatments for obesity and weight main-
4
tenance are still strongly demanded. In actual fact, some patients
taking currently available drugs are suffering from variable efficacy
Recently, Guan et al. have reported a potent and brain-pene-
trant BRS-3 agonist as clinical candidate 1a (MK-5046, Fig. 1)
which showed anti-obesity effects in rats and dogs, while 1a also
caused side effects, such as an increase of body temperature, heart
⇑
Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5436 8563.
E-mail address: matsufuji.tetsuyoshi.nf@daiichisankyo.co.jp (T. Matsufuji).
9
rate and blood pressure in animals. In addition, it raised blood
http://dx.doi.org/10.1016/j.bmc.2014.11.018
968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
0

9
0
T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
O
R
CF
3
3
F CO
N
O
O
F
3
C
OH N
SO
N
A
B
N
2
OH
N
N
N
H
N
R
A
N
B
B
A
N
C
F
3
C
N
N
N
N
H
H
3
F C
N
O
Bicyclic diazepines
from 1b and 2a,2b
1
a (MK-5046)
hEC50 = 14 nM
mEC50 = 21 nM
1b (MK-7725)
hEC50 = 73 nM
mEC50 = 130 nM
2a (R = Me) 2b (R =
)
N
H
hEC50 = 916 nM
mEC50 = 256 nM
hEC50 = 131 nM
mEC50 = 34 nM
Figure 1. Brain penetrant imidazole 1a (MK-5046), benzodiazepine sulfonamide 1b (MK-7725) and our hit compounds 2a and 2b with BRS-3 EC50 values.
pressure in humans in the clinical trial.10 Hence, lowering exposure
of the compound to the brain is expected to avoid these side effects
due to the absence of the activation of the sympathetic nervous
system by stimulating BRS-3 in the CNS. In addition, to balance
stimulation of BRS-3 in the intestine as a peripheral receptor and
reduce brain exposure, we adopted one approach to significantly
reducing systemic exposure including CNS: the ‘antedrug’
approach. The terms ‘antedrug’ and ‘soft drug’ were introduced
to describe drugs which act topically at the site of application
but that are transformed into inactive metabolites upon entry into
the systemic circulation.11 Therefore, we speculated that more
potent and safer BRS-3 agonists would be obtained by use of the
antedrug approach. Furthermore, our approach could be helpful
to identify an unknown mechanism of BRS-3 signaling by the
peripheral simulation if any physiological effect was observed.
Initially, we designed and synthesized a novel benzodiazepine
BRS-3 agonist based on compounds 2a, 2b and a benzodiazepine
sulfonamide 1b. Compounds 2a and 2b were identified by a high
to obtain the diazepine 6 by the reduction of a corresponding lac-
tam ring with lithium aluminum hydride (LAH) under heating, but
failed probably because of the strong distortion of the ring. As a
result, we succeeded at procuring diazepine 6 using the following
4 steps: (1) a commercially available pyridyl carboxylic acid 3 was
reduced with borane–THF complex to pyridylmethyl alcohol, (2)
ethylenediamine addition by S
dation with MnO furnished the 7-membered imine without iso-
lating the corresponding aldehyde in a moderate yield, and (4)
hydrogenation by a Pd/C catalyst under an H atmosphere in ethyl
N
Ar reaction upon heating, (3) oxi-
2
2
acetate yielded the desired 6. As a final step, amidation of 6 with
various carboxylic acids yielded 7a–7d. Aliphatic derivatives 7e–
7h were synthesized via S 2 reaction using various alkyl halides
N
and the phenol intermediate which was prepared by direct amida-
tion of 6 and [3-(benzyloxy)phenyl]acetic acid.
A polar derivative 11 with an acetic acid moiety on the diazepine
ring was synthesized as shown in Scheme 2. First, the aldehyde 8
was condensed with N-Boc-ethylenediamine in anhydrous media,
and the resulting imine was reacted with an allyl Grignard reagent
to afford 9. Then, a deprotection of the Boc group and an HCl salt for-
mation by addition of 4 N HCl in ethylacetate, followed by cycliza-
tion upon heating at 160 °C and Boc protection of a nitrogen atom
on the diazepine ring provided 10. The allyl group of 10 was con-
verted to methyl ester after oxidative cleavage to aldehyde by using
1
2
throughput screening campaign of our corporate library, and
1
7
b was previously reported as a potent BRS-3 agonist MK-
725. Inspired by a structural similarity, we surmised the sulfo-
1
3
nyl group of 1b could be replaced by another linker and the ring, C,
in Figure 1 could be removed from the rigid benzodiazepine struc-
ture. Then, after exploring a novel potent scaffold, we planned to
use an antedrug method by introducing a labile functional group
onto the molecule.
4 4
an OsO catalyst and NaIO as an oxidant, Klaus oxidation reaction to
1
4
carboxylic acid, and methyl esterification with methyl iodide.
Finally, deprotection of the Boc group of 10, and amidation with
2
-(3-phenoxyphenyl)acetic acid afforded a methyl ester of 11.
2
. Chemistry
Subsequent hydrolysis under a basic condition gave 11 as a racemic
compound, while chiral separation of the methyl ester of 11 using
chiral column chromatography (CHIRALPAK IC) and the following
hydrolysis yielded each enantiomer (R)-11 and (S)-11, respectively.15
The general procedure for the synthesis of chiral diazepine
derivatives 15a–15c, 17a–17f and 19a–19e is depicted in Scheme 3.
The preparation of diazepine derivatives 5a–5d and 7a–7h is
shown in Scheme 1. The formation of a tricyclic benzodiazepine
3a
4
was performed in a similar manner reported for 1b.1 Subse-
quent amidation with the corresponding carboxylic acid or acyl
chloride provided the desired compounds 5a–5d. New scaffold
compounds (7a–7h) were synthesized after synthetic examination
for a formation of a 7-membered diazepine ring. Initially, we tried
2
First, Ellman’s chiral sulfinamide ((S)-t-BuS(O)NH ) was adopted to
16
stereospecifically introduce an allyl group to the benzyl position.
O
t-Bu
O
R1=
R1
H
N
N
t-Bu
OPh
COOH
a, b
c
5a
5b
F
3
C
N
N
H
F
3
C
N
N
H
O
O
F
3
C
N
Cl
R1=
OPh
5
a-5d
3
4
5c
5d
O
H
N
2
R
7a (R2 = Ph)
7e (R = i-Pr)
2
d-g
h or i-k
N
O
2
7f (R2 = n-Bu)
7
b (R2 = 4-F-Ph)
F
3
C
N
N
H
7
c (R = 4-tolyl)
7
g (R2 = i-Bu)
F
3
C
N
N
2
2
6
H
7d (R = 6-Me-3-Py) 7h (R = neopentyl)
7
a-7h
Scheme 1. Reagents and conditions: (a) 1,2-diaminobenzene, ethylene glycol monobutylether, 160 °C, 64%; (b) BH
butylbenzoylchloride, Et N, CH Cl , or arylacetic acid, HATU, (i-Pr) EtN, CH Cl , 72–92% (d) BH –THF complex, THF, 0 °C–rt, quant.; (e) ethylenediamine (neat), 120 °C, 62%; (f)
MnO , CH Cl /MeOH, 76%, (g) H , Pd–C, AcOEt, 82%; (h) arylacetic acid, HATU, (i-Pr) EtN, CH Cl , 34–96%; (i) [3-(benzyloxy)phenyl]acetic acid, (i-Pr) EtN, HATU, CH Cl , 99%;
j) H , Pd–C, EtOH, 98%; (k) alkylhalide, K CO , DMF, 80 °C, 31–83%. HATU: (dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methaniminium
hexafluorophosphate.
3
–THF complex, THF, 0 °C–rt, 66%; (c) 4-tert-
3
2
2
2
2
2
3
2
2
2
2
2
2
2
2
2
2
(
2
2
3

T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
91
O
O
Boc
N
HO
CHO
Cl
N
O
a, b
c-e
f-l for 11
N
H
NHBoc
F
3
C
N
F
3
C
N
N
H
10
f-k, m, l
for (R)-11 and (S)-11
F
3
C
N
N
H
F
3
C
N
Cl
8
9
11, (R)-11, and (S)-11
Scheme 2. Reagents and conditions: (a) N-Boc-ethylenediamine, Na
NMP, 160 °C; (e) di-tert-butyl dicarbonate, Et N, CH Cl , 59%, 3 steps; (f) 2.5% OsO
NaH PO O, 2-methyl-2-butene, t-BuOH/H O; (i) iodomethane, K CO , DMF, 50 °C, 77%, 4 steps; (j) 4 N HCl/AcOEt, 92%; (k) 2-(3-phenoxyphenyl)acetic acid, (i-Pr)
HATU, CH
2
SO
4
, CH
2
Cl
2
, rt, 69%; (b) allylmagnesium bromide/Et
2
O, THF, 0 °C, 73%; (c) 4 N HCl/AcOEt; (d) (i-Pr)
2
EtN,
2
,
3
2
2
4
/i-PrOH, N-methylmorpholine oxide, acetone; (g) NaIO
4
, THF/H
2
O; (h) NaClO
2
4
Á2H
2
2
2
3
2
EtN,
2
Cl
2
, 91%; (l) 2 N NaOH aq, THF/MeOH, 88–97%; (m) chiral separation (CHIRALPAK IC, EtOH/hexane = 30/70). NMP: N-methylpyrrolidinone.
O
NHBoc
e ,f
·2HCl
R1
O
S
Boc
O
H
N
a, b
c, d
N
g-j
N
H
t-Bu
N
H
8
F
3
C
N
Cl
F
3
C
N
Cl
F
3
C
N
N
H
F
3
C
N
N
H
14a (R1 = Me)
1
1
2
13
(R)-10
4b (R1 = Bn)
O
15a (R2 = 4-F-Ph)
O
R2 15b (R = i-Bu)
HO
2
k,l
14a
N
O
5c (R2 = 6-Me-3-Py)
1
F
3
C
N
N
H
O
O
1
1
7a (R3 = H)
O
O
7b (R3 = CO-i-Pr)
3
m
p
BnO
n,o
HO
R
17c (R3 = CO-i-Bu)
N
O
O
N
OH
1
1
7d (R3 = CO-n-Bu)
F
3
C
N
N
H
F
3
C
C
N
N
H
7e (R3 = COCH -c-Pr)
2
1
7a-17f
14b
16
17f (R3 = COCH -t-Bu)
2
O
O
9a (R4 = H)
19b (R4 = i-Pr)
1
O
q
O
BnO
O
R3
HO
O
R4
N
N
1
1
9c (R4 = i-Bu)
9d (R4 = n-Bu)
O
F
3
C
N
N
H
F
3
N
N
H
19e (R4 = CH -c-Pr)
18
2
1
9a-19f
1
9f (R4 = CH -t-Bu)
2
Scheme 3. Reagents and conditions: (a) (S)-t-BuS(O)NH
oxoethyl)carbamate, NaBH(OAc) , CH Cl , quant.; (e) 4 N HCl/AcOEt; (f) (i-Pr)
N-methylmorpholine oxide, acetone/H O, 0 °C–rt; (h) NaIO , THF/H O; (i) NaClO , NaH
CO , acetone, 60 °C, then 4 N HCl/1,4-dioxane, 5 steps, 64–80%; (k) arylacetic acid, (i-Pr)
3-hydroxyphenyl)acetic acid, DMT-MM, MeOH, 77%; (n) alkylacyl chloride, Et N, CH Cl ; (o) H , Pd–C, EtOH, 46–96%, 2 steps; (p) arylacetic acid, (i-Pr)
, Pd–C, EtOH, 74–80%, 2 steps. NMP, N-methylpyrrolidinone. HATU: (dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methaniminium hexafluoro-
2
, Ti(OEt)
4
, THF, reflux, 99%; (b) allyl bromide, in, satd NaBr aq, 94%; (c) 4 N HCl/1,4-dioxane, 82%; (d) tert-butyl N-(2-
EtN, NMP, 160 °C, then di-tert-butyl dicarbonate, 59%, 3 steps; (g) 2.5% OsO /i-PrOH,
PO O, 2-methyl-2-butene, t-BuOH/H O; (j) TMSCH , THF/MeOH, 0 °C or BnBr,
Á2H
EtN, HATU, CH Cl ; (l) 2 N NaOH aq, THF/MeOH, 69–97%, 2 steps; (m)
EtN, HATU, CH Cl2, (q)
3
2
2
2
4
2
4
2
2
2
4
2
2
2 2
N
K
2
3
2
2
2
(
3
2
2
2
2
2
H
2
phosphate; DMT-MM: (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate.
After imine formation with the aldehyde 8 and the Ellman’s sulfin-
amide, an effective asymmetric addition of an allyl group to the
imine moiety was carried out under the condition of using an
indium catalyst in aqueous NaBr, which was reported by Sun
et al.1 A pure desired (R,S)-diastereomer 12 was easily isolated
3. Results and discussions
3.1. In vitro evaluation for structure–activity relationship (SAR)
study
7
18
by silica gel column chromatography in a high yield. Deprotec-
tion of the sulfoxide under an acidic condition, and the subsequent
For an efficient derivatization, we initiated the SAR study by
adopting the 4-tert-butylphenyl group as an upper moiety by
reference to the reported compound 1c as an (S)-atropisomer by
reductive amination using NaBH(OAc)
ethyl)carbamate gave 13. Then, deprotection of the Boc group of
3 and cyclization in NMP at 160 °C yielded a chiral key interme-
3
and tert-butyl N-(2-oxo-
1
3a
Liu et al.,
as shown in Table 1. Although replacement of the sul-
1
fonyl group with acyl group 5a resulted in no activity in human
and mouse BRS-3 expressing cells, aryl acetyl group 5b showed a
good result. While the replacement of the tert-butyl group with a
para-phenoxy group 5c showed a sharp loss of potency, the
synthesis of meta-phenoxy compound 5d by reference to a partial
structure of 2b led to a dramatic improvement in both activities.
Encouraged by the result, we continued further modifications of
compound 5d by removing the phenyl ring to improve its
physical properties such as rigidity and lipophilicity. Consequently,
this reduction turned out to further enhance the in vitro
activity against both species (7a; human EC50 = 8.5 nM, mouse
EC50 = 12.2 nM).
1
9
diate (R)-10. Next, the allyl group of (R)-10 was converted to
4a or 14b after oxidative cleavage to carboxylic acid as depicted
in Scheme 3, methyl or benzyl esterification of the carboxy group
with alkylation by an S 2 reaction, and deprotection of the Boc
1
N
group in acidic solvent. The versatile phenol 16 was directly
obtained by amidation with 14b and 3-hydroxybenzene acetic
acid using DMT-MM as a condensation agent.20 The desired
phenol esters 17b–17f were provided by debenzylation after the
corresponding acylation, whereas the benzoic acid esters of
1
9b–19f were obtained by debenzylation after amidation of the
corresponding carboxylic acid.

9
2
T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
Table 1
EC50 values of BRS-3 agonists 1c, 5a–5d and 7a
R
N
7
A
N
B
F3C
N
C
8
H
10
9
a
a
Compound
R
Ring C
Human BRS-3 EC50 (nM)
Mouse BRS-3 EC50 (nM)
O
S
O
1
5
c ((S)-isomer)
7,8-Dimethylphenyl
Phenyl
97b
33b
t-Bu
t-Bu
O
O
a
>10,000
>10,000
t-Bu
5
5
b
c
Phenyl
Phenyl
48
227
O
O
O
O
368
>10,000
5
7
d
a
Phenyl
None
25
46
O
8.5
12.2
O
a
Data are averages of at least 3 repeated measurements.
Data are cited from Ref. 13a.
b
Table 2
EC50 values of BRS-3 agonists 7a–7h, 11 and 15a–15c
R2
O
R1
N
O
F
3
C
N
N
H
a
a
LogDb
Compound
R1
R2
Human BRS-3 EC50 (nM)
Mouse BRS-3 EC50 (nM)
7a
7b
7c
Ph
H
H
H
8.5
3.6
1.3
12.2
2.7
1.0
4.5
4.6
>4.8
p-F-Ph
p-Tolyl
N
7
d
H
9.5
2.8
3.6
7
7
7
e
f
g
i-Pr
n-Bu
i-Bu
H
H
H
381
59
70
569
52
62
NDc
NDc
4.3
7
1
h
1
H
199
121
NDc
Ph
Ph
Ph
p-F-Ph
i-Bu
N
CH
(R)-CH
(S)-CH
(R)-CH
(R)-CH
2
CO
2
H
CO
CO
CO
CO
13
15
1.2
1.2
1.2
1.2
0.9
(
(
R)-11
S)-11
2
2
H
H
1.6
2071
1.7
17
3.4
3124
2.7
62
2
2
1
1
5a
5b
2
2
H
H
2
2
1
5c
2
(R)-CH CO
2
H
1.7
3.5
0.3
a
b
c
Data are averages of repeated measurements at least 3 times.
The distribution coefficients (logD) were measured between 1-octanol and phosphate buffered saline (pH 7.4).
Not determined.
Derivatization of the right terminal phenyl ring of 7a was
on the right phenyl ring (7b). Also para-tolyl substituent 7c
indicated improved activity with an increase of the logD value
(>4.8). Meanwhile, the combination of para-methyl and 3-pyridyl
group 7d provided an excellent potency with a modest lipophilicity
(logD = 3.6). We also examined a replacement of the R1 part with
aliphatic groups. Relatively small alkyl derivative 7e decreased the
activity presumably because the pharmacophore of the right ter-
minal moiety required a certain size of lipophilic functional group.
focused on next, as described in Table 2, because this part was cru-
cial for in vitro activity from the results in Table 1, and could be
efficiently converted by amidation of the diazepine moiety with
various carboxylic acids. Also 5b with a 4-tert-butylphenyl group
was relatively potent, but we did not derivatize it further in light
of its rigidity, lipophilicity and synthetic variety. An increase of
potency was observed by the installment of a para-fluoro atom

T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
93
Figure 2. Calculated molecular model structures of compound A, B, and C for the most stable conformers (hydrogen atoms are omitted for clarity).
In fact, in vitro activity was retained in cases where the number of
carbons was more than four (7f–7h). These results suggested 7g
should be excellent in terms of activity and lipophilicity at this
stage.
under the ambient condition. Thus, we were successful not only
at lowering lipophilicity of 7a, but also at improving its potency
by having (S)-configuration exist mainly due to the chiral (R)-acetic
acid group on the neighboring carbon atom of the 7-membered
ring.
As a next step, an introduction of a polar group onto the mole-
cule was examined to obtain a potent compound with a lowered
logD value aiming for low brain penetration. After a thorough syn-
thetic examination, we succeeded at finding an acetic acid group at
the R2 position that retained potency (11). A chiral separation was
achieved by using chiral column chromatography to yield each
pure enantiomer (R)-11 and (S)-11. As a result, it turned out that
only (R)-11 retained the in vitro activity (compound (R)-11;
human EC50 = 1.6 nM, mouse EC50 = 3.4 nM). The absolute configu-
ration of (R)-11 was determined by the asymmetric synthesis men-
tioned in Scheme 3. Sequentially, further SAR studies were pursued
to improve potency and to decrease the logD value by the intro-
duction of potent functional groups on the R1 position and a car-
boxyl unit on the R2 position. This combination enabled us to
obtain 15a–15c with good activity and low logD values, and was
especially effective for 15c, which indicated a good activity in both
species with the lowest logD value (compound 15c; human
EC50 = 1.7 nM, mouse EC50 = 3.5 nM, logD = 0.3).
Next, we decided to incorporate 15b and 15c into an antedrug
approach to avoid the adverse effect derived from the slightest
amount of their exposure to CNS. In particular, we installed various
kinds of ester groups, which were easily metabolically hydrolyzed
in blood, onto the right terminal moiety because it was suggested
by the results of Table 2 that their pharmacophore required a cer-
tain size of lipophilic functional group. Thus, the phenol esters
17b–17f and benzoic acid esters 19b–19f were designed with the
expectation that they would be rapidly metabolized by reaching
into the blood stream after they acted on BRS-3s in the intestine
in order to reduce the exposure to the brain as much as possible.
First, we confirmed that the phenol ester 17b, which was sug-
gested from 15b, retained the in vitro activity (Table 3), while
17a, a metabolite from 17b, had no activity as expected. In con-
trast, we could not apply this method of ester group installment
to the diaryl ether 15c because it resulted in a complete loss of
potency (data not shown). As a next step, ester compounds 17c–
17f with various length and bulkiness of alkyl groups were synthe-
sized. It turned out that compounds with more than 4 carbon
atoms retained the activity. Similarly, the benzoic acid ester 19b
showed excellent activity in both species, and the metabolite 19a
lost the activity (Table 4). Also, benzoic acid esters 19c–19f were
found to possess a similar structure–activity relationship and bet-
ter potency compared to 17b–17f.
To elucidate a significant activity difference between (R)-11 and
(S)-11, we investigated the possible reason for the difference
from the aspect of conformers of simplified diazepine models by
using computational calculations (Fig. 2). Compound A, depicted
as a model compound of 11 with no functional group on the
7
-membered ring, showed (S)-configuration on the nitrogen atom
21
as the lowest energy conformer, while the ring can easily be
flipped from (S) to (R)-configuration (relative potential energy for
the inversion:
D
E = E(R) À E(S) = 0.022 kcal/mol). Likewise, com-
3.2. Pharmacokinetic studies
pound B, regarded as an active form with the (R)-acetic acid group
on the ring, also provided (S)-configuration on the nitrogen atom.
Given MK-7725 (1b), reported by Chobanian et al., also possessed
the active (S)-atropisomer on the nitrogen atom, this configuration
should be suitable for exerting a high affinity for a BRS-3. Addition-
ally, since the ring inversion of compound B is more difficult to
ascertain than compound A due to the effect of the (R)-acetic
Pharmacokinetic (PK) parameters of compounds 17b–17f and
19b–19f in mice were evaluated to select the most suitable com-
pound for an antedrug approach: a highly active compound on
peripheral BRS-3, and was rapidly metabolized before it reached
the systemic circulation (Table 5).
Although the benzoic acid esters 19b–19f indicated better
in vitro activity than the phenol esters 17b–17f on the whole,
active compounds were also slightly detected (the ratio of
Active/Inactive (A/I) was at least 0.014). On the other hand, the
phenol esters 17b–17f provided preferable results: while the active
forms of 17d (n-butyl ester) and 17f (neopentyl ester) were slightly
acid group (
improved. In contrast, the inactive enantiomer, compound C with
S)-acetic acid group indicated (R)-configuration on the nitrogen
atom (
DE = 2.2 kcal/mol), the activity was supposed to be
(
D
E = À2.2 kcal/mol). Meanwhile, we assumed that some
diazepine analogs existed as a mixture of conformers according
1
to the H NMR spectra because the ring inversion easily occurred
detected (Cmax of 17d: 0.0285 lM, 17f: 0.0058 lM), more labile

9
4
T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
Table 3
EC50 values of BRS-3 agonists 15b and 17a–17f
O
O
O
O
O
metabolization
hydrolysis)
HO
HO
(
N
O
R
N
OH
F
3
C
N
N
H
F
3
C
N
N
H
17b-17f
17a
a
a
Compound
R
Human BRS-3 EC50 (nM)
Mouse BRS-3 EC50 (nM)
15b
17a
17b
17c
17d
17e
17f
—
—
i-Pr
i-Br
n-Br
17
>10,000
68
12
19
62
>10,000
282
43
35
81
CH
CH
2
c-Pr
t-Bu
42
8.7
2
20
a
Data are averages of repeated measurements at least 3 times.
Table 4
EC50 values of BRS-3 agonists 19a–19f
O
O
O
O
metabolization
(hydrolysis)
HO
HO
O
OH
N
R
N
O
O
F
3
C
N
N
H
F
3
C
N
N
H
19b-19f
19a
a
a
Compound
R
Human BRS-3 EC50 (nM)
Mouse BRS-3 EC50 (nM)
19a
19b
19c
19d
19e
19f
H
>10,000
40
4.2
4.0
3.4
>10,000
45
5.4
5.6
3.7
i-Pr
i-Br
n-Br
CH
CH
2
c-Pr
t-Bu
2
5.6
8.1
a
Data are averages of repeated measurements at least 3 times.
Table 5
Mouse PK parameters of 17b–17f and 19b–19f
O
O
HO
N
R
F
3
C
N
N
H
Active compound (A)
Inactive metabolite (I)
Mouse PK (po, 30 mg/kg)a
M) of A
C
max
(
l
C
max
(
lM) of I
Ratio of A/I
1
1
1
1
1
1
1
1
1
1
7b (R = O
2
CiPr)
CiBu)
CnBu)
CCH cPr)
CCH tBu)
9b (R = CO iPr)
iBu)
nBu)
CH cPr)
CH tBu)
17a (R = OH)
17a (R = OH)
17a (R = OH)
17a (R = OH)
17a (R = OH)
0b
0
0.68
1.26
1.63
0b
0
b
b
7c (R = O
7d (R = O
2
2
0.0285
0.0175
b
b
7e (R = O
7f (R = O
2
2
0
0.607
0
2
2
0.0058
1.24
0.0308
0.0285
0.0393
0.0127
0.0325
0.918
2.12
1.63
0.300
0.197
0.179
1.35
0.0145
0.0175
0.131
0.0643
2
19a (R = CO
19a (R = CO
19a (R = CO
19a (R = CO
19a (R = CO
2
H)
2
H)
2
H)
2
H)
2
H)
9c (R = CO
9d (R = CO
9e (R = CO
9f (R = CO
2
2
2
2
2
2
a
Average of 2 or 3 mice dosed at 30 mg/kg po. Each dose was administered with 0.5%MC (methyl cellulose) as a solvent.
Under limit of qualification.
b
esters 17b, 17c and 17e provided no detection of active com-
pounds. Particularly, 17c possessed the largest amount of exposure
of an inactive form among them (Cmax: 1.26 lM). As shown in
Figure 3, the PK profile of 17c was ideal for an antedrug approach:
high exposure of an inactive form and no exposure of an active
form in mouse blood. On the other hand, in the case of oral
administration of 17a itself, it was hardly detected at 30 mg/kg
(Cmax = 0.04 l
M).22 Namely, 17c existed during absorption by the
gastrointestinal (GI) tract after oral administration, and then was
quickly metabolized by the time it reached the systemic circula-
tion. Therefore, we selected 17c as a promising antedrug candidate.
Physical properties and PK parameters of compound 17c and its
metabolite 17a in mice were summarized (Table 6). An active par-
ent compound 17c showed a low logD value, good solubility in JP2

T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
95
Figure 4. Effect of 17c on food intake in C57BL/6N mice. Mice were fasted for 16 h
and fed normal chow one hour after the compound administration. Cumulative
food intake of each mouse over 6 h was measured. Data are mean ± SEM n = 4–5.
Figure 3. PK profile of 17c (po, 30 mg/kg in mice, n = 2).
⁄
P < 0.05, Dunnett’s test.
(
Japanese pharmacopoeia second test fluid, pH = 6.8), and low pro-
due to the absence of the detection of the active compound 17c in
blood. Also, this result suggested that a part of the food intake
suppression observed in brain penetrant BRS-3 agonists such as
tein binding (PB) ability. We presumed that these properties could
enhance drug efficacy of 17c incorporating an antedrug approach
as seen in Table 5. Fairly high polar surface area (PSA) values of
1
a (MK-5046) and 1b (MK-7725) was comprised of the activation
1
7a and 17c are also preferable to avoid acting on BRS-3 in the
2
3
of BRS-3 in the intestine.
CNS from the aspect of lowering brain-blood barrier penetration.
3
.4. Cardiovascular safety evaluation in dogs
3
.3. In vivo pharmacological evaluation for food-intake
suppression in mice
We turned our attention to a cardiovascular (CV) safety evalua-
tion of 17c using halothane anaesthetized dogs by iv infusion
From the most preferable PK profile of 17c in mice as an ante-
drug, we evaluated the effect of 17c on food intake in B6 mice to
2
4
(Fig. 5). In this test, 15b with low brain penetration and no labile
ester group as a counterpart of 17c was also evaluated to confirm
the value of an antedrug approach aiming for removing CV change
including heart rate (HR) and blood pressure (BP) because CV
change is used as typical and reliable index for the CNS adverse
confirm anti-obesity efficacy as
a peripheral BRS-3 agonist
(Fig. 4). When 1, 3, 10, 30 mg/kg of the ester 17c was orally
administered to mice at a single dose, 17c showed a tendency of
a dose-dependent anorectic effect and the 30 mg/kg group showed
a statistically significant reduction of food intake (P < 0.05) com-
pared to the vehicle-treated group. We also confirmed these effects
were not observed in BRS-3 deficient mice as a preliminary data.
Taken together, the novel chiral diazepine 17c was found to pos-
sess a food-intake suppression effect by stimulating peripheral
BRS-3. Since a single dose of 17c at 30 mg/kg resulted in about
1
0
effects. Each compound was administered to 2 dogs at 3 mg/kg/
mL for 30 min by iv infusion. In case of 15b, both dogs showed
3
the increase of HR and BP by the time the infusion was almost fin-
ished (30 min). Since the dog shown as animal #2 especially indi-
cated a sharp increase of HR and seemed to have difficulty with
anesthetic maintenance, we had to stop the experiment at
4
6
8 min. Blood concentration of 15b and 17c at 30 min and
0 min was also measured. At 30 min, 15b indicated relatively high
3
0% food intake suppression, it is expected that a repeated admin-
istration at the same dose would show an anti-obesity effect from
the perspective of a suppressant for energy intake, and the effect
was supposed to be derived from BRS-3 activation in the intestine
blood concentration compared to 17c (15b; animal #1 : 16.85 lM,
#2 : 18.67
l
M, 17b; animal #1 : 1.73
lM, #2 : 1.39
lM), and the
Table 6
Physical properties and mouse PK parameters of 17c and its metabolite 17a
O
O
O
O
HO
N
F
3
C
N
N
H
17c
LogDa Solubility JP1/JP2 (
lM/
PB freec
(%)
PSAd
T
(h)
e
C
mL)
e
(
lg/
AUCe (hr
mL)
l
g/
T
1/2
(h)
e
Compound
In vitro EC50 (human/mouse,
nM)
max
max
⁄
mL)b
1
1
7c (active
12/43
0.7
62/900
10.8
101.5 NDf
LOQg
LOQg
LOQg
compound)
7a (inactive
meabolite)
>10,000/>10,000
À0.8
750/780
>30.0
101.0 0.25h
1.26h
1.23h
0.25h
a
b
c
The distribution coefficients (logD) were measured between 1-octanol and phosphate buffered saline (pH 7.4).
JP1/JP2: Japanese pharmacopoeia first/second test fluid (pH = 1.2/6.8).
Unbound fractions (%) in mouse plasma. PB: Protein Binding.
d
e
f
2
Polar surface area (Å ).
Average of 2 mice dosed at 30 mg/kg po. Each dose was administered with 0.5% MC (methyl cellulose) as a solvent.
Not determined.
Under limit of qualification.
Each PK parameter of 17a was observed in the administration of 17c at 30 mg/kg po.
g
h

9
6
T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
Figure 5. Effect of 15b (animal #1, #2, red) and 17c (animal #1, #2, blue) on (a) heart rate (HR) and (b) blood pressure (BP) in dogs (n = 2/group). The compound 15b and
antedrug 17c were administered to halothane anaesthetized dogs at 3 mg/kg/3 mL for 30 min by iv infusion (the duration of the iv infusion is shown as a light blue zone).
Cardiovascular (CV) change was normalized by the pre-administration values (at 0 min) for the heart rate and blood pressure. The observation of the animal #2 was forcibly
stopped at 48 min (18 min after completing dosage) due to the difficulty of anesthetic maintenance.
measurement at 60 min provided similar results (15b; animal #1 :
.01 M, #2 : 1.28 M, 17b; animal #1 : 0 M, #2 : 0 M). This
(dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-
3-yloxy)methaniminium hexafluorophosphate; DMT-MM, (4-(4,
6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
n-hydrate.
1
l
l
l
l
suggested that the antedrug approach installed into 17c worked
well as we expected: the terminal ester group of 17c was rapidly
hydrolyzed during and after iv infusion in dogs as well, and an
unnecessary accumulation of 17c in the systemic circulation
including exposure to CNS was avoided. On the other hand, given
the low brain penetration of 15b, minimizing brain penetration
alone might be insufficient for avoiding CV change. Actually, the
HR was not affected at all by 17c infusion in contrast to the abrupt
increase in the case of 15b, and also 17c did not show a clear BP
change during and after iv infusion. For these reasons, it can be said
that the antedrug approach applied to 17c is certainly effective for
obtaining a potent peripheral BRS-3 agonist without an adverse
effect derived from acting on CNS.
5.1.1. 2-(Trifluoromethyl)-6,11-dihydro-5H-pyrido[3,2-
c][1,5]benzodiazepine (4)
A mixture of 2-chloro-6-(trifluoromethyl)pyridine-3-carboxylic
acid 3 (400 mg, 1.77 mmol) and 1,2-diaminobenzene (196 mg.
1.81 mmol) in ethylene glycol monobutylether (5 mL) was stirred
at 160 °C for 6 h. The cooled reaction mixture was diluted with
water, and the resulting suspension was filtered off. The obtained
solid was washed with water and dissolved in AcOEt (30 mL).
2 4
The organic solution was washed with brine, dried (Na SO ), con-
centrated and purified by silica gel column chromatography (hex-
ane/AcOEt = 1:1) to provide the amide (317 mg, 64%) as a colorless
1
solid. H NMR (CDCl
3
) d: 8.42 (1H, d, J = 8.2 Hz), 7.54 (1H, br s), 7.24
4
. Conclusion
(
(
(
1H, d, J = 7.8 Hz), 7.10–7.03 (2H, m), 6.91–6.89 (1H, m), 6.87–6.85
1H, m), 6.68 (1H, br s). A solution of borane–THF complex in THF
1.0 M, 3.3 mL, 3.3 mmol) was added to a solution of the obtained
amide (307 mg, 1.10 mmol) in THF (5 mL) under N
°C, and the mixture was stirred at the same temperature for 2 h.
In summary, we obtained a chiral diazepine 17c as a novel and
potent BRS-3 agonist incorporating metabolic inactivation.
Through a strenuous synthetic exploration, development of an effi-
cient chiral synthetic route, and adopting the antedrug (metabolic
inactivation) approach with an installment of ester groups onto an
optimal position, we were able to concurrently achieve both an
anorectic effect and a reduction of the risk of adverse effects
derived from CNS by selective stimulation of peripheral BRS-3s.
In fact, 17c showed statistically significant food intake suppression
in mice, and no clear adverse effect such as heart rate or blood
pressure change, arose from the CNS in dog iv infusion. These
results have provided an initial first step for developing a highly-
anticipated drug with a safer profile for the treatment of diabetes
and obesity, as well as valuable insight about a physiological effect
due to the activation of BRS-3 in the intestine.
2
atmosphere at
0
The reaction mixture was raised to room temperature and further
stirred for 16 h. After addition of methanol (1 mL), the reaction
mixture was concentrated under reduced pressure and the residue
was purified by silica gel column chromatography (hexane/
AcOEt = 1:1) to provide 4 (192 mg, 66%) as a colorless solid. 1
H
3
NMR (CDCl ) d: 7.37 (1H, d, J = 7.4 Hz), 7.22 (1H, br s), 7.00 (1H,
d, J = 7.4 Hz), 6.91–6.87 (1H, m), 6.85–6.81 (2H, m), 6.76 (1H, d,
J = 7.4 Hz), 4.23–4.19 (3H, m).
5
.1.2. (4-tert-Butylphenyl)-[2-(trifluoromethyl)-5,11-
dihydropyrido[3,2-c][1,5]benzodiazepin-6-yl]methanone (5a)
-tert-Butylbenzoyl chloride (103 L, 0.57 mmol) was added to
a solution of 4 (100 mg, 0.38 mmol) and triethylamine (158 L,
.13 mmol) in CH Cl (5 mL) at room temperature, and the reaction
mixture was stirred for 3 h. The mixture was diluted with CH Cl
30 mL), washed with brine, dried (Na SO ), concentrated and
purified by silica gel column chromatography (hexane/AcOEt =
:1). The obtained solid was triturated in hexane/CH Cl , and
filtered to provide 5a (116 mg, 72%) as a colorless solid. H NMR
CDCl ) d: 7.67 (1H, br s), 7.45 (1H, s), 7.19–7.09 (6H, m),
.98 (1H, d, J = 7.8 Hz), 6.70–6.68 (2H, m), 5.89–5.85 (1H, m),
4
l
l
5
5
. Experimental
1
2
2
2
2
.1. Chemistry
(
2
4
Unless otherwise noted, materials were obtained from commer-
3
2
2
cial suppliers and used without further purification. NMR spectra
were recorded on a Varian Mercury 400 or 500 spectrometer with
tetramethylsilane as an internal reference. Mass spectra were
recorded on a Agilent Technologies Agilent 1100 series LC/MS.
Optical rotations were measured on an Autopol V Plus. TLC analysis
was performed on 60F254 plates (Merck). Flash column chroma-
tography was performed on Shoko scientific cartridge series (SI-
1
(
6
4
3
+
+
.10–4.06 (1H, m), 1.22 (9H, s). MS (ESI ) m/z: 426 ((M+H) .
5.1.3. 2-(4-tert-Butylphenyl)-1-[2-(trifluoromethyl)-5,11-
dihydropyrido[3,2-c][1,5]benzodiazepin-6-yl]ethanone (5b)
4-tert-Butylphenylactic acid (75 mg, 0.39 mmol) was added to a
solution of the diazepine 4 (80 mg, 0.30 mmol), HATU (126 mg,
6
0). The following abbreviations are used: AcOEt, ethylacetate;
MeOH, methanol; EtOH, ethanol; THF, tetrahydrofurane; DMF,
N,N-dimethylformamide; NMP, N-methylpyrrolidinone; HATU,

T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
97
0
.33 mmol) and (i-Pr)
room temperature, and the reaction mixture was stirred overnight.
The mixture was diluted with CH Cl (20 mL), washed with satu-
rated NaHCO aq and brine, dried (Na SO ), concentrated and puri-
fied by silica gel column chromatography (hexane/AcOEt = 3:1).
The obtained solid was triturated in hexane/CH Cl , and filtered
to provide 5b (120 mg, 91%) as a colorless solid. H NMR (CDCl
d: 7.59 (1H, d, J = 7.4 Hz), 7.22–7.16 (2H, m), 7.13–7.09 (2H, m),
2
EtN (165
l
L, 0.90 mmol) in CH
2
Cl
2
(5 mL) at
J = 7.4 Hz), 5.11 (1H, s), 3.91 (2H, br s), 3.49 (1H, s), 3.28–3.25
(2H, m), 3.11–3.09 (2H, m).
2
2
3
2
4
5.1.7. 2-(3-Phenoxyphenyl)-1-[8-(trifluoromethyl)-1,2,3,5-
tetrahydropyrido[2,3-e][1,4]diazepin-4-yl]ethanone (7a)
2-(3-Phenoxyphenyl)acetic acid (41 mg, 0.18 mmol) was added
to a solution of the diazepine 6 (30 mg, 0.14 mmol), HATU (63 mg,
2
1
2
3
)
0.17 mmol) and (i-Pr)
room temperature, and the reaction mixture was stirred for 15 h.
The mixture was diluted with CH Cl (20 mL), washed with brine,
dried (Na SO ), concentrated and purified by silica gel column
chromatography (hexane/AcOEt = 2:3). The obtained solid was
triturated in hexane/CH Cl , and filtered to provide 7a (49 mg,
85%) as a colorless solid. H NMR (CDCl ) a mixture of conformers
2 2 2
EtN (75 lL, 0.48 mmol) in CH Cl (3 mL) at
7
1
.05 (1H, d, J = 7.4 Hz), 6.99–6.95 (2H, m), 6.81 (1H, dd, J = 8.0,
.4 Hz), 6.73–6.70 (2H, m), 5.40 (1H, d, J = 15.2 Hz), 3.86 (1H, d,
2
2
+
+
J = 14.9 Hz), 3.52 (2H, s), 1.24 (9H, s). MS (ESI ) m/z: 440 (M+H) .
2
4
5
.1.4. 2-(4-Phenoxyphenyl)-1-[2-(trifluoromethyl)-5,11-
2
1
2
dihydropyrido[3,2-c][1,5]benzodiazepin-6-yl]ethanone (5c)
3
Compound 5c was prepared in a similar manner described for
d: 7.66–7.30 (3H, m), 7.25–7.10 (3H, m), 6.98–6.83 (5H, m), 5.21
(0.4H, br s), 5.03 (0.6H, br s), 4.61 (1.2H, s), 4.48 (0.8H, s),
3.91–3.88 (0.8H, m), 3.76–3.74 (1.2H, m), 3.73 (0.8H, s), 3.70
1
5
7
(
6
3
3
b. Yield: 92%. H NMR (CDCl ) d: 7.64 (1H, d, J = 7.4 Hz), 7.34–
.29 (2H, m), 7.25–7.21 (3H, m), 7.10–7.07 (2H, m), 7.03–7.01
1H, m), 6.98–6.96 (2H, m), 6.91 (1H, dd, J = 8.0, 1.4 Hz), 6.82–
+
(1.2H, s), 3.44–3.40 (0.8H, m), 3.31–3.27 (1.2H, m). MS (ESI )
+
.77 (4H, m), 5.45 (1H, d, J = 14.9 Hz), 3.94 (1H, d, J = 14.5 Hz),
m/z: 428 ((M+H) .
+
+
.57 (2H, s). MS (ESI ) m/z: 476 ((M+H) .
5
.1.8. 2-[3-(4-Fluorophenoxy)phenyl]-1-[8-(trifluoromethyl)-
5
.1.5. 2-(3-Phenoxyphenyl)-1-[2-(trifluoromethyl)-5,11-
1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-4-yl]ethanone (7b)
dihydropyrido[3,2-c][1,5]benzodiazepin-6-yl]ethanone (5d)
Compound 7b was prepared in a similar manner described for
1
Compound 5d was prepared in a similar manner described for
7a. Yield: 89%. H NMR (CDCl
3
) a mixture of conformers d: 7.65
1
5
b. Yield: 89%. H NMR (CDCl
3
) d: 7.63 (1H, d, J = 7.8 Hz), 7.32–
(0.6H, d, J = 7.3 Hz), 7.25–7.09 (2H, m), 7.05–6.88 (5.4H, m),
6.83–6.78 (2H, m), 5.21 (0.4H, br s), 5.03 (0.6H, br s), 4.61 (1.2H,
s), 4.48 (0.8H, s), 3.92–3.89 (0.8H, m), 3.76–3.74 (1.2H, m), 3.74
(0.8H, s), 3.71 (1.2H, s), 3.44–3.41 (0.8H, m), 3.32–3.29 (1.2H, m).
7
.28 (2H, m), 7.24–7.17 (2H, m), 7.12–7.07 (4H, m), 6.99–6.95
(
1H, m), 6.92 (2H, d, J = 8.6 Hz), 6.85 (1H, d, J = 8.2 Hz), 6.80 (1H,
d, J = 8.2 Hz), 6.61 (1H, d, J = 8.2 Hz), 6.48 (1H, s), 5.45 (1H, d,
+
+
+
J = 14.9 Hz), 3.93 (1H, d, J = 14.9 Hz), 3.57 (2H, s). MS (ESI ) m/z:
MS (ESI ) m/z: 446 ((M+H)
+
4
76 ((M+H) .
5
.1.9. 2-[3-(4-Methylphenoxy)phenyl]-1-[8-(trifluoromethyl)-
5
.1.6. 8-(Trifluoromethyl)-2,3,4,5-tetrahydro-1H-pyrido[2,3-
1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-4-yl]ethanone (7c)
e][1,4]diazepine (6)
Compound 7c was prepared in a similar manner described for
1
A solution of borane–THF complex in THF (0.9 M, 70 mL,
7a. Yield: 96%. H NMR (CDCl
3
) a mixture of conformers d: 7.65
6
3 mmol) was added to
methyl)pyridine-3-carboxylic acid (5.10 g, 22.1 mmol) in THF
100 mL) under N atmosphere at 0 °C, then the mixture was grad-
a
solution of 2-chloro-6-(trifluoro-
(0.6H, d, J = 7.4 Hz), 7.24–7.09 (4H, m), 7.00–6.81 (5.4H, m), 5.21
(0.4H, br s), 5.02 (0.6H, br s), 4.60 (1.2H, s), 4.47 (0.8H, s), 3.90–
3.88 (0.8H, m), 3.74–3.72 (1.2H, m), 3.72 (0.8H, s), 3.72–3.70 (2H,
(
2
+
ually raised to room temperature and stirred for 20 h. After addi-
tion of methanol (10 mL), the reaction mixture was diluted with
m), 3.44–3.26 (1.2H, m), 2.34–2.33 (3H, m). MS (ESI ) m/z: 442
+
((M+H) .
2 4
AcOEt (100 mL), washed with brine, dried (Na SO ), concentrated
under reduced pressure and the residue was purified by silica gel
5.1.10. 2-[3-[(6-Methyl-3-pyridyl)oxy]phenyl]-1-[8-(trifluoromethyl)-
column chromatography (hexane/AcOEt = 1:1) to obtain the alco-
1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-4-yl]ethanone (7d)
1
hol (4.66 g, quant.) as a colorless oil. H NMR (CDCl
3
) d: 8.12 (1H,
The synthesis of 7d was prepared in a similar manner described
1
d, J = 7.8 Hz), 7.69 (1H, d, J = 7.8 Hz), 4.87 (2H, d, J = 5.5 Hz), 2.06
1H, t, J = 5.5 Hz). The obtained alcohol (42.4 g, 201 mmol) was dis-
for 7a. Yield: 34%. H NMR (CDCl
3
) a mixture of conformers d:
(
8.28–8.26 (1H, m), 7.64 (0.6H, d, J = 7.4 Hz), 7.24–7.09 (3.4H, m),
7.00–6.82 (4H, m), 5.22 (0.4H, br s), 5.04 (0.6H, br s), 4.62 (1.2H,
s), 4.48 (0.8H, s), 3.92–3.89 (0.8H, m), 3.76–3.74 (1.2H, m), 3.72
(0.8H, s), 3.70 (1.2H, s), 3.46–3.43 (0.8H, m), 3.35–3.32 (1.2H, m),
solved in ethylenediamine (161 ml, 1.20 mol), and the reaction
mixture was stirred at 120 °C for 12 h. The cooled mixture was
diluted with brine, extracted with AcOEt (300 mL Â 3). The organic
+
+
layer was dried over Na
2
SO
4
, concentrated and purified by silica gel
2.54 (3H, s). MS (ESI ) m/z: 443 ((M+H) .
column chromatography (hexane/AcOEt = 3:1) to provide the
1
amino alcohol (29.4 g, 62%) as a colorless oil. H NMR (CDCl
7
4
3
) d:
.33 (1H, d, J = 7.4 Hz), 6.85 (1H, d, J = 7.4 Hz), 6.03 (1H, br s),
.59 (2H, s), 3.72 (2H, q, J = 5.9 Hz), 3.48 (2H, t, J = 6.3 Hz). MnO
5.1.11. 2-(3-Isopropoxyphenyl)-1-[8-(trifluoromethyl)-1,2,3,5-
tetrahydropyrido[2,3-e][1,4]diazepin-4-yl]ethanone (7e)
The diazepine 6 (230 mg, 1.06 mmol) was added to a solution of
[3-(benzyloxy)phenyl]acetic acid (256 mg, 1.06 mmol), HATU
2
(
(
98.8 g, 1.00 mol) was added to a solution of the amino alcohol
29.4 g, 125 mmol) in CH Cl /MeOH (700 mL/100 mL), and the
reaction mixture was stirred at room temperature for 2 h. The mix-
2
2
(443 mg, 1.16 mmol) and (i-Pr)
(10 mL) at room temperature, and the reaction mixture was stirred
for 15 h. The mixture was diluted with CH Cl (50 mL), washed
with saturated NaHCO aq and brine, dried (Na SO ), concentrated
2 2 2
EtN (578 lL, 3.18 mmol) in CH Cl
ture was filtered through a Celite pad, concentrated to provide the
2
2
1
imine (20.4 g, 76%). H NMR (CDCl
3
) d: 8.27 (1H, t, J = 1.6 Hz), 7.75
3
2
4
(
(
1H, d, J = 7.8 Hz), 7.04 (1H, d, J = 7.8 Hz), 5.99 (1H, br s), 4.14–4.11
2H, m), 3.49–3.46 (2H, m). 10% Pd/C (8.00 g) was added to a solu-
and purified by silica gel column chromatography (hexane/
AcOEt = 7:3) to provide the benzyl compound (465 mg, 99%) as a
1
tion of the imine (20.4 g, 94.8 mmol) in AcOEt (250 mL), and the
reaction mixture was stirred under H atmosphere at room tem-
perature for 4 h. The mixture was filtered through a Celite pad,
concentrated, and purified by silica gel column chromatography
colorless solid. H NMR (CDCl
3
) a mixture of conformers d: 7.67
2
(0.6H, d, J = 7.8 Hz), 7.40–7.32 (5.4H, m), 7.23–7.09 (2H, m),
6.93–6.77 (3H, m), 5.19 (0.4H, s), 5.03–4.99 (2.6H, m), 4.60 (1.2H,
s), 4.43 (0.8H, s), 3.91–3.89 (0.8H, m), 3.72–3.70 (3.2H, m), 3.46–
3.43 (0.8H, m), 3.25–3.22 (1.2H, m). 10% Pd/C (140 mg) was added
to a solution of the benzyl compound (465 mg, 1.05 mmol) in EtOH
(
MeOH/CH
2
Cl
2
= 1:8) to provide the diazepine 6 (16.9 g, 82%) as a
1
yellow oil. H NMR (CDCl ) d: 7.46 (1H, d, J = 7.4 Hz), 7.07 (1H, d,
3

9
8
T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
(
15 mL), and the reaction mixture was stirred under H
2
atmo-
5.1.15. tert-Butyl N-[2-[1-[2-chloro-6-(trifluoromethyl)-3-
pyridyl]but-3-enylamino]ethyl]carbamate (9)
sphere at room temperature for 4 h. The mixture was filtered
through a Celite pad and concentrated to provide the phenol com-
2-Chloro-6-(trifluoromethyl)pyridine-3-carbaldehyde 8 (11.96 g,
57.1 mmol) was added to a solution of N-Boc ethylenediamine
1
pound (361 mg, 98%) as a colorless solid. H NMR (CDCl
3
) a mixture
of conformers d: 7.66 (0.6H, d, J = 7.4 Hz), 7.21–7.14 (1.4H, m),
.08–6.97 (1H, m), 6.74–6.70 (3H, m), 5.26 (0.4H, br s), 5.04
0.6H, br s), 4.60 (1.2H, s), 4.49 (0.8H, s), 3.91–3.88 (0.8H, m),
2 4 2 2
(5.83 mL, 58.2 mmol) and Na SO (50 g) in CH Cl (400 mL) at
room temperature, and the reaction mixture was stirred for 17 h.
After the reaction mixture was concentrated, the residue was puri-
7
(
3
3
.77–3.74 (1.2H, m), 3.70–3.68 (2H, m), 3.46–3.43 (0.8H, m),
.31–3.27 (1.2H, m). The phenol compound (60 mg, 0.17 mmol)
fied by silica gel column chromatography (hexane/AcOEt = 3:2) to
1
provide the imine (13.87 g, 69%) as a yellow oil. H NMR (CDCl
3
)
was dissolved in a mixture of 2-iodopropane (34
and K CO (35 mg, 0.26 mmol) in DMF (2 mL), and the reaction
mixture was stirred at 80 °C for 5 h. The mixture was diluted with
Et O (30 mL), washed with brine, dried (Na SO ), concentrated and
purified by silica gel column chromatography (MeOH/CH Cl
:9). The obtained solid was triturated in hexane/Et O, and filtered
to provide 7e (28 mg, 42%) as a colorless solid. H NMR (CDCl ) a
mixture of conformers d: 7.68 (0.6H, d, J = 7.4 Hz), 7.17–7.13
l
L, 0.34 mmol)
d: 8.67 (1H, s), 8.53 (1H, d, J = 7.8 Hz), 7.68 (1H, d, J = 7.8 Hz),
4.79 (1H, br s), 3.83 (2H, t, J = 5.5 Hz), 3.50 (2H, q, J = 5.7 Hz),
2
3
1.44 (9H, s). A solution of allylmagnesium bromide in Et
(0.7 M, 60 mL, 42 mmol) was added to a solution of the imine
(13.44 g, 38.2 mmol) in THF (150 mL) under N atmosphere at
0 °C, and the mixture was stirred at the same temperature for
45 min. Then, the reaction mixture was diluted with
(200 mL), extracted with AcOEt (400 mL), washed with brine, dried
(Na SO ), concentrated. The residue was purified by silica gel col-
umn chromatography (hexane/AcOEt = 7:3) to provide 9 (10.91 g,
2
O
2
2
4
2
2
=
2
1
2
1
3
2
H O
(
(
1.4H, m), 7.10 (0.6H, d, J = 7.4 Hz), 6.95 (0.4H, d, J = 7.4 Hz), 6.73
3H, m), 5.20 (0.4H, br s), 4.99 (0.6H, br s), 4.61 (1.2H, s), 4.48–
2
4
1
4
.45 (1.8H, m), 3.93–3.90 (0.8H, m), 3.75–3.72 (1.2H, m), 3.72
3
73%) as a yellow oil. H NMR (CDCl ) d: 8.12 (1H, d, J = 8.2 Hz),
(
1
3
0.8H, s), 3.70 (1.2H, s), 3.47–3.44 (0.8H, m), 3.21–3.18 (1.2H, m),
7.64 (1H, d, J = 7.8 Hz), 5.82–5.71 (1H, m), 5.17–5.11 (2H, m),
4.72 (1H, br s), 4.21–4.10 (2H, m), 3.21–3.14 (2H, m), 2.65–2.59
(1H, m), 2.57–2.52 (1H, m), 2.47–2.41 (1H, m), 2.24–2.16 (1H,
m), 1.44 (9H, s).
+
.29 (2.4H, d, J = 5.9 Hz), 1.25 (3.6H, d, J = 5.9 Hz). MS (ESI ) m/z:
+
94 ((M+H) .
5
.1.12. 2-(3-Butoxyphenyl)-1-[8-(trifluoromethyl)-1,2,3,5-
tetrahydropyrido[2,3-e][1,4]diazepin-4-yl]ethanone (7f)
The phenol compound obtained in the procedure of 7e (60 mg,
5.1.16. tert-Butyl 5-allyl-8-(trifluoromethyl)-1,2,3,5-
tetrahydropyrido[2,3-e][1,4]diazepine-4-carboxylate (10)
Compound 9 (12.22 g, 31.0 mmol) was dissolved in 4 N HCl/
AcOEt (50 mL) at room temperature, and the reaction mixture
was stirred for 5 h. The mixture was concentrated and triturated
0
.17 mmol) was dissolved in a mixture of 1-iodobutane (29
lL,
0
.16 mmol) and K CO (35 mg, 0.26 mmol) in DMF (2 mL), and
2
3
the reaction mixture was stirred at 80 °C for 3 h. The mixture
was diluted with Et O (30 mL), washed with brine, dried (Na SO ),
concentrated and purified by silica gel column chromatography
hexane/AcOEt = 7:3). The obtained solid was triturated in hex-
ane/Et O, and filtered to provide 7f (58 mg, 83%) as a colorless
2
2
4
in (i-Pr)
the obtained solid was washed with (i-Pr)
to provide the diamine HCl salt (10.3 g). The obtained diamine
HCl salt (10.3 g) was added to a solution of (i-Pr) EtN (15.0 mL,
2
O (100 mL). Then, after the suspension was filtered off,
2
O and dried in vacuo
(
2
2
1
solid. H NMR (CDCl
3
) a mixture of conformers d: 7.67 (0.6H, d,
82.2 mmol) in NMP (100 mL) at 160 °C, and the reaction mixture
J = 7.8 Hz), 7.19 (1.4H, t, J = 7.8 Hz), 7.10 (0.6H, d, J = 7.3 Hz), 6.95
was stirred for 8 h. After the reaction mixture was diluted with
(
0.4H, d, J = 7.8 Hz), 6.77–6.71 (3H, m), 5.20 (0.4H, br s), 5.00
2
AcOEt/Et O (150 mL/150 mL), washed with brine (300 mL), dried
(
0.6H, br s), 4.61 (1.2H, s), 4.47–4.44 (0.8H, s), 3.95–3.81 (2.8H,
(Na SO ), and concentrated. The residue was purified by silica gel
2
4
m), 3.76–3.73 (1.2H, m), 3.72 (0.8H, s), 3.70 (1.2H, s), 3.46–3.44
0.8H, m), 3.24–3.21 (1.2H, m), 1.76–1.68 (2H, m), 1.50–1.41 (2H,
m), 0.98–0.94 (3H, m). MS (ESI ) m/z: 408 ((M+H) .
column chromatography (hexane/AcOEt = 3:7) to provide the dia-
zepine including NMP (11.8 g) as a brown oil. Di-tert-butyl dicar-
bonate (7.98 g, 36.5 mmol) was added to a solution of the
diazepine including NMP (11.8 g) and triethylamine (12.7 mL,
(
+
+
5
.1.13. 2-(3-Isobutoxyphenyl)-1-[8-(trifluoromethyl)-1,2,3,5-
91.4 mmol) in CH
tion mixture was stirred for 15 h. Then, the reaction mixture was
diluted with H O (200 mL), extracted with Et
O (200 mL Â 2),
washed with brine, dried (Na SO ), concentrated and purified by
silica gel column chromatography (hexane/AcOEt = 4:1) to provide
2 2
Cl (80 mL) at room temperature, and the reac-
tetrahydropyrido[2,3-e][1,4]diazepin-4-yl]ethanone (7g)
Compound 7g was prepared in a similar manner described for
2
2
1
7
f. Yield: 31%. H NMR (CDCl
3
) a mixture of conformers d: 7.67
2
4
(
6
(
3
0.6H, d, J = 7.4 Hz), 7.22–7.17 (1.4H, m), 7.10 (0.6H, d, J = 7.4 Hz),
.97 (0.4H, d, J = 7.4 Hz), 6.78–6.72 (3H, m), 5.20 (0.4H, br s), 5.01
0.6H, br s), 4.61 (1.2H, s), 4.48 (0.8H, s), 3.92–3.90 (0.8H, m),
.75–3.73 (1.2H, m), 3.72 (0.8H, s), 3.70 (1.2H, s), 3.52–3.50 (2H,
m), 3.48–3.44 (0.8H, m), 3.26–3.22 (1.2H, m), 2.02 (1H, m), 1.00
1
3
10 (6.53 g, 59%, 3 steps) as a yellow oil. H NMR (CDCl ) a mixture
of conformers d: 7.56 (0.5H, d, J = 7.4 Hz), 7.44 (0.5H, d, J = 7.4 Hz),
7.10 (1H, d, J = 7.4 Hz), 5.68–5.59 (1H, m), 5.33 (0.5H, br s), 5.08–
4.99 (3.5H, m), 4.14–4.00 (1H, m), 3.38–3.31 (2H, m), 3.19–3.08
(1H, m), 2.86–2.72 (1H, m), 2.53–2.46 (1H, m), 1.45 (9H, s).
+
+
(
6H, d, J = 8.2 Hz). MS (ESI ) m/z: 408 ((M+H) .
5
.1.14. 2-[3-(2,2-Dimethylpropoxy)phenyl]-1-[8-
5.1.17. 2-[4-[2-(3-Phenoxyphenyl)acetyl]-8-(trifluoromethyl)-1,
(
trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-5-yl]acetic acid (11)
4
-yl]ethanone (7h)
A mixture of 10 (550 mg, 1.54 mmol), 2.5% OsO
4
in i-PrOH
Compound 7h was prepared in a similar manner described for
(20 20 mol), and N-methylmorpholine oxide (360 mg.
3.08 mmol) in acetone (15 mL) was stirred at room temperature
l
L
l
1
7
3
f. Yield: 39%. H NMR (CDCl ) a mixture of conformers d: 7.67
(
6
(
3
0.6H, d, J = 7.4 Hz), 7.22–7.17 (1.4H, m), 7.10 (0.6H, d, J = 7.4 Hz),
.97 (0.4H, d, J = 7.4 Hz), 6.78–6.72 (3H, m), 5.20 (0.4H, br s), 5.01
0.6H, br s), 4.61 (1.2H, s), 4.48 (0.8H, s), 3.93–3.91 (0.8H, m),
.76–3.74 (1.2H, m), 3.72 (0.8H, s), 3.70 (1.2H, s), 3.52–3.50 (2H,
m), 3.48–3.44 (0.8H, m), 3.26–3.22 (1.2H, m), 1.01 (3.6H, s), 0.99
for 18 h. The mixture was diluted with AcOEt (30 mL), washed with
brine, dried (Na
(600 mg). A mixture of the diol (600 mg), sodium periodate
(491 mg. 2.30 mmol) in THF/H O (9 mL/3 mL) was stirred at room
temperature for 2 h. The mixture was diluted with AcOEt (30 mL),
washed with brine, dried (Na SO ), concentrated in vacuo to
2 4
SO ), concentrated to provide the diol as brown oil
2
+
+
(
5.4H, s). MS (ESI ) m/z: 422 ((M+H) .
2
4

T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
99
provide the aldehyde as colorless oil (495 mg). A mixture of the
obtained aldehyde, NaH PO O (2.15 g, 13.8 mmol), NaClO
312 mg, 3.45 mmol), 2-methyl-2-butene (7.33 mL, 69.0 mmol) in
The organic layer was washed with saturated NaHCO
dried (Na SO
triturated in Et
11 as colorless solid (47 mg, 88%), [
(S)-11 as colorless solid (54 mg, 97%), [
The absolute configurations were determined by the correspon-
dence of the oprical rotation of (R)-11 obtained from the stereospe-
cific route in Scheme 3.
3
aq and brine,
2
4
Á2H
2
2
2
4
) and concentrated in vacuo. The obtained residue was
O/hexane, filtered and dried in vacuo to provide (R)-
(
2
2
1.0
t-BuOH/H
2
O (5 mL/5 mL) was stirred at room temperature for
a]
D
À43.5 (c 0.49, CHCl
3
), and
2
1.0
2
h. After concentration, the residue was diluted with AcOEt
SO ), concentrated in
a
]
D
+42.7 (c 0.48, CHCl ).
3
(
50 mL Â 2), washed with brine, dried (Na
2
4
vacuo to provide the carboxylic acid as brown oil (560 mg). A mix-
ture of the obtained carboxylic acid, iodomethane (1.82 mL,
2
.93 mmol), K
2 3
CO (303 mg, 2.20 mmol) in DMF (8 mL) was stirred
at 50 °C for 4 h. Then, the reaction mixture was diluted with H
2
O
5.1.19. N-[(1R)-1-[2-Chloro-6-(trifluoromethyl)-3-pyridyl]but-
3-enyl]-2-methylpropane-2-sulfinamide (12)
(
30 mL), extracted with Et
2
O (30 mL Â 2), washed with brine, dried
(
Na SO ), concentrated and purified by silica gel column chroma-
2
4
A mixture of 2-chloro-6-(trifluoromethyl)pyridine-3-carbalde-
tography (hexane/AcOEt = 2:1) to provide the methyl ester
4
hyde 8 (19.0 g, 90.8 mmol), Ti(OEt) (41.4 g, 182 mmol), and (S)-
1
(
460 mg, 77%, 4 steps) as a brown oil. H NMR (CDCl
of conformers d: 7.72–7.59 (1H, m), 7.13 (1H, d, J = 7.4 Hz), 5.76
0.4H, s), 5.57 (0.6H, s), 5.02–4.98 (1H, m), 4.12–3.99 (2H, m),
.63 (3H, s), 3.50–3.33 (2H, m), 3.18–2.99 (2H, m), 1.44 (9H, s).
The methyl ester (460 mg, 1.18 mmol) was dissolved in 4 N HCl/
AcOEt (5 mL) and stirred at room temperature for 2 h. (i-Pr)
30 mL) was added to the resulting suspension. The deposited
3
) a mixture
(À)-tert-butylsulfinamide (11.2 g, 92.6 mmol) in THF (200 mL)
was stirred at reflux for 5 h. The cooled reaction mixture was con-
centrated, and dissolved in AcOEt (500 mL). The resulting suspen-
sion was filtered through a Celite pad, and the filtrate was
concentrated and purified by column chromatography (hexane/
(
3
2
O
AcOEt = 4:1) to provide the imine (28.9 g, 99%) as a light yellow
1
(
solid. H NMR (CDCl
3
) d: 9.00 (1H, s), 8.53 (1H, d, J = 7.8 Hz), 7.73
HCl salt was separated by filtration and dried in vacuo to provide
the diamine HCl salt as colorless solid (395 mg, 92%). H NMR
(1H, d, J = 7.8 Hz), 1.29 (9H, s). A mixture of the imine (28.9 g,
90.0 mmol), allyl bromide (55.9 g, 0.46 mol), and indium (42.5 g,
0.37 mol) was vigorously stirred in saturated NaBr aq at room tem-
1
(
3
CD OD) d: 7.85 (1H, d, J = 7.8 Hz), 7.26 (1H, d, J = 7.8 Hz), 5.10
(
1H, t, J = 7.0 Hz), 3.72 (3H, s), 3.57–3.40 (4H, m), 3.24–3.13 (2H,
3
perature for 21 h. After addition of saturated NaHCO aq (500 mL),
m). A mixture of the diazepine (300 mg, 0.83 mmol), 2-(3-phen-
the reaction mixture was extracted with AcOEt (250 mL Â 3). The
oxyphenyl)acetic acid (236 mg, 1.04 mmol), (i-Pr)
.31 mmol), HATU (378 mg, 0.99 mmol) in CH Cl
2
EtN (0.60 mL,
(10 mL) was
organic layers were filtered through a Celite pad, and dried
3
2
2
(Na
2
SO
4
), concentrated and purified by column chromatography
N) to provide 12 as a colorless
solid (30.7 g, 94%). H NMR (CDCl ) d: 7.95 (1H, d, J = 7.8 Hz),
stirred at room temperature for 17 h. The mixture was diluted in
CH Cl (20 mL), washed with saturated NaHCO aq and brine, dried
SO ), concentrated and purified by column chromatography
AcOEt/hexane = 1:1) to provide the acylated methyl ester as color-
(hexane/AcOEt = 1:4 with 5% of Et
3
1
2
2
3
3
(
(
Na
2
4
7.64 (1H, d, J = 7.8 Hz), 5.79–5.68 (1H, m), 5.24 (2H, t,
J = 14.1 Hz), 5.03–4.99 (1H, m), 3.78 (1H, s), 2.79–2.73 (1H, m),
2.51–2.43 (1H, m), 1.24 (9H, s).
1
less oil (375 mg, 91%). H NMR (CDCl
3
) a mixture of conformers d:
7
7
5
.77 (0.6H, d, J = 7.8 Hz), 7.33–7.29 (2H, m), 7.25–7.23 (1.2H, m),
.16–7.07 (1.2H, m), 7.00–6.77 (6H, m), 6.17 (0.6H, t, J = 7.8 Hz),
.44 (0.4H, t, J = 7.6 Hz), 5.08 (0.4H, s), 4.92 (0.6H, s), 4.70 (0.6H,
5.1.20. tert-Butyl N-[2-[[(1R)-1-[2-chloro-6-(trifluoromethyl)-3-
pyridyl]but-3-enyl]amino]ethyl]carbamate (13)
d, J = 14.5 Hz), 4.04–3.90 (1H, m), 3.85–3.67 (2.4H, m), 3.64–3.60
3H, m), 3.40–3.28 (1H, m), 3.21–2.70 (3H, m). A mixture of the
acylated methyl ester (200 mg, 0.40 mmol), 2 N NaOH aq (4 mL)
in THF/MeOH (2 mL/2 mL) was stirred at room temperature for
The sulfinamide 12 (22.8 g, 64.3 mmol) was dissolved in 4 N
HCl/1,4-dioxane (100 mL) and stirred at room temperature for
2 h. After the reaction mixture was concentrated, the residue was
(
added to Et
2
O (300 mL). The resulting suspension was filtered,
2
h. The mixture was acidified with 2 N HCl aq and extracted with
AcOEt. The organic layer was washed with brine, dried (Na SO ),
concentrated and purified by silica gel column chromatography
MeOH/CH Cl = 1:12). The obtained solid was triturated in Et O/
hexane, filtered and dried in vacuo to provide 11 as colorless solid
and dried in vacuo to provide the amine HCl salt (15.1 g, 82%) as
a colorless solid. H NMR (CD OD) d: 8.24 (1H, d, J = 7.8 Hz), 7.97
3
(1H, d, J = 8.2 Hz), 5.83–5.72 (1H, m), 5.25 (1H, d, J = 5.1 Hz), 5.22
(1H, s), 4.93 (1H, t, J = 7.2 Hz), 2.81 (2H, t, J = 7.2 Hz). A mixture
of the amine HCl salt (15.1 g, 52.8 mmol), tert-butyl N-(2-oxoeth-
1
2
4
(
2
2
2
1
(
183 mg, 94%). H NMR (CDCl
3
) a mixture of conformers d: 7.77
yl)carbamate (12.6 g, 79.3 mmol), and NaBH(OAc)
95.1 mmol) in CH Cl (250 mL) was stirred at room temperature
for 4 h. After addition of saturated NaHCO aq (300 mL), the reac-
tion mixture was extracted with CH Cl
(250 mL Â 2). The organic
layers were washed with brine, dried (Na SO ), concentrated and
purified by column chromatography (hexane/AcOEt = 2:3) to pro-
3
(25.2 g,
(
0.6H, d, J = 7.4 Hz), 7.33–7.28 (2H, m), 7.25–7.21 (1.2H, m),
2
2
7
5
.15–7.06 (1.2H, m), 6.97–6.76 (6H, m), 6.16 (0.6H, t, J = 7.4 Hz),
.41 (0.4H, t, J = 7.6 Hz), 5.15 (0.4H, s), 5.02 (0.6H, s), 4.71 (0.6H,
3
2
2
d, J = 14.5 Hz), 3.97 (1H, m), 3.84–3.63 (2.4H, m), 3.40–3.27 (1H,
m), 3.21–2.70 (3H, m). MS (ESI ) m/z: 486 ((M+H) .
2
4
+
+
1
3
vide 13 as a colorless solid (20.8 g, quant.). H NMR (CDCl ) d
5
1
.1.18. 2-[(5R)-4-[2-(3-Phenoxyphenyl)acetyl]-8-(trifluoromethyl)-
,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-5-yl]acetic acid ((R)-11)
and 2-[(5S)-4-[2-(3-phenoxyphenyl)acetyl]-8-(trifluoromethyl)-
,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-5-yl]acetic acid
(S)-11)
Each enantiomer of the acylated methyl ester obtained in the
procedure of 11 (120 mg, 0.240 mmol) was separated by using
chiral HPLC: column, t of (R)-enantiomer = 6.56 min, (S)-enantio-
mer = 7.47 min; temperature, 27 °C, Chiral Pack IC (4.6 Â 2
0 mm); eluent, ethanol/hexane = 30/70; flow rate = 1.0 mL/min.
8.12 (1H, d, J = 7.8 Hz), 7.64 (1H, d, J = 8.2 Hz), 5.81–5.71 (1H, m),
5.13 (2H, t, J = 12.3 Hz), 4.72 (1H, br s), 4.21–4.17 (1H, m), 3.19
(2H, br s), 2.65–2.59 (1H, m), 2.57–2.51 (1H, m), 2.47–2.41 (1H,
m), 2.24–2.16 (1H, m), 2.05 (1H, s), 1.44 (9H, s).
1
(
5.1.21. tert-Butyl (5R)-5-allyl-8-(trifluoromethyl)-1,2,3,5-
tetrahydropyrido[2,3-e][1,4]diazepine-4-carboxylate ((R)-10)
Compound 13 (20.8 g, 52.8 mmol) was dissolved in 4 N HCl/
AcOEt (100 mL) and stirred at room temperature for 1 h. After
the reaction mixture was concentrated, the residue was dissolved
R
5
Then, the obtained (R)-enantiomer (55 mg, 0.11 mmol) and (S)-
enantiomer (56 mg, 0.11 mmol) was respectively dissolved in a
mixture of 2 N NaOH aq (2 mL), THF (1 mL), and MeOH (1 mL),
and the mixture was stirred at room temperature for 2 h. The
mixture was acidified with 2 N HCl aq and extracted with AcOEt.
in CH
dried (Na
raphy (MeOH/CH
solid (12.7 g). A mixture of the diamine (12.7 g, 43.3 mmol) and
(i-Pr) EtN (23.7 mL, 130 mmol) in N-methylpyrrodinone (250 mL)
2
Cl
2
(300 mL), washed with saturated NaHCO
SO ), concentrated and purified by column chromatog-
Cl = 1:12) to provide the diamine as colorless
3
aq and brine,
2
4
2
2
2

1
00
T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
was stirred at 160 °C for 8 h. Subsequently, di-tert-butyl dicar-
bonate (14.2 g, 65.0 mmol) was added to the cooled mixture.
After stirring at room temperature for 1 h. The mixture was
5.1.24. 2-[(5R)-4-[2-[3-(4-Fluorophenoxy)phenyl]acetyl]-8-
(trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
5-yl]acetic acid (15a)
diluted with Et
2
O, washed with brine, dried (Na
2
SO
4
), concen-
A mixture of 14a (120 mg, 0.32 mmol), 2-[3-(4-fluorophen-
trated and purified by column chromatography (AcOEt/hex-
oxy)phenyl]acetic acid (94 mg, 0.38 mmol), (i-Pr)
0.64 mmol), HATU (146 mg, 0.38 mmol) in CH Cl
red at room temperature overnight. The mixture was diluted in
CH Cl (20 mL), washed with saturated NaHCO aq and brine, dried
(Na SO ), concentrated and purified by column chromatography
(AcOEt/hexane = 1:1) to provide the acylated diazepine as colorless
2
EtN (0.116 mL,
(5 mL) was stir-
ane = 1:4) to provide (R)-10 as brown oil (11.1 g, 59%, 3 steps).
2
2
1
3
H NMR (CDCl ): a mixture of conformers d 7.56 (0.5H, d,
J = 7.4 Hz), 7.44 (0.5H, d, J = 7.4 Hz), 7.10 (1H, d, J = 7.4 Hz),
2
2
3
5
4
.68–5.59 (1H, m), 5.33 (0.5H, br s), 5.08–4.99 (3.5H, m), 4.14–
.00 (1H, m), 3.38–3.31 (2H, m), 3.19–3.08 (1H, m), 2.86–2.72
2
4
1
(
1H, m), 2.53–2.46 (1H, m), 1.45 (9H, s).
3
oil (165 mg, quant.). H NMR (CDCl ) a mixture of conformers d:
7
.77 (0.6H, d, J = 7.8 Hz), 7.24–7.15 (1.4H, m), 7.03–6.88 (5H, m),
5
1
.1.22. Methyl 2-[(5R)-8-(trifluoromethyl)-2,3,4,5-tetrahydro-
H-pyrido[2,3-e][1,4]diazepin-5-yl]acetate dihydrochloride
6.83–6.71 (3H, m), 6.16 (0.4H, t, J = 8.0 Hz), 5.43 (0.6H, t,
J = 7.4 Hz), 5.08 (0.4H, d, J = 5.5 Hz), 4.92 (0.6H, d, J = 4.3 Hz),
4.72–4.67 (0.6H, m), 4.03–3.90 (1H, m), 3.86–3.69 (2.4H, m),
3.65–3.61 (3H, m), 3.42–3.29 (1H, m), 3.20–2.70 (3H, m). A mixture
of the acylated diazepine (170 mg, 0.33 mmol), 2 N NaOH aq
(4 mL) in THF/MeOH (2 mL/2 mL) was stirred at room temperature
for 2 h. The mixture was acidified with 2 N HCl aq and extracted
with AcOEt. The organic layer was washed with brine, dried
(
14a)
A mixture of (R)-10 (11.1 g, 31.1 mmol), 2.5% OsO
8.00 mL, 0.79 mmol), and N-methylmorpholine oxide (4.74 g,
0.5 mmol) was dissolved in acetone/H O (90 mL/30 mL) at 0 °C,
then stirred at room temperature for 18 h. The mixture was
diluted with AcOEt (300 mL), washed with brine, dried (Na SO ),
concentrated and purified by column chromatography (MeOH/
CH Cl = 1:8) to provide the diol as brown oil (12.9 g). A mixture
of the diol (12.9 g), sodium periodate (13.3 g, 62.3 mmol) in THF/
O (150 mL/50 mL) was stirred at room temperature for 1.5 h.
The mixture was diluted with AcOEt (300 mL), washed with
brine, dried (Na SO ), concentrated in vacuo to provide the alde-
hyde as colorless oil (11.9 g). A mixture of the aldehyde (11.9 g),
NaH PO O (36.4 g, 234 mmol) and NaClO (7.04 g, 78 mmol),
-methyl-2-butene (33.1 mL, 311 mmol) in t-BuOH/H O (150 mL/
50 mL) was stirred at room temperature for 17 h. After concen-
4
in i-PrOH
(
4
2
2
4
(Na
triturated in Et
15a as colorless solid (160 mg, 97%) H NMR (CDCl
2
SO
4
) and concentrated in vacuo. The obtained residue was
2
2
2
O/hexane, filtered and dried in vacuo to provide
1
3
) a mixture of
H
2
conformers d: 7.77 (0.6H, d, J = 7.8 Hz), 7.23 (1H, d, J = 8.2 Hz),
7.16 (0.4H, d, J = 7.4 Hz), 7.03–6.89 (5H, m), 6.83–6.71 (3H, m),
6.16 (0.6H, t, J = 8.0 Hz), 5.43 (0.4H, t, J = 7.4 Hz), 5.08 (0.6H, d,
J = 5.5 Hz), 4.92 (0.4H, d, J = 5.5 Hz), 4.72–4.67 (0.6H, m), 4.03–
3.91 (1H, m), 3.85–3.61 (2.4H, m), 3.42–3.28 (1H, m), 3.21–2.70
2
4
2
4
Á2H
2
2
+
+
2
1
2
(3H, m). MS (ESI ) m/z: 504 ((M+H) .
tration, the residue was diluted with AcOEt (300 mL Â 2), washed
with brine, dried (Na SO ), concentrated in vacuo to provide the
carboxylic acid as brown oil (16.1 g). A mixture of the carboxylic
acid (15.2 g), 2.0 M (trimethylsilyl)diazomethane/hexane
18.0 mL, 36.0 mmol) in THF/MeOH (50 mL/50 mL) was stirred
5.1.25. 2-[(5R)-4-[2-(3-Isobutoxyphenyl)acetyl]-8-
(trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
5-yl]acetic acid (15b)
2
4
15b was prepared in a similar manner described for 15a. Yield:
1
(
72% (2 steps from 14a). H NMR (CDCl
3
): a mixture of conformers d
at 0 °C for 1 h. The mixture was concentrated and purified by
column chromatography (AcOEt/hexane = 1:3) to provide the
methyl ester as colorless oil (8.38 g). The methyl ester (8.82 g,
7.79 (0.6H, d, J = 7.4 Hz), 7.19–7.11 (1.2H, m), 6.91 (0.6H, d,
J = 7.4 Hz), 6.84 (0.6H, d, J = 7.4 Hz), 6.77–6.68 (3H, m), 6.19
(0.6H, t, J = 7.6 Hz), 5.44 (0.4H, t, J = 7.6 Hz), 5.14 (0.4H, br s), 4.98
(0.6H, br s), 4.78–4.63 (0.6H, m), 4.03–3.91 (1H, m), 3.87–3.82
(0.4H, m), 3.77–3.41 (4H, m), 3.25–2.73 (4H, m), 2.05–1.95 (1H,
2
2.7 mmol) was dissolved in 4 N HCl/1,4-dioxane (50 mL) and
stirred at room temperature for 1 h (i-Pr) O (300 mL) was added
2
+
+
À
to the resulting suspension. The deposited HCl salt was separated
m), 0.99–0.95 (6H, m). MS (ESI ) m/z: 466 ((M+H) . HRMS (ESI )
À
by filtration and dried in vacuo to provide 14a as colorless solid
m/z: 464.1793 (MÀH) (calcd for
23 26 3 4 3
C H N O F : 464.1797).
1
21.0
(
7.07 g, 64%, 5 steps). H NMR (CDCl
3
): a mixture of conformers d
[a]
D
À40.2 (c 1.00, CHCl
3
).
7
.85 (1H, d, J = 7.8 Hz), 7.26 (1H, d, J = 7.8 Hz), 5.10 (1H, t,
J = 7.0 Hz), 3.72 (3H, s), 3.66 (1H, s), 3.58–3.40 (4H, m), 3.25–
5.1.26. 2-[(5R)-4-[2-[3-[(6-Methyl-3-pyridyl)oxy]phenyl]acetyl]-
8-(trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
3
.22 (2H, m).
5
-yl]acetic acid (15c)
5
1
.1.23. Benzyl 2-[(5R)-8-(trifluoromethyl)-2,3,4,5-tetrahydro-
H-pyrido[2,3-e][1,4]diazepin-5-yl]acetate dihydrochloride
Compound 15c was prepared in a similar manner described for
1
15a. Yield: 69% (2 steps from 14a). H NMR (CD
3
OD) a mixture of
(
14b)
A mixture of the carboxylic acid obtained in the procedure of 14a
16.5 g, 36.0 mmol), benzyl bromide (5.56 mL, 46.8 mmol), K CO
7.47 g, 54.0 mmol) in acetone (250 mL) was stirred at 60 °C for
h. Then, the reaction mixture was filtered through a Celite pad,
concentrated, and purified by silica gel column chromatography
hexane/AcOEt = 1:1) to provide the benzyl ester (17.0 g) as a brown
oil. The benzyl ester (16.9 g) was dissolved in 4 N HCl/1,4-dioxane
80 mL) and stirred at room temperature for 0.5 h. Et O (300 mL)
conformers d: 8.13–8.09 (1H, m), 7.75 (0.5H, d, J = 7.3 Hz), 7.34–
7.24 (3H, m), 7.13 (0.5H, d, J = 7.3 Hz), 7.06–7.02 (1.5H, m), 6.90
(1.5H, d, J = 7.2 Hz), 6.81 (0.5H, d, J = 8.3 Hz), 6.70 (0.5H, s), 6.12
(0.5H, t, J = 7.6 Hz), 5.53 (0.5H, t, J = 7.3 Hz), 4.60–4.56 (0.5H, m),
4.16–4.13 (0.5H, m), 3.99–3.96 (0.5H, m), 3.91–3.78 (1.5H, m),
3.72–3.67 (0.5H, m), 3.50–3.36 (1.5H, m), 3.23–2.78 (4H, m),
(
(
7
2
3
+
+
À
(
2.52–2.48 (3H, m). MS (ESI ) m/z: 501 ((M+H) . HRMS (ESI ) m/z:
À
21.0
499.1597 (MÀH) (calcd for C25
23 4 4 3 D
H N O F : 499.1593). [a]
(
2
3
+32.9 (c 1.00, CHCl ).
was added to the resulting suspension. The deposited hydrochloride
salt was separated by filtration and dried in vacuo to provide
5.1.27. Benzyl 2-[(5R)-4-[2-(3-hydroxyphenyl)acetyl]-8-
(trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
5-yl]acetate (16)
A mixture of 14b (500 mg, 1.14 mmol), 2-(3-hydroxyphenyl)ace-
tic acid (208 mg, 1.37 mmol), DMT-MM (573 mg, 1.71 mmol) in
1
4b as colorless solid (12.8 g, 80%, 5 steps). 1H NMR (CD
3
OD)
d: 7.77 (1H, d, J = 7.8 Hz), 7.34–7.29 (5H, m), 7.16 (1H, d, J = 7.8 Hz),
.16 (2H, s), 5.11 (1H, t, J = 7.0 Hz), 3.56–3.40 (4H, m), 3.26–3.23
2H, m).
5
(

T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
101
+
+
MeOH (10 mL) was stirred at room temperature for 24 h. The
mixture was diluted in AcOEt (50 mL), washed with saturated
(1H, m), 1.07–1.03 (6H, m). MS (ESI ) m/z: 494 ((M+H) . HRMS
+
+
27 3 5 3
(ESI ) m/z: 494.1908 ((M+H) (calcd for C24H N O F : 494.1903).
2
1.0
NaHCO
3
aq and brine, dried (Na
2
SO
4
), concentrated and purified
[a
]
D
À21.6 (c 1.00, CHCl
3
).
by column chromatography (AcOEt/hexane = 1:1) to provide 16 as
1
colorless solid (441 mg, 77%). H NMR (CDCl
3
) a mixture of
5.1.31. 2-[(5R)-4-[2-(3-Pentanoyloxyphenyl)acetyl]-8-
(trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
5-yl]acetic acid (17d)
Compound 17d was prepared in a similar manner described for
17b. Yield: 96% (2 steps from 16). H NMR (CDCl
conformers d: 7.72 (0.6H, d, J = 7.6 Hz), 7.38–7.33 (3H, m),
7
6
.29–7.27 (1.4H, m), 7.17–7.07 (2H, m), 6.90 (0.6H, d, J = 7.6 Hz),
.81 (0.4H, d, J = 7.8 Hz), 6.73–6.60 (3H, m), 6.18 (0.6H,
1
t, J = 8.1 Hz), 5.89 (0.4H, s), 5.45–5.41 (1H, m), 5.18 (0.4H,
d, J = 4.9 Hz), 5.11–4.88 (3H, m), 4.60–4.57 (0.6H, m), 3.88–3.58
3
) a mixture of con-
formers d: 7.76 (0.6H, d, J = 7.6 Hz), 7.31–7.25 (1.4H, m), 7.15–6.88
(
3H, m), 3.27–2.77 (4H, m).
(4H, m), 6.25–6.23 (0.6H, m), 5.34–5.32 (0.4H, m), 5.19 (0.4H, br s),
5
.11 (0.6H, br s), 4.75–4.73 (0.4H, m), 4.02–3.63 (3.6H, m), 3.43–
5
1
.1.28. 2-[(5R)-4-[2-(3-Hydroxyphenyl)acetyl]-8-(trifluoromethyl)-
,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-5-yl]acetic acid (17a)
0% Pd/C (60 mg) was added to a solution of 16 (300 mg,
.60 mmol) in EtOH (5 mL), and the reaction mixture was stirred
atmosphere at room temperature for 5 h. The mixture
was filtered through a Celite pad, concentrated, and purified by
silica gel column chromatography (MeOH/CH Cl = 1:9) to provide
the diazepine 17a (213 mg, 87%) as a colorless solid. H NMR
CD OD) a mixture of conformers d: 7.77 (0.6H, d, J = 7.4 Hz),
.13–6.82 (2.4H, d, J = 7.8 Hz), 6.67–6.53 (3H, m), 6.16 (0.6H,
t, J = 7.8 Hz), 5.52 (0.4H, t, J = 7.4 Hz), 4.64–4.58 (0.4H, m),
3.27 (1.4H, m), 3.21–3.14 (1.4H, m), 3.05–2.97 (0.6H, m), 2.75–
2.68 (0.6H, m), 2.58–2.54 (2H, m), 1.74–1.72 (2H, m), 1.43 (2H, td,
J = 15.1, 7.3 Hz), 0.99–0.95 (3H, m). MS (ESI ) m/z: 494 ((M+H) .
+
+
1
0
under H
2
5.1.32. 2-[(5R)-4-[2-[3-(2-Cyclopropylacetyl)oxyphenyl]acetyl]-
8-(trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
5-yl]acetic acid (17e)
2
2
1
Compound 17e was prepared in a similar manner described for
1
(
7
3
17b. Yield: 57% (2 steps from 16). H NMR (CDCl
3
) a mixture of
conformers d: 7.77 (0.6H, d, J = 7.8 Hz), 7.32–7.20 (1.4H, m),
7.14–6.91 (4H, m), 6.24–6.22 (0.6H, m), 5.35–5.33 (0.4H, m), 5.20
(0.4H, br s), 5.11 (0.6H, br s), 4.77–4.75 (0.4H, m), 4.01–3.99
(0.6H, m), 3.89–3.88 (0.4H, m), 3.85–3.77 (2H, m), 3.68–3.62
(0.6H, m), 3.45–3.27 (1H, m), 3.23–3.00 (2H, m), 2.72 (0.4H, dd,
J = 15.3, 7.6 Hz), 2.56 (0.6H, dd, J = 15.7, 5.5 Hz), 2.48–2.46 (2H,
m), 1.20–1.10 (1H, m), 0.66–0.58 (2H, m), 0.29–0.24 (2H, m). MS
4
3
3
.10–4.06 (0.6H, m), 3.97–3.92 (0.4H, m), 3.85–3.81 (0.6H, m),
.75 (1H, s), 3.70–3.63 (0.4H, m), 3.45 (0.6H, dd, J = 13.9, 4.5 Hz),
.25–3.02 (2H, m), 2.95–2.76 (2H, m). MS (ESI ) m/z: 410 ((M+H) .
+
+
5
8
.1.29. 2-[(5R)-4-[2-[3-(2-Methylpropanoyloxy)phenyl]acetyl]-
-(trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]
+
+
(ESI ) m/z: 492 ((M+H) .
diazepin-5-yl]acetic acid (17b)
A mixture of 16 (100 mg, 0.20 mmol), 2-methylpropanoyl chlo-
5.1.33. 2-[(5R)-4-[2-[3-(3,3-Dimethylbutanoyloxy)phenyl]
acetyl]-8-(trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e]
[1,4]diazepin-5-yl]acetic acid (17f)
ride (256 mg, 0.24 mmol), Et
10 mL) was stirred at room temperature for 1.5 h. The mixture
was diluted in AcOEt (30 mL), washed with water and brine, dried
3 2 2
N (56 lL, 0.40 mmol) in CH Cl
(
Compound 17f was prepared in a similar manner described for
1
(
Na
2
SO
4
), concentrated and purified by column chromatography
17b. Yield: 46% (2 steps from 16). H NMR (CDCl
3
) a mixture of
(
AcOEt/hexane = 1:1) to provide the benzyl ester (114 mg, quant.).
H NMR (CDCl ) a mixture of conformers d: 7.69 (0.6H, d,
3
conformers d: 7.76 (0.6H, d, J = 7.8 Hz), 7.34–7.28 (1.4H, m),
7.15–6.89 (4H, m), 6.27–6.24 (0.6H, m), 5.32–5.30 (0.4H,m), 5.13
(0.4H, br s), 5.04 (0.6H, br s), 4.75–4.73 (0.4H,m), 4.01–3.97
(0.6H, m), 3.89–3.76 (2H, m), 3.64 (1H, t, J = 11.3 Hz), 3.41–3.04
(3H, m), 2.66 (0.6H, dd, J = 15.1, 6.1 Hz), 2.51 (0.4H, dd, J = 15.1,
1
J = 7.8 Hz), 7.37–7.28 (5.4H, m), 6.80–6.79 (5H, m), 6.18 (0.6H, t,
J = 7.8 Hz), 5.40 (0.4H, t, J = 7.4 Hz), 5.09–4.88 (3H, m), 4.72–4.69
(
m), 3.28–3.07 (2H, m), 2.99–2.71 (2H, m), 1.31–1.25 (6H, m). 10%
Pd/C (50 mg) was added to a solution of the benzyl ester
0.4H, m), 3.96–3.88 (0.6H, m), 3.79–3.59 (2H, m), 3.42–3.30 (2H,
+
4.9 Hz), 2.44 (2H, s), 1.13 (9H, d, J = 3.5 Hz). MS (ESI ) m/z: 508
+
((M+H) .
(
114 mg, 0.200 mmol) in EtOH (5 mL), and the reaction mixture
was stirred under H atmosphere at room temperature for 5 h.
The mixture was filtered through a Celite pad, concentrated, and
purified by silica gel column chromatography (MeOH/CH Cl
1:9). The obtained was triturated in Et O/hexane, filtered and
dried in vacuo to provide 17b (75 mg, 78%) as a colorless solid.
2
5.1.34. Isopropyl 3-[2-[(5R)-5-(2-benzyloxy-2-oxo-ethyl)-8-
(trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
4-yl]-2-oxo-ethyl]benzoate (18, R = i-Pr)
Compound 14b (229 mg, 0.52 mmol) was added to a solution of
2-(3-isopropoxycarbonylphenyl)acetic acid (110 mg, 0.48 mmol),
3
2
2
=
2
1
H NMR (CDCl
J = 7.6 Hz), 7.31–7.24 (1.4H, m), 7.15–6.88 (4H, m), 6.27–6.24
0.6H, m), 5.35–5.33 (0.4H, m), 5.18 (0.4H, br s), 5.09 (0.6H, br s),
.76–4.74 (0.4H, m), 4.01–3.99 (0.6H, m), 3.88 (0.4H, d,
J = 15.6 Hz), 3.74 (1H, s), 3.68–3.62 (0.6H, m), 3.43–3.28 (2H, m),
3
) a mixture of conformers d: 7.76 (0.6H, d,
HATU (217 mg, 0.58 mmol) and (i-Pr)
CH Cl (10 mL) at room temperature, and the reaction mixture
was stirred for 5 h. The mixture was diluted with CH Cl (20 mL),
washed with saturated NaHCO aq and brine, dried (Na SO ), con-
2
EtN (325 lL, 1.90 mmol) in
2
2
(
4
2
2
3
2
4
centrated and purified by silica gel column chromatography (hex-
3
2
.21–3.14 (1.6H, m), 3.03–3.01 (0.4H, m), 2.83–2.78 (1H, m),
.69–2.54 (1H, m), 1.32–1.28 (3.6H, m), 0.90–0.86 (2.4H, m). MS
ane/AcOEt = 5:2) to provide 18 (212 mg, 78%) as a colorless solid.
H NMR (CDCl ) a mixture of conformers d: 7.93–7.85 (1H, m),
3
1
+
+
(
ESI ) m/z: 480 ((M+H) .
7.80 (1H, s), 7.70 (0.6H, d, J = 7.4 Hz), 7.38–7.28 (7H, m), 7.07
0.4H, d, J = 7.8 Hz), 7.01–6.83 (1H, m), 6.18 (0.6H, t, J = 6.3 Hz),
(
5
.1.30. 2-[(5R)-4-[2-[3-(3-Methylbutanoyloxy)phenyl]acetyl]-8-
5.44 (0.4H, t, J = 7.0 Hz), 5.25–5.18 (1H, m), 5.09–4.99 (2H, m),
4.91–4.69 (1H, m), 3.95 (1H, s), 3.83–3.64 (2H, m), 3.42–2.79
(4H, m), 1.35–1.32 (6H, m).
(
trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
5
-yl]acetic acid (17c)
Compound 17c was prepared in a similar manner described for
1
7b. Yield: 71% (2 steps from 16). 1H NMR (CDCl
3
) a mixture of
5.1.35. 3-[2-[(5R)-5-(Carboxymethyl)-8-(trifluoromethyl)-
1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-4-yl]-2-oxo-
ethyl]benzoic acid (19a)
conformers d: 7.76 (0.6H, d, J = 7.8 Hz), 7.34–7.29 (1.4H, m),
.16–6.90 (4H, m), 6.28–6.24 (0.6H, m), 5.32–5.28 (0.4H, m), 5.12
7
(
(
0.6H, br s), 5.02 (0.4H, br s), 4.78–4.73 (0.4H, m), 3.99–3.60
3.6H, m), 3.40–2.60 (4H, m), 2.47–2.45 (2H, m), 2.28–2.18
A mixture of 18 (110 mg, 0.223 mmol), 2 N NaOH aq (0.4 mL) in
MeOH (2 mL) was stirred at room temperature for 6 h. The mixture

1
02
T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
was acidified with 2 N HCl aq, and concentrated in vacuo. The
aqueous phase was extracted with i-PrOH in CHCl (25%). The com-
bined organic extract was washed with brine, dried over Na SO
filtered and concentrated in vacuo. The residue was triturated
upon Et O in hexane, collected by filtration to afford 19a as color-
less solid (97 mg, quant.) H NMR (CDCl
d: 8.13–8.03 (1H, m), 8.00–7.95 (1.6H, m), 7.64–7.42 (2.4H, m),
2.81–2.73 (1H, m), 1.32–1.21 (1H, m), 0.88 (2H, t, J = 6.8 Hz),
0.38–0.33 (2H, m). MS (ESI ) m/z: 492 ((M+H) .
+
+
3
2
4
,
5.1.40. 2-[(5R)-4-[2-[3-(2,2-
2
Dimethylpropoxycarbonyl)phenyl]acetyl]-8-(trifluoromethyl)-
1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-5-yl]acetic acid
(19f)
1
3
) a mixture of conformers
7
5
.22–7.15 (1H, m), 6.56–6.51 (0.6H, m), 5.65 (0.4H, t, J = 6.7 Hz),
.20 (0.4H, br s), 5.07 (0.6H, br s), 4.71–4.68 (0.4H, m), 4.13–4.09
Compound 19f was prepared in a similar manner described for
1
19b. Yield: 75% (2 steps from 14b). H NMR (CDCl
3
) a mixture of
(
0.6H, m), 3.90–3.77 (2H, m), 3.60–3.37 (2H, m), 3.27–2.99 (2H,
conformers d: 7.93–7.90 (0.6H, m), 7.86–7.80 (2H, m), 7.42–7.30
(2H, m), 7.19–6.91 (1.4H, m), 6.37–6.33 (0.6H, m), 5.48–5.44
+
+
m), 2.86–2.60 (1H, m). MS (ESI ) m/z: 438 ((M+H) .
(
0.4H, m), 5.12 (0.4H, br s), 5.01 (0.6H,br s), 4.78–4.73 (0.4H, m),
5
(
5
.1.36. 2-[(5R)-4-[2-(3-Isopropoxycarbonylphenyl)acetyl]-8-
trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
-yl]acetic acid (19b)
0% Pd/C (60 mg) was added to a solution of the benzyl ester 18
212 mg, 0.372 mmol) in EtOH (5 mL), and the reaction mixture
was stirred under H atmosphere at room temperature for 2 h.
The mixture was filtered through a Celite pad, concentrated, and
purified by silica gel column chromatography (MeOH/CH Cl
:1). The obtained was triturated in Et O/hexane, filtered and dried
in vacuo to provide 19b (174 mg, 98%) as colorless solid. H NMR
CDCl ) a mixture of conformers d: 7.91–7.87 (0.6H, m), 7.85–
.79 (2H, m), 7.38–7.29 (2H, m), 7.15–6.88 (1.4H, m), 6.28 (0.6H,
t, J = 7.8 Hz), 5.46 (0.4H, t, J = 7.8 Hz), 5.28–5.17 (1.2H, m), 4.77–
.72 (0.4H, m), 4.08–3.99 (0.4H, m), 3.82 (2H, s), 3.76–3.64 (1H,
m), 3.45–3.03 (3H, m), 2.84–2.72 (1H, m), 1.37–1.28 (4H, m),
4.08–3.96 (1.6H, m), 3.84 (2H, s), 3.67–3.64 (2H, m), 3.41–3.35
(1H, m), 3.26–3.17 (1H, m), 3.12–3.06 (1H, m), 2.75–2.69 (1H,
m), 1.04–1.02 (9H, m). MS (ESI ) m/z: 508 ((M+H) .
+
+
1
(
5.2. Biological assays
2
5.2.1. In vitro BRS-3 agonist activity
2
2
=
The 384-well IP-One HTRFÒ Assay (Cisbio, Bedford, MA) was
performed as described by the manufacturer’s protocol. CHO-K1
stably expressing human and mouse BRS-3 was plated at 200,000
9
2
1
(
7
3
cells per well in 50
at 37 °C for 24 h. On the next day, the media were removed and
25 L of compounds diluted in HAM containing 0.1% BSA and
2
lL HAM and incubated in the CO incubator
l
4
20 mM LiCl were added to each well and serial diluted IP1 stan-
dards (Cisbio) were also added to corresponding wells for this step.
After the cells were incubated for 1 h in the CO incubator at 37 °C,
2
+
+
0
.90–0.87 (3H, m). MS (ESI ) m/z: 480 ((M+H) .
all the media were removed again and d2-labeled IP1 and cryptate-
labeled anti-IP1 monoclonal antibody diluted in lysis buffer were
added sequentially. The assay plates were kept in the dark at
4 °C, overnight. Ratiometric measurements of fluorescence emis-
sion at 665 nm and 620 nm were obtained using a RUBY star fluo-
rometer (BMG Labtech, Ortenberg, Germany). IP1 levels in each
well were calculated according to the standard curves on each
plate. EC50 values were obtained by fitting data to a nonlinear
curve-fitting program (GraphPad Software, Inc., La Jolla CA).
5
.1.37. 2-[(5R)-4-[2-(3-Isobutoxycarbonylphenyl)acetyl]-8-
(
trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
5
-yl]acetic acid (19c)
Compound 19c was prepared in a similar manner described for
1
9b. Yield: 75% (2 steps from 14b). 1H NMR (CDCl
3
) a mixture of
conformers d: 7.93–7.89 (0.6H, m), 7.86–7.80 (2H, m), 7.41–7.29
2H, m), 7.19–6.89 (1.4H, m, 6.35–6.31 (0.6H, m), 5.46 (0.4H, t,
(
J = 7.4 Hz), 5.15 (0.4H, br s), 5.06 (0.6H, br s), 4.78–4.73 (0.4H,
m), 4.13 (1.6H, d, J = 6.3 Hz), 4.08–4.02 (1H, m), 3.83 (2H, s),
3
2
.68–3.66 (1H, m), 3.40–3.34 (1H, m), 3.25–3.04 (2H, m), 2.76–
5.2.2. Food intake evaluation in mice
+
.74 (1H, m), 2.12–2.02 (1H, m), 1.02–0.99 (6H, m). MS (ESI ) m/
Male C57BL/6N mice 7 weeks of age were purchased from
Charles River Laboratories (Kanagawa, Japan). All animals were
held under standard laboratory conditions (12 h light per day, light
on at 7:00 AM, 23 ± 2 °C, 55 ± 10% humidity) with food and water
available ad libitum. On the day of testing, mice had been fasted
for 16 h were weighed and placed individually in cages. After 1 h
habituation to the new environment, mice were orally adminis-
tered vehicle (0.5% methylcellulose) or compounds. One hour after
compound administration, pre-weighed normal chow diet (FR2,
purchased from Funabashi farm) was fed to the mice. Cumulative
food intake of each mouse over 6 h was measured. All experimen-
tal procedures were reviewed and approved by the Institutional
Animal Care and Use Committee at Daiichi Sankyo. Statistical anal-
ysis using Dunnett’s test was performed to compare the vehicle-
treated and compound-administered groups. P values <0.05 were
considered significant.
+
z: 494 ((M+H) .
5
.1.38. 2-[(5R)-4-[2-(3-Butoxycarbonylphenyl)acetyl]-8-
(
trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
5
-yl]acetic acid (19d)
Compound 19d was prepared in a similar manner described for
9b. Yield: 80% (2 steps from 14b). 1H NMR (CDCl
1
3
) a mixture of
conformers d: 7.91–7.89 (0.6H, m), 7.87–7.81 (2H, m), 7.41–7.29
2H, m), 7.17–6.91 (1.4H, m), 6.36–6.32 (0.6H, m), 5.46 (0.4H, t,
(
J = 7.4 Hz), 5.12 (0.4H, br s), 5.06 (0.6H, br s), 4.78–4.74 (0.4H,
m), 4.08–3.98 (0.4H, m), 3.83 (2H, s), 3.68–3.65 (1H, m), 3.39–
3
.35 (1H, m), 3.25–3.04 (2H, m), 2.78–2.72 (1H, m), 1.78–1.63
(
2H, m), 1.35–1.33 (2H, m), 1.32–1.16 (2H, m), 0.99–0.88 (3H,
+
+
m). MS (ESI ) m/z: 494 ((M+H) .
5
.1.39. 2-[(5R)-4-[2-[3-
5.3. Pharmacokinetic evaluation in mice
(
(
5
Cyclopropylmethoxycarbonyl)phenyl]acetyl]-8-
trifluoromethyl)-1,2,3,5-tetrahydropyrido[2,3-e][1,4]diazepin-
-yl]acetic acid (19e)
Male C57BL/6N mice were purchased at 5 weeks old from
Charles River Laboratories Japan, Inc (Kanagawa, Japan). For accli-
mation, they were housed in stainless steel cages for 7–11 days
in the controlled animal area. The mice were allowed free access
to FR2 laboratory food (Funabashi Farm Co., Ltd) and tap water.
Compound 17b–17f and 19b–19f were suspended in a 0.5 (w/v)
% methyl cellulose 400 solution (Wako pure chemical industries,
Osaka, Japan) for oral administration. For the administration,
Compound 19e was prepared in a similar manner described for
1
9b. Yield: 74% (2 steps from 14b). 1H NMR (CDCl
3
) a mixture of
conformers d: 7.96–7.93 (0.6H, m), 7.89–7.80 (2H, m), 7.41–7.31
2H, m), 7.16–6.90 (1.4H, m), 6.30 (0.6H, t, J = 7.4 Hz), 5.46 (0.4H,
(
t, J = 7.4 Hz), 5.20 (0.4H, br s), 5.10 (0.6H, br s), 4.78–4.72 (0.4H,
m), 4.17–4.12 (2H, m), 4.08–3.98 (1.6H, m), 3.83–3.05 (5H, m),

T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
103
2
mL/kg of the solution at a concentration of 5 mg/mL was used.
51, 305; (e) Allison, D. B.; Gadde, K. M.; Garvey, W. T.; Peterson, C. A.; Schwiers,
M. L.; Najarian, T.; Tam, P. Y.; Troupin, B.; Day, W. W. Obesity 2012, 20, 330; (f)
Davenport, R. J.; Wright, S. Drug Discovery Today 2014, 19, 845.
. Bays, H. E. Obes. Res. 2004, 12, 1197.
5. For example, a cannabinoid-1 receptor (CB1R) inverse agonist, rimonabant was
withdrawn from the US market due to serious psychiatric effects in 2007. Also,
The solutions of compounds were administered to male C57BL/
6
0
N mice, after overnight fasting. A blood sample of approximately
.2 mL was collected from the jugular vein with a heparinized syr-
4
inge. The blood was centrifuged at 14,000 rpm for 3 min at 4 °C
himac CR15D, Hitachi Koki Co., Ltd rotor: RT15A2) to obtain the
a
serotonin-norepinephrine reuptake inhibitor (SNRI), sibtramine once
(
approved by FDA in 1997, was reported to increase cardiovascular events
and strokes, then withdrawn in 2010. See details below: (a) Fong, T. M.;
Heymsfield, S. B. Int. J. Obes. 2009, 33, 947; (b) Faulconbridge, L.; Wadden, T. A.;
Berkowitz, R. I.; Sarwer, D. B.; Womble, L. G.; Hesson, L. A.; Stunkard, A. J.;
Fabriatore, A. N. Obesity 2009, 1009, 17; (c) Warren, K. R.; Buchanan, R. W.;
Feldman, S.; Conley, R. R.; Linthicum, J.; Ball, M. P.; Liu, F.; McMahon, R. P.;
Gorelick, D. A.; Huestis, M. A.; Kelly, D. L. J. Clin. Psycophamacol. 2013, 33, 118;
plasma. The plasma was stored frozen at À20 °C until use for mea-
surement of plasma concentration. The determination of the
plasma concentration was performed by LC–MS/MS method using
API 4000QTRAP (Applied Biosystems/MDS SCIEX). PK parameters
were calculated by a non-compartmental model.
(
d) James, W. P. T.; Caterson, I. D.; Coutinho, W.; Finer, N.; Gaal, L. F. V.;
Maggioni, A. P.; Torp-P, C.; Sharma, A. M.; Shepherd, G. M.; Rode, R. A.; Renz, C.
L. N. Eng. J. Med. 2010, 363, 905.
(a) Ohki-Hamazaki, H.; Wada, E.; Matsui, K.; Wada, K. Brain Res. 1997, 762, 165;
5
.4. Safety evaluation in dogs
6
7
.
.
(
b) Liu, J.; Lao, Z. J.; Zhang, J.; Schaeffer, M. T.; Jiang, M. M.; Guan, X. M.; Van der
Female beagle dogs were obtained from Nosan Corporation
Ploeg, L. H.; Fong, T. M. Biochemistry 2002, 41, 8954; (c) Porcher, C.; Juhem, A.;
Peinnequin, A.; Bonaz, B. Cell Tissue Res. 2005, 320, 21; (d) Feng, Y.; Guan, X.-M.;
Li, J.; Metzger, J. M.; Zhu, Y.; Juhl, K.; Zhang, B. B.; Thornberry, N. A.; Reitman, M.
L.; Zhou, Y.-P. Endocrinology 2011, 152, 4106.
(a) Ohki, H.; Watase, K.; Yamamoto, K.; Ogura, H.; Yamano, M.; Yamada, K.;
Maeno, H.; Imaki, J.; Kikuyama, S.; Wada, E.; Wada, K. Nature 1997, 390, 165;
(b) Ladenheim, E. E.; Hamilton, N. L.; Behles, R. R.; Bi, S.; Hampton, L. L.; Battey,
J. F.; Moran, T. H. Endocrinology 2008, 149, 971. (c) Metzger, J. M.; Gagen, K.;
Raustad, K. A.; Yang, L.; White, A.; Wang, S. –P.; Craw, S.; Liu, P.; Lanza, T.; Lin, L.
S.; Nargund, R. P.; Guan, X. –M.; Strack, A. M.; Reitman, M. L. Am. J. Physiol.
Endocrinol. Metab. 2010, 299, E816.
BRS-3 was reported to be expressed by the enteric nervous system and by
interstitial cells of Cajal in the rat GI tract (see Ref. 6c). Also, there are some
papers reporting that continuous vagal neuromodulation on GI function in rats
caused food-intake suppression and a similar effect was observed in human.
See details below: (a) Krolczyk, D. G.; Zurowski, J.; Sobocki, J.; Slowiaczek, M.
P.; Laskiewicz, J.; Matyja, A.; Zaraska, K.; Thor, P. J. J. Physiol. Pharmacol. 2001,
(
2
Kanagawa, Japan). Animals were housed in a temperature—(22–
5 °C) and humidity controlled (35–75%) environment with a 12-h
light/dark cycle. All experimental procedures were performed in
accordance with the in-house guidelines of the Institutional Ani-
mal Care and Use Committee of Daiichi Sankyo Co., Ltd, Tokyo,
Japan. Test compounds were dispersed in 20% v/w hydroxypro-
pyl-b-cyclodextrin solution, at the concentration of 1 mg/mL. Dogs
were anesthetized with halothane (0.5–1.5% end-tidal concentra-
tion) proceeded by intravenous injection of thiopental (30 mg/kg
of body weight). Two catheters were placed in the femoral artery
and vein, for blood pressure recording and test substance adminis-
tration, respectively. Heart rate and mean blood pressure were
analyzed by the hemodynamic analysis software (SBP2000, Softron
Co., Ltd, Tokyo). After the recording of pre- administration value,
test substance was administered via the catheter in the femoral
vein with syringe driver, at 3 mg/kg/3 mL for 30 min (adjusted by
infusion speed). Blood pressure was recorded for 60 min from the
administration start, except for the one animal (#2, 15b
administered).
8
9
.
.
5
2, 705; (b) Pardo, J. V.; Sheikh, S. A.; Kuskowski, M. A.; Surerus, J. C.; Hagen, M.
C.; Lee, J. T.; Rittberg, B. R.; Adson, D. E. Int. J. Obes. 2007, 31, 1756.
(a) Sebhat, I. K.; Franklin, C.; Lo, M. M.-C.; Chen, D.; Jewell, J. P.; Miller, R.; Pang,
J.; Palyha, O.; Kan, Y.; Kelly, T. M.; Guan, X.-M.; Marsh, D. J.; Kosinski, J. A.;
Metzger, J. M.; Lyons, K.; Dragovic, J.; Guzzo, P. R.; Henderson, A. J.; Reitman, M.
L.; Nargund, R. P.; Wyvratt, M. J.; Lin, L. S. ACS Med. Chem. Lett. 2011, 2, 43; (b)
Guan, X.-M.; Metzger, J. M.; Yang, L.; Raustad, K. A.; Wang, S.-P.; Spann, S. K.;
Kosinski, J. A.; Yu, H.; Shearman, L. P.; Faidley, T. D.; Palyha, O.; Kan, Y.; Kelly, T.
M.; Sebhat, I.; Lin, L. S.; Dragovic, J.; Lyons, K. A.; Craw, S.; Nargund, R. P.;
Marsh, D. J.; Strack, A. M.; Reitman, M. L. J. Pharmacol. Exp. Ther. 2011, 336, 356;
(c) Lateef, D. M.; Abreu-Vieira, G.; Xiao, C.; Reitman, M. L. Am. J. Physiol.
Endocrinol. Metab. 2014, 306, E681.
5
.5. Computational calculation for the conformational analysis
1
1
0. Reitman, M. L.; Dishy, V.; Moreau, A.; Denney, W. S.; Liu, C.; Kraft, W. K.; Mejia,
A. V.; Matson, M.; Stoch, S. A.; Wagner, J. A.; Lai, E. J. Clin. Pharmacol. 2012, 52,
1306.
3
D coordinates of compound (a–c) illustrated in Figure 5 were
generated using Program Ligprep and used for Conformational
Search with Macromodel (Schrodinger, LLC, New York, NY, 2010).
Relative energy
as the energy barrier for the N-atom inversion. E(R)/E(S) is defined
as the potential energy in OPLS-2005 forcefield of the lowest
energy conformation with (R)/(S) ring configuration.
1. (a) Shimizu, K.; Kawase, A.; Haneishi, T.; Kato, Y.; Kinoshita, K.; Ohmori, M.;
Furuta, Y.; Emura, T.; Kato, N.; Mitsui, T.; Yamaguchi, K.; Morita, K.; Sekiguchi,
N.; Yamamoto, T.; Matsushita, T.; Shimaoka, S.; Sugita, A.; Morikawa, K. Bioorg.
Med. Chem. Lett. 2006, 16, 3323; (b) Kurimoto, A.; Hashimoto, K.; Nakamura, T.;
Norimura, K.; Ogita, H.; Takaku, H.; Bonnert, R.; McInally, T.; Wada, H.; Isobe, Y.
J. Med. Chem. 2010, 53, 2964; (c) Procopiou, P. A.; Barrett, V. J.; Ford, A. J.;
Looker, B. E.; Lunniss, G. E.; Needham, D.; Smith, C. E.; Somers, G. Bioorg. Med.
Chem. 2011, 19, 6026; (d) Hasumi, K.; Sato, S.; Saito, T.; Kato, J.; Shirota, K.;
Sato, J.; Suzuki, H.; Ohta, S. Bioorg. Med. Chem. 2014, 22, 4162.
2. We confirmed a selective binding ability of some key compounds to human
BRS-3 by a radio-ligand receptor binding assay including other bombesin
receptors such as GRP (gastrin releasing peptide) receptor and NMB
(neuromedin B) receptor (data not shown).
3. (a) Liu, P.; Lanza, T. J., Jr.; Chioda, M.; Jones, C.; Chobanian, H. R.; Guo, Y.; Chang,
L.; Kelly, T. M.; Kan, Y.; Palyha, O.; Guan, X.-M.; Marsh, D. J.; Metzger, J. M.;
Ramsay, K.; Wang, S.-P.; Strack, A. M.; Miller, R.; Pang, J.; Lyons, K.; Dragovic, J.;
Ning, J. G.; Schafer, W. A.; Welch, C. J.; Gong, X.; Gao, Y.-D.; Hornak, V.; Ball, R.
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D
E = E(R) À E(S) was calculated in each compound
Acknowledgments
1
1
We thank the members of Daiichi Sankyo RD NOVARE Co., Ltd
for the high-resolution mass analysis and the measurement of
optical rotation.
Reference and notes
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.
(a) Obesity: Preventing and Managing the Global Epidemic, Report of a WHO
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138; (b) Guh, D. P.; Zhang, W.; Bansback, N.; Amarsi, Z.; Birmingham, C. L.;
Anis, A. H. BMC Public Health 2009, 9, 88.
1
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4. Kraus, G. A.; Roth, B. J. Org. Chem. 1980, 45, 4825.
(a) McNeely, W.; Benfield, P. Drugs 1998, 56, 241; (b) Torgerson, J. S.; Boldrin,
M. N.; Hauptman, J.; Sjostrom, L. Diabetes Care 2004, 27, 155; (c) Padwal, R. S.;
Majumdar, S. R. Lancet 2007, 369, 71; (d) Smith, B. M.; Smith, J. M.; Tsai, J. H.;
Schultz, J. A.; Gilson, C. A.; Estrada, S. A.; Chen, R. R.; Park, D. M.; Prieto, E. B.;
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Whelan, K. T.; Grottick, A. J.; Menzaghi, F.; Thomsen, W. J. J. Med. Chem. 2008,
5. Each enantiomer (R)-11 and (S)-11 was obtained by hydrolysis after chiral
HPLC separation of 11: column, Chiral Pack IC (4.6 Â 250 mm); eluent, ethanol/
hexane = 30/70. The ee value of the methyl esters of (R)-11 and (S)-11 were
determined by chiral HPLC analysis: column, Chiral Pack IC (4.6 Â 250 mm);
R R
flow rate 1.0 mL/min; temperature, 27 °C; t of (R)-enantiomer = 6.56 min; t
of (S)-enantiomer = 7.47 min. The retention time of the methyl ester of (R)-

1
04
T. Matsufuji et al. / Bioorg. Med. Chem. 23 (2015) 89–104
enantiomer ((R)-11) via chiral separation was well corresponded to the one
synthesized by the stereospecific route in Scheme 3.
6. (a) Liu, G.; Cogan, D. A.; Ellman, J. A. J. Am. Chem. Soc. 1997, 119, 9913; (b)
Ellman, J. A.; Owens, T. D.; Tang, T. P. Acc. Chem. Res. 2002, 35, 984.
carbon atom on the carbonyl group is designated as the pilot atom. Therefore,
compound A and B have an (S)-configuration, and compound C has an (R)-
configuration.
1
22. PK profile of 17a (po, 30 mg/kg in mice, 0.5% methyl cellulose as a solvent,
1
1
1
7. Sun, X.-W.; Liu, M.; Xu, M.-H.; Lin, G.-Q. Org. Lett. 2008, 10, 1259.
n = 3): Tmax = 0.42 hr, Cmax = 0.04 lM, T1/2 = 1.79 hr, AUC = 0.07 hr * lg/mL.
8. An undesired (S,S)-diastereomer was also obtained in 2.0% yield (30 g scale).
9. The ee value of the key intermediate (R)-10 was determined to 98.1% ee by
chiral HPLC analysis: column, Chiral Pack IA (4.6 Â 150 mm); eluent, ethanol/
23. (a) Doan, K. M. M.; Humphereys, J. E.; Webster, L. O.; Wring, S. A.; Shampine, L.
J.; Serabjit-S, C. J.; Adkinson, K. K.; Polli, J. W. J. Pharmacol. Exp. Ther. 2002, 303,
1029; (b) Clark, D. E. Drug Discovery Today 2003, 8, 927.
isopropylalcohol = 50/50; flow rate, 1.0 mL/min; temperature, 27 °C; t
R
of (R)-
24. The brain to plasma concentration ratios (Kp brain) of 15b was fairly low
enantiomer = 3.56 min;
t
R
of (S)-enantiomer = 4.11 min. The absolute
(Kp = 0.043). Kp value was determined after a single administration of
configuration of (R)-10 was inferred by the reaction mechanism proposed by
the Ellman’s papers (see Ref. 16).
0. Kunishima, K.; Kawachi, K.; Hioki, K.; Terao, K.; Tani, S. Tetrahedron 2001, 57,
compound at 30 mg/kg po to mice. The measurement of the concentration
was conducted at 2.5 h after administration. See details below: Matsufuji, T.;
Shimada, K.; Kobayashi, S.; Kawamura, A.; Fujimoto, T.; Arita, T.; Hara, T.;
Konishi, M.; Abe, O. R.; Izumi, M.; Sogawa, Y.; Nagai, Y.; Yoshida, K.; Takahashi,
H. Bioorg. Med. Chem. Lett. 2014, 22, 750.
2
2
1
551.
1. The assignment of absolute stereochemistry is based on the Cahn-Ingold-
Prelog convention. The diazepine plane is regarded as a chiral plane, and a