Synthesis and optimization of novel (3S,5R)-5-(2,2-dimethyl-5-oxo-4- phenylpiperazin-1-yl)piperidine-3-carboxamides as orally active renin inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.06.057

We report synthesis and optimization of a series of (3S,5R)-5-(2,2- dimethyl-5-oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides as renin inhibitors. Chemical modification of P₁, P₂ and P ₃ portions led to a promising 3

Full text of DOI:10.1016/j.bmc.2013.06.057

Accepted Manuscript
Synthesis and optimization of novel (3S,5R)-5-(2,2-dimethyl-5-oxo-4-phenyl‐
piperazin-1-yl)piperidine-3-carboxamides as orally active renin inhibitors
Yutaka Mori, Yasuyuki Ogawa, Akiyoshi Mochizuki, Yuji Nakamura, Teppei
Fujimoto, Chie Sugita, Shojiro Miyazaki, Kazuhiko Tamaki, Takahiro
Nagayama, Yoko Nagai, Shin-ichi Inoue, Katsuyoshi Chiba, Takahide Nishi
PII:
S0968-0896(13)00597-X
http://dx.doi.org/10.1016/j.bmc.2013.06.057
BMC 10950
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
26 May 2013
22 June 2013
24 June 2013
Please cite this article as: Mori, Y., Ogawa, Y., Mochizuki, A., Nakamura, Y., Fujimoto, T., Sugita, C., Miyazaki,
S., Tamaki, K., Nagayama, T., Nagai, Y., Inoue, S-i., Chiba, K., Nishi, T., Synthesis and optimization of novel
(3S,5R)-5-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides as orally active renin inhibitors,
Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.06.057
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Synthesis and optimization of novel (3S,5R)-
5-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-
yl)piperidine-3-carboxamides as orally active
renin inhibitors
Leave this area blank for abstract info.
Yutaka Mori, Yasuyuki Ogawa, Akiyoshi Mochizuki, Yuji Nakamura, Teppei Fujimoto, Chie Sugita, Shojiro Miyazaki,
Kazuhiko Tamaki, Takahiro Nagayama, Yoko Nagai, Shin-ichi Inoue, Katsuyoshi Chiba, Takahide Nishi
fumaric acid
H
N
H
N
O
N
N
O
IC50 in purified human renin = 1.6 nM
IC50 in human plasma renin = 3.6 nM
N
32o
F

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Synthesis and optimization of novel (3S,5R)-5-(2,2-dimethyl-5-oxo-4-
phenylpiperazin-1-yl)piperidine-3-carboxamides as orally active renin inhibitors
Yutaka Moria, , Yasuyuki Ogawab, Akiyoshi Mochizukic, Yuji Nakamurac, Teppei Fujimotoc, Chie Sugitac,
Shojiro Miyazakic, Kazuhiko Tamakic, Takahiro Nagayamad, Yoko Nagaie, Shin-ichi Inouee, Katsuyoshi
Chibaf, Takahide Nishig
a New Modality Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Venture Science Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Medicinal Chemistry Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Cardiovascular-Metabolics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
e Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
f Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
g Daiichi Sankyo Life Science Research Center in India, Daiichi Sankyo India Pharma Pvt. Ltd, Village Sehroul, Sector 18, Udyog Vihar Industrial Area,
Gurgaon-122015, Haryana, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We report synthesis and optimization of a series of (3S,5R)-5-(2,2-dimethyl-5-oxo-4-
phenylpiperazin-1-yl)piperidine-3-carboxamides as renin inhibitors. Chemical modification of
P1’, P2’ and P3 portions led to a promising 3,5-disubstituted piperidine 32o showing high renin
inhibitory activity and favorable oral exposure in both rats and cynomolgus monkeys with
acceptable CYP and hERG current inhibition. Compound 32o exhibited a significant blood
pressure lowering effect by oral administration in two hypertensive animal models, double
transgenic rats and furosemide pretreated cynomolgus monkeys.
Available online
Keywords:
Hypertension
Renin
2009 Elsevier Ltd. All rights reserved.
Piperidine
Ketopiperazine
1. Introduction
Hypertension is a major risk factor for cardiovascular disease,
including chronic heart and kidney failure, myocardial infarction
and stroke, and is one of the leading causes of death in the
developed world.1 The renin-angiotensin-aldosterone system
(RAAS) plays an important role in the regulation of blood
pressure (BP) and fluid homeostasis.2 The inhibition of either the
formation or the action of angiotensin II (Ang II), the main
product of the RAAS, represents a major therapeutic approach in
the treatment of hypertension and the prevention of associated
comorbidities. It has long been hypothesized that inhibition of
renin, which is the rate-limiting enzyme in the RAAS cascade,
may represent the most attractive therapeutic strategy to block
the RAAS.3
During the 1980s, most of the renin inhibitors were based on
the modified peptides that incorporated peptide hydrolysis
transition state isosteres. However, because of their poor oral
bioavailability, none of these inhibitors were ultimately deemed
suitable for full development.4 Beginning in the middle of the
1990s, several novel nonpeptidic renin inhibitors were reported
and have entered human clinical trials, such as Aliskiren
hemifumarate (1)5, ACT-077825 (MK-8141) (2)6, and VTP-
27999 (3)7 (Figure 1). However, only Aliskiren has reached the
market for the treatment of essential hypertension.
———
Corresponding author. Tel.: +81 3 3492 3131; Fax: +81 3 5436 8563.
E-mail address: mori.yutaka.c7@daiichisankyo.co.jp (Y. Mori).

Figure 1. Chemical structures of Aliskiren hemifumarate (1), ACT-077825 (MK-8141) (2), and VTP-27999 (3).
0.5 fumaric acid
Aliskiren hemifumarate (
)
1
ACT-077825, MK-8141 (
)
2
VTP-27999 ( )
3
Figure 2. Chemical structures of originally designed renin inhibitors containing 2,2-dimethyl-4-phenylpiperazin-5-one.
P1'
P1'
H
N
O H
H
N
H2
N
N
O H
H
N
O
N
O
O
5
3
P2'
N
O
O
N
P1
7
fumaric acid
2
P1
P2'
fumaric acid
Cl
H O
・
2
Cl
P3
P3
DS-8108b (
)
4
5
IC50 in purified human renin = 0.9 nM
IC50 in monkey plasma renin = 6.3 nM
IC50 in purified human renin = 1.3 nM
IC50 in monkey plasma renin = 1.4 nM
10
(a)
(b)
(c), (d)
(e), (f)
6
8
9
11
-
13 15
(g)
(h)
(i), (j)
-
-
-
22 24
12
16 18
19 21
n HCl
(n = 0, 1, or 2)
,
-
28 29a n
(m), (n)
(k)
(l)
fumaric acid
-
,
-
, -
5 32a p
25 27
30 31a p
Scheme 1. Synthetic pathway leading to (3S,5R)-5-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides 5 and 32a-p. Reagents and
conditions: (a) 7, MeOH, THF, −20 °C, 1 day, 69%, 94% ee; (b) diphenylphosphoryl azide, Et3N, toluene, benzyl alcohol, 90 °C, 5 h, 96%; (c) H2, Pd on carbon,
MeOH, rt, 1.5 h; (d) 2-bromo-2-methylpropanal 10, DMF, water, rt, 1 day, 73% (2 steps); (e) 4N-HCl in 1,4-dioxane, rt, 0.5 h; (f) NsCl, NaHCO3, 1,4-dioxane,
water, 0 °C, 1.5 h, 86% (2 steps); (g) aniline 13-15, AcOH, toluene, 130 °C, then NaBH(OAc)3, rt; (h) bromoacetyl bromide, Et3N, CH2Cl2, 0 °C, then 40 °C; (i)
PhSH, Cs2CO3, CH3CN, rt; (j) (Boc)2O, NaHCO3, AcOEt, water, rt; (k) LiOH-H2O, THF, water, 0 °C; (l) amine 28 or 29a-n, HBTU, N,N-diisopropylethylamine,
DMF, 0 °C; (m) TFA, CH2Cl2, rt; (n) fumaric acid, MeOH, rt.

(a)
(b)
(c)
33
34
35
28
Scheme 2. Synthetic pathway leading to chiral amine 28. Reagents and conditions: (a) BnBr, K2CO3, EtOH, rt, 4 days, 85%; (b) EtI, NaH, THF, rt, 18 h, 81%;
(c) H2, 20% Pd(OH)2 on carbon, MeOH, rt, 6 h, 98%.
HCl
(a)
(b), (c)
36
37
29b
Scheme 3. Synthetic pathway leading to chiral amine 29b. Reagents and conditions: (a) diphenylphosphoryl azide, DBU, toluene, rt, 2 days, 28%; (b) H2, Pd on
carbon, AcOEt, EtOH, rt, 3 h; (c) 4N-HCl in 1,4-dioxane, rt, 63% (2 steps).
O
NH2
S
39
H
N
R1
O
R1
H2N
R1
O
(a), (b)
(c)
S
n HCl
(n = 1 or 2)
R2
R2
R2
,
-
, -
29a 29d n
38a 38d n
40a, 40d-n
Scheme 4. Synthetic pathway leading to chiral amines 29a and 29d-n. Reagents and conditions: (a) (S)-t-BuSONH2 39, Ti(OEt)4, THF, 70 °C; (b) L-selectride,
THF, 0 °C; (c) 4N-HCl in 1,4-dioxane, MeOH, rt.
amides 41 and 42 are described in the Supplementary Material.
Chiral carboxylic acid 8 was successfully obtained by the
In the preceding paper,8 starting from our clinical candidate
enantioselective ring opening of known carboxylic acid
DS-8108b (4)9,10, we discovered a new type of renin inhibitor
anhydride 614 with methanol in the presence of the chiral sulfonyl
which is a unique 3,5-disubstituted piperidine derivative (Figure
amide 715 as an asymmetric catalyst. Then, Curtius rearrangement
2). Direct connection with 2,2-dimethyl-4-phenylpiperazin-5-one
of the carboxylic acid (8) led to N-benzyloxycarbonyl (Cbz)
at the 5-position of the piperidine ring is essential to afford high
protected amine 9. Removal of the Cbz group of 9 gave the
renin inhibitory activities. Furthermore, introduction of the
corresponding amine derivative. Conversion of the amine
secondary amide part at the 3-position contributed to improve
derivative into aldehyde 11 was achieved by using 2-bromo-2-
their renin inhibitory activities. The most potent compound (5)
methylpropanal 10. Replacement of the N-tert-butoxycarbonyl
among the initialy optimized compounds demonstrated favorable
(Boc) group with the N-2-nitrobenzenesulfonyl (Ns)16 group,
oral bioavailability in rats (F = 45%) and a significant BP
provided 12. Compound 12 was converted into anilines 16-18 by
lowering effect by oral administration in hypertensive double
reductive amination with various aniline derivatives 13-15.
transgenic rats (dTG rats) harboring both the human
Subsequent ring formation with bromoacetyl bromide yielded
angiotensinogen and the human renin genes.11 Encouraged by
ketopiperazines 19-21. Replacement of the N-Ns groups with N-
these results, we evaluated the oral efficacy of 5 in cynomolgus
Boc groups gave 22-24. The condensation of various amines (28
monkeys as a further characterization. Unfortunately, compound
and 29a-n) and carboxylic acids 25-27 obtained by the hydrolysis
5 did not show any oral efficacy in cynomolgus monkeys. As far
of 22-24 afforded the corresponding amide derivatives 30 and
as we know, only a few renin inhibitors have been reported to
31a-p. Finally, removal of the N-Boc groups and then addition of
show oral efficacy in both rodent and non-rodent animal
fumaric acid gave 5 and 32a-p as fumarate salts.
models.12,13 We do not have enough information to explain which
animal model is the most appropriate for predicting human
efficacy. However, under this situation, we considered that
obtaining the compounds showing in vivo efficacy in at least two
different animal species would be necessary for acquiring a
clinical candidate. Herein, we report synthesis and optimization
The synthetic pathway leading to chiral amine 28 used for the
synthesis of 5 was outlined in Scheme 2. Compound 35 was
prepared by N-benzyl (Bn) protection of amine 33 and then
ethylation of the hydroxyl group. Subsequent removal of the two
N-Bn groups gave chiral amine 28. The synthetic route of chiral
amine 29b used for the synthesis of 32b was outlined in Scheme
3. Alcohol 36 was converted into azide 37 with
diphenylphosphoryl azide. Reduction of the azide group and then
treatment with hydrogen chloride in 1,4-dioxane gave chiral
amine 29b as hydrochloride salt.17 On the other hand, the
synthetic route of chiral amines 29a and 29d-n used for the
synthesis of 32a and 32d-p was outlined in Scheme 4.
Sulfinimine formation of ketones 38a and 38d-n with chiral
of
(3S,5R)-5-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-
yl)piperidine-3-carboxamides to demonstrate favorable oral
efficacy in both cynomolgus monkeys and rats.
2. Chemistry
The synthetic pathway leading to (3S,5R)-5-(2,2-dimethyl-5-
oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides
5
and
32a-p is outlined in Scheme 1. The synthetic methods for other

Table 1. In vitro renin inhibitory activities (IC50 and ratio) and oral exposures of previously synthesized compoundsa,b
Cmpd
R
Purified human renin
IC50 (nM)
Monkey plasma renin
Cynomolgus monkey PK (1 mg/kg, po)d
IC50 (nM)
Ratioc
AUCpo,0-4h (ng*h/ml)
ne
5
1.3
2.3
1.6
2.1
2.5
2.1
1.4
1.4
0.5
0.8
2.8
1.0
2.0
1.8
3.6
0.9
14
4
41f
4.8
10
10
3
4
3
3
3
3
3
42
25
32a
32b
32c
32d
9.0
18
51
137
60
6.6
17
52
32e
2.1
4.2
1.6
a
b
c
Compounds were obtained as fumarate salts. Assay results of renin inhibitory activity are the average of at least two replicates. Ratio = IC50 (nM) of
compound / IC50 (nM) of 1. d The data were obtained by cassette dosing. e Number of compounds dosed in cassette. f Compound 41 is a single diastereoisomer .
Stereochemistry is not determined.
sulfinyl amine 39 in the presence of Ti(OEt)4, and subsequent
diastereoselective reduction of the sulfinimines afforded
sulfinamides 40a and 40d-n.18 Compounds 40a and 40d-n were
Table 2. Oral exposures of compounds 5 and 32c in cynomolgus monkeys
or ratsa
Cmpd
Rat PK (10 mg/kg, po),
AUC0-24h (ng*h/ml)
Cynomolgus monkey PK (3
mg/kg, po), AUC0-24h (ng*h/ml)
5
1940
1500
60
32c
730
a The data were obtained by individual dosing.
obtained as the single diastereoisomer by the separation with
silica gel column chromatography. Then, removal of the N-
sulfinyl groups with hydrogen chloride in 1,4-dioxane was

carried out as described in ref. 18. As a result, chiral amines 29a
and 29d-n were obtained as hydrochloride salts.
Table 3. In vitro renin inhibitory activities (IC50 and ratio) and oral exposures of compounds with modification of the P2’ phenyl ring a,b
Cmpd
R1
R2
Purified human renin
Monkey plasma renin
Cynomolgus monkey PK (1 mg/kg, po)d
IC50 (nM)
2.1
IC50 (nM)
Ratioc
1.0
2.0
1.8
2.3
2.8
3.7
5.0
11
AUCpo,0-4h (ng*h/ml)
ne
3
3
3
3
3
3
3
3
32a
32b
32c
32f
32g
32h
32i
Isobutyl
Propyl
Ethyl
Ethyl
Ethyl
Ethyl
Ethyl
Ethyl
Phenyl
9.0
18
6.6
11
14
19
25
55
51
137
60
90
57
52
83
Phenyl
2.5
Phenyl
2.1
2-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
3-Chlorophenyl
4-Chlorophenyl
3.3
1.8
2.4
2.3
32j
5.2
130
a
b
c
Compounds were obtained as fumarate salts. Assay results of renin inhibitory activity are the average of at least two replicates. Ratio = IC50 (nM) of
compound / IC50 (nM) of 1. d The data were obtained by cassette dosing. e Number of compounds dosed in cassette.
Table 4. In vitro renin inhibitory activities (IC50 and ratio) and oral exposures of compounds with modification of the P2’ portiona,b
Cmpd
R2
Purified human renin
Monkey plasma renin
Cynomolgus monkey PK (1 mg/kg, po)d
IC50 (nM)
1.4
IC50 (nM)
17
Ratioc
AUCpo,0-4h (ng*h/ml)
ne
3
3
3
3
3
3
32d
32k
32e
32l
2-Pyridyl
3.6
2.1
0.9
0.8
0.6
1.4
52
53
1.6
5.4
11
15
5-Fluoro-2-pyridyl
3-Pyridyl
1.0
9.7
2.1
4.2
5-Fluoro-3-pyridyl
5-Isoxazolyl
2-Oxazolyl
1.7
6.9
32m
1.1
3.3
32n
1.9
8.3
a
b
c
Compounds were obtained as fumarate salts. Assay results of renin inhibitory activity are the average of at least two replicates. Ratio = IC50 (nM) of
compound / IC50 (nM) of 1. d The data were obtained by cassette dosing. e Number of compounds dosed in cassette.
value of 5 (AUCpo,0-24h = 60 ng*h/ml, 3 mg/kg, po) was revealed
to be only about one-tenth of that of orally active DS-8108b (4)
(AUCpo,0-24h = 685 ng*h/ml, 3 mg/kg, po).10 Based on this result,
we speculated that the oral exposure of 5 did not reach a
sufficient level to show oral efficacy in cynomolgus monkeys.
3. Results and discussions
To identify the reason why compound 5 did not show any oral
efficacy in cynomolgus monkeys, pharmacokinetics (PK) study
of 5 in cynomolgus monkeys was performed. The AUCpo,0-24h

Thus, we attempted to explore 3,5-disubstituted piperidines
showing appropriate oral exposure in cynomolgus monkeys.
Same as in the case of discovering DS-8108b (4), the cassette
dosing method (po, 0-4 h) was used to increase the throughput of
PK screening.10 Initially, we evaluated compounds 5, 32a-e, 41,
and 42 (Table 1). These compounds are potent renin inhibitors
and have been described previously.8 The AUCpo,0-4h values of
compounds containing the ether (5), hydroxyl (41), alkyl (42), or
3-pyridyl (32e) group were unfortunately low in cynomolgus
monkeys. Meanwhile, compounds 32a-d containing the phenyl
or 2-pyridyl ring exhibited higher AUCpo,0-4h values than 5.
Specifically, the P2’ phenyl derivatives (32a-c) showed high
renin inhibitory activities against monkey plasma renin.
Table 5. In vitro renin inhibitory activities (IC50 and ratio), oral exposures, BP lowering effect, and CYP and hERG current inhibition of compounds with
modification of the P3 portiona,b
32k
32o
32p
Cmpd
Purified
human
renin
Monkey plasma renin
Cynomolgus
BP lowering effect, AUC0-24h
CYP direct
inhibition (%
at 10 μM)
hERG
monkey PK (10
mg/kg, po), AUCpo,0-
24h (ng*h/mL)d
(mmHg·h)e
current
inhibition
(% at 30
μM)f
IC50 (nM)
IC50 (nM) Ratioc
dTG rat (30 Cynomolgus monkey
3A4
2D6
mg/kg, po)
(10 mg/kg, po)
32k
32o
1.0
1.6
1.3
9.7
7.0
16
2.1
0.9
2.1
2580
3190
NTg
−535
−194
22
30
44
8.3
1.0
11
27
5.2
40
−581
−267
32p
−606
NTg
a
b
c
Compounds were obtained as fumarate salts. Assay results of renin inhibitory activity are the average of at least two replicates. Ratio = IC50 (nM) of
d
e
f
compound / IC50 (nM) of 1. The data were obtained by individual dosing. Area under the change in MAP (mmHg) versus time curve. hERG currents were
measured using the patch clamp technique on IonWorks® QuattroTM system. g NT = not tested.
potency with sustained good oral exposure (32k vs. 32d). On the
other hand, introduction of the fluoro group to the 3-pyridyl ring
In order to verify our strategy, PK studies of 32c in rats and
of 32e showed high in vitro potency, however did not have an
cynomolgus monkeys were conducted by individual dosing
attractive impact on the plasma drug concentration (32l).
(Table 2). The AUCpo,0-24h value of 32c in rats was almost similar
Unfortunately, other evaluated hetero-aromatic rings, the 5-
to that of 5. Moreover, as expected from the result of cassette
isoxazolyl (32m) and 2-oxazolyl (32n) rings, showed low
dosing, compound 32c exhibited 10-fold higher AUCpo,0-24h value
AUCpo,0-4h values. In addition to the good in vitro potency and
than 5 by individual dosing in cynomolgus monkeys.
oral exposure, compound 32k exhibited improvement in the
inhibitory activity against CYP 3A4 (22%), CYP 2D6 (8.3%) at
10 μM, and hERG current (27%) at 30 μM compared to 32a.
Therefore, we selected the 5-fluoro-2-pyridyl ring as a favorable
P2’ portion and decided to revisit the P3 part modification.
Since 3,5-disubstituted piperidines with the phenyl ring at the
P2’ position were suggested to have suitable properties in both
renin inhibition and oral exposure, we started to evaluate the
substituent effect on the P2’ phenyl ring as the next step (Table 3).
The ethyl group was used as substituent at R1 for this SAR study
because the corresponding starting materials, ketones 38f-j, were
readily available from commercial sources unlike the case of R1
isobutyl or propyl analogs. Introduction of the halogen group
onto the phenyl ring did not affect the AUCpo,0-4h values (32f-j),
however, inhibitory activities against monkey plasma renin were
deteriorated. Especially, compounds 32i and 32j containing the
chloro group exhibited lower renin inhibitory activity than
compounds 32g and 32h containing the fluoro group. Based on
these SAR results, very limited space was suggested to be left for
substitution on the P2’ phenyl ring.
Considering the previous SAR of the P3 portion,10,21 2-chloro
(32k), 2-methyl (32o), and 5-fluoro-2-methyl (32p) phenyl
derivatives were prepared. Both in vitro and in vivo profiles of
these compounds were evaluated and listed in Table 5.
Compound 32k showed suitable oral exposure in cynomolgus
monkeys and reduction in mean arterial blood pressure (MAP) in
cynomolgus monkeys as well as dTG rats with acceptable CYP
and hERG current inhibition. Compound 32p also showed BP
lowering effect in dTG rats, however, strong hERG current
inhibition was observed. Thus, further evaluation of 32p was
discontinued. Compound 32o exhibited the most potent
inhibitory activities against monkey plasma renin among three
compounds, suitable oral exposure in cynomolgus monkeys and
stronger BP lowering effects in both dTG rats and cynomolgus
monkeys than 32k. In addition, hERG current inhibition of 32o
was weaker than that of 32k. From these results, we finally
selected 32o as the promising 3,5-disubstituted piperidine
derivative for further evaluation.
For further characterization, we evaluated the in vitro safety
profile of 32a, which showed the highest inhibitory activity
against monkey plasma renin among 32a-c. However, direct
inhibition of CYP 3A4 (49%) and CYP 2D6 (51%) at 10 μM, and
hERG current inhibition (36%) at 30 μM were observed. These
undesired interactions are known to be often lowered by
increasing polarity of compounds.19,20 Therefore, our attention
was shifted to P2’ hetero-aromatic ring derivatives (Table 4).
Compound 32k, which has an additional fluoro group at the 5-
position of the 2-pyridyl ring of 32d, exhibited restored in vitro
The key characteristics of 32o are summarized in Table 6.
Compound 32o showed high renin inhibitory activity in the

presence of human plasma as well as monkey plasma, and also
showed favorable metabolic stability in human and monkey liver
microsomes. With regard to the initial safety profile, effects on
the cardiac action potential, CYP, and hERG current of 32o were
considered to be acceptable and the Ames test was negative.
Regarding the PK profile, compound 32o exhibited moderate oral
bioavailability in both SD rats (20%) and cynomolgus monkeys
(25%). The antihypertensive efficacy of 32o was displayed in
Figures 3 and 4. At first, vehicle or 30 mg/kg of 32o was orally
administered to dTG rats (Figure 3). Compound 32o induced a
significant reduction in MAP sustained over a period of 24 h.
Next, vehicle, or 3 and 10 mg/kg of 32o were orally administered
to cynomolgus monkeys pretreated with furosemide (Figure 4).
Compound 32o showed a significant reduction in MAP in a dose-
dependent manner sustained over a period of 24 h.
4. Conclusion
We have synthesized and optimized a novel type of renin
inhibitor based on
phenylpiperazin-1-yl)piperidine-3-carboxamides
a
(3S,5R)-5-(2,2-dimethyl-5-oxo-4-
template.
Chemical modification of P1’, P2’ and P3 portions led to a
promising 3,5-disubstituted piperidine 32o showing high renin
inhibitory activity and favorable oral exposure in both rats and
cynomolgus monkeys with acceptable CYP and hERG current
inhibition. Compound 32o achieved the significant BP lowering
effect over a period of 24 h in both cynomolgus monkeys
pretreated with furosemide and dTG rat models by oral
administration. This promising compound 32o was selected for
further evaluation.
Table 6. In vitro, primary safety, and PK profiles of compound 32oa,b
Purified human renin
Human plasma renin
IC50 (nM)
IC50 (nM)
Ratioc
1.6
30
20
10
0
3.6
1.2
Monkey plasma renin
IC50 (nM)
Ratioc
7.0
0.9
-10
-20
Molecular weight
510
-30
Lipophilicity (LogD7.4
)
2.0
Vehicle (0.5% MC, n = 9)
-40
30 mg/kg of 32o (po, n = 5)
Metabolic stability (human, monkey, rat)d
Plasma protein binding ratio (human, monkey, rat)e
CYP 3A4, direct inhibition (10 μM)
100%, 84%, 89%
38%, 55%, 63%
30%
-50
0
10
20
30
40
50
Time (h)
Figure 3. Effect of compound 32o on MAP in dTG rats.
CYP 2D6, direct inhibition (10 μM)
1.0%
hERG current inhibition (30 μM)f
5.2%
Guinea pig right ventricular
papillary muscle-cardiac
action potential (30 μM)g,h
APD90
−1.0%
(Shortening)
20
10
0
dV/dt max
−11.6%
(Decrease)
Ames test (Salmonella typhimurium TA98, TA100)
Negative
-10
F (%)
20
-20
-30
-40
Vehicle (1% MC, n = 6)
3 mg/kg of 32o (po, n = 6)
Cmax (ng/mL)
204
1100
4.8
10 mg/kg of 32o (po, n = 6)
SD rat, female
AUCpo,0-24h (ng*h/mL)
CL (L/h/kg)
Vss (L/kg)
(30 mg/kg p.o., 0.5% MC)
(1 mg/kg i.v., Saline)
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (h)
11
Figure 4. Effect of compound 32o on MAP in cynomolgus monkeys
t1/2 (h)
5.4
pretreated with furosemide.
F (%)
25
Cmax (ng/mL)
AUCpo,0-24h (ng*h/mL)
CL (L/h/kg)
Vss (L/kg)
604
3190
0.57
5.4
Cynomolgus monkey, male
(10 mg/kg p.o., 0.5% MC)
(1 mg/kg i.v., Saline)
t1/2 (h)
7.9
a
b
Compound 32o was obtained as fumarate salt. Assay results of renin
c
inhibitory activity are the average of at least two replicates. Ratio = IC50
d
(nM) of compound / IC50 (nM) of 1. Percent remaining after 30 min in
human, monkey, and rat liver microsomes. e The plasma protein binding ratio
was determined by a micro-scale ultracentrifugation method (436,000g, 2 h
f
20 min, at 4 °C). hERG currents were measured using the patch clamp
technique on IonWorks® QuattroTM system.
Action potentials were
g
measured using the glass microelectrode method. h % change from baseline.

1252, 1147, 819, 627 cm-1. HRMS (ESI−): m/z calcd for
C13H21NO6−H: 286.1291; found: 286.1298.
5.1.2 1-tert-Butyl 3-methyl (3S,5R)-5-
(benzyloxycarbonylamino)piperidine-1,3-dicarboxylate (9)
Et3N (11.3 mL, 80.8 mmol) and diphenylphosphoryl azide
(20.4 g, 74.1 mmol) were added to a solution of 8 (19.4 g, 67.4
mmol) in toluene (335 mL) at rt, and the mixture was stirred at
90 °C for 1.5 h. After cooling, benzyl alcohol was added to the
reaction mixture, and mixture was stirred at 90 °C for 3.5 h. The
reaction mixture was cooled to room temperature and poured into
water, followed by extraction with AcOEt. Then, the organic
layer was washed with brine, and dried over anhydrous Na2SO4.
After filtration, the solvent was evaporated under reduced
pressure, and the residue was purified by silica gel column
chromatography (eluent, n-hexane/AcOEt = 4/1) to obtain 9 (25.5
25.0
1
g, 96%) as a colorless liquid. [α]D −4.16 (c 1.01, MeOH). H
NMR (400 MHz, CDCl3): δ 7.37-7.26 (m, 5H), 5.11 (br s, 3H),
4.06-4.03 (m, 2H), 3.70-3.63 (m, 5H), 3.09-3.05 (m, 1H), 2.60
(br s, 1H), 2.31-2.27 (m, 1H), 1.61-1.56 (m, 1H), 1.45 (s, 9H).
13C NMR (125 MHz, CDCl3): δ 173.4, 155.5, 154.5, 128.4, 128.2,
127.6, 127.0, 80.3, 66.8, 65.3, 52.0, 46.4, 45.0, 40.0, 32.8, 28.3.
IR: 3342, 2976, 1720, 1671, 1522, 1435, 1239, 1157, 1047, 737
cm-1. HRMS (ESI+): m/z calcd for C20H28N2O6+H: 393.2026;
found: 393.2023.
5. Experimental
5.1. Synthesis
Starting reagents were purchased from commercial suppliers
and used without further purification unless otherwise specified.
Flash column chromatography was performed on silica gel 60 N
(spherical, neutral), 40-50 mesh, purchased from Kanto Chemical
Co., Inc., or NH silica gel, 100-200 mesh, purchased from Fuji
Silysia Chemical Ltd. 1H NMR and 13C NMR spectra were
obtained on a Varian Unity 400 or 500 spectrometer, or a Bruker
Avance III 500 spectrometer. Spectra were taken in the indicated
solvent at ambient temperature, and chemical shifts are reported
in parts per million (ppm (δ)) relative to the lock of the solvent
used. Resonance patterns are recorded with the following
notations: br (broad), s (singlet), d (doublet), t (triplet), q
(quartet), and m (multiplet). Mass spectra were obtained on a
JEOL JMS-LCmate or an LC-MS system composed of Waters
Xevo Q-Tof MS and Acquity UPLC systems. Optical rotations
were measured on an Autopol V Plus. Elemental analyses for
CHN and ClF were determined on a Microcorder JM10 and a
Dionex ICS-1500, respectively. Infrared spectrum was recorded
in a KBr disc with a Jasco FT/IR-6100.
5.1.3 1-tert-Butyl 3-methyl (3S,5R)-5-[(1,1-dimethyl-2-
oxoethyl)amino]piperidine-1,3-dicarboxylate (11)
To a suspension of 9 (50.0 g, 127 mmol) in MeOH (300 mL),
10% Pd on carbon (50% wet, 5.00 g) were added. The suspension
was stirred under H2 atmosphere at room temperature for 1.5 h.
H2 in the reaction vessel was replaced by N2, and then Pd on
carbon was separated by filtration. The solvent was evaporated
under reduced pressure to obtain crude 1-tert-butyl 3-methyl
(3S,5R)-5-aminopiperidine-1,3-dicarboxylate as
a
colorless
liquid. 2-Bromo-2-methylpropanal 10 (112 g, 792 mmol) was
added to a solution of the crude 1-tert-butyl 3-methyl (3S,5R)-5-
aminopiperidine-1,3-dicarboxylate in a mixed solvent of DMF
(211 mL) and water (52.0 mL) at room temperature, and the
mixture was stirred at the same temperature for 1 day. Saturated
NaHCO3 aqueous solution was added to the reaction mixture,
followed by extraction with AcOEt. Then, the organic layer was
washed with water, 10% NaCl aqueous solution, and brine, and
dried over anhydrous Na2SO4. After filtration, the solvent was
evaporated under reduced pressure, and the residue was purified
by silica gel column chromatography (eluent, n-hexane/AcOEt =
1/1) to obtain 11 (31.8 g, 73%, 2 steps) as a light yellow liquid.
5.1.1 (3R,5S)-1-tert-Butoxycarbonyl-5-
methoxycarbonylpiperidine-3-carboxylic acid (8)
Methanol (43.0 mL, 105 mmol) was added to a solution of
tert-butyl
carboxylate 6 (27.0 g, 105 mmol) and N-[(S)-(6-methoxy-4-
quinolyl)-(5-vinylquinuclidin-2-yl)methyl]-3,5-
2,4-dioxo-3-oxa-7-azabicyclo[3.3.1]nonane-7-
25.0
1
[α]D
−9.47 (c 1.05, MeOH). H NMR (400 MHz, CDCl3): δ
bis(trifluoromethyl)benzenesulfonamide 7 (8.30 g, 13.8 mmol) in
THF (1.00 L) at −20 °C, and the mixture was stirred at the same
temperature for 1 day. 1N HCl aq. was added to the reaction
mixture, followed by extraction with Et2O-AcOEt. Then, the
organic layer was washed with brine and dried over anhydrous
MgSO4. After filtration, the solvent was concentrated under
reduced pressure, and the residue was purified by silica gel
column chromatography (eluent, n-hexane/AcOEt = 1/5 to 0/1) to
obtain 8 (20.7 g, 69%, 94% ee) as a colorless solid. The ee value
of 8 was determined by chiral HPLC analysis: column, Chiral
Pack AD-H (4.6 x 250 mm); eluent, n-hexane/ethanol = 95/5;
9.43 (s, 1H), 4.32-4.21 (m, 1H), 4.11-4.01 (m, 1H), 3.68 (s, 3H),
2.71-2.61 (m, 1H), 2.54-2.41 (m, 2H), 2.30 (dd, J = 12.7, 10.7
Hz, 1H), 2.20 (br d, J = 12.5 Hz, 1H), 1.45 (s, 9H), 1.40-1.33 (m,
1H), 1.22 (s, 3H), 1.17 (s, 3H). 13C NMR (125 MHz, CDCl3): δ
203.8, 172.9, 154.6, 80.1, 61.3, 51.9, 50.8, 48.9, 44.9, 41.2, 36.5,
28.4, 22.7, 21.9. IR: 3334, 2974, 1726, 1679, 1425, 1326, 1252,
1172, 937, 899, 772, 661 cm-1. HRMS (ESI+): m/z calcd for
C16H28N2O5+H: 329.2076; found: 329.2071.
5.1.4 Methyl (3S,5R)-5-[(1,1-dimethyl-2-oxoethyl)amino]-1-(2-
nitrophenyl)sulfonylpiperidine-3-carboxylate (12)
flow rate = 0.5 mL/min; tR of (3R,5S)-isomer = 29.6 min; tR 1of
4N-HCl in 1,4-dioxane (362 mL, 1.45 mmol) was added to 11
(31.8 g, 96.6 mmol) at room temperature, and the mixture was
stirred at the same temperature for 0.5 h. The solvent was
evaporated under reduced pressure to obtain crude methyl
(3S,5R)-5-[(1,1-dimethyl-2-oxoethyl)amino]piperidine-3-
25.0
(3S,5R)-isomer = 33.3 min. [α]D
+5.77 (c 1.01, MeOH). H
NMR (500 MHz, CDCl3): δ 4.37 (br s, 2H), 3.71 (s, 3H), 2.72 (br
s, 2H), 2.55-2.45 (m, 3H), 1.72 (q, J = 12.7 Hz, 1H), 1.47 (s, 9H).
13C NMR (125 MHz, CDCl3): δ 177.8, 172.9, 154.5, 80.7, 52.0,
45.0, 40.7, 40.6, 30.0, 28.4. IR: 3197, 2982, 1739, 1670, 1435,
carboxylate. NaHCO3 (40.6 g, 483 mmol) and 2-
nitrobenzenesulfonyl chloride (21.2 g, 95.6 mmol) were added to

a solution of the crude methyl (3S,5R)-5-[(1,1-dimethyl-2-
oxoethyl)amino]piperidine-3-carboxylate in a mixed solvent of
1,4-dioxane (250 mL) and water (250 mL) at 0 °C, and the
mixture was stirred at the same temperature for 1.5 h. Water was
added to the reaction mixture, followed by extraction with
AcOEt. Then, the organic layer was washed with brine, and dried
over anhydrous Na2SO4. After filtration, the solvent was
evaporated under reduced pressure, and the residue was purified
by silica gel column chromatography (eluent, n-hexane/AcOEt =
1/1 to 0/1) to obtain 12 (34.4 g, 86%, 2 steps) as a colorless solid.
6.23 (dd, J = 11.2, 2.4 Hz, 1H), 4.26 (br s, 1H), 4.07-4.05 (m,
1H), 3.69-3.64 (m, 4H), 2.89-2.84 (m, 2H), 2.80-2.70 (m, 3H),
2.36-2.32 (m, 1H), 2.21-2.17 (m, 1H), 2.07 (s, 3H), 1.31 (q, J =
12.0 Hz, 1H), 1.22 (s, 3H), 1.20 (s, 3H).
5.1.8 Methyl (3S,5R)-5-[4-(2-chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-1-(2-nitrophenyl)sulfonylpiperidine-3-
carboxylate (19)
A solution of bromoacetyl bromide (24.4 g, 121 mmol) in
CH2Cl2 (80 mL) was added dropwise to a solution of 16 (16.0 g,
30.5 mmol) and Et3N (24.8 g, 245 mmol) in CH2Cl2 (320 mL)
under ice-cooling over a period of 2.5 h. The reaction mixture
was stirred at room temperature for 45 min, and then stirred at
40 °C for 3 h. After cooling, water was added to the reaction
mixture, followed by extraction with CH2Cl2. Then, the organic
layer was washed with brine, and dried over anhydrous Na2SO4.
After filtration, the solvent was evaporated under reduced
pressure, and the residue was purified by silica gel column
chromatography (eluent, n-hexane/AcOEt = 7/3 to 0/1) to obtain
25.0
1
[α]D
+22.0 (c 1.04, MeOH). H NMR (500 MHz, CDCl3): δ
9.44 (s, 1H), 8.01-7.99 (m, 1H), 7.74-7.69 (m, 2H), 7.66-7.63 (m,
1H), 4.01-3.98 (m, 1H), 3.83-3.80 (m, 1H), 3.69 (s, 3H), 2.79-
2.64 (m, 3H), 2.38 (dd, J = 12.7, 10.7 Hz, 1H), 2.28 (br d, J =
12.7 Hz, 1H), 1.32 (q, J = 12.2 Hz, 1H), 1.24 (s, 3H), 1.18 (s,
3H). 13C NMR (125 MHz, CDCl3): δ 203.7, 172.2, 148.0, 133.8,
132.1, 131.8, 130.9, 124.3, 61.4, 52.6, 52.1, 49.0, 46.6, 41.1, 35.9,
23.0, 21.9. IR: 3331, 2694, 1729, 1707, 1553, 1378, 1357, 1172,
1017, 782, 584 cm-1. HRMS (ESI+): m/z calcd for
C17H23N3O7S+H: 414.1335; found: 414.1330.
1
19 (13.5 g, 78%) as a colorless liquid. H NMR (500 MHz,
CDCl3): δ 8.03-8.01 (m, 1H), 7.76-7.70 (m, 2H), 7.67-7.65 (m,
1H), 7.48-7.46 (m, 1H), 7.33-7.23 (m, 3H), 4.03 (br d, J = 11.2
Hz, 1H), 3.87-3.81 (m, 1H), 3.72 (s, 3H), 3.71-3.22 (m, 5H),
2.76-2.68 (m, 3H), 2.24-2.17 (m, 1H), 1.73-1.66 (m, 1H), 1.41-
1.35 (m, 6H).
5.1.5 Methyl (3S,5R)-5-{[2-(2-chloroanilino)-1,1-
dimethylethyl]amino}-1-(2-nitrophenyl)sulfonylpiperidine-3-
carboxylate (16)
To a single-neck flask upon which there was placed a Dean-
Stark, 12 (17.1 g, 41.4 mmol), 2-chloroaniline 13 (7.87 g, 61.7
mmol), toluene (400 mL), and acetic acid (8.67 g, 144 mmol)
were added. The mixture was stirred at 130 °C for 4 h. After
cooling in an ice bath, NaBH(OAc)3 (17.5 g, 82.6 mmol) was
added to the reaction mixture. The mixture was further stirred at
room temperature for 16 h. Saturated NaHCO3 aqueous solution
was added to the reaction mixture, followed by extraction with
AcOEt. Then, the organic layer was washed with brine, and dried
over anhydrous Na2SO4. After filtration, the solvent was
evaporated under reduced pressure, and the residue was purified
by silica gel column chromatography (eluent, n-hexane/AcOEt =
4/1 to 7/3) to obtain 16 (16.0 g, 74%) as a light colorless liquid.
1H NMR (500 MHz, CDCl3): δ 7.74 (d, J = 8.3 Hz, 1H), 7.64-
7.58 (m, 2H), 7.52-7.48 (m, 1H), 7.25-7.23 (m, 1H), 7.17-7.14
(m, 1H), 6.65-6.62 (m, 2H), 4.85 (br s, 1H), 4.07 (br d, J = 9.3
Hz, 1H), 3.75-3.71 (m, 1H), 3.69 (s, 3H), 2.97 (d, J = 11.7 Hz,
1H), 2.94 (d, J = 11.7 Hz, 1H), 2.81-2.69 (m, 3H), 2.34 (dd, J =
12.5, 10.5 Hz, 1H), 2.21 (br d, J = 12.5 Hz, 1H), 1.32 (q, J = 12.2
Hz, 1H), 1.22 (s, 3H), 1.21 (s, 3H).
5.1.9 Methyl (3S,5R)-5-[2,2-dimethyl-4-(o-tolyl)-5-
oxopiperazin-1-yl]-1-(2-nitrophenyl)sulfonylpiperidine-3-
carboxylate (20)
The title compound was prepared from 17 in a manner similar
to that described for 19 as a colorless solid (84%). 1H NMR (500
MHz, CDCl3): δ 8.03-8.01 (m, 1H), 7.76-7.71 (m, 2H), 7.67-7.65
(m, 1H), 7.27-7.21 (m, 3H), 7.13-7.06 (m, 1H), 4.05-4.00 (m,
1H), 3.87-3.81 (m, 1H), 3.72 (s, 3H), 3.68-3.15 (m, 5H), 2.79-
2.66 (m, 3H), 2.24-2.18 (m, 4H), 1.75-1.66 (m, 1H), 1.37-1.34
(m, 6H).
5.1.10 Methyl (3S,5R)-5-[4-(5-fluoro-2-methylphenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-1-(2-
nitrophenyl)sulfonylpiperidine-3-carboxylate (21)
The title compound was prepared from 18 in a manner similar
to that described for 19 as a colorless solid (84%). 1H NMR (500
MHz, CDCl3): δ 8.03-8.02 (m, 1H), 7.76-7.71 (m, 2H), 7.67-7.65
(m, 1H), 7.27-7.20 (m, 1H), 6.95 (dq, J = 8.3, 2.4 Hz, 1H), 6.87-
6.81 (m, 1H), 4.05-3.99 (m, 1H), 3.87-3.80 (m, 1H), 3.72 (s, 3H),
3.68-3.14 (m, 5H), 2.80-2.66 (m, 3H), 2.32-2.17 (m, 4H), 1.74-
1.66 (m, 1H), 1.37-1.34 (m, 6H).
5.1.6 Methyl (3S,5R)-5-{[1,1-dimethyl-2-(2-
methylanilino)ethyl]amino}-1-(2-
nitrophenyl)sulfonylpiperidine-3-carboxylate (17)
5.1.11 1-tert-Butyl 3-methyl (3S,5R)-5-[4-(2-chlorophenyl)-
2,2-dimethyl-5-oxopiperazin-1-yl]piperidine-1,3-
dicarboxylate (22)
The title compound was prepared from 12 and 2-methylaniline
14 in a manner similar to that described for 16 as a colorless solid
(98%). 1H NMR (500 MHz, CDCl3): δ 7.67 (dd, J = 8.3, 1.0 Hz,
1H), 7.64-7.58 (m, 2H), 7.48-7.45 (m, 1H), 7.16-7.13 (m, 1H),
7.05 (br d, J = 7.3 Hz, 1H), 6.68-6.65 (m, 1H), 6.58 (br d, J = 8.3
Hz, 1H), 4.08-4.05 (m, 1H), 3.73-3.69 (m, 4H), 2.96 (d, J = 11.7
Hz, 1H), 2.91 (d, J = 11.7 Hz, 1H), 2.81-2.70 (m, 3H), 2.32 (dd, J
= 12.2, 10.7 Hz, 1H), 2.21-2.17 (m, 1H), 2.12 (s, 3H), 1.31 (q, J
= 12.0 Hz, 1H), 1.22 (s, 3H), 1.21 (s, 3H).
Cesium carbonate (9.31 g, 28.6 mmol) was added to a solution
of 19 (13.5 g, 23.9 mmol) and thiophenol (3.64 mL, 35.7 mmol)
in CH3CN (400 mL) under ice-cooling, and the mixture was
stirred at room temperature for 2.5 h. After concentration under
reduced pressure, water was added to the reaction mixture,
followed by extraction with CH2Cl2. Then, the organic layer was
washed with brine, and dried over anhydrous Na2SO4. After
filtration, the solvent was evaporated under reduced pressure, and
the residue was purified by silica gel column chromatography
(eluent, CH2Cl2/MeOH = 1/0 to 7/3) to afford methyl (3S,5R)-5-
[4-(2-chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-
5.1.7 Methyl (3S,5R)-5-{[2-(5-fluoro-2-methylanilino)-1,1-
dimethylethyl]amino}-1-(2-nitrophenyl)sulfonylpiperidine-3-
carboxylate (18)
The title compound was prepared from 12 and 5-fluoro-2-
methylaniline 15 in a manner similar to that described for 16 as a
colorless solid (98%). 1H NMR (500 MHz, CDCl3): δ 7.75 (dd, J
= 7.8, 1.5 Hz, 1H), 7.66-7.59 (m, 2H), 7.51 (dt, J = 7.8, 1.5 Hz,
1H), 6.95 (br t, J = 7.3 Hz, 1H), 6.32 (dt, J = 8.3, 2.4 Hz, 1H),
yl]piperidine-3-carboxylate (9.10 g, quant.). Di-t-butyl
dicarbonate (6.00 g, 27.5 mmol) was added to a solution of
methyl
(3S,5R)-5-[4-(2-chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]piperidine-3-carboxylate (9.10 g, 23.9 mmol)

obtained above and NaHCO3 (6.20 g, 73.8 mmol) in a mixed
solvent of AcOEt (200 mL) and water (200 mL), and the mixture
was stirred at room temperature for 0.5 h. The reaction mixture
was extracted with AcOEt, washed with brine, dried over
anhydrous Na2SO4, and purified by silica gel column
chromatography (eluent, n-hexane/AcOEt = 7/3 to 1/9) and NH
silica gel column chromatography (eluent, n-hexane/AcOEt = 1/0
(500 MHz, CDCl3): δ 7.22 (dd, J = 8.3, 6.3 Hz, 1H), 6.98-6.94
(m, 1H), 6.87-6.83 (m, 1H), 4.29 (br s, 1H), 4.09 (br s, 1H), 3.78-
3.61 (m, 2H), 3.42-3.18 (m, 2H), 3.01 (br s, 1H), 2.74-2.54 (m,
3H), 2.32-2.16 (m, 4H), 1.78-1.71 (m, 1H), 1.47 (s, 9H), 1.36-
1.34 (m, 6H).
5.1.17 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-1-(ethoxymethyl)-3-
methylbutyl]carbamoyl}piperidine-1-carboxylate (30)
1
to 1/1) to afford 22 (7.80 g, 68%) as colorless solid. H NMR
(500 MHz, CDCl3): δ 7.47 (dd, J = 7.3, 1.5 Hz, 1H), 7.34-7.22
(m, 3H), 4.29 (br s, 1H), 4.12 (br s, 1H), 3.74-3.52 (m, 5H), 3.36-
3.29 (m, 2H), 3.02-2.98 (m, 1H), 2.68-2.56 (m, 3H), 2.14 (br s,
1H), 1.84-1.73 (m, 1H), 1.48 (s, 9H), 1.44-1.31 (m, 6H).
HBTU (180 mg, 0.475 mmol) was added to a solution of 25
(148 mg, 0.318 mmol) and (2R)-1-ethoxy-4-methylpentan-2-
amine 28 (92.0 mg, 0.633 mmol) and N,N-diisopropylethylamine
(0.166 mL, 0.953 mmol) in DMF (3.00 mL) under ice-cooling,
and then the reaction mixture was stirred at room temperature for
18 h. Water was added to the reaction mixture, followed by
extraction with AcOEt. Then, the organic layer was washed with
brine, and dried over anhydrous Na2SO4. After filtration, the
solvent was evaporated under reduced pressure, and the residue
was purified by silica gel column chromatography (eluent, n-
hexane/AcOEt = 3/1 to 0/1) to obtain 30 (137 mg, 73%) as a
colorless solid. 1H NMR (500 MHz, CDCl3): δ 7.48-7.46 (m, 1H),
7.33-7.22 (m, 3H), 5.64 (br d, J = 4.9 Hz, 1H), 4.22-4.10 (m, 2H),
3.77-3.70 (m, 1H), 3.62-3.31 (m, 8H), 2.98 (br s, 1H), 2.74-2.68
(m, 2H), 2.34 (br s, 1H), 1.99-1.91 (m, 2H), 1.58 (br s, 1H), 1.48
(s, 9H), 1.46-1.31 (m, 8H), 1.20 (t, J = 6.8 Hz, 3H), 0.93-0.91 (m,
6H).
5.1.12 1-tert-Butyl 3-methyl (3S,5R)-5-[2,2-dimethyl-4-(o-
tolyl)-5-oxopiperazin-1-yl]piperidine-1,3-dicarboxylate (23)
The title compound was prepared from 20 in a manner similar
1
to that described for 22 as a colorless solid (79%, 2steps). H
NMR (500 MHz, CDCl3): δ 7.27-7.21 (m, 3H), 7.12-7.08 (m,
1H), 4.30 (br s, 1H), 4.08 (br s, 1H), 3.72-3.52 (m, 5H), 3.39-
3.35 (m, 1H), 3.21 (br d, J = 11.2 Hz, 1H), 3.01-2.97 (m, 1H),
2.70-2.54 (m, 3H), 2.24-2.23 (m, 3H), 2.12 (br d, J = 12.7 Hz,
1H), 1.83-1.74 (m, 1H), 1.48 (s, 9H), 1.36-1.32 (m, 6H).
5.1.13 1-tert-Butyl 3-methyl (3S,5R)-5-[4-(5-fluoro-2-
methylphenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]piperidine-
1,3-dicarboxylate (24)
The title compound was prepared from 21 in a manner similar
to that described for 22 as a colorless liquid (72%, 2steps). H
5.1.18 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-3-methyl-1-
phenylbutyl]carbamoyl}piperidine-1-carboxylate (31a)
1
NMR (500 MHz, CDCl3): δ 7.21 (dd, J = 8.3, 6.8 Hz, 1H), 6.97-
6.93 (m, 1H), 6.86-6.83 (m, 1H), 4.29 (br s, 1H), 4.09 (br s, 1H),
3.71-3.51 (m, 5H), 3.37-3.33 (m, 1H), 3.19 (br d, J = 11.7 Hz,
1H), 3.01-2.97 (m, 1H), 2.69-2.53 (m, 3H), 2.18-2.10 (m, 4H),
1.81-1.74 (m, 1H), 1.48 (s, 9H), 1.36-1.32 (m, 6H).
The title compound was prepared from 25 and (1R)-3-methyl-
1-phenylbutan-1-amine hydrochloride 29a in a manner similar to
1
that described for 30 as a colorless solid (74%). H NMR (500
MHz, CDCl3): δ 7.45 (br d, J = 7.8 Hz, 1H), 7.35-7.23 (m, 8H),
5.92 (br s, 1H), 4.99-4.96 (m, 1H), 4.20-3.96 (m, 2H), 3.67-3.48
(m, 2H), 3.31-3.25 (m, 2H), 2.93-2.66 (m, 3H), 2.34 (br s, 1H),
1.88-1.26 (m, 20H), 0.94-0.90 (m, 6H).
5.1.14 (3S,5R)-1-tert-Butoxycarbonyl-5-[4-(2-chlorophenyl)-
2,2-dimethyl-5-oxopiperazin-1-yl]piperidine-3-carboxylic
acid (25)
A solution of 22 (7.80 g, 16.3 mmol) in a mixed solvent of
THF (150 mL) and water (75.0 mL) was cooled in an ice bath,
and then lithium hydroxide monohydrate (1.37 g, 32.6 mmol)
were added. The mixture was stirred at the same temperature for
2 h. 1N HCl aqueous solution was added to the reaction mixture
to acidify it (pH = 2-3). The reaction mixture was extracted with
CH2Cl2, washed with brine, and dried over anhydrous Na2SO4.
After filtration, the solvent was evaporated under reduced
5.1.19 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-1-
phenylbutyl]carbamoyl}piperidine-1-carboxylate (31b)
The title compound was prepared from 25 and (1R)-1-
phenylbutan-1-amine hydrochloride 29b in a manner similar to
that described for 30 as a colorless solid (85%).1H NMR (500
MHz, CDCl3): δ 7.47-7.43 (m, 1H), 7.34-7.21 (m, 8H), 6.00 (br s,
1H), 4.91-4.84 (m, 1H), 4.10-3.93 (m, 2H), 3.69-3.62 (m, 1H),
3.58-3.47 (m, 1H), 3.32-3.25 (m, 2H), 2.94 (br s, 1H), 2.83-2.64
(m, 2H), 2.36 (br s, 1H), 1.96-1.68 (m, 6H), 1.46 (s, 9H), 1.35-
1.21 (m, 6H), 0.90 (t, J = 7.6 Hz, 3H).
1
pressure to obtain 25 (7.70 g, quant.) as a colorless solid. H
NMR (500 MHz, CDCl3): δ 7.47 (dd, J = 7.8, 1.5 Hz, 1H), 7.34-
7.25 (m, 3H), 4.30 (br s, 1H), 4.12 (br s, 1H), 3.76 (br d, J = 17.6
Hz, 1H), 3.66 (br d, J = 17.6 Hz, 1H), 3.38-3.29 (m, 2H), 3.03-
2.99 (m, 1H), 2.69-2.56 (m, 3H), 2.22-2.15 (m, 1H), 1.81-1.69
(m, 1H), 1.47 (s, 9H), 1.43-1.32 (m, 6H).
5.1.20 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-1-
5.1.15 (3S,5R)-1-tert-Butoxycarbonyl-5-[2,2-dimethyl-4-(o-
phenylpropyl]carbamoyl}piperidine-1-carboxylate (31c)
tolyl)-5-oxopiperazin-1-yl]piperidine-3-carboxylic acid (26)
The title compound was prepared from 25 and (1R)-1-
phenylpropan-1-amine 29c in a manner similar to that described
for 30 as a colorless solid (55%). 1H NMR (500 MHz, CDCl3): δ
7.46 (dd, J = 7.8, 1.5 Hz, 1H), 7.37-7.22 (m, 8H), 5.80 (br s, 1H),
4.88 (br q, J = 7.5 Hz, 1H), 4.26-4.00 (m, 2H), 3.74-3.50 (m, 2H),
3.34-3.29 (m, 2H), 2.95 (br s, 1H), 2.78-2.70 (m, 2H), 2.34 (br s,
1H), 1.93-1.78 (m, 4H), 1.48 (s, 9H), 1.39-1.27 (m, 6H), 0.89 (t,
J = 7.6 Hz, 3H).
The title compound was prepared from 23 in a manner similar
1
to that described for 25 as a colorless solid (quant.). H NMR
(500 MHz, CDCl3): δ 7.28-7.23 (m, 3H), 7.12-7.08 (m, 1H), 4.30
(br s, 1H), 4.05 (br s, 1H), 3.79-3.63 (m, 2H), 3.44-3.18 (m, 2H),
3.00 (br s, 1H), 2.73-2.54 (m, 3H), 2.24-2.17 (m, 4H), 1.79-1.70
(m, 1H), 1.47 (s, 9H), 1.36-1.33 (m, 6H).
5.1.16 (3S,5R)-1-tert-Butoxycarbonyl-5-[4-(5-fluoro-2-
methylphenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]piperidine-
3-carboxylic acid (27)
5.1.21 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-3-methyl-1-(2-
pyridyl)butyl]carbamoyl}piperidine-1-carboxylate (31d)
The title compound was prepared from 24 in a manner similar
to that described for 25 as a colorless solid (quant.). H NMR
1

The title compound was prepared from 25 and (1R)-3-methyl-
1-(2-pyridyl)butan-1-amine dihydrochloride 29d in a manner
similar to that described for 30 as a colorless solid (67%). H
NMR (500 MHz, CDCl3): δ 8.55 (br d, J = 4.4 Hz, 1H), 7.45 (br t,
J = 7.3 Hz, 1H), 7.46-7.45 (m, 1H), 7.32-7.18 (m, 5H), 6.60 (br d,
J = 7.8 Hz, 1H), 5.12 (br q, J = 7.8 Hz, 1H), 4.26-4.00 (m, 2H),
3.71-3.50 (m, 2H), 3.29 (br s, 2H), 2.97 (br s, 1H), 2.78-2.70 (m,
2H), 2.40 (br s, 1H), 1.90 (br s, 2H), 1.70-1.27 (m, 18H), 0.95-
0.93 (m, 6H).
chlorophenyl)propyl]carbamoyl}piperidine-1-carboxylate
(31i)
1
The title compound was prepared from 25 and (1R)-1-(3-
chlorophenyl)propan-1-amine hydrochloride 29i in a manner
similar to that described for 30 as a colorless solid (69%). H
NMR (500 MHz, CDCl3): δ 7.46 (dd, J = 7.8, 2.0 Hz, 1H), 7.32-
7.21 (m, 6H), 7.17-7.14 (m, 1H), 6.34 (br s, 1H), 4.83 (q, J = 7.6
Hz, 1H), 4.26-3.96 (m, 2H), 3.73-3.50 (m, 2H), 3.34-3.28 (m,
2H), 2.95 (br s, 1H), 2.80-2.68 (m, 2H), 2.41 (br s, 1H), 2.01-
1.74 (m, 4H), 1.49 (s, 9H), 1.36-1.24 (m, 6H), 0.89 (t, J = 7.5 Hz,
3H).
1
5.1.22 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-3-methyl-1-(3-
pyridyl)butyl]carbamoyl}piperidine-1-carboxylate (31e)
5.1.27 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-1-(4-
chlorophenyl)propyl]carbamoyl}piperidine-1-carboxylate
(31j)
The title compound was prepared from 25 and (1R)-3-methyl-
1-(3-pyridyl)butan-1-amine dihydrochloride 29e in a manner
similar to that described for 30 as a colorless solid (54%). H
1
NMR (500 MHz, CDCl3): δ 8.58-8.44 (m, 2H), 7.60-7.54 (m,
1H), 7.47-7.40 (m, 1H), 7.32-7.18 (m, 4H), 6.23 (br s, 1H), 5.07-
4.98 (m, 1H), 4.24-3.90 (m, 2H), 3.72-3.46 (m, 2H), 3.35-3.21
(m, 2H), 3.00-2.89 (m, 1H), 2.81-2.60 (m, 2H), 2.36 (br s, 1H),
1.97-1.79 (m, 2H), 1.74-1.18 (m, 18H), 0.95-0.86 (m, 6H).
The title compound was prepared from 25 and (1R)-1-(4-
chlorophenyl)propan-1-amine hydrochloride 29j in a manner
similar to that described for 30 as a colorless solid (55%). H
NMR (500 MHz, CDCl3): δ 7.46 (dd, J = 7.8, 1.5 Hz, 1H), 7.31-
7.19 (m, 7H), 6.10 (br s, 1H), 4.83 (q, J = 7.6 Hz, 1H), 4.26-3.95
(m, 2H), 3.73-3.50 (m, 2H), 3.35-3.26 (m, 2H), 2.94 (br s, 1H),
2.81-2.65 (m, 2H), 2.37 (br s, 1H), 2.02-1.72 (m, 4H), 1.48 (s,
9H), 1.41-1.30 (m, 6H), 0.88 (t, J = 7.3 Hz, 3H).
1
5.1.23 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-1-(2-
fluorophenyl)propyl]carbamoyl}piperidine-1-carboxylate
(31f)
5.1.28 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-1-(5-fluoro-2-pyridyl)-3-
methylbutyl]carbamoyl}piperidine-1-carboxylate (31k)
The title compound was prepared from 25 and (1R)-1-(2-
fluorophenyl)propan-1-amine hydrochloride 29f in a manner
1
similar to that described for 30 as a colorless solid (70%). H
The title compound was prepared from 25 and (1R)-1-(5-
fluoro-2-pyridyl)-3-methylbutan-1-amine dihydrochloride 29k in
a manner similar to that described for 30 as a colorless solid
(60%). 1H NMR (500 MHz, CDCl3): δ 8.41 (br d, J = 2.9 Hz, 1H),
7.47-7.45 (m, 1H), 7.39-7.35 (m, 1H), 7.31-7.22 (m, 4H), 6.41
(br d, J = 8.3 Hz, 1H), 5.12 (br q, J = 8.3 Hz, 1H), 4.26-4.00 (m,
2H), 3.71-3.50 (m, 2H), 3.29 (br s, 2H), 2.97 (br s, 1H), 2.75-
2.69 (m, 2H), 2.39 (br s, 1H), 1.92-1.86 (m, 2H), 1.69-1.27 (m,
18H), 0.95-0.92 (m, 6H).
NMR (400 MHz, CDCl3): δ 7.48-7.43 (m, 1H), 7.32-7.20 (m,
5H), 7.12-7.01 (m, 2H), 6.14 (br s, 1H), 5.02 (q, J = 8.0 Hz, 1H),
4.25-3.95 (m, 2H), 3.74-3.46 (m, 2H), 3.34-3.24 (m, 2H), 2.96
(br s, 1H), 2.76-2.68 (m, 2H), 2.38 (br s, 1H), 1.96-1.74 (m, 4H),
1.48 (s, 9H), 1.35-1.23 (m, 6H), 0.89-0.86 (m, 3H).
5.1.24 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-1-(3-
fluorophenyl)propyl]carbamoyl}piperidine-1-carboxylate
(31g)
5.1.29 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-1-(5-fluoro-3-pyridyl)-3-
methylbutyl]carbamoyl}piperidine-1-carboxylate (31l)
The title compound was prepared from 25 and (1R)-1-(3-
fluorophenyl)propan-1-amine hydrochloride 29g in a manner
1
similar to that described for 30 as a colorless solid (64%). H
The title compound was prepared from 25 and (1R)-1-(5-
fluoro-3-pyridyl)-3-methylbutan-1-amine dihydrochloride 29l in
a manner similar to that described for 30 as a colorless solid
NMR (400 MHz, CDCl3): δ 7.46 (dd, J = 7.8, 2.2 Hz, 1H), 7.33-
7.22 (m, 4H), 7.05 (d, J = 7.8 Hz, 1H), 6.98-6.93 (m, 2H), 6.24
(br s, 1H), 4.86 (q, J = 7.6 Hz, 1H), 4.25-3.98 (m, 2H), 3.73-3.50
(m, 2H), 3.34-3.27 (m, 2H), 2.96 (br s, 1H), 2.79-2.70 (m, 2H),
2.41 (br s, 1H), 1.99-1.86 (m, 2H), 1.81-1.74 (m, 2H), 1.49 (s,
9H), 1.36-1.23 (m, 6H), 0.89 (t, J = 7.6 Hz, 3H).
1
(46%). H NMR (500 MHz, CDCl3): δ 8.41-8.34 (m, 2H), 7.46
(dd, J = 7.6, 1.7 Hz, 1H), 7.34-7.22 (m, 4H), 5.92 (br s, 1H), 5.06
(q, J = 8.0 Hz, 1H), 4.25-3.96 (m, 2H), 3.73-3.47 (m, 2H), 3.37-
3.25 (m, 2H), 2.96 (br s, 1H), 2.85-2.64 (m, 2H), 2.38 (br s, 1H),
1.99-1.82 (m, 2H), 1.73-1.27 (m, 18H), 0.98-0.94 (m, 6H).
5.1.25 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-1-(4-
fluorophenyl)propyl]carbamoyl}piperidine-1-carboxylate
(31h)
5.1.30 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-1-isoxazol-5-yl-3-
methylbutyl]carbamoyl}piperidine-1-carboxylate (31m)
The title compound was prepared from 25 and (1R)-1-(4-
fluorophenyl)propan-1-amine hydrochloride 29h in a manner
similar to that described for 30 as a colorless solid (65%). H
NMR (500 MHz, CDCl3): δ 7.46 (dd, J = 7.8, 1.5 Hz, 1H), 7.32-
7.21 (m, 5H), 7.05-6.98 (m, 2H), 6.11 (br s, 1H), 4.84 (q, J = 7.6
Hz, 1H), 4.25-3.98 (m, 2H), 3.75-3.49 (m, 2H), 3.35-3.26 (m,
2H), 2.95 (br s, 1H), 2.78-2.70 (m, 2H), 2.37 (br s, 1H), 1.99-
1.72 (m, 4H), 1.47 (s, 9H), 1.35-1.23 (m, 6H), 0.88 (t, J = 7.1 Hz,
3H).
The title compound was prepared from 25 and (1R)-1-
isoxazol-5-yl-3-methylbutan-1-amine hydrochloride 29m in a
manner similar to that described for 30 as a colorless solid (80%).
1H NMR (500 MHz, CDCl3): δ 8.21-8.16 (m, 1H), 7.46 (dd, J =
7.6, 1.2 Hz, 1H), 7.34-7.24 (m, 3H), 6.17-6.10 (m, 1H), 5.93 (br s,
1H), 5.35 (q, J = 8.0 Hz, 1H), 4.24-3.97 (m, 2H), 3.73-3.51 (m,
2H), 3.37-3.25 (m, 2H), 2.96 (br s, 1H), 2.81-2.66 (m, 2H), 2.36
(br s, 1H), 1.95-1.87 (m, 2H), 1.77-1.22 (m, 18H), 0.98-0.92 (m,
6H).
1
5.1.26 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-1-(3-
5.1.31 tert-Butyl (3R,5S)-3-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-5-{[(1R)-3-methyl-1-oxazol-2-yl-
butyl]carbamoyl}piperidine-1-carboxylate (31n)

The title compound was prepared from 25 and (1R)-3-methyl-
1-oxazol-2-yl-butan-1-amine hydrochloride 29n in a manner
similar to that described for 30 as a colorless solid (37%). H
493.2945; found: 493.2963. Anal. C26H41ClN4O3·C4H4O4·H2O;
calcd: C 57.45, H 7.55, N 8.93, Cl 5.65; found: C 57.43, H 7.50,
N 8.82, Cl 5.54.
1
NMR (400 MHz, CDCl3): δ 7.62-7.59 (m, 1H), 7.46 (dd, J = 7.8,
1.6 Hz, 1H), 7.34-7.24 (m, 3H), 7.09-7.04 (m, 1H), 6.16 (br s,
1H), 5.35-5.27 (m, 1H), 4.27-4.08 (m, 2H), 3.76-3.50 (m, 2H),
3.35-3.28 (m, 2H), 2.97 (br s, 1H), 2.79-2.68 (m, 2H), 2.42 (br s,
1H), 2.01-1.92 (m, 2H), 1.80-1.29 (m, 18H), 0.96-0.91 (m, 6H).
5.1.35 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-3-methyl-1-
phenylbutyl]piperidine-3-carboxamide fumarate (32a)
The title compound was prepared from 31a in a manner
similar to that described for 5 as a colorless solid (54%, 2 steps).
1H NMR (500 MHz, CD3OD): δ 7.52-7.22 (m, 9H), 6.70 (s, 2H),
4.94 (br dd, J = 9.3, 5.4 Hz, 1H), 3.63-3.22 (m, 7H), 3.05-2.88 (m,
3H), 2.06-2.00 (m, 1H), 1.87-1.69 (m, 2H), 1.61-1.54 (m, 2H),
1.38-1.35 (m, 6H), 0.97-0.91 (m, 6H). HRMS (ESI+): m/z calcd
for C29H39ClN4O2+H: 511.2840; found: 511.2850.
5.1.32 tert-Butyl (3R,5S)-3-[2,2-dimethyl-4-(o-tolyl)-5-
oxopiperazin-1-yl]-5-{[(1R)-1-(5-fluoro-2-pyridyl)-3-
methylbutyl]carbamoyl}piperidine-1-carboxylate (31o)
The title compound was prepared from 26 and (1R)-1-(5-
fluoro-2-pyridyl)-3-methylbutan-1-amine dihydrochloride 29k in
a manner similar to that described for 30 as a colorless solid
(67%). 1H NMR (500 MHz, CDCl3): δ 8.40 (br s, 1H), 7.39-7.35
(m, 1H), 7.26-7.21 (m, 4H), 7.11-7.05 (m, 1H), 6.49-6.48 (m,
1H), 5.13 (br q, J = 7.7 Hz, 1H), 4.26-4.00 (m, 2H), 3.71-3.49 (m,
2H), 3.35 (br d, J = 11.7 Hz, 1H), 3.19-3.16 (m, 1H), 2.96-2.69
(m, 3H), 2.40 (br s, 1H), 2.23 (s, 1.5H), 2.20 (s, 1.5H), 1.89 (br s,
2H), 1.69-1.28 (m, 18H), 0.95-0.92 (m, 6H).
5.1.36 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-1-phenylbutyl]piperidine-3-
carboxamide fumarate (32b)
The title compound was prepared from 31b in a manner
similar to that described for 5 as a colorless solid (63%, 2 steps).
1H NMR (500 MHz, CD3OD): δ 7.53-7.50 (m, 1H), 7.48-7.23 (m,
8H), 6.70 (s, 2H), 4.87-4.82 (m, 1H), 3.62-3.21 (m, 7H), 3.05-
2.86 (m, 3H), 2.08-2.00 (m, 1H), 1.94-1.93 (m, 1H), 1.87-1.70
(m, 4H), 1.42-1.31 (m, 6H), 0.94 (t, J = 7.3 Hz, 3H). HRMS
(ESI+): m/z calcd for C28H37ClN4O2+H: 497.2683; found:
497.2695.
5.1.33 tert-Butyl (3R,5S)-3-[4-(5-fluoro-2-methylphenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-5-{[(1R)-1-(5-fluoro-2-
pyridyl)-3-methylbutyl]carbamoyl}piperidine-1-carboxylate
(31p)
The title compound was prepared from 27 and (1R)-1-(5-
fluoro-2-pyridyl)-3-methylbutan-1-amine dihydrochloride 29k in
a manner similar to that described for 30 as a colorless solid
(61%). 1H NMR (400 MHz, CDCl3): δ 8.41 (br s, 1H), 7.39-7.35
(m, 1H), 7.26-7.18 (m, 2H), 6.96-6.91 (m, 1H), 6.85-6.80 (m,
1H), 6.46 (br d, J = 8.2 Hz, 1H), 5.13 (br q, J = 7.8 Hz, 1H),
4.29-4.00 (m, 2H), 3.70-3.48 (m, 2H), 3.35 (br d, J = 10.5 Hz,
1H), 3.18-3.14 (m, 1H), 2.96-2.69 (m, 3H), 2.39 (br s, 1H), 2.18
(s, 1.5H), 2.14 (s, 1.5H), 1.88 (br s, 2H), 1.70-1.28 (m, 18H),
0.95-0.92 (m, 6H).
5.1.37 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-1-phenylpropyl]piperidine-3-
carboxamide fumarate (32c)
The title compound was prepared from 31c in a manner
similar to that described for 5 as a colorless solid (68%, 2 steps).
25.0
1
[α]D +19.5 (c 1.02, MeOH). H NMR (500 MHz, CD3OD): δ
7.53-7.51 (m, 1H), 7.41-7.21 (m, 8H), 6.70 (s, 2H), 4.73 (br t, J =
7.3 Hz, 1H), 3.63-3.42 (m, 4H), 3.39-3.20 (m, 3H), 3.06-3.01 (m,
1H), 2.96 (br t, J = 12.2 Hz, 1H), 2.90-2.85 (m, 1H), 2.08-2.02
(m, 1H), 1.89-1.77 (m, 3H), 1.39-1.35 (m, 6H), 0.92 (t, J = 7.3
Hz, 3H). 13C NMR (125 MHz, CD3OD): δ 170.6, 168.0, 166.0,
143.5, 139.2, 135.0, 131.4, 129.9, 129.4, 129.2, 128.2, 128.1,
126.5, 126.2, 61.5, 54.0, 52.9, 49.1, 45.9, 43.9, 31.4, 29.2, 23.9,
23.1, 22.6, 21.1, 10.9. IR: 3318, 2968, 1648, 1551, 1493, 1334,
986, 757, 702 cm-1. HRMS (ESI+): m/z calcd for
5.1.34 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-1-(ethoxymethyl)-3-
methylbutyl]piperidine-3-carboxamide fumarate (5)
Trifluoroacetic acid (0.350 mL, 4.71 mmol) was added to a
solution of 30 (135 mg, 0.228 mmol) in CH2Cl2 (0.700 mL) at
room temperature, and the mixture was stirred at the same
temperature for 15 min. Saturated NaHCO3 aqueous solution was
added to the reaction mixture under ice-cooling, followed by
extraction with CH2Cl2. Then, the organic layer was washed with
brine, and dried over anhydrous Na2SO4. After filtration, the
solvent was evaporated under reduced pressure, and the residue
was purified by NH silica gel column chromatography (eluent,
CH2Cl2/MeOH = 20/1 to 7/3) to obtain the free base of 5 (94.0
mg, 0.190 mmol). Fumaric acid (22.0 mg, 0.190 mmol) was
added to a solution of the free base of 5 (94.0 mg, 0.190 mmol)
in MeOH (1.00 mL) at room temperature, and the mixture was
stirred at the same temperature for 5 min. The solvent was
evaporated under reduced pressure, and then 5 (97.0 mg, 70%, 2
steps) was obtained as a colorless solid by crystallization from
C27H35ClN4O2+H:
483.2527;
found:
483.2541.
Anal.
C27H35ClN4O2·C4H4O4·H2O; calcd: C 60.33, H 6.70, N 9.08, Cl
5.74; found: C 60.61, H 6.57, N 9.13, Cl 5.75.
5.1.38 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-3-methyl-1-(2-
pyridyl)butyl]piperidine-3-carboxamide fumarate (32d)
The title compound was prepared from 31d in a manner
similar to that described for 5 as a colorless solid (71%, 2 steps).
1H NMR (500 MHz, CD3OD): δ 8.51 (br d, J = 4.9 Hz, 1H), 7.79
(br t, J = 7.6 Hz, 1H), 7.52-7.51 (m, 1H), 7.41-7.35 (m, 3H),
7.31-7.28 (m, 2H), 6.70 (s, 2H), 5.03 (dd, J = 9.3, 5.9 Hz, 1H),
3.64-3.44 (m, 4H), 3.39-3.22 (m, 3H), 3.06-3.01 (m, 1H), 2.98-
2.89 (m, 2H), 2.17-2.11 (m, 1H), 1.87-1.78 (m, 1H), 1.76-1.58
(m, 3H), 1.41-1.36 (m, 6H), 0.98 (d, J = 6.8 Hz, 3H), 0.95 (d, J =
6.3 Hz, 3H). HRMS (ESI+): m/z calcd for C28H38ClN5O2+H:
512.2792; found: 512.2813.
25.0
1
CH2Cl2 and Et2O. [α]D +18.3 (c 1.05, MeOH). H NMR (400
MHz, CD3OD): δ 7.54-7.52 (m, 1H), 7.43-7.32 (m, 3H), 6.69 (s,
2H), 4.12-4.08 (m, 1H), 3.69-2.94 (m, 13H), 2.84-2.78 (m, 1H),
2.16-2.10 (m, 1H), 1.98-1.89 (m, 1H), 1.67-1.58 (m, 1H), 1.54-
1.32 (m, 8H), 1.17 (t, J = 7.0 Hz, 3H), 0.95-0.90 (m, 6H). 13C
NMR (125 MHz, CD3OD): δ 173.0, 171.4, 169.4, 140.1, 136.3,
133.3, 131.5, 130.9, 130.5, 129.5, 73.8, 67.5, 63.5, 54.8, 50.3,
47.7, 47.0, 45.6, 41.5, 32.6, 26.0, 25.1, 24.3, 23.7, 23.1, 22.3,
21.7, 15.6. IR: 2967, 2870, 1648, 1556, 1386, 1272, 1174, 984,
755 cm-1. HRMS (ESI+): m/z calcd for C26H41ClN4O3+H:
5.1.39 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-3-methyl-1-(3-
pyridyl)butyl]piperidine-3-carboxamide fumarate (32e)
The title compound was prepared from 31e in a manner
similar to that described for 5 as a colorless solid (66%, 2 steps).
1H NMR (500 MHz, CD3OD): δ 8.51 (br s, 1H), 8.45 (br d, J =

5.1 Hz, 1H), 7.82-7.79 (m, 1H), 7.53-7.51 (m, 1H), 7.45-7.26 (m,
4H), 6.72 (s, 2H), 4.99 (dd, J = 9.4, 5.5 Hz, 1H), 3.63-3.22 (m,
7H), 3.09-2.85 (m, 3H), 2.10-2.01 (m, 1H), 1.87-1.75 (m, 2H),
1.64-1.57 (m, 2H), 1.39-1.36 (m, 6H), 0.99 (d, J = 6.3 Hz, 3H),
0.94 (d, J = 6.3 Hz, 3H). HRMS (ESI+): m/z calcd for
C28H38ClN5O2+H: 512.2792; found: 512.2813.
The title compound was prepared from 31j in a manner
similar to that described for 5 as a colorless solid (64%, 2 steps).
1H NMR (500 MHz, CD3OD): δ 7.54-7.50 (m, 1H), 7.40-7.26 (m,
7H), 6.70 (s, 2H), 4.71 (t, J = 7.3 Hz, 1H), 3.64-3.22 (m, 7H),
3.07-2.86 (m, 3H), 2.09-2.01 (m, 1H), 1.89-1.77 (m, 3H), 1.41-
1.34 (m, 6H), 0.92 (t, J = 7.5 Hz, 3H). HRMS (ESI+): m/z calcd
for C27H34Cl2N4O2+H: 517.2137; found: 517.2144.
5.1.40 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-1-(2-
fluorophenyl)propyl]piperidine-3-carboxamide fumarate
(32f)
5.1.45 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-1-(5-fluoro-2-pyridyl)-3-
methylbutyl]piperidine-3-carboxamide fumarate (32k)
The title compound was prepared from 31f in a manner
similar to that described for 5 as a colorless solid (68%, 2 steps).
1H NMR (500 MHz, CD3OD): δ 7.52 (br d, J = 7.8 Hz, 1H),
7.41-7.26 (m, 5H), 7.16 (dt, J = 7.3, 1.0 Hz, 1H), 7.08 (ddd, J =
10.7, 8.3, 1.0 Hz, 1H), 6.71 (s, 2H), 5.01 (br t, J = 7.6 Hz, 1H),
3.63-3.43 (m, 4H), 3.39-3.22 (m, 3H), 3.07-3.02 (m, 1H), 2.99-
2.89 (m, 2H), 2.11-2.04 (m, 1H), 1.89-1.79 (m, 3H), 1.40-1.36
(m, 6H), 0.93 (t, J = 7.3 Hz, 3H). HRMS (ESI+): m/z calcd for
C27H34ClFN4O2+H: 501.2433; found: 501.2440.
The title compound was prepared from 31k in a manner
similar to that described for 5 as a colorless solid (78%, 2 steps).
25.0
1
[α]D +16.6 (c 1.16, MeOH). H NMR (500 MHz, CD3OD): δ
8.41 (br d, J = 2.0 Hz, 1H), 7.58-7.50 (m, 2H), 7.42-7.35 (m, 3H),
7.33-7.30 (m, 1H), 6.71 (s, 2H), 5.05 (dd, J = 9.3, 6.4 Hz, 1H),
3.64-3.44 (m, 4H), 3.39-3.21 (m, 3H), 3.04 (br t, J = 11.7 Hz,
1H), 2.99-2.89 (m, 2H), 2.15-2.09 (m, 1H), 1.86-1.78 (m, 1H),
1.76-1.64 (m, 2H), 1.61-1.55 (m, 1H), 1.40-1.36 (m, 6H), 0.97 (d,
J = 6.8 Hz, 3H), 0.94 (d, J = 6.3 Hz, 3H). 13C NMR (125 MHz,
CD3OD): δ 172.8, 171.5, 169.4, 161.2, 159.1, 140.1, 138.3, 136.3,
133.3, 131.5, 130.8, 130.4, 129.5, 125.0, 123.8, 63.4, 54.8, 53.7,
50.3, 47.7, 47.0, 45.5, 41.2, 32.6, 26.1, 25.1, 24.2, 23.3, 22.3,
21.8. IR: 3279, 2961, 1649, 1552, 1485, 1387, 1228, 983, 752,
633 cm-1. HRMS (ESI+): m/z calcd for C28H37ClFN5O2+H:
5.1.41 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-1-(3-
fluorophenyl)propyl]piperidine-3-carboxamide fumarate
(32g)
530.2698;
found:
530.2700.
Anal.
The title compound was prepared from 31g in a manner
similar to that described for 5 as a colorless solid (70%, 2 steps).
1H NMR (500 MHz, CD3OD): δ 7.52-7.51 (m, 1H), 7.40-7.27 (m,
4H), 7.12 (br d, J = 7.8 Hz, 1H), 7.06-7.04 (m, 1H), 7.00-6.96 (m,
1H), 6.70 (s, 2H), 4.74 (br t, J = 7.3 Hz, 1H), 3.64-3.42 (m, 4H),
3.39-3.22 (m, 3H), 3.07-2.89 (m, 3H), 2.10-2.02 (m, 1H), 1.89-
1.78 (m, 3H), 1.39-1.36 (m, 6H), 0.93 (t, J = 7.3 Hz, 3H). HRMS
(ESI+): m/z calcd for C27H34ClFN4O2+H: 501.2433; found:
501.2444.
C28H37ClFN5O2·C4H4O4·0.5H2O; calcd: C 58.66, H 6.46, N 10.69,
Cl 5.41, F 2.90; found: C 58.70, H 6.58, N 10.73, Cl 5.43, F 2.86.
5.1.46 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-1-(5-fluoro-3-pyridyl)-3-
methylbutyl]piperidine-3-carboxamide fumarate (32l)
The title compound was prepared from 31l in a manner similar
to that described for 5 as a colorless solid (72%, 2 steps). H
1
NMR (400 MHz, CD3OD): δ 8.39-8.37 (m, 2H), 7.62-7.59 (m,
1H), 7.51 (br d, J = 7.8 Hz, 1H), 7.41-7.35 (m, 2H), 7.31-7.27
(m, 1H), 6.70 (s, 2H), 5.02 (dd, J = 9.5, 5.6 Hz, 1H), 3.64-3.42
(m, 4H), 3.39-3.21 (m, 3H), 3.06-3.00 (m, 1H), 2.98-2.86 (m,
2H), 2.09-2.01 (m, 1H), 1.88-1.76 (m, 2H), 1.67-1.58 (m, 2H),
1.39-1.36 (m, 6H), 0.99 (d, J = 6.4 Hz, 3H), 0.96 (d, J = 6.4 Hz,
3H). HRMS (ESI+): m/z calcd for C28H37ClFN5O2+H: 530.2698;
found: 530.2725.
5.1.42 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-1-(4-
fluorophenyl)propyl]piperidine-3-carboxamide fumarate
(32h)
The title compound was prepared from 31h in a manner
similar to that described for 5 as a colorless solid (67%, 2 steps).
1H NMR (400 MHz, CD3OD): δ 7.53-7.51 (m, 1H), 7.38-7.28 (m,
5H), 7.09-7.04 (m, 2H), 6.70 (s, 2H), 4.73 (br t, J = 7.4 Hz, 1H),
3.64-3.22 (m, 7H), 3.07-2.86 (m, 3H), 2.08-2.00 (m, 1H), 1.89-
1.77 (m, 3H), 1.38-1.36 (m, 6H), 0.91 (t, J = 7.4 Hz, 3H). HRMS
(ESI+): m/z calcd for C27H34ClFN4O2+H: 501.2433; found:
501.2435.
5.1.47 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-1-isoxazol-5-yl-3-
methylbutyl]piperidine-3-carboxamide fumarate (32m)
The title compound was prepared from 31m in a manner
similar to that described for 5 as a colorless solid (66%, 2 steps).
1H NMR (500 MHz, CD3OD): δ 8.33-8.31 (m, 1H), 7.53-7.51 (m,
1H), 7.42-7.28 (m, 3H), 6.70 (s, 2H), 6.31-6.29 (m, 1H), 5.26 (dd,
J = 9.3, 6.3 Hz, 1H), 3.68-3.23 (m, 7H), 3.08-2.86 (m, 3H), 2.14-
2.08 (m, 1H), 1.95-1.87 (m, 1H), 1.82-1.74 (m, 2H), 1.68-1.61
(m, 1H), 1.41-1.33 (m, 6H), 0.99 (d, J = 6.8 Hz, 3H), 0.95 (d, J =
6.8 Hz, 3H). HRMS (ESI+): m/z calcd for C26H37ClN5O3+H:
502.2585; found: 502.2588.
5.1.43 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-1-(3-
chlorophenyl)propyl]piperidine-3-carboxamide fumarate
(32i)
The title compound was prepared from 31i in a manner similar
to that described for 5 as a colorless solid (50%, 2 steps). H
1
NMR (500 MHz, CD3OD): δ 7.53-7.50 (m, 1H), 7.39-7.23 (m,
7H), 6.70 (s, 2H), 4.71 (t, J = 7.6 Hz, 1H), 3.73-3.49 (m, 4H),
3.47-3.42 (m, 1H), 3.40-3.22 (m, 2H), 3.06-2.88 (m, 3H), 2.12-
2.02 (m, 1H), 1.88-1.77 (m, 3H), 1.42-1.35 (m, 6H), 0.93 (t, J =
7.3 Hz, 3H). HRMS (ESI+): m/z calcd for C27H34Cl2N4O2+H:
517.2137; found: 517.2132.
5.1.48 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-3-methyl-1-oxazol-2-yl-
butyl]piperidine-3-carboxamide fumarate (32n)
The title compound was prepared from 31n in a manner
similar to that described for 5 as a colorless solid (34%, 2 steps).
1H NMR (500 MHz, CD3OD): δ 7.88-7.86 (m, 1H), 7.53 (dd, J =
7.6, 1.7 Hz, 1H), 7.41-7.35 (m, 2H), 7.33-7.30 (m, 1H), 7.14-7.12
(m, 1H), 6.70 (d, J = 2.9 Hz, 2H), 5.17 (dd, J = 8.5, 7.1 Hz, 1H),
3.69-3.22 (m, 7H), 3.08-2.95 (m, 2H), 2.92-2.84 (m, 1H), 2.23-
2.15 (m, 1H), 1.94-1.85 (m, 1H), 1.82-1.77 (m, 2H), 1.65-1.60
5.1.44 (3S,5R)-5-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-N-[(1R)-1-(4-
chlorophenyl)propyl]piperidine-3-carboxamide fumarate
(32j)

(m, 1H), 1.43-1.36 (m, 6H), 0.98 (d, J = 6.8 Hz, 3H), 0.94 (d, J =
6.8 Hz, 3H). HRMS (ESI+): m/z calcd for C26H37ClN5O3+H:
502.2585; found: 502.2603.
(eluent, n-hexane/AcOEt = 1/0 to 5/1) to obtain 35 (1.78 g, 81%)
as a colorless liquid. 1H NMR (400 MHz, CDCl3): δ 7.37 (br d, J
= 7.4 Hz, 4H), 7.30-7.26 (m, 4H), 7.22-7.17 (m, 2H), 3.75 (d, J =
13.7 Hz, 2H), 3.66-3.61 (m, 3H), 3.45 (q, J = 7.0 Hz, 2H), 3.38
(dd, J = 9.8, 5.1 Hz, 1H), 2.89-2.83 (m, 1H), 1.84-1.74 (m, 1H),
1.46 (ddd, J = 13.7, 8.2, 5.4 Hz, 1H), 1.22 (t, J = 7.0 Hz, 3H),
1.10 (ddd, J = 13.7, 8.2, 5.4 Hz, 1H), 0.82 (d, J = 6.3 Hz, 3H),
0.59 (d, J = 6.3 Hz, 3H).
5.1.49 (3S,5R)-5-[2,2-Dimethyl-4-(o-tolyl)-5-oxopiperazin-1-
yl]-N-[(1R)-1-(5-fluoro-2-pyridyl)-3-methylbutyl]piperidine-
3-carboxamide fumarate (32o)
The title compound was prepared from 31o in a manner
similar to that described for 5 as a colorless solid (74%, 2 steps).
5.1.53 (2R)-1-Ethoxy-4-methylpentan-2-amine (28)
25.0
1
[α]D +15.3 (c 1.19, MeOH). H NMR (500 MHz, CD3OD): δ
8.41 (br s, 1H), 7.59-7.54 (m, 1H), 7.41-7.38 (m, 1H), 7.30-7.24
(m, 3H), 7.12-7.09 (m, 1H), 6.70 (s, 2H), 5.06-5.03 (m, 1H),
3.64-3.46 (m, 4H), 3.41-3.19 (m, 3H), 3.04 (t, J = 12.2 Hz, 1H),
2.98-2.87 (m, 2H), 2.19 (s, 1.5H), 2.17 (s, 1.5H), 2.13-2.09 (m,
1H), 1.86-1.57 (m, 4H), 1.38-1.36 (m, 6H), 0.98-0.93 (m, 6H).
13C NMR (125 MHz, DMSO-d6): δ 170.8, 168.1, 165.9, 158.9,
158.7, 156.9, 141.0, 136.6, 136.4, 135.1, 130.5, 127.4, 126.8,
123.6, 121.8, 61.9, 52.9, 51.7, 48.9, 45.9, 45.6, 43.9, 43.7, 31.3,
24.4, 24.0, 23.0, 22.7, 21.6, 21.0, 17.3. IR: 3296, 2960, 1643,
1485, 1387, 1228, 983, 749, 647 cm-1. HRMS (ESI+): m/z calcd
for C29H40FN5O2+H: 510.3244; found: 510.3235. Anal.
C29H40FN5O2·C4H4O4·0.5H2O; calcd: C 62.44, H 7.15, N 11.03, F
2.99; found: C 62.47, H 7.41, N 10.97, F 2.88.
To a suspension of 35 (1.78 g, 5.47 mmol) in MeOH (25.0
mL), 20% Pd(OH)2 on carbon (50% wet, 360 mg) were added.
The suspension was stirred under 1 MPa H2 at room temperature
for 6 h. H2 in the reaction vessel was replaced by N2, and then
Pd(OH)2 on carbon was separated by filtration. The solvent was
evaporated under reduced pressure to obtain 28 (799 mg, 98%) as
a colorless liquid. [α]D −11.8 (c 1.03, MeOH). H NMR (500
MHz, CDCl3): δ 3.55-3.45 (m, 2H), 3.39 (dd, J = 9.0, 3.7 Hz, 1H),
3.12 (t, J = 8.6 Hz, 1H), 3.05-3.00 (m, 1H), 1.77-1.69 (m, 1H),
1.22-1.13 (m, 5H), 0.93 (d, J = 6.8 Hz, 3H), 0.90 (d, J = 6.8 Hz,
3H). HRMS (ESI+): m/z calcd for C8H19NO+H: 146.1545; found:
146.1552.
25.0
1
5.1.54 [(1R)-1-Azidobutyl]benzene (37)
5.1.50 (3S,5R)-5-[4-(5-Fluoro-2-methylphenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-N-[(1R)-1-(5-fluoro-2-pyridyl)-3-
methylbutyl]piperidine-3-carboxamide fumarate (32p)
DBU (2.40 mL, 16.1 mmol) and diphenylphosphoryl azide
(3.5 mL, 16.3 mmol) were added to a solution of (1S)-1-
phenylbutan-1-ol 36 (2.00 g, 13.3 mmol) in toluene (20 mL) at rt,
and the mixture was stirred at the same temperature for 2 days.
Water was added to the reaction mixture, followed by extraction
with AcOEt. Then, the organic layer was washed with brine, and
dried over anhydrous Na2SO4. After filtration, the solvent was
evaporated under reduced pressure, and the residue was purified
by silica gel column chromatography (eluent, n-hexane/AcOEt =
The title compound was prepared from 31p in a manner
similar to that described for 5 as a colorless solid (72%, 2 steps).
1H NMR (500 MHz, CD3OD): δ 8.41 (br s, 1H), 7.59-7.52 (m,
1H), 7.43-7.38 (m, 1H), 7.31-7.28 (m, 1H), 7.04-7.00 (m, 1H),
6.96-6.93 (m, 1H), 6.69 (s, 2H), 5.04 (br dd, J = 8.4, 6.5 Hz, 1H),
3.64-3.18 (m, 7H), 3.02 (br t, J = 12.1 Hz, 1H), 2.98-2.90 (m,
2H), 2.16-2.07 (m, 4H), 1.89-1.56 (m, 4H), 1.37-1.35 (m, 6H),
0.97 (d, J = 6.7 Hz, 3H), 0.94 (d, J = 6.7 Hz, 3H). HRMS (ESI+):
m/z calcd for C29H39F2N5O2+H: 528.3150; found: 528.3162.
1
1/0 to 9/1) to obtain 37 (0.650 g, 28%) as a colorless liquid. H
NMR (500 MHz, CDCl3): δ 7.40-7.36 (m, 2H), 7.33-7.29 (m,
3H), 4.41 (t, J = 7.3 Hz, 1H), 1.87-1.79 (m, 1H), 1.76-1.68 (m,
1H), 1.45-1.38 (m, 1H), 1.35-1.27 (m, 1H), 0.93 (t, J = 7.4 Hz,
3H).
5.1.51 (2R)-2-(Dibenzylamino)-4-methylpentan-1-ol (34)
K2CO3 (14.7 g, 107 mmol) was added to a solution of (R)-
leucinol 33 (5.00 g, 42.7 mmol) and benzyl bromide (11.2 mL,
93.9 mmol) in ethanol (200 mL) at room temperature, and the
mixture was stirred at the same temperature for 4 days. The
reaction mixture was concentrated under reduced pressure and
diluted with water, followed by extraction with AcOEt. Then, the
organic layer was washed with brine and dried over anhydrous
Na2SO4. After filtration, the solvent was evaporated under
reduced pressure, and the residue was purified by silica gel
column chromatography (eluent, n-hexane/AcOEt = 10/1 to 1/1)
5.1.55 (1R)-1-Phenylbutan-1-amine hydrochloride (29b)
To a suspension of 37 (650 mg, 3.71 mmol) in AcOEt (6.00
mL) and EtOH (6.00 mL), 10% Pd on carbon (50% wet, 130 mg)
were added. The suspension was stirred under H2 atmosphere at
room temperature for 3 h. H2 in the reaction vessel was replaced
by N2, and then Pd on carbon was separated by filtration. The
solvent was evaporated under reduced pressure. 4N-HCl in 1,4-
dioxane (3.60 mL, 14.4 mmol) was added to the residue at room
temperature, and the mixture was evaporated under reduced
pressure. The residue was washed with AcOEt to obtain 29b (430
1
to obtain 34 (10.8 g, 85%) as a colorless liquid. H NMR (400
1
MHz, CDCl3): δ 7.33-7.21 (m, 10H), 3.81 (d, J = 13.3 Hz, 2H),
3.48 (dd, J = 10.6, 5.1 Hz, 1H), 3.42 (d, J = 10.6 Hz, 1H), 3.37 (d,
J = 13.3 Hz, 2H), 3.19 (br s, 1H), 2.84 (ddt, J = 9.8, 5.1, 2.4 Hz,
1H), 1.55-1.46 (m, 2H), 1.20-1.11 (m, 1H), 0.92 (d, J = 6.3 Hz,
3H), 0.86 (d, J = 6.3 Hz, 3H).
mg, 63%, 2 steps) as a colorless solid. H NMR (500 MHz,
CD3OD): δ 7.48-7.40 (m, 5H), 4.24 (dd, J = 9.3, 6.3 Hz, 1H),
1.98-1.91 (m, 2H), 1.35-1.28 (m, 1H), 1.26-1.18 (m, 1H), 0.95 (t,
J = 7.6 Hz, 3H).
5.1.56 (SS)-2-Methyl-N-[(1R)-3-methyl-1-
5.1.52 (2R)-N,N-Dibenzyl-1-ethoxy-4-methylpentan-2-amine
phenylbutyl]propane-2-sulfinamide (40a)
(35)
Ti(OEt)4 (6.82 g, 29.9 mol) was added to a solution of 3-
methyl-1-phenylbutan-1-one 38a (27.0 g, 105 mmol) and (S)-(−)-
2-methylpropane-2-sulfinamide 39 (2.00 g, 16.5 mmol) in THF
(36.0 mL) at rt, and the mixture was stirred at 70 °C for 5.5 h.
After cooling, brine was added to the reaction mixture. The
mixture was filtered, and then the solvent was extracted with
AcOEt. Then, the organic layer was washed with brine, and dried
over anhydrous Na2SO4. After filtration, the solvent was
evaporated under reduced pressure, and the residue was purified
by silica gel column chromatography (eluent, n-hexane/AcOEt =
NaH (60%, dispersion in paraffin liquid) (349 mg, 8.74 mmol)
was added to a solution of 34 (2.00 g, 6.72 mmol) and EtI (0.700
mL, 8.74 mmol) in THF (30.0 mL) at room temperature over a
period of 10 min. The reaction mixture was stirred at room
temperature for 18 h. Water was added to the reaction mixture,
followed by extraction with AcOEt. Then, the organic layer was
washed with brine, and dried over anhydrous Na2SO4. After
filtration, the solvent was evaporated under reduced pressure, and
the residue was purified by silica gel column chromatography

1/0 to 7/3) to obtain (SS)-2-methyl-N-[(1E)-3-methyl-1-
5.1.62 (SS)-N-[(1R)-1-(3-Chlorophenyl)propyl]-2-
phenylbutylidene]propane-2-sulfinamide (2.31 g, 47%) as a
colorless liquid. H NMR (500 MHz, CDCl3): δ 7.82-7.80 (m,
methylpropane-2-sulfinamide (40i)
1
The title compound was prepared from 1-(3-
chlorophenyl)propan-1-one 38i in a manner similar to that
described for 40a as a colorless liquid (57%, 2 steps). H NMR
(500 MHz, CDCl3): δ 7.29-7.24 (m, 3H), 7.19-7.16 (m, 1H),
4.29-4.26 (m, 1H), 3.38 (br s, 1H), 1.87-1.74 (m, 2H), 1.20 (s,
9H), 0.85 (t, J = 7.4 Hz, 3H).
2H), 7.48-7.40 (m, 3H), 3.35-3.31 (m, 1H), 3.08-3.05 (m, 1H),
2.10-2.02 (m, 1H), 1.32 (s, 9H), 0.98-0.96 (m, 6H). 1M L-
selectride in THF (26.0 mL, 26.0 mmol) was added to a solution
of (SS)-2-methyl-N-[(1E)-3-methyl-1-phenylbutylidene]propane-
2-sulfinamide (2.31 g, 8.70 mmol) in THF (20 mL) under ice-
cooling over a period of 5 min, and the mixture was stirred at the
same temperature for 1 h. The reaction mixture was evaporated
under reduced pressure, and the residue was purified by silica gel
column chromatography (eluent, n-hexane/AcOEt = 1/0 to 1/1) to
1
5.1.63 (SS)-N-[(1R)-1-(4-Chlorophenyl)propyl]-2-
methylpropane-2-sulfinamide (40j)
1
The title compound was prepared from 1-(4-
chlorophenyl)propan-1-one 38j in a manner similar to that
described for 40a as a colorless liquid (60%, 2 steps). 1H NMR
(400 MHz, CDCl3): δ 7.32-7.29 (m, 2H), 7.24-7.21 (m, 2H),
4.30-4.26 (m, 1H), 3.37 (br s, 1H), 1.86-1.72 (m, 2H), 1.18 (s,
9H), 0.84 (t, J = 7.3 Hz, 3H).
obtain 40a (1.93 g, 83%) as a colorless liquid. H NMR (500
MHz, CDCl3): δ 7.34-7.25 (m, 5H), 4.47-4.43 (m, 1H), 3.33 (br s,
1H), 1.72-1.63 (m, 2H), 1.48-1.40 (m, 1H), 1.16 (s, 9H), 0.94 (d,
J = 6.4 Hz, 3H), 0.89 (d, J = 6.4 Hz, 3H).
5.1.57 (SS)-2-Methyl-N-[(1R)-3-methyl-1-(2-
pyridyl)butyl]propane-2-sulfinamide (40d)
5.1.64 (SS)-N-[(1R)-1-(5-Fluoro-2-pyridyl)-3-methylbutyl]-2-
methylpropane-2-sulfinamide (40k)
The title compound was prepared from 3-methyl-1-(2-
pyridyl)butan-1-one 38d in a manner similar to that described for
40a as a colorless liquid (63%, 2 steps). H NMR (500 MHz,
1
The title compound was prepared from 1-(5-fluoro-2-pyridyl)-
3-methylbutan-1-one 38k22 in a manner similar to that described
for 40a as a colorless liquid (53%, 2 steps). 1H NMR (400 MHz,
CDCl3): δ 8.43 (d, J = 2.7 Hz, 1H), 7.36 (dt, J = 8.6, 2.7 Hz, 1H),
7.29-7.26 (m, 1H), 4.55 (dt, J = 7.4, 5.5 Hz, 1H), 3.73 (br d, J =
5.5 Hz, 1H), 1.84-1.72 (m, 2H), 1.54-1.44 (m, 1H), 1.15 (s, 9H),
0.95 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H).
CDCl3): δ 8.58-8.57 (m, 1H), 7.65 (dt, J = 7.6, 1.8 Hz, 1H), 7.27-
7.24 (m, 1H), 7.18 (ddd, J = 7.6, 4.9, 1.2 Hz, 1H), 4.54-4.50 (m,
1H), 3.85 (d, J = 5.9 Hz, 1H), 1.85-1.76 (m, 2H), 1.56-1.51 (m,
1H), 1.15 (s, 9H), 0.95 (d, J = 7.0 Hz, 3H), 0.92 (d, J = 7.0 Hz,
3H).
5.1.58 (SS)-2-Methyl-N-[(1R)-3-methyl-1-(3-
pyridyl)butyl]propane-2-sulfinamide (40e)
5.1.65 (SS)-N-[(1R)-1-(5-Fluoro-3-pyridyl)-3-methylbutyl]-2-
methylpropane-2-sulfinamide (40l)
The title compound was prepared from 3-methyl-1-(3-
The title compound was prepared from 1-(5-fluoro-3-pyridyl)-
3-methylbutan-1-one 38l in a manner similar to that described for
40a as a colorless liquid (41%, 2 steps). 1H NMR (400 MHz,
CDCl3): δ 8.41-8.40 (m, 2H), 7.40-7.37 (m, 1H), 4.54 (dt, J = 7.2,
1.7 Hz, 1H), 3.56 (d, J = 2.0 Hz, 1H), 1.76-1.63 (m, 2H), 1.53-
1.42 (m, 1H), 1.19 (s, 9H), 0.96 (d, J = 6.6 Hz, 3H), 0.93 (d, J =
6.6 Hz, 3H).
pyridyl)butan-1-one 38e in a manner similar to that described for
1
40a as a colorless liquid (45%, 2 steps). H NMR (400 MHz,
CDCl3): δ 8.57-8.54 (m, 2H), 7.64-7.61 (m, 1H), 7.30-7.27 (m,
1H), 4.50 (dt, J = 7.4, 2.3 Hz, 1H), 3.40 (d, J = 1.5 Hz, 1H), 1.70
(t, J = 7.3 Hz, 2H), 1.48-1.43 (m, 1H), 1.17 (s, 9H), 0.95 (d, J =
6.3 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H).
5.1.59 (SS)-N-[(1R)-1-(2-Fluorophenyl)propyl]-2-
methylpropane-2-sulfinamide (40f)
5.1.66 (SS)-N-[(1R)-1-Isoxazol-5-yl-3-methylbutyl]-2-
methylpropane-2-sulfinamide (40m)
The title compound was prepared from 1-(2-
The title compound was prepared from 1-isoxazol-5-yl-3-
methylbutan-1-one 38m22 in a manner similar to that described
for 40a as a colorless liquid (69%, 2 steps). 1H NMR (500 MHz,
CDCl3): δ 8.18 (d, J = 1.9 Hz, 1H), 6.18 (d, J = 1.9 Hz, 1H), 4.70
(dt, J = 7.6, 5.0 Hz, 1H), 3.38 (d, J = 4.9 Hz, 1H), 1.90-1.77 (m,
2H), 1.64-1.59 (m, 1H), 1.19 (s, 9H), 0.98-0.94 (m, 6H).
fluorophenyl)propan-1-one 38f in a manner similar to that
1
described for 40a as a colorless liquid (49%, 2 steps). H NMR
(500 MHz, CDCl3): δ 7.31-7.23 (m, 2H), 7.14-7.10 (m, 1H),
7.05-7.01 (m, 1H), 4.62-4.58 (m, 1H), 3.44 (d, J = 4.4 Hz, 1H),
1.99-1.84 (m, 2H), 1.17 (s, 9H), 0.89 (t, J = 7.6 Hz, 3H).
5.1.60 (SS)-N-[(1R)-1-(3-Fluorophenyl)propyl]-2-
methylpropane-2-sulfinamide (40g)
5.1.67 (SS)-2-Methyl-N-[(1R)-3-methyl-1-oxazol-2-yl-
butyl]propane-2-sulfinamide (40n)
The title compound was prepared from 1-(3-
The title compound was prepared from 3-methyl-1-oxazol-2-
yl-butan-1-one 38n in a manner similar to that described for 40a
as a colorless liquid (58%, 2 steps). 1H NMR (400 MHz, CDCl3):
δ 7.61 (s, 1H), 7.08 (s, 1H), 4.68-4.62 (m, 1H), 3.53 (d, J = 5.9
Hz, 1H), 1.97-1.82 (m, 2H), 1.63-1.57 (m, 1H), 1.17 (s, 9H), 0.95
(d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H).
fluorophenyl)propan-1-one 38g in a manner similar to that
described for 40a as a colorless liquid (60%, 2 steps). H NMR
(400 MHz, CDCl3): δ 7.32-7.28 (m, 1H), 7.09-7.05 (m, 1H),
7.02-6.94 (m, 2H), 4.32-4.28 (m, 1H), 3.38 (br s, 1H), 1.88-1.74
(m, 2H), 1.19 (s, 9H), 0.85 (t, J = 7.4 Hz, 3H).
1
5.1.61 (SS)-N-[(1R)-1-(4-Fluorophenyl)propyl]-2-
methylpropane-2-sulfinamide (40h)
5.1.68 (1R)-3-Methyl-1-phenylbutan-1-amine hydrochloride
(29a)
The title compound was prepared from 1-(4-
4N-HCl in 1,4-dioxane (3.60 mL, 14.4 mmol) was added to a
solution of 40a (1.93 g, 7.22 mmol) in methanol (3.60 mL) at
room temperature, and the mixture was stirred at the same
temperature for 40 min. Et2O was added to the reaction mixture,
and then the precipitate was collected by filtration to obtain 29a
(1.30 g, 90%) as a colorless solid. 1H NMR (500 MHz, CD3OD):
δ 7.49-7.41 (m, 5H), 4.31 (dd, J = 10.0, 5.6 Hz, 1H), 1.93 (ddd, J
fluorophenyl)propan-1-one 38h in a manner similar to that
1
described for 40a as a colorless liquid (70%, 2 steps). H NMR
(400 MHz, CDCl3): δ 7.28-7.23 (m, 2H), 7.05-7.00 (m, 2H),
4.31-4.25 (m, 1H), 3.38 (br s, 1H), 1.87-1.72 (m, 2H), 1.18 (s,
9H), 0.83 (t, J = 7.3 Hz, 3H).

25.0
1
= 15.6, 10.3, 5.4 Hz, 1H), 1.81-1.75 (m, 1H), 1.44-1.36 (m, 1H),
0.96 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H).
[α]D −4.39 (c 1.00, MeOH). H NMR (500 MHz, CD3OD): δ
8.56 (d, J = 2.9 Hz, 1H), 7.67 (dt, J = 8.6, 2.9 Hz, 1H), 7.53 (dd,
J = 8.8, 4.4 Hz, 1H), 4.51-4.48 (m, 1H), 1.86 (ddd, J = 13.7, 8.3,
6.4 Hz, 1H), 1.76 (ddd, J = 13.7, 7.8, 6.4 Hz, 1H), 1.51-1.43 (m,
1H), 1.00 (d, J = 6.4 Hz, 3H), 0.94 (d, J = 6.4 Hz, 3H). HRMS
(ESI+): m/z calcd for C10H15FN2+H: 183.1298; found: 183.1294.
5.1.69 (1R)-3-Methyl-1-(2-pyridyl)butan-1-amine
dihydrochloride (29d)
The title compound was prepared from 40d in a manner
1
similar to that described for 29a as a colorless solid (89%). H
5.1.77 (1R)-1-(5-Fluoro-3-pyridyl)-3-methylbutan-1-amine
dihydrochloride (29l)
NMR (500 MHz, CD3OD): δ 8.81-8.79 (m, 1H), 8.28 (dt, J = 7.8,
1.5 Hz, 1H), 7.86 (br d, J = 7.8 Hz, 1H), 7.75 (ddd, J = 7.8, 5.4,
1.0 Hz, 1H), 4.66 (t, J = 7.3 Hz, 1H), 1.99-1.87 (m, 2H), 1.57-
1.49 (m, 1H), 1.02 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H).
The title compound was prepared from 40l in a manner similar
to that described for 29a as a colorless solid (88%). H NMR
1
(500 MHz, CD3OD): δ 8.71 (d, J = 2.0 Hz, 1H), 8.67 (br s, 1H),
8.03 (dt, J = 8.8, 2.0 Hz, 1H), 4.58 (dd, J = 9.3, 6.4 Hz, 1H),
2.00-1.94 (m, 1H), 1.91-1.86 (m, 1H), 1.49-1.41 (m, 1H), 0.99 (d,
J = 6.4 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H).
5.1.70 (1R)-3-Methyl-1-(3-pyridyl)butan-1-amine
dihydrochloride (29e)
The title compound was prepared from 40e in a manner
similar to that described for 29a as a colorless solid (84%). H
NMR (500 MHz, CD3OD): δ 9.16-9.15 (m, 1H), 8.99-8.98 (m,
1H), 8.87-8.84 (m, 1H), 8.24 (dd, J = 8.3, 5.9 Hz, 1H), 4.76 (dd,
J = 8.8, 6.8 Hz, 1H), 2.07-1.96 (m, 2H), 1.54-1.46 (m, 1H), 1.01
(d, J = 6.8 Hz, 3H), 0.99 (d, J = 6.8 Hz, 3H).
1
5.1.78 (1R)-1-Isoxazol-5-yl-3-methylbutan-1-amine
hydrochloride (29m)
The title compound was prepared from 40m in a manner
1
similar to that described for 29a as a colorless solid (87%). H
NMR (500 MHz, CD3OD): δ 8.48 (d, J = 1.5 Hz, 1H), 6.63 (d, J
= 1.5 Hz, 1H), 4.75 (dd, J = 9.8, 5.9 Hz, 1H), 2.04-1.97 (m, 1H),
1.86-1.80 (m, 1H), 1.56-1.49 (m, 1H), 0.99 (d, J = 6.3 Hz, 3H),
0.96 (d, J = 6.8 Hz, 3H).
5.1.71 (1R)-1-(2-Fluorophenyl)propan-1-amine hydrochloride
(29f)
The title compound was prepared from 40f in a manner
1
similar to that described for 29a as a colorless solid (89%). H
5.1.79 (1R)-3-Methyl-1-oxazol-2-yl-butan-1-amine
hydrochloride (29n)
NMR (400 MHz, CD3OD): δ 7.51-7.45 (m, 2H), 7.33-7.29 (m,
1H), 7.25-7.21 (m, 1H), 4.47 (dd, J = 9.3, 5.9 Hz, 1H), 2.12-1.94
(m, 2H), 0.92 (t, J = 7.3 Hz, 3H).
The title compound was prepared from 40n in a manner
similar to that described for 29a as a colorless solid (quant.). 1H
NMR (500 MHz, CD3OD): δ 8.01 (s, 1H), 7.26 (s, 1H), 4.62 (dd,
J = 9.3, 5.9 Hz, 1H), 2.05-1.98 (m, 1H), 1.83-1.77 (m, 1H), 1.63-
1.55 (m, 1H), 0.99 (d, J = 6.3 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H).
5.1.72 (1R)-1-(3-Fluorophenyl)propan-1-amine hydrochloride
(29g)
The title compound was prepared from 40g in a manner
1
similar to that described for 29a as a colorless solid (80%). H
NMR (500 MHz, CD3OD): δ 7.52-7.47 (m, 1H), 7.27-7.16 (m,
3H), 4.21 (dd, J = 9.3, 5.9 Hz, 1H), 2.08-1.90 (m, 2H), 0.90 (t, J
= 7.3 Hz, 3H).
5.2. Biological Assays
5.2.1. IC50 in buffer
5.1.73 (1R)-1-(4-Fluorophenyl)propan-1-amine hydrochloride
(29h)
The activity of renin inhibitors against purified enzyme was
measured using the following protocol: All reactions were carried
out in a flat bottom black opaque microtiter plate. Test
compounds in DMSO (2 μL) were mixed with 100 L of the
assay buffer (50 mM Tris-HCl (pH 7.9), 100 mM NaCl)
containing 5 L of trypsin-activated recombinant human renin
(final enzyme concentration of 50 μM), and the solution was pre-
incubated at room temperature for 10 min. Next, 2 μM of the
substrate (Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-
His-Thr-Lys(DABCYL)-Arg) in 100 μL of the assay buffer was
added, and the resulting mixture was incubated at 37 °C for 90
min. After completion of incubation, the concentration of
generated angiotensin I was measured by fluorescence at 492 nm
(excitation at 340 nm) using a multilabel reader (PerkinElmer
Inc.). The slope of the linear portion of the plot of fluorescence
increase as a function of time was then determined, and the rate
was used to calculate % inhibition in relation to uninhibited
control. The % inhibition values were plotted as a function of
inhibitor concentration, and the IC50 value was determined by
probit analysis. The IC50 value is defined as the concentration of
a particular inhibitor that reduces the formation of product by
50% relative to a control sample containing no inhibitor.
The title compound was prepared from 40h in a manner
1
similar to that described for 29a as a colorless solid (quant.). H
NMR (400 MHz, CD3OD): δ 7.50-7.44 (m, 2H), 7.23-7.18 (m,
2H), 4.19 (dd, J = 9.2, 5.7 Hz, 1H), 2.09-1.88 (m, 2H), 0.89 (t, J
= 7.4 Hz, 3H).
5.1.74 (1R)-1-(3-Chlorophenyl)propan-1-amine hydrochloride
(29i)
The title compound was prepared from 40i in a manner similar
1
to that described for 29a as a colorless solid (90%). H NMR
(500 MHz, CD3OD): δ 7.50-7.44 (m, 3H), 7.38-7.36 (m, 1H),
4.19 (dd, J = 9.0, 6.1 Hz, 1H), 2.06-1.91 (m, 2H), 0.90 (t, J = 7.3
Hz, 3H).
5.1.75 (1R)-1-(4-Chlorophenyl)propan-1-amine hydrochloride
(29j)
The title compound was prepared from 40j in a manner
1
similar to that described for 29a as a colorless solid (59%). H
NMR (500 MHz, CD3OD): δ 7.49-7.47 (m, 2H), 7.45-7.42 (m,
2H), 4.19 (dd, J = 9.3, 5.9 Hz, 1H), 2.09-1.89 (m, 2H), 0.89 (t, J
= 7.6 Hz, 3H).
5.2.2. IC50 in plasma
5.1.76 (1R)-1-(5-Fluoro-2-pyridyl)-3-methylbutan-1-amine
dihydrochloride (29k)
The activity of renin inhibitors in vitro in cynomolgus monkey
plasma was measured by the decrease in plasma renin activity
(PRA) levels observed in the presence of the compounds.
Compounds and Aliskiren hemifumarate (1) were dissolved in
The title compound was prepared from 40k in a manner
similar to that described for 29a as a colorless solid (quant.).

DMSO and the final concentration of DMSO was 1%. Incubation
mixtures were contained in the final volume of 20 μL of test
compound solution, 200 μL of pooled mixed-gender human or
cynomolgus monkey plasma stabilized with EDTA, 20 μL of pH
adjusting solution, and 10 μL of Inhibitor A solution. Reaction
mixture was incubated at 37 °C for 60 min. After incubation,
Acknowledgments
We would like to thank Masao Yoshida, Kazuki Mori,
Mitsuhiro Iwamoto, Sayaka Suzuki, Masayoshi Asano, Akifumi
Kurata, Makoto Mizuno, Masumi Ueno, Mina Nishi, Kiyoshi
Takasuna, and Naoyuki Maeda, for their technical assistance and
helpful discussions.
angiotensin
I in the reaction mixture was measured by
competitive radioimmunoassay using a commercial available
RIA kit, RENIN RIABEAD (Yamasa Co.). An uninhibited tube
containing 1% DMSO and control tube incubated at 4 °C were
used to derive the % inhibition for each concentration of
inhibitors. The % inhibition values were plotted as the function
of inhibitor concentration, and the IC50 value was determined
from a fit of this data to a four parameter equation. The IC50
value is defined as above.
Supplementary Material
Experimental details and characterization data for compounds
38k, 38m, 41, and 42 are available on-line.
5.3. Animal Studies
5.3.1. Blood pressure study in dTG rats
Double transgenic rats (dTGRs) were bred at PhoenixBio Co.,
Ltd. from parents who were homozygous for hREN or
hAOGEN.13c Male and female hypertensive dTGRs (150-200 g)
>8 weeks of age were instrumented with telemetric devices
(TA11PA-C40, Data Sciences International Inc.) using a sterile
technique. Baseline values were recorded for 24 h prior to
administration. Following measurement of baseline values, rats
were dosed by oral gavage with vehicle (0.5% methylcellulose, n
= 9) or compound 32o at 30 mg/kg (n = 5) after which animals
were monitored for 48 h post-dose. Blood pressure signals were
digitized and analyzed using Dataquest Art Software (Data
Sciences International Inc.) for the derivation of MAP. These
MAP values are reported as 1 h average values and also as
changes from the corresponding time-matched baseline values.
References and notes
1.
2.
3.
4.
Kearney, P. M.; Whelton, M.; Reynolds, K.; Muntner, P.;
Whelton, P. K.; He, J. Lancet 2005, 365, 217.
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5.3.2. Blood pressure study in cynomolgus monkeys pre-
treated with furosemide
5.
6.
Rahuel, J.; Rasetti, V.; Maibaum, J.; Rüeger, H.; Göschke, R.;
Cohen, N.-C.; Stutz, S.; Cumin, F.; Fuhrer, W.; Wood, J. M.;
Grütter, M. G. Chem. Biol. 2000, 7, 493.
Arterial pressure was measured by a telemetry system in
conscious, freely moving cynomolgus monkeys (n = 6). Pressure
transmitters (TL11M2-D70-PCT, Data Sciences International
Inc., USA) were implanted into the peritoneal cavity under
aseptic conditions and anesthesia, and the sensor catheter was
placed in the left femoral artery. Cynomolgus monkeys were
allowed to recover for at least 1 week before any experiment. The
animals were fasted from the morning on the dosing day. Feeding
on the dosing day was conducted 8 hours after dosing or later.
The animals were allowed free access to water the whole time.
Furosemide at 5 mg/kg/day was intramuscularly administered for
3 days before drug administration. Cynomolgus monkeys orally
received 32o at doses of 3 and 10 mg/kg, or vehicle (1%
methylcellulose). Arterial pressure was continuously measured
telemetrically from 3 h before administration to 24 h after
administration with the data collection and real-time analysis
system (Dataquest™ OpenART™, Data Sciences International,
USA). The mean value for 1 h of MAP was calculated.
Bezençon, O.; Bur, D.; Weller, T.; Richard-Bildstein, S.; Remeň,
L.; Sifferlen, T.; Corminboeuf, O.; Grisostomi, C.; Boss, C.; Prade,
L.; Delahaye, S.; Treiber, A.; Strickner, P.; Binkert, C.; Hess, P.;
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