Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)- 4-hydroxyhexanamides to reduce a cardiac safety issue: Discovery of DS-8108b, an orally active renin inhibitor

Article abstract of DOI:10.1016/j.bmc.2013.03.022

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure-activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.

Full text of DOI:10.1016/j.bmc.2013.03.022

Bioorganic & Medicinal Chemistry 21 (2013) 3175–3196
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpi-
perazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety
issue: Discovery of DS-8108b, an orally active renin inhibitor
a
a
a
a
Yuji Nakamura a, , Teppei Fujimoto , Yasuyuki Ogawa , Hidenori Namiki , Sayaka Suzuki ,
Masayoshi Asano a, Chie Sugita a, Akiyoshi Mochizuki b, Shojiro Miyazaki a, Kazuhiko Tamaki a,
Yoko Nagai c, Shin-ichi Inoue c, Takahiro Nagayama d, Mikio Kato e, Katsuyoshi Chiba f,
Kiyoshi Takasuna f, Takahide Nishi a
⇑
a Lead Discovery & Optimization Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Lead Discovery & Optimization Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Drug Metabolism & Pharmacokinetics 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 Frontier 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
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim to address an undesired cardiac issue observed with our related compound in the recently
disclosed novel series of renin inhibitors, further chemical modifications of this series were performed.
Extensive structure–activity relationships studies as well as in vivo cardiac studies using the electrophys-
iology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b)
as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3
and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction
of mean arterial blood pressure for more than 12 h.
Received 10 February 2013
Revised 11 March 2013
Accepted 15 March 2013
Available online 26 March 2013
Keywords:
Renin inhibitor
Ketopiperazine
Plasma renin activity
Hypertension
Na channel
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
resources from the pharmaceutical industry have been invested to-
ward the discovery of orally active renin inhibitors.4 During the
The renin–angiotensin–aldosterone system (RAAS) is well
established as an endocrine system in regulating blood pressure
and extracellular fluid volume.1 In the RAAS, the aspartyl protease
renin is the first and rate-limiting enzyme, which cleaves angioten-
sinogen to produce angiotensin I (Ang I). Ang I is further converted
by angiotensin converting enzyme (ACE) to Ang II, which acts on
the angiotensin receptor type 1 (AT1), leading to pharmacological
effects such as vasoconstriction, inflammation and fibrosis.2 Direct
renin inhibitors are expected to provide better kidney and heart
protection while offering superior blood pressure lowering, but
also cause fewer mechanism-based adverse events than existing
ACE inhibitors and AT1 blockers.3 Significant research efforts and
past few decades, most of the scaffolds were based on peptidic or
peptidomimetic that were designed to mimic the N-terminal se-
quence of angiotensinogen, however, these scaffolds exhibited
poor pharmacokinetic properties and low oral bioavailabilities.5
Continued efforts for research on these scaffolds led to the discov-
ery of aliskiren hemifumarate (1) (Fig. 1).6 After disclosure of
non-peptidic 1, novel renin inhibitors on diverse scaffolds such
as ACT-077825 (MK-8141) (2)7 and VTP-27999 (3)8 were reported
(Fig. 1). Although these have advanced to human clinical trials,
only aliskiren has been launched to the market for the treatment
of hypertension to date.9
Recently, we have reported the discovery and initial optimiza-
tion of a novel series of renin inhibitors, (2S,4S,5S)-5-amino-6-
(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxy-2-isopropyl
hexanamides as exemplified by compound 4 (Fig. 2).10 Compound
4 exhibited 2.0 and 25 nM inhibitory activities in purified human
and monkey plasma renin, 18.5% oral bioavailability in cynomolgus
Abbreviations: HR, heart rate; PR, PR interval; QRS, QRS width; QTc, corrected QT
interval.
⇑
Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5436 8563.
E-mail address: nakamura.yuji.mf@daiichisankyo.co.jp (Y. Nakamura).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.022

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Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
H
N
H
N
H
H
O
OH
H
N
H2N
N
NH2
O
O
O
O
O
Cl
O
0.5 fumaric acid
O
Cl
Aliskiren hemifumarate (1)
ACT-077825, MK-8141 (2)
O
HN
O
H
N
H
O
N
O
O
N
H
H
H
Cl
VTP-27999 (3)
Figure 1. Representative non-peptidic renin inhibitors.
P1'
P2'
P1'
P2'
OH
OH
H
N
H
N
H2N
N
H2N
N
O
O
O
O
F
N
N
P1
P1
Cl
Cl
P3
P3
4
5
IC50 in purified human renin: 2.0 nM
IC50 in monkey plasma renin: 25 nM
Bioavailability: 18.5%
IC50 in purified human renin: 1.0 nM
IC50 in monkey plasma renin: 3.0 nM
Bioavailability: 26.4%
Figure 2. Structures of compounds 4 and 5.
monkeys, and specificity over other proteases without interacting
to the Ss3p pocket of renin.11 During the course of further modifica-
tion of 4, we identified neopentyl derivative 5, which demon-
strated excellent in vitro potencies in both purified human and
monkey plasma assays, while maintaining the bioavailability sim-
ilar to 4 (Fig. 2). Oral administration of 5 at a 3 mg/kg dose in cyno-
molgus monkeys pre-treated with furosemide showed significant
reduction in plasma renin activity (PRA) and mean arterial blood
pressure (MAP) (data not shown). Encouraged by these results, 5
was advanced to safety profiling, but unfortunately a serious car-
diac issue was revealed. After administration of a single oral dose
of 50 mg/kg of 5 to telemetered cynomolgus monkeys, ventricular
tachycardia (VT) and fibrillation (VF) were observed. Concurrently,
compound 5 was found to reduce the maximum rate of rise and
shorten the action potential duration (APD) at 90% repolarizations
possibility of other channels’ contributions. Therefore, we decided
to use in vivo and ex vivo cardiac studies for assessing VT/VF con-
cern of this series. In this paper, we report our efforts to address
this issue and the discovery of compound 56 (DS-8108b).13
2. Strategy to obtain compounds with reduced
arrhythmogenicity
Under the circumstance described above, we used a more time
and resource sparing rodent alternative to large animal models.14
As a result, we figured that the electrophysiology (EP) rat model
was applicable for assessing a given compound’s potential to in-
duce QRS complex abnormality mainly based on the action of the
Na channel. Iv bolus injections of 5 at 3 and 10 mg/kg doses caused
QRS complex abnormalities in a dose-dependent manner in elec-
trocardiogram (ECG) and finally resulted in deaths (Fig. 3). We con-
sidered that these ECG changes were related to VT/VF observed in
monkeys, and decided to initiate rat in vivo screening. In this
screening, the QRS complex abnormality was qualitatively classi-
fied to the three levels of ECG change; no (Fig. 3A), moderate
(Fig. 3B) or severe (Fig. 3C) in the order of seriousness. After the
ECG screening in rats, selected compounds were evaluated in an
from 10
fect hERG and hCav1.2 currents at 100
27.2% inhibition against hNav1.5 current at 10
l
M in guinea pig papillary muscle. Although 5 did not af-
M, it was found to show
M.12 Compound
l
l
5 also showed 87% inhibition against Site 2 Na channel binding as-
say in rats. Based on these data, VT/VF observed in monkeys was
supposed to be mainly derived from the action of 5 on the Na
channel in the myocardium, however, we could not exclude the

Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
3177
(A) Pre-dose
(B) 3 mg/kg
(C) 10 mg/kg
Figure 3. ECG after iv bolus injection of compound 5 in rats: (A) pre-dose, no change; (B) 3 mg/kg, moderate change; (C) 10 mg/kg, severe change.
O
N
NH
P2'
P1'
Lidocaine (6)
Severe ECG change
OH
H
N
H2N
N
O
N
N
O
O
P3
Modification site
N
P1
Cl
Cl
5
7
No ECG change
Figure 4. Structures of 6 and 7, and modification site.
isolated Langendorff-perfused rabbit heart or APD in guinea pig pap-
illary muscle to confirm the reduction of arrhythmogenicity at the
ex vivo level. Final assessment of VT/VF concern was planned to be
evaluated in telemetered monkeys in addition to rabbits and dogs.
Compounds 1 and 5 utilize the same transition-state mimetic
Et3N, followed by SN2 substitution of the methanesulfonyloxy
groups with the azide groups were accomplished by treating with
NaN3 in DMPU to obtain azides 14. Then, hydrogenation of 14 in
the presence of HCl, and the subsequent reaction with NsCl19, fol-
lowed by crystallizationgave N-Ns-alcohols 10a–c as a single diaste-
reomer. Using these three components, derivatives are prepared as
shown in Scheme 2. N-Ns-aziridines 15 were formed by the Mitsun-
obu reaction of 10 with DEAD and Ph3P. The ring opening of aziri-
dines 15 with ketopierazines 8 afforded lactones 16 in excellent
yields. After replacement of the N-Ns groups with the N-Boc protect-
ing group, sequential N-terminal amide bond formations with
amines 9 under neat conditions in the presence of 2-hydroxypyri-
dine as a catalyst20, removal of the N-Boc protecting groups with
TFA, and treatment with fumaric acid afforded the desired com-
pounds as fumarate salts. In the case of aromatic amines 9, the amide
formation with 17 proceeded in the presence of Me2AlCl.21
Preparation of derivatives, which were difficult to synthesize
via direct lactone-opening with amine under the established con-
ditions, is outlined in Scheme 3. After conversion of lactones 17a
and 17b to the methyl esters by the hydrolysis with NaOH, fol-
lowed by the treatment with TMSCHN2, reaction with 2,2-dime-
thoxypropane and PPTS gave N,O-acetals 18. Acetals 18 were
hydrolyzed with NaOH, and then coupling of amines 9 with result-
ing carboxylic acids using HATU as an activating reagent afforded
carboxamides 19. Deprotection of the N,O-acetal and N-Boc pro-
tecting groups was achieved smoothly by reaction with 10% HCl/
MeOH. Finally, treatment with fumaric acid afforded desired deriv-
atives as fumarate salts.
0
0
scaffold and interact with S3, S1, S1 and S2 regions of renin. Since
cardiac concern of aliskiren has not been reported in humans, we
surmised that at least one of the structurally different portions be-
0
tween 1 and 5, P3–P1 or P2 portion, was associated with arrhyth-
mogenicity observed with 5. Initially, the structural similarity of
Na channel blocker lidocaine (6) suggested to us that the P3–P1
portion of 5 might be attributed to the action on the Na channel
(Fig. 4). Lidocaine caused QRS complex abnormality based on Na
channel inhibition in the EP rat model (n = 2, 10 mg/kg, bolus injec-
tion), whereas compound 7 did not affect the ECG signal. From
these results, we assumed that the contribution of the P3–P1 por-
tion to the action on the Na channel would be low. Hence, we
0
decided to focus on the modification of the P2 portion to reduce
arrhythmogenicity of this series.
3. Chemistry
All derivatives evaluated in this report are prepared from three
key components depicted in Figure 5, except compound 26 (see
Supplementary data). First component, 4-phenylketopiperazines 8
were prepared by the same procedure reported previously.10 Second
component, amines 9 (R4-NH2) were either obtained commercially
or prepared according to the literature methods.9a,15 Third compo-
nent, N-2-nitrobenzenesulfonyl (N-Ns) protected alcohols 10 were
prepared as shown in Scheme 1.13,16 Diastereoselective alkylation
of 11 with benzyl (2E)-4-bromobut-2-en-1-yl ether17 proceeded
smoothly, and the chiral auxiliaries were removed with
LiOH–H2O2 to afford the chiral carboxylic acids 12. Shi’s asymmetric
epoxidation18 of 12 and subsequent intramolecular lactonization
produced alcohols 13 directly. Mesylation of 13 with MsCl and
4. Results and discussion
0
4.1. Structure–activity relationships of P2 portion
A broad range of derivatives bearing simple alkyl (4, 5, 20–26),
functionalized alkyl (27–34), or aromatic (35–39) group, were

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Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
OH R3
H
N
H2N
N
R4
O
O
NH
O
R3
R2
N
O
O
R4 NH2
NsHN
HO
+
+
R2
N
R1
R1
8
9
10
Figure 5. Key components for the preparation of derivatives.
O
R3
Bn
O
R3
a, b
c
OH
HO
BnO
N
O
R3
O
BnO
O
O
11a: R3 = i-Pr
11b: R3 = Et
11c: R3 = Me
12a-c
13a-c
O
O
O
O
R3
R3
d, e
f, g
N3
BnO
NsHN
HO
14a-c
10a-c
Scheme 1. Reagents and conditions: (a) NaN(TMS)2, (E)-BnOCH2CH@CHCH2Br, THF, ꢀ78 to ꢀ40 °C; (b) LiOH, 30% H2O2, THF–H2O (3:1), 0 °C to rt; (c) 1,2:4,5-di-O-
isopropylidene-b-
-erythro-2,3-hexodiulo-2,6-pyranose, oxone, K2CO3, aq. Na2(EDTA)2, Na2B4O7ꢁ10H2O, dimethoxymethane-MeCN (2:1), 0 °C; (d) MsCl, Et3N, CH2Cl2, 0 °C; (e)
NaN3, DMPU, 60 °C; (f) cat. Pd–C, H2, HCl, EtOH, rt; (g) (i) NsCl, Et3N, THF–H2O (10:1), rt; (ii) crystallization in diisopropylether–AcOEt.
D
O
O
NH
R2
N
O
a
b
10a-c
R3
+
R1
Ns
N
15a-c
8a-c
O
O
R3
OH
R3
H
N
PGHN
N
PGHN
R4
e, f, g
O
O
O
N
R2
N
R2
N
n fumaric acid
R1
R1
16a-h: PG = Ns
17a-h: PG = Boc
4-5, 20-25, 27-31, 33-58, 61-64
c, d
Scheme 2. Reagents and conditions: (a) DEAD, Ph3P, THF, 0 °C; (b) toluene, 110 °C; (c) PhSH, Cs2CO3, DMF, rt; (d) Boc2O, Et3N, CH2Cl2, rt; (e) amines 9, cat. 2-hydroxypyridine,
80–120 °C, or Me2AlCl, aromatic amines 9, CH2Cl2, rt; (f) TFA, CH2Cl2, rt; (g) fumaric acid, MeOH, rt.
examined to investigate the impact on renin inhibition and the ECG
signal. Table 1 summarizes the result of invitroIC50 dataagainstpuri-
fied human and monkey plasma renin, and classification of ECG
changes in rats. The ratios of IC50 values between the tested com-
pounds and 1 in a same monkey plasma batch are also shown since
the IC50 values varied from batch to batch with in-house in vitro as-
say. The values of the ratio lower than 1.0 indicated that inhibitory
activities of those compounds are superior to that of 1. Higher renin
inhibitory activities than lead compound 4 were detected for most
of the simple alkyl derivatives, in which neopentyl analogues (5,
23–25) were the most potent compounds. Of functionalized alkyl
analogues, alcohol analogues (28, 29, 33) showed improved renin
inhibitory activities in both purified human and monkey plasma as-
says compared to 4. Other functionalized alkyl analogues such as
amide (30), methoxy carbonyl (31), and ether (34) exhibited almost
the same level of monkey plasma renin inhibitory activities as 4,
but a marked decrease in renin inhibition was observed in carboxylic
acid analogue 32. Meanwhile, in the case of aromatic analogues,

Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
3179
O
O
R3
O
R3
N
N
O
Boc
BocHN
a, b, c
O
O
d, e
O
N
N
N
Cl
Cl
17a: R3 = i-Pr
17b: R3 = Et
18a,18b
R3
R3
OH
O
N
H
N
H
N
H2N
N
R4
R4
Boc
f, g
O
O
O
O
N
N
N
fumaric acid
Cl
Cl
19a,19b
59, 60
Scheme 3. Reagents and conditions: (a) NaOH, THF–MeOH–H2O, rt; (b) TMSCHN2, toluene–MeOH, rt; (c) 2,2-dimethoxypropane, PPTS, DMF, 65 °C; (d) NaOH, THF–MeOH–
H2O, 65 °C; (e) amines 9, HATU, Et3N, DMF, rt ꢀ40 °C; (f) 10% HCl–MeOH, rt; (g) fumaric acid, MeOH, rt.
installation of the 2-pyridyl group (36) into P20 portion improved re-
nin inhibitory activity, but phenyl (35), 3- and 4-pyridyl analogues
(37–39) showed similar or decreased renin inhibitory activities in
monkey plasma compared to 4. Next, our attention was turned to
the cardiac effects of these compounds in the EP rat model. Simple al-
kyl analogues (20–23, 25, 26) were found to show a similar undesired
potent inhibitory activities in both purified human and monkey
plasma renin assays (36 vs 46, 47), but with further modifications
(48–51), better compounds could not be found compared to 46 and
47. ECG screening in rats revealed that 2-pyridyl analogues showed
improved ECG signals from the severe to moderate state, but none
of the compounds listed in Table 3 could accomplish the elimination
of ECG change. Thus, we decided to stop further efforts on this series.
0
0
effect on ECG signals as 5 regardless of the P1 and P2 structures.
0
Intriguingly, P2 geminal methyl analogue 26 provided the same ef-
fect on ECG as 5 despite the weak renin inhibitory activity, suggesting
that the observed effect on the ECG signal did not correlate with the
intension of renin inhibition. Thus, we decided to stop further inves-
4.3. Cardiac study on isolated Langendorff-perfused rabbit
heart
0
tigation of P2 simple alkyl analogues because of the unveiled high
To investigate whether the improvement in in vivo rat screen-
ing would be reflected on the reduction of arrhythmogenicity at
the ex vivo level, cardiac study on isolated Langendorff-perfused
rabbit heart was conducted using compounds 29 and 46, which
showed no and moderate ECG changes with potent renin inhibitory
activities, respectively (Table 3). As a result, compound 46 was
found to prolong QRS width, PR, and QTc intervals significantly at
risk of arrhythmogenicity. By contrast, functionalized alkyl ana-
logues (27–30, 33, 34) showed a tendency to improve the effect on
ECG, except methoxycarbonyl analogue 31. In particular, alcohol
analogues (27, 29, 33) and amide analogue 30, which possessed the
same P20 residue as 1, did not cause ECG changes. Similarly, aromatic
analogues (35, 38, 39) exhibited improved effects on ECG compared
0
to P2 simple alkyl analogues. Based on these results and the
10 lM, and caused VT/VF at 30 lM similar to 5. On the other hand,
comparison of the clogP value,22 it is suggested that not only the
undesired signals were not observed with 29 compared to 5 and
46. Taken these results together, we concluded that only com-
pounds free from ECG change might have a chance to reduce
arrhythmogenicity at the ex vivo level.
lipophilicity of the molecules but also the structural feature of the
0
P2 part plays an important role for determining the effect on ECG
in rats.
0
4.2. Modification of P2 portion of aromatic analogues
4.4. Optimization of alcohol and amide analogues
Functionalized alkyl derivatives (29, 30, 33) realized potent re-
nin inhibition as well as free from ECG change, but poor PK profiles
in cynomolgus monkeys emerged as the next issue; for example
AUC0–24 of 33 (75 ng h/mL) versus AUC0–24 of 5 (460 ng h/mL).
Meanwhile, regarding aromatic analogues (35–39), milder ECG
changes compared to simple alkyl derivatives in spite of their high-
er lipophilicity than functionalized alkyl derivatives are worth fur-
Encouraged by the result of the cardiac study on an isolated
Langendorff-perfused rabbit heart, we focused our attention on an-
other approach; adjustment of lipophilicity of alcohol and amide
analogues (29, 30, 33) to obtain compounds with good PK profiles
(Table 4). Exposure levels of designed compounds were mainly
measured by cassette-dosing assay in cynomolgus monkeys. At
first, increase of the bulkiness and the lipophilicity of the geminal
methyl group of 29 improved human renin inhibitory activities
(52–54). However, these modifications affected the ECG signals
and the exposure levels were low. Next, with an aim of improving
oral exposure as well as keeping its desirable attributes of cyclo-
hexanol analogue 33, adamantanol analogues (55–59) were de-
signed and evaluated. Introduction of the adamantanol groups
(55–58) improved human renin inhibitory activities compared to
0
ther exploration (Table 2). Fluorination at the 4-position on the P2
phenyl group of 35 (40, 41) showed high renin inhibitory activities,
whereas substitution of the fluoro group of 41 with the methyl or
methoxy group (42, 43) and installation of the second fluoro group
0
at the 2- or 3-position on the P2 phenyl group of 40 (44, 45) weak-
ened renin inhibitions. Meanwhile, introduction of the fluoro
group at the 5-position onto the 2-pyridine ring of 36 exhibited

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Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
Table 1
SAR of modification of P2 portiona
0
OH R3
5 H
R
H2N
N
N
R4
O
O
R2
N
R1
Compound
R1
R2
R3
R4
R5
Purified human renin
IC50 (nM)
Monkey plasma renin
ECG change in ratsc
clogPd
IC50 (nM)
Ratiob
4
Cl
Cl
F
i-Pr
i-Pr
H
H
2.0
1.0
25
1.2
0.8
NTe
C
5.33
5.19
20
H
9.0
21
22
Cl
Cl
H
H
i-Pr
i-Pr
H
H
1.4
2.9
4.0
33
0.7
2.7
C
C
5.06
4.44
5
Cl
H
H
H
F
i-Pr
Et
H
1.0
0.8
1.0
1.1
97
3.0
2.3
0.5
0.3
0.5
0.6
132
1.9
C
5.46
5.06
4.53
4.46
4.67
3.98
23
24
25
26
27
Cl
H
C
Cl
Me
Me
Me
i-Pr
H
2.1
NT
C
Me
Cl
H
3.4
F
Me
H
738
15
C
Cl
H
2.4
A
OH
OH
28
29
Cl
Cl
H
H
i-Pr
H
H
1.7
1.6
9.6
5.2
0.8
0.8
B
A
4.34
3.94
Et
OH
NH2
30
31
32
33
Cl
H
H
F
i-Pr
i-Pr
i-Pr
i-Pr
H
H
H
H
1.4
1.8
16
7.0
23
1.2
1.6
16
A
3.72
4.88
2.06
3.55
O
O
O
O
Cl
C
OH
OH
Me
Cl
39
NT
A
H
1.4
4.3
0.8
34
35
36
Cl
Cl
Cl
H
H
H
i-Pr
i-Pr
i-Pr
H
H
H
2.0
1.4
1.8
9.8
9.2
3.4
1.4
1.3
0.7
B
B
C
3.23
5.60
4.93
O
N
37
38
Cl
H
F
i-Pr
i-Pr
H
H
2.1
2.4
19
33
1.9
3.3
NT
B
4.93
4.86
N
N
Me

Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
3181
Table 1 (continued)
Compound
R1
Cl
R2
H
R3
R4
R5
H
Purified human renin
IC50 (nM)
Monkey plasma renin
ECG change in ratsc
clogPd
IC50 (nM)
Ratiob
39
i-Pr
1.6
15
1.5
B
4.93
N
a
Compound 32 was obtained as a succinate salt. Other compounds were obtained as fumarate salts. Assay results are the average of at least two replicates.
Relative activity of inhibition of monkey plasma renin: IC50 of compound/IC50 of 1.
ECG change (n = 2, 10 mg/kg, iv bolus injection): A = no change; B = moderate change; C = severe change. See Figure 3.
See Ref. 22.
b
c
d
e
NT = not tested.
33 except 3-hydroxyadamantane (59). In particular, replacement
of the P1 isopropyl group of 55 with ethyl group (56) exhibited
5. Conclusion
0
sub-nanomolar inhibition in purified human renin assay. However,
twofold decrease of renin inhibitory activity in monkey plasma
was observed when the P1 ethyl group of 56 was changed to the
We have described our efforts to address arrhythmogenicity ob-
served with the related compound in the recently disclosed novel
series of renin inhibitors. Utilizing the EP rat model as an in vivo
first screening, optimizations of the P3, P1 , and P2 portions of this
series led to the identification of clinical candidate trans-adaman-
tan-1-ol analogue 56 (DS-8108b), as a potent renin inhibitor with
reduced cardiac potential risk. Oral administration of single doses
of 3 and 10 mg/kg of 56 in cynomolgus monkey pre-treated with
furosemide elicited sustained reductions mean arterial blood pres-
sure in a dose-dependent manner.
0
0
0
methyl group (57). Regarding the ECG evaluation, only trans-ada-
mantan-1-ol analogues (55–57) did not affect the signal. As cis-
adamantan-1-ol analogue 58 having almost the same clogP value
as 56 affected the ECG signal, we assumed that the structural fea-
ture of trans-adamantan-1-ol portion would play an important role
to prevent an undesired effect on ECG in rats. Although oral expo-
sure levels of 55, 58, and 59 were poor in cynomolgus monkeys,
pleasingly, compound 56 exhibited drastically improved exposure
levels in both cassette- and single-dosing assays (Tables 4 and 5).
Further chemical modification of the terminal hydroxyl group to
carboxamide group (60) showed potent renin inhibition, but the
exposure was poor (Table 5). Having obtained potent compound
56 with good exposure, the PRA suppressive effect of 56 was eval-
uated in cynomolgus monkeys pre-treated with furosemide. As a
result, compound 56 exhibited excellent suppressive effects in a
dose-dependent manner at a dose of 1, 3 and 10 mg/kg (Fig. 6).13
Since trans-adamantan-1-ol analogue 56 was obtained, our final
6. Experimental
6.1. 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 Sily-
sia 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 ambi-
ent temperature, and chemical shifts are reported in parts per mil-
lion (ppm (d)) 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 sys-
tems. Optical rotations were measured on an Autopol V Plus. Ele-
0
approach was to select the best combination of P3 and P1 portions
for further evaluation (Table 6). All compounds (61–64) exhibited
high in vitro potencies in monkey plasma without any effect on
ECG. As a result of evaluation of PRA suppressive effect of the
trans-adamantan-1-ol analogues (61–64) in ex vivo assay, com-
pound 56 showed the most potent efficacy within this series (data
not shown). Oral administration of single doses of 1, 3 and 10 mg/
kg of 56 in cynomolgus monkeys pre-treated with furosemide re-
duced MAP dose-dependently, and the reduction was sustained
over a period of 12 h (Fig. 7).13 This reduction in blood pressure
was almost paralleled by the suppression on ex vivo PRA within
that time, indicating that the pharmacological effect was mecha-
nistically related to renin inhibition.
mental analyses for CHN and Cl 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.
4.5. Cardiac assessments of 56
6.1.1. (2R,4E)-6-(Benzyloxy)-2-methylhex-4-enoic acid (12c)
A solution of sodium bis(trimethylsilyl)amide in n-hexane
(1.03 M, 164 mL, 169 mmol) was added to a solution of 11c
(32.9 g, 141 mmol) in THF (330 mL) under N2 atmosphere and at
ꢀ78 °C over 45 min, and the mixture was stirred at the same tem-
perature for 30 min. Then, a solution of benzyl (2E)-4-bromobut-
2-en-1-yl ether (35.5 g, 148 mmol) in THF (80 mL) was added to
the above solution over 30 min, and the mixture was stirred at
the same temperature for 30 min. The reaction mixture was raised
to ꢀ40 °C and further stirred for 4 h. Saturated NH4Cl aqueous
solution (100 mL) was added to the reaction mixture, and the mix-
ture was further stirred at room temperature for 1 h. The reaction
mixture was concentrated under reduced pressure and diluted
with water (500 mL), followed by extraction with AcOEt. Then,
the organic layer was washed with water and brine, and dried over
anhydrous MgSO4. After filtration, the solvent was evaporated under
Finally, we evaluated the cardiac safety of 56. In agreement
with the result of the EP rat model, 56 did not cause significant
prolongation of QRS width, PR, or QTc intervals in the ex vivo
Langendorff rabbit heart, and in in vivo rabbits, dogs, and monkeys
(Table 7). In addition, compound 56 showed no effects on the Site 2
Na channel (10
100 M). These data support that the potential cardiac risk of 56
l
M),23 hNav1.5 current or hERG current (up to
l
is considered to be low. Compound 56 also demonstrated low po-
tential of drug-drug interactions risk based on the combination of
our single time- and concentration-dependent CYP3A4 inhibition
assay (77.9% remaining at 100 l
M)24 and estimated therapeutic
blood concentration (data not shown). Based on its overall
in vitro/in vivo profile, compound 56 (DS-8108b) was selected as
a clinical candidate.

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Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
Table 2
SAR of modifications of P1 and P2 portions of aromatic analoguesa
0
0
R3
OH
H
N
H2N
N
R4
O
O
N
Cl
Compound
R3
R4
Purified human renin
IC50 (nM)
Monkey plasma renin
IC50 (nM)
Ratiob
ECG change in ratsc
clogPd
40
i-Pr
1.4
7.2
1.2
B
6.00
F
F
41
42
43
Et
Et
Et
1.6
3.2
1.4
3.4
0.9
4.5
2.1
B
B
B
5.60
5.70
5.28
28
11
21
14
O
F
44
i-Pr
1.9
4.5
B
6.17
F
45
46
47
i-Pr
i-Pr
Et
2.0
0.9
1.0
3.0
0.5
0.6
B
B
B
5.64
5.19
4.79
F
F
F
F
3.4
N
N
2.1
3.3
F
48
i-Pr
1.7
0.7
B
4.77
N
F
O
49
50
51
Et
6.7
5.6
3.8
33
13
NTe
4.97
5.43
4.38
N
N
i-Pr
Et
47
15
7.3
2.9
B
B
N
a
b
c
Compounds were obtained as fumarate salts. Assay results are the average of at least two replicates.
Relative activity of inhibition of monkey plasma renin: IC50 of compound/IC50 of 1.
ECG change (n = 2, 10 mg/kg, iv bolus injection): B = moderate change. See Figure 3.
See Ref. 22.
d
e
NT = not tested.
reduced pressure, and the residue was purified by silica gel
column chromatography (eluent, n-hexane/AcOEt = 7:1–2:1) to
obtain (4S)-4-benzyl-3-[(2R,4E)-6-(benzyloxy)-2-methylhex-4-en-
oyl]-1,3-oxazolidin-2-one (37.9 g, 69%, 99% ee) as a colorless
liquid. 1H NMR (400 MHz, CDCl3): d 7.40–7.14 (m, 10H), 5.75 (dt,
1H, J = 15.6, 6.3 Hz), 5.69 (dt, 1H, J = 15.6, 5.4 Hz), 4.71–4.63
(m, 1H), 4.49 (s, 2H), 4.22–4.11 (m, 2H), 3.98 (d, 2H, J = 5.5 Hz),
3.92–3.81 (m, 1H), 3.28 (dd, 1H, J = 13.3, 3.1 Hz), 2.67 (dd,
1H, J = 13.3, 10.2 Hz), 2.58–2.49 (m, 1H), 2.30–2.21 (m, 1H), 1.19
(d, 3H, J = 6.7 Hz).
A
solution of (4S)-4-benzyl-3-[(2R,4E)-

Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
3183
Table 3
Summary data of compounds on the Langendorff rabbit heart
Compound
HR (%)a
PR (%)a
QRS (%)a
QTc (%)a
10
lM
30
lM
10
l
M
30
lM
10
lM
30
lM
10
l
M
30 lM
5
46
29
ꢀ4.6
ꢀ3.7
ꢀ3.5
VT/VF
VT/VF
ꢀ4.3
68.9
64.1
ꢀ7.8
VT/VF
VT/VF
3.4
15.6
74.7
3.8
VT/VF
VT/VF
1.7
ꢀ1.5
13.5
ꢀ2.1
VT/VF
VT/VF
ꢀ5.4
a
% Change from baseline.
Table 4
SAR of modifications of P1 and P2 portionsa
0
0
R3
OH
H
N
H2N
N
R4
O
O
N
Cl
Compound
R3
R4
Purified human renin
IC50 (nM)
Monkey plasma renin
ECG change in ratsc
AUC0-4 in monkeysd
clogPf
IC50 (nM)
Ratiob
(ng h/mL)
NTg
ne
29
52
Et
Et
1.6
1.0
5.2
0.8
A
C
––
––
3.94
5.00
OH
OH
3.0
0.8
NT
53
54
Et
Et
1.1
1.1
3.0
3.4
0.7
0.6
B
B
5
4
3
3
4.70
5.26
OH
OH
33
i-Pr
1.4
4.3
0.8
A
11
4
3.55
OH
55
56
57
i-Pr
Et
1.3
0.9
1.1
2.2
6.3
7.3
0.9
0.8
1.6
A
A
A
17
67
NT
3
4.17
3.78
3.25
OH
3
OH
OH
Me
––
OH
58
Et
1.1
1.8
0.7
B
7
3
3.76
59
60
i-Pr
2.0
1.3
6.5
1.2
0.7
0.5
B
2
3
5.04
3.43
OH
NH2
Et
NT
NT
––
O
a
b
c
d
e
f
Compounds were obtained as fumarate salts. Assay results are the average of at least two replicates.
Relative activity of inhibition of monkey plasma renin: IC50 of compound/IC50 of 1.
ECG change (n = 2, 10 mg/kg, iv bolus injection): A = no change; B = moderate change; C = severe change. See Figure 3.
Cassette-dosing assay at a 1 mg/kg oral dose in cynomolgus monkeys.
Number of compounds dosed in cassette.
See Ref. 22.
NT = not tested.
g

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Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
Table 5
Mean PK parameters in cynomolgus monkeys after the single administration
a
a
a
b
Compound
AUC0–24 (ng h/mL)
Cmax (ng/mL)
T1/2 (h)
Vss (L/kg)
CLb (mL/min/kg)
F (%)
5
460
75
685
90
111
19
102
19
15.3
NTc
5.1
23.5
NT
1.12
NT
28.4
NT
3.59
NT
26.4
NT
4.1
NT
33
56d
60
NT
a
b
c
3 mg/kg PO.
1 mg/kg iv.
NT = not tested.
See Ref. 13.
d
60
50
40
30
20
10
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (h)
Figure 6. Ex vivo PRA suppressive effects of 56. Vehicle (1% MC solution, s), 1 mg/kg ( ), 3 mg/kg ( ) and 10 mg/kg ( ) of 56 were orally administered to cynomolgus
monkeys pre-treated with furosemide. PRA was measured 1, 2, 4, 8, and 24 h after oral administration. See Ref. 13.
Table 6
Optimizations of P3 and P portions of trans-adamantan-1-ol analoguesa
0
1
OH R3
H
OH
H2N
N
N
O
O
R2
N
R1
Compound
R1
R2
R3
Purified human renin
IC50 (nM)
Monkey plasma renin
ECG change in ratsc
clogPd
IC50 (nM)
Ratiob
56
61
62
63
64
Cl
Cl
Cl
Me
Me
H
F
F
F
F
Et
i-Pr
Et
i-Pr
Et
0.9
1.9
1.4
1.7
1.6
6.3
7.8
3.6
10
0.8
0.7
0.7
0.9
0.9
A
A
A
A
A
3.78
4.32
3.92
4.10
3.70
4.6
a
b
c
Compounds were obtained as fumarate salts. Assay results are the average of at least two replicates.
Relative activity of inhibition of monkey plasma renin: IC50 of compound/IC50 of 1.
ECG change (n = 2, 10 mg/kg, iv bolus injection): A = no change. See Figure 3.
See Ref. 22.
d
6-(benzyloxy)-2-methylhex-4-enoyl]-1,3-oxazolidin-2-one
was concentrated under reduced pressure, diluted with water
(500 mL) and washed with AcOEt. Then, the aqueous layer was
made acidic with sodium dihydrogenphosphate (30.0 g), followed
by extraction with AcOEt. The organic layer was washed with
brine and dried over anhydrous MgSO4. After filtration, the sol-
vent was evaporated under reduced pressure to obtain crude
12c (11.0 g) as a colorless liquid. 1H NMR (400 MHz, CDCl3): d
7.40–7.25 (m, 5H), 5.73–5.62 (m, 2H), 4.49 (s, 2H), 4.03–3.93
(m, 2H), 2.61–2.51 (m, 1H), 2.50–2.40 (m, 1H), 2.28–2.17 (m,
1H), 1.19 (d, 3H, J = 7.0 Hz).
(18.7 g, 47.5 mmol) in a mixed solvent of THF (700 mL) and
water (230 mL) was cooled in an ice bath, and then 30% hydro-
gen peroxide aqueous solution (30.0 mL) and lithium hydroxide
monohydrate (4.15 g, 95.3 mmol) were added. The mixture was
stirred at the same temperature for 30 min, and then raised to
room temperature and further stirred for 16 h. After cooling in
an ice bath, 1.5 M sodium thiosulfate aqueous solution
(250 mL) was added to the reaction mixture. The mixture was
further stirred at room temperature for 1 h. The reaction mixture

Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
3185
100
90
80
70
60
50
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (h)
Figure 7. Changes in mean arterial pressure (MAP). 1% MC (s), 1 mg/kg ( ), 3 mg/kg ( ) and 10 mg/kg ( ) of 56 were orally administered to cynomolgus monkeys pre-
treated with furosemide. Data are expressed as mean SEM. See Ref. 13.
Table 7
Electrophysiological profile of compound 56
HRa (%)
PR(PQ)a (%)
QRSa (%)
QTca (%)
Drug concn (lg/mL)
Ex vivo Langendorff rabbit heart (100
l
M)
ꢀ5.8
3.9
1.7
ꢀ1.3
ꢀ1.5
0.3
0.1
ꢀ1.3
5.8
ꢀ7.6
ꢀ3.2
2.7
––
Anesthetized rabbit (1 mg/kg/min, iv infusion) at 10 min
Anesthetized dog (0.5 mg/kg/min, iv infusion) at 30 min
Conscious monkey (1000 mg/kg, po) at 4 h
NTb
38.1
5.4
ꢀ5.4
ꢀ1.4
ꢀ4.2
ꢀ0.4
a
% Change from baseline.
NT = not tested.
b
6.1.2. (3R,5S)-5-[(1R)-2-(Benzyloxy)-1-hydroxyethyl]-3-
methyldihydrofuran-2(3H)-one (13c)
the organic layer was washed with brine and dried over anhy-
drous MgSO4. After filtration, the solvent was concentrated under
reduced pressure, and the residue was purified by silica gel col-
umn chromatography (eluent, n-hexane/AcOEt = 4:1–1:1) to
To a 2 L three-neck round-bottom flask, the buffer (0.05 M so-
dium tetraborate decahydrate in 0.4 mM aqueous ethylenedi-
aminetetraacetic acid disodium salt, 400 mL), dimethoxymethane
(333 mL), acetonitrile (167 mL), carboxylic acid 12c (10.1 g,
43.2 mmol), tetra-n-butylammonium hydrogen sulfate (648 mg,
obtain
a
diastereomeric mixture of (1R)-2-(benzyloxy)-1-
[(2S,4R)-4-methyl-5-oxotetrahydrofuran-2-yl]ethyl methanesulfo-
nate (9.90 g, 97%, 88% de) as a major product and a colorless
liquid. 1H NMR (400 MHz, CDCl3): major isomer d 7.41–7.28 (m,
5H), 4.87–4.80 (m, 1H), 4.69–4.63 (m, 1H), 4.56 (s, 2H),
3.80–3.71 (m, 2H), 3.05 (s, 3H), 2.82–2.70 (m, 1H), 2.58 (ddd,
1H, J = 13.3, 9.4, 3.9 Hz), 1.99 (dt, 1H, J = 13.3, 8.2 Hz), 1.29 (d,
3H, J = 7.4 Hz). Sodium azide (2.93 g, 45.1 mmol) was added
to a solution of (1R)-2-(benzyloxy)-1-[(2S,4R)-4-methyl-5-oxote
trahydrofuran-2-yl]ethyl methanesulfonate (9.90 g, 30.1 mmol)
in 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU)
(100 mL) at room temperature, and the mixture was stirred at
60 °C for 3 days. The reaction mixture was cooled to room tem-
perature and poured into ice water, followed by extraction with
Et2O. Then, the organic layer was washed with water and brine,
and dried over anhydrous MgSO4. After filtration, the solvent
was evaporated under reduced pressure, and the residue was
purified by silica gel column chromatography (eluent, n-hexane/
AcOEt = 5:1) to obtain a diastereomeric mixture of 14c (7.56 g,
91%, 88% de) as a major product and a colorless liquid. 1H NMR
1.91 mmol), and 1,2:4,5-di-O-isopropylidene-b-D-erythro-2,3-hex-
odiuro-2,6-pyranose (11.1 g, 43.0 mmol) were added. The reaction
mixture was cooled with an ice bath. A solution of oxone (36.7 g,
59.6 mmol) in 0.4 mM aqueous ethylenediaminetetraacetic acid
disodium salt (200 mL) and a solution of potassium carbonate
(34.4 g, 247 mmol) in water (200 mL) were added dropwise
through two separate additional funnels over a period of 8 h. The
mixture was stirred at the same temperature for 1 h and then
the reaction was quenched by the addition of AcOEt and water.
The mixture was extracted with AcOEt, washed with brine, and
dried over anhydrous MgSO4. After filtration, the solvent was evap-
orated under reduced pressure, and the residue was purified by
flash chromatography (eluent, n-hexane/AcOEt = 6:1–1:1) to afford
a diastereomeric mixture of 13c (7.82 g, 72%, 88% de, two-steps) as
a major product and a colorless oil. 1H NMR (400 MHz, CDCl3): ma-
jor isomer d 7.42–7.29 (m, 5H), 4.59 (d, 1H, J = 11.7 Hz), 4.55 (d, 1H,
J = 11.7 Hz), 4.48 (ddd, 1H, J = 8.2, 6.3, 3.9 Hz), 3.91–3.84 (m, 1H),
3.64 (dd, 1H, J = 9.8, 3.9 Hz), 3.56 (dd, 1H, J = 9.8, 6.3 Hz), 2.81–
2.69 (m, 1H), 2.53 (ddd, 1H, J = 13.2, 9.4, 3.9 Hz), 2.47 (d, 1H,
J = 5.1 Hz), 1.93 (dt, 1H, J = 13.2, 8.2 Hz), 1.28 (t, 3H, J = 7.0 Hz).
(500 MHz, CDCl3):
d 7.40–7.29 (m, 5H), 4.62–4.54 (m, 3H),
3.78–3.72 (m, 2H), 3.69–3.64 (m, 1H), 2.91–2.82 (m, 1H), 2.38
(ddd, 1H, J = 13.2, 9.8, 3.9 Hz), 2.00 (dt, 1H, J = 13.2, 8.3 Hz), 1.27
(d, 3H, J = 7.3 Hz).
6.1.3. (3R,5S)-5-[(1S)-1-Azido-2-(benzyloxy)ethyl]-3-
methyldihydrofuran-2(3H)-one (14c)
6.1.4. N-{(1S)-1-[(2S,4R)-4-Methyl-5-oxotetrahydrofuran-2-yl]-
2-hydroxyethyl}-2-nitrobenzenesulfonamide (10c)
To a solution of 14c (7.56 g, 27.5 mmol) in EtOH (150 mL), a
solution of 4 N HCl in dioxane (15.0 mL, 60.0 mmol) and 10% palla-
dium–carbon (50% wet, 1.88 g) was added. The suspension was
stirred under H2 atmosphere at room temperature for 6 h. H2 in
Methanesulfonyl chloride (5.36 g, 47.0 mmol) was added to a
solution of 13c (7.80 g, 31.2 mmol) and triethylamine (9.45 g,
93.6 mmol) in CH2Cl2 (200 mL) under ice-cooling, and the mixture
was stirred at the same temperature for 3 h. Water was added to
the reaction mixture, followed by extraction with CH2Cl2. Then,

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Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
the reaction vessel was replaced by N2, and then the
reaction mixture was diluted with EtOH (100 mL). Palladium–
carbon was separated by filtration and washed with EtOH. The
solvent was evaporated from the filtrate under reduced pressure
to obtain crude (3R,5S)-5-[(1S)-1-amino-2-hydroxyethyl]-3-meth-
yldihydrofuran-2(3H)-one. Triethylamine (8.66 g, 85.7 mmol) and
2-nitrobenzenesulfonyl chloride (9.67 g, 41.3 mmol) were added
to a solution of the crude (3R,5S)-5-[(1S)-1-amino-2-hydroxy-
ethyl]-3-methyldihydrofuran-2(3H)-one in a mixed solvent of
THF (120 mL) and water (12 mL) at 0 °C, and the mixture was
stirred at the same temperature for 2 h. The reaction mixture
was concentrated under reduced pressure, and water (200 mL)
was added, followed by extraction with AcOEt. Then, the organic
layer was washed with water, saturated sodium bicarbonate
aqueous solution and brine, and dried over anhydrous MgSO4.
After filtration, the solvent was evaporated under reduced pres-
sure, and the residue was purified by silica gel column chroma-
tography (eluent, AcOEt) to obtain 10c (5.27 g, 56%, >99% ee,
two-steps) as a single stereoisomer and a colorless solid after
crystallization from diisopropyl ether (10 mL) and AcOEt
(20 mL). The ee value of 10c was determined by chiral HPLC anal-
ysis: column, Chiral Pack AD-H (4.6 ꢂ 250 mm); eluent, ethanol;
flow rate = 0.8 mL/min; tR of (1S,2S,4R)-isomer = 4.9 min; tR of
6.1.7. N-{(1S)-2-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-1-[(2S,4R)-4-ethyl-5-oxotetrahydrofuran-2-
yl]ethyl}-2-nitrobenzenesulfonamide (16b)
In the same manner as the preparation of 16a, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz, CDCl3):
a mixture of rotamers d 8.17 (br d, 1H, J = 7.8 Hz), 7.93 (br s, 1H),
7.79 (br s, 2H), 7.45 (br d, 1H, J = 8.2 Hz), 7.33–7.26 (m, 2H),
7.19–7.10 (m, 1H), 5.90 (br s, 0.5H), 5.51 (br s, 0.5H), 4.89 (br s,
1H), 3.63 (br s, 1H), 3.30–3.10 (m, 3H), 2.91-2.45 (m, 4.5H), 2.15
(br s, 1.5H), 1.92–1.82 (m, 1H), 1.64–1.51 (m, 1H), 1.17 (br s, 3H),
1.07 (br s, 3H), 1.04 (br t, 3H, J = 7.4 Hz). IR: 1777, 1649, 1539,
1353, 1332, 1168, 1122 cmꢀ1
.
6.1.8. N-{(1S)-2-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-1-[(2S,4R)-4-methyl-5-oxotetrahydrofuran-
2-yl]ethyl}-2-nitrobenzenesulfonamide (16c)
In the same manner as the preparation of 16a, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz, CDCl3):
a mixture of rotamers d 8.17 (br d, 1H, J = 7.0 Hz), 7.93 (br s, 1H),
7.79 (br s, 2H), 7.44 (dd, 1H, J = 7.4, 2.0 Hz), 7.35–7.09 (m, 3H),
5.87 (br s, 0.5H), 5.53 (br s, 0.5H), 4.90 (br s, 1H), 3.63 (br s, 1H),
3.29–3.10 (m, 3H), 2.90–2.56 (m, 4.5H), 2.20–2.05 (m, 1.5H), 1.33
(br d, 3H, J = 7.0 Hz), 1.16 (br s, 3H), 1.07 (br s, 3H).
(1R,2R,4S)-isomer = 6.0 min.
½
a 2D3:8
ꢃ
+56.0 (c 1.00, MeOH).
Mp = 131.0–131.2 °C. 1H NMR (500 MHz, CDCl3): d 8.16–8.09 (m,
1H), 7.94–7.87 (m, 1H), 7.79–7.71 (m, 2H), 5.89 (br d, 1H,
J = 6.8 Hz), 4.72–4.64 (m, 1H), 3.74–3.58 (m, 3H), 2.92–2.81 (m,
1H), 2.63 (ddd, 1H, J = 13.2, 9.8, 4.9 Hz), 2.03 (dt, 1H, J = 13.7,
8.3 Hz), 2.01–1.96 (m, 1H), 1.28 (d, 3H, J = 7.3 Hz). MS (FAB+):
m/z = 345 (M+H)+.
6.1.9. N-{(1S)-2-[4-(2-Chloro-5-fluorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-1-[(2S,4S)-4-isopropyl-5-
oxotetrahydrofuran-2-yl]ethyl}-2-nitrobenzenesulfonamide
(16d)
In the same manner as the preparation of 16a, the title com-
pound was obtained as a colorless solid. 1H NMR (500 MHz, CDCl3):
a mixture of rotamers d 8.18–8.16 (m, 1H), 7.94 (br s, 1H), 7.83–
7.77 (m, 2H), 7.42 (dd, 1H, J = 8.8, 5.4 Hz), 7.05–6.89 (m, 2H),
5.92 (br s, 0.6H), 5.57 (br s, 0.4H), 4.86–4.83 (m, 1H), 3.62 (br s,
1H), 3.30–3.10 (m, 3H), 2.88–2.40 (m, 4.4H), 2.27–2.15 (m, 2.6H),
1.17 (br s, 3H), 1.08 (br s, 3H), 1.03 (d, 3H, J = 6.8 Hz), 0.97 (d,
3H, J = 6.6 Hz).
6.1.5. (3R,5S)-3-Methyl-5-{(2S)-1-[(2-
nitrophenyl)sulfonyl]aziridin-2-yl}dihydrofuran-2(3H)-one
(15c)
A solution of diethyl azodicarboxylate in toluene (40%, 1.60 mL,
3.48 mmol) was added to a solution of 10c (1.00 g, 2.90 mmol) and
triphenylphosphine (912 mg, 3.48 mmol) in THF (30 mL) under
ice-cooling over 5 min, and the mixture was stirred at the same
temperature for 5 min. The reaction mixture was concentrated un-
der reduced pressure, and the residue was purified by silica gel col-
umn chromatography (eluent, toluene/acetone = 5:1) to obtain 15c
(823 mg, 87%) as a colorless oil. 1H NMR (400 MHz, CDCl3): d 8.14
(dd, 1H, J = 7.4, 1.5 Hz), 7.83–7.73 (m, 3H), 4.76 (dt, 1H, J = 8.8,
2.0 Hz), 3.26–3.23 (m, 1H), 2.97–2.88 (m, 1H), 2.83 (d, 1H,
J = 7.0 Hz), 2.65–2.60 (m, 2H), 2.18–2.10 (m, 1H), 1.26 (d, 3H,
J = 6.8 Hz). MS (FAB+): m/z = 327 (M+H)+.
6.1.10. N-{(1S)-2-[4-(2-Chloro-5-fluorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-1-[(2S,4R)-4-ethyl-5-oxotetrahydrofuran-2-
yl]ethyl}-2-nitrobenzenesulfonamide (16e)
In the same manner as the preparation of 16a, the title com-
pound was obtained as a colorless solid. 1H NMR (500 MHz, CDCl3):
a mixture of rotamers d 8.18–8.16 (m, 1H), 7.94 (br s, 1H), 7.83–
7.78 (m, 2H), 7.42 (dd, 1H, J = 8.8, 5.4 Hz), 7.05–7.01 (m, 1H),
6.94–6.87 (m, 1H), 5.87 (br s, 0.6H), 5.54 (br s, 0.4H), 4.89 (br s,
1H), 3.62 (br s, 1H), 3.30–3.10 (m, 3H), 2.92–2.53 (m, 4.5H), 2.14
(br s, 1.5H), 1.91–1.82 (m, 1H), 1.63–1.56 (m, 1H), 1.17 (br s, 3H),
1.08 (br s, 3H), 1.03 (t, 3H, J = 7.6 Hz).
Experimental details for the synthesis and characterization of
15a and 15b are available in Refs.13,16.
6.1.6. N-{(1S)-2-[4-(2-Chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-1-[(2S,4S)-4-isopropyl-5-
oxotetrahydrofuran-2-yl]ethyl}-2-nitrobenzenesulfonamide
(16a)
6.1.11. N-{(1S)-2-[4-(5-Fluoro-2-methylphenyl)-2,2-dimethyl-5-
oxo-1-piperazinyl]-1-[(2S,4S)-4-isopropyl-5-oxotetrahydro-2-
furanyl]ethyl}-2-nitrobenzenesulfonamide (16f)
In the same manner as the preparation of 16a, the title compound
was obtained as a colorless solid. 1H NMR (400 MHz, CDCl3): a mix-
ture of rotamers d 8.18–8.16 (m, 1H), 7.98–7.90 (m, 1H), 7.85–7.78
(m, 2H), 7.20 (dd, 1H, J = 8.2, 6.7 Hz), 6.96 (dt, 1H, J = 8.2, 2.7 Hz),
6.82–6.67 (m, 1H), 5.89 (br d, 0.6H, J = 6.7 Hz), 5.60 (br d, 0.4H,
J = 7.0 Hz), 4.88–4.79 (m, 1H), 3.68–3.58 (m, 1H), 3.37–3.27 (m,
1H), 3.16–2.17 (m, 9H), 2.11 (s, 3H), 1.16–0.93 (m, 12H).
A solution of N-Ns-aziridine 15a (192 mg, 0.541 mmol) and
1-(2-chlorophenyl)-5,5-dimethylpiperazin-2-one (8a) (181 mg,
0.758 mmol) in toluene (7 mL) was stirred at 110 °C for 90 min. After
cooling, the reaction mixture was concentrated under reduced pres-
sure and the residue was purified by silica gel column chromatogra-
phy (eluent, CH2Cl2/AcOEt = 5:1) to obtain 16a (299 mg, 93%) as a
colorless solid. 1H NMR (400 MHz, CDCl3): a mixture of rotamers d
8.18–8.16 (m, 1H), 7.93 (br s, 1H), 7.83–7.76 (m, 2H), 7.46 (dd, 1H,
J = 7.8, 2.0 Hz), 7.36–7.27 (m, 2H), 7.22–7.11 (m, 1H), 5.93 (br s,
0.5H), 5.53 (br s, 0.5H), 4.88–4.84 (m, 1H), 3.62 (br s, 1H), 3.30–
3.10 (m, 3H), 2.92–2.69 (m, 2.5H), 2.56 (br s, 1H), 2.44 (ddd, 1H,
J = 13.7, 10.6, 5.9 Hz), 2.27–2.14 (m, 2.5H), 1.17 (br s, 3H), 1.08 (br
s, 3H), 1.04 (d, 3H, J = 6.6 Hz), 0.98 (d, 3H, J = 6.6 Hz).
6.1.12. N-{(1S)-1-[(2S,4R)-4-Ethyl-5-oxotetrahydrofuran-2-yl]-
2-[4-(5-fluoro-2-methylphenyl)-2,2-dimethyl-5-oxopiperazin-
1-yl]ethyl}-2-nitrobenzenesulfonamide (16g)
In the same manner as the preparation of 16a, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz, CDCl3):
a mixture of rotamers d 8.19–8.15 (m, 1H), 7.98–7.90 (m, 1H),

Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
3187
7.83–7.78 (m, 2H), 7.22–7.18 (m, 1H), 6.96 (dt, 1H, J = 8.2, 2.7 Hz),
6.82–6.66 (m, 1H), 5.85 (br d, 0.6H, J = 6.7 Hz), 5.57 (br d, 0.4H,
J = 8.2 Hz), 4.93–4.83 (m, 1H), 3.69–3.59 (m, 1H), 3.37–2.47 (m,
7.6H), 2.21–2.11 (m, 4.4H), 1.90–1.82 (m, 1H), 1.62–1.54 (m, 1H),
1.17–1.00 (m, 9H).
a mixture of rotamers d 7.49–7.46 (m, 1H), 7.35–7.21 (m, 3H), 4.90
(br s, 1H), 4.43 (br s, 1H), 3.88–3.82 (m, 1H), 3.58–3.24 (m, 4H),
2.80–2.40 (m, 4H), 2.04–1.96 (m, 1H), 1.45 (s, 9H), 1.31 (br d, 3H,
J = 7.0 Hz), 1.26 (br s, 3H), 1.23 (br s, 3H).
6.1.17. tert-Butyl {(1S)-2-[4-(2-chloro-5-fluorophenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-1-[(2S,4S)-4-isopropyl-5-
oxotetrahydrofuran-2-yl]ethyl}carbamate (17d)
In the same manner as the preparation of 17a, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz, CDCl3):
a mixture of rotamers d 7.43 (dd, 1H, J = 8.8, 5.3 Hz), 7.05–6.99 (m,
2H), 4.82 (br s, 1H), 4.44 (br s, 1H), 3.87–3.81 (m, 1H), 3.59–2.39
(m, 7H), 2.31–2.11 (m, 3H), 1.45 (br s, 9H), 1.25 (br s, 6H), 1.03
(br d, 3H, J = 6.7 Hz), 0.97 (br d, 3H, J = 7.0 Hz).
6.1.13. N-{(1S)-2-[4-(5-Fluoro-2-methylphenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-1-[(2S,4R)-4-methyl-5-oxotetrahydrofuran-
2-yl]ethyl}-2-nitrobenzenesulfonamide (16h)
In the same manner as the preparation of 16a, the title com-
pound was obtained as a colorless solid. 1H NMR (500 MHz, CDCl3):
a mixture of rotamers d 8.18–8.16 (m, 1H), 7.99–7.78 (m, 3H),
7.22–7.18 (m, 1H), 6.98–6.93 (m, 1H), 6.81 (br d, 0.4H, J = 7.0 Hz),
6.66 (br d, 0.6H, J = 7.0 Hz), 5.83 (br d, 0.6H, J = 7.4 Hz), 5.57 (br
d, 0.4H, J = 7.8 Hz), 4.94 (br s, 0.4H), 4.85 (br s, 0.6H), 3.70–3.26
(m, 2H), 3.12–2.50 (m, 7H), 2.18–2.05 (m, 4H), 1.34–1.32 (m,
3H), 1.17–1.05 (m, 6H).
6.1.18. tert-Butyl {(1S)-2-[4-(2-chloro-5-fluorophenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-1-[(2S,4R)-4-ethyl-5-
oxotetrahydrofuran-2-yl]ethyl}carbamate (17e)
6.1.14. tert-Butyl {(1S)-2-[4-(2-chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-1-[(2S,4S)-4-isopropyl-5-
oxotetrahydrofuran-2-yl]ethyl}carbamate (17a)
Cesium carbonate (197 mg, 0.605 mmol) was added to a solu-
tion of 16a (299 mg, 0.504 mmol) and thiophenol (110 lL,
1.00 mmol) in DMF (5 mL) under N2 atmosphere at room tempera-
ture, and the mixture was stirred at the same temperature for 1 h.
Brine was added to the reaction mixture, followed by extraction
with CH2Cl2. Then, the organic layer was 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 = 20:1–10:1) to afford
4-{(2S)-2-amino-2-[(2S,4S)-4-isopropyl-5-oxotetrahydrofuran-2-
In the same manner as the preparation of 17a, the title com-
pound was obtained as a colorless solid. 1H NMR (500 MHz, CDCl3):
a mixture of rotamers d 7.45–7.42 (m, 1H), 7.05–6.96 (m, 2H), 4.87
(br s, 1H), 4.56 (br s, 1H), 3.85–3.84 (m, 1H), 3.57–2.36 (m, 8H),
2.08–2.02 (m, 1H), 1.90–1.82 (m, 1H), 1.61–1.52 (m, 1H), 1.46 (br
s, 9H), 1.24 (br s, 6H), 1.03 (t, 3H, J = 7.3 Hz).
6.1.19. tert-Butyl {(1S)-2-[4-(5-fluoro-2-methylphenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-1-[(2S,4S)-4-isopropyl-5-
oxotetrahydrofuran-2-yl]ethyl}carbamate (17f)
In the same manner as the preparation of 17a, the title com-
pound was obtained as a colorless solid. 1H NMR (500 MHz, CDCl3):
a mixture of rotamers d 7.21 (dd, 1H, J = 8.3, 6.4 Hz), 6.95 (dt, 1H,
J = 8.3, 2.4 Hz), 6.86–6.80 (m, 1H), 4.83–4.76 (m, 1H), 4.47–4.44
(m, 1H), 3.87–3.82 (m, 1H), 3.55–3.16 (m, 4H), 2.75–2.41 (m,
3H), 2.30–2.11 (m, 6H), 1.45 (br s, 9H), 1.22 (br s, 6H), 1.03 (br d,
3H, J = 6.8 Hz), 0.98–0.96 (m, 3H).
yl]ethyl}-1-(2-chlorophenyl)-5,5-dimethylpiperazin-2-one as
a
colorless solid. Triethylamine (210 L, 1.51 mmol) and di-t-butyl
l
dicarbonate (132 mg, 0.605 mmol) were added to a solution of
4-{(2S)-2-amino-2-[(2S,4S)-4-isopropyl-5-oxotetrahydrofuran-2-
yl]ethyl}-1-(2-chlorophenyl)-5,5-dimethylpiperazin-2-one ob-
tained above in CH2Cl2 (5 mL), and the mixture was stirred at room
temperature for 15 h. Brine was added to the reaction mixture, fol-
lowed by extraction with CH2Cl2. Then, the organic layer was 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/AcOEt = 2:1) to obtain
6.1.20. tert-Butyl {(1S)-1-[(2S,4R)-4-ethyl-5-
oxotetrahydrofuran-2-yl]-2-[4-(5-fluoro-2-methylphenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]ethyl}carbamate (17g)
In the same manner as the preparation of 17a, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz, CDCl3):
a mixture of rotamers d 7.22 (dd, 1H, J = 8.2, 6.3 Hz), 6.96 (dt, 1H,
J = 8.2, 2.7 Hz), 6.87–6.80 (m, 1H), 4.89–4.82 (m, 1H), 4.46–4.42
(m, 1H), 3.88–3.82 (m, 1H), 3.56–3.15 (m, 4H), 2.77–2.36 (m,
4H), 2.19 (br s, 1.2H), 2.18 (br s, 1.8H), 2.08–2.01 (m, 1H), 1.92–
1.81 (m, 1H), 1.62–1.51 (m, 1H), 1.45 (br s, 9H), 1.22 (br s, 6H),
1.03 (br t, 3H, J = 7.2 Hz).
17a (205 mg, 80%, two-steps) as
a
colorless solid. 1H NMR
(400 MHz, CDCl3): a mixture of rotamers d 7.47 (dd, 1H, J = 7.8,
2.0 Hz), 7.34-7.20 (m, 3H), 4.86–4.78 (m, 1H), 4.47–4.42 (m, 1H),
3.87–3.81 (m, 1H), 3.58–3.24 (m, 4H), 2.79–2.32 (m, 0.5H), 2.62–
2.57 (m, 2H), 2.44–2.39 (m, 0.5H), 2.31–2.24 (m, 1H), 2.21–2.11
(m, 2H), 1.45 (s, 9H), 1.26–1.22 (m, 6H), 1.03 (d, 3H, J = 7.0 Hz),
0.97 (d, 3H, J = 6.6 Hz).
6.1.21. tert-Butyl {(1S)-2-[4-(5-fluoro-2-methylphenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-1-[(2S,4R)-4-methyl-5-
oxotetrahydrofuran-2-yl]ethyl}carbamate (17h)
In the same manner as the preparation of 17a, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz, CDCl3):
a mixture of rotamers d 7.22 (dd, 1H, J = 8.6, 6.3 Hz), 6.98–6.93 (m,
1H), 6.87–6.79 (m, 1H), 4.92–4.85 (m, 1H), 4.45–4.41 (m, 1H),
3.88–3.82 (m, 1H), 3.56–3.16 (m, 4H), 2.77–2.41 (m, 4H), 2.19 (br
s, 1.8H), 2.18 (br s, 1.2H), 2.04–1.96 (m, 1H), 1.45 (br s, 9H), 1.32
(br d, 3H, J = 7.4 Hz), 1.23 (br s, 6H).
6.1.15. tert-Butyl {(1S)-2-[4-(2-chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-1-[(2S,4R)-4-ethyl-5-oxotetrahydrofuran-2-
yl]ethyl}carbamate (17b)
In the same manner as the preparation of 17a, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz, CDCl3):
a mixture of rotamers d 7.47 (br d, 1H, J = 7.4 Hz), 7.34–7.20 (m,
3H), 4.91–4.83 (m, 1H), 4.46–4.40 (m, 1H), 3.88–3.81 (m, 1H),
3.58–3.24 (m, 4H), 2.80–2.36 (m, 4H), 2.08–2.01 (m, 1H), 1.91–
1.81 (m, 1H), 1.62–1.50 (m, 1H), 1.45 (s, 9H), 1.26 (br s, 3H), 1.22
(br s, 3H) 1.03 (br t, 3H, J = 7.4 Hz).
6.1.22. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-N-(2,2-dimethylpropyl)-4-hydroxy-2-
isopropylhexanamide hemifumarate (5)
2-Hydroxypyridine (11.2 mg, 0.118 mmol) was added to a solu-
tion of 17a (300 mg, 0.590 mmol) and neopentylamine (700 lL,
6.1.16. tert-Butyl {(1S)-2-[4-(2-chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-1-[(2S,4R)-4-methyl-5-oxotetrahydrofuran-
2-yl]ethyl}carbamate (17c)
In the same manner as the preparation of 17a, the title com-
5.94 mmol), and the mixture was stirred at 80 °C for 3 h. After
cooling, water was added to the reaction mixture, followed by
pound was obtained as a colorless solid. 1H NMR (400 MHz, CDCl3):

3188
Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
extraction with AcOEt. 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 puri-
fied by silica gel column chromatography (eluent, CH2Cl2/
MeOH = 40:1–20:1) to obtain tert-butyl {(2S,3S,5S)-1-[4-(2-chloro-
phenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]-5-[(2,2-dimethylpropyl)
carbamoyl]-3-hydroxy-6-methylheptan-2-yl}carbamate (216 mg,
(s, 1H), 3.67–3.60 (m, 2H), 3.52–3.46 (m, 1H), 3.37–3.11 (m, 4H),
2.97–2.89 (m, 1.6H), 2.80–2.67 (m, 0.8H), 2.53–2.49 (m, 0.6H),
2.37–2.32 (m, 1H), 1.88–1.68 (m, 4H), 1.31–1.26 (m, 6H), 1.00–
0.93 (m, 12H). MS (FAB+): m/z = 481 (M+H)+.
6.1.26. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxo-piperazin-1-yl]-4-hydroxy-N,2-diisopropylhexanamide
fumarate (22)
61%) as a colorless solid. Trifluoroacetic acid (837 lL, 10.9 mmol)
was added to a solution of tert-butyl {(2S,3S,5S)-1-[4-(2-chloro-
phenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]-5-[(2,2-dimethylpropyl)
carbamoyl]-3-hydroxy-6-methylheptan-2-yl}carbamate (216 mg,
0.363 mmol) in CH2Cl2 (1.7 mL) at room temperature, and the mix-
ture was stirred at the same temperature for 30 min. After concen-
tration under reduced pressure, saturated sodium bicarbonate
aqueous solution was added to the reaction mixture, followed by
extraction with CH2Cl2. Then, the organic layer was dried over
anhydrous Na2SO4. After filtration, the solvent was evaporated un-
der reduced pressure, and the residue was purified by silica gel col-
umn chromatography (eluent, CH2Cl2/MeOH/Et3N = 100:10:1) to
obtain the free base of 5. Fumaric acid (21.0 mg, 0.181 mmol)
was added to a solution of the free base of 5 in MeOH (5 mL),
and the mixture was stirred at room temperature for 5 min. The
solvent was evaporated under reduced pressure to obtain 5
(163 mg, 81%, two-steps) as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 7.92 (br s, 1H), 7.55–7.33 (m,
1H), 7.44–7.31 (m, 3H), 6.67 (s, 1H), 3.67–3.59 (m, 2H), 3.52–
3.47 (m, 1H), 3.39–3.11 (m, 4H), 2.97–2.90 (m, 1.6H), 2.80–2.66
(m, 0.8H), 2.51 (dd, 0.6H, J = 13.7, 3.9 Hz), 2.43–2.38 (m, 1H),
1.91–1.80 (m, 2H), 1.74–1.68 (m, 1H), 1.31–1.26 (m, 6H), 1.01–
0.99 (m, 6H), 0.94 (s, 9H). MS (FAB+): m/z = 495 (M+H)+. HPLC
In the same manner as the preparation of 5, the title compound
was obtained as a colorless solid. 1H NMR (500 MHz, CD3OD): a
mixture of rotamers d 7.90 (br d, 1H, J = 7.8 Hz), 7.55–7.54 (m,
1H), 7.43–7.32 (m, 3H), 6.68 (s, 2H), 4.06–3.98 (m, 1H), 3.66–3.60
(m, 2.4H), 3.53–3.46 (m, 1.4H), 3.37–3.35 (m, 0.8H), 3.23–3.19
(m, 0.8H), 3.17–3.13 (m, 0.6H), 2.97–2.92 (m, 0.6H), 2.80–2.69
(m, 0.8H), 2.54–2.51 (m, 0.6H), 2.29–2.25 (m, 1H), 1.85–1.78 (m,
2H), 1.74–1.68 (m, 1H), 1.31–1.26 (m, 6H), 1.19–1.15 (m, 6H),
1.00–0.96 (m, 6H). MS (FAB+): m/z = 467 (M+H)+.
6.1.27. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-N-(2,2-dimethylpropyl)-2-ethyl-4-
hydroxyhexanamide hemifumarate (23)
In the same manner as the preparation of 5, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 7.98 (br t, 1H, J = 5.7 Hz),
7.56–7.53 (m, 1H), 7.44–7.32 (m, 3H), 6.67 (s, 1H), 3.66–3.50
(m, 3H), 3.37–3.09 (m, 4H), 2.95–2.89 (m, 1.6H), 2.78–2.66 (m,
0.8H), 2.61–2.48 (m, 1.6H), 1.90–1.83 (m, 1H), 1.72–1.48 (m,
3H), 1.30–1.26 (m, 6H), 0.97 (t, 3H, J = 7.4 Hz), 0.93 (s, 9H). MS
(FAB+): m/z = 481 (M+H)+.
tR = 4.14 min
(>99.9%
purity;
column,
Inertsil
ODS-3,
6.1.28. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-N-(2,2-dimethylpropyl)-4-hydroxy-2-
methylhexanamide hemifumarate (24)
4.6 ꢂ 250 mm; eluent, acetonitrile/0.1% ammonium acetate aque-
ous solution = 70:30; flow rate, 1 mL/min; wave length, 220 nm).
In the same manner as the preparation of 5, the title compound
was obtained as a colorless solid. 1H NMR (400 MHz, CD3OD): a
mixture of rotamers d 7.99 (br t, 1H, J = 5.9 Hz), 7.55–7.53 (m,
1H), 7.44–7.32 (m, 3H), 6.67 (s, 1H), 3.67–3.52 (m, 3H), 3.38–
3.11 (m, 4H), 2.98–2.91 (m, 1.6H), 2.78–2.68 (m, 1.8H), 2.51 (dd,
0.6H, J = 13.3, 3.9 Hz), 1.94–1.87 (m, 1H), 1.59–1.52 (m, 1H),
1.31–1.26 (m, 6H), 1.21 (d, 3H, J = 7.0 Hz), 0.92 (s, 9H). MS
(FAB+): m/z = 467 (M+H)+.
6.1.23. (2S,4S,5S)-5-Amino-N-butyl-6-[4-(2-chloro-5-
fluorophenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]-4-hydroxy-2-
isopropylhexanamide fumarate (4)
In the same manner as the preparation of 5, the title compound
was obtained as a colorless solid. 1H NMR (400 MHz, CD3OD): a
mixture of rotamers d 8.04 (br s, 1H), 7.59–7.56 (m, 1H), 7.22–
7.18 (m, 2H), 6.71 (s, 2H), 3.63–3.59 (m, 2H), 3.49–3.44 (m, 1H),
3.36–3.18 (m, 5H), 2.95 (br t, 0.6H, J = 13.3 Hz), 2.80–2.69 (m,
0.8H), 2.52–2.48 (m, 0.6 H), 2.32–2.28 (m, 1H), 1.85–1.78 (m,
2H), 1.74–1.68 (m, 1H), 1.55–1.48 (m, 2H), 1.41–1.33 (m, 2H),
1.31–1.26 (m, 6H), 1.00–0.93 (m, 9H). MS (FAB+): m/z = 499
(M+H)+.
6.1.29. (2R,4S,5S)-5-Amino-N-(2,2-dimethylpropyl)-6-[4-(5-
fluoro-2-methylphenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]-4-
hydroxy-2-methylhexanamide hemifumarate (25)
In the same manner as the preparation of 5, the title compound
was obtained as a colorless solid. 1H NMR (400 MHz, CD3OD): a
mixture of rotamers d 7.33–7.29 (m, 1H), 7.06–6.93 (m, 2H), 6.66
(s, 1H), 3.68–3.48 (m, 3H), 3.39–3.11 (m, 4H), 2.95–2.89 (m,
1.6H), 2.81–2.73 (m, 1.4H), 2.66–2.62 (m, 0.4H), 2.50–2.46 (m,
0.6H), 2.19 (s, 3H), 1.93–1.87 (m, 1H), 1.58–1.51 (m, 1H), 1.27–
1.25 (m, 6H), 1.21 (d, 3H, J = 7.0 Hz), 0.92 (s, 9H). MS (FAB+): m/
z = 465 (M+H)+.
6.1.24. (2S,4S,5S)-5-Amino-N-butyl-6-[4-(2-chlorophenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-4-hydroxy-2-
isopropylhexanamide fumarate (20)
In a similar manner as the preparation of 5, the title compound
was obtained as a colorless solid. 1H NMR (400 MHz, CD3OD): a
mixture of rotamers d 8.03 (br s, 1H), 7.56–7.53 (m, 1H), 7.44–
7.32 (m, 3H), 6.68 (s, 2H), 3.68–3.60 (m, 2H), 3.51–3.45 (m, 1H),
3.37–3.18 (m, 5H), 2.95 (br t, 0.6H, J = 11.0 Hz), 2.82–2.67 (m,
0.8H), 2.52 (dd, 0.6H, J = 13.7, 3.9 Hz), 2.33–2.28 (m, 1H), 1.87–
1.78 (m, 2H), 1.75–1.68 (m, 1H), 1.55–1.48 (m, 2H), 1.42–1.33
(m, 2H), 1.31–1.26 (m, 6H), 1.00–0.93 (m, 9H). MS (FAB+): m/
z = 482 (M+H)+.
6.1.30. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-4-hydroxy-N-(2-hydroxy-2-
methylpropyl)-2-isopropylhexanamide hemifumarate (27)
In the same manner as the preparation of 5, the title compound
was obtained as a colorless solid. 1H NMR (400 MHz, CD3OD): a
mixture of rotamers d 7.91–7.87 (m, 1H), 7.56–7.53 (m, 1H),
7.44–7.32 (m, 3H), 6.70 (s, 1H), 3.67–3.60 (m, 2H), 3.55–3.48 (m,
1H), 3.38–3.13 (m, 5H), 2.98–2.92 (m, 0.6H), 2.80–2.68 (m, 0.8H),
2.54–2.50 (m, 0.6H), 2.42–2.36 (m, 1H), 1.91–1.69 (m, 3H), 1.31–
1.27 (m, 6H), 1.21 (s, 6H), 1.00 (d, 3H, J = 4.3 Hz), 0.98 (d, 3H,
J = 4.3 Hz). MS (FAB+): m/z = 497 (M+H)+.
6.1.25. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-4-hydroxy-N-isobutyl-2-
isopropylhexanamide hemifumarate (21)
In the same manner as the preparation of 5, the title compound
was obtained as a colorless solid. 1H NMR (400 MHz, CD3OD): a
mixture of rotamers d 7.56–7.53 (m, 1H), 7.44–7.32 (m, 3H), 6.67

Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
3189
6.1.31. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
6.1.34. Methyl 3-{[(2S,4S,5S)-5-amino-6-[4-(2-chlorophenyl)-
5-oxopiperazin-1-yl]-4-hydroxy-N-(3-hydroxy-2,2-
2,2-dimethyl-5-oxopiperazin-1-yl]-4-hydroxy-2-
dimethylpropyl)-2-isopropylhexanamide fumarate (28)
In the same manner as the preparation of 5, the title compound
was obtained as a colorless solid. 1H NMR (500 MHz, CD3OD): a
mixture of rotamers d 8.08–8.05 (m, 1H), 7.56–7.55 (m, 1H),
7.44–7.34 (m, 3H), 6.70 (s, 2H), 3.67–3.61 (m, 2H), 3.52–3.45 (m,
1H), 3.39–3.15 (m, 6H), 3.05–3.02 (m, 1H), 2.98–2.93 (m, 0.6H),
2.80–2.70 (m, 0.8H), 2.55–2.52 (m, 0.6H), 2.43–2.39 (m, 1H),
1.89–1.83 (m, 2H), 1.74–1.69 (m, 1H), 1.32–1.27 (m, 6H), 1.02–
1.00 (m, 6H), 0.91–0.90 (m, 6H). MS (FAB+): m/z = 511 (M+H)+.
isopropylhexanoyl]amino}-2,2-dimethylpropanoate
hemifumarate (31)
In the same manner as the preparation of 5, the title compound
was obtained as a colorless solid. 1H NMR (400 MHz, CD3OD): a
mixture of rotamers d 7.55–7.54 (m, 1H), 7.42–7.32 (m, 3H), 6.67
(s, 1H), 3.68 (s, 3H), 3.63–3.61 (m, 2H), 3.51–3.48 (m, 1H), 3.42–
3.12 (m, 5H), 2.96 (br t, 0.6H, J = 12.2 Hz), 2.81–2.69 (m, 0.8H),
2.55–2.51 (m, 0.6H), 2.37–2.32 (m, 1H), 1.87–1.78 (m, 2H), 1.72–
1.66 (m, 1H), 1.31–1.27 (m, 6H), 1.20–1.18 (m, 6H), 0.99–0.96
(m, 6H). MS (FAB+): m/z = 539 (M+H)+.
6.1.32. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-2-ethyl-4-hydroxy-N-(3-hydroxy-2,2-
dimethylpropyl)hexanamide hemifumarate (29)
6.1.35. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-4-hydroxy-N-(trans-4-
In the same manner as the preparation of 5, the title
compound was obtained as a colorless solid. 1H NMR (500 MHz,
CD3OD): a mixture of rotamers d 8.10–8.06 (m, 1H), 7.57–7.52
(m, 1H), 7.44–7.33 (m, 3H), 6.68 (s, 1H), 3.73–3.68 (m, 2H), 3.67–
3.60 (m, 2H), 3.53–3.45 (m, 1H), 3.39–3.13 (m, 6H), 3.04–3.01
(m, 1H), 2.97–2.92 (m, 0.6H), 2.80–2.72 (m, 0.8H), 2.55–2.50 (m,
0.6H), 1.89–1.83 (m, 1H), 1.72–1.60 (m, 2H), 1.32–1.26 (m, 6H),
0.99–0.96 (m, 3H), 0.90–0.89 (m, 6H). MS (FAB+): m/z = 497
(M+H)+.
hydroxycyclohexyl)-2-isopropylhexanamide fumarate (33)
In the same manner as the preparation of 30, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 7.95–7.93 (m, 1H), 7.56–7.54
(m, 1H), 7.45–7.32 (m, 3H), 6.69 (s, 2H), 3.69–3.59 (m, 3H), 3.55–
3.44 (m, 2H), 3.37–3.12 (m, 3H), 2.97–2.91 (m, 0.6H), 2.81–2.67
(m, 0.8H), 2.54–2.50 (m, 0.6H), 2.29–2.23 (m, 1H), 1.98–1.68 (m,
7H), 1.37–1.26 (m, 10H), 0.99–0.95 (m, 6H). MS (FAB+): m/z = 522
(M+H)+.
6.1.33. (2S,4S,5S)-5-Amino-N-(3-amino-2,2-dimethyl-3-
oxopropyl)-6-[4-(2-chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-4-hydroxy-2-isopropylhexanamide
hemifumarate (30)
6.1.36. (2S,4S,5S)-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-4-hydroxy-2-isopropyl-N-(tetrahydro-2H-
pyran-4-yl)hexanamide fumarate (34)
In the same manner as the preparation of 5, the title compound
was obtained as a colorless solid. 1H NMR (400 MHz, CD3OD): a mix-
ture of rotamers d 7.56–7.54 (m, 1H), 7.44–7.38 (m, 2H), 7.35–7.32
(m, 1H), 6.70 (s, 2H), 3.95–3.92 (m, 3H), 3.67–3.59 (m, 2H), 3.51–
3.45 (m, 4H), 3.36–3.14 (m, 2H), 2.97–2.93 (m, 0.6H), 2.81–2.68
(m, 0.8H), 2.52 (dd, 0.6H, J = 13.4, 3.7 Hz), 2.32–2.28 (m, 1H), 1.86–
1.81 (m, 4H), 1.75–1.70 (m, 1H), 1.60–1.49 (m, 2H), 1.31–1.27 (m,
6H), 1.00–0.97 (m, 6H). MS (FAB+): m/z = 509 (M+H)+.
2-Hydroxypyridine (38.3 mg, 0.404 mmol) was added to a solu-
tion of 17a (205 mg, 0.404 mmol) and 3-amino-2,2-dimethylpro-
panamide (140 mg, 1.21 mmol) in triethylamine (4 mL), and the
mixture was stirred at 80 °C for 4 h. The reaction mixture was con-
centrated under reduced pressure, and then stirred at 80 °C for an
additional 14 h. After cooling, water was added to the reaction mix-
ture, followed by extraction with CH2Cl2. The organic layer was dried
over anhydrous MgSO4. After filtration, the solvent was evaporated
under reduced pressure, and the residue was purified by silica gel
column chromatography (eluent, CH2Cl2/MeOH = 40:3) to obtain
tert-butyl {(2S,3S,5S)-5-[(3-amino-2,2-dimethyl-3-oxopropyl)car-
bamoyl]-1-[4-(2-chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]-
3-hydroxy-6-methylheptan-2-yl}carbamate (167 mg, 66%) as a
6.1.37. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-2-ethyl-N-[3-ethyl-3-
(hydroxymethyl)pentyl]-4-hydroxyhexanamide fumarate (52)
In the same manner as the preparation of 5, the title compound
was obtained as a colorless solid. 1H NMR (500 MHz, CD3OD): a
mixture of rotamers d 8.04–7.99 (m, 1H), 7.56–7.53 (m, 1H),
7.45–7.33 (m, 3H), 6.72 (s, 2H), 3.67–3.60 (m, 2H), 3.56–3.45 (m,
1H), 3.39–3.15 (m, 6H), 3.06–3.02 (m, 1H), 2.98–2.92 (m, 0.6H),
2.77–2.70 (m, 0.8H), 2.60–2.50 (m, 1.6H), 1.90–1.83 (m, 1H),
1.70–1.51 (m, 3H), 1.34–1.21 (m, 10H), 0.99–0.95 (m, 3H), 0.87–
0.84 (m, 6H). MS (FAB+): m/z = 525 (M+H)+.
yellow solid. Trifluoroacetic acid (412 lL, 5.35 mmol) was added
to a solution of tert-butyl {(2S,3S,5S)-5-[(3-amino-2,2-dimethyl-
3-oxopropyl)carbamoyl]-1-[4-(2-chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]-3-hydroxy-6-methylheptan-2-yl}carbamate
(167 mg, 0.268 mmol) in CH2Cl2 (0.82 mL) at room temperature, and
the mixture was stirred at the same temperature for 50 min. After
concentration under reduced pressure, saturated sodium bicarbon-
ate aqueous solution was added to the reaction mixture, followed by
extraction with CH2Cl2. Then, the organic layer was dried over anhy-
drous Na2SO4. After filtration, the solvent was evaporated under re-
duced pressure, and the residue was purified by silica gel column
chromatography (eluent, CH2Cl2/MeOH/Et3N = 100:10:1) to obtain
the free base of 30. Fumaric acid (10.0 mg, 0.0861 mmol) was added
to a solution of the free base of 30 in MeOH (2 mL), and the mixture
was stirred at room temperaturefor 5 min. The reaction mixture was
concentrated under reduced pressure, and CH2Cl2 (0.8 mL) was
added to the residue. Et2O (8 mL) were further added and the solid
was collected by filtration to obtain 30 (90.0 mg, 58%, two-steps)
as a colorless solid. 1H NMR (400 MHz, CD3OD): a mixture of
rotamers d 7.56–7.53 (m, 1H), 7.44–7.32 (m, 3H), 6.67 (s, 1H),
3.86–3.59 (m, 2H), 3.52–3.12 (m, 6H), 2.95 (br t, 0.6H, J = 12.5 Hz),
2.81–2.67 (m, 0.8H), 2.51 (dd, 0.6H, J = 13.5, 4.1 Hz), 2.38–2.33 (m,
1H), 1.88–1.67 (m, 3H), 1.31–1.27 (m, 6H), 1.22 (s, 3H), 1.20 (s,
3H), 0.99–0.96 (m, 6H). MS (FAB+): m/z = 525 (M+H)+.
6.1.38. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-2-ethyl-4-hydroxy-N-{2-[1-
(hydroxymethyl)cyclopentyl]ethyl}hexanamide fumarate (53)
In a similar manner as the preparation of 30, the title compound
was obtained as a colorless solid. 1H NMR (500 MHz, CD3OD): a mix-
ture of rotamers d 7.54 (d, 1H, J = 7.3 Hz), 7.43–7.37 (m, 2H), 7.35–
7.32 (m, 1H), 6.69 (s, 2H), 3.86–3.81 (m, 1H), 3.65–3.58 (m, 2H),
3.52–3.48 (m, 1H), 3.38–3.20 (m, 7H), 3.02–2.99 (m, 0.6H), 2.86–
2.82 (m, 0.4H), 2.74–2.70 (m, 0.4H), 2.52 (dd, 0.6H, J = 13.4,
4.2 Hz), 1.97–1.85 (m, 5H), 1.76–1.71 (m, 1H), 1.34–1.24 (m, 12H),
1.05–1.02 (m, 1H), 0.86–0.79 (m, 2H). MS (ESI+): m/z = 523 (M+H)+.
6.1.39. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-2-ethyl-4-hydroxy-N-{2-[1-
(hydroxymethyl)cyclohexyl]ethyl}hexanamide fumarate (54)
In a similar manner as the preparation of 30, the title compound
was obtained as a colorless solid. 1H NMR (500 MHz, CD3OD): a

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Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
mixture of rotamers d 8.06 (t, 1H, J = 5.9 Hz), 7.54 (d, 1H, J = 7.3 Hz),
7.43–7.33 (m, 3H), 6.69 (s, 2H), 3.67–3.60 (m, 2H), 3.55–3.48 (m,
1H), 3.38–3.11 (m, 3H), 2.97–2.92 (m, 0.6H), 2.79–2.70 (m, 0.6H),
2.59–2.50 (m, 1.8H), 1.89–1.85 (m, 1H), 1.69–1.46 (m, 10H),
1.35–1.27 (m, 12H), 1.05–1.02 (m, 1H), 0.97 (t, 3H, J = 7.3 Hz). MS
(ESI+): m/z = 537 (M+H)+.
(m, 0.6H), 2.80–2.59 (m, 1.8H), 2.53 (dd, 0.6H, J = 13.2, 3.4 Hz),
2.20 (br s, 1H), 2.16 (br s, 1H), 2.10 (br s, 1H), 1.95–1.84 (m, 3H),
1.74–1.48 (m, 12H), 1.31–1.26 (m, 6H), 0.97 (t, 3H, J = 7.6 Hz). MS
(ESI+): m/z = 561 (M+H)+.
6.1.44. (2S,4S,5S)-5-Amino-6-[4-(2-chloro-5-fluorophenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-4-hydroxy-N-[(2s,5s)-5-
hydroxyadamantan-2-yl]-2-isopropylhexanamide fumarate
(61)
6.1.40. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-4-hydroxy-N-[(2s,5s)-5-
hydroxyadamantan-2-yl]-2-isopropylhexanamide fumarate
(55)
In a similar manner as the preparation of 30, the title compound
was obtained as a colorless solid. 1H NMR (400 MHz, CD3OD): a
mixture of rotamers d 7.78 (br d, 1H, J = 7.3 Hz), 7.58–7.55 (m,
1H), 7.21–7.18 (m, 2H), 6.69 (s, 2H), 3.97 (br s, 1H), 3.65–3.46
(m, 3H), 3.38–3.13 (m, 3H), 2.96–2.91 (m, 0.6H), 2.78–2.70 (m,
0.8H), 2.55-2.51 (m, 0.6H), 2.47–2.43 (m, 1H), 2.11 (br s, 2H),
2.06 (br s, 1H), 1.98–1.70 (m, 11H), 1.51–1.48 (m, 2H), 1.31–1.26
(m, 6H), 1.01–0.97 (m, 6H). MS (FAB+): m/z = 593 (M+H)+.
In a similar manner as the preparation of 30, the title compound
was obtained as a colorless solid. 1H NMR (500 MHz, CD3OD): a
mixture of rotamers d 7.78 (br s, 1H), 7.54 (br d, 1H, J = 7.8 Hz),
7.43–7.32 (m, 3H), 6.69 (s, 2H), 3.97 (br s, 1H), 3.65–3.47 (m,
3H), 3.36–3.26 (m, 2H), 3.21–3.12 (m, 1H), 2.96–2.91 (m, 0.6H),
2.79–2.70 (m, 0.8H), 2.55–2.52 (m, 0.6H), 2.45 (br s, 1H), 2.11 (br
s, 2H), 2.06 (br s, 1H), 1.98–1.70 (m, 11H), 1.50–1.48 (m, 2H),
1.31–1.26 (m, 6H), 1.00 (d, 3H, J = 6.8 Hz), 0.98 (d, 3H, J = 6.4 Hz).
MS (FAB+): m/z = 575 (M+H)+.
6.1.45. (2R,4S,5S)-5-Amino-6-[4-(2-chloro-5-fluorophenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-2-ethyl-4-hydroxy-N-[(2s,5s)-5-
hydroxyadamantan-2-yl]hexanamide fumarate (62)
6.1.41. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-2-ethyl-4-hydroxy-N-[(2s,5s)-5-
hydroxyadamantan-2-yl]hexanamide monofumarate dihydrate
(56)
In a similar manner as the preparation of 30, the title compound
was obtained as a colorless solid. 1H NMR (500 MHz, CD3OD): a
mixture of rotamers d 7.80 (m, 1H), 7.58–7.55 (m, 1H), 7.21–7.18
(m, 2H), 6.69 (s, 2H), 3.97 (br s, 1H), 3.64–3.12 (m, 6H), 2.95–
2.91 (m, 0.6H), 2.75–2.61 (m, 1.8H), 2.53–2.51 (m, 0.6H), 2.11 (br
s, 2H), 2.07 (br s, 1H), 1.97–1.82 (m, 5H), 1.77–1.75 (m, 4H),
1.68–1.48 (m, 5H), 1.30–1.26 (m, 6H), 0.96 (t, 3H, J = 7.3 Hz). MS
(FAB+): m/z = 579 (M+H)+.
Experimental details for the synthesis and characterization of
56 are available in Ref. 13 ½a D25:0
ꢃ
ꢀ31.6 (c 1.07, MeOH). 1H NMR
(500 MHz, CD3OD):
a mixture of rotamers d 7.84 (d, 1H,
J = 7.3 Hz), 7.55–7.54 (m, 1H), 7.43–7.32 (m, 3H), 6.68 (s, 2H),
3.97 (br s, 1H), 3.65–3.26 (m, 5H), 3.22–3.12 (m, 1H), 2.96–2.91
(m, 0.6H), 2.79–2.63 (m, 1.8H), 2.53 (dd, 0.6H, J = 13.7, 3.9 Hz),
2.12 (br s, 2H), 2.06 (br s, 1H), 1.98–1.82 (m, 5H), 1.77–1.74 (m,
4H), 1.69–1.47 (m, 5H), 1.31–1.26 (m, 6H), 0.95 (t, 3H, J = 7.3 Hz).
13C NMR (125 MHz, CD3OD): a mixture of rotamers d 177.7,
171.5, 168.9, 168.7, 140.1, 139.9, 136.3, 133.33, 133.27, 131.6,
131.5, 130.9, 130.5, 130.3, 129.6, 129.5, 68.5, 68.3, 67.9, 61.9,
55.7, 54.9, 54.7, 54.45, 54.41, 54.0, 53.2, 50.7, 50.2, 45.9, 45.50,
45.47, 45.4, 45.2, 37.9, 35.2, 35.1, 31.4, 31.2, 31.1, 28.0, 24.8,
22.5, 20.2, 17.9, 12.2. IR: 3431, 2916, 1642, 1627, 1588, 1531,
6.1.46. (2S,4S,5S)-5-Amino-6-[4-(5-fluoro-2-methylphenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-4-hydroxy-N-[(2s,5s)-5-
hydroxyadamantan-2-yl]-2-isopropylhexanamide fumarate
(63)
In a similar manner as the preparation of 30, the title compound
was obtained as a colorless solid. 1H NMR (500 MHz, CD3OD): a
mixture of rotamers d 7.79 (br d, 1H, J = 7.8 Hz), 7.33–7.30 (m,
1H), 7.06–6.93 (m, 2H), 6.69 (s, 2H), 3.97 (br s, 1H), 3.68–3.46
(m, 3H), 3.38–3.15 (m, 3H), 2.95–2.90 (m, 0.6H), 2.81–2.76 (m,
0.4H), 2.68–2.65 (m, 0.4H), 2.52–2.44 (m, 1.6H), 2.20 (br s, 3H),
2.12 (br s, 2H), 2.06 (br s, 1H), 1.99–1.71 (m, 11H), 1.51–1.48 (m,
2H), 1.27–1.25 (m, 6H), 1.01–0.97 (m, 6H). MS (FAB+): m/z = 573
(M+H)+.
1493, 1318, 1121, 750 cmꢀ1
C30H46N4O4Cl [M+H]+: 561.3208; found [M+H]+: 561.3217. Anal.
30H45N4O4ClꢁC4H4O4ꢁ2H2O (713.26); calcd: C 57.25, H 7.49, N
7.86, Cl 4.97; found: C 57.21, H 7.55, N 7.89, Cl, 4.88. HPLC
tR = 2.88 min (>99.5% purity; column, Inertsil ODS-3,
.
HRMS (ESI+): m/z calcd for
C
4.6 ꢂ 250 mm; eluent, acetonitrile/0.1% ammonium acetate aque-
6.1.47. (2R,4S,5S)-5-Amino-6-[4-(5-fluoro-2-methylphenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-2-ethyl-4-hydroxy-N-[(2s,5s)-5-
hydroxyadamantan-2-yl]hexanamide fumarate (64)
ous solution = 50:50; flow rate, 1 mL/min; wave length, 220 nm).
6.1.42. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-4-hydroxy-N-[(2s,5s)-5-
In a similar manner as the preparation of 30, the title compound
was obtained as a colorless solid. 1H NMR (500 MHz, CD3OD): a
mixture of rotamers d 7.80 (m, 1H), 7.33–7.30 (m, 1H), 7.06–7.02
(m, 1H), 6.98–6.93 (m, 1H), 6.69 (s, 2H), 3.97 (br s, 1H), 3.67–
3.13 (m, 6H), 2.94–2.90 (m, 0.6H), 2.80–2.75 (m, 0.4H), 2.69–2.62
(m, 1.4H), 2.50 (dd, 0.6H, J = 13.4, 4.2 Hz), 2.20 (br s, 3H), 2.11 (br
s, 2H), 2.07 (br s, 1H), 1.97–1.82 (m, 5H), 1.78–1.75 (m, 4H),
1.68–1.49 (m, 5H), 1.27–1.25 (m, 6H), 0.96 (t, 3H, J = 7.3 Hz). MS
(FAB+): m/z = 559 (M+H)+.
hydroxyadamantan-2-yl]-2-methylhexanamide fumarate (57)
In a similar manner as the preparation of 30, the title compound
was obtained as a yellow solid. 1H NMR (CD3OD, 400 MHz): a mix-
ture of rotamers d 7.82 (br d, 1H, J = 7.4 Hz), 7.56–7.54 (m, 1H),
7.44–7.32 (m, 3H), 6.71 (s, 2H), 3.93 (br s, 1H), 3.66–3.13 (m,
6H), 2.98–2.72 (m, 2.4H), 2.53 (dd, 0.6H, J = 13.3, 4.3 Hz), 2.10 (br
s, 3H), 1.97–1.73 (m, 9H), 1.60–1.48 (m, 3H), 1.31–1.26 (m, 6H),
1.20 (d, 3H, J = 7.0 Hz). MS (FAB+): m/z = 547 (M+H)+.
6.1.48. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-4-hydroxy-2-isopropyl-N-
phenylhexanamide hemifumarate (35)
6.1.43. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-2-ethyl-4-hydroxy-N-(cis-5-
hydroxyadamantan-2-yl)hexanamide fumarate (58)
A solution of Me2AlCl in n-hexane (1.0 M, 2.70 mL, 2.70 mmol)
In a similar manner as the preparation of 30, the title compound
was obtained as a colorless solid. 1H NMR (500 MHz, CD3OD): a
mixture of rotamers d 7.81 (br d, 1H, J = 6.3 Hz), 7.54 (br d, 1H,
J = 7.8 Hz), 7.43–7.32 (m, 3H), 6.69 (s, 2H), 3.86 (br s, 1H), 3.65–
3.52 (m, 3H), 3.37–3.27 (m, 2H), 3.23–3.13 (m, 1H), 2.97–2.92
was added to a solution of aniline (243 lL, 2.68 mmol) in CH2Cl2
(3 mL) under N2 atmosphere at room temperature, and the mixture
was stirred at room temperature for 1 h. A solution of 17a (271 mg,
0.533 mmol) in CH2Cl2 (5 mL) was added to the solution obtained
above, and the mixture was stirred at room temperature for

Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
3191
3.5 h. A 10% potassium sodium tartrate aqueous solution was
added to the reaction mixture, followed by dilution with AcOEt.
Then, the mixture was stirred at room temperature for 30 min.
The reaction mixture was extracted with AcOEt, and then the or-
ganic layer was washed with brine and dried over anhydrous
MgSO4. After filtration, the solvent was evaporated under reduced
pressure, and the residue was purified by silica gel column chro-
matography (eluent, CH2Cl2/MeOH = 40:1) to obtain tert-butyl
[(2S,3S,5S)-1-[4-(2-chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-
yl]-3-hydroxy-6-methyl-5-(phenylcarbamoyl)heptan-2-yl]carbamate
1H), 6.70 (s, 2H), 3.67–3.48 (m, 3H), 3.36–3.15 (m, 3H), 2.96–
2.91 (m, 0.6H), 2.81–2.77 (m, 0.4H), 2.68–2.62 (m, 1.4H), 2.50
(dd, 0.6H, J = 13.2, 3.9 Hz), 2.18 (s, 3H), 2.00–1.91 (m, 2H), 1.81
(ddd, 1H, J = 13.7, 10.7, 2.9 Hz), 1.26–1.24 (m, 6H), 1.05 (d, 6H,
J = 6.6 Hz). MS (FAB+): m/z = 500 (M+H)+.
6.1.52. ((2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-4-hydroxy-2-isopropyl-N-
(pyridin-4-yl)hexanamide fumarate (39)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 8.39 (dd, 2H, J = 4.6, 1.5 Hz),
7.70 (dd, 2H, J = 5.0, 1.5 Hz), 7.56–7.52 (m, 1H), 7.44–7.36 (m,
2H), 7.34–7.28 (m, 1H), 6.68 (s, 2H), 3.67–3.51 (m, 3H), 3.37–
3.18 (m, 3H), 2.97–2.91 (m, 0.6H), 2.81–2.64 (m, 1.8H), 2.54–2.50
(m, 0.6H), 2.02–1.92 (m, 2H), 1.83–1.76 (m, 1H), 1.30–1.25 (m,
6H), 1.04 (d, 3H, J = 2.3 Hz), 1.03 (d, 3H, J = 2.3 Hz). MS (FAB+): m/
z = 502 (M+H)+.
as a colorless solid. Trifluoroacetic acid (488
lL, 6.21 mmol) was
added to solution of tert-butyl [(2S,3S,5S)-1-[4-(2-chloro-
a
phenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]-3-hydroxy-6-methyl-
5-(phenylcarbamoyl)heptan-2-yl]carbamate (125 mg, 0.207 mmol)
in CH2Cl2 (1 mL) at room temperature, and the mixture was stirred
at the same temperature for 1 h. After concentration under re-
duced pressure, saturated sodium bicarbonate aqueous solution
was added to the reaction mixture, followed by extraction with
CH2Cl2. Then, the organic layer was dried over anhydrous Na2SO4.
After filtration, the solvent was evaporated under reduced pres-
sure, and the residue was purified by silica gel column chromatog-
raphy (eluent, CH2Cl2/MeOH = 20:1 - 8:1) to obtain the free base of
35. Fumaric acid (12.0 mg, 0.103 mmol) was added to a solution of
the free base of 35 in MeOH (5 mL), and the mixture was stirred at
room temperature for 5 min. The solvent was evaporated under re-
duced pressure to obtain 35 (99.0 mg, 84%, two-steps) as a color-
less solid. 1H NMR (500 MHz, CDCl3): a mixture of rotamers d
7.80 (d, 2H, J = 8.3 Hz), 7.55–7.53 (m, 1H), 7.43–7.37 (m, 2H),
7.33–7.28 (m, 3H), 7.11 (t, 1H, J = 7.3 Hz), 6.69 (s, 1H), 3.66–3.56
(m, 3H), 3.36–3.17 (m, 3H), 2.97–2.92 (m, 0.6H), 2.79–2.70 (m,
0.8H), 2.60–2.50 (m, 1.6H), 1.98–1.89 (m, 2H), 1.83–1.78 (m, 1H),
1.30–1.25 (m, 6H), 1.07–1.05 (m, 6H). MS (FAB+): m/z = 501
(M+H)+.
6.1.53. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-N-(4-fluorophenyl)-4-hydroxy-2-
isopropylhexanamide hemifumarate (40)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 7.61–7.58 (m, 2H), 7.55–7.53
(m, 1H), 7.42–7.37 (m, 2H), 7.34–7.27 (m, 1H), 7.07–7.03 (m,
2H), 6.67 (s, 1H), 3.65–3.52 (m, 3H), 3.34–3.15 (m, 3H), 2.96–
2.91 (m, 0.6H), 2.78–2.68 (m, 0.8H), 2.59–2.49 (m, 1.6H), 1.97–
1.88 (m, 2H), 1.82–1.78 (m, 1H), 1.30–1.24 (m, 6H), 1.05–1.03
(m, 6H). MS (FAB+): m/z = 519 (M+H)+.
6.1.54. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-2-ethyl-N-(4-fluorophenyl)-4-
hydroxyhexanamide fumarate (41)
6.1.49. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-4-hydroxy-2-isopropyl-N-(pyridin-2-
yl)hexanamide fumarate (36)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 7.62–7.53 (m, 2H), 7.55–7.53
(m, 1H), 7.43–7.37 (m, 2H), 7.33–7.28 (m, 1H), 7.08–7.03 (m,
2H), 6.69 (s, 2H), 3.66–3.56 (m, 3H), 3.34–3.16 (m, 3H), 2.97–
2.92 (m, 0.6H), 2.79–2.69 (m, 1.8H), 2.51 (dd, 0.6H, J = 13.7,
3.9 Hz), 1.97–1.92 (m, 1H), 1.79–1.58 (m, 3H), 1.30–1.24 (m, 6H),
1.03 (t, 3H, J = 7.3 Hz). MS (FAB+): m/z = 505 (M+H)+.
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 8.32–8.30 (m, 1H), 8.12–8.10
(br d, 1H, J = 8.6 Hz), 7.81–7.76 (m, 1H), 7.55–7.52 (m, 1H), 7.44–
7.27 (m, 3H), 7.14–7.11 (m, 1H), 6.69 (s, 2H), 3.66–3.54 (m, 3H),
3.35–3.18 (m, 3H), 2.97–2.91 (m, 0.6H), 2.76–2.60 (m, 1.8H),
2.55–2.50 (m, 0.6H), 2.00–1.90 (m, 2H), 1.84–1.78 (m, 1H), 1.29–
1.24 (m, 6H), 1.05 (d, 6H, J = 7.0 Hz). MS (FAB+): m/z = 502 (M+H)+.
6.1.55. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-2-ethyl-4-hydroxy-N-(4-
methylphenyl)hexanamide fumarate (42)
6.1.50. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-4-hydroxy-2-isopropyl-N-(pyridin-3-
yl)hexanamide fumarate (37)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 7.56–7.52 (m, 1H), 7.46 (d, 2H,
J = 8.2 Hz), 7.41–7.38 (m, 2H), 7.33–7.27 (m, 1H), 7.13 (d, 2H,
J = 7.8 Hz), 6.69 (s, 2H), 3.66–3.55 (m, 3H), 3.35–3.12 (m, 3H),
2.94 (dd, 0.6H, J = 13.3, 11.3 Hz), 2.75–2.69 (m, 1.8H), 2.51 (dd,
0.6H, J = 13.5, 4.1 Hz), 2.29 (s, 3H), 1.94 (ddd, 1H, J = 13.9, 11.2,
2.7 Hz), 1.78–1.58 (m, 3H), 1.29–1.24 (m, 6H), 1.01 (t, 3H,
J = 7.4 Hz). MS (FAB+): m/z = 501 (M+H)+.
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 8.81 (s, 1H), 8.28–8.27 (m, 1H),
8.15–8.13 (m, 1H), 7.55–7.53 (m, 1H), 7.43–7.29 (m, 4H), 6.68 (s,
2H), 3.66–3.53 (m, 3H), 3.36–3.18 (m, 3H), 2.98–2.91 (m, 0.6H),
2.81–2.62 (m, 1.8H), 2.55–2.51 (m, 0.6H), 2.02–1.91 (m, 2H),
1.84–1.77 (m, 1H), 1.30–1.25 (m, 6H), 1.05 (d, 6H, J = 6.6 Hz). MS
(FAB+): m/z = 502 (M+H)+.
6.1.56. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-2-ethyl-4-hydroxy-N-(4-
methoxyphenyl)hexanamide fumarate (43)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 7.55–7.27 (m, 6H), 6.90–6.86
(m, 2H), 6.69 (s, 2H), 3.76 (m, 3H), 3.65–3.55 (m, 3H), 3.35–3.15
(m, 3H), 2.98–2.92 (m, 0.6H), 2.75–2.68 (m, 1.8H), 2.51 (dd, 0.6H,
J = 13.5, 4.1 Hz), 1.93 (ddd, 1H, J = 13.8, 11.2, 2.3 Hz), 1.79–1.55
6.1.51. (2S,4S,5S)-5-Amino-6-[4-(5-fluoro-2-methylphenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-4-hydroxy-2-isopropyl-N-
(pyridin-3-yl)hexanamide fumarate (38)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (500 MHz,
CD3OD): a mixture of rotamers d 8.81 (s, 1H), 8.27 (d, 1H,
J = 2.9 Hz), 8.14 (d, 1H, J = 7.3 Hz), 7.41 (dd, 1H, J = 8.3, 4.4 Hz),
7.31 (t, 1H, J = 7.3 Hz), 7.04 (td, 1H, J = 8.3, 2.4 Hz), 6.98–6.90 (m,

3192
Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
(m, 3H), 1.29–1.24 (m, 6H), 1.01 (t, 3H, J = 7.4 Hz). MS (ESI+): m/
z = 517 (M+H)+.
CD3OD): a mixture of rotamers d 8.11 (d, 1H, J = 5.9 Hz), 7.75 (br
s, 1H), 7.55–7.52 (m, 1H), 7.43–7.28 (m, 3H), 6.73 (dd, 1H, J = 5.9,
2.3 Hz), 6.68 (s, 2H), 3.87 (s, 3H), 3.65–3.55 (m, 3H), 3.36–3.18
(m, 3H), 2.94 (dd, 0.6H, J = 13.5, 11.1 Hz), 2.80–2.71 (m, 1.8H),
2.53 (dd, 0.6H, J = 13.5, 4.1 Hz), 1.96 (ddd, 1H, J = 13.9, 11.1,
2.7 Hz), 1.81–1.59 (m, 3H), 1.29–1.24 (m, 6H), 1.01 (t, 3H,
J = 7.4 Hz). MS (ESI+): m/z = 518 (M+H)+.
6.1.57. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-N-(3,4-difluorophenyl)-4-hydroxy-2-
isopropylhexanamide hemifumarate (44)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (500 MHz,
CD3OD): a mixture of rotamers d 7.78–7.74 (m, 1H), 7.55–7.52
(m, 1H), 7.42–7.17 (m, 5H), 6.67 (s, 1H), 3.64–3.50 (m, 3H), 3.34–
3.10 (m, 3H), 2.91 (t, 0.6H, J = 12.2 Hz), 2.73–2.68 (m, 0.8H),
2.58–2.48 (m, 1.6H), 1.95–1.88 (m, 2H), 1.82–1.76 (m, 1H), 1.29–
1.24 (m, 6H), 1.04–1.02 (m, 6H). MS (FAB+): m/z = 537 (M+H)+.
6.1.63. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-4-hydroxy-N-(6-methylpyridin-2-yl)-2-
isopropylhexanamide hemifumarate (50)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 7.91 (d, 1H, J = 8.2 Hz), 7.66 (t,
1H, J = 7.8 Hz), 7.55–7.52 (m, 1H), 7.43–7.27 (m, 3H), 7.00 (d, 1H,
J = 7.6 Hz), 6.70 (s, 1H), 3.65–3.55 (m, 3H), 3.35–3.18 (m, 3H),
2.96–2.90 (m, 0.6H), 2.75–2.73 (m, 0.6H), 2.63–2.58 (m, 1.2H),
2.52 (dd, 0.6H, J = 13.4, 4.1 Hz), 2.45 (s, 3H), 2.01–1.78 (m, 3H),
1.28–1.24 (m, 6H), 1.05–1.03 (m, 6H). MS (FAB+): m/z = 516
(M+H)+.
6.1.58. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-N-(2,4-difluorophenyl)-4-hydroxy-2-
isopropylhexanamide hemifumarate (45)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 7.76–7.72 (m, 1H), 7.55–7.52
(m, 1H), 7.44–7.28 (m, 3H), 7.07–7.02 (m, 1H), 6.98–6.93 (m,
1H), 6.67 (s, 1H), 3.66–3.57 (m, 3H), 3.37–3.14 (m, 3H), 2.95 (dd,
0.6H, J = 13.2, 11.0 Hz), 2.79–2.62 (m, 1.8H), 2.55–2.50 (m, 0.6H),
1.98–1.88 (m, 2H), 1.84–1.77 (m, 1H), 1.30–1.25 (m, 6H), 1.06 (d,
6H, J = 6.9 Hz). MS (FAB+): m/z = 537 (M+H)+.
6.1.64. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-2-ethyl-4-hydroxy-N-(3-methylpyridin-2-
yl)hexanamide fumarate (51)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 8.14 (d, 1H, J = 2.3 Hz), 7.98 (d,
1H, J = 8.6 Hz), 7.62 (dd, 1H, J = 8.4, 2.2 Hz), 7.55–7.52 (m, 1H),
7.44–7.37 (m, 2H), 7.34–7.27 (m, 1H), 6.68 (s, 2H), 3.65–3.55 (m,
3H), 3.35–3.17 (m, 3H), 2.94 (dd, 0.6H, J = 13.3, 11.3 Hz), 2.79–
2.72 (m, 1.8H), 2.51 (dd, 0.6H, J = 13.3, 3.9 Hz), 2.30 (s, 3H), 1.95
(ddd, 1H, J = 13.8, 11.2, 2.7 Hz), 1.82–1.57 (m, 3H), 1.29–1.24 (m,
6H), 1.01 (t, 3H, J = 7.4 Hz). MS (ESI+): m/z = 502 (M+H)+.
6.1.59. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-N-(5-fluoropyridin-2-yl)-4-hydroxy-2-
isopropylhexanamide fumarate (46)
In the same manner as the preparation of 35, the title compound
was obtained as a colorless solid. 1H NMR (500 MHz, CD3OD): a mix-
ture of rotamers d 8.21 (d, 1H, J = 2.9 Hz), 8.17 (dd, 1H, J = 9.0, 4.2 Hz),
7.61–7.57 (m, 1H), 7.54–7.53 (m, 1H), 7.42–7.37 (m, 2H), 7.33–7.29
(m, 1H), 6.68 (s, 2H), 3.65–3.54 (m, 3H), 3.34–3.19 (m, 3H), 2.96–
2.91 (m, 0.6H), 2.76–2.61 (m, 1.8H), 2.52 (dd, 0.6H, J = 13.4,
4.2 Hz), 2.00–1.91 (m, 2H), 1.83–1.78 (m, 1H), 1.29–1.24 (m, 6H),
1.05–1.03 (m, 6H). MS (FAB+): m/z = 521 (M+H)+.
6.1.65. tert-Butyl (4S,5S)-4-{[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]methyl}-5-[(2S)-2-(methoxycarbonyl)-3-
methylbutyl]-2,2-dimethyl-1,3-oxazolidine-3-carboxylate (18a)
2 N aqueous NaOH (2.95 mL, 5.83 mmol) was added to a solution
of 17a (1.48 g, 2.91 mmol) in a mixed solvent of MeOH (10 mL) and
THF (10 mL) at room temperature and the mixture was stirred at the
same temperature for 75 min. The mixture was neutralized with 2 N
aqueous HCl (2.95 mL, 5.83 mmol), followed by extraction with
AcOEt, and then the organic layer was washed with brine and dried
over anhydrous Na2SO4. After filtration, the solvent was evaporated,
and the crude product was dissolved in a mixed solvent of toluene
(30 mL) and MeOH (10 mL). TMSCHN2 (2.0 M n-hexane solution,
2.20 mL, 4.40 mmol) was added to the mixture at 0 °C, and stirred
at the same temperature for 30 min. Then, the mixture was concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography (eluent, n-hexane/AcOEt = 7:3–1:4)
to obtain methyl 5-[(tert-butoxycarbonyl)amino]-6-[4-(2-chloro-
phenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]-2,3,5,6-tetradeoxy-
6.1.60. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-2-ethyl-N-(5-fluoropyridin-2-yl)-4-
hydroxyhexanamide fumarate (47)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 8.21–8.16 (m, 2H), 7.61–7.53
(m, 2H), 7.43–7.28 (m, 3H), 6.69 (s, 2H), 3.66–3.56 (m, 3H), 3.35–
3.22 (m, 3H), 2.95 (t, 0.6H, J = 11.9 Hz), 2.80–2.72 (m, 1.8H), 2.51
(dd, 0.6H, J = 13.3, 3.5 Hz), 1.97 (m, 1H), 1.80–1.59 (m, 3H), 1.29–
1.24 (m, 6H), 1.00 (t, 3H, J = 7.4 Hz). MS (FAB+): m/z = 507 (M+H)+.
6.1.61. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-N-(3,5-difluoropyridin-2-yl)-4-hydroxy-2-
isopropylhexanamide fumarate (48)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 8.25 (d, 1H, J = 2.7 Hz), 7.74–
7.69 (m, 1H), 7.56–7.53 (m, 1H), 7.44–7.29 (m, 3H), 6.68 (s, 2H),
3.69–3.60 (m, 3H), 3.36–3.19 (m, 3H), 2.98 (dd, 0.6H, J = 13.5,
11.2 Hz), 2.84–2.62 (m, 1.8H), 2.53 (dd, 0.6H, J = 13.9, 4.1 Hz),
2.01–1.89 (m, 2H), 1.85–1.78 (m, 1H), 1.31–1.26 (m, 6H), 1.10–
1.06 (m, 6H). MS (ESI+): m/z = 538 (M+H)+.
2-propan-2-yl-L-lyxo-hexonate (1.38 g, 88%, two-steps) as a
colorless solid. To a solution of methyl 5-[(tert-butoxycarbonyl)
amino]-6-[4-(2-chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]-
2,3,5,6-tetradeoxy-2-propan-2-yl-
L
-lyxo-hexonate
(995 mg,
1.84 mmol) in DMF (20 mL) were added 2,2-dimethoxypropane
(3.40 mL, 27.6 mmol) and PPTS (1.16 g, 4.60 mmol) at room temper-
ature. After stirring at 65 °C for 90 min, water was added, and the
water layer was extracted with AcOEt. The combined organics were
washed with brine, dried over anhydrous Na2SO4, and evaporated.
The residue was purified by silica gel column chromatography (elu-
ent, n-hexane/AcOEt = 4:1-45:55) to obtain 18a (727 mg, 68%) as a
colorless solid. 1H NMR (400 MHz, CDCl3): a mixture of rotamers
d 7.48–7.46 (m, 1H), 7.33–7.25 (m, 3H), 4.02–3.96 (m, 1H),
3.69–1.46 (m, 29H), 1.26–1.20 (m, 6H), 0.97–0.94 (m, 6H).
6.1.62. (2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-2-ethyl-4-hydroxy-N-(4-methoxypyridin-
2-yl)hexanamide fumarate (49)
In the same manner as the preparation of 35, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,

Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
3193
6.1.66. tert-Butyl (4S,5S)-4-{[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]methyl}-5-[(2R)-2-(methoxycarbonyl)
butyl]-2,2-dimethyl-1,3-oxazolidine-3-carboxylate (18b)
In the same manner as the preparation of 18a, the title com-
pound was obtained as a colorless solid. 1H NMR (500 MHz, CDCl3):
a mixture of rotamers d 7.47 (d, 1H, J = 7.8 Hz), 7.34–7.26 (m, 3H),
4.05–4.01 (m, 1H), 3.70–1.56 (m, 30H), 1.27–1.21 (m, 6H), 0.94–
0.91 (m, 3H).
free base of 59 in MeOH (2 mL), and the mixture was stirred at
room temperature for 5 min. The solvent was evaporated under re-
duced pressure to obtain 59 (93.0 mg, 60%, two-steps) as a color-
less solid. 1H NMR (400 MHz, CD3OD): a mixture of rotamers d
7.56–7.51 (m, 1H), 7.44–7.32 (m, 3H), 6.69 (s, 2H), 3.66–2.73 (m,
9.6H), 2.52 (dd, 0.4H, J = 13.5, 4.5 Hz), 2.00–1.91 (m, 2H), 2.01–
1.97 (m, 6H), 1.79–1.57 (m, 8H), 1.32–1.27 (m, 6H), 1.01–0.96
(m, 6H). MS (FAB+): m/z = 575 (M+H)+.
6.1.70. trans-4-({(2R,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-
dimethyl-5-oxopiperazin-1-yl]-2-ethyl-4-hydroxyhexanoyl}amino)
adamantane-1-carboxamide fumarate (60)
6.1.67. tert-Butyl (4S,5S)-4-{[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]methyl}-5-{(2S)-2-[(3-hydroxyadamantan-
1-yl)carbamoyl]-3-methylbutyl}-2,2-dimethyl-1,3-oxazolidine-
3-carboxylate (19a)
In the same manner as the preparation of 59, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz,
CD3OD): a mixture of rotamers d 7.89 (d, 1H, J = 7.0 Hz), 7.56–
7.54 (m, 1H), 7.44–7.31 (m, 3H), 6.69 (s, 2H), 4.00 (br s, 1H),
3.66–3.46 (m, 3H), 3.37–3.12 (m, 3H), 2.97–2.91 (m, 0.8H), 2.81–
2.61 (m, 1.4H), 2.53 (dd, 0.8H, J = 13.3, 4.3 Hz), 2.06–1.85 (m,
12H), 1.70–1.51 (m, 5H), 1.31–1.26 (m, 6H), 0.98–0.94 (m, 3H).
MS (FAB+): m/z = 589 (M+H)+.
2 N aqueous NaOH (590 lL, 1.18 mmol) was added to a solution
of 18a (172 mg, 0.296 mmol) in a mixed solvent of MeOH (1.5 mL)
and THF (1.5 mL) at room temperature and the mixture was stirred
at 65 °C for 11 h. After cooling to room temperature, the mixture was
neutralized with 2 N aqueous HCl (590 lL, 1.18 mmol), followed by
extraction with AcOEt, and 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, AcOEt/
MeOH = 99:1–9:1) to obtain (2S)-2-{[(4S,5S)-3-(tert-butoxycar-
bonyl)-4-{[4-(2-chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]-
methyl}-2,2-dimethyl-1,3-oxazolidin-5-yl]methyl}-3-methylbutanoic
acid (150 mg, 89%) as a colorless solid. To a solution of (2S)-2-
{[(4S,5S)-3-(tert-butoxycarbonyl)-4-{[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]methyl}-2,2-dimethyl-1,3-oxazolidin-5-yl]-
methyl}-3-methylbutanoic acid (150 mg, 0.256 mmol) and
1-amino-3-hydroxyadamantane (128 mg, 0.768 mmol) in DMF
6.2. Biological assays
6.2.1. IC50 in buffer
The activity of renin inhibitors against purified enzyme was
measured using the following protocol: All reactions were car-
ried out in a flat bottom black opaque microtiter plate. Test
compounds in DMSO (2
assay buffer (50 mM Tris–HCl (pH7.9), 100 mM NaCl) containing
L of trypsin-activated recombinant human renin (final en-
zyme concentration of 50 M), and the solution was pre-incu-
bated at room temperature for 10 min. Next, of the
substrate (Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-
Thr-Lys (DABCYL)-Arg) in 100 L of the assay buffer was added,
lL) were mixed with 100 lL of the
5
l
l
(3 mL) were added Et3N (214
lL, 1.54 mmol) and HATU (389 mg,
2 lM
1.02 mmol) at room temperature, and the mixture was stirred at
40 °C for 2 h. Water was added, and the water layer was extracted
with AcOEt. The combined organics were washed with brine,
dried over anhydrous Na2SO4, and evaporated. The residue was
purified by silica gel column chromatography (eluent, CH2Cl2/
l
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 (excita-
tion at 340 nm) using a multilabel reader (Perkin–Elmer Inc.).
The slope of the linear portion of the plot of fluorescence in-
crease as a function of time was then determined, and the rate
was used to calculate% inhibition in relation to uninhibited con-
trol. The % inhibition values were plotted as a function of inhib-
itor concentration, and the IC50 value was determined by probit
analysis. The IC50 value is defined as the concentration of a par-
ticular inhibitor that reduces the formation of product by 50%
relative to a control sample containing no inhibitor.
MeOH = 99:1–91:9) to obtain 19a (160 mg, 87%) as
a pale
yellow solid. 1H NMR (400 MHz, CDCl3): a mixture of rotamers d
8.02 (s, 1H), 7.47–7.24 (m, 4H), 5.44 (br s, 1H), 4.04–2.60
(m, 8H), 2.24–1.50 (m, 33H), 1.25–1.21 (m, 6H), 0.95–0.94
(m, 6H).
6.1.68. tert-Butyl (4S,5S)-5-{(2R)-2-[(trans-5-
carbamoyladamantan-2-yl)carbamoyl]butyl}-4-{[4-(2-
chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]methyl}-2,2-
dimethyl-1,3-oxazolidine-3-carboxylate (19b)
In a similar manner as the preparation of 19a, the title com-
pound was obtained as a colorless solid. 1H NMR (400 MHz, CDCl3):
a mixture of rotamers d 7.46 (d, 1H, J = 7.8 H), 7.36–7.26 (m, 3H),
6.11–6.01 (m, 1H), 5.70–5.61 (m, 1H), 5.28–5.17 (m, 1H), 4.11–
4.01 (m, 2H), 3.69–3.18 (m, 6H), 2.68–1.44 (m, 34H), 1.23–1.15
(m, 6H), 0.92 (t, 3H, J = 7.2 Hz).
6.2.2. IC50 in plasma
The activity of renin inhibitors in vitro in cynomolgus mon-
key plasma was measured by the decrease in plasma renin activ-
ity (PRA) levels observed in the presence of the compounds.
Compounds and aliskiren hemifumarate (1) were dissolved in
DMSO and the final concentration of DMSO was 1%. Incubation
6.1.69. (2S,4S,5S)-5-Amino-6-[4-(2-chlorophenyl)-2,2-dimethyl-
5-oxopiperazin-1-yl]-4-hydroxy-N-(3-hydroxyadamantan-1-
yl)-2-isopropylhexanamide fumarate (59)
mixtures were contained in the final volume of 20
compound solution, 200 L of pooled mixed-gender cynomolgus
monkey plasma stabilized with EDTA, 20 L of pH adjusting
solution, and 10 L of Inhibitor A solution. The reaction mixture
was incubated at 37 °C for 1 h. After incubation, angiotensin I in
the reaction mixture was measured by competitive radioimmu-
noassay using commercial available RIA kit RENIN RIABEAD (Ya-
masa Co.). An uninhibited tube containing 1% DMSO and a
control tube incubated at 4 °C were used to derive the % inhibi-
tion for each concentration of inhibitors. The % inhibition values
were plotted as a 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.
lL of test
l
l
Ten percent HCl in MeOH (3.0 mL) was added to 19a (160 mg,
0.224 mmol) at room temperature and the mixture was stirred at
the same temperature for 12 h. After concentration under reduced
pressure, saturated sodium bicarbonate aqueous solution was
added to the reaction mixture, followed by extraction with CH2Cl2.
Then, the organic layer was dried over anhydrous Na2SO4. After fil-
tration, the solvent was evaporated under reduced pressure, and
the residue was purified by NH silica gel column chromatography
(eluent, CH2Cl2/MeOH = 1:0–95:5) to obtain the free base of 59. Fu-
maric acid (19.8 mg, 0.171 mmol) was added to a solution of the
l

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Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
6.3. Animal studies
performed continuously during the experiment. An interpulse
interval of 20 s allowed the recovery of hCav1.2 at ꢀ80 mV. Test
compounds were incubated with the cells between 2 and 5 min
until the current reached a steady state level.
6.3.1. Ex vivo PRA study in cynomolgus monkeys pre-treated
with furosemide
A single oral dose administration of compound 56 was per-
formed to furosemide-pretreated cynomolgus monkeys to deter-
mine the ex vivo plasma renin activity. Animals were dosed
orally with compound 56 at doses of 1, 3, and 10 mg/kg, or vehicle
(1% methylcellulose). Venous blood was collected into a Venoject
tube (EDTA-2Na) and cooled immediately on ice. Subsequently,
all the blood was centrifuged with a cooled centrifuge to obtain
plasma. The plasma samples were stored in a deep freezer until
the measurements. Plasma renin activity (PRA) was measured by
a radioimmunoassay kit (Renin-Riabead, TFB, Japan). The plasma
concentrations of the free form of 56 after administration of 56
at 1, 3, and 10 mg/kg were increased in a dose-dependent manner.
6.4.3. hNav1.5 currents assay
Sodium currents in HEK293 cells stably expressing the hNav1.5
sodium channel were measured using the patch clamp technique
on PatchXpress 7000A system. hNav1.5 channels were activated
by 20 ms pulses to ꢀ10 mV from a holding potential of ꢀ120 mV,
and peak sodium currents were measured. This voltage-clamp
pulse protocol was performed continuously during the experiment
at a frequency of 1 Hz. Test compounds were incubated with the
cells for 2 min.
6.4.4. Sodium channel (Site 2) binding assay
Wister rat brain membranes in modified Tris–HCl buffer pH 7.4
6.3.2. Blood pressure study in cynomolgus monkeys pre-treated
with furosemide
were incubated with 5 nM [3H]batrachotoxin for 1 h at 37 °C. Non-
specific binding was estimated in the presence of 100
lM veratri-
Arterial pressure was measured by a telemetry system in con-
scious, freely moving cynomolgus monkeys (n = 6). Pressure trans-
mitters (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 fem-
oral 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 h after dosing or later. The animals were allowed free
access to water whole time. Furosemide at 5 mg/kg/day was intra-
muscularly administered for 3 days before drug administration.
Cynomolgus monkeys received 56 at doses of 1, 3 and 10 mg/kg,
or vehicle (1% methylcellulose), orally in a crossover design. Arte-
rial 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 mean
arterial blood pressure (MAP) was calculated.
dine. Binding affinity of test compounds (10 M) was expressed as
a percent inhibition referred to the vehicle control.
l
6.4.5. Guinea pig right ventricular papillary muscle action
potential assay
Action potentials were measured using the glass microelectrode
method. Male Hartley guinea pigs were sacrificed by cervical dislo-
cation and exsanguination. The heart was immediately excised. A
strand of free-running papillary muscle from the right ventricle
was dissected and immediately mounted into a perfusion chamber.
The perfusing Tyrode solution was oxygenated with a gas mixture
of 5% CO2 and 95% O2, and kept at 36.5 °C with a temperature-con-
trolled circulator. Transmembrane action potentials were recorded
using a 3 M KCl glass microelectrode with a tip resistance of 5–30
megohms, which was coupled to an Ag-AgCl bath electrode and
connected to a microelectrode amplifier (MEZ-8300 or MEZ-
8301, Nihon Kohden, Japan). The tissue preparation was electri-
cally driven at 1 Hz through bipolar electrodes using a stimulator
(SEN-3201 or SEN-3301, Nihon Kohden). The stimulation pulse
was square in shape, 1 ms duration, with an intensity of about
1.3 times the diastolic threshold. The action potentials were dis-
played on an oscilloscope (CS-4025 or DSC-7040, Kenwood, Japan),
and recorded and analyzed using a computer system (WinCA-
PA1.6.6.Aui, Physio-Tech, Japan). Resting membrane potential, ac-
tion potential amplitude, maximal upstroke velocity, and action
potential duration at 30%, 60% and 90% repolarization levels were
quantified. After confirming that these parameters were stabilized,
test compounds were incubated with the tissue preparation for
30 min.
6.3.3. Pharmacokinetics
The pharmacokinetics of compounds was determined in cyno-
molgus monkeys. Compounds were administered intravenously
as a saline solution or by oral gavage in 0.5% methylcellulose.
Following dosing, blood samples were taken over 24 h with a min-
imum of 7 time points, and plasma concentrations were measured
by the HPLC-MS/MS method. PK parameters were calculated from
non-compartmental analysis of the plasma concentration-time
curves.
6.4. Cardiac studies
6.4.6. Isolated Langendorff-perfused rabbit heart model
6.4.1. hERG currents assay
Female NZW rabbits were anesthetized with thiopental (30 mg/
kg, iv). The heart was immediately excised and transferred to
chilled Krebs Henseleit solution. The isolated hearts were attached
to a Langendorff apparatus via an aortic cannula, perfused with
Krebs Henseleit solution at 37 °C, gassed with 95% O2 and 5%
CO2, and pressurised to approximately 70 mmHg. Electrocardio-
gram (ECG) and monophasic action potential (MAP) electrodes
were placed on the surface of the heart. The ECG and MAP were
continuously monitored using a polygraph system (RMP-6008M,
Nihon Kohden), and recorded on a thermal array recorder (RTA-
1300M, Nihon Kohden). Heart rate (HR), PR interval, QRS width,
QT interval, QTc (corrected with Bazett’s formula), and monophasic
action potential duration at 90% repolarization levels were quanti-
fied. After confirming that these parameters were stabilized, test
Potassium currents in CHO-K1 cells stably expressing the hERG
potassium channel were measured using the patch clamp tech-
nique on an IonWorks Quattro system (Molecular Devices, USA).
hERG channels were activated from
a holding potential of
ꢀ80 mV by a step to +50 mV for 2 s, followed by a step to
ꢀ40 mV for 2 s, and then tail peak currents were measured. Test
compounds were incubated with the cells for 5 min.
6.4.2. hCav1.2 currents assay
Calcium currents in HEK293 cells stably expressing the hCav1.2
calcium channel were measured using the patch clamp technique
on a PatchXpress 7000A system (Molecular Devices). hCav1.2 chan-
nels were activated by 125 m pulses to +25 mV from a holding po-
tential of ꢀ80 mV. Peak calcium currents were recorded upon
repolarization to ꢀ40 mV. This voltage-clamp pulse protocol was
compounds (10, 30, and 100
for 20 min.
lM) were perfused through the heart

Y. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 3175–3196
3195
6.4.7. Electrophysiological rat model (anesthetized condition)
Male Sprague-Dawley rats were anesthetized with inactin
(100 mg/kg, ip). The surface lead II ECG was continuously recorded
on a recticorder (RJG-4124, Nihon Kohden). A 30-gauge needle
connected with PE-10 polyethylene tube was inserted into the
right jugular vein to infuse the test compounds. After the stabiliza-
tion, test compounds (3 and 10 mg/kg) were injected for the
assessment of QRS complex abnormality.
The animals were allowed free access to water whole time. HR,
PR interval, QRS width, QT interval, QTc and systemic blood pres-
sure were monitored by the telemetry method, and analyzed
before administration and at 1, 2, 4, 7, and 24 h after administra-
tion. Data were analyzed using data acquisition and a real-time
analysis system (Dataquest™ OpenART™, Data Sciences Interna-
tional). Test compounds (30, 100, and 1000 mg/kg) were orally
administered with a dose escalation design at 6 day intervals.
6.4.8. Electrophysiological rabbit model (anesthetized
condition)
Acknowledgments
Female NZW rabbits were anesthetized with ketamine (35 mg/
kg, ip) and xylazine (5 mg/kg, ip). A tracheotomy was performed to
control the respiration using a volume-limited ventilator (SN-480-
6, Shinano, Japan) with room air. Tidal volume and respiratory rate
were set at 5–10 mL/kg and 35 strokes/min, respectively. The sur-
face lead AB ECG was obtained from the electrodes. Catheters were
inserted into the right femoral artery and vein to monitor the sys-
temic blood pressure and to infuse the test compounds, respec-
tively. The ECG and systemic blood pressure were continuously
monitored using a polygraph system (RMP-6008M, Nihon Kohden),
and recorded on a thermal array recorder (RTA-1300M, Nihon
Kohden). HR, PR interval, QRS width, QT interval, QTc, and systemic
blood pressure were quantified. After confirming that these
parameters were stabilized, test compounds (1 mg/kg/min) were
continuously infused for 1 h.
We would like to thank Masaki Meguro, Tsuyoshi Nakamura,
Yukiko Sekiguchi, Yumiko Mizuno, Keiji Saito, Masao Yoshida, Mas-
umi Ueno, Mina Nishi, Kotaro Sugimoto, Kenichi Manabe, Makoto
Mizuno, Noriko Masubuchi, and Naoyuki Maeda for their technical
assistance and helpful discussions.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.022.
References and notes
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6.4.9. Electrophysiological dog model (anesthetized condition)
Female beagle dogs were initially anesthetized with thiopental
(30 mg/kg, iv). After incubation with a cuffed endotracheal tube, 1%
halothane vaporized with 100% O2 was inhaled with a volume-lim-
ited ventilator (SN-480-3, Shinano). The tidal volume and respira-
tory rate were set at 20 mL/kg and 15 strokes/min, respectively.
The surface lead II ECG was obtained from the limb electrodes.
The systemic blood pressure was measured at the right femoral ar-
tery. A pig tail catheter was positioned at the left ventricle through
the right femoral artery to measure the left ventricular pressure.
The ECG, systemic blood pressure, and left ventricular pressure
were continuously monitored using a polygraph system (RMP-
6008M, Nihon Kohden), and recorded on a thermal array recorder
(RTA-1300M, Nihon Kohden). A 7.5 French Swan-Ganz catheter
(Baxter Healthcare, USA) connected with a CEDV monitor (Model:
VGSVSYS, Baxter Healthcare) was inserted into the femoral vein,
and positioned in the pulmonary artery for measuring cardiac out-
put and mixed venous blood oxygen saturation. HR, PR interval,
QRS width, QT interval, QTc, systemic blood pressure, left ventric-
ular pressure, and cardiac output were quantified. After confirming
that these parameters were stabilized, test compounds (0.5 mg/kg/
min) were continuously infused for 1 h. A volume of 2 mL of blood
was drawn from the left femoral artery to measure the plasma
drug concentration.
ˇ
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