An efficient synthesis of (3S,5S)-5-[3,3-dimethyl-1-(o-tolyl)-6-OXO-2H- pyridin-4-YL]- piperidine-3-carboxamide as potent renin inhibitor

Article abstract of DOI:10.3987/COM-14-12988

We report synthesis and biological evaluation of (3S,5S)-5-[3,3- dimethyl-1-(o-tolyl)-6-oxo-2H-pyridin-4-yl]piperidine-3-carboxamide as renin inhibitor. This effective synthetic route involves a zinc mediated Barbier reaction and an intramolecular Horner-Wadsworth-Emmons reaction of sterically hindered ketone as key reactions. The prepared compound 4 exhibited both potent renin inhibitory activity and significant in vivo efficacy in furosemide pretreated cynomolgus monkeys.

Full text of DOI:10.3987/COM-14-12988

HETEROCYCLES, Vol. 89, No. 6, 2014
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HETEROCYCLES, Vol. 89, No. 6, 2014, pp. 1413 - 1426. © 2014 The Japan Institute of Heterocyclic Chemistry
Received, 23rd March, 2014, Accepted, 22nd April, 2014, Published online, 2nd May, 2014
DOI: 10.3987/COM-14-12988
AN EFFICIENT SYNTHESIS OF
(3S,5S)-5-[3,3-DIMETHYL-1-(o-TOLYL)-6-OXO-2H-PYRIDIN-4-YL]-
PIPERIDINE-3-CARBOXAMIDE AS POTENT RENIN INHIBITOR
Yutaka Mori,a,* Mitsuhiro Iwamoto,a Kazuki Mori,a Masao Yoshida,b
Takeshi Honda,a Takahiro Nagayama,c and Takahide Nishid
aNew Modality Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi,
Shinagawa-ku, Tokyo 140-8710, Japan. bMedicinal Chemistry Research
Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo
c
140-8710, Japan. Cardiovascular-Metabolics Research Laboratories, Daiichi
Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.
dDaiichi Sankyo Life Science Research Center in India, Daiichi Sankyo India
Pharma Pvt. Ltd, Village Sehroul, Sector 18, Udyog Vihar Industrial Area,
Gurgaon-122015, Haryana, India. E-mail: mori.yutaka.c7@daiichisankyo.co.jp
Abstract – We report synthesis and biological evaluation of (3S,5S)-5-[3,3-
dimethyl-1-(o-tolyl)-6-oxo-2H-pyridin-4-yl]piperidine-3-carboxamide as renin
inhibitor. This effective synthetic route involves a zinc mediated Barbier reaction
and an intramolecular Horner-Wadsworth-Emmons reaction of sterically hindered
ketone as key reactions. The prepared compound 4 exhibited both potent renin
inhibitory activity and significant in vivo efficacy in furosemide pretreated
cynomolgus monkeys.
INTRODUCTION
Hypertension is a major risk factor for cardiovascular disease, including chronic heart and kidney failures,
myocardial infarction and stroke and is one of the leading causes of death in the developed world.1 The
renin-angiotensin-aldosterone system (RAAS) plays an important role in the regulation of blood pressure
(BP) and fluid homeostasis.2 The inhibition of either the formation or the action of angiotensin II (Ang II),
the main product of the RAAS, represents a major therapeutic approach in the treatment of hypertension
and the prevention of associated comorbidities.3a,b It has long been hypothesized that inhibition of renin,
which is the rate-limiting enzyme in the RAAS cascade, may represent the most attractive therapeutic

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HETEROCYCLES, Vol. 89, No. 6, 2014
strategy to block the RAAS.4 However, only Aliskiren hemifumarate (1) has reached the market for the
treatment of essential hypertension (Figure 1).5,6
In the preceding papers,7,8 starting from our clinical candidate DS-8108b (2),9,10 we discovered a
3,5-disubstituted piperidine derivative with 2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl group at the
5-position as a new type of renin inhibitor (Figure 1). The potent compound 3 obtained by the chemical
modification of the P1’, P2’ and P3 portion showed a significant BP lowering effect by oral administration
in two hypertensive animal models, double transgenic rats11 and furosemide pretreated cynomolgus
monkeys. The renin inhibitory activity of 3 was almost the same level as that of DS-8108b (2), however,
the potential for chemical modification of 5,5-dimethylpiperazin-2-one ring of the P1 position remains
due to the limitation on the synthesis and due to the requirement to keep intact our original partial
structure. Regarding the P1 portion, we previously reported the existence of hydrophobic interaction of
gem-dimethyl group on 5,5-dimethylpiperazin-2-one ring with S1 pocket of human renin as well as
hydrogen bond interaction between the carboxamide oxygen on the ring and Tyr77 by X-ray
crystallography.9,12 However, the interaction between basic nitrogen on 5,5-dimethylpiperazin-2-one ring
and the target enzyme was not observed. With consideration of these findings, we designed 4 having
3,3-dimethyl-1,2-dihydropyridine-6-one part as the P1 portion (Scheme 1). Herein, we report an efficient
synthesis of (3S,5S)-5-[3,3-dimethyl-1-(o-tolyl)-6-oxo-2H-pyridin-4-yl]piperidine-3-carboxamide that
demonstrates both potent renin inhibitory activity and significant in vivo efficacy in furosemide pretreated
cynomolgus monkeys.
P1'
P1'
P1'
P2'
fumaric acid
5
3
P2'
P1
fumaric acid
P2'
P1
0.5 fumaric acid
2 H O
・
P3
P1
2
P3
P3
Aliskiren hemifumarate ( )
DS-8108b ( )
1
2
3
IC50 in purified human renin = 0.9 nM
IC50 in monkey plasma renin = 6.3 nM
IC50 in purified human renin = 1.6 nM
IC50 in monkey plasma renin = 7.0 nM
Figure 1. Chemical structures of Aliskiren hemifumarate (1) and originally designed renin inhibitors,
DS-8108b (2) and 3 containing 2,2-dimethyl-4-phenylpiperazin-5-one part
RESULTS AND DISCUSSION
The retrosynthetic analysis of 4 is shown in Scheme 1. Compound 4 would be produced from 5, which
would be synthesized from ketone 6 by using the inter- or intramolecular Horner-Wadsworth-Emmons
(HWE) reaction as the key step. Ketone 6 would be obtained through construction of quaternary carbon
atom by γ-regioselective prenylation of aldehyde derivative prepared from chiral carboxylic acid 7, which

HETEROCYCLES, Vol. 89, No. 6, 2014
1415
is available on a large scale as described in previous paper.8
P1'
fumaric acid
HWE reaction
P1
P2'
P3
4
5
regioselective
γ-
prenylation
6
7
Scheme 1. Retrosynthetic analysis of 4
The synthetic pathway leading to the intermediate 6 is outlined in Scheme 2. Initially, known chiral
carboxylic acid 78 was reduced with borane-THF complex to give alcohol 8 in 91% yield. The following
Swern oxidation of alcohol 8 yielded aldehyde 9. Next, γ-regioselective prenylation of the aldehyde 9 by
prenyl bromide was investigated. At first, a CrCl2 mediated Nozaki-Hiyama reaction was attempted,
however, the yield was moderate and the reproducibility was poor (45~68%, 2 steps). Then, a Barbier
reaction using other metal was conducted alternatively. As a result, γ-regioselective prenylation
proceeded smoothly by using zinc dust in the mixed solvent of NH4Cl aq. and THF.13 Finally, Swern
oxidation of 10 led to the intermediate 6 (87%, 3 steps).
(a)
(b)
(c)
(d)
7
8
9
10
6
Scheme 2. Synthetic pathway leading to 6. Reagents and conditions: (a) BH3-THF, THF, −78 °C to rt, 1 h,
91%; (b) (COCl)2, DMSO, CH2Cl2, then Et3N, −78 °C to 0 °C, 2 h; (c) prenyl bromide, zinc dust, NH4Cl
aq., THF, rt, 1.5 h; (d) (COCl)2, DMSO, CH2Cl2, then Et3N, −78 °C to 0 °C, 2 h, 87% (3 steps)
As a next step, an intermolecular HWE reaction of ketone 6 with 11 was conducted (Scheme 3). However,
desired compound 12 was not obtained and most of the ketone 6 was recovered (89% recovery). This
suggested that steric bulkiness around the carbonyl group of 6 was too high to accomplish the
intermolecular HWE reaction. Thus, we focused on an alternative route involving the intramolecular
HWE reaction which was reported to be effective for the substrates containing sterically hindered

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HETEROCYCLES, Vol. 89, No. 6, 2014
carbonyl groups.14
11
NaH
THF
6
12
Scheme 3. Intermolecular HWE reaction of 6 with 11
The synthetic pathway leading to the target compound 4 is outlined in Scheme 4. Ozonolysis of the
terminal vinyl group of 6 delivered aldehyde 13 in 81% yield. The following reductive amination between
the aldehyde 13 and o-toluidine yielded aniline 14 (91%). The aniline 14 was acylated with freshly
prepared CH2Cl2 solution of phosphorylacetyl chloride 15,15 providing the phosphono acetanilide 16 in
88% yield. Next, the key intramolecular HWE reaction of 16 was investigated. In our initial experiment,
using NaH as a base produced 5 in 36% yield. After our efforts to improve the yield, compound 5 was
obtained in 72% by using the KHMDS in the presence of 18-crown-6. Hydrolysis of ester 5 with
LiOH·H2O led carboxylic acid 17 in 92% yield. Amidation of 17 with chiral amine 188 quantitatively
proceeded to give 19. Finaly, removal of the N-tert-butoxycarbonyl (Boc) group of 19 was followed by
addition of fumaric acid to give the target compound 4 as fumarate salt (81%, 2 steps).
Boc
N
O
O
P
Boc
N
OEt
OEt
Boc
N
Cl
O
OMe
15
O
H
OMe
(a)
(b)
(c)
O
OMe
H
N
O
O
O
O
6
13
14
Boc
Boc
N
Boc
N
EtO OEt
N
O
P
OMe
O
OMe
O
O H
O
(d)
(e)
O
N
O
N
O
N
O
16
5
17
H2N
Boc
N
H
N
fumaric acid
2 HCl
N
H
N
H
N
O
N
O
18
O
F
N
O
N
N
(f)
(g), (h)
F
F
19
4
Scheme 4. Synthetic pathway leading to 4. Reagents and conditions: (a) O3, CH2Cl2, −78 °C, 3 h, then
PPh3, 4 °C, 18 h, 81%; (b) o-toluidine, AcOH, toluene, 80 °C, 1 h, then NaBH(OAc)3, rt, 1.5 h, 91%; (c)
15 in CH2Cl2, DMA, rt, 1 h, 88%; (d) 0.5 M KHMDS in toluene, 18-crown-6, THF, −78 °C to −20 °C, 3
h, 72%; (e) LiOH·H2O, THF, water, 0 °C, 1 h, 92%; (f) amine 18, HBTU, N,N-diisopropylethylamine,
DMF, 0 °C, 1 h, quant.; (g) TFA, CH2Cl2, rt, 40 min; (h) fumaric acid, MeOH, rt, 5 min, 81% (2 steps)

HETEROCYCLES, Vol. 89, No. 6, 2014
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With 4 in hand, we carried out biological evaluation of 4. Initially, renin inhibitory activity was measured
(Table 1). Compound 4 showed high renin inhibitory activity. Especially, inhibitory activity against
purified human renin (0.7 nM) was higher than that of 1 (1.5 nM, in-house data)9 or 3 (1.6 nM). Next, to
evaluate the in vivo efficacy of 4, vehicle or 10 mg/kg of 4 were orally administered to cynomolgus
monkeys pretreated with furosemide (Figure 2). Compound 4 showed a significant mean arterial blood
pressure (MAP) reduction which was comparable to that of 3. (AUC0-24h of BP lowering effect of 4 and 3
was −242 mmHg·h and −267 mmHg·h8 respectively.)16
Table 1. In vitro renin inhibitory activities (IC50 and ratio) of 3 and 4a,b
Compound Purified human renin
IC50 (nM)
Monkey plasma renin
IC50 (nM) Ratioc
3
4
1.6
0.7
7.0
2.7
0.9
0.8
a Compounds were obtained as fumarate salts. b Assay results of renin inhibitory activity are the average
of at least two replicates. c Ratio = IC50 (nM) of compound / IC50 (nM) of 1.
20
Vehicle (1% MC, n = 6)
10 mg/kg of 3 (po, n = 6)
10
10 mg/kg of 4 (po, n = 6)
0
-10
-20
-30
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (h)
Figure 2. Effect of compound 3a and 4 on MAP in cynomolgus monkeys pretreated with furosemide
a The data of compound 3 was cited from ref. 8.
In summary, we have synthesized and evaluated (3S,5S)-5-[3,3-dimethyl-1-(o-tolyl)-6-oxo-2H-pyridin-4-
yl]piperidine-3-carboxamide as novel renin inhibitor. The synthesis was achieved by using a zinc
mediated Barbier reaction and an intramolecular HWE reaction of sterically hindered ketone as key steps.
The prepared compound 4 exhibited potent renin inhibitory activity. In addition, 4 showed significant BP
lowering effect by oral administration in furosemide pretreated cynomolgus monkeys. Thus, we selected

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HETEROCYCLES, Vol. 89, No. 6, 2014
this newly designed 3,5-disubstituted piperidine derivative as appropriate candidate for further
optimization and evaluation.
EXPERIMENTAL
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
1
13
Fuji Silysia Chemical Ltd. H NMR and C NMR spectra were obtained on a Varian Unity 400 or 500
spectrometer, or a Bruker Avance III 500 spectrometer. Spectra were taken in the indicated solvent at
ambient temperature, and chemical shifts are reported in parts per million (ppm (δ)) relative to the lock of
the solvent used. Resonance patterns are recorded with the following notations: br (broad), s (singlet), d
(doublet), t (triplet), q (quartet), and m (multiplet). Mass spectra were obtained on a JEOL JMS-LCmate
or an LC-MS system composed of Waters Xevo Q-Tof MS and Acquity UPLC systems. Optical rotations
were measured on an Autopol V Plus. Infrared spectra were recorded in a KBr disc or ATR mode with a
Jasco FT/IR-6100.
1-tert-Butyl 3-methyl (3S,5R)-5-hydroxymethylpiperidine-1,3-dicarboxylate (8)
BH3-THF (133 mL, 1.09 M THF solution, 145 mmol) was added dropwise to a solution of 78 (27.7 g,
96.4 mmol) in THF (500 mL) at −78 °C over a period of 25 min. After warming the reaction mixture to
room temperature (rt), the mixture was stirred at the same temperature for 1 h. After cooling to 0 °C,
saturated NaHCO3 aq. was added to the reaction mixture, followed by extraction with AcOEt. Then, the
organic layer was washed with brine, and dried over anhydrous Na2SO4. After filtration, the solvent was
evaporated under reduced pressure, and the residue was purified by silica gel column chromatography
(eluent, n-hexane/AcOEt = 3/1 to 1/3) to obtain 8 (24.1 g, 91%) as a colorless liquid. [α]D25.0 23.4 (c 1.06,
1
MeOH). H NMR (500 MHz, CDCl3): δ 4.38-4.26 (br m, 1H), 4.23-4.16 (br m, 1H), 3.69 (s, 3H),
3.57-3.50 (m, 2H), 2.77-2.72 (m, 1H), 2.53-2.46 (m, 1H), 2.42 (dd, J = 15.9, 8.5 Hz, 1H), 2.15 (d, J =
13.7 Hz, 1H), 1.76-1.70 (m, 1H), 1.53-1.48 (m, 1H), 1.45 (s, 9H), 1.37-1.30 (m, 1H). 13C NMR (125 MHz,
CDCl3): δ 173.3, 154.7, 80.1, 65.1, 51.8, 46.3, 45.8, 41.3, 38.1, 30.4, 28.4. IR: 3440, 2932, 2870, 1733,
1689, 1667, 1422, 1253, 1145, 881, 767 cm-1. HRMS (ESI+): m/z calcd for C13H23NO5+H: 274.1654;
found: 274.1655.
1-tert-Butyl 3-methyl (3S,5R)-5-(2,2-dimethylbut-3-enoyl)piperidine-1,3-dicarboxylate (6)

HETEROCYCLES, Vol. 89, No. 6, 2014
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A solution of DMSO (14.4 mL, 203 mmol) in CH2Cl2 (100 mL) was added dropwise to a solution of
oxalyl chloride (8.70 mL, 101 mmol) in CH2Cl2 (300 mL) at −78 °C over a period of 20 min, and then the
mixture was stirred at the same temperature for 20 min. To the reaction mixture, a solution of 8 (24.1 g,
88.2 mmol) in CH2Cl2 (150 mL) was added dropwise at −78 °C over a period of 25 min, and then the
mixture was stirred at the same temperature for 20 min. To the reaction mixture, Et3N (68.0 mL, 488
mmol) was added dropwise at −78 °C over a period of 15 min. After warming the reaction mixture to
0 °C, the mixture was stirred at the same temperature for 2 h. H2O was added to the reaction mixture,
followed by extraction with CH2Cl2. Then, the organic layer was washed with 1N HCl aq., saturated
NaHCO3 aq., and brine, and dried over anhydrous Na2SO4. After filtration, the filtrate was diluted with
AcOEt. The solution was filtered through the silica gel pad. The filtrate was evaporated under reduced
pressure to obtain crude 9 (23.3 g) as a colorless liquid. Saturated NH4Cl aq. (60.0 mL) was added to a
suspension of the crude 9 (23.3 g), prenyl bromide (14.4 mL, 125 mmol), and zinc dust (11.0 g, 168
mmol) in THF (500 mL) at 0 °C, and then the mixture was stirred at rt for 1.5 h. The reaction mixture was
filtered through the celite pad. The filtrate was evaporated under reduced pressure until the volume of the
solution was about 250 mL. The residue was extracted with AcOEt. Then, the organic layer was washed
with 1N HCl aq., saturated NaHCO3 aq., and brine, and dried over anhydrous Na2SO4. After filtration, the
solution was filtered through the silica gel pad. The filtrate was evaporated under reduced pressure to
obtain crude 10 (28.1 g) as a colorless solid. A solution of DMSO (14.0 mL, 197 mmol) in CH2Cl2 (100
mL) was added dropwise to a solution of oxalyl chloride (8.50 mL, 99.1 mmol) in CH2Cl2 (300 mL) at
−78 °C over a period of 20 min, and then the mixture was stirred at the same temperature for 20 min. To
the reaction mixture, a solution of crude 10 (28.1 g) in CH2Cl2 (150 mL) was added dropwise at −78 °C
over a period of 20 min, and then the mixture was stirred at the same temperature for 30 min. To the
reaction mixture, Et3N (63.0 mL, 452 mmol) was added dropwise at −78 °C over a period of 20 min.
After warming the reaction mixture to 0 °C, the mixture was stirred at the same temperature for 2 h. H2O
was added to the reaction mixture, followed by extraction with CH2Cl2. Then, the organic layer was
washed with 1N HCl aq., saturated NaHCO3 aq., and brine, and dried over anhydrous Na2SO4. After
filtration, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel
column chromatography (eluent, n-hexane/AcOEt = 9/1 to 2/1) to obtain 6 (25.9 g, 87%, 3 steps) as a
colorless solid. [α]D25.0 −31.0 (c 1.00, MeOH). 1H NMR (500 MHz, CDCl3): δ 5.91 (dd, J = 17.6, 10.7 Hz,
1H), 5.24 (d, J = 10.7 Hz, 1H), 5.22 (d, J = 17.6 Hz, 1H), 4.38-4.29 (m, 1H), 4.13-4.01 (m, 1H), 3.68 (s,
3H), 2.98-2.93 (m, 1H), 2.81-2.70 (m, 1H), 2.69-2.60 (m, 1H), 2.49-2.42 (m, 1H), 2.06-2.01 (m, 1H),
1.80-1.72 (m, 1H), 1.46 (s, 9H), 1.26 (s, 3H), 1.24 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 212.2, 173.0,
154.3, 141.1, 115.8, 80.2, 51.9, 51.6, 47.0, 45.3, 43.2, 40.4, 31.1, 28.4, 23.0, 22.9. IR: 2978, 1731, 1689,
1421, 1163, 1149, 963, 770 cm-1. HRMS (ESI+): m/z calcd for C18H29NO5+H: 340.2124; found:

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HETEROCYCLES, Vol. 89, No. 6, 2014
340.2153.
1-tert-Butyl 3-methyl (3S,5R)-5-(2,2-dimethyl-3-oxopropanoyl)piperidine-1,3-dicarboxylate (13)
Ozonized oxygen was bubbled through a solution of 6 (25.9 g, 76.3 mmol) in CH2Cl2 (380 mL) at −78 °C
for 3 h. The end of ozonolysis was indicated by the blue color appearance in the reaction mixture. The
ozone stream was then stopped and the solution was flushed with N2 for 1.5 h to remove excess ozone.
Triphenylphospine (26.0 g, 99.1 mmol) was then added at −78 °C and the reaction mixture was allowed
to reach 4 °C and was stirred for additional 18 h. Silica gel (260 g) and Et2O (380 mL) was added to the
reaction mixture. After filtration, the solvent was evaporated under reduced pressure, and the residue was
purified by silica gel column chromatography (eluent, n-hexane/AcOEt = 19/1 to 3/2) to obtain 13 (21.1 g,
25.0
1
81%) as a colorless solid. [α]D
−49.8 (c 1.04, MeOH). H NMR (400 MHz, CDCl3): δ 9.60 (s, 1H),
4.39-4.27 (m, 1H), 4.17-4.06 (m, 1H), 3.69 (s, 3H), 2.87-2.61 (m, 3H), 2.50-2.42 (m, 1H), 2.16-2.09 (m,
13
1H), 1.80-1.71 (m, 1H), 1.47 (s, 9H), 1.39 (s, 3H), 1.36 (s, 3H). C NMR (125 MHz, CDCl3): δ 209.2,
200.5, 172.7, 154.2, 80.5, 61.0, 52.0, 46.6, 45.6, 44.8, 40.3, 30.4, 28.4, 18.9, 18.8. IR: 3411, 2979, 1730,
1693, 1425, 1254, 1147, 960, 892 cm-1. HRMS (ESI+): m/z calcd for C17H27NO6+H: 342.1917; found:
342.1918.
1-tert-Butyl
3-methyl
(3S,5R)-5-[2,2-dimethyl-3-(2-methylanilino)propanoyl]piperidine-1,3-
dicarboxylate (14)
To a solution of 13 (10.0 g, 29.3 mmol) in toluene (75.0 mL), o-toluidine (4.71 mL, 44.4 mmol) and
AcOH (2.51 mL, 43.9 mmol) were added at rt, and the mixture was stirred at the same temperature for 15
min. Then, the mixture was stirred at 80 °C for 1 h. After cooling in an ice bath, NaBH(OAc)3 (19.6 g,
92.5 mmol) and AcOH (5.28 mL, 92.2 mmol) were added to the reaction mixture. The mixture was
further stirred at rt for 1.5 h. After cooling in an ice bath, saturated NaHCO3 aq. was added to the reaction
mixture, followed by extraction with AcOEt. Then, the organic layer was washed with 1N HCl aq.,
saturated NaHCO3 aq., and brine, and dried over anhydrous Na2SO4. After filtration, the solvent was
evaporated under reduced pressure, and the residue was purified by silica gel column chromatography
(eluent, n-hexane/AcOEt = 19/1 to 4/1) to obtain 14 (11.5 g, 91%) as a light yellow liquid. [α]D25.0 −37.1
1
(c 1.01, MeOH). H NMR (400 MHz, CDCl3): δ 7.14-7.10 (m, 1H), 7.04 (d, J = 6.4 Hz, 1H), 6.69-6.64
(m, 2H), 4.40-4.30 (m, 1H), 4.16-4.04 (m, 1H), 3.67 (s, 3H), 3.30-3.22 (m, 2H), 3.07-2.99 (m, 1H),
2.80-2.64 (m, 2H), 2.49-2.42 (m, 1H), 2.10 (s, 3H), 2.08-2.05 (m, 1H), 2.04-2.02 (m, 1H), 1.80 (q, J =
12.6 Hz, 1H), 1.45-1.36 (m, 9H), 1.31 (s, 6H). 13C NMR (125 MHz, CDCl3): δ 215.1, 172.9, 154.3, 146.1,
130.2, 127.1, 122.4, 117.3, 109.9, 80.3, 51.9, 51.7 49.5, 47.0, 45.4, 42.8, 40.5, 30.9, 28.3, 22.5, 22.4, 17.4.
IR: 2972, 1735, 1689, 1419, 1251, 1144, 858, 745 cm-1. HRMS (ESI+): m/z calcd for C24H36N2O5+H:

HETEROCYCLES, Vol. 89, No. 6, 2014
1421
433.2702; found: 433.2683.
1-tert-Butyl
3-methyl
(3S,5R)-5-{3-[N-(2-diethoxyphosphorylacetyl)-2-methylanilino]-2,2-
dimethylpropanoyl}piperidine-1,3-dicarboxylate (16)
Oxalyl chloride (3.35 mL, 38.5 mmol) was added to a solution of (diethoxyphosphinoyl)acetic acid (9.20
g, 46.9 mmol) in CH2Cl2 (120 mL) at 0 °C, and then the mixture was stirred at rt for 5 d. The solvent was
evaporated under reduced pressure. After azeotropic drying with CH2Cl2, CH2Cl2 (38.0 mL) was added to
the residue to obtain a crude 15 (ca. 1.00 M CH2Cl2 solution). The crude 15 (32.7 mL, ca. 1.00 M CH2Cl2
solution) was added to a solution of 14 (9.29 g, 21.5 mmol) in DMA (70.0 mL) at 0 °C for 3 h, and then
the mixture was stirred at rt for 1 h. After cooling, saturated NaHCO3 aq. was added to the reaction
mixture, followed by extraction with AcOEt. Then, the organic layer was washed with brine, and dried
over anhydrous Na2SO4. After filtration, the solvent was evaporated under reduced pressure, and the
residue was purified by silica gel column chromatography (eluent, AcOEt) to obtain 16 (11.6 g, 88%) as a
25.0
1
colorless liquid. [α]D −17.9 (c 1.00, MeOH). H NMR (400 MHz, CDCl3): δ 7.28-7.18 (m, 4H), 4.46
(dd, J = 33.8, 13.9 Hz, 1H), 4.32-4.19 (m, 1H), 4.17-4.02 (m, 5H), 3.96-3.92 (m, 1H), 3.68-3.65 (m, 3H),
3.47-3.25 (m, 1H), 2.96-2.82 (m, 1H), 2.76-2.56 (m, 3H), 2.50-2.29 (m, 2H), 2.24-2.22 (m, 3H),
1.86-1.81 (m, 1H), 1.46-1.44 (m, 9H), 1.36-1.24 (m, 12H). 13C NMR (125 MHz, CDCl3): δ 213.1, 213.0,
173.0, 172.9, 166.5, 166.4, 154.2, 141.5, 141.4, 135.5, 132.0, 131.8, 130.1, 129.8, 128.8, 128.7, 127.3,
127.0, 80.3, 62.5, 62.4, 62.3, 54.2, 54.0, 51.8, 49.8, 49.7, 45.4, 42.9, 40.5, 33.5, 32.4, 32.3, 30.5, 30.3,
28.6, 28.4, 23.8, 22.3, 22.1, 21.0, 17.8, 16.4, 16.3. IR: 2978, 1734, 1691, 1659, 1248, 1146, 1022, 957,
773 cm-1. HRMS (ESI+): m/z calcd for C30H47N2O9P+H: 611.3097; found: 611.3141.
1-tert-Butyl
3-methyl
(3S,5S)-5-[3,3-dimethyl-1-(o-tolyl)-6-oxo-2H-pyridin-4-yl]piperidine-1,3-
dicarboxylate (5)
After azeotropic drying of 16 (11.4 g, 18.7 mmol) with toluene, 18-crown-6 (4.93 g, 18.7 mmol) and THF
(350 mL) were added to the residue at rt, and then the mixture was stirred at −78 °C for 30 min. KHMDS
(37.3 mL, ca. 0.5 M in toluene) was added to the reaction mixture at −78 °C, and then the mixture was
stirred at the same temperature for 1 h. After warming the reaction mixture to −20 °C, the mixture was
stirred at the same temperature for 3 h. Saturated NH4Cl aq. and AcOEt were added to the reaction
mixture, followed by extraction with AcOEt. Then, the organic layer was washed with saturated NH4Cl
aq. and brine, and dried over anhydrous Na2SO4. After filtration, the solvent was evaporated under
reduced pressure, and the residue was purified by silica gel column chromatography (eluent,
25.0
1
n-hexane/AcOEt = 2/1) to obtain 5 (5.92 g, 72%) as a colorless solid. [α]D −24.3 (c 1.00, MeOH). H
NMR (400 MHz, CDCl3): δ 7.28-7.19 (m, 3H), 7.14-7.10 (m, 1H), 5.85 (d, J = 4.3 Hz, 1H), 4.46-4.35 (br

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m, 1H), 4.28-4.15 (br m, 1H), 3.71 (s, 3H), 3.51 (dd, J = 12.1, 8.2 Hz, 1H), 3.41 (dd, J = 12.1, 5.1 Hz,
1H), 2.83-2.75 (m, 1H), 2.59-2.49 (m, 2H), 2.34-2.18 (m, 5H), 1.75-1.64 (m, 1H), 1.48 (s, 9H), 1.30-1.24
(m, 6H). 13C NMR (125 MHz, CDCl3): 172.9, 163.7, 162.1, 154.3, 141.3, 135.7, 131.1, 127.6, 127.1,
126.3, 120.3, 80.3, 61.9, 52.0, 49.6, 45.5, 41.5, 36.8, 36.5, 34.5, 28.4, 24.4, 24.3, 18.2. IR: 2973, 1736,
1694, 1667, 1474, 1421, 1255, 1150, 874, 748 cm-1. HRMS (ESI+): m/z calcd for C26H36N2O5+H:
457.2702; found: 457.2702.
(3S,5S)-1-tert-Butoxycarbonyl-5-[3,3-dimethyl-1-(o-tolyl)-6-oxo-2H-pyridin-4-yl]piperidine-3-
carboxylic acid (17)
LiOH·H2O (1.09 g, 26.0 mmol) was added to a solution of 5 (5.92 g, 13.0 mmol) in THF (80.0 mL) and
H2O (40.0 mL) at 0 °C, and then the mixture was stirred at the same temperature for 1 h. 1 N HCl aq.
(26.0 mL, 26.0 mmol) and CH2Cl2 were added to the reaction mixture, followed by extraction with
CH2Cl2. Then, the organic layer was washed with H2O, and dried over anhydrous Na2SO4. After filtration,
the solvent was evaporated under reduced pressure, and then Et2O was added to the residue. The resulting
25.0
solid was collected by filtration to obtain 17 (5.27 g, 92%) as a colorless solid. [α]D
−31.1 (c 1.01,
1
MeOH). H NMR (400 MHz, CD3OD): δ 7.32-7.23 (m, 3H), 7.22-7.16 (m, 1H), 5.84 (s, 1H), 4.40-4.34
(m, 1H), 4.23-4.14 (m, 1H), 3.63-3.45 (m, 2H), 2.90-2.80 (m, 1H), 2.72-2.62 (m, 1H), 2.61-2.52 (m, 1H),
2.49-2.42 (m, 1H), 2.26-2.20 (m, 4H), 1.77-1.68 (m, 1H), 1.48 (s, 9H), 1.33-1.29 (m, 6H). 13C NMR (125
MHz, CD3OD): 175.9, 166.2, 166.0, 156.1, 142.4, 136.9, 132.0, 128.9, 128.3, 127.5, 119.9, 81.6, 62.7,
51.4, 46.3, 42.4, 38.3, 37.7, 35.5, 28.7, 24.6, 24.5, 18.3. IR: 3418, 2979, 2928, 1717, 1686, 1649, 1425,
1268, 1150, 751 cm-1. HRMS (ESI+): m/z calcd for C25H34N2O5+H: 443.2546; found: 453.2545.
tert-Butyl (3S,5S)-3-[3,3-dimethyl-1-(o-tolyl)-6-oxo-2H-pyridin-4-yl]-5-{[(1R)-1-(5-fluoro-2-pyridyl)-
3-methylbutyl]carbamoyl}piperidine-1-carboxylate (19)
A solution of HBTU (0.62 g, 1.64 mmol) in DMF (4.00 mL) was added to a solution of 17 (0.600 g, 1.36
mmol), (1R)-1-(5-fluoro-2-pyridyl)-3-methylbutan-1-amine dihydrochloride 188 (0.420 g, 1.65 mmol),
and N,N-diisopropylethylamine (0.946 mL, 5.42 mmol) in DMF (12.0 mL) under ice-cooling, and then
the reaction mixture was stirred at the same temperature for 1 h. Water was added to the reaction mixture,
followed by extraction with AcOEt. Then, the organic layer was washed with 1 N HCl aq., saturated
NaHCO3 aq., and brine, and dried over anhydrous Na2SO4. After filtration, the solvent was evaporated
under reduced pressure, and the residue was purified by silica gel column chromatography (eluent,
25.0
n-hexane/AcOEt = 1/1) to obtain 19 (0.823 g, quant.) as a colorless solid. [α]D 17.9 (c 1.00, MeOH).
1H NMR (400 MHz, CDCl3): δ 8.40 (d, J = 2.7 Hz, 1H), 7.39-7.34 (m, 1H), 7.26-7.19 (m, 4H), 7.12-7.08
(m, 1H), 6.55-6.49 (m, 1H), 5.84 (d, J = 4.3 Hz, 1H), 5.13 (q, J = 7.8 Hz, 1H), 4.38-4.29 (m, 1H),

HETEROCYCLES, Vol. 89, No. 6, 2014
1423
4.19-4.11 (m, 1H), 3.51-3.46 (m, 1H), 3.41-3.36 (m, 1H), 2.89-2.83 (m, 1H), 2.64-2.51 (m, 1H),
2.42-2.34 (m, 1H), 2.30-2.22 (m, 4H), 1.99-1.94 (m, 1H), 1.86-1.75 (m, 1H), 1.71-1.58 (m, 2H),
1.50-1.44 (m, 10H), 1.29-1.26 (m, 3H), 1.21 (d, J = 4.7 Hz, 3H), 0.96-0.92 (m, 6H). 13C NMR (125 MHz,
CDCl3): 171.2, 163.7, 159.6, 157.6, 156.6, 154.4, 141.3, 137.6, 135.7, 131.1, 127.6, 127.1, 126.3, 123.5,
123.2, 120.3, 80.3, 61.8, 51.5, 50.6, 46.4, 45.7, 43.5, 36.9, 36.5, 34.5, 28.4, 24.9, 24.4, 22.7, 22.6, 18.2.
IR: 3307, 2960, 1659, 1481, 1255, 1153, 849, 753, 558 cm-1. HRMS (ESI+): m/z calcd for
C35H47FN4O4+H: 607.3660; found: 607.3696.
(3S,5S)-5-[3,3-Dimethyl-1-(o-tolyl)-6-oxo-2H-pyridin-4-yl]-N-[(1R)-1-(5-fluoro-2-pyridyl)-3-
methylbutyl]piperidine-3-carboxamide fumarate (4)
Trifluoroacetic acid (5.00 mL) was added to a solution of 19 (0.823 g, 1.36 mmol) in CH2Cl2 (10.0 mL) at
0 °C, and the mixture was stirred at rt for 40 min. Saturated NaHCO3 aq. was added to the reaction
mixture under ice-cooling, followed by extraction with CH2Cl2. Then, the organic layer was washed with
brine, and dried over anhydrous Na2SO4. After filtration, the solvent was evaporated under reduced
pressure, and the residue was purified by NH silica gel column chromatography (eluent, CH2Cl2/MeOH =
20/1 to 7/3) to obtain the free base of 4 (0.610 g, 1.20 mmol). Fumaric acid (0.139 g, 1.20 mmol) was
added to a solution of the free base of 4 (0.610 g, 1.20 mmol) in MeOH (1.00 mL) at room temperature,
and the mixture was stirred at the same temperature for 5 min. The solvent was evaporated under reduced
pressure, and then Et2O was added to the residue. The resulting solid was collected by filtration to obtain
4 (0.690 g, 81%, 2 steps) as a colorless solid. [α]D25.0 35.9 (c 1.01, MeOH). 1H NMR (400 MHz, CD3OD):
δ 8.41 (d, J = 2.3 Hz, 1H), 7.56 (td, J = 8.5, 3.0 Hz, 1H), 7.39 (dd, J = 8.8, 4.5 Hz, 1H), 7.30-7.23 (m, 3H),
7.18-7.14 (m, 1H), 6.70 (s, 2H), 5.85 (d, J = 2.3 Hz, 1H), 5.05 (dd, J = 9.0, 6.3 Hz, 1H), 3.59 (dd, J = 12.5,
11.3 Hz, 1H), 3.52-3.44 (m, 2H), 3.41-3.35 (m, 1H), 3.13-3.06 (m, 1H), 3.00-2.88 (m, 2H), 2.83-2.76 (m,
1H), 2.23-2.14 (m, 4H), 1.76-1.55 (m, 4H), 1.32-1.28 (m, 6H), 0.97 (d, J = 6.4 Hz, 3H), 0.94 (d, J = 6.4
13
Hz, 3H). C NMR (125 MHz, CD3OD): 172.8, 171.5, 165.6, 164.5, 161.2, 159.2, 142.2, 138.2, 136.9,
136.3, 132.1, 129.0, 128.3, 127.5, 125.0, 123.8, 121.3, 62.6, 53.8, 45.8, 45.4, 41.5, 37.6, 35.2, 34.8, 26.2,
24.6, 24.5, 23.3, 22.3, 18.3. IR: 3293, 2960, 1658, 1482, 1388, 1252, 983, 751, 646. HRMS (ESI+): m/z
calcd for C30H39FN4O2+H: 507.3135; found: 507.3148.
Biological Assays
IC50 in buffer
The activity of renin inhibitors against purified enzyme was measured using the following protocol: All
reactions were carried out in a flat bottom black opaque microtiter plate. Test compounds in DMSO (2
μL) were mixed with 100 μL of the assay buffer (50 mM Tris-HCl (pH 7.9), 100 mM NaCl) containing 5

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μL of trypsin-activated recombinant human renin (final enzyme concentration of 50 μM), and the solution
was pre-incubated at room temperature for 10 min. Next, μM of the substrate
2
(Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(DABCYL)-Arg) in 100 μL of the
assay buffer was added, and the resulting mixture was incubated at 37 °C for 90 min. After completion of
incubation, the concentration of generated angiotensin I was measured by fluorescence at 492 nm
(excitation at 340 nm) using a multilabel reader (Perkin-Elmer Inc.). The slope of the linear portion of the
plot of fluorescence increase as a function of time was then determined, and the rate was used to
calculate % inhibition in relation to uninhibited control. The % inhibition values were plotted as a
function of inhibitor concentration, and the IC50 value was determined by probit analysis. The IC50 value
is defined as the concentration of a particular inhibitor that reduces the formation of product by 50%
relative to a control sample containing no inhibitor.
IC50 in plasma
The activity of renin inhibitors in vitro in cynomolgus monkey plasma was measured by the decrease in
plasma renin activity (PRA) levels observed in the presence of the compounds. Compounds and Aliskiren
hemifumarate (1) were dissolved in DMSO and the final concentration of DMSO was 1%. Incubation
mixtures were contained in the final volume of 20 μL of test compound solution, 200 μL of pooled
mixed-gender human or cynomolgus monkey plasma stabilized with EDTA, 20 μL of pH adjusting
solution, and 10 μL of Inhibitor A solution. The reaction mixture was incubated at 37 °C for 60 min.
After incubation, angiotensin I in the reaction mixture was measured by competitive radioimmunoassay
using a commercial available RIA kit, RENIN RIABEAD (Yamasa Co.). An uninhibited tube containing
1% DMSO and control tube incubated at 4 °C were used to derive the % inhibition for each concentration
of inhibitors. The % inhibition values were plotted as the function of inhibitor concentration, and the IC50
value was determined from a fit of this data to a four parameter equation. The IC50 value is defined as
above.
Animal Studies
Blood pressure study in cynomolgus monkeys pre-treated with furosemide
Arterial pressure was measured by a telemetry system in conscious, freely moving cynomolgus monkeys
(n = 6). Pressure transmitters (TL11M2-D70-PCT, Data Sciences International Inc., USA) were
implanted into the peritoneal cavity under aseptic conditions and anesthesia, and the sensor catheter was
placed in the left femoral artery. Cynomolgus monkeys were allowed to recover for at least 1 week before
any experiment. The animals were fasted from the morning on the dosing day. Feeding on the dosing day
was conducted 8 hours after dosing or later. The animals were allowed free access to water the whole

HETEROCYCLES, Vol. 89, No. 6, 2014
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time. Furosemide at 5 mg/kg/day was intramuscularly administered for 3 days before drug administration.
Cynomolgus monkeys orally received 4 at dose 10 mg/kg, or vehicle (1% methylcellulose). Arterial
pressure was continuously measured telemetrically from 3 h before administration to 24 h after
administration with the data collection and real-time analysis system (Dataquest™ OpenART™, Data
Sciences International, USA). The mean value for 1 h of MAP was calculated.
ACKNOWLEDGEMENTS
We would like to thank Dr. Yasuyuki Ogawa, Dr. Akiyoshi Mochizuki, Dr. Yuji Nakamura, Dr. Teppei
Fujimoto, Chie Sugita, Dr. Shojiro Miyazaki, Dr. Kazuhiko Tamaki, and Masumi Ueno for their technical
assistance and helpful discussions.
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