Design and discovery of new (3S,5R)-5-[4-(2-chlorophenyl)-2,2-dimethyl-5- oxopiperazin-1-yl]piperidine-3-carboxamides as potent renin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.09.103

Utilizing X-ray crystal structure analysis, (3S,5R)-5-[4-(2-chlorophenyl)- 2,2-dimethyl-5-oxopiperazin-1-yl]piperidine-3-carboxamides were designed and identified as renin inhibitors. The most potent compound 15 demonstrated favorable pharmacokinetic and

Full text of DOI:10.1016/j.bmcl.2012.09.103

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7677–7682
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and discovery of new (3S,5R)-5-[4-(2-chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]piperidine-3-carboxamides as potent renin inhibitors
Yutaka Mori a, Yasuyuki Ogawa a, Akiyoshi Mochizuki b, Yuji Nakamura a, Chie Sugita a, Shojiro Miyazaki a,
Kazuhiko Tamaki a, Yumi Matsui b, Mizuki Takahashi b, Takahiro Nagayama c, Yoko Nagai d, Shin-ichi Inoue d,
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 Cardiovascular-Metabolics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Utilizing X-ray crystal structure analysis, (3S,5R)-5-[4-(2-chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-
yl]piperidine-3-carboxamides were designed and identified as renin inhibitors. The most potent com-
pound 15 demonstrated favorable pharmacokinetic and pharmacodynamic profiles in rat.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 11 September 2012
Revised 25 September 2012
Accepted 28 September 2012
Available online 17 October 2012
Keywords:
Renin
Ketopiperazine
Piperidine
X-ray crystal structure analysis
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 devel-
oped world.1 The renin–angiotensin–aldosterone system (RAAS)
plays an important role in the regulation of blood pressure and
fluid homeostasis.2 The inhibition of either the formation or the
action of angiotensin II (Ang II), the main product of the RAAS, rep-
resents a major therapeutic approach in the treatment of hyperten-
sion and the prevention of associated comorbidities. Presently,
inhibitors of the angiotensin-converting enzyme (ACE) and antag-
onists of the Ang II type-1 receptor (AT1R) are known as major
drugs which modulate the RAAS.3 It has long been hypothesized
that inhibitors of renin, which is the rate-limiting enzyme in the
RAAS cascade, may represent the most attractive therapeutic strat-
egy to block the RAAS.4 Despite the fact that many pharmaceutical
companies have researched and developed renin inhibitors suit-
able for clinical development during the past few decades, only Ali-
skiren (1) has reached the market for the treatment of essential
hypertension (Fig. 1).5,6
tory activity and specificity against other aspartic proteases with-
out using the S3 interaction, which is suggested to play an
sp
important role in the potent renin inhibitory activity and the spec-
ificity of Aliskiren.8 Further chemical modifications of this series
successfully led to the identification of a clinical candidate, DS-
8108b (4).9 Encouraged by this success, we explored a structurally
new type of renin inhibitor possessing 2,2-dimethyl-4-phenylpi-
perazin-5-one in the P3–P1 portion to improve efficacy and oral
bioavailability. Herein, we report our efforts to design and charac-
terize the novel (3S,5R)-5-[4-(2-chlorophenyl)-2,2-dimethyl-5-
oxopiperazin-1-yl]piperidine-3-carboxamides that demonstrate
both favorable pharmacokinetic and pharmacodynamic profiles
as renin inhibitors.
When we started our design of structurally new types of renin
inhibitors, we focused on Roche’s piperidine-based renin inhibitor
2 (Fig. 1).10 The scaffold of compound 2 has served as the inspira-
tion for the design of piperidine-based renin inhibitors, repre-
sented by 3 (ACT-077825, MK-8141) which has entered human
clinical trials (Fig. 1).11 Based on the reported structural analysis
of piperidine-based inhibitors complexed with human renin,12 it
appears that the naphthyl group in compound 2 is located in the
S3–S1 region. The large phenyl substituent at the 4-position of
the piperidine ring is assumed to occupy the flap region, which is
formed by the break of the hydrogen bond between Tyr75 and
Trp39. The remaining substituent at the 3-position potentially
Recently, we discovered a novel class of renin inhibitors which
contain our original 2,2-dimethyl-4-phenylpiperazin-5-one part in
the P3–P1 portion.7 These inhibitors showed potent renin inhibi-
⇑
Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5436 8563.
E-mail address: nishi.takahide.xw@daiichisankyo.co.jp (T. Nishi).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.103

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Y. Mori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7677–7682
1 (Aliskiren)
2
fumaric acid
2 H2O
3 (ACT-077825, MK-8141)
4 (DS-8108b)
Figure 1. Chemical structures of 1 (Aliskiren), piperidine-based compounds 2 and 3 (ACT-077825, MK-8141), and our renin inhibitor 4 (DS-8108b).
P1'
4
5
3
P3
P2'
P3
P2'
5
3
P1
fumaric acid
2 H2O
P1
P1
P2'
X = CO, (CH2)0-1
P3
2
4 (DS-8108b)
General structure A
Figure 2. Design of a new series of 3,5-disubstituted piperidines having 2,2-dimethyl-4-phenylpiperazin-5-one.
extends into the S2’ pocket. In the case of 1 or 4, the flap region is
closed and the N-terminal secondary amide part of the molecule is
known to be docked in the S2’ pocket.8b,9 Taking this into consider-
ation, we initially designed a 3,5-disubstituted piperidine (General
structure A) that possessed a tethered characteristic S3–S1 favored
2,2-dimethyl-4-phenylpiperazin-5-one substituent at the 5-posi-
tion of the piperidine ring (Fig. 2). The large phenyl substituent
at the 4-position was removed not only to reduce the molecular
weight and lipophilicity, but to close the flap region similar to
was located spatially close to the ketopiperazine ring (Fig. 3). Based
on this result, the direct connection between the piperidine ring
and ketopiperazine part may be helpful at eliminating the pre-
sumed unstable conformation. Thus, we redesigned and synthe-
sized compound 7 with an aim to find a more appropriate lead
compound by locating the ketopiperazine part at a more suitable
place in the binding pocket of human renin (Table 1). Piperidine
analog 7, which is directly substituted by ketopiperazine, had po-
tent human renin inhibitory activity (IC50 = 7.7 nM in purified hu-
man renin). X-ray crystal structure analysis of 7 in complex with
human renin indicated that the piperidine-5-yl group adopted a
favorable pseudo-equatorial conformation against the ketopiper-
azine ring as expected (Fig. 4). This suggests that this favorable
conformation contributed to the potent inhibitory activity of 7
against purified human renin.
0
the case of 1 or 4. Thus, the preference of the S2 pocket for a sec-
ondary amide group, (3-methylbutylamino)carbonyl group, would
be applicable to the substituent at the 3-position.
To evaluate the newly designed 3,5-disubstituted piperidine,
the in vitro renin inhibitory activities of compounds 5 and 6 were
initially measured (Table 1).13 Compound 5, which has a carbonyl
group as a linker of ketopiperazine and piperidine, did not show
any interesting renin inhibitory activity. However, the conforma-
tionally more flexible methylene-tethered compound 6 exhibited
weak inhibitory activity for purified human renin, although the
activity was insufficient. To identify the reason for the weak affin-
ity, the X-ray crystal structure of 6 in complex with human renin
was obtained. From the X-ray structure, it was found that the
piperidine moiety of compound 6 interacted to the catalytic Asp
residues as previously reported piperidine-based renin inhibi-
tors.11,12 However, the substituent on the 5-position of piperidine
was revealed to adopt a presumed unstable pseudo-axial confor-
mation relative to the ketopiperazine ring, and the piperidine ring
After identifying the appropriate position of the 2,2-dimethyl-
4-phenylpiperazin-5-one part relative to the piperidine ring, we fo-
cused on modifying the amide part at the 3-position of the piper-
idine ring to obtain a more potent lead compound. We obtained
0
0
the X-ray crystal structure (S1 and S2 regions) of compound 7
(Fig. 5. A). The S2 pocket was occupied by the isobutyl group (R2)
0
0
as expected. In addition, the existence of the empty S1 pocket lo-
cated around the R3 moiety encouraged us to evaluate compounds
having alkyl groups at R3 (8 and 9). Installation of ethyl (8) or the
slightly larger isobutyl (9) group improved the renin inhibitory
activity. To confirm the substitution effect at R3, benzyl amide
derivatives were also synthesized (10–12). Compounds having an

Y. Mori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7677–7682
7679
Table 1
In vitro renin inhibitory activities (IC50) of initial compoundsa
5
3
Compound
R1
Purified human renin IC50 (nM)
>1000
Monkey plasma renin IC50 (nM)
5
>1000
>1000
54
6
7
406
7.7
a
Compounds were obtained as fumarate salts.
conducted to further characterize the compound. As listed in
Table 4, good oral bioavailability was observed in SD rat
(F = 45%). The antihypertensive efficacy of compound 15 was
investigated in double transgenic rat (dTG rat) harboring both
the human angiotensinogen and the human renin gene (Fig. 6).15
Vehicle or 10 mg/kg of compound 15 was orally administered.16
Compound 15 induced a significant reduction in mean arterial
blood pressure (MAP) sustained over a period of 24 h. Based on
these results, we concluded compound 15 was an attractive new
lead compound for further exploration.
The synthetic pathway leading to compounds 7–17 is outlined
in Scheme 1. Chiral carboxylic acid 20 was successfully obtained
by the enantioselective ring opening of the known carboxylic acid
anhydride 1817 with methanol in the presence of the sulfonyl
amide 1918 as an asymmetric catalyst. Conversion of the carboxylic
acid 20 to the N-allyloxycarbonyl protected amine by the Curtius
rearrangement, replacement of the N-(tert-butoxycarbonyl) (Boc)
group with the N-(2-nitrobenzenesulfonyl) (Ns) group19, and N-
allyloxycarbonyl group deprotection led to the corresponding
amine 21. Condensation of isobutylaldehyde with the amine 21
Figure 3. X-ray crystal structure of 6 in complex with human renin.14 Pseudo-axial
bond between the piperidine ring and ketopiperazine ring is indicated.
and chlorination with N-chlorosuccinimide yielded
a-chloro
ethyl (10), propyl (11), or isobutyl (12) group showed about the
same inhibitory activity as compounds 8 and 9 (Table 2). To verify
the interaction of the alkyl substituent at R3 with the S10 pocket, the
X-ray crystal structure of 9 bound to renin was obtained. The re-
sults indicated that, as expected, the isobutyl substituent at R3
0
occupied the S1 pocket (Fig. 5. B).
We then investigated the variation of the functionality at the
P2 portion (Table 3). While retaining the isobutyl group as a sub-
0
stituent of R3, we evaluated the compounds having a hydroxyl
(13 and 14), ether (15), dimethyl amide (16), phenyl (12), or
3-pyridyl (17) group at R2. Although the dimethyl amide (16) de-
creased renin inhibitory activity, other compounds (13–15, 12,
and 17) exhibited similar IC50 values against purified human re-
nin as compound 9. In particular, compound 15 containing an
ether functionality showed potent inhibitory activity against
monkey plasma renin (IC50 = 1.4 nM) as well as purified human
renin (IC50 = 1.3 nM).
Since compound 15 appeared to possess potent inhibitory activ-
ity in both the purified human and monkey plasma renin assays, a
pharmacokinetic study and a blood pressure lowering study were
Figure 4. X-ray crystal structure of 7 in complex with human renin.14

7680
Y. Mori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7677–7682
Figure 5. Crystal structures of human renin (S1 and S2 regions) in complex with 7 (A) or 9 (B).14
0
0
Table 2
Table 3 (continued)
In vitro renin inhibitory activities (IC50) with modification of the amide parta
Compound R2
Purified human
renin IC50 (nM)
Monkey plasma
renin IC50 (nM)
5
3
17
2.1
4.2
a
b
c
Compounds were obtained as fumarate salts.
Stereochemistry is not determined.
Compound 14 is the diastereomer of compound 13.
Compound
R2
R3
Purified human
renin IC50 (nM)
Monkey plasma
renin IC50 (nM)
7
8
9
10
11
12
Isobutyl
Isobutyl
Isobutyl
Ph
Ph
Ph
H
Ethyl
Isobutyl
Ethyl
Propyl
Isobutyl
7.7
2.8
1.6
2.1
2.5
2.1
54
16
10
6.6
18
9.0
Table 4
PK profile of compound 15 in SD rat
a
Compounds were obtained as fumarate salts.
Table 3
In vitro renin inhibitory activities (IC50) with modification of the P2 portiona
0
SD rat, male
(10 mg/kg p.o., 0.5%
MC)(1 mg/kg i.v., saline)
F (%)
45
540
1940
31
Cmax (ng/mL)
AUCpo,0–24 (ng⁄h/mL)
CL (mL/min/kg)
Vss (L/kg)
5.8
3.2
t1/2 (h)
Compound R2
Purified human
renin IC50 (nM)
Monkey plasma
renin IC50 (nM)
40
20
9
1.6
2.3
10
more polar
less polar
13b
4.8
*
*
0
14b,c
15
2.5
1.3
8.4
7.5
1.4
23
-20
-40
-60
Vehicle (n=17)
16
Compound 15, 10 mg/kg p.o. (n=10)
0
10
20
30
Time (h)
40
50
12
2.1
9.0
Figure 6. Effect of compound 15 on mean arterial pressure (MAP) in dTG rat.

Y. Mori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7677–7682
7681
19
(a)
(b), (c), (d), (e)
(f), (g), (h)
(i), (j)
18
20
21
22
(k), (l)
(m), (n)
(o), (p)
fumaric acid
23
24
25
7-17
Scheme 1. Synthetic pathway leading to (3S,5R)-5-[4-(2-chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]piperidine-3-carboxamides 7–17. Reagents and conditions: (a) 19,
CH3OH, THF, ꢀ20 °C, 1 d, 69%, 94% ee; (b) diphenylphosphoryl azide, Et3N, toluene, allyl alcohol, 90 °C, 15 h, 90%; (c) TFA, CH2Cl2, r.t., 1 h; (d) NsCl, Et3N, CH2Cl2, r.t., 1 h, 75% (2
steps); (e) (PPh3)4Pd, morpholine, THF, r.t., 1 h, 81%; (f) isobutylaldehyde, CH2Cl2, r.t., 2 h; (g) N-chlorosuccinimide, CH2Cl2, r.t., 2 h; (h) CH3OH, 60 °C, 18 h, 49% (3 steps); (i)
conc. HCl aq., CH2Cl2, 40 °C, 4 h; (j) SOCl2, CH3OH, r.t., 1 h, 68% (2 steps); (k) 2-chloroaniline, AcOH, toluene, 110 °C, 2 h, then NaBH(OAc)3, r.t., 6 h, 76%; (l) bromoacetyl
bromide, Et3N, CH2Cl2, 0 °C, then 40 °C, 5 h, 66%; (m) 1N NaOH aq., CH3OH, 50 °C, 0.5 h, quant.; (n) amine (R-NH2), HBTU, diisopropylethylamine, DMF, r.t., 5 h; (o) PhSH,
Cs2CO3, CH3CN, r.t., 1.5 h; (p) fumaric acid, CH3OH, r.t., 1 min.
3. (a) Zaman, M. A.; Oparil, S.; Calhoun, D. A. Nat. Rev. Drug Disc. 2002, 1, 621; (b)
aldimine. Treatment of the a-chloro aldimine with methanol gave
Schmieder, R. E.; Hilgers, K. F.; Schlaich, M. P.; Schmidt, B. M. W. Lancet 2007,
369, 1208.
4. Hershey, J. C.; Steiner, B.; Fischli, W.; Feuerstein, G. Drug Discovery Today:
Therapeutic Strategies 2005, 2, 181.
the rearranged product 22 via the ring opening of the correspond-
ing aziridine intermediate.20 The dimethyl acetal and methyl ester
in 22 were hydrolyzed with concentrated hydrochloric acid, then
esterification of the carboxylic acid with thionyl chloride and
methanol provided aldehyde 23. The aldehyde 23 was converted
into ketopiperazine 24 by reductive amination with 2-chloroani-
line and subsequent ring formation with bromoacetyl bromide.
The condensation of various amines21 and carboxylic acid resulting
from the hydrolysis of compound 24 afforded the amides 25. Final-
ly, removal of the N-Ns groups, then addition of fumaric acid gave
compounds 7–17 as fumarate salts.
5. (a) Greenlee, W. J. Med. Res. Rev. 1990, 10, 173; (b) Rosenberg, S. H. Prog. Med.
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In summary, we have described the design and discovery of
(3S,5R)-5-[4-(2-chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-
yl]piperidine-3-carboxamides as new types of renin inhibitors by
using X-ray crystal structure analysis. The application of the con-
0
0
cept of using interactions with both S1 and S2 pockets resulted
in an improvement of the renin inhibitory activities. The most po-
tent compound 15 demonstrated 45% oral bioavailability in SD rat
and was orally efficacious in a dTG rat model of hypertension. From
these encouraging results, we selected compound 15 as a new lead
compound. Further optimization to acquire a more promising com-
pound is under investigation. These results will be reported in due
course.
9. Nakamura, Y.; Fujimoto, T.; Ogawa, Y.; Sugita, C.; Miyazaki, S.; Tamaki, K.;
Takahashi, M.; Matsui, Y.; Nagayama, T.; Manabe, K.; Mizuno, M.; Masubuchi,
N.; Chiba, K.; Nishi, T. ACS Med. Chem. Lett. 2012, 3, 754.
10. Märki, H. P.; Binggeli, A.; Bittner, B.; Bohner-Lang, V.; Breu, V.; Bur, D.;
Coassolo, Ph.; Clozel, J. P.; D’Arcy, A.; Doebeli, H.; Fischli, W.; Funk, Ch.;
Foricher, J.; Giller, T.; Grüninger, F.; Guenzi, A.; Güller, R.; Hartung, T.; Hirth, G.;
Jenny, Ch.; Kansy, M.; Klinkhammer, U.; Lave, T.; Lohri, B.; Luft, F. C.; Mervaala,
E. M.; Müller, D. N.; Müller, M.; Montavon, F.; Oefner, Ch.; Qiu, C.; Reichel, A.;
Sanwald-Ducray, P.; Scalone, M.; Schleimer, M.; Schmid, R.; Stadler, H.; Treiber,
A.; Valdenaire, O.; Vieira, E.; Waldmeier, P.; Wiegand-Chou, R.; Wilhelm, M.;
Wostl, W.; Zell, M.; Zell, R. Il Farmaco 2001, 56, 21.
Acknowledgments
We thank Dr. Masao Yoshida, Dr. Kazuki Mori, Dr. Mitsuhiro
Iwamoto, Sayaka Suzuki, Dr. Akifumi Kurata, Dr. Makoto Mizuno,
Masumi Ueno and Mina Nishi for their technical assistance and
helpful discussions.
ˇ
11. Bezençon, O.; Bur, D.; Weller, T.; Richard-Bildstein, S.; Remen, L.; Sifferlen, T.;
Corminboeuf, O.; Grisostomi, C.; Boss, C.; Prade, L.; Delahaye, S.; Treiber, A.;
Strickner, P.; Binkert, C.; Hess, P.; Steiner, B.; Fischli, W. J. Med. Chem. 2009, 52,
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12. (a) Oefner, C.; Binggeli, A.; Breu, V.; Bur, D.; Clozel, J.-P.; D’Arcy, A.; Dorn, A.;
Fischli, W.; Grüninger, F.; Güller, R.; Hirth, G.; Märki, H. P.; Mathews, S.; Müller,
M.; Ridley, R. G.; Stadler, H.; Vieira, E.; Wilhelm, M.; Winkler, F. K.; Wostl, W.
Chem. Biol. 1999, 6, 127; (b) Vieira, E.; Binggeli, A.; Breu, V.; Bur, D.; Fischli, W.;
Güller, R.; Hirth, G.; Märki, H. P.; Müller, M.; Oefner, C.; Scalone, M.; Stadler, H.;
Wilhelm, M.; Wostl, W. Bioorg. Med. Chem. Lett. 1999, 9, 1397.
13. Assays were performed with the same procedure described by in Ref. 9. Assay
results are the average of at least two replicates.
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Y. Mori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7677–7682
14. The X-ray crystallographic studies were accomplished according to the
procedure described in Ref. 9. Coordinates and statistics are available
from the PDB using accession code 3VYD (complex with 6), 3VYE (7),
and 3VYF (9).
(a)
(b)
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16. 0.5% MC was used as the vehicle.
26
27
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28
29
aReagents and conditions: (a) BnBr, K2CO3, ethanol, r.t., 4 d, 85%; (b) ethyl
iodide, NaH, THF, r.t., 1 d, 81%; (c) H2, 20% Pd(OH)2 on carbon, CH3OH, r.t., 1 d,
98%.
21. Chiral amine 29 used for the synthesis of compound 15 was prepared as
follows.a