Structure-based design of substituted piperidines as a new class of highly efficacious oral direct renin inhibitors

Article abstract of DOI:10.1021/ml500137b

A cis-configured 3,5-disubstituted piperidine direct renin inhibitor, (syn,rac)-1, was discovered as a high-throughput screening hit from a target-family tailored library. Optimization of both the prime and the nonprime site residues flanking the central piperidine transition-state surrogate resulted in analogues with improved potency and pharmacokinetic (PK) properties, culminating in the identification of the 4-hydroxy-3,5-substituted piperidine 31. This compound showed high in vitro potency toward human renin with excellent off-target selectivity, 60% oral bioavailability in rat, and dose-dependent blood pressure lowering effects in the double-transgenic rat model.

Full text of DOI:10.1021/ml500137b

Letter
pubs.acs.org/acsmedchemlett
Structure-Based Design of Substituted Piperidines as a New Class of
Highly Efficacious Oral Direct Renin Inhibitors
Takeru Ehara,†,§ Osamu Irie,† Takatoshi Kosaka,† Takanori Kanazawa,† Werner Breitenstein,‡
Philipp Grosche,‡ Nils Ostermann,‡ Masaki Suzuki,† Shimpei Kawakami,† Kazuhide Konishi,†
Yuko Hitomi,† Atsushi Toyao,† Hiroki Gunji,† Frederic Cumin,‡ Nikolaus Schiering,‡ Trixie Wagner,‡
Dean F. Rigel,∥ Randy L. Webb,∥ Jurgen Maibaum,‡ and Fumiaki Yokokawa*,†,⊥
̈
†Novartis Institutes for BioMedical Research, Ohkubo 8, Tsukuba, Ibaraki 300-2611, Japan
‡Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland
∥Novartis Pharmaceuticals Corp., Institutes for BioMedical Research, East Hanover, New Jersey 07936, United States
S
* Supporting Information
ABSTRACT: A cis-configured 3,5-disubstituted piperidine direct renin inhibitor, (syn,rac)-1, was discovered as a high-
throughput screening hit from a target-family tailored library. Optimization of both the prime and the nonprime site residues
flanking the central piperidine transition-state surrogate resulted in analogues with improved potency and pharmacokinetic (PK)
properties, culminating in the identification of the 4-hydroxy-3,5-substituted piperidine 31. This compound showed high in vitro
potency toward human renin with excellent off-target selectivity, 60% oral bioavailability in rat, and dose-dependent blood
pressure lowering effects in the double-transgenic rat model.
KEYWORDS: Renin inhibitor, structure-based design, aspartic protease, 3,5-piperidines
renin inhibitors (DRIs) suffered from lack of oral bioavailability
ypertension is a major risk factor for cardiovascular
Hdiseases (CVDs), such as congestive heart failure, stroke, and poor antihypertensive efficacy in clinical trials. To improve
the unfavorable pharmacokinetic (PK) properties, a combina-
tion of crystallographic structure analyses and computational
molecular modeling had been employed by Novartis to design
nonpeptidic DRIs. This approach has led to the identification of
aliskiren (Tekturna, Rasilez), a highly potent, selective, and
long lasting antihypertensive DRI, which to date is the only
marketed DRI for the treatment of hypertension.5,6 Sub-
sequently, a number of DRIs based on diverse transition-state
surrogate structural motifs and scaffolds with different renin
active-site binding topology have been reported.7−9
As part of our continued research efforts to identify novel
lead scaffolds, we have successfully applied complementary
approaches including enzymatic high-throughput screens
(HTSs), a combined NMR/X-ray fragment based screen,10
and in silico three-dimensional pharmacophore searches.11
Compound 1, a racemic cis-configured 3,5-disubstituted
and myocardial infarction, which are the leading causes of death
in industrialized countries. However, despite several classes of
therapeutic drugs available, less than 30% of hypertensive
patients achieve currently recommended blood pressure
goals.1,2
The renin-angiotensin-aldosterone system (RAAS) has long
been established as being the key cascade in the regulation of
blood pressure and homeostasis of body fluid volume. Renin,
an aspartic protease, cleaves angiotensinogen to release inactive
peptide angiotensin I (Ang-I). Cleavage of Ang-I by angiotensin
converting enzyme (ACE) then generates angiotensin II (Ang-
II), which induces the principal physiological effects of the
RAAS, i.e. an increase of blood pressure by vasoconstriction,
sodium retention and altering vascular resistance.3 Renin
controls the first and rate-limiting step of the RAAS and has
high specificity for its substrate angiotensinogen. Blockade of
renin has been considered to be a highly attractive paradigm to
treat hypertension and to protect from end-organ damage.4
Since the early 1980s, substantial efforts had centered on
peptidic or peptidomimetic scaffolds. However, these direct
Received: April 5, 2014
Accepted: April 21, 2014
Published: April 21, 2014
© 2014 American Chemical Society
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Letter
piperidine, originated as a weakly active HTS hit from a
piperidine-based combinatorial library that had been specifically
designed for aspartic proteases at Novartis (Figure 1).12 We
We next investigated the optimization of the C5-amide
moiety with various α-branched amines occupying the S1′-S2′
pockets (Table 1). Incorporation of an H-bond donor or
Table 1. Optimization of the C5-Amide Moiety Filling the
S1′-S2′ Pockets
in vitro CLint
renin IC50 (nM)
(buffer/plasma)
(μL/min/mg,
rat F (%) (i.v./p.o.
a
cmpd
human/rat)
2/6 mg/kg)
7
3/40
3/8
194/141
157/111
173/109
679/533
29
8
0.4
<0.1
nd
9
0.4/1
0.4/2
10
a
All compounds were used as their free base.
acceptor into the prime-site amide moiety was anticipated to
target an H-bond with Gly34 as well as to reduce lipophilicity,
thereby increasing plasma renin potency. Indeed, compounds 8
and 9 displayed IC50 values of 8 and 1 nM, respectively, in the
presence of plasma. However, these compounds suffered from
low oral bioavailability in rat (<1%) with medium clearance in
rat liver microsomes (RLM). An o,o-disubstituted aniline
moiety was also found to fill the prime-site efficiently, however,
compound 10 showed high in vitro clearance. In addition, we
noticed that the potential for metabolism of the 3-amino-
methylindole moiety could lead to generation of a reactive
electrophilic intermediate.16 We therefore returned our focus to
alternative P3−P1 moieties.
At this stage, the development of a versatile synthetic route
was required to facilitate optimization of both the prime- and
the nonprime binding motifs (Scheme 1). We established a
stereoselective synthesis of both (S,R)- and (R,S)-configured
cis-piperidine enantiomers 12 and 13 based on an asymmetric
desymmetrization approach starting from the meso-cyclic
anhydride 11.17 Cinchona base-catalyzed methanolysis of 11,
followed by recrystallization with (R)- and (S)-1-phenylethyl-
amine provided the enantiomerically pure 3,5-piperidine
carboxylic acids (3S,5R)-12 and (3R,5S)-13 with >99% ee,
respectively.18 Subsequent amide coupling of (3S,5R)-12 with
sterically hindered N-cyclopropyl anilines required extensive
optimization. A mixed anhydride coupling method using
TcBocCl (2,2,2-trichloro-1,1-dimethylethyl chloroformate) in
the presence of magnesium bromide proved optimal in
providing amides 14. Alkaline hydrolysis followed by coupling
to the prime-site moiety afforded intermediates 16. N-Boc
deprotection using HCl gave the target products 17. Similarly,
the reversed order of coupling steps starting from (3R,5S)-13
could be employed.
Figure 1. Discovery of (rac)-6 from the HTS hit (syn,rac)-1.
previously reported that X-ray crystallography employing (rac)-
1 and recombinant human renin (rh-renin) resulted in a
complex structure with only the (3S,5R)-enantiomer of 1
bound in the active site and with the flap β-hairpin loop in a
closed conformation (see Supporting Information, Figure S1).
Subsequently, merging this piperidine scaffold with a second
fragment-based screening hit led to (3S,5R)-2 with enhanced in
vitro potency (IC50 = 3 nM).10 We report herein the results of
an alternative approach to optimize screening hit 1, which led
to the discovery of several potent and orally bioavailable DRIs.
Initially, the highly lipophilic diphenylmethylamine P3−P1
pharmacophore of 1 was replaced by the N-cyclopropyl N-
dichlorobenzyl amide motif, previously reported for 3,4-
piperidine-based DRIs to fill the large contiguous S3−S1
binding pocket,13 to provide (rac)-3 (Figure 1). Replacement of
the C5-sulfonamide spacer by a carbamate linker identified the
10-fold more potent (rac)-4. The X-ray cocrystal structure of
(rac)-4 with rh-renin showed only the (3R,5S)-enantiomer
bound, opposite to the observation made for (rac)-1 (see
Supporting Information, Figure S1). With the aim of improving
potency by filling the nonsubstrate S3 subpocket (S3sp),
extending perpendicular from the S3 pocket, we next replaced
the dichlorophenyl moiety by an indole scaffold substituted at
the ring nitrogen by a methoxypropyl side chain14 to afford
(rac)-5. Further optimization of (rac)-5 by introduction of the
reversed amide spacer at the 5-position of the central piperidine
provided (rac)-6, exhibiting a marked increase in potency.
Computational modeling suggested that the higher inhibitory
affinity is due to the reversed amide carbonyl group forming an
H-bond to the backbone nitrogen of Ser76 of the flap β-
hairpin.15
Next, we revisited the N-cyclopropyl N-dichlorobenzyl amide
P3−P1 binding motif. Modeling and SAR studies led to
replacement of the 3-aminomethylindole with 4-isopropyl-3-(3-
methoxypropoxy)anilide. Compound 21 afforded IC50s of 0.3
and 2.0 nM against rh-human renin in buffer and in the
presence of plasma, respectively (Table 2). The potency of 21
is believed to be a result of the fluorine atom forming an H-
bond with the hydroxyl group of Thr77. Unfortunately, inhibitor
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ACS Medicinal Chemistry Letters
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21 suffered from high clearance in RLM as well as hERG
binding, presumably due to the more lipophilic character of the
molecule. We therefore explored the less hydrophobic 1,4-
benzoxazinone motif as a P3 pharmacophore.19 Inhibitor 22
(Table 2) showed not only high in vitro potency but also high
clearance in RLM, translating into high in vivo clearance in rat.
Replacement of the 6-fluoroaniline with 6-aminopyridine
offered an alternative H-bond acceptor for Thr77, and also
reduced the lipophilicity. As a result, the 6-aminopyridine P3
pharmacophore in 23 retained high potency while improving in
vitro and in vivo clearance in rats. The regioisomeric 2-
aminopyridine analogue 24 was found to be less potent and
metabolically less stable than 23. We suspected that the S3sp-
filling alkoxy side chain of 23 potentially constitutes a
metabolically weak spot, and furthermore that its high number
of rotatable bonds may limit cell permeability. Truncation of
the S3sp side chain provided 25 and 26, which were found to
retain high in vitro potency despite the suboptimal occupancy of
S3sp. Most remarkably, the P3-aminopyridine analogue 27
bearing no side chain at the C4 position still exhibited high in
vitro potency, while the corresponding P3-aniline analogue 28
was 4 and >1000-fold less active in the buffer and plasma renin
assays, respectively. These results suggested that the H-bond
interaction of the P3-pyridyl nitrogen atom with the OH of
Thr77 fully compensates for the lack of binding interactions to
the S3sp cavity. In addition, the more hydrophilic P3-
aminopyridine moiety led to a reduction in the plasma IC50
shift. Compound 27 also showed improved in vitro metabolic
stability and oral bioavailability in the rat. However, 27 (logD
(pH 6.8) = 4.9) showed modest affinity to the hERG channel
as determined by a dofetilide binding assay,20 as compared to
the less hydrophobic compounds 25 (logD (pH 6.8) = 2.2) and
26 (logD (pH 6.8) = 2.1). Therefore, introduction of a polar
Scheme 1. Stereoselective Synthesis of 3,5-Piperidine
Analogues
a
a
Reagents and conditions: (a) MeOH, (DHQ)2AQN, 95%, 63% ee;
(b) recrystallization with (R)-1-phenylethylamine, >99% ee; (c)
MeOH, (DHQD)2AQN, 91%, 75% ee; (d) recrystallization with
(S)-1-phenylethylamine, >99% ee; (e) TcBocCl, Et3N, CH3CN then
ArR1-NH, MgBr2; (f) aq. LiOH, THF; (g) R2-NH2, EDCI·HCl,
HOAt, DMF; (h) HCl in dioxane; (i) R2-NH2, EDCI·HCl, HOAt,
DMF; (j) aq. LiOH, THF; (k) ArR1-NH, BopCl, Et3N, CH2CH2; (l)
HCl in dioxane.
Table 2. SAR Data for Inhibitors Modified at P3−P1, including in Vivo Rat Pharmacokinetics (PK) (i.v. 2 mg/kg, p.o. 6 mg/kg)
b
b
i.v. PK parameters
p.o. PK parameters
renin IC50 (nM)
a
in vitro CLint (μL/min/mg,
CL
Vdss
T1/2
(h)
oral Cmax/dose
(nM/mg/kg)
oral AUCinf./dose
(nM*h/mg/kg)
F
(%)
hERG
IC50
bind.a
(μM)
a
cmpd
(buffer/plasma)
human/rat)
(L/h/kg) (L/kg)
21
22
23
24
25
26
27
28
29
30
31
0.3/2.0
0.3/3.0
0.4/1.0
0.8/6.0
0.2/0.4
0.2/0.6
0.9/1.1
4.0/5340
0.8/2.0
0.6/2.5
0.2/0.6
115/214
61/198
53/96
nd
nd
12
11
nd
4.3
4. 6
7.2
nd
6.1
2.6
4
nd
nd
12
nd
nd
20
19
nd
39
44
45
nd
61
60
61
5.5
7.3
4.8
nd
1.9
4.3
nd
49
>30
>30
3.3
18
93
289/252
15/101
46/62
nd
68
nd
2.4
1.4
3.6
nd
7.0
7.4
6.5
nd
324
690
296
nd
>30
>30
7
155
63
<3.4/13
nd/nd
nd
74
nd
22/142
22/54
2.4
0.74
2.4
7.7
5.9
6.8
511
1630
552
>30
>30
>30
252
136
<3.4/52
a
b
Determined for the HCl salt of each compound. The following salts were used for in vivo PK studies in rat: 22, HCl; 23, HCl; 25, tartrate; 26,
tartrate; 27, fumarate; 30, fumarate; 31, succinate; inhibitor 29 was administered as its free base.
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hydroxyl group to the 4-position of the central piperidine ring
was investigated. Inhibitors 29, 30 and 31 (logD (pH 6.8) =
0.93) all retained the in vitro inhibitory potency against human
renin as well as a favorable hERG profile. These 4-OH
analogues also showed better oral bioavailability compared to
their corresponding nonhydroxylated analogues (25, 26 and 27,
respectively, Table 2).
The synthesis of inhibitors bearing the core (3S,4S,5R)-4-
hydroxy-3,5-piperidine scaffold started from (3S,4S,5R)-32,
which was obtained by the published procedure,21 followed
by recrystallization with (R)-1-phenylethylamine to increase the
enantiomeric excess to >99%. Amide coupling of 32 with the
respective P3−P1 anilines was carried out using acid chloride or
TcBocCl mediated activation of the carboxylic acid function.
Alkaline ester hydrolysis of the resulting intermediate 33,
followed by the second amide coupling step to attach the
prime-site moiety and deprotection afforded inhibitors 36
(Scheme 2).
of S9 mix. Compound 31 exhibited weak inhibitory activity
against hERG, as well as the cardiac sodium (Nav1.5) and L-
type calcium (Cav1.2) channels (IC50s of >30 μM) as measured
by in vitro patch clamp assays. The succinate salt is crystalline
(mp 122 °C), chemically stable in bulk during 3 days at 80 °C,
and highly soluble in water at the pH range between 1 and 9
(>10 mg/mL). Single oral dosing of 31 as its succinate salt to
telemetered double-transgenic rats (dTGRs)22 at 1, 3, 10, 30,
and 100 mg/kg resulted in a dose-dependent and long-lasting
reduction in mean arterial blood pressure (MAP) with peak
changes in MAP of 33, 42, 52, 63, and 66 mmHg, respectively
(Figure 2). Remarkably, a significant blood pressure lowering
effect was still observed 42 h after the 100 mg/kg oral dose was
administered.
The crystal structure of rh-renin in complex with 31 revealed
the ligand to bind in an extended conformation, occupying the
S3 to S2′ site of the enzyme active site (Figure 3). The basic
Scheme 2. Stereoselective Synthesis of 4-Hydroxy-3,5-
a
piperidine Analogues
a
Reagents and conditions: (a) (i) (1-chloro-2-methylpropenyl)-
dimethylamine, CH2Cl2 or TcBocCl, Et3N, THF, (ii) P1−P3 amine,
MgBr2 (for TcBoc method); (b) aq. LiOH, THF; (c) R′-NH2, EDCI·
HCl, HOAt, DMF; (d) HCl in dioxane.
Figure 3. Crystal structure of 31 (shown as a stick model in cyan
color; PDB code 4Q1N) bound to rh-renin. Shown is the solvent
accessible surface of the active site of renin, the catalytic aspartate
residues Asp32 and Asp215, as well as the residues of the flap β-hairpin
with carbon atoms colored in gray. The flap residues Tyr75, Ser76,
Thr77, and Gly78 have been omitted from the protein surface
calculation. The oxygen and nitrogen atoms are colored in red and
blue, respectively.
Inhibitor 31 was selected for further in vitro and in vivo
evaluation based on its overall attractive profile. The compound
was highly selective against a panel of recombinant human
aspartic proteases comprising pepsins A and C, cathepsins D
and E, as well as BACE 1 and BACE 2 (all IC50s >30 μM).
Furthermore, high selectivity over a panel of >60 receptors, ion
channels, and enzymes including CYP3A4, 2C9, and 2D6 (all
IC50 >30 μM) was achieved. Parent 31 as well as the
corresponding aminopyridine P3−P1 fragment were non-
mutagenic in the Ames test, both in the presence and absence
nitrogen atom of the piperidine ring interacts with both
catalytic aspartic acid residues, Asp32 and Asp215, which
Figure 2. In vivo blood-pressure lowering oral efficacy of 31 (succinate salt) in telemetered dTGRs.
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functions as a transition-state surrogate. The 4-hydroxyl group
is H-bonded to Ser76 of the flap being in a closed
conformation.23 The N-cyclopropyl group and the isopropyl-
substituted pyridyl moiety are positioned in the large
contiguous hydrophobic S1/S3 pocket, leaving the S3sp
unoccupied. The pyridyl nitrogen is in close H-bond distance
to the side chain hydroxyl of Thr77 located at the tip of the flap
domain. The data support the notion that this interaction is
indeed crucial for strong binding affinity, in particular for
inhibitors lacking interactions with the S3sp site. The carbonyl
oxygen of the prime-site amide linker forms an H-bond to the
backbone nitrogen of the flap Ser76, while the amide NH
functions as an H-bond donor to the carbonyl oxygen of Gly34.
The sec-butyl makes hydrophobic interactions to S1′, and the
terminal ethoxyethane group occupies the S2′ pocket.
In summary, novel highly potent cis-configured (3R,5S)-
piperidine-based DRIs were derived from a weakly active HTS
hit (compound 1). Structure-based modification of the prime-
site linker and optimization of the P3−P1 and P1′-P2′ portions
guided by X-ray crystallography significantly improved the in
vitro potency. The discovery of the N-alkyl substituted 6-pyridyl
as a novel high affinity P3 scaffold even in the absence of a P3sp
side chain constituted a breakthrough of our extensive
optimization efforts. The additional introduction of a hydroxyl
group on the piperidine core furnished several inhibitors with
improved PK profiles in rats. Inhibitor 31 demonstrated high in
vitro potency and off-target selectivity, an attractive phys-
icochemical and in vitro ADME profile, as well as excellent oral
bioavailability and long elimination half-life in rat. Inhibitor 31
showed high efficacy in the dTGR model with a dose-
dependent sustained blood pressure reduction observed for
more than 24 h after single oral dose administration.
their support in NMR studies. We thank Leslie Bell, Susanne
Glowienke, Martin Traebert, Steven Whitebread, and their
team members for their help in getting the profiling data of 31.
We thank Jason Elliott and Christopher M. Adams for their
critical reading of the manuscript.
ABBREVIATIONS
■
RAAS, renin−angiotensin−aldosterone system; ACE, angio-
tensin converting enzyme; ADME, absorption, distribution,
metabolism, and excretion; DRI, direct renin inhibitor; HTS,
high-throughput screening; rh-renin, recombinant human
renin; Ac2O, acetic anhydride; Boc, tert-butoxycarbonyl;
(Boc)2O, di-tert-butyl dicarbonate; BopCl, bis(2-oxo-3-
oxazolidinyl)phosphinic chloride; DMF, dimethylformamide;
dTGR, double transgenic rat; EDCI·HCl, 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide hydrochloride; Et3N,
triethylamine; EtOAc, ethyl acetate; EtOH, ethyl alcohol;
̀
hERG, the human ether-a-go-go-related gene; HOAT, 1-
hydroxy-7-azabenzotriazol; mp, melting point; PK, pharmaco-
kinetics; RLM, rat liver microsomes; TcBocCl, 2,2,2-trichloro-
1,1-dimethylethyl chloroformate
REFERENCES
■
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ASSOCIATED CONTENT
* Supporting Information
Full experimental details for compounds synthesized, exper-
imental procedures for biological assays, in vivo pharmacoki-
netics, in vivo pharmacology, and X-ray crystallographic
information for the rh-renin−inhibitor complexes. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
*Tel: +65-6722-2931. Fax +65-6722-2982. E-mail: fumiaki.
yokokawa@novartis.com.
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■
Present Addresses
§Novartis Institutes for BioMedical Research, 100 Technology
Square, Cambridge, Massachusetts 02139, United States
⊥Novartis Institute for Tropical Diseases, 10 Biopolis Road,
#05-01 Chromos, Singapore 138670.
(8) Webb, R. L.; Schiering, N.; Sedrani, R.; Maibaum, J. Direct Renin
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(10) Ostermann, N.; Ruedisser, S.; Ehrhardt, C.; Breitenstein, W.;
Marzinzik, A.; Jacoby, E.; Vangrevelinghe, E.; Ottl, J.; Klumpp, M.;
Hartwieg, J. C. D.; Cumin, C.; Hassiepen, U.; Trappe, J.; Sedrani, R.;
Geisse, S.; Bernd Gerhartz, B.; Richert, R.; Francotte, E.; Wagner, T.;
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Hans-Peter Baum and Markus Sanner for IC50
determinations of renin inhibition and Marie-Helene Bellance,
Corinne Durand, and Paul Schultheiss for their technical
assistance. We thank Florence Zink and Allan D’Arcy for
protein crystallization. We also thank Masashi Furuichi and
Oliver Grosche for measuring the melting point and aqueous
solubility of 31 succinate. We acknowledge Virginia Casadas for
her dTGR study with 31, and Charles Babu and Bin Wang for
Kromer, M.; Kosaka, T.; Randy, L.; Webb, R. L.; Rigel, D. F.;
̈
Maibaum, J.; Baeschlin, D. K. A Novel Class of Oral Direct Renin
Inhibitors: Highly Potent 3,5-Disubstituted Piperidines Bearing a
Tricyclic P3−P1 Pharmacophore. J. Med. Chem. 2013, 56, 2196−2206.
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dx.doi.org/10.1021/ml500137b | ACS Med. Chem. Lett. 2014, 5, 787−792