Structural modification of the P2′ position of 2,7-dialkyl- substituted 5(S)-amino-4(S)-hydroxy-8-phenyl-octanecarboxamides: The discovery of aliskiren, a potent nonpeptide human renin inhibitor active after once daily dosing in marmosets

Article abstract of DOI:10.1021/jm070316i

Due to its function in the rate limiting initial step of the renin-angiotensin system, renin is a particularly promising target for drugs designed to control hypertension, a growing risk to health worldwide. Despite vast efforts over more than two decades

Full text of DOI:10.1021/jm070316i

4832
J. Med. Chem. 2007, 50, 4832-4844
Structural Modification of the P2′ Position of 2,7-Dialkyl-Substituted
5(S)-Amino-4(S)-hydroxy-8-phenyl-octanecarboxamides: The Discovery of Aliskiren, a Potent
Nonpeptide Human Renin Inhibitor Active after Once Daily Dosing in Marmosets
Ju¨rgen Maibaum,* Stefan Stutz,† Richard Go¨schke, Pascal Rigollier, Yasuchika Yamaguchi,‡ Fre´de´ric Cumin, Joseph Rahuel,
Hans-Peter Baum, Nissim-Claude Cohen,§ Christian R. Schnell, Walter Fuhrer, Markus G. Gruetter,| Walter Schilling, and
Jeanette M. Wood
NoVartis Institutes for BioMedical Research, NOVARTIS Pharma AG, WKL-136.683, CH-4002 Basel, Switzerland
ReceiVed March 19, 2007
Due to its function in the rate limiting initial step of the renin-angiotensin system, renin is a particularly
promising target for drugs designed to control hypertension, a growing risk to health worldwide. Despite
vast efforts over more than two decades, no orally efficacious renin inhibitor had reached the market. As a
result of a structure-based topological design approach, we have identified a novel class of small-molecule
inhibitors with good oral blood-pressure lowering effects in primates. Further lead optimization aimed for
improvement of in vivo potency and duration of action, mainly by P2′ modifications at the hydroxyethylene
transition-state isostere. These efforts resulted in the discovery of aliskiren (46, CGP060536B, SPP100), a
highly potent, selective inhibitor of renin, demonstrating excellent efficacy in sodium-depleted marmosets
after oral administration, with sustained duration of action in reducing dose-dependently mean arterial blood
pressure. Aliskiren has recently received regulatory approval by the U.S. Food and Drug Administration for
the treatment of hypertension.
Introduction
vasoconstrictor agent and the principal mediator of the RAS
mainly through its interaction with the AT1 receptor. Ang II
also exerts direct action on the proximal tubule to promote
sodium reabsorption and, furthermore, stimulates the adrenal
cortex to secrete aldosterone, which in turn acts upon the distal
nephron to retain sodium, leading to fluid retention.6
Despite the wide availability of many antihypertensive
treatments, hypertension currently affects approximately 1 billion
people worldwide,1 and in 2004, an estimated 45% of patients
being treated in the United States remained uncontrolled.2,3 This
lack of control is particularly concerning because hypertension
is widely accepted today as a key prevalent risk factor for
cardiovascular diseases including stroke and heart and kidney
failure.
As a key regulator of blood pressure and fluid homeostasis,
the renin-angiotensin system (RASa) has long been recognized
as highly attractive for therapeutic intervention and associated
control of blood pressure.4,5 Upon stimulation of the RAS by
various mechanisms, such as decrease in blood pressure,
circulating volume, or plasma sodium, renin is released from
the kidney and acts by cleaving a decapeptide from the
N-terminus of the substrate angiotensinogen to form angiotensin
I (Ang I). Angiotensin converting enzyme (ACE) then acts upon
Ang I, cleaving an additional two amino acids to produce the
octapeptide angiotensin II (Ang II), which is a highly potent
Realized as early as 1956 by Skeggs et al.,7 blockade of the
interaction of renin with its substrate, angiotensinogen, appears
to be the most attractive of the three major targets within the
RAS offering intervention strategies aimed at producing effec-
tive treatments to control hypertension. Angiotensinogen cleav-
age is the first and rate limiting step of the RAS cascade, which
could be fully blocked even in situations of elevated circulating
or tissue active renin, and moreover, renin displays remarkable
specificity for its substrate.5 First introduced to the market for
the effective treatment of hypertension,8 angiotensin converting
enzyme inhibitors (ACEi) may give rise to incidences of
persistent cough and angioedema associated with the broader
substrate specificity of ACE,9 and in addition, ACEi do not block
Ang II formation via ACE-independent pathways.10 Angiotensin
AT1 receptor blockers (ARBs), which interfere at the final stage
of the RAS cascade and inhibit the effects of Ang II independent
of its source (non-renin and non-ACE Ang II generation), have
also become established for the effective management of
cardiovascular diseases.11 However, long-term ARB treatment
results in chronically elevated levels of Ang II and its
biodegradation products, which may exert their own pharma-
cological activities in vivo.12
* To whom correspondence should be addressed. Phone: +41 61
6965560. Fax: +41 61 6961163. E-mail: juergen_klaus.maibaum@
novartis.com.
† Current affiliation: Speedel Experimenta AG, Gewerbestrasse 14, CH-
4123 Allschwil, Switzerland.
‡ Current affiliation: Faculty of Pharmaceutical Sciences, Nagasaki
International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-
3298, Japan.
§ Current affiliation: Synergix, Ltd., Technology Park Malha, Building
1, Jerusalem 91487, Israel.
| Current affiliation: Prof. Markus Gruetter, Biochemisches Institut,
Winterthurerstr. 190, CH-8057 Zu¨rich, Switzerland.
Several peptide-like, second-generation renin inhibitors such
as enalkiren,13 remikiren,14 and zankiren15 were shown in early
investigational clinical trials to be potent in reducing blood
pressure similar to ACEi and ARBs, indicating furthermore that
renin inhibition would be a safe and efficacious way to treat
hypertension. Hence, enalkiren reduced blood pressure to a
slightly greater extent than enalapril, whereas remikiren dis-
played similar efficacy to captopril.16 While these results do
Current affiliation: F. Hoffmann-La Roche, Ltd., Pharmaceuticals
Division, Grenzacherstrasse 124, CH-4070 Basel, Switzerland.
a Abbreviations: ACE, angiotensin converting enzyme; ACEi, ACE
inhibitors; Ang I, angiotensin I; Ang II, angiotensin II; ARBs, angiotensin
AT1 receptor blockers; AT1, angiotensin II type I receptor; dTGR, double-
transgenic rat; MAP, mean arterial blood pressure; ∆MAP, change in mean
arterial blood pressure; RAS, renin-angiotensin system; rh-renin, recom-
binant human renin.
10.1021/jm070316i CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/08/2007

Aliskiren, A Nonpeptide Human Renin Inhibitor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4833
Figure 1. Inhibitor 1 and modification sites.
not make a conclusive case for the benefits of renin inhibitors,
some further preclinical17,18 and clinical19 studies suggest that
renin inhibitors may have also better efficacy against blood-
pressure-independent renal organ damage as compared to
treatment with either an ACEi or an Ang II receptor blocker.
Furthermore, co-treatment with an ACEi and an ARB has
demonstrated enhanced clinical benefits in chronic renal failure
conditions20 and chronic heart failure patients,21 despite no
enhanced effects on lowering blood pressure. It, therefore, could
be hypothesized that a more complete and sustained blockage
of the RAS by a single mechanism, for example through renin
inhibition, may offer a superior therapeutic approach than the
currently available ACEis or ARBs.1,18
reagent and its addition to the N-Boc amino aldehyde 2 of
defined absolute stereochemistry28 resulted in a 4:6 ratio of the
two diastereoisomers 2(S)-3a and 2(R)-3b (53% total yield),
which were inseparable by chromatography on silica gel. The
mixture 3a/3b was completely converted to the mixture of N,O-
acetals 4a/4b by reaction with 2,2-dimethoxy-propane/cat.
p-TsOH over 24 h at rt, which could be separated only with
major effort by chromatography. However, we found that N,O-
acetal formation can be achieved under mild reaction conditions
(15 °C for 1 h) in a highly selective fashion on large scale,
yielding almost exclusively the desired 5(S)-4a (97:3 crude
mixture with 5(R)-4b). This allowed expeditious removal of the
major unreacted 2(R)-3b and traces of 5(R)-4b by flash
chromatography to afford the pure stereoisomer 5(S)-4a. The
absolute 5(S)- and 5(R)-stereochemistry for 4a and 4b, respec-
tively, was tentatively supported by proton NMR, based on
Further clinical development of these and other second
generation renin inhibitors has been hampered in most cases
by their limited oral bioavailability in humans as well as
technical hurdles due to complexity in synthesis of drug
substance.22,23 In a quest for the identification of more promising
renin inhibitor lead scaffolds, an early medium throughput
screening of a smaller corporate compound collection did not
provide attractive hits as new entries into lead optimization, in
contrast to reports by other groups.24,25 Instead, we successfully
developed, as the result of a target enzyme structure-oriented
computational design approach and extensive structure-activity
relationship (SAR) work, several novel, structurally distinct
series of in vitro potent and selective topologically P3-P1-
tethered transition-state mimetic renin inhibitors.26 Common to
all these inhibitors is their important binding interaction to a
narrow nonsubstrate pocket extending from the P3 site of renin,
as discovered by X-ray crystallography of several ligand-
enzyme complexes.27 In the preceding paper, the optimization
of a highly attractive small-molecule lead series has been
described, yielding the first preclinical candidate renin inhibitor
1 of this structure class (Figure 1), which demonstrated potent
blood-pressure lowering activities in sodium-depleted marmosets
after oral dosing.28 We report here a fully detailed account29,30
of our extended SAR exploration in the quest for analogues of
1 with increased in vivo potency and improved oral bioavail-
ability. As a first step toward this goal, a P1′ isopropyl residue
was incorporated into the transition-state isostere moiety of 1,
as this would mimic more closely the scissile dipeptide Leu-
Val portion of the renin substrate7 and, hence, would potentially
lead to improved intrinsic potency in vitro. The stereoselective
access to the advanced N,O-protected acid intermediate 7 then
enabled us to conveniently investigate broadly on modifications
at the P2′ position. Among several potent and selective
analogues, the novel peptidomimetic inhibitor 46 was identified
to be the most efficacious, long-acting, and orally active renin
inhibitor in primates, as reported to date.
3
decoupling/NOE experiments and the vicinal JH,H coupling
constants for the 1,3-oxazolidine moiety,34 as well as by the
observed differences in the chemical reactivity as described
herein; we reasoned that the 5(S)-configured trans-oxazolidine
4a is formed more readily from 2(S)-3a, as compared to the
sterically more demanding cis-isomer 4b from 3(R)-3b.
The benzyl ether 4a was cleaved by hydrogenolysis to afford
alcohol 5, which was then oxidized in a stepwise fashion first
with N-methylmorpholine-N-oxide/TPAP to provide aldehyde
6 and, subsequently, to the carboxylic acid 7 by reaction with
KMnO4/Bu4N+Br- in 58% yield (two steps). Based on various
optimization efforts, this protocol in our hands proved to be
advantageous over the direct transformation of 5 to 7, in
particular, when performed on a larger reaction scale.
The final renin inhibitors (Tables 1-3) were conveniently
prepared by the sequence representatively shown in Scheme 2.
Thus, standard mixed anhydride coupling of a variety of P2′
amines to carboxylic acid 7, using either diethyl cyanophos-
phonate (DEPC) as the activating reagent, or HBTU35 for the
reaction with sterically hindered secondary amines, afforded
carboxamides 8a-f. Final N,O-deprotection was achieved
smoothly in a sequential manner under mild conditions, first
by reaction with catalytic p-TsOH in MeOH to give intermedi-
ates 9, followed by removal of the N-Boc group in 4 N HCl/
dioxane to afford the hydroxyethylene dipeptide isosteres as
amorphous HCl salts. Alternatively, aminolysis of a carboxylic
ester precursor was employed for efficient elaboration of the
SAR at the N-terminal P2′ position, as exemplified by the
transformation of 8c to the carboxamide 8d (Scheme 2).
The carba-analogue inhibitors 59 and 60 (Table 4), both
bearing a 4-methoxybutyl side chain at the phenyl moiety, were
prepared by a different synthetic route (Scheme 3).36 Wittig
reaction of aldehyde 11 with phosphonium bromide 10 afforded
olefin 12 as a mixture of E/Z isomers, which was hydrogenated
in the presence of 5% platinum on carbon to give bromide 13.
Lithiation with n-butyllithium (THF, -78 °C) and subsequent
metal exchange by addition of Mg/1,2-dibromoethane at low
temperature generated the Grignard species of 13. Condensation
with the azido-lactone aldehyde 1436 proceeded to about a 3:1
diastereomeric mixture of inseparable secondary alcohols 15.
Chemistry
The synthesis of the N,O-protected key intermediate acid 7
is detailed in Scheme 1. The tetrahedral hydroxyethylene
dipeptide isostere portion was assembled29,31 by employing the
method reported previously.32 Conversion of the enantiopure
2(S)-bromomethyl-3-methylbutyl-benzyl ether33 to the Grignard

4834 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Scheme 1. Synthesis of the Carboxylic Acid Intermediate 7a
Maibaum et al.
a Reagents and conditions: (a) 2(S)-bromomethyl-3-methylbutyl-benzyl ether, Mg, 1,2-dibromoethane, THF (53%); (b) 2,2-dimethoxypropane, p-TsOH,
CH2Cl2, 24 h, rt (36% for 5(R)-4b and 50% for 5(S)-4a; isolated yields); (c) H2, Pd/C, THF, 15 min (91%); (d) N-methylmorpholine-N-oxide, TPAP,
CH2Cl2, 30 min, quant.; (e) KMnO4, Bu4N+Br-, toluene-H2O, 48 h, 0 °C (58%).
followed by desilylation with Bu4N+F-, afforded 19 and, after
Table 1. In Vitro Activity of Renin Inhibitors with P1′ Methyl versus
i-Propyl Moietya
subsequent removal (4 N HCl/dioxane, 0 °C) of the N-Boc
group, the final inhibitor 60 as an amorphous HCl salt.
The preparation of the P2′ primary amines 23a,b and 27 is
shown in Scheme 4 (see Supporting Information). Aminolysis
of the Cbz-protected ethyl ester 21, readily obtained from
cyanoester 20 via Ra-Ni hydrogenation and subsequent Cbz
protection, with ammonia gas gave the carboxamide 22a in
binding affinity IC50 (nM),
moderate but acceptable yields, although the reaction proceeded
very sluggishly at 70 °C under pressure in an autoclave reactor
renin (pH 7.2)
human
marmoset
plasmac
with methanol as the solvent (no reaction observed in EtOH).
Similarly, 21 was reacted under pressure with NH2Me (60 °C)
in EtOH to afford 22b. Hydrogenation of compounds 22a,b was
performed under pH control (pH 6) to give the crystalline HCl
salts 23a,b. Transformation of the chiral hydroxy ester 24 was
achieved in three steps by O-tosylation, azide displacement, and
H2/Pd reduction to afford amine 27 as HCl salt.
cmpd
R1 )
Me
R2 )
purifiedb plasmac
1d
28
(CH2)3CH3
1
1
1
4
3
8
i-propyl (CH2)3CH3
Me (CH2)2CONH2
i-propyl (CH2)2CONH2
Me (CH2)3CONH2
i-propyl (CH2)3CONH2
29d
30
7
1
17
3
13
6
31d
32
1
0.7
3
0.9
11
3
a Compounds obtained as HCl salts; for details, see Experimental Section.
b Purified human renin, measured at pH 7.2. c Human or marmoset plasma
renin assay; for details, see ref 28. d See ref 28.
Results and Discussion
Replacement of Methyl versus i-Propyl at P1′. The IC50
data shown in Table 1 for a set of representative analogues
bearing either an aliphatic n-butyl or an N-terminal carboxamide
P2′ residue indicated that replacing the methyl group at P1′ of
the dipeptide transition-state isostere by the more bulky iso-
propyl residue (1 and 28, 29 and 30, 31 and 32) resulted in
compounds with at least similar or improved in vitro potencies.
For example, a 3-5-fold increase in binding affinity was
observed for 30 as compared to 29 against human renin. Most
importantly, the more lipophilic P1′ isopropyl analogues re-
vealed very similar enzyme affinities in the human and
marmoset plasma assays, with an only up to 4-fold increase in
IC50 as compared to the purified human renin buffer assay. We
considered the retention of high binding affinity under these
physiologically more relevant assay conditions as a key require-
Removal of the benzylic OH required O-acetylation prior to
hydrogenation, which proceeded to completion in the presence
of Pd/C 5% in EtOH after four days with concomitant reduction
of the azide group. N-Boc protection of the primary amine
afforded intermediate 16 in 48% yield over three steps.
Aminolysis of the lactone 16 with 2-aminoethyl-morpholine in
the presence of catalytic acetic acid37 provided 17 in 86% yield,
which after N-deprotection eventually gave inhibitor 59 as its
dihydrochloride salt.
In contrast, direct lactone-opening with amines 23 (Scheme
4) as their free base under identical conditions surprisingly
proved unsuccessful. Therefore, we adopted the approach
reported by Evans et al.,38 to form the O-TBDMSi-protected
γ-hydroxy carboxylic acid 18 after alkaline hydrolysis of 16
(66% yield, two steps). HBTU coupling of 18 with 23a,

Aliskiren, A Nonpeptide Human Renin Inhibitor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4835
Table 3. In Vitro Activity of Renin Inhibitorsa
Table 2. In Vitro Activity of Renin Inhibitors Bearing Carboxamides at
P2′ Positiona
binding affinity IC50 (nM),
renin (pH 7.2)
human
marmoset
plasma
cmpd
R )
configb purified plasma
33
34
35
36
37
38
39
40
41
42
43
44
45
46
CH2CONH2
CHMeCONH2
CHEtCONH2
CH(CH2OH)CONH2
CMe2CONH2
(CH2)2CONHMe
(CH2)2CONMe2
CHMeCH2CONH2
CHMeCH2CONH2
CH2CHMeCONH2
CH2CHMeCONH2
CH2CHMeCONHMe
CH2CHMeCONHMe
3
4
0.8
2
13
3
3
1
6
1
0.9
1
0.9
0.6
10
12
0.6
3
100
2
7
2
57
1
2
16
13
1
6
200
4
6
3
28
3
2
S
S
S
S
R
S
R
S
2
3
0.6
10
1
2
R
47
0.4
0.7
2
48
49
(CH2)3CONHMe
(CH2)3CONMe2
0.8
1
3
4
3
3
50
51
S
0.6
2
0.9
1
3
2
a Compounds obtained as HCl salts; for details, see Experimental Section.
b Purified human renin, measured at pH 7.2. c Human or marmoset plasma
renin assay; for details, see ref 28. d nd ) not determined.
R
a Compounds obtained as HCl salts; for details, see Experimental Section.
Scheme 2. Final Reaction Sequence to Renin Inhibitorsa
b Absolute configuration at the P2′ moiety.
ment for the identification of inhibitors with high in vivo potency
in our animal model. The new compounds 28, 30, and 32 were
furthermore highly active against marmoset plasma renin (Table
1). As will be discussed later, the P1′ isopropyl inhibitors were
generally found to have a more pronounced potency and longer
duration of action, as compared to their P1′ methyl congeners,
in sodium-depleted renin-activated marmosets after oral admin-
istration.
In Vitro Structure-Activity Relationship at P2′ Position.
We noted in the course of our work28 that N-terminal carboxa-
mides at the P2′ position were particularly favorable in providing
highly potent inhibitors in vitro in the presence of plasma, as
well as oral potency in our pharmacodynamic primate model.
Therefore, and based on the in vitro results for the initial P1′
isopropyl analogues 30 and 32, we focused our attention on
further tuning such P2′ carboxamide moieties, mainly with the
intention of optimizing their in vivo potency and duration of
action. The IC50 values for human and marmoset renin for a
range of closely related unsubstituted versus N-methylated
analogues with modified length and branching of the aliphatic
chain, are summarized in Table 2. The vast majority of these
inhibitors demonstrated very high in vitro binding affinities,
which were even in the subnanomolar range in several cases.
The glycine- and (S)-alanine-derived carboxamides 33 and 34
showed similar activities, whereas for the (S)-ethyl branched
analogue 35 a significant improvement in potency was observed
in particular in the plasma assay. The physiological substrate
of renin, angiotensinogen, contains an Ile residue at P2′,
a Reagents and conditions: (a) H2N-R (HCl salt or free base),
(EtO)2P(O)CN, Et3N, DMF, rt; (b) H2N-R, HBTU, Et3N, MeCN-DMF
(90% for 8f); (c) 8c, NH2Me 33% in EtOH, 40 °C, 40 h (83%); (d) p-TsOH
(hydrate), MeOH, 0 °C or rt; (e) 4 N HCl-dioxane, 0 °C.
suggesting that analogues of 35 with larger and more lipophilic
substituents at this position could provide more potent in vitro
inhibitors. However, we did not investigate this possibility
further in the specific context of this compound series to keep
the molecular weight and overall lipophilicity low. Introducing
a side-chain OH group (36 vs 34) had no impact on the binding
affinity. On the other hand, a marked drop of in vitro potency

4836 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Maibaum et al.
Table 4. In Vitro Activity of Side-Chain Carba Analogue Inhibitors 59
and 60a
mediated H-bonding of the phenoxy ether oxygen to the side
chain hydroxyl of Ser219. In order to probe its contribution to
the binding affinity, the carba-analogues 59 and 60, both bearing
a methoxybutyl chain at P3sp, were synthesized and tested
against human and marmoset renin (Table 4). Both compounds
displayed very similar in vitro potencies in both the buffer and
the plasma enzyme assays as compared to the ether analogues
52 and 46, respectively, thus indicating that the “proximal” ether
oxygen is not critical for binding to the enzyme. Furthermore,
both 59 and 60 were found to be highly potent in vivo with a
long duration of action in reducing blood pressure in marmosets
(vide infra).29
binding affinity IC50 (nM),
renin (pH 7.2)
human
purifiedb
marmoset
plasmac
cmpd
R )
plasmac
0.9
Enzyme Selectivity. All new inhibitors were routinely tested
for their in vitro selectivity against porcine pepsin and bovine
cathepsin D40 and found to be completely inactive at 10 µM
concentrations (data not shown). Inhibitor 46 displayed very
high enzyme selectivity for rh-renin, with IC50 values > 10 µM
for human pepsin A, cathepsin D, cathepsin E, BACE-1, and
other human enzyme family members,41 as well as for the viral
HIV-1 aspartyl protease. Furthermore, 46 revealed high species
specificity for primate renin (human, monkey) and was less
active against plasma renin from various other mammalians,
such as dog, rabbit, pig, guinea pig, and rat plasma renin (IC50
) 7, 11, 150, 63, and 80 nM, respectively).30
Physicochemical Parameters. Experimental data for the
aqueous solubility, the logP octanol-water partition coefficient
at physiological and weakly acidic pH, as well as pKa values
of several potent inhibitors, are listed in Table 5. All compounds
are characterized by a low to moderate lipophilicity, with
logPpH7.4 ranging from 2.1 to 3.1, and furthermore by very high
solubility of their salts in water or in aqueous buffer at neutral
pH 7.4 (>100 g/L for 46 as its hemi-fumarate salt) as well as
high intrinsic solubility for 46 of 0.44 g/L. In this respect, our
newly designed structural class of renin inhibitors revealed an
improved physicochemical profile clearly distinct from that of
many of the previous generation, peptide-like transition-state
mimetics.37 We reasoned that such compound characteristics
of low lipophilicity (particularly low logD) and high intrinsic
aqueous solubility could be of favorable impact on the drug
metabolism and pharmacokinetics (DMPK) profile of these renin
inhibitors. The large volume of distribution observed for 46 in
some species, including human, could be beneficial for a more
target organ specific tissue distribution, such as the kidney, and
hence may, at least partially, contribute to the excellent in vivo
efficacy of 46 (vide infra).42
59
2
1
1
4
60
3
a Compounds obtained as HCl salts; for details, see Experimental Section.
b Purified human renin, measured at pH 7.2. c Human or marmoset plasma
renin assay; for details, see ref 28.
was observed for the isobutyramide 37, which was most striking
when determined in both plasma renin assays.
Extension of the P2′ moiety by one carbon and N-substitution
by one (38) or two methyl groups (39) was of little influence
on the affinities to the enzyme as compared to 33. Similarly,
the impact of branching the â-position by methyl was only
minor, with the expected preference for the (S)- over the (R)-
configuration (40 vs 41). Introduction of a methyl group at the
R-carboxamide position, either with absolute (S)- (42 and 44)
or (R)-configuration (43 and 45) resulted in virtually equipotent
inhibitors independent of N-substitution. However, and most
intriguingly, the corresponding geminal-dimethyl-substituted
carboxamides 46 and 47 demonstrated subnanomolar binding
affinities for both purified and plasma human renin.
Further extension of the linear P2′ chain by one more carbon
(48 and 49) retained the high enzyme affinity (compared to 33
and 39). The conformationally more rigid (S)- and (R)-
configured lactams 50 and 51, respectively, showed very similar
potencies in vitro. It is important to note that most of the
inhibitors listed in Table 2 demonstrated very comparable in
vitro binding affinities toward marmoset renin with low-
nanomolar IC50 values in the presence of plasma.
In vitro data for a series of analogues bearing a basic P2′
morpholine moiety tethered by different alkyl spacers is listed
in Table 3. The selection of these compounds was based on the
results of a more extended SAR undertaken in the context of
the related P1′ methyl dipeptide isostere series.28,39 The ethyl
side-chain derivative 52 appeared to be slightly more potent in
vitro than the propyl analogue 53 toward human renin, but was
equipotent against marmoset renin. The R,R-dimethylated
derivative 54 turned out to have similar affinity to human renin
as 52, albeit being somewhat less active against marmoset renin.
Similar to the findings in the P2′ carboxamide sub-series, a
tertiary carbon adjacent to the transition-state mimetic amide
nitrogen, as in inhibitor 55, dramatically reduced the binding
affinity when compared to the monomethylated derivative 56,
most likely as result of an unfavorable conformational constraint.
As was demonstrated previously,28 acidic functionalities are
well accommodated at the P2′ position. Accordingly, the
carboxylic acid derivative 57 and the tetrazole 58 of the P1′
isopropyl series (Table 3) yielded potent in vitro inhibitors.
P3sp Side-Chain Carba Analogues. The X-ray structure of
46 bound to recombinant human renin (rh-renin; at 2.2 Å
resolution, vide infra) revealed an indirect, water molecule
A broader salt screening program was performed for the
preclinical candidate inhibitor 46 from which the crystalline
hemi-fumarate salt (CGP60536B, SPP100, aliskiren) was identi-
fied to be suitable for formulation.
X-ray Crystallography. The crystal structure of compound
46 has been resolved by cocrystallization of the inhibitor with
recombinant glycosylated human renin at 2.2 Å resolution
(Figure 2a-c).27 We wish to illustrate here some of the key
features of the binding interactions of 46 in more detail, also in
view of the in vitro SAR data that has emerged within this
compound series. In contrast to known second generation
peptide-based renin inhibitors, compound 46 does not interact
with the S2 or S4 binding sites. Instead, its hydrophobic P3-
P1 pharmacophore, comprising of the P1 isopropyl group and
the phenyl spacer substituted by the small polar P3 methoxy
group forming contacts to the side chains of Phe117 and Pro111
at approximately 4 Å, is accommodated nicely by the large
contiguous S3-S1 “superpocket”. The methoxypropoxy side
chain of 46 is binding to the nonsubstrate cavity S3sp, extending

Aliskiren, A Nonpeptide Human Renin Inhibitor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4837
Scheme 3. Synthesis of P3sp Side-Chain Carba-Analogues 59 and 60a
a Reagents and conditions: (a) MeO(CH2)3P+Ph3Br- (10), NaHMDS, 0 °C (80%); (b) Pt/C 5%, THF (76%); (c), 13, n-BuLi, THF, -75 °C, then Mg/
1,2-C2H4Br2, -75 °C; then add 14; (d) Ac2O, Et3N, 4-DMAP, CH2Cl2; (e) Pd/C 5%, EtOH, 4 days; (f) BOC2O, Et3N, CH2Cl2 (48%, 3 steps); (g) NH2-R,
AcOH cat., 80 °C, 24 h (86%); (h) 4 N HCl-dioxane, 0 °C; (i) LiOH, DME-H2O (97%); (j) TBDMSiCl, imidazole, DMF, 6 days (68%); (k) 23a, HBTU,
Et3N, MeCN; (l) n-Bu4NF, THF (87%, 2 steps); (m) 4 N HCl-dioxane, 0 °C.
Scheme 4a
a Reagents and conditions: (a) H2, RaNi, NH3/MeOH 4% (67%); (b) BnOCOCl, 1 M NaHCO3, EtOAc (85%); (c) NH3/MeOH, 70 °C, 300 h (53%), or
NH2Me 33% in EtOH, 70 °C, 8 days (49%); (d) H2, Pd/C 10%, MeOH, dilut. aq HCl (pH stat control; pH 6); (e) TsCl, Et3N, CH2Cl2 (80%); (f) NaN3, DMF,
40-45 °C; (g) H2, Pd/C 10%, THF (35%, 2 steps).
Table 5. Physicochemical Characteristicsa
by approximately 9 Å from the S3 site toward the protein core,
in a similar fashion as reported for inhibitor 1.28 An enzyme-
bound water (W092) at the entrance to S3sp forms a bridging
H-bond to the phenolic oxygen of 46 (3 Å) and the OH of
Ser219 (2.8 Å). However, the similar in vitro potency of the
related inhibitor 60 in which the ether oxygen has been replaced
by a carbon atom (Table 4; compare also 52 vs 59) suggests
only a minor contribution to the overall binding affinity.
Figure 2b illustrates the binding interactions of the hydroxy-
ethylene dipeptide isostere portion of 46 in comparison to the
super-positioned close analogue 1. Strikingly, the complex
structure of 46 revealed a marked shift in the position of the
carbon backbone of the mimetic portion along the axis of the
catalytic cleft as well as significant differences in the conforma-
compound
39b
46c
51
59
2.85
60
log P (pH 7.4)d
log D (pH 6.8)
intrinsic solubility [g/L]
solubilitye [g/L]
pKa
2.48
2.5
0.1
0.44
2.06
3.31
>100 >100 >100
9.4g 9.2 f
9.09
6.01/9.55 9.44
a Determined for amorphous HCl salts, if not otherwise indicated. b As
amorphous monosodium citrate salt. c As crystalline hemi-fumarate salt.
d Apparent octanol-aqueous phosphate buffer partition coefficient at 22 °C.
Determined for 46 by dual-phase potentiometric titration in 0.15 M KCl at
25 °C.53 e Determined in water at 22 °C; for 46, determined in aqueous
phosphate buffer (pH 7.4) at 22 °C. f Potentiometric titration in 0.15 M
KCl at 25 °C.54 g pKa ) 5.7 for citrate.

4838 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Maibaum et al.
Figure 2. (a) X-ray structure of inhibitor 46 (green color) with rh-renin.27 (b) Superposition of the hydroxyethylene transition-state dipeptide
isostere portions (spanning P1 to P1′) of inhibitors 46 (green color) and 1 (orange color) in the catalytic site of renin. Inhibitors are shown in a stick
model with oxygen and nitrogen atoms in red and blue, respectively. The catalytic aspartic acids (D32 and D215) as well as Gly217 are shown in
gray color, selected H-bond interactions are shown as dotted lines using the color code corresponding to that of the inhibitor. (c) Binding interactions
of 46 (green color) to the S2′ subpocket. The n-butyl P2′ group of 1 is superimposed in orange color. All graphs were generated using the PyMOL
software package.43
tion of the center scaffold. As a consequence, the mimetic OH
group gets positioned in a more symmetrical fashion to both
aspartates D32 (H-bond distances to oxygen atoms 3.1 and 2.9
Å, respectively) and D215 (3.5 Å), in contrast to 1 with a
H-bond only to D32.28 Also, the primary NH2 in 46 is within
H-bond distance both to Gly217 (3.0 Å) and Asp215 (2.8 Å).
The P1′ isopropyl group is now ideally positioned to closely fit
to and deeply penetrate into the hydrophobic S1′ site; this is
again in contrast to what is observed for inhibitor 1, which binds
only partially to S1′ (Figure 2b).
The binding mode of the R,R-diMe â-alanine carboxamide
of 46 within the S2′ pocket is shown in Figure 2c. The terminal
CONH2 forms a tight network of direct and solvent-mediated
H-bonds to several amino acids of this pocket, for example,
Arg74 of the flap, Gly34, Gln128, and Thr295, involving two
enzyme-bound water molecules (W010 and W096). The geminal
methyl groups provide additional hydrophobic van der Waals
Figure 3. Changes in ∆MAP after single oral administration of renin
inhibitors at a dose of 3 mg/kg, or vehicle control, to Na+-depleted
freely moving telemetered marmosets. Values are means ( SEM of
the change (4) in MAP from baseline over 24 h after p.o. dosing (time
0) with 1 (HCl salt; n ) 4, orange triangle), 28 (HCl salt; n ) 4, gray
circle), 39 (citrate salt; n ) 6, upside-down blue triangle), 46 (HCl
salt; n ) 6, green square), compound 50 (HCl salt; n ) 3, red diamond),
and vehicle control (n ) 20, black square). Mean baseline arterial blood
pressure and heart rate values in all groups were similar to 81 ( 2
mmHg and 205 ( 8 beats/min observed in the 10 mg/kg po dose group.
contacts to the S2′ site, as compared to the aliphatic n-butyl
chain of 1. Overall, the specific hydrophobic and polar binding
interactions of the unique P2′ pharmacophore are thought to
contribute substantially to the remarkably strong enzyme affinity
of 46, being in our hands one of the most potent inhibitors of
rh-renin discovered to date.
In Vivo Activity in Na+-Depleted Marmosets. The in vitro
potent renin inhibitors from this series were routinely admin-
istered orally, at a 3 mg/kg dose, to conscious, unrestrained Na+-
depleted marmosets to evaluate their efficacy as blood pressure
(BP) reducing agents. In this pharmacological model, systolic,
diastolic, and mean arterial pressures (MAPs) and heart rate
were noninvasively and continuously recorded over a 24 h time
period using an implanted telemetry device.44 Time courses of
changes (∆) in MAP values from baseline for the test com-
pounds and vehicle were compared. Several compounds, as
exemplified in Figure 3 for inhibitors 28, 39, 46, and 50, were
found to be highly potent in this renin-dependent normotensive
primate model, leading to a pronounced and sustained reduction
in ∆MAP. Inhibitor 28 bearing a P1′ isopropyl residue showed
a peak ∆MAP of -20 mmHg 1-3 h post-dose, which appeared
to be significantly more potent, albeit not longer acting, as
compared to the corresponding P1′ methyl analogue 1. Interest-

Aliskiren, A Nonpeptide Human Renin Inhibitor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4839
ingly, we observed a consistent trend for a markedly stronger
effect on BP and a longer duration of action for the respective
P1′ isopropyl versus methyl analogues (data not shown), which
were considered to be likely due to differences in the pharmaco-
kinetics profiles of the corresponding analogues.
primate model, which used conscious, unrestrained, sodium-
depleted marmosets, the oral administration of inhibitor 46
resulted in a very pronounced (-30 mmHg), sustained (at 24
h) drop in MAP. This reduction in blood pressure was paralleled
by a complete blockage of PRA that lasted for at least 24 h.
Inhibitor 46 was found to display very high enzyme selectivity
for rh-renin, as well as for primate renin and was less active
against plasma renin from other mammalian sources. Many of
the newly designed class of inhibitors, described herein,
exhibited low to medium lipophilicity with high aqueous
solubility and were, therefore, clearly distinct from many of
the previous generations of described peptide-like mimetics. We
conclude that these physical characteristics may, at least in part,
be responsible for the excellent observed in vivo efficacies.
In summary, inhibitor 46 proved to be among the most potent
renin inhibitors after oral administration that has as yet been
described. Based upon its excellent overall pharmacological
profile, 46 has been selected for clinical trials, has recently
received regulatory approval by the U.S. Food and Drug
Administration (FDA) for the treatment of hypertension, and
has become the first marketed orally active renin inhibitor.
Some of the most potent in vivo and long-acting inhibitors
were identified from the P2′ carboxamide sub-series. Inhibitors
39 and 50 (and similarly 43, 45, 47, as well as the P3sp side
chain carba analogues 59 and 60; data not shown) induced
similar peak BP reduction as 1 and 28, but revealed a clearly
more sustained pharmacological activity, because blood pressure
levels did not fully recover to baseline even 24 h post-dose
(Figure 3). Compound 46 (as its HCl salt) was found to be most
potent, with a rapid onset of action leading to a remarkable peak
∆MAP of -30 mmHg 1 h after dosing. This inhibitor also
revealed a very prolonged activity, with MAP being still lowered
by -10 mmHg at the 24 h time point and, hence, appeared to
be one of the longest acting inhibitors in this primate model.
The absolute oral bioavailability of 46 in non-Na+-depleted
marmosets, dog, and rat was determined to be 16, 32, and 2.4%,
respectively, as determined from plasma concentrations using
an enzymatic assay.45-47 All analogues bearing an acidic
functionality at P2′ (cf. Table 3) were completely inactive in
vivo, suggesting a poor bioavailability profile in marmosets.
The extent of ∆MAP reduction measured in Na+-depleted
marmosets paralleled the % inhibition of plasma renin activity
(PRA),48 thus strongly indicating that the pharmacological effect
was mechanistically related to renin inhibition. For example,
PRA was completely blocked over the initial 6 h after giving a
3 mg/kg oral dose of inhibitor 28, with significant reduction in
∆MAP observed (Figure 3), and was only partially inhibited
by 79% at 24 h when BP had basically recovered to normal
levels. On the other hand, PRA was completely inhibited over
the 24 h period for all inhibitors having a longer duration of
action including 46, which can be most likely attributed to more
extended in vivo plasma and/or tissue exposures of these
compounds.
The blood pressure lowering effects of inhibitor 46 as its
hemi-fumarate salt has been more extensively investigated in
sodium-depleted marmosets after single and multiple doses over
8 days,30,46 in spontaneously hypertensive rats (SHR) following
subchronic administration for 14 days,46 and in double-trans-
genic rats (dTGR) expressing human renin and human angio-
tensinogen.49 In these studies in sodium-depleted marmosets,
orally administered 46 reduced blood pressure dose-dependently
and proved to be more effective and long-lasting than two
known orally active peptide-like renin inhibitors that have been
in clinical trials previously. Furthermore, 46 was shown to be
at least as effective in reducing MAP as an ACE inhibitor and
an AT1 receptor blocker.46 Based on its overall characteristics
and preclinical in vitro/in vivo profile, inhibitor 46 (hemi-
fumarate salt) was selected as a candidate for further develop-
ment in clinical trials.
Experimental Section
General methods for experimental and analytical procedures and
for purifications can be found in the Supporting Information.
Noncommercial amines used for the coupling to acid intermediate
7 or for aminolysis of 8c and related analogues, and which are not
mentioned explicitly in the section below, were prepared according
to our preceding paper28 or to standard literature procedures.
(1S,2S,4S,2′S and 1S,2R,4S,2′S)-(4-Benzyloxymethyl-2-hy-
droxy-1-{2′-[4-methoxy-3-(3-methoxy-propoxy)-benzyl]-3-meth-
yl-butyl}-5-methyl-hexyl)-carbamic Acid tert-Butyl Ester (3a
and 3b). To Mg turnings (51.1 g, 2.10 mol) in THF (1.4 L) at 55
°C was added over 30 min a solution of 2(S)-bromomethyl-3-
methyl-butyl-benzylether33 (380 g, 1.40 mol) and 1,2-dibromoethane
(30.2 mL, 0.35 mol) in THF (0.8 L). Stirring was continued at 55
°C for 20 min, followed by the dropwise addition of crude aldehyde
228 (190 g, theoretical 0.42 mol) in THF (0.7 L) at 5 °C. The mixture
was stirred for 3 h at ambient temperature and, after cooling to 5
°C, was quenched by the addition of a saturated NH4Cl solution (2
L). Extractive workup with Et2O and FC purification (EtOAc/
hexane l:3) afforded a 4:6 ratio of inseparable diastereomers
(1S,2S,4S,2′S)-3a and (1S,2R,4S,2′S)-3b (139 g, 53%) as a colorless
oil: TLC Rf 0.26 (AcOEt/hexane l:2); HPLC tR 22.7/22.8 min (peak
1
ratio 6:4); H NMR (CDCl3, 360 MHz, most of the signals were
doubled) δ 0.78 (m, 12H), 1.1-1.7 (m, 9H), 2.00 (m, 2H), 2.28
(m, 1H), 2.60 (m, 1H), 3.27 (s, 3H), 3.3-3.6 (m, 4H), 3.75 (s,
3H), 3.95-4.2 (m, 4H), 4.4-4.5 (m, 2H), 4.65 (d, J ) 12, 0.6H),
4.73 (d, J ) 12, 0.4H), 6.6-6.8 (m, 3H), 7.1-7.3 (m, 5H).
(4S,5S,2′S,2′′S)- and (4S,5R,2′S,2′′S)-5-(2′-Benzyloxymethyl-
3-methyl-butyl)-4-{2′′-[4-methoxy-3-(3-methoxy-propoxy)-ben-
zyl]-3-methyl-butyl}-2,2-dimethyl-oxazolidine-3-carboxylic Acid
tert-Butyl Ester (4a and 4b). A solution of 3a, 3b (7.0 g, 11.1
mmol), 2,2-dimethoxypropane (10.9 mL, 88.9 mmol), and catalytic
p-TsOH‚H2O (5 mg) in CH2Cl2 (70 mL) was stirred for 24 h at
ambient temperature. Evaporation and FC purification of the residue
(CH2Cl2/EtO2 96:4) afforded both pure diastereoisomers 4a (3.72
g, 50%) and 4b (2.68 g, 36%) as colorless oils. Data for
(4S,5S,2′S,2′′S)-4a: TLC Rf 0.35 (CH2Cl2/EtO2 95:5); HPLC tR 27.7
Conclusions
The structural modification of the P1′ and P2′ group of the
parent nonpeptide transition state renin inhibitor 1 had little
influence on the observed in vitro IC50 values (plasma assays),
however, this did provide compounds with significantly im-
proved oral potency when measured in vivo. Several inhibitors
that exhibited moderate lipophilicity and high water solubility
were found to be very efficacious in reducing mean arterial
blood pressure in salt-depleted, telemetered marmosets with a
long duration of action. In particular, in our renin-dependent
1
min; H NMR (CDCl3, 360 MHz) δ 0.8-0.9 (m, 12H), 1.15-1.9
(m, 10H), 1.42 (s, 3H, CH3), 1.49 (s, 9H, C(CH3)3), 1.55 (s, 3H,
CH3), 2.07 (m, 2H, CH2CH2O), 2.3-2.6 (m, 2H, ArHaHb), 3.35
(s, 3H, OCH3), 3.41 (m, 1H, CHOH), 3.38 (t, J ) 7, 2H, OCH2),
3.6-3.8 (br m, 1H, CHN), 3.81 (s, 3H, OCH3), 4.06 (t, J ) 7, 2H,
OCH2), 4.44 (s, 2H, OCH2C6H5), 6.6-6.8 (m, 3H, arom), 7.2-
7.35 (m, 5H, arom). Data for (4S,5R,2′S,2′′S)-4b: TLC Rf 0.46
1
(CH2Cl2/EtO2 95:5); HPLC tR 28.1 min; H NMR (CDCl3, 360
MHz) δ 0.78 (d, J ) 7, 3H, CH3), 0.79 (t, J ) 7, 3H, CH3), 0.86

4840 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Maibaum et al.
(d, J ) 7, 3H, CH3), 0.91 (d, J ) 7, 3H, CH3), 1.17 (m, 1H, CH),
1.25-1.8 (m, 20H), 1.91 (m, 1H, ArCH2CH), 2.09 (m, 2H,
CH2CH2O), 2.20 (m, 1H), 2.47 (br m, 1H, ArHaHb), 2.95 (m, 1H,
ArHaHb), 3.35 (s, 3H, OCH3), 3.41 (m, 1H, CHOH), 3.45 (m, 1H,
CHN), 3.57 (t, J ) 7, 2H, OCH2), 3.82 (s, 3H, OCH3), 4.05-4.2
(m, 2H, CHCH2O), 4.12 (t, J ) 7, 2H, OCH2), 4.49 (br s, 2H,
OCH2C6H5), 6.65-6.8 (m, 2H, arom), 6.97 (br s, 1H, arom), 7.2-
7.4 (m, 5H, arom).
1.45 (s, 9H), 1.49 (s, 3H), 1.93 (t, J ) 7, 2H), 1.25-2.00 (m, 7H),
2.25-2.3 (m, 1H), 2.3-2.6 (m, 2H), 3.00 (br s, 1H), 3.26 (s, 3H),
3.49 (t, J ) 7, 2H), 3.6-3.7 (m, 2H), 3.75 (s, 3H), 4.01 (t, J ) 7,
2H), 6.69 (dd, J ) 2, 8, 1H), 6.77 (d, J ) 2, 1H), 6.86 (d, J ) 8,
1H). Anal. (C33H55NO8) C, H, N.
Compound 9e. The mixture of 7 (8.9 g, 15.0 mmol), Et3N (5.02
mL, 36.0 mmol), 3-amino-2,2-dimethyl-propionamide, HCl salt
(23a; 2.75 g, 18.0 mmol), and DEPC (95%, 2.87 mL, 18.0 mmol)
in DMF (360 mL) was reacted overnight at rt as described for 8a
(see Supporting Information). The mixture was concentrated, and
the residue was partitioned between a saturated aqueous NaHCO3
solution (40 mL) and EtOAc (3 × 40 mL). The combined organics
were washed with brine, dried (MgSO4), and concentrated in vacuo.
FC purification (EtOAc/hexane 1:2) afforded 8e (10.4 g) as a white
Selective N,O-protection of the desired diastereoisomer 3a on a
larger reaction scale was achieved by the following procedure: To
a cooled solution of stereoisomers 3a,b (4:6 ratio; 186 g, 0.295
mol) in CH2Cl2 (1.86 L) were added 2,2-dimethoxypropane (290
mL, 2.362 mol) and p-TsOH‚H2O (250 mg) by keeping the reaction
temperature at 15 °C (slightly exothermic; the proceeding of the
reaction was checked by analytical RP-HPLC to keep formation
of 4b to a minimum). After stirring for 60 min, Et3N (0.5 mL) was
added to the mixture, which was then concentrated at 30 °C under
reduced pressure. The residue was flash chromatographed on silica
gel (2.4 kg, CH2Cl2/t-BuOMe gradient 96:4 to 1:2) to afford a 93:7
mixture of products 4a and 4b (total 128 g), respectively, besides
unreacted 3b (55 g, pale yellow oil). From this product mixture,
diastereometrically pure 4a (97.2 g, 49%) and 4b (7.5 g) were
isolated by repeated FC on silica gel (CH2Cl2/t-BuOMe 96:4).
(4S,5S,2′S,2′′S)-5-(2′-Hydroxymethyl-3-methyl-butyl)-4-{2′′-
[4-methoxy-3-(3-methoxy-propoxy)-benzyl]-3-methyl-butyl}-2,2-
dimethyl-oxazolidine-3-carboxylic Acid tert-Butyl Ester (5). A
solution of 4a (3.70 g, 5.52 mmol) in THF (50 mL) was
hydrogenated in the presence of 5% palladium on charcoal (1.0 g)
for 15 min at ambient temperature. The catalyst was removed by
filtration over Hyflo, the combined filtrates were concentrated, and
the residue was purified by FC (EtOAc/hexane 1:2) to afford 5
1
foam: TLC Rf 0.27 (CH2Cl2/MeOH 95:5); H NMR (DMSO-d6,
80 °C; 360 MHz) δ 0.85-0.91 (m, 12H, 4CH3), 1.07 (s, 3H, (CH3)2-
CONH2), 1.08 (s, 3H, (CH3)2CONH2), 1.2-1.85 (m, 7H, aliphat),
1.33 (s, 3H, CH3), 1.45 (s, 9H, C(CH3)3), 1,51 (s, 3H, CH3), 1.93
(m, 2H, CH2CH2O), 2.24 (m, 1H, CHCONH), 2.33 (m, 1H,
ArCHaHb,), 2.45-2.5 (m, 1H, ArCHaHb; signal overlapped), 3.15
(dd, J ) 6, 13, 1H, CONHCHa), 3.26 (s, 3H, OCH3), 3.33 (dd, J
) 6, 13, 1H, CONHCHb), 3.49 (t, J ) 6.4, 2H, OCH2), 3.57 (m,
1H, CHOH), 3.55 m, 1H, CHNH), 3.74 (s, 3H, OCH3), 4.03 (t, J
) 6.5, 2H, ArOCH2), 6.66 (br s, 2H, CONH2), 6.71 (dd, J ) 2, 8,
1H, arom), 6.79 (d, J ) 2, 1H, arom), 6.84 (d, J ) 8, 1H, arom),
7.20 (br t, 1H, CONH).
A solution of 8e (10.3 g, 14.9 mmol) and a catalytic amount of
p-TsOH‚H2O (0.10 g) in absolute MeOH (210 mL) was stirred at
0 °C overnight. Extractive workup, FC purification on silica gel
(400 g; eluant: CH2Cl2-MeOH 9:1), and recrystallization from
Et2O-hexane (1:1, 40 mL) afforded the title compound 9e (7.70
g, 79% over 2 steps) as a white solid: mp 141-142 °C; TLC Rf
0.45 (CH2Cl2/MeOH 9:1); 1H NMR (DMSO-d6, 360 MHz) δ 0.74
(d, J ) 6.6, 3H, CH3), 0.75 (d, J ) 6.6, 3H, CH3), 0.84 (d, J )
6.6, 6H, 2CH3), 1.04 (s, 6H, (CH3)2CONH2), 1.15-1.7 (m, 7H,
aliphat), 1.40 (s, 9H, C(CH3)3), 1.91 (m, 2H, CH2CH2O), 2.18-
2.25 (m, 2H, ArCHaHb, CHCONH), 2.61 (m, 1H, ArCHaHb), 3.12
(dd, J ) 6, 13.3, 1H, CONHCHa), 3.24 (s, 3H, OCH3), 3.25-3.35
(m, 2H, CH, CONHCHb; signals overlapped), 3.46 (t, J ) 6.3, 2H,
OCH2), 3.45-3.55 (m, 1H, CHOH), 3.71 (s, 3H, OCH3), 3.95 (t,
J ) 6.5, 2H, ArOCH2), 4.30 (d, J ) 6.2, 1H, OH), 6.15 (d, J )
9.8, 1H, BOC-NH), 6.64 (m, 1H, arom), 6.75-6.85 (m, 3H, arom,
CONHaHb), 7.10 (br s, 1H, CONHaHb), 7.38 (t, J ) 6, 1H, CONH).
Anal. (C35H61N3O8) C, H, N.
(2S,4S,5S,7S)- 5-Amino-4-hydroxy-2-isopropyl-7-[4-methoxy-
3-(3-methoxy-propoxy)-benzyl]-8-methyl-nonanoic Acid Butyl-
amide, Hydrochloride Salt (28). A solution of 9a (2.45 g, 4.02
mmol) in dioxane (10 mL) was concentrated to a volume of 5 mL,
cooled to 0 °C in an ice bath, and 4 N HCl-dioxane (30 mL) was
added dropwise. Stirring was continued for 3 h at 0 °C, followed
by lyophilisation of the white suspension in high vacuo overnight.
The residue was dissolved in CH2Cl2, filtered through a glass fiber
filter, the solvent was evaporated, and the residue was dried in high
vacuo at rt to give title compound 28 (2.15 g, 96%) as an amorphous
solid: TLC Rf 0.31 (CH2Cl2/MeOH 9:1); HPLC tR 14.7 min (100%);
1H NMR (DMSO-d6, 500 MHz) δ 0.7-0.9 (m, 15H, 5CH3), 1.2-
1.45 (m, 7H, aliphat), 1.5-1.7 (m, 3H, aliphat), 1.78 (m, 1H,
ArCH2CH), 1.92 (m, 2H, CH2CH2O), 2.16 (m, 1H, CHCONH),
2.38 (dd, J ) 8, 13.5, 1H, ArCHa), 2.44 (dd, J ) 7, 13.5, 1H,
ArCHb), 2.68 (m, 1H, CHNH3+), 2.96 (m, 1H, CONHCHa), 3.11
(m, 1H, CONHCHb), 3.20-3.25 (m, 1H, CHOH), 3.27 (s, 3H,
OCH3), 3.46 (t, J ) 6.3, 2H, OCH2), 3.71 (s, 3H, OCH3), 3.97 (t,
J ) 6.5, 2H, ArOCH2), 5.31 (br s, 1H, OH; exchangeable), 6.69
(dd, J ) 1.7, 8, 1H, arom), 6.80 (d, J ) 1.7, 1H, arom), 6.82 (d, J
) 8, 1H, arom), 7.3-7.9 (br s, 3H, NH3+), 7.83 (t, J ) 5.5, 1H,
NH amide); HRMS m/z 509.3973 [(M + H)+ calcd for C29H53N2O5+,
509.3954]. Anal. (C29H52N2O5‚HCl‚0.64H2O) C, H, N; Cl: calcd,
6.37; found, 7.09.
(2.92 g, 91%) as a colorless oil: TLC Rf 0.28 TLC (AcOEt/hexane
1
1:2); HPLC tR 23.1 min; [R]20 +44.3 (c 1.3, CHCl3); H NMR
D
(CDCl3) δ 0.8-1.05 (m, 12H), 1.48 (s, 9H), 1.2-1.9 (m, 15H),
2.09 (m, 2H), 2.2-2.8 (m, 2H), 3.35 (s, 3H), 3.45-3.65 (m, 6H),
3.84 (s, 3H), 4.10 (m, 2H), 6.67 (d, J ) 8, 1H), 6.73 (s, 1H), 6.78
(d, J ) 8, 1H). Anal. (C33H57NO7) H, N; C: calcd, 68.36; found,
67.78.
(4S,5S,2′S,2′′S)-5-(2′-Formyl-3-methyl-butyl)-4-{2′′-[4-meth-
oxy-3-(3-methoxy-propoxy)-benzyl]-3-methyl-butyl}-2,2-dimethyl-
oxazolidine-3-carboxylic Acid tert-Butyl Ester (6). To a solution
of 5 (53.0 g, 91.4 mmol) in CH2Cl2 (1.8 L) was added N-methyl-
morpholine-N-oxide (16.6 g, 137 mmol) and molecular sieves 0.3
nm (100 g). After 10 min, tetrapropylammonium-perruthenate (1.60
g, 4.55 mmol) was added, and the mixture was stirred for another
30 min at ambient temperature. After removal of the molecular
sieves by filtration, the mixture was diluted with CH2Cl2 and then
subsequently washed with a 2 M solution of Na2SO3, brine, a 1 M
solution of CuSO4, and again with brine. The dried (MgSO4)
organics were evaporated and dried in vacuo to yield crude aldehyde
6 (53 g, used in the next step without further purification): TLC
Rf 0.43 (EtOAc/hexane 1:2); HPLC tR 23.8 min; [R]20 +76.1 (c
D
1.2, CHCl3); 1H NMR (CDCl3) δ 0.9-1.0 (m, 12H), 1.48 (s, 9H),
1.2-1.9 (m, 14H), 2.09 (m, 2H), 2.15-2.65 (m, 3H), 3.35 (s, 3H),
3.45-3.7 (m, 1H), 3.57 (t, J ) 7, 2H), 3.83 (s, 3H), 4.10 (m, 2H),
6.65-6.8 (m, 3H), 9.67 (s, 1H).
(4S,5S,2′S,2′′S)-5-(2′-Carboxy-3-methyl-butyl)-4-{2′′-[4-meth-
oxy-3-(3-methoxy-propoxy)-benzyl]-3-methyl-butyl}-2,2-dimethyl-
oxazolidine-3-carboxylic Acid tert-Butyl Ester (7). To a solution
of 6 (53 g, theor. 91.4 mmol) in toluene (470 mL) was added water
(470 mL), KMnO4 (79.1 g, 500 mmol), and n-Bu4NBr (9.7 g, 30
mmol) at 0 °C. After 48 h, a 10% aqueous solution of Na2SO3 (1.2
L) was added dropwise by keeping the temperature at 0-10 °C.
The colored mixture was stirred for 30 min (aqueous phase pH ∼
12), followed by addition of a 10% solution of citric acid
monohydrate (1.95 L) and water (1.2 L). The aqueous layer was
extracted with EtOAc (3 × 2.5 L), and the combined organics were
washed with water and brine, dried (MgSO4), and concentrated.
Purification by FC (EtOAc/hexane 3:7) afforded 7 (31.7 g, 58%)
as an oil: TLC Rf 0.27 (EtOAc/hexane 1:2); HPLC tR 22.3 min;
1H NMR (DMSO-d6, 80 °C) δ 0.9-0.95 (m, 12H), 1.33 (s, 3H),
Compound 39, Mono-Na-Citrate Salt. Compound 9b (0.86 g,
1.32 mmol), dissolved in 4 N HCl-dioxane (17 mL), was stirred
at 0 °C for 2 h, followed by freeze-drying, to afford the

Aliskiren, A Nonpeptide Human Renin Inhibitor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4841
hydrochloride salt of compound 39 as a white solid. This material
was dissolved in ice-cold 1 N NaOH (50 mL), and the aqueous
layer was extracted with Et2O (3 × 70 mL). The combined organics
were washed with water and brine, dried (MgSO4), and concentrated
to afford the free base of 39 as a pale yellow oil. To a solution of
this material (0.72 g, 1.31 mmol) in EtOH (50 mL) was added citric
acid monohydrate (0.274 g, 1.31 mmol) at rt. After stirring for 10
min, water (50 mL) and 1 N NaOH (1.31 mL, 1.31 mmol) were
added. Then volatiles were removed in vacuo at 30 °C, the water
phase (ca. 60 mL) was filtered through a glass fiber filter to remove
remaining particles, and the filtrates were lyophilised overnight.
The residue was then taken up in Et2O (60 mL) and stirred for 3 h
at rt and the precipitate was filtered. After drying in high vacuo at
30 °C overnight, the title compound 39, monosodium citrate salt,
(0.70 g, 69%) was obtained as white powder: TLC Rf 0.38 (CH2Cl2/
MeOH 8:2); HPLC tR 11.4 min (99.2%); 1H NMR (DMSO-d6, 80
°C; 500 MHz) δ 0.81-0.87 (m, 12H), 1.27-1.37 (m, 3H), 1.6-
1.68 (m, 2H), 1.70 (m, 1H), 1.76 (m, 1H), 1.92 (m, 2H), 2.08 (m,
1H), 2.4-2.58 (m, 14H), 3.12-3.18 (m, 1H), 3.25 (s, 3H), 3.2-
3.3 (m, 1H; overlapped), 3.3-3.4 (m, 2H), 3.48 (t, J ) 6.3, 2H),
3.73 (s, 3H), 4.00 (t, J ) 6.5, 2H), 6.71 (dd, J ) 2, 8, 1H), 6.77 (d,
J ) 2, 1H), 6.83 (d, J ) 8, 1H), 7.36 (br t, 1H); HRMS m/z
552.4014 [(M + H)+ calcd for C30H53N3O6+, 552.4012]. Anal.
(C36H60NaN3O13‚0.59H2O) C, H, N.
continued overnight at rt. The mixture was concentrated and the
residue was partitioned between EtOAc and satd aq NaHCO3
solution. The combined organics were washed with brine, dried
(MgSO4), and concentrated, followed by FC purification (CH2Cl2/
MeOH 95:5), to afford compound 8f as a colorless oil (132 mg,
90%): TLC Rf 0.47 (CH2Cl2/MeOH 95:5); HPLC tR 19.0 min
1
(97.8% purity); H NMR (DMSO-d6, 500 MHz) δ 0.8-0.9 (m,
12H), 1.19 (s, 3H), 1.20 (s, 3H), 1.15-1.55 (m, 3H), 1.32 (s, 3H),
1.42 (s, 9H), 1.48 (s, 3H), 1.6-1.75 (m, 4H), 1.90 (m, 2H), 2.14
(m, 1H), 2.2-2.55 (m, 8H), 3.22 (s, 3H), 3.45 (t, J ) 6.5, 2H),
3.45-3.65 (m, 6H), 3.70 (s, 3H), 3.98 (m, 2H), 6.66 (dd, J ) 1.5,
8, 1H), 6.77 (d, J ) 1.5, 1H), 6.80 (d, J ) 8, 1H), 7.06 (s, 1H,
NH). FAB-MS m/z 734 (M + H)+. The thus obtained 8f (128 mg,
0.174 mmol), dissolved in MeOH (5 mL), was stirred in the
presence of p-TsOH‚H2O (32 mg, 0.20 mmol) overnight at rt. After
evaporation of the solvent, the residue was taken up in CH2Cl2,
the organic layer was washed with ice-cold 0.1 N NaOH, filtered
through cotton wool, and concentrated. FC purification (CH2Cl2/
MeOH 95:5) gave the title compound 9f (99 mg, 82%) as colorless
foam: TLC Rf 0.54 (CH2Cl2/MeOH 9:1); HPLC tR 15.4 min (>98%
1
purity); H NMR (DMSO-d6, 500 MHz) δ 0.75 (t, J ) 6.5, 6H),
0.84 (t, J ) 6.5, 6H), 1.10 (m, 1H), 1.20 (s, 3H), 1.21 (s, 3H),
1.15-1.6 (m, 5H), 1.39 (s, 9H), 1.64 (m, 1H), 1.90 (m, 2H), 2.11
(m, 1H), 2.25 (m, 1H), 2.4-2.6 (m, 7H), 3.2-3.3 (m, 1H), 3.23
(s, 3H), 3.43-3.5 (m, 1H), 3.45 (t, J ) 6.5, 2H), 3.53 (m, 4H),
3.70 (s, 3H), 3.95 (t, J ) 6.5, 2H), 4.18 (d, J ) 6, 1H, OH), 6.04
(d, J ) 9.8, 1H, NH), 6.64 (dd, J ) 1.5, 8, 1H), 6.75 (d, J ) 1.5,
1H), 6.78 (d, J ) 8, 1H), 6.92 (s, 1H, NH). FAB-MS m/z 695 (M
+ H)+.
(2S,4S,5S,7S)-5-Amino-4-hydroxy-2-isopropyl-7-[4-methoxy-
3-(3-methoxy-propoxy)-benzyl]-8-methyl-nonanoic Acid (2-Car-
bamoyl-2-methyl-propyl)-amide, Hydrochloride Salt (46). A
solution of 9e (7.60 g, 11.7 mmol) in 4 N HCl-dioxane (100 mL)
was stirred at 0 °C for 2 h. The mixture was concentrated under
reduced pressure at rt to a small volume, which was lyophilised
overnight. The residue was dissolved in water (200 mL), filtered
through a glass fiber filter, and the combined filtrates were freeze-
dried in high vacuo overnight at ambient temperature to yield the
title compound 46, HCl salt, (7.00 g) as a hygroscopic white
powder: TLC Rf 0.33 (CH2Cl2/MeOH 8:2); HPLC tR 12.7 min
(3S,5S,1′S,3′S,1′′R and 3S,5S,1′S,3′S,1′′S)-5-(1′-Azido-3′-{1′′-
hydroxy-[4-methoxy-3-(4-meth-oxy-butyl)-phenyl]-methyl}-4-
methyl-pentyl)-3-isopropyl-dihydro-furan-2-one (15). To the
solution of bromide 13 (8.94 g, 32.7 mmol) in THF (70 mL) was
added over 15 min at -75 °C a 1.5 M solution of n-BuLi in hexane
(20.8 mL, 31.2 mmol), followed by stirring for 1 h at -75 °C. To
a stirred suspension of Mg powder (1.14 g, 46.7 mmol) in THF
(18 mL) was added over 10 min a solution of 1,2-dibromoethane
(4.03 mL 46.7 mmol) in THF (40 mL) at rt, and stirring was
continued for 1 h. To this suspension was added, after cooling to
-75 °C, via syringe, the clear solution obtained above. After 1 h,
a solution of aldehyde 1436 (4.38 g, 15.58 mmol) was added
dropwise by keeping the temperature below -65 °C. After 40 min
at -65 °C, the reaction mixture was poured into an ice-cooled
saturated aqueous NH4Cl solution (400 mL), followed by extraction
of the water layer with EtOAc. Standard workup and FC purification
(EtOAc/hexane 1:4) gave the title compound in the form of a 3:1
ratio of inseparable diastereoisomers (7.41 g) as a colorless oil:
TLC Rf 0.24 (EtOAc/hexane 1:4); HPLC tR 19.1 min (26%) and
20.1 min (72%); IR (CH2Cl2) 3600 (OH), 2110 (N3), 1770 (CO);
1H NMR (CDCl3, 300 MHz) δ 0.78-2.43 (m, 24H), 2.61-2.68
(m, 3H), 3.33 (s, 3H), 3.40 (t, J ) 6, 2H), 3.64-3.70 (m, 0.75H),
3.82 (s, 3H), 3.98-4.02 (m, 0.25H), 4.35-4.42 (m, 1H), 4.44 (dd,
J ) 9, 3.5, 0.25H), 4.59 (dd, J ) 8, 2, 0.75H), 6.80-6.84 (m, 1H),
7.09-7.18 (m, 2H); FAB-MS, m/z 430 [M - N2 - OH]+.
(1S,3S,2′S,4′S)-{1-(4-Isopropyl-5-oxo-tetrahydro-furan-2-yl)-
3-[4-methoxy-3-(4-methoxy-butyl)-benzyl]-4-methyl-pentyl}-car-
bamic Acid tert-Butyl Ester (16). The mixture of 15 (7.41 g, 15.6
mmol), Et3N (3.26 mL, 23.4 mmol), acetic anhydride (2.21 mL,
23.4 mmol), and a catalytic amount of 4-DMAP (95 mg) in CH2Cl2
was stirred for 30 min at 0 °C, and for 15 h at rt. Then, CH2Cl2
(150 mL) was added and the organic phase was washed with 1 N
HCl, a saturated NaHCO3 solution, and brine, dried (Na2SO4), and
evaporated. FC (EtOAc/hexane 1:3) gave the crude O-acetyl
derivative of 15 (8.21 g, >100%) as a colorless oil: TLC Rf 0.45
1
(99.4%); H NMR (DMSO-d6, 360 MHz): δ 0.7-0.9 (m, 12H,
4CH3), 1.05 (s, 6H, (CH3)2CONH2), 1.2-1.45 (m, 3H, aliphat),
1.5-1.7 (m, 3H, aliphat), 1.78 (m, 1H, ArCH2CH), 1.92 (m, 2H,
CH2CH2O), 2.26 (m, 1H, CHCONH), 2.36-2.47 (m, 2H, ArCH2),
2.68 (m, 1H, CHNH3+), 3.07 (dd, J ) 6, 13.3, 1H, CONHCHa),
3.20-3.25 (m, 1H, CHOH), 3.27 (s, 3H, OCH3), 3.31 (dd, J ) 6,
13.3, 1H, CONHCHb), 3.46 (t, J ) 6.3, 2H, OCH2), 3.71 (s, 3H,
OCH3), 3.98 (t, J ) 6.5, 2H, ArOCH2), 5.35 (d, J ) 5.5, 1H, OH),
6.69 (dd, J ) 1.5, 8, 1H, arom), 6.80-6.82 (m, 3H, arom,
CONHaHb), 7.13 (br s, 1H, CONHaHb), 7.54 (t, J ) 6, 1H, CONH),
7.65 (br s, 3H, NH3+); HRMS m/z 552.4015 [(M + H)+ calcd for
C30H54N3O6+, 552.4013]. Anal. (C30H54N3O6‚HCl‚0.32H2O) C, H,
Cl, N.
Compound 46, Hemi-Fumarate Salt (Aliskiren). To a solution
of 46, HCl salt, (0.24 g, 0.40 mmol) in water (2.5 mL) were added
crushed ice (2 g) and 2 N NaOH (1.2 mL, 2.4 mmol). The white
suspension was extracted with Et2O (3 × 15 mL), and the combined
organics were dried (MgSO4) and evaporated. The free base (220
mg) was dissolved in i-PrOH, and a solution of fumaric acid (23
mg, 0.20 mmol) in i-PrOH (2 mL) was added. Insoluble materials
were removed by filtration through a glass fiber filter, and the filtrate
was concentrated to a volume of 1.5 mL. Et2O (2 mL) was slowly
added, followed by the addition of a seed crystal, and stirring of
the gradually forming fine suspension was continued overnight at
rt. The white precipitate was filtered, washed with a small volume
of i-PrOH-Et2O, and dried in high vacuo at 50 °C to afford the
title compound (150 mg) as a white solid; mp 108-115 °C; HPLC
tR 12.7 min (99.1%). Anal. (C30H53N3O6‚C2H2O2‚0.88H2O) C, H,
N.
General MethodsCoupling of Sterically Hindered Amines.
Compound 9f. To a solution of acid 7 (119 mg, 0.20 mmol) in
MeCN (5 mL) were subsequently added NEt3 (34 µL, 0.24 mmol),
1,1-dimethyl-2-morpholin-4-yl-ethylamine (114 mg, 0.72 mmol),50
and O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluo-
rophosphate (HBTU) (91 mg, 0.24 mmol). After a 30 min reaction
time, DMF (2 mL) was added to the suspension and stirring was
1
(EtOAc/hexane 1:2); HPLC tR 21.8 min; H NMR (CDCl3, 300
MHz) δ 0.83-2.45 (m, 26H), 2.58-2.65 (m, 3H), 3.17-3.20 (m,
1H), 3.33 (s, 3H), 3.38-3.42 (m, 2H), 3.80 (s, 3H), 4.00-4.05
(m, 0.25H), 4.32-4.35 (m, 0.75H), 5.61 (d, J ) 9, 0.25H), 5.73
(d, J ) 8, 0.75H), 6.78-6.83 (m, 1H), 7.04-7.18 (m, 2H).
This material, dissolved in EtOH (400 mL), was hydrogenated
in the presence of 5% palladium on charcoal (Engelhardt) for 4

4842 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Maibaum et al.
days under atmospheric pressure at rt. The catalyst was removed
by filtration over Celite, followed by rigorous washing with EtOH
(1 L). Concentration of the combined filtrates and drying in vacuo
afforded a yellow oil (TLC Rf 0.56 (CH2Cl2/MeOH 10:1)). Without
further purification, this material was dissolved in CH2Cl2 (80 mL),
and BOC2O (5.10 g, 23.4 mmol) and Et3N (5.6 mL, 32.7 mmol)
were added at 0 °C. After warming to rt, stirring was continued
for 16 h before removing volatiles under reduced pressure. The
residue was purified by FC (EtOAc/hexane 1:4) to afford title
compound 16 (4.00 g, 48%; 3 steps) as a white crystalline solid:
mp 81-82 °C; TLC Rf 0.36 (EtOAc/hexane 1:4); HPLC tR 23.1
min; IR (CH2Cl2) 1765 (CO), 1710 (BOC); 1H NMR (CDCl3, 300
MHz) δ 0.82 (d, J ) 7, 6H), 0.93 (d, J ) 7, 3H), 1.01 (d, J ) 7,
3H), 1.23-1.32 (m, 1H), 1.47 (s, 9H), 1.52-1.69 (m, 7H), 2.04-
2.20 (m, 3H), 2.31-2.39 (m, 1H), 2.52-2.66 (m, 4H), 3.33 (s,
3H), 3.37-3.41 (m, 2H), 3.79 (s, 3H), 3.80-3.88 (m, 1H), 4.37-
4.40 (m, 2H), 6.72 (d, J ) 8, 1H), 6.91-6.95 (m, 2H); FAB-MS
m/z 534 [M + H]+, 434 [M - BOC]+.
afforded 18 (0.46 g, 68%) as an amorphous solid: TLC Rf 0.45
(EtOAc/hexane 1:2); IR (CH2Cl2) 1710 (CO); H NMR (CDCl3,
1
300 MHz) δ 0.02 (s, 3H), 0.07 (s, 3H), 0.81-0.97 (m, 21H), 1.48
(s, 9H), 1.10-1.93 (m, 12H), 2.30-2.42 (m, 2H), 2.50-2.66 (m,
3H), 3.34 (s, 3H), 3.41-3.44 (m, 2H), 3.55-3.60 (m, 1H), 3.65-
3.74 (m, 1H), 3.78 (s, 3H), 4.58 (d, J ) 10, 1H), 6.70-6.73 (m,
1H), 6.89-6.96 (m, 2H); FAB-MS m/z 689 [M + Na]+, 666 [M +
H]+, 586 [M - BOC]+.
(1S,2S,4S,2′S)-(4-(2-Carbamoyl-2-methyl-propylcarbamoyl)-
2-hydroxy-1-{2′-[4-methoxy-3-(4-methoxy-butyl)-benzyl]-3-methyl-
butyl}-5-methyl-hexyl)-carbamic Acid tert-Butyl Ester (19). To
a solution of acid 18 (0.45 g, 0.68 mmol) and 3-amino-2,2-dimethyl-
propionamide, HCl salt (23a; 0.114 g, 0.746 mmol), in acetonitrile
(20 mL) were added Et3N (0.45 mL, 3.19 mmol) and, after 5 min,
coupling reagent HBTU (0.309 g, 0.814) at 0 °C. Stirring was
continued for 30 min at 0 °C and then at rt for 15 h. Volatiles were
removed under reduced pressure at <35 °C, brine (50 mL) was
added, and the water layer was extracted with EtOAc. The combined
organics were washed with 1 N HCl, a saturated NaHCO3 solution,
and brine, dried (Na2SO4), and evaporated. FC purification (EtOAc/
hexane 2:1) gave the O-TBDMSi-protected coupling product (0.54
g, >100%) as an amorphous solid: TLC Rf 0.48 (CH2Cl2/MeOH
(1S,2S,4S,2′S)-[2-Hydroxy-1-{2′-[4-methoxy-3-(4-methoxy-but-
yl)-benzyl]-3-methyl-butyl}-5-methyl-4-(2-morpholin-4-yl-eth-
ylcarbamoyl)-hexyl]-carbamic Acid tert-Butyl Ester (17). To the
mixture of lactone 16 (4.00 g, 7.49 mmol) and 4-(2-aminoethyl)-
morpholine (15 mL) was added a catalytic amount of acetic acid
(4 µL), followed by heating for 24 h at 80 °C. After cooling, excess
amine was removed under reduced pressure, and the residue was
purified by FC on silica gel (CH2Cl2/MeOH 10:1) and recrystal-
lization from diisopropyl ether-hexane (1:1, 60 mL) to afford 17
(4.28 g, 86%) as a white solid: mp 108-110 °C; TLC Rf 0.36
1
10:1); H NMR (CDCl3, 300 MHz) δ 0.06 (s, 3H), 0.09 (s, 3H),
0.79 (d, J ) 6, 6H), 0.89 (s, 9H), 0.92-0.96 (m, 6H), 1.21 (s, 3H),
1.22 (s, 3H), 1.48 (s, 9H), 1.10-1.80 (m, 11H), 1.95-2.00 (m,
1H), 2.27-2.32 (m, 1H), 2.51-2.70 (m, 3H), 3.18-3.23 (m, 1H),
3.33 (s, 3H), 3.38-3.41 (m, 2H), 3.50-3.58 (m, 2H), 3.68-3.76
(m, 1H), 3.78 (s, 3H), 4.53 (d, J ) 9.5, 1H), 5.21 (m, 1H), 6.71 (d,
J ) 8, 1H), 6.27-6.33 (m, 1H), 6.58-6.62 (m, 1H), 6.92-6.97
(m, 2H); FAB-MS m/z 786 [M + Na]+, 764 [M + H]+, 664 [(M
- BOC]+.
1
(CH2Cl2/MeOH 10:1); HPLC tR 17.4 min; H NMR (CDCl3, 500
MHz) δ 0.80 (d, J ) 7, 3H), 0.82 (d, 3H, J ) 7, 3H), 0.92 (d, 3H,
J ) 7, 3H), 0.94 (d, J ) 7, 3H), 1.11-1.17 (m, 1H), 1.48 (s, 9H),
1.45-1.78 (m, 10H), 1.85-1.92 (m, 1H), 2.00-2.05 (m, 1H),
2.30-2.68 (m, 10H), 3.20-3.28 (m, 1H), 3.32 (s, 3H), 3.39 (m,
2H), 3.52-3.60 (m, 3H), 3.68-3.76 (m, 4H), 3.79 (s, 3H), 4.74
(br d, J ) 10, 1H), 6.00 (br t, J ) 5, 1H), 6.72 (d, J ) 8, 1H),
6.95-6.97 (m, 2H); FAB-MS m/z 664 [M + H]+. Anal.
(C37H65N3O7) C, H, N.
To the solution of the above O-silyl ether (0.52 g, 0.67 mmol)
in THF (10 mL) was added a 1 M solution of n-Bu4NF in THF
(0.71 mL, 0.71 mmol) at 0 °C. After warming to rt, the mixture
was stirred for 36 h, followed by the addition of 0.71 mL of 1 M
n-Bu4NF in THF. After completion of the reaction (TLC), the
mixture was poured into water (60 mL), followed by extractive
workup with EtOAc. FC purification (eluent: EtOAc) gave the title
compound 19 (0.38 g, 87%) as an amorphous glass: TLC Rf 0.33
(2S,4S,5S,7S)-5-Amino-4-hydroxy-2-isopropyl-7-[4-methoxy-
3-(4-methoxy-butyl)-benzyl]-8-methyl-nonanoic Acid (2-Mor-
pholin-4-yl-ethyl)-amide, Dihydrochloride Salt (59). A solution
of 17 (4.25 g, 6.40 mmol) in 4 N HCl-dioxane (20 mL) was stirred
for 2 h at 0 °C. The mixture was lyophilized in high vacuo to give
the title compound 59 (4.07 g) as an amorphous solid: TLC Rf
1
(EtOAc); HPLC tR 19.7 min; H NMR (CDCl3, 300 MHz) δ 0.83
(d, J ) 7, 6H), 0.92 (d, J ) 6, 6H), 1.45 (s, 9H), 1.00-1.70 (m,
16H), 1.87-1.91 (m, 1H), 2.00-2.10 (m, 1H), 2.42 (m, 1H), 2.53-
2.60 (m, 3H), 3.33 (s, 3H), 3.35-3.53 (m, 6H), 3.59-3.65 (m,
1H), 3.78 (s, 3H), 4.66 (d, J ) 9, 1H), 5.43 (m, 1H), 6.15 (m, 1H),
6.38-6.42 (m, 1H), 6.72 (d, J ) 8, 1H), 6.91-6.96 (m, 2H); FAB-
MS m/z 672 [M + Na]+, 650 [M + H]+, 550 [M - BOC]+.
(2S,4S,5S,7S)-5-Amino-4-hydroxy-2-isopropyl-7-[4-methoxy-
3-(4-methoxy-butyl)-benzyl]-8-methyl-nonanoic Acid (2-Car-
bamoyl-2-methyl-propyl)-amide, Hydrochloride Salt (60). A
solution of 19 (0.37 g, 5.77 mmol) in 4 N HCl-dioxane (2 mL)
was stirred for 2 h at 0 °C. The mixture was lyophilised in high
vacuo to give the title compound (0.33 g) as an amorphous solid:
TLC Rf 0.28 (CH2Cl2/MeOH/NH4OH 350:50:1); HPLC tR 14.3 min
(98% purity); 1H NMR (CDCl3, 300 MHz) δ 0.83 (d, J ) 6, 3H),
0.89 (d, 9H, J ) 6, 9H), 1.21 (s, 3H), 1.23 (s, 3H), 0.95-1.84 (m,
11H), 1.90-1.95 (m, 1H), 2.36-2.58 (m, 6H), 2.77-2.84 (m, 1H),
2.93-2.98 (m, 1H), 3.31 (s, 3H), 3.35-3.38 (m, 2H), 3.44-3.51
(m, 1H), 3.75 (s, 3H), 3.65-3.86 (m, 3H), 6.71 (d, J ) 8, 1H),
6.98 (d, J ) 1.5, 1H), 7.07 (dd, J ) 1.5, 8, 1H), 7.17 (br s, 1H),
7.29 (br s, 3H), 7.92 (br s, 2H); MS FAB-MS m/z 550 [M + H]+.
Anal. (C31H55N3O5‚HCl‚1.5H2O) C, H, N.
1
0.21 (CH2Cl2/MeOH 10:1); HPLC tR 12.7 min (96% purity); H
NMR (CDCl3, 300 MHz) δ 0.87-0.94 (m, 12H), 1.23-2.05 (m,
18H), 2.22-2.30 (m, 1H), 2.51-2.61 (m, 3H), 2.80-2.95 (m, 2H),
3.20-3.32 (m, 1H), 3.32 (s, 3H), 3.37-3.41 (m, 2H), 3.44-3.75
(m, 4H), 3.77 (s, 3H), 3.94-4.20 (m, 7H), 6.74 (d, J ) 8, 1H),
7.01 (d, J ) 1.5, 1H), 7.09 (dd, J ) 8, 1.5, 1H), 7.92-8.20 (m,
2H), 8.42 (m, 1H); FAB-MS m/z 564 [M + H]+. Anal. (C32H57N3O5‚
2HCl‚0.5H2O) C, H, N.
(2S,4S,5S,7S)-5-tert-Butoxycarbonylamino-4-(tert-butyl-di-
methyl-silanyloxy)-2-isopropyl-7-[4-methoxy-3-(4-methoxy-bu-
tyl)-benzyl]-8-methyl-nonanoic Acid (18). To a solution of 16
(0.54 g, 1.01 mmol) in 1,2-dimethoxyethane-water (2:1, 30 mL)
was added a 1 M aqueous solution of LiOH (4.05 mL, 4.04 mmol)
at rt. After 1 h, the reaction was complete according to TLC control.
The volatiles were removed under reduced pressure, and the water
phase was cooled to 0-5 °C and adjusted to pH 4 by the addition
of a 10% aqueous solution of citric acid (ca. 11 mL). Repeated
extraction with EtOAc, drying of the combined organics (Na2SO4),
and evaporation afforded the crude γ-hydroxy acid (0.54 g, 97%)
as a colorless gum: TLC Rf 0.43 (CH2Cl2/MeOH 10:1). This
material was dissolved in DMF (8.0 mL), TBDMSiCl (0.335 g,
2.22 mmol) and imidazole (0.303 g, 4.45 mmol) were added, and
the mixture was stirred for 6 days at rt. The solvent was removed
in vacuo at ambient temperature, and the residue was taken up in
ice-water (50 mL) followed by repeated extraction with EtOAc.
The combined organics were washed with ice-water (50 mL), ice-
cold 10% citric acid (50 mL), ice-water (50 mL), and brine, dried
(Na2SO4), and evaporated. FC purification (EtOAc/hexane 1:4)
Enzyme Inhibition Measurements. Human BACE-1 activity
was assayed in 100 mM acetate buffer, pH 4.5, containing 0.1%
CHAPS, using Mca-SEVNLDAEFK(DNP)-OH (3 µM), as de-
scribed.51 The HIV-1 protease assay was performed as described
previously.52 IC50 determination against nonprimate plasma renin
of various species is described elsewhere.48
Acknowledgment. We thank Prof. John Kay (University of
Cardiff, U.K.) for inhibitor IC50 determination against human
pepsin A, cathepsin D, and cathepsin E. Dr. Bernard Faller and

Aliskiren, A Nonpeptide Human Renin Inhibitor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4843
(20) Nakao, N.; Yoshimura, A.; Morita, H.; Takada, M.; Kayano, T.;
Ideura, T. Combination Treatment of Angiotensin-II Receptor Blocker
and Angiotensin-Converting-Enzyme Inhibitor in Non-Diabetic Renal
Disease (COOPERATE): A Randomised Controlled Trial. Lancet
2003, 361, 117-124.
(21) McMurray, J. J. V.; Ostergren, J.; Swedberg, K.; Granger, C. B.;
Held, P.; Michelson, E. L.; Olofsson, B.; Yusuf, S.; Pfeffer, M. A.;
CHARM Investigators, and Committees. Effects of Candesartan in
Patients with Chronic Heart Failure and Reduced Left-Ventricular
Systolic Function Taking Angiotensin-Converting-Enzyme Inhibi-
tors: The CHARM-Added Trial. Lancet 2003, 362, 767-771.
(22) Greenlee, W. J. Renin Inhibitors. Med. Res. ReV. 1990, 10, 173-
236.
Dr. Hans-Ulrich Gremlich are acknowledged for the determi-
nation of physicochemical data of the renin inhibitors. The
authors thank Dr. Nils Ostermann for providing the illustrations
in Figure 2 and for critical discussions. R. Go¨schke is indebted
to Ms. Nicole Hasler and Ms. Florence Kummli-Lugrin, and
Y. Yamaguchi is indebted to Mr. Thomas Stebler and Reto
Wicki for their excellent technical assistance. Technical and
editorial assistance was provided by Geoff Davison (ACUMED,
Macclesfield, U.K.).
Supporting Information Available: Additional data on analyti-
cal characterization of the final inhibitor compounds and experi-
mental procedures for the preparation of 8a-d, 9a, 9b, 9d, 10, 12,
13, 23a, 23b, 27, and the P2′ amine of inhibitor 49. This material
is available free of charge via the Internet at http://pubs.acs.org.
(23) Rosenberg, S. H. Renin Inhibitors. In Progress in Medicinal
Chemistry; Ellis, G. P., Luscombe, D. K., Eds.; Elsevier Science:
New York, 1995; Vol. 32, pp 37-115.
(24) Oefner, C.; Binggeli, A.; Breu, V.; Bur, D.; Clozel, J.-P.; D’Arcy,
A.; Dorn, A.; Fischli, W.; Gru¨ninger, F.; Gu¨ller, R.; Hirth, G.; Ma¨rki,
H. P.; Mathews, S.; Mu¨ller, M.; Ridley, R. G.; Stadler, H.; Vieira,
E.; Wilhelm, M.; Winkler, F. K.; Wostl, W. Renin Inhibition by
Substituted Piperidines: A Novel Paradigm for the Inhibition of
Monomeric Aspartic Proteinases. Chem. Biol. 1999, 6, 127-131.
(25) Ma¨rki, 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.; Gru¨ninger, F.; Guenzi, A.;
Gu¨ller, R.; Hartung, T.; Hirth, G.; Jenny, Ch.; Kansy, M.; Klinkham-
mer, U.; Lave, T.; Lohri, B.; Luft, F. C.; Mervaala, E. M.; Mu¨ller,
D. N.; Mu¨ller, 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. Piperidine Renin
Inhibitors: From Leads to Drug Candidates. Il Farmaco 2001, 56,
21-27.
(26) Maibaum, J.; Rasetti, V.; Ru¨eger, H.; Cohen, N. C.; Go¨schke, R.;
Mah, R.; Rahuel, J.; Gruetter, M. G.; Cumin, F.; Wood, J. M. Design
and Synthesis of Novel Potent, Non-Peptide and Orally Active Renin
Inhibitors. In Medicinal Chemistry: Today and Tomorrow, Proceed-
ings of the AFMC International Medicinal Chemistry Symposium,
Tokyo, Japan,September 3-8, 1995; Mikio Yamazaki, Ed.; Blackwell
Science: Oxford, U.K., 1997; pp 155-162.
(27) Rahuel, J.; Rasetti, V.; Maibaum, J.; Ru¨eger, H.; Go¨schke, R.; Cohen,
N. C.; Stutz, S.; Cumin, F.; Fuhrer, W.; Wood, J. M.; Gruetter, M.
G. Structure-Based Drug Design: The Discovery of Novel Nonpep-
tide Orally Active Inhibitors of Human Renin. Chem. Biol. 2000, 7,
493-504.
(28) Go¨schke, R.; Stutz, S.; Rasetti, V.; Cohen, N.-C.; Rahuel, J.; Rigollier,
P.; Baum, H.-P.; Forgiarini, P.; Schnell, C. R.; Wagner, T.; Gruetter,
M. G.; Fuhrer, W.; Schilling, W.; Cumin, F.; Wood, J. M.; Maibaum,
J. Novel 2,7-Dialkyl-Substituted 5(S)-Amino-4(S)-Hydroxy-8-Phenyl-
Octanecarboxamide Transition State Peptidomimetics are Potent and
Orally Active Inhibitors of Human Renin. J. Med. Chem. 2007, 50,
4818-4831.
(29) For a preliminary account of this work, see: Maibaum, J.; Stutz, S.;
Go¨schke, R.; Rigollier, R.; Yamaguchi, Y.; Schilling, W.; Wood, J.
M. Structural Modification of the P2′ Position of 2,7-Dialkyl-
Substituted 5(S)-Amino-4(S)-Hydroxy-8-Phenyl-Octanecarboxam-
ides: Discovery of a Potent Nonpeptide Renin Inhibitor Active After
Once Daily Dosing in Marmosets. Proceedings of the XVth EFMC
International Symposium on Medicinal Chemistry, Edinburgh, U.K.,
September 6-10, 1998; Abstract Book P230.
(30) Wood, J. M.; Maibaum, J.; Rahuel, J.; Gruetter, M. G.; Cohen, N.-
C.; Rasetti, V.; Ru¨ger, H.; Go¨schke, R.; Stutz, S.; Fuhrer, W.;
Schilling, W.; Rigollier, P.; Yamaguchi, Y.; Cumin, F.; Baum, H.-
P.; Schnell, C. R.; Herold, P.; Mah, R.; Jensen, C.; O’Brien, E.;
Stanton, A.; Bedigian, M. P. Structure-Based Design of Aliskiren, a
Novel Orally Effective Renin Inhibitor. Biochem. Biophys. Res.
Commun. 2003, 308, 698-705.
(31) For, a modification of this synthetic route, see the following
account: Dong, H.; Zhang, Z.-L.; Huang, J.-H.; Ma, R.; Chen, S.-
H.; Li, G. Practical Synthesis of an Orally Active Renin Inhibitor
Aliskiren. Tetrahedron Lett. 2005, 46, 6337-6340.
(32) (a) Holladay, M. W.; Rich, D. H. Synthesis of Hydroxyethylene and
Ketomethylene Dipeptide Isosteres. Tetrahedron Lett. 1983, 24,
4401-4404. (b) Holladay, M. W.; Salituro, F. G.; Rich, D. H.
Synthetic and Enzyme Inhibition Studies of Pepstatin Analogues
Containing Hydroxyethylene and Ketomethylene Dipeptide Isosteres.
J. Med. Chem. 1987, 30, 374-383.
References
(1) Hershey, J. C.; Steiner, B; Fischli, W.; Feuerstein, G. Renin
inhibitors: An Antihypertensive Strategy on the Verge of Reality.
Drug DiscoVery Today: Therapeutic Strategies 2005, 2, 181-185.
(2) American Heart Association, Heart Disease and Stroke Statisticss
2004 Update; American Heart Association: Dallas, TX, 2004.
(3) White, W. B. Update on the Drug Treatment of Hypertension in
Patients with Cardiovascular Disease. Am. J. Med. 2005, 118, 696-
705.
(4) MacGregor, G. A.; Markandu, N. D.; Roulston, J. E.; Jones, J. C.;
Morton, J. J. Maintenance of Blood Pressure by the Renin-
Angiotensin System in Normal Man. Nature 1981, 291, 329-331.
(5) Wood, J. M.; Stanton, J. L.; Hofbauer, K. G. Inhibitors of Renin as
Potential Therapeutic Agents. J. Enzyme Inhib. 1987, 1, 169-185.
(6) Hackenthal, E.; Paul, M.; Ganten, D.; Taugner, R. Morphology,
Physiology, and Molecular Biology of Renin Secretion. Physiol. ReV.
1990, 70, 1067-1116.
(7) Skeggs, L. C.; Kahn, J. R.; Lentz, K.; Shumway, N. P. Preparation,
Purification and Amino Acid Sequence of a Polypeptide Renin
Substrate. J. Exp. Med. 1957, 106, 439-453.
(8) Fyhrquist, F. Clinical pharmacology of the ACE inhibitors. Drugs
1986, 32 (Suppl 5), 33-39.
(9) Cugno, M.; Nussberger, J.; Cicardi, M.; Agostoni, A. Bradykinin
and the Pathophysiology of Angioedema. Int. J. Immunopharmacol.
2003, 3, 311-317.
(10) Hollenberg, N. K.; Fisher, N. D. L.; Price, D. A. Pathways for
Angiotensin II Generation in the Tissues of Intact Humans: Evidence
from Comparative Pharmacological Interruption of the Renin System.
Hypertension 1998, 32, 387-392.
(11) Ruilope, L. M.; Rosei, E. A.; Bakris, G. L.; Mancia, G.; Poulter, N.
R.; Taddei, S.; Unger, T.; Volpe, M.; Waeber, B.; Zannad, F.
Angiotensin Receptor Blockers: Therapeutic Targets and Cardio-
vascular Protection. Blood Pressure 2005, 14, 196-209.
(12) Harding, J. W.; Wright, J. W.; Swanson, G. N.; Hanesworth, J. M.;
Krebs, L. T. AT4 Receptors: Specificity and Distribution. Kidney
Int. 1994, 46, 1510-1512.
(13) Neutel, J. M.; Luther, R. R.; Boger, R. S.; Weber, M. A. Immediate
Blood Pressure Effects of the Renin Inhibitor Enalkiren and the
Angiotensin-Converting Enzyme Inhibitor Enalaprilat. Am. Heart J.
1991, 122, 1094-1100.
(14) Himmelmann, A.; Bergbrant, A.; Svensson, A.; Hansson, L.; Aurell,
M. Remikiren (Ro 42-5892)sAn Orally Active Renin Inhibitor in
Essential Hypertension. Effects on Blood Pressure and the Renin-
Angiotensin-Aldosterone System. Am. J. Hypertens. 1996, 9, 517-
522.
(15) Boger, R. S.; Glassman, H.; Thys, R.; et al. Absorption and Blood
Pressure Response to the New Orally Active Renin Inhibitor,
A-72517, in Hypertensive Patients. Am. J. Hypertens. 1993, 6, 103A.
(16) Azizi, M.; Guyene, T. T.; Chatellier, G.; Me´nard, J. Blood Pressure
Effects of Acute Intravenous Renin or Oral Angiotensin Converting
Enzyme Inhibition in Essential Hypertension. J. Hypertens. 1994,
12, 419-427.
(17) El Amrani, A. I.; Menard, J.; Gonzales, M. F.; Michel, J. B. Effects
of Blocking the Angiotensin II Receptor, Converting Enzyme, and
Renin Activity on the Renal Hemodynamics of Normotensive Guinea
Pigs. J. CardioVasc. Pharmacol. 1993, 22, 231-239.
(18) Azizi, M.; Webb, R.; Nussberger, J.; Hollenberg, N. K. Renin
inhibition with aliskiren: where are we now, and where are we going?
J. Hypertens. 2006, 24, 243-256.
(19) Hollenberg, N. K.; Fisher, N. D. Renal Circulation and Blockade of
the Renin-Angiotensin System. Is Angiotensin-Converting Enzyme
Inhibition the Last Word? Hypertension 1995, 26, 602-609.
(33) Go¨schke, R.; Stutz, S.; Heinzelmann, W.; Maibaum, J. The Nonchiral
Bislactim Diethoxy Ether as a Highly Stereo-inducing Synthon for
Sterically Hindered, γ-Branched R-Amino Acids: A Practical, Large-
scale Route to an Intermediate of the Novel Renin Inhibitor Aliskiren.
HelV. Chim. Acta 2003, 86, 2848-2870.

4844 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Maibaum et al.
(34) Footnote: T. Winkler, unpublished results. Final experimental
confirmation was achieved in the course of this work by X-ray
crystallographic analysis of chiral intermediates prepared on a
different synthetic route and their transformation into the common
intermediate 9e (cf. Scheme 2; Go¨schke, R. et al., unpublished
results).
(35) Carpino, L. A.; Imazumi, H.; El-Faham, A.; Ferrer, F. J.; Zhang, C.;
Lee, Y.; Foxman, B. M.; Henklein, P.; Hanay, C.; Mugge, C.;
Wenschuh, H.; Klose, J.; Beyermann, M.; Bienert, M. The Uronium/
Guanidinium Peptide Coupling Reagents: Finally the True Uronium
Salts. Angew. Chem., Int. Ed. 2002, 41, 441-445.
(46) Wood, J. M.; Schnell, C. R.; Cumin, F.; Menard, J.; Webb, R. L.
Aliskiren, a Novel, Orally Effective Renin Inhibitor, Lowers Blood
Pressure in Marmosets and Spontaneously Hypertensive Rats. J.
Hypertens. 2005, 23, 417-426.
(47) Footnote: Absolute oral bioavailability (BAV) of inhibitor 46 in fed
sodium-replete marmosets, based on 24 h plasma exposures after 1
mg/kg intravenous and 3 mg/kg peroral doses and using an LC-MS
analytical detection method of the parent compound in plasma
samples, was determined to be 30% with a long estimated terminal
half-life T0.5 ) 8.7 h, a total peak plasma concentration, Cmax ) 3130
( 1462 ng/mL (3 mg/kg oral dose) and a volume of distribution Vss
) 456 mL/kg (1 mg/kg iv dose). Similarly, BAV in marmoset for
inhibitors 39, 47, 50, 59, and 60 was determined to be 14, 25, 22,
30, and 8%, respectivelysHandan He et al., unpublished results.
(48) Poulsen, K.; Jorgensen, J. An Easy Radioimmunological Microassay
of Renin Activity, Concentration and Substrate in Human and Animal
Plasma and Tissues Based on Angiotensin I Trapping by Antibody.
J. Clin. Endocrinol. Metab. 1974, 39, 816-825.
(49) Pilz, B.; Shagdarsuren, E.; Wellner, M.; Fiebeler, A.; Dechend, R.;
Gratze, P.; Meiners, S.; Feldman, D. L.; Webb, R. L.; Garrelds, I.
M.; Danser, A. H. J.; Luft, F. C.; Mueller, D. N. Aliskiren, a Human
Renin Inhibitor, Ameliorates Cardiac and Renal Damage in Double-
Transgenic Rats. Hypertension 2005, 46, 569-576.
(50) Clapp, L. B. Reactions of Ethylenimines: With Ammonia and
Amines. J. Am. Chem. Soc. 1948, 70, 184-186.
(51) Hanessian, S.; Yun, H.; Hou, Y.; Yang, G.; Bayrakdarian, M.;
Therrien, E.; Moitessier, N.; Roggo, S.; Veenstra, S.; Tintelnot-
Blomley, M.; Rondeau, J.-M.; Ostermeier, C.; Strauss, A.; Ramage,
P.; Paganetti, P.; Neumann, U.; Betschart, C. Structure-Based Design,
Synthesis, and Memapsin 2 (BACE) Inhibitory Activity of Carbocy-
clic and Heterrocyclic Peptidomimetics J. Med. Chem. 2005, 48,
5175-5190.
(52) Fa¨ssler, A.; Bold, G.; Capraro, H.-G.; Cozens, R.; Mestan, J.;
Poncioni, B.; roesel, J.; Tintelnot-Blomley, M.; Lang, M. Aza-
Peptide Analogs as Potnet Human Immunodeficiency Virus Type1
Protease Inhibitors with Oral Bioavailability. J. Med. Chem. 1996,
39, 3203-3216.
(53) Avdeef, A. pH-Metric log P. II. Refinement of Partition Coefficients
and Ionization Constants of Multiprotic Substances. J. Pharm. Sci.
1993, 82, 183-190.
(54) Avdeef, A. pH-Metric Solubility. 1. Solubility-pH Profiles from
Bjerrum Plots. Gibbs Buffer and pKa in the Solid State. Pharm.
Pharmacol. Commun. 1998, 4, 165-178.
(36) Go¨schke, R.; Maibaum, J. K.; Schilling, W.; Stutz, S.; Rigollier, P.;
Yamaguchi, Y.; Cohen, N. C.; Herold, P. EP0678503A1, 1995.
(37) Boyd, S. A.; Fung, A. K. L.; Baker, W. R.; Mantei, R. A.; Stein, H.
H.; Cohen, J.; Barlow, J. L.; Klinghofer, V.; Wessale, J. L.; et al.
Nonpeptide Renin Inhibitors with Good Intraduodenal Bioavailability
and Efficacy in Dog. J. Med. Chem. 1994, 37, 2991-3007.
(38) Evans, B. E.; Rittle, K. E.; Homnick, C. F.; Springer, J. P.; Hirshfield,
J.; Veber, D. F. A Stereocontrolled Synthesis of Hydroxyethylene
Dipeptide Isosteres Using Novel, Chiral Aminoalkyl Epoxides and
γ-(Aminoalkyl) γ-Lactones. J. Org. Chem. 1985, 50, 4615-4625.
(39) Boyd, S. A.; Fung, A. K. L.; Baker, W. R.; Mantei, R. A.; Armiger,
Y.-L.; Stein, H. H.; Cohen, J.; Egan, D. A.; Barlow, J. L.; Klinghofer,
V.; Verburg, K. M.; Martin, D. L.; Young, G. A.; Polakowski, J. S.;
Hoffman, D. J.; Garren, K. W., Perun, T. J.; Kleinert, H. D.
C-Terminal Modifications of Nonpeptide Renin Inhibitors: Improved
Oral Bioavailability via Modification of Physicochemical Properties.
J. Med. Chem. 1992, 35 (10), 1735-1746.
(40) Wood, J. M.; Gulati, N.; Forgiarini, P.; Fuhrer, W.; Hofbauer, K. G.
Effects of a Specific and Long-Acting Renin Inhibitor in the
Marmoset. Hypertension 1985, 7, 797-803.
(41) Footnote: IC50 values for the human aspartyl proteases mentioned
in the main text, as well as pepsin C (gastricsin) and BACE-2, were
determined to be >30 µM using corresponding FRET assays with
fluorescence-labelled substrates; F. Cumin et al., unpublished results.
(42) Feldman, D. L.; Persohn, E.; Schu¨tz, H.; Jin, L.; Miserendino-Moltini,
R.; Xuan, H.; Zhuang, S.; Zhou, W. Renal Localisation of the Renin
Inhibitor Aliskiren. Proceedings of The American Society of
Hypertension, New York, NY, May 16-20, 2006; Poster P-178.
(43) DeLano, W. L. The PyMOL Molecular Graphics System; DeLano
Scientific LLC: San Carlos, CA, http://www.pymol.org.
(44) Schnell, C. R.; Wood, J. M. Measurement of Blood Pressure and
Heart Rate by Telemetry in Conscious, Unrestrained Marmosets. Am.
J. Physiol. 1993, 264, H1509-H1516.
(45) Cumin, F.; de Gasparo, M.; Wood, J.; Schnell, C.; Frueh, F.; Graf,
P. Assays to Measure Nanomolar Levels of the Renin Inhibitor CGP
38 560 in Plasma. Hypertension 1989, 14, 379-384.
JM070316I