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

Article abstract of DOI:10.1021/jm070314y

The action of renin is the rate-limiting step of the renin-angiotensin system (RAS), a key regulator of blood pressure. Effective renin inhibitors directly block the RAS entirely at source and, thus, may provide a vital weapon for hypertension therapy. Our efforts toward identifying novel small-molecule peptidomimetic renin inhibitors have resulted in the design of transition-state isosteres such as 1 bearing an all-carbon 8-phenyl-octanecarboxamide framework. Optimization of the extended P3 portion of 1 and extensive P2′ modifications provided analogues with improved in vitro potencies in the presence of plasma. X-ray resolution of rh-renin/38a in the course of SAR work surprisingly unveiled the exploitation of a previously unexplored pocket (S3^sp) important for strong binding affinities. Several inhibitors demonstrated oral efficacy in sodium-depleted marmosets. The most potent, 38a, induced dose-dependently a pronounced reduction in mean arterial blood pressure, paralleled by complete blockade of active plasma renin, up to 8 h post-dose. Oral bioavailability of 38a was 16% in marmosets.

Full text of DOI:10.1021/jm070314y

4818
J. Med. Chem. 2007, 50, 4818-4831
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
Richard Go¨schke, Stefan Stutz,† Vittorio Rasetti, Nissim-Claude Cohen,‡ Joseph Rahuel, Pascal Rigollier, Hans-Peter Baum,
Peter Forgiarini, Christian R. Schnell, Trixie Wagner,§ Markus G. Gruetter,| Walter Fuhrer, Walter Schilling, Fre´de´ric Cumin,
Jeanette M. Wood, and Ju¨rgen Maibaum*
NoVartis Institutes for BioMedical Research, NOVARTIS Pharma AG, CH-4002 Basel, Switzerland
ReceiVed March 19, 2007
The action of renin is the rate-limiting step of the renin-angiotensin system (RAS), a key regulator of blood
pressure. Effective renin inhibitors directly block the RAS entirely at source and, thus, may provide a vital
weapon for hypertension therapy. Our efforts toward identifying novel small-molecule peptidomimetic renin
inhibitors have resulted in the design of transition-state isosteres such as 1 bearing an all-carbon 8-phenyl-
octanecarboxamide framework. Optimization of the extended P3 portion of 1 and extensive P2′ modifications
provided analogues with improved in vitro potencies in the presence of plasma. X-ray resolution of rh-
renin/38a in the course of SAR work surprisingly unveiled the exploitation of a previously unexplored
pocket (S3sp) important for strong binding affinities. Several inhibitors demonstrated oral efficacy in sodium-
depleted marmosets. The most potent, 38a, induced dose-dependently a pronounced reduction in mean arterial
blood pressure, paralleled by complete blockade of active plasma renin, up to 8 h post-dose. Oral
bioavailability of 38a was 16% in marmosets.
Introduction
prorenin,3 is released from the kidney where it is synthesized
in the juxtaglomerular apparatus.4 In common with all aspartyl
proteases, the superfamily to which it belongs, renin’s active
site is a long, deep cleft that can accommodate seven amino
acid residues of the substrate angiotensinogen. Renin controls
the first and rate-limiting step of the RAS and catalyzes the
specific hydrolysis of the Leu10-Val11 peptide bond of the amino-
terminal portion of angiotensinogen to release the decapeptide
angiotensin I (Ang I). Angiotensin converting enzyme (ACE),
a monomeric zinc metalloenzyme, then converts Ang I to the
key component of the RAS, which is angiotensin II (Ang II)
an octapeptide. Ang II acts upon two main receptor subtypes
Ang II type I (AT1) and Ang II type II (AT2) and also upon
Ang type IV (AT4) to bring about the main physiological effects
of the RAS, namely, vasoconstriction and increase of BP by
altering vascular resistance.5 Ang II also inhibits renin secretion
by the juxtaglomerular cells acting as a negative feedback
mechanism. Despite its important contribution to regulating BP
homeostasis, excessive RAS activity and chronic production of
Ang II is believed to form the basis of many pathological states.
This is because Ang II increases BP and has growth promoting
effects that lead to organ damage, particularly in the heart and
kidneys.6
Hypertension is currently estimated to cause 4.5% of the total
global disease burden1 and is seen as one of the most modifiable
risk factors for cardiac disease. An already highly prevalent risk
factor for cardiovascular disease (CVDa) throughout the indus-
trialized world, hypertension is becoming an increasingly
common health problem worldwide, estimated to cause 7.1
million premature deaths annually.1 Despite the large number
of people who suffer from hypertension worldwide, many (30%)
are not aware they are hypertensive, 25% are taking medication,
but their blood pressure (BP) is not controlled, 34% are taking
medication and their BP is controlled, and 11% are aware but
are not taking medication.2 Consequently, well-tolerated effec-
tive treatments to control hypertension and associated CVDs
are required.
The renin-angiotensin system (RAS) has long been established
as being a key regulator of BP and body fluid volume. The
RAS in normotensive humans is stimulated by a number of
signals including decreased sodium plasma concentration, body
fluid volume depletion, or a reduction in BP. Upon stimulation,
renin, a 340 amino acid residue protein formed from pre-
* To whom correspondence should be addressed. Phone: +41 61
6965560. Fax: +41 61 6961163. E-mail: juergen_klaus.maibaum@
novartis.com.
A number of approaches have been taken to control the RAS
and two strategies have focused upon blockading the system
by inhibiting ACE and AT1 receptors. These approaches have
led to successful treatments for hypertension and heart failure.7
However, ACE inhibitors lead to increased levels of Ang I and
increased levels of bradykinin (which can result in cough or
rarely life threatening angioedema) and will not suppress the
non-ACE-dependent production of Ang II.5 AT1 receptor
blockade results in increased levels of Ang II, resulting in
stimulation of AT2 and AT4 receptors. Due to the fact that the
interaction of renin with its substrate is the rate-limiting step in
the RAS and that renin’s substrate specificity for angiotensi-
nogen is unique, successful renin inhibitors are expected to have
† Current affiliation: Speedel Experimenta AG, Gewerbestrasse 14, CH-
4123 Allschwil, Switzerland.
‡ Current affiliation: Synergix, Ltd., Technology Park Malha, Building
1, Jerusalem 91487, Israel.
§ NIBR Discovery Technologies, Lichstrasse 35, WSJ-503.12.08, CH-
4056 Basel, Switzerland.
| Current affiliation: Prof. Markus Gruetter, Biochemisches Institut,
Winterthurerstr. 190, CH-8057 Zu¨rich, Switzerland.
Current affiliation: F.Hoffmann-La Roche Ltd., Pharmaceuticals
Division, Grenzacherstrasse 124, CH-4070 Basel, Switzerland.
a Abbreviations: ACE, angiotensin converting enzyme; Ang I, angio-
tensin I; Ang II, angiotensin II; AT1, angiotensin II type I receptor; AT2,
angiotensin II type II receptor; AT4, angiotensin II type IV receptor; BP,
blood pressure; CVD, cardiovascular disease; ∆MAPmax, maximal change
in mean arterial blood pressure; RAS, renin-angiotensin system; rh-renin,
recombinant human renin.
10.1021/jm070314y CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/08/2007

Potent and Orally ActiVe Inhibitors of Human Renin
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4819
Figure 1. Peptidomimetic inhibitor 1 and previous clinical candidate 2.
Scheme 1. Synthesis of Aldehyde Intermediatesa
a Reagents and conditions: (a) Me3SiBr, CHCl3, rt, 99%; (b) 4-(R)-benzyl-3-isovaleroyl-oxazolidin-2-one, LiHMDS, -75 to 0 °C, 83%; (c) BnSH,
n-BuLi, THF, 0 °C; then 5, LiAlH4, 0 °C, 98%; (d) NBS, Ph3P, CH2Cl2, rt, 93%; (e) (2R)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine, n-BuLi (1.6 M
in hexane), -75 to -20 °C, 94%; (f) 1 N HCl, MeCN; (g) BOC2O, DIPEA, CH2Cl2, 97% (2 steps); (h) DIBAH, toluene, -75 °C (g97%).
the potential to become “best in class” RAS inhibitors,5 as they
offer a more selective means for blocking the RAS.8
studies to identify analogues of this novel compound class,
which display improved in vitro potency in the presence of
plasma, good oral bioavailability and in vivo efficacy in animal
models. Part of this work has been published as a preliminary
account24 when we described the discovery of highly potent
5(S)-amino-4(S)-hydroxy-8-phenyl-octanecarboxamide ana-
logues derived from 1.
While the attractiveness of renin as a target to inhibit the
RAS has been realized for many years, effective strategies to
achieve this have not yet progressed beyond phase III clinical
trials, despite the numerous approaches toward the design of
potent and selective renin inhibitors investigated over more than
two decades. Peptide-like renin inhibitors derived from the
substrate angiotensinogen amino acid sequence by replacing the
scissile peptide bond have been demonstrated to generally
exhibit high enzyme specificity, inhibit plasma renin activity,
lower BP in marmosets,9 and lower BP at least as effectively
as ACE inhibitors in human trials.10-12 However, the molecular
properties and insufficient pharmacodynamic efficacy of these
renin inhibitors mimicking the extended â-strand conformation
of the renin substrate cleavage fragment have limited their
clinical developability.13-17 The design of fully nonpeptidic and
orally potent renin inhibitors, following a novel target-structure
based topological concept, derived from the binding conforma-
tionofthepeptide-likepreviousclinicalcandidate2(CGP038560),18
initially predicted by molecular modeling and subsequently
resolved by X-ray crystallography, was recently described by
us.19-23 This led to the development of several series of unique,
low-molecular weight peptidomimetic inhibitors of renin, such
as 1, comprised of a core aminoalcohol transition-state dipeptide
isostere, structurally related to the hydroxyethylene mimetic of
inhibitor 2, which is tethered to a combined P3-P1 pharma-
cophore (Figure 1). It was found that potent inhibition of human
renin was related to a hitherto unprecedented binding mode to
a distinct nonsubstrate binding site S3sp.22 Exploitation of this
previously unrecognized binding site resulted in dramatically
increased binding affinity and excellent selectivity against related
enzymes of the aspartic protease family. Encouraged by these
initial results, and described herein, we have expanded our
Chemistry
The syntheses of the key intermediates 14 and 15 bearing
the transition-state mimetic structural framework was ac-
complished by a similar route, described by us previously,20
starting from the enantiopure N-Boc R-amino aldehydes 10a
and 10b (Schemes 1 and 2). For efficient structural modification
of the meta-aryl side chain at a late synthetic stage, the
corresponding O-benzyl protected amino ester precursor 9a was
prepared by the Scho¨llkopf method.25 Thus, diastereoselective
alkylation of 4-(R)-benzyl-3-isovaleroyl-oxazolidin-2-one with
benzyl bromide 4 afforded crystalline 5 in 98% yield on large
scale (Scheme 1). Cleavage of the auxiliary with benzyl
mercaptan/n-BuLi followed by LiAlH4 reduction of the resulting
thioester gave the enantiomeric alcohol 6, which was trans-
formed with NBS/Ph3P to bromide 7. The Scho¨llkopf adduct 8
was obtained by highly diastereoselective alkylation26 to give,
after mild acidic hydrolysis and N-Boc protection, derivative
9a in excellent overall yield. DIBAH reduction then afforded
crude aldehyde 10a, and in a similar fashion 10b from known
9b,26 which were used in the next reaction step without further
purification.
For efficient generation of the dipeptide transition-state
mimetic portion, we adapted the protocol by D. J. Kempf27
(Scheme 2). Reaction of aldehyde 10a with the Li-dianion of
N-butylmethacrylamide (see Supporting Information) in the
presence of Ti(Oi-Pr)3Cl afforded a mixture of 11a/12a in a

4820 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Scheme 2. Synthesis of the Transition-State Mimeticsa
Go¨schke et al.
a Reagents and conditions: (a) N-butylmethacrylamide, n-BuLi (1.6 M in hexane), Ti(Oi-Pr)3Cl (1 M in hexane), THF, -78 °C; (b) H2, MeOH, Ru2Cl4[(S)-
BINAP2]NEt3, 20 h, rt, 25 bar; (c) H2, 5% Pd/C, MeOH, quant.; (d) p-TsOH, CHCl3, rt, 75%; (e) LiOH (1 M in water), 1,2-DME/water 2:1, rt; (f) (1)
TBDMSiCl, imidazole, DMF, 6 days, rt; (2) AcOH, THF/water, 68% (2 steps from 15); (g) 4 N HCl in EtOAc, 0-2 °C, 18 h.
Scheme 3. Synthesis of Inhibitors via O-Alkylation or Direct Lactone-Openinga
a Reagents and conditions: (a) NaH, MeO(CH2)3I, DMF, 86% (13b); (b) NaH, n-C5H11I, DMF, rt, 70% (18); (c) 4 N HCl/dioxane, 0 °C; (d) NH2(CH2)3OH
(neat), 80 °C, 82%.
0.7:1 diastereomeric ratio and >60% overall yield, which were
separated by repeated silica gel chromatography. Subsequent
hydrogenation of the desired 2(S)-11a using Noyori’s (S)-
BINAP ruthenium catalyst28 afforded 4(R)-configured 13a with
high diastereoselectivity (de g 95% by RP-HPLC). The minor
4(S)-epimer could be removed by flash chromatography (FC)
and recrystallization from EtOAc/hexane to give the single
isomer 13a (mp 124-125.5 °C). Hydrogenation on 10% Pd/C
afforded the phenolic 14 as the key intermediate, which allowed,
via O-alkylation and final N-Boc deprotection with 4 N HCl in
dioxane according to Scheme 3, the preparation of the inhibitors
listed in Tables 1 and 2. Similarly, the analogous methoxypro-
poxy derivative 13b was prepared as a single isomer from 10b
after recrystallization (mp 106.5-107 °C) via a 2:3 ratio of
separable 11b and 12b and subsequent diastereoselective
catalytic hydrogenation of 11b (Scheme 2). Because we
envisaged a broader exploration of the structure-activity
relationship related to the P2′ position of the new inhibitors,

Potent and Orally ActiVe Inhibitors of Human Renin
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4821
Table 1. In Vitro Activity of Renin Inhibitors with para-OMe versus
para-tButyl at the P3 Aryl Positiona
removal of the O-TBDMSi protecting group and final N-Boc
cleavage (Scheme 4), afforded inhibitors 47, 48, 59-62, 64-
69, and 75-77 (see Tables 3-5). Inhibitor 58 bearing a terminal
carboxylic acid at P2′ was prepared from the ethyl ester 22 by
alkaline saponification to give 23, followed by final N-Boc
cleavage, whereas the corresponding ethyl ester analogue 60
was obtained directly from 22.
Inhibitor 38a was initially prepared on a small scale from 14
by O-alkylation with 3-methoxy-propyliodide (Scheme 3) and
final N-deprotection with 4 N HCl in dioxane. Further preclinical
studies then required multigram quantities of this compound,
which were more advantageously obtained from intermediate
13b (Scheme 2) by N-Boc deprotection with 4 N HCl in EtOAc
at 0-2 °C to afford 38a as its amorphous monohydrochloride
salt in high purity.
The structure and absolute configuration of 13b was con-
firmed by single-crystal X-ray analysis (Figure 2). To demon-
strate that the 4(R) configuration at the P1′ position of the
dipeptide mimetic portion is required for strong affinity to
recombinant human renin (rh-renin) also for this novel class of
inhibitors, the 4(S)-epimer 38b was prepared (Scheme 5). The
4(S,R)-diastereomeric mixture (ca. 1:1 ratio), obtained from the
mother liquors after purification of 13b by recrystallization, was
transformed to the corresponding N,O-acetals 24 and 25, which
were readily separated by silica gel chromatography. Stepwise
deprotection of 25 gave 26 and, subsequently, the pure 4(S)-
configured inhibitor 38b (Table 2). Likewise, transformation
of 24 afforded compound 38a, which was identical with the
material obtained as described above.
binding affinity
IC50 (nM), renin (pH 7.2)
human
marmoset
cmpd
R1 )
tert-butyl CH2CO2Me
MeO CH2CO2Me
tert-butyl CH2CONH2
R2 )
purifiedb plasmac
plasmac
27d
28
6
4
210
340
460
nde
95
160
220
nd
230
190
230
nd
40
230
90
nd
21
5
1d
20
92
42
13
50
26
29
MeO
MeO
CH2CONH2
CH2CONHMe
30
31d
32
tert-butyl CH2SO2Me
MeO
CH2SO2Me
33d
tert-butyl
MeO
MeO
34
35
5
0.6
23
13
a Compounds were obtained as HCl salts according to the General
Procedures A or B; see Experimental SectionsBiological Materials and
Methods. b Purified human renin, measured at pH 7.2. c Human or marmoset
plasma renin assay; see Experimental Section. d Reference 20; about a 1:1
mixture of 4(R)- and 4(S)-configured diastereomers. e Not determined.
Table 2. Modification of the P3sp Aryl Side Chaina
Results and Discussion
Inhibitor Design Concept. The basic target-structure topo-
graphical concept upon which the design of novel chemotype
renin inhibitors such as 1 and several related inhibitor series
was based has been described in detail previously.20,21 Initially,
no X-ray crystal complex structures with rh-renin were available
to us to probe the enzyme-bound conformation for our prototype
lead inhibitors. In particular, the incorporation of H-bonding
groups into the combined P3-P1 pharmacophore, targeted
toward Ser219 for mimicking a key binding interaction of
peptide-like renin inhibitors, turned out to be a promising
approach leading to in vitro highly potent inhibitors.
Computational docking of inhibitor 1 suggested to us that
the large contiguous hydrophobic S3-S1 specificity site of the
human enzyme is fully occupied by the complementary hydro-
phobic P1 isopropyl and the P3 tert-butyl groups, respectively.20
Furthermore, the side chain carboxamide tethered to the phenyl
spacer was anticipated to be located at a distance that would
facilitate H-bonding to the Ser219 backbone NH. In the
predicted binding mode, an N-alkyl residue would be directed
toward the S4 binding site. Accordingly, we considered
structural modifications that could mimic the P3-P4 backbone
of a classical peptide-like inhibitor with the potential to extend
further into the S4 pocket, in an attempt to find more effective
renin inhibitors. In the course of this SAR work, we discovered
inhibitors such as 38a, bearing an extended substituent at the
aryl spacer position, that showed improved in vitro potencies
toward rh-renin. Eventually, the X-ray structure of 38a bound
to rh-renin was resolved (vide infra)22 and revealed the existence
of an unexpected interaction to a distinct binding site extending
from the S3 pocket of rh-renin. In addition to this unprecedented
binding mode to the nonsubstrate S3sp pocket, the crystal
structure also demonstrated that the alkyl ether side chain of
38a was not interacting directly with Ser219 as initially
hypothesized.
binding affinity
IC50 (nM), renin (pH 7.2)
human
marmoset
plasmac
cmpd
R )
purifiedb
plasmac
36
37
38a
38bd
39
40
41
42
43
CH2CH2OMe
CH2CH2OEt
CH2CH2CH2OMe
11
4
1
120
3
6
2
19
4
38
32
1
15
31
3
nd
25
9
18
70
140
nde
20
36
22
90
70
CH2CH2CH2OEt
CH2CH2CH2OH
CH2CH2CH2CH2OMe
CH2CH2OCH2CH2OMe
(CH2)4CH3
a Compounds were obtained as HCl salts according to the General
Procedures A or B; see Experimental SectionsBiological Materials and
Methods. b Purified human renin, measured at pH 7.2. c Human or marmoset
plasma renin assay; see Experimental Section. d 4(S)-epimer of 38a, see
Scheme 4. e Not determined.
13b was efficiently transformed by the reaction with equimolar
p-TsOH in CHCl3 to the corresponding lactone intermediate 15.
Alternatively, alkaline hydrolysis of 15 to the γ-hydroxy acid
16 and subsequent O-silylation provided the acid intermediate
1729 (Scheme 2).
Lactone 15 underwent direct aminolysis in the presence of
various amines (neat), as exemplified in Scheme 3. Hence,
heating of 15 in aminopropanol at 80 °C afforded intermediate
20 in high yield, which was transformed under standard
conditions to the inhibitor 44 as its hydrochloride salt. The
standard coupling reaction of carboxylic acid 17 to a number
of P′2 amines using (EtO)2P(O)CN/NEt3 in DMF, followed by

4822 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Go¨schke et al.
Scheme 4. Synthesis of Renin Inhibitors via Amide Coupling to Acid Intermediate 17a
a Reagents and conditions: (a) H2N(CH2)2CO2Et (HCl), Et3N, DMF, (EtO)2P(O)CN, rt, 94%; (b) n-Bu4NF (hydrate), DMF, rt, 80%; (c) 1 N NaOH,
MeOH, 90%; (d) 4 N HCl/dioxane, 0 °C.
Table 3. In Vitro Activity of Renin Inhibitors with Modified
Substitution at the P2′ Positiona
Table 4. In Vitro Activity of Renin Inhibitors with Modified
Substitution at the P2′ Positiona
binding affinity
IC50 (nM), renin (pH 7.2)
human
marmoset
cmpd
R )
(CH2)2CO2H
(CH2)3CO2H
(CH2)2CO2Et
(CH2)3CO2Me
(CH2)4OCOMe
(CH2)3CN
(CH2)2CONH2
(CH2)3CONH2
(CH2)3CONHMe
(CH2)3CONH(CH2)2OMe
(CH2)4CONH2
purifiedb plasmac
plasmac
58
59
60
61
62
63
64
65
66
67
68
69
12
3
4
1
6
1
7
1
3
2
2
2
17
10
11
2
36
3
17
3
2
23
13
13
3
9
3
13
11
11
10
9
4
1
2
4
70
71
72
73
(CH2)2NHCOMe
(CH2)2SO2Et
(CH2)3SO2tBu
(CH2)2SO2NMe2
0.3
4
6
20
16
7
18
ndd
25
9
2
5
a Compounds were obtained as HCl salts according to the General
Procedures A or B; see Experimental SectionsBiological Materials and
Methods. b Purified human renin, measured at pH 7.2. c Human or marmoset
plasma renin assay; see Experimental Section. d Not determined.
and marmoset renin in a modified in vitro enzyme assay in the
presence of plasma (Table 1). Marked reductions of in vitro
potencies in the presence of plasma have been reported for renin
inhibitors from different structural classes, which could be due
to interference with plasma components, rendering these inhibi-
tors significantly less effective in vivo.32 Renin binding affinity
in the presence of plasma can be affected, at least in part, by
the lipophilicity of the compound, and large potency discrep-
ancies are often observed for highly hydrophobic inhibitors.33,34
We, therefore, reasoned that replacing the highly lipophilic tert-
a Compounds were obtained as HCl salts according to the General
Procedures A or B; see Experimental SectionsBiological Materials and
Methods. b Purified human renin, measured at pH 7.2. c Human or marmoset
plasma renin assay; see Experimental Section.
In Vitro Biological Data and Structure-Activity Relation-
ships. Modification of the P3 Aryl Substitution. Inhibitors 1,
27, and 31 bearing a lipophilic tert-butyl residue at P3 showed
high affinity to purified human renin in a buffer assay at pH
7.2, but were found to be only poor inhibitors of both human

Potent and Orally ActiVe Inhibitors of Human Renin
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4823
Table 5. In Vitro Activity of Renin Inhibitors with Modified
Substitution at the P2′ Positiona
indicating that a H-bonding functionality was not necessarily
required at this position for strong binding to the enzyme (vide
infra).
Variation of the Tethered P3 Aryl Side Chain. In line with
our results from SAR studies on related topographical renin
inhibitors,21 substitution of the phenyl spacer with various alkyl
ether and hydroxylated alkyl residues ortho to the OMe group
at the P3 site, afforded a series of highly potent in vitro
analogues 36-43, as represented in Table 2. The influence of
varying the length of the linear chain from five up to eight
atoms, as well as the position of the oxygen atom along the
chain, was extensively investigated. As discussed above, we
again placed particular emphasis upon producing inhibitors that
demonstrated high binding affinities to human renin, which were
also retained in the presence of plasma, as a key requirement
for in vivo potency. This turned out to be one of the major
challenges during lead optimization, because such discrepancies
in binding affinities were dependent on even minor structural
changes. This is most strikingly illustrated by a comparison of
the in vitro data of inhibitor 38a, bearing a terminal methyl
ether group, with that of the n-pentyloxy-substituted inhibitor
43 of the same chain length. Both compounds were equipotent
in the buffer assay, however, 43 showed a greater than 20-fold
loss in potency against primate plasma renin, whereas compound
38a retained its low-nanomolar binding affinity under the
physiological assay conditions. Furthermore, a shift in the
position of the ether oxygen, as in 37, or extension of the side
chain by one carbon (39 and 41) provided similar binding
affinities in buffer, but caused a significant, albeit less dramatic,
increase in IC50 values when determined in plasma. The terminal
OH group in 40 slightly reduced the in vitro affinity, particularly
against human plasma renin. Inhibitor 36, with a shorter
substituent, was less active, as was 42, bearing a further extended
and highly flexible side chain. The 4(S)-configured P1′ epimer
38b was more than 50-fold less active than 4(R)-38a against
rh-renin.
a Compounds were obtained as HCl salts according to the General
Procedures A or B; see Experimental SectionsBiological Materials and
Methods. b Purified human renin, measured at pH 7.2. c Human or marmoset
plasma renin assay; see Experimental Section.
In Vitro SAR of P2′ Moiety. With the methoxypropoxy side
chain of inhibitor 38a as the optimal P3sp ligand in hand, we
next turned to the extensive variation of the P2′ position, with
the aim of identifying compounds with equally high or further
improved affinities to primate plasma renin, as well as of high
in vivo oral potency in sodium-depleted marmosets. Previously,
the C-terminal P2′ pharmacophore of hydroxyethylene dipeptide
isostere renin inhibitors has been shown to be less sensitive
toward structural modifications, thus allowing the incorporation
of a variety of functional groups to tune their physicochemical
properties.35,36 A broad range of analogues bearing neutral
(inhibitors 44-46, 60-73), basic (47-57), or acidic (58, 59,
76) functional groups, as well as heteroaryl residues (74, 75,
77), tethered by a two- to four-carbon spacer to the dipeptide
isostere carboxamide were examined. Tables 3-5 summarize
the in vitro IC50 data against human (buffer and plasma assay)
and marmoset plasma renin. In general, low nanomolar binding
affinities toward buffered renin were determined for most
inhibitors, with the methyl ether derivative 45 and the inversed
acetamide 70 being the most potent analogues. A marked
decrease in the binding affinity of greater than 10-fold as
compared to 38a was observed only for a few analogues, in
particular for amines 47-49 (Table 3). Interestingly, incorpora-
tion of a heteroatom into the piperidine ring of 49 restored high
affinity to the enzyme (e.g., 50-52, 54-55), whereas hydroxy-
lation at the 4-position was less effective (56, 57). Table 4
presents inhibition data for carboxylic ester (60-62), nitrile (63),
carboxamide (64-70), sulfone (71, 72), and sulfonamide (73)
Figure 2. Structure of compound 13b in the crystal (atomic displace-
ment ellipsoids drawn at the 50% probability level, hydrogen atoms
drawn as spheres of arbitrary radius30). For the enantiomer shown (C16S,
C21S, C30S, C33R), the Flack x parameter is refined to 0.01(11).31
butyl by smaller and more polar residues would increase
potencies of the new chemotype inhibitors under more physi-
ologically relevant in vitro assay conditions.
Several inhibitors bearing a less lipophilic para-methoxy
group at the putative P3 position were prepared from readily
available starting materials (vide supra) and were shown to have
very similar in vitro affinities toward purified renin as compared
to their respective tert-butyl analogues (Table 1). As noted
previously,20 replacement of the terminal carboxylic ester of
highly potent inhibitor 28 by a carboxamide (29 and 30) or a
sulfone (32) as H-bond acceptors caused a drop in binding
affinity up to 20-fold. Intriguingly, the OMe analogues 36 and
38 exhibited only slightly lower IC50 values toward human and
marmoset plasma renin. The considerably reduced potency of
28 in the presence of plasma was attributed to potential ester
cleavage under the assay conditions. Surprisingly, the most
potent inhibitor in this series was the benzyloxy derivative 35
with subnanomolar IC50 in the buffer assay, albeit with slightly
lower affinity in plasma. The 4-pyridyl analogue 34, designed
to potentially interact with Ser219, showed a remarkable 10-
fold lower affinity toward renin, as compared to 35, thus

4824 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Go¨schke et al.
Scheme 5. Synthesis of Epimeric 2(S)-Configured Inhibitor 38ba
a Reagents and conditions: (a) 2,2-DMP, p-TsOH (hydrate), CH2Cl2, rt (quant.); (b) separation by silica gel chromatography; (c) p-TsOH (hydrate),
MeOH, rt (72% for 26); (d) 4 N HCl/dioxane, 0 °C (quant.).
derivatives, demonstrating that these functionalities are well
accommodated at the P2′ position. The carboxylic acid 59 was
of equivalent potency, as was the tetrazole 76 (Table 5), whereas
a minor drop in affinity was seen for acid 58 bearing a shorter
2-carbon spacer. All new renin inhibitors were found to be
highly selective against porcine pepsin and bovine cathepsin D
(no inhibition at 10 µM concentrations; data not shown).9
As discussed above, retention of high affinity of inhibitors
in the presence of plasma was considered as a key requirement.
With the striking exception of the acetamide 70 and, to a lesser
extent, the alkylether 45, all of the inhibitors listed in Tables
3-5 exhibited similar or only slightly reduced (less than 5-fold)
potencies in the presence of plasma. We were gratified that a
number of analogues from this series were identified that
revealed single-digit nanomolar IC50 values toward human
plasma renin, such as alcohols 44 and 46, the P2′ morpholines
50 and 52, and, in particular, inhibitors 65-69 bearing a terminal
primary, secondary or tertiary amide group. There was, though,
a trend for a slightly reduced in vitro affinity toward marmoset
renin for some of these analogues when measured under plasma
assay conditions.
X-ray Structure of rh-Renin with 38a. In the course of our
SAR work, the crystal structure of rh-renin soaked with inhibitor
38a was determined at 3.1 Å resolution (Figures 3 and 4).22
The overlay of enzyme-bound 38a with the cocrystal conforma-
tion of the peptide-like inhibitor 2 revealed an overall good
agreement of the positioning of the dipeptide transition-state
mimetic encompassing the P1′-P2′ portion, as well as a tight
occupancy of the large contiguous S1-S3 specificity pocket
of rh-renin. Hence, the bulky P1 isopropyl of 38a deeply
penetrates into the hydrophobic S1 pocket, whereas the small
para-methoxy group of the phenyl spacer appears to sufficiently
fill the complementary S3 site, with the Me group pointing
toward Leu114, Ala115, and Phe117. In contrast to the
transition-state mimetic OH group of inhibitor 2, which forms
a H-bond network with both Asp32 and Asp215, the dipeptide
isostere OH of 38a adopts a shifted position and interacts only
with Asp32 (distances to the outer oxygen atom 3.1 Å, and inner
oxygen atom 3.0 Å, respectively). Furthermore, it is interesting
to note that the basic NH2 of 38a is not within H-bond distance
to any of the two aspartates, but instead forms a H-bond to the
backbone carbonyl of Gly217 (3.0 Å), similar to the interaction
observed for the P2-P1 carboxamide NH of the peptidic
inhibitor 2 (Figure 3).22 As a consequence of the slight positional
shift and minor conformational changes of the dipeptide isostere
of 38a (as compared to 2), the P1′ Me group is occupying its
complementary site only suboptimally. The adjacent amide
carbonyl still can form a H-bond to Gly77 important for strong
binding, due to a conformation adaptation of the flexible enzyme
flap loop, which positions the aliphatic butyl chain into the S2′
pocket (Figure 4).
The generally very low discrepancies in IC50s against purified
buffer versus plasma renin could be attributed, at least in part,
to a favorable physicochemical profile of these inhibitor
molecules, which is in general characterized by very high
intrinsic aqueous solubility and high solubility in acidic buffer,
as well as a relatively low lipophilicity range. For example,
inhibitor 38a as its HCl salt showed a reasonably low n-octanol/
water coefficient logP ) 1.96 at pH 7.4 and remarkably high
solubility of 11.0 g/L at neutral pH in phosphate buffer (22 °C)
and of 56 g/L at pH 6.0, with a pKa of 9.13. In this respect, this
structurally novel class of renin inhibitors clearly differs from
the previous peptide-like inhibitors, such as 2, which were in
many cases of high lipophilicity and poor solubility, hence
limiting their overall pharmaco-kinetic properties.
Surprisingly, and deviating from the original design concept
based on modeling considerations (vide supra), the X-ray
structure of 38a revealed the very close fit of the methoxypro-
poxy side chain to a distinct, narrow nonsubstrate pocket, which
extends from the S3 site perpendicular to the axis of the enzyme

Potent and Orally ActiVe Inhibitors of Human Renin
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4825
Figure 3. Stereoview with the super-positioned X-ray crystal structures of the peptide-like inhibitor 2 (green color)38 and the nonpeptide inhibitor
38a (orange color) complexed to rh-renin. Inhibitors are shown in ball-and-stick model, with oxygen and nitrogen atoms in red and blue, respectively.
The catalytic aspartic acids (Asp32 and Asp215) as well as Gly217 are shown as stick model in gray color. H-bond interactions between 38a and
Asp32 and Gly217 are shown as dotted yellow lines. The S3 and S3sp pockets are shown as a solvent accessible surface in gray. The graphs in
Figures 3 and 4 were generated using the PyMOL software package.37
Figure 4. Stereo picture with close-up view of the amino acids (in blue) comprising the S3sp subpocket of rh-renin accommodating the methoxypropoxy
side chain of inhibitor 38a. Superimposed are the same amino acids (gray) flanking the S3sp pocket of rh-renin in complex with 2. The two water
molecules 370 and 410 (magenta spheres) were observed in the X-ray complex with 2 only.
cleft and which is flanked by Tyr14, Tyr155, Val30 and Ala303
(Figure 3). Furthermore, no direct binding interaction to Ser219
is observed for 38a in its complex structure (the distances of
the Ser219 hydroxyl to the ligand side chain ether oxygens were
found to be 5.4 and 4.7 Å, respectively). The terminal ether
oxygen of the inhibitor tightly interacts with the backbone
nitrogen of Tyr14 (Figure 4, dotted yellow line) and replaces
the two water molecules W370 and W410 bound in the complex
structure with the peptide-like inhibitor 2 and similarly in the
crystal structure of apo-rh-renin.38 This H-bond is likely to be
a favorable interaction as it is within a binding site completely
shielded from solvent space.39 On the other hand, this interaction
appeared not to be essential for strong binding, as demonstrated
by the similar in vitro potency of inhibitor 43 bearing an all-
carbon chain of the same length. No attempts were made to
obtain an X-ray structure of renin-bound 43, as the common
binding mode of several related inhibitors from different sub-
series and with modified side-chains suggested a very similar
positioning of the respective side chains of 38a and 43 within
the S3sp site.22 Shortening of the side chain by one carbon, as
for 36, reduced the binding affinity 10-fold, which could be
restored by adding one terminal carbon atom (37), again
emphasizing the importance of hydrophobic interactions for
potent ligand affinity. Extending the side chain further (42)
would force the inhibitor to adopt a disfavored conformation
due to the limited depth of the rigid S3sp pocket of about 9 Å,
hence resulting in a markedly weaker inhibitor. The amino acids
flanking the S3sp site undergo at most very minor conformational
changes upon binding of 38a to the active site of rh-renin (Figure
3). This is in line with results we have obtained with inhibitors
from other series,21-24 indicating that this is a rather rigid pocket
of limited size. Still, this channel can accommodate a suitably
positioned phenyl residue of an inhibitor molecule as shown
previously by X-ray;22 retrospective computational docking of
the benzyloxy inhibitor 35 suggested a similar binding mode
by interacting with the S3sp pocket (data not shown), which
would explain its strong binding affinity. The independent
discovery,33,40-42 and exploitation for potent inhibitor design43-45
of this nonsubstrate binding site by other groups was disclosed
after we had completed our structure-based design work.
In Vivo Activity in Conscious Telemetered Na+-Depleted
Marmosets. The oral potency of inhibitors that demonstrated
high in vitro affinities for human and marmoset plasma renin
was evaluated in conscious telemetered sodium-depleted and
normotensive marmosets by constantly measuring the effect on
systolic, diastolic, and mean arterial blood pressures (MAPs)
and heart rate.46 Time courses of changes (∆) in MAP values
from baseline for the test compounds and vehicle were
compared. In addition, the plasma renin activity was determined
1.5, 3, 6, and 24 h post-dosing in nontelemetered marmosets.
Table 6 summarizes the results for selected inhibitors. Com-
pounds 45, 52, 54, and 65 showed a rapid onset of action and
exhibited significant reductions of ∆MAP at peak (1-2 h after
dosing); however, the duration of action was relatively short
for these compounds, with ∆MAP recovering to vehicle-treated
baseline values within 5 h post-dose. Compound 66, a very close
analogue of 65 (Table 4), with a terminal P2′ N-Me carboxa-
mide, showed a similar ∆MAP lowering effect but a signifi-
cantly longer duration of action of up to 7 h. The carboxylic
ester analogue 61 showed only a very minor and short-lasting

4826 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Go¨schke et al.
Table 6. In Vivo Oral Potency, Duration of Action on Mean Arterial
Blood Pressure (MAP), and Inhibition of Plasma Renin Activity (PRA)
of Selected Renin Inhibitors in Na+-Depleted Marmosets
Vdss ) 0.6 L/kg, which is greater than the expected blood
volume, suggesting an extravascular distribution, and plasma
clearance CLp was 5.2 ( 0.4 mL/min/kg. The weaker oral
potencies of related inhibitors 45, 52, 65, and 66 in the Na+-
depleted marmoset model, as compared to 38a, could be
attributed to reduced oral plasma exposures of the respective
inhibitors in nondepleted marmosets (preliminary data, not
shown), suggesting a limited oral bioavailability for these
compounds in monkeys.
single oral
dosea
(mg/kg)
duration
of actionc
(hrs)
peak ∆MAPb
(mmHg)
% inhibition
cmpd
of PRA at 6 hd
38a
45
52
54
59
61
65
66
3
3
10
3
3
3
3
3
-9
8
5
4
3
98
90
88
28
-21
-24
-26
inactive
minor
-22
<1
2
7
Conclusions
95
74
The optimization of the P3 and P3sp regions of novel
nonpeptide transition state isosteres with an all-carbon 8-phenyl-
octanecarboxamide framework, described herein, has provided
inhibitors with high in vitro potencies for human and marmoset
renin. Importantly, high binding affinities were retained in the
presence of plasma, and this was seen as a key requirement to
produce inhibitors exhibiting high affinities in vivo. The
retention of such affinities was demonstrated using a modified
in vitro assay. These new inhibitors are highly soluble in aqueous
buffer, in contrast to peptide-based inhibitors that exhibit high
lipophilicity and poor aqueous solubility, thus limiting phar-
macokinetic properties. Determination of the X-ray structure
of the rh-renin/38a complex revealed a close interaction of the
MeO(CH2)3O residue with the previously discovered S3sp
pocket, proving the latter to be a common binding site for
different series of tethered P3-P1 topographical renin inhibitors.
We have discovered a similar, hitherto unprecedented, binding
mode for several other (P3-P1)-tethered transition-state mimetic
renin inhibitors and have used this structural information to
further optimize their in vitro potencies.21,22
For the first time, high in vivo potency has been demonstrated
for this novel class of renin inhibitors. In particular, inhibitor
38a (HCl salt, CGP054061A) induced in a dose-dependent
manner (over the range of 1 to 30 mg/kg single dose) a
pronounced BP reduction after oral administration to telemetered
Na+-depleted normotensive marmosets, which was shown to
be specifically induced by inhibition of plasma renin. Compound
38a and related analogues are physico-chemically characterized
by their low lipophilicity (logP) and remarkably high intrinsic
solubility in water, in contrast to previous peptide-like inhibitors.
Absolute oral bioavailability of 38a was still found to be
moderate (16%) in non-depleted marmosets, indicating that other
multiple factors may play a role in limiting bioavailability of
this novel compound class. Overall, these encouraging results
stimulated continued efforts toward the design of more potent
in vivo analogues with even greater oral bioavailability.51
-17
a Compounds were administered by oral gavage as clear solutions of
their HCl salts in 5% glucose. b Maximal ∆MAP: change in mean arterial
blood pressure relative to pretreatment period values, reported as the mean
of n > 4 animals. c Time until ∆MAP values returned to baseline levels
after oral dosage. d PRA: plasma renin activity (see Experimental Section
for details).
effect, which was attributed to rapid metabolic ester cleavage
in plasma to the corresponding acid 59 being completely inactive
orally.
The most potent inhibitor in this series was 38a, with a long
duration of action on MAP of up to 8 h and with plasma renin
activity fully blocked at least 6 h after dosing. For compounds
45, 52, 65, and 66, plasma renin activity was inhibited by more
than 74% during at least 6 h following oral administration,
whereas during this period ∆MAP returned to baseline. The
dissociation between pharmacodynamic effects and ex vivo
plasma renin activity measurements might be explained by
displacement of the fraction of inhibitor bound to plasma protein
under the in vitro assay conditions, leading to a higher free
fraction of the inhibitor and, hence, an overestimation of renin
inhibition.47 Alternatively, the trapping assay, although more
appropriate to measure plasma renin activity compared to a
conventional assay,48 may still overestimate the effectiveness
of the compound ex vivo, as plasma Ang I could not be fully
protected from degradation. Furthermore, the recovery of BP
to baseline observed in vivo could be mediated by an extra-
plasmatic pool of renin that is not readily accessible to inhibitors
and is playing an important role in the control of systemic BP.49
Oral administration of single doses of 38a (1-30 mg/kg, HCl
salt in 5% glucose) caused dose-dependent reductions of ∆MAP
in sodium-depleted telemetered marmosets (Figure 5). At the 1
mg/kg threshold dose, the peak effect was only moderate and
transient, with BP recovering to baseline levels after 4 h. A
more pronounced efficacy and a longer duration of action was
observed at the 3 mg/kg dose, and a maximum fall in ∆MAP
of ∼20-25 mmHg (compared with vehicle control) was
achieved at the 10 mg/kg dose. With the higher 30 mg/kg dose
of 38a, the magnitude of the MAP reduction did not further
increase, however, its duration markedly increased, with sig-
nificant effects observed even 24 h after dosing. No significant
effects on heart rate were observed at any given doses of 38a.
The ∆MAP lowering was paralleled by the inhibition of plasma
renin activity, which was completely blocked over the 24 h
period after administration of the 10 and 30 mg/kg doses.
The absolute oral bioavailability for 38a in marmosets was
16%, as estimated from plasma inhibitor concentrations (and
possibly of any circulating potent in vivo metabolites) after 3
mg/kg intravenous and 10 mg/kg oral administration by using
a renin activity assay.50 The terminal elimination half-life t0.5
for 38a was 2.0 ( 0.06 h, with biphasic elimination kinetics
(first distribution-elimination phase followed by a second
elimination phase). The steady-state volume of distribution was
Experimental Section
General methods for experimental and analytical procedures can
be found in the Supporting Information. Synthetic amine P2′
building blocks were either commercially available, or were
prepared according to literature protocols, if not otherwise indicated.
2-Benzyloxy-4-bromomethyl-1-methoxy-benzene (4). To a
solution of benzylic alcohol 3 (59.5 g, 0.244 mol)52 in CHCl3 (0.8
L) was rapidly added Me3SiBr (47.2 mL, 0.365 mol) in a dropwise
manner at ambient temperature. After stirring for 15 min, the
reaction mixture was concentrated under reduced pressure. The pale-
colored solid was dissolved in warm EtOAc (200 mL), followed
by the addition of hexane (150 mL) and submission of the mixture
to a silica gel column (500 g silica gel) to give, after washing with
EtOAc/hexane (1:3) and evaporation of solvent, compound 4 (74.4
g, 99%) as a beige solid [Caution: any contact of this material
with metal equipment (eg., spatula) should be avoided to prevent
rapid decomposition]: mp 95-97 °C (lit. [ref 1] mp 94.5 °C); 1H
NMR (300 MHz, CDCl3) δ 3.85 (s, 3H), 4.43 (s, 2H), 5.13 (s,

Potent and Orally ActiVe Inhibitors of Human Renin
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4827
Figure 5. Values are means ( SEM of the change (4) in MAP from baseline over 24 h after p.o. dosing (time 0) with inhibitor 38a, 1 mg/kg (n
) 4; gray circles), 3 mg/kg (n ) 4; orange triangles), 10 mg/kg (n ) 5; green diamond square), 30 mg/kg (n ) 4; blue square), or vehicle control
(n ) 20; black square) to Na+-depleted freely moving telemetered marmosets. 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 p.o. dose group.
2H), 6.8-7.0 (m, 3H), 7.25-7.5 (m, 5H). This material was used
in the next step without further purification.
3.43 (t, J ) 6, 2H), 3.86 (s, 3H), 5.17 (s, 2H), 6.7-6.85 (m, 3H),
7.25-7.45 (m, 5H). Anal. (C20H26O3) C, H.
(2S,4R)-4-Benzyl-3-[2-(3-benzyloxy-4-methoxy-benzyl)-3-meth-
yl-butyryl]-oxazolidin-2-one (5). To a 1 M solution of LiHMDS
in dry THF (291 mL, 0.291 mol), cooled to -75 °C, was added
dropwise a solution of 4(R)-benzyl-3-isovaleroyl-oxazolidin-2-one
(76.0 g, 0.291 mol) in THF (0.9 L). After stirring for 1 h, a solution
of 4 (74.4 g, 0.242 mol) in THF (250 mL) was added dropwise at
-70 °C. The reaction temperature was gradually raised from -70
to 0 °C over 2 h, and stirring was continued for 18 h at 0 °C. Then,
a 10% aqueous NH4Cl solution (115 mL) was added with cooling
and volatiles were removed under reduced pressure. The residue
was partitioned between Et2O and water, and the aqueous layer
was extracted with Et2O. The combined organics were washed with
brine and dried (MgSO4), and the residue was purified by FC
(EtOAc/hexane 1:5) to yield 5 (97.6 g, 83%) as white solid: mp
90-92 °C; TLC Rf ) 0.25 (EtOAc/hexane 1:5); HPLC tR 22.0 min;
[R]20D -13.6 ( 1.0 (c 1.0, CH2Cl2); IR (CH2Cl2) 3050, 2962, 1589,
1453, 1442, 1158, 1101, 807; 1H NMR (200 MHz, CDCl3) δ 1.02
(t, J ) 6, 6H), 2.0 (m, 1H), 2.18 (m, 1H), 2.7-3.0 (m, 3H), 3.78
(s, 3H), 4.02 (m, 2H), 4.25 (m, 1H), 4.60 (m, 1H), 5.10 (s, 2H),
6.7-7.0 (m, 5H), 7.15-7.45 (m, 8H). Anal. (C30H33NO5) C, H, N.
(2S)-2-(3-Benzyloxy-4-methoxy-benzyl)-3-methyl-butan-1-ol
(6). To a solution of benzyl-mercaptane (22.1 mL, 0.188 mol) in
dry THF (200 mL), cooled to 0 °C, was added dropwise 1.6 M
n-BuLi in hexane (88 mL, 0.141 mol) over 10 min. To the mixture
was then dropwise added 5 (45.8 g, 0.094 mmol) in THF (200 mL).
Stirring was continued at 0 °C for 1 h, followed by the addition of
a precooled (0 °C) suspension of LiAlH4 (14.2 g, 0.376 mol) in
THF (0.8 L). After reaction for 2 h at 0 °C, EtOAc (52 mL) was
added dropwise to the mixture at 0 °C, which was stirred for 10
min before adding a 1:1 mixture of THF-water (112 mL;
Caution: vigorous gas evolution), followed by subsequent addition
of 2 N H2SO4 (790 mL), water and Et2O. After separation of the
layers, the aqueous phase was extracted with Et2O, and the
combined organics were washed with water, dried (Na2SO4), and
concentrated. FC purification (EtOAc/hexane 1:4) yielded 6 (29.0
g, 98%) as an oil: TLC Rf ) 0.18 (EtOAc/hexane 1:3); HPLC tR
(2S)-2-Benzyloxy-4-(2-bromomethyl-3-methyl-butyl)-1-meth-
oxy-benzene (7). To a solution of 6 (61.4 g, 0.195 mol) in dry
CH2Cl2 (1.2 L) was added Ph3P (61.4 g, 0.234 mol) and,
subsequently, NBS (41.6 g, 0.234 mol) in portions at 0 to 10 °C.
After stirring for 18 h at ambient temperature, the mixture was
concentrated and the residue was purified by FC (EtOAc/hexane
1:4) give 7 (68.7 g, 93%) as pale colored solid: mp 61.5-63 °C;
TLC Rf ) 0.56 (EtOAc/hexane 1:3); HPLC tR 24.0 min; IR (CH2-
Cl2) 2960, 1588, 1464, 1442, 1386, 811; 1H NMR (CDCl3) δ 0.9-
1.0 (2d, 6H), 1.50 (m, 1H), 1.78 (m, 1H), 2.43 (dd, J ) 10, 15,
1H), 2.72 (dd, J ) 6, 15, 1H), 3.23 (dd, J ) 6, 10, 1H), 3.33 (dd,
J ) 6, 10, 1H), 3.87 (s, 3H), 5.15 (s, 2H), 6.70-6.85 (m, 3H),
7.2-7.5 (m, 5H). Anal. (C20H25BrO2) C, H; Br: calcd, 21.18; found,
20.74.
(2R,5S,2′S)-2-[2′-(3-Benzyloxy-4-methoxy-benzyl)-3-methyl-
butyl]-5-isopropyl-3,6-dimethoxy-2,5-dihydro-pyrazine (8). To
a solution of (2R)-2-isopropyl-3,6-dimethoxy-2,5-dihydro-pyrazine
(51.3 g, 0.273 mol) in THF (700 mL), cooled to -75 °C, was added
dropwise 1.6 M n-BuLi in hexane (170 mL, 0.273 mol) over 45
min. After 30 min at -75 °C, a solution of 7 (68.7 g, 0.182 mol)
in THF (0.3 L) was added dropwise over 30 min. The reaction
mixture was stirred for 2 h at -75 °C and overnight at -20 °C.
The mixture was concentrated, and the residue was partitioned
between EtOAc (3 × 1 L) and water (3 × 1 L). The combined
organics were washed with brine (1 L), dried (MgSO4), and
concentrated. FC purification (gradient CH2Cl2/hexane 1:1 to 2:1
to 10:0) and repeated chromatography of enriched fractions afforded
8 (82.1 g, 94%) as a pale yellow oil: TLC Rf ) 0.22 (CH2Cl2); 1H
NMR (300 MHz, CDCl3) δ 0.68 (d, J ) 7, 3H), 0.75 (m, 6H),
1.05 (d, J ) 7, 3H), 1.2-1.85 (m, 4H), 2.27 (m, 1H), 2.35 (dd, J
) 10, 15, 1H), 2.68 (dd, J ) 6, 15, 1H), 3.66 (s, 6H), 3.86 (s, 3H),
3.97 (m, 2H), 5.12 (s, 2H), 6.7-6.85 (m, 3H), 7.25-7.45 (m, 5H);
MS (EI) m/z 480 [M+].
(2S,4S)-4-(3-Benzyloxy-4-methoxy-benzyl)-2-tert-butoxycar-
bonylamino-5-methyl-hexanoic Acid Methyl Ester (9a). The
solution of 8 (82.1 g, 0.171 mol) in MeCN (0.68 L) and 1 N HCl
(0.68 L) was stirred at ambient temperature for 2 h. The mixture
was poured into an ice-cold saturated aqueous solution of NaHCO3
(2.5 L) and extracted with CH2Cl2 (3 × 1 L). The organic layers
were washed with water (2 × 1 L), dried (Na2SO4), and evaporated.
19.0 min; [R]20 +6.2 ( 1.8 (c 0.55, CHCl3); IR (CH2Cl2) 3618,
D
1
2958, 1588, 1514, 1464, 1369, 1232, 1139, 1028; H NMR (300
MHz, CDCl3) δ 0.92 (d, J ) 8, 6H), 1.52 (m, 1H), 1.55 (s, 1H),
1.76 (m, 1H), 2.40 (dd, J ) 10, 15, 1H), 2.60 (dd, J ) 10, 15, 1H),

4828 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Go¨schke et al.
FC purification (CH2Cl2/MeOH/NH3 concd 100:5:0.1) yielded
(2S,4S)-2-amino-4-(3-benzyloxy-4-methoxy-benzyl)-5-methyl-hex-
anoic acid methyl ester (55.5 g, 84%) as a pale yellow oil: TLC Rf
) 0.38 (CH2Cl2/MeOH/NH3 concd 700:10:10); HPLC tR 14.8 min;
1H NMR (300 MHz, CDCl3) δ 0.80-0.85 (m, 6H), 1.2-1.8 (m,
6H), 2.43 (d, J ) 7, 2H), 3.30 (m, 1H), 3.65 (s, 3H), 3.86 (s, 3H),
5.15 (s, 2H), 6.65-6.80 (m, 3H), 7.25-7.50 (m, 5H); MS (EI) m/z
385 [M]+.
To a solution of the above product (55.0 g, 0.143 mol) in CH2-
Cl2 (1.0 L), cooled to 0 °C, were subsequently added DIPEA (34.1
mL, 0.200 mol) and a solution of BOC2O (40.4 g, 0.185 mol) in
CH2Cl2 (200 mL). After warming to room temperature, the mixture
was stirred overnight and then concentrated to give an oil that was
FC purified (CH2Cl2/Et2O 10:0 to 8:2). Compound 9a (66.9 g, 97%)
was obtained as pale yellow oil: TLC Rf ) 0.66 (CH2Cl2/Et2O
8:2); HPLC tR 22.2 min; IR (CH2Cl2) 3433, 2956, 1588, 1437, 1367,
1026; 1H NMR (300 MHz, CDCl3) δ 0.7-0.8 (m, 6H), 1.35-1.65
(m, 13H), 2.35 (m, 1H), 2.62 (m, 1H), 3.70 (s, 3H), 3.86 (s, 3H),
4.35 (m, 1H), 4.84 (d, J ) 8, 1H), 5.16 (s, 2H), 6.65-6.85 (m,
3H), 7.25-7.50 (m, 5H). Anal. (C28H39NO6) C, H, N.
5.40 (s, 1H), 5.56 (s, 1H), 6.08 (t, 1H), 6.65-6.85 (m, 3H). Anal.
(C32H54N2O7) C, H, N.
Data for 12b: TLC Rf ) 0.16 (EtOAc/hexane 1:1); HPLC tR
1
25.6 min (solvent gradient within 35 min); H NMR (CDCl3) δ
0.85 (d, J ) 8, 6H), 0.93 (t, J ) 8, 3H), 1.2-1.75 (m, 8H), 1.43
(s, 9H), 2.09 (m, 2H), 2.25-2.5 (m, 3H), 2.6-2.7 (m, 1H), 3.30
(q, J ) 8, 2H), 3.33 (s, 3H), 3.56 (t, J ) 7, 2H), 3.5-3.75 (m,
2H), 3.82 (s, 3H), 4.10 (t, J ) 7, 2H), 4.75 (d, J ) 11, 1H), 4.90
(s, 1H), 5.34 (s, 1H), 5.60 (s, 1H), 6.27 (t, 1H), 6.66-6.83 (m,
3H).
(1S,2S,4R,2′S)-(4-Butylcarbamoyl-2-hydroxy-1-{2′-[4-meth-
oxy-3-(3-methoxy-propoxy)-benzyl]-3′-methyl-butyl}-pentyl)-
carbamic Acid tert-Butyl Ester (13b). A solution of 11b (81.0 g,
0.140 mol) in dry MeOH (800 mL) was hydrogenated at 25 bar
pressure in the presence of Ru2Cl4[(S)-BINAP)2]NEt3 (0.557 g) for
20 h at ambient temperature. Removal of solvent in vacuo gave
the crude product as about a 9:1 mixture of the (4R)- and (4S)-
diastereomers. Repeated recrystallization from Et2O/hexane (0.45:
1, 1.5 L) afforded the single isomer 13b (57 g, 70%) as a white
solid: mp 106.5-107 °C; TLC Rf ) 0.19 (EtOAc/hexane 2:1);
HPLC tR 18.2 min; IR (CH2Cl2) 3437, 2959, 1703, 1667, 1588,
1513, 1465, 1391, 1367, 1234; 1H NMR (DMSO-d6; 80 °C) δ 0.79
(t, J ) 8.0, 6H), 0.88 (t, J ) 7.3, 3H), 1.02 (d, J ) 6.9, 3H), 1.15-
1.7 (m, 10H), 1.42 (s, 9H), 1.93 (m, 2H), 2.32 (m, 1H), 2.42 (m,
1H), 2.60 (m, 1H), 3.06 (m, 2H), 3.26 (s, 3H), 3.34 (m, 1H), 3.47
(m, 1H), 3.49 (t, J ) 7, 2H), 3.70 (s, 3H), 4.00 (t, J ) 7, 2H), 4.14
(d, J ) 6.0, 1H), 5.72 (br s, 1H), 6.69 (dd, J ) 2, 8, 1H), 6.78 (d,
J ) 2, 1H), 6.81 (d, J ) 8, 1H), 7.23 (m, 1H). Anal. (C32H56N2O7)
C, H, N.
(1S,2S,4R,2′S)-{4-Butylcarbamoyl-2-hydroxy-1-[2′-(3-hydroxy-
4-methoxy-benzyl)-3-methyl-butyl]-pentyl}-carbamic Acid tert-
Butyl Ester (14). A solution of 13a (4.50 g, 7.51 mmol) in MeOH
(60 mL) was hydrogenated in the presence of 5% palladium on
charcoal (E 101N, Degussa; 0.9 g) for 60 min at ambient
temperature. Removal of the catalyst over Celite and evaporation
of the filtrates afforded 14 (3.78 g, quant.) as a colorless foam:
mp 113-114 °C (Et2O/hexane); TLC Rf ) 0.21 (CH2Cl2/MeOH
9:1); HPLC tR 18.0 min; FAB-MS m/z 509 [M + H]+; IR (CH2-
Cl2) 3535, 3436, 2960, 1666, 1591, 1456, 1366, 1239, 1170, 1029;
1H NMR (500 MHz, DMSO-d6, 70 °C) δ 0.75 (q, J ) 7, 6, 6H),
0.86 (t, J ) 7, 3H), 1.00 (d, J ) 7, 3H), 1.1-1.7 (m, 10H), 1.39
(s, 9H), 2.20 (m, 1H), 2.45 (m, 1H), 2.55 (dd, J ) 5, 15, 1H), 3.03
(q, J ) 6, 13, 2H), 3.30 (m, 1H), 3.48 (m, 1H), 3.72 (s, 3H), 4.21
(d, J ) 6, 1H), 5.88 (m, 1H), 6.53 (dd, J ) 2, 8, 1H), 6.59 (d, J )
2, 1H), 6.75 (d, J ) 8, 1H), 7.38 m, 1H), 8.33 (br s, 1H). Anal.
(C28H48N2O6) C, H, N.
(1S,3S,2′S,4′R)-[4-Methoxy-3-(3-methoxy-propoxy)-benzyl]-
4-methyl-1-(4′-methyl-5-oxo-tetrahydro-furan-2′-yl)-pentyl]-car-
bamic Acid tert-Butyl Ester (15). The mixture of 13b (5.64 g,
9.64 mmol) and p-TsOH‚H2O (2.02 g, 10.6 mmol) in CHCl3 (240
mL) was kept for 18 h at ambient temperature, followed by
evaporation to a small volume. Et2O was added, and the organic
layer was washed with 0.1 M HCl and brine, dried (MgSO4), and
evaporated. FC purification (EtOAc/hexane 1:1) and recrystalliza-
tion from Et2O/hexane yielded 15 (3.67 g, 75%): mp 86-88 °C;
TLC Rf ) 0.47 (EtOAc/hexane 2:1); HPLC tR 17.2 min; 1H NMR
(CDCl3) δ 0.82 (d, J ) 8, 6H), 1.26 (d, J ) 8, 3H), 1.45 (s, 9H),
1.4-1.75 (m, 4H), 1.90 (m, 1H), 2.10 (t, J ) 6, 2H), 2.3-2.45 (m,
2H), 2.6-2.75 (m, 2H), 3.35 (s, 3H), 3.56 (t, J ) 7, 2H), 3.75-
3.9 (m, 1H), 3.83 (s, 3H), 4.10 (t, J ) 7, 2H), 4.37 (d, J ) 2, 1H),
4.45 (m, 1H), 6.67 (dd, J ) 2, 8, 1H), 6.72 (d, J ) 2, 1H), 6.78 (d,
J ) 8, 1H); MS(FAB) m/z 507 [M + H]+. Anal. (C28H45NO7) C,
H, N.
(2R,4S,5S,7S)-5-tert-Butoxycarbonylamino-4-(tert-butyl-dim-
ethyl-silanyloxy)-7-[4-methoxy-3-(3-methoxy-propoxy)-benzyl]-
2,8-dimethyl-nonanoic Acid (17). To a solution of 15 (3.60 g,
7.12 mmol) in 1,2-DME/H2O (2:1; 210 mL) was added 1 M LiOH
(28.5 mL, 28.5 mmol). After 1 h at room temperature, volatiles
were removed in vacuo, followed by addition of an ice-cold 10%
aqueous solution of citric acid (60 mL). Extraction of the water
phase with CHCl3 (4 × 150 mL), drying of the combined organics
(1S,3S)-{1-Formyl-3-[4-methoxy-3-(3-methoxy-propoxy)-ben-
zyl]-4-methyl-pentyl}-carbamic Acid tert-Butyl Ester (10b). The
stirred solution of 9b26 (193 g, 0.40 mol) in toluene (2 L) was cooled
to -70 °C, and a precooled 20% solution of DIBAH in toluene
(800 mL, 0.96 mol) was added over 5 min at -75 to -80 °C (liquid
ammonia cooling bath). Stirring at -75 °C was continued for 45
min, followed by addition of MeOH (80 mL) over 80 min while
maintaining the temperature between -70° and -60 °C (Caution:
vigorous gas evolution). The reaction mixture was poured with
stirring into a mixture of 1 N HCl (4 L) and ice-water (2 kg),
followed by addition of EtOAc (3 L). After 10 min, the organic
layer was separated, and the aqueous phase was extracted twice
with EtOAc (3 L). The combined organics were washed with water
(2 × 4 L) and brine (1 × 4 L) and filtered over Hyflo. The filtrates
were concentrated, taken up in toluene (1 L), concentrated, and
dried in high vacuo at ambient temperature over 2 h to afford crude
10b (190 g, g97%) as a pale yellow oil: TLC Rf ) 0.44 (EtOAc/
hexane 1:1); [R]20D +14.6 ( 1.3 (c 0.78, CHCl3); IR (CH2Cl2) 3431,
1
2959, 1710, 1589, 1514; H NMR (CDCl3) δ 0.86 (m, 6H), 1.44
(s, 9H), 1.55-1.8 (m, 4H), 2.09 (m, 2H), 2.4-2.7 (m, 2H), 3.35
(s, 3H), 3.57 (t, J ) 7, 2H), 3.83 (s, 3H), 4.10 (t, J ) 7, 2H),
4.05-4.4 (m, 1H), 4.89 (br d, J ) 9, 1H), 6.8-6.8 (m, 3H), 9.50
(s, 1H).
(1S,2S,2′S)- and (1S,2R,2′S)-(4-Butylcarbamoyl-2-hydroxy-
1-{2′-[4-methoxy-3-(3-methoxy-propoxy)-benzyl]-3′-methyl-bu-
tyl}-pent-4-enyl)-carbamic Acid tert-Butyl Ester (11b) and (12b).
To a solution of N-butylmethacrylamide,20 (see also Supporting
Information; 82.8 g, 0.587 mol) in THF (1.3 L) was added a 1.6 M
solution of n-BuLi in hexane (0.765 L, 1.22 mol) over 15 min at
-78 °C. After warming to 0 °C for 30 min and recooling to -78
°C, the red-orange solution was subsequently treated with a 1 M
solution of Ti(Oi-Pr)3Cl in hexane (0.931 L, 0.931 mol) for 55 min
and a solution of 10b (126 g, 0.266 mol) in THF (0.7 L) for 15
min. Stirring of the dark colored solution for an additional 75 min
at -78 °C was followed by quenching of the reaction by adding a
saturated aqueous solution of NH4Cl (1.6 L) at -20 °C. Then Et2O
(2 L) was added, and the mixture was stirred at 0-5 °C for 20
min. The organic layer was separated, and the aqueous phase was
extracted with Et2O (2 × 3 L). The combined organics were washed
with water (2 × 4 L) and brine (3 L), dried (MgSO4), and
concentrated to provide the crude product as a mixture of the (2S)-
and (2R)-configured diastereoisomers 11b/12b in about a 2:3 ratio.
Repeated FC purification on silica gel (EtOAc/hexane, gradient from
1:3 to 1:1) provided the pure isomers 11b (76 g, 16.4%) and 12b
(188 g, 40%) as colorless oils.
Data for 11b: TLC Rf ) 0.21 (EtOAc/hexane 1:1); HPLC tR
1
26.4 min (solvent gradient within 35 min); H NMR (CDCl3) δ
0.83 (m, 6H), 0.93 (t, J ) 8, 3H), 1.05-1.8 (m, 8H), 1.43 (s, 9H),
2.09 (m, 2H), 2.28-2.51 (m, 3H), 2.65-2.75 (m, 1H), 3.30 (q, J
) 8, 2H), 3.33 (s, 3H), 3.56 (t, J ) 7, 2H), 3.5-3.7 (m, 2H), 3.82
(s, 3H), 4.10 (t, J ) 7, 2H), 4.85 (d, J ) 11, 1H), 5.10 (s, 1H),

Potent and Orally ActiVe Inhibitors of Human Renin
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4829
(MgSO4), and evaporation of solvents gave the crude product 16,
which was used directly in the next reaction step.
3H), 1.15-1.5 (m, 11H), 1.55 (m, 1H), 1.65-1.75 (m, 3H), 1.80
(m, 1H), 2.35-2.5 (m, 2H), 2.55 (m, 1H), 2.70 (m, 1H), 3.03 (m,
2H), 3.30 (m, 1H), 3.70 (s, 3H), 3.92 (t, J ) 7, 2H), 5.47 (br s,
1H), 6.70 (dd, J ) 2, 10, 1H), 6.8-6.85 (m, 2H), 7.66 (br s, 3H),
7.92 (m, 1H); HRMS m/z 479.3883 [(M + H)+ calcd for
C28H50N2O4+, 479.3849].
Compound 38a‚HCl. Compound 38a‚HCl was obtained as
described above by dissolving 13b (1.13 g, 1.94 mmol; obtained
by method B) in 4 N HCl/dioxane (40 mL), cooled to 0 °C, followed
by reaction at room temperature for 30 min and subsequent freeze-
drying in high vacuo to afford the product (0.99 g, 99%) as a pale
beige foam: TLC Rf ) 0.13 (CH2Cl2/MeOH 9:1); HPLC tR 23.7
min (97.0% purity); HRMS m/z 481.3641 [(M + H)+ calcd for
C27H48N2O5+, 481.3615].
C. Direct Aminolysis of Lactone 15 (Scheme 3). (1S,2S,4R,2′S)-
(2-Hydroxy-4-(3-hydroxy-propylcarbamoyl)-1-{2′-[4-methoxy-
3-(3-methoxy-propoxy)-benzyl]-3-methyl-butyl}-pentyl)-car-
bamic Acid tert-Butyl Ester (20). Lactone 15 (0.102 g, 0.20 mmol)
and neat 3-amino-1-propanol (0.50 mL, 6.57 mmol) were heated
at 80 °C for 1 h. To the reaction mixture was added ice-cold 1 N
HCl (30 mL), followed by extraction of the water phase with EtOAc
(2 × 25 mL). The combined organics were washed with H2O (1 ×
30 mL) and a saturated solution of NaHCO3 (1 × 20 mL) and dried
(MgSO4). FC purification (CH2Cl2/MeOH 9:1) and recrystallization
from Et2O yielded 20 (0.095 g, 82%) as a white solid: mp 100-
101 °C; TLC Rf ) 0.43 (CH2Cl2/MeOH 9:1); HPLC tR 16.5 min;
IR (CH2Cl2) 3436, 2959, 2935, 1702, 1654, 1514, 1237, 1163; 1H
NMR (DMSO-d6) δ 0.73 (t, J ) 7, 6H), 0.95-1.5 (m, 7H), 0.98
(d, J ) 7, 3H), 1.37 (s, 9H), 1.90 (m, 2H), 2.21 (m, 1H), 2.4-2.55
(m, 3H), 2.63 (m, 1H), 3.07 (m, 2H), 3.26 (s, 3H), 3.38 (m, 2H),
3.44 (m, J ) 7, 2H), 3.48 (m, 1H), 3.70 (s, 3H), 3.94 (m, 2H),
4.38 (m, 2H), 6.17 (d, J ) 10, 1H), 6,64 (dd, J ) 2, 8, 1H), 6.76
(d, J ) 2, 1H), 6.78 (d, J ) 8, 1H), 7.64 (t, J ) 6, 1H). Anal.
(C31H54N2O8) C, H, N.
To a solution of the above material in dry DMF (45 mL) was
added TBDMSiCl (2.36 g, 15.66 mmol) and imidazole (2.03 g,
29.9 mmol), and the mixture was kept for six days at room
temperature. The solvent was removed under reduced pressure, and
the residue was partitioned between EtOAc and a 10% aqueous
solution of citric acid. The layers were separated, and the organic
phase was washed with water and brine and dried (MgSO4). After
evaporation of the solvent, the residue (TLC Rf ) 0.82; EtOAc/
hexane 1:1) was dissolved in THF (20 mL), water (8 mL) and acetic
acid (20 mL), and the mixture was stirred at ambient temperature
overnight. After removal of the solvent, ice/water was added to
the residue, followed by extraction with EtOAc (3 × 250 mL).
The combined organics were washed with water and brine, dried
(MgSO4), and evaporated. FC purification (EtOAc/hexane (1:1)
gave 17 (3.11 g, 68%) as a colorless oil: TLC Rf ) 0.25 (EtOAc/
1
hexane 1:1); HPLC tR 24.0 min; H NMR (DMSO-d6; 80 °C) δ
0.08 (s, 6H), 0.80 (m, 6H), 0.88 (s, 9H), 1.09 (d, J ) 7, 3H), 1.25
(m, 2H), 1.40 (m, 1H), 1.42 (s, 9H), 1.55-1.7 (m, 2H), 1.88 (m,
1H), 1.92 (m, 2H), 2.31 (m, 1H), 2.45 (m, 1H), 2.57 (m, 1H), 3.26
(s, 3H), 3.49 (t, J ) 7, 2H), 3.61 (m, 1H), 3.70 (m, 1H), 3.74 (s,
3H), 4.00 (t, J ) 7, 2H), 5.76 (br s, 1H), 6.68 (d, J ) 8, 1H), 6.76
(s, 1H), 6.82 (d, J ) 8, 1H); FAB-MS m/z 640 [M + H]+. Anal.
(C34H61NO8Si) C, H, N.
General Procedures. A. O-Alkylation of Intermediate 14
(Scheme 3). The general procedure is exemplified by the synthesis
of compound 18: To a suspension of NaH (60-65% in mineral
oil; 14 mg, 0.324 mmol) in DMF (2 mL) was added dropwise a
solution of 14 (0.150 g, 0.295 mmol) in DMF (1 mL) at ambient
temperature. After 30 min, a solution of n-pentyliodide (117 mg,
0.590 mmol) in DMF (1 mL) was added and stirring was continued
for 16 h. Solvents were evaporated, the residue was dissolved in
H2O (15 mL) followed by extraction of the aqueous layer with Et2O
(2 × 20 mL). The organics were dried (MgSO4) and evaporated,
and the residue was purified by flash chromatography (CH2Cl2/
Et2O 2:1). Recrystallization from Et2O/hexane afforded 18 (0.120
g, 70%) as a white solid: mp 104-105 °C; TLC Rf ) 0.21 (CH2-
Cl2/MeOH 9:1); HPLC tR 23.7 min; IR (CH2Cl2) 3437, 2960, 2934,
Inhibitors 44-46, 48-57, 60, 63, and 70-74 (Tables 3-5) were
synthesized in a similar manner as described above.
D. Coupling to Acid 17 and Removal of the O-TBDMSi
Protecting Group (Scheme 4). (2R,4S,5S,7S)-3-{5-tert-Butoxy-
carbonylamino-4-(tert-butyl-dimethyl-silanyloxy)-7-[4-methoxy-
3-(3-methoxy-propoxy)-benzyl]-2,8-dimethyl-nonanoylamino}-
propionic Acid Ethyl Ester (21). To a solution of 17 (0.160 g,
0.25 mmol) in DMF (10 mL) at 0 °C was added subsequently Et3N
(0.084 mL, 0.60 mmol), â-alanine ethylester HCl (0.046 g, 0.30
mmol), and diethyl cyanophosphonate (0.048 mL, 0.30 mmol). After
stirring for 20 h at room temperature, the mixture was concentrated
and a 10% aqueous solution of citric acid (20 mL) was added.
Extraction with EtOAc (2 × 20 mL) was followed by washing of
the organics with a saturated NaHCO3 (20 mL) and brine (20 mL).
FC purification (EtOAc/hexane 1:1) afforded 21 (0.174 g, 94%)
as a colorless oil: TLC Rf ) 0.28 (EtOAc/hexane 1:1); HPLC tR
1
1703, 1514, 1236, 1164; H NMR (DMSO-d6) δ 0.71 (m, 6H),
0.86 (m, 6H), 0.97 (d, J ) 7, 3H), 1.0-1.7 (m, 16H), 1.34 (s, 9H),
2.20 (dd, J ) 10, 15, 1H), 2.43 (m, 1H), 2.61 (dd, J ) 5, 15, 1H),
3.01 (m, 2H), 3.25 (m, 1H), 3.49 (m, 1H), 3.69 (s, 3H), 3.88 (m
2H), 4.38 (d, J ) 7, 1H), 6.17 (d, J ) 10, 1H), 6.63 (dd, J ) 2, 10,
1H), 6.76 (d, J ) 2, 1H), 6.78 (d, J ) 10, 1H), 7.60 (m, 1H). Anal.
(C33H58N2O6) C, H, N.
Compound 13b (Method B). Compound 13b obtained as
described above from 14 (1.2 g, 2.36 mmol), NaH (60-65% in
mineral oil; 103 mg, 2.59 mmol) and 3-methoxypropyliodide (0.63
g, 3.16 mmol) in DMF (80 mL) followed by FC purification on
silica gel (CH2Cl2/MeOH 95:5) afforded the product as a white
foam (1.18 g, 86%): TLC Rf ) 0.52 (CH2Cl2/MeOH 95:5); FAB-
MS m/z 582 [M + H]+.
The N-Boc derivatives of compounds 28, 29, 34 (Table 1), and
36-42 (Table 2) were obtained in a similar manner by using the
corresponding alkyl halides. The N-Boc derivative 30 was obtained
by the reaction of N-Boc-28 (28.8 mg, 0.05 mmol) with methy-
lamine (1 mL) in DMF (3 mL) in a sealed tube for 60 h at room
temperature to give, after FC purification (CH2Cl2/MeOH 9:1), a
colorless oil (18.6 mg, 62%).
B. N-Boc-Deprotection. The renin inhibitors listed in Tables
1-5 were obtained, if not otherwise stated, as their hydrochloride
salts by final N-Boc-deprotection under standard reaction conditions
as described for the following examples:
(2R,4S,5S,7S)-5-Amino-4-hydroxy-7-(4-methoxy-3-pentyloxy-
benzyl)-2,8-dimethyl-nonanoic Acid Butylamide, Hydrochloride
Salt (43‚HCl). A solution of 18 (53 mg, 0.092 mmol) in 4 N HCl/
dioxane (1 mL) was rigorously stirred for 2 h at 0 °C. Subsequent
freeze-drying in high vacuo gave 43‚HCl (47 mg) as an amorphous
solid: TLC Rf ) 0.22 (CH2Cl2/MeOH 9:1); HPLC tR 16.7 min; 1H
NMR (DMSO-d6) δ 0.80 (m, 6H), 0.86 (m, 6H), 1.01 (d, J ) 7,
1
27.2 min; H NMR (DMSO-d6) δ 0.02 (s, 3H), 0.05 (s, 3H), 0.74
(t, J ) 7, 6H), 0.85 (s, 9H), 0.99 (d, J ) 7, 3H), 1.05-1.6 (m,
5H), 1.18 (t, J ) 7, 3H), 1.42 (s, 9H), 1.8-2.65 (m, 5H), 1.90 (m,
2H), 3.2-3.65 (m, 5H), 3.26 (s, 3H), 3.44 (t, J ) 7, 2H), 3.69 (s,
3H), 3.93 (t, J ) 7, 2H), 4.04 (m, 2H), 6.31 (d, J ) 10, 1H), 6.63
(dd, J ) 2, 8, 1H), 6.71 (d, J ) 2, 1H), 6.78 (d, J ) 8, 1H), 7.64
(t, J ) 6, 1H). Anal. (C39H70N2O9Si) C, H , N.
(2R,4S,5S,7S)-3-{5-tert-Butoxycarbonylamino-4-hydroxy-7-[4-
methoxy-3-(3-methoxy-propoxy)-benzyl]-2,8-dimethyl-nonanoy-
lamino}-propionic Acid Ethyl Ester (22). A solution of 21 (0.155
g, 0.210 mmol) and tetrabutyl-ammonium-fluoride trihydrate (0.132
g, 0.420 mmol) in DMF (10 mL) was stirred for 2 h at ambient
temperature. The mixture was concentrated, and the residue was
partitioned between EtOAc (2 × 30 mL) and water-brine (1:1, 30
mL). The usual workup and FC purification (EtOAc/hexane 2:1)
gave 22 (0.105 g, 80%) as a colorless oil: mp 85-86 °C
(recrystallized from Et2O/hexane); TLC Rf ) 0.12 (EtOAc/hexane
2:1); HPLC tR 17.5 min; 1H NMR (CDCl3) δ 0.83 (m, 6H), 1.05-
1.75 (m, 6H), 1.15 (d, J ) 7, 3H), 1.27 (t, J ) 7, 3H), 1.45 (s,
9H), 2.08 (m, 2H), 2.35-2.65 (m, 5H), 3.35 (s, 3H), 3.4-3.6 (m,
5H), 3.57 (t, J ) 7, 2H), 3.82 (s, 3H), 4.10 (t, J ) 7, 2H), 4.15 (m,

4830 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Go¨schke et al.
2H), 4.67 (d, J ) 12, 1H), 6.32 (br m, 1H), 6.70 (dd, J ) 2, 8,
1H), 6.77 (d, J ) 8, 1H), 6.78 (m, 1H). Anal. (C33H56N2O9) C, H,
N.
after administration of the inhibitor) by the antibody trapping
method48 from blood samples collected by direct puncture of a
femoral vein using EDTA as an anticoagulant. For each animal,
percentage inhibition of PRA at each time point was calculated
using the pretreatment PRA as the baseline.
(2R,4S,5S,7S)-3-{5-tert-Butoxycarbonylamino-4-hydroxy-7-[4-
methoxy-3-(3-methoxy-propoxy)-benzyl]-2,8-dimethyl-nonanoy-
lamino}-propionic Acid (23). To a solution of 22 (70 mg, 0.112
mmol) in MeOH (2 mL) was added 1 N NaOH (0.224 mL, 0.224
mmol). After 18 h at ambient temperature, the volatiles were
removed under reduced pressure. The residue was taken up in water
(15 mL) and the aqueous phase was acidified by adding 1 N HCl
(0.25 mL). Extractive workup with EtOAc (2 × 15 mL) and FC
purification (CH2Cl2/MeOH 8:2) afforded 23 (60 mg, 90%) as a
white amorphous solid: TLC Rf ) 0.12 (CH2Cl2/MeOH 9:1); HPLC
Acknowledgment. We are grateful to Dr. Eric Francotte and
Mr. Paul Richert for analytical chiral HPLC separations. The
authors thank Dr. Nils Ostermann for providing the illustrations
in Figures 3 and 4 and for critical discussions. Richard Go¨schke
is indebted to Ms. Nicole Hasler and Ms. Florence Kummli-
Lugrin for their excellent technical assistance. Technical and
editorial assistance was provided by Geoff Davison (ACUMED,
Macclesfield, U.K.).
1
tR 16.8 min; H NMR (DMSO-d6) δ 0.74 (t, J ) 7, 6H), 0.97 (d,
J ) 7, 3H), 1.0-1.65 (m, 5H), 1.39 (s, 9H), 1.95 (m, 2H), 2.1-
2.25 (m, 3H), 2.43 (m, 1H), 2.64 (m, 1H), 3.15-3.55 (m, 6H),
3.23 (s, 3H), 3.30 (s, 1H, COOH + H2O), 3.44 (t, J ) 7, 2H), 3.70
(s, 3H), 3.94 (t, J ) 7, 2H), 6.16 (d, J ) 10, 1H), 6.65 (dd, J ) 2,
8, 1H), 6.75 (d, J ) 2, 1H), 6.78 (d, J ) 8, 1H), 7.64 (br m, 1H).
Inhibitors 47-48, 58-62, 64-69, and 75-77 (Tables 3-5) were
synthesized in a similar manner as described above.
Supporting Information Available: Additional data on analyti-
cal characterization of the final inhibitor compounds, experimental
procedures for the preparation of N-butyl-2-methyl-acrylamide,
compounds 10a, 11a, 12a, 13a, 24-26, 44, 48, 58, and 60, as well
as for P2′ amines used in the synthesis of inhibitors 57, 67, 71-
73, and 77, and X-ray crystallographic data for compound 13b.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Large-Scale Preparation of Inhibitor 38a, Hydrochloride Salt.
To a solution of 13b (57 g, 0.098 mol) in EtOAc (300 mL) was
added 4 N HCl in EtOAc (600 mL) at -5 °C. The mixture was
stirred for 18 h at 0-2 °C, followed by removal of the solvent
under reduced pressure at room temperature. The residue was dried
in high vacuo at 35 °C over 48 h to yield the title compound (50.8
g) as a white amorphous powder: mp 60-70 °C; TLC Rf ) 0.24
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(CH2Cl2/MeOH 9:1); HPLC tR 11.8 min; [R]20 -29.4 ( 0.8 (c
D
1.2, CHCl3); IR (CH2Cl2) 2959, 1661, 1515, 1465, 1388, 1235; 1H
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