Design and optimization of a substituted amino propanamide series of renin inhibitors for the treatment of hypertension

Article abstract of DOI:10.1016/j.bmcl.2010.02.036

The discovery and SAR of a new series of substituted amino propanamide renin inhibitors are herein described. This work has led to the preparation of compounds with in vitro and in vivo profiles suitable for further development. Specifically, challenges p

Full text of DOI:10.1016/j.bmcl.2010.02.036

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2204–2209
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and optimization of a substituted amino propanamide series of
renin inhibitors for the treatment of hypertension
a
c
c
a
Austin Chen a, , Christopher Bayly , Olivier Bezençon , Sylvia Richard-Bildstein , Daniel Dubé ,
Laurence Dubé a, Sébastien Gagné a, Michel Gallant a, Mireille Gaudreault a, Erich Grimm a, Robert Houle a,
Patrick Lacombe a, Sébastien Laliberté a, Jean-François Lévesque a, Suzanna Liu a, Dwight MacDonald a,
*
a
a
a
a
b
ˇ c
Bruce Mackay , David Martin , Dan McKay , David Powell , L’uboš Remen , Stephen Soisson ,
Sylvie Toulmond a
a Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Highway, Kirkland, Québec, Canada H9H 3L1
b Merck Research Laboratories, Sumneytown Pike, PO Box 4, West Point, PA 19486, United States
c Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, CH-4123 Allschwil, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and SAR of a new series of substituted amino propanamide renin inhibitors are herein
described. This work has led to the preparation of compounds with in vitro and in vivo profiles suitable
for further development. Specifically, challenges pertaining to oral bioavailability, covalent binding and
time-dependent CYP 3A4 inhibition were overcome thereby culminating in the identification of com-
pound 50 as an optimized renin inhibitor with good efficacy in the hypertensive double-transgenic rat
model.
Received 18 November 2009
Accepted 8 February 2010
Available online 18 February 2010
Keywords:
Renin
Inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
Anti-hypertensive
Protease
The renin–angiotensin–aldosterone system (RAAS, Fig. 1) plays
a key role in the regulation of extracellular fluid volume and blood
pressure.1 Over the past decades, agents capable of antagonizing
the RAAS pathway at its various steps (i.e., angiotensin-converting
enzyme inhibitors, and angiotensin II receptor blockers) have
emerged as effective treatments for hypertension.2 Despite these
significant advances and the availability of therapies that operate
distinctly from the RAAS pathway (i.e., diuretics, b-blockers and
calcium channel blockers)3, hypertension remains poorly con-
trolled in many patients and often requires combination therapy
to achieve the targeted blood pressure lowering.4 Since the cleav-
age of angiotensinogen by renin represents the first and rate-lim-
iting step of the RAAS cascade, the maximum benefit that can be
gleaned from the modulation of this pathway could come in the
form of a direct inhibitor of renin.5,6
Despite significant research investments from the pharmaceuti-
cal industry directed towards the discovery of renin inhibitors suit-
able for clinical development,7,8 only Aliskiren (1, Fig. 2) has gained
FDA approval for the treatment of hypertension.9
All of the first generation renin inhibitors synthesized during
the 1980s such as remikiren (RO 42-5892, 2, Fig. 2), were pepti-
* Corresponding author. Tel.: +1 514 428 2770.
E-mail address: austin_chen@merck.com (A. Chen).
Figure 1. The renin–angiotensin–aldosterone system.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.036

A. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2204–2209
2205
Figure 2. Representative renin inhibitors.
domimetics and possessed identical peptide hydrolysis transition
state isostere.10 Consequently, these compounds often suffered
from high molecular weight, limited metabolic stability and low
oral bioavailability. While efforts to decrease the peptidic character
eventually culminated in the discovery of second generation renin
inhibitors such as Aliskiren,9 vestiges of the peptide backbone and
side chains present in the first generation renin inhibitors
remained as key structural features. Consequently, these second
generation compounds were also plagued by low oral exposure
in preclinical species and man. The first significant structural
departure from these original scaffolds came from the recognition
that the nitrogen of 3,4-disubstituted piperidines (3),11 when pro-
tonated, would bind strongly to the aspartic acid dyad at the renin
active site. Further structural diversifications have since revealed
that morpholine (4),12 piperazine (5)13 and pyrrolidine (6),14 as
well as their bicyclic analogues (8, 9, 10),15,16 along with diami-
no-pyrimidine (7),17 could also serve as suitable warheads for
renin inhibition.
centers, we were intrigued by the idea that compounds bearing a
simple methylene amine warhead could serve as a suitable starting
scaffold. For the initial proof-of-concept experiment, direct map-
ping of this functionality onto the known diaza-bicyclononene re-
nin inhibitor 9 was envisioned (Fig. 3). Indeed, using well-known,
high-yielding reactions, compound 11 could be rapidly accessed
from the known bromoarene 13 in 7 steps (Scheme 1). Pd-cata-
lyzed formylation of 13 under standard conditions with sodium
formate as the reducing agent afforded aldehyde 14 in 81% yield.18
Subsequent Knoevenagel condensation of 14 with methyl cyano-
acetate in the presence of catalytic amount of piperidine was car-
ried out quantitatively.
a,b-Unsaturated cyanoester 15 thus ob-
tained could then be hydrogenated in CHCl3-spiked MeOH with
Pd black as the catalyst before the resulting amine was protected
as the Boc-carbamate 16 in 42% overall yield. Hydrolysis to carbox-
ylic acid 17 with aq lithium hydroxide followed by coupling with
amine 18 both proceeded without incidents. Finally, removal of
the Boc-protecting group from amide 19 was accomplished with
a large excess of HCl and afforded 11 in 85% yield over three
steps.15 Synthesis of compound 12 on the other hand, commenced
Motivated by the need to simplify the often laborious synthesis
of typical renin inhibitors; many of which display multiple stereo-

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A. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2204–2209
Figure 3. Design of amino methylene warhead scaffold.
Scheme 1. Synthesis of 11 and 12. Reagents and conditions: (a) Pd(PPh3)2Cl2, CO, NaCO2H, DMF, 110 °C, 8 h, 81%; (b) methyl cyano-acetate, piperidine, PhH, 80 °C, 5 h, >99%;
(c) Pd/C, H2, MeOH, CHCl3, 14 h; (d) BOC2O, 1 N aq NaOH, CH2Cl2, 14 h, 42% over two steps; (e) 2 N aq LiOH, MeOH, THF, 16 h, >99%; (f) 18, HATU, Hunig’s base, DMF, 5 h, 75%;
(g) 4 M HCl in dioxane, CH2Cl2, 4 h, 85%; (h) 18, HATU, Hunig’s base, DMF, 15 h, 95%; (i) piperidine, PhH, 80 °C, 18 h, >99%; (j) MeCN, nBuLi, À78 °C, 45 min, then CuI, À30 °C,
30 min, then 21, À30 °C to rt, 2 h, 66%; (k) 1 N aq LiOH, MeOH, reflux, 24 h, 90%; (l) Pt/C, H2, AcOH, 23%.
with the coupling of ethyl malonate with amine 18. Knoevenagel
condensation between amide 20 and aldehyde 14 then afforded
We were pleased to observe that compound 11, synthesized as
a racemate, was a potent inhibitor of renin in both the absence or
presence of human plasma, with an IC50 of 0.79 nM and 21 nM,
respectively (Table 1). On the other hand, its constitutional isomer
12 was >800-fold less potent in the renin buffer assay. While toler-
ated, methylation of the primary amine in 11 using standard
reductive amination conditions did not further improve the intrin-
sic potency against renin and the resulting compound was found to
be more plasma-shifted. On the other hand, methylation of the
amide-bearing carbon in 11 to furnish 26 led to a precipitous drop
in intrinsic potency.
a,b-unsaturated amide 21 as a ꢀ1.2:1 mixture of geometric iso-
mers. Amide 21 readily underwent conjugate addition when trea-
ted with the cuprate prepared in situ from lithiated acetonitrile
and copper(I) iodide.19 When the resulting amide 22, a ꢀ2.2:1 mix-
ture of diastereomers, was heated to reflux in the presence of aque-
ous lithium hydroxide in methanol, ester hydrolysis followed by
decarboxylation took place and afforded amide 23 in 90% yield.
Finally, reduction of the cyano group could be accomplished in ace-
tic acid with Pt/C as the hydrogenation catalyst.

A. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2204–2209
2207
Table 1
Table 3
SAR of select renin compounds: core modifications
SAR of select renin compounds: P3 arene modifications
R1
R2
R3
X
Renin IC50 (nM)
a,b
Compound
R1
R2
Renin IC50 (nM)
Buffer Plasma
a,b
Compd
Buffer
Plasma
27
28
29c
30
31
32
33
34
35
36c
37
38
39
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
Cl
H
H
H
H
H
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
0.40
0.26
0.09
0.21
0.19
2.9
29
11
12
24
25
26
H
—
Me
CH2Ph
H
H
—
H
H
0.79
650
1.9
260
210
21
NDc
350
NDc
NDc
(CH2)3OMe
(CH2)3OMe
O(CH2)2OMe
(CH2)2OMe
CH2N(Me)Ac
(CH2)3N(Me)2
(CH2)3OMe
(CH2)2CN
5.8
2.4
18
7.0
250
103
31
Me
a
b
c
See Ref. 20 for assay protocols.
Average of at least two replicates.
ND = not determined.
1.5
0.26
0.27
0.11
0.09
0.19
0.3
10
(CH2)3CN
4.6
6.4
2.2
1.8
(CH2)3OH
(CH2)3OMe
(CH2)3OMe
N+–OÀ
The discovery that related compounds possessing the 2-chloro-
a
b
c
3,6-difluorophenol residue can bind covalently to protein in the
presence of NADPH and liver microsomes15 necessitated a re-opti-
mization of the northern chain. Gratifyingly, it was found that a
switch to the 2-(2,6-dichloro-4-methylphenoxy)ethoxy northern
terminus led to a decrease in the compound’s covalent binding po-
tential (Table 2) but without jeopardizing its potency against renin
(vide infra).
Computer modeling suggested that re-optimization of the ben-
zyl amine aromatic plate would offer the best opportunity for
improving renin potency. Namely, we hypothesized that the intro-
duction of an aliphatic chain of an appropriate length and function-
ality at the meta position of the benzyl amide residue would better
anchor the substrate to the renin enzyme by filling the still vacant
S3 sub-pocket. In this task, a 3-methoxypropyl tail proved to be
optimal (Table 3). Furthermore, since the renin inhibition observed
for racemate 28 should be ascribed specifically to one of its two
antipodes, a stereoselective synthesis of the (S)-enantiomer was
See Ref. 20 for assay protocols.
Average of at least two replicates.
(S)-Enantiomer only.
carried out via an adaptation of a sequence previously developed
by Hintermann and Seebach for the synthesis of unnatural, homol-
ogated amino acids.22 Indeed, the ꢀ2-fold increase in potency ob-
served on going from 28 to 29 confirmed that the active
enantiomeric form to be of the (S)-configuration.
Compound 29 was chosen for further profiling. The requisite
amine 49 could be accessed from ester 44 using the chemistry dis-
closed in Scheme 2. Specifically, after an initial Suzuki-coupling of
44 with commercially available pinacolboronate 45, subsequent
hydrogenation of the double bond in 46 was best accomplished
with Wilkinson’s catalyst on a Parr shaker (50 psi H2). Ester 47 thus
obtained was then converted into aldehyde 48 via a two-step se-
quence: reduction with DIBAl-H followed by Swern oxidation.23 Fi-
nally, reductive amination with cyclopropylamine afforded amine
49. When compound 29 was formulated either as a hemifumarate
or HCl salt, moderate to good oral exposure was observed in SD
rats, and beagle dogs (Table 4). When tested in a telemetered dou-
ble-transgenic rat (dTGR) cohort (n = 8)24, maximum blood pres-
sure lowering of 27 mm Hg could be achieved at a P.O. dose of
3 mpk. Duration of action was found to exceed 48 h. Although
compound 29 did exhibit worrisome affinity for the hERG channel
(220 nM), none of the key CV intervals (i.e., QTc, PR, QRS) were sig-
nificantly prolonged (<5%) when 29 was given intravenously to
Table 2
Covalent binding potential of different northern terminia
Terminus
A
Species
Rat
Additive
pmol equiv/(mg @ 1 h)
beagle dogs at up to 12 lM plasma exposure. Unfortunately, com-
pound 29 was found to inhibit CYP 3A4 activity in a time-depen-
dent manner and thus possesses the potential to perpetrate
drug–drug interaction when co-dosed with drugs known to be
cleared mainly by CYP 3A4 metabolism (e.g., simvastatin).
Given the promiscuous nature of the CYP 3A4 pocket, it proved
highly challenging to completely dial out the time-dependent inhi-
bition associated with this structural series while at the same time
maintaining potency against renin. After much optimization, it was
observed that the installation of a second appendage onto the east-
ern aromatic plate (e.g., 50, Table 4) did lead to a significant
improvement in CYP profile but without jeopardizing the
None
NADPH
None
82
415
71
Human
NADPH
508
B
Rat
None
NADPH
None
25
111
22
Human
NADPH
159
a
COBILM assay (covalent binding in liver microsomes): incubations (n = 3) were
done with rat or human liver microsomes at 10 M, with 1 mg protein/mL, for
l
60 min with or without NADPH (1 mM) as additive. For complete protocol, see Ref.
21.

2208
A. Chen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2204–2209
Scheme 2. Synthesis of 49 and 56. Reagents and conditions: (a) Pd(PPh3)4, 2 N aq Na2CO3, nPrOH, 80 °C, 1 h, 90%; (b) Rh(PPh3)3Cl, 50 psi H2, EtOH, 24 h, 85%; (c) DIBAl-H,
CH2Cl2, À78 °C to rt, 3 h; (d) (COCl)2, DMSO, TEA, CH2Cl2, 0 °C to rt, 1 h, 82% over two steps; (e) cyclopropylamine, MgSO4, CH2Cl2, 18 h, then NaBH4, MeOH, 1.5 h, 95% for 49,
88% for 56; (f) Br(CH2)2OMe, Cs2CO3, DMF, 80 °C, 16 h, 90%; (g) nBuLi, DMF, THF, À78 °C, 30 min, 45%; (h) Pd(PPh3)2Br2, 45, 2 N aq Na2CO3, DMF, 80 °C, 3 h, 77%; (i) Pd/C, H2,
EtOAc, 75%.
Table 4
Table 4 (continued)
Key profiles of compounds 29 and 50
29
50
Efficacy in dTGR (3 mpk
P.O.)
Max. BP ; (mm Hg)
Duration (h)
27
35
ꢀ2 days ꢀ2 days
CYP 3A4 IC50 (nM)
Reversible
30 min pre-incubation
14000
130
2000
1300
a
See Ref. 23 for assay protocols.
Average of at least two replicates.
COBILM assay (covalent binding in liver microsomes): incubations (n = 3) were
M, with 1 mg protein/mL, for 60 min
b
c
done with human liver microsomes at 10
l
with or without NADPH (1 mM) as additive. For complete protocol, see Ref. 21.
29
50
compound’s potency against renin. An X-ray of 50 bound (Fig. 4) to
renin revealed that the second appendage was threaded through a
small channel in the renin S3 pocket into the solvent. Compound
50 was then selected for further profiling and the synthesis of
the requisite amine 56 used for its preparation is shown in Scheme
2. Starting with 3,5-tribromophenol (51), O-alkylation with 2-
bromoethyl methyl ether with cesium carbonate as base furnished
ether 52 in 90% yield. Initial treatment of 52 with 0.8 equiv of n-bu-
tyl lithium, followed by quenching with DMF afforded aldehyde 53
in 45% yield. Using a similar end game (e.g., Suzuki-coupling with
45, hydrogenation and reductive amination with cyclopropyl-
amine), aldehyde 53 was converted to amine 56 in 51% yield over
three steps. Compound 50 also exhibited good oral bioavailability
in both SD rats (48%) and beagle dogs (36%). Robust blood pressure
lowering at 3 mpk was achieved with 50 when the compound was
tested in the dTGR model (n = 8) with a duration of action also
exceeding 48 h.
a,b
Renin IC50 (nM)
Buffer
0.09
2.4
0.02
3.5
Plasma
SD Rat (30 mpk P.O.,
5 mpk I.V.)
F (%)
P.O. AUC (lM h)
41
8.3
35
6
48
27.5
13
4
Cl (mL/min/kg)
T1/2 (h)
Vdss (L/kg)
12
3.6
Beagle Dog (3 mpk P.O.,
1 mpk I.V.)
F (%)
P.O. AUC (lM h)
32
1.3
16
17
8.4
36
0.7
35
7.5
15
Cl (mL/min/kg)
T1/2 (h)
Vdss (L/kg)
COBILMc, human
With NADPH pmol equiv/
(mg @ 1 h)
115
90
Without NADPH
pmol equiv/(mg @ 1 h)
44
32

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EDTA, 0.1% BSA, pH = 7.4), human tetradecapeptide (1–14) substrate (5
10 mM HCl), hydroxyquinoline sulfate (30 mM in H2O) and assay buffer were
pre-mixed at 4 °C at a ratio of 100:30:10:145. 47.5 L per well of this pre-mix
was transferred into polypropylene plates. Test compounds were dissolved and
diluted in DMSO and 25 L added to the pre-mix, then incubated at 37 °C for
3 h. At the end of the incubation period, 5 L of the renin reaction (or standards
l
L) in assay buffer (PBS1X, 1 mM
Figure 4. X-ray structure of 50 bound to renin.
l
M in
l
In conclusion, we have described a novel series of potent and
orally-bioavailable renin inhibitors. Modification of the northern
terminus led to compounds with lower potential to bind covalently
to proteins when incubated with liver microsomes. To address the
pre-incubation dependent CYP 3A4 inhibition observed with com-
pound 29, further optimization of the eastern aromatic plate affor-
ded compound 50, a renin inhibitor with a profile suitable for
further clinical development.
l
l
in the assay buffer) were measured for AngI accumulation. The percentage of
renin inhibition (AngI decrease) was calculated for each concentration of
compound and an IC50 was determined by curve fitting software. Plasma assay:
citrate-plasma from human volunteers was pooled, aliquoted and stored
frozen at À20 °C. Renin activity in pooled plasma was supplemented with
recombinant human renin (150 pg/mL final concentration) in order to increase
the readout of the assay. Five microlitre of renin inhibitors, at various
concentrations in DMSO, was added to 80 lL of a mixture (7:1) of human
plasma and a fast trapping primary AngI antibody (anti-AngI: AS1, bleed 6, pre-
diluted 1:10 in horse serum) diluted initially 1:26.5 in assay buffer (PBS1X,
1 mM EDTA, 0.1% BSA, pH 7.4) and then diluted 1:3.3 in 3 M Tris, 200 mM
EDTA, pH 7.2 (final anti-serum dilution 1:50,000) and was incubated at 37 °C
for 2 h. As was done in the buffer assay, 12 lL of the renin reaction (or
standards in the assay buffer) were measured for AngI accumulation by
immunoassay.
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