Discovery of novel non-peptidic ketopiperazine-based renin inhibitors

Article abstract of DOI:10.1016/j.bmc.2005.01.048

Ketopiperazine 2 was designed from a previously published analog. Compound 2 was shown to be a novel, potent inhibitor of renin that, when administered orally, lowered blood pressure in a hypertensive double transgenic (human renin and angiotensinogen) mouse model. Compound 2 was further optimized to sub-nanomolar potency by designing an analog that addressed the S3 sub-pocket of the renin enzyme (16).

Full text of DOI:10.1016/j.bmc.2005.01.048

Bioorganic & Medicinal Chemistry 13 (2005) 2657–2664
Discovery of novel non-peptidic ketopiperazine-based
renin inhibitors
Daniel D. Holsworth,a,* Noel A. Powell,a Dennis M. Downing,a Cuiman Cai,a
Wayne L. Cody,a J. Michael Ryan,b Robert Ostroski,b Mehran Jalaie,c
John W. Bryantb and Jeremy J. Edmundsa
aDepartment of Chemistry, Pfizer Global Research and Development, Michigan Laboratories, Ann Arbor, MI 48105, USA
bDepartment of Pharmacology, Pfizer Global Research and Development, Michigan Laboratories, Ann Arbor, MI 48105, USA
cDepartment of Discovery Technologies, Pfizer Global Research and Development, Michigan Laboratories, Ann Arbor, MI 48105, USA
Received 29 October 2004; accepted 8 January 2005
Abstract—Ketopiperazine 2 was designed from a previously published analog. Compound 2 was shown to be a novel, potent inhib-
itor of renin that, when administered orally, lowered blood pressure in a hypertensive double transgenic (human renin and angio-
tensinogen) mouse model. Compound 2 was further optimized to sub-nanomolar potency by designing an analog that addressed the
S3 sub-pocket of the renin enzyme (16).
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
fluid electrolyte balance (Scheme 1).3 The RAS system
is stimulated by a number of signals, including a drop
in BP, a decrease in circulating volume, or a reduction
in plasma sodium concentration. These signals work to
stimulate the release of renin, an enzyme synthesized
in the juxtaglomerular apparatus of the kidney. Renin
cleaves angiotensinogen (a hepatic a-2 globular protein)
to form the hemodynamically inactive angiotensin I
(AI). Angiotensin converting enzyme (ACE), a mono-
meric zinc metalloenzyme found in the vascular endo-
thelium, then converts this pro-hormone to
angiotensin II (AII), the final active messenger in the
RAS pathway. AII inhibits renin secretion by acting di-
rectly on the juxtaglomerular cells, forming the basis of
a short loop negative feedback mechanism. AII has a
number of physiological effects, most importantly as a
powerful vasoconstrictor, increasing BP by altering
peripheral vascular resistance.
Hypertension (high blood pressure) in humans is defined
as a systolic pressure of 140 mmHg or higher, or dia-
stolic pressure of 90 mmHg or higher or both. Pre-
hypertensive is defined as having a systolic pressure of
120–139 mmHg or diastolic pressure of 80–90 mmHg
or both. Approximately 1 in 4 adults in the United
States suffer from hypertension and 45 million suffer
pre-hypertension conditions. Interestingly, of the indi-
viduals who have hypertension, 30% are not aware they
are hypertensive, 25% are on medication but their blood
pressure (BP) is not controlled, 34% are taking medica-
tion and their BP is under control, and 11% are not tak-
ing medication.1 Hypertension has been shown to
precede the development of congestive heart failure in
91% of cases.2 Further, hypertension is a major risk fac-
tor for other cardiovascular diseases such as stroke,
myocardial infarction, and is the leading cause of death
in the Western World.
Since angiotensinogen is the only known substrate for
renin and cleavage of angiotensinogen by renin is the
rate determining step in the renin–angiotensin endocrine
system, it is of general consensus that inhibition of renin
would be an attractive strategy for the control of hyper-
tension. Furthermore, renin inhibitors would prevent
the formation of AI and AII, and, therefore, may act
differently from angiotensin receptor blockers (ARBꢀs)
and angiotensin converting enzyme (ACE) inhibitors,
The renin angiotensin system (RAS) is well established
as an endocrine system involved in BP regulation and
Keywords: Renin–angiotensin system; Renin inhibitor; Ketopiperazi-
none; Double transgenic mouse model; Blood pressure.
*
Corresponding author. Fax: +1 734 622 3909; e-mail:
daniel.holsworth@pfizer.com
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.048

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D. D. Holsworth et al. / Bioorg. Med. Chem. 13 (2005) 2657–2664
Angiotensinogen (alpha-2 Globular Protein)
Renin (aspartic acid proteinase)
Renin inhibitor
Angiotensin I (10-mer)
ACE
ACE inhibitor
AII antagonists
Chymase
Angiotensin II (8-mer)
AT2
AT1A
AT1B
+ other sub-types
Peripheral Resistance
Blood Volume
Vessel Wall Injury
Cell proliferation
Stimulate aldosterone release
Sodium re-absorption
Thirst
Vasoconstriction
Enhanced transmitter release
Central pressor activation
Baroreceptor blunting
Endothelial dysfunction
Adhesion molecule induction
Monocyte activation
Scheme 1. The Renin Angiotensin System (RAS). Renin is the rate limiting step in the RAS cascade.
H
N
O
O
O
OH
O
OH
H
N
O
H2N
NH2
O
O
O
O
O
O
O
A
B (Aliskiren)
Figure 1. Renin inhibitors by Roche (A) and Novartis (B).
which increase AI levels but do not block ACE-indepen-
dent AII production. It has been suggested that renin
inhibitors may provide better kidney and heart protec-
tion than ARB and ACE inhibitors.4
however, compounds with improved properties are
needed for this approach to be useful in the clinic.
Recently, Roche6 and Novartis7 (Fig. 1) have disclosed
non-peptidic renin inhibitors. Although only limited
data is available about the Roche series, Novartisꢀs renin
inhibitor, Aliskiren, entered phase III clinical trials in
March 2004.4,8
Inhibitors of renin were pursued by many companies in
the 1980s and 1990s. However, the early inhibitors were
peptide based, synthetically challenging, and exhibited
poor pharmacokinetic properties such as low oral bio-
availability and high clearance, which limited the clinical
utility of these agents. Most importantly, however, stud-
ies in humans did show that intravenously administered
renin inhibitors lowered BP at least as effectively as ACE
inhibitors.5 Therefore, renin remains a viable target,
2. Chemistry
Recently, we have developed a new class of potent non-
peptidic renin inhibitors for the treatment of hyperten-

D. D. Holsworth et al. / Bioorg. Med. Chem. 13 (2005) 2657–2664
2659
H
N
H
N
H
N
O
O
N
O
N
O
O
O
O
O
O
O
OMe
OMe
OMe
C, IC50: 2 nM
1, IC50: 180 nM
2, IC50: 54 nM
Scheme 2. Evolution of a new class of renin inhibitors based on piperidine template. Ketopiperazine 2 was best in series due to stability and ease of
synthesis. Compound 2 was taken further into the optimization process.
sion.9,10 A previously reported compound6b,d (C), an
analog of A, served as the starting point of our efforts
(Scheme 2). In an attempt to reduce the length and com-
plexity of the synthesis of the chiral 3,4-disubstituted
piperidine (C) scaffold, we proposed to prepare com-
pound 1, which eliminated a chiral center. To avoid syn-
thesis of hemi-aminals, the naphthylmethoxy substituent
of C was replaced with a naphthyloxy-methylene substi-
tuent in 1 (Scheme 2). The synthetic route to produce 1
is outlined in Scheme 3.
iable with increasing scale, and required the slow addi-
tion of DIAD to the chilled reaction mixture.
Reduction of the ketopiperazine carbonyl was accom-
plished by treatment of 4 with BH3ÆSMe2 to afford
piperazine 5 in good yield. Aryl bromide 7 and piper-
azine 5 were coupled in the presence of catalytic
Pd(t-Bu3P)2 and KOt-Bu to yield the protected aryl
piperazine 8. The N-Boc group was removed by treat-
ment with anhydrous HCl generated in situ through
the addition of acetyl chloride to anhydrous MeOH to
give compound 1 in good yield.
Treatment of the chiral ketopiperazine alcohol 39 with 2-
naphthol in the presence of DIAD and polymer-sup-
ported PPh3 provided the desired ether 4 in 49% yield.
The yields of the Mitsunobu reaction proved to be var-
Compound 1 had modest potency against renin (IC50:
180 nM), and represented a reasonable starting point
for the development of more potent analogs. Table 1
Boc
N
Boc
N
Boc
N
(i)
(ii)
O
OH
O
O
N
H
4
O
N
H
N
H
5
3
Br
Br
O
(iii)
(iv)
O
OH
O
6
7
Boc
N
H
N
O
O
(v)
N
N
O
O
O
O
O
O
1
8
Scheme 3. Synthetic route to compound 1. Reagents and conditions: (i) 2-naphthol, DIAD, PS–PPh3, CH2Cl2, 0–25 ꢁC, 49%; (ii) BH3ÆSMe2, THF,
50 ꢁC, 77%; (iii) 1-(3-iodopropoxymethyl)-2-methoxybenzene, K2CO3, CH3CN, reflux, 71%; (iv) Pd(t-Bu3P)2, KOt-Bu, toluene, 60 ꢁC, 35%; (v) AcCl,
MeOH, 0–25 ꢁC, 89%.

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D. D. Holsworth et al. / Bioorg. Med. Chem. 13 (2005) 2657–2664
Table 1. SAR of piperazine series based on compound 19a
2-fold. An N-[2–(7-methyl-3,4-dihydro-2H-quinolin-1-
yl)-ethyl]-acetamide side chain has demonstrated an
increase in potency within the piperidine series.6e Based
on X-ray results with the piperidine series, the acetamide
moiety penetrates the S3 sub-pocket of renin, providing
the increased affinity. We found this to also translate
to the piperazine series (compound 15), where sub-
nanomolar potency was achieved.
H
N
O
N
R
O
O
O
Compound 1 and related analogs (Table 1) were found
to exhibit a degree of instability over time, possibly as
a consequence of air oxidation of the electron rich phe-
nyl ring. In addition, the synthesis was still lengthy, and
the Buchwald coupling was problematic on a large scale.
Therefore, compound 2 was designed (Scheme 2). It was
envisioned that the ketopiperazine template would still
possess the basic nitrogen needed to interact with the
catalytic aspartic acids within the renin active site,6d
yet the lone pair of the second nitrogen atom would
be delocalized through the carbonyl group. This
arrangement was hypothesized to reduce the electron
density on the para-substituted phenyl ring, and would,
therefore, impart additional stability.
Compound ID
R Side chain
IC50 (nM)
1700
12
13
14
15
1020
440
O
N
H
The synthesis of compound 2 began with intermediate 9
(Scheme 4).11 Alkylation of the phenol with 1-(3-iodo-
propoxymethyl)-2-methoxybenzene9b in the presence of
K2CO3 provided 10 in excellent yield. Mitsunobu cou-
pling of 10 with 2-naphthol in the presence of DIAD
and polymer supported PPh3 afforded the desired ether
11 in 81% yield. In contrast to our experience with the
Mitsunobu coupling of 3, the reaction between alcohol
10 and 2-naphthol was quite robust, scalable, and
high-yielding, indicating that the acidity of the amide
NH in 3 played a large role in the non-linear reaction
scale variability and lower yields of 4 (Scheme 3).
Deprotection of the N-Boc protecting group was again
accomplished with in situ generated HCl in cold MeOH
0.50
N
Compound 15, an analog designed to penetrate the S3 sub-pocket of
renin achieved sub-nanomolar potency.
depicts some SAR within the piperazine series. Installa-
tion of a 2-naphthyloxy methylene unit or methoxy
biphenyl group (compounds 12 and 13, respectively),
resulted in a drastic decrease in potency. Installation
of the 6-methyl- 1,2,3,4-tetrahydro-naphthalene ring
(compound 14) also lowered potency approximately
Boc
N
Boc
N
OH
OH
O
N
O
N
(i)
(ii)
O
OH
9
O
O
10
Boc
N
H
N
O
O
O
N
(iii)
O
N
O
O
O
O
O
O
11
2
Scheme 4. Synthetic route to compound 2. Reagents and conditions: (i) 1-(3-iodopropoxymethyl)-2-methoxybenzene, K2CO3, CH3CN, reflux, 89%;
(ii) 2-naphthol, DIAD, PS–PPh3, CH2Cl2, 0–25 ꢁC, 81%; (iii) AcCl, MeOH, 0–25 ꢁC, 78%.

D. D. Holsworth et al. / Bioorg. Med. Chem. 13 (2005) 2657–2664
2661
to provide compound 2 in good yield. Compound 2 was
found to be 3-fold more active against renin in vitro
than compound 1 (Scheme 2), as well as more stable
and amenable to large scale synthesis.11 Therefore,
compound 2 was chosen as a new framework for the
creation of more potent non-peptidic renin inhibitors.
lowered blood pressure in the double transgenic (human
renin and angiotensinogen) mouse when dosed orally.
Further efforts toward the discovery of optimized com-
pounds based on this template will be reported in due
course.
Insertion of the R side chain of compound 15 into the
ketopiperazine core, formed compound 16,9b a very po-
tent inhibitor of renin. Based on these data, compounds
that possess R side chains designed to address the S3
sub-pocket of renin will be the focus of future work.
5. Experimental
5.1. General
All chemicals, reagents, and solvents were purchased
from commercial sources (e.g., Aldrich Chemical Co.,
Inc., Milwaukee, WI; Mallinckrodt Baker, Inc., Paris,
KY, etc.) were available and used without further puri-
O
HN
H
1
fication. H NMR spectra were obtained on a Varian
N
Unity 400 MHz spectrometer. Elemental analyses were
determined on a Perkin–Elmer model 240C instrument.
Mass spectral data were obtained on a VG Analytical
7070 E/HF mass spectrometer. Flash column chroma-
tography was performed on silica gel 60, 230–400 mesh,
purchased from Mallinckrodt.
O
N
O
N
O
O
O
Compound 16, IC50: 0.30 nM
5.2. Chemistry
Experimentals for compounds 12, 13, 14, 15, and 16 are
listed in Ref. 9b.
3. In vivo activity
5.2.1.
(3R)-3-(Naphthalen-2-yloxymethyl)-5-oxopiper-
The next crucial step was to determine if whether com-
pound 2 or compound H lowered blood pressure in an
experimental, whole animal model of renin-dependent
hypertension. In this case, compound 2 was evaluated
in the in vivo study. Double transgenic mice (h-Ang
204/1 h-Ren 9) that constitutively express both human
renin and its substrate angiotensinogen were equipped
with telemetry implants for 24 h blood pressure mea-
surements. The mice have mean arterial blood pressures
(MABP) that average 155 mmHg, ꢀ45 mmHg higher
than in mice lacking the human genes. To establish a
blood pressure control, mice were given vehicle (3%
DMA, 97% SBE-CD (40% w/v) in 50 mM lactic acid
(10 mL/kg) on two consecutive days before dosing com-
pound 2. Blood pressures obtained at 15 min intervals
from the two vehicle treatment days were then averaged
to establish a baseline at each time point. The com-
pound was given by oral gavage (30 mg/kg) on day 3.
The change in MABP at each 15 min time point was ob-
tained by subtracting the MABP from the mice treated
with compound 2 from the baseline MABP. Compound
2 lowered blood pressure by a maximum of 20 mmHg
after 1 h post dose (results not shown). Blood pressure
reached baseline levels at 3 h post dose. The observed
blood pressure reduction suggests the template may
have the potential to be developed into a novel antihy-
pertensive agent.
azine-1-carboxylic acid tert-butyl ester (4). An oven-
dried, 25 mL round bottom flask was cooled under a
N2 stream and charged with (3R)-3-hydroxymethyl-5-
oxopiperazine-1-carboxylic
acid
tert-butyl
ester
(100mg, 0.434 mmol), 2-naphthol (94 mg, 0.65 mmol),
and 5 mL of anhydrous dichloromethane. Polymer-sup-
ported triphenylphosphine (383 mg, 0.521 mmol,
1.36 mmol/g loading) was added, and the resulting slur-
ry was stirred at rt for 20 min. The mixture was cooled
in an ice bath, and diisopropyl azodicarboxylate
(141 mg, 0.695 mmol) was added dropwise via a syringe.
The yellow slurry was stirred overnight, allowing to
warm to rt. The resin was collected on a medium frit
and rinsed with dichloromethane (3 · 4 mL). The com-
bined filtrates were washed with aqueous 1 N HCl and
10% NaOH, dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. Purification
by flash column chromatography (gradient: 60% ethyl
acetate/hexanes to 100% ethyl acetate) afforded (3R)-3-
(naphthalen-2-yloxymethyl)-5-oxopiperazine-1-carbox-
ylic acid tert-butyl ester (76.3 mg, 49%) as a clear viscous
1
oil that exists as a mixture of carbamate rotamers. H
NMR (400 MHz, CDCl3) d 1.39–1.50 (m, 9H), 3.53
(m, 1H), 3.85–4.00 (m, 3H), 4.07–4.20 (m, 3H), 6.60 (s,
1H), 7.11 (m, 2H), 7.36 (dd, J = 7.4, 7.4 Hz, 1H), 7.45
(dd, J = 7.4, 7.4 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H) 7.76
(d, J = 7.6 Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H).
4. Conclusion
5.2.2. (3R)-3-(Naphthalen-2-yloxymethyl)-piperazine-1-
carboxylic acid tert-butyl ester (5). (3R)-3-(Naphthalen-
2-yloxymethyl)-5-oxopiperazine-1-carboxylic acid tert-
butyl ester (1.55 g, 4.34 mmol) was dissolved in 40 mL
of anhydrous tetrahydrofuran under a N2 atmosphere.
A 2.0 M solution of borane dimethyl sulfide complex
From a previously reported template, a novel non-
peptidic ketopiperazine-based renin inhibitor has been
created. The ketopiperazine framework (i.e., compound
2) displays good affinity toward the renin enzyme and

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D. D. Holsworth et al. / Bioorg. Med. Chem. 13 (2005) 2657–2664
in tetrahydrofuran (6.5 mL, 13.0 mmol) was added via
syringe, and the reaction mixture was heated in a
50 ꢁC oil bath for 2 h. After cooling to rt, excess hydride
was quenched by the careful addition of methanol. The
mixture was concentrated under reduced pressure, and
then re-dissolved in 75 mL of methanol. An aqueous
solution of 10% potassium sodium tartrate (50 mL)
was added, and the resulting slurry was heated at reflux
for 2 h and then stirred at rt for 18 h. The mixture was
concentrated under reduced pressure and partitioned
between water and ethyl acetate (3·). The combined
organic layers were washed with brine, dried over
anhydrous MgSO4, filtered, and concentrated under re-
duced pressure. Purification by flash column chromato-
graphy (65/33/2 ethyl acetate/hexanes/methanol then 70/
28/2 ethyl acetate/hexanes/methanol) provided (3R)-3-
(naphthalen-2-yloxymethyl)-piperazine-1-carboxylic acid
back-filled with N2 twice. Palladium bis(tri-tert-butyl-
phosphine) (125 mg, 0.24 mmol), and KOt-Bu (301 mg,
2.69 mmol) were added, and the flask was again purged
and back-filled with N2. Anhydrous tetrahydrofuran
(10 mL) was added, and the resulting orange slurry
was heated in a 75 ꢁC oil bath for 18 h. After cooling
to rt, the mixture was diluted with diethyl ether and fil-
tered through a Celite pad, rinsing with diethyl ether.
The combined filtrates were concentrated under reduced
pressure and purified by flash column chromatography
(gradient: 10% ethyl acetate/hexanes gradient to 20%
ethyl acetate/hexanes, then 70/28/2 ethyl acetate/hex-
anes/methanol) to give (3R)-4-4-[3-(2-methoxybenzyl-
oxy)-propoxy]-phenyl-3-(naphthalen-2-yloxymethyl)-pip-
erazine-1-carboxylic acid tert-butyl ester (530 mg, 35%)
as a yellow oil that exists as a mixture of carbamate
1
rotamers. H NMR (400 MHz, CDCl3) d 1.25 (s, 4H),
1
tert-butyl ester (1.149 g, 77%) as a clear glassy solid. H
1.27 (s, 5H), 2.07 (quintet, J = 5.9 Hz, 2H), 3.16 (br s,
2H), 3.24 (br s, 1H), 3.47 (br s, 1H), 3.69 (t,
J = 6.1 Hz, 2H), 3.77 (s, 3H), 3.90 (m, 3H), 4.06 (t,
J = 6.1 Hz, 2H), 4.06 (m, 1H), 4.23 (m, 1H), 4.55 (s,
2H) 6.82 (d, J = 8.3 Hz, 1H), 6.87 (m, 2H), 6.91 (d,
J = 7.6 Hz, 1H), 6.94 (m, 3H), 7.06 (dd, J = 9.0,
1.8 Hz, 1H), 7.23 (ddd, J = 7.6, 7.6, 1.5 Hz, 1H), 7.31
(dd, J = 7.3, 7.3 Hz, 1H), 7.34 (d, J = 7.3 Hz, 1H), 7.39
(dd, J = 7.8, 7.8 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.69
(d, J = 8.8 Hz, 1H), 7.73 (d, J = 8.3 Hz, 1H). MS: m/z
613.4 (M+1).
NMR (400 MHz, CDCl3) d 1.48 (s, 9H), 2.82 (m, 2H),
2.94 (m, 1H), 3.02 (m, 1H), 3.15 (m, 1H), 3.97 (dd,
J = 9.1, 7.7 Hz, 1H), 3.98 (br s, 2H), 4.07 (dd, J = 9.2,
4.1 Hz, 1H), 7.14 (m, 2H), 7.34 (dd, J = 6.9, 6.9 Hz,
1H), 7.44 (ddd, J = 8.0, 6.8, 1.2 Hz, 1H), 7.72 (d,
J = 7.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.76 (d,
J = 8.8 Hz, 1H). HRMS calcd for [C20H26N2O3 + 1]
343.2. Found: 343.2 (M+1).
5.2.3. 1-Bromo-4-(3-(2-methoxybenzyloxy)-propyloxy)-
benzene (7). A 100 mL round bottom flask was charged
with 4-bromophenol (1.87 g, 10.78 mmol), 1-(3-iodo-
propoxymethyl)-2-methoxybenzene (3.00 g, 9.80 mmol),
acetonitrile (20 mL), and K2CO3 (1.63 g, 11.8 mmol).
The resulting slurry was heated to reflux for 18 h. After
cooling to rt, the mixture was filtered through a Celite
pad, which was rinsed with additional acetonitrile. The
combined filtrates were concentrated under reduced
pressure. The residue was dissolved in ethyl acetate,
and washed with aqueous 10% NaOH and brine. The
organic layer was dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. Purification
by flash column chromatography (gradient: 5% ethyl
acetate/hexanes to 10% ethyl acetate/hexanes) afforded
1-bromo-4-(3-(2-methoxybenzyloxy)-propyloxy)benzene
(2.45 g, 71%) as a clear oil. 1H NMR (400 MHz, CDCl3)
d 7.37 (d, J = 9.0 Hz, 2H), 7.29–7.25 (m, 2H), 6.94 (t,
J = 7.4 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.78 (d,
J = 9.0 Hz, 2H), 4.57 (s, 2H), 4.07 (t, J = 6.2 Hz, 2H),
3.81 (s, 3H), 3.70 (t, J = 6.1 Hz, 2H), 2.10 (quintet,
J = 6.1 Hz, 2H). MS calcd for [C17H19BrO3 + 1]:
351.1 (79Br), 353.0 (81Br). Found 351.1 (79Br),
353.0 (81Br).
5.2.5. (2R)-1-{4-[3-(2-Methoxybenzyloxy)-propoxy]-phen-
yl}-2-(naphthalen-2-yloxymethyl)-piperazine (1). (3R)-4-
4-[3-(2-Methoxybenzyloxy)-propoxy]-phenyl-3-(naphtha-
len-2-yloxymethyl)-piperazine-1-carboxylic acid tert-
butyl ester (530 mg, 0.864 mmol) was suspended in
15 mL of anhydrous methanol under a N2 atmosphere,
and cooled in an ice bath. Acetyl chloride (0.62 mL,
8.64 mmol) was added dropwise over a 2 min interval.
The N2 inlet needle was removed and the resulting green
solution was stirred for 12 h, allowing the ice bath to
warm to room temperature. Excess acid was quenched
by the addition of aqueous saturated NaHCO3. The
aqueous layer was extracted with ethyl acetate
(3 · 50 mL). The combined organic layers were dried
over anhydrous MgSO4, filtered, and concentrated un-
der reduced pressure. Purification by flash column chro-
matography (95/5 dichloromethane/methanol then 90/10
dichloromethane/methanol) provided (2R)-1-4-[3-(2-
methoxybenzyloxy)-propoxy]-phenyl-2-(naphthalen-2-
yloxymethyl)-piperazine (393 mg, 89%) as a brown vis-
cous oil. [a]D +89.4 (c 1.7, CH2Cl2); IR (ATR/diamond)
2950, 2833, 1508, 1461, 1240, 1216, 1180, 1087, 1028,
1
816 cmÀ1; H NMR (400 MHz, CDCl3) d 1.98 (br s,
5.2.4. (3R)-4-{4-[3-(2-methoxybenzyloxy)-propoxy]-phen-
yl}-3-(naphthalen-2-yloxymethyl)-piperazine-1-carboxylic
acid tert-butyl ester (8). A 50 mL round bottom flask
was charged with 1-bromo-4-(3-(2-methoxybenzyloxy)-
propyloxy)benzene (1.72 g, 4.88 mmol) and (3R)-3-
(naphthalen-2-yloxymethyl)-piperazine-1-carboxylic acid
tert-butyl ester (836 mg, 2.44 mmol). The mixture was
dissolved in 10 mL of anhydrous toluene and concen-
trated under reduced pressure to remove residual water.
After the flask was equipped with a magnetic stir bar
and a reflux condenser, the flask was purged and
1H), 2.08 (quintet, J = 6.2 Hz, 2H), 3.02 (q, J = 6.6 Hz,
1H), 3.12 (m, 3H), 3.26 (m, 2H), 3.69 (t, J = 6.1 Hz,
2H), 3.78 (s, 3H), 3.85 (dddd, J = 7.3, 3.7, 3.7,
3.7 Hz, 1H), 3.97 (dd, J = 9.0, 3.4 Hz, 1H), 4.06 (t,
J = 6.2 Hz, 2H), 4.29 (t, J = 9.0 Hz, 1H), 4.56 (s, 2H),
6.82 (d, J = 8.1 Hz, 1H), 6.86 (m, 2H), 6.91 (dd,
J = 7.4, 7.4 Hz, 1H), 6.98 (m, 3H) 7.06 (dd, J = 8.9,
2.6 Hz, 1H), 7.23 (ddd, J = 7.8, 7.8, 1.7 Hz, 1H), 7.30
(ddd, J = 7.3, 6.8, 1.2 Hz, 1H), 7.35 (dd, J = 7.4,
1.6 Hz, 1H), 7.39 (ddd, J = 8.3, 7.1, 1.2 Hz, 1H), 7.63
(d, J = 8.1 Hz, 1H), 7.69 (d, J = 9.0 Hz, 1H), 7.73 (d,

D. D. Holsworth et al. / Bioorg. Med. Chem. 13 (2005) 2657–2664
2663
J = 8.1 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 157.3,
156.7, 154.3, 143.9, 134.6, 129.5, 129.2, 129.0, 128.8,
127.8, 156.9, 126.5, 123.8, 120.6, 120.4, 119.0, 115.6,
110.4, 107.0, 68.0, 67.3, 65.5, 64.4, 56.3, 55.5, 48.1,
47.4, 45.9, 30.0; HRMS calcd for [C32H36N2O4 + 1]:
513.2753. Found: 513.2742 (M+1).
a mixture of carbamate rotamers. [a]D +43.5 (c 2.6,
CHCl3); H NMR (400 MHz, CDCl3) d 1.19–1.37 (m,
1
9H), 2.01 (quintet, J = 6.0 Hz, 2H), 3.56 (m, 1H), 3.61
(t, J = 6.0 Hz, 2H), 3.71 (s, 3H), 3.64 (m, 1H), 4.02 (t,
J = 6.0 Hz, 2H), 4.08–3.98 (m, 3H), 4.42–4.51 (m, 1H),
4.47 (s, 2H), 4.54 (m, 1H), 6.75 (d, J = 8.2 Hz, 1H),
6.83 (d, J = 7.4 Hz, 1H), 6.87 (d, J = 8.4 Hz, 2H), 7.00
(dd, J = 8.9, 2.4 Hz, 1H), 7.09 (d, J = 8.7 Hz, 2H), 7.16
(dd, J = 7.6, 7.6 Hz, 1H), 7.26 (m, 2H), 7.35 (dd,
J = 7.3, 7.3 Hz, 1H), 7.56 (br s, 1H) 7.64 (d,
J = 9.0 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H); LRMS calcd
for [C37H42N2O7 + 1]: 627.1. Found 627.1 (M+1).
5.2.6. (3R)-3-Hydroxymethyl-4-(4-[3-(2-methoxybenzyl-
oxy)-propoxy]-phenyl)-5-oxopiperazine-1-carboxylic acid
tert-butyl ester (10). A solution of 5.0 g (15.5 mmol) of
(3R)-3-hydroxymethyl-4-(4-hydroxyphenyl)-5-oxopiper-
azine-1-carboxylic acid tert-butyl ester in 30 mL of ace-
tonitrile was treated with 5.70 g (18.6 mmol) of 1-(3-
iodopropoxymethyl)-2-methoxybenzene, and 3.22 g
(23.3 mmol) of K2CO3. The reaction mixture was heated
to reflux for 18 h. The reaction mixture was then cooled
to rt, diluted with 100 mL of water, and partitioned with
200 mL of ethyl acetate. The organic layer was washed
with brine (2100 mL), dried over anhydrous MgSO4, fil-
tered, and concentrated under reduced pressure. Purifi-
cation by flash column chromatography (gradient:
50% ethyl acetate/hexanes to 100% ethyl acetate) affor-
ded 6.94 g (89%) of (3R)-3-hydroxymethyl-4-(4-[3-(2-
methoxybenzyloxy)-propoxy]-phenyl)-5-oxopiperazine-
1-carboxylic acid tert-butyl ester that exists as a mixture
of carbamate rotamers. [a]D À19.6 (c 5.1, CHCl3); IR
(ATR/diamond) 3338, 2972, 2873, 1687, 1639, 1627,
1512, 1462, 1415, 1245, 1150, 1107, 1024, 835,
5.2.8. (6R)-1-{4-[3-(2-Methoxybenzyloxy)-propoxy]-phenyl}-
(2).
-6-(naphthalen-2-yloxymethyl)-piperazin-2-one
(3R)-4-(4-[3-(2-Methoxybenzyloxy)-propoxy)-phenyl])-
3-(naphthalene-2-yloxymethyl)-5-oxopiperazine-1-carbo-
xylic acid tert-butyl ester (55 mg, 87.7 mmol) was dis-
solved in methanol (2 mL) under nitrogen and cooled
to 0 ꢁC. Acetyl chloride (68.9 mg, 0.877 mmol) was
added dropwise. The nitrogen inlet needle was removed,
and the reaction mixture allowed to warm to rt over a
period of 18 h. The reaction mixture was quenched with
aqueous saturated NaHCO3. The mixture was extracted
with ethyl acetate (3 · 10 mL). The organic layer was
dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure. The resultant solid was purified
by flash column chromatography (gradient: 100%
dichloromethane then 95/5 dichloromethane/methanol
to 90/10 dichloromethane/methanol) to afford 36.1 mg
(78.1%) of 1-{4-[3-(2-methoxybenzyloxy)-propoxy]-phen-
yl}-6-(naphthalen-2-yloxymethyl)-piperazin-2-one as a
semi-solid. [a]D +36.4 (c 1.4, CHCl3); IR (neat) 3353,
2859, 1640, 1509, 1465, 1239, 1176, 1088, 1027,
1
754 cmÀ1; H NMR (400 MHz, CDCl3) d 1.50 (br s,
9H), 2.09 (quintet, J = 6.2 Hz, 2H), 2.88 (m, 1H), 3.41
(m, 2H), 3.68 (t, J = 6.1 Hz, 2H), 3.75 (m, 1H), 3.80 (s,
3H), 3.97 (d, J = 18.6 Hz, 1H), 4.08 (t, J = 6.1 Hz,
2H), 4.43 (m, 1H), 4.55 (s, 2H), 6.85 (d, J = 8.1 Hz,
1H), 6.91 (d, J = 9.0 Hz, 2H), 6.92 (m, 1H), 7.07 (d,
J = 8.8 Hz, 2H), 7.25 (ddd, J = 8.3, 8.3, 1.6 Hz, 1H),
7.34 (d, J = 7.3 Hz, 1H); 13C NMR (100 MHz, CDCl3)
d 28.5 (3), 29.8, 55.5, 65.4, 67.0, 68.0, 105.0, 110.4,
115.5, 116.6, 120.6, 126.8, 128.5, 128.8, 129.1, 133.1,
157.3, 158.6; HRMS calcd for [C27H36N2O7 + 1]:
501.2601. Found: 501.2608.
1
841 cmÀ1; H NMR (400 MHz, CDCl3) d 2.08 (quintet,
J = 6.1 Hz, 2H), 3.38 (d, J = 13.4 Hz, 1H), 3.52 (d,
J = 13.8 Hz, 1H), 3.67 (dd, J = 6.3, 1.7 Hz, 1H), 3.70
(dd, J = 6.3, 1.7 Hz, 1H), 3.77 (s, 3H), 4.03 (dd,
J = 7.2, 2.2 Hz, 1H), 4.08 (ddd, J = 6.3, 6.3, 2.0 Hz,
1H); 4.27 (t, J = 8.8 Hz, 1H), 4.54 (s, 2H), 6.81 (d,
J = 7.6 Hz, 1H), 6.92 (m, 4H), 7.07 (ddd, J = 8.9, 2.7,
2.7 Hz, 1H), 7.15 (dd, J = 8.8, 2.0 Hz, 2H), 7.21 (m,
1H), 7.33 (m, 2H), 7.41 (t, J = 7.4 Hz, 1H), 7.62 (d,
J = 8.5 Hz, 1H), 7.74 (t, J = 9.0 Hz, 2H); 13C NMR
(100 MHz, CDCl3) d 29.7 (CH2), 45.8 (CH2), 50.78
(CH2), 55.3 (CH3), 58.6 (CH), 65.2 (CH2), 66.2 (CH2),
66.9 (CH2), 67.8 (CH2), 106.8 (CH), 110.2 (CH), 115.4
(2 CH), 118.5 (CH), 120.4 (CH), 124.0 (CH), 126.6
(CH), 126.7 (C), 126.8 (CH), 127.7 (CH), 128.7 (CH),
128.8 (2 CH), 128.9 (CH), 129.2 (C), 129.7 (CH), 132.7
(C), 134.4 (C), 156.0 (C), 157.1 (C), 158.4 (C), 169.1
(C); HRMS (ESI) calcd for C32H35N2O5 527.2546.
Found: 527.2547. Anal. Calcd for C32H34N2O5: C,
72.86; H, 6.52; N, 5.31. Found: C, 72.57; H, 6.53; N,
5.27.
5.2.7. (3R)-4-(4-[3-(2-Methoxybenzyloxy)-propoxy)-phen-
yl])-3-(naphthalene-2-yloxymethyl)-5-oxopiperazine-1-
carboxylic acid tert-butyl ester (11). A N2-flushed, round
bottom flask, was charged with (3R)-3-hydroxymethyl-
4-(4-[3-(2-methoxybenzyloxy)-propoxy]-phenyl)-5- oxo-
piperazine-1-carboxylic acid tert-butyl ester (0.500 g,
0.999 mmol), 2-naphthol (216 mg, 1.498 mmol), and
dichloromethane (15 mL). Triphenylphosphine sup-
ported on polystyrene (0.975 g, 1.598 mmol, 1.64
mmol/g) was added at rt, and the resulting slurry
was stirred for 10 min. The reaction mixture was cooled
in an ice bath, and diisopropyl azodicarboxylate
(0.236 mL, 1.199 mmol) was added dropwise over
2 min. The orange-red reaction mixture was stirred for
18 h, slowly warming to rt. The reaction mixture was fil-
tered, and the resin rinsed with dichloromethane (3·).
The crude product was purified by flash column chro-
matography (gradient: 20% ethyl acetate/hexanes to
50% ethyl acetate/hexanes) to afford 0.507 g (81%) of
(3R)-4-(4-[3-(2-methoxybenzyloxy)-propoxy)-phenyl])-
3-(naphthalene-2-yloxymethyl)-5-oxopiperazine-1-car-
boxylic acid tert-butyl ester as a white solid that exists as
5.3. In vitro renin IC50 determinations
The renin assay utilized a tandem green fluorescent pro-
tein (t-GFP) substrate (175 nM) that was hydrolyzed by
renin human (50.4 IU/well). The t-GFP substrate con-
tained a nine amino acid (Ile-His-Pro-Phe-His-Leu-
Val-Ile-His) recognition sequence for human renin

2664
D. D. Holsworth et al. / Bioorg. Med. Chem. 13 (2005) 2657–2664
2004, 116, 263; (c) Nicholls, M. G.; Robertson, J. S. J.
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4. Stanton, A. JRAAS 2003, 4, 6.
5. Zimmerman, B. G. Clin. Exp. Pharmacol. Physiol. 2000,
27, 370.
flanked by two GFP proteins (W1B and Topaz). Human
renin cleaves the leucine–valine site of the substrate lin-
ker. Tandem GFP Fret assays were carried out in a reac-
tion buffer containing 50 mM Hepes (pH 7.4), 1.0 mM
EDTA, 1% PEG (MW800), 1.0 mM DTT, and 0.10%
BSA. Once cleaved, the emission ratio changes. The
change was monitored by the ratio of 530 nM (topaz)
over 475 nM (W1B) with the excitation set at 432 nM
and the cutoff at 515 nM. The assay used a 384 well
plate format that was read using a Gemini XS fluoro-
metric plate reader (Molecular Devices). Compounds
were screened at a starting concentration of 10 lM
and used a 4-fold 11-point dilution regimen.
6. (a) Bittner, B.; Chou, R.; Schleimer, M.; Morgenroth, B.;
Zell, M.; Lave, T. Arzneimittel-Forsch./Drug Res. 2002, 52,
593; (b) Vieira, E.; Binggeli, A.; Breu, V.; Bur, D.; Fischli,
W.; Guller, R.; Hirth, G.; Marki, H. P.; Muller, M.;
Oefner, C.; Scalone, M.; Stadler, H.; Wilhelm, M.; Wostl,
W. Bioorg. Med. Chem. Lett. 1999, 9, 1397; (c) Oefner, C.;
Binggeli, A.; Breu, V.; Bur, D.; Clozel, J.-P.; DꢀArcy, A.;
Dorn, A.; Fischli, W.; Gruninger, F.; Guller, R.; Hirth,
G.; Markl, H. P.; Mathews, S.; Muller, M.; Ridley, R. G.;
Stadler, H.; Vieira, E.; Wilhelm, M.; Winkler, F. K.;
Wostl, W. Chem. Biol. 1999, 6, 127; (d) Guller, R.;
Binggeli, A.; Breu, V.; Bur, D.; Fischli, W.; Hirth, G.;
Jenny, C.; Kansy, M.; Montavon, F.; Muller, M.; Oefner,
C.; Stadler, H.; Viera, E.; Wihelm, M.; Wostl, W.; Markl,
H. P. Bioorg. Med. Chem. Lett. 1999, 9, 1403; (e) Marki,
H. P.; Binggeli, A.; Bittner, B.; Bohner-Lang, V.; Breu, V.;
Bur, D.; Coassolo, Ph.; Clozel, J. P.; DꢀArcy, A.; Doebeli,
H.; Fischli, W.; Funk, Ch.; Foricher, J.; Giller, T.;
Gruninger, F.; Guenzi, A.; Guller, R.; Hartung, T.; Hirth,
G.; Jenny, Ch.; Kansy, M.; Klinkhammer, U.; Lave, T.;
Lohri, B.; Luft, F. C.; Mervaala, E. M.; Muller, D. N.;
Muller, M.; Montavon, F.; Oefner, Ch.; Qiu, C.; Reichel,
A.; Sanwald-Ducray, P.; Scalone, M.; Schleimer, M.;
Schmid, R.; Stadler, H.; Treiber, A.; Valdenaire, O.;
Vieira, E.; Waldmeier, P.; Wiegand-Chou, R.; Wilhelm,
M.; Wostl, W.; Zell, M.; Zell, R. Il Farmaco 2001, 56, 21.
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tre, J. Drugs Future 2001, 26, 1139; (b) Wood, J. M.;
Maibaum, J.; Rahuel, J.; Grutter, M. G.; Cohen, N.-C.;
Rasetti, V.; Vuger, H.; Goschke, 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.; OꢀBrien, E.; Stanton, A.; Bedigian, M. P. Biochem.
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42, 1137.
5.4. In vivo efficacy studies
Blood pressure data was obtained by telemetry in con-
scious, free moving three- to four-month-old double
transgenic mice that expressed both human angiotensin-
ogen and renin. The double transgenic mice were de-
rived from a founder colony of eight male mice
expressing human angiotensinogen (h-Ang 204/1) and
eight female mice expressing human renin (h-Ren 9)
obtained through a breeding program conducted at
Charles River Laboratories (Wilminton, MA). The dou-
ble transgenic mice were hypertensive with mean arterial
blood pressures (MABP) of 140 mmHg. Both males and
females were used. MABP was measured via radiotrans-
mitter (model TA11PA-20, Data Sciences International,
Saint Paul, Minnesota) implanted subcutaneously, be-
tween the left fore and hind limbs. The radiotransmitter
catheter was placed in the left carotid artery. To obtain
baseline blood pressure data the mice were dosed via
oral gavage with vehicle (3% volume dimethyl acetam-
ide, 97% sulfobutylether-beta-cyclodextrin (40% w/v)
in 50 nM lactic acid) for two consecutive days. On the
third day, either CI-992 (N-(4-morpholinylsulfonyl)-
L-phenylalanyl-3-(2-amino-4-thiazoyl)-N-[(1S,2R,3S)-
1-(cyclo-hexyl-methyl)-2,3-dihydroxy-5-methylhexyl]-
(HCL))12,13 or compound 2 was administered at 30 mg/
kg. Delta MABP was obtained by subtracting CI-992 or
compound 2 dosed blood pressure from baseline blood
pressure. Animals were allowed food and water ad libi-
tum. The maximum antihypertensive response occurred
within 1 h of a 30 mg/kg oral dose of compound 2. At
the nadir for compound 2, MABP was normalized,
but returned to baseline hypertensive levels approxi-
mately 3–4 h later.
8. www.speedelpharma.com, press releases, July 13, 2004.
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