Synthesis and docking of novel piperidine renin inhibitors

Article abstract of DOI:10.1007/s00706-012-0903-5

A series of 4-triazolyl-substituted piperidine derivatives were synthesized from N-Boc protected trans-4-ethynyl-3-hydroxypiperidine and tested as novel renin inhibitors. Piperidine derivatives containing a 1-substituted 1,2,3-triazol-5-yl substituent were found to be most active. Molecular docking experiments provide a rank order that is in agreement with experimental data. Furthermore, all compounds explore the S1 and the S3 subpockets through the piperidine substituents.

Full text of DOI:10.1007/s00706-012-0903-5

Monatsh Chem (2013) 144:479–494
DOI 10.1007/s00706-012-0903-5
ORIGINAL PAPER
Synthesis and docking of novel piperidine renin inhibitors
•
Rianne A. G. Harmsen Annfrid Sivertsen Davide Michetti
•
•
•
Bjørn Olav Brandsdal Leiv K. Sydnes Bengt Erik Haug
•
Received: 28 September 2012 / Accepted: 9 December 2012 / Published online: 1 February 2013
Ó Springer-Verlag Wien 2013
Abstract A series of 4-triazolyl-substituted piperidine
derivatives were synthesized from N-Boc protected trans-
4-ethynyl-3-hydroxypiperidine and tested as novel renin
inhibitors. Piperidine derivatives containing a 1-substituted
1,2,3-triazol-5-yl substituent were found to be most active.
Molecular docking experiments provide a rank order that is
in agreement with experimental data. Furthermore, all
compounds explore the S1 and the S3 subpockets through
the piperidine substituents.
catalyzes the rate-determining step in the formation of
angiotensin-II and for several decades has been an estab-
lished therapeutic target for drug development in relation to
hypertension [1].
Renin inhibitors that were discovered early on were
often found to exhibit poor oral absorption and low bio-
availability because of their peptidic nature, and further
development into non-peptide peptidomimetic drugs has
been sought [2–4]. At the turn of the century, substituted
piperidine derivatives were reported as a new class of renin
inhibitors [5–8]. In later years, substituted piperidines have
proven to be efficient scaffolds for the development of
novel non-peptide aspartic protease inhibitors, particularly
toward renin [9–11].
Keywords Alkyne ꢀ Triazole ꢀ Renin inhibitor ꢀ
Molecular docking
Introduction
Previously, renin inhibitors based on piperidine deriva-
tives bearing C-4-substituted 1,2,3-triazol-1-yl moieties
have been reported [8]. Herein, we report the synthesis
of a series of piperidine derivatives with N-substituted
1,2,3-triazol-4-yl or -5-yl substituents in the 4-position,
evaluation of their biological activity and selectivity towards
renin, and the development of a computational docking
protocol to predict their activity.
Renin is a highly selective aspartic protease that catalyzes
the hydrolysis of angiotensinogen, a protein secreted from
the liver, to the decapeptide angiotensin-I [1]. Angiotensin-
I is further processed by the angiotensin-converting
enzyme (ACE) to give the octapeptide angiotensin-II, a
potent vasoconstrictor and the dominant signal peptide
produced by the renin-angiotensin system (RAS). Renin
Results and discussion
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-012-0903-5) contains supplementary
material, which is available to authorized users.
Chemistry
R. A. G. Harmsen ꢀ L. K. Sydnes ꢀ B. E. Haug (&)
We have recently reported the regioselective ring opening
of racemic oxirane 1 (Scheme 1) using the lithium acetylide
of ethynyltrimethylsilane with addition of trimethylalu-
minium and activation of the epoxide using boron
trifluoride-diethyl ether complex [12]. This reaction cleanly
gave the 3-hydroxy-4-(trimethylsilylethynyl)piperidine deri-
vative in 80 % yield. Subsequent removal of the TMS
Department of Chemistry, University of Bergen,
´
Allegaten 41, 5007 Bergen, Norway
e-mail: bengt-erik.haug@farm.uib.no
A. Sivertsen ꢀ D. Michetti ꢀ B. O. Brandsdal
Department of Chemistry, The Norwegian Structural Biology
Centre and The Centre for Theoretical and Computational
Chemistry, University of Tromsø, 9037 Tromsø, Norway
123

480
R. A. G. Harmsen et al.
was carried out using iron powder in acetic acid, followed
by acetylation using acetyl chloride, only about 5 % of the
chlorinated analogs of 13 and 14 were observed. The two
bromides could then be converted to the desired azides 15
and 16 by treatment with sodium azide in DMSO [14]. The
corresponding chlorides were unreactive under these con-
ditions. N-Boc protected trans-4-ethynylpiperidin-3-ol (2)
was alkylated with 2-(bromomethyl)naphthalene using
method C to give N-Boc protected trans-4-ethynyl-3-
(naphthalen-2-ylmethoxy)piperidine (17) in 80 % yield
(see experimental part). This material was allowed to react
with azides 15 or 16 under ruthenium catalysis (method B),
which followed by Boc deprotection gave compounds 9da
and 9ea (Fig. 2). In order to also introduce an alkyl group
on the acetamide groups, ruthenium catalyzed cycloadditon
(method B) was carried out first, followed by concurrent N-
and O-alkylation using method C, which after Boc depro-
tection gave compounds 9fa and 9ga (Fig. 2).
1)
, n-BuLi,
TMS
Me3Al, Et2O, 0 °C,
O
OH
(then 1)
.
2) BF3 OEt2, Et2O, -78 °C
3) TBAF, THF
N
N
Boc
Boc
( )-1
76%
( )-2
Scheme 1
group can either be carried out using TBAF in THF in
95 % yield (76 % combined yield from 1) as previously
reported [12] or by use of K2CO3 in methanol to give piperi-
dine building block 2 in 92 % yield for the deprotection step.
We have now utilized this new piperidine building block
for the preparation of a number of novel trans-3,4-disubsti-
tuted piperidine derivatives (Scheme 2). The terminal alkyne
of piperidine ( )-2 was converted into either 1,4-disubsti-
tuted 1,2,3-triazoles ( )-3 (method A) or 1,5-disubstituted
1,2,3-triazoles ( )-4 (method B) through copper and ruthe-
nium catalysis, respectively. Either of these 4-triazolyl-
substituted piperidines was further derivatized by conversion
of the secondary alcohol at the piperidine 3-position into
ether derivatives (method C) or ester derivatives (method D).
Finally, trifluoroacetic acid-mediated deprotection of the
piperidine Boc-protecting group furnished 11 piperidine
derivatives (Fig. 1). We were also interested in introducing
an acetamide-substituted phenyl ring of the R1 side chain
of 9aa (see Fig. 1), and toward this end two new azides
were prepared (Scheme 3).
Inhibitory activity toward renin
The 15 piperidine derivatives (see Figs. 1 and 2) were
tested for their ability to inhibit recombinant human renin
using the fluorimetric assay SensoLyteÒ520 Renin Assay
Kit. All compounds were tested as the racemic mixture,
initially at a concentration of 75 lM and then at 5 lM for
the most active compounds. The use of two different
endpoint concentrations is motivated by the need to dis-
tinguish the activity of the inhibitors. The endpoint
concentration of 75 lM enabled us to separate the activity
of the initial ligand series, whereas the use of 5 lM pro-
vided more details of the most active compounds. The
inhibitory activity expressed as the remaining renin proteolytic
It turned out that reduction of the nitro group of 11 and
12 using stannous chloride in concentrated hydrochloric
acid as described by Evans and Walker [13] gave a high
degree of halogen exchange. Unfortunately, the chlorides
were inseparable from the bromides. When the reduction
R1
R1
Scheme 2
R1-N3
Cu-cat.
Method A
N N
N
N N
N
Br
R2
OH
Method C
O
R2
OH
N
Boc
N
N
O
R
Boc
( )-2
R2
HO
Method D
( )-5 R = Boc
( )-8 R = H
( )-3
TFA
CH2Cl2
R1-N3
Ru-cat.
Method B
R1
N N
N
O
R2
N N
N
N N
N
O
R1
Br
R2
R1
N
R
O
R2
Method C
OH
( )-7 R = Boc
( )-10 R = H
TFA
CH2Cl2
N
N
R
Boc
( )-6 R = Boc
( )-9 R = H
( )-4
TFA
CH2Cl2
123

Synthesis and docking of novel piperidine renin inhibitors
481
OMe
OMe
N
N
N
N
N
N
N
N
N
N N
N
N
N
N
O
O
O
O
O
N
N
N
N
N
H
H
H
H
H
( )-8ca
( )-8aa
( )-8ab
( )-8ba
( )-8bb
N
N
N
N
N
N
MeO
O
O
N
N
H
H
( )-9ba
( )-9aa
OMe
OMe
N
N
N
N
N
N
N
N
N
N N
N
O
O
O
O
O
O
O
O
N
N
N
N
H
H
H
H
( )-10bb
( )-10ba
( )-10ca
( )-10cb
Fig. 1 Structures of the initial piperidine derivatives
Scheme 3
Br
Br
N3
1) Fe, HOAc,
MeOH, 60 °C
NaN3, DMSO
80-86%
2) Et3N, AcCl, reflux
R'
R'
R'
R
17-30%
R
R
15, 16
13, 14
11 R = H, R' = NO2
12 R = NO2, R' = H
13, 15 R = H, R' = NHAc
14, 16 R = NHAc, R' = H
activity at these concentrations is compiled in Table 1. All
the 4-triazolyl-substituted piperidine derivatives were
found to inhibit renin with varying potency. As has been
found for other piperidine-based renin inhibitors, a large
substituent at the piperidine C-3 results in higher inhibitory
activity, i.e. derivative 8aa shows higher activity than 8ab.
Conversion of the secondary hydroxyl group of the initial
piperidine building block into an ester instead of an ether
did not influence the inhibitory activity to any large extent.
Inhibitors containing 1,5-disubstituted 1,2,3-triazoles were
found to be more active than their isomers containing
1,4-disubstituted 1,2,3-triazoles. 9ga, which contains a
tertiary aryl amide in the R1 side chain, was found to be the
most active compound, with 93 % inhibition of renin at
5 lM. For this compound the IC50 was determined to be
631 nM for the racemic mixture, with a narrow 95 %
confidence interval of 580–687 nM. The remaining com-
pounds with 1,5-disubstituted triazolyl substituents were all
found to inhibit renin to approximately 50 % at 5 lM. All
compounds were also tested for activity against a second
aspartic protease, beta-secretase 1 (BACE1). All com-
pounds in this study showed more than 50 % remaining
BACE1 activity at 300 lM compound concentration (data
not shown), indicating that our substituted piperidines
have selectivity for renin over other members of this pro-
tease family.
123

482
R. A. G. Harmsen et al.
Fig. 2 Piperidine derivatives
containing an acetamide group
N
N
N
N
N
N
O
N
H
O
O
NH
N
O
N
H
H
( )-9da
( )-9ea
N
N
N
N
N
N
N
O
O
O
N
O
N
N
H
H
( )-9fa
( )-9ga
orientation of the flap has been found to depend on the
nature of the ligand [15]. For a recent and comprehensive
review, please see [16]. When the ligand is able to form
strong interactions with residues in the flap, especially
through hydrogen bonding, the flap will be in a closed
form. In contrast, ligands that interact weakly with the flap
will lead to a complex with an open form. Ligand-induced
effects on active site residues must also be considered. In
order to take the uncertainties with respect to ligand-
induced effects on the flap and active site residues into
account, a selection of experimental crystal structures were
included as targets in the docking protocol. The following
complexes were used: 3OAG [9], 2FS4 [17], 3OAD [9],
and 3O9L [9]. To assess the docking protocol, the four
experimental ligands in these complexes were docked into
their respective target structures using Glide [18]. RMSD
Table 1 Renin inhibitory activity and scoring of piperidine
derivatives
Compound
Remaining renin activity/%
Scoring
kJ/mol
5 lMa
95
75 lMb
8aa
8ab
8ba
8bb
8ca
9aa
9ba
9da
9ea
9fa
32
71
21
77
20
2
-31.8
-29.7
-32.7
-29.7
-31.4
-32.7
-31.4
-28.1
-30.1
-30.1
-36.8
-32.7
-30.1
-31.4
-28.5
–
84
–
71
37
51
48
43
35c
7d
89
–
–
–
–
–
9ga
10ba
10bb
10ca
10cb
a
–
˚
values lower than 0.5 A were obtained for the top ranked
22
73
30
66
docking solutions, and the docking scores were within
0.85 kJ/mol of the reported experimental IC50 values.
Based on the reproducibility of the complex crystal struc-
tures and agreement of the predicted binding affinities with
experimental values, we conclude that the docking protocol
is satisfactory for our system. However, when performing
cross-docking of the four ligands, variations in the binding
mode as well as the scoring are observed, but the rank
order of the ligands is still reproduced. The difference
between the docking score and experimental data increased
slightly to 2.5 kJ/mol.
91
–
Average of three parallels (\10 % spread)
Average of two parallels (\8 % spread)
Tested at 4 lM concentration
b
c
d
IC50 = 631 nM for the racemic mixture
Molecular docking study
Four piperidine-based renin inhibitors with known binding
modes and affinities were selected to develop a docking
procedure suitable for the compounds in this study. An
important aspect in ligand-binding modeling with aspartic
protease targets is the orientation of the characteristic
flap that covers the active site cleft of these proteases. This
particular region closes upon substrate binding to accom-
modate the required spatial arrangement during catalysis
and opens up to release the products after catalysis.
However, when inhibitors bind to aspartic proteases, the
Comparison of the region underneath the flap in crystal
structures of the two aspartic proteases renin (2FS4) and
BACE1 (2OHN) reveals that the former has residues with
smaller side chains in this region. Consequently, more
space is available to accommodate larger ligand moieties in
the S1 site of renin compared to BACE1 and is a plausible
explanation for the observed selectivity between the two
proteases. Attempts to dock our compounds to BACE1
(2OHN) produced solutions with docking scores corre-
sponding to low mM affinity (-21 kJ/mol), consistent with
123

Synthesis and docking of novel piperidine renin inhibitors
483
the kinetic assay results. In the in silico docking with the
established docking protocol for renin described above, the
closed form of the flap produced fewer docking solutions
and with significantly lower affinities compared with the
reported inhibition activities in Table 1. However, docking
of the substituted piperidines shown in Figs. 1 and 2 into
the active site cleft of renin (PDB code: 2FS4) gave
binding affinities ranging from approximately -16.8 to
-42.0 kJ/mol depending on the substitution pattern of the
ligand and its stereochemistry. Our findings support that
the (3R,4R) enantiomer was favored over the (3S,4S)
enantiomer for all compounds, possibly because of a less
favorable interaction with the catalytic aspartate dyad of
the latter. This is also consistent with the orientation of the
piperidine substituents in the crystal structures examined.
The docking scores for all compounds are given in Table 1
along with the experimental inhibition results. The energies
reported in Table 1 are averages of the top five docking
solutions of the (3R,4R) enantiomers. It is clear that most of
the compounds are correctly predicted in terms of scoring
value. In the experimental assay all compounds were
evaluated as racemic mixtures, and it is to be expected that
one of the enantiomers will have a stronger binding affinity
than the other because of the tendency of handedness in
biological systems. The substituted piperidine with the
highest inhibition potency was 9ga, where we reported an
IC50 value of 631 nM. In view of the docking observations
the (3R,4R) enantiomer would most likely obtain a sig-
nificantly better inhibition activity in the lower nM range
than the (3S,4S) enantiomer.
The positively charged nitrogen atom is in an optimal
position relative to the two catalytic aspartates, with
˚
hydrogen bond distances of 3.1 A to OD1 of Asp219 and to
OD2 of Asp33. This is in agreement with crystal structures
of renin where ligands containing piperidine derivatives are
bound. The top-ranked docking pose of 9ga has one of the
naphthyl groups placed in the S3 pocket, and the other,
which is attached to the triazole branch, is tucked under the
flap (see Fig. 3). Tyr78 at the tip of the flap interacts
through stacking interactions with the triazole and the
benzyl ring. We did not observe any hydrogen bonds
between 9ga and the flap region, hence indicating only
weaker van der Waals interactions. Some of the other
docking poses of 9ga explore the S1 site differently, but
maintain the S3 interactions as well as a similar orientation
of the piperidine. In this second observed binding mode,
the naphthyl previously tucked underneath the flap partially
explores the S10 site. However, the docking score for this
orientation is lower in comparison to the top-ranked solu-
tion. In addition, stacking interactions are potentially
present between the phenyl group and Trp 40, His 40, and
Phe 114 in the S1 site, as well as with Phe119 in the S3
subsite for this latter docking pose.
Conclusions
In this study, we utilized a new piperidine building block
toward the synthesis of a series of novel 4-triazolyl-
substituted piperidine renin inhibitors. Piperidines with
N-substituted 1,2,3-triazol-5-yl substituents were generally
found to be more active than the isomeric compounds
containing a 1,4-disubstituted 1,2,3-triazole moiety. The
most active inhibitor was found to inhibit renin with an
IC50 in the mid nM range as the racemic mixture, and all
compounds presented in this study were more potent
toward renin compared to BACE1. Through molecular
docking of all final compounds, we were able to establish a
method to predict inhibitory potency toward renin for these
molecules. The insight gained in this study will be applied
toward the design and synthesis of new inhibitors based on
the presented scaffold.
The docking experiments reveal that our compounds
explore the S1 and S3 binding pockets of renin. The tria-
zole side chain is placed under the flap in subsite S1,
whereas the ether-linked naphthyl explores the S3 pocket.
Figure 3 shows the top-ranked docking solution of com-
pound 9ga.
Experimental
All starting materials, reagents, and solvents were obtained
from Sigma-Aldrich and used without purification unless
otherwise noted. 2-(Azidoethyl)benzene (R1 side chain a)
and 1-(2-azidoethyl)-4-methoxybenzene (R1 side chain b)
were prepared following the literature procedures [14].
Alkyne 2 and triazoles 3a, 3b, and 4a were prepared as
previously reported by us [12]. Anhydrous solvents were
Fig. 3 Top-ranked docking solution of 9ga to renin (PDB code:
2FS4). The triazole is located in subsite S1, the triazole-linked
naphthyl group is tucked under the flap, and the ether linked naphthyl
group is located in S3. The piperidine nitrogen forms hydrogen bonds
to the aspartate dyad
123

484
R. A. G. Harmsen et al.
residue was co-evaporated with toluene. CH2Cl2 (10 cm3)
was added to the residue, and the resulting solution was
washed with 5 cm3 concentrated NH3 solution and 5 cm3
saturated aqueous NaCl solution. The organic phase was
dried over Na2SO4 and the solvent removed under reduced
pressure. The resulting product was dissolved in 0.4 cm3
dry CH2Cl2, 0.13 cm3 Et3N (0.95 mmol) and 1.6 cm3
acetyl chloride (0.95 mmol) were added, and the mixture
was stirred under reflux for 2.5 h. The reaction mixture
was then quenched with 1 cm3 0.1 M NaOH, layers were
separated, and the aqueous mixture was extracted
with 5 cm3 CH2Cl2. The organic phase was washed with
saturated aqueous NaCl solution, dried (MgSO4), and the
solvent removed under reduced pressure. Purification by
flash column chromatography (hexane/EtOAc, 2:1) gave
13 as a yellow solid (17 mg, 17 %). M.p.: 86.5–87.3 °C;
Rf = 0.22 (hexane/EtOAc, 2:1); 1H NMR (400 MHz,
CDCl3): d = 7.45 (d, J = 8.3 Hz, 2H), 7.26 (s, 1H), 7.16
(d, J = 8.3 Hz, 2H), 3.54 (t, J = 7.6 Hz, 2H), 3.12 (t,
J = 7.6 Hz, 2H), 2.17 (s, 3H) ppm; 13C NMR (101 MHz,
CDCl3): d = 168.9, 137.1, 135.1, 129.5, 120.5, 39.1, 33.4,
24.8 ppm; HRMS (ESI): m/z [M ? H]? calcd for
C10H13BrNO 242.0184, found 242.0184 and 244.0161.
obtained from an anhydrous solvent delivery system
(mBraun SPS-800 system). Flash column chromatography
was performed using silica gel from J.T. Baker (‘‘Baker
Analyzed,’’ 0.063–0.200 mm) supplied by Chiron AS
(Norway). TLC analysis was done using Macherey-Nagel
aluminum plates coated with silica gel 60 with fluorescent
indicator UV254 supplied by Chiron AS (Norway) with
visualization using ultraviolet light or a solution of 2 %
ninhydrin in ethanol containing 10 drops of conc. sulfuric
acid per 100 cm3 of the solution.
Purification by reversed-phase high performance liquid
chromatography (RP-HPLC) was performed using a C18
column (AscentisÒ C18, 5 lm, 21.2 9 250 mm, Supelco
Corp., Bellefonte, PA, USA) with a mixture of water and
acetonitrile (both containing 0.1 % TFA) as mobile phase
and UV detection at 220 nm. Analytical RP-HPLC was
performed using a C18 column (AscentisÒ C18, 5 lm,
4.6 9 250 mm, Supelco Corp., Bellefonte, PA, USA).
NMR spectra were recorded using a Bruker Spectrospin
AC. The chemical shifts are reported in parts per million
relative to TMS, and the coupling constants are given in
Hz. 13C peaks marked with * were identified from cross
peaks in HSQC spectra. Accurate mass determinations
were performed using a Thermo Scientific LTQ Orbitrap
MS (University of Tromsø).
N-[3-(2-Bromoethyl)phenyl]acetamide
(14, C10H12BrNO)
The title compound was prepared from 1.10 g 3-nitrophe-
nylethyl bromide (4.78 mmol) as described for bromide 13.
Purification by flash column chromatography (hexane/
EtOAc, 2:1) gave the title compound as a yellow solid
(0.347 g, 30 %). M.p.: 136.0–137.3 °C; Rf = 0.2 (hexane/
EtOAc, 2:1); 1H NMR (400 MHz, CDCl3): d = 7.45
(s, 1H), 7.38–7.23 (m, 3H), 6.96 (d, J = 7.4 Hz, 1H),
3.56 (t, J = 7.5 Hz, 2H), 3.14 (t, J = 7.5 Hz, 2H), 2.18
(s, 3H) ppm; 13C NMR (101 MHz, CDCl3): d = 168.8,
140.2, 138.5, 129.5, 124.9, 120.4, 118.7, 39.6, 33.1,
24.9 ppm; HRMS (ESI): m/z [M ? H]? calcd for
C10H13BrNO 242.0184, found 242.0187 and 244.0165.
Azidobenzene (R1 side chain c)
A suspension of 0.50 cm3 aniline (5.49 mmol) in 6 cm3
acetic acid and conc. sulfuric acid (ratio 2.25:1) was cooled
to 0 °C in an ice bath. A mixture of 2.9 cm3 NaNO2 (2 M
in H2O) was added dropwise to the suspension, and the
resulting mixture was stirred at 0 °C for 1 h. To the
obtained solution 2.9 cm3 urea (2 M in H2O) and 2.4 cm3
NaN3 (2.5 M in H2O) were added and the mixture was
stirred for another 3 h at 0 °C. The reaction was quenched
with 50 cm3 ice-cold H2O and made basic with 40 % aq.
NaOH. This aqueous mixture was extracted with hexane
(3 9 50 cm3), dried over MgSO4, and filtered, and the
solvent was removed under reduced pressure. The crude
product was purified by flash column chromatography
(hexane) to give the title compound as a yellow liquid
(0.49 g, 75 %); Rf = 0.53 (hexane). Analytical data are in
accordance with those reported in the literature [19].
N-[4-(2-Azidoethyl)phenyl]acetamide
(15, C10H12N4O) (R1 side chain d)
To a solution of 0.153 g bromide 13 (0.63 mmol) in
0.6 cm3 DMSO was added 2.20 cm3 of a solution of NaN3
in DMSO (0.5 M, 1.10 mmol). The resulting mixture was
stirred overnight at room temperature before 25 cm3 H2O
was added. The aqueous mixture was extracted three times
with Et2O (3 9 15 cm3), and the combined organic layers
were washed with H2O (2 9 25 cm3) and 25 cm3 saturated
aqueous NaCl solution before it was dried over MgSO4.
After filtration the solvent was evaporated under reduced
pressure to give the title compound as a yellow solid
(0.110 g, 86 %). M.p.: 89.2–90.8 °C; Rf = 0.20 (hexane/
N-[4-(2-Bromoethyl)phenyl]acetamide
(13, C10H12BrNO)
4-Nitrophenylethyl bromide (0.100 g, 0.43 mmol) was
dissolved in a mixture of 1.6 cm3 acetic acid and
0.75 cm3 methanol and heated to 65 °C. Fe powder
(87 mg) was added slowly to this solution. The reaction
mixture was stirred at 65 °C for 20 h, after which the
solvent was removed under reduced pressure. The resulting
1
EtOAc, 2:1); H NMR (400 MHz, CDCl3): d = 7.45 (d,
J = 8.2 Hz, 2H), 7.20–7.16 (apparent d, J = 8.2 Hz, 3H),
123

Synthesis and docking of novel piperidine renin inhibitors
485
3.68 (t, J = 7.4 Hz, 2H), 3.03 (t, J = 7.4 Hz, 2H), 2.17 (s,
3H) ppm; 13C NMR (101 MHz, CDCl3): d = 168.6, 136.7,
134.1, 129.4, 120.3, 52.6, 34.9, 24.7 ppm; HRMS (ESI):
m/z [M ? H]? calcd for C10H13N4O 205.1089, found
205.1104, m/z [2 M ? H]? calcd for C20H25N8O2
409.2101, found 409.2091.
119.3, 80.3, 70.7, 50.0, 43.6, 41.8, 29.7, 28.8 ppm; HRMS
(ESI): m/z [M ? Na]? calcd for C18H24N4NaO3 367.1746,
found 367.1740.
General procedure for ruthenium-catalyzed
cycloadditions: method B
N-[3-(2-Azidoethyl)phenyl]acetamide (16, C10H12N4O)
(R1 side chain e)
A solution of alkyne 2 (1.0 mmol) in dry THF (15 cm3/
mmol) was heated in an oil bath to 80 °C. To this mixture
Cp*RuCl(PPh3)2 (0.01 mmol) and azide (1.1 mmol) were
added. The reaction mixture was stirred at 80 °C until TLC
analysis showed full conversion of the starting material.
The solvent was removed using a rotary evaporator. The
crude reaction mixture was purified by flash column
chromatography.
The title compound was prepared from 0.107 g bromide 14
(0.44 mmol) as described for azide 15 and was obtained as
a colorless oil (71 mg, 80 %). Rf = 0.33 (hexane/EtOAc,
1
1:1); H NMR (400 MHz, CDCl3): d = 8.79 (s, 1H), 7.52
(s, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H),
6.93 (d, J = 7.5 Hz, 1H), 3.47 (t, J = 7.2 Hz, 2H), 2.84 (t,
J = 7.2 Hz, 2H), 2.14 (s, 3H) ppm; 13C NMR (101 MHz,
CDCl3): d = 168.8, 139.4, 138.6, 129.5, 125.0, 120.5,
118.5, 52.6, 35.6, 24.9 ppm; HRMS (ESI): m/z [M ? Na]?
calcd for C10H12N4NaO 227.0903, found 227.0897.
( )-tert-Butyl trans-3-hydroxy-4-[1-(4-methoxyphenethyl)-
1H-1,2,3-triazol-5-yl]piperidine-1-carboxylate
(4b, C21H30N4O4)
The title compound was prepared from 0.114 g alkyne 2
(0.51 mmol) and 98 mg 1-(2-azidoethyl)-4-methoxyben-
zene (0.56 mmol) using method B. The reaction mixture
was stirred for 20 h at 80 °C, after which TLC analysis
indicated full conversion of the starting material. Purifica-
tion by flash column chromatography (hexane/EtOAc, 1:4)
gave 4b as a colorless solid (0.173 g, 84 %). M.p.:
General procedure for copper-catalyzed
cycloadditions: method A
To a solution of alkyne 2 (1.0 mmol) and an azide
(1.0 mmol) in a mixture of H2O and CH2Cl2 (1:1,
0.05 mmol/cm3) were added CuSO4ꢀ5H2O (0.05 mmol)
and sodium ascorbate (0.15 mmol). The reaction mixture
was stirred at room temperature until TLC showed full
conversion of the starting material after which the mixture
was filtered, and the precipitate was washed with H2O. The
filtrate was extracted with EtOAc (3 9 10 cm3), and the
organic layers were combined with the precipitate. These
combined layers were washed with 10 cm3 H2O and
10 cm3 saturated aqueous NaCl solution, dried over
MgSO4, and filtered, and the solvent was removed under
reduced pressure. The crude product was purified by flash
column chromatography.
1
142.4–144.3 °C; Rf = 0.3 (hexane/EtOAc, 1:4); H NMR
(400 MHz, CDCl3): d = 7.28 (s, 1H), 6.94 (d, J = 8.3 Hz,
2H), 6.79 (d, J = 8.3 Hz, 2H), 4.65–4.46 (m, 2H),
4.31–4.19 (m, 1H), 4.10–3.98 (m, 1H), 3.75 (s, 3H),
3.48–3.37 (m, 1H), 3.21–3.08 (m, 2H), 2.65 (s, 1H),
2.57–2.42 (m, 2H), 2.24–2.13 (m, 1H), 1.53–1.43 (m,
10H), 1.39–1.28 (m, 1H) ppm; 13C NMR (101 MHz,
CDCl3): d = 159.0, 154.7, 139.6, 130.5, 130.3, 120.0,
114.4, 80.5, 71.6, 55.7, 51*, 50.1, 44*, 40.5, 36.4, 30.3,
28.7 ppm; HRMS (ESI): m/z [M ? Na]? calcd for
C21H30N4NaO4 425.2165, found 425.2159.
( )-tert-Butyl trans-3-hydroxy-4-(1-phenyl-1H-1,2,3-tria-
zol-4-yl)piperidine-1-carboxylate (3c, C18H24N4O3)
( )-tert-Butyl trans-4-[1-(4-acetamidophenethyl)-1H-
1,2,3-triazol-5-yl]-3-(naphthalen-2-ylmethoxy)piperidine-
1-carboxylate (6da, C33H39N5O4)
Triazole 3c was prepared from 0.308 g alkyne
2
(1.37 mmol) and 0.163 g azidobenzene (1.37 mmol) using
method A. The reaction mixture was stirred at room
temperature for 3 h, after which TLC analysis indicated
full conversion of the starting material. The crude product
was purified by flash column chromatography (hexane/
EtOAc, 1:1) to give the title compound as a colorless solid
(0.420 g, 89 %). M.p.: 130.4–132.3 °C; Rf = 0.19 (hex-
The title compound was prepared from 37 mg alkyne 17
(0.10 mmol) and 23 mg azide 15 (0.11 mmol) using
method B. The reaction was stirred at 80 °C for 20 h.
Purification by flash column chromatography (hexane/
EtOAc, 1:9) gave the title compound as a colorless oil
1
(37 mg, 65 %). Rf = 0.25 (hexane/EtAOc, 1:9); H NMR
(400 MHz, CDCl3): d = 7.80–7.61 (m, 3H), 7.58–7.52
(m, 1H), 7.51–7.41 (m, 3H), 7.38–7.30 (m, 3H), 6.99–6.90
(m, 1H), 6.84 (d, J = 7.9 Hz, 1H), 4.72–4.25 (m, 5H),
4.10–3.84 (m, 1H), 3.21–2.94 (m, 3H), 2.89–2.71 (m, 1H),
2.55–2.39 (m, 1H), 2.31–2.18 (m, 1H), 2.12 (s, 3H),
1.61–1.19 (m, 11H) ppm; 13C NMR (101 MHz, CDCl3):
d = 168.7, 154.6, 146*, 137.4, 135.0, 134.8, 133.4, 133.2,
1
ane/EtOAc, 1:1); H NMR (400 MHz, CDCl3): d = 7.84
(s, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.52 (t, J = 8.0 Hz, 2H),
7.48–7.41 (m, 1H), 4.48–4.33 (m, 1H), 4.31–4.07 (m, 1H),
3.83–3.69 (m, 1H), 2.94–2.66 (m, 3H), 2.15–2.05 (m, 1H),
1.85–1.69 (m, 1H), 1.47 (s, 9H) ppm; 13C NMR (101 MHz,
CDCl3): d = 155.1, 146*, 137.5, 130.1, 129.2, 120.9,
123

486
R. A. G. Harmsen et al.
132.4, 132.3, 129.7, 129.4, 128.9, 128.8, 128.2, 128.0,
126.6, 125.7, 120.4, 80.5, 79.0, 72.2, 49.9, 47*, 43*, 38.7,
36.5, 28.7, 24.8 ppm; HRMS (ESI): m/z [M ? H]? calcd
for C33H40N5O4 570.3080, found 570.3078.
21 h, after which TLC analysis indicated full conversion of
the starting material. Purification by flash column chroma-
tography (hexane/EtOAc, 1:9) gave the title compound as a
colorless oil (0.197 g, 82 %). Rf = 0.26 (hexane/EtOAc,
1:9); 1H NMR (400 MHz, CDCl3): d = 7.64 (s, 1H),
7.52–7.00 (m, 3H), 6.80 (d, J = 7.4 Hz, 1H), 4.61 (t,
J = 6.7 Hz, 2H), 4.36–4.18 (m, 1H), 4.20–3.95 (m, 1H),
3.49–3.34 (m, 1H), 3.30–3.05 (m, 2H), 2.60–2.44 (m, 2H),
2.26–2.20 (m, 1H), 2.15 (s, 3H), 1.55–1.34 (m, 10H),
1.39–1.16 (m, 1H) ppm; 13C NMR (101 MHz, CDCl3):
d = 169.0, 154.8, 150*, 138.9, 138*, 137*, 130*, 129.7,
125.2, 118.9, 80.5, 71.4, 50*, 49.5, 43*, 40*, 36.9, 30.5,
28.7, 24.9 ppm; HRMS (ESI): m/z [M ? H]? calcd for
C22H32N5O4 430.2454, found 430.2457.
( )-tert-Butyl trans-4-[1-(3-acetamidophenethyl)-1H-
1,2,3-triazol-5-yl]-3-(naphthalen-2-ylmethoxy)piperidine-
1-carboxylate (6ea, C33H39N5O4)
The title compound was prepared from 0.12 g alkyne 17
(0.33 mmol) and 74 mg azide 16 (0.36 mmol) using method
B. The reaction was stirred at 80 °C for 22 h. Purification by
flash column chromatography (hexane/EtOAc, 1:6) gave the
title compound as a colorless oil (0.142 g, 76 %). Rf = 0.27
(hexane/EtAOc, 1:6); 1H NMR (400 MHz, CDCl3):
d = 7.88–7.64 (m, 3H), 7.58–7.42 (m, 2H), 7.39–7.32 (m,
2H), 7.30–7.24 (m, 2H), 7.14 (t, J = 7.8 Hz, 1H), 7.03–6.88
(m, 1H), 6.80–6.53 (m, 1H), 4.67–4.42 (m, 4H), 4.31 (d,
J = 11.4 Hz, 1H), 4.07–3.94 (m, 1H), 3.18–2.95 (m, 3H),
2.63–2.34 (m, 2H), 2.32–2.19 (m, 1H), 2.13 (s, 3H),
1.58–1.49 (m, 1H), 1.43 (s, 9H), 1.33–1.19 (m, 1H) ppm;
13C NMR (101 MHz, CDCl3): d = 168.8, 154.6, 135.0,
133.4, 133.3, 132.4, 132.3, 130.6, 129.6, 128.9, 128.6,
128.2, 128.0, 126.8, 126.6, 126.4, 125.7, 124.9, 120.5,
118.6, 80.5, 79.1, 72.1, 49.6, 47*, 43*, 38.6, 37.1, 29*, 28.7,
24.9 ppm; HRMS (ESI): m/z [M ? Na]? calcd for
C33H39N5NaO4 592.2893, found 592.2900.
General procedure for the formation of ether linkages:
method C
To a secondary alcohol (0.2 mmol) was added a suspension
of NaH (60 wt % in mineral oil, 0.4 mmol) in 5 cm3 DMF.
After stirring for 30 min at room temperature, a mixture of
tetrabutylammonium iodide (TBAI, 0.02 mmol) and an
alkyl bromide (0.2 mmol) in 1 cm3 DMF was added, and
the reaction mixture was stirred at room temperature until
TLC analysis indicated full conversion of the alcohol. The
reaction was quenched by addition of 10 cm3 H2O, and
the aqueous mixture was extracted with 20 cm3 EtOAc.
The organic layer was washed with 10 % aqueous
HCl (2 9 10 cm3), saturated aqueous NaHCO3 solution
(2 9 10 cm3), and H2O (2 9 10 cm3) and dried over
MgSO4. After removal of the drying agent by filtration, the
solvent was removed under vacuum. The crude product
was purified by flash column chromatography.
( )-tert-Butyl trans-4-[1-(4-acetamidophenethyl)-1H-
1,2,3-triazol-5-yl]-3-hydroxypiperidine-1-carboxylate
(18, C22H31N5O4)
The title compound was prepared from 43 mg alkyne 2
(0.19 mmol) and 43 mg azide 15 (0.21 mmol) using
method B. The reaction mixture was stirred at 80 °C for
21 h, after which TLC analysis indicated full conversion of
the starting material. Purification by flash column chroma-
tography (EtOAc) gave the title compound as a colorless
solid (73 mg, 90 %). M.p.: 208.7–209.5 °C; Rf = 0.24
(EtOAc); 1H NMR (400 MHz, CDCl3): d = 7.36
(d, J = 8.1 Hz, 2H), 7.31–7.13 (m, 2H), 6.98 (d, J =
8.1 Hz, 2H), 4.57 (t, J = 7.4 Hz, 3H), 4.30–4.18 (m, 1H),
4.09–3.90 (m, 1H), 3.53–3.35 (m, 1H), 3.24–3.03 (m, 2H),
2.74–2.42 (m, 3H), 2.23–1.98 (m, 4H), 1.46 (s, 9H),
1.38–1.32 (m, 1H) ppm; 13C NMR (101 MHz, CDCl3):
d = 170.2, 156.1, 147*, 135.6, 130.5, 131.1, 129.7, 120.6,
80.6, 71.5, 50*, 49.3, 44*, 40*, 36.5, 29*, 28.6, 24.5 ppm;
HRMS (ESI): m/z [M ? H]? calcd for C22H32N5O4
430.2454, found 430.2459.
( )-tert-Butyl trans-3-(naphthalene-2-ylmethoxy)-4-(1-
phenethyl-1H-1,2,3-triazol-4-yl)piperidine-1-carboxylate
(5aa, C31H36N4O3)
The title compound was prepared from 55 mg alcohol 3a
(0.15 mmol) and 66 mg 2-(bromomethyl)naphthalene
(0.30 mmol) using method C. The reaction mixture was
stirred for 20 h, after which TLC analysis indicated full
conversion of the starting material. Purification by flash
column chromatography (hexane/EtOAc, 1:1) gave 5aa as a
colorless oil (39 mg, 70 %). Rf = 0.18 (hexane/EtOAc,
1:1); 1H NMR (400 MHz, CDCl3): d = 7.83–7.74 (m, 3H),
7.60 (s, 1H), 7.48–7.40 (m, 2H), 7.29–7.15 (m, 4H),
7.05–6.97 (m, 2H), 6.93 (s, 1H), 4.78–4.63 (m, 1H),
4.54–4.29 (m, 4H), 4.06–3.97 (m, 1H), 3.63–3.52 (m, 1H),
3.09 (t, J = 7.1 Hz, 2H), 2.96–2.75 (m, 3H), 2.09–1.98 (m,
1H), 1.89–1.76 (m, 1H), 1.44 (s, 9H) ppm; 13C NMR
(101 MHz, CDCl3): d = 155.0, 148.7, 137.6, 136.2, 133.7,
133.4, 129.1, 129.0, 128.4, 128.3, 128.0, 127.4, 126.9, 126.5,
126.27, 126.25, 122.0, 80.1, 77.6, 72.2, 51.7, 47*, 43*, 40.4,
( )-tert-Butyl trans-4-[1-(3-acetamidophenethyl)-1H-
1,2,3-triazol-5-yl]-3-hydroxypiperidine-1-carboxylate
(19, C22H31N5O4)
The title compound was prepared from 0.12 g alkyne 2
(0.33 mmol) and 74 mg azide 16 (0.36 mmol) using
method B. The reaction mixture was stirred at 80 °C for
123

Synthesis and docking of novel piperidine renin inhibitors
487
37.0, 30.5, 28.8 ppm; HRMS (ESI): m/z [M ? Na]? calcd
for C31H36N4NaO3 535.2685, found 535.2693.
starting material. Purification by flash column chromatog-
raphy (hexane/EtOAc, 1:1) gave 5bb as a colorless oil
(0.106 g, 89 %). Rf = 0.31 (hexane/EtOAc, 1:1); 1H NMR
(400 MHz, CDCl3): d = 7.30–7.20 (m, 3H), 7.14 (d,
J = 6.5 Hz, 2H), 7.01–6.94 (m, 3H), 6.78 (d, J =
8.5 Hz, 2H), 4.60–4.41 (m, 3H), 4.31 (d, J = 11.6 Hz,
2H), 4.00 (d, J = 13.4 Hz, 1H), 3.74 (s, 3H), 3.59–3.46 (m,
1H), 3.09 (t, J = 7.2 Hz, 2H), 2.96–2.83 (m, 2H),
2.83–2.72 (m, 1H), 2.11–1.95 (m, 1H), 1.90–1.77 (m,
1H), 1.46 (s, 9H) ppm; 13C NMR (101 MHz, CDCl3):
d = 159.2, 155.0, 148.6, 138.7, 130.0, 129.6, 128.7, 128.1,
128.0, 122.0, 114.7, 80.2, 77.6, 72.1, 55.6, 52.0, 47*, 43*,
40.3, 36.3, 30.5, 28.8 ppm; HRMS (ESI): m/z [M ? H]?
calcd for C28H37N4O4 493.2815, found 493.2813.
( )-tert-Butyl trans-3-benzyloxy-4-(1-phenethyl-1H-1,2,3-
triazol-4-yl)piperidine-1-carboxylate
(5ab, C27H34N4O3)
The title compound was prepared from 60 mg alcohol 3a
(0.18 mmol) and 43 mm3 benzyl bromide (0.36 mmol)
using method C. The reaction mixture was stirred for 20 h,
after which TLC analysis indicated full conversion of the
starting material. Purification by flash column chromatog-
raphy (hexane/EtOAc, 2:1) gave 5ab as a colorless oil
1
(53 mg, 64 %). Rf = 0.33 (hexane/EtOAc, 1:2); H NMR
(400 MHz, CDCl3): d = 7.32–7.19 (m, 6H), 7.14 (d,
J = 6.9 Hz, 2H), 7.08 (d, J = 6.7 Hz, 2H), 7.00 (s, 1H),
4.61–4.42 (m, 4H), 4.31 (d, J = 11.1 Hz, 1H), 4.02 (d,
J = 13.2 Hz, 1H), 3.58–3.49 (m, 1H), 3.15 (t, J = 7.2 Hz,
2H), 2.93–2.56 (m, 3H), 2.08–1.97 (m, 1H), 1.89–1.76 (m,
1H), 1.47 (s, 9H) ppm; 13C NMR (101 MHz, CDCl3):
d = 155.0, 148.5, 138.6, 137.5, 129.1, 129.0, 128.6, 128.1,
128.0, 127.4, 122.0, 80.1, 77.4, 72.0, 51.8, 48*, 43*, 40.2,
37.1, 30.4, 28.8 ppm; HRMS (ESI): m/z [M ? Na]? calcd
for C27H34N4NaO3 485.2529, found 485.2526.
( )-tert-Butyl trans-3-(naphthalen-2-ylmethoxy)-4-(1-phe-
nyl-1H-1,2,3-triazol-4-yl)piperidine-1-carboxylate
(5ca, C29H32N4O3)
The title compound was prepared from 80 mg alcohol 3c
(0.23 mmol) and 0.101 g 2-(bromomethyl)naphthalene
(0.46 mmol) using method C. The reaction mixture was
stirred for 5 h, after which TLC analysis indicated full
conversion of the starting material. Purification by flash
column chromatography (hexane/EtOAc, 2:1) gave 5ca as a
colorless oil (98 mg, 88 %). Rf = 0.31 (hexane/EtOAc, 2:1);
1H NMR (400 MHz, CDCl3): d = 7.76–7.64 (m, 3H), 7.61
(d, J = 11.4 Hz, 2H), 7.52 (d, J = 7.2 Hz, 2H), 7.46–7.33
(m, 5H), 7.24 (d, J = 7.2 Hz, 1H), 4.80 (d, J = 11.4 Hz,
1H), 4.63–4.41 (m, 2H), 4.18–4.06 (m, 1H), 3.70–3.59 (m,
1H), 3.08–2.96 (m, 1H), 2.95–2.73 (m, 2H), 2.19–2.08 (m,
1H), 2.04–1.87 (m, 1H), 1.47 (s, 9H) ppm; 13C NMR
(101 MHz, CDCl3): d = 155.0, 149.5, 137.6, 136.0, 133.6,
133.4, 130.0, 128.7, 128.5, 128.2, 128.0, 127.1, 126.5,
126.32, 126.28, 120.6, 120.0, 80.2, 77.6, 72.3, 47.7, 43.9,
40.6, 30.5, 28.8 ppm; HRMS (ESI): m/z [M ? H]? calcd for
C29H33N4O3 485.2553, found 485.2552.
( )-tert-Butyl trans-4-[1-(4-methoxyphenethyl)-1H-1,2,3-
triazol-4-yl]-3-(naphthalene-2-ylmethoxy)piperidine-1-
carboxylate (5ba, C32H38N4O4)
The title compound was prepared from 0.229 g alcohol 3b
(0.57 mmol) and 0.252 g 2-(bromomethyl)naphthalene
(1.14 mmol) using method C. The reaction mixture was
stirred for 21 h, after which TLC analysis indicated full
conversion of the starting material. Purification by flash
column chromatography (hexane/EtOAc, 1:1) gave 5ba as
a colorless oil (0.245 g, 79 %). Rf = 0.17 (hexane/EtOAc,
1:1); 1H NMR (400 MHz, CDCl3): d = 7.83–7.71 (m, 3H),
7.61 (s, 1H), 7.49–7.21 (m, 2H), 7.27–7.21 (m, 1H), 6.95
(s, 1H), 6.91 (d, J = 8.5 Hz, 2H), 6.74 (d, J = 8.5 Hz,
2H), 4.77–4.62 (m, 1H), 4.53–4.30 (m, 4H), 4.07–3.97 (m,
1H), 3.71 (s, 3H), 3.64–3.52 (m, 1H), 3.02 (t, J = 7.2 Hz,
2H), 2.96–2.74 (m, 3H), 2.09–1.96 (m, 1H), 1.90–1.76 (m,
1H), 1.44 (s, 9H) ppm; 13C NMR (101 MHz, CDCl3):
d = 158.9, 154.9, 148.5, 136.0, 133.5, 133.3, 130.0, 129.4,
128.3, 128.2, 128.0, 126.9, 126.5, 126.3, 126.2, 122.0,
114.4, 80.1, 77*, 72.2, 55.5, 51.9, 48*, 44*, 40.3, 36.2,
30.5, 28.7 ppm; HRMS (ESI): m/z [M ? Na]? calcd for
C32H38N4NaO4 565.2791, found 565.2772.
( )-tert-Butyl trans-3-(naphthalene-2-ylmethoxy)-4-(1-
phenethyl-1H-1,2,3-triazol-5-yl)piperidine-1-carboxylate
(6aa, C31H36N4O3)
The title compound was prepared from 60 mg alcohol 4a
(0.16 mmol) and 71 mg 2-(bromomethyl)naphthalene
(0.32 mmol) using method C. The reaction mixture was
stirred for 19 h, after which TLC analysis indicated full
conversion of the starting material. Purification by flash
column chromatography (hexane/EtOAc, 1:1) gave 6aa as a
colorless solid (45 mg, 55 %). M.p.: 108.8–112.6 °C;
Rf = 0.17 (hexane/EtOAc, 1:1); 1H NMR (400 MHz,
CDCl3): d = 7.81–7.69 (m, 3H), 7.49–7.41 (m, 2H), 7.37
(s, 1H), 7.34 (s, 1H), 7.23–7.14 (m, 3H), 6.98 (d,
J = 8.3 Hz, 1H), 6.93 (d, J = 6.9 Hz, 2H), 4.66–4.37 (m,
4H), 4.31 (d, J = 8.0 Hz, 1H), 4.06–3.94 (m, 1H), 3.20–3.07
(m, 3H), 2.53–2.36 (m, 2H), 2.25–2.13 (m, 1H), 1.59–1.49
(m, 1H), 1.42 (s, 9H), 1.31–1.20 (m, 1H) ppm; 13C NMR
( )-tert-Butyl trans-3-benzyloxy-4-[1-(4-methoxyphenethyl)-
1H-1,2,3-triazol-4-yl]piperidine-1-carboxylate
(5bb, C28H36N4O4)
The title compound was prepared from 98 mg alcohol
3b (0.24 mmol) and 57 mm3 benzylbromide (0.48 mmol)
using method C. The reaction mixture was stirred for 22 h,
after which TLC analysis indicated full conversion of the
123

488
R. A. G. Harmsen et al.
(101 MHz, CDCl3): d = 154.7, 139.6, 138.4, 135.1, 133.6,
133.5, 130.7, 129.3, 129.0, 128.7, 128.3, 128.1, 127.2,
126.9, 126.6, 126.5, 125.8, 80.6, 79.3, 72.4, 49.9, 48*, 44*,
38.9, 37.3, 30.3, 28.8 ppm; HRMS (ESI): m/z [M ? Na]?
calcd for C31H36N4NaO3 535.2685, found 535.2670.
(0.28 mmol) using method C. Purification by flash column
chromatography (hexane/EtOAc, 1:6) gave the title com-
pound as a colorless oil (70 mg, 71 %). Rf = 0.27 (hexane/
1
EtOAc, 1:6); H NMR (400 MHz, CDCl3): d = 7.88–7.63
(m, 6H), 7.53 (s, 1H), 7.50–7.32 (m, 7H), 6.99–6.91 (m,
1H), 6.85 (d, J = 7.6 Hz, 2H), 6.79 (d, J = 7.6 Hz, 2H),
4.97 (s, 2H), 4.74–4.56 (m, 2H), 4.55–4.40 (m, 2H), 4.34
(d, J = 11.6 Hz, 1H), 4.20–4.00 (m, 1H), 3.34–2.98 (m,
3H), 2.70–2.30 (m, 3H), 1.85 (s, 3H), 1.70–1.53 (m, 2H),
1.44 (s, 9H) ppm; 13C NMR (101 MHz, CDCl3):
d = 170.7, 154.6, 150*, 141.9, 139.1, 137*, 135.3, 134.8,
133.6, 133.4, 133.1, 131.0, 130.2, 128.7, 128.6, 128.2,
128.1, 128.0, 127.6, 127.03, 126.95, 126.6, 126.5, 126.4,
126.3, 126.2, 125.8, 80.8, 79.0, 72.3, 53.2, 49.2, 48*,
44*, 38.9, 36.1, 30*, 28.7, 23.1 ppm; HRMS (ESI):
m/z [M ? H]? calcd for C44H48N5O4 710.3706, found
710.3711.
( )-tert-Butyl trans-4-[1-(4-methoxyphenethyl)-1H-1,2,3-
triazol-5-yl]-3-(naphthalene-2-ylmethoxy)piperidine-1-
carboxylate (6ba, C32H38N4O4)
The title compound was prepared from 0.116 g alcohol 4b
(0.29 mmol) and 0.128 g 2-(bromomethyl)naphthalene
(0.58 mmol) using method C. The reaction mixture was
stirred for 18 h, after which TLC analysis indicated full
conversion of the starting material. Purification by flash
column chromatography (hexane/EtOAc, 1:1) gave the title
compound as a colorless oil (0.141 g, 89 %). Rf = 0.35
(hexane/EtOAc, 1:1); 1H NMR (400 MHz, CDCl3):
d = 7.84–7.69 (m, 3H), 7.50–7.42 (m, 2H), 7.39–7.33 (m,
2H), 7.01–6.92 (m, 1H), 6.83 (d, J = 8.2 Hz, 2H), 6.73 (d,
J = 8.2 Hz, 2H), 4.67–4.52 (m, 2H), 4.52–4.40 (m, 2H),
4.35–4.27 (m, 1H), 4.10–3.96 (m, 1H), 3.73 (s, 3H),
3.18–3.01 (m, 3H), 2.53–2.37 (m, 2H), 2.24–2.14 (m, 1H),
( )-tert-Butyl trans-3-(naphthalen-2-ylmethoxy)-4-[1-[3-
[N-(naphthalen-2-ylmethyl)acetamido]phenethyl]-1H-
1,2,3-triazol-5-yl]piperidine-1-carboxylate
(6ga, C44H47N5O4)
1.62–1.44 (m, 1H), 1.42 (s, 9H), 1.34–1.22 (m, 1H) ppm; 13
C
The title compound was prepared from 20 mg alcohol 19
(0.035 mmol) and 23 mg 2-(bromomethyl)naphthalene
(0.11 mmol) using method C. Purification by flash column
chromatography (hexane/EtOAc, 1:9) gave the title com-
pound as a colorless oil (18 mg, 72 %). Rf = 0.26 (hexane/
NMR (101 MHz, CDCl3): d = 158.8, 154.6, 139.6, 135.0,
133.4, 133.3, 130.7, 130.3, 130.1, 128.6, 128.2, 128.0, 126.8,
126.6, 126.4, 125.7, 114.3, 80.6, 79.0, 72.2, 55.6, 50.1, 48*,
44*, 38.7, 36.3, 30.2, 28.7 ppm; HRMS (ESI): m/z
[M ? H]? calcd for C32H39N4O4 543.2971, found 543.2973.
1
EtOAc, 1:9); H NMR (400 MHz, CDCl3): d = 7.88–7.63
(m, 6H), 7.53–7.51 (m, 1H), 7.50–7.39 (m, 4H), 7.38–7.31
(m, 3H), 7.11 (t, J = 7.8 Hz, 1H), 6.97–6.90 (m, 1H), 6.86
(d, J = 7.5 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 6.60 (s,
1H), 5.00 (d, J = 14.6 Hz, 1H), 4.89 (d, J = 14.6 Hz, 1H),
4.66–4.47 (m, 2H), 4.45–4.34 (m, 2H), 4.29 (d, J =
11.6 Hz, 1H), 4.05–3.98 (m, 1H), 3.22–3.08 (m, 2H),
3.07–2.96 (m, 1H), 2.69–2.33 (m, 3H), 1.82 (s, 3H),
1.65–1.52 (m, 1H), 1.49–1.37 (m, 10H) ppm; 13C NMR
(101 MHz, CDCl3): d = 171.5, 154.5, 147*, 143.5, 139.6,
135.3, 134.8, 133.5, 133.4, 133.0, 130.9, 128.9, 128.7,
128.5, 128.2, 128.1, 128.02, 127.97, 127.72, 127.67,
126.99, 126.96, 126.7, 126.5, 126.4, 126.2, 125.8, 123.0,
122.5, 121.1, 80.7, 79.0, 72.3, 60.7, 49.2, 49*, 45*,
38.8, 36.4, 30*, 28.7, 23.1, 21.4 ppm; HRMS (ESI):
m/z [M ? H]? calcd for C44H48N5O4 710.3706, found
710.3681.
( )-tert-Butyl trans-4-ethynyl-3-(naphthalen-2-ylmethoxy)-
piperidine-1-carboxylate (17, C23H27NO3)
The title compound was prepared from 60 mg alcohol 2
(0.30 mmol) and 0.132 g 2-(bromomethyl)naphthalene
(0.60 mmol) using method C. Purification of the crude
product by flash column chromatography (hexane/EtOAc,
9:1) gave 17 as a colorless solid (88 mg, 80 %). M.p.:
1
60.9–64.7 °C; Rf = 0.30 (hexane/EtOAc, 9:1); H NMR
(400 MHz, CDCl3): d = 7.86–7.77 (m, 4H), 7.52–7.41 (m,
3H), 4.87 (d, J = 12.0 Hz, 1H), 4.77 (d, J = 12.0 Hz, 1H),
3.81–3.68 (m, 1H), 3.61–3.51 (m, 1H), 3.50–3.40 (m, 2H),
3.39–3.30 (m, 1H), 2.77–2.69 (m, 1H), 2.15 (d,
J = 2.1 Hz, 1H), 2.10–2.00 (m, 1H), 1.61–1.50 (m, 1H),
1.43 (s, 9H) ppm; 13C NMR (101 MHz, CDCl3):
d = 155.2, 136.2, 133.8, 133.5, 128.5, 128.3, 128.1,
126.8, 126.4, 126.2, 84.9, 80.0, 75.8, 72.0, 71.4, 45*,
41*, 33.3, 28.8, 28.4 ppm; HRMS (ESI): m/z [M ? Na]?
calcd for C23H27NNaO3 388.1889, found 388.1870.
General procedure for the formation of ester linkages:
method D
( )-tert-Butyl trans-3-(naphthalen-2-ylmethoxy)-4-[1-[4-
[N-(naphthalen-2-ylmethyl)acetamido]phenethyl]-1H-
1,2,3-triazol-5-yl]piperidine-1-carboxylate
To a solution of an alcohol (0.5 mmol) in 5 cm3 dry
CH2Cl2 were added a carboxylic acid (0.75 mmol), DMAP
(0.9 mmol), and DCC (0.9 mmol). The reaction mixture
was stirred at room temperature until TLC analysis indi-
cated full conversion of the alcohol. The reaction mixture
(6fa, C44H47N5O4)
The title compound was prepared from 60 mg alcohol 18
(0.14 mmol) and 62 mg 2-(bromomethyl)naphthalene
123

Synthesis and docking of novel piperidine renin inhibitors
489
was diluted with 25 cm3 Et2O and washed with 25 cm3
5 % citric acid and 25 cm3 saturated aqueous NaCl solu-
tion before it was dried over MgSO4. After removal of the
drying agent by filtration, the solvent was removed under
reduced pressure. The crude product was purified by flash
column chromatography.
( )-tert-Butyl trans-3-(2-naphthoyloxy)-4-(1-phenyl-1H-
1,2,3-triazol-4-yl)piperidine-1-carboxylate
(7ca, C29H30N4O4)
The title compound was prepared from 73 mg alcohol 3c
(0.21 mmol) and 55 mg 2-naphthoic acid (0.32 mmol)
using method D. The reaction mixture was stirred for 20 h,
after which TLC analysis indicated full conversion of the
starting material. Purification by flash column chromatog-
raphy (hexane/EtOAc, 2:1) gave 7ca as a colorless solid
(75 mg, 72 %). M.p.: 174.9–177.1 °C; Rf = 0.25 (hexane/
( )-tert-Butyl trans-3-(2-naphthoyloxy)-4-[1-(4-methoxy-
phenethyl)-1H-1,2,3-triazol-4-yl]piperidine-1-carboxylate
(7ba, C32H36N4O5)
1
The title compound was prepared from 90 mg alcohol 3b
(0.40 mmol) and 57 mg 2-naphthoic acid (0.33 mmol)
using method D. The reaction mixture was stirred for 20 h,
after which TLC analysis indicated full conversion of the
starting material. Purification by flash column chromatog-
raphy (hexane/EtOAc, 1:1) gave 7ba as a colorless oil
EtOAc, 2:1); H NMR (400 MHz, CDCl3): d = 8.56 (s,
1H), 8.01 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 7.9 Hz, 1H),
7.87–7.80 (m, 3H), 7.62 (d, J = 7.9 Hz, 2H), 7.59–7.48
(m, 2H), 7.47–7.40 (m, 2H), 7.39–7.33 (m, 1H), 5.41–5.29
(m, 1H), 4.38–4.25 (m, 1H), 4.12–3.99 (m, 1H), 3.55–3.43
(m, 1H), 3.40–3.20 (m, 2H), 2.39–2.28 (m, 1H), 2.07–1.89
(m, 1H), 1.46 (s, 9H) ppm; 13C NMR (101 MHz, CDCl3):
d = 166.1, 155.0, 148.8, 137.3, 136.0, 132.8, 131.6, 130.0,
129.7, 129.0, 128.7, 128.6, 128.1, 127.1, 125.5, 120.8,
119.2, 80.5, 71.8, 49.5, 48*, 42*, 38.7, 30*, 28.7 ppm;
HRMS (ESI): m/z [M ? Na]? calcd for C29H30N4NaO4
521.2165, found 521.2167.
1
(96 mg, 77 %). Rf = 0.22 (hexane/EtOAc, 1:1); H NMR
(400 MHz, CDCl3): d = 8.54 (s, 1H), 7.99 (d, J = 8.7 Hz,
1H), 7.93 (d, J = 8.2 Hz, 1H), 7.88–7.81 (m, 2H),
7.62–7.49 (m, 2H), 7.10 (s, 1H), 6.86 (d, J = 8.2 Hz,
2H), 6.69 (d, J = 8.2 Hz, 2H), 5.25–5.15 (m, 1H), 4.45 (t,
J = 7.2 Hz, 2H), 4.26–4.14 (m, 1H), 3.98–3.87 (m, 1H),
3.70 (s, 3H), 3.40–3.15 (m, 3H), 3.03 (t, J = 7.2 Hz, 2H),
2.27–2.17 (m, 1H), 1.91–1.78 (m, 1H), 1.44 (s, 9H) ppm;
13C NMR (101 MHz, CDCl3): d = 166.1, 158.9, 155.0,
147.8, 136.0, 132.8, 131.6, 129.9, 129.7, 129.2, 128.7,
128.5, 128.1, 127.4, 127.1, 125.6, 121.2, 114.4, 80.4, 71.9,
55.5, 52.2, 47*, 43*, 38.5, 36.2, 29*, 28.7 ppm; HRMS
(ESI): m/z [M ? Na]? calcd for C32H36N4NaO5 579.2583,
found 579.2580.
( )-tert-Butyl trans-3-benzoyloxy-4-(1-phenyl-1H-1,2,3-
triazol-4-yl)piperidine-1-carboxylate (7cb, C25H28N4O4)
The title compound was prepared from 0.105 g alcohol 3c
(0.30 mmol) and 56 mg benzoic acid (0.46 mmol) using
method D. The reaction mixture was stirred for 18 h, after
which TLC analysis indicated full conversion of the
starting material. Purification by flash column chromatog-
raphy (hexane/EtOAc, 2:1) gave the 7cb as a colorless
solid (0.130 g, 96 %). M.p.: 155.3–157.4 °C; Rf = 0.34
(hexane/EtOAc, 2:1); 1H NMR (400 MHz, CDCl3):
d = 8.01 (d, J = 7.6 Hz, 2H), 7.83 (s, 1H), 7.64 (d,
J = 7.8 Hz, 2H), 7.54 (t, J = 7.3 Hz, 1H), 7.48 (t,
J = 7.7 Hz, 2H), 7.44–7.36 (m, 3H), 5.34–5.23 (m, 1H),
4.19–4.15 (m, 1H), 4.07–3.96 (m, 1H), 3.49–3.37 (m, 1H),
3.35–3.12 (m, 2H), 2.36–2.23 (m, 1H), 2.03–1.88 (m, 1H),
1.45 (s, 9H) ppm; 13C NMR (101 MHz, CDCl3):
d = 165.9, 157.2, 148.8, 137.3, 133.5, 130.1, 130.03,
130.01, 129.0, 128.7, 120.8, 119.1, 80.5, 71.6, 46.7, 42.4,
38.5, 30.0, 28.7 ppm; HRMS (ESI): m/z [M ? Na]? calcd
for C25H28N4NaO4 471.2008, found 471.2004.
( )-tert-Butyl trans-3-benzoyloxy-4-[1-(4-methoxyphen-
ethyl)-1H-1,2,3-triazol-4-yl]piperidine-1-carboxylate
(7bb, C28H34N4O5)
The title compound was prepared from 98 mg alcohol 3b
(0.24 mmol) and 46 mg benzoic acid (0.38 mmol) using
method D. The reaction mixture was stirred for 21 h, after
which TLC analysis indicated full conversion of the
starting material. Purification by flash column chromatog-
raphy (hexane/EtOAc, 1:2) gave 7bb as a colorless oil
1
(95 mg, 75 %). Rf = 0.35 (hexane/EtOAc, 1:2); H NMR
(400 MHz, CDCl3): d = 8.12 (d, J = 7.3 Hz, 1H), 7.98 (d,
J = 7.4 Hz, 2H), 7.51–7.37 (m, 2H), 7.09 (s, 1H), 6.89 (d,
J = 8.5 Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H), 5.19–5.06 (m,
1H), 4.47 (t, J = 7.0 Hz, 2H), 4.26–4.00 (m, 1H),
3.95–3.84 (m, 1H), 3.75 (s, 3H), 3.38–3.11 (m, 3H), 3.05
(t, J = 6.9 Hz, 2H), 2.24–2.13 (m, 1H), 1.87–1.74 (m, 1H),
1.43 (s, 9H) ppm; 13C NMR (101 MHz, CDCl3):
d = 165.8, 158.9, 155.0, 147.7, 133.5, 130.0, 129.9,
129.2, 128.74, 128.71, 121.2, 114.5, 80.4, 77.6, 71.7,
55.6, 52.2, 46.6, 38.3, 36.2, 30.0, 28.7 ppm; HRMS (ESI):
m/z [M ? H]? calcd for C28H35N4O5 507.2608, found
507.2600.
General procedure for Boc deprotection: method E
The Boc-protected piperidine derivative was dissolved in a
mixture of CH2Cl2 and TFA (1:1, 2 cm3/mmol), and the
reaction mixture was stirred at room temperature for
30 min, after which all starting material was consumed.
The solvents were removed under reduced pressure, and
the residue was co-evaporated with toluene (2 9 25 cm3),
CH3CN (2 9 25 cm3), and CH2Cl2 (2 9 25 cm3) to afford
123

490
R. A. G. Harmsen et al.
3.59–3.43 (m, 1H), 3.38–3.23 (m, 1H), 3.18–3.09 (m, 1H),
3.09–2.92 (m, 4H), 2.41–2.25 (m, 1H), 2.11–1.96 (m, 1H)
ppm; 13C NMR (101 MHz, DMSO-d6): d = 158.8, 147.1,
136.4, 133.6, 133.4, 130.6, 130.3, 128.7, 128.6, 128.5,
127.1, 127.0, 126.9, 126.7, 123.5, 114.7, 74.7, 71.9, 55.8,
51.6, 45.7, 42.8, 36.9, 35.8, 26.9 ppm; HRMS (ESI): m/z
[M ? H]? calcd for C27H31N4O2 443.2447, found
443.2438.
the amine as its TFA salt. The crude product was purified
by semi-preparative HPLC using a C18 column. Fractions
of equal purity were pooled and concentrated under vac-
uum before the aqueous solution was lyophilized. All final
compounds were found to be of higher than 95 % purity
(HPLC).
( )-trans-3-(Naphthalene-2-ylmethoxy)-4-(1-phenethyl-
1H-1,2,3-triazol-4-yl)piperidine (8aa, C26H28N4O)
( )-trans-3-Benzyloxy-4-[1-(4-methoxyphenethyl)-1H-
1,2,3-triazol-4-yl]piperidine (8bb, C23H28N4O2)
The title compound was prepared from 50 mg Boc-
protected piperidine 5aa (0.1 mmol) using method E.
Purification by semi-preparative RP-HPLC afforded 8aa as
The title compound was prepared from 0.100 mg Boc-
protected piperidine 5bb (0.20 mmol) using method E.
Purification by semi-preparative RP-HPLC afforded 8bb as
a colorless solid (50 mg, 50 %). 1H NMR (400 MHz,
CDCl3): d = 9.75–9.56 (m, 1H), 9.39–9.20 (m, 1H),
7.33–7.24 (m, 3H), 7.21–7.13 (m, 2H), 7.07 (s, 1H), 6.99
(d, J = 8.6 Hz, 2H), 6.80 (d, J = 8.6 Hz, 2H), 4.57–4.43
(m, 3H), 4.36 (d, J = 11.6 Hz, 1H), 3.99–3.89 (m, 1H),
3.75 (s, 3H), 3.60–3.49 (m, 1H), 3.43–3.30 (m, 1H),
3.20–2.93 (m, 5H), 2.40–2.29 (m, 1H), 2.14–1.99 (m, 1H)
ppm; 13C NMR (101 MHz, MeOH-d4): d = 159.1, 146*,
137.5, 130.0, 129.1, 128.8, 128.4, 128.3, 122.2, 114.6,
73.8, 72.4, 55.6, 52.3, 45.8, 42.7, 36.7, 36.1, 26.0 ppm;
HRMS (ESI): m/z [M ? H]? calcd for C23H29N4O2
393.2291, found 393.2283.
1
a colorless powder (16 mg, 40 %). H NMR (400 MHz,
MeOH-d4): d = 7.87–7.79 (m, 3H), 7.71 (s, 1H), 7.52
(s, 1H), 7.51–7.45 (m, 2H), 7.36–7.30 (m, 1H), 7.21–7.09
(m, 3H), 7.05 (d, J = 6.9 Hz, 2H), 4.70 (d, J = 9.8 Hz,
1H), 4.65–4.47 (m, 3H), 3.94–3.85 (m, 1H), 3.52–3.42 (m,
1H), 3.35–3.28 (m, 1H), 3.26–3.18 (m, 1H), 3.17–3.04 (m,
4H), 2.33–2.21 (m, 1H), 2.09–1.96 (m, 1H) ppm; 13C NMR
(101 MHz, MeOH-d4): d = 148.4, 139.5, 137.2, 135.52,
135.45, 130.6, 130.5, 130.1, 129.8, 129.6, 128.9, 128.8,
128.2, 128.1, 127.8, 125.3, 75.5, 74.0, 53.4, 47.3, 44.2,
38.2, 37.7, 27.2 ppm; HRMS (ESI): m/z [M ? H]? calcd
for C26H29N4O 413.2341, found 413.2339.
( )-trans-3-Benzyloxy-4-(1-phenethyl-1H-1,2,3-triazol-4-yl)-
piperidine (8ab, C22H26N4O)
( )-trans-3-(Naphthalen-2-ylmethoxy)-4-(1-phenyl-1H-
1,2,3-triazol-4-yl)piperidine (8ca, C24H24N4O)
The title compound was prepared from 52 mg Boc-
protected piperidine 5ab (0.11 mmol) using method E. Puri-
fication by semi-preparative RP-HPLC afforded 8ab as a
The title compound was prepared from 47 mg Boc-
protected piperidine 5ca (0.097 mmol) using method E.
Purification by semi-preparative RP-HPLC afforded 8ca as
a colorless solid (28 mg, 57 %). 1H NMR (400 MHz,
DMSO-d6): d = 9.02–8.93 (m, 1H), 8.85–8.73 (m, 1H),
8.68 (s, 1H), 7.89–7.82 (m, 3H), 7.82–7.74 (m, 2H), 7.70 (s,
1H), 7.60 (t, J = 7.8 Hz, 2H), 7.53–7.43 (m, 3H),
7.33–7.26 (m, 1H), 4.78 (d, J = 12.2 Hz, 1H), 4.63 (d,
J = 12.2 Hz, 1H), 4.04–3.93 (m, 1H), 3.65–3.55 (m, 1H),
3.35–3.23 (m, 2H), 3.16–3.01 (m, 2H), 2.26–2.16 (m, 1H),
2.14–2.01 (m, 1H) ppm; 13C NMR (101 MHz, DMSO-d6):
d = 148.5, 137.6, 136.4, 133.6, 133.4, 130.8, 129.5, 128.7,
128.54, 128.47, 127.12, 127.06, 126.9, 126.7, 122.0, 120.8,
74.6, 71.8, 45.7, 42.8, 37.1, 26.7 ppm; HRMS (ESI): m/z
[M ? H]? calcd for C24H25N4O 385.2028, found 385.2021.
1
colorless oil (34 mg, 65 %). H NMR (400 MHz, MeOH-
d4): d = 8.03–8.00 (m, 1H), 7.58 (s, 1H), 7.34–7.27 (m,
3H), 7.25–7.14 (m, 5H), 7.09 (d, J = 6.6 Hz, 2H),
4.67–4.56 (m, 2H), 4.53 (d, J = 11.8 Hz, 1H), 4.45 (d,
J = 11.8 Hz, 1H), 3.90–3.83 (m, 1H), 3.49–3.38 (m, 1H),
3.34–3.25 (m, 1H), 3.24–3.15 (m, 3H), 3.15–3.02 (m, 2H),
2.33–2.21 (m, 1H), 2.08–1.96 (m, 1H) ppm; 13C NMR
(101 MHz, MeOH-d4): d = 148.4, 139.8, 139.6, 130.7,
130.5, 130.4, 130.3, 130.0, 129.9, 128.8, 125.3, 75.5, 73.9,
53.5, 47.2, 44.1, 38.3, 37.5, 27.0 ppm; HRMS (ESI): m/z
[M ? H]? calcd for C22H27N4O 363.2185, found 363.2184.
( )-trans-4-[1-(4-Methoxyphenethyl)-1H-1,2,3-triazol-4-yl]-
3-(naphthalen-2-ylmethoxy)piperidine
(8ba, C27H30N4O2)
( )-trans-3-(Naphthalen-2-ylmethoxy)-4-(1-phenethyl-1H-
1,2,3-triazol-5-yl)piperidine (9aa, C26H28N4O)
The title compound was prepared from 0.100 g Boc-
protected piperidine 5ba (0.18 mmol) using method E.
Purification by semi-preparative RP-HPLC afforded 8ba as
a colorless solid (62 mg, 72 %). 1H NMR (400 MHz,
CDCl3): d = 10.04–9.81 (m, 1H), 9.46–9.25 (m, 1H),
7.84–7.70 (m, 3H), 7.64 (s, 1H), 7.51–7.42 (m, 2H),
7.33–7.22 (m, 1H), 7.00–6.87 (m, 3H), 6.77 (d, J = 8.5 Hz,
2H), 4.66 (d, J = 11.7 Hz, 1H), 4.51 (d, J = 11.7 Hz, 1H),
4.47–4.27 (m, 2H), 4.02–3.93 (m, 1H), 3.72 (s, 3H),
The title compound was prepared from 75 mg Boc-
protected piperidine 6aa (0.15 mmol) using method E.
Purification by semi-preparative RP-HPLC afforded 9aa as
a colorless solid (38 mg, 45 %). 1H NMR (400 MHz,
MeOH-d4): d = 7.88–7.82 (m, 1H), 7.82–7.73 (m, 2H),
7.55–7.44 (m, 4H), 7.28–7.17 (m, 3H), 7.06–6.96 (m, 3H),
4.74–4.55 (m, 3H), 4.43 (d, J = 11.9 Hz, 1H), 3.80–3.68
123

Synthesis and docking of novel piperidine renin inhibitors
491
(m, 1H), 3.58–3.47 (m, 1H), 3.37–3.31 (m, 1H), 3.26–3.09
(m, 2H), 2.98–2.89 (m, 1H), 2.89–2.78 (m, 1H), 2.78–2.67
(m, 1H), 1.85–1.71 (m, 1H), 1.57–1.46 (m, 1H) ppm; 13C
NMR (101 MHz, MeOH-d4): d = 140.8, 140.0, 136.4,
135.5, 135.4, 132.1, 131.0, 130.6, 130.3, 129.8, 129.6,
129.0, 128.8, 128.3, 128.2, 127.5, 77.5, 74.1, 51.6, 48.0,
45.2, 38.4, 38.1, 28.3 ppm; HRMS (ESI): m/z [M ? H]?
calcd for C26H29N4O 413.2341, found 413.2334.
HPLC and lyophilization gave the TFA salt of 9ea as a
colorless solid (0.100 g, 78 %). 1H NMR (400 MHz,
MeOH-d4): d = 7.84–7.78 (m, 1H), 7.78–7.68 (m, 2H),
7.50–7.44 (m, 2H), 7.42 (s, 2H), 7.33 (s, 1H), 7.26 (d,
J = 8.0 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 6.97 (d,
J = 8.2 Hz, 1H), 6.65 (d, J = 7.4 Hz, 1H), 4.75–4.51
(m, 3H), 4.38 (d, J = 12.0 Hz, 1H), 3.76–3.63 (m, 1H),
3.55–3.41 (m, 1H), 3.35–3.24 (m, 1H), 3.22–3.12 (m, 1H),
3.12–3.01 (m, 1H), 2.90 (t, J = 11.4 Hz, 1H), 2.85–2.73
(m, 1H), 2.73–2.62 (m, 1H), 2.09 (s, 3H), 1.84–1.69 (m,
1H), 1.50–1.40 (m, 1H) ppm; 13C NMR (101 MHz,
MeOH-d4): d = 171.8, 140.1, 140.0, 135.6, 134.6, 134.5,
130.2, 129.4, 128.9, 128.7, 128.0, 127.4, 127.3, 126.6,
126.1, 122.4, 120.2, 76.7, 73.2, 50.8, 47.2, 44.2, 37.7, 37.2,
27.4, 23.8 ppm; HRMS (ESI): m/z [M ? H]? calcd for
C28H32N5O2 470.2556, found 470.2553.
( )-trans-4-[1-(4-Methoxyphenethyl)-1H-1,2,3-triazol-5-
yl]-3-(naphthalen-2-ylmethoxy)piperidine
(9ba, C27H30N4O2)
The title compound was prepared from 50 mg Boc-protected
piperidine 6ba (0.092 mmol) using method E. Purification
by semi-preparative RP-HPLC afforded 9ba as a colorless
solid (30 mg, 74 %). 1H NMR (400 MHz, MeOH-d4):
d = 7.84–7.79 (m, 1H), 7.78–7.69 (m, 2H), 7.50–7.41 (m,
4H), 6.99 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 8.4 Hz, 2H),
6.74 (d, J = 8.5 Hz, 2H), 4.67 (d, J = 11.9 Hz, 1H),
4.63–4.47 (m, 2H), 4.39 (d, J = 11.9 Hz, 1H), 3.77–3.66
(m, 4H), 3.57–3.46 (m, 1H), 3.38–3.28 (m, 1H), 3.15–2.97
(m, 2H), 2.96–2.77 (m, 2H), 2.75–2.64 (m, 1H), 1.86–1.72
(m, 1H), 1.62–1.49 (m, 1H) ppm; 13C NMR (101 MHz,
MeOH-d4): d = 160.1, 140.0, 135.6, 134.6, 134.5, 131.1,
130.9, 129.4, 128.9, 128.7, 128.1, 127.4, 127.3, 126.6, 115.1,
76.6, 73.2, 55.7, 51.1, 47.2, 44.3, 37.2, 36.7, 27.6 ppm;
HRMS (ESI): m/z [M ? H]? calcd for C27H31N4O2
443.2447, found 443.2435.
( )-N-[4-[2-[5-[trans-3-(Naphthalen-2-ylmethoxy]piperi-
din-4-yl]-1H-1,2,3-triazol-1-yl]ethyl]phenyl]-N-(naphtha-
len-2-ylmethyl)acetamide (9fa, C39H39N5O2)
The title compound was prepared from 50 mg 6fa
(0.07 mmol) using method E. Purification by semi-
preparative HPLC and lyophilization gave the TFA salt
of the title compound as a colorless solid (24 mg, 47 %).
1H NMR (400 MHz, MeOH-d4): d = 7.84–7.66 (m, 5H),
7.65 (d, J = 8.6 Hz, 1H), 7.51–7.37 (m, 7H), 7.35–7.23
(m, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 7.6 Hz,
2H), 6.83 (d, J = 7.9 Hz, 2H), 4.95 (s, 3H), 4.67 (d,
J = 11.8 Hz, 1H), 4.51 (t, J = 7.4 Hz, 2H), 4.37 (d,
J = 11.7 Hz, 1H), 3.86–3.71 (m, 1H), 3.65–3.54 (m, 1H),
3.28–3.20 (m, 1H), 3.16–3.05 (m, 3H), 3.03–2.89 (m, 1H),
1.85–1.73 (m, 5H) ppm; 13C NMR (101 MHz, MeOH-d4):
d = 173.0, 142.3, 139.7, 139.1, 136.0, 135.6, 134.7, 134.6,
134.5, 134.2, 131.1, 129.4, 129.3, 129.2, 129.0, 128.8,
128.7, 128.3, 128.2, 127.40, 127.37, 127.3, 127.0, 126.8,
76.8, 73.3, 53.8, 50.1, 47.1, 44.2, 37.1, 36.2, 27.9,
22.6 ppm; HRMS (ESI): m/z [M ? H]? calcd for
C39H40N5O2 610.3182, found 610.3177.
( )-N-[4-[2-[5-[trans-3-(Naphthalen-2-ylmethoxy)piperi-
din-4-yl]-1H-1,2,3-triazol-1-yl]ethyl]phenyl]acetamide
(9da, C28H31N5O2)
The title compound was prepared from 30 mg 6da
(0.053 mmol) using method E. Purification by semi-
preparative HPLC and lyophilization gave the TFA salt
1
of 9da as a colorless solid. H NMR (400 MHz, MeOH-
d4): d = 7.85–7.78 (m, 1H), 7.77–7.69 (m, 2H), 7.68–7.59
(m, 2H), 7.49–7.42 (m, 3H), 7.34 (d, J = 7.8 Hz, 2H),
7.06–6.97 (m, 1H), 6.91 (d, J = 7.8 Hz, 2H), 4.70–4.52
(m, 3H), 4.49–4.28 (m, 1H), 3.73–3.61 (m, 1H), 3.61–3.48
(m, 1H), 3.21–3.00 (m, 3H), 3.00–2.76 (m, 2H), 2.72–2.51
(m, 1H), 2.06 (s, 3H), 1.82–1.67 (m, 1H), 1.68–1.52 (m,
1H) ppm; 13C NMR (101 MHz, MeOH-d4): d = 171.6,
138.6, 135.6, 134.6, 133.1, 133.0, 130.4, 130.1, 129.4,
128.9, 128.7, 128.0, 127.4, 127.3, 127.0, 126.6, 122.1,
76.8, 73.5, 50.8, 47.3, 44.5, 37.4, 37.0, 27.5, 23.7 ppm;
HRMS (ESI): m/z [M ? H]? calcd for C28H32N5O2
470.2556, found 470.2537.
( )-N-[3-[2-[5-[trans-3-(Naphthalen-2-ylmethoxy]piperi-
din-4-yl]-1H-1,2,3-triazol-1-yl]ethyl]phenyl]-N-(naphtha-
len-2-ylmethyl)acetamide (9ga, C39H39N5O2)
The title compound was prepared from 17 mg 6ga
(0.024 mmol) using method E. Purification by semi-
preparative HPLC and lyophilization gave the TFA salt
of the title compound as a colorless solid. 1H NMR
(400 MHz, MeOH-d4): d = 7.82–7.77 (m, 2H), 7.77–7.68
(m, 4H), 7.52–7.38 (m, 7H), 7.31 (d, J = 8.4 Hz, 1H), 7.17
(t, J = 7.8 Hz, 1H), 7.06 (d, J = 7.8 Hz, 1H), 7.01–6.92
(m, 1H), 6.90 (d, J = 7.7 Hz, 1H), 6.63 (s, 1H), 5.00–4.84
(m, 4H), 4.62 (d, J = 11.8 Hz, 1H), 4.55–4.42 (m, 2H),
4.35 (d, J = 11.8 Hz, 1H), 3.80–3.68 (m, 1H), 3.60–3.51
(m, 1H), 3.17–3.00 (m, 3H), 3.00–2.87 (m, 2H), 1.86–1.70
(m, 5H) ppm; 13C NMR (126 MHz, MeOH-d4): d = 172.9,
( )-N-[3-[2-[5-[trans-3-(Naphthalen-2-ylmethoxy]piperi-
din-4-yl]-1H-1,2,3-triazol-1-yl]ethyl]phenyl]acetamide
(9ea, C28H31N5O2)
The title compound was prepared from 0.127 g 6ea
(0.22 mmol) using method E. Purification by semi-preparative
123

492
R. A. G. Harmsen et al.
143.8, 140.8, 139.6, 136.0, 135.5, 134.7, 134.6, 134.5,
134.2, 131.5, 130.8, 129.8, 129.7, 129.5, 129.3, 128.9,
128.8, 128.7, 128.7, 128.4, 128.1, 127.8, 127.5, 127.5,
127.4, 127.3, 127.0, 126.7, 76.8, 73.3, 53.8, 50.2, 47.1,
44.2, 37.1, 36.5, 27.8, 22.8 ppm; HRMS (ESI): m/z
[M ? H]? calcd for C39H40N5O2 610.3182, found
610.3207.
2H), 6.68 (t, J = 7.4 Hz, 1H), 5.05–4.66 (m, 1H),
2.87–2.79 (m, 2H), 2.65–2.40 (m, 3H), 1.68–1.58 (m,
1H), 1.58–1.50 (m, 1H) ppm; 13C NMR (101 MHz,
DMSO-d6): d = 164.8, 147.5, 137.5, 136.1, 132.9, 132.1,
130.8, 130.3, 129.8, 129.6, 129.3, 128.7, 128.0, 127.3,
125.9, 122.1, 120.8, 69.8, 44.9, 42.2, 35.7, 25.8 ppm;
HRMS (ESI): m/z [M ? H]? calcd for C24H23N4O2
399.1821, found 399.1820.
( )-trans-4-[1-(4-Methoxyphenethyl)-1H-1,2,3-triazol-4-
yl]piperidin-3-yl 2-naphthoate (10ba, C27H28N4O3)
The title compound was prepared from 95 mg Boc-
protected piperidine 7ba (0.18 mmol) using method E.
Purification by semi-preparative RP-HPLC afforded 10ba
( )-trans-4-(1-Phenyl-1H-1,2,3-triazol-4-yl)piperidin-3-yl
benzoate (10cb, C20H20N4O2)
The title compound was prepared from 50 mg Boc-
protected piperidine 7cb (0.11 mmol) using method E.
Purification by semi-preparative RP-HPLC afforded 10cb
1
as a colorless solid (50 mg, 40 %). H NMR (400 MHz,
1
DMSO-d6): d = 9.18–8.95 (m, 2H), 8.67 (s, 1H), 8.14
(d, J = 8.0 Hz, 1H), 8.11–7.98 (m, 4H), 7.74–7.61 (m,
2H), 6.87 (d, J = 8.4 Hz, 2H), 6.60 (d, J = 8.4 Hz, 2H),
5.47–5.38 (m, 1H), 4.48 (t, J = 7.0 Hz, 2H), 3.63–3.54 (m,
4H), 3.54–3.43 (m, 1H), 3.37–3.24 (m, 2H), 3.24–3.12 (m,
1H), 2.96 (t, J = 6.9 Hz, 2H), 2.31–2.19 (m, 1H),
2.19–2.04 (m, 1H) ppm; 13C NMR (101 MHz, DMSO-
d6): d = 164.8, 157.8, 145.4, 135.2, 132.0. 131.1, 129.5,
129.4, 129.3, 128.9, 128.4, 127.8, 127.2, 126.4, 124.9,
122.5, 113.6, 69.1, 54.8, 50.9, 44.2, 41.7, 34.9, 25.7 ppm;
HRMS (ESI): m/z [M ? H]? calcd for C27H29N4O3
457.2240, found 457.2235.
as a colorless solid (28 mg, 55 %). H NMR (400 MHz,
MeOH-d4): d = 8.50 (s, 1H), 8.05 (d, J = 7.2 Hz, 2H),
7.76 (d, J = 7.9 Hz, 2H), 7.63 (t, J = 7.4 Hz, 1H), 7.55 (t,
J = 7.6 Hz, 2H), 7.52–7.43 (m, 3H), 5.61–5.51 (m, 1H),
3.84–3.75 (m, 1H), 3.67–3.52 (m, 2H), 3.43–3.24 (m, 2H),
2.54–2.41 (m, 1H), 2.41–2.27 (m, 1H) ppm; 13C NMR
(101 MHz, MeOH-d4): d = 167.4, 148.7, 139.2, 135.8,
131.8, 131.7, 131.2, 131.0, 130.6, 123.3, 122.4, 71.0, 46.9,
44.3, 37.4, 27.6 ppm; HRMS (ESI): m/z [M ? H]? calcd
for C20H21N4O2 349.1665 found: 349.1661.
In vitro fluorimetric assays
( )-trans-4-[1-(4-Methoxyphenethyl)-1H-1,2,3-triazol-4-
yl]piperidin-3-yl benzoate (10bb, C23H26N4O3)
The SensoLyteÒ520 Renin Assay Kit Fluorimetric (Ana-
Spec, CA, USA, supplied by Biosite, Sweden) was used to
determine the inhibitory activity of the final compounds at
5 lM and 75 lM following the endpoint protocol for
96-well microtiter plate experiments as described by the
manufacturer. Two different endpoint concentrations were
used in order to differentiate the activity of the compounds.
Briefly, test compounds were dissolved in DMSO and
diluted with the assay buffer provided with the kit. The final
DMSO level in the assay did not exceed 1 %. Test com-
pounds and renin were added to the wells of a black 96-well
microtiter plate (Non-Binding Surface, Corning, NY, USA)
and incubated while protected from light for 30 min at
37 °C. Pre-incubated substrate, 30 min at 37 °C, was then
added to each well before being gently shaken and further
incubated for 15 min at 37 °C. The fluorescence signal was
then immediately read at Ex/Em wavelengths 490/520 nm
with a SpectraMax M2e (Molecular Devices Corp., CA,
USA) and the results processed using SOFTMAXÒPRO
software, version 5.2 (Molecular Devices Corp., CA, USA).
Controls included in every experiment were the signal from
the test compound alone, inhibition control by the provided
kit inhibitor, a 100 % activity control, and a vehicle control
containing DMSO at the same concentration as in the test
compound solution. The mean of the substrate back-
ground was subtracted from the RFU of all parallels. The
The title compound was prepared from 95 mg Boc-
protected piperidine 7bb (0.19 mmol) using method E.
Purification by semi-preparative RP-HPLC afforded 10bb
1
as a colorless solid (55 mg, 53 %). H NMR (400 MHz,
DMSO-d6): d = 9.13–8.91 (m, 2H), 8.04–7.95 (m, 3H),
7.69 (t, J = 7.4 Hz, 1H), 7.55 (t, J = 7.7 Hz, 2H), 6.91 (d,
J = 8.5 Hz, 2H), 6.67 (d, J = 8.5 Hz, 2H), 5.39–5.30 (m,
1H), 4.49 (t, J = 7.0 Hz, 2H), 3.66 (s, 3H), 3.58–3.50 (m,
1H), 3.45–3.37 (m, 1H), 3.34–3.09 (m, 3H), 2.98 (t,
J = 7.0 Hz, 2H), 2.26–2.15 (m, 1H), 2.14–2.00 (m, 1H)
ppm; 13C NMR (101 MHz, DMSO-d6): d = 165.6, 158.8,
146.3, 134.7, 130.5, 130.4, 130.3, 130.0, 129.7, 123.4,
114.6, 69.9, 55.9, 51.8, 45.1, 42.6, 35.8, 35.8, 26.6 ppm;
HRMS (ESI): m/z [M ? H]? calcd for C23H27N4O3
407.2083, found 407.2077.
( )-trans-4-(1-Phenyl-1H-1,2,3-triazol-4-yl)piperidin-3-yl
2-naphthoate (10ca, C24H22N4O2)
The title compound was prepared from 75 mg Boc-
protected piperidine 7ca (0.15 mmol) using method E.
Purification by semi-preparative RP-HPLC afforded 10ca
1
as a colorless solid (35 mg, 46 %). H NMR (400 MHz,
DMSO-d6): d = 8.65 (s, 1H), 8.05 (s, 1H), 7.97 (s, 1H),
7.35 (d, J = 8.0 Hz, 1H), 7.28–7.22 (m, 3H), 7.06 (d,
J = 8.0 Hz, 2H), 6.96–6.82 (m, 2H), 6.78 (t, J = 7.8 Hz,
123

Synthesis and docking of novel piperidine renin inhibitors
493
SensoLyteÒ520 assay was also used for determining the
IC50 value for compound 9ga. The assay protocol was fol-
lowed as stated in the manual for kinetic reading with the
exception that data were collected every 30 s instead of
every 60 s as recommended in the instructions. The same
instrument and wavelength settings as described for the
endpoint assay were applied. The concentration range
applied for the IC50 determination was 10 nM to 15 lM,
with 12 different concentrations in total, each tested in 3
parallels. The DMSO concentration did not exceed 1 %.
The inhibitor supplied with the assay kit was used as the
100 % inhibition of renin at a concentration set to 100 lM,
and the 100 % activity assay control was used as the 0 %
inhibition value in the analysis and set to a concentration of
1 nM. The data were normalized and analyzed with non-
linear regression, one site Fit IC50 using GraphPad Prism
v5.00 (GraphPad Software Inc., CA, USA).
Preparation Wizard in Maestro [20]. Crystallographic water
molecules were removed, and all hydrogen atoms added.
Hydrogen-bonding patterns were analyzed and optimized,
and Asp219 was protonated on the innermost oxygen, as
suggested by Friedman and Caflisch [21]. Finally, the
molecular structures were energy minimized prior to
docking experiments. The ligand library was built in Lig-
Prep [17], and the generated docking grids were prepared in
Glide [17] by centering on the catalytic aspartate dyad.
Molecular docking was carried out using the Glide
program [11]. Based on information from experimental
crystal structures of renin complexed with piperidine-based
inhibitors, three constraints were applied to reduce the
number of false poses. The charged nitrogen atom in the
piperidine ring was required to be placed within a sphere of
˚
1-A radius of the corresponding nitrogen position in the
2FS4 structure, and the two hydrogens of the piperidine
nitrogen were required to be within hydrogen-bonding
distance to the two catalytic aspartates. The grid generation
and docking were repeated to obtain a more customized
constraint system for our system.
The BACE1 (b-secretase) FRET Assay Kit, Red (Pan-
Vera), was used to determine the inhibition effect of the
final compounds at 300 lM against b-secretase. The man-
ufacturer’s protocol for endpoint assay was followed, with
the exception of an increase to four times the recommended
volumes of all solutions to achieve appropriate signals. The
test compounds were dissolved in DMSO and diluted with
50 mM sodium acetate pH 4.5 buffer. The final DMSO
content in the assay did not exceed 10 %. The substrate and
test compounds were added to a black 96-well microtiter
plate (Non-Binding Surface, Corning, NY, USA) and
mixed. BACE1 enzyme was added to start the reactions,
and the plate was gently shaken. The microtiter plate was
incubated at room temperature for 60 min protected from
light before 2.5 M sodium acetate was added to immedi-
ately stop the enzymatic reaction. The signals were read on
a SpectraMax M2e (Molecular Devices Corp., CA, USA) at
Ex/Em wavelength 545/585 nm. All resulting data were
processed using the SOFTMAXÒPRO software, version 5.2
(Molecular Devices Corp., CA, USA). The mean substrate
background was subtracted from all signals, along with the
compounds’ own signals. Assay controls included a 100 %
activity control with the same amount of DMSO as the
test compounds, an inhibitor control with the commer-
cial inhibitor b-secretase inhibitor IV (CalbiochemÒ, no.
565788, Merck), and a test of the signal from the com-
pounds alone. All endpoint data were normalized using
GraphPad Prism, v5.00 (GraphPad Software Inc., CA,
USA).
Acknowledgments The authors gratefully acknowledge financial
support from the Research Council of Norway through the KOSK
programme (PhD fellowship to R.A.G.H). The Norwegian Structural
Biology Centre (NorStruct) is supported by the Functional Genomics
Program (FUGE) of the Research Council of Norway.
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