The discovery of novel potent trans-3,4-disubstituted pyrrolidine inhibitors of the human aspartic protease renin from in silico three-dimensional (3D) pharmacophore searches

Article abstract of DOI:10.1021/jm3017078

The small-molecule trans-3,4-disubstituted pyrrolidine 6 was identified from in silico three-dimensional (3D) pharmacophore searches based on known X-ray structures of renin-inhibitor complexes and demonstrated to be a weakly active inhibitor of the human enzyme. The unexpected binding mode of the more potent enantiomer (3S,4S)-6a in an extended conformation spanning the nonprime and S1′ pockets of the recombinant human (rh)-renin active site was elucidated by X-ray crystallography. Initial structure-activity relationship work focused on modifications of the hydrophobic diphenylamine portion positioned in S1 and extending toward the S2 pocket. Replacement with an optimized P3-P1 pharmacophore interacting to the nonsubstrate S3^sp cavity eventually resulted in significantly improved in vitro potency and selectivity. The prototype analogue (3S,4S)-12a of this new class of direct renin inhibitors exerted blood pressure lowering effects in a hypertensive double-transgenic rat model after oral administration.

Full text of DOI:10.1021/jm3017078

Article
pubs.acs.org/jmc
The Discovery of Novel Potent trans-3,4-Disubstituted Pyrrolidine
Inhibitors of the Human Aspartic Protease Renin from in Silico Three-
Dimensional (3D) Pharmacophore Searches
Edwige Lorthiois,*,† Werner Breitenstein,† Frederic Cumin,† Claus Ehrhardt,† Eric Francotte,†
Edgar Jacoby,† Nils Ostermann,† Holger Sellner,† Takatoshi Kosaka,‡,⊥ Randy L. Webb,§ Dean F. Rigel,§
Ulrich Hassiepen,† Paul Richert,† Trixie Wagner,† and Jurgen Maibaum*,†
̈
†Novartis Pharma AG, Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland
‡Novartis Institutes for BioMedical Research, Ohkubo 8, Tsukuba, Ibaraki 300-2611, Japan
§Novartis Pharmaceuticals Corp., Institutes for BioMedical Research, East Hanover, New Jersey, United States
jm301706j
S
* Supporting Information
ABSTRACT: The small-molecule trans-3,4-disubstituted pyr-
rolidine 6 was identified from in silico three-dimensional (3D)
pharmacophore searches based on known X-ray structures of
renin−inhibitor complexes and demonstrated to be a weakly
active inhibitor of the human enzyme. The unexpected binding
mode of the more potent enantiomer (3S,4S)-6a in an extended
conformation spanning the nonprime and S1′ pockets of the
recombinant human (rh)-renin active site was elucidated by X-
ray crystallography. Initial structure−activity relationship work
focused on modifications of the hydrophobic diphenylamine
portion positioned in S1 and extending toward the S2 pocket.
Replacement with an optimized P3−P1 pharmacophore
interacting to the nonsubstrate S3sp cavity eventually resulted in significantly improved in vitro potency and selectivity. The
prototype analogue (3S,4S)-12a of this new class of direct renin inhibitors exerted blood pressure lowering effects in a
hypertensive double-transgenic rat model after oral administration.
renin inhibitors (DRIs) may provide unique therapeutic
benefits5,8−11 as a result of more complete blockade of the
RAAS at source by preventing the formation of AngI and AngII
at compensatory elevated circulating renin levels and hence the
formation of AngI derived products12 without affecting kinin
metabolism.13 Moreover, clinical studies have demonstrated the
potential of direct renin inhibition for end organ protection
beyond blood pressure lowering effects.14
Our current perspective on designing orally active DRIs has
experienced a remarkable transformation during more than
three decades starting from peptide-like transition-state
inhibitors and subsequently progressing toward various distinct
classes of nonpeptide inhibitors (1−4, Figure 1).15,16 Inhibitor
1 (aliskiren, Tekturna, Rasilez) has received recently marketing
approval as the first DRI for the treatment of hypertension. The
compound has emerged from a S3−S1 topological design
concept, which notably exploited in addition tight binding
interactions to the nonsubstrate S3 subpocket (S3sp) of human
renin.17,18 The early class of 3,4-piperidine-based “group
INTRODUCTION
■
Chronic elevation of systemic blood pressure, known as
hypertension, is an important risk factor for cardiovascular
disease such as congestive heart failure, stroke, and myocardial
infarction and is the leading cause of death in the western
world.1 The renin−angiotensin−aldosterone system (RAAS)2
plays a fundamental role in normal blood pressure control and
body fluid volume homeostasis, while excessive stimulation of
the RAAS contributes directly to cardiorenal diseases.3 The
highly substrate-specific aspartic protease renin found in the
circulation following synthesis and release from juxtaglomerular
cells of the kidney4 catalyzes the first and rate limiting cleavage
step of the RAAS, generating the biologically inactive
decapeptide angiotensin I (AngI) from the α2-globulin
angiotensinogen substrate.5 AngI is further processed by the
angiotensin-converting enzyme (ACE) to generate the
octapeptide AngII. The interaction of AngII with the AT1
receptor triggers various hemodynamic effects, such as sodium
and water retention, stimulation of aldosterone release, and
potent vasoconstriction.6 Furthermore, AngII exerts direct
growth factor-like effects via several pathways mainly down-
stream of AT1 inducing cell proliferation or fibrosis.7 Direct
Received: November 20, 2012
Published: February 20, 2013
© 2013 American Chemical Society
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Figure 1. Diverse classes of nonpeptide direct inhibitors.
replacement assisted binding (GRAB) peptidomimetics”,19 first
reported by Hoffmann-La Roche,20,21 has further advanced to
3,9-diazabicyclo[3.3.1]nones, such as 2 (ACT-077825) (Figure
1), which has undergone phase II clinical trials.22 The basic
amine mono- or bicyclic center scaffolds of such GRAB
peptidomimetic inhibitors form favorable charge-enforced H-
bond interactions to the catalytic Asp32 and Asp215. More
intriguingly, these DRIs bind to a fundamentally different
enzyme active site topography characterized by an open
conformation of the flap β-hairpin and formation of a large
hydrophobic pocket (Tyr75 flap pocket) that is unrecognized by
the renin substrate. A series of potent 2,4-diaminopyrimidines,
such as 3, bearing a P3−P1 benzoxazinone pharmacophore and
interacting with the basic heterocyclic headgroup to the
catalytic dyad, have been reviewed elsewhere.16,23 Most
recently, the alkylamine class of DRIs has attracted much
interest with compound 4 (VTP-27999) (Figure 1) having
been reported to be under clinical investigation.24
followed by Unity 3D flexible pharmacophore searching within
the Novartis compound database.26a The selected X-ray
complexes included that of the low-affinity trans-3,4-piperidine
527 (34% inhibition of rh-renin at 10 μM concentration in the
FRET enzymatic assay;26a Figures 2 and 3), which was resolved
Figure 2. trans-3,4-Piperidine (3R,4R)-5 used for 3D pharmacophore
search and pharmacophore hit 6.
in our group at 2.4 Å resolution.26a In brief, the key elements of
the three-point pharmacophore derived from (3R,4R)-5 were
We have embarked on a broad integrated screening program
encompassing multiple approaches in parallel in the quest for
identifying attractive novel-class druglike scaffolds, potentially
leading to orally efficacious DRIs with improved oral
bioavailability and reduced cost of goods. These efforts
included two large-scale HTS campaigns using both an
enzymatic and a competitive ligand binding assay format, a
combined NMR/X-ray fragment-based screen of an aspartic
protease-family directed library, as well as virtual searches
utilizing the renin−inhibitor X-ray structure knowledge. The
various screening approaches that were utilized proved to be
remarkably complementary in identifying attractive hits of high
structural diversity from different sources.25 We report herein
the discovery of the novel 3,4-disubstituted pyrrolidine 6 from a
computational pharmacophore search and our initial SAR work
guided by X-ray crystallography, leading to the orally active
(3S,4S)-12a with significantly improved potency and selectivity.
Figure 3. X-ray crystal structure of the trans-3,4-pyrrolidine 6a (shown
as stick model in green color) bound to the active site of glycosylated
rh-renin (refined at 2.6 Å resolution) and superimposed with inhibitor
1 (left-hand side picture; orange color; PDB code 2V0Z)17b,28 and
inhibitor 5 (right-hand side picture; magenta color) in complex with
rh-renin. Shown is the solvent accessible surface of renin. Residues of
the flap β-hairpin (residues Tyr75, Ser76, Thr77, and Gly78) are depicted
as thin lines in green, orange, and magenta for the rh-renin/6a, rh-
renin/1, and rh-renin/5 complexes, respectively, and have been
omitted from the protein surface calculation. The oxygen and nitrogen
atoms of the ligands are colored in red and blue, respectively. The
absolute (3S,4S)-configuration of 6a was tentatively assigned based on
the observed difference electron density and was subsequently
confirmed by synthesis and chemical correlation with an authentic
sample (see text for discussion).
RESULTS AND DISCUSSION
■
A virtual screening approach was developed based on three-
dimensional (3D) pharmacophore models constructed from
the X-ray crystal structures of renin−inhibitor complexes
representing a diversity of active site binding topographies,
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defined by the basic piperidine nitrogen atom interacting to the
catalytic Asp32 and Asp215, the geometric center of the S3−S1
binding naphthyl, and of the C4 phenyl residue positioned in
the Tyr75 flap pocket. Both hydrophobic centers were defined
with a positional constraint of 1.5 Å.
phenyl of piperidine 5, as was anticipated from the 3D
pharmacophore search and suggested by additional modeling
work. The basic pyrrolidine nitrogen atom of 6a occupied the
pivotal position almost symmetrical between the catalytic Asp32
and Asp215 of renin, as was predicted by modeling, most likely
forming electrostatically enforced H-bond interactions to both
carboxylates. The very close superposition of the five-
membered ring transition-state surrogate of 6a with the center
piperidine scaffold of renin-bound 5 was quite noteworthy.
The difference electron density map observed for 6a was
only compatible with absolute (3S,4S)-configuration. The
ligand was bound in an energetically less favored trans-
pseudoaxial conformation which allowed the hydrophobic 3(S)-
benzyl residue to be positioned in the S1′ pocket in an unusual
orientation almost perpendicular to the axis of this binding site
(Figure 3). Interestingly, a significant conformational move-
ment of Pro292 toward the phenyl C4 of 6a was noted. On the
other hand, the lipophilic N,N-diphenylamino moiety was
bound to the nonprime site with the unsubstituted N-phenyl
interacting with S1 pocket. The N-4-chlorophenyl was located
at the entrance of the S2 pocket of rh-renin and in close
distance to Ser219, with the chlorine atom pointing toward the
aryl side chain of Tyr220 within 3.4 Å binding distance. The 4-
chlorophenyl portion nicely overlapped with the P3/P2
carboxamide group of peptide-like peptidomimetic DRIs28 in
a perpendicular relative conformation. Notably, the closed flap
β-hairpin adopted a significantly different conformation as was
observed for the S3−S1 topological inhibitor 1 (Figure 2). This
was attributed to the fact that a second molecule of 6a was
observed in the crystal structure, binding to the S3 and S4
nonprime sites with the chlorophenyl residue being in close
contact with the flap region and forming a direct H-bond
interaction to the side chain of Tyr220 via its pyrrolidine
nitrogen. Two entities of renin were present in the asymmetric
unit of the crystal, each complexed with two molecules of
inhibitor 6a. The unsubstituted phenyl ring of each “second
site” inhibitor molecules was engaged in intermolecular
contacts with the Phe242 side chains of both renin entities in
the crystal packing.26c Interestingly, the electron density was
well-defined and the inhibitor occupancy was identical for both
ligands binding to the active site of renin in the S1′ and S2 or
the S3 and S4 nonprime sites, respectively. On the basis of these
observations, a significant contribution of the second ligand to
the binding affinity of 6a to the S1′/S2 active site cannot be
ruled out. Quantification of the energetic contributions of the
“second ligand” would require additional experiments.
The primary in silico hit list comprising a total of 3717
compounds was further refined by adding Unity volume
constraints for the van der Waals surfaces of the protein atoms
calculated with MOLCAD.26a The surface constraints were
generated and applied independently by using both the X-ray
crystal complex structure of 5 and that of a related analogue
bearing an extended ortho-chlorobenzyloxymethyl moiety at the
C4′ phenyl position (PDB code 1PR8).20 Triaging by visual
inspection of the combined remaining hits removed com-
pounds with an apparent structural alert, and physical
availability in the corporate database was then checked. A
total of 328 compounds were finally tested for renin inhibition
using a fluorescence resonance energy transfer (FRET)
biochemical assay identifying four weakly active nonpeptide
hits. (Rac)-6 was the most potent, with an IC50 of 14 μM
(Figure 2, Table 1), representing the first attractive analogue of
a
Table 1. In Vitro Inhibition of rh-Renin
a
Half−maximal inhibition of rh-renin determined in a FRET-based
enzymatic assay using (RE(EDANS)IHPFHLVIHTK(Dabcyl)R as the
substrate in 100 mM Tris−HCl buffer pH 7.4.26a Data represent mean
b
values from duplicates. All compounds prepared as HCl salts.
c
Racemic mixture.
a new chemotype DRI. Racemic resolution of 6 by chiral HPLC
(Chiracel OD) afforded the single enantiomer 6a (enantio-
meric excess ee >99.9%), with an IC50 of 7.6 μM toward rh-
renin and being slightly more potent compared to its antipode
6b (IC50 = 18 μM, Table 1). Also, compound 6a only weakly
inhibited other human aspartic proteases with IC50 = 11 μM for
both rh-cathepsin D and E and IC50 = 4.5 and 29 μM for rh-
pepsin A and C, respectively. The absolute configuration of
(3S,4S)-6a was subsequently confirmed by synthesis and
chemical correlation with an authentic sample (vide infra).26b
We were very gratified that the X-ray crystal structure of the
weakly active 6a in its complex with rh-renin could be rapidly
resolved by the soaking method,26a revealing an unexpected
ligand binding orientation along the axis of the enzyme
substrate cleft (Figure 3). In its complex, 6a adopted an
extended conformation spanning both the prime and nonprime
recognition sites. Most notably, 6a lacked any binding
interaction to the open flap Tyr75 pocket similar to the C4
Retrospectively, the 3D pharmacophore-assisted discovery of
6a as a weakly active inhibitor of human renin is quite
remarkable in view of its unexpected binding pose unveiled by
X-ray crystallography. The two hydrophobic constraints in
combination with the basic amine constraint of our
pharmacophore model derived from the GRAB peptidomimetic
5 were fully matched by the two aromatic substituents of the
N,N-diphenylamino moiety and the pyrrolidine template of 6a,
respectively. In this model, the unsubstituted N-phenyl moiety
of 6a matched with the C4 phenyl group of 5 binding to the
open-flap conformation, and the N-para-Cl-phenyl corre-
sponded to the S1−S3 naphthalene scaffold of 5. Notably, the
exclusion volume derived from the published X-ray structure
(1PR8) of a GRAB peptidomimetic binding deeply into the
open flap pocket accommodated 6 in its pharmacophore-
derived orientation and conformation. On the other hand, there
was not sufficient space within the exclusion volume derived
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from the human renin/5 crystal complex structure because the
open-conformation flap pocket in this case was too narrow in
order to identify 6 as a potential ligand of the enzyme.
The insight gained by X-ray crystallography into the binding
interactions of (3S,4S)-6a was crucial for developing an efficient
hit-to-lead optimization strategy. Initially, we focused our
attention on exploring the potential offered by the
unprecedented N,N-diphenylamine nonprime site binding
motif to improve the binding affinity to rh-renin. Specifically,
major efforts addressed modifications of the “upper” N-4-
chlorophenyl portion with the aim to induce a H-bond to the
Ser219 at the entrance of the nonsubstrate S3sp cavity and/or to
tether this aryl portion by a short linker and to further expand
into the unoccupied S2 pocket. Combined attempts were also
made to fill the large hydrophobic S3−S1 pocket by
appropriately substituting the S1 binding N-phenyl residue.
However, while more than 150 analogues of 6 with such
modifications of both N-aryl groups were synthesized, no
significant improvement in potency was accomplished (data not
shown).
Figure 4. X-ray crystal structure of (3S,4S)-12a (shown as stick model
in green color) bound in the rh-renin active site (2.8 Å resolution), see
Experimental Section. Shown is the solvent accessible surface of the
active site of rh-renin. Residues of the flap β-hairpin are depicted in
yellow as thin lines and have been omitted for protein surface
calculation. The catalytic aspartates are shown as stick models. Oxygen
and nitrogen atoms are shown in red and blue color, respectively.
pharmacophore residues of inhibitor 1. The methoxypropyloxy
side chain of 12a was bound in an extended conformation into
sp
the nonsubstrate S3 channel of rh-renin, forming an H-bond
The superimposed X-ray crystal structures of (3S,4S)-6a and
117,28 revealed the N,N-diphenylamine nitrogen atom of 6a to
be positioned very closely to the chirality center bearing the S1
isopropyl group of 1 (Figure 3). This prompted us to merge the
unique key structural elements of both inhibitor chemotypes by
combining the optimized topological P3/P3sp−P1 pharmaco-
phore of 1 with the 3,4-pyrrolidine center transition-state
surrogate of 6a. Analogues of 1 bearing either a basic N-
isopropyl tertiary amine or the related neutral N-isopropyl
benzamide P3−P1 motif had been previously discovered to
display high in vitro potency toward human renin.29
Compounds (rac)-7 and (rac)-8 in which the N-phenyl of 6
between the distal ether oxygen and the NH of the Tyr14
backbone, while the para-methoxy group fills the S3 pocket.
The N-isopropyl of 12a was penetrating more deeply into S1
compared to the N-phenyl of (3S,4S)-6a,30 thereby forming
closer van der Waals contacts to the hydrophobic surface of this
pocket. The benzamide moiety of 12a was bridging the
contiguous S3−S1 “superpocket” by adopting a distorted
conformation in which the carbonyl group is rotated by a 90°
angle relative to the plane of the phenyl portion. In addition,
the carbonyl of the P3−P1 carboxamide spacer was in H-bond
distance (2.8 Å) to the hydroxyl of the flap Thr77 residue.29 The
flap β-hairpin loop was in a closed conformation with the
backbone and side chains of Tyr75−Ser76−Thr77−Gly78, being
in a very similar orientation as observed for inhibitor 1. The
only major difference was found for the hydroxyl group of
Ser76, which was flipped toward solvent space in the case of rh-
renin bound 6a. On the other hand, the center pyrrolidine
scaffold as well as the hydrophobic 3(S)-benzyl group binding
to the S1′ site of both 12a and the initial hit 6a (Figure 4) were
in a virtually identical position within the rh-renin active cleft.
Interestingly, the basic pyrrolidine nitrogen atom of 12a was
located almost symmetrically between the primary amine NH2
and the hydroxyl group of the aminohydroxyethylene dipeptide
isostere of 1, forming a H-bond network to the catalytic dyad
which is partially distinct from that of classical hydroxyethylene
(HE) isosteres of peptide-derived peptidomimetics.28
With the discovery of the 3,4-piperidine based GRAB
peptidomimetic renin inhibitors, basic cyclic secondary amine
skeletons have been recognized as privileged core structural
entities interacting with the enzyme family specific catalytic
dyad for the conceptual design of novel aspartic protease
inhibitors.20,31−34 The five-membered 3,4-di(aminomethyl)-
pyrrolidine template has been utilized for the design of potent
HIV-1 protease inhibitors as well as for cathepsin D and was
predicted by modeling to bind into the S2−S2′ specificity
pockets.31 X-ray crystallography confirmed the basic pyrrolidine
nitrogen atom to be positioned in close binding distance to
both catalytic aspartates of the HIV protease and, most notably,
unveiled an unexpected inhibitor pose characterized by an
altered occupation of the recognition pockets and a major
conformational distortion of the flap region.31 3,4-Trisubsti-
tuted pyrrolidine-based BACE-1 inhibitors have been reported
recently and were demonstrated by X-ray crystallography to
sp
is replaced by an unsubstituted benzyl and a P3 -tethered
dialkoxybenzyl group, respectively, were found to be equipotent
to (3S,4S)-6a (Table 1). Furthermore, the N-isopropyl
analogue (rac)-9 surprisingly did not show improved inhibitory
affinity in the FRET biochemical assay.
Next, we investigated the introduction of a carbonyl linker in
order to avoid a basic amine functional group at P1 and to
constrain the ligand conformational flexibility. In addition,
computational docking predicted a H-bond interaction between
the spacer carbonyl and Thr77 of the flap β-hairpin,29
anticipating a closed binding conformation similar to that of
the renin−inhibitor 1 complex. A ∼5-fold improvement in
potency was observed for the N-chlorophenyl-N-benzamide
(rac)-10 (IC50 = 1.7 μM) and its des-chloro analogue (rac)-11
(Table 1). Most notably, a further significant increase in
inhibitory affinity was accomplished with the N-isopropyl
analogue (rac)-12 (IC50 = 0.45 μM). This result was considered
to be in line with previous findings30 indicating that the N-
phenyl residue would be suboptimal for this inhibitor series to
be smoothly accommodated by the S1 pocket. The pure
enantiomer (3S,4S)-12a showed an IC50 of 170 nM and was 15
times more active compared to the distomer (3R,4R)-12b
(Table 1). Importantly, the potency of (3S,4S)-12a toward
renin was retained when measured in the presence of human
plasma (IC50 = 300 nM). Furthermore, (3S,4S)-12a was highly
selective against human pepsins A, C, human cathepsins D, E,
as well as human BACE-1 and BACE-2 (all IC50 >30 μM).
The X-ray crystal structure of (3S,4S)-12a in complex with
rh-renin (Figure 4) revealed a strikingly close overlay of the 3,4-
sp
dialkoxy phenyl portion accommodated by the S3−S3 cavity
and of the S1 N-isopropyl group with the respective
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a
form a bidentate interaction between the pyrrolidine nitrogen
and the two aspartate residues from the catalytic dyad.35
Notably, the spiro-tetrahydronaphthalene moiety in position 4
binding in the S1 pocket and the 2,4-diphenyl substituted
piperidine moiety attached to the C3 occupied a newly formed
pocket under the flap with one phenyl group pointing toward
the S2′ pocket.35 The trans-3,4-pyrrolidine 12a reported herein
represents the first prototype of a new class of DRIs that
interacts to the extended β-strand substrate binding topography
with the flap β-hairpin in a closed conformation and by
spanning the S3−S1′ specificity pockets in a topological
fashion.36
Scheme 2
An efficient synthesis of the key trans-pyrrolidine-3-
carbaldehyde intermediate (rac)-16 was established as outlined
in Scheme 1, providing flexible access to various nonprime side
a
(a) R−CH2Br, K2CO3, DMF, 80°C or 4-methoxy-3-(3-methoxypro-
poxy)-benzaldehyde, NaBH(OAc)3, 1,2-dichloroethane, RT; (b) TFA,
CH2Cl2 and formation of HCl salt,26b or 4 M HCl−dioxane; (c) 4-
methoxy-3-(3-methoxypropoxy)-benzoylchloride, Et3N, CH2Cl2, RT;
(d) HCl−iPrOH, RT; (e) [Pd(μ-Br)(t-Bu3P)]2, tBuOK, PhBr, toluene
110 °C.
a
Scheme 1
bound 6a as determined by X-ray crystallography (vide supra).
Furthermore, both enantiomeric inhibitors (3S,4S)-12a and
(3R,4R)-12b were prepared from (3S,4S)-15a and (3R,4R)-
15b, respectively, according to Scheme 2.
To further assess the lead potential of the prototype inhibitor
(3S,4S)-12a, the in vitro selectivity against a broad safety
pharmacology panel of receptors and ion channels, the
pharmacokinetic profile in rat and the in vivo efficacy in
hypertensive double-transgenic rats (dTGRs) overexpressing
both human renin and human angiotensinogen37 were
evaluated. (3S,4S)-12a was found to be inactive against 52
receptors (all IC50 >10 μM) and only weakly active against 5-
HT1A, 2A, 2B, and the α1-adrenoceptor (IC50s ranging
between 1 and 10 μM), respectively. The compound had
modest affinity to the hERG channel with IC50 = 14.5 μM
(mean value from two independent measurements using an in
vitro dofetilide binding assay). Absolute oral bioavailability was
53% in cannulated fed Sprague−Dawley rats when dosed at 2
mg/kg intravenously and 6 mg/kg orally (estimated t1/2 = 2 h;
ClP = 1.5 L/h/kg; Vdss = 2.8 L/kg; oral Cmax = 1310 nM; oral
AUC(0−24 h) = 4810 nM·h). Single oral dosing of (3S,4S)-12a to
telemetered dTGRs at 6 and 30 mg/kg resulted in a dose-
dependent moderate reduction in mean arterial blood pressure
(MAP) with peak changes in MAP of 15 and 35 mmHg,
respectively. Blood pressure returned to baseline pretreatment
levels after 12 h, demonstrating a sustained pharmacological
effect of the compound in this model.
a
(a) N-Benzyl-N-(methoxymethyl)trimethylsilyl methylamine, TFA,
toluene, 0°C, 88%; (b) H2, Pd(OH)2, (Boc)2O, EtOH, RT, 95%; (c)
LiBH4, THF, reflux, 60%; (d) Dess−Martin periodinane, wet CH2Cl2,
89%; (e) R1−NH2, NaBH(OAc)3, 1,2-dichloroethane, RT.
chain modifications. Ethyl trans-4-phenyl-2-butenoate 13 read-
ily obtained from phenylacetaldehyde by Wittig−Horner
olefination was converted via a 1,3-dipolar cycloaddition
reaction to afford the trans-configured pyrrolidine (rac)-14.26b
Reductive debenzylation and in situ N-Boc protection followed
by LiBH4 reduction of the ester furnished the alcohol derivative
(rac)-15. Dess−Martin oxidation to the aldehyde (rac)-16 was
followed by reductive amination to afford the secondary amine
derivatives (rac)-17 to (rac)-19, respectively. Inhibitors (rac)-7
to (rac)-9 were prepared from (rac)-17 or (rac)-19 by N-
alkylation with the respective benzylbromides in the presence
of K2CO3 or by reductive amination using 4-methoxy-3-(3-
methoxypropoxy)-benzaldehyde/NaBH(OAc)3, followed by
removal of the N-Boc protecting group (Scheme 2). N-
Acylation of amines (rac)-17 to (rac)-19 with 4-methoxy-3-(3-
methoxypropoxy)-benzoylchloride and final deprotection af-
forded inhibitors (rac)-10, (rac)-11, and (rac)-12, respectively
(Scheme 2).
Resolution of the alcohol intermediate (rac)-15 was
accomplished by preparative chiral HPLC (Chiracel OJ) to
afford the single enantiomers (3S,4S)-15a and (3R,4R)-15b
with high enantiomeric excess of ee ≥ 99.8%. The absolute
configuration of both (3S,4S)-15a and (3R,4R)-15b was
unambiguously assigned by their transformation to the
crystalline aniline derivatives (3S,4R)-17a and (3R,4S)-17b
according to Scheme 1 and subsequent X-ray diffraction
analysis.26a Intermediate (3S,4R)-17a served as starting material
for the preparation of the pure enantiomer (3S,4S)-6a
according to Scheme 2, thereby confirming the initially
tentative assignment of the absolute configuration of rh-renin
CONCLUSION
■
In summary, a small-molecule trans-3,4-disubstituted pyrroli-
dine derivative was discovered from an in silico 3D
pharmacophore approach as a weakly active screening hit
toward rh-renin. Utilizing X-ray crystal structure information,
the combination of the center basic amine transition-state
surrogate with an optimized P3/P3sp−P1 scaffold derived from 1
resulted in (3S,4S)-12a with submicromolar in vitro potency.
The druglike lead compound of a novel class of DRIs
demonstrated promising pharmacokinetic and pharmacody-
namic properties in rat. Results from continued work focusing
on prime site modifications and leading to highly potent clinical
candidates will be presented in due course.
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(t, J = 7.1 Hz, 3H), 1.36 (s, 9H), 2.62 (m, 2H), 2.81 (m, 2H), 2.95 (m,
1H), 3.29−3.36 (m, 2H), 3.57 (m, 1H), 3.98 (q, J = 7.1 Hz, 2H), 7.19
(m, 3H), 7.27 (m, 2H).
EXPERIMENTAL SECTION
■
Chemistry General Procedures. Unless otherwise specified, all
solvents and reagents were obtained from commercial suppliers and
used without further drying or purification. Normal-phase flash
chromatography was performed using Merk silica gel 60 (230−400
mesh). Rf values for TLC are measured on 5 cm × 10 cm TLC plates,
silica gel F254, Merck, Darmstadt, Germany. Purity was determined by
analytical HPLC from the integration of the area under the UV
(3S*,4S*)-3-Benzyl-4-hydroxymethyl-pyrrolidine-1-carboxylic
Acid tert-Butyl Ester ((rac)-15). To a solution of (3S*,4S*)-4-
benzylpyrrolidine-1,3-dicarboxylic acid 1-tert-butyl ester 3-ethyl ester
(0.15 g, 0.45 mmol) in THF (4 mL) was slowly added a solution of
LiBH4 (2N in THF, 0.225 mL, 0.450 mmol) at 0 °C under an N2
atmosphere. The reaction mixture was stirred overnight at RT, then
refluxed for 2 h and subsequently carefully poured into an aqueous
saturated NaHCO3 solution. EtOAc was added, the layers were
separated, and the aqueous phase was back-extracted twice with
EtOAc. The combined organics were dried over Na2SO4, filtered, and
concentrated. The crude material was purified by flash chromatog-
raphy on silica gel (eluent: c-hexane−EtOAc 100:0 to 80:20) to give
(rac)-15 (0.79 g, 60%) as a colorless oil. TLC, Rf (c-hexane−EtOAc
80:20) = 0.20. MS (LC−MS): 236.1 [M-tBu + H]+. tR (HPLC, Waters
Symmetry C18, 3.5 μm, 2.1 mm × 50 mm, 20−95% CH3CN−H2O/
3.5 min, 95% CH3CN/2 min, CH3CN and H2O containing 0.1% TFA;
flow, 0.6 mL/min): 3.4 min. 1H NMR (DMSO-d6, 600 MHz): δ 1.35
(s, 9H), 1.96 (m, 1H), 2.14−2.24 (m, 1H), 2.46 (m, 1H), 2.81 (m,
1H), 2.90 (m, 1H), 3.05 (m, 1H), 3.20 (m, 1H), 3.30 (m, 1H), 3.37−
3.45 (m, 2H), 4.65 (t, J = 5.3 Hz, 1H, OH), 7.18 (m, 3H), 7.27 (m,
2H). 13C NMR (DMSO-d6, 150 MHz, 2 rotamers): δ 28.14, 37.73,
37.85, 40.35, 41.09, 44.76, 45.68, 48.33, 48.78, 50.61, 50.64, 61.29,
61.40, 78.08, 125.97, 128.28, 128.60, 140.24, 153.47, 153.50.
absorption curve at λ = 254 or 214 nm signals. H NMR and 13C
1
NMR spectra were recorded on a Bruker DPX400 BBO spectrometer
and a Bruker Avance 600 spectrometer using a 13C−1H dual
1
cryoprobe. H and 13C NMR spectra were recorded at 394 K on a
Bruker DPX400 TXI spectrometer. Chemical shift (δ) values were
referenced to tetramethylsilane as internal standard and are reported in
parts per million (ppm). Mass spectral HRMS were obtained using
electrospray ionization in positive ion modus. The elemental
composition was derived from the averaged mass spectra acquired at
the high resolution of about 30000 on an 9.4T APEX-III FT-MS
(Bruker Daltonics). The high mass accuracy below 1 ppm was
obtained using an internal calibration.
Preparation of Intermediates 13, 14, 15(a,b), 16, 17(a,b),
19(a,b). (E)-4-Phenyl-but-2-enoic Acid Ethyl Ester (13). To a
solution of triethylphosphonoacetate (9.00 mL, 45.4 mmol) in THF
(90 mL) was added sodium hydride (60% dispersion in oil, 1.81 g,
45.4 mmol) at RT under an N2 atmosphere. The reaction mixture was
stirred at 25 °C for 45 min, and phenylacetaldehyde (4.87 mL, 41.0
mmol) was added. After 30 min at RT, the reaction was partitioned
between Et2O and water. The aqueous layer was extracted three times
with Et2O, dried over Na2SO4, filtered, and concentrated. The crude
material was purified by flash chromatography on silica gel (eluent: c-
hexane−EtOAc 90:10) to give 13 (7.0 g, 81%) as a colorless oil. TLC,
Rf (c-hexane−EtOAc 90:10) = 0.55. MS (LC-MS): 191.0 [M + H]+. tR
(HPLC, Waters Symmetry C18, 3.5 μm, 2.1 mm × 50 mm, 5−95%
CH3CN−H2O/3.5 min, 95% CH3CN/2 min, CH3CN and H2O
containing 0.1% TFA; flow, 0.6 mL/min): 3.6 min. 1H NMR (DMSO-
d6, 400 MHz): δ 1.17 (t, J = 7.1 Hz, 3H), 3.53 (dd, J = 7.0, 1.3 Hz,
2H), 4.08 (q, J = 7.1 Hz, 2H), 5.83 (dt, J = 15.6, 1.3 Hz, 1H), 6.97 (dt,
J = 15.6, 7.0 Hz, 1H), 7.20−7.24 (m, 3H), 7.30−7.33 (m, 2H).
(3S*,4S*)-1,4-Dibenzyl-pyrrolidine-3-carboxylic Acid Ethyl Ester
((rac)-14). To a stirred solution of 13 (4.70 g, 24.7 mmol) in toluene
(60 mL) was added N-benzyl-N-(methoxymethyl)trimethylsilylamine
(6.54 mL, 25.6 mmol) at 0 °C under an N2 atmosphere.
Trifluoroacetic acid (0.19 mL, 2.47 mmol) was added at 0 °C, and
the mixture was stirred for 30 min at RT overnight.38 The reaction was
quenched by addition of a saturated aqueous solution of NaHCO3 and
was the extracted three times with EtOAc. The combined organics
were dried over Na2SO4 and concentrated. The crude material was
purified by flash chromatography on silica gel (eluent: hexane−EtOAc
90:10) to give (rac)-14 (7.0 g, 88%) as a colorless oil. TLC, Rf (c-
hexane−EtOAc 90:10) = 0.25. MS (LC−MS): 324.2 [M + H]+. tR
(HPLC, Waters Symmetry C18, 3.5 μm, 2.1 mm × 50 mm, 20−95%
CH3CN−H2O/3.5 min, 95% CH3CN/2 min, CH3CN and H2O
containing 0.1% TFA; flow, 0.6 mL/min): 2.7 min. 1H NMR (DMSO-
d6, 400 MHz): δ 1.08 (t, J = 7.1 Hz, 3H), 2.22 (dd, J = 8.85, 5.8 Hz,
1H), 2.55 (m, 1H), 2.66−2.70 (m, 5H), 2.76 (m, 1H), 3.47 (d, J =
13.1 Hz, 1H), 3.56 (d, J = 13.1 Hz, 1H), 3.95 (q, J = 7.1 Hz, 2H),
7.12−7.30 (m, 10H).
(3S,4S)-3-Benzyl-4-hydroxymethyl-pyrrolidine-1-carboxylic Acid
tert-Butyl Ester (15a) and (3R,4R)-3-Benzyl-4-hydroxymethyl-
pyrrolidine-1-carboxylic Acid tert-Butyl Ester (15b). (Rac)-15 (1.2
g) was separated into its enantiomers by preparative chiral HPLC
column: Chiracel OJ, Diacel Chemical Industries Ltd., 10 cm × 50 cm,
20 μm; eluent, hexane−EtOH 85:15; flow rate, 120 mL/min;
detection, UV at 210 nm. (3S,4S)-15a: tR 40.9 min (preparative chiral
HPLC), tR (HPLC, Chiracel OJ (1099), 0.46 cm × 25 cm, hexane/
EtOH 90−10; flow, 1 mL/min): 7.1 min; enantiomeric excess ee =
99.8%, [α]D20 −48.4 (c = 0.99, MeOH). The absolute configuration of
the 2 stereogenic centers was determined based on X-ray structure
crystallography of the corresponding (3S,4R)-3-benzyl-4-[(4-chloro-
phenylamino)-methyl]-pyrrolidine-1-carboxylic acid tert-butyl ester
17a26a prepared in 2 steps as described below. (3R,4R)-15b: tR 32.5
min (preparative chiral HPLC), tR (HPLC, Chiracel OJ (1099), 0.46
cm × 25 cm, hexane−EtOH 90−10; flow, 1 mL/min): 5.2 min;
20
enantiomeric excess ee = 100%. [α]D +46 (c = 1.02, MeOH). The
absolute configuration of the 2 stereogenic centers was determined
based on X-ray structure crystallography of the corresponding
(3R,4S)-3-benzyl-4-[(4-chloro-phenylamino)-methyl]-pyrrolidine-1-
carboxylic acid tert-butyl ester 17b26a prepared in two steps as
described below.
(3S*,4S*)-3-Benzyl-4-formyl-pyrrolidine-1-carboxylic Acid tert-
Butyl Ester ((rac)-16). To a well stirred mixture of (rac)-15 (10.0 g,
34.3 mol) and Dess−Martin periodinane (14.6 g, 34.3 mmol) in
CH2Cl2 (200 mL) was slowly added wet CH2Cl2 (0.68 mL of water in
50 mL of CH2Cl2, 37.7 mmol). The clear solution turned cloudy
toward the end of wet CH2Cl2 addition. The mixture was diluted with
Et2O and then concentrated by rotary evaporation to a small volume.
The residue was taken up in Et2O, washed with a 1:1 mixture of a 10%
aqueous Na2S2O3 solution and a saturated aqueous NaHCO3 solution,
followed by washing with H2O and brine. The combined aqueous
phases were back-extracted with Et2O. The combined organics were
washed with H2O and brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude material was purified
by flash chromatography on silica gel (eluent: c-hexane−EtOAc 2:1) to
give (rac)-16 as a slightly yellow oil (8.8 g, 89%). TLC, Rf (c-hexane−
EtOAc 2:1) = 0.50. MS (LC−MS): 234.1 [M-tBu + H]+. tR (HPLC,
Waters Symmetry C18, 3.5 μm, 2.1 mm × 50 mm, 20−95% CH3CN−
H2O/3.5 min, 95% CH3CN/2 min, CH3CN and H2O containing 0.1%
TFA; flow, 0.6 mL/min): 3.55 min. 1H NMR (DMSO-d6, 600 MHz):
δ 1.36 (s, 9H), 2.65−2.78 (m, 3H), 2.83 (m, 1H), 3.05 (m, 1H), 3.20
(m, 1H), 3.42 (m, 1H), 3.50 (m, 1H), 7.21 (m, 3H), 7.29 (m, 2H),
9.45 (m, 1H). 13C NMR (DMSO-d6, 150 MHz, 2 rotamers): δ 28.1,
(3S*,4S*)-4-Benzylpyrrolidine-1,3-dicarboxylic Acid 1-tert-Butyl
Ester 3-Ethyl Ester. A mixture of (rac)-14 (6.57 g, 20.3 mmol), di-
tert-butylcarbonate (4.43 g, 20.3 mmol) and Pd(OH)2 (0.6 g, 50%
wet) in EtOH (300 mL) was stirred under an hydrogen atmosphere
for 20 h. After filtration through a pad of Celite, the filtrate was dried
over Na2SO4 and concentrated. Purification of the residue by
chromatography on silica gel (eluent: c-hexane−EtOAc 90:10)
afforded (3S*,4S*)-4-benzylpyrrolidine-1,3-dicarboxylic acid 1-tert-
butyl ester 3-ethyl ester (6.42 g, 95%). TLC, Rf (c-hexane−EtOAc
80:20) = 0.35. MS (LC−MS): 356.1 [M + Na]+. tR (HPLC, Waters
Symmetry C18, 3.5 μm, 2.1 mm × 50 mm, 20−95% CH3CN−H2O/
3.5 min, 95% CH3CN/2 min, CH3CN and H2O containing 0.1% TFA;
1
flow, 0.6 mL/min): 4.1 min. H NMR (DMSO-d6, 400 MHz): δ 1.14
2212
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37.54, 39.99, 40.54, 44.43, 44.60, 49.91, 49.95, 53.64, 54.46, 78.55,
126.31, 128.82, 139.49, 153.35, 201.81, 201.87.
Preparation of Compounds 6(a,b), (rac) 7−11, 12(a,b).
((3S,4S)-4-Benzylpyrrolidin-3-ylmethyl)-(4-chlorophenyl)-phenyl-
amine Hydrochloride Salt (6a) and ((3R,4R)-4-Benzylpyrrolidin-3-
ylmethyl)-(4-chlorophenyl)-phenylamine Hydrochloride Salt (6b).
((3S*,4S*)-4-Benzyl-pyrrolidin-3-ylmethyl)-(4-chlorophenyl)-
phenylamine, (rac)-6,40 (215 mg) was separated into its enantiomers
by preparative chiral HPLC column: Chiracel OD prep, Diacel
Chemical Industries Ltd., 50 mm × 500 mm (20 μm); eluent, hexane−
EtOH 95:5 + 0.1% TFA; flow rate, 80 mL/min; detection, UV at 210
nm. (3S,4S)-6a: The product (contaminated with residual trifluoro-
acetic acid) was isolated as peak 1 with tR 52.7 min (preparative chiral
HPLC). This material was dissolved in CH2Cl2 and subsequently
washed with a saturated aqueous solution of NaHCO3. The organics
were dried over Na2SO4, filtered, and concentrated. The residue was
dissolved in dioxane (1 mL), followed by addition of 4 M HCl in
dioxane (1 equiv) and lyophilization to afford (3S,4S)-6a, HCl salt,
(3S*,4R*)-3-Benzyl-4-[(4-chlorophenylamino)-methyl]-pyrroli-
dine-1-carboxylic Acid tert-Butyl Ester ((rac)-17). A mixture of (rac)-
16 (0.50 g, 1.72 mmol) and 4-chloroaniline (0.220 g, 1.69 mmol) in
1,2-dichloroethane (7 mL) was treated with sodium triacetoxybor-
ohydride39 (0.510 g, 2.42 mmol) with stirring at RT under a N2
atmosphere overnight. The reaction was quenched by addition of
aqueous saturated NaHCO3 solution (10 mL), followed by extraction
with CH2Cl2. The organic layer was dried over Na2SO4, filtered, and
concentrated. The residual brown oil was purified by flash
chromatography on silica gel (eluent: c-hexane−EtOAc 90:10) to
give (rac)-17 (0.66 g, 95%). TLC, Rf (c-hexane−EtOAc 90:10) = 0.20.
MS (LC−MS): 445.1 [M + HCOOH]+. tR (HPLC, Waters Symmetry
C18, 3.5 μm, 2.1 mm × 50 mm, 20−95% CH3CN−H2O/3.5 min, 95%
CH3CN/2 min, CH3CN and H2O containing 0.1% TFA; flow, 0.6
mL/min): 4.38 min. 1H NMR (DMSO-d6, 600 MHz): δ 1.35 (s, 9H),
2.10−2.15 (m, 1H), 2.20−2.28 (m, 1H), 2.52 (m, 1H), 2.84−2.97 (m,
3H), 3.02 (dd, J = 10.8, 7.6 Hz, 1H), 3.08 (m, 1H), 3.24 (m, 1H), 3.50
(m, 1H), 5.89 (m, 1H, NH), 6.51 (m, 2H), 7.05 (m, 2H), 7.19 (m,
3H), 7.28 (m, 2H). 13C NMR (DMSO-d6, 150 MHz, 2 rotamers): δ
28.1, 37.69, 37.76, 41.67, 41.85, 42.59, 44.77, 44.84, 49.49, 49.94,
50.48, 50.56, 78.23, 113.18, 118.70, 126.03, 128.31, 128.52, 128.65,
140.13, 147.68, 153.45.
1
(67 mg). H NMR (DMSO-d6, 600 MHz): δ 2.25 (m, 1H), 2.31 (m,
1H), 2.65 (dd, J = 13.5, 8.3 Hz, 1H), 2.75 (dd, J = 13.5, 6.6 Hz, 1H),
2.83 (m, 1H), 2.91 (m, 1H), 3.18 (m, 1H), 3.26 (m, 1H), 3.64 (dd, J =
14.8, 5.6 Hz, 1H), 3.72 (dd, J = 14.8, 9.2 Hz, 1H), 6.79 (d, J = 8.9 Hz,
2H), 6.95 (d, J = 7.9 Hz, 2H), 7.04 (t, J = 7.6 Hz, 1H), 7.14 (d, J =
7.25 Hz, 2H), 7.23 (m, 3H), 7.30 (m, 4H), 9.03 (m, 1H), 9.1 (m, 1H).
13C NMR (DMSO-d6, 150 MHz): δ 37.43, 41.12, 42.22, 48.14, 49.16,
53.37, 120.95, 122.66, 122.96, 124.31, 126.40, 128.51, 128.78, 129.02,
129.64, 139.33, 146.81, 146.99. tR (HPLC, Waters Symmetry C18, 3.5
μm, 2.1 mm × 50 mm, 20−95% CH3CN−H2O/3.5 min, 95%
CH3CN/2 min, CH3CN and H2O containing 0.1% TFA; flow, 0.6
mL/min): 3.46 min, purity 99.3%. tR (Waters XBridge C18, 2.5 μm, 3
mm × 50 mm, 10−98% CH3CN−H2O/8.6 min, 98% CH3CN/1.4
min, CH3CN and H2O containing 0.1% TFA; flow, 1.4 mL/min,
temperature 40 °C): 4.69 min, purity 99%. tR (HPLC, Daicel OD-H,
250 mm × 4.6 mm (5 μm), hexane−EtOH 95:5 + 0.1% TFA, flow 1
mL/min, UV 210 nm, injection volume 10 μL of a 0.1% solution in
EtOH): 14.5 min; enantiomeric excess ee > 99.9%. HRMS calcd for
C24H25ClN2 [M + H]+, 377.1779; found, 377.1778. (3R,4R)-6b: The
product (contaminated with residual trifluoroacetic acid) was isolated
as peak 2 with tR 76.4 min (preparative chiral HPLC). This material
was dissolved in CH2Cl2 and subsequently washed with a saturated
aqueous solution of NaHCO3. The organics were dried over Na2SO4,
filtered, and concentrated. The residue was dissolved in dioxane (1
mL), followed by addition of 4 M HCl in dioxane (1 equiv) and
lyophilization to afford (3R,4R)-6b, HCl salt, (80 mg). tR (HPLC,
Waters Symmetry C18, 3.5 μm, 2.1 mm × 50 mm, 20−95% CH3CN−
H2O/3.5 min, 95% CH3CN/2 min, CH3CN and H2O containing 0.1%
TFA; flow, 0.6 mL/min): 3.44 min, purity 100%. tR (Waters XBridge
C18, 2.5 μm, 3 mm × 50 mm, 10−98% CH3CN-H2O/8.6 min, 98%
CH3CN/1.4 min, CH3CN and H2O containing 0.1% TFA; flow, 1.4
mL/min, temperature 40 °C): 4.70 min, purity 99%. tR (HPLC, Daicel
OD-H, 250 mm × 4.6 mm (5 μm), hexane−EtOH 95:5 + 0.1% TFA,
flow 1 mL/min, UV 210 nm, injection volume 10 μL of a 0.1%
solution in EtOH): 18.7 min; enantiomeric excess ee > 99.9%. HRMS
calcd for C24H25ClN2 [M + H]+, 377.1779; found, 377.1779.
Crystallographic data (excluding structure factors) for (3S,4R)-17a
and (3R,4S)-17b have been deposited with the Cambridge Crystallo-
graphic Data Centre as supplementary publication number CCDC
904172 (17a) and CCDC 904173 (17b). Copies of the data can be
obtained, free of charge, on application to CCDC, 12 Union Road,
Cambridge CB2 1 EZ, UK [fax: +44-(0)1223−336033 or email:
deposit@ccdc.cam.ac.uk].
(3S,4R)-3-Benzyl-4-[(4-chlorophenylamino)methyl]-pyrrolidine-1-
carboxylic Acid tert-Butyl Ester ((3S,4R)-17a). The title compound
20
was prepared as described for (rac)-17 from (3S,4S)-15a. [α]D
−
33.1 (c = 1.17, MeOH). Absolute configuration determined by X-ray
crystallography.26a
(3R,4S)-3-Benzyl-4-[(4-chlorophenylamino)methyl]-pyrrolidine-1-
carboxylic Acid tert-Butyl Ester ((3R,4S)-17b). The title compound
was prepared as described for (rac)-17 from (3R,4R)-15b. [α]D20 + 33
(c = 0.96, MeOH). Absolute configuration determined by X-ray
crystallography.26a
(3S*,4R*)-3-Benzyl-4-(isopropylamino-methyl)-pyrrolidine-1-car-
boxylic Acid tert-Butyl Ester ((rac)-19). A mixture of (rac)-16 (2.64 g,
9.12 mmol), isopropylamine (2.35 mL, 27.4 mmol), and 1,2-
dichloroethane (150 mL) was stirred at RT for 10 min. Sodium
triacetoxyborohydride39 (4.83 g, 22.8 mmol) was added, and the
mixture was stirred for 20 h at RT. The reaction was quenched by
addition of a saturated aqueous NaHCO3 solution. After separation of
the organic layer, the aqueous phase was extracted twice with CH2Cl2.
The combined organics were dried over Na2SO4, and volatiles were
removed in vacuo to afford (rac)-19 as a colorless oil (3.1 g,
quantitative yield). tR (HPLC, Waters Symmetry C18, 3.5 μm, 2.1 mm
× 50 mm, 20−95% CH3CN−H2O/3.5 min, 95% CH3CN/2 min,
CH3CN and H2O containing 0.1% TFA; flow, 0.6 mL/min): 2.82 min.
1H NMR (DMSO-d6, 600 MHz): δ 0.94 (m, 6H), 1.35 (m, 9H), 1.94
(m, 1H), 2.11−2.19 (m, 1H), 2.39 (m, 1H), 2.45 (m, 1H), 2.64 (m,
2H), 2.82 (m, 1H), 2.89 (m, 1H), 2.98 (dd, J = 10.5, 7.9 Hz, 1H), 3.20
(m, 1H), 3.40 (m, 1H), 7.19 (m, 3H), 7.27 (m, 2H). 13C NMR
(DMSO-d6, 150 MHz, 2 rotamers): δ 21.28, 22.26, 22.50, 22.57, 28.16,
37.77, 37.84, 39.99, 42.00, 42.65, 42.73, 43.69, 48.37, 48.52, 49.99,
50.40, 50.58, 50.63, 78.12, 125.98, 128.28, 128.30, 128.64, 140.24,
153.44, 153.47, 172.17.
The synthesis of (3S,4S)-6a, HCl salt, was also accomplished in two
steps starting from enantiomerically pure (3S,4R)-17a of known
absolute configuration (vide infra) according to the following
procedure:
(3S,4R)-3-Benzyl-4-{[(4-chlorophenyl)-phenylamino]-methyl}-pyr-
rolidine-1-carboxylic Acid tert-Butyl Ester. A dry three-necked flask
equipped with a magnetic stirring bar, septum, and condenser with an
argon inlet−outlet was charged with [Pd(μ-Br)(t-Bu3P)]241 (9.70 mg,
0.0125 mmol), sodium tert-butoxide (35.5 mg, 0.37 mmol),
bromobenzene (59 mg, 0.37 mmol), and dry toluene (0.5 mL). A
solution of (3S,4R)-17a (100 mg, 0.25 mmol) in dry degassed toluene
(1 mL) was added, and the mixture was stirred at RT for 15 min,
followed by heating to 110 °C (gentle reflux) overnight. After cooling
to RT, the mixture was poured into water and the water phase was
extracted twice with EtOAc. The combined organic extracts were dried
over Na2SO4, filtered, and concentrated. The crude product was
purified by flash chromatography on silica gel (eluent: c-hexane−
EtOAc 90:10) to afford the title compound (91 mg, 76%). TLC, Rf (c-
(3S,4R)-3-Benzyl-4-(isopropylamino-methyl)-pyrrolidine-1-car-
boxylic Acid tert-Butyl Ester ((3S,4R)-19a). The title compound was
prepared as described for (rac)-19 in two steps from (3S,4S)-15a.
20
[α]D − 44.9 (c = 1.18, MeOH).
(3R,4S)-3-Benzyl-4-(isopropylamino-methyl)-pyrrolidine-1-car-
boxylic Acid tert-Butyl Ester ((3R,4S)-19b). The title compound was
prepared as described for (rac)-19 in two steps from (3R,4R)-15b.
20
[α]D + 40.9 (c = 1.16, MeOH).
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hexane−EtOAc 80:20) = 0.50. MS (LC−MS): 421 [M-tBu + H]+, 477
[M + H]+, 499 [M + Na]+. tR (HPLC, Waters Symmetry C18, 3.5 μm,
2.1 mm × 50 mm, 20−95% CH3CN−H2O/3.5 min, 95% CH3CN/2
min, CH3CN and H2O containing 0.1% TFA; flow, 0.6 mL/min): 5.16
min. 1H NMR (CDCl3, 400 MHz): δ 1.43 (s, 9H), 2.25 (m, 1H), 2.33
(m, 1H), 2.60 (m, 1H), 2.78 (m, 1H), 3.04−3.21 (m, 2H), 3.43 (m,
1H), 3.53 (dd, J = 14.6, 8.85 Hz, 1H), 3.59 (m, 1H), 3.73 (m, 1H),
6.76 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 7.8 Hz, 2H), 7.0 (t, J = 7.3 Hz,
1H), 7.09 (m, 2H), 7.16 (m, 2H), 7.24−7.28 (m, 5H).
((3S,4S)-4-Benzyl-pyrrolidin-3-ylmethyl)-(4-chlorophenyl)-phenyl-
amine, (3S,4S)-6a, Hydrochloride Salt. To a solution of (3S,4R)-3-
benzyl-4-{[(4-chlorophenyl)-phenylamino]-methyl}-pyrrolidine-1-car-
boxylic acid tert-butyl ester (91 mg, 0.19 mmol) in dioxane (2 mL) was
added 1 M HCl in dioxane (2 mL), and the reaction mixture was
stirred at RT for 4 h followed by lyophilization to afford (3S,4S)-6a,
HCl salt (78 mg, quantitative yield). The product thus obtained
showed the same retention time as the corresponding enantiomer
(peak 1, vide supra) by analytical chiral HPLC. tR (HPLC, Daicel OD-
H, 250 mm × 4.6 mm (5 μm), hexane−EtOH 95:5 + 0.1% TFA, flow
1 mL/min, UV 210 nm, injection volume 10 μL of a 0.1% solution in
EtOH): 14.5 min.
compound was prepared using the same procedure as described for
(rac)-7 using 4-bromomethyl-1-methoxy-2-(3-methoxypropoxy)-ben-
zene40 for the alkylation reaction. The crude product obtained after
aqueous workup was purified by preparative HPLC (Waters Sunfire
C18-OBD, 5 μm, 19 mm × 50 mm; eluent, 20% CH3CN/1 min, 20−
100% CH3CN−H2O/16 min, 100% CH3CN/4 min, CH3CN and
H2O containing 0.1% HCOOH; flow, 20 mL/min). The HPLC
fractions were collected, concentrated, diluted with EtOAc, and
washed with a saturated aqueous solution of NaHCO3. The organic
layer was dried over Na2SO4, filtered, and concentrated to give the title
compound (73 mg, 48%). TLC, Rf (CH2Cl2−MeOH 95:5) = 0.45. MS
(LC−MS): 609.2 [M + H]+. tR (HPLC, Waters Symmetry C18, 3.5
μm, 2.1 mm × 50 mm, 5−95% CH3CN−H2O/3.5 min, 95% CH3CN/
2 min, CH3CN and H2O containing 0.1% TFA; flow, 0.6 mL/min):
4.7 min. 1H NMR (DMSO-d6, 400 MHz): δ 1.33 (s, 9H), 1.87 (p, J =
6.4 Hz, 2H), 2.20−2.31 (m, 2H), 2.45−2.53 (m, 1H), 2.80 (dd, J =
13.6, 5.3 Hz, 1H), 2.94 (m, 1H), 3.05 (m, 1H), 3.20 (s, 3H), 3.25 (m,
1H), 3.30−3.35 (m, 1H), 3.41 (t, J = 6.4 Hz, 2H), 3.41−3.46 (m, 1H),
3.48 (m, 1H), 3.68 (s, 3H), 3.89 (t, J = 6.4 Hz, 2H), 4.44 (m, 2H),
6.60 (m, 1H), 6.63 (d, J = 8.8 Hz, 2H), 6.73 (m, 1H), 6.84 (d, J = 8.1
Hz, 1H), 7.08 (d, J = 8.8 Hz, 2H), 7.16 (m, 3H), 7.26 (m, 2H). 13C
NMR (DMSO-d6, 150 MHz, 2 rotamers): δ 28.09, 28.94, 37.82, 37.94,
40.96, 41.61, 41.98, 42.72, 49.26, 50.30, 50.40, 53.04, 53.24, 54.00,
54.06, 55.51, 57.91, 65.22, 68.47, 69.76, 78.19, 78.25, 111.80, 112.04,
114.25, 114.31, 118.59, 118.64, 119.61, 126.04, 128.31, 128.50, 128.60,
128.63, 130.52, 140.10, 146.96, 147.03, 147.81, 147.98, 153.40.
N-((3S*,4S*)-4-Benzylpyrrolidin-3-yl-methyl)-(4-chlorophenyl)-[4-
methoxy-3-(3-methoxypropoxy)-benzyl]-amine (rac)-8, Hydro-
chloride Salt. To a solution of (3S*,4R*)-3-benzyl-4-({(4-chloro-
phenyl)-[4-methoxy-3-(3-methoxypropoxy)-benzyl]-amino}-methyl)-
pyrrolidine-1-carboxylic acid tert-butyl ester (52 mg, 0.085 mmol) in
dioxane (2 mL) was added 4 M HCl in dioxane (1 mL) followed by
stirring for 2 h at RT. Lyophilization afforded (rac)-8, HCl salt, as a
solid (50 mg, 100%). MS (LC−MS): 509.1 [MH]+. tR (Waters
XBridge C18, 2.5 μm, 3 mm × 30 mm, 10−98% CH3CN/H2O/3 min,
98% CH3CN/0.5 min, CH3CN and H2O containing 0.1% TFA; flow,
1.4 mL/min; temperature 40 °C): 2.18 min, purity 99%. tR (Waters
XBridge C18, 2.5 μm, 3 mm × 50 mm, 10−98% CH3CN−H2O/8.6
min, 98% CH3CN/1.4 min, CH3CN and H2O containing 0.1% TFA;
Preparation of (rac)-7, HCl Salt. (3S*,4R*)-3-Benzyl-4-{[benzyl-
(4-chlorophenyl)-amino]-methyl}-pyrrolidine-1-carboxylic Acid tert-
Butyl Ester. To a solution of (rac)-17 (80 mg, 0.20 mmol) in DMF (1
mL) were added K2CO3 (0.062 g, 0.44 mmol) and benzylbromide
(0.052 mL, 0.44 mmol). Stirring was continued at 80 °C for 5 h before
the reaction mixture was quenched with a saturated aqueous NaHCO3
solution. The water phase was extracted with EtOAc, and the
combined organics were dried over Na2SO4, filtered, and concentrated.
The crude residue was purified by flash chromatography (eluent: c-
hexane−EtOAc 95:5 to 90:10) to give the title compound (64 mg,
1
65%). TLC, Rf (c-hexane−EtOAc 80:20) = 0.55. H NMR (CD3OD,
400 MHz): δ 1.39 (s, 9H), 2.18 (m, 1H), 2.32 (m, 1H), 2.50 (m, 1H),
2.73 (dd, J = 17.9, 8.9 Hz, 1H), 3.0 (dd, J = 14.8, 8.6 Hz, 1H), 3.23−
3.06 (m, 2H), 3.34−3.49 (m, 3H), 4.42 (m, 2H), 6.55 (d, J = 12.1 Hz,
2H), 6.99−7.31 (m, 12H).
Benzyl-((3S*,4S*)-4-benzylpyrrolidin-3-ylmethyl)-(4-chlorophen-
yl)-amine (rac)-7, Hydrochloride Salt. To a solution of (3S*,4R*)-3-
benzyl-4-{[benzyl-(4-chlorophenyl)-amino]-methyl}-pyrrolidine-1-car-
boxylic acid tert-butyl ester (64 mg, 0.13 mmol) in CH2Cl2 (2 mL)
was added trifluoroacetic acid (0.05 mL), and the mixture was stirred
at RT overnight. Volatile were removed under reduced pressure. The
mixture was then poured into a saturated aqueous solution of
NaHCO3, the water phase was extracted with EtOAc, and the
combined organics were dried over Na2SO4 and concentrated. The
crude residue was purified by Isolute Flash NH2 ion exchange
chromatography (eluent: CH2Cl2−MeOH 97:3 to 90:10 + 1% of
NH4OH) to give (rac)-7 as the free base (44.2 mg, 87%). This
material was dissolved in dioxane (2 mL), followed by addition of 4 M
HCl in dioxane (28 μL, 0.113 mmol) and lyophilization to afford
(rac)-7, HCl salt (94% purity by HPLC using two analytical methods).
MS (LC-MS): 391, 393 [M + H]+. tR (Waters XBridge C18, 2.5 μm, 3
mm × 30 mm, 10−98% CH3CN/H2O/3 min, 98% CH3CN/0.5 min,
CH3CN and H2O containing 0.1% TFA; flow, 1.4 mL/min;
temperature 40 °C): 2.34 min, purity 99%. tR (Waters XBridge C18,
2.5 μm, 3 mm × 50 mm, 10−98% CH3CN−H2O/8.6 min, 98%
CH3CN/1.4 min, CH3CN and H2O containing 0.1% TFA; flow, 1.4
mL/min; temperature 40 °C): 4.73 min, purity 99%. 1H NMR
(DMSO-d6, 600 MHz): δ 2.31 (m, 1H), 2.41 (m, 1H), 2.68 (m, 1H),
2.78 (m, 1H), 2.86 (m, 1H), 3.02 (m, 1H), 3.23 (m, 1H), 3.30 (m,
1H), 3.42−3.49 (m, 2H), 4.55 (bs, 2H), 6.50 (d, J = 8.95 Hz, 2H),
7.05 (d, J = 8.95 Hz, 2H), 7.08 (d, J = 7.3 Hz, 2H), 7.18−7.24 (m,
4H), 7.28−7.32 (m, 4H), 9.05 (m,1H), 9.12 (m,1H). 13C NMR
(DMSO-d6, 150 MHz): δ 37.78, 40.71, 42.17, 47.86, 49.21, 53.09,
54.15, 114.01, 119.77, 126.38, 126.69, 128.48, 128.52, 128.59, 128.80,
138.23, 139.49, 146.37. HRMS calcd for C25H28ClN2 [M + H]+,
391.1936; found, 391.1933.
1
flow, 1.4 mL/min, temperature 40 °C): 4.58 min, purity 99%. H
NMR (DMSO-d6, 600 MHz): δ 1.87 (p, J = 6.3 Hz, 2H), 2.31 (m,
1H), 2.38 (m, 1H), 2.66 (dd, J = 13.5, 8.2 Hz, 1H), 2.76 (dd, J = 13.5,
7.0 Hz, 1H), 2.86 (m, 1H), 3.0 (m, 1H), 3.21 (s, 3H), 3.22 (m, 1H),
3.28 (m, 1H), 3.38 (m, 2H), 3.41 (t, J = 6.3 Hz, 2H), 3.68 (s, 3H),
3.89 (m, 2H), 4.44 (d, J = 16.8 Hz, 1H), 4.48 (d, J = 16.8 Hz, 1H),
6.53 (d, J = 8.9 Hz, 2H), 6.55 (m, 1H), 6.71 (m, 1H), 6.82 (d, J = 8.2
Hz, 1H), 7.06 (d, J = 8.9 Hz, 2H), 7.19−7.23 (m, 3H), 7.31 (m, 2H),
8.98 (m, 1H), 9.05 (m, 1H). 13C NMR (DMSO-d6, 150 MHz): δ
28.95, 37.74, 40.70, 42.20, 47.96, 49.23, 52.98, 54.05, 55.52, 57.93,
65.25, 68.46, 111.77, 112.05, 114.29, 118.51, 119.84, 126.38, 128.51,
128.54, 128.78, 130.39, 139.49, 146.53, 147.84, 147.99. HRMS calcd
for C30H38ClN2O3 [M + H]+, 509.2566; found, 509.2563. HRMS
calcd for C30H37ClN2O3Na [M + Na]+, 531.2385; found, 531.2385.
Preparation of (rac)-9, HCl Salt. (3S*,4R*)-3-Benzyl-4-({iso-
propyl-[4-methoxy-3-(3-methoxypropoxy)-benzyl]-amino}-methyl)-
pyrrolidine-1-carboxylic Acid tert-Butyl Ester. A mixture of (rac)-19
(300 mg, 0.9 mmol) and 4-methoxy-3-(3-methoxypropoxy)-benzalde-
hyde40 (202 mg, 0.90 mmol) in 1,2-dichloroethane (5 mL) was treated
with sodium triacetoxyborohydride39 (765 mg, 3.61 mmol) with
stirring at RT under a N2 atmosphere overnight and was then poured
into an aqueous saturated solution of NaHCO3. The layers were
separated, and the water phase was extracted twice with CH2Cl2. The
combined organics were dried over Na2SO4, filtered, and concentrated.
The residue was purified by preparative HPLC (YMC-Pack C18-ODS-
AQ 5 μm, 20 mm × 50 mm; eluent, 5% CH3CN/2.5 min, 5−100%
CH3CN−H2O/10.5 min, 100% CH3CN/5 min, CH3CN and H2O
containing 0.1% HCOOH; flow, 20 mL/min). The compound
containing HPLC fractions were collected, diluted with EtOAc, and
washed with a saturated aqueous solution of NaHCO3. The organic
layer was dried over Na2SO4, filtered, and concentrated to give the title
Preparation of (rac)-8, HCl Salt. (3S*,4R*)-3-Benzyl-4-({(4-
chlorophenyl)-[4-methoxy-3-(3-methoxypropoxy)-benzyl]-amino}-
methyl)-pyrrolidine-1-carboxylic Acid tert-Butyl Ester. The title
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compound (72 mg, 15%). MS (LC−MS): 541 [M + H]+. H NMR
(DMSO-d6, 400 MHz): δ 0.90 (m, 6H), 1.32 (m, 9H), 1.89 (m, 2H),
1.99 (m, 1H), 2.08−2.22 (m, 2H), 2.31 (m, 1H), 2.44 (m, 1H), 2.75
(m, 2H), 2.85−2.94 (m, 2H), 3.08 (m, 1H), 3.20 (s, 3H), 3.35 (m,
2H), 3.39 (m, 1H), 3.42 (t, J = 6.1 Hz, 2H), 3.70 (s, 3H), 3.93 (m,
2H), 6.75 (m, 1H), 6.85 (m, 2H), 7.15 (m, 3H), 7.26 (m, 2H).
((3S*4S*)-4-Benzylpyrrolidin-3-ylmethyl)-isopropyl-[4-methoxy-
3-(3-methoxypropoxy)benzyl]-amine, (rac)-9, Hydrochloride Salt.
To a solution of (3S*,4R*)-3-benzyl-4-({isopropyl-[4-methoxy-3-(3-
methoxypropoxy)-benzyl]-amino}-methyl)-pyrrolidine-1-carboxylic
acid tert-butyl ester (72.0 mg, 0.133 mmol) in CH2Cl2 (2 mL) was
added trifluoroacetic acid (0.1 mL), and the mixture was stirred at RT
for 1 h. The mixture was then poured into a saturated aqueous
solution of NaHCO3, extracted with EtOAc, dried over Na2SO4, and
concentrated. The crude residue was purified by Isolute Flash NH2 ion
exchange chromatography (eluent: CH2Cl2−MeOH 100:0 to 90:10 +
5% of NH4OH) to give (rac)-9 as the free base (47.7 mg). This
material was dissolved in dioxane (2 mL), followed by addition of 4 M
HCl in dioxane (27 μL, 0.11 mmol) and lyophilization to afford (rac)-
9, HCl salt (48 mg, 86%). MS (LC−MS): 441 [M + H]+. tR (HPLC,
Waters Symmetry C18, 3.5 μm, 2.1 mm × 50 mm, 20−95% CH3CN−
H2O/3.5 min, 95% CH3CN/2 min, CH3CN and H2O containing 0.1%
TFA; flow, 0.6 mL/min): 2.18 min; purity 100%. tR (HPLC, MN
Nucleosil C18HD, 3 μm, 70 mm × 4 mm, 20−95% CH3CN−H2O/
3.5 min, 95% CH3CN/2 min, CH3CN and H2O containing 0.1% TFA;
flow, 0.6 mL/min): 3.57 min; purity 100%. 1H NMR (DMSO-d6, 400
MHz, 394 K): δ 1.12 (m, 6H), 1.94 (m, 2H), 2.25 (m, 2H), 2.52 (m,
1H), 2.80−2.90 (m, 2H), 3.12 (dd, J = 17.0, 10.6 Hz, 1H), 3.25 (s,
3H), 3.25 (m, 1H), 3.46 (m, 1H), 3.48 (t, J = 9.5 Hz, 2H), 3.51 (m,
1H), 3.77 (s, 3H), 3.78 (m, 2H), 4.04 (m, 2H), 6.90 (m, 1H), 6.94−
7.10 (m, 2H), 7.19 (m, 3H), 7.27 (m, 2H), 9.19 (m, 2H). HRMS calcd
for C27H41N2O3 [M + H]+, 441.3112; found, 441.3110.
(m, 5H), 3.90 (dd, J = 14.0, 8.2 Hz, 1H), 3.96 (dd, J = 14.0, 6.1 Hz,
1H), 6.69 (d, J = 2.0 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 6.82 (dd, J =
8.4, 2.0 Hz, 1H), 6.99 (d, J = 7.4 Hz, 2H), 7.11 (m, 2H), 7.17−7.22
(m, 2H), 7.25 (m, 4H), 8.94 (m, 2H). 13C NMR (DMSO-d6, 150
MHz): δ 28.7, 36.77, 41.20, 42.19, 48.07, 48.82, 50.40, 55.38, 57.39,
65.16, 68.35, 110.62, 113.53, 122.19, 126.34, 126.62, 127.61, 128.47,
128.86, 128.94, 129.24, 139.14, 142.91, 146.49, 149.96, 169.31.
HRMS: 489.27470 [M + H]+ (calcd 489.27478 for C30H36N2O4).
Preparation of (rac)-12, (3S,4S)-12a, (3R,4R)-12b, HCl Salt.
(3S*,4R*)-3-Benzyl-3-({isopropyl-[4-methoxy-3-(3-methoxypro-
poxy)-benzoyl]-amino}-methyl)-pyrrolidine-1-carboxylic Acid tert-
Butyl Ester. A mixture of (rac)-19 (600 mg, 1.80 mmol), 4-methoxy-3-
(3-methoxypropoxy)-benzoyl chloride40 (512 mg, 1.98 mmol), and
triethylamine (326 μL, 2.34 mmol) in CH2Cl2 (6 mL) was stirred at
RT overnight and then quenched by the addition of an aqueous
NaHCO3 solution. The organic layer was separated, and the aqueous
phase was extracted three times with CH2Cl2. The combined organic
extracts were dried (Na2SO4), and volatiles were removed in vacuo.
The crude product was purified by flash chromatography (eluent: c-
hexane−EtOAc 2:1) to afford the title compound (791 mg, 79%). MS
1
1
(LC-MS): 455 [M-Boc + H]+. H NMR (MeOD, 400 MHz): δ 1.12
(m, 6H), 1.45 (s, 9H), 2.03 (m, 2H), 2.32−2.43 (m, 2H), 2.62 (m,
1H), 2.84 (m, 1H), 3.09 (m, 1H), 3.23 (m, 2H), 3.25 (s, 3H), 3.46−
3.56 (m, 3H), 3.57 (m, 2H), 3.86 (s, 3H), 4.07 (m, 3H), 6.90 (m, 2H),
7.19 (m, 4H), 7.27 (m, 2H).
N-((3S*,4S*)-4-Benzylpyrrolidin-3-ylmethyl)-N-isopropyl-4-me-
thoxy-3-(3-methoxypropoxy)benzamide, (rac)-12, Hydrochloride
Salt. To a solution of (3S*,4R*)-3-benzyl-4-({isopropyl-[4-methoxy-
3-(3-methoxypropoxy)-benzoyl]-amino}-methyl)-pyrrolidine-1-car-
boxylic acid tert-butyl ester (343.4 mg, 0.619 mmol) in 2-propanol (2
mL) was added 6 M HCl in 2-propanol (1 mL), and stirring was
continued for 2 h at RT. The reaction mixture was concentrated,
diluted with CH2Cl2, and neutralized by the addition of 1 N NaOH.
The aqueous phase was extracted three times with CH2Cl2 , and the
combined organics were dried over Na2SO4, filtered, and concentrated.
The crude residue was purified by preparative HPLC (Macheney-
Nagel C18 Nucleosil-100 10 μm, 40 mm × 250 mm; eluent, 20−100%
CH3CN/10 min, 100% CH3CN/10 min, CH3CN and H2O containing
0.1% TFA; flow, 40 mL/min). The compound containing fractions
were combined and concentrated in vacuo. The residue was diluted
with CH2Cl2, neutralized by addition of a saturated aqueous solution
of NaHCO3, and extracted three times with CH2Cl2. The combined
organic extracts were dried over Na2SO4, filtered, and concentrated to
afford (rac)-12 as the free base (214 mg, 76%). 1H NMR (DMSO-d6,
400 MHz, 394 K): δ 1.07 (d, J = 6.7 Hz, 3H), 1.11 (d, J = 6.7 Hz, 3H),
1.93 (p, J = 6.3 Hz, 2H), 2.10−2.22 (m, 2H), 2.59 (m, 2H), 2.78 (m,
1H), 2.97 (dd, J = 8.0, 7.5 Hz, 1H), 3.09 (dd, J = 11.2, 7.5 Hz, 1H),
3.25 (s, 3H), 3.26 (m, 2H), 3.48 (t, J = 6.3 Hz, 2H), 3.80 (s, 3H), 3.96
(m, 1H), 4.02 (t, J = 6.3 Hz, 2H), 6.86 (m, 2H), 6.96 (m, 1H), 7.18
(m, 3H), 7.27 (m, 2H).
To a solution of (rac)-12, free base, in dioxane was added 4N HCl
in dioxane (117 μL, 0.470 mmol) followed by lyophilization to afford
the (rac)-12, HCl salt. MS (LC−MS): 455 [M + H]+. tR (HPLC,
Waters Symmetry C18, 3.5 μm, 2.1 mm × 50 mm, 20−95% CH3CN−
H2O/3.5 min, 95% CH3CN/2 min, CH3CN and H2O containing 0.1%
TFA; flow, 0.6 mL/min): 2.75 min, purity 96%. tR (HPLC, MN
Nucleosil C18HD, 3 μm, 70 mm × 4 mm, 20−95% CH3CN−H2O/
3.5 min, 95% CH3CN/2 min, CH3CN and H2O containing 0.1% TFA;
flow, 0.6 mL/min): 4.15 min, purity 96.5%. 1H NMR (DMSO-d6, 600
MHz): δ 0.96 (m, 3H), 1.04 (m, 3H), 1.92 (m, 2H), 2.34 (m, 2H),
2.64 (m, 1H), 2.88 (m, 2H), 3.04 (m, 1H), 3.16 (m, 1H), 3.22 (s, 3H),
3.32 (m, 2H), 3.37 (m, 1H), 3.44 (m, 2H), 3.77 (s, 3H), 3.90 (m, 1H),
3.98 (m, 2H), 6.86 (m, 2H), 6.98 (m, 1H), 7.22 (m, 1H), 7.25 (m,
2H), 7.31 (m, 2H), 8.95 (m, 2H). HRMS calcd for C27H39N2O4,
455.2904 [M + H]+; found, 455.2904.
Preparation of (rac)-10, HCl Salt. N-((3S*,4S*)-4-Benzylpyrro-
lidin-3-ylmethyl)-N-(4-chlorophenyl)-4-methoxy-3-(3-
methoxypropoxy)benzamide, (rac)-10. The title compound was
prepared as described for (rac)-12 in two steps from (rac)-17. MS
(LC−MS): 523.4 [M + H]+. tR (HPLC, Waters Symmetry C18, 3.5
μm, 2.1 mm × 50 mm, 20−95% CH3CN/H2O/3.5 min, 95%
CH3CN/2 min, CH3CN and H2O containing 0.1% TFA; flow, 0.6
mL/min): 3.01 min; purity 97%. tR (Waters XBridge C18, 2.5 μm, 3
mm × 50 mm, 10−98% CH3CN−H2O/8.6 min, 98% CH3CN/1.4
min, CH3CN and H2O containing 0.1% TFA; flow, 1.4 mL/min,
1
temperature 40 °C): 3.96 min, purity 96%. H NMR (DMSO-d6, 600
MHz): δ 1.78 (m, 2H), 2.09 (m, 1H), 2.32 (m, 1H), 2.59 (dd, J = 13.3,
7.9 Hz, 1H), 2.69 (dd, J = 13.3, 6.6 Hz, 1H), 2.85 (m, 1H), 3.0 (m,
1H), 3.22 (m, 1H), 3.23 (s, 3H), 3.30−3.39 (m, 3H), 3.69 (s, 3H),
3.73 (m, 2H), 3.90 (bd, J = 7.0 Hz, 2H), 6.72 (m, 1H), 6.80 (m, 2H),
7.0 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 7.3 Hz, 2H), 7.21 (m, 1H), 7.25
(m, 2H), 7.30 (d, J = 8.4 Hz, 2H), 8.91 (m, 2H). 13C NMR (DMSO-
d6, 150 MHz): δ 28.68, 36.73, 39.98, 41.03, 42.11, 48.05, 48.87, 50.26,
55.41, 57.95, 65.23, 68.34, 110.71, 113.57, 122.22, 126.38, 127.33,
128.47, 128.86, 129.16, 129.36, 130.94, 139.11, 141.80, 146.60, 150.12,
169.30. HRMS calcd for C30H36ClN2O4 [M + H]+, 523.2358; found,
523.2358.
Preparation of (rac)-11, HCl Salt. N-((3S*,4S*)-4-Benzylpyrro-
lidin-3-ylmethyl)-N-phenyl-4-methoxy-3-(3-methoxypropoxy)-ben-
zamide, (rac)-11, Hydrochloride Salt. The title compound was
prepared as described for (rac)-12 in two steps from (rac)-18
(prepared as described for (rac)-17 using aniline instead of 4-
chloroaniline). MS (LC−MS): 489 [M + H]+. tR (Waters XBridge
C18, 2.5 μm, 3 mm × 30 mm, 10−98% CH3CN/H2O/3 min, 98%
CH3CN/0.5 min, CH3CN and H2O containing 0.1% TFA; flow, 1.4
mL/min, temperature 40 °C): 1.81 min, purity 100%. tR (Waters
XBridge C18, 2.5 μm, 3 mm × 50 mm, 10−98% CH3CN−H2O/8.6
min, 98% CH3CN/1.4 min, CH3CN and H2O containing 0.1% TFA;
1
flow, 1.4 mL/min; temperature 40 °C): 3.57 min, purity 100%. H
N-((3S,4S)-4-Benzylpyrrolidin-3-ylmethyl)-N-isopropyl-4-me-
thoxy-3-(3-methoxypropoxy)benzamide, (3S,4S)-12a. The title
compound was prepared in two steps as described for (rac)-12 from
(3S,4R)-19a. tR (Waters XBridge C18, 2.5 μm, 3 mm × 30 mm, 10−
98% CH3CN/H2O/3 min, 98% CH3CN/0.5 min, CH3CN and H2O
NMR (DMSO-d6, 600 MHz): δ 1.77 (m, 2H), 2.12 (m, 1H), 2.34 (m,
1H), 2.60 (dd, J = 13.55, 8.1 Hz, 1H), 2.70 (dd, J = 13.55, 6.6 Hz, 1H),
2.84 (dd, J = 11.5, 7.6 Hz, 1H), 3.0 (dd, J = 11.9, 7.6 Hz, 1H), 3.21
(dd, J = 11.5, 7.4 Hz, 1H), 3.23 (s, 3H), 3.37 (t, J = 6.2 Hz, 3H), 3.68
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containing 0.1% TFA; flow, 1.4 mL/min; temperature 40 °C): 1.78
min, purity 99%. tR (Waters XBridge C18, 2.5 μm, 3 mm × 50 mm,
10−98% CH3CN−H2O/8.6 min, 98% CH3CN/1.4 min, CH3CN and
H2O containing 0.1% TFA; flow, 1.4 mL/min; temperature 40 °C):
(2) Scott, B. B.; McGeehan, G. M.; Harrison, R. R. Development of
inhibitors of the aspartyl protease renin for the treatment of
hypertension. Curr. Protein Pept. Sci. 2006, 7, 241−254.
(3) Nicholls, M. G.; Robertson, J. I. S.; Inagami, T. The renin−
angiotensin system in the twenty-first century. Blood Pressure 2001, 10,
327−343.
20
3.50 min, purity 99%. [α]D −11.1 (c = 1.15, MeOH). HRMS calcd
for C27H39N2O4 [M + H]+, 455.2904; found, 455.2905.
N-((3R,4R)-4-Benzylpyrrolidin-3-ylmethyl)-N-isopropyl-4-me-
thoxy-3-(3-methoxypropoxy)benzamide, (3R,4R)-12b. The title
compound was prepared in two steps as described for (rac)-12 from
(3R,4S)-19b. tR (Waters XBridge C18, 2.5 μm, 3 mm × 30 mm, 10−
98% CH3CN/H2O/3 min, 98% CH3CN/0.5 min, CH3CN and H2O
containing 0.1% TFA; flow, 1.4 mL/min; temperature 40 °C): 1.78
min, purity 99%. tR (Waters XBridge C18, 2.5 μm, 3 mm × 50 mm,
10−98% CH3CN−H2O/8.6 min, 98% CH3CN/1.4 min, CH3CN and
H2O containing 0.1% TFA; flow, 1.4 mL/min; temperature 40 °C):
3.50 min, purity 100%. [α]D20 +10.6 (c = 0.95, MeOH). HRMS calcd
for C27H39N2O4 [M + H]+, 455.2904; found, 455.2904.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Description of the 3D pharmacophore searching protocol,
experimental procedures for biological assays, in vivo
pharmacokinetics, in vivo pharmacology, X-ray crystallographic
information for the rh-renin−inhibitor complexes with 5, 6a,
and 12a, X-ray crystal structure of two ligands trans-3,4-
pyrrolidine 6a bound to the active site of glycosylated rh-renin.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
The crystal structures of rh-renin in complex with 5, 6a, and
12a have been deposited at the Protein Data Bank RSCB PDB
with the PDB IDs 4GJ5, 4GJ6, and 4GJ7, respectively.
AUTHOR INFORMATION
Corresponding Author
■
*For E.L.: phone, +41-61-6961955; fax, +41-61-6967155; E-
mail, edwige.lorthiois@novartis.com. For J.M.: phone, +41-61-
6965560; fax, +41-61-6967155; E-mail, juergen_klaus.
maibaum@novartis.com.
(15) Webb, R. L.; Schiering, N.; Sedrani, R.; Maibaum, J. Direct
Renin Inhibitors as A New Therapy for Hypertension. J. Med. Chem.
2010, 53, 7490−7520.
Present Address
⊥Novartis Institutes for BioMedical Research, Horsham, West
Sussex RH12 5AB, UK.
(16) Tice, C. M. and Singh S. B. Evolution of Diverse Classes of
Renin Inhibitors through the Years. In Aspartic Acid Proteases as
Therapeutic Targets, Ghosh, A. K., Ed.; Methods and Principles in
Medicinal Chemistry, Mannhold, R., Kubinyi, H., Folkers, G., Eds.;
Wiley-VCH: Weinheim, Germany, 2010, Vol. 45, pp 297−324.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(17) (a) Wood, J. M.; Maibaum, J.; Rahuel, J.; Grutter, M. G.; Cohen,
̈
We thank Anne Rogemont, Jean-Francois Bulber, and Florence
Zink for excellent technical assistance and Hans-Peter Baum
and Markus Sanner for IC50 determinations. We are grateful to
Gabrielle Lecis and Claude Hager for chiral separations and to
N.-C.; Rasetti, V.; Rueger, 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.; O’Brien, E.; Stanton,
A.; Bedigian, M. P. Structure-based design of aliskiren, a novel orally
effective renin inhibitor. Biochem. Biophys. Res. Commun. 2003, 308,
Markus Kromer for X-ray data depositions.
̈
698−705. (b) Maibaum, J.; Stutz, S.; Goschke, R.; Rigollier, P.;
̈
ABBREVIATIONS USED
Yamaguchi, Y.; Cumin, F.; Rahuel, J.; Baum, H.-P.; Cohen, N.-C.;
■
Schnell, C. R.; Fuhrer, W.; Grutter, M. G.; Schilling, W.; Wood, J. M. J.
̈
AngI, angiotensin I; AngII, angiotensin II; AT1 receptor,
angiotensin I receptor; DRI, direct renin inhibitor; FRET,
fluorescence resonance energy transfer; GRAB, group replace-
ment assisted binding; HE, hydroxyethylene; RAAS, renin−
angiotensin−aldosterone system; rh-renin, recombinant human
renin; S3sp, S3 subpocket; dTGR, double transgenic rats
Med. Chem. 2007, 50, 4832−4844.
(18) Maibaum, J.; Feldman, D. L. Case history on Tekturna/Rasilez
(aliskiren), a highly efficacious direct oral renin inhibitor as a new
therapy for hypertension. Annu. Rev. Med. Chem. 2009, 44, 105−127.
(19) Bursavich, M. G.; Rich, D. H. Designing non-peptide
peptidomimetics in the 21st century: inhibitors targeting conforma-
tional ensembles. J. Med. Chem. 2002, 45, 541−558.
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