A novel class of oral direct renin inhibitors: Highly potent 3,5-disubstituted piperidines bearing a tricyclic P3 - P1 pharmacophore

Article abstract of DOI:10.1021/jm301706j

A small library of fragments comprising putative recognition motifs for the catalytic dyad of aspartic proteases was generated by in silico similarity searches within the corporate compound deck based on rh-renin active site docking and scoring filters. Subsequent screening by NMR identified the low-affinity hits 3 and 4 as competitive active site binders, which could be shown by X-ray crystallography to bind to the hydrophobic S₃-S ₁ pocket of rh-renin. As part of a parallel multiple hit-finding approach, the 3,5-disubstituted piperidine (rac)-5 was discovered by HTS using a enzymatic assay. X-ray crystallography demonstrated the eutomer (3S,5R)-5 to be a peptidomimetic inhibitor binding to a nonsubstrate topography of the rh-renin prime site. The design of the potent and selective (3S,5R)-12 bearing a P ₃^sp-tethered tricyclic P₃-P₁ pharmacophore derived from 3 is described. (3S,5R)-12 showed oral bioavailability in rats and demonstrated blood pressure lowering activity in the double-transgenic rat model.

Full text of DOI:10.1021/jm301706j

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Article
A Novel Class of Oral Direct Renin Inhibitors: Highly Potent 3,5-
Disubstituted Piperidines bearing a Tricyclic P3–P1 Pharmacophore
Nils Ostermann, Simon Ruedisser, Claus Ehrhardt, Werner Breitenstein, Andreas L.
Marzinzik, Edgar Jacoby, Eric Vangrevelinghe, Johannes Ottl, Martin Klumpp, Constanze
Hartwieg, Frederic Cumin, Ulrich Hassiepen, Jörg Trappe, Richard C Sedrani, Sabine Geisse,
Bernd Gerhartz, Paul Richert, Eric Francotte, Trixie Wagner, Markus Krömer, Takatoshi
Kosaka, Randy L Webb, Dean F Rigel, Juergen Klaus Maibaum, and Daniel K Baeschlin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301706j • Publication Date (Web): 29 Jan 2013
Downloaded from http://pubs.acs.org on February 20, 2013
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Institutes for BioMedical Research
Webb, Randy; Novartis Pharmaceuticals Corp., Novartis Institutes for
BioMedical Research
Rigel, Dean; Novartis Pharmaceuticals Corp., Novartis Institutes for
BioMedical Research
Maibaum, Juergen; Novartis Pharma AG, Novartis Institutes for BioMedical
Research
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Baeschlin, Daniel; Novartis Pharma AG, Novartis Institutes for BioMedical
Research
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NH2
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OH
MeO
O
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O
OMe
O
Aliskiren (1): IC50 (plasma renin) = 0.6 nM
2: IC50 (plasma renin) = 8.9 nM
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(rac)-4
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(
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OH
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FBS-hit
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(
rac)-3
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SBDD
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O O
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Lead compound (3R,5S)-12
Renin, IC50 = 3 nM
N
H
NH
HTS-hit
rac)-5
(
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HO C
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a - d
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(3S,5R)-11
R
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(
2 4
3S,5R)-12: R = (CH ) OCH3
Boc
(3R,5S)-12: R = (CH ) OCH
2
4
3
HO C
N
2
H
(3R,5S)-11
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NH2
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CO CH3
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e -h
i, j
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O
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A Novel Class of Oral Direct Renin Inhibitors:
Highly Potent 3,5ꢀDisubstituted Piperidines bearing
a Tricyclic P –P Pharmacophore
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a
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a
a
Nils Ostermann, Simon Ruedisser, Claus Ehrhardt, Werner Breitenstein, Andreas Marzinzik,
a
a
a
a
Edgar Jacoby, Eric Vangrevelinghe, Johannes Ottl, Martin Klumpp, J. Constanze D.
a
a
a
a
a
Hartwieg, Frederic Cumin, Ulrich Hassiepen, Jörg Trappe, Richard Sedrani, Sabine
a
a
a
a
a
a
Geisse, Bernd Gerhartz, Paul Richert, Eric Francotte, Trixie Wagner, Markus Krömer,
b
c
c
a
Takatoshi Kosaka, Randy L. Webb, Dean F. Rigel, Jürgen Maibaum, and Daniel K.
a
Baeschlin*
a
Novartis Pharma AG, Institutes for BioMedical Research, Novartis Campus, CHꢀ4056 Basel,
b
Switzerland, Novartis Institutes for BioMedical Research, Horsham, West Sussex RH12 5AB,
c
UK. Novartis Pharmaceuticals Corp., Novartis Institutes for BioMedical Research, East
Hanover, New Jersey
KEYWORDS. Renin inhibitor, fragment based screening, 3,5ꢀdisubstituted piperidines
ABSTRACT. A small library of fragments comprising putative recognition motifs for the
catalytic dyad of aspartic proteases was generated by in silico similarity searches within the
corporate compound deck based on rhꢀrenin active site docking and scoring filters. Subsequent
screening by NMR identified the lowꢀaffinity hits 3 and 4 as competitive active site binders,
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which could be shown by Xꢀray crystallography to bind to the hydrophobic S ꢀS pocket of rhꢀ
3
1
renin. As part of a parallel multiple hit–finding approach, the 3,5ꢀdisubstituted piperidine (rac)-5
was discovered by HTS using a enzymatic assay. Xꢀray crystallography demonstrated the
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eutomer (3S,5R)-5 to be a peptidomimetic inhibitor binding to a nonsubstrate topography of the
sp
rhꢀrenin prime site. The design of the potent and selective (3S,5R)-12 bearing a P ꢀtethered
3
tricyclic P –P pharmacophore derived from 3 is described. (3S,5R)-12 showed oral
3
1
bioavailability in rats and demonstrated blood pressure lowering activity in the doubleꢀtransgenic
rat model.
Hypertension, the chronic elevation of systemic blood pressure, is a major health risk in the
Western world with approximately 29% of the adult population worldwide projected to develop
1
this condition by 2025. High blood pressure increases the risk of stroke, heart disease, and
2
kidney failure causing approximately 277,000 deaths in the US in 2002 alone. In a majority of
hypertension patients, blood pressure is still controlled inadequately or not at all despite the
existence of a variety of drug treatments and as a consequence, a high unmet medical need for
more efficient drug therapy remains. The renin–angiotensin–aldosterone system (RAAS) plays a
central role in the regulation of blood pressure as revealed by multiple preclinical and clinical
studies employing angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor (AT)
3
antagonists and direct renin inhibitors (DRIs). The first step in this regulatory cascade is the
cleavage of angiotensinogen as the only known substrate for the aspartic protease renin to
generate the decapeptide angiotensin I. Subsequent cleavage by ACE produces the octapeptide
angiotensin II, which is the principal mediator of RAAS and which exerts its major effects
through the AT and AT receptors. Blockade of the RAAS at its source by direct inhibition of
1
2
the first and rateꢀlimiting step involving renin, has been recognized for many years as an
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attractive approach to treat hypertension. Hence, DRIs have been considered to provide
potential clinical benefits over other antihypertensive drugs interfering with the RAAS, such as
5
ACEi and AT receptor antagonists. Major efforts and substantial progress made in the design
1
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of potent and selective nonꢀpeptide DRIs have culminated in the discovery of the firstꢀinꢀclass
orally active 1 (aliskiren, Rasilez, Tekturna; Figure 1) now approved for the once daily treatment
6
ꢀ8
for hypertension.
H
N
O
OH
H
O
O
OMe
H N
N
NH2
2
OH
MeO
O
O
O
O
O
OMe
O
Aliskiren (1): IC50 (plasma renin) = 0.6 nM
2: IC50 (plasma renin) = 8.9 nM
Figure 1. Potent nonꢀpeptide direct renin inhibitors.
Several diverse classes of nonꢀpeptide DRIs have evolved through recent years, as the
topographical space of the renin active site targetable for inhibitor design has significantly
8,9
expanded. The hydroxyethylene dipeptide isostere analogue 1, designed based on a S –S
3
1
topological concept, emulates the extended βꢀstrand binding active site conformation of renin
sp
10
and, in addition, tightly interacts with the nonsubstrate S3 pocket. The class of 3,4ꢀ
1
1
disubstituted piperidine GRAB peptidomimetics (cf. 2, Figure 1), first reported in 1999, bind to
a fundamentally distinct active site topography characterized by a newly formed hydrophobic
pocket resulting from major concerted movements of the βꢀhairpin loop to an “open flap” ligandꢀ
1
2
binding conformation. The center piperidine interacts as a basic transitionꢀstate surrogate with
the catalytic Asp and Asp by forming favorable chargeꢀenforced Hꢀbonds. A second
3
2
215
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generation GRAB peptidomimetic bearing a 3,9ꢀdiazabicyclo[3.3.1]nonene center scaffold has
1
3
undergone clinical trials. Furthermore, the 2,4ꢀdiaminopyrimidine and most recently the
8
,9,14
alkylamine class of DRIs have attracted attention.
Our continued interest in the discovery of
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a “bestꢀinꢀclass” DRI prompted a broad screening program toward new druglike leads by
applying multiple approaches in parallel. These included highꢀthroughput screening (HTS) using
an enzymatic and a competitive ligand binding assay, a combined NMR/Xꢀray fragmentꢀbased
screen of focused libraries, as well as in silico 3D pharmacophore searches. These integrated
efforts turned out to be remarkably complementary in identifying attractive diverse chemotype
15
hits from different sources. We report herein the discovery of 3,5ꢀdisubstituted piperidines
bearing a tricyclic P3–P1 unit as a novel and potent direct renin inhibitor class through the
combination of structural features of a HTS hit and a fragment binding weakly to renin identified
by a NMR/Xꢀray fragment based screen.
OH
OH
N
N
N
S
H
H
(
rac)-3
(rac)-4
N
O O
S
3
5
O
N
H
NH
(
rac)-5
Figure 2. Fragmentꢀbased screening hits (rac)-3 and (trans,rac)-4, and HTS hit (rac)ꢀ5.
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5
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6
We designed a small fragment screening library intended to target specifically the family of
aspartic proteases and comprising “privileged” recognition motifs related to transitionꢀstate
mimetics, such as amino alcohols, as well as basic 5ꢀ and 6ꢀmembered cycloalkylamine
transitionꢀstate (TS) surrogates inspired by the structures of known aspartic protease inhibitors.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
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9
0
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9
0
16
2D and 3D in silico database mining tools were applied for substructure and similarity searches
within the Novartis deck of physically available compounds. To further reduce the fragment
1
7
library to a size compatible with NMR screening capacity, docking and scoring using GOLD
1
8
and subsequent rescoring analyses by Cscore were performed. A total of 113 compounds were
finally selected and initially screened by NMR for binding to rhꢀrenin, using ligand observation
techniques such as T1ρ relaxation experiments. Several members of the biased fragment library,
including (rac)-3 and (trans,rac)-4 (Figure 2; NMR K >500 ꢁM; IC >100 ꢁM in the FRET
d
50
biochemical assay), were found to have very weak binding affinities to rhꢀrenin and, in addition,
1
9
to be active site ligands as determined by NMR competition experiments in the presence of 1.
Cocrystallization as well as soaking experiments using different crystal forms of recombinant
1
9
human (rh) apoꢀrenin, were subsequently performed for more than twenty fragment hits
validated by NMR as lowꢀaffinity binders. We were very gratified that crystal structures of two
of these fragments, i.e., the competitive active site binders (rac)-3 and (trans,rac)-4, in complex
with rhꢀrenin could be solved thereby elucidating their unexpected binding mode to the nonprime
active site of the enzyme (Figure 3). Both the hydrophobic 9Hꢀthioxanthene moiety of 3 and the
5
Hꢀdibenzoazepine scaffold of 4 were found to be in close van der Waals contact and to
completely fill the open contiguous hydrophobic S –S binding site of human renin with the flap
3
1
βꢀhairpin (residues Tyr , Ser , Thr and Gly ) in a closed conformation. The adopted bent
7
5
76
77
78
“butterfly” conformations of the heteroaryl portions of 3 and 4 allow to bridge both hydrophobic
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phenyl moieties accommodating a position considerably deeper inside the S and S pockets,
3
1
respectively, compared to the P and P binding motifs of known peptide peptidomimetic renin
3
1
1
0
inhibitors as well as the topological transitionꢀstate mimetic 1 (Figure 3b). As a consequence of
the binding poses of the tricylic scaffolds of 3 and 4 within the S –S cavity, both the
10
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3
1
hydroxylated side chains and the nitrogen atoms of the pyrrolidine moieties of the ligands were
in remote positions relative to the Asp and Asp carboxylates. The amino alcohol side chain
3
2
215
of 3, adopting an axial conformation in the crystal structure obtained by using the racemate for
soaking, exhibited different binding orientations for the two rhꢀrenin molecules in the
asymmetric unit of the crystal which have been fitted with the Rꢀ and Sꢀenantiomers and were
located in large distance from the enzyme catalytic dyad (Figure 3a). This was surprising to us in
view of the targetꢀbiased fragment library design based on the query for privileged transitionꢀ
state surrogate motifs for aspartic proteases. The observed binding mode suggests to us that the
binding of 3 and 4 is mainly entropy–driven by hydrophobic interactions to the hydrophobic S3–
S1 cavity of renin. This specific binding site is now well recognized as a “hydrophobic hot spot”
8ꢀ13
and a key topography to target for the design of potent direct renin inhibitors.
sp
Most strikingly, the 2ꢀhydroxypropyl side chain of 4 extended into the nonsubstrate S3 cavity
by extruding the ordered water of apoꢀrenin and forming a Hꢀbond to the carbonyl of Thr216
located close to the bottom of this distinct rigid channel (Figure 3b). To our knowledge, a Hꢀ
bonding opportunity involving Thr216 has not been observed previously for other chemotype
8
renin inhibitors. It should be noted that (trans,rac)-4 was build with the (R)ꢀconfiguration of the
hydroxyl group. A fit with the (S)ꢀconfiguration of the hydroxyl group would be possible and
would result in an Hꢀbond between the hydroxyl group of 4 and the NH of the Tyr backbone.
1
4
As a result, fragment screening by NMR and Xꢀray has allowed two key design principles to be
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1
1
1
1
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1
1
1
1
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2
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2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
sp
“
rediscovered”, i.e., targeting of the S –S hydrophobic hot spot and exploitation of the S
1
3
3
binding site of human renin: both principles have been critical for the conception of the
10b
topological transitionꢀstate mimetic inhibitor 1, and have been subsequently applied to the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
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7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
8ꢀ13
design of other nonꢀpeptide classes of DRIs. In general, this work represents an impressive
illustration of the power of biophysical screening approaches for active site mapping by lowꢀ
affinity ligands of renin as a member of the aspartic protease family.
In particular 3 gained our attention as an attractive scaffold for further optimization by a
fragment growing approach. We sought to expand the heteroaryl scaffold by targeting binding
interactions to the catalytic Asp and/or Asp as well as the prime site specificity pockets of
3
2
215
human renin in the quest to substantially gain binding affinity by several orders of magnitude. In
the course of our initial exploratory work on the structure–activity relationship (SAR) for 3, a
highꢀthroughput biochemical screen of the Novartis compound deck using a miniaturized
fluorescence–polarization assay identified the racemic cisꢀconfigured 3,5ꢀdisubstituted piperidine
5
(IC of 1.2 ꢁM, Figure 2, Table 1) as a novel chemotype renin inhibitor. (Rac)-5 originated
50
from a split–and–mix library comprising some 950 compounds which had been specifically
2
0
designed to target aspartic proteases. The general library concept had been based on the
8
,12
hypothesis that the positively charged nitrogen of the piperidine “privileged” scaffold could
interact with the enzyme catalytic dyad, and that the NH of the (sulfon)amide residues are
potentially involved in Hꢀbonds with backbone atoms of the flap region. The crystal structure of
rhꢀrenin in complex with (rac)-5 indicated that only the (3S,5R)ꢀenantiomer was bound in the
2
1
active site (Figure 3a). The basic 3,5ꢀdisubstituted center piperidine was indeed confirmed to
function as a transitionꢀstate surrogate with the nitrogen atom being placed symmetrically
between Asp and Asp to form a charge–charge interaction. Inhibitor (3S,5R)ꢀ5 emulates the
3
2
215
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5
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7
8
9
extended substrate βꢀstrand binding topography of the renin active site by spanning the nonprime
S –S recognition sites and the prime region with the flap βꢀhairpin in a closed conformation.
3
1
This is in contrast to the class of 3,4ꢀdisubstituted piperidine GRAB peptidomimetics, such as 2,
10
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1
2
binding to a very distinct “open flap” topography of human renin. The carboxamide at the
piperidine C3 of 5 forms Hꢀbonds to Gly217 and Ser of the flap βꢀhairpin, whereas the
76
sulfonamide group is involved in two water–mediated Hꢀbonds with the NH in close distance to
the flap Arg and one sulfonyl oxygen atom interacting with Gly of the prime site. The
7
4
34
diphenylmethane pharmacophore of (3S,5R)ꢀ5 resides in the large S –S cavity in a very similar
1
3
fashion as observed for (rac)-3 and (rac)-4 (Figure 3).
Figure 3. (a) Crystal structures of the (S)ꢀconfigured 9Hꢀthioxanthene 3 (shown as stick model
in green color) and (R)ꢀ3 (orange color), both superimposed with the 3,5ꢀpiperidine inhibitor
19
(
3S,5R)ꢀ5 (grayish color) bound to rhꢀrenin. Shown is the solvent accessible surface of the
active site of renin. Residues of the flap βꢀhairpin (residues Tyr , Ser , Thr and Gly ) in a
7
5
76
77
78
closed conformation are depicted for the complex of renin with (3S,5R)ꢀ5 in yellow as thin lines
and have been omitted from the protein surface calculation. The oxygen and nitrogen atoms of
the ligands are colored in red and blue, respectively. (b) Superposition of the crystal structure of
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1
1
1
1
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1
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1
1
1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the 5Hꢀdibenzazepine 4 (shown as stick model in green color) and the topological transitionꢀstate
mimetic 1 (orange color; PDB code 2v0z) in complex with rhꢀrenin. The crystal structure
obtained with (trans,rac)-4 was build with the (R)ꢀconfiguration of the hydroxyl group in 4 (as
shown). Residues of the flap βꢀhairpin (residues Tyr , Ser , Thr and Gly ) in a closed
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
5
76
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78
conformation are depicted for the complex of rhꢀrenin with 4 in yellow as thin lines and have
been omitted from the protein surface calculation.
A particularly intriguing feature of this new class of topological peptidomimetic inhibitors is the
unprecedented binding mode to the prime site of rhꢀrenin. First, the sulfonamide oxygens of 5
were found to bind into the S ’ pocket, which is considered to preferentially accommodate
1
2
2
10
hydrophobic residues. Superposition with the Xꢀray structure of 1 revealed a close overlap of
both sulfonyl oxygens with the S ’ isopropyl group of the hydroxyethylene dipeptide isostere (cf.
1
Figure 4b for 9). The dual character of weakly polar sulfonyl groups as a Hꢀbond acceptor and as
a hydrophobic group capable to form van der Waals contacts with nonpolar atoms in a
2
3
hydrophobic environment has been discussed recently for ligand–protein interactions.
Secondly, the geometry of the sulfonamide spacer positions the tolyl residue between the
prolineꢀrich loop (Met –Thr ) and the flap region (Thr –Ser ), thereby leaving the S ’ site
2
89
298
72
81
2
unoccupied. This nonsubstrate binding topography at the prime site of the renin catalytic cleft,
which further extends toward solvent space, is primarily the result of a slight conformational
movement of the proline loop, which has not been recognized previously to the best of our
knowledge.
The Xꢀray structural information gained for the polycyclic heteroaryl ligands 3 and 4 and the
piperidine (3S,5R)ꢀ5 suggested to us several avenues for further optimization by merging key
structural features. The rigid tricyclic framework tightly binding into the hydrophobic S –S1
3
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5
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9
cavity was considered to offer some advantage over the more flexible diphenylmethane moiety.
More importantly, the Xꢀray data provided reasonable evidence for the three–atom carboxamide
linker of 5 to be also suitable to connect the tricyclic portion of 3 to a TS surrogate targeting the
catalytic dyad of the enzyme active site. In order to probe the concept, the readily accessible 9Hꢀ
xanthene derivative (rac)-6 replacing the 9Hꢀthioxanthene heterocycle of (rac)-1 was initially
synthesized (Scheme 1). In brief, the commercial carboxylic acid 7 was transformed in 3 steps to
the primary amine 8, followed by PyBOP–mediated coupling to (rac)-1ꢀBocꢀpiperidineꢀ3ꢀ
carboxylic acid and final Nꢀdebocylation to afford (rac)-6 in 13% yield. Compound 6 was found
to be completely inactive toward rhꢀrenin in the FRET biochemical assay at the highest
concentration tested (100 ꢁM; Table 1). However, we were much intrigued by the fact that the
crystal structure of 6 in complex with rhꢀrenin could be solved (Figure 4a), despite the very low
apparent binding affinity of the ligand. As was expected by modeling, the observed binding
mode of 6 was very similar to the interactions of the corresponding substructures of rhꢀrenin–
bound (3S,5R)ꢀ5. The tricyclic scaffolds of both ligands accommodated by the S –S cavity
10
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3
1
closely overlapped, apart from minor geometrical differences due to the nature of the ring
heteroatoms, as was the case for the center piperidines with the basic nitrogen atoms in the
pivotal position between the catalytic aspartates. Also, the geometry and lengths of the Hꢀbonds
exerted by the carboxamide spacer of 6 to Gly217 and Ser of the flap βꢀhairpin were comparable
76
to those observed for 5. The Xꢀray data demonstrated to us that it is indeed possible to grow from
the S –S fragment toward the catalytic Asp and Asp by the proper choice of a linear spacer
3
1
32
215
group and connecting to a transitionꢀstate analog, albeit this had not resulted in a significant
improvement in the in vitro potency for 6.
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9
1
1
1
1
1
1
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1
1
1
2
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H
N
O
O
O
OH
NH2
NH
a - c
d, e
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
7
8
(rac)-6
Scheme 1. Synthesis of (rac)-6: (a) EEDQ, benzylamine, CH Cl , RT, 40%; (b) LiAlH , AlCl ,
2
2
4
3
THF, 0°C to 50°C, 72%; (c) H , 10% Pd/C, EtOH, RT, 99%; (d) 1ꢀBocꢀpiperidineꢀ3ꢀcarboxylic
2
acid, PyBOP, Et N, CH CN, RT, 79%; (e) 4 M HCl–dioxane, RT, 56%.
3
3
H
N
Fmoc
N
O O
S
O
O
N
a, b
c - f
N
O
O
H
NH
HO C
N
H
O
HO C
N
O
2
2
H
(
rac)-9
1
0
(rac)-11
Scheme 2. Synthesis of (rac)-9: (a) Nishimura catalyst, 25% aq. NH OH–H O, RT, 24 h ,
4
2
4
0%; (b) Fmocꢀsuccinimide, THF–H O 1:1, NaHCO , 88%; (c) 4M HCl–dioxane, RT, 96%; (d)
2 3
tolueneꢀ4ꢀsulfonyl chloride, aq. K CO , dioxane, 0°C, 54%; (e) 8, HCTU, DIPEA, CH Cl –
2
3
2
2
CH CN 1:1, 0°C; (f) CH Cl –piperidine 4:1, RT, 5% (two steps).
3
2
2
Next, we envisaged the introduction of the sulfonamide side chain to the piperidine C3 of (rac)-6
as suggested by the structural overlay of renin–bound 5 and 6 (overlay not shown). Accordingly,
(rac)-9 was prepared starting from commercial 5ꢀBocꢀaminoꢀnicotinic acid 10 (Scheme 2) via
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9
hydrogenation, subsequent NꢀFmoc protection and precipitation from water to provide the
versatile building block (rac,cis)ꢀ11 in 35% yield. NꢀBoc deprotection followed by sulfonamide
formation and coupling with amine 8 afforded Fmoc protected 9. The amide coupling reaction
and the final Fmoc deprotection step proceeded to give (rac)-9, although in only 5% overall
yield. Notably, (rac)ꢀ9 demonstrated to be a potent inhibitor of rhꢀrenin (IC of 0.09 ꢁM) in the
10
11
12
13
14
15
16
17
18
19
20
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44
45
46
47
48
49
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55
56
57
58
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60
50
FRET assay (Table 1).
Figure 4. Crystal structures of rhꢀrenin in complex with (a) compound 6 (shown as stick model
in green color); (b) inhibitor 9 (stick model in green color) superimposed with 1 (orange color;
PDB code 2v0z); (c) compound 12 (as stick model in green). Shown is the solvent accessible
surface of the active site of rhꢀrenin. Residues of the flap βꢀhairpin are only depicted for the rhꢀ
renin–9 complex in grayish as thin lines and have been omitted for protein surface calculation.
The catalytic aspartates are shown as stick model. Oxygen, nitrogen and sulfur atoms are shown
in red, blue and yellow color, respectively. The ligand electron density of crystal structures
obtained with (rac)-6, 9 and 12 was better compatible with the piperidine ring in the 3S, 5R
configuration and was therefore build as 3S for 6 and 3S, 5R for 9 and 12.
The crystal structure obtained by soaking of (rac)ꢀ9 into rhꢀrenin crystals confirmed the expected
binding pose (Figure 4b) and revealed a perfect overlap with the tricyclic scaffold of 6 on one
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hand, by filling the S –S cavity (Figure 4a), as well as with the tolylsulfonamide portion of
3
1
(3S,5R)ꢀ5, interacting to the prime site (Figure 3a). Also, both phenyl residues of the
diphenylmethyl moiety of renin–bound (3S,5R)ꢀ5 were in almost identical positions when
compared to the tricycle of (rac)ꢀ9, with only a very minor difference in the planar orientations
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
observed for the respective P phenyl groups. The 13ꢀfold potency increase for (rac)ꢀ9 suggested
1
the conformationally rigid P –P scaffold to be superior as compared to the more flexible
1
3
diphenylmethyl pharmacophore.
Fmoc
N
H
a - d
N
H
O O
S
N
O
O
O O
Boc
Boc
N
HO C
N
2
H
O
O
S
H
NH
N
H
(3S,5R)-11
R
NH
R
Fmoc
N
a - d
(3S,5R)-12: R = (CH ) OCH3
2 4
(
3R,5S)-12: R = (CH ) OCH
2 4 3
HO C
N
2
H
(
3R,5S)-11
Scheme 3. Synthesis of (3S,5R)ꢀ12 and (3R,5S)ꢀ12: (a) 13, HCTU, DIPEA, CH Cl –CH CN
2
2
3
1:1, RT, 42%; (b) 0.4 M HCl–dioxane, RT, 68%; (c) tolueneꢀ4ꢀsulfonyl chloride, DMAP,
pyridine; (d) CH Cl –piperidine 4:1, RT, 39% (two steps).
2
2
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7
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9
H
N
O
O
O
O
O
NH2
NH
CO CH
a -d
2
3
e -h
i, j
10
11
12
13
14
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
O
O
14
13
(rac)-15
Scheme 4. Synthesis of (rac)-15: (a) Cs CO , CH I, DMF, RT, 97%; (b) LDA, 4ꢀbromoꢀ1ꢀ
2
3
3
butene, HMPA, THF, –78°C to 0°C, 81%; (c) 9ꢀBBN, THF, 40°C; then H O , 2 M NaOH, 63%;
2
2
(
d) MeI, NaH, DMF, 0°C to RT, 90%; (e) 2 M NaOH, dioxane, 60°C; (f) PyBOP, benzylamine,
Et N, DMF, 0° to RT, 83% (two steps); (g) BH ·Me S, THF, 150°C, microwave, 86%; (h) 10%
3
3
2
Pd/C, EtOH, RT, 78%; (i) 1ꢀBocꢀpiperidineꢀ3ꢀcarboxylic acid, HCTU, Et N, 0°C to RT, 87%; (j)
3
4
M HCl–dioxane, RT, 84%.
Most notably, the Xꢀray structure of the rhꢀrenin–inhibitor 9 complex revealed the unsubstituted
equatorial C9 position of the tricyclic portion to be located at the entrance of the nonsubstrate
sp
sp
S3 pocket, thereby providing an attractive trajectory for flexible P3 side chain extensions
toward this important binding site. Inspection of the superimposed crystal structure of rhꢀrenin
bound inhibitor 1 (Figure 4b) and molecular modeling suggested a methoxybutyl chain attached
to the central ring to be optimal in length in order to fill the cavity and to potentially induce a Hꢀ
bond interaction to Tyr by the terminal methoxy group similarly to 1. Other options for
1
4
sp
introducing a suitable P3 substituent, for example at the P phenyl of the tricycle, appeared to be
3
plausible as well, but were not considered at this stage due to the expected higher synthetic
complexity.
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Journal of Medicinal Chemistry
Page 28 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In order to probe the design concept, the target compound 12 was initially prepared as the
racemate from readily available (rac)ꢀ11 and amine 13 (Schemes 3 and 4). Side chain alkylation
of the methyl ester of 7 via deprotonation using LDA and reaction with 4ꢀbromoꢀ1ꢀbutene was
followed by 9ꢀBBN–mediated hydroboration and O–methylation to afford intermediate 14 in
45% yield (Scheme 4). Transformation of 14 to the amine 13 was accomplished in overall 4
steps (56% yield) via reduction of the corresponding benzylamide intermediate with boraneꢀ
dimethylsulfide under microwave conditions and hydrogenolysis of the resulting benzylamine
derivative. A reversed reaction sequence by first starting with HCTU coupling of 13 to (rac)ꢀ11
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(42% yield), followed by Nꢀdebocylation, sulfonylation and Fmoc deprotection was found to be
more advantageous and afforded (rac)ꢀ12 in acceptable overall yield. (Rac)ꢀ12 inhibited rhꢀrenin
with an IC of 0.004 ꢁM, and hence was >20ꢀfold more potent in vitro as compared to (rac)ꢀ9
5
0
lacking the methoxybutyl side chain at the tricyclic P –P scaffold (Table 1). This prompted us to
3
1
prepare the pure enantiomers (3S,5R)ꢀ12 and (3R,5S)ꢀ12 from (3S,5R)ꢀ11 and (3R,5S)ꢀ11
(Scheme 3), respectively, which were accessible from racemate 11 by chiral chromatography
19
separation. (3S,5R)ꢀ12 was found to be a highly potent inhibitor of rhꢀrenin (IC = 0.003 ꢁM),
5
0
while the distomer (3R,5S)ꢀ12 was ~200ꢀfold less active in vitro (Table 1). The crystal structure
of (3S,5R)ꢀ12 bound to rhꢀrenin (Figure 4c) revealed a perfect overlay with enzyme–bound 9 and
sp
moreover confirmed the flexible methoxybutyl side chain to penetrate deeply into the S3 cavity
in an extended conformation. The terminal methoxy group was positioned at the bottom of the
channel, closely overlaying with the side chain methoxy of 1 and forming a Hꢀbond to the Tyr14
sp
backbone nitrogen. Limited SAR investigation by modifying P3 showed its length and shape to
be critical for binding affinity, with the methoxybutyl residue leading to maximal potency (data
not shown).
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Page 29 of 52
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Finally, (rac)-15 lacking the C5 prime site extension was synthesized (Scheme 4) and tested in
the FRET biochemical assay (IC of 4.0 ꢁM, Table 1) in order to estimate the relative
50
sp
contributions of the tolylsulfonamide moiety vs the P3 side chain for inhibitory binding affinity
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
in comparison to the most potent compound (3S,5R)ꢀ12 of this novel class of renin inhibitors.
sp
The impact of adding the P3 methoxybutyl side appeared to be very significant as suggested by
the more than 100ꢀfold difference in the IC for (rac)-15 compared to the complete lack of renin
5
0
inhibition at 100 ꢁM in the biochemical assay for (rac)-6 (Table 1) but is less pronounced for
tolylsulfonamide analogues (rac)-9 vs (rac)-12 (23ꢀfold difference in IC s). Most notably, (rac)-
5
0
15 was found to be 1000ꢀfold less potent than (3S,5R)ꢀ12, the latter bearing the C5 sulfonamide
1
0b
portion. This result corroborates previous findings indicating the importance of efficient
ligand–protein interactions at the extended prime site for strong inhibitory activity for different
classes of S –S topological peptidomimetic renin inhibitors.
3
1
a
Table 1. In vitro inhibition of rhꢀrenin.
(
rac)-5 (rac)-6 (rac)-9 (rac)-12 (3S,5R)-12
(3R,5S)-12
0.5
(rac)-15
4.0
a
b
IC (ꢀM)
1.2
>100
0.09
0.004
0.003
5
0
StdDev
±0.5
±0.05
±0.002
±0.002
±0.2
±2.5
a
Half–maximal inhibition of rhꢀrenin in a fluorescence–polarization enzymatic assay using
16
(RE(EDANS)IHPFHLVIHTK(Dabcyl)R as the substrate in 50 mM TrisꢀHCl buffer pH 7.4.
b
Data represent mean values from ≥4 independent experiments. 0% inhibition at the maximal
100 ꢀM ligand concentration tested.
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