Discovery and optimization of a new class of potent and non-chiral indole-3-carboxamide-based renin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.08.092

Selective inhibition of the aspartyl protease renin has gained attraction as an interesting approach to control hypertension and associated cardiovascular risk factors given its unique position in the renin-angiotensin system. Using a combination of high-throughput screening, parallel synthesis, X-ray crystallography and structure-based design, we identified and optimized a novel series of potent and non-chiral indole-3-carboxamides with remarkable potency for renin. The most potent compound 5k displays an IC₅₀ value of 2 nM.

Full text of DOI:10.1016/j.bmcl.2010.08.092

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6268–6272
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of a new class of potent and non-chiral
indole-3-carboxamide-based renin inhibitors
Bodo Scheiper a, , Hans Matter a, Henning Steinhagen a, , Ulrich Stilz a, Zsolt Böcskei b, Valérie Fleury c,
⇑
Gary McCort c
a Sanofi-Aventis Deutschland GmbH, Chemical and Analytical Sciences, Building G878, D-65926 Frankfurt, Germany
b Sanofi-Aventis, Chemical and Analytical Sciences, 16, Rue d’Ankara, F-67080 Strasbourg, France
c Sanofi-Aventis, Cardiovascular Department, 1, Avenue Pierre Brossolette, F-91385 Chilly-Mazarin, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Selective inhibition of the aspartyl protease renin has gained attraction as an interesting approach to
control hypertension and associated cardiovascular risk factors given its unique position in the renin–
angiotensin system. Using a combination of high-throughput screening, parallel synthesis, X-ray crystal-
lography and structure-based design, we identified and optimized a novel series of potent and non-chiral
indole-3-carboxamides with remarkable potency for renin. The most potent compound 5k displays an
IC50 value of 2 nM.
Received 22 June 2010
Revised 16 August 2010
Accepted 17 August 2010
Available online 21 August 2010
Keywords:
Hypertension
Ó 2010 Elsevier Ltd. All rights reserved.
Renin inhibitors
Indole
Structure based design
X-ray crystallography
Hypertension is one of the major risk factors for cardiovascular
diseases, which are the leading cause of mortality in the Western
world.1 Lowering blood-pressure can considerably reduce the risk
of myocardial infarction, stroke, heart failure and end-stage kidney
disease.2 However, despite available therapies, approximately 70%
of patients with hypertension do not reach their target blood pres-
sure levels. Some of them yet do not respond fully to a combination
of treatments. Consequently, opportunities remain for designing
and developing well-tolerated effective medicines to control
hypertension and associated cardiovascular diseases.
The renin–angiotensin system (RAS) is well-established as an
endocrine system involved in regulation of blood pressure and
fluid electrolytes.3 Activation of the RAS is initiated by several sig-
nals including lowering of blood pressure, decrease in circulating
volume or decrease in plasma-sodium concentration. These signals
stimulate the release of the aspartyl protease renin, which cleaves
angiotensinogen to produce angiotensin I. Since renin forms the
rate-limiting step in this cascade and angiotensinogen is its only
known substrate,4 inhibition of this step would be a very effective
antihypertensive strategy. Any appropriate medication affecting
the RAS might also result in optimal end-organ protection, in par-
ticular for heart and kidney as shown in animal models.2 This
might also be accompanied by a diminished potential for cough
side effects, affecting 5–35% of patients treated with ACE
inhibitors.5
Accordingly, substantial efforts were reported over the last dec-
ades to discover renin inhibitors for clinical use, for example, those
structures shown in Figure 1 and to overcome issues like low oral
bioavailability.6 Aliskiren (SPP100), an orally active renin inhibitor
with four chiral centers (Fig. 1), is currently the only compound,
which has reached the market.2b,7 Consequently, several research
groups have reported novel renin inhibitors on diverse scaffolds
with different renin active-site binding topologies.6
In this publication, we describe the identification, structure–
activity relationship and optimization of a novel series of indole-
3-carboxamides as potent and non-chiral renin inhibitors.8 The
first active molecule 19 was discovered from high-throughput
screening (HTS) of our internal collection and rapidly optimized
to result in phenoxy- and benzyl-derivatives 2a and 2m at the in-
dole-2 position, displaying increased binding activity (Fig. 2). Con-
sequently these derivatives were used as starting point for further
inhibitor design.
Docking10–12 these molecules into public renin X-ray structures
(e.g., PDB 1rne)13 in addition to our internal experience with other
aspartyl-proteases led us to postulate that the piperazine moiety is
⇑
Corresponding author. Tel.: +49 69 305 28306; fax: +49 69 305 943099.
E-mail address: Bodo.Scheiper@sanofi-aventis.com (B. Scheiper).
Present address: Grünenthal Pharma GmbH, Global Drug Discovery, Zieglerstr. 6,
52099 Aachen, Germany.
situated close to both catalytic aspartate residues Asp32 and Asp215
,
engaged in ionic hydrogen-bonding interactions. The internal
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.092

B. Scheiper et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6268–6272
6269
N
NH
O
OH
OH
H
N
H
N
NH2
O
O
S
N
H
OH
O
O
O
NH2
O
O
O
Aliskiren
H
N
Remikiren
N
O
S
HO
O
N
O
OH
H
N
N
S
N
H
OH
O
O
O
O
O
O
RO-66-1168
Zankiren
Figure 1. Chemical structures of potent renin inhibitors Remikiren, Aliskiren, Zankiren and RO-66-1168.
Gly34
Ser35
NH
O
Cpd
R=
Renin IC50 [µM]
N
Asp215
Ser76
1
2a
2m
N-piperidinyl- 3.800
R
Asp32
Phenoxy-
Benzyl-
0.420
0.091
N
Tyr75
Met289
Val120
Figure 2. Initial indole-3-carboxamides as basis for design. IC50 data was obtained
as described in the text and Ref. 8.
His287
Trp39
Ser219
Phe112
Thr77
Tyr220
X-ray structures of 2a and 2m at resolutions of 2.9 and 2.75 Å ni-
cely confirmed these assumptions, thus supporting further lead
optimization (Fig. 3).14
Phe117
Gln13
Thr12
Pro111
The protein-binding pocket adopts the ‘closed-flap’ conforma-
tion, as also observed in renin X-ray structures of peptidomimetics
(1rne)13 or Aliskiren (2v0z).16 The protonated piperazine amine is
in hydrogen-bonding interactions with the carboxylates of both
Asp32 and Asp215. A further hydrogen-bond interaction is present
-
OOC-Asp215
Thr77-Oγ
O
NH
Tyr75
N
-
between Thr77-O
cY and the carboxamide oxygen in 2a. This side-
OOC
Asp32
Met289
chain hydroxyl-functionality is also in a slightly longer hydrogen-
bonding distance to the phenoxy-oxygen; although di-aromatic
ether oxygens normally do not reveal any hydrogen-bond acceptor
tendency. Interestingly, replacement of this oxygen in 2a results in
improved binding affinity,17 which could be due to a more favorable
conformational effect of the benzyl substituent in 2m, as deduced
from conformational analyzes. This preference might be attributed
to the close 3.12 Å distance between carbonyl oxygen and ether
oxygen atoms in 2a, resulting in repulsive electrostatic interactions
of their lone pair electrons, which is not possible in 2m.
O
N
Val120
Trp39
Ser219
Phe117
Pro111
Figure 3. Upper: X-ray crystal structures of two indole-3-carboxamides 2a (gray
carbons, 2.9 Å resolution) and 2m (orange carbons, 2.75 Å resolution) in complex
with human renin. The protein binding site is indicated using
hydrogen bond surface (red = ligand acceptor expected, blue = donor expected).
Crystallographic water is indicated by cyan spheres. Lower: Schematic view of
interactions between 2a and renin.
a
MOLCAD15
The phenoxy- or benzyl substituents are located in the hydro-
phobic renin S1 pocket, lined by the lipophilic side-chains of
residues Phe117, Trp39, Val120, Tyr75 and Val30. This aromatic substi-
tuent is in optimal arrangement for favorable p–p
interactions18, in
particular an edge-to-face interaction with Tyr75. This pocket is
filled with an isopropyl group by Aliskiren. The N1-Phenyl-substi-
tuent occupies the slightly more polar S3 pocket, favorably inter-
acting with Pro111 and Phe117 side chains. Aliskiren fills this
region with a methoxy-phenyl group. The indole core is engaged
The synthetic pathway for preparation of compounds 2–5 is
outlined in Scheme 1, here exemplified for compounds 5c and 5j.
Indole 6 was first arylated under Copper catalysis, followed by
chlorination with NCS and subsequent hydrolysation under acidic
conditions.19 Vilsmeyer–Haack formylation with concomitant
chlorination led to aldehyde 7. Oxidation to the carboxylic acid
and coupling with N-Boc-piperazine provided key intermediate 8.
To gain compound 5j 3-fluoro-2-methylbenzyl bromide was trans-
formed into the benzyl zinc compound, transmetallated to the
in lipophilic interactions to Ser219Cb, Met289
Ce and Thr77Cc moie-
ties. These X-ray structures suggest that the indole-3-carboxamide
scaffold could serve as template for exploration of both the S1 and
S3 pockets.

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B. Scheiper et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6268–6272
Table 1
NBoc
In vitro renin activity (IC50) for compounds 2a–2oa
O
O
N
O
O
O
NH
O
a-d
e, f
N
Cl
Cl
N
H
N
N
R
6
7
8
N
j, h, i
g, h, i
N
a
Compds
R
Renin IC50
0.420
(lM)
2a
Phenoxy
NH
O
NH
O
N
CH3
N
HO
HO
2b
2c
3.160
9.000
O
N
N
F
O
5c
5j
F
Scheme 1. Synthesis of compounds 5c and 5j. Reagents and conditions: (a) CuI,
proline, Phenyliodide, K2CO3, DMSO, 125 °C, microwave, 63%; (b) NCS, DCM, rt; (c)
HOAc, H3PO4, 70 °C?130 °C, 65% over both steps; (d) POCl3, DMF, CHCl3, pyridine,
0 °C?rt, 68%; (e) NaClO2, NaH2PO4, t-BuOH, 0 °C?80 °C; (f) N-Boc-piperazine, EDC,
HOAt, NMM, DMF, rt, quant. over both steps; (g) (i) 3-fluoro-2-methylbenzyl
bromide, Zn, THF, 0 °C, (ii) 9-MeO-9-BBN, ꢀ78 °C?rt, (iii) Pd(OAc)2, S-Phos, DMF,
100 °C, 53%; (h) TFA, DCM, rt, quant.; (i) BBr3, DCM, ꢀ78 °C?rt, 47–58%; (j)
5-fluoro-2-methylphenol, NaH, NMP, rt?150 °C, microwave, 50%.
CF3
O
O
S
CH3
O
2d
2e
2f
0.680
0.021
0.071
CH3
CH3
corresponding organoboron species and finally coupled with 8 in a
Palladium-mediated reaction. Deprotection of the piperidine and
hydroxyl group let to 5j. Alternatively 8 submitted to nucleophilic
substitution, followed by deprotection to provide the arylether 5c.
Initial variations of the phenoxy-group in 2a group led to a sig-
nificant improvement of renin binding affinity by optimally filling
the hydrophobic S1 subsite. A representative collection of synthe-
sized molecules and experimentally determined renin inhibition
O
2g
0.773
CH3
IC50 values [lM] is given in Table 1. To calculate this IC50 value,
O
the activity of an inhibitor was evaluated in a 10 point concentra-
tion ranging, in duplicate (intraplate), and two to four independent
experiments were performed. The IC50 is then expressed as geo-
metric mean standard deviation (SD),8 which in general was
low for this assay.
2h
2i
0.063
0.064
0.005
0.016
F
O
H3C
CH3
In particular the favorable effect of ortho-Methyl-substitution
on affinity directed towards a small subpocket near Val30 and
Gly217 was remarkable (2e: 0.021
l
M). Adding another fluorine-
substituent further enhances binding (2l with meta-F: 0.011 M;
2j with meta0-F: 0.005
M) for both the phenoxy- and benzyl-in-
dole scaffolds (e.g., 2n with benzyl and meta-F: 0.009 M). Further
O
O
l
CH3
2j
l
l
F
F
molecular modeling suggested that the favorable effect of the
meta0-F could be attributed to a close contact to the aromatic plane
of the Tyr75 ring system.20 The meta-F substituent on the other
CH3
2k
hand interacts with another subpocket, in particular with Val30
Cc
(3.4 Å) and the side-chain carbon atoms of Asp32 (3.7 Å). Represen-
tative distances are taken from the X-ray structure of 5k in Figure 4
(see below).
O
CH3
2l
0.011
0.091
Initial modifications at the piperazine ring did not result in in-
creased binding affinity, as shown in Table 2. Hence we then inves-
tigated the S3 pocket by variation of the indole N1-substituent.
Representative variations of synthesized molecules are shown in
Table 3. While 2-pyridyl and 4-fluorophenyl groups attached to
the N1 position were less efficient for binding, the incorporation
of a methoxy–propoxy substituent increased affinity, in particular
if added to the ortho-position at the N1-phenyl ring pointing to-
wards the renin S3sp subpocket, which itself is accessible from
F
2m
Benzyl
CH3
F
2n
2o
0.009
0.018
Cl
F
the S3 pocket.13 This is obvious from comparing 2a (0.420
with 4c (0.175 M) in Table 3. A similar substitution is present in
lM)
a
IC50 data were obtained as described in the text and in Ref. 8.
l

B. Scheiper et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6268–6272
6271
Table 3
In vitro activity for compounds 4a–4ia
Asp215
Asp32
NH
O
O
N
Tyr75
CH3
F
R2
Met289
N
R1
R2 = A
R2 = B
Val120
a
Compds
R1
Phenyl
2-Pyridyl
4-Fluorphenyl
R2
Renin IC50 (lM)
Trp39
Phe112
His287
2a
4a
4b
A
A
A
0.420
4.970
0.707
Thr77
Ser219
Thr12
Tyr220
O
O
4c
CH3
A
0.175
Phe117
3
Pro111
4d
A
0.763
CH3
O
O
Figure 4. X-ray crystal structure of indole-3-carboxamide 5k (orange carbons, 2.5 Å
resolution) in complex with human renin resulting from rational compound
optimization. The protein binding site is indicated using a MOLCAD15 hydrogen-
bonding surface. Crystallographic water is indicated by cyan spheres.
3
2n
4e
4f
Phenyl
B
B
B
B
0.009
0.034
0.020
0.752
Cyclohexyl
Cyclopentyl
4-Pyranyl
4g
Table 2
4h
4i
B
9.350
In vitro activity for compounds 3a–3ea
N
R3
OH
R2
R1
NH
O
N
N
B
0.360
O
O
N
CH3
a
IC50 data were obtained as described in the text and in Ref. 8.
R1
R2
R3
Renin IC50 (lM)
a
Compds
2a
3a
3b
3c
3d
3e
H
H
H
H
H
H
CH3
H
H
0.420
6.460
7.300
4.500
1.600
3.410
Table 4
rac-CH3
CH3
H
In vitro activity for compounds 5a–5oa
NH
O
rac-Benzyl
H
Homopiperazine
N
O
rac-CH2OH
CH3
CH3
F
R1
R2
a
IC50 data were obtained as described in the text and in Ref. 8.
N
F
R2 = A
R2 = B
Aliskiren in this area.7 However, molecular modeling suggests that
the corresponding vector for the indole scaffold is slightly deviat-
ing from the narrow entrance into this subpocket, which might ex-
plain the less pronounced effect of this substitution compared to
other series like Aliskiren analogs. Furthermore, the replacement
of phenyl by aliphatic rings did not result in a dramatic loss of
affinity, underscoring the hydrophobic nature of the favorable
Compds
R1
R2
Renin IC50
(l
M)
a
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
6-OCH3
5-OCH3
5-OH
5-CONH2
6-CO2H
6-OCH2CH2N(CH3)2
6-OCH2CO2H
6-OCH3
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
0.024
1.350
0.002
0.006
0.122
0.029
0.130
0.012
0.004
0.004
0.002
0.084
0.024
0.031
0.044
interactions in this S3 site (4f: 0.020 lM vs 2n with 0.009 lM).
6-OH
5-OH
Finally we focused on variations at the indole core to explore
polar and hydrophobic interactions with the binding site to
increase affinity. The resulting molecules are summarized in
Table 4. Several polar substitutions display a slightly favorable ef-
4-CH3, 5-OH
7-F
6-OCH2CH2OPh
6-OCH2CH2OH
6-SO2CH3
fect on affinity, especially the 5-OH substitution in 5c (0.002
lM),
when compared to its parent 2j (0.005 M). Small, polar groups
l
a
like hydroxyl in this area in the protein pocket are acceptable,
IC50 data were obtained as described in the text and in Ref. 8.
while a 5-methoxy group resulted in a reduction of binding affinity
(5b: 1.350 lM). As docking and the subsequent X-ray structure of
the close analog 5k suggests (see below), this 5-hydroxyl-group
favorably interacts with the imidazole ring of His287, while there
is not enough space in the binding site to accommodate an addi-
tional methyl group in 5b.
Other tolerated substitutions are the 5-carboxamide (5d:
0.006 M) and 6-OH group (5i: 0.004 M). Interestingly, the 6-
methoxy substitution in 5h (0.012 M) is significantly more active
compared to the 5-methoxy-substitution in 5b. This was explained
l
l
l

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B. Scheiper et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6268–6272
by docking and the subsequent X-ray structure of 5k (see below),
revealing that the His287 side chain limits the cavity size for 5-
OMe substitution in this region. In contrast, substituents at the in-
dole-6 position point to a more open area limited by Ser219 and
Tyr220, where slightly larger and lipophilic substitutions might be
accommodated. The further inspection of available X-ray struc-
tures also suggests that more lipophilic substituents might be tol-
erated in position 6 in addition to the small polar substitution in
position 5. This resulted in synthesis of 5k with an IC50 of
References and notes
1. Kearney, P. M.; Whelton, M.; Reynolds, K.; Muntner, P.; Whelton, P. K.; He, J.
Lancet 2005, 365, 217.
2. (a) Amin Zaman, M.; Oparil, S.; Calhoun, D. A. Nat. Rev. Drug Disc. 2002, 1, 621;
(b) Jensen, C.; Herold, P.; Brunner, H. R. Nat. Rev. Drug Disc. 2008, 7, 399.
3. MacGregor, G. A.; Markandu, N. D.; Roulston, J. E.; Jones, J. E.; Morton, J. J.
Nature 1981, 291, 329.
4. Wood, J. M.; Stanton, J. L.; Hofbauer, K. G. J. Enzyme Inhib. Med. Chem. 1987, 1,
169.
5. (a) Dicpinigaites, P. V. Chest 2006, 129, 169S; (b) Ravid, D.; Lishner, M.; Lang, R.;
Ravid, M. J. Clin. Pharmacol. 1994, 3, 1116.
0.002 lM.
6. (a) Yokokawa, F.; Maibaum J. Exp. Opin. Ther. Pat. 2008, 18, 581; (b) Tice, C. M.
Annu. Rep. Med. Chem. 2006, 41, 155.
We then solved the X-ray structure of compound 5k in complex
with human renin by X-ray structure analysis at 2.5 Å (Fig. 4), thus
validating our hypothesis about favorable protein–ligand interac-
tions for biological affinity.14 In addition to the interaction of the
7. (a) Göschke, R.; Stutz, S.; Rasetti, V.; Cohen, N.-C.; Rahuel, J.; Rigollier, P.; Baum,
H.-P.; Forgiarini, P.; Schnell, C. R.; Wagner, T.; Grütter, M. G.; Fuhrer, W.;
Schilling, W.; Cumin, F.; Wood, J. M.; Maibaum, J. J. Med. Chem. 2007, 50, 4818;
(b) Maibaum, J.; Stutz, S.; Göschke, R.; Rigollier, P.; Yamaguchi, Y.; Cumin, F.;
Rahuel, J.; Baum, H.-P.; Cohen, N.-C.; Schnell, C. R.; Fuhrer, W.; Grütter, M. G.;
Schilling, W.; Wood, J. M. J. Med. Chem. 2007, 50, 4832.
8. Steinhagen, H.; Scheiper, B.; Matter, H.; Stilz, H. U.; McCort, G. PCT Int. Appl.,
WO 2009095163, 2009.
9. (a) Lattrell, R.; Bartmann, W.; Kaiser, J. DE 2557341, 1977.; (b) Lattrell, R.;
Bartmann, W.; Kaiser, J. U.S. 4148895, 1979.
piperazine with the catalytic aspartates, the Thr77-O
cH hydrogen
bond and the lipophilic interactions of both phenyl rings in S1
and S3, several other interactions further contribute to the ob-
served high binding affinity of 5k.
The 2-methyl-3-fluoro substituted phenol optimally fills the S1
pocket with fluorine directed towards a small lipophilic subpocket
formed by side-chain carbon atoms of Val30, Val120 and Asp32 with
10. McMartin, C.; Bohacek, R. S. J. Comput. Aided Mol. Des. 1997, 11, 333.
11. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.
distances of 3.4 and 3.7 Å to Val30 and Asp32
Cc Cc@O), respectively.
12. Modeling studies were done using Sybyl 7.0–8.0 (Tripos). Energy calculations
were based on the MMFF94s force field (Halgren, T. J. Comput. Chem. 1999, 20,
720.). Protein–ligand complexes were minimized using the quasi-Newton–
Raphson procedure. For manual docking, ligands were placed in the binding
site and optimized treating all side chains within 4 Å flexible. Automated
The close distance and geometric arrangement between fluorine
and Asp32 carboxylate also suggests an involvement of orthogonal
multipolar interactions to complex stabilization.21,22 The ortho-
methyl group is pointing towards Gly217 , Val30
Ca Cc and the aro-
docking was carried out using QXP10 or Glide11
.
matic ring in Phe117, thus also contributing to binding.
13. Rahuel, J.; Priestle, J. P.; Grütter, M. G. J. Struct. Biol. 1991, 107, 227.
14. The renin–ligand complexes for compounds 2a, 2m and 5k were prepared by
soaking using renin apo-crystals crystallized in hanging drops from conditions
described by Lim et al. J. Mol. Biol. 1989, 210, 239. Data were collected at beam-
line ID 14-3 of the European Synchrotron Radiation Facility (ESRF) in Grenoble
or in house. The data were processed with hkl2000 (Otwinowski, Z.; Minor, W.
In Carter, C. W., Jr.; Sweet, R. M., Eds.; Methods Enzymol.; 1997, 276, 307.). The
crystal diffracted to 2.9 (2a), 2.75 (2m) and 2.5 (5k) Å. The space group was
P213 in all cases. The inhibitor was fitted using Quanta (Accelrys, 1986–2010)
and refined using Refmac (Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta
Cryst. 1997, D53, 240.) and Buster 2.9.5 (Global Phasing 2010). The coordinates
for the complexes were deposited in the Brookhaven protein database (PDB:
3OOT, 3OQK, 3OQF).
The substitutions at the indole core also favorably contribute to
binding affinity. The 5-OH substituent is engaged in an additional
hydrogen-bonding interaction to the imidazole-nitrogen in
His287, which also lower activity, when adding a methyl group to
the phenolic OH (5b: 1.350
in lipophilic contacts to the side-chain atoms of Thr77
somewhat more distant Ser76Cb and Ala218
. This combination
lM). The 4-methyl group is involved
Cc
, and
Ca
of favorable interactions thus accounts for the improved binding
affinity of 5k in comparison to the template 2m.
In summary, we have described the discovery and structure–
activity relationship of a novel series of potent and non-chiral renin
inhibitors with various modifications around an indole scaffold.
The incorporation of substituted phenoxy- or benzyl substituents
in S1 combined with an adequate substitution at the indole core
led to a significant improvement of biological activity. The best
compounds (e.g., 5k) display IC50 values of 2 nM against renin
without significant inhibition of related proteases. Thanks to the
availability of experimental information from several X-ray struc-
tures of renin-inhibitor complexes during our optimization, we
were able to understand the influence of key structural elements
to affinity, which might allow for further design around this versa-
tile scaffold. Hence, this series constitutes a promising area for lead
optimization supported by structure-based design. Further details
on these studies will be reported in due time.
15. Heiden, W.; Goetze, T.; Brickmann, J. J. Comput. Chem. 1993, 14, 246.
16. Rahuel, J.; Rasetti, V.; Maibaum, J.; Rüeger, H.; Göschke, R.; Cohen, N.-C.; Stutz,
S.; Cumin, F.; Fuhrer, W.; Wood, J. M.; Grütter, M. G. Chem. Biol. 2000, 7, 493.
17. Inhibitory activity expressed as inhibition constant IC50 toward recombinant
human renin was determined using an assay in which the non-endogenous
fluorogenic substrate Dabcyl-c-Abu-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-
EDANS is cleaved at the Leu-Val bond in analogy to angiotensinogen. Human
renin at a concentration of 5 nM is incubated with the test compound at
various concentrations and the substrate at a concentration of 10 lM (2 h at rt)
in 0.05 M Tris buffer (pH 8). The increase in fluorescence due to fluorescence
resonance energy transfer at an emission wavelength of 485 nm is recorded in
a microplate spectrofluorometer.8
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Acknowledgments
22. Paulini, R.; Müller, K.; Diederich, F. Angew. Chem., Int. Ed. 2005, 44, 1788.
The authors thank Sabrina Berger, Holger Gaul, Silvia Hein, Lae-
titia Martin, Andrea Müller, Mathilde Pierre and Katja Wittmann
for their technical assistance.