Capped dipeptide phenethylamide inhibitors of the HCV NS3 protease

Article abstract of DOI:10.1016/j.bmcl.2004.02.032

The N-terminal aminoacid of phenethylamide tripeptide inhibitors of the hepatitis C virus NS3 protease can be replaced with an α-hydroxy acid to obtain more 'drug like' inhibitors with low micromolar activity. The preferred S-configuration of the capping

Full text of DOI:10.1016/j.bmcl.2004.02.032

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2151–2154
Capped dipeptide phenethylamide inhibitors of the HCV NS3
protease
Emanuela Nizi, Uwe Koch, Jesus M. Ontoria, Antonella Marchetti, Frank Narjes,
Savina Malancona, Victor G. Matassa and Cristina Gardelli*
Department of Chemistry, IRBM, MRL Rome, Via Pontina Km 30,600, 00040 Pomezia, Rome, Italy
Received 28 October 2003; revised 6 February 2004; accepted 9 February 2004
Abstract—The N-terminal aminoacid of phenethylamide tripeptide inhibitors of the hepatitis C virus NS3 protease can be replaced
with an a-hydroxy acid to obtain more Ôdrug likeÕ inhibitors with low micromolar activity. The preferred S-configuration of the
capping residue can be explained by molecular modeling studies.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Inhibitors described in the literature are predominantly
based on the substrates of NS32 and due to their pep-
tidic nature they will likely suffer from poor oral bio-
availability. Nevertheless, enormous progress has been
made in the peptidomimetic area, where macrocyclic
inhibitors showed a discrete oral absorption and an
antiviral effect in clinical trials.7
The hepatitis C virus (HCV) represents the chief etio-
logic agent in non-A and non-B hepatitis, a disease that
causes liver cirrhosis and hepatocellular carcinoma1;2
and affects more than 170 million people worldwide.3
Although the introduction of a combination therapy,
pegylated interferon-a and ribavirin, has markedly
improved clinical outcomes, less than 50% of infected
individuals can be expected to have a favourable
response to this treatment.4 The HCV NS3/4A protease
is an important target to treat this disease and has been
intensively studied because of its central role in the viral
replication. A recent important study has shown that
NS3 protease is necessary for HCV replication in
chimpanzee.5
Only a few other small molecule inhibitors whose
structure is not based on the peptide substrates of the
NS3 protease have been reported:2 their mechanism of
action is not clear and none of these are competitive
inhibitors of NS3.
Recently we reported8 the evolution of potent capped
dipeptide a-ketoacid inhibitors of HCV NS3 protease
based on the primary discovery that the a-ketoacid
moiety is a particularly effective serine trap for NS3
protease9 and that 4,4-difluoro-2-aminobutyric acid
(difluoroAbu)10 is an excellent and chemically inert
replacement for the canonical P1 cysteine found in most
of the cleavage sequences of NS3 protease.
The identification of clinically relevant compounds––
low molecular weight, orally bioavailable, cell-penetrant
entities––is exceptionally challenging and the reason can
be found in the unique structural characteristics of the
target itself, the NS3/NS4 protease/co-factor complex:
the featureless and highly polar substrate binding region
causes the need for unusually large, polyanionic deca-
peptide substrate molecules.2;6
Capped dipeptides showed that the entire P3 residue of the
N-protected tripeptides can be replaced by a small
hydroxylated residue (e.g., 1, IC50 3 lM) whose chirality is
critical for binding recognition (Fig. 1). Contrary to the S-
configuration found in the P3 aminoacids, a-hydroxy acids
with R-configuration were more potent than those with S-
configuration. We explained this with a conformational
preference for a coplanar arrangement of the C–O and N–
H bonds, obtained by an intramolecular hydrogen bond
between these groups, as shown in Figure 1.
Keywords: HCV NS3 protease; Phenethylamide dipeptide.
* Corresponding author. Tel.: +39-06-91093279; fax: +39-06-910936-
54; e-mail: cristina_gardelli@merck.com
Present address: Graffinity Pharmaceuticals AG, Im Neuenheimer
Feld 518, Heidelberg 69120, Germany.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.032

2152
E. Nizi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2151–2154
F
Combining these informations, we made a series of
F
O
H
compounds aimed towards potent reversible capped
dipeptide phenethylamides as HCV NS3/4A protease
inhibitors.
O
H
N
O
N
H
OH
O
O
1
Figure 1. Capped dipeptide a-ketoacid lead 1 (IC50 3.00 lM).
2. Synthesis
The synthesis of the capped dipeptide 12 from the
intermediate 412 is shown in Scheme 1 and is represen-
tative for other analogues. Condensation of intermedi-
ate 4 with unprotected (S)-isovaleric acid 5, followed by
acid promoted removal of the tert-butyl ester, gave the
desired capped dipeptide phenethylamide 12.
F
F
O
O
Cl
H
N
H
N
O
N
H
N
H
O
O
COOH
2
All crude phenethylamides were purified by HPLC and
when necessary the two diastereoisomers, resulting from
the use of racemic a-hydroxy acids, were separated at
this stage as well. The more active diastereoisomer
always eluted first and was later shown by use of chiral
fragments, such as (R)- or (S)-isovaleric acids and (S)-
hexahydromandelic acid, to have the S-configuration at
the capping residue. The l-configuration at P1, and the
2S,4S-configuration at P2 was in accord with the results
from tripeptide work.11
Figure 2. Tripeptide phenethylamide lead (IC50 2.4 lM).
The more recent discovery11 that the a-ketoacid moiety
of potent tripeptides can be replaced by an amide resi-
due (e.g., 2, IC50 2.4 lM, Fig. 2), led us to develop a new
series of reversible, competitive inhibitors of HCV NS3
protease, which are characterized by the presence of a
phenethylamide group in the P10 position. These inhib-
itors, even if they are slightly less potent than the cor-
responding tripeptide ketoacids, have the clear
advantage of lacking the electrophilic carbonyl group.
They also explore novel interactions in the prime side of
the NS3/4A protease, which renders them highly specific
against related proteases as for example elastase.
As far as the P2 residues reported in Table 2, they have
been synthesized as described in Scheme 2. Following a
procedure described in literature,14 direct alkylation of
the sodium dianion of 6, in the presence of NaI, fol-
lowed by exchange of the protecting group gave the
desired intermediate in moderate to good yields.
These properties led us to investigate, replacing the
N-terminal aminoacid of the phenethylamide, inhibitors
with an a-hydroxy acid in a manner analogous to that in
the serine trap ketoacid series.8
Compounds were then evaluated as inhibitors using the
full length HCV NS3/NS4A protease complex as
described in Ref. 15.
In the investigation of the SAR in the tripeptide series,
we achieved the highest potencies with the 4-(2-amino-
ethyl)-3,5-difluorobenzoic acid as phenethylamine12 and
the (S,S)-4-phenylproline in P2,13 and a corresponding
optimized tripeptide is shown in Figure 3.
F
F
OH
O
F
H
N
H
a, b, c
OH
N
+
N
H
O
O
4
F
COOtBu
The combination of an a-hydroxyamide in P3 with the
phenylproline in P2 removes the amide N–H of this
residue, which favoured a coplanar arrangement with
the P3 C–O and thus the R-configuration of the a-
hydroxyamide (Fig. 1). Accordingly, we expected in this
case the S-configuration to be favoured as for the
aminoacids.
5
OH
F
F
O
O
F
H
N
N
N
H
O
F
COOH
12
Scheme 1. Reagents and conditions: (a) HATU, 2,6-lutidine, DCM–
DMF (55%); (b) TFA–DCM–H2O (99%); (c) RP-HPLC.
O
F
Boc
N
Fmoc
N
NH
F
O
O
COOH
COOH
a,b,c
O
F
H
N
N
N
H
HO
RO
O
7
6
F
COOH
Scheme 2. Reagents and conditions: (a) NaH, THF, RBr, NaI (25–
76%); (b) HCl, dioxane (67–98%); (c) Fmoc-OSuc, NaHCO3, H2O/
acetone (78–95%).
3
Figure 3. Optimized phenethylamide tripeptide (IC50 0.9 lM).

E. Nizi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2151–2154
2153
3. Results and discussion
Modeling of the compounds in the active site of NS316
show that the a-hydroxy group superimposes with the
P3 amide NH participating in the same P3–S3 hydrogen
bonds to Ala157 as the tripeptide 2 (Fig. 4). In this
conformation the lipophilic cyclopentyl side chain is
orientated towards the S3 site, which it is capable of
occupying completely.
A selection of capped dipeptide analogues was prepared
to investigate the hypothesis of replacing an amide or a
carbamate by an alcohol, wherein the lipophilic sub-
stituents R and the stereochemistry of the alcohol were
varied (Table 1).
Of these, certain alkyl or cycloalkyl analogues were
active at the micromolar level.
Compounds 19 and 20 lacking the hydroxy group, have
been prepared as well (Fig. 5). The 3-fold lower activity
(IC50 34 and 58 lM, respectively) confirm the impor-
tance of the hydroxy group for the activity of the capped
dipeptides.
Comparing the two isopropyl a-hydroxy acetic acid
derivatives 11 and 12 it was found that one isomer was
active, whereas the other one did not show any inhibi-
tion even up to 100 lM. In the case of the two cyclo-
pentyl a-hydroxy acetic acid derivatives 13 and 14 one
isomer was almost 5-fold more potent than the other
one. The most active diastereoisomer has the (S)-con-
figuration at the hydroxy alcohol residue, in agreement
with our expectations.
Having discovered that the commercially available (S)-
hydroxy-isovaleric residue in P3 was able to replace the
N-protected aminoacid we tried to optimize the P2
residue and the compounds reported in Table 2 were
made. Only compounds 22 and 24, containing, respec-
tively, the p-Br-benzyloxy and the 3-phenylpropyloxy
moieties instead of the phenyl group in the 4-position of
the pyrrolidine ring gave a small increase in potency.
Previously we had used the a-hydroxy acid replacement
in the design of phenethylamide-Boehringer hybrid
structures to explore the role of bigger P2 aryloxypro-
lines.17
Table 1. SAR of capped dipeptide phenethylamides
OH
F
F
O
R
O
F
H
N
N
N
H
O
F
COOH
Compds
R
P3 conf.
IC50, lMa
8
(R)
na
na
na
na
18
9
(S)
10
11
12
2 diaster.
(R)
(S)
13
14
15
16
(R)
62
13
18
50
Figure 4. Superposition of 14 (blue) and 2 (only P2, P3 and capping
group shown, white).
(S)
(S)
F
F
O
R
O
F
2 diaster.
H
N
N
N
H
O
F
COOH
17
18
(S)
14
na
R = cyclopentyl 19, IC50 = 34 µM
= isopropyl 20, IC50 = 58µM
2 diaster.
Figure 5. Capped dipeptide phenethylamides without OH in the cap-
ping group.
a na means IC50 > 100 lM.

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E. Nizi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2151–2154
Table 2. P2 SAR of capped dipeptide phenethylamides
€
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OH
F
F
O
O
F
H
N
N
N
H
O
F
COOH
IC50, lMa
27
R
Compds
R
O
21
O
22
12
Br
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Cl
O
O
23
24
na
11
ꢀ
A.-M.; Goudreau, N.; Kawai, S. H.; Kukolj, G.; Lagace,
O
L.; LaPlante, S. R.; Narjes, H.; Poupart, M.-A.; Rancourt,
J.; Sentjens, R. E.; George, R. St.; Simoneau, B.; Stein-
mann, G.; Thibeault, D.; Tsantrizos, Y. S.; Weldon, S. M.;
Yong, C.-L.; Llinas-Brunet, M. Nature 2003, 426, 186–
189.
25
32
a na means IC50 > 100 lM.
8. Nizi, E.; Koch, U.; Ponzi, S.; Matassa, V. G.; Gardelli, C.
Bioorg. Med. Chem. Lett. 2002, 12, 3325–3328.
9. Narjes, F.; Koehler, K. F.; Koch, U.; Gerlach, B.;
In summary, we have shown that also in the non-
covalent series of tripeptide phenethylamide inhibitors,
an a-hydroxy acid is a viable replacement for the P3-
aminoacid, obtaining compounds with lower peptidic
character. The preferred (S)-configuration was correctly
inferred from molecular modeling. Compounds like 14,
17, 22 and 24 are active at the low micromolar level and
no longer contain the serine trap present in 1.
€
Colarusso, S.; Steinkuhler, C.; Brunetti, M.; Altamura,
S.; De Francesco, R.; Matassa, V. G. Bioorg. Med. Chem.
Lett. 2002, 12, 701–704.
10. Narjes, F.; Brunetti, M.; Colarusso, S.; Gerlach, B.; Koch,
U.; Biasiol, G.; Fattori, D.; De Francesco, R.; Matassa,
€
V. G.; Steinkuhler, C. Biochemistry 2000, 39, 1849–1861.
11. Colarusso, S.; Koch, U.; Gerlach, B.; Steinkuhler, C.; De
€
Francesco, R.; Altamura, S.; Matassa, V. G.; Narjes, F.
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12. Malancona, S.; Colarusso, S.; Ontoria, J. M.; Marchetti,
A.; Poma, M.; Stansfield, I.; Laufer, R.; Di Marco, A.;
Taliani, M.; Gonzalez-Paz, O.; Matassa, V. G.; Narjes, F.,
submitted for publication.
Acknowledgements
The authors gratefully acknowledge Sergio Altamura
€
13. Summa, V.; Pace Napoleone, M. P.; Petrocchi, A.
Unpublished results.
and Christian Steinkuhler for the assay data, Fabio
14. Smith, E. M.; Swiss, G. F.; Neustadt, B. R.; Gold, E. H.;
Sommer, J. A.; Brown, A. D.; Chiu, P. J. S.; Moran, R.;
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885.
Bonelli and Francesca Naimo for the analytical data,
Silvia Pesci and Daniel Cicero for NMR spectra, Clau-
dio Giuliano for synthetic support and Michael Rowley
for valuable discussions. This work was supported in
part by a grant from the MIUR.
15. Gallinari, P.; Paolini, C.; Brennan, D.; Nardi, C.;
€
Steinkuhler, C.; De Francesco, R. Biochemistry 1999, 38,
5620–5632.
16. Di Marco, S.; Rizzi, M.; Volpari, C.; Walsh, M. A.;
Narjes, F.; Colarusso, S.; De Francesco, R.; Matassa, V.
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9 of this reference containing an unoptimized phenethyl-
amide has an IC50 of 11 lM).
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