Glycine α-ketoamides as HCV NS3 protease inhibitors

Article abstract of DOI:10.1016/S0960-894X(03)00031-3

Using a tetrapeptide-based α-ketoamide template, various amines and amino acids were incorporated to explore the prime side of the HCV NS3 protease catalytic site. Glycine carboxylic acid was found to be the most effective prime group. Further optimizatio

Full text of DOI:10.1016/S0960-894X(03)00031-3

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1111–1114
Glycine ꢀ-Ketoamides as HCV NS3 Protease Inhibitors
Wei Han,* Zilun Hu, Xiangjun Jiang, Zelda R. Wasserman and Carl P. Decicco
Department of Discovery Chemistry, Pharmaceutical Research Institute, Bristol-Myers Squibb Company,
PO Box 5400, Princeton, NJ 08543, USA
Received 29 October 2002; accepted 3 January 2003
Abstract—Using a tetrapeptide-based a-ketoamide template, various amines and amino acids were incorporated to explore the
prime side of the HCV NS3 protease catalytic site. Glycine carboxylic acid was found to be the most effective prime group. Further
optimization yielded an inhibitor with IC50 of 0.060 mM.
# 2003 Elsevier Science Ltd. All rights reserved.
The hepatitis C virus (HCV) is the principal etiologic
agent of both parenterally transmitted and sporadic
non-A non-B hepatitis.1 HCV infection most commonly
results in chronic hepatitis that eventually develops into
cirrhosis, hepatocellular carcinoma or liver failure.
Current therapies for HCV infection include treatment
with interferon-a alone and in combination with riba-
virin.2 These therapies have limited efficacy and fre-
quently are accompanied by side effects. Given these
drawbacks, new treatments for the disease are currently
being explored through multiple approaches. One prom-
ising approach is the inhibition of the serine protease
associated with the NS3 protein.3 In a chimpanzee
study, the NS3 protease has been shown to be required for
HCV infectivity, supporting NS3 as a viable drug dis-
covery target.4 Moreover, the NS3 protease is well
characterized5 and its crystal structure has been obtained.6
reported using peptide carboxamides,9 information on
prime site interactions of the a-ketoamide series is lim-
ited. This communication discloses our SAR efforts
exploring the prime site interactions using a-ketoamide
2 as a template, and brief optimization of P1 group.
We and others reported the use of a-ketoamides as tem-
plates for HCV NS3 protease inhibitors.7,8 Our original
hexapeptide lead 1 has an IC50 of 0.42 mM. Truncation and
modification led to tetrapeptide 2, which has an IC50 of 2.7
mM. In the proposed binding mode of 2 with HCV NS3
protease, the ketoamide forms a covalent bond with the
catalytic serine and projects the side chains of Cha, Ile,
and Leu to the S2, S3, and S4 sites, respectively. The
ethyl substituent on the ketoamide is believed to occupy
the S1 site. The ketoamide group offers an advantage of
extending into the prime side through the amide linker.
Even though prime site binding inhibitors have been
Several simple amide derivatives were first synthesized
and tested to explore the prime site interaction. As
shown in Table 1,10 amides derived from small primary
amines, such as allyl-, ethyl-, propargyl- and cyclopro-
pylamine, gave comparable potency in the NS3 protease
assay (2.7, 1.8, 1.6 and 2.2 mM, respectively, entries 1–
4). The more bulky N-benzyl amide resulted in sig-
nificant loss of potency (11 mM, entry 5). a-Ketoamides
derived from pyrrolidine and morpholine were even less
active (42 and >60 mM, entries 6 and 7).
*Corresponding author. Fax: +1-609-818-6570; e-mail: wei.han1@
bms.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00031-3

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W. Han et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1111–1114
Table 2. SAR of a-ketoamides with amino acids as prime groups
Table 1. SAR of analogues with simple amide groups
P0
IC50 (mM)
Entry
1
P0
IC50 (mM)
Entry
1
5.8
2.7
2
3
4
5
6
7
1.8
1.6
2
3
4
5
6
7
8
4.3
0.23
12
2.2
11
>60
1.3
42
>60
>60
>60
A series of amino acids was coupled with the ketoamide
to further explore the prime site interaction. The methyl
ester and the primary amide of the glycine derivatives
gave inhibitors at IC50 around 5 mM (entries 1 and 2,
Table 2). In contrast, the free acid afforded a 0.23 mM
inhibitor, a 20-fold enhancement (entry 3). The ketoa-
mide of b-alanine was less potent, and that of
N-methylglycine was totally inactive (entries 4 and 5). The
l-alanine analogue was 5-fold less potent than the glycine
compound (entries 6 vs 3), indicating that the methyl side
chain was tolerated but not preferred. d-Alanine and ami-
noisobutyric acid derivatives were not tolerated at the
prime site and gave inactive compounds (entries 7 and 8).
l-Amino acids with side chains larger than methyl groups
further attenuated the activity, regardless of the electronic
properties of the side chain (entries 9–11).
9
3.6
5.3
7.7
10
11
To help understanding the structure–activity relation-
ships, the a-ketoamide analogue with glycine free acid
(Table 2, entry 3) was modeled in the active site of NS3
protease (Fig. 1). According to the model, the car-
boxylic acid of the glycine appears to interact with
Lys136 and Arg 109 of the protein.11 Loss of binding
affinity of the glycinamide compared to the glycine free
acid suggested that the carboxylic acid group of glycine
serves either as hydrogen bond acceptor or forms charge
comlex with Lys136 and Arg109. The narrow channel of
the prime site adjacent to the catalytic serine could
explain the preference for more linear groups, such as
allylamine and glycine, over branched groups such as
pyrrolidine and a-substituted amino acids.
and cysteine residue is one of the most preferred P1
groups.12 Attempts at replacing the ethyl P1 group with
disubstituted analogues (a,a0-dimethyl or a,a0-cyclized)
proved detrimental to activity (entries 2 and 3, Table 3).
In contrast, the a,a0-cyclized (ethylenyl) P1 was reported
to be tolerated in carboxylate series.13 Significantly,
replacement of ethyl P1 with 2,2-difluoroethyl, a known
CH2SH mimetic,14 increased the potency to 0.060 mM
(entry 4). This potency enhancement was also seen in
other peptide-based HCV NS3 protease inhibitors.14
In summary, prime site interactions were investigated
using a tetrapeptide-based a-ketoamide template. Gly-
cine carboxylic acid is the preferred prime group. Fur-
ther modification of P1 group yielded a potent HCV
NS3 protease inhibitor with IC50 of 0.060 mM.
Keeping the glycine carboxylic acid constant, we briefly
investigated the effect of P1 substitution. NS3 protease
is known to have a shallow and hydrophobic S1 pocket

W. Han et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1111–1114
1113
Figure 1. Sectional view of a computer generated model of the glycine
carboxylic acid analogue (in stick presentation) in the active site of
HCV NS3 protease (in surface presentation).
Table 3. SAR of glycine a-ketoamide with different P1 group
Scheme 1. (a) Boc-Ile-OSu, DIEA, DMF, 0 ꢀC to rt, 8 h (92%); (b) (i)
TFA, CH2Cl2 (>95%); (ii) Boc-Leu-OSu, DIEA, DMF, 0 ꢀC to rt, 8 h
(90%); (c) (i) TFA, CH2Cl2 (>95%); (ii) pyrazinecarbonyl-OSu,
DIEA, DMF, 0 ꢀC to rt, 4 h (79%); (d) PyBOP, DIEA, DMF, 0 ꢀC to
rt, 4 h (71–84%); (e) LiOH, THF, H2O, 0 ꢀC, 1.5 h (90%); (f)
NHR1R2, BOP, DIEA, 0 ꢀC to rt, 4 h (78–88%); (g) Dess–Martin
reagent, CH2Cl2, 2–8 h (50–83%); (h) H-Gly-OtBu, BOP, DIEA,
DMF, 0 ꢀC to rt, 4 h (65–87%); (i) TFA, CH2Cl2, rt, 1 h (74–91%).
Entry
R1
R2
IC50 (mM)
1
2
3
4
H
Me
Et
Me
0.23
>60
>60
–CH2CH2–
CHF2CH2
H
0.060
Scheme 1 outlines the synthesis of ketoamides 7 and 8.
Cyclohexylalanine (3) was coupled sequentially with
appropriately functionalized isoleucine, leucine and
pyrazinecarboxylic acid to give tripeptide 4. Upon cou-
pling with 5a or 5b,4 the corresponding a-hydroxyl ester
6 was obtained. Saponification of ester 6 provided the
corresponding carboxylic acid, which was coupled with
various amines to give the desired amides. Dess–Martin
oxidation then completed the synthesis of a-ketoamides
7. Alternatively, the acid derived from ester 6 was cou-
pled with glycine t-butyl ester. Oxidation and acid
deprotection provided glycine a-ketoamides 8.
Preparation of glycine a-ketoamides with a,a0-cyclized
P1 group (14) is depicted in Scheme 2. The amino-
cyclopropane carboxylic acid 10 was activated with
EDCI and displaced with (cyanomethylene)-triphenyl-
phosphorane to give cyano keto ylide 11.15 Ozonolysis
followed by in situ trapping with glycine t-butyl ester
provided the glycine ketoamide 12. The a-carbonyl was
reduced to alcohol to prevent interference with sub-
sequent coupling reaction. After removal of Cbz, the
resultant amine was coupled with tripeptide 15 to give
Scheme 2. (a) Ph3P¼CHCN, EDCI, DMAP, CH2Cl2, 0 ꢀC to rt, 8 h
(63%); (b) O3, CH2Cl2, ꢁ78 ꢀC, then H-Gly-OtBu HCl, 1 h (84%); (c)
(i) NaBH4, THF, 0 ꢀC, 0.5 h; (ii) H2, Pd/C, MeOH, 0.5 h (56% for two
steps); (d) pyrazinyl-CO-Leu-Ile-Cha-OH (15), PyBOP, DIEA, DMF,
0 ꢀC to rt, 4 h (90%); (e) Dess–Martin, CH2Cl2, rt, 8 h (45%); (f) TFA,
CH2Cl2, rt, 1 h (90%).

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W. Han et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1111–1114
a-hydroxy amide 13, which was oxidized and depro-
tected to give glycine a-ketoamide 14. The same route
was used to prepare the dimethyl P1 analogue.
7. (a) Han, W.; Hu, Z.-L.; Jiang, X.-J.; Decicco, C. P. Bioorg.
Med. Chem. Lett. 2000, 10, 711 and references therein. (b)
Han, W.; Jiang, X.-J.; Hu, Z.-L.; Wasserman, Z. R.; Decicco,
C. P. Abstract of Presentation, National Meeting of the
American Chemical Society, San Diego, Apr. 2001; MEDI-
119 and MEDI-121.
8. (a) Bennett, J. M.; Campbell, A. D.; Campbell, A. J.; Carr,
M. G.; Dunsdon, R. M.; Greening, J. R.; Hurst, D. N.; Jen-
nings, N. S.; Jones, P. S.; Jordan, S.; Kay, P. B.; O’Brien,
M. A.; King-Underwood, J.; Raynham, T. M.; Wilkinson,
C. S.; Wilkinson, T. C. I.; Wilson, F. X. Bioorg. Med. Chem.
Acknowledgements
We would like to thank Drs. Bruce D. Korant and
Marina G. Bukhtiyarova for providing HCV protease,
Dr. James L. Meek and Ms. Lorraine Gorey-Feret for
determination of IC50 values.
Lett. 2001, 11, 355. (b) Llinas-Brunet, M.; Bailey, M.; Deziel,
´
R.; Fazal, G.; Gorys, V.; Goulet, S.; Halmos, T.; Maurice, R.;
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