Novel P4 truncated tripeptidyl α-ketoamides as HCV protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.09.076

We describe herein the synthesis and evaluation of two series of P-4 truncated tripeptidyl α-ketoamides as HCV serine protease inhibitors. The most promising compound disclosed in this communication 7b demonstrated enzyme binding affinity (K_i)

Full text of DOI:10.1016/j.bmcl.2003.09.076

Bioorganic & MedicinalChemistry Letters 14 (2004) 263–266
Novel P4 truncated tripeptidyl ꢀ-ketoamides as HCV
protease inhibitors
Jason Lamar, Frantz Victor, Nancy Snyder, Robert B. Johnson,
Q. May Wang, John I. Glass and Shu-Hui Chen*
Lilly Research Laboratory, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 4 August 2003; accepted 8 September 2003
Abstract—We describe herein the synthesis and evaluation of two series of P-4 truncated tripeptidyl a-ketoamides as HCV serine
protease inhibitors. The most promising compound disclosed in this communication 7b demonstrated enzyme binding affinity (Ki)
at 0.27 uM.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
Figure 1. Inhibitors 4–7 contain isopropylcarbamate
moiety as their P3 N-capping group, whereas inhibitors
8 and 9 carry 2-hydroxyl-3-methylbutyric acid as the P3
capping group. It is hoped that the isopropylmoieties
embedded in these P3 capping groups could mimic the
P4 valine seen in the tetrapeptidyl ketoamides such as 2
and 3. As can be seen in Figure 1, bc-proline moieties
(deoxy and gem-difluoro) and nor-valine were incorpo-
rated at the respective P2 and P1 sites in all of the target
molecules. In addition, Val and t-Leu were chosen for
Hepatitis C virus (HCV) infection is widely recognized
today as a huge public health concern, with more than
170 million people infected worldwide, most of them
unknowingly.1 Majority of the HCV infected patients
developed slowly progressing liver diseases which can
lead to liver cirrhosis and hepatocellular carcinoma.
Unfortunately, neither a generally effective treatment
nor a preventive vaccine is available so far.2 To address
this unmet medicalneed, we embarked on the discovery
of novelketoamide based inhibitors for HCV serine
protease, a key enzyme involved in viral polyprotein
cleavage and maturation.3 As depicted in Figure 1, in
contrast to rather large ketoamide inhibitor 1 reported
by DuPont,4 we focused our SAR effort on the design
and evaluation of novel bicycloproline P2 bearing tet-
rapeptidylketoamides such as 2.5 Replacement of the
P3 valine in 2 with tert-Leu led to another series of more
impressive inhibitors including 3a.6 As documented in
our recent publications, inhibitors 2 and 3a were identi-
fied as promising HCV serine protease inhibitors
endowed with excellent overall antiviral activity, cyto-
toxicity, and pharmacokinetics profiles. To make fur-
ther improvement on these inhibitors, we decided to
carry out (1) P4 and P4 cap modifications,7 and (2)
P4 truncation to reduce synthetic complexity.8 In
this communication, we will discuss two types of P4
truncated tripeptidylketoamide inhibitors as shown in
0
P3 pocket. Both neutraland acidic P1 moieties were
incorporated into each set of tripeptidylketoamides ( a
or b).
2. Series 1
Tripeptidylketoamides 4–7 were prepared according
to the synthetic route outlined in Scheme 1 for com-
pound 5a. HOAt/DIC mediated coupling9 between
isopropylcarbamate protected t-leucine 10a with the
previously reported deoxy-bicycloproline derivative
11a5,10 provided the dipeptide ester 14 (64%), which
was then converted to its corresponding carboxylic
acid 15 (81%) via NaOH mediated hydrolysis. Fur-
ther reaction of 15 with the known P1–P10 amino-
alcohol 12b11 was promoted by HOAt/DIC and
yielded the expected adduct 16 (82%), which was
then oxidized with Dess–Martin periodinane to pro-
vide the desired tripeptidyl a-ketoamide inhibitor 5a in
49% yield. The reaction yields for other inhibitors
within this series (4a, 4b, 5b, 6a, 6b, 7a, and 7b) are
included in ref. 18.
* Corresponding author. Tel.: +1-317-276-2076; fax: +1-317-276-
1177; e-mail: chen_shu-hui@lilly.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.076

264
J. Lamar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 263–266
Figure 1. Representative structures of HCV protease inhibitors.
Scheme 1. Synthesis of inhibitor 5a.
3. Series 2
three-step sequence: (1) N-acylation, (2) O-silyl protec-
tion, and (3) NaOH mediated hydrolysis. HOAt/DCC
mediated coupling9 of 13 with the known difluoro-bc-
Pro-OEt 11b afforded the adduct 19 (66%), which was
saponified to provide the corresponding carboxylic acid
20 (90%). PyBOP mediated coupling12 of 20 with the
The representative synthetic route employed for the
2-hydroxy-3-methylbutyric acid capped inhibitors 8 and
9 is shown in Scheme 2. The requisite O-silyl-acid 13
was prepared from HCl-Val-OMe via the following

J. Lamar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 263–266
265
requisite P1–P10 unit 12a11 provided the carbinoladduct
Table 1. Biological activity of tripeptidyl a-ketoamides
Compd P-3 Ki (pNA) Replicon inhibition Cytotoxicity
P-10
21 (94%), which was further converted to the O-silyl
protected ketoamide 22 (92%). Further treatment of a
dichloromethane solution of 22 with TFA yielded the
target HCV protease inhibitor 9a in 92% yield.
(mM)
@50 mM
(mM)
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
2
Val(s)MeBn
ValPhe
t-Leu (s)MeBn
t-Leu Phe
Val(s)MeBn
ValPhe
t-Leu (s)MeBn
1.91
0.42
1.75
0.82
6.7
2.7
7%
Not active
>100
>100
>100
Not tested
>100
>100
>100
>100
>100
>100
53%
Not active
22%
All other a-ketoamides discussed herein were prepared
according to the identicalsequences described in
Scheme 1 or 2. The overall yields for each inhibitor are
listed in ref 18. The structures of these tripeptidyl
a-ketoamides (4–9) were secured on the basis of their
proton NMR and mass spectra analyses.
Not active
2.1
0.27
51%
Not active
Not active
Not active
Not active
Not active
t-Leu
Phe
Valc-Pr
ValPhe
Valc-Pr
ValPhe
Valc-Pr
t-Leu
14.5
1.8
16.5
2.0
not tested
>100
>100
>100
>100
>100
All tripeptidyl a-ketoamides synthesized (4–9) were
evaluated in the following bioassays: (1) enzyme binding
assay against truncated NS3 enzyme;13 (2) HCV repli-
cation surrogate assay (replicon) for cellular activity;14
(3) cytotoxicity assay in a liver cell line (Huh-7 cells).15
The testing results obtained with the newly synthesized
inhibitors along with four previously reported tetra-
peptidyl a-ketoamides (2 and 3) are listed in Table 1.
0.123
IC
50=7.0 mM
3a
3b
3c
c-Pr
0.084
0.19
0.023
IC50=2.2 mM
IC50=0.78 mM
16%
t-Leu (s)MeBn
t-Leu Phe
reported difluoroAbu P1 bearing tripeptidyl a-ketoacid
(Ki=0.38 mM).16 (2) P3-isopropylcarbamate capped
analogues such as 4b (Ki=0.42 mM) and 7b (Ki=0.27
mM) showed superior activity to that found with
2-hydroxy-3-methylbutyric acid capped analogues 8b
(Ki=1.8 mM) and 9b (Ki=2.0 mM), respectively.
(3) Carefulcomparison of the Ki data generated for
three pairs of inhibitors (8a vs 2, 5a vs 3b, and 5b vs 3c)
clearly showed that the tetrapeptidyl a-ketoamides were
10- to 100-fold more potent than their corresponding P4
truncated tripeptidylketoamide based inhibitors.
4. Enzyme inhibition assay
All tripeptidyl a-ketoamides were found to be tight-
binding inhibitors for HCV NS3 protease. Therefore,
the inhibition constant, Ki, was determined using Mor-
rison equation.13 Carefulinspection of the data ilsted in
Table 1 revealed the following trends: (1) All P10 Phe
bearing inhibitors were found to be more potent than
their corresponding neutralP1 0 containing counterparts.
A number of P10 Phe containing analogues (e.g., 4b, 5b,
and 7b) displayed Ki values less than 1 uM, with com-
pound 7b being the most potent one (Ki=0.27 mM). It is
worthwhile to mention that the enzyme inhibitory
activity demonstrated by 7b is comparable to a recently
5. Replicon assay and cytotoxicity assay14,15
In view of the data listed in Table 1, we discovered the
following SAR trends: (1) All of the P10 Phe bearing
inhibitors (4b–9b) were devoid of cellular activity. This
Scheme 2. Synthesis of inhibitor 9a.

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J. Lamar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 263–266
is likely due to their poor membrane permeability. (2) P3
t-Leu bearing analogues 5a and 7a displayed better
activity than their respective P3 Valcontaining counter-
parts 4a and 6a. (3) When compared with their corre-
sponding tetrapeptidyl a-ketoamides 3b (IC50=0.78 mM)
and 2 (IC50=7.0 mM), both tripeptidyl a-ketoamides 5a
(IC50 ꢀ50 mM) and 8a (inactive at 50 mM) showed sig-
nificantly reduced activity. (4) Judged from the IC50
values listed in Table 1, all newly prepared inhibitors
(4–9) were found to be non-cytotoxic with IC50 values
greater than 100 mM.
5. For LY514962: Yip, Y.; Victor, F.; Lamar, J.; Johnson,
R.; Wang, Q. M.; Barket, D.; Glass, J.; Jin, L.; Liu, L.;
Venable, D.; Wakulchik, M.; Xie, C.; Heinz, B.; Villar-
real, E.; Colacino, J.; Yumibe, N.; Tebbe, M.; Munroe,
J.; Chen, S.-H. Bioorg. Med. Chem. Lett. 2004, 14,
preceding paper in this issue. doi: 10.1016/
j.bmcl.2003.09.074.
6. For LY526181: Victor, F.; Lamar, J.; Snyder, N.; Yip, Y.;
Guo, D.; Yumibe, N.; Johnson, R. B.; Wang, Q. M.;
Glass, J. I.; Chen, S.-H. Bioorg. Med. Chem. Lett. 2004, 14,
preceding paper in this issue. doi: 10.1016/
j.bmcl.2003.09.075.
7. Chen, S.-H., unpublished results.
8. Personalconversation with chemists at Vertex Pharma-
ceuticals, Inc.
6. Conclusion
9. Carpino, L. A.; El-Faham, A. Tetrahedron 1999, 55, 6815.
10. Monn, J. A.; Valli, M. J. J. Org. Chem. 1994, 59, 2773.
11. For the syntheses of various a-aminohydroxylamide
building blocks (P1–P10), see: PCT (Eli Lilly): WO/02/
18369 A2, Mar. 7, 2002.
12. PyBOP: Costa, J.; Le-Nguyen, D.; Castro, B. Tetrahedron
Lett. 1990, 31, 205.
13. The detailed description of HCV protease enzyme assay is
provided in ref 5 (ref 19 therein).
14. (a) Replicon assay: Lohmann, V.; Korner, F.; Koch, J.-
O.; Herian, U.; Theilmann, L.; Bartenschlager, R. Science
1999, 285, 110. (b) Blight, K.; Kolykhalov, A.; Rice, C.
Science 2000, 290, 1972.
15. XTT cytotoxicity assay: Roehm, N. W.; Rodgers, G. H.;
Hatfield, S. M.; Glasebrook, A. L. J. Immunol. Methods
1991, 142, 257.
16. (a) Colarusso, S.; Gerlach, B.; Koch, U.; Muraglia, E.;
Conte, I.; Stansfield, I.; Matassa, V. G.; Narjes, F. Bioorg.
Med. Chem. Lett. 2002, 12, 705. (b) For a recent review on
P1 diFluoroAbu bearing a-ketoacids, see: Steinkuhler, C.;
Koch, U.; Narjes, F.; Matassa, V. G. Curr. Med. Chem.
2001, 8, 919.
17. (a) A number of papers describing various tripeptide
based HCV protease inhibitors have appeared since
completion of this manuscript. These include: Colar-
usso, S.; Koch, U.; Gerlach, B.; Steinkuhler, C.; De
Francesco, R.; Altamura, S.; Mastassa, V. G.;
Narjes, F. J. Med. Chem. 2003, 46, 345. (b) Nizi, E.;
Koch, U.; Ponzi, S.; Matassa, V. G.; Gardelli, C. Bioorg.
Med. Chem. Lett. 2002, 12, 3325. (c) Andrews, D. M.;
Jones, P. S.; Mills, G.; Hind, S. L.; Slater, M. J.; Trivedi,
N.; Wareing, K. J. Bioorg. Med. Chem. Lett. 2003, 13,
1657. (d) Slater, M. J.; Andrews, D. M.; Baker, G.;
Bethell, S. S.; Carey, S.; Chaignot, H.; Clarke, B.; Coom-
ber, B.; Ellis, M.; Good, A.; Gray, N.; Hardy, G.; Jones,
P.; Mills, G.; Robinson, E. Bioorg. Med. Chem. Lett.
2002, 12, 3359.
We prepared and evaluated two series of P4 truncated
tripeptidyl a-ketoamide inhibitors 4–9. The most potent
inhibitor discussed in this manuscript was 7b with Ki value
of 0.27M. Despite its enzyme inhibitory activity, com-
pound 7b was inactive in the replicon assay. Compared
with their respective tetrapeptidylketoamide counter-
parts, the newly prepared tripeptidyl a-ketoamides pos-
sessed significantly reduced enzyme and cellular activity.17
Acknowledgements
We shall thank the chemists at Vertex Pharmaceuticals
for sharing their expertise in connection to HCV pro-
tease inhibitor and the replicon assay design with us. We
thank D. Barket, L. Jin, L. Liu, D. Venable, M. Walk-
ulchik, C.-P. Xie for performing HCV replicon and
XTT cytotoxicity assays. We are also indebted to Drs. J.
Munroe, J. Colacino, C. Lopez and G. Cassell for
helpful discussions and encouragement.
References and notes
1. (a) For recent reviews, see: Dymock, B. W. Emerg. Drugs
2001, 6, 13. (b) Dymock, B. W.; Jones, P. P.; Wilson, F. X.
Antiviral Chem. Chemother. 2000, 11, 79.
2. Current therapies for HCV infection include IFN-a-2a,
IFN-a-2b and their combination with ribavirin. More
recently, a number of pegylated-IFNs such as Pegasys,
PEG-Intron and their respective combination with ribavirin.
3. Kwong, A. D.; Kim, J. L.; Rao, G.; Lipovsek, D.; Ray-
buck, S. Antiviral Res. 1998, 40, 1.
18. Four- or five-step overall yields for the preparation of 4a:
28%; 4b: 23%; 5a: 21%; 5b: 25%; 6a: 20%; 6b: 17%; 7a:
12%; 7b: 16%; 8a: 35%; 8b: 46%; 9a: 51%; 9b: 34%.
4. Han, W.; Hu, Z.; Jiang, X.; Decicco, C. P. Bioorg. Med.
Chem. Lett. 2000, 10, 711. The IC50 value of 340 nM was
reported for DuPont’s ketoamide 1.