P4 and P1′ optimization of bicycloproline P2 bearing tetrapeptidyl α-ketoamides as HCV protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.07.007

We describe herein the design, synthesis, and antiviral activity of a series of P4 modified tetrapeptidyl α-ketoamides as HCV protease inhibitors. The most promising analog identified through this SAR, 5a, 5c, and 5e demonstrated excellent enzyme inhibitory potency, enzyme selectivity, cellular activity, and acceptable therapeutic indexes. With the aim of improving HCV protease inhibitors reported in our previous manuscripts, we synthesized and evaluated a series of 1a-based tetrapeptidyl α-ketoamides with additional P4 modification. The promising analog discovered through this SAR, 5a, was further derivatized at P1′ or P1 position. As a result of these efforts, we found that replacement of the P4 valine as seen in 1a with cyclohexylglycine (Chg) resulted in the discovery of 5a, 5c, and 5e endowed with improved cellular activity in comparison to 1a.

Full text of DOI:10.1016/j.bmcl.2004.07.007

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5007–5011
P4 and P10 optimization of bicycloproline P2 bearing tetrapeptidyl
a-ketoamides as HCV protease inhibitors
Yvonne Yip, Frantz Victor, Jason Lamar, Robert Johnson, Q. May Wang, John I. Glass,
Nathan Yumibe, Mark Wakulchik, John Munroe and Shu-Hui Chen*
Lilly Research Laboratory, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 1 June 2004; accepted 2 July 2004
Available online 31 July 2004
Abstract—With the aim of improving HCV protease inhibitors reported in our previous manuscripts, we synthesized and evaluated
a series of 1a-based tetrapeptidyl a-ketoamides with additional P4 modification. The promising analog discovered through this
SAR, 5a, was further derivatized at P10 or P1 position. As a result of these efforts, we found that replacement of the P4 valine
as seen in 1a with cyclohexylglycine (Chg) resulted in the discovery of 5a, 5c, and 5e endowed with improved cellular activity in
comparison to 1a.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
derivatives 1a, 1b, and 1c as shown in Figure 1. We
are pleased to find that compound 1a exhibited very
good in vitro potency (enzyme and cellular), toxicity
profiles, as well as adequate liver exposure upon oral
administration.8 To further optimize this novel series
inhibitors, we embarked on P4 modification either alone
or in combination with additional P10optimization. On
the basis of substrate preference determined for a set
of hexapeptides (P1–P6) by Pessi and his collaborators,9
we decided to incorporate several aliphatic and aromatic
residues (e.g., IIe, Cha, and Phg, etc.) at the P4 position
as listed in Figure 1. Moreover, in order to maintain
good membrane permeability, we excluded charged res-
idues at that pocket. It should be mentioned that each of
the tetrapeptidyl a-ketoamide inhibitor discussed in this
manuscript incorporates a diastereomeric center at P1
position due to synthetic convenience.
Heptatitis C virus (HCV), the major etiological agent of
the non-A non-B hepatitis, was identified at the end of
1980s. Currently, HCV has infected about 170 million
people worldwide and has become a major health prob-
lem. About 70% of HCV infected patients become
chronically infected, approximately 20% of those
patients are at the risk of developing cirrhosis that can
eventually lead to hepatocellular carcinoma.1 Current
interferon-based therapies are far less than ideal espe-
cially for those infected with HCV genotype 1. The sub-
optimal efficacy along with poor tolerance associated
with current treatments necessitates the need for devel-
opment of new therapeutics.2 The HCV-encoded NS3
serine protease is essential for viral replication and thus
has been considered as an attractive target for therapeu-
tic intervention for the treatment of HCV-infected
patients.3–5 In order to address the unmet medical need,
we became interested in the discovery of novel HCV
protease inhibitors with therapeutic potentials. As docu-
mented in a series of recent publications from Lilly6 and
Vertex,7 we focused our research efforts on the design
and subsequent optimization of the bicycloproline P2
incorporated a-ketoamides as HCV protease inhibitors
including a number of tert-butylglycine P3 bearing
In this communication, we report our recent progress
achieved through such P4 optimization, which has led
to the identification of a number of promising HCV
NS3 protease inhibitors such as 5a, 5c, and 5e endowed
with improved activity in both the enzyme inhibition
and replicon assays relative to 1a.
2. Chemical synthesis
*
The representative synthetic route devised for the
P4 modified peptidyl a-ketoamides 2a through 7a is
Corresponding author. Fax: +86-21-50461087; e-mail: chen_shuhui@
pharmatechs.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.007

5008
Y. Yip et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5007–5011
P3 (t-Leu)
P2
N
N
N
O
H
O
H
O
N
N
H
N
O
P4 modification
N
N
N
H
N
N
H
H
N
H
N
O
O
N
P1'
P4
H
O
O
O
O
O
O
1:1 mixture
P4 (Val)
1a P1' = c-Pr
1b P1' = (s)-sec-butyl
P1 (Nva)
2a P4 = IIe;
4a P4 = tert-Leu; 5a P4 =Chg
6a P4 = Cha; 7a P4 = Phg
3a P4 = Thr-OMe
1:1 mixture
1c P1' = (s)-α-methylbenzyl
P1' modifications
P1 modification
N
O
H
N
O
N
N
H
O
N
N
H
H
N
N
P1'
H
O
N
N
O
O
H
N
N
H
N
N
O
O
H
O
O
O
O
F
1:1 mixture
5b P1' = (s)-sec-butyl
5c P1 = (s)-MeBn
5d P1' = (s)-sec-MeButyl
F
1:1 mixture
5e
Figure 1. P4 and P10-modified a-ketoamide HCV protease inhibitors.
outlined in Scheme 1. In general, a four-step sequence
(Step A–Step D) was used for the preparation of all final
products discussed in this manuscript. As shown in
Scheme 1, the P4–P3 dipeptidyl acid unit 14 was assem-
bled from HCl salt of H2NChg-(t-Bu)GlyOMe 12 via N-
acylation and subsequent ester hydrolysis. Compound
12 was in turn prepared by HOBt/EDCI mediated cou-
pling10 of BocHNChgOH 9 and amine 10, followed by
removal of the N-Boc protective group. Condensation
of 14 (P4–P3 acid) with the previously described P2
amine 156 was promoted by HOAt/DCC11 and afforded
the tripeptide ester 16, thereafter the corresponding acid
17, upon aqueous hydrolysis. PyBOP mediated cou-
pling12 of 17 with the requisite P1–P10 hydroxylamine
186 provided the expected adduct 19 (88%), which was
then oxidized with Dess–Martin periodinane to afford
the desired a-ketoamide inhibitor 5a in 50% yield. The
structure of 5a along with other P4 modified analogs
(2a, 3a, 4a, 6a, and 7a) were confirmed on the basis of
their proton NMR and mass spectra analyses.
various P4 modified inhibitors listed in Table 1 revealed
the following SAR trends: (1) replacement of the P4 Val.
in 1a with IIe or cyclohexylglycine (Chg) led to 2a and
5a with similar NS3 binding affinity; (2) incorporation
of either ThrOMe or t-Leu at the P4 pocket produced
inhibitors 3a, or 4a displaying 2-fold reduced enzyme
binding affinity; and (3) replacing of the Val moiety at
P4 with either Cha or Phg yielded inhibitors 6a or 7a,
both of which showed at least 8-fold weaker enzyme
inhibitory potency relative to that found with 1a.
Further P10 SAR: Having completed the first round of
P4 SAR exploration, we synthesized and evaluated a
few more tetrapeptidyl a-ketoamides with additional
modification at either P10 or P1 position. Judging from
the data shown in Table 2, it is clear that incorporation
of (S)-sec-butyl (5b) or (S)-MeBn (5c) functionality as
the P10 group retained the enzyme binding affinity in
comparison to that obtained with 5a. Introduction of
an additional methyl group at the P10 position as seen
in 5d resulted in ꢀ3x reduction in enzyme potency rela-
tive to 5a. In contrast to this finding, replacing the P1
Nva in 5a with difluoroAbu led to 5e displaying ꢀ3x en-
hanced enzyme binding affinity (Ki = 25nM).
It should be pointed out that four P10 or P1 modified
analogs of 5a, namely 5b–5e, were designed according
to the rationale articulated in the previous manu-
scripts.6,8 The syntheses of these analogs were accom-
plished using appropriate P1–P10 building blocks
described in Ref. 8 cited in this manuscript.
Replicon and cytotoxicity assays:15,16 All newly synthe-
sized HCV NS3 protease inhibitors disclosed in this
manuscript were also evaluated in a modified replicon
assay as well as XTT cytotoxicity assay in Huh-7 liver
cells. Careful inspection of the IC50 values determined
in the replicon assay as listed in Table 1 showed the fol-
lowing trends: (1) three out of seven inhibitors, 2a, 4a,
and 5a, demonstrated improved cellular potency relative
to 1a. The P4 Chg incorporated inhibitor, 5a, demon-
strated the best potency with IC50 value of 0.55lM,
which is about 4-fold more potent than that obtained
with 1a; (2) consistent with the reduced enzyme affinity,
inhibitors 3a, 6a, and 7a showed weaker potencies in the
replicon assay relative to 1a; (3) when compared with 5a,
whilst two P10 modified compounds 5b and 5d showed
2-fold weaker activity than 1a, inhibitors 5c
3. Biological evaluation
All P4 modified peptidyl a-ketoamides synthesized (2–7)
were evaluated in the following bioassays: (1) enzyme
inhibition assay against truncated NS3 enzyme;13 (2) en-
zyme selectivity assays against a panel of relevant cellu-
lar proteases;14 (3) HCV replicon assay for cellular
activity;15 (4) cytotoxicity assay in a liver cell line
(Huh-7 cells).16
Enzyme inhibition assay:13 Careful comparison of the Ki
values determined in the pNA based inhibition assay for

Y. Yip et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5007–5011
5009
O
O
HOBt/EDCI
BocHN
O
BocHN
N
OH
O
H
THF, 30%
HCl-H2N
+
O
O
11
10
9
N
N
O
O
4N HCl/dioxane
H
HOBt/EDCI
N
H2N
HCl
O
N
OR
N
N
THF
H
H
O
O
O
CO2H
N
12
36% (2-step)
13 R = Me
14 R = H
2N NaOH/MeOH
~100%
Step A
N
N
N
O
O
H
H
HOAt/DCC
CH2Cl2/THF
~100%
N
OH
N
N
+
N
N
N
N
H
H
OR
H
O
O
O
O
O
O
14
15
O
16 R = Et
17 R = H
2N NaOH
then 1N HCl
~100%
Step B
OH
H
N
H2N
PyBOP/EtPr2N
Step C 88%
O
18
1:1 mixture
N
N
O
H
N
O
Dess-Martin
O
H
N
N
H
H
N
N
OH
H
N
N
N
H
H
Step D
50%
N
N
O
O
N
N
H
O
O
O
O
O
O
19
5a
1:1 mixture
1:1 mixture
Scheme 1. Synthesis of 5a.
Table 1. Enzyme binding affinity Ki, and replicon IC50 and cytotox-
icity
As far as in vitro cytotoxicity is concerned, all P4 mod-
ified inhibitors listed in Table 1, except for 6a, were con-
sidered to be noncytotoxic with CC50 values > 50lM
and therapeutic indexes of greater than 20-fold. In addi-
tion, in light of the cytotoxicity data obtained with a set
of 5a based P10 or P1 modified analogs, it is interesting
to note that although the absolute CC50 values deter-
mined for 5c and 5d were in the 20–30lM range, the
actual CC50/IC50 (replicon) ratio reached at least 30-
fold. It is encouraging to see that compound 5e with
difluoroAbu incorporated at P1 exhibited minimal cyto-
toxicity (CC50 > 100lM) and an excellent selectivity
index of 250 (see Table 2). As also shown in Table 2,
the therapeutic indexes (CC50/ IC50) values obtained
with 6a and 7a were found to be around 20.
Comp HCV Ki pNA Replicon IC50 Cytotoxicity CC50/IC50
(lM)
(lM)
CC50 (lM)
ratio
1a
2a
3a
4a
5a
6a
7a
0.084
0.089
0.151
0.162
0.070
0.655
0.655
2.21
1.44
4.35
1.29
0.55
2.81
2.81
>100
>100
>100
>100
86.5
>45
>69
>22
>77
157
16
45.5
58.6
21
Table 2. 5a based P10 or P1 modifications
Comp HCV Ki pNA Replicon
Cytotoxicity CC50/IC50
IC50 (lM) CC50 (lM) ratio
(lM)
Enzyme selectivity:14 Based upon the encouraging activ-
ities determined in the enzyme and replicon assays,
seven P4 modified inhibitors were chosen for further
evaluation in the enzyme selectivity assays against a
panel of related cellular enzymes. As shown in Table
3, all of the P2 bicycloproline bearing a-ketoamides
discussed herein exhibited excellent selectivity against
kallikrein, thrombin, plasmin, trypsin, and chymotry-
psin with Ki values greater than 50lM. Generally
5a
5b
5c
5d
5e
0.070
0.084
0.090
0.254
0.025
0.55
1.07
0.21
1.0
86.5
69.2
22
157
65
105
32
31.7
>100
0.40
250
(IC50 = 0.21lM) and 5e (IC50 = 0.40lM) exhibited en-
hanced cellular potency (see Table 2).

5010
Y. Yip et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5007–5011
Table 3. Enzyme binding affinity KiÕs (lM) or x-fold of enzyme selectivity
Compd
HCVNS3
Elastase
Cathep. B
Cathep. L
Thromb.
Chymotrp.
Trypsin
Plasmin
Kallikrein
1a
2a
3a
4a
5a
5b
5c
5e
0.084
0.089
0.151
0.162
0.070
0.084
0.090
0.025
139x
36x
126x
102x
62
1.6x
10x
7.3x
3.9x
3x
154x
15x
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
146x
123x
134x
107x
51x
81x
29x
392
13x
283x
40x
760x
2. Kwong, A. D.; Kim, J. L.; Rao, G.; Lipovsek, D.;
Raybuck, S. A. Antiviral Res. 1998, 40, 1.
3. (a) Dymock, B. W. Emerging Drugs 2001, 6, 13–42; (b)
Dymock, B. W.; Jones, P. S.; Wilson, F. X. Antiviral
Chem. Chemother. 2000, 11, 79–96.
4. (a) Perni, R. B.; Kwong, A. D. In Progress in Medicinal
Chemistry; King, F. D., Oxford, A. W., Eds.; Elsevier
Science, 2002; pp 215–255; (b) Walker, M. P.; Appleby, T.
C.; Zhong, W.; Lau, J. Y. N.; Hong, Z. Antiviral Chem.
Chemother. 2003, 14, 1.
speaking, all P4 modified inhibitors (except for 2a)
showed at least 30-fold or 50-fold selectivity against
elastase or cathepsin L. On the other hand, relatively
low selectivity (610x) towards cathepsin B, an enzyme
involved in cancer metastases, was observed with 1a
through 5a. Interestingly, replacing the P10 c-Pr moiety
in 5a with a more bulky (S)-MeBn as seen in 5c resulted
in significant improvement (90x) in selectivity against
cathepsin B. Likewise, the P1 difluoroAbu bearing ana-
log 5e was found to be ꢀ6-fold more selective towards
elastase, cathepsin B and cathepsin L in comparison to
its P1 Nva containing counterpart 5a. Thus, in light of
the data listed in Table 3, it is reasonable to claim that
the P4 modified tetrapeptidyl a-ketoamides discussed
in this manuscript are rather specific for HCV NS3
protease.
5. Tan, S.-L.; Victor, F.; Chen, S.-H. In Frontiers of
Biotechnology
& Pharmaceuticals; Reiner, J., Chen,
S. H., Guo, M., Zhao, K., Eds.; Science: New York,
2004; Vol. 4, pp 123–146.
6. For the first report of bicycloproline P2 bearing peptidyl
a-ketoamide based HCV protease inhibitors, see: Yip, Y.;
Victor, F.; Lamar, J.; Johnson, R. B.; Wang, Q. M.;
Barket, D.; Glass, J. I.; Jin, L.; Liu, L.; Venable, D.;
Wakulchik, M.; Xie, C.; Heinz, B.; Villarreal, E.; Cola-
cino, J.; Yumibe, N.; Tebbe, M.; Munroe, J.; Chen, S. H.
Bioorg. Med. Chem. Lett. 2004, 14, 251; Also see: Lamar,
J.; Victor, F.; Snyder, N.; Johnson, R. B.; Wang, Q. M.;
Glass, J. I.; Chen, S. H. Bioorg. Med. Chem. Lett. 2004,
14, 263.
7. (a) Vertex patent regarding a-ketoamide based HCV
protease inhibitors: US 6,265,380 2001; (b) Perni, R. B.;
Pitlik, J.; Britt, S. D.; Court, J. J.; Courtney, L. F.;
Deininger, D. D.; Farmer, L. J.; Gates, C. A.; Harbeson,
S. L.; Levin, R. B.; Lin, C.; Lin, K.; Moon, Y.-C.; Luong,
Y.-P.; OÕMalley, E. T.; Rao, B. G.; Thomson, J. A.; Tung,
R. D.; Van Drie, J. H.; Wei, Y. Bioorg. Med. Chem. Lett.
2004, 14, 1441; (c) Perni, R. B.; Farmer, L. J.; Cottrell, K.
M.; Court, J. J.; Courtney, L. F.; Deininger, D. D.; Gates,
C. A.; Harbeson, S. L.; Kim, J. L.; Lin, C.; Lin, K.;
Luong, Y.-P.; Maxwell, J. P.; Murcko, M. A.; Pitlik, J.;
Rao, B. G.; Schairer, W. C.; Tung, R. D.; Van Drie, J. H.;
Wilson, K.; Thomson, J. A. Bioorg. Med. Chem. Lett.
2004, 14, 1939.
4. Conclusions
Starting from the previously identified P4 Val. bearing
HCV NS3 protease inhibitor 1a, we incorporated fur-
ther structural modification at P4 alone or in combina-
tion with additional modification at P10 or P1. On the
basis of biological data shown in Table 1, it is clear that
the P4 Chg bearing analog 5a exhibited improved en-
zyme (Ki = 70nM) and cellular potency (IC50 = 0.55M)
relative to 1a, thus it was selected for further P10 and
P1 modification. This continued effort led to the discov-
ery of 5c and 5e endowed with excellent enzyme and cel-
lular potency and desirable therapeutic indexes (see
Table 2). Thus, in view of the data presented in Tables
1–3, it is evident that the newly prepared P4 Chg bearing
inhibitors 5a, 5c, and 5e represent new promising P4
modified a-ketoamide based HCV protease inhibitors
endowed with very good enzyme binding affinity,
enzyme specificity, and replicon activity.
8. 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, 257.
9. Ingallinella, P.; Altamura, S.; Bianchi, E.; Taliani, M.;
Ingenito, R.; Cortese, R.; De Francesco, R.; Steinkuhler,
C.; Pessi, A. Biochemistry 1998, 37, 8906.
Acknowledgements
10. Carpino, L. A.; El-Faham, A. J. Org. Chem. 1995, 60,
3561.
We shall thank B. Perni, A. Kwong and their co-work-
ers from Vertex and R. Miller, J. Audia, J. Colacino, C.
Lopez, and G. Cassell from Eli Lilly and Company for
helpful discussions and encouragements.
11. HOAT/DCC(DIC) was used to minimize racemization
occurred at a-carbon during peptide coupling cf. Carpino,
L. A.; El-Faham, A. Tetrahedron 1999, 55, 6813.
12. Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett.
1990, 31, 205.
13. For detailed conditions used for the pNA based NS3 · 4A
inhibition assay, see: Landro, J. A.; Raybuck, S. A.;
Luong, Y. P. C.; OÕMalley, E. T.; Harbeson, S. L.;
Morgenstern, K. A.; Rao, G.; Livingston, D. J. Biochem-
istry 1997, 36, 9340. The Ki data reported in this
References and notes
1. For an overview, see: Bartenschlagar, R. Antiviral Chem.
Chemother. 1997, 8, 281.

Y. Yip et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5007–5011
5011
manuscript were acquired essentially using the Vertex
optimized kinetic assay conditions.
15. Replicon assay: (a) 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.
16. XTT cytotoxicity assay: Roehm, N. W.; Rodgers, G. H.;
Hatfield, S. M.; Glasebrook, A. L. J. Immunol. Meth.
1991, 142, 257.
14. A panel of 10 human serine and cysteine proteases
including elastase, chymotrypsin, trypsin, kallikrein, plas-
min, thrombin, Factor Xa, and cathepsins B, G, and L
were selected. Assays were performed using the conditions
substrates suggested by manufacturer.