P1 and P3 optimization of novel bicycloproline P2 bearing tetrapeptidyl α-ketoamide based HCV protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.09.075

With the aim of discovering potent and selective HCV protease inhibitors, we synthesized and evaluated a series of 1a based tetrapeptidyl ketoamides with additional modification(s) at P1′, P1, and P3 positions. As a result of this effort, we found that re

Full text of DOI:10.1016/j.bmcl.2003.09.075

Bioorganic & Medicinal Chemistry Letters 14 (2004) 257–261
P1 and P3 optimization of novel bicycloproline P2 bearing
tetrapeptidyl ꢀ-ketoamide based HCV protease inhibitors
Frantz Victor, Jason Lamar, Nancy Snyder, Yvonne Yip, Deqi Guo, Nathan Yumibe,
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—With the aim of discovering potent and selective HCV protease inhibitors, we synthesized and evaluated a series of 1a
based tetrapeptidyl ketoamides with additional modification(s) at P10, P1, and P3 positions.As a result of this effort, we found that
replacement of the P3 valine with tert-leucine resulted in the discovery of a series of inhibitors (e.g., 3a, 3c, and 4c) endowed with
improved enzyme and/or cellular activity relative to 1a.When dosed to F-344 rats orally at 50 mg/kg, 3a achieved 2.5Â higher liver
and plasma exposure in comparison to that detected with 1a.
# 2003 Elsevier Ltd.All rights reserved.
1. Introduction
discovered that the deoxy bc-Proline P2 containing
inhibitor LY514962 (1a) displayed best overall profiles
(enzyme and cellular activity and liver drug exposure)
within the inhibitors discussed therein.7a
Heptatitis C virus (HCV) infection has become a major
health problem.It has been estimated that about 170
million people worldwide are infected with HCV,
including 3.9 million (ꢀ1.8% population) in the USA.
About 70% of HCV infections become chronically
infected, approximately 20% of those patients will
develop cirrhosis that can eventually lead to hepatocel-
lular carcinoma.1,2 The common routes of HCV trans-
mission include blood transfusion, sexual activity,
intravenous drug abuse, needle-stick infection in health
care workers or those occurring as a result of acu-
puncture, tattoos, routine dental care, or surgery.The
best treatment options for HCV are pegylated-INFs
such as Pegasys and PEG-Intron.3,4 The response rate
thus obtained with HCV genotype-1 patients is far less
than ideal.In view of this unmet medical need, we
launched a research program aiming at designing potent
and selective inhibitors to suppress the function of the
HCV serine protease, an essential enzyme responsible
for viral replication.5,6 As disclosed in our previous
publication, we focused our research efforts on the
design and evaluation of a novel class of bicycloproline
P2 bearing a-ketoamides as HCV NS3 protease inhibi-
tors.7 As documented in our previous paper detailing
the comparative evaluation of various P2 modified
bicycloproline bearing peptidyl ketoamides, we
To further improve upon 1a, we decided to take advan-
tage of the findings reported by Pessi and Narjes on P3
and P1 SAR, respectively.Recently Pessi discovered
that other hydrophobic P3 moieties (e.g., IIe, Abu, Nva)
can be used as P3 Val replacements.8a On the other
hand, Narjes reported that diFAbu can mimic the P1
Cys moiety very well.Thus, a number of P1 diFAbu
bearing peptidyl a-ketoacids exhibited very potent
enzyme inhibitory activity against HCV NS3 protease
inhibitors (see Fig.1 ).8b As can be seen in Figure 1, we
incorporated tert-leucine at P3 (as seen in 3 vs valine in
1) and diFAbu at P1 (as seen in 2 vs nvaline in 1) in
hopes of increasing hydrolytic stability and/or inhibi-
tory activity of the resulting HCV protease inhibitors.
In conjunction with P10 variation, further combination
of P1 and P3 modifications led to another set of enzyme
inhibitors 4a–c.
In this communication, we report herein our recent
progress achieved through 1a based SAR modifications
at P1, P3, and P10 positions as depicted in Figure 1.
These efforts have culminated in the discovery of
LY526181 3a, a very promising HCV protease inhibitor
endowed with balanced enzyme activity, enzyme selec-
tivity, cellular activity, liver exposure and favorable
toxicity profiles.
* Corresponding author.Te:l. +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.2003.09.075

258
F. Victor et al. / Bioorg. Med. Chem. Lett. 14 (2004) 257–261
Figure 1. P1 and P3 modified bc-proline P2 bearing inhibitors.
The syntheses of both P10 and/or P3 modified inhibitors
were accomplished via sequential coupling of the build-
ing blocks AA, BB, and CC/CC0 as shown in Scheme 2.
The preparation of the deoxy-bc-proline P2 unit (BB) as
well as the N-pyrazine capped Val-Val-OH (AA0) were
described previously.6 The synthetic routes employed
for the preparation of AA and CC and CC0 are outlined
in Scheme 1.Towards these ends, HOAt/DCC mediated
coupling9 of Z-Val-OH and tert-Leu-OMe afforded III,
thereafter the free amine IV, upon removal of the Cbz
protective group.Acylation of the resulting amino moi-
ety in IV with pyrazine carboxylic acid provided the
methyl ester V, which was converted to the requisite
acid AA via base hydrolysis.The P1–P1 0 building blocks
CC and CC0 were prepared in two steps (PyBOP pro-
moted peptide coupling and subsequent hydrogenation)
from the known a-hydroxyacids VI10 and VIII,11
respectively.The diFAbu bearing a-hydroxyacid VIII
was prepared in turn from another known acid VII
according to the published patent literature from Insti-
tuto Di Ricerche Di Biologia Melecolare.11
With the key building blocks in hand, we then moved on
to assembling various a-ketoamides depicted in Figure 1.
In general, all of the HCV protease inhibitors discussed
herein (1–4) were synthesized via a four-step successive
coupling procedure using AA, BB, and CC (or their
closely related analogues) as the key building blocks.
The overall yields of the final products and their
respective intermediates are listed in ref 16.The struc-
tures of the HCV NS3 protease inhibitors disclosed in
this manuscript were secured on the basis of their pro-
ton NMR and mass spectra analyses.
The syntheses of the HCV NS3 protease inhibitors 1a–
1c were reported previously.7 The representative syn-
thetic routes used for the preparation of inhibitors 3 and
4 are exemplified for compounds 3a and 4c.Inhibitors
2a–c were prepared in a similar fashion to that descri-
bed for 4.
As can be seen in Scheme 2, the HOAt/DCC mediated
coupling between the dipeptidyl acid AA and the bc-
Scheme 1. Syntheses of the key building blocks AA, CC, and CC0.

F. Victor et al. / Bioorg. Med. Chem. Lett. 14 (2004) 257–261
259
Scheme 2. Synthesis of 3a and 4c.
proline P2 unit BB provided the desired adduct IX,
which was next converted to its corresponding tripepti-
dyl acid X via base hydrolysis.Subsequent PyBOP
mediated coupling of the acid X with the P1-Nva bear-
ing building block CC afforded the a-hydroxyamide
intermediate XI, which was then oxidized (Dess–Martin
reagent) to the final a-ketoamide inhibitor 3a.On the
other hand, the coupling of the tripeptidyl acid X with
the diFluroAbu-P1 bearing unit CC0 gave rise to the
corresponding intermediate XII, which was further
converted to the final product 4c upon Dess–Martin
periodinane mediated oxidation.
(1) Generally speaking, the cyclopropyl-P10 bearing inhi-
bitors (1a, 2a, and 3a) displayed higher potency relative to
their respective sec-butyl and a-MeBn P10 bearing coun-
terparts.(2) Similar Ki values obtained for inhibitors
bearing different P1 moieties [e.g., 3a–c vs 4a–c] suggest
that there is no clear preference at P1 position (diFAbu vs
Nva).(3) As far as P3 SAR is concerned, the results
obtained from P1-Nva series indicate that P3-t-Leu bear-
ing inhibitors 3a–c exhibited higher potency relative to the
P3-Val containing counterparts 1a–c.On the other hand,
the results obtained with P1-diFluoroAbu series (2 and 4)
did not show any preference at P3 (Table 1).
All peptidyl ketoamides synthesized (1–4) were eval-
uated in the following bioassays: (1) enzyme inhibition
assay against truncated NS3 enzyme; (2) selected potent
HCV NS3 protease inhibitors were evaluated against a
panel of relevant cellular proteases; (3) HCV sub-
genomic replicon assay for cellular activity; (4) cyto-
toxicity assay in a liver cell line (Huh-7 cells).Promising
inhibitors emerging from this set of testing were further
evaluated in vivo (rat) for drug exposure level in liver
and systemic circulation.
3. Enzyme selectivity13
Three P3 tert-Leu containing and one diFAbu P10
bearing inhibitors (3a–c, and 4a) were selected, on the
basis of their activities demonstrated in enzyme and
replicon assays as shown in Table 1, for further testing
against a panel of related cellular serine/cysteine pro-
teases.As can be seen in Table 2, with the exception of
3c, all of the HCV NS3 protease inhibitors exhibited at
least 10-fold selectivity against chymotrypsin, elastase,
cathepsins G and L.Lower selectivity (except for 3c)
against cathepsin B (an enzyme invoked in cancer
metastases) was observed for our inhibitors.In addi-
tion, no significant inhibition against kallikrein, throm-
bin, plasmin, and trypsin was detected at 100 mM for all
2. Enzyme inhibition assay12
Careful analysis of the Ki values generated from NS3
enzyme inhibition assay revealed the following trends:

260
F. Victor et al. / Bioorg. Med. Chem. Lett. 14 (2004) 257–261
Table 1. Enzyme binding affinity Ki, replicon IC50 and XTT cyto-
toxicity
improved activity in replicon assay.For example, com-
pound 3a (IC50=2.2mM) demonstrated ꢀ3-fold higher
potency than 1a (IC50=7.0 mM).Less pronounced
improvements were noted with the P1 diFAbu bearingpairs
(4 vs 2).(4) The XTT cytotoxicity assay indicated that none
of the inhibitors evaluated had significant cytotoxicity.All
IC50 values were greater than 100 mM.(5) Based on their
cellular activity and cytotoxicity data, compounds 2c, 3a,
3c, and 4c were identified as potent HCV NS3 protease
inhibitors with at least >45-fold selectivity index.
Compd
HCV Ki
PNA (mM) IC50 (mM)
Replicon
Cytotoxicity CC50/IC50 ratio
CC50 (mM)
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
0.12
1.80
2.66
0.22
0.38
0.40
0.084
0.21
0.19
0.20
0.48
0.12
7.0
>10
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>14
N/A
>30
>26
>25
>110
>45
>29
>128
>34
>38
>159
3.36
3.78
3.94
0.91
2.21
3.48
0.78
2.90
2.61
0.63
In vivo target tissue drug exposure.With the aim of
identifying orally active HCV protease inhibitors with
improved drug exposures in liver relative to that found
with 1a, compounds 2a, 3a, 3b, and 4a were dosed orally
to male Sprague–Dawley rats at a dose of 50 mg/kg.The
compounds were dosed as suspensions in a wet granu-
lation vehicle (povidone/lactose/polysorbate).Blood
was collected by orbitakl sinus and cardiac puncture
(terminal) bleeding procedures at selected time points
following dosing.Liver samples were obtained after
intracardiac perfusion of the livers with saline to remove
residual blood in the liver.Plasma and liver drug
concentrations were determined by LC/MS/MS.
five compounds listed in Table 3 (data not shown).In
view of the enzyme selectivity data generated thus far, it
is fair to say that the peptidyl ketoamide based inhibi-
tors discussed in this manuscript are quite selective
against HCV protease.
4. Replicon assay and cytotoxicity assay
Analysis of the exposure data listed in Table 3 revealed
the following in vivo SAR trends: (1) for P3 site: com-
parison of the exposure data (both liver and plasma)
obtained with 1a/3a as well as 2a/4a indicated that the
P3-tert-Leu bearing inhibitors (3a and 4a) achieved at
least 2.5-fold higher liver exposure than their corre-
sponding P3-Val bearing counterparts (1a and 2a).(2)
for P1 site: comparing the exposure data generated for
two sets of inhibitors (1a vs 2a and 3a vs 4a) clearly
showed that P1-Nva bearing inhibitors (1a and 3a) were
able to deliver at least 4-fold more drug to liver than
their respective P1-diFAbu bearing inhibitors (2a and
4a).Similar SAR trend was also observed in systemic
circulation.(3) for P1 0 site: when compared with its P10-
cyclopropyl bearing inhibitor 3a, the P10-(s)-sec-butyl
bearing derivative 3b achieved similarliver and plasma drug
exposure.(4) in order to identify promising HCV protease
All NS3 protease inhibitors discussed herein (1–4) were
also tested in a modified HCV subgenomic replicon
assay14 as well as in the XTT cytotoxicity assay in Huh-
7 liver cells.15 Careful reviewing of the IC50 values from
replicon assay shown in Table 1 indicated the following
trends: (1) In all cases, the P10 (s)-MeBn bearing inhibi-
tors (1c–4c) were found to be the most potent ones
within each series (c vs a or b).Compounds 2c, 3c, and
4c demonstrated impressive IC50 values less then 1 uM.
(2) In terms of P1 SAR, the diFAbu P1 bearing inhibi-
tors 2a–c were proved to be superior to their Nva P1
bearing counterparts 1a–c.In contrast to this finding,
however, we did not observe any P1 preference for the t-
Leu P3 bearing series inhibitors (3 and 4).(3) When
compared with their P3 Val bearing counterparts (1 and
3), all t-Leu P3 containing inhibitors (2 and 4) exhibited
Table 2. Enzyme binding affinity Ki’s (nM) or x-fold of selectivity
Compd
HCV NS3
Elastase
Cathepsin B
Cathepsin G
Cathepsin L
Chymotrypsin
1a
3a
3b
3c
4a
123
84
210
190
200
38x
41x
36x
6x
4x
1.6x
3.7x
52x
5x
683x
492x
1061x
229x
1370x
12x
154x
52x
29x
30x
583x
IC50>100 ꢀM
IC50>100 mM
62x
15x
303x
Table 3. In vivo exposure of 1a, 3a, 3b, and 4a following oral administration @50 mg/kg
Compd
Liver exposure
Cave.(0À8 h) (mM)
Plasma exposure
AUC (0–8 h) (mgÂh/mL)
C liver/IC50 (replicon)
C8 h (mM)
C8 h (mg/mL)
1a
2a
3a
3b
4a
6.29
ꢁ0.88
16
14
3.64
0.50
BDLa
3.67
8.68
0.67
2.86
0.145
25.83
22.27
1.88
0.01
0.02
0.52
0.92
0.04
0.9
ꢁ0.23
7.2
4.0
1.3
a BDL, Below Detection Limit.

F. Victor et al. / Bioorg. Med. Chem. Lett. 14 (2004) 257–261
261
inhibitors for further ADME and toxicology evaluations,
References and notes
we ranked the compounds on the basis of the ratio of
Cliver ave:ð0À8 hÞ=IC50ðrepliconÞ calculated for inhibitors
included in Table 3.Our preference decreases according to
the following order: 3a >3b >4a >1a >2a.The best
compound disclosed in this manuscript, 3a, was about 7
times better than the previously reported inhibitor 1a.
1.For an overview of HCV, see: Bartenschlagar, R. Anti-
viral Chem. Chemother. 1997, 8, 281.
2.(a) For recent reviews, see: Dymock, B.W. Emerg. Drugs
2001, 6, 13. (b) Dymock, B. W.; Jones, P. S.; Wilson, F. X.
Antiviral Chem. Chemother. 2000, 11, 79.
3. Zeuzem, S.; Feinman, S. V.; Rasenack J. New Engl. J.
Med. 2000, 343, 1666.
4. Heathcote, E. J; Shiffman, M. L.; Cooksley, W. G. E.;
Dusheiko, G. M.; Lee, S. S.; Balart, L.; Reindollar, R.;
Reddy, R. K.; Wright, T. L.; Lin, A.; Hoffman, J.; De
Pamphilis, J. New Engl. J. Med. 2000, 343, 1673.
5. Kwong, A. D.; Kim, J. L.; Rao, G.; Lipovsek, D.; Ray-
buck, S.A. Antiviral Res. 1998, 40, 1.
6. Attwood, M. R.; Bennett, J. M.; Campbell, A. D.; Can-
ning, G. G. M.; Carr, M. G.; Conway, E.; Dunsdon,
R. M.; Greening, J. R.; Jones, P. S.; Kay, P. B.; Handa,
B. K.; Hurst, D. N.; Jennings, N. S.; Jordan, S.; Keech,
E.; O’Brien, M. A.; Overton, H. A.; King-Underwood, J.;
Raynham, T. M.; Stenson, K. P.; Wilkinson, C. S.; Wilk-
5. Conclusion
Starting from the previously identified bicycloproline P2
bearing HCV protease inhibitor 1a, we incorporated
further structural modifications at P1 (diFAbu as
replacement for Nva) or/and at P3 (tert-Leu as replace-
ment for Val).On the basis of enzyme inhibition data
shown in Table 2, it is clear that neither P1 nor P3
modification had significant impact on enzyme binding
affinity (Ki).However, when compared with their P1-
Nva bearing counterparts 1a–c, all three P1-diFAbu
bearing inhibitors 2a–c exhibited improved (2-fold)
activity in the replicon assay.Parallel with this finding,
all three P3-t-Leu containing inhibitors 3a–c demon-
strated enhanced cellular activity (3-fold) than their
corresponding P3-Val analogues 1a–c.In view of the
exposure data shown in Table 3, it is evident that
incorporation of P1-diFAbu moiety resulted in 4-(3a vs
4a) to 7-(1a vs 2a) fold reduction in liver drug exposure.
In contrast to this observation, introduction of the t-
Leu at P3 (as seen in 3a) led to ꢀ2.5-fold improvement
in drug exposure in liver relative to that found with the
P3-Val bearing counterpart 1a.More pronounced
enhancement in liver drug exposure was detected for 4a
relative to 2a.Careful inspection of the data presented
in Table 2 clearly indicates that compound 3a was the
most promising one endowed with good enzyme binding
affinity, replicon activity, acceptable enzyme specificity,
and excellent drug liver exposure yet without inherent
cytotoxicity.On the basis of these data, compound 3a
was selected for further evaluation at NovaScreen.
When tested against 80 different receptors and enzyme
targets, compound 3a only showed >50% inhibitory
effect against elastase (at 10 mM).Thus, in light of the
encouraging data generated for 3a, we are convinced
that further optimization of the bicycloproline P2 bear-
ing peptidyl ketoamide series could lead to novel HCV
protease inhibitors with therapeutic utilities.
inson, T.C.I;. Wilson, F.X.
1999, 10, 259.
Antiviral Chem. Chemother.
7. (a) Yip, Y.; Victor, F.; Lamar, J.; Johnson, R.; Wang,
Q. M.; Barket, D.; Glass, J.; Jin, L.; Lui, L.; Venable,
D.; Wakulchik, M.; Xie, C.; Heinz, B.; Villarreal, E.;
Colacino, J.; Yumibe, N.; Tebbe, M.; Munroe, J.; Chen,
S.-H. Bioorg. Med. Chem. Lett 2004, 14, preceeding
paper in this issue. doi: 10.1016/j.bmcl.2003.09.074.
(b) PCT Patent (Eli Lilly): WO/02/18369 A2, Mar.7,
2002.(c) For a recent paper describing tetrapeptidyl a-
ketoamides, see: Han, W.; Hu, Z.; Jiang, X.; Wasserman,
Z.R;. Decicco, C.P. Bioorg. Med. Chem. Lett. 2003, 13,
1111.
8. (a) Ingallinella, P.; Altamura, S.; Bianchi, E.; Taliani, M.;
Ingenito, R.; Cortese, R.; De Francesco, R.; Steinkuhler,
C.; Pessi, A. Biochemistry 1998, 37, 8906.(b) Narjes, F;.
Brunetti, M.; Colarusso, S.; Gerlach, B.; Koch, U.; Bia-
siol, G.; Fattori, D.; De Francesco, R.; Matassa, V. G.;
Steinkuhler, C. Biochemistry 2000, 39, 1849.
9.HOAT/DCC(DIC) was used to minimize racemization
occurred at a-carbon during peptide coupling; cf.: Car-
pino, L.A;. El-Faham, A. Tetrahedron 1999, 55, 6813.
10.P1-Nva a-hydroxyacid was prepared from Z-Val(H) via
its corresponding cyanohydrin intermediate.
11. (a) Matassa, V.; Narjes, F.; Koehler, K.; Ontoria, J.; Poma,
M.; Marchetti, A. PCT Patent WO 99/64442, Dec. 16,
1999.(b) Winkler, D;. Burger, K. Synthesis 1996, 1419.
12.The detailed protocol for HCV NS3 inhibition assay was
performed according to that described in ref 19 of the
proceeding manuscript. doi: 10.1016/j.bmcl.2003.09.076.
13.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.
Acknowledgements
We would like to thank the chemists at Vertex Pharma-
ceuticals for sharing their expertise in connection to HCV
protease inhibitor and HCV replicon assay design with us.
K.Case, J.Catlow and J.Eckstein are acknowledged for
technical assistance.We thank D.Barket, L.Jin, L.Liu,
D.Venable, M.Wakulchik, C-.P.Xie for performing
HCV replicon and XTT cytotoxicity assays.We are also
indebted to Drs.M.Tebbe, J.Munroe, J.Audia, B.
Heinz, E.Villarreal, J.Colacino, C.Lopez, and G.Cassell
for helpful discussions and encouragement.
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.;
J. Immunol. Methods
Hatfield, S.M;. Glasebrook, A.L.
1991, 142, 257.
16.Overall yields for the preparation of 1a: 51%; 1b: 31%;
1c: 16%; 2a: 7%; 2b: 8%; 2c: 5%; 3a: 41%; 3b: 63%; 3c:
48%; 4a: 33%; 4b: 21%; 4c: 7%.