Depeptidization efforts on P3-P′2 α-ketoamide inhibitors of HCV NS3-4A serine protease: Effect on HCV replicon activity

Article abstract of DOI:10.1016/j.bmcl.2005.12.013

Depeptidization efforts of the P₃-P₂ region of P ₃ capped α-ketoamide inhibitor of HCV NS3 serine protease 1 are reported. We clearly established that N-methylation of the P₂ nitrogen and modification of the P2′

Full text of DOI:10.1016/j.bmcl.2005.12.013

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1621–1627
Depeptidization efforts on P3–P02 a-ketoamide inhibitors
of HCV NS3-4A serine protease: Effect on HCV replicon activity
*
´
Stephane L. Bogen, Sumei Ruan, Rong Liu, Sony Agrawal,
John Pichardo, Andrew Prongay, Bahige Baroudy,
Anil K. Saksena, Viyyoor Girijavallabhan and F. George Njoroge
Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
Received 9 September 2005; revised 3 November 2005; accepted 7 December 2005
Available online 4 January 2006
Abstract—Depeptidization efforts of the P3–P2 region of P3 capped a-ketoamide inhibitor of HCV NS3 serine protease 1 are report-
ed. We clearly established that N-methylation of the P2 nitrogen and modification of the P02 carboxylic acid terminus were essential
for activity in the replicon assay.
Ó 2005 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV), a small (+)-RNA virus belong-
Arg
155
P2
ing to the Flaviviridae family, infects 200 million people
worldwide.1 Untreated HCV infections can progress to
liver cirrhosis and hepatocellular carcinoma.2 Currently,
antiviral drug ribavarin in combination with the im-
mune system booster a-interferon is the only available
treatment.3 Although combination therapy is reason-
ably successful with genotypes 2 and 3, its efficacy
against the predominant genotype 1 is moderate at best.
Therefore, several research groups have been working
toward the development of a more effective, convenient,
and tolerable treatment. Because of its vital role in viral
replication,4 HCV NS3 serine protease has been actively
pursued as a viral protein target.5 Oligopeptide deriva-
tives containing a-ketoamide electrophilic trap have
been reported by our group6 and others7 to be potent
inhibitors of HCV NS3 serine protease.
O
H
N
O
HN
H
N
1, Ki* = 0.015 µM
O
O
HN
O
O
Lys
136
O
NH
O
P1
P3
OH
P2'
Figure 1.
Ala 157
Ar 155
P2
Serine trap
O
O
O
P2'
H
H
H
N
N
N
OH
N
H
N
H
P3
O
P1
O
O
More recently, we demonstrated, through inhibitor 1,
that introduction of a cyclopropyl alanine side chain
both at P2 and P1 combined with a phenylglycine residue
at P02 improved the binding potency of our earlier P3
capped inhibitors by about 10-fold (Fig. 1).8 However,
we reasoned that less peptidic inhibitors would be more
desirable not only for cellular activity but also to get
benefits such as oral bioavailability and improved phar-
macokinetics. From the hydrogen bond model depicted
Gly 137
Ser 139
P3-P2 Area
Thr 42
Figure 2.
in Figure 2, it is clear that substrate and inhibitor were
bonded through a combination of hydrogen bonds
and side-chain hydrophobic interactions. In designing
our peptide modification, we had to make sure that
parts of the ligand that formed crucial H-bonds with
the enzyme backbone were left in place. Therefore, the
P3–P2 area of inhibitor 1 was targeted for depeptidiza-
tion (Fig. 2).
*
Corresponding author. Tel.: +1 908 740 4642; fax: +1 908 740
7152; e-mail: stephane.bogen@spcorp.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.013

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S. L. Bogen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1621–1627
Table 1.
Entry
Compound
Structure
Kꢀi (lM)
Replicon
IC90 (lM)
O
O
O
H
N
H
N
H
N
O
O
OH
1
2
1
0.015
>5.0
>5.0
N
N
H
H
O
O
O
O
O
OH
OH
O
O
O
O
H
N
H
N
H
N
OH
29
30
31
32
33
34
35
42.0
10.0
2.1
N
H
N
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
N
H
N
H
N
O
O
O
O
O
O
N
N
N
OH
OH
OH
OH
3
4
5
6
7
8
>5.0
>5.0
>5.0
>5.0
0.95
N
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
N
H
N
H
N
N
H
O
H
N
H
N
H
N
0.23
0.12
0.06
0.05
N
N
H
H
O
O
O
H
N
H
N
H
N
N
N
H
Me
Me
N
H
N
H
N
H
N
N
N
H
Me
Me
Me
N
H
N
H
N
H
N
> 5.0
N
H
N
H
Me
The preparation of compound 29 (Table 1) is depicted in
Schemes 1 and 2. The protected c-amino acid 5 used in
the synthesis of 29 was prepared as shown in Scheme 1.
Thus, Boc-L-cyclohexylglycine 2 was converted to the
corresponding Weinreb amide that was reduced with
LiAlH4 to give carbinol 3 in high yield. The protocol

S. L. Bogen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1621–1627
1623
OH
Preparation of the a-hydroxyl amide core of our inhib-
itors is depicted in Scheme 2. Carbinol 7, prepared fol-
lowing the same methodology used for the preparation
of 3, was subjected to the Passerini reaction condi-
tions.10 Thus, use of methyl-isocyanoacetate and acetic
acid in CH2Cl2 generated the acetamides 8 as a mixture
of diastereomers in good yield. The acetate protecting
group and the methyl ester moiety of 8 were removed
in one pot with LiOH. The resulting carboxylic acid
was then subjected to standard coupling conditions
(EDCI, HOOBt, and NMM) with protected phenylgly-
cine amino acid to deliver the P1–P02 intermediate 9.
After chemoselective removal of the t-Boc-protecting
group of 9, the cyclopropylalanine moiety 6 was incor-
porated at P2 using the coupling and deprotection con-
ditions described above to deliver the HCl salt 10 for
final assembly. Thus, intermediate 10 and protected
c-amino acid 5 prepared earlier were subjected to
aforementioned coupling conditions to provide, after
Dess–Martin’s periodinane oxidation,11 the desired
a-ketoamide 11 in excellent yield. One-pot deprotection
of the acetonide and the terminal tert-butyl ester moie-
ties was performed with a 50% TFA in CH2Cl2 solution
and provided the desired target 29 in high yield (Scheme
2).12
O
BocHN
CO2Et
BocHN
CO2H
BocHN
H
a
b
3
4
2
O
CO2H
c
i-BocN
5
Scheme 1. Reagents and conditions: (a) i—HClÆHN(OMe)Me, EDCI,
NMM, CH2Cl2, ꢁ10 °C (100%); ii—LAH, THF, ꢁ30 °C (100%); (b)
LDA, MeCO2Et, ZnBr2, THF, ꢁ78 °C, (49% yield, 80% de); (c) i—
4 M HCl, dioxane, (100%); ii—i-BuOCOCl, Na2CO3, dioxane, H2O,
(74%); iii—DMP, Et2OÆBF3, (78%); iv—aq 1 N LiOH, THF/H2O
(95%).
O
OAc
O
H
BocHN
BocHN
CO2H
BocHN
N
CO2Me
H
b
a
8
6
7
OH
O
d
H
c
Preparation of compound 30 (Table 1) is depicted in
Schemes 3 and 4. A different approach for the synthesis
of this compound was adopted to avoid extensive protect-
ing group manipulation. Thus, the threo carbamate 12
(Scheme 3) was constructed from Boc-L-cyclohexylgly-
cine 2 in four steps following the methodology developed
by Luly et al.13 Addition of aminomethylcyclopropane to
epoxide 12 was achieved by heating both components
together in MeOH at 60 °C providing the amine 13 in
85% yield. The preparation of the a-hydroxyl amide core
of inhibitor 30 was anticipated to proceed again via a
Passerini reaction, albeit on a more elaborate intermedi-
ate 15 (Scheme 4). Thus, conversion of the Boc-L-cyclo-
propylalanine 6 to the corresponding Weinreb amide
followed by removal of the t-Boc-protecting group under
acidic condition provided the HCl salt 14. Amino alcohol
13 (Scheme 3), treated with carbonyl-diimidazole and
Et3N in CH2Cl2, readily reacted with amine 14 to give
urea peptoid 15. The corresponding aldehyde of 15, ob-
tained by LAH reduction, was successfully subjected to
the Passerini conditions as described earlier to provide
acetates 16 in 72% yield. Protecting group manipulation
on the amino end of 16 was performed as shown in
Scheme 4 to introduce the i-Boc and the acetonide func-
tionalities. Acetate and methyl ester hydrolysis was per-
BocHN
N
N
CO2t-Bu
H
9
O
OH
O
e
H
H
N
N
HCl.H2N
i-BocN
N
CO2t-Bu
H
O
O
10
O
O
O
O
f
H
H
29
N
N
N
N
H
CO2t-Bu
H
O
O
11
Scheme 2. Reagents and conditions: (a) i—HClÆHN(OMe)Me, EDCI,
NMM, CH2Cl2, ꢁ10 °C (100%); ii—LAH, THF, ꢁ30 °C (95%); (b)
AcOH, methyl-isocyanoacetate, CH2Cl2, (70% yield, 2 to 1 ratio);
(c) i—aq 1 N LiOH, THF/H2O; (100%); ii—H-PhG-O-t-Bu, EDCI,
HOOBt, NMM, CH2Cl2, ꢁ20 °C, (95%); (d) i—4 M HCl in dioxane,
(100%); ii—6, EDCI, HOOBt, NMM, CH2Cl2, ꢁ20 °C, (52%); iii—
4 M HCl in dioxane, (100%); (e) i—5, EDCI, HOOBt, NMM, CH2Cl2,
(41%); ii—Dess–Martin’s periodinane, CH2Cl2, (100%); (f) 50% TFA
in CH2Cl2, 1 h, then n-heptane, (95%).
developed by Roberts et al.9 was applied to 3 to intro-
duce the hydroxyethylene scaffold. Thus, condensation
of aldehyde 3 with the zinc enolate of ethyl acetate at
ꢁ78 °C furnished the 3S,4S b-hydroxy-c-amino ester
5. Protecting group manipulation on the amino end
was performed to introduce the i-Boc functionality of
the P3 capped inhibitors. The hydroxyl and the carba-
mate amino groups were tied up as acetonide using
DMP and Et2OÆBF3 to provide, after base hydrolysis
of the methyl ester group, the desired protected c-amino
acid 5 in 27% overall yield (Scheme 1).
OH
O
BocNH
BocNH
NH
a
12
13
Scheme 3. Reagents and conditions: (a) aminomethylcyclopropane,
MeOH, 60 °C, 16 h, (85%).

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S. L. Bogen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1621–1627
Scheme 4. Reagents and conditions: (a) 4 M HCl in dioxane, (100%); (b) 13, CDI, Et3N, CH2Cl2, (47%); (c) i—LAH, THF, ꢁ30 °C; ii—AcOH,
methyl-isocyanoacetate, CH2Cl2, (72% over two steps); (d) i—4 M HCl in dioxane, (100%); ii—i-BuOCOCl, CH2Cl2, DIPEA, (83%); iii—DMP,
Et2OÆBF3, (70%); iv—aq 1 N LiOH, THF/H2O (100%); v—H-PhG-O-t-Bu, HATU, DMF, DIPEA, ꢁ20 °C, (95%); (e) i—Dess–Martin’s
periodinane, CH2Cl2, (91%); ii—50% TFA in CH2Cl2, 1 h, then n-heptane (100%).
formed as described earlier and the resulting carboxylic
b
a
acid was subjected to coupling conditions with protected
phenylglycine amino acid to deliver the P3–P02 intermedi-
ate 17 in high yield. Dess–Martin’s periodinane oxidation
of a-ketoamide 17 followed by a one-pot deprotection of
the acetonide and the terminal tert-butyl ester moieties,
with a 50% TFA in CH2Cl2 solution, provided the desired
target 30.
NH
OSO2Ar
20
OH
BocNH
21
19
c
d
O
OAc
O
O
H
H
H
N
N
N
N
N
BocNH
N
O
BocNH
O
O
O
Preparation of compound 31 (Table 1) is outlined in
Scheme 5. Thus, the acetonide moiety of a-ketoamide
17 was removed chemoselectively with a 0.5 M solution
of TFA in CH2Cl2. Dess–Martin’s periodinane oxida-
tion of the resulting diol provided a-ketoamide 18 in
good yield. Deprotection of the terminal tert-butyl ester
group with a 50% TFA in CH2Cl2 solution yielded the
desired target 31.
22
23
e
f
OH
O
H
H
N
N
N
Ot-Bu
HCl.H2N
N
H
O
O
O
24
O
O
O
g
H
H
N
Preparation of compound 32 (Table 1) is depicted in
Scheme 6. First attempts to prepare hydrazine 21 via
reduction of the corresponding hydrazone were unsuc-
cessful and led only to the dicyclopropyl adduct.14 Syn-
thesis of the desired hydrazine 21 was achieved via the
displacement of tosylate 20 in 60% yield. About 20 equiv
of tert-butyl carbazate were required to observe good
chemoselectivity. Using conditions similar to those de-
scribed earlier, carbonyl-diimidazole was used to couple
amine 14 and the hydrazine 21. The resulting Weinreb
amide 22 was reduced to the corresponding aldehyde
that was immediately subjected to the Passerini condi-
32
i-BocNH
N
N
Ot-Bu
N
N
H
H
O
O
O
25
Scheme 6. Reagents and conditions: (a) TosCl, pyridine, 0 °C, (66%);
(b) tert-butylcarbazate, EtOH, 0–40 °C, (60%); (c) 14, CDI, Et3N,
CH2Cl2, (30%); (d) i—LAH, THF, ꢁ30 °C; ii—AcOH, methyl-
isocyanoacetate, CH2Cl2, (70% over two steps); (e) i—aq 1 N LiOH,
THF/H2O (77%); ii—H-PhG-O-t-Bu, HATU, DMF, DIPEA, ꢁ20 °C,
(90%); iii—4 M HCl in dioxane, (100%); (f) i—i-Boc-cyclohexylglycine,
EtOCOCl, THF, Et3N, ꢁ5 °C, (50%); ii—Dess–Martin’s periodinane,
CH2Cl2, (95%); (g) 50% TFA in CH2Cl2, 1 h, then n-heptane (100%).
tions to yield 23. As described earlier, after acetate
and methyl ester hydrolysis, the resulting carboxylic acid
was subjected to coupling conditions with protected
phenylglycine amino acid which delivered, after chemo-
selective removal of the Boc-protecting group, the P2–P20
hydrazine intermediate 24 in high yield. However, incor-
poration of the last residue at P3 was problematic. Thus,
standard coupling condition (HATU and DIPEA) with
i-Boc-cyclohexylglycine did proceed, albeit in very poor
yield. Fortunately, preparation of the mixed anhydride
of the cyclohexylglycine moiety followed by displace-
O
O
O
a
H
H
H
17
O
N
N
N
N
OtBu
N
H
O
O
O
O
18
b
31
Scheme 5. Reagents and conditions: (a) i—0.5 M TFA in CH2Cl2; ii—
Dess–Martin’s periodinane, CH2Cl2, (73%); (b) 50% TFA in CH2Cl2,
1 h, then n-heptane (95%).

S. L. Bogen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1621–1627
1625
ment with the hydrazine 24 led, after Dess–Martin oxi-
dation, to the desired azapeptide 25 in 47% isolated
yield. Deprotection of the terminal tert-butyl ester group
with a 50% TFA in CH2Cl2 solution yielded the desired
target 32.
Introduction of a hydroxyethylene spacer between P3
and P2 was detrimental for the activity of 29 with a Kiꢀ
of 42 lM. The non-peptidic linkage at the bond between
P2 and P3 building blocks, longer than the correspond-
ing peptide bond, displaced the P3 side chain outwardly
relative to a native peptide substrate. The loss of interac-
tion with Ala 157 certainly contributed to the loss in
activity. Thus, we decided to keep the length of the P3
side chain identical to that of the native peptide sub-
strate for later compounds. We then turned our efforts
toward the incorporation of a urea peptoid moiety in
the P3–P2 region. Replacement of the a-carbon by a tri-
valent nitrogen was realized and led to the urea peptoid
30. We envisioned that the hydroxyl group of 30 was
well positioned to interact with Ala 157 but unfortunate-
ly, compound 30, with a Kꢀi ¼ 10 lM, lacked good affin-
ity with the enzyme backbone. Importance of the
interaction with Ala 157 was further demonstrated with
the oxidation of hydroxyl group of compound 30. Thus,
incorporation of a carbonyl function at P3 had a positive
effect on the potency: inhibitor 31, with a Kꢀi ¼ 2:1 lM,
show a 5-fold improvement in potency compared to 30.
Since lack of inhibitory activity of peptidomimetics 30
and 31 could also be attributed to the loss of asymmetry
associated with this transformation, we decided to intro-
duce an aza-amino acid moiety in that position. While
some researchers have considered that the loss of asym-
metry associated with this transformation could lead to
a conformation that can be considered intermediate be-
tween the D- or L-amino acid, others have shown that
the adjacent nitrogen could confer a sp3 character to
the a-trivalent nitrogen that could largely preserve the
original peptide conformation in the resulting peptido-
mimetic.17 Thus, incorporation of cyclopropyl aza-
amino acyl fragment at P2 led to inhibitor 32. While
azapeptide 32 ðKꢀi ¼ 0:23 lMÞ was substantially less po-
tent than inhibitor 1 ðKꢀi ¼ 0:015 lMÞ, incorporation of
the cyclopropyl aza-amino acid motif at P2 resulted in
10-fold improvement in activity compared to urea pep-
toid 31 ðKꢀi ¼ 2:1 lMÞ. X-ray crystal structure of the
inhibitor 32 bound to the protease is shown in Figure
3.18 It can be seen that the peptidic core binds to the
protease through a series of hydrogen bonding interac-
Preparation of compounds 33 and 34 (Table 1) is out-
lined in Scheme 7. Thus, Boc-L-cyclopropylalanine 6
was converted to the N-methyl carbamate 26 following
a two-step, high-yielding, sequence. Removal of the
Boc-protecting group under acidic condition provided
the HCl salt 27. Coupling with i-Boc cyclohexylglycine
followed by hydrolysis of the methyl ester moiety pro-
vided intermediate 28. Following a similar methodology
as shown in Scheme 2 for the preparation of target 29,
compound 9 was used to deliver inhibitor 33. Standard
coupling conditions with dimethylamine hydrochloride
provided the dimethyl amide P02 capped inhibitor 34.
Recently, we reported the discovery of inhibitor 1 as a
potent inhibitor of the hepatitis C virus NS3-4A serine
protease.8 While 1 showed very good enzyme inhibitory
activity ðKꢀi ¼ 0:015 lMÞ, it obviously lacked drug-like
properties. Alteration of peptides to peptidomimetics
could afford compounds with improved biological
potencies and increased resistance to enzymatic degra-
dation. It was therefore decided to modify inhibitor 1
to bring in these desirable attributes. After analyzing
the H-bond network outlined in Figure 2, we targeted
the P3–P2 area of inhibitor 1 for depeptidization.
HCV NS3 serine protease inhibitory activity and HCV
replicon inhibitory activity for the targets synthesized
were obtained using previously reported assays.15,16
Me
HCl.HN
O
Me
BocN
O
OMe
a
b
OMe
6
26
27
Me
OH
d
N
c
i-BocHN
O
O
28
O
O
e
H
N
H
34
Me
N
OH
N
N
H
i-BocHN
O
O
O
O
33
Scheme 7. Reagents and conditions: (a) i—NaH (3 equiv), MeI
(8 equiv), THF; ii—CH2N2, Et2O (90%); (b) 4 M HCl in dioxane,
(100%); (c) i—i-Boc-cyclohexylglycine, DEPBT, DIPEA, THF (50%);
ii—aq 1 N LiOH, THF/H2O (83%); (d) i—9, 4 M HCl in dioxane,
(100%); ii—EDCI, HOOBt, NMM, CH2Cl2, ꢁ20 °C, (92%); iii—Dess–
Martin’s periodinane, CH2Cl2, (100%); iv—50% TFA in CH2Cl2, 1 h,
then n-heptane, (95%); (e) HClÆHNMe2, EDCI, HOOBt, NMM,
CH2Cl2, ꢁ20 °C, (45%).
Figure 3. X-ray structure of 32 bound to the protease.

1626
S. L. Bogen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1621–1627
aimed at the incorporation and optimization of proline
moieties at P2 is under progress and will be reported
shortly.
References and notes
1. Consensus Panel. EASL International Consensus Confer-
ence on Hepatitis C, Paris, February 26–28, 1999, Con-
sensus Statement. J. Hepatol. 1999, 30, 956.
1: sp3 center
32: Planarity of azapetide
2. (a) Cohen, J. Science 1999, 285, 26; (b) Houghten, M. In
Virology; Fields, B. N., Knipe, D. M., Howley, P. M.,
Eds.; Raven Press: New York, 1996; pp 1035–1058; (c)
Cuthbert, J. A. Clin. Microbiol. Rev. 1994, 7, 505.
3. Dymock, B. W. Emerg. Drugs 2001, 6, 13, and references
cited therein.
4. (a) Kolykhalov, A. A.; Mihalik, K.; Feinstone, S. M.;
Rice, C. M. J. Virol. 2000, 74, 2046; (b) Bartenschlager,
R.; Lohmann, V. J. Gen. Virol. 2000, 81, 1631.
5. (a) De Francesco, R.; Tomei, L.; Altamura, S.; Summa,
V.; Migliaccio, G. Antiviral Res. 2003, 58, 1; (b) Stein-
kuhler, C.; Koch, U.; Narjes, F.; Matassa, V. G. Curr.
Med. Chem. 2001, 8, 919; (c) Kwong, A. D.; Kim, J. L.;
Rao, G.; Lipovsek, D.; Raybuck, S. A. Antiviral Res.
1998, 40, 1.
6. (a) Arasappan, A.; Njoroge, F. G.; Chan, T. Y.; Bennett, F.;
Bogen, S.; Chen, K.; Gu, H.; Hong, L.; Jao, E.; Liu, Y.-T.;
Lovey, R. G.; Parekh, T.; Pike, R. E.; Pinto, P.; Santhanam,
S.; Venkatraman, V.; Vaccaro, H.; Wang, H.; Yang, X.;
Zhu, B.; Mckittrick, B.; Saksena, A. K.; Girijavallabhan,
V.; Pichardo, J.; Butkiewicz, N.; Ingram, R.; Malcolm, B.;
Prongay, A.; Yao, N.; Marten, B.; Madison, V.; Kemp, S.;
Levy, O.; Lim-Wilby, M.; Tamura, S.; Ganguly, A. K.
Bioorg. Med. Chem. Lett. 2005, 15, 4180; (b) Arasappan, A.;
Njoroge, F. G.; Parekh, T. N.; Yang, X.; Pichardo, J.;
Butkiewicz, N.; Prongay, A.; Yao, N.; Girijavallabhan, V.
Bioorg. Med. Chem. Lett. 2004, 14, 5751; (c) Saksena, A. K.,
et al., U.S. Patent 6800434 B2, 2004.
Figure 4. P2–P1 region geometry of compounds 1 and 32 bound to the
protease.
tions. In the P2–P1 region, analysis of the X-ray struc-
ture revealed a planar geometry for the azapeptide motif
compared to the sp3 geometry of the P2 amino acid residue
of 18 (Fig. 4). Consequently, we decided to keep the ste-
reochemistry of the P2 side chain identical to that of the
native peptide substrate and turned our efforts toward
the replacement of the a-hydrogen of the P2 amino acid.
Replacements of the a-hydrogen of the common amino
acids by a methyl group had been reported as another
example of depeptidization usually referred to as a-alkyl
modification.19 N-Methylation of the amide linkage at
P2 was found to be not as detrimental as some of the
earlier modifications. Compound 33, with a Kꢀi ¼
0:12 lM, was the most active peptidomimetic synthe-
sized. Targets 1 and 29–33 included in Table 1 were pre-
pared with a carboxylic acid residue at P02. In spite of
the fact that these compounds had good enzyme inhib-
itory activity, none of the derivatives showed cellular
activity with IC90 values less than 5 lM against HCV
replicon.16 This large difference was certainly related
to the physicochemical features of these compounds
such as solubility and cell penetration. According to
the H-bond model depicted in Figure 2, the hydroxyl
group of the carboxylic acid residue at P02 did not form
any crucial interactions with the enzyme backbone. We
carried out modifications aimed at removing the
charged residue.
7. (a) Bennett, J. M.; Campbell, A. D.; Campbell, A. J.; Carr,
M. G.; Dunsdon, R. M.; Greening, J. R.; Hurst, D. N.;
Jennings, 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. Lett. 2001, 11, 355; (b) Han, W.; Hu,
Z.; Jiang, X.; Decicco, C. P. Bioorg. Med. Chem. Lett.
`
´
2000, 10, 711; (c) Llinas-Brunet, M.; Bailey, M.; Deziel,
R.; Fazal, G.; Gorys, V.; Goulet, S.; Halmos, T.; Maurice,
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We discovered that incorporation of a dimethyl amide
cap at the terminal carbonyl provided, for the first time,
activity in the replicon assay. Thus, inhibitor 34 (Table
1) with a Kꢀi of 0.06 lM exhibited the best cellular poten-
cy in that series with IC90 = 0.95 lM. To further estab-
lish the importance of masking the NH at P2, we also
prepared compound 35. Inhibitor 35, with a Kꢀi of
0.05 lM and a replicon IC90 > 5 lM, clearly established
that removal of the charged residue at P02 alone was not
enough to observe inhibition of the HCV replicon.
8. Bogen, S.; Saksena, A. K.; Arasappan, A.; Gu, H.;
Njoroge, F. G.; Girijavallabhan, V.; Pichardo, J.; But-
kiewicz, N.; Prongay, A.; Madison, V. Bioorg. Med.
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9. Roberts, D. A.; Bradbury, R. H.; Brown, D.; Faull, A.;
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Ratcliffe, A. H.; Revill, J.; Waterson, D. J. Med. Chem.
1990, 33, 2326.
In summary, depeptidization of our earlier P3 capped
inhibitor led to the identification of a potent inhibitor of
the HCV NS3 serine protease and with good activity
against HCV replicon. N-Methylation at P2 and replace-
ment of the charged residue at P02 with a dimethyl amide
cap did not produce any substantial increase in enzyme
inhibitory activity but were essential for inhibition of
the HCV replicon system. N-Methylation at P2 seemed
to overcome an apparent ‘defect at P2’ conferred by the
absence of a proline residue. Consequently, further work
10. (a) Passerini, M.; Ragni, G. Gazz. Chim. Ital. 1931, 61,
964; (b) Marquarding, D.; Gokel, G.; Hoffmann, P.; Ugi,
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7277.
12. Use of large excess of heptane is required during work-up
to avoid concentration of TFA and subsequent elimina-
tion of the hydroxyl group.

S. L. Bogen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1621–1627
1627
13. Luly, J. R.; Dellaria, J. F.; Plattner, J. J.; Soderquist, J. L.;
Yi, N. J. Org. Chem. 1987, 52, 1487.
14. tert-Butylcarbazate and cyclopropylcarboxyaldehyde were
used to prepare the corresponding hydrazone. Reduction
with NaBH4, NaBH3CN, or NaBH(OAc)3 led only to the
dicyclopropyl adduct below.
Njoroge, F. G.; Windsor, W. T.; Malcolm, B. A. Anal.
Biochem. 1999, 270, 268, For the present study, the
substrate Ac-DTEDVVP(Nva)-O-PAP was employed.
16. Replicon assay: (i) Lohmann, V.; Korner, F.; Koch, J.-O.;
Herian, U.; Theilmann, L.; Bartenschlager, R. Science
1999, 285, 110; (ii) Blight, K.; Kolykhalov, A.; Rice, C.
Science 2000, 290, 1972.
17. (i) Dutta, A. S.; Giles, M. J. Chem. Soc., Perkin Trans. 1
1976, 244; (ii) Gante, J. Synthesis 1989, 405.
18. Crystallographic data for the structures in this paper have
been deposited with the RCSB Protein Data Bank as PDB
ID 2F9V. The structural details can be viewed at
www.rcsb.org using the ID number above.
19. Spatola, A. F. In Chemistry and Biochemistry of Amino
Acids, Peptides, and Proteins; Weinstein, B., Ed.; Marcel
Dekker: New York, 1983; pp 267–357.
O
N
O
N
H
15. Enzyme assay: the HCV NS3 serine protease inhibitory
value is provided as Kꢀi . For a definition of Kiꢀ and
discussion, see: (i) Morrison, J. F.; Walsh, C. T. In
Meister, A., Ed. Adv. Enzymol. 1988, Vol. 61, 201–301;
(ii) Zhang, R.; Beyer, B. M.; Durkin, J.; Ingram, R.;