Hepatitis C virus NS3-4A serine protease inhibitors: Use of a P 2-P1 cyclopropyl alanine combination for improved potency

Article abstract of DOI:10.1016/j.bmcl.2005.07.009

Modification of the P₂ and P₁ side chains of earlier P₃-capped α-ketoamide inhibitor of HCV NS3 serine protease 1 resulted in the discovery of compound 24 with about 10-fold improvement in potency.

Full text of DOI:10.1016/j.bmcl.2005.07.009

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4515–4519
Hepatitis C virus NS3-4A serine protease inhibitors: Use of a P2–P1
cyclopropyl alanine combination for improved potency
S. Bogen,a,* A. K. Saksena,a A. Arasappan,a H. Gu,a F. G. Njoroge,a V. Girijavallabhan,a
J. Pichardo,b N. Butkiewicz,b A. Prongayc and V. Madisonc
aChemical Research, Schering Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
bAntiviral Therapy, Schering Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
cStructural Chemistry, Schering Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
Received 18 May 2005; revised 6 July 2005; accepted 6 July 2005
Available online 19 August 2005
Abstract—Modification of the P2 and P1 side chains of earlier P3-capped a-ketoamide inhibitor of HCV NS3 serine protease 1 result-
ed in the discovery of compound 24 with about 10-fold improvement in potency.
Ó 2005 Elsevier Ltd. All rights reserved.
P2
1. Introduction
O
H
Hepatitis C virus (HCV), a blood-borne virus previously
referred to as non-A, non-B hepatitis, infects 200 million
people worldwide.1 Untreated HCV infections can pro-
gress to liver cirrhosis and hepatocellular carcinoma.2
Currently, a-interferon and PEG-interferon monothera-
pies or in combination with the antiviral drug ribavirin
are the only approved treatment options.3 Although
combination therapy is reasonably successful with geno-
types 2 and 3, its efficacy against the predominant geno-
type 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 derivatives containing
a-ketoamide electrophilic trap have been reported by
our group6 and others7 to be potent inhibitors of HCV
NS3 serine protease.
N
O
HN
H
1
O
N
O
HN
O
O
P1
Lys
136
O
NH
O
P3
OH
P2'
Figure 1.
1 bound to the protease, it was evident that the P1–P20
side chains formed a C-clamp around Lys136 of the pro-
tease and thus provided extensive hydrophobic interac-
tion that resulted in improved potency. X-ray crystal
structure analysis also revealed a close proximity of P2
side chain to arginine 155. Unsuccessful attempts from
our group6 and others9 to make a salt bridge between
Arg155 and a variety of charged species at P2 prompted
us to evaluate the possibility of hydrophobic interac-
tions with the side chain of that residue and hydropho-
bic moieties of the P2 position. To assess the effect of
replacement of leucine at P2 with more lypophilic resi-
dues, we decided to take advantage of hydrophobic sul-
fide moiety by introducing thioketals in the P2 position.
Better hydrophobic contact could also be accomplished
via incorporation of larger alkyl and cycloalkyl chains at
that position (Fig. 2).
More recently, we demonstrated that introduction of a
phenylglycine residue at P02 position improved the bind-
ing potency of P3-capped ketoamide inhibitor of type 1
(Fig. 1).8 From the X-ray crystal structure of compound
Keywords: Hepatatis C; P3 capped ketoamide; Inhibitor.
*
Corresponding author. Tel.: +1 908 7404642; fax: +1 908 7407152;
e-mail: stephane.bogen@spcorp.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.009

4516
S. Bogen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4515–4519
Aldehyde 3 generated through ozonolysis of the allyl
Arg
155
Arg
155
P2
P2
moiety could provide, after ketalization and subsequent
oxidation, inhibitors of type 4 bearing desirable lypo-
philic P2 surrogates.
S
n
n
S
H
H
N
N
HN
HN
Synthesis of a-hydroxyl amide 2 and inhibitors of type 4
are depicted in Scheme 2. Thus, dipeptide 6,6 bearing the
hydroxyl amide moiety, was reacted with P2 allylglycine
residue 5 under standard coupling procedure (EDCI,
HOOBt, NMM) to provide allyl ester 7. After removal
of the t-Boc protecting group of 7, the i-Boc protected
cyclohexyl glycine moiety at P3 was incorporated, via
8, using the coupling conditions described earlier.
Saponification of the allyl ester 9 followed by incorpora-
tion of the P02 moiety by coupling to phenylglycine ami-
no acid derivative gave key intermediate 2 (Scheme 2).
Ozonolysis of 2 generated the desired aldehyde interme-
diate 3 as the minor product with methoxy ketals as the
major products of the reaction as established by NMR
and mass spectrometry analysis. The mixture was then
subjected to transketalization procedure; thus, use of
Et2OÆBF3 efficiently promoted thioketal formation but
unfortunately cleaved the tert-butyl ester moiety at P02.
Control of temperature, reaction time or amount of
Et2OÆBF3 did not afford the desired chemoselectivity.
However, the side reaction was totally eliminated by
employing a milder Lewis acid for activation. Use of
1.1 equiv of zinc triflate and 2.5 equiv of 1,2-ethanedith-
iol afforded in <24 h the desired thioketal 10 in high
yield.
O
O
n= 0, 1
Figure 2.
OH
O
H
H
N
N
OtBu
HN
N
H
N
H
O
O
O
O
O
2
O
O3
S
n= 0, 1
O
H
n
S
O
OH
H
N
H
H
H
N
1) ketalizations
2) oxidations
N
N
HN
HN
O
O
O
O
3
4
Scheme 1.
Hydroxyl amide 2, containing P3–P20 residues, was
identified as a key intermediate for the synthesis of
ketals at P2 (Scheme 1).
OH
O
OH
O
H
b
H
H
a
HCl.H2N
N
OH
N
N
Oallyl
t-BocHN
t-BocHN
Oallyl
100%
86%
O
O
O
O
5
6
7
O
OH
O
H
OH
H
O
H
H
e
c
d
i-BocHN
N
N
N
N
2
H N
Oallyl
HCl.
N
Oallyl
2
> 99%
53%
78%
H
O
O
O
O
8
9
n= 0, 1
X=S, O
O
X
X
O
OH
O
O
OH
O
H
N
H
N
f
i-BocHN
H
H
N
OtBu
i-BocHN
N
OtBu
N
N
N
H
N
H
H
H
O
O
O
O
O
O
X
3
n= 0, 1
X=O, n=0, 12 (89%)
X=O, n=1, 13 (85%)
X=S, n=0, 10 (98%)
X=S, n=1, 11 (89%)
X=S, O
O
X
O
O
H
N
H
i-BocHN
N
OH
g
N
N
H
H
O
O
O
X=S, n=0, 15 (70%)
X=S, n=1, 16 (60%)
X=O, n=0, 18 (75%)
X=O, n=1, 19 (83%)
Scheme 2. Reagents and conditions: (a) EDCI, HOOBt, NMM, DMF/CH2Cl2; (b) 4 M HCl, dioxane; (c) i-Boc-cyclohexyl glycine, EDCI, HOOBt,
NMM, DMF; (d) (i) aq 1 N LiOH, THF/H2O; (ii) H-PhG-O-t-Bu, EDCI, HOOBt, DIPEA, DMF; (e) (i) O3, CH2Cl2/MeOH, ꢀ78 °C; (ii) DMS, rt,
12 h; (f) X = S, di-thiols (2.5 equiv), Zn(OTf)2, (1.1 equiv), CH2Cl2, X = O, diols (10 equiv), molecular sieves, TsOH cat, CH2Cl2, (g) (i) Swern
(X = S), Dess–Martin (X = O), (ii) 50% TFA/CH2Cl2.

S. Bogen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4515–4519
4517
2
The aromatic ring of the benzenedimethane thioketal
17 did not provide any additional hydrophobic interac-
tion with the enzyme backbone as demonstrated by its
poorer potency ðKꢁi ¼ 2.3 lMÞ. The smaller the ring,
the better was the activity; thus, compound 15, with a
five-membered thioketals group, turned out to be the
most potent ðKꢁi ¼ 0.09 lMÞ of all the inhibitors evalu-
ated in this series. For the oxygenated compounds, the
same trend was also observed with the five-membered
ketal 18 ðKꢁi ¼ 0.30 lMÞ being more potent than the
six-membered analog 19 ðKꢁi ¼ 0.43 lMÞ. In the two
series evaluated, the thioketal showed better activity
than the oxygen counterpart with more than 3-fold loss
in activity for the five-membered ketal 18 compared to
the thioketals analog 15. This was in good agreement
with the hydrophobic character of sulfide versus ether
and confirmed the need for a hydrophobic side chain
at P2.
CH2N2, Pd(OAc)2
Et2O, 100%
OH
O
H
H
N
N
OtBu
HN
N
H
H
O
N
O
O
O
O
O
14
Scheme 3.
Oxidation of 10 to generate the corresponding a-keto-
amide 15 was rather challenging. Thus, with a stoichi-
ometric amount of Dess–Martinꢀs periodinane,10 the
reaction was totally chemoselective toward the sulfide
oxidation. Use of tetrapropyl ammonium perruthe-
nate11 (TPAP) or SO3–pyridine12 complex as oxidizing
reagent led mainly to degradation products. Moffatt13
oxidation furnished the desired ketoamide albeit with
a M+16 adduct that was detected by mass spectrometry
analysis. Interestingly, Swern14 oxidation of thioketal 10
provided, chemoselectively, the corresponding a-ketoa-
mide in good yield (70%). Finally, hydrolysis of the ter-
minal tert-butyl ester group with 50% TFA in CH2Cl2
gave the desired target 15. Compounds 16 and 17 were
prepared in a similar fashion using 1,2-propanethiol
and 1,2-benzenedime-thanethiol, respectively. The oxy-
gen analogs 18 and 19 were also prepared from common
intermediate 3. In the case of the oxygen analogs, trans-
ketalization was performed in the presence of molecular
sieves and a catalytic amount of TsOH. Dess–Martinꢀs
periodinane oxidation followed by TFA deprotection
of the terminal tert-butyl ester group furnished the de-
sired a-ketoamides 18 and 19 without affecting the ketal
group. P2 alkyl and cycloalkyl chain analogs 20, 21, and
22 were prepared via solid-phase synthesis8 using Sasrin
resin. Inhibitor 23, bearing a cyclopropyl alanine at P2
was prepared from key intermediate 2. As described in
Scheme 3, cyclopropanation of the allyl group was effi-
ciently performed with CH2N2 and a catalytic amount
of Pd(OAc)2.15 Dess–Martin oxidation of 14 followed
by deprotection of the tert-butyl ester moiety with
TFA provided 23 in high yield.
To achieve better hydrophobic contact with Arg155, we
also tried to incorporate bulkier leucine analogs at P2.
From the HCV protease inhibitory data obtained for
compounds 20 and 21 ðKiꢁ ¼ 0.31 and 0.30 lM, respec-
tively) compared to compound 1 ðKꢁi ¼ 0.12 lMÞ, it
was clear that steric factors were critical for activity.
Incorporation of long side chains homo-leucine in 20
and homo-norleucine in 21 resulted in about 3-fold loss
in activity. Although we anticipated that hydrophobic
moiety should be well accommodated at this position,
we noticed that larger substitutions at P2 did not provide
enhancement in activity. SAR of the ketals series indi-
cated that the smaller the size of P2 modification, the
better was the activity. Using those observations, we
prepared compound 22 bearing a cyclobutyl alanine
moiety at P2. Inhibitor 22, with a Kꢁi of 0.14 lM, was
equipotent to the leucine analog 1 ðKꢁi ¼ 0.12 lMÞ.
Incorporation of the smaller cyclopropyl ring in 23
resulted in more than 2-fold improvement in activity
ðKꢁi ¼ 0.05 lMÞ. From the X-ray analysis (Fig. 4), we
could see that the cyclopropyl ring exhibited good van
der Waals contacts with the two terminal NH groups
˚
of Arg155. Distances were 3.52 and 3.26 A from the
nearest methylene group of the ring. The Cc of Arg
˚
155, the nearest methylene group, was 4.41 A from the
same methylene group of the cyclopropyl ring. These
observations were in good agreements with the cyclo-
propyl ring filling the S2 pocket in an optimum manner.
2. Results and discussion
Targets included in Table 1 were prepared using n-valine
as the P1 residue. We carried out modifications aimed at
optimizing the P1 residue by retaining cyclopropyl ala-
nine at P2 since it had previously demonstrated
Because of the close proximity of the P2 side chain to
Arg155, we anticipated that introduction of larger
hydrophobic moieties off the P2 position of our inhibi-
tors would present favorable interactions with this resi-
due. Toward this end, we investigated replacement of
leucine at P2 with more lipophilic moieties. Thus, with
a Kꢁi of 0.12 lM, inhibitor 1 was a good candidate for
P2 optimization. HCV NS3 serine protease inhibitory
activity16 for the targets synthesized was obtained using
the continuous spectrophotometric assay previously
reported.17 For the thioketals series, the steric factor
seemed to be critical for the activity, thus, the five-mem-
bered thioketals 15 with a Kꢁi of 0.09 lM was more po-
tent than the six-membered analog 16 ðKꢁi ¼ 0.25 lMÞ.
O
O
H
H
N
N
OH
HN
N
H
H
O
N
O
O
O
O
O
24, Ki* = 0.015 µM
Figure 3.

4518
S. Bogen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4515–4519
Packing Distances
A = 3.52Å
Arg155
B = 4.11Å
C = 3.26Å
D = 3.26Å
D
C
B
A
Figure 5. X-ray structure of 24 bound to the protease.
Figure 4. Packing distances of cyclopropyl alanine at P2 with Arg 155.
peptidic core binds to the protease through a series of
hydrogen bonding interactions. The cyclopropyl alanine
residues at P2 and P1 fit well in the S2 and S1 pockets,
respectively, making tight hydrophobic contacts. The
cyclohexyl moiety at P3 provided additional hydropho-
bic contacts in comparison with smaller groups at P3.8
The P1–P02 residues form a ꢁC-clampꢀ that wraps over
the side chain of lys136.
improved potency. We discovered that incorporation of
a cyclopropyl alanine at P1 also provided a real boost in
activity. Thus, inhibitor 24 (Fig. 3) exhibited the best
potency in that series with Kꢁi ¼ 0.015 lM.
X-ray crystal structure of the inhibitor 24 bound to the
protease is shown in Figure 5.18 It can be seen that the
In summary, we have identified potent inhibitors of the
HCV NS3 serine protease. Incorporation of a cyclopro-
pyl alanine side chain at both P2 and P1 improved the
binding potency by about 10-fold compared to our ear-
lier P3-capped inhibitors 1. Further work aimed at the
depeptidization of 24 is under progress and will be
reported shortly.
Table 1. Synthesis of inhibitors 15–23
R
O
O
H
H
N
N
OH
HN
N
H
H
O
N
O
O
O
O
O
References and notes
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Entry
1
Compound no.
R
Kꢁi (lM)
S
S
15
0.09
S
2
16
0.25
3. Dymock, B. W. Emerging Drugs 2001, 6, 13, and refer-
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S
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S
3
4
5
17
18
19
2.3
S
O
O
0.30
0.43
O
O
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6
7
8
9
20
21
22
23
0.31
0.30
0.14
0.05

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4519
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18. Crystallographic data for the structures in this article have
been deposited with the RCSB Protein Data Bank as PDB
ID 2A4R. The structural details can be viewed at
www.rcsb.org using the ID number above.