Novel 2-oxoimidazolidine-4-carboxylic acid derivatives as Hepatitis C virus NS3-4A serine protease inhibitors: Synthesis, activity, and X-ray crystal structure of an enzyme inhibitor complex

Article abstract of DOI:10.1016/j.bmcl.2004.09.058

Synthesis and HCV NS3 serine protease inhibitory activity of some novel 2-oxoimidazolidine-4-carboxylic acid derivatives are reported. X-ray structure of an inhibitor, 15c bound to the protease is presented. Synthesis and HCV NS3 serine protease inhibitor

Full text of DOI:10.1016/j.bmcl.2004.09.058

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5751–5755
Novel 2-oxoimidazolidine-4-carboxylic acid
derivatives as Hepatitis C virus NS3-4A serine protease
inhibitors: synthesis, activity, and X-ray crystal structure
of an enzyme inhibitor complex
Ashok Arasappan,* F. George Njoroge, Tejal N. Parekh, Xiaozheng Yang,
John Pichardo, Nancy Butkiewicz, Andrew Prongay, Nanhua Yao
and Viyyoor Girijavallabhan
Schering Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
Received 21 July 2004; revised 15 September 2004; accepted 17 September 2004
Abstract—Synthesis and HCV NS3 serine protease inhibitory activity of some novel 2-oxoimidazolidine-4-carboxylic acid deriva-
tives are reported. Inhibitors derived from this new P2 core exhibited activity in the low lM range. X-ray structure of an inhibitor,
15c bound to the protease is presented.
Ó 2004 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) is the etiologic agent of non-A,
non-B hepatitis leading to liver cirrhosis, heptacellular
carcinoma, and liver failure in humans.1 Ithas been esti-
mated that 3% of the human population worldwide is in-
fected by HCV.2 Currently, a-interferon monotherapy
and a-interferon–ribavirin combination therapy are the
only approved treatment regimen.3 Recently pegylated
version of a-interferon, PEG-INTRON and PEGASYS
has been approved by the FDA as improved therapy.
Due to the seriousness of the disease there is an urgent
need for new and more effective protocol for the treat-
mentof HCV infecitons.
line based7 analogs. Replacementof naturally occurring
proline residue with a non-natural proline surrogate
may impartbeneficial biological and pharmacological
properties to the peptide inhibitors. Herein, we report
the incorporation of one such proline surrogate, 2-oxo-
imidazolidine-4-carboxylic acid derivative,8 as a promis-
ing P2 scaffold in the design and synthesis of potent
HCV NS3 serine protease inhibitors.9 Noteworthy fea-
ture of this scaffold is the ability to introduce additional
functionality off the N-1 position; a mission that was
undertaken in the SAR development of potential inhib-
itors. In the present study, an a-ketoamide functional
group was incorporated as electrophilic serine trap (see
Fig. 1), since this entity facilitated probing the P prime
region of the protease.10
Since identification of this virus, the NS3 serine protease
contained within the N-terminal region of the NS3 pro-
tein has been studied extensively.4 This chymotrypsin-
like serine protease is implicated in the viral replication
and hence is an attractive target for HCV antiviral thera-
peutics.5 While a number of HCV NS3 serine protease
inhibitors have been reported by different groups, most
of these are peptide-like molecules derived from the
cleavage site sequences. Based on the P2 moiety, these
reported inhibitors could be classified as leucine6 or pro-
Synthetic approach for the preparation of the designed
inhibitors was initiated from 2-oxoimidazolidine deriva-
tive 1 prepared by the method of Hayashi et al.12
(Scheme 1). Migration of the benzyloxycarbonyl (Z)
protecting functionality was carried out in DMF using
sodium hydride as the base at room temperature to af-
ford 2. N-Acylation of the potassium salt of 2 with the
activated ester derived from N-Boc-Val-OH at ꢀ40°C
provided the benzyl ester 3. Removal of the benzyloxy-
carbonyl and benzyl protecting groups were carried out
in a single step under hydrogenation conditions to give
*
Corresponding author. Tel.: +1 9087402607; fax: +1 9087407152;
e-mail: ashok.arasappan@spcorp.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.058

5752
A. Arasappan et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5751–5755
Selective removal of the Z functionality was achieved by
O
P2
H
N
H
N
treatment with 30% HBr in glacial acetic acid to provide
ester 11. Installation of the P3 valine residue and further
processing to afford the targets 15 essentially followed
procedures described in Scheme 1.
P'
P6
O
O
P4
N
H
N
H
N
O
P1
O
CapHN
CapHN
N
H
O
O
O
P5
O
O
P3
A
HCV NS3 serine protease inhibitory data13 for the imid-
azolidineone targets were obtained using the continuous
spectrophotometric assay described earlier14 and the
results are summarized in Table 1. The parentmolecule
8 containing the unsubstituted imidazolidineone moiety
at P2 and the corresponding diester 7 exhibited moder-
ate inhibition with Ki* of 46 and 69lM, respectively.
It has been previously reported that ethers derived from
trans-4-hydroxyproline as P2 core improved the potency
considerably.15 Hence, we decided to investigate the ef-
fect of substitution on the ring nitrogen of our imidazol-
idineone P2 core. Introduction of the methylene
carbethoxy moiety at the N-1 position improved the
activity significantly; while the protected compound
14a displayed an activity of 30lM againstthe NS3 ser-
ine protease, the corresponding fully deprotected target
15a exhibited an improved activity of 4lM. Importance
of the P6-P5 residue is clearly evident from the data for
the truncated derivatives 16 and 17 (synthesis not
shown), which were less potent. Installation of the ben-
zyl group at the N-1 position on the ring was well toler-
ated as exemplified by compound 15b, which displayed
Ki* of 1lM. In an attempt to probe the P prime region,
compound 15b0 (glycine allyl ester as P0 residue) was
synthesized. This P prime extended inhibitor 15b0 was
found to be equipotent (Ki* = 1lM) as the allyl amide
15b. Further exploration of the structure–activity rela-
tionship (SAR) established that substitution of the aro-
matic ring as 3,4-dichloro derivative in the P prime
extended series enhanced the potency further, resulting
in 15c with HCV NS3 serine protease inhibitory activity
of 0.31lM.
R1
O
N
H
N
H
N
O
P'
P6
P4
N
H
N
H
O
P1
O
N
N
H
O
B
P5
P3
Figure 1.
acid 4. Installation of the P1-P0 residue was done by cou-
pling the acid 4 with amine 5 under EDCI, HOBtcondi-
tions. The resulting mixture of hydroxyl amide
diastereomers was oxidized using Dess–MartinÕs period-
inane to provide the ketoamide 6. Deprotection of the
Boc group of 6 and subsequent elaboration of the P re-
gion was then accomplished by treatment with the P6-P4
tripeptide, Ac-Glu(t-Bu)-Glu(t-Bu)-Val-OH [prepared
from commercially available protected amino acid
derivatives employing standard protocol] using peptide
coupling methodology, which afforded the protected
ketoamide 7. The target compound 8 was obtained by
deprotection of the ester functionalities using 4M HCl
in dioxane.
Introduction of substitution on the N-1 position of the
P2 imidazolidinone core and subsequentconversion ot
target compounds are described in Scheme 2. Thus, N-
alkylation of 1 to give 10 was carried outin DME (or
acetone) using potassium carbonate as the base rather
than NaH, to avoid migration of the Z protecting group.
R1
O
N
HN
ZN
H
N
H
N
HN
O
O
a
b
d
O
O
CO2R
N
N
CO2Bn
CO2Bn
N
H
N
Z
O
O
BocHN
BocHN
O
O
2
1
6
3, R = Bn, R1 = Z
4, R, R1 = H
OH
H
N
c
HCl.H2N
O
5
O
O
CO2tBu
HN
CO2H
HN
H
N
H
N
H
N
H
N
O
O
O
H
N
O
N
H
H
N
H
e
f
O
O
O
O
N
N
N
AcHN
N
H
O
AcHN
N
O
H
O
O
O
O
CO2tBu
7
CO2H
8
Scheme 1. Reagents and conditions: (a) NaH, DMF (66%); (b) 2, KOt-Bu, THF; add Boc-Val-OSu, THF (80%); (c) H2, 10% Pd/C, EtOH (quant);
(d) (i) 5, EDCI, HOBt, NMM, CH2Cl2; (ii) Dess–MartinÕs periodinane, CH2Cl2 (60%); (e) (i) 4M HCl, dioxane; (ii) Ac-Glu(t-Bu)-Glu(t-Bu)-Val-OH,
EDCI, HOOBt, NMM, CH2Cl2 (31%); (f) 4M HCl, dioxane (quant).

A. Arasappan et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5751–5755
5753
R1
R1
N
N
HN
O
CO2R
N
a
e
O
c
O
b
CO2Bn
N
Z
CO2Bn
N
BocHN
O
R2
1
10a,b,c: R2 = Z
11a,b,c: R2 = H
12a,b,c: R = Bn
13a,b,c: R = H
OH
O
H
N
d
HCl.H2N
Oallyl
O
9
R1
N
R1
N
O
N
H
O
O
CO2tBu
CO2H
H
N
H
N
H
N
H
N
O
P'
P'
O
N
O
f
H
N
H
H
O
O
O
O
N
N
N
AcHN
N
H
O
AcHN
N
H
O
O
O
O
O
CO2tBu
CO2H
14a,b,b',c
15a,b,b',c
For Series a and b, P' =
For Series b' and c, P' =
Cl
Cl
EtO
R1 =
O
O
O
a
b, b'
c
Scheme 2. Reagents and conditions: (a) R1Br, K2CO3, TBAI (cat), DME (62–83%); (b) 30% HBr/AcOH (50–70%); (c) 11, KOt-Bu, THF; add Boc-
Val-OSu, THF (50–55%); (d) for series a and b—H2, 10% Pd/C, EtOH; for series c—aq 1M LiOH, THF, MeOH (quant); (e) for 14a see method A,
for 14b see method B, for 14b0 see method C, for 14c see method D;11 (f) 4M HCl, dioxane (quant).
Table 1.
R1
N
O
H
N
H
N
O
P'
N
H
O
O
N
Cap
O
Compound
Cap
R1
P0
Ki* (lM)a
7
Ac-Glu(t-Bu)-Glu(t-Bu)-Val-
Ac-Glu-Glu-Val-
H
H
Allyl
Allyl
69
46
8
14a
15a
16
Ac-Glu(t-Bu)-Glu(t-Bu)-Val-
Ac-Glu-Glu-Val-
i-Boc-Val-
CH2CO2EtAllyl
CH2CO2EtAllyl
CH2CO2EtAllyl
CH2CO2EtAllyl
CH2Ph
30
4
78
17
t-Boc-
Ac-Glu(t-Bu)-Glu(t-Bu)-Val-
Ac-Glu-Glu-Val-
150
Allyl
14b
15b
14b0
15b0
14c
15c
61
1
CH2Ph
CH2Ph
Allyl
Ac-Glu(t-Bu)-Glu(t-Bu)-Val-
Ac-Glu-Glu-Val-
Gly-Oallyl
Gly-Oallyl
Gly-Oallyl
Gly-Oallyl
>100
1
CH2Ph
CH2Ph(3,4-dichloro)
CH2Ph(3,4-dichloro)
Ac-Glu(t-Bu)-Glu(t-Bu)-Val-
Ac-Glu-Glu-Val-
>100
0.31
a Ki* value is reported as a weighted mean of at least three independent determinations.
X-ray crystal structure of the inhibitor 15c bound to the
protease is shown in Figure 2. It can be seen that the
peptidic core binds to the protease through a series of
hydrogen bonding interactions. The norvaline residue
at P1 occupies the shallow hydrophobic S1 pocket.
The P0 residue wraps over the side chain of lysine 136,
thus resulting in some hydrophobic interaction. Most
notably, the dichlorobenzyl group off the N-1 position
of the imidazolidineone ring extends towards glutamine
41, and thereby causing a slight increase in the size of the
S10 pocket.
In summary, we have discovered that 2-oxoimidazol-
idine-4-carboxylic acid derivatives are suitable P2

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9. The HCV NS3 serine protease inhibitory activity of
similar compounds (i.e., P6-P0 ketoamide derivative) with
P2 proline moiety is described in Ref. 7f.
10. Ingallinella, P.; Bianchi, E.; Ingenito, R.; Koch, U.;
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11. Method A: (i) 5, EDCI, HOBt, NMM (47%); (ii) 4M HCl/
dioxane; (iii) Ac-Glu(t-Bu)-Glu(t-Bu)-Val-OH, EDCI,
HOOBt, NMM (81%); (iv) Dess–MartinÕs periodinane
(73%).
Figure 2. X-ray structure of 15c bound to the protease.
surrogates with potent HCV NS3 serine protease inhib-
itory activity. While truncated inhibitors were not as po-
tent, targets containing the P6 residues exhibited potent
activity. X-ray crystal structure of one of the inhibitors
(15c) bound to the protease revealed that the substituent
off the N-1 position of the P2 ring extends towards glu-
tamine 41 in an unique fashion. We are currently in the
process of using this information in the design of other
analogs in this series.
Acknowledgements
We thank the Structural Chemistry group for providing
NMR and Mass Spectral assistance. We also thank Dr.
Bruce Malcolm for helpful discussion.
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(iii)
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