Novel inhibitors of hepatitis C NS3-NS4A serine protease derived from 2-aza-bicyclo[2.2.1]heptane-3-carboxylic acid

Article abstract of DOI:10.1016/j.bmcl.2005.12.046

Prolonged hepatitis C infection is the leading cause for cirrhosis of the liver and hepatocellular carcinoma. The etiological agent HCV virus codes a single polyprotein of ～3000 amino acids that is processed with the help of a serine protease NS3A to prod

Full text of DOI:10.1016/j.bmcl.2005.12.046

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1628–1632
Novel inhibitors of hepatitis C NS3–NS4A serine protease
derived from 2-aza-bicyclo[2.2.1]heptane-3-carboxylic acid
Srikanth Venkatraman,* F. George Njoroge, Wanli Wu, Viyyoor Girijavallabhan,
Andrew J. Prongay, Nancy Butkiewicz and John Pichardo
Schering Plough Research Institute, K-15, MS-3545, 2015 Galloping Hill road, Kenilworth, NJ 07033, USA
Received 17 August 2005; revised 10 September 2005; accepted 7 December 2005
Available online 18 January 2006
Abstract—Prolonged hepatitis C infection is the leading cause for cirrhosis of the liver and hepatocellular carcinoma. The etiological
agent HCV virus codes a single polyprotein of ꢀ3000 amino acids that is processed with the help of a serine protease NS3A to
produce structural and non-structural proteins required for viral replication. Inhibition of NS3 protease can potentially be used
to develop drugs for treatment of HCV infections. Herein, we report the development of a series of novel NS3 serine protease inhib-
itors derived from 2-aza-bicyclo[2.2.1]-heptane carboxylic acid with potential therapeutic use for treatment of HCV infections.
Ó 2005 Elsevier Ltd. All rights reserved.
Hepatitis C virus is the primary etiological agent respon-
sible for chronic HCV infections of liver leading to cir-
rhosis and hepatocellular carcinoma. An estimated 170
million people worldwide are infected with HCV and
up to 80% of these infections turn chronic.1 Current
therapy includes combination of a-interferon and ribavi-
rin, which is effective only in 25–40% of patients for sus-
tained response.2 Introduction of pegylated interferon
has reduced the frequency of injections enabling lower
viral titers and improved histological growth particular-
ly in relapse patients after interferon therapy.2 Lack of
efficient therapies necessitates discovery of new agents
for treatment of chronic hepatitis C.
HCV NS3 protease is a serine protease; with the assis-
tance of co-factor NS4A catalyzes the hydrolysis of a
cysteine serine amide bond. The hydrolysis proceeds
via attack of Ser-139 followed by stabilization of tetra-
hedral intermediate in oxy-anion hole. Most potent
inhibitors reported to date contain either a 4-substituted
proline or a 3,4-disubstituted proline as P2 amino acid
residue.5 In an attempt to depeptidize these inhibitors
we decided to explore bicyclic amino acid 2-aza-
bicyclo[2.2.1]-heptane carboxylic acid 4 as a P2 surro-
gate. This amino acid could be visualized either as a
trans-3,5-disubstituted proline or as a conformationally
rigid pipecolinic acid derivative that presented the piper-
idine ring in a rigid boat form enabling maximum con-
tact with the surface of the enzyme. Our previous
studies with pipecolinic acid as the P2 amino acid had
produced inhibitors less potent than the inhibitors de-
rived from proline.
HCV, a Flaviviridae family virus, encodes a polyprotein
of about 3000 amino acids from a positive strand RNA
genome. This polyprotein is post translationally spliced
to produce the various proteins essential for viral repli-
cation.3 Autocatalytic cleavage at the NS2-NS3 junction
followed by cleavage of NS3–NS4A, NS4A–NS4B,
NS4B–NS5A, and NS5A–NS5B by the NS3 serine pro-
tease produces functionally mature virions. The inhibi-
tion of this pivotal protease would possibly impair the
ability of the enzyme to process the polyprotein arrest-
ing maturation of virus and viral reproduction.4
Synthesis of amino acid 2-aza-bicyclo[2.2.1]-heptane
carboxylic acid was accomplished following the proce-
dure of Stella et al. as outlined in Scheme 1.6 Thus, con-
densation of ethylglyoxalate with (R)-phenethylamine in
refluxing benzene yielded chiral imine 2 that underwent
hetero-Diels–Alder reaction with cyclopentadiene to
yield aza-bicyclo[2.2.1]heptene derivative 3 as a mixture
of diastereomers that were easily separable by chroma-
tography. Hydrogenation of the double bond with con-
comitant removal of N-methylbenzyl group yielded the
amino ester 4 that was coupled with Boc-tert-butylgly-
Keywords: HCV NS3 protease; Ketoamide.
*
Corresponding author. Tel.: +1 908 740 3758; fax: +1 908 740
7152; e-mail: Srikanth.Venkatraman@spcorp.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.046

S. Venkatraman et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1628–1632
1629
Me
OH
OH
a
Me
O
b
Me
O
N
O
O
1
2
d
O
Me
c
O
Me
N
N
O
Me
H
O
4
3
OH
O
Me
N
f
N
e
O
BocHN
Me
O
BocHN
Me
O
7
O
6
5
Me
Me
Me
Me
O
OH
H
N
H
NHR
N
NHR
g
N
N
O
O
BocHN
Me
O
O
BocHN
Me
O
O
Me
Me
Me
Me
9
Me
Me
8
Scheme 1. Reagents and conditions: (a) (R)-phenethylamine, benzene, PPTS, reflux, 2 h; (b) cyclopentadiene, BF3ÆO(C2H5)2, TFA, ꢂ78 °C; (c) H2/
Pd/C, CH3OH, 3 h; (d) Boc-tert-butylglycine EDCIÆHCl, HOOBt, NMM, CH2Cl2/DMF (1:1) 0 °C, 12 h; (e) LiOHÆH2O, H2O/THF, rt; (f) NH2-
CH(C3H7)-CH(OH)-CONHRÆHCl, (7) EDCIÆHCl, HOOBt, NMM, CH2Cl2/DMF (1:1) 0 °C, 12 h; (g) Dess–Martin reagent, CH2Cl2, rt, 1 h (when
R 5 H) or EDCIÆHCl, Cl2CHCOOH, DMSO/toluene (when R = H).
cine using standard peptide coupling conditions with
EDCI and HOOBt to yield dipeptide 5 in 60% yield.
Table 1.
O
Hydrolysis of ethyl ester 5 with aq LiOH resulted in acid
6, which was coupled with norvaline segment 77 using
EDCI and HOOBt. The resulting hydroxy amides of
type 8 were oxidized with either Dess–Martin reagent8
or Moffat conditions to yield the ketoamide inhibitors
of type 9. The inhibitors synthesized were evaluated
for their ability to inhibit the hydrolysis of chromogenic
4-phenylazophenyl (PAP) ester from the peptide frag-
ment Ac-DTEDVVP(Nva)-O-4-PAP in a HCV protease
continuous assay.9 The dependence of inhibitory activity
of the P0 residue is summarized in Table 1.
H
H
N
N
R
N
O
O
BocHN
Me
P'1
O
Me
Me
Me
Compound
R
H
Kꢁi (lM)
10
6.9
CH2
O
11
12
5.5
1.5
C6H5
O
O
Replacement of the P01 hydrogen in 10 with allyl group
resulted in compound 11 with a minimal improvement
in binding from Kꢁi ¼ 6:9 to 5:5 lM. Incorporation of
glycine benzyl ester at P01 gave compound 12
ðKꢁi ¼ 1:5 lMÞ, which was 4-fold more potent than 10.
Hydrogenation of 12 gave glycine acid 13, which was
slightly more potent than 10 with a Kꢁi ¼ 3:8 lM.
OH
13
14
3.8
O
N
H
N
Me
0.11
O
Me
To further improve the potency of our inhibitor, we
decided to incorporate phenylglycine dimethylamide as
a P02 residue, since it had been previously shown to be
an excellent P02 group.10 Functionalization of 9 with
glycine-phenylglycine segment as the P01–P20 residues
resulted in compound 14 with Kꢁi ¼ 0:11 lM, a 14-fold
improvement in activity over inhibitor 12.
of these inhibitors are outlined in Scheme 2. Thus,
Boc-protected P1 amino acids 15 were converted to their
corresponding Weinreb amides and reduced to corre-
sponding aldehydes 16 using LiAlH4. These aldehydes
were converted to hydroxy acids 17 by treatment with
acetone cyanohydrin followed by acid hydrolysis.
Coupling of hydroxy acid with glycine-phenylglycine
dimethylamide yielded P1–P02 segment 18, which was
coupled with P2 acid 19 and oxidized using Dess–Martin
reagent to yield inhibitors 22–39. The effect of variation
of P1 and P3 groups is shown in Table 2.
Having established inhibitors of type 14 containing
glycine phenylglycine derivative gave potent inhibitors
against HCV NS3 protease, we decided to modify the
P1 and P3 residues to improve potency. The syntheses

1630
S. Venkatraman et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1628–1632
Table 2.
O
O
b
a
BocHN
BocHN
OH
P2'
H
P1
15
P1
16
P2
O
O
Me
N
OH
COOH
H
H
OH
O
Me
N
N
N
c
H
BocHN
N
Me
N
HCl.H2N
N
H
N
Me
P1
O
P1
P1
O
O
BocHN
H
P1
O
O
O
P1'
18
17
21
P3
P3
O
O
Me
d
OH
H
H
N
N
N
N
N
Me
H
N
O
X
N
P1
O
O
Compound
P1
P3
Kꢁi (lM)
H
H
tBu
O
O
X
N
tBu
O
O
19
P3
Me
Me
20
X= O, NH
O
P3
22
23
24
25
26
27
28
29
30
0.081
Me
Me
Scheme 2. Reagents and conditions: (a) i. EDCIÆHCl, CH3ON(CH3)-
HÆHCl, HOOBt, NMM, CH2Cl2, rt, 12 h; ii. LiAlH4, Et2O, 0 °C to rt,
1 h; (b) i. (CH3)2C(OH)CN, Et3N, CH2Cl2, rt; ii. 6 M methanolic HCl
reflux; iii. aq NaOH, THF, Boc2O; iv. aq LiOH, THF rt; (c) i. EDCI,
HOOBt, NMM, HClÆH2N-Gly-Phg-CON(Me)2, DMF/CH2Cl2, rt;
ii.4 M HCl/dioxane; (d) i. 18, EDCI, HOOBt, NMM, DMF/CH2Cl2,
rt; ii. Dess–Martin periodinane, CH2Cl2 rt, 1 h.
Me
Me
Me
0.040
0.019
0.027
0.090
0.041
0.053
0.020
0.051
Me
Me
Me
Me
Me
Me
Me
Me
As shown in Table 2 changing the P1 residue from
norvaline to norleucine yielded 22 which marginally
improved the binding from Kꢁi ¼ 0:11 to 0:08 lM.
Incorporation of a b-branched amino acid leucine yield-
ed 23 with further improvement in Kꢁi ¼ 0:040 lM. This
enhancement in binding was encouraging and prompted
us to investigate cyclopropylalanine and cyclobutylala-
nine at P1 position. The binding of cyclopropylalanine
derived inhibitor 24 and that of cyclobutylalanine
derived inhibitor 25 were superior to that of norvaline,
providing compounds with Kꢁi ¼ 0:019 and 0:027 lM,
respectively, a 4- to 5-fold improvement in potency in
comparison to 14. The poor binding of cyclobutylala-
nine compared to that of cyclopropylalanine suggested
that the S1 pocket was shallow and tolerated small
changes in sterics. Having established cyclopropylala-
nine as the optimal P1 residue, we explored the effect
of varying the P3 amino acid to cyclohexylglycine (inhib-
itors 26–30) from tert-butylglycine. As shown in Table 2,
this provided little improvement in binding activity. In
P3-cyclohexylglycine-derived series norleucine-derived
inhibitor 27 was more potent than cyclobutylalanine
derived inhibitor 30 in contrast to P3 tert-butylglycine
series. Once again incorporation of cyclopropylalanine
at P1 provided an inhibitor with activity better than that
of noravline or norleucine; thus, compound 29 with
Kꢁi ¼ 0:020 lM was more potent than compound 26
with a Kꢁi ¼ 0:090 lM.
Me
Me
Me
Me
H57
R155
K136
To establish the mode of binding of these inhibitors,
X-ray crystal structure of inhibitor 27 bound to NS3
protease was solved.11 From Figure 1 it is clear that
the P2 bicyclic amino acid binds to the enzyme with
the concave face of piperidine making surface contact
to the enzyme and the bridging methylene of bicyclic
amino acid is solvent exposed. The P01–P20 glycine-phe-
nylglycine segment forms a ‘C-clamp’ around lysine-
136 restricting its mobility. The carbonyl oxygen of
phenylglycine dimethylamide forms a hydrogen bond
with Thr-42 of protein backbone and aryl group of
phenylglycine forms hydrophobic interactions with al-
C159
I132
Figure 1. X-ray structure of inhibitor 27 bound to NS3 protease.
kyl chain of lysine-136. The ketoamide moiety makes
covalent bond with serine-139 with amide carbonyl
occupying oxy-anion hole. The concave portion of
the bicyclic P2 overlaps with alanine-156 further
enhancing binding.

S. Venkatraman et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1628–1632
1631
Table 3.
Analysis of the X-ray of inhibitor 27 bound to the en-
zyme suggested that functionalization at 4 or 5 position
of the bicyclic ring with aliphatic groups could potential-
ly make additional contact with Arg-155 and Ala-156
with a possibility of improving potency. To evaluate
the effect of substitution at 4 and 5 positions cycloprop-
anation of 3 was explored. Intermediate 3 obtained from
the cycloaddition of ethylglyoxalate and imine 2 was
cyclopropanated using Pd(OAc)2 and diazomethane to
form the amino ester 32 (Scheme 3). The cyclopropyl
group was exclusively delivered from the less hindered
exo-face.
O
O
Me
N
H
H
N
N
N
Me
N
H
H
H
O
P1
35
O
O
Me
Me
N
N
O
Me
O
P3
P1
Compound
P3
Kꢁi (lM)
36
0.019
This tricyclic amino ester 32 was incorporated into the
ketoamide inhibitors using a similar procedure as out-
lined in Scheme 2 to provide inhibitors 33 and 34 (Fig. 2).
Me
Me
37
38
39
0.010
0.021
0.013
Me
Me
As it can be seen from Figure 2 incorporation of cyclo-
propyl ring on the aza-bicyclo[2.2.1]heptane ring provid-
ed compounds 33 and 34 with HCV protease activity of
Kꢁi ¼ 0:090 and 0:027 lM, respectively. This was a mar-
ginal improvement in potency in comparison to com-
pounds 26 and 27. Analysis of X-ray structure of these
inhibitors bound to NS3 protease clearly showed that
derivatization of the bicyclic ring system from the exo-
face placed the cyclopropyl functionality away from
the protein into the solvent, thus rendering minimal
overlap to the surface of the enzyme. The effect of this
interaction was small and made a low contribution to
the binding that is clearly reflected in the case of the
inhibitors 33 and 34. Since the exo-side was the sterically
more accessible face most functionalizations of the dou-
ble bond would form exo-product providing small
improvement in potency.
Me
Me
From the results indicated in Table 3 it was clear that
the replacement of the Boc group with a urea enhanced
the binding affinity by 2- to 3-fold. Inhibitor 37 contain-
ing cyclopropylalanine at P1 had Kꢁi ¼ 0:010 lM and
inhibitor 39 with cyclobutylalanine at P1 and tert-butyl-
glycine at P3 had a Kꢁi ¼ 0:013 lM. Both these inhibitors
were 10-fold better than 14.
The selectivity of inhibitor 37 and 39 to HCV protease
was evaluated by comparing its binding affinity to a
structurally homologous serine protease, human neutro-
phil elastase (HNE). Inhibitor 37 had a HNE activity of
Kꢁi ¼ 340 nM with a HNE/HCV selectivity of 35 and
inhibitor 39 had a HNE activity of Kꢁi ¼ 5300 nM with
a HNE/HCV selectivity of 420. In a replicon-based cel-
lular assay, inhibitor 27 demonstrated a EC50 = 0.6 lM
and inhibitor 39 had a EC50 = 2.0 lM.
Further efforts were focused on modification of capping
functionality on our most potent inhibitors. Replace-
ment of tert-butyl carbamate to tert-butyl urea, an isos-
teric replacement that changes hydrogen bond acceptor
oxygen to hydrogen bond donor nitrogen lead to inhib-
itors 36–39 (Table 3).
In conclusion, we have effected the development of
potent HCV protease inhibitors with non-proline P2
amino acid obtained from a 2-aza-bicyclo[2.2.1]-heptane
carboxylic acid. The binding affinity of these inhibitors
was optimized from high micromolar to low double digit
nanomolar potencies. The constriction of pipecolinic
acid with a bridged methylene group constrains the
piperidine ring in a six-membered boat conformation
enabling it to overlap effectively with the enzyme.
Further modifications and evaluation of DMPK proper-
ties of inhibitors 36–39 are in progress.
O
Me
a
b
O
Me
N
3
(S)
N
O
H
O
Me
31
32
Scheme 3. Reagents and conditions: (a) CH2N2, Pd(OAc)2, ether, 0 °C
to rt; (b) H2/Pd/C, CH3OH, 3 h.
References and notes
O
O
Me
N
H
H
N
N
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N
Me
N
H
O
R1
O
O
BocHN
O
Ki*= 0.090 µM
Ki*= 0.027 µM
33 R1=
34 R1=
Me
Me
2. (a) McHutchison, J. G.; Gordon, S. C.; Schiff, E. R.;
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Figure 2.

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11. Crystallographic data for the inhibitor 27-HCV NS3
protease complex shown in Figure 1 have been deposited
with the RCSB (code rcsb035631) Protein Data Bank as
PDB ID 2F9U. The structural details can be viewed at
www.rscb.org using the ID number above.