Design and synthesis of depeptidized macrocyclic inhibitors of hepatitis C NS3-4A protease using structure-based drug design

Article abstract of DOI:10.1021/jm0489556

Hepatitus C virus (HCV) NS3, when bound to NS4A cofactor, facilitates development of mature virons by catalyzing cleavage of a polyprotein to form functional and structural proteins of HCV. The enzyme has a shallow binding pocket at the catalytic site, ma

Full text of DOI:10.1021/jm0489556

5088
J. Med. Chem. 2005, 48, 5088-5091
Letters
HCV, a Flaviviridae family virus, produces a single
positive strand of RNA genome with a single open
reading frame of 9.6 KB and encodes a polyprotein of
approximately 3000 amino acids.3 The encoded polypro-
tein is cleaved cotranslationally and posttranslationally
to produce the core proteins, envelope proteins, and
nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A,
and NS5B.4 Posttranslational modification leading to
mature virus is accomplished by autocatalytic cleavage
of the NS2-NS3 junction followed by cleavage of NS3-
NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B
with the assistance of a single NS3 serine protease.
These processed proteins are indispensable for develop-
ment of mature virus, and inhibition of any one of these
enzymes makes it an attractive target for development
of a drug for hepatitis C.3b,5 However, the central role
of NS3 protease in the replication cycle makes it
particularly attractive for development of inhibitors for
the treatment of chronic hepatitis C.6
HCV NS3 protease is a serine enzyme with a catalytic
site that is located on the surface and is characteristi-
cally featureless, solvent-exposed, and shallow.7 The
enzyme catalyzes the cleavage of a cysteine-serine
amide bond with the assistance of a 52 amino acid
cofactor NS4A. The enzyme comprises of eight-stranded
distorted â-barrels with the NS4A bound between two
â-strands in the N-terminal subdomain. The active site
of the enzyme consists of the catalytic triad Ser-139,
His-57, and Asp-81 that is characteristic of all serine
proteases. The P1 amino acid cysteine and P1′ serine
residue are conserved in all the enzyme substrates.5c,8
The S1 pocket is hydrophobic, containing Phe-154, Ile-
135, and Ala-157 where the sulfhydryl group of cysteine
from the substrate makes an efficient interaction with
the aromatic group of Phe-154. The scission of the
cysteine-serine amide bond is initiated by a nucleo-
philic attack of serine-139 with the formation of a
tetrahedral intermediate, which is stabilized by the
histidine present in the oxyanion hole. The hydrolytic
cycle is completed by the collapse of the tetrahedral
intermediate followed by nucelophilic attack of water
to hydrolyze the acylserine ester. A suitably placed
electrophilic trap such as trifluoromethyl ketones, tri-
carbonyls, or ketoamides in inhibitors could reversibly
trap serine by the formation of a tetrahedral adduct,
thus inhibiting the enzyme catalytic cycle.9 This strat-
egy has been well-explored and used for the identifica-
tion of various serine protease inhibitors.
Design and Synthesis of Depeptidized
Macrocyclic Inhibitors of Hepatitis C
NS3-4A Protease Using Structure-Based
Drug Design
Srikanth Venkatraman,* F. George Njoroge,
Viyyoor M. Girijavallabhan, Vincent S. Madison,
Nanua H. Yao, Andrew J. Prongay,
Nancy Butkiewicz, and John Pichardo
Schering Plough Research Institute, K-15, MS-3545, 2015
Galloping Hill Road, Kenilworth, New Jersey 07033
Received December 23, 2004
Abstract: Hepatitus C virus (HCV) NS3, when bound to NS-
4A cofactor, facilitates development of mature virons by
catalyzing cleavage of a polyprotein to form functional and
structural proteins of HCV. The enzyme has a shallow binding
pocket at the catalytic site, making development of inhibitors
difficult. We have designed, preorganized, and depeptidized
macrocyclic inhibitors from P4 to P2′ and optimized binding to
0.1 µM. The structure of an inhibitor bound to the enzyme was
also solved.
Chronic hepatitis C virus (HCV) infections are the
leading cause of liver cirrhosis and hepatocellular
carcinoma.1 An estimated 170 million people worldwide
are infected with HCV, making it a major public health
concern. More than 80% of HCV infections turn chronic,
of which 20% develop hepatocellular carcinoma, making
it one of the leading reasons for liver transplantions.
In the U.S., ∼72% of the patients are infected with
genotype 1, a more resistant strain for treatment using
existing therapy that includes three or four injections
of R-interferon (IFN) per week and combination therapy
of IFN with ribavirin.2 Although approximately 30-50%
of infected patients initially respond to IFN mono-
therapy, only 8-20% have sustained response. Admin-
istration of a combination of ribavirin and interferon
has increased the sustained response rate to 14-40%
particularly in patients who relapse after IFN therapy.
Introduction of pegylated interferon has reduced the
frequency of injection to once a week with a better
pharmacokinetics profile while providing sustained
response to 50%. Patients administered with a combina-
tion of ribavirin and pegylated interferon have markedly
lower viral titers and enhanced histological growth. The
use of pegylated interferon has a very high response rate
of up to 80% in patients infected with genotypes 2 and
3 and is less effective with patients infected with
genotype 1 (40%). Even though IFN and combination
therapies have allowed the management of HCV infec-
tions in a number of patients, an effective treatment of
existing genotypes of HCV that could be orally admin-
istered is vital.
In our HCV program we identified the R-ketoamide
functionality as a good serine trap that provided potent
inhibitors of HCV NS3-NS4A protease. In an attempt
to depetidize these inhibitors, various strategies were
explored. A close analysis of the X-ray structure of the
NS3 protease revealed the proximity of the S2 and the
S4 pockets. We reckoned that a suitable linker that
would tether residues, which efficiently bound to the S2
and S4 pockets, could potentially provide depeptidized
* To whom correspondence should be addressed. Phone: 908-740-
3758. Fax: 908-740-7152. E-mail: Srikanth.Venkatraman@spcorp.com.
10.1021/jm0489556 CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/14/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5089
Scheme 1a
Figure 1.
inhibitors with high affinity and selectivity to HCV
protease. Further modeling and structure-activity re-
lationship studies suggested biaryl ether of type 1
(Figure 1) as an excellent scaffold for the generation of
NS3 protease inhibitors. Previously, a group from IRBM
reported a biaryl scaffold as potential HCV NS3 pro-
tease inhibitors that spanned from P4 to P1, which
lacked the electrophilic ketoamide moiety. However, the
group, to our knowledge, did not disclose the inhibitory
activity of these compounds. We therefore wanted to
further explore this scaffold by incorporating a keto-
amide trap and to evaluate their potential as HCV
inhibitors.10
The structure shown in Figure 1 contains a 17-
membered biaryl macrocyclic ether derived from (L)-
tyrosine and (L)-m-tyrosine as the P4 and P2 residues.
Cyclohexylglycine was chosen as the P3 amino acid
because this moiety had been shown to be an excellent
group at this position in our acyclic series of inhibitors
(data not shown). Similarly, norvaline and glycine were
deemed as appropriate P1 and P1′ residues, respectively,
thus making 1 the desired macrocyclic inhibitor candi-
date. Although various methods have been reported for
the construction of cyclic biaryl ethers reported in the
context of the total syntheses of vancomycin, ristocetin
A, teicoplanin, OF4949III, and K-1310 macroetherifi-
cation using η6-ruthenium chemistry pioneered by Pear-
son and Rich was our method of choice.11
The synthesis of macrocycle 1 began with the coupling
of Boc protected cyclohexylglycine 2 with m-tyrosine
using the mixed-anhydride method to produce the
dipeptide 3 in 73% yield (Scheme 1). The Boc group of
3 was deprotected using 4 M HCl in dioxane, and the
resulting amine was coupled with the η6-ruthenium
complex of Boc-4-chlorophenylglycine to yield 4 in 69%.
Macrocycliclization of 4 was effected using Cs2CO3 as
the base followed by photolytic decomplexation of Ru
at λ ) 350 nm, giving macrocycyclic biaryl ether 5 in
47% yield as a colorless solid. Completion of the syn-
thesis of 1 was achieved by hydrolysis of the methyl
ester with aqueous LiOH followed by coupling with a
norvaline-hydroxyamide12 segment using EDCI and
HOOBt. Dess-Martin oxidation13 of the hydroxyamide
6 yielded R-ketoamide 1 in excellent yield.
The synthesized macrocycle 1 was evaluated for its
ability to inhibit the hydrolysis of chromogenic 4-phe-
nylazophenyl (PAP) ester from the peptide fragment Ac-
DTEDVVP(Nva)-O-4-PAP in a HCV protease continu-
ous assay.14 The inhibitory activity of 1 was found to
be Ki* ) 12 µM. Modeling studies clearly indicated that
the arene rings of P2 m-tyrosine and P4 tyrosine oc-
cupied the S2 and S4 pockets of the enzyme appropri-
ately. However, attempts to obtain a crystal structure
of 1a bound to the HCV NS3 protease were unsuccess-
ful. Further modeling and SAR studies from our acyclic
inhibitors prompted us to incorporate a phenylglycine
at the P2′ site of inhibitor,15 resulting in the synthesis
a Reagents and conditions: (a) m-Tyr-OH, IBCF, NMM, -20
f 5 °C, 1 h, 53%; (b) (i) 4 M HCl in dioxane, (ii) 7f or 8a, EDCI,
HOBt, DMF, room temp, 24 h; (c) (i) 5.0 equiv of Cs2CO3, DMF,
12 h, room temp, (ii) CH3CN, hν, λ ) 350 nm, 48 h, 47%, (iii)
LiOH‚H2O, THF/H2O, 100%; (d) EDCI, HOOBt, H-Nva(OH)-C(O)-
Gly-OBn•HCl, (iPr)2EtN, (e) (i) Dess-Martin reagent, CH2Cl2, (ii)
H2/Pd/C, CH3OH.
of 7 (Scheme 1). Inhibitor 7 had a binding of Ki* ) 7.8
µM. Because the incorporation of phenylglycine at P2′
slightly improved the binding, which was encouraging,
we therefore decided to further refine our subsequent
targets. To further improve potency, various possible
sites for modification of 1 were evaluated and changes
at the P3 site and P4 capping group seemed appropriate.
The BocNH (P4 capping) group of 1 was replaced with
an acetamide (AcNH) moiety to afford inhibitor 8
(Scheme 1). Synthesis of 8 was initiated from acetyl
protected 4-chlorophenylalanine 8a and synthesized as
shown in Scheme 1 in good yields. Inhibitor 8 was found
to have a binding of Ki* ) 0.8 µM, which was 15 times
more potent than 1. This improvement in activity by
the replacement of the Boc group with an acetamide
group was ascribed to the smaller steric requirement
of the acetyl group in comparison to the bulky tert-
butoxycarbonyl moiety. We therefore reasoned that the
complete removal of the P4 capping group would further
enhance binding efficiency, and thus, we designed
macrocyclic inhibitor 9 (Scheme 2).

5090 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Letters
Scheme 2a
Figure 2. X-ray crystal structure of inhibitor 9 bound to active
site of HCV NS3 protease.
Figure 3. Pictorial representation of hydrogen bonding
network of inhibitor 9 to HCV NS3 protease.
inhibitor, we wanted to understand the mode of binding
of these macrocycles to HCV NS3A. The X-ray crystal
structure of inhibitor 9 bound to the protease was
obtained (Figure 2).
As shown in the Figure 2, the inhibitor 9 bound to
the protease with the aryl groups of m-tyrosine in the
S2 domain and the phenylpropionic acid in the S4 pocket.
The arene rings effectively made contact with the
surface of the protein and wrap around the methyl
group side chain of alanine-156, thereby maximizing
surface contact. The biaryl ether adopted a 90° confor-
mation, with the two aryl rings being orthogonal to each
other. The cyclohexyl ring of cyclohexylglycine (P3 amino
acid) adopted a chair conformation with excellent van
der Waals contact with the protein in the S3 pocket. The
propyl chain of the norvaline (P1 residue) was buried
deeply into the S1 pocket, making excellent contact with
Phe-154 located at the base of the S1 pocket. The P2′
phenylglycine and P1′ glycine form a part of a “C clamp”
around the extended aliphatic chain of lysine-136, thus
minimizing the mobility of the lysine side chain residue.
The arene ring of P2′ phenylglycine also made excellent
hydrophobic contact with the carbon chain of lysine,
thus maximizing binding of the individual residues.
Additionally, the inhibitor 9 is held in close contact
to the surface of the protein by an array of specific
hydrogen bonds to the backbone. A schematic represen-
tation of these specific hydrogen bonds are shown in
Figure 3.
a Reagents and Conditions: (a) (i) 4 M HCl in dioxane, (ii) EDCI,
HOBt, DMF, room temp, 24 h, (b) (i) 5.0 equiv of Cs2CO3, DMF,
12 h, room temp, (ii) CH3CN, hν, λ ) 350 nm, 48 h; (d) (i)
LiOH‚H2O, THF/H2O, 100%, (ii) EDCI, HOOBt, H-Nva(OH)-C(O)-
Gly-Phg-OtBu‚HCl, (iPr)2EtN; (e) (i) Dess-Martin reagent, CH2Cl2;
(ii) 4 M HCl, dioxane.
The synthesis of 9 was initiated with 4-chlorophenyl-
propionic acid and is shown in Scheme 2. Inhibitor 9
was assayed for its inhibitory activity and was found to
have a Ki* ) 0.11 µM, which was 100 times more potent
than 1. To evaluate the specificity of binding of macro-
cycle 9 to HCV protease in comparison with other serine
proteases, binding studies with a structurally closely
related protease, human neutrophil elastase (HNE), was
evaluated.16 The binding constant of macrocycle 9 to
HNE was 2.9 µM, providing a selectivity of HNE/HCV
of 27. In addition, selectivities against other serine and
cysteine proteases chymotripsin, cathepsin G, and cathe-
psin H were determined to be BPC/HCV ) 26, HNC-
G/HCV ) 250 and HLC-H/HCV > 380, respectively.
After a potent, selective HCV protease inhibitor was
developed, the correlation of inhibitory activity (Ki*) on
the macrocyclic ring size was evaluated. The 18-mem-
bered inhibitor 10 and 19-membered inhibitor 11 were
synthesized as shown in Scheme 2 starting from 4-chlo-
rophenylbutyric acid and 4-chlorophenylpentanoic acid,
respectively. These inhibitors were also very potent,
with a binding activity of Ki* ) 0.3 µM and Ki* ) 0.16
µM, respectively. Thus, changing the ring size did not
enhance the binding efficiency of the resulting inhibitor.
Having optimized the ring size of the macrocyclic
The amide nitrogens of P2′ phenylglycine, P3 cyclo-
hexylglycine, and P1 norvaline donate hydrogen bonds
to threonine-42, alanine-157, and arginine-155 of the
protease, and the carbonyl group of the ketoamide is
attacked by the hydroxyl group of serine-139. In addi-
tion, the oxygen of the ketoamide accepts a hydrogen
bond from the NH group of glycine-137 from the
backbone, making the binding of 9 extremely specific.

Letters
In conclusion we have developed a novel biaryl P2-
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5091
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P4 macrocyclic depeptidized inhibitor of HCV NS3
protease with excellent in vitro binding potencies. The
binding efficiency of the inhibitor was optimized from
12 to 0.11 µM activity through the optimization of
appropriate moieties. An X-ray crystal structure of the
inhibitor 9 bound to the protease provided information
about the various hydrogen bondings and hydrophobic
interactions of the inhibitor with the protease. The X-ray
clearly showed a “C clamp” formation between the
phenylglycine and glycine that immobilizes lysine-139,
thus enhancing binding efficiency. The P2-P4 macro-
cyclic inhibitors are selective toward HCV protease and
are being further evaluated for its pharmacokinetics
properties.
Supporting Information Available: Experimental and
characterization data for intermediates and final compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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JM0489556