Design, synthesis, and biological activity of m-tyrosine-based 16- and 17-membered macrocyclic inhibitors of hepatitis C virus NS3 serine protease

Article abstract of DOI:10.1021/jm050323b

The limited efficacy and considerable side effects of currently available therapies for the treatment of hepatitis C virus (HCV) infection have prompted significant efforts toward the development of safe and effective new therapeutics. The pentapeptide α-

Full text of DOI:10.1021/jm050323b

J. Med. Chem. 2005, 48, 6229-6235
6229
Design, Synthesis, and Biological Activity of m-Tyrosine-Based 16- and
17-Membered Macrocyclic Inhibitors of Hepatitis C Virus NS3 Serine Protease
Kevin X. Chen,* F. George Njoroge, John Pichardo, Andrew Prongay, Nancy Butkiewicz, Nanhua Yao,
Vincent Madison, and Viyyoor Girijavallabhan
Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-3-3545, Kenilworth, New Jersey 07033
Received April 8, 2005
The limited efficacy and considerable side effects of currently available therapies for the
treatment of hepatitis C virus (HCV) infection have prompted significant efforts toward the
development of safe and effective new therapeutics. The pentapeptide R-ketoamides of type 1
were weak HCV inhibitors with a binding constant, Ki*, above 5 µM. We envisioned that
cyclization of a P2 phenyl side chain to a P3 capping group could enhance binding through an
interaction of the resulting macrocycle with the methyl group of Ala156 on the enzyme backbone.
The macrocyclic dipeptide moiety would also decrease the peptidic nature of the inhibitors.
The synthesis of macrocyclic HCV inhibitors started from m-tyrosine methyl ester. Two
consecutive couplings, first, with Boc-cyclohexylglycine and, then, with hept-6-enoic acid,
provided compound 6. The alkene was converted to an alcohol via hydroboration. The key
macrocyclization of phenol alcohol 7 was achieved through a Mitsunobu reaction. Both 16-
and 17-membered macrocycles (8 and 21) were prepared. After hydrolysis, the macrocyclic acids
(15 and 22) were coupled to the right-hand tripeptide (14) to afford R-hydroxyamides, which
upon Dess-Martin periodinane oxidation furnished the desired R-ketoamides. Esters, acids,
and amides were incorporated at the C-terminal of these peptides. These inhibitors were tested
in an HCV protease continuous assay. The binding constants (Ki*) indicated that the
16-membered macrocyclic inhibitors (23 and 24) were less potent than the 17-membered
analogues (16-19). It was also evident that C-terminal acids (i.e., 17) and amides (18 and 19)
(Ki* range: 0.16-0.31 µM) were much better inhibitors than tert-butyl esters (16 and 23).
The X-ray crystal structure of compound 17 bound to the enzyme revealed that the macrocycle
formed a “donut”-shaped ring around the methyl group of Ala156. P2′ phenyl and P1 propyl
groups wrapped around the Lys136 side chain, forming a “C”-shaped clamp. The 17-membered
macrocyclic inhibitors 17-19 were significantly more potent than the acyclic pentapeptide 1.
Introduction
target in drug design. Nonetheless, considerable efforts
have been targeting the NS3 serine protease and a
number of potent inhibitors have been identified by
various research laboratories around the world.4
The hepatitis C virus (HCV) has infected more than
170 million people worldwide. It is the major cause of
chronic liver disease leading to cirrhosis, hepatocellular
carcinoma, and, ultimately, liver failure. HCV is emerg-
ing as a global health crisis.1 The only currently avail-
able therapies are subcutaneous R-interferon or pegyl-
ated R-interferon monotherapy or R-interferon and oral
ribavirin combination therapy. These therapeutics are
only effective in up to 50% of the patients and have
considerable side effects in certain patients.2 Given the
prevalence of HCV infections, a small molecule drug is
highly desirable.
In the hepatitis C virus, the virally encoded protease
responsible for processing the nonstructural (NS) por-
tion of the polyprotein is located in the N-terminal
portion of the NS3 protein. Studies have confirmed that
the NS3 protease belongs to the trypsin or chymotrypsin
superfamily of serine proteases and that it is essential
for viral replication.3 However, the NS3 protease is
unique, in that it requires a small polypeptide cofactor
(NS4A) for efficient processing. The fact that the binding
pockets of the NS3 protease are very shallow, solvent-
exposed, and relatively featureless makes it a difficult
Many protease inhibitors developed in recent years
are peptides because their design is based on the
cleavage products of the substrates. Peptides are easily
cleaved by peptidases and, thus, suffer from low bio-
availability and poor pharmacological profiles.5 Major
efforts have been undertaken to design peptidomimetics
with less peptide character. One such approach that is
showing considerable promise is through macrocycliza-
tion.6 Many biologically active macrocyclic molecules
exist in nature. The vancomycin family of antibiotics
and chloropeptins are well-known examples.7 Macro-
cycles offer certain advantages over peptides as drug
candidates. They are conformationally preorganized for
stronger binding. They are also potentially more stable
toward degradation by peptidases.
It has been shown that N-terminal cleavage products
(e.g., DDIVPC-OH) of the peptide substrates are com-
petitive inhibitors of HCV NS3 protease.8 We and others
have been using the hexapeptide as the starting point
in developing more potent and smaller HCV inhibitors.
Among the different classes of inhibitors discovered,
R-ketoacids and ketoamides, with their electrophilic
* To whom correspondence should be addressed. Tel: (908) 740-
7556. Fax: (908) 740-7152. E-mail: kevin.chen@spcorp.com.
10.1021/jm050323b CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/31/2005

6230 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Chen et al.
Scheme 1a
Figure 1. Pentapeptide HCV inhibitor.
“war-heads”, have demonstrated their potential in VX-
950, a clinical candidate in development from Vertex
Pharmaceuticals.9 In the course of our search for potent
HCV inhibitors, we found that compound 1 (Figure 1),
a pentapeptide R-ketoamide, had modest potency against
NS3 protease (Ki* ) 8 µM).10 We envisioned that
cyclization between the P2 phenyl group and the N-
terminal P3 capping group would provide a macrocycle
which could have enhanced binding with the protease.
Macrocyclization would also reduce the peptidic nature
of the inhibitor. Computer modeling using an enzyme
surface from the X-ray crystal structure of the protein
confirmed that the strategy was feasible and that the
appropriate ring size would be 16 or 17.
Synthesis
The key step in the synthesis of a compound such as
8 is macrocyclization. Among various synthetic meth-
odologies available, we envisioned that ring closure
through an aryl alkyl ether formation from a phenol
alcohol precursor could be accomplished under Mit-
sunobu reaction conditions.11
a Reaction conditions: (a) DhBtOH, EDC, NMM, CH2Cl2/DMF,
-20 °C, 88%; (b) 4 N HCl, p-dioxane, rt, 96%; (c) hept-6-enoic acid,
DhBtOH, EDC, NMM, CH2Cl2/DMF, -20 °C, 94%; (d) BH3‚HF,
THF, 0 °C, 68%; (e) (n-Bu)3P, ADDP, CH2Cl2, rt, 68%.
Our synthesis started with commercially available
N-Boc-cyclohexylglycine 2 and m-tyrosine methyl ester
hydrochloride 3 (Scheme 1). Standard coupling between
2 and 3 using 3-hydroxy-1,2,3-benzotriazin-4(3H)-one
(DhBtOH), 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide (EDC) hydrochloride, and 4-methylmorpholine
(NMM) afforded dipeptide 4. The Boc protecting group
was removed, and the resulting amine hydrochloride 5
was coupled with hept-6-enoic acid under the same
coupling conditions to provide compound 6. The termi-
nal alkene was then converted to primary alcohol 7
through hydroboration using borane-THF. With the
macrocycle precursor in hand, macrocyclization of 7 was
attempted under Mitsunobu reaction conditions.11 The
reaction was first conducted in benzene using tri-n-
butylphosphine and 1,1′-(azodicarbonyl)dipiperidine
(ADDP). Under these conditions, a very low yield of 8
was obtained, possibly due to low solubility of the
substrate in benzene. When the solvent was changed
to dichloromethane, the desired macrocycle (8) was
obtained in good yield.
Scheme 2a
The right-hand tripeptide segment of the inhibitors
was prepared starting from 1-nitrobutane 9 (Scheme 2).
Thus, when treated with triethylamine, the reaction
between 9 and glyoxylic acid afforded compound 10 as
a diastereomeric mixture. The nitro group was reduced
to an amine via low-pressure catalytic hydrogenation.
The resulting amino acid was converted to a Boc
protected norvaline R-hydroxyacid (11). Coupling of 11
with glycine benzyl ester under standard conditions
gave dipeptide 12, which, upon hydrogenation, provided
a Reaction conditions: (a) glyoxylic acid monohydrate, Et3N,
MeOH, 0 °C, then rt, 99%; (b) H2, 59 psi, Pd/C, AcOH, rt, 66%; (c)
NaOH, (Boc)2O, dioxane/H2O, rt, 89%; (d) glycine benzyl ester
hydrochloride, DhBtOH, EDC, NMM, DMF/CH2Cl2, -20 °C, 95%;
(e) H2, Pd/C, EtOH, rt, 98%; (f) phenylglycine tert-butyl ester
hydrochloride, DhBtOH, EDC, NMM, DMF/CH2Cl2, -20 °C, 80%;
(g) 2 M HCl in EtOAc/dioxane (1:1), rt, quantitative.
carboxylic acid 13. Compound 13 was coupled to phen-
ylglycine tert-butyl ester to afford a tripeptide. Treat-

Inhibitors of Hepatitis C Virus NS3 Serine Protease
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6231
Scheme 3a
Scheme 4a
a Reaction conditions: (a) LiOH, MeOH/THF/H2O, rt, 81%; (b)
14, DhBtOH, EDC, NMM, DMF/CH2Cl2, -20 °C, 74%; (c) Dess-
Martin periodinane, CH2Cl2, rt, 35%; (d) TFA, CH2Cl2, rt, quan-
titative; (e) methylamine hydrochloride, DhBtOH, EDC, NMM,
DMF/CH2Cl2, -20 °C, 58%; (f) dimethylamine, DhBtOH, EDC,
NMM, DMF/CH2Cl2, -20 °C, 46%.
a Reaction conditions: (a) 6-hydroxyhexanoic acid, DhBtOH,
EDC, NMM, LiOH, DMF/CH2Cl2, -20 °C, 39%; (b) n-Bu3P, ADDP,
CH2Cl2, rt, 74%; (c) LiOH, MeOH/THF/H2O, rt, 88%; (d) 14,
DhBtOH, EDC, NMM, DMF/CH2Cl2, -20 °C, 48%; (e) Dess-
Martin periodinane, CH2Cl2, rt, 70%; (f) TFA, CH2Cl2, rt, quan-
titative.
ment of this product with 2 M hydrochloric acid selec-
tively removed the Boc protecting group in the presence
of tert-butyl ester to give the desired amine (14).
With both left-hand macrocycle and right-hand trip-
eptide fragments in hand, the stage was set to assemble
the final targets. Thus, methyl ester 8 was hydrolyzed
to acid 15 using lithium hydroxide (Scheme 3). Com-
pound 15 was coupled with amine 14 to afford an
R-hydroxyamide, which was oxidized to R-ketoamide 16
using Dess-Martin periodinane.12 Under low-temper-
ature coupling conditions, no racemization occurred at
the phenylalanine center. Treatment of 16 with trifluo-
roacetic acid provided carboxylic acid 17. Coupling of
17 with methylamine or dimethylamine under standard
conditions gave methyl amide 18 and dimethyl amide
19, respectively. The compounds were isolated as an
inseparable mixture of two diastereomers. The ratio of
the two diastereomers was in the range of 4:6 to 6:4 but
was compound specific.
To determine the effect of ring size on the potency of
the inhibitors, 16-membered macrocycle 21 was also
prepared through a reaction sequence similar to that
described in the synthesis of 17-membered macrocycle
8 in Scheme 1. Starting from amine 5, the ultimate
product R-ketoamide 23 was obtained after a number
of operations. Treatment of 23 with trifluoroacetic acid
converted it to carboxylic acid 24 (Scheme 4).
Table 1. Inhibitors of HCV NS3 Protease
compound
R
macrocycle ring size
Ki* (µM)
1
NH2
8.0 ( 1.5
5.5 ( 1.3
0.16 ( 0.02
0.17 ( 0.02
0.31 ( 0.11
>8.9
1.2 ( 0.10
16
17
18
19
23
24
OtBu
OH
17
17
17
17
16
16
NHMe
NMe2
OtBu
OH
formed between the ketone and serine hydroxyl, and
other interactions between the inhibitor and enzyme.15
The Ki* values along with standard errors for these
compounds are listed in Table 1. It was evident that
17-membered macrocyclic inhibitors were more potent
than 16-membered analogues. tert-Butyl ester 16 (17-
membered) was a modest inhibitor with a Ki* of 5.5 µM,
while 23 (16-membered) was inactive. Presumably, the
bulky tert-butyl group not only prevented hydrogen
bonding of the P2′ carbonyl with the enzyme backbone
but also had some detrimental steric interactions with
the protein surface. Seventeen-membered acid 17 (Ki*
) 0.16 µM) was a potent inhibitor that was eight times
more active than 16-membered derivative 24 (Ki* ) 1.2
µM). Both monomethyl and dimethyl amides (18 and
19) were also potent inhibitors (Ki* ) 0.17 and 0.31 µM,
respectively). This study clearly demonstrated that, with
the right ring size, macrocyclization increases the
potency of these inhibitors over that of acyclic analogue
1 26-50 times (17-19 vs 1).
Results and Discussions
Macrocyclic compounds 16-19, 23, and 24 were
tested in an HCV continuous assay13 using the NS4A-
tethered single chain NS3 serine protease.14 The Ki*
value is a collective reflection of the equilibrium con-
stant, determined by the reversible covalent bond
The selectivity of compound 17 was measured against
human neutrophil elastase (HNE), which is the closest
serine protease to human hepatitis C virus NS3 pro-

6232 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Chen et al.
Ser139 hydroxyl of the protease. It also revealed that
the macrocycle adopted a donut-shaped conformation on
top of Ala156. A C-shaped clamping around the Lys136
side chain by the P1 norvaline and the P2′ phenylglycine
was also observed.
Experimental Section
General Methods. Reagents and solvents, including an-
hydrous tetrahydrofuran (THF), dichloromethane, and DMF,
were purchased from Aldrich or other commercial sources and
were used without further purification. Reactions that were
moisture sensitive or used anhydrous solvents were performed
under either a nitrogen or an argon atmosphere. Analytical
thin-layer chromatography (TLC) was performed on precoated
silica gel plates obtained from Analtech. Visualization was
accomplished with UV light or via staining with basic KMnO4
solution, ethanolic H2SO4, or Vaughn’s reagent. Compounds
were purified by flash chromatography either on a glass
column using Merck silica gel 60 (230-400 mesh) or on an
ISCO RediSep disposable silica gel column. NMR spectra were
recorded at 300, 400, or 500 MHz for 1H and at 75, 100, or
125 MHz for 13C on a Bruker or Varian spectrometer with
CDCl3 or DMSO-d6 as a solvent. The chemical shifts are given
in ppm and referenced to the internal TMS or deuterated
solvent signal.
Figure 2. X-ray structure of compound 17 bound to HCV NS3
protease.
tease. The ratio of Ki* values (HNE/HCV) was 5. This
indicated that compound 17 has a moderate selectivity
against another closely related protease.
The X-ray crystal structure of inhibitor 17 bound to
HCV NS3/NS4A protease was obtained (Figure 2). As
expected, the P1 (S)-diastereomer was more potent and
was observed in the crystal structure. A reversible
covalent bond was formed between the enzyme active
site serine (Ser139) hydroxyl and the ketone carbonyl
of the inhibitor. The resulting oxygen anion was stabi-
lized by hydrogen bonding with His57. The macrocycle
encircled the methyl group of Ala156. This established
a donut-shaped conformation that provided extra in-
teraction between the inhibitor and the protease. The
inhibitor-protease complex indicated that the smaller
16-membered ring was not large enough to properly
interact with the Ala156 residue. The n-propyl side
chain of the P1 norvaline fit very well within the S1
pocket. In addition, the aromatic ring of the P2′ phen-
ylglycine reaches to the other side of the Lys136 side
chain such that it formed a C-shaped clamp around the
lysine residue along with a P1 propyl group. Although
this phenomenon is not unique for macrocyclic inhibi-
tors, the clamping is part of the overall binding that
contributes to the potency of the compound. The inhibi-
tor also made multiple hydrogen bonds with the pro-
tease through its amide chain.
N-tert-Butoxycarbonyl-(S)-cyclohexylglycine-(S)-m-ty-
rosine Methyl Ester (4). To a solution of (S)-m-tyrosine
methyl ester hydrochloride 3 (2.00 g, 8.63 mmol), N-tert-
butoxycarbonyl-(S)-cyclohexylglycine (2.30 g, 8.94 mmol), 3,4-
dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (DhBtOH) (1.50
g, 9.20 mmol), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiim-
ide (EDC) hydrochloride (1.99 g, 10.38 mmol) in anhydrous
DMF (100 mL) and CH2Cl2 (100 mL) at -20 °C was added
4-methylmorpholine (NMM) (3.00 mL, 27.3 mmol). After
stirring at this temperature for 30 min, the reaction mixture
was kept in a freezer for 18 h. It was then allowed to warm to
room temperature. EtOAc (300 mL), brine (100 mL), and 5%
aqueous H3PO4 solution (100 mL) were added. After separation
of the layers, the organic solution was washed consecutively
with 5% aqueous H3PO4 solution (150 mL), aqueous saturated
sodium bicarbonate solution (2 × 150 mL), water (150 mL),
and brine (150 mL). It was then dried over magnesium sulfate,
filtered, and concentrated in vacuo. Flash chromatography (2-
5% MeOH/CH2Cl2) afforded 4 (3.43 g, 88% yield) as a white
1
foam. H NMR (500 MHz, CDCl3) δ 7.42 (s, 1H), 7.12 (t, J )
7.6 Hz, 1H), 6.76 (dd, J ) 8.1, 1.7 Hz, 1H), 6.58-6.54 (m, 2H),
6.47 (d, J ) 7.5 Hz, 1H), 5.28 (d, J ) 9.1 Hz, 1H), 4.89-4.85
(m, 1H), 3.96 (dd, J ) 9.0, 7.0 Hz, 1H), 3.73 (s, 3H), 3.13 (dd,
J ) 13.7, 5.7 Hz, 1H), 3.04 (dd, J ) 13.7, 4.2 Hz, 1H), 1.75-
1.58 (m, 6H), 1.46 (s, 9H), 1.28-0.93 (m, 5H). 13C NMR (125
MHz, CDCl3) δ 171.5, 171.4, 156.5, 156.3, 136.3, 129.7, 120.4,
117.0, 114.5, 80.6, 59.8, 53.0, 52.4, 40.3, 36.8, 29.5, 28.4, 28.3,
25.9, 25.8. HRMS calcd for C23H35N2O6 (M + H)+: 435.2495.
Found: 435.2491.
Conclusion
H-(S)-Cyclohexylglycine-(S)-m-tyrosine Methyl Ester
Hydrochloride (5). A solution of 4 (10.0 g, 23.0 mmol) was
dissolved in a 4 M HCl solution in dioxane (100 mL) and stirred
at room temperature for 4 h. The reaction mixture was
concentrated in vacuo to give a solid residue, which was
suspended in ether (200 mL). The suspension was filtered, and
the solid was washed with ether and dried under vacuum to
give a white solid 5 (8.2 g, 96% yield). It was used in
subsequent reactions without further purification. 1H NMR
(400 MHz, CD3OD) δ 7.09 (t, J ) 8.0 Hz, 1H), 6.71-6.36 (m,
3H), 4.69 (dd, J ) 6.0, 3.2 Hz, 1H), 3.69 (s, 3H), 3.66 (d, J )
5.2 Hz, 1H), 3.15-3.10 (dd, J ) 5.6, 4.0 Hz, 1H), 1.87-1.69
(m, 6H), 1.32-1.10 (m, 5H).
N-6-Heptenoyl-(S)-cyclohexylglycine-(S)-m-tyrosine
Methyl Ester (6). The coupling of amine hydrochloride 5 (1.20
g, 3.23 mmol) and hept-6-enoic acid (0.610 g, 4.68 mmol) was
carried out in a manner similar to that described for the
preparation of 4. The crude product was purified by flash
chromatography (2-5% MeOH/CH2Cl2), which gave compound
A concise synthesis of both 16- and 17-membered
macrocycles 8 and 21 has been accomplished. The
macrocyclization of phenol alcohols was achieved through
a Mitsunobu protocol using tri-n-butylphosphine and
1,1′-(azodicarbonyl)dipiperidine. The macrocycles have
been incorporated into HCV protease inhibitors as
peptidomimetics for the P2-P3 dipeptide moiety. When
tested in an HCV continuous assay, 17-membered
macrocyclic inhibitors were more potent than their 16-
membered analogues. Carboxylic acid 17 and amides
18 and 19 were potent inhibitors with Ki* values in the
range of 0.16-0.31 µM. They were 26-50 times more
active than acyclic analogue 1. tert-Butyl esters (16 and
23) were much less potent. The X-ray structure of
compound 17 bound to enzyme confirmed the formation
of a covalent bond between the ketone carbonyl and the

Inhibitors of Hepatitis C Virus NS3 Serine Protease
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6233
6 (1.36 g, 94% yield) as a white solid. 1H NMR (500 MHz,
CDCl3) δ 7.62 (s, 1H), 7.11 (t, J ) 7.7 Hz, 1H), 7.78 (dd, J )
8.2, 2.6 Hz, 1H), 6.51 (d, J ) 7.2 Hz, 1H), 6.45 (d, J ) 1.5 Hz,
1H), 6.36 (d, J ) 7.9 Hz, 1H), 6.26 (d, J ) 9.2 Hz, 1H), 5.81-
5.73 (m, 1H), 5.01-4.93 (m, 2H), 4.84-4.81 (m, 1H), 4.22 (t, J
) 8.7 Hz, 1H), 3.75 (s, 3H), 3.13 (dd, J ) 14.0, 5.4 Hz, 1H),
3.01 (dd, J ) 13.8, 4.3 Hz, 1H), 2.30 (t, J ) 7.6 Hz, 2H), 2.06
(q, J ) 7.0 Hz, 2H), 1.81-1.64 (m, 8H), 1.46-1.39 (m, 2H),
1.26-0.96 (m, 5H). 13C NMR (125 MHz, CDCl3) δ 174.4, 171.2,
170.8, 171.1, 156.7, 138.2, 135.9, 129.7, 120.3, 117.8, 114.9,
114.8, 58.7, 53.1, 52.5, 39.7, 36.5, 33.3, 29.6, 29.1, 28.4, 26.0,
25.7, 25.0. HRMS calcd for C25H37N2O5 (M + H)+: 445.2702.
Found: 445.2683.
N-7-Hydroxyheptanoyl-(S)-cyclohexylglycine-(S)-m-
tyrosine Methyl Ester (7). To the solution of 6 (1.46 g, 3.28
mmol) in anhydrous THF (60 mL) under nitrogen at 0 °C was
added a borane-THF solution (12 mL, 1.0 M, 12 mmol)
cautiously. The resulting solution was stirred at 0 °C for 1 h
and 40 min. Ethanol (4 mL) and a pH 7 buffer (8 mL) were
added, followed by aqueous 30% H2O2 solution (7.5 mL). After
stirring at 0 °C for 20 min, the mixture was warmed to room
temperature and stirred for 2 h. EtOAc (200 mL) and brine
(100 mL) were added, and the layers were separated. An
aqueous solution was extracted with EtOAc (2 × 150 mL). The
combined organic solution was dried with magnesium sulfate,
filtered, and concentrated under reduced pressure. Flash
chromatography (2-5% MeOH/CH2Cl2) afforded 7 (1.05 g, 2.18
mmol, 68% yield) as a white solid. 1H NMR (500 MHz, DMSO-
d6) δ 9.28 (s, 1H), 8.35 (d, J ) 7.3 Hz, 1H), 7.73 (d, J ) 9.2 Hz,
1H), 7.04-7.00 (m, 1H), 6.60-6.58 (m, 3H), 4.40-4.33 (m, 2H),
4.20 (dd, J ) 8.8, 7.3 Hz, 1H), 3.54 (s, 3H), 3.36-3.33 (m, 2H),
2.89 (dd, J ) 13.9, 6.3 Hz, 2H), 2.81 (dd, J ) 13.9, 8.7 Hz,
2H), 2.16-2.02 (m, 2H), 1.65-1.34 (m, 10H), 1.27-0.88 (m,
9H). 13C NMR (125 MHz, DMSO-d6) δ 172.9, 172.7, 172.1,
158.1, 139.3, 130.0, 120.4, 116.7, 114.4, 61.5, 57.3, 54.4, 52.6,
40.9, 37.4, 35.9, 33.3, 29.8, 29.4, 28.8, 26.7, 26.4, 26.3, 26.1.
HRMS calcd for C25H39N2O6 (M + H)+: 463.2813. Found:
463.2808.
Methyl 11(S)-Cyclohexyl-9,12-dioxo-2-oxa-10,13-diaza-
bicyclo[14.3.1]eicosa-1(20),16,18-triene-14(S)-carboxyl-
ate (8). To a solution of phenol alcohol 7 (1.00 g, 2.16 mmol)
and tri-n-butylphosphine (1.10 mL, 4.28 mmol) in anhydrous
CH2Cl2 (100 mL) and THF (40 mL) at 0 °C under an argon
atmosphere was added 1,1′-(azodicarbonyl)dipiperidine (ADDP)
(1.08 g, 4.28 mmol). After stirring at 0 °C for 1 h, the solution
was warmed to room temperature and stirred for another 3
h. TLC indicated complete consumption of the starting mater-
ial. After removal of solvent in vacuo, the residue was purified
by flash chromatography (0-3% MeOH/CH2Cl2) to afford
compound 8 (650 mg, 1.46 mmol, 68% yield). An analytical
sample was obtained through a second flash column chroma-
tography eluted with a gradient of 0-80% acetone/hexanes.
1H NMR (500 MHz, DMSO-d6) δ 8.61 (d, J ) 8.3 Hz, 1H), 7.81
(d, J ) 9.4 Hz, 1H), 7.15 (t, J ) 7.9 Hz, 1H), 6.74 (d, J ) 7.6
Hz, 1H), 6.69 (dd, J ) 8.2, 2.2 Hz, 1H), 6.63 (s, 1H), 4.75-
4.71 (m, 1H), 4.29 (t, J ) 8.6 Hz, 1H), 3.96-3.92 (m, 1H), 3.79-
3.74 (m, 1H), 3.66 (s, 3H), 3.15 (d, J ) 14.2 Hz, 1H), 2.94 (dd,
J ) 16.0, 12.0 Hz, 2H), 2.30-2.25 (m, 1H), 2.00-1.96 (m, 1H),
1.70-0.83 (m, 19H). 13C NMR (125 MHz, DMSO-d6) δ 172.1,
171.7, 171.5, 158.9, 139.0, 129.1, 121.1, 113.0, 111.9, 66.4, 56.1,
52.1, 50.7, 40.7, 34.9, 34.2, 28.8, 28.4, 26.8, 26.3, 26.0, 25.6,
25.5, 25.3, 24.2. HRMS calcd for C25H37N2O5 (M + H)+:
445.2685. Found: 445.2702.
3-tert-Butoxycarbonylamino-2-hydroxyhexanoic Acid
(11). To a stirred solution of 1-nitrobutane (16.5 g, 0.160 mol)
and glyoxylic acid monohydrate (28.1 g, 0.305 mol) in MeOH
(120 mL) at 0 °C was added triethylamine (93.0 mL, 0.667 mol)
dropwise over 2 h. The solution was warmed to room temper-
ature, stirred overnight, and concentrated to an oil. The oil
was dissolved in water (100 mL), and the solution was acidified
to pH ) 1 with 10% aqueous HCl solution and extracted with
EtOAc (3 × 200 mL). The combined organic solution was
washed with brine, dried over Na2SO4, filtered, and concen-
trated to dryness to give 3-nitro-2-hydroxyhexanoic acid (28.1
g, 99% yield). To a stirred solution of the nitro-acid (240 g,
1.35 mol) in acetic acid (1.25 L) was added 10% Pd/C (37 g).
The resulting solution was hydrogenated at 59 psi for 20 h.
The acetic acid was then evaporated, and the residue was
azeotroped three times with toluene. It was then triturated
with MeOH and ether. The solid that formed was separated
by filtration and azeotroped twice with toluene to give the
amino acid product as an off-white solid (131 g, 66% yield).
To a stirred solution of this amino acid (2.0 g, 13.6 mmol) in
dioxane (10 mL) and H2O (5 mL) at 0 °C was added a 1 N
aqueous NaOH solution (14.0 mL, 14.0 mmol). After stirring
for 10 min, di-tert-butyl dicarbonate (3.10 g, 14.0 mmol) was
added and the mixture was stirred for 15 min at 0 °C. It was
then warmed to room temperature and stirred for an ad-
ditional 45 min before it was placed in a refrigerator overnight.
The reaction was then concentrated to dryness. The residue
was dissolved in EtOAc (100 mL). Ice (∼50 g), KHSO4 (3.36
g), and H2O (32 mL) were added, and the mixture was stirred
for 5 min. The layers were then separated, and the aqueous
layer was extracted twice with EtOAc (2 × 50 mL); the
combined organic solution was washed with water and brine,
dried (Na2SO4), filtered, and concentrated to dryness to yield
the product, 11 (3.0 g, 89% yield), as a mixture of four
diastereomers. HRMS calcd for C11H22NO5 (M + H)+: 248.1498.
Found: 248.1510.
(3-tert-Butoxycarbonylamino-2-hydroxyhexanoylami-
no)acetic Acid Benzyl Ester (12). The coupling of carboxylic
acid 11 (3.00 g, 12.0 mmol) and glycine benzyl ester hydro-
chloride (2.56 g, 13.0 mmol) was carried out in a manner
similar to that described for the preparation of 4. The crude
product was purified by flash chromatography (25-50%
acetone/hexanes) to afford dipeptide 12 (4.50 g, 95% yield) as
a mixture of four diastereomers. HRMS calcd for C20H31N2O6
(M + H)+: 395.2182. Found: 395.2199.
(3-tert-Butoxycarbonylamino-2-hydroxyhexanoylami-
no)acetic Acid (13). To a solution of the benzyl ester 12 (4.50
g, 11.4 mmol) in ethanol (50 mL) was added 5% Pd/C (1.0 g).
The mixture was stirred vigorously under a hydrogen atmo-
sphere at room temperature for 3 h before it was filtered
through a Celite pad. The filter cake was washed with EtOAc
(2 × 30 mL). The solution was concentrated under reduced
pressure to afford the product, 13 (3.40 g, 98% yield), as a
mixture of four diastereomers. LRMS m/z MH+: 305. HRMS
calcd for C13H25N2O6 (M + H)+: 305.1713. Found: 305.1699.
[2-(3-Amino-2-hydroxyhexanoylamino)acetylamino]-
phenylacetic Acid tert-Butyl Ester Hydrochloride (14).
The coupling of carboxylic acid 13 (2.00 g, 6.57 mmol) and
phenyl glycine tert-butyl ester hydrochloride (1.76 g, 6.57
mmol) was carried out in a manner similar to that described
for the preparation of 4. The crude product was purified by
flash chromatography (1:1 EtOAc/hexanes) to afford a tripep-
tide product (2.60 g, 80% yield) as a mixture of four
diastereomers. HRMS calcd for C25H40N3O7: 494.2866. Found:
494.2863. A solution of this tert-butyl ester (2.6 g, 5.30 mmol)
in 2 M HCl in EtOAc/dioxane (1:1) was stirred at room
temperature for 15 min. The reaction mixture was concen-
trated in vacuo to yield 14 (2.11 g, quantitative yield) as a
pale yellow solid, which was used in subsequent reactions
without further purification. HRMS calcd for C20H32N3O5 (M
+ H)+: 394.2342. Found: 394.2336.
11(S)-Cyclohexyl-9,12-dioxo-2-oxa-10,13- diazabicyclo-
[14.3.1]eicosa-1(20),16,18-triene-14(S)-carboxylic Acid (15).
To a solution of methyl ester 8 (330 mg, 0.742 mmol) in THF
(20 mL) and ethanol (10 mL) at room temperature was added
an aqueous solution of lithium hydroxide (70 mg, 15 mL of
H2O, 2.92 mmol). The mixture was stirred at room tempera-
ture for 3 h. The progress of the reaction was monitored by
TLC. After the solution was concentrated in vacuo to one-third
of its original volume, EtOAc (60 mL), 1 N HCl solution (10
mL), and water (20 mL) were added and the layers were
separated. The aqueous solution was extracted with EtOAc
(2 × 50 mL). Organic solutions were combined, dried with
magnesium sulfate, filtered, and concentrated to afford 15 (260
1
mg, 81% yield) as a white solid. H NMR (500 MHz, DMSO-

6234 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Chen et al.
d6) δ 12.7 (bs, 1H), 8.46 (d, J ) 8.2 Hz, 1H), 7.78 (d, J ) 9.4
Hz, 1H), 7.16-7.13 (m, 1H), 6.75 (d, J ) 7.5 Hz, 1H), 6.69 (dd,
J ) 8.2, 1.9 Hz, 1H), 6.63 (s, 1H), 4.64-4.60 (m, 1H), 4.28 (t,
J ) 8.5 Hz, 1H), 3.96-3.92 (m, 1H), 3.78-3.74 (m, 1H), 3.14
(d, J ) 14.3 Hz, 1H), 3.92 (dd, J ) 16.2, 11.7 Hz, 1H), 2.30-
2.25 (m, 1H), 2.00-1.96 (m, 1H), 1.69-1.39 (m, 10H), 1.34-
0.85 (m, 9H). 13C NMR (125 MHz, DMSO-d6) δ 173.3, 171.6,
171.3, 158.8, 139.4, 129.1, 121.2, 113.1, 111.8, 66.4, 56.2, 50.8,
40.8, 35.1, 34.2, 28.8, 28.3, 26.9, 26.3, 26.0, 25.6, 25.5, 25.3,
24.2. HRMS calcd for C24H35N2O5 (M + H)+: 431.2564.
Found: 431.2546.
1,1-Dimethylethyl-R-(S)-[[[[3-[[(11(S)-cyclohexyl-2-oxa-
10,13-diazabicyclo[14.3.1]eicosa-1(20),16,18-trien-14(S)-
yl)carbonyl]amino]-1,2-dioxohexyl]amino]acetyl]amino]-
benzeneacetate (16). The coupling of macrocyclic acid 15
(0.215 g, 0.499 mmol) and tripeptide amine hydrochloride 14
(0.220 g, 0.513 mmol) was carried out in a manner similar to
that described for the preparation of 4. The intermediate
product, R-hydroxyamide, was obtained as a mixture of dia-
stereomers (0.270 g, 0.335 mmol, 74% yield), which was used
in subsequent reactions without further purification. To the
solution of this product (0.290 g, 0.36 mmol) in anhydrous CH2-
Cl2 (60 mL) at room temperature was added 1,1,1-tris-
(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one (Dess-Mar-
tin reagent) (0.450 g, 1.06 mmol). The mixture was stirred for
3 h. Saturated aqueous sodium bicarbonate and sodium
thiosulfate solutions (40 mL each) were added. After stirring
for 10 min, the layers were separated. The aqueous solution
was extracted with CH2Cl2 (2 × 50 mL). The organic solutions
were combined, dried with magnesium sulfate, filtered, and
concentrated in vacuo. Flash chromatography (1-5% MeOH/
CH2Cl2) afforded 16 (100 mg, 0.124 mmol, 35% yield) as a
mixture of diastereomers. The two diastereomers were insepa-
rable under a variety of solvent combinations. The ratio of the
two isomers was in the range of 4:6 to 6:4 but was compound
specific and depended on the reaction conditions. HRMS calcd
for C44H61N5O9 (M + H)+: 804.4542. Found: 804.4548.
1,1-Dimethylethyl-R-(S)-[[[[3-[[(11(S)-cyclohexyl-2-oxa-
10,13-diazabicyclo[14.3.1]eicosa-1(20),16,18-trien-14(S)-
yl)carbonyl]amino]-1,2-dioxohexyl]amino]acetyl]amino]-
benzeneacetic Acid (17). A solution of tert-butyl ester 16
(56.8 mg, 0.0706 mmol) in trifluoroacetic acid (15 mL) and CH2-
Cl2 (15 mL) was stirred at room temperature for 4 h. The
mixture was concentrated in vacuo. The residue was redis-
solved in 1:1 MeOH/CH2Cl2 (3 mL) and concentrated to dryness
to afford product 17 (53 mg, 0.0707 mmol, quantitative) as a
mixture of diastereomers. HRMS calcd for C40H54N5O9 (M +
H)+: 748.3947. Found: 748.3922.
11(S)-Cyclohexyl-N-[1-[2-[[2-[[2-(methylamino)-2-oxo-
1(S)-phenylethyl]amino]-2-oxoethyl]amino]-1,2-dioxo-
ethyl]butyl]-9,12-dioxo-2-oxa-10,13-diazabicyclo[14.3.1]-
eicosa-1(20),16,18-triene-14(S)-carboxamide (18). Coupling
of acid 17 (60 mg, 0.080 mmol) and methylamine hydrochloride
(7.0 mg, 0.104 mmol) was carried out in a manner similar to
that described for the preparation of 4. The product was
purified by flash chromatography (0-5% MeOH/CH2Cl2) to
afford compound 18 as a mixture of diastereomers (35 mg, 58%
yield). HRMS calcd for C41H57N6O8 (M + H)+: 761.4238.
Found: 761.4230.
was carried out in a manner similar to that described for the
preparation of 4. The product was purified by flash chroma-
tography (10-50% EtOAc/CH2Cl2) to afford compound 20
(0.430 g, 39% yield). 1H NMR (500 MHz, CDCl3) δ 8.09 (s, 1H),
7.11 (t, J ) 8.0 Hz, 1H), 6.78-6.76 (m, 1H), 6.52-6.49 (m, 2H),
6.43-6.35 (m, 2H), 4.88-4.83 (m, 1H), 4.18 (t, J ) 8.6 Hz, 1H),
4.09-4.05 (m, 1H), 3.75 (s, 3H), 3.68-3.66 (m, 1H), 3.11 (dd,
J ) 13.5, 5.3 Hz, 1H), 3.04 (dd, J ) 13.5, 4.4 Hz, 1H), 2.68 (bs,
1H), 2.36-2.28 (m, 1H), 1.81-1.56 (m, 12H), 1.46-1.37 (m,
2H), 1.25-0.94 (m, 5H). 13C NMR (125 MHz, CDCl3) δ 174.5,
171.2, 171.0, 156.8, 136.0, 129.8, 120.2, 117.5, 114.9, 62.1, 58.9,
53.0, 52.5, 39.6, 36.7, 36.0, 31.5, 29.6, 29.1, 26.0, 25.8, 25.7,
24.8, 24.3. HRMS calcd for C24H36N2O6: 449.2652. Found:
449.2641.
10-Cyclohexyl-8,11-dioxo-2-oxa-9,12-diazabicyclo[13.3.1]-
nonadeca-1(19),15,17-triene-13-carboxylic Acid Methyl
Ester (21). Macrocyclization of 20 (0.420 g, 0.936 mmol) was
carried out in a manner similar to that described for the
preparation of 8. The product was purified by flash chroma-
tography (1-5% MeOH/CH2Cl2) to afford compound 21 (0.300
1
g, 74% yield). H NMR (500 MHz, CDCl3) δ 7.18 (t, J ) 7.8
Hz, 1H), 6.72 (dd, J ) 7.5, 2.2 Hz, 1H), 6.69 (d, J ) 7.5 Hz,
1H), 6.58 (d, J ) 2.0 Hz, 1H), 6.37 (d, J ) 7.8 Hz, 1H), 5.99 (d,
J ) 9.2 Hz, 1H), 4.86 (td, J ) 8.1, 4.0 Hz, 1H), 4.25 (t, J ) 8.5
Hz, 1H), 4.06-4.02 (m, 2H), 3.82 (s, 3H), 4.32 (dd, J ) 14.3,
3.8 Hz, 1H), 2.83 (dd, J ) 14.3, 8.5 Hz, 1H), 2.31 (ddd, J )
14.2, 7.3, 5.0 Hz, 1H), 2.12 (ddd, J ) 14.2, 9.2, 4.6 Hz, 1H),
1.82-1.39 (m, 14H), 1.29-0.96 (m, 5H). 13C NMR (125 MHz,
CDCl3) δ 173.0, 172.2, 171.2, 159.0, 137.9, 130.1, 121.6, 117.7,
113.0, 68.2, 58.3, 53.4, 53.2, 41.5, 38.2, 36.5, 29.8, 29.3, 28.3,
26.6, 26.3, 26.2, 25.8, 25.2. HRMS calcd for C24H34N2O5 (M +
H)+: 431.2546. Found: 431.2545.
10-Cyclohexyl-8,11-dioxo-2-oxa-9,12-diazabicyclo[13.3.1]-
nonadeca-1(19),15,17-triene-13-carboxylic Acid (22). Hy-
drolysis of methyl ester 21 (0.280 g, 0.650 mmol) was carried
out in a manner similar to that described for the preparation
of compound 15 to afford carboxylic acid 22 (0.238 g, 88%
1
yield). H NMR (500 MHz, DMSO-d6) δ 12.74 (bs, 1H), 8.47
(d, J ) 8.8 Hz, 1H), 7.72 (d, J ) 9.4 Hz, 1H), 7.14 (t, J ) 7.9
Hz, 1H), 6.78-6.76 (m, 1H), 6.68 (s, 1H), 4.50-4.45 (m, 1H),
4.24 (t, J ) 9.1 Hz, 1H), 4.08-4.03 (m, 1H), 4.01-3.96 (m, 1H),
3.07 (dd, J ) 14.3, 2.0 Hz, 1H), 2.77 (dd, J ) 14.3, 11.8 Hz,
1H), 2.26-2.20 (m, 1H), 1.93-1.88 (m, 1H), 1.63-1.43 (m,
10H), 1.38-1.22 (m, 4H), 1.20-0.84 (m, 5H). 13C NMR (125
MHz, DMSO-d6) δ 173.1, 171.7, 171.0, 158.2, 139.7, 129.2,
121.3, 116.8, 112.0, 66.9, 56.5, 52.1, 40.5, 35.9, 34.6, 28.9, 28.6,
27.7, 26.0, 25.5, 25.4, 24.9, 24.4. HRMS calcd for C23H32N2O5
(M + H)+: 417.2389. Found: 417.2379.
1,1-Dimethylethyl-R-(S)-[[[[3-[[(10(S)-cyclohexyl-8,11-
dioxo-2-oxa-9,12-diazabicyclo[13.3.1]nonadeca-1(19),15,-
17-trien-13(S)-yl)carbonyl]amino]-1,2-dioxohexyl]ami-
no] acetyl]amino]benzene Acetate (23). Coupling of
carboxylic acid 22 (0.230 g, 0.552 mmol) and tripeptide amine
hydrochloride 14 (0.262 g, 0.609 mmol) was carried out in a
manner similar to that described for the preparation of 4 to
give an R-hydroxy amide pentapeptide intermediate (0.210 g,
48% yield). This crude product (0.200 g, 0.253 mmol) was
oxidized with Dess-Martin periodinane in a manner similar
to that described for the preparation of compound 16. The
product was purified by flash chromatography (1-5% MeOH/
CH2Cl2) to afford compound 23 (0.140 g, 70% yield) as a
mixture of diastereomers. HRMS calcd for C43H60N5O9 (M +
H)+: 790.4391. Found: 790.4426.
11(S)-Cyclohexyl-N-[1-[2-[[2-[[2-(dimethylamino)-2-oxo-
1(S)-phenylethyl]amino]-2-oxoethyl]amino]-1,2-dioxo-
ethyl]butyl]-9,12-dioxo-2-oxa-10,13-diazabicyclo[14.3.1]-
eicosa-1(20),16,18-triene-14(S)-carboxamide (19). Coupling
of acid 17 (65 mg, 0.087 mmol) and dimethylamine (6.0 µL,
0.113 mmol) was carried out in a manner similar to that
described for the preparation of 4. The product was purified
by flash chromatography (0-5% MeOH/CH2Cl2) to afford
compound 19 as a mixture of diastereomers (31 mg, 46% yield).
HRMS calcd for C42H59N6O8 (M + H)+: 775.4394. Found:
775.4406.
R-(S)-[[[[3-[[(10(S)-Cyclohexyl-8,11-dioxo-2-oxa-9,12-
diazabicyclo[13.3.1]nonadeca-1(19),15,17-trien-13(S)-yl)-
carbonyl]amino]-1,2-dioxohexyl]amino]acetyl]amino]-
benzeneacetic Acid (24). Carboxylic acid 24 was obtained,
as a mixture of diastereomers, in a manner similar to that
described for the preparation of compound 17 in quantitative
yield. HRMS calcd for C39H52N5O9 (M + H)+: 734.3765.
Found: 734.3747.
2-[2-Cyclohexyl-2-(5-hydroxypentanoylamino)acetyl-
amino]-3-(3-hydroxyphenyl)propionic Acid Methyl Ester
(20). Coupling of 6-hydroxyhexanoic acid (0.620 g, 75% pure,
3.52 mmol) and amine hydrochloride 5 (0.922 g, 2.37 mmol)

Inhibitors of Hepatitis C Virus NS3 Serine Protease
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6235
Nonconventional Stereochemical Issues in the Design of the
Synthesis of the Vancomycin Antibiotics: Challenges Imposed
by Axial and Nonplanar Chiral Elements in the Heptapeptide
Aglycons. Angew. Chem., Int. Ed. 1998, 37, 2704-2708. (d) Rama
Rao, A.; Garjar, M.; Reddy, K.; Rao, A. Studies directed toward
the synthesis of vancomycin and related cyclic peptides. Chem.
Rev. 1995, 95, 2135-2167. (e) Mtsuzaki, K.; Ikeda, H.; Ogino,
T.; Matsumoto, A.; Woodruff, H. B.; Tanaka, H.; Omura, S.
Chloropeptins I and II, Novel Inhibitors Against gp120-CD4
Binding from Streptomyces sp. J. Antibiot. 1994, 47, 1173-1174.
(f) Matsuzaki, K.; Ogino, T.; Sunazuka, T.; Tanaka, H.; Omura,
S. Chloropeptins, New Anti-HIV Antibiotics Inhibiting gp120-
CD4 Binding from Streptomyces sp. II. Structure Elucidation of
Chloropeptin I. J. Antibiot. 1997, 50, 66-69.
References
(1) (a) Cohen, J. The Scientific Challenge of Hepatitis C. Science
1999, 285, 26-30. (b) Alter, M. J.; Kruszon-Moran, D.; Nainan,
O. V.; McQuillan, G. M.; Gao, F.; Moyer, L. A.; Kaslow, R. A.;
Margolis, H. S. The Prevalence of Hepatitis C Virus Infection
in The United States, 1988 Through 1994. N. Engl. J. Med. 1999,
341, 556-562. (c) Cuthbert, J. A. Hepatitis C: Progress and
Problems. Clin. Microbiol. Rev. 1994, 7, 505-532.
(2) (a) Neumann, A. U.; Lam, N. P.; Dahari, H.; Gretch, D. R.; Wiley,
T. E.; Layden, T. J.; Perelson, A. S. Hepatitis C Virus Dynamics
in vivo and the Antiviral Efficacy of Interferon-R Therapy.
Science 1998, 282, 103-107. (b) Rosen, H. R.; Gretch, D. R.
Hepatitis C Virus: Current Understanding and Prospects for
Future Therapies. Mol. Med. Today 1999, 5, 393-399. (c) Di
Bisceglie, A. M.; McHutchison, J.; Rice, C. M. New therapeutic
strategies for hepatitis C. Hepatology 2002, 35, 224-231.
(3) (a) Kwong, A. D.; Kim, J. L.; Rao, G.; Lipovsek, D.; Raybuck, S.
A. Hepatitis C Virus NS3/4A Protease. Antiviral Res. 1998, 40,
1-18. (b) Yan, Y.; Li, Y.; Munshi, S.; Sardana, V.; Cole, J. L.;
Sardana, M.; Steinkuehler, C.; Tomei, L.; De Francesco, R.; Kuo,
L. C.; Chen, Z. Complex of NS3 protease and NS4A Peptide of
BK strain Hepatitis C Virus: a 2.2 A resolution structure in a
hexagonal crystal form. Protein Sci. 1998, 7, 837-847.
(4) (a) Tan, S.-L.; He, Y.; Huang, Y.; Gale, M., Jr. Strategies for
Hepatitis C Therapeutic Intervention: Now and Next. Curr.
Opin. Pharmacol. 2004, 4, 465-470. (b) Malancona, S.; Colar-
russo, S.; Ontoria, J. M.; Marchetti, A.; Poma, M.; Stansfield, I.;
Laufer, R.; Marco, A. D.; Taliani, M.; Verdirame, M.; Gonzalez-
paz, O.; Matassa, V. G.; Narjes, F. SAR and Pharmacokinetics
Studies on Phenethylamide Inhibitors of the Hepatitis C Virus.
Bioorg. Med. Chem. Lett. 2004, 14, 4575-4579. (c) Llinas-Brunet,
M.; Bailey, M. D.; Ghiro, E.; Gorys, V.; Halmos, T.; Poirier, M.;
Rancourt, J.; Goudreau, N. A Systematic Approach to the
Optimization of Substrate-Based Inhibitors of the Hepatitis C
Virus NS3 Protease: Discovery of Potent and Specific Tripeptide
Inhibitors. J. Med. Chem. 2004, 47, 6584-6594. (d) Zhang, X.
Inhibitors of Hepatitis C - A Review of the Current Patent
Literature. IDrugs 2002, 5, 154-158. (e) Dymock, B. W. Emerg-
ing Therapies for Hepatitis C Virus Infection. Expert Opin.
Emerging Drugs 2001, 6, 13-42. (f) Dymock, B. W.; Jones, P.
S.; Wilson, F. X. Novel Approaches to the Treatment of Hepatitis
C Virus. Antiviral. Chem. Chemother. 2000, 11, 79-96. (g)
Zhang, R.; Durkin, J. P.; Windsor, W. T. Azapeptides as
Inhibitors of the Hepatitis C Virus NS3 Serine Protease. Bioorg.
Med. Chem. Lett. 2002, 12, 1005-1008. (h) Ingallinella, P.;
Fattori, D.; Altamura, S.; Steinkuhler, C.; Koch, U.; Cicero, D.;
Bazzo, R.; Cortese, R.; Bianchi, E.; Pessi, A. Prime Site Binding
Inhibitors of a Serine Protease: NS3/4A of Hepatitis C Virus.
Biochemistry 2002, 41, 5483-5492. (i) Beevers, R.; Carr, M. G.;
Jones, P. S.; Jordan, S.; Kay, P. B.; Lazell, R. C.; Raynham, T.
M. Solution and Solid-Phase Synthesis of Potent Inhibitors of
Hepatitis C Virus NS3 Protease. Bioorg. Med. Chem. Lett. 2002,
12, 641-643.
(8) (a) Llinas-Brunet, M.; Bailey, M. D.; Fazal, G.; Goulet, S.;
Halmos, T.; LePlante, S.; Maurice, R.; Poirier, M.; Poupart, M.-
A.; Thibeault, D.; Wernic, D.; Lamarre, D. Peptide-Based Inhibi-
tors of the Hepatitis C Virus Serine Protease. Bioorg. Med.
Chem. Lett. 1998, 8, 1713-1718. (b) Steinkuhler, C.; Biasiol, G.;
Brunetti, M.; Urbani, A.; Koch, U.; Cortese, R.; Pessi, A.; De
Francesco, R. Product Inhibition of the Hepatitis C Virus NS3
Protease. Biochemistry 1998, 37, 8899-8905.
(9) (a) Lin, C.; Lin, K.; Luong, Y.-P.; Rao, B. G.; Wei, Y.; Brennan,
D. L.; Fulghum, J. R.; Hsiao, H.-M.; Ma, S.; Maxwell, J. P.;
Cottrell, K. M.; Perni, R. B.; Gates, C. A.; Kwong, A. D. In Vitro
Resistance Studies of Hepatitis C Virus Serine Protease Inhibi-
tors, VX-950 and BILN 2061. J. Biol. Chem. 2004, 279, 17508-
17514. (b) Sun, D. X.; Liu, L.; Heinz, B.; Kolykhalov, A.; Lamar,
J.; Johnson, R. B.; Wang, Q. M.; Yip, Y.; Chen, S.-H. P4 Cap
Modified Tetrapeptidyl R-Ketoamides as Potent HCV NS3
Protease Inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 4333-
4338. (c) Yip, Y.; Victor, F.; Lamar, J.; Johnson, R.; Wang, Q.
M.; Glass, J. I.; Yumibe, N.; Wakulchik, M.; Munroe, J.; Chen,
S.-H. P4 and P1′ Optimization of Bicycloproline P2 Bearing
Tetrapeptidyl R-Ketoamides as HCV Protease Inhibitors. Bioorg.
Med. Chem. Lett. 2004, 14, 5007-5011. (d) Perni, R. B.; Farmer,
L. J.; Cottrell, K. M.; Court, J. J.; Courtney, L. F.; Deininger, D.
D.; Gates, C. A.; Harbeson, S. L.; Kim, J. L.; Lin, C.; Lin, K.;
Luong, Y.-P.; Maxwell, J. P.; Murcko, M. A.; Pitlik, J.; Rao, B.
G.; Schairer, W. C.; Tung, R. D.; Van Drie, J. H.; Wilson, K.;
Thomson, J. A. Inhibitors of Hepatitis C Virus NS3/4A Protease.
Part 3: P2 Proline Variants. Bioorg. Med. Chem. Lett. 2004, 14,
1939-1942.
(10) Schering-Plough Research Institute: unpublished results.
(11) (a) Hughes, D. L. The Mitsunobu Reaction. Org. React. 1992,
42, 335. (b) Mitsunobu, O. The Use of Diethyl Azodicarboxylate
and Triphenylphosphine in Synthesis and Transformation of
Natural Products. Synthesis 1981, 1-28.
(12) Dess, D. B.; Martin, J. C. A Useful 12-I-5 Triacetoxyperiodinane
(the Dess-Martin Periodinane) for the Selective Oxidation of
Primary or Secondary Alcohols and a Variety of Related 12-I-5
Species. J. Am. Chem. Soc. 1991. 113, 7277-7287.
(13) Zhang, R.; Beyer, B. M.; Durkin, J.; Ingram, R.; Njoroge, F. G.;
Windsor, W. T.; Malcolm, B. A. A Continuous Spectrophotometric
Assay for the Hepatitis C Virus Serine Protease. Anal. Biochem.
1999, 270, 268-275; The substrate Ac-DTEDVVP(Nva)-O-PAP
was used in the present study.
(14) Taremi, S. S.; Beyer, B.; Maher, M.; Yao, N.; Prosise, W.; Weber,
P. C.; Malcolm, B. A. Construction, Expression, and Character-
ization of a Novel Fully Activated Recombinant Single-Chain
Hepatitis C Virus Protease. Protein Sci. 1998, 7, 2143-1249.
(15) For a definition of Ki* and discussions, see: Morrison, J. F.;
Walsh, C. T. The Behavior and Significance of Slow-binding
Enzyme Inhibitors. Adv. Enzymol. 1998, 61, 201-301.
(5) West, M. L.; Fairlie, D. P. Targeting HIV-1 protease: A Test of
Drug-Design Methodologies. Trends Pharm. Sci. 1995, 16, 67-
75.
(6) Fairlie, D. P.; West, M. L.; Wong, A. K. Towards Protein Surface
Mimetics. Curr. Med. Chem. 1998, 5, 29-62.
(7) (a) Boger, D. L.; Kim, S. H.; Miyazaki, S.; Strittmatter, H.; Weng,
J.-H.; Mori, Y.; Rogel, O.; Castle, S. L.; McAtee, J. J. Total
Synthesis of the Teicoplanin Aglycon. J. Am. Chem. Soc. 2000,
122, 7416-7417. (b) Evans, D. A.; Wood, M. R.; Trotter, B. W.;
Richardson, T. I.; Barrow, J. C.; Katz, J. L. Total Syntheses of
Vancomycin and Eremomycin Aglycons. Angew. Chem., Int. Ed.
1998, 37, 2700-2704. (c) Evans, D. A.; Dinsmore, C. J.; Watson,
P. S.; Wood, M. R.; Richardson, T. I.; Trotter, B. W.; Katz, J. L.
JM050323B