Novel potent hepatitis C virus NS3 serine protease inhibitors derived from proline-based macrocycles

Article abstract of DOI:10.1021/jm050820s

The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline

Full text of DOI:10.1021/jm050820s

J. Med. Chem. 2006, 49, 995-1005
995
Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors Derived from Proline-Based
Macrocycles
Kevin X. Chen,* F. George Njoroge, Ashok Arasappan, Srikanth Venkatraman, Bancha Vibulbhan, Weiying Yang,
Tejal N. Parekh, John Pichardo, Andrew Prongay, Kuo-Chi Cheng, 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 August 17, 2005
The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for
intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that
macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue
and the S4 pocket. Thus, a number of P2 proline-based macrocyclic R-ketoamide inhibitors were prepared
and investigated in an HCV NS3 serine protease continuous assay (Ki*). The biological activity varied
substantially depending on factors such as the ring size, number of amino acid residues, number of methyl
substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The
pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones
(24, Ki* ) 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered
macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active.
gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45,
Ki* ) 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47
realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC50 of 130 nM in a
cellular replicon assay, while IC50 for 24 was 400 nM. Several compounds had excellent subcutaneous
AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger
pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45
bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group
and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency
(>20-fold greater than that of 1) and in structural depeptization.
Introduction
process, intensive efforts have been focused on NS3 serine
protease, and a number of novel inhibitors have been reported.7
Among them, two compounds have been progressed into phase
I clinical trials in human beings: BILN-2061 from Boehringer-
Ingelheim and VX-950 from Vertex Pharmaceuticals.8
Hepatitis C virus (HCV) is a human pathogen affecting nearly
3% of the world’s population. The disease was described as a
“silent epidemic” because only about 20% of patients develop
acute clinical hepatitis when infected with HCV. Most infections
progress to a chronic state that can lead to cirrhosis, liver failure,
or liver cancer.1 Currently, the most effective therapies for HCV
infection involve treatment with pegylated R-interferon, either
alone or in combination with the antiviral agent ribavirin. These
therapeutics have limited efficacy with a sustained response rate
of only about 50%.2 They are also accompanied by considerable
side effects in certain patients. Hence, more effective antiviral
agents for HCV with fewer side effects are in urgent need.
The HCV RNA genome encodes a polyprotein of ap-
proximately 3000 amino acids, which must be processed by host
and viral proteases into structural and nonstructural (NS)
polypeptides. The NS3 protease is located at the N-terminal
portion of the NS3 protein. It is responsible for processing four
cleavage sites of the nonstructural region and, thus, is essential
for viral replication.3 It has been determined that the HCV NS3
protease belongs to the trypsin or chymotrypsin superfamily of
serine proteases.4 For efficient processing, the protease forms
a complex with a small polypeptide cofactor, NS4A.5 The
structure data of the protease have revealed a shallow and
solvent-exposed substrate binding region, where the binding
energy is mainly derived from weak lipophilic and electrostatic
interactions.6 Despite tremendous difficulty encountered in the
The cleavage products of the substrates have been frequently
used as the starting point for the development of many protease
inhibitors. However, these strategies resulted in compounds that
are mostly peptidic in nature. It has been difficult to obtain
desirable pharmacological profiles because peptides are sus-
ceptible to cleavage by peptidases.9 Various peptidomimetics
have been designed to develop drug candidates with less peptide
character. Macrocyclization of a substrate has proven to be a
promising strategy,10 with BILN-2061 as a recent example.8a,b
Many naturally occurring macrocyclic molecules, such as the
vancomycin family of antibiotics and chloropeptins,11 are
biologically active. Potentially, macrocycles could offer certain
advantages over acyclic peptides as drug candidates. For
example, they are conformationally restrained for enhanced
binding, and they are less prone to degradation by peptidases.12
The hexapeptides, N-terminal cleavage products from the
substrates, have been shown to be competitive inhibitors of the
NS3 protease.13 These peptides have been used as lead
compounds and templates in developing more potent and smaller
HCV inhibitors. Various inhibitors with diverse structural types
have been discovered.8 One class of compounds employs a
reactive electrophile to form a reversible covalent bond with
active site serine hydroxyl. These compounds, exemplified by
R-ketoacids and ketoamides, have demonstrated their potential
as hepatitis C therapeutics.14 In the course of our search for
* To whom correspondence should be addressed. Phone: (908) 740-
7556. Fax: (908) 740-7152. E-mail: kevin.chen@spcorp.com.
10.1021/jm050820s CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/07/2006

996 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Chen et al.
Scheme 1a
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) (for tert-butyl ester) t-BuOH, EDC, DMAP,
CH2Cl2, rt, 34%; (for amides) amide coupling using one of the following
amines, NH4Cl, methylamine hydrochloride, allylamine, glycine benzyl ester
hydrochloride, glycine-phenylglycine tert-butyl ester hydrochloride, or
glycine-phenylglycine dimethylamide hydrochloride, and HOOBt, EDC,
NMM, DMF/CH2Cl2, -20 °C, 45-95%; (e) 4 M HCl in dioxane for 9-12
and 14, 2 M HCl in EtOAc/dioxane for 8 and 13, quantitative.
Figure 1. Pentapeptide HCV inhibitor.
Scheme 2a
Figure 2. Macrocyclic left-hand fragment.
potent HCV inhibitors, we discovered a pentapeptide R-keto-
amide, 1 (Figure 1). As a lead, compound 1 shows moderate
potency against NS3 protease (Ki* ) 220 nM).15 Modeling
studies on the enzyme surface from the X-ray crystal structure7
revealed the close proximity of the tert-butyl group of P2 proline
and the Boc capping group. We reasoned that it would be
interesting to investigate the potency of P2- and cap-cyclized
analogues such as a structure of type 2. The macrocycle would
be expected to provide enhanced binding with the methyl group
of Ala156 of the enzyme backbone. We also reckoned that
synthesis would be more facile through the use of simple amide
capping at P3. To possibly further improve binding in the S4
pocket, the tert-butyl group was also replaced with a phenyl
group. On the basis of modeling results, we estimated that the
appropriate ring size would be between 15 and 17.
a Reaction conditions: (a) amine hydrochlorides 8 and 10-14, HOOBt,
EDC, NMM, DMF/CH2Cl2, -20 °C, 50-95%; (b) Dess-Martin periodi-
nane, CH2Cl2, rt, 60-95%; then (for 16) CF3CO2H, CH2Cl2, rt, quantitative;
(for 20) 19, LiOH, THF/MeOH/water, rt; (for 21, 26, or 27) 20, BnNH2,
HCl‚H2NCH(Ph)CN, or H2NCH(Ph)CH2OH, HOOBt, EDC, NMM, DMF/
CH2Cl2, -20 °C, 85%; (for 23) 22, CF3CO2H, CH2Cl2, rt, quantitative.
amino acid, 6. The acid was converted to a tert-butyl ester, a
primary amide, a methylamide, or an allylamide under ester-
or amide-forming conditions. Treatment of intermediates of type
7 with hydrochloric acid removed the Boc protecting group to
provide the desired right-hand subunits 8-11. On the other hand,
acid 6 was coupled to glycine benzyl ester, glycine-phenyl-
glycine tert-butyl ester, or glycine-phenylglycine dimethyl-
amide to give the corresponding di- or tripeptide compounds,
which upon removal of the Boc protecting group afforded
necessary amine hydrochloride intermediates 12-14.
Synthesis of Macrocyclic Inhibitors
The synthesis of the left-hand-side macrocyclic methyl esters
starting from 4-hydroxyprolines has been reported in our earlier
publications.16 A number of macrocyclic acid intermediates (3,
Figure 2) with different ring sizes (15-17), heteroatoms (X )
O, N, S), and substituents (R ) H, Me) were prepared through
hydrolysis of these esters.
The right-hand segments of the inhibitors were prepared from
R-hydroxy-â-amino acid 6, which in turn was synthesized from
1-nitrobutane (4) (Scheme 1). Thus, when treated with triethyl-
amine, the reaction between 4 and glyoxylic acid afforded
compound 5 as a mixture of four diastereomers. The nitro group
was reduced to give an amine, which was protected as a Boc-
With both left-hand macrocyclic acids and right-hand mono-,
di-, and tripeptide amine intermediates in hand, the stage was
set to put together the final target molecules (Scheme 2). Thus,
carboxylic acid 15 was coupled to amine hydrochlorides 8 and
10-14 under standard conditions (HOOBt, EDC, NMM) to
afford a series of R-hydroxyester or R-hydroxyamide intermedi-

Hepatitis C Virus NS3 Serine Protease Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 997
Scheme 4a
Scheme 3a
a Reaction conditions: (a) 14, HOOBt, EDC, NMM, DMF/CH2Cl2, -20
°C, 69%; (b) Dess-Martin periodinane, CH2Cl2, rt, 80%.
a Reaction conditions: (a) 14, HOOBt, EDC, NMM, DMF/CH2Cl2, -20
°C, 50-95%; (b) (for 35) DMSO, Cl2CHCO2H, EDC, toluene, rt, 60%;
(for all others) Dess-Martin periodinane, CH2Cl2, rt, 60-95%; (c) 9, 11-
14, HOOBt, EDC, NMM, DMF/CH2Cl2, -20 °C, 48-90%; (d) (for 39)
DMSO, Cl2CHCO2H, EDC, toluene, rt, 35%; (for 40, 42, and 44-45, Dess-
Martin periodinane, CH2Cl2, rt, 55-88%; (for 41) 40, LiOH, THF/MeOH/
water, rt, 98%; (for 43) 41, BnNH2, HOOBt, EDC, NMM, DMF/CH2Cl2,
-20 °C, 80%.
ates. These coupling products were then converted to R-ke-
toesters or R-ketoamides (17-19, 22, 24, 25) using Dess-
Martin periodinane.17 The R-keto-tert-butyl ester was treated
with trifluoroacetic acid (TFA) to give the R-ketoacid 16. The
glycine benzyl ester product 19 was hydrolyzed to its corre-
sponding carboxylic acid 20. Compound 20 was then converted
to glycine benzylamide 21. Treatment of pentapeptide tert-butyl
ester 22 with TFA provided phenylglycine acid derivative 23.
Finally, coupling of compound 20 with (S)-R-aminophenylac-
etonitrile and phenylglycinol gave R-aminonitrile analogue 26
and â-amino alcohol analogue 27, respectively. The R-ketoester
or R-ketoamide products were a mixture of two diastereomers
at the P1 R-center, and in most cases, they are inseparable.
However, the dimethylamide derivative was separated by flash
chromatography to give rise to pure P1 S-isomer 24 and
R-isomer 25.
Similarly, when tert-leucine was used as the P3 moiety and
synthesis carried out as previously described,16 macrocyclic acid
29 was obtained (Scheme 3). Coupling of acid 29 and amine
14 with subsequent oxidation afforded inhibitor 30. The same
reaction sequence with 4-cis-hydroxyproline 31 as the starting
material provided the final target 32. Compounds 30 and 32
were isolated as a single S-isomer at the P1 R-center.
To determine the optimal ring size of the macrocycle, 15-
and 17-membered rings were also prepared using a previously
reported synthetic sequence.16 The macrocyclic acids were
coupled to amine hydrochloride 14 under standard conditions
to afford the desired pentapeptide compounds 33 and 34
(Scheme 4).
macrocyclic acid 38 was prepared (Scheme 4).16 The coupling
of 38 to amine hydrochlorides 9 and 11-14 under standard
conditions provided R-hydroxyamide intermediates which were
oxidized to the respective ketoamides. Primary ketoamide 39
was obtained through a modified Moffatt oxidation.21 Secondary
ketoamides 40-45 were obtained through a Dess-Martin
periodinane oxidation.
Variation of the P3 moiety was also of interest. The amino
acid residue can be varied readily. A series of macrocyclic acids
(46, Scheme 5) with a number of P3 hydrocarbon substituents
were prepared starting from 4-trans-hydroxyproline 28 following
previously described procedures.16 These acids were coupled
to amine 14 under standard conditions. The intermediate
R-hydroxyamides were oxidized to R-ketoamides 47-51 with
Dess-Martin periodinane.
Results and Discussion
Sixteen-Membered Macrocyclic HCV Inhibitors: P′-
Section SAR. All macrocyclic inhibitors described above were
tested in an HCV continuous assay18 using the NS4A-tethered
single-chain NS3 serine protease.19 The Ki* values in the assay
reflected the equilibrium constant determined by the reversible
covalent bond formed between the ketone and serine and other
interactions between the inhibitors and the enzyme.20 Most
compounds were isolated as an inseparable mixture of two
diastereomers at the P1 R-center. The ratio of two isomers varied
between 2:1 and 1:2. It should be noted that all our P2′ amides
were dimethylamides, instead of a primary amide as in
compound 1. Compound 1 was prepared through a solid-state
synthesis. During the course of our SAR study, we found that
compounds with dimethylamides were equally potent with
To explore the potential electronic or substitution effect from
the proline C-4 substituent (“X” in Scheme 4), the ether
functionality was replaced with a sulfide, sulfone, or sulfonamide
moiety. Thus, 16-membered macrocyclic acids with X ) S, SO2,
and NHSO2Ph were coupled to amine 14 to provide, after Dess-
Martin periodinane oxidation, R-ketoamide inhibitors 35-37
(Scheme 4).
The good potency realized from (tert-butyloxy)proline deriva-
tive 1 prompted us to investigate the tert-alkyl ether linked
proline macrocycle. Thus, dimethyl-substituted 16-membered

998 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Scheme 5. Inhibitors of HCV NS3 Proteasea
Chen et al.
Table 2. Inhibitors of HCV NS3 Protease
ring
Ki*
compd
n
size
X
(nM)
33
24
34
35
36
37
0
1
2
1
1
1
15
16
17
16
16
16
O
O
O
S
30b
8a
7a
10a
SO2
NSO2Ph
3300b
36b
a Single isomer (S) at the P1 R-center. b Mixture of S- and R-isomers at
the P1 R-center.
Table 3. Inhibitors of HCV NS3 Proteasea
P1-P2
moiety
Ki*
P1-P2
moiety
Ki*
(nM)
compd
(nM) compd
39
40
41
42
NH2
NH-allyl
NH-Gly-OH
NH-Gly-OBn
220
530
720
88
43
44
1
NH-Gly-NHBn
NH-Gly-Phg-OtBu
see Figure 1
269
16
220
6
45
NH-Gly-Phg-NMe2
a All compounds were a mixture of S- and R-isomers at the P1 R-center.
to S in 32, its Ki* increased more than 8-fold to 130 nM. The
R-substitution was clearly preferred to S-substitution.
Ring Size and P2 Substituent Variations. Compounds with
15- to 17-membered macrocycles were examined to evaluate
the effect of ring size on potency (Table 2). As a mixture of
two isomers at P1, the 15-membered inhibitor 33, with a Ki*
value of 30 nM, was less potent than 16-membered compound
24 (Ki* ) 8 nM), assuming that Ki* of a single isomer is
approximately half of that of a mixture. However, 17-membered
analogue 34 had an excellent potency of 7 nM, which was
similar to that achieved by 24. These results demonstrated that
a 16- or 17-membered ring was the preferred ring size rather
than a 15-membered ring.
a Reaction conditions: (a) 14, HOOBt, EDC, NMM, DMF/CH2Cl2, -20
°C, 50-95%; (b) Dess-Martin periodinane, CH2Cl2, rt, 60-95%.
Table 1. Biological Activity of HCV Inhibitors 16-27, 30, and 32
Ki*
Ki*
compd
P1-P2 moiety
OH
NHMe
NH-allyl
NH-Gly-OBn
NH-Gly-OH
(nM) compd
P1-P2 moiety
(nM)
16
17
18
19
20
6100
9900
10000
430
23
24
25
26
27
NH-Gly-Phg-OH
NH-Gly-Phg-NMe2
NH-Gly-Phg-NMe2
10
8a
500b
NH-Gly-NHCH(Ph)CN 310
7400
NH-Gly-NHCH(Ph)-
CH2OH
NH-Gly-Phg-NMe2
NH-Gly-Phg-NMe2
60
Table 2 also lists the assay results for compounds 35-37.
The linker from proline C-4 was changed from oxygen to sulfur
and nitrogen to explore the effects these different heteroatoms
would have on potency. The sulfide analogue 35 was nearly as
potent (Ki* ) 10 nM) as ether analogue 24. However,
introduction of a sulfone moiety as in compound 36 resulted in
greatly reduced activity (Ki* ) 3300 nM). The phenylsulfona-
mide derivative 37 was an active inhibitor (36 nM), but not as
potent as either the ether or the sulfide analogues. Apparently,
more polar functionalities such as sulfone and sulfonamide were
less tolerated in this region. The presence of a larger group
(phenyl) did not provide any improvement to the potency.
Effect of gem-Dimethyl Substitution: tert-Alkoxyproline
Derivatives. Inspired by tert-butoxyproline P2 in compound 1,
a number of tert-alkoxy-substituted proline macrocyclic inhibi-
tors were prepared and tested. The enzyme assay results for
these compounds are listed in Table 3. The smallest tripeptides,
compounds 39 and 40, were active inhibitors (220 and 530 nM,
respectively), realizing an at least 10-fold improvement in
activity over analogues of similar size listed in Table 1. The
same trend held for tetrapeptide inhibitors 41-43, albeit with
a smaller magnitude of improvement (5-10-fold) in activity
when compared to compounds 19-21. The larger pentapeptides
also demonstrated superiority in Ki*, with that of tert-butyl ester
44 being 16 nM and that of dimethylamide 45 being 6 nM.
Considering the fact that all these compounds were tested as a
mixture of two diastereomers at P1, a 3-fold enhancement in
these potencies was achieved in 44 and 45 when compared to
the activities of compounds 22 and 24, respectively. These
results have clearly demonstrated that gem-dimethyl substituents
provided additional binding which contributed to better potency.
The magnitude of improvement was more pronounced with
21
22
NH-Gly-NHBn
NH-Gly-Phg-OtBu
1100
44
30
32
15a
130a
a Single isomer (S) at the P1 R-center. b Single isomer (R) at the P1
R-center. All others are a mixture of S- and R-isomers at the P1 R-center.
primary amides.15 But primary amides were less metabolically
stable, which led to poor PK.
Results for compounds 16-27 derived from 16-membered
macrocycle 15 are presented in Table 1. Two other inhibitors
(30, 32) with tert-leucine P3 are also included. All but four
compounds were tested as P1 diastereomeric mixtures. Com-
pounds 24, 25, 30, and 32 were tested as single isomers. Clearly,
the general trend was that larger inhibitors were more potent
than the smaller analogues. All tripeptides (16-18) were
modestly active and had high single-digit micromolar Ki*. The
results for three tetrapeptides (19-21) were mixed, with the
benzyl ester 19 activity being in the submicromolar range (0.43
µM), that of acid 20 in the high micromolar range, and that of
benzylamide 21 in between (1.1 µM). The pentapeptides 22-
27, 30, and 32 were all more potent inhibitors than their
truncated analogues. The carboxylic acid 23 and dimethylamide
24 had excellent Ki* (10 and 8 nM, respectively). Compound
24 was a single diastereomer with S-configuration at the P1
R-center. The corresponding R-diastereomer 25 was much less
potent (500 nM), a 63-fold increase in Ki*. Replacement of
cyclohexyl with a tert-butyl group at P3 slightly increased Ki*
of dimethylamide 30 to 15 nM. Other P2′ phenylglycine
derivatives such as tert-butyl ester 22, nitrile 26, and amino
alcohol 27 were found to be less potent than their acid and amide
counterparts, presumably due to the diminished or loss of
hydrogen bond acceptor capability. On the other hand, when
the stereocenter at C-4 of the P2 proline was changed from R

Hepatitis C Virus NS3 Serine Protease Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 999
Table 4. SAR at the P3 Positiona
Table 6. HNE Selectivity of Representative HCV NS3 Protease
Inhibitors
24
45
30
47
R
H
Me
H
Me
R3
cyclohexyl
cyclohexyl
t-Bu
t-Bu
Ki*HCV (nM)
Ki*HNE/Ki*HCV
8a
6b
15a
17b
46
107
147
306
a Single isomer (S) at the P1 R-center. b Mixture of S- and R-isomers at
the P1 R-center.
Table 7. PK of Selected Compounds
24
16
1.5
23.1
3
34
17
0.40
5.5
45
16
0.46
2.2
39
16
0.3 0
40
16
26.3
13.6
97
a All compounds were a mixture of S- and R-isomers at the P1 R-center.
Table 5. Results from a Cell-Based Replicon Assay
macrocycle ring size
AUC(PO) (µM‚h)
AUC(IV) (µM‚h)
F(PO) (%)
0.8
2.1
AUC(SC) (µM‚h)
F(SC) (%)
41.6
90
34.7
63
16.5
77
34.7
24a
30a
40b
45b
47b
Ki* (nM)
IC50 (nM)
8
400
15
700
530
400
6
130
17
200
there was a slight advantage (∼2-fold) with a cyclohexyl group
at the P3 position rather than a tert-butyl group. Finally, the
IC50 of tripeptide 40 (400 nM) was very intriguing. It was
unusual that IC50 was similar to its Ki* value. A possible
explanation is that smaller molecules such as 40 have better
cell permeability than the larger analogues. Overall, the replicon
potency of compounds 24 and 40 was in the same range as that
of VX-950 (IC50 ) 350 nM),8d and 45 and 47 were more potent
than VX-950.
a Single isomer (S) at the P1 R-center. b Mixture of S- and R-isomers at
the P1 R-center.
smaller tri- and tetrapeptide inhibitors (18-21 vs 40-43). A
possible explanation is that less potent compounds were more
sensitive to any additional binding, thus giving rise to a larger
enhancement. Extra binding was not as important to the most
potent inhibitors. If compared with acyclic analogue compound
1, the macrocyclic pentapeptide 45 achieved a 36-fold improve-
ment in potency.
Selectivity of the Inhibitors against Human Neutrophil
Elastase. The ubiquitous existence of serine proteases presents
a challenge for any protease inhibitor with regard to selectivity.
The macrocyclic inhibitors were screened against human
neutrophil elastase (HNE), which is a serine protease structurally
closely related to HCV NS3 serine protease. Our goal was to
achieve a minimum selectivity of 100. Representative data for
16-membered macrocyclic inhibitors are shown in Table 6.
SAR of the P3 Amino Acid Residue. The S3 region of HCV
NS3 protease is highly lipophilic. The P3 moiety binds the
enzyme mainly through hydrophobic interactions. Six com-
pounds (45 and 47-51) with P3 side chains of various sizes
were examined in an HCV protease continuous assay, and the
results are included in Table 4. They were all tested as a mixture
of diastereomers at P1. It is evident that cyclohexylglycine
remained as the best moiety at the P3 position (45, 6 nM). Two
other amino acid (tert-leucine and valine) derivatives, 47 and
48, were 2-3-fold less potent (17 and 19 nM, respectively).
The analogues derived from isoleucine (49), phenylglycine (50),
and indanylglycine (51) were all significantly less active (7-
12-fold) than compound 45. These data suggested that cyclo-
hexylglycine is the amino acid of choice for the P3 position.
Potency of Selected Compounds in a Cell-Based Replicon
Assay. To further evaluate the potency and the “in vivo”
characteristics of these inhibitors, a few compunds were tested
in a replicon-based cellular assay,22 and the results are included
in Table 5. IC50, the concentration required for inhibition of
50% of virus replication, was recorded as a measure of cell-
based replicon potency, which is a more accurate reflection of
the potency of the inhibitors in a “real” living environment.
The IC50 values of pentapeptides 24 and 30 were 400 and 700
nM, respectively. However, compounds 45 and 47 were superior
with IC50 values of 130 and 200 nM, respectively. Apparently,
as was observed in the enzyme assay, the gem-dimethyl
substituents had a positive impact of about 3-fold on replicon
potency. Comparing compound 24 with 30, and 45 with 47,
After examination of the HNE inhibition data, an SAR was
observed for various combinations of linker dimethyl substit-
uents and P3 residues. When R3 was cyclohexyl and R was
hydrogen, the selectivity (HNE/HCV) of compound 24 was 46
with respect to HNE. The presence of two methyl groups in
the linker (45, R ) Me) improved not only the potency to 6
nM, but also the selectivity to 107. Replacement of cyclohexyl
with a tert-butyl group at P3 further enhanced the selectivity
against HNE. Thus, compounds 30 and 47 had an approximately
3-fold improvement in selectivity over 24 and 45, respectively.
As a mixture of two diastereomers, compound 47 exhibited both
good potency and good selectivity.
Pharmacokinetic Properties of Selected Compounds. A
number of the macrocyclic HCV protease inhibitors described
were evaluated in various PK studies. Results from these studies,
on selected compounds, are listed in Table 7. As expected, oral
bioavailability was quite low (0.8-3.0%) for the larger pen-
tapeptide compounds 24, 34, and 45. However, rat oral (PO)
AUC of compound 24 was reasonable (1.5 µM‚h) while IV
AUC was excellent (23.1 µM‚h). The fact that 24 had very good
IV AUC but low PO AUC implied that absorption could be a

1000 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Chen et al.
The P1 n-propyl group extended snugly into the S1 pocket. The
cyclohexyl moiety fit comfortably in the lipophilic S3 region.
In addition, the inhibitor also made multiple hydrogen bonds
with the enzyme backbone through its amide chain. The
enhancement in potency from the dimethyl substituents seemed
to come from the contact between one of the methyl groups
and the aromatic ring of the His57 residue.
Both structures demonstrated that the macrocycles had
excellent interaction with the protease, especially with the
Ala156 methyl group and S4 pocket. The concept of macro-
cyclization was demonstrated to be successful in achieving
compounds with better potency and that were more depeptized.
A 36-fold improvement in potency was accomplished from
compound 1 to compound 45. Although the oral bioavailability
was still not satisfactory for the most potent and selective
compounds, some exciting PK data in the subcutaneous study
and from a smaller compound such as 40 indicated that the
future is promising for some of these macrocyclic inhibitors.
Conclusion
In summary, a number of P2 proline-based macrocyclic
R-ketoamide inhibitors were prepared and investigated in an
HCV NS3 serine protease continuous assay. The biological
activity varied substantially depending on several factors such
as the ring size, number of amino acid residues, number of
methyl substituents, type of heteroatom in the linker, P3 residue,
and configuration at the proline C-4 center. The pentapeptide
inhibitors were very potent, with the C-terminal acid and amide
analogues being the most active ones. Single-digit nanomolar
potency (Ki*) was achieved with some of the macrocyclic
compounds (45, 6 nM). The tetrapeptides were less potent, while
the tripeptides were only moderately active. The 16- and 17-
membered macrocyclic compounds were equally potent, while
15-membered analogues were less active. A sulfide linker from
proline C-4 was as effective as an ether linkage. However, the
corresponding sulfone and nitrogen analogues were less effec-
tive. The dimethyl substituents at the carbon adjacent to the
oxygen improved the potency of all inhibitors across the board.
Compound 45 had very good potency in a cell-based replicon
assay (IC50 ) 130 nM). The tert-leucine at P3 and dimethyl
substituents at the linker enhanced the selectivity of the inhibitors
against HNE. The combination of these two factors in compound
47 realized a good selectivity of 307. Several compounds had
excellent subcutaneous AUC and bioavailability in rats. A less
potent smaller inhibitor, 40, had a remarkable oral bioavailability
of 97%. The X-ray crystal structures of compounds 24 and 45
bound to the protease confirmed the excellent potency of these
compounds resulted from good binding of the macrocyclic ring
with the Ala156 methyl group and S4 pocket. They also revealed
the formation of a covalent bond between the inhibitor ketone
carbonyl and the Ser139 hydroxyl of the protease. A C-shaped
clamping around the Lys136 side chain by n-propyl at P1 and
phenyl at P2′ was also observed. All these and other interactions
contributed to the enhanced activity of these inhibitors. The
strategy of macrocyclization has been proved to be successful
in both improving potency and structural depeptization.
Figure 3. X-ray crystal structures of compounds 24 and 45 bound to
HCV NS3 protease.
potential problem due to the size of the molecules. Though
smaller in size, the P1 primary ketoamide 39 was among the
compounds with very poor PK (rat PO AUC ) 0.30 µM‚h).
Presumably, the stability of the primary amide moiety was an
issue in compound 39. The tripeptide allylamide 40, however,
demonstrated excellent AUC values (PO, 26.3 µM‚h; IV, 13.6
µM‚h) and an exceptional oral bioavailability of 97%. It was
also encouraging to observe that AUCs in rat were all very good
(16.5-41.6 µM‚h) when the compounds were administered
subcutaneously (sc). Excellent subcutaneous bioavailability (63-
90%) provides a viable alternative way of administering the
compound if it becomes necessary.
X-ray Crystal Structures of Compounds 24 and 45 Bound
to Protease. The X-ray crystal structures of two compounds,
24 and 45, bound to the HCV NS3 protease active site were
obtained through soaking the compounds to enzyme crystals
(Figure 3). They bound to the enzyme surface with similar
conformations. As expected, one of the major interactions was
the formation of a reversible covalent bond between the active
site serine (Ser139) hydroxyl and the ketone carbonyl of the
inhibitor. However, as a result of the nucleophilic attack on the
face opposite that usually observed for serine protease, the
oxyanion hole was occupied by the P1 carboxylamide carbonyl,
rather than by the hemiketal oxygen atom. Instead, the hemiketal
oxygen was interacting with and stabilized by a catalytic
histidine residue (His57). The macrocycles encircled the methyl
group of alanine 156, forming a nice doughnut-shaped crown.
The phenyl group in the ring also had good contact within the
S4 pocket. Another interesting feature was that the n-propyl
side chain of the P1 norvaline and the aromatic ring of the P2′
phenylglycine wrapped around the side chain of lysine 136 in
such a manner that they formed a C-shaped clamp around it.
Experimental Section
General Methods. Reagents and solvents, including anhydrous
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.

Hepatitis C Virus NS3 Serine Protease Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1001
Visualization was accomplished with UV light or by 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
3.53-3.48 (m, 2H), 3.36 (d, J ) 15.6 Hz, 1H), 2.92 (s, 3H), 2.84
(s, 3H), 2.33 (dd, J ) 7.6, 13.6 Hz, 1H), 1.84-1.59 (m, 10H),
1.47-1.13 (m, 6H), 0.90-0.76 (m, 5H); 13C NMR (100 MHz,
DMSO-d6) δ 196.7, 171.2, 169.69, 169.67, 169.2, 166.9, 160.7,
158.3, 138.2, 137.5, 129.1, 128.6, 127.84, 127.76, 121.7, 115.4,
112.1, 76.9, 64.9, 63.4, 57.4, 54.4, 53.7, 53.3, 53.0, 41.6, 41.3, 36.6,
35.3, 33.7, 31.7, 28.9, 28.5, 28.3, 26.0, 25.3, 18.7, 13.5. HRMS
calcd for C42H58N6O9(M + H)+: 789.4187. Found: 789.4184. Data
for 25: 1H NMR (500 MHz, DMSO-d6) δ 8.75 (t, J ) 6.2 Hz,
1H), 8.56 (d, J ) 7.5 Hz, 1H), 8.43 (d, J ) 9.8 Hz, 1H), 8.07 (d,
J ) 7.8 Hz, 1H), 7.38-7.29 (m, 5H), 7.15 (t, J ) 7.8 Hz, 1H),
6.80 (s, 1H), 6.72 (app d, J ) 8.1 Hz, 2H), 5.82 (d, J ) 7.7 Hz,
1H), 5.04 (ddd, J ) 3.9, 7.9, 9.4 Hz, 1H), 4.78-4.43 (m, 1H),
4.28 (dd, J ) 7.9, 9.5 Hz, 1H), 4.20-4.00 (m, 4H), 3.96 (d, J )
10.8 Hz, 1H), 3.81 (t, J ) 5.8 Hz, 1H), 3.76 (d, J ) 6.3 Hz, 1H),
3.70-3.64 (m, 2H), 3.52-3.48 (m, 1H), 3.39-3.35 (m, 1H), 2.92
(s, 3H), 2.84 (s, 3H), 2.38-2.34 (m, 1H), 1.84-1.59 (m, 10H),
1.43-1.11 (m, 6H), 0.91-0.77 (m, 5H). 13C NMR (125 MHz,
DMSO-d6), δ, 196.6, 171.3, 169.9, 169.6, 169.2, 166.9, 160.9,
158.4, 138.1, 137.4, 129.1, 128.6, 128.3, 127.8, 127.75, 121.7,
115.3, 112.1, 76.7, 64.9, 63.3, 58.1, 54.5, 53.7, 53.1, 53.0, 41.6,
41.4, 36.5, 35.3, 33.8, 31.9, 29.1, 28.5, 28.3, 26.0, 25.3, 18.6, 13.4.
HRMS calcd for C42H58N6O9(M + H)+: 789.4187. Found: 789.4192.
12-Cyclohexyl-11,14-dioxo-2,6-dioxa-10,13-diazatricyclo[14.
3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxylic Acid ((([1-
(Cyanophenylmethyl)carbamoyl]methyl)aminooxalyl)butyl)-
amide (26). Compound 26 was prepared through standard coupling
of 20 and (S)-R-aminophenylacetonitrile hydrochloride according
to standard coupling procedures. It was isolated as a mixture of
two diastereomers: 1H NMR (500 MHz, DMSO-d6) δ 9.33-9.29
(m, 1H), 8.9 and 8.84 (t, J ) 6.0 Hz, 1H), 8.43 (d, J ) 10.0 Hz,
1H), 8.27 (d, J ) 7.0 Hz) and 8.11 (dd, J ) 5.5, 3.0 Hz, 1H),
7.50-7.40 (m, 5H), 7.15 (t, J ) 8.0 Hz, 1H), 6.80 (m, 1H), 6.73
(m, 2H), 6.21-6.18(m, 1H), 5.11-5.07 and 5.03-4.99 (m, 1H),
4.46 (dt, J ) 3.5, 10 Hz, 1H), 4.36 and 4.30 (t, J ) 9.5 and 8.5
Hz, 1H), 4.17-4.15 (m, 4H), 3.88-3.85 (m, 2H), 3.71-3.64 (m,
2H), 3.55-3.49 (m, 2H), 2.34-2.33 (m, 1H), 2.00-1.40 (m, 10H),
1.38-0.98 (m, 7H), 0.91-0.84 (m, 5H); 13C NMR (125 MHz,
DMSO-d6) δ 197.3, 197.2, 172.2, 170.8, 170.6, 168.7, 161.8, 161.7,
159.3, 159.2, 139.1, 139.0, 134.9, 130.0, 129.8, 127.9, 122.6, 119.3,
116.3, 113.0, 77.8, 77.7, 65.8, 64.2, 59.0, 58.3, 55.4, 55.3, 54.6,
54.3, 54.0, 44.3, 44.2, 42.6, 42.5, 42.3, 42.2, 34.7, 34.6, 33.0, 32.6,
30.0, 29.8, 29.4, 29.2, 26.9, 26.2, 19.6, 19.5, 14.4; HRMS m/z calcd
for C40H51N6O8 (M + H)+ 743.3768, found 743.3773.
12-Cyclohexyl-11,14-dioxo-2,6-dioxa-10,13-diazatricyclo[14.
3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxylic Acid (([1-
(2-Hydroxy-1-phenylethylcarbamoyl)methyl]aminooxalyl)butyl)-
amide (27). Compound 27 was prepared through standard coupling
of 20 and (S)-2-amino-2-phenylethanol: 1H NMR (500 MHz,
DMSO-d6) δ 8.71 and 8.65 (t, J ) 6.0 and 6.2 Hz, 1H), 8.42 (dd,
J ) 3.3, 9.3 Hz, 1H), 8.34 (dd, J ) 8.4, 11.1 Hz, 1H), 8.27 and
8.08 (d, J ) 6.8, 8.2 Hz, 1H), 7.31-7.21 (m, 5H), 7.15 (t, J ) 7.7
Hz, 1H), 6.79 (br s, 1H), 6.72 (dd, J ) 2.1, 8.1 Hz, 2H), 5.09-
4.98 (m, 1H), 4.84 (q, J ) 6.8 Hz, 1H), 4.44 (dt, J ) 5.5, 9.7 Hz,
1H), 4.35 and 4.28 (dd, J ) 7.7, 9.6 and 8.2, 9.4 Hz, 1H), 4.18-
4.02 (m, 2H), 3.97-3.83 (m, 2H), 3.81-3.63 (m, 2H), 3.59-3.47
(m, 4H), 3.38-3.35 (m, 1H), 2.35 (ddd, J ) 7.5, 13.2, 21.2 Hz,
1H), 1.84-1.59 (m, 10H), 1.48-1.23 (m, 3H), 1.17-1.09 (m, 3H),
0.90-0.78 (m, 5H); 13C NMR (125 MHz, DMSO-d6) δ 197.53,
197.50, 172.19, 172.16, 170.8, 170.57, 170.55, 168.2, 161.7, 161.5,
159.3, 159.2, 141.79, 141.76, 139.1, 139.0, 130.0, 128.92, 128.90,
127.76, 127.75, 127.66, 122.6, 116.3, 113.0, 77.8, 77.6, 65.81,
65.76, 65.48, 65.46, 64.22, 64.17, 59.0, 58.2, 55.98, 55.95, 55.4,
55.3, 54.62, 54.57, 54.2, 54.0, 42.7, 42.6, 42.3, 42.2, 34.7, 34.6,
32.9, 32.6, 30.5, 30.0, 29.8, 29.4, 29.2, 26.9, 26.2, 19.6, 19.5, 14.40,
14.36; HRMS m/z calcd for C40H54N5O9 (M + H)+ 748.3922, found
748.3903.
1
at 300, 400, or 500 MHz for H and at 75, 100, or 125 MHz for
13C on a Bruker or Varian spectrometer with CDCl3 or DMSO-d6
as solvent. The chemical shifts are given in parts per million,
referenced to the internal TMS or deuterated solvent signal.
All characterization data for macrocyclic acids, other intermedi-
ates, and some inhibitors and general reaction conditions are
included in the Supporting Information.
(2-{3-[(12-Cyclohexyl-11,14-dioxo-2,6-dioxa-10,13-diazatricyclo-
[14.3.1.17,10]henicosa-1(19),16(20),17-triene-9-carbonyl)amino]-
2-oxohexanoylamino}acetylamino)phenylacetic Acid tert-Butyl
Ester (22). For detailed reaction conditions for the preparation of
the inhibitor, see the Supporting Information. The product 22 (78%
yield, two steps) was isolated as a mixture of two diastereomers:
1H NMR (500 MHz, DMSO-d6) δ 8.75 (t, J ) 6.0 Hz) and 8.64-
8.63 (m, 1H), 8.71 (d, J ) 7.0 Hz, 1H), 8.42 (dd, J ) 2.4, 9.6 Hz,
1H), 8.26 and 8.08 (d, J ) 7.0 and 7.9 Hz, 1H), 7.41-7.33 (m,
5H), 7.15 (app t, J ) 7.8 Hz, 1H), 6.80 (s, 1H), 6.72 (app d, J )
7.6 Hz, 1H), 5.27 (dd, J ) 4.0, 7.1 Hz, 1H), 5.07-4.97 (m, 1H),
4.45 (dt, J ) 3.0, 9.7 Hz, 1H), 4.35 and 4.28 (t, J ) 8.5 and 8.6
Hz, 1H), 4.19-3.80 (m, 7H), 3.68 (d, J ) 16.9 Hz, 1H), 3.53-
3.50 (m, 2H), 2.39-2.32 (m, 1H), 1.84-1.59 (m, 10H), 1.46-
1.23 (m, 12H), 1.19-1.06 (m, 3H), 0.91-0.74 (m, 5H); 13C NMR
(125 MHz, DMSO-d6) δ 196.7, 196.6, 171.7, 171.30, 171.25, 170.0,
169.7, 169.41, 169.38, 168.5, 168.4, 167.60, 167.58, 160.9, 160.7,
158.5, 158.4, 138.2, 138.15, 138.12, 136.7, 136.5, 129.7, 128.71,
128.69, 128.6, 128.1, 127.42, 127.35, 127.3, 121.7, 115.4, 112.2,
112.1, 81.32, 81.29, 76.9, 76.8, 64.94, 64.89, 64.87, 63.4, 63.3,
58.1, 57.4, 56.92, 56.87, 56.81, 56.76, 56.7, 54.6, 54.52, 54.45,
53.8, 53.70, 53.67, 53.4, 53.2, 49.6, 41.8, 41.44, 41.39, 33.9, 33.7,
31.7, 29.2, 29.1, 29.0, 28.5, 28.3, 27.5, 26.0, 25.3, 18.7, 18.63,
18.60, 13.82, 13.75, 13.5; HRMS m/z calcd for C44H61N5O10 (M
+ H)+ 820.4497, found 820.4493.
(2-{3-[(12-Cyclohexyl-11,14-dioxo-2,6-dioxa-10,13-diazatricyclo-
[14.3.1.17,10]henicosa-1(19),16(20),17-triene-9-carbonyl)amino]-
2-oxohexanoylamino}acetylamino)phenylacetic Acid (23). A
solution of the tert-butyl ester 22 (26 mg, 0.032 mmol) in
trifluoroacetic acid (2 mL) and CH2Cl2 (2 mL) was stirred at room
temperature for 3 h. After the volatiles were removed in vacuo,
the residue was dissolved in 50% MeOH-CH2Cl2 and concentrated
to dryness in vacuo to afford an off-white solid (24 mg, quantita-
tive): 1H NMR (400 MHz, DMSO-d6) δ 8.73-8.65 (m, 2H), 8.40
(dd, J ) 9.5, 2.6 Hz, 1H), 8.24-8.05 (1 H), 7.64-7.55 (m, 1H),
7.41-7.32 (m, 5H), 7.15 (t, J ) 7.8 Hz, 1H), 6.80-6.71 (m, 3H),
5.35 (dd, J ) 7.5, 1.9 Hz, 1H), 5.04-4.96 (m, 1H), 4.48-4.43 (m,
1H), 4.37-4.22 (m, 1H), 4.16-3.27 (m, 11H), 2.35-2.31 (m, 1H),
1.84-0.70 (m, 21 H); 13C NMR (100 MHz, DMSO-d6) δ 196.7,
171.7, 171.4, 171.3, 170.0, 169.7, 167.5, 161.0, 160.7, 158.5, 158.5,
158.4, 138.2, 138.2, 137.1, 137.0, 132.1, 132.1, 131.6, 131.5, 131.5,
129.2, 128.8, 128.7, 128.7, 128.6, 128.0, 127.7, 127.5, 127.5, 121.8,
115.4, 112.2, 76.9, 76.8, 65.0, 64.9, 63.4, 63.3, 58.2, 5 7.4, 56.3,
56.2, 56.2, 54.6, 54.5, 53.8, 53.4, 53.2, 41.5, 41.5, 41.4, 40.2, 33.9,
33.7, 31.9, 31.7, 29.2, 29.0, 28.6, 28.3, 26.1, 25.3, 18.7, 18.6, 13.5;
HRMS m/z calcd for C40H51N5O10 (M + H)+ 762.3714, found
762.3705.
12-Cyclohexyl-11,14-dioxo-2,6-dioxa-10,13-diazatricyclo[14.
3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxylic Acid ((([1-
(Dimethylcarbamoylphenylmethyl)carbamoyl]methyl)aminoox-
alyl)butyl)amide (24 and 25). The S-isomer 24 and R-isomer 25
(82% combined yield, 2 steps) were separated by flash chroma-
1
tography (2-4% MeOH/CH2Cl2). Data for 24: H NMR (500 MHz,
DMSO-d6) δ 8.70 (t, J ) 5.2 Hz, 1H), 8.56 (d, J ) 7.7 Hz, 1H),
8.42 (d, J ) 6.6 Hz, 1H), 8.25 (d, J ) 6.6 Hz, 1H), 7.36-7.29 (m,
5H), 7.16-7.13 (m, 1H), 6.79-6.78 (m, 2H), 6.71 (app dd, J )
2.0, 7.9 Hz, 1H), 5.81 (d, J ) 7.8 Hz, 1H), 5.00-4.96 (m, 1H),
4.46-4.42 (m, 1H), 4.35 (dd, J ) 7.6, 9.8 Hz, 1H), 4.18-3.99 (m,
5H), 3.86-3.74 (m, 2H), 3.67 (app d, J ) 15.1 Hz, 1H),
12-tert-Butyl-11,14-dioxo-2,6-dioxa-10,13-diazatricyclo[14.
3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxylic Acid ((([1-
(Dimethylcarbamoylphenylmethyl)carbamoyl]methyl)amino-
oxalyl)butyl)amide (30). The products were separated into the

1002 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Chen et al.
S-isomer and R-isomer (57% combined yield, two steps). Data for
the S-isomer: 1H NMR (500 MHz, DMSO-d6) δ 8.72 (t, J ) 6.3
Hz, 1H), 8.57 (d, J ) 7.5 Hz, 1H), 8.37 (d, J ) 9.8 Hz, 1H), 8.26
(d, J ) 6.9 Hz, 1H), 7.38-7.35 (m, 4H), 7.34-7.30 (m, 1H), 7.16
(dd, J ) 7.2 and 8.9 Hz, 1H), 6.74-6.71 (m, 3H), 5.83 (d, J ) 7.7
Hz, 1H), 5.07-5.03 (m, 1H), 4.69 (d, J ) 9.7 Hz, 1H), 4.39 (dd,
J ) 7.6 and 10.2 Hz, 1H), 4.14-4.04 (m, 3H), 3.97 (d, J ) 10.4
Hz, 1H), 3.88-3.75 (m, 3H), 3.54-3.48 (m, 2H), 3.40-3.34 (m,
2H), 2.93 (s, 3H), 2.85 (s, 3H), 2.34-2.30 (m, 1H), 1.87-1.79
(m, 1H), 1.77-1.67 (m, 3H), 1.47-1.35 (m, 3H), 0.95 (s, 9H),
0.86 (t, J ) 7.3 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ 196.6,
171.2, 170.1, 169.6, 169.2, 166.9, 160.6, 158.2, 138.4, 137.4, 129.0,
128.5, 127.8, 127.7, 121.6, 115.1, 111.7, 76.7, 65.0, 63.1, 57.3,
56.2, 54.8, 54.1, 53.1, 53.0, 41.5, 41.2, 36.5, 35.3, 34.9, 33.7, 31.6,
28.5, 26.2, 18.5, 13.4; HRMS m/z calcd for C40H55N6O9 (M + H)+
763.4031, found 763.4047.
58.7, 55.9, 55.1, 54.8, 54.5, 43.9, 42.6, 42.1, 37.3, 36.5, 34.0, 32.7,
29.6, 29.3, 27.1, 26.6, 26.3, 19.2, 14.0; HRMS m/z calcd for
C43H59N6O9 (M + H)+ 803.4344, found 803.4347.
12-Cyclohexyl-11,14-dioxo-2-oxa-6-thia-10,13-diazatricyclo-
[14.3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxylic Acid
((([1-(Dimethylcarbamoylphenylmethyl)carbamoyl]methyl)amino-
oxalyl)butyl)amide (35). Standard coupling of 3c and 14 provided
an R-hydroxyamide intermediate: HRMS m/z calcd for C42H59N6O8
(M + H)+ 807.4115, found 807.4103. To a solution of this
intermediate (180 mg, 0.220 mmol) in dichloromethane at room
temperature were added sequentially DMSO (0.313 mL, 4.40
mmol), DCC (908 mg, 4.40 mmol), and dichloroacetic acid (0.0364
mL, 0.440 mmol). The reaction mixture was stirred overnight (16
h) and was then quenched by addition of a 5% aqueous citric acid
solution (5 mL) and MeOH (1 mL), and the resulting mixture was
stirred for 30 min. The solid material was filtered off, and the filtrate
was washed with saturated aqueous sodium bicarbonate solution
and brine, dried over Na2SO4, and concentrated in vacuo. The crude
product was purified by flash column chromatography using 0-2%
methanol in dichloromethane to give the product (105 mg, 60%)
as a mixture of diastereomers. One part of the mixture (36 mg)
was purified again by column chromatography to provide more
polar pure P1 S-isomer (8 mg) and its less polar R-isomer (6 mg).
Data for the S-isomer: 1H NMR (500 MHz, DMSO-d6) δ 7.56
(app t, J ) 5.3 Hz, 1H), 7.51-7.49 (m, 1H), 7.41-7.32 (m, 7H),
7.21 (app t,, J ) 7.9 Hz, 1H), 7.03 (s, 1H), 6.82-6.77 (m, 2H),
5.85 (d, J ) 6.9 Hz, 1H), 5.23-5.19 (m, 1H), 4.61-4.55 (m, 2H),
4.32-4.28 (m, 1H), 4.18-4.13 (m, 1H), 4.06-3.91 (m, 2H), 3.81-
3.78 (m, 1H), 3.64-3.61 (m, 2H), 3.47-3.42 (m, 2H), 3.01 (s,
3H), 2.91 (s, 3H), 2.61 (app t, J ) 7.1 Hz, 2H), 2.52-2.47 (m,
1H), 2.13-2.04 (m, 1H), 1.92-1.54 (m, 11H), 1.37-1.31 (m, 1H),
1.24-1.15 (m, 1H), 0.90 (t, J ) 7.3 Hz, 3H); 13C NMR (125 MHz,
DMSO-d6) δ 172.4 171.4, 170.4, 169.6, 166.6, 159.1, 137.1, 137.0,
130.2, 129.6, 129.5, 129.0, 128.3, 122.3, 116.3, 114.9, 67.8, 59.2,
56.3, 54.8, 54.6, 54.0, 43.9, 42.9, 42.7, 41.1, 37.3, 36.5, 33.8, 30.0,
29.9, 29.2, 27.2, 26.6, 26.10, 26.06, 19.3, 14.1; HRMS m/z calcd
for C42H57N6O8S (M + H)+ 805.3959, found 805.3950.
12-tert-Butyl-11,14-dioxo-2,6-dioxa-10,13-diazatricyclo[14.
3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxylic Acid ((([1-
(Dimethylcarbamoylphenylmethyl)carbamoyl]methyl)amino-
oxalyl)butyl)amide (32). The macrocyclic methyl ester was
prepared according to the published procedures.16 It was hydrolyzed
1
to its corresponding carboxylic acid. On the basis of H NMR
analysis, the proline R-center was partially racemized (ca. 20%)
during hydrolysis: 13C NMR (DMSO-d6) δ 172.6, 172.1, 169.6,
159.9, 139.4, 129.3, 122.0, 115.7, 112.8, 74.6, 65.9, 63.7, 59.1,
58.1, 51.6, 43.6, 38.9, 36.5, 30.3, 27.4; HRMS m/z calcd for
C22H31N2O6 (M + H)+ 419.2182, found 419.2176. The coupling
and oxidation were carried out according to general procedures.
The S-isomer and R-isomer were isolated (35% combined yield,
two steps). Data for the S-isomer: 13C NMR (DMSO-d6) δ 197.3,
171.7, 170.1, 169.5, 167.8, 161.6, 159.4, 138.7, 138.4, 130.3, 129.5,
128.72, 128.66, 123.3, 116.3, 114.4, 110.0, 76.8, 65.9, 65.1, 58.4,
57.8, 55.8, 53.9, 51.3, 42.6, 42.4, 37.5, 36.2, 35.5, 34.8, 32.43 29.4,
27.5, 19.4, 14.4; HRMS calcd for C40H55N6O9 (M + H)+ 763.4031,
found 763.4040.
11-Cyclohexyl-10,13-dioxo-2,5-dioxa-9,12-diazatricyclo[13.
3.1.16,9]icosa-1(18),15(19),16-triene-8-carboxylic Acid ((([1-(Di-
methylcarbamoylphenylmethyl)carbamoyl]methyl)aminooxalyl)-
butyl)amide (33). Compound 33 was isolated as an inseparable
mixture of two diastereomers: 1H NMR (500 MHz, DMSO-d6) δ
8.73 and 8.68 (t, J ) 6.1 and 6.0 Hz, 1H), 8.55 (d, J ) 7.6 Hz,
1H), 8.46 (t, J ) 10.0 Hz, 1H), 8.23 and 7.98 (d, J ) 6.6 and 7.9
Hz, 1H), 7.47-7.29 (m, 5H), 7.11 (t, J ) 7.8 Hz, 1H), 7.04 and
7.01 (s, 1H), 6.75 (d, J ) 7.2 Hz, 1H), 6.72-6.70 (m, 1H), 5.84
and 5.82 (d, J ) 7.8 and 7.7 Hz, 1H), 5.04-4.94 (m, 1H), 4.38-
4.10 (m, 3H), 4.03 (br s, 1H), 3.88-3.85 (m, 1H), 3.83 (d, J ) 6.3
Hz, 1H), 3.79 (d, J ) 5.9 Hz, 1H), 3.76-3.72 (m, 1H), 3.65 (d, J
) 14.2 Hz, 1H), 3.47-3.40 (m, 2H), 3.24 (d, J ) 13.9 Hz, 1H),
2.92 (s, 3H), 2.84 (s, 3H), 2.45 (dd, J ) 7.4, 13.4 Hz, 1H), 1.75-
1.53 (m, 8H), 1.50-1.24 (m, 3H), 1.22-1.14 (m, 3H), 0.89-0.76
(m, 5H); 13C NMR (125 MHz, DMSO-d6) δ 196.5, 173.2, 171.2,
170.2, 169.4, 169.2, 169.1, 166.9, 160.6, 159.3, 138.3, 137.4, 128.7,
128.5, 127.8, 127.7, 122.5, 116.0, 113.6, 77.5, 68.6, 67.7, 57.3,
54.4, 53.31, 53.27, 53.2, 53.0, 52.9, 41.8, 41.5, 38.7, 38.5, 36.5,
35.3, 32.9, 31.6, 29.3, 28.0, 26.0, 25.3, 25.2, 18.6, 13.5, 13.4; HRMS
m/z calcd for C41H54N6O9 (M + H)+ 775.4031, found 775.4048.
5-Cyclohexyl-3,6-dioxo-11,16-dioxa-4,7-diazatricyclo[15.3.1.17,10]-
docosa-1(21),17,19-triene-8-carboxylic Acid ((([1-(Dimethylcar-
bamoylphenylmethyl)carbamoyl]methyl)aminooxalyl)butyl)-
amide (34). Compound 34 was isolated as the S-isomer: 1H NMR
(500 MHz, DMSO-d6) δ 7.61 (d, J ) 7.2 Hz, 1H), 7.57 (app t, J
) 5.2 Hz, 1H), 7.39-7.33 (m, 7H), 7.18 (app t, J ) 7.9 Hz, 1H),
6.79-6.70 (m, 3H), 5.85 (d, J ) 7.6 Hz, 1H), 5.29-5.25 (m, 1H),
4.70 (app t, J ) 8.8 Hz, 1H), 4.48 (dd, J ) 7.9 and 9.6 Hz, 1H),
4.10-3.94 (m, 4H), 3.83-3.80 (m, 1H), 3.62-3.54 (m, 3H), 3.50-
3.46 (m, 1H), 3.43-3.39 (m, 1H), 3.02 (s, 3H), 2.92 (s, 3H), 2.34-
2.29 (m, 1H), 2.23-2.17 (m, 1H), 1.84-1.56 (m, 13H), 1.32-
1.00 (m, 7H), 0.84 (t, J ) 7.5 Hz, 3H); 13C NMR (125 MHz,
DMSO-d6) δ 195.5, 172.6, 171.5, 170.7, 166.7, 159.5, 159.2, 137.1,
136.9, 130.1, 129.5, 129.0, 128.3, 121.7, 116.9, 111.7, 68.8, 68.6,
12-Cyclohexyl-6,6,11,14-tetraoxo-2-oxa-6λ6-thia-10,13-diaza-
tricyclo[14.3.1 .17,10]henicosa-1(19),16(20),17-triene-9-carboxylic
Acid ((([1-(Dimethylcarbamoylphenylmethyl)carbamoyl]methyl)-
aminooxalyl)butyl)amide (36). Compound 36 was obtained as a
mixture of P1 diastereomers: 1H NMR (500 MHz, DMSO-d6) δ
8.74 (dd, J ) 5.8, 10.6 Hz, 1H), 8.54 (d, J ) 7.6 Hz, 1H), 8.49-
8.45 (m, 1H), 8.33 and 8.10 (d, J ) 7.1 and 8.0 Hz, 1H), 7.38-
7.29 (m, 5H), 7.22-7.19 (m, 1H), 6.91-6.88 (m, 2H), 6.82-6.80
(m, 1H), 5.82 and 5.81 (d, J ) 7.6 and 8.0 Hz, 1H), 4.99 (ddd, J
) 3.6, 7.8, 9.3 Hz, 1H), 4.52-4.45 (m, 1H), 4.42-4.32 (m, 2H),
4.27-4.22 (m, 1H), 4.08 (q, J ) 5.3 Hz, 1H), 3.98-3.79 (m, 5H),
3.64 (d, J ) 14.4 Hz, 1H), 3.17-3.12 (m, 1H), 3.01-2.95 (m,
1H), 2.92 (s, 3H), 2.84 (s, 3H), 2.61-2.56 (m, 1H), 2.19-2.13
(m, 1H), 1.99-1.93 (m, 2H), 1.75-1.60 (m, 7H), 1.49-1.24 (m,
3H), 1.18-1.12 (m, 3H), 0.97-0.84 (m, 5H); 13C NMR (125 MHz,
DMSO-d6) δ 196.4, 196.2, 170.4, 170.3, 170.2, 170.1, 169.28,
169.25, 169.1, 166.8, 160.7, 160.5, 156.95, 156.88, 138.0, 137.9,
137.4, 129.3, 129.2, 128.4, 127.65, 127.60, 127.59, 122.7, 122.6,
115.8, 115.7, 115.4, 65.7, 65.6, 61.0, 60.5, 59.5, 59.0, 58.4, 55.1,
54.7, 53.4, 52.9, 48.4, 47.5, 47.3, 45.44, 45.41, 41.5, 41.4, 41.1,
40.9, 36.4, 35.2, 31.5, 28.9, 28.7, 28.4, 28.3, 28.21, 28.16, 26.9,
25.8, 25.2, 20.6, 19.8, 19.7, 18.5, 18.4, 13.9, 13.30, 13.26; HRMS
m/z calcd for C42H57N6O10S (M + H)+ 837.3857, found 837.3865.
6-Benzenesulfonyl-12-cyclohexyl-11,14-dioxo-2-oxa-6,10,13-
triazatricyclo[14.3.1.17,10]henicosa-1(19),16(20),17-triene-9-car-
boxylic Acid ((([1-(Dimethylcarbamoylphenylmethyl)carbamoyl]-
methyl)aminooxalyl)butyl)amide (37). Compound 37 was obtained
as a mixture of P1 diastereomers: 1H NMR (500 MHz, DMSO-
d6) δ .86 and 8.73 (t, J ) 6.3 and 6.2 Hz, 1H), 8.62 and 8.60 (d,
J ) 7.7 and 7.4 Hz, 1H), 8.30 and 8.25 (d, J ) 9.7 and 10.0 Hz,
1H), 8.17 and 8.03 (d, J ) 6.8 and 7.8 Hz, 1H), 7.95 (d, J ) 8.5
Hz, 1H), 7.82 (d, J ) 9.6 Hz, 1H), 7.71-7.60 (m, 3H), 7.38-7.30
(m, 5H), 7.20-7.16 (m, 1H), 6.81 (d, J ) 7.0 Hz, 1H), 6.76-6.74
(m, 2H), 5.85 (d, J ) 8.8 Hz, 1H), 5.04 and 4.99 (m, 1H), 4.57-

Hepatitis C Virus NS3 Serine Protease Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1003
4.48 (m, 2H), 4.40 and 4.30 (t and d, J ) 9.9 and 10.9 Hz, 1H),
4.18-4.10 (m, 1H), 3.95-3.74 (m, 3H), 3.63-3.59 (d, 2H), 3.36-
3.33 (m, 1H), 3.12-3.01 (m, 3H), 2.94 and 2.93 (s, 3H), 2.853
and 2.850 (s, 3H), 1.88-1.60 (m, 11H), 1.45-1.31 (m, 2H), 1.17-
1.08 (m, 3H), 0.89-0.83 (m, 5H); 13C NMR (125 MHz, DMSO-
d6) δ 197.3, 197.1, 172.1, 171.8, 171.51, 171.50, 170.1, 169.9,
169.7, 167.89, 167.87, 161.4, 161.3, 158.0, 157.9, 139.9, 139.8,
139.2, 139.1, 138.4, 133.97, 133.96, 130.6, 130.5, 130.28, 130.27,
129.5, 128.74, 128.69, 128.67, 127.8, 127.7, 123.2, 123.1, 116.7,
113.6, 113.5, 110.0, 65.1, 59.2, 58.2, 55.2, 54.3, 54.1, 53.93, 53.91,
42.69, 42.67, 42.6, 42.5, 37.5, 36.2, 32.4, 32.0, 31.2, 30.9, 30.3,
29.7, 28.8, 27.1, 26.9, 26.5, 26.31, 26.25, 20.0, 19.6, 18.8, 18.7,
14.4, 14.3; HRMS m/z calcd for C48H62N7O10S (M + H)+ 928.4279,
found 928.4290.
12-Cyclohexyl-5,5-dimethyl-11,14-dioxo-2,6-dioxa-10,13-diaza-
tricyclo[14.3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxyl-
ic Acid (1-Aminooxalylbutyl)amide (39). Oxidation of interme-
diate coupling product R-hydroxyamide to 39 through a modified
Moffatt oxidation was described above in the preparation of
compound 35 (35% yield, two steps): 1H NMR (300 MHz, CDCl3)
δ 7.44 (br s, 1H), 7.27 (br s, 1H), 7.14 (app t, J ) 12.7 Hz, 1H),
6.72-6.65 (m, 4H), 6.51 (t, J ) 8.6 Hz, 1H), 4.79 (dd, J ) 4.5,
8.5 Hz, 1H), 4.69 (dd, J ) 9.8, 17.5 Hz, 1H), 4.27-4.07 (m, 3H),
3.97 (d, J ) 11.2 Hz, 1H), 3.70-3.50 (M, 4H), 2.48-2.41 (m,
1H), 2.06-1.96 (m, 2H), 1.84-1.49 (m, 11H), 1.43-1.11 (m, 11H),
0.95-0.90 (m, 3H); 13C NMR (75 MHz, CDCl3) δ 195.6, 195.5,
172.8, 170.9, 170.5, 170.2, 160.9, 158.9, 156.4, 151.8, 145.8, 136.5,
132.5, 129.5, 121.2, 116.2, 110.5, 76.3, 74.9, 69.3, 63.9, 58.2, 58.1,
56.8, 56.7, 55.4, 55.3, 54.1, 54.0, 53.4, 43.1, 41.8, 41.0, 40.9, 35.8,
33.4, 29.2, 29.1, 29.0, 26.1, 25.8, 25.73, 25.68, 19.0, 18.9, 13.7;
HRMS m/z calcd for C32H47N4O7 (M + H)+ 599.3445, found
599.3456.
110.6, 81.21, 81.19, 74.6, 74.0, 69.54, 69.48, 63.2, 63.1, 58.4, 57.8,
56.83, 56.79, 55.8, 55.6, 54.4, 54.3, 53.3, 53.0, 41.4, 41.31, 41.28,
41.25, 40.7, 40.6, 37.7, 37.6, 31.9, 31.5, 28.8, 28.6, 28.4, 27.4, 26.2,
26.1, 25.9, 25.25, 25.16, 25.0, 24.9, 18.6, 18.5, 13.4; HRMS m/z
calcd for C46H64N5O10 (M + H)+ 846.4653, found 846.4644.
12-Cyclohexyl-5,5-dimethyl-11,14-dioxo-2,6-dioxa-10,13-diaza-
tricyclo[14.3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxyl-
ic Acid ((([1-(Dimethylcarbamoylphenylmethyl)carbamoyl]-
methyl)aminooxalyl)butyl)amide (45). Compound 45 was obtained
as a mixture of two inseparable P1 diastereomers: 1H NMR (300
MHz, CDCl3) δ 7.83 (app t, J ) 8.5 Hz, 1H), 7.68 and 7.60 (t, J
) 4.9 and 4.9 Hz, 1H), 7.45-7.22 (m, 6H), 7.20-6.74 (m, 2H),
6.67-6.64 (m, 3H), 5.86 (d, J ) 7.4 Hz, 1H), 5.29 (dt, J ) 4.7,
7.6 Hz, 1H), 4.82-4.63 (m, 2H), 4.26-3.93 (m, 6H), 3.64 (s, 2H),
3.56 (dd, J ) 3.3, 11.0 Hz, 1H), 2.97 (s, 3H), 2.90 (s, 3H), 2.37-
1.97 (m, 3H), 1.86-1.47 (m, 9H), 1.36-0.97 (m, 13H), 0.88 and
0.84 (t, J ) 7.1 and 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ
195.1, 195.0, 172.6, 172.5, 170.4, 169.4, 166.4, 165.0, 162.6, 159.2,
158.7, 142.5, 136.7, 136.6,129.4, 129.0, 128.8, 128.4, 128.2, 127.7,
127.6, 127.5, 125.4, 121.1, 116.0, 110.5, 103.4, 82.0, 80.2, 74.74,
74.68, 69.4, 63.8, 58.4, 58.3, 56.7, 55.3, 55.1, 54.1, 53.9, 42.8, 42.3,
41.4, 41.3, 41.0, 36.9, 36.4, 36.0, 33.4, 32.5, 30.3, 29.1, 28.9, 28.6,
26.3, 26.1, 26.0, 25.7, 18.8, 13.7; HRMS m/z calcd for C44H61N6O9
(M + H)+ 817.4500, found 817.4528.
12-tert-Butyl-5,5-dimethyl-11,14-dioxo-2,6-dioxa-10,13-diaza-
tricyclo[14.3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxyl-
ic Acid [1-(Dimethylcarbamoylphenylmethyl)carbamoyl]methyl)-
aminooxalyl)butyl)amide (47). Compound 47 was prepared through
the same general procedures as described above, except that Boc-
tert-butylglycine was substituted for Boc-cyclohexylglycine at P3.
It was isolated as a mixture of two diastereomers: 1H NMR (300
MHz, CDCl3) δ 7.54-7.23 (m, 8H), 7.13 (t, J ) 7.7 Hz, 1H), 6.70-
6.64 (m, 3H), 6.58 (br s, 1H), 5.80 (d, J ) 7.0 Hz, 1H), 5.34-5.20
(m, 1H), 4.92 (t, J ) 8.9 Hz, 1H), 4.72 and 4.65 (t, J ) 8.9 and
8.9 Hz, 1H), 4.24-3.92 (m, 6H), 3.68 and 3.59 (ABq, J ) 15.3
Hz, 1H), 3.47 (dt, J ) 2.6, 12.0 Hz, 1H), 2.98 and 2.95 (s, 3H),
2.88 and 2.87 (s, 3H), 2.49-2.33 (m, 1H), 2.09-1.80 (m, 3H),
1.65-1.29 (m, 4H), 1.23 (s, 3H), 1.21 (s, 3H), 1.03 and 1.00 (s,
9H), 0.88 (q, J ) 7.5 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 197.9,
197.4, 171.0, 170.5, 170.3, 170.1, 169.1, 167.9, 166.1, 165.9, 159.0,
158.8, 158.7, 145.4, 136.6, 129.4, 129.0, 128.9, 128.5, 127.8, 127.4,
121.10, 121.07, 116.1, 116.0, 110.15, 110.09, 74.8, 69.3, 69.1, 67.9,
63.9, 63.7, 58.4, 57.9, 57.5, 57.4, 57.2, 54.24, 54.17, 43.1, 42.2,
41.0, 40.9, 36.8, 36.3, 36.2, 36.0, 35.6, 33.6, 33.1, 32.5, 26.4, 26.3,
25.9, 25.7, 25.4, 25.1, 18.9, 18.8, 13.63, 13.57; HRMS m/z calcd
for C42H59N6O9 (M + H)+ 791.4344, found 791.4342.
12-Isopropyl-5,5-dimethyl-11,14-dioxo-2,6-dioxa-10,13-diaza-
tricyclo[14.3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxyl-
ic Acid ((([1-(Dimethylcarbamoylphenylmethyl)carbamoyl]-
methyl)aminooxalyl)butyl)amide (48). Compound 48 was prepared
by the same general procedures using Boc-valine at P3. It was
isolated as a mixture of two diastereomers: 1H NMR (500 MHz,
DMSO-d6) δ 8.74 and 8.68 (t, J ) 6.2 and 6.2 Hz, 1H), 8.56 (dd,
J ) 6.1, 7.6 Hz, 1H), 8.50 (d, J ) 9.4 Hz, 1H), 8.28 and 8.19 (d,
J ) 6.9 and 8.2 Hz, 1H), 7.39-7.27 (m, 5H), 7.15-7.12 (m, 1H),
6.71-6.70 (m, 2H), 6.66-6.64 (m, 1H), 5.825 and 5.818 (d, J )
7.5 and 7.6 Hz, 1H), 5.06-5.02 (m, 1H), 4.44-4.25 (m, 4H), 4.11-
4.04 (m, 1H), 3.89-3.57 (m, 5H), 3.38 and 3.37 (d, J ) 15.4 and
15.1 Hz, 1H), 3.31 (d, J ) 3.6 Hz, 1H), 2.92 and 2.90 (s, 3H),
2.84 and 2.83 (s, 3H), 2.12-1.80 (m, 4H), 1.70-1.58 (m, 2H),
1.45-1.14 (m, 9H), 0.89-0.77 (m, 9H); 13C NMR (125 MHz,
DMSO-d6) δ 196.6, 196.5, 171.29, 171.25, 170.0, 169.8, 169.64,
169.61, 169.2, 166.9, 160.8, 158.2, 138.25, 138.18, 137.4, 129.1,
128.5, 127.8, 127.72, 127.70, 121.2, 115.3, 110.8, 73.98, 73.96,
69.5, 63.2, 58.2, 57.7, 55.8, 55.7, 54.8, 53.2, 53.0, 52.9, 41.53,
41.46, 41.35, 41.28, 40.7, 40.6, 37.7, 36.5, 35.3, 31.8, 30.6, 30.5,
28.3, 26.3, 26.2, 26.1, 24.9, 24.8, 19.0, 18.9, 18.8, 18.74, 18.68,
18.6, 18.5, 13.41, 13.37; HRMS m/z calcd for C41H57N6O9 (M +
H)+ 777.4187, found 777.4171.
12-Cyclohexyl-5,5-dimethyl-11,14-dioxo-2,6-dioxa-10,13-diaza-
tricyclo[14.3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxyl-
ic Acid (1-Allylaminooxalylbutyl)amide (40). Product 40 was
1
obtained as a mixture of two P1 diastereomers: H NMR (500 MHz,
DMSO-d6) δ 8.86 and 8.82 (t, J ) 6.0 and 6.0 Hz, 1H), 8.43 (dd,
J ) 1.9, 9.5 Hz, 1H), 8.29 and 8.19 (d, J ) 6.6 and 7.9 Hz, 1H),
7.13 (t, J ) 7.9 Hz, 1H), 6.70-6.69 (m, 2H), 6.64 (d, J ) 7.6 Hz,
1H), 5.83-5.75 (m, 1H), 5.13-5.05 (m, 3H), 5.02 and 5.4.94 (ddd,
J ) 3.8, 8.0, 9.6 and 2.7, 5.7, 9.2 Hz, 1H), 4.48 (t, J ) 9.6 Hz,
1H), 4.43-4.35 (m, 2H), 4.30-4.24 (m, 1H), 4.10-4.04 (m, 1H),
3.86 (dd, J ) 10.7, 19.5 Hz, 1H), 3.77-3.72 (m, 3H), 3.66 and
3.56 (dd, J ) 3.6, 10.6 and 3.3, 10.5 Hz, 1H), 3.36 (d, J ) 15.5
Hz, 1H), 2.12-2.06 (m, 1H), 1.89-1.80 (m, 2H), 1.78-1.55 (m,
7H), 1.49-1.23 (m, 4H), 1.21-1.12 (m, 8H), 0.95-0.79 (m, 6H);
13C NMR (125 MHz, DMSO-d6) δ 197.1, 197.0, 171.30, 171.26,
169.72, 169.58, 169.1, 160.9, 160.8, 158.1, 138.25, 138.20, 134.1,
129.1, 121.2, 121.1, 115.49, 115.46, 115.29, 115.27, 110.7, 110.6,
73.99, 73.96, 69.50, 69.47, 63.1, 58.3, 57.7, 54.3, 53.4, 53.1, 41.3,
41.2, 40.8, 40.7, 40.66, 40.59, 40.58, 37.7, 37.6, 36.6, 31.9, 31.5,
28.8, 28.4, 26.2, 26.1, 25.9, 25.3, 25.2, 25.04, 24.98, 24.89, 22.5,
22.3, 22.0, 18.6, 18.5, 13.4; HRMS calcd for C35H51N4O7S (M +
H)+ 639.3758, found 639.3765.
(2-{3-[(12-Cyclohexyl-5,5-dimethyl-11,14-dioxo-2,6-dioxa-10,-
13-diazatricyclo[14.3.1.17,10]henicosa-1(19),16(20),17-triene-9-
carbonyl)amino]-2-oxohexanoylamino}acetylamino)phenylace-
tic Acid tert-Butyl Ester (44). Compound 44 was obtained as a
mixture of two inseparable P1 diastereomers: 1H NMR (500 MHz,
DMSO-d6) δ 8.74-8.67 (m, 2H), 8.42 (d, J ) 9.5 Hz, 1H), 8.27
and 8.13 (d, J ) 7.0 and 7.8 Hz, 1H), 7.41-7.33 (m, 5H), 7.14-
7.11 (m, 1H), 6.70-6.69 (m, 2H), 6.65 (d, J ) 7.5 Hz, 1H), 5.27
(dd, J ) 3.4, 7.3 Hz, 1H), 5.08-4.98 (m, 1H), 4.51-4.25 (m, 4H),
4.10-4.04 (m, 1H), 3.92-3.78 (m, 3H), 3.74 (d, J ) 3.4 Hz, 1H),
3.70-3.64 and 3.58-3.56 (m, 1H), 3.36 (d, J ) 15.8 Hz, 1H),
2.14-2.08 (m, 1H), 1.91-1.52 (m, 10H), 1.51-1.38 (m, 2H), 1.341
and 1.336 (s, 9H), 1.27-1.03 (m, 10H), 0.95-0.76 (m, 5H); 13C
NMR (125 MHz, DMSO-d6) δ 196.64, 196.55, 171.30, 171.26,
169.9, 169.7, 169.61, 169.58, 169.4, 169.3, 167.5, 160.8, 160.6,
158.1, 138.3, 138.2, 136.5, 136.4, 133.0, 132.2, 132.0, 129.1, 128.7,
128.6, 128.1, 127.37, 127.35, 121.2, 121.1, 115.30, 115.27, 110.7,

1004 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Chen et al.
12-sec-Butyl-5,5-dimethyl-11,14-dioxo-2,6-dioxa-10,13-diaza-
tricyclo[14.3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxyl-
ic Acid ((([1-(Dimethylcarbamoylphenylmethyl)carbamoyl]-
methyl)aminooxalyl)butyl)amide (49). Compound 49 was prepared
by the same general procedures using Boc-isoleucine at P3. It was
isolated as a mixture of two diastereomers: 1H NMR (500 MHz,
DMSO-d6) δ 8.74 and 8.69 (t, J ) 6.2 and 6.1 Hz), 8.56 (t, J )
7.1 Hz, 1H), 8.50 (dd, J ) 1.5, 9.4 Hz, 1H), 8.28 and 8.20 (d, J )
6.9 and 8.2 Hz, 1H), 7.37-7.28 (m, 5H), 7.13 (dt, J ) 1.6, 7.9 Hz,
1H), 6.70-6.69 (m, 2 H), 6.65 (d, J ) 7.6 Hz, 1H), 5.82 (d, J )
7.6 Hz, 1H), 5.06-4.99 (m, 1H), 4.48-4.25 (m, 4H), 4.11-4.05
(m, 1H), 3.90 (dd, J ) 10.8, 20.6 Hz, 1H), 3.83-3.72 (m, 3H),
3.66 and 3.57 (dd, J ) 3.4, 10.8 and 3.2, 10.6 Hz, 1H), 3.37 (d, J
) 15.4 Hz, 1H), 2.92 (s, 3H), 2.842 and 2.838 (s, 3H), 2.14-2.08
(m, 1H), 1.90-1.58 (m, 4H), 1.52-1.33 (m, 3H), 1.29-1.01 (m,
9H), 0.89-0.76 (m, 9H); 13C NMR (125 MHz, DMSO-d6) δ 196.6,
196.5, 171.29, 171.26, 170.1, 169.9, 169.52, 169.51, 169.2, 166.9,
160.8, 160.6, 158.1, 138.23, 138.17, 137.4, 129.1, 128.5, 127.8,
127.71, 127.70, 121.2, 121.1, 115.3, 110.7, 110.6, 74.0, 73.9, 69.6,
69.5, 63.1, 58.3, 57.7, 55.8, 55.6, 54.8, 54.2, 54.1, 53.2, 52.95,
52.93, 41.54, 41.47, 41.3, 41.2, 40.7, 40.6, 37.7, 37.6, 36.5, 36.4,
35.3, 31.8, 31.5, 31.2, 28.3, 26.1, 26.0, 25.0, 24.9, 24.3, 22.0, 18.6,
18.5, 15.0, 14.9, 13.9, 13.41, 13.38, 10.65, 10.63; HRMS m/z calcd
for C42H59N6O9 (M + H)+ 791.4340, found 791.4340.
5,5-Dimethyl-11,14-dioxo-12-phenyl-2,6-dioxa-10,13-diaza-
tricyclo[14.3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxyl-
ic Acid ((([1-(Dimethylcarbamoylphenylmethyl)carbamoyl]-
methyl)aminooxalyl)butyl)amide (50). Compound 50 was prepared
by the same general procedures using Boc-phenylglycine at P3. It
was isolated as a mixture of two diastereomers: 1H NMR (500
MHz, DMSO-d6) δ 9.26 (dd, J ) 6.4, 9.1 Hz) and 9.20 (t, J ) 8.2
Hz, 1H), 8.74-8.70 (m, 1H), 8.68 (t, J ) 7.0 Hz, 1H), 8.58-8.54
(m, 1H), 7.37-7.13 (m, 11H), 6.86-6.81 (m, 1H), 6.77-6.67 (m,
2H), 5.92 (dd, J ) 5.5, 8.7 Hz, 1H), 8.22 (dd, J ) 6.3, 8.0 Hz,
1H), 5.03-4.88 (m, 2H), 4.49-4.06 (m, 4H), 3.95-3.58 (m, 5H),
3.33-3.30 (m) and 3.22 (dt, J ) 2.6, 11.9 Hz, 1H), 2.93 and 2.92
(s, 3H), 2.841 and 2.836 (s, 3H), 2.37-2.34 (m, 1H), 1.95-1.62
(m, 4H), 1.44-1.13 (m, 9H), 0.87-0.75 (m, 3H); 13C NMR (125
MHz, DMSO-d6) δ 196.5, 196.2, 172.0, 171.1, 170.1, 170.0, 169.24,
169.17, 166.87, 166.85, 160.6, 158.53, 158.46, 138.62, 138.59,
138.2, 138.0, 137.42, 137.39, 129.2, 129.1, 128.5, 127.84, 127.79,
127.75, 127.73, 127.70, 127.64, 127.61, 127.4, 127.1, 127.0, 121.6,
121.4, 115.7, 115.3, 111.6, 111.1, 110.8, 74.3, 73.9, 73.8, 69.5,
68.0, 63.9, 63.6, 58.1, 54.8, 53.4, 53.3, 52.9, 41.55, 41.50, 41.47,
41.45, 41.1, 40.9, 40.5, 37.7, 37.6, 36.5, 35.3, 31.4, 31.2, 28.3, 27.2,
27.0, 25.2, 24.7, 24.5, 24.4, 22.0, 18.7, 18.6, 13.9, 13.4; HRMS
calcd for C44H55N6O9 (M + H)+ 811.4031, found 811.4026.
12-Indan-2-yl-11,14-dioxo-2,6-dioxa-10,13-diazatricyclo[14.
3.1.17,10]henicosa-1(19),16(20),17-triene-9-carboxylic Acid ((([1-
(Dimethylcarbamoylphenylmethyl)carbamoyl]methyl)aminoox-
alyl)butyl)amide (51). Compound 51 was prepared by the same
general procedures using Boc-2-indanylglycine at P3. It was isolated
as a mixture of two diastereomers: 1H NMR (500 MHz, DMSO-
d6) δ 8.78 and 8.73 (t and m, 2H), 8.59 and 8.58 (d, J ) 6.0 and
5.6 Hz, 1H), 8.31 and 8.13 (d, J ) 7.2 and 8.0 Hz, 1H), 7.65-7.61
and 7.58-7.54 (m, 1H), 7.38-7.31 (m, 5H), 7.20-7.10 (m, 4H),
6.75-6.72 (m, 2H), 6.68 (t, J ) 7.9 Hz, 1H), 5.84 and 5.82 (d, J
) 3.8 and 3.4 Hz, 1H), 5.11-5.07 and 5.04-5.00 (m, 1H), 4.71
and 4.66 (t, J ) 9.6 and 9.6 Hz, 1H), 4.44 and 4.32 (t and m, J )
8.4 Hz, 1H), 4.12-4.05 (m, 1H), 3.85-3.68 (m, 5H), 3.58-3.53
(m, 1H), 3.44-3.41 (m, 1H), 2.98-2.90 (m, 1H), 2.93 and 2.92
(s, 3H), 2.88-2.79 (m, 1H), 2.86 and 2.85 (s, 3H), 2.76-2.69 (m,
2H), 2.12-2.06 (m, 1H), 1.89-1.84 (m, 2H), 1.76-1.68 (m, 1H),
1.66-1.62 (m, 1H), 1.50-1.38 and 1.32-1.28 (m, 3H), 1.19 and
1.18 (s, 3H), 1.17 and1.15 (s, 3H), 0.91-0.87 (m, 3H); 13C NMR
(125 MHz, DMSO-d6) δ 197.7, 172.3, 170.9, 170.8, 170.4, 170.3,
170.1, 167.8, 161.8, 161.6, 159.3, 159.2, 143.4, 143.2, 139.0, 138.9,
138.4, 138.4, 132.4, 132.3, 130.1, 129.7, 129.6, 129.5, 128.7, 128.7,
127.0, 127.0, 127.0, 125.3, 125.3, 125.2, 125.1, 122.3, 122.2, 116.3,
112.4, 75.0, 73.2, 70.5, 70.3, 64.6, 64.5, 64.2, 58.8, 54.4, 54.22,
54.17, 54.1, 53.9, 53.9, 42.4, 41.7, 37.5, 36.2, 36.2, 32.6, 32.4, 27.8,
27.4, 25.8, 25.7, 19.7, 19.6, 14.4, 14.4; HRMS (FAB) m/z calcd
for C47H59N6O9 851.4344 (M + H)+, found 851.4149.
Acknowledgment. We thank the Structural Chemistry
Department at the Schering-Plough Research Institute for NMR
and MS analysis for all new compounds. We also thank Ms.
Wanli Wu and Regina Huelgas for their assistance in the
preparation and characterization of some compounds.
Supporting Information Available: Compound characteriza-
tion data for intermediates and general procedures for hydrolysis
and amide coupling. This material is available free of charge via
the Internet at http://pubs.acs.org.
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) Lindenbach, B. D.; Rice, C. M. Unravelling hepatitis C virus
replication from genome to function. Nature 2005, 436, 933-938.
(b) Kolykhalov, A. A.; Mihalik, K.; Feinstone, S. M.; Rice, C. M.
Hepatitis C Virus-Encoded Enzymatic Activities and Conserved RNA
Elements in the 3′ Nontranslated Region Are Essential for Virus
Replication In Vivo. J. Virol. 2000, 74, 2046-2051.
(4) Lesk, A. M.; Fordham, W. D. Conservation and Variability in the
Structure of Serine Proteinase of the Chymotrypsin Family. J. Mol.
Biol. 1996, 258, 501-537.
(5) De Francesco, R.; Steinkuhler, C. Structure and Function of the
Hepatitis C Virus NS3-NS4A Protease. Curr. Top. Microbiol.
Immunol. 2000, 242, 149-169.
(6) (a) 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. (b) Di Marco, S.; Rizzi, M.; Volpari,
C.; Walsh, M. A.; Narjes, F.; Colarusso, S.; De Francesco, R.;
Matassa, V. G.; Sollazzo, M. Inhibition of Hepatitis C Virus NS3/
NS4A Protease: The Crystal Structures of Two Protease-Inhibitor
Complexes. J. Biol. Chem. 2000, 275, 7152-7157.
(7) (a) De Francesco, R.; Migliaccio, G. Challenges and Successes in
Developing New Therapies for Hepatitis C. Nature 2005, 436, 953-
960. (b) 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. (c) Malancona, S.; Colarrusso, 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 Phenethyl-
amide Inhibitors of the Hepatitis C Virus. Bioorg. Med. Chem. Lett.
2004, 14, 4575-4579. (d) 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. (e) Andrews, D. M.; Barnes, M. C.; Dowle, M. D.; Hind, S.
L.; Johnson, M. R.; Jones, P. S.; Mills, G.; Patikis, A.; Pateman, T.
J.; Redfern, T. J.; Ed Robinson, J.; Slater, M. J.; Trivedi, N.
Pyrrolidine-5,5-trans-Lactams. 5. Pharmacokinetic Optimization of
Inhibitors of Hepatitis C Virus NS3/4A Protease. Org. Lett. 2003, 5,
4631-4634. (f) Zhang, X. Inhibitors of Hepatitis CsA Review of
the Current Patent Literature. Idrugs 2002, 5, 154-158. (g) Dymock,
B. W. Emerging Therapies for Hepatitis C Virus Infection. Expert
Opin. Emerging Drugs 2001, 6, 13-42. (h) 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. (i) 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. (j) Ingallinella, P.; Fattori, D.; Altamura, S.;

Hepatitis C Virus NS3 Serine Protease Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1005
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. (k)
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.
(14) (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 Inhibitors, 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 Bicyclo-
proline 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.
(15) Schering-Plough Research Institute. Unpublished results.
(16) (a) Chen, K. X.; Arasappan, A.; Njoroge, F. G.; Vibulbhan, B.;
Buevich, A.; Chan, T.-M.; Girijavallabhan, V. Syntheses of Novel
4-tert-Alkyl Ether Proline-Based 16- and 17-Membered Macrocyclic
Compounds. J. Org. Chem. 2002, 67, 2730-2733. (b) Arasappan,
A.; Chen, K. X.; Njoroge, F. G.; Parekh, T. N.; Girijavallabhan, V.
Novel Dipeptide Macrocycles from 4-Oxo, -Thio, and -Amino-
Substituted Proline Derivatives. J. Org. Chem. 2002, 67, 3923-3926.
(17) 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.
(18) 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.
(19) Taremi, S. S.; Beyer, B.; Maher, M.; Yao, N.; Prosise, W.; Weber,
P. C.; Malcolm, B. A. Construction, Expression, and Characterization
of a Novel Fully Activated Recombinant Single-Chain Hepatitis C
Virus Protease. Protein Sci. 1998, 7, 2143-1249.
(20) For a definition of Ki* and discussions, see: Morrison, J. F.; Walsh,
C. T. In AdVances in Enzymology; Meister, A., Ed.; 1988; Vol. 61,
pp 201-301.
(21) (a) Tidwell, T. T. Oxidation of Alcohols to Carbonyl Compounds
via Alkoxysulfonium Ylides: The Moffatt, Swern and Related
Oxidations. Org. React. 1990, 39, 297. (b) Epstein, W. W.; Sweat,
F. W. Dimethyl Sulfoxide Oxidations. Chem. ReV. 1967, 67, 247-
260.
(8) For BILN-2061, see: (a) Lamarre, D.; Anderson, P. C.; Bailey, M.;
Beaulieu, P.; Bolger, G.; Bonneau, P.; Bo¨s, M.; Cameron, D. R.;
Cartier, M.; Cordingley, M. G.; Faucher, A.-M.; Goudreau, N.;
Kawai, S. H. Kukolj, G.; Lagace´, L.; LaPlante, S. R. Narjes,H.;
Poupart, M.-A Rancourt, J.; Sentjens, R. E.; George, T, S. Simoneau,
B.; Steinmann, G.; Thibeault, D.; Tsantrizos, Y. S. Weldon, S. M.;
Yong, C-L.; and Llina`s-Brunet M. An NS3 protease inhibitor with
antiviral effects in humans infected with hepatitis C virus Nature
2003, 426, 186-189. (b) Tsantrizos, Y. S.; Bolger, G.; Bonneau, P.;
Cameron, D. R.; Gordreau, N.; Kukolj, G.; LaPlante, S. R.; Llinas-
Brunet, M.; Nar, H.; Lamarre, D. Macrocyclic Inhibitors of the NS3
Protease as Potential Therapeutic Agents of Hepatitis C Virus
Infection. Angew. Chem., Int. Ed. 2003, 42, 1355-1360. For VX-
950, see: (c) Yip, Y.; Victor, F.; Lamar, J.; Johnson, R.; Wang, Q.;
May; G.; John I.; Yumibe, N.; Wakulchik, M.; Munroe, J.; Chen,
S.-H. P4 and P1′ optimization of bicycloproline P2 bearing tetrapep-
tidyl R-ketoamides as HCV protease inhibitors. Bioorg. Med. Chem.
Lett. 2004, 14, 5007-5011. (d) Perni, R. The Discovery of VX-950,
an Inhibitor of the HCV NS3-4A Protease in Clinical Development.
Gordon Research Conference, New London, NH, Aug 7-12, 2005.
(9) West, M. L.; Fairlie, D. P. Targeting HIV-1 protease: A Test of
Drug-Design Methodologies. Trends Pharm. Sci. 1995, 16, 67-75.
(10) Fairlie, D. P.; West, M. L.; Wong, A. K. Towards Protein Surface
Mimetics. Curr. Med. Chem. 1998, 5, 29-62.
(11) 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. Nonconventional
Stereochemical Issues in the Design of the Synthesis of the
Vancomycin Antibiotics: Challenges Imposed by Axial and Non-
planar Chiral Elements in the Heptapeptide Aglycons Angew. Chem.,
Int. Ed. 1998, 37, 2704-2708. (e) 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. (f)
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. (g) 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.
(22) (a) Chung, v.; Carroll, A. R.; Gray, N. M.; Parry, N. R.; Thommes,
P. A.; Viner, K. C.; D′Souza, E. A. Development of Cell-Based
Assays for In Vitro Characterization of Hepatitis C Virus NS3/4A
Protease Inhibitors. Antimicrob. Agents Chemother. 2005, 49, 1381-
1390. (b) Blight, K. J.; Kolykhalov, A. A.; Rice, C. M. Science 2000,
290, 1972-1974. (c) Lohmann, V.; Ko¨rner, F.; Koch, J.-O.; Herian,
U.; Theilmann, L. Bartenschlager, R. Replication of subgenomic
Hepatitis C virus RNAs in a Hepatoma cell line. Science 1999, 285,
110-113.
(12) McGeary, R. P.; Fairlie, D. P. Macrocyclic Peptidomimetics: Po-
tential for Drug Development. Curr. Opin. Drug DiscoVery DeV.
1998, 1, 208-217.
(13) (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 Inhibitors 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.
JM050820S