Second-generation highly potent and selective inhibitors of the hepatitis C virus NS3 serine protease

Article abstract of DOI:10.1021/jm801238q

The hepatitis C virus (HCV) infection is a leading cause of chronic liver disease. The moderate efficacy along with side effects of the current pegylated interferon and ribavirin combination therapy underscores the need for more effective and safer new tr

Full text of DOI:10.1021/jm801238q

1370
J. Med. Chem. 2009, 52, 1370–1379
Second-Generation Highly Potent and Selective Inhibitors of the Hepatitis C Virus NS3 Serine
Protease
Kevin X. Chen,* Latha Nair, Bancha Vibulbhan, Weiying Yang, Ashok Arasappan, Stephane L. Bogen, Srikanth Venkatraman,
Frank Bennett, Weidong Pan, Melissa L. Blackman, Angela I. Padilla, Andrew Prongay, Kuo-Chi Cheng, Xiao Tong,
Neng-Yang Shih, and F. George Njoroge
Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-A3545, Kenilworth, New Jersey 07033
ReceiVed September 30, 2008
The hepatitis C virus (HCV) infection is a leading cause of chronic liver disease. The moderate efficacy
along with side effects of the current pegylated interferon and ribavirin combination therapy underscores
the need for more effective and safer new treatment. In an effort to improve upon our current clinical candidate,
Boceprevir (SCH 503034), extensive SAR studies were performed on the P3 capping moieties. This led to
the discovery of tert-leucinol derived cyclic imides as a potent series of novel P3 capping groups. Thus, the
introduction of these imide caps improved the cell-based replicon EC90 by more than 10-fold. A number of
imides with various substitutions, ring sizes, bicyclic systems, and heterocyclic rings were explored. The
4,4-dimethyl substituted glutarimide emerged as the best cap as exemplified in compound 21 (Ki* ) 4 nM,
EC90 ) 40 nM). Systematic optimization of different positions (P′, P3, and P1) of the inhibitor resulted in
the identification of the lead compound 46, which had an excellent potency (Ki* ) 4 nM, EC90 ) 30 nM)
and good pharmacokinetic profile (22% and 35% bioavailability in rats and dogs, respectively). X-ray structure
of inhibitor 46 bound to the enzyme revealed that there was an additional hydrogen bonding interaction
between one of the imide carbonyls and Cys159.
Introduction
Hepatitis C virus (HCV) has infected an estimated 3% of
the world’s population, over 170 million people.1 The slow
progression and mild symptoms of the HCV infection make it
easily undetected. Most infections progress to a chronic state
that persists for decades and eventually lead to cirrhosis, liver
failure, or liver cancer.2 Currently, the most effective therapies
involve the treatment with pegylated R-interferon in combination
with antiviral agent ribavirin.3 The infection is considered cured
when there is a sustained virologic response (SVR) defined as
the absence of HCV RNA in serum at the end of treatment and
six-months post treatment. The SVR rate for patients with
genotype-1 HCV is about 40% with the current therapy.3 Lack
of efficacy along and the side effects with these therapies
necessitates the need for new and more effective HCV treatments.
Hepatitis C virus (HCV) is a positive strand RNA virus with
Figure 1. Compound 1: the HCV protease inhibitor that is currently
in phase III clinical trials.
a single open frame of ∼9600 nucleosides. Located at N-
terminal portion of nonstructural (NS) protein NS3 is a serine
protease. The NS3 protease has demonstrated a vital role in the
replication of the HCV virus.4 There is compelling evidence to
suggest that the inhibition of NS3 protease is a viable strategy
for the development of small molecule antiviral agents. It has
been determined that the HCV NS3 protease belongs to the
trypsin or chymotrypsin super family of serine proteases.5 A
small polypeptide cofactor, NS4A, is essential for the protease’s
activity.6 The X-ray structure of the protease has revealed a
shallow and solvent exposed substrate binding region, and the
inhibitor binding energy is mainly derived from weak lipophilic
and electrostatic interactions.7 Despite tremendous difficulty
encountered in the process, intensive efforts have been focused
on NS3 serine protease and a number of novel inhibitors have
been reported.8 Several drug candidates have or are being
progressed into clinical trials in human beings. The earliest entry,
BILN-2061,9 an NS3 protease inhibitor from Boehringer-
Ingelheim, failed in phase I clinical trials due to cardiac toxicity.
Currently, the most advanced candidates are Telapravir (VX-
950)10 from Vertex and 1 (Boceprevir, SCH 503034, Figure
1)11 from Schering-Plough. Both of these drugs are in phase
III clinical trials.
The first-generation HCV NS3 protease inhibitor (1) was
shown to be a potent inhibitor of the NS3 protease with a
enzymatic continuous assay Ki* of 14 nM and a cell-based
replicon assay EC90 of 350 nM.11 It had a favorable pharma-
cokinetic (PK) profile in rats and dogs (26% and 34% bioavail-
ability, respectively) but low bioavailability in monkeys (4-11%).
The goal for a second-generation inhibitor was to improve the
potency by at least 10-fold while maintaining or improving upon
PK profile. Modeling studies based on X-ray crystal structure
of compound 1 revealed that there was ample room for the P3
* To whom correspondence should be addressed. Phone: 1-908-740-7556.
Fax: 1-908-740-7152. E-mail: kevin.chen@spcorp.com.
10.1021/jm801238q CCC: $40.75
2009 American Chemical Society
Published on Web 02/05/2009

Second-Generation Inhibitors of HCV NS3 Serine Protease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1371
Scheme 2a
Scheme 1a
a Reaction conditions: (a) DIAD, PPh3, THF; (b) 4 M HCl, p-dioxane;
(c) phosgene, NaHCO3, H2O /CH2Cl2; (d) phthalimide, DIAD, PPh3, THF;
(e) NH2NH2, EtOH; (f) reflux, toluene, then Ac2O, Et3N, 90 °C; (g) Ac2O,
100 °C.
a Reaction conditions: (a) Boc-P3-OH, EDC, HOOBt; DMF/CH2Cl2; (b)
4 M HCl; p-dioxane; (c) appropriate isocyanate, i-Pr2NEt, CH2Cl2; (d) LiOH,
THF/MeOH/H2O; (e) P1-R-hydroxyamide, EDC, HOOBt, DMF/CH2Cl2;
(f) Dess-Martin periodinane.
depicted in Scheme 2. A Mitsunobu reaction14 of a commercially
available cyclic imide 7 and Boc-t-leucinol 8 gave the key
intermediate 9. Removal of the Boc-protecting group and
subsequent reaction with phosgene would provide the desired
isocyanate intermediate (12) for further capping. If cyclic imide
was not available, then 9 could be obtained through a two-step
procedure from cyclic anhydride 10. Thus, Boc-t-leucine amine
11 was prepared from alcohol 8 through a Mitsunobu reaction
with phthalimide followed by removal of the phthalyl group
using hydrazine. After opening of the cyclic anhydride 10 with
11, the cyclic imide of type 9 could be obtained by the treatment
with acetic anhydride. Finally, if neither 7 nor 10 were available,
then the cyclic anhydride could be accessed via cyclization of
a diacid of type 13.
cap to be extended beyond the tert-butyl group. The Cys159
amino acid residue of the protein backbone in the S4 region
provided an opportunity for additional hydrogen bonding
interaction. It was envisioned that an extended P3 cap having
carbonyl or sulfonyl groups at appropriate distance could engage
in a hydrogen bonding with Cys159. In the course of an
extensive search for the optimal functionality to establish the
hydrogen bond, it was discovered that cyclic imides derived
from tert-leucinol provided the most potent P3 cappings. With
optimized imide capping in hand, systematic investigation of
structure-activity relationship (SAR) was also conducted at
other sites of the inhibitor such as P3, P1, and P′. The
combination of the optimal moiety in each position gave rise
to a potent inhibitor with favorable PK profile. Herein, we report
the SAR development and the discovery of the second-
generation HCV NS3 protease inhibitor.
Synthesis of HCV Protease Inhibitors. The general syn-
thesis of the inhibitors is outlined in Scheme 1 and has been
described previously.11,12 Starting from the unique gem-dim-
ethylcyclopropylproline P2 core (2), coupling with a Boca-
protected P3 amino acid gave a dipeptide (3). Removal of the
Boc group and reaction with an isocyanate of the capping moiety
afforded compound 4. Hydrolysis of methyl ester to carboxylic
acid 5 and its subsequent coupling with a P1 R-hydroxyamide
amine provided a tripeptide. Dess-Martin periodinane oxida-
tion13 of this R-hydroxyamide furnished the desired R-ketoamide
6. Earlier experience indicated the difficulty in obtaining
acceptable PK from primary R-ketoamides (R′ ) H), it was
thus decided to perform all subsequent SAR studies on
secondary ketoamides. Among various R′ groups investigated,
allyl group was one of the best moieties for providing good
potency and PK. It was used as the R′ group for initial
explorations.
Results and Discussions
P3 Capping SAR: Discovery of Cyclic Imides as Potent
Capping Moiety. All inhibitors synthesized were tested in HCV
continuous enzymatic assay15 using the NS4A-tethered single
chain NS3 serine protease.16 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.17 Com-
pounds with good potency were then evaluated in a replicon-
based cellular assay.18 EC90, the concentration required for
inhibition of 90% of replicon replication, was obtained as a
measure of replicon cellular potency. Both enzyme and replicon
assays were performed in the absence of human serum. To
address the issue of selectivity among serine proteases, all
inhibitors were assayed against human neutrophil elastase
(HNE), which is a serine protease structurally most closely
related to HCV NS3 serine protease. All compounds were
assayed as a mixture of two diastereomers at P1 R-center. The
ratio of two isomers varied between 2:1 and 1:2. Rapid
equilibrium was observed under enzyme and cellular assay
conditions, which deemed the separation of the two isomers
less meaningful.
The capping moiety, focus of the current SAR development,
was synthesized according to one of the general procedures
Guided by X-ray structures of inhibitors bound to the protein,
an extensive search for the P3 capping moieties that could form
strong hydrogen bonding with Cys159 was conducted. Among
the structures that were capable of hydrogen bonding, cyclic
a Abbreviations: Boc, tert-butoxy carbonyl; EDC, N-(3-dimethylamino-
propyl)-3-ethyl carbodiimide hydrochloride; HOOBt, 3-hydroxy-1,2,3-
benzotriazin-4(3H)-one; DIAD, diisopropyl azodicarboxylate; HPBCD,
hydroxypropyl ꢀ cyclodextrin.

1372 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Table 1. Cyclic Imides as P3 Capping
Chen et al.
Table 2. Heterocyclic Imides as P3 Cappings
imides were identified as a promising moiety (Table 1). The
most potent cyclic imides were those derived from tert-leucine
amine. The first compound in this series, phthalimide derivative
14 (Table 1), had a enzyme assay potency (Ki*) of 30 nM and
cell-based replicon assay potency (EC90) of 750 nM. Its
selectivity against elastase (HNE/HCV) was 500. On the basis
of the size of the S4 pocket, it was speculated that a smaller
group might have a better fit. Encouragingly, changing the
capping from phthalimide to succinimide analogue (15) im-
proved potency and selectivity 2-4 fold. To make the ring more
flexible, six-membered glutarimide was introduced. To our
delight, compound (16) further improved potency about 2-fold
to Ki* of 8 nM and EC90 of 150 nM. The selectivity (HNE/
HCV ) 69000) was more than 30-fold higher than that of 15.
Encouraged by these results, we prepared a number of deriva-
tives (17-22) based on the six-membered imide ring system.
Three bicyclic imides (17-19) and three substituted glutarimides
(20-22) are shown in Table 1. Compound 17 had a fused 5,3-
bicyclic imide capping. It was significantly more potent than
16 in both enzyme and replicon assays (5 and 70 nM,
respectively). Although its selectivity was lower than 16, at
9500-fold, it was still extremely high. The spiro-5,6-bicyclic
imide cap in compound 18 maintained excellent binding (Ki*
) 6 nM) but lost some cellular potency (200 nM) and selectivity
(3800). The profile of [3,2,1]-bicyclic imide capped compound
19 was similar to that of 17, with excellent potency and very
high selectivity. Substitutions on the glutarimide improved
replicon potency even further. Thus, the 3,3-dimethylglutarimide
cap lowered EC90 of compound 20 by another 2-fold to 35 nM
while maintaining good enzyme activity and selectivity. On the
other hand, the 4,4-dimethyl analogue 21 achieved best potency
in both enzyme and replicon assays (4 and 40 nM, respectively).
It also had outstanding selectivity of 9300-fold against HNE.
The ethyl analogue of 21 did not provide any further enhance-
ment in potency in 22 (Ki* ) 8 nM, EC90 ) 40 nM), but
decreased the selectivity by almost 4-fold to 2500.
Besides the carbocyclic imides P3 cappings shown in Table
1, a parallel SAR study of heterocyclic imides with variations
in ring size and substitutions was also conducted (Table 2).
Replacing C-4 of the glutarimide with an oxygen gave rise to
the morpholine-dione analogue 23, which upheld enzyme
activity (Ki* ) 8 nM) but lost considerable cellular potency
(500 nM) and selectivity. The corresponding methylated 1,4-
dinitrogen analogue (piperazine-dione) 24 demonstrated excel-
lent potency and selectivity with a profile that was similar to
compound 21. The N-phenyl analogue (25), however, was much
less potent and selective. The 1,3-biazo cyclic imides (26, 27)
were also prepared. Although they had good Ki*s, with or
without N-methyl substitution, the cellular potency and selectiv-

Second-Generation Inhibitors of HCV NS3 Serine Protease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1373
Table 4. P3 Residue Investigation
Table 3. SAR of P′ Moieties
ity were not as good as their carbocyclic and 1,4-dinitrogen
analogues. Because of their cyclic imide-type structures, several
hydantoin derivatives (28-31) were also investigated. The
unsubstituted hydantoin 28 was essentially inactive in the
cellular assay. Although N-methyl and 4,4-dimethyl analogues
had improved replicon activity (both with EC90 ) 250 nM),
they were still far less potent than glutarimide capped inhibitors.
Finally, the trimethyl substituted hydantoin analogue 31 achieved
a respectable EC90 of 60 nM but much less selective.
In summary, we have achieved great success in the cyclic
imide SAR studies (Tables 1-2): a 10-fold improvement in
potency and extremely high selectivity had been accomplished.
The 4,4-dimethyl glutarimide (21) and 4-N-methyl piperazine-
dione (24) analogues were the most potent in both enzyme and
replicon assays and were also among the most selective against
elastase. Because of the more favorable PK property (discussed
in a later section) of compound 21, glutarimide capping was
used in further SAR investigations below.
Modifications of P′ Moiety. Earlier SAR’s were performed
with allyl amide at prime side. Although it was one of the best
group discovered as P′ moiety, further evaluation was still
needed to determine whether there was a better alternative.
Several potential alkyl and aromatic groups were examined, and
the results are given in Table 3. The shorter ethyl group in
compound 33 decreased both enzyme and cellular potency by
about 2-fold compared to 21. In compound 34, the allyl group
was replaced by saturated n-propyl chain, when the potency
and selectivity deteriorated even further, demonstrating that
saturated alkyl moieties were not as good as allyl group at P′
position. Next, a cyclopropyl group was introduced. It had some
sp2 characteristics in the ring system and conceivably could
mimic the allyl moiety. Indeed, compound 35 had an excellent
EC90 of 30 nM, the best replicon potency achieved so far. Its
selectivity against elastase (HNE/HCV ) 6100) was somewhat
lower that of 21. Then, three aromatic moieties were tested
(36-38). The benzyl analogue was equally potent in cellular
assay (40 nM), but the enzyme activity and selectivity were
significantly lower. Both thiophene and pyridine derivatives (37
and 38) showed 2-5 fold loss in Ki* or EC90 and much lower
selectivity. In summary, this P′ residue investigation confirmed
that allyl group was still one of the best moiety at that position.
Although cyclopropyl analogue 35 was slightly more potent, it
was less selective. As a result, further SAR studies were
performed with allyl amide.
P3 Residue Optimization. It is evident from previous SAR
studies and X-ray structure that the S3 region of HCV NS3
protease is highly lipophilic and the P3 moiety binds to the
enzyme mainly through hydrophobic interactions.7 On the basis
of earlier experience, modifications of the P3 side chain were
limited to branched alkyl groups or 5-6-membered aliphatic
rings. In this study, five different P3 moieties were incorporated
and the test results are summarized in Table 4. In contrast to
the tert-leucine P3 residue used in 21, all four other inhibitors
had cyclic ring structures at P3 position. Cyclohexylglycine in
39 provided better enzyme activity of 2 nM, but its replicon
potency and selectivity were worse. Considered a hybrid of
cyclohexylglycine and tert-leucine, the ꢀ-methyl cyclohexyl-
glycine derivative 40 did not improve either potency or
selectivity. The P3 2-indanylglycine analogue 41 maintained
enzyme activity but lost significant cellular potency and
selectivity. Finally, the ꢀ-methyl indanylglycine P3 derived 42
was also quite disappointing in both potency and selectivity.
This limited evaluation of these P3 residues proved the
superiority of the tert-butyl group as P3 side chain.
SAR Study of P1 Residue. From our previous studies and
the X-ray structure of the NS3 protease,7 it was also apparent

1374 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Table 5. P1 Residue Optimization
Chen et al.
Table 6. Rat PO AUC of Selected Compounds
compd
Ki*
EC90
rat PO AUCa (µM·h)
16
21
24
39
46
47
8
4
6
2
4
7
150
40
40
45
30
30
0.44
1.58
0.36
0.45
1.2
0.50
a 10 mg/kg administered with 20% HPBCD.
Table 7. PK Parameters of Compound 46
compd 46
rat
monkey
dog
AUC(PO) (µM·h)
AUC(IV) (µM·h)
t1/2 (IV) (h)
bioavailability (%)
rat liver conc (ng/g)
0.9a
0.1b
0.7d
2.3
4
1.2b
2.2c
2.7
35
NA
1.6b
7.4
22
1796
NA
a 10 mg/kg. b 3 mg/kg. c 2 mg/kg. d 1 mg/kg. All administered with 0.4%
MC.
that the S1 pocket was small and narrow. The preferred P1 side
chain was either a short linear alkyl chain or a small ring
attached to a short alkyl chain. Compound 1 had a cyclobutyl
methyl P1 side chain, which was one of the largest group the
S1 pocket could accommodate. Several aliphatic side chains
with different lengths and ring sizes were investigated, and the
results are listed in Table 5. The norvaline derivative 43, which
had a short n-propyl side chain, was found to be equally potent
to compound 21 but much less selective against elastase (HNE/
HCV ) 1600). On the other hand, P1 side chain of compound
44 had the same number of carbons but a smaller terminal
alkyne. It also showed excellent potency in enzyme and replicon
assays. However, its selectivity was on the lower side at 230-
fold. Compound 1, the clinical candidate, had cyclobutylalanine
at P1 position. When this same residue was put into current
combination, the resulting inhibitor (45) demonstrated an
excellent but similar profile compared with 21. When the smaller
cyclopropylalanine was used at P1 position, improvement was
observed in all properties. Thus, compound 46 had an excellent
Ki* of 4 nM, the best replicon EC90 of 30 nM, and very high
selectivity of 7100-fold. With the success of the cyclopropyl
ring system, the one-carbon longer homocyclopropylalanine P1
analogue (47) was prepared. It was also an excellent inhibitor
of HCV protease with the best cellular activity and selectivity.
After these extensive SAR developments, two compounds (46,
47) emerged as the leads with excellent overall profile. The goal
of improving replicon activity by more than 10-fold over the
compound 1 has been achieved.
Figure 2. X-ray structure of compound 46 bound to HCV NS3 protease
surface.
6. Compounds 16, 24, 39, and 47 have lower PO AUC’s that
were below 1.0 µM·h. Two compounds (21 and 46) stood out
with better rat PK (AUC of 1.58 and 1.2 µM·h, respectively),
but compound 46 had better activity in replicon. Although
compounds 46 and 47 were equally potent, the former had better
PK in rats. On the basis of its overall superior profile, inhibitor
46 was selected for further PK studies.
The results of full PK studies of compound 46 in rats,
monkeys, and dogs are shown in Table 7. All samples were
administered with 0.4% hydroxypropyl methylcellulose (MC).
Compound 46 had a respectable 0.9 µM·h PO AUC and 22%
bioavailability in rats. The PK data in monkeys were less
impressive with 0.1 µM·h AUC and 4% bioavailability. The
results were, however, very good in dogs with 1.2 µM·h AUC
and 35% bioavailability. It was also highly concentrated in rat
liver after 6 h (1796 ng/g), which is our target organ. It was
quite encouraging that the overall PK profile was very similar
to that of compound 1, our clinical candidate, which had
demonstrated good pharmacokinetic properties in human.
Compound 46 was 12 times more potent than 1.
Pharmacokinetic Property of Selected Compounds. The
promising compounds from above SAR studies were evaluated
in rats for their PK properties. The oral area under the curve
(PO AUC) results for selected compounds are listed in Table
X- Ray Structure of Compound 46. The X-ray crystal
structure of inhibitor 46 bound to the enzyme surface was
determined (Figure 2). The bindings of the P1, P2, and P3
residues with their respective pockets/surface were similar to

Second-Generation Inhibitors of HCV NS3 Serine Protease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1375
that reported for compound 1.11 The R-ketoamide formed a
reversible covalent bond with Ser139 hydroxyl group. The prime
side allyl group wrapped around the surface and had some
interaction with the alkyl portion of the side chain of the
backbone residue Lys136. The newly discovered cyclic
imide capping group occupied the S4 pocket with its tert-butyl
group. The glutarimide moiety formed a strong hydrogen
bonding with residue Cys159, between one of the two imide
carbonyls and amide N-H. This critical interaction provided
the observed enhancement in potency.
3.90-3.53 (m, 6 H), 3.47 (t, J ) 12.6 Hz, 1 H), 1.74-1.62 (m, 1
H), 1.55-1.37 (m, 2 H), 1.35-1.10 (m, 6 H), 1.01-0.58 (m, 26
H). 13C NMR (125 MHz, DMSO-d6) δ 198.35, 171.76, 171.30,
171.17, 168.66, 161.70, 158.67, 158.49, 135.02, 135.00, 134.88,
132.51, 123.71, 116.45, 116.34, 109.99, 60.09, 57.29, 57.22, 55.34,
54.49, 41.67, 39.46, 34.96, 34.38, 31.45, 30.20, 30.01, 28.37, 28.19,
27.45, 27.19, 27.04, 26.95, 26.90, 26.73, 22.58, 22.55, 19.20, 14.55,
13.19, 13.14.
(1R,5S)-N-[1-[1,2-dioxo-2-(2-propenylamino)ethyl]-pentyl]-3-
[2(S)-[[[[1(S)-[(2,5-dioxo-1-pyrrolidinyl)methyl]-2,2-dimethylpro-
pyl]-amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]-6,6-dimethyl-
3-azabicyclo[3.1.0]hexane-2(S)-carboxamide (15). The compound
was isolated as a mixture of two diastereomers. 1H NMR (500 MHz,
DMSO-d6) δ 8.32 and 8.18 (d, J ) 6.93, 7.25 Hz, 1H), 8.02 (d, J
) 13.9 Hz, 1 H), 7.76 (s, 1 H), 7.35-7.22 (m, 1 H), 6.16-6.06
(m, 2 H), 5.87 (d, J ) 9.46 Hz, 2 H), 5.00-4.92 (m, 1 H),
4.32-4.09 (m, 3 H), 4.03-3.80 (m, 2 H), 3.77-3.69 (m, 1 H),
3.63 (t, J ) 9.62 Hz, 2 H), 2.47-2.41 (m, 4 H), 1.74-1.62 (m, 2
H), 1.62-1.02 (m, 11 H), 0.99-0.70 (m, 22 H). 13C NMR (125
MHz, DMSO-d6) δ 197.73, 197.11, 177.23, 170.96, 170.74, 170.52,
170.30, 163.06, 162.77, 157.91, 157.87, 59.53, 59.23, 58.87, 56.67,
56.58, 54.84, 54.82, 53.70, 53.66, 53.07, 53.04, 47.27, 47.22,, 34.17,
34.15, 33.31, 31.70, 31.56, 30.54, 30.48, 29.51, 27.77, 26.69, 26.55,
26.06, 26.01, 25.97, 20.83, 18.63, 18.54, 18.29, 18.07, 13.88, 13.81,
13.49, 13.42, 13.33, 12.31, 12.25. HRMS calcd for C35H56N6O7,
673.4289 (M + H)+; found, 673.4286.
Conclusion
Starting from current clinical candidate, compound 1, exten-
sive SAR studies were performed on the P3 capping moieties.
This effort led to the discovery of tert-leucine amine derived
cyclic imides as a potent series of novel P3 capping groups. A
number of cyclic imides were explored with variations in
substitutions, ring sizes, ring numbers, ring types (spiro and
fused), and various heteroatom incorporations. The introduction
of these imide cappings improved the cell-based replicon EC90
by more than 10-fold in many compounds. The 4,4-dimethyl
substituted glutarimide cap emerged as the best as exemplified
in compound 21 (Ki* ) 4 nM, EC90 ) 40 nM). Systematic
optimization of P′, P3, and P1 residues resulted in the identifica-
tion of the best moiety for each position. The combination of
these optimal moieties led to compound 46, which had an
excellent potency (Ki* ) 4 nM, EC90 ) 30 nM) and good
pharmacokinetic profiles (22% and 35% bioavailabilities in rats
and dogs, respectively). X-ray structure of the inhibitor 46 bound
to the enzyme revealed that there was a strong hydrogen bonding
interaction between one of the imide carbonyls and Cys159.
Compared with compound 1, compound 46 provided a 12-fold
improvement in cellular potency and maintained a good PK
profile.
(1R,2S,5S)-N-(1-(allylamino)-1,2-dioxoheptan-3-yl)-3-((2S)-2-(3-
((2R)-1-(2,4-dioxo-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethylbu-
tan-2-yl)ureido)-3,3-dimethylbutanoyl)-6,6-dimethyl-3-
azabicyclo[3.1.0]hexane-2-carboxamide (17). The product was
1
isolated as a mixture of two diastereomers. H NMR (500 MHz,
DMSO-d6) δ 8.88 and 8.82 (t, J ) 5.99 and 6.93 Hz, 1 H), 8.37
and 8.30 (d, J ) 6.62 and 7.25 Hz, 1 H), 6.15-6.11 (m, 1 H),
5.89-5.86 (m, 1 H), 5.82-5.75 (m, 1 H), 5.12-5.03 (m, 2 H),
4.97-4.93 and 4.86-4.16 (m, 1 H), 4.26 (d, J ) 8.19 Hz, 1 H),
4.19-4.16 (m, 1 H), 3.92 (t, J ) 10.1 Hz, 1 H), 3.78-3.67 (m, 4
H), 3.45 (t, J ) 10.1 Hz, 1 H), 3.12 (t, J ) 12.92 Hz, 1 H),
2.48-2.41 (m, 2 H), 1.76-1.21 (m, 10 H), 0.99-0.73 (m, 27 H).
13C NMR (125 MHz, DMSO-d6) δ 198.17, 197.56, 175.89, 175.81,
171.95, 171.75, 171.51, 171.30, 161.98, 161.61, 159.00, 158.96,
135.03, 134.99, 116.43, 116.33, 60.09, 59.81, 57.73, 57.65, 55.92,
55.87, 54.50, 48.24, 41.69, 38.86, 38.79, 35.01, 34.99, 34.51, 31.47,
30.19, 30.01, 28.37, 28.19, 27.69, 27.61, 27.09, 27.02, 26.97, 26.93,
26.82, 22.61, 22.56, 20.45, 20.27, 19.26, 14.55, 13.33, 13.28. HRMS
calcd for C36H56N6O7, 685.4289 (M + H)+; found, 685.4296.
(1R,5S)-3-[2(S)-[[[[1(S)-[(7,9-Dioxo-8-Azaspiro[4.5]dec-8-yl)methyl]-
2,2-dimethylpropyl]amino]carbonyl]amino]-3,3-dimethyl-1-oxobu-
tyl]-N-[1-[1,2-dioxo-2-(2-propenylamino)ethyl]pentyl]-6,6-dimethyl-
3-azabicyclo[3.1.0]hexane-2(S)-Carboxamide (18). The product was
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 using 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 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 at 300,
1
isolated as a mixture of two diastereomers. H NMR (500 MHz,
DMSO-d6) δ 8.88 and 8.82 (t, J ) 6.30 and 5.99 Hz, 1 H), 8.37
and 8.32 (d, J ) 6.62 and 7.25 Hz, 1 H), 6.12 (t, J ) 11.1 Hz, 1
H), 5.82-5.74 (m, 2 H), 5.11-5.03 (m, 2 H), 4.97-4.82 (m, 1 H),
4.25-4.07 (m, 3 H), 3.90-3.54 (m, 6 H), 2.54 (bs, 4 H), 1.74-1.09
(m, 16 H), 0.98-0.95 (m, 3 H), 0.91-0.78 (m, 21 H), 0.75-0.72
(m, 3 H).
1
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 ppm, referenced to the internal
TMS or deuterated solvent signal.
General experimental procedures were described in earlier
publications11,12 for the synthesis of P2 core, peptide coupling, Boc
group deprotection, hydrolysis of ester to carboxylic acid, and
Dess-Martin periodinane oxidation. The syntheses of cyclic imide
from various starting material were performed according to literature
procedures with some minor modifications.
(1R,5S)-3-[2(S)-[[[[1(S)-[(2,4-dioxo-3-azabicyclo[3.2.1]oct-3-yl)-
methyl]-2,2-dimethylpropyl]amino]carbonyl]amino]-3,3-dimethyl-
1-oxobutyl]-N-[1-[1,2-dioxo-2-(2-propenyl-amino)ethyl]pentyl]-6,6-
dimethyl-3-azabicyclo-[3.1.0]hexane-2(S)-carboxamide (19). The
compound is isolated as a mixture of two diastereomers. 1H NMR
(500 MHz, DMSO-d6) δ 8.87 and 8.80 (t, J ) 5.99 Hz, 1 H), 8.36
and 8.30 (d, J ) 6.94 and 7.25 Hz, 1 H), 6.12 (q, J ) 10.4 and
2.52 Hz, 1 H), 5.82-5.74 (m, 2 H), 5.11-5.03 (m, 2 H), 4.96-4.92
and 4.85-4.81 (m, 1 H), 4.25 (d, J ) 7.88 Hz, 1 H), 4.16-4.07
(m, 1 H), 3.92 (t, J ) 9.46 Hz, 1 H), 3.78-3.58 (m, 5 H), 2.96 and
2.89 (s, 2 H), 2.04 (d, J ) 11.35 Hz, 1 H), 1.94 (s, 2 H), 1.77-1.67
(m, 3 H), 1.54-1.19 (m, 8 H), 0.98-0.74 (m, 28 H). 13C NMR
(125 MHz, DMSO-d6) δ 197.28, 196.66, 176.15, 176.07, 171.13,
170.94, 170.81, 170.61, 161.12, 160.74, 134.12, 134.08, 115.56,
115.47, 59.23, 58.94, 53.59, 53.57, 47.34, 47.24, 44.33, 40.79,
(1R,5S)-3-[2(S)-[[[[1(S)-[(1,3-Dihydro-1,3-dioxo-2H-iso-indol-2-
yl)-methyl]-2,2-dimethylpropyl]amino]carbonyl]-amino-3,3-dimethyl-
1-oxobutyl]-N-[1-[1,2-dioxo-2-(2-propenylamino)ethyl]pentyl]-6,6-
dimethyl-3-azabicyclo-[3.1.0]hexane-2(S)-carboxamide (14). The
1
product was isolated as a mixture of two diastereomers. H NMR
(500 MHz, DMSO-d6) δ 8.86 and 8.81 (t, J ) 5.99 Hz, 1 H), 8.33
and 8.20 (d, J ) 6.62 and 7.25 Hz, 1H), 7.77 (bs, 3 H), 6.10-5.92
(m, 2 H), 5.80-5.73 (m, 1H), 5.19-5.02 (m, 2 H), 4.93-4.88 (m,
2 H), 4.85-4.80 (m, 1H), 4.29-4.26 and 4.21-4.18 (m, 1 H),

1376 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Chen et al.
34.00, 33.98, 30.59, 30.58, 29.26, 29.09, 27.47, 27.27, 26.90, 26.83,
26.55, 26.30, 26.08, 26.02, 21.70, 21.65, 18.40, 13.67, 13.65, 12.57,
12.53. HRMS calcd for C38H60N6O7, 713.4602 (M + H)+; found,
713.4612.
3-azabicyclo[3.1.0]hexane-2(S)-carboxamide (25). The product was
isolated as a mixture of two diastereomers. H NMR (500 MHz,
1
DMSO-d6) δ 8.87 and 8.81 (t, J ) 5.99 and 6.30 Hz, 1 H),
8.37-8.34 and 8.28-8.28 (m, 1 H), 7.59-7.23 (m, 3 H), 7.10-6.87
(m, 2 H), 6.13-6.07 (m, 1 H), 5.85-5.74 (m, 2 H), 5.13-5.03
(m, 2 H), 4.28-4.01 (m,5 H), 3.93-3.60 (m, 8 H), 1.78-1.14 (m,
8 H), 0.98-0.71 (m, 27 H). 13C NMR (125 MHz, DMSO-d6) δ
198.17, 197.56, 175.36, 171.98, 171.78, 171.52, 171.30, 170.13,
163.89, 161.98, 161.62, 158.97, 148.47, 135.03, 134.99, 130.06,
125.72, 123.25, 121.23, 116.59, 116.43, 116.34, 60.12, 59.81, 57.98,
57.66, 55.45, 54.50, 52.98, 48.19, 41.68, 34.97, 34.54, 34.28, 34.00,
32.12, 31.47, 30.18, 30.00, 29.23, 28.36, 28.19, 27.71, 27.02, 25.45,
22.97, 22.59, 19.27, 14.87, 14.56, 14.55, 13.33, 13.27. HRMS calcd
for C41H61N7O7, 764.4711 (M + H)+; found, 764.4723.
(1R,5S)-3-[2(S)-[[[[1(S)-[(4,4-dimethyl-2,6-dioxo-1-piperidinyl)-
methyl]-2,2-dimethylpropyl]amino]-carbonyl]amino]-3,3-dimethyl-
1-oxobutyl]-N-[1-[1,2-dioxo-2-(2-propenylamino)ethyl]pentyl]-6,6-
dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-carboxamide. (21). The
compound is isolated as a mixture of two diastereomers. 1H NMR
(500 MHz, DMSO-d6) δ 8.85 and 8.79 (t, J ) 5.99 Hz, 1 H), 8.35
and 8.28 (d, J ) 6.94 and 7.25 Hz, 1 H), 6.12 (t, J ) 11.35 Hz, 2
H), 5.81-5.73 (m, 2 H), 5.12-5.02 (m, 2 H), 4.96-4.92 and
4.85-4.80 (m, 1 H), 4.24 (d, J ) 9.46 Hz, 1 H), 4.10 and 4.08
(dd, J ) 4.41 and 10.09 Hz, 1 H), 3.88 (t, J ) 8.99 Hz, 1 H),
3.81-3.55 (m, 6 H), 2.44-2.35 (m, 4 H), 1.75-1.66 (m, 1 H),
1.53-1.19 (m, 6 H), 0.97-0.73 (m, 33 H). 13C NMR (100 MHz,
DMSO-d6) δ 197.31, 196.70, 171.87, 171.19, 171.00, 170.82,
170.62, 161.16, 160.77, 158.18, 158.13, 134.15, 134.10, 115.61,
115.52, 115.03, 59.30, 59.01, 57.08, 56.98, 56.93, 54.95, 54.90,
53.65, 53.62, 47.38, 47.37, 45.50, 40.84, 35.34, 34.26, 34.24, 34.14,
34.11, 30.62, 29.30, 29.14, 28.38, 27.52, 27.33, 27.23, 26.93, 26.85,
26.79, 26.33, 26.12, 26.08, 21.74, 21.69, 18.45, 13.71, 13.69, 12.61,
12.56. HRMS calcd for C38H62N6O7, 715.4758 (M + H)+; found,
715.4763.
(1R,5S)-3-[2(S)-[[[[2,2-Dimethyl-1(S)-[(tetrahydro-2,6-dioxo-
1(2H)-pyrimidinyl)methyl]propyl]amino]carbonyl]amino]-3,3-di-
methyl-1-oxobutyl]-N-[1-[1,2-dioxo-2-(2-propenylamino)ethyl]pentyl]-
6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-carboxamide (26). The
1
product was isolated as a mixture of two diastereomers. H NMR
(500 MHz, DMSO-d6) δ 8.88 and 8.83 (t, J ) 8.19 and 6.30 Hz,
1H), 8.36 and 8.27 (d, J ) 7.88 and 7.25 Hz, 1 H), 7.64 (bs, 1 H),
6.15-6.09 (m, 1 H), 5.82-5.74 (m, 2 H), 5.11-4.83 (m, 3 H),
4.26-4.15 (m, 2 H), 3.98-3.56 (m, 7 H), 3.06 (m, 2 H), 2.48-2.40
(m, 2 H), 1.74-1.07 (m, 8 H), 0.98-0.74 (m, 27 H). 13C NMR
(125 MHz, DMSO-d6) δ 198.18, 197.58, 152.01, 171.80, 171.66,
171.45, 170.55, 161.98, 161.62, 159.14, 154.90, 135.04, 135.00,
116.44, 116.35, 60.09, 59.78, 57.76, 57.67, 56.23, 54.52, 48.23,
41.69, 35.14, 35.04, 34.44, 32.51, 31.44, 30.13, 30.05, 28.43, 28.14,
27.74, 27.62, 27.09, 26.96, 22.60, 19.24, 14.57, 13.37, 13.33. HRMS
calcd for C35H57N7O7, 688.4398 (M + H)+; found, 688.4418.
(1R,5S)-3-[2(S)-[[[[2,2-Dimethyl-1(S)-[(tetrahydro-3-methyl-2,6-
dioxo-1(2H)-pyrimidinyl)methyl]propyl]amino]carbonyl]amino]-
3,3-dimethyl-1-oxobutyl]-N-[1-[1,2-dioxo-2-(2-propenylamino)-
ethyl]pentyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-
carboxamide (27). The product was isolated as a mixture of two
diastereomers. 1H NMR (500 MHz, DMSO-d6) δ 8.88 and 8.83 (t,
J ) 6.30 and 5.67 Hz, 1 H), 8.37 and 8.29 (d, J ) 6.62 and 7.56
Hz, 1 H), 6.13-6.08 (m, 1 H), 5.82-5.75 (m, 2 H), 5.11-5.02
(m, 2 H), 4.97-4.93 and 4.87-4.83 (m, 1 H), 4.26 (d, J ) 9.14
Hz, 1 H), 4.19-4.14 (m, 2 H), 3.94-3.89 (m, 2 H), 3.78-3.58
(m, 4 H), 3.21 (t, J ) 6.62 Hz, 2 H), 2.86 (s, 3 H), 2.57-2.46 (m,
2 H), 1.76-1.07 (m, 8 H), 0.98-0.74 (m, 27 H). HRMS calcd for
C36H59N7O7, 702.4554 (M + H)+; found, 702.4531.
(1R,5S)-N-[1-[1,2-Dioxo-2-(2-propenylamino)ethyl]pentyl]-3-
[2(S)-[[[[1(S)-[(4-ethyl-4-methyl-2,6-dioxo-1-piperidinyl)methyl]-
2,2-dimethylpropyl]amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]-
6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-Carboxamide (22). The
1
product was isolated as a mixture of two diastereomers. H NMR
(500 MHz, DMSO-d6) δ 8.88 and 8.82 (t, J ) 5.99 and 6.62 Hz,
1 H), 8.37 and 8.31 (d, J ) 6.93 and 6.30 Hz, 1 H), 6.15-6.10 (m,
1 H), 5.82-5.75 (m, 2 H), 5.15-5.04 (m, 2 H), 4.97-4.81 (m, 1
H),4.25-3.56 (m, 9 H), 2.47-2.29 (m, 4 H), 1.72-1.20 (m,10 H),
0.98-0.72 (m, 33 H). HRMS calcd for C39H64N6O7, 751.4734 (M
+ Na)+; found, 751.4768.
(1R,5S)-3-[2(S)-[[[[1(S)-[(3,5-Dioxo-4-morpholinyl)methyl]-2,2-
dimethylpropyl]amino]-carbonyl]amino]-3,3-dimethyl-1-oxobutyl]-
N-[1-[1,2-dioxo-2-(2-propenylamino)ethyl]pentyl]-6,6-dimethyl-3-
azabicyclo[3.1.0]hexane-2(S)-carboxamide (23). The compound is
1
isolated as a mixture of two diastereomers. H NMR (500 MHz,
DMSO-d6) δ 8.87 and 8.82 (d, J ) 5.99 Hz, 1 H), 8.36 and 8.26
(d, J ) 6.93 and 7.25 Hz, 1H), 6.13-6.08 (m, 1 H), 5.85-5.75
(m, 2 H), 5.12-5.03 (m, 2 H), 4.97-4.93, 4.87-4.83 (m, 1 H),
4.31-4.14 (m, 6 H), 3.91 (t, J ) 10.08 Hz, 1 H), 3.80-3.62 (m,
6 H), 1.75-1.46 (m, 1 H), 1.40-1.22 (m, 7 H), 0.99-0.75 (m, 27
H). 13C NMR (100 MHz, DMSO-d6) δ 198.19, 197.58, 171.99,
171.79, 171.55, 171.34, 170.42, 161.97, 161.63, 158.92, 158.88,
135.04, 135.00, 116.44, 116.35, 67.74, 60.12, 59.80, 57.69, 57.62,
55.64, 54.51, 41.68, 35.07, 35.04, 34.53, 31.47, 30.19, 30.01, 28.37,
28.19, 27.69, 27.60, 27.05, 26.99, 26.94, 22.60, 22.56, 19.27, 14.55,
13.30, 13.24. HRMS calcd for C35H56N6O8, 689.4238 (M + H)+;
found, 689.4213.
(1R,5S)-3-[2(S)-[[[[1(S)-[(2,5-Dioxo-imidazolidinyl)-methyl]-2,2-
dimethylpropyl]amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]-
N-[1-[1,2-dioxo-2-(2-propenyl-amino)ethyl]pentyl]-6,6-dimethyl-3-
azabicyclo[3.1.0]-hexane-2(S)-carboxamide (28). The product was
1
isolated as a mixture of two diastereomers. H NMR (500 MHz,
DMSO-d6) δ 8.88 and 8.83 (t, J ) 5.99 and 5.99 Hz, 1H), 8.37
and 8.27 (d, J ) 6.62 and 7.25 Hz, 1 H), 7.88 (s, 1 H),
6.10-6.04(m, 1 H), 5.89-5.72 (m, 2 H), 5.11-5.00 (m, 2 H),
4.97-4.92 and 4.87-4.82 (m, 1 H), 4.26 (d, J ) 11.66 Hz, 1 H),
4.18-4.14 (m, 1 H), 3.91-3.60 (m, 8 H), 3.23 (t, J ) 13.24 Hz,
1 H), 1.74-1.21 (m, 8 H), 0.98-0.74 (m, 27 H). 13C NMR (125
MHz, DMSO-d6) δ 198.18, 197.58, 172.64, 171.91, 171.80, 171.53,
171.35, 161.96, 161.60, 158.75, 158.71, 158.43, 135.03, 134.99,
130.88, 129.02, 116.44, 116.36, 60.12, 59.78, 59.79, 57.52, 56.00,
54.50, 48.19, 46.54, 41.69, 35.15, 35.13, 34.41, 31.50, 31.47, 30.45,
30.17, 30.02, 28.38, 28.20, 27.65, 27.56, 27.01, 26.93, 22.60, 22.56,
19.27, 14.57, 13.29, 13.22. HRMS calcd for C34H55N7O7, 674.4241
(M + H)+; found, 674.4246.
(1R,5S)-3-[2(S)-[[[[2,2-Dimethyl-1(S)-[(4-methyl-2,6-dioxo-1-pip-
erazinyl)methyl]propyl]amino]carbonyl]-amino]-3,3-dimethyl-1-ox-
obutyl]-N-[1-[1,2-dioxo-2-(2-propenylamino)ethyl]pentyl]-6,6-di-
methyl-3-azabicyclo[3.1.0]hexane-2(S)-carboxamide (24). The
compound was isolated as a mixture of two diastereomers. 1H NMR
(500 MHz, DMSO-d6) δ 8.88 and 8.82 (d, J ) 5.99 and 5.67 Hz,
1H), 8.36 and 8.28 (d, J ) 6.62 and 7.25 Hz, 1 H), 6.14-6.09 (m,
1 H), 5.82-5.76 (m, 2 H), 5.12-5.04 (m, 3 H), 4.97-4.93 and
4.86-4.82 (m, 1 H), 4.26-3.58 (m, 12 H), 2.22 (s, 3 H), 1.76-1.14
(m, 8 H), 0.99-0.74 (m, 27 H). 13C NMR (100 MHz, DMSO-d6)
δ 198.19, 197.25, 171.99, 171.38, 170.85, 170.88, 158.90, 158.85,
135.04, 135.00, 116.44, 116.36, 89.96, 59.90, 59.70, 58.34, 57.63,
55.58, 54.51, 48.23, 43.84, 41.69, 35.08, 34.80, 31.49, 30.11, 28.45,
28.21, 27.14, 26.96, 26.89, 22.58, 19.24, 14.61, 13.40, 13.27. HRMS
calcd for C36H59N7O7, 702.4554 (M + H)+; found, 702.4531.
(1R,5S)-3-[2(S)-[[[[2,2-Dimethyl-1(S)-[(3,4,4-trimethyl-2,5-dioxo-
1-imidazolidinyl)methyl]propyl]amino]-carbonyl]amino]-3,3-di-
methyl-1-oxobutyl]-N-[1-[1,2-dioxo-2-(2-propenylamino)ethyl]pentyl]-
6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-carboxamide (31). The
1
product was isolated as a mixture of two diastereomers. H NMR
(500 MHz, DMSO-d6) δ 8.88 and 8.83 (t, J ) 5.99 and 6.30 Hz,
1 H), 8.37 and 8.29 (d, J ) 6.93 and 7.25 Hz, 1 H), 6.11-6.05 (m,
1 H), 5.93-5.89 (m, 1 H), 5.82-5.74 (m, 1 H), 5.11-5.03 (m, 2
H), 4.96-4.92 and 4.86-4.82 (m, 1 H), 4.25 (d, J ) 8.82 Hz, 1
H), 4.17-4.08 (m, 2 H), 3.91-3.63 (m, 4 H), 3.47-3.42 (m, 1 H),
(1R,5S)-3-[2(S)-[[[[1(S)-[(2,6-Dioxo-4-phenyl-1-piperazinyl)methyl]-
2,2-dimethylpropyl]amino]-carbonyl]amino]-3,3-dimethyl-1-oxobu-
tyl]-N-[1-[1,2-dioxo-2-(2-propenylamino)ethyl]-pentyl]-6,6-dimethyl-

Second-Generation Inhibitors of HCV NS3 Serine Protease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1377
3.23-3.18 (m, 1 H), 2.71 (s, 3 H), 1.75-1.19 (m, 14 H), 0.98-0.74
(m, 27 H). 13C NMR (125 MHz, DMSO-d6) δ 198.17, 197.56,
177.10, 171.99, 171.78, 171.55, 171.35, 161.97, 161.61, 158.79,
158.74, 155.46, 135.02, 134.98, 116.44, 116.35, 61.20, 60.12, 59.79,
57.72. 57.62, 56.07, 55.72, 54.48, 48.14, 41.68, 35.05, 35.01, 34.50,
31.45, 30.19, 29.97, 28.35, 28.19, 27.67, 27.56, 27.09, 26.92, 24.94,
24.85, 22.59, 22.55, 22.36, 21.74, 19.26, 14.55, 13.36, 13.28. HRMS
calcd for C37H61N7O7, 716.4711 (M + H)+; found, 716.4718.
(1R,5S)-3-[2(S)-[[[[1(S)-[(4,4-Dimethyl-2,6-dioxo-1-piperidinyl)-
methyl]-2,2-dimethylpropyl]amino]-carbonyl]amino]-3,3-dimethyl-
1-oxobutyl]-N-[1-[2-(ethylamino)-1,2-dioxoethyl]pentyl]-6,6-dimethyl-
3-azabicyclo[3.1.0]hexane-2(S)-carboxamide (33). This compound
was isolated as a mixture of two diastereomers. 1H NMR (500 MHz,
DMSO-d6) δ 8.71 and 8.67 (t, J ) 5.67 and 5.99 Hz, 1 H), 8.36
and 8.27 (d, J ) 6.93 and 7.56 Hz, 1 H), 6.13 (t, J ) 10.08 Hz, 1
H), 5.79 (d, J ) 10.4 Hz, 1 H), 4.98-4.94 and 4.89-4.85 (m, 1
H), 4.25 (d, J ) 5.99 Hz, 1 H), 4.11 (m, 1 H), 3.89 (t, J ) 10.08
Hz, 1 H), 3.80 (t, J ) 11.66 Hz, 1 H), 3.74-3.63(m, 2 H), 3.58 (d,
J ) 11.66 Hz, 1H), 2.44-2.36 (m, 4 H), 1.76-1.20 (m, 8 H), 1.03
(t, J ) 7.25 Hz, 3 H), 0.99 (d, J ) 9.14 Hz, 3 H), 0.94 (s, 6 H),
0.86-0.75 (m, 21 H). 13C NMR (125 MHz, DMSO-d6) δ 198.39,
197.81, 172.67, 172.02, 171.80, 171.65, 171.44, 161.74, 161.29,
159.0, 158.95, 130.24, 116.08, 60.15, 59.83, 57.91, 57.81, 55.75,
54.44, 48.09, 46.36, 35.10, 34.97, 34.29, 31.57, 31.52, 30.16, 30.06,
29.24, 28.43, 28.24, 28.03, 27.75, 27.66, 27.21, 26.95, 22.56, 19.30,
15.11, 14.57, 13.48, 13.43. HRMS calcd for C37H62N6O7, 703.4758
(M + H)+; found, 703.4726.
(1R,5S)-3-[2(S)-[[[[1(S)-[(4,4-Dimethyl-2,6-dioxo-1-piperidinyl)-
methyl]-2,2-dimethylpropyl]amino]carbonyl]amino]-3,3-dimethyl-
1-oxobutyl]-N-[1-[1,2-dioxo-2-(propylamino)ethyl]-pentyl]-6,6-di-
methyl-3-azabicyclo-[3.1.0]hexane-2(S)-carboxamide (34). The com-
pound is isolated as a mixture of two diastereomers. 1H NMR (500
MHz, DMSO-d6) δ 8.68 and 8.63 (t, J ) 5.99 Hz, 1 H), 8.32 and
8.24 (d, J ) 6.93 and 7.25 Hz, 1H), 6.15-6.10 (m, 1 H), 5.79 and
5.77 (dd, J ) 3.15 and 10.72 Hz, 1 H), 4.97-4.93 and 4.88-4.84
(m, 1 H), 4.25 (d, J ) 7.25 Hz, 1 H), 4.12-4.07 (m, 1 H), 3.88,
3.79 (t, J ) 9.62 and 11.82 Hz, 2 H), 3.73-3.55 (m, 4 H),
3.08-3.01 (m, 2 H), 2.46-2.34 (m, 5 H), 1.75-1.66 (m, 1 H),
1.51-1.13 (m, 8 H), 0.97-0.85 (m, 32 H). 13C NMR (100 MHz,
DMSO-d6) δ 197.50, 196.89, 171.81, 171.80, 171.12, 170.89,
170.77, 170.55, 161.15, 160.76, 158.11, 158.09, 158.06, 59.26,
58.95, 57.01, 56.92, 54.87, 54.82, 53.56, 53.52, 47.31, 45.46, 34.21,
34.19, 34.07, 34.05, 30.64, 30.61, 29.26, 29.16, 28.33, 27.48, 27.28,
27.18, 26.86, 26.77, 26.29, 26.28, 26.07, 26.04, 21.91, 21.88, 21.67,
21.63, 18.40, 13.66, 13.64, 12.57, 12.51, 11.22. HRMS calcd for
C38H64N6O7, 717.4915 (M + H)+; found, 717.4914.
141.30, 134.97, 116.45, 116.36, 60.42, 59.90, 55.91, 55.78, 54.65,
54.27, 47.77, 47.56, 46.36, 41.68, 35.07, 31.58, 30.17, 29.82, 29.61,
29.25, 28.44, 28.20, 28.05, 27.61, 27.53, 26.96, 26.20, 22.58, 22.53,
19.38, 14.55, 13.52, 13.43. HRMS calcd for C40H64N6O7, 741.4915
(M + H)+; found, 741.4890.
(1R,5S)-3-[2(S)-[[[[1(S)-[(4,4-Dimethyl-2,6-dioxo-1-piperidinyl)-
methyl]-2,2-dimethylpropyl]amino]-carbonyl] amino]-2-(1-methyl-
cyclohexyl)acetyl]-N-[1-[1,2-dioxo-2-(2-propenylamino)-ethyl]pen-
tyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-carboxamide (40).
1
The product was isolated as a mixture of two diastereomers. H
NMR (500 MHz, DMSO-d6) δ 8.88 and 8.82 (t, J ) 5.99 Hz, 1
H), 8.34 and 8.21 (d, J ) 6.94 and 7.25 Hz, 1 H), 6.15 (q, J )
10.09 and 4.10 Hz, 1 H), 5.82-5.74 (m, 2 H), 5.11-5.03 (m, 2
H), 4.96-4.92 and 4.86-4.82 (m, 1 H), 4.24 (d, J ) 15.13 Hz, 1
H), 4.17 (q, J ) 7.25 and 2.52 Hz, 1 H), 3.89 (t, J ) 10.09 Hz, 1
H), 3.81-3.56 (m, 7 H), 2.48-2.34 (m, 4 H), 1.76-1.67 (m, 1 H),
1.51-1.06 (m, 8 H), 0.97-0.93 (m, 11 H), 0.89 (s, 4 H), 0.86-0.72
(m, 20 H). 13C NMR (125 MHz, DMSO-d6) δ 197.29, 196.70,
171.76, 171.74, 171.11, 170.90, 170.60, 170.35, 161.11, 160.75,
158.08, 158.03, 134.12, 134.08, 115.55, 115.45, 59.36, 58.98, 54.89,
54.84, 53.61, 47.35, 47.33, 45.49, 40.79, 40.77, 36.83, 36.79, 34.14,
33.55, 33.47, 33.38, 33.33, 30.55, 30.52, 29.30, 29.16, 28.35, 27.49,
27.27, 27.16, 26.83, 26.74, 26.05, 26.00, 25.87, 25.86, 21.73, 21.68,
21.30, 21.16, 19.18, 18.39, 18.36, 13.66, 12.58, 12.51. HRMS calcd
for C41H66N6O7, 755.5071 (M + H)+; found, 755.5086.
(1R,5S)-3-[2(S)-(2,3-Dihydro-1H-inden-2-yl)-2-[[[[1(S)-[(4,4-di-
methyl-2,6-dioxo-1-piperidinyl)methyl]-2,2-dimethylpropyl]ami-
no]carbonyl]amino]acetyl]-N-[1-[1,2-dioxo-2-(2-propenylamino)-
ethyl]pentyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-
carboxamide (41). The product was isolated as mixture of two
diastereomers. 1H NMR (500 MHz, DMSO-d6) δ 8.91 and 8.81 (t,
J ) 6.36 and 11.98 Hz, 1 H), 8.43 and 8.29 (d, J ) 6.93 and 9.14
Hz, 1 H), 7.16-7.10 (m, 4 H), 6.31 (t, J ) 8.19 Hz, 1 H), 5.83-5.76
(m, 1 H), 5.67(d, J ) 6.62 Hz, 1H), 5.13-4.95 (m, 3 H), 4.25 (d,
J ) 15.44 Hz, 1 H), 4.16-4.08 (m, 1 H), 3.82-3.76 (m, 4 H),
3.67-3.54 (m, 3 H), 2.86-2.56 (m, 5 H), 2.44 (bs, 4 H), 1.78-1.48
(m, 2 H), 1.38-1.21 (m, 6 H), 0.97 (bs, 9 H), 0.88-0.77 (m, 15
H). 13C NMR (125 MHz, DMSO-d6) δ 198.19, 197.75, 172.77,
172.05, 171.85, 171.59, 171.55, 161.85, 161.67, 158.73, 143.40,
143.01, 134.98, 130.87, 129.02, 127.09, 127.00, 125.16, 116.46,
116.38, 60.60, 60.12, 55.90, 54.70, 54.43, 53.88, 47.65, 47.08,
46.50, 41.70, 36.89, 35.05, 30.05, 29.30, 28.47, 28.29, 28.02, 27.58,
26.97, 26.95, 22.55, 19.44, 14.57, 13.48, 13.55. HRMS calcd for
C43H62N6O7, 775.4758 (M + H)+; found, 775.4777.
(1R,5S)-3-[2(S)-(2,3-Dihydro-2-methyl-1H-inden-2-yl)-2-[[[[1(S)-
[(4,4-dimethyl-2,6-dioxo-1-piperidinyl)methyl]-2,2-dimethylpropyl]-
amino]carbonyl]amino]acetyl]-N-[1-[1,2-dioxo-2-(2-propenylami-
no)ethyl]pentyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-car-
boxamide (42). The product was isolated as a mixture of two
diastereomers. 1H NMR (500 MHz, DMSO-d6) δ 8.89 and 8.83 (t,
J ) 5.99 and 5.99 Hz, 1 H), 8.40 and 8.35 (d, J ) 6.93 and 7.56
Hz, 1 H), 7.15-7.09 (m, 4 H), 6.32 (t, J ) 11.66 Hz, 1 H),
5.82-5.74 (m, 2 H), 5.12-5.03 (m, 2 H), 4.97-4.93 and 4.89-4.84
(m, 1 H), 4.36 (t, J ) 10.40 Hz, 1 H), 4.27 (d, J ) 9.77 Hz, 1 H),
3.86-3.56 (m, 7 H), 3.12-3.08 (m, 2 H), 2.46-2.35 (m, 6 H),
1.75-1.22 (m, 8 H), 1.02-0.97 (m, 3 H), 0.87-0.77 (m, 24 H).
(1R,5S)-3-[2(S)-[[[[1(S)-[(4,4-Dimethyl-2,6-dioxo-1-piperidinyl)-
methyl]-2,2-dimethylpropyl]amino]carbonyl]-amino]-3,3-dimethyl-
1-oxobutyl]-N-[1-[1,2-dioxo-2-(2-propenylamino)ethyl]butyl]-6,6-
dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-carboxamide (43). The
(1R,5S)-3-[2(S)-[[[[1(S)-[(4,4-Dimethyl-2,6-dioxo-1-piperidinyl)-
methyl]-2,2-dimethylpropyl]amino]-carbonyl]amino]-3,3-dimethyl-
1-oxobutyl]-N-[1-[1,2-dioxo-2-[(2-thienylmethyl)amino]ethyl]pen-
tyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-carboxamide (37).
1
The product was isolated as a mixture of two diastereomers. H
NMR (500 MHz, DMSO-d6) δ 9.33 and 9.28 (d, J ) 7.56 Hz, 1
H), 8.37and 8.29 (d, J ) 7.25 Hz, 1 H), 7.39 (d, J ) 5.04 Hz, 1
H), 6.97-6.94 (m, 2 H), 6.14 (t, J ) 12.61 Hz, 1 H), 5.79 (d, J )
10.71 Hz, 1 H), 5.01-4.97 and 4.89-4.84 (m, 1 H), 4.47-4.08
(m, 4 H), 3.91-3.56 (m, 5 H), 2.45-2.36 (m, 4 H), 1.74-1.15
(m, 8 H), 0.98-0.93 (m, 9 H), 0.86 (s, 9 H), 0.82-0.74 (m, 15 H).
HRMS calcd for C40H62N6O7S, 771.4479 (M + H)+; found,
771.4478.
(1R,5S)-3-[2(S)-Cyclohexyl-2-[[[[1(S)-[(4,4-dimethyl-2,6-dioxo-1-
piperidinyl)methyl]-2,2-dimethylpropyl]amino]-carbonyl]ami-
no]acetyl]-N-[1-[1,2-dioxo-2-(2-propenyl-amino)ethyl]pentyl]-6,6-
dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-carboxamide (39). The
1
product was isolated as a mixture of two diastereomers. H NMR
(500 MHz, DMSO-d6) δ 8.86 and 8.81 (t, J ) 6.15 and 5.99 Hz,
1 H), 8.36 and 8.31 (d, J ) 6.93 and 7.25 Hz, 1 H), 6.12 (t, J )
9.93 Hz, 1 H), 5.82-5.74 (m, 2 H), 5.11-5.03 (m, 2 H), 4.98-4.93
and 4.87-4.83 (m, 1 H), 4.25 (d, J ) 5.36 Hz, 1 H), 4.09 (q, J )
4.10 and 5.68 Hz, 1 H), 3.88 (q, J ) 7.88 and 2.52 Hz, 1 H),
3.82-3.64 (m, 5 H), 3.58 (d, J ) 12.61 Hz, 1 H), 2.41 (q, J )
15.76 Hz and 7.88 Hz, 4 H), 1.72-1.62 (m, 1 H), 1.53-1.14 (m,
6 H), 0.98-0.93 (m, 8 H), 0.85-0.74 (m, 24 H). 13C NMR (125
MHz, DMSO-d6) δ 197.23, 196.61, 171.68, 171.67, 171.02, 170.81,
170.65, 170.49, 161.05, 160.67, 158.04, 157.99, 134.08, 134.03,
1
product was isolated as a mixture of two diastereomers. H NMR
(500 MHz, DMSO-d6) δ 9.88 and 8.84 (d, J ) 5.99 and 6.30 Hz,
1 H), 8.38 and 8.24 (d, J ) 7.56 and 6.62 Hz, 1 H), 6.05 (t, J )
7.56 Hz, 1H), 5.83-5.75 (m, 1H), 5.64-5.61 (m, 1 H), 5.10-5.04
(m, 2 H), 4.93-4.89 (m, 1 H), 4.23 (d, J ) 12.92 Hz, 1 H),
4.00-3.57 (m, 8 H), 2.45-2.36 (m, 4 H), 1.72-1.07 (m, 19 H),
0.98-0.76 (m, 22 H). 13C NMR (125 MHz, DMSO-d6) δ 198.18,
197.67, 172.72, 172.01, 171.77, 171.65, 161.87, 161.63, 158.58,

1378 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Chen et al.
115.48, 115.39, 114.97, 68.39, 59.14, 58.88, 56.98, 56.89, 55.72,
54.78, 54.73, 53.37, 53.33, 47.23, 45.41, 40.71, 38.66, 38.59, 34.14,
34.11, 34.00, 33.97, 31.52, 30.52, 30.50, 29.48, 28.25, 27.11, 26.82,
26.74, 26.24, 26.22, 26.00, 25.97, 18.59, 18.44, 18.33, 18.32, 13.46,
13.35, 12.50, 12.46. HRMS calcd for C37H60N6O7, 701.4602 (M +
H)+; found, 701.4617.
1.72-1.23 (m, 6 H), 1.03(s, 3 H), 0.97-0.86 (m, 24 H), 0.76 (bs,
3 H), 0.41 (d, J ) 8.05 Hz, 2 H), 0.06-0.0 (m, 2 H).
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(1R,5S)-N-[1-(Cyclobutylmethyl)-2,3-ioxo-3-(2-propenylamino)-
propyl]-3-[2(S)-[[[[1(S)-[(4,4-dimethyl-2,6-dioxo-1-piperidinyl)-
methyl]-2,2-dimethylpropyl]amino]carbonyl]amino]-3,3-dimethyl-
1-oxobutyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-carbox-
amide (45). The product was obtained as a mixture of two
diastereomers. 1H NMR (500 MHz, DMSO-d6) δ 8.86 and 8.83 (t,
J ) 13.24 and 5.99 Hz, 1 H), 8.33 and 8.29 (d, J ) 8.82 and 7.25
Hz, 1 H), 6.14 (d, J ) 12.61 Hz, 1 H), 5.78-5.75 (m, 2 H),
5.11-5.03((m, 2 H), 4.94-4.90 and 4.78-4.75 (m, 1 H), 4.24-4.23
(m, 1 H), 4.11-4.08 (m, 2 H), 3.90-3.56 (m, 6 H), 2.48-2.34
(m, 4 H), 1.97-1.91 (m, 2 H), 1.79-1.72 (m, 3 H), 1.65-1.57
(m, 3 H), 1.39-1.37(m, 1 H), 1.24-1.14 (m, 2 H), 0.98-0.75 (m,
31 H). 13C NMR (125 MHz, DMSO-d6) δ 198.14, 197.50, 172.67,
172.66, 171.85, 171.67, 171.65, 171.46, 161.84, 161.54, 159.03,
158.96, 135.06, 135.01, 116.47, 116.39, 60.07, 59.89, 57.95, 57.85,
55.74, 55.69, 53.25, 53.03, 48.19, 46.36, 41.70, 37.59, 37.38, 35.12,
35.10, 34.97, 34.94, 33.03, 32.96, 31.46, 31.38, 29.24, 28.80, 28.62,
28.24, 28.22, 28.08, 27.83, 27.71, 27.20, 26.95, 19.30, 18.73, 18.56,
13.47, 13.44. HRMS calcd for C39H62N6O7, 727.4758 (M + H)+;
found, 727.4772.
(1R,5S)-N-[1-(Cyclopropylmethyl)-2,3-dioxo-3-(2-propenylamino)-
propyl]-3-[2(S)-[[[[1(S)-[(4,4-dimethyl-2,6-dioxo-1-piperidinyl)-
methyl]-2,2-dimethylpropyl]amino]-carbonyl]amino]-3,3-dimethyl-
1-oxobutyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-carbox-
amide (46). The product was isolated as a mixture of two
diastereomers. 1H NMR (500 MHz, DMSO-d6) δ 8.84 and 8.80 (t,
J ) 6.15 Hz, 1 H), 8.41 and 8.38 (d, J ) 6.62 and 6.94 Hz, 1 H),
6.12 (t, J ) 11.19 Hz, 1 H), 5.81-5.73 (m, 2 H), 5.10-4.94 (m,
3 H), 4.26 (s, 1 H), 4.12-4.08 (m, 1 H), 3.88 (t, J ) 9.14 Hz, 1
H), 3.81-3.55 (m, 7 H), 2.41 (q, J ) 16.39 and 11.35 Hz, 4 H),
1.72-1.67 and 1.63-1.57 (m, 1 H), 1.55-1.36 (m, 2 H), 1.29 and
1.25 (d, J ) 7.57 and 7.88 Hz, 1 H), 0.97-073 (m, 30 H), 045-0.33
(m, 2 H), 0.12-0.0 (m, 2 H). 13C NMR (125 MHz, DMSO-d6) δ
197.08, 196.53, 171.04, 170.77, 170.76, 170.60, 160.85, 160.46,
158.12, 158.08, 134.13, 134.09, 115.59, 115.53, 59.15, 58.95, 57.06,
56.93, 54.87, 54.84, 54.42, 54.38, 47.33, 45.47, 40.80, 34.98, 34.83,
34.21, 34.19, 34.08, 34.05, 30.62, 30.50, 28.33, 27.19, 26.83, 26.77,
26.31, 26.67, 26.09, 26.05, 18.41, 18.39, 12.58, 12.55, 7.86, 7.63,
4.99, 4.33, 3.99. HRMS calcd for C38H60N6O7, 713.4602 (M + H)+;
found, 713.4612.
(1R,5S)-N-[1-(2-Cyclopropylethyl)-2,3-dioxo-3-(2-propenylamino)-
propyl]-3-[2(S)-[[[[1(S)-[(4,4-dimethyl-2,6-dioxo-1-piperidinyl)-
methyl]-2,2-dimethylpropyl]amino]-carbonyl]amino]-3,3-dimethyl-
1-oxobutyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2(S)-carbox-
amide (47). The product was isolated as a mixture of two
(9) 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.;
1
diastereomers. H NMR (300 MHz, CDCl3) δ 7.99-7.64 (m, 2
H), 5.88-5.77 (m, 1 H), 5.72-5.66 (m, 1 H), 5.63-5.56 (m, 1 H),
5.24-5.13 (m, 2 H), 5.05-4.99 (m, 1 H), 4.54-4.40 (m, 2 H),
4.04-3.81 (m, 7 H), 2.53-2.41 (m, 4 H), 2.12-2.04 (m, 1 H),

Second-Generation Inhibitors of HCV NS3 Serine Protease
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1379
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