Potent and selective small molecule NS3 serine protease inhibitors of Hepatitis C virus with dichlorocyclopropylproline as P2 residue

Article abstract of DOI:10.1016/j.bmc.2007.11.002

Starting from a pentapeptide Hepatitis C virus NS3 protease inhibitor, a number of α-ketoamide inhibitors based on novel dichlorocyclopropylproline P2 core were synthesized and investigated for their HCV NS3 serine protease activity. The key intermediate 3,4-dichlorocyclopropylproline was obtained through a dichloro carbene insertion to 3,4-dehydroproline. The size of the molecules was reduced significantly through a series of truncations of the initial pentapeptide. By varying P1 side chain in length and size, potency and selectivity were improved. A variety of aliphatic carbamate and urea capping groups were examined. In general, compounds with urea cappings were more potent and selective than their carbamate counterparts. The most potent compound was a tert-butyl urea analog. Variations at P3 position were also investigated. Among the three residues incorporated, tert-leucine was clearly superior, leading to compounds that had excellent enzyme potency and selectivity. The most potent compound achieved cell-based replicon assay EC₅₀ of 40 nM. The most promising compound of all had excellent potency in both enzyme (K_i^* = 9 nM) and replicon assays (EC₅₀ = 100 nM). Its bioavailabilities were above 10% in all three animal species (rats, monkeys, and dogs). It has provided a lead for future investigations.

Full text of DOI:10.1016/j.bmc.2007.11.002

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1874–1883
Potent and selective small molecule NS3 serine protease
inhibitors of Hepatitis C virus with dichlorocyclopropylproline
as P2 residue
Kevin X. Chen,* Bancha Vibulbhan, Weiying Yang, Kuo-Chi Cheng, Rong Liu,
John Pichardo, Nancy Butkiewicz and F. George Njoroge
Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-3-3545, Kenilworth, NJ 07033, USA
Received 16 August 2007; revised 27 October 2007; accepted 1 November 2007
Available online 5 November 2007
Abstract—Starting from a pentapeptide Hepatitis C virus NS3 protease inhibitor, a number of a-ketoamide inhibitors based on
novel dichlorocyclopropylproline P2 core were synthesized and investigated for their HCV NS3 serine protease activity. The key
intermediate 3,4-dichlorocyclopropylproline was obtained through a dichloro carbene insertion to 3,4-dehydroproline. The size
of the molecules was reduced significantly through a series of truncations of the initial pentapeptide. By varying P1 side chain in
length and size, potency and selectivity were improved. A variety of aliphatic carbamate and urea capping groups were examined.
In general, compounds with urea cappings were more potent and selective than their carbamate counterparts. The most potent com-
pound was a tert-butyl urea analog. Variations at P3 position were also investigated. Among the three residues incorporated, tert-
leucine was clearly superior, leading to compounds that had excellent enzyme potency and selectivity. The most potent compound
achieved cell-based replicon assay EC50 of 40 nM. The most promising compound of all had excellent potency in both enzyme
(Kꢀi = 9 nM) and replicon assays (EC50 = 100 nM). Its bioavailabilities were above 10% in all three animal species (rats, monkeys,
and dogs). It has provided a lead for future investigations.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
The HCV RNA genome is processed by host and viral
proteases into structural and nonstructural (NS) poly-
peptides.3 The NS3 protease is located at N-terminal
portion of NS3 protein. It is responsible for processing
four cleavage sites of the nonstructural region and is
essential for viral replication.4 It has been determined
that the HCV NS3 protease belongs to the trypsin or
chymotrypsin superfamily of serine proteases.5 For effi-
cient processing, the protease forms a complex with a
small polypeptide co-factor NS4A.6 The structural data
of the protease have revealed a shallow and solvent ex-
posed substrate binding region, where the binding en-
ergy is mainly derived from weak lipophilic and
electrostatic interactions.7 Intensive efforts have been fo-
cused on NS3 serine protease as a target. Despite tre-
mendous difficulties encountered in the research, a
number of novel inhibitors have been reported.8
Hepatitis C virus (HCV) is a human pathogen affecting
nearly 170 million, or 3% of the world’s population.
HCV infection is described as a ‘silent epidemic’ because
only about 20% of patients develop acute clinical hepa-
titis. In most cases, chronic progress of the infection
leads to cirrhosis, liver failure or liver cancer.1 Cur-
rently, the most effective therapies for HCV infection in-
volve treatment with pegylated a-interferon, either alone
or in combination with antiviral agent ribavirin.2 These
therapeutics have limited efficacy with sustained re-
sponse rate of only about 50%. They are also accompa-
nied by considerable side effects in certain patients.
More effective antiviral agents for HCV with fewer side
effects are in urgent need.
Similar to other proteases, the products from the cleav-
age of the substrates by HCV NS3 protease have been
shown to be competitive inhibitors.9 The hexapeptide
products have been used as leads to develop more potent
and smaller HCV inhibitors. Inhibitors with diverse
Keywords: HCV; Protease inhibitor; Ketoamide; Peptide.
*
Corresponding author. Tel.: +1 908 740 7556; fax: +1 908 740
7152; e-mail: kevin.chen@spcorp.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.002

K. X. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1874–1883
1875
structure types have been discovered.8 In one class of
2. Synthesis of HCV protease inhibitors
compounds, a reactive electrophile that can form a
covalent bond with active site serine hydroxyl has been
introduced. These compounds, exemplified by a-ketoac-
ids and ketoamides, have demonstrated their potential
use as hepatitis C therapeutics. VX-950 and SCH-
503034, two examples in this class, are in clinical devel-
opment.10 In the course of our search for potent HCV
inhibitors, we discovered a pentapeptide a-ketoamide 1
(Fig. 1). Compound 1 had excellent potency against
NS3 protease (Kꢀi = 3.8 nM) and in cell-based replicon
assay (EC50 = 40 nM).11 However, its pharmacokinetic
(PK) properties were poor: 4% and 1% bioavailability
in rats and monkeys, respectively. To improve PK pro-
file of this type of peptic molecules, it was desirable to
reduce molecular weight through truncation of the pen-
tapeptide to a smaller tetra- or tripeptide. The result of
our early effort was the discovery of the current clinical
candidate SCH 503034 (Boceprevir, Fig. 1), which had
decent potency and good PK. Parallel to the SAR devel-
opment using dimethylcyclopropylproline as P2 residue,
the analogs with alternative P2 moieties were also inves-
tigated. Since the dimethylcyclopropylproline P2 had
been demonstrated to be vital for potency,11 we decided
to structurally stay as close to this core as possible. It
was envisioned that the two methyl groups at P2 could
be replaced by two chloro substituents. The chloro
groups resemble the steric bulk of the methyls, but have
different electronic characteristics. They could poten-
tially provide improved potency and/or better PK. We
believe that the SAR of inhibitors using this new di-
chloro P2 residue should follow the same pattern as
those using dimethyl P2 moiety, because modifications
at other sites are similar. Herein, we report our effort
and progress in these endeavors.
The general synthesis of the HCV protease inhibitors is
summarized in Scheme 1. Preparation of the 3,4-trans-
dichlorocyclopropylproline P2 core was the key step in
the synthesis. Thus, N-Boc-3,4-dehydroproline 2 was
converted to its corresponding tert-butyl ester.12 The
cyclopropanation was achieved through the addition
of dichloro carbene13 onto the dehydroproline to give
the desired product 3. The reaction was conducted in a
bi-phasic system of chloroform and 50% aqueous so-
dium hydroxide. The cyclopropanation occurred exclu-
sively trans to the tert-butyl ester moiety as evidenced
by proton NMR analysis. However, the C-2 stereocenter
of proline was partially racemized under the strongly ba-
sic conditions used during the course of the reaction. Re-
moval of the Boc group, cleavage of the tert-butyl ester
and formation of a methyl ester were accomplished in
one pot through refluxing in methanolic hydrochloric
acid to provide the key intermediate 4. Standard cou-
pling (HOOBt, EDC, NMM) between 4 and a N-Boc-
protected amino acid P3 afforded the dipeptide 5. The
desired compound with the right P2 stereocenter was
isolated as the major isomer out of the two diastereo-
mers of 5. Hydrolysis of the methyl ester provided the
carboxylic acid of type 6 which served as the left-hand
side section core the inhibitor.
Preparation of the right-hand fragments of the inhibi-
tors (from P1 ketoamide up to P20) has been described
in earlier publications.14 Coupling of the carboxylic acid
Cl Cl
a, b
c
CO2H
N
Boc
CO2tBu
N
Boc
3
2
P2'
P2
Cl Cl
Cl Cl
O
O
CO2R
N
d
H
H
N
N
NMe2
BocHN
N
N
O
H
CO2Me
N
H
O
O
N
O
O
R3
R = Me, 5
R = H,
O
H.HCl
O
e
P1'
4
6
P1
Cap
P3
Cl Cl
OH
HCl.H2N
R'
1
Ki* = 3.8 nM, EC50 = 40 nM
O
H
R1
O
7
N
R'
N
H
O
N
O
O
H
R1
O
Cap
N
NH2
f, (g, h), i
R3
N
H
H
O
N
O
N
8-26
O
O
Scheme 1. Reagents and conditions: (a) (Boc)2O, DMAP, CH3CN; (b)
CHCl3, 50% NaOH, 40%; (c) HCl, MeOH, quant.; (d) appropriate N-
Boc-amino acid, HOOBt, EDC, NMM, DMF/CH2Cl2, 50–95%; (e)
LiOH, THF/MeOH/water; (f) 6, HOOBt, EDC, NMM, DMF/
CH2Cl2, 50–95%; (g) 4 M HCl, dioxane; (h) appropriate isocyanate,
saturated NaHCO3/CH2Cl2, 40–75%; (i) Dess–Martin periodinane,
CH2Cl2, 60–90%; or DMSO, Cl2CHCO2H, EDC, toluene, 45–70%.
SCH 503034 (Boceprevir)
Ki* = 14 nM, EC50 = 200 nM
Figure 1. Pentapeptide HCV protease inhibitor 1 and SCH 503034
(Boceprevir).

1876
K. X. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1874–1883
Table 1. P0 and P3-capping variations of the inhibitors
6 with the amine 7 afforded a series of a-hydroxyamide
intermediates, which upon oxidation using Dess–Martin
periodinane15 (for secondary amides) or a modified
Moffatt protocol16 (for primary amides) gave rise to
the desired final target a-ketoamides 8–26.
Cl Cl
O
H
N
R
N
H
X
N
O
O
O
3. Results and discussion
3.1. Enzyme assay and selectivity against HNE
8-12
Compound
X
O
R
Kiꢀ
(nM)
HNE/HCV
All the inhibitors described above were tested in HCV
continuous assay17 using theꢀNS4A-tethered single chain
NS3 serine protease.18 The Ki values in the assay reflected
the equilibrium constant determined by the reversible
covalent bond formed between the ketone and serine hy-
droxyl group and by other interactions between the inhib-
itors and the enzyme.19 All compounds were measured as
a mixture of two diastereomers at P1 a-center. The ratio of
the two isomers varied slightly based on specific com-
pound. In cases where single isomers were obtained, race-
mization at P1 was observed for most of them during
in vitro enzyme assay and in vivo pharmacokinetics stud-
ies. The selectivity of HCV serine protease inhibitor
against other serine proteases was an important consider-
ation since it is relevant to potential side effect of the ther-
apy. In this case, the selectivity was measured against
human neutrophil elastase (HNE), which is a serine pro-
tease structurally closely related to HCV NS3 serine pro-
tease. Our goal was to achieve a selectivity index of one
hundred or greater.
Ph
O
H
N
NMe2
8
15
4
N
H
O
H
N
9
O
90
1
4
10
NH
100
11
12
O
NH
36
20
3
9
NH2
between a primary amide N–H and the backbone. These
results demonstrated that truncation was feasible and
smaller molecule inhibitors with reasonable potency
were achievable.
3.3. P1 side chain variations
3.2. The P0 side SAR
Modifications were also made at P1 position. The S1
pocket has been shown to be small but lipophilic. It
can only accommodate short alkyl straight chains or
small ring systems. Thus, the n-propyl of norvaline P1
was expanded to cyclopropylmethyl, cyclobutylmethyl,
and butenyl groups. Inhibitors with varying number of
amino acid residues were prepared (Table 2). For penta-
peptide series (8, 13, and 14), from P1 norvaline
(Kꢀi = 15 nM) to P1 cyclopropyl alanine (7 nM), a two-
fold improvement in potency was obtained. A Kꢀi of
9 nM was observed for P1 cyclobutylalanine analog
14. However, the selectivity against elastase improved
dramatically from 8, 13 to 14 (HNE/HCV = 4, 70, 280,
respectively) with the bulkier cyclobutylalanine having
the best selectivity. The two allyl amide compounds (9
and 15) had norvaline and butenyl glycine as P1. The
longer butenyl group had certain advantages in potency
(44 nM vs 99 nM) and selectivity (6 vs 1). The primary
amide series (11, 16–17) had similar trend in potency
and selectivity to two series discussed above. The larger
or longer side chain at P1 gave better potency and selec-
tivity. The analog with cyclopropylalanine P1 had a Kiꢀ
of 11 nM and selectivity of 23, while those for cyclobu-
tylalanine derivative were 17 nM and 200, respectively.
These data clearly indicated that cyclopropylalanine
was the group that gave best potency at P1 position.
On the other hand, cyclobutylalanine P1 analogs were
the most selective against elastase. The results also dem-
onstrated that the tripeptide primary amides (11, 16–17)
were equally potent and selective compared with larger
pentapeptide analogs (8, 13–14).
First, we explored the effect of the truncation on potency
at P0 side of the pentapeptide (8–12). The enzyme assay
results for these compounds are presented in Table 1.
Similar to compound 1, inhibitor 8 is a pentapeptide
with a glycine-phenylglycine dipeptide piece at the prime
side. It was a potent inhibitor of HCV protease with Kiꢀ
of 15 nM. The selectivity (HNE/HCV) was modest at 4.
Compound 9 (tert-butyl carbamate capped) and com-
pound 10 (tert-butyl urea capped), both truncated as al-
lyl tripeptides, demonstrated significantly diminished
potency with Kꢀi of 90 and 100 nM, respectively. Surpris-
ingly, if the molecule was further shortened to a primary
amide such as that in compounds 11 and 12, the potency
was regained. Thus, the two short tripeptides had Kꢀi of
36 and 20 nM for tert-butyl carbamate and urea capped
inhibitors, respectively. In both allyl and primary amide
pairs, the tert-butyl urea capped inhibitors were more
selective than the tert-butyl carbamate capped analogs.
From X-ray and modeling analysis, the good potency
of compounds 1 and 8 came in part from the extra
hydrogen-bonding of P20 amide N–H and hydrophobic
interaction between the phenyl group and the backbone
Lys136 side chain. The loss of these bindings and the
unfavorable steric interaction between the allyl group
and the Gln41 side chain in allyl amide analogs 9 and
10 explained their lack of potency. The tripeptide pri-
mary amides (11 and 12) regained most of the potency
probably because they did not have unfavorable steric
contacts with Gln41 and a stronger hydrogen bonding

K. X. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1874–1883
1877
Table 2. P1 side chain modifications
3.5. SAR of P3 amino acid residue
Cl Cl
The S3 region of HCV NS3 protease is shallow but
highly lipophilic, thus its P3 group binds to the enzyme
surface through hydrophobic interactions. From our
earlier experience, side chains that had sizable alkyl or
aromatic hydrocarbon moieties interacted favorably
with S3 pocket.11 Three amino acid residues were exam-
ined at P3 position: cyclohexylglycine, tert-leucine, and
2-indanylglycine (Table 4). They were capped either
with a Boc or a tert-butyl urea group, which, as previ-
ously shown, had proved to be the best groups at that
position. The results for compounds (17 and 20) with
cyclohexylglycine P3 were discussed earlier. When tert-
butylglycine was used as P3 residue, both Boc and
tert-butyl urea capped compounds (23–24) had excellent
potency (Kꢀi of 9 and 7 nM, respectively). Interestingly,
both compounds exhibited great selectivity against elas-
tase (HNE/HCV = 2800 and 2400, respectively). The po-
tency and selectivity achieved by these small molecules
were remarkable. On the other hand, with the larger
aromatic indanylglycine at P3 position, both potency
and selectivity were not as good as the other two groups,
with Kꢀi 4- to 10-fold higher and HNE/HCV 3- to
40-fold lower.
O
H
N
R
N
H
O
O
N
O
R1
O
O
8, 9, 11, 13-17
Compound R1
R
Kiꢀ
(nM) HCV
HNE/
8
15
7
4
70
280
1
Ph
O
H
N
NMe2
13
14
9
N
H
O
9
90
44
36
11
17
3.6. Cell-based replicon assay potency and pharmacoki-
netic properties of representative compounds
H
N
15
11
16
17
6
The potency of inhibitors as measured in cell-based rep-
licon assay20 has been a very important criterion in
selecting the compounds for further advancement. Due
to the lack of a good animal model to evaluate HCV
NS3 protease, it is the next best assessment of the poten-
tial of all these compounds as antiviral agents. A num-
ber of the compounds described above were tested in
this assay. Results from these studies on selected com-
pounds are listed in Table 5. Compounds 16 and 17 were
Boc-capped primary a-ketoamide inhibitors that dif-
fered at P1 residue. They had relatively similar potency
and PK profiles. They had good Kꢀi of 11 and 17 nM,
and good replicon EC50 of 500 and 300 nM, respec-
tively. Compound 17 had better selectivity than 16
(HNE/HCV, 200 vs 23). They both had good rat PO
AUC (2.7 and 2.4 lM h, respectively) and 16 was mod-
erately bioavailable in rats (26%). Unfortunately, they
both had poor PK in monkey and dogs with PO AUC’s
at 0.1 lM h or less and bioavailability of less than 10%.
Primary a-ketoamides 19 and 20 had cyclobutylalanine
at P1 and differed at P3 capping site. One was norbornyl
carbamate (19) and the other was tert-butyl urea (20).
The latter was better in enzyme potency (60 nM vs
6 nM) and selectivity (15 vs 460) but had no advantage
in replicon potency (200 nM vs 300 nM). Inhibitor 21
had the best replicon potency (EC50 = 40 nM) but did
not give any rat PO AUC in PK study. The last two
compounds (23 and 24) were the most interesting of
all. With tert-leucine as P3 residue, they were both extre-
mely potent with single digit nanomolar potency in
enzymatic assay. Both also had excellent selectivity
against elastase. In replicon assay, compound 23 was
better than 24, with EC50 of 100 nM. Compound 24 is
similar in structure to SCH 503034. It was more potent
3
NH2
23
200
3.4. P3 capping modifications
Based on the results from P0 and P1 investigations, we
decided to focus our SAR development on primary keto-
amides with cyclobutylalanine substituent at P1. The ef-
fect of P3 capping groups was explored next. Thus,
several alkyl groups were incorporated to P3 nitrogen
through either a carbamate or a urea connection (Table
3). Compounds 17–19 were carbamate capped analogs.
The trifluoro-Boc 18 and norbonyloxycarbonyl 19
capped derivatives (Kꢀi of 65 and 60 nM, respectively)
were less potent than Boc capped inhibitor 17 (17 nM).
They were also less selective than 17. On the other hand,
all three urea capped compounds (20–22) were quite po-
tent with Kꢀi between 6 and 15 nM. The tert-butyl urea
analog 20 was the most potent of all the inhibitors. Com-
pounds 20 and 21 also had excellent selectivity (HNE/
HCV = 460 and 570, respectively). With the improved
potency, we clearly achieved our original goal of truncat-
ing pentapeptide 1 and maintaining its potency.

1878
K. X. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1874–1883
Table 3. Capping modifications
PK profile, with bioavailability above 10% in all three
animal species (rat, monkey, and dog). Although the
AUC’s were moderate, it had an impressive 50% bio-
availability in monkeys. Compound 23 had much-im-
proved PK compared to 1. It was more potent than
SCH 503034 in both enzyme and cell-based replicon as-
says. It has become a lead for future SAR development.
Cl Cl
O
H
N
NH2
N
H
N
O
Cap
O
O
O
17-22
4. Conclusion
Compound
Cap
Kꢀi (nM)
HNE/HCV
200
In summary, a number of a-ketoamide inhibitors based
on dichlorocyclopropylproline P2 core were synthesized
and investigated for their HCV NS3 serine protease
activity. The size of the molecules was reduced signifi-
cantly through a series of truncations of the initial lead
pentapeptide 1. The P1 primary ketoamides were found
to be potent inhibitors. The P1 residue was modified
from norvaline to cyclopropylalanine, and to cyclobu-
tylalanine. Both potency and selectivity were improved
as a result of these modifications. A group of aliphatic
carbamate and urea capping groups were examined. In
general, the urea cappings were more potent than
carbamate ones. The most potent compound (20) was
a tert-butyl urea analog. Variations at P3 were also
investigated. Among the three residues (cyclohexylgly-
cine, tert-leucine, and indanylglycine) incorporated,
compounds with tert-leucine P3 (23 and 24) were clearly
superior in potency and selectivity. Selected compounds
were evaluated in cell-based replicon assay and in PK
studies. Inhibitors 16 and 17 had good PO AUC’s in
rats, but were poor in monkeys and dogs. Compound
21 was the most potent in replicon with EC50 of
40 nM. Compound 23 was the most promising of all
the inhibitors, had better PK than compound 1, and
was more potent than SCH 503034. It had excellent po-
tency in both enzyme (Kiꢀ = 9 nM) and replicon assays
(EC50 = 100 nM). It also demonstrated good overall
PK profile with bioavailability above 10% in all three
species (rat, monkey, and dog). This compound is a lead
for our future investigations.
O
O
17
17
18
19
20
65
60
6
80
15
CF3
O
H
N
460
H
N
21
22
10
15
570
80
H
N
Table 4. SAR at P3 position
Cl Cl
O
O
H
N
NH2
N
H
O
X
N
O
O
R3
17, 20, 23-26
Kꢀi (nM)
HNE/HCV
5. Experimental
Compound
X
R3
5.1. General methods
17
20
23
O
17
200
460
Reagents and solvents, including anhydrous THF,
dichloromethane, and DMF, were purchased from Al-
drich or other commercial sources and were used without
further purification. Reactions that were moisture sensi-
tive or using anhydrous solvents were performed under
either a nitrogen or an argon atmosphere. Analytical thin
layer chromatography (TLC) was performed on pre-
coated silica gel plates obtained from Analtech. Visuali-
zation was accomplished with UV light or by staining
with basic KMnO4 solution, ethanolic H2SO4 or Vau-
ghn’s reagent. Compounds were purified by flash chroma-
tography either on a glass column using Merck silica gel
60 (230–400 mesh) or on an ISCO RediSep disposable sil-
ica gel column. NMR spectra were recorded at 300, 400 or
500 MHz for 1H and at 75, 100 or 125 MHz for 13C on a
Bruker or Varian spectrometer with CDCl3 or d6-DMSO
NH
O
6
9
2800
24
25
NH
O
7
2400
60
70
26
NH
30
170
than SCH 503034 in enzyme assay, but equally potent in
replicon assay. It had poor PK in rats. On the other
hand, compound 23 demonstrated a superior overall

K. X. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1874–1883
Table 5. Cell-based replicon assay potency and PK of selected compounds
1879
Compound
16
17
19
20
21
23
24
Kꢀi (nM)
EC50 (nM, Replicon)
HNE/HCV
11
17
60
6
10
40
9
7
200
500
23
300
200
200
15
300
460
100
2800
570
2400
Rats
PO AUC (lM h)
2.7a
10.7a
4.4
2.4a
0.75a
0.32a
0.0a
0.30a
2.6a
5.6
0.12f
1.9g
2.7
h
h
h
h
IV AUC (lM h)
t1/2 (h)
F% (PO)
h
h
h
h
h
h
h
h
26%
12%
5%
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
Monkeys
Dogs
PO AUC (lM h)
IV AUC (lM h)
t1/2 (h)
0.13b
1.2c
9.6
0.06d
0.6e
1.9
0.77b
1.2c
1.1
F% (PO)
8%
4%
50%
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
h
PO AUC (lM h)
IV AUC (lM h)
t1/2 (h)
0.07b
2.6c
16
0.10d
2.0e
4.7
0.61b
4.5c
6.3
F% (PO)
2%
2%
10%
a 10 mg/kg.
b 3 mg/kg.
c 2.2 mg/kg.
d 1.5 mg/kg.
e 0.6 mg/kg.
f 7.5 mg/kg.
g 6 mg/kg.
h Data not obtained.
as solvent. The chemical shifts are given in ppm, refer-
enced to the internal TMS or deuterated solvent signal.
concentrated under reduced pressure. The crude product
was purified by flash chromatography to give the desired
a-ketoamide in 60–90% yield.
5.2. General method for coupling of an N-Boc amino acid
and an amine
5.4. 6,6-Dichloro-3-aza-bicyclo[3,1,0]hexane-2,3-dicar-
boxylic acid di-tert-butyl ester (3)
To a solution of the N-Boc-amino acid (1.0 mmol),
amine methyl ester hydrochloride (1.2 mmol), 3-hydro-
xy-1,2,3-benzotriazin-4(3H)-one (HOOBt) (1.2 mmol),
and N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride (EDC) (1.3 mmol) in DMF/CH2Cl2 (1:1,
100 mL) at ꢁ20 ꢁC was added N-methylmorpholine
(NMM) (4.5 mmol). After stirred at this temperature
for 30 min, the mixture was stored in a refrigerator over-
night (16 h). Then 5% aqueous H3PO4 solution (50 mL),
brine (50 mL), and EtOAc (150 mL) were added. After
two layers were separated, the organic solution was
washed with 5% H3PO4 (150 mL), saturated aqueous so-
dium bicarbonate solution (2· 150 mL), water (150 mL),
and brine (150 mL). It was then dried (magnesium sul-
fate), filtered, and concentrated under reduced pressure.
The crude product was purified by flash chromatogra-
phy (0–60%) eluting with acetone/hexanes to give the de-
sired product in 50–95% yield.
The N-Boc-3,4-dehydroproline 2 was converted to its
tert-butyl ester according to a literature procedure.12
The tert-butyl ester (1.0 mmol) was dissolved in chloro-
form (20 mL). To the mixture was added 50% aqueous so-
dium hydroxide solution (20 mL). The bi-phasic mixture
was vigorously stirred at rt for 18 h. Dichloromethane
(50 mL) and water (50 mL) were added and layers were
separated. The aqueous solution was extracted with
dichloromethane (2· 50 mL). The combined organic
solution was dried (magnesium sulfate), filtered, and con-
centrated under reduced pressure. The crude product was
purified by flash chromatography to give product 3 (40%
yield). Two sets of peaks were observed due to the exis-
1
tence of two rotamers. H NMR (500 MHz, d6-DMSO)
d 4.38 and 4.25 (s, 1H), 3.76–3.75 and 3.72–3.70 (m,
2H), 2.34–2.31 (m, 2H), 1.47 and 1.46 (s, 9H), 1.41 and
1.38 (s, 9H). 13C NMR (125 MHz, d6-DMSO) d 169.34,
169.32, 153.20, 152.76, 82.29, 82.21, 80.44, 80.33, 62.58,
61.49, 47.54, 47.46, 37.53, 36.72, 36.58, 34.03, 33.25,
28.26, 28.20, 28.06, 27.92, 24.62. HR-MS Calcd for
C15H23NO4Cl2: 352.1082 (M+H)+. Found: 352.1085.
5.3. General method for Dess–Martin periodinane
oxidation
To a solution of a-hydroxyamide (1.0 mmol) in dichlo-
romethane (50 mL) was added Dess–Martin period-
inane (2.0 mmol). The mixture was stirred at rt for 3 h.
Saturated aqueous sodium thiosulfate solution (50 mL)
was added and the mixture stirred for 5 min. The layers
were separated, the aqueous solution was extracted with
dichloromethane (2· 50 mL). The combined organic
solution was dried (magnesium sulfate), filtered, and
5.5. 6,6-Dichloro-3-aza-bicyclo[3.1.0]hexane-2-carboxylic
acid methyl ester (4)
The N-Boc proline tert-butyl ester (0.4 mmol) was dis-
solved in methanol (30 mL) and 1 N aqueous HCl solu-
tion (0.6 mL) was added. The resulted mixture was
heated to reflux in an oil bath for 18 h before it was

1880
K. X. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1874–1883
concentrated under reduced pressure to give proline
methyl ester hydrochloride 4. H NMR (500 MHz, d6-
(m, 1H), 1.73–1.42 (m, 9H), 1.34 (d, J = 3.1 Hz, 9H),
1.15–1.08 (m, 3H), 0.91–0.85 (m, 5H). 13C NMR
(125 MHz, d6-DMSO) d 196.15, 196.00, 169.15, 166.82,
166.77, 155.39, 154.08, 137.42, 137.37, 128.51, 127.77,
127.70, 77.80, 61.19, 56.50, 53.78, 53.36, 52.89, 41.46,
36.50, 36.4, 35.33, 33.50, 31.61, 28.70, 28.45, 28.03,
18.54, 13.48, 13.36. HR-MS Calcd for C37H52N6O8Cl2:
779.3302 (M+H)+. Found: 779.3304.
1
DMSO) d 4.42 (d, J = 1.8 Hz, 1H), 3.8 (ddd, J = 2.8,
9.0, 13.2 Hz, 1H), 3.8 (s, 3H), 3.34 (dd, J = 1.85, 3.77
and 1.8, 3.8, 1 H), 3.12 (d, J = 2.01, 1.60, 1H), 3.06 (d,
J = 1.6 and 1.8, 1H), 2.97–2.94 (m, 1H). 13C NMR
(125 MHz, d6-DMSO) d 167.5, 61.7, 61.4, 53.5, 47.5,
37.8, 35.3. HR-MS Calcd for C7H10NO2: 210.0090
(M+H)+. Found: 210.0090.
5.10. 1,1- Dimethylethyl [1-cyclohexyl-2-[(1S,5R)-6,6-di
chloro-2(S)-[[[1-[1,2-dioxo-2-(2-propenylamino) ethyl]bu-
tyl]-amino]carbonyl]-3-azabicyclo[3.1.0]hexan-3-yl]-2-oxo-
ethyl]carbamate (9)
5.6. 3-(2-tert-Butoxycarbonylamino-2-cyclohexyl-acetyl)-
6,6-dichloro-3-aza-bicyclo[3.1.0]hexane-2-carboxylic acid
(6: R3 is cyclohexyl) 1H NMR (500 MHz, CDCl3) d 6.96
(d, J = 9.3 Hz, 1H), 4.39 (s, 1H), 4.26 (d, J = 11.3 Hz,
1H), 3.96–3.89 (m, 2H), 2.76–2.74 (m, 1H), 2.68 (d,
J = 8.6 Hz, 1H), 2.09 (s, 1H), 2.04 (s, 1H), 1.84 (s, 1H),
1.74–1.58 (m, 4H), 1.33 (s, 9H), 1.17–1.10 (m, 5H),
0.95–0.84 (m, 2H). 13C NMR (125 MHz, d6-DMSO) d
170.43, 170.40, 155.4, 153.8, 124.1,77.9, 63.2, 60.7, 56.3,
55.8, 47.7, 38.6, 35.1, 33.6, 31.5, 29.5, 29.2, 29.1, 28.6,
28.3, 28.11, 28.05, 25.9, 25.4, 25.3. HR-MS Calcd for
C19H28N2O5Cl2: 435.1454 (M+H)+. Found: 435.1460.
1H NMR (500 MHz, d6-DMSO) d 8.92 and 8.88 (t, J = 6.0
and 6.3 Hz, 1H), 8.67 and 8.59 (d, J = 6.8 and 7.7 Hz, 1H),
6.92 (q, J = 4.5 Hz, 1H), 5.82–5.75 (m, 1H), 5.11–5.04 (m,
2H), 4.97–4.93 (m, 1H), 4.61 (d, J = 10.2 Hz, 1H), 4.23–
4.20 (m, 1H), 3.96–3.86 (m, 2H), 3.78–3.68 (m, 3H), 2.75–
2.70 (m, 1H), 2.42–2.36 (m, 1H), 1.75–1.49 (m, 8H), 1.41–
1.29 (m, 10H), 1.14–1.08 (m, 3H), 0.90–0.85 (m, 5H). 13C
NMR (125 MHz, d6-DMSO) d 197.1, 196.4, 168.9, 160.5,
155.4, 134.8, 134.03, 134.00, 115.6, 115.4, 109.0, 77.84,
77.80, 60.9, 56.5, 56.3, 53.9, 53.5, 40.7, 33.6, 33.5, 31.7,
31.5, 28.0, 25.8, 25.39, 25.35, 25.2, 18.6, 18.5, 13.5, 13.4.
HR-MS Calcd for C28H42N4O6Cl2: 560.2407 (M+H)+.
Found: 560.2416.
5.7. 3-(2-tert-Butoxycarbonylamino-3,3-dimethyl-butyryl)-
6,6-dichloro-3-aza-bicyclo[3.1.0]hexane-2-carboxylic acid
(6: R3 is tert-butyl) 1H NMR (500 MHz, d6-DMSO) d 6.69
(d, J = 9.7 Hz, 1H), 4.44 (s, 1 H), 4.17 (d, J = 11.4 Hz,
1H), 4.09 (d, J = 9.7 Hz, 1H), 3.96 (dd, J = 5.1, 11.4 Hz,
1H), 2.75 (dd, J = 5.1, 8.5 Hz, 1H), 2.70 (d, J = 8.6 Hz,
1H), 1.91 (s, 2H), 1.35 (s, 9H), 0.92 (s, 9H); 13C NMR
(125 MHz, d6-DMSO) d 171.9, 170.3, 169.9, 155.6, 78.1,
63.2, 60.5, 58.2, 48.3, 35.1, 34.3, 33.5, 28.12, 28.10, 28.0,
26.15, 26.10, 21.0, 14.0. HR-MS Calcd for
C17H27N2O5Cl2: 409.1294 (M+H)+. Found: 409.1297.
5.11. (1S,5R)-6,6-Dichloro-3-[cyclohexyl[[[(1,1-dimethyl-
ethyl)amino]carbonyl]amino]acetyl]-N-[1-[1,2-dioxo-2-(2-
propenylamino)ethyl]butyl]-3-azabicyclo[3.1.0]hexan-2(S)-
carboxamide (10)
1H NMR (500 MHz, d6-DMSO) d 7.70–7.66 (m, 1H),
7.09 (t, J = 6.1Hz, 1H), 6.98 (t, J = 6.0 Hz, 1H), 5.86–
5.77 (m, 2H), 5.24–5.15 (m, 3H), 4.81 (d, J = 7.8 Hz,
1H), 4.49–4.47 (m, 1H), 4.36–4.32 (m, 1H), 3.95–3.87
(m, 3H), 2.72 (dd, J = 8.5, 11.7 Hz, 1H), 2.48–2.42 (m,
1H), 1.90–1.53 (m, 9H), 1.41–1.29 (m, 3H), 1.27 (br s,
9H), 1.23–1.07 (m, 3H), 0.94–0.89 (m, 3H). 13C NMR
(125 MHz, d6-DMSO) d 196.1, 195.5, 174.3, 174.1,
168.4, 168.2, 158.8, 156.7, 151.8, 132.5, 117.5, 85.6,
61.9, 61.5, 61.3, 56.0, 55.8, 54.2, 50.3, 48.9, 48.7, 41.6,
40.8, 35.2, 35.1, 34.0, 33.3, 33.1, 32.5, 29.5, 29.4, 28.7,
26.1, 25.8, 18.9, 13.6. HR-MS Calcd for
C28H44N5O5Cl2: 600.2719 (M+H)+. Found: 600.2720.
5.8. 3-(2-tert-Butoxycarbonylamino-2-indan-2-yl-acetyl)-
6,6-dichloro-3-aza-bicyclo[3.1.0]hexane-2-carboxylic acid
1
(6: R3 is 2-indanyl) H NMR (500 MHz, d6-DMSO) d
7.30 (d, J = 8.5 Hz, 1H), 7.19–7.09 (m, 4H), 4.44 (s,
1H), 4.25 (d, J = 11.0 Hz, 1H), 4.13 (t, J = 9.2 Hz,
1H), 3.89 (dd, J = 5.0, 11.5 Hz, 1H), 2.93 (dd, J = 7.2,
15.4 Hz, 1H), 2.83 (dd, J = 7.2, 15.2 Hz, 1H), 2.76–
2.63 (m, 5H), 2.50–2.49 (m, 1H), 1.36 (s, 9H). 13C
NMR (125 MHz, d6-DMSO) d 170.3, 170.2, 155.4,
142.3, 142.1, 126.13, 126.11, 124.4, 124.06, 78.1, 63.2,
60.7, 54.8, 47.6, 40.9, 35.3, 35.1, 35.0, 33.6, 30.3, 29.2,
29.1, 28.0. HR-MS Calcd for C22H26N2O5Cl2:
469.1297 (M+H)+. Found: 469.1310.
5.12. 1,1-Dimethylethyl [2-[(1S,5R)-2(S)-[[[1-(2-amino-1,
2-dioxoethyl)butyl]amino]carbonyl]-6,6-dichloro-3-azabicy-
clo[3.1.0]hexan-3-yl]-1(S)-cyclohexy;-2-oxoethyl]- carba-
mate (12)
5.9. 1,1- Dimethylethyl [1-cyclohexyl-2-[(1S,5R)-6,6-
dichloro-2(S)-[[[1-[2-[[2-[[2-(dimethylamino)-2-oxo-1(S)-
phenylethyl]amino]-2-oxoethyl]amino]-1,2-dioxoethyl]-
butyl]amino]carbonyl]-3-azabicyclo[3.1.0]hexan-3-yl]-2-
oxoethyl]carbamate (8)
1H NMR (500 MHz, CDCl3) d 7.62–7.57 (m, 1H), 5.97–
5.83 (m, 1H), 5.31–5.22 (m, 2H), 5.14–5.10 (m, 2H),
4.84 (d, J = 9.33 Hz, 1H), 4.32 (d, J = 11.8 Hz, 1H),
4.20–4.12 (m, 1H), 3.94–3.86 (m, 1H), 2.77 (t,
J = 7.72 Hz, 1H), 2.51–2.41 (m, 2H), 1.80–1.45 (m,
11H), 1.39 (br s, 9H), 1.29–1.08 (m, 2H), 0.98–0.88 (m,
3H). 13C NMR (125 MHz, CDCl3) d 195.40, 172.97,
172.73, 168.45, 168.33, 160.95, 155.50, 85.62, 79.85,
61.91, 61.49, 61.31, 56.97, 56.87, 54.08, 53.99, 48.68,
40.59, 40.61, 34.96, 34.87, 33.93, 33.81, 33.11, 29.53,
28.40, 28.22, 26.04, 25.80, 25.64, 19.00, 18.90, 13.65,
1H NMR (500 MHz, d6-DMSO) d 8.79–8.73 (m, 1H),
8.58–8.51 (m, 1H), 7.36–7.35 (m, 5H), 7.32–7.30 (m
1H), 6.91–6.89 (m, 1H), 5.82 (d, J = 7.8 Hz, 1H), 5.00–
4.97 (m, 1H), 4.62 (d, J = 11.3 Hz, 1H), 4.23–4.20 (m,
1H), 3.96–3.76 (m, 5H), 2.92–2.84 (m, 7H), 2.75–2.71

K. X. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1874–1883
1881
13.56. HR-MS Calcd for C25H38N5O6Cl2: 561.2238
(M+H)+. Found: 561.2247.
34.90, 34.86, 29.1, 28.8, 27.4, 26.4. HR-MS Calcd
for C27H37N4O6Cl2F3: 641.2112 (M+H)+. Found:
641.2121.
5.13. 1,1-Dimethylethyl[2-[(1S,5R)-6,6-dichloro-2(S)-
[[ [1-(cyclopropyl-methyl)-3-[[2-[[2-(dimethylamino)-2-oxo-
1-phenylethyl]amino]-2-oxoethyl]amino]-2,3-dioxopropyl]-
amino]carbonyl]-3-azabicyclo[3.1.0]-hexan-3-yl]-1-cyclo-
hexyl-2-oxoethyl]carbamate (13)
5.16. 6,6-Dichloro-3-[2-cyclohexyl-2-(3-cyclohexyl-ureido)-
acetyl]-3-aza-bicyclo[3.1.0]hexane-2-carboxylic acid(2-car-
bamoyl-1-cyclobutylmethyl-2-oxo-ethyl)-amide (22)
1H NMR (500 MHz, d6-DMSO) d 8.56 and 78.42 (d,
J = 7.3 and 7.5 Hz, 1H), 8.05–8.01 (m, 1H), 7.80 (s,
1H), 5.87–5.84 (m, 2H), 4.94–4.90 (m, 1H), 4.61–4.59
(m, 1H), 4.16–4.08 (m, 2H), 3.97–3.90 (m, 1H), 2.73–
2.71 (m, 1H), 2.43–2.38 (m, 1H), 2.01–1.95 (m, 2H),
1.84–1.37 (m, 17H), 1.27–0.83 (m, 12H); 13C NMR
(125 MHz, d6-DMSO) d 35.0, 34.0, 33.8, 33.7, 32.6,
32.4, 29.6, 28.8, 28.5, 28.2, 28.0, 26.1, 25.84, 25.82,
24.9, 24.8, 18.4, 18.3. HR-MS Calcd for
C29H43N5O5Cl2: 612.2714 (M+H)+. Found: 612.2720.
1H NMR (500 MHz, CDCl3) d 7.72–7.66 (m, 2H), 7.62–
7.53 (m, 1H), 7.40–7.31 (m, 5H), 5.84 (d, J =7.14 Hz,
1H), 5.38–5.29 (m, 1H), 5.20–5.07 (m, 1H), 4.86 (d,
J = 8.79 Hz, 1H), 4.31 (d, J = 11.57 Hz, 1H), 4.22–3.84
(m, 3H), 2.97 (s, 3H), 2.88 (s, 3H), 2.81–2.72 (m, 1H),
2.49–2.41 (m, 1H), 2.31–2.06 (m, 1H), 1.83–1.53 (m,
8H), 1.39 (br s, 9H), 1.24–0.62 (m, 6H), 0.45–0.40 (m,
2H), 0.11–0.01 (m, 2H). 13C NMR (125 MHz, d6-
DMSO) d 194.9, 194.6, 172.8, 172.5, 168.0, 166.3,
159.2, 155.5, 136.6, 129.0, 128.5, 127.8, 85.6, 79.8,
62.0, 61.4, 61.2, 56.9, 56.8, 55.1, 54.9, 54.1, 48.6, 42.2,
40.6, 36.8, 36.3, 36.0, 35.1, 34.8, 33.9, 33.7, 29.5, 28.2,
26.0, 25.6, 7.2, 5.2, 5.1, 4.5. HR-MS Calcd for
C38H52N6O8Cl2: 791.3302 (M+H)+. Found: 791.3310.
5.17. {1-[2-(2-Carbamoyl-1-cyclobutylmethyl-2-oxo-ethyl-
carbamoyl)-6,6-dichloro-3-aza-bicyclo[3.1.0]hexane-3-car-
bonyl]-2,2-dimethyl-propyl}-carbamic acid tert-butyl ester
(23)
5.14. 1,1-Dimethylethyl[2-[(1S,5R)-6,6-dichloro-2(S)-[[[1-
(cyclobutylmethyl)-3-[[2-[[2-(dimethylamino)-2-oxo-1(S)-
phenylethyl]amino]-2-oxomethyl]amino]-2,3-dioxopropyl
amino]carbonyl]-3-azabicyclo[3.1.0]hexan-3-yl]-1(S)-cyclo-
hexyl-2-oxomethyl]carbamate (14)
1H NMR (500 MHz, d6-DMSO) d 8.57 and 8.51 (d,
J = 7.4 and 7.3 Hz, 1H), 8.04 and 7.79 (d, J = 11.9 and
18.9 Hz, 1H), 5.93 (s, 1H), 5.87 (q, J = 4.46 Hz, 1H),
4.97–4.86 (m, 1H), 4.64 (d, J = 3.2 Hz, 1H), 4.23–4.14
(m, 2H), 3.96–3.92 (m, 1H), 2.74–2.70 (m, 1H), 2.40
(dd, J = 8.7, 8.5 Hz, 1H), 2.01–1.93 (m, 2H), 1.84–1.72
(m, 2H), 1.67–1.56 (m, 3H), 1.24 (br s, 1H), 1.17 (d,
J = 1.9 Hz, 9H), 1.10 ( s, 1H) 0.87 (d, J = 6.9 Hz, 9H);
13C NMR (125 MHz, d6-DMSO) d 198.4, 197.7, 171.9,
171.4, 169.7, 169.3, 163.7, 163.5, 157.93, 157.90, 64.2,
61.7, 61.6, 57.44, 57.40, 53.2, 49.8, 49.6, 37.5, 37.3,
37.1, 35.2, 34.6, 34.4, 33.1, 33.0, 32.1, 30.0, 28.9, 28.7,
28.32, 28.30, 27.1, 23.0. HR-MS Calcd for
C25H38N4O6Cl2: 561.2240 (M+H)+. Found: 561.2247.
1H NMR (500 MHz, d6-DMSO) d 8.80–8.76 (m, 1H),
8.62–8.55 (m, 2H), 7.36 (s, 5H), 6.92 (t, J = 8.5 Hz,
1H), 5.82 (d, J = 6.9 Hz, 1H), 4.94 (m, 1H), 4.61 (d,
J = 10.7 Hz, 1H), 4.23–4.20 (m, 1H), 3.95–3.77 (m,
4H), 2.96 (s, 3H), 2.80 (s, 3H), 2.74–2.71 (m, 1H),
2.49–2.33 (m, 3H), 1.98–1.97 (m, 2H), 1.85–1.61 (m,
12H), 1.34 (s, 9H), 1.09 (m, 2H), 0.865 (m, 2H). 13C
NMR (125 MHz, d6-DMSO) d 196.5, 196.1, 195.9,
170.13, 170.06, 169.1, 168.8, 168.4, 166.83, 166.80,
160.6, 160.4, 155.39, 155.37, 137.45, 137.40, 128.5,
127.8, 127.7, 77.84, 77.80, 63.40, 63.37, 61.2, 60.9,
56.5, 56.4, 53.0, 52.9, 52.5, 52.4, 48.2, 48.1, 41.6, 41.5,
36.6, 36.5, 36.3, 36.2, 35.3, 33.6, 33.5, 32.1, 28.7, 28.6,
28.4, 28.3, 28.03, 27.97, 27.8, 27.7, 27.2, 25.9, 25.44,
25.36, 25.30, 25.26, 17.80, 17.69. HR-MS Calcd for
C39H54N6O8Cl2: 805.3458 (M+H)+. Found: 805.3472.
5.18. 3-[2-(3-tert-Butyl-ureido)-3,3-dimethyl-butyryl]-6,6-
dichloro-3-aza-bicyclo[3.1.0]hexane-2-carboxylic acid(2-
carbamoyl-1-cyclobutylmethyl-2-oxo-ethyl)-amide (24)
1H NMR (500 MHz, d6-DMSO) d 8.60 (d, J = 7.1 Hz,
1H), 8.06 (s, 1H), 7.81 (s, 1H), 6.63 (d, J = 9.8 Hz,
1H), 4.97–4.93 (m, 1H), 4.65 (s, 1H), 4.14 (d,
J = 11.4 Hz, 1H), 4.05 (d, J = 9.7 Hz, 1H), 3.95 (dd,
J = 5.3, 11.0 Hz, 1H), 2.74 (dd, J = 8.6, 8.8 Hz, 1H),
2.41 (d, J = 8.9 Hz, 1H), 2.00–1.92 (m, 2H), 1.84–1.70
(m, 3H), 1.66–1.55 (m, 3H), 1.41 (br s, 1H), 1.35 (s,
9H), 1.24 (br s, 1H), 0.90 (s, 9H). 13C NMR
(125 MHz, d6-DMSO) d 198.4, 170.5, 169.7, 163.5,
156.6, 79.0, 64.3, 61.8, 59.3, 53.2, 49.7, 37.5, 37.3, 35.1,
34.6, 33.0, 32.1, 29.0, 28.9, 28.7, 28.2, 27.1, 23.0, 18.7,
18.6, 14.8. HR-MS Calcd for C25H39N5O5Cl2:
560.2339 (M+H)+. Found: 560.2407.
5.15. 2,2,2-Trifluoro-1,1-dimethylethyl[2-[(1S,5R)-2(S)-
[[[3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]amino]-
carbonyl]-6,6-dichloro-3-azabicyclo[3.1.0]hexan-3-yl]-1(S)-
cyclohexyl-2-oxoethyl]carbamate (18)
1H NMR (500 MHz, d6-DMSO) d 8.60 and 8.51 (d, J = 7.5
and 7.2 Hz, 1H), 8.05 (d, J = 15.2 Hz, 1H), 7.81 (d,
J = 6.3 Hz, 1H), 7.68–7.63 (m, 1H), 4.95–4.91 (m, 1H),
4.62 (d, J = 6.6 Hz, 1H), 4.20–4.16 (m, 1H), 3.98–3.86 (m,
2H), 2.76–2.75 (m, 1H), 2.36–2.33 (m, 2H), 1.98–1.94 (m,
2H), 1.82–1.48 (m, 17H), 1.09–1.08 (m, 3H), 0.94–0.83 (m,
3H). 13C NMR (125 MHz, d6-DMSO) d 207.1, 206.7,
206.5, 179.31, 179.30, 178.7, 178.3, 177.9, 172.3, 173.2,
163.2, 70.9, 70.7, 66.5, 66.4, 62.5, 61.8, 57.90, 57.89, 57.8,
48.1, 47.9, 46.4, 46.2, 46.0, 45.9, 43.2, 43.1, 41.8, 38.1,
38.03, 37.98, 37.5, 37.4,37.0, 36.9, 35.4, 35.04, 35.00,
5.19. {2-[2-(2-Carbamoyl-1-cyclobutylmethyl-2-oxo-ethyl-
carbamoyl)-6,6-dichloro-3-aza-bicyclo[3.1.0]hex-3-yl]-1-
indan-2-yl-2-oxo-ethyl}-carbamic acid tert-butyl ester (25)
1H NMR (500 MHz, d6-DMSO) d 8.62 and 8.49 (d,
J = 7.3 and 7.5 Hz, 1H) 8.07 (d, J = 13.8 Hz, 1H), 7.83

1882
K. X. Chen et al. / Bioorg. Med. Chem. 16 (2008) 1874–1883
F. Bioorg. Med. Chem. Lett. 2004, 14, 4575; (c) Llinas-
Brunet, M.; Bailey, M. D.; Ghiro, E.; Gorys, V.; Halmos,
T.; Poirier, M.; Rancourt, J.; Goudreau, N. J. Med. Chem.
2004, 47, 6584; (d) Zhang, X. Idrugs 2002, 5, 154; (e)
Dymock, B. W. Expert Opin. Emerging Drugs 2001, 6, 13;
(f) Dymock, B. W.; Jones, P. S.; Wilson, F. X. Antiviral
Chem. Chemother. 2000, 11, 79; (g) Zhang, R.; Durkin, J.
P.; Windsor, W. T. Bioorg. Med. Chem. Lett. 2002, 12,
1005; (h) Ingallinella, P.; Fattori, D.; Altamura, S.;
Steinkuhler, C.; Koch, U.; Cicero, D.; Bazzo, R.; Cortese,
R.; Bianchi, E.; Pessi, A. Biochemistry 2002, 41, 5483; (i)
Beevers, R.; Carr, M. G.; Jones, P. S.; Jordan, S.; Kay, P.
B.; Lazell, R. C.; Raynham, T. M. Bioorg. Med. Chem.
Lett. 2002, 12, 641.
(d, J = 8.0 Hz, 1H) 7.29–7.25 (m, 1H), 7.18–7.09 (m,
4H), 5.00–4.95 (m, 1H), 4.64 (d, J = 13.9 Hz, 1H),
4.21–4.08 (m, 1H), 3.95–3.86 (m, 1H), 3.36 (s, 1H),
2.94–2.64 (m, 7H), 2.47–2.38 (m, 2H), 2.03–1.97 (m,
1H), 1.88–1.59 (m, 6H), 1.37–1.36 (m, 9H); 13C NMR
(125 MHz, d6-DMSO) d 207.1 206.5, 179.6, 178.38,
178.00, 172.3, 172.2, 165.1, 165.0, 152.0, 151.9, 151.8,
135.73, 135.70, 134.0, 133.7, 87.71, 87.66, 71.0, 70.7,
64.7, 64.4, 62.1, 61.8, 57.8, 50.5, 50.4, 46.3, 46.7, 46.0,
45.8, 45.7, 44.91, 44.86, 44.66, 44.56, 43.2, 43.1, 41.8,
37.6, 37.5, 37.4, 36.9, 35.9, 27.40, 27.36. HR-MS Calcd
for C30H38N4O6Cl2: 621.2235 (M+H)+. Found:
621.2247.
9. (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.
Bioorg. Med. Chem. Lett. 1998, 8, 1713; (b) Steinkuhler,
C.; Biasiol, G.; Brunetti, M.; Urbani, A.; Koch, U.;
Cortese, R.; Pessi, A.; De Francesco, R. Biochemistry
1998, 37, 8899.
5.20. 3-[2-(3-tert-Butyl-ureido)-2-indan-2-yl-acetyl]-6,6-
dichloro-3-aza-bicyclo[3.1.0]hexane-2-carboxylic acid(2-
carbamoyl-1-cyclobutylmethyl-2-oxo-ethyl)-amide (26)
1H NMR (500 MHz, d6-DMSO) d 8.62 and 8.49 (d,
J = 7.3 and 7.5 Hz, 1H) 8.07 (d, J = 13.8 Hz, 1H), 7.83
(s, 1H) 7.18–7.09 (m, 4H), 6.07–6.04 (m, 1H), 5.83 (s,
1H), 5.00–4.95 (m, 1H), 4.64 (d, J = 13.9 Hz, 1H),
4.31–4.28 (m, 1H), 4.15–4.12 (m, 1H), 3.95–3.86 (m,
1H), 2.94–2.64 (m, 7H), 2.47–2.38 (m, 2H), 2.03–1.97
(m, 1H), 1.88–1.59 (m, 6H), 1.37–1.36 (m, 9H); 13C
NMR (125 MHz, d6-DMSO) d 207.1 206.5, 180.4,
180.3, 178.4, 178.0, 172.3, 166.4, 152.0, 151.8, 135.7,
134.0, 133.7, 72.9, 70.9, 70.5, 62.6, 62.1, 61.8, 58.62,
58.60, 57.7, 57.5, 51.3, 51.1, 46.2, 46.0, 45.7, 44.7, 44.6,
44.2, 44.1, 43.1, 43.0, 41.8, 39.1, 38.7, 37.42, 37.40,
37.0, 36.9, 27.37. HR-MS Calcd for C30H39N5O5Cl2:
620.2400 (M+H)+. Found: 620.2407.
10. (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. J. Biol. Chem. 2004, 279, 17508; (b) Sun, D.
X.; Liu, L.; Heinz, B.; Kolykhalov, A.; Lamar, J.;
Johnson, R. B.; Wang, Q. M.; Yip, Y.; Chen, S.-H.
Bioorg. Med. Chem. Lett. 2004, 14, 4333; (c) Yip, Y.;
Victor, F.; Lamar, J.; Johnson, R.; Wang, Q. M.; Glass, J.
I.; Yumibe, N.; Wakulchik, M.; Munroe, J.; Chen, S.-H.
Bioorg. Med. Chem. Lett. 2004, 14, 5007; (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.
Bioorg. Med. Chem. Lett. 2004, 14, 1939; (e) Venkatr-
aman, S.; Bogen, S. L.; Arasappan, A.; Bennett, F.; Chen,
K.; Jao, J.; Liu, Y.-T.; Lovey, R.; Hendrata, S.; Huang,
Y.; Pan, W.; Parekh, T.; Pinto, P.; Popov, V.; Pike, R.;
Ruan, S.; Santhanam, B.; Vibulbhan, B.; Wu, W.; Yang,
W.; Kong, J.; Liang, X.; Wong, J.; Liu, R.; Butkiewicz, N.;
Chase, R.; Hart, A.; Agrawal, S.; Ingravallo, P.; Pichardo,
J.; Kong, R.; Baroudy, B.; Malcolm, B.; Guo, Z.;
Prongay, A.; Madison, V.; Broske, L.; Cui, X.; Cheng,
K.-C.; Hsieh, Y.; Brisson, J.-M.; Prelusky, D.; Korfm-
acher, W.; White, R.; Bogdanowich-Knipp, S.; Pavlovsky,
A.; Bradley, P.; Saksena, A. K.; Ganguly, A.; Piwinski, J.;
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