Peptide-Based Inhibitors of the Hepatitis C Virus NS3 Protease: Structure-Activity Relationship at the C-Terminal Position

Article abstract of DOI:10.1021/jm030573x

The structure-activity relationship at the C-terminal position of peptide-based inhibitors of the hepatitis C virus NS3 protease is presented. The observation that the N-terminal cleavage product (DDIVPC-OH) of a substrate derived from the NS5A/5B cleavag

Full text of DOI:10.1021/jm030573x

J . Med. Chem. 2004, 47, 2511-2522
2511
P ep tid e-Ba sed In h ibitor s of th e Hep a titis C Vir u s NS3 P r otea se:
Str u ctu r e-Activity Rela tion sh ip a t th e C-Ter m in a l P osition
J ean Rancourt,* Dale R. Cameron,† Vida Gorys, Daniel Lamarre, Martin Poirier, Diane Thibeault, and
Montse Llina`s-Brunet
Research and Development, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard Street, Laval (Que´bec), Canada H7S 2G5
Received November 12, 2003
The structure-activity relationship at the C-terminal position of peptide-based inhibitors of
the hepatitis C virus NS3 protease is presented. The observation that the N-terminal cleavage
product (DDIVPC-OH) of a substrate derived from the NS5A/5B cleavage site was a competitive
inhibitor of the NS3 protease was previously described. The chemically unstable cysteine residue
found at the P1 position of these peptide-based inhibitors could be replaced with a norvaline
residue, at the expense of a substantial drop in the enzymatic activity. The fact that an
aminocyclopropane carboxylic acid (ACCA) residue at the P1 position of a tetrapeptide such as
1 led to a significant gain in the inhibitory enzymatic activity, as compared to the corresponding
norvaline derivative 2, prompted a systematic study of substituent effects on the three-
membered ring. We report herein that the incorporation of a vinyl group with the proper
configuration onto this small cycle produced inhibitors of the protease with much improved in
vitro potency. The vinyl-ACCA is the first reported carboxylic acid containing a P1 residue
that produced NS3 protease inhibitors that are significantly more active than inhibitors
containing a cysteine at the same position.
In tr od u ction
It is estimated that up to 200 million people around
the world are infected with the hepatitis C virus
(HCV).1,2 From this population, approximately 70% of
individuals suffering from persistent infection will
develop chronic hepatitis C. This progressive liver
disease may lead to cirrhosis and hepatocellular carci-
nomas3 and results in around 10 000 deaths annually
in the United States alone. Standard therapies involve
the use of pegylated interferon in combination with the
broad spectrum antiviral agent ribavirin and are char-
acterized by a limited efficacy (low sustained response
rate) and severe side effects.4
F igu r e 1.
The NS3 protease is essential for viral replication, as
shown by the lack of infectivity of an HCV RNA clone
harboring a mutation in its catalytic domain (Ser-139-
Ala), and therefore represents a prime target for drug
discovery.11
The HCV was discovered in 1989 as the etiological
agent for non-A and non-B hepatitis infections.5 This
enveloped RNA virus belongs to the Flaviviridae family.
Its genome is composed of approximately 9600 base
pairs and encodes a precursor polyprotein (ca. 3011
amino acids) that undergoes proteolytic maturation.6,7
The NS3 is one of a number of mature proteins that
result from the polypeptide processing. The N-terminal
region of this NS3 protein exhibits proteolytic activity
and can be considered as a chymotrypsin-like serine
protease. The C-terminal portion of the protein contains
an ATP-dependent RNA helicase. The NS3 protease is
responsible for cleaving four sites on the polypeptide.8-10
It possesses one structural zinc-binding domain and
functions as a heterodimer complexed with the NS4A
protein. This small polypeptide is an essential cofactor
for efficient processing of the polyprotein by the NS3
protease.10
We,12 and others,13 discovered that the N-terminal
cleavage product (DDIVPC-OH) of a substrate derived
from the NS5A/5B cleavage site is a competitive inhibi-
tor of the NS3 protease. Early efforts using this hexapep-
tide as a starting point focused on finding a replacement
for the unstable cysteine residue found at the P114
position (dimerization reaction). A structure-activity
study performed on this cleavage product eventually led
to the discovery of tetrapeptide 1 (Figure 1), a moder-
ately potent (IC50 ) 14 µM), highly specific and competi-
tive inhibitor,15 possessing a chemically stable cysteine
replacement at the C-terminal position. Related work
carried out by Narjes et al.16 led to the identification of
4,4-difluoro-2-aminobutyric acid (difluoro-Abu) as one
possible P1 cysteine mimetic.
In this paper we present how the inhibitory activity
of compound 1 was further improved through a struc-
ture-activity study carried out on the cyclopropane ring
of the P1 position.
* Corresponding author. Telephone: (450) 682-4640. Telefax: (450)
682-4189. E-mail: jrancourt@lav.boehringer-ingelheim.com.
† Present address: Micrologix Biotech Inc., BC Research Building,
3650 Wesbrook Mall,Vancouver, BC, Canada V6S 2L2.
10.1021/jm030573x CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/01/2004

2512 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Rancourt et al.
Ta ble 1. ACCA Optimization
IC50 (µm)a
entry
compd
R
n
a
b
1
2
3
4
5
6
7
8
2
1
Et
H
Et
Me
n-Pr
i-Pr
Bn
0
1
1
1
1
1
1
1
23
14
14
15
16
17
18
19
4.8
22
5.5
5.5
14
22
32
180
41
640
81
F igu r e 2.
PhCH2CH2
90
a
See main text for a comment on the establishment of the
relative configuration at C1 and C2 on the cyclopropane ring.
3b (with the 1S configuration as with norvaline).15d
However, a closer look at how the carboxylate group of
both 3a and 3b would most likely bind in the active site
of the enzyme suggested that this prediction was
probably incorrect. The binding mode of the C-terminus
of the helicase domain in the active site of the protease
was revealed by the first crystal structure of the full-
length NS3 protein with its N-terminus covalently
linked to the NS4A peptide cofactor.17 This X-ray crystal
structure of a C-terminal carboxylate bound to the active
site of the NS3/NS4A complex provided an atomic view
of the interactions between the protease domain and the
P-side product of the cis cleavage reaction (as high-
lighted in Figure 4A). In this structure, the P1 φ angle
is -127.6°, properly positioning the carboxylate in the
active site, where it is stabilized via several hydrogen
bonds. These involve the side chains of catalytic residues
His 57 and Ser 139, as well as the backbone amide
protons of the oxyanion hole residues Gly 137 and Ser
139 (Figure 4A).
Application of the same carboxylate binding mode to
both 3a and 3b (panels C and B, respectively) suggested
that 3b may not be the preferred rigidified norvaline
mimic, as this analogue would thrust the ethyl side
chain too deep into the small S1 binding pocket, result-
ing in unfavorable interactions with the Phe 154 side
chain. On the other hand, the unnatural (1R,2R) con-
figuration of 3a allowed a better fit of the ethyl side
chain into the pocket of interest (as illustrated in Figure
4C), resulting in a more potent analogue. This turned
out to be the case when the 3a - and 3b-containing
peptides were tested (Table 1, entry 3). The correspond-
ing “anti” isomers (4a ,b) were also prepared (see next
section for a brief description of their synthesis) but did
not offer any advantages over the “syn” ACCA deriva-
tives and required two additional synthetic steps for
their preparation (data not shown). The above results
obtained for 3a prompted us to carry out a more
extensive structure-activity study centered around the
cyclopropane residue that eventually culminated in the
identification of more potent inhibitors of the NS3
protease.
F igu r e 3.
Resu lts a n d Discu ssion
We have previously shown that norvaline15a and
aminocyclopropane carboxylic acid15b,c (ACCA) (Figure
2) are two effective replacements for the cysteine residue
at P1 of peptide-based inhibitors. It was anticipated that
a gain in activity would be observed upon combining
the beneficial effect of the cyclopropane ring with the
propyl side chain of norvaline.
As depicted in Figure 3, the substituted cyclic amino
acid resulting from this exercise can be viewed as a
rigidified norvaline group (four possible diastereomers).
Isomers 3a ,b, which possess an ethyl substituent on
a three-membered ring syn to the carboxylic acid group,
were synthesized first. When these two “syn” methano-
norvaline derivatives were introduced in a tetrapeptide
series analogous to compound 1, we discovered that one
of them was well tolerated, giving a moderated increase
in potency, while the other diastereomer was signifi-
cantly less active (entry 3 in Table 1). The absolute
configuration of these substituted ACCA derivatives was
determined subsequently (see Chemistry section). As
outlined in Figure 3, it was expected that the more
active three-membered-ring isomer would correspond to

Peptide-Based Inhibitors of HCV NS3 Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2513
F igu r e 4. (A) Ribbon representation of the active site of the crystal structure of the engineered full-length NS3 protease-
helicase protein (PDB entry 1CU1).17 This structure provides an atomic view of the binding of the C-terminal carboxylic acid
moiety of the helicase domain to the active site of the protease domain. (B) Binding model of the inhibitor P1 (1S)-amino-(2S)-
ethylcyclopropanecarboxylic acid derivative (3b) to the active site of the NS3 protease domain. (C) Binding model of the inhibitor
P1 (1R)-amino-(2R)-ethylcyclopropanecarboxylic acid derivative (3a ) to the active site of the NS3 protease domain. In this model,
the NS3 protein is represented as a Conolly surface, while the inhibitor P1 residue is represented in sticks and is colored by atom
type (oxygen is red, nitrogen is blue, carbon is green, and hydrogen is light gray). For clarity, most of the hydrogen atoms were
omitted.
Sch em e 1a
Ta ble 2. Further Optimization of ACCA Derivatives
entry
compd
R1
R2
IC50 (µm)
1
2
3
4
1
14
20a
21
H
Et
Et
vinyl
H
H
Et
H
14
4.8
310
0.63
a
Reagents and conditions: (a) di-tert-butyl malonate, 50%
aqueous NaOH, BnEt3N+Cl-; (b) tBuOK, H2O, Et2O, 0 °C to room
temperature; (c) Et3N, DPPA, benzene; 2-(trimethylsilyl)ethanol,
reflux; (d) 1.0 M TBAF, THF, room temperature reflux.
a
Only the result for the more potent diastereomer is shown.
The relative configuration at C1 and C2 on the cyclopropane ring
is tentatively assigned.
Ch em istr y. (i) Syn th esis of ACCA Der iva tives.
The ethyl-substituted cyclopropane derivatives pre-
sented above were prepared according to Scheme 1. This
approach is not limited to the formation of the ethyl-
substituted three-membered ring and is actually quite
general for the preparation of several other derivatives
(see Table 1), simply by changing the structure of the
bis-electrophile. As reported,18 cyclic sulfate derivatives
have been found to be a useful alternative to the
dibromide in this cyclization reaction (see Experimental
Section).
2,2-diethyl ACCA derivative 20 (see entry 3 in Table
2).
All P1’s were incorporated into the tripeptide Ac-Chg-
Val-Pro-(4R)-(1-naphthylmethoxy)-OH 9 (left-hand side
of compound 1) using standard solution-phase peptide-
coupling reaction conditions.
(ii) Absolu te Con figu r a tion Deter m in a tion of
ACCA Der iva tives. The determination of the absolute
configuration of the most active ethyl ACCA derivative
was done as shown in Schemes 2 and 3. Carbamate 7
(racemic) was deprotected under standard conditions,
and the resulting amine was coupled to (4R)-1-(tert-
butoxycarbonyl)-4-(1-naphthylmethoxy)-L-proline 10
(homochiral). The mixture of dipeptide diastereomers
was purified by flash chromatography to afford a pure
sample of both isomers 11 and 12. Independently,
dipeptide 11 was prepared using a homochiral cyclo-
propane derivative of known absolute configuration23
(Scheme 3). Thus, compound 13 (homochiral, 1R,2R
isomer) was treated with a solution of hydrogen chloride
in ethyl acetate in order to selectively remove the acid-
sensitive carbamate in the presence of the tert-butyl
ester group.24 The resulting hydrochloride salt was
Thus, the acid ester 6 was obtained from di-tert-butyl
malonate after dialkylation19 followed by a selective
saponification reaction.20 The racemic “syn” aminoester
8 was prepared via a Curtius rearrangement followed
by a fluoride-induced deprotection performed on the
intermediate carbamate 7.21 In the case of the “anti”
isomer, the intermediate acid 6 was first converted to
the corresponding allyl ester and then treated with
anhydrous HCl in order to deprotect the tert-butyl ester.
The revealed carboxylic acid was finally transformed
into an amine functionality using the sequence of
reactions exposed above for compound 7.
Conversely, simple 2,2-dialkylated derivatives could
be generated using the procedure of Verhe´.22 This
alternative methodology allowed us to synthesize the
1
coupled to 10. HPLC, TLC, and H NMR comparison of
this authentic sample with the dipeptide 11 allowed us

2514 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Rancourt et al.
Sch em e 2
Sch em e 3
carbon atom chain appears to be slightly preferred for
activity (entry 3, R ) Et). At this point, a more focused
SAR study aiming at the optimization of the best R
group identified above was performed.
The model presented earlier in Figure 4 (panel C)
suggests that introduction of another substituent adja-
cent to the ethyl group already present on the cyclopro-
pane ring may be tolerated and possibly better fill the
S1 pocket. To evaluate this possibility, disubstitution
was examined (Table 2). Unfortunately, the introduction
of a gem-diethyl group led to a less potent inhibitor
(entry 3). Another simple, two-carbon atom group was
considered: a small vinyl substituent. When this par-
ticular group was introduced, we observed an appreci-
able gain in potency (entry 4). Thus, the formal replace-
ment of one hydrogen atom of the three-membered ring
by a small planar group (vinyl) produced an inhibitor
with a 22-fold improved inhibitory activity (entries 1 and
4). The difference in potency observed between the ethyl
and the vinyl substituents (entries 2 and 4) may be due,
at least in part, to a beneficial electronic interaction
between the π electrons of the vinyl group and the
phenyl group of Phe 154, which is in close proximity
(see Figure 4).
Further evaluation of this newly discovered P1 frag-
ment was then carried out by introducing it in three
different series of inhibitors that we reported previ-
ously25,26 (see Table 3). These related series differ only
by the nature of the substituent located at the 4 position
of the P2 proline residue.
As can be seen from Table 3, the introduction of vinyl-
ACCA residue in three different series provides a 29-
to 34-fold improvement in potency over the simple
cyclopropane derivative.
to establish that the absolute configuration of the most
active P1 fragment is (1R,2R) (Figure 3, structure 3a ).
Str u ctu r e-Activity Stu d y on ACCA Der iva tives.
As mentioned previously, the encouraging biological
results obtained with an inhibitor having fragment 3a
in the P1 position prompted us to expand further on this
observation. We were particularly interested by sub-
stituent effects on the cyclopropane ring. Thus, various
ACCA derivatives were synthesized, and the results
obtained are collected in Table 1.
Two different diastereomers (a and b) were made and
tested for each R group. Notice that the relative con-
figuration at C1 and C2 on the cyclopropane ring of
these isomers has been proven only for compounds
14a ,b (Table 1), as described above. In all the other
cases, the configuration is tentatively assigned on the
basis of the observation that the fast-eluting material
on TLC at the tetrapeptide ester stage will always
provide the most potent inhibitor after the ester saponi-
fication step. For the ethyl case, this would correspond
to the isomer a (entry 3). An ethyl group (14a ) afforded
a modest 3-fold improvement of potency over the simple
cyclopropane analogue 1. A small decrease or increase
in the length of the R group, from an ethyl to a methyl
or a propyl substituent (entries 4 and 5), did not result
in an improvement of potency. The effect of branching
was subsequently evaluated. When an isopropyl group
was introduced, an equally potent compound was pro-
duced (entry 6, compound 17a ). The incorporation of a
larger substituent such as a phenyl group onto the
cyclopropane ring, with two different tethers incorpo-
rated between the rings, led to a decrease in potency
(entries 7 and 8). From the above observations, a two-
The importance of the vinyl-ACCA27 residue in these
series of inhibitors can be further demonstrated if one
compares with the activity found for the corresponding
cysteine derivative (which is the amino acid found in
the natural substrate of the NS3 protease) and the two
other residues that were the starting points for this
study (Table 4). IC50 results indicate that not only is
the vinyl-ACCA derivative (entry 4) more potent than
the corresponding norvaline and ACCA residues (entries
2 and 3, respectively), but it is also around 14 times
more active than cysteine itself (entry 1)! Recent direct
comparison with the P1 cysteine mimetic developed by
the IRBM/Merck group (difluoro-Abu16) also demon-
strated the superiority of the vinyl-ACCA residue.28
Con clu sion
The approach of combining a cyclopropane ring and
the side chain of norvaline to produce novel P1 residues

Peptide-Based Inhibitors of HCV NS3 Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2515
Ta ble 3. Effects of the Introduction of Vinyl-ACCA in Two
Other Inhibitor Series
Reagents and solvents, including anhydrous THF and CH2-
Cl2, were purchased from Aldrich Chemical Co. or VWR
Scientific of Canada. The final compounds were purified by
preparative C18 reversed-phase HPLC on a Whatman Partisil
10 ODS-3 column (2.2 × 50 cm, λ ) 230 nm) using a linear
gradient from 5% aqueous CH3CN (containing 0.06% TFA) to
100% CH3CN. HPLC homogeneities were determined under
reversed-phase conditions using a C18 Vydac column (0.46 ×
12.5 cm, 5 µM), a 35 min linear gradient at 1.5 mL/min flow
from 5% CH3CN-H2O) (0.06% TFA) to 100% CH3CN (0.06%
TFA), and UV detection at 230 nm. Capillary electrophoresis
was performed on a P/ACE MDQ capillary electrophoresis
system with a PDA (purchased from Beckman Coulter, Ful-
lerton, CA) and a fused-silica capillary, 50 cm length to
detector, 60 cm total length, 50 µm internal diameter (pur-
chased from Polymicro Technologies, Phoenix, AZ). The elution
was monitored at 220 nm using a photodiode array detector
and a data acquisition rate of 8 Hz. The capillary temperature
and the sample temperature were 20 and 10 °C, respectively.
The separation voltage was set at 30 kV. The separation
electrolyte consisted of 50 mM Borax (pH 9.4). Mass spectral
data were obtained in FAB mode on an MF 50 TATC instru-
ment operating at 6 kV and 1 mA using thioglycerol or
nitrobenzyl alcohol (NBA) as a matrix support. High-resolution
mass spectrometry (HRMS) was preformed on a Micromass
AutoSpecTOF apparatus using metastable atom bombardment
ionization.
a
Prepared by sequential couplings using racemic vinyl-ACCA
and diastereomer separation at the dipeptide stage by flash
chromotography.
Flash chromatography was performed on Merck silica gel
60 (0.040-0.063 mm). Analytical thin-layer chromatography
(TLC) was carried out on precoated (0.25 mm) Merck silica
gel F-254 plates.
Ta ble 4. Vinyl-ACCA in Comparison with Various P1 Residues
Ch em istr y. Syn th esis of ter t-Bu tyl (1R,2R)/(1S,2S)-1-
Am in o-2-eth ylcyclop r op a n eca r boxyla te (8).
(a ) P r ep a r a tion of Di-ter t-bu tyl 2-Eth ylcyclop r op a n e-1,1-
d ica r boxyla te (5). To a suspension of benzyltriethylammo-
nium chloride (21.0 g, 92.2 mmol) in a 50% aqueous NaOH
solution (92.4 g in 185 mL of H2O) were successively added
di-tert-butyl malonate (20.0 g, 92.5 mmol) and 1,2-dibromo-
butane (30.0 g, 138.9 mmol). The reaction mixture was
vigorously stirred overnight at room temperature, and a
mixture of ice and water was then added. The crude product
was extracted with CH2Cl2 (3×) and sequentially washed with
water (3×) and brine. The organic layer was dried (MgSO4),
filtered, and concentrated. The residue was flash chromato-
graphed (7 cm, 2-4% Et2O in hexane) to afford the desired
cyclopropane derivative 5 (19.1 g, 70.7 mmol, 76% yield): 1H
NMR (CDCl3) δ 1.78-1.70 (m, 1H), 1.47 (s, 9H), 1.46 (s, 9H),
1.44-1.39 (m, 1H), 1.26-1.16 (m, 3H), 1.02 (t, 3H, J ) 7.6
Hz); MS 293 (M + Na).
(b) P r ep a r a tion of (1S,2R)/(1R,2S)-1-(ter t-Bu toxyca r -
bon yl)-2-eth ylcyclop r op a n eca r boxylic Acid (6). To a sus-
pension of potassium tert-butoxide (6.71 g, 59.8 mmol, 4.4
equiv) in dry ether (100 mL) at 0 °C was added H2O (270 µL,
15.0 mmol, 1.1 equiv). After 5 min, diester 5 (3.68 g, 13.6 mmol)
in ether (10 mL) was added to the suspension. The reaction
mixture was stirred overnight at room temperature and then
poured in a mixture of ice and water and washed with ether
(3×). The aqueous layer was acidified with a 10% aqueous
citric acid solution at 0 °C and extracted with AcOEt (3×). The
combined organic layer was successively washed with water
(2×) and brine. After the usual treatment (Na2SO4, filtration,
and concentration), the desired acid 6 was isolated as a pale
yellow oil (1.86 g, 8.68 mmol, 64% yield): 1H NMR (CDCl3) δ
2.09-2.01 (m, 1H), 1.98 (dd, J ) 3.8, 9.2 Hz, 1H), 1.81-1.70
(m, 1H), 1.66 (dd, J ) 3.0, 8.2 Hz, 1H), 1.63-1.56 (m, 1H),
1.51 (s, 9H), 1.0 (t, J ) 7.3 Hz, 3H).
led, after optimization, to the discovery of a vinyl-ACCA,
a derivative with improved potency compared to the
starting point (between 22- and 34-fold). Its beneficial
effect was demonstrated in four different series and
allowed for the identification of a nanomolar tetrapep-
tide inhibitor (compound 27, 19 nM). The vinyl-ACCA
is the first reported carboxylic acid containing a P1
residue that produced inhibitors that are significantly
more active than inhibitors containing a cysteine at the
same position.
Exp er im en ta l Section
Gen er a l Meth od s. 1H NMR spectra were obtained on a
Bruker AMX400 spectrometer, and the chemical shifts are
given in ppm, referenced to the internal deuterated solvent.
(c) P r ep a r a tion of ter t-Bu tyl (1R,2R)/(1S,2S)-2-Eth yl-
1-({[2-(t r im e t h ylsilyl)e t h oxy]ca r b on yl}a m in o)cyclo-

2516 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Rancourt et al.
p r op a n eca r boxyla te (7). To the acid 6 (2.02 g, 9.41 mmol)
in dry benzene (32 mL) were successively added Et3N (1.5 mL,
10.8 mmol, 1.14 equiv) and DPPA (2.2 mL, 10.2 mmol, 1.08
equiv). The reaction mixture was refluxed for 3.5 h, and then
2-(trimethylsilyl)ethanol (2.7 mL, 18.8 mmol, 2.0 equiv) was
added. The reflux was maintained overnight, and then the
reaction mixture was diluted with Et2O and successively
(mixture of rotamers ca. 1:2) δ 8.04 (d, J ) 8 Hz, 1H), 7.87 (d,
J ) 8 Hz, 1H), 7.82 (d, J ) 8 Hz, 1H), 7.55-7.41 (m, 4H), 5.95-
5.85 (m, 1H), 5.34-5.21 (m, 2H), 5.03-4.88 (m, 2H), 4.70-
4.56 (m, 2H), 4.48 (dd, J ) 7.3, 7.3 Hz) and 4.39 (dd, J ) 7.6,
7.6 Hz) (1H total), 4.28-4.23 (m, 1H), 3.81-3.55 (m, 2H), 2.46-
2.36 (m, 1H), 2.13-2.05 (m, 1H), 1.44 and 1.41 (s, 9H); MS
(FAB) 412 MH+.
washed with
a 10% aqueous citric acid solution, water,
(b) P r ep a r a tion of Boc-Va l-P r o-(4R)-(1-Na p h th ylm eth -
oxy) Allyl Ester . (4R)-1-(tert-Butoxycarbonyl)-4-(1-naphth-
ylmethoxy)-L-proline allyl ester (2.89 g, 7.02 mmol) was treated
with 4 N HCl/dioxane (30 mL). After 30 min at room temper-
ature, the mixture was concentrated, and the crude hydro-
chloride salt was coupled to Boc-Val-OH (1.53 g, 7.73 mmol)
with NMM (3.1 mL, 28.1 mmol) and TBTU (2.71 g, 8.43 mmol)
in DCM (35 mL) for 3.5 h. The reaction mixture was diluted
with EtOAc and successively washed with 10% aqueous citric
acid (2×), saturated aqueous NaHCO3 (2×), water (2×), and
brine (1×). The AcOEt layer was dried (MgSO4), filtered, and
evaporated to dryness to provide the crude desired dipeptide
as an ivory oil-foam (ca. 3.60 g, 100% yield): 1H NMR (CDCl3)
δ 8.04 (bd, J ) 8 Hz, 1H), 7.87 (bd, J ) 7 Hz, 1H), 7.82 (d, J
) 8 Hz, 1H), 7.56-7.40 (m, 4H), 5.93-5.85 (m, 1H), 5.34-
5.28 (m, 1H), 5.24-5.19 (m, 2H), 5.04 (d, J ) 12 Hz, 1H), 4.92
(d, J ) 12 Hz, 1H), 4.67-4.60 (m, 3H), 4.31-4.26 (m, 2H),
4.11-4.09 (m, 1H), 3.72 (dd, J ) 4, 11 Hz, 1H), 2.48-2.41 (m,
1H), 2.07-1.99 (m, 1H), 1.44-1.36 (m, 1H), 1.37 (s, 9H), 1.01
(d, J ) 7 Hz, 3H), 0.93 (d, J ) 7 Hz, 3H); MS (FAB) 509.3
(MH-), 511.3 (MH+), and 533.2 (M + Na).
(c) P r ep a r a tion of Boc-Ch g-Va l-P r o-(4R)-(1-n a p h th yl-
m eth oxy) Allyl Ester . The crude dipeptide obtained above
(ca. 7.02 mmol) was treated with 4 N HCl/dioxane (30 mL) as
described above. The crude hydrochloride salt was coupled to
Boc-Chg-OH‚H2O (2.13 g, 7.73 mmol) with NMM (3.1 mL,
28.09 mmol) and TBTU (2.71 g, 8.43 mmol) in CH2Cl2 (35 mL)
as described for the synthesis of the above dipeptide fragment
to provide the crude desired tripeptide as an ivory foam (ca.
4.6 g, 100% yield): 1H NMR (CDCl3) δ 8.06 (bd, J ) 8 Hz,
1H), 7.87 (bd, J ) 7.5 Hz, 1H), 7.82 (bd, J ) 8 Hz, 1H), 7.57-
7.40 (m, 4H), 6.46 (bd, J ) 8.5 Hz, 1H), 5.94-5.84 (m, 1H),
5.31 (dd, J ) 1, 17 Hz, 1H), 5.23 (dd, J ) 1, 10.5 Hz, 1H), 5.03
(d, J ) 12 Hz, 1H), 5.00-4.97 (m, 1H), 4.93 (d, J ) 12 Hz,
1H), 4.63-4.59 (m, 4H), 4.29-4.27 (m, 1H), 4.10-4.07 (m, 1H),
3.92-3.86 (m, 1H), 3.72 (dd, J ) 5, 11 Hz, 1H), 2.48-2.41 (m,
1H), 2.10-1.99 (m, 1H), 1.76-1.57 (m, 6H), 1.43 (s, 9H), 1.20-
0.92 (m, 6H), 1.00 (d, J ) 7 Hz, 3H), 0.93 (d, J ) 7 Hz, 3H);
MS (FAB) 648.5 (MH-) and 672.4 (M + Na).
(d ) P r ep a r a tion of Ac-Ch g-Va l-P r o-(4R)-(1-n a p h th yl-
m eth oxy) Allyl Ester . The crude tripeptide obtained above
(ca. 7.02 mmol) was treated with 4 N HCl/dioxane (30 mL)
under standard conditions. The crude hydrochloride salt was
further treated with acetic anhydride (1.33 mL, 14.1 mmol)
and NMM (3.1 mL, 28.1 mmol) in CH2Cl2 (35 mL) as described
above for the dipeptide fragment. The crude product was flash
purified (eluent, hexane-AcOEt, 30:70) to provide the acet-
ylated protected tripeptide as a white foam (3.39 g, 81% yield
over three steps): 1H NMR (CDCl3) (mainly one rotamer) δ
8.06 (d, J ) 8 Hz, 1H), 7.88 (bd, J ) 8 Hz, 1H), 7.83 (d, J ) 8
Hz, 1H), 7.58-7.41 (m, 4H), 6.37 (d, J ) 9 Hz, 1H), 5.97 (d, J
) 8.5 Hz, 1H), 5.94-5.84 (m, 1H), 5.31 (dd, J ) 1, 17 Hz, 1H),
5.24 (dd, J ) 1, 10.5 Hz, 1H), 5.05 (d, J ) 12 Hz, 1H), 4.94 (d,
J ) 12 Hz, 1H), 4.66-4.57 (m, 4H), 4.31-4.22 (m, 2H), 4.11-
4.05 (m, 1H), 3.73 (dd, J ) 4.5, 11 Hz, 1H), 2.50-2.43 (m, 1H),
2.09-2.01 (m, 2H), 2.00 (s, 3H), 1.68-1.55 (m, 5H), 1.15-0.89
(m, 6H), 0.99 (d, J ) 7 Hz, 3H), 0.91 (d, J ) 7 Hz, 3H); MS
(FAB) 590.3 (MH-), 592.4 (MH+), and 614.4 (M + Na)+.
(e) P r ep a r a tion of Ac-Ch g-Va l-P r o-(4R)-(1-n a p h th yl-
m eth oxy)-OH (9). To the acetylated tripeptide (3.39 g, 5.73
mmol) in a 1:1 mixture of anhydrous CH3CN-DCM (30 mL)
were successively added tetrakis(triphenylphosphine)palladium-
(0) catalyst (172 mg, 0.15 mmol), triphenylphosphine (78 mg,
0.30 mmol), and pyrrolidine (516 µL, 6.19 mmol). The reaction
mixture was stirred overnight at room temperature and then
concentrated in vacuo. The residue was dissolved in a mixture
of AcOEt and a 10% aqueous citric acid solution. The organic
saturated aqueous NaHCO3, water (2×), and brine. After the
usual treatment (MgSO4, filtration, and concentration), the
residue was purified by flash chromatography (5 cm, 10%
AcOEt-hexane) to afford the desired carbamate 7 (2.6 g, 7.88
mmol, 84% yield) as a pale yellow oil: 1H NMR (CDCl3) δ 5.1
(bs, 1H), 4.18-4.13 (m, 2H), 1.68-1.38 (m, 4H), 1.45 (s, 9H),
1.24-1.18 (m, 1H), 1.00-0.96 (m, 5H), 0.03 (s, 9H); MS (FAB)
330 (MH+).
(d ) P r ep a r a tion of ter t-Bu tyl (1R,2R)-1-Am in o-2-eth yl-
cyclop r op a n eca r boxyla te (8). To carbamate 7 (258 mg,
0.783 mmol) was added a 1.0 M TBAF solution in THF (940
µL, 0.94 mmol, 1.2 equiv). After 4.5 h, an additional amount
of 1.0 M TBAF was added (626 µL, 0.63 mmol, 0.8 equiv). The
reaction mixture was stirred overnight at room temperature,
refluxed for 30 min, and then diluted with AcOEt. The solution
was successively washed with water (2×) and brine. After the
usual treatment (MgSO4, filtration, and concentration), the
desired amine 8 was isolated (84 mg, 0.453 mmol, 58% yield)
as a pale yellow liquid: 1H NMR (CDCl3) δ 1.96 (bs, 2H), 1.60-
1.40 (m, 2H), 1.47 (s, 9H), 1.31-1.20 (m, 1H), 1.14 (dd, J )
4.1, 7.3 Hz, 1H), 1.02 (dd, J ) 4.1, 9.2 Hz, 1H), 0.94 (t, J ) 7.3
Hz, 3H).
Syn th esis of (4R)-1-(ter t-Bu toxyca r bon yl)-4-(1-n a p h -
th ylm eth oxy)-L-p r olin e (10).
Commercially available (4R)-1-(tert-butoxycarbonyl)-4-hydroxy-
L-proline (5.0 g, 21.6 mmol) was dissolved in THF (100 mL)
and cooled to 0 °C. Sodium hydride (60% dispersion in oil, 1.85
g, 45.4 mmol) was added portionwise over 10 min, and the
suspension was stirred at room temperature for 1 h. 1-(Bromo-
methyl)naphthalene29 (8.0 g, 36.2 mmol) was then added, and
the mixture was heated at reflux for 18 h. The mixture was
poured into water (300 mL) and washed with hexane. The
aqueous layer was acidified with 10% aqueous HCl and
extracted twice with AcOEt. The organic layers were combined
and washed with brine. After the usual treatment (MgSO4,
filtration, and concentration), the residue was purified by flash
chromatography (49:49:2 hexane-AcOEt-AcOH) to give the
desired compound 10 as a colorless oil (4.51 g, 56% yield): 1H
NMR (DMSO-d6) (indicating the presence of two rotamers) δ
8.07-7.87 (m, 3H), 7.62-7.46 (m, 4H), 5.64-5.53 (m, 2H), 5.06
(bs, 1H), 4.31-4.20 (m, 2H), 3.41-3.31 (m, 1H), 2.16-2.05 (m,
1H), 1.36 and 1.21 (s, 9H).
P r ep a r a tion of th e Tr ip ep tid e Acid F r a gm en t Ac-Ch g-
Va l-P r o-(4R)-(1-n a p h th ylm eth oxy)-OH (9). (a ) P r ep a r a -
tion of (4R)-1-(ter t-Bu toxyca r bon yl)-4-(1-n a p h th ylm eth -
oxy)-L-p r olin e Allyl Ester . The acid 10 obtained above (4.45
g, 11.98 mmol) was dissolved in anhydrous CH3CN (60 mL).
DBU (2.2 mL, 14.4 mmol) and allyl bromide (1.1 mL, 13.2
mmol) were added successively, and the reaction mixture was
stirred for 24 h at room temperature. The mixture was
concentrated, and the resulting oil was diluted with AcOEt
and water and successively washed with water (2×) and brine.
The AcOEt layer was dried (MgSO4), filtered, and evaporated
to dryness. The yellow oil was purified by flash chromatogra-
phy (eluent, hexane-AcOEt, 90:10 to 85:15) to provide (4R)-
1-(tert-butoxycarbonyl)-4-(1-naphthylmethoxy)-L-proline allyl
ester as a yellow oil (4.17 g, 85% yield): 1H NMR (CDCl3)

Peptide-Based Inhibitors of HCV NS3 Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2517
layer was successively washed with 10% aqueous citric acid
(2×), water (2×), and brine (2×). After the usual treatment
(MgSO4, filtration, and concentration), a crude light yellow
foam product was obtained. This material was triturated in
Et2O-DCM (85:15) to provide, after filtration, the title tri-
peptide as an off-white solid (3.0 g, 95% yield): 1H NMR
(CDCl3) δ 8.08 (d, J ) 8 Hz, 1H), 8.04 (bd, J ) 9 Hz, 1H), 7.88
(bd, J ) 7.5 Hz, 1H), 7.82 (d, J ) 8 Hz, 1H), 7.58-7.37 (m,
5H), 5.05 (d, J ) 12 Hz, 1H), 4.94 (d, J ) 12 Hz, 1H), 4.61 (t,
J ) 9.5, 19.5 Hz, 1H), 4.46-4.37 (m, 2H), 4.27 (bs, 1H), 4.17
(d, J ) 11 Hz, 1H), 3.74 (dd, J ) 4, 11 Hz, 1H), 2.49 (bdd, J )
7.5, 13 Hz, 1H), 2.17-2.09 (m, 1H), 2.04 (s, 3H), 2.03-1.94
(m, 1H), 1.79 (bd, J ) 12.5 Hz, 1H), 1.62-1.43 (m, 5H), 1.08-
0.85 (m, 5H), 1.00 (d, J ) 7 Hz, 3H), 0.90 (d, J ) 7 Hz, 3H);
MS (FAB) 550.3 (MH-).
Hz, 3H), 0.75 (dd, J ) 6.7, 14.0 Hz, 1H); MS 685 (M + Na);
HRMS calcd for C37H51N4O7 663.375776, found 663.375321.
Compound 14b: 1H NMR (DMSO-d6) δ 8.45 (s, 1H), 8.05
(d, J ) 8.1 Hz, 1H), 7.94 (d, J ) 7.8 Hz, 1H), 7.88 (d, J ) 8.3
Hz, 1H), 7.83-7.80 (m, 2H), 7.58-7.44 (m, 4H), 4.99 (d, J )
12.1 Hz, 1H), 4.90 (d, J ) 11.8 Hz, 1H), 4.37-4.32 (m, 2H),
4.26 (dd, J ) 8.0, 15.9 Hz, 1H), 4.19 (dd, J ) 7.3, 16.5 Hz,
1H), 4.11 (d, J ) 10.5 Hz, 1H), 3.70 (dd, J ) 4.4, 11.1 Hz, 1H),
2.25-2.17 (m, 1H), 2.04-1.90 (m, 2H), 1.84 (s, 3H), 1.62-1.32
(m, 8H), 1.29-1.22 (m, 4H), 1.03-0.88 (m, 9H), 0.85 (d, J )
6.7 Hz, 3H), 0.75 (dd, J ) 6.4, 16.5 Hz, 1H); MS 685 (M +
Na); HRMS calcd for C37H51N4O7 663.375776, found 663.375977.
Ch em ica l Resolu tion of th e Dip ep tid es 11 a n d 12. The
amine 8 was dissolved in anhydrous CH2Cl2 (120 mL). NMM
(8.5 mL, 77.6 mmol), (4R)-1-(tert-butoxycarbonyl)-4-(1-naph-
thylmethoxy)-L-proline 10 (10.1 g, 27.2 mmol), and HATU
(11.79 g, 31.03 mmol) were added successively. The reaction
mixture was stirred at room temperature overnight and then
worked up as described previously. The crude diastereomeric
mixture was separated by flash chromatography (eluent,
hexane-Et2O, 25:75) to provide the dipeptide 11 (the less polar
eluting spot) as a white foam (4.42 g, 64% of the theoretical
yield) and 12 (the more polar eluting spot) as an ivory foam
(4.0 g, 57% of theoretical yield). At this time, both isomers were
separated but the absolute configuration was still not known.
Compound 11: 1H NMR (CDCl3) (ca. 1:1 mixture of rotam-
ers) δ 8.05 (d, J ) 8.0 Hz, 1H), 7.86 (d, J ) 7.5 Hz, 1H), 7.81
(d, J ) 8.0 Hz, 1H), 7.55-7.40 (m, 4 H), 7.37 and 6.45 (bs,
1H), 5.20-4.91 (m, 2H), 4.41-3.44 (m, 4H), 2.70-2.02 (m, 2H),
1.61-1.25 (m, 5H), 1.44 (s, 9H), 1.41 (s, 9H), 0.96 (t, J ) 7.5
Hz, 3H); MS (FAB) 539 (MH+).
P r ep a r a tion of Com p ou n d s 14a a n d 14b. A partially
resolved27 mixture of (1R,2R) and (1S,2S) (2.9:1 ratio, respec-
tively) forms of Boc-protected methyl 1-amino-2-ethylcyclo-
propanecarboxylate (44 mg, 0.18 mmol) was treated with a 4
N HCl/dioxane solution (2 mL). After 30 min at room temper-
ature, the reaction mixture was concentrated. The residue was
dissolved in CH2Cl2 (2 mL), and then NMM (80 µL, 0.73 mmol),
the acid tripeptide component 9 (100 mg, 0.18 mmol), and
HATU (83 mg, 0.22 mmol) were added. The reaction mixture
was stirred overnight at room temperature, diluted with
AcOEt, and successively washed with 10% aqueous citric acid
(2×), saturated aqueous NaHCO3 (2×), H2O (2×), and brine.
After the usual treatment, the residue was flash chromato-
graphed (1 cm, 90% AcOEt-hexane) to afford a mixture of the
two desired diastereomers (corresponding methyl ester of 14a
and 14b) (5:1 ratio as estimated by HPLC at 220 nM) (58 mg,
0.086 mmol, 47% yield): 1H NMR (CDCl3) δ 8.05 (d, J ) 8.3
Hz, 1H), 7.87 (d, J ) 8.3 Hz, 1H), 7.82 (d, J ) 8.0 Hz, 1H),
7.56-7.40 (m, 5H), 6.56 (bs, 1H), 5.97 (d, J ) 8.6 Hz, 1H),
5.01 (d, J ) 12.1 Hz, 1H), 4.96 (d, J ) 11.8 Hz, 1H), 4.68-
4.61 (m, 2H), 4.42 (dd, J ) 4.1, 8.3 Hz, 1H), 4.28 (dd, J ) 8.3,
15.3 Hz, 1H), 3.94 (dd, J ) 3.2, 10.8 Hz, 1H), 3.67 (dd, J )
4.8, 10.5 Hz, 1H), 3.64 (s, 3H), 2.63-2.55 (m, 1H), 2.15-2.05
(m, 1H), 2.01 (s, 3H), 1.71-1.47 (m, 13H), 1.17-1.02 (m, 3H),
0.97-0.87 (m, 10H); MS 699 (M + Na).
The methyl esters (56 mg, 0.083 mmol) obtained above were
dissolved in a mixture of absolute MeOH (1 mL) and THF (2
mL). The solution was cooled at 0 °C for the addition of
hydrated lithium hydroxide (10 mg, 0.24 mmol) in water (1
mL). The reaction mixture was stirred for 30 min at 0 °C and
then overnight at room temperature. Another quantity of
hydrated lithium hydroxide (11 mg, 0.26 mmol) dissolved in
water (0.5 mL) was added. An HPLC analysis of the mixture
done 5 h later indicated that around 20% of starting ester was
still present in the reaction mixture. To force the reaction to
go to completion, a further 2.0 equiv of lithium hydroxide (7
mg, 0.17 mmol) in water (0.25 mL) was added. After 48 h at
room temperature, the reaction mixture was diluted with
water (5 mL) and extracted with ether (2×). The aqueous layer
was acidified with citric acid and extracted with AcOEt (3×).
The combined organic layer was successively washed with
water (2×) and brine (1×). After the usual treatment, the crude
mixture was purified by HPLC (Whatman10-ods-3, 2.2 × 50
cm, λ ) 230 nm, linear gradient at 15 mL/min, 5% CH3CN-
H2O for 10 min and then 5-58% CH3CN-H2O for 65 min and,
finally, 58% CH3CN-H2O for 15 min) to afford compounds 14a
(29 mg, 0.044 mmol, 53% yield) and 14b (3.5 mg, 0.0053 mmol,
6.4% yield). Homogeneity for 14a : 93% by HPLC (contains
6% of 14b) and 99.2% as evaluated by CE. Homogeneity for
14b: 95% by HPLC (contains 1.6% of 14a ) and 95.6% by CE.
Compound 12: 1H NMR (CDCl3) (ca. 1:1 mixture of rotam-
ers) δ 8.04 (d, J ) 8.0 Hz, 1H), 7.86 (d, J ) 8.1 Hz, 1H), 7.81
(d, J ) 7.9 Hz, 1H), 7.54-7.32 (m, 4H), 7.32 and 6.37 (bs, 1H),
5.03-4.87 (m, 2H), 4.42-3.42 (m, 4H), 2.71-2.02 (m, 2H),
1.64-1.10 (m, 5H), 1.44 (s, 9H), 1.41 (s, 9H), 1.00 (t, J ) 7.3
Hz, 3H); MS (FAB) 539 (MH+).
Deter m in a tion of th e Absolu te Con figu r a tion of Com -
p ou n d s 11 a n d 12 by Cor r ela tion w ith Kn ow n ter t-Bu tyl
(1R-Am in o-2R)-eth ylcyclop r op a n eca r boxyla te. tert-Butyl
(1R,2R)-1-[(tert-butoxycarbonyl)amino]-2-ethylcyclopropane-
carboxylate 1323 (13.2 mg, 0.046 mmol) was dissolved in 1 M
HCl/EtOAc (240 µL) and stirred for approximately 48 h. The
mixture was evaporated to dryness to provide its corresponding
hydrochloride salt as a light yellow paste. This material was
coupled to (4R)-1-(tert-butoxycarbonyl)-4-(1-naphthylmethoxy)-
L-proline 10 (18 mg, 0.049 mmol) as described above, using
NMM (20.3 µL, 0.185 mmol) and HATU (21.1 mg, 0.056 mmol)
in CH2Cl2. The crude material was purified by flash chroma-
tography (eluent, hexane-Et2O, 50:50) to provide a pure
dipeptide as an oil (7.7 mg, 31% yield). By TLC, HPLC, and
1H NMR comparison, this dipeptide was found to be identical
to the less polar compound 11 obtained above, thus identifying
the absolute configuration of 11 as (1R,2R).
P r ep a r a tion of Com p ou n d s 20a a n d 20b. (a ) P r ep a r a -
tion of 2,2-Dieth yl-1-(m eth oxyca r bon yl)cyclop r op a n e-
ca r boxylic Acid . To dimethyl 2,2-diethylcyclopropane-1,1-
dicarboxylate22 (1.24 g, 5.8 mmol) in methanol (6 mL) was
added a 1 N aqueous NaOH solution (5.79 mL, 5.79 mmol,
0.97 equiv). The reaction mixture was stirred for 5 days at
room temperature, and then the methanol was removed in
vacuo. The aqueous layer was washed with ether (2×),
acidified with a 10% aqueous citric acid solution, and extracted
with ethyl acetate. After the usual treatment (MgSO4, filtra-
tion, and concentration), the desired acid, 2,2-diethyl-1-(meth-
oxycarbonyl)cyclopropanecarboxylic acid, was isolated as a
colorless oil (1.08 g, 5.4 mmol, 93% yield): 1H NMR (CDCl3) δ
1.79 (d, J ) 5.1 Hz, 1H), 1.69 (d, J ) 5.1 Hz, 1H), 1.65-1.49
(m, 3H), 1.47-1.39 (m, 1H), 0.90-0.84 (m, 6H).
Compound 14a : 1H NMR (DMSO-d6) δ 8.37 (s, 1H), 8.04
(d, J ) 7.3 Hz, 1H), 7.94 (dd, J ) 2.9, 7.3 Hz, 1H), 7.88 (d, J
) 8.0 Hz, 1H), 7.84-7.80 (m, 2H), 7.57-7.51 (m, 3H), 7.47 (d,
J ) 8.0 Hz, 1H), 4.99 (d, J ) 11.8 Hz, 1H), 4.90 (d, J ) 11.8
Hz, 1H), 4.36 (dd, J ) 8.3, 16.8 Hz, 1H), 4.34-4.31 (m, 1H),
4.20 (dd, J ) 8.0, 16.2 Hz, 1H), 4.10 (d, J ) 11.1 Hz, 1H), 3.75
(dd, J ) 4.5, 11.4 Hz, 1H), 2.22-2.13 (m, 1H), 2.03-1.93 (m,
2H), 1.84 (s, 3H), 1.63-1.42 (m, 8H), 1.35-1.22 (m, 2H), 1.20
(dd, J ) 4.5, 7.6 Hz, 1H), 1.03-0.88 (m, 11H), 0.85 (d, J ) 6.7
(b) P r ep a r a tion of Meth yl 1-Am in o-2,2-d ieth ylcyclo-
p r op a n eca r boxyla te. To the acid obtained above (300 mg,
1.50 mmol) in dry THF (10 mL) were successively added
triethylamine (229 µL, 1.65 mmol, 1.1 equiv) and DPPA (348
µL, 1.62 mmol, 1.08 equiv). The reaction mixture was refluxed

2518 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Rancourt et al.
for 2 h, and then 2-(trimethylsilyl)ethanol (430 µL, 3.00 mmol,
2.0 equiv) was added. After 16 h under reflux, the reaction
mixture was concentrated and then diluted with ethyl acetate.
The organic layer was successively washed with 10% aqueous
citric acid, (2×), saturated aqueous NaHCO3 (2×), and brine.
After the usual treatment (MgSO4, filtration, and concentra-
tion), the residue was flash chromatographed (3 cm, 10%
AcOEt-hexane) to afford the desired carbamate (300 mg, 1.04
mmol, 70% yield) as a colorless oil: 1H NMR (CDCl3) δ 5.1
(bs, 1H), 4.18-4.11 (m, 2H), 3.71 (s, 3H), 1.68-1.59 (m, 3H),
1.53-1.40 (m, 2H), 1.00-0.95 (m, 6H), 0.82 (t, J ) 7.3 Hz, 3H),
0.03 (s, 9H).
To the carbamate obtained above (300 mg, 1.04 mmol) in
dry THF (4 mL) was added a 1.0 M TBAF solution in THF
(2.1 mL, 2.1 mmol, 2.0 equiv). The reaction mixture was
refluxed for 30 min, and then AcOEt was added and the
mixture was successively washed with sodium bicarbonate
(2×) and brine. After the usual treatment (MgSO4, filtration,
and concentration), methyl 1-amino-2,2-diethylcyclopropan-
ecarboxylate was isolated (135 mg, 0.788 mmol, 75% yield) as
a yellow oil: 1H NMR (CDCl3) δ 3.65 (s, 3H), 1.74-1.62 (m,
1H), 1.51-1.39 (m, 2H), 1.32 (d, J ) 4.8 Hz, 1H), 0.94 (t, J )
7.3 Hz, 3H), 0.93-0.89 (m, 1H), 0.81 (t, J ) 7.6 Hz, 3H), 0.67
(d, J ) 4.4 Hz, 1H).
(c) P r ep a r a tion of th e Cor r esp on d in g Meth yl Ester of
Com p ou n d s 20a a n d 20b. Coupling of the methyl 1-amino-
2,2-diethylcyclopropanecarboxylate obtained above (45 mg,
0.26 mmol) to the tripeptide fragment 9 (145 mg, 0.26 mmol)
was done as described for the preparation of compounds 14a
and 14b. The crude material was flash chromatographed (3
cm, 100% AcOEt-hexane) to afford a mixture of the desired
tetrapeptides (164 mg, 88% yield): 1H NMR (CDCl3) (mixture
of two diastereomers) δ 8.05 (d, J ) 8.0 Hz, 1H), 7.87 (d, J )
8.0 Hz, 1H), 7.82 (d, J ) 8.0 Hz, 1H), 7.70-7.64 (m, 1H), 7.57-
7.40 (m, 4H), 6.51-6.43 (m, 1H), 5.95 (d, J ) 8.6 Hz, 1H),
5.03-4.94 (m, 2H), 4.72-4.55 (m, 2H), 4.45-4.38 (m, 1H), 4.26
(dd, J ) 7.0, 7.0 Hz, 1H), 3.96-3.90 (m, 1H), 3.69-3.58 (m,
1H), 3.65 and 3.61 (s, 3H), 2.62-2.47 (m, 1H), 2.07-1.96 (m,
2H), 2.04 and 2.01 (s, 3H), 1.71-1.57 (m, 10H), 1.50-1.30 (m,
1H), 1.19-1.14 (m, 2H), 1.01-0.79 (m, 16 H).
(d ) P r ep a r a tion of Com p ou n d s 20a a n d 20b. The
mixture of esters (101 mg, 0.14 mmol) was saponified using
the conditions described for the preparation of compounds 14a
and 14b. The crude mixture was dissolved in acetic acid and
purified by HPLC to afford the less polar isomer 20a (96%
homogeneous by HPLC and 99.2% by CE) and the more polar
isomer 20b (99% homogeneous by HPLC and 99.9% by CE).
Note that the compound represented as 20 in Table 2, entry
2, corresponds to 20a (20b was omitted for clarity).
Compound 20a : 1H NMR (DMSO-d6) δ 8.30 (s, 1H), 8.05
(d, J ) 7.6 Hz, 1H), 7.94 (d, J ) 8.3 Hz, 1H), 7.88 (d, J ) 8.6
Hz, 1H), 7.86 (d, J ) 9.5 Hz, 1H), 7.81 (d, J ) 8.9 Hz, 1H),
7.58-7.44 (m, 4H), 4.99 (d, J ) 11.8 Hz, 1H), 4.89 (d, J ) 11.8
Hz, 1H), 4.43 (dd, J ) 8.0, 8.0 Hz, 1H), 4.37 (dd, J ) 8.0, 8.0
Hz, 1H), 4.34-4.29 (m, 1H), 4.20 (dd, J ) 7.6, 7.6 Hz, 1H),
4.11 (d, J ) 11.4 Hz, 1H), 3.71 (dd, J ) 4.5, 10.8 Hz, 1H), 2.15-
2.06 (m, 1H), 2.01-1.92 (m, 2H), 1.84 (s, 3H), 1.68-1.42 (m,
9H), 1.34 (d, J ) 5.1 Hz, 1H), 1.23-1.14 (m, 1H), 1.05-0.84
(m, 15H), 0.80 (t, J ) 7.3 Hz, 3H), 0.71 (d, J ) 5.1 Hz, 1H);
MS 713 (M + Na); HRMS calcd for C39H55N4O7 691.407076,
found 691.406387.
Compound 20b: 1H NMR (DMSO-d6) δ 8.33 (s, 1H), 8.06
(d, J ) 8.0 Hz, 1H), 7.94 (d, J ) 8.3 Hz, 1H), 7.88 (d, J ) 8.3
Hz, 1H), 7.82 (dd, J ) 8.3, 16.9 Hz, 1H), 7.58-7.45 (m, 5H),
4.99 (d, J ) 12.1 Hz, 1H), 4.91 (d, J ) 12.1 Hz, 1H), 4.39 (dd,
J ) 7.6, 7.6 Hz, 1H), 4.36-4.32 (m, 2H), 4.19 (dd, J ) 7.3, 7.3
Hz, 1H), 4.12 (d, J ) 11.1 Hz, 1H), 3.71 (dd, J ) 4.1, 11.1 Hz,
1H), 2.27-2.19 2.02-1.90 (m, 2H), 1.84 (s, 3H), 1.71 (dd, J )
7.0, 14.0 Hz, 1H), 1.64-1.39 (m, 10 H), 1.30 (dd, J ) 7.0, 14
Hz, 1H), 1.04-0.70 (m, 18H); MS 713 (M + Na); HRMS calcd
for C39H55N4O7 691.407076, found 691.407126.
propane was used instead of 1,2-dibromobutane for the cyclo-
propane ring elaboration. The diastereomers were separated
at the ester stage.
Compound 15a (91% homogeneous by HPLC; contains 5.7%
of 15b, 98.9% homogeneous by CE): 1H NMR (DMSO-d6) δ
8.32 (s, 1H), 8.04 (d, J ) 8.3 Hz, 1H), 7.94 (dd, J ) 2.9, 7.3
Hz, 1H), 7.88 (d, J ) 8.3 Hz, 1H), 7.85-7.78 (m, 2H), 7.57-
7.45 (m, 4H), 4.98 (d, J ) 11.8 Hz, 1H), 4.90 (d, J ) 12.1 Hz,
1H), 4.38-3.32 (m, 2H), 4.25 (dd, J ) 8.0, 15.9 Hz, 1H), 4.19
(dd, J ) 8.0, 16.2 Hz, 1H), 4.08 (d, J ) 11.1 Hz, 1H), 3.76 (dd,
J ) 4.5, 11.1 Hz, 1H), 2.20-2.15 (m, 1H), 2.03-1.97 (m, 2H),
1.84 (s, 3H), 1.60-1.45 (m, 6H), 1.38-1.27 (m, 1H), 1.15 (d, J
) 6.4 Hz, 3H), 1.05-0.90 (m, 6H), 0.89 (d, J ) 6.7 Hz, 3H),
0.85 (d, J ) 6.7 Hz, 3H), 0.76 (dd, J ) 6.7, 13.0 Hz, 1H); MS
649 (MH+), 671 (M + Na); HRMS calcd for C36H49N4O7
649.360125, found 649.361314.
Compound 15b (91% homogeneous by HPLC; contains 8.4%
of 15a , 99.0% homogeneous by CE ): 1H NMR (DMSO-d6) δ
8.35 (bs, 1H), 8.05 (d, J ) 7.6 Hz, 1H), 7.94 (dd, J ) 2.5, 7.3
Hz, 1H), 7.95-7.81 (m, 4H), 4.99 (d, J ) 12.1 Hz, 1H), 4.89 (d,
J ) 11.8 Hz, 1H), 4.37-4.09 (m, 5H), 3.70 (dd, J ) 4.1, 11.1
Hz, 1H), 2.23-2.15 (m, 1H), 2.05-1.92 (m, 2H), 1.84 (s, 3H),
1.65-1.17 (m, 6H), 1.13 (d, J ) 6.0 Hz, 3H), 1.07-0.9 (m, 10H),
0.90 (d, J ) 6.7 Hz, 3H), 0.85 (d, J ) 6.7 Hz, 3H), 0.75 (dd, J
) 6.7, 18.1 Hz, 1H); MS 649 (M + H), 671 (M + Na); HRMS
calcd for C36H49N4O7 649.360125, found 649.360923.
P r ep a r a tion of Com p ou n d s 16a a n d 16b. (a ) P r ep a r a -
tion of 4-P r op yl-1,3,2-d ioxa th iola n e 2,2-Dioxid e. To 1,2-
pentanediol (5.0 g, 48.0 mmol) in carbon tetrachloride (48 mL)
was added thionyl chloride (4.2 mL, 57.6 mmol). The reaction
mixture was refluxed for 30 min and then cooled to 0 °C for
the successive addition of CH3CN (48 mL), RuCl3‚H2O (8 mg,
0.038 mmol), NaIO4 (15.4 g, 72.0 mmol), and H2O (72 mL).
After being stirred at 0 °C for 15 min and for 1 h at room
temperature, the reaction mixture was diluted with ether (380
mL) and successively washed with H2O (20 mL), saturated
aqueous NaHCO3 (2 × 20 mL), and brine (20 mL). The solution
was dried over MgSO4 and filtered through a short pad of silica
gel. After concentration, a quantitative yield of the desired
cyclic sulfate was obtained (8.0 g, 48.0 mmol).
(b ) P r ep a r a t ion of Dim et h yl 2-P r op ylcyclop r op a n e-
1,1-d ica r boxyla te. The crude intermediate prepared above
(6.29 g, 37.8 mmol) was then added to a suspension of sodium
hydride (3.2 g of a 60% dispersion in mineral oil, 80.0 mmol)
and dimethyl malonate (5.0 g, 37.8 mmol) in dry THF (190
mL). The reaction mixture was refluxed for 36 h and then
concentrated. To the residue was added water and AcOEt. The
layers were separated, and the aqueous phase was further
extracted with AcOEt (2×). The combined organic layer was
successively washed with water (2×) and brine. After the usual
treatment, the desired dimethyl 2-propylcyclopropane-1,1-
dicarboxylate was isolated (7.61 g of crude material): 1H NMR
(CDCl3) δ 3.76 (s, 3H), 3.72 (s, 3H), 1.93-1.85 (m, 1H), 1.52-
1.35 (m, 4H), 1.31-1.09 (m, 3H), 0.92 (t, J ) 7.0 Hz, 3H).
(c) P r ep a r a tion of (1R,2R)/(1S,2S)-1-(Meth oxyca r bo-
n yl)-2-p r op ylcyclop r op a n eca r boxylic Acid . To the cyclo-
propane derivative obtained above (5.0 g, 24.97 mmol) in
absolute MeOH (11 mL) was added a water solution (27 mL)
of sodium carbonate (2.7 g, 25.47 mmol). The reaction mixture
was stirred for 3 days at room temperature and then refluxed
overnight. After removal of the MeOH in vacuo, the aqueous
layer was extracted with ether (3×), acidified at 0 °C with a
cold 10% aqueous HCl solution, and extracted with AcOEt
(3×). The combined organic layer was washed with brine (2×).
After the usual treatment, the desired monoacid was isolated
as a yellow oil (3.37 g, 18.08 mmol, 72% yield): 1H NMR
(CDCl3) δ 3.83 (s, 3H), 2.16-2.07 (m, 1H), 1.98 (dd, J ) 4.1,
9.2 Hz, 1H), 1.73 (dd, J ) 4.1, 8.6 Hz, 1H), 1.59-1.52 (m, 2H),
1.48-1.32 (m, 2H), 0.92 (t, J ) 7.3 Hz, 3H).
(d ) P r ep a r a tion of Com p ou n d s 16a a n d 16b. The above
acid was successively treated with DPPA and TBAF and then
coupled to the tripeptide fragment 9 as described above in the
preparation of compounds 14a and 14b.
P r ep a r a tion of Com p ou n d s 15a a n d 15b. These tetra-
peptides were prepared using the procedure already described
for the preparation of compounds 14a and 14b. 1,2-Dibromo-

Peptide-Based Inhibitors of HCV NS3 Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2519
Com p ou n d 16a (95% homogeneous by HPLC; 98.0% ho-
mogeneous by CE): 1H NMR (DMSO-d6) δ 8.38 (s, 1H), 8.04
(d, J ) 7.6 Hz, 1H), 7.94 (s, J ) 7.6 Hz, 1H), 7.90-7.79 (m,
3H), 7.59-7.42 (m, 4H), 4.98 (d, J ) 12.1 Hz, 1H), 4.89 (d, J
) 12.1 Hz, 1H), 4.39-4.15 (m, 4H), 4.10 (d, J ) 10.8 Hz, 1H),
3.79-3.71 (m, 1H), 2.21-2.12 (m, 1H), 2.04-1.92 (m, 1H), 1.84
(s, 3H), 1.62-1.41 (m, 7H), 1.39-1.28 (m, 2H), 1.21-1.16 (m,
1H), 1.07-0.81 (m, 15 H); MS 677 (MH+); HRMS calcd for
solid (24.4 mg, 0.036 mmol, 59% yield; 99% homogeneous by
HPLC and 99.1% homogeneous by CE): 1H NMR (DMSO-d6)
δ 8.25 (bs, 1H), 8.04 (d, J ) 7.6 Hz, 1H), 7.94 (d, J ) 8.3 Hz,
1H), 7.88 (d, J ) 8.3 Hz, 1H), 7.88-7.78 (m, 2H), 7.57-7.44
(m, 4H), 4.99 (d, J ) 11.8 Hz, 1H), 4.89 (d, J ) 12.1 Hz, 1H),
4.37 (dd, J ) 8.3, 8.3 Hz, 1H), 4.32 (bs, 1H), 4.27 (dd, J ) 8.0,
8.0 Hz, 1H), 4.19 (dd, J ) 7.9, 7.9 Hz, 1H), 4.09 (d, J ) 11.8
Hz, 1H), 3.74 (dd, J ) 4.4, 11.1 Hz, 1H), 2.18-2.13 (m, 1H),
2.05-1.93 (m, 1H), 1.84 (s, 3H), 1.72-1.43 (m, 7H), 1.08-0.71
(m, 21H); MS 699 (M + Na); HRMS calcd for C38H53N4O7
677.391426, found 677.390801.
C
38H53N4O7 677.391426, found 677.392437.
Com p ou n d 16b (87% homogeneous by HPLC; contains 12%
of 16a , 94.1% homogeneous by CE): 1H NMR (DMSO-d6) δ
8.47 (s, 1H), 8.05 (d, J ) 7.0 Hz, 1H), 7.94 (d, J ) 8.6 Hz, 1H),
7.91-7.79 (m, 3H), 7.58-7.44 (m, 4H), 4.99 (d, J ) 12.1 Hz,
1H), 4.90 (d, J ) 12.1 Hz, 1H), 4.37-4.29 (m, 2H), 4.26 (dd, J
) 7.6, 7.6 Hz, 1H), 4.19 (dd, J ) 7.6, 7.6 Hz, 1H), 4.13 (d, J )
11.5 Hz, 1H), 3.69 (dd, J ) 3.8, 10.8 Hz, 1H), 2.25-2.16 (m,
1H), 2.03-1.89 (m, 2H), 1.84 (s, 3H), 1.62-1.22 (m, 12 H),
1.05-0.79 (m, 15 H); MS 677 (MH+); HRMS calcd for
The enantiomer of Boc-methano-leucine18b was converted to
compound 17b using the conditions described for the prepara-
tion of compound 17a .
Compound 17b (98% homogeneous by HPLC and 98.2%
homogeneous by CE): 1H NMR (DMSO-d6) δ 8.41 (bs, 1H),
8.05 (d, J ) 7.6 Hz, 1H), 7.94 (d, J ) 7.6 Hz, 1H), 7.88 (d, J )
8.0 Hz, 1H), 7.84-7.78 (m, 2H), 7.58-7.44 (m, 4H), 4.99 (d, J
) 11.8 Hz, 1H), 4.91 (d, J ) 12.1 Hz, 1H), 4.37-4.30 (m, 2H),
4.27 (dd, J ) 7.6, 7.6 Hz, 1H), 4.19 (dd, J ) 7.3, 7.3 Hz, 1H),
4.10 (d, J ) 11.5 Hz, 1H), 3.74-3.67 (m, 1H), 2.25-2.16 (m,
1H), 2.05-1.89 (m, 2H), 1.84 (s, 3H), 1.62-1.40 (m, 7H), 1.30-
1.22 (m, 1H), 1.16-0.86 (m, 12H), 0.96 (d, J ) 6.7 Hz, 3H),
0.85 (d, J ) 6.7 Hz, 3H), 0.77 (dd, J ) 6.7, 15.9 Hz, 1H). MS
675 (M - H); HRMS calcd for C38H53N4O7 677.391426, found
677.388923.
C
38H53N4O7 677.391426, found 677.392265.
P r ep a r a tion of Com p ou n d s 17a a n d 17b. (a ) P r ep a r a -
tion of Boc-2,3-m eth a n o-leu cin e Allyl Ester . To a suspen-
sion of Boc-2,3-methano-leucine18b (53 mg, 0.22 mmol) in
CH3CN (2.0 mL) were successively added DBU (39 µL, 0.26
mmol) and allyl bromide (75 µL, 0.87 mmol). After 6 h at room
temperature, another amount of DBU (10 µL, 0.067 mmol) and
allyl bromide (38 µL,0.44 mmol) was added. The reaction
mixture was stirred overnight at room temperature and then
concentrated. The residue was dissolved in AcOEt and suc-
cessively washed with 10% aqueous citric acid, 10% aqueous
Na2CO3, water (2×), and brine. After the usual treatment
(MgSO4, filtration, and concentration), the residue was flash
chromatographed (1 cm, 5% AcOEt-hexane) to afford the
desired allyl ester as a colorless oil (50 mg, 0.176 mmol, 81%
yield): 1H NMR (CDCl3) δ 5.94-5.85 (m, 1H), 5.34 (dd, J )
1.6, 17.2 Hz, 1H), 5.22 (d, J ) 10.5 Hz, 1H), 5.12 (bs, 1H),
4.67-4.56 (m, 2H), 1.64-1.59 (m, 1H), 1.44 (s, 9H), 1.28-1.17
(m, 2H), 1.02 (dd, J ) 6.4, 14.0 Hz, 1H), 1.01 (d, J ) 6.7 Hz,
3H), 0.91 (d. J ) 6.7 Hz, 3H); MS 284 (MH+), 306 (M + Na).
(b) P r ep a r a tion of th e Cor r esp on d in g Allyl Ester of
Com p ou n d s 17a a n d 17b. The ester obtained above (24.4
mg, 0.086 mmol) was treated with a 4 N HCl/dioxane solution
(1 mL). After 30 min at room temperature, the reaction
mixture was concentrated. The residue was dissolved in CH2-
Cl2 (1 mL) and successively treated with NMM (38 µL, 0.344
mmol), the tripeptide fragment 9 (50 mg, 0.90 mmol), and
HATU (39 mg, 0.10 mmol) as for compounds 14a and 14b.
After flash chromatography (1 cm, 80% AcOEt-hexane), the
desired tetrapeptide was isolated (46.4 mg, 0.065 mmol, 75%
yield): 1H NMR (CDCl3) δ 7.99 (d, J ) 8.3 Hz, 1H), 7.81 (d, J
) 8.0 Hz, 1H), 7.76 (d, J ) 8.0 Hz, 1H), 7.49-7.35 (m, 4H),
6.42 (bs, 1H), 6.10 (d, J ) 8.6 Hz, 1H), 5.89 (d, J ) 8.9 Hz,
1H), 5.81-5.71 (m 1H), 5.19 (dd, J ) 1.3, 17.2 Hz, 1H), 5.10
(d, J ) 1.3, 11.4 Hz, 1H), 4.95 (d, J ) 12.4 Hz, 1H), 4.91 (d, J
) 12.1 Hz, 1H), 4.64-4.42 (m, 3H), 4.39-4.31 (m, 1H), 4.27-
4.16 (m, 1H), 4.14-4.02 (m, 2H), 3.92-3.83 (m, 1H), 3.63-
3.57 (m, 1H), 2.61-2.51 (m, 2H), 2.02-1.95 (m, 1H), 1.95 (s,
3H), 1.63-1.48 (m, 6H), 1.21-0.72 (m, 20H); MS 739 (M +
Na).
(c) P r ep a r a tion of Com p ou n d s 17a a n d 17b. The tetra-
peptide obtained above (44.2 mg, 0.062 mmol) was dissolved
in CH3CN (1 mL) and successively treated with triphenylphos-
phine (0.84 mg, 0.0032 mmol), Pd(PPh3)4 (1.9 mg, 0.0016
mmol), and pyrrolidine (5.6 mL, 0.067 mmol). The reaction
mixture was stirred overnight at room temperature. Since the
reaction was not completed, the same amount of reagents was
added again. The reaction mixture was stirred for another 4
h at room temperature, and then the CH3CN was evaporated.
The residue was dissolved in AcOEt and successively washed
with 10% aqueous citric acid (2×), water (2×), and brine. After
the usual treatment (MgSO4, filtration, and concentration), the
residue was flash chromatographed (1 cm, AcOEt and then
eluted with a mixture of CHCl3, MeOH, andAcOH). The
product obtained was dissolved in a mixture of CH3CN and
water and lyophilized to afford the desired acid 17a as a white
P r ep a r a tion of Com p ou n d s 18a a n d 18b. Using 1,2-
dibromopropylbenzene and the same conditions used for the
generation of tetrapeptides 14a and 14b, the desired acids 18a
and 18b were prepared.
Compound 18a (92% homogeneous by HPLC; contains 5%
of 18b; 98.2% homogeneous by CE): 1H NMR (DMSO-d6) δ
8.33 (bs, 1H), 8.04 (d, J ) 8.9 Hz, 1H), 7.93 (d, J ) 8.5 Hz,
1H), 7.87 (d, J ) 8.3 Hz, 1H), 7.89-7.78 (m, 1H), 7.56-7.44
(m, 4H), 7.30-7.13 (m, 5H), 4.98 (d, J ) 11.8 Hz, 1H), 4.88 (d,
J ) 11.8 Hz, 1H), 4.36 (dd, J ) 8.3, 8.3 Hz, 1H), 4.33-4.25
(m, 2H), 4.19 (dd, J ) 7.6, 7.6 Hz, 1H), 4.08 (d, J ) 11.1 Hz,
1H), 3.74 (dd, J ) 4.5, 11.5 Hz, 1H), 2.94-2.80 (m, 2H), 2.20-
2.11 (m, 1H), 2.04-1.93 (m, 2H), 1.84 (s, 3H), 1.63-1.42 (m,
7H), 1.38-1.30 (m, 1H), 1.12-0.81 (m, 6H), 0.89 (d, J ) 6.4
Hz, 3H), 0.84 (d, J ) 6.4 Hz, 3H), 0.75 (dd, J ) 6.7, 18.8 Hz,
1H); MS 747 (M + Na); HRMS calcd for C42H53N4O7 725.391426,
found 725.393120.
Compound 18b (91% homogeneous by HPLC; contains 9%
of 18a ; 97.5% homogeneous by CE): 1H NMR (DMSO-d6) δ
8.48 (bs, 1H), 8.04 (d, J ) 7.6 Hz, 1H), 7.93 (d, J ) 7.3 Hz,
1H), 7.88 (d, J ) 8.3 Hz, 1H), 7.84-7.77 (m, 2H), 7.58-7.44
(m, 4H), 7.32-7.15 (m, 5H), 4.98 (d, J ) 12.1 Hz, 1H), 4.89 (d,
J ) 12.1 Hz, 1H), 4.38-4.25 (m, 2H), 4.18 (dd, J ) 7.6, 7.6
Hz, 1H), 4.11 (d, J ) 11.1 Hz, 1H), 3.74-3.67 (m, 1H), 2.91-
2.75 (m, 2H), 2.25-2.16 (m, 1H), 2.04-1.90 (m, 2H), 1.83 (s,
3H), 1.69-1.40 (m, 7H), 1.29-1.22 (m, 1H), 1.09-0.92 (m, 6H),
0.89 (d, J ) 6.4 Hz, 3H), 0.84 (d, J ) 6.7 Hz, 3H), 0.73 (dd, J
) 7.0, 15.3 Hz, 1H); MS 747 (M + Na); HRMS calcd for
C
42H53N4O7 725.391426, found 725.393184.
P r ep a r a tion of Com p ou n d s 19a a n d 19b. 4-Phenyl-1-
butene was treated with bromine in CCl4 to produce 1,2-
dibromo-4-phenylbutane. This dibromide was used to synthe-
size the title compounds using the procedure already described
for compounds 14a and 14b.
Compound 19a (97% homogeneous by HPLC; 98.9% homo-
geneous by CE): 1H NMR (DMSO-d6) δ 8.37 (s, 1H), 8.04 (d,
J ) 7.3 Hz, 1H), 7.94 (d, J ) 7.3 Hz, 1H), 7.88 (d, J ) 8.0 Hz,
1H), 7.85-7.80 (m, 2H), 7.58-7.45 (m, 4H), 7.28-7.23 (m, 2H),
7.19-7.13 (m, 3H), 4.98 (d, J ) 12.1 Hz, 1H), 4.90 (d, J ) 12.1
Hz, 1H), 4.39-4.30 (m, 2H), 4.27 (dd, J ) 8.0, 8.0 Hz, 1H),
4.20 (dd, J ) 8.3, 8.3 Hz, 1H), 4.10 (d, 11.4 Hz, 1H), 3.75 (dd,
J ) 4.1, 11.1 Hz, 1H), 2.69-2.59 (m, 2H), 2.22-2.14 (m, 1H),
2.05-1.94 (m, 2H), 1.84 (s, 3H), 1.82-1.73 (m, 2H), 1.63-1.33
(m, 6H), 1.26-1.20 (m, 1H), 1.07-0.91 (m, 6H), 0.89 (d, J )
6.7 Hz, 3H), 0.84 (d, J ) 6.7 Hz, 3H), 0.76 (dd, J ) 6.7, 14.0
Hz, 1H); MS 761 (M + Na); HRMS calcd for C43H55N4O7
739.407076, found 739.405613.

2520 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Rancourt et al.
Compound 19b (85% homogeneous by HPLC; contains 15%
of 19a ; 98.1% homogeneous by CE): 1H NMR (DMSO-d6) δ
8.46 (s, 1H), 8.05 (d, J ) 7.3 Hz, 1H), 7.94 (d, J ) 7.31 Hz,
1H), 7.88 (d, J ) 7.9 Hz, 1H), 7.84-7.77 (m, 2H), 7.59-7.44
(m, 4H), 7.31-7.23 (m, 2H), 7.21-7.13 (m, 3H), 4.99 (d, J )
11.8 Hz, 1H), 4.90 (d, J ) 11.8 Hz, 1H), 4.39-4.30 (m, 2H),
4.29 (dd, J ) 8.0, 8.0 Hz, 1H), 4.19 (dd, J ) 7.3, 7.3 Hz, 1H),
4.12 (d, J ) 11.1 Hz, 1H), 3.74-3.67 (m, 1H), 2.71-2.53 (m,
2H), 2.26-2.15 (m, 1H), 2.04-1.92 (m, 2H), 1.84 (s, 3H), 1.79-
1.70 (m, 2H), 1.65-1.35 (m, 6H), 1.32-1.20 (m, 1H), 1.07-
0.92 (m, 6H), 0.89 (d, J ) 6.4 Hz, 3H), 0.85 (d, J ) 6.4 Hz,
3H), 0.75 (dd, J ) 6.0, 13.7 Hz, 1H); MS 761 (M + Na); HRMS
calcd for C43H55N4O7 739.407076, found 739.408618.
The acid obtained above (16.35 g, 66.4 mmol) was converted
to the corresponding trimethylsilyl carbamate using the condi-
tions already described for the preparation of compounds 20a
and 20b. The crude material was flash chromatographed (8.5
cm, 10-15% AcOEt-hexane) to afford the desired carbamate
(11.0 g, 47% yield) as a yellow oil: 1H NMR (CDCl3) δ 7.33
(bs, 5H), 5.79-5.70 (m, 1H), 5.28 (d, J ) 17.2 Hz, 1H), 5.26
(bs, 1H), 5.18 (d, J ) 12.4 Hz, 1H), 5.16 (d, J ) 12.4 Hz, 1H),
5.10 (d, J ) 10.8 Hz, 1H), 4.15-4.11 (m, 2H), 2.21-2.15 (m,
1H), 1.86-1.83 (m, 1H), 1.58-1.52 (m, 1H), 0.99-0.88 (m, 2H),
0.018 (s, 9H); MS 384 (M + Na).
To the carbamate (10.4 g, 28.8 mmol) was added a 1.0 M
solution of tetrabutylammonium fluoride in THF (65 mL, 65.0
mmol). The reaction mixture was stirred for 20 h at room
temperature, and then the THF was removed in vacuo. The
residue was diluted with AcOEt and successively washed with
water (2×) and brine. After the usual treatment (MgSO4,
filtration, and concentration), the crude amine was isolated
(6.82 g).
P r ep a r a tion of Com p ou n d 21. (a ) P r ep a r a tion of
Diben zyl 2-Vin ylcyclop r op a n ed ica r boxyla te.
(d ) P r ep a r a tion of Dip ep tid e F r a gm en ts.
To a THF solution (850 mL) of potassium tert-butoxide (22 g,
196.04 mmol, 1.1 equiv) at -30 °C was slowly added dibenzyl
malonate (50 g, 175.86 mmol). The internal temperature was
maintained between -30 and - 10 °C throughout the addition.
After 30 min, 1,4-dibromobutene (45.1 g, 210.84 mmol, 1.2
equiv) was added. The cooling bath was then removed. After
1.5 h, the reaction mixture was cooled to - 10° C for the second
addition of potassium tert-butoxide (22.0 g, 196.0 mmol, 1.1
equiv). The cooling bath was again removed. One hour later,
a mixture of ice and water was added to the reaction mixture.
The two layers were separated, and the organic one was
concentrated. The residue was combined with the initial
aqueous layer, and the mixture was extracted with ether (3×).
The combined organic layer was then washed with brine. After
the usual treatment (MgSO4, filtration, and concentration), the
residue was flash chromatographed (12 cm, 3-6% AcOEt-
hexane) to afford dibenzyl 2-vinylcyclopropanedicarboxylate
as a colorless oil (30.87 g, 92 mmol, 52% yield): 1H NMR
(CDCl3) δ 7.32-7.25 (m, 10H), 5.45-5.36 (m, 1H), 5.29-5.08
(m, 6H), 2.65-2.59 (m, 1H), 1.75 (dd, J ) 4.8, 8.0 Hz, 1H),
1.60 (dd, J ) 4.8, 9.2 Hz, 1H); MS 337 (MH+).
To (4R)-1-(tert-butoxycarbonyl)-4-(1-naphthylmethoxy)-L-pro-
line 10 (11.8 g, 31.8 mmol) in acetonitrile (100 mL) was added
TBTU (10.7 g, 33.3 mmol). To the crude amine obtained above
(ca. 28.8 mmol) in acetonitrile (80 mL) was added NMM (9.5
mL, 86.4 mmol). This solution was added to the acid solution,
and the reaction mixture was stirred overnight at room
temperature. The acetonitrile was then removed in vacuo, and
the residue was dissolved in AcOEt and successively washed
with 10% aqueous citric acid (2×), saturated aqueous NaHCO3
(2×), and brine. After the usual treatment (MgSO4, filtration,
and concentration), the residue was flash chromatographed
(9 cm, 25-35% AcOEt-hexane) to afford the less polar
diastereomer (4.33 g, 7.6 mmol), the more polar diastereomer
(4.05 g, 7.1 mmol), and a mixture of both compounds (0.93 g,
1.6 mmol).
(b) P r ep a r a tion of (1R,2S)/(1S,2R)-1-[(Ben zyloxy)ca r -
bon yl]-2-vin ylcyclop r op a n eca r boxylic Acid .
(e) P r ep a r a tion of Com p ou n d 21. Sequential coupling
of the less polar diastereomer with Boc-Val-OH, Boc-Chg-OH,
and acetic anhydride, followed by a saponification reaction of
the benzyl ester under the conditions already described above,
afforded the desired acid 21.
Compound 21 (>99% homogeneous by HPLC; 95.8% homo-
geneous by CE): 1H NMR (DMSO-d6) δ 8.52 (s, 1H), 8.06-
7.77 (m, 5H), 7.58-7.43 (m, 4H), 5.74-5.64 (m, 1H), 5.26-
4.88 (m, 4 H), 4.39-3.83 (m, 5H), 3.76 (dd, J ) 4.2, 10.8 Hz,
1H), 2.21-2.16 (m, 1H), 2.06-1.93 (m, 3H), 1.84 (s, 3H), 1.69-
1.42 (m, 7H), 1.22 (dd, J ) 4.8, 9.2 Hz, 1H), 1.12-0.92 (m,
4H), 0.9 (d, J ) 6.7 Hz, 3H), 0.86 (d, J ) 6.7 Hz, 3H), 0.75 (dd,
J ) 6.7, 13.4 Hz, 1H); MS 661 (MH+), 683 (M + Na); HRMS
calcd for C37H49N4O7 661.360125, found 661.360768.
To the diester obtained above (29.38 g, 87.3 mmol) in a mixture
of THF (145 mL), tert-butyl alcohol (72 mL) and water (72 mL)
at 0 °C was added lithium hydroxide monohydrate (4.03 g, 96.0
mmol, 1.1 equiv). The reaction mixture was stirred for 18.5 h
at room temperature, and then an additional amount of
hydroxide was added (916 mg, 0.25 equiv). After 3 days at room
temperature, most of the THF and the tert-butyl alcohol were
removed in vacuo. A 10% aqueous citric acid solution was
added to the residue for acidification. The aqueous layer was
extracted with ethyl acetate (3×). The combined organic
extract was successively washed with water (2×) and brine.
After the usual treatment (MgSO4, filtration, and concentra-
tion), the desired monoacid was obtained as a colorless oil
(16.35 g, 66.4 mmol, 76% yield): 1H NMR (CDCl3) δ 7.39-
7.33 (m, 5H), 5.61-5.52 (m, 1H), 5.34 (d, J ) 17.2 Hz, 1H),
5.22 (s, 2H), 5.14 (d, J ) 10.4 Hz, 1H), 2.77-2.71 (m, 1H),
2.08 (dd, J ) 4.8, 9.2 Hz, 1H), 1.98 (dd, J ) 4.8, 8.4 Hz, 1H).
(c) P r ep a r a tion of Ben zyl (1S,2S)/(1R,2R)-1-Am in o-2-
vin ylcyclop r op a n eca r boxyla te.
P r ep a r a tion of Com p ou n d 22. This inhibitor was pre-
pared using commercially available building blocks and stan-
dard coupling conditions (TBTU).
Compound 22 (>99% homogeneous by HPLC; 99.4% homo-
geneous by CE): 1H NMR (DMSO-d6) δ 8.73 (s, 1H), 7.89 (d,
J ) 8.4 Hz, 1H), 7.85 (d, J ) 8.8 Hz, 1H), 7.38-7.25 (m, 5H),
4.53 (d, J ) 11.9 Hz, 1H), 4.45 (d, J ) 11.7 Hz, 1H), 4.33-
4.15 (m, 4H), 4.04 (d, J ) 11.1 Hz, 1H), 3.68 (dd, J ) 4.2, 11.2
Hz, 1H), 2.23-2.15 (m, 1H), 2.02-1.89 (m, 2 H), 1.84 (s, 3H),
1.69-1.42 (m, 6H), 1.39-1.31 (m, 1H), 1.28-1.20 (m, 1H),
1.13-0.82 (m, 6H), 0.88 (d, J ) 6.4 Hz, 3H), 0.84 (d, J ) 6.7
Hz, 3H), 0.78 (dd, J ) 7.4, 7.4 Hz, 1H); MS 585 (MH+); HRMS
calcd for C31H45N4O7 585.328825, found 585.331500.
P r ep a r a tion of Com p ou n d 23. This inhibitor (98% ho-
mogeneous by HPLC; 95.7% homogeneous by CE) was pre-

Peptide-Based Inhibitors of HCV NS3 Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2521
pared using racemic vinyl-ACCA (see the preparation of
compound 21 above). After coupling to the Boc-Hyp(O-Bn)-
OH, the less polar diastereomer was isolated and converted
to the final compound using sequential coupling and standard
conditions (TBTU).
Compound 23 (98% homogeneous by HPLC; 95.7% homo-
geneous by CE): 1H NMR (DMSO-d6) δ 8.52 (s, 1H), 7.85 (d,
J ) 8.6 Hz, 1H), 7.82 (d, J ) 8.9 Hz, 1H), 7.38-7.24 (m, 5H),
5.74-5.63 (m, 1H), 5.19 (dd, J ) 1.6, 17.5 Hz, 1H), 5.06 (d, J
) 11.1 Hz, 1H), 4.52 (d, J ) 12.1 Hz, 1H), 4.45 (d, J ) 11.8
Hz, 1H), 4.35-4.15 (m, 4H), 4.00 (d, J ) 11.1 Hz, 1H), 3.72
(dd, J ) 4.1, 10.8 Hz, 1H), 2.21-2.11 (m, 1H), 2.05-1.91 (m,
3H), 1.84 (s, 3H), 1.71-1.43 (m, 6H), 1.22 (dd, J ) 5.1, 9.5 Hz,
1H), 1.12-0.82 (m, 5H), 0.89 (d, J ) 6.4 Hz, 3H), 0.85 (d, J )
6.7 Hz, 3H), 0.79 (dd, J ) 7.0, 1.3 Hz, 1H); MS 611 (MH+),
633 (M + Na+); HRMS calcd for C33H46N4O7 611.344475, found
611.342000.
concentration (IC50) was calculated through the use of SAS
(Statistical Software System, SAS Institute Inc., Cary, NC).
Ack n ow led gm en t. We thank Mr. Ewald Welchner
for all the enzymatic assays. We also thank Mr. Michael
Little and Mr. Sylvain Bordeleau for analytical support.
Professor Charette (Universite´ de Montre´al), who kindly
provided us with a sample of compound 13 in a homo-
chiral form, is gratefully acknowledged. We are grateful
to Dr. Nathalie Goudreau for her input in the compu-
tational chemistry section of this document and to Ms.
Elise Ghiro for the preparation of compound 21. Finally,
we also thank Drs. Pierre Beaulieu and Peter White for
proofreading the manuscript.
Refer en ces
P r ep a r a tion of Com p ou n d 24. This compound has been
(1) Di Bisceglie, A. M. Hepatitis C. Lancet 1998, 351, 351-355.
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S144.
described previously.25
P r ep a r a tion of Com p ou n d 25. This compound was pre-
pared using conditions already described above.
Compound 25 (99% homogeneous by HPLC; 97.6% homo-
geneous by CE): 1H NMR (DMSO-d6) δ 9.06 (d, J ) 6.5 Hz,
1H), 8.59 (s, 1H), 8.26 (d, J ) 10.0 Hz, 1H), 7.97 (d, J ) 7.6
Hz, 1H), 7.67 (d, J ) 8.6 Hz, 1H), 7.54-7.40 (m, 3H), 5.77-
5.61 (m, 2H), 5.21 (d, J ) 17.4 Hz, 1H), 5.07 (d, J ) 11.4 Hz,
1H), 4.50 (d, J ) 11.9 Hz, 1H), 4.38 (dd, J ) 8.4, 8.4 Hz, 1H),
4.19 (dd, J ) 8.0, 8.0 Hz, 1H), 4.13 (dd, J ) 7.8, 7.8 Hz, 1H),
4.06 (dd, J ) 2.9, 11.9 Hz, 1H), 3.99 (s, 3H), 2.05-1.94 (m,
2H), 1.83 (s, 3H), 1.71-1.36 (m, 7H), 1.24 (dd, J ) 4.9, 9.2 Hz,
1H), 1.15-0.84 (m, 6H), 0.93 (d, J ) 6.5 Hz, 3H), 0.89 (d, J )
6.4 Hz, 3H), 0.78 (dd, J ) 6.0, 14.0 Hz, 1H); MS 611 (MH+),
633 (M + Na+); HRMS calcd for C33H46N4O7 611.344475, found
611.342000.
(5) Houghton, M. Hepatitis C Viruses. In Fields’ Virology, 3rd ed.;
Fields, B. N., Knipe, D. M., Howley, P. M., Eds.; Lippincott-
Raven: Philadelphia and New York, 1996; pp 1035-1058,
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strategies for hepatitis C. Hepatology 2002, 35, 224-231.
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structure, polyprotein processing, and protein properties. Curr.
Top. Microbiol. Immunol. 2000, 242, 55-84.
P r ep a r a tion of Com p ou n d s 26 a n d 27. These compounds
have been described previously.26
(9) Kwong, A. D.; Kim, J . L.; Rao, G.; Lipovsek, D.; Raybuck, S. A.
Hepatitis C virus NS3/4A protease. Antiviral Res. 1998, 40,
1-18.
(10) De Francesco, R.; Steinku¨hler, C. Structure and function of the
Hepatitis C virus NS3-NS4A proteinase. Curr. Top. Microbiol.
Immunol. 2000, 242, 149-169.
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Hepatitis C virus-encoded enzymatic activities and conserved
RNA elements in the 3′ nontranslated region are essential for
virus replication in vivo. J . Virol. 2000, 74, 2046-2051.
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LaPlante, S.; Maurice, R.; Poirier, M.; Poupart, M.-A.; Thibeault,
D.; Wernic, D.; Lamarre, D. Peptide-based inhibitors of the
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Com p u ta tion a l Ch em istr y (F igu r e 4). The binding mod-
els for the inhibitor P1 residues (Figure 4) were generated
using the INSIGHT II software package (Accelerys Inc., San
Diego, CA). Both (R,R) and (S,S) isomers of the P1 ACCA were
built and docked into the substrate-binding region of the X-ray
crystal structure of the apo NS3 protease.30 Specifically, the
P1 residues were appropriately oriented in order to mimic the
conformation of the C-terminal threonine amino acid observed
in the crystal structure of the full-length NS3 protease-
helicase protein. In this conformation, the P1 φ angle is 127.6°,
and the carboxylate is properly oriented to make hydrogen
bonds with the side chains of the active-site residues His 57
and Ser 139 as well as with the backbone amide protons of
the oxyanion hole residues Gly 137 and Ser 139.
Biologica l Assa y. Reported IC50 values are averages of
duplicate experiments on two separate samples (four measure-
ments) and were determined using the radiometric assay
described below.
(14) For a protease subsite nonmenclature, see: Schechter, I.; Berger,
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(15) (a) Llina`s-Brunet, M.; Bailey, M.; Fazal, G.; Ghiro, E.; Gorys,
V.; Goulet, S.; Halmos, T.; Maurice, R.; Poirier, M.; Poupart, M.-
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When the configuration of the (1S)-norvaline was changed to
(1R), a dramatic drop in the enzymatic activity was observed
(unpublished result). (e) Notice that the difference observed for
the reported IC50 value for compound 1 in ref 15b (3.5 µM)
compared to ours (14 µM) comes from the fact that two different
assays were used (the details of the first assay can be found in
ref 12 and, for the second one, in the Experimental Section of
the present article).
NS3-NS4A P r otein Ra d iom etr ic Assa y. The NS3-
NS4A protein was produced using a recombinant baculovirus
system and purified as previously described.31 The enzymatic
assay was performed in 50 mM Tris-HCl, pH 8.0, 0.25 M
sodium citrate, 0.01% n-dodecyl-â-D-maltoside, 1 mM TCEP.
A 25 µM concentration of the substrate DDIVPC-SMSYTW-
OH, a 1 nM concentration of biotin-DDIVPC-SMSY[125I]TW-
OH, and various concentrations of inhibitor were incubated
with 6 nM NS3-NS4A protein for 2 h at 23 °C. The reaction
was terminated by the sequential addition of 0.5 N NaOH and
1 M MES, pH 5.8. The separation of the biotinylated substrate
from products was performed by adding avidin-coated agarose
beads (Pierce) to the assay mixture and incubating for 1 h at
23 °C. Following filtration on a Millipore MADP N65 plate,
the amount of SMS[125I-Y]TW-OH product found in the filtrate
was quantitated using a TopCount detector (Perkin-Elmer)
and allowed for the calculation of the percentage of inhibition.
A nonlinear curve fit using the Hill model was then applied
to the % inhibition-concentration data, and 50% effective

2522 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Rancourt et al.
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