Novel azapeptide inhibitors of hepatitis C virus serine protease

Article abstract of DOI:10.1021/jm049864b

Azapeptides are known inhibitors of several serine and cysteine proteases. In seeking different classes of inhibitors for the HCV serine protease, a series of novel azapeptide-based inhibitors were investigated which incorporated noncleavable P1/P1′ aza-a

Full text of DOI:10.1021/jm049864b

3788
J . Med. Chem. 2004, 47, 3788-3799
Novel Aza p ep tid e In h ibitor s of Hep a titis C Vir u s Ser in e P r otea se
Murray D. Bailey,* Ted Halmos, Nathalie Goudreau, Ewen Lescop, and Montse Llina`s-Brunet
Boehringer Ingelheim (Canada) Ltd. Research and Development, 2100 Cunard Street, Laval, Quebec (Canada) H7S 2G5
Received February 16, 2004
Azapeptides are known inhibitors of several serine and cysteine proteases. In seeking different
classes of inhibitors for the HCV serine protease, a series of novel azapeptide-based inhibitors
were investigated which incorporated noncleavable P1/P1′ aza-amino acyl residues. Extensive
SAR studies around the P1/P1′ aza-amino acyl fragment resulted in the identification of potent
and selective inhibitors. Using NMR studies, we have shown that this series of inhibitors bind
in a noncovalent competitive fashion to the NS3 protease active site. The bound conformation
of one of these new azapeptide-based inhibitors was determined using the transfer NOE
technique. Incorporation of these new aza-amino acyl functionalities in the P1 position provided
a handle to probe for new interactions in the S′ region of the enzyme.
In tr od u ction
ment for the cysteine residue with a 5-fold loss in
potency but chemically more stable. We have also
reported that, in general, replacement of the P1 termi-
nal carboxylic acid by activated carbonyl functionalities
(R-ketoamides or fluorinated ketones) did not produce
any substantial increase in potency but did in turn
impart a loss in specificity against other serine pro-
teases.10,11 This unusual characteristic of the HCV
serine protease have encouraged us to explore other
nonacylating functionalities at the P1 position.
The hepatitis C virus (HCV) is the major agent
responsible for non-A and non-B viral hepatitis world-
wide. This viral infection is the leading cause of chronic
hepatitis, liver cirrhosis, and hepatocellular carcinoma.1
The World Health Organization estimates that about
200 million people are infected with this virus world-
wide.2 The HCV RNA genome encodes a single polypro-
tein precursor of about 3000 amino acids which is
proteolytically processed into four structural and six
nonstructural (NS) proteins. One of the most studied
targets for antiviral therapy against HCV is the virally
encoded NS3 serine protease.3 Recently this target has
led to the first reported protease inhibitor to have
entered clinical trials, BILN 2061.4a,b The NS3 protease
has been established as a chymotrypsin/trypsin-like
serine protease as shown by structural determination.5
The protease domain is located at the N-terminal
portion of the NS3 protein and is responsible for the
proteolytic cleavage of the nonstructural proteins found
downstream from it, namely the NS3/NS4A, NS4A/
NS4B, NS4B/NS5A, and NS5A/NS5B junctions.6 It has
also been shown that the activity of the NS3 protease
is enhanced by the NS4A protein which is an essential
cofactor required for polyprotein maturation.7 We have
previously reported our findings that the NS3 protease
complexed with an NS4A cofactor peptide is inhibited
by the N-terminal cleavage product derived from a
modified NS5A/5B peptide substrate.8 This unusual
N-terminal cleavage product inhibition has also been
observed with substrates corresponding to the NS4A-
NS4B and NS4B-NS5A cleavage sites.9 Early work
from our group made use of hexapeptide Ac-DDIVPC-
OH (a modified N-terminal cleavage product from
NS5A/5B) as our original lead (IC50 ) 43 µM). We
reported that the P1 cysteine residue which is found in
three of the natural cleavage sites of the substrate could
be replaced by lipophilic amino acids but not without a
loss in potency.10 In particular, a linear three-carbon
chain (norvaline) proved to be an acceptable replace-
In h ibitor Design . In our continuing efforts to dis-
cover new classes of inhibitors of the HCV NS3 serine
protease, a strategy which avoided the use of traditional
electrophilic carbonyl groups in the P1 position was
investigated. In addition to C-terminal carboxylic acids,
one approach we considered consisted of substitution
of the R-carbon atom of the P1 amino acid residue by a
trivalent nitrogen atom to yield an azapeptide P1
residue. This strategy seemed particularly appealing
since it has been demonstrated that this replacement
largely preserves the original peptide conformation in
the resulting biomimetic peptide.12 Moreover, the in-
corporation of a central P1 aza-amino acyl moiety would
fulfill a number of objectives: (1) allow exploration of
the S′ region of the enzyme to identify new binding
interactions, (2) remove the C-terminal charged residue,
and (3) reduce the peptidic character of the inhibitors
since azapeptides are known to be metabolically more
stable to enzymatic degradation13 than their amino acid
counterparts.
Azapeptide-based inhibitors have been investigated
for several serine proteases such as the porcine pan-
creatic14,15 and human leukocyte elastases,15,16 R-chy-
motrypsin,17,18 subtilisins,18 and human leukocyte cathe-
psin G.18 We have reported on the use of azapeptides
as inhibitors of the HCV serine protease.19 Recently,
others have reported on the use of azapeptides as
inhibitors of HCV serine protease which involved the
introduction of a variety of groups in the P1′ position
with binding affinities varying by >500-fold depending
on the nature of the group.20 One observation was that
the potencies of these compounds correlated with the
leaving group ability of the P1′ functionality. Regardless
* To whom correspondence should be addressed. E-mail: mbailey@
lav.boehringer-ingelheim.com.
10.1021/jm049864b CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/17/2004

Azapeptide Inhibitors of Hepatitis C Virus Serine Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3789
Sch em e 1. Final Assembly and Deprotectiona
a
Reaction conditions: (a) TBTU, i-Pr2NEt, DMF, rt; (b) 10% Pd/C, EtOH, NH4OAc;(c) 4 N HCI/dioxane, 30 min, rt; (d) isocyanate,
NEt3, THF, 0 °C; (e) carboxylic acid, TBTU, DIPEA, DMF; (f) chloroformate, NEt3, THF; (g) isothiocyanate, NEt3, THF, 0 °C; (h) neat
TFA, 1.5 h.
of this, these azapeptides consistently seemed to display
competitive inhibition kinetics.20 In general however,
the approach of introducing labile groups in the P1′
position in azapeptide analogues have resulted in the
inhibition through the acylation of the active site serine
residue (the active site serine residue nucleophilically
attacks the activated carbonyl and forms an acyl-
enzyme).16 The acyl-enzyme intermediate has also been
shown to be quite stable, resulting in a slow deacylation
rate.16 In contrast, we envisioned the possibility of
introducing a noncleavable aza-amino acyl fragment in
the P1-P1′ position to generate noncovalent, reversible
inhibitors. In this report we describe extensive SAR
studies around the P1 azapeptide moiety of these
inhibitors which have lead to the discovery of new
interactions within the S1′ region of the enzyme and
the generation of a novel series of azapeptide-based
inhibitors of the HCV serine protease.21
Ch em istr y. The synthesis of the various aza-amino
acyl fragments and the final inhibitors are described in
Schemes 1-3. The final assembly and deprotection
routes to the azapeptide inhibitors followed a straight-
forward and divergent approach as outlined in Scheme
1. Specifically, three assembly methods were used to
prepare the azapeptide-based inhibitors. In method A,
the aza-amino acyl P1/P1′ unit 3 was coupled to the
protected pentapeptide 1 (P6-P2) using standard pep-
tide coupling protocols. Removal of the benzyl ester
groups under hydrogenation conditions afforded the
final inhibitors 4. This approach was conveniently used
to study various alkyl groups on the central P1 nitrogen
atom. To probe the importance of other structural
features of the aza-amino acyl fragment in the P1′
position, Method B was followed (Scheme 1). This
method installs a common carbazate fragment 5 with a
defined N-alkyl group to pentapeptide 1 to give inter-
mediate 6. This versatile intermediate is well suited to
allow for the exploration of either the P1-P1′ linkage
or variations of the P′ group. Derivatization of the
carbazate intermediate 6 was conducted by first removal
of the tert-butyl carbamate group, followed by subse-
quent treatment with a variety of reactive electrophiles.
Final deprotection as before yielded the azapeptide
inhibitors 4. These two strategies proved to be both
practical and expedient for SAR studies when X ) C or
NH. A modification of Method A was utilized to prepare
the benzyl carbamate analogues (X ) O) since hydro-
genation is incompatible with this group. Method C
utilized 2-(trimethylsilyl)ethyl ester groups to protect
the P5 and P6 acid functionalities in the pentapeptide
fragment 2. This protecting group is extremely useful
when using Boc-protected peptides since it survives
anhydrous HCl conditions used to cleave tert-butyl
carbamate groups, while being readily liberated with
either TFA or n-BuNF treatment.22 The preparation of
the aza-amino acyl fragments 3 used in Methods A and
C followed analogous protocols as previously reported
by others23 (Scheme 2). Starting with commercially
available tert-butylhydrazine 7, the desired P1 side
chains were readily incorporated via a two-step se-
quence by first formation of hydrazones 8a -g with the
appropriate aldehyde or ketone followed by subsequent
reduction with DIBAL to produce the N-alkyl hydra-
zines 9a -g. The various P1/P1′ linkages were then
obtained by condensation of 9a -g with isocyanates,
chloroformates, carboxylic acids, carbamoyl chlorides,
or thioisocyanates as shown in Scheme 2 to give the
corresponding C(O)NHR3 (10a -g), C(O)OBn (11b),
C(O)CH2CH2Ph (12b), C(O)NR′R′′ (13b, 14b), or C(S)-
NHBn (15b) substituted analogues. The N-Me and
N-CH2CF3 analogues were prepared from the available
N-methylhydrazine and 2,2,2-trifluoroethylhydrazine
via an analogous sequence. All of the aza-amino acyl
intermediates (10a -g, 11b, 12b, 13b, 14b, 15b) were

3790 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
Sch em e 2. Preparation of the Aza P1/P1′ Fragmentsa
Bailey et al.
a
Reaction conditions: aldehyde or ketone, toluene, rt; (b) DIBAL, THF/toluene, -78 °C to rt; (c) R3-NdCdO, i-Pr2NEt, THF, 0 °C to
rt; (d) ClCOOBn, i-Pr2NEt, THF, pyridine 0 °C to rt; (e) PhCH2CH2CO2H, TBTU, i-Pr2NEt, 0 °C to rt; (f) phosgene/toluene, i-Pr2NEt,
THF, 0 °C, 45 min; (g) NH(Me)Ph or indoline, THF, i-Pr2NEt, 0 °C to rt, 16 h; (h) Bn-NdCdS, i-Pr2NEt, THF, 0 °C to rt.
Ta ble 1. Optimization of Compound 20
Sch em e 3. Alternate Approach for Aza P1/P1′
Fragmentsa
a
Reaction conditions: (a) propionaldehyde, toluene, rt, NEt3,
THF; (b) DIBAL, THF/toluene, -78 °C to rt; Boc2O, NEt3, THF;
(d) 10% Pd/C,EtOH, H2.
subsequently deprotected (removal of the tert-butyl
carbamate group) before being incorporated into the
inhibitor as in Scheme 1, Method A or C. Finally, the
preparation of the protected hydrazine 5 used in Method
B (Scheme 1) is shown in Scheme 3. Starting from
commercially available benzyloxy-carbonyl hydrazine
16, a reductive alkylation sequence was used to prepare
hydrazone 17, which was subsequently reduced to
hydrazine 18 and protected as the tert-butyl carbamate
derivative 19. Selective removal of the benzyloxy-
carbonyl group afforded hydrazine 5 which was readily
incorporated into pentapeptide 1.
by us and are shown in Table 1.11 One of the most
important findings was the introduction of substituents
on the P2 proline moiety of compound 20. Incorporation
of a (R)-benzyloxy group in the 4-position of proline
resulted in a 21-fold improvement in potency as il-
lustrated with compound 21. Further improvement in
potency was realized with the incorporation of a D-
glutamic acid residue into the P5 position to give peptide
22 with approximately a 10-fold improvement in potency
(IC50 ) 0.60 µM). Having established an acceptable
potency with the terminal carboxylic acid series, we then
set out to investigate the effects of changes to the P1
residue. The following observations were made (Table
2). As expected, incorporation of the D isomer of norva-
line as in compound 23 resulted in a substantial loss in
Resu lts a n d Discu ssion
It is well-known that the P1 residues of serine
protease inhibitors are critical for enzyme specificity and
recognition. In an earlier study, we had shown that the
naturally occurring P1 cysteine residue could be re-
placed by the chemically more stable norvaline amino
acid. While this replacement is tolerated, a 5-fold loss
in potency was realized.9 Further optimizations to these
early hexapeptide analogues have since been described

Azapeptide Inhibitors of Hepatitis C Virus Serine Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3791
Ta ble 3. SAR at the P′ Position
Ta ble 2. SAR at the P1 Position
potency (57-fold). More surprisingly however, was the
loss in potency realized with the introduction of a
terminal amide group of L-norvaline as in compound 24.
Even with the natural amino acid configuration, this
simple substitution resulted in greater than a 40-fold
loss in potency. Substitution of the R-carbon atom in
24 for a trivalent nitrogen atom afforded the azapeptide-
based inhibitor 25, which was of similar potency to the
amino acid analogue 24. The acceptability of this
backbone atom replacement is based on the assumption
that the orientation of the N-alkyl side chain can adopt
the same basic structure as the corresponding peptide,
although this has been shown to be structure depend-
ent.24,25 Other features of the azapeptide-based analogue
may also be responsible for maintaining the potency of
25 in relation to amide 24. It has been shown that the
acidity of the P1-NH is enhanced over its amino acid
counterpart14,26 and might improve the hydrogen bond-
ing interaction with the enzyme. As well, the electronic
nature of the urea carbonyl in the azapeptide is expected
to be different than that of the corresponding amide
carbonyl in amide 24. While azapeptide 25 was sub-
stantially less potent than the C-terminal carboxylic
acid inhibitor 22 (29-fold less potent), the potential to
explore the S′ binding domain encouraged us to inves-
tigate this novel series further. Having validated the
concept that the P1 amino acid could be replaced by the
corresponding aza-amino acyl counterpart, our attention
was focused on the introduction of residues into the P′
position. Our objective was to probe the S′ region for
new binding interactions necessary to improve the
intrinsic potency. A dramatic improvement was ob-
served when hydrophobic residues were incorporated.
As shown in Table 3, incorporation of a terminal benzyl
group as in compound 26 resulted in a 30-fold improve-
ment in potency over 25. Interestingly, this more
elaborate P1′ residue produced the first azapeptide
analogue with equivalent potency to the corresponding
terminal acid inhibitor (compound 22) but without the
associated charge. Addition of substituents on the
phenyl ring was also well tolerated as shown by the
introduction of the piperonylamine group in compound
27. N-Methylation or rigidification of the terminal amide
linkage as in compounds 28 and 29 were, however,
found to be detrimental to the potency. Disubstitution
on the terminal nitrogen atom likely results in an
unfavorable conformational change which is not well
suited for the active site.
Ta ble 4. Importance of the P1′-NH and P1-CO Group
The importance of the linkage between the P1-P1′
residue was also investigated (Table 4). First, the
importance of the P1 carbonyl group was established
by the simple substitution of the carbonyl in carbamate
26 by the thiocarbonyl group as in 30 which resulted in
greater than a 7-fold drop in potency. The nature of the
linking atom between P1-P1′ was shown to have little
or no effect on potency. Replacement of the terminal
monosubstituted nitrogen atom by either an oxygen
(compound 31) or carbon atom (compound 32) were
equally well tolerated. Analogue 32 is of particular
interest since this compound clearly rules out the

3792 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
Ta ble 5. Effect of A-Branching in the P1′ Position
Bailey et al.
However, substitution with the enantiomeric R-(S)-
methyl group as in compound 34 resulted in more than
a 7-fold improvement in potency relative to 26. The
preference for the (S)-methyl group in the P′ region is
presumably a result of a beneficial orientation of the
aryl group. It is interesting to note that the stereospeci-
ficity of the P1′ position as observed here has also been
observed for other protease targets.27 Further investiga-
tion into the effect of R substitution revealed that while
lengthening of the side chain as in 35 was not well
tolerated, rigidification as in 36 was well accepted.
Replacement of the R stereocenter by the N-methyl
phenylhydrazine moiety as in 37 resulted in a compound
which loses the beneficial effect of the R-substitution and
is similar in potency to 26. The necessity of the phenyl
ring was next examined. Saturation of the aromatic ring
as in compound 38 or incorporation of a tetrazole ring
as in compound 39 was not beneficial. Increasing the
size of the aryl group such as in 40 was also detrimental.
It was found, however, that substitution is tolerated in
the para position of the phenyl ring as in 41 which was
equipotent (IC50 ) 0.072 µM) to compound 34. From this
table, the optimal modifications in the P′ region were
found for compound 34 which was over 7-fold more
potent than the starting benzyl analogue 26. It also
appears from compound 41 that further modifications
are possible on the phenyl ring.
P 1 Sid e Ch a in Op tim iza tion . The replacement of
the R-carbon in an amino acid by a trivalent nitrogen
atom has a number of consequences. One of these is that
the conformation adopted by the aza-amino acid moiety
around the R-nitrogen atom can be significantly affected
depending on the peptide sequence. While some have
considered the conformation somewhere intermediate
between the D- or L-amino acid,13 others have shown
that the R-nitrogen atom can be nearly planar.24 These
possible conformational effects around the R-aza-
substitution prompted us to investigate whether the
n-propyl side chain (aza-norvaline) was fully optimal in
our case. Three issues were examined, the length of the
side chain, the effect of branching, and the effect of
fluorine substitution (Table 6). Chain lengths of two,
three, or four carbon atoms, as in compounds 43, 34,
and 44, were found to be more or less equipotent against
the protease. Incorporation of shorter or longer chain
lengths as in compounds 42 or 45 were much less
favorable, showing that the enzyme requires at least a
two- to four-carbon side chain. The effect of branching
was also investigated. Branching at various positions
along the side chain were not well tolerated (compounds
46, 47, 48), indicating that larger alkyl groups are not
well suited for this enzyme. In addition, the introduction
of a trifluoromethyl group as in derivative 49 was also
found not to be optimal for binding.
Sp ecificity. A number of these azapeptide-based
analogues were screened for their activity against other
serine or cysteine proteases. In particular, special
attention was paid to human leukocyte elastase (HLE)
which has a high substrate sequence homology to that
of the NS3 protease in the P3 through P1 residues
(Table 7). The selectivity between the NS3 serine
protease and HLE was found to be dependent on the
P1 side chain. The least selective compound was found
to be compound 34 having a three-carbon side chain.
possibility of having inhibition through the enzyme
acylation pathway as described for reactive azapeptides.
From this study it appears that while the NH(P1′) is
nonessential for potency, the carbonyl (P1) contributes
significantly to the activity of these compounds. Having
established that a benzyl group in the P′ position was
beneficial to potency (Table 3), further modifications
were investigated (Table 5). Incorporation of an R-meth-
yl group in the benzylic position with the “R” configu-
ration (compound 33) resulted in a 7-fold loss in potency.

Azapeptide Inhibitors of Hepatitis C Virus Serine Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3793
Ta ble 6. SAR on the P1 Side Chain
Ta ble 7. Comparison between HLE and HCV Protease with
Various P1 Residues
F igu r e 1. Subregion of 1H spectra of inhibitor 25 recorded in
the absence (blue) and in the presence (red) of NS3 protease
at an inhibitor-to-protease ratio of 34:1. Upon addition of the
protease, significant line broadening was observed for specific
resonances.
intrinsic potencies of these compounds and the slow on/
off rates. Our initial NMR investigations focused on
determining which parts of azapeptide 25 interact with
the NS3 protease. This was accomplished using dif-
ferential line-broadening techniques which we have
previously described.28-30 Figure 1 presents a region of
the 1D 1H NMR spectrum of 25 recorded in absence
(blue trace) and in the presence (red trace) of the NS3
protease. In the absence of enzyme, the inhibitor has
sharp resonance line shapes while in the presence of
the NS3 protease specific differential line broadening
effects are observed. Indeed, the P1 side chain reso-
nances (γ-CH3 at 0.88 ppm and â-CH2 at 1.52 ppm), and
the P3 Val and P4 Ile methyl resonances (P3 γ-CH3 at
0.93 ppm and P4 γ-CH3 at 0.79 ppm/δ-CH3 at 0.81 ppm)
experience significant line broadening, while no signifi-
cant change is observed for other resonances such as
the P4 Ile â-CH (1.86 ppm) and γ-CH2 (1.42 ppm) and
the P5 D-Glu â-CH2 (1.93 ppm). The specific nature of
these protease-induced perturbations suggests that the
segments of 25 which have their resonances affected by
the addition of NS3 are likely at or near the binding
interface. In a second step, the transfer NOE technique
was applied to azapeptide 25 in order to establish the
NS3 bound conformation. This technique has previously
This compound exhibited a 65-fold window between IC50
values. The most selective inhibitor from this study
(compound 44) exhibited more than a 400-fold window
between IC50 values and incorporated the slightly longer
butyl side chain. This difference in selectivity between
34 and 44 comes about by maintaining the excellent
potency level against the HCV serine protease while
decreasing the affinity toward the human leukocyte
elastase enzyme. The methyl and n-pentyl analogues
(42 and 45) also showed selectivity against HLE but
these were also much less potent against the desired
HCV serine protease. Additionally, compounds 34, 42-
45 were also screened against chymotrypsin and cathe-
psin B and were shown to be completely selective (>150
µM).
NMR Stu d ies. To get a better understanding of how
the azapeptide-based inhibitors bind to the NS3 pro-
tease, NMR studies were conducted using compound 25
(Table 3). Unfortunately, the study of our more potent
inhibitors which extend into the P1′ region using the
transfer-NOE approach was not possible due to the high

3794 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
Bailey et al.
F igu r e 2. Stereoview of the superposition (P1-P4 backbone atoms only) of the eight best structures generated by restrained
simulated annealing and derived from the NMR data of inhibitor 25 when bound to the NS3 protease. The structures are colored
by atom type (oxygen is red, nitrogen is blue, carbon is dark gray, and hydrogen is light gray). Most of the hydrogen atoms are
not shown.
been successfully used for hexapeptide 22.30 Part A of
the figure in Supporting Information shows a region of
the 2D NOESY spectrum of inhibitor 25 recorded in the
absence of the NS3 protease where only a few NOEs
are observed as expected for a small molecule of this
size. In contrast, the NOESY spectrum of inhibitor 25
recorded after the addition of the NS3 protease (34:1
inhibitor-to-protease ratio) reveals an extensive set of
new, negative transferred NOE cross-peaks (part B).
The detection of these transferred NOEs also confirms
the reversibility and the fast exchange binding of 25 to
the NS3 protease. To establish that these transferred
NOEs come from the binding of 25 to the enzyme active
site, a competition experiment was carried out. In this
experiment, a potent covalent and reversible R-keto-
amide inhibitor (serine-trap based) (IC50 ) 0.023 µM)
which is known to block the NS3 active site was added
to the above azapeptide 25/NS3 protease sample (at four
times the concentration of the enzyme) and a NOESY
spectrum was reacquired (part C). The resulting disap-
pearance of the previous set of transferred NOE cross-
peaks and the restoration of the spectrum to that of the
free inhibitor (part C versus part A) is a clear indication
of the displacement of 25 from the NS3 active site by
the potent serine-trap-based inhibitor and supports the
claim that these compounds are noncovalent competitive
inhibitors of the protease.
Having established that the transferred NOE cross-
peaks in part B arise from 25 when bound to the active
site of the NS3 protease, we then focused our efforts on
determining the bound conformational properties of 25.
For this, the distance restraints derived from the
volumes of the transferred NOEs cross-peaks were used
in a simulated annealing protocol in order to generate
an ensemble of bound conformations. Figure 2 displays
a stereoview of the final eight low energy, transferred
NOE-consistent structures of 25 that are superimposed
from P1 to P4. A well defined, extended conformation
is clearly evident (from P1-P4) with a root-mean-square
deviation for the P1-P4 backbone atoms of 0.21 Å. The
side-chain conformations are also rather well defined
for most residues with the exception of P5 and P6
residues which show a larger degree of variation due to
a smaller number of restraints involving their side
chains. Moreover, the P2 and P4 side chains lie close to
each other in the bound state as evidenced by the
observation of transferred NOEs between the P4 γ-CH3
and â-CH and the P2 aromatic protons.
Altogether, this NMR study clearly demonstrates that
the azapeptide 25 binds to the NS3 protease in an
overall extended conformation that is very similar to
that of the C-terminal carboxylic acid series of inhibitors
such as hexapeptide 22.30 In particular the P1 aza-
amino acyl group of inhibitor 25 adopts a conformation
that is quite comparable to the one adopted by the P1
norvaline residues of hexapeptide 22. In addition,
similar differential line broadening patterns were also
observed for both of these P1 residues, therefore cor-
roborating the concept that the P1 amino acid can be
replaced by its aza-amino acid counterpart.
Con clu sion
We have successfully identified a new series of potent
NS3 serine protease inhibitors that incorporate an aza-
amino acyl fragment at the P1/P1′ position. While the
P1/P1′ NH linkage was found to be nonessential, the
P1 carbonyl group was shown to play an important role
in binding to the enzyme. The introduction of an R-(S)-
methyl benzyl group in the P1′ position of the inhibitor
was critical for improving potency and presumably helps
orient the aryl group into the enzyme. Benzylic residues
were found to be the most preferred groups in the S′
region of the enzyme since the larger biaryl substitution
resulted in reduced activity. Transferred NOE experi-
ments have shown that compound 25 binds in an
extended conformation, similar to that observed for the
C-terminal carboxylic acid inhibitor series previously
reported. Through NMR displacement studies with the
enzyme, this family of azapeptide-based inhibitors were
shown to act through a noncovalent competitive mode
of inhibition since a potent serine-trap-based inhibitor
displaced the azapeptide inhibitor from the active site.
The choice of the P1 side chain was very important for
providing specificity against other serine proteases. Our
best inhibitor in terms of potency and specificity was
compound 44, with an IC50 ) 0.099 µM against the HCV
NS3 serine protease, and a window of greater than 400-
fold when counterscreened against human leukocyte
elastase. Continued efforts with this inhibitor class
would include the further optimization of the P1-P4
residues and reduction in size through truncation stud-
ies.
Exp er im en ta l Section
Ch em istr y. Unless otherwise noted, materials were ob-
tained from commercial sources and used without further
purification. The purity of each inhibitor was determined by

Azapeptide Inhibitors of Hepatitis C Virus Serine Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3795
1
HPLC using two systems. H NMR spectra were obtained on
colorless oil after chromatography (SiO2, 3:1 hexane/EtOAc).
MS (FAB) 182 (M + Na+).
P r ep a r a tion of N′-Bu tylh yd r a zin eca r boxylic Acid ter t-
Bu tyl Ester 9c. Obtained from 8c (2.22 g, 12 mmol) using
the procedure described for 9b afforded 9c (2.32 g, 100%) as a
colorless oil after chromatography (SiO2, 3:1 hexane/EtOAc).
MS (FAB) 187 (M-H+).
P r ep a r a tion of N′-P en tylh yd r a zin eca r boxylic Acid
ter t-Bu tyl Ester 9d . Obtained from 8d (2.0 g, 10 mmol) using
the procedure described for 9b afforded 9d (0.78 g, 38%) as a
white solid after chromatography (SiO2, 3:1 hexane/EtOAc).
MS (FAB) 203.1 (MH+).
P r ep a r a tion of N′-(1-Eth yl-p r op yl)h yd r a zin eca r boxy-
lic Acid ter t-Bu tyl Ester 9e. Obtained from 8e (3.5 g, 17.5
mmol) using the procedure described for 9b afforded 9e (2.17
g, 61%) after chromatography (SiO2, 3:1 hexane/EtOAc). MS
(FAB) 203.1 (MH+).
P r ep a r a tion of N′-Isobu tylh yd r a zin eca r boxylic Acid
ter t-Bu tyl Ester 9f. Obtained from 8f (1.26 g, 6.8 mmol) using
the procedure described for 9b afforded 9f (0.79 g, 62%) as a
white solid after chromatography (SiO2, 3:1 hexane/EtOAc).
MS (FAB) 189.1 (MH+).
P r ep a r a tion of N′-(3-Meth yl-bu tyl)h yd r a zin eca r boxy-
lic Acid ter t-Bu tyl Ester 9g. Obtained from 8g (3.8 g, 18.9
mmol) using the procedure described for 9b afforded 9g (2.0
g, 52%) as a white solid after chromatography (SiO2, 3:1
hexane/EtOAc). MS (FAB) 203.1 (MH+).
Gen er a l P r ep a r a tion of Com p ou n d s 10a -g (r ep r esen -
ta tive exa m p les). P r ep a r a tion of (3-ter t-BSu tylca r bon yl-
2-p r op ylca r ba zoyl)-(S)-m eth ylben zyla m in e 10b (w h er e
R3 ) CH(“S”-Me)P h ). The hydrazine 9b (0.20 g, 1.15 mmol)
was combined with (S)-(-)-1-methylphenyl isocyanate (0.162
g, 1.15 mmol) in CH2Cl2 (2.4 mL) with DIPEA (0.44 mL, 2.52
mmol) and stirred at 0 °C for 1 h and then at RT for 3 h. The
mixture was concentrated in vacuo to give a white solid which
was purified by flash chromatography to give the protected
intermediate 10b (0.25 g, 68%). MS (FAB) 322.3 (MH+).
P r ep a r a tion of (3-ter t-Bu tylca r bon yl-2-p r op ylca r ba -
zoyl)a m in e 10b (w h er e R3 ) H). To intermediate 9b (0.20
g, 1.15 mmol) dissolved in dioxane/H2O (4 mL, 1:1 mix) was
added potassium cyanate (0.19 g, 2.3 mmol) portionwise over
30 min followed by 4 N HCl (aq) (0.28 mL, 1.15 mmol). The
solution was stirred at RT (16 h) before the volatiles were
removed in vacuo and the residue extracted into EtOAc (50
mL), washed sequentially with 10% citric acid and brine. Then
the organic phase was dried (Na2CO3), filtered, and concen-
trated before being purified (SiO2, 7:3 EtOAc/hexane, Rf ) 0.20)
to give 10b (R1 ) H) as a white solid (0.15 g, 60%). MS (FAB)
218.1 (MH+).
P r ep a r a tion of (3-ter t-Bu tylca r bon yl-2-p r op ylca r ba -
zoyl)ben zyl Alcoh ol 11b. Intermediate 9b (0.50 g, 2.87
mmol) was dissolved in anhydrous THF (6 mL) and cooled to
0 °C with anhydrous pyridine (0.29 mL, 3.6 mmol). The
solution was treated dropwise with benzyl chloroformate (0.42
mL, 2.95 mmol). The mixture was allowed to slowly warm to
RT and stirred 16 h before being diluted with EtOAc (50 mL)
and washed with brine. After drying (Na2SO4), the material
was purified (SiO2, 4:1 hexane/EtOAc) to give the precursor
to 11b as a white solid (0.80 g, 90%). MS (FAB) 309 (MH+),
209 (M - Boc)+.
a Bruker AMX 400 spectrometer and can be found in the
Supporting Information. FAB mass spectra were recorded on
an Autospec, VG spectrometer. Column chromatography was
performed either on silica gel (10-40 µm or 230-400 mesh
ASTM, E. Merck) or by preparative HPLC using a Partisil 10
ODS-3, C18 preparative column (50 cm × 22 mm). Analytical
HPLC were carried out on the following systems: System A:
Vydac C18, 10 µm analytical column (24 cm × 4.6 mm); mobile
phase, acetonitrile/0.06% trifluoroacetic acid (TFA) in water/
0.06% TFA; System B: ODS-AQ, Combiscreen, 5 µm analytical
column (50 mm × 4.6 mm); mobile phase, acetonitrile/0.06%
trifluoroacetic acid in water/0.06% TFA; System C: Symmetry
C8, 5 µm analytical column (2.1 × 150 mm); mobile phase,
acetonitrile/50mM NaH2PO4, pH ) 4.4.
Gen er a l P r otocol for th e P r ep a r a tion of Com p ou n d s
8a -g. P r ep a r a tion of N′-P r op ylid en eh yd r a zin eca r boxy-
lic Acid ter t-Bu tyl Ester 8b. To a solution of Boc-hydrazine
7 (3.0 g, 22.6 mmol) in toluene (42 mL) was added propional-
dehyde (1.8 mL, 24.9 mmol). The solution was heated to 50
°C for 1 h and then stirred at RT for 24 h. The mixture was
concentrated to give 8b as a white solid (3.70 g, 95%) which
was homogeneous by analytical HPLC (97.5%). MS (FAB)
173.1 (MH+).
P r epar ation of N′-Eth yliden eh ydr azin ecar boxylic Acid
ter t-Bu tyl Ester 8a . Obtained from 7 (2.0 g, 12.6 mmol) using
the procedure described for 8b but utilizing acetaldehyde (0.78
mL, 13.9 mmol) afforded 8a (1.99 g, 100%) as a colorless oil.
HPLC homogeneity (99%); MS (FAB) 159 (MH+).
P r epar ation of N′-Bu tyliden eh ydr azin ecar boxylic Acid
ter t-Bu tyl Ester 8c. Obtained from 7 (2.64 g, 20 mmol) using
the procedure described for 8b but utilizing butraldehyde (1.8
mL, 20 mmol) afforded 8c (2.25 g, 60%) as a white solid after
purification by chromatography (SiO2, 2:1 hexane/EtOAc).
HPLC homogeneity (99%); MS (FAB) 187 (MH+).
Preparation of N′-Pentylidenehydrazinecarboxylic Acid tert-
Butyl Ester 8d.
Obtained from 7 (2.5 g, 18.9 mmol) using the procedure
described for 8b but utilizing n-valeraldehyde (2.2 mL, 21
mmol) afforded 8d (3.78 g, 100%) as a colorless oil. MS (FAB)
201.1 (MH+). This material was sufficiently clean to be used
as is in the next step.
P r ep a r a tion of N′-(1-Eth ylp r op ylid en e)h yd r a zin eca r -
boxylic Acid ter t-Bu tyl Ester 8e. Obtained from 7 (2.5 g,
18.9 mmol) using the procedure described for 8b but utilizing
3-pentanone (3.2 mL, 21 mmol) afforded 8e (3.5 g, 92%) as a
colorless oil. MS (FAB) 201.1 (MH+).
P r ep a r a tion of N′-[2-Meth ylp r op ylid en e]h yd r a zin e-
ca r boxylic Acid ter t-Bu tyl Ester 8f. Obtained from 7 (2.5
g, 18.9 mmol) using the procedure described for 8b but utilizing
isobutyraldehyde (1.9 mL, 21 mmol) afforded 8f (3.5 g, 100%)
as a colorless oil. MS (FAB) 187.1 (MH+), 209.1 (M + Na)+.
P r ep a r a tion of N′-[3-Meth ylbu tylid en e]h yd r a zin eca r -
boxylic Acid ter t-Bu tyl Ester 8g. Obtained from 7 (2.5 g,
18.9 mmol) using the procedure described for 8b but utilizing
isovaleraldehyde (1.3 mL, 21 mmol) afforded 8g (3.8 g, 100%)
as a colorless oil. MS (FAB) 201.4 (MH+), 223.3 (M + Na)+.
Gen er a l P r oced u r e for P r ep a r a t ion of Com p ou n d s
9a-g. P r epar ation of N′-P r opylh ydr azin ecar boxylic Acid
ter t-Bu tyl Ester 9b. To imine 8b (3.7 g, 21.48 mmol) in THF
(80 mL) at -78 °C was added DIBAL (31 mL, 47.25 mmol) as
a 1.5 M solution in toluene. The reaction was maintained at
-78 °C for 2 h and then -40 °C for 2 h. The mixture was then
warmed to RT before Rochelle’s salt (aqueous potassium
sodium tartrate) solution was added and the reaction mixture
stirred at RT overnight. The organic phase was separated and
the aqueous phase extracted with Et2O (2 × 75 mL). The
combined organic extracts were washed with brine, dried
(Na2SO4), filtered, and concentrated in vacuo. Purification by
flash chromatography on silica gel gave intermediate 9b as a
colorless oil (3.4 g, 91%). MS (CI-NH3) 175.2 (MH+).
P r ep a r a tion of N′-Eth ylh yd r a zin eca r boxylic Acid ter t-
Bu tyl Ester 9a . Obtained from 8a (2.4 g, 15 mmol) using the
procedure described for 9b afforded 9a (1.1 g, 46%) as a
P r ep a r a tion of N′-(3-P h en yl-p r op ion yl)-N′-p r op ylh y-
d r a zin eca r boxylic Acid ter t-Bu tyl Ester 12b. To interme-
diate 9b (0.20 g, 1.15 mmol) were added hydrocinnamic acid
(0.19 g, 1.26 mmol), TBTU (0.40 g, 1.26 mmol), and DIPEA
(0.44 mL, 2.53 mmol) in DMF (5 mL) at RT. The coupling
reaction was left 16 h before the solvent was removed in vacuo
and the product purified (SiO2, 3:1 hexane/EtOAc) to give the
Boc intermediate 12b as a white solid (0.29 g, 81%). MS (FAB)
307 (MH+), 207 (M - Boc)+; HPLC homogeneity (system A;
95.5%).
P r ep a r a tion of (3-ter t-Bu tylca r bon yl-2-p r op ylca r ba -
zoyl)-N-m eth yla n ilin e 13b. To a solution of phosgene in
toluene (1.93M, 5.1 mL, 9.9 mmol) in dry THF (5 mL) at 0 °C
was added a solution of intermediate 9b (0.43 g, 2.47 mmol)

3796 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
Bailey et al.
and diisopropylethylamine (0.94 mL, 5.42 mmol) in THF (5
mL) over 15 min. The mixture was stirred at 0 °C for 30 min
before the excess phosgene and solvents were removed in
vacuo. The residue was redissolved in THF (5 mL) with DIPEA
(0.94 mL, 5.42 mmol) and cooled to 0 °C before a solution of
N-methylbenzylamine (0.32 mL, 2.47 mmol) with DIPEA (0.94
mL, 5.42 mmol) was added all at once. The reaction was
maintained at 0 °C (1 h) and then warmed to RT (16 h). The
mixture was poured into EtOAc (100 mL) and washed sequen-
tially with HCl (1 N), sat. NaHCO3, and brine before being
dried (MgSO4), filtered, and concentrated. The product was
purified by flash chromatography (SiO2, 2:1 hexane/EtOAc) to
afford 13b as a white solid (0.485 g, 64%). MS (FAB) 322.1
(MH+).
P r ep a r a tion of (3-ter t-Bu tylca r bon yl-2-p r op ylca r ba -
zoyl)in d olin e 14b. Following the same protocol as 13b except
using indoline as the amine, 9b (0.43 g, 2.47 mmol) was
converted into compound 14b (0.56 g, 74%) as a white solid.
MS (FAB) 320.1 (MH+), 342.1 (M + Na)+.
P r ep a r a tion of (3-ter t-Bu tylca r bon yl-2-p r op ylth ioca r -
ba zoyl)ben zyla m in e 15b. To intermediate 9b (16.4 mg, 0.094
mmol) under an inert atmosphere in dry THF (4 mL) at 0 °C
was added benzyl isothiocyanate (14 mg, 0.094 mmol) contain-
ing DIPEA (36 uL, 0.21 mmol) in THF (2 mL). The mixture
was stirred at 0 °C for 1 h and then at RT (16 h). The mixture
was partitioned between EtOAc and brine and then washed
with 1 N HCl (aq), sat. NaHCO3, and brine. The organic phase
was dried (MgSO4), filtered, and concentrated before being
purified by chromatography (SiO2, 2/1 hexane/EtOAc) to give
15b (17 mg, 56%). MS (electrospray) 346 (M + Na)+. Used as
such to prepare the final inhibitor according to Scheme 1.
P r ep a r a tion of N-P r op ylh yd r a zin eca r boxylic Acid
ter t-Bu tyl Ester 5. Commercially available benzyl carbazate
16 (12.5 g, 75.3 mmol) in toluene (140 mL) was treated with
propionaldehyde (6.0 mL, 83.2 mmol) and heated at 60 °C for
2 h. The solution was cooled and left at RT (16 h). The volatiles
were removed in vacuo to afford the Schiff base 17 as a white
solid (19.5 g). Part of 17 (10 g, 48.5 mmol) was dissolved in
THF (180 mL) and then cooled to -78 °C before DIBAL in
toluene (1.5 M, 47 mL, 70.5 mmol) was added from a dropping
funnel over 30 min. The solution was stirred an additional 2
h before warming to -40 °C for 2 h. To the solution at -10 °C
was added diethyl ether (180 mL) followed by dropwise
addition of Rochelle’s salt solution (70 mL). The mixture was
stirred at RT (16 h), and the solvents were removed. The
residue was extracted with diethyl ether (3 × 150 mL), and
the combined extracts were washed with sat. brine, dried (Na2-
SO4), filtered, and concentrated to give hydrazine 18 (9.5 g,
94%) as a colorless oil. Hydrazine 18 (5.0 g, 24 mmol) was
dissolved in dioxane (20 mL) and added to a solution of 2 N
NaOH (13.2 mL, 26.4 mmol) containing di-tert-butyl dicar-
bonate (5.2 g, 24 mmol) with stirring at RT (16 h). The mixture
was diluted with EtOAc (200 mL) and washed with water (2
× 200 mL) and brine (1 × 100 mL). The extract was dried
(Na2SO4), filtered, and concentrated before being purified
(SiO2, 3/1 hexane/EtOAc) to afford compound 19 as a white
solid (1.78 g). Finally, removal of the Cbz group was ac-
complished by dissolving 19 (640 mg, 2.07 mmol) in ethanol
corresponding hydrazinyl HCl salt in quantitative yield. In a
separate operation, to a solution of phosgene/toluene (1.93 M,
0.3 mL, 0.57 mmol) in dry THF (2 mL) with DIPEA (59 µL,
0.46 mmol) at 0 °C was added (S)-(+)-1-cyclohexylethylamine
(21 µL, 0.142 mmol) over 15 min. After being stirred at 0 °C
for 30 min, the solvents were removed in vacuo and subse-
quently redissolved in THF (2 mL) with DIPEA (59 µL, 0.46
mmol). This solution was added to the above-mentioned
hydrazinyl HCl salt (dissolved in 2 mL of THF containing 59
µL of DIPEA) at 0 °C and stirred for 1 h and then at RT (16
h). The mixture was concentrated to dryness and dissolved into
EtOAc (75 mL) before being washed with 1 N HCl (aq) and
brine. The organic phase was dried (Na2SO4), filtered, and
concentrated to give a pale yellow solid (0.12 g, 0.11 mmol).
This crude material was finally deprotected in EtOH (9 mL)
with ammonium acetate (12 mg, 0.17 mmol) being added. This
mixture was treated with 10% Pd/C (12 mg) and hydrogenated
under an atmosphere of H2 (1 atm) over 16 h. The reaction
was flushed and the catalyst removed by filtration through
Celite. The solvent was removed in vacuo and the resulting
inhibitor purified by preparative HPLC (H2O/AcCN) to afford
compound 38 (43 mg, 42%). HPLC (system A: 99%, system
B: 98%); MS (FAB) 929.5 (MH+); HRMS calcd for C46H72N8O12
(MH+) 929.53479, found: 929.53720.
Gen er a l P r oced u r e for P r ep a r a tion of In h ibitor s Ac-
cor d in g to Sch em e 1 (Meth od s A a n d C): P r ep a r a tion
of 34 (Meth od C). The HCl salt of 10b after deprotection
(where R3 ) CH(“S”-Me)Ph (77 mg, 0.24 mmol) was combined
with the pentapeptide 2 (0.20 g, 0.22 mmol), TBTU (85 mg,
0.26 mmol), and diisopropylethylamine (0.13 mL, 0.73 mmol)
in DMF (2.5 mL) at RT for 16 h. The reaction mixture was
concentrated and the residue dissolved in EtOAc and washed
sequentially with saturated aqueous NaHCO3, 10% aqueous
HCl, and brine before being dried (MgSO4), filtered, and
concentrated in vacuo. Purification (SiO2, gradient 7:3 EtOAc/
hexane to neat EtOAc) gave the precursor to 4 (R3 ) CH(“S”-
Me)Ph) as a white solid (80 mg, 33%). This peptide (75 mg,
0.067 mmol) was treated with neat TFA (1.5 mL) for 1.5 h
before being concentrated in vacuo. Purification by preparative
HPLC gave compound 34 as a white solid (17 mg, 27%). HPLC
(system A: 97%, system C: 95%); MS (FAB) 923.6 (MH+);
HRMS calcd for
923.49097.
C
46H66N8O12 (MH+) 923.48785, found:
Com p ou n d 25 (Meth od C). Following method C (Scheme
1), the final compound was purified by preparative HPLC to
give compound 25 as a white solid (85 mg, 73%). HPLC (system
A: 98%, system C: 100%); MS (FAB) 819.3 (MH+); HRMS
calcd for C38H58N8O12 (MH+) 819.42160, found: 819.42523.
Com p ou n d 26 (Meth od C). Following method C (Scheme
1), the final compound was purified by preparative HPLC to
give compound 26 as a white solid (69 mg, 35%). HPLC (system
A: 99%, system B: 97%); MS (FAB) 909.4 (MH+); HRMS calcd
for C45H64N8O12 (MH+) 909.47217, found: 909.47500.
Com p ou n d 27 (Meth od B). Following method B (Scheme
1), the final compound was purified by preparative HPLC to
give compound 27 as a white solid (17 mg, 19%). HPLC (system
A: 100%, system C: 97%); MS (FAB) 953.0 (MH+); HRMS
calcd for C46H64N8O14 (MH+) 953.46680, found: 953.46207.
with 10% Pd/C (63 mg) under
a balloon atmosphere of
Com p ou n d 28 (Meth od A). Following method A (Scheme
1), the final compound was purified by preparative HPLC to
give compound 28 as a white solid (40 mg, 20%). HPLC (system
A: 95%, system B: 96%); MS (FAB) 923.5 (MH+); HRMS calcd
for C46H66N8O12 (MH+) 923.49100, found: 923.48785.
hydrogen (16 h). The vessel was evacuated and the catalyst
filtered off using Celite as a filter aid. Concentration in vacuo
gave the desired Boc protected hydrazine 5 (344 mg, 95%) as
a colorless liquid. MS (FAB) 175 (MH+), 197 (M + Na)+.
Gen er a l P r oced u r e for P r ep a r a tion of In h ibitor s Ac-
cor d in g to Sch em e 1 (Meth od B): P r ep a r a tion of 38
(Meth od B). To pentapeptide 1 (1.75 g, 1.95 mmol) in DMF
(20 mL) with the Boc-protected hydrazine 5 (0.34 g, 1.95 mmol)
and O-(7-azabenzotriazol-1-yl)hexafluorophosphate (HATU)
(1.26 g, 2.53 mmol) was added DIPEA (1.36 mL, 7.8 mmol).
The reaction was allowed to stir at RT (16 h) before being
concentrated in vacuo and purified (SiO2, neat EtOAc) to afford
the fully protected peptide 5 (1.4 g, 67%). Following this,
peptide 6 (0.15 g, 0.14 mmol) was treated with 4 N HCl/dioxane
(3 mL, 30 min, RT) and concentrated to dryness to give the
Com p ou n d 29 (Meth od A). Following method A (Scheme
1), the final compound was purified by preparative HPLC to
give compound 29 as a white solid (4 mg, 2%). HPLC (system
A: 99%, system C: 97%); MS (FAB) 923.5 (MH+); HRMS calcd
for C46H64N8O12 (MH+) 921.472117, found: 921.46970.
Com p ou n d 30 (Meth od B). Following method B (Scheme
1), the final compound was purified by preparative HPLC to
give compound 30 as a white solid (8 mg, 9%). HPLC (system
A: 99%, system C: 95%); MS (FAB) 925.4 (MH+); HRMS calcd
for C45H64N8O11S (MH+) 925.44934, found: 925.44510.

Azapeptide Inhibitors of Hepatitis C Virus Serine Protease
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3797
Com p ou n d 31 (Meth od C). Following method C (Scheme
1), the final compound was purified by preparative HPLC to
give compound 31 as a white solid (56 mg, 69%). HPLC (system
A: 98%, system C: 97%); MS (FAB) 877.1 (MH+).
Com p ou n d 32 (Meth od C). Following method C (Scheme
1), the final compound was purified by preparative HPLC to
give compound 32 as a white solid (47 mg, 24%). HPLC (system
A: 95%, system B: 97%); MS (FAB) 908.4 (MH+); HRMS calcd
for C46H65N7O12 (MH+) 908.47693, found: 908.47693.
Com p ou n d 33 (Meth od C). Following method C (Scheme
1), the final compound was purified by preparative HPLC to
give compound 33 as a white solid (27 mg, 13%). HPLC (system
A: 99%, system C: 95%); MS (FAB) 923.6 (MH+); HRMS calcd
for C46H66N8O12 (MH+) 923.48785, found: 923.49004.
Com p ou n d 35 (Meth od C). Following method C (Scheme
1), the final compound was purified by preparative HPLC to
give compound 35 as a white solid (3 mg, 2%). HPLC (system
A: 98%, system C: 98%); MS (FAB) 937.5 (MH+); HRMS calcd
for C47H68N8O12 (MH+) 937.50398, found: 937.50630.
Com p ou n d 36 (Meth od A). Following method A (Scheme
1), the final compound was purified by preparative HPLC to
give compound 36 as a white solid (75 mg, 78%). HPLC (system
A: 99%, system C: 97%); MS (FAB) 921.3 (MH+); HRMS calcd
for C46H65N8O12 (MH+) 921.47212, found: 921.46970.
Com p ou n d 37 (Meth od A). Following method A (Scheme
1), the final compound was purified by preparative HPLC to
give compound 37 as a white solid (41 mg, 20%). HPLC (system
A: 97%, system B: 97%); MS (FAB) 924.4 (MH+); HRMS calcd
for C45H65N9O12 (MH+) 924.48309, found: 924.47820.
Com p ou n d 39 (Meth od B). Following method B (Scheme
1), the final compound was purified by preparative HPLC to
give compound 39 as a white solid (7.5 mg, 6%). HPLC (system
A: 98%, system C: 97%); MS (FAB) 915.5 (MH+); HRMS calcd
for C41H62N12O12 (MH+) 915.47250, found: 915.46881.
Com p ou n d 40 (Meth od C). Following method C (Scheme
1), the final compound was purified by preparative HPLC to
give compound 40 as a white solid (28 mg, 13%). HPLC (system
A: 98%, system C: 98%); MS (FAB) 974.3 (MH+); HRMS calcd
for C50H68N8O12 (MH+) 973.50398, found: 973.50580.
system B: 99%); MS (FAB) 937.5 (MH+); HRMS calcd for
C
Com p ou n d 48 (Meth od A). Following method A (Scheme
47H68N8O12 (MH+) 937.50348, found: 937.50730.
1), the final compound was purified by preparative HPLC to
give 48 as a white solid (100 mg, 48%). HPLC (system A: 95%,
system C: 97%); MS (FAB) 951.4 (MH+); HRMS calcd for
C
48H70N8O12 (MH+) 951.51917, found: 951.52300.
Com p ou n d 49 (Meth od A). Following method A (Scheme
1), the final compound was purified by preparative HPLC to
give 49 as a white solid (4 mg, 2%). HPLC (system A: 98%,
system B: 91%); MS (electrospray) 961.4 (M-H)-; HRMS calcd
for C45H61F3N8O12 (MH+) 963.44391, found: 963.44740.
Biologica l Assa ys. The IC50 values were obtained from our
in house protease assay which has been previously described
and represent an average of at least four determinations.7 The
specificity of selected compounds was determined against a
variety of serine proteases (human leukocyte and porcine
pancreatic elastases (HLE and PPE), bovine pancreas R-chy-
motrypsin) and one cysteine protease (human liver cathepsin
B) as described previously.27b
Exp r ession a n d P u r ifica tion of NS3 P r otea se. The NS3
protease domain was expressed in the pET29b vector (Novagen
Inc.) and encoded a modified BK strain sequence that spanned
amino acids 1-180 and contained a C-terminal solubilization
motif (ASKKKK).30 The expression and purification protocol
was exactly as previously described.32
NMR Sa m p le P r ep a r a tion . NMR samples were prepared
by adding 24 µL of concentrated solution of inhibitor 25 in
DMSO-d6 to an aqueous buffer composed of 50 mM Na2PO4,
300 mM NaCl, 3 mM dithiothreitol-d10 and 10% (v/v) D2O
(spiked with 3-(trimethylsilyl)-propionic 2,2,3,3-d4) at pH 6.5.
The final volume of these solutions was 600 µL with an
inhibitor concentration ranging between 1.1 and 1.5 mM. To
these samples was added a concentrated stock solution of NS3
protease (9.2 mg/mL, ∼450 µM) in a buffer identical to that
described above such that an inhibitor/protease ratio of 30:1
to 35:1 was typically achieved. For the competition experiment,
a stock solution of a potent activated carbonyl inhibitor (IC50
) 23.5 nM) in DMSO-d6 (3.6 mM) was added to one of the
samples described above such that
a final inhibitor 25/
Com p ou n d 41 (Meth od B). Following method B (Scheme
1), the final compound was purified by preparative HPLC to
give compound 41 as a white solid (7 mg, 7%). HPLC (system
A: 98%, system C: 95%); MS (FAB) 1001.5 (MH+) and 1003.5
(MH+2)+.
protease/potent inhibitor ratio of 34:1:4 was achieved.
NMR Meth od s. All spectra were acquired on a Bruker DRX
600 MHz NMR spectrometer equipped with a 5 mm TXI probe
at 27 °C. Two-dimensional (2D) double quantum-filtered COSY
(DQF-COSY), TOCSY, NOESY, and ROESY spectra were
acquired using standard pulse sequences with time propor-
tional phase incrementation (TPPI) method. Suppression of
the solvent signal was achieved by the use of presaturation or
by inserting a 3-9-19 WATERGATE module prior to data
acquisition.33 The 2D transferred-NOESY experiments were
recorded with mixing time of 70, 100, 150, and 200 ms. In these
experiments, a 25 ms spin-lock pulse was applied prior to t1
delay in order to eliminate protein background signals.34 The
ROESY experiment was recorded with a 300 ms spin-lock
period. The 2D data sets were typically acquired with 2048
points in t2, 350-420 points in t1 and 128 scans. The data were
processed and analyzed using XWinNMR and WinNMR soft-
ware (Bruker Canada, Milton, Ontario) and Felix software
(Molecular Simulations Inc., San Diego, CA). Shifted sine-bell
apodization and zero-filling (2048 × 1048 real points) were
applied prior to Fourier transformation, and subsequent
baseline corrections were applied in one or both dimensions.
Com p u ta tion a l Meth od s. The NS3 bound conformation
of inhibitor 25 was modeled by a simulating annealing protocol
using Discover 95.0 and the CFF95 force field (Molecular
Simulations Inc., San Diego, CA). The dynamics were per-
formed without cross-terms and nonbonded cutoffs and with
a dielectric constant of 1.0. A total of 60 NMR-derived distance
restraints were generated from transferred-NOESY volume
buildup rates using the Assign module of Felix software
(Molecular Simulations Inc., San Diego, CA). These NMR
distance restraints were applied as strong (1.8-2.5 Å), medium
(1.8-3.5 Å), or weak (1.8-5.0 Å) flat-bottomed potentials
having force constants of 15 kcal/mol‚Å2. Pseudoatoms defining
Com p ou n d 42 (Meth od A). Following method A (Scheme
1), the final compound was purified by preparative HPLC to
give compound 42 as a white solid (153 mg,39%). HPLC
(system A: 100%, system B: 99%); MS (FAB) 895.4 (MH+);
HRMS calcd for
895.45654.
C
44H62N8O12 (MH+) 895.45970, found:
Com p ou n d 43 (Meth od C). Following method C (Scheme
1), the final compound was purified by preparative HPLC to
give 43 as a white solid (18 mg, 14%). HPLC (system A: 98%,
system B: 97%); MS (FAB) 909.9 (MH+); HRMS calcd for
C
45H64N8O12 (MH+) 909.47217, found: 909.46860.
Com p ou n d 44 (Meth od C). Following method C (Scheme
1), the final compound was purified by preparative HPLC to
give 44 as a white solid (15 mg, 8%). HPLC (system A: 97%,
system B: 96%); MS (FAB) 937.5 (MH+); HRMS calcd for
C
47H69N8O12 (MH+) 937.50348, found: 937.50630.
Com p ou n d 45 (Meth od A). Following method A (Scheme
1), the final compound was purified by preparative HPLC to
give 45 as a white solid (89 mg, 43%). HPLC (system A: 99%,
system B: 100%); MS (FAB) 951.5 (MH+); HRMS calcd for
C
48H70N8O12 (MH+) 951.51917, found: 951.52110.
Com p ou n d 46 (Meth od A). Following method A (Scheme
1), the final compound was purified by preparative HPLC to
give 46 as a white solid (9 mg, 8%). HPLC (system B: 95%,
system C: 93%); MS (FAB) 951.1 (MH+); HRMS calcd for
C
48H70N8O12 (MH+) 951.51917, found: 951.52110.
Com p ou n d 47 (Meth od A). Following method A (Scheme
1), the final compound was purified by preparative HPLC to
give 47 as a white solid (104 mg, 46%). HPLC (system A: 96%,

3798 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
Bailey et al.
a Synthetic NS4A Cofactor Peptide. Cell 1996, 87, 343-355. (c)
Yan, Y.; Li. Y.; Munshi, S.; Sardana, V.; Cole, J . L.; Sardana,
M.; Steinku¨hler, C.; Tomei, L.; De Francesco, Kuo, L.; Chen, Z.
Complex of NS3 Protease and NS4A Peptide of BK Strain
Hepatitis C Virus: A 2.2A Resolution Structure in a Hexagonal
Crystal Form. Protein Sci. 1998, 7, 837-847.
the centroids of the phenyl aromatic ring and the methyl
groups were introduced in the definition of the NOE restraints,
and the interproton distances were corrected accordingly.35
A
single, high temperature unrestrained dynamics run was
performed at 900 K using a time step of 1 fs, with 50 structures
collected at 1 ps intervals to generate a starting set of
conformations. Each structure was then retrieved, cooled, and
minimized using the following simulated annealing protocol.
The temperature was initially lowered to 750 K at a rate of
30 K/ps where only strong restraints were applied. The
remaining restraints were added, and additional cooling to first
500 K (25 K/ps) and then 300 K (20 K/ps) was performed,
followed by restrained minimization (including cross-terms)
to a final gradient of 0.005 kcal/mol‚Å. A total of 14 low energy,
NMR-consistent structures were isolated at the end of this
protocol. These 14 structures were further divided into two
subfamilies of structures (eight and six members, respectively)
according to their slightly different P1 conformations. The first
subfamily of structures (eight-members) was finally selected
as the final representative set of NS3 bound conformations
based on additional knowledge of the three-dimensional
structure of the NS3 protease bound to an inhibitor (unpub-
lished data). These final eight structures are shown superim-
posed (P1-P4 backbone atoms only) in Figure 2. The root-
mean-square deviation for the backbone atoms of P1-P4 is
0.21 Å. None of these structures have distance violations
greater than 0.2 Å and the average total restraint violation
energy is 0.43 kcal/mol with a S. D. ) 0.21 kcal/mol.
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Complex Formation between the NS3 Serine-type Proteinase of
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Su p p or tin g In for m a tion Ava ila ble: 1H NMR data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Ack n ow led gm en t. We gratefully acknowledge Co-
lette Boucher and Sylvain Bordeleau for providing
analytical support. We also thank Diane Thibeault,
Roger Maurice, Pierre Bonneau, and Ce´line Plouffe for
assay support in this study. Finally, we thank Paul
Anderson, Daniel Lamarre, and Michael Cordingley for
their encouragement and support.
(9) Steinku¨hler, C.; Biasiol, G.; Brunetti, M.; Urbani, A.; Koch, U.;
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