The design and enzyme-bound crystal structure of indoline based peptidomimetic inhibitors of hepatitis C virus NS3 protease

Article abstract of DOI:10.1021/jm049435d

The design of a series of peptidomimetic inhibitors of the hepatitis C virus NS3 protease is described. These inhibitors feature an indoline-2-carboxamide as a novel heterocyclic replacement for the P3 amino acid residue and N-terminal capping group of tr

Full text of DOI:10.1021/jm049435d

J. Med. Chem. 2004, 47, 6443-6446
6443
The Design and Enzyme-Bound Crystal
Structure of Indoline Based
Peptidomimetic Inhibitors of Hepatitis C
Virus NS3 Protease
Jesus M. Ontoria,† Stefania Di Marco,‡
Immacolata Conte,† M. Emilia Di Francesco,†
Cristina Gardelli,† Uwe Koch,† Victor G. Matassa,§
Marco Poma,† Christian Steinku¨hler,‡
energy being generated by hydrophobic contacts and
electrostatic interactions along the length of this sur-
face.10 Short synthetic substrates for the enzyme have
not been found, and only peptides containing 10 or more
amino acids (spanning the P6-P4′ region) are pro-
cessed.11,12 Remarkably, the N-terminal hexapeptide
cleavage products of these substrates inhibit NS313 and
have served as a starting point for drug discovery
efforts. Sequence optimization14 and the design of a non-
nucleophilic mimetic for the canonical P1 cysteine
residue15 led to a series of potent tripeptide “serine trap”
inhibitors16 (e.g. 1) that became the lead structures in
our medicinal chemistry program. Here we report our
efforts aimed at reducing the peptidic character of 1
through the design and optimization of a novel replace-
ment for the N-terminal amino acid and capping group.
We additionally disclose the enzyme bound crystal
structure of one compound from this series that vali-
dates a C2-alkylated indoline ring as a peptidomimetic
replacement for the P3 residue of tripeptide NS3 pro-
tease inhibitors.
Cinzia Volpari,‡ and Steven Harper*,†
IRBM-MRL Rome, Via Pontina km 30,600,
00040 Roma, Italy
Received July 16, 2004
Abstract: The design of a series of peptidomimetic inhibitors
of the hepatitis C virus NS3 protease is described. These
inhibitors feature an indoline-2-carboxamide as a novel hetero-
cyclic replacement for the P3 amino acid residue and N-
terminal capping group of tripeptide based inhibitors. The
crystal structure of the ternary NS3/NS4A/inhibitor complex
for the most active molecule in this series highlights its
suitability as an N-terminal capping group of a dipeptide
inhibitor of the NS3 protease.
Despite improved therapeutic regimens for hepatitis
C virus1 (HCV) the disease remains the leading cause
of liver transplantation in the United States2 and affects
over 170 million individuals worldwide.3 Current treat-
ments based on interferon-R dosed in combination with
ribavarin are often unsuccessful due to reemergence of
virus following cessation of therapy, creating a pressing
need for new antivirals to tackle HCV infection.4
The NS3 gene product encoded by the (+)-stranded
RNA genome of HCV contains a trypsin-like serine
protease enzyme that plays a key role in the viral life
cycle.5 After liberation of its N-terminus from the
initially translated HCV poly-protein, NS3 is respon-
sible for four crucial poly-protein cleavages that release
the viral nonstructural proteins NS3-NS5B. The cata-
lytic activity of NS3 is augmented by formation of a
complex with the viral NS4A protein, and heterodimeric
NS3-NS4A is believed to be the physiologically relevant
form of the enzyme.6,7 There is now compelling evidence
to suggest that inhibition of NS3 is a viable strategy
for the development of antiviral agents,8 validating the
search for novel, potent, and drug-like inhibitors of this
enzyme.
Figure 1. Design of indoline based peptidomimetic replace-
ments for the N-terminal amino acid of 1.
To replace the N-terminal capping group and P3
residue of 1, we sought heterocyclic scaffolds that could
(i) retain the double hydrogen bonding interaction with
Ala157 observed in the enzyme bound crystal struc-
ture17 of peptide based inhibitors; (ii) orient a P2-P1
amino acid sequence as an active site “anchor”; (iii)
direct pendant functionality to engage the P3/S3 side
chain interaction; and (iv) function as a bridge to
auxiliary binding regions, such as those accessed by the
N-terminal capping group in the peptide series. Using
the enzyme bound crystal structure of tripeptide keto-
acid inhibitors as a starting point, a range of aromatic
and nonaromatic nitrogen heterocycles were evaluated.
Aromatic heterocycles appeared unattractive in view of
the difficulty of accessing the S3 site from a substituent
on an sp2 hybridized ring carbon atom, leading to the
selection of an indoline-2-carboxylic acid (Figure 1) as
a promising scaffold. When the indoline engages the
Ala157 NH and CO bonds in a twin hydrogen-bonding
interaction, the substituent on its sp3 hybridized C2
atom projects perpendicular to the plane of the indoline
ring and (in one diastereoisomer) can access the S3
Medicinal chemistry efforts toward inhibitors of NS3
have been complicated by the unusual structural fea-
tures of the enzyme. Crystal structures7,9 reveal that
while the overall fold of the enzyme and the orientation
of its catalytic machinery are in register with those of
mammalian serine proteases, the viral enzyme differs
markedly in structure at its substrate-binding site.
Substrates bind in an extended conformation in a
shallow and solvent exposed channel, the binding
* To whom correspondence should be addressed. Phone: +39 06
91093444. Fax: +39 06 91093654. E-mail: Steven_Harper@Merck.com.
† Department of Medicinal Chemistry.
‡ Department of Biochemistry.
§ Present address: Graffinity Pharmaceuticals AG, Im Neuenheimer
Feld 518, Heidelberg, Germany 69120.
10.1021/jm049435d CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/13/2004

6444 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Letters
Table 1. Structure and Biological Activity of Indole Based
Ketoacid Inhibitors of HCV NS3 Protease
Figure 2. The crystal20 structure of 7i (purple) with the NS3/
NS4 complex at 2.3 Å resolution. Superimposed is the crystal
structure with the tripeptide inhibitor Boc-Glu-Leu-difluoro-
Abu-COCO2H (white).17 Both inhibitors are shown in stick
representation with heteroatoms colored as follows: F ) cyan;
O ) red; S ) yellow; N ) blue. Atoms on the enzyme are shown
in space-filled representation, with C ) green; N ) blue; O )
red; S ) yellow; solvent accessible protein surface ) white.
respect to the side chain of the P2 (L)-leucine residue.
The S absolute stereochemistry at each of the three
chiral centers in 7i is in line with the requirement for
(L)-amino acids at P1-P3 in the peptide series and
suggests that, for the C2-alkylated indolines in entries
2-3, the active component in the diastereoisomer
mixtures (7d and 7f) will likely also have S configura-
tion at P1 and the same relative stereochemistry at P3,
(i.e. S for 7d and R for 7f). Analogous stereochemistry
is proposed for 7h (entry 4), the only ketoacid isomer
obtained in sufficient purity for biological testing from
6d. In light of the good potency shown by 7h and the
strong drop in activity seen for 7i with respect to 7j-l,
we speculate that 7h likely has the all-S stereochem-
istry that led to activity in entry 5. Surprisingly, in entry
6 two active isomers for the C2 unsubstituted indoline
were found. In view of the usual preference for S
absolute configuration at P1, we presume that both R
and S stereochemistry is tolerated at P3.
The C2 unsubstituted indoline 7b showed weak
inhibition of the NS3 protease. Surprisingly, only mod-
est gains in potency were achieved by introducing alkyl
substituents at the C2 position of the indoline. Both
R-branched alkyl chains (entry 2) and simple â-branched
alkyl groups (entry 3) gained only around 2-fold in
potency: the IC50’s for the postulated single active
isomers for both 7d and 7f would be in the 15-20 µM
range. More significant gains in potency were achieved
by introduction of benzylic substituents at C2, and we
were attracted to the thiophene ring as a means of
engaging Cys159, a prominent residue in a convex
lipophilic patch that constitutes the S3 site of the
enzyme. Donor acceptor interactions21 with the electron
deficient sulfur atom of the thiophene itself, or substit-
uents such as halogen atoms, were envisaged. Chloro-
thiophene 7h gained an order of magnitude in activity
over the unsubstituted indoline and illustrates that low
micromolar potency can be attained with no acidic
functionality in the inhibitor other than the keto-acid
moiety at the C-terminus. The combination of thienyl
and carboxylic acid functionality at P3 (7i) did bring a
further 5-fold gain in activity, however, and resulted in
the most potent inhibitors in this study. Improvements
a The use of (R,S)-difluoroAbu in this work results in the
inhibitors being obtained as four diastereoisomers differing in
stereochemistry at the P3 (indoline) and at P1 residues. The isomer
ratio (as determined by 1H NMR) of the biological testing sample
is denoted. b IC50 values (µM) are determined after 30 min
preincubation and are the mean of 2 independent measurements.
Standard deviations are within 35% of the mean. c n/a ) not active
(below 50% inhibition at 100 µM). d 1:1 mixture of diastereoisomers
at the P1 difluoroAbu residue. 7f and 7g differ in stereochemistry
at the P3 (indoline) residue.
surface. In this orientation the aromatic ring of the
indoline lies somewhat askew to the plane of the enzyme
with the C4 and C5 atoms exposed to solvent.
The C6 position of the indoline was envisioned as a
site that could project substituents toward the enzyme
surface in search of interactions such as those which
enhance potency in the context of the N-terminal
capping group of tripeptide inhibitors. The design
retained the R-ketoacid functionality that proved an
exceptional serine trap in the peptide series.18
The activities19 of indolines 7a-p against the NS3
protease domain in the presence of a synthetic NS4A
cofactor peptide are summarized in Table 1. In entry 5,
relevant biological activity is shown by a single dia-
stereoisomer of the inhibitor, with 7j-l showing around
a 40-fold or more loss in affinity with respect to the
active component 7i. The stereochemistry of 7i was
unambiguously assigned from the crystal structure of
an NS3/NS4A/7i complex (Figure 2).20 Both the difluo-
roAbu side chain at P1 and the thienylmethyl substitu-
ent at P3 lie on the opposite face of the inhibitor with

Letters
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6445
Scheme 1. Synthesis of Indoline Based Peptidomimetic
in activity could also be achieved through introduction
of functionality on the aromatic ring of the indoline, the
ether 7m showing a 5-fold gain over its unsubstituted
counterpart 7b.
Inhibitors of HCV NS3 Proteasea
The enzyme bound crystal structure of a representa-
tive molecule (7i, Figure 2) from the current series of
peptidomimetics retains many of the features observed
previously for the tripeptide keto-acid inhibitors17 from
which they were designed. The inhibitor combines with
the E2 strand of the enzyme to form an antiparallel
â-sheet, with the expected covalent bond between the
keto group and the active site serine having been formed
through nucleophilic attack on the opposite face to that
usually observed for serine proteases. As a consequence
the oxyanion hole is occupied by the P1 carboxylic acid
rather than the hemiketal oxygen atom (which is
instead stabilized through hydrogen bonding with the
catalytic histidine residue). The difluoromethyl group
binds snugly in the S1 specificity pocket and is in
contact with the P3 substituent through contact of a
fluorine atom with the thiophene C4 position (3.34 Å).
This interaction, which may be considered a hydropho-
bic collapse that serves to preorganize the inhibitor for
binding in its extended conformation, results in efficient
filling of the lipophilic enzyme surface at S1-S3. The
indoline ring engages in the expected P3/S3 hydrogen
bonding that was inherent in our design, and presents
the thienylmethyl group in an orientation suitable for
interaction with Val132. There is an additional close P3/
S3 contact between the thiophene carboxylic acid with
Cys159. The orientation of the Lys136 side chain, a
residue that plays a crucial role in stabilizing the
transition states of peptide ketoacids, is also worthy of
note. Here we find this chain in hydrogen bonding
distance to both the P1 carboxylic acid (2.94 Å) and the
P2 carbonyl group (3.15 Å), an orientation which places
the aliphatic part of the side chain above the difluo-
roAbu P1 residue and encloses the S1 specificity pocket.
The importance of inhibitor-enzyme interactions at
the S3 site of the HCV NS3 protease is now well
appreciated. The S3 region is an open, lipophilic patch
that stretches to S1 and in which Cys159 and Val132
are prominent. Optimal interaction at S3 in the peptide
series16 was achieved with P3 amino acids that contain
an R-branched aliphatic side chain such as cycloalkyl
glycine, an approach which failed in the current setting.
The restricted conformational space available to an
R-branched substituent when attached to the indoline
(7d) apparently precludes a favorable interaction at the
S3 site. In contrast, the potency of inhibitors such as
7h over the unsubstituted indoline 7a is likely due in
large part to an increase in the lipophilic contact area
at S3. We have shown in previous work that the S3
region is characterized by a positive electrostatic po-
tential created by a number of basic residues, including
Lys136.10 Amphiphilic substituents at the indoline C2
position were therefore attractive, adding an apparently
complementary electrostatic component to the lipophilic
contact surface of the inhibitor. This substitution indeed
proved effective, leading to sub-micromolar inhibitors
of the enzyme (7i).
a Reagents: (i) HATU, DIEA, H-Leu-(()-difluoroAbu-CH(OH)-
CO2Me; (ii) HATU, DIEA, H-Leu-OBn; (iii) Pd/C (10%), H2 (1 atm);
(iv) EDC, HOBt, DIEA, (()-H-difluoroAbu-CH(OH)CO2Me; (v)
Dess-Martin periodinane, tert-BuOH; (vi) TFA, CH2Cl2, H2O; (vii)
NaOH, MeOH; (viii) reversed-phase HPLC.
tively, aliphatic (Cbz-Ile-Leu-difluoroAbu-COCO2H, IC50
) 1.7 µM) or acidic (Boc-Glu-Leu-difluoroAbu-COCO2H,
IC50 ) 0.33 µM) functionality at P3. The achievement
of this level of potency through optimization of the P3/
S3 interaction alone is notable given the significant
contribution to binding that derives from the N-terminal
capping group of the P3 amino acid in a peptide
sequence.16 Figure 2 illustrates that the indoline ring
does not superimpose well with the peptide capping
group, and while the introduction of oxyacetic acid
functionality on the aromatic ring of the indoline (7m)
did bring a modest improvement of activity (potentially
through interaction with Arg123 in the S6 region of the
enzyme), there likely remains significant scope for
further optimization. In contrast to our expectation,
more direct access to the protein surface may in fact be
achievable through substitution of the indoline C7
position. Optimization of both the aromatic ring of the
indoline and its C2 substituent in tandem may also
bring further gains in activity, and the combination of
this work with P1/P2 modifications that lead to non-
covalent inhibitor series22 offers an attractive strategy
toward the development of more drug-like inhibitors of
the HCV NS3 protease.
Indoline based inhibitors of the NS3 protease (Table
1) were accessed using the synthetic strategy illustrated
in Scheme 1. The required 2-substituted indoline-2-
carboxylic acids 5b-e were obtained from the corre-
sponding esters 3b-e available via alkylation of 2a or
2b using KHMDS as base (Table 2).
The ester of the 6-substituted indoline 3i was pre-
pared from methyl 6-benzyloxyindole-2-carboxylate as
outlined in Scheme 2. Thus, Boc protection of the indole
nitrogen was followed by concomitant hydrogenolysis of
the benzyl ether and reduction of the indole ring to
afford 4, which was alkylated with tert-butyl bromoac-
etate.
The acids 5a-f were elaborated to the keto-acid
precursors 6 either by direct HATU mediated peptide
coupling with H-Leu-(()-difluoroAbu-CH(OH)CO2Me16
or through stepwise elongation using the corresponding
single amino acids. The latter route was used for 5b,
allowing hydrogenation of the cyclohexene ring in
tandem with hydrogenolytic cleavage of the leucine
Compounds 7h and 7i, which may be considered
capped dipeptides, show similar inhibition of NS3 as
their related tripeptide counterparts containing, respec-

6446 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Table 2. Preparation of 2-Alkylindoline-2-carboxylic Acids
Letters
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a peptide sequence.
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(20) The coordinates have been deposited in the PDB. Coordinate
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Supporting Information Available: Synthetic proce-
dures and spectral data for compounds 3b-f and 7a-p.
Experimental protocols for the collection and refinement of
X-ray structure data. This material is available free of charge
via the Internet at http://pubs.acs.org.
JM049435D