Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02352493 2001-05-25
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PHARMACEUTICAL COMPOUNDS FOR THE INHIBITION
OF HEPATITIS C VIRUS NS3 PROTEASE
Technical Field
This invention relates to compounds which can act as
inhibitors of the hepatitis C virus (HCV) NS3 protease,
to uses of such compounds and to their preparation.
Background Art
The hepatitis C virus (HCV) is the major causative
agent of parenterally-transmitted and sporadic non-A,
non-B hepatitis (NANB-H). Some 1~ of the human
population of the planet is believed to be affected.
Infection by the virus can result in chronic hepatitis
and cirrhosis of the liver, and may lead to
hepatocellular carcinoma. Currently no vaccine nor
established therapy exists, although partial success has
been achieved in a minority of cases by treatment with
recombinant interferon-a, either alone or in combination
with ribavirin. There is therefore a pressing need for
new and broadly-effective therapeutics.
Several virally-encoded enzymes are putative targets
for therapeutic intervention, including a metalloprotease
(NS2-3), a serine protease (NS3), a helicase (NS3), and
an RNA-dependent RNA polymerase (NS5B). The NS3 protease
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is located in the N-terminal domain of the NS3 protein,
and is considered a prime drug target since it is
responsible for an intramolecular cleavage at the NS3/4A
site and for downstream intermolecular processing at the
NS4A/4B, NS4B/5A and NSSA/5B junctions.
Previous research has identified classes of
peptides, in particular hexapeptides, showing degrees of
activity in inhibiting the NS3 protease. The aim of the
present invention is to provide further compounds which
exhibit similar, and if possible improved, activity.
According to the nomenclature of Schechter & Berger
(1967, Biochem. Biophys. Res. Commun. 27, 157-162)
cleavage sites in substrates for the NS3 protease are
designated P6-P5-P4-P3-P2-P1 . . . P1'-P2'-P3'-P4'-, with each P
representing an amino acid, and the scissile bond lying
between P1 and P1'. Corresponding binding sites on the
enzyme are indicated as S6-S5-S4-S3-S2-S1...S1'-S2'-S3'-S4'.
The present applicant has previously disclosed so
called product inhibitors which are based on the P-region
of the natural cleavage sites and which have been
optimised to low nanomolar potency ((1998) Biochemistry
37: 8899-8905 and (1998) Biochemistry 37: 8906-8914).
These inhibitors extract much of their binding energy
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from the C-terminal carboxylate, the remaining
interactions with NS3 being similar to the ones used by
the natural substrates, including binding in the S1 pocket
and the prominent electrostatic interaction of the P6-P5
acidic couple.
At variance with the P region, the P' region of
the substrate, while being important for catalysis, does
not influence significantly ground-state binding to the
enzyme as expressed by the Km value. In other words,
binding energy released by the substrate interaction with
the enzyme to form an initial non-covalent complex is
essentially due to the interaction of the residues of the
P region; the P'region residues contribute to a lesser
extent to the binding energy. Accordingly, peptides
based on the P'region of the. natural substrates (spanning
residues P1'-Plo) do not inhibit NS3 to any significant
extent. This notwithstanding, inspection of the crystal
structure of NS3 with ar without 4A (and more recently of
the NMR structure of NS3) shows the presence of binding
pockets in the S' region which might be exploited for the
binding of active-site directed inhibitors. S'-binding
ligands would therefore display a range of interactions
with the enzyme different from the ones used by the
substrate, and represent a novel class of NS3 inhibitors.
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Landro et al in (1997) Biochemistry 36, 9340-9348
synthesized certain non-cleavable decapeptides based on
the NSSA/5B cleavage site by substituting the P1' serine
by a bulky cyclic aromatic (tetrahydroisoquinoiine-3-
carboxylic acid) or smaller cyclic alkyl compound
(proline or pipecolinic acid). They then investigated
the interaction of these decapeptides with the substrate
binding site of NS3 either in the presence or absence of
NS4A cofactor. By looking at the effect of truncation at
either the P or P' side of the molecule they concluded
that most of the binding energy of the decapeptide is due
to interactions with NS3-NS4A complex on the P side of
the molecule. Truncation on the P' side produced a
relatively large effect in the presence of NS4A cofactor,
but less when NS4A was absent. They concluded that the
P4' substrate Tyr residue present in their molecules was
in close proximity, or in direct contact with NS4A and
that this residue contributes significantly to binding in
the presence of NS4A.
The present inventors have developed inhibitors
which are more powerful than those described by Landro et
al because they have better binding on their P' side. In
other words, the inhibitors.take advantage of binding to
the S' region in addition to binding to the S-region of
NS3. By varying the P' amino acid residues, the present
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inventors have shown that the binding energy which may be
extracted from S'-region binding is substantial, since
inhibitors with optimised and non-optimised P'-regions
differ in potency > 1000-fold. Since no activity was
present in any of the peptides corresponding to the
isolated P'-region, optimisation of an S'-binding fragment
was pursued in the context of non-cleavable decapeptides
spanning P6-PQ .
The inventors found that, by replacing Landro's P4'
Tyr residue by leucine the effectiveness of the
decapeptides as NS3 protease inhibitors could be
enhanced. Although it had been previously shown that
leucine in position P4' is better than tyrosine in a
decapeptide substrate cleavable by N53 (Urbani et al
(1997) J. Biol. Chem 272, 9204-9209), this is the first
showing that the same applies to decapeptide inhibitors
which are not cleaved under the influence of the enzyme.
By optimising the P4' residue and then the P2'-P3' fragment
and using these together with an optimised P region the
inventors have arrived at oligopeptides which show
potency in the low nanomolar-subnanomolar range.
Disclosure of the Invention
According to a first aspect of the present invention
there is provided a compound having the formula (I)
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(written from N-terminus to C-terminus):
Pep-A'-B'-C'-D' ( I )
wherein "Pep" is a peptide or peptide analogue
capable of binding to HCV NS3 protease; in particular, it
is capable of binding in the S-region of the protease;
A' is proline which is optionally substituted, for
instance with one to three substituent groups;
B' is an amino acid or amino acid analogue having a
non polar side chain. Preferably, the side chain is an
alkyl, aryl or aralkyl group containing 3 to 10,
particularly 4 to 8 carbon atoms;
C' is an amino acid or amino acid analogue having a
polar side group. Examples of polar side group may
contain between 2 and 10, preferably 2 to 6 carbon atoms;
D' is leucine, or less preferably another amino acid
with a non-polar aliphatic side chain, such as valine,
isoleucine, norleucine or methionine. Alternatively, it
is a short peptide or peptide analogue having one of
these amino acids, especially leucine at its N terminus.
The short peptide on peptide analogue may, for instance
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comprise 2 to 6, preferably 2 to 4 amino acids or amino
acid analogues.
As used herein, the term "amino acid analogue"
includes organic compounds containing an amino and a
carboxylic acid group, for instance arranged a- to each
other, and which do not necessarily occur in nature.
The Pep-A bond of the compound of formula (I) is
substantially uncleavable by HCV NS3 protease. For
instance, it is preferable that no cleavage be detectable
using the assay described below under the heading
"Substrate Assay".
Pharmaceutically acceptable salts of the compound of
formula (I), as well as derivatives, such as esters are
within the scope of the present invention.
Preferably, the compound of formula (I) is N-
terminally acylated, especially acetylated, although
other derivatives of the N-terminus are also possible,
for instance N-terminal sulphoxide, sulphonamide,
urethane or urea derivatives.
Preferably, the compound of formula (I) is C-
terminally amidated. However, the C-terminus may be an
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underivatised carboxylic acid group. Alternatively,
other C-terminal groups may be present.
Assuming no substitution of the proline residue at A'
is present, then a preferred C-terminal portion of the
compound of formula I is:
Pro-B'-C'-Leu
possibly with a short C terminal extension at Leu.
Preferred examples of the amino acid, or analogue, B'
for inclusion in compounds of the first aspect of the
invention, include:
~-cyclohexylalanine, phenylglycine,
homophenylalanine and norleucine; other possibilities,
though less preferred, are leucine, methionine,
norvaiine, and (3-cyclopropylalanine. Of all these,
cyclohexylalanine and phenyl glycine are, most preferred.
Examples of the amino acid or analogue, C' include
aspartic acid, glutamic acid, Y-carboxyglutamic acid,
glutamine, asparagine, and hydroxyproline. Slightly less
preferred are N-(3-Aloc-diaminobutyric acid,
thiazolylalanine, methionine sulfoxide, pyridylalanine
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and serine. Of all of these aspartic acid is most
preferred.
The following combinations of amino acid residues at
B' and C' are preferred, of which the combination of
cyclohexylalanine and aspartic acid is especially
preferred.
TABLE 1
B' C'
Cha Ser
Cha Asp
Nle Asp
Hof Asp
Phg Asp
Cha Gln
Nle Gln
Hof Gln
Cha Hyp
Nle Hyp
Hof Hyp
Nle Ser
Notes: Cha - ~3-cyclohexylalanine.
Nle - norleucine.
Phg - phenylglycine.
Hof - homophenylalanine.
Hyp - hydroxyproline.
When the residue D' is leucine (or. other amino acid)
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with a small peptide as C-terminal extension the peptide
may be chosen by comparison with the corresponding P'
portion of natural substrates.
The residues A', B', C' and D' may have D- or L-
stereochemistry, although L-stereochemistry is, in
general, preferred for each.
As regards the Pep part of the compound of formula
(I) this is particularly preferably a peptide or peptide
analogue capable of binding to HCV NS3 protease, even in
the absence of the C-terminal residues A'-B'-C'-D', for
instance when Pep carries just a carboxylic acid group at
the C terminus. For example, when tested in the
inhibition assay described below the fragment Pep-OH
preferably has an ICSO below 100uM, e.g. below 20uM,
particularly below lOUM and, optimally, of less than lpM.
Preferably, Pep is a hexa-, penta- or tetra peptide
having formula II below:
F-E-D-C-B-A (II)
wherein: A is an amino acid or amino acid analogue
having a relatively small (C1-C6) aliphatic side chain.
Possible choices for this group include cysteine,
aminobutyric acid (Abu) (including di- and tri-fluoro
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Abu), norvaline, allylglycine and alanine, any of which
may be N-methylated. Of these, cysteine and the
fluorinated aminobutyric acids are preferred choices for
A.
S
B is an amino acid or analogue having a non-polar or
acidic side chain. Some amino acids having polar but
uncharged side groups may also be suitable. Examples of
suitable amino acids include glutamic and aspartic acid,
glycine and methyl glycine, 2-amino butyric acid,
alanine, isoleucine, valine, leucine, cysteine,
naphthylalanine and a-cyclohexylalanine. Of these,
cyclohexylalanine is particularly preferred.
C is an amino acid or amino acid analogue having a
non-polar or acidic side chain. For instance, the
examples of such amino acids given above for B apply also
to C. In this case isoleucine and glutamic acid are
particularly preferred.
D is usually an amino acid or amino acid analogue
having a hydrophobic side group such ~as methionine,
isoleucine, leucine, norleucine, valine, methyl valine,
phenylglycine or, diphenylalanine. Among these leucine
and, particularly, diphenylalanine are preferred. Some
polar amino acids which include hydrophobic portions,
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such as tyrosine, thienylalanine, and chlorophenylalanine
may be suitable.
E together with F may be absent, but if present is
generally an amino acid or amino acid analogue having an
acidic side chain. Preferred examples are glutamic and
aspartic acid, with the former being preferred. E may,
alternatively, be an amino acid or analogue having a non-
polar, or polar but uncharged side chain. Of the non-
polar amino acids, phenylalanine, diphenylalanine,
isoleucine and valine are preferred, especially the D-
enantiomers. Among the polar amino acids suitable
examples are tyrosine and 4-nitrophenylalanine.
Alternatively, where F is absent (see below), E may be a
dicarboxylic acid containing up to 6 carbon atoms and
lacking the amino group of acidic amino acids. Suitable
examples are glutaric and succinic acid.
F may be absent (either by itself, or together with
E), but when present is an amino acid or analogue having
an acidic side chain. Aspartic acid is preferred,
although glutamic acid is another possibility. Like E, F
may also be a dicarboxylic acid containing up to 6 carbon
atoms, and lacking the amino group of acidic amino acids.
Examples are glutaric and succinic acid.
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Of residues E and F preferably at least E is
present. Particularly preferably both are present.
The amino acids and analogues A-F may be either L-
or D- enantiomers though L- is generally preferred for
all residues. In some cases it may be beneficial for one
or other of the residues to be D- while the rest are L-.
In particular it may be advantageous for E to be D-glu.
Preferred examples of the peptide "Pep" are listed
below in Tables 2 and 3 together with their ICSO values
when unextended at the C-terminus. Except for the
compounds having a succinyl residue at the N-terminus,
all compounds tested were N-acetylated at the N-terminus.
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TABLE 2
__ ... ,_..._..~......_.__....._.....
Exp
No. Se uence a
q ; ICSO (~'1)
.
1 Asp Glu Met Glu Glu Cys I 1.0
S 2 Asp Glu Met Glu Glu D-Cys 4.0
3 Asp Glu Met Glu Glu Abu 5.8
4 Met Glu Glu Cys 150.0
5 Glu Met Glu Glu Cys 21.0
6 Glu Asp Val Val Cys Cys 5.3
7 Glu Asp Val Val Abu Cys 2.8
8 Asp Glu Val Val Cys Cys 2.1
9 Glu Asp Val Val Gly Cys 20.0
10 Asp Glu Met Glu Glu Alg 12.0
11 Glu Asp Val Val MGly Cys 21.0
12 Glu Asp MVal Val Abu Cys 1.3
13 GluS Met Glu Glu Cys 1.3
14 AsGlu Met Glu Glu Cys 0.6
15 Asp Glu Met Glu Leu Cys 1.1
16 Asp Glu Met Glu Cha Cys 0.3
17 Asp Glu Met Glu Nap Cys 0.8
18 Asps Val Val Abu Cys 4.6
19 Asp Glu Met Glu Glu Cys(Me) 16.7
20 Asp Glu Val Glu Cha Cys 0.33
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21 Asp Glu Ile Glu Cha Cys 0.12
22 Asp Glu Tyr Glu Cha Cys 0.24
23 Asp Glu Phe Glu Cha Cys 0.42
24 Asp Glu Leu Glu Cha Cys 0.12
25 Asp Glu Cha Glu Cha Cys 0.14
26 Asp Glu Nle Glu Cha Cys 0.22
27 Asp Glu Tha Glu Cha Cys 0.87
28 Asp Glu FCI Glu Cha Cys 0.3
29 Asp Glu Phg Glu Cha Cys 0.12
30 Asp 3.4
Glu
Dif
Glu
Cha
D-Cys
31 Glu f u 1.4
Di Gl Cha
Cys
32 Dif Glu Cha Cys 30.0
33 Asp MGluLeu Glu Cha Cys 1.0
34 Asp 7.1
Glu
Dif
Glu
Cha
DHAla
35 Asp Glu Met Glu Glu Cpc 9.0
36 G lu e 2.5
Dif Cha
Il Cys
37 Dif Ile Cha Cys 100.0
38 Asp 19.0
Glu
Met
Glu
Glu
CnAla
39 Asp Glu Leu Glu Cha Abu 1.6
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40 Asp Glu Leu Glu Cha Val 4.0
41 Asp Glu Leu Glu Cha Nva 1.3
42 Asp-Asp-Leu-Glu-Cha-Cys 0.290
43 Asp-Fno-Leu-Glu-Cha-Cys 0.240
44 Asp-Tyr-Leu-Glu-Cha-Cys 0.135
45 Asp-(D)Phe-Leu-Glu-Cha-Cys 0.820
46 Asp-(D)Tyr-Leu-Glu-Cha-Cys 0.680
47 Asp-(D)Val-Leu-Glu-Cha-Cys 0.470
48 Asp-(D)Ile-Leu-Glu-Cha-Cys 0.330
49 Asp-(D)Dif-Leu-Glu-Cha-Cys 0,276
50 Asp-(D)Asp-Leu-Glu-Cha-Cys 0,122
51 Asp-Glu-Dap(N-b-Dns)-Glu-Cha-Cys 0,4
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Particularly preferred examples of Pep, together
with their ICSOs (in pM) are set out below in Table 3 are:
TABLE 3
Most preferred:
1 Asp Glu Dif Glu Cha Cys 0.05
2 Asp Glu Leu Val Cha Cys 0.08
3 Asp Glu Leu Ile Cha Cys 0.06
4 Asp Glu Dif Ile Cha Cys 0.06
5 Asp-Gla-Leu-Glu-Cha-Cys 0.055
6 Asp-(D)Glu-Leu-Glu-Cha-Cys 0.045
7 Asp-(D)Gla-Leu-Ile-ChawCys 0.0015
8 Glu-Leu-Glu-Cha-Cys 1.3
9 (D)Glu-Leu-Glu-Cha-Cys-(Pro-Cha-Asp-Leu) 0.080*
10 Succinyl Glu-Leu-Ile-Cha-Cys
11 Succinyl (D)Glu-Leu-Glu-Cha-Cys-(Pro-Cha- 0.0040*
Asp-Leu)
12 ~ Asp-(D)Glu-Leu-Ile-Cha-Cys
~ 13 ~ Asp-(D)Glu-Leu-Ile-Cha-Cys-(Pro-Cha-Asp- ~<0.0002*~
Leu)
* Tested only as decapeptides
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In these compounds:
Alg - allylglycine.
MGly - methylglycine.
MVal - methylvaline.
Abu - 2-aminobutyric acid.
GluS - N-succinylglutamic acid.
AsGlu - Glutamic acid having N-terminal
acylsulphonamide.
Cha - (3-cyclohexylalanine.
Nap - naphthylalanine.
Asps - N-succinylaspartic acid.
Nle - norleucine.
Dif - 3,3-diphenylalanine.
Tha - ~ 2-thienylalanine.
FCI - 4-chlorophenylalanine.
Phg - phenylglycine.
CysMe - S-methylcysteine.
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Cys(ACS) - Cysteine with C-terminal
acylsulphonamide.
DHAla - dehydroalanine.
Cpc - 1-amino-1-cyclopentane carboxylic
acid.
CnAla - cyanoalanine.
MGlu - N-methylglutamic acid.
Fno - ~ 4-nitrophenylalanine.
Gla - y-carboxyglutamic acid.
Dap - ~i-diaminopropionic acid.
Dns - dansyl(5-dimethylamino-1-
naphthalene-sulfonyl).
Examples of compounds of the present invention may
be effective as inhibitors of NS3 protease at micromolar
or nanomolar levels. Preferably, the ICSO, as measured in
the assay described below is less than 100nM,
particularly preferably less than 20nM and, optimally,
less than SnM.
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According to a second aspect, the present invention
provides a compound, salt or derivative according to the
first aspect, for use in any therapeutic method,
preferably for use in inhibiting the HCV N53 protease,
and/or for use in treating or preventing hepatitis C or a
related condition. By "related condition" is meant a
condition which is or can be caused, directly or
indirectly, by the hepatitis C virus, or with which the
HCV is in any way associated.
According to a third aspect the present invention
provides the use of a compound or derivative according to
the first aspect in the manufacture of a medicament for
the treatment or prevention of hepatitis C or a related
condition.
A fourth aspect of. the invention provides a
pharmaceutical composition which includes one or more
compounds or derivatives according to the first aspect.
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The composition may also include pharmaceutically
acceptable adjuvants such as carriers, buffers,
stabilisers and other excipients. It may additionally
include other therapeutically active agents, in
particular those of use in treating or preventing
hepatitis C or related conditions.
The pharmaceutical'composition may be in any
suitable form, depending on the intended.method of
administration. It may for example be in the form of a
tablet, capsule or liquid for oral administration, or of
a solution or suspension for administration parenterally.
According to a fifth aspect of the invention, there
is provided a method of inhibiting HCV NS3 protease
activity, and/or of treating or preventing hepatitis C or
a related condition, the method ~involving~ administering
to a human or animal (preferably mammalian) subject
suffering from the condition a therapeutically or
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prophylactically effective amount of a composition
according to the fourth aspect of the invention, or of a
compound or derivative according to the first aspect.
"Effective amount" means an amount sufficient to cause a
benefit to the subject or at least to cause a change in
the subj ect's condition.
The dosage rate at~which the compound, derivative or
composition is administered will depend on the nature of
the subject, the nature and severity of the condition,
the administration method used, etc. Appropriate values
can be selected by the trained medical practitioner.
Preferred daily doses of the compounds are likely to be
of the order of about 1 to 100 mg. The compound,
derivative or composition may be administered alone or in
combination with other treatments, either simultaneously
or sequentially. It may be administered by any suitable
route, including orally, intravenously, cutaneously,
subcutaneously, etc. Intravenous administration is
preferred. It may be administered directly to a suitable
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site or in a manner in which it targets a particular
site, such as a certain type of cell - suitable targeting
methods are already known.
A sixth aspect of the invention provides a method of
preparation of a pharmaceutical composition, involving
admixing one or more compounds or derivatives according
to the first aspect of 'the invention with one or more
pharmaceutically acceptable adjuvants, and/or with one or
more other therapeutically or prophylactically active
agents.
According to a seventh aspect of the invention there
is provided a method of producing the compounds of
formula I. These compounds may be generated wholly or
partly by chemical synthesis beginning from individual,
preferably protected, amino acids or oligopeptides and
using known peptide synthesis methods.
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Modes for Carryina Out the Invention
Embodiments of the invention are exemplified below
by way of illustration only.
EXAMPLES
(1) Synthesis
The synthesis of one of the compounds of the present
invention is described below. Other compounds may be
synthesized by an analogous method.
Synthesis of Ac-Asp-(D)Glu-Leu-IIe-Cha-Cys-Pro-Cha-
Asp-Leu-Pro-Tyr-Lys(Ng-Ac)-NH2
The synthesis was performed on solid phase by the
continuous-flow Fmoc-polyamide method (Atherton, E. and
Sheppard, R. C. (1989) Solid phase peptide synthesis. A
practical approach, IRL Press, Oxford.). The resin used
was Tentagel~~ derivatised with a modified Rink amide
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linker p-[(R,S)-a-[1-(9H-Fluoren-9-yl)-methoxyformamido]-
2,4-dimethoxybenzyl]-phenoxyacetic acid (Rink, H. (1987)
Tetrahedron Lett. 28, 3787-3789; Bernatowicz, M. S.,
Daniels, S. B. and Koster, H. (1989) Tetrahedron Lett.
30, 4645-4667). All the coupling reactions were performed
for 30 min with 5-fold excess of activated amino acid
over the resin free amino groups, using FSmoc-amino
acid/PyBOP/HOBt/DIEA (1:1:1:2) activation; double
coupling was used for the cysteine residue. At the end of
the assembly, the dry peptide-resin was treated with
trifluoroacetic acid/water/triisopropylsilane
(92.5:5:2.5) for 1.5h at room temperature; the resin was
filtered out and the peptide precipitated with cold
methyl t-Bu ether; the precipitate was redissolved in 50%
water/acetonitrile containing 0.1%TFA and lyophillised.
Purification to >98% homogeneity was achieved
through preparative HPLC on a Waters RCM (C-18) column
(100 X 25 mm, l5mm) using as eluents (A) 0.1%
trifluoroacetic acid in water and (B) 0.1~
CA 02352493 2001-05-25
WO 00/31129 PCT1EP99/09207
trifluoroacetic acid in acetonitrile. The gradient used
was 40~B isocratic for 5 min, then 40-60~B over 20 min,
flow rate 30 ml/min; the fractions were analysed by HPLC
(column: Beckman Ultrasphere, C-18, 25 X 4.6 mm, 5mm;
gradient: 35-65~B in 20 min, same eluents as the
preparative run, flow lml/min) and those containing the
pure material were pooled and lyophilised (yield=50~).
The Mass spectrum was acquired on a Perkin-Elmer API-100
spectrometer: MS= 1695.03 (calc.) 1694.6 (found).
(2) Inhibition Assay
The ability of the compounds to inhibit NS3 protease
was evaluated using an NS3/4A complex comprising the NS3
protease domain and a modified form of the NS4A peptide,
Pep 4AK [KKKGSWIVGRIILSGR(NH2)]. As substrate, a
substrate peptide 4AB [DEMEECASHLPYK) based on the
sequence of the NS4A/NS4B cleavage site of the HCV
polyprotein, was used.
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Cleavage assays were performed in 57u1 50 mM Hepes
pH7.5, 1 ~ CHAPS, 15 ~ glycerol, 10 mM DTT (buffer A), to
which 3u1 substrate peptide were added. As protease co-
factor a peptide spanning the central hydrophobic core
(residues 21-34) of the NS4A protein, Pep4AK
[KKKGSWIVGRIILSGR(NH2)] was used. Buffer solutions
containing 80 uM Pep4AK were preincubated for 10 minutes
with 10-200 nM protease' and reactions were started by
addition of substrate. Six duplicate data points at
different substrate concentrations were used to calculate
kinetic parameters. Incubation times were chosen in
order to obtain <7~ substrate conversion and reactions
were stopped by addition of 40 ul 1 ~ TFA. Cleavage of
peptide substrates was determined by HPLC using a Merck-
Hitachi chromatograph equipped with an autosampler. 80
ul samples were injected on a Lichrospher C18 reversed
phase cartridge column (4 x 74mm, Sum, Merck) and
fragments were separated using a 10-40 ~ acetonitrile
gradient a 5~/min using a flow rate of 2.5m1/min. Peak
detection was accomplished by monitoring both the
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absorbance at 220nm and tyrosine fluorescence (l~eY = 260
nm, l~em = 305nm). Cleavage products were quantitated by
integration of chromatograms with respect to appropriate
standards. Kinetic parameters were calculated from
nonlinear least-squares fit of initial rates as a
function of substrate concentration with the help of a
Kaleidagraph software, assuming Michaelis-Menten
kinetics.
K1 values of peptide inhibitors were calculated from
substrate titration experiments performed in the presence
of increasing amounts of inhibitor. Experimental data
sets were simultaneously fitted to eq.l using a
multicurve fit macro with the help of a Sigmaplot
software:
V = (Vmars) / (K,n(1+K;/I) +S) ~ (eq.l)
Alternatively, Ki values were derived from IC50
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values, calculated using a four-parameter logistic
function, according to eq.2:
IC50 - ( 1+S/Km) Ki ( eq. 2 )
The table below sets out the IC~o values for a
variety of peptides tested in this assay and establishes
that several optimised compounds of the present invention
are active at nanomolar or subnanomolar levels. All the
compounds tested - except for compound 26 which has a
succinyl residue at the N-terminus- were tested as their
N-acetyl derivatives.
Some of these compounds are the most potent in vitro
inhibitors of HCV protease described to date. They are
reversible, non covalent inhibitors which do not contain
an electrophilic ("serine-trap") moiety in the molecule.
They bind to both the S and S' region of the enzyme, and
this makes them suitable for developing competition
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binding assays, since they would be competitive with
compounds binding to either the S or the S' region of the
enzyme.
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Table 4
Ex. Sequence ICso
No (nM)
1 Glu Asp Val Val Abu Cys Pro Nle Ser 8500
Tyr
2 Glu Asp Val Val Abu Cys (Me)Ala Nle 3500
Ser Tyr
3 Asp Glu Dif Ile Cha Abu AIa Ser His 29000
Leu
4 Asp Glu Dif Ile Cha Abu (Me)Ala Ser 29000
His Leu
5 Asp Glu Dif Ile Cha (Me)Abu Ala Ser 8000
His Leu
6 Asp Glu Dif 11e Cha (Me)Abu (Me)Ala 3800
Ser His Leu
7 Asp (D)Glu Dif Ile Cha (Me)Abu (Me)Ala3100
Ser His Leu
8 Asp (D)Glu Leu Ile Cha Abu (Me)Ala 5000
Ser His Leu
9 Asp Glu Dif Ile Cha Cys Pro Nle Ser 876
Tyr
10 Glu Dif Ile Cha Cys Pro Nle Ser Leu 64
11 Asp Glu Dif ile Cha Cys Pro Cha Ser 23
Leu
12 Asp Glu Dif Ile Cha Cys Pro Cha Asp 1.3
Leu
13 Asp Glu Dif Ile Cha Cys Pro Phg Asp 7
Leu
14 Asp Glu Dif Ile Cha Cys Pro Nle Asp 1.8
Leu
15 Asp Glu Dif Ile Cha Cys Pro Hof Asp 1.8
Leu
16 Asp Glu Dif Ile Cha Cys Pro Cha Gln 14
Leu
17 Asp Glu Dif Ile Cha Cys Pro Nle Gln 32
Leu
18 Asp Glu Dif Ile Cha Cys Pro Hof Gln 18
Leu
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19 Asp Glu Dif Ile Cha Cys Pro Cha Hyp 11
Leu
20 Asp Glu Dif Ile Cha Cys Pro Nle Hyp 26
Leu
21 Asp Glu Dif Ile Cha Cys Pro Hof Hyp 15
Leu
22 Asp (D)Glu Leu lle Cha Cys Pro Nle 10
Ser Leu
23 Asp Glu Dif Ile Cha Cys Pro Cha Asp 0.85
Leu PYK(Ac)
24 Asp (D)Glu Leu Ile Cha Cys Pro Cha < 0.2
Asp Leu PYK(Ac)
25 Asp (D)Glu Leu Ile Cha Cys Pro Cha < 0.2
Asp Leu
26 Suc-(D)Glu Leu (le Cha Cys Pro Cha 4
Asp Leu
27 Asp (D)Glu Leu Glu Cha Cys Pro Cha 0.63
Asp Leu
28 (D)Glu Leu Glu Cha Cys Pro Cha Asp 80
Leu
29 Asp (D)Glu Leu Glu Cha Ala Pro Cha 17
Asp Leu
30 Asp (D)Glu Leu Ile Cha Cys Pro Nle 10
Ser Leu
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Abbreviations used in Table I:
Abu = aminobutyric acid
Cha = a-cyclohexylalanine
Hof = homophenylalanine
Hyp = hydroxyproline
Lys(Ac) or K(Ac) - Ne-Acetyl-Lysine
Nle = norleucine
Phg = phenylglycine
Sta = statine [(3S,4S)-4-amino-3-hydroxy-6-
methylheptanoic acid]
Dif = 3,3-diphenylalanine
Suc=succinyl
N-methylation is indicated as (Me) preceding the three-
letter code of the amino acid
PYK = proline-tyrosine-lysine
(3) Substrate Assay
In order to determine whether or not an inhibitor
molecule was a substrate for HCV NS3 protease a modified
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version of the cleavage assay described above was
employed using, as before, an NS3/4A complex comprising
the N53 protease domain and a modified form of the NS4A
peptide, Pep4AK [KKKGSWIVGRIILSGR(NH2)].
luM of the enzyme complex was incubated for l6hrs in
the presence of lOUM inhibitor as a candidate substrate
peptide. Assays were performed in 57u1 50 mM Hepes
pH7.5, 1~ CHAPS, 15~ glycerol, 10 mM DTT.
After this time HPLC was used to separate any
peptides resulting from cleavage and separated cleavage
products detected.
Samples were analysed by HPLC on a Beckman 0.46 x 25
cm C18 reversed phase column equilibrated in 95~ solvent
A (O.lo TFA in H20) and S~ solvent B (O.lo TFA in
acetonitrile) at a flow rate of 1 ml/min. Samples were
eluted from this column with a linear gradient from 5~ to
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90~ of B in 45 minutes. Peak detection was accomplished
by monitoring absorbance at 220 nm.
35
