WO2000031129A1

WO2000031129A1 - Pharmaceutical compounds for the inhibition of hepatitis c virus ns3 protease

Info

Publication number: WO2000031129A1
Application number: PCT/EP1999/009207
Authority: WO
Inventors: Antonello Pessi; Paola Ingallinella; Elisabetta Bianchi
Original assignee: Istituto di Ricerche di Biologia Molecolare P Angeletti SpA
Current assignee: Istituto di Ricerche di Biologia Molecolare P Angeletti SpA
Priority date: 1998-11-26
Filing date: 1999-11-24
Publication date: 2000-06-02
Anticipated expiration: 2001-05-26
Also published as: JP2002530430A; CA2352493A1; GB9825946D0; EP1144446A1; AU1387100A; AU764589B2

Abstract

Peptidic inhibitors of hepatitis C virus NS3 protease are disclosed which are based on the P and P' regions of the natural substrate. The P' part of the inhibitor is optimised to achieve maximum binding energy through interaction with the S' region of the enzyme. By selecting amino acids such that the inhibitor is substantially not cleavable by the NS3 protease inhibitors having potency in the low nanomolar to sub-nanomolar range can be achieved.

Description

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-trans itted 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-α, 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 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 NS5A/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...Pl'-P2'-P3'-P4'-, with each P representing an amino acid, and the scissile bond lying between PI and Pi'. Corresponding binding sites on the enzyme are indicated as S6-S5-S4-S3-S2-S1... Sl'-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 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 P^-P^') do not inhibit NS3 to any significant extent. This notwithstanding, inspection of the crystal structure of NS3 with or 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. Landro et al in (1997) Biochemistry 3_6, 9340-9348 synthesized certain non-cleavable decapeptides based on the NS5A/5B cleavage site by substituting the P serine by a bulky cyclic aromatic (tetrahydroisoquinoline-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 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-P₄'.

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 NS3 (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) (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 or peptide analogue may, for instance 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 - 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 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:

β-cyclohexylalanine, phenylglycine, homophenylalanine and norleucine; other possibilities, though less preferred, are leucine, methionine, norvaline, and β-cyclopropylalanine . Of all these, cyclohexylalanine and phenyl glycine are most preferred.

Examples of the amino acid or analogue, C' include aspartic acid, glutamic acid, γ-carboxyglutamic acid, glutamine, asparagine, and hydroxyproline . Slightly less preferred are N-β-Aloc-diaminobutyric acid, thiazolylalanme, methionine sulfoxide, pyridylalanine 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

Notes : Cha β-cyclohexylalanine , Nle norleucine. Phg phenylglycine . Hof homophenylalanine . Hyp hydroxyproline .

When the residue D' is leucine (or other amino acid) 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 IC₅₀ below lOOuM, e.g. below 20μM, particularly below lOμM and, optimally, of less than lμM. Preferably, Pep is a hexa-, penta- or tetra peptide having formula II below:

F-E-D-C-B-A ill)

wherein: A is an amino acid or amino acid analogue having a relatively small (Ci-C aliphatic side chain. Possible choices for this group include cysteine, aminobutyric acid (Abu) (including di- and tri-fluoro 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.

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 β-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, 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. 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 IC₅₀ 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 .

TABLE 2

10

15

10

Particularly preferred examples of Pep, together

with their IC₅₀s (in μM) are set out below in Table 3 are:

TABLE 3

Most preferred:

* Tested only as decapeptides ese compounds:

Alg allylglycine .

MGly methylglycine .

MVal methylvaline .

Abu 2-aminobutyric acid.

GluS N-succinylglutamic acid.

AsGlu Glutamic acid having N-terminal

acylsulphonamide .

Cha β-cyclohexylalanine .

Nap naphthylalanine .

AspS N-succinylaspartic acid,

Nle norleucine.

Dif 3, 3-diphenylalanine .

Tha 2-thienylalanine .

FCI 4-chlorophenylalanine .

Phg phenylglycine .

CysMe S-methylcysteine . 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 γ-carboxyglutamic acid.

Dap β-diaminopropionic acid.

Dns dansyl (5-dimethylamino-l-

naphthalene-sulfonyl ]

Examples of compounds of the present invention may

be effective as inhibitors of NS3 protease at micromolar

or nanomolar levels. Preferably, the IC₅₀, as measured in

the assay described below is less than lOOnM,

particularly preferably less than 20nM and, optimally,

less than 5nM. 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 NS3 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 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 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 subject'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 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. Modes for Carrying 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-Ile-Cha-Cys-Pro-Cha-

Asp-Leu-Pro-Tyr-Lys (N^G-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 linker p- [ (R, S) - - [1- (9H-Fluoren-9-yl) -methoxyformamido] -

2, -dimethoxybenzyl] -phenoxyacetic acid (Rink, H. (1987)

Tetrahedron Let t . 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 Fmoc-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/triisopropyIsilane

(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, 15mm) using as eluents (A) 0.1%

trifluoroacetic acid in water and (B) 0.1% 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 [KKKGSWIVGRHLSGR (NH₂) ] . As substrate, a

substrate peptide 4AB [DEMEECASHLPYK] based on the

sequence of the NS4A/NS4B cleavage site of the HCV

polyprotein, was used. Cleavage assays were performed in 57μl 50 rtiM Hepes

pH7.5, 1 % CHAPS, 15 % glycerol, 10 mM DTT (buffer A), to

which 3μl substrate peptide were added. As protease co-

factor a peptide spanning the central hydrophobic core

(residues 21-34) of the NS4A protein, Pep4AK

[KKKGSWIVGRHLSGR(NH₂) ] was used. Buffer solutions

containing 80 μM 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 μl 1 % TFA. Cleavage of

peptide substrates was determined by HPLC using a Merck-

Hitachi chromatograph equipped with an autosampler. 80

μl samples were injected on a Lichrospher C18 reversed

phase cartridge column (4 x 74mm, 5μm, Merck) and

fragments were separated using a 10-40 % acetonitrile

gradient a 5%/min using a flow rate of 2.5ml/min. Peak

detection was accomplished by monitoring both the absorbance at 220nm and tyrosine fluorescence (λ_ex = 260

nm, λ_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 .

K_α 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 = (V_naxS)/(K_m(l+K₁/I)+S); (eq.l

Alternatively, K_λ values were derived from IC50 values, calculated using a four-parameter logistic

function, according to eq.2:

IC50 l+S/K K, [eq.2!

The table below sets out the IC₅₀ 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 binding assays, since they would be competitive with

compounds binding to either the S or the S' region of the

enzyme .

Table 4

10

15

20

10

Abbreviations used in Table I :

Abu = aminobutyric acid

Cha = β-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 version of the cleavage assay described above was

employed using, as before, an NS3/4A complex comprising

the NS3 protease domain and a modified form of the NS4A

peptide, Pep4AK [KKKGSWIVGRIILSGR (NH₂) ] .

lμM of the enzyme complex was incubated for 16hrs in

the presence of lOμM inhibitor as a candidate substrate

peptide. Assays were performed in 57μl 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 (0.1% TFA in H20) and 5% solvent B (0.1% TFA in

acetonitrile) at a flow rate of 1 ml/min. Samples were

eluted from this column with a linear gradient from 5% to 90% of B in 45 minutes. Peak detection was accomplished

by monitoring absorbance at 220 nm.

Claims

CLAIMS :

1. A compound of formula (I), or a pharmaceutically acceptable salt or derivative thereof: Pep-A'-B'-C'-D¹ (I) wherein formula (I) is written from N-terminus on the left to C-terminus at the right and:

"Pep" is a peptide or peptide analogue capable of binding to HCV NS3 protease; A' is a proline residue which is optionally substituted;

B' is an amino acid or amino acid analogue having a non-polar side chain;

C is an amino acid or amino acid analogue having a polar side chain; and

D' is selected from leucine, other amino acids or amino acid analogues having a non-polar aliphatic side chain, and peptides of 2 to 6 amino acids having leucine or other amino acid or amino acid analogue with a non- polar aliphatic side chain as N-terminal residue; and wherein the bond between Pep and A' is substantially uncleavable by HCV NS3 protease.

2. The compound of claim 1, a pharmaceutically acceptable salt or derivative thereof which is C- terminally amidated.

3. The compound of claim 1 or claim 2, a pharmaceutically acceptable salt or derivative thereof which is N-terminally acylated.

4. A compound, salt or derivative according to any one of claims 1 to 3 wherein A'-B'-C'-D' is a tetrapeptide of formula: Pro-B'-C'-Leu wherein B' and C are as defined in claim 1.

5. A compound, salt or derivative according to any one of the previous claims wherein B' is selected from: β- cyclohexylalanine, phenylglycine, homophenylalanine, norleucine, leucine, methionine, norvaline and β- cyclopropylalanine .

6. A compound, salt or derivative according to claim 5 wherein B' is selected from cyclohexylalanine and phenylglycine .

7. A compound, salt or derivative according to any one of the previous claims wherein C is selected from: aspartic acid, glutamic acid, γ-carboxyglutamic acid, glutamine, asparagine, hydroxyproline, N-β-Aloc- diaminobutyric acid, thiazolylalanme, methionine sulfoxide, pyridylalanine and serine.

8. A compound, salt or derivative according to claim 7 wherein C is aspartic acid.

9. A compound, salt or derivative according to any one of the preceding claims, wherein the combination of amino acids B'C is selected from:

Cha-Ser

Cha-Asp

Nle-Asp

Hof-Asp Phg-Asp

Cha-Gln

Nle-Gln

Hof-Gln

Cha-Hyp Nle-Hyp

Hof-Hyp

Nle-Ser

10. A compound, salt, or derivative according to any one of the preceding claims wherein Pep-OH is capable of binding HCV NS3 protease, in the absence of the C- terminal residues A'-B'-C'-D', and has an IC₅₀ below lOOμM in an inhibition assay.

11. A compound, salt, or derivative according to any one of the preceding claims wherein Pep is a hexa-, penta- or tetra-peptide having formula (II) below:

F-E-D-C-B-A wherein: A is an amino acid or amino acid analogue having an aliphatic side chain of form 1 to 6 carbon atoms ;

B is an amino acid or analogue having a non-polar, acidic, or polar but uncharged side group;

C is an amino acid or amino acid analogue having a non-polar or acidic side chain;

D is an amino acid or amino acid analogue having a hydrophobic side group;

E together with F may be absent, but if present is an amino acid or amino acid analogue having an acidic side chain, non-polar side chain or polar, but uncharged side chain, or is a dicarboxylic acid containing up to 6 carbon atoms and lacking the amino group of acidic amino acids; and 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 or is a dicarboxylic acid containing up to 6 carbon atoms .

12. A compound according to claim 11 wherein: A is selected from: cysteine, aminobutyric acid, di- and tri-fluoro aminobutyric acid, norvaline, allylglycine and alanine;

B is selected from: glutamic acid, aspartic acid, glycine, methyl glycine, 2-amino butyric acid, alanine, isoleucine, valine, leucine, cysteine, naphthylalanine and β-cyclohexylalanine;

C is selected from: glutamic acid, aspartic acid, glycine, methyl glycine, 2-amino butyric acid, alanine, isoleucine, valine, leucine, cysteine, naphthylalanine and β-cyclohexylalanine;

D is selected from: methionine, isoleucine, leucine, norleucine, valine, methylvaline, phenylglycine, diphenylalanine, tyrosine, thienylalanine, and chlorophenylalanine; E is selected from: glutamic acid, aspartic acid, phenylalanine, diphenylalanine, isoleucine, valine, tyrosine, 4-nitrophenylalanine, glutaric acid and succinic acid; and F is selected from: aspartic acid, glutamic acid, glutaric acid and succinic acid.

13. A compound, salt, or derivative, according to any one of the preceding claims for use in therapy.

14. A pharmaceutical composition comprising a compound, salt or derivative according to any one of the preceding claims and a pharmaceutically acceptable excipient, diluent or carrier.

15. Use of a compound, salt or derivative according to any one of the preceding claims in the manufacture of a medicament for the treatment or prevention of hepatitis C or a related condition.

16. A method of inhibiting HCV NS3 protease activity, and/or of treating or preventing hepatitis C or a related condition, comprising administering to a human or mammalian subject suffering from the condition a therapeutically or prophylactically effective amount of a composition according to claim 14, or of a compound of any one of claims 1 to 12.