WO2004022582A1

WO2004022582A1 - Peptidomimetic drugs

Info

Publication number: WO2004022582A1
Application number: PCT/AU2003/001149
Authority: WO
Inventors: Marie-Isabel Aguilar; Patrick Perlmutter; Alexander Ian Smith; Romila Devi
Original assignee: Monash University; Baker Medical Research Institute
Priority date: 2002-09-04
Filing date: 2003-09-04
Publication date: 2004-03-18
Also published as: AU2002951215A0

Abstract

The present invention relates generally to peptidomimetic compounds which inhibit Aminopeptidase P and their use in treating disorders and diseases associated with aberrant peptidase activity. More particularly, the present invention relates to peptidomimetic compounds comprising a β-amino acid. The peptidomimetic compounds of the invention may be useful in a range of therapeutic, prophylactic and diagnostic applications.

Description

PEPTIDOMIMETIC DRUGS

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to in this specification are collected alphabetically at the end of the description.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Biomolecular interactions such as hormone-receptor, enzyme-substrate and antigen- antibody interactions underpin normal cellular functions. The understanding of these interactions is not only essential in knowing how the bio-system works, but also in the development of drugs for the treatment of diseases associated with abnormal functioning of these cellular processes.

It is now well established that proteolytic reactions play an important role in many cellular events such as apoptosis, blood clotting and hormonal regulation to name just a few examples. Indeed, aberrant proteolytic events are known to be involved in many disease states such as hypertension, thrombosis, autoimmunity and viral invasion. Given the importance of proteases and peptidases in these various inter- and intracellular processes, inhibitors of these enzymes continue to serve as both molecular probes of major cellular networks as well as potential therapeutic agents for various human diseases. In spite of the advances and successes in enzyme inhibitor design, new approaches to the development of inhibitors of different classes of enzymes are required, particularly for the design of molecules which can specifically inhibit one of a family of very closely-related enzymes are needed.

While peptides are exquisitely designed to exert very specific biological activity, peptide- based drugs have suffered various therapeutic limitations such as poor oral bio-availability and short half-life in vivo (Boumendjel A, Roberts J C, Hu E, Pallai P V and Rebek J Jr, 1996, J Org Chem, 61, 4434-4438). Physiologically active peptides are rapidly degraded by luminal, pancreatic, cytosolic and lysosomal proteases and are subsequently removed from the system (Mϋller A, Schumann F, Koksch M and Sewald N, 1997, Letters in Peptide Science, 4, 275-281) thereby reducing the effect of the drugs.

There are also restrictions on the permeability of peptides either due to their size or barriers by the tissues and organs (Fix J A, 1996, J Pharmaceutical Sciences, 85(12), 1282-1285).

Peptide-based drugs need to reach the target organ or tissue in order to exhibit its effects.

The size of the peptide drug is sometimes too bulky to effectively move through the cells.

Thus, the peptides move through the circulation system and are eventually removed without even reaching the target area.

The biological efficacy of peptides is also affected by their conformational flexibility. In particular the peptide drug may not adopt the conformation of the native peptide in vivo and will not interact with the target protein. Due to these limitations, the effectiveness of the peptide drugs is significantly reduced. In order to ensure the drugs administered reach the target area or organ, peptide drugs need to be designed so that they are stable to enzymatic metabolism, small in size for easier movement through the barriers and adopt conformations similar to the native peptide. All these factors ensure that the drug interacts with the target protein. -Aminopeptidase P (APP) is a specific metallopeptidase enzyme capable of cleaving the N- terminal amino acid of peptides with an X-pro amino acid sequence at their N-terminus, where X is any amino acid. While the presence of a proline residue normally makes a peptide resistant to proteolytic attack, due to the cyclic structure which introduces conformational constraints (Turner A J, Hyde R J, Lim J and Hooper NM, 1997, Advanced Experimental Med Biology, 421, 7-16), APP is one enzyme that is able to cleave these peptides if the proline residue is attached to the N-terminus amino acid. APP is found in organisms as diverse as bacteria, yeast, fish and mammals and occurs in both cytosolic and membrane bound forms. APP is capable of degrading a wide variety of substrates such as bradykinin, allatostatin I, Substance P, peptide YY and neuropeptide Y (Venema R C, Ju H, Zou R, Venema V J and Ryan J W, (1997), Biochimca Et Biophysica Acta, 1354, 45- 48). It may also degrade interleukin-6 and collagen with a high degree of Gly-Pro-Y (Orawski AT and Simmons WH, 1995, Biochemistry, 34,11227-11236; Yoshimoto T, Orawski A T and Simmons W H, 1994, Archives of Biochemistry and Biophysics, 311(1), 28-34; Turner et al 1997, supra).

APP is not strongly inhibited by a broad range of known metallopeptidase inhibitors. Based on the knowledge of the substrate specificities of APP, it was reported that Pro-Tyr (Ki 7.5μM), Pro-Leu (Ki 21μM) and Pro-Phe (8.2μM) acted as competitive inhibitors (Yoshimoto et al, 1994, supra). Apstatin, (N-[2S,3R-3-amino-2-hydroxy-4-phenyl- butanoyl]-L-prolyl-L-prolyl-L-alaninamide, is a tetrapeptide with proline in the second and third positions. Apstatin has been found to have a Ki of 2.6μM with rat APP and 0.64μM with human membrane APP (Pretchel M M, Orawski A T, Maggiora L L and Simmons W H, 1995, J Pharm Exp Ther, 275, 1136-1142). An apstatin analogue (CH₃)₂CHCH₂CH(NH₂)CH(OH)CO-Pro-Ala-NH₂ was reported to have an IC₅₀ of 0.23 μM with human APP (Maggiora. L L, Orawski A T and Simmons W H, 1999, J . Medical Chemistry, 42, 2394-2402). Thioxo amino acids, such as a compound of the formula:

Ki = 0.445μM

have also been reported as inhibitors of E.coli APP (Stockel-Maschek A, Mrestani-Klaus C, Stiebitz B, Demuth H, andNeubert K, 2000, Biochim et Biophys Acta, 1479, 15-31).

There is a need for potent and selective inhibitors of APP as therapeutics and diagnostics. Furthermore, potent and selective inhibitors of APP may provide useful information defining the physiological role of APP leading to the development of further potential inhibitors.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a method of inl ibiting aminopeptidase P comprising contacting aminopeptidase P with a peptidomimetic compound of formula (I) or a derivative thereof,

X-Y-Z (I) wherein X is a proline residue or β-proline residue;

Y is an α-amino acid residue or a β-amino residue; and Z is absent or is from 1 to 8 amino acid residues; and wherein at least one of X and Y is a β-amino acid residue.

Another aspect of the present invention relates to a pharmaceutical composition comprising a peptidomimetic compound of formula (I) or a derivative thereof and a pharmaceutically acceptable carrier, diluent or excipient, X-Y-Z (I) wherein X is a proline residue or β-proline residue;

Another aspect of the present invention relates to a method of treating or preventing a disease or disorder modulated by aminopeptidase P in a mammal, comprising administering to the mammal a peptidomimetic compound of formula (I) or a derivative thereof, X-Y-Z (I) wherein X is a proline residue or β-proline residue;

Y is an α-amino acid residue or a β-amino residue; and Z is absent or is from 1 to 8 amino acid residues; and wherein at least one of X and Y is a β -amino acid residue. In a further aspect of the present invention there is provided a use of a peptidomimetic compound of formula (I) or a derivative thereof,

X-Y-Z (I) wherein X is a proline residue or β-proline residue; Y is an α-amino acid residue or a β-amino residue; and Z is absent or is from 1 to 8 amino acid residues; and wherein at least one of X and Y is a β -amino acid residue, in the preparation of a medicament for treating or preventing a disease or disorder modulated by aminopeptidase P.

In yet another aspect of the invention relates to a peptidomimetic compound of formula (I) or a derivative thereof,

X-Y-Z (I) wherein X is a proline residue or β-proline residue; Y is an α-amino acid residue or β-amino acid residue; and Z is absent or is from 1 to 8 amino acid residues; and wherein at least one of X and Y is a β-amino acid residue.

Amino acid structure and single and three letter abbreviations used throughout the specification are defined in Table 1. Table 1

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the determination that peptidomimetic compounds of formula (I) are APP inhibitors. The peptidomimetic compounds may be useful in therapy and diagnosis of diseases and disorders modulated by aminopeptidase P.

Accordingly, one aspect of the present invention relates to a method of inhibiting aminopeptidase P comprising contacting aminopeptidase P with a peptidomimetic compound of formula (I) or a derivative thereof, X-Y-Z (I) wherein X is a proline residue or β-proline residue;

Y is an α-amino acid residue or a β-amino residue; and

Z is absent or is from 1 to 8 amino acid residues; and wherein at least one of X and Y is a β-amino acid residue.

According to another aspect of the present invention there is provided a peptidomimetic compound of formula (I) or a derivative thereof,

X-Y-Z (I) wherein X is a proline residue or β-proline residue; Y is an α-amino acid residue or a β-amino residue; and

As used herein, the term "peptidomimetic compound" refers to a compound designed to perform the function of a peptide. Such functions include eliciting a specific biological effect through agonist/antagonist activity or inhibition of peptide/protein metabolizing enzymes.

As used herein, the term "amino acid" refers to an α-amino acid or a β-amino acid and may be a L- or D- isomer. The amino acid may have a naturally occurring side chain (see

Table 1 above) or a non-naturally occurring side chain (see Table 2 below). The amino acid may also be further substituted in the α-position or the β-position with a group selected from -Cι-Cι₀alkyl, -C₂-Cι₀alkenyl, -C₂-Cι₀alkynyl, -(CH₂)_nCOR_b -(CH₂)_nR₂, -PO₃H, -(CH₂)_nheterocyclyl or -(CH₂)_naryl where Ri is -OH, -NH₂, -NHCι-C₃alkyl, -OCi- C₃alkyl or -Cι-C₃alkyl and R₂ is -OH, -SH, -SCι-C₃alkyl, -OCι-C₃alkyl, -C₃-Cι₂cycloalkyl, -C₃-Cι₂cycloalkenyl, -NH₂, -NHCι-C₃alkyl or -NHC(C=NH)NH₂, n is 0 or an integer from 1 to 10 and where each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl or heterocyclyl group may be substituted with one or more groups selected from -OH, -NH₂, -NHCι-C₃alkyl, -OC_rC₃alkyl, -SH, -SCι-C₃alkyl, -CO₂H, -C0₂Ci-C₃alkyl, -CONH₂ or -CONHC_rC₃alkyl.

The term "α-amino acid" as used herein, refers to a compound having an amino group and a carboxyl group in which the amino group and the carboxyl group are separated by a single carbon atom, the α-carbon atom. An α-amino acid includes naturally occurring and non-naturally occurring L-amino acids and their D-isomers and derivatives thereof such as salts or derivatives where functional groups are protected by suitable protecting groups. The α-amino acid may also be further substituted in the α-position with a group selected from -Ci-Cioalkyl, -C₂-Cι₀alkenyl, -C₂-Cι₀alkynyl, -(CH₂)_nCORι, -(CH₂)_nR₂, -PO₃H, -(CH₂)_nheterocyclyl or -(CH₂)_naryl where Rj is -OH, -NH₂, -NHC C₃ alkyl, -OC C₃alkyl or Cι-C₃alkyl and R₂ is -OH, -SH, -SCι-C₃alkyl, -OCι-C₃alkyl, -C₃-Cι₂cycloalkyl, -C₃- Cπcycloalkenyl, -NH₂, -NHCι-C₃alkyl or -NHC(C=NH)NH₂, n is 0 or an integer from 1 to 10 and where each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl or heterocyclyl group may be substituted with one or more groups selected from -OH, -NH₂, -NHCi- C₃alkyl, -OCι-C₃alkyl, -SH, -SCι-C₃alkyl, -C0₂H, -CO₂Cι-C₃alkyl, -CONH₂ or -CONHC C₃alkyl.

As used herein, the term "β-amino acid" refers to an amino acid that differs from an α- amino acid in that there are two (2) carbon atoms separating the carboxyl terminus and the amino terminus. As such, β-amino acids with a specific side chain can exist as the R or S enantiomers at either of the α (C2) carbon or the β (C3) carbon, resulting in a total of 4 possible isomers for any given side chain. The side chains may be the same as those of naturally occurring α-amino acids (see Table 1 above) or may be the side chains of non- naturally occurring amino acids (see Table 2 below).

Furthermore, the β-amino acids may have mono-, di-, tri- or tetra-substitution at the C2 and C3 carbon atoms. Mono-substitution may be at the C2 or C3 carbon atom. Di- substitution includes two substituents at the C2 carbon atom, two substituents at the C3 carbon atom or one substituent at each of the C2 and C3 carbon atoms. Tri-substitution includes two substituents at the C2 carbon atom and one substituent at the C3 carbon atom or two substituents at the C3 carbon atom and one substituent at the C2 carbon atom. Tetra-substitution provides for two substituents at the C2 carbon atom and two substituents at the C3 carbon atom. Suitable substituents include -Cι-Cιoalkyl, -C₂-Cι₀alkenyl, -C₂- Cioalkynyl, -(CH₂)_nCORι, -(CH₂)„R₂, -PO₃H, -(CH₂)_nheterocyclyl or -(CH₂)_naryl where Ri is -OH, -NH₂, -NHCι-C₃alkyl, -OCι-C₃alkyl or -Cι-C₃alkyl and R₂ is -OH, -SH, -Sd- C₃alkyl, -OCι-C₃alkyl, -C₃-Cι₂cycloalkyl, -C₃-Cι₂cycloalkenyl, -NH₂, -NHCι-C₃alkyl or -NHC(C=NH)NH₂, n is 0 or an integer from 1 to 10 and where each alkyl, alkenyl, alkynyl cycloalkyl, cycloalkenyl, aryl or heterocyclyl group may be substituted with one or more groups selected from -OH, -NH₂, -NHCι-C₃alkyl, -OCι-C₃alkyl, -SH, -SCι-C₃alkyl, -CO₂H, -CO₂Cι-C₃alkyl, -CONH₂ or -CONHC₁-C₃alkyl.

Other suitable β-amino acids include conformationally constrained β -amino acids. Cyclic β-amino acids are conformationally constrained and are generally not accessible to enzymatic degradation. Suitable cyclic β-amino acids include, but are not limited to, cis and trans 2-amino-C₃-Cι₀-cycloalkyl-l-carboxylic acids, 2-amino-C₃-Cι₀cycloalkenyl-l- carboxylic acids, 2-amino-norbornane-l -carboxylic acids and their unsaturated derivatives and heterocyclic and bicyclic heterocyclic saturated or unsaturated carboxylic acid compounds where the β-amino nitrogen appears in the ring. Non limiting examples of suitable conformationally constrained β-amino acids include 2-aminocyclopropyl carboxylic acids, 2-aminocyclobutyl and cyclobutenyl carboxylic acids, 2- aminocyclopentyl and cyclopentenyl carboxylic acids, 2-aminocyclohexyl and cyclohexenyl carboxylic acids, 2-amino-norbornane carboxylic acids and tropane amino acids, and their derivatives, some of which are shown below:

Suitable derivatives of β-amino acids include salts and may have functional groups protected by suitable protecting groups.

The term "non-naturally occurring amino acid" as used herein, refers to amino acids having a side chain that does not occur in the naturally occurring L-α-amino acids. Examples of non-natural amino acids and derivatives include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6- methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids that may be useful herein is shown in Table 2.

TABLE 2

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine Nle

D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D -α-methylvaline Dmval N-cylcododecylglycine Ncdod

D -N-methy lalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3,3-diρhenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3 -guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-( 1 -hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-( 1 -methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-( 1 -methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyl-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine Mgln L-α-methylglutamate Mglu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine Mmet L-α-methylnorleucine Mnle

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr

L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine

1 -carboxy- 1 -(2,2-diphenyl- Nmbc ethylamino)cyclopropane The term "alkyl" as used herein refers to straight chain or branched hydrocarbon groups. Suitable alkyl groups include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl. For example, Cι-C₃alkyl refers to methyl, ethyl, propyl and isopropyl.

The term "alkenyl" as used . herein refers to straight chain or branched unsaturated hydrocarbon groups containing one or more double bonds. Suitable alkenyl groups include, but are not limited to ethenyl, propenyl, isopropenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl and decenyl.

The term "alkynyl" as used herein refers to straight chain or branched hydrocarbon groups containing one or more triple bonds. Suitable alkynyl groups include, but are not limited to ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl.

The term "cycloalkyl" as used herein, refers to cyclic hydrocarbon groups. Suitable cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl.

The term "cycloalkenyl" as used herein, refers to cyclic unsaturated hydrocarbon groups having at least one double bond in the ring. Suitable cycloalkenyl groups include, but are not limited to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl, cyclodecenyl, cycloundecenyl and cyclododecenyl.

The term "heterocyclyl" as used herein refers to 5 or 6 membered cyclic hydrocarbon groups in which at least one carbon atom has been replaced by N, O or S. Optionally, the heterocyclyl group may be fused to a phenyl ring. Suitable heterocyclyl groups include, but are not limited to pyrrolidinyl, piperidinyl, pyrrolyl, thiophenyl, furanyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridinyl, quinolinyl, isoquinolinyl, indolyl, benzofuranyl, benzothiophenyl, oxadiazolyl, tetrazolyl, triazolyl and pyrimidinyl. The term "aryl" as used herein, refers to C₆-Cιo aromatic hydrocarbon groups, for example phenyl and naphthyl.

It will also be recognised that the compounds of formula (I) possess asymmetric centres and are therefore capable of existing in more than one stereoisomeric form. The invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centres eg., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof. Such isomers may be naturally occurring or may be prepared by asymmetric synthesis, for example using chiral intermediates, or by chiral resolution.

The compounds of formula (I) are useful in treating or preventing diseases or disorders modulated by aminopeptidase P. The term "diseases or disorders modulated by aminopeptidase P" refers to diseases or disorders where inhibition or partial inhibition of aminopeptidase P provides a therapeutic effect. APP may contribute to the degradation of bradykinin, Allostatin (I), Substance P, Peptide YY, neuropeptide Y, interleukin-6 and collagen in vivo, as well as other peptides and proteins having an N-terminal sequence X- Pro-. Therapeutic inhibition of APP may be useful for the treatment of inflammatory conditions such as artliritis or asthma, vascular disease such as cardiovascular disease, hypertension or stroke, collagen-degeneration diseases, hormone regulated diseases, blood clotting disorders, Alzheimer's disease and Alzheimer's-like diseases, tumor related diseases, insulin resistance, and bacterial diseases, such as sepsis, (Murphey LJ, Hachey DL, Oates JA, Morrow JD, Brown NJ; J Pharmacology and Experimental Therapeutics, 294, 2000, 263-269).

The peptidomimetic compound of formula (I) may contain up to 10 amino acid residues. Preferably the group Z is absent or comprises 1 to 6 amino acids Preferably Z is absent or comprises one or two amino acids so that the compound of formula (I) is a di-, tri- or tetra- peptide. Especially preferred compounds of formula (I) are di- or tri-peptides. The peptidomimetic compound is optionally derivatised by the incorporation of functional groups that enhance the binding of the inhibitor to the metal ion in the active site of APP.

Functional groups include any groups that are able to coordinate to a metal ion. Suitable functional groups include, but are not limited to mercapto groups (SH), phosphorus containing derivatives, for example -OPO₃H, carboxyl groups (CO₂H), aldehydes or ketones. Preferably, such functional groups are incorporated in the N-terminal amino acid residue, for example, at C2, C3 or the terminal nitrogen atom. For example, a suitable N- terminal functional derivative may include a (2S,3R)-3-amino-2-hydroxy-4-phenyl butanoic acid (AHPB) group which may form an amide with the terminal amino group of the compound of formula (I) :

In a preferred embodiment, Y is an α-amino acid residue or a β-amino acid residue having a side chain comprising 4 or less carbon atoms.

In another preferred embodiment, Y is an α-amino acid residue or β-amino acid residue having a non-polar side chain.

In another preferred embodiment, Y is α-Ala, α-Gly, α-Ile, α-Leu, α-Phe, α-Pro, α-Tyr, α-Val, β-Ala, β-gly, β-Ile, β-Leu, β-Phe, β-Pro, β-Tyr or β-Val. Especially preferred are α-Leu, α-Pro, α-Val, β-Leu and β-Pro.

Preferably, X is β-Pro.

Preferred tripeptide compounds of formula (I) include βPro-Leu-Z, Pro-βLeu-Z, βPro- βLeu-Z, βPro-Pro-Z, Pro-βPro-Z, βPro-βPro-Z, βPro-Phe-Z, βPro-Ala-Z, βPro-Val-Z or a derivative thereof, where Z is an amino acid residue. Preferred dipeptide compounds of formula (I) include βPro-Leu, Pro-βLeu, βPro-βLeu, βPro-Pro, Pro-βPro, βPro-βPro, βPro-Phe, βPro-Ala, βPro-Val or a derivative thereof.

Especially preferred dipeptide compounds of formula (I) include βPro-αLeu, βPro-βLeu, βPro-αPro and βPro-αVal or a derivative thereof.

β-amino acid compounds may be prepared by using the methods depicted or described herein or known in the art. It will be understood that minor modifications to methods described herein or known in the art may be required to synthesize particular β-amino acid compounds. General synthetic procedures applicable to synthesis of compounds may be found in standard references such as Comprehensive Organic Transformations, R. C. Larock, 1989, VCH publishers and Advanced Organic Chemistry, J. March, 4^th Edition (1992), Wiley InterScience, and references therein. It will also be recognised that certain reactive groups may require protection and deprotection during the synthetic process preparing β-amino acids or peptides containing them. Suitable protecting and deprotecting methods for reactive functional groups are known in the art, for example in Protective Groups in Organic Synthesis, T. W. Green & P. Wutz, John Wiley & Son, 3^rd Edition, 1999 and Amino Acid and Peptide Synthesis, John Jones, Oxford Science Publications, 1992.

β-amino acids having the R side chain group at C2 may be prepared with the exemplified general method depicted in Scheme 1. Suitable starting materials can be obtained commercially or prepared using methods known in the art.

N-deprotection

Scheme 1

β-amino acids having the R side chain group at C3 may be prepared with the exemplified general method depicted in Scheme 2. Suitable starting materials can be obtained commercially or prepared using methods lαiown in the art.

,CO₂CH₃ benzylamine

N-deprotection

Scheme 2

Alternatively, β-amino acids having the R side chain group at C3 may be prepared with the exemplified general method depicted in Scheme 3 using the Amdt-Eistert reaction. Suitable starting materials can be obtained commercially, for example, α-amino acids or prepared using methods known in the art.

CH₂N₂

Scheme 3

Peptides containing the β-amino acids may be synthesized using lαiown solution peptide synthesis techniques or solid phase techniques. A typical solid phase synthesis is shown in Scheme 4.

fmoc deprotection 20%Pip/DMF 20 minutes

H,N. -Resin

R' O

Scheme 4 The term "derivative thereof includes salts or prodrugs which include any pharmaceutically acceptable salt, ester, solvate, hydrate or any other compound which, upon administration to the recipient is capable of providing (directly or indirectly) a compound of Formula (I) as described herein. The term "pro-drug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compounds of the invention. Such derivatives would readily occur to those skilled in the art, and include, for example, compounds where a free hydroxy group is converted into an ester, such as an acetate, or where a free amino group is converted into an amide. Procedures for acylating hydroxy or amino groups of the compounds of the invention are well known in the art and may include treatment of the compound with an appropriate carboxylic acid, anhydride or acylchloride in the presence of a suitable catalyst or base.

Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.

Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.

APP may contribute to the degradation of bradykinin, Allostatin (I), Substance P, Peptide YY, neuropeptide Y, interleukin-6 and collagen in vivo, as well as other peptides and proteins having an N-terminal sequence X-Pro-. Therapeutic inhibition of APP is useful for the treatment of inflammatory conditions such as arthritis and asthma, vascular disease such as cardiovascular disease, hypertension or stroke, collagen-degeneration diseases, hormone regulated diseases, blood clotting disorders, Alzheimer's disease and Alzheimer's- like diseases, tumor related diseases, insulin resistance, and bacterial diseases, such as sepsis, (Murphey LJ, Hacey DL, Oates JA, Morrow JD, Brown NJ; J Pharmacology and Experimental Therapeutics, 294, 2000, 263-269).

Accordingly, another aspect of the present invention relates to a method of treating or preventing a disease or disorder modulated by aminopeptidase P in a mammal, comprising administering to the mammal a peptidomimetic compound of formula (I) or a derivative thereof,

X-Y-Z (I) wherein X is a proline residue or β-proline residue; Y is an α-amino acid residue or a β-amino residue; and Z is absent or is from 1 to 8 amino acid residues; and wherein at least one of X and Y is a β -amino acid residue.

As used herein, the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired APP inhibiting or treatment or therapeutic activity, or disease/condition prevention. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. An APP inhibiting amount is an amount which will at least partially inhibit the activity of APP. A therapeutic, or treatment, effective amount is an amount of the compound which, when administered according to a desired dosing regimen, is sufficient to at least partially attain the desired therapeutic effect, or delay the onset of, or inhibit the progression of or halt or partially or fully reverse the onset or progression of a particular disease condition being treated. A prevention effective amount is an amount of compound which when administered according to the desired dosing regimen is sufficient to at least partially prevent or delay the onset of a particular disease or condition. A diagnostic effective amount of compound is an amount sufficient to bind to APP to enable detection of the APP-compound complex such that diagnosis of a disease or condition is possible.

Yet another aspect of the present invention provides a use of a peptidomimetic compound of formula (I) or a derivative thereof,

X-Y-Z (I) wherein X is a proline residue or β-proline residue; Y is an α-amino acid residue or a β-amino residue; and Z is absent or is from 1 to 8 amino acid residues; and wherein at least one of X and Y is a β-amino acid residue, in the preparation of a medicament for treating or preventing a disease or disorder modulated by aminopeptidase P.

Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage. The dosage is preferably in the range of 1 μg to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage is in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage is in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another preferred embodiment, the dosage is in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per kg of body weight per dosage. In yet another embodiment, the dosage is in the range of lμg to lmg per kg of body weight per dosage.

Suitable dosage amounts and dosing regimens can be determined by the attending physician or veterinarian and may depend on the desired level of inhibiting activity, the particular condition being treated, the severity of the condition as well as the general age, health and weight of the subject. The active ingredient may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition.

The formulation of such compositions is well lαiown to those skilled in the art. The composition may contain pharmaceutically acceptable additives such as carriers, diluents or excipients. These include, where appropriate, all conventional solvents, dispersion agents, fillers, solid carriers, coating agents, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.

The carrier must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. Compositions include those suitable for oral, rectal, inhalational, nasal, transdermal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intraspinal, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Depending on the disease or condition to be treated, it may or may not be desirable for a compound of Formula (I) to cross the blood/brain barrier. Thus the compositions for use in the present invention may be formulated to be water or lipid soluble.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg inert diluent, preservative, disintegrant (eg. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose)) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

The compounds of Formula (I) may also be administered intranasally or via inhalation, for example by atomiser, aerosol or nebulizer means.

Compositions suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Transdermal devices, such as patches, may also be used to administer the compounds of the invention.

Compositions for rectal administration may be presented as a suppository with a suitable carrier base comprising, for example, cocoa butter, gelatin, glycerin or polyethylene glycol.

Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are lαiown in the art to be appropriate.

Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage compositions are those containing a daily dose or unit, daily sub- dose, as herein above described, or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the active ingredients particularly mentioned above, the compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents, disintegrating agents, coatmg agents, preservatives, lubricants and/or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

Still another aspect of the present invention relates to a pharmaceutical composition comprising a peptidomimetic compound of formula (I) or a derivative thereof and a pharmaceutically acceptable carrier, diluent or excipient,

X-Y-Z (I) wherein X is a proline residue or β-proline residue; Y is an α-amino acid residue or a β-amino residue; and Z is absent or is from 1 to 8 amino acid residues; and wherein at least one of X and Y is a β-amino acid residue.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

The invention will now be described with reference to the following examples which are included for the purpose of illustration only and are not intended to limit the generality of the invention hereinbefore described. EXAMPLE 1

SYNTHESIS OF FMOC-β3-PROLINE

Synthesis of Fmoc-Proline Diazoketone

To a solution of Fmoc-N-α-Proline (2.86g, 2.83mmol) in THF (60ml) was added NEt (3.1 lmmol) and ClCO₂Et (3.1 lmmol) at -25 °C and the mixture was stirred for 20 minutes. Diazomethane in ether (30ml) was added and the reaction was stirred for 17 hours at room temperature. The solvent was removed to yield Fmoc-N-α-proline diazoketone after which flash chromatography gave bright yellow solid (2.27g, 73%). 1H NMR (300Hz, CDC1₃) 57.75 (d, J=3, 2H), 7.57 (d, J=3, 2H), 7.44 -7.26 (m, 4H), 5.33- 4.99 (s,lH), 4.55 (m, 1H), 4.4 - 4.18 (m, 2H),3.9 (d-br, J=3.97Hz, 1H), 63.45 (m, 2H), 52.16-1.67 (m,4H).

Synthesis of Fmoc-β3-Proline

Fmoc-N-α-proline diazoketone (2.27g, 6.27mmol) was dissolved in THF/water (25.0ml:2.5ml). A solution of silver trifluoroacetate (152mg, 0.69mmol) and NMM (1.72ml, 15.67mmol) was added and the reaction was left stirring for 17 hours. The solvent was removed to yield Fmoc-N-β-proline (1.32g, 44%). ¹HNMR (300Hz,CDCl₃) 57.75 (d, J=7.0, 2H), 7.59 (d, J=7.0, 2H), 7.38 -7.26 (m, 4H), 4.55 (d, J=5.0, 1H), 4.4 - 4.18 (m, 2H), 3.98 (d-br, J=4.0Hz, 1H), 3.45 (m, 2H),2.16-1.67 (m, 4H).

EXAMPLE 2

SYNTHESIS OF FMOC-β3-TYROSINE

Synthesis of Fmoc-Tyrosine Diazoketone

To a solution of Fmoc-N-α-tyrosine (1.30g, 2.83mmol) in THF (20ml) was added NEt₃ (3.1 lmmol) and ClCO₂Et (3.1 lmmol) at -25°C and the mixture was stirred for 20 minutes. Diazomethane in ether (10ml) was added and the reaction was stirred for 17 hours at room temperature. The solvent was removed and after chromatography yielded Fmoc-N-α- tyrosine diazoketone as a bright yellow solid (0.834g, 61%). 'HNMR (300HZ, CDC1₃) 57.75 (d, J=7.3, 2H), 7.56 (d, J=7.3, 2H), 7.44 - 7.26 (d, 4H), 7.23 (s, IH), 7.06 (d,2H), 6.9 (d, 2H), 5.33, (m,lH), 5.05 (s, IH), 4.4 (d, 2H), 4.27 (t, IH), 2.95 (d, 2H), 1.25 (s, 9H).

Synthesis of Fmoc-β3-Tyrosine

Fmoc-N-α-tyrosine diazoketone (0.83g, 1.72mmol) was dissolved in THF/water (7 ml:0.7ml). A solution of silver trifluoroacetate (41.8mg, 0.189mmol) andNMM (0.473ml, 4.3mmol) was added and the reaction was left stirring for 17 hours. The solvent was removed to yield Fmoc-N-β-tyrosine (0.29g, 22%). ¹HNMR (300Hz, CDC1₃) 57.76-7.74 (d, J=7.3, 2H), 7.57-7.55 (d, J=7.3, 2H), 7.41 -7.26 (m, 4H), 7.1(d, J=5.0, 2H), 6.9 (d, 2H), 5.3 (d, IH), 4.37 (m, J=6.4,1H), 2.16-1.67 (m, 4H), 4.2 (m, 2H), 2.96 (d, IH), 2.64-2.55 (m, IH), 1.3 (s, 9H).

EXAMPLE 3

SYNTHESIS OF FMOC-β3-LEUCINE

Synthesis of Fmoc-Leucine Diazoketone

To a solution of Fmoc-N-α-leucine (l.OOg, 2.83mmol) in THF (30ml) was added NEt₃ (3.1 lmmol) and ClCO₂Et (3.1 lmmol) at -25°C and the mixture was stirred for 20 minutes. Diazomethane in ether (10ml) was added and the reaction was stirred for 17 hours at room temperature. The solvent was removed after chromatography yielded Fmoc-N-α-leucine diazoketone as a bright yellow solid (1.31g, 122%). ¹HNMR (300Hz, CDC1₃) 57.77 (d, J=7.5, 2H), 7.59 (d, J=7.5, 2H), 7.34 (d, 4H), 7.25 (s, IH), 5.3 (s,lH), 5.16 (d-br, IH), 4.46 (m, 2H), 4.2 (t, IH), 1.6 (m, 2H), 0.92 (d, 6H), 0.85 (m, IH). Synthesis of Fmoc-β3-Leucine

Fmoc-N-α-leucine diazoketone (1.3 lg, 3.47mmol) was dissolved in THF/water (14.0ml: 1.4ml). A solution of silver trifluoroacetate (84.24mg, 0.38mmol) and NMM (0.88ml, 8.69mmol) was added and the reaction was left stirring for 17 hours. The solvent was removed to yield Fmoc-N-β-leucine (0.54g, 52%). H*NMR (300Hz,CDCl₃) 57.75 (d, J=7.4, 2H), 7.6 (d, J=7.4, 2H), 7.4 -7.2 (m, 4H), 5.5, 5.1 (d, J=9.3, IH), 4.45 (d, IH), 4.2 (t, 2H), 4.05 (m, IH), 2.6 (m, 2H), 1.65-1.45 (m, 2H), 1.3 (m, IH), 0.9 (d, 6H).

EXAMPLE 4

PEPTIDE SYNTHESIS

The Wang resin (1.19mmol of amino acid/g of resin) was first washed with DMF (3 times for 1 minute each). The first protected amino acid (1 mol equiv) was dissolved in a small amount of DMF while DMAP (O.lmol equiv) was dissolved in DIC (Imol equiv). The solutions were combined and added to the resin and shaken for 4 hours. Unreacted amino acids and reagents were washed away with DMF. The Fmoc protecting group on the attached amino acid was removed by washing with 20%) piperidine in DMF once for a minute and again for 20 minutes. The next amino acid (Imol equiv), PyBOP (Imol equiv), HOBt (Imol equiv) and NMM (2mol equiv) were dissolved in DMF and added to the resin from the previous coupling and shaken for another 90 minutes.

The unreacted amino acids and the protecting groups were removed as above and the peptide resin complex was washed with DCM and the peptide cleaved from the resin using 95%o TF A/water. For peptides containing tyrosine, 95% TFA/2.5% water and 2.5% mercaptoethanol was used as a cleavage solution. The solvents were then removed under the stream of nitrogen gas and the dipeptides were precipitated in 1 :1 mixture of ether/hexane. The peptides were redissolved in 1 : 1 acetonitrile/water mixture and lyophilised in a freeze dryer. The peptides were characterised by mass spectrometry and RP-HPLC. Some peptides were also purified. EXAMPLE 5

APP INHIBITION ASSAY USING HPLC

Standard Assay

For the optimisation of conditions for the enzyme in the inhibition assay, serial dilutions (1/5, 1/10, 1/20, 1/50, 1/100, 1/1000) were made from the enzyme stock solution of 1.28μ g/ml. Bradykinin (1 μg/μl) was used as the substrate. The reaction protocol that was followed is summarised in Table 3. From this degradation assay, the amount of enzyme (APP) and the time required to degrade at least 60% of the substrate was determined and was used in the subsequent inhibition assays.

Table 3 Protocol for HPLC-based inhibition assay.

At times 0, 15, 30, 60 and 120 minutes, 20μl of the reaction mixture was taken and the reaction was stopped by the addition of 200μl of 80% MeOH/0.1 %> TFA. The reaction mixtures were dried, reconstituted with 120μl of 0.1%> TFA, vortexed, spun (14x1000 mm ¹) for 10 minutes and analysed by LCMS. Degradation assays for all samples were carried out in duplicate.

Lineweaver-Burk Plots for Peptide Inhibitors

Different volumes of 1 μg/μl of bradykinin were added to the reaction tubes to give concentrations of 0.3623, 0.2717, 0.1811, 0.0916 and 0.0453 nmol/μl. To each reaction tube 10 μl of 1.28μg/ml APP was added. Enzyme inhibition was then monitored at either two or three different concentrations of peptide and the samples were analysed by HPLC. The rate of degradation of bradykinin (velocity) with time, was calculated from the plot of percentage degradation. Lineweaver-Burk plots were constructed using the reciprocal of the rate of degradation versus reciprocal of the substrate concentration to calculate Km and Km_app. The linear graph obtained was extrapolated and the point of intercept at X-axis with the control gives -1/Km while plots while the point of intercept on X-axis with inhibitors gives -1/Km_app. Km_app is the apparent michaelis constant of the substrate in the presence of inhibitor. The results obtained were compared to the control with no inhibitor.

The percentage degradation of bradykinin with time was carried out to determine the standard conditions for the assay using bradykinin as a substrate, ie determining the concentration of bradykinin that would be used to give the optimal activity of the enzyme. The best results were obtained with lOμl "stock" APP (0.0128μg/ml), which degrades about 70% of bradykinin in 30 minutes.

Lineweaver-Burk Plots of the Inhibitors

In this assay bradykinin was used as a substrate. The bradykinin concentration used was 0.377μM per tube and the average Km calculated was 0.29μM. The Km was then used to calculate the inhibition constant (Ki) using the expression Km_app = Km (1+ [I]/Ki). The inliibition constants, and the IC₅₀ values determined are listed in Table 4.

Table 4 Inhibition constants (Ki) and IC₅₀ values for the peptides.

From the Lineweaver Burk Plots, all peptides were seen to exhibit competitive inhibition. This suggests that all the peptides bound to the same site as the substrate. EXAMPLE 6

PEPTIDE STABILITY ASSAY

Rabbit kidney was prepared as previously described by Hooper & Turner, 1988. 40μl of the synthetic peptides (0.4 nmol) were incubated with 200μg of rabbit kidney membranes in lOOμl of TBS buffer. The reaction mixtures were incubated at 37°C and 25μl aliquots were removed at times 0 and 30 minutes, 1, 2, 5 and 24 hours. The reactions were stopped using 200μl of methanol/0.1 %> TFA. The solvent was then dried using vacuum centrifugation (Speed-Vac, Savant) and was analysed by RP- LCMS.

All peptides were stable to degradation by peptidases in kidney membranes after 24 hours.

EXAMPLE 7

Computational Docking

The structure of APP from Protein Data Bank, Biology Department, Brookhaven National Laboratory, Upton, NY 11973 (reference no 1AZ9, Wilce M C J, Bond C S, Dixon N E,

Freeman H C, Guss J M, Lilley P E and Wilce J A, 1998, Structure and Mechanism of a

Proline Specific Aminopeptidase from E. coli, Proc. Natl. Acad. Sci, USA, 95, 3472-3477) was used as the enzyme structure. All water molecules were then removed from the enzyme except for the water molecules that ligate with the two manganese (Mn²⁺) ions at the catalytic site. Protons were added to all atoms and the structure minimised within

Sybyl using the Tripos forcefield (Tripos Inc, St.Louis, MO, USA). All atom charges were loaded with the added manganese atom in to Sybyl with +2 charge to the manganese.

Atom types for Mn with appropriate atomic radii were then added to the Conolly

Molecular Surface Program. All the residues within 13 A of the catalytic water molecules were surfaced and used to prepare site points for the dock. The resulting sphere file was trimmed by hand to remove any extraneous spheres. Scoring Grid Setup

A grid resolution of 0.35 A was created and centred on the sphere site with a 6 A overlap. Grids were then obtained using an All Atom Model and the distance dependent dielectric function E=4r with a cutoff distance of 10 A.

Inhibitors Setup

Atomic coordinates of the crystalised inhibitor Pro-Leu was used as a basis for modelling the proline ring conformation, since the Sybyl force field does not contain any ring bending forces. The ligands were built upon this scaffold which was minimised within Sybyl. The Gasteiger-Huckel charges were then loaded to the ligand.

A flexible docking of all inhibitors was performed using DOCK 4.01 torsion drive and energy minimisation with a maximum of 1000 orientations were determined. The top scoring 25 orientations based on the dock energy functions were written. A normalised rank was used to select the best orientation for further rescoring and analysis. The Scoring Functions based on Chemscore, Smog and Potential of mean force (PMF) was used to select the best orientation to be stored in the database. The final orientations were then ranked using a Rank by Rank procedure including the dock energy function.

Through these docking procedures, 25 top scoring orientations were ranked, and the lowest energy orientation based on the scoring functions were selected for analysis.

The results are shown in Tables 5-9 below. Table 5 The distances that determine the orientation of the Pro-Leu family of inhibitors at the active site of APP.

A) The Ki and the distances between the inhibitors and the OH^" ion at the active site of APP.

B) Distances (A) between first amino acid in the dipeptides and APP active site residues:

C) Distances (A) between second amino acid in the dipeptides and active site residues:

Table 6 The distances that determine the orientation of the Pro-Pro family of inhibitors at the active site of APP.

A) The Ki and the distances between the proline and the nucleophilic OH^" ion.

B) Distances (A) from the first amino acid in the dipeptides to active site residues:

Table 7 The distances of Pro-Phe and βPro-Phe to the residues in the active site.

A) The Ki and the distances between the inhibitors and the nucleophile OH^" ion.

B) Distances (A) between the first amino acid of the dipeptides and the active site residues:

C) Distances (A) between the second amino acid of the dipeptides and the active site residues:

Table 8 Modelling data of Pro-Ala and βPro-Ala at the active site of APP

A) Ki value of the peptides.

B) Distances (A) from first amino acid in the dipeptides to active site residues:

C) Distances (A) from second amino acid in the dipeptides to active site residues:

Table 9 The distances that determine the orientation of the inhibitors Pro-Val and βPro-Val at the active site of APP.

A) The Ki and the distances between inhibitors and the nucleophilic OH^" ion.

Comparison between peptide inhibitory activity and their interaction in the active site revealed a correlation between Ki of the compound of formula (I) and the distances between the compounds of formula (I) and the active site residues. This modelling may be used to assess the potential of other compounds of formula (I) as inhibitors of APP activity. Abbreviations, excluding abbreviations given for amino acids in Tables 1 and 2, used herein are set out below:

AHPB 2S,3R-3-amino-2-hydroxy-4-phenyl butanoic acid APP aminopeptidase P DIC 1 ,3 -diisopropylcarbodiimide DMAP N,N-dimethylaminopyridine DMF dimethylformamide fmoc fluorenylmethoxycarbonyl HOBt N-hydroxybenzotrazol.H₂O HPLC High Performance Liquid Chromatography Ki competitive inhibitor constant Km Michaelis constant

Kmapp apparent Michaelis constant LCMS Liquid Chromatography Mass Spectrometry

NMM N-methylmorpholine

Pip piperidine

PyBOP phosphonium hexafluorophosphate

RP-HPLC Reverse Phase High Performance Liquid Cliromatography TFA trifluoroacetic acid

THF tetrahydrofuran

REFERENCES:

Boumendjel A, Roberts J C, Hu E, Pallai P V and Rebek J Jr, 1996, Design and Asymmetrical Synthesis of β-Strand Peptidomimetics, J Org Chem, 61, 4434-4438.

Fix J A, 1996, Strategies for Delivery of Peptides Utilizing Absorption Enhancing Agents, J Pharmaceutical Sciences, 85(12), 1282-1285

Green, T W & Wutz, P, Protective Groups in Organic Synthesis, John Wiley & Son, 3^rd Edition, 1999

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Claims

CLAIMS:

1. A method of inhibiting aminopeptidase P comprising contacting aminopeptidase P with a peptidomimetic compound of formula (I) or a derivative thereof:

X-Y-Z (I)

wherein X is an α-proline residue or β-proline residue; Y is an α-amino acid or a β-amino acid residue; and Z is absent or is from 1 to 8 amino acid residues; and wherein at least one of X and Y is a β-amino acid residue.

2. The method according to claim 1 wherein X is a β-proline residue.

3. The method according to claim 1 wherein Y is an α-amino acid residue or a β-amino acid residue having a side chain comprising 4 or less carbon atoms.

4. The method according to claim 1 wherein Y is an α-amino acid residue or a β- amino acid residue having a non-polar side chain.

5. The method according to claim 1 wherein Y is selected from the group consisting of α-Ala, α-Gly, α-Ile, α-Leu, α-Phe, α-Pro, α-Tyr, α-Val, β-Ala, β-Gly, β-Ile, β-Leu, β- Phe, β-Pro, β-Tyr or β-Val.

6. The method according to claim 1 wherein Z is absent or comprises 1 to 6 amino acid residues.

7. The method according to claim 1 wherein Z is absent or comprises 1 or 2 amino acid residues.

8. The method according to claim 1 wherein Z is absent or comprises 1 amino acid residue.

9. The method according to claim 1 wherein the compound of formula (I) is a tripeptide selected from the group consisting of βPro-Leu-Z, Pro-βLeu-Z, β-Pro-β-Leu-Z, βPro-Pro-Z, βPro-Pro-Z, Pro-β-Pro-Z, βPro-βPro-Z, βPro-Phe-Z, βPro-Ala-Z and βPro- Val-Z, or a derivative thereof, wherein Z is an amino acid residue.

10. The method according to claim 1 wherein the compound of formula (I) is a dipeptide selected from the group consisting of βPro-Leu, Pro-βLeu, βPro-βLeu, βPro-Pro, Pro-βPro, βPro-βPro, βPro-Phe, βPro-Ala and βPro-Val or a derivative thereof.

11. The method according to claim 10 wherein the dipeptide is selected from the group consisting of βPro-αLeu, βPro-βLeu, βPro-αPro and βPro-αVal or a derivative thereof.

12. A pharmaceutical composition comprising a peptidomimetic compound of formula (I) or a derivative thereof, and a pharmaceutically acceptable carrier, diluent or excipient,

X-Y-Z (I)

13. The pharmaceutical composition according to claim 12, wherein X is a β-proline residue.

14. The pharmaceutical composition according to claim 12, wherein Y is an α-amino acid residue or a β-amino acid residue having a side chain comprising 4 or less carbon atoms.

15. The pharmaceutical composition according to claim 12, wherein Y is an α-amino acid residue or a β-amino acid residue having a non-polar side chain.

16. The pharmaceutical composition according to claim 12, wherein Y is selected from the group consisting of α-Ala, α-Gly, α-Ile, α-Leu, α-Phe, α-Pro, α-Tyr, α-Val, β-Ala, β- Gly, β-Ile, β-Leu, β-Phe, β-Pro, β-Tyr or β-Val.

17. The pharmaceutical composition according to claim 12, wherein Z is absent or comprises 1 to 6 amino acid residues.

18. The pharmaceutical composition according to claim 12, wherein Z is absent or comprises 1 or 2 amino acid residues.

19. The pharmaceutical composition according to claim 12, wherein Z is absent or comprises 1 amino acid residue.

20. The pharmaceutical composition according to claim 12, wherein the compound of formula (I) is a tripeptide selected from the group consisting of βPro-Leu-Z, Pro- βLeu-Z, β-Pro-β-Leu-Z, βPro-Pro-Z, βPro-Pro-Z, Pro-β-Pro-Z, βPro-βPro-Z, βPro-Phe-Z, βPro- Ala-Z and βPro-Val-Z, or a derivative thereof, wherein Z is an amino acid residue.

21. The pharmaceutical composition according to claim 12, wherein the compound of formula (I) is a dipeptide selected from the group consisting of βPro-Leu, Pro-βLeu, βPro- βLeu, βPro-Pro, Pro-βPro, βPro-βPro, βPro-Phe, βPro-Ala and βPro-Val or a derivative thereof.

22. The pharmaceutical composition according to claim 21, wherein the dipeptide is selected from the group consisting of βPro-αLeu, βPro-βLeu, βPro-αPro and βPro-αVal or a derivative thereof.

23. A method of treating or preventing a disease or disorder modulated by aminopeptidase P in a mammal, comprising administering to the mammal a peptidomimetic compound of formula (I) or a derivative thereof,

X-Y-Z (I)

24. A method according to claim 23, wherein X is a β-proline residue.

25. A method according to claim 23, wherein Y is an α-amino acid residue or a β-amino acid residue having a side chain comprising 4 or less carbon atoms.

26. A method according to claim 23, wherein Y is an α-amino acid residue or a β- amino acid residue having a non-polar side chain.

27. A method according to claim 23, wherein Y is selected from the group consisting of α-Ala, α-Gly, α-Ile, α-Leu, α-Phe, α-Pro, α-Tyr, α-Val, β-Ala, β-Gly, β-Ile, β-Leu, β- Phe, β-Pro, β-Tyr or β-Val.

28. A method according to claim 23, wherein Z is absent or comprises 1 to 6 amino acid residues.

29. A method according to claim 23, wherein Z is absent or comprises 1 or 2 amino acid residues.

30. A method according to claim 23, wherein Z is absent or comprises 1 amino acid residue.

31. A method according to claim 23, wherein the compound of formula (I) is a tripeptide selected from the group consisting of βPro-Leu-Z, Pro- βLeu-Z, β-Pro-β-Leu-Z, βPro-Pro-Z, βPro-Pro-Z, Pro-β-Pro-Z, βPro-βPro-Z, βPro-Phe-Z, βPro-Ala-Z and βPro- Val-Z, or a derivative thereof, wherein Z is an amino acid residue.

32. A method according to claim 23, wherein the compound of formula (I) is a dipeptide selected from the group consisting of βPro-Leu, Pro-βLeu, βPro-βLeu, βPro-Pro, Pro-βPro, βPro-βPro, βPro-Phe, βPro-Ala and βPro-Val or a derivative thereof.

33. A method according to claim 32, wherein the dipeptide is selected from the group consisting of βPro-αLeu, βPro-βLeu, βPro-αPro and βPro-αVal or a derivative thereof.

34. A method according to claim 23, wherein the disease or disorder modulated by aminopeptidase P is a vascular disease, hypertension, stroke, a collagen-degeneration disease, a hormone regulated disease, a blood clotting disorder, Alzheimer's disease, an Alzheimer's-like disease, a tumor related disease, an inflammatory disease, insulin resistance or a bacterial disease.

35. A method according to claim 34, wherein the disease or disorder modulated by aminopeptidase P is hypertension, insulin resistance, sepsis, arthritis or asthma.

36. Use of a peptidomimetic compound of formula (I) or a derivative thereof,

X-Y-Z (I) wherein X is an α-proline residue or β-proline residue;

Y is an α-amino acid or a β-amino acid residue; and

Z is absent or is from 1 to 8 amino acid residues; and wherein at least one of X and Y is a β-amino acid residue in the preparation of a medicament for treating or preventing a disease or disorder modulated by aminopeptidase P.

37. Use according to claim 36, wherein X is a β-proline residue.

38. Use according to claim 36, wherein Y is an α-amino acid residue or a β-amino acid residue having a side chain comprising 4 or less carbon atoms.

39. Use according to claim 36, wherein Y is an α-amino acid residue or a β-amino acid residue having a non-polar side chain.

40. Use according to claim 36, wherein Y is selected from the group consisting of α- Ala, α-Gly, α-Ile, α-Leu, α-Phe, α-Pro, α-Tyr, α-Val, β-Ala, β-Gly, β-Ile, β-Leu, β-Phe, β-Pro, β-Tyr or β-Val.

41. Use according to claim 36, wherein Z is absent or comprises 1 to 6 amino acid residues.

42. Use according to claim 36, wherein Z is absent or comprises 1 or 2 amino acid residues.

43. Use according to claim 36, wherein Z is absent or comprises 1 amino acid residue.

44. Use according to claim 36, wherein the compound of formula (I) is a tripeptide selected from the group consisting of βPro-Leu-Z, Pro- βLeu-Z, β-Pro-β-Leu-Z, βPro-Pro-

Z, βPro-Pro-Z, Pro-β-Pro-Z, βPro-βPro-Z, βPro-Phe-Z, βPro-Ala-Z and βPro-Val-Z, or a derivative thereof, wherein Z is an amino acid residue.

45. Use according to claim 36, wherein the compound of formula (I) is a dipeptide selected from the group consisting of βPro-Leu, Pro-βLeu, βPro-βLeu, βPro-Pro, Pro- βPro, βPro-βPro, βPro-Phe, βPro-Ala and βPro-Val or a derivative thereof.

46. Use according to claim 45, wherein the dipeptide is selected from the group consisting of βPro-αLeu, βPro-βLeu, βPro-αPro and βPro-αVal or a derivative thereof.

47. Use according to claim 36, wherein the disease or disorder modulated by aminopeptidase P is a vascular disease, hypertension, stroke, a collagen-degeneration disease, a hormone regulated disease, a blood clotting disorder, Alzheimer's disease, an Alzheimer's-like disease, a tumor related disease, an inflammatory disease, insulin resistance or a bacterial disease.

48. Use according to claim 47, wherein the disease or disorder modulated by aminopeptidase P is hypertension, insulin resistance, sepsis, arthritis or asthma.

49. A compound of formula (I) or a derivative thereof;

X-Y-Z (I)

50. A compound of formula (I) according to claim 49, wherein X is a β-proline residue.

51. A compound of formula (I) according to claim 49, wherein Y is an α-amino acid residue or a β-amino acid residue having a side chain comprising 4 or less carbon atoms.

52. A compound of formula (I) according to claim 49, wherein Y is an α-amino acid residue or a β-amino acid residue having a non-polar side chain.

53. A compound of formula (I) according to claim 49, wherein Y is selected from the group consisting of α-Ala, α-Gly, α-Ile, α-Leu, α-Phe, α-Pro, α-Tyr, α-Val, β-Ala, β-Gly, β-Ile, β-Leu, β-Phe, β-Pro, β-Tyr or β-Val.

54. A compound of formula (I) according to claim 49, wherein Z is absent or comprises 1 to 6 amino acid residues.

55. A compound of formula (I) according to claim 49, wherein Z is absent or comprises 1 or 2 amino acid residues.

56. A compound of formula (I) according to claim 49, wherein Z is absent or comprises 1 amino acid residue.

57. A compound of formula (I) according to claim 49, which is a tripeptide selected from the group consisting of βPro-Leu-Z, Pro- βLeu-Z, β-Pro-β-Leu-Z, βPro-Pro-Z, βPro- Pro-Z, Pro-β-Pro-Z, βPro-βPro-Z, βPro-Phe-Z, βPro-Ala-Z and βPro-Val-Z, or a derivative thereof, wherein Z is an amino acid residue.

58. A compound of formula (I) according to claim 49, which is a dipeptide selected from the group consisting of βPro-Leu, Pro-βLeu, βPro-βLeu, βPro-Pro, Pro-βPro, βPro- βPro, βPro-Phe, βPro-Ala and βPro-Val or a derivative thereof.

59. A compound of formula (I) according to claim 58, wherein the dipeptide is selected from the group consisting of βPro-αLeu, βPro-βLeu, βPro-αPro and βPro-αVal or a derivative thereof.