WO1984000365A1

WO1984000365A1 - Synthetic peptides and their preparation

Info

Publication number: WO1984000365A1
Application number: PCT/GB1983/000177
Authority: WO
Inventors: Ian James Galpin; Anna Halina Wilby
Original assignee: Nat Res Dev
Priority date: 1982-07-19
Filing date: 1983-07-18
Publication date: 1984-02-02
Also published as: GB8319305D0; GB2124233B; GB2124233A

Abstract

The enzyme chymotrypsin has many biological functions. It would be useful to inhibit this enzyme for diagnostic and therapeutic purposes. However it is a nuisance to synthesise because it contains the amino acid capreomycidine. The present invention provides peptides which are alternative inhibitors of chymotrypsin and are more readily synthesised than chymostatin. The peptides of the invention are compounds of formula (Org). CO. (BAA). (NAA). X' (al), wherein: Org represents an organic residue, preferably a hydrocarbyl or oxyhydrocarbyl group; BAA represents the residue of a basic acyclic or aromatic amino acid which imparts to the amino acid a pKa of from about 10.5 to about 12.5 and is preferably a lysine, arginine or ornithine residue; NAA represents the residue of a neutral amino acid (being one with no charged side-chain) and is preferably leucine, valine, isoleucine or phenylalanine; X' (al) represents a phenylalanine, tyrosine or tryptophan residue in which the terminal carboxyl group has been replaced by an aldehyde group, and the stereochemical configuration of at least the X' group, and preferably throughout, is of the L-amino acid; derivatives thereof having a functionally equivalent derivative of the aldehyde group, in place of the aldehyde group and the acid addition salts of any of these compounds. The peptides can be synthesised by simple conventional methods. They are useful for diagnosis of chymotrypsin and for therapy, e.g. in treatment of muscular dystrophy.

Description

SYNTHETIC PEPTIDES AND THEIR PREPARATION This invention relates to synthetic peptides and their preparation.

Certain micro-organisms (species of actinomycetes) have been found to produce both proteinase enzymes, e.g. papain, pepsin, trypsin and chymotrypsin, and inhibitors of these enzymes. Such naturally produced inhibitors are described by H Umezawa and T Aoyagi in "Proteinases in Mammalian Cells and Tissues", edited by Barrett, Elsevier/North-Holland Biomedical Press 1977, pages 637-646. In contrast to the proteinase inhibitors produced in animal and plant tissue, which are proteins, these inhibitors are low molecular weight peptides.

The structures of the microbial proteinase inhibitors described by Umezawa are shown below:- (i) Leupeptin Alk. CO.Leu.Leu.Arg(al); Alk. = CH₃ or C₃H₅

(ii) Antipain

(iii) Chymostatin

*In a minor proportion of molecules, Val or Leu replaces lie

(iv) Elastinal

In the above formulae, the left-hand amino acid is "reverse bonded" via its amino group to the carbonyl group shown. The amino acids are in the form of their L - enantiomers. ["(al)" denotes an aldehyde group in place of a carboxylic acid group]. These proteinase inhibitors have considerable specificity in their inhibitory action. For example, chymostatin inhibits the hydrolysis of casein by chymotrypsins. It inhibits the hydrolysis of casein by papain but not the hydrolysis of the test peptide Bz-Arg-NH₂ (Bz = C₆H₅-CH₂-) by papain. It inhibits cathepsins but not plasmin or trypsin.

Leupeptin, antipain, chymostatin and elastin contain a C-terminal aldehyde group which is functionally essential to their inhibitory activity. The nature of this activity depends on the kind of terminal amino acid residue bearing the aldehyde function. This has been confirmed by the synthesis of the peptides Ac - Leu - Leu - Phe (al) Ac - Leu — Leu - Tyr (al) Ac - Leu - Leu - Trp (al) (where Ac = acetyl). These compounds are leupeptin analogues differing in their right-hand terminal amino acid residue and, unlike leupeptin, all are reported to be chymotrypsin inhibitors, Ito et al., Biochem. Biophys. Res. Commun. , 49 , 343-349 (1972). The synthetic peptide Ac-Pro-Ala-Pro-Ala (al) is an elastase inhibitor, see R C Thompson, Biochemistry, 12, 47-51 (1973). Because of the specificity of their inhibitory action these proteinase inhibitors are useful in diagnosing the presence of the enzymes, which are involved in a variety of biological functions. Many proteinases are involved in the metabolic pathways associated with degradative diseases. Thus, chymotrypsin cleaves the carboxyl link of phenylalanine in peptides and this cleavage is inhibited by chymostatin. The present invention relates to synthetic peptides having inhibitory activity towards (inter alia) chymotrypsin and therefore useful for the above purposes, and in particular for the diagnosis and suppression of chymotrypsin and like enzymes in vitro and in vivo. It is expected that they will be useful in the treatment or muscular dystrophy which is brought about through the action of chymotrypsin on certain peptides in the pathway to this disease.

According to the present invention there are provided peptides which are compounds of formula (Org). CO. (BAA). (NAA). X' (al) (1) wherein:

Org represents an organic residue, preferably a hydrophobic residue;

BAA represents the residue of a basic acyclic or aromatic amino acid which imparts to the amino acid a pKa of from about 10.5 to about 12.5;

NAA represents the residue of a neutral amino acid (being one with no charged side-chain);

X' (al) represents a phenylalanine, tyrosine or tryptophan residue in which the terminal carboxyl group has been replaced by an aldehyde group; and the stereochemical configuration of the X' group is of the L-amino acid kind; derivatives thereof having a functionally equivalent derivative of the aldehyde group, for example a semicarbazone (sc) , in place of the aldehyde group and the acid addition salts of any of these compounds. Preferably the stereochemical configuration throughout is L, but any one or possibly more of the other groups, i.e. Org, BAA or NAA, can have the D-configuration. While the L-configuration is likely to impart greater binding activity to the peptide, the D- con figuration may serve to block degradation of the peptide by proteinases in vivo. Racemates are of course included in the invention.

An important distinction of the peptides of the invention over the above-described known peptides is that they contain the residue of a basic acyclic or aromatic amino acid, preferably arginine. The basic amino acid will usually have the formula where R¹ represents an alkylene chain terminated by an

amino or guanidine group. These amino acids include Lysine : R¹ = -CH₂CH₂CH₂CH₂NH₂ Arginine :

Ornithine : R¹ - -CH₂CH₂CH₂NH₂

The acyclic amino acids are more readily obtainable than capreomycidine, which is the cyclic amino acid present in chymostatin. The neutral amino acid is preferably of formula

where R² represents a polar or hydrophobic group, normally a hydrocarbyl group, especially alkyl or phenylalkyl. Examples of such amino acids are

Leucine : R² = -CH₂ CH(CH₃)₂ Valine R² = -CH (CH₃)₂

Isoleucine R² = -CH (CH₃) (C₂H₅) (R or S configuration) Phenylalanine R² = -CH₂Ph, i.e. benzyl.

The nature of the neutral amino acid, and in particular of the group R², Is believed not to be critical to the inhibitory action of the molecule against chymotrypsin.

The organic residue "Org" is preferably a hydrophobic residue which will often be a hydrocarbyl group or an oxyhydrocarbyl group, conveniently a group of formula - O - hydrocarbyl as in two preferred groups, viz. benzoxy and t-butoxy groups. Such groups can arise, of course, through use of the benzyloxycarbonyl and t-butoxycarbonyl protective groups In synthesis.

The organic residue can be aliphatic or aromatic and can contain polar groups, e.g. amino or carboxyl, and one preferred such class of residues is amino acids linked via their terminal amino group to the CO group shown in formula (1) . Phenylalanine, leucine, isoleucine and t-butylglycine are examples.

A lower alkyl group, especially methyl, is a suitable hydrocarbyl group; phenethyl (Ph-CH₂-CH₂-) and adamantyl are others. It can also be a heterocyclic ring-containing group. Conveniently the length of the main chain of the organic residue does not exceed 10 atoms and is preferably from 1 to 6 atoms, counting the shortest pathway along any ring as part of the chain.

The X' group is preferably a phenylalanine-derived residue.

It is believed that the aldehyde terminal group or its func tionally equivalent derivative is essential to the inhibitory properties of the compounds. The functional mechanism responsible appears to be formation of a hemiacetal linkage between a hydroxyl group in the active site of the enzyme and the aldehyde group, i.e. of the form:

where "Enz" represents the enzyme residue and "Pep" the residue of the peptide of the invention. In the case of chymotrypsin, the hydroxyl group for serine residue (No. 195) is believed to be so bom.!. Semicarbazones, hemiacetals and diacetals are considered functionally equivalent derivatives of the aldehyde group. The preferred synthetic peptides of the invention are

Z-Arg-Leu-Phe (al) , where Z represents a benzyloxycarboxyl group

Ph-CH₂-O-CO-, and its acid addition salts. The formula of the free base written out in full is

The acid addition salts can be any desired or convenient. The strongly basic character of the free base makes It easy to acquire acid addition salts in acidic solution and generally the anion can be of any organic or inorganic acid, for example, chloride, bromide, iodide or p-toluene sulphonate. The peptides of the invention are conveniently prepared from a starting compound of formula

X - O - Y or PTG¹ - X - O - Y where: PTG¹ represents a protecting group for the amine group of the residue X

X is an amino acid residue from phenylalanine, tyrosine or tryptophan Y represents hydrogen (completing a terminal carboxyl group) or an ester-forming group, especially a methyl or ethyl group. The starting compound, if necessary with its amine group protected, is reduced to the aldehyde in one or more steps to give a compound of formula X' (al) or PTG - X' (al) , respectively, from which the protecting group PTG¹, if present, is removed.

The aldehyde group is protected by any known means, conveniently as the semicarbazone, diacetal or hemiacetal, and the amino acid protecting group is removed. The peptide chain is then built-up by any of the general methods of peptide synthesis which are described in the literature for build-up from the amino acid end. Preferably the build-up is carried out in stages, using In the first stage an amino acid of formula PTG² - (NAA)-OH where:

PTG² is a protecting group for the amino group, and NAA is as defined above, or its functional equivalent, i.e. an activated derivative effective for peptide synthesis, preferably a mixed or symmetrical anhydride or an active ester. The mixed anhydride is suitably formed with pivalic (trimethylacetlc) acid. An active ester would be, for example, an ester formed with p-nitrophenol or 2,4,5-trichlorophenol. The product peptide has the formula: PTG² - (NAA) - X'(al) - (AP) where AP is the aldehyde-protecting group. Again, the amine protecting group PTG² is removed and the final amino acid or its functional equivalent is added. This amino acid has the formula

PTG³ - (BAA) - OH where PTG³ is a protecting group for the amine group, and BAA is as defined above. The product is the tripeptide of formula PTG³ - (NAA) - X'(al) - (AP) which is also part of the present invention, as a useful interme diate. Preferably PTG³ is the group Org. CO. in formula (1) or Is easily convertible thereinto. The aldehyde protecting group is then removed to give the peptide of formula (1) :

Org. CO. (BAA). (NAA). X'(al) In this synthesis PTG¹, PTG² and PTG³ are preferably the same, e.g. benzyloxycarbonyl, and this group can be liberated by hydro genolysis in the presence of p-toluenesulphonic acid (Tos.OH) giving the p-toluene sulphonate quaternary salt (Tos.O-H⁺) amine, which is the functional equivalent of the amine. The condensations are preferably carried out in a customary way of using dicyclohexyl carbodiimide (DCCI) with the addition of hydroxybenzotriazole (HO Bt) to suppress racemization. The free aldehyde is conveniently liberated from its semicarbazone at the end of the synthesis using formaldehyde and acid conditions. For purification of the product and the intermediates, gel filtration on "Sephadex LH20" followed by elution with N,N-dimethylformamide (DMF) is very effective.

Instead of adding the NAA and BAA amino acids one by one it is of course possible to prepare a dipeptide of these acids by conventional methods and react the dipeptide of formula:

PTG³ - (BAA) - (NAA) - OH with the compound of formula

X' (al) -(AP) the symbols being as used above. The protecting groups PTG and

AP are then removed in any customary way, e.g. as described above. It is also possible to synthesise the tripeptide of the invention by building up from the other end, i.e. from a starting amino acid derivative of formula PTG³ - (BAA) - OH In such a reverse synthesis, the carboxyl group of the incoming amino acid or dipeptide would need to be protected. C-terminal protecting groups well known in peptide chemistry can be used, e.g. methyl, ethyl, phenyl, benzyl or picolyl esters and substituted or unsubstituted amide or hydrazide groups. Additives for preventing racemization are preferably included e.g. hydroxybenzotriazole or N-hydroxysuccinimide. Alternatively the azide method can be used. When the incoming reagent is a dipeptide or when the X amino acid group (preferably phenylalanine) is to be added, the reagent is conveniently terminated by a protected aldehyde group, i.e. is of formula

H- (NAA) - X' (al)-(AP) (dipeptide) or

H - X' (al) - (AP) (to add the X amino acid in the final step of the build-up) It is, however, also possible to use the corresponding reagents conventionally terminated by protected carboxyl, as mentioned above, and reduce the tripeptides with protected carboxyl into the corresponding aldehydes in one or more stages.

All these methods described above follow the general principles of building up the peptide from one terminus (N- or C-) with single amino acids or peptides, the terminus of the incoming or added peptide which it is desired should not react (correspondingly N- or C-) being protected by a protecting group which is removed before adding the next amino acid or in order to liberate the product.

The reaction conditions for the synthesis can be any customary in peptide synthesis and in the reactions of aldehydes, as appropriate. The amino acid build-up can be carried out in solution or with the terminal amino acid or aldehyde attached to a resin.

It will be appreciated that the methods of synthesis described above are not the only ones which may be used for the preparation of peptides according to the present invention and that obvious chemical equivalents of these methods or other methods known in the art for effecting similar reactions and obvious chemical equivalents thereof may be employed.

The peptides are normally prepared as their acid addition salts, usually with a monovalent anion and therefore of formula HA where A denotes the anion. The free base would, of course, not be prepared directly from the aldehyde (for fear of It undergoing a Cannizzaro reaction). Rather, the aldehyde would have to be generated as the last step of the synthesis.

Peptides according to the present invention are of particular interest for use in the treatment of various pathological conditions in human patients. Accordingly, the present invention also includes a pharmaceutical composition comprising a peptide as defined above as an active component thereof. The compositions may take various forms, including those suitable for both oral and parenteral administration which are of Interest. For oral administration the peptide is usually formulated with some form of carrier material such as starch, lactose, dextrin, magnesium stearate and the like, whilst for the latter type of composition parenteral administration is of rather more interest, for example Intravenously, intramuscularly or subcutaneously, the peptide then usually being formulated with a diluent which is conveniently sterile and pyrogen-free. Other forms of administration are possible, for example nasally when a diluent is again used, although not necessarily of a sterile and pyrogen-free nature.

In the accompanying drawings: Figure 1 in a graph in which various concentrations of

Z-Arg-Leu-Phe (al) and, for comparison, chymostatin are plotted on the abscissa against the degree of inhibition which they exert on chymotrypsin, on the ordinate. (The action of chymotrypsin is monitored by its hydrolysis of a compound "Glutaryl Phe 7 NMec" described hereinafter); and

Figure 2 is a similar graph to Figure 1 but with degree of displacement of chymotrypsin-bound proflavin plotted on the ordinate.

The following Examples illustrate the preparation of peptides of the invention and their use for chymotrypsin. In the Examples, the following mixtures of organic compounds were used as solvents in the thin layer chromatography. Proportions are by volume. (1) CHCl₃/MeOH 97/3

(2) CHCl₃/MeOH 95/5 (3) ⁿBuOH/toluene 20/35

(4) ⁿBuOH/toluene 3/1 (5) CHCl₃/MeOH 9/1

(6) CHCl₃/MeOH/AcOH/H₂O 60/18/2/3

(7) CHCl₃/ⁱPrOH 1/1

(8) CHCl₃/MeOH 1/1

(9) ⁱPrOH/AcOH/H₂O 5/1/1

(10) CHCl₃ /MeOH/AcOH 2/1/0.1/

(11) CHCl₃/MeOH 4/1

The ratio Ve/Vt refers to the ratio of volume of solvent eluted to the total bed volume of the column. In some preparations an acid addition salt of mixed anions

(bromide and p-toluene sulphonate) was generated and the product was regarded prlnicipally as the p-toluene sulphonate. Clearly, one could generate either salt by basifying and re-acidifying appropriately, and the choice of anion is not important. The structures are confirmed by n.m.r. data (not reported). Temperatures are in ºC.

EXAMPLE 1

Preparation of Z. Phe semicarbazone

The title compound was prepared by reduction of Z.Phe.OMe with diisobutylaluminium hydride using the general method described by A. Ito et al., Chem. Pharm. Bull, 23, 3106-3113 (1975). Yields of the pure semicarbazone varied from 30 to 55%. It had mp 142-143°, (c = 4.0, MeOH), R_f(1) 0.2, R_f(2) 0.3,

(Found: C, 63.37; H, 5.83; N, 16.62. C₁₈H₂₀N₄O₃ requires: C, 63.51; H, 5.92; N, 16.46%.) Ito et al. obtained Z. Phe semicarbazone. 0.25 H₂O, mp 132-134°, (c = 1.01,

MeOH) . Preparation of TosO-H₂ ⁺.Phe semicarbazone

Z.Phe semicarbazone prepared above (6.0g, 17.6mM) and p-toluenesulphonic acid (Tos.OH.H₃O, 3.35g, 17.6mM) were dissolved In DMF (50ml) and 5% Pd/C (0.88g) added. Hydrogenolysis was carried over 2h. , being monitored by the layer chromatography, then the catalyst was removed and solvent evaporated. The residue was dissolved in MeOH and the title compound was precipitated by the addition of Et₂O giving (5.45g, 82%), mp 200-202, (c = 2.0, MeOH), R_f(2) 0.3, R_f(1) 0.2 (Found: C, 53.85; H, 6.04; N, 14.67. C₁₇H₂₂N₄O₄S requires: C, 53.95; H, 5.85; N, 14.80%). Preparation of Z. Leu. Phe semicarbazone

Z.Leu.OH (1.24g, 4.6mM) and Tos O-H₂ ⁺ Phe semicarbazone (1.51g, 4.0mM) were dissolved in DMF (50ml) and the solution cooled to -4°. HOBt (0.76g, 5.6mM), N-methylmorpholine (0.47g, 0.51ml, 4.7mM) and DCCI (0.96g, 4.7mM) were then added and the reaction mixture stirred overnight. The resulting dicyclohexylurea (DCU) was removed by filtration and the concentrated filtrate applied directly to Sephadex LH20 eluting with DMF. Combination of appropriate fractions with Ve/Vt=0.5 and evaporation gave the title compound (1.38g, 76.4%), mp 148-150°,

(c = 2.0, MeOH), R_f (3) 0.6, R_f (4) 0.5, R_f (5) 0.3, (Found:

C, 62.79; H, 6.97; N, 15.15. C₂₄H₃₁N₅O₄. 0.25 H₂O requires: C, 62.94; H, 6.93; N, 15.29%).

Preparation of Tos O-H₂ ⁺. Leu. Phe semicarbazone

Z. Leu. Phe semicarbazone (0.75g, 1.7mM) and Tos.OH H₂O (0.32g, 1.7mM) were dissolved in DMF (50ml) and 5% Pd/C (84mg) added. After a 2h. hydrogenolysis the catalyst was removed and the solvent was evaporated to give a residue which was crystallised from MeOH/Et₂O giving the title compound (0.39g, 47%), mp 128-129°, (Found: C, 53.23;

H, 6.78; N, 12.96. C₂₃H₃₃N₅O₅S. 1.5 H₂O requires: C, 53.26; H, 6.70; N, 13.50%). Preparation of Z. Arg (Tos OH). Leu. Phe semicarbazone

Z. Leu. Phe semicarbazone (0.65g, 1.4mM) and TosOH. H₂O (0.28g, 1.4mM) were dissolved in DMF (50ml) and hydrogenolysed in the present of 5% Pd/C (75mg) . After 3h. the catalyst was removed and Z. Arg (HBr) . OH (0.56g, 1.4mM), HOBt (0.38g, 2.8mM) NMM (0.15g, 0.16ml, 1.4mM) added to the filtrate. The solution was cooled to -10° and DCCI (0.39, 1.5mM) added. After 2 days the reaction mixture was filtered and the solvent volume reduced before gel filtration on Sephadex LH20, eluting with DMF. The major product had Ve/Vt = 0.42; combination of the appropriate fractions and evaporation gave the title compound (0.39g, 39%), mp 160° (dec), (c = 1.9, MeOH), R_f (6) 0.4, amino

acid analysis: Arg 1.00 Leu, 1.00, (Found: C, 55.04; H, 6.93; N, 15.43. C₃₇H₅₁N₉O₈S. 1.5 H₂O requires: C, 54.93; H, 6.67; N, 15.58%). Preparation of Z. Arg (HCl) . Leu. Phe (al)

Z. Arg (Tos OH). Leu. Phe semicarbazone (100mg, 1.45mM) was dissolved In MeOH (3.5ml) and 0.5M HCl (1.5ml) and 40% formaldehyde (0.33ml) added. After 4.5h. the solution was evaporated and the residue purified by gel filtration on Sephadex LH20, eluting with DMF. Isolation of the product from the peak at Ve/Vt = 0.45 gave the title compound, (40mg, 43%) mp 170° (dec.),

(c = 1.02, MeOH), R_f (7) 0.7, R_f (8) 0.8, (Found: C, 53.80;

H, 6.90; N, 13.41. C₂₉H₄₁N₆O₅ Cl. 3H₂O requires: C, 54.15; H, 7.15; N, 13.07%). EXAMPLE 2

Preparation of Z. ILe. Phe semicarbazone

Z. lie. OH (1.3g, 5mM) was dissolved in Et OAc (28ml) and a solution of Tos O-H₂ ⁺. Phe semicarbazone (1.8g, 4.8mM) was added. HOBt (0.8g, 6mM) and N-methylmorpholine (0.51g, 0.55ml, 5mM) were then added along with DMF (20ml) which was required to maintain solubility. The solution was cooled to -4º and DCCI (1.03g, 6mM) added, the reaction mixture was then stirred for 48h. After this period the resulting urea was removed by filtration and the concentrated filtrate applied directly to Sephadex LH20, eluting with DMF. Evaporation of the fractions with Ve/Vt=0.53 gave the title compound as a white solid (1.18g, 55%), mp 196-198°, (c = 2, MeOH), R_f (5) 0.4, (Found: C, 63.30; H, 6.92;

N, 15.45. C₂₄H₃₁N₅O₄ requires: C, 63.56; H, 6.89; N, 15.44%).

Preparation of TosO-H₂ ⁺. lie. Phe semicarbazone

The Z. lie. Phe semicarbazone (3.0g, 6.6mM) and Tos.OH.H₂O (1.16g, 6.1mM) were dissolved in DMF (50ml) and hydrogen passed through the solution for 1.5h. in the presence of 5% Pd/C (0.3g). The catalyst was removed and the solvent evaporated to give a residue which was crystallised fro MeOH/Et₂O yielding the title compound (1.1g, 43%), mp 130-131°, 10.9° (c = 2.0, MeOH),

R_f (6) 0.4, (Found: C, 55.00; H, 6.90; N, 14.07. C₂₃H₃₃N₅O₅S. 0.5 H₂O requires: C, 55.18; H, 6.84; N, 13.99%). Preparation of Z. Arg (Tos OH) . lie. Phe semicarbazone

Z. Arg (HBr). OH (0.95g, 2.44mM), TosO-H₂ ⁺. lie. Phe semicar bazone (1.2g, 2.44mM), HOBt (0.66g, 4.88mM) and NMM (0.27ml, 2.44mM) were dissolved in DMF (60ml) and the solution cooled to -20º. DCCI (0.6g, 2.93mM) in CH₂Cl₂ (50ml) was added and the reaction allowed to proceed for 2 days at room temperature. The resulting DCU was removed by filtration and the solution volume was reduced before gel filtration on Sephadex LH20, eluting with DMF. The appropriate fractions with Ve/Vt = 0.46 were combined and evaporated to give the title compound as a white powdery solid (0.84g, 50%), mp 128 (dec), [α]J³- 26.7° (c = 3.0, MeOH), R_f (6) 0.2. Found: C, 55.11; H, 6.82; N, 15.88. C^H^^OgS. 1.25 H₂0 requires C, 55.24; H, 6.70; N, 15.67%). Preparation of Z. Arg (HC1). lie. Phe (al)

Z. Arg (Tos OH). lie. Phe semicarbazone (0.62g, 0.9mM) was dissolved in a mixture of 40% formaldehyde (2.0ml), 0.5 molar HC1 (9.0ml) and MeOH (18.6ml). After stirring for 24h. the solution was evaporated to give a residue which was purified on Sephadex LH20, eluting with DMF. The fractions making up the single peak at Ve/Vt=0.51 were pooled and evaporated to give the title compound (0.29g, 51%), mp 115°

(c = 0.68, MeOH, R_f (9) 0.5, R_f (10) 0.5. Amino acid analysis (6M HCl, 110°, 24h) Arg,0.96 Leu, 1.05; Mass spectrum FAB (positive ion mode) m/e : 553

(Z.Arg.lie.Phe(al) + 1); 376 (Z.Arg.lie + 1); 291 (Z.arg + 1), ion also observed at 599 indicating dimethyl acetal present.

EXAMPLE 3 Preparation of Z. Val. Phe semicarbazone z. Val. OH (1.32g, 5.3mM), the p-toluenesulphonate salt,

Tos O-H₂ ⁺. Phe semicarbazone (2.0g, 5.3mM) and HOBt (0.85g, 6.3mM) were dissolved in DMF (50ml) and cooled to 0°. NMM (0.52g, 0.57ml, 5.3mM) and DCCI (1.09g, 5.3mM) were then added and the reaction mixture stirred for 24h. The reaction mixture was concentrated and filtered to remove DCU, and the filtrate applied directly to

Sephadex LH20, eluting with DMF. The fractions making up the peak at Ve/Vt=0.50 were pooled and evaporated giving the title compound (2.23g, 96%), mp 197.5-200, (c - 2.0, MeOH), R_f (5) 0.4,

(Found: C, 61.59; H, 6.81; N, 15.76. C₂₃H₂₉N₅O₄. 0.5 H₂O requires: C, 61.59, H, 6.74; N, 15.61%). Preparation of TosO-H₂ ⁺. Val. Phe semicarbazone

Z. Val. Phe semicarbazone (1.5g, 3.4mM) was dissolved in DMF (75ml) and Tos.OH.H₂O (0.65g, 3.4mM) and 5% Pd/C (85mg) added. Hydrogenolysis was carried out for 1.5h, being monitored by tic. The catalyst was removed by filtration and the filtrate evaporated to leave a residue which was crystallised from MeOH/Et₂O giving the title compound mp 128-130, (c - 2.0, MeOH), R_f

(6) 0.4, R_f (11) 0.2, (Found: C, 52.81; H, 6.96; N, 13.61. C₂₂H₃₁N₅O₅S. 1.5 H₂O requires: C, 52.37; H, 6.79; N, 13.82%). Preparation of Z. Arg (Tos OH). Val. Phe semicarbazone The p-toluenesulphonate TosO-H₂ ⁺. Val. Phe semicarbazone

(0.9g, 1.9mM) and Z. Arg (HBr) . OH (1.1g, 2.8mM) were dissolved in a mixture of DMF (25ml) and CH₂Cl₂ (25ml) . After cooling to 0°, HOBt (0.4g, 2.8mM), NMM (0.21ml, 1.9mM) and DCCI (0.59g, 2.8mM) were added and the reaction mixture stirred for 24h. at room temperature. The reaction mixture was concentrated and filtered before gel filtration on Sephadex LH20, eluting with DMF. The appropriate fractions making up the peak at Ve/Vt = 0.49 were pooled and evaporated giving the title compound (0.9, 70%), mp 98° dec, (c - 2.0, MeOH), R_f (6) 0.3;

Found: C, 55.71; H, 6.72; N, 16.57 C₃₆H₄₉N₉O₈S. 0.5H₂O requires: C, 55.66; H, 6.48; N, 16.23%). Preparation of Z.Arg(HCl) .Val.Phe(al)

The Z.Arg (Tos OH) .Val.Phe semicarbazone (1.35g, 2mM) was dissolved in MeOH (42ml) and 0.5M HCl (20ml) and a 40% solution of formaldehyde (4.4ml) added. The solution was stirred for 4% hours and the course of the reaction monitored by tic The solution was diluted with brine and extracted with ethyl acetate and n-butanol, and combined extracts were then dried (MgSO₄) and the solution evaporated. This crude product was purified by gel filtration on Sephadex LH20, eluting with DMF. The purified material was obtained by combination of the fractions making up the peak at Ve/Vt = 0.63, giving (0.30g, 25%), mp 85 - 87°,

amino acid analysis (6M, HCl, 110 ; Phe, trace. FAB mass spectrum (positive ion mode): m/e 539 (Z.Arg.Val.Phe(al) + 1); 362 (Z.Arg.Val + 1); 291 (Z.Arg + 1); ions also observed at 669 and 595 indicating that some dibutyl acetal was present. ASSAYS

The ability of the preferred synthetic tripeptides of the invention and their semicarbazones to bind and inhibit chymo trypsin has been investigated, and compared with the ability shown by chymostatin itself and by corresponding synthetic dipeptides not within the scope of the invention. The effectiveness of the test peptides in binding to and inhibiting chymotrypsin was judged by their effect on the chymotryptic hydrolysis of glutaryl phenylalanine-7-amino-4-methylcoumarin (hereinafter "glutaryl-Phe 7 N Mec" for short) and their ability to displace chymotrypsin bound proflavin.

The chymotrypsin used was alpha-chymotrypsin from bovine pancreas and proflavin bought from the Sigma Chemical Company. The N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid (HEPES) , glutaryl-Phe 7 N Mec and 7-amino-4 methylcoumarin were bought from Cambridge Research Biochemicals Limited. Chymostatin was bought from the Peptide Research Institute, Osaka, Japan. All the synthetic peptides tested were prepared by the present inventors. A Perkin Elmer 3000 Fluorescent Spectrophotometer was used. (1) Inhibiton Assay A series of fluorimeter cuvettes were prepared, each containing:-

(a) 2 ml 0.05M HEPES/0.01M CaCl buffer, pH 8.0, at about 30°C.

(b) 56 nM of chymotrypsin (c) Various amounts of test peptide dissolved in very small amounts of dimethyl sulphoxide (DMSO) . The cuvettes were incubated for five minutes, after which 10 microlitres of (0.02 M) glutaryl-Phe 7 N Mec in DMSO was added. The rate of increase of fluorescence in unit time at an excitation wavelength of 380 nm and an emission wavelength of 460 nm was recorded for each concentration of inhibitor, and calibrated using standard solutions of the hydrolysis product 7-amino-4-methylcoumarin.

The rates of increase of fluorescence (in the presence of increasing concentrations of test peptide) were expressed as a percentage of the rate in the absence of test peptide, and then subtracted from 100% to give percentage inhibitions of the chymo tryptic hydrolysis of glutaryl-Phe 7 N Mec.

Table 1 below shows the results. Of the synthetic peptides tested only the preferred synthetic peptides of the invention (with aldehyde terminal groups) produced inhibition of the chymo tryptic hydrolysis of glutaryl-Phe 7 N Mec approaching that of chymostatin. Of the other synthetic peptides tested, the tri peptide semicarbazones of the invention and the dipeptides Z.Leu.Phe aldehyde inhibited the hydrolysis, but only at a far higher concentration. The other semicarbazones, lacking the Arg. residue, had no inhibitory effect on chymotrypsin and when added in a high enough concentration actually increased the rate of hydrolysis.

a no inhibition observed at inhibitor concentrations of 50 micromolar) + semicarbazones of the Invention * aldehydes of the invention

More detailed results for the comparison of Z.Arg.Leu.Phe(al) with chymotrypsin are shown in Figure 1 of the drawings. (2) Proflavin Displacement Assay

The addition of chymotrypsin to free solution proflavin has a quenching affect on the fluorescence of the proflavin. The degree of quenching is dependent on the concentration of chymotrypsin and will reach saturation. For the concentration of proflavin used here, the concentration at which quenching by chymotrypsin reaches saturation is around 200 micromolar. The binding constant is about 35.1 micromolar of chymotrypsin. The fluorescence of proflavin having been decreased by its binding to the added chymotrypsin, it may be increased again by the addition of inhibitors that, in binding to chymotrypsin, displace the proflavin.

A series of three fluorimetric cuvettes were prepared each containing 2 ml of 0.01 micromole proflavin in 0.05 M HEPES/ 0.01 M CaCl₂ buffer, pH 8.0 (stored in the total absence of light). In addition:-

(a) contained 0.2 ml of 1 mM HCl.

(b) contained 0.2 ml of chymotrypsin solution (about 13 micromolar) In 1 mM HCl, and aliquots of a solution of test peptide in varied concentrations in DMSO. (c) contained 0.2 ml of chymotrypsin solution (about 13 micromolar) in 1 mM HCl, and aliquots of DMSO only. The level of fluorescence was recorded at an excitation wavelength of 445 nm and an emission wavelength of 504 nm for each concentration of test peptide.

The level of fluorescence of a solution of proflavin and chymotrypsin in the presence of increasing concentrations of test peptide was recorded. The percentage displacement of proflavin was then calculated for each concentration of test peptide using the following equation:-

The percentage displacement of proflavin was plotted versus the concentration of test peptide.

Results of the assay are shown In Table 2 below. Chymostatin, Z-Arg-Leu-Phe aldehyde and Z-Leu-Phe aldehyde were all quite effective at displacing proflavin, though the concentrations of each required to produce 50% displacement varied in the ratio 1:2:4. Z-Arg-Leu-Phe semicarbazone was less effective and has to be present in approximately ten times the concentration of Z-Leu-Phe aldehyde, the least effective of the other three, to produce the same degree of proflavin displacement.

More detailed results for the comparison of Z.Arg.Leu.Phe(al) with chymotrypsin are shown in Figure 2 of the drawings.

The results of the proflavin displacement assays have been corrected for the effect of the inhibitor solvent dimethyl sulphoxide (DMSO) on the fluorescence of proflavin, and the results for the displacement of proflavin by Z-Arg-Leu-Phe aldehyde have also been corrected for the effect of an interaction between Z-Arg-Leu-Phe aldehyde and free solution proflavin that leads to an additional fluorescence yield of up to 25%. (3) Determination of inhibition constants

The inhibition constants (K_i) for the binding of the three tripeptide aldehydes were determined at 25ºC by the spectrophoto metric measurement at 405 nm of the release of 4-nItroaniline from the substrate N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide, in 0.05M HEPES buffer, pH 8.0 containing 0.01M CaCl₂. Chymotrypsin (20 nM) was preincubated for 10 minutes with various concentrations of each inhibitor (added as solutions in dimethylsulphoxide; final concentration 1.0% (v/v). At the end of this period the reaction was initiated by the addition of substrate to give a final volume of 1.0 ml and the rate of Increase in absorbance at 405 nm was compared with that obtained in equivalent incubations without inhibitor. The hyperbolic inhibition data were analysed by non-linear curve fitting using a microcomputer to a parameter accuracy of better than 0.1%. Results are shown in Table 3.

DISCUSSION OF RESULTS

The test enzyme chymotrypsin is a 23,000 molecular weight, pancreatic produced, digestive endopeptidase. As such it is specific for peptide linkages where the carbonyl function is contributed by aromatic amino acid residues, e.g. tyrosine, tryptophan and phenylalanine; but it also catalyses the hydrolysis of esters and amides of such amino acids. It may best be described therefore, as a transferer of acyl groups to acyl receptors. It possesses a hydrophobic binding site for the aromatic amino acid, and. a catalytic site for the removal and transfer of the acyl group. This catalytic site consists of three main residues forming a charge relay complex. Serine 195 possesses a hydroxyl group which forms a hydrogen bond with the imidazole ring of Histidine 57, and this in turn forms another hydrogen bond with the carboxylate group of Aspartate 102. These interactions make use of the hydroxyl group on Serine 195 as a nucleophile. The hydrophobic binding site first recognises and binds to an aromatic amino acid, and then the nucleophilic hydroxyl group attacks the carbon atom involved in the peptide linkage. This leads to the breaking of the link, the formation of an acyl enzyme complex and the release of a leaving group, which in the case of glutaryl-Phe 7 N Mec is the fluorescent product 7-amino-4-methylcoumarin. The acyl enzyme complex is later hydrolysed to give back the enzyme and release an acyl group. The phenylalanine aldehyde group possessed by chymostatin, and two of the synthetic peptides, Z-Arg-Leu-Phe aldehyde and Z-Leu-Phe aldehyde, apparently forms a hemiacetal linkage with the hydroxyl group of Serine 195 and thus prevents chymotrypsin from catalysing substrate hydrolysis. However, if the only requirement for effective inhibition was the presence of a C terminal aromatic amino acid aldehyde residue, one would expect chymostatin, Z-Arg-Leu-Phe aldehyde and Z-Leu-Phe aldehyde all to inhibit chymotrypsin activity to the same degree, and Z-Arg-Leu-Phe semicarbazone to have no inhibitory effect. Z-Arg-Leu-Phe aldehyde and Z-Leu-Phe aldehyde differ only in the presence of the basic amino acid arginine, yet Z-Arg-Leu-Phe aldehyde is approximately seventy times more effective as an inhibitor of chymotrypsin activity (Table 1).

The possession of this basic amino acid improves the effeciency of inhibition, and possibly it is the presence of arginine that allows the trlpeptide semicarbazones to function as a chymotrypsin inhibitor. Thus, it appears possible that there is a secondary binding mechanism at work linking arginine to the enzyme. In chymostatin the basic amino acid present is capreomycidine, but this cannot conveniently be synthesized. It is therefore an important achievement of the present invention to find that the capreomycidine can be replaced by a basic amino acid which can be synthesized easily.

Claims

1. Peptides which are compounds of formula (Org). CO. (BAA). (NAA). X' (al) wherein:

Org represents an organic residue;

X' (al) represents a phenylalanine, tyrosine or tryptophan residue in which the terminal carboxyl group has been replaced by an aldehyde group, and the stereochemical configuration of at least the X' group is of the L-amino acid; derivatives thereof having a functionally equivalent derivative of the aldehyde group, in place of the aldehyde group and the acid addition salts of any of these compounds.

2. Peptides according to Claim 1, wherein the stereochemical configuration throughout is L.

3. Peptides according to Claim 1, wherein BAA represents an lysine, arginine or ornithine residue.

4. Peptides according to Claim 1, wherein NAA represents the residue of an amino acid formula

where R² represents a hydrocarbyl group.

5. Peptides according to Claim 4, wherein NAA represents the residue of leucine, valine, isoleucine or phenylalanine.

6. Peptides according to Claim 1, wherein "Org" represents a hydrocarbyl or oxyhydrocarbyl group.

7. Peptides according to Claim 1, wherein "Org" represents a benzoxy or t-butoxy group.

8. Peptides according to Claim 1, wherein "Org" has a main chain of length from 1 to 6 atoms, counting the shortest pathway along any ring as part of the chain.

9. Peptides according to Claim 1, wherein X' represents a phenylalanine residue.

10. Peptides according to Claim 1 of formula

(Ph = phenyl) and its acid addition salts.

11. A process of preparing a peptide claimed in Claim 1 in the form of an acid addition salt, from a starting compound of formula

X - O - Y or PTG¹ - X - O - Y where:

PTG¹ represents a protecting group for the amine group of the residue X defined below;

X is an amino acid residue from phenylalanine, tyrosine or tryptophan; Y represents hydrogen (completing a terminal carboxyl group) or an ester-forming group, which process comprises (1) reducing the starting compound, if necessary with its amine group protected, to the aldehyde in one or more steps to give a compound of formula X' (al) or PTG - X' (al) , respectively, and removing the protecting group PTG¹, if present; (2) protecting the aldehyde group;

(3) removing the amino acid protecting group; (4) reacting the resulting amino acid with an amino acid of formula PTG² - (NAA) - OH where PTG² is a protecting group for the amino group, and NAA is as defined above, or an activated derivative thereof effective for peptide synthesis, to give a protected dipeptide of formula:

PTG² - (NAA) - X'(al) - (AP) where AP is an aldehyde-protecting group; (5) removing the amine protecting group PTG²; (6) reacting the resultant amine with an amino acid of the formula:

PTG³ - (BAA) - OH where PTG³ is a protecting group for the amine group and BAA is as defined above, to give a tripeptide of formula:

PTG³ - (NAA) - X'(al) - (AP) where the symbols are as defined above and if the group PTG is not a group of formula Org. CO as defined in formula (1), converting it thereinto by de-protection and re-protection of the amine function; and (8), where the aldehyde itself is to be prepared, removing the aldehyde protecting group AP to give the peptide of formula

Org. CO - (BAA) - (NAA) - X'(al) (1) and, if AP is not a functionally equivalent derivative of the aldehyde group, derivatising the aldehyde function.

12. A pharmaceutical composition comprising a peptide claimed in Claim 1, in association with a pharmaceutically acceptable carrier or diluent.