WO1990005141A1 - Amino acid and peptide inhibitors of human leukocytic elastase and collagenase - Google Patents

Abstract

Potent and specific inhibitor compounds for collagenase and/or elastase - type enzymes, especially for human leucocytic elastase (HLE), are provided by combining an amino acid or short peptide with a hydrocarbonyl-oxycarbonyl protecting group having at least 4 carbon atoms at the N-terminal of the amino acid or peptide, and a hydrophobic hydrocarbon chain of 8 to 16 carbon atoms at the carbonyl group of the amino acid or peptide, the protecting group and the hydrophobic group being physiologically innocuous components, but providing the required inhibition. The inhibitor compounds can be expected to be useful in the treatment of collagenase or elastase - type implicated diseases such as arthritis, tumour growth, epidermolysis bullosa, pulmonary emphysema, and chronic obstructive lung disease.

Description

AMINO ACID AND PEPTIDE INHIBITORS OF HUMAN LEUKOCYTIC ELASTASE AND COLLAGENASE

Field of the Invention

This invention provides potent and specific inhibitor compounds for elastase-type enzymes, especially for human leukocytic elastase (HLE) (also known as human granulocyte elastase or HGE), and for collagenase. These proteolytic enzymes are involved in cartilage and tissue degradation, and have been implicated in the growth and metastasis of

cancerous tumours; degradation of tissues in rheumatoid

arthritis, osteoarthritis and epidermolysis bullosa; and

destruction of lung tissues in pulmonary emphysema or

chronic obstructive lung disease. The molecular structure of the inhibitor compounds of the invention is designed

to have an expected absence of undesirable side-effects

in use.

The invention also provides: processes for the preparation of said inhibitor compounds; pharmaceutical/veterinary

compositions containing said inhibitor compounds; and methods for the clinical application of said inhibitor compounds

and said pharmaceutical/veterinary compositions.

Description of the Prior Art :

There is evidence to implicate the neutral proteases of human leukocytes (polymorphonuclear leukocytes) in the degradation of cartilage in both rheumatoid and osteoarthritis. The chronic destruction of the elastic component of lung

connective tissues by elastase-type enzymes, in particular by HLE and cathepsin G, is

currently believed to result in the onset of chronic

obstructive lung disease. These proteases are primarily inhibited by the major serum protease inhibitor α ₁ - proteinase inhibitor (α₁-PI), which is also a normal

constituent of bronchioalveolar lavage fluid (BAL).

However, α ₁-Pl is readily inactivated by oxidants such as those present in cigarette smoke or oxidative enzymes (i.e. myeloperoxidase) that normally function in phagocytic cells during inflammatory states. In addition, some persons are genetically deficient in α ₁-Pl, with levels of the inhibitor which are 25% less than normal. Thus, persons with a compromised inhibitor screen are prime candidates for chronic obstructive lung disease.

There are many classes of compounds reported in the literature as inhibitors of HLE. Some of these compounds are the chloromethyl ketones, vide: J.C. Powers, B.F.

Lupton, A.D. Harley, N. Nishino, R.J. Whitley, Biochem.

and Biophys. Acta. 485, 156 (1977) and K. Haveman & A.

Janoff: "Neutral proteases of Human Polymorphonuclear

Leukocytes", p.221, Urban and Schwartzenberg, Baltimore (1977); the sulfonyl fluorides, vide: K. Havemen & A.

Janoff: "Neutral proteases of Human Polymorphonuclear

Leukocytes", p.221, Urban and Schwartzenberg, Baltimore (1977); the imidazole-N-carboxamides, vide: W.C. Croutas, R.C. Budger, T.D. Ocain, D. Felter, J. Frankson, M.

Theodorakis, Biochem. and Biophys. Research Commun. 95, 1890 (1980); the azapeptides, vide: K. Haveman & A.

Janoff, "Neutral proteases of Human Polymorphonuclear Leukocytes", p.221, Urban and Schwartzenberg, Baltimore (1977); cyclohexylamide, vide: CH. Hassall, W.H.

Johnson, N.A. Roberts: Bio-Organic Chem. 8, 299 (1979); the adamantane sulphenyl peptides, vide: A.M.J. Blow, Biochem. J. 161, 13 (1977); the cis-unsaturated fatty acids, vide: B.M. Ashe, M. Zimmerman, Biochem. and

Biophys. Res. Commun. 75, 94 (1977); and the gold

complexes such as gold thiomalate, vide: A. Baici, P.

Salgam, K. Fehr, A. Boni, Biochem. Pharmac 30, 903

(1981).

The chloromethyl ketones, the sulfonyl fluorides, the imidazole-N-carboxamides, the azapeptides, the cisunsaturated fatty acids, and the adamantane sulphenyl peptides all have reactive groups which make them

inadvisable, if not dangerous, to use. In general, it can be said that those compunds bearing highly reactive functional groups are difficult, if not impossible, to targe t onto receptor sites, as they are likely to react with the many components of the body available between the point of administration and the target receptor site.

Oleic acid, a cis-unsaturated carboxylic acid, has been shown to be an acceptably good specific inhibitor of HLE, but not of porcine pancreatic elastase (PPE), trypsin, chymo-trypsin or cathepsin G, which has

indicated to us that the principal difference between HLE and the other serine proteases could be due to the different hydrophobic character of a site near the active site, noting that HLE has not been fully sequenced, its 3-dimensional structure has not been determined, and its active site-stereochemistry is unknown.

These proteases apparently prefer certain amino acids or short peptide sequences as substrates, and previous workers in the field have sought to utilize this preference, either by using the synthetic substrate as a competitive inhibitor, or by functionalizing those substrates so as to cause them to react with the active site, thereby inactivating the enzyme.

Cathepsin G is a serine protease present in

azurophil granules. The enzyme is a chymotrypsin-like neutral proteinase, with a molecular weight of

approximately 28,000 daltons.

Cathepsin G is capable of hydrolysing cartilage proteoglycans and insoluble collagen. Cathepsin G also causes conversion of angiotensin I to angiotensin II, which is associated with inflammatory processes. The enzyme is classified as chymotrypsin-like due to its ability to act on come synthetic substrates of

chymotrypsin (especially of α-chymotrypsin). Cathepsin G shows a preference for phenylalanine and tyrosine amino acids at the S₁ site of the catalytic centre. The enzyme is inhibited by soybean trypsin inhibitor, aprotinin, α₁-antichymotrypsin,α₂-macroglobulin and α₁-antitrypsin.

Collagenase is a metalloenzyme, located in specific granules of human polymorphonuclear leukocytes, and is active at physiological pH. The enzyme requires Ca²⁺ or Zn²⁺ for activity. The enzyme has a molecular weight of between 45,000 and 76,000 daltons. The wide variation in the molecular weight values of the enzyme have been attributed to the conditions utilized for its

determination. It has been shown that denatured and reduced collagenase in the SDS-polyacrylamide

electophores is system revealed two components with molecular weights of 42,000 and 33,000. The enzyme shows a preference for type I collagen. The action of the enzyme on collagen appears to be on the extra-helical peptides and involves elimination of cross-links between the strands. The enzyme is inhibited by EDTA, cysteine and the serum proteins α₂-macroglobulin and α ₁ - antitrypsins

Summary of the Invention

We have now conceived the idea of making inhibitors for collagenase and elastase-type enzymes by combining a hydrocarbonyl-oxycarbonyl or hydrocarbonyl-carbonyl protecting group, having at least 4 carbon atoms, and an optionally substituted hydrophobic hydrocarbon group of 6 to 16 carbon atoms, optionally via an amino acid or short peptide chain, the protecting group, the hydrophobic group and the amino acid or peptide being physiologically innocuous components, but providing the required

inhibition. The advantage of this concept resides in that enzyme cleavage of the inhibitor would merely result in end products which are physiologically innocuous

components.

According to a first aspect of the present

invention, there are provided collagenase inhibitor compounds consisting of amino acid derivatives or peptide derivatives as defined by the general formula (I)

and physiologically acceptable salts thereof, wherein: R₁ is a hydrocarbonyl-oxycarbonyl group with at least 4 carbon atoms, the hydrocarbyl moiety of which may be substituted or unsubstituted, for protecting the N-terminal of the amino acid or peptide, which protecting group, if cleaved in vivo, forms a physiologically innocuous compound; R₂ and R₃, which may be the same or different and

may differ from unit to unit when-n is greater than

1, are independently selected from hydrogen, alkyl of 1 to 10 carbon atoms, substituted alkyl of 1 to 10 carbon atoms in which the substituents are those present in natural or non-natural amino acids, provided that they are physiologically innocuous, or

R₂ and R₃ taken together with the adjacent

nitrogen and carbon atoms may form a 5-membered ring with 4 carbon atoms, which ring may optionally be substituted by substituents which are present in natural or non-natural amino acids, provided that they are physiologically innocuous;

R₄ is an optionally substituted hydrophobic hydrocarbon group of 6 to 16 carbon atoms which, if cleaved in vivo, forms a physiologically innocuous compound, said hydrophobic hydrocarbon group being bonded direct to the carbonyl carbon or to the carbonyl carbon through an oxygen, sulphur or nitrogen hetero atom; and

n is an integer from 1 to 6.

The group R ₁ , which is preferably a bulky,

hydrophobic, apolar, amino-protecting group having steric influence on the collagenase enzyme and materially contributing to the inhibitory effect of the present

inhibitors, may be of the formula R' - O - C - wherein R' is selected from:

(i) straight-chain alkyl of 1 to 10 carbon atoms,

straight-chain alkenyl of 1 to 10 carbon atoms, or straight-chain alkynyl of 1 to 10 carbon atoms, each of which is optionally substituted with one or more of alkyl of 3 to 10 carbon atoms, alkenyl of 3 to 10 carbon atoms, and alkynyl of 3 to 10 carbon atoms to form a branched-chain; or substituted with one or more of cycloalkyl of 3 to 10 carbon atoms,

cycloalkenyl of 3 to 10 carbon atoms, aryl of 6 to 10 carbon atoms, adamantyl, or heterocyclic

radical (s) such as pyryl, furyl, thiophenyl or pyrazolyl, any of which may be substituted, for example, with one or more of halo, hydroxy, nitro, alkyl of 1 to 5 carbon atoms, or alkoxy of 1 to 5 carbon atoms; or

(ii) branched-chain alkyl of 3 to 10 carbon atoms,

branched-chain alkenyl of 3 to 10 carbon atoms, branched-chain alkynyl of 3 to 10 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, cycloalkenyl of 3 to 10 carbon atoms, aryl of 6 to 10 carbon atoms, adamantyl, or a heterocyclic radical such as pyryl, furyl, thiophenyl or pyrazolyl, any of which may be substituted, for example, with one or more of halo, hydroxy, nitro, alkyl of 1 to 5 carbon atoms, or alkoxy of 1 to 5 carbon atoms. Thus, the amino-protecting group R₁ may be: (a) branched-chain alkoxycarbonyl of 4 to 11 carbon atoms, such as t-butyloxycarbonyl, isopentyloxycarbonyl or isohexyloxycarbonyl; (b) aryl substituted straight-chain alkoxycarbonyl in which the alkoxy moiety has 1 to 10 carbon atoms, such as benzyloxycarbonyl; (c)

cycloalkyloxy-carbonyl in which the cycloalkyl moiety has 3 to 10 carbon atoms, such as cyclohexyl-oxycarbonyl; (d) aryloxycarbonyl in which the aryl moiety has 6 to 10 carbon atoms, such as phenyloxycarbonyl; or (e)

substituted aryloxycarbonyl in which the aryl moiety has 6 to 10 carbon atoms, such as tolyloxycarbonyl or

xylyloxycarbonyl.

The groups R₂ and R₃, which are constituent

parts of natural or non-natural amino acids, may be the same or different and are selected from hydrogen; alkyl of 1 to 10 carbon atoms, for example, methyl, isopropyl or isobutyl; substituted alkyl of 1 to 10 carbon atoms in which the substituents are those present in natural or non-natural amino acids, for example, benzyl, p-hydroxy phenylmethyl or 4-hydroxy-3, 5-diiodobenzyl; or R₂ and R₃ taken together with the adjacent nitrogen and carbon atoms may form a 5-membered ring with 4 carbon atoms, for example, the 5-membered ring of proline, which 5-membered ring may optionally be substituted by substituents which are present in natural or non-natural amino acids, for example, the 5-membered ring of either hydroxyproline or trifluoromethylproline. The group R₄ is a hydrophobic hydrocarbon group of 6 to 16 carbon atoms, preferably 10 to 14 carbon atoms, more preferably 12 carbon atoms, which, as indicated, if cleaved in vivo, forms a physiologically innocuous compound, said hydrophobic hydrocarbon group being bonded direct to the carbonyl carbon or to the carbonyl carbon through an oxygen, sulphur or nitrogen hetero atom, preferably through nitrogen. For example, the group R₄ may be alkyl, alkenyl, alkynyl, alkylamino, alkenylamino, hydrocarbyloxy or hydrocarbylthio, each of 6 to 16 carbon atoms, preferably 10 to 14 carbon atoms, more preferably 12 carbon atoms. Moreover, the hydrophobic hydrocarbon group R₄ may be substituted, for example, with one or more of halo, hydroxy, carboxy, amino, nitro, alkyl of 1 to 5 carbon atoms, or alkoxy of 1 to 5 carbon atoms.

Thus, the hydrophobic hydrocarbon group R₄ may be octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl, octylamino, nonylamino, undecylamino,

decylamino, dodecylamino, tetradecylamino,

hexadecylamino, or optionally substituted phenylamino.

Many of the compounds of general formula (I) are known, but only as inhibitors of HLE and other elastasetype enzymes (see International Patent Specification No. PCT/AU85/000317). Usefulness as collagenase inhibitors has not previously been suggested. According to a second aspect of the present

invention, there is provided a second group of compounds, which are inhibitors of collagenase, HLE and other elastase-type enzymes . These compounds are defined by the general formula (IA)

and include physiolog ically accep table sa l ts thereof , where in :

m is an integer from 0 to 6;

R₅ represents a hydrocarbonyl-carbonyl protecting group or, if m is 0, R₅ may also represent a

hydrocarbonyl-oxycarbonyl protecting group, each of said protecting groups having at least 4 carbon atoms, and the hydrocarbyl moiety of each of said protecting groups being substituted or

unsubstituted;

R₂ and R₃, which may be the same or different and

may differ from unit to unit when m is greater than 1, are as hereinbefore defined; and

R₄ is as hereinbefore defined.

The group R₅, which is preferably a bulky,

hydrophobic, apolar, protecting group having steric influence on the elastase-type enzyme and

materially contributing to the inhibitory effect of the

present inhibitors, may be of the formula R' - C- or

(if m is 0) R' -O - C- , wherein R' is as hereinbefore defined. Thus, the protecting group R₅ may

represent (a) branched-chain alkylcarbonyl of 4 to 11 carbon atoms, such as t-butylcarbonyl, isopentylcarbonyl or isohexylcarbonyl; (b) aryl substituted straight-chain alkylcarbonyl in which the alkyl moiety has 1 to 10 carbon atoms, such as benzylcarbonyl; (c) cycloalkyl- carbonyl in which the cycloalkyl moiety has 3 to 10 carbon atoms, such as cyclohexylcarbonyl; (d)

arylcarbonyl in which the aryl moiety has 6 to 10 carbon atoms, such as benzoyl; or (e) substituted arylcarbonyl in which the aryl moiety has 6 to 10 carbon atoms, such as toluoyl, dimethylbenzoyl, hydroxybenzoyl and benzoyl substituted by amino and hydroxy groups; and, if m is 0, R₅ may also represent (f) branched-chain alkoxycarbonyl of 4 to 11 carbon atoms, such as t-butyloxycarbonyl, isopentyloxycarbonyl or isohexyloxycarbonyl; (g) aryl substituted straight-chain alkoxycarbonyl in which the alkoxy moiety has 1 to 10 carbon atoms, such as

benzyloxycarbonyl; (h) cycloalkyloxy-carbonyl in which the cycloalkyl moiety has 3 to 10 carbon atoms, such as cyclohexyl-oxycarbonyl; (i) aryloxycarbonyl in which the aryl moiety has 6 to 10 carbon atoms, such as

phenyloxycarbonyl; or (j) substituted aryloxycarbonyl in which the aryl moiety has 6 to 10 carbon atoms, such as tolyloxycarbonyl or xylyloxycarbonyl.

The present invention also provides

pharmaceutical/veterinary compositions comprising at least one amino acid derivative or peptide derivative of the general formula (I) or (IA) above, or a

physiologically acceptable salt thereof, in associat ion with one or more non-toxic physiologically acceptable carriers.

The present invention further provides a method for inhibiting the growth or metastasis of tumors, the degradation of tissues in arthritis or epidermolysis bullosa, or the destruction of lung tissue in emphysema, which comprises administering to an animal, including humans, suffering from any one of such conditions, an effective amount of at least one compound of the general formula (I) or (IA) above, or a physiologically

acceptable salt thereof, or a pharmaceutical/veterinary composition comprising at least one such compound or salt.

The amino acid derivatives or peptide derivatives defined by the general formula (I) or (IA) above, and physiologically acceptable salts thereof, may be produced by methods comprising: A: For the preparation of compounds when n or m is 1, as set out in the following reaction scheme A:

(i) reacting an amino acid of formula (II) with a source of an R₁ or R₅ protecting group such as di-t-butylcarbonate to form an amino acid of formula (III) so protected at its N-terminal; and

(ii) reacting a source of an optionally substituted hydrophobic hydrocarbon group of 6 to 16 carbon atoms, such as dodecylamine, with the carboxyl group of the amino acid of formula (III) to form the desired compound of formula (IV).

Reaction Scheme A

a source of an R ₁ or R₅ protecting

↓ group, such as di-t-butylcarbonate

a source of an R ₄ optionally

substituted hydrophobic hydrocarbon group of 6 to 16 carbon atoms , such

as dodecylamine

wherein R₆ represents R₁ or Re, and R₁, R₂, R₃, R₄ and R₅ are as defined in formulae (I) and (IA) above.

B: For the preparation of compounds when n or m is 2 to 6, as set out in the following reaction scheme B:

(i) reacting an amino acid or peptide of formula

(V) with a source of a protecting group such as an alcohol, exemplified by methanol, to form an amino acid or peptide of formula (VI) protected at its carboxyl terminal;

(ii) reacting an amino acid or peptide of formula (VII) with a source of an R₁ or R ₅ protecting group, such as di-t-butylcarbonate, to form an amino acid or peptide of formula (VIII), so protected at its N-terminal;

(iii) reacting the amino acid or peptide of formula

(VI) with the amino acid or peptide of formula (VIII) to form a peptide of formula (IX) with both the N-terminal and the carboxyl terminal of the peptide so protected;

(iv) removing the protecting group from the carboxyl terminal of the peptide of formula (IX); and (v) then reacting the peptide with a source of an optionally substituted hydrophobic hydrocarbon group of 6 to 16 carbon atoms, such as dodecylamine, to form the desired peptide of formula (X) with the required R₁ or R₅ protecting group at the N-terminal of the peptide and the required optionally

substituted hydrophobic hydrocarbon group at the carboxyl terminal of the peptide.

Reaction Scheme B f

removal of the R protecting group, followed by reaction with a source of an R₄ optionally substituted hydrophobic hydrocarbon group of 6 to 16 carbon atoms, such as ^ dodecylamine

(X)

wherein R₂, R₃, R₄ and R₆ are as hereinbefore defined; and a and b are integers of 1 to 5, provided that a + b is no greater than 6.

For the preparation of compounds of formula (IA) above, wherein m is 0, as set out in the following reaction scheme C:

(i) reacting a source of an optionally substituted hydrocarbon group of 6 to 16 carbon atoms (formula (XII)), such as dodecylamine, with a compound of formula (XI) to form the desired compound of formula (XIII)

R₅ - X + Y - R₄,

(XI) (XII

^R5 ^{" R}4

(xIII) wherein R₄ and R₅ are as hereinbefore

defined, and (a) X is hydroxy or a leaving group and Y is a leaving group or, if compound (XII) is an amine, alcohol or thiol, (b) X is a leaving group and Y is hydrogen. Thus, (a) compound (XII), terminating in an

activated amino or thiol group or in a leaving group, is condensed with compound (XI) having a free or activated terminal carboxyl group; or (b) compound (XI), having an activated terminal carboxyl group, is condensed with compound (XII), having a free terminal amino, hydroxy or thiol group.

Activation of the carboxyl group can take place, for example, by converting the carboxyl group into an acyl halide, azide, anhydride or imidazolide, or into an activated ester such as the cyanomethyl ester or p-nitrophenyl ester.

The amino group can be activated, for example, by converting the amino group into a phosphate amide.

Preferred embodiments of the invention

Preferred inhibitors defined by the general formula (I) above are those wherein R₁ is t- butyloxycarbonyl or benzyloxycarbonyl, more preferably, R₁ is t- butyloxycarbonyl; R₂ and R₃ are selected from

hydrogen, methyl, isopropyl and isobutyl, or R₂ and

R₃, taken together with the adjacent nitrogen and

carbon atoms, form a 5-membered ring of proline, more preferably R₂ and R₃ are selected from hydrogen,

methyl and isopropyl; R₄ is hydrocarbyl of 6 to 16

carbon atoms, more preferably 10 to 14 carbon atoms, most preferably 12 carbon atoms; and n is 1 to 4, more preferably 1 to 3; and physiologically acceptable salts thereof.

Particularly preferred inhibitors, defined by the general formula (I) above, are selected from the group N-tert-butyloxycarbonyl-valylamidyl decane,

N-tert-butyloxycarbonyl-valylamidyl dodecane,

N-tert-butyloxycarbonyl-valyl-valylamidyl decane,

N-tert-butyloxycarbonyl-valyl-valylamidyl dodecane,

N-tert-butyloxycarbonyl-valyl-valyl-valylamidyl decane, N-tert-butyloxycarbonyl-valyl-valyl-valylamidyl dodecane, N-tert-butyloxycarbonyl-alanylamidyl decane,

N-tert-butyloxycarbonyl-alanylamidyl dodecane,

N-tert-butyloxycarbonyl-alanyl-alanylamidyl decane,

N-tert-butyloxycarbonyl-alanyl-alanylamidyl dodecane, N-tert-butyloxycarbonyl-alanyl-alanyl-alanylamidyl decane, N-tert-butyloxycarbonyl-alanyl-alanyl-alanylamidyl

dodecane, N-tert-butyloxycarbonyl-alanyl-prolyl-va lylamidyl

decane ,

N-tert-butyloxycarbonyl-alanyl-prolyl-valylamidyl

dodecane ,

N-tert-butyloxycarbonyl-alanyl-alanyl-prolyl-valylamidyl decane ,

N-tert-butyloxycarbonyl-alanyl-alanyl-prolyl-valylamidyl dodecane ,

N-tert-butyloxycarbonyl-valylamidyl tetradecane,

N-tert-butyloxycarbonyl-phenylalanyl-amidyl dodecane,

N-tert-butyloxycarbonyl-leucylamidyl dodecane,

or a physiologically acceptable salt of any thereof.

The following compounds are potent inhibitors of

HLE. The rationale of these compounds is that the benzene residue binds to the S'₁ site of the enzyme, which has been shown to bind the p-nitroaniline group of the substrates very effectively.

Aryl and alkylaryl amides

Boc-Val-Ph,

Boc-Val-Ph-(CH₂)₉CH₃,

or a physiologically acceptable salt of any thereof

(wherein Ph is aniline).

Further preferred inhibitors of collagenase and/or human elastase are selected from the following alkyl esters :

Boc-Phe-O (CH ₂ ) _{1 1}CH ₃ , Boc-Val-Phe-O(CH₂)₉CH₃,

Boc-Pro-Leu-O(CH₂)₁₁CH₃,

Boc-Pro-Leu-Gly-O(CH₂)₁₁CH₃,

Boc-Pro-Gly-Leu-O(CH₂)₁₁CH₃,

Boc-Gly-Leu-O(CH₂)₁₁CH₃,

Boc-Gly-Pro-Leu-O(CH₂)₁₁CH₃,

Boc-Gly-Leu-Pro-O(CH₂)₁₁CH₃,

Boc-Ala-Pro-Val-O(CH₂)₁₁CH₃,

and the following alkyl, aryl and alkylaryl amides:

Boc-Val-Ph,

Boc-Val-Ph-CH₂CH₃,

Boc-Val-Phe-NH-(CH₂)₉CH₃,

Boc-Pro-Leu-NH-(CH₂)₁₁CH₃,

Boc-Pro-Leu-Gly-NH-(CH₂)₁₁CH₃,

Boc-Pro-Gly-Leu-NH-(CH₂)₁₁CH₃,

Boc-Gly-Leu-NH-(CH₂)₁₁CH₃,

Boc-Gly-Pro-Leu-NH-(CH₂)₁₁CH₃,

Boc-Val-SA/N,

Boc-Gly-Leu-Pro-NH-(CH₂)₁₁CH₃,

or a physiologically acceptable salt of any thereof

(wherein Boc is t-butyloxycarbonyl, Ph is aniline and

SA/N is 5-aminosalicylic acid).

Ethylaniline, decylaniline, aniline, and 5- aminosal icylic acid groups are joined to the adjacent amino acid residue via an amide linkage.

We have found that the amino acid valine is

important in the inhibition of the enzyme human elastase Therefore, the following compounds, and their

physiologically acceptable salts, are also of interest:

Alkyl esters

Boc-Val-O(CH₂)₁₁CH₃,

Boc-Val-O(CH₂)₁₃CH₃,

Boc-Val-Leu-O(CH₂)₁₁CH₃,

Boc-Gly-Val-O{CH₂)₁₁CH₃,

Boc-Pro-Val-Gly-O(CH₂)₁₁CH₃,

Boc-Val-Gly-Leu-O(CH₂)₁₁CH₃,

Boc-Gly-Val-Leu-O(CH₂)₁₁CH₃,

Boc-Val-Val-O(CH₂)₁₁CH₃,

Boc-Pro-Val-O(CH₂)₁₁CH₃,

Boc-Val-Leu-Gly-O(CH₂)₁₁CH₃,

Boc-Pro-Gly-Val-O(CH₂)₁₁CH₃,

Boc-Gly-Pro-Val-O(CH₂)₁₁CH₃.

Alkyl amides

Boc-Val-Leu-NH-(CH₂)₁₁CH₃,

Boc-Gly-Val-NH-(CH₂)₁₁CH₃,

Boc-Pro-Val-Gly-NH-(CH₂) ₁₁CH_3,

Boc-Val-Gly-Leu-NH-(CH₂)₁₁CH_3,

Boc-Gly-Val-Leu-NH-(CH₂)₁₁CH₃,

Boc-Pro-Val-NH-(CH₂)₁₁CH₃,

Boc-Val-Leu-Gly-NH-(CH₂)₁₁CH₃,

Boc-Pro-Gly-Val-NH-(CH₂)₁₁CH₃,

Boc-Gly-Pro-Val-NH-(CH₂)₁₁CH₃,

(In the above formulae, Boc is t-butyloxycarbonyl.) Another series of compounds, with the peptide sequence containing an aromatic amino acid residue (e.g. phenylalanine or tyrosine), are potential inhibitors of human cathepsin G (one of the three main enzymes involved in tissue degradation). Preferred inhibitors of human cathepsin G are:

Alkyl esters

Z-Ala-Phe-O(CH₂)₁₁CH₃,

Z-Ala-Tyr-O(CH₂)₁₁CH₃,

Alkyl amides

Z-Ala-Phe-NH-(CH₂)₁₁CH₃,

Z-Ala-Tyr-NH-(CH₂)₁₁CH₃,

or a physiologically acceptable salt of any thereof.

(In the above formulae, Z is benzyloxycarbonyl.)

Preferred inhibitors, defined by the general formula

(IA) above, are based on a substituted aromatic residue, e.g. salicylic acid (SA).

Examples are as follows:

Esters

SA-O(CH₂)₁₁CH₃,

SA-Gly-Val-O(CH₂)₁₁CH₃,

Amides

SA-NH-(CH₂)₁₁CH₃,

SA-Gly-Val-NH-(CH₂)₁₁CH₃,

or a physiologically acceptable salt of any thereof.

(In the above formulae, SA is salicyloyl.) Possible salts of compounds of the general formulae (I) and (IA) are all the acid addition salts. The

physiologically acceptable salts may be derived from inorganic or organic acids. Physiologically unacceptable salts, which may initially be obtained as process

products, for example in the preparation of the compounds according to the invention on an industrial scale, are converted into physiologically acceptable. salts by processes which are known to the skilled person. Examples of such suitable physiologically acceptable salts are water-soluble and water-insoluble acid addition salts, such as the hydrochloride, hydrobromide, hydriodide, phosphate, nitrate, sulfate, acetate, citrate, gluconate, benzoate, butyrate, sulfosalicylate, maleate, laurate, malate, fumarate, succinate, oxalate, tartrate, stearate, tosylate, mesylate and salicylate.

The most conventional methods for the condensation of the amino acid or peptide units of formulae (I) and (IA) are:

The carbodiimide method, the azide method, the anhydride method and the method of the activated esters, described in, for example, "THE PEPTIDES", Volume I, 1965 (Academic Press) by E. Schroder and K. Lubke. Furthermore the so-called "solid phase" method of Merrifield, described in J. Am. Chem. Soc. 85,2149 (1963), can be used for the manufacture of the present peptides. The free functional groups in the amino acid or peptide, which should not participate in the condensation reaction, are protected effectively by the so-called protecting groups, which can be removed again quite easily by hydrolysis or

reduction. Thus, for example, the carboxyl group can be protected effectively by, for example,

esterification with methanol, ethanol, tertiary butanol, benzyl alcohol or p-nitrobenzyl alcohol or by forming an amide. The latter group, however, is very difficult to remove so that it is to be

preferred to use it only to protect the terminal hydroxyl group in the ultimate peptide. The N- protecting groups are generally acid groups, for example, an acid group derived from an aliphatic, aromatic, araliphatic or heterocyclic acid such as acetic acid, chloro-acetic acid, butyric acid, benzoic acid, phenyl-acetic acid, pyridine- carboxylic acid, or an acid group derived from carbonic ac id such as ethoxy-carbonyl , benzyloxy- carbonyl , t-butyloxy-carbonyl or p-methoxy- benzyloxy-carbonyl, or an acid derived from a sulphonic acid such as benzenesulfonyl or p-toluene- sulfonyl, but other groups, too, can be used, such as substituted or unsubstituted aryl or aralkyl groups, for example, benzyl and triphenyl methyl. The guanidine group of arginine should preferably be protected by a nitro group, while the imino group of histidine should preferably be protected by a benzyl or trityl group. Generally it is preferred to use a tertiary butylester to protect the carboxyl group and a butyloxy-carbonyl or benzyloxy-carbonyl group or a tosyl group to protect the amino group.

The protecting groups can be split off by various conventional methods dependent upon the nature of the relative group, for example, by means of trifluoro-acetic acid or by mild reduction, for example with hydrogen and a catalyst such as palladium, or with HBr in glacial acetic acid.

The preferred amino acid derivatives or peptide derivatives within the general formulae (I) and (IA) above, and physiologically acceptable salts thereof, may be produced by methods comprising:

A: for the preparation of compounds when n or m is 1:

(i) reacting an amino acid such as alanine or valine with a source of an R₁ or R₅

protecting group such as di-t-butylcarbonate to form an amino acid so protected at its N-terminal, such as t-butyloxycarbonyl alanine or t-butyloxycarbonyl valine; and (ii) reacting a source of an optionally substituted hydrophobic hydrocarbon group of 6 to 16 carbon atoms, such as dodecylamine, with the carboxyl group of the amino acid of A (i) above to form the desired compound;

B : for the preparation of compounds when n or m is 2:

(i) reacting an amino acid such as valine with a source of a protecting group such as an alcohol, exemplified by methanol, to form an amino acid protected at its carboxyl terminal, exemplified by valine methyl ester;

(ii) reacting an amino acid such as valine with a source of an R₁ or R₅ protecting group, such as di-t-butylcarbonate, to form an amino acid so protected at its N-terminal, exemplified by t-butyloxycarbonyl valine;

(iii) reacting the product of B (i) above with the product of B (ii) above to form a dipeptide with both the N-terminal and the carboxyl terminal of the dipeptide so protected;

(iv) removing the protecting group from the

carboxyl terminal of the dipeptide of B (iii) above; and

(v) reacting the dipeptide of B (iv) above with a source of an optionally substituted hydrophobic hydrocarbon group of 6 to 16 carbon atoms, such as dodecylamine, to form the desired dipeptide with the required R₁ or R₅ protecting group at the N-terminal of the dipeptide and the required optionally substituted hydrophobic hydrocarbon group at the carboxyl terminal of the dipeptide;

C: for the preparation of compounds when n or m is 3:

(i) reacting a dipeptide such as valylvaline with a source of a protecting group such as an alcohol, exemplified by methanol, to form a dipeptide protected at its carboxyl terminal, exemplified by valylvaline methyl ester;

(ii) reacting an amino acid such as valine with a source of an R₁ or R₅ protecting group

such as di-t-butylcarbonate, to form an amino acid so protected at its N-terminal, exemplified by t-butyloxycarbonyl valine;

(iii) reacting the product of C (i) above with the product of C (ii) above to form a tripeptide with both the N-terminal and the carboxyl terminal of the peptide so protected;

(iv) removing the protecting group from the

carboxyl terminal of the tripeptide of C (iii) above; and

(v) reacting the tripeptide of C ((v) above with a source of an optionally substituted hydrophobic hydrocarbon group of 6 to 16 carbon atoms, such as dodecylamine, to form the desired tripeptide with the required R₁ or R₅ protecting group at the N-terminal of the tripeptide and the required optionally substituted hydrophobic hydrocarbon group at the carboxyl terminal of the tripeptide, or alternatively:

(vi) reacting an amino acid such as valine with a source of a protecting group such as an alcohol, exemplified by methanol, to form an amino acid protected at its carboxyl terminal, exemplified by valine methyl ester;

(vii) reacting a dipeptide such as valylvaline with a source of an R₁ or R₅ protecting group, such as di-t-butyl-carbonate, to form a dipeptide so protected at its N-terminal, exemplified by t-butyloxycarbonylvalylvaline;

(viii) reacting the product of C (vi) above with

the product of C (vii) above to form a tripeptide with both the N-terminal and the carboxyl terminal of the tripeptide so protected;

(ix) removing the protecting group from the

carboxyl terminal of the tripeptide of C (viii) above; and (x) reacting the tripeptide of C (ix) above with a source of an optionally substituted hydrophobic hydrocarbon group of 6 to 16 carbon atoms, such as dodecylamine, to form the desired tripeptide with the required R₁ or R₅ protecting group at the N-terminal of the tripeptide and the required optionally substituted hydrophobic hydrocarbon group at the carboxyl terminal of the tripeptide;

D: for the preparation of compounds when n or m is 4:

(i) reacting a tripeptide such as valylvalylvaline with a source of a protecting group such as an alcohol, exemplified by methanol, to form a tripeptide protected at its carboxyl terminal, exemplified by valylvalylvaline methyl ester;

(ii) reacting an amino acid such as valine with a source of an R₁ or R₅ protecting group

such as di-t-butylcarbonate, to form an amino acid so protected at its N-terminal, exemplified by t-butyloxycarbonyl valine;

(iii) reacting the product of D (i) above with the product of D (ii) above to form a tetrapeptide with both the N-terminal and the carboxyl terminal of the tetrapeptide so protected; (iv) removing the protecting group from the carboxyl terminal of the tetrapeptide of D (iii) above; and (v) reacting the tetrapeptide of D (iv) above with a source of an optionally substituted

hydrophobic hydrocarbon group of 6 to 16 carbon atoms, such as dodecylamine, to form the desired tetrapeptide with the required

R₁ or R₅ protecting group at the N- terminal of the tetrapeptide and the required optionally substituted hydrophobic hydrocarbon group at the carboxyl terminal of the

tetrapeptide,

or alternatively:

(vi) reacting an amino acid such as valine with a source of a protecting group such as an alcohol, exemplified by methanol, to form an amino acid protected at its carboxyl terminal, exemplified by valine methyl ester;

(vii) reacting a tripeptide such as valylvalylvaline with a source of an R₁ or R₅ protecting group, such as di-t-butylcarbonate, to form a tripeptide so protected at its N-terminal, exemplified by t-butyloxycarbonylvalylvalylvaline;

(viii) reacting the product of D (vi) above with the product of D (vii) above to form a

tetrapeptide with both the N-terminal and the carboxyl terminal of the tetrapeptide so protected; (ix) removing the protecting group from the

carboxyl terminal of the tetrapeptide of D

(viii) above; and

(x) reacting the tetrapeptide of D (ix) above with a source of an optionally substituted

hydrophobic hydrocarbon group of 6 to 16 carbon atoms, such as dodecylamine, to form the desired tetrapeptide with the required R₁ or R₅ protecting group at the N- terminal of the tetrapeptide and the required optionally substituted hydrophobic hydrocarbon group at the carboxyl terminal of the

tetrapeptide,

or, alternatively:

(xi) reacting a dipeptide such as valylvaline with a source of a protecting group such as an alcohol, exemplified by methanol, to form a dipeptide protected at its carboxyl terminal, exemplified by valylvaline methyl ester;

(xii) reacting a dipeptide such as valylvaline with a source of an R₁ or R₅ protecting group such as di-t-butylcarbonate, to form a

dipeptide so protected at its N-terminal, exemplified by t-butyloxycarbonyl valylvaline; (xiii) reacting the product of D (xi) above with the product of D (xii) above to form a tetrapeptide with both the N-terminal and the carboxyl terminal of the peptide so protected;

(xiv) removing the protecting group from the

carboxyl terminal of the tetrapeptide of D (xiii) above; and

(xv) reacting the tetrapeptide of D (xiv) above with a source of an optionally substituted hydrophobic hydrocarbon group of 6 to 16 carbon atoms, such as dodecylamine, to form the desired tetrapeptide with the required R₁ or R₅ Protecting group at the N- terminal of the tetrapeptide and the required optionally substituted hydrophobic hydrocarbon group at the carboxyl terminal of the

tetrapeptide.

In carrying out the methods set out immediately above, the source of the optionally substituted

hydrophobic hydrocarbon group of 6 to 16 carbon atoms is preferably an alkylamine or alkenylamine of 8 to 16 carbon atoms.

The compounds according to the invention are

initially obtained either as such or as their salts, depending on the nature of the starting compounds and depending on the reaction conditions. Moreover, salts are obtained by dissolving the free compounds in a suitable solvent, for example in a chlorinated hydrocarbon, such as methylene chloride or chloroform, or a low-molecular weight aliphatic alcohol (ethanol or isopropanol), which contains the desired acid or base, or to which the desired acid or base is subsequently added, if necessary in the precisely calculated stoichiometric amount. The salts are isolated by filtration, reprecipitation or precipitation or by evaporation of the solvent.

Resulting salts can be converted into the free compounds by treatment with bases or acids, for example, with aqueous sodium bicarbonate or with dilute

hydrochloric acid, and the compounds can in turn be converted into their salts. By this means, the compounds can be purified, or physiologically unacceptable salts can be converted into physiologically acceptable salts. Practical Embodiments of the Invention

The following non-limitative practical examples are provided as illustrative of the preparation of the compounds of the invention:

Example 1

Preparation of t-butyloxycarbonyl-L-valyl-L-valylamido- dodecane

(a) Valine methyl ester hydrochloride (Val-OMe HCl)

Valine (11.7g, 0.1 mole) was suspended in

100ml dry MeOH in a condenser to which was added dropwise thionyl chloride (15ml, 0.2 mole). The solution was refluxed for 6 hrs and the solvent evaporated to yield a white solid in 98% yield

(13. Ig) mp. 174-175°C. ¹H nmr (DMSO-d₆) δ: C- α -Val (1H, 3.9, q), C-β-Val (1H,2.4, m), C-γ-Val (6H, 1.0, d), OCH₃ (3H, 3.9, s).

(b) t-Butyloxycarbonyl-L-valine (Boc-Val)

Valine (11.7g, 0.1 mole) was dissolved in 300ml of dioxane/water (2:1) and 100 ml IM NaOH. The solution was cooled to 5°C and di-t-butylcarbonate (24g, 0.11 mole) was added over a period of 10 min. The solution was then stirred at room temperature for 3 hrs.

The dioxane was evaporated and the aqueous solution was ice-cooled and mixed with 75ml ethyl acetate. The mixture was acidified to pH 2-3 with 0.5M KHSO₄. The organic layer was separated and the aqueous layer was extracted with a further 2 x 60ml ethyl acetate. The combined organic layer was dried (Na₂SO₄) and the solvent evaporated to

yield a light yellow oil. The oil was left at 4°C to crystallize in 76% yield (16.5g ) mp. 76-78°C. 1H nmr (DMSO-d₆) 5: C-α-Val (1H, 3.9 q), C-β-Val (1H, 2.4, m), C-γ-Val (6H, 1.0 q), (CH₃)₃(9H,

1.4, s). (c) t-Butyloxycarbonyl-L-valyl-L-valine-methyl ester (Boc-Val-Val-OMe)

Boc-Val (lg, 4.6 mmole ) was dissolved in 20ml dry THF and triethy1 amine (0.7ml, 5.0 mmole). The solution was cooled to -10°C with stirring before the addition of ethyl chloroformate (0.48ml, 5.0 mmole). The solution was kept at -10°C for 20 min with continuous stirring. To this solution was added, dropwise, a solution of Val-OMe (0.655g, 5.0 mmole) in 15ml THF, 2ml H₂O and triethyl amine

(0.7ml, 5.0 mmole). The solution was then stirred for 15 hrs at room temperature. The solution was then placed in a separating funnel and 70ml ethyl acetate and 25ml H₂O were added. The organic layer was collected and the aqueous layer was extracted with a further 2 x 40ml of ethyl acetate. The combined organic layers were then washed with 30ml of IM HCl, H₂O, sat.NaHCO₃ and H₂O, dried

(Na₂SO₄) and the solvent evaporated to yield a white solid, 85% yield (1.29g) mp. 132-134°C. ¹H nmr (DMSO-d₆) δ : C-α-Val (2H, 3.9, m), C-β-Val

(2H, 2.5, m), C- γ-Val (12H, 0.9, d), O-CH₃ (3H, 3.9, s), (CH₃)₃ (9H, 1.4, s), NH-1 (1H, 7.0,

broad), NH-2 (1H, 8.2, broad).

(d) t-Butyloxycarbonyl-L-valyl-L-valine (Boc-Val-Val-OH)

Boc-Val-Val-OMe (1.19g, 3.6 mmole) was

dissolved in 30ml dioxane. With stirring at room temperature was added 10ml IM NaOH, dropwise. The solution was then stirred for 5 hrs at room

temperature. The solution was acidified with

concentrated HCl and extracted with 3 x 40ml of ethyl acetate, dried (Na₂SO₄) and the solvent evaporated to yield a white solid. 95% yield (1.09g) mp. 144-147°C. ¹H nmr (DMSO-d₆) δ: C-α-Val

(2H, 4.0, m), C-β-Val (2H, 2.4, m), G-γ-Val (12H, 0.9, d),(CH₃)₃ (9H, 1.4, s), COOH (1H, 11.2,

broad), NH-1 (1H, 6.9, broad), NH-2 (1H, 7.8, broad).

(e ) t-Butyloxycarbonyl-L-valyl-L-valylamido dodecane

(Boc-Val-Val-NH (CH₂)₁₁CH₃)

Boc-Val-Val-OH (0.3g, 0.95 mmole) was dissolved in 20ml dry THF and triethyl amine

(0.14ml, 1.0 mmole). The solution was cooled to -10°C before ethyl chloroformate (0.1ml, 1.0

mmole) was added. The solution was then stirred for 20 min at -10°C before a solution of dodecyl amine (0.185g, 1.0 mmole) dissolved in 20ml THF and triethylamine (0.14ml, 1.0 mmole) was added

dropwise. The solution was then stirred at room temperature for 15 hrs. The solvent was then

evaporated to yield a white solid. This was

dissolved in 75ml ethyl acetate and 25ml 5%

NaHCO₃. The organic layer was washed with 30ml

H₂O, IM HCl and H₂O, dried (Na₂SO₄) and the solvent evaporated to yield a white solid.

Recrystallized from ethyl acetate/hexane. 94% yield (0.43g) mp. 108-110°C. ¹H nmr (DMSO-d₆) δ : C-α -Val (2H, 4.0, m), C-β-Val (2H, 2.5, m), C-γ-Val (12H, 0.9, d), (CH₃)₃ (9H, 1.4, s), C-1 (2H,

3.0, t), CH₂'s (20H, 1.3, s), C-T (3H, 1.0, s), NH-1 (1H, 6.9, broad), NH-2 (2H, 7.8, broad), analysis: C₂₇H₅₃N₃O₄ requires C, 67.04; H,

11.04; N, 8.69; found: C, 67.21; H, 11.22; N, 8.66. Example 2

Preparation of t-Butyloxycarbonyl-L-alanylamido dodecane (t-Boc-Ala-NH(CH₂ ) _{1 1}CH₃)

t-Boc-Ala-OH(0.95g, 5 mmoles), and dodecylamine (2.8g, 15 mmoles), was stirred at

-5°C for 8 minutes (activation time) in THF (12.5 mis). After allowing the solution to warm to room temperature, the solvent was removed under vacuum. To the white crystalline solid was added ethyl acetate (75mls) and 5% NaHCO₃ (25mls). Following thorough shaking and then separation, the ethyl acetate layer was washed with water (25mls), lM.HCl(25mls), and finally with water (25nrtls). After drying over anhydrous Na₂SO₄, evaporation of the ethyl acetate yielded a white crystalline solid (1.25g, 70%) mp 67-68°C. Recrystallizing solvent: ethyl acetate/hexane (1:3), 'Hnmr(CDCl₃)δ: C-α -Ala (1H,4.1q), t-CH₃ (9H , 1.44s), C-1 (2H, 3.29),

CH₂'s(20H, 1.33, broad s), C-T (3H, 0.9t), N-H(1H, 5.3d, 6.5 broad), IR(KBr ) (cm^-1 ) N-Hstr (3330,

3350), Amide I (1600, 1695), Amide II (1525), C-N (1260), C-Hstr (2900-3000), C-13nmr (CDCl₃)ppm C-α -Ala (49.93), C-β-Ala (18.59), CONH (172.64,

173.54), C-O (155.47),C-1 (31.78), C-T (13.97), CH₂'s (29.52). Anal. Calc. C₂₀H₃₉N₂O₃

required C, 67.56, H, 11.06, N, 7.87; found C, 67.54, H, 11.40, N, 7.65.

Example 3

Preparation of compounds with benzyloxy as the protecting group

(a ) Benzyloxycarbonyl-L-alanylamido dodecane (Z-Ala- NH(CH₂)₁₁CH₃)

Z-Ala-OH (1.1g, 5 mmoles) and dodecylamine (2.8g, 15 mmoles), under the reaction conditions of Example 2, yielded a white crystalline solid (1.5g, 77%), mp 78-80°C. Recrystall izing solvent: ethyl acetate/hexane 'Hnmr (CDCl₃)δ :C-α-Ala (1H, 4.1 q), C-β-Ala (3H, 1.43 d), C-1 (2H, 3.2 t), CH₂'s (20H, 1.38 broad s), C-T (3H, 0.96 t), CH₂ ( 2H , 5.1 s), phenyl (H) (5H, 7.3 s), IR( KBr ) (cm^-1 ) N-Hstr

(3300), C-Hstr (2900-3000), Amide I (1655, 1700), Amide II (1550), C-N (1270) C-13nmr (CDCl₃)ppm C- α-Ala (50.51), C-β-Ala (18.77), CONH (172.23, 171.85), C-O (156.03), phenyl (C) (128.47, 127.13, 127.91, 136.21), HN-Cl (66.86), CT (14.05), CH₂'s (29.59).

(b) Benzyloxycarbonyl-L-valyl-L-alanylamido dodecane (Z- Val-Ala-NH (CH₂)₁₁CH₃)

Z-Val-Ala-OH (0.6g, 3.7 mmoles) and dodecylamine (1.05g, 11 moles), under the reaction conditions described in Example 2, yielded a white crystalline solid (0.35g, 34%) mp 46-48°C.

Recrystallizing solvent : ethyl

acetate/hexane, 'Hnmr (CDCl₃)δ: C-cd-Ala (1H, 4.1 g), C- α-Val (1H, 4.7 m), C-5-Val (6H, 1.0 d, 0.98 d), CH₂-O (2H, 5.07 s), phenyl (H) (5H, 7.24 s), C-1 (2H, 3.2 t), C-T (3H, 0.93 t), CH₂'s (20H, 1.33 broad s), IR (KBr)(cm^-1) N-Hstr (3300), C-

Hstr (2900-3000), Amide I (1645, 1695, 1750), Amide II (1540), C-N (1260).

(c) Benzyloxycarbonyl-L-valylamido dodecane (Z-Val-NH- (CH₂)₁₁ CH₃)

Z-Val-OH (1.26 g, 5 mmoles) and dodecylamine

(2.8 g, 15 mmoles), under the reaction conditions described in Example 2, yielded a white crystalline solid (1.36 g, 65%), mp 90-92°C. Recrystallizing solvent : ethyl acetate/hexane. 'Hnmr (CDCl₃)δ : C-α- Val (1H, 4.0 d), C-β-Val (1H, 2.0m), C-γ-Val (6H, 0.98 d, 0.91 d), CH₂-O (2H, 5.0 s), phenyl (H) (5H, 7.2 s), C-1 (2H, 3.16 t), IR( KBr ) (cm^-1 ) N-Hstr (3300), C-Hstr (2900-2980), Amide I (1650, 1695), Amide II (1550), C-N (1255) C-13nmr (CDCl₃)ppm C- α-Val (60.58), C-β-Val (31.07), C-γ-Val (19.17, 17.98), phenyl (C)(128.39, 128.03, 127.81, 140.51), HN-Cl (66.83), C-T (14.03), CONH (171.21, 156.49). (d ) Benzyloxycarbonyl-L-valyl-L-valylamido dodecane (Z- Val-Val-NH-(CH₂)₁₁CH₃)

Z-Val-Val-OH (0.91g, 2.6 mmoles), and

dodecylamine (1.4g, 7.8 mmoles), under the reaction conditions described in Example 2 , yielded a white crystalline solid (0.89g, 70%) mp 134-136°C.

Recrystallizing solvent : ethyl acetate/hexane

'Hnmr(CDCl₃)δ: C-α-Val (1H, 4.0 m), C-β-Val (2H, 2.18m), C-γ-Val (12H, 0.98 d, 0.91 broad s), CH₂-O

(2H, 4.96 s), phenyl(H) (5H, 7.1s), C-1 (2H, 3.1 t), C-T (3H, 0.71 t), IR(KBr) (cm^-1) N-Hstr (2995), C- Hstr (2900-2980), Amide I (1640, 1695, 1725), Amide II (1565), C-N-(1260). C-13nrnr (CDCl₃ ) ppm C- α- Val(60.58, 58.91), C-β-Val ( 31.12, 31.90), C-V-Val (17.89, 18.07, 19.23, 19.35), phenyl (C) (128.47, 128.08, 127.89, 132.26), CONH (172.06, 171.25

171.19), C-O (156.69), NH-Cl (66.95), C-T (14.07), CH₂'s (29.62).

I Analytical techniques

To aid in analysis of the present compounds in biological fluids, it was necessary to develop a method to monitor their Dresence in solution It was shown that these compounds had a λ_max at

210 nm. Subsequently the extinction coefficients for a series of the compounds were determined at 210 nm.

II Solubility studies

In preparing compounds for pharmacological testing, it was necessary to determine the solubility of such compounds in solution. The solution chosen was the Krebs solution. This buffer is the standard solution used in tissue culture experiments due to the similarity in the amount and type of salts used in the buffer and those present in biological fluid.

Testing was carried out on two compounds :Boc-Val- NH(CH₂)₁₁CH₃ and Boc-Val-Val-NH(CH₂)₁₁CH₃.

The amount of solubility was determined by the filtration method. Basically, a known excess of compound is added to a known volume of the buffer solution, then the mixture is shaken for 6 hours at various temperatures, filtered and weighed. The amount of sample collected is

proportional to the amount that dissolved in the

solution. In each experiment, approximately 15 mg of sample was added to 50 ml of buffer.

At room temperature (20°C), both of the above compounds were found to be insoluble in the buffer. At physiological temperature (37°C) it was found that Boc- Val-Val-NH(CH₂)₁₁CH₃ was slightly soluble (5.4

mg/litre). At 100°C, all 15 mg (300 mg/litre) of both compounds were solubilized in 50 ml Krebs Buffer. Due to the long period of time required to determine the solubility of the present compounds in Krebs buffer by the filtration method, it was decided to use the technique developed in Part I (i.e. UV spectroscopy). The method still involved shaking a known amount of sample in a known volume of buffer at 37°C for at least 48

hours. After this time the solution was filtered and an aliquot of the filtrate was extracted with an organic solvent , evaporated , and redissolved in methanol . The absorbance of this solution was subsequently measured at 210 nm against methanol as reference.

The solubility of some of the compounds measured using the above technique is as follows:

Boc-Val-C12 24.24 mg/litre

Boc-Val-Val-C12 8.70 mg/litre

Boc-Ala-Pro-Val-C12 1.76 mg/litre

The compounds according to the present invention are very hydrophobic and therefore not expected to be soluble in aqueous media to any great extent. Of course, this is not a great problem as many drugs currently on the market are practically insoluble in water. Solubiliz tion studies indicate that biologically compatible detergents e.g. Brij-35 or small amounts of harmless organic

solvents, such as alcohol, can solubilize these compounds sufficiently for a successful formulation to be achieved. At any rate oral or transdermal applications do not present problems on the basis of present indications. To increase the amount of the compounds of the present invention which can be solubilized into aqueous solution, the use of detergents was investigated. Upon the formation of micelles by the detergents, the

compounds would be incorporated within these complexes, hence increasing the amount of compound solubilized.

Before the above work could be performed, it was

necessary to determine at what concentration of detergent micelles were formed (i.e. critical micelle

concentration, CMC). Two non-ionic detergents were investigated, i.e. Brij-35 (a polyoxyethylene alcohol) and Triton X-100 (polyethylene glycol p-isooctylphenyl ether). The CMC for each of these detergents was

subsequently determined.

Results:

detergent CMC (μM )

Brij-35 45

Triton X-100 200

It was found that, to achieve 95-100% micelles in solution, Brij-35 required a concentration of at least 100 μM, while Triton X-100 required a concentration of 400μM or greater. Preliminary results of the present compounds, in the presence of these detergents, indicate a dramatic increase in their solubility in an aqueous solution. Other methods of solubilizat ion have also been investigated. To aid in the solubility of the present compounds for in vitro pharmacological testing, the use of an organic solvent/aqueous solution mixture has been investigated. If has been found that a solution

containing 1% methanol or dioxane (which would have little effect on the biological fluid) can contain a 10μM amount of sample without the compound precipitating or forming a suspension.

Studies have concentrated on Boc-Val-C12. The solubility of Boc-Val-C12 was measured in water in the presence of different concentrations of methanol or dioxane, and with or without the presence of Brij-35. It was found that at a concentration of 10μM of Boc-Val-C12 in the presence or absence of Brij-35, the compound is soluble in concentration of less than 0.5% methanol or dioxane. Present studies on the stability of Boc-Val-C12 (10/μM) in 1% methanol or dioxane with or without Brij-35 have shown that the compound is stable for over 5 days. No signs of degradation products have been detected. The result suggests that, in an aqueous solution, the alkyl peptides are stable.

Ill Inhibitory activity against proteolytic enzymes

(a) Elastase

Gelatin plates:

In our aim of developing potent inhibitors of the proteolytic enzymes, which degrade cartilage and tissue, methods of observing the activity of such compounds on these enzymes are required. To simulate the extracellular matrix found in the body, tissue culture plates containing a 5% gelatin solution (solidifying with time) were developed. Such a solid phase would mimic the extracellular matrix. The enzyme and drug are added to a well, and the amount of solid material degradation is proportional to the activity of the proteolytic enzymes. Preliminary results suggest that the present compounds are capable of arresting the advance of the enzyme

(Human Leukocyte Elastase) through the matrix, and therefore prevent degradation of the matrix

material.

Even though this assay is time-consuming, i.e. one week in duration, it does give some insight into the influence of these compounds against elastase in an environment mimicking connective tissue.

Results:

Boc-Val-C12 56% inhibition

Boc-Val-C14 33% inhibition

Boc-Val-Val-C12 67% inhibition

(all compounds were tested at 10μM ) .

Other connective tissue degrading enzymes

To determine the specificity of these type of compounds towards elastase, other connective tissue degrading enzymes were investigated, in particular human collagenase. This enzyme is involved in the degradation of collagen within connective tissue.

This enzyme has no commercially available chromophoric synthetic substrate, which is required for a spectrophotometric analysis of the activity of the enzyme. To undertake an analysis of this

enzyme's activity in the presence of our compounds, a new analytical procedure was developed. The method involves measuring the change in viscosity of a solution containing a known amount of the natural substrate of the enzyme, in the presence of the enzyme, and with or without our compounds.

The procedure was found to be effective to monitor collagenase activity. Using this procedure a preliminary study of several compounds was

undertaken. Below is a list of several compounds which were tested for activity against human

collagenase.

Compound Concentration % Inhibition

(μM )

Boc-Pro-Leu-Gly-C12 50 82

Boc-Gly-Leu-Pro-C12 50 44

Boc-Pro-Gly-Leu-C12 50 38 Boc-Val-Val -C 12 50 100

1 100

Boc -Val -C 12 50 68 Preliminary results appear to suggest a correlation between elastase and collagenase inhibitors. It appears that our compounds are able to control the activity of at least two of the most important enzymes involved in tissue degradation.

IV Stability testing

It has been shown that, in the solid state at least, the present compounds are stable for at least 4 months at 50°C.

V Pharmacology

Pharmacological testing has given outstandingly good results.

(a) Tracheal strip

None of the compounds tested had any measurable effect, indicating total lack of side-effects in the rat.

(b) Respiratory muscle

Rat diaphragm preparations gave no reaction with the compounds, indicating no measurable side-effects. (c) Intestinal movement

The isolated rat ileum showed absolutely no response to the compounds tested, indicating no detectable side-effects,

( d ) Toxicity

No LD₅₀ in rats was measurable, due to the

extremely low toxicity of the compounds. The

compounds were fed to the rats at dosage rates up to 5 gm/kg body weight (i.e. the highest dosage which the rats were physically capable of ingesting). At that dosage rate, there were still no deaths amongst the test* animals, and hence the LD₅₀ could not be calculated.

About 250 tests so far indicate a total lack of side-effects.

Comparative tests have indicated that the addition of small amounts of metal salts, especially copper salts, to compositions containing the compounds of the present invention is beneficial as an adjuvant.

Inhibitors in accordance with the present invention as set out above have been found not only to be very effective inhibitors of HLE and/or collagenase but also to be:

(a) substantially non-hydrolysed after incubation with human plasma for one hour at 37°C; and

(b) extremely non-toxic, the LD_{5 0} values of the

inhibitors being greater than 5 g/kg of body weight in rats.

Thus , the inh ibi tor compounds and sa lts of the invention can be expected to be useful in the treatment of HLE and collagenase implicated diseases such as arthritis, tumor growth and emphysema. The possibility of inhaling a selected inhibitor compound or salt thereof as an aerosol, in the treatment of emphysema, makes the exploitation of that area of use attractive. The invention thus also relates to a method of treating animals suffering from any of the above- mentioned diseases. The method is characterised in that a therapeutically active and physiologically acceptable amount of one or more of the inhibitors defined above is administered to the animal.

The invention also relates to pharmaceutical/ veterinary compositions which contain one or more of the inhibitors defined by the general formula (I) or (IA) and/or their physiologically acceptable salts.

The pharmaceutical/veterinary compositions are produced by processes which are known per se and with which the skilled person is familiar. The physiologically active compounds according to the invention are used either as such or, preferably, in combination with suitable pharmaceutical auxiliaries, in the form of tablets, dragees, capsules, suppositories, emulsions, suspensions or solutions.

The skilled person is familiar with the auxiliaries which are suitable for the desired pharmaceutical

formulations. Besides solvents, gelling agents,

suppository bases, tableting auxiliaries and other excipients for active ingredients, it is also possible to use, for example, antioxidants, dispersing agents, emulsifiers, anti-foaming agents, flavor correctants, preservatives, solubilizing agents and colorants. The optimum dosage and method of administration of the active compound required in each particular case can easily be determined by any skilled person.

If the compounds according to the invention and/or their salts are to be used for treatment of the above- mentioned diseases, the pharmaceutical formulations can also contain one or more physiologically active members of other groups of medicaments, such as steroidal. and/or non-s teroidal anti-inflammatory agents, immuno- suppressants, sulfated glycosamines/glycans and other sulfated carbohydrates, analgesics and antipyretics.

In clinical use, the inhibitor compounds and salts of the present invention may be administered orally, rectally, by injection or percutaneously, for example, by transdermal application for the treatment of arthritis, in the form of pharmaceutical preparations comprising at least one of said compounds or salts thereof in

association with a pharmaceutically acceptable carrier, which may be a solid or semi-solid or liquid diluent or capsule or aerosol applicator. Usually the active

substance will constitute between 0.1 and 99% by weight of a solid/semi-solid/ liquid preparation, more

particularly, between 0.5 and 20% by weight for

preparations suitable for oral administration.

Dosage unit pharmaceutical preparations containing at least one compound or salt thereof in accordance with the invention, for oral application, may be prepared by mixing the selected compound or salt with a solid

pulverulent carrier such as lactose, saccharose,

sorbitol, mannitol, starches such as potato starch, corn starch or amylopectin, cellulose derivatives, or

gelatine, and a lubricant such as magnesium stearate, calcium stearate or polyethylene glycol waxes, then compressed to form tablets. Coated tablets can be

prepared by coating the tablets, prepared as described above, with a concentrated sugar solution which may contain components such as gum arabic, gelatine, talcum, or titanium dioxide, or the tablet can be coated with a lacquer dissolved in a readily volatile organic solvent or mixture of organic solvents.

Soft gelatine capsules can be prepared "by enclosing the selected compound or salt, mixed with a vegetable oil, in a soft gelatine shell. Hard gelatine capsules may contain the selected compound or salt in admixture with solid, pulverulent carriers such as lactose, saccharose, sorbitol, mannitol, starches such as potato starch, corn starch or amylopectin, cellulose derivatives or gelatine.

Dosage unit preparations for rectal application can be prepared in the form of suppositories comprising the active substance in admixture with a neutral fatty base, or gelatine rectal capsules comprising the active substance in admixture with vegetable oil or paraffin oil. Liquid preparations for oral application can be in the form of syrups or suspensions, such as solutions containing from about 0.2% to about 20% by weight of the selected compund, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol.

Solutions for parenteral application by injection can be prepared as an aqueous solution of the selected compound or the selected compounds preferably in a concentration of from about 0.5% to about 10% by weight. These solutions may also contain stabilizing agents and/or buffering agents and may conveniently be provided in various dosage unit ampoules.

Suitable transdermal daily-dose administration of the selected compounds or salts in accordance with the invention can be 100-500 mg, preferably 200-300 mg, whilst weekly-dose administration can be in dosage of 25-2000 mg every 1 to 3 weeks.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A collagenase and/or elastase-type enzyme inhibitor compound consisting of an amino acid derivative or peptide derivative as defined by the general formula (I)

and physiologically acceptable salts thereof, wherein:

R₁ is a hydrocarbonyl-oxycarbonyl group with at least 4 carbon atoms, which may be substituted or

unsubstituted, for protecting the N-terminal of the amino acid or peptide, which protecting group, if cleaved in vivo, forms a physiologically innocuous compound;

R₂ and R₃, which may be the same or different and may differ from unit to unit when n is greater than 1, are independently selected from hydrogen, alkyl of 1 to 10 carbon atoms, substituted alkyl of 1 to 10 carbon atoms in which the substituents are those present in natural or non-natural amino acids, provided that they are physiologically innocuous, or R₂ and R₃ taken together with the adjacent nitrogen and carbon atoms may form a 5-membered ring with 4 carbon atoms, which ring may optionally be substituted by substituents which are present in natural or non-natural amino acids, provided that they are physiologically innocuous;

R₄ is an optionally substituted hydrophobic hydrocarbon group of 6 to 18 carbon atoms which, if cleaved in vivo, forms a physiologically innocuous compound, said

hydrophobic hydrocarbon group being bonded direct to the carbonyl carbon or to the carbonyl carbon through an oxygen, sulphur or nitrogen hetero atom; and

n is an integer from 1 to 6.

2. A compound according to Claim 1, wherein R₁ is of the formula R' -

- and R' is selected from

(i) straight-chain alkyl of 1 to 10 carbon atoms, straight-chain alkenyl of 1 to 10 carbon atoms, or straight-chain alkynyl of 1 to 10 carbon atoms, each of which is optionally substituted with one or more of alkyl of 3 to 10 carbon atoms, alkenyl of 3 to 10 carbon atoms, and alkynyl of 3 to 10 carbon atoms to form a branched-chain; or substituted with one or more of cycloalkyl of 3 to 10 carbon atoms, cycloalkenyl of 3 to 10 carbon atoms, aryl of 6 to 10 carbon atoms, adamantyl, or heterocyclic radical (s) such as pyryl, furyl, thiophenyl or pyrazolyl, any of which may be substituted, for example, with one or more of halo, hydroxy, nitro, alkyl of 1 to 5 carbon atoms, or alkoxy of 1 to 5 carbon atoms; or (ii) branched-chain alkyl of 3 to 10 carbon atoms, branched-chain alkenyl of 3 to 10 carbon atoms, branched-chain alkynyl of 3 to 10 carbon atoms, cycloalkyl of 3 to 10 carbon atoms, cycloalkenyl of 3 to 10 carbon atoms, aryl of 6 to 10 carbon atoms, adamantyl, or a heterocyclic radical such as pyryl, furyl, thiophenyl or pyrazolyl, any of which may be substituted, for example, with one or more of halo, hydroxy, nitro, alkyl of 1 to 5 carbon atoms, or alkoxy of 1 to 5 carbon atoms.

3. A compound according to Claim 1 or Claim 2 in which R₂ and R₃ may be the same or different, and are selected from hydrogen; alkyl of 1 to 10 carbon atoms, substituted alkyl of 1 to 10 carbon atoms in which the substituents are those present in natural or non-natural amino acids; or R₂ and R₃ taken together with the adjacent nitrogen and carbon atoms may form a 5-membered ring with 4 carbon atoms,

4. A compound according to Claim 3 in which R₂ and R₃ are independently selected from hydrogen, methyl, isopropyl and isobutyl.

5. A compound according to Claim 3 in which R₂ and R₃ taken together with the adjacent nitrogen and carbon atoms form the 5-merαbered ring of proline, which 5-membered ring may optionally be substituted by substituents which are present in natural or non-natural amino acids.

6. A compound according to any one of the preceding claims in which R₄ comprises 10 to 14 carbon atoms.

7. A compound according to Claim 6 wherein R ₄ comprises 12 carbon atoms.

8. A compound according to any one of Claims 5 to 7, in which R₄ is selected from alkyl, alkenyl, alkynyl, alkylamino, alkenylamino, hydrocarbonyloxy or hydrocarbonylthio,

optionally substituted with one or more groups selected from halo, hydroxy, carboxy, amino, nitro, alkyl of 1 to 5 carbon atoms, and alkoxy of 1 to 5 carbon atoms.

9. A compound according to Claim 5 in which R₄ is selected from the group consisting of octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl, octylamino,

nonylamino, undecylamino, decylamino, dodecylamino,

tetradecylamino, hexadecylamino, and optionally substituted phenylamino.

10. A compound according to any one of the preceding claims in which n is 1 to 3.

11. A method of inhibiting the activity of one or more enzymes selected from the group consisting of collagenase and elastase type enzymes, comprising the step of administering to an animal in need of such treatment an effective dose of at least one compound of general formula

n wherein R₁ is a hydrocarbonyl-oxycarbonyl group with at least 4 carbon atoms, which may be substituted or unsubstituted, for protecting the N-terminal of the amino acid or peptide, which protecting group, if cleaved in vivo, forms a physiologically innocuous compound, and has the structure

R', R₂, R₃ and R₄ are as hereinbefore defined, and n is an integer from 1 to 6 or a physiologically acceptable salt thereof, or a pharmaceutical or veterinary composition

comprising at least one such compound or salt.

12. A method according to Claim 11 in which R₁ is selected from the group consisting of (a) branched-chain alkylcarbonyl of 4 to 11 carbon atoms, such as

t-butylcarbonyl, isopentylcarbonyl or isohexylcarbonyl; (b) aryl substituted straight-chain alkylcarbonyl in which the alkyl moiety has 1 to 10 carbon atoms, such as benzylcarbonyl; (c) cycloalkylcarbonyl in which the cycloalkyl moiety has 3 to 10 carbon atoms, such as cyclohexylcarbonyl; (d) arylcarbonyl in which the aryl moiety has 6 to 10 carbon atoms, such as benzoyl; (e) substituted arylcarbonyl in which the aryl moiety has 6 to 10 carbon atoms, such as toluoyl, dimethylbenzoyl, hydroxybenzoyl and benzoyl substituted by amino and hydroxy groups; (f) branched-chain alkoxycarbonyl of 4 to 11 carbon atoms, such as t-butyloxycarbonyl, isopentyloxycarbonyl or isohexyloxycarbonyl; (g) aryl substituted straight-chain alkoxycarbonyl in which the alkoxy moiety has 1 to 10 carbon atoms, such as benzyloxycarbonyl; (h) cycloalkyloxy-carbonyl in which the cycloalkyl moiety has 3 to 10 carbon atoms, such as cyclohexyl-oxycarbonyl; (i) aryloxycarbonyl in which the aryl moiety has 6 to 10 carbon atoms, such as

phenyloxycarbonyl; or (j) substituted aryloxycarbonyl in which the aryl moiety has 6 to 10 carbon atoms, such as

tolyloxycarbonyl or xylyloxycarbonyl.

13. A method according to Claim 11 or Claim 12 in which the compound is selected from the group consisting of

N-tert-butyloxycarbσnyl-valylamidyl decane,

N-tert-butyloxycarbonyl-valylamidyl dodecane,

N-tert-butyloxycarbonyl-valyl-valylamidyl decane,

N-tert-butyloxycarbonyl-valyl-valylamidyl dodecane,

N-tert-butyloxycarbonyl-valyl-valyl-valylamidyl decane,

N-tert-butyloxycarbonyl-valyl-valyl-valylamidyl dodecane, N-tert-butyloxycarbonyl-alanylamidyl decane,

N-tert-butyloxycarbonyl-alanylamidyl dodecane,

N-tert-butyloxycarbonyl-alanyl-alanylamidyl decane

N-tert-butyloxycarbonyl-alanyl-alanylamidyl dodecane,

N-tert-butyloxycarbonyl-alanyl-alanyl-alanylamidyl decane, N-tert-butyloxycarbonyl-alanyl-alanyl-alanylamidyl dodecane,

N-tert-butyloxycarbonyl-alanyl-prolyl-valylamidyl decane

N-tert-butyloxycarbonyl-alanyl-prolyl-valylamidyl dodecane,

N-tert-butyloxycarbonyl-alanyl-alanyl-prolyl-valylamidyl decane,

N-tert-butyloxycarbonyl-alanyl-alanyl-prolyl-valylamidyl dodecane,

N-tert-butyloxycarbonyl-valylamidyl tetradecane

N-tert-butyloxycarbonyl-phenylalanyl-amidyl dodecane, and

N-tert-butyloxycarbonyl-leucylamidyl dodecane,

or a physiologically acceptable salt of any thereof.

14. A method for inhibiting the growth or metastasis of tumours, the degradation of tissues in arthritis or

epidermolysis bullosa, or the destruction of lung tissue in pulmonary emphysema or chronic obstructive lung disease, which comprises the step of administering to an animal, suffering from such a condition, an effective amount of at least one compound as defined in any one of Claims 1 to 13.

15. A method of manufacture of a medicament for the treatment of a condition selected from the group consisting of growth or metastasis of tumours, rheumatoid arthritis, osteoarthritis, epidermolysis bullosa, chronic obstructive lung disease, and pulmonary emphysema, comprising the step of incorporating as active ingredient a compound as defined in any one of Claims 1 to 13.

16. A method of synthesis of a compound of general formula I as defined in Claim 1, comprising the following steps:

A: For the preparation of compounds when n is 1, as set out in the following reaction scheme A:

(i) reacting an amino acid of formula (II) with a source of an R₁ protecting group such as di-t-butylcarbonate to form an amino acid of formula (III) so protected at its N-terminal; and (ii) reacting a source of an optionally

substituted hydrophobic hydrocarbon group of 6 to 16 carbon atoms, such as dodecylamine, with the carboxyl group of the amino acid of formula (III) to form the desired compound of formula (IV).

Reaction Scheme A

L

( IV)

and R₁, R₂, R₃ , R ₄ are as defined

in formula (I) above. For the preparation of compounds when n is 2 to 6, in the following reaction scheme B:

(i) reacting an amino acid or peptide of formula

(V) with a source of a protecting group such as an alcohol, exemplified by methanol, to form an amino acid or peptide of formula (VI) protected at its carboxyl terminal;

(ii) reacting an amino acid or peptide of formula

(VII) with a source of an R₁ protecting group, such as di-t-butylcarbonate, to form an amino acid or peptide of formula (VIII), so protected at its N-terminal;

(iii) reacting the amino acid or peptide of formula

(VI) with the amino acid or peptide of formula (VIII) to form a peptide of formula (IX) with both the N-terminal and the carboxyl terminal of the peptide so

protected;

(iv) removing the protecting group from the

carboxyl terminal of the peptide of formula (IX); and

(v) then reacting the peptide with a source of an optionally substituted hydrophobic

hydrocarbon group of 6 to 18 carbon atoms, such as dodecylamine, to form the desired peptide of formula (X) with the required R₁ protecting group at the N-terminal of the peptide and the required optionally

substituted hydrophobic hydrocarbon group at the carboxyl terminal of the peptide.

Reaction Scheme B

a source of an R₁ a source of a protecting protecting group, group (R), such as such as di-t-butyl- methanol

carbonate

a+b

removal of the R

protecting group, followed by reaction with a source of an R₄ optionally substituted hydrophobic

hydrocarbon group of 6 to

18 carbon atoms, such as

dodecylamine

(X)

wherein R₁, R₂, R₃ and R₄ are as hereinbefore defined; and a and b are integers of 1 to 5 provided that a + b is no greater than 6.

17. A pharmaceutical or veterinary composition

comprising at least one amino acid derivative or peptide derivative according to any one of the preceding claims, or a physiologically acceptable salt thereof, in association with one or more non-toxic physiologically acceptable carriers, excipients or diluents.

18. A composition according to Claim 17 which

additionally comprises a biocompatible detergent or

pharmaceutically acceptable organic solvent.

19. A composition according to Claim 18 which comprises one or more of Brij-35, Triton X-100, ethanol, dioxane or methanol.

20. A composition according to any one of Claims 17, 18 or 19 which additionally comprises a pharmaceutically

acceptable metal salt.

21. A composition according to Claim 20 in which the metal is copper or zinc.

22. A composition according to any one of Claims 17 to 21 which is suitable for oral, percutaneous or inhalable aerosol administration.

23. A method according to any one of Claim 11 to 13 in which both collagenase and an elastase-type enzyme are inhibited.

24. A compound according to any one of Claims 1 to 10 which inhibits both collagenase and elastase-type enzymes.