WO1988010258A1

WO1988010258A1 - Human leucocyte elastase inhibitor compounds

Info

Publication number: WO1988010258A1
Application number: PCT/AU1988/000155
Authority: WO
Inventors: Bela Ternai; Maria Luisa Cook
Original assignee: La Trobe University
Priority date: 1987-06-17
Filing date: 1988-05-24
Publication date: 1988-12-29
Also published as: EP0365539A4; EP0365539A1

Abstract

A compound of formula (III) or physiologically acceptable salts thereof, wherein: Z is a bond, -O- or -NH-; R1 and R3 are independently selected from C5-12 substituted of unsubstituted hydrocarbon radicals; R2 is hydrogen or a C1-5 substituted or unsubstituted hydrocarbon radical; said substituted hydrocarbon radicals are substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes; provided that: when R1 is oxohexyl and R2 is hydrogen, then R3 cannot be pentyl; when R1 is oxoheptyl and R2 is hydrogen, then R3 cannot be hexyl; when R1 is oxononyl and R2 is hydrogen, then R3 cannot be octyl; when R1 is oxodecyl and R2 is hydrogen, then R3 cannot be nonyl; and when R1 is oxododecyl and R2 is hydrogen, then R3 cannot be undecyl. These compounds are potent and specific inhibitor compounds fo elastase-type enzymes, especially for human leucocytic elastase (HLE) and can be expected to be useful in the treatment of elastase-type implicated diseases such as arthritis, tumor growth and emphysema. Production, method of use and pharmaceutical compositions containing these compounds are disclosed.

Description

HUMAN LEUCOCYTE ELASTASE INHIBITOR COMPOUNDS BACKGROUND OF THE INVENTION

Field of the Invention:

This invention provides potent and specific inhibitor compounds for elastase-type enzymes, especially for human leucocytic elastase (HLE), which enzyme is implicated in the growth of tumors, degradation of tissues in arthritis and in the destruction of lung tissues in emphysema, the molecular structure of the inhibitor compounds of the invention being 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:

Elastase-type enzymes, especially human leucocyte elastase (HLE), have been implicated in many inflammatory disease states such as pulmonary emphysema, acute arthritis and the destruction of connective tissue ^{1-4 .}

Thus, there is evidence to implicate the neutral proteases of human leucocytes (polymorphonuclear leucocytes) 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 α₁ - proteinase inhibitor

(α₁ -PI), which is also a normal constituent of bronchioal- veolar lavage fluid (BAL).

However, α₁-PI 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 α₁ -PI with levels of the inhibitor which are 25% of 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. Haveman & A. Janoff: "Neutral proteases of Human Polymorphonuclear Leukocytes", p. 221, Urban and Schwartzenberg, Baltimore (1977); the imidazole-N-carboxa- mides, 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: C.H. 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 thiomal- ate, 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 aza peptides, the cis- unsaturated 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 compounds bearing highly reactive functional groups are difficult, if not impossible, to target 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 inhibitors of HLE, but not of porcine pancreatic elastase (PPE), trypsin, chymotrypsin 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. Omura et al⁵ reported that elasnin (I), an alkylated

2-pyrone of microbial origin, was a good inhibitor of HLE.

(I)

Groutas et al¹⁷ , Spencer et al¹⁸ and Groutas et al¹⁹ disclose HLE inhibitors of the formula II

(II)

wherein: (a) in Groutas et al¹⁷, R¹ is hydrogen and R is methyl, propyl, heptyl or undecyl;

(b) in Spencer et al ¹⁸, R¹ is methyl when R is methyl or - CH₂ COPh, and R₁ is hydrogen when R is hydrogen or - CH₂ COPh; and

(c) in Groutas et al ¹⁹, R¹ is hydrogen when R is propyl, butyl, pentyl, hexyl or undecyl, and R¹ is

alkyl in which the alkyl group is ethyl, propyl, pentyl, undecyl or pentadecyl, when R is methyl.

Kogel et al ²⁹ studied the activity of pyrones of the formula (IIA):

(IIA).

wherein R¹ is pentyl, hexyl, octyl, nonyl or undecyl, when R is pentyl, hexyl, octyl, nonyl or undecyl, respectively, in hindering the growth of bacteria.

SUMMARY OF THE INVENTION

The present invention provides as potent and specific inhibitors of elastase-type enzymes, especially for HLE, a novel compound of the formula (III):

(III)

or physiologically acceptable salts thereof, wherein:

R₁ and R₃ are independently selected from hydrocarbon-chain radicals of 5 to 12 carbon atoms in length which are bonded directly to the pyrone ring or bonded indirectly to the pyrone ring through an oxygen or nitrogen atom, said hydro- carbon-chain radicals being independently unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes, the total number of carbon atoms of the longest chain length in each of the groups R₁ ana R₃ being 5 to 12; and R₂ is hydrogen or hydrocarbon radical of 1 to 5 carbon atoms unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes;

provided that: when R₁ is oxohexyl and R₂ is hydrogen, then R₃ cannot be pentyl; when R₁ is oxoheptyl and R₂ is hydrogen, then R₃ cannot be hexyl; when R₁ is oxononyl and R₂ is hydrogen, then R₃ cannot be octyl; when R₁ is oxo- decyl and R₂ is hydrogen, then R₃ cannot be nonyl; and when R₁ is oxododecyl and R₂ is hydrogen, then R₃ cannot be undecyl.

The present invention also provides a pharmaceutical or veterinary composition comprising as active ingredient at least one compound of formula (IV):

(IV)

or physiologically acceptable salts thereof, wherein:

R₁ and R₃ are independently selected from hydrocarbon-chain radicals of 5 to 12 carbon atoms in length which are bonded directly to the pyrone ring or bonded indirectly to the pyrone ring through an oxygen or nitrogen atom, said hydrocarbon-chain radicals being independently unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes, the total number of carbon atoms of the longest chain length in each of the groups R₁, and R₃ being 5 to 12; and

R₂ is hydrogen or hydrocarbon radical of 1 to 5 carbon atoms unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes; in association with a pharmaceutical or veterinary adjuvant or carrier.

The present invention further provides the use of an effective amount of at least one compound or physiologi- cally acceptable salt thereof according to formula (III), or a composition according to formula (IV), as an inhibitor for elastase-type enzymes.

The present invention still further provides a method for inhibiting the growth of tumors or the degradation of tissues in arthritis or the destruction of lung tissues in emphysema, which comprises administering to an animal suffering from any one or such conditions , a therapeutical ly effective amount of at least one compound or physiologically acceptable salt thereof according to formula (III), or a composition according to formula (IV).

PREFERRED EMBODIMENTS OF THE INVENTION

As generally indicated above, the groups R₁ and R₃ of the compounds of general formulae (III) and (IV) above may be independently selected from unsubstituted alkyl, alkenyl, alkynyl or alkoxy, of 5 to 12 carbon atoms, or alkyl, alkenyl, alkynyl or alkoxy, of 5 to 12 carbon atoms, substituted with alkyl, alkenyl, alkynyl, alkoxy, carboxy, oxo, amino, or other such physiologically innocuous substituents which do not interfere with the binding of said compounds with elastase-type enzymes.

Preferably, the groups R₁ and R₃ of the compounds of general formulae (III) and (IV) above may be independently selected from:

(i) alkyl, alkenyl or alkynyl of 5 to 12 carbon atoms, unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compounds with elastase-type enzymes, exemplified by oxoalkyl of 5 to 12 carbon atoms or oxoalkyl of 5 to 12 carbon atoms substituted with said physiologically innocuous substituents exemplified by

wherein n is zero or an integer 1 to 11 and R', is hydrogen or alkyl, alkenyl or alkynyl of 1 to 11 carbon atoms unsubstituted or substituted with alkyl, alkenyl, alkoxy, carboxy, oxo, amino, or other such physiologically innocuous substituents which do not interferewith the binding of said compounds with elastase-type enzymes, provided that the total number of carbon atoms of the longest chain length in the groups R₁ and R₃ is 5 to 12;

(ii) alkoxy of 5 to 12 carbon atoms, unsubstituted or substituted with physiologically innocuous substituents which do not interferewith the binding of said compounds with elastase-type enzymes, exemplified by

-

wherein n is zero or an integer 1 to 11 and R'' is hydrogen or alkyl, alkenyl or alkynyl of 1 to 11 carbon atoms unsubstituted or substituted with alkyl, alkenyl, alkoxy, carboxy, oxo, amino or other such physiologically innocuous substituents which do not interferewith the binding of said compounds with elastase-type enzymes, provided that the total number of carbon atoms of the longest chain length in the groups R₁ and R₃ is 5 to 12; and

(iii) hydrocarbon radicals of 5 to 12 carbon atoms bonded to the pyrone ring through a nitrogen atom, unsubstituted or substituted with physiologically innoc- uous substituents which do not interferewith the binding of said compounds with elastase-type enzymes, exemplified by

wherein n is zero or an integer 1 to 11 and R''' is hydrogen or alkyl, alkenyl or alkynyl of 1 to 11 carbon atoms unsubstituted or substituted with alkyl, alkenyl, alkoxy, carboxy, oxo, amino or other physiologically innocuous substituents which do not interfere with the binding of said compounds with elastase-type enzymes, provided that the total number of carbon atoms of the longest chain length in the groups R₁ . and R₃ is 5 to 12.

The group R₁ in the compounds of general formulae (III) and (IV) above is more preferably oxoalkyl of 5 to 12 carbon atoms, most preferably oxoalkyl of 7 to 10 carbon atoms.

The group R₃ in the compounds of general formulae (III) and (IV) above is more preferably alkyl of 5 to 12 carbon atoms, most preferably alkyl of 7 to 10 carbon atoms. The group R₂ in the compounds of general formulae (III) and (XV) above is preferably selected from hydrogen or alkyl, alkenyl or alkynyl, inclusive of straight and branched chain radicals and cyclic radicals, exemplified by methyl, ethyl, n-propyl, isopropyl, cyclopropyl, tertiary butyl, and n-butyl. The group R₂ in the compounds of general formulae

(III) and (IV) above is more preferably selected from hydrogen, C_1-5 alkyl, C_1-5 alkenyl, or C_1-5 alkynyl, most preferably from hydrogen or methyl.

Possible salts of compounds of the general formulae (III) and (XV) 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, hydroiodide, phosphate, nitrate, sulfate, acetate, citrate, gluconate, benzoate, butyrate, sulfosalicylate, maleate, laurate, malate, fumarate, succinate, oxalate, tartrate, stearate, tosylate, mesylate and salicylate.

Especially preferred compounds of the present invention are:

3-(1'- oxononyl)-4-hydroxy-6-octyl-2-pyrone;

3- (1'- oxodecyl)-4-hydroxy-6-nonyl-2-pyrone;

3- (1'- oxononyl)-4-hydroxy-6-nonyl-2-pyrone;

3-(1'- oxodecyl)-4-hydroxy-6-octyl-2-pyrone; 3-(1'- oxononyl)-4-methoxy-6-octyl-2-pyrone; 3-(1'- oxodecyl)-4-methoxy-6-nonyl-2-pyrone; 3-(1'- oxononyl)-4-methoxy-6-nony1-2-pyrone; 3-(1' - oxodecyl)-4-methαxy-6-octyl-2-pyrone; 3-(1'- oxononyl)-4-hydroxy-6-pentyl-2-pyrone; 3-(1'- oxononyl)-4-hydroxy-6-hexyl-2-pyrone; 3-(1'- oxononyl)-4-hydroxy-6-hepty1-2-pyrone; 3-(1'- oxodecyl)-4-hydroxy-6-pentyl-2-pyrone; 3-(1'- oxodecyl)-4-hydroxy-6-hexyl-2-pyrone; 3-(1'- oxodecyl)-4-hydroxy-6-heptyl-2-pyrone; 3-(1'- oxooctyl)-4-hydroxy-6-heptyl-2-pyrone; 3-(1'- oxooctyl)-4-hydroxy-6-octyl-2-pyrone; 3-(1 '- oxooctyl)-4-hydroxy-6-nonyl-2-pyrone; 3-(1 '- oxononyl)-4-methoxy-6-hexyl-2-pyrone; 3-(1'- oxononyl)-4-methoxy-6-heptyl-2-pyrone; 3-(1 '- oxodecyl)-4-methoxy-6-hexyl-2-pyrone; 3-(1'- oxodecyl)-4-methoxy-6-heptyl-2-pyrone; 3-(1 '- oxooctyl)-4-methoxy-6-octyl-2-pyrone; and 3-(1 '- oxooctyl)-4-methoxy-6-nonyl-2-pyrone.

As will be apparentfrom a reading of the subsequent description under the heading 'Practical Embodiments of the Invention', compounds of the general formula (III) and (IV) above may be prepared by: (A) reacting an appropriate acid imidazolide with an appropriate salt of an alkyl hydrogen malonate to form the corresponding 3-oxocarboxylate ester; hydrolysing said 3-oxocarboxylate ester to afford the corresponding 3-oxocarboxylic acid; and cyclizing said 3-oxocarboxylic acid with carbonyl- diimidazole to afford the corresponding 3-(1 ' -oxoalkyl)-4-hydroxy-6-alkyl-2-pyrones of the formula (V):

(V)

wherein R₁ and R₃ are independently alkyl of 5 to 12 carbon atoms in length and optionally substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes, the total number of carbon atoms of the longest chain length in each of the groups R₁ and R₃ being 5 to 12; and/or

(B) replacing by conventional techniques, the hydrogen of the hydroxy group at position-4 of the pyrone ring of formula (V), with a hydrocarbon radical of 1 to 5 carbon atoms unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes; and/or

(C) replacing by conventional techniques, the 1'-oxoalkyl and/or the alkyl, respectively, at positions -3 and

-6, of the pyrone ring of formula (V), with required hydrocarbon-chain radicals of 5 to 12 carbon atoms in length which are bonded as required directly to the pyrone ring or bonded indirectly to the pyrone ring through an oxygen or nitrogen atom, said hydrocarbon- chain radicals being independently unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes, the total number of carbon atoms of the longest chain length in each of the groups R₁ and R₃ being 5 to 12; and/or

(D) converting the compounds of formula (III) or formula (IV) above so obtained to a physiologically acceptable salt thereof.

A further preferred embodiment of the present invention comprises a process for the preparation of a compound of the formula (X):

(X)

or physiologically acceptable salts thereof, wherein:

R₁ and R₃ are independently alkyl of 5 to 12 carbon atoms in length, optionally substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes, the total number of carbon atoms of the longest chain length in the groups R₁, and R₃ being 5 to 12; and

R₂ is hydrogen or alkyl of 1 to 5 carbon atoms unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes;

which comprises reacting an appropriate acid imidazolide with an appropriate salt of an alkyl hydrogen malonate to form the corresponding 3-oxocarboxylate ester of formula (XI):

R₃COCH₂COOR (XI) wherein R₃ is as defined above and R is alkyl, then hydro- lysing the ester of formula (XI) to form the corresponding 3-oxocarboxylic acid of formula (XII):

R₃COCH₂COOH: (XII)

wherein R₃ is as defined above, followed by cyclizing the 3-oxocarboxylic acid of formula XII with carbonyldiimida- zole to form the corresponding 3-(1'-oxoalkyl)-4-hydroxy-6- alkyl-2-pyrone of the formula (Xi):

(XIII)

where R₁ and R₃ are as defined above, and optionally methylating the 4-hydrόxy group of the 3-(1'-oxoalkyl)-4- hydroxy-6-alkyl-2-pyrone of the formula (XIII), with dialkyl sulfate to form the corresponding 3-(1'-oxoalkyl)-4- alkyl-6-alkyl-2-pyrone of the formula (X), whereafter, if desired, the compound of 'formula (X) so obtained is converted to a physiologically acceptable salt thereof.

Thus, 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 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 stoichio- metric 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 treating 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, in which reference is made to compounds 1 to 19 of Table 1 below, are provided as illustrative of the present invention.

CHEMISTRY

The substituted 2-pyrones were prepared according to Scheme I. The 3-oxocarboxylate⁶ was formed by reacting an acid imidazolide (formed in situ) with the neutral magnesium salt of ethyl hydrogen malonate⁷. Hydrolysis of the ester in 1M NaOH afforded the 3-oxocarboxylic acid, which was cyclized using 1.1 eq. carbonyldiimidazole in THF⁸ to afford the 3-(1'-oxoalkyl)-4-hydroxy-6-alkyl-

2-pyrones (compounds 9-13). Deacylation was easily carried out⁹ by heating the 2-pyrone at 130ºC in 90% H₂SO₄, producing compounds 5-8. The 4-hydroxy group was methylated¹⁰ using dimethyl sulfate and anhyd. K₂CO₃ in pet. spirit (60-80°C), yielding compounds 14 and 15.

Acylation of 4-hydroxy-6-methyl-2-pyrone with the appropriate acid chloride in CF₃COOH yielded the desired 3-(1'-oxoalkyl)-4-hydroxy-6-methyl-2-pyrone¹¹

(compounds 1-4). The contaminating carboxylic acid was removed by dissolving the crude product in di- methylsulfoxide and adding lead acetate in dioxan. Addition of water precipitated the pure 2-pyrone only, which was collected by filtration.

4-Methoxy-6-methyl-2-pyrone was converted to the 6-carboalkoxyvinyl derivative by treatment with dialkyl oxalate in the presence of metallic sodium¹¹ (compounds

16-18). Acid hydrolysis of the ethyl derivative¹¹ (compound 16) yielded the carboxy derivative (compound 19). Compounds 17 and 18 were purified by triturating the crude brown oil with acetone, which yielded fine yellow crystals after filtration.

KINETIC ANALYSIS

Each compound was tested at 3 or more concentrations, at 5-7 different concentrations of substrate. To determine the mechanism of inhibition the data were plotted as [S]/v vs [S]¹², 1/v vs 1/[S]¹³, [s]/v vs I¹⁴ and 1/v vs

[ I] ¹⁵. To determine whether the inhibition was pure (inactive ESI complex or partial (active ESI complex), the data were plotted as 1/V_max i against [i]. If the plot was linear then the inhibitor exhibited pure inhibition: that is, the ESI complex is inactive, and hence cannot dissociate to El and P (β=O, Scheme II). For linear mixed-type inhibitors (K_sapp and V_maxi change) and pure non-competi- tive inhibitors (Vmaxi changes), a plot of K_s app/V_maxi vs [I] was linear and for pure uncompetitive inhibitors

(K_sapp/V_max i constant) a plot of 1/K_s app vs [I]was linear. A hyperbolic plot of 1/V_max i vs [I] indicated partial inhibition, with β<1 and ESI is active but less so than ES.

In this case a replot of ΔV_maxi vs 1/[I] gave a linear plot

(ΔV_maxi=(1/V_maxi-1/V_max)^-1). Using this plot and ΔV_max/K_s app vs 1/ [I] α, β & K_i could be calculated. All the equations and their derivations can be found in the excellent book by I.H. Segal¹⁶.

Results and Discussion There were three major aspects to this work: a) mapping of the binding region of HLE by determining structure/activity relationships using K_i and K_i' data (Scheme II), for the substituted 2-pyrones; b) determination of the mechanism of inhibition, followed by speculation as to where the binding region might lie; c) evaluation of these compounds as specific inhibitors of KLE, compared with related serine proteases.

The inhibitory activity of each compound, reported as K_i and K_i', are included in Table 1. K_i is of primary interest because it gives a measure of the affinity of I for E; however, for some inhibitors an ESI and not an El complex is formed therefore it is necessary to compare K_i' values.

The effect of substitution in the 3-position was evaluated using 3-(1'-oxoalkyl)-4-hydroxy-6-methyl- 2-pyrones (compounds 1-4), which have values of K_i ranging from 1995 to 54 μM. An increase in the number of methylenes was accompanied by a decrease in the magnitude of K_i; that is, an increase in hydrophobicity resulted in greater affinity between E and I. K_i did not reach a lower limit, so we can presume that the binding region is at least large enough to accomodate a 2-pyrone nucleus and a dodecyl group. Insolubility of the higher homologs in the assay medium prevented us from carrying out further studies on longer chain analogues. The effect of substitution in the 6 position was evaluated using 4-hydroxy-6-alkyl-2-pyrones (compounds 5-8) which have values of K_i ranging from 1709 to 83 μM. An increase in the number of methylenes was accompanied by an increase in affinity between E and I, indicating the binding region is hydrophobic. We have evidence from mechanistic data (vide infra) that indicates that the 3- and 6- substituents do not bind in the same region. Based on these results we conclude that the 4-hydroxy-2-pyrone binds to a particular region, and that there are two hydrophobic regions either side of the 2-pyrone binding region. Groutas et al ¹⁷ have suggested that hydrophobic groups in the 6- position are of primary importance, while Spencer et al ¹⁸ have argued that binding of the 3-substituent is of major importance. Our results indicate thathydrophobic substituents at either the 3 or 6 position can bind equally well to HIE.

Based on these results, compounds that featured a minimum of five methylene units at both the 3 and 6 posi- tions were therefore of great interest. Compounds 9-13 were prepared and found to have K_i's ranging from 67 to 4.6 uM. The observed K_i values are much lower than those observed for the analogous 3-(1'-oxoalkyl)- and 4-hydroxy-6- alkyl-2-pyrones. For example, compound 10 has a K_i of 15 μM, while compounds 2 and 6 have K_i's of 442 and 1128 μM, respectively. The K. has apparently improved because of the increased hydrophobicity and presumably because the 2-pyrone nucleus is held in the correct orientation by the two alkyl chains resulting in maximum binding. Groutas et al¹⁹ have shown that alkylated phenyl compounds do not bind to HLE, whereas the analogous 2-pyrone derivatives have considerable affinity for HLE. Saturated fatty acids have been shown to be inactive. These results indicate that both features are necessary for inhibition. Another interesting feature of the dialkyl compounds 9-13 is that, unlike the monoalkyl compounds 1-4 and 5-8, K_i does reach a limit. Based on the results for the long chain derivatives, compounds 4 and 8 and the assumption that the 4-hydroxy-2- pyrone binds to a specific region of HLE we would expect compound 13 to be the most potent compound. This was not observed; instead, the shorter chain homolog, compound 11 was the most potent. This gives an upper limit to the length of the inhibitor binding region, equivalent to the extended length of compound 11. Using standard bond lengths and angles this was calculated to be 24A°. The results obtained for compounds 4 and 8 can be explained if we assume that the potential binding region for the 4-hydroxy-2-pyrone nucleus is somewhat extended. A slight shift of the 2- pyrone would allow all methylenes to bind to E, resulting in the maximum number of enzyme-inhibitor hydrophobic interactions. Compound 11 (K_i=4.6 μM) was found to be thirty times as effective as elasnin (I)K_i = 140 μM¹⁸ in binding to HLE. Hydrophobicity had been shown to be an important factor in binding of I to E, so we wished to see if activity could be improved by increasing hydrophobicity by methylating the 4-hydroxy group. Methylation was not successful when the 3-(1'-oxoalkyl) functionality was present, although it proceeded quite smoothly for the 6- alkyl derivatives. Therefore, for this initial study on the binding capability of the 4-methoxy group, t:wo compounds were prepared, compounds 14 and 15 with K_i'=136 and 127 μM, respectively. Note that since these are uncompetitive inhibitors, it is necessary to compare values of K_i'. The analogous non-methylated compounds 6 and 7, have K_i' values of 790 and 566 μM, respectively. Compounds 14 and 15 are different from compounds 6 and 7 in three ways: they are unable to bind to E; they have greater affinity for ES; and the affinity of I for ES is independent of the number of methylenes at the 6 position. This can be interpreted to mean that because of steric hindrance and the loss of the hydrophilic 4-hydroxy group, the 4-methoxy-2-pyrone is unable to bind to E. A change in conformation when S binds to E would allow I to bind to ES, hydrophobic interactions would be enhanced, resulting in the greater affinity. The independence of K_i' with 6-alkyl chain length indicates that the binding region might be large enough for a heptyl or nonyl residue.

Groutas et al reported that methylation of the 4-hydroxy group resulted in a compound with reduced inhibitory activity. This is the converse to our ob- servations and an explanation for this disparity may lie in the use of percentage inhibition as a measure of the relative order of true inhibitory activity. They observed a decrease in percentage inhibition upon methyl- ation of the 4-hydroxy group. We have observed percentage inhibition to be dependent on [s] for some enzyme-inhibitor interactions. Results for compounds 7 and 14 demonstrate this quite well. At S = 250μM, compound 7 (1= 250 μM) inhibits HLE by 24%, while compound 14, (I = 250 μM) inhibits HLE by only 13%. The conclusion arrived at is that the 4-hydroxy derivative (compound 7) is the better inhibitor. However, if the experiment is performed at a higher S, (750 μM for both cases), then the reverse result is observed: compound 7 inhibits HLE by 34%, while compound 14 inhibits HLE by 42%. This result is of course expected, because compound 14 is an uncompetitive inhibitor, that is, it can only bind to ES, and as [ES] is increased, the [ESI] increases and consequently the observed percentage inhibition is higher. These results indicate that using percentage inhibition as a measure of relative inhibitory activity can lead to incorrect conclusions. Determination of the dissociation constants, ' K_i and K_i', gives a reliable result since they are independent of [s]. Maintaining the 4-methoxy-2-pyrone nucleus, the substituent in the 6-position was then modified. An increase in the number of ester methylenes resulted in a decrease in inhibitory activity (compounds 16-18; Ki'=104, 138 and 204 μM, respectively). We have suggested that the binding region for the 6-substituent of the 4-methoxy-2- pyrone might be large enough for 7-9 methylenes. This conclusion is substantiated by the result obtained for compound 17, which has 8 "methylene-type" units and has optimal inhibitory activity for this series. Comparison between compounds 15 and 18, with K_i' = 127 and 204 μM, respectively, shows that the hydrophobic derivative has greater affinity for ES, than does the oxygenated, and hence hydrophilic compound 18. This indicates that the 6- substituent binds to a particularly hydrophobic region. The inactivity of the free carboxyl derivative, compound 19, is then not surprising, since the results indicate that the binding region for the 6-substituent is hydro- phobic. A structure/activity relationship has been established, by comparison of the dissociation constants K_i and

K_i', and based on this we predict that the inhibitor binding region is 24Aº in length and is hydrophobic in nature. From the mechanistic data we can speculate where this region might lie. Mechanistic studies show that these substituted 2-pyrones do not act directly at the active site. We do, however, have reason to believe that they bind to the extended substrate binding region. A discussion of the relevant data is presented below.

Binding of the 3-(1'-oxoalkyl)-2-pyrones, compounds 1-4, is unaffected by S binding, whereas binding of the 6- alkyl-2-pyrones, compounds 5-8, is influenced by the presence of S on E. The normally accepted nomenclature describes the former class of compounds as pure noncompetitive inhibitors, while the latter are called mixed-type inhibitors. The mechanism of action differs for the two types of compounds, therefore we conclude that there is a specific binding region for the 4-hydroxy-2-pyrone nucleus and for each of the two alkyl fragments at positions 3 and 6. A further point of interest is that for compounds 1-4, the mechanism is not influenced by the number of methylenes, whereas for compounds 5-8 the reverse is true. The long chain homologs, compounds 7 and 8, have K_i < K_i', that is, I has greater affinity for E than for ES. We interpret this to mean that the inhibitor and substrate binding regions are overlapping, and that the long alkyl chain ( ≥ nonyl) cannot form as many hydrophobic interactions with E, when S is already bound, hence the decreased affinity for ES. For the shorter homologs, (compounds 5 and 6) the reverse is true, (K_i' < K_i) that is they have greater affinity for ES. Binding of S would create a srraller hydrophobic pocket, and consequently hydrophobic inter- actions would be increased, leading to the enhanced affinity. The 3,6-dialkyl-2-pyrones, compounds 9-13 would by comparison with the 6-alkyl 2-pyrones, compounds 5-8, be expected to be mixed-type inhibitors. This result was observed, although for the latter the ESI complex is active. The 4-methoxy-6- alkyl-2-pyrones, compounds 14 and 15, are uncompetitive inhibitors, which can be considered to be an extreme form of mixed-type inhibition, where K_i >> K_i' ind binding of I to ES is not favourable, presumably because of the loss of the hydrophilic group and the increased bulk of the 4-methoxy group. A different result was observed for the hydrophilic 4-methoxy-derivatives (compounds 16-18)-these compounds are mixed-type inhibitors and hence can bind to E, presumably because the hydrophilic substituent binds to a different part of the enzyme.

Interpretation of the dependence of mechanism on the substituent and pattern of substitution puts us in the position of being able to speculate on the location of this binding region. Similarities between our results and those of Marossy et al²¹ suggest to us that the substituted 2- pyrones are binding to the extended substrate binding region of HLE. They found that a tetrapeptide substrate,

Z-D-Phe-Pro-Ala-pNA was a very good substrate for HLE, and that removal or replacement of the P₄ residue ²², benzyioxy- carbonyl (Z), resulted in a decrease in affinity (Scheme

III). These results indicate that there is a region, designated subsite S ₄ , that is large enough to accomodate a hydrophobic residue such as a phenyl and therefore, also a 4-hydroxy-2-pyrone nucleus. The mechanistic data for the 6-alkyl derivatives, compounds 5-8, provides evidence that the 4-hydroxy-2-pyrone nucleus can bind at the S₄ subsite. The change in mechanism that is observed as the number of methylenes is increased, suggests that for the short chain homologs, compounds 5 and 6, the alkyl chain is bound to S₄ and S₃, with the substrate residues, boc and ala bound to S₂ and S₁ respectively. The alkyl chain of the longer homologs, compounds 7 and 8, would be bound at subsites S₂ and S₁ and so hindrance to S binding would be observed, hence the change in mechanism and the greater preference for binding to E and not ES. Spencer et al¹⁸ have suggested that the pyrone carbonyl might be bound to an "oxyanion hole"²³ and if so, the substituent at position 3 could be placed in the P₁, site. Our data do not support such a suggestion. Instead, we think that the 3-substituent could be placed at the P₅. site, that is, it is bound at the S₅ subsite.

None of the hydrophobic compounds were found to have inhibitory activity when tested against three related serine proteases, porcine pancreatic elastase, bovine α- chymotrypsin and bovine trypsin, when they were tested at the approximate limit of their solubility (25 μM for compounds 9-12, 10 μM for compound 13, 1000 μM for compound

19 and 200 μM for the remaining compounds). The hydrophilic derivatives (compounds 16-18) were nonspecific, inhibiting all four enzymes. These results indicate that

HLE has an unusually hydrophobic binding cleft and that better inhibitors can be developed by increasing the hydrophobicity of the substituents. The mechanism of action and a possible binding site have been established and based on these results it would be unlikely that these compounds were irreversible inhibitors. A further point in favour of reversible action is that the interaction is very rapid, as measured by UV spectroscopy, and is not dependent on incubation time of E and I, unlike the 6-chloropyrones²⁴. However, we decided to prove that this was the case. Spencer et al ¹⁸ used UV difference spectra to show that α-chymo- trypsin was not acylated, and hence they concluded that the interaction was reversible. We chose dialysis to study this problem. Two representative compounds were chosen; compound 10, a 3,6-dialkyl 2-pyrone, and compound 17, a 6-carbobutoxyvinyl 2-pyrone. In both cases, full recovery of enzyme activity was observed after dialysis in buffer for 4 days, indicating that the interaction is fully reversible.

In summary, the present study has shown that these substituted 2-pyrones bind to a hydrophobic region which overlaps the extended substrate binding region. We have evidence that indicates that the 4-nydroxy-2- pyrone can bind to the S ₄ subsite, with the 6-alkyl substituent binding to S₄ - S₁ subsites for the higher homologs. Compound 11 was found to be the most potent compound, with a K_i of 4.6 μM, 30 times more potent than elasnin, K_i= 140 μM. The hydrophobic compounds were found to be specific for HLE and the interaction with E is fully reversible.

Inhibitors in accordance with the present invention as set out above have been found not only to be very effective inhibitors of HLE but also to be relatively non-toxic, the LD₅₀ values of the inhibitors being of the order greater than 3g/Kg of body weight in both mice and rats.

Thus, typical LD₅₀ values of the inhibitors of the present invention have been found to be as set out immediately below, where the LD₅₀ value is in g of compound per Kg of body weight:

R LD₅₀

^C11^H23 5.0

^C8^H17 3.0

The inhibitor compounds and salts of the invention can be expected to be useful in the treatment of HLE 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 one of the above-mentioned diseases. The method is characterized 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 (IV) 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, antioxidents, 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/salts or compositions according to the invention 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-steroidal anti-inflamatory agents, immunosuppressants, sulfated glycosamines/glycans and other sulfated carbohydrates, analgesics and antipyretics.

In clinical use, the inhibitor compounds/salts or compositions of the present invention may be administered orally, rectally or by injection, for example, by transdermal application for the treatment of arthritis, in the form of pharmaceutical preparations comprising at least one active compound or salt 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 intended for injection, and between 2 and 50% 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, polyethlene 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, titanium dioxide, or the tablet can be coated with a lacquer dissolved in a readily volatile organic solvent or mixture or 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 or 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 compound, the balance being sugar and a mixture or 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.

Experimental Section

A . Lentini (La Trobe University , Bundoora, Australia) isolated HLE from lung washings (kindly donated by the Department of Medicine, Austin Hospital, Victoria, Australia), using a modification²⁵ of the methods of

Andrews et al²⁶ , Viscarello et al²⁷ and Martodam et al²⁸.

N-t-Boc-L-ala-p-nitrophenyl ester (BAN), N-(2-hydroxy- ethyl)-piperazine-N'-ethanesulfonic acid (HEPES), porcine pancreatic elastase, α-chymotrypsin and trypsin were purchased from Sigma Co. Spectrophotometric grade dimethyl sulfoxide (DMSO) was purchased from Aldrich. The 1H NMR spectra were recorded on a Perkin-Elmer R-32 spectrophotometer at 90 MHz using tetramethylsilane. (TMS) as internal standard. ¹³C NMR spectra were recorded using a JEOL FX-200 NMR spectrometer at 50.1 MHz with CDCl₃ as internal lock. CDCl₃ (77.00ppm) or TMS (O.OOppm) were used as internal standards. Melting points were determined on a Reichert microscope melting point apparatus. Elemental analyses were performed by Amdel Australian Microanalytical Service (Fisherman's Bend, Victoria). A Varian DMS 100 UV/visible spectrophotometer coupled with a DS-15 data station was used to store the assay data. Lines of best fit were calculated by linear regression.

Enzyme Inhibition Studies 1. Assay of HLE

HLE (1.8 μg/50 μl) was made up in 50mM sodium acetate, 0.5M NaCl and 0.1% brij-35 at pH 5.5. The assay conditions were 0.1M HEPES, 0.5M NaCl and 14.8% DMSO at pH 7.5 and 37ºC. All inhibitor and substrate solutions were made up in 5% aq. DMSO to retard hydrolysis. HLE (50 μl), DMSO/inhibitor (100 μl) and buffer (800 μl) were added to the thermostatted cuvette. The reference cell contained DMSO/inhibitor (100 μl) and buffer (850 μl). BAN (50 ul, final concentration 50-1000 μM) was added to each cuvette. After a lag time of 10 sec, the production of p-nitrophenol was followed at 400nm. Varian software:Kinetics Storage P/N 85-100541-00 version 3 1984 and Kinetics Calculations (enhanced) P/N 85-100542-00 version 4 1984 and the DS-15 data station was used to store the data. Initial velocities were calculated using an extinction coefficient of 11580cm M^-1 for p-nitrophenol, determined under these assay conditions. 2. Assay of porcine pancreatic elastase, α-chymotrypsin and trypsin

The assay conditions were identical to the HLE assays, except that each inhibitor was tested at one concentration with one concentration of substrate. Inhibitors were tested at their approximate limit of solubility:

25 μM for compounds 9-12, 10 μM for compound 13, 1000 μM for compound 19 and 200 uM for the remaining 2-pyrones. Each concentration was repeated at least twice. Since we normally use 100 μM BAN with HLE to screen new compounds, this was used as the standard and the required [s] was calculated for each of the other enzymes, so that there was a constant ratio of [E] to [ES]. Dialysis Experiment

To an aliquot of HLE (100 μl) was added 300 μl of DMSO (as control), or compound 3 (300 μM), or compound 16 (3000 μM) . Sufficient inhibitor was added to inactivate the enzyme by at least 90%. The solutions were pipetted into pre-soaked dialysis tubing (retention 10 K daltons) . The enzyme-complex was dialysed in 1L of 50mM sodium acetate, 0.45M NaCl and 0.1% brij-35 at pH 5.5 and magnetically stirred at 4°C for 4 days. The activity of the enzyme was determined in the normal way using 100 μM BAN.

Preparation of Compounds:

Synthesis of RCOCH₂COOC₂H₅

Solid magnesium methoxide (0.49g, 5mmol) was added to a solution of ethyl hydrogen malonate (1.2g, lOmmol) in THF aiid stirred for 1 h. The solvent was removed under reduced pressure to give a white slightly hygroscopic salt, Mg (OOCOCH₂COOEt), which was used directly. Carbonyldiimidazole was added to a solution of carboxylic acid (10 mmol) in. THF. After stirring at room temperature for six h the prepared Mg (OOCCH₂COOEt)₂ was added. The mixture was stirred for 18 h at 25ºC, the solvent was then removed at reduced pressure. The residue was partitioned between ether and aq. 0.5M HCl. The ether extract was washed with aq. sat. NaHCO₃, dried (Na₂SO₄) and con- centrated under reduced pressure to yield a yellow oil, yield > 95%. Synthesis of RCOCH₂COOH

The crude ester was stirred with 1 eq. IM NaOH overnight. Any remaining ester was removed by washing with ether. The aq. layer was cooled and acidified with 32% HCl. The precipitated product was collected and thoroughly dried before being used in the next step. Further purification was not necessary.

Synthesis of 3-(1'oxoalkyl)-4-hydroxy-6-alkyl-2-pyrone Solid carbonyldiimidazole was added to a THF solution of the 3-oxocarboxylic acid. The reaction was stirred under N₂ for 24 h, then acidified to pH 1 with 0.5M HCl. The reaction mixture was then extracted with ethylacetate, the organic layer was washed with brine, dried (NaSO₄) and concentrated under reduced pressure to yield the desired 2-pyrone (yield > 85%) as an orange solid. Recrystallisation from MeOH yielded white needles.

3-(1'-Oxohexyl)-4-hydroxy-6-pentyl-2-pyrone (compound 9) Anal. (C₁₆H₂₄O₄) CH mp 59.5-60º (MeOH) lit. mp 52º (MeOH) . 'H NMR (CDCl₃) 1.05 (t, J = 3Hz, 6H, CH₃); 1.36-1.92 (m, 12H, CH₂); 2.60 (t, J = 5Hz, 2H, CH₂C=); 3.18 (t, J = 5Hz, 2H, CH₂C=); 5.94 (s, 1H, CH=). 1³C NMR (CDCl₃) 13.79, 13.88 (CH₃); 22.23, 22.44, 23.66, 26.03, 31.05 (8CH₂); 99.61 (C3); 100.69 (C₅); 161.01 (C₆); 172.49 (C₄); 181.25 (COO); 207.97 (COCH₂).

3-(1'-Oxooctyl)-4-hydroxy-6-heptyl-2-pyrone (compound 10) Anal. (C₂₀H₃₂O₄) CH mp.64.5° (MeOH) (lit mp 68°) 1H NMR (CDCl₃) 0.86 (br t, 6H, CH₃); 1.32 (br s, 16H, CH₂); 1.67 (m, 4H, CH₂); 2.44 (t, 2H, J = 7Hz, CH₂ C=); 3.00 (t, J = 7Hz, 2H , CH₂CO); 5.82 (s, 1H , CH=) . 1³C NMR (CDCl₃) 13.99, 14.05 (CH₃); 22.54, 22.60, 24.00, 26.37, 28.82, 28.80, 29.08, 29.20, 31.57, 31.68, 34.28 (CH₂); 41.64 (CH₂C=); 99.60 (C₃); 100.71 (C₅); 161.06 (C₆) ; 172.51 (C₄); 181.27 (COO); 207.99 COCH₂).

3-(1'-Oxononyl)-4-hydroxy-6-octyl-2-pyrone (compound 11)

Anal. (C₂₂H₃₆O₄) CH mp. 68-5-69º (MeOH) (lit 65°) ¹H NMR (CDCl₃) 0.87 (t, J = 6Hz, 6H, CH₃); 1.27

(br s, 20H, CH₂); 1.68 (m, 4H, CH₂); 2.51 (t, J = 7Hz, 2H, CH₂=); 3.09 (t, J = 7Hz, 2H, CH₂CO); 5.96 (s, 1H, CH=) . ¹³C NMR (CDCl₃) 14.16 (2CH₃); 22.54, 23.92, 26.31, 29.09, 29.35, 31.77, 34.22, 41.55 (14CH₂); 99.51 (C₃); 100.62 (C₅); 160.92 (C₆); 172.45 (C₄); 181.18 (COO); 207.87 (COCH₂).

3-(1'-Oxodecyl)-4-hydroxy-6-nonyl-2-pyrone (compound 12) Anal. (C₂₄H₄₀O₄) CH mp . 73.5-74° (MeOH) (lit mp 65°) ¹H NMR (CDCl₃) 0.88 (br t, 6H, CH₃); 1.27 (br s, 24H, CH₂); 1.71 (m, 4H, CH₂); 2.49 (t, J = 7Hz , 2H,

CH₂C=); 3.07 (t, J = 7Hz, 2H , CH₂CO); 5.90 (s, 1H , CH=).

¹³C NMR (CDCl₃) 14.13 (2CH₃); 22.72, 24.03, 26.43, 28.99,

29.29, 29.43, 29.52, 31.89, 31.94, 34.34, 41.70 (16CH₂);

99.63 (C₃); 100.74 (C₅); 161.03 (C₆); 172.57 (C₄); 181.30 (COO); 207.9 (COCH₂). 3-(1'-Oxododecyl)-4-hydroxy-6-undecyl-2-pyrone (compound 13)

Anal. (C₂₈H₄₈O₄) CH mp. 81-5-82° (MeOH) (lit mp 82-83°) ¹H NMR (CDCl₃) 0.89 (br t, 6H, CH₃); 1.30 (br s, 32H, CH₂); 1.60 (m, 4H, CH₂); 2.51 (t, J = 7Hz, 2H, CH C=); 3.11 (t, J = 7Hz, 2H, CH₂CO); 5.98 (s, 1H, CH=) . ¹³C NMR (CDCl₃) 14.05 (2CH₃); 22.63, 23.95, 26.34, 28.91, 29.14, 29.29, 29.52, 31.86, (19CH₂); 41.61 (CH₂C=); 99.57 (C₃); 100.65 (C₅) 161.01 (C₆); 172.48 (C₄); 181.21 (COO): 207.93 (COCH₂).

Synthesis of 4-hydroxy-6-alky1-2-pyrone

The 2-pyrone was added to 5 equivalents (by weight) of 90% H₂SO₄ and heated at 130°C for 15 min. Ice was added to the cooled black solution with vigorous stirring. Recrystallisation from MeOH₂:O(1:1 ) yielded white needles.

4-hydroxy-6-pentyl-2-pyrone (compound 5)

Anal. (C₁₀H₁₄O₃) CH mP 48-50° (MeOH/H₂O) (lit mp 46-47) ¹H NMR (CDCl₃/dg-DMSO) 0.92 (t, J=7Hz, 3H,CH₃); 1.38 (m, 4H, CH₂); 1.66 (m, 2H, CH₂); 2.44 (t, J=8Hz, 2H, CH₂=); 5.38 (d, J=2Hz, 1H, CH=, C₃). ¹³C NMR (CDCl₃)

13.87 (CH₃); 22.28, 26.37, 31.07, 33.61 (4CH₂); 89.76 (C₃); 101.38 (C₅); 167.34 (C₆); 168.57 (C₂); 172.80 (C₄).

4-Hydroxy-6-heptyl-2-pyrone (compound 6)

Anal. (C₁₂H₁₈O₃) CH mp 71-71.5º (MeOH/H₂O) (lit mp 71°) ¹H NMR (CDCl₃) 0.89 (br t, 3H, CH₃); 1.27 (br s,

6H CH₂) 1.67 (m, 4H, CH₂) 2.49 (t, J = 7Hz, 2H, CH₂CO) ; 5.59 (d, J = 2Hz, 1H, CH=, C₃); 6.00 (d, J = 2Hz, 1H, CH=, C₅). ¹³C NMR (CDCl₃) 14.06 (CH₃); 22.62, 26.73, 28.92, 31.67, 33.66 (6CH₂); 89.81 (C₃); 101.46 (C₅); 167.33 (C₆); 168.67 (COO); 172.93 (C₄).

4-Hydroxy-6-nonyl-2-pyrone (compound 7)

Anal. (C₁₄H₂₂O₃) CH mp 75.5-76° (MeOH/H₂O) (lit 79°) ¹H NMR (CDCl₃) 0.88 (br t, 3H, CH₃); 1.27 (br s, 12H, CH₂); 1.64 (m, 4H , CH₂); 2.48 (t, J = 7Hz, 2H, CH₂C=); 5.62 (d, J = 2Hz, 1H, CH=, C₃) 6.00 (d, J = 2Hz, 1H, CH=, C₅). ¹³C NMR (CDCl₃) 14.11 (CH₃); 22.70, 26.73, 29.00, 29.28, 29.46, 31.89, 33.67 (8CH₂); 89.83 (C₃); 101.39 (C₅); 167.33 (C₆); 168.62 (C₂); 172.83 (C₄).

4-Hydroxy-6-undecyl-2-pyrone (compound 8)

Anal. (C₁₆H₂₆O₃) mp 82-83° (MeOH/H₂O) (lit mp 80°) ¹HMR (CDCl₃) 0.87 (br t, 3H, CH₃); 1.27 (s, 14H,

CH₂; 1.69 (br s, 4H, CH₂); 2.47 (t, J=6Hz, 2H, CH₂C=); 5.59 (s, 1H, CH=, C₃); 5.99 (s, 1H, CH= , C₅). ¹³C NMR (CDCl₃) 14.10 (CH₃); 22.71, 22.74; 29.02, 29.28, 29.49 (5CH₂); 29.63 (2CH₂); 31.94, 33.69 (CH₂); 89.81 (C₃); 101.34 (C₅) 167.33 (C₆); 168.44 (C₂); 172.74 (C₄).

Synthesis of 4-methoxy-6-alkyl-2-pyrone

Dimethyl sulfate (2.85 mmoles) was added to a methyl ethyl ketone solution of the required 4-hydroxy- 6-alkyl-2-pyrone (2.85mmoles) with anhydrous K₂CO₃ (8.84 mmoles, 3:1 excess). The reaction mixture was heated under reflux for 22 h. After cooling, K₂CO₃ was removed by filtration and the filtrate concentrated under reduced pressure. The orange oil crystallised on cooling. The product was recrystallised from pet.spirit (60-80°) to yield pale yellow crystals.

4-Methoxy-6-heptyl-2-pyrone (compound 14) Anal. (C₁₃H₂₀O₃) CH mp 44-45° (MeOH) ¹H NMR

(CDCl₃) 0.87 (br, t, 3H, CH₃); 1.31 (br s, 8H, CH₂); 1.62 (m, 2H, CH₂); 2.44 (t, J = 8Hz, 2H, CH₂C=); 3.81 (s, 3H, O.CH₃); 5.42 (d, J = 2Hz, 1H, CH=, C₃); 5.89 (d, J = 2Hz, 1H, CH=, C₅). ¹³C NMR (CDCl₃) 14.01 (CH₃); 22.66, 26.68, 28.93, 29.26, 29.43, 31.85, 33.64 (6CH₂); 55.80 (OCH₃); 87.48 (C₃); 99.62 (C₅) ; 165.03 (C₂); 165.85 (C₆); 171.34

(c₄).

4-Methoxy-6-nonyl-2-pyrone (compound 15)

Anal. (C₁₅H₂₄O₃) CH mp 56.5-57° (MeOH) (lit mp 58-59°) ¹H NMR (CDCl₃) 0.88 (br t, 3H, CH₃); (br s, 8H, CH₂); 1.62 (m, 2H, CH₂); 2.44 (t, J = 8Hz , 2H, CH₂ C=); 3.81 (s, 3H, OCH₃); 5.42 (d, J = 2Hz, 1H, CH= (C )); 5.78 (s, 1H, CH= (C₅)). ¹³C NMR (CDCl₃) 14.04 (CH₃);

22.60, 26,68, 28.94, 31.68, 33.64, (8CH₂); 55.83 (OCH₃); 87..48 (C₃); 99.66 (C₅); 164.98 (C₂); 165.82 (C₆);

171.37 (C₄).

Synthesis of 3-(1'-oxoalkyl)-4-hydroxy-2-pyrone

4-Hydroxy-6-methyl-2-pyrone (0.005 moles) was added to trifluoroacetic acid (3ml) and the appropriate acid chloride (0.01 moles) The mixture was stirred and heated under reflux for 3 hours then cooled and poured into ice-water (15ml) The collected crude product was dissolved in dimethylsulfoxide, to this was added lead acetate in dioxin to complex with the free carboxylic acid. Addition of water precipitated the pure 2-pyrone which was recrystallised from MeOH.

3- (1'-Oxohexyl)-4-hydroxy-6-methyl-2-pyrone (compound 1)

Anal. (C₁₂H₁₆O₄) CH mp. 66-66.5°C (MeOH) (lit mp 65°). ¹H NMR (CCl₄) 0.86 (m, 3H, alkyl-CH₃); 1.21- 1.59 (m, 10H, CH₂); 2.18 (s, 3H, CH₃C=); 2.89 (t, 2H COCH₂); 5.59 (s, 1H, CH=, C₅). ¹³C NMR (CDCl₃) 13.98 (CH₃-alkyl); 22.56 (CH₃C=); 22.54, 29.06, 31.64 (CH₂); 41.58 (COCH₂); 99.44 (C₃); 101.47 (C₅); 160.86 (C₆); 168.71 (C₄); 181.21 (C₂); 207.96 (COCH₂).

3-(1'-Oxooctyl)-4-hydroxy-6-methyl-2-pyrone (compound 2)

Anal. (C₁₄H₂₀O₄) CH mp 67-5-68ºc (MeOH) ¹H NMR (CCl₄) 0.86 (br t, 3H, CH₃CH₂; 1.21 - 1.59 (m, 10H,

CH₂); 2.18 (s, 3H, CH₃C=); 2.89 (t, J = 7Hz, 2H, COCH₂);

5.59 (s, 1H, CH=) . ¹³C NMR (CDCl₃) 13.98 (CH₃CH₂);

20.56 (CH₃C=); 22.54, 23.95, 29.06, 29.14, 31.63, 41.58

(6CH₂); 99.43 (C₃); 101.47 (C₅); 160.86 (C6); 168.71 (C₄); 181.21 (COO); 207.96 (COCH₂).

3-(1'-Oxodecyl)-4-hydroxy-6-methyl-2-pyrone (compound 3) Anal. (C₁₆H₂₄O₄) CH mp 74-74.5° (MeOH) ¹H NMR (CDCl₃) 0.87 (br t, 3H, CH₃); 1.29 (s, 12H, CH₂); 1.60, (br m, 2H, CH₂); 2.24 (s, 3H , CH₃C=); 3.14 (t, J = 7Hz, 2H, COCH₂); 5.89 (s, 1H, CH= , C₅). ¹³C NMR (CDCl₃ ) 14 . 10 (CH₃-alkyl) ; 20 . 61 (CH₃C=) ; 22 . 72 (CH₂); 29.29 (CH₂); 29.52 (3CH₂); 31.91 (CH₂); 41.67 (COCH₂); 99.42 (C₃); 101.50 (C₅); 160.80 (C₆); 168.80 (C₄); 181.27 (COO); 207.93 (COCH₂).

3-(1'-Qxododecyl)-4-hydroxy-6-methyl-2-pyrone (compound 4) Anal. (C₁₈HSO₄) CH mp. 82-82.5°C (MeOH) . ¹H

NMR (CCl₄) 0.92 (t, 3H, J = 3Hz, CH₃-alkyl), 1.23-1.46 (m,20H, CH ), 2.27 (s, 3H, CH₃C=), 3.04 (t, 2H, J = 5Hz, COCH₂), 5.85 (s, 1H, CH=). ¹³C NMR (CDCl₃) 14.07 (CH₃ CH₂); 20.62 (CH₃C=); 22.66, 23.97, 29.23, 24.30, 29.46, 29.47, 29.59, 31.88, (10CH₂); 41.61 (CH₂CO); 99.48 (C₃), 101.52 (C₅); 160.92 (C₆); 168.73 (C₄); 181.26 (COO); 208.011 (COCH₂) .

Synthesis of 4-methoxy-6-(2'-hydroxy-2'carboalkoxyvinyl)- 2-pyrone Prepared according to the method of Douglas

& Money¹¹ . The butoxy and pentoxy derivatives were purified by triturating the crude brown oil with acetone. Recrystallization from ethyl acetate yielded yellow crystals.

4-Methoxy-6-(2'-hydroxy-2'-carbobutoxyvinyl)-2-pyrone (compound 17)

Anal. (C₁₁H₁₂O₆) CH mp. ¹H NMR (CDCl₃/DMSO) 0.96 (t, J = 6Hz, 3H, CH₃); 1.51 (m, 4H, CH₂); 3.81 (s, 3H, OCH₃); 4.22 (t, J = 7Hz, CH₂O); 5.40 (d, J =

2Hz, 1H, CH=, C₃); 5.99 (s, 1H, CH, C₅); 6.77 (d, J = 2Hz, 1H, CH = COH, C₂). ¹³C NMR CDCl₃/DMSO 13.57 (CH₃); 18.90 (CH₂); 30.26 (CH₂); 55.92 (OCH₃); 65.92 (OCH₂); 88.20 (C₃); 101.41 (C₅); 103.16 (CH = COH); 146.41 (COH=); 157.07 (C₆); 163.51 (CO); 163.68 (CO); 171.20

(c₄).

Scheme I:

a) carbonyldiimidazole, THF, RT; b) Mg (OOCCH₂COOC₂H₅) ; c) H⁺; d) IM NaOH; e) H⁺; f) carbonyldiimidazole, THF, RT; g) H⁺; h) 90% H₂SO₄, 130°; i) (CH₃)₂SO₄, K₂CO₃, pet spirit (60-80°); j) (R'OOC)₂, Na; k) H⁺; 1) R"COCl,

CF₃COOH

Scheme II:

E=enzyme, S=substrate, I=inhibitor, P=p-nitrophenol,

K_S =[E] [S]/[ES] , αK_S =[EI] [S]/[ESI] ,

K_i=[E] [I] /[El], ακ_i=K_i' = [ES] [I]/[ESI], k_p=catalytic constant. Pure noncompetitive inhibition : K_i=K_i' , K_s=αk_s , β=0 Mixed-type linear inhibition : K_i≠K_i' K_s=αK_s, β=0 Mixed-type hyperbolic inhibition : K_i≠K_i' ^, K_s≠αk_s , β≠0 Pure uncompetitive inhibition : K_i=∞ , k_s=∞ β= 0 Scheme III :

^S4 ^S3 ^S2 ^S1 ^S1 ' K_s (UM)

Z D-Phe Pro Ala NA^a 9.24^b

D-Phe Pro Ala NA 100^b Suc D-Phe Pro Ala NA 461^b

Boc Ala NP^C 333^d

a: NA=p-nitroanilide; b: Ref 21; c: NP=p-nitrophenol; d: this work

Following is a bibliography of the numbered publications referred to herein. The disclosures of those publications are incorporated herein by reference. 1. Janoff, A., Ann. Rev. Med., 1972, 23, 177. 2. Muttman, C, Ed. "Pulmonary Emphysema and Prote- olysis". Academic Press, New York, 1972.

3. Lieberman, J., Chest, 1976, 70, 62.

4. Janoff, A.; Feinstein, G.; Malemund, C; Elias, J.M. J. Clin. Invest., 1976, 57, 615. 5. Omura, S.; Ohno, H.; Saheki, T.; Yoshida, M.;

Nakagawa, A.; Biochem. Biophys. Res. Commun.

1978, 83, 704. 6. Brooks, D.W.; Lu, L.D-L.; Masamune, S., Angew.

Chem. Int. Eng. Edn., 1979, 18, 72. 7. Hauser, C.R.; Breslow, D.; Baumgarten, E. J. Amer.

Chem. Soc, 1944, 66, 1286.

8. Ohta, S.; Tsujimura, A.; Okamoto, M. Chem. Pharm. Bull. 1981, 29, 2762.

9. Collie, J.N. J. Chem. Soc. 1891, 59, 609. 10. Bu'Lock, J.D.; Smith, H.G. J. Chem. Soc. 1960, 502.

11. Douglas, J.L.; Money, T. Can. J. Chem. 1968, 46, 695.

12. Hanes, C.S. Biochem. J. 1932, 26, 1406.

13. Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 56, 658.

14. Cornish-Bowden, A. Biochem. J. 1974, 137, 143.

15. Dixon, M. Biochem. J. 1953, 55, 1970.

16. Segel, J. H. "Enzyme Kinetics Behaviour Analysis of Rapid Equilibrium and Steady-State Enzyme Systems", John Wiley and Sons, New York, 1975, Chaps. 3 & 4.

17. Groutas, W.C.; Abrams, W.R.; Carroll, R.T.; Moi, M.K.; Miller, K.E.; Margolis, M.T. Experientia

1984, 40, 361.

18. Spencer, R.W; Copp, L.S; Pfister, J.R. J. Med. Chem. 1985, 28, 1828.

19. Groutas, W.C.; Stanga, M.A; Brubaker, M.J.; Huang, T.L.; Moi, M.K.; Caroll, R.T. J. Med. Chem.

1985, 28, 1106.

20. Cook, L. unpublished results.

21. Marossy, K. Szabo, G.C.; Pozsgay, M.; Elodi, P. Biochem. Biophys. Res. Commun. 1980, 96, 762. 22. Notation of Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27 157.

23. Kraut, J. Am. Rev. Biochem. 1977, 46, 331.

24. Westkaemper R.B.; Abeles, R.H. Biochemistry. 1983, 22 3256. 25. Lentini, A.; Ternai, B. unpublished results.

26. Andrews, J.L Ghosh, P.; Lentini, A.; Ternai, B. Chem-Biol. Interactions, 1983, 47, 157.

27. Viscarello, B.R.; Stein, R.L.; Kusner, E.J.; Holsclaw, D.; Krell, R.D. Prep. Biochem. 1983, 13, 57.

28. Martodam, R.R.; Baugh, R.J.; Twumasi, D.Y.; Leiner, I.E. Prep. Biochem. 1979, 9, 15.

29. Kogel, F.; Salemink, C.A. Rec. Trav. Chim. 1952, 71, 779.

Claims

CLAIMS:

1. A compound of the formula (III):

(III)

or physiologically acceptable salts thereof, wherein:

R₁ and R₃ are independently selected from hydrocarbon-chain radicals of 5 to 12 carbon atoms in length which are bonded directly to the pyrone ring or bonded indirectly to the pyrone ring through an oxygen or nitrogen atom, said hydrocarbon-chain radicals being independently unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes, the total number of carbon atoms of the longest chain length in each of the groups R₁ and R₃ being 5 to 12; and

R₂ is hydrogen or hydrocarbon radical of 1 to 5 carbon atoms unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes;

provided that: when R₁ is oxohexyl and R₂ is hydrogen, then R₃ cannot be pentyl; when R₁ is oxoheptyl and R₂ is hydrogen, then R₃ cannot be hexyl; when R₁ is oxononyl and R₂ is hydrogen, then R₃ cannot be octyl; when R₁ is oxodecyl and R₂ is hydrogen, then R₃ cannot be nonyl; and when R₁ is oxododecyl and R₂ is hydrogen, then R₃ cannot be undecyl.

2. A compound or physiologically acceptable salt thereof according to claim 1, wherein R₁ and R₃ are independently selected from unsubstituted alkyl, alkenyl, alkynyl or alkoxy, of 5 to 12 carbon atoms, or alkyl, alkenyl, alkynyl or alkoxy of 5 to 12 carbon atoms substituted with alkyl, alkenyl, alkynyl, alkoxy, carboxy, oxo, amino, or other such physiologically innocuous substituents.

3. A compound or physiologically acceptable salt thereof according to claim 1 or 2 wherein R₁ and R₃ are independently selected from:

(i) alkyl, alkenyl or alkynyl of 5 to 12 carbon atoms, unsubstituted or substituted with physiologically innocuous substituents exemplified by oxoalkyl of 5 to 12 carbon atoms or oxoalkyl of 5 to 12 carbon atoms substituted with said physiologically innocuous substituents exemplified by

wherein n is zero or an integer 1 to 11 and R' is hydrogen or alkyl, alkenyl or alkynyl of 1 to 11 carbon atoms unsubstituted or substituted with alkyl, alkenyl, alkoxy, carboxy, oxo, amino, or other such physiologically innocuous substituents, provided that the total number of carbon atoms of the longest chain length in the groups R₁ and R₃ is 5 to 12;

(ii) alkoxy of 5 to 12 carbon atoms, unsubstituted or substituted with physiologically innocuous substituents exemplified by

wherein n is zero or an integer 1 to 11 and R'' is hydrogen or alkyl, alkenyl or alkynyl of 1 to 11 carbon atoms unsubstituted or substituted with alkyl, alkenyl, alkoxy, carboxy, oxo, amino or other such physiologically innocuous substituents, provided that the total number of carbon atoms of the longest chain length in the groups R₁ and R₃ is 5 to 12; and

(iii) hydrocarbon radicals of 5 to 12 carbon atoms bonded to the pyrone ring through a nitrogen atom , unsubstituted or substituted with physiologically innocuous substituents exemplified by

wherein n is zero or an integer 1 to 11 and R'" is hydrogen or alkyl, alkenyl or alkynyl of 1 to 11 carbon atoms unsubstituted or substituted with alkyl, alkenyl, alkoxy, carboxy, oxo, amino or other physiologically innocuous substituents, provided that the total number of carbon atoms of the longest chain length in the groups R₁ and R₃ is 5 to 12.

4. A compound or physiologically acceptable salt thereof according to any one of claims 1 to 3 wherein R₂ is selected from hydrogen or alkyl, alkenyl or alkynyl, inclusive of straight and branched chain radicals and cyclic radicals, exemplified by methyl, ethyl, n-propyl, isopropyl, cyclopropyl, tertiary butyl, and n-butyl.

5. A compound or physiologically acceptable salt thereof according to any one of claims 1 to 4 wherein R₁, is oxoalkyl of 5 to 12 carbon atoms.

6. A compound or physiologically acceptable salt thereof according to any one of claims 1 to 5 wherein

R₃ is alkyl of 5 to 12 carbon atoms.

7. A compound or physiologically acceptable salt thereof according to any one of claims 1 to 6 wherein R₂ is selected from hydrogen, C_{1 -5} alkyl, C_{1 -5} alkenyl or C_1-5 alkynyl.

8. A compound or physiologically acceptable salt thereof according to any one of claims 1 to 7 wherein R₁ is oxoalkyl of 7 to 10 carbon atoms.

9. A compound or physiologically acceptable salt thereof according to any one of claims 1 to 8 wherein R₃ is alkyl of 7 to 10 carbon atoms.

10. A compound or physiologically acceptable salt thereof according to any one of claims 1 to 9 wherein R₂ is hydrogen or methyl.

11. A compound according to any one of claims 1 to 6 selected from the group:

3-(1' - oxononyl -4-hydroxy-6-nonyl-2-pyrone; 3-(1'- oxodecyl -4-hydroxy-6-octyl-2-pyrone; 3-(1'- oxononyl -4-methoxy-6-octyl-2-pyrone; 3-(1'- oxodecyl -4-methoxy-6-nonyl-2-pyrone; 3-(1'- oxononyl -4-methoxy-6-nonyl-2-pyrone; 3-(1'- oxodecyl -4-methoxy-6-octyl-2-pyrone; 3-(1'- oxononyl -4-hydroxy-6-pentyl-2-pyrone; 3-(1'- oxononyl -4-hydroxy-6-hexy1-2-pyrone; 3-(1'- oxononyl -4-nydroxy-6-heptyl-2-pyrone; 3-(1'- oxodecyl -4-hydroxy-6-pentyl-2-pyrone; 3-(1'- oxodecyl -4-hydroxy-6-hexyl-2-pyrone; 3-(1'- oxodecyl -4-hydroxy-6-heptyl-2-pyrone; 3-(1'- oxooctyl -4-hydroxy-6-heptyl-2-pyrone; 3-(1'- oxooctyl -4-hydroxy-6-octyl-2-pyrone; 3-(1'- oxooctyl -4-hydroxy-6-nony1-2-pyrone; 3-(1'- oxononyl -4-methoxy-6-hexyl-2-pyrone; 3-(1'- oxononyl -4-methoxy-6-heptyl-2-pyrone; 3-(1'- oxodecyl -4-methoxy-6-hexyl-2-pyrone; 3-(1'- oxodecyl -4-methoxy-6-heptyl-2-pyrone; 3-(1'- oxooctyl -4-methoxy-6-octyl-2-pyrone; 3-(1'- oxooctyl -4-methoxy-6-nonyl-2-pyrone;

or a physiologically acceptable salt of any thereof.

12. A compound according to any one of claims 1 to 6 selected from the group:

3-(1'- oxopentyl)-4-hydroxy-6-pentyl-2-pyrone; 3-(1'- oxoheptyl)-4-hydroxy-6-heptyl-2-pyrone; 3-(1'- oxooctyl)-4-hydroxy-6-octyl-2-pyrone; 3-(1'- oxononyl)-4-hydroxy-6-nonyl-2-pyrone;

or a physiologically acceptable salt of any thereof.

13. A process for the preparation of a compound according to claim 1, which comprises:

(A) reacting an appropriate acid imidazolide with an appropriate salt of an alkyl hydrogen malonate to form the corresponding 3-oxocarboxylate ester; hydrolysing said 3-oxocarboxylate ester to afford the corresponding 3-oxocarboxylic acid; and cyclizing said 3-oxocarboxylic acid with carbonyldiimidazole to afford the corresponding 3-(1' -oxoalkyl)-4-hydroxy-6-alkyl-2-pyrones of the formula (V):

(V)

wherein R₁ and R₃ are independently alkyl of 5 to 12 carbon atoms in length and optionally substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes, the total number of carbon atoms of the longest chain length in each of the groups R₁ and R₃, being 5 to 12; and/or (B) replacing by conventional techniques, the hydrogen of the hydroxy group at position-4 of the pyrone ring of formula (V), with a hydrocarbon radical of 1 to 5 carbon atoms unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes; and/or

(c) replacing by conventional techniques, the 1'- oxoalkyl and/or the alkyl, respectively, at positions -3 and -6, of the pyrone ring, of formula (V), with required hydro- carbon-chain radicals of 5 to 12 carbon atoms in length which are bonded as required directly to the pyrone ring or bonded indirectly to the pyrone ring through an oxygen. or nitrogen atom, said hydrocarbon-chain radicals being independently unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase- type enzymes, the total number of carbon atoms of the longest chain length in each of the groups R₁ and R₃ being 5 to 12; and/or

(E) converting the compounds of claim 1 so obtained to a physiologically acceptable salt thereof.

14. A process according to claim 13, which comprises the preparation of a compound according to any one of claims 2 to 12.

15. A. process for the preparation of a compound of the formula (X):

(X)

or physiologically acceptable salts thereof, wherein: R₁ and R₃ are independently alkyl of 5 to 12 carbon atoms in length, optionally substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes, the total number of carbon atoms of the longest chain length in the groups R1 and R₃ being 5 to 12; and

provided that: when R₁ is pentyl and R₂ is hydrogen, then R₃ cannot be pentyl; when R₁ is hexyl and R₂ is hydrogen, then R₃ cannot be hexyl; when R₁ is octyl and R₂ is hydrogen, then R₃ cannot be octyl; when R₁ is nonyl and R₂ is hydrogen, then R₃ cannot be nonyl; and when R₁ is undecyl and R₂ is hydrogen, then R₃ cannot be undecyl,

which comprises reacting an appropriate acid imidazolide with an appropriate salt of an alkyl hydrogen malonate to form the corresponding 3-oxocarboxylate ester of formula (XI): R₃COCH₂COOR (XI)

wherein R₃ is as defined above and R is alkyl, then hydro- lysing the ester of formula (XI) to form the corresponding 3-oxocarboxylic acid of formula (XII):

R₃COCH₂COOH (XII)

wherein R₃ is as defined above, followed by cyclizing the 3-oxocarboxylic acid of formula XII with carbonyldiimidazole to form the corresponding 3-(1'-oxoalkyl)-4-hydroxy- 6-alkyl-2-pyrone of the formula (XI): (XIII)

where R₁ and R₃ are as defined above, and optionally methylating the 4-hydroxy group of the 3- (1'-oxoalkyl)-4- hydroxy-6-alkyl-2-pyrone of the formula (XEII), with dialkyl sulfate to form the corresponding 3- (1'-oxoalkyl)-4- alkyl-6-alkyl-2-pyrone of the formula (X), whereafter, if desired, the compound of formula (X) so obtained is converted to a physiologically acceptable salt thereof.

16. A process according to claim 13 or 14, which comprises the preparation of a compound according to any one of claims 1 to 12.

17. A process according to claim 15, which comprises the preparation of a compound according to any one of claims 8 to 12.

18. A compound according to any one of claims 1 to 12 when obtained by the process of any one of claims 13 to 17, respectively.

19. A pharmaceutical or veterinary composition comprising as active ingredient at least one compound of formula (IV):

(IV)

or physiologically acceptable salts thereof, wherein:

R₁ and R₃ are independently selected from hydrocarbon-chain radicals of 5 to 12 carbon atoms in length which are bonded directly to the pyrone ring or bonded indirectly to the pyrone ring through an oxygen or nitrogen atom, said hydro- carbon-chain radicals being independently unsubstituted or substituted with physiologically innocuous substituents which do not interfere with the binding of said compound with elastase-type enzymes, the total number of carbon atoms of the longest chain length in each of the groups R₁ and R₃ being 5 to 12; and

in association with a pharmaceutical or veterinary adjuvant or carrier.

20. A composition according to claim 19 wherein R₁ and R₃ are independently selected from unsubstituted alkyl, alkenyl, alkynyl or alkoxy, of 5 to 12 carbon atoms, or alkyl, alkenyl, alkynyl or alkoxy of 5 to 12 carbon atoms substituted with alkyl, alkenyl, alkynyl, alkoxy, carboxy, oxo, amino, or other such physiologically innocuous substituents.

21. A composition according to claim 19 or 20 wherein R₁ and R₃ are independently selected from: (i) alkyl, alkenyl or alkynyl of 5 to 12 carbon atoms, unsubstituted or substituted with physiologically innocuous substituents exemplified by oxoalkyl of

5 to 12 carbon atoms or oxoalkyl of 5 to 12 carbon atoms substituted with said physiologically innocuous substituents exemplified by

(ii) alkoxy of 5 to 12 carbon atoms, unsubstituted or substituted with physiologically innocuous sub- stituents exemplified by

(iii) hydrocarbon radicals of 5 to 12 carbon atoms bonded to the pyrone ring through a nitrogen atom, unsubstituted or substituted with physiologically innocuous substituents exemplified by

22. A composition according to any one of claims 19 to 21 wherein R₂ is selected from alkyl, alkenyl or alkynyl, inclusive of straight and branched chain radicals and cyclic radicals, exemplified by methyl, ethyl, n-propyl, isopropyl, cyclopropyl, tertiary butyl, and n-butyl.

23. A composition according to any one of claims 19 to 22 wherein R₁ is oxoalkyl of 5 to 12 carbon atoms.

24. A composition according to any one of claims 19 to 23 wherein R₃ is alkyl of 5 to 12 carbon atoms.

25. A composition according to any one of claims 19 to 24 wherein R₂ is selected from hydrogen, C_1-5 alkyl, C_1-5 alkenyl, or C_1-5 alkynyl.

26. A composition according to any one of claims 19 to 22 wherein R₁ is oxoalkyl of 7 to 10 carbon atoms.

27. A composition according to any one of claims 19 to 23 wherein R₃ is alkyl of 7 to 10 carbon atoms.

28. A composition according to any one of claims 19 to 24 wherein R₂ is hydrogen or methyl.

29. A composition according to any one of claims 19 to 22 comprising as active ingredient at least one compound selected from the group: 3-(1'- oxononyl))-4-hydroxy-6-octyl-2-pyrone; 3-(1'- oxodecyl))-4-hydroxy-6-nonyl-2-pyrone; 3-(1' - oxononyl))-4-hydroxy-6-nonyl-2-pyrone; 3-(1'-oxodecyl)-4-hydroxy-6-octyl-2-pyrone; 3-(1'- oxononyl))-4-methoxy-6-octyl-2-pyrone; 3-(1'- oxodecyl))-4-methoxy-6-nonyl-2-pyrone; 3-(1'- oxononyl))-4-methoxy-6-nonyl-2-pyrone; 3-(1'- oxodecyl))-4-methoxy-6-octyl-2-pyrone; 3-(1'- oxononyl))-4-hydroxy-6-pentyl-2-pyrone; 3- (1'- oxononyl))-4-hydroxy-6-hexyl-2-pyrone; 3-(1'- oxononyl))-4-hydroxy-6-heptyl-2-pyrone; 3-(1'- oxodecyl))-4-hydroxy-6-pentyl-2-pyrone; 3-(1 '- oxodecyl))-4-hydroxy-6-hexyl-2-pyrone; 3-(1'- oxodecyl))-4-hydroxy-6-heptyl-2-pyrone; 3-(1'- oxooctyl))-4-hydroxy-6-heptyl-2-pyrone; 3-(1'- oxooctyl))-4-hydroxy-6-octyl-2-pyrone; 3-(1'- oxooctyl))-4-hydroxy-6-nonyl-2-pyrone; 3-(1'- oxononyl))-4-methoxy-6-hexyl-2-pyrone; 3-(1 '- oxononyl))-4-methoxy-6-heptyl-2-pyrone; 3-(1 '- oxodecyl))-4-methoxy-6-hexyl-2-pyrone; 3-(1 '- oxodecyl))-4-methoxy-6-heptyl-2-pyrone; 3-(1 '- oxooctyl))-4-methoxy-6-octyl-2-pyrone;

3- (1 '- oxooctyl))-4-methoxy-6-nonyl-2-pyrone;

or a physiologically acceptable salt of any thereof, in association with a pharmaceutical or veterinary adjuvant or carrier.

30. A composition according to any one of claims 19 to 22 comprising as active ingredient at least one compound selected from the group:

3-(1'-oxopentyl)-4-hydroxy-6-pentyl-2-pyrone; 3-(1'-oxoheptyl)-4-hydroxy-6-heptyl-2-pyrone; 3-(1'-oxooctyl)-4-hydroxy-6-octyl-2-pyrone; 3-(1'-oxononyl)-4-hydroxy-6-nonyl-2-pyrone, or a physiologically acceptable salt of any thereof, in association with a pharmaceutical or veterinary adjuvant or carrier.

31. The use of a compound or physiologically acceptable salt of any one of claims 1 to 12 and 18, or of a composition of any one of claims 19 to 30, as an inhibitor for elastase-type enzymes.

32. A method for inhibiting the growth of tumors or the degradation of tissues in arthritis or the destruction of lung tissues in emphysema, which comprises administering to an animal suffering from any one of such conditions, a therapeutically effective amount of at least one compound or physiologically acceptable salt according to any one of claims 1 to 12 and 18, or a composition according to any one of claims 19 to 30.

33. A compound or physiologically acceptable salt thereof; a process; a pharmaceutical or veterinary composition; and a method, as claimed in any one of claims 1 to 32, respectively, and substantially as described herein.