HUMAN LEUCOCYTE ELASTASE INHIBITOR COMPOUNDS

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 prep¬ aration of said inhibitor compounds; pharmaceutical/veteri¬ nary compositions containing said inhibitor compounds; and methods for the clinical application of said inhibitor com¬ pounds 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 α, - proteinase inhibitor (α,-PI) , which is also a normal constituent of bronchioal- veolar lavage fluid (BAL) . However, c -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 α,-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. 9_5, 1890 (1980) ; the azapeptides, vide: K. Haveman & A. Janoff, "Neutral proteases of Human Poly¬ morphonuclear Leukocytes", p. 221, Urban and Schwartzenberg, Baltimore (1977) ; cyclohexylamide, vide: C.H. Hassall, W.H. Johnson, N.A. Roberts: Bio-Organic Chem. , 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. 3_0, 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 inad¬ visable, 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 charac¬ ter 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. 5 Omura et al reported that elasnin (I) , an alkylated 2-pyrone of microbial origin, was a good inhibitor of HLE. 17 18 19 Groutas et al , Spencer et al and Groutas et al disclose HLE inhibitors of the formula II 17 1 wherein: (a) in Groutas et al , R is hydrogen and R is methyl, propyl, heptyl or undecyl; (b) in Spencer et al 18, R1 is methyl when R is methyl or - CH 2 COPh, and R, is hydrogen when R is hydrogen or - CH 2 COPh; and (c) in Groutas et al 19, R1 is hydrogen when R is propyl, butyl, pentyl, hexyl or undecyl, and 0 1 " R is - C - 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) : wherein R 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) : or physiologically acceptable salts thereof, wherein: , and R. 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 R, ana 3 being 5 to 12; and 2 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 R, is oxohexyl and R 2 is hydrogen, then R 3 cannot be pentyl; when R, is oxoheptyl and R 2 is hydrogen, then R.-. cannot be hexyl; when R, is oxononyl and R 2 is hydrogen, then R 3 cannot be octyl; when R, is oxo- decyl and R 2 is hydrogen, then R 3 cannot be nonyl; and when R, is oxododecyl and R 2 is hydrogen, then 3 cannot be undecyl. The present invention also provides a pharmaceut¬ ical or veterinary composition comprising as active ingredient at least one compound of formula (IV) : or physiologically acceptable salts thereof, wherein: R, and R_ 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 R-, and R 3 being 5 to 12; and R 2 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 suffer¬ ing from any one or such conditions, a therapeutically 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 R, and R_ 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 sub¬ stituents which do not interfere with the binding of said compounds with elastase-type enzymes. Preferably, the groups R, and 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 -(CH 2.,.)n-C-R' ,-(.CB Δln-C-O-R 1 or -(CH-.,i)n-C-NH-R 1 , 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 R, and R 3 is 5 to 12; (ii) alkoxy of 5 to 12 carbon atoms, unsubstituted or substituted with physiologically innocuous sub¬ stituents which do not interferewith the binding of said compounds with elastase-type enzymes, exemplified by 0 0 -O-(CH 2^)n-R•' ' ,-0-(CH 2- > )„n-C-O-R' • or -0-(CH 2. TΪ-C-NH-R' • 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 R, and R 3 is 5 to 12; and (iii) hydrocarbon radicals of 5 to 12 carbon atoms bonded to the pyrone ring through a nitrogen atom, unsub¬ stituted or substituted with physiologically innoc- uous substituents which do not interferewith the binding of said compounds with elastase-type enzymes, exemplified by 0 -NH- (CH 2 l n -R « • ' , -NH- CCH 2 ). n -C-0-R ' ' ' or -NH- O - (CH.,) -C-NH-R 1 1 ' . n wherein n is zero or an integer 1 to 11 and R 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 physio¬ logically 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 R, . and R 3 is 5 to 12. The group R-, 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 R 3 in the compounds of general formulae (HI) and (IV) above is more preferably alkyl of 5 to 12 carbon atoms, most preferably alkyl of 7 to 10 carbon atoms. The group R 2 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 R 2 in the compounds of general formulae (III) and (IV) above is more preferably selected from hydrogen, C, 5 alkyl, C, 5 alkenyl, or C, 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 physio¬ logically 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 physio¬ logically acceptable salts by processes which are known to the skilled person. Examples of such suitable physiologi¬ cally 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, esylate and salicylate. Especially preferred compounds of the present invention are: 3- -d' - oxononyl) -4-hydroxy-6-octyl-2-pyrone; 3- -(I' - oxodecyl)-4-hydroxy-6-nony1-2-pyrone; 3- -d' - oxononyl)-4-hydroxy-6-nonyl-2-pyrone; 3- -d' - oxodecyl) -4-hydroxy-6-octyl-2-pyrone; 3- -d' - oxononyl) -4-methoxy-6-octyl-2-pyrone; 3- -d' - oxodecyl) -4-methoxy-6-nonyl-2-pyrone; 3- -d' - oxononyl)-4-methoxy-6-nony1-2-pyrone; 3- -(! ■ - oxodecyl)-4-methαxy-6-octyl-2-pyrone; 3- ■ d' - oxononyl)-4-hydroxy-6-pentyl-2-pyrone; 3- -d" - oxononyl) -4-hydroxy-6-hexyl-2-pyrone; 3- -d « - oxononyl)-4-hydroxy-6-hepty1-2-pyrone; 3- ■ d' - oxodecyl)-4-hydroxy-6-pentyl-2-pyrone; 3- ■ d' - oxodecyl)-4-hydroxy-6-hexyl-2-pyrone; 3- ■ d' - oxodecyl) -4-hydroxy-6-heptyl-2-pyrone; 3- ■ d'- • oxooctyl)-4-hydroxy-6-heptyl-2-pyrone; 3- •d' - oxooctyl)-4-hydroxy-6-octyl-2-pyrone; 3- d' - oxooctyl) -4-hydroxy-6-nony1-2-pyrone; 3- d' - oxononyl) -4-methoxy-6-hexyl-2-pyrone; 3- (1* - oxononyl) -4-methoxy-6-heptyl-2-pyrone; 3- d- - oxodecyl) -4-methoxy-6-hexyl-2-pyrone; 3- (1* - oxodecyl) -4-methoxy-6-heptyl-2-pyrone; 3- d' - oxooctyl)-4-methoxy-6-octyl-2-pyrone; and 3- d' - oxooctyl)-4-methoxy-6-nonyl-2-pyrone. As will be apparentfroma 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 i idazolide with an appropriate salt of an alkyl hydrogen alonate to form the corresponding 3-oxocarboxylate es-ter; 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-d* -oxo¬ alkyl) -4-hydroxy-6-alkyl-2-pyrones of the formula (V) : wherein R, and 3 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 R, and R 3 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 R, and R 3 being 5 to 12; and/or (D) converting the compounds of formula (ITU 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 or physiologically acceptable salts thereof, wherein: R, and R 3 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 R-, and R 3 being 5 to 12; and R 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): R 3 COCH 2 COOR (XI) wherein R 3 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 (XID : R 3 CCCH 2 COOH: (XΓI) wherein R 3 is as defined above, followed by cyclizing the 3-oxocarboxylic acid of formula XIX with carbonyldiimida- zole to form the corresponding 3-(!•-oxoalkyl)-4-hydroxy-6- alkyl-2-pyrone of the formula (XX) : where R, and R 3 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 (XCH) , with dialkyl sulfate to form the corresponding 3-(!'-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, depend¬ ing 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-oxocarboxylate 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 THF to afford the 3-(1'-oxoalkyl)-4-hydroxy-6-alkyl- 2-pyrones (compounds 9-13).. Deacylation was easily carried 9 o out by heating the 2-pyrone at 130 C in 90% H 2 S0 4 , producing compounds 5-8. The 4-hydroxy group was methylated using dimethyl sulfate and anhyd. K 2 C0 3 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 CF^COOH yielded the desired 3-(1'-oxoalkyl) -4-hydroxy-6-methyl-2-pyrone 11 (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 sodium (compounds 16-18) . Acid hydrolysis of the ethyl derivative (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 concentra¬ tions, 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 l/[Sj 1 ?, [sj/v vs I 14 and 1/v vs rl_I-jrl5. To determine whether the inhibition was pure (in¬ active ESI complex or partial (active ESI complex) , the data were plotted as 1/V i against [ij. If the plot was linear then the inhibitor exhibited pure inhibition: that is, the ESI complex is inactive, and hence cannot dissoc¬ iate to El and P (3=0, Scheme II) . For linear mixed-type inhibitors (Ksapp and Vmaxi change) and pure non-competi- tive inhibitors (Vmaxi chanqes) ,' a p c lot of Ks a c pp/Vmaxi vs i was linear and for pure unco petitive inhibitors (K app/ i constant) a plot of 1/K app vs [ij as linear. A hyperbolic plot of 1/V i vs indicated partial in¬ hibition, with β<l and ESI is active but less so than ES. In this case a rep c lot of ΔVmaxi vs 1/' fLlJlσ--ave a linear p r lot . Using y this p e lot and ΔVmax/Ks app vs 1/ [i] α, B & K. could be calculated. All the equa¬ tions and their derivations can be found in the excellent book by I.H. Segal 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. and K. ' 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. and K. ', are included in Table 1. K. 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. ' 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. ranging from 1995 to 54 μM. An increase in the number of methylenes was accompanied by a decrease in the magni¬ tude of K. ; that is, an increase in hydrophobicity resulted in greater affinity between E and I. K. 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. ranging from 1709 to 83 uM. An increase in the number of methylenes was accompanied by an increase in affinity between E and I, indicating the bind¬ ing region is hydrophobic. We have evidence from mechanis¬ tic 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 reg¬ ions 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 substitu¬ ents ateither the 3 or 6position can bind equally wellto 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. 's ranging from 67 to 4.6 uM. The observed K. values are much lower than those ob¬ served for the analogous 3-(1'-oxoalkyl)- and 4-hydroxy-6- alkyl-2-pyrones. For example, compound 10 has a K. of 15 uM " , while compounds 2 and 6 have K.'s of 442 and 1128 uM, 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 20 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 inter¬ esting feature of the dialkyl compounds 9-13 is that, unlike the monoalkyl compounds 1-4 and 5-8, K. does reach a limit. Based on the results for the long chain derivatives, com¬ pounds 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 obtain¬ ed 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.=4.6 uM) was found to be 18 thirty times as effective as elasnin (I)K.= 140 uM 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 activ¬ ity 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. '=136 and 127 j iM, respectively. Note that since these are uncompetitive inhibitors, it is necessary to compare values of K. ' . The analogous non-methylated compounds 6 and 7, have K. 1 values of 790 and 566 uM, 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 en¬ hanced, resulting in the greater affinity. The inde¬ pendence of K. ■ 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 eth lation 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 = 250 μM, compound 7 (1= 250 uM) inhibits HLE by 24%, while compound 14, (I = 250 uM) 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 pK 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 [ESIJ 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, ' K1. and Ki.', 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; K. '=104, ■ 138 and 204 uM, 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. ' = 127 and 204 uM, 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 esta¬ blished, by comparison of the dissociation constants K. and K.', and based on this we predict that the inhibitor binding region is 24A 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 mech¬ anism of action differs for the two types of compounds, there¬ fore 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. < K. ', 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, (com¬ pounds 5 and 6) the reverse is true, (K. 1 < K.) 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- alky1-2-pyrones, compounds 14 and 15, are uncompetitive inhibitors, which can be considered to be an extreme form of mixed-type inhibition, where K. >> K. ' and 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 21 of Marossy et al 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 P . residue 22 , benzyioxy- carbonyl (Z) , resulted in a decrease in affinity (Scheme III) . These results indicate that there is a region, desig¬ nated subsite S . , 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 S 4 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 S. and S 3 , with the substrate residues, boc and ala bound to S„ and S, respectively. The alkyl chain of the longer homologs, compounds 7 and 8, would be bound at subsites S 2 and S, 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. 18 Spencer et al have suggested that the pyrone 23 carbonyl might be bound to an "oxyanion hole" and if so, the substituent at position 3 could be placed in the P-, site. Our data do not support such a suggestion. Instead, we think that the 3-substituent could be placed at the P j . site, that is, it is bound at the S^ 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 uM for compounds 9-12, 10 uM for compound 13, 1000 uM for compound 19 and 200 uM for the remaining compounds) . The hydro- philic 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 24 time of E and I, unlike the 6-chloropyrones . 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 . subsite, with the 6-alkyl sub¬ stituent binding to S. - S-, subsites for the higher homologs. Compound 11 was found to be the most potent compound, with a K. of 4.6 uM, 30 times more potent than elasnin, K.= 140 uM. The hydrophobic compounds were found to be specific for HLE and the interaction with E is fully reversible. 24 Table 1 Inhibition of HLE in vitro by 4-hydroxy- and 4-methoxy-2- pyrones of the formula: _ Cαπpαund No. R 3 R 4 R 6 K^uM) mechanism 1 COCgH j , OH CH 3 1995 a 1. 0 noncompetitive 2 COC 7 H 15 OH CH 3 442 a 1. 0 noncompetitive 3 OH CH 3 248 a 0 noncompetitive COC 9 H 19 1. 4 COC n H 23 OH CH 3 54 b 1. 0 noncompetitive 5 H OH 1709 a 0. C 5 H 11 2 mixed-type 6 H OH C 7 H 15 1128 a 0. 7 mixed-type 7 H OH 404 C 1. C 9 H 19 4 mixed-type 8 H OH C 11 H 23 83 6. 5 mixed-type 9 COCgH., OH 67 C 1.0 noncompetitive C 5 H 11 10 C0C 7 H 15 OH C 7 H 15 15 c 0. 7 mixed-type 11 COC 8 H 17 OH C 8 H 17 4.6 C 0. 8 mixed-type 12 COC g H 19 OH 7.9 C C 9 H 19 0.7 mixed-type 13 COC u H 23 OH NIA d - C 11 H 23 14 H OCH 3 136 C C 7 H 15 - uncompetitive 15 H OCH 3 127 e C 9 H 19 - uncompetitive 16 a OCH 3 CH=C(OH)COOC 2 H 5 115 a 0. 9 .mixed-type 17 Ξ OCH 3 ' CH=C(OH)COOC 4 H g 345 a 0. 4 mixed-type 18 H OCH 3 CH=C(OH)COOC 5 H I;L 412 c 0. 5 mixed-type 19 H OCH, CH-C(OH)iCOOH NIA f — a: 5% error b: 15% error c: 10% error d: NIA = * no inhibitory activity at 10 uM (aolubility limit) e: 20% error f: NIA= no inhibitory activity at 1000 uM - 25 - 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 LDj- n values of the inhibitors being of the order greater than 3g/Kg of body weight in both mice and rats. Thus, typical LD 5Q values of the inhibitors of the present invention have been found to be as set out immediately below, where the LD 5Q value is in g of compound per Kg of body weight: Compound LD 50 The inhibitor compounds and salts of the invention can be expected to be useful in the treatment of HLE im¬ plicated 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. - 26 - 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 pro¬ duced by processes which are known per se and with which the skilled person is familiar. The physiologically active com¬ pounds 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 con¬ taining 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 concen¬ tration 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, 25 Australia) , using a modification of the methods of 26 27 28 Andrews et al , Viscarell et al and Martodam et al 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 spectro¬ photometer 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 CDCl 3 as internal lock. CDCl., (77.00ppm) or TMS (O.OOppm) were used as internal standards. Melting points were deter¬ mined 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 ug/50 ul) 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 37 C. All inhibitor and substrate solutions were made up in 5% aq. DMSO to retard hydrolysis. HLE (50 ul) , 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 uM) 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 ~ for p-nitrophenol, determined under these assay con¬ ditions. 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 con¬ centration 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 uM for compound 19 and 200 uM for the remaining 2-pyrones. Each concentration was repeated at least twice. Since we normally use 100 M 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 ul of DMSO (as control) , or compound 3 (300 μM) , or compound 16 (3000 M) . 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 ace¬ tate, 0.45M NaCl and 0.1% brij-35 at pH 5.5 and magneti¬ cally 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 RCOCH 2 COOC 2 H 5 Solid magnesium methoxide (0.49g, 5mmol) was added to a solution of ethyl hydrogen malonate (1.2g, lOmmol) in THF aid stirred for 1 h. The solvent was re¬ moved under reduced pressure to give a white slightly hygroscopic salt, Mg (OOCOCH 2 COOEt) , 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(OOCCH 2 COOEt) „ was added. The mixture was stirred for 18 h at 25 C, the solvent was then removed at reduced pressure. The residue was partit¬ ioned between . ether and aq. 0.5M HC1. The ether extract was washed with aq. sat. NaHC0 3 , dried (Na 2 S0 4 ) and con- centrated under reduced pressure to yield a yellow oil, yield > 95%. Synthesis of RCOCH 2 COOH 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% HC1. The precipitated product was collected and thoroughly dried before being used in the next step. Further puri¬ fication was not necessary. Synthesis of 3-(1Oxoalkyl) -4-hydroxy-6-alkyl-2-pyrone Solid carbonyldiimidazole was added to a THF solution of the 3-oxocarboxylic acid. The reaction was stirred under N~ for 24 h, then acidified to pH 1 with 0.5M HC1. The reaction mixture was then extracted with ethylacetate, the organic layer was washed with brine, dried (NaSO.) and concentrated under reduced pressure to yield the desired 2-pyrone (yield > 85%) as an orange solid. Recrystallisation from MeOH yielded white needles. 3- (I'-Qxohexyl) -4-hydroxy-6-pentyl-2-pyrone (compound 9) Anal. ( 16 H 24 0 4 ) CH mp 59.5-60° (MeOH) lit. mp 52° (MeOH) . 1 H NMR (CDCl 3 ) 1.05 (t, J = 3Hz, 6H, CH 3 ) ; 1.36-1.92 (m, 12H, CH 2 ) ; 2.60 (t, J = 5Hz, 2H, CH 2 C=) ; 3.18 (t, J = 5Hz , 2H, CH 2 C=) ; 5.94 (s, 1H, CH=) . 13 C NMR (CDC1 3 ) 13.79, 13.88 (CH 3 ) ; 22.23, 22.44, 23.66, 26.03, 31.05 (8CH 2 ) ; 99.61 (C3) ; 100.69 (C 5 > ; 161.01 (C 6 ) ; 172.49 (C 4 ) ; 181.25 (COO) ; 207.97 (COCH 2 ) . 3-(1'-Oxooctyl) -4-hydroxy-6-heptyl-2-pyrone (compound 10) Anal. (C 2Q H 32 0 4 ) CH mp.64.5° (MeOH) (lit mp 68°) 1H NMR (CDC1 3 ) 0.86 (br t, 6H, CH.,) ; 1.32 (br s, 16H, CH 2 ) ; 1.67 (m, 4H, CH 2 ) ; 2.44 (t, 2H, J = 7Hz, CH 2 C=) ; 3.00 (t, J = 7Hz, 2H, CH 2 C0) ; 5.82 (s, 1H, CH=) . 13 C NMR (CDC1 3 ) 13.99, 14.05 (CH 3 ) ; 22.54, 22.60, 24.00, 26.37, 28.82, 28.80, 29.08, 29.20, 31.57, 31.68, 34.28 (CH 2 ) ; 41.64 (CH 2 C=) ; 99.60 (C 3 ) ; 100.71 (Cg) ; 161.06 (Cg) ; 172.51 (C 4 ) ; 181.27 (COO) ; 207.99 COCH 2 ) . 3- (1 '-Oxononyl) -4-hydroxy-6-octyl-2-pyrone (compound 11) Anal. ( C 22 H 36°4 ) CH mp * 68 - 5-69 ° ( MeOH) ( lit 65°) " 4 * 1 NMR (CDC1 3 ) 0.87 (t, J = 6Hz, 6H, CH 3 ) ; 1.27 (br s, 20H, CH 2 ) ; 1.68 (m, 4H, CH 2 ) ; 2.51 (t, J = 7Hz , 2H, CH 2 =) ; 3.09 (t, J = 7Hz, 2H, CH 2 CO) ; 5.96 (s, 1H, CH=) . 13 C NMR (CDC1 3 ) 14.16 (2CH 3 ) ; 22.54, 23.92, 26.31, 29.09, 29.35, 31.77, 34.22, 41.55 (14CH 2 ) ; 99.51 (C 3 ) ; 100.62 (C 5 ) ; 160.92 (Cg) ; 172.45 (C 4 ) ; 181.18 (COO) ; 207.87 (COCH 2 ) . 3-(1 '-Oxodecyl) -4-hydroxy-6-nonyl-2-pyrone (compound 12) • Anal. ( 24 H 4Q 0 4 ) CH mp. 73.5-74° (MeOH) (lit mp 65°) 1 H NMR (CDCl 3 ) 0.88 (br t, 6H, CH 3 ) ; 1.27 (br s, 24H, CH 2 ) ; 1.71 Cm, 4H, CH 2 ) ; 2.49 (t, J = 7Hz , 2H, CH 2 C=) ; 3.07 (t, J = 7Hz, 2H, CH 2 CO) ; 5.90 (s, 1H, CH=) . 13 C NMR (CDCi 3 ) 14.13 (2CH 3 ) ; 22.72, 24.03, 26.43, 28.99, 29.29, 29.43, 29.52, 31.89, 31.94, 34.34, 41.70 (16CH 2 ) ; 99.63 (C 3 ) ; 100.74 (C 5 ) ; 161.03 (Cg) ; 172.57 (C 4 ) ; 181.30 (COO) ; 207.9 (COCH 2 ) . 3-(l'-Oxododecyl) -4-hydroxy-6-undecyl-2-pyrone (compound 13) Anal. CH mp * 81 - 5 ~ 82 ° (MeOH) ( lit mp 82-83°) 1 H NMR (CDCl 3 ) 0.89 (br t, 6H, CH 3 ) ; 1.30 (br s, 32H, CH 2 ) ; 1.60 (m, 4H, CH 2 ) ; 2.51 (t, J = 7Hz, 2H, CH C=) ; 3.11 (t, J = 7Hz, 2H, CH 2 CO) ; 5.98 (s, 1H, CH=) . 13 C NMR (CDC1 3 ) 14.05 (2CH 3 ) ; 22.63, 23.95, 26.34, 28.91, 29.14, 29.29, 29.52, 31.86, (19CH 2 ) ; 41.61 (CH 2 C=) ; 99.57 (C 3 ) ; 100.65 (C 5 ) 161.01 (C g ) ; 172.48 (C 4 ) ; 181.21 (COO) : 207.93 (COCH 2 ) . Synthesis of 4-hydroxy-6-alky1-2-pyrone The 2-pyrone was added to 5 equivalents (by weight) of 90% H 2 S0 4 and heated at 130°C for 15 min. Ice was added to the cooled black solution with vigorous stirring. Recrystallisation from MeOH:H O(l:l) yielded white needles. 4-hydroxy-6-pentyl-2-pyrone (compound 5) Anal. (C 10 H 14°3 ) CH m P 48 "50° ( MeOH/H 2 0) ( lit mp 46-47) " 1 NMR (CDCl 3 /dg-DMSO) 0.92 (t, J=7Hz , 3H,CH 3 ) ; 1.38 (m, 4H, CH 2 ) ; 1.66 (m, 2H, CH 2 ) ; 2.44 (t, J=8Hz, 2H, CH 2 =) ; 5.38 (d, J=2Hz, 1H, CH=, C 3 ) . 13 C NMR (CDC1.-.) 13.87 (CH 3 ) ; 22.28, 26.37, 31.07, 33.61 (4CH 2 ) ; 89.76 (C 3 ) ; 101.38 (C 5 ) ; 167.34 (.Cg) ; 168.57 (C 2 ) ; 172.80 (C 4 ) . 4-Hydroxy-6-heptyl-2-pyrone (compound 6) Anal. (C 12 H lg 0 3 ) CH mp 71-71.5° (MeOH/H 2 0) (lit mp 71°) R NMR (CDC1 3 ) 0.89 (br t, 3H, CH 3 ) ; 1.27 (br s, 6H CH 2 ) 1.67 (m, 4H, CH 2 ) 2.49 (t, J = 7Hz, 2H, CH 2 CO) 5.59 (d, J = 2Hz, 1H, CH= ; C 3 ) ; 6.00 (d, J = 2Hz , 1H , CH=, C 5 ) . 13 C NMR (CDC1 3 ) 14.06 (CH 3 ) ; 22.62, 26.73, 28.92, 31.67, 33.66 (6CH 2 ) ; 89.81 (C 3 ) ; 101.46 (Cg) ; 167.33 (Cg) ; 168.67 (COO) ; 172.93 (C 4 ) . 4-Hydroxy-6-nonyl-2-pyrone (compound 7) Anal. ( 14 H 22 0 3 ) CH mp 75.5-76° (MeOH/H 2 0) (lit 79°) 1 H NMR (CDC1 3 ) 0.88 (br t, 3H, CH 3 ) ; 1.27 (br s, 12H, CH 2 ) ; 1.64 (m, 4H, CH 2 ) ; 2.48 (t, J = 7Hz , 2H, CH 2 C=) ; 5.62 (d, J = 2Hz , 1H , CH=, C 3 ) 6.00 (d, J = 2Hz , 1H, CH=, Cg) . 13 C NMR (CDClg) 14.11 (CH 3 ) ; 22.70, 26.73, 29.00, 29.28, 29.46, 31.89, 33.67 (8CH 2 ) ; 89.83 (C 3 ) ; 101.39 (C 5 ) ; 167.33 (C g ) ; 168.62 (C 2 ) ; 172.83 (C 4 ) . 4-Hydroxy-6-undecyl-2-pyrone (compound 8) Anal. (C 16 H 2 g0 3 ) mp 82-83° (MeOH/H 2 0) (lit mp 80°) 1 HMR (CDC1 3 ) 0.87 (br t, 3H , CH 3 ) ; 1.27 (s, 14H, CH 2 ; 1.69 (br s, 4H, CH 2 ) ; 2.47 (t, J=6Hz , 2H , CH 2 C=) ; 5.59 (s, 1H, CH=, C 3 ) ; 5.99 (s, 1H, CH=, Cg) . 13 C NMR (CDC1 3 ) 14.10 (CH 3 ) ; 22.71, 22.74; 29.02, 29.28, 29.49 (5CH 2 ) ; 29.63 (2CH 2 ) ; 31.94, 33.69 (CH 2 ) ; 89.81 (C 3 ) ; 101.34 (Cg) ; 167.33 (Cg) ; 168.44 (C 2 ) ; 172.74 (C 4 ) . 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 K 2 C0 3 (8.84 mmoles, 3:1 excess) . The reaction mixture was heated under reflux for 22 h. After cooling, K-CO-, was removed by filtration and the filtrate concentrated under reduced —....—i 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-py one (compound 14) Anal. (C 13 H 20 O 3 ) CH mp 44-45° (MeOH) 1 H NMR (CDC1 3 ) 0.87 (br, t, 3H, CH 3 ) ; 1.31 (br s, 8H, CH 2 ) ; 1.62 (m, 2H, CH 2 ) ; 2.44 (t, J = 8Hz, 2H, CH 2 C=) ; 3.81 (s, 3H, O.CH 3 ) ; 5.42 (d, J = 2Hz, 1H, CH=, C 3 ) ; 5.89 (d, J = 2Hz, 1H, CH=, Cg) . 13 C NMR (CDC1 3 ) 14.01 (CH.-.) ; 22.66, 26.68, 28.93, 29.26, 29.43, 31.85, 33.64 (6CH 2 ) ; 55.80 (OCH 3 ) ; 87.48 (C 3 ) ; 99.62 (Cg) ; 165.03 (C 2 ) ; 165.85 (Cg) ; 171.34 (c 4 ) .- 4- ethoxy-6-nonyl-2-pyrone (compound 15) Anal. ( 15 H 24 0 3 ) CH mp 56.5-57° (MeOH) (lit mp 58-59°) H NMR (CDCl 3 ) 0.88 (br t, 3H, CH 3 ) ; (br s, 8H, CH 2 ) ; 1.62 (m, 2H, CH 2 ) ; 2.44 (t, J = 8Hz , 2H, CH 2 C=) ; 3.81 (s, 3H, OCH 3 ) ; 5.42 (d, J = 2Hz, 1H, CH= (C ) ) ; 5.78 (s, 1H, CH= (Cg)). 13 C NMR (CDC1 3 ) 14.04 (CH 3 ) ; 22.60, 26,68, 28.94, 31.68, 33.64, (8CH 2 ) ; 55.83 (OCH 3 ) ; 87..48 (C 3 ) ; 99.66 (Cg) ; 164.98 (C 2 ) ; 165.82 (Cg) ; 171.37 ( c 4 > 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. ( 12 H 16 0 4 ) CH mp. 66-66.5°C (MeOH) (lit mp 65°) . 1 H NMR (CC1 4 ) 0.86 ( , 3H, alkyl-CH 3 ) ; 1.21- 1.59 (m, 10H, CH 2 ) ; 2.18 (s, 3H, CH 3 C=) ; 2.89 (t, 2H COCH 2 ) ; 5.59 (s, 1H , CH= , Cg) . 13 C NMR (CDC1 3 ) 13.98 (CH 3 -alkyl) ; 22.56 (CH 3 C=) ; 22.54, 29.06, 31.64 (CH 2 ) ; 41.58 (COCH 2 ) ; 99.44 (C 3 ) ; 101.47 (Cg) ; 160.86 (C g ) ; 168.71 (C 4 ) ; 181.21 (C 2 ) ; 207.96 (COCH 2 ) . 3-(1 '-Oxooctyl) -4-hydroxy-6-methyl-2-pyrone (compound 2) Anal. (C 1 4 H 20°4 ) CH mp 67 - 5 - 68 ° ( MeOH ) 1 H NMR (CC1 4 ) 0.86 (br t, 3H, CH_ 3 CH 2 ; 1.21 - 1.59 (m, 10H, CH 2 ) ; 2.18 (s, 3H, CH 3 C=) ; 2.89 (t, J = 7Hz , 2H, COCH 2 ) ; 5.59 (s, 1H, CH=) . 13 C NMR (CDCl 3 ) 13.98 (CH 3 CH 2 ) ; 20.56 (CH 3 C=) ; 22.54, 23.95, 29.06, 29.14, 31.63, 41.58 (6CH 2 ) ; 99.43 (C 3 ) ; 101.47 (Cg) ; 160.86 (Cg) ; 168.71 (C 4 ) ; 181.21 (COO) ; 207.96 (COCH 2 ) . 3-(1 ' -Oxodecyl) -4-hydroxy-6-methyl-2-pyrone (compound 3) Anal. (C lg H 24 0 4 ) CH mp 74-74.5° (MeOH) 1 H NMR (CDC1 3 ) 0.87 (br t, 3H, .CH 3 ) ; 1.29 (s, 12H, CH 2 ) ; 1.60, (br m, 2H, CH 2 ) ; 2.24 (s, 3H, CH 3 C=) ; 3.14 (t, J = 7Hz , 2H, COCH 2 ) ; 5.89 (s, 1H, CH=, Cg) . 13 C NMR (CDC1 3 ) 14.10 (CH 3 -alkyl) ; 20.61 (CH 3 C=) ; 22.72 (.CH 2 ) ; 29.29 (CH 2 ) ; 29.52 (3CH 2 ) ; 31.91 (CH 2 ) ; 41.67 (C0CH 2 ) ; 99.42 (C 3 ) ; 101.50 (Cg) ; 160.80 (Cg) ; 168.80 (C 4 ) ; 181.27 (COO); 207.93 (COCH 2 ) . 3-(l'-Qxododecyl) -4-hydroxy-6-methyl-2-pyrone (compound 4) Anal. (C lg HS0 4 ) CH mp. 82-82.5°C (MeOH) . " hi NMR (CC1 4 ) 0.92 (t, 3H, J = 3Hz, CH 3 -alkyl) , 1.23-1.46 (m,20H, CH ) , 2.27 (s, 3H, CH 3 C=) , 3.04 (t, 2H, J = 5Hz, COCH 2 ) , 5.85 (s, 1H, CH=) . 13 C NMR (CDC1 3 ) 14.07 (CH_ 3 CH 2 ) ; 20.62 (CH 3 C=) ; 22.66, 23.97, 29.23, 24.30, 29.46, 29.47, 29.59, 31.88, (10CH 2 ) ; 41.61 (CH 2 CO) ; 99.48 (C 3 ) , 101.52 (Cg) ; 160.92 (Cg) ; 168.73 (C 4 ) ; 181.26 (COO); 208.011 (COCH 2 ) . Synthesis of 4-methoxy-6-(2 r -hydroxy-2'carboalkoxyvinyl) - 2-pyrone Prepared according to the method of Douglas & Money . 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. ( 11 H 12 Og) CH mp. _ 1 H NMR (CDC1 3 /DMS0) 0.96 (t, J = 6Hz, 3H, CH 3 ) ; 1.51 (m, 4H, CH 2 ) ; 3.81 (s, 3H, OCH 3 ) ; 4.22 (t, J = 7Hz, CH 2 0) ; 5.40 (d, J = 2Hz, 1H, CH=, C 3 ) ; 5.99 (s, 1H, CH, Cg) ; 6.77 (d, J = 2Hz, 1H, CH = COH, C 2 ) . 13 C NMR CDCl 3 /DMSO 13.57 (CH 3 ) ; 18.90 (CH 2 ) ; 30.26 (CH 2 ) ; 55.92 (OCH 3 ) ; 65.92 (OCH 2 ) ; 88.20 (C 3 ) ; 101.41 (Cg) ; 103.16 (CH = COH) ; 146.41 (COH=) ; 157.07 (C g ) ; 163.51 (CO) ; 163.68 (CO) ; 171.20 (c 4 ). Scheme I: a,b,c d,e a) carbonyldiimidazole, THF, RT; b) Mg (OOCCH 2 COOC 2 Hg) ; c) H + ; d) IM NaOH; e) H + ; f) carbonyldiimidazole, THF, RT; g) H + ; h) 90% H 2 S0 4 , 130°; i) (CH 3 ) 2 S0 4 , K 2 C0 3 , pet spirit (60-80°); j) (R'OOC) 2,,' Na; k) H + ; 1) R"C0C1, CF 3 COOH Scheme II S, ' ES + El = ■ ESI- - El + c.κ E=enzyme, S=substrate, I=inhibitor, P=p-nitrophenol , K =[E] [S]/[ES] , O.K =[EI] [S]/[ESI] , S i^ K i =[E] [I]/[El] , ακ i =K i '=[ES] [I]/CESIJ , k =catalytic constant. Pure noncompetitive inhibition Mixed-type linear inhibition Mixed-type hyperbolic inhibition κ ≠κ • ≠ακs /3≠0 Pure uncompetitive inhibition K.=oo /3=0 1 Scheme III ; S 4 S 3 . S 2 S l K s (UM) Z D- -Phe Pro Ala NA a 9.24 b D- -P ' he Pro Ala NA 100 b uc 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. .; 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.; Tsuji ura, 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 E! 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. Westkae per, 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.; einer, 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) : or physiologically acceptable salts thereof, wherein: R, and R 3 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 R, and R 3 being 5 to 12; and R 2 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 R, is oxohexyl and R 2 is hydrogen, then R 3 cannot be pentyl; when R, is oxoheptyl and R 2 is hydrogen, then R 3 cannot be hexyl; when R, is oxononyl and R 2 is hydrogen, then R 3 cannot be octyl; when R, is oxo¬ decyl and R- is hydrogen, then R 3 cannot be nonyl; and when R, is oxododecyl and R 2 is hydrogen, then R 3 cannot be undecyl. 2. A compound or physiologically acceptable salt thereof according to claim 1, wherein R- and R_ are in¬ dependently 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 sub¬ stituents. 3. A compound or physiologically acceptable salt thereof according to claim 1 or 2 wherein R, and R_ 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 0 0 0 -(CH Δ,) χι-C-R',-(CH_s)n-C-O-R 1 or -(CH Δ)n-C-NH-R' , 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 R, and R 3 is 5 to 12; (ii) alkoxy of 5 to 12 carbon atoms, unsubstituted or substituted with physiologically innocuous sub¬ stituents exemplified by 0 0 -0-(CH Δ)n-R" ,-0-(CH Δ) xx-C-O-R' 1 or -O-(CH.n-C-NH-R' ' 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 R, and R 3 is 5 to 12; and (iii) hydrocarbon radicals of 5 to 12 carbon atoms bonded to the pyrone ring through a nitrogen atom, unsub¬ stituted or substituted with physiologically innocuous substituents exemplified by O 0 -NH-(CH 2 ) n -R"', -NH-fαy -C-O-R" 1 or -NH-fO * ^) -C-NH-R'" 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 R, and R-. is 5 to 12. 4. A compound or physiologically acceptable salt thereof according to any one of claims 1 to 3 wherein R 2 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 R-, 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 3 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 R~ is selected from hydrogen, C, g alkyl, C- _ r alkenyl or C-_ c- alkynyl. 8. A compound or physiologically acceptable salt thereof according to any one of claims 1 to 7 wherein R, 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 R 3 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 R 2 is hydrogen or methyl. 11. A compound according to any one of claims 1 to 6 selected from the group: 3- oxononyl ■4-hydroxy-6 -nonyl-2-pyrone; 3- oxodecyl •4-hydroxy-6-octyl-2-pyrone; 3- oxononyl ■4-methoxy-6-octyl-2—pyrone; 3- oxodecyl ■4-methoxy-6-nonyl-2-pyrone; 3- oxononyl -4-methoxy-6-nonyl-2-pyrone; 3- oxodecyl -4-methoxy-6-octyl-2-pyrone; 3- oxononyl -4-hydroxy-6-pentyl-2-pyrone; 3- oxononyl -4-hydroxy-6-hexy1-2-pyrone; 3- oxononyl -4-nydroxy-6-heptyl-2-pyrone; 3- oxodecyl -4-hydroxy-6-pentyl-2-pyrone; 3- oxodecyl •4-hydroxy-6-hexyl-2-pyrone; 3- oxodecyl -4-hydroxy-6-heptyl-2-pyrone; 3- oxooctyl -4-hydroxy-6-heptyl-2-pyrone; 3- oxooctyl -4-hydroxy-6-octyl-2-pyrone; 3- oxooctyl -4-hydroxy-6-nony1-2-pyrone; 3- oxononyl -4-methoxy-6-hexyl-2-pyrone; 3- oxononyl -4-methoxy-6-he tyl-2-pyrone; 3- oxodecyl -4-methoxy-6-hexy1-2-pyrone; 3- oxodecyl -4-methoxy-6-he yl-2-pyrone; 3- oxooctyl -4-methoxy-6-octyl-2-pyrone; 3- 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-(l'- 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 alonate 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 carbonyldi¬ imidazole to afford the corresponding 3-(l' -oxo¬ alkyl) -4-hydroxy-6-alkyl-2-pyrones of the formula (V) : wherein R. and R_* 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 elas¬ tase-type enzymes, the total number of carbon atoms of the longest chain length in each of the groups R, and R-, 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 physiolo¬ gically innocuous substituents which do not interfere 251 with the binding of said compound with elastase-type enzymes; and/or (O 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- 30- 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 35- 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 R and R 3 being 5 to 12; and/or 40. (E) converting the compounds of claim 1 so obtained to a physiologically acceptable salt thereof. 141-.. 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) t or physiologically acceptable salts thereof, wherein: R, and R 3 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 R, and R 3 being 5 to 12; and R 2 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; provided that: when R-, is pentyl and R„ is hydrogen, then R 3 cannot be pentyl; when R, is hexyl and R„ is hydrogen, then R~ cannot be hexyl; when R, is octyl and R~ is hydrogen, then R 3 cannot be octyl; when R-, is nonyl and 2 is hydrogen, then R 3 cannot be nonyl; and when R, is undecyl and R_ is hydrogen, then R-. 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) : R 3 COCH 2 COOR (XI) wherein R 3 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) : R 3 COCH 2 COOH (XII) wherein R-. 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) : ' • - - where R, and R_ 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 com¬ prising as active ingredient at least one compound of formula (IV) : or physiologically acceptable salts thereof, wherein: R-, and R 3 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 R, and R 3 being 5 to 12; and R 2 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. 20. A composition according to claim 19 wherein R, and R 3 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 sub¬ stituents. 21. A composition according to claim 19 or 20 wherein R, and R-. 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 0 0 o -(CH Δ) XX-C-R', -(CH Δ. XI-C-O-R' or -(CH- Δ) XI-C-NH-R', 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 R, and R 3 is 5 to 12; (ii) alkoxy of 5 to 12 carbon atoms, unsubstituted or substituted with physiologically innocuous sub- stituents ' exemplified by 0 0 -0-(CH Δ)n-R", -O-(CH^)n-C-O-R" o-r -0-(CH Δ)n-C-NH-R" 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 R, and R 3 is 5 to 12; and (iii) hydrocarbon radicals of 5 to 12 carbon atoms bonded to the pyrone ring through a nitrogen atom, unsub¬ stituted or substituted with physiologically innoc¬ uous substituents exemplified by -NH- ( CH Δ- ) n -R"' , -NH- ( CH Δ ) n -C-O-R"' or -NH- ( CH Δ ) n -C-NH-R "' wherein n is zero or an integer 1 to 11 and R 1 " 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 physio¬ logically innocuous substituents, provided that the total number of carbon atoms of the longest chain length in the groups R, and R_ is 5 to 12. 22. A composition according to any one of claims 19 to 21 wherein R 2 is selected from alkyl, alkenyl or alkynyl, in¬ clusive 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 R, is oxoalkyl of 5 to 12 carbon atoms. 24. A composition according to any one of claims IS to 23 wherein R 3 is alkyl of 5 to 12 carbon atoms. 25. A composition according to any one of claims 19 to 24 wherein R 2 is selected from hydrogen, C, 5 alkyl, C, 5 alken¬ yl, or C, g alkynyl. 26. A composition according to any one of claims 19 to 22 wherein R, is oxoalkyl of 7 to 10 carbon atoms. 27. A composition according to any one of claims 19 to 23 wherein R 3 is alkyl of 7 to 10 carbon atoms. 28. A composition according to any one of claims 19 to 24 wherein R 2 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-d'- oxononyl)) -4-hydroxy-6-octy1-2-pyrone; 3-d' - oxodecyl)) -4-hydroxy-6-nonyl-2-pyrone; 3- -d' - oxononyl)) -4-hydroxy-6-nonyl-2-pyrone; 3-d' -oxodecyl) - 4-hydroxy-6-octyl-2-pyrone; 3-d' - oxononyl)) -4-methoxy-6-octyl-2-pyrone; 3-d' - oxodecyl)) -4-methoxy-6-nonyl-2-pyrone; 3- ■ d' - oxononyl)) -4-methoxy-6-nonyl-2-pyrone; 3- •d' - oxodecyl)) -4-methoxy-6-octyl-2-pyrone; 3- •d' - oxononyl)) -4-hydroxy-6-pentyl-2-pyrone; 3- - oxononyl)) -4-hydroxy-6-hexyl-2-pyrone; 3- -d - oxononyl)) -4-hydroxy-6-heptyl-2-pyrone; 3- -d - oxodecyl)) -4-hydroxy-6-pentyl-2-pyrone; 3- ■ d - oxodecyl)) -4-hydroxy-6-hexy1-2-pyrone; 3- -d - oxodecyl)) -4-hydroxy-6-heptyl-2-pyrone; 3- -d - oxooctyl)) -4-hydroxy-6-heptyl-2-pyrone; 3- -d - oxooctyl)) -4-hydroxy-6-octyl-2-pyrone; 3- -d - oxooctyl)) -4-hydroxy-6-nonyl-2-pyrone; 3- -d - oxononyl)) -4-methoxy-6-hexyl-2-pyrone; 3- -d '- oxononyl)) -4-methoxy-6-hept 1-2-pyrone; 3- -d 1 - oxodecyl)) -4-methoxy-6-hexyl-2-pyrone; 3- -d '- oxodecyl)) -4-methoxy-6-heptyl-2-pyrone; 3- -d '- oxooctyl)) -4-methoxy-6-octyl-2-pyrone; 3- -d '- 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 composi¬ tion 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 administer¬ ing 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.

Inventors: COOK MARIA LUISA, TERNAI BELA, COOK, MARIA, LUISA, TERNAI, BELA
Applicants: UNIV TROBE, LA TROBE UNIVERSITY
Application year: 1988
Main classification: C07D309
IPC codes: C07D309