NOVEL COMPOUNDS

Abstract: Hydroxamic acid derivatives for use as medicaments, of formula (1), or a salt, solvate or hydrate thereof, in which R1 is hydrogen, C1-6alkyl, or -(CH2)n-A-R4 where n is from 0 to 6, A is a bond or an oxygen or sulphur heteroatom and R4 is an optionally substituted aryl or optionally substituted heteroaryl group; R2 is C3-6alkyl; and R3 is -(CH2)p-X-(CH2)q- where p is an integer from 1 to 9, q is an integer from 2 to 10, and the moiety -(CH2)p- is adjacent to the carbon atom marked with an asterisk in formula (1), and X is -NR5- where R5 is selected from hydrogen, C1-6 alkyl, C2-6 alkanoyl, C1-6 alkoxycarbonyl and aryl, aralkyl or aralkyloxycarbonyl in each of which the aryl moiety is optionally substituted.

Description: AZALACTAM HYDROXAMIC ACID DERIVATIVES AS COLLAGENASE INHIBITORS The present invention relates to novel hydroxamic acids, processes for their preparation and their use in medicine. In particular, the present invention relates to their use as inhibitors of enzymes of the collagenase family of neutral metalloproteases, for treating arthritic and other diseases. The mammalian collagenase family of enzymes comprises a number of proteases, exemplified by interstitial (type I) collagenase itself, the stromelysins (also known as proteoglycanases or transins) , fibroblast and polymorphonuclear leucocyte gelatinases (also known as collagen-IV-ases) , and 'pump-1' (putative metalloprotease 1, uterine metalloprotease) [Goldberg et al, J. Biol. Chem. 2610, 6600, 1986; hitham et al, Biochem. J. 240, 913, 1986; Breathnach βt al. Nucleic Acids Res., 15, 1139, 1987; Muller βt al, Biochem. J., 253, 187, 1988; Collier et al, J. Biol. Chem., 263, 6579, 1988; Murphy et al ' , Biochem. J., 258, 463, 1989; Quantin et al, Biochem. (N.Y.), 28, 5327, 1989; Birkedal-Hansen, J. Oral Pathol., 17, 445, 1988]. Membershi of the mammalian collagenase family of proteases is evident by possession of a number of highly characteristic and experimentally verifiable properties, as described in EPA 401963 (Beecham Group) which can be adopted as criteria for allocation to this family of enzymes. As a particular example of the therapeutic value of inhibitors of the collagenase family of enzymes such as are disclosed in the present invention, chronic arthritic diseases leading to extensive loss of the collagen, proteoglycan and elastin components of the cartilage, bone and tendons within the joints, should be amenable to treatment with inhibitors of the collagenases, proteoglycanases (stromelysins) and gelatinases currently thought to be the major enzymes involved. These enzymes have been detected in extracts of synovial an cartilage tissue, and have also been extensively studied in tissue cultures of a wide range of connective tissues. Apa from control of the biosynthesis, secretion and activation the enzymes, the most important natural regulation of these enzymes in normal and diseased states, is considered to be the endogenous production of inhibitors such as the Tissue Inhibitor of Metalloproteases and alpha-2 macroglobulin. A imbalance between the local levels of the proteolytic enzym and natural inhibitors will allow destruction of connective tissue components to occur. The compounds described in the present invention, being synthetic and low molecular weight inhibitors of this famil of enzymes, offer a therapeutically useful way in which a more normal or non-pathological balance between inhibition and enzymic activity can be restored: they thus act to complement and supplement the endogenous enzyme inhibitors. Indeed, because these enzymes usually act only within restricted pericellύlar environments, before being inactivated by inhibitors circulating in the blood and present in most inflammatory exudates, the low molecular weight inhibitors disclosed here may be more effective than endogenous proteinaceous inhibitors that are excluded by their size from the localized regions of connective tissue destruction. A novel class of azalactam hydroxamic acid derivatives has now been discovered, which are collagenase inhibitors and thus of potential utility in the treatment of diseases in which activity of members of the collagenase family of neutral metalloproteases is implicated. 3 - According to the present invention there is provided a compound of the general formula (1) , or a salt, solvate or hydrate thereof: (1) in which, R]_ is hydrogen, C^-galkyl, or -(CH2) n -A-R where n is from to 6, A is a bond or an oxygen or sulphur heteroatom and R is an optionally substituted aryl or optionally substitute heteroaryl group; R2 is C3_galkyl; and R3 is - (CH2)p-X- (CH2)q- where p is an integer from 1 to 9, is an integer from 2 to 10, and the moiety -(CH2)p- is adjacent to the carbon atom marked with an asterisk in formula (1), and X is -NR5- where R5 is selected from hydrogen, C^-g alkyl, C2- alkanoyl, C]__g alkoxycarbonyl a aryl, aralkyl or aralkyloxycarbonyl in each of which the a moiety is optionally substituted. Unless otherwise specified, each alkyl or alkoxy group is CI- Q group, more preferably a Cι_g group, and may be strai chain or branched. Values for aryl groups include naphthyl and phenyl, preferably phenyl. Heteroaryl groups include thienyl, fur and pyridyl. Optional substituents for aryl and heteroaryl groups will b apparent to those skilled in the art and may be selected fr -OH, Cι_galkyl, Cι_galkoxy and halogen. Preferred values for R^ include hydrogen; C^-^alkyl; and (CH2) n _A-R 4 where n is 1, A is a bond or sulphur atom and R is an aryl or heteroaryl group. Most preferably R]_ is hydrogen or methyl. R2 is preferably a C4 alkyl group, most preferred are C4alk groups such as n-butyl, iso-butyl or sec-butyl, especially iso-butyl. Values for R3 include - (CH2) p -X-(CH2)q- wherein p and q hav values such that R3 forms part of an 11- to 16-membered azalactam structure, and X is a group -NR5- where R5 is hydrogen, methyl, benzyl, t-butoxycarbonyl or benzyloxycarbonyl. Most preferred are compounds where R3 is -(CH2) p - - (CH2)q- where p is 4 and q is 5, or p is 4 and q is 6 and X is -NR5 where R5 is hydrogen. The compounds of formula (1) may form salts with bases e.g. sodium hydroxide. When a basic nitrogen atom is present, t compounds of formula (1) may form acid addition salts e.g. with hydrochloric acid. Such compounds form part of the present invention. Where compounds of formula (1) , or pharmaceutically acceptable salts thereof, form solvates such as hydrates, these also form an aspect of the invention. The compounds of formula (1) have at least three asymmetric centres and therefore exist in more than one stereoisomeric form. The invention extends to all such forms and to mixtures thereof, including racemates, and diastereoisomeric mixtures. Preferred isomers are those having the (S)-configuration at the chiral centre marked with an asterisk in formula (1) . The present invention provides the compounds of formula (1) or pharmaceutically acceptable salts thereof for use as active therapeutic agents, particularly as agents for treatment of musculo-skeletal disorders resulting from collagenolytic activity, particularly arthritic diseases, and tissue remodelling. Compounds of formula (1) also have potential utility in the treatment of cancer; for preventing myelin degradation in th central and peripheral nervous system; and in other conditions in which members of the collagenase family of neutral metalloproteases have pathological or other roles. The present invention also provides a process for the preparation of a compound of formula (1) , which process comprises : (a) deprotecting a compound of the formula (2) (2) in which R is an O-protecting group and R^, R2 and R3 are as hereinbefore defined; or (b) coupling a compound of the formula (3) or an activated derivative thereof : (3) in which Rl, R2 and R3 are as hereinbefore defined with hydroxylam or a salt thereof; and (c) optionally after step (a) or step (b) converting compound of the formula (1) into another compound of the formula (1) . Suitably R in a compound of the formula (2) is benzyl or a substituted benzyl group, preferably R is benzyl. Cleavag of the group R may be carried out by standard deprotection procedures known in the art, including hydrogenolysis. Suitably, a compound of the formula (2) in which R is benz or substituted benzyl is hydrogenated in the presence of a metal catalyst such as palladium on charcoal in a suitable organic solvent such as a Cι_4alcohol at ambient or elevat (e.g. 20-60°C) temperature. A compound of the formula (1) may be prepared by reacting compound of the formula (3) with hydroxylamine or a salt thereof using standard coupling techniques. For example a compound of the formula (3) may be reacted with a coupling reagent such as ethyl chloroformate or l-ethyl-3-[3- (dimethylamino)-propyl]carbodiimide hydrochloride (EDC) in the presence of l-hydroxybenzotriazole in a suitable solven such as dichloromethane, followed by reaction with hydroxylamine or a salt thereof, for example hydroxylamine hydrochloride. A compound of the formula (2) may be prepared by coupling, for example by conventional coupling techniques, a compound of the formula (3) with an O-protected hydroxylamine, for example benzylhydroxylamine. Suitably a compound of the formula (3) may be treated with a standard coupling agent such as EDC/1-hydroxybenzotriazole or 1,3-dicyclohexyl- carbodiimide/1-hydroxybenzotriazole in a suitable inert solvent such as dichloromethane or acetonitrile in the presence of a base such as triethylamine or diisopropyl- ethylamine followed by addition of benzylhydroxylamine or a salt thereof. Compounds of the formula (3) may be prepared by hydrolysis a compound of the formula (4) : (4) in which R l' R 2 anc R 3 are as hereinbefore defined and Rg is an ester forming group. Suitably Rg in a compound of the formula (4) is C]__galkyl especially methyl or an optionally substituted benzyl gro Suitably a compound of the formula (4) is hydrolysed by reaction with an aqueous base such as sodium hydroxide or potassium hydroxide optionally in a co-solvent such as a Cι_ alcohol or 1,4 dioxane at ambient temperature. Alternatively a compound of the formula (3) in which R^ is -(CH2) n -A-R where n is 1, and A is oxygen or sulphur and is as hereinbefore defined may be prepared by reacting a compound of the formula (5) : (5) in which R2 and R3 are as hereinbefore defined with the appropriate thiol or phenol, R4-A-H in which A is oxygen or sulphur an R4 is as hereinbefore defined. Suitably a compound of the formula (5) is reacted with a thiol or phenol optionally in the presence of an organic solvent such as toluene at ambient or elevated temperature (e.g. 40-100°C) , preferably at 60°C. A compound of the formula (5) may be prepared from a compo of the formula (6) : (6) in which R2 and R3 are as hereinbefore defined, R7 is a C]__galkyl o an optionally substituted benzyl group, and Rg is a Cι_galkoxycarbonyl or benzyloxycarbonyl group. Suitably a compound of the formula (6) in which R7 is optionally substituted benzyl and Rg is benzyloxycarbonyl be hydrogenated to the corresponding malonic acid, derivative, which is then treated with aqueous formaldehyde and piperidine to give a compound of the formula (5) as hereinbefore defined. A compound of the formula (6) may be prepared by a procedur similar to that outlined in PCT/GB89/01398. A compound of the formula (4) may be prepared by coupling a acid of the formula (7) : (7) in which R^, R2 and Rg are as hereinbefore defined with an a ine o the formula (8) : (8) in which R3 is as hereinbefore defined. Suitably a compound of the formula (7) is converted to its corresponding acid chlorid using a standard chlorinating agent, such as thionyl chlor or oxalyl chloride, optionally in an organic solvent such dichloromethane at ambient or reduced temperature (e.g. -1 to 0°C) followed by addition of an amine of the formula (8) Where R5 in R3 is hydrogen, the secondary amine is suitabl in protected form, for example as a benzyloxycarbonyl derivative. Alternatively, the reaction may be carried out in the presence of a coupling agent, such as dicyclohexyl- carbodiimide or l-ethyl-3-[3-(dimethylamino)propyl]- carbodiimide hydrochloride in the presence of 1-hydroxy- benzotriazole, or using 1,1'-carbonyldiimidazole, in an in solvent such as dichloromethane or acetonitrile. Compounds of formula (8) may be prepared by oxidising the primary alcohol function in a compound of formula (9) : (9) wherein p and q are as defined for R3 in formula (1) , Y is nitrogen protection group, and Z is R5, to give the corresponding aldehyde, followed by removal of Z when R5 is an acyl group; cyclisation and reduction; and thereafter, a necessary, removing the nitrogen protection group Y and interconverting R5. Suitable values for Y include t-butoxycarbonyl (BOC) and benzyloxycarbonyl groups, preferably t-butoxycarbonyl. The oxidation may be carried out using pyridinium chlorochromate or under Swern oxidising conditions, for example by treatment with di ethylsulphoxide and an acyl halide followed by triethylamine, as described by D. Swern al., J. Org. Chem., 43, 2480 (1978) . The cyclisation and reduction step may be effected by catalytic hydrogenation over a suitable noble metal catalyst, for example palladium on carbon, or by reaction with sodium cyanoborohydride or sodium borohydride. In some cases the yield of azalactams may be increased by carrying out the reductive amination ste under acidic conditions. Nitrogen protection groups may be removed by standard methods. A Y or Z t-butoxycarbonyl group may be removed by treatment with trifluoroacetic acid at reduced temperature. A Y or Z benzyloxycarbonyl group may be removed by conventional hydrogenation over palladium on carbon, or by treatment with formic acid-methanol and palladium black. group Z may be selected to undergo concomitant cleavage during the cyclisation reaction to give a compound in whic R5 is hydrogen. For example, when Z is a benzyloxycarbony group, it will be readily removed by catalytic hydrogenati An R5 hydrogen may be interconverted to an R5 C^-g alkyl, aralkyl or aryl group. The secondary amine group may be alkylated, to form an R5 alkyl group. For example the ami group may be methylated to form an R5 methyl group. The methylation step may be effected by catalytic hydrogenation over a suitable noble metal catalyst, for example palladium on carbon, in the presence of aqueous formaldehyde. Other suitable methylation procedures are described by E. Askitoglu et al., Helv. Chi . Acta., 68, 750, (1985); E. Engler et al., Helv. Chim. Acta., 68, 789, (1985); and M. Lennon et al., J. Chem. Soc. (Perkin I), 622, (1975). Compounds of formula (9) may be prepared by reacting a compound of formula (10) : O (10) wherein p, Y and Z are as defined for formula (9), with a compound of formula (11) : H 2 N-(CH 2 ) q -OH (11) wherein q is as defined for formula (9) . The reaction may be carried out using standard procedures f forming an amide from a carboxylic acid and an amine, for example using a coupling agent such as 1, 1'-carbonyl- diimidazole, 1,3-dicyclohexyl-carbodiimide or l-ethyl-3-[3- (dimethylamino)propyl]carbodiimide hydrochloride in the presence of l-hydroxybenzotriazole. Compounds of formula (10) are di-aminoalkanoic acid derivatives. These are known compounds or may be prepared from known starting materials by standard methods. For example the compound of formula (8) in which R3 is - (CH2) p -X- (CH2)q- where p is 3, q is 3 to 8 and X is -NH- is prepared from a compound of formula (10) derived from ornithine which is commercially available. The compound of formula (8) in which R3 is - (CH2)p-X- (CH2)q- where p is 4, q is 3 to 8 and X is -NH- is prepared from a compound of formula (10) derived from the amino acid lysine. The compound of formula (10), derived from (S)-lysine, in which Y is t-butoxycarbonyl and Z is benzyloxycarbonyl, is commercially available. Similarly, the compound of formula (8) in which R3 is - (CH2)p-X- (CH2) q - where p is 1, q is 3 to 8. and X is -NH- ma be prepared from 2,3-diaminopropionic acid. The amino alcohols of formula (11) are known compounds. Compounds of the formula (7) are either commercially available or may be prepared by standard techniques from commercially available starting materials. For example a compound of the formula (7) in which R ] _ is hydrogen may be prepared by procedures analogous to those described in EPA 274453. A compound of the formula (7) in which R^ is a methyl grou may be prepared from a compound of the formula (12) : R_ (12) in which R^ is a methyl group, R2 and Rg are as hereinbefo defined and R9 is an ester forming group. Suitably a compound of the formula (12) is treated with an acid, such as trifluoroacetic acid, at ambient or reduced temperature (e.g. -5 to 0°C) . Suitably the groups Rg and R9 are chosen so that R9 is selectively cleaved in the presence of acid producing a compound of the formula (7) in which Rg is retained. Preferably R9 is a t-butyl group. A compound of the formula (12) in which R ] _ is methyl may be prepared from a compound of the formula (13) : (13) in which R2 and Rg are as hereinbefore defined. Suitably a compound of formula (13) is treated with an alcohol RgOH in the presence of an acid catalyst in an alcohol solvent at elevated temperature, for example at the reflux temperature of the reaction mixture followed by hydrogenation in the presence of a noble metal catalyst. Alternatively a compound of formula (12) in which R ] _ and Rg are methyl may be prepared by treating a compound of formula (13) with diazomethane followed by hydrogenation of the resulting ester. It will be appreciated that a compound of formula (13) may b converted to a compound of formula (12) by initial lydrogenation followed by esterification with an alcohol RgOH, or diazomethane. Compounds of the formula (13) may be prepared by standard procedures, for example using the procedures analogous to those described in PCT/GB89/01399. Where obtainable, pharmaceutically acceptable salts of the compounds of formula (1) may be formed conventionally by reaction with the appropriate acid or base. Solvates may be formed by crystallization from the appropriate solvent. As mentioned previously, the compounds of formula (1) exist in more than one diastereoisomeric form. Where the processes of the invention produce mixtures thereof, the individual isomers may be separated one from another by chromatography, e.g. column chromatrography or HPLC. Alternatively, separate diastereoisomeric compounds of formula (1) can be obtained by using stereoisomerically pure starting materials or by separating desired isomers of intermediates at any stage in the overall synthetic process, and converting these intermediates to compounds of formu (1) • It will be appreciated that although the absolute configuration at a particular chiral centre may not be k it is possible to characterise a given diastereoisomer relative to its epi er or to another diastereoisomer usi NMR spectroscopy or optical rotation. The present invention further provides a pharmaceutical composition, which comprises a compound of formula (1) , pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. A composition of this invention is useful in the treatme musculo-skeletal disorders, particularly arthritic disea and for modulation of tissue remodelling. A composition of the invention, which may be prepared by admixture, may contain a diluent, binder, filler, disintegrant, flavouring agent, colouring agent, lubrica preservative as described in EPA 401963. A composition of the invention may be adapted for oral, topical, rectal or parenteral administration but oral administration is preferred. Parenteral compositions ma administered intravenously, intramuscularly or intra-articularly. The preferred route for administrat will depend upon the disorder for which treatment is required, and is preferably in unit dosage form or in a that a human patient may administer to himself in a singl dosage. Suitable compositions for the compounds of this invention have been described in EPA 401963. The suitable dosage range for the compounds of the invent may vary from compound to compound and may depend on the condition to be treated. It will also depend, inter alia, upon the relation of potency to absorbability and the mode administration chosen. Compositions may, for example, be in the form of tablets, capsules, sachets, vials, powders, granules, lozenges, reconstitutable powders, or liquid preparations, for exampl solutions or suspensions, or suppositories. For topical and percutaneous administration, the preparatio may also be presented as an ointment, cream, lotion, gel, spray, aerosol, wash, skin paint or patch. A unit dose for treating diseases and physiological phenome in which enzymes from the collagenase family are involved will generally contain from 10 to 1000 mg and preferably wi contain from 10 to 500 mg, in particular 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 mg. The composition ma be administered once or more times a day, for example 2, 3 4 times daily, so that the total daily dose for a 70 kg adu will normally be in the range 10 to 3000 mg. Such a dose corresponds to approximately 0.15 to 50 mg/kg per day. Alternatively, in particular for injection, the unit dose will contain from 2 to 20 mg of a compound of the invention and be administered in multiples, if desired, to give the desired daily dose. The present invention additionally provides a method of treating conditions in which degradation of connective tiss and other proteinaceous components of the body occurs, such as rheumatism and/or arthritic conditions in mammals, such humans, which comprises administering to the mammal in need of such treatment an effective amount of a compound of formula (1) or a pharmaceutically acceptable salt thereof. The present invention also provides the use of a compound of formula (1) or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of conditions in which degradation of connective tissue and other proteinaceous components of the body occurs such as rheumatism and/or arthritic conditions. Description 1 N α -tert-Butoxycarbonyl-N ε -benzyloxycarbonyl-(S)-lysine-(5- hydroxy)pentylamide (Dl) To a solution of N -tert-butoxycarbonyl-N- ε -benzyloxy- carbonyl-(S)-lysine (7.8g, 21 mmol) in anhydrous dichloromethane (150 ml) maintained at 0°C was added 1-eth 3-[3- (dimethylamino)propyl]carbodiimide hydrochloride (4. 26.5 mmol). The mixture was stirred for 0.5 h at 0°C, 5- aminopentan-1-ol (2.3g, 22.5 mmol) added and stirring continued at room temperature. After 3h the mixture was washed with saturated aqueous aHCθ3 ( 60 ml ) • * dr i ed over anhydrous magnesium sulphate and evaporated in vacuo to afford a viscous oil. Purification by flash chromatograp [CHCl3:MeOH) (20:1) v/v] gave the title compound (Dl) as a clear oil (8.01g) . Observed FAB (M+H) + 466. C24H39θgN3 requires M 465. Description 2 N α -tert-Butoxycarbonyl-N ε -benzyloxycarbonyl-(S)-lysine-(4- formyl)butyla de (D2) To a stirred solution of oxalyl chloride (1.47g, 12 mmol) anhydrous dichloromethane (40 ml) maintained under an atmosphere of nitrogen at -60°C was added dimethyl sulphox (1.21g, 15 mmol) dropwise, such that the temperature remai below -50°C. The mixture was left stirring at -60°C for 1 mins, alcohol (Dl) (3.6g, 7.7 mmol) diluted in anhydrous dichloromethane (10 ml) was added, and allowed to warm up -25°C over lh. The mixture was then cooled down to -60°C, triethylamine (4.7g, 46 mmol) added slowly such that the internal temperature remained below -50°C. On completion addition, the mixture was gradually warmed up to room temperature, washed with water (30 ml) and sat. aq. NaCl (30 ml) . The aqueous washes were back extracted with dichloromethane (2x30 ml) and the combined organic fractions were dried over anhydrous magnesium sulphate and evaporated in vacuo to yield a viscous clear oil. Purification by flas chromatography [ (EtOAc:MeOH) (20:1)v/v] afforded the title compound (D2) as an oil (2.8g). Observed FAB (M+H) + 464 . C24H 37 0 N3 requires M 463 . Description 3 (S) -3- (N-tert-Butoxycarbonyl) a ino-8- (N-benzyloxycarbonyl) 1 , 8-diazacyclotridecan-2-one (D3) Method A The aldehyde (D2) (1.8g, 3.88 mmol) was dissolved in ethanol (180 ml) and hydrogenated over 5% palladium-carbon (200 mg) at atmospheric pressure and 35°C for 72h. The suspension was filtered through Kieselguhr and evaporated in vacuo to give crude (S)-3-(N-tert-butoxycarbonyl)amino-1, 8-diazacyclo- tridecan-2-one. The crude amine was dissolved in a mixed solvent system of tetrahydrofuran/water, (6:20 v/v) cooled to 0°C and treated with benzyl chloroformate (0.66g, 3.88 mmol) and excess sodium carbonate to maintain a pH between 10 and 11. The mixture was left stirring at room temperature overnight, washed with ethyl acetate (3x25 ml) , and the combined organic fractions dried over anhydrous magnesium sulphate and evaporated in vacuo to afford a clear oil. Purification by flash chromatography [ (EtOAc:MeOH) (20:1)v/v] yielded the title compound (D3) as a white solid (0.2g) . Observed FAB (M+H) + 447. C24H37O5N3 requires M 447 Method B The aldehyde (D2) was hydrogenated at about 100 psi over 5% palladium on carbon in methanol, and then in acidic methanol to afford crude (S)-3- (N-tert-butoxycarbonyl) amino-1, 8- diazacyclotridecan-2-one. The amine was treated with benzyl chloroformate and purified as described in Method A to yiel the identical title compound (D3) . Description 4 (S)-3-Amino-8-(N-benzyloxycarbonyl)-1,8-diazacyclotridecan-2 one triflυoroacetate (D4) A cooled (0°C) solution of the lactam (D3) (1.21g) in dichloromethane (30 ml) was treated with trifluoroacetic aci (12 ml) . After 0.5h the solvent was evaporated to dryness i vacuo to give crude title compound (D4) as an oil. This was used as such without further purification in the preparation of (D6) . Description 5 3-Methoxycarbonyl-2-(2-_nethylpropyl)propanoic acid (D5) This compound was prepared from 3,4-dihydro-3-(2-methyl- propylidene)-2,5-furandione (EPA 274453) by heating in methanol followed by hydrogenation over 10% palladium-carbon in methanol, as a colourless oil. δ (CDCI3) : 0.92 (3H, d, J=6Hz) , 0.96 (3H, d, J=6Hz) , 1.33 (1H, m) , 1.65 (2H, m) , 2.45 (1H, dd, J=16, 5Hz) , 2.6 (1H, dd J=16, 10Hz), 2.92 (1H, m) and 3.70 (3H, s) . Description 6 (S)-3-N-[3-Methoxycarbonyl-2-(2-n_ethylpropyl)propanoyl]amin 8-(N-benzyloxycarbαnyl)-1,8-diazacyclotridecan-2-one (D6) A solution of 3-methoxycarbonyl-2- (2-methylpropyl)propanoic acid (D5) (0.56g) in dichloromethane (20 ml) was treated wit an excess of oxalyl chloride (0.75g) and one drop of dimethy formamide and the solution was stirred at 0°C for 0.5h and then evaporated to dryness in vacuo to afford 3-methoxy- carbonyl-2-(2-methylpropyl)propanoyl chloride. (υ max . 1785 cm -1 ) . This acid chloride, dissolved in dichloromethane (20 ml) at 0°C, was then treated with a solution of dichloromethane (5 ml) containing a mixture of (S)-3-amino-8-(N-benzyloxy- carbonyl)-1, 8-diazacyclotridecan-2-one trifluoroacetate (D4) [the crude product from (D3) (1.21g)] and diisopropyl- ethylamine (1.05g), and the resulting solution was stirred a 0°C for lh, and then at room temperature for a further 2h. The reaction mixture was evaporated to dryness in vacuo and dissolved in chloroform. The chloroform was washed twice with 5% aqueous citric acid, 10% sodium carbonate, and brine It was dried ( a2Sθ4) and evaporated to dryness in vacuo. The crude product (1.3g) was chromatographed on silica gel 6 (120g) , eluting with ethyl acetate - pentane (1:1) and finally ethyl acetate. Collection of early fractions from the column, followed by evaporation to dryness in vacuo and trituration with ether, gave the title compound as a single diastereoisomer (D6A) (0.6g), as a white solid, m.p. 154-158°C. Observed FAB (M+H) + 518. C 2 H 4 3N 3 0 requires M 517. Found: C, 64.95; H,8.34; N,8.12. C2 H 4 3N 3 0 requires C, 64.97; N,8.37; N,8.12%. Collection of later fractions from the column, followed by evaporation to dryness in vacuo and trituration with ether, gave the title compound as a single diastereoisomer (D6B) (0.44g) as a white solid, m.p. 173-176°C. Observed FAB (M+H) + 518. C28H43N3θ requires M 517. Found: C, 65.85; H,8.34; N,8.16. C2sH 3N 3 0 requires C, 64.9 H,8.37; N,8.12%. Description 7 (S)-3-N-[3-Carboxy-2-(2-methylρropyl)propanoyl]a ino-8-(N- benzyloxycarbonyl)-1,8-diazacyclotridecan-2-one (D7) To a stirred solution of (S)-3-N-[3-methoxycarbonyl-2-(2- methylpropyl)propanoyl]amino-8-(N-benzyloxycarbonyl)-1, 8- diazacyclotridecan-2-one (D6A) (0.51g) in 1,4-dioxane (5 ml at 0°C was added, dropwise, a solution of potassium hydroxi (83 mg) in water (2 ml), and the solution was stirred at 0° for lh. It was acidified with 2N hydrochloric acid and extracted with chloroform. The chloroform extract was wash with brine, dried (MgS04), and evaporated to dryness in vac to afford the title compound, as a single diastereoisomer (D7A) (0.5g), that was used without purification in the nex step. Following the same procedure, the ester (D6B) (0.4g) gave t title compound (D7B) (0.39g) as a single diastereoisomer, that was used without purification. Description 8 (S)-3-N-[3-(N-Benzyloxycarboxamido)-2-(2-methylpropyl)- propanoyl]amino-8-(N-benzyloxycarbonyl)-1,8-diazacyclo- tridecan-2-one (D8) To a solution of (S)-3-N-[3-carboxy-2-(2-methylpropyl)- propanoyl]amino-8-(N-benzyloxycarbonyl)-1, 8-diazacyclo- tridecan-2-one (D7A) (0.48g) in dichloromethane (40 ml) at 0°C was added l-ethyl-3-[3-(dimethylamino)propyl]carbodiimid hydrochloride (0.22g) and the solution was stirred at 0°C fo 0.5h. O-Benzylhydroxylamine hydrochloride (0.198g) and diisopropylethylamine (0.26g) were added and the mixture wa stirred at 0°C for lh and then left at room temperature overnight. The solution was evaporated to dryness in vacuo, and the residue dissolved in chloroform. The chloroform solution wa washed with IN hydrochloric acid, 10% sodium carbonate, brine, and then dried ( a2S04) and evaporated to dryness in vacuo. The product was chromatographed on silica gel 60 (lOOg) , eluting with ethyl acetate, followed by methanol- ethyl acetate (1:49) to give the title compound as a single diastereoisomer (D8A) (0.22g), as a white solid, m.p. 198- 201°C. Observed FAB (M+H) + 609. C3 H 8 N 4 0g requires M 608. Found: C, 66.89; H,7.85; N,9.23. C^^g^O requires C, 67.08 H,7.95; N,9.20%. Following the same procedure, the carboxylate (D7B) (0.35g) afforded the title compound as a single diastereoisomer (D8B (0.18g), as a white solid, m.p. 203-206°C. Observed FAB (M+H) + 609. C3 H 8N4O6 requires M 608 Found: C, 66.70; H,7.84; N, 9.20. C^^g^Og requires C, 67.08; H,7.95; N,9.20%. Description 9 3-(R)-(tert-Butoxycarbonyl)-2-methylene-5-methylhexanoic aci (D9) Piperidine (4.8g) was added to a solution of benzyl (2- benzyloxycarbonyl)-3 (R)- (tert-butoxycarbonyl) -5-methyl- hexanoate 1 (13.5g) in ethanol (100 ml) and the solution was stirred at room temperature for 15 min. A solution of 40% aqueous formaldehyde (23 ml) was then added and the solution was stirred at room temperature for 48h, evaporated to dryness in vacuo and dissolved in ethyl acetate. It was washed with 5% citric acid, brine, and dried (MgSθ4) . The product was chromatographed on silica gel 60 (700 g) eluting with pentane, and then ethyl acetate-pentane (1:4), to afford the title compound (D9) (7.1g) as a colourless oil. Found: C, 64.01; H,9.21. C!3H22θ4 requires: C, 64.44; H, 9.15%. δ (CDC1 3 ) : 0.9 (3H,d,J=6Hz) , 0.95 (3H,d,J=6Hz) , 1.42 (9H,s), 1.5-1.8 (3H,m), 3.5 (1H,t,J=6Hz) , 5.82 (lH,s) and 6.45 (lH,s) . 1 PCT/GB89/01399. Description 10 Methyl 3-(R)-(tert-butoxycarbonyl)-2-(S)-methyl-5- methylhexanoate (D10) A solution of 3-(R)-(tert-butoxycarbonyl)-2-methylene-5- methylhexanoic acid (D9) (6.5g) in ethanol (150 ml) was hydrogenated over 10% palladium-carbon (l.Og) at room temperature and atmospheric pressure until uptake of hydrogen ceased (6h) . The solution was filtered (Kieselguhr) and evaporated to dryness in vacuo. The product, dissolved in ether (50 ml) , was then treated with an excess of ethereal diazomethane and evaporated to dryness in vacuo. The product was chromatographed on silica gel 60 (400g) , eluting with pentane followed by ether-pentane (1:4). Early fractions from the column were combined and evaporated to dryness in vacuo to afford the title compound (D10) as a single diastereoisomer, in the form of a colourless oil (1.4g). δ (CDC1 3 ) : 0.89 (3H,d,J=6Hz), 0.93 (3H,d,J=6Hz) , 1.5-1.7 (ca.3H,m), 2.65 (2H,m) and 3.7 (3H,s). Later fractions from the column (2.8g) contained the title compound contaminated with approx. 10% of a second diastereoisomer. Description 11 N α -tert-Butoxycarbonyl-N ε -benzyloxycarbonyl-(S)-lysine-(6- hydroxy)hexylami.de (Dll) A solution of N α -tert-butoxycarbonyl-N ε -benzyloxycarbonyl- (S)-lysine (15.8g, 0.042 mol) in anhydrous dichloromethane (200 ml) maintained at 0°C, was treated sequentially with 1- ethyl-3-[3- (dimethylamino)propyl]carbodiimide hydrochloride (9.96g, 0.051 mol) and l-hydroxybenzotriazole (7.0g, 0.051 mol) . The solution was stirred at 0°C for lh, treated with 6-aminohexan-l-ol (4.7g, 0.046 mol), and left stirring overnight at room temperature. The mixture was then washed with saturated aqueous aHCθ3, dried over anhydrous magnesium sulphate, and evaporated in vacuo to afford a viscous oil. Purification by flash chromatography [ (CHCl3:MeOH) (20:1 v/v) gave the title compound (Dll) as a clear oil (16g) . Observed (M+H) + 480. requires M 479 Description 12 N α -tert-Butoxycarbonyl-N ε -benzyloxycarbonyl-(S)-lysine-(5- formy1)pentylamide (D12) A stirred solution of dimethyl sulphoxide (3.63g, 0.046 mol) in anhydrous dichloromethane (100 ml) maintained at -60°C, was treated with oxalyl chloride (2.58g, 0.0198 mol) diluted in dichloromethane (10 ml) at such a rate, so as to ensure the temperature remained below -50°C. After stirring for 20 mins, the alcohol (Dll) (6.35g, 0.013 mol) dissolved in dichloromethane (50 ml) was added dropwise over 5 mins. The mixture was stirred at -60°C for 15 mins, warmed up to -35°C, stirred for a further 10 mins then cooled down to -60°C. The solution was treated with triethylamine (8g, 0.08 mol), warmed up to room temperature, washed with water (2x100 ml) , dried over anhydrous magnesium sulphate and solvent evaporated under reduced pressure to afford a viscous oil. Purification by flash chromatography [ (EtOAc:MeOH) (30:1) v/v] gave the title compound (D12) as an oil (5g) . Observed (M+H) + 478. C25H 39 N 3 0 requires M 477. Description 13 (S)-3-(N-tert-Butoxycarbonyl)amino-8-(N-benzyloxycarbonyl)- 1,8-diazacyclotetradecan-2-one (D13) The aldehyde (D12) (5.0g) in methanol (450 ml) was treated with 5% palladium-carbon (5.5g) . The suspension was hydrogenated at 140 psi and ambient temperature for 48h, treated with 2.5M aqueous hydrochloric acid (3 ml) and hydrogenation continued at the said pressure for a further 24h. The suspension was filtered through Kieselguhr and evaporated in vacuo to give crude (S)-3-(N-tert- butoxycarbonyl)amino-1, 8-diazacyclotetradecan-2-one. The crude amine was dissolved in a mixed solvent system of tetrahydrofuran/water (10:40 v/v) cooled to 0°C, and treated with benzyl chloroformate (2.85g) and excess sodium carbonate to maintain a pH between 10 and 11. The mixture was left stirring at room temperature for 4h, solvent partially evaporated in vacuo and the residue extracted with dichloromethane (3x100 ml) . The organic fraction was dried over anhydrous magnesium sulphate and evaporated in vacuo to afford a clear oil. Purification by flash chromatography [EtOAc:MeOH) (50:1) v/v] yielded the title compound (D13) as a white solid (2.0g) m.p. 131.5 - 134.0°C. Observed M + 461. C25H39N3O5 requires M 461. Description 14 (S)-3-Amino-8-(N-benzyloxycarbonyl)-1,8-diazacyclotetradecan 2-one trifluoroacetate (D14) A cooled (0°C) solution of the lactam (D13) (1.78g) in dichloromethane (50 ml) was treated with trifluoroacetic aci (20 ml) . After lh the solvent was evaporated under reduced pressure, to afford the crude title compound (D14) as an oil. This was used as such without further purification. Description 15 (S)-3-N-[3-(S)-Methoxycarbonyl-2-(R)-(2-methylpropyl)- butanoyl]amino-8-(N-benzyloxycarbonyl)-1,8-diazacyclo- tetradecan-2-one (D15) Methyl 3-(R)-(tert-butoxycarbonyl)-2-(S)-methyl-5-methyl hexanoate (D10) (lg) was added to 5% aqueous trifluoroacetic acid (15 ml) at 0°C and the solution was stirred at 0°C for 8h and then evaporated to dryness in vacuo. The resulting carboxylic acid was dissolved in dichloromethane (20 ml) , cooled to 0°C, and then the solution was treated with 1- ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochlorid (0.74g) and l-hydroxybenzotriazole (0.52g). After stirrin for 0.5h, the reaction mixture was sequentially treated wi the crude lactam salt (D14) (3.87 mmol) and N,N-diisopropy ethylamine (lg) and stirring was continued at room temperature overnight. The mixture was then washed with water, sodium bicarbonate solution, dried (MgSθ4) and evaporated to dryness in vacuo. The product was chromatographed by flash chromatography (1% methanol in chloroform) to give the title compound (D15) as a single diastereoisomer. Observed M + 545. C3øH47θgN3 requires M 545. Description 16 (S)-3-N-[3-Carboxy-2-(2-methylpropyl)butanoyl]amino-8-(N- benzyloxycarbonyl)-1,8-diazacyclotetradecan-2-one (D16) The title compound was prepared as a mixture of diastereoisomers from (S)-N-[3-(S)-methoxycarbonyl-2- (R)- ( methylpropyl)butanoyl]amino-8-(N-benzyloxycarbonyl)-1, 8- diazacyclotetradecan-2-one (D15) by the procedure describe for (D7) . Observed M + 531. C2 H4 5 0gN 3 requires M 531. Description 17 (S)-3-N-[3-(N-Benzyloxycarboxamido)-2-(2-methylpropyl)- butanoyl]amino-8-(N-benzyloxycarbonyl)-1,8-diazacyclo- tetradecan-2-one (D17) To a solution of (S)-3-N-[3-carboxy-2-(2-methylpropyl)- butanoyl]amino-8-(N-benzyloxycarbonyl)-1, 8-diaza- cyclotetradecan-2-one (D16) (0.467g) in anhydrous dichloromethane (15ml) at 0° was added l-ethyl-3-[3- dimethylamino)propyl]carbodiimide hydrochloride (0.168g) an l-hydroxybenzotriazole (O.llδg). After stirring for 1 h, t reaction mixture was sequentially treated with O-benzylhydroxylamine hydrochloride (0.154g) and N,N- diisopropylethylamine (0.27g) and stirring was continued at room temperature overnight. The mixture was then washed wi brine, dried and evaporated under reduced pressure to give foam. Purification by column chromatography eluting with 5 methanol in chloroform gave the title compound as a mixture of diastereoisomers (D17) . Observed (M+H)+637. C3 H52N 4 0g requires M 636. The diastereomeric mixture (D17) can be separated into individu diastereomers D17A, Rf (chloroform/methanol 9:1) 0.3 follow by diastereomer D17B, Rf (chloroform/methanol 9:1) 0.26, by column chromatography on silica gel with initially 2% methanol/chloroform as eluent, rising to 5% methanol/chloroform. Example 1 (S)-3-N-[3-(N-Hydroxycarboxamido)-2-(2-methylpropyl)- propanoyl]amino-1,8-diazacyclotridecan-2-one (El) A mixture of (S)-N-[3-(N-benzyloxycarboxamido) -2-(2- methylpropyl)propanoyl]amino-8- (N-benzyloxycarbonyl)-1, 8- diazacyclotridecan-2-one (D8A) (O.lβg) and 10% palladium- carbon (80 mg) in ethanol (50 ml) was hydrogenated at room temperature and atmospheric pressure until uptake of hydrog had ceased (18 hours) . The mixture was filtered through Kieselguhr, and then evaporated to dryness in vacuo. The product was triturated with ether to afford the title compound as a single diastereoisomer (E1A) (0.105g), as a white solid, m.p. 176-170°C. Observed FAB (M+H) + 385. C!9H3gN θ4 requires M 384. Hydrogenation of diastereoisomer (D8B) (0.16g) over 10% palladium-carbon by the same procedure afforded the title compound as a single diastereoisomer (E1B) (88 mg) , as a white solid, m.p. 122-126°C. Observed FAB (M+H) + 385. CιgH3 N 4 θ4 requires M 384. Example 2 (S)-3-N-[3-(N-Hydroxycarboxamido)-2-(2-methylpropyl) butanoyl]amino-1,8-diazacyclotetradecan-2-one (E2) A solution of the diastereoisomeric mixture (S)-3-N-[3-N- benzyloxycarboxamido)-2-(2-methylpropyl)butanoyl]amino-8- (N benzyloxycarbonyl)-1, 8-diazacyclotetracdecan-2-one (35mg) (D17) in methanol was hydrogenated over 5% palladium on charcoal (15mg) at atmospheric pressure for 24h. The solution was filtered through Kieselguhr and the solvent evaporated in vacuo. The residue was triturated with dieth ether (2ml) to give the title compound (E2) as a white soli (19mg) . δ (CH 3 0D) : 0.8-0.95(6H,m) , 1.05-1.95(20H,m) , 2.5 (lH,m), 2. 3.3(6H,m), 3.65 (lH,m), 4.35 (lH,m) . Hydrogenation of diastereoisomer (D17A) and (D17B) individually over 5% palladium-carbon by the same procedure afforded the-title compound as a single diastereoisomer (E2 (Observed FAB (M+H)+397. C21H 0 4O3 requires M 396) and (E2B) respectively (observed FAB (M+H)+397. C21H40N4O3 requires M 396) . Example 3 (S)-3-N-[3-(N-Hydroxycarboxamido)-2-(2-methylpropyl)- propanoyl]amino-l,8 diazacyclotridecan-2-one hydrochloride (E3) (S)-3-N-[3-(N-Hydroxycarboxamido)-2-(2-methylpropyl)- propanoyl]amino-1,8-diazacyclotridecan-2-one (ElA) (0.03g) wa dissolved in 0.1M aqueous hydrochloric acid (5ml) then the solution freeze-dried to leave the title compound (E3A) as a white solid. Observed FAB (M+H) + 385. Ci9H3gN4U4 (free base) requires M 384 Treatment of diastereoisomer (ElB) (0.02g) with 0.1M aqueous hydrochloric acid (4ml) then freeze-drying the solution gave the title compound (E3B) as a white solid. Observed FAB (M+H)+385. C 19 H 3 gN θ4 (free base) requires M 384 Example 4 Pharmaceutical compositions for oral administration may be prepared by combining the following: 1) Solid Dosage Formulation % w/w Compound of Example 1 10% Magnesium stearate 0.5% Starch 2.0% HPM cellulose 1.0% Microcrystalline cellulose 86.5% The mixture may be compressed to tablets, or filled into har gelatin capsules. The tablet may be coated by applying a suspension of film former (e.g. HPM cellulose), pigment (e.g. titanium dioxide) and plasticiser (e.g. diethyl phthalate) and drying the film by evaporation of the solvent. The film coat can comprise 2.0% to 6.0% of the tablet weight, preferably about 3.0%. 2) Capsule %w/w Compound of Example 1 20% Polyethylene glycol 80% The medicinal compound is dispersed or dissolved in the liquid carrier, with a thickening agent added, if required. The formulation is then enclosed in a soft gelatin capsule b suitable technology. Example 5 A pharmaceutical composition for parenteral administration may be prepared by combining the following: Preferred Level Compound of Example 3 1.0% Saline 99.0% The solution is sterilised and sealed in sterile containers COLLAGENASE INHIBITOR ASSAY The test is performed essentially as in Cawston and Barre Anal. Biochem. 99, 340-345 (1979). Compounds for testin are dissolved in methanol by sonication and added to collagenase (purified from culture supernatants from the human lung fibroblast cell line, WI-38) in buffer. Afte 5 in pre-incubation at 37°C, the assay tubes are cooled t 4°C and ^H-acetylated rat skin type I collagen is added. The assay tubes are incubated at 37°C overnight. The 3 H- collagen forms insoluble fibrils, which are the substrate the enzyme. To terminate the assay, the assay tubes are spun at 12000 for 15 minutes. Undigested ^H-collagen . s pelleted, whi digested ^π-collagen is found as soluble peptides in the supernatant. A sample of the supernatant is taken for liquid scintillation counting. The activity of collagenase inhibitors (IC50: 50% inhibit concentration) is expressed as that concentration of comp that inhibits a known (standard) concentration of enzyme 50%. The compounds of Examples 1 and 2 had IC5Q values in the range 8xl0 "7 - 8xl0 _9 M.

Claims: Claims : 1. A compound of the formula (1), or a salt, solvate or hydrate thereof : (1) in which R^ is hydrogen, C^-galkyl, or -(CH2)n~ A-R 4 where n is from 0 to 6, A is a bond or an oxygen or sulphur heteroatom and R4 is an optionally substituted aryl or optionally substituted heteroaryl group; R2 is C3_galkyl; and R3 is -(CH2)p-X-(CH2)q~ where p is an integer from 1 to 9, q is an integer from 2 to 10, and the moiety -(CH2)p- is adjacent to the carbon atom marked with an asterisk in formula (1) , and X is -NR5- where R5 is selected from hydrogen, C^-g alkyl, C2-g alkanoyl, C _g alkoxycarbonyl and aryl, aralkyl or aralkyloxycarbonyl in each of which the aryl moiety is optionally substituted. 2. A compound according to claim 1 in which R } is hydrogen, Cι_ alkyl or - (CH2) n _A-R 4 where n is 1, A is a bond or sulphur atom and R4 is an aryl or heteroaryl group; R2 is n-butyl, iso-butyl or sec-butyl; R3 is -(CH2)p-X-(CH2)q- in which p and q have values such that R3 forms part of an 11- to 16-membered azalactam structure and X is a group NR5 in which R5 is hydrogen, methyl, benzyl, t-butoxycarbonyl or benzyloxycarbonyl. 3. A compound according to any one of claims 1 to 2 which Ri is hydrogen or methyl, R2 is iso-butyl, R3 is -(CH2)p-X-(CH2)q- in which p is 4 and q is 5, or p is 4 an is 6, and X is a group NR5 in which R5 is hydrogen. 4. A compound according to any one of claims 1 to 3 which the chiral centre marked with an asterisk in formula (1) has the (S) -configuration. 5. A compound according to claim 1 which is : (S)-3-N-[3-(N-hydroxycarboxamido)-2- (2-methylpropyl) - propanoyl]amino-1, 8-diazacyclotridecan-2-one, or (S)-3-N-[3-(N-hydroxycarboxamido)-2-(2-methylpropyl)- butanoyl]amino-1, 8-diazacyclotetradecan-2-one, and salts, solvates and hydrates thereof. 6. A process for the preparation of a compound according to claim 1, which process comprises : (a) deprotecting a compound of the formula (2) : (2) in which R is an O-protecting group and R]_, R2 and R3 are as hereinbefore defined; or (b) coupling a compound of the formula (3; (3) in which R l' R 2 anc R 3 are as hereinbefore defined with hydroxylamine or a salt thereof; and (c) optionally after step (a) or step (b) converting a compound of the formula (1) into another compound of the formula (1) . 7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, solvate or hydrate thereof, and a pharmaceutically acceptable carrier. 8. A compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, solvate or hydrate thereof, for use as an active therapeutic substance. 9. A compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, solvate or hydrate thereof, for use in treating collagenolytic conditions such as connective tissue disorders in mammals. 10. A method of treatment of conditions in which degradation of connective tissue and other proteinaceous components of the body occurs which comprises administration to a host in need thereof an effective amount of a compound according to any one of claims 1 to 5 or a pharmaceutica acceptable salt, solvate or hydrate thereof.

Inventors: MARKWELL ROGER EDWARD, RAHMAN SHAHZAD SHAROOQ, WARD ROBERT WILLIAM, MARKWELL, ROGER, EDWARD, RAHMAN, SHAHZAD, SHAROOQ, WARD, ROBERT, WILLIAM
Applicants: SMITHKLINE BEECHAM PLC
Application year: 1992
Main classification: A61K31
IPC codes: A61K31, A61P3, C07D245