THIOL-CARBOXYLIC ACID DERIVATIVES AS COLLAGENASE INHIBITORS

Abstract: A compound of formula (I) or a salt, solvate or hydrate thereof in which, R1 is -OH; alkoxy; aryloxy or aralkyloxy; -NR6R7, where each of R6 and R7 is independently hydrogen or alkyl, or R6 and R7 together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring with an additional oxygen, sulphur or optionally substituted nitrogen atom in the ring; or a group (II) where R8 is hydrogen; alkyl optionally substituted by -OH, alkoxy, -NR6R7, guanidine, -CO2H, CONH2, SH, or S-alkyl; or -CH2-Ar where Ar is optionally substituted aryl; and R9 is alkoxy; OH; or -NR6R7; R2 is hydrogen; or acyl, such as (a) or (b)-, where Z is optionally substituted aryl; R3 is C3-6 alkyl; R4 is -(CH2)nNR10R11, -(CH2)nNHCOR12, -(CH2)nNR13C(=NR14)NR10R11, -(CH2)nCONH(CH2)qNR10R11 or -(CH2)n-R15 where n is an integer from 1 to 6, and q is an integer from 2 to 4, and each of R10 and R11 is independently hydrogen or alkyl, or R10 and R11 together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring with an optional oxygen or sulphur atom or an optionally substituted second nitrogen atom in the ring, R12 is alkyl, alkoxy, aralkyloxy or -(CH2)mNR10R11, where m is 1 or 2, R13 is hydrogen or alkyl, R14 is hydrogen or alkyl or R14 and R10 together with the nitrogen atoms to which they are bonded form an optionally substituted 5-, 6- or 7-membered ring, and R15 is an optionally substituted piperidyl ring; and R5 is hydrogen, alkyl, -CH2-(CH2)nOR16, -CH2-(CH2)n-OCOR17 or (III) where n is an integer from 1 to 6, R16, R17 and R18 are independently hydrogen or C1-6 alkyl, and R19 is hydroxy, -O-C1-6alkyl or NR10R11 where R10 and R11 are as defined for R4, for use in the treatment of collagenolytic conditions.

Description: Th1ol-carboxyl1c add derivatives as collagenase inhibitors. The present invention relates to novel thiol-carboxylic acid derivatives, 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 βt al, J. Biol. Chem. 2610, 6600, 1986; hitham et al, Biochem. J. 240, 913,.1986; Breathnach et al, Nucleic Acids Res., 15, 1139, 1987; Muller et 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; P. Basset et al. Nature 348, 699, 1990] . Membership 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 and cartilage tissue, and have also been extensively studied in tissue cultures of a wide range of connective tissues. Apart from control of the biosynthesis, secretion and activation of 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 Metalloproteinases and alpha-2 macroglobulin. An imbalance between the local levels of the proteolytic enzymes and of their 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 family 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 pericellular 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. EPA 273689 (Beecham Group) and US 4235885 (Squibb) disclose thiol-carboxylic acid derivatives having activity as inhibitors of collagenase and useful in the treatment of rheumatoid arthritis and related diseases in which collagenolytic activity is a contributing factor. A novel class of thiol-carboxylic 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. According to the present invention there is provided a compound of general formula (I) , or a salt, solvate or hydrate thereof: (I) in which, R-j_ is -OH; alkoxy; aryloxy or aralkyloxy; -NRgR7, where each of Rg and R7 is independently hydrogen or alkyl, or Rg and R7 together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring with an additional oxygen, sulphur or optionally substituted nitrogen atom in the ring; or a group: where Rs is hydrogen; alkyl optionally substituted by -OH, alkoxy, -NRgR7, guanidine, -CO2H, CONH2, SH, or S-alkyl; or -CH2~Ar where Ar is optionally substituted aryl; and Rg is alkoxy; OH; or -NR R7; 0 0 Ii ii R2 is hydrogen; or acyl, such as -C-alkyl or - C - Z, where Z is optionally substituted aryl; R 3 is C 3 _g alkyl; R is -(CH2) n NR 10 R 11 , -(CH 2 ) n NHC0Rι 2 , -(CH 2 ) n CONH(CH 2 ) q NR 10 Rn or -(CH2) n -Ri5 where n is an integer from 1 to 6, and q is an integer from 2 to 4, and each of R^n and R]^ is independently hydrogen or alkyl, or R^g and Rχι together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring with an optional oxygen or sulphur atom or an optionally substituted second nitrogen atom in the ring, R12 is alkyl, alkoxy, aralkyloxy or -(CH2) m NRιo R ll/ where m is 1 or 2, R13 is hydrogen or alkyl, R-^4 is hydrogen or alkyl or R14 and R-J_Q together with the nitrogen atoms to which they are bonded form an optionally substituted 5-, 6— or 7-membered ring, and R 5 is an optionally substituted piperidyl ring; and R5 is hydrogen, alkyl, -CH2-(CH2) n ORιg, -CH2-(CH2) n -0C0Ri7 or where n is an integer from 1 to 6, Ri , R17 and R 8 are independently hydrogen or C^-galkyl, and R^g is hydroxy, -0-Ci_galkyl or NR-]_o R ll wheire R-^Q and Rχι are as defined for R . Unless otherwise specified, each alkyl or alkoxy group is a Cι_8 group, more preferably C^-g, and may be a straight chain or branched. An aryl moiety is preferably phenyl. Optional substituents for aryl, phenyl and heteroaryl groups may be selected from -OH, C^- alkyl, C-^-g alkoxy and halogen. Examples of heterocyclic rings formed by the groups R^n and Rn include pyrrolidine, piperidine, piperazine and morpholine rings. Examples of R^ are hydroxy; Cι_g alkoxy, such as methoxy, ethoxy, iso-propoxy or t-butoxy; benzyloxy; and -NRgR7 in which Rg is hydrogen and R7 is hydrogen or such as methyl or ethyl, or -NRgR7 may be N'-methyl-N-piperazinyl or N-morpholinyl. Other examples of R^ are -NH-CH2COOH r -NH-CH2-CONH2, -NH-CH2-Cθ2Et, -NH-CH2-CO2- t Bu, -NH-CH-CO2H and -NH-CH-Cθ2 t B . -i-Bu ---Bu Rl is preferably hydroxy, Cι_ al oxy especially methoxy or iso-propoxy, or amino. Most preferably R-]_ is iso-propoxy. 0 11 When R2 is -C-Z, Z is preferably an optionally substituted phenyl group. Preferred examples of R2 are hydrogen, acetyl and benzoyl. Most preferably R2 is hydrogen or acetyl. R3 is preferably a C4 alkyl group, such as n-butyl, iso-butyl or sec-butyl. Most preferably R3 is iso-butyl. Preferably R4 is -(CH2) n NR lO R ll where R-j_o and R * j_ι are hydrogen or methyl, -(CH2) n HC0Ri2 where R12 s aralkyloxy or -(CH2)χpNRιo R ll in which m is 1 and R^Q and R-]_ are hydrogen, -(CH2) n CONH(CH2)q Rιo R ll where q is 2 and RIQ and Rn together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring, R , Rι_3 and R14 are all hydrogen, where R-j_o and R14 together with the nitrogen atoms to which they are bonded form an optionally substituted 2-imidazolinyl group, -(CH2) n -R 15 where R 5 is optionally substituted piperidyl and, in each of the above, n is an integer from 1 to 4. Most preferably R4 is -(CH2)4NH2 or -(CH2> NHCOR 2 where Rχ2 is benzyloxy. Preferred values for R5 are hydrogen, methyl or hydroxyethyl. Most preferably R5 is methyl. The compounds of formula (I) may form salts with bases e.g. sodium hydroxide. When a basic nitrogen atom is present, the compounds of formula (I) may form acid addition salts e.g. with hydrochloric acid. Such compounds form part of the present invention. Where compounds of formula (I) , or salts thereof, form solvates such as hydrates, these also form an aspect of the invention. The compounds of formula (I) have at least one asymmetric centre 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 (I) . The compounds of formula (I) or their salts, solvates or hydrates are preferably in pharmaceutically acceptable or substantially pure form. By pharmaceutically acceptable form is meant, inter alia, of a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and including no material considered toxic at normal dosage levels. The present invention provides the compounds of formula (I) or pharmaceutically acceptable salts or solvates 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, and also for the systemic chemotherapy of cancer. The present invention also provides a process for the preparation of a compound of formula (I) in which R2 is hydrogen, which process comprises cleaving a group L from a compound of formula (II) : (ID wherein L is L- ] _ which is a conventional sulphur protecting group or __2 which is a group R-S- where R is any organic residue such that the group R-S- provides a cleavable disulphide bond, and R-^, R3, R4 and R5 are as defined for formula (I) . Typically a protecting group L is a substituted benzyl group, such as alkoxybenzyl, e.g. 4-methoxybenzyl, or an aliphatic or aryl acyl group such as acetyl or benzoyl. When __χ is acyl it is of course identical to R2, so these compounds of formula (II) are themselves compounds of the invention. When L is R—S- then typically the compound of formula (II) is a dimer of the compound of formula (I) in which R2 is hydrogen. When L is ]_■ •■ _, and L-j_ is a substituted benzyl sulphur protecting group, such as 4-methoxybenzyl, then L^ may be removed by treatment with mercury acetate in trifluoroacetic acid containing anisole, followed by reaction with hydrogen sulphide in dimethylformamide, in a procedure analogous to that described in Chem. Pharm. Bull., 1978, 26, 1576. When l>ι is an acyl group it may be removed by treatment with a base, for example aqueous ammonia or dilute aqueous sodium hydroxide, or by treatment with an acid, for example methanolic hydrochloric acid. When L is L2 the dimerized compound may be split at the disulphide link by treatment with zinc and hydrochloric acid or by passing hydrogen sulphide through the solution. Other conventional methods for removing sulphur protecting groups or cleaving disulphide bridges may also be used. Compounds of the formula (II) in which L is Lj may be prepared by treating a compound of formula (III) : (III) in which R-j_, R3, R4 and R5 are as defined in formula (I) with a thiol of formula (IV) : ■ SH (IV) in which __ •■ _ is as defined in formula (II) . When L^ is R2, the compounds of formula (II) thereby produced are compounds of the invention. Compounds of formula (II) may also be prepared by treating a compound of formula (V) : (V) in which L, R^ and R3 are as defined in formula (II) , with a compound of formula (VI) : R. (VI) in which R4 and R5 are as defined in formula (I) . The reaction is preferably carried out in the presence of a coupling agent, such as 1,1'-carbonyldiimidazole, or 1-ethyl- 3-[3-(dimethylamino)propyl]carbodiimide hydrochloride in the presence of 1—hydroxybenzotriazole. Compounds of formula (II) in which L is 1-2 may also be prepared by oxidative coupling, with iodine or oxygen, of a compound of formula (I) in which R2 is hydrogen. Compounds of formula (II) can be converted to further compounds of formula (II) while retaining the same group L, which group in turn can be cleaved to form compounds of the invention in which R2 is hydrogen. For example, those compounds of formula (II) in which R-^ is hydroxy may be prepared under acid conditions by hydrolysis of compounds in which R-^ is alkoxy, aryloxy or aralkyloxy or by hydrogenolysis of compounds in which R-^ is benzyloxy or substituted benzyloxy in the presence of a catalyst such as palladium black. Compounds of formula (I) in which R*]_ is alkoxy may be prepared from compounds in which R-]_ is hydroxy by esterification. For example by treatment with an appropriate alcohol in the presence of an acid catalyst such as BF 3 -Et2θ (Synthesis, 316, 1972) . Those compounds of formula (II) in which R-^ is -NR 7 may be prepared from compounds in which R]_ is hydroxy by treating the latter compounds with an amine of formula NHR R7 in the presence of a coupling agent such as N,N-dicyclohexyl- carbodiimide or l-ethyl-3-[3-(dimethylamino)propyl]- carbodiimide in the presence of 1-hydroxybenzotriazole, or ethyl chloroformate. Compounds of formula (II) in which R-^ is -NH-CH(Rg)-COR9 may be similarly prepared from compounds in which R-^ is OH by treatment with amine derivatives of formula NH2CH(Rg)COR9 where R9 is an alkoxy or amine group, followed by hydrolysis to give an R9 hydroxy group, if desired. In addition, compounds of the invention in which R2 is acyl • can be converted to further compounds of the invention with concomitant cleavage of the acyl group to give compounds of formula (I) in which R2 is hydrogen. For example, those compounds of formula (I) in which R^ is -OH and R2 is hydrogen may be prepared by hydrolysis of compounds in which R^ is alkoxy, aryloxy or aralkyloxy and R2 is acyl under basic conditions such as treatment with dilute sodium hydroxide. The intermediate compounds of formula (III) may be prepared by treating a compound of formula (VII) : (VII) in which R]_ and R3 are as defined in formula (I) , with a compound of formula (VI) as defined above. The thiols of formula (IV) are known compounds. The intermediate compounds of formula (V) may be prepared by treating a compound of formula (VII) , as defined above, with a thiol of formula (IV) . It may be necessary or convenient to protect the carboxyl function in compounds of formula (VII) , for example by esterification, prior to treatment with the thiol of formula (IV) and subsequently remove the protecting group under acid conditions. The preparation of certain compounds of formula (VII) and (V) has been described in EPA 273689 (Beecham Group) . Compounds of formula (VI) may be prepared from amino acid derivatives, many of which are commercially available, by conventional alkylation or coupling reactions. Thus a compound of formula (VI) may be prepared from a compound of formula (VIII) : (VIII) in which Q is -(CH 2 ) n -Z, -(CH 2 ) n NH 2 , -(CH2) n NR 13 C(=NH)NH2, -(CH2) n NR 1 3C(= H) θ2 or -(CH2) n Cθ2H, and n, R5 and R-^ are as defined in formula (I) , Z is optionally substituted pyridyl and Y is a nitrogen protection group, by conversion of Q to R4 and removal of the nitrogen protection group, Y. It will be appreciated that the converion of Q to R4 may be most readily effected at a later stage, for example conversion of (CH2) n NHC ( =NH)NHN0 2 to ( CH2 ) n NHC( = NH)NH 2 b y hydrogenation. A compound of formula (VI) in which R4 is -(CH2) n Rio n may be prepared by alkylation of a compound of formula (VIII) in which Q is -(CH2) n NH2 using standard alkylation procedures. A compound of formula (VI) in which R is -(CH2) n NHC0Rχ2 ma y be prepared by reaction of a compound of formula (VIII) in which Q is -(CH2) n NH2 with a carboxylic acid R12CO2H, in the presence of a coupling agent. A compound of formula (VI) in which R4 is -(CH2) n CONH(CH2)q Rιo R l l ma -° e prepared by reaction of a compound of formula (VIII) in which Q is -(CH2) n c θ2 H with an amine derivative, H2 (CH2)q NR iθ R ll ^ n t e presence of a coupling agent, and thereafter if Rχo or Rn is hydrogen optionally protecting the basic nitrogen atom. A compound of formula (VI) in which R4 is - ( CH2 ) n NR 13 c(=NR 1 )NR 10 R ll ma y -° e prepared from a compound of formula (VIII) in which Q is -(CH 2 ) n NR 13 c (=NH)NH 2 or -(CH2) n NHR 13 by N-alkylation and optionally thereafter protecting the basic nitrogen atoms. A compound of the formula (VI) in which R4 is _ ( H2)n~" R 15 where R15 is an optionally substituted piperidyl group may be prepared by hydrogenation of a compound of formula (VIII) in which Q is -(CH2) n -z and optionally thereafter protecting the piperidyl nitrogen atom. Suitable nitrogen protection groups for Y and for any primary amino function in R4 include t-butoxycarbonyl (BOO and benzyloxycarbonyl groups. When R4 is -(CH2) n ~ R 15 where R15 is piperidyl, suitable nitrogen protecting groups include benzyloxycarbonyl groups. Nitrogen protection groups may be removed by standard methods. A t-butoxycarbonyl group may be removed by treatment with trifluoroacetic acid at reduced temperature. Benzyloxycarbonyl groups may be removed by catalytic hydrogenation. A compound of formula (VIII) may be prepared from a compound of formula (IX) : (IX) wherein Q' is Q in protected form or Q 1 is a precursor to Q and Y and Q are as defined for formula (VIII) . The reaction may be carried out by reaction with an amine, NH2 5, using standard procedures for forming an amide from a carboxylic acid and an amine, for example using a coupling agent such as 1,l'-carbonyldii idazole, or l-ethyl-3-[3- (dimethylamino)propyl]carbodiimide in the presence of 1-hydroxybenzotriazole, or ethyl chloroformate. Compounds of formula (IX) are known compounds or may be prepared from known starting materials by standard methods. For example the compound of formula (IX) in which Q' is (CH2) NHCθ2CH2Ph and Y is t-butoxycarbonyl is derived from lysine and is commercially available. The compound of formula (IX) in which Q' is CH2CO2CH2PΪ1 and Y is t-butoxycarbonyl is derived from aspartic acid and is commercially available. Where obtainable, pharmaceutically acceptable salts of the compounds of formula (I) 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 (I) 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 chromatography or HPLC. Alternatively, separate diastereoisomeric compounds of formula (I) 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 formula (I) . The present invention further provides a pharmaceutical composition, which comprises a compound of formula (I) , or a pharmaceutically acceptable salt, solvate or hydrate thereof, and a pharmaceutically acceptable carrier. A composition of this invention is useful in the treatment of rheumatism and arthritis and in the treatment of other collagenolytic conditions. A composition of the invention, which may be prepared by admixture, may contain a diluent, binder, filler, disintegrant, flavouring agent, colouring agent, lubricant or preservative in conventional manner. These conventional excipients may be employed in conventional manner, for example as in the preparation of compositions of related peptide enzyme inhibitors, such as the ACE inhibitor captopril. A composition of the invention may be adapted for oral, topical, percutaneous, rectal or parenteral - intravenous, intramuscular, sub-cutaneous, intradermal or intra-articular administration, but oral administration is preferred. The preferred route for administration will depend upon the disorder for which treatment is required, and is preferably in unit dosage form or in a form that a human patient may administer to himself in a single dosage. The suitable dosage range for the compounds of the invention 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 of administration chosen. Compositions may, for example, be in the form of tablets, capsules, sachets, vials, powders, granules, lozenges, reconstitutable powders, or liquid preparations, for example solutions or suspensions, or suppositories. The compositions, for example those suitable for oral administration, may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinyl¬ pyrrolidone, sodium starch glycollate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulphate. Solid compositions may be obtained by conventional methods of blending, filling, tabletting or the like. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. When the composition is in the form of a tablet, powder, or lozenge, any carrier suitable for formulating solid pharmaceutical compositions may be used, examples being magnesium stearate, starch, glucose, lactose, sucrose, rice flour and chalk. Tablets may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating. The composition may also be in the form of an ingestible capsule, for example of gelatin containing the compound, if desired with a carrier or other excipients. For example, a hard gelatin capsule containing the required amount of a compound of the invention in the form of a powder or granulate in intimate mixture with a lubricant, such as magnesium stearate, a filler, such as microcrystalline cellulose, and a disintegrant, such as sodium starch glycollate. Compositions for oral administration as liquids may be in the form of, for example, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; aqueous or non-aqueous vehicles, which include edible oils, for example almond oil, fractionated coconut oil, oily esters, for example esters of glycerine, or propylene glycol, or ethyl alcohol, glycerine, water or normal saline; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and if desired conventional flavouring or colouring agents. The compounds of this invention may also be administered by a non-oral route. In accordance with routine pharmaceutical procedure, the compositions may be formulated, for example for rectal administration as a suppository or for parenteral administration in an injectable form. For injection, the compounds of the invention may be presented in an aqueous or non-aqueous solution, suspension or emulsion in a pharmaceutically acceptable liquid, e.g. sterile pyrogen-free water or a parenterally acceptable oil or a mixture of liquids, which may contain bacteriostatic agents, anti-oxidants or other preservatives, buffers or solutes to render the solution isotonic with the blood, thickening agents, suspending agents or other pharmaceutically acceptable additives. Such forms will be presented in sterile unit dose form such as ampoules or disposable injection devices or in multi-dose forms such as a bottle from which the appropriate dose may be withdrawn or a solid form or concentrate which can be used to prepare an injectable formulation. For topical and percutaneous administration, the preparations may also be presented as an ointment, cream, lotion, gel, spray, aerosol, wash, or skin paint or patch. A unit dose for inflammatory diseases will generally contain from 10 to 1000 mg and preferably will contain from 10 to 500 mg, in particular 10, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 mg. The composition may be administered one or more times a day, for example 2, 3 or 4 times daily, so that the total daily dose for a 70 kg adult 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 suitably 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 a collagenolytic condition such as rheumatism and/or arthritic conditions or cancer, or other diseases in which enzyme-mediated breakdown of connective tissue components plays a role in mammals, such as humans, which comprises administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof, to the mammal. The present invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof, for the manufacture of a medicament for use as an active therapeutic substance, particularly in the treatment of collagenolytic conditions such as rheumatism, cancer, bone disorders, skin diseases, periodontal disease or corneal ulceration, in mammals. The following Descriptions and Examples illustrate the preparation of compounds of the invention. Description 1 4-Isopropoxycarbonyl-2-(2-methylpropyl)but-2-enoic acid (Dl) 5 A solution of 2-(2-methylpropyl)pent-2-enedicarboxylic anhydride (prepared as in EP-A-273689) in 2-propanol (100 ml) was heated under reflux for 8h. The solvent was evaporated in vacuo to give the title compound as a brown oil (31.7g, 98%) . 0 δ (CDCI3) : 0.88 (6H,d,J=7Hz) , 1.25 (6H,d,J=7Hz) , 1.80 (IH, septuplet, J=7Hz) , 2.18 (2H,d,J=7Hz) , 3.60 (2H,d,J=7Hz) , 5.05 (IH, septuplet, J=7Hz) and 6.30 (lH,t,J=7Hz) . 5 Description 2 N α -tert-Butoxycarbonyl-N ε -benzyloxycarbonyl-(S) -lysine methylamide (D2) 0 A solution of N α -tert-butoxycarbonyl-N ε -benzyloxycarbonyl- (S)-lysine (50g, 0.13 mol) in dichloromethane (500 ml) was cooled in ice and treated with 1, 1 '-carbonyldiimidazole (22g, 0.136 mol) . After 30 min the mixture was warmed to room temperature for 15 min then cooled again in ice. A solution 5 of methylamme (4.5g, 0.145 mol) in dichloromethane (100 ml) was added dropwise and the mixture was stirred at room temperature overnight. The mixture was washed successively with 5N hydrochloric acid, water, 10% sodium carbonate, water and brine, then was dried (Na2S04) and evaporated to give the 0 title compound as a white solid (37g, 72%) . δ (CDCI3) : 1.3-1.85 (6H,m), 1.42 (9H,s), 2.78 (3H,d), 3.19 (2H,q), 4.05 (lH,m) , 4.88 (lH,m), 5.08 (2H,s), 5.15 (lH,m) , 6.2 (lH,m) , 7.35 (5H,m) . f ** Description 3 N ε -Benzyloxycarbonyl- (S) -lysine methylamide bistrifluoroacetate (D3) An ice-cooled solution of the amide (D2) (31.7g, 81 mmol) in dichloromethane (150 ml) was treated with trifluoroacetic acid (150 ml) . After stirring at 0-5° for lh then at room temperature for 3h, the solution was evaporated in vacuo, and azeotroped with toluene to give the crude product (45.5g) which was used in the following step without purification. Description 4 4-[[5-(Benzyloxycarbonylamino)-1-(S)-methylaminocarbonyl)- pentyl]aminocarbonyl]-6-methylhept-2(and 3)-enoic acids, isopropyl esters (D4) A solution of 4-isopropoxycarbonyl-2-(2-methylpropyl)but-2- enoic acid (Dl) (17.6g, 78 mmol) in acetonitrile (250 ml) was cooled in ice then was treated with 1, 1'-carbonyldiimidazole (13.9g, 85 mmol). The solution was stirred in ice for 30 min, at room temperature for 15 min, then cooled in ice again. A solution of the lysine derivative (D3) (45.5g, 0.81 mmol) and diisopropylethylamine (33 ml, 190 mmol) in acetonitrile (150 ml) was added to the above solution. The mixture was stirred with ice cooling for lh, then at room temperature for 18h. The mixture was filtered and evaporated in vacuo to leave an oil which was dissolved in ethyl acetate (500 ml) and was washed successively with 10% sodium carbonate, water, 10% citric acid and brine, then was dried (Na2Sθ ) and evaporated in vacuo to leave a dark red oil. Chromatography on silica gel (700 g) eluting with 5% isopropanol/chloroform gave the product as a gummy oil (16.9g, 43%) . δ (CDCI3) : 0.88 (6H,m), 1.22 (6H,m), 1.3-2.1 (8H,m), 2.20 (lH,d, J=7Hz) , 2.36 (0.25H, d, J=7Hz) , 2.78 (3H,m), 3.15 (4H,_ι), 4.45 (lH,m), 5.03 (2H,m), 5.08 (2H, s) , 5.84 (0.25H, , J=llHz) , 5.90 (0.25H,d, J=llHz) , 6.40 (0.5H, t, J=7Hz) , 6.65 (2H,m) , 6.83 (0.25H,dd, J=3,8Hz) , 6.9 (0.25H, dd, J=3, 8Hz) and 7.33 (5H,s) . Example 1 3-Acetylmercapto-4-[[5-(benzyloxycarbonylamino)-1-(S)- (methylaminocarbonyl)pentyl]aminocarbonyl]-6-methylheptanoic acid, isopropyl ester (El) A solution of the unsaturated amides (D4) (16.9g, 33.6 mmol) in thiolacetic acid (100 ml) was set aside for 23 days, then the solvent was evaporated and the residue was azeotroped with toluene. Column chromatography of the crude product on silica (750 g) , eluting first with ether, then with 20%, rising to 50% ethyl acetate/ether, gave a mixture of isomers A and B (6.1g) of the title compound. Alternatively, a solution of the mercaptan (E2) (l_8g, 3.3 mmol) in ice-cooled, nitrogen-purged chloroform (100 ml) was treated with acetic anhydride (1.5 ml, 15.9 mmol) and N- methylmorpholine (1.8 ml, 16.3 mmol). The reaction was stirred in ice for lh then at room temperature overnight. The solution was washed with 10% citric acid and brine, then dried ( a2S04) and evaporated in vacuo to give the title compound as a foam (1.8g, 93%) . δ (CDCI3) : 0.85 (6H,m), 1.20 (6H,m), 1.2-2.0 (9H,m), 2.3 (2H,s), 2.33 (IH, s), 2.5-2.9 (3H,m), 2.78 (3H,m), 3.2 (2H,m), 4.0 (lH,m), 4.35 (lH,m), 4.9-5.1 (2H,m), 5.08 (2H,s), 6.25 (lH,m), 6.43 (lH,m) and 7.35 (5H,s). Example 2 4-[[5-(Benzyloxycarbonylamino)-1-(S)-(methylaminocarbonyl)- pentyl]aminocarbonyl]-3-mercapto-6-methylheptanpic acid, isopropyl ester (E2) A solution of isomers A and B of the acetylmercapto compound (El) (6.1g, 10.5 mmol) in isopropanol (500 ml) was purged with nitrogen for 15 min, then was cooled in ice and treated with 35% aqueous ammonia (50 ml) . After stirring in ice for lh and at room temperature for 2h the solvent was evaporated and the residue was azeotroped with toluene, then ether to give a foam. Trituration with ether gave a solid (3.02g), and, on evaporation of the ether, an oil (3.28g). Column chromatography of the solid on silica (lOOg) , eluting with ethyl acetate, and then trituration of the product with hexane gave a 3:1 mixture of isomers A and B of the title compound as a white powder (2.20g), mp 87-89°C. Found: C, 60.16; H,8.10; N,7.76. C27H 3N 3 0gS requires: C, 60.31; H,8.06; N,7.81%. δ (CDCI3) : 0.85 (6H,m), 1.23 (6H, d, J=7Hz) , 1.3-2.0 (9H,m), 1.85 (lH,d, =8Hz) , 2.5 (2H,m), 2.7-2.85 (lH,m), 2.77 (3H,d, J=5Hz) , 3.08 (2H, ), 3.30 (lH,m), 4.38 (lH,m) , 4.9-5.1 (2H,m), 5.10 (2H,s), 6.3-6.55 (lH,m), 6.6 (lH,br.d) and 7.33 (5H,s) . Example 3 3-Acetylmercapto-4- [ [5-amino-l- (S) - (methylaminocarbonyl) - pentyl]aminocarbonyl]-6-methylheptanoic acid, isopropyl ester hydrochloride (E3) A solution of the benzyloxycarbonylamino compound (El) (190 mg) in 5% formic acid/methanol (3 ml) was added to a stirred suspension of palladium black (200 mg) in the same solvent (10 ml) . After 2h the mixture was filtered through Kieselguhr and evaporated to dryness. A solution of the residue in chloroform was treated with IN ethereal hydrogen chloride and the solvents were evaporated and the residue was triturated with ether to give the title compound as a solid (50 mg) , mp 151-157°C. Alternatively, a solution of the benzyloxycarbonylamino compound (El) , (100 mg) in glacial acetic acid (1 ml) was treated with 45% hydrogen bromide in glacial acetic acid (0.4 ml) . After 4h the mixture was diluted with ether and the precipitated gum was triturated with ether to give the title compound as its hydrobromide salt. δ (CDCI3) : 0.90 (6H,m), 1.25 (6H,m), 1.2-2.2 (9H,m) , 2.35 (3H,s), 2.5-3.1 (3H,m), 2.95 (3H,br.s), 3.18 (2H,m) , 4.12 (lH,m) , 4.77 (lH,m) , 5.0 (lH,m) and 7.9 (3H,br.s) . Example 4 4-[[5-Amino-l-(S)-(methylaminocarbonyl)pentyl]amino- carbonyl]-3-mercapto-6-methylheptanoic acid, isopropyl ester hydrochloride (E4) A mixture of the acetylmercapto compound (E3; hydrobromide) and isopropanol was purged with nitrogen for 15 min then cooled in ice and treated with 35% aqueous ammonia. After 3h the solvent was evaporated and the residue was dissolved in chloroform and treated with IN ethereal hydrogen chloride. The solvent was evaporated. Column chromatography on silica, eluting with 20% methanol/ethyl acetate gave the title compound. δ (CD3OD) : 0.9 (6H,m), 1.25 (6H,d,J=7Hz) , 1.3-1.9 (9H,m), 2.3-2.8 (3H,m), 2.72 (3H,s) , 2.93 (2H,t,J=8Hz) , 3.20 (lH,m), 4.30 (lH,m) and 5.0 (lH,m). Example 5 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 hard 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 by suitable technology. Example 6 A pharmaceutical composition for parenteral administration may be prepared by combining the following: Preferred Level Compound of Example 4 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 Barrett, Anal. Biochem. 99, 340-345 (1979) . Compounds for testing 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. To ensure that thiol collagenase inhibitors remain unoxidised, β-mercaptoethanol may be incorporated in the methanol solvent and/or the diluent buffers to give a final concentration of 9.6 x 10~*^M. The minimal direct effect of β-mercaptoethanol on the degradation of collagen by human collagenase is controlled for. After a 5 min pre-incubation at 37°C, the assay tubes are cooled to 4°C and ^H-acetylated rat skin type I collagen is added. The assay tubes are incubated at 37°C overnight. The ^H-collagen forms insoluble fibrils, which are the substrate for the enzyme. To terminate the assay, the assay tubes are spun at 12000 rpm for 15 minutes. Undigested -^H-collagen is pelleted, while digested -^H-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% inhibitory concentration) is expressed as that concentration of compound that inhibits a known (standard) concentration of enzyme by 50%. The compound of Example 4 had an IC50 value of l.lμM.

Claims: Claims 1. A compound of the formula (I) or a salt, solvate or hydrate thereof: (I) in which, R]_ is -OH; alkoxy; aryloxy or aralkyloxy; - RgR7, where each of Rg and R7 is independently hydrogen or alkyl, or Rg and R7 together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring with an additional oxygen, sulphur or optionally substituted.nitrogen atom in the ring; or a group: 0 ii -NH-CH-C-R9 8 where Rg is hydrogen; alkyl optionally substituted by -OH, alkoxy, -NRgR7, guanidine, -CO2H, CONH2, SH, or S-alkyl; or -CH2~Ar where Ar is optionally substituted aryl; and R9 is alkoxy; OH; or - R R7; 0 II R2 is hydrogen; or acyl, such as -C-alkyl or - C - Z, where is optionally substituted aryl; R 3 is C 3 _g alkyl; R 4 is -(CH 2 ) n N R iO R llf - ( CH 2 ) n NHCOR 12 , -(CH 2 ) n CONH(CH 2 )qNR 10 Rn or -(CH2) n -Ri5 where n is an integer from 1 to 6, and q is an integer from 2 to 4, and each of R-J_Q and R is independently hydrogen or alkyl, or Rχo an & R ll together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring with an optional oxygen or sulphur atom or an optionally substituted second nitrogen atom in the ring, R-]_2 is alkyl, alkoxy, aralkyloxy or ~(CH2) ιrι NRιo R ll where is 1 or 2, R13 is hydrogen or alkyl, R1 is hydrogen or alkyl or R14 and Rχo together with the nitrogen atoms to which they are bonded form an optionally substituted 5-, 6- or 7-membered ring, and R 5 is an optionally substituted piperidyl ring; and R5 is hydrogen, alkyl, -CH2-(CH2) n ORχg, -CH2-(CH2) n -O.CORχ7 or where n is an integer from 1 to 6, Rig, R17 and Ri are independently hydrogen or Cι_galkyl, and R19 is hydroxy, -0-Ci_galkyl or NRIQRH where RI Q and Rn are as defined for R4. 2. A compound according to claim 1 in which R is hydroxy, Cι_4alkoxy, or amino. 3. A compound according to claims 1 or 2 in which R2 is hydrogen, acetyl, or benzoyl. . A compound according to any one of claims 1 to 3 in which R3 is n-butyl, iso-butyl or sec-butyl. 5. A compound according to any one of claims 1 to 4 in which R4 is -(CH2)n NR 10 R ll where Rχo and Rn are hydrogen or methyl, -(CH2) n NHC0R 2 where R12 is aralkyloxy or -(CH2) m NR o R n in which m is 1 and RI Q and Rn are hydrogen, -(CH2) n CONH(CH2)qNR oRn where q is 2 and RIQ and Rn together with the nitrogen atom to which they are bonded form a 5-, 6- or 7-membered ring, -(CH2) n NR 13 c ( =NR 14) NR 10 R 11 where R 10' R ll' R 13 anci R 14 are a H hydrogen, ~(CH2) n NR 13 c(=NR 14) NR 10 R ll where RIQ and R14 together with the nitrogen atoms to which they are bonded form an optionally substituted 2-imidazolinyl group, _ (CH2) n - 5 where R15 is optionally substituted piperidyl and, in each of the above, n is an integer from 1 to 4. 6. A compound according to any one of claims 1 to 5 in which R5 is hydrogen, methyl, or hydroxyethyl. 7. A compound according to any one of claims 1 to 6 in which Ri is iso-propoxy, R2 is hydrogen or acetyl, R3 is iso-butyl; R4 is -(CH2)4~ H2 or -(CH2) 4NHCOR12 where Rχ2 is benzyloxy and R5 is methyl. 8. A compound according to any one of claims 1 to 7 in which the chiral centre marked with an asterisk in formula (1) has the (S)-configuration. 9. A compound according to claim 1 which is: 3-acetylmercapto-4-[ [5-(benzyloxycarbonylamino)-1-(S)- (methylaminocarbonyl)pentyl]aminocarbonyl]-6-methylheptanoic acid, isopropyl ester, 4-[ [5-(benzyloxycarbonylamino)-1-(S)- (methylaminocarbonyl)pentyl]aminocarbonyl]-3-mercapto-6- methylheptanoic acid, isopropyl ester, 3-acetylmercapto-4-[ [5-amino-l-(S)- (methylaminocarbonyl)pentyl]aminocarbonyl]—6—methylheptanoic acid, isopropyl ester hydrochloride, and 4-[ [5-amino-l-(S)-(methylaminocarbonyl)pentyl]amino¬ carbonyl]-3-mercapto-6-methylheptanoic acid, isopropyl ester hydrochloride. 10. A process for the preparation of a compound as claimed in claim 1, which process comprises: (a) The reaction of a compound of formula (III) : (III) in which Ri, R3, R4 and R5 are as defined in formula (I) with a thiol of formula (IV) : Li-SH (IV) in which Li is a conventional sulphur protecting group; or (b) the reaction of a compound of formula (V) (V) in which Ri and R3 are as defined in formula (I) and L is which is a conventional sulphur protecting group, or L2 which is a group R-S- where R is any organic residue such that the group R-S- provides a cleavable disulphide bond, with a compound of formula (VI) : R_ (VI) in which R4 and R5 are as defined in formula (I) , to give a compound of formula (II) : (ID in which Ri, R3, R4 and R5 are as defined in formula (I) and L is I_ι or I_2; and subsequently, where R2 in a compound of formula (I) is hydrogen, cleaving the group L. 11. A process according to claim 10 in which L is Li where Li is acetyl or benzoyl. 12. A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 9 and a pharmaceutically acceptable carrier. 13. A compound according to any one of claims 1 to 9 for use as an active therapeutic substance. 14. A compound according to any one of claims 1 to 9 for use in the treatment of collagenolytic conditions such as connective tissue disorders, in mammals. 15. A method of treatment of conditions in which degradation of connective tissue or 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 9 or a salt, solvate or hydrate thereof.

Inventors: MARKWELL ROGER EDWARD, RATCLIFFE STEVEN JOHN, WARD ROBERT WILLIAM, MARKWELL, ROGER, EDWARD, RATCLIFFE, STEVEN, JOHN, WARD, ROBERT, WILLIAM
Applicants: SMITHKLINE BEECHAM PLC
Application year: 1992
Main classification: A61K31
IPC codes: A61K31, A61P29, A61P43, C07C323, C07C327, C07D295