CARBOXYALKYL PEPTIDE DERIVATIVES

Abstract: This invention encompasses novel carboxyalkyl pep­ tide derivatives which are collagenase inhibitors.

Description: BACKGROUND OF THE INVENTION This-application is a continuation-in-part application of prior copending application Ser.No. 59937, filed April 12, 1984. This invention relates to novel compounds having pharmacological activity, to the production thereof, to compositions containing them, and to their use in the treatment or management of conditions or diseases, e.g., rheumatoid arthritis, in which collagenase promoted collagen breakdown is a causative factor. We have now found a group of compounds which act as inhibitors of mammalian collagenase which initiates collagen breakdown. Extensive proteolytic enzyme promoted degradation of articular cartilage is associated with joint destruction in rheumatoid arthritis. Collagen is one of the major components of the protein matrix of joint cartilage and bone. Histological observations of rheumatoid lesions have established that such lesions are characterized by the proliferation of synovial lining cells with subsequent neovascularization and infiltration by lymphocytes, macrophages and plasma cells, collectively referred to as soft tissue or wpannus". The importance of such soft tissue in cartilage erosion has been well demonstrated. Evanson and coworkers, for example, found that large amounts of neutral collagenase are produced by pannus tissue LEvanson, J.M., et al., Arthritis & Rheum., 21, 2639-2651, 1968]. More recently, others have confirmed that neutral collagenase plays an important degradative role in the arthritic joints of experimental animals Lsee Cambray, et al., Rheumatol Int. 1, 11-16 and 17-28, 1981] and in humans [Cawston, et al., Arthritis & Rheum., 27, 285-29, 1984]. A mono-specific antiserum to purified synovial collagenase has been used to localize the enzyme in rheumatoid tissues [Wooley, et al., Eur. J. Biochem., 69, 421-428, 19761. Immunoreactive collagenase was detected in high density at the cartilage-pannus junction -- the site of joint erosion --but not in the cartilage matrix or synovial tissue remote from the cartilage-pannus junction [Wooley, et al., Arthritis & Rheumatism, 20, 1231-1349.] Wooley, et al., (Science, 200, 773-775, 1978) have further identified a sub-population of synovial cells responsible for collagenase production. Thus, the foregoing observations have provided conclusive evidence that collagenase is directly involved in the cartilage erosion process seen in rheumatoid arthritis. Accordingly, the compounds of the present invention which specifically inhibit mammalian collagenase are pharmacologically useful in the treatment of rheumatoid arthritis and related diseases in which collagenolytic activity is a contributing factor, such as corneal ulceration, peridontal disease, tumor invasion, dystrophic epidermolysis bullosa, etc. These compounds have substantially no angiotensin converting enzyme (ACE)-inhibiting activity. ACE inhibitors are described in European Appl. No. A-0012401. ACE is a carboxydipeptidase -- it cleaves a peptide substrate two residues from the C-terminus. Consequently the C-terminal carboxylic acid is a prime recognition site for both substrates and inhibitors; removal of this ionic binding group drastically reduces inhibitory potency. Collagenase, on the other hand, is an endopeptidase and, as such, has no prerequisite for this binding interaction. Additionally, the structure of collagen differs essentially from angiotensin-I which is a decapeptide and is cleaved at a phenylalanine-histidine bond to give an octapeptide (angiotensin-II) and a dipeptide (histidylleucine). Collagen is much more complex, in being a triple helix, each strand of the helix containing of the order of 1,000 amino acid residues, the sequence of amino acids around the site cleaved by collagenase being completely different from that around the cleavage site of Angiotensin I. Collagenase cleaves this triple helix at a single locus on each chain approximately two-thirds of the way along the chain from the N-terminus. The amide bond which is cleaved by collagenase is either a glycine-leucine or a glycine-isoleucine bond. BRIEF DESCRIPTION OF THE INVENTION The present invention provides compounds of the general formula I: EMI3.1 EMI3.2 and the pharmaceutically acceptable acid addition salts thereof wherein R'2 represents hydroxy, alkoxy, cycloalkoxy, aralkoxy or substituted alkoxy wherein the substituent is selected from alkylaminocarbonyl or the group EMI3.3 R 3 represents alkylamino or aralkylamino and the stereochemistry of the carbon atom marked by the asterisk is R, S or a diastereomeric mixture thereof with R configuration being preferred. DETAILED DESCRIPTION OF THE INVENTION The term alkyl as used herein to designate a group or a part thereof includes reference to both straight and branched alkyl groups and to cycloalkyl groups which may contain from I to 1, preferably 1 to 6, carbon atoms in the case of straight or branched chain non-cyclic alkyl groups (for example methyl, ethyl, propyl,isopropyl) and from 3 to 10, preferably 3 to 7 in the case of cyclic alkyl groups (for example cyclopentyl, norbornyl). By the term aryl, is meant phenyl or naphthyl. The terms aralkyl and aralkoxy include in particular those groups containing 1 to 4 carbon atoms in the alkyl portion, and those groups in which aryl has the meaning just given. Typical pharmaceutically acceptable addition salts are those derived from mineral and organic acids such as hydrochloric, hydrobromic, hydroiodic, p-toluene sulphonic, sulphuric, perchloric, acetic, benzoic, trifluoroacetic and the like. There are several chiral centres in the compounds according to the invention because of the presence of asymmetric carbon atoms. These centres may be racemised or in any optically active form, as well as comprise R, S or diastereomeric mixture. The compounds according to the invention exhibit inhibitory action against collagenase. This was determined following the procedure of Cawston and Barrett, Anal. Biochem., 99, 340-345 (1979) whereby the lmM of the inhibitor being tested or dilutions thereof are incubated at 37 0C for 16 hours with native collagen and collagenase (buffered with Tris BCl-CaC12; 14 pH 7.6). The collagen is acetyl C collagen. The samples are centrifuged to sediment undigested collagen and an aliquot of the radiactive supernatant removed for assay on a scintillation counter as a measure of hydrolysis. The collagenase activity in the presence of lmM inhibitor, or a dilution thereof, is compared to activity in a control devoid of inhibitor and the results reported as that inhibitor concentration effecting 5% inhibition of the collagenase. Table II illustrates the activity of compounds of this invention. For use in treatment of rheumatoid arthritis the compounds of this invention can be administered by any convenient route preferably in the form of a pharmaceutical composition adapted to such route and in a dose effective for the intended treatment. In the treatment of arthritis administration may conveniently be by the oral route or by injection intraarticularly into the affected joint. The daily dosage for a 7 kilogram mammal will be in the range of 1B milligrams to 1 gram. The compounds of this invention can be formulated in compositions such as tablets, capsules or elixirs for oral administrationor in sterile solutions or suspensions for parenteral administration. About 90 to 500 og of a compound according to the invention is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavour, etc., in a unit dosage from as called for by accepted pharmaceutical practice. (See for example, Remingtonts Pharmaceutical Science Mach Publishing Co., Easton, Penn. 1965). The amount of active substance in these compositions or preparations is such that a suitable dosage in the range indicated is obtained. The compounds according to the invention may be made by methods which are generally known in peptide chemistry for analogous compounds. In particular it is to be understood that reactive groups not involved in a particular reaction (e.g. amino, carboxy, hydroxy etc.,) may be protected by methods standard in peptide chemistry prior to reactions of other groups and subsequently deprotected. The intermediates of use in the production of the end-products are either knawn compounds or can be made by known methods, as described in the Examples. In order that the invention may be more fully understood the following Exarrples are given by way of illustration and should not limit the invention in spirit or scope. All temperatures herein are degrees C. Example 1 N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-Lleucyl-O-methyl-L-tyrosine N-Methylamide This was prepared in two stages from methyl 4-N-(benzyloxycarbonyl)amino-2-bromo-butanoate and L-leucyl-O-methyl-L-tyrosine N-methylamide as described. below: (a) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonyl propyl]-L-leucyl-O-methyl-L-tyrosine N-Methylamide Methyl 4-N-(benzyloxycarbonyl)amino-2-bromobutanoate (30g), L-leucyl-O-methyl-L-tyrosine N-methylamide (30g) and N-methyl morpholine (9.4g) in acetonitrile (250ml) was stirred and heated under reflux overnight. A further portion of the amine (1.1g) was then added and the solution was heated under reflux for a further 4h. The reaction mixture was then concentrated in vacuo, dissolved in chloroform and the solution washed with saturated aq. sodium bicarbonate solution. The material isolated from the organic layer was chromatographed on silica with ethyl acetate as eluant to yield N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosine N-methylamide (11.7g); (Found: C,63.9; H,7.46, N,9.59. C30H42N4O7 requires C, 63.16; H, 7.37; N.9.83%); #max (CHCl3) 340±), 1720 and 166D cm-1; C (CDC13) 0.86 (6H,m,CH(CH3)2); 1.2-2.1(6H,m,NHCH2CH2CH, CH2CH(CH3)2,NH); 2.77(3H,d,J=5Hz,NCH3); 2.95-3.45 (5H,m,NHCH2, CH2C6H4(CH); 3.66 and 3.76 (each 3E, each S, 2x0CH3); 3.8 and 4.61 (each lH, each rn, 2x CIj); 5.18(2H,m,CH2C6H5); 5.21(1H,m,OCONH); 6.72 (1H,m,CONH); 6.81(2H,d,J=8.6Hz, Tyr H-3 and H-5); 7.12 (2H,d,J=8.SHz, Tyr H-2 and H-6); 7.35 (SH,s,C6H5); 7.55(1H,d,J=8Hz,CONH); m/e 571 (100% [m+1]+) The methyl 4-N-(benzyloxycarbonyl)amino-2bromobutanoate required in this preparation was made from L-glutamic acid as described in the following paragraphs: L-Glutamic acid (185, 8.713 M) was dissolved in concentrated sulphuric acid (300ml) and to this was added chloroform 380ml). To the stirred bi-phasic mixture at 8 was added portionwise over 30 min. sodium azide (60g,0.9 mole). The reaction mixture was stirred at 5-10 for 30 min. and was then allowed to slowly warm to room terperature. The reaction mixture was then slowly heated to 80 for one hour the reaction was cooled, poured into water (1.5 1) and the aqueous layer was separated. The aqueous extract was diluted (to 20 litres) and was then applied to Dowex 50WX8, 16-40 mesh, H+ resin. The column was washed with water and then with 1:1 880 Ammonia/Water and the fractions containing the product were lyophilised. The crude product obtained above was dissolved in water (1 litre) and to this was added basic copper carbonate (1039). The stirred mixture was heated under reflux for 40 min. and the hot solution was filtered. The solution was cooled to 350 and NaHCO3 (60g) and CHCl3 (300 ml) were added. After stirring for 30 min. at room temperature, benzyloxycarbonyl chloride (75m1) was added and the mixture was then allowed to stir at room temperature overnight. A further portion of benzyloxycarbonyl chloride (30ml) was then added and stirring was continued for a further 24h. The crystalline copper complex which had precipitated was then filtered, washed with water and added to a solution of EDTA (di Na salt) (120g) in water (1.5 litre). The resulting mixture was stirred and heated under reflux for 3h and was then cooled to 5 . After 4Eh at 50 the crystalline product was collected by filtration, washed with water and acetone and dried in vacuo at 450. The 4-z-diamino-butyric acid from above (120g) was suspended in a mixture cf dilute sulphuric acid (lM, 600ml), water (200ml) and potassium bromide (240g) Sufficient water (200ml) was then added to forn a single phase. To the resulting solution stirred at -7 to -9 , was added a solution of scdium nitrite (44g) in H20 dropwise over lh. After 30 min at -7 , the mixture was warmed to rocn temperature over lh. Diethyl ether (1.5 litres) was acted and the separated aqueous layer was washed with a further portion of ether. The dried ethereal extracts were concentrated in vacuo and the residue in methanol (1 litre) was cooled to 0 and treated dropwise with thionyl chloride (65 ml). The reaction was then concentrated in vacuo and the residue was partitioned between diethyl ether and saturated aq. sodium bicarbonate. The material isolated from the ether was chromatographed on silica eluting with a gradient of ethyl acetate in hexane to give methyl 4-N-(benzyloxycarbonyl)amino-2-bromo-butanoate (90g) as an oil which crystallised on standing, m.p. 46-580; (Found: C, 47.17; H, 5.01; N, 4.16. C13H16BrNO4 requires C,47.29; B14.88; N,4.24%) ; #(CDCl3) 2.8-2.45 (2H,m,CH2); 3.37(2H,m,NHCH2); 3.76(3H,s,OCHµ); 4.32 (1H,dd,J=10Hz and 6Hz, Cli); 4.97 (lH,broad s, OCONH); 5.09 (2H,s,OCH2) and 7.34 (5H,s,C6H5). (b) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl]-L-leucyl-O-methyl-L-tyrosine N-Methylamide To a solution of the preceeding ester (171mg, 0.3mM) in methanol (10ml) stirred at 0 was added dilute NaOH After .6ml). After stirring overnight at 0 a further portion of NaOH (1N, 0.3ml) was added and the solution was then stirred for 6h at roou temperature. The reaction mixture was s then acidified with acetic acid and concentrated to a solid in vacuo. Recrystallisatien of this material from methanol/H2O gave the title compound (150 mg); m.p. 170-172 ; (Found: C, 60.97; H, 7.11; N, 9.68. C29H48N4O7+C.8 H2O requires C, 60.99; H, 7.34; N, 9.81%); #max (Nujol) 3330, 1690 and 1640 cm-1; #(CD3OD) 0.88(6H,dd,J=14Hz and 8Hz, CH(CH3)2); 1.2-1.95 (5H,m,NHCH2, CH2CH(CH3)2); 2.69 (3H,s,NCH3); 2.75-3.65(6H,m,NHCH2,CH2C6H4, and 4-Cfix2); 3.74 (3H,s,OCH3); 4.54 (1H,dd,J=10Hz and 6Hz, &alpha;-CH); 5.08 (2H,m,CH2C6H5); 6.82(2H,d,J=8.6Hz, Tyr H-3 and H-5); 7.12 (2H,d,J=8.6Hz, Tyr H-2 and H-6); 7.35(5H,m,C6H5). EXAMPLE 2 (a) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)carboxypropyl]-L-leucyl-L-tyrosine N-Methylamide To a stirred solution of the ester from example 1 (.35g, 8.63 mM) in methanol (10 ml) was added dilute sodium hydroxide (1.3 ml, 1N). After stirring for 42 h at room temperature the reaction mixture was neutralised with acetic acid, concentrated invacuo and partitioned between ethyl acetate and water to give the title compound as a solid (121 mg); m.p. 179-182 , (Found: [m+1]+=557.2985. C28H38N4O7 requires 557.2975); #(d6 DMSO) 0.8(6H,m,CH(CH3)2); 1.18(2H,m,CH2CH(CH3)2); 1.46-1.9(3H,m,NHCH2CH2, CH2CH(CH3)2); 2.55(3H,d, J=5Hz, CONHCH3); 2.6-3.8(6H,m,NHCH2CH2CHCO2H, &alpha;-CH, CH2Tyr); 4.38(1H,dd,J=7 and 15Hz, &alpha;-CH); 5.02 (2H,s, CH2C6H5); 6.63(2H,d,J=8Hz,Tyr); 6.97(2H,d,J=8Hz, Tyr); 7.28(1H,br,CONH); 7.36(5H,s,C6H); 7.84(1H, 5 br,CONH); 8.14(1H,d,J=12Hz,CONH). (b) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-L-tyrosine N-Methylamide To a cold (0 ) stirred solution of N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpro pyl]-L-leucine (422mg, l.Bl mM) and N-methylmorpholine (204mg) in dichloromethane (10 ml) was added l-hydroxybenzotriazole (154 mg, l.l mM) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (194 mg, 1.01 mM). After 15 minutes at a solution of L-tyrosine N-methylamide hydrochloride (235mg, l.l mM) in dichloromethane/dimethylformamide (11 ml, 10:1) was added followed by N-methylmorpholine (102 mg, l.lmM). The reaction mixture was then allowed to warm and stir to room temperature overnight. The reaction mixture after washing with water, 3N citric acid, saturated aqueous sodium bicarbonate solution and water was dried and concentrated in vacuo to a foam (0.35g)#(CDCl3) .85 (6H, m, CH2(CH3)2); 1.1-2.0 (6H, m, NHCH2CH2, CH2CH(CH3)2); 2.74(3H,d,J=5Hz,CONHCH3); 2.95-3.4(6H,m,NHCH2CH2CH,&alpha;-CH,CH2C6H4); 3.64(3H,s, OCH3); 4.62(1H,dd,J=7 and 15Hz,&alpha;-CH); 5.10(2H,s, CH2C6H5); 5.32 (111, br, CONH); 6.78 (2H, d, J=8Hz, C6H4); 6.86 (111, br, CONH); 7.2 (2H, d, J=8Hz, C6H4); 7.35(5H,s,C6H5); 7.65(1H,d,J=10Hz,CONH). L-tyrosine N-methylamide hydrochloride used in this preparation was prepared as follows: (c) L tyrosine N-Methylamide hydrochloride To a solution of O-benzyl-L-tyrosine N-methylamide HC1 (1.13g) in methanol (50 ml) was added 10% Pd/C and the mixture was stirred in an atmosphere of hydrogen at room temperature and atmospheric pressure for 4 h. The catalyst was then filtered and the solvent was removed in vacuo to yield L-tyrosine N-methylamide HCl as a foam (0.85g). (d) N-[3-N-(Benzyloxycarbonyl)amino-1-(R) methoxycarbonylpropyl]-L-leucine N-[3-N-(Benzyloxycarbonyl)amino-1-(R)methoxycarbonylpropyl)-L-leucine t-butyl ester (4.2g) was treated with Trifluoroacetic acid (5 ml) and water (5ml) for one hour at room temperature. Volatiles were then removed in vacuo and the residue was co-evaporated with ethereal HC1. Recrystallisation of the residue from sethanol/ether gave the title compound as its hydrochloride salt (2.2 g) m.p. 103-109 ; (Found: C, 54.55; H, 7.37; N, 6.37. ClgE29N206Cl requires C, 54.74; H, 7.01; $N, 6.72%); #(d6 DMSO) 0.9(6H,m,CH(CH3)2); 1.72 (3H, m,CH2CH(CH3)2); 2.5 (2H, ii, NHCH2CH2); 3.12 (2H, m,NHCH2); 3.74(3H,s,OCH3); 3.82(1H,m,&alpha;-CH); 4.02 (1H,m,&alpha;-CH); 5.02(2H,s,CH2C6H5); 7.35(5H,br s, C6H5). The foregoing t-Butyl ester was prepared as described below: (e) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)methoxycarbonylpropyl]-L-leucine t-Butyl ester To a solution of methyl 4-N-(benzyloxycarbonyl) amino-2-bromo-butanoate (64g) and L-leucine t-butyl ester (34g) in acetonitrile (3 ml) was added N-methylmorpholine (20g) and the solution was heated under reflux for 48 h. The reaction mixture was then cooled, concentrated invacuo , taken up in water and extracted with ether. The combined ethereal extracts were then dried and concentrated invacuo to an oil. Column chromatography of this oil on silica eluting with a gradient of ethyl acetate in hexane gave the title compound as an oil (20g); #(CDCl3) .87 (6H, m, CH(CH3)2); 1.44 (9H, s, OC(CH3)3); 1.4-1.95 (5H, m, NHCH2CH2, CH2CH(CH2); 3.05-3.5 (4H,m,NHCH2,&alpha;-CH % 2); 3.70(3H,s,OCH3); 5.10(2H,s,CH2C6H5); 5.73(1H, 5 m, CONH); 7.36 (5H, br s, C6H5). EXAMPLE 3 (a) N-(3-N-(Benzyloxycarbonyl)amino-1-(R) carboxypropyl)-L-leucyl-O-propyl-L-tyrosine N-methyl amide (a) N-(3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxy carbonypropyl)-L-leucyl-O-propyl-L-tyrosine N-methyl amide (210 mg, B.35 mM) in methanol (lBml) was treated with aqueous sodium hydroxide (1.1 ml, 0.5 M) at room temperature for 48h. The reaction was then neutralised with acetic acid and concentrated in vacuo to afford N-(3-N-Benzyloxycarbonyl)amino-l-(R)-carboxylpropyl)-L leucyl-O-propyl-L-tyrosine N-methyl amide which crystallised from methanol/water as needles m.p. 160-167 C (Found: C, 63.2; H, 7.8; N, 9.7. C31H44N4O7+ 8.3 H20 requires: C,63.7; H,7.6; N,9.6%); #(CDCl3) 8.8 (9H,m,(C3)2CH,CE3CH2); 8.88-1.88 (SH,m,CH,CH2,CH2,CH2CH2); 2.58 (3H,d,J=4Hz CH3NH); 2.2-3.Ç6 (5H,m,CH2,CH,CH2); 3.84, (2H,t,J=6Hz,CH2-o); 4.4(1H,m,NHCHCO); 5.02(2H,s,O-CH2C6H5); 5.6 (lH,m,NH); 6.76, and 7.08 (4H,each d,each J=7Hz,C6H4); 7.24(1H,m,NH); 7.32(5H,m,C6H5); 7.8(1H,m,NH); 8.14 (1H,m,NH). The preceeding methyl ester was prepared as follows: (b) N-(3-N-(Benzyloxycarbonyl)amino-1-(R)methoxy carbonypropyl)-L-leucyl-O-propyl-L-tyrosine N-methyl amide N-Tertiary butyloxycarbonyl-O-propyl-L-tyrosine N-methyl amide (37 mg, 1.1 mM) in dichloromethane (8 ml) was treated with trifluoroacetic acid (2 ml) at 280C for 2h. The solvent was removed invqcno and the residue redissolved in ether saturated with hydrogen chloride and this procedure repeated twice more to afford O-propyl-L-tyrosine-N-methyl amide hydrochloride (1.1 mM) which was used directly in the next step. N- (3-N- (Benzyloxycarbonyl) amino-l- (R) -methoxycarbonyl- propyl)-I±-leucine hydrochloride (prepared as described in Example 1) (416 mg, 1 mM) in dichloromethane (18 ml) and dimethyl formamide (2 ml) was treated with l-hydroxybenzotriazole (159 mg, 1.4 mM), O-propyl-Ltyrosine-N-methyl amide hydrochloride (1.1 mM), N-methyl morpholine (315 mg, 3.3 mM) and N-ethyl-N'-(3dimethylaminopropyl)-carbodiimide hydrochloride (200 mg, 1.04 mM) at 0 C. The reaction mixture was stirred continuously as it was allowed to warm to 200C over 16h. The reaction was diluted with dichloromethane (3 ml) and washed successively with water, aqueous saturated sodium hydrogen carbonate and aqueous citric acid (1 M) dried over anhydrous sodium sulphate and concentrated in vacuo to afford N-(3-N-Benzyloxycarbonyl)amino-l-(R) methoxycarbonylpropyl)-L-leucyl-O-propyl-L-tyrosine-N methyl amide (.22g, .36 mM) which crystallised from ethyl acetate/hexane as needles m.p. 135-140 C (Found: C, 62.8; H, 7.8; N, 9.2. C32H46N4O7+0.5H2O requires: C, 63.2; H, 7.8; N, 9.2%); #(CDCl3) 0.86(6H,d,d,J=4Hz J=4Hz (CH3)2C); 1.02(3H,t,J=7Hz CH3CH2); 1.1-1.98 (9H,m,CH,CH2,CH2CH2); 2.74 (3H,d,J=4Hz Ca3NH); 2.86-3.48, (5H,m,CH2CH2,CH); 3.64 (3H,s,OCH3); 3.86 (2H,t,J=6Hz,CH2O); 4.58(1H,m,NHCHCO); 5.06 (2H,s,CH2C6H5); 5.14(1H,m,NH) 6.54(1H,m,NH); 6.76, and 7.86 (4H,each d,each J=8Hz,C6H4); 7.32 (5E,m,C6H5); 7.42(1H,m,NH). The intermediate used in the preceeding section was prepared as follows: (c) N-Tertiarybutyloxycarbonyl-O-propyl-L-tyrosine N methyl amide Tertiarybutyloxycarbonyl-L-tyrosine (14.1g, 50 mM) in dimethyl formamide (200 ml) was cooled to 10 C and treated with sodium hydride (80%, 3.3g, 118 mM) with continuous stirring for 1h. Propyl bromide (6.15g, 50 mM) was added and the reaction allowed to warm to 20 C over 16h. Water (58 ml) was added and the reaction concentrated in vacuo to 188 ml, water 388 ml was added and the solution washed twice with ethyl acetate (388 ml). The reaction was then acidified to pHl with hydrochloric acid (6N) and extracted twice with ethyl acetate. The organic extract was dried over anhydrous sodium sulphate and concentrated invacuo to a gum. Column chromatography on silica in ethyl acetate afforded N-tertiarybutyl oxycarbonyl-O-propyl-L-tyrosine (15.lg 46.7 mM) as a foam which was used directly in the next step. To (13g, 48 mM) of this foam in dichloromethane (258 ml) was added l-hydroxybenzotriazole (6.8 g, 45 mM); methylamine hydrochloride (2.5 g, 88 mM); N-methyl morpholine (8g, 80 mM) and dicyclohexylcarbodiimide (9.1g, 45 mM) at 0 C. The reaction was stirred continuously whilst allowed to warm to 20 C over 6h. The reaction was filtered and the filtrate washed with water, aqueous saturated sodium hydrogen carbonate and aqueous citric acid (1M), dried over anhydrous sodium sulphate and concentrated in vacuo to afford N-tertiarybutyloxycarbonyl-O-propyl-L-tyrosine -N-methyl-amide (8.5g, 25 mM) which crystallised from ethyl acetate as needles m.p. 134-135 (Found: C,64.5; H, 8.7; N, 8.4.C18H28N2O4 requires: C, 64.3; H, 8.4; N,8.3%; & CDc13) 0.96 (3H,t,J=8Hz,CH3CH2); 1.36 (9H,s,(CH3)3C); 1.74 (2H,t,q,J=7Hz,J=7Hz,CH2); 2.68 (3H,d,J=4Hz,CH3NH); 2.92(2H,m,CH2C6H4); 3.82 (2H,t,J=7Hz,CH2-O); 4.16 (lH,m,NHCECO); 4.98 (lH,m,NH); 5.68 (lH,m,NH); 6.7.4 and 7.8 (4H, each 2H, each d, each J=8Hz,C6H4). EXAMPLE4 (a) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)carbonypropyl)-L-leucyl-O-propyl-L-tyrosine N-methylamide N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl)-L-leucyl-O-isopropyl-L-tyrosine N-methylamide (8.4g 8.65 mM) in methanol (15 ml) was treated with aqueous sodium hydroxide (2ml, 0.5M, 0.75 mM) at 20 C for 24h. The solution was then adjusted to pH7 with acetic acid and concentrated to low volume. The white solid which crystallised was recrystallised from methanol/water 1:1 to afford N-[3-N-Benzyloxyzarbonyl)amino-l-(R)-carboxypropyl]-L- leucyl-O-isopropyl-L-tyrosine-N-methyl amide (100 mg, 8.17 mM) m.p. 148-160 C (Found: C,63.3; H,7.6; N,9.6. C31H44N40 requires: C,63.7; N,7.8; N,9.3%) ?(CDCl3) 8.8 (6H,m, (CH3)2C); 8.98-1.84 (5H,m,CH2,CH2,CH); 1.22 (6H,d,J=6Hz, (CZ3)2CHO); 2.56 (3H,d,J=4Hz CH3NH); 2.64-3.66 (7H,m,CH2,CH2,CH2,CH); 4.4 (lH,m,NbCACO); 4.52 (1H,m,NHCHCO); 5.8 (2H,s,OCH2C6H5); 6.72, and 7.86 (each 2H,each d,each J=8Hz C6H4); 7.32 (5H,m,C6H5); 7.88 (lH,m,NH); 8.22 (lH,m,NH). The preceeding methyl ester was prepared as follows: (b) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxy carbonylpropyl)-L-leucyl-O-isopropyl-L-tyrosine N-nethylamide N-Tertiary butyloxycarbonyl-O-isopropyl L-tyrosine-N-methylamide (378 mg, 1.1 znM); in dichloromethane (18 ml) was treated with trifluoroacetic acid (10 ml) at 20 C for lh. The resulting trifluoroacetate was converted to the hydrochloride salt by concentrating to a gum in vacuo and redissolving the residue in ether saturated with hydrogen chloride. This procedure was repeated three times. To the product in dichloromethane (18mI) was added N-13-N- (Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl] L-leucine hydrochloride (416 mg, 1 mM) (prepared as described in Example 1) 1-hydroxybenzotriazole (159 mg, 1.04 mM), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (288 mg, 1.54 mM) at 0 C. The stirred reaction mixture was adjusted to pH7 with N-methylmorpholine and allowed to warm to room temperature over 16h. The reaction was diluted with dichloromethane and washed with water, aqueous saturated sodium hydrogen carbonate, and aqueous citric acid (1M), dried over sodium sulphate and concentrated invacuo to a gum. (Found: [M+H]+ = 599.3446. C32H46N4O7 requires: [M+H]+=599.3445); #(CDCl3) 0.84(6H,m,(CH3)2CH) 1.3 (6H,d,J=7Hz, (CH3)2CHO) 1.1-1.96 (5H,m,2xCH2,CH) 2.72 (3H,d,J=4Hz,CH3NH); 3.84 - 3.36 (6H,m,2xCH2,2xCH); 3.64(3H,s,CH3O); 4.46(1H,m,NHCHCO); 4.56 (1H,m,NHCHCO); 5.06(2H,s,O-(CH2C5H6); 5.32(1H,m,NH); 6.76, and 7.84 (each 2H,each d,each J=8Hz,C6H4); 6.98 (lH,m,NH); 7.3 (5H,m,C6H5); 7.52 (lH,m,NH). The intermediate used in the preceeding section was prepared as follows: (c) N-Tertiarybutyloxycarbonyl-O-isopropyl-L-tyrosine N-methylamide N-Tertiarybutyloxycarbonyl-L-tyrosine (14.lg 58 mM) in dry dimethylformamide (288 ml) was treated with sodium hydride (3.45g, 115 mM) with vigorous stirring under an atmosphere of argon at 1BOC for lh. 2-Bromopropane (6.15g, 58 mM) was added and the stirred solution allowed to warm to 2BOC over 16h. Water (288 ml) was added and the solution extracted with ethyl acetate (2 x 200 ml). The reaction was adjusted to pHl with hydrochloric acid (6M) and extracted with dichloromethane 2 x 300 ml. The combined extracts were columned on silica in ethyl acetate to afford N-tertiarybutyloxycarbonyl-O-isopropyl-L-tyrosine (13.6g, 42 mM) as a foam. The foam was dissolved in dichloromethane (288 ml) and treated with methylamine hydrochloride (2.5g, 88 mM), l-hydroxybenzotriazole (6.75g, 44 mN), dicyclohexylcarbodiimide (9.lg, 44 mM) and N-methyl morpholine (2.5g, 88 mM) at BOC with continuous stirring. The solution was allowed to warm to room temperature over 16h and filtered. The filtrate was washed with water, aqueous saturated sodium hydrogen carbonate solution and aqueous citric acid (1N), dried over sodium sulphate and concentrated in vacuo to afford N-tertiarybutyloxycarbonyl-O- isopropyl-L-tyrosine-N-methyl amide (6.lg, 18 mM) which crystallised from ethyl acetate/hexane as needles m.p. 118-1140C (Found: C,63.5; H,8.4; N,8.7. C18H28N204 0.25 H20 requires: C,63.4; H,8.4; N,8.2%) 6' (CDCl3) 1.32(6H,d,J=7Hz,(CH3)2CH); 1.4(9H,s,(CH3)3C); 2.72 (3H,d,J=4Hz NHCH3); 2.98, (2H,d,J=7Hz CH2C6H5); 4.26 (1H,m,NBCHCO); 4.5 (lH, heptet, J=SHz, CH(CH3)2): 5.88 (1H,m,MH); 5.92(1H,m,NH); 6.78, and 7.04 (each 2H,each d,each J=8Hz C6H4). EXAMPLES (a) N-[3-N-(Benzyloxycarbonyl)(amino-1-(R) carboxvropyl0-L-leucvl-O tertiarv hutyl-L-tyrosine. N-methyl amide N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl)-L-leucyl-O-tertiary butyl-L-tyrosine N-methyl amide (8.26g, 0.42mM) in methanol (18 ml) was treated with aqueous sodium hydroxide (1.3 ml, 0.5M) at 20 C for 24h. The reaction was then adjusted to pH 7 with hydrochloric acid (6N) and concentrated in vacuo to low volume. The white solid which crystallized out was washed with ether and water and dried invacuo to afford N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxy propyl-t-leucyl-O-tertiary butyl-L-tyrosine-N-methyl amide m.p. 1680C dec. (Found: C,62.51; H,7.67; N,9.24; C32H46N407.H20 requires: C,62.32; H,7.84; N, 9.08%). #(CDCl3) 0.78(6H,m(CH3)2C); 1.08-1.86 (SH,m,CH2,CH2CH); 1.24 (9H,s,(CH3)3C); 2.56 (3H,d,J=4Hz,CH3NH); 2.44-3.2(6H,m,2xCH2,2xCH); 4.4 (lH,m,NHCACO); 5.8 (2H,s,CH2C5H6); 6.82 and 7.88 (each 2H,each d,each J=8Hz,C6H4); 7.22 (lR,m,NH); 7.3 (SH,m,C6H5); 7.84 (lH,m,NH); and 8.2 (lH,m,NH). The preceeding methyl ester was prepared as follows: (b) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxy carbonyl propyl]-L-leucyl-O-tertiary butyl-L tyrosine N-methyl amide N-Benzyloxycarbonyl-O-tertiarybutyl-L-tyrosine N-methylamide (2.4g 6.2mM) in ethanol (lml) was heated under reflux with cyclo hexene (4 ml) acetic acid (8.38g 6.2 mM) and palladium on carbon (18%, lg) under an atmosphere of argon for 20m. The solvents were then removed in vacuo to afford O-tertiarybutyl-L-tyrosine N-methylamide acetate m.p. 121-123 C. This was dissolved in dichloromethane (58ml) and shaken with aqueous saturated sodium hydrogen carbonate (50 ml). The organic layer was dried over anhydrous sodium sulphate and concentrated in vacuo to afford O-tertiary butyl-L-tyrosine N-methyl amide as a crude gum which was used directly in the next step. N-13-N-Benzyloxy carbonyllamino-l-(R)-methoxycarbonylpropyl]-L-leucine hydrochloride (418 mg, lmM) in dichloromethane (18 ml) was treated with l-hydroxybenzotriazole (159 mg, 1.8 mM) N-methylmorpholine (218 mg, 2mM), O-tertiarybutyl-L-tyrosine-N-methyl amide (275 mg, 1 IsiM) and N- (3-dimethylaminopropyl) -N' -ethyl carbodiimide hydrochloride (288 mg, 1.04 mM) at BOC with continuous stirring. The reaction mixture was adjusted to pH7 with N-methylmorpholine and was allowed to warm to 2BOC over 16h. The reaction was diluted with dichloromethane and washed successively with water, aqueous saturated sodium hydrogen carbonate and aqueous 3N citric acid, dried over sodium sulphate and concentrated in vacuo to afford N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonypropyl)-L-leucyl-O-tertiary butyl-L-tyrosine-Nmethyl amide (358 mg, 8.6mM) which crystallised from ethyl acetate/hexane as needles m.p. 66-680C (Found: C,64.7; H, 8.1; N,9.2. C33H48N407 requires: C,4.7; H, 7.9; N, 9.1%); #(CDCl3) 0.88(6H,d,J=7Hz, (CH3)2Cl; 1.32 (9H,s, (CH3)3C); 1.16-2.8 (SH,m,CH2,CH2CH); 2.72 (3H,d,J=5Hz, CH3NH); 3.82 to 3.4 (6H,m,2xCH,2xCH2); 3.66 (3H,s,CH3O); 4.58 (lH,m,NHCACO); 5.88 (2H,s,CH2C5H6); 5.2 (lH,m,NH); 6.54 (lH,m,NH); 6.88 and 7.86 (each 2H,each d,each J=8Hz,C6H4); 7.32 (5H,m,C6H5); 7.52(1H,m,NH). The foregoing intermediate was prepared as follows: (c) N-Benzyloxycarbonyl-O-tertiarybutyl-L-tyrosine N-methyl amide Benzyloxycarbonyl-O-tertiarybutyl-L-tyrosine dicyclohexyclamine salt (5g, 8.9 mM) was dissolved in aqueous citric acid (25 ml, 1M) and extracted with ethyl acetate (3 x 50 ml). The organic layer was dried over sodium sulphate and concentrated invacuo to afford the free acid (4.1 g) as a gum. This was dissolved in dichloromethane (100 ml) and treated with hydroxy benzotriazole (1.5 g, 10mM), methylamine hydrochloride (.56g, 18 mM) and dicyclohexyl carbodiimide (2.lg 18 mM) at BOC with continuous stirring. The reaction was allowed to warm to 20 C over 16h and then filtered. The filtrate was diluted with dichloromethane and washed with water. aqueous saturated sodium hydrogen carbonate and aqueous citric acid (1M), dried over sodium sulphate and concentrated in vacuo to afford Benzyloxycarbonyl O-tertiary butyl-L-tyrosine N-methylamide (2.5g) which crystallised from ethyl acetate/hexane as needles m.p. 124-125 C (Found: C, 68.7; H, 7.9; N, 7.4. C22H28N2O4 requires: C, 68.7, H, 7.3; N, 7.3%); #(CDCl3) 1.34 (9H,s,(CH3)3C); 2.68 (3B,d,J=4Hz NHCil3); 2.94 and 3.1 (2H,each dd, each J=15Hz,J=6Hz,CH2C6H4); 4.3(1H,q,J=5Hz) NHCECO); 5.8 (2H,s,CH2C6H5); 5.34 (lH,m,NH); 5.58 each (1H,m,NH); 6.88, and 7.06 (each 2H,each d,each J=8Hz,C6H4); 7.3(5H,m,C6H5). EXAMPLE (a) N-[3-N-(Benzyloxycarbonyl)amino-1-(R) carboxypropyl)-L-leucyl-O-pentyl-L-tyrosine N-Methylamide To a stirred solution of 1 (28 mg, .47 mM) in methanol (10 ml) was added dilute sodium hydroxide (.7 ml, 1M). After stirring for 48 h at room temperature the reaction was neutralised with acetic acid and was concentrated invacuo to a solid. Recrystallisation from methanol/water gave the title compound as a solid (107 mg); m.p. 165-170 ; #(d6 DMSO) 0.8(6H,dd,J=5Hz, J=CH2CH(CH3)2); 0.87(3H,t,J=7Hz,CH2CH3); 1.1-1.25 (2H, m, CH2CH(CH3)2); 1.26-1.85 (9H, m, OCH2(2)3CH3), NHCH2CH2CH, CH2CH(CH3)); 2.56(3H,d,J=8Hz,CONHCH3); 2.6-3.65(6H,m,NHCH2CH2CHCO2H,&alpha;-CH,CH2Tyr); 3.87 (2H, ti J=7Hz, OCH2CH2); 4.42 (1H, dd, J=7 and 15Hz, &alpha;-CH); 5.02(2H,s,CH2C6H5); 6.77(2H,d,J=8Hz,Tyr); 7.10 (2H, d, J=8Hz, Tyr); 7.26 (1H, br, CONH); 7.35 (5H, s, C6H5); 7.85 (1H, br, CONH); 8.14 (lH, d, J=8Hz, CONH). The preceeding ester was synthesised as described below: (a) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxy carbonyl propyl)-L-leucyl-O-pentyl-L-tyrosine N-Methylamide To a solution of N-[3-N-(Benzyloxycarbonyl)amino- 1-(R)-methoxycarbonylpropyl]-L-leucine hydrochloride (418 mg, 1mM) in dichloromethane (10 ml), stirred and cooled to was added N-methylmorpholine (101 mg), 1-hydroxybenzotriazole (153 mg, 1mM) and dicyclohexylcarbodiimide (26 mg, lmM). After 15 minutes at SO a solution of O-pentyl-L-tyrosine N-iethylamide (265 mg) (which had been prepared from the N-benzyloxycarbonyl precursor in the usual manner) was added dissolved in dichloromethane (10 ml). The reaction after being allowed to warm and stir to room temperature overnight was filtered, washed with water, 3N citric acid , saturated aqueous sodium bicarbonate and water. The washed organic extract was dried and concentrated to an oil invacuo. Chromatography on silica eluting with ethyl acetate gave N-[3-N-(benzyloxycarbonyl)amino-1-(R)-methoxycarbonyl propylh-l-leucyl-O-pentyl-l-tyrosine N-Methylamide as a solid (359 mg); m.p. 81-85 ; (Found: C, 64.78; H, 8.55; N,8.82. C34H52N407 requires C, 64.94; H, 8.34; N, 8.91%); #(CDCl3) 0.83(3H,d,J=5Hz,CH2CH(CH3); 0.87 (3H,d,J=5Hz,CH2CH(CH3); 0.92(3H,d,J=7Hz,CH2CH3); 1.0-2.0(12H,NHCH2CH2,NH,CH2CH(CH3)2,OCH2(CH2)3CH3); 2.75 (3H, d, J=5Hz, CONHCH3); 2.9-3.6 (6H, m, NHCH2CH2CHCO2H,&alpha;-CH,CH2Tyr); 3.66(3H,s,OCH3), 3.9( 2H, t, J=7Hz, s, 4.58 (1H, dd, J=7 and 15Hz, CH); 5.11 (3H, br, 22C6H5, CONH); 6.58 (lH, br, CONH); 6.81 (2H, d, J=8Hz, C6H4); -7.18 (2H, d, J=8Hz, C6H4); 7.36 (5H, s, C6H5); 7.47 (1H, d, J=8Hz, CONH). N-Benzyloxycarbonyl-O-pentyl-L-tyrosine N-methylamide used in this preparation was synthesised as follows: (c) N-(Benzyloxycarbonyl)-O-pentyl-L-tyrosine N-Methylamide To a cold (0 ) stirred solution of N-benzyloxycarbonyl-O-pentyl-L-Tyrosine (8.3g, 22mM) in THF (120 ml) was added l-hydroxybenzotriazole (3.6g, 24mM) and dicyclohexylcarbodiimide (4.9g, 24mM). After stirring for 25 minutes at 0 a solution of methylamine in THF (7.2 ml, 5M) was added and the reaction was stirred and warmed to room temperature overnight. The reaction mixture was then filtered, evaporated in vacuo and dissolved in dichloromethane (150 ml). The dichloromethane extract after washing with water, 3N citric acid, saturated sodium bicarbonate solution and water was dried, filtered and concentrated in vacuo to a solid. Recrystallisation from ethyl acetate gave the required N-Methylamide as needles (6.43g); m.p. 132-135 ; (Found: C, 69.3; H, 7.63; N, 7.43. C23H30N2O4 requires C, 69.32; H, 7.59; N, 7.3%); #(CDCl3) .92 (3H,t,J=7Hz,CH2CH3); 1.25-2.0(6H,m,OCH2(CH2)3CH3); 2.71 (3H, t, J=5Hz, CONCHCH3); 3.0 (2h, m, CH2Tyr); 3.92 (3H, t, J=7Hz, OCH2CH3); 4.29 (1H, dd, J=7 and 15 Hz, &alpha;-CH); 5.09 (2H, s, CH2C6H5); 5.37 (1H, br, CONH); 5.69 (1H, br, CONH); 6.82 (2H, d, J=8Hz, Tyr); 7.08 (2H, d, J=8Hz, Tyr); 7.33 (5H, m, C6H5). The preceeding acid was synthesised as described below. (d) N-(Benzyloxycarbonyl)-O-pentyl-L-tyrosine To a cold (10 ) stirred solution of N-benzyloxycarbonyl-L-tyrosine (12.61g ,40 mM) in dry DMF (150 ml) under argon was added sodium hydride (2.76g , 88% dispersion in oil). After stirring for one hour at 18 n-bromopentane (6.04g, 40 mM) was added and the reaction mixture was allowed to stir and warm to room temperature overnight. Water (800 ml) and ethyl acetate (800 ml) were then added and the separated organic phase was acidified and extracted with ethyl acetate (200 ml x 3). This organic extract was dried, filtered and concentrated to an oil in vacuo. Chromatography on silica eluting with ethyl acetate gave N-benzyloxycarbonyl-O-pentyl-L-Tyrosine which was recrystallised from ether/hexane to give a solid (8.5g), m.p. 77-81 ; (Found: C, 68.58; H, 7.19; N, 3.77. C22H27NO5 requires C, 68.55; H, 7.06; N, 3.63%); #(CDCl3) 0.92 (3H, t, J=7Hz, CH2CH3); 1.29-1.59 (4H, m, CH2 alkyl); 1.66-1.86 (2H, m, CH2 alkyl); 3.0-3.12 (2H, m, CH2Tyr); 3,93 (2H, t, J=7Hz, OCH2CH3); 4.67 (1H, m, CH); 5.12 (2H, 8, CH2C6H5); 5.2 (1H, d, J=8Hz, CONH); 6.82 (2H, d, J=8Hz, C6H4); 7.7 (2H, d, J=8Hz, C6H4); 7.35 (5H, s, C6H5). EXAMPLE 7 (a) N[3-N-(Benzyloxycarbonyl)amino-1-(R)carboxypropyl]-L-leucyl-O-3(methyl)butyl-L-tyrosine N-methyl amide N-[3-N-(Benzyloxycarbonyl)amino-1-(R)methoxycarbonylpropyl]-L-leucyl-O-3(methyl)butyl-L-tyrosine Nmethyl amide (27 mg .43 znM) in methanol (18 ml) was treated with aqueous sodium hydoxide (1.3 ml, 0,5 M) at 20 C for 40h. The reaction was adjusted to pH4 with acetic acid and the solvent removed invacuo to afford N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-carboxypropyl)-Lleucyl-O-3(methyl)butyl-L-tyrosine N-methylamide (121 mg) which crystallised from methanol/water as needles m.p. 155-160 C (Found: C,63.7; H,7.7; N,9.2. C33H48N4O7 requires: C, 64.7; H, 7.9; N, 9.1%); #((CD3)2SO) 0.8 and 0.92 (12H, each m, 2x(CH3)2C); 1.8-1.9 (8H, m, 3xCH2, 2xCH) 2.56 (3H,d,J=4Hz CE3NH) 2.76-3.8 (6H, m, 2xCH2, 2xCH); 3.92 (2H, m, CH2O); 4.52 (1H, m, NHCHCO); 5,0 (2H, s, CH2C6H5); 6.76 and 7.1 (each 2H,each d,each J=8Hz,C6H4); 7.34 (5H,m,C6H5); 7.9 (1H, m, NH); 0.1 (1H, m, NH). The preceeding methyl ester was prepared as described below: (b) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxy carbonylpropyl]-L-leucyl-O-3(methyl)butyl-L tyrosine N-methyl amide Tertiarybutyloxycarbonyl-0-3-methylbutyl-Ltyrosine-N-methylamide (41 mg, 1.1 mM) in dichloromethane (l ml) was treated with trifluoroacetic acid (1 ml) at 280C for 1.5h. The solution was concentrated invacuo to a gum and dissolved in ether saturated with hydrogen chloride, and this procedure repeated twice to afford O-3(methyl)butyl-L-tyrosine-N-methylamide hydrochloride which was used directly in the next step. N (3-N- (Benzyloxycarbonyl) amino-l- (R) -methoxycarbonyl propyl)-L-leucine Hydrochloride (418 mg 1 mM) in dichloromethane (2 ml) and dimethyl formamide (2 ml) was treated with N-methyl morpholine (42 mg, 4 mM), l-hydroxybenzo- triazole (159 mg 1.4 mM), 0-3-Methylbutyl-L-tyrosine N-methyl amide hydrochloride (1.1 mM) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (2 mg, 1.4 mM) at BOC with continuous stirring. The reaction was allowed to warm to 2BOC over 16h. The reaction was diluted with dichloromethane (3 ml) and washed successively with water, aqueous saturated sodium hydrogen carbonate and aqueous citric acid (1M), dried over sodium sulphate and concentrated in vacuo to afford N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxy carbonylpropyl)-L-leucyl-O-3(methyl)butyl-L-tyrosine Nmethylamide (36 mg, 0.57 mM) which crystallised from ethyl acetate/hexane as needles m.p. 78-810C. (Found: C,65.8; H, 8.0; N,8.9 C34H5N407 requires: C,64.8; H, 8,3; N, 8,9); #(CDCl3) 0.86 (6H, dd, J=6Hz J=1Hz (CH3)2C) .94 (6H,d,J=6Hz, (CH3)2C); 1.02-2.0 (8H, m, 3x(CH2), 2xCH); 2.74 (3H, d, J=4Hz, CH3NH); 2.92-3.42 (6H, m, 2xCH2, 2xCH); 3.66 (3H,s,CH3O); 3.92 (2H, t, J=6Hz, CH2O); 4.58 (1H, m, NHCHCO); 5.1 (2H, s, CH2C6H5); 5.16 (lH,m,NH); 6.52 (lH,m,NE); 6.78 and 7.6 (each 2H, each d,each J=8Hz,C6H4); 7.3 (5H, m, C6H5); 7.74 (1H, m, NH). The intermediate used in the preceeding section was prepared as described.below: (c) Tertiarybutyl-oxycarbonyl-O-3(methyl)butyl-L tyrosine-N-methylamide Tertiarybutyloxycarbonyl-L-tyrosine (14.lg, 50 mM) in dry dimethyl formamide (2 ml) was treated with sodium hydride (3.45g, 115 mM) under an atmosphere of argon at 10 C for 1h. 3(Methyl)butyl bromide (8.3g, 55 mM) was added and the reaction allowed to warm to 20 C over 2h. Water (2 ml) was added and the solution washed with ethyl acetate (2 x 3 ml) and adjusted to pHl with 6N HCl. The solution was extracted with ethyl acetate and the extract dried over sodium sulphate and concentrated invacuo to afford tertiary butyl oxycarbonyl-O-3(methyl)butyl-L-tyrosine (15g, 35 mM) as a gum. The gum was dissolved in dichloromethane (2 ml) and treated with l-hydroxybenzotriazole (5.8g, 38 mM) methylamine hydrochloride (2.2g, 70 mM), dicyclohexyl carbodiimide (7.8g, 38 mM), adjusted to pH7 with N-methyl morpholine and stirred continuously as the reaction was allowed to warm from BOC to 20 C over 16h. The reaction was filtered and washed with water, aqueous saturated sodium hydrogen carbonate and aqueous saturated citric acid, dried over sodium sulphate and concentrated invacuo to afford tertiarybutyloxycarbonyl-0-3 (methyl) butyl-L-tyrosine Nmethyl amide (6.45g, 17 mM) which crystallised from ethyl acetate/hexane as needles m.p. 115-1160C (Found: C,66.3; H, 9.3; N,8. C2H32N204 requires: C,65.9; H,8.9; N,7.7%); # (CDCl3) .94 (6H,d,J=6Hz,(CH3)2C)i 1.42 (9H,s,(CH3)3); 1.6-2. (3H,m,CH2CH) 2.74 (3H, d, J=4Hz, CH3NH); 3.0 (2H, m, CH2C6H4); 3.9 (2H, t, J=8Hz, CH2O); 4.24 (1H, m, NHCHCO); 5.08 (1H, m, NH) 5.8 (lH,m,NH) 6.8, and 7.6 (each 2H,each d,each J=8Hz,C6H4). EXAMPLE 8 (a) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)carboxypropyl]-L-leucyl-O-6(methyl)heptyl- L-tyrosine N-methyl amide N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxy carbonylpropyl]-l-leucyl-O-6-methyl heptyl-L-tyrosine N-methyl amide (.16 g, .24 mM) in methanol (1 ml) was treated with aqueous sodium hydroxide (.7 ml, .5M) with continuous stirring at 2BOC for 24h. The reaction was adjusted to pH7 with acetic acid and concentrated in vacuo to afford a white solid which was washed with ethyl acetate and water and dried invacuo to yield N-[3-N-(Benzyloxycarbonyl)amino-1-(R)carboxypropyl]-L leucyl-O-6(methyl)heptyl-l-tyrosine N-methylamide m.p. 176-1770c (Found: C,66.3; H,8.2; N,8.4; C36H54N47+ H20 requires: C,64.3; H,8.4; N, 8.4%); d'(CDCl3) .84 (12H, m, 2x(CH3)2C); 1.0-1.8 (14H, m, 6xCH2 2xCH); 2.56 (3H, d, J=CH3NH); 2.4-3.64 (6H,m,2xCH2,2xCH); 3.88 (2H, m, CH2O); 4.82 (1H, m, NHCHCO); 5.0 (2H, s, CH2C6H5); 6.74, and 7.6 (each 2H,each d,each J=8Hz,C6H4); 7.18 (1H, m, NH); 7.32 (5H, m, C6H5); 7.8, (1H, m, NH); 6.08 (lH,m,NH). The methyl ester used in the preceeding section was prepared as described below: (b) N-[3-N-(Benzoyloxycarbonyl)amino-1-(R)-methoxy carbonylpropyl]-L-leucyl-O-(methyl)heptyl-L tyrosine N-methyl amide N-Tertiarybutyl oxycarbonyl-0-6-methyl heptyl-t- tyrosine-N-methylamide (447 mg, 1.1 mM) in CH2C12 (1 ml) was treated with trifluoroacetic acid (1 ml) at 2BOC for 2h. The solvent was removed in vacuo and the residue redissolved in ether saturated with hydrogen chloride. This was repeated twice to afford 0-6-methyl heptyl-L-tyrosine-N-methylamide hydrochloride and treated with N-methyl morpholine (42 mg, 4.4 mM), l-hydroxybenzotriazole (159 mg, 1.4 mM), N-[3-N-(Benzoyloxycarbonyl) amino-1-(R)-methoxycarbonylpropyl)-L-leucine hydrochloride (418 mg, 1mM) prepared as described in Example (1) and N-Ethyl-N'-3'dimethyl aminopropyl carbodiimide hydrochloride (2 mg, 1.4 mM) at BOC. The reaction was adjusted to pH7 with N-methylmorpholine, stirred continuously and allowed to warm to 20 C over lh. The reaction was diluted with dichloromethane and washed successively with water, aqueous saturated sodium hydrogen carbonate and aqueous citric acid (1M) dried over sodium sulphate and concentrated invacuo to afford a gum. Column chromatography on silica in ethyl acetate/hexane 1:1 afforded N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxy carbonylpropyl1-L-leucyl-0-6(methyl)heptyl-L-tyrosine N-methylamide (8.17 g, 8.25 mM) as a colourless oil (Found: lM+HI+ = 694.213. C37H56N407 requires: [M+H] + = 699.4227); # (CDCl3) 0.88 (12H, m, 2x(CH3)2CH); 1.12-1.92 (14H, m, 6xCH2, 2xCH) 2.74 (3H, d, J=4HzCH3NH); 2.9-3.42 (6H,m,CH2,2xCH); 3.64 (3H,s,CH30); 3.9 (2H, t, J=5HzCH2NH); 4.6 (1H, q, NHCHCO) 5.1 (2H,s,CH2C6H5); 5.3 (lH,m,NH); 6.7 (lH,m,NH); 6.76, and 7.6 (each 2H,each d,each J=8Hz,C6H4); 7.3 (5H, s, C6H5); 7.4 (1H, m, NH). The preceeding intermediate was prepared as described below: (c) N-Tertiary butvl oxycarbonyl-0-6-methyl heptyl-L tyrosine-N-methylamide M-Tertiarybutyloxycarbonyl-L-tyrosine (14.1 g, 50 mM) in dry dimethylformamide (2 ml) was treated with sodium hydride (3.45g, 115 m) at 10 C under an atmosphere of argon for 2h. 6-Methyl hexyl bromide (13.1 g, 50 mM) was added and the reaction stirred at 20 C for 16h. Water (200 ml) was added and the reaction extracted twice with ethyl acetate (488 ml). The aqueous phase was adjusted to pHl with hydrochloric acid (6M) and extracted twice with ethyl acetate (488 ml) and the ethyl acetate extracts combined, dried over sodium sulphate and concentrated invacuo to a gum. Column chromatography on silica in ethyl acetate afforded crude N-tertiarybutyloxycarbonyl-L-tyrosine (4.9 g) as a colourless foam which was used directly in the next step. This acid (4.9 g) in dichloromethane (150 ml) was treated with methylamine hydrochloride (8.8 g, 25 mM), 1-hydroxybenzotriazole (2.1 g, 14 mM), dicyclohenylcarbodiimide (2.8g, 14 mM) adjusted to pH7 with N-methylmorpholine at 0 C and stirred continuously as the reaction was allowed to warm to 20 C over 16h. The reaction was filtered, washed with water, aqueous saturated sodium hydrogen carbonate and aqueous citric acid (1M), dried over sodium sulphate and concentrated invacuo to a gum. Column chromatography on silica in ethyl acetate/hexane 1:1 afforded N-tertiarybutyloxycarbonyl-O-6(methylheptyl)-Ltyrosine-N-methyl amide which crystallised from ethyl acetate as needles m.p. 183-1860c (Found: C,67.5; H,9.5; . C23H38N204 requires: C,68.8; H, 9.4; N,6.9%); # (CDCl3) 8.88 (6H,d,J=6Hz, (CH3)2C); 1.12-1.86 (9H,m,(CH2)4CH); 1.4 (9H,s,(CH3)3C); 2.72 (3H,d,J=4Hz, ClI3NH); 2.98 (2H, m, CH2C6H4); 3.9 (2H,t,J=6Hz,0-CH2); 4.2 (1H, m, NHCHCO); 5.84 (lH,m,NH); 5.72 (lH,m,NH); 6.78, and 7.26 (each 2H,each d,each J=9Hz, C6H4). EXAMPLE 9 (a) N-[3-N-(Benzyloxycarbonyl)amino-1-(R) carboxypropyl]-L-leucyl-O-benzyl-L-tyrosine N-Methylamide To a stirred solution of the ester from example (8.36g) in methanol (18 ml) was added dilute sodium hydroxide (1ml, 1M). After stirring for 48 h at room temperature the reaction mixture was neutralised with acetic acid and the product was filtered off. Recrystallisation from methanol/water gave the title compound as a solid (159mg); m.p. 172-175 ; (Found: C, 65.70; H, 6.91; N, 8.40. C35H44N4O7.0.5H2O requires C, 65.50; H, 7.06; N, 8.73%); # (d6 DMSO) 0.80 (6H, m, CH(CH3)2); 1.0-1.35 and 1.44-1.84 (together 5H, NHCH2CH, CH2CH(CH3)2); 2.4-3.5 (9H, NHCH2CH2CHCO2H, &alpha;-CH, CH2Tyr, CONHCH3); 4.43 (1H, m, &alpha;-CH); 5.02 (2H, s, CH2C6H5); 6.87 (2H, d, J=8Hz, Tyr); 7.11 (2H, d, J=8Hz, Tyr); 7.2-7.6 (llH, m, C6H5 x 2, CONH); 7.86 (1H, m, CONH); 8.16 (1H, d, J=8Hz, CONH). (b) N-[3-N-(Benzyloxycarbonyl)amino-1-(R) methoxycarbonylpropyl]-L-leucyl-O-benzyl-L-tyrosine N-Methvlamide To a cold (0 ) solution of N-[3-N-(Benzyloxycarbonyl)amino-l-(R)-methoxyCarbonylpro pyl]-L-leucine (418 mg, 1mM) in dichloromethane (50 ml) was added N-methylmorpholine (0.1g), l-hydroxybenzotriazole (153 mg, 1 mM) and dicyclohexylcarbodiimide (286 mg, 1mM). After stirring for 15 minutes at 80 O-benzyl L-tyrosine N-Methylamide hydrochloride (321 mg, 1mM) and N-methylmorpholine (8.1 g) were added. The reaction mixture after stirring and warming to room temperature over 4.5 h was filtered and washed with water, 3N citric acid, saturated aqueous sodium bicarbonate solution and water. The organic extract was dried and concentrated invacuo to an oil. Column chromatography on silica eluting with 1:1 ethyl acetate/hexane in an increasing ethyl acetate gradient then gave the title compound as a foam (8.37g); (Found: [m+1]+ = 647.3417. C36H49N4O7 requires 647.3445); #(CDCl3) 8.88 (6H, m, CH(CH3)2); 1.85-2.2 (6H, m, NHCH2CH2, CH2CH(CH3)2); 2.74 (3H, d, J=5Hz, CONHCH3); 2.96-3.44 (6H, m, NHCH2CH2CHCO2, ,o(-CH, CH2Tyr); 3.66 (3H, s, OCH3); 4.68 (1H, dd, J=7 and lSHz,OC-CH); 5.82 (2H, s, SE2C6H5)i 5.89 (2H, S, Ìj2C6H5); 6.56 (1H, br, CONH); 6.98 (2H, d, J=8Hz, Tyr); 7.12 (2H, d, J=8Hz, Tyr); 7.25-7.6 (12H, m, CONH x 2, C6H5 x 2). O-benzyl-L-tyrosine N-Methylamide hydrochloride used in this preparation was synthesised as follows: N-tertiarybutoxycarbonyl-O-benzyl-L-tyrosine N-methylamide (3g) was added to a mixture of TFA and CH2C12 (1:1 188 ml) at room temperature. After 15 minutes volatiles were removed ill vacua and the residue was dissolved in water. Neutralisation with solid sodium bicarbonate, extraction into CH2Cl2 and evaporation of the organic extract in vacuo then gave a solid. Co-evaporation of this with ethereal HCl gave O-benzyl-L-tyrosine N-Methylamide hydrochloride. (c) N"feriarvbutoxvcarbonvl-O-benzvl-l-tvro N-methylamide To a solution of N-tertiarybutoxycarbonyl-O-benzyl-L-tyrosine (7.4g, 28mM), 1-hydroxybenzotriazole (3g, 28 mM) methylamine HCl (1.3g, 28 mM) and N-methylmorpholine (2g, 28 mM) in CH2C12 (28 ml) stired and cooled to 80 was added DCC (4.2g, 28 mM). After being allowed to stir and warm to room temperature overnight the reaction mixture was filtered and washed with saturated aqueous sodium bicarbonate solution, 3N citric acid and brine. The dried organic extract was then concentrated invacuo to give N-tertiarybutoxycarbonyl-O-benzyl-L-tyrosine N-methylamide as a solid. Recrystallisation from dichloromethane/hexane gave (4.5g); m.p. 165-172 ; (Found: C, 68.85; H, 7.43; N, 7.39. C22H28N204 requires C, 68.73; H, 7.34; N, 7.29%); #(CDCl3) 1.48 (9H, 5, OC(CH3)3); 2.73 (3H, d, J=SHz, CONHCH3); 2.96 (2H, m, CH2Tyr); 4.25 (1H, m, &alpha;-CH); 5.03 (2H, s, CH2C6H5); 5.80 (1H, br, CONH); 6.86 (2H, d, J=8Hz, Tyr); 7.12 (2H, d, J=8Hz, Tyr); 7.3-7.5 (5H, m, C6H5). EXAMPLE 10 (a) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)carboxypropyl]-L-leucyl-O-cyclopentyl-L-tyrosine N-Methylamide To a solution of N-[3-N-(Benzyloxycarbonyl)amino- l-(R)-methoxycarbonylpropyll-L-Leucyl-O-cyclopentyl-L- tyrosine N-Methylamide (536 mg, 0.86 mM) in methanol (18 ml) was added dilute sodium hydroxide (1.8 ml, 1M), After stirring for 24 h at room temperature the reaction mixture was filtered, neutralised with acetic acid and concentrated in vacuo to a solid. Purification from ethyl acetate/water gave the title compound as a solid (245 mg); m.p. 160-170 ; (Found: C, 64.36; H, 7.71; N, 9.18. C33H46N407+8.25H20 requires C, 64.42; H, 7.62; N, 9.11%); #(d6DMSO) 0.8 (6H, M, CH(CH3)2); 1.17 (2H, m, CH2CH(CH3)); 1.45-2.0 (11H, m, NHCH2CH2, CH2CH(CH3)2, CH-alkyl); 2.58 (3H, d, J=SHz, CONHCH3); 2.6-3.65 (6H, m, NHCH2CH2CHCO2H, &alpha;-CH, CH2C6H4); 4.42 (1H, q, J=7 and 15Hz, &alpha;-CH); 4.73 (lH, m, O-CH(cyclopentyl)); 5.02 (2H, s, CH2C6H5); 6.77 (2H, d, J=8Hz, C6H4); 7.88 (2H, d, J=8zz, C6H4); 7.25 (lH, br, CONH); 7.35 (5H, s, C6H5); 7.85 (1H, br, CONH); 8.15 (lH, d, J= Hz, CONH). The preceeding ester was synthesised as described below: (b) N-[3-N-(Benzyloxycarbonyl)amino-1-(R) methoxycarbonylpropyl]-L-leucyl-O-cyclopentyl L-tyrosine N-Methylamide To a solution of N-[3-N-(Benzyloxycarbonyl)amino-l-(R)-methoxycarbonylpropyl]-L-leucine (418 mg, 1mM) in dichloromethane (10 ml) was added N-methylmorpholine (106 mg, lmM) and the solution was stirred and cooled to 0 l-hydroxybenzotriazole (161 mg, 1.55 mM), N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (252 mg, 1.55 mM) and N-methylmorpholine (156 mg, 1mM) were then added. After 15 minutes a solution of O-cyclopentyl-L-tyrosine N-methylamide (1.1 mM) in dichloromethane (5 ml) (which had been prepared by treatment of the N-tertiarybutoxycarbonyl precursor with trifluroacetic acid and subsequent neutralisation with N-methylmorpholine) was added. The reaction after being allowed to stir and warm to room temperature overnight was diluted with dichloromethane (30 ml), washed with water, saturated aqueous sodium bicarbonate and water. The dried organic extract was concentrated in vacuo to to give the title compound as an oil (0.55g); (Found: [m+l]+ = 625.3658. C34H49N407 requires 625.3601); #(CDCl3) 0.82 (6H, m, CH(CH3)2); 1.0-2.0 (11H, m, CH2CH(CH3)2, CH-cyclopentyl); 2.7(3H, d, J= Hz, CONHCH3); 2.9-3.5 (6H, m, NHCH2CH2CHCO2H, &alpha;-CH, CH2Tyr); 3.65 (3H, s, OCH3); 4.5 (1H, dd, J=7 and 15Hz, &alpha;-CH); 4.68 (lH, br, ?); 5.58 (2H, s, CH2C6H5); 5.22 (1H, br, CONH); 6.68 (1H, br, CONH); 6.78 (2H, d, J=8Hz, Tyr); 7.15 (2H, d, J=8Hz, Tyr); 7.32 (5H, s, C6H5); 7.52( 1H, d, J=8Hz, CONH). The N-(Tertiarybutoxycarbonyl)-O-cyclopentyl-L-tyrosine N-Methylamide used in this preparation was prepared as follows: (c) N-(Tertiarybutoxycarbonyl)-O-cyclopentyl-L-tyrosine N-Methylamide To a cold (0 ) solution of N-(Tertiarybutoxycarbonyl)-O-cyclopentyl-L-tyrosine (6.89g, 25 mM) in dichloromethane (40 ml) was added l-hydroxybenzotriazole (3.32g, 21mM) and dicyclohenylcarbodiimide (4.47g, 21 mM). After 25 minutes at (0 a solution of methylamine (1.34g, 45 mM) in dichloromethane (20 ml) was added and the reaction mixture was allowed to stir and warm to room temperature overnight. The reaction mixture afer filtration and washing with water, dilute (1M) sodium hydroxide, 3N citric acid and water was dried and concentrated in vacuo to an oil. Recrystallisation from ethyl acetate/hexane gave the title compound as white crystals (Ig); m.p. 152-154 ; (Found: C, 66.77; H, 8.47; N, 7.83. C20H30N2O4 requires C, 66.27; H, 8.34; N, 7.73%); #(CDCl3) 1.40 (9J, s, C(CH3)3); 1.5-2.0 (8H, CH-cyclopentyl); 2.72 (3H, d, J= 5Hz, CONHCH3); 2.95 2H, m, CH2Tyr); 4.22 (lH, dd, J=7 and 15Hz, &alpha;-CH); 4.72 (lH, br, O-CH (cyclopentyl)); 5.57 (lH, br, CONH); 5.75( 1H, br, CONH); 6.78 (2H, d, J=8Hz, C6H4); 7.15 (2H, d, J=8Hz, C6H4). The N-(Tertiarybutoxycarbonyl)-O-cyclopentyl-L-tyrosine used in this preparation was prepared as follows: (d) N-Tertiarybutoxycarbonyl)-O-cyclopentyl-L-tyrosine To a cold (10 ) stirred solution of N-tertiarybutoxycarbonyl-L-tyrosine (11.25g, 45 mM) in dry DMF (150 ml) under argon was added sodium hydride (2.76g, 80% dispersion in oil, 92 mM). After one hour at 10 cyclopentyl bromide (5.96g, 40 mM) was added and the reaction was allowed to warm and stir to room temperature overnight. Water (255 ml) and dichloromethane (355 ml) were then added and the aqueous layer was separated. The separated aqueous layer was washed with dichloromethane, acidified with hydrochloric acid and re-extracted with dichloromethane (355 ml x 2). These latter dichloromethane extracts were dried and concentrated invacuo to an oil. Column chromatography of this material on silica eluting with ethyl acetate gave the title compound as a foam (7.S5g). This was used directly in the next step without further purification. EXAMPLE 11 (a) N-[3-N-(Benzyloxycarbonyl)amino-1-(R)carboxypropyl]-L-leucyl-O-(2-aminobutyl-2oxo]ethyl-L-tyrosine N-Methylamide To a solution of N-[3-N-(Benzyloxycarbonyl)amino-l-(R)-methoxycarbonylpro pyl]-L-leucyl-O-(2-aminobutyl-2-oxo)ethyl-L-tyrosine N-Methylamide (199 mg, 5.3 mM) in methanol (10 ml) was added dilute sodium hydroxide (5.5 ml, 1M). After stirring for 95 h at room temperature the reaction mixture was neutralised with acetic acid and concentrated invacuo. Purification from ethyl acetate/water gave the title compound as a solid (143 mg); m.p. 164-170 ; (Found: C, 59.44; H, 7.39; N, 15.49 C34H49N508+1.5H20 requires C, 59.81; H, 7.68; N, 10.26%); #(d6 DMSO) 0.7-1.0 (9H, m, CH2CH3, CH(CH3)2); 1.2-2.0 (9H, m, NHCH2CH2, NHCH2(CH2)2CH3, CH2CH(CH3)); 2.62-3.64 (9H, NHCH2CHCO2H, CH, CH2Tyr, CONHCH3; 4.64 (2H, s, OCH2CO); 4.56 (1H, m, &alpha;-CH ); 5.57 (2H, s, CH2C6H5); 6.95 (2H, d, J=BHz, Tyr); 7.17 (2H, d, J=8Hz, Tyr); 7.34 (5H, br s, C6H5). (b) N-[3-N-(Benzyloxycarbonyl)amino-1-(R) methoxycarbonyl]propyl]-L-leucyl-O-(2aminobutyl-2-oxo]ethyl-L-tyrosine N-Methylamide To a cold (0 ) stirred solution of N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpro pyl]-L-leucine HC1 in CH2Cl2/DMF (11 ml, 15:1) was added N-methylmorpholine (240 mg, 2.4mM), l-hydroxybenzotriazole (180 mg, 1.2mM) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (5.24g, 1.2mM). After stirring for 15 min. at E a solution of n-butylamine (90mg, 1.2mM) in CH2C12 was added and the reaction mixture was allowed to warm and stir to room temperature overnight. The reaction mixture after dilution with CH2C12 (30 ml) was washed (water (10ml), 3N citric acid (15 ml) and saturated aqueous sodium bicarbonate (15 ml)), dried, filtered and concentrated invacuo to an oil. Crystallisation from ethyl acetate/hexane then gave the title compound as a white solid (5.27g); m.p. 101-107 ; (Found: C, 62.13; H, 7.84; N, 15.42. C35H51N508+S.5 H20 requires C, 61.93; H, 7.72; N, 10.32%); #(CDCl3) 0.87 (6H, m, CH2CH(CH3)2); 0.94 (3H, t, J=7 Hz, CH2±fi3); 1.04-2.0 (9H, m, NHCH2CH2, CH2CH(CH3)2, NHCH2(CH2)2CH3); 2.75 (3H, d, J=5Hz, CONHCH3); 2.85-3.45 (9H, M, NHCH2CH2CHCO2H, NHCH2, &alpha;-CH, CH2Tyr); 3.66 (3H, s, OCH3); 4.42 (2H, s, OCH2CO), 4.63 (lH, dd, J=7 and 15 Hz, &alpha;-CH); 5.57 (2H, s, CH2C6H5); 5.26 (1H, br, CONH); 6.63 (lH, br, COHN); 6.82 (2E, d, J=8Hz, Tyr); 7.15 (2H, d, J=8Hz, Tyr); 7.34 (5E, s, C6H5); 7.58 (1H, d, J=10 Hz, CONH). EXAMPLE 12 (a) N-(3-N-(Benzyloxycarbonyl)amino-1-(R) carboxypropyl]-L-leucyl-O-(2'-carboxyethyl-L leucyl N-methylamide)-L-tyrosine N-Methylamide To a solution of N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-(2'-carboxyethyl-L-leucyl N-Methylamide)-L-tyrosine N-Methylamide (355 mg, 5.4 mM) in methanol (10 ml) was added dilute sodium hydroxide (1.2 ml, 0.5M). After stirring for 48 h at room temperature the reaction mixture was neutralised with acetic acid and concentrated invacuo. Recrystallisation from methanol/water gave the title compound as a solid (115 mg); m.p. 162-163 ; (Found: C, 65.26; H, 7.49; N, 11.59 C37H54N6O9+0.5H2O requires C, 60.39; H, 7.53; N, 11.42%); 6(d6DMSO) 5.7-5.9 (12H, m, CH(CH3)2 x 2); 0.95-1.85 (8H, m, CH2CH(CH3) x 2, NHCH2CH2); 2.45-3.7 (13H, m, NHCH2CH2CHCO2H, CONHCH3 x 2, oC-CH x 2, CH2Tyr); 4.34 (1H, m, -CH); 4.46 (2H, s, OCH2CO); 5.55 (2H, s, CH2C6H5); 6.82 (2H, d, J=8Hz, Tyr); 7.13 (2H, d, J=8Hz, Tyr); 7.24 (1H, br, CONH); 7.35 (5H, s, C6H5); 7.85 (1H, br, CONH); 7.98 (2H, br, CONH x 2 ); 8.15 (1H, d, J= 10Hz, CONH). The ester required in this preparation was prepared as follows: (b) N-[3-N-(Benzyloxycarbonyl)amino-1-(R) methoxycarbonylpropyl]-L-leucyl-O-(2'-carboxyethyl L-leucyl N-Methylamide]-L-tyrosine N-Methylamide To a cold (0 ) solution of N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpro pyl]-L-leucyl-O-(2'-carboxyethyl)-L-tyrosine N-Methylamide (S.6g, 1.1 mM) in CH2C12/DMF (llml 10: :1= was added l-hydroxybenzotriazole (S.18g, 1.2 mM), N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.24g, 1.2 mM) and N-methylmorpholine (S.24g, 2.4 mM). After 5 min. at O L-leucine N-methylamide HCl (S.22g, 1.2 mM) and N-methylmorpholine (120 mg, 1.2 mM) were added and the reaction was allowed to stir and warm to room temperature overnight. The reaction mixture after diluting with CH2Cl2 (30 ml) was washed with water (20 ml), 3N citric acid (30 ml) and saturated aqueous sodium bicarbonate solution. The organic extract was dried and concentrated in vacuo to an oil. Recrystallisation from ethyl acetate/hexane gave the title compound as crystals (350mg); m.p. 122-123 ; (Found: C,61.16; H,7.45; N, 11.14. C38H56N609 requires C, 61.60; H, 7.62; N, 11.34%); #(CDCl3) 0.8-1.0 (12H, m, CH(CH3)2 x 2); 1.05-2.05 (8H, m, CH2CH(CH3)2 x 2, NHCH2CH2); 2.75 (3H, d, J= 5Hz, CONHCH3); 2.85 (3H, d, J=5Hz, CONHCH3); 2.8-3.5 (7H, m, NHCH2CHCO2, CH2Tyr, &alpha;-CH x 2); 3.68 (3H, s, OCH3); 4.44 (2H, s, OCH2CO); 4.4- 4.7 (2H, m, &alpha;-CH x 2); 5.15 (2H, s, CH2C6H5); 5.34( lH, t,J=6Hz, CONH);6.42 (1H, br, CONH); 6.73 (lH, br, CONH); 6.82 (2H, d, J=8Hz, Tyr); 7.0 (1H, br, CONH); 7.15 (2H, d, J=8Hz, Tyr), 7.36 (5H, s, C6H5); 7.56 (lH, br, CONH). EXAMPLE 13 (a) N-[3-N-(Benzyloxycarbonyl)amino-1-(R) carboxypropyl]-L-leucyl-O-methyl-L-tyrosine N-Phenethvlamide To a solution of N-[3-(Benzyloxycarbonyl)amino-l-(R)-methoxycarbonylpropy 1]-L-leucyl-O-methyl-L-tyrosine N-Phenethylamide (210 mg, 5.33 mM) in methanol (5ml) was added dilute sodium hydroxide (0,5 ml, 1M). After stirring for 5 h at room temperature the reaction mixture was concentrated in vacuo and diluted with water. The aqueous extract after washing with ether was acidifed to pH 3 with dilute (1N) HCl. The resulting solid was filtered and dried to give the title compound as a solid (llB mg); (Found: C, 65.5; H, 7.0; N, 9.1. .0.5H2O requires C, 65.5; H, 7.5; N, 8.7%). (b) N-[3-N-(Benzyloxycarbonyl)amino-1-(R) methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosine N-Phenethylamide To a cold (0 ) solution of N-13-N-(BenzyloxycarbonylÇamino-l-(R)-methoxyzarbonylpro pyl]-L-leucyl-O-methyl-L-tyrosine Hydrochloride (455 mg, B.67 mM) in DMF was added N-methylmorpholine (68 mg, 0.67 mM), 1-hydroxybenzotriazole (157 mg, 5.7 mM) and benzylamine (72 mg, 0.67 mM). The reaction mixture was then cooled to -10 and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (134 mg, 5.7 mM) and N-methylmorpholine (71 mg, 5.7 mM) were added. After being allowed to warm and stir to room temperature over 25 h the reaction mixture was concentrated in vacuo. The resultant residue after dissolving in CH2C12 and washing (water, saturated aqueous sodium bicarbonate solution) was concentrated in vacuo Recrystallisation from ethyl acetate/hexane gave the title compound (290 mg); (Found: C, 66.9; H, 7.1; N, 8.7. required C, 67.2; H, 7.3; N, 8.6%); #(CDCl3) 0.86 (6H, M, CH(3)2); 1.1-1.9 (6H, m, NHCH2CH2, CH2CH(CH3)2); 2.95-3.42 (6H, m, HNCH2CH2CHCO2CH3, &alpha;-CH, CH2Tyr); 3.45 (3H, s, OCH3); 3.73 (3H, s, OCH3); 4.38 (2H, m, CH2C6H5); 4.65 (1H, q, J= 7and 15Hz, &alpha;-CH); 5.54 (1H, br, CONH); 5.58 (2H, s, CH2C6H5); 6.78 (2H, d, J=8Hz, Tyr); 6.96 (1H, br, CONH); 7.11 (2H, d, J=8Hz, Tyr); 7.10-7.30 (5H, m, C6H5); 7.34 (5H, s, C6H5); 7.54 (1H, d, J=8Hz, CONH). The starting material for this reaction was prepared as follows: (c) N-[3-N-(Benzyloxycarbonyl)amino-1-(R) methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosine N-[3-N-(Benzyloxycarbonyl)amino-1-(R)-methoxy carbonylpropyl]-L-leucyl-O-methyl-L-tyrosine t-butyl ester (3g) was dissolved in TFA (30 ml) and water (1.5 ml). After 4 h at room temperature volatiles were removed invacuo and the residue was co-evaporated twice with toluene. Trituation of the resulting solid with ethereal HCl then gave N-[3-N-iBenzyloxycarbonyl1amino-1-(R)-methoxycarbonylpro pyl-L-leucyl-O-methyl-L-tyrosine as its hydrochloride salt. (d) N-[3-N-(Benzyloxycarbonyl)amino-1-(R) methoxycarbonylpropyl-L-leucyl-O-methyl-L-tyrosine tButyl ester A solution of methyl 4-N-(benzyloxycarbonyl)amino2-bromo-butanoate (2.5g, 7.5 mM),L-leucyl-O-methyl-L- tyrosine t-butyl ester (1.82g, 5 mM),(which had been prepared by hydrogenolysis of the N- benzyloxycarbonyl precursor in the usual manner), and N-methylmorpholine (0.76g, 7.5 mM) in acetonitrile (15 ml) was heated under reflux for 35 h. The reaction mixture was then concentrated invacuo t dissolved in CH2C12, washed (water, 3N citric acid and saturated aqueous sodium bicarbonate solution), dried and evaporated in vacuo to yield an oil. Column chromatography on silica eluting with 45:65 ethyl acetate/hexane gave the title compound as an oil (670 mg);S(CDcl3) 5.88 (6H, ddt J=7Hz, CH(CH3)2); 1.42 (9H, s, OC(CH3)3), 1.62 (3H, m, 2(CH3)2); 1.86 (2H, m, NHCH2CH2); 2.9-3.36 (6H, m, NHCH2CH2CHCO2CH3, &alpha;-CH, CH2Tyr); 3.66 (3H, s, OCH3); 3.78 (3H, s, OCH3); 4.68 (1H, dd, J= 7 and 15Hz, &alpha;-CH)M 5.59 (2H, S, CH2C6H5); 5.16 (1H, br, CONH); 6.85 (2H, d, J=8Hz, Tyr); 7.51 (1H, br, CONH); 7.57 (2H, d, J=8HZ, Tyr); 7.35 (5H, s, C6H5). The N-(benzyloxycarbonyl)-L-leucyl-O-methyl-L-tyrosine t-butyl ester used in this preparation was prepared as follows: (e) N-(Benzyloxycarbonyl)-L-leucyl-O-methyl-L tyrosine tButyl ester To a cold (0 ) solution of N-tertiarybutoxycarbonyl-L-leucine (4g, 15.1 mM), O-methyl-L-tyrosine t-butyl ester (3.8g, 15.1 mM) and l-hydroxybenzotriazole (2.43g, 15.9 mM) in DMF (30 ml) was added N-methylmorpholine (1.61g, 15.9 mM) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride 3.04g, 15.9 mM). After stirring at 80 for 1 h the solution was allowed to warm to room temperature overnight. The reaction mixture was then concentrated in vacuo, dissolved in ethyl acetate, washed (water, 0.5 mole citric acid, brine ,saturated aqueous sodium bicarbonate solution and brine), dried and evaporated in vacuo to give an oil. Chromatography on silica eluting with 25% ethyl acetate in hexane then gave the title compound as a foam (4.65g); (Found: [m+l] + = 499.2853. C20H39N2O6 requires 499.2808); #(CDCl3) 0.92 (6H, m, CH(Cz3)3); 1.42 (9H, s, OC(CH3)3); 1.4-1.83 (3H, m, CH2CH(CH3)2); 3.53 2H, m, CH2Tyr); 3.76 (3H, 5, OCH3); 4.18 (1H, m, &alpha;-CH); 4.69 (1H, dd, J=7 and 15Hz, &alpha;-CH); 5.12 (2H, s, CH2C6H5); 5.17 (1H, m, CONH); 6.43 (1H, m, CONH); 6.79 (2H, d, J=8Hz, Tyr); 7.06 (2H, d, J=8Hz, Tyr); 7.36 (5H, s, C6H5). The O-methyl-L-tyrosine t-butyl ester used in this synthesis was prepared as follows: (f) O-methyl-L-tyrosine t-butyl ester To a cold. (-5 ) ,stirred solution of N-benzyloxycarbonyl-O-methyl-L-tyrosine (6.58g, 25 mM) in t-butanol/pyridine (70 ml, 5:2) was added phosphorus oxychloride (3.35g, 22 mM) dropwise over 5.5 h. After a further 5.5 h at -5 the reaction was warmed to room temperature and was then left to stir overnight. The reaction mixture, after diluting with ethyl acetate and washing (5.SM citric acid, brine, saturated aqueous sodium bicarbonate solution and brine) was concentrated in vacuo to give the required t-butyl ester (7.54g). To a portion of this (7.4g, 19.2 mM) in methanol (100 ml) was added 10% Pd/C (Ig) and acetic acid (1.16g, 19.2 mM) After stirring the reaction mixture under hydrogen at room temperature for 4 h it was filtered and evaporated in vacuo. The residue was then taken up in dichlromethane and saturated aqueous sodium bicarbonate -solution and the organic phase was separated, washed with brine, dried and evaporated invacuo to yield O-methyl-L-tyrosine t-butyl ester as a solid (3.95g). The activities of representative compounds according to the invention are given below in Table I. TABLE I. EMI62.1 <tb> Example <SEP> No. <SEP> 50 <SEP> ( M) <SEP> <tb> <SEP> Human <SEP> Rheumatoid <tb> <SEP> Synovial <SEP> Collagenase <tb> <SEP> 1 <SEP> 0.81 <tb> <SEP> 2 <SEP> 1.2 <tb> <SEP> 3 <SEP> 0.74 <tb> <SEP> 4 <SEP> 2.1 <tb> <SEP> 5 <SEP> 3.6 <tb> <SEP> 6 <SEP> 0.54 <tb> <SEP> 7 <SEP> 1.1 <tb> <SEP> 8 <SEP> 2.5 <tb> <SEP> 9 <SEP> 0.91 <tb> <SEP> 10 <SEP> 0.74 <tb> <SEP> 11 <SEP> 2.6 <tb> <SEP> 12 <SEP> 2.4 <tb> <SEP> 13 <SEP> 1.0 <tb> One of the preferred compounds in accordance with the present invention was tested in vivo to demonstrate the antiarthritic activity thereof in a standardized animal model test and compared against prednisolone which has been shown to be clinically effective in the treatment of rheumatoid arthritis. The therapeutic effectiveness is assessed in the rat model of inflammatory arthritis described by Trentham et al. (1977). In this model, the arthritis, induced by an intradermal injection of Type II collagen, generally develops in both hind paws and the progression and severity of the lesion are monitored by physical measurements of the malleolar width and ankle extensibility. The compound of Example II in the present specification was administered by intraperitoneal injections at 100 mg/kg twice daily for 21 days commencing seven days after the injection of the disease. The results of the foregoing test are set forth in Table Ilibelow. TABLE II The effect of N-[3-N-(benzyloxycarbonyl)amino-1-(R)- carboxypropyll-L-leucyl-O-methyl-L-tyrosine-N-methylamide (Example 1 ) on the Physical Parameters of Arthritic Rats' Paws PARAMETER PERCENTAGE CHANGE FROM CONTROL EXPERIMENT NUMBER MEAN 1 2 3 x MALLEOLAR WIDTH Example 1 65 43 41 55 Prednisolone 98 96 97 97 Example 1 66 39 39 48 Predoisolone 100 96 95 97 Drugs (Example 1 or prednisolone) and vehicle were administered to groups of 15 rats each. Physical measurements on the rat paws were made weekly for four weeks and consisted of (a) the distance between the medial and lateral malleolus (malleolar width) and (b) the ankle extension which was measured by applying a standard force to the rat paw and measuring the angle formed between the flexor surface of the foot and the tibia. Evidence of a therapeutic effect was denoted by a significant reduction in malleolar width and a significant increase in ankle extension. Both measurements were carried out on apparatus designed for the purpose. Area-under-the-curve data from tbese time course experiments was examinaed by non-parametric statistical analysis. The prednisolone standard was given at a dose of 5.62mg/kg b.i.d. Prednisolone was chosen as a standard since steroids are known to be effective both clinically in Rheumatoid Arthritis and in animal models of arthritis. References Trentham, D.E., Townes, A.S. & Kang, A.B. (1977) "Autoimmunity to Type II Collagen: An Experimental Hodel of Arthritis' J. Expt. Med. lik. 857-868.

Claims: What is claimed is: 1. A compound of the formula: EMI65.1 and the pharmaceutically acceptable acid addition salts thereof wherein R2 represents hydroxy, alkoxy, cycloalkoxy, aralkoxy or substituted alkoxy wherein the substituent is selected from alkylaminocarbonyl or the group EMI65.2 R'.3 represents alkylamino or aralkylamino; and the stereochemistry of the carbon atom marked by the asterisk is R or S or a diastereomeric mixture thereof. 2. A compound according to Claim 1 wherein R'2 is straight or branched chain alkoxy of from about 1 to 6 carbon atoms. 3. A compound according to Claim 2 wherein R'2 is selected from n-propoxy or n-pentoxy. 4. A compound according to Claim 1 wherein R'3 is alkylamino. 5. A compound according to-Claim 4 wherein said alkylamino is methylamino. --6. A compound according to Claim 1 wherein tne F;tereocN'emistry of the -- carbon atom marked by the asterisk is R. 7. A compound according to Claim 1 of the formula. EMI66.1 or a pharmaceutically acceptable acid addition salt thereof. 8. A compound according to Claim 1 of the formula EMI66.2 or a pharmaceutically acceptable acid addition salt thereof. 9. A method of promoting an antiarthritic effect in a mammal in need thereof comprising administering thereto a collagenase inhibiting effective amount of a compound according to Claim 1. 10. A sethod according to Claim S wherein said compound is of the formula: EMI66.3

Inventors: HANN, MICHAEL, MCCULLAGH, KEITH, WADSWORTH, HARRY
Applicants: G.D. SEARLE & CO.
Application year: 1984
Main classification: C07K5
IPC codes: C07K5, A61K38