Carboxyalkyl Peptide Derivatives

This invention relates to novel compounds having pharmacological activity, to the production thereof, to compositions containing them, and to their use in pharmacy.

A number of compounds have been described which are competitive reversible inhibitors of zinc-containing metalloproteinase enzymes. Such competitive reversible inhibitors are for example those which are inhibitors for the angiotensin converting enzymes (ACE). The utility of such an inhibitor is that it acts to block conversion of the decapeptide angiotensin I to antiotensin II, this last-mentioned compound being a potent pressor substance. ACE inhibitors are therefore potentially of use in the treatment of hypertension. Compounds of this type are for example described in European Patent Application A-0012401. Related inhibitors of the enzyme enkephalinase are described in EPA 0054862.

We have found a group of compounds which act as inhibitors of mammalian collagenase [EC 3.4.24.7] which initiates collagen breakdown. There is now compelling evidence [see for example Arthritis and Rheumatism, 20, 1231, (19770] implicating the involvement of the zinc metalloproteinase, collagenase, as one of the key enzymes in the degradation of articular cartilage and bone in rheumatoid arthritis. Collagen is ont of the major components of the protein matrix of cartilage and bone. Potent inhibitors of collagenase are useful in the treatment of rheumatoid arthritis and associated diseases in which collagenolytic activity is a contributing factor. These diseases include corneal ulceration, periodontal disease, tumour invasion and dystrophic epidermolysis bullosa.

These compounds have substantially no ACE-inhibiting-activity. 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 as noted above 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.

The present invention provides compounds of the general formula I: ##STR1## and pharmaceutically acceptable salts thereof in which

n=1-4

R.sup.1 represents hydroxy, alkoxy, aralkoxy or hydroxy-amino;

R.sup.2 represents hydrogen or alkyl;

R.sup.3 represents

hydrogen,

alkyl,

substituted alkyl wherein the substituent may be one or more of the groups selected from hydroxy, alkoxy, aryloxy, aralkoxy, mercapto, alkylthio, arylthio, alkylsulphinyl (e.g. SOCH.sub.3), alkylsulphonyl (e.g. SO.sub.2 CH.sub.3), carboxy, carboxamido (e.g. CONH.sub.2), carboxyalkyl (e.g. CO.sub.2 CH.sub.3), carboxyaralkyl (e.g. CO.sub.2 CH.sub.2 Ph), aralkoxycarbonylamino (e.g. NHCOOCH.sub.2 Ph), amino, dialkylamino, acylamino (e.g. NHCOCH.sub.3), aroylamino (e.g. NHCOPh) and trihalomethyl (e.g. CF.sub.3),

aralkyl,

substituted aralkyl wherein the substituent on the aryl moiety may be one or more groups selected from halogen (e.g. fluorine, chlorine, bromine, iodine), alkyl, hydroxy, alkoxy, aralkoxy, amino, aminomethyl (CH.sub.2 NH.sub.2), cyano, alkylamino, dialkylamino, carboxy, sulphonamido, alkylthio, nitro and phenyl,

or heteroaralkyl;

Y represents NR.sup.4 wherein R.sup.4 represents H or alkyl; or for certain values of A.sup.1, A.sup.2 may alternatively be a direct chemical bond.

When Y represents NR.sup.4,

A.sup.1 represents a group of formula R.sup.5 wherein

R.sup.5 may be

hydrogen,

alkyl,

aralkyl,

aryl,

substituted aryl wherein the substituent may be one or more groups selected from halogen alkyl, hydroxy, alkoxy, aralkoxy, aralkoxyamino, aminomethyl, cyano, acylamino, dialkylamino, carboxy, sulphonamido, alkylthio, nitro and phenyl,

acyl (e.g. CH.sub.3 CO),

aroyl (e.g. PhCO),

aralkylacyl (e.g. PhCH.sub.2 CO),

alkoxycarbonyl (e.g. (CH.sub.3).sub.3 OCO),

or aralkoxycarbonyl (e.g. PhCH.sub.2 OCO);

A.sup.1 may also represent a group of the formula: ##STR2##

wherein R.sup.6 represents a group having the meanings defined above for R.sup.5 ;

R.sup.7 and R.sup.8 which may be the same or different represent hydrogen, alkyl or aralkyl; or

R.sup.7 and R.sup.8 may together represent an alkylene chain of 2-4 carbon atoms so to form with the adjacent nitrogen atom a nitrogen-containing ring having 4-6 atoms;

R.sup.9 represents

hydrogen,

alkyl,

substituted alkyl wherein the substituent is exactly as defined for this moiety above,

aralkyl,

substituted aralkyl wherein the substituent is exactly as defined for this moiety above, or heteroaralkyl;

A.sup.2 represents a group of the formula ##STR3## wherein

R.sup.10 and R.sup.11 which may be the same or different represent groups having the meanings given above for R.sup.7 or together represent an alkylene chain of 2-4 carbon atoms so as to form with the adjacent nitrogen a nitrogen-containing ring having 4 to 6 atoms;

R.sup.12 represents a group having the meanings given above for R.sup.9.

Additionally, A.sup.1 and A.sup.2 taken together may represent

hydrogen,

alkyl,

aralkyl,

heteroaralkyl,

alkylsulphonyl,

arylsulphonyl,

aralkylsulphonyl,

or a group R.sup.13 CO wherein R.sup.13 represents

hydrogen,

alkyl,

alkoxy,

aryl,

aralkyl,

aralkoxy,

substituted aryl (as defined in R.sup.3), substituted aralkyl (as defined in R.sup.3) and substituted aralkoxy wherein the substituent on the aromatic moiety of the aralkoxy is as defined for aralkyl

phenethenyl (PhCH.dbd.CH--),

phenethynyl (PhC.dbd.C--),

alkylamino,

arylamino,

aralkylamino, P1 or dialkylamino;

In a further aspect of this invention, Y may also represent a direct chemical bond. In this instance, A.sup.1 and A.sup.2 taken together represent

hydrogen,

alkyl,

aryl,

alkoxy,

aralkoxy,

substituted aryl (as in R.sup.3) and substituted aralkoxy (as in R.sup.3) wherein the substituent on the aromatic moiety of the aralkoxy is as defined for aralkyl,

hydroxy,

mercapto,

alkylthio,

arylthio,

aralkylthio, P1 carboxy,

or carboxyalkyl;

A.sup.3 represents a group of the formula

or ##STR4## wherein R.sup.14 represents amino,

alkylamino,

dialkylamino,

hydroxyamino,

or aralkylamino,

and R.sup.15, R.sup.16 and R.sup.17 which may be the same or different represent groups having the meaning given above for R.sup.10,

R.sup.11 and R.sup.12 respectively and

R.sup.18 represents amino,

alkylamino,

dialkylamino,

substituted alkylamino wherein the substituent is amino, hydroxy, alkoxy, carboxy, carboxamido, carboxyalkyl, alkylthio, alkylsulphinyl or alkylsulphonyl,

hydroxyamino,

alkoxyamino,

aralkylamino,

alkoxy,

aralkoxy,

or alkylaminoalkoxy.

all with the exception that when A.sup.3 is alkylamino one of R.sup.2 and R.sup.3 is not hydrogen and the other alkyl or hydroxyalkyl.

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 1 to 10, 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.

By the term heteroaralkyl we mean in particular groups containing 1 to 4 carbon atoms in the alkyl moiety. The term heteroaryl includes for example, pyridyl, thienyl, furyl, indolyl, imidazolyl and thiazolyl.

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. We have found surprisingly that those compounds in which the chiral centre indicated below by an asterisk in the group shown is in the R form are preferred. ##STR5##

Certain groups of compounds according to the invention are preferred, these including the following. A group of preferred compounds are those in which the group A.sup.3 has the following meaning ##STR6## in which R.sup.17 represents substituted alkyl (wherein the substituent is alkoxy, aralkoxy, alkoxycarbonylamino, aralkoxycarbonylamino, carboxyalkyl or carboxyaralkyl); or substituted aralkyl (wherein the aryl substituent is one or more groups selected from alkyl, alkoxy, alkyl thio or aralkoxy). In this preferred group of compounds, R.sup.3 should have the meanings described hereinbefore but excluding aralkyl or heteroalkyl. Within this definition of A.sup.3, there is a preferred subclass of compounds in which A.sup.1 +A.sup.2 taken together represent H, Y is a direct chemical bond, R.sup.2 represents H, and R.sup.3 represents alkyl or substituted alkyl where the substituent(s) is one or more trifluoromethyl groups.

Therefore this first sub-class of preferred compounds may be defined by the formula ##STR7## wherein R.sup.3 and R.sup.17 are as defined above. A most preferred set of compounds within this group are those in which R.sup.17 is benzyloxymethyl (PhCH.sub.2 OCH.sub.2 --), 1-benzyloxyethyl (PhCH.sub.2 OCH(CH.sub.3)--), 4-benzyloxyphenylmethyl (4--PhCH.sub.2 OC.sub.6 H.sub.4 CH.sub.2 --) or 4-methoxyphenylmethyl (4--CH.sub.3 OC.sub.6 H.sub.4 CH.sub.2 --).

In a second preferred sub-class of compounds within the preferred definition of A.sup.3, Y represents NR.sup.4, and A.sup.1 +A.sup.2 represent a group R.sup.13 CO wherein R.sup.13 represents

alkyl, P1 aryl,

aralkyl,

aralkoxy,

substituted aryl, substituted aralkyl and substituted aralkoxy wherein the substituent on the aromatic moiety is exactly as defined hereinbefore,

alkylamino,

arylamino,

aralkylamino

or dialkylamino.

Therefore this second sub-class of preferred compounds may be defined by the formula ##STR8##

Particularly preferred examples are those in which R.sup.4 is H and R.sup.3, R.sup.17 and R.sup.18 are as defined for the first preferred sub-class of compounds. A most preferred series of compounds within this sub-class is where n is 2, R.sup.13 is benzyloxy (PhCH.sub.2 O), substituted benzyloxy (where the aromatic substituent is selected from 4-chloro, 2-chloro, 4-methyl, 4-nitro or 4-amino), benzylamino (PhCH.sub.2 NH), phenyl or substituted phenyl (where the aromatic substituent is selected from 4-chloro, 2-chloro, 4-methyl, 4-nitro or 4-amino).

A further preferred embodiment of the invention is a compound of the formula ##STR9## and the pharmaceutically acceptable acid addition salts thereof wherein x represents hydrogen, alkoxy or benzyloxy; y represents a radical selected from alkyl, alkylthioalkyl, ##STR10## wherein v is 2 or 3, ##STR11## wherein z represents hydrogen or nitro; W.sub.1 and W.sub.2 represent methyl or trifluoromethyl; and R.sup.1 represents hydroxy or alkoxy and the stereochemistry of the carbon marked by the asterisk is R.

Specific compounds according to the invention are those, the preparation of which is described in the Examples.

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 1 mM of the inhibitor being tested or dilutions thereof are incubated at 37.degree. C. for 16 hours with native collagen and collagenase (buffered with Tris HCl--CaCl.sub.2 ; pH 7.6). The collagen is acetyl .sup.14 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 1 mM inhibitor, or a dilution thereof is compared to activity in a control devoid of inhibitor and the results reported as that inhibitor concentration effecting 50% 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 70 kilogram mammal will be in the range of 10 milligrams to 1 gram.

The compounds of this invention can be formulated in compositions such as tablets, capsules or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. About 10 to 500 mg 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, Remington's Pharmaceutical Science Mach Publishing Co., Easton, Pa. 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 known compounds or can be made by known methods, as described in the Examples.

The following description of the preparative methods indicates generally the routes which may be used for the production of the compounds according to the invention.

This process involves reductive amination ##STR12## A keto acid (or derivative) of formula II is condensed with a peptide of formula III. This condensation is conveniently carried out in a suitable solvent (e.g. aqueous tetrahydrofuran, methanol) at a pH between 6 and 7 in the presence of sodium cyanoborohydride which effects reduction to give the desired compound of formula I. Alternatively, II and III may be reacted in the solvent medium to form a Schiff's Base as an intermediate and this may then be reduced catalytically to yield the desired compound of formula I for example by hydrogenation in the presence of Raney Nickel or palladium on charcoal.

As an alternative to Process 1, Route A, the compound of formula II can be condensed with an amino acid of formula IV below (or protected derivative thereof) under the same conditions as given in Process 1 to yield an intermediate of formula V. This intermediate is then subsequently coupled with an amino acid or peptide derivative of the formula A.sup.3 to give the compound of formula I. ##STR13## The known processes for peptide bond formation referred to above and also in the following processes encompass reactive group protection during the coupling reaction, e.g., for amines employing N-t-butyloxycarbonyl or N-benzyloxycarbonyl followed by their removal. Condensing agents are typically those useful in peptide chemistry such as dicyclohexylcarbodiimide, water soluble carbodiimide [N-ethyl-N.sup.1 -(3-dimethylaminopropyl)-carbodiimide], diphenyl phosphoryl azide or V may be activated via the intermediary of active esters such as those derived from 1-hydroxybenzotriazole, 4-nitro phenol, 4-picolyl alcohol.

In an alternative reductive amination process as shown below, the starting materials providing the groups A.sup.1 --A.sup.2 on the one hand and the group A.sup.3 on the other are reversed. Otherwise the process is the same as

This process is applicable to the production of compounds in which R.sup.4 =H. ##STR14## The amino acid (or derivative) VI is condensed with the ketone (VII) under the conditions given in Route A.

As an alternative to Process 1, Route B the synthesis can be performed in a step wise manner by condensing VI with the keto acid (or derivative) VIII to yield the intermediate IX. By known processes (summarised above), IX can then be condensed with an amino acid or peptide derivative A.sup.3 to give I. ##STR15##

This process is essentially an alkylation. ##STR16## In this process the peptide III is alkylated with the appropriate .alpha.-haloacid (or derivative) X or .alpha.-sulphonyloxy acid in a suitable solvent (e.g. CH.sub.2 Cl.sub.2, CH.sub.3 CN, etc.) in the presence of a base (e.g. triethylamine).

As an alternative to this process, the synthesis can be performed in a stepwise fashion firstly to produce an intermediate IX which is then condensed by standard processes above with a peptide derivative A.sup.3 to give the compound of formula I, as described above for the alternative for Process 1, Route A. ##STR17##

In an alternative alkylation shown below the starting materials providing the groups A.sup.1 --A.sup.2 -- on the one hand and A.sup.3 on the other are reversed. Otherwise the method is the same as Process 2, Route A. ##STR18## The amino acid (or derivative) VI is alkylated with the .alpha.-haloacetyl or .alpha.-sulphonyloxyacetyl peptide derivative XI under the conditions described in Route A.

As an alternative to Process 2, Route B the synthesis can be performed in a stepwise fashion by condensing an amino acid (or derivative) VI with a substituted .alpha.-haloacetic acid or .alpha.-sulphonyloxy acetic acid (XII) to yield the intermediate IX which by standard processes is condensed with a peptide derivative A.sup.3 to give the compound of formula I ##STR19##

It should be noted that when A.sup.1 and/or A.sup.2 represent amino acid residues, that these residues may be introduced by standard coupling procedures at any convenient stage of the synthesis.

The starting materials which are required for the above processes are either known in the literature, or can be made by standard processes from known starting materials, or are described in the Examples.

When R.sup.1 in I represents hydroxy, these compounds may be derived from those described above (wherein R.sup.1 =alkoxy or aralkoxy) by hydrolysis in a suitable solvent (such as aqueous methanol) containing a base such as sodium or lithium hydroxide. Alternatively, when R.sup.1 =aralkoxy (such as PhCH.sub.2 O), this group may be removed by hydrogenolysis.

As mentioned above there are various potentially asymmetric centres in the amide derivatives of this invention. In particular the carbon atom which bears the groups (CH.sub.2).sub.n, COR.sup.1 and NH is asymmetric as is that which bears the groups NH, COA.sup.3, R.sup.2 and R.sup.3 (when R.sup.2 and R.sup.3 are not simultaneously hydrogen). The above synthesis can utilize racemates, enantiomers or diastereoisomers as starting materials, the products can therefore exist in racemic or optically active forms. The invention therefore encompasses the racemic form as well as any other optically active forms. As noted above, however, and in contrast to inhibitors of other zinc metalloproteinases (such as angiotensin converting enzyme), the preferred isomer has R-stereochemistry at the carbon atom bearing the groups (CH.sub.2).sub.n, COR.sup.1 and NH whilst having the stereochemistry of the natural amino acids at the other asymmetric centres.

The compounds according to the invention include pharmaceutically acceptable salts of the compounds of formula I. Such salts may include acid addition salts as well as amine salts, etc., and the processes described above for the production of the compounds may include as a final step the conversion of the compound I into such a salt, or the compound may be isolated as such salt.

It is understood that the compounds which bind most effectively to collagenase have R.sup.1 equal to either hydroxy or hydroxyamino. When R.sup.1 is alkoxy or aralkoxy, these compounds function as orally active prodrugs of the parent carboxylic acids; once absorbed these esters are rapidly hydrolysed by non specific plasma esterases to yield the active species.

In order that the invention may be more fully understood the following Examples are given by way of illustration and should not limit the invention in spirit or scope.

N-(Tertiarybutyloxycarbonyl)-L-leucyl-L-valine-N-Hexylamide (2 g) was treated with trifluoroacetic acid (20 ml) at room temperature for forty-five minutes. The excess trifluoroacetic acid was removed in vacuo and the residue dissolved in methanol (20 ml). The solution was adjusted to pH7 with triethylamine. Dried 3A molecular sieve (10 g), sodium cyanoborohydride (0.75 g) and methyl pyruvate (1.5 g) were added and the reaction mixture stirred at room temperature for 2 days. The reaction mixture was then filtered and the filtrate concentrated in vacuo to a gum. The residue was taken up in dichloromethane and the organic phase washed in turn with saturated sodium hydrogen carbonate solution and then 1M citric acid solution and dried over sodium sulphate. The material isolated after evaporation of the dichloromethane was chromatographed on silica, developed in a gradient of 20% ethyl acetate in hexane to 60% ethyl acetate in hexane. Elution with 40% ethyl acetate hexane afforded

Elution with 50% ethyl acetate hexane afforded N[1-(R)-methoxycarbonylethyl]-L-leucyl-L-valine N-hexylamide, (0.5 g) which crystallised from methanol/water as needles m.p. 98.degree.-101.degree. C.; [.alpha.].sub.D.sup.20 =-43.degree. (C=0.2, MeOH); (Found: C,62.7; H,10.2; N,10.5. C.sub.21 H.sub.41 N.sub.3 O.sub.4 requires C,63.1; H,10.3; N,10.5%); .nu..sub.max (Nujol) 3250, 3060, 1730 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.9 (15H,m, 2xCH(CH.sub.3).sub.2 and CH.sub.2 CH.sub.3); 1.3 (3H,d,J=6 Hz, CH.sub.3 CH); 1.2-2.4 (12H,m, 2xCH(CH.sub.3).sub.2, CHCH.sub.2 CH and (CH.sub.2).sub.4); 3.0-3.3 (4H,m, 2xNHCHCO,CH.sub.2); 3.44(1H,q,J=7 Hz, val .alpha.--CH); 3.7(3H,s CH.sub.3 --O); 4.28 (1 H, q. J=7 Hz,NH); 7.16 (1H,m,NH); and 7.92 (1H,d, J=8 Hz,NH).

The N-(t-butyloxycarbonyl)-L-leucyl-L-valine N-hexylamide used as a starting material was prepared as follows:

N-Tertiarybutyloxycarbonyl-L-valine N-hexylamide (15 g) in dichloromethane (30 ml) was treated with trifluoroacetic acid (30 ml) at room temperature for 45 minutes. The excess trifluoroacetic acid was removed in vacuo and the residue redissolved in dichloromethane. The solution was adjusted to pH7 with triethylamine, N-tertiarybutyloxycarbonyl-L-leucine (13 g), 1-hydroxybenzotriazole (7 g) and DCC (10 g) were added and the reaction mixture stirred at room temperature overnight. The reaction mixture was filtered and the organic phase washed with aqueous sodium hydrogen carbonate, 1M citric acid and then water, dried over sodium sulphate and concentrated in vacuo to a gum. The gum was chromatographed on silica developed in a gradient of 20% ethyl acetate to 50% ethyl acetate in petrol to afford N-tertiarybutyloxycarbonyl-L-leucyl-L-valine N-hexylamide (19 g) which crystallised from ether hexane as needles; m.p. 115.degree.-116.degree. C.; (Found: C,63.2; H, 10.3; N,10.1. C.sub.22 H.sub.43 N.sub.3 O.sub.4.1/4H.sub.2 O requires C,63.2; H,10.5; N 10.1%); .nu..sub.max (nujol) 3300, 3080, 1680, 1630 and 1520 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.9 (15H,m, 2xCH(CH.sub.3).sub.2 and CH.sub.2 CH.sub.3); 1.1-2.3 (12H,m, (CH.sub.2).sub.4, CH.sub.2 CH(CH.sub.3).sub.2, CHCH(CH.sub.3).sub.2); 1.45 (9H,s,C(CH.sub.3).sub.3); 3.25 (2H,m,NHCH.sub.2); 4.12 (1H,m, .alpha.--CH from leucyl residue); 4.2 (1H,t, J=5 Hz., .alpha.--CH from valyl residue); 5.07 (1H,m,NH); 6.55 (1H,m,NH) and 6.80 (1H,d,J=10 Hz, NH).

The N-t-butyloxycarbonyl-L-valine N-hexylamide required as a starting material in the preparation above was synthesised as follows:

Tertiarybutyloxycarbonyl-L-valine (25 g) in dichloromethane (200 ml) was treated with 1-hydroxybenzotriazole (15.5 g) hexylamine (11.6 g) and DCC (26 g) at room temperature for 2 days. The solution was filtered and the organic phase washed with aqueous sodium hydrogen carbonate, aqueous citric acid (1M) and water, dried over sodium sulphate and concentrated in vacuo to afford tertiary butyloxy-carbonyl-L-valine N-hexylamide (28 g) which crystallised from methanol-water as needles; m.p. 74.degree.-76.degree. C.; (Found: C,63.8; H,10.6; N,9.4. C.sub.16 H.sub.32 N.sub.2 O.sub.3 requires C,64.0; H,10.7; N,9.32%); .nu..sub.max (Nujol): 3280 and 1630 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.8-1 (9H,m, 3xCH.sub.3); 1.3 (8H,m,(CH.sub.2).sub.4); 1.45 (9H,s, (CH.sub.3).sub.3 C); 2.1 (1H, m,CH(CH.sub.3).sub.2); 3.3 (2H,m, NHCH.sub. 2); 3.9 (1H,dd, J=8 Hz and 5 Hz., .alpha.--CH); 5.2 (1H,d, J=8 Hz, CONH) and 6.26 (1H,m,NH).

N-Boc-O-benzyl-L-tyrosine methylamide (3 g, 7.7 mM) was dissolved in 1:1 TFA/CH.sub.2 Cl.sub.2 (100 ml). After 15 min. the solvent was removed in vacuo and the residue taken up in H.sub.2 O (100 ml), neutralised with NaHCO.sub.3 and extracted into CH.sub.2 Cl.sub.2 (3.times.100 ml). The organic extract was dried and evaporated in vacuo to yield a white solid (2.2 g). This material is CH.sub.2 Cl.sub.2 (50 ml) and DMF (5 ml) was treated at 0.degree. with N-[1-(R)-methoxycarbonylethyl]-L-leucine (1.3 g, 6 mM), 1-hydroxybenzotriazole (960 mg, 6.4 mM) and dicyclohexylcarbodiimide (1.3 g, 6.5 mM) and the mixture allowed to warm to room temperature over 2 h. After a further 12 h the reaction mixture was filtered, washed with sat. NaHCO.sub.3 and then brine, dried and then evaporated in vacuo to yield a solid, 2.5 g (68%). Recrystallisation from CH.sub.2 Cl.sub.2 /hexane gave the title compound; m.p. 65.degree.-68.degree.; [.alpha.].sub.D.sup.20 =-3.3.degree. (C=0.2, MeOH); (Found: C, 66.72; H, 7.61; N, 8.72. C.sub.27 H.sub.37 N.sub.3 O.sub.5 requires C, 67.02; H, 7.71; N, 8.69%); .nu..sub.max (nujol) 3280 br, 1735, 1635 and 1510 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.87 and 0.9 (each 3H, each d, J=4 Hz. and 2.5 Hz, (CH.sub.3).sub.2 CH); 1.1(1H,m, (CH.sub.3).sub.2 CH.sub.2 CH); 1.3 (3H,d,J=8.5 Hz., CH.sub.3 CH); 1.45 (2H,m, (CH.sub.3).sub.2 CH.sub.2 CH); 1.58 br (1H,s, CHNHCH, exch); 2.77 (3H,d,J=6 Hz, NHCH.sub.3); 3.0 (1H,dd,J=12 and 8 Hz, CH.sub.2 C.sub.6 H.sub.4); 3.07 (1H,m, (CH.sub.3).sub.2 CH.sub.2 CH); 3.18 (1H,dd,J=12 and 6 Hz, CHCH.sub.2 C.sub.6 H.sub.4); 3.38 (1H,q,J=8.5 Hz, CH.sub.3 CH); 3.68 (3H,s,OCH.sub.3), 4.62 (1 H,q,J=7 Hz, NHCH(CH.sub.2 C.sub.6 H.sub.4)CO); 5.02 (2H,s,OCH.sub.2 C.sub.6 H.sub.5); 6.71 br (1H,q,J ca 5 Hz, exch. NHCH.sub.3); 6.89 and 7.12 (each 2H, each d, each J=8 Hz, C.sub.6 H.sub.4); 7.4 (5H,m,C.sub.6 H.sub.5) and 7.75 (1H,d, J=9 Hz, exch, CONHCHCO); m/e 484 (100%, [M+1].sup.+), 381 (27) and 172 (28).

The syntheses for the two starting materials required in the preparation above are described in the following paragraphs.

N-t-Butyloxycarbonyl-O-benzyl-L-tyrosine (7.4 g, 20 mM), 1-hydroxybenzotriazole (3 g, 20 mM), methylamine hydrochloride (1.3 g, 20 mM) and N-methyl morpholine were dissolved in CH.sub.2 Cl.sub.2 (200 ml) and cooled to 0.degree. C. Dicyclohexylcarbodiimide (4.2 g, 20 mM) was added and the reaction allowed to warm to room temperature over 4 h. After a further 12 h the reaction mixture was filtered; the filtrate was washed with sat NaHCO.sub.3, 3N citric acid and brine, dried and evaporated in vacuo to give the required N-methyl-amide which was recrystallised from CH.sub.2 Cl.sub.2 and hexane (4.5 g, 58%); m.p. 165.degree.-172.degree.; [.alpha.].sub.D.sup.20 =+15.2.degree. (C=0.2, MeOH); (Found: C, 68.85; H, 7.43; N, 7.39. C.sub.22 H.sub.28 N.sub.2 O.sub.4 requires C, 68.73; H, 7.34; N,7.29%); .nu..sub.max (nujol) 3330, 1685, 1672, 1655 and 1520 cm.sup.-1 ; .delta. (CDCl.sub.3) 1.4 (9H,s, (CH.sub.3).sub.3 C); 2.91 (3H,d, J=5 Hz, NHCH.sub.3); 3.0 (2H,m,CH.sub.2 C.sub.6 H.sub.4); 4.26 (1H,q, J=7.5 Hz, NHCH(CH.sub.2)CO); 5.04 (2H,s, OCH.sub.2 C.sub.6 H.sub.5); 5.08 br (1H,s, exch, NH); 5.84 br (1H,s,exch); 6.91 and 7.09 (each 2H, each d, each J=8 Hz., C.sub.6 H.sub.4) and 7.4 (5H,m,C.sub.6 H.sub.5); m/e 385 (68%, [M+1].sup.+), 329 (100), 285 (66) and 267 (58).

This was prepared in two steps from L-leucine benzyl ester as illustrated below:

L-Leucine benzyl ester, para-toluene sulphonic acid salt (120 g, 0.3M) was dissolved in dry methanol (300 ml) and the pH (moist pH paper) adjusted to 6 using Et.sub.3 N and acetic acid. Methyl pyruvate (62.4 g, 0.6M) in dry methanol (10.0 ml) and 3A molecular sieves were added; the mixture was cooled to 5.degree. and then NaBH.sub.3 CN (100 g, 1.58M) in methanol (600 ml) added. After stirring for 3 days the reaction mixture was filtered and evaporated in vacuo. The residual white solid was partitioned between H.sub.2 O (500 ml) and CH.sub.2 Cl.sub.2 (4.times.200 ml); the organic phase was evaporated to a yellow oil and then partitioned between hexane (250 ml) and 1M oxalic acid (4.times.250 ml). The aqueous phase was neutralised with NaHCO.sub.3 and extracted into CH.sub.2 Cl.sub.2 (4.times.250 ml). The organic phase was dried and evaporated in vacuo to yield a yellow oil (90 g), which was chromatographed on SiO.sub.2 using a gradient of EtOAc in hexane as eluant. The faster running diastereoisomer, N-[1-(R)-methoxycarbonylethyl]-L-leucine benzyl ester, was isolated as an oil (22 g, 20%); [.alpha.].sub.D.sup.20 =-49.5.degree. (C=0.2, MeOH); (Found: C, 66.06; H, 8.19; N, 4.75. C.sub.17 H.sub.26 NO.sub.4 requires C, 66.42; H, 8.19; N, 4.54); .nu..sub.max (nujol) 1735 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.89 and 0.92 (each 3H, each d, each J=3.5 Hz., (CH.sub.3).sub.2); 1.29 (3H,d,J=7 Hz; CH.sub.3); 1.5 (2H,m,CH.sub.2 CH): 1.74 (2H,m,NH and CH.sub.2 CH(CH.sub.3).sub.2); 3.34 (1H,q, J=7 Hz, CHCH.sub.3), 3.39 (1H,t,J=7 Hz, CH.sub.2 CH(NH)CO); 3.69 (3H, s, OCH.sub.3); 5.15 (2H,m,OCH.sub.2 C.sub.6 H.sub.5) and 7.35 (5H,s,C.sub.6 H.sub.5); m/e 308 (100%, [M+1].sup.+); 232 (53) and 172 (44).

The slow running diastereoisomer, N-[1-(S)-methoxycarbonylethyl)-L-leucine benzyl ester, was isolated as an oil (11.3 g, 10%); [.alpha.].sub.D.sup.20 =1.73.degree. (C=0.2, MeOH); (Found: C, 66.42; H, 8.30; N, 4.54. C.sub.17 H.sub.26 NO.sub.4 requires C, 66.42; H, 8.19; N, 4.55%) .nu..sub.max (film) 1730 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.87 and 0.9 (each 3H, each d, each J=5.5 Hz, (CH.sub.3).sub.2 CH); 1.27 (3H,d,J=7 Hz, CH.sub.3 CH); 1.49 (2H,m, (CH.sub.3).sub.2 -CHCH.sub.2); 1.74 (1H, heptet, J=7 Hz, (CH.sub.3).sub.2 CH); 2.2 br (1H,s,NH), 3.3 (2H,m,CHNHCH), 3.65 (3H,s,OCH.sub.3); 5.13 (2H,s,OCH.sub.2 C.sub.6 H.sub.5) and 7.35 (5 H,s,C.sub.6 H.sub.5); m/e 308 (100%, [M+1].sup.+) and 172 (100).

The R-benzyl ester (13 g, 42 mM) was dissolved in methanol (300 ml) and hydrogenated over 10% palladium on charcoal at atmospheric pressure. The catalyst was removed by filtration through celite and the filtrate evaporated in vacuo to yield a white gum, which was crystallised from MeOH/Et.sub.2 O to give the required leucine derivative as a white crystalline solid (7.5 g, 82%); mp 150.degree.-151.degree.; (Found: C, 55.27; H, 8.72; N, 6.43. C.sub.10 H.sub.19 NO.sub.4 requires C, 55.3; H, 8.81; N, 6.45%); [.alpha.].sub.D.sup.20 8.4 (C=0.2, MeOH); .nu..sub.max (nujol) 3400 br, 2500 br and 1755 cm.sup.-1 ; .delta.(d.sup.6 DMSO) 0.85 (6H, m, (CH.sub.3).sub.2 CH.sub.2); 1.17 (3H,d,J=7 Hz, CH.sub.3 CH); 1.38 (2H,m, (CH.sub.3).sub.2 CHCH.sub.2); 1.74 (1H, heptet, J=6 Hz, (CH.sub.3).sub.2 CH); 3.14 (1H, t, J=7 Hz, NHCH(CH.sub.2)CO.sub.2 H); 3.29 (1H,q,J=7 Hz, CH.sub.3 CH) and 3.6 (3H, s,OCH.sub.3); m/e 218 (100%, [M+1].sup.+), 172 (27) and 158 (17).

This was prepared starting from methyl N-t-butyloxycarbonyl-N-benzyloxycarbonyl(R)-2,3-diaminopropionate and benzyl 4-methyl-2-oxo-pentanoate in the steps described in the following paragraphs.

To a stirred solution of methyl N-t-butyloxycarbonyl-N-benzyl oxycarboxyl-(R)-2,3-diaminopropionate (25 g) in THF (150 ml) and acetic acid (8 ml) was added palladised charcoal (10%, 2 g) and the mixture hydrogenated at 25.degree. and 760 mmHg for 2 h. The catalyst was removed by filtration and to the filtrate was added THF (50 ml), benzyl 4-methyl-2-oxopentanoate (50 g, from the corresponding acid by treatment at reflux with benzyl alcohol in the presence of para-toluene sulphonic acid and azeotropic removal of water) in THF (50 ml) and finally water (70 ml). The pH of the rapidly stirred solution was adjusted to 6.5 with triethylamine and sodium cyanoborohydride (4.5 g) was added portionwise over 0.5 h. The pH was maintained at 6.5 by periodic addition of acetic acid. After 16 h at 20.degree., a further portion of sodium cyanoborohydride (2 g) was added and stirring continued for 24 h. The reaction mixture was concentrated in vacuo and the residue was partitioned between CH.sub.2 Cl.sub.2 (200 ml) and water (100 ml). The aqueous layer was washed with fresh CH.sub.2 Cl.sub.2 (2.times.100 ml) and the combined organic extracts washed successively with 3N-citric acid solution water and finally saturated aqueous sodium hydrogen carbonate solution and then dried over MgSO.sub.4. The oil isolated from the CH.sub.2 Cl.sub.2 was purified by chromatography on silica eluting with CH.sub.2 Cl.sub.2 in an increasing ethyl acetate gradient to give the required benzyl ester (9.6 g) as an oil which slowly crystallised, m.p. 59.5.degree.-61.degree. (from ether-hexane); [.alpha.].sub.D.sup.25 =22.1.degree. (C=1.1, MeOH); (Found: C, 62.40; H, 8.08; N, 6.57. C.sub.22 H.sub.34 N.sub.2 O.sub.6 requires C, 62.54; H, 8.11; N, 6.36%); .nu..sub.max (CHCl.sub.3) 1730 and 1705 cm.sup.-1 ; .delta. (CDCl.sub.3) 0.89 (6H, t, J=6.3 Hz, CH(CH.sub.3).sub.2); 1.43 (9H, s, C(CH.sub.3).sub.3); 1.50 (2H, m, CH.sub.2 CH); 1.76 (2H,m,CH.sub.2 CH(CH.sub.3).sub.2 and NH); 3.35 (4H,m,CH.sub.2 N and 2x.alpha.--CH); 3.67 (3H,s,OCH.sub.3); 4.98 br (1H,s,NHCOO), 5.12 (2H,d, J=11.5 Hz, CH.sub.2 Ph) and 7.36 (5H,m,C.sub.6 H.sub.5); m/e 423 ([M+1].sup.+).

The foregoing benzyl ester (6 g) in methanol (50 ml) was hydrogenated at S.T.P. over 10% palladised charcoal (100 mg) for 0.5 h. The catalyst was removed by filtration and the material recovered from the methanol was recrystallised from methanol-ether to give the intermediate carboxylic acid (4.5 g), m.p. 147.degree.-148.degree.. A portion of this material (2.8 g) in CH.sub.2 Cl.sub.2 (100 ml) and DMF (20 ml) was treated at 0.degree. with 1-hydroxybenzotriazole (1.3 g), and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.61 g). After 0.5 h at 0.degree., L-O-benzyl-threonine N-methylamide (1.86 g) in CH.sub.2 Cl.sub.2 (10 ml) was added and the mixture allowed to warm to 20.degree. over 1 h. After 36 h at 20.degree., the reaction mixture was washed in turn with saturated sodium hydrogen carbonate solution, 3N-citric acid solution and finally brine and then dried and evaporated in vacuo. Crystallisation of the resulting oil from ether-pentane gave N-[2-N-(t-butyloxycarbonyl)amino-1-(R)-methoxycarbonylethyl]-L-leucyl-O-be nzyl-L-threonine N-methylamide (3 g), m.p. 95.degree.-97.degree.; (Found; C, 60.42; H, 8.20; N, 10.44. C.sub.27 H.sub.44 N.sub.4 O.sub.7 requires C, 60.43; H, 8.26; N, 10.44%); .delta. (CDCl.sub.3) 0.94 (6H,d, J=6.2 Hz., CH(CH.sub.3).sub. 2); 1.13 (3H,d,J=6.4 Hz, CHCH.sub.3); 1.43 (9H,s,C(CH.sub.3).sub.3); 1.54-1.85 (3H,m,CH.sub.2 CH); 1.95 broad (1H,s, NH); 2.82 (3H,d, J=4.8 Hz, NHCH.sub.3); 3.1-3.62 (4H,m,NCH.sub.2, OCH and .alpha.CH), 3.65 (3H,s,OCH.sub.3), 4.3 (1H,m,.alpha.--CH), 4.45 (1H,dd, J=6.3 and 2.3 Hz, .alpha.--CH); 4.54 and 4.62 (each 1H, each d, each J=11.6 Hz, CH.sub.2 Ph); 5.05 broad (1H,s, NHCOO), 7.05 (1H,m,NHCH.sub.3), 7.32 (5H,m,C.sub.6 H.sub.5) and 7.88 (1H,d, J=8.4 Hz, NH).

To a stirred solution of the foregoing t-butyloxycarbonyl protected peptide (536 mg) in CH.sub.2 Cl.sub.2 (3 ml) was added trifluoroacetic acid (3 ml) at 0.degree.. The solution was allowed to warm to 20.degree. and then stirred at this temperature for 2 h. The residue after evaporation of the organic solvents was taken into CH.sub.2 Cl.sub.2 and the solution washed with saturated sodium hydrogen carbonate solution, dried (Na.sub.2 SO.sub.4) and evaporated in vacuo to yield N-[2-amino-1-(R)-methoxycarbonylethyl]-L-leucyl-O-benzyl-L-threonine N-methylamide (330 mg). This material in CH.sub.2 Cl.sub.2 (10 ml) was added to a solution of N-[N-(2,4-dinitrophenyl)-L-prolyl]-L-leucine (330 mg) in CH.sub.2 Cl.sub.2 (10 ml) containing 1-hydroxybenzotriazole (132 mg) and N-ethyl-N'-(3-dimethylamino-propyl)carbodiimide hydrochloride (191 mg) stirred at 5.degree.. After 16 h at 4.degree. the solvent was removed in vacuo and the residue in ethyl acetate washed in turn with water, saturated sodium hydrogen carbonate solution and finally 3N-citric acid solution. The material isolated from the ethyl acetate was recrystallised from CH.sub.2 Cl.sub.2 -- ether to give the required peptide (440 mg), m.p. 138.degree.-142.degree.; (Found: C, 57.34; H, 6.92; N, 13.61. C.sub.39 H.sub.56 N.sub.8 O.sub.11 requires C, 57.62; H, 6.94; N, 13.78%); .nu..sub.max (CHCl.sub.3) 3295, 1730 and 1635 cms.sup.-1 ; .delta. (CDCl.sub.3) 0.95 (12H,m, 2xCH(CH.sub.3).sub.2); 1.14 (3H,d,J=6.3 Hz, CH.sub.3 CH); 1.3-2.15 and 2.45 (10H,m,CH.sub.2 CH.sub.2 and 2xCH.sub.2 CH); 2.74 (3H,s,NHCH.sub.3); 3.3 (3H,m,CH.sub.2 N and CHO); 3.56 (3H,m,CH.sub.2 N and .alpha.--CH); 3.63 (3H,s,OCH.sub.3); 4.06, 4.25 and 4.56 (1H,2H and 1H respectively, each m, 4x.alpha.CH); 4.43 and 4.55 (each 1H, each d, J=11.7 Hz, CH.sub.2 Ph); 7.00 (1H,d, J=9.5 Hz, 6-H in C.sub.6 H.sub.3); 7.28 (5H,s,C.sub.6 H.sub.5); 8.16 (1H,dd, J=9.5 and 2.8 Hz, 5-H in C.sub.6 H.sub.3) and 8.54 (1H,d, J=2.8 Hz, 3-H in C.sub.6 H.sub.3); m/e 813 ([M+1].sup.+).

O-Benzyl-1-threonine N-methylamide used in step (b) above was prepared from N-t-butyloxycarbonyl-O-benzyl-L-threonine N-methylamide by exposure to trifluoroacetic acid in CH.sub.2 Cl.sub.2. This in turn was prepared from N-t-butyloxycarbonyl-O-benzyl-L-threonine and methylamine using the procedure described in Example 2 for the tyrosine analogue.

N-[N-(2,4-Dinitrophenyl)-L-prolyl]-L-leucine used as starting material in stage (c) was prepared from N-(2,4-dinitrophenyl)-L-proline and leucine methyl ester using the coupling procedure involving N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride as the condensing agent in the presence of 1-hydroxybenzotriazole (as illustrated in Example 3) followed by hydrolysis of the methyl ester with 2N-sodium hydroxide solution (see Example 5).

Methyl N.beta.-t-butyloxycarbonyl-N.alpha.-benzyloxycarbonyl-(R)-2,3-diaminopropi onate, used as the starting material in stage (a), was prepared as follows:

To a stirred suspension of N-benzyloxycarbonyl-(R)-2,3-diaminopropionic acid [19.5 g; from N.alpha.-benzyloxycarbonyl-D-asparagine exactly as described for the L-isomer in Synthesis, 266, (1981)] in dry methanol (60 ml) at -20.degree. was added thionyl chloride (30 g) dropwise over 40 min. The reaction mixture was allowed to warm to 20.degree. over 1 h and then heated at 50.degree. for 1 h. The residue after removal of the solvent was recrystallised from methanol-ether to give methyl N.alpha.-benzyloxycarbonyl-R-2,3-diaminopropionate hydrochloride (22.5 g); m.p. 170.degree.-172.degree.; (Found: C,49.86; H, 5.89; N, 9.53. C.sub.12 H.sub.17 N.sub.2 O.sub.4 Cl requires C, 49.91; H, 5.93; N, 9.70%); .nu..sub.max (Nujol) 3305, 1735 and 1688 cm.sup.-1 ; .delta. (d.sup.6 DMSO) 3.00-3.32 (2H,m,CH.sub.2 NH.sub.2); 3.7 (3H,s,OCH.sub.3); 4.45 (1H,m,.alpha.--CH); 5.09 (2H,s,CH.sub.2 Ph); 7.36 (5H,s,C.sub.6 H.sub.5); 7.95 (1H,d,J=7.5 Hz, NHCOO) and 8.28 broad (3H,s, NH.sub.3); m/e 253 ([M+1].sup.+). A portion of this material (22.5 g) in DMF (150 ml) was treated with Et.sub.3 N until the pH was 10. Di-t-butyl dicarbonate (16.8 g) was added to the solution stirred at 5.degree.. After a further 2 h at 20.degree., the reaction mixture was filtered and evaporated in vacuo and the residue partitioned between ether and water. The aqueous layer was extracted twice more with fresh ether and the combined organic extracts washed in turn with ice cold 1N-hydrochloric acid, saturated sodium hydrogen carbonate solution and finally water. The oil isolated from the ether was crystallised from ethyl acetate-hexane to give methyl N.beta.-t-butyloxycarbonyl-N.alpha.-benzyloxycarbonyl-(R)-2,3-diaminopropi onate (22.5 g); m.p. 89.degree.-91.degree.; (Found: C, 57.86; H, 6.95; N, 7.93. C.sub.17 H.sub.24 N.sub.2 O.sub.6 requires C, 57.94; H, 6.86; N, 7.95%); .nu..sub.max (CHCl.sub.3) 3600 and 1700 cm.sup.-1 ; .delta.1.4 (9H,s,C(CH.sub.3).sub.3); 3.5 broad (2H, s, CH.sub.2 N); 3.72 (3H, s, OCH.sub.3); 4.4 (1H, .alpha.--CH); 5.09 (2H, s, CH.sub.2 Ph); 5.2 broad (1H,s, NHCOO); 6.06 (1H, d, J=7.3 Hz, NHCOO) and 7.32 (5H, s, C.sub.6 H.sub.5).

Leucine benzyl ester para-toluene sulphonic acid salt (113 g) in dry acetonitrile (800 ml) was treated with methyl 2-bromopropionate (62.7 ml) and N-methyl morpholine (100 ml) under reflux for 16 h. The reaction mixture was concentrated in vacuo and the residue in ethyl acetate washed with brine, dried (Na.sub.2 SO.sub.4) and evaporated. Chromatography of the resulting oil on silica in 1:4 ethyl acetate-hexane gave N-[1-(R)-methoxycarbonylethyl]-L-leucine benzyl ester (45 g) as the faster running fraction. This material was treated exactly as described above in Example 2 to give the title compound.

The methyl ester (1.5 g, 3.1 mM) from Example 2 was dissolved in methanol (20 ml) and treated with 1N sodium hydroxide (3.5 ml, 3.5 mM). After 18 h, the pH was adjusted to 5 with acetic acid and the solvent removed in vacuo to yield a white solid. Recrystallisation first from water and then from methanol-ether yielded the N-[1-(R)-carboxyethyl]-L-leucyl-O-benzyl-L-tyrosine N-methylamide as a white powder (1.02 g), m.p. 195.degree.; [.alpha.].sub.D.sup.20 =+7.2.degree. (C=0.2, MeOH); (Found: C, 63.76; H, 7.64; N, 8.57. C.sub.26 H.sub.35 N.sub.3 O.sub.5.H.sub.2 O requires C, 64.05; H, 7.65; N, 8.62%); .nu..sub.max (Nujol) 3540 (br), 3330 and 1680 cm.sup.-1 ; .delta. (d.sup.6 DMSO) 0.82 (3H,d, J=6 Hz, (CH.sub.3).sub.2 CH); 0.87 (3H,d, J=6 Hz, (CH.sub.3).sub.2 CH); 1.13 (3H,d, J=7 Hz., CH.sub.3 CH); 1.24 (2H,t, J=6 Hz., CHCH.sub.2 CH: 1.59 (1H,m, (CH.sub.3).sub.2 CHCH.sub.2); 2.63 (3H,d, J= 5 Hz, NHCH.sub.3); 2.72 (1H,dd, J=11 and 12 Hz, CHCH.sub.2 C.sub.6 H.sub.4); 2.8 (1H,q, J=7 Hz, CH.sub.3 CH(NH)CO.sub.2 H); 2.95 (1H,dd, J=12 and 5 Hz, CHCH.sub.2 C.sub.6 H.sub.4); 3.21 (1H,t, J=7.5 Hz, NHCH(CH.sub.2 CH(CH.sub.3).sub.2 CO); 4.53 br (1H,m, NHCH(CH.sub.2 C.sub.6 H.sub.4)CO); 5.08 (2H,s,C.sub.6 H.sub.4 OCH.sub.2 C.sub.6 H.sub.5); 6.93 and 7.17 (each 2H, each d, each J=7.5 Hz, C.sub.6 H.sub.4 O); 7.48 (5H,m,C.sub.6 H.sub.5); 7.98 br (1H,q, J=5 Hz, NHCH.sub.3, exch) and 8.1 (1H,d, J=9 Hz., CHCONHCH, exch); m/e 470 (88% [M+1] .sup.+), 452 (51), 424 (29), 285 (100) and 158 (49).

N[1-(R)-Ethoxycarbonylethyl]-L-Leucine N-phenethylamide (710 mg, 2.1 mM) was dissolved in MeOH (50 ml) and treated with IN NaOH (3 ml, 3 mM) at room temperature. After 12 h, the reaction mixture was acidified with AcOH and evaporated in vacuo to a solid which was washed with H.sub.2 O and dried to yield the title compound (400 mg); m.p. 201.degree.-205.degree.; (Found: C,66.44; H,8.55; N,9.11; C.sub.17 H.sub.26 N.sub.2 O.sub.3 requires C,66.64; H,8.55; N,9.14%); .nu..sub.max (Nujol) 3330, 1660 and 1530 cm.sup.-1 ; .delta.(d.sup.6 DMSO) 0.825 (6H,t,J=6.2 Hz, (CH.sub.3).sub.2 CH), 1.15 (3H,d, J=6.8 Hz, CH.sub.3 CH), 1.29 (2H,m,CH.sub.2 CH), 1.55 (1H, heptet,J=7 Hz, CH(CH.sub.3).sub.2), 2.71 (2H,t,J=7 Hz, CH.sub.2 C.sub.6 H.sub.5), 3.0 (1H,q,J=7 Hz, CHCH.sub.3), 3.14 (1H,t,J=7 Hz, .alpha.--CH), 3.32 (2H,q,J=6 Hz, NHCH.sub.2 CH.sub.2), 7.12 (5H,m,C.sub.6 H.sub.5), 7.5 (2H,br s, OH and CHNHCH) and 8.15 (2H,t,J=5 Hz, NHCH.sub.2).

The N-[1-(R)-ethoxycarbonylethyl]-L-leucine N-phenethylamide required in the preparation above was synthesised as follows:

N[1-(R)-Ethoxycarbonylethyl]-L-leucine (1.39 g, 6 mM), N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (1.16 g, 6 mM), 1-hydroxybenzotriazole (0.93 g, 6 mM) and phenethylamine (0.7 g, 6 mM) were dissolved in DMF (50 ml) at -6.degree.. N-Methyl-morpholine (0.62 g, 6.2 mM) was added and the reaction mixture allowed to warm to room temperature. After 12 h the solvent was removed in vacuo. The residue in EtOAc (150 ml) was washed with H.sub.2 O (2.times.100 ml), dried and evaporated in vacuo to yield an oil which was purified by chromatography on SiO.sub.2 in EtOAc to give N[1-(R)-ethoxycarbonylethyl]-L-leucine N-phenethylamide as an oil (1.84 g). For analysis, a portion of this material was dissolved in MeOH, treated with anhydrous HCl in Et.sub.2 O and evaporated in vacuo to yield the corresponding hydrochloride as a foam; (Found: C,60.28; H,8.54; N,7.35; C.sub.19 H.sub.30 N.sub.2 O.sub.3.HCl.0.4H.sub.2 O (requires C,60.35; H,8.48; N,7.41%); [.alpha.].sub.D.sup.20 =+9.4.degree. (c=0.2, MeOH); .nu..sub.max (Nujol) 3400 (br), 3100 (br), 2510 (br), 2400 (br), 1740, 1670 and 1550 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.92 and 0.95 (6H, each d, each J=7 Hz, (CH.sub.3).sub.2 CH), 1.27 (3H, t, J=6.5 Hz, CH.sub.3 CH.sub.2), 1.28 (3H,d,J=7 Hz, CH.sub.3 CH), 1.3-1.7 (3H,m,CH.sub.2 CH), 2.85 (2H,t,J=6 Hz,CH.sub.2 CH.sub.2 C.sub.6 H.sub.5), 3.21 (1H,dd,J=10 Hz and 4 Hz, .alpha.--CH), 3.27 (3H,q,J=6.5 Hz, CH.sub.3 CH), 3.54 (2H,q,J=7 Hz, NHCH.sub.2 CH.sub.2), 4.16 and 4.18 (2H, each q, each J=6.5 Hz, OCH.sub.2 CH.sub.3) and 7.25 (6H,m,C.sub.6 H.sub.5 and NHCO).

The starting material required in the preparation given above was synthesised in two steps from leucine benzyl ester as follows:

L-Leucine benzyl ester (186.65 g, 0.843M), ethyl 2-bromopropionate (153.1 g, 0.846M) and N-methylmorpholine (165 ml, 1.5M) were dissolved in dry CH.sub.3 CN (800 ml) and refluxed for 12 h. The solvent was removed in vacuo and the residue partitioned between H.sub.2 O (21) and EtOAc (3.times.11). The organic phase was washed with brine, dried and evaporated in vacuo. The resulting oil was chromatographed on SiO.sub.2 in 7.5% EtOAc in hexane to give the title compound (70 g) as the faster running fraction; (Found: C,67.02; H,8.42; N,4.25; C.sub.18 H.sub.27 NO.sub.4 requires C,67.26; H,8.47; N,4.36%); .nu..sub.max film 1730 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.88 and 0.9 (each 3H, each d, each J=6.5 Hz, CH(CH.sub.3).sub.2) 1.23 (3H,t,J=7 Hz,CH.sub.3 CH.sub.2 O), 1.27 (3H,d,J=7 Hz,CH.sub.3 CH), 1.5 (2H,m,CHCH.sub.2), 1.7 (1H, heptet, J=7 Hz, CH(CH.sub.3).sub.2), 2.2 (1H, br, s,NH), 3.32 (2 H,m,CHNHCH), 4.10 and 4.12 (each 1H, each q, each J=7 Hz, OCH.sub.2 CH.sub.3), 5.12 (2H,s, OCH.sub.2 C.sub.6 H.sub.5) and 7.34 (5H,s, C.sub.6 H.sub.5); m/e 322 (100%; [m+1]).sup.+, 260 (15), 186 (26) and 112 (28).

N[1-(R)-Ethoxycarbonylethyl]-L-leucine benzyl ester (69.09 g, 0.215M) was dissolved in MeOH (300 ml) and hydrogenated at 1 atmosphere over 5% palladium on charcoal (5 g). After 1.5 h, the catalyst was removed by filtration and the filtrate evaporated in vacuo to yield a solid (46.8 g) which was recrystallized from MeOH/Et.sub.2 O to yield the title compound (24 g); m.p. 149.degree.-150.degree.; [.alpha.].sub.D.sup.20 =8.8.degree. (C=1.4, MeOH); (Found: C,57.14; H,9.06; N,6.02; C.sub.11 H.sub.21 NO.sub.4 requires C,57.12; H,9.15; N,6.06%); .nu..sub.max (Nujol) 3090 (br), 2300 (br), 1755 and 1560 cm.sup.-1 ; .delta.(d.sup.6 DMSO) 0.86 and 0.87 (6H, each d, each J=6.5 Hz, (CH.sub.3).sub.2 CH), 1.19 (6H,m,OCH.sub.2 CH.sub.3 and CHCH.sub.3), 1.36 (2H,m, CHCH.sub.2 CH), 1.74 (1H, heptet, J=6.5 Hz, CH.sub.2 CH(CH.sub.3).sub.2), 3.14 (1H,t,J=6.2 Hz,.alpha.--CH), 3.27 (1H,q,J=6.8 Hz,NHCHCH.sub.3) and 4.07 (2H,q,J=7 Hz,OCH.sub.2 CH.sub.3); m/e 232 (100%, [m+1].sup.+), 186 (3) and 158 (7).

This was prepared from D-methionine methyl ester hydrochloride, 2-oxo-4-methylpentanoic acid and O-methyl-2-tyrosine in the steps described in the following paragraphs.

D-Methionine methyl ester hydrochloride (10 g, 50 mM) and 2-oxo-4-methylpentanoic acid t-butyl ester (9.3 g, 50 mM) were dissolved in THF (75 ml) and H.sub.2 O (25 ml). The pH was adjusted to 6.5 with N-methylmorpholine, NaCNBH.sub.3 (630 mg, 10 mM) was added, followed after 2 h by a further portion (400 mg). After 18 h the reaction mixture was evaporated in vacuo and then partitioned between EtOAc (100 ml) and sat.NaHCO.sub.3 solution (2.times.100 ml). The oil isolated from the organic layer was chromatographed on SiO.sub.2 using a gradient of 5-10% EtOAc in hexane. The faster running fraction afforded the required isomer as an oil (1.4 g). .delta.(CDCl.sub.3) 0.96 (6H,m, (CH.sub.3).sub.2 CH), 1.5 (9H,m, (CH.sub.3).sub.3 C), 1.4-2.0 (5H,m, CH.sub.2 CH and SCH.sub.2 CH.sub.2 CH), 2.1 (3H,S,CH.sub.3 S), 2.62 (2H,m,SCH.sub.2 CH.sub.2), 3.17 and 3.38 (each 1H, t, each J=7 Hz, CHNHCHO and 3.7 (3H,s,OCH.sub.3)]. The slower running isomer was also obtained as an oil (1.2 g). .delta.(CDCl.sub.3) 0.95 (6H,m, (CH.sub.3).sub.2 CH), 1.5 (9H,s, (CH.sub.3).sub.3 C), 1.5-2.1 (5H,m, SCH.sub.2 CH.sub.2 CH and CH.sub.2 CH), 2.09 (3H,s,CH.sub.3 S), 2.6 (2H,m, SCH.sub.2 CH.sub.2), 3.08 and 3.22 (each 1H, each dd, each J=7 Hz, CHNHCH) and 3.7 (3H,s,OCH.sub.3)].

The faster running t-butyl ester (2.9 g, 9 mM) from above was dissolved in TFA (50 ml) and H.sub.2 O (0.5 ml). After 3 h the mixture was evaporated in vacuo, toluene (50 ml) was added and the solution was reevaporated in vacuo. The resulting oil was dissolved in CH.sub.2 Cl.sub.2 (100 ml) and the pH adjusted to 7 (moist pH paper). O-Methyl-L-tyrosine N-methylamide (2.0 g, 10 mM) and 1-hydroxybenzotriazole (1.5 g, 10 mM) were added. The reaction mixture was cooled to 0.degree., treated with dicyclohexylcarbodiimide (2.1 g, 10 mM) and then allowed to warm slowly to room temperature. After 18 h, the mixture was filtered and the filtrate washed with H.sub.2 O and sat. NaHCO.sub.3 solution. After drying, the solvent was removed in vacuo to yield an oil which was chromatographed on SiO.sub.2 in 1:1 EtOAc/hexane. The relevant fractions yielded, after recrystallisation from Et.sub.2 O/hexane, the title compound (1.4 g); m.p. 108.degree.-111.degree.; (Found: C,58.62; H,7.91; N,8.85; C.sub.23 H.sub.35 N.sub.3 O.sub.5 S requires C,59.07; H,7.97; N,8.96%); .nu..sub.max (Nujol) 3380 (br), 1740, 1610 and 1560 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.86 and 0.87 (each 3H, each d, each J=6 Hz, (CH.sub.3).sub.2 CH), 1.15 and 1.4 (each 1H, each m, CH.sub.2 CH(CH.sub.3).sub.2), 1.6 (1H,m,CH), 1.90 (2H,m,SCH.sub.2 CH.sub.2), 2.08 (3H,s,CH.sub.3 S), 2.5 (2H,m, SCH.sub.2), 2.77 (3H,d,J=6 Hz, NHCH.sub.3), 3.05 (3H,m,CH.sub.2 C.sub.6 H.sub.5 and .alpha.--CH) 3.47 (1H,t,J=5 Hz, .alpha.--CH), 3.7 (3H,s,OCH.sub.3), 3.8 (3H,s,CO.sub.2 CH.sub.3), 4.63 (1H,q,J=7 Hz, .alpha.--CH), 6.69 (1H,brq,J=6 Hz, NHCH.sub.3), 6.82 and 7.13 (each 2H, each d, J=9 Hz,C.sub.6 H.sub.4) and 7.53 (1H,d,J=9 Hz, CONHCH); m/e 468 (100%, [m+1].sup.+) and 232 (27).

N[1-(R)-Carbomethoxy-3-methylthiopropyl]-L-leucyl-O-methyl-L-tyrosine N-methylamide (100 mg, 0.2 mM) was dissolved in MeOH (10 ml) and treated with IN NaOH (0.25 ml, 0.25 mM). After 18 h another portion of IN NaOH (0.5 ml, 0.5 mM) and H.sub.2 O (2 ml) were added. After a further 18 h the reaction mixture was acidified with AcOH and evaporated in vacuo. The resulting white solid was chromatographed on C.sub.18 -Silica eluting with a gradient of 10% to 40% MeOH in H.sub.2 O. The relevant fractions were pooled and evaporated in vacuo; the residue was recrystallised from hot H.sub.2 O to yield the title compound (20 mg); m.p. 170-180; (Found: C,56.88; H,7.49; N,9.05; calculated for C.sub.22 H.sub.35 N.sub.3 O.sub.5 S,.0.6H.sub.2 O: C,56.90; H,7.86; N,9.0 5%); .nu..sub.max (Nujol) 3340, 1650 and 1625 cm.sup.-1 ; .delta.(d.sup.6 DMSO) 0.82 (6H,t,J=7 Hz, (CH.sub.3).sub.2 CH), 1.17 and 1.5-1.9 (5H,m, CH.sub.2 CH(CH.sub.3).sub.2 and SCH.sub.2 CH.sub.2), 2.04 (3H,s,CH.sub.3 S), 2.3 (2H,m,--SCH.sub.2), 2.6 (3H,d,J=5 Hz, NHCH.sub.3), 2.6-2.95 (3H,m,CH.sub.2 C.sub.6 H.sub.4 and .alpha.--CH),), 3.14 (1H,t,J=7 Hz,.alpha.--CH), 3.7 (3H,s,OCH.sub.3), 4.25 (1H,m, --CH), 6.8 and 7.12 (2.times.2H, each d, each J=9 Hz,C.sub.6 H.sub.4), 7.88 (1H,q,J=5 Hz, NHCH.sub.3) and 8.18 (1H,d,J=9 Hz, NHCH); m/e 454 (100%, [m+1].sup.+).

O-Methyl-L-tyrosine N-methylamide used in stage (a) above was prepared from Z-L-tyrosine as follows:

Z-L-Tyrosine (150 g, 0.476M) was dissolved with stirring in dilute aqueous sodium hydroxide (42 g, 1.05M in 750 ml H.sub.2 O). Dimethyl sulphate (51 ml, 0.54M) was then added dropwise over 30 min. to this solution at room temperature. After 2 h further NaOH was added (4.2 g, 0.105M in 40 ml H.sub.2 O) followed by dimethyl sulphate (5.1 ml) after which the reaction was allowed to stir overnight at room temperature. The reaction was then acidified to pH 2, extracted with CH.sub.2 Cl.sub.2 and the CH.sub.2 Cl.sub.2 layer washed with aqueous sodium chloride, dried (MgSO.sub.4) and concentrated in vacuo to yield the crude product. Recrystallisation from ethyl acetate/hexane gave the required methyl ether (155 g); m.p. 114.degree.-115.degree.; (Found: C,65.84; H,5.82; N,4.22. C.sub.18 H.sub.19 NO.sub.5 requires C,65.64; H,5.81; N,4.25%); .nu..sub.max (CHCl.sub.3) 3412 and 1715 cm.sup.-1 ; .delta.(CDCl.sub.3) 3.1 (2H,m, CH.sub.2 C.sub.6 H.sub.4); 3.76 (3H,s,OCH.sub.3); 4.66 (1H,dd,J=8 and 3 Hz, .alpha.--CH); 5.1 (2H,m,CH.sub.2 C.sub.6 H.sub.5); 5.23 (1H,d,J=8 Hz, NH); 6.8 (2H,d,J=8.6 Hz,Tyr H-3,H-5); 7.05 (2H,d,J=8.6 Hz,Tyr H-2,H-6); 7.33 (5H, broad s,C.sub.6 H.sub.5); m/e 330 (68% [M+1].sup.+), 285 (100% [M--CO.sub.2 H].sup.+).

To a stirred solution of N-(Benzyloxycarbonyl)-O-methyl-L-tyrosine (155 g, 0.471M) in dry CH.sub.2 Cl.sub.2 was added 1-hydroxybenzotriazole (63.6 g, 0.471M) followed by a solution of DCC (97.2 g, 0.0471M) in CH.sub.2 Cl.sub.2 (100 ml) added slowly at 0.degree. C. After warming to room temperature over 1 hr, a solution of methylamine (30 g) in CH.sub.2 Cl.sub.2 (250 ml) was added dropwise to the reaction mixture which was then stirred overnight at room temperature. The reaction was then filtered, washed with saturated aqueous sodium bicarbonate (x2), dried (MgSO.sub.4) and concentrated in vacuo to give a solid. Recrystallisation from ethyl acetate/hexane gave the desired amide (142 g); m.p. 167.degree.-170.degree.; (Found: C,66.72; H,6.58; N,8.29. C.sub.19 H.sub.22 N.sub.2 O.sub.4 requires C,66.65; H,6.48; N,8.18%).nu..sub.max (CHCl.sub.3) 3440, 1710 and 1672 cm.sup.-1 ; .delta. (CDCl.sub.3) 2.70 (3H,d,J=5 Hz,NCH.sub.3); 2.98 (2H,m,CH.sub.2 C.sub.6 H.sub.4); 3.77 (3H,s,OCH.sub.3); 4.30 (1H,dd,J=7.6 and 3 Hz, .alpha.--CH); 5.06 (2H,m,OCH.sub.2 C.sub.6 H.sub.5); 5.43 (1H,m,OCONH); 5.84 (1H,m,CONH); 6.80 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.15 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.32 (5H,m,C.sub.6 H.sub.5); m/e 343 (100%, [m+1].sup.+).

To a solution of N-(Benzylocycarbonyl)-O-methyl-L-tyrosine N-methylamide (15.6 g, 0.056M) in ethanol (200 ml) and DMF (200 ml) was added 10% Pd/C (1 g) and trifluoroacetic acid (4 ml). Hydrogen was then passed through the solution for 3 h after which the reaction was filtered and concentrated in vacuo. The residue was dissolved in H.sub.2 O (150 ml), neutralised with sodium bicarbonate and extracted into CH.sub.2 Cl.sub.2 (150 ml.times.5). The combined organic extracts were dried (Na.sub.2 SO.sub.4) and concentrated in vacuo to yield an oil which subsequently crystallised. Recrystallisation from ethyl acetate/hexane gave O-methyl-L-tyrosine N-methylamide (9.0 g), m.p. 90.degree.-91.degree.; (Found: C,63.49; H,7.71; N,13.44 C.sub.11 H.sub.16 N.sub.2 OA requires C,63.44; H,7.74; N,13.45%) .nu..sub.max (CHCl.sub.3) 3350 and 1660cm.sup.-1 ; .delta.(CDCl.sub.3) 1.3 (2H,br,NH.sub.2); 2.64 (1 H,dd,J=13.8 and 9.2 Hz,CHC.sub.6 H.sub.4); 2.80 (3H,d,J=5 Hz,NCH.sub.3); 3.18 (1H,dd,J=13.8 Hz and 4 Hz, CHC.sub.6 H.sub.4); 3.55 (1H,dd,J=9 Hz and 4 Hz, .alpha.--CH) 3.78 (3H,s,OCH.sub.3); 6.85 (2H,d,J=8,2 Hz,Tyr H-3 and H-5); 7.12 (2H,d,J=8,2 Hz, Tyr H-2 and H-6); 7.28 (1H,br,CONH).

To a solution of methyl 5-N-(benzyloxycarbonyl)amino-2-bromo-pentanoate (10.3 g, 0.03M), L-leucyl-O-methyl-L-tyrosine N-methylamide (9.6 g, 0.03M) and N-methyl morpholine in dry acetonitrile (100 ml) was added sodium iodide (4.5 g, 0.03M). The mixture was then stirred and heated under reflux for 24 hr. The cooled reaction mixture was then filtered and evaporated in vacuo to yield an oil. Chromatography on silica eluting with dichloromethane in an increasing ethyl acetate gradient gave the title compound (2.8 g); m.p. 124.degree.-127.degree.; (Found: C,63.7; H,7.52; N,9.56. C.sub.31 H.sub.44 N.sub.4 O.sub.7 requires C,63.68; H,7.58; N,9.58%); .nu..sub.max (CHCl.sub.3) 3400, 1718 and 1660 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.85 and 0.87 (each 3H, each d, each J=6 Hz, CH)CH.sub.3).sub.2) 1.0-1.85 (8H,m,NHCH.sub.2 CH.sub.2 CH.sub.2, CH.sub.2 CH and NH); 2.74 (3H,d,J=5 Hz,NCH.sub.3); 2.96-3.42 (6H,m,NHCH.sub.2, .alpha.--CHx2, CH.sub.2 C.sub.6 H.sub.4); 3.66 (3H,s,OCH.sub.3); 3.75 (3H,s,OCH.sub.3); 4.6 (1H,dd,J=13 Hz and 6 Hz, .alpha.--CH); 5.0 (1H,m,OCONH); 5.1 (2H,s,CH.sub.2 C.sub.6 H.sub.5); 6.71 (1H,br,CONH); 6.80 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.10 (2H,d,J=8.6 Hz, Tyr H-2and H-6); 7.35 (5H,m,C.sub.6 H.sub.5); 7.56 (1H,m,CONH); m/e 585 (100% [m+1].sup.+).

The starting materials used in this preparation were synthesised as follows:

To a solution of BOC-L-Leucine (5.26 g, 0.021M) in CH.sub.2 Cl.sub.2 (40 ml) and DMF (10 ml) stirred at 0.degree. was added N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (4 g, 0.021M). After 15 min., N-methyl morpholine (0.021M) was added followed by, after a further 10 min. at 0.degree., a solution of O-methyl-L-tyrosine N-Methylamide (4.3 g, 0.019M) in CH.sub.2 Cl.sub.2. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction was then concentrated in vacuo, and the residue in CH.sub.2 Cl.sub.2, washed in turn with H.sub.2 O (200 ml), saturated aq. NaHCO.sub.3 (200 ml), dilute HCl (1M; 200 ml), saturated aq. NaHCO.sub.3 (200 ml) and water (150 ml), dried (Na.sub.2 SO.sub.4) and evaporated in vacuo to a solid. Recrystallisation from ethyl acetate/hexane gave N-(Tertiarybutoxycarbonyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide as a white crystalline solid, (4.5 g), m.p. 159-161; (Found: C,62.65; H,8.33; N,9.96. C.sub.22 H.sub.35 N.sub.3 O.sub.5 requires C,62.69; H,8.37; N9.97%). .nu..sub.max (CDCl.sub.3) 3400, 1700 and 1662 cm.sup.-1 ; (.delta.(CDCl.sub.3) 0.91 (6H,dd,J=7 and 14 Hz, CH(CH.sub.3).sub.2); 1.37 (9H,s, OC(CH.sub.3).sub.3); 1.47-1.7 (3H,m,CH.sub.2 CH(CH.sub.3).sub.2); 2.71 (3H,d,J=4.7 Hz, NHCH.sub.3), 2.98 and 3.14 (each 1H, each m, CH.sub.2 C.sub.6 H.sub.4); 3.78 (3H,s,OCH.sub.3); 4.0 and 4.61 (each 1H, each m, 2x.alpha.--CH); 4.86, (1H,br s,OCONH); 6.40 and 6.55 (each 1H, each br s, CONHx2); 6.82 (2H,d,J=8.4 Hz, Tyr H-3 and H-5); 7.08 (2H,d,J=8.4 Hz, Tyr H-2and H-6); m/e 422 (70%, [m+1].sup.+), 365 (70%, [m-58].sup.+).

To a stirred solution of N-Tertiarybutoxycarbonyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide (7.0 g, M) in CH.sub.2 Cl.sub.2 (40 ml) cooled at 10.degree. was added trifluoroacetic acid (70 ml) and the resulting solution stirred at room temperature for 1 h. The reaction was then concentrated in vacuo, and the residue dissolved in water, neutralised with sodium bicarbonate and extracted with CH.sub.2 Cl. The organic extracts were dried (Na.sub.2 SO.sub.4), filtered and concentrated in vacuo to give L-leucyl-O-methyl-L-tyrosine N-methylamide (5.2 g); m.p. 128.degree.-132.degree.; (Found: C,60.04; H,8.72; N,12.26 C.sub.17 H.sub.27 N.sub.3 O.sub.3 requires C,60.16; H,8.61; N,12.38%); .nu..sub.max (CDCl.sub.3) 3325 and 1655 cm.sup.-1 ; [.alpha.].sub.D.sup.20 =10.2.degree. (C=2.00, MeOH); .delta.(CD.sub.3 OD) 0.88 and 0.92 (each 3H, each d; 1.2-1.4 (1H,m,CH.sub.2 CH(CH.sub.3).sub.2); 1.44-1.8 (2H,m,CHCH(CH.sub.3).sub.2); 2.73 (3H,d,J=5 Hz, NCH.sub.3); 2.82-3.3 (4H,m,NH.sub.2,CH.sub.2 C.sub.6 H.sub.4); 3.46 (1H,m,CH); 3.76 (3H,s,OCH.sub.3 ); 4.58 (1H,q,dd,J=8 and 3 Hz, .alpha.--CH); 6.56 (1H,br,CONH); 6.82 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.13 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.96 (1H,d,J=8 Hz,CONH); m/e 322 (100% [m+1].sup.+).

To a stirred solution of .epsilon.-Z-ornithine (53.2 g, 0.1M) in dilute H.sub.2 SO.sub.4 (2.5N, 500 ml) at 0.degree. was added KBr (60 g, 0.5M). To this solution was then added portionwise sodium nitrite (7.6 g, 0.11M) whilst the reaction temperature was maintained at 0.degree. by the addition of ice. After stirring for 1 h at 0.degree. the reaction mixture was allowed to warm to room temperature over 2 h. Diethyl ether (500 ml) was then added and the aqueous layer was re-extracted with diethylether (500,;.times.3). The combined ethereal extracts, were washed with water and then brine, dried (MgSO.sub.4), filtered and concentrated to an oil in vacuo.

To the crude bromo-acid (45 g, 0.136M) in dry methanol (300 ml) cooled to -30.degree. was added dropwise thionyl chloride (33.7 ml, 0.405M) at such a rate that the temperature did not exceed -15.degree.. The reaction mixture was warmed to 10.degree. over 2 h and stirred at room temperature for 30 min. and then at 40.degree. for 30 min. The resulting solution was then concentrated in vacuo, dissolved in CH.sub.2 Cl.sub.2 and washed in turn with water, saturated aq. NaHCO.sub.3 and water. The residue isolated from the organic layer was chromatographed on silica in 5% ethylacetate in CH.sub.2 Cl.sub.2 to give the title compound as an oil (10.3 g), (Found: C,48.61; H,5.61; N,4.00. C.sub.14 H.sub.18 BrNO.sub.4 requires C,48.85; H,5.27; N,4.07%); .delta.(CDCl.sub.3) 1.5-1.8 and 1.9-2.2 (each 2H, each m, CH.sub. 2 CH.sub.2), 3.23 (2H,q,J=6 Hz,NCH.sub.2), 3.77 (3H,s,OCH.sub.3), 4.25 (1H,dd,J=7 and 14 Hz, .alpha.--CH), 4.8-4.9 (1H, broad s, NH), 5.10 (2H,s,OCH.sub.2) and 7.35 (5H, broad s, C.sub.6 H.sub.5).

To a solution of the ester (650 mg, 1.14M) from Example 8 in methanol/water (10:1, 11 ml) was added dilute NaOH (IN, 2.3 ml). The reaction mixture was stirred for 6 h at room temperature, acidified with acetic acid and then concentrated to a semi-solid in vacuo. This was partitioned between ethyl acetate and water and the resulting solid was filtered, washed with water and ethyl acetate and dried in vacuo to give the title compound (585 mg); m.p. 164.degree.-169.degree.; (Found: C,61.59; H,7.24; N,9.40. C.sub.30 H.sub.42 N.sub.4 O.sub.7 requires C,61.21; H,7.53; N,9.52%); .nu..sub.max (Nujol) 3320, 1690 and 1645 cm.sup.-1 ; .delta.(d.sup.6 DMSO) 0.85 (6H,m,CH(CH.sub.3).sub.2); 0.96-1.8 (7H,m,CH.sub.2 CH(CH.sub.3).sub.2, NHCH.sub.2 CH.sub.2 CH.sub.2); 2.57 (3H,d,J=5 Hz,NCH.sub.3); 2.5-3.2 (6H,m,NHCH.sub.2, CH.sub.2 C.sub.6 H.sub.4, .alpha.--CHx2); 3.70 (3H,s,OCH.sub.3); 4.42 (1H,m,.alpha.--CH); 5.0 (2H,s,CH.sub.2 C.sub.6 H.sub.5); 6.78 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.10 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.20 (1H,m,CONH); 7.35 (5H,m,C.sub.6 H.sub.5); 7.88 (1H,m,CONH); 8.18 (1H,m,CONH).

This was synthesised from Z-proline, leucine methyl ester and N-[4-N-(benzyloxycarbonyl)amino-1-(R)-methoxycarbonylbutyl]-L-leucyl-O-met hyl-L-tyrosine N-methylamide as described in the following paragraphs:

To a stirred solution of Z-L-proline (12.7 g, 0.051M) in CH.sub.2 Cl.sub.2 (200 ml) cooled to 0.degree. was added 1-hydroxy benzotriazole (7.0 g) followed by a solution of DCC (10.6 g) in CH.sub.2 Cl.sub.2 (50 ml). After 30 min. at 0.degree. L-leucine ethyl ester (10.0 g, 0.051 molM) was added followed by triethylamine (15 ml) and the reaction mixture was then left to stir and warm up to room temperature overnight. The reaction mixture was then filtered and washed in turn with saturated aq. NaHCO.sub.3 (250 ml.times.3), H.sub.2 O (250 ml), dilute aq. HCl (1M, 250 ml.times.3) and water (250 ml.times.2). The organic layer was dried (Na.sub.2 SO.sub.4), filtered and concentrated in vacuo to an oil which subsequently crystallised. Recrystallisation from ethyl acetate/hexane gave N-(benzyloxycarbonyl)-L-prolyl-L-leucine ethyl ester as a white crystalline solid, 15.5 g, (78%); m.p. 67.degree.-68.degree.; (Found: C,64.55; H,7.79; N,7.22. C.sub.21 H.sub.30 N.sub.2 O.sub.5 requires C,64.61; H,7.74; N,7.17%); .nu..sub.max (CHCl.sub.3) 1740 and 1680 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.7-0.95 (6H,m,CH(CH.sub.3).sub.2); 1.18 (3H,m,OCH.sub.2 CH.sub.3); 1.3-1.95 and 2.05-2.25 (7H,m,CH.sub.2 CH.sub.2, CH.sub.2 CH(CH.sub.3).sub.2); 3.4 (2H,m,CH.sub.2 N); 4.05 (2H,m,OCH.sub.2 CH.sub.3); 4.25 (2H,m,.alpha.--CH); 4.98 and 5.05 (together 2H, respectively q,J=7 Hz, and m, CH.sub.2 C.sub.6 H.sub.5); 7.35 (5H, broad s, C.sub.6 H.sub.5) and 8.26 (1H,m,CONH); m/e 391 (100%, [m+1].sup.+).

To a solution of N-(benzyloxycarbonyl)-L-prolyl-L-leucine ethyl ester (7.5 g, 0.02 mM) in methanol (100 ml) was added acetic acid and 10% Pd/C (0.8 g). After stirring under hydrogen for 3 h at room temperature the reaction was filtered and concentrated to an oil in vacuo. Trituation of the residue with ether and recrystallisation from ethyl acetate/hexane gave L-prolyl-L-leucyl ethyl ester as the acetate salt (5.0 g), m.p. 87.degree.-89.degree.. .nu..sub.max 1760 and 1660 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.94 (6H,m,CH(CH.sub.3).sub.2); 1.27 (3H,t,J=7 Hz,OCH.sub.2 CH.sub.3); 1.45-2.35 (7H,m,CH.sub.2 CH.sub.2, CH.sub.2 CH(CH.sub.3).sub.2); 2.2 (3H,s,CH.sub.3 CO.sub.2); 3.1 (2H,m,CH.sub.2 N); 4.15 (1H,m,.alpha.--CH); 4.19 (2H,q,J=7 Hz, OCH.sub.2 CH.sub.3); 4.55 (1H,m,.alpha.--CH); 7.24 (2H,br,NH,CO.sub.2 H); 7.87 (1H,d,J=7 Hz,CONH); m/e (100% [m+1].sup.+)].

To a solution of the foregoing amine (3.0 g, 11.7 mM) in CH.sub.2 Cl.sub.2 (50 ml) was added p-nitrophenylacetate (2 g, 12 MM). After stirring the reaction mixture at room temperature for 3 days it was diluted with CH.sub.2 Cl.sub.2 (350 ml), washed with water, dried (Na.sub.2 SO.sub.4) and concentrated to an oil in vacuo. Chromatography on silica in 1:1 CH.sub.2 Cl.sub.2 /EtOAc followed by 9:1 CH.sub.2 Cl.sub.2 /MeOH yielded N-acetyl-L-prolyl-L-leucine ethyl ester as a pale yellow oil (2.2 g); (Found [m+1].sup.+ =299.19704. C.sub.15 H.sub.27 N.sub.2 O.sub.4 requires [m+1].sup.+ =299.19707); .nu..sub.max (CHCl.sub.3) 3600-3100 (broad), 1735, 1675 and 1625 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.95 (6H,m,CH(CH.sub.3).sub.2); 1.25 (3H,t,J=7 Hz,OCH.sub.2 CH.sub.3 ); 1.44-2.5 (7H,m,CH.sub.2 CH.sub.2, CH.sub.2 CH(CH.sub.3).sub.2); 2.12 (3H,s,CH.sub.3 CO); 3.36-3.7 (2H,m,CH.sub.2 N); 4.18 (2H,t,J=7 Hz,OCH.sub.2 CH.sub.3); 4.25-4.55 (1H,m, CH Pro); 4.6 (1H, CH Leu); 6.38 and 7.35 (1H, each d,J=7 Hz,CONH).

To a solution of N-[4-N-(benzyloxycarbonyl)amino-1-(R)-methoxycarbonylbutyl]-L-leucyl-O-met hyl-L-tyrosine N-Methylamide (570 mg, 0.97 mM) in methanol (8 ml) was added 10% Pd/C and dilute HCl (1M, 2 ml). After stirring the reaction mixture under hydrogen for 2 h at room temperature it was filtered and concentrated in vacuo to a solid, (490 mg) (100%), which was used as such in the next step.

N-Acetyl-L-prolyl-L-leucine (271 mg, 1.06 MM; obtained from the foregoing ethyl ester by hydrolysis in methanol with one equivalent of IN-sodium hydroxide solution at 20.degree. over 16 h followed by neutralisation with dilute HCl) in CH.sub.2 Cl.sub.2 (2 ml) and DMF (2 ml) was stirred at 0.degree. and 1-hydroxy benzotriazole (162 mg, 1.06 mM) and N-ethyl-N'-(dimethylaminopropyl)carbodiimide hydrochloride (240 mg, 1.06 mM) were then added. After 5 min N-methylmorphine (107 mg, 1.06 mM) was added followed, after 15 min, by the amine hydrochloride (prepared above) (485 mg, 0.96 mM). After stirring overnight at 0.degree.-4.degree., the reaction mixture was concentrated in vacuo, dissolved in CH.sub.2 Cl.sub.2 and washed in turn with water, saturated aq. NaHCO.sub.3 and dilute HCl. The acid layer was separated, neutralised with NaHCO.sub.3 and extracted with CH.sub.2 Cl.sub.2. The organic extracts were dried, (Na.sub.2 SO.sub.4) and evaporated in vacuo to yield the title compound as a foam (570 mg); m.p. 68.degree.-72.degree.; (Found: C,59.99; H,8.35; N,11.65. C.sub.36 H.sub.56 N.sub.6 O.sub.8.1H.sub.2 O requires C,59.98; H,8.39; N,11.66%); .delta.(d.sup.6 DMSO) 0.82 (12H,m, CH(CH.sub.3).sub.2 x2); 1.0-2.34 (14H,m,CH.sub.2 CH.sub.2 x2, CH.sub.2 CHL (CH.sub.3).sub.2 x2); 1.98 and 2.0 (together 3H, each s, CH.sub.3 CO); 2.50-3.08 (8H,m,CH.sub.2 C.sub.6 H.sub.4, CH.sub.2 Nx2 and 2x.alpha.--CH); 2.56 (3H,d,J=5 Hz,CH.sub.3 N); 3.54 (3H,s,OCH.sub.3); 3.70 (3H,s,OCH.sub.3); 4.0-4.5 (3H,m,.alpha.--CH); 6.78 (2H,d,J=8 Hz Tyr H-3 and H-5); 7.11 (2H,d,J=8.6 Hz, Tyr H-2and H-6); 7.5-8.35 (4H,m,CONH).

To a solution of the preceeding ester (380 mg, 0.54 mM) in methanol (5 ml) was added dilute NaOH (1M, 1 ml). After stirring overnight at room temperature, the reaction mixture was neutralised with acetic acid and concentrated in vacuo. Chromatography on reverse phase silica in 1:1 MeOH/H.sub.2 O gave the title compound (280 mg); m.p. 97.degree.-101.degree.; (Found: C,58.52; H,7.93; N,11.46. C.sub.36 H.sub.56 N.sub.6 O.sub.8.1.5H.sub.2 O requires C,58.72; H,8.31; N,11.74%). .nu..sub.max (Nujol) 3700-3140 (broad) and 1635 cm.sup.-1 ; .delta.(CD.sub.3 OD) 0.9 (12H,M,2xCH(CH.sub.3).sub.2); 1.4-2.25 (14H,M,2xCH.sub.2 CH.sub.2, 2xCH.sub.2 CH(CH.sub.3).sub.2); 1.98 and 2.0 (together 3H, each s, CH.sub.3 CO); 2.68 and 2.72 (together 3H, each s, CH.sub. 3 N), 2.75-3.8 (8H,m,CH.sub.2 C.sub.6 H.sub.5, CH.sub.2 Nx2, 2xCH); 3.75 (3H,s,OCH.sub.3); 4.25-4.65 (3H,m,.alpha.CH), 6.78 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.11 (2H,d,J=8.6 Hz, Tyr H-2 and H-6).

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:

Methyl 4-N-(benzyloxycarbonyl)amino-2-bromobutanoate (30 g), L-leucyl-O-methyl-L-tyrosine N-methylamide (30 g) and N-methyl morpholine (9.4 g) in acetonitrile (250 ml) was stirred and heated under reflux overnight. A further portion of the amine (1.1 g) was then added and the solution was heated under reflux for a further 4 h. 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-me thyl-L-tyrosine N-methylamide (11.7 g); (Found: C,63.09; H,7.46; N,9.59. C.sub.30 H.sub.42 N.sub.4 O.sub.7 requires C,63.16; H,7.37; N.9.83%); .nu..sub.max (CHCl.sub.3) 3400, 1720 and 1660 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.86 (6H,m,CH(CH.sub.3).sub.2); 1.2-2.1 (6H,m,NHCH.sub.2 CH.sub.2 CH, CH.sub.2 CH(CH.sub.3).sub.2,NH); 2.77 (3H,d,J=5 Hz,NCH.sub.3); 2.95-3.45 (5H,m,NHCH.sub.2, CH.sub.2 C.sub.6 H.sub.4,.alpha.CH); 3.66 and 3.76 (each 3H, each s, 2xOCH.sub.3); 3.8 and 4.61 (each 1H, each m, 2xCH); 5.10 (2H,m,CH.sub.2 C.sub.6 H.sub.5); 5.21 (1H,m,OCONH); 6.72 (1H,m,CONH); 6.81 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.12 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.35 (5H,s,C.sub.6 H.sub.5); 7.55 (1H,d,J=8 Hz,CONH); m/e 571 (100% [m+1].sup.+).

The methyl 4-N-(benzyloxycarbonyl)amino-2-bromobutanoate required in this preparation was made from L-glutamic acid as described in the following paragraphs:

L-Glutamic acid (105 g, 0.713M) was dissolved in concentrated sulphuric acid (300 ml) and to this was added chloroform (300 ml). To the stirred bi-phasic mixture at 0.degree. was added portionwise over 30 min. sodium azide (60 g, 0.9 mole). The reaction mixture was stirred at 5.degree.-10.degree.for 30 min. and was then allowed to slowly warm to room temperature. The reaction mixture was then slowly heated to 80.degree. for one hour the reaction was cooled, poured into water (1.5 l) and the aqueous layer was separated. The aqueous extract was diluted (to 20 liters) and was then applied to Dowex 50WX8, 16-40 mesh, H.sup.+ 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 liter) and to this was added basic copper carbonate (100 g). The stirred mixture was heated under reflux for 40 min. and the hot solution was filtered. The solution was cooled to 35.degree. and NaHCO.sub.3 (60 g) and CHCl.sub.3 (300 ml) were added. After stirring for 30 min. at room temperature, benzyloxycarbonyl chloride (75 ml) was added and the mixture was then allowed to stir at room temperature overnight. A further portion of benzyloxycarbonyl chloride (30 ml) was then added and stirring was continued for a further 24 h. The crystalline copper complex which had precipitated was then filtered, washed with water and added to a solution of EDTA (di Na salt) (120 g) in water (1.5 liter). The resulting mixture was stirred and heated under reflux for 3 h and was then cooled to 5.degree.. After 40 h at 5.degree. the crystalline product was collected by filtration, washed with water and acetone and dried in vacuo at 45.degree..

The 4-Z-diamino-butyric acid from above (120 g) was suspended in a mixture of dilute sulphuric acid (1M, 600 ml), water (200 ml) and potassium bromide (240 g). Sufficient water (200 ml) was then added to form a single phase. To the resulting solution stirred at -7.degree. to -9.degree., was added a solution of sodium nitrite (44 g) in H.sub.2 O dropwise over 1 h. After 30 min at -7.degree., the mixture was warmed to room temperature over 1 h. Diethyl ether (1.5 liters) was added 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 liter) was cooled to 0.degree. 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 (90 g) as an oil which crystallised on standing, m.p. 46.degree.-50.degree.; (Found: C,47.17; H,5.01; N,4.16. C.sub.13 H.sub.16 BrNO.sub.4 requires C,47.29; H,4.88; N,4.24%); .delta.(CDCl.sub.3) 2.08-2.45 (2H,m,CH.sub.2); 3.37 (2H,m,NHCH.sub.2); 3.76 (3H,s,OCH.sub.3); 4.32 (1H,dd,J=10 Hz and 6 Hz, CH); 4.97 (1H,broad s, OCONH); 5.09 (2H,s,OCH.sub.2) and 7.34 (5H,s,C.sub.6 H.sub.5).

To a solution of the preceeding ester (171 mg, 0.3 mM) in methanol (10 ml) stirred at 0.degree. was added dilute NaOH (1N, 0.6 ml). After stirring overnight at 0.degree. a further portion of NaOH (1N, 0.3 ml) was added and the solution was then stirred for 6 h at room temperature. The reaction mixture was then acidified with acetic acid and concentrated to a solid in vacuo. Recrystallisation of this material from methanol/H.sub.2 O gave the title compound (150 mg); m.p. 170.degree.-172.degree.; (Found: C,60.97; H,7.11; N,9.68. C.sub.29 H.sub.40 N.sub.4 O.sub.7 +0.8 H.sub.2 O requires C,60.99; H,7.34; N,9.81%); .nu..sub.max (Nujol) 3300, 1690 and 1640 cm.sup.-1 ; .delta.(CD.sub.3 OD) 0.88 (6H,dd,J=14 Hz and 7 Hz, CH(CH.sub.3).sub.2); 1.2-1.95 (5H,m,NHCH.sub.2 CH.sub.2, CH.sub.2 CH(CH.sub.3).sub.2); 2.69 (3H,s,NCH.sub.3); 2.75-3.65 (6H,m,NHCH.sub.2, CH.sub.2 C.sub.6 H.sub.4, and .alpha.--CHx2); 3.74 (3H,s,OCH.sub.3); 4.54 (1H,dd,J=10 Hz and 6 Hz, .alpha.--CH); 5.08 (2H,m,CH.sub.2 C.sub.6 H.sub.5); 6.82 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.12 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.35 (5H,m,C.sub.6 H.sub.5).

To a solution of N-(Tertiarybutoxycarbonyl)-L-leucyl-O-methyl-L-tyrosyl N-methylamide (4.2 g, 0.01M) in CH.sub.2 Cl.sub.2 (5 ml) at 18.degree. was added trifluoroacetic acid (8 ml). After stirring for 2 h at room temperature the reaction was concentrated in vacuo and was then trituated with dry ether to yield a gum. This was taken up in methanol (25 ml), methyl 4-N-(benzyloxycarbonyl)amino-2-oxo-butanoate (4.0 g; 0.015M; Synthesis, (1982), 41) was added and the pH of the solution adjusted to 6.5 with triethylamine. To this solution stirred at 0.degree. was added sodium cyanoborohydride (400 mg) portionwise whilst the pH was periodically re-adjusted to 6.5 by the addition of acetic acid. After 1 h further sodium cyanoborohydride (400 mg) was added and the reaction was stirred overnight at room temperature. After concentration in vacuo the residue was partitioned between CH.sub.2 Cl.sub.2 (100 ml) and water (50 ml). The CH.sub.2 Cl.sub.2 layer was separated, washed in turn with dilute HCl (1M, 20 ml), water (25 ml), saturated sodium bicarbonate solution (2.times.30 ml), dried and evaporated to an oil. Chromatography on silica in CH.sub.2 Cl.sub.2 in an increasing ethyl acetate gradient gave the title compound as a foam (1.0 g) which had physical data identical to that given above in Example 11.

The acid (320 mg, 0.56 mM) from Example 11 in methanol (10 ml) was treated with dilute HCl (1M, 1 ml). This solution was hydrogenated over 10% palladium on charcoal (60 mg) for 90 min. at room temperature, filtered and then concentrated in vacuo to give the title compound as its dihydrochloride salt; m.p. 149.degree.-152.degree. (from CH.sub.2 Cl.sub.2 -ether); (Found: C,48.17; H,6.98; N,10.49. C.sub.21 H.sub.34 N.sub.4 O.sub.5.2HCl+0.5CH.sub.2 Cl.sub.2 requires C,48.01; H,6.93; N,10.42%); .nu..sub.max (Nujol) 3650-2400 (br), 1730 and 1650 cm.sup.-1 ; .delta.(CD.sub.3 OD) 0.92 and 0.95 (each 3H, each d, each J=15 Hz,CH(CH.sub.3).sub.2); 1.45-1.90 (3H,m,CH.sub.2 CH(CH.sub.3).sub.2); 2.25 (2H,m,NHCH.sub.2 CH.sub.2); 2.68 3H,s,OCH.sub.3); 3.04 (4H,m,NHCH.sub.2 and CH.sub.2 C.sub.6 H.sub.4); 3.58 (1H,dd,J=8 Hz and 6 Hz,.alpha.--CH); 3.77 (3H,s,OCH.sub.3); 3.94 (1H,dd,J=8 Hz and 4 Hz,.alpha.--CH); 4.64 (1H,dd,J=13 Hz and 6 Hz,.alpha.--CH); 6.88 (2H,d,J=8.6 Hz, Tyr H-3 and H-5) and 7.10 (2H,d,J=8.6 Hz, Tyr H-2 and H-6).

A solution of N-[3-N-(benzyloxycarbonyl)amino-1-(R)-methoxycarbonyl propyl]-L-leucyl-O-methyl-L-tyrosine N-methylamide (1.24 g, mM) in methanol (25 ml) containing ethereal HCl (1 ml of a 2.6M solution) was hydrogenated over 10% palladised charcoal (0.3 g) for 6h at 20.degree.. The solution was filtered and concentrated in vacuo to give N-[3-N-amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosine N-methylamide dihydrochloride as a foam (1.2 g) which was used in the next step without further purification.

To a suspension of N-[3N-amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosine N-methylamide dihydrochloride (400 mg, 0.808 mM) in dry CH.sub.2 Cl.sub.2 (6 ml) cooled in an ice bath, was added p-nitrobenzyloxycarbonyl chloride (400 mg) in dry CH.sub.2 Cl.sub.2. To this was then added dropwise a solution of N-methyl morpholine (270 mg, 2.67 mM) in dry CH.sub.2 Cl.sub.2 (2 ml). After 30 min at 0.degree., a further portion of p-nitrobenzyloxycarbonyl chloride (400 mg) in dry CH.sub.2 Cl.sub.2 (1 ml) was added followed by a solution of NMM (100 mg) in dry CH.sub.2 Cl.sub.2 (1 ml). After a further 0.5 h at 0.degree. the reaction mixture was diluted with CH.sub.2 Cl.sub.2 (20 ml), washed in turn with water (20 ml), aq. citric acid solution (20 ml) and saturated aq. NaHCO.sub.3 (20 ml). The organic extract was concentrated in vacuo and purified by chromatography on silica eluting with CH.sub.2 Cl.sub.2 in a rapidly increasing ethyl acetate gradient to give the title compound as a foam (450 mg, 90%); (Found: [m+1].sup.+ =616.3012. C.sub.30 H.sub.42 N.sub.5 O.sub.9 requires [m+1].sup.+ =616.2983); .sub.max (CHCl.sub.3) 3380, 1742 and 1660 cm.sup.-1 ; m/e 616 (5% [m+1].sup.+); 153 (100% [O.sub.2 NC.sub.6 H.sub.4 CH.sub.2 OH].sup.+). .delta.(CDCl.sub.3 0.87 (6H,m,CH(CH.sub.3); 1.1-2.0 (5H,m,NHCH.sub.2 CH.sub.2, CH.sub.2 CH(CH.sub.3).sub.2,NH) 2.76 (3H,d,J=5 Hz,NCH.sub.3); 2.9-3.5 (6H,m,NHCH.sub.2,CH.sub.2 C.sub.6 H.sub.4,.alpha.--CHx2) 3.68 and 3.77 (each 3H; each s, 2xOCH.sub.3); 4.60 (1H,dd,J=13 Hz and 6 Hz,.alpha.--CH); 5.10 (2H,s,CH.sub.2 C.sub.6 H.sub.4 NO.sub.2); 5.45 (1H,m,OCONH); 6.50 (1H, broad s, OCONH); 6.82 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.11 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.45 (1H,d,J=8 Hz, CONH); 7.52 (2H,d,J=9 Hz, benzoyl H-2 and H-6); 8.21 (2H,d,J=9 Hz, benzoyl H-3 and H-5).

To a solution of the preceeding ester (360 mg, 0.58 mM) in methanol (6 ml) at 0.degree. was added dilute NaOH (1N, 1.2 ml). After standing at 0.degree. for 48 h, the solution was acidified with acetic acid and concentrated to a solid in vacuo. Trituration with ethyl acetate and water gave the title compound (56 mg); m.p. 167.degree.-170.degree.; (Found: C,56.56; H,6.58; N,11.21. C.sub.29 H.sub.39 N.sub.5 O.sub.9 +0.8H.sub.2 O requires C,56.54; H,6.64; N,11.37%); .nu..sub.max (Nujol) 3250, 1690 and 1642 cm.sup.-1 ; .delta.(d.sup.6 DMSO) 0.8 (6H,m,CH(CH.sub.3).sub.2 ; 1.1-2.0 (5H,m,NHCH.sub.2 CH.sub.2, CH.sub.2 CH(CH.sub.3).sub.2); 2.57 (3H,d,J=5 Hz,NCH.sub.3); 2.62-3.85 (7H,m,NCH.sub.2, .alpha.--CHx2, CH.sub.2 C.sub.6 H.sub.4,OH); 3.67 (3H,s,OCH.sub.3); 4.43 (1H,m,.alpha.--CH); 5.10 (2H,s,OCH.sub.2); 6.78 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.13 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.95 (1H,m,CONH); 8.07 (2H,d,J=8.6 Hz, Benzoyl H-2 and H-6); 8.25 (1H,m,CONH); 8.31 (2H,d,J=8.6 Hz, Benzoyl H-3 and H-5); 9.12 (1H,m,CONH).

This was prepared in two steps from N-[3-N-amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tryosine N-methylamide as described below:

To a stirred suspension of N-[3-N-amino-1-(R)-methoxycarbonylpropyl]-L-leucyl-O-methyl-L-tyrosine N-methylamide dihydrochloride (539 ml, 1 mM) and benzoyl chloride (186 mg, 1 mM) in dry CH.sub.2 Cl.sub.2 (30 ml) at 0.degree. was added dropwise N-methyl morphline (439 mg, 4.3 mM). The reaction mixture was then stirred overnight, concentrated in vacuo and chromatographed on silica eluting with ethyl acetate in an ethyl acetate/methanol gradient to yield the title compound (350 mg); m.p. 145.degree.-148.degree.; (Found: C,63.97; H,7.38; N,10.20. C.sub.29 H.sub.40 N.sub.4 O.sub.6 +0.2H.sub.2 O requires C,64.00; H,7.48; N,10.29%). .delta.(CDCl.sub.3) 0.85 and 0.86 (each 3H, each d, each J=6.5 Hz, CH(CH.sub.3).sub.2); 1.18-1.80 (4H,m,NHCH.sub.2 CH.sub.2 CH and CH.sub.2 CH(CH.sub.3).sub.2,NH); 2.0 (2H,dd,J=13 and 6 Hz, CH .sub.2 CH(CH.sub.3).sub.2); 2.75 (3H,d,J=5 Hz,NCH.sub.3); 3.06 and 3.4-3.7 (6H,m,NHCH.sub.2, CH.sub.2 C.sub.6 H.sub.4 and .alpha.--CHx2); 3.64 and 3.74 (each 3H, each s, 2xOCH.sub.3) 4.60 (1H,dd,J=15 Hz and 6 Hz, .alpha.--CH); 6.5 and 6.75 (each 1H, each m, 2xCONH); 6.82 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.15 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.5 (5H,m,C.sub.6 H.sub.4) and 7.77 (1H,d,J=8 Hz,CONH).

To the proceeding ester (150 mg, 0.27 mM) in methanol (10 ml) was added dilute NaOH (1N, 1 ml) and the solution was then stirred at room temperature for 3 days. The reaction mixture was acidified with acetic acid and was concentrated in vacuo. Recrystallisation of the residue from methanol-H.sub.2 O gave the title compound (110 mg); m.p. 175.degree.-177.degree.; (Found: C,61.41; H,7.71; N,10.17. C.sub.28 H.sub.38 N.sub.4 O.sub.6 +1.2H.sub.2 O requires C,61.34; H,7.34; N,10.22%); .nu. max (Nujol) 3320 and 1640 cm.sup.-1 ; .delta.(d.sup.6 DMSO) 0.82 (6H,m,CH(CH.sub.3).sub.2); 1.05-2.0 (5H,m,NHCH.sub.2 CH.sub.2,CH.sub.2 CH(CH.sub.3).sub.2); 2.58 (3H,d,J=5 Hz,NCH.sub.3); 3.65-4.55 (6H,m,NHCH.sub.2), CH.sub.2 C.sub.6 H.sub.4 and .alpha.--CHx2); 3.68 (3H,s,OCH.sub.3); 4.42 (1H,m,.alpha.--CH); 6.78 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.11 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.46 (3H,m,CONH and 2 protons from C.sub.6 H.sub.5); 7.86 (3H,m, 3 protons from C.sub.6 H.sub.5); 8.20 (2H,d,J=8 Hz,CONH); 8.51 (1H,m,CONH).

This was prepared exactly as described for the N-benzoyl derivative in Example 15 except that p-nitrobenzoyl chloride was used in place of benzoyl chloride in the first step. After hydrolysis of the intermediate ester, the resulting solid was recrystallized from methanol-water to give the title compound, (450 mg); m.p. 170.degree.-180.degree.; (Found: C,57.38; H,6.82; N,11.86. C.sub.28 H.sub.37 N.sub.5 O.sub.8 +0.8H.sub.2 O requires C,57.39; H,6.64; N,11.95%; .nu..sub.max (Nujol) 3340 and 1645 cm.sup.-1 ; .delta.(d.sup.6 DMSO) 0.82 (6H,m,CH(CH.sub.3).sub.2 ; 1.05-2.05 (5H,m,NCH.sub.2 CH.sub.2 CH, CH.sub.2 CH(CH.sub.3).sub.2); 2.58 (3H,m,NCH.sub.3); 2.6-3.65 (6H,m,NHCH.sub.2 .alpha.--CHx2 and CH.sub.2 C.sub.6 H.sub.4); 3.7 (3H,m,OCH.sub.3); 4.45 (1H,m,.alpha.--CH); 6.8 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.12 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.88 (1H,m,CONH); 8.08 (2H,d,J=8 Hz, Benzoyl H-2 and H-6); 8.2 (1H,d,J=8 Hz,CONH); 8.33 (2H,d,J=8 Hz, Benzoyl H-3 and H-5) and 8.88 (1H,m,CONH).

The acid (351 mg), from Example 16 was dissolved in methanol (25 ml) and to this solution was added 10% Pd/C (400 mg) and dilute ethereal HCl (2.6M, 2 ml). After stirring the reaction mixture under hydrogen for 2.5 h at room temperature it was filtered and concentrated in vacuo to yield the title compound as a foam (290 mg); m.p. 155.degree.-160.degree.; (Found: C,50.39; H,6.68; N,10.23. C.sub.28 H.sub.39 N.sub.5 O.sub.6 3HCl+1H.sub.2 O requires C,50.26; H,6.62; N,10.46%); .nu..sub.max (Nujol) 3650-2120 (broad), 1730 and 1645 cm.sup.-1 ; .delta.(d.sup.6 DMSO) 0.81 and 0.87 (each 3H, each s, CH(CH.sub.3).sub.2); 1.3-1.8 (3H,m,CH(CH.sub.3).sub.2); 2.05 (2H,m,NHCH.sub.2 CH.sub.2 CH); 2.58 (3H,d,NCH.sub.3); 2.75 and 2.98 (together 2H, each m, CH.sub.2 C.sub.6 H.sub.4); 3.2-3.5 (3H,m,NHCH.sub.2 and .alpha.--CH); 3.7 (3H,s,OCH.sub.3); 3.97 (1H,m,.alpha.--CH); 4.58 (1H,m,.alpha.--CH); 6.83 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.01 (2H,d,J=8 Hz, benzoyl H-3 and H-5); 7.10 (2H,d,J-6.8 Hz, Tyr H-2 and H-6); 7.81 (2H,d,J=8 Hz, benzoyl H-2 and H-6); 8.17 (1H,m,CONH); 8.67 (1H,m,CONH); 9.11 (1H,d,J=8 Hz,CONH) and 9.5 (3H,br,NH.sub.3).

This was prepared according to the following steps:

To a stirred suspension of N-[3-N-amino-1-(R)-methoxy-carbonylpropyl]-L-leucyl-O-methyl-L-tyrosyl N-Methylamide dihydrochloride (406 mg, 0.78 mM) in dry CH.sub.2 Cl.sub.2 (10 ml) at 0.degree. was added benzyl isocyanate (104 .mu.l, 1.56 mM). A solution of N-methyl morpholine (189 mg, 1.87 mM) in dry CH.sub.2 Cl.sub.2 (5 ml) was then added dropwise over 5 min. After 30 min at 0.degree., a further portion of benzyl isocyanate (25 .mu.l) was added and this was repeated after an additional 30 min. at 0.degree.. The reaction mixture was then allowed to warm to room temperature over 3 h. Water (50 ml) and CH.sub.2 Cl.sub.2 (50 ml) were then added and the material isolated from the organic extracts was chromatographed on silica in 5% MeOH in CH.sub.2 Cl.sub.2 to afford the title compound (223 mg); 61.degree.-69.degree.; (Found: C,62.77; H,7.64; N,12.03. C.sub.30 H.sub.43 N.sub.5 O.sub.6 +0.3H.sub.2 O requires C,62.65; H,7.64; N,12.18%); .delta.(CDCl.sub.3) 0.87 (6H,m,CH(CH.sub.3).sub.2) 1.10-2.0 (6H,m,NHCH.sub.2 CH.sub.2,CH.sub.2 CH(CH.sub.3).sub.2 and NH) 2.64 (3H,d,J=5 Hz,NCH.sub.3); 2.85-3.54 (6H,m,NHCH.sub.2, CH.sub.2 C.sub.6 H.sub.4 and .alpha.--CHx2); 3.67 and 3.78 (each 3H, each s, 2xOCH.sub.3); 4.37 (2H,dd,15 Hz and 2 Hz, CH.sub.2 C.sub.6 H.sub.5); 4.56 (1H,dd,J=13 Hz and 6 Hz,.alpha.--CH); 5.16, 5.42 and 6.44 (each 1H, each broad s, 3xCONH) 6.80 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.08 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.3 (5H,m,C.sub.6 H.sub.5) and 7.7 (1H,d,J=8 Hz,CONH).

To a solution of the preceeding ester (240 mg, 0.42 mM) in methanol (25 ml) at room temperature, was added dilute NaOH (1N, 1.5 ml). After standing overnight at room temperature the reaction mixture was acidified with acetic acid and concentrated in vacuo. Chromatography on reverse phase silica eluting with a methanol/H.sub.2 O gradient gave the title compound (107 mg); m.p. 104.degree.-108.degree.; (Found: C,60.88; H,7.44; N,12.12. C.sub.29 H.sub.41 N.sub.5 O.sub.6 H.sub.2 O requires C,60.71; H,7.55; N,12.20%); .nu..sub.max (Nujol) 3300 and 1640 cm.sup.-1 ; .delta.(d.sup.6 DMSO) 0.8 (6H,m,CH(CH.sub.3).sub.2) 0.95-1.85 (5H,m,NHCH.sub.2 CH.sub.2 and CH.sub.2 CH(CH.sub.3).sub.2); 2.2-3.4 (6H,m,NHCH.sub.2, .alpha.--CHx2 and CH.sub.2 C.sub.6 H.sub.4); 2.56 (3H,d,J=5 Hz, NHCH.sub.3); 3.70 (3H,s,OCH.sub.3); 4.22 (2H,m,CH.sub.2 C.sub.6 H.sub.5); 4.45 (1H,m,.alpha.--CH); 6.0 and 6.42 (each 1H, each m, 2xCONH); 6.82 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.12 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.28 (5H,m,C.sub.6 H.sub.5); 7.94 (1H,m,CONH) and 8.25 (1H,d,J=Hz,CONH).

N-(Tertiarybutoxycarbonyl)-L-leucine (10 g, 0.04M in a mixture of CH.sub.2 Cl.sub.2 (100 ml) and DMF (10 ml) was cooled to 0.degree.. To this was added 1-hydroxybenzotriazole (6.2 g, 0.04M) followed dropwise by a solution of DCC (8.2 g, 0.04 mole) in CH.sub.2 Cl.sub.2. After 10 min. at 0.degree. a solution of phenethylamine (4.84 g, 0.04M) in CH.sub.2 Cl.sub.2 (15 ml) was added dropwise and the stirred solution was then allowed to warm to room temperature overnight. The reaction mixture was then filtered, concentrated in vacuo and dissolved in ethyl acetate (150 ml). The ethyl acetate solution was washed in turn with water (40 ml), saturated aq. NaHCO.sub.3 (50 ml.times.2), aqueous citric acid (50 ml) and saturated aq. NaHCO.sub.3 (50 ml). The residue after evaporation of the solvent was recrystallised from ethyl acetate/hexane to give N-(tertiarybutoxycarbonyl)-L-leucyl-N-phenethylamide as a white powder (9.6 g); m.p. 86.degree.-88.degree.; .nu..sub.max (CHCl.sub.3) 3415 and 1670 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.85 (6H,m,CH(CH.sub.3).sub.2); 1.35 (9H,s,OC(CH.sub.3).sub.3); 1.3-1.75 (3H,m,CH.sub.2 CH(CH.sub.3).sub.2); 2.69 (2H,t,J=7.2 Hz, CH.sub.2 C.sub.6 H.sub.5); 3.3-3.6 (2H,m,NCH.sub.2); 4.05 (1H,m,.alpha.--CH); 4.9 (1H,m,OCONH); 6.2 (1H,m,CONH); 7.2-7.4 (5H,m,C.sub.6 H.sub.5).

N-(tertiarybutoxycarbonyl)-L-leucine N-phenethylamide (6.17 g, mole) was dissolved in a 1:1 TFA/CH.sub.2 Cl.sub.2 mixture (60 ml). After stirring for 6 h at 20.degree. the reaction mixture was concentrated in vacuo and the residue in CH.sub.2 Cl.sub.2 (50 ml) washed with saturated aq. NaHCO.sub.3 (100 ml). The aqueous extract was back extracted with CH.sub.2 Cl.sub.2 (50 ml.times.3) and the combined organic extracts concentrated to an oil in vacuo. The crude L-leucine N-phenethylamide so obtained was used as such in the next step.

To a solution of methyl 4-N-(benzyloxycarbonyl)amino-2-bromo-butanoate (330 mg, 1 mmole) in dry acetonitrile (10 ml) was added L-leucine N-phenethylamide (235 mg, 1 mM) and N-methyl morpholine (110 mg, 1 mM). The solution was heated at reflux overnight, sodium iodide (150 mg, 1 mM) was added and the reaction was reheated to reflux for a further 7 h. The reaction mixture was then filtered and concentrated to an oil in vacuo. Chromatography of the residue on silica in 1:1 EtOAc/Hexane gave N-[3-N-(benzyloxycarbonyl)amino-1-(R,S)-methoxycarbonylpropyl]-L-leucine N-phenethylamide (310 mg). Rechromatography on silica then gave the R diastereoisomer as an oil.

To a solution of the foregoing R-isomer (110 mg) in methanol (4 ml) was added dilute NaOH (1N, 0.5 ml). After standing overnight at 20.degree. the reaction mixture was acidified with acetic acid and concentrated to a solid in vacuo. Chromatography on reverse phase silica eluting with 1:1 MeOH/H.sub.2 O gave the title compound as a white powder (55 mg), m.p. 130.degree.-135.degree.; (Found: C,65.62; H,7.59; N,8.85. C.sub.28 H.sub.35 N.sub.3 O.sub.5 +0.3H.sub.2 O requires C,65.75; H,7.55; N,8.85%); .nu..sub.max (Nujol) 1690, 1655 and 1630 cm.sup.-1 ; .delta.(d.sup.6 DMSO) 0.83 (6H,m,CH(CH.sub.3).sub.2); 1.1-1.85 (6H,m,NCH.sub.2 CH.sub.2 CH.sub.2 (CH.sub.3).sub.2 and NH); 2.69 (2H,t,J=7.2 Hz, CH.sub.2 C.sub.6 H.sub.5); 3.0-3.6 (7H,NCH.sub.2 x2,.alpha.--CHx2, CO.sub.2 H); 5.0 (2H,s,OCH.sub.2 C.sub.6 H.sub.5); 7.1-7.5 (10H,m,C.sub.6 H.sub. 5 x2); 8.05 (1H,m,CONH).

To a stirred solution of crude methyl 6-N-(benzyloxycarbonyl)amino-2-oxo-hexanoate (7.03 g, 24 mM; Tet.Lett., (1982), 23, 1875) and L-leucyl-O-methyl-L-tyrosine N-Methylamide (1.86 g, 6 mM) is methanol (50 ml) was added acetic acid to bring the pH to 6.5. Sodium cyanoborohydride (400 mg, 6.5 mM) was then added portionwise whilst the pH of the solution was continually re-adjusted to 6.5 by the addition of acetic acid. After 1.5 h at room temperature a further portion of sodium cyanoborohydride (400 mg) was added and the pH was again re-adjusted to 6.5 with acetic acid. After a further 1 h at room temperature, the reaction mixture was concentrated in vacuo and the residue in CH.sub.2 Cl.sub.2 (50 ml) was washed in turn with water (30 ml), dilute HCl (1M, 30 ml) and saturated aq.NaHCO.sub.3. The material isolated from the organic layer was purified by column chromatography on silica in CH.sub.2 Cl.sub.2 in an increasing ethyl acetate gradient to give the title compound as an oil (360 mg); (Found: [m+1].sup.+ =xxx.xxxx.C.sub.32 H.sub.44 N.sub.4 O.sub.7 requires [m+1].sup.+ =xx.xxxx); .delta.(CDCl.sub.3) 0.88 CH(CH.sub.3).sub.2); 1.0-1.86 (10H,m,NHCH(CH.sub.2).sub.3), CH.sub.2 CH(CH.sub.3).sub.2 and NH); 2.74 (3H,d,J=5 Hz,NCH.sub.3); 2.85-3.4 (6H,m,NHCH.sub.2,CH.sub.2 C.sub.6 H.sub.4 and .alpha.--CHx2); 3.65 and 3.75 (each 3H, each s, 2xOCH.sub.3); 4.64 (1H,dd,J=13 Hz and 6 Hz, .alpha.--CH); 5.10 (2H,s,CH.sub.2 C.sub.6 H.sub.5); 6.78 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.10 (2H,d,J=8.6 Hz, Tyr H-2 and H-6); 7.35 (5H,m,C.sub.6 H.sub.5) and 7.64 (1H,d,J=10 Hz,CONH).

To a solution of the ester from Example 20 (140 mg, 0.23 mM) in methanol (10 ml) at 0.degree. was added dilute NaOH (1N, 0.5 ml). After 48 h at 0.degree., a further portion of NaOH (1N, 0.4 ml) was added and the solution stirred at 20.degree. for a further 24 h. The reaction mixture was then acidified with acetic acid and concentrated in vacuo to give a semi-solid which was purified by partition between ethyl acetate and water at 0.degree.. The resulting solid was filtered, washed with water and ethyl acetate and was dried in vacuo to give the title compound (110 mg); 122.degree.-128.degree.; (Found: [m+1].sup.+ =585.3290 C.sub.31 H.sub.44 N.sub.4 O.sub.7 requires [m+1].sup.+ =585.3288) .nu..sub.max (Nujol) 3340, 1688 and 1640 cm.sup.-1 ; .delta.(CD.sub.3 OD) 0.88 (6H,m,CH(CH.sub.3).sub.2); 1.0-1.86 (9H,m,NHCH.sub.2 (CH.sub.2).sub.3 and CH.sub.2 CH(CH.sub.3).sub.2); 2.74 (3H,s,NCH.sub.3); 2.8-3.6 (6H,m,NHCH.sub.2,CH.sub.2 C.sub.6 H.sub.4 and .alpha.--CHx2); 3.77 (3H,s,OCH.sub.3); 4.60 (1H,m,.alpha.--CH); 5.10 (2H,s,CH.sub.2 C.sub.6 H.sub.5); 6.78 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.05 (1H,m,CONH); 7.10 (2H,d,J= 8.6 Hz Tyr H-2 and H-6) and 7.35 (5H,m,C.sub.6 H.sub.5); m/e 585 (1%, {m+1].sup.+), 567 (20% [m+1-H.sub.2 O].sup.+).

N-[5-N-(Benzyloxycarbonyl)amino-1-(R)-methoxycarbonylpentyl)-L-leucyl-O-met hyl-L-tyrosine N-methylamide (400 mg, 0.66 mM) in methanol (20 ml) was treated with dilute HCl (1N, 1.2 ml) and PdCl.sub.2 (50 mg). The reaction mixture was stirred under hydrogen for 20 min. at room temperature and was then filtered. Concentration of the resulting solution in vacuo gave N-[5-amino-1-(R)-methoxycarbonylpentyl]-L-leucyl-O-methyl-L-tyrosine N-methylamide hydrochloride as an oil. This was dissolved in CH.sub.2 Cl.sub.2 (20 ml) and DMF (5 ml) and to the resulting solution was added N-methyl morpholine (300 mg) and N-acetyl-L-proline p-nitrophenyl ester (191 mg). After standing at 20.degree. for 72 h, the reaction mixture was concentrated in vacuo and the residue in ethyl acetate (20 ml) was washed with aq. citric acid solution. These aqueous washings were concentrated in vacuo and the resultant oil was purified by chromatography on reverse phase silica eluting with a gradient of methanol in H.sub.2 O to give N-[5-N-(N-acetyl-L-prolyl)amino-1-(R)-methoxycarbonylpentyl]-L-leucyl-O-me thyl-L-tyrosine N-methylamide (350 mg) .delta.(CDCl.sub.3) 0.84 (6H,dd,J=14 Hz and 7 Hz,CH(CH.sub.3).sub.2); 1.05-2.4 (13H,m,NHCH.sub.2 (CH.sub.2).sub.3, CH.sub.2 CH(CH.sub.3).sub.2 and CH.sub.2 CH.sub.2); 2.08 (3H,s,COCH.sub.3); 2.70 (3H,s,NCH.sub.3); 2.76-3.82 (8H,m,NCH.sub.2 NHCH.sub.2 C.sub.6 H.sub.4 and .alpha.--CHx2); 3.66 and 3.74 (each 3H, each s, 2xOCH.sub.3); 4.32 (1 H,m,.alpha.--CH); 4.56 (1H,dd,J=13 Hz and 6 Hz,.alpha.--CH); 6.80 (2H,d,J=8.6 Hz, tyr H-3 and H-5) and 7.12 (2H,d,J=8.6 Hz, Tyr H-2 and H-6).]

A portion of this material (130 mg) in methanol (5 ml) was treated at 0.degree. with dilute NaOH (1N, 0.5 ml). After standing overnight at room temperature, a further portion of NaOH was added (1N, 0.2 ml) and this was then repeated 6 h later. After a further 18 h at 20.degree. the reaction mixture was acidified with acetic acid and concentrated to an oil in vacuo. Chromatography on reverse phase silica eluting with water in an increasing methanol gradient gave the title compound (100 mg); m.p. 97.degree.-101.degree.; (Found: [m+1].sup.+ =590.3552 C.sub.30 H.sub.47 N.sub.5 O.sub.7 requires [m+1].sup.+ =590.3554); .nu..sub.max (Nujol 3280 (br) and 1625 (br) cm.sup.-1 ; .delta.(CD.sub.3 OD) 0.94 (6H,m,CH(CH.sub.3).sub.2); 1.2-2.4 (13H,m,NHCH.sub.2 (CH.sub.2).sub.3, CH.sub.2 CH(CH.sub.3).sub.2 and CH.sub.2 C.sub.2); 2.12 (3H,s,COCH.sub.3); 2.68 (3H,s,NCH.sub.3); 2.75-4.1 (8H,m,NCH.sub.2,NHCH.sub.2, CH.sub.2 C.sub.6 H.sub.4 and .alpha.--CHx2); 3.77 (3H,s,OCH.sub.3); 4.33 and 4.58 (each 1H, each m, 2x2CH); 6.85 (2H,d,J=8.6 Hz, Tyr H-3 and H-5); 7.16 (2H,d,J=8.6 Hz, Tyr H-2 and H-6) and 8.03 (1H,m,CONH); m/e 590 (2%, [m+1].sup.+, 572 (10% [m+1-H.sub.2 O.sup.+).

N-[2-(S)-N(1-(R)-Methoxycarbonylethyl)amino-4,4-dimethylpentanoyl]-L-alanin e N-butylamide (65 mg) in methanol (30 ml) was treated with IN-sodium hydroxide (3 ml) at 20.degree. for 6 h. Excess acetic acid was then added and the solvent evaporated in vacuo. The residue was chromatographed on reverse phase silica (RF 18) in a gradient of 20%-80% methanol in water. Elution in 70% methanol in water afforded the title compound (30 mg) as a freeze-dried powder, m.p. 137.degree.-138.degree.; (Found: [m+1].sup.+ =344.2548. C.sub.17 H.sub.34 N.sub.3 O.sub.4 requires [m+1].sup.+ =344.2549); .delta.(D.sub.2 O) 0.9 (3H,t,J=6 Hz,CH.sub.2 CH.sub.3); 0.94 (9H,s,C(CH.sub.3).sub.3); 1.2-1.8 (6H,m,(CH.sub.2).sub.2 and CH.sub.2); 1.4 (3H,d,J=8 Hz,CH.sub.3); 1.52 (3H,d,J=7 Hz,CH.sub. 3); 3.18 (2H,t,J=6 Hz,NHCH.sub.2); 3.66 (1H,q,J=5 Hz,CHCO); 3.88 (1H,d,J=10 Hz,CHCH.sub.2) and 4.38 (1H,q,J=5 Hz,CHCH.sub.3).

The starting material required in the preceeding preparation was synthesised as described in the following paragraphs:

4,4-Dimethylpentanoic acid (40 g; Chem Lett, (1980), 571) was treated at 20.degree. for 16 h with thionyl chloride (40 g) and the mixture distilled under reduced pressure to yield 4,4-dimethylpentanoyl chloride (38 g) b.p. 52.degree.-58.degree. at 10 mm Hg; .delta. (CDCl.sub.3) 0.94 (9H,s,C(CH.sub.3).sub.3 ; 1.66 (2H,t,J=9 Hz,CH.sub.2) and 2.88 (2H,t,J=9 Hz,CH.sub.2 CO).

A portion of this material (20 g) was treated at 110.degree. with bromine (20 g) for 4 h. Further bromine (5 g) was then added and the reaction continued for 1 h. Distillation under reduced pressure afforded 2-bromo-4,4-dimethylpentanoyl chloride (26 g), b.p. 92.degree.-96.degree. at 10 mmHg; .delta. (CDCl.sub.3) 1.0 (9H,s,C(CH.sub.3).sub.3); 1.94 (1H,dd,J=15 and 5 Hz, CHCHBr); 2.42 (1H,dd,J=15 and 8 Hz, CHCHBr) and 4.64 (1H,dd,J=8 and 5 Hz, CHBr).

The bromo-acid chloride (12 g) in CH.sub.2 Cl.sub.2 (100 ml) was treated with benzyl alcohol (8.8 g) and N-methyl morpholine (4.06 g) at 0.degree. for 16 h. The solution was then washed successively with dilute HCl and Sat.aq.NaHCO.sub.3 solution. The residue after evaporation of the solvent was purified by chromatography on silica in 20% ether-hexane to give the desired bromo ester (11.2 g) as an oil; (Found: C,56.3; H,6.4; Br,26.8; C.sub.14 H.sub.19 Br,O requires C,56.2; H,6.4; Br,26.7%); .nu..sub.max 2940 and 1730 cm.sup.-1 .delta.(CDCl.sub.3) 0.888 (9H,s,(CH.sub.3).sub.3 C); 1.92 (1H,dd,J=15 and 4 Hz,CHCHBr); 2.38 (1H,dd,J=15 and 10 Hz,CHCHBr); 4.34 (1H,dd,J=10 and 4 Hz CHBr); 5.2 (2H,s,OCH.sub.2 --C.sub.6 H.sub.5) and 7.4 (5H,m,C.sub.6 H.sub.5).

Benzyl-2-bromo-4,4-dimethylpentanoate (20 g) in dry dimethyl sulphoxide (250 ml) was treated with D-alanine methylester hydrochloride (9.33 g), N-methyl morpholine (6.78 g) and tetrabutyl ammonium iodide (24.7 g) at 90.degree. under an atmosphere of argon for 2 days. The reaction mixture was allowed to cool to room temperature, poured into water (500 ml) and the products recovered by extraction into dichloromethane (3.times.250 ml). The material isolated from the organic extracts was purified by chromatography on silica developed in a gradient of hexane-ether. Elution with 30% ether-hexane afforded benzyl 4,4-dimethylpent-2-enoate (14 g). Elution with 40% ether in hexane afforded the title compound (350 mg) as a gum; (Found: [m+1].sup.+ =322.2022. C.sub.18 H.sub.27 N.sub.1 O.sub.4 requires [m+1].sup.+ =322.2018); .nu..sub.max (film) 1735 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.90 (9H,s,C(CH.sub.3).sub.3); 1.28 (3H,d,J=7 Hz CHCH.sub.3); 2.46 and 2.68 (2H, each dd,J=12 and 5 Hz,CH.sub.2 (CH.sub.3).sub.3); 3.30 (1H,q,J=5 Hz CH--CH.sub.3); 3.36 (1H,t,J=5 Hz, CH--CH.sub.2); 3.66 (3H,s,OCH.sub.3); 5.12 (2H,s,OCH.sub.2) and 7.36 (5H,s,C.sub.6 H.sub.5). Elution with 45% ether in hexane afforded benzyl 2-(R)-N-(1-(R)-methoxycarbonylethyl)-amino-4,4-dimethylpentanoate (340 mg); (Found: [m+1].sup.+ =322.2022. C.sub.11 H.sub.27 NO.sub.4 requires 322.2018); .nu..sub.max (film) 3360 and 1735 cms.sup.-1 ; .delta.(CDCl.sub.3) 0.90 (9H,s, C(CH.sub.3).sub.3); 1.28 (3H,d,J=6 Hz, CHCH.sub.3); 1.44 and 1.72 (2H, each dd,J=5 and 12.5 Hz,CH.sub.2); 3.32 (1H,q,J=7 Hz, CHCH.sub.3); 3.44 (1H,t,J=6 Hz, CHCH.sub.2); 3.69 (3H,s,OCH.sub.3), 5.24 (2H,s,OCH.sub.2) and 7.36 (5H,m,C.sub.6 H.sub.5).

The foregoing benzyl ester (450 mg) in methanol (50 ml) was treated with palladium on charcoal (10% 400 mg) under 1 atmosphere of hydrogen with continuous stirring. When the uptake of hydrogen had ceased (15 min) the solution was filtered and the filtrate concentrated in vacuo to afford 2-(S)-N-(1-(R)-methoxycarbonylethyl)amino-4,4-dimethylpentanoic acid (210 mg); m.p. 120.degree.-124.degree. (from ether).

This material (200 mg) in CH.sub.2 Cl.sub.2 (50 ml) was treated with L-alanine N-butylamide hydrochloride (220 mg), N-ethyl-N'-(3-dimethylamino propyl) carbodiimide hydrochloride (200 mg) and 1-hydroxybenzotriazole (120 mg) at 0.degree. C. The pH of the reaction mixture was adjusted to 7 by the addition of N-methyl morpholine. After 16 h at 20.degree., the solution was washed in turn with saturated sodium hydrogen carbonate solution and 1M citric acid solution. The material isolated after evaporation of the dichloromethane was chromatographed on silica developed in a gradient of 20% ethyl acetate in dichloromethane to 60% ethyl acetate in dichloromethane to afford the title compound (110 mg) as a colourless oil, (Found: [m+1].sup.+ =358.2705. C.sub.18 H.sub.35 N.sub.3 O.sub.4 requires [m+1].sup.+ =358.2706); (CDCl.sub.3) 0.92 (3H,t,J=7.5 Hz,CH.sub.2 CH.sub.3); 1.0 (9H,s,C(CH.sub.3).sub.3); 1.36 and 1.40 (each 3H, each t, J=6 Hz, 2xCH.sub.3); 1.2-1.9 (6H,m,3xCH.sub.2); 3.24 (2H,m,NHCH.sub.2); 3.46 (1H,q,J=6 Hz,CH); 3.77 (3H,s,OCH.sub.3), 4.46 (1H,t,J=6 Hz,CHCH.sub.2); 4.5 (1H,q,J=6 Hz,CH), 7.15 (1H,m,NH) and 7.73 (1H,d,J=8 Hz,NH).

The L-alanine N-butylamide hydrochloride used in step (c) was prepared from N-tertiarybutoxycarbonyl-L-alanine N-butylamide by exposure to TFA in CH.sub.2 Cl.sub.2 followed by treatment with ethereal HCl. This in turn was prepared from N-tertiarybutoxy-L-alanine and n-butylamine following the procedure described in Example 2 for N-tertiarybutoxy-O-benzyl-L-tyrosine N-methylamide except that butylamine was used in place of methylamine hydrochloride.

This was prepared from tertiarybutoxycarbonyl-L-norleucine, L-alanine N-butylamide and 2-bromopropionic acid methyl ester as described in the following steps:

Tertiarybutoxycarbonyl-L-norleucine (13.2 g) in CH.sub.2 Cl.sub.2 (200 ml) was treated at 0.degree. with L-alanine N-butylamide (5.25 g), DCC (7.77 g) and 1-hydroxybenzotriazole (5 g). The pH of the reaction mixture was adjusted to 7 with N-methyl morpholine and allowed to warm to room temperature overnight. The precipitated urea was filtered off and the filtrate washed successively with saturated aqueous sodium hydrogen carbonate, water and 1M citric acid. The organic phase was dried over sodium sulphate and the solvent evaporated in vacuo. The residue was chromatographed on silica in a gradient of 30-70% ethyl acetate in dichloromethane. Elution with 50% ethyl acetate in dichloromethane afforded the title compound (7.6 g) which crystallised from ethyl acetate as needles m.p. 108.degree.-112.degree.; (Found: C,60.8; H,9.8; N,11.8. C.sub.18 H.sub.35 N.sub.3 O.sub.4 requires C,60.5; H,9.9; N,11.75%); .nu..sub.max (Nujol) 3280, 3340 1675 and 1640 cms.sup.-1 .delta. (CDCl.sub.3) 0.9 and 0.91 (each 3H, each t, each J=5 Hz,2xCH.sub.3); 1.1-1.9 (10H,m,(CH.sub. 2).sub.3 and (CH.sub.2).sub.2); 1.38 (3H,d,J=5 Hz,6H.sub.2 CHCH.sub.3); 1.44 (9H,s, C(CH.sub.3).sub.3); 3.24 (2H,tt,J=5 Hz NHCH.sub.2) and 4.1 and 4.48 (each 1H, each m, 2x CH).

Tertiarybutonycarbonyl-L-norleucine-L-alanine N-butylamide (5 g) in dichloromethane (20 ml) was treated with trifluroacetic acid (20 ml) at room temperature for 2 h. The solvents were evaporated in vacuo and the residue in water was treated with excess sodium hydrogen carbonate and the free amine recovered in dichloromethane. Evaporation of the CH.sub.2 Cl.sub.2 and crystallisation of the residue from ether-hexane gave the title compound (3.1 g); m.p. 83.degree.-84.degree.; (Found: C,60.7; H,10.4; N,16.0. C.sub.13 H.sub.27 N.sub.3 O.sub.2 requires C,60.6; H,10.6; N,16.3%); .nu..sub.max (Nujol): 3360, 3280, 1635 and 1675 cm.sup.-1 ; .delta.(CDCl.sub.3) 0.94 (6H,t,J=5 Hz,2xCH.sub.2 CH.sub.3); 1.40 (3H,d,J=6 Hz CH--CH.sub.3); 1.4-1.9 (10H,m,(CH.sub.2).sub.3 and (CH.sub.2).sub.2); 3.26 (2H,dt, each J=5 Hz,NH--CH.sub.2 --); 3.35 (1H,dd,J=4 and 8 Hz, CH-CH.sub.2); 4.50 (1H,dq, each J=6 Hz, CH--CH.sub.3); 6.9 (1H,m,NH); 7.86 (1H,d,J=7 Hz,NH).

L-Norleucine-L-alanine N-butylamide (1 g) in acetonitrile (10 ml) was treated with N-methyl morpholine (0.4 g) and methyl 2-bromopropionate (0.64 g) under reflux for 16 h. The solvent was removed in vacuo and the residue in dichloromethane washed successively with 1M citric acid, water and saturated aqueous sodium hydrogen carbonate. The residue after evaporation of the CH.sub.2 Cl.sub.2 was chromatographed on silica in a gradient of ethyl acetate in CH.sub.2 Cl.sub.2. Elution with 60% ethyl acetate in CH.sub.2 Cl.sub.2 afforded N-(1-(S)-methoxy-carbonylethyl)-S-norleucyl-S-alanine N-butylamide (210 mg); (Found: [m+1].sup.+ =344.2547. C.sub.17 H.sub.34 N.sub.3 O.sub.4 requires [m+1].sup.+ =344.2582); .nu..sub.max (Nujol) 3320 and 1740 cms.sup.-1 ; .delta.(CDCl.sub.3) 0.95 (6H,t,J=7 Hz,2xCH.sub.2 CH.sub.3); 1.36 and 1.40 (each 3H, each d, each J=6 Hz, 2xCHCH.sub.3); 1.2-1.8 (10H,m,(CH.sub.2).sub.2 and (CH.sub.2).sub.3); 2.98 (1H,dd,J=4 and 5 Hz, CHCH.sub.2); 3.24 (3H,m,NHCH.sub.2 and CHCO); 3.7 (3H,s,OCH.sub.3); 4.56 (1H,dq,J=5 Hz,CH) and 7.04 and 7.9 (each 1H, each m, 2xNH).

Continued elution with 65% ethyl acetate in CH.sub.2 Cl.sub.2 bave the title compound (190 mg), m.p. 84.degree.-88.degree. (from ethyl acetate); (Found: C,59.2; H,9.5; N,12.2. C.sub.17 H.sub.33 N.sub.3 O.sub.4 requires C,59.6; H,9.4; N,12.3%); .nu..sub.max (Nujol) 3280 and 1740 cm.sup.-1 ; .delta. (CDCl.sub.3) 0.94 (6H,t,J=6 Hz,2xCH.sub.2 CH.sub.2); 1.38 and 1.42 (each 3H, each d, each J=5 Hz, 2xCHCH.sub.3); 1.3-1.9 (10H,m, (CH.sub.2).sub.2); 3.06 (1H,dd,J=5 and 8 Hz, CHCH.sub.2); 3.24 (2H,dt,J=5 and 6 Hz,NHCH.sub.2); 3.46 (1H,q,J=6 Hz,CHCO); 3.72 (3H,s,OCH.sub.3); 4.67 (1H,dq,J=5 and 7 Hz,CHCH.sub.3); 6.84 (1H,m,NH) and 7.82 (1H,d,J=7 Hz,NH).

The foregoing methyl ester (150 mg) in CH.sub.3 OH (50 ml) was treated with 1M NaOH (1 ml) at room temperature for 72 h. Excess acetic acid was added and the solvents evaporated in vacuo. The residue was chromatographed on reverse phase silica (RP18) in a gradient of 0-60% methanol in water. Elution with 50% methanol in water afforded the title compound (110 mg) as needles from ether/hexane; m.p. 185.degree.-190.degree.; (Found: C,56.7; H,9.2; N,12.4. C.sub.16 H.sub.31 N.sub.3 O.sub.4.H.sub.2 O requires C,56.8; H,9.5; N,12.4%); .nu..sub.max (Nujol) 3200 and 1650 cm.sup.-1 ; .delta.(CD.sub.3 OD) 0.92 and 0.94 (each 3H, each t, each J=6 Hz,2xCH.sub.2 CH.sub.3); 1.36 and 1.48 (each 3H, each d, each J=6 Hz,2xCHCH.sub.3); 1.2-1.9 (10H,m,(CH.sub.2).sub.2 and (CH.sub.2).sub.3); 3.20 (2H,t,J=6 Hz NH-CH.sub.2); 3.56 (1H,q,J=6 Hz,CHCO.sub.2 H); 3.88

The compounds of Examples 25 to 131 and their routes of preparation are exemplified within the following Tables.

Using the methods illustrated in examples 1-24 further examples 25-131 in Table 1 are prepared.

Compounds N-[2-(S)-N-(1-(R)-carboxyethyl) amino-4,4-di-(trifluoromethyl)butanoyl]-O-methyl-L-tyrosine N-methylamide and N-[2-(S)-N-(3-(benzyloxycarbonyl) amino-1-(R)-carboxypropyl)amino-4,4-di-(trifluoromethyl)butanoyl]-O-methyl -L-tyrosine N-methylamide are likewise prepared by methods described in examples 1-24. STEREO-.sup.1 MP. .degree.C..sup.2 MP. .degree.C..sup.2 No PROCESS A.sup.1 A.sup.2 Y n R.sup.2 R.sup.3 A.sup.3 CHEM R.sup.1 = OCH.sub.3 R.sup.1 = OH 25 1A -- H -- 1 H CH(CH.sub.3)CH.sub.2 CH.sub.3 GlyNHC.sub.4 H.sub.9.sup.n RS 82-84 74-77 26 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 GlyNHC.sub.4 H.sub.9.sup.n RS 87-95 27 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 GlyNHC.sub.4 H.sub.9.sup.n SS 175-18.0. 28 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 ValNHC.sub.6 H.sub.13.sup.n RSS 19.0.-193 29 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 ValNHC.sub.6 H.sub.13.sup.n SSS 2.0..0.-2.0.3 3.0. 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 LeuNHC.sub.4 H.sub.9.sup.n RSS 138-139 18.0.-185 31 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 LeuNHC.sub.4 H.sub.9.sup.n SSS 18.0.-185 183-185 32 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 LeuNHC.sub.4 H.sub.9.sup.n RSR 1.0.3-1.0.7 15.0.-16.0. 33 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 LeuNHC.sub.4 H.sub.9.sup.n SSR 94-98 185-188 34 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHC.sub.4 H.sub.9.sup.n RSSR 62-67 145-148 35 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHC.sub.4 H.sub.9.sup.n SSSR 61-64 147-152 36 1A -- H -- 3 H CH.sub.2 CH(CH.sub.3).sub.2 ValGlyOCH.sub.3 RS 87-92 37 1A -- H -- 3 H CH.sub.2 CH(CH.sub.3).sub.2 ValGlyOCH.sub.3 SS 177-18.0. 38 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 ValGylOCH.sub.3 RS 39 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RSSR 72-76 194-197 4.0. 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NH(CH.sub.2).sub.2SCH.sub.2 CH.sub.3 RSSR .sup. 162-164.sup.3 1.0.5-1.0.9 41 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NH(CH.sub.2).sub.2SOCH.sub.2 CH.sub.3 RSSR 8.0.-85 42 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NH(CH.sub.2).sub.3CONH.sub.2 RSSR 97 193 43 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NH(CH.sub.2).sub.5CONH.sub.2 RSSR foam 115-12.0. 44 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)N(CH.sub.3)C.sub.4 H.sub.9.sup.n RSSR oil 56-57 45 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NH(CH.sub.2).sub.2SO.sub.2 CH.sub.2 CH.sub.3 RSSR .sup. 157-161.sup.3 9.0.-95 46 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NH(CH.sub.2).sub.5CO.sub.2 H RSSR .sup. 131-133.sup.3 1.0.5-1.0.7 47 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 NHCH(CONHCH.sub.3)CH.sub.2NHCO.sub.2 C(CH.sub.3).sub.3 RSS 1.0.7-112 174-182 48 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Ser(OBZ)NHCH.sub.3 RSS 61-64 188-19.0. 49 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 TyrNHCH.sub.3 RSS 212-217 5.0. 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 NHCH(CONHCH.sub.3)CH.sub.2NHCOPh RSS 71-74 186-191 51 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 NHCH(CONHCH.sub.3)CH.sub.2NHZ RSS 11.0.-112 163-166 52 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 ThrNH(CH.sub.2).sub.3 CONH.sub.2 RSSR 235 53 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 AlaNHC.sub.4 H.sub.9.sup.n RSS .sup. 174-176.sup.3 158-162 54 1A -- Z NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 AlaNHCH.sub.3 SSS 176-182 55 1A -- Z NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 AlaNHCH.sub.3 RSS 166-168 56 1A -- H -- 6 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 SSSR 62-64 112-12.0. 57 1A -- H -- 6 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RSSR 79-8.0. 63-66 58 1A -- Z NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RSSR 92-94 16.0.-164 59 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 AlaNHCH.sub.3 RSS 87-9.0. 84-88 6.0. 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 PheNHCH.sub.3 RSS 116-119 115-116 61 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 SarNHCH.sub.3 RS 16.0.-175 77-8.0. 62 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 ProNHCH.sub.3 RSS 99-1.0.2 1.0..0.-1.0.5 63 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 NHCH(CONHCH.sub.3)(CH.sub.2).sub.6 CH.sub.3 RS(RS) .sup. 155-159.sup.3 186-191 64 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 NCH.sub.3Tyr(OBZ)NHCH.sub.3 RSS .sup. 115-12.0..sup.3 115-121 65 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 iso-AbaNHCH.sub.3 RS .sup. 15.0.-153.sup.3 177-179 66 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 NZLysNHCH.sub.3 RSS .sup. 17.0.-172.sup.3 162-164 67 1A Z Leu NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 SRSS 145-15.0. 68 1A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 SerNHCH.sub.3 RSS 191-198 69 1A -- Z NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 AlaNHCH.sub.3 SSS 176-182 7.0. 1A -- Z NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 AlaNHCH.sub.3 RSS 166-168 71 1B -- H NH 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RSSR 1.0.5-1.0.7 72 1B -- CH.sub.3 CO NH 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RSSR 131-132 1.0.8-112 73 1B -- CH.sub.3 CO NH 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RRSR 133-134 1.0..0.-1.0.2 74 1B -- (CH.sub.3).sub.3 COCO NH 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RSSR 95-97 114-118 75 1B -- (CH.sub.3).sub.3 COCO NH 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 RRSR 123-124 8.0.-9.0. 76 1B DnpPro Leu NH 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 SSRSSR 112-115 77 1B DnpPro Leu NH 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OBZ)NHCH.sub.3 SSRRSR 1.0.8-115 78 1B -- Ph(CH.sub.2).sub.2 NHCO -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 R(RS)S 124-128 79 1B -- HO.sub.2 C -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 64-66 13.0.-132 8.0. 2A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OBZ)NH.sub.2 RSS .sup. 117-119.sup.4 193-196 81 2A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS .sup. 96-97.sup.4 2.0..0.-2.0.2 82 2A -- H -- 1 H CH(CH.sub.3).sub.2 AlaNHC.su b.4 H.sub.9.sup.n SSS 6.0.-62 1.0..0.-1.0.5 83 2A -- H -- 1 H CH(CH.sub.3).s ub.2 AlaNHC.sub.4 H.sub.9.sup.n RSS 165-17.0. 84 2A -- H -- 1 H CH.sub.3 AlaNHC.sub.4 H.sub.9.sup.n (RS)SS 22.0.-223 85 2A -- H -- 1 H CH.sub.3 AlaNHC.sub.4 H.sub.9.sup. n (SR)SS 231-234 86 2A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 His(BZ)NHCH.sub.3 RSS 95-1.0.3 87 2A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 AlaNHOCH.sub.3 RSS 192-196 88 2A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Thr(OC.sub.4 H.sub.9.sup.t)NHCH.sub.3 RSSR 98-1.0.8 89 2A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 TyrNH.sub.2 RSS 219-23.0. 9.0. 2A -- H -- 1 H (CH.sub.2).sub.2 CH.sub.3 AlaNHC.sub.4 H.sub.9.sup.n R(RS)S 84-85 199-2.0.1 91 2A -- CH.sub.3 CO NH 3 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 1.0.1-1.0.4 15.0. 92 2A -- H -- 1 H (CH.sub.2).sub.2 SCH.sub.3 AlaNHC.sub.4 H.sub.9.sup.n RSS foam 155-159 93 2A -- H -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 NHCH(CH.sub.3)CH.sub.2 Ph RS(RS) 94 2A -- H -- 1 H CH.sub.2 C.sub.6 H.sub.5 AlaNHC.sub.4 H.sub.9.sup.n (RS)SS 173-178 95 2A -- H -- 1 H CH.sub.2 OCH.sub.2 Ph AlaNHC.sub.4 H.sub.9.sup.n RSS 158-162 96 2A -- H -- 1 H CH(CH.sub.3)CH.sub.2 CH.sub.3 AlaNHC.sub.4 H.sub.9.sup.n RSS 9.0.-94 162-164 97 2A -- Ph(CH.sub.2).sub.2 CO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 114-118 162-163 98 2A -- H -- 1 (CH.sub.2).sub.5 AlaNHC.sub.4 H.sub.9.sup.n (RS)S 95-1.0.5 99 2 A -- (CH.sub.3).sub.2 CHCH.sub.2 CO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 168-172 1.0..0. 2A -- CH.sub.3 OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3 NHCH.sub.3 RSS 149-153 1.0.1 2A Z Pro NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 SRSS 1.0.2-1.0.3 174-179 1.0.2 2A -- (CH.sub.3).sub.2 CHCH.sub.2 OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 171-175 1.0.3 2A -- PhCHCHCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 188-191 1.0.4 2A -- 2-ClC.sub.6 H.sub.4 CO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 1.0.9 158-163 1.0.5 2A -- 4-ClC.sub.6 H.sub.4 CO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 1.0.5-1.0.8 187-192 1.0.6 2A -- Z NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)OC.sub.4 H.sub.9.sup.t RSS 1.0.1-1.0.2 1.0.7 2A -- 4-CH.sub.3C.sub.6 H.sub.4 CH.sub.2OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 141-143 164-167 1.0.8 2A -- 4-ClC.sub.6 H.sub.4 CH.sub.2 OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 167-171 1.0.9 2A -- HO.sub.2 C(CH.sub.2).sub.2 CO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 5.0.-55 16.0.-171 11.0. 2A -- 4-CH.sub.3C.su b.6 H.sub.4 CO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 98 19.0.-194 111 2A -- PhCH.sub.2 CO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 173-179 112 2A -- 2-ClC.sub.6 H.sub.4 CH.sub.2 OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 168-174 113 2A -- 4-CH.sub.3 OC.sub.6 H.sub.4 CH.sub.2OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 162-166 114 2A -- Bornyl-OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 145-150 115 2A -- 2-CH.sub.3C.sub. 6 H.sub.4 CH.sub.2OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 17.0.-173 116 2A -- Ph(CH.sub.2).sub.2 OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 147-151 117 2A -- PhCH.sub.2 SO.sub.2 NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 5.0.-60 174-178 118 2A -- PhCH.sub.2 N(CH.sub.3)CO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 65-7.0. 9.0.-95 119 2A -- 2-NaphthylCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 148-153 162-172 12.0. 2A -- 1-NaphthylCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 67-71 167-173 121 2A -- Ph -- 1 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 161-166 122 2A -- 1-NaphthylCH.sub.2OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 85-86 182- 184 123 2A -- 2-NaphthylCH.sub.2OCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 92-98 168-171 124 2A -- PhCCCO NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 125 2A -- H -- 1 H CH.sub.2 CH(CF.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 126 2A -- Z NH 2 H CH.sub.2 CH(CF.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 127 2A -- Z NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OBZ)NHCH.sub.3 RSS 172-175 128 2A -- Z NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OC.sub.5 H.sub.11.sup.n)NHCH.sub.3 RSS 81-85 155-157 129 2A ZPro Leu NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 SSRSS 13.0. 2A ZPro Pro NH 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 SSRSS 131 2B -- HO.sub.2 C -- 2 H CH.sub.2 CH(CH.sub.3).sub.2 Tyr(OCH.sub.3)NHCH.sub.3 RSS 133-135 11.0.-12.0. Notes for TABLE 1: 1. Stereochemistryoptical centres labelled from left to right. 2. Of hydrated form where appropriate. 3. m.p. of HCl salt. 4. R.sup.1 = OC.sub.2 H.sub.5 not OCH.sub.3 Gly = glycyl = NHCH.sub.2 CO ##STR20## ##STR21## Ph = phenyl = C.sub.6 H.sub.5 Bz = CH.sub.2 C.sub.6 H.sub.5 Z = PhCH.sub.2 O.CO DNP = 2,4dinitrophenyl ##STR22## Sar = Sarcosyl = N(CH.sub.3)CH.sub.2 CO ##STR23## ##STR24## ##STR25## ##STR26## ##STR27## ##STR28##

The activities of representative compounds according to the invention are given below in Table II.