GB2058077A - Enkephalin analogues - Google Patents

Abstract

Compounds corresponding in structure to enkephalin or polypeptide analogues thereof, wherein one or more peptide links of the enkephalin or analogue is represented by a group or groups the same or different selected from dimethylene, methylene-imino, hydroxydimethylene and keto- methylene groups or by adjacent peptide links cyclised to give a hydantoin or thiohydantoin derivative.

Description

APPENDIX I - Structure

Code 1 NO' 2 ~Tyr X2 Gly3 34 5 z No. R T r X Cl B Y CH2Ph Zt C0H2CH2SCH3 12 I 12 H 224 H - Tyr - DAla - Gly - NH-CH-CH2 N-CH-CO - N-CH-CH2 OH.HCl ClH2Ph C1H2CH2SCH3 H 225 H - Tyr - DAla - Gly - NH-CH-CH2 - NH-CH-CH2 OH. 2HC1 CH Ph Z1 CH CH2SCH3 i2 , 12 H 226 H - Tyr - Gly - Gly - NH-CH-CH2 - N-CH-CH2 OH.HC1 12 1 2C 2 H3 H 227 H - Tyr - Gly - Gly - NH-CH-CH2 - NH-CH-CH2 OH.2HC1 Z1 CH Ph 12 H 228 H - Tyr -DAla-NHCH2CH2- N-CH-CO - Met NH2.HCl CH2 Ph 12 CH CH SCH CHCO ClH2CH2SCH3 H 229 H - Tyr -DAla-NHCH2CH2- N N - CH-CO ------ NH2.HC1 CO CH Ph j2 ,CHCO\ CH2CH2SCH3 H 230 H - Tyr - Gly -NHCH2CH2- N N - CH-CO NH2 HCl CO CH CH SCH 12 2 3 H 231 H - Tyr -DAla-NHCH2CH2 - Phe - NH-CH-CH2 OH.2HC1 Z1 CH Ph CH CH SCH I i2 2 2 2 3 H 232 H - Tyr -DAla-NHCH2CH2 - N-CH-CO - NH-CH-CH2 OH. 2HC1 - CH Ph CH CH SCH Met H - Tyr - Gly-NHCH2CO - i2 i2 2 N-CH-CO 3 NH-CH-CO - Gly-NHCH,CO - NH-CH-CO - N-CH-CO ----- OH enkephalin 2 (Gly) (Phe) (Met)

Activity Relation to Met5 enkephalin or Met5 amide - enkephalin % *' (APPENDIX I cont'd) 100/270 Reduced Phe4 ; reduced and substituted Met DAla2 200/520 Reduced Phe4 and Met5; DAla2 3.0/11 As H 224 but Gly as enkephalin 36/37 As H 225 but Gly as enkephalin 1162*/N.D. D Ala reduced Gly3; substituted Phe4 5.0/5.0 D Ala ; reduced Gly ; Phe4 and Met5 cyclised 0.6/5 As H 229 but Gly as enkephalin 10/120 D Ala ; reduced Gly3 and Met5 180/75 As H 231 but Phe4 substituted also Notes

*' Two assays: (benzyloxycarbonyl) i) 3H naloxone receptor (ii) guinea pig # Nie -enkephalin ileun = 100% APPENDIX II Structure (with residues numbered)

Code 1 2 3 4 5 No. R Tyr X Gly B Y Z OH C2CH2 SCH3 H 236 H - Tyr - NHCH2CH - CH2CH2CO - Phe - NH-CH-CH2 - OH.HC1 - ZI H 237 H - Tyr - NHCH2CH2- N CH2CO - Phe Met - NH2HC1 CH2-C=O CH Ph I 1 I 12 H 238 H - Tyr - NHCH2CH -N N - CH-CO - Met - NH2HC1 2' C 6, 0 Z1CH Ph I H 239 H - Tyr - DAla - NHCH2CH2 - N-CH-CO - Met - OH.HC1 H-CO CH2Ph 1 12 H 240 H - Tyr - DAla - NHCH2CH2 - N - CH - CO - Met - OH. HCl H-CO H-FO C1H2Ph H 241 H - Tyr - DAla - NHCH CH - N - CH - CO - Met ~ NH2,HC1 Notes i) Z1 is as in Appendix I (Benzyloxycarbonyl) ii) Preliminary results indicate biological activity of the above six compounds comparable to that of the compounds of Appendix I. APPENDIX II (cont'd) Relation to Met - enkephalin or Met - enkephalin amide Gly carbonvl reduced to

3 Gly -NH- replaced with -CH2-; Met5 reduced to methioninol (new type of isostere) Gly reduced ; Gly substituted with Z Gly reduced ;Gly and Phe4 cyclised DAla ; Gly reduced and Phe4 substituted with Z DAla ; Gly reduced and Phe4 substituted with HCO DAla ; Gly reducedand Phe4 substituted with HCO SPECIFICATION Enkephalin analogues Background The invention relates to enkephalin analogues or, as they are also referred to herein, isosteres.

Enkephalin, specifically methionine enkephalin, is the pentapeptide H-Tyr-Gly2-Gly3-Phe4-Met5-OH Since its discovery a great deal of work has been done synthesising analogues with a view to elucidation of the mechanism of action of enkephalin itself and clinical use of the analogues.

The past decade has witnessed an unprecedented growth of knowledge in the field of biologically active peptides. The discovery of a new naturally occurring peptide is usually followed by the synthesis of analogues in order to obtain more potent, more selective or longer acting compounds, or antagonists of the parent peptide. Since peptides are synthesized by linking successive amino acids, it is technically easy and therefore tempting to synthesize analogues in which some of the native amino acid residues are replaced by others, sometimes of the unnatural D-configuration. The structure-activity correlations derived in this way reflectthe contribution of individual amino acid residues.

We have essentially taken a different approach, and in the present application and a preceding application No. 28598/78 (2,000,783) set out to investigate the role the backbone plays in the biological properties of peptides. This backbone, consisting of the monotonous polymer -(NH-CH-CO)n-, is common to all peptides.

Attached to it at the cr-carbon atoms are the amino acid side-chains, and it is the sequence and variety of these that endow the peptide with its peculiar physical, chemical and biological properties. In practical terms, our broad aim has been to synthesize isosteric analogues, in which the amino acid side-chains of the parent peptide are retained but parts of the peptide backbone are replaced with other, stereo-chemically similar residues.

Since the metabolic instability of peptides resides in the backbone, isosteric substitutions that are resistant to proteolysis may endow the analogue with increased stability and a prolonged duration of action provided, of course, that they are replacing susceptible parts of the backbone. The variations we propose are fully set out below, but for example reduced analogues -CH2-NH-, obtained by chemical reduction of the amide carbonyl to methylene, introduce a basic centre into the backbone and slightly alter the orientation of the side-chains. Hydrocarbon analogues, in which the peptide bond -CO-NH- is replaced by -CH2-CH2 show a stereochemically very slight change, but the backbone is more lipophilic and is incapable of forming a hydrogen bond at the site of replacement.

In U.K. patent application No. 28598/78 (published as No. 000 783) filed by us and others there are disclosed compounds corresponding in structure to enkephalin or polypeptide analogues thereof, wherein one or more peptide links of the enkephalin or analogue are represented by a group or groups the same or different selected from dimethylene, methylene-imino and keto-methylene groups. The resulting isosteres are referred to as 'hydrocarbon', 'reduced', and 'keto' analogues. Also disclosed are 'hydroxy' analogues where the keto group can be regarded as present in the form of the link -CH(OH)-CH2-.

The invention The present invention is a development of the above and in one aspect provides compounds corresponding in structure to enkephalin or polypeptide analogues thereof, possibly containing different numbers of residues, wherein one or more of the peptide groups -CO-NH- are represented by a group or groups, the same or different, selected from i) protected methylene-imino groups -CH2-NP-, P being a protective grouping defined below, ii) groups -CH(OA)-CH2-, A being a grouping also defined below, and iii) groups wherein there is a replacement of any adjacent pair of peptide bonds as shown below:

that is to say a carbonyl orthiocarbonyl group links the peptide-bond nitrogen atoms of the nth and the (n+1 )th residues of which Rn and Rn+ are the side chains.

A general formula for such compounds is for example: A-Tyr-X-G Iy-B-Y-Z where (a) -X- is Gly, or aza-Gly or aza-Ala, or any D-amino acid particularly D-AIa, D-Thr, D-Ser or D-Met; -B- is Phe or N-methyl-Phe or dehydro-Phe, as such or substituted with hydroxy, halogen or nitro groups in the aromatic ring, or alternatively cyclohexylalanine;; -Y- is any D- or L-amino acid residue, particularly Leu, Nle and Met the most preferred, or the sulphoxide of Met or aza derivatives of these, Pro or Hypro (not in the cyclised compounds), or homoserine lactone, or formal derivatives of any of these in which the terminal

group is replaced by -CH2-Z or a hydrogen atom -Z- is NH2, NHA, N(A)2, OH or OA (b) the -CO-NH- link between one or more pairs of residues is represented by a group or groups as set out above, optionally in conjunction with i) other peptide bond modifications

as such or in the form of hydroxydimethylene -CH(OH)-CH2-, as described in the earlier application No.

28598/78 referred to above, andlor ii) the presence of substituted peptide bonds

The group P may be an oxygen atom or an organic group, selected from aryl groups such as phenyl, benzyl or the like, or simple halogenated (in particular fluorinated) derivatives such as chloroacetyl or trifluoroacetyl, or substituted derivatives such as

where R1 may be the same as R (below) or phenyl or benzyl or ring substituted phenyl or benzyl derivatives, tertiary butyl or substituted derivatives such as phenylisopropyl, diphenylisopropyl, or fluorenylmethylene or simple chemical modifications of these alternatives.

Preferred combinations for R" and Run 1 in the cyclised compounds are i) R" as the side chain of Gly, aza-Gly, aza-Ala or any D-amino acid residue particularly D-AIa, D-Thr, D-Ser or D-Met and Rn+1 as the side chain of Gly.

ii) R" as the side chain of Gly and Pin*1 as the side chain of Phe, N-substituted (particularly N-methyl)-Phe or dehydro-Phe all optionally substituted in the ring by hydroxy, halo, nitro or other groups, or alternatively cyclohexylalanine.

iii) R" as the side chain of Phe, N-substituted (particularly N-methyl) -Phe or dehydro-Phe all optionally substituted in the ring by hydroxy, halo, nitro or other groups, or alternatively cyclohexylalanine, and Rn+' as any D- or L-amino acid residue particularly Leu, Nle, Met or the sulphoxide of Met all as such or in aza form; or homoserine lactone; or formal derivatives of any of these in which the terminal

group is replaced by -CH2-Z (Z is -N H2, -NHA, -N(A)2, -OH or -OA where A is an aliphatic or other protective group as above) or by hydrogen.

It will be understood that the amino-terminal residue of the compounds, tyrosine in the case of enkephalin itself, should not be involved in the linking unless there is further modification to provide, still, a positively charged terminal corresponding to that given by tyrosine.

The group R may be a methyl, ethyl, n-or iso-propyl, cyclo-propyl, cyclopropyl-methyl, n-, iso-, sec- or tert-butyl, allyl or like group. The group A may be the same as R or the organic groups listed for P.

Compounds within the general formula where a peptide group is represented by -CH2-NP- are H224, H226, H228, H237, H239, H240 and H241 in the Examples later in the specification, P being benzyloxycarbonyl or formyl. Other groups may be attached similarly. The cyclised compounds are represented by H229, H230 and H238.A compound where a peptide group is replaced by derivatives of -CH(OH)-CH2- is H236 and these compounds may be made generally following the reaction scheme set out at pages 10 and 11 of specification No. 28598/78 (pages 5 and 6 as printed in No.2000783) but directed to compounds such as (10) or (11), for example generally following the procedure of Example 5 thereof but for example selectively alkylating with an alkyl halide such as methyl iodide and a base such as potassium carbonate or silver oxide, and the carboxyl protecting group R3 removed by hydrolysis.

The invention extends further to new compounds using the peptide bond modifications of application No.

28598/78, namely compounds A-Tyr-X-G Iy-B-Y-Z where the peptide bond modifications of that application are used and a) one or more of the following is satisfied: -X- being aza-Gly or aza-Ala, D-Thr or D-Ser -B- being dehydro-Phe as such or substituted with hydroxy or halogen, or nitro-substituted Phe, N-methyl-Phe or dehydro-Phe, or alternatively cyclohexylalanine -Y- being norleucine (Nle); or Leu, Nle, Met or the sulphoxide of Met all in the form of aza derivatives; or homoserine lactone; or formal derivatives of D- or L-amino acid residues particularly Leu, Met, the sulphoxide of Met or aza derivatives of these, or Pro or Hypro, or homoserine lactone, wherein the terminal

group is replaced by a hydrogen atom: b) X, B, Y and Z are otherwise as previously set out herein.

Compounds according to this last general formula with variations in -X- and -B- are available by the synthetic methods of specification No. 2 000 783 where for example substituted Phe groups may be incorporated by the methods given for Phe itself in the specific compounds of the Examples of that specification. For variations at -Y- the method exemplified in the preparation of compound H232 herein where Y is Metol, is general.

The invention extends still further to compounds A-Tyr-X-G ly-B-Y-Z where a) the B to Y or the Glyto B peptide link is replaced by -CH2-NH- and b) X is D-Ala or Gly in the first case and D-Ala in the second, and A is H, B is Phe, Y is Metol and Z is OH in both cases. (strictly Y+Z = Metol) that is to say the compounds

(2) the corresponding Gly2 compound H227, and

It will be understood that any of the compounds may be in salt form or suitably protected at amino or other groups and that bare reference to a compound includes as to the scope of the invention the compound in any such form or as made up with a suitable pharmaceutical carrier or diluent for administration.

Throughout, the side chains are selected preferably from those of the natural amino acids or their D-forms.

Protected or otherwise modified side chains may however be used where the desired biological properties are not deleteriously affected.

It will further be understood throughout the above that while compounds containing five amino acid or like residues, the same number as enkephalin itself, are primarily in mind, further residues, for example one or two, may be present and compounds containing them are regarded as derivatives within the spirit and scope of the invention as defined in the claims hereof. To retain the biological activity of the compounds such further residues are at the amino or carboxyl terminal, and do not interrupt the portion of the structure that corresponds to enkephalin. If present at the amino terminal the further residue should be such as to retain a positive charge, or the capability of it at physiological pH's, and should thus be lysine or arginine or a residue with a free amino (-NH2) group.

Methods of synthesis: biological activity In general the methods of synthesis described in published U.K. specification No. 2 000 783 are applicable in the present invention and the disclosure of that specification is incorporated herein by reference. The biological activities and methods of testing them are further in general those described therein.

Table ofexamples The specific synthesis of a number of compounds is given in the Examples hereafter, which Examples are summarised in a table accompanying this specification as Appendix i. The table sets out the relation of the compounds to Met5-enkephalin and gives the activity of the compounds in a test method essentially that of Bradbury et al. Nature 260 793-795 (1976), an optiate binding assay.

Activity test A hypotonically lysed (1 OmM Tris-HCI, pH 7.4), extensively washed crude synaptosome preparation was made from whole supratentorial rat brain. The membranes were incubated with the peptides in 0.05M Tris-HCI buffer pH 7.6 with 0.1 M NaCI, 0.1% bovine serum albumin and 0.1% bacitracin containing tritiated naloxone (New England Nuclear, 20 Ci mmole-1,1 x 10-9M).After 20 minutes incubation at 25 the suspension was centrifuged (15,0009, 1.5 minutes), the pellets rapidly and superficially washed with 0.1 m NaCI in 0.05M Tris-HCI buffer pH 7.6, the pellets resuspended in water (0.5ml) and solubilised with scintillator (6g/litre of PPO in toluene containing 20% (v/v Triton X 100), counted at 30% efficiency to determine the bound counts. Specific binding was defined as that fraction of the bound radioactivity displaced by morphine (10-5M). Incubations were carried out in triplicate, in at least two separate experiments. Standards of naloxone, Met5-enkephalin and Nle5-enkephalin were used.

Activity in the above test procedure is pharmacologically significant whether higher or lower than naturally occurring Met5-enkephalin. The structure of the compounds of the invention renders them more resistant than enkephalin itself to enzymic degradation in the body and therefore of longer lasting effect.

Natural enkephalin is very highly potent, and thus weaker as well as stronger effects are of value. Further, compounds with a low opiate activity but possessing other activities (e.g. prolactin or growth hormone releasing activity) are also of value. Both prolonged action and a lack of addictive properties may be shown and, generally, the activities of the compounds of the present invention are as discussed in general terms in specification No. 2 000 783 for the compounds disclosed there.

In relation to the structures it will be noted for example that in H224-H227 the Phe4-Met5 peptide bond is replaced by a methylene-imino isostere, whereas in H228 the Gly3-Ph4 peptide bond is altered in the same way. The relation of the compounds to Met5-enkephalin is shown in the tables. All the compounds exhibit significant levels of activity, three of them being more potent than Met5-enkephalin used as a standard.

(Nle5-enkephalin with which compound H228 was compared is itself more active in this test system than Met5-enkephalin,the natural material.) Examples The following examples illustrate the invention.

Example 7 H224 (1) Boc-Phe-Met-OMe Boc-Phe-OH (0.95g, 3.5 mmole) and HCI.H-Met-OMe (0.719,3.6 mmol) were coupled together at 0 in CH2CI2 in the presence of DCCI (0.819,3.9 mmol) and NEt3 (0.5 ml, 3.6 mmole). After 1 day the reaction mixture was worked up by normal acid-base wash procedures.Crystallisation from ether-40 -60 petrol gave 1 .08g (76%) fine needles, m.p. 70-71 . t.l.c. (silica): Rf 0.83 chloroform/methanol (95:5); Rf 0.56 benzene/ dioxan/acetic acid (95:25:4); NMR spectrum in accord T (CDCl3) 2.7 (5H multiplet, ArH) 3,4(1 H, 4, d, J=8Hz, D2O exchangeable, amide NH), 4.9 (1 H, d, J=7Hz, D20 exchangeable, urethane NH), 5.5 (2H, complex, 2x -CH),6,3 (3H, s, -OCH3), 6.95 (2H, d, J=6Hz, Phe (3-CH2), 7.8 (4H, complex, -CH2-CH2-), 7.95 (3H, S, S-CH3), 8.55 (9H, S, Boc Bu).

The dipeptide (1) (0.7g. 1.7 mmol) in dry benzene (10 ml) was treated with 70% SDA solution in toluene (4ml) at room temperature, then refluxed for 45 minutes. The mixture was cooled to 0 and added to ice cold 10% citric acid (50ml), and then the whole mixture neutralised to pH 8 and extracted into ethyl acetate (3X).

The combined washed organic phases were evaporated and the residue chromatographed on a 30 x 2.5cm column of SP Sephadex C25 (Pyrform). The column was run in MeOH/nBuOH/H20 (13:10:7) and eluted with 100 l(of this system then 100 ml ml each of 20% pyridine and finally 1M NEt3, Evaporation of the triethylamine wash gave an oily residue which was crystallised from methanol/water to give 0.369 (57%) of white needles; t.l.c. (silica) Rf 0.1 CHCI3/MeOH (95:5), Rf 0.05 benzene/dioxan/acetic acid (95:25:4). Nmr: T (CDCI3) 2.75 (5H, multiplet, ArH), 5.25 (1 H, d, J=8Hz, D2O exchangeable, urethane NH), 6.2 (2H, complex, 2xNH-CHCH2), 7.9 (3H, S, SCH3), 7.0 - 8.5 (11 H, complex 5 x CH2 + NH).

The reduced isostere (0.18g, 0.49 mmol) and KHCO3 (0.5g, 5 mmol) were stirred at r.t. in peroxide-free dioxan (5 ml) and water (5ml). Benzyl chloroformate (250mg, 1.5 mmol) was added and the mixture stirred overnight. Unsymmetrical dimethylethylenediamine (0.16ml, 1.5 mmol) was added and the mixture stirred for a further 6 hours. The solution was evaporated and partitioned between ethyl acetate and cold 10% citric acid. The organic layer was washed twice more with citric acid solution, once with water, twice with saturated sodium bicarbonate solution, dried and evaporated to give 270mg (100%) of a colourless oil; t.l.c.

(silica) Rf 0.71 chloroform/methanol (95:5); Rf 0.49 benzene/dioxan/acetic acid (95:25:4); Nmr: T (CDCl3), 2.5 (5H, s, C6H5CH2-O-CO-), 2.6 (5H, multiplet, Phe, ArH), 4.7 (2H, s, C6H5CH2-OCO-), 5.25 (1 H, d, J=7Hz, D20 exchangeable, urethane NH), 6.2 (2H, complex, 2 x NHCHCH2, 7.9 (3H, s, SCH3), 7.0-8.5 (11 H, complex, 5 x CH2, + NH)

The total product from (3) (0.54 mmol) was dissolved in 80% aqueous trifluoroacetic acid (30 ml) under N2.

After 30 minutes the solvents were evaporated and the residue dried in vacua. This was dissolved in ethyl acetate and the solution washed twice with 1 M sodium bicarbonate, once with sat. brine, dried over MgSO4 and evaporated to give the free base of the isostere as an oil, 211 mg (98%), t.l.c. (silica) Rf 0.63 n-butanol/acetic acid/H2O (3:1:1).

Approximately one half of this product (94mg, 0.23 mmol) in DMF (1 ml) was cooled to 0 and added to a preactivated mixture at 0 of Boc-Tyr-DAla-Gly-OH (94mg, 0.23 mmol), HOBt (70mg, 0.46 mmol) and DCCI (0.3 mmol in DMF (1.5ml). The reaction was allowed to warm to room temperature and stirred for two days.

After filtration, the mixture was applied to a column (95 x 2.5cm) of Sephadex LH20 and eluted with DMF at 15 ml/hourcollecting 190 drop (6 ml)fractions. Fractions 35-39 were pooled and evaporated to give 130 mg of product as a colourless glass; t.l.c, (silica) Rf 0.62 chloroform/methanol/acetic acid (85:10:5).

The total amount of fully protected peptide was dissolved in 80% aq. trifluoroacetic acid (25 ml) under N2.

After 30 minutes, the solvents were evaporated and the residue dried in vacuo. Most of this product was used in the synthesis of H225 (see below). One fifth of the material was applied to a column (92 x 1.6 cm) of Sephadex G25 SF in 50% acetic acid run at 10 ml/hr collecting 130 drop (4 ml) fractions. Fractions 24-29 were pooled and chromatographed on a 40 x 1cm column of Whatman CM 52 run at 10 ml/ hour collecting 100 drop (6ml) fractions with a linear gradient over 2 days from 0.01 M NH4OAC pH 7 to 0.2 M NH40Ac pH 7.

Fractions 37-40 were pooled and lyophilised, then relyophilised from 0.05M HCl to give 17 mg of fluffy peptide; t.l.c. (silica) Rf 0.80 EtOAc/n-BuOH/AcOH/H20 (1:1:1:1), Rf 0.85 EtOAc/Pyr/AcOH/H2O (50:20;6:11) ; electrophoresis (cellulose):- pH 6.5, 1000 V, 18mA, 30 minutes, mobility 3.6; pH 2.1, 1000 V, 12 mA, 30 minutes, mobility 3.2cm; amino acid analysis (after hydrolysis with 6N HCI + Phenol, 110 , 18 hours) Tyr 0.95, Gly 1.03, AIA 1.02, peptide content 75%.

Example 2 H225 The remaining crude sample of H224from above and methionine (250mg) were treated with anhydrous liquid HF (1 Oml) in the presence of anisole (1 ml) at 0 for 30 minutes. The solvents were carefully evaporated and the residue dried extensively over KOH in vacuo. The residue was dissolved in deaerated 50% aq. acetic acid (2ml), filtered, and chromatographed in this system on a column (93 x 2.3 cm) of Sephadex G25 SF run at 12 ml/hour collecting 190 drop (6ml) fractions.Fractions 56 - 59 were pooled and evaporated and the residue lyophilised twice from deaerated 0.05M HCI to give 45.4mg of white fluffy compound; t.l.c. (silica) Rf 0.66 Et OAc/n-BuOH/AcOH/H2O) (1:1:1:1); Rf 0.70 EtOAc/Pyr/AcOH/H20 (50:20:6:11); electrophoresis (cellulose):pH 6.5, 1000 V, 18mA, 30 minutes, mobility 6.2 cm; pH 2.1, 1000 V, 12mA, 30 minutes, mobility 5.2cm; amino acid analysis (after hydrolysis with 6NCHI + Phenol) Tyr 0.98, Gly 1.01, Ala 1.01, Peptide content 80%.

Example 3 H226 The free base of the isostere (4) from the preparation of H-224 (1 17mg, 0.29 mmol) in DMF (1 ml) at 0" was added to a mixture of Boc-Tyr-Gly-Gly-OH (120 mg, 0.29 mmol) and HOBt (88mg, 0.58 mmol) in DMF (1 ml) at 00to which two minutes earlier had been added DCCI (0.4 mmol). After two days the reaction mixture was filtered and applied directly to Sephadex LH20 in DMF eluted at 12 ml/hr collecting 190 drop (6ml)fractions.

Fractions 36-39 gave 181 mg of a colourless glass; t.l.c. (silica) Rf 0.58 in chloroform/methanol/acetic acid (85:10:5).

The total product was dissolved in 80% TFA (25ml) under N2. After 30 minutes the solvent was evaporated and the residue dried. One half of the product was applied to a 93 x 2.5cm column of Sephadex G25 SF in 50% acetic acid eluted at 1 5m1/hr collecting 190 drop (6ml) fractions. Fractions 51 - 55 were pooled, evaporated and lyophilised twice from 0.1M HCI to give 56.7mg of white fluffy peptide; t.l.c. (silica) Rf 0.87 EtOAc/n-BuOH/AcOH/H2O (1 :1:1:1); Rf 0.75 EtOAc/Pyr/AcOH/H2O (60:20:6:11); electrophoresis (cellulose): pH 6.5, 1000 V, 18mA, mobility 3.0cm; pH 2.1, 1000V, 12mA, mobility 3.1 cm . Amino acid analysis (after hydrolysis in 6NCHI + Phenol at 110 C, 18 hours) Tyr 0.98, Gly 2.01 (Peptide content 92%).

Example 4 H227 The remaining crude sample of H226 was deprotected at 00for30 minutes with liquid HF (10 ml) in the presence of methionine (250mg) and anisole (1 ml). The peptide was isolated as described for H225; from Sephadex G25 SF chromatography, fractions 50 - 56 gave 48 mg of white fluffy peptide; t.l.c. (silica) Rf 0.72 EtOAc/n-BuOH/AcOH/H2O (1:1:1:1) : Rr0.31 EtoAc/Pyr/AcOH/H2O (60:20:6:11) ; electrophoresis (cellulose) pH 6.5, 1000 V, 18mA, 30 minutes, mobility 6.1 cm; pH 2.1, 1000 V, 12mA, 30 minutes, mobility 5.2cm; amino acid analysis (after hydrolysis with 6NHCI + Phenol, 110", 18 hours) Tyr 1.02 Gly 1.98. Peptide content 84%.

Example 5 H229

(1) Boc-NH(CH2)2Br [1.62g, 7.25mmol ; prep. in 66% yield by reaction of Boc-azide (1.6g, 11.2 mmol) and 2-bromoethylamine hydrochloride (3.079, 15 mmol) in DMF (150ml) in the presence of NEt3 (3.6 ml, 26 mmol)], phenylalanine ethyl ester hydrochloride (3.36g, 14.5 mmol) and triethylamine (3.0 ml, 21.75 mmol) in dry DMSO (12 ml) were stirred vigorously at r.t. for 9 days. The mixture was partitioned between ethyl acetate and water, and the organic layer washed with 1 M NaHCO3 (6 X). The ethyl acetate layer was dried and evaporated, the residue dissolved in methanol and chromatographed on a column of Sephadex LH20 (90 x 2.5 cm), eluted with methanol at 1 Oml/hr collecting 5ml fractions. Fractions 88 - 92 were combined to give the substitution product, 0.30g as a colourless oil; t.l.c. (silica) Rf 0.58 n-butanol/acetic acid/H20 (3:1:1).

Nmr: r (CDCl3) 2.7 (5H multiplet, ArH), 5.0 (1 H, broad, D2O exchangeable, -O-CO-NH-), 4.9 (2H, quartet, J=Hz OCH2-CH3), 6.7 - 7.5 (7H, complex, 3 x CH2 + CH), 8.35 (1 H, multiplet, D20 exchangeable, -CH2NH-), 8.6 (9H, s, tBu), 8.9 (3H, triplet, J=7Hz,CH2CH3).

(2) The reduced isostere from (1) above was reacted overnight with benzyl chloroformate (0.19 ml) in dioxan (semi) and 1 M KHCO3 solution (5ml). Unsym. dimethylethylenediamine (0.1 ml) was added and after 1 day the reaction mixture partitioned between water (after the pH was adjusted to 3.0 by the addition of citric acid) and ethyl acetate. The aqueous phase was extracted twice more with ethyl acetate and the combined organic layers washed with water (2X) and brine, then dried over an hydros MgSO4 and evaporated to give 0.38g of a colourless oil; t.l.c. (silica): Rf 0.72 n-butanol/acetic acid/H20 (3:1 :1); NMR shows additional peaks to above spectrum: T (CDCI3) 2.5 (5H, s, ArH), 3.75 (2H, s, -CH2-O-), with loss of 8.35 peak.

(3) The ester from (2) was dissolved in methanol (5ml) and 0.2M NaOH (5ml) added slowly to the stirred solution. After 18 hours, water (20 ml) was added and the solution extracted with ethyl acetate (2 x 10 ml).

The aqueous phase was acidified to pH 3.0 with citric acid and extracted with ethyl acetate (3 x 10ml). The combined organic layers were washed with water (2 x 10ml), brine (1 x 10 ml), and dried over MgSO4.

Evaporation gave the product (A) (0.259) as a colourless gum; t.l.c. (silica) Rf 0.47 n-butanol/acetic acid/H2O (3:1:1); Nmr: T (CDCI3) 0.0 (1 H, s, D20 exchangeable, -CO2H), 2.5 (5H, s, ArH), 2.7 (5H, multiplet, Arll), 4.7 - 4.8 (3H, complex, 1 H D20 exchangeable PhCH2-O- + NH) 5.85 (1 H, broad, CH), 6.5-7.2 (6H, complex, 3 x CH2), 8.5 (9H, s, tBu).

(B) Boc-Methionine was coupled to phenolic resin (0.5g) as usual. After standard acetylation, TFA deprotection and base wash cycles; (A) from above (145mg, 0.3 mmol) in 1:1 DMF/CH2CI2 (6ml) was activated with DCCI (0.45 mmol) and HOBt (92mg, 0.6 mmol) and added to the resin. After 4- hours, the resin was thoroughly washed, the base wash cycle repeated and the resin treated with acetyl imidazole overnight.

After thorough washing, the resin was dried (0.9g; > 100% increase in weight; still containing DCU).

One half of the resin (0.459, = 0.15 mmol) was replaced in the synthesis vessel, and deprotected with 33% TFA in CH2CI2 containing 2% diethyl phosphite and 2% ethane dithiol (1 minute and 30 minutes). After base wash, Boc-DAla-OH (0.115g, 0.6 mmol) was coupled in 1:1 DMF/CH2Cl2 (4mI) in the presence of HOBt (0.16g, 1.2 mmol) with DCCI (0.9 mmol). After 2B hours, the resin was thoroughly washed; TFA and base wash cycles were repeated, and Boc-Tyr-OH (0.179, 0.6 mmol) was coupled as previously. After thorough washing, the resin was suspended in 1:1 DMF/MeOH (30 ml) and saturated with anhydrous ammonia. The flask was tightly stoppered and the suspension stirred gently for 2 days. The resin beads were filtered and washed thoroughly with DMF.The combined filtrates were evaporated and chromatographed on Sephadex LH 20 (92 x 2.5 cms) in DMF at 18 ml/hour collecting 190 drop (6ml) fractions. Fractions 35 - 39 were pooled and evaporated to give 1 10mug of a glassy residue; t.l.c. (silica) showed a main component in CHCI3/MeOH/AcOH (85:10:5) with Rf 0.53; and a minor component with Rf 0.70. The total product was dissolved in 80% aqueous TFA (25ml) under N2. After 30 minutes the solvents were evaporated and the residue dried in vacuo over NaOH pellets. This was dissolved in methanol (5ml) and a sample (1 ml) removed, evaporated and used in the preparation of H228.The remaining material was thoroughly dried, then treated for 30 minutes at 0" with anhydrous liquid hydrogen fluoride (10 ml) in the presence of methionine (250mg) and anisole (1 ml). The solvents were evaporated and the residue carefully dried, then chromatographed on Sephadex G25 SF (92 x 2.5 cm) in 50% acetic acid at 18ml/hr collecting 190 drop (6ml) fractions. Fractions 53 - 57 were pooled and evaporated to give the hydantoin derivative (50.0mg) as a colourless glass; this was lyophilised several times from 0.05M HCI to give a white fluffy powder; t.l.c. (silica) Rf 0.72 in EtOAc/n-BuOH/AcOH/H20 (1:1:1:1);Rf 0.78 in EtOAc/Pyr/AcOH/H2O (50:20:6:11); electrophoresis (cellulose) pH 6.5, 1000 V, 18mA, 30 minutes, mobility 4.0 cm; pH 2.1, 1000 V, 1 2mA, 30 minutes, mobility 3.3cm; Amino acid analysis Tyr 1.00, Ala 1.00; Met 0.22.

Example 6 H228 The sample of peptide removed from the preparation of H-229 (see above) was dissolved in 50% deaerated acetic acid and applied to a 93 x 1.6cm column of Sephadex G25 SF and eluted at 10 ml/hr collecting 130 drop (4my) fractions. The desired Z'-protected derivative was not completely separated on this column and so fractions 27 - 31 containing both products were pooled and evaporated. The residue was dissolved in 0.01 M ammonium acetate (pH 7) and applied to a 35 x 1.5cm column of Whatman CM 52 which was eluted at 15 ml/hour with a linear gradient over two days from 0.01 M to 0.5M NH40Ac (pH 7) collecting 100 drop fractions. Again inadequate resolution was obtained; fractions 41 - 47 were pooled and lyophilised.The residue was chromatographed on Sephadex G25 SF (2.5 x 93 cm) in 50% deaerated acetic acid at 15 ml/hr collecting 190 drop (6ml) fractions. Fractions 49 - 51 gave 0.6mg of the desired product, whereas fractions 52 -55 gave 9.5mg of H229; t.l.c. (silica): Rf 0.81 EtOAc/Pyr/AcOH/H20 (60:20:6:11); Rf 0.91 in EtOAc/n-BuOH/ AcOH/H20 (1:1:1:1); electrophoresis (cellulose) pH 6.5, 1000V, 15 mA, 30 minutes, mobility 4.0cm.

Example 7 H230 Preparation similar to H229. Boc-glycine (0.1 1g 0.6 mmol) was coupled to remaining half of isostere-Met resin. After completion of the synthesis, the peptide resin was transamidated and the product chromatographed on Sephadex LH20 in DMF as before: fractions 36 - 39 gave 118mg; t.l.c. in chloroform/methanol/ acetic acid (85:10:5) Rf 0.45 (single spot). The total peptide was dissolved in 80% TFA (25ml) under nitrogen.

After ten minutes a sample (5ml) was removed and evaporated; after3 hours the remaining solution was evaporated. T.l.c. showed the two samples to be identical. The main batch was chromatographed on q 94 x 2.5cm column of Sephadex G25 SF in 50% acetic acid and eluted at 18ml/hr collecting 190 drop (6ml) fractions. Fractions 64-69 were pooled, evaporated and the residue dried. This was treated with liquid HF as before, again with no change, and was chromatographed on Sephadex G25 SF (93 x 2.5 cm column) in 50% acetic acid.Fractions 54 - 58 were pooled, evaporated and repeatedly lyophilised from 0.05M HCI to give 48mg of white solid; t.l.c. (silica) Rf 0.61 in ethyl acetate/n-butanol/acetic acid/water (1:1:1:1); Rf 0.50 in ethyl acetate/pyridine/acetic acid/water (60:70:6:11); electrophoresis (cellulose) pH 6.5, 1000 V, 15 mA, 30 minutes, mobility 4.0 cm; pH 2.1, 1000 V, 12 mA, 30 minutes, mobility 3.3 cm. Amino acid analysis: Tyr 0.99, Gly 1.01, Met 0.05.

Example 8 H232

(46mg, 0.13 mmol), L-methioninol (27 mg, 0.2 mmol) and HOBt (40mg, 0.26 mmol) were dissolved in DMF (1 ml) and the stirred solution cooled to 0". A solution of DCCI in methylene chloride (0.1 ml, 0.19 mmol) was added and the reaction stirred for one day, gradually warming to room temperature. The solution was filtered and the filtrate applied directly to a 92 x 2.5cm column of Sephadex LH 20, and eluted with DMF at 12ml/hr collecting 190 drop (6ml) fractions.Fractions 40 - 43 were pooled, evaporated, and the residue dried to give 48.2mg of a colourless glass; Rf (silica) 0.40 in benzene/dioxan/ acetic acid (95:25:4); Nmr: t (CDCl3) 2.6 (5H, s, ArH), 2.8 (5H, multiplet, ArH) (3H, complex, 1 H D2O exchangeable, ArCH2-O-CO- + NH), 5.8(1 H, t, α -CH), 6.4-8.3 (12H, complex, 6X - CH2-), 8.0 (3H, s, -CH3); 8.6 (9H, s, tBu).

The total product (47mg, 0.084 mmol) was dissolved in 80% TFA (20ml) under N2. After 30 minutes the solvents were evaporated and the residue partitioned between ethyl acetate and 1 M sodium bicarbonate.

The organic layer was separated and washed with 1 M sodium bicarbonate and saturated brine; each aqueous wash was back-extracted with ethyl acetate. The combined organic layers were dried and evaporated to give 35.4mg of an oily residue. This was dissolved in DMF (1 ml) and Boc-Tyr-DAla-OH (30mg, 0.11 mmol) and HOBt (33.6mg, 0.22 mmol) added. The stirred solution was cooled to0" and treated with DCCI solution (0.16 mmol). The reaction was allowed to slowly warm to room temperature. After two days, the solution was filtered and applied to a column of Sephadex LH 20 in DMF as described previously.

Fractions 36 - 39 were pooled and evaporated to give 59mg of a glassy residue; Nmrt (d4MeOH) 2.7 (5H, s, ArH), 2.9 (5H, broad ArH), TA 3.02 T5 3.25 (4H, A2B2, J=9Hz, 2 x 2 ortho ArH), 4.9 (2H, s, ArCH2-O-CO-), 5.7 - 6.2 (4H, complex, 4x α -CH), 6.4 - 8.3 (14H, complex, 7x-CH2-1.8.0 (3H, s, S-CH3), 8.6 (9H, s, tBu), 8.95 (3H, d, J=7Hz, =CH-CH3), The total product was dissolved in 80% TFA (20ml) under N2. After 30 minutes, the solvents were evaporated and the residue dried. Approximately 40% of this material was chromatographed on Sephadex G25 SF in 50% acetic acid as before.Fractions 48 - 53 gave 20.9mg of the desired Z-protected derivative which was lyophilised several times from 0.1 MHCI to give the hydrochloride as a white solid. The NMR (d4MeOH) spectrum was very similar to that of the protected precursor but lacking the 9H, s, tBu peak at 8.6; t.l.c. (silica): Rf 0.86 in ethyl acetate/n-butanol/acetic acid/H2O (1:1:1:1); Rf 0.98 in ethyl acetate/Pyr/acetic acid/H2O (60:20:6:11); electrophoresis (cellulose): pH 6.5, 1000V,30 minutes, mobility 5.1 cm; pH 2.1, 18mA, 1000 V, 30 minutes, mobility 4.5 cm, Amino acid analysis: Tyr 0.98, Ala 1.02.

Example 9 The remaining 60% of crude H232 was deprotected as usual with HF at 0", and chromatography on Sephadex G25 SF gave, after lyophilisation from 0.1M HC1, 18.6mg of white solid; NMR (d4MeOH) similar to that of H232 but lacking the t 2.7 (5H, s, ArH) and 4.9 (2H, s, ArCH2-O-CO-) peaks; t.l.c. (silica) Rf 0.71 in ethyl acetate/n-butanol/acetic acid/H20 (1:1:1:1); Rf 0.73 in ethyl acetate/pyridine/acetic acid/H20 (60:20:6:11); electrophoresis (cellulose) pH 6.5, 25mA, 1000 V, 30 minutes, mobility 7.3cm; pH 2.1, 18mA, 1000 V, 30 minutes, mobility 7.5cm; amino acid analysis: Tyr 0.99, Ala 1.01.

Analogue 11236 Example 10 Structure

Synthesis a) Boc-Tyr-NHCH2COCH2CH2CO-Phe-Metol Boc-Tyrosine was coupled to Cl NH3CH2COCH2CH2CO2Me (prepared by esterification of either b-aminoacid described in scheme 2 or deprotected keto-acid VI in scheme 1) in DMF using DCCI in the presence of 1-hydroxy-benzotriazole (HOBT).After ester hydrolysis a solution of Boc-Tyr-NH-CH2COCH2CH2CO2H (55mg, 0.12 mmol) in DMF (1.5ml) was treated at 0"C with HOBT.H2O (46mg, 0.30 mmol) in DCCI (0.20 mmol in 0.11 ml DMF). CI H2-Phe-Met-OI (prepared from Boc-Phe-Met-OMe by sodium borohydride reduction, removal of the N-protecting group using 80% aqueous TFA under N2, and lyophilisation from dilute HCI) (45mg, 0.14 mmol) was added followed by NEt3 (20yl 0.14 mmol). The mixture was stirred at 25"C for 65 hours and evaporated. The residue was dissolved in EtoAc and washed with KHSO4 solution, H20, NaHCO3 and brine, dried and evaporated.Trituration with EtoAc afforded Boc-Tyr-NHCH2COCH2CH2-Phe-Met-ol (53mg):tic (silica) Rf 0.46 in chloroform/methanol/acetic acid (85:10:5).

b) Boc-Tyr-NHCH2CH(OH)CH2CH2CO-Phe-Metol, H236 A solution of Boc-Tyr-NHCH2COCH2CH2CO-Phe-Met-ol (30mg) in MeOH (20ml) and H20 (7ml) was treated with sodium borohydride (60mg). After 1.0 hour at 25"C the solvents were evaporated and the residue treated with 80% aqueous TFA under N2 for 0.5 hour. Evaporation and chromatography on Sephadex G25-SF in 50% acetic acid gave, after lyophilisation from dilute HCI, H236 (19.0mg) :tic (silica): Rf 0.50 in n-propanol/water (7:3); Rf 0.60 in ethylacetate/pyridine/acetic acid/water (60:20:6:11). Electrophoretically homogeneous at pH 2.1 and 6.5. Amino-acid analysis:Tyr 0.98, Phe 1.02 (methionine absent).

Analogues H237 and H238, Examples 11 and 12 Structures

A suspension of Boc-Tyr-NHCH2CH2N(Z')CH2COPheMet-phenyl ester resin (described in the preparation of H218, H219 and H220) (0.209) in 1:1 DMF-MeOH (20ml) was saturated at 0" with dry NH3 and kept at 25"C for 2 days. Evaporation, deprotection with 80% aqueous TFA under N2, followed by chromatography on SP Sephadex C25 (30% acetic acid, 0.01 - 1.00M NaCI) and desalting on Sephadex G25 SF in 50% acetic acid gave after lyophilisation from dilute HCI, (i) H237 (23.6mg): tic (silica) Rf 0.87 in ethylacetate/pyridine/acetic acid/water (60:20:6:11), Rf 0.78 in ethyl acetate/n-butanol/acetic acid/water (1:1:1:1). Electrophoretically homogeneous at pH 2.1 and 6.5.Aminoacid analysis Tyr 1.00, Phe 1.03, Met 0.97; and (ii) H238 (1.4mg):tle (silica) Rf 0.76 in ethyl acetate/pyridine;acetic acid/water(60:20:6: 11), Rf 0.76 in ethylacetate/n-butanol/acetic acidiwater (1:1:1:1). Electrophoretically homogeneous at pH 2.1 and 6.5.

Amino-acid analysis Tyr 1.10, Phe 0.28 (caused by acid stability of hydantoin), Met 0.90.

Analogue H239, Example 13 Structure

Synthesis A suspension of

ester resin (described in the preparation of H229) (0.57g) in 1:1 DMF-dimethylaminoethanol (30 mls) was stirred gently at 25"C for 24 hours. The dimethylaminoethyl ester was hydrolysed overnight at pH 9.7 in 1:1 DMF-H2O. Deprotection with 80% aqueous TFA under N2 followed by chromatography on Sepha-dex G25 SF in 50% acetic acid gave, after lyophilisation from dilute HCI,H239 (75.0mg) :tlc (silica) Rf 0.97 in ethyl acetate/pyridine/acetic acid/water (60:20:6:11), Rf 0.77 in ethyl acetate/n-butanol/acetic acid/water (1:1:1:1). Electrophoretically homogeneous at pH 2.1 and 6.5. Amino-acid analysis Tyr 0.99, Ala 1.01, Met. 0.88.

Analogue H-240 - Example 14 Structure:

Synthesis: (a) Formylation of the isostere ester.

(187mg. 0.56 mmol, prepared as described under H-229) was dissolved in THF and treated at -10"C with formic-pivalic mixed anhydride (obtained by reacting formic acid with pivaloyl chloride in THF in the presence of triethylamine). Unchanged starting material was removed by partitioning the crude product between ethyl acetate and 10% w/v aqueous citric acid solution. Evaporation of the ethyl acetate solution gave the formyl derivative as a colourless gum, 124.7 mg (60%). TLC (silica) in benzene-dioxan-acetic acid (95:25:4 v/v) Rf 0.43. NMR spectrum in accord with structure.

(b) Saponification of the isostere ester. The above formyl compound (124.7 mg., ca 0.34 mmol) was hydrolysed with 0.2M-NaOH solution (2ml) in methanol (4ml) for 105 mins. After partial evaporation in vacuo, the reaction mixture was partitioned between cold 10% w/v aqueous citric acid solution and ethyl acetate. The free acid was extracted from the ethyl acetate phase with 3% w/v aqueous ammonia, the ammonia solution was acidified with citric acid and the product extracted into ethyl acetate. Drying and evaporation of the ethyl acetate solution gave the formylated isostere acid as a colourless gum, 73 mg (65%).

TLC (silica) in benzene-dioxan-acetic acid (95:25:4, v/v) gave Rf 0.12, in chloroform-methanol-acetic acid (85:10:5, v/v) Rf 0.80. NMR spectrum in accord with structure.

(c) Incorporation of the isostere. The product from (b) was coupled to methionine resin and the synthesis completed as forth corresponding benzloxycarbonyl derivative H-229.

(d) Removal from the resin. One half of the completed peptide resin (0.42 g) was transesterified for 24 hours with N,N-dimethylaminoethanol (10 ml) in DMF (10 ml). After filtration and evaporation of the DMF solution, the resulting ester was dissolved in DMF-water (1:1, v/v) and hydrolysed at pH 9.7 with 0.2M NaOH solution for 16 hours. After acidification to pH 3 the resulting peptide acid was dissolved in DMF (2 ml) and chromatographed in DMF on Sephadex LH-20 (2.5 x 90 cm). The fractions containing the product were pooled and evaporated.

(e) Deprotection. The product from (d) was dissolved in trifluoroacetic acid (20 ml) and dimethylsulphide (1 ml) under nitrogen. After 30 mins. the solution was evaporated and the residue chromatographed on Sephadex G-25-8F in 50% (v/v) acetic acid. The fractions containing the product were combined and evaporated. The residue was redissolved in 0.05M HCI and lyophilised to give H-240 (18.2 mg) as a white flocculous solid. TLC (silica) in n-propanol-water (7:3, v/v) Rf 0.67; in ethyl acetate-n-butanol-acetic acid-water (1:1:1:1, v/v) Rf 0.69; in ethylacetate-pyridine-acetic acid-water (60:20:6:11, v/v) Rf 0.61.

Electrophoresis (on cellulose) at pH 2.1, 1000 V, 15 mA, 30 mins.: mobility 2.8 cm: at pH 6.5, 1000 V, 25 mA, 30 min., mobility 4.8 cm. NMR in accord with structure. Amino acid analysis (after hydrolysis with (6N-HCl + phenol, 110", 18 hrs) Tyr, 1.04; Ala, 1.01; Met. 0.95. Peptide content 80%.

Analogue 11-241-Example 15 Structure:

Synthesis: The remaining half of the peptide resin from H-240/section (c) above was dissolved in DMF-methanol (1:1, viv,20 ml) and the solution was saturated with ammonia at 0". After stirring at 22" for 18 hrs., the resin was filtered off, the filtrate evaporated and the residue chromatographed on Sephadex LH-20 in DMF. Fractions 35-38 were pooled and evaporated and the colourless glassy residue dissolved in anhydrous trifluoroacetic acid (20 ml) containing dimethylsulphide (1 ml) under N2. After 30 mins the solvents were evaporated and the residue chromatographed on Sephadex G25 SF in 50% (v/v) acetic acid as previously described.Fractions 35-38 were pooled and evaporated and the residue (71.1 mg) further chromatographed, as previously, on SP Sephadex C25 in 30% (v/v) acetic acid with a gradient from 0.01 M to 1M sodium chloride. Fractions 41-45 were pooled, evaporated and the residue extracted several times into glacial acetic acid. These extracts were pooled and evaporated and the residue lyophilised from 0.1 M hydrochloric acid to give H-241, 42.4mg as a white solid; t.l.c. (silica): n-propanol/water (7:3 v/v) Rf 0.62; ethyl acetate/n-butanol/acetic acid/water (1:1:1:1 v/v) Rf 0.65; electrophoresis (cellulose):- pH 2.1,1000 V, 15mA, 30 mins mobility 3.2 cm; pH 6.5, 1000 V, 25mA, 30 min., mobility 6.6 cm; NMR in accord with structure; amino acid analysis (hydrolysis in 6M hydrochloric acid + phenol 110", 18 h) Tyr 1.01, Ala 1.03, Met 0.95, peptide content 75%.

Claims (19)

1. Compounds corresponding in structure to enkephalin or polypeptide analogues thereof wherein an adjacent pair of peptide bonds is modified by linking the nitrogen atoms of the bonds through a carbonyl or thiocarbonyl group, forming a five membered hydantoin orthiohydantoin ring.

2. Compounds according to claim 1 wherein, in the ring structure

R" and Run+1 being the side chains of adjacent amino acid residues: i) R" is the side chain of Gly, aza-Gly, aza-Ala or any D-amino acid residue particularly D-AIa, D-Thr, D-Ser or D-Met, and Run+1 is the side chain of Gly; or ii) R" is the side chain of Gly and Rn is the side chain of Phe, N-substituted (particularly N-methyl)-Phe or dehydro-Phe all optionally substituted in the ring by hydroxy, halo, nitro or other groups, or alternatively cyclohexylalanine.

iii) R" is the side chain of Phe, N-substituted (particularly N-methyl)-Phe or dehydro-Phe all optionally substituted in the ring by hydroxy, halo, nitro or other groups, or alternatively cyclohexylalanine, and Run+1 is any D- or L-amino acid residue particularly Leu, Nle, Met or the sulphoxide of Met all as such or in aza form; or homoserine lactone; or formal derivatives of any of these in which the terminal

group is replaced by -CH2-Z (Z is -N H2, -NHA, -N(A)2, -OH or -OA where A is an aliphatic or other protective group as defined in claim 3) or by hydrogen.

3. Compounds of the general formula: A-Tyr-X-Gly-B-Y-Z where a) the peptide link between one or more of the residues is represented by a group or groups the same or different selected from protected methylene-imino -CH2-NP- and protected hydroxymethylene -CH(OA)-CH2groups, optionally in conjunction with one or more other links represented by methylene imino -CH2-NH- or -CH2-NR-, keto methylene

(as such or in the form of hydroxydimethylene -CH(OH)-CH2-), or dimethylene -CH2-CH2- groups, - P being oxygen or an organic group, selected from aryl groups such as phenyl, benzyl or the like, or simple halogenated (in particular fluorinated) derivatives thereof; formyl, acetyl or other acyl groups or simple halosubstituted derivatives thereof such as chloroacetyl or trifluoroacetyl; or substituted derivatives such as

where R1 may be the same as R or phenyl or benzyl or ring substituted phenyl or benzyl derivatives, tertiary butyl or substituted derivatives such as phenylisopropyl, diphenylisopropyl, orfluoreny Imethylene or simple chemical modifications of these alternatives.

- R being a methyl, ethyl, n-or iso-propyl, cyclopropyl, n-,iso-, sec- ortert-butyl, allyl or like group, and - A being the same as R or selected from the above organic groups given for P b) -X- is Gly, or aza-Gly or aza-Ala, or any D-amino acid particularly D-AIa, D-Thr, D-Ser or D-Met; -B- is Phe or N-substituted particularly N-methyl-Phe or dehydro-Phe, as such or substituted with hydroxy, halogen nitro or other groups in the aromatic ring, or alternatively cyclohexylalanine;; -Y- is any D- or L-amino acid residue, particularly Leu, Nie and Met (the most preferred) or the sulphoxide of Met, or aza derivatives of any of these, or Pro or Hypro, or homoserine lactone, or formal derivatives of any of these in which the terminal

group is replaced by -CHrZ or a hydrogen atom -Z- is -NH2, -NHA, -N(A)2, -OH or-OA or, a') an adjacent pair of peptide bonds is modified by linking the nitrogen atoms of the bonds through a carbonyl orthiocarbonyl group, forming a five-membered hydantoin orthiohydantoin ring, as set out in claim 2 or otherwise, optionally in conjunction with modification of one or more other peptide links as set out at a) above b') X,B,YandZareassetoutatb)aboveor a") the peptide link between one or more of the residues is represented by a group or groups the same or different selected from methylene imino, ketomethylene as such or in the form of hydroxydimethylene, or dimethylene one one or more of the following is satisfied, namely -X- is aza-Gly or aza-Ala, D-Thr or D-Ser -B- is dehydro-Phe as such or substituted with hydroxy or halogen, or nitro-substituted Phe, N-methyl-Phe or dehydro-Phe, or alternatively cyclohexyalanine -Y- is norleucine (Nle); or Leu, Nle, Met or the sulphoxide of Met all in the form of aza derivatives; or homoserine lactone; or formal derivatives of D- or L-amino acid residues particularly Leu, Met, the sulphoxide of Met or aza derivatives of these, or Pro or Hypro, or homoserine lactone, wherein the terminal

group is replaced by a hydrogen atom; c) X, B, Y and Z are otherwise as set out at b) above or a"') the B to Y or the Gly to B peptide link is replaced by -CH2-NH- and b"') X is D-Ala or Gly in the first case and D-Ala in the second, and A is H, B is Phe, Y is Metol and Z is OH in both cases and where optionally, one or more remaining peptide links and/or any methylene imino group(s) present, is in N-substituted form carrying a group A.

4. The compound

wherein Z' is benzyloxycarbonyl

5. The compound:

6. The compound:

(Z' as claim 4)

7. The compound:

8. The compound:

(Z' as claim 4)

9. The compound:

10. The compound:

11. The compound:

12. The compound:

(Z' as claim 4)

13. The compound:

14. The compound:

(Z' as claim 4)

15. The compound:

16. The compound:

(Z' as claim 4)

17. The compound

18. The compound

19. Any compound of the preceding claims, as such or in pharmaceutically acceptable N-protected or salt form, when made up with a pharmaceutically acceptable diluent or carrier for administration.