Psychopharmacologically Active Tetra-, Penta-, Hexa-, And Heptapeptides

Abstract

The invention relates to certain psychopharmacological peptides with the formula: A--L--Glu(X)--L--His--L--Phe--L--Arg(or L--Lys)--Y in which A represents: H--l--met; H--D--Met; H--L--Met(.fwdarw.O); H--D--Met(.fwdarw.O); H--L--Met(.fwdarw.O.sub.2); H--D--Met(.fwdarw.O.sub.2); desamino-Met; desamino-Met(.fwdarw.O), desamino-Met(.fwdarw.O.sub.2) or the moiety: H.sub.2 N--B--CO; in which B is a branched or unbranched alkylene group with 1-6 carbon atoms, X represents the group OH or NH.sub.2 and Y represents the group L--Phe--OH, L--Phe--Gly--OH or a (N-phenyl-alkyl)amino group of the formula ##SPC1## In which Alk represents a branched or unbranched alkylene group with 1-6 carbon atoms and R represents hydrogen, halogen, hydroxy, a lower alkyl (1-4 C) or lower alkoxy (1-4 C) group, As well as to functional derivatives of these peptides. The above compounds inhibit the extinction of conditioned avoidance response, that means that they can be used, in general, as antidepressant agents. More particularly they can be used for the treatment of mental disorders whereby a stimulation of the mental performance is desired.

Description

The invention relates to peptides and peptide derivatives with valuable psychopharmacological activities.

From European Journal of Pharmacology 2, 14 (1967) certain peptide fragments of the natural adrenocortiocotrophic hormones (ACTH) are known to inhibit the extinction of the conditioned avoidance response. Especially the peptide having the amino acid sequence 1-10 of ACTM proved to be active in this respect. Moreover it was found that the first three amino acids (Ser--Tyr--Ser) could even entirely be omitted without much loss of activity. The article ends with the conclusion that the peptide with the amino acid sequence 4-10 of ACTH, viz. H--Met--Glu--His--Phe--Arg (or Lys)--Trp--Gly--OH, is the shortest peptide, and perhaps the key sequence for the said activity.

The peptide with the amino acid sequence 4-10 of ACTH does not only exert the psychopharmacological property mentioned above, but also a slight MSH activity, as usual in this type of ACTH fragments. Although the effect of a low dose administration of a MSH active peptide in men is still unknown, a search was made for peptides having at least the same psychopharmacological activity, but no or a reduced MSH-activity.

In our co-pending Netherlands Pat. application No. 72,02,278 and corresponding copending U.S. application Ser. No. 331,945, filed Feb 12, 1973, it is already indicated that the N-terminal amino acid L--Met of the 4-10 ACTH peptide can be replaced by the amino acid or acid moieties: D--MET, L-- or D--Met(.fwdarw.O), L-- or D--Met (.fwdarw.O.sub.2), desamino--Met, desamino--Met (.fwdarw.O), desamino-Met (.fwdarw.O.sub.2) or the group ##SPC2##

In which B represents a branched or unbranched alkylene group with 1-6 carbon atoms, such as glycyl, valyl, alanyl, .beta.-alanyl or (.alpha.--Me)--Alanyl, without any loss of activity. The modification (from L--Met) into D--Met, methionylsufoxide, methionylsulfone, desamino-methionyl, desamino-methionylsulfoxide, desamino-methionylsulfone and .beta.-alanyl even increases the said psychopharmacological activity.

Surprisingly it has now been found that a replacement of the C-terminal peptide residue "Trp--Gly--OH" of the original peptide: 4-10 ACTH by L--Phe--OH, L--Phe--Gly--OH, or a (N-phenylalkyl)amino moiety, causes a considerable increase of the inhibition of the extinction of the conditioned avoidance response, in comparison with 4-10 ACTH.

The present invention therefore comprises the manufacture and use of peptides and peptide derivatives of the general formula:

A--L--Glu(X)--L--His--L--Phe--L--Arg(or L--Lys)--Y,

in which A represents:

H--l-- or D--Met, H--L-- or D--Met(.fwdarw.O), H--L-- or D--Met(.fwdarw.O.sub.2), desamino-Met, desamino-Met(.fwdarw.O) desamino-Met (.fwdarw.O.sub.2) or the moiety: H.sub.2 N--B--Co--, in which B is a branched or unbranched alkylene group with 1-6 carbon atoms,

X represents: the group OH or NH.sub.2, and

Y represents: the group L--Phe--OH, L--Phe--Gly--OH or an (N-phenylalkyl)amino group of the formula: ##SPC3##

in which Alk represents a branched or unbranched alkylene group with 1-6 carbon atoms, and R represents hydrogen, halogen, hydroxy, or a lower alkyl or alkoxy group, the alkyl group contains 1-4 carbon atoms,

as well as the functional derivatives thereof.

By replacing the C-terminal peptide residue --Trp--Gly--OH of the original 4-10 ACTH peptide by the groupings represented by "Y" the psychopharmacological activity obtained is roughly about three times stronger. This activity can be raised further by replacing the N-terminal amino acid residue L--Met (of the original 4-10 ACTH peptide) by another suitable moiety, in particular, .beta.--Ala, D--Met, L-- or D--Met(.fwdarw.O), L-- or D--Met(.fwdarw. O.sub.2), desamino--Met or the corresponding sulfoxide or sulfone.

The peptides and peptide derivatives according to the invention are prepared by a process commonly used in peptide chemistry. The processes that are employed usually for the manufacture of the present compounds can be summarized as follows:

a. condensation of a compound (amino acid, peptide) having a free carboxyl group and protected other reactive groups, with a compound (amino acid, peptide or amine) having a free amino group and protected other reactive groups, in the presence of a condensation agent;

b. condensation of a compound (amino acid, peptide) having an activated carboxyl group and optionally protected other reactive groups, with a compound (amino acid, peptide, amine) having a free NH.sub.2 group and optionally protected other reactive groups;

c. condensation of a compound (amino acid, peptide) having a free carboxyl group and optionally protected other reactive groups, with a compound (amino acid, peptide, amine) having an activated amino group and optionally protected other reactive groups, after which the protecting groups are removed, if necessary.

Activation of the carboxyl group can be effected, for example, by converting the carboxyl group into an acid halide, an azide, anhydride, imidazolide, or an activated ester such as the N-hydroxy-succinimido ester, or the p-nitro-phenyl ester.

The amino group can be activated by converting it into a phosphite amide or by the "phosphor-azo" method.

Methods usually employed for the above condensation reactions are: the carbodiimide method, the azide method, the mixed anhydride method and the method of the activated esters as described in "The Peptides," vol. I, 1965 (Acad. Press), by E. Schroder and K. Lubke. Moreover Merrifield's so-called Solid Phase method, described in J. Am. Chem. Soc. 85, 2149 (1963), can be applied for the manufacture of the present peptides and peptide derivatives.

The reactive groups that are not allowed to participate in the condensation reaction are protected effectively by the so-called protecting groups, which can be easily removed again, for example, by hydrolysis or reduction. Thus, for example, a carboxyl group can be protected effectively by esterification with methanol, ethanol, tertiary butanol, benzylalcohol or p-nitrobenzylalcohol, or by conversion into an amide. This latter protecting group is very hard to remove, however, so that it is recommendable to use this group only to protect the carboxyl group of the C-terminal amino acid in the ultimate peptide or the .UPSILON.-carboxyl group of glutamic acid. In this case the peptide synthesis leads direct to the amide of a peptide according to formula I.

Groups that are capable of protecting an amino group effectively are usually acid groups, for example an acid group derived from an aliphatic, aromatic, araliphatic or heterocyclic carboxylic acid, such as acetic acid, benzoic acid, or pyridine-carboxylic acid, or an acid group derived from carbonic acid such as the group ethoxy-carbonyl, benzyloxy-carbonyl, t-butyloxy-carbonyl or p-methyloxy-benzyloxy-carbonyl, or an acid group derived from a sulfonic acid, such as the group benzene-sulfonyl or p-toluene-sulfonyl, but also other groups can be employed, such as substituted or unsubstituted aryl or aralkyl groups, for example benzyl and triphenylmethyl, or groups such as ortho-nitro-phenyl-sulfenyl and 2-benzoyl-1-methylvinyl.

It is mostly recommendable also to protect the guanidine group of arginine, the .epsilon.-amino group of lysine, and the imidazole group of histidine, but this protection is not absolutely necessary. Conventional protecting groups in this connection are a tertiary butyloxy-carbonyl, or a tosyl group for the .epsilon.-amino group of lysine, a nitro group for the guanidine group of arginine, and a benzyl, dinitro-phenyl or a trityl group for the imidazole group of histidine.

The protecting groups can be split off by various conventional methods, depending upon the nature of the group in question, for example with trifluoro acetic acid, or by mild reduction, for example with hydrogen and a catalyst, such as palladium, or with HBr in glacial acetic acid.

Peptides according to the present invention having as the N-terminal moiety a methionylsulfoxide or desaminomethionylsulfoxide group, may be prepared from the corresponding Met- or Desamino-Met peptide by means of a mild oxidation known per se, for example with dilute hydrogenperoxide or a peracid. Such as oxidation yields a mixture of the S-- and R-sulfoxide (=d- or 1-sulfoxide), which mixture may be split off into the separate diastereomeric compounds in a conventional manner.

By coupling the S- or R-sulfoxide (= d- or 1-sulfoxide) or methionine or desaminomethionine with the peptide H--Glu (X)--His--Phe--Arg(or Lys)--Y, in which X and Y have the meanings indicated above, the separate enantiomers can also be obtained in a direct way.

The peptides according to the invention having as the N-terminal residue a methionylsulfone (Met.fwdarw.O.sub.2) or desaminomethionylsulfone (desamino-Met.fwdarw.O.sub.2) group may be prepared most conveniently by an oxidation known per se of the corresponding Met- or Desamino-Met peptide, for example with H.sub.2 O.sub.2 or a peracid.

By functional derivatives of the peptides and peptide-derivatives according to the invention are meant:

1. the pharmaceutically acceptable acid addition salts of the peptides and peptide derivatives,

2. peptides or peptide derivatives in which one or more free amino groups have been substituted by an acyl group derived from an aliphatic carboxylic acid with 1-6 carbon atoms, such as 6 acetyl group;

3. unsubstituted amides or lower alkyl (1-6 C) substituted amides of those peptides and peptide derivatives according to the invention having a free carboxyl group, such as a N(CH.sub.3).sub.2 or N(C.sub.2 H.sub.5).sub.2 group,

4. esters of the present peptides derived from aliphatic or araliphatic alcohols with 1-18 carbon atoms; in particular, the lower aliphatic (1-6 C) alcohols, such as methanol, ethanol, butanol, pentanol or cyclohexanol, and the lower araliphatic (7-10 C) alcohols, such as benzylalcohol, phenylethylalcohol, phenylpropylalcohol, or cinnamylalcohol,

5. metal complexes formed by contacting the peptides or peptide derivatives with a sparingly soluble salt, hydroxide or oxide of a metal, preferably zinc, or preparations obtained by associating the present peptides with organic, mostly polymeric, compounds, such as gelatine, polyphloretinphosphate or polyglutamic acid, to obtain a prolonged mode of action.

The acid addition salts are obtained by reacting the present compounds with a pharmaceutically acceptable organic or inorganic acid, such as HCl, phosphoric acid, acetic acid, maleic acid, tartaric acid or citric acid.

As already briefly said the present peptides and peptide derivatives as well as their functional derivatives defined above have valuable psychopharmacological activities. The present compounds inhibit the extinction of conditioned avoidance response, that means that they can be used, in general, as antidepressant agents. More particularly they can be used for the treatment of certain mental disorders whereby a stimulation of the mental performance is desired, such as in certain types of neurosis and in old-age infirmities (senility).

The peptides according to the invention and the functional derivatives defined above can be administered orally, parenterally or intranasally. Preferably the peptides are employed as an injection preparation, for which purpose they are dissolved, suspended or emulsified in a suitable liquid, but mixed with suitable auxiliaries and fillers they can also be placed in a form suitable for oral administration, such as pills, tablets or coated tablets. The present peptides can also be administered in the form of suppositories or sprays.

The peptides or peptide derivatives according to the invention are preferably administered in daily dosages of from 0.001 to 1 mg per kg body weight, dependent upon the peptid's activity level and the form in which they are administered.

Exceedingly valuable preparations are obtained if the present peptides are placed in a form in which they have a prolonged activity, for example, incorporated into gelatin, polyphloretinphosphate or polyglutamic acid, or preferably as metal complexes. These metal complexes can be obtained by contacting the peptides with sparingly soluble metal salts, metal hydroxides or metal oxides. As sparingly soluble metal salts the metal phosphates, metal pyrophosphates and metal polyphosphates are commonly used.

Metals than can be used in this process are the metals belonging to the b-groups of the periodic system, for example cobalt, nickel, copper, iron, and preferably zinc, as well as the metals belonging to the main groups of the periodic system and capable of forming complexes, such as magnesium and aluminum. The preparation of the said metal complexes takes place in the conventional manner.

Thus, for example, a metal complex can be obtained by adding the peptide and a poorly soluble metal salt, metal hydroxide or metal oxide to an aqueous medium. The metal complex can also be obtained by adding an alkaline medium to an aqueous solution of the peptide and an insoluble metal salt to form the insoluble peptide/metal hydroxide complex.

Moreover, the metal complex can be obtained by adding the peptide, a soluble metal salt and a soluble salt to an aqueous, preferably alkaline medium to form an insoluble peptide/metal salt complex in situ.

The metal complexes can be employed at once as suspensions, or for example be lyophilized and afterwards suspended again.

Biological activity: Extinction of the conditioned avoidance response.

Male white rats weighing approximately 150 grams were conditioned by means of the so-called pole-jumping test. The conditioned stimulus was a light presented over the cage for 5 seconds, whereupon the unconditioned stimulus of shock was delivered through the grid floor of the cage.

For 3 consecutive days 10 tests were run every day with an average interval of 60 seconds. The day after this acquisition period the extinction was studied in sessions of 10 trials. All animals that made 8 or more positive responses in the first extinction session were treated with the substance to be tested or with a placebo. After that, extinction sessions of 10 trials each were carried out 2 and 4 hours after the treatment of the animals with the substance to be tested.

In the following table the results of the known peptide 4-10 ACTH are compared with some peptides according to this invention.

__________________________________________________________________________ Dosage Estimated in .mu.gm First Second Third potency Peptide per session session session ratio animal after after compared s.c. 0 hour 2 hours 4 hours with 4-10 ACTH (= 1) __________________________________________________________________________ H--Met--Glu--His--Phe-- Arg--Trp--Gly--OH 100 8 8 7 (4-10 ACTH) 30 8 5 3 1 H--Met--Glu--His--Phe-- 30 8 8 7 Arg--Phe--Gly--OH 10 8 5 4 3 H--Met(.fwdarw. 0)--Glu--His-- 10 9 9 7 Phe--Arg--Phe--Gly--OH 3 9 9 4 >10 .beta.--Ala--Glu--His--Phe-- 30 9 9 8 Lys--Phe--OH 10 8 8 7 3 9 6 4 10 H--Met--Glu--His--Phe-- 30 9 9 8 Lys--Amf 10 9 7 5 >3 __________________________________________________________________________

With regard to the various abbreviations used throughout the specification, examples and claims, the following remarks are made:

I. If no optical configuration has been stated the L-form is meant.

II. The following abbreviations have been used for the protecting or activating groups:

Z = benzyloxy-carbonyl Boc = tertiary butyloxy-carbonyl tBu = tertiary butyl Me = methyl ONP = p-nitrophenyloxy Su = succinimido

III. For the solvents or reagents the following abbreviations have been used:

Bz = benzene EtOH = ethanol Bu = butanol Py = pyridine Ac or HAc = acetic acid Fo = formic acid Am = amyl alcohol iPro = isopropanol DMF = dimethylformamide THF = tetrahydrofuran DCCI = dicyclohexyl-carbodiimide DCHU = dicyclohexyl-urea TAA = triethylamine TFA = trifluoro acetic acid Wa = water

IV. FOr the amino acid residues the following abbreviations have been used:

Met = methionyl Met(.fwdarw.O) = methionylsulfoxide (rac.) Met(d,.fwdarw.O) = methionyl(d)sulfoxide Met(1,.fwdarw.O) = methionyl(l)sulfoxide Met(.fwdarw.O.sub.2) = methionylsulfone Glu(X) = glutamyl (X = OH) = Glu Gln(X) = glutaminyl (X = NH.sub.2) = Gln His = histidyl Phe = phenylalanyl Arg = arginyl Lys = lysyl Gly = glycyl Val = valyl Ala = alanyl alanyl-Ala = .beta. (.alpha.-Me)Ala methylalanyl = .alpha.

V. Abbreviations used for other residues:

PPA = (N-phenylpropyl)amino group PEA = (N-phenylethyl)amino group HPEA = (N-p.hydroxyphenylethyl)amino group Amf = (N-l-phenylisopropyl)amino group (derived from amfetamine) Desamino-Met = desaminomethionyl (= .gamma. methylthiobutyryl) group Desamino-Met(.fwdarw.O) = desaminomethionyl-sulfoxide Desamino-Met(.fwdarw.O.sub.2) = desaminomethionyl-sulfone.

Preparation starting substances

A. Preparatin Boc--Met--Glu(OtBu)--His--N.sub.2 H.sub.3

1. Boc--Met--Glu(OtBu)--His--OMe

Boc--Met--N.sub.2 H.sub.3 (10.52 g), dissolved in 75 ml of DMF, is cooled down to 0.degree.C, after which 23.6 ml of 3.4 N HCl in THF are added, and at -20.degree.C 5.85 ml (43.3 mmol) of isoamyl nitrite. The mixture is stirred for 7 minutes at -20.degree.C and then added to a solution of 17.05 g of H--Glu(OtBu)--His--OMe.2 HCl in 50 ml of dimethylformamide. Then enough triethylamine is added to adjust the final pH of the mixture to 6.9. Then the mixture is stirred for 3 days at 0.degree.C. The triethylamine.HCl formed is then filtered off and the filtrate evaporated to dryness. The residue is dissolved in 150 ml of ethyl acetate/water. The water layer is separated and the ethyl acetate layer washed twice with water. Then the water layers are combined and extracted again with ethyl acetate (2 .times. 25 ml). The ethyl acetate layers are dried, after which the solution is evaporated to about 100 ml and set aside at 0.degree.C.

Melting point: 138.degree.-142.degree.C.

Rf in Bu:Ac:Wa (4:1:1) = 0.59 on SiO.sub.2.

2. Boc--Met--Glu(OtBu)--His--N.sub.2 H.sub.3

Of the above methyl ester 3.7 g are dissolved in 70 ml of methanol, after which 3.7 ml of hydrazine hydrate are added. The mixture is stirred for 5 hours at room temperature. The solution is evaporated to dryness and then stirred with water and dried.

Rf in Am:iPro:Wa (10:4:5) = 0.39 on SiO.sub.2.

B. preparation of Boc--D--Met--Glu(OtBu)--His--N.sub.2 H.sub.3

1. Boc--D--Met--Glu(OtBu)--His--OMe

Boc--D--Met--N.sub.2 H.sub.3 (10.52 g), dissolved in 75 ml of DMF, is cooled down to 0.degree.C, after which 23.6 ml of 3.4 N hydrochloric acid in THF and, at -20.degree.C, 5.85 ml of isoamyl nitrite are added. The mixture is stirred for 7 minutes, after which 17.05 g of H--Glu (OtBu)--His--OMe.2 HCl in 50 ml of DMF are added and the pH is adjusted to 6.9 with triethylamine. The mixture is stirred for 3 days at 0.degree. and filtered, and the filtrate evaporated to dryness in vacuum. The residue is taken up in 150 ml of ethyl acetate/water and washed with water. The organic phase is dried, after which it is evaporated to 100 ml and set aside at 0.degree.. The crystals obtained are dried.

Rf in Bu:Ac:Wa (4:1:1) = 0.63 on SiO.sub.2.

Melting point: 69.degree.-71.degree.C.

2. Boc--D--Met--Glu(OtBu)--His--N.sub.2 H.sub.3

Of the above methyl ester 3.2 g (5.45 mmol) are dissolved in 60 ml of methanol, after which 3.7 ml of hydrazine hydrate are added. The mixture is stirred for 5 hours at room temperature, after which the methanol is distilled off in vacuum and the residue stirred with water. After being dried, the hydrazide is immediately processed further.

Rf in Am:iPro:Wa (10:4:5) = 0.37 (SiO.sub.2).

C. preparation of Boc--Val--Glu(OtBu)--His--N.sub.2 H.sub.3

1. Boc--Val--Glu(OtBu)--His--OMe

Boc--Val--OH (3.26 g; 15 mmol) is dissolved in 20 ml of methylene chloride, after which 1.73 g of N-hydroxy-succinimide are added. The mixture is cooled down to -20.degree.C. after which 3.09 g of DCCI, dissolved in 20 ml of cooled methylene chloride, are added and the resulting solution is stirred for 1 hour at -20.degree.C and then for 20 hours at +20.degree.C.

The resulting DCHU is filtered, after which the filtrate is evaporated to dryness and the residue dissolved in 30 ml of DMF. Then 7.33 g of Z-Glu(OtBu)-His-OMe, prepared according to Kappler Helv. 44, 1991, 1961) and 1.4 g of 10% palladium/charcoal are added. Then hydrogen is bubbled through the solution for 5 hours, after which the solution is stirred for 1 night, filtered and the filtrate evaporated to dryness.

The residue is dissolved in aqueous ethyl acetate and washed with water, sodium bicarbonate and water. The organic phase is dried, after which the ethyl acetate is evaporated in vacuum. The residue is recrystallised from ethyl acetate/petroleum ether.

yield: 3.95 g; melting point: 117.degree.-119.degree.C.

Rf in Bz:EtOH (8:2) = 0.55 (SiO.sub.2).

2. Boc--Val--Glu(OtBu)--His--N.sub.2 H.sub.3

Of the above methyl ester 3.73 g are dissolved in 85 ml of methanol, after which 3.72 g of hydrazine hydrate are added. The mixture is stirred for 7 hours at room temperature, after which the solution is evaporated to dryness

Rf the residue triturated with ether. Rf in AM:iPro:Wa (10:4:5) = 0.33 on SiO.sub.2.

D. preparation of Boc--.beta.--Ala--Glu(OtBu)--His--N.sub.2 H.sub.3

1. Boc--.beta.--Ala--Glu(OtBu)--His--OMe

Boc--.beta.--Ala--OH (3.78 g) is dissolved in methylene chloride. The solution is cooled down to 0.degree.C, after which 2.3 g of N-hydroxy-succinimide are added. The mixture is cooled down further to -22.degree.C, after which 4.12 g of DCCI are added. The mixture is stirred for 30 minutes at -22.degree.C, for 3 hours at 0.degree.C and for 12 hours at room temperature. The precipitate formed (DCHU) is filtered off, the filtrate evaporated to dryness and the residue taken up in dimethylformamide. To this mixture 9.77 g of z--Glu(OtBu)--His--OMe are added and also 1.5 g of palladium (10%) on charcoal as a catalyst. Then hydrogen gas is bubbled through the mixture for 6 hours. Then the mixture is stirred for 12 hours. Then the catalyst is filtered off and the filtrate evaporated to dryness. The residue is taken up in ethyl acetate and the solution washed successively with a sodium bicarbonate solution (5%) and water. The ethyl acetate phase is dried, after which the solution is evaporated to dryness and the residue recrystallised from ethyl acetate/petroleum ether (1:1).

Melting point: 93.degree.-95.degree.C.

Rf in Bz:EtOH (8:2) = 0.25 on SiO.sub.2.

2. Boc--.beta.--Ala--Glu(OtBu)--His--N.sub.2 H.sub.3

The methyl ester obtained in (1) (3 g) is dissolved in 60 ml of methanol, after which 3 ml of hydrazine hydrate are added. The mixture is stirred for 6.5 hours at room temperature, after which the solution is evaporated to dryness and the residue triturated with dry ether. The substance was immediately used for further reactions.

Rf in Am:iPro:Wa (10:4:5) = 0.42 on SiO.sub.2.

E. preparation of Boc--Gly--Glu(OtBu)--His--N.sub.2 H.sub.3

1. Boc--Gly--Glu(OtBu)--His--OMe

In the same manner as described in (C.1) Boc--Gly--Glu(OtBu)--His--OMe is prepared by reacting Boc--Gly--OH with H--Glu(OtBu)--His--OMe.

Melting point: 103.degree.-108.degree.C.

Rf in Bz:EtOH (8:2) = 0.43 on SiO.sub.2.

2. Boc--Gly--Glu(OtBu)--His--N.sub.2 H.sub.3

By reacting this substance with hydrazine hydrate as described in (A.2) the Boc--Gly--Glu(OtBu)--His--N.sub.2 H.sub.3 is prepared.

Rf in AM:iPro:Wa (10:4:5) = 0.32 on SiO.sub.2.

F. prepared in the same manner as described in C. and D.

1. Boc--Ala--Glu(OtBu)--His--N.sub.2 H.sub.3 ;

Rf = 0.33 (Am:iPro:Wa = 10:4:5)

2. Boc--(.alpha.--Me)Ala--Glu(OtBu)--His--N.sub.2 H.sub.3 ;

Rf = 0.31 (Am:iPro:Wa = 10:4:5).

3. Desamino--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 ;

Rf = 0.32 (Am:iPro:Wa = 10:4:5).

G. preparation of Boc--Met--Gln--His--N.sub.2 H.sub.3

In the same manner as described in (C.1) the amino acid derivative Boc--Met--OH is coupled to H--Gln--His--OMe, obtained from the corresponding Z-protected peptide (Helv. 44, 476, 1961) by hydrogenation with 10% palladium on charcoal, yielding the protected peptide ester: Boc--Met--Gln--His--OMe, which peptide is immediately processed into the corresponding hydrazide by the method described in C.2).

Rf in Am:iPro:Wa (10:4:5) = 0.28 on SiO.sub.2.

H. preparation of H--Phe--Arg--Phe--Gly--OH.HAc

1. Boc--Phe--Gly--OBzl

To a solution of 14.5 g of Boc--Phe--OH in 135 ml of methylene chloride are added 9.93 g of H--Gly--OBzl.HCl, while stirring. The suspension is cooled to -5.degree.C, after which 7.1 ml of triethyl amine are added. The mixture is stirred for 5 minutes, after which enough triethyl amine is added to adjust the pH of the mixture to 7-7.2 (about 3 ml). Then the temperature of the mixture is reduced to -22.degree.C, after which 10.1 g (49.3 mmol) DCCI in 55 ml of methylene chloride are added. The mixture is stirred for 1 hour at -20.degree.C and for 12 hours at room temperature, after which it is left to stand in a refrigerator for some time. Then the precipitate formed (DCHU) is filtered off, and the filtrate is washed with a sodium bicarbonate solution (5%), water, 0.1 N HCl, again with water and a saturated NaCl solution. After that the organic phase is dried and evaporated to dryness. The residue is recrystallised from ethyl acetate.

Melting point: 130.degree.-132.degree.C.

Rf in Bz:EtOH (9:1) = 0.86 on SiO.sub.2.

2. H--Phe--Gly--OBzl.HCl

Of the peptide obtained in (1) 12.5 g are dissolved in a mixture of 85 ml of ethyl acetate, 33 ml of nitromethane and 45 ml of methylene chloride. After that 54 ml of 5.66 N HCl in ethyl acetate are added at room temperature. Then the mixture is stirred and evaporated to half of the volume, after which dry ether (about 250 ml) is gently added. The resulting white oil is separated from the liquid layer. Then the oil is stirred with ether.

3. Boc--Arg(NO.sub.2)--Phe--Gly--OBzl

Of the above oil 10.03 g are dissolved in 100 ml of dioxane, after which successively 4.3 ml of triethyl amine, 2.21 ml of glacial acetic acid and 9.75 g of t-butyloxycarbonyl-(nitro)arginine-acetoxim are added. The mixture is stirred for 40 hours at room temperature, after which it is evaporated to dryness in vacuum. The residue is then taken up in 150 ml of ethyl acetate and washed successively with 0.1 N HCl, water, a sodium bicarbonate solution (5%), water and a saturated NaCl solution. The organic layer is dried and evaporated, and the residue recrystallised from dioxane.

Melting point: about 66.degree.C (transition into a thick oil).

Rf in Bz:EtOH (9:1) = 0.35 on SiO.sub.2.

4. H--Arg(NO.sub.2)--Phe--Gly--OBzl.HCl

Of the peptide obtained in (3) 12.5 g are dissolved in 85 ml of ethyl acetate and 40 ml of nitromethane. Then 35.5 ml of 5.7 N HCl in ethyl acetate are added, after which the mixture is treated as described in (2).

Rf in Am:Py:Wa (5:3:2) = 0.66 on SiO.sub.2.

5. Boc--Phe--Arg(NO.sub.2)--Phe--Gly--OBzl

To a solution of 9.14 g of the peptide obtained in (4) in 35 ml of dimethylformamide 3.42 ml of triethyl amine are added at 0.degree.C, after which the solution is cooled down to -20.degree.C. This solution is immediately added to a solution of 6.8 g of Boc--Phe--ONP in 75 ml of dimethylformamide, also cooled down to -20.degree.C. Then the mixture is stirred for 1 hour at -20.degree.C and for 20 hours at room temperature. The excess of active ester is then removed by stirring for 2 hours in the presence of 147 mg of 2-dimethyl aminoethyl amine. The solvent is removed in vacuum. The oily residue obtained is taken up in a mixture of ethyl acetate (200 ml) and water (35 ml). The ethyl acetate layer is washed successively with 0.1 N HCl, water, a potassium carbonate solution (5%), water and a saturated NaCl solution. The solid substance formed during the whole process is collected and dried in vacuum. The ethyl acetate layer is completed with ether to a volume of about 1 litre whereby additional solid substances crystallize out.

Melting point: 175.degree.-177.degree.C.

Rf in Bz:EtOH (9:1) = 0.32 on SiO.sub.2.

6. H--Phe--Arg(NO.sub.2)--Phe--Gly--OBzl.TFA

Of the protected peptide-ester obtained in (5) 10.4 g are dissolved, while stirring, in 36 ml of 90% trifluoro-acetic acid (TFA), cooled down beforehand to about 10.degree.C. The reaction mixture was stirred for 1.5 hours and then added to 400 ml of ether, while stirring vigorously. After 2.5 hours the resulting jelly-like white substance is filtered off in vacuum and washed with ether.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.78 on SiO.sub.2.

7. H--Phe--Arg--Phe--Gly--OH.acetate

Of the peptide obtained in (6) 9.9 g are dissolved in 500 ml of 90% acetic acid. Then 1.8 g of 10% palladium/charcoal are added to this solution, after which hydrogen is bubbled through for 48 hours, while stirring. The catalyst is filtered off and washed with glacial acetic acid. The filtrate is evaporated to dryness in vacuum to obtain a colourless oil, which crystallizes out at 0.degree.C after standing under ether for 24 hours.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.27 on SiO.sub.2.

K. preparation of H--Phe--Lys(Boc)--(N-phenylpropyl)amide

1. Z--Lys(Boc)--PPA

Z--Lys(Boc)--ONP (10.33 g; 20.6 mmol) is dissolved in 80 ml of methylene chloride at about 0.degree.C. Then 2.7 g of 3-phenylpropylamine are added to this solution, after which the mixture is stirred for 1.5 hours at 0.degree.C and for 18 hours at room temperature. The solvent is evaporated and the residue dissolved in 200 ml of ethyl acetate. The ethyl acetate solution is washed successively with a sodium carbonate solution (10%), a NaCl solution (30%), 0.1 N HCl and a 30% NaCl solution. Then the ethyl acetate layer is dried and evaporated to a volume of about 80 ml. Then enough ether is added to cause a turbidity, after which the mixture is stored in a refrigerator. After 2 hours the precipitate formed is filtered off.

Melting point: 78.degree.-79.degree.C.

Rf in Bz:EtOH (9:1) = 0.53 on SiO.sub.2.

2. H--Lys(Boc)--PPA

Of the compound obtained in (1) 8.75 g are dissolved in 120 ml of methanol to which 1.2 g of 10% palladium/charcoal are added. While stirring, hydrogen is bubbled through the solution for 3.5 hours, after which the catalyst is filtered off. The filtrate is evaporated to dryness to obtain a practically colourless oil, which is used at once for further reactions.

Rf in Am:Fo:Wa (7:2:1) 0.58 on SiO.sub.2.

3. Z--Phe--Lys(Boc)--PPA

Of the protected amino acid derivative obtained in (2) 6.39 g are dissolved in 68 ml of dimethylformamide after which a solution of 7.61 g of Z--Phe--ONP in 20 ml of dimethylformamide is added. The mixture is stirred at room temperature for 20 hours, after which the solvent is evaporated in vacuum. The residue is dissolved in 170 ml of ethyl acetate and washed successively with a potassium carbonate solution (5%), a NaCl solution (30%), 0.1 N HCl and a NaCl solution (30%). The ethyl acetate layer is then dried on Na.sub.2 SO.sub.4 and evaporated to about 100 ml. The solution is stored in a refrigerator for 3 days, during which time the peptide crystallizes out completely.

Melting point: 134.degree.-136.degree.C.

Rf in Bz:EtOH (8.2) = 0.72 on SiO.sub.2.

4. H--Phe--Lys(Boc)--PPA

Of the peptide derivative obtained in (3) 9.07 g are dissolved in 300 ml of dimethylformamide to which 4 ml of 4N HCl and 1.5 g of 10% palladium/charcoal have been added. Hydrogen is bubbled through the solution for 3.5 hours, while stirring, after which the catalyst is filtered off and the filtrate evaporated to dryness to a practically colourless oil.

Rf in Bu:Ac:Wa (4:1:1) = 0.63 on SiO.sub.2.

L. in the same manner as described in example K are prepared:

1. H--Phe--Lys(Boc)--Amf (the starting amine in this synthesis is L-amfetamine)

Rf in Bu:Ac:Wa (4:1:1) = 0.60 on SiO.sub.2.

2. H--Phe--Lys(Boc)--PEA (the starting amine is phenylethylamine)

Rf in Bu:Ac:Wa (4:1:1) = 0.65 on SiO.sub.2

3. H--Phe--Lys(Boc)--HPEA (the starting amine is p-hydroxyphenylethylamine) Rf = 0.57.

M. preparation of H--Phe--Lys(Boc)--Phe derivatives

1. Z--Phe--Lys(Boc)--Phe--OMe

H--Lys(Boc)--Phe--OMe (4.24 g) is dissolved in 25 ml of dimethylformamide, after which 4.77 g (11.4 mmol) of Z--Phe--ONP are added to this solution. The mixture is stirred for about 24 hours, after which the solvent is evaporated in vacuum. The residue is dissolved in a mixture of 120 ml of ethyl acetate and 30 ml of water, after with the ethyl acetate phase is washed successively with 0.1 N HCl, water, a sodium carbonate solution (5%) and water. The ethyl acetate is distilled off and the residue recrystallised from ethyl acetate to which a little petroleum ether has been added.

Rf in Bz:EtOh (8:2) = 0.76 on SiO.sub.2.

2. H--Phe--Lys(Boc)--Phe--OMe

of the peptide obtained in (1) 1.9 g are dissolved in 50 ml of methanol to which 0.5 g of 10% palladium/charcoal is added. Then hydrogen gas is bubbled through the solution for 3 hours, after which the catalyst is filtered off and the filtrate evaporated to dryness.

Rf in Bz:EtOH (8:2) = 0.35 on SiO.sub.2.

3. H--Phe--Lys(Boc)--Phe--NH.sub.2

Of the ester obtained in (1) 500 mg are dissolved in methanol, after which the solution is saturated with ammonia. The mixture is stirred for 24 hours. The Z-protected peptide amide crystallizes from the solution.

In the manner described in (2) the protecting Z-group is removed from this amide.

Rf in Bz:EtOH (8:2) = 0.23 on SiO.sub.2.

4. H--Phe--Lys(Boc)--Phe--OH

Of the peptide ester obtained in (2) 0.5 g is dissolved in 6 ml of methanol, after which 1 equiv. of NaOH is added. The mixture is stirred for 1 hour, after which it is gently acidified to precipitate the tripeptide acid.

Rf in Bz:EtOH (8:2) = 0.18 on SiO.sub.2.

5. H--Phe--Lys(Boc)--Phe--O(CH.sub.2).sub.3 --C.sub.6 H.sub.5

1 g of Z--Phe--Lys(Boc)--Phe--OH, obtained by saponification of the corresponding methylester described in (1) by the method described in (4) is dissolved in 15 ml DMF after which 1.1 equiv. phenylpropylbromide and 1.1 equiv. dicyclohexylamine is added to that solution. After two days of stirring the suspension obtained is cooled to 0.degree.C and filtered off. The filtrate is evaporated after which aqueous ethyl acetate is added to the residue. The mixture is then washed with 0.1 n HCl, water, 5% sodium bicarbonate and water, whereupon the ethyl acetate phase is dried and evaporated. The residue obtained is partially deprotected (removal of the Z-group) by means of the method described in 2).

Rf in Bz:EtOH (8:2) = 0.40 on SiO.sub.2.

N. h--phe--Arg--Phe derivatives

1. Z--Phe--Arg(NO.sub.2)--Phe--OMe

Z--Phe--ONP (2.1 g), dissolved in 25 ml of DMF, is added to a solution of 2.3 g of H--Arg(NO.sub.2)--Phe--OMe.HBr, (melting point: 163.degree. dec.) cooled down to 0.degree. and 0.73 ml of triethyl amine. The reaction mixture is stirred for 2 hours at 0.degree. and for 20 hours at 20.degree.C, after which it is evaporated and the filtrate stirred three times with dry ether. The residue is recrystallised from methanol-ether (2:1).

Rf in Bz:EtOH (8:2) = 0.68 on SiO.sub.2.

2. Z--Phe--Arg(NO.sub.2)--Phe--NH.sub.2 or Z--Phe--Arg(NO.sub.2) --Phe--N(CH.sub.3).sub.2

To a solution of 0.8 g of tripeptide ester of 1) in 30 ml of methanol at about 0.degree.C is added dry ammonia (saturated solution) or about 10 equiv. dimethylamine.

After 20 hours' stirring at 0.degree.C, the temperature is allowed to rise to room temperature. After stirring for 1 hour the resulting suspension is filtered.

Rf in Bz:EtOH (8:2) = 0.53 on SiO.sub.2 for the unsubstituted amide.

Rf in Bz:EtOH (8:2) = 0.62 on SiO.sub.2 for the dimethylamide.

3. Z--Phe--Arg(NO.sub.2)--Phe--OH

Of the tripeptide ester of (1) 1.28 g are suspended in 2 ml of 1 N sodium hydroxide and 10 ml of methanol. When a clear solution is obtained, this solution is stirred for another 15 minutes, after which it is acidified with hydrochloric acid to pH 3. The resulting precipitate is filtered and washed with water.

Rf in Bz:EtOH (8:2) = 0.43 on SiO.sub.2.

4. Deprotection

Of the tripeptides 1, 2 or 3 0.5 g is dissolved in 25 ml of 90% acetic acid. Then 10% palladium on charcoal is added, after which the mixture is hydrogenated for 2 days. The resulting black suspension is filtered off, after which the filtrate is evaporated to a dryness in vacuum and the residue taken up in water.

The filtrate is filtered again, after which it is lyophilised.

______________________________________ 1. H--Phe--Arg--Phe--OMe.acetate Rf* = 0.25 on SiO.sub.2 2. H--Phe--Arg--Phe--NH.sub.2.acetate Rf* = 0.18 on SiO.sub.2 3. H--Phe--Arg--Phe--N(CH.sub.3).sub.2.acetate Rf* = 0.22 on SiO.sub.2 4. H--Phe--Arg--Phe--OH.acetate Rf* = 0.12 on SiO.sub.2 ______________________________________ *Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1)

P. h--phe--Lys(Boc)--Phe--Gly derivatives

1. Z--Phe--Lys(Boc)--N.sub.2 H.sub.3

Twelve grams of Z--Phe--Lys(Boc)--OMe are dissolved in 30 ml of methanol and 6ml of hydrazine hydrate. The solution is left to stand for 24 hours, after which it is stirred at 0.degree.C for 2 hours. The resulting precipitate is filtered off, washed with cold methanol and dried.

Rf in Bz:EtOH (8:2) = 0.12 on SiO.sub.2.

2. Z--Phe--Lys(Boc)--Phe--Gly--OBzl

Of the above hydrazide 5.42 g are dissolved in 20 ml of DMF. The solution is cooled down to 0.degree., after which 2 equiv. of hydrochloric acid/THF are added and the mixture is cooled down to -20.degree.C. Then 1.35 ml iso isoamyl nitrite are added, after which the mixture is stirred for 7 minutes at -20.degree.C and added to a solution of 3.5 g of H--Phe--Gly--OBzl.HCl (H.2) and 4.2 ml of triethyl amine (pH7). The mixture is left to stand for 70 hours at 0.degree., after which the solvent is distilled off and the residue taken up in ethyl acetate/water. Then the organic phase is washed with 0.1 N HCl, sodium bicarbonate (5%) and water. The ethyl acetate layer is dried over sodium sulphate, after which the ethyl acetate is distilled off in vacuum.

Rf in Bz:EtOH (8:2) = 0.71 (SiO.sub.2).

3. H--Phe--Lys(Boc)--Phe--Gly--OH

One gram of the peptide obtained in (2) is dissolved in methanol/water 1:1 (20 ml). Then 10% palladium on charcoal is added, after which hydrogen is bubbled through the mixture for 5 hours. The mixture is filtered over hyflo, after which the filtrate is evaporated and the residue stirred into dry ether.

Rf in Bu:Ac:Wa (4:1:1) = 0.43 (SiO.sub.2).

EXAMPLE I

Preparation of H--Met--Glu--His--Phe--Arg--Phe--Gly--OH

A. Boc--Met--Glu(OtBu)--His--Phe--Arg--Phe--Gly--OH

8.39 g of Boc--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 (see A.2) are dissolved in 80 ml of dimethylformamide. To this solution 26.2 ml of 1.6 N HCl/THF are added at 0.degree.C, while stirring, after which 1.88 ml of isoamyl nitrite are added at -20.degree.C. Then the mixture is stirred for 20 minutes at -20.degree.C, after which successively 5.8 ml of triethyl amine, 10 ml of dimethylformamide and a solution of 8.14 g of H--Phe--Arg--Phe--Gly--OH.acetate (H.7) in 90 ml of dimethylformamide titrated with triethyl amine till pH 7, are added. By adding extra triethyl amine the pH of the whole reaction mixture is adjusted to 7. The mixture is stirred for 30 minutes at -20.degree.C, after which it is stored in a refrigerator. At set times, the pH is measured and adjusted to 7, if required. After 48 hours the precipitate formed (triethyl ammonium chloride) is filtered off and the filtrate evaporated to dryness in vacuum. The residue is taken up in 450 ml of ethyl acetate. The mixture is cooled down, after which greyish-white material crystallizes out, which is removed by centrifugation and successively washed with ethyl acetate, ether, 0.07 N HCl and water, and finally dried.

Rf in Bu:Py:Ac:Wa (4:0.75:0.25:1) = 0.41 on SiO.sub.2.

B. H--Met--Glu--His--Phe--Arg--Phe--Gly--OH.acetate

Of the protected heptapeptide obtained in (A) 3.71 g (3.42 mmol) are dissolved in 40 ml of 90% trifluoro acetic acid and stirred for 2 hours at room temperature. Then the mixture is poured, while stirring ring, into 350 ml of ether. After 1 hour's stirring the white precipitate formed is filtered off and washed with ether. After being dried, the substance is dissolved in a mixture of butanol and water (1:1), after which about 2 g of Dowex X 8 ionexchanger in the acetate form are added. Then the mixture is stirred for 30 minutes, after which again 2 g of Dowex are added. Then the ionexchanger is filtered off and the filtrate lyophilised.

Rf in Bu:Py:Ac:Wa (2:0.75:0.25:1) = 0.30 on SiO.sub.2.

Finally the product obtained is subjected to a purification according to the counter current principle. System Bu:Ac:Wa = 4:1:5. Amino acid composition:

His 1.04

Arg 1.00

Glu 1.00

Gly 1.00

Met 0.98

Phe 1.99

EXAMPLE II

Preparation of A--Glu--His--Phe--Arg--Phe--Gly--peptides

Condensation of the peptide H--Phe--Arg--Phe--Gly--OH.HAc, described in H.7, with one of the peptides mentioned in B.2, C.2, D.2, F.2 and F.3 by the process described in example I yields after removal of the protecting group(s) the following peptide acetates:

1. H--D--Met--Glu--His--Phe--Arg--Phe--Gly--OH Rf* 0.31 2. H--Val--Glu--His--Phe--Arg--Phe--Gly--OH Rf* 0.28 3. H--.beta.-Ala--Glu--His--Phe--Arg--Phe--Gly--OH Rf* 0.28 4. H--(.alpha.--Me)Ala--Glu--His--Phe--Arg--Phe--Gly--OH Rf* 0.30 5. Desamino--Met--Glu--His--Phe--Arg--Phe--Gly--OH Rf* 0.33 *Rf in Bu:Py:Ac:Wa (2:3/4:1/4:1)

EXAMPLE III ##SPC4##

(a = met, Desamino-Met or Gly; X = OH or NH.sub.2

1. Boc--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--Gly--OH 2.1 of Boc--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 (see A.2) are dissolved and converted into the azide with isoamyl nitrite by the process described in example I A) and then condensed with H--Phe--Lys(Boc)--Phe--Gly--OH (P.3). The reaction mixture obtained is stirred for 70 hours at 0.degree.C and then poured into water, after which the precipitate formed is dissolved in ethylacetate/ethanol (1:1). This solution is slowly added to ether. The precipitate is filtered off.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.54 on SiO.sub.2

2. In the same manner are obtained:

a. Boc--D--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--Gly--OH by condensation of the peptide B.2 with the peptide P.3;

b. Desamino--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--Gly--OH by condensation of the peptide F.3 with the peptide P.3;

c. Boc--Met--Gln--His--Phe--Lys(Boc)--Phe--Gly--OH by condensation of peptide G with the peptide obtained in P.3;

d. Boc--Gly--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--Gly--OH by condensation of the peptide E.2 with the peptide of P.3.

3. Deprotection of the peptides obtained in (1) and (2)

The peptides are dissolved in 90% TFA and stirred for 1 hour at room temperature. The solvent is distilled off, and the residue taken up in water and lyophilised. After being dried over solid KOH, the residue is dissolved in tBu/Wa (1:1); Dowex X 8 in the acetate form is added, after which the mixture is stirred for about 1.5 hours. Then the ionexchanger is filtered off and the filtrate is lyophilised.

Obtained the acetates of:

Peptides Rf* ______________________________________ a) H--Met--Glu--His--Phe--Lys--Phe--Gly--OH 0.27 b) H--D--Met--Glu--His--Phe--Lys--Phe--Gly--OH 0.28 c) Desamino--Met--Glu--His--Phe--Lys--Phe--Gly--OH 0.31 d) H--Met--Gln--His--Phe--Lys--Phe--Gly--OH 0.29 e) H--Gly--Glu--His--Phe--Lys--Phe--Gly--OH 0.25 ______________________________________ *Rf in Bu:Py:Ac:Wa (2:3/4:1/4:1) on SiO.sub.2.

EXAMPLE IV

Preparation of A--Glu--His--Phe--Arg--Phe--derivatives

1.a. Boc--Met--Glu(OtBu)--His--Phe--Arg--Phe--OH

1.17 g of Boc--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 (A.2) are dissolved in 20 ml of DMF. This solution is cooled down, after which 3 ml of 2 N HCl in THF are added at 0.degree.C and 0.27 ml of isoamyl nitrite at -20.degree.C and the mixture is stirred for 7 minutes.

Then 1.58 g of the peptide H--Phe--Arg--Phe--OH.acetate (N.4.4) are added, and the pH is adjusted to 7 with TAA.

Then the mixture is stirred for 70 hours at 0.degree.C, after which the solvent is evaporated in vacuum and the residue taken up in ethyl acetate and washed with water. The organic layer is evaporated to dryness and the residue recrystallised from ethylacetate (Rf in

Bz:EtOH (8:2) = 0.20).

1.b. Boc--Met--Glu(OtBu)--His--Phe--Arg--Phe--derivatives

By condensation of Boc--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 (A.2) with H--Phe--Arg--Phe--NH.sub.2 (N.4.2); H--Phe--Arg--Phe--N(CH.sub.3).sub.2 (N.4.3) or H--Phe--Arg--Phe--OMe (N.4.1) by the process described in (a) are obtained:

Boc--Met--Glu(OtBu)--His--Phe--Arg--Phe--NH.sub.2 Rf = 0.29 Boc--Met--Glu(OtBu)--His--Phe--Arg--Phe--N(CH.sub.3).sub.2 Rf = 0.32 Boc--Met--Glu(OtBu)--His--Phe--Arg--Phe--OMe Rf = 0.37

1.c. Boc--A--Glu(OtBu)--His--Phe--Arg--Phe--OH (A = Val or Ala)

By condensation of Boc--Val--Glu(OtBu)--His--N.sub.2 H.sub.3 (C.2) or Boc--Ala--Glu(OtBu)--His--N.sub.2 H.sub.3 (F.1) with H--Phe--Arg--Phe-- OH (N.4.4) by the process described above are obtained:

Boc--Val--Glu(OtBu)--His--Phe--Arg--Phe--OH, and

Boc--Ala--Glu(OtBu)--His--Phe--Arg--Phe--OH.

1.d. Desamino--Met--Glu(OtBu)--His--Phe--Arg--Phe--OH

Condensation of Desamino--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 (F.3) with H--Phe--Arg--Phe--OH (N.4.4) by the azide method described in a), yields the protected peptide: Desamino--Met--Glu(OtBu)--His--Phe--Arg--Phe--OH.

2. Removal of protecting group(s)

Of the peptides prepared in (1) 250 mg are dissolved in 10 ml of 90% trifluoro acetic acid and stirred for 1 hour at 20.degree.C. The solvent is distilled off, after which the residue is taken up in water and lyophilised. After being dried over solid potassium hydroxide, the residue is dissolved in t-butanol/water (1:1) and the solution stirred with Dowex X 8 in the acetate form to exchange the trifluoro acetate for acetate. When the pH of the solution has become 4 or 5, the ionexchanger is filtered off and the filtrate lyophilised. After counter current distribution the acetates are obtained of:

Rf* ______________________________________ H--Met--Glu--His--Phe--Arg--Phe--OH 0.22 H--Met--Glu--His--Phe--Arg--Phe--NH.sub.2 0.27 H--Met--Glu--His--Phe--Arg--Phe--N(CH.sub.3).sub.2 0.29 H--Met--Glu--His--Phe--Arg--Phe--OMe 0.33 H--Val--Glu--His--Phe--Arg--Phe--OH 0.21 H--Ala--Glu--His--Phe--Arg--Phe--OH 0.21 Desamino--Met--Glu--His--Phe--Arg--Phe--OH 0.26 ______________________________________ *Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1).

EXAMPLE V

Synthesis of A--Glu--His--Phe--Lys--Phe--derivatives

1.a. Boc--D--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Phe derivatives.

1.17 g of Boc--D--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 (B.2) are dissolved in 20 ml of DMF. The solution is cooled down, after which 3 ml of 2 N HCl in THF are added at 0.degree.C and 0.27 ml of isoamyl nitrite at -20.degree.C, after which the mixture is stirred for 7 minutes. After that 3 mmol of the peptide prepared in M.2, M.3, M.4 or M.5 (H--Phe--Lys(Boc)--Phe--derivatives) are added and the pH is adjusted to 7.3 with TAA. After 70 hours' stirring at 0.degree.C the solvent is distilled off in vacuum and the residue taken up in ethyl acetate and washed with water. Then the organic layer is evaporated to dryness. The residue is pure enough for further processing. Obtained in this manner:

Rf* ______________________________________ Boc--D--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--OH 0.44 Boc--D--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--OMe 0.62 Boc--D--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--NH.sub.2 0.50 Boc--D--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--O(CH.sub.2).sub.3 -- C.sub.6 H.sub.5 0.67 ______________________________________ *Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1).

1.b. By using the peptide Boc--.beta.--Ala--Glu(OtBu)--His--N.sub.2 H.sub.3 (D.2) instead of Boc--D--Met--Glu(OtBu)--His--N.sub.2 H.sub.3, is obtained:

Boc--.beta.--Ala--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--OH Rf = 0.42.

1.c. By using the peptide derivative Desamino--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 (F.3) instead of Boc--D--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 in the process described in (a) the following peptides are obtained:

Desamino--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--OH

Desamino--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--OMe

Desamino--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--NH.sub.2

Desamino--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Phe--O(CH.sub.2).sub.3 --C.sub.6 H.sub.5.

2. Deprotection

In the manner described in Example IV.2 the peptides obtained in (1) are deprotected. The acetates of the following peptides are obtained:

Peptide: Rf* ______________________________________ H--D--Met--Glu--His--Phe--Lys--Phe--OH 0.21 H--D--Met--Glu--His--Phe--Lys--Phe--OMe 0.30 H--D--Met--Glu--His--Phe--Lys--Phe--NH.sub.2 0.24 H--D--Met--Glu--His--Phe--Lys--Phe--O(CH.sub.2).sub.3 --C.sub.6 H.sub.5 0.33 H--.beta.--Ala--Glu--His--Phe--Lys--Phe--OH 0.20 Desamino--Met--Glu--His--Phe--Lys--Phe--OH 0.25 Desamino--Met--Glu--His--Phe--Lys--Phe--OMe 0.36 Desamino--Met--Glu--His--Phe--Lys--Phe--NH.sub.2 0.29 Desamino--Met--Glu--His--Phe--Lys--Phe--O(CH.sub.2).sub.3 --C.sub.6 H.sub.5 0.38 ______________________________________ *Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) on SiO.sub.2.

EXAMPLE VI ##SPC5##

By the process, described in example I.a, one of the peptide hydrazides prepared in A - G is converted into the azide and condensed with H--Phe--Lys(Boc)--PPA (K.4), H--Phe--Lys(Boc)--Amf. (L.1), H--Phe--Lys(Boc)--PEA (L.2) or H--Phe--Lys(Boc)--HPEA (L.3).

Deprotection of the peptides obtained in this manner with TFA 90%, followed by exchange for acetate by means of Dowex X 8 in the acetate form, yields the acetate of the following peptide derivatives:

Starting Peptide: products: Rf* ______________________________________ H--Met--Glu--His--Phe--Lys--PPA A.2 + K.4 0.40 H--Desamino--Met--Glu--His--Phe--Lys--Amf F.3 + L.1 0.45 H--.beta.--Ala--Glu--His--Phe--Lys--PPA D.2 + K.4 0.38 H--Met--Glu--His--Phe Lys--Amf A.2 + L.1 0.39 H--Val--Glu--His--Phe--Lys--PEA C.2 + L.2 0.43 H--Ala--Glu--His--Phe--Lys--PEA F.1 + L.2 0.42 H--Met--Gln--His--Phe--Lys--HPEA G + L.3 0.41 ______________________________________ *Rf in Bu:Py:Ac:Wa (2:3/4:1/4:1).

EXAMPLE VII

Sulfoxides

1. Of the peptide H--Met--Glu--His--Phe--Arg--Phe--OH (Ex. IV.2) 45 mg are dissolved in 4 ml of acetic acid, after which 15 ml of 30% H.sub.2 O.sub.2 are added. The mixture is stirred for 1 hour at about 20.degree.C, after which a suspension of 20 mg of platinum black in 2.5 ml of glacial acetic acid is added and stirring is continued for 30 minutes. Then the mixture is filtered and the filtrate evaporated to dryness in vacuum. The residue is taken up in 10 ml of t.butanol/water and lyophilised.

Rf of the peptide obtained: H--Met (.fwdarw.O)--Glu--His--Phe--Arg--Phe--OH in Bu:Py:Ac:Wa (4:3/4:1/4:1) 0.20 on SiO.sub.2.

2. In the same manner the following peptide-sulfoxides are obtained:

H--Met(.fwdarw.O)--Glu--His--Phe--Lys--PPA Rf 0.36 H--Met(.fwdarw.O)--Glu--His--Phe--Arg--Phe--Gly--OH Rf 0.28 H--D--Met(.fwdarw.O)--Glu--His--Phe--Lys--Phe--OH Rf 0.19 Desamino--Met(.fwdarw.O)--Glu--His--Phe--Lys--Amf. Rf 0.41 Desamino--Met(.fwdarw.O)--Glu--His--Phe--Arg--Phe--OMe Rf 0.34

EXAMPLE VIII

Sulfones

1. Of the peptide H-D-Met-Glu-Hos-Phe-Lys-Phe-OH (example V.2) 175 mg are dissolved in a mixture of 0.5 ml of water, 0.1 ml of 4 N perchloric acid, 0.02 ml of 0.5 M ammonium molybdate, after which 0.06 ml of 30% H.sub.2 O.sub.2 is added.

The mixture is stirred for 2 hours at a temperature of about 10.degree.C, after which Dowex X 8 in the acetate form is added. After 30 minutes' stirring the ionexchanger is filtered off and the filtrate lyophilised.

Rf in Bu:Ac:Wa (4:1:1) of the peptide: H--D--Met (.fwdarw.O.sub.2)--Glu--His--Phe--Lys--Phe--OH is 0.22 on SiO.sub.2.

2. In the same manner the following sulfones are prepared:

Rf* ______________________________________ H--Met(.fwdarw.O.sub.2)--Glu--His--Phe--Arg--Phe--Gly--OH 0.29 Desamino--Met(.fwdarw.O.sub.2)--Glu--His--Phe--Lys--Phe--Gly--OH 0.29 Desamino--Met(.fwdarw.O.sub.2)--Glu--His--Phe--Lys--Phe--OMe 0.38 H--Met(.fwdarw.O.sub.2)--Glu--His--Phe--Lys--PPA 0.38 H--Desamino--Met(.fwdarw.O.sub.2)--Glu--His--Phe--Lys--Amf. 0.43. ______________________________________ *Rf in Bu:Ac:Wa (4:1:1)

EXAMPLE IX

Zinc complexes

Of a solution of zinc chloride containing 50 mg of zinc per ml, 1.5 ml are added to a solution of 31.5 mg of Na.sub.2 HPO.sub.4.2 H.sub.2 O in 30 ml of distilled water. The precipitate of zinc phosphate formed during this addition is dissolved by adding 4 N HCl. Then 175 mg of NaCl and 0.5 g of benzyl alcohol are added to this mixture, after which 150 mg of the hexapeptide H--L--Met--L--Glu--L--His--L--Phe--L--Lys--L--Phe--OH are dissolved in this mixture. Then enough 1 N sodium hydroxide is added to obtain a pH of 8.5, after which the volume is completed with distilled water to 50 ml. 1 ml suspension contains:

3 mg of hexapeptide 1.5 mg of zinc 0.63 mg of Na.sub.2 HPO.sub.4.2 H.sub.2 O 3.5 mg of NaCl 10 mg of benzylalcohol

EXAMPLE X

Preparation of H--Met (1,.fwdarw.O)--Glu--His--Phe--Arg--Phe--OH

Boc--L--Met (1.fwdarw.O)--OH is prepared by reacting Boc-azide with H--L--Met (1,.fwdarw.O)--OH, described in J. Biol. Chem. 169, 477 (1947). Boc--L--Met(1,.fwdarw.O)--OH: m.p. 68.degree.C; [.alpha.].sub.D.sup.20 = -58.degree. (c = 1, DMF). This N-protected amino acid is converted into the corresponding N-hydroxy-succinimido ester by treatment with DCCI and HOSu. The active ester obtained is used at once for a coupling reaction with H--Glu(OtBu)--His--Phe--Arg--Phe--OH (obtained by the condensation of Z--Glu(OtBu)--His--azide with H--Phe--Arg--Phe--OH, followed by hydrogenation of the Z-moiety) in the presence of N-ethylmorpholine.

The resulting peptide derivative: Boc--L--Met(1,.fwdarw.O)--Glu(OtBu)--His--Phe--Arg--Phe--OH, Rf in Bz:EtOH (8:2) = 0.18 on SiO.sub.2, is deprotected in the manner described before by means of TFA and converted into the acetate. H--L--Met(1,.fwdarw.O)--Glu--His--Phe--Arg--Phe--OH.acetate:

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.28.

In the same manner the peptide-sulfoxide:

H--L--Met(d,.fwdarw.O)--Glu--His--Phe--Arg--Phe--OH.acetate is prepared starting from Boc--L--Met(d,.fwdarw.O)--OH, m.p. 135.degree.C.

Claims

What is claimed is:

1. A peptide of the formula:

A--L--Glu(X)--L--His--L--Phe--Q--Y

in which A is selected from the group consisting of H--L--Met, H--D--Met, H--L--Met (.fwdarw.O), H--D--Met (.fwdarw.O), H--L--Met (.fwdarw.O.sub.2), H--D--Met (.fwdarw.O.sub.2), desamino-Met, desamino --Met (.fwdarw.O), desamino-Met (.fwdarw.O.sub.2), and the moeity: H.sub.2 N--B--CO, in which B is alkylene having 1-6 carbon atoms,

X is selected from the group consisting of OH and NH.sub.2,

Q is selected from the group consisting of L--Arg and L--Lys, and Y is selected from the group consisting of L--Phe--OH, L--Phe--Gly--OH, and (N-phenyl-alkyl) amino of the formula ##SPC6##

in which ALK is alkylene with 1-6 carbon atoms and R is selected from the group consisting of hydrogen and hydroxy, and functional derivatives of said peptide selected from the group consisting of pharmaceutically acceptable acid addition salts, derivatives in which one or more free amino groups are substituted by acyl derived from an aliphatic carboxylic acid with 1-6 carbon atoms, unsubstituted amides or lower alkyl (1-6 C) substituted amides of those peptides having a free carboxyl group, esters derived from aliphatic or araliphatic alcohols with 1-18 carbon atoms, and metal complexes thereof.

2. A peptide according to claim 1 of the formula:

A--L--Glu(X)--L--His--L--Phe--Q--L--Phe--OH

in which A, X and Q have the meanings indicated in claim 1.

3. A peptide according to claim 1 in which A is selected from .beta.-Ala, Desamino-Met, D--Met, and the sulfoxide and sulfone of L--Met, D--Met and Desamino-Met.

4. Metal complexes of the peptides and peptide derivatives as claimed in claim 3.