Psychopharmacologically Active Peptides Related To Acth

Abstract

The invention relates to psychopharmacologically active peptides with the general formula: A--L--Glu--L--His--X, in which A represents: 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 stands for a branched or unbranched alkylene group with 1-6 carbon atoms, and In which X represents a hydroxy group, a (N-phenylalkyl)amino group or the group L--Phe--Y, in which Y represents a hydroxy group, a (N-aminoalkyl)amino group or the group L--Lys--Z or L--Arg--Z, in which Z means a hydroxy group, the group L--Trp--OH, L--Trp--Gly--OH or a (N--3-indolylalkyl)amino group, As well as to the functional derivatives of these peptides. These compounds inhibit the extinction of the 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).

Description

The present invention relates to psychopharmacologically active peptides and peptide derivatives.

From European Journal of Pharmacology 2, 14 (1967) certain peptide fragments of the natural adrenocorticotrophic hormones (ACTH) are known to inhibit the extinction of the conditioned avoidance response. Especially the peptide having the amino acid sequence 1-10 of ACTH 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 a peptide with the amino acid sequence 4-10 of ACTH is the shortest peptide, and perhaps the key sequence for the said activity.

However, 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 fragments of ACTH. Although the effect of a low dosage of an MSH active peptide in men is still unknown, a search was made for peptides having the same psychopharmacological activity, but no or at least reduced MSH activity.

In first instance it was found in our laboratories, that 4-10 ACTH is not the key sequence for the said psychopharmacological activity, but a much smaller peptide having the amino acid sequence 4-6 ACTH.

The peptide H--L--Met--L--Glu--L--His--OH proved to be the shortest peptide inhibiting the extinction of the conditioned avoidance response at practically the same level as the known 4-10 ACTH, while the tri-peptide does not possess a demonstrable MSH activity.

Extension of the chain length at the C-terminal side of the tri-peptide with --Phe--OH (4-7 ACTH), --Phe--Lys(or Arg)--OH (4-8 ACTH), --Phe--Lys(or Arg)--Trp--OH (4-9 ACTH), or --Phe--Lys(or Arg)--Trp--Gly--OH (4-10 ACTH) turned out to cause no marked increase of activity.

It was further observed that the C-terminal aminoacid residues of the 4-7, 4-8 and 4-9 ACTH peptides could be replaced by groups which are chiefly distinguished from the said C-terminal amino acid radicals by the absence of the carboxyl group, without a marked decrease of activity.

Thus, for example, the C-terminal amino acid residue Phe in the tetrapeptide 4-7 ACTH (Met--Glu--His--Phe) could be replaced by a (N-phenylalkyl) amino group of the general formula: ##SPC1##

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

The C-terminal amino acid residue Lys or Arg in the pentapeptide 4-8 ACTH (Met--Glu--His--Phe--Lys or --Arg) could be replaced by a (N-aminoalkyl)amino group of the general formula: ##SPC2##

in which Alk represents a branched or unbranched alkylene group with 2-6 carbon atoms, R.sub.2 hydrogen or a lower alkyl group, and R.sub.3 hydrogen, a lower alkyl group or an amidine group.

The C-terminal amino acid residue Trp in the hexapeptide 4-9 ACTH (Met--Glu--His--Phe--Lys (or Arg)--Trp) could be replaced by a (N-3-indolylalkyl)amino group of the general formula: ##SPC3##

in which "Alk" represents a branched or unbranched alkylene group with 1-6 carbon atoms. The latter modification appeared to be particularly valuable.

Surprisingly it has now been found that the N-terminal amino acid (L--Met) in the above-mentioned peptides and peptide derivatives, that was thought to be essential for psychopharmacological activity, can be replaced by various other groups, which groups do not affect the biological activity of the peptides in question, but even cause in certain cases a considerable increase of activity.

It is the subject of this invention to modify the N-terminal amino acid residue L--Met of the key-peptide 4-6 ACTH and of the (at the C-terminal side) extended peptides and peptide derivatives mentioned above into the following groups:

D-methionyl, L- or D-methionyl-sulfoxide(Met.fwdarw.O), L- or D-methionyl-sulfone(Met.fwdarw.O.sub.2), desaminomethionyl or the corresponding sulfoxide or sulfone thereof or the moiety ##SPC4##

in which B represents a branched or unbranched alkylene group with 1-6 carbon atoms.

The latter moiety ##SPC5##

encompasses amino acid residues as well as amino-substituted carboxylic acid residues, such as Gly, Val, Ala, .beta.-Ala and (.alpha.-Me)Ala.

From merely synthetic-chemical considerations, the peptides in which L--Met has been replaced by one of the above-mentioned groupings, that have no asymetric centre, such as desamino-methionyl, desamino-methionyl-sulfone, Gly or .beta.--Ala, are preferred because these peptides can be prepared more conveniently.

Apart from these synthetic-chemical considerations the modifications of L--Met into methionyl-sulfoxide (L-- or D--Met.fwdarw.O), methionyl-sulfone (L-- or D-Met.fwdarw.O.sub.2), desaminomethionyl, desaminomethionylsulfoxide, desaminomethionylsulfone and .beta.--Ala give a considerable increase of activity in comparison with the corresponding L--Met peptide, so that, in particular, these modifications are preferred.

Summarizing, it is the subject of the present invention to provide new and valuable psychopharmacological peptides or peptide derivatives with the general formula:

A--L--Glu--L--His--X I

in which A represents 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--NH--B--CO--, in which B stands for a branched or unbranched alkylene group with 1-6 carbon atoms, and

in which X represents a hydroxy group, a (N-phenylalkyl)amino group of the general formula: ##SPC6##

(in which R.sub.1 stands for hydrogen, halogen, hydroxy, alkyl (1-4 C) or alkoxy (1-4 C) and "Alk" means a branched or unbranched alkylene group with 1-6 carbon atoms) or the group L--Phe--Y, in which Y represents a hydroxyl group, a (N-aminoalkyl) amino group with the general formula: --NH--Alk--NR.sub.2 R.sub.3 (in which "Alk" stands for a branched or unbranched alkylene group with 2-6 carbon atoms, R.sub.2 for hydrogen or a lower alkyl group (1-6 C) and R.sub.3 for hydrogen, a lower alkyl (1-6 C) or an amidine group) or the group L--Lys--Z or L--Arg--Z, in which Z represents a hydroxyl group, the group L--Trp--OH, L--Trp--Gly--OH or a (N-3-indolylalkyl)amino group, the alkyl group of which contains 1-6 carbon atoms,

as well as functional derivatives of these peptides and peptide derivatives thereof.

The peptides and peptide derivatives according to the invention are prepared by a process commonly used in peptid-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 protected other reactive groups;

c. condensation of a compound (amino acid, peptide) having a free carboxyl group and 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-succinimide 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 Y-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 an oxidation yields a mixture of the S- and R-sulfoxide (= 1- or d-sulfoxide), which mixture may be split off into the separate diastereomeric compounds in a conventional manner.

By coupling the S- or R-sulfoxide (= 1- or d-sulfoxide) of methionine or desaminomethionine with the peptide Glu--His--X, in which X has 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 an 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,

4. esters of the present peptides derived from aliphatic or araliphatic alcohols with 1-18 carbon atoms, such as methanol, ethanol, pentanol, hexanol, cyclohexanol, octylalcohol, undecylalcohol, hexadecylalcohol, oleylalcohol, octadecylalcohol, 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 aluminium. 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.

__________________________________________________________________________ Peptide or peptide Dosage Estimated derivative in .mu.gm First Second Third potency per session session session ratio animal after after (4-10 ACTH s.c. 0 hour 2 hrs. 4 hrs. =1) __________________________________________________________________________ H-Met-Glu-His-Phe- Arg-Trp-Gly-OH 100 9 8 7 30 9 7 5 10 9 6 3 1 H-D-Met-Glu-His-OH 30 8 6 5 1 H-Val-Glu-His-OH 30 8 6 4 1 H-.beta.-Ala-Glu-His-OH 30 9 8 7 10 9 7 6 3 H-Met(.fwdarw.0)-Glu-His-OH 30 8 8 8 10 8 8 7 3 8 4 2 >3 H-D-Met-Glu-His- Phe-OH 30 8 7 5 10 8 6 2 1 H-D-Met-Glu-His Phe-Lys-OH 30 8 7 5 1 H-D-Met-Glu-His- Phe-Lys-Tra 30 10 10 8 10 8 8 7 3 9 8 7 >10 H-Met(.fwdarw.O)-Glu-His- Phe-Arg-Trp-Gly-OH 10 8 8 8 3 8 4 2 >3 H-Met(d,.fwdarw.O)-Glu-His- Phe-Arg-Trp-Gly-OH 5 9 7 6 1.5 9 7 3 6 H-.beta.-Ala-Glu-His-Phe- Arg-Trp-Gly-OH 30 9 8 8 10 9 8 6 >3 H-D-Met-Glu-His-Amf 30 8 8 6 10 9 7 3 >1 Desamino-Met-Glu-His- Phe-Lys-Trp-OH 30 9 8 8 10 9 7 6 3 9 5 3 3 Desamino-Met(.fwdarw.O.sub.2)- Glu-His-Phe-Lys-Trp- OH 10 8 8 8 3 9 6 5 1 8 5 2 10 __________________________________________________________________________

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 = benzyloxycarbonyl Boc = tertiary butyloxycarbonyl tBu = tertiary butyl Me = methyl ONP = p-nitrophenyloxy ONB = p-nitrobenzyloxy 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 Wa = water Am = amylalcohol iPro = isopropanol DMF = dimethylformamide THF = tetrahydrofuran DCCI = dicyclohexylcarbodiimide TAAU = = tri-ethylamine TFA = trifluoro acetic acid Iv. for the amino acid residues the following abbreviations have been used:

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

V. abbreviations for other residues:

Tra = (N-3-indolylethyl)amino group (derived from tryptamine) Desamino- = desamino-methionyl (or .gamma.-methylthiobutyryl) Met group Amf = (N-1-phenylisopropyl)amino group (derived from amfetamine).

EXAMPLE I

A. h--met(.fwdarw.O)--Glu--His--OH

25 mg of the tripeptide H--Met--Glu--His--OH are dissolved in 2.5 ml of acetic acid, after which 15 .mu.l of 30 percent hydrogen peroxide are added. After stirring for 1 hour at 20.degree. C, a suspension of 20 mg of platinum black in 2.5 ml of glacial acetic acid is added and the mixture is stirred for 30 minutes. Then the mixture is filtered and the solvent distilled off in vacuum. The resulting residue is taken up in 10 ml of tertiary butanol/water (1:1) and lyophilised.

Rf of the sulfoxide = 0.14 in Bu:Py:Ac:Wa (4:3/4:1/4:1) on SiO.sub.2.

EXAMPLE II

Synthesis D--Met--Glu--His derivatives and Desamino--Met--Glu--His derivatives

A. 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 are added, and at -20.degree. C 5.85 ml of isoamyl nitrite. 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. Stirring is continued for 3 days at 0.degree., after which the mixture is 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, evaporated to 100 ml and set aside at 0.degree..

Rf in Bu:Ac:Wa (4:1:1) = 0.63 (SiO.sub.2). Melting point: 69.degree.-71.degree.C.

B. desamino--Met--Glu(OtBu)--His--OMe

In the same manner as described in II.A, the hydrazide desamino--Met--N.sub.2 H.sub.3 is converted into the azide and then coupled to H--Glu(OtBu)--His--OMe.2 HCl, whereafter the compound obtained is isolated and purified as indicated in A.

Rf in Bu:Ac:Wa (4:1:1) = 0.50 on SiO.sub.2.

C. boc--D--Met--Glu(OtBu)--His--OH

Tripeptide ester (1.17 g; example II.A) is dissolved in 50 ml of 50 percent dioxane. To this solution 2.5 ml of 1 N sodium hydroxide are added, after which the mixture is stirred for 30 minutes at 20.degree. C. The reaction mixture is neutralised to pH 7, after which it is evaporated to dryness in vacuum. The residue is taken up in 40 ml of ethyl acetate, acidified with 1.4 ml of 2 N HCl to pH 4 and washed with a little water, after which the layers are separated.

The ethyl acetate layer is dried and evaporated to dryness. The residue is an oil.

Rf in Am:Py:Wa (5:3:2) = 0.19 (SiO.sub.2).

D. boc--D--Met--Glu(NH.sub.2)--His--OH

1 gram of tripeptide ester is dissolved, while heating, in 20 ml of dioxane/water (1:1). The mixture is cooled down to 20.degree. C, after which 1.9 milli equiv. of barium hydroxide are added and the mixture is stirred for 30 minutes. After neutralisation to pH 5.4, the mixture is carefully evaporated. The residue is dissolved in methanol and filtered, and the filtrate diluted with ethyl acetate.

The precipitate is filtered and recrystallised from methanol/ethyl acetate.

Rf in Am:iPro:Wa (10:4:5) = 0.59 (SiO.sub.2).

E. boc--D--Met--Glu(OtBu)--His--NH

In a cooled solution of dry ammonia gas in 25 ml of methanol 1 g of Boc--D--Met--Glu(OtBu)--His--OMe is dissolved (example II.A). The mixture is stirred for 25 hours, after which the ammonia is evaporated, the residue taken up in methanol and the solution diluted with ether. The precipitate formed is filtered off.

Melting point: 72.degree.-75.degree. C. Rf in Bz:EtOH (8:2) = 0.43 (SiO.sub.2).

F. desamino--Met--Glu(OtBu)--His--OH

The ester obtained in II.B is hydrolysed in the same manner as described in II.C.

Rf in Am:Py:Wa (5:3:2) = 0.16 (SiO.sub.2).

G. removal of protecting group(s) from the peptides obtained in A-F

100 mg of the peptide (A, B, C, D, E or F) are dissolved in 5 ml of 90 percent TFA. The solution is left to stand for 1 hour, after which the solvent is distilled off, the residue stirred twice with ether and dried over solid potassium hydroxide. The trifluoro acetate is dissolved in 20 ml of t-butanol/water (1:1), after which it is converted into acetate by stirring the solution with so much Dowex X-8 in the acetate form to obtain a final pH of 5.4. After filtration the filtrate is lyophilised and the white residue stored over solid phosphor pentoxide.

______________________________________ Substance obtained starting Rf in Bu:Py: peptide Ac:Wa (4:3/4:1/4:1) on SiO.sub.2 ______________________________________ 1. H-D-Met-Glu-His-OMe.HAc II.A 0.18 2. H-D-Met-Glu-His-OH.HAc II.C 0.14 3. Desamino-Met-Glu-His-OMe II.B 0.25 4. Desamino-Met-Glu-His-OH II.F 0.21 5. H-D-Met-Gln-His-OH.HAc II.D 0.17 6. H-D-Met-Glu-His-NH.sub.2.HAc II.E 0.16 ______________________________________

H. sulfoxide of H--D--Met--Glu--His--OH

25 mg of the tripeptide H--D--Met--Glu--His--OH (G. 2) are dissolved in 2.5 ml of acetic acid, after which 15 .mu.l of 30 hydrogen peroxide are added. The mixture is treated in the manner described in example I. The residue is then taken up in 10 ml of t-butanol/water (1:1), after which this solution is lyophilised.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.12 on SiO.sub.2.

K. sulfoxide of desamino--Met--Glu--His--OH

25 mg of the peptide obtained in G. 4 is oxidized in the same manner as described in H.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.18 on SiO.sub.2.

EXAMPLE III

Synthesis of A--Glu--His derivatives (A=Val, Gly, Ala, .beta.-Ala or (.alpha.-Me)Ala)

A. 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 solution 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, whereupon 7.33 g of Z--Glu(OtBu)--His--OMe (prepared in accordance with Kappler Helv. 44, 1991, 1961) and 1.4 g of 10 percent palladium/carbon are added. Then hydrogen is bubbled through for 5 hours, after which the mixture is stirred for 1 night and filtered, and the filtrate evaporated to dryness. The residue is dissolved in aqueous ethyl acetate and washed with citric acid, 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-petroleumether. Yield 3.95 g; melting point: 117.degree.-119.degree. C; [.alpha.].sub.D.sup.25 = -8.4 (c = 1 DMF); Rf in Bz:EtOH (8.2) = 0.55 (SiO.sub.2).

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

Starting from 2.63 g of Boc--Gly--OH the tripeptide Boc--Gly--Glu (OtBu)--His--OMe is prepared by the process described in example A. Yield: 1.47 g; melting point: 103.degree.-108.degree. C; [.alpha.].sub.D.sup.25 = -5.2 (C = 2, DMF).

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

C. boc--Ala--Glu(OtBu)--His--OMe

Boc--Ala--OH (3.78 g) is converted into the "active ester" with 2.3 g of N-hydroxy-succinimide and 4.12 g of DCCI. The active ester, dissolved in 40 ml of DMF, is condensed with H--Glu(OtBu)--His--OMe, obtained from 9.77 g of Z--Glu(OtBu)--His--OMe as described in example III.A.

Yield: 5.56 g; melting point: 97.5.degree.-101.degree. C.

Rf in Bz:EtOH (8:2) = 0.36 (SiO.sub.2).

D. boc--.beta.--Ala--Glu(OtBu)--His--OMe

Starting from 3.78 g of Boc--.beta.--Ala--OH (melting point 77.degree.-78.degree. C) the peptide ester Boc--.beta.--Ala--Glu(OtBu)--His--OMe is prepared in the same manner as described in example III.C.

Yield: 7.4 g; [.alpha.].sub.D.sup.22 = -8.7 (c = 2, DMF).

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

E. boc--(.alpha.-Me)Ala--Glu(OtBu)--His--OMe

Boc--(1-methyl)alanine (1.83 g; m.p. 118.degree.-119.5.degree. C) is dissolved in 40 ml of methylene chloride, after which 3.51 g of H--Glu(OtBu)--His--OMe.2 HCl, prepared by hydrogenation of the Z-peptide in methanol, in the presence of 2 equiv. hydrochloric acid, and 2.29 ml of TAA are added to this solution. The mixture is cooled down to 0.degree., after which 2.07 g of N-hydroxy-succinimide are added and, finally, after the mixture has been cooled down to -20.degree., 1.86 g of DCCI. In the manner described before (example A) the reactionmix ture is treated further after being stirred for 10 min at -20.degree.C, for 2 hours at 0.degree. C and for 20 hours at 20.degree. C. Yield: 3.02 g of oil.

Rf in Bu:Ac:Wa (4:1:1) = 0.61 (SiO.sub.2).

F. hydrolysis of the peptide esters A, B, C, D and E

1.15 g of the peptide ester A, B, C, D or E is dissolved in 50 ml of 50 percent dioxane. After the addition of 2.5 ml of 1 N sodium hydroxide, the mixture is stirred for 30 minutes at 20.degree. C. The reaction mixture is neutralised to pH 7, after which it is evaporated to dryness, the residue taken up in 40 ml of ethyl acetate, and the solution washed with 0.7 ml of 2 N hydrochloric acid (pH 4). The ethyl acetate is dried and evaporated to dryness.

______________________________________ Rf in Am:Py:Wa (5:3:2) (SiO.sub.2 ______________________________________ 1. Boc-Val-Glu(OtBu)-His-OH 0.24 2. Boc-Gly-Glu(OtBu)-His-OH 0.23 3. Boc-Ala-Glu(OtBu)-His-OH 0.23 4. Boc-.beta.-Ala-Glu(OtBu)-His-OH 0.26 5. Boc-(.alpha.-Me)Ala-Glu(OtBu)-His-OH 0.25 ______________________________________

G. removal of protecting groups of the peptides obtained in F.1-F.5.

In the manner as described in Example II.G the peptides mentioned in III.F are deprotected with TFA and converted into the acetate.

______________________________________ Acetate of: Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) on SiO.sub.2 ______________________________________ 1. H-Val-Glu-His-OH 0.15 2. H-Gly-Glu-His-OH 0.15 3. H-Ala-Glu-His-OH 0.17 4. H-.beta.-Ala-Glu-His-OH 0.17 5. H-(.alpha.-Me)Ala-Glu-His-OH 0.16 ______________________________________

H. removal of protecting groups from the peptide esters obtained in A-E.

In the manner as described in example II.G the tripeptide esters obtained in A, B, C, D and E are deprotected and converted into the acetate.

______________________________________ Acetate of: Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) on SiO.sub.2 ______________________________________ 1. H-Val-Glu-His-OMe 0.20 2. H-Gly-Glu-His-OMe 0.18 3. H-Ala-Glu-His-OMe 0.21 4. H-.beta.-Ala-Glu-His-OMe 0.22 5. H-(.alpha.-Me)Ala-Glu-His-OMe 0.18 ______________________________________

EXAMPLE IV

Synthesis of H--(.alpha.--Me)Ala--Glu--His--R (R = --NH.sub.2, --N(CH.sub.3).sub.2 or --NHC.sub.2 H.sub.5)

A. Boc--(.alpha.--Me)Ala--Glu(OtBu)--His--OMe (0.7 g; example III.E) is dissolved in a cooled solution of dry ammonia gas in 20 ml of methanol. The mixture is stirred for 20 hours, after which the ammonia is evaporated, the residue taken up again in methanol and the solution diluted with ether. The precipitate formed is filtered off (0.41 g); m.p. 93.degree.-95.degree. C. The precipitate is then dissolved in 20 ml of 90 percent TFA. The solution is left to stand for 1 hour, after which the solvent is distilled off in vacuum, the residue obtained stirred into ether and dried over KOH tablets. The trifluoro acetate is then exchanged for the acetate by adding enough Dowex X-8 in the acetate form to a solution of the peptide in t-butanol/water (1:1) to reach a pH of about 5.4. Filtration of the mixture and lyophilisation of the filtrate yields an almost white power, being the pure peptideamide H--(.alpha.--Me)Ala--Glu--His--NH.sub.2.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.20 on SiO.sub.2.

B. If instead of dry ammonia gas, dimethylamine or ethylamine is used, the corresponding N,N-dimethyl-peptide-amide or N-ethyl-peptide-amide is obtained.

EXAMPLE V

Synthesis of D--Met--Glu--His--Phe derivatives

A. z--glu(OtBu)--His--Phe--OMe

Z--Glu(OtBu)--His--N.sub.2 H.sub.3 (9.76 g) is dissolved in 30 ml of DMF, after which the solution is cooled to 0.degree. C.

After the addition of 14.4 ml of 4.8 N hydrochloric acid/THF, the solution is cooled further to -20.degree. C, after which 3.24 ml of isoamyl-nitrite are added dropwise. The solution is stirred for 7 minutes, after which it is added to a solution of 4.32 g of H--Phe--OMe.HCl in 40 ml of DMF and 12.6 ml of TAA, pre-cooled to 0.degree. C. The mixture is left to stand for 3 days at 0.degree. C, after which it is filtered. The filtrate is taken up in ethyl acetate/water. The organic phase is washed successively with water, sodium bicarbonate and water, and after that dried, whereupon the solvent is distilled off in vacuum. The residue is recrystallised from hot ethyl acetate.

Melting point: 183.degree., [.alpha.].sub.D.sup.25 = -21.degree. (c = 1 DMF).

Rf in Bz:EtOH (8:2) (SiO.sub.2) = 0.81.

B. z--glu(OtBu)--His--Phe--OH

To an equal quantity of the azide Z--Glu(OtBu)--His--N.sub.3 as prepared in A, are added 3.3 g of phenylalanine and 12.6 ml of triethylamine, dissolved in 15 ml of DMF of 0.degree. C. The mixture is left to stand for 3 days at 0.degree. C, after which it is filtered. The filtrate is evaporated to dryness, taken up in ethyl acetate/water, acidified with 5 percent citric acid to pH 3 and then washed with water. The layers are separated, and the organic layer is dried and evaporated.

The residue is recrystallised from acetonitril/ether.

Rf in Am:Py:Wa (5:3:2) = 0.73 (SiO.sub.2).

C. z--glu(OtBu)--His--(N--phenylethyl)amide

The phenylalanine used in B is replaced by .beta.-phenylethylamine to obtain the peptide Z--Glu(OtBu)--His--(N-phenylethyl)amide.

Rf in Bz:EtOH (9:1) = 0.47 (SiO.sub.2).

D. z--glu(OtBu)--His--Phe--NH.sub.2

1 gram of the tripeptide ester as described in A is dissolved in 20 ml of methanol, after which ammonia gas is bubbled through the mixture. Then the mixture is stirred for 1 night, after which the resulting precipitate is filtered. After crystallisation from methanol/ether the amide is obtained.

Melting point: 119.degree.--120.degree. C.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.70 (SiO.sub.2).

E. deprotection of the peptides or peptide derivatives A-D (incl.)

0.7 g of the peptide obtained in A, B, C or D is dissolved in 18 ml of pure methanol, after which 10 percent palladium on charcoal is added. Hydrogen is bubbled through the mixture till no CO.sub.2 escapes anymore, after which the mixture is filtered and the filtrate evaporated in vacuum at a bath temperature of 30.degree. C. The partially deprotected peptide is immediately processed further.

______________________________________ Peptide: Rf in Am:Py:Wa (5:3:2) (SiO.sub.2) ______________________________________ 1. H-Glu(OtBu)-His-Phe-OMe 0.35 2. H-Glu(OtBu)-His-Phe-OH 0.23 3. H-Glu(OtBu)-His-Phe-NH.sub.2 0.47 4. H-Glu(OtBu)-His-(N-phenylethyl)amide 0.41 ______________________________________

F. condensation of D-Met with peptides prepared in E

Boc--D--Met--N.sub.2 H.sub.3 (3.92 g) is dissolved in 75 ml of DMF. The solution is cooled down to 0.degree. C, after which 7.5 ml of 4N HC1/THF are added and the solution is cooled down further to -20.degree. C. Isoamyl-nitrite (2.1 ml) is added, after which the mixture is stirred for 5 minutes and the volume made up to 90 ml ("solution A").

Meanwhile 5 mmol of one of the tripeptides prepared in E has been dissolved in 20 ml of DMF and 2 ml of water. This solution is cooled down to 0.degree. C.

Of "solution A" 30 ml are added to the tripeptide solution. The mixture is left to stand for 72 hours at 0.degree. C, after which the DMF is distilled off and the residue taken up in ethyl acetate/water.

The organic phase is washed with bicarbonate and water, after which the organic phase is dried and the solvent distilled off in vacuum.

The residue is crystallised from methanol-ether.

______________________________________ Peptide: Rf in Bz:EtOH (8:2) on SiO.sub.2 ______________________________________ 1. Boc-D-Met-Glu(OtBu)-His-Phe-OMe 0.42 2. Boc-D-Met-Glu(OtBu)-His-Phe-OH 0.14 3. Boc-D-Met-Glu(OtBu)-His-Phe-NH.sub.2 0.20 4. Boc-D-Met-Glu(OtBu)-His-(N-phenylethyl) 0.40 amide ______________________________________

G. condensation of desamino--Met with the peptides prepared in E

In the same manner as described in F, desaminomethionylhydrazide (5 mmol) is converted into the corresponding azide by means of isoamylnitrite, after which the azide formed is coupled to one of the peptides in E.

______________________________________ Peptide: Rf in Bz:EtOH (8.2) on SiO.sub.2 ______________________________________ 1. Desamino-Met-Glu(OtBu)-His-Phe-OMe 0.36 2. Desamino-Met-Glu(OtBu)-His-Phe-OH 0.11 3. Desamino-Met-Glu(OtBu)-His-Phe-NH.sub.2 0.17 4. Desamino-Met-Glu(OtBu)-His-(N-phenyl- ethyl) amide 0.34 ______________________________________

H. boc--D--Met--Glu(OtBu)--His--Phe--OC.sub.11 H.sub.23

Of the peptide Boc--Met--Glu(OtBu)--His--Phe--OH prepared in F.2, 0.25 g is dissolved in 1.4 ml of undecanol and 0.3 g of Dowex X-8.HCl (basic resin). The mixture is stirred for 7 days at room temperature and filtered, and the filtrate evaporated in vacuum. The undecyl ester is isolated by chromatography of the residue obtained over SiO.sub.2 by means of the eluent benzene/ethanol (9:1). Rf in Bz:EtOH (8:2) = 0.68 (SiO.sub.2).

K. deprotection of the peptides obtained in F, G and H

In a similar manner as described in example II.G the peptides obtained in F, G and H are deprotected.

______________________________________ Peptide Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) on SiO.sub.2 ______________________________________ 1. H-D-Met-Glu-His-Phe-OH 0.27 2. H-D-Met-Glu-His-Phe-OMe 0.35 3. H-D-Met-Glu-His-(N-phenylethyl) 0.36 amide 4. H-D-Met-Glu-His-Phe-NH.sub.2 0.33 5. H-D-Met-Glu-His-Phe-OC.sub.11 H.sub.23 0.39 6. Desamino-Met-Glu-His-Phe-OMe 0.40 7. Desamino-Met-Glu-His-Phe-OH 0.31 8. Desamino-Met-Glu-His-amide 0.38 9. Desamino-Met-Glu-His-(N-phenyl- 0.42 ethyl)amide ______________________________________

L. sulfoxide

Of the tetrapeptide H--Met--Glu--His--Phe--OH 30 mg are dissolved in 2.5 ml of acetic acid after which 15 .mu.l of 30 percent H.sub.2 O.sub.2 are added. The mixture is processed further in the manner described in Example I. The residue is then taken up in 5 ml of t-butanol/water (1:1), after which the mixture is lyophilised.

In the same manner the peptides obtained in K.1, K.2 and K.7 are also converted into the corresponding sulfoxide.

______________________________________ Peptide: Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) on SiO.sub.2 ______________________________________ H-Met (.fwdarw.O)-Glu-His-PheOH 0.26 H-D-Met(.fwdarw.O)-Glu-His-Phe-OH 0.26 H-D-Met(.fwdarw.O)-Glu-His-Phe-OMe 0.33 Desamino-Met(.fwdarw.O)-Glu-His-Phe-OH 0.29 ______________________________________

Example VI

Synthesis of Val--Glu--His--Phe derivatives

A. boc--Val--Glu(OtBu)--His--Phe--NH.sub.2

Boc--Val--OH (0.33 g) is dissolved in 20 ml of DMF, after which 0.81 g of H--Glu(OtBu)--His--Phe--NH.sub.2 (example V.E.3) is added and the pH adjusted to 7.2. After the addition of 0.31 g of DCCI, the mixture is stirred for 5 hours at 0.degree. C and for 20 hours at 20.degree. C. The resulting DCHU is filtered off, after which the filtrate is poured into 100 ml of 0.1 N sodium bicarbonate and the precipitate stirred for 1 hour at 0.degree. C. After filtration the residue is dried and recrystallised twice from ethyl acetate/ether. Yield: 0.51 g; m.p. 137.degree. (dec.). Rf in Bz:EtOH (8:2) = 0,27 on SiO.sub.2.

B. h-val-Glu-His-Phe-NH.sub.2

Deprotection of the protected tetrapeptide-amide of A by the process described in example II.G yielded the acetate of the above peptide.

EXAMPLE VII

Synthesis of D-Met-Glu-His-(N-phenylalkyl)amides

Boc-D-Met-Glu(OtBu)-His-N.sub.2 H.sub.3 (1.17 g; example IX.A) is dissolved in 20 ml of DMF. This solution is cooled down, after which 3 ml of 2 N HCl/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 at -20.degree. C.

To the azide solution 3 mmol of the required amine are added and adjusted with TAA to pH 7.

The reaction mixture is stirred for 70 hours at 0.degree. C, after which it is evaporated to dryness in vacuum and the residue dissolved in aqueous ethyl acetate.

The organic phase is washed with water, bicarbonate and water, and dried.

The residue is evaporated to dryness, then stirred with ether/petroleumether, and dried again.

By the process described in example II.G the protecting groups are removed.

______________________________________ Amine used: H-D-Met-Glu-His(N-phenylalkyl) amide.HAc Rf in Bu:Ac:Wa (4:1:1) ______________________________________ benzylamine 0.20 phenylethylamine *.sup.) 0.21 1-amphetamine 0.19 p-hydroxyphenylethylamine 0.19 p-methylbenzylamine 0.20 ______________________________________ *.sup.) cf. Example V.K.3

EXAMPLE VIII

Synthesis of A--Glu--His--(N--phenylalkyl)amides (A = Val or .beta.-Ala)

A. Boc--Val--Glu(OtBu)--His--OMe (1.11 g; example III.A) is dissolved in 20 ml of methanol, after which 1 ml of hydrazine hydrate is added. The mixture is stirred for 6 hours at room temperature, after which the methanol is distilled off in vacuum, the remaining residue stirred with water and then dried.

The dry residue is then dissolved in DMF and coupled to 1-amphetamine(1-phenylisopropylamine) by the azide method as described in example VII.

In the manner described in example II.G, the resulting compound is deprotected to obtain the peptide H--Val--Glu--His--(N--1-phenylisopropyl)amide.acetate, which peptide is lyophilised.

Rf in Bu:Ac:Wa (4:1:1) = 0.18 on SiO.sub.2. Starting substance 0.53.

B. boc--.beta.--Ala--Glu(OtBu)--His--OMe is converted into the corresponding H--.beta.--Ala--Glu--His--(N--benzyl)amide in the same manner as described in A on the understanding that instead of 1-amphetamine now benzylamine is used.

Rf in Bu:Ac:Wa (4:1:1) = 0.16 on SiO.sub.2.

EXAMPLE IX

Synthesis of D--Met--Glu--His--Phe--Arg

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

Boc--D--Met--Glu(OtBu)--His--OMe (3.2 g; example II.A) is 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, after which the methanol is distilled off in vacuum and the residue stirred with water.

After being dried, the hydrazide is at once processed further.

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

B. boc--D--Met--Glu(OtBu)--His--Phe--Arg--OH

Of the tripeptide-hydrazide (A; 1.17 g) is dissolved in 20 ml of DMF. After the addition of 3 ml of 2N HCl in THF, at 0.degree. C, the mixture is cooled down further to -20.degree. C, after which 0.27 ml of isoamyl-nitrite is added. The mixture is stirred for 6 minutes after which the azide solution is added to 2 mmol of the peptide H-Phe-Arg-OH in 5 ml of DMF, after which the pH is adjusted to 7.3 with TAA.

The reaction mixture is stirred for 70 hours at 0.degree. C and then evaporated. The residue is stirred into ethyl acetate. Evaporation of this ethyl acetate gives an amorphous pentapeptide.

Rf in Bz:EtOH (8:2) = 0.17 on SiO.sub.2.

C. h--d--met--Glu--His--Phe--Arg--OH

Deprotection of the pentapeptide obtained in B with trifluoro acetic acid yielded the pentapeptide.diacetate after the exchange with Dowex X-8 in the acetate form.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.18 on SiO.sub.2.

EXAMPLE X

Sulfoxide of H--Met--Glu--His--Phe--Arg--OH, H--Met--Glu--His--Phe--Lys--OH and H--D--Met--Glu--His--Phe--Arg--OH

In the manner described in example I the pentapeptides H--Met--Glu--His--Phe--Arg--OH, H--Met--Glu--HIs--Phe--Lys--OH and H--D--Met--Glu--His--Phe--Arg--OH (example IX.C) are oxidised by means of 30% H.sub.2 O.sub.2 in acetic acid.

______________________________________ Peptide: Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) on ______________________________________ SiO.sub.2 sulfoxide of H-Met-Glu-His-Phe-Arg-OH 0.13 sulfoxide of H-Met-Glu-His-Phe-Lys-OH 0.21 sulfoxide of H-D-Met-Glu-His-Phe-Arg-OH 0.17 ______________________________________

EXAMPLE XI

Synthesis of A--Glu--His--Phe--Lys derivatives (A = .alpha.-Me--Ala, Ala, .beta.--Ala or Desamino--Met)

A. h--phe--Lys(Boc)--OtBu

Coupling of 4.2 g of Z--Phe--ONP to H--Lys(Boc)--OtBu yielded the dipeptide Z--Phe--Lys(Boc)--OtBu as a viscous oil. Rf in Bz:EtOH (9:1) = 0.57 on SiO.sub.2 ; yield 79 percent. The dipeptide H--Phe--Lys(Boc)--OtBu is obtained in 95 percent yield by hydrogenation of the peptide Z--Phe--Lys(Boc)--OtBu in methanol with 10 percent palladium on charcoal.

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

B. boc--(.alpha.--Me)Ala--Glu(OtBu)--His--N.sub.2 H.sub.3

Of the protected peptide Boc--(1--Me)--Ala--Glu(OtBu)--His--OMe (example III.E; 2.9 g) is converted into the hydrazide with hydrazine hydrate in the manner described in example IX.A. melting point: 218.degree.--225.degree. C dec.

C. In the same manner as described in B the methyl esters: Boc--Ala--Glu(OtBu)-- His--OMe, (Example III.C), Boc--.beta.--Ala--Glu(OtBu)--His--OMe (Example III.D) and Desamino--Met--Glu(OtBu)--His--OMe (Example II.B) are converted into the corresponding hydrazides.

D. h--(.alpha.--me)Ala--Glu--His--Phe--Lys--OH

The peptide-hydrazide of B (1 mmol) is dissolved in 10 ml of DMF. The mixture is cooled, after which 3 ml of 1N HCl in THF and 0.13 ml of isoamyl-nitrite are added. The mixture is then stirred for 6 minutes at -20.degree. C. The thus obtained azide is added to a solution of 1 mmol of H--Phe--Lys(Boc)--OtBu (from A) in 5 ml of DMF. After the pH has been adjusted to 7.4 with TAA, the mixture is stirred for 70 hours at 0.degree. C. The DMF is distilled off in vacuum, the residue dissolved in aqueous ethyl acetate and the organic phase washed with water, bicarbonate and water. The ethyl acetate is dried and then distilled off in vacuum. The residue is chromatographed over SiO.sub.2 and the peptide Boc--(.alpha.--Me)Ala--Glu(OtBu)--His--Lys(Boc)--OtBu isolated.

Rf in Bz:EtOH (8:2) = 0.74 on SiO.sub.2.

Deprotection of this protected peptide in the manner described in example II.G, gives the above pentapeptide.acetate in 63 percent yield.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.18 on SiO.sub.2.

In the same manner as described in D the peptide methyl esters obtained in C are converted into the corresponding azide and coupled with H--Phe--Lys(Boc)--OtBu (A).

Removal of the protecting groups, according to the manner described in Example II.G, gives the following unprotected peptides as the acetates in 60-70 percent yield:

Peptide: Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) on SiO.sub.2 ______________________________________ H-.beta.-Ala-Glu-His-Phe-Lys-OH 0.18 H-Ala-Glu-His-Phe-Lys-OH 0.20 Desamino-Met-Glu-His-Phe-Lys-OH 0.24 H-D-Met-Glu-His-Phe-Lys-OH 0.18 ______________________________________

EXAMPLE XII

Synthesis of A--Glu--His--Phe--NH--(CH.sub.2).sub.5 --NH.sub.2 (A = D--Met,, Gly or Desamino--Met)

A. h--d--met--Glu--His--Phe--NH--(CH.sub.2).sub.5 --NH.sub.2

Of the tripeptide-hydrazide Boc--D--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 (example IX.A) 1.17 g are dissolved in 20 ml of DMF. After the addition of 3 ml of 2 N HCl in THF, at 0.degree. C, the mixture is cooled further to -20.degree. C. Then 0.27 ml of isoamyl-nitrite is added to this mixture, whereupon the mixture is stirred for some time. The azide solution obtained is then added to 2 mmol of the compound H--Phe--NH--(CH.sub.2).sub.5 --N.sub.H --Boc (obtained by hydrogenation of the corresponding compound Z--Phe--NH--(CH.sub.2).sub.5 --NH--Boc, m.p. 131.degree.-133.degree. C) in 5 ml of DMF, after which the pH is adjusted to 7.5 with TAA.

The reaction mixture is stirred for 70 hours at 0.degree. C, after which it is evaporated and the residue stirred into ethyl acetate. After evaporation of the ethyl acetate a yellowish white amorphous powder is obtained, which is treated further in the manner described in example II.G. After treatment with Dowex X-8 in the acetate form, the H--D--Met--Glu--His--Phe--NH--(CH.sub.2).sub.5 --NH.sub.2.diacetate is obtained.

Rf in Bu:Py:Ac:Wa (2:3/4:1/4:1) = 0.35 on SiO.sub.2.

B. h--gly--Glu--His--Phe--NH(CH.sub.2).sub.5 --NH.sub.2

In the same manner as described (in A) Boc--Gly--Glu(OtBu)--His--N.sub.2 H.sub.3 (obtained from the corresponding methylester) is converted into the diacetate of H--Gly--Glu--His--Phe--NH-(CH.sub.2).sub.5 -NH.sub.2.

Rf in Bu:Py:Ac:Wa (2:3/4:1/4:1) = 0.32 on SiO.sub.2.

C. desamino--Met--Glu--His--Phe--NH--(CH.sub.2).sub.5 --NH.sub.2

In the same manner as described (in A) Desamino--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 (see Example XI.c) is converted into the acetate of Desamino--Met--Glu--His--Phe--NH--(CH.sub.2).sub.5 --NH.sub.2.

Rf in Bu:Py:Ac:Wa (2:3/4:1/4:1) = 0.38.

EXAMPLE XIII

Synthesis of Val--Glu--His--Phe--Arg--Tra

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

Starting from the tripeptide ester (example III.A) the hydrazide Boc--Val--Glu(OtBu)--His--N.sub.2 H.sub.3 is obtained in the manner described in example VIII.A.

Rf in Am:iPro:Wa (10:4:5) = 0.33 on SiO.sub.2.

B. boc--Arg(NO.sub.2)--Tra

Boc--Arg(NO.sub.2)--OH (5.76 g) is dissolved in 70 ml of DMF and 2.52 ml. of TAA. This solution is cooled to -10.degree. C, after which 2,38 ml of isobutyl chloroformate are added and the mixture is stirred for 10 minutes at -10.degree. C. To this solution is added a solution of 2.9 g of tryptamine in 10 ml of DMF and 2.32 ml of TAA, the temperature being maintained at about -10.degree. C.

The mixture is stirred for 30 minutes at -10.degree. C, for 2 hours at 0.degree. C and for 18 hours at 20.degree. C, after which the solvent is evaporated in vacuum and the residue taken up in ethyl acetate/water. The organic phase is washed with water, bicarbonate (5 percent) and water, and then dried and evaporated.

Yield 7.4 g of oil; Rf in Bz:EtOH (8:2) = 0.54 on SiO.sub.2.

C. z--phe--Arg(NO.sub.2)--Tra and H--Phe--Arg--Tra

Z--Phe--ONP (2.1 g) is dissolved in 10 ml of DMF and 10 ml of ethyl acetate. Then 1.81 g of H--Arg(NO.sub.2)--Tra (obtained by cleavage of the amide of B in the manner described in example II.G) are added, after which the mixture is stirred for 30 minutes at -10.degree. C and for 20 hours at 20.degree. C. The reaction mixture is processes as described in B.

Yield: 1.3 g; melting point: 131.degree.-133.degree. C.

Rf in Bz:EtOH (8:2) = 0.47 on SiO.sub.2.

Of the compound obtained 1.3 g are converted into H--Phe--Arg--Tra by hydrogenation over palladium 10 percent on charcoal. Yield 72 percent.

D. h--val--Glu--His--Phe--Arg--Tra

The hydrazide prepared in A is converted into the azide by means of isoamyl-nitrite, in a conventional manner, and coupled to the peptide H--Phe--Arg--tryptamide of C. The reaction mixture is stirred for 70 hours at 0.degree. C and poured out into 0.1 N sodium bicarbonate to precipitate the peptide.

After filtration the precipitate is dissolved in DMF and solution poured out into a tenfold quantity of ethyl acetate. Filtration gives a residue with Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.54 on SiO.sub.2.

Cleavage in the manner described before (example II.G) gives the acetate of H-Val-Glu-His-Phe-Tra in 33 percent yield.

Rf in the same solvent mixture = 0.18.

EXAMPLE XIV

D--met--Glu--His--Phe--Lys--tryptamide derivatives

A. z--phe--Lys(Boc)--Tra

Starting from 3.4 g of Z--Phe--ONP and 3 g of H--Lys(Boc)--Tra, prepared from Z--Lys(Boc)--Tra (melting point: 77.degree.-80.degree. C), the Z--Phe--Lys (Boc)--Tra is obtained in the manner described in example XIII.C. Yield: 65 percent. Melting point: 125.degree.-129.degree. C.

Rf in Bz:EtOH (8:2) = 0.70 on SiO.sub.2.

B. h--phe--Lys(Boc)--Tra

2 grams of the peptide described above (of A) are dissolved in 25 ml of methanol and hydrogenated in a conventional manner, in the presence of 10 percent palladium/charcoal. After evaporation to dryness a foam is obtained. Yield: 95 percent.

Rf in Bu:Ac:Wa (4:1:1) = 0.80 on SiO.sub.2.

C. boc--A--Glu(OtBu)--His--Phe--Lys(Boc)--Tra (A = Met, D-Met or .beta.---Ala)

The tryptamide derivative of B in DMF (1.5 g) and the hydrazide Boc--A--Glu(OtBu)--His--N.sub.2 H.sub.3 are coupled by means of the azide method described in previous examples. After evaporation of the DMF, the residue is taken up in aqueous ethyl acetate and the organic phase washed with water and bicarbonate (5 percent).

The organic phase is distilled off, after which the residue is recrystallised from ethyl acetate/alcohol/ether.

______________________________________ Peptide: Rf in Bz:EtOH (8:2) ______________________________________ Boc-Met-Glu(OtBu)-His-Phe-Lys(Boc)-Tra 0.52 Boc-D-Met-Glu(OtBu)-His-Phe-Lys(Boc)-Tra 0.52 Boc-.beta.-Ala-Glu(OtBu)-His-Phe-Lys(Boc)-Tra 0.49 ______________________________________

D. desamino--Met--Glu(OtBu)--His--Phe--Lys(Boc)--Tra

In the same manner as described in C, the hydrazide Desamino--Met--Glu(OtBu)--His--N.sub.2 H.sub.3, obtained from the corresponding methylester (II B) is coupled to H--Phe--Lys(Boc)--Tra (B) by means of the azide method previously described.

Rf in Bz:EtOH (8:2) = 0.45 on SiO.sub.2.

E. removal of protecting groups from the peptides obtained in C and D

In the manner described in example II.G the above peptides are deprotected by means of TFA, after which the TFA molecules of the peptide salt thus obtained is exchanged for acetic acid molecules by means of an ionexchanger in the acetate form (e.g. Dowex X-8 in acetate form).

______________________________________ Peptide: Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) on SiO.sub.2 ______________________________________ H-Met-Glu-His-Phe-Lys-Tra 0.20 H-D-Met-Glu-His-Phe-Lys-Tra 0.18 H-.beta.-Ala-Glu-His-Phe-Lys-Tra 0.17 Desamino-Met-Glu-His-Phe-Lys-Tra 0.25 ______________________________________

F. sulfoxides

45 mg of one of the peptide derivatives, obtained in E, are dissolved in 2.5 ml of acetic acid, after which 15 .mu.l of 30 percent hydrogen peroxide are added. The mixture is treated in the manner described in example I. The residue is taken up in butanol/water (1:2) and lyophilised.

______________________________________ Peptide Rf in Bu:Py:Ac:Wa (4:33/4:1/4:1) on SiO.sub.2 ______________________________________ H-D-Met(.fwdarw.O)-Glu-His-Phe-Lys-Tra 0.16 H-L-Met(.fwdarw.O)-Glu-His-Phe-Lys-Tra 0.18 Desamino-Met(.fwdarw.O)-Glu-His-Phe-Lys-Tra 0.22 ______________________________________

EXAMPLE XV

Synthesis of H--A--Glu--His--Phe--Arg--Trp derivatives

A. boc--Arg(NO.sub.2)--Trp--ONB

Boc--Arg(NO.sub.2)--OH (5.76 g) are dissolved in 70 ml of DMF and 2.52 ml of triethylamine. The solution is cooled down to -10.degree. C, after which 2.38 ml of isobutyl chloroformate are added and the mixture is stirred for 10 minutes at -10.degree. C. To this solution a solution of 6.14 g of H--Trp--ONB.HCl in 40 ml of cooled DMF and 3.01 ml of triethylamine are added.

The mixture is stirred for 30 minutes at -10.degree. C, for 3 hours at 0.degree. C and for 20 hours at 20.degree. C, after which the solvent is evaporated in vacuum and the residue taken up in ethyl acetate/water (1:1). The organic phase is washed with water, 5 percent sodium bicarbonate and water, dried and evaporated to dryness in vacuum. Yield: 8.5 g of oil.

Rf in Bz:EtOH (8:2) = 0.79 (SiO.sub.2).

B. h--arg(NO.sub.2)--Trp--ONB

1 gram of the peptide prepared in A is dissolved in 20 ml of methylene chloride, after which hydrogen chloride gas is bubbled through, while cooling. The solution is stirred for 1 hour, after which the precipitate is filtered off and thoroughly washed with dry methylene chloride. The precipitate was immediately processed further.

Rf in Am:Py:Wa (5:3:2) = 0.66 on SiO.sub.2 (Rf starting substance 0.93).

C. z--phe--Arg(NO.sub.2)--Trp--ONB

3 grams of the dipeptide of B are dissolved in 20 ml of DMF. This solution is cooled down to 0.degree. C, after which 1.12 ml of TAA and 2.12 g of Z--Phe--ONP are added. The reaction mixture is stirred for 2 hours at 0.degree. C and for 20 hours at 20.degree. C, and evaporated in vacuum.

The oily residue is dissolved in ethyl acetate/water (2:1) and processed as described in A.

The organic phase is evaporated, the residue dissolved in 5 ml of methanol and poured slowly into 100 ml of dry ether to precipitate the peptide.

Yield: 2.9 g; [.alpha.].sub.D.sup.22 = -15.degree. (c = 1, DMF); Rf in Bz:EtOH (8:2) 0.48 on SiO.sub.2.

D. h--phe--Arg--Trp--OH

2 grams of the above-mentioned peptide of C are dissolved in 40 ml of acetic acid, after which 1 g of 10 percent palladium/charcoal is added and the mixture hydrogenated for 2 days (Parr.). After filtration the acetic acid is evaporated in vacuum, after which the residue is stirred into dry ether. Yield: 98 percent. The slightly coloured foam is dried over solid potassium hydroxide. The substance contains 1.1 molecule of acetic acid.

Rf in Am:Py:Wa (5:3:2) = 0.17 on SiO.sub.2.

E. boc--D--Met--Glu(OtBu)--His--Phe--Arg--Trp--OH

Of the tripeptide Boc--D--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 (example IX.A) 1.17 g are dissolved in 20 ml of DMF. After the addition of 3 ml of 2 N HCl in THF at 0.degree.C the mixture is cooled to -20.degree. C. Then 0.27 ml if isoamyl-nitrite is added. The mixture is stirred for a few minutes, after which this "azide" solution is added to 8 ml of DMF, in which 2 mmol of the tripeptide of D (H--Phe--Arg--Trp--OH.acetate) have been dissolved. The pH of the mixture is then adjusted to 7.3 with TAA. The reaction mixture is stirred for 70 hours at 0.degree. C, after which it is evaporated at reduced pressure, and the residue stirred into aqueous ethyl acetate.

Then the organic phase is washed with water, after which the organic layer is dried. After filtration of this organic layer, petroleum ether is added to precipitate the peptide.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.52 on SiO.sub.2.

F. boc--Gly--Glu(OtBu)--His--Phe--Arg--Trp--OH

Of the tripeptide Boc--Gly--Glu(OtBu)--His--N.sub.2 H.sub.3, prepared from Boc--Gly--Glu(OtBu)--His--OMe by conversion with hydrazine hydrate, 2 mmol are coupled to the peptide H--Phe--Arg--Trp--OH in the manner described in E. The reaction mixture is stirred for 70 hours, after which it is poured out into 100 ml of ethyl acetate and extracted twice with saliferous water.

The organic phase is evaporated to dryness. The residue is stirred with dry ethyl acetate/petroleum ether (9:1) and then dried.

Melting point: 214.degree.-217.degree. (dec.).

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.48 on SiO.sub.2.

______________________________________ G. In the same manner are prepared: Rf*.sup.) ______________________________________ 1. Boc-Val-Glu(OtBu)-His-Phe-Arg-Trp-OH 0.49 2. Boc-.beta.-Ala-Glu(OtBu)-His-Phe-Arg-Trp-OH 0.48 3. Boc-Ala-Glu(OtBu)-His-Phe-Arg-Trp-OH 0.52 4. Boc-(.alpha.-Me)Ala-Glu(OtBu)-His-Phe-Arg-Trp-OH 0.54 5. Desamino-Met-Glu(OtBu)-His-Phe-Arg-Trp-OH 0.48 ______________________________________ *.sup.) Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) on SiO.sub.2.

______________________________________ H. The peptides described in E, F and G are deprotected in the manner described in example II.G. The acetates of the following peptides are obtained: Rf*.sup.) ______________________________________ 1. H-Val-Glu-His-Phe-Arg-Trp-OH 0.20 2. H-Gly-Glu-His-Phe-Arg-Trp-OH 0.18 3. H-.beta.-Ala-Glu-His-Phe-Arg-Trp-OH 0.19 4. H-D-Met-Glu-His-Phe-Arg-Trp-OH 0.19 5. H-Ala-Glu-His-Phe-Arg-Trp-OH 0.20 6. H-(.alpha.-Me)Ala-Glu-His-Phe-Arg-Trp-OH 0.21 7. Desamino-Met-Glu-His-Phe-Arg-Trp-OH 0.23 ______________________________________ *.sup.) Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) on SiO.sub.2.

EXAMPLE XVI

Synthesis of D--Met--Glu--His--Phe--Lys--Trp

A. z--phe--Lys(Boc)--Trp--OH

Z--Phe--ONP ester (2.1 g) is coupled to 2.24 g of H--Lys(Boc)--Trp--OMe as described in example XIII.C. The oily ester is isolated and then saponified with 1.1 equivalent of sodium hydroxide in 10 ml of methanol. On acidification the tripeptide acid precipitates, which is recrystallised from methanol/water. Yield: 2.61 g.

Rf in Bz:EtOH (8.2) = 0.23 (SiO.sub.2).

B. h--phe--Lys(Boc)--Trp--OH

Hydrogenation of the tripeptide derivative of A gives a foam in quantitative yield. Crystallisation from water/methanol gives the tripeptide in 71 percent yield.

Rf in Bz:EtOH (8:2) = 0.05 (SiO.sub.2).

C. r--glu(OtBu)--His--Phe--Lys(Boc)--Trp--OH (R = Desamino--Met or Boc--D--Met)

In the manner described in example VII, R--Glu(OtBu)--His--N.sub.2 H.sub.3, in which R means: Desamino--Met or Boc--D-Met, is coupled to the tripeptide prepared in B by means of the azide method. The DMF solution is poured into a tenfold quantity of water containing acetic acid (pH 3-4) to obtain a precipitate. After filtration and stirring with water the peptide is isolated as an amorphous substance in 55-60 percent yield.

D. r--glu--His--Phe--Lys--Trp--OH (R = Desamino--Met or H--D--Met)

Cleavage of the protected peptides prepared in C, in the manner described in example II.G, gives the acetate of the corresponding unprotected peptides.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.21 on SiO.sub.2 (for the H--D--Met--peptide) and 0.25 (for the Desamino--Met--peptide).

E. sulfoxide of R--Glu--His--Phe--Lys--Trp--OH (R = Desamino--Met or H--D--Met)

In the manner described in example I the peptides obtained in D are oxidized in acetic acid containing 30 percent H.sub.2 O.sub.2.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.19 on SiO.sub.2 for the D--Met(.fwdarw.O) peptide and 0.22 for the Desamino--Met(.fwdarw.O) peptide.

EXAMPLE XVII

Sulfoxide of H--Met--Glu--His--Phe--Arg--Trp--OH, and of H--Met--Glu--His--Phe--Lys--Trp--OH

By oxidizing H--Met--Glu--His--Phe--Arg--Trp--OH and H--Met--Glu--His--Phe--Lys--Trp--OH with 30 percent hydrogenperoxide in acetic acid (see example I) the corresponding sulfoxides are obtained which are purified via counter current.

System in counter current: Bu:Ac:Wa (4:1:5).

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.17 for the sulfoxide of the "Lys-peptide" and 0.20 for the sulfoxide of the "Arg-peptide."

EXAMPLE XVIII

Synthesis of A--Glu--His--Phe--Arg--Trp--Gly peptides

A. boc--D--Met--Glu(OtBu)--His--Phe--Arg--Trp--Gly--OH

In the manner described in example IX.B the Boc--D--Met--Glu(OtBu)--His--N.sub.2 H.sub.3 is coupled to H--Phe--Arg--Trp--Gly--OH.acetate.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.49 on SiO.sub.2.

B. h-d-met-Glu-His-Phe-Arg-Trp-Gly-OH

Cleavage of the protected peptide obtained in A in the manner described in example II.G, gives the acetate of H--D--Met--Glu--His--Phe--Arg--Trp--Gly--OH.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.16 on SiO.sub.2.

C. sulfoxide of H--D--Met--Glu--His--Phe--Arg--Trp--Gly--OH

The peptide prepared in B is oxidized by means of acetic acid and 30 percent H.sub.2 O.sub.2 in the manner described in example I and then lyophilised.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.14 on SiO.sub.2.

D. in the same manner the following peptides are prepared:

H--gly--Glu--His--Phe--Arg--Trp--Gly--OH

H--.beta.--ala--Glu--His--Phe--Arg--Trp--Gly--OH

H--(.alpha.--me)Ala--Glu--His--Phe--Arg--Trp--Gly--OH

H--val--Glu--His--Phe--Arg--Trp--Gly--OH

Desamino--Met--Glu--His--Phe--Arg--Trp--Gly--OH

Desamino--Met(.fwdarw.O)--Glu--His--Phe--Arg--Trp--Gly--OH

EXAMPLE XIX

Sulfoxide of H--Met--Glu--His--Phe--Arg--Trp--Gly--OH

In the manner described in example I the heptapeptide H--Met--Glu--His--Phe--Arg--Trp--Gly--OH is oxidized and then lyophilised.

Rf in Bu:Py:Ac:Wa (4:3/4:1/4:1) = 0.16 on SiO.sub.2.

EXAMPLE XX

Sulfone of H--Met--Glu--His--Phe--Lys--Trp--Gly--OH.acetate

Of the peptide H--Met--Glu--His--Phe--Lys--Trp--Gly--OH 200 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 percent hydrogen peroxide is added.

The mixture is stirred for 2 hours at a temperature of about 10.degree. C. Then the mixture is stirred with Dowex X-8 in the acetate form, after which the mixture is filtered and the filtrate lophilised.

Rf in Bu:Ac:Wa (4:1:1) = 0.21 on SiO.sub.2.

In the same manner were prepared:

H-met(.fwdarw.O.sub.2)--Glu--His--Phe--Lys--Tra

desamino-Met(.fwdarw.O.sub.2)--Glu--His--Phe--Lys--Trp--OH

desamino-Met(.fwdarw.O.sub.2)--Glu--His--Phe--OH.

EXAMPLE XXI

Preparation of H--Met(d,.fwdarw.0)--Glu--His--Phe--Arg--Trp--Gly--OH

A. boc--Met(d,.fwdarw.O)--OSu

26 grams of L-methionine are oxidized to the corresponding sulfoxide according to the procedure of Toennies and Kolb cf. Biol. Chem. 128, 399 (1939), yield: 24 g, m.p. 250.degree. C (dec.). Both diastereomeric sulfoxides are resolved following the method as described by Lavine, J. Biol. Chem. 169, 477 (1947).

Yield:

L-methionine(d,.fwdarw.O) 8.7 g [.alpha.].sub.D.sup.22 + 110.degree. (c = 1, H.sub.2 O)

L-methionine(1,.fwdarw.O) 1.8 g [.alpha.].sub.D.sup.22 -75.degree. (c = 1, H.sub.2 O).

These isomers were then convertedto the Boc-protected derivatives by means of Boc-N.sub.3 in a conventional manner:

Boc-Met(d,.fwdarw.O)--OH m.p. 135.degree.-137.degree. C [.alpha.].sub.D.sup.22 + 42.degree. (c = 1, DMF)

Boc-Met(1,.fwdarw.O)--OH m.p. 68.degree. C [.alpha.].sub.D.sup.22 - 58.degree. (c = 1, DMF)

and thereupon converted into the corresponding N-hydroxy succinimide ester by treatment with DCCI and HOSu. These active ester derivatives are used at once for the following coupling reaction.

B. h-met(d,.fwdarw.O)--Glu--His--Phe--Arg--Trp--Gly--OH

929 Boc-Met(d--sulfoxide)--OSu are dissolved in 20 ml DMF. The solution is cooled to 0.degree. C whereupon is added a cooled solution of 2.38 g of H--Glu(OtBu)--His--Phe--Arg--Trp--Gly--OH and 0.83 ml of N-ethyl morpholine in 25 ml of DMF.

The reaction mixture is stirred for 3 hours at 0.degree.C, 70 hours at room temperature and then evaporated. The residue is stirred with 40 ml of ethyl acetate and 10 ml of water, filtered, washed with petroleum ether and dried.

Yield: 2.1 g; Rf 0.44 (Bu:Py:Ac:Wa -- 4:3/4:1/4:1) on SiO.sub.2.

2 grams of Boc-Met(d-sulfoxide)--Glu(OtBu)--His--Phe--Arg--Trp--Gly--OH are treated with 20 ml of 90 percent TFA for 30 minutes. The solution is then stirred into 300 ml of ether whereupon the precipitate is filtered off, washed with ether and dried over solid potassium hydroxide. The trifluoro acetate is dissolved in 50 ml of t-butanol/water (1:1) and converted into the acetate by treatment with Dowex X-8 in the acetate form. After filtration the filtrate is lyophilised.

Yield 1.45 g; Rf 0.24 in Bu:Py:Ac:Wa (2:3/4:1/4:1) on SiO.sub.2.

Claims

What is claimed is:

1. A peptide of the formula:

A--L--Glu--L--His--X,

in which A is a member of the group consisting of 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 moiety H.sub.2 N--B--CO--, in which B is alkylene having 1-6 carbon atoms, and in which X is selected from the group consisting of hydroxy, (N-phenylalkyl)-amino of the formula ##SPC7##

in which R.sub.1 is selected from the group consisting of hydrogen and hydroxy and Alk is alkylene with 1-6 carbon atoms, and the group L--Phe--Y in which Y is selected from the group consisting of hydroxy and (N-aminoalkyl)-amino selected from the group consisting of descarboxy-lysyl and descarboxy-arginyl, the groups L--Lys--Z and L--Arg--Z, in which Z is selected from the group consisting of hydroxy, the group L--Trp--OH, the group L--Trp--Gly--OH, and a (N-3-indolylethyl) amino group, 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 (1-6C) substituted amides of those peptides having a free carboxyl group, esters derived from aliphatic or phenylaliphatic alcohols with 1-18 carbon atoms, and metal complexes thereof.

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

A.sub.1 --L--Glu--L--His--X

in which A.sub.1 is selected from a sulfoxide and a sulfone of H-Met and X has the meanings indicated in claim 1.

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

A.sub.2 --L--Glu--L--His--X

in which A.sub.2 is selected from desamino--Met, desamino--Met(.fwdarw.O) and desamino--Met(.fwdarw.O.sub.2) and X has the meanings indicated in claim 1.

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

A.sub.3 --L--Glu--L--His--X

in which A.sub.3 stands for .beta.--Ala and X has the meanings indicated in claim 1.

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

A--L--Glu--L--His--L--Phe--L--Lys--(N-3-indolyl ethyl)amine

in which A has the meanings indicated in claim 1.

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

A--L--Glu--L--His--L--Phe--L--Arg--(N--3--indolyl ethyl)amine

in which A has the meanings indicated in claim 1.

7. Metal complexes of the peptides claimed in claim 1.