CS237319B2 - A method for producing gonadotropin release peptides from pituitary mammals - Google Patents

Abstract

A method for producing peptides inhibiting the release of gonadotropins from the pituitary gland of mammals of the general formula X-Ri-Rz-Rs-Ser-Tyr-Rd-Rs-Arg-Pro-Rs, where the individual substituents have the meaning given in the definition of the subject of the invention, as well as non-toxic salts of these peptides, in which at least one group X* 1 to X5 is cleaved from the intermediate of the general formula X4-Ri-R2-R3-Ser(χ2) -Tyr-(X3 J -R4-R5- -Arg(X4)-Pro-X5 and optionally the resulting peptide is converted into a non-toxic salt.

Description

The invention relates to a method for producing peptides that inhibit the release of gonadotropins from the pituitary gland of mammals, including humans; said peptides can prevent ovulation and/or inhibit the release of steroids. These are peptides that cause inhibition of the function of the gonads and thus the release of steroid hormones, progesterone and testosterone.

The pituitary gland is attached by a stalk to the base of the brain at a place called the hypothalamus. The pituitary gland has two lobes, the anterior and posterior. The posterior lobe of this gland produces and releases into the circulation two hormones that are also produced in the hypothalamus, namely vasopressin and oxytocin. The anterior lobe of the pituitary gland secretes a number of hormones that are complex proteins or glycoprotein molecules and are carried by the blood to various organs and promote the secretion of other hormones from peripheral organs. These hormones are mainly follicle-stimulating hormone FSH and luteinizing hormone LH, which are also usually called gonadotropins or gonadotropic hormones. These hormones control the function of the gonads, especially the production of testosterone in the testes and progesterone and estrogen in the ovaries, and they also control the maturation of gametes.

Hormone release by the anterior pituitary usually requires the prior release of another hormone produced in the hypothalamus. One of the hypothalamic hormones directly triggers the release of gonadotropic hormones, particularly LH. This hypothalamic hormone that releases LH is called LRF, although it is sometimes also referred to as LH-RH and GnRH. LRF has been isolated and analyzed as a decapeptide with the structure p-Glu/his-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2.

Peptides are compounds that contain 2 or more amino acids, in which the carboxyl group of one amino acid is bonded to the amino group of another. The LRF formula, as shown above, corresponds to the common type of peptide, where the amino group is on the left and the carboxyl group on the right. The position of the amino acid residue is identified by the number of amino acid residues from left to right. In the case of LRF, the hydroxyl group of the carboxyl group of glycine has been replaced by an amino group.

Abbreviations for individual amino acid residues are commonly used and are based on the names of the amino acids, p-Glu is pyroglutamic acid, His is histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, Gly is glycine, Leu is leucine, Arg is arginine and Pro is proline. With the exception of glycine, the amino acids in the peptides of the invention are in the L-configuration unless otherwise indicated.

It is known that the substitution of D-amino acids for glycine at position 6 of the decapeptide LRF yields a peptide material that is 1 to 35 times more potent than LRF in releasing LH and other gonadotropins in the mammalian pituitary gland. This effect occurs when the analog is administered to mammals intravenously, subcutaneously, intramuscularly, orally, nasally, or vaginally.

It is also known that the substitution of various amino acids for His or the omission of H,is at position 2 of the decapeptide produces an analogue with inhibitory activity on the release of LH and other gonadotropins by the mammalian pituitary gland. Different degrees of inhibition of LH release are achieved when His is omitted (des His) or replaced by D-Ala, D-Phe or Gly. The inhibitory activity of peptides modified at position 2 can be further increased by introducing a D-amino acid instead of Gly at position 6 of the decapeptide. For example, a peptide of the formula p-Glu-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH2 is four times more effective as an inhibitor of gonadotropin release than the same peptide containing Gly at position 6.

Some female mammals that do not have an ovulatory cycle and are completely healthy will begin to secrete normal amounts of the gonadotropins LH and FSH after administration of LRF. This means that administration of LRF is probably appropriate for those cases of sterility and infertility that are caused by a disorder in the hypothalamus.

It is also conceivable that ovulation may be prevented by administration of LRF analogs. For this reason, LRF analogs are being investigated as potential contraceptives that can control conception. It would be desirable to produce peptides that have a strong antagonistic effect and inhibit LH secretion and steroid release from mammalian gonads.

The subject of the invention is a method of producing peptides that inhibit the release of gonadotropin in mammals including humans and thus can be used to inhibit the release of steroids from the gonads of both males and females. The improved LRF analogs antagonize LRF and thus have an inhibitory effect on the reproductive processes in mammals. They can be used to inhibit the production of gonadotropins and sex hormones in various circumstances including premature puberty, hormone-dependent tumors, dysmenorrhea and endometriosis.

Thus, the method of the invention provides peptides that potently inhibit gonadotropins and inhibit their release by the pituitary gland of mammals, including humans, and/or inhibit steroid release by the gonads. These peptides are LRF analogs, where dehydroproline or meta-thiazolidine-2-carboxylic acid is present in position 1 and preferably substituents are also present in positions 2, 3 and Θ. The substituent in position 1 can be modified so that an acyl group, for example formyl, acetyl, acrylyl, vinylacetyl or benzoyl, is attached to the amino group in position a. In position 1, dehydro L-Pro is preferred.

In position 2, modified D-Phe is preferably present and its presence increases the antagonistic activity, especially depending on the specific modifications of the benzene ring. Usually, ss replaces a single hydrogen atom in the para position or in position 4, when replacing two hydrogen atoms, the substitution is preferably carried out in the 2,4- or 3,4-positions. The substituents are preferably selected from the group of dichloro, methyl, fluorine, difluoro, trifluoromethyl, methoxy, bromine, dibromo, nitro, dintro, acetylamino and methylmercapto. In position 3, D-Trp is preferred and in position 6, D-His or D-Trp or some other lipophilic aromatic D-amino acid is preferred, although Gly or any D-isomer of the amino acid, for example D-Leu and D-Ser (O-tert.but) can also be used. Substitution at positions 7 and 10 is used only in special cases.

Because these peptides are highly effective in inhibiting LH, they are often called LRF antagonists. These peptides inhibit ovulation in mammals when administered at very low doses in the womb and are also effective in resorbing fertilized eggs when administered very shortly after conception.

The subject of the invention is a method for producing peptides inhibiting the release of gonadotropins from the pituitary gland of mammals of the general formula

X-R1-R2-R3-Sor-Tyr-R1-R)-Arg-Pro-R6 where

X represents a hydrogen atom or an acyl group of up to 7 carbon atoms,

R1 represents dehydro Pro, dehydro D-Pro, Thz or D-Thz,

Rž stands for D-Phe, D-H's, D-Trp, Trp, Cl-D-Phe, dichloro-D-Phe, CFs-D-Phe, F-D-Phe, difluoro-D-Phé, AcNH-D-Phe, where '

Ac means acetyl,

NOz-Ď-Phe, dinitro-D-Phe, Br-D-Fhe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe or CH3-D-Phe,

R3 is D-Trp, Trp, D-Phe, or D-His,

Ri D-isomer of lipophilic «-amino acids from the group Arg, Lys, Trp, Phe, His, Tyr and C-phenylglycine,

Rs means Leu or N"-Me-Leu where

Me means methyl,

Re means Gly-NH2 or NHCH2CH3, as well as non-toxic salts of these peptides, characterized in that from the intermediate of the general formula

Xi-Rl-R2-R3-Ser (X ² )-Tyr-(X ³ )-R4-R5-Arg(X ⁴ )-Pro-X ⁵ , where

X ¹ represents a protecting group on the α-amino group,

X ² represents a protecting group on the alcoholic hydroxyl group of Ser,

X ³ represents a protecting group on the phenolic hydroxyl group of Tyr,

X ⁴ means a protecting group on the nitrogen atoms of Arg and

X ³ is selected from the groups Gly-O-CH 2 -(resin), O-CH 5 -(resin), Gly-NH-(resin), Gly-NH 2 and NHCH 2 Cl-I 3 ,

R1, R2, R5, R4 and R6 have the above-mentioned meaning, at least one group ^X1 to ^X5 is cleaved off and optionally the resulting peptide is converted into a non-toxic salt.

The term dehydro Pro means 3,4-dihydroproline of formula C5H7O2N, in the case where X represents an acyl residue, this residue is bound to the nitrogen atom. Thz is metath ^: azolidine-2-carboxylic acid of formula C4H7O2NS, which can be prepared by reacting cysteine hydrochloride with formaldehyde and, for example, Ac-Thz can be obtained by reacting Thz with acetic anhydride.

The peptides of the invention can be obtained in solid phase using a chloromethylated resin, methylbenzhydrylan resin (MBHA) or benzhydrylamine resin (BHA). The reaction is carried out by sequentially adding amino acids as detailed in U.S. Patent No. 4,211,693. Side chain protecting groups, as is commonly known in the art, are preferably added to Ser, Tyr, Arg and His before these amino acids are coupled to the resin-based rs ^x sigma.

In this way, a fully protected resin-bound peptide intermediate is formed.

The criterion for the selection of the side chain and the protecting groups X ² to X ⁴ is that the protecting groups must be stable to the reagents under the reaction conditions chosen to remove the α-amino group and the protecting group on the α-amino group at each stage of the synthesis. The protecting group must be cleaved during the coupling and must be capable of being removed after synthesis of the desired amino acid sequence under reaction conditions that do not alter the peptide chain.

The fully protected peptide can be cleaved from the chloromethyl resin by ammonolysis in a known manner to give a fully protected intermediate. The removal of the protecting groups and the cleavage of the peptide from the benzhydrylamine resin is carried out at 0 °C by the action of hydrofluoric acid (HF). Anisole is added to the peptide before the action of this acid. After the removal of the hydrofluoric acid in vacuo, the cleaved peptide freed from the protecting groups is treated with ether, the resulting product is isolated by decantation, dissolved in dilute acetic acid and lyophilized.

Peptide purification is performed by ion exchange chromatography on a carboxymethylcellulose column followed by partition chromatography using a mixture of n-butanol and 0.1 N acetic acid in a volume ratio of 1:1 on a column packed with Sephadex G-25 or by high-pressure liquid chromatography.

The peptides of the invention are effective at doses of less than 200 mg/kg body weight when administered at midday during proestrus and in this way can effectively prevent ovulation in female rats. If it is necessary to suppress ovulation for a longer period, doses of 0.1 to 5 mg/kg should be used. These substances are also effective as contraceptives when administered to males.

Because the compounds produced by the method of the invention reduce testosterone levels, which is undesirable in a normal male, it may be necessary to administer testosterone together with an effective dose of these agents.

The invention will be illustrated by the following examples.

Example

Peptides of general formula

X-dehydro Pro-R2-D-Trp-Ser-Trp-D-Trp-R5-Arg-Pro-Gly-NH2 can be prepared in solid phase by the above method.

Table I

Peptide X R2 Rs. Kd ²² ° 1 Ac 3,4-Cl-D-Phe Leo —79.9 2 Ac 4 CF3-D-Phe Leo —69.1 3 Ac 4 F-D-Phe Leo —78.2 4 Ac 4 AvNH-D-Phe Leo —76.5 5 Ac 4 KNIFE-S-Phe Leo —89.6 6 Ac 4 Br-D-Phe Leo —76.0 7 Ac 4 CHsS-D-Phe Leo —79.8 3 Ac 4 OCH3-D-Phe Leo. —82.7 9 Ac 4 CH3-D-Pihe Leo —81.7 10 Ac 2,4 Cl2-D-Phe Leo —77.2 11 Air 3,4 Cl2-D-Phe Leo —72.5 12 Ac 4 OCH3-D-Phe N ^K MeLeu —113.4 13 Ac 4 CH3-S-Phe N“MeLeu —106.5 14 Ac 3,4 Cl2-D-Phe N“MeLeu —110,—

As an example, the synthesis of peptide No. 1 will be given below, which can be expressed by the formula [Ac-dehydro Pro ¹ , 3,4 Ch-D-Phe ² , D-Trp ³ ' ^ó ]-LRE or in more detail _; . ......

Ac-dehydro Pro-3,4 Ch-D-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2

BHA resin is used and Gly is coupled to the resin with a Boc protecting group over 2 hours in CH2Cl2 using a three-fold excess of Boc-derivative and DGC as the activating reagent. The glycine residues are linked to the BHA residue by an amide bond.

After binding each amino acid residue, washing, removing the protecting group, and binding the next amino acid residue, the next amino acid is bound according to the following scheme, using an automated device and starting with 5 g of resin:

Stage Reagent and procedure

Reaction time (min)

1 CH2CI2 wash 80 ml twice 3 2 methanol (MeOH) wash 30 ml 3 3 CH2CI2 wash 80 ml three times 3 4 50% TFA with 5% 1,2-ethanedithiol in CH2Cl2 — 70 ml, twice 10 5 CH2CI2 — 80 ml, twice 3 6 TEA 12.5% in CH2Cl2 — 70 mL, twice 5 7 MeOH — 40 ml, twice 2 8 CH2CI2 — 80 ml three times 3 9 Boc-amino acid (10 mmol) in 30 ml DMF or CH2CI2 depending on the solubility of the protected amino acid, once and DCC (10 mmol) in 7- GH2CI2 30 10 MeOH — 40 ml, twice 3 11 TEA 12.5% in CH2Cl2 — 70 mL), once 3 12 MeOH — 30 ml, twice 3 13 CH2CI2 — 80 ml, twice 3

237313

After step 13, a sample of the reaction mixture is taken for the ninhydrin test. If this test is negative, it is necessary to start again at step 1 for the binding of another amino acid. If the test is positive or weakly positive, steps 9 to 13 must be repeated.

The above scheme is used for the coupling of amino acids in the peptide of the invention after the first amino acid has been coupled. The N ^K Boc protecting group is used for each subsequent amino acid during the synthesis. The Arg side chain is protected with Tos. OBZ1 is used as the protecting group for the Ser hydroxyl group and 2,6-dichlorobenzyl is used as the protecting group for the Tyr side chain hydroxyl group. N-acetyldehydro-Pro is introduced as the last amino acid, Boc-Arg(Tos) and Boc-D-Trp with low solubility in CH2Cl2 are coupled using a mixture of dimethylformamide and methylene dichloride.

Cleavage of the peptide from the resin and complete removal of the side chain protecting groups can be easily accomplished by treatment with hydrofluoric acid at 0°C. Anisole is added prior to the acid treatment. After removal of the acid in vacuo, the resin is extracted with 50% acetic acid and the washings are lyophilized to yield the crude peptide as a powder.

The peptide is then purified by ion exchange chromatography on carboxymethylcellulose (Whatman CM 32) using 0.05 to 0.3 M ammonium acetate in a 50:50 mixture of methanol and water followed by gel filtration chromatography using a 1:1 mixture of n-butanol and 0.1 N acetic acid (v/v).

The peptides listed in the table were investigated in vitro and in vivo. In the in vitro test, rat pituitary cells were used, which were grown in culture for 4 days before the experiment. LH levels were a measure of the response to the application of peptides, the results were determined radioimmunologically. Control cell samples contained 3 mol LRF, experimental cells contained 3 mol LRF and 0.01 to 3 mol of the peptide obtained by the method of the invention. The amount of LH secreted by the control samples and experimental samples was then compared. The results were calculated and are shown in Table IA as the molar concentration ratio of the peptide under study to LRF, which is required to reduce the amount of LH released by 3 mol LRF to 50% of the control value (ICR50).

The peptides described above were used to inhibit ovulation in female rats. In this test, adult female Sprague-Dowley rats weighing 225 to 250 g were administered 5 or 10 pg of the peptide in corn oil at noon during proestrus. Proestrus is the afternoon before ovulation. A separate control group was left without peptide administration. Ovulation occurred in each of the control rats. As can be seen from the following table, the peptides effectively suppressed ovulation in female rats at very low doses and all peptides were fully effective at a dose of 1 mg.

Table IA

In vitro

Peptide 1'ČRso

In vivo (10 ^g) number of ovulating rats

In vivo (5 µg) number of ovulating rats

1 0.039 0/10 4/7 2 0.070 7/9 3 0.021 0/10 0/10 4 9/10 5 0.011 0/10 2/10 6 0.010 5/7 7 0.030 1/10 7/10 8 0.125 3/10 9 0.044 1/7 9/9 10 5/10 4/7 11 0.048 1/10 4/10 12 0.29 5/10 13 0.11 3/4 14 0.05 1/3 8/10 Example 2

can be obtained in the solid phase by the method according to

Peptides of the general formula of Example 1 with the exception of peptide No. 20, which is obtained on a chloromethylated resin.

X-Ri-pCl-D-Phe-D-Trp-Ser-Tyr-Rí-Rí-Arg-Pro-Re

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The peptides listed in the table were investigated in vitro and in vivo. The in vitro experiment was again performed using dissociated rat pituitary cells that had been grown in culture for 4 days prior to the experiment. The levels of LH produced in response to the application of the peptides were determined radioimmunologically.

Control samples were grown in a medium with 3 mol LRF, experimental cells in a medium with 3 mol LRF and in addition 1 to 100 mol of the test peptide. The amount of LH secreted by the control samples was compared with the amount secreted by the experimental samples. The results were calculated and are shown in Table IIA as the ratio of the molar concentration of the test peptide to the concentration of LRF required to reduce the amount of LH released to 50% of the control value. (ICR50).

Some peptides have been used to inhibit ovulation in female rats. In this experiment, 4, 7, 9 or 10 adult Sprague-Dawley rats weighing 225 to 250 g were used, and the rats were given 0.02 mg of the peptide, unless otherwise indicated, in corn oil at noon during proestrus. Proestrus is the afternoon before ovulation. The control group consisted of 10 female rats that were not given the peptide. All rats in the control group ovulated. As shown in the following table, the peptides were effective in inhibiting ovulation in female rats at very low doses and all peptides were fully effective at a dose of 1 mg.

Table IIA

In vitro ICRso

Peptide in vivo number of ovulating rats

15 0.043 0/10 16 0.058 0/7 17 0.19 3/8 18 0.27 5/10 19 0.03 0/9 20 0.2 21 0.14 5/8 22 0.13 9/10 23 0.1 24 0.12 25 0.042 4/10 26 0.2 10/10 27 0.15 7/10 28 0.1 7/10

* 0.025 mg **0.010 mg

Example 3

Peptides formula

X-R1-R2-R3-Ser-Tyr-D-Trp-R5-Arg-Pro-R6 can be obtained in the solid phase of Example 1 with the exception of peptide No. 37, in which case a chloromethylated resin is used.

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The peptides listed in the table were investigated in vitro and in vivo. The in vitro experiment was again carried out using dissociated rat pituitary cells that had been grown in culture for 4 days prior to the experiment. The levels produced in response to the application of the peptides were determined radioimmunologically. Control cell samples were grown in medium with 3 mol LRF, experimental cells in medium with 3 mol LRF and 1 to 100 mol of the test peptide. The amount of LH secreted by the control samples was compared with the amount secreted by the experimental samples. The results were calculated and are shown in Table IIIA as the ratio of the molar concentration of the test peptide to the concentration of LRF required to reduce the amount of LH released to 50% of the control value (ICR50).

Some peptides were used to inhibit ovulation in female rats. In this experiment, 4, 7, 9 or 10 adult Sprague-Dawley rats weighing 225 to 250 g were used. The rats were given 0.2 mg of the peptides, unless otherwise indicated, in corn oil at noon during proestrus. Proestrus is the afternoon before ovulation. The control group consisted of 10 female rats that were not given pepbd. All rats in the control group ovulated. As shown in the following table, the peptides were effective in inhibiting ovulation in female rats at very low doses and all peptides were fully effective at a dose of 1 mg.

Table IIIA

Peptide

In vitro ICRso in vivo number of ovulating rats * 0.025 mg

These peptides can be administered to mammals intravenously, subcutaneously, intramuscularly, orally, intranasally, or vaginally to achieve inhibition and/or control of fertility. The effective dose varies depending on the route of administration and the species of mammal. An example of a typical form for administration is a physiological saline solution containing the peptides. This solution is administered in such a way that it is possible to

0.5 : 1 4/10 0.8:1 7/10 0.3 : 1 1/10 0.4 : 1 2/10 0.6 : 1 0/4

A dose of 0.1 to 5 mg/kg can be guaranteed. Peptides can be administered orally in solid or liquid form.

Although the invention has been described in terms of preferred embodiments, it is clear that a number of modifications may be devised that also fall within the scope of the invention, for example, other substituents may be used that do not substantially alter the activity of the resulting peptide.

Claims (11)

SUBJECT A process for the production of gonadotropin release inhibiting peptides from the pituitary of mammals of the general formula X-R1-R2-R3-Ser-Tyr-R4-R5-Arg-Pro-R6 wherein X represents a hydrogen atom or an acyl group of up to 7 carbon atoms, R1 denotes dehydro Pro, dehydr-o D-Pro, Thz or D-Thz, R2 is D-Phe, D-His, D-Trp, Trp, Cl-D-Phe, dichloro-D-Phe, CF3-D-Phe, FD-Phe, difluoro-D-Phe, AcNH-D-Phe, where Ac is acetyl, NO-D-Phe, dinitro-D-Phe, Br-D-Phe, dibromo-D-Phe, CH3S-D-Phe, OCH3-D-Phe, or CH3-D-Phe, R3 is D-Trp, Trp, D-Phe or D-His, R4 D-isomer of lipophilic α-amino acids from the group of Arg, Lys, Trp, Phe, His, Tyr and C-phenylglycine, Rs stands for Leu or N-Me-Leu Me means methyl, R 6 is Gly-NH 2 or NHCH 2 CH 3, as well as the non-toxic salts of these peptides, characterized in that it is derived from an intermediate of the general formula X ¹ -R ¹ -R ² -R ³ -Ser (X ² ) -Tyr- (X ³ ) -R ¹ -R ⁵ -Arg (X ⁴ ) -Pro-X ⁵ where X ¹ represents a protecting group on the α-amino group, X ² represents a protecting group on the alcohol hydroxyl group Ser, X ³ represents a protecting group on the phenol hydroxyl group Tyr, X ⁴ represents a protecting group on the Arg and nitrogen atoms X ⁵ is selected from Gly-G-CH 2 (resin), O-CH 2 - (resin), Gly-NH- (resin), Gly-NH 2 and NHCH 2 CH 3, R 1, R 2, R 3, R 4 and R 5 are as defined above, At least one X ¹ to X ⁵ group is cleaved and optionally the resulting peptide is converted to a non-toxic salt. 2. Method according to claim 1, characterized in that starting from an intermediate in which X ¹ is an acyl group of up to 7 carbon atoms, R is 3,4-Cl-D-Phe and R represents Leu, and the remaining substituents are as defined in point 1. 3. The process of claim 1, wherein X ¹ is an acyl group of up to 7 carbon atoms, R 2 is 4 FD-Phe and R 5 is Leu, and the remaining substituents are as defined in 1. 4. Method according to claim 1, characterized in that starting from an intermediate in which X ¹ is an acyl group of up to 7 carbon atoms, R2 is 4-NO2 -D-Phe, and Leu and R represents the remaining substituents are as defined in point 1. 5. Method according to claim 1, characterized in that starting from an intermediate in which X ¹ is an acyl group of up to 7 carbon atoms, R2 is 2,4-Cl2 -D-Phe and R represents Leu, and the remaining substituents are as defined in point 1. 6. Method according to claim 1, characterized in that starting from an intermediate in which X ¹ is an acyl group of up to 7 carbon atoms, R 2 is 3,4 Cl-D-Phe and R represents Leu, and the remaining substituents are as defined in point 1. ltí 7. A method according to claim 1, characterized in that starting from an intermediate in which X ¹ is an acyl group of up to 7 carbon atoms, R 2 is 3,4-Cl2 -D-Phe and R is N "Mel and the remaining substituents have a the meaning referred to in point 1. 8. A method according to claim 1, characterized in that starting from the intermediate V, wherein X ¹ is an acyl group of up to 7 carbon atoms, Ri is dehydro-Pro, R4 znamemená D-Trp, R₅ is Leu, and X ⁵ represents Gly-NH2 and the remaining substituents are as defined in 1. 9. The method according to claim 1, characterized in that starting from an intermediate in which X ¹ is an acyl group of up to 7 carbon atoms, Ri is dehydro-Pro, R4 is D-Trp, R₅ is MeLeu N ^N, and X ⁵ is Gly-NH2 and the remaining substituents are as defined in 1. 10. The method according to claim 1, characterized in that starting from an intermediate in which X ¹ is an acyl group of up to 7 carbon atoms, Ri is dehydro Pro, R4 is D-Trp, R₅ is n "MeLeu, and X ⁵ represents Gly-NH2 and the remaining substituents are as defined in 1. 11. The method according to claim 1, characterized in that starting from an intermediate in which X ¹ is an acyl group of up to 7 carbon atoms, Ri is dehydro Pro, R4 is (imBzl) D-His, R₅ is Leu and X ⁵ is Gly-NH2 and the remaining substituents are as defined in 1.