HU189504B - Process for preparing decapeptide derivatives inhibiting the release of luteinizing hormone - Google Patents

Abstract

LRF ANTAGONISTS Peptides which inhibit the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads. The peptides have the structure: X-R1-R2-R3-Ser-Tyr-R4-R5-Arg-Pro-R6 wherein X is hydrogen or an acyl group having 7 or less carbon atoms; R1 is dehydro Pro, dehydro D-Pro, Thz or D-Thz; R2 is D-Phe, D-His, D-Trp, Trp, Cl-D-Phe, dichloro-D-Phe, CF3-D-Phe, F-D-Phe, difluoro-D-Phe, AcNH-D-Phe, NO2-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 is Gly or a D-isomer amino acid; R5 is Leu or N.alpha.Me-Leu; and R6 is Gly-NH2 or NHCH2CH3.

Description

The present invention relates to novel peptides which inhibit pituitary (pituitary) gonadotropin production and inhibit ovulation (rupture of mature follicles and ovum release) and / or release of steroid hormones in mammals, including man. More particularly, the present invention provides peptides that inhibit gonadal function and the production of steroid hormones such as progesterone and testosterone.

The pituitary gland attaches to the pituitary gland, known as the hypothalamus. The pituitary gland consists of two lobes, anterior and posterior. The dorsal lobe stores and transmits to the general circulation the two hormones produced in the hypothalamus, vasopressin and oxytocin. The anterior pituitary gland produces several hormones that are complex protein or glycoprotein molecules; these pass through the bloodstream to various organs and stimulate the peripheral organs to secrete hormones into the bloodstream. The anterior pituitary gland produces, among other things, follicle-stimulating hormone (FSH) and luteinising hormone (LH), also called gonadotropic hormones or gonadotropins. These hormones together regulate the function of the gonads when they produce testosterone in the testis and progesterone and estrogen in the ovaries, and also regulate the production and maturation of gametes (germ cells).

Hormone production in the anterior pituitary gland usually requires prior release of another group of hormones formed in the hypothalamus. One of the hypothalamic hormones acts as a factor that triggers the release of gonadotropic hormones, especially LH. The hypothalamic hormone acting as a releasing factor for LH is referred to herein as LRF ("LH-releasing factor"), although both LHRH and GnRH are used.

LRF was isolated and characterized as a decapeptide of the following structure:

p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH ₂ .

Peptides are compounds containing two or more amino acids in which the carboxyl group of one amino acid is linked to the amino group of another. The above formula of LRF is consistent with the usual labeling of peptides with the amino group on the left and the carboxyl group on the right. To identify the position of the amino acid residue, the amino acid residues are numbered from left to right. In LRF, the hydroxyl moiety of the carboxyl group of glycine is replaced by an amino group (NH ₂ ). Abbreviations for the above amino acid residues are common in peptide chemistry and based on the trivial name of amino acids; Thus p-Glu = pyroglutamic acid

His = histidine

Trp = tryptophan

Ser = serine

Tyr = tyrosine

Gly = glycine

Leu = leucine

Arg = arginine and

Pro = proline.

. (Other abbreviations not given in the description: Thz = metathiazolidine-2-carboxylic acid Al - alanine

Phe = phenylalanine

Me = methyl im Bzl = benzyl group attached to His imino group).

The amino acids of the peptides of the invention, except for glycine, are in the L-configuration unless otherwise indicated.

It is known that when Gly is substituted at the 6-position of the LRF decapeptide with D-amino acids, the resulting peptide material stimulates the pituitary release of LH and other gonadotropins by about 1 to 35 fold in mammals. This release effect can be produced by administering to mammals the LRF analog intravenously, subcutaneously, intramuscularly, orally, intranasally, or intravaginally.

It is also known that the substitution of His for a different amino acid at position 2 of the decapeptide of LRF (or the deletion of His) results in analogs that inhibit pituitary release of LH and other gonadotropic hormones in mammals. In particular, varying degrees of inhibition of LH release can be achieved when His is omitted (des-His) or replaced by D-Ala, D-Phe or Gly. The inhibitory effect of such peptides modified at the 2-position can be further enhanced by replacing the Gly at the 6-position of the decapeptide with a D-amino acid. For example, the peptide p-Glu-Trp-Ser-Tyr-D-Ala-Leu-Arg-ProGly-NH _{2 is} four times more potent in inhibiting the release of gonadotropins than the similar peptide, where Gly rather than D-Ala is present.

6 position.

Some female mammals that do not have an ovulation cycle and have no pituitary or ovarian abnormalities begin to secrete normal amounts of LH and FSH gonadotropin after appropriate use of LRF. Thus, LRF appears to be useful in the treatment of infertility in cases where there is a functional defect in the hypothalamus.

It is often desirable to prevent ovulation in female mammals; this can be achieved by using LRF analogs that antagonize the normal function of LRF. To this end, the potential use of LRF antagonist LRF analogues for contraception or control of conception periods is being extensively studied. It would be desirable to provide peptides that are potent antagonists of endogenous LRF and inhibit LH secretion and steroid production of the gonads in mammals.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a peptide that inhibits gonadotropin release in mammals, including man, and to provide a method for inhibiting the gonadal steroid production of male and female mammals. The improved LRF analogs of the present invention are potent LRF antagonists and inhibit the mammalian reproductive process. The analogs of the present invention can be used to inhibit the formation of gonadotropins and sex hormones in 189,504 different conditions, including premature sexual maturation, hormone-dependent malignancies, menstrual disorders, and irregular mucosal positioning (endometriosis).

In general, the invention provides peptides that are potent inhibitors of pituitary secretion of gonadotrophins by mammals, including humans, and / or inhibit steroid release of the gonads. These peptides are LRF analogs wherein the 1-position substitution is in the form of dehydroproline or metathiazolidine-2-carboxylic acid, and preferably also has substituents at the 2-, 3- and 6-positions. The substituent at the 1-position can be modified such that its α-amino group contains an acyl group such as formyl, (Male) acetyl, (Ac) acrylic, (Acr) vinylacetyl or benzoyl (Bzl). In the 1-position, dehydro-L-Pro is preferred. Preferably, D-Phe is present at the 2-position, resulting in enhanced antagonist activity due to specific modification of the benzene ring. Hydrogen is preferably substituted singly at the para or 4 position and twice preferably at the 2,4 or 3,4 position. Preferred substituents are dichloro, methyl, fluoro, difluoro, trifluoromethyl, methoxy, bromo, dibromo, nitro, dinitro, acetylamino and methyl mercapto. In position 3, D-Trp and

In position 6, imBz1 is D-His or D-Trp. The substitution at the 7- and 10-positions is optional.

Because these peptides are very effective at inhibiting LH release, they are often referred to as LRF antagonists. These peptides inhibit ovulation in female mammals when used at a very low dose level in the pre-natal cycle (proestrus) and induce resorption of the fertilized egg and applied shortly after conception.

In particular, the peptides of the invention may be represented by the following formula: XR, -R ₂ -D-Trp-Ser-Tyr-R ₄ -R ₅ -Arg-ProR ₆ , wherein

X is hydrogen, (C 1 -C 4) alkanoyl, (C 2 -C 5) alkenoyl, or benzoyl;

R _t is dehydro-Pro, dehydro-D-Pro, dehydro-D-Pro, Thz or D-Thz;

R ₂ is D-Phe, 4-chloro-D-Phe, dichloro-D-Phe,

4-trifluoromethyl-D-Phe, 4-fluoro-D-Phe, difluoro-DPhe, 4-acetylamino-D-Phe, 4-nitro-D-Phe, 4-bromo-D-Phe, dibromo-D- Phe, 4-methylthio-D-Phe, 4-methoxy-D-Phe or 4-methyl-D-Phe;

R ₄ is D-Trp or (imBzl) D-His;

_Rs is Leu or N ° Me-Leu; and

R ₆ is Gly-NH ₂ or -NHCH ₂ CH, with the proviso that when R _{6 is} -NHCH ₂ CH ₃ , it is other than R, D-Thz and R ₄ (imBzl) -D-His.

Dehydro-Pro refers to 3,4-dehydroproline (C ₁ H ₇ O ₂ N) and when X represents an acyl group, dehydroproline is attached to the nitrogen atom. Thz means metathiazolidine-2-carboxylic acid (C ₄ H ₇ O ₂ NS) which may be prepared from cysteine hydrochloride by treatment with formaldehyde; For example, Ac-Thz is prepared by reacting Thz with acetic anhydride.

The peptides of the invention may be prepared by solid-phase peptide synthesis using a conventional chloromethylated resin, a methylbenzhydrylamine resin (MBHA) or a benzhydrylamine resin (BHA) commonly used in peptide chemistry. Synthesis step by step! The method is carried out by the method of step one, increasing the chain one by one by linking the next amino acid (described in detail in U.S. Patent No. 4,211,693). Preferably, side chain protecting groups, which are well known in peptide chemistry, are added to Ser, Tyr, Arg, and His before being linked to the resin-based chain.

Such a process results in a fully protected intermediate peptide resin. The intermediates of the invention are represented by the following formula: X'-R ₁ -R ₂ -D-Trp-Ser (X ² ) -Tyr (X ³ ) -R ₄ R ₅ -Arg (X ⁴ ) -Pro-X ⁵ where

X ^{1 is} a type of α-amino protecting group well known in the art for stepwise synthesis of peptides; if X is an acyl group in the peptide to be prepared, said acyl group can be used as a protecting group. The classes of α-amino protecting groups corresponding to X 'include (1) acyl-type protecting groups such as formyl (For), trifluoroacetyl, phthalyl, p-toluenesulfonyl (Tos), benzoyl (Bzl), benzenesulfonyl-. o-nitrophenylsulfenyl (Nps), tritylsulfenyl, o-nitro-phenoxyacetyl, acrylyl (Acr), chloroacetyl, acetyl (Ac) and γ-chlorobutyryl;

(2) aromatic urethane-type protecting groups such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl such as ρ-chlorobenzyloxycarbonyl, pnitrobenzyloxycarbonyl, p-bromo benzyloxycarbonyl and p-methoxybenzyloxycarbonyl;

(3) aliphatic urethane-type protecting groups such as tert-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl and allyloxycarbonyl;

(4) cycloaliphatic urethane-type protecting groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl;

(5) thiourethane-type protecting groups such as phenylthiocarbonyl;

(6) alkyl-type protecting groups such as allyl (Aly), phenylmethyl (trityl) and benzyl (Bzl);

(7) trialkylsilane groups such as trimethylsilane.

A preferred α-amino protecting group is Boc when X is hydrogen.

X ^{2 is} a protecting group for the alcoholic hydroxyl group of Ser and is acetyl, benzoyl, tetrahydropyranyl, tert-butyl, trityl, benzyl or 2,6-dichlorobenzyl. The benzyl group is preferred.

X ^{3 is} a protecting group for the phenolic hydroxy group of Tyr and is tetrahydropyranyl, tert-butyl, trityl, benzyl, benzyloxycarbonyl, 4-bromobenzyloxycarbonyl or 2,6-dichlorobenzyl.

X ⁴ is a nitrogen protecting group for Arg and represents nitro, Tos, benzyloxycarbonyl, adamantyloxycarbonyl or Boc; However, X ⁴ may also be hydrogen, which means that they are absent

189,504 protecting groups on Arg side chain nitrogen atoms.

X ⁵ is Gly-O-CH ₂ - [resin], O-CH ₂ - [guar] or Gly-NH- [resin].

The criterion for the selection of the side chain protecting groups X ² to X * is that the protecting group is stable to the reagent under the reaction conditions used at each step of the synthesis to remove the α-amino group. The protecting group must not be cleaved under the coupling conditions and must be removable upon completion of synthesis of the desired amino acid sequence under reaction conditions that do not affect the peptide chain.

When X ⁵ is Gly-O-CH ₂ - [resin], the ester portion of one of the many functional groups of the polystyrene resin carrier is involved in the bond formation. When the X ⁵ group Gly-NH- [resin support], such amide bond connects Gly to BHA resin or MBHA resin.

When X is acetyl, formyl, acrylyl, vinylacetyl, benzoyl or an up to 7 carbon atoms other acyl, X may be used as the X ^{1 * 3 4 5 6 7} protecting group R _is amino the amino group, and in this case may be used prior to the attachment of the last amino acid to the peptide chain. However, it is also possible to carry out the reaction with the peptide on the resin, for example with acetic acid in the presence of dicyclohexylcarbodiimide (DCC), or preferably with acetic anhydride.

The fully protected peptide can be cleaved from the chloromethylated resin by known methods of ammonolysis to provide a fully protected amide intermediate. Deprotection of the peptide as well as cleavage of the peptide from the benzhydrylamine resin is accomplished at 0 ° C with hydrogen fluoride (HF). Anisole is added to the peptide before treatment with HF. After removal of HF in vacuo, the cleaved deprotected peptide is treated with ether, decanted, taken up in dilute acetic acid and lyophilized.

The peptide was purified by ion exchange chromatography on a carboxymethylcellulose (CMC) column followed by partition chromatography on a Sephadex G-25 column using n-butanol-0.1N acetic acid (1: 1 by volume) or by high performance liquid chromatography (HPLC). ). The peptides of the invention are effective in inhibiting ovulation of female rats at doses of less than 200 µg / kg body weight when applied at noon on the day of the proestrus. Dose levels of about 0.1 to about 5 mg / kg body weight are required to permanently suppress ovulation. The antagonists of the invention are also effective contraceptives when used at a regular rate in male mammals.

Because these compounds reduce testosterone levels (an undesirable consequence in normal sexually active males), it may be advisable to use testosterone boost doses in combination with the LRFantagonist.

The following examples illustrate further aspects of the invention without limiting the scope of the invention.

Example 1

By the above solid phase technique, the following peptides of the general formula are prepared: X-dehydro-Pro-R ₂ -D rp-Ser-Tyr-DT rp-R ₅ -ArgPro-Gly-NH ₂

Table I (50% acetic acid, c = 1)

Pulp- tid X R ₂ r ₅ a ²² u _D First Ac 3,4-dichloro-D-Phe Leu -79.9 ° Second Ac 4-trifluoromethyl-D-Phe Leu -69,1 ' Third Ac 4-fluoro-D-Phe Leu -78,2 ' 4th Ac 4-acetylamino-D-Phe Leu -76.5 ° 5th Ac 4-nitro-D-Phe Leu -29,6 ' 6th Ac 4-bromo-D-Phe Leu -76,0 ' 7th Ac 4-methylthio-D-Phe Leu -79.8 ' 8th Ac 4-D-Phe Leu -82.7 ° 9th Ac 4-methyl-D-Phe Leu -81.7 ° 10th Ac 2,4-dichloro-D-Phe Leu -77.2 ⁰ 11th Acr 3,4-dichloro-D-Phe Leu -72.5 ° 12th Ac 4-D-Phe N "MeLeu -113.4 ° 13th Ac 4-methyl-D-Phe N "MeLeu -106.5 ' 14th Ac 3,4-dichloro-D-Phe N ° MeLeu -110 '

By way of example of Peptide No. 1 above - that [Ac-dehydro Pro ^1, 3,4-dichloro-D-Phe ^2, D-Trp ^"6] LRF has called - solid phase synthesis of the characteristics described below.

The peptide has the formula: Ac-dehydro-Pro-3,4-dichloro-D-Phe-DT rp-Ser-Ty rD-Trp-Leu-Arg-Pro-Gly-NH ₂ .

BHA resin was used and Boc-protected Gly was coupled to the resin in methylene chloride (CH ₂ Cl ₂ ) for 2 hours; the Boc derivative was used in triple excess and dicyclohexylcarbodiimide (DCC) was used as activator. The glycine moiety is linked to the BHA moiety by an amide bond.

After attachment of each amino acid, the washing, deprotection and coupling of the following amino acid is carried out according to the following procedure using an automated apparatus and starting from approximately 5 g of resin:

Mixing Reagents and Operations (minutes)

1. CH ₂ Cl ₂ wash 80 ml (twice) 3

- Methanol (MeOH) wash - 30 ml (twice)

3. CH ₂ Cl ₂ Wash - 80 ml (three times) 3

4. 50% triethanolamine (TEA) + 5% 1,2-ethanedithiol in CH ₂ Cl ₂ - 70 mL (2 times)

5. CH ₂ Cl ₂ Wash - 80 mL (twice) 3

6. 12.5% TEA in CH ₂ Cl ₂ -70 ml (two times)

7. MeOH Wash - 40 mL (twice) 2

189,504

Juice- pés Reagents and operations Sheaf- filtering time (minute) 8th CH ₂ Cl ₂ wash - 80 ml (three times) 3 9th Boc-amino acid (10 millimoles) in 30 ml of dimethylformamide (DMF) or CH ₂ Cl ₂ , depending on the protected 30-300 10th amino acid solubility (once) plus DCC (10 millimol) in CH ₂ Cl ₂ MeOH wash - 40 mL (twice) 3 11th 12th 12.5% TEA in CH ₂ Cl ₂ - 70 mL (once) MeOH wash - 30 mL (twice) 3 3 13th CH ₂ Cl ₂ wash - 80 ml (twice) 3

After step 13, an aliquot is removed for the ninhydrin test: if the probe is negative, we return to step 1 for the next amino acid coupling; if the assay is positive or slightly positive, repeat steps 9-13. step.

Each of the peptides of the invention is coupled to each amino acid using the above procedure after the first amino acid is coupled to the carrier. N 'Boc protection is applied to all remaining amino acids during synthesis. The Arg side chain is protected with a Tos group. The side chain protecting group used is the OBzl group for the Ser hydroxyl group, and the 2.6 dichlorobenzyl group is used as the side chain protecting group for the Tyr hydroxy group. N-acetyldehydro-Pro was incorporated as the final amino acid. Sparingly soluble in CH ₂ Cl _2, Boc-Arg (Tos) and Boc-D-Trp coupling is carried out in DMF, CH ₂ C1 ₂ mixture.

Cleavage of the peptide from the resin and complete deprotection of the side chains occurs very easily at 0 ° C under HF. Anisole is used as a decontaminant prior to HF treatment. After the HF was removed in vacuo, the resin was extracted with 50% acetic acid and lyophilized to give crude peptide powder.

The peptide was then purified by ion exchange chromatography on a CMC column (Whatman CM 32) and eluted with a 0.05-0.3 M ammonium acetate gradient in methanol / water 50:50, followed by gel column chromatography on n-butanol. -0.1 N acetic acid (1: 1 by volume) using an eluent system.

The peptides described in the table above were tested in vitro and in vivo. Dissociated rat pituitary cells were used for the in vitro assay and maintained in cell culture for 4 days prior to the assay. LH levels in response to peptide application are determined by radioimmunoassay specific for rat LH. Control wells containing cell cultures were supplemented with only 3 nanomolar LRF, while experimental wells contained 3 nanomolar LRF plus 0.01-3 nanomolar peptide of interest. The amount of LH secreted in LRF-treated samples alone is compared to the amount of LH secreted in peptide plus LFR-treated samples. The results are shown in IA. Table 1 shows the In Vitro column, where the molar concentration of test peptide to LRF is given (antagonist per LRF), which is required to reduce the amount of LH released by 3 nanomolar LRF to 50% of control (ICR _S0 ).

In vivo testing of the above peptides to inhibit ovulation in female rats was performed. In this assay, a defined number of mature female Sprague-Dawley rats weighing 225-250 g were inoculated with 5 or 10 pg of peptide in corn oil at noon on the day of the proestrus. The proestrus falls on the afternoon before estrus (ovulation). A separate group of female rats to which no peptide was administered was used as a control. All control female rats ovulate at the time of estrus (oestrus). As shown in FIG. As shown in the In Vivo column of Table I, peptides are very effective at inhibiting ovulation of female rats at very low doses, and all peptide formulations are considered to be perfectly effective at a dose of 1 mg.

IA. spreadsheet

peptide In Vitro icr ₅₀ In Carrier (0 Pg) Ovulating rats In the Carrier (5 pgs) ovulating rats First 0,039 0.10 4.7 z. 0,070 7 9 Third 0,021 0 10 0 10 4th 9/10 5th 0.011 0 10 2 10 6th 0,010 5/7 Ί t. 0,030 1 10 7.10 8th 0,125 3/10 9th 0,044 1.7 9.9 10th 5/10 4 7 11th 0,048 1.10 4 10 12th 0.29 5.10 13th 0.11 3/4 14th 0.05 1.3 Second example Next, X-R , P-chloro-D-P he-D-Trp-Ser

Tyr-R ₄ -R ₅ -Arg-Pro-R ₆ peptides are prepared by the solid phase procedure described in Example 1 except for peptide 20 which is prepared on a chloromethylated resin.

-5II. spreadsheet

139,504

peptide X R R ₄ r ₅ a ^{20 U} D (50% acetic acid, c = 1) 15th Ac dehydro-Pro D-Trp Leu Gly-NH ₂ 92.3 ° 16th Ac dehydro-Pro D-Trp "MeLeu Gly-NH ₂ -110.5 ° 17th Ac dehydro D-Pro D-Trp Leu Gly-NH ₂ + 5.2 ° 18th Ac dehydro D-Pro D-Trp N ° MeLeu Gly-NH ₂ - 21.1 ° 19th acr dehydro-Pro D-Trp N "MeLeu Gly-NH ₂ 69.0 ° 20th Ac dehydro D-Pro D-Trp Leu NHCH ₂ CH ₃ + 2.4 ° 21st Ac thz D-Trp Leu Gly-NH ₂ - 51.9 ° 22nd 23rd 24th Ac Ac Ac thz D-Thz D-Thz D-Trp D-Trp D-Trp N "MeLeu N'MeLeu Leu Gly-NH ₂ Gly-NH ₂ Gly-NH ₂ 79.0 ° - 46.5 ° - 17.7 ° + 18.2 ° + 18.2 ° - 41.3 ° - 7.7 ° 25th Ac dehydro-Pro (ImBzl) D-His Leu Gly-NH ₂ 26th Ac dehydro D-Pro (ImBzl) D-His Leu Gly-NH ₂ 27th Ac thz (ImBzl) D-His Leu Gly-NH ₂ 28th Ac D-Thz (ImBzl) D-His Leu Gly-NH ₂

II. The peptides listed in Table II are tested in vitro and in vivo. Dissociated rat pituitary cells were used for the in vitro assay and maintained in cell culture for 4 days prior to assay ₂₅ .

LH levels in response to peptide application are determined by the rat LH specific radoimmune assay. Only 3 nanomoles of _gQ LRF were added to _dishes containing control cultures, while 3 nanomoles of LRF plus 1-100 nanomolar peptides of interest were added to the test dishes. The amount of LH secreted in LRF-treated samples alone is compared to the amount of ₃₅ LH secreted in peptide plus LRF-treated samples. The results are shown in Table IIA. Table shown Vitro column where a solution of the ratio of the peptide and the LRF analyzed molar concentration at (antagonist per LRF), which is released by the 3 nanomolar LRF, LH amount of _{four (one} control value required to reduce 50% (ICR ₅₀₎ .

The efficacy of the peptides described above to inhibit ovulation in female rats is also examined. In this test, injected into four, seven, nine or ten mature, 225-250 g female Sprague-Dawley rats (MEG other marking almost ^b) 0.02 mg of peptide in corn oil at about noon on the day of proestrus. Proestrus falls in the afternoon before estrosis (ovulation). As a control, a separate group of female rats to which ^o0 peptide was not administered was used. All control female rats ovulate during estrus. As shown in the In Vivő column, peptides are very effective at inhibiting ovulation of female rats at very low doses and all peptide formulations are considered to be perfectly effective at a dose of 1 mg.

IIA. spreadsheet

peptide In Vitro ICR _S0 In the Carrier Ovulating rats 15th 0,043 0/10 * 16th 0,058 0/7 17th 0.19 3/8 18th 0.27 5/10 ** 19th 0.03 0/9 20th 0.2 21st 0.14 5/8 22nd 0.13 9/10 ** 23rd 0.1 24th 0.12 25th 0.042 4/10 26th 0.2 10/10 27th 0.15 7/10 ** 28th 0.1 7/10 **

* 0.025 mg ** 0.010 mg

Example 3

The following peptides of the formula XR, -R ₂ -D-Trp-Ser-Tyr-D-TrpR ₅ -Arg-Pro-R _{6 were} prepared by the solid phase method generally described in Example 1.

189,504

III. spreadsheet

peptide X R Ra r ₅ R ' a '(50% acetic acid, c = 1) 29th H dehydro-DL-Pro D-Phe N "MeLeu Gly-NH ₂ -57.7 ' 30th H dehydro-DL-Pro D-Phe Leu Gly-NH ₂ 31st Ac dehydro-DL-Pro D-Phe Leu Gly-NH ₂ -22.7 ' 32. H dehydro-DL-Pro D-Phe N "MeLeu Gly-NH ₂ 33rd Ac Dehydro-Pro D-Phe Leu Gly-NH ₂ -77.7 ' 34th Ac dehydro D-Pro D-Phe Leu Gly-NH ₂ 35th H dehydro-Pro D-Phe Leu Gly-NH ₂ 36th H dehydro D-Pro D-Phe Leu Gly-NH ₂ 37th H dehydro-Pro D-Phe Leu NHCH ₂ CH 3 38th bz dehydro-Pro D-Trp Leu Gly-NH ₂ 39th for dehydro-Pro D-Trp Leu Gly-NH ₂ 40th for dehydro-Pro D-His Leu Gly-NH ₂ 41st acr dehydro-Pro D-His Leu Gly-NH ₂ 42nd bz dehydro-Pro Trp Leu Gly-NH ₂

In III. The peptides listed in Table II are tested in vitro and in vivo. A 4-day primary culture of dissociated rat pituitary cells was used in the in vitro assay. LH levels in response to peptide application are determined by radioimmunoassay specific for rat LH. Only 3 nanomolar LRF was added to the wells containing the control cultures, while 3 nanomolar LRF plus 1-100 nanomolar peptide to be tested was added to the experimental dishes. The amount of LH secreted in LRF-treated samples alone is compared to the amount of LH secreted in peptide plus LRF-treated samples. The results are shown in IIIA. Table II shows the In Vitro column where the molar concentration (antagonist per LRF) of the test peptide and LRF is required to reduce the amount of LH released by 3 nanomolar LRF to 50% of control (ICR ₅₀ ).

The above peptides are also tested for their ability to inhibit ovulation in female rats. In this assay, 0.02 mg of peptide taken from corn oil of 225-250 g of mature female Sprague-Dawley rats is injected at noon on the day of the proestrus. The proestrus falls on the afternoon before estrus (ovulation). As a control, a separate group of female rats is administered to which no peptide is administered. All control female rats ovulate during estrus. As shown in the In Vivő column, peptides are very effective at inhibiting ovulation of female rats at very low doses and all peptide formulations are considered to be perfectly effective at a dose of 1 mg. ,,,,

IIIA. spreadsheet

peptide In Vitro icr ₅₀ In the Carrier Ovulating rats 29th 0.5: 1 4/10 30th 0.8: 1 7/10 31st 0.3: 1 1/10 32 0.4: 1 2/10 33rd 0.6: 1 0/4 *

* 0.025 mg dose

These peptides to mammals intravenously, subcutaneously, intramuscularly, orally, intranasally or intravaginally ²⁵ may be used to fertile ity inhibition and / or control. The effective doses will depend on the route of administration and the species of mammal being treated. A typical dosage forms include peptide is dissolved in physiological saline, which is employed from about 0.1 to 5 mg / kg body weight range ^{of 30} invention. Orally, the peptide may be administered in solid or liquid form.

Although the invention has been described with reference to its preferred embodiment, changes and modifications will be apparent to those skilled in the art without departing from the spirit of the invention. For example, other substitutions known in peptide chemistry that do not significantly reduce the efficiency of the peptides may also be used in the peptides of the invention.

Claims (42)

Claims A process for the preparation of decapeptide derivatives of the formula XR ₎ -R ₂ -D-Trp-Ser-Tyr-R ₄ -R ₅ -Arg 45 Pro-R ₆ and their pharmacologically acceptable acid addition salts, wherein X is hydrogen, C 1-4 alkanoyl, C 2-5 alkenoyl or benzoyl 50 represents; R 1 is dehydro-Pro, dehydro-D-Pro, dehydro-DL-Pro, Thz or D-Thz; R ₂ is D-Phe, 4-chloro-D-Phe, dichloro-D-Phe, 4-trifluoromethyl-D-Phe, 4-fluoro-D-Phe, difluoro-D55-Phe, 4-acetylamino-D-Phe, 4-nitro-D-Phe, 4-bromo-D-Phe, dibromo-D-Phe, 4-methylthio-D-Phe, 4-methoxy-D-Phe or 4-methyl-D-Phe; D-Trp or (imBzl) D-His; _Rs is Leu or N "Me-Leu; and 60 _R6 is _Gly-NH2 or - NHCH ₂ CH _3, with the proviso that when R ₆ NHCH ₂ CH _3, R _is different from D and R ₄ Thz (imBzl) D-His onwards, wherein egyX -R ^1, -R 2 -D-Trp-Ser ^(X2) -Tyr ^(X3) -R4-R _s -Arg ^{(X4) -Pro-X5} közben65 salt compound of formula, wherein J89 504 R "usual R _2, R ₄ and R _s as defined above, X ¹ in the peptide α-amino protecting group; X ^{2 is} the conventional protecting group for the alcoholic hydroxyl group of Ser in peptide chemistry; X ^{3 is} the conventional protecting group for the phenolic hydroxy group of Tyr in peptide chemistry; X ⁴ is a conventional protecting group for the nitrogen atom of Arg in peptide chemistry; and X ⁵ is Gly-O-CH ₂ [resin], -O-CH ₂ - [resin], -Gly-NH- [resin], -Gly-NH _2, or -NHCH ₂ CH ₃ ; wherein the resin is customary in peptide chemistry, preferably chloromethylated, methyl benzhydryl amine or carrying benzhydrylamine resin with the X'-X ⁵ protecting groups and the peptide resin was cleaved known way, and - if desired - the resulting peptide is a pharmaceutically acceptable acid addition salt thereof transforming it. (Priority: April 14, 1981) The process for the preparation of the decapeptide derivatives of the formula XR, -R ₂ -Drp-Ser-Tyr-R ₄ -R ₅ -Arg-Pro-Re and their pharmacologically acceptable acid addition salts according to claim 1, wherein X is hydrogen, acetyl or acridyl; R 1 is dehydro-Pro, dehydro-D-Pro, Thz or D-Thz; R ₂ is 4-chloro-D-Phe; R ₄ is D-Trp or (imBzl) D-His; R ₅ is Leu or N "Me-Leu; and R _e is -Gly-NH ₂ or -NHCH ₂ CH ₃ , characterized in that an X 1 R 1, -R ₂ -D-Trp-Ser (X ² ) -Tyr (X ³ ) -R ₄ -R ₅ -Arg (X ⁴ ) -ProX ⁵ , wherein R2, R4 and _R5 as defined above, X ¹ customary in peptide α-amino protecting group; X ^{2 is} the conventional protecting group for the alcoholic hydroxyl group of Ser in peptide chemistry; X ^{3 is} the conventional protecting group for the phenolic hydroxy group of Tyr in peptide chemistry; X ⁴ is a conventional protecting group for the nitrogen atom of Arg in peptide chemistry; and X ⁵ is -Gly-O-CH ₂ - [resin], -O-CH ₂ - [resin], -Gly-NH- [resin], -Gly-NH _2, or -NHCH ₂ CH ₃ ; wherein the resin of the peptide standard, preferably chloromethylated, methyl benzhydryl amine or a benzhydrylamine resin support, the X'-X ⁵ deprotected known in the peptide art, and - if desired - the resulting peptide is converted into pharmacologically acceptable acid addition salt thereof. (Priority: April 15, 1980) The process for the preparation of the decapeptide derivatives of the formula XR, -R ₂ -D-Trp-Ser-Tyr-R ₄ -R ₅ -Arg-Pro-R ₆ and their pharmacologically acceptable acid addition salts according to claim 1, wherein X is hydrogen, acetyl or acridyl; R 1 is dehydro-Pro, R ₂ is dichloro-D-Phe, 4-trifluoromethyl-D-Phe, 4-fluoro-D-Phe, 4-acetylamino-D-Phe, 4-nitro-D-Phe, 4-bromo-D-Phe, 4-methylthio-D-Phe, 4-methoxy-D-Phe or 4-methyl-D-Phe; R ₄ is D-Trp, RS _is Leu or N "Me-Leu; and R ₆ is Gly-NH _2, characterized in that an ^{X'-R-R-D-Trp-2} SerjX) -Tyr ^(X3) _{-R4 -R5} -Arg ^(X4) -Pro X is an intermediate of formula ⁵ wherein Rj, _R2 conventional, R ₄ and R _s as defined above, X ¹ in the peptide α-amino protecting group; X ^{2 is} the conventional protecting group for the alcoholic hydroxyl group of Ser in peptide chemistry; X ^{3 is} the conventional protecting group for the phenolic hydroxy group of Tyr in peptide chemistry; X ⁴ is a conventional protecting group for the nitrogen atom of Arg in peptide chemistry; and X ⁵ is -Gly-O-CH ₂ [resin], -O-CH ₂ - [resin], -Gly-NH- [resin], or -Gly-NH ₂ ; wherein the resin of the conventional peptide chemistry, preferably chloromethylated, methyl benzhydrylamine support resin or benzhydrylamine, ^X! -X ^and deprotected using known in the peptide art, and - if desired, the resulting peptide is converted into pharmacologically acceptable acid addition salt thereof. (Priority: August 1980 29.) The process for preparing decapeptides of formula I according to claim 1, wherein R 1 is dehydro-Pro and X, R ₂ , R ₄ , R ₅ and R _{6 are} as defined in claim 1, characterized in that peptides wherein R _{n is} R ₂ , R ₄ , R ₅ and R ₆ as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 15, 1980) 5. The method of claim 1 decapeptides are formula I compounds wherein R ₂ is dichloro-D-Phe, and X, R ", R _4, R _s and R ₆ are as defined in claim 1, characterized in that a corresponding , starting from the protected peptide in which R _n R _2, R _4, R _s and R _e as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ⁵ in the first claim. (Priority: August 29, 1980) 6. The method of claim 1 decapeptides are formula I compounds wherein _R2 is 4-trifluoromethyl-D-Phe, and X, R ,, R _4, R _s and R ₆ are as defined characterized in Claim 1 by starting with a peptide in accordance with the meaning of R "R _2, R _4, R ₅ and R ₆ as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ^s in the first claim. (Priority: August 29, 1980) The process for preparing decapeptides of the formula I according to claim 1, wherein R ₂ is 4-acetylamino-D-Phe, and X, R ₄ , R ₅ and R _{6 are} as defined in claim 1, characterized in that by starting from the corresponding peptide in which R ,, R _2, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ⁵ are as defined in claim 1. (Priority: August 29, 1980) The process for the preparation of the decapeptides of the formula I according to claim 1, wherein R ₂ is 4-fluoro-D-Phe, and X, R ₁ , R ₄ , R ₅ and R _{6 are} as defined in claim 1, by starting from the corresponding peptide in which R ,, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ⁵ are as defined in claim 1. (Priority: August 29, 1980) A process for the preparation of a decapeptide of formula I according to claim 1, wherein R ₂ is 4-nitro-D-Phe and X, R 1, R ₄ , R ₅ and R _{6 are} as defined in claim 1, by starting from the corresponding peptide in which R ,, R _2, R _3, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ⁵ are as defined in claim 1 . (Priority: August 29, 1980) The process for the preparation of decapeptides of the formula I according to claim 1, wherein R ₂ is 4-bromo-D-Phe and X, R 1, R ₄ , R ₅ and R _{6 are} as defined in claim 1, by starting from the corresponding peptide in which R], R _2, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X ⁴ 189,504 and ^X5 are as defined in claim 1. (Priority: August 29, 1980) The process for preparing decapeptides of formula I according to claim 1, wherein R ₂ is 4-methylthio-D-Phe and X, R _n R ', R ₅ and R _{6 are} as defined in claim 1, by starting with a peptide in accordance with the meaning of R "R _2, R _4, R ₅ and R _s as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ⁵ in the first claim. (Priority: August 29, 1980) 12. The method of claims 1 or I decapeptide of formula wherein R ₂ is 4-methoxy-D-Phe, and X, R, R _4, R _s and R ₆ are as defined in claim 1, characterized in that starting from the corresponding peptide in which R _{u R2, R4, R5} and _R6 are as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ⁵ are as defined in claim 1. (Priority: August 29, 1980) The process for preparing decapeptides of formula I according to claim 1, wherein R ₂ is 4-methyl-D-Phe, and X, R 1, R ₄ , R ₅ and R _{6 are} as defined in claim 1, by starting with a peptide in accordance with the meaning of R "R _2, R _4, R _s and R _e as defined above, and X ^1, X ^2, X ^3, X ⁴ 'and X ⁵ in the first claim. (Priority: August 29, 1980) 14. The method of claims 1 or I decapeptide of formula where X akrililcsoportot, and R "R _2, R _4, R _s and R _ö to claim 1 as defined above, characterized in that starting with a peptide in accordance off, R ₁ , R ₂ , R ₄ R ₅ and R _{6 are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 14, 1981) 15. The method of claims 1 or I decapeptide of formula wherein X is Ac, R ,, R _2, R _4, indicated R _s and R _he is the first claim, characterized in that a peptide in accordance wherein R ', R ₂ , R ₄ , R ₅ and R _{6 are} as defined above and X', X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 14, 1981) The process for preparing decapeptides of the formula I according to claim 1, wherein R ₂ is 2,4-dichloro-D-Phe, 3,4-dichloro-D-Phe, 4-fluoro-D-Phe, 4-nitro -D-Phe, 4-bromo-D-Phe or 4metiltio-D-Phe, X is hydrogen, acetyl or akridilcsoport and R is as defined _n R _4, R _s and R ₆ in claim 1, characterized in that a corresponding , starting from the protected peptide in which R ,, R _2, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ⁵ are as defined in claim 1. (Priority: August 29, 1980) 17. The method of claims 1 or I decapeptide of formula wherein R ₄ is D-Trp, and X, R "R _2, R _s and R ₆ are as defined in claim 1, characterized in that a corresponding protected starting from peptides, where R ,, R _2, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ⁵ are as defined in claim 1. (Priority: April 14, 1981) A process for the preparation of a decapeptide of formula I according to claim 1 or claim 16, wherein R ₄ (imBz 1) is D-His and X, R 1, R ₂ , R ₅ and R _{6 are} as defined in claim 1, characterized in that by starting from the corresponding peptide in which R, R _2, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ⁵ are as defined in claim 1. (Priority: April 14, 1981) A process for the preparation of a decapeptide of formula I according to claim 1 or claim 16, wherein R ₅ is Leu and X, R 1 _; R ₂ , R _4, and R _{6 are} as defined in claim 1, wherein R 1, R ₂ , R ₄ , R ₅ and R _{6 are} as defined above, and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 14, 1981) A process for the preparation of a decapeptide of formula I according to claim 1 or claim 16, wherein R ₆ is -Gly-NH ₂ , and X, R 1, R ₂ , R ₄ and R _{5 are} as defined in claim 1, characterized in that starting from suitable protected peptides wherein R 1, R ₂ , R ₄ , R ₅ and R _{6 are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 14, 1981) 21. A process for the preparation of a decapeptide of formula I according to any one of claims 1 to 4 or 16 wherein X is acetyl, R ₄ is D-Trp, R _e is -Gly-NH ₂ , and R 1, R ₂ and R _{5 are} as defined in claim 1, characterized in that starting from suitable protected peptides wherein R 1, R ₂ , R ₄ , R ₅ and R _{6 are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1; . (Priority. April 14, 1981) 22. A process for the preparation of a decapeptide of formula I according to any one of claims 1 to 4 or 16, wherein X is acrylyl, R ₄ is D-Trp, R _e is -Gly-NH ₂ , and R ₁ , R ₂ and R _{5 are} as defined in claim 1, characterized in that starting from suitable protected peptides wherein R _{15 is} R ₂ , R ₄ , R ₅ and R _{e are} as defined above and X ', X ² , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 14, 1981) 23. The method according to any one of claims 21 or 22, a decapeptide of the formula I compounds wherein R _s is Leu, X is acetyl vgy akrililcsoport, _R4 is D-Trp, R ₆ is Gly-NH _2, and R and _R2 are as defined in claim 1, characterized in that starting from the corresponding peptide in which R ,, R _2, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 1981 IA) A process for the preparation of a decapeptide of formula I according to claim 1, wherein R ₂ is 4-chloro-D-Phe, and X, R 1, R ₄ , R ₅ and R _{6 are} as defined in claim 1, starting from suitable protected peptides wherein R ₁ , R ₂ , R ₄ , R ₅ and R _{6 are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 15, 1980) 25. The process of claim 24 for the preparation of decapeptides of formula I wherein R ₄ is D-Trp, R ₂ is 4-chloro-D-Phe, and X, R, R _s and R are as defined in claim 25, characterized in that starting from the corresponding peptide in which R "R _2, R _4". R ₅ and R <are as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{s are} as defined in claim 1. (Priority: April 15, 1980) 26. The method of claim 24 for the preparation of a decapeptide of formula 1 wherein R _{4 is} 189504 is (imBzl) D-His, R ₂ is 4-chloro-D-Phe, and X, R _l R _s and R e are as defined in claim 22, characterized by starting from the corresponding peptide in which R given _t, R _2, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X * and X ^s in the first claim. (Priority: April 15, 1980) The process for preparing decapeptides of formula I according to claim 24, wherein R * (imBzl) D-His, R ₂ is 4-chloro-Phe, and X, R ₅ , R ₅ and R _{6 are as} defined in claim 24, characterized in that starting from the corresponding peptide in which R _{B is} R _2, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ⁵ in claim 1. (Priority: April 15, 1980) 28. A 24-27. A method according to any preceding an I decapeptide of formula wherein R _s is Leu, R ₂ is 4-chloro-D-Phe, and X, R _n R ₄ and R ₆ are as defined in claim 22, characterized in that a corresponding starting from protected peptides, wherein R ₁ , R ₂ , R ₄ , R ₅ and R _{6 are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 15, 1980) 29. A 24-28. A method according to any preceding an I decapeptide of formula wherein R _e represents Gly-NH _3, R ₂ is 4-chloro-D-Phe, and X, R ,, R ₄ and R _s are as defined in claim 22, wherein characterized in that starting from suitable protected peptides wherein R 1, R ₂ , R ₄ , R ₅ and R _{6 are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 15, 1980) 30. A 24-29. A process for the preparation of a decapeptide of the formula I as claimed in any one of claims 1 to 4, wherein X is acetyl, R ₂ is 4-chloro-D-Phe, and R _x , R ₄ , R ₅ and R _{e are} as defined in claim 24, characterized in that starting with suitable protected peptides, wherein R _{15 is} R ₂ , R ₄ , R ₅ and R ₆ as above, and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 15, 1980) 31. A 24-29. A process for the preparation of a decapeptide of the formula I as claimed in any one of claims 1 to 4, wherein X is acrylic, R ₂ is 4-chloro-D-Phe, and R 1, R ₄ R ₅ and R _{6 are} as defined in claim 24, characterized in that they are derived from suitable protected peptides wherein R _{1 is;} R ₂ , R ₄ , R ₅ and R _{6 are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 1980 15.) 32. A 24-31. A process for the preparation of a decapeptide of formula I according to any one of claims 1 to 4, wherein R 1 is dehydro-Pro, R ₂ is 4-chloro-D-Phe, and X, R ₄ R ₅ and R _{e are} as defined in claim 24, characterized in that starting with suitable protected peptides wherein R ₃ , R ₂ , R ₄ , R 1 and R _{6 are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 1980 15.) 33. A 24-31. A method according to any preceding an I decapeptide of formula wherein R represents Thz, R ₂ is 4-chloro-D-Phe, and X, R _4, R _s and R ₆ are as defined in claim 24, characterized in that a corresponding , starting from the protected peptide in which R ,, R _2, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ⁵ are as defined in claim 1. (Priority: April 15, 1980) 34. A 24-31. A process for the preparation of a decapeptide of the formula I as claimed in any one of claims 1 to 3, wherein R 1 is D-Thz, R ₂ is 4-chloro-D-Phe, and X, R ₄ and R _{e are} as defined in claim 24, characterized in that they are derived from suitable protected peptides wherein R _{1 is;} R ₂ , R ₄ , R ₅ and R _{e are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 15, 1980) The process for preparing a decapeptide of formula I according to claim 1, wherein R ₂ is D-Phe and X, R _1; R _4, R _s and R ₆ are as defined in claim 1, characterized in that starting from the corresponding peptide in which R "R _2, R _4, R ₅ and R ₆ as defined above, and X ^1, X ^2, X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 15, 1980) 36. The process of claim 35 for the preparation of decapeptides of the formula wherein X is hydrogen, R _{2 is} as defined in claim 35, and R 1, R ₄ , R ₅ and R _{6 are} as defined in claim 1, wherein , starting from the protected peptide in which R _x, R _2, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ⁵ are as defined in claim 1. (Priority: April 15, 1980) The process for preparing decapeptides of formula I according to claim 35, wherein X is acetyl, R _{2 is} as defined in claim 35, and R 1, R ₄ , R ₅ and R _{6 are} as defined in claim 1, characterized in that starting from suitable protected peptides wherein R 1, R ₂ , R ₄ , R ₅ and R _{6 are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 15, 1980) 38. The method of claim 35 for the preparation of decapeptides of formula I wherein X is acrylic, R _{2 is} as defined in claim 35, and R 1, R ₄ , R ₅ and R _{6 are} as defined in claim 1, characterized in that starting from suitable protected peptides wherein R ₁ , R ₂ , R ₄ , R ₅ and R _{6 are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 15, 1980) A process for the preparation of a decapeptide of the formula I as claimed in any one of claims 35-38, wherein R ₅ is Leu, R ₂ in claim 35, and X, R 1, R ₄ and R ₆ in claim 1, starting from suitable protected peptides wherein R 1, R ₂ , R ₄ , R ₅ and R _{6 are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 15, 1980) 40. A 35-38. A process for the preparation of a decapeptide of formula I according to any one of claims 1 to ₅ , wherein R ₅ is N 'Me-Leu, R _{2 is} as defined in claim 35, and X, R 1, R ₄ and R _{6 are} as defined in claim 1, characterized in that starting with suitable protected peptides wherein R _x , R ₂ , R ₄ , R j and R _{e are} as defined above and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 1980 15.) A process for the preparation of a decapeptide of formula I according to claim 39 or 40, wherein R ₆ is -Gly-NH ₂ , R ₅ is Leu or n 'Me-Leu, R _{2 is} in claim 35, and X, R R ₄ and R ₅ as defined in claim 1, which are suitable protected peptides10 -101 189504 starting vesicle, where R "R _2, R _4, R _s and R ₆ are as defined above, and X ^1, X ^2, X ^3, X ⁴ and X ^s is given in claim 1. (Priority: April 15, 1980) 42. A process for the preparation of a decapeptide of formula I according to claim 39 or 40, wherein R ₆ is -NHCH ₂ CH ₃ , R ₅ is Leu or N "Me-Leu, R ₂ as defined in claim 35 and X, R" R ₄ and R ₅ as defined in claim 1, characterized in that they are derived from suitable protected peptides wherein R 1, R ₂ , R ₄ , R ₅ and R _{6 are} as defined above, ⁵ and X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} as defined in claim 1. (Priority: April 15, 1980)