DE2447097A1 - P-GLU-D-PHE-TRP-SER-TYR-D-ALA-LEUARG-PRO-GLY-NH TIEF 2, P-GLU-D-PHE-TRPSER-TYR-D-LEU-LEU-ARG-PRO- GLY-NH TIEF 2 AND INTERMEDIATE PRODUCTS - Google Patents

Description

patent attorney 244 / Uw /

PROF. DR. DR. J.- REITSTÖTTER DR.-ING. WOLFRAM BUNTE DR. WERNER KINZEBACH

D-8OOO MUNICH AO. BAUERSTRASSE 22 ■ FERNRUF (089) 37 63 83 ■ TELEX S21S2O8 ISAR D POSTAL ADDRESS: D-6OOO MUNICH 43. POST BOX 7ΘΟ

Munich, October 2nd. 1974 M / 15 489

AMERICAN HOME PRODUCTS CORPORATION 685, Third-Avenue, New York, New York 10017, USA

P-GLU-D-PHE-TRP-SER-TYR-D-ALA-LEU-ARG-PRO-GLY-Nh ₂ , P-GLU-D-PHE-TRP-SER-TYR-D-LEU-LEu-ARG -PRO-GLY-NH ₀

and intermediates

The invention relates to the new decapeptides p-GLU-D-Phe-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NHp, p-Glu-D-Phe-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-Gly-NHp » their manufacturing processes and new intermediates involved in such a synthesis formed.

The releasing factor for the luteinizing hormone (im hereinafter referred to as LRF) is the decapeptide L- (5.-0xoprolyl) -L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-glycyl- L-leucyl-L-arginyl-L-prolyl-glycine amide. This decapeptide is excreted through the hypothalamus and carried to the adenohypophysis where the release of the luteini

509815/1278

stimulating hormone. The present invention is concerned with structural modifications of LRF which have anti-ovulatory activity.

The new peptides according to the invention are obtained by the compounds of the formula:

p-Glu-D-Phe-Trp-Ser-Tyr-DX-Leu-Arg-Pro-Gly-NH ₂

(D

shown, where X has the meaning AIa or Leu, and their non-toxic salts. All chiral amino acid residues used in Formula I above and those below Other formulas given are of natural- or L-configuration, unless otherwise noted.

The intermediates also fall within the scope of the present invention the formula:

R ⁴ -P-Glu-D-Phe-Trp-Ser (R ³ ) -Tyr (R ² ) -D ~ X-Leu-Arg- (N ⁰ ^ -R ¹ ) -Pro-Gly-R

(II)

wherein:

X has the meaning AIa or Leu,

R is selected from NH ₂ , OH, O- (lower) alkyl, in which (lower) alkyl is C ₁ to Cg (for example methyl, ethyl, pentyl, hexyl, etc.) and O-benzyl,

N denotes the side chain nitrogen atoms ^{of arginine, R 1} a protecting group for the N ^, N ^ω and N ^{6 ^ nitrogen}

- 2 509815/1278

represents atoms of arginine selected from nitro, tosyl, benzyloxycarbonyl and adamantyloxycarbonyl; or where R is hydrogen, which means that there are no protecting groups on the side chain nitrogen atoms of the arginine. Where the protecting group means nitro or tosyl, the protection is on one of the Ν ^ώ , Ν ^Φ nitrogens and in the case of benzyloxycarbonyl or adamantyloxycarbonyl the protection is on the N · nitrogen and one of the N ^ω , Ν '^ nitrogen atoms. The preferred protecting group represented by R is the nitro group;

ρ
R is a protective group for the phenolic hydroxyl group of tyrosine, which is selected from acetyl, tosyl, benzoyl, tetrahydropyranyl, tert-butyl, trityl, benzyl, 2,4-dichlorobenzyl and benzyloxycarbonyl. The preferred protecting group is

2
Benzyl; or R is hydrogen, which means that there is no protecting group on the phenolic hydroxyl function;

R is a protecting group for the alcoholic hydroxyl group des

Represents serine and is selected from the meanings given for R; or R stands for hydrogen, which means that there is no protective group on the alcoholic oxygen atom. Preferably R is benzyl;

4th
R is preferably hydrogen or an α-amino protecting group

represents. In the case of the α-amino protective one denoted by R The group is those known from the prior art to be useful in the step-wise synthesis of polypeptides Groups. Among the classes of α-amino protective agents encompassed by R Groups include (1) protecting groups of the Acy1 type, which are illustrated by the following groups: Formyl, Trifluoroacetyl, phthalyl, toluenesulfonyl (tosyl), benzenesulfonyl, nitrophenylsulfenyl, tritylsulfenyl, o-nitrophenoxyacetyl, Chloroacetyl, acetyl, γ-chlorobutyryl, and the like,

- 3 -B09B1B / 1 278

(2) urethane-type aromatic protecting groups represented by Benzyloxycarbonyl and substituted benzyloxycarbonyl are illustrated, such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl; (3) aliphatic urethane protecting groups, illustrated by tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, Isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; (4) Cycloalkyl urethane type protecting groups by cyclopentyloxycarbonyl, AdamantyIoxycarbonyl, Cyclohexyloxycarbonyl; (5) thiourethane-type protecting groups such as phenylthiocarbonyl; (6) Alkyl-type protecting groups by triphenylmethyl (trityl), benzyl; (7) trialkylsilane groups, like .trimethylsilane. The preferred α-amino protecting groups represented by R are selected among t-butyloxycarbonyl, cyclopentyloxycarbonyl, tert-amyloxycarbonyl and d-isobornyloxycarbonyl.

12 In formula II, at least one of the radicals R, R or R is a protecting group.

Another aspect of the present invention relates to intermediates that are supported on a solid resin. These intermediates are by the formula:

R ⁵ -D-Phe-L-Trp-L-Ser (R ³ ) -L-Tyr (R ² ) -DXL-Leu-Arg (N ⁰ ^ R ¹ ) -L-Pro-Gly-A

(III)

shown in which:

X is AIa or Leu;

12 3
R, R and R have the same meanings as in formula II;

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5 4

R is selected from R -p-Glu, an oc-amino-protecting one Group or hydrogen (deprotected oc-amino group). The α-amino protecting group is preferably to tert-butyloxycarbonyl (tert-Boc), this protecting group also preferred to protect the α-amino group of all in Amino acids added to the stepwise solid phase synthesis is used. However, other oc-amino-protecting groups can can be used, such as o-nitrophenylsulfenyl, t-amyloxycarbonyl and biphenylisopropyloxycarbonyl ;;

"A" is the anchoring connector that is used in solid phase synthesis is used in bond to a solid resin support. "A" is selected under

H H

t!

'' Resin- ^N -

N_C - φ _ 'carrier

t /

Resin-\

and

(IHa)

O - CH ₂ φ

(HIb)

carrier i

The symbol φ means "phenyl".

When choosing a specific side chain protecting group to be used in the synthesis of the peptides according to formula I. the following rules should be followed: (a) The protecting group must be against the reactant and among those contributing to the removal of the α-amino protecting group The reaction conditions chosen at each stage of the synthesis must be stable, (b) the protective group must retain its protective properties retained (i.e. it must not be split off under coupling conditions) and (c) the side chain protection group must be retained Termination of the synthesis after the desired amino acid sequence has been obtained, removable under reaction conditions that do not change the peptide chain.

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-6.

To illustrate pharmaceutically acceptable, non-toxic Salts of the formula I include the hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, Succinate, malate, ascorbate and the like.

The peptides of Formula I are made using solid phase synthesis. The synthesis is from the C-terminal End of the peptide using an oc-amino protected Resin started. Such a starting material can be prepared by adding an α-amino protecting glycine to a benzhydrylamine resin, a chlorinated ethylated resin or a hydroxymethyl resin attaches, the aforesaid being preferred. The manufacture of a benzhydrylamine resin is by P. Rivaille et al., HeIv. 54, 2772 (1971) and the preparation of the hydroxymethyl resin is described by Bodanszky et al., Chem. Ind. (London) 38, 1597-98 (1966). A chloromethylated resin is from Bio Rad Laboratories Richmondj California im Commercially available. When the benzhydrylamine resin is used, it becomes amide anchoring as follows. Chain with the a-amino protected Glycine formed:

HOH ι hi

R ⁴ - N - CH ₅ - C - NH - C - 0 - 'carrier

This allows the C-terminal amide function right after the amino acid sequence in the synthesis is complete by cleavage of the resin support to form the glycine amide am C-terminal part of the desired peptide of formula I can be obtained. If other resins are used, act the anchoring chain is the benzyl ester group, as defined in formula (HIb) above, which occurs after the cleavage of the peptide from the resin carrier into the C-terminal amide

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M / 15 489

must be grounded. The preferred method is to use the protected Remove peptide from the resin by ammonolysis and then by hydrogenolysis or hydrogen fluoride cleavage remove the protecting group. An alternative method would be cleavage by transesterification with methanol / (Et) Ji and then converting the ester obtained into an amide and again subsequent cleavage of the protective group, as described above. Compare with J.M. Stewart "Solid Phase Peptide Synthesis ", pages 42-46 (W.H. Freeman & Co. 1969)

The a-amino protected. Glycine is coupled to the benzhydrylamine resin by means of a carboxyl activating compound such as dicyclohexylcarbodiimide. Following the coupling of the cx-amino-protected glycine to the resin support, the σ-amino-protecting group is removed, such as by using trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone, or HCl in dioxane. The protecting group is removed at a temperature between about zero C. and room temperature Other standard cleavage reagents and conditions for removing specific α-amino protecting groups can be used as described in Schröder and Lubke, "The Peptides", I, 72-75 (Academic Press 1965). After removal of the oc-amino-protecting group, the remaining α-amino-protected amino acids are coupled stepwise in the desired order to obtain a compound of the formula I. However, as an alternative to adding each amino acid separately to the reaction, some of them can be added before the addition If the C-terminal end of the peptide unit is connected by glycine or proline is shown and the coupling is carried out with DCC, a minimal racemization occurs with proline ¹ and with glycine, which does not have an asymmetric center, no problems arise. Any protected amino acid or amino acid sequence is in the solid phase reactor in approximately

_ 7 _ 509815/1278

·? ■

introduced a four-fold excess and the clutch is carried out in a medium of dimethylformamide: methylene chloride (1: 1) or in dimethylformamide or methylene chloride alone. In cases where an incomplete coupling has occurred, the coupling procedure will be performed prior to removal the a-amino-protecting group, before the coupling of the next amino acid to the solid phase reactor, repeated. The success of the coupling reaction at any stage of the synthesis is confirmed by the ninhydrin reaction, as described by E. Kaiser et al., Analyte. Biochem, 34, 595 (1970).

After the desired amino acid sequence has been synthesized, the peptide is removed from the resin support by treating with a reactant, such as hydrogen fluoride, which not only cleaves the peptide from the resin, but also all of them remaining side chain protecting groups and the α-amino protecting group, (if present) removed on the pyroglutamic acid, a compound of formula I being obtained directly in the case in which the benzhydrylamine resin was used. Where a chloromethylated resin has been used, the peptide can be obtained from Resin are separated by methanolysis, whereupon the recovered product is chromatographed on silica gel and the collected Fraction is subjected to ammonolysis to convert the methyl ester into the C-terminal amide. Every Side chain protecting groups can then be used as previously described or by other methods such as catalytic reduction (e.g. Pd on C) can be cleaved under conditions in which the Trp unit remains intact. Used if hydrogen fluoride is used for cleavage, anisole is added to the reaction vessel to prevent the oxidation of labile amino acids, (for example tryptophan) to prevent.

The solid phase synthesis discussed above is well known in the art and was essentially carried out by M. Monahan

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et al. CR. Acad. May be. Paris, 273, 508 (1971).

The nomenclature used for peptide synthesis is by Schroder and Lubke, supra, pp. viii-xxix and in Biochemistry 11, 1726-1732 (1972).

The following examples explain the preparation of the compounds of the formulas I to III.

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489 ^2U7097

example 1

L-Pyroglutamyl-D-phenylalanyl-L-tryptophyl-O-benzyl-L-seryl-0-benzyl-L-tyrosyl-D-alanyl-L-leucyl-N ^guan -N0 ₂ -L- arginyl-L-prolylglycyl- benzhydrylamine resin

20.0 g of benzhydrylamine resin are placed in a Merrifield reaction vessel with a capacity of 300 ml and subjected to the following wash cycle: A) methylene chloride, b) trifluoroacetic acid, (three times for 10 minutes each time); c) methylene chloride} d) methanol} e) triethylamine 12.5 % in dimethylformamide (twice 10 minutes each time), f) methanol (twice), g) methylene chloride (twice)} · with a contact time of at least 3 minutes each time, if not otherwise stated, is allowed.

The resin produced in this way is then mixed with 3.65 g (21 mmol) t-Butyloxycarbonylglycine in 1: 1 methylene chloride-dimethylformamide Shaken gently and 25.6 ml of the dicyclohexylcarbodiimide in methylene chloride are added over the course of 30 Added in 3 portions for minutes. Shake at ambient temperature for a total of 18 hours. The peptide resin will then washed successively with methanol, methylene chloride, methanol (twice) and methylene chloride (twice). To examine the completeness of the reaction, the peptide-resin is subjected to a ninhydrin test according to the method of E. Kaiser et al., Analytical Biochemistry 34, 595 (1970).

The removal of the protective group of the attached amino acid is carried out as follows: The peptide resin is with a 1: 1 solution of trifluoroacetic acid-methylene chloride (three times for 15 minutes each time), then the stages are treated (c) to (g) as described above for the washing cycle. Again, a sample of the peptide resin is a nin-

- 10 -

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M / 15 «9 ^2U7097

-AA s

Subject to hydrin test to check the completeness of the reaction. The sample is now strongly positive, which means the Eatschützun g of the glycine molecule bound to the resin is displayed.

The following amino acid residues are then introduced in sequence: t-Boc-L-proline (21 mmol, 25.6 mmol DCC), t-Bocnitro-L-arginine (21 mmol, 25.6 mmol DCC), t-Boc-L-leucine (21 mmol, 25.6 mmol DCC), t-Boc-D-alanine (21 mmol, 25.6 mmol DCC) and t-Boc-O-benzyl-L-tyrosine (21 mmol, 25.6 mmol DCC). Each coupling step is carried out in a medium of methylene chloride, dimethylformamide (1: 1) and the α-amino protecting agents are removed Group, at each stage is carried out as described for the deprotection of the t-Boc-glycine resin.

At this point the washed hexapeptide resin is dried, weighed (36.0 g) and synthesis is continued with one half (18.0 g) of the hexapeptide resin. The next amino acid added is t-Boc-O-benzyl-L-serine (10.4 mmol, 13 mmol DCC), followed by deprotection, then t-Boc-L-tryptophan (10.4 mmol, 13 "mmol DCC) . the washed peptide resin is dried again (20.8 g) and the synthesis is reacted with 2.0 g DC-OE peptide resin continued. However, upon addition of the t-Boc-tryptophane the deprotection reaction by the addition of 5% 1,2-ethanedithiol , which was added to the trifluoroacetic acid-methylene chloride medium, then t-BOC-D-phenylalanine (1.36 mmol, 2 mmol DCC) is added, followed by L-2-pyrrolidone-5-carboxylic acid (2.7 mmol, 3.4 mmol DCC) The washed peptide-resin is dried in vacuo.

- 11 -

50981 5/1278

Example 2

Pyroglutamyl-D-phenylalanyl-L-tryptophyl-L-seryl-L-tyrosyl-D-alanyl-L-leucyl-L-arginyl-L-prolylglycine amide

Removal of the protective groups and cleavage of the decapeptide from the resin is carried out by treating the dried peptide resin according to Example 1 in vacuo with liquid hydrogen fluoride (25 ml) and anisole (10 ml) for one hour at ice bath temperature. The hydrogen fluoride is removed by vacuum distillation and the anisole is removed by washing with ether. The peptide is dissolved in 10 % acetic acid and removed from the resin by filtration. Lyophilization provides the desired decapeptide in the form of a white, flaky powder.

example

Purification and characterization of pyroglutamyl-D-phenylalanyl-L-tryptophyl-L-seryl-L-tyrosyl-D-alanyl-L-leucyl- L-arginyl-L-prolylglycine amide acetate

The crude peptide is 0.2 η acetic acid in a minimal volume dissolved, applied to a Biogel P-2 0.074 mm to 0.037 mm (200 to 400 mesh) gel filtration column (2.6 cm 90 cm) and eluted with the same solvent. Fractions of 9 ml are collected in each case. The one you want Fractions containing peptide are determined by the Pauly spot test and by UV analysis. After putting together and Lyophilization gives 252 mg of a white, flaky powder.

- 12 -

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A distribution column with Sephadex G-25 fine (2.6 cm 90 cm) is achieved by equilibrating with the lower phase and then the upper phase of the BAW solvent system (n-butanol: acetic acid; Water, 4: 1: 5, V ^ = 165 ml).

The above lyophilized peptide is applied in a minimal volume of upper phase. Elute with upper phase (6 ml fractions) provides the desired product, which is localized as described above. After putting together and Lyophilization gives 142.8 mg of a white, flaky powder.

The optical rotation is performed on a Carl Zeiss LEP A-2

η f.

photoelectric precision polarimeter determined; [a] _D = -36.54 (c = 1.024, 1 % acetic acid); the amino acid analysis gives the following ratios: Ser (0.55), GIu (1.04), Pro (0.88), GIy (1.00, AIa (1.02), Leu (0.96), Tyr ( 1.02), Phe (1.04), NH ₃ (0.68), Trp (0.65), Arg (1.04); R _f (on silica): 0.28 (n-butanol: acetic acid : Water, 4: 1: 5).

The peptide (batch of 20 µg) is in three thin layer chromatography systems (TLC systems) (acidic, neutral and basic) when examined under ultraviolet light, iodine vapor and Pauly reagent homogeneous.

Example 4

L-Pyroglutamyl-D-phenylalanyl-L-tryptophyl-O-benzyl-L-seryl-0-benzyl-L-tyrosyl-D-leucyl-L-leucyl-N ^ ^uan -NOp-L-arginyl-L-prolylglycyl- benzhydrylamine resin

20.0 g of benzhydrylamine resin are placed in a Merrifield reaction vessel with a capacity of 300 ml and through the the following washing cycle performed: a) methylene chloride, b) 1: 1

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Trifluoroacetic acid methylene chloride (3 times for 10 minutes each time), c) methylene chloride, d) methanol, e) 12.5 % triethylamine in dimethylformamide (2 times for 10 minutes each time), f) methanol (twice), g) methylene chloride (twice ), whereby a contact time of at least 3 minutes is allowed, unless otherwise stated.

The resin produced in this way is then mixed with 3.65 g (21 mmol) t-Butyloxycarbonylglycine in 1: 1 methylene chloride-dimethylformamide Shaken gently and add 25.6 ml of the dicyclohexylcarbodiimide in methylene chloride over the course of 30 minutes added in 3 portions. Shaking is done at ambient temperature continued for a total of 18 hours. The peptide resin is then sequentially treated with methanol, methylene chloride, and methanol washed (twice) and methylene chloride (twice). The peptide resin is used to examine the completeness of the reaction subjected to a ninhydrin test according to the method of E. Kaiser et al., Analytical Biochemistry 34, 595 (1970).

The deprotection of the attached amino acid is carried out as follows: The peptide resin is treated with a 1: 1 solution of Trifluoroacetic acid methylene chloride (three times, for 15 minutes each time) treated, then steps (c) "to (g), v / ie described above for the washing cycle. Again, a sample of the peptide-resin is subjected to a ninhydrin test to determine whether the reaction is complete to investigate. The sample is now strongly positive, thereby deprotecting the glycine molecule bound to the resin is shown.

The following amino acid residues are then introduced in sequence: t-Boc-L-proline (21 mmol, 25.6 mmol DCC), t-Boc-nitro-L-arginine (21 mmol, 25.6 mmol DCC), t-Boc-L-leucine (21 mmol, 25.6 mmol DCC). Each coupling stage is in a medium of Methylene chloride-dimethylformamide (1: 1) carried out and the ent-

- 14 509815/1 278

removed the α-amino protecting group at each stage v / i ο described for the deprotection of the t-Boc-glycine resin, carried out.

At this point the washed tetrapeptide is dried and synthesis is continued with 6 g of the tetrapeptide resin. The next amino acid added is t-Boc-D-leucine (6.8 mmol, 8.5 mmol DCC), followed by deprotection, then t-Boc-O-benzyl-L-tyrosine (6.8 mmol, 8, 5 mmol DCC) was added, followed by deprotection, then t-Boc-O-benzyl-L-seriri (6.8 mmol, 8.5 mmol DCC) was added, followed by deprotection., Then t-Boc-L- tryptophan (6.8 mmol, 8.5 mmol DCC) added, followed by deprotection. The washed peptide-resin is dried again (7.37 g) and synthesis is continued with 2.45 g of the peptide-resin. However, after the addition of t-Boc-tryptophan, the deprotection reaction is carried out by adding 5 % 1,2-ethanedithiol, which is added to the trifluoroacetic acid-methylene chloride medium. Next, t-Boc-D-phenylalanine (2.4 mmol, 3 mmol DCC) is added, followed by deprotection, then L-2-pyrrolidone-5-carboxylic acid (2.4 mmol, 3 mmol DCC) is added. The washed peptide resin is dried in vacuo.

Example 5

Pyroglutamyl-D-phenylalanyl-L-tryptophyl-L-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-L-prolylglycine amide

Removal of the protecting groups and cleavage of the decapeptide from the resin (2.5 g) is carried out by treating the dried peptide-resin according to Example 4 in a vacuum with 25 ml of liquid hydrogen fluoride and 10 ml of anisole for one hour at ice bath temperature carried out. The hydrogen fluoride is through

- 15 50981 5/1278

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Vacuum distillation is removed and the anisole is removed by washing with ether. The peptide is dissolved in 10 % acetic acid and removed from the resin by filtration. Lyophilisieron delivers the desired decapeptide in the form of a white, flaky powder.

example

Purification and characterization of pyroglutamyl-D-phenylalanyl-L-tryptophyl-L-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-prolylglycine amide acetate

The crude peptide is dissolved in a minimal volume of 0.2 η acetic acid, there is 0.074 mm - 0.037 mm on a Bio-Gel P-2 (200-400 mesh) gel filtration column (2.6 cm 90 cm) and eluted with the same solvent. There will be each Fractions of 9 ml collected. The parliamentary groups (32 to 40), which contain the desired peptide are determined by the Pauly spot test and by UV analysis. After putting them together and lyophilization, 460 mg of a white, flaky powder.

A distribution column with Sephadex G-25 fine (2.6 cm 90 cm) is equilibrated with the lower phase and then with the upper phase of the BAW solvent system (n-butanol: acetic acid: water, 4: 1: 5, V _R = l65 ml).

The above lyophilized peptide is applied in a minimal volume of upper phase. Elute with upper phase (6 ml fractions) provides the desired product, which is localized as described above. After combining and lyophilizing 237 mg of a white, flaky powder are obtained.

- 16 509815/1278

The optical rotation is measured on a Carl Zeiss LEP A-2 photoelectric precision polarimeter; [α] β = -26.0 ° (c = 0.798, 1 % acetic acid); the amino acid analysis gives the following ratios: Ser (0.97), GIu (1.03), Pro (0.9), GIy (1.0), Leu plus D-Leu (2.0), Try (1, 2), D-Phe (1.0), Trp (0.73), Arg (1.0), R _f (on silica): 0.34 (n-butanol: acetic acid: water 4: 1: 5) ·

The peptide (20 g batch) is in three TLC systems (acidic, neutral and basic) for! examination under ultraviolet Light, homogeneous with iodine vapor and with Pauly's reagent.

The compounds of the formula I have anti-ovulatory properties Activity and are "therefore potentially useful in fertility inhibition in female mammals. In tests carried out on female rats (225 to 250 g body weight) complete ovulation inhibition was achieved at a dose of 12 mg / kg with the compound according to Example 3 and obtained with the compound according to Example 6 at a dose of 24 mg / kg. The test was made with mature Sprague-Dawley Rats with the normal cycle, non-anesthetized, pre-fevered rats. In the afternoon During the pre-oestrus, each rat in the test group receives six subcutaneous injections of the acetate salt of formula I in corn oil, each injection given half an hour after the previous injection. The rats are the next day killed and the number of ovulating animals and the number of eggs shed according to the method described by E.S. France, Neuroendocrinology, 6, pp. 77-89 (1970). The absence or a significant decrease the number of eggs is the criterion for an anti-ovulation effect. At a dose of 500 µg per injection of the compound according to Example 3 and 1 ml per injection of the compound according to Example 3 Example 6 complete inhibition of ovulation is achieved. . ,

- 17 509815/1278

The compounds according to formula I can be used intravenously, subcutaneously, intramuscularly or orally to inhibit fertility and -Control administered to mammals. The effective dose will vary with the mode of administration and the particular one Type of mammal to be treated. A typical dosage is a physiological saline solution, which is a Compound of formula I contains, and in a dosage range of between 8 and 30 mg / kg of body weight and in particular 8 to 12 mg / kg of compound according to Example 3 and 20 to 30 mg / kg of compound according to Example 6 are administered will. Oral administration can be in either solid or liquid form.

- 18 -509815/1278

Claims (2)

Claims 1. Compound selected from LP-Glu-D-Phe-L-Trp-L-Scr-L-Tyr-DXL-Leu-L-Arg-L-PrO-GIy-NH ₂ R ⁴ _L-P-Glu-D-Phe-L-Trp-L-Ser (R ³ ) -L-Tyr- (R ² ) -DXL-Leu-L-Arg- (N ¹ ^ -R ¹ ) - L-Pro-Gly-R and their non-toxic salts; wherein X is AIa or Leu; R is selected from NH ₉ , OH, O- (lower) alkyl and O-benzyl; R is a protecting group for the N ^, N ^ and Ν * "'nitrogen atoms des arginine and is selected from nitro, tosyl, benzyloxycarbonyl and adamantyloxycarbonyl; or where R represents hydrogen; 2
R represents a protective group for the phenolic hydroxyl group of tyrosine, which is selected from acetyl, tosyl, benzoyl, tert-butyl, tetrahydropyranyl, trityl, benzyl, 2,4-dichlorobenzyl and benzyloxycarbonyl; or in which R is hydrogen; R is a protecting group for the alcoholic hydroxyl group des Represents serine and is selected from acetyl, tosyl, benzoyl, tetrahydropyranyl, tert-butyl, trityl, 2,4-dichlorobenzyl, Benzyl and benzyloxycarbonyl; or wherein R is hydrogen; - 19 509815/1278 R is selected from hydrogen or an α-amino-protecting one Group; provided that at least one of the groups 12 3
R, R and R represent a protecting group. 2. A compound according to claim 1, wherein R is NH ₉ . 3. A compound according to claim 1, wherein R is NH ₀ , 1 2 3 R is nitro, R is benzyl, R 1 is benzyl and in which R is hydrogen. 4. Compound according to claim 1, selected from L-pyroglutamyl-D-phenylalanyl-L-tryptophyl-L-seryl-L-tyrosyl-D-alanyl-L-leucyl-L-arginyl-L-prolyl-glycine amide, L-pyroglutamyl-D-phenylalanyl-L-tryp tophyl-L-s eryl-L-tyrο sy1-D-1eucyl-L-leucyl-L-arginyl-L-prolyl-glycine amide and their non-toxic acid addition salts. 5. Compound of the formula: R ⁴ -D-Phe-L-Trp-L-Ser (R ³ ) -L-Tyr (R ² ) -DXL-Leu-L-Arg (N ^G -R ¹ ) -L-Pro-Gly-A wherein: X is AIa or Leu; R, R, R and R have the same meanings as in claim 1 own; - 20 5 0 9815/1278 5 4 R- is selected from R -p-Glu-, an α-amino-protecting one Group or hydrogen;
and A for HH f " ~ X ¹ '/' resin / resin N - C - 0 —j carriers and -0 - CH ₉ - 0 -! Carriers v / V stands. 6. A compound according to claim 5, wherein R is α-amino protecting Represents a group selected from tert-butyloxycarbonyl, o-nitrophenylsulfenyl, tert-amyloxycarbonyl and biphenylisopropyloxycarbonyl. 7. A compound according to claim t>, wherein A is a benzhydrylamine resin!
substance means. 5 4 4 represents amine resin and R stands for R -p-Glu and R stands for water- 8. A compound according to claim 7, wherein R is nitro 2 3
and R and R represent benzyl. - 21 - 509815/1278