HU190207B - Process for production of new gonadoliberine derivatives - Google Patents

Abstract

Gonadoliberin analogues of formula (I) and their salts and complexes are new. - Glp-His-Trp-Ser-Tyr-X1-X2-X3-Pro-X4 (II) X1=Gly or a D-amino acid; X2=L-Phe, L-Trp or an L-amino acid with a 1-4C side chain; X3=an L-amino acid with a 1-4C alkyl side chain or a 2-4C alkanoylamide gp.; X4=Gly-NH2 or 1-4C alkylamino; provided that X3 is not Leu when X1 is other than Gly and X2=Trp.

Description

The present invention relates to a novel general formula (I)

Glp-His-Trp-Ser-Tyr-X, -X ₂ -X ₃ -Pro-X ₄ - wherein

X 1 is glycyl, D-alanyl, D-leucyl, O-tert-butyl D-seryl, D-tryptophyl or D-phenylalanyl,

X ₂ is leucyl, tryptophyl or phenylalanyl;

X ₃ is leucyl or glutaminyl,

X ₄ is a glycylamide or ethyl amide, with the proviso that when X 1 is Dalanyl,

X ₂ is tryptopyl, X ₃ is leucyl, then X ₄ may be other than ethyl amide only for the preparation of nonapeptide ethyl amide or decapeptide amide and salts thereof.

The abbreviations used in the formulas correspond to the nomenclature accepted in peptide chemistry, e.g. Chem., 241, 527 (1966). Accordingly, the abbreviations used in this description have the following meanings:

x - x Glp: Pyroglutamic acid (Pyroglutamil)

His: histidine (histidyl)

Trp: tryptophan (tryptophan)

Ser: Serine (Seril)

Tyr: Tyrosine (Tyrosyl)

Gly: glycine (glycyl)

Phe: phenylalanine (phenylalanil)

Leu: leucine (leucil)

Pro: Proline (Proli)

Glu: glutamic acid (glutamil)

Gin: glutamine (glutamine)

Me: methyl

Et: ethyl

Boc: tert-butyloxycarbonyl

Bzl: Benzyl

UV: ultraviolet

Tos: tosyl

ONP: p-nitrophenyl

DNP: Dinitrophenyl

EA: Ethyl amide

Z: Benzyloxycarbonyl

DMF: dimethylformamide

THF: tetrahydrofuran

TFA: trifluoroacetic acid

TEA: triethylamine

DCC: dicyclohexylcarbodiimide

DIC: Diisopropylcarbodiimide

DCU: dicyclohexylurea

HPLC: high performance liquid chromatography TLC: thin layer chromatography.

Gonadoliberin (also known in the art as gonadotropin releasing hormone, Gn RH, luteinizing and follicle stimulating hormone, releasingl hormone, LH / FSH-RH) and its derivatives are also commonly known to possess the ability to luteinize and follicle stimulate. to release hormones (LH and FSH).

In the early 1980s, it became known that the structure of gonadoliberin in some fish is different from that of mammalian gonadoliberin (J.A.

'Ring and RP Milari, J. Bioi. Chem. 257: 10722-28 (1982); South African Journal of Science 78, 124 (1982); N. Sherwood at al. Proc. Natl. Acad. Sci. 80, 2794-2798 (1983). These differences affect the amino acids at positions 7 and ⁵ , respectively.

It is also known in the literature that gonadoliberin derivatives containing certain D-amino acids instead of glycine at position 6 show a ^10- fold increase in activity relative to gonadoliberin, J. Sandow et al., Control of Ovulation, Butterworth, London, 1971. pp. 49-70.

It is also known that by replacing the glycine amide group at position 10 with aliphatic amide groups, the biological activity of gonadoliberin can be further enhanced (M. Fujino et al., J. Med. Chem. 16, 1144-1147 (1973)).

U.S. Patent No. 4,410,514 discloses [D-Ala ⁶ , Tro ⁷ , Leu ⁸ , desGly ¹⁰ ] -Gonadoliberin ethylamide as a compound for breeding fish.

It is an object of the present invention to provide novel gonadoliberin derivatives which are useful in fish breeding.

²⁵ is another object of the present invention, these compounds are derivatives of the preparation, which exhibit improved biological activity of the parent compounds.

The invention is based on the discovery that replacement of the gona ³⁰ dofiberin 7- and 8-position of the amino acids of certain amino acids and certain combinations of these substitutions can be made from fish propagation gonadoliberin derivatives.

A further basis ³⁵ of the invention is the recognition that biological effectiveness of gonadoliberin derivatives of gonadoliberin 7- and 8-position the amino acids of the exchange can be increased by replacing some to ⁴⁰ D-amino acids in the 6-position of glycine group, and the 10 by replacing the glycinamide at position 3 with an alkylamide.

Accordingly, the process of the invention is represented by the formula (I)

Glp-His-Trp-Ser-Tyr-X ₁ -X ₂ -X ₃ -Pro-X ₄ (I)

wherein X 1, X ₂ , X ₃ and X ₄ are as defined above

- nonapeptide ethyl amide or decapeptide amide and salts thereof.

According to the invention, the process is carried out by:

a) employing solid-phase peptide synthesis, stepwise condensation of protected amino acids on a solid support by carbodiimide or active ester coupling, respectively, and removal of the final product from the solid support by acidic or basic cleavage and simultaneous removal or deprotection of the resin. or after removal, or

b) using stepwise and appropriate combinations of fragment condensation, the protected amino acids are condensed and deprotected.

As a solid support, it is convenient to use benzhydrylamine resin or chloromethylated polystyrene resin.

190 207

It is advantageous to remove the protecting group-containing final product from the resin by hydrogen fluoride cleavage and / or ethyl ammonolysis.

Among the combinations according to variant b), the following variants are particularly preferred:

a compound of formula II

Glp-His-Trp-Ser-Tyr-N ₃ (II) pentapeptide azide is a compound of formula (III)

X, -X ₂ -X ₃ -Pro-X ₄ (III) are condensed with a tetra- or pentapeptide.

In formula (III) above, X ₁ , X ₂ , X ₃ and X ₄ are as defined above.

The resulting nona or decapeptide amide is optionally reacted with a pharmaceutically acceptable acid to form an acid addition salt, or optionally reacted with a base to liberate the free base.

The invention further relates to a process for the preparation of fish breeding compositions from the compounds of formula (I). This process is carried out by mixing a compound of formula (I) or an acid addition salt thereof with a conventional carrier and / or excipient to form a fish breeding composition.

The gonadoliberin derivatives of formula (I) are effective in fish in a dose range of 0.1 pg to 5 mg when administered intramuscularly or subcutaneously.

The main advantage of the process according to the invention is that the new gonadoliberin derivatives produced by it contain new amino acid combinations at the 7 and 8 position and are advantageously used for fish breeding.

In order to carry out the process according to the invention, the following examples are given.

In Examples 1-4, 6-7 and 9-12, the compounds were prepared according to the commonly used methods of solid phase synthesis (Merrifield, R. B., J. Am. Chem. Soc. 85, 2149-2151 (1963)). Depending on the structure of the desired compound, chloromethylated polystyrene divinylbenzene was used for peptide alkylamide and benzhydrylamine resin for peptide acid amide. The individual amino acids were stepwise coupled to the resin in the form of their N-t-butyloxycarbonyl (Boc) derivatives via dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC) or by active ester coupling.

The degree of coupling reactions was investigated by the ninhydrin assay (Kaiser, E., Colescott, R. L., Bossinger, C. D., Cook, P. I., Anal. Biochem. 34, 595-598 (1970)). Acylation was repeated if a free amino group was detected. The duration of the coupling varied from 1 to 16 hours depending on the amino acids.

Deprotection and deprotection of the peptide from the resin were performed in one step using liquid anhydrous hydrogen fluoride (Sakakibara S., Shimonishi Y., Kishida Y., Okada M., Sugikara H., Bull. Soc. Japan. 40, 2164-2167). / 1967 /). In the case of N ^im- dinitrophenyl, the protecting group was removed before the HF cleavage. The protected peptide resin was mixed with propylamine-containing dimethylformamide, after removal of the solvent, the peptide resin was mixed with propylamine-containing dimethylformamide, and after removal of the solvent, the peptide resin was treated with HF in the usual manner. In the case of a peptide alkyl amide type compound, the peptide was removed from the resin by alkyl ammonolysis followed by catalytic hydrogenation and / or acid hydrolysis, depending on the nature of the protecting groups.

The crude product obtained after HF cleavage was purified by gel filtration on a Sephadex G25 column using acetic acid solutions as eluent.

Further purifications were carried out on a preparative HPLC column and / or silica gel chromatography. The purity of the peptides was tested by amino acid analysis and TLC. The Rf value _{of R} were determined by silica gel plates (DC Alufolien, Merck) in the following solvent systems:

Ethyl acetate-pyridine-acetic acid-water 30: 20: 6: 11 • 2 Ethyl acetate-pyridine-acetic acid-water 60: 20: 6: 11

Ethyl acetate-pyridine-acetic acid-water 120: 20: 6: 11

Ethyl acetate-pyridine-acetic acid-water 240120: 6: 11 t-Butanol-acetic acid-water-ethyl acetate 1: 1: 1: 1 n-butanol-acetic acid water 4: 1: 1 i-Propanol-1mole acetic acid 2: 1

Ethyl acetate-pyridine-acetic acid-water 5: 5: 1: 3

Example 1 Preparation of [D-Phe ⁶ , Gln ⁸ ] gonadoliberin M: 1243

a) Synthesis

1.25 g (1 mmol) of benzhydrylamine-HCl resin [0.8 mM / g, Pierce] was swollen in dichloromethane for 2 hours and the following cycle was repeated during acylation with amino acids:

Step Reagent, Action Mixing Time (Minutes)

1. CH ₂ Cl ₂ , Wash 3 times 1

2. A mixture of 33% trifluoroacetic acid (TFA) and 1% CH ₂ Cl ₂

3. 33% trifluoroacetic acid (TFA) and 25%

67% CH ₂ Cl ₂

4. CH ₂ Cl ₂ , Wash 3 times 1

5. CHCl ₃ , Wash twice 1

6. 10% triethylamine (TEA) and 2

90% CHCl3 ₃ , Wash twice

7. _CHCl3, wash twice with 1

8. Ethanol, Wash twice 1

9. CH ₂ Cl ₂ , Wash twice 1

10. 3 eq. Boc-amino acid, dissolved in CH ₂ Cl ₂ * eq. DCC v. DIC CH ₂ Cl ₂ dissolved in 60 variables

11. CH ₂ Cl ₂ , Wash twice 1

12. Ethanol, Wash twice with 1 * Gin coupled with Boc-Gln-ONP active ester coupling; no protecting group is used for pyroglutamic acid.

After each step, a ninhydrin test is performed; if a positive result is obtained, the cycle is the

Repeat from row 5.

190 207

After the final step, the peptide resin Glp-His (Tos) -TrpSer (OBzl) -Tyr (OBzl) -D-Phe-Leu-Gln-Pro-Gly weighed 2.28 kg.

AW: 1.05 g (66%) (Protected peptide 1,577)

b) HF cleavage

To the resin was added 3 ml of anisole, 100 mg of dithiothreitol, 30 ml of HF was condensed and stirred at 0 ° C for 1 hour. The HF is then removed with a stream of N ₂ , the residue is suspended in absolute ether and filtered. The solid residue was washed with 50% acetic acid and the filtrate was concentrated at 37 ° C. The HF salt thus obtained is directly subjected to gel chromatography

(c) Gel chromatography

The material from the previous point was purified on a Sephadex G25 column (2.5 x 100 cm) using 50% acetic acid as eluent. Separation was monitored by UV absorbance at 280 nm and TLC. At a flow rate of 15 ml / h, fractions ranging from 300 ml to 350 ml are collected and lyophilized. Weight: 690 mg (55%).

d) Preparative HPLC

A CG silica gel LRP-1 (13-24 pm) (Whatman) preparative HPLC column (2.5 x 45 cm) was used. The equilibration was performed with a mixture of 25% methanol and 75% 30% acetic acid. After equilibration, 230 mg of the gel-purified material is dissolved in this mixture. Elution was performed with a linear gradient of 25% methanol to 75% 30% acetic acid and 40% methanol to 60% 30% acetic acid. Flow rate 2 ml / min, the pressure is 3,5 10 ⁵ Pa. The fractions were monitored at 280 nm and their content is monitored by TLC. The final product obtained after lyophilization had a pure [D-Phe ⁶ , Gln ⁸ ] GnRH of 133 mg. This corresponds to 399 mg (32%) of the purified final product based on the total amount of material.

R _f = 0.76 R 0.68 ⁵ R = [R _f = 0.33 ⁷ = 0.93 R = 0.83

Amino acid analysis: Gly: 1.02; Pro: 0.95; Leu: 1.00; Phe: 1.02 Tyr: 1.01; Ser: 0.93; His: 0.99; Glu: 1.96

Example 2 Preparation of [D-Phe ⁶ , Trp ⁷ , Leu ⁸ ] Gonadoliberin M: 1301

a) Synthesis

Starting from 2.5 g (1 mmol) of benzhydrylamine HCl resin [0.4 mmol / g], Glp-His (Tos) -TrpSer (OBzl) -Tyr (OBzl) -DPhe was synthesized as described in Example 1 (a). -Trp-Leu-Pro-Gly-deca peptide amide. Weight of peptide resin obtained 3.8 g AW: 1.3 g (80%) ( ^M protected peptide: 1635)

b) HFos cleavage

The procedure is the same as described in Example 1 (b). The residue obtained after evaporation of the 50% acetic acid filtrate and lyophilization weighed 965 mg (74%).

(c) Gel chromatography

Gel chromatography was performed on a Sephadex G25 column using the conditions described in 1 / c. The fraction collected and lyophilized weighed 500 mg (38%).

d) SiO ₂ chromatography

Silica gel chromatography was performed on a Kieselgel 60 (230-400 mesh) (Merck) column (2x100 cm). Equilibration and elution were performed with ethyl acetate-acetic acid-pyridine-water = = 30: 6: 20: 11 at a flow rate of 8 ml / h. The content of each fraction was examined by TLC in solvent mixture 2 followed by amino acid analysis. Based on the amino acid content, fractions ranging from 470 ml to 500 ml correspond to pure [D-Phe ⁶ , Trp ⁷ -Leu ⁸ ] gonadoliberin. The peptide weight was 375 mg (29%). R f: 0.58 R ⁷ : 0.24, R f: 0.72

Amino acid analysis: Glu 0.97; Gly 0.99; Pro 1.02; Leu 1.00; Tyr 1.03; Phe 1.01; Ser 0.93; Trp 1.87; His 0.95

Example 3 Preparation of [Trp ¹ , Gln ⁸ ] gonadoliberin M: 1226

a) Synthesis

Starting from 2.5 g [1 mmol] of bihydrylamine HCl resin [0.4 meq / g] and preparing the GlpHis (DNP) -Trp-Ser- ( OBzl) _{-Tyr-Gly-Trp-Gln-Pro-Gly-NH2} decapeptide. Peptide Resin Weight 3.62 g ·

AW: 1.12 g (76%) (Protected Peptide · 1468)

b) Deprotection and cleavage of the peptide from the resin

DNP (dinitrophenyl) protecting group deprotection:

The peptide resin was stirred in 20 ml of DMF and 1 ml of propylamine at room temperature for 1 hour. The resin was then filtered and washed with dichloromethane and ethanol.

HF cleavage

After removal of the dinitrophenyl protecting group, the peptide is removed from the resin and the protecting groups by the method described in Example 1 (b).

The material obtained after two steps weighed 0.8 g (65%).

(c) Gel chromatography

The procedure is the same as described in Example I (c). The weight of the collected and lyophilized peptide was 520 mg (42%).

d) Preparative HPLC

The procedure was carried out in a similar manner to that described in Example I (d) except that the equilibration was carried out with a mixture of 18% methanol and 82% 30% acetic acid. The material purified by gel chromatography was 18% methanol - 82% 30%

Elute from the column with a linear gradient of 190 acetic acid and 35% methanol to 65% 30% acetic acid. Other conditions for fractionation are the same as described in 1 / d. The weight of the lyophilized final product was 380 mg (31%). ^!

Rf: 0.66 Rf: 0.57 Rf: 0.78,

Amino acid analysis: Glu 1.93; Gly 2.04; Pro

1.00; Tyr 0.98; Ser 0.93; Trp 1.86; His, 03

Example 4 [T rp ^7, Leu ^B desGlyNHi °] -gonadoliberin ethylamide Preparation M: 1198 ₁₅

a) Synthesis

The first step in the synthesis, Boc-Pro-is coupled to chloromethylated polystyrene resin is known in the art and commonly used method serum Rint ₂ θ (Merrifield, RB, J. Am. Chem. Soc. 86, 304/1964 /). 2.0 g (1 mmol) of the resulting Boc-Pro resin [0.5 meq / g] was swelled in dichloromethane for 2 hours and then coupled stepwise to the resin according to the reaction described in Example 1 (a). adequately protected amino acids. The weight of GlpHis (DNP) -Trp-Ser (OBzI) -Tyr (OBzI) -Gly-Trp, Leu-Pro-nonapeptide resin obtained after the last cycle was 3.02 g.

AW: 1.02 g (83%) "( _{Protected peptide} M, _d : 1486)

b) Deprotection and cleavage of the peptide from the resin

By cleavage of the dinitrophenyl protecting group ₃₅ compares the histidine as described in Example 3, paragraph b). The peptide is then cleaved from the resin in the form of ethyl amide (Coy, DH et al., Biochemistry 13, 323-326 (1974)). For this purpose, 15 ml of ethylamine were condensed onto the protected peptide resin and stirred at 0 ° C for 3 hours. The excess amine was evaporated using ^Q N ₂ gas. This is followed by washing with methanol followed by dimethylformamide. After evaporation of the dimethylformamide solution, the oil obtained is triturated with ether and the precipitate is filtered off.

The resulting solid was treated with HF gas as described in Example 1 (b) ( ⁴⁵ ) to remove the protecting groups. These operations afforded 710 mg (59%) of nonapeptide ethylamide.

(c) Gel chromatography

Molecular filtration was performed as described in Example 1 (c) using a Sephadex G25 column, eluting with 30% acetic acid.

The fraction collected and lyophilized weighed 490 mg (41%).

d) SiO 2 chromatography

Silica gel chromatography was performed as described in Example 2 (d). The fraction corresponding to [Trp ⁷ , Leu ⁸ , - 60 desGlyNHf °] -Gn-RH-EA is determined from the amino acid content.

The lyophilized material weighed 320 mg (26%).

Rf: 0.61; Rf: 0.42; Rf: 0.75

Amino acid analysis: Glu 0.96; Gly 0.97; Pro

0.95; Leu 1.00; Ser 0.89; l Tyr 1.02; Trp 1.88; His 1.03

Example 5 Preparation of [D-Phe ⁶ , Gln ⁸ , desGlyNHf °] -gonadoliberin-ethylamide M: 1198

a) BOC-His (DNP) -Trp ~ OMe M. 622.62

21.12 g (50 mmol) of BOC-IIis (DNP) -OH are dissolved in 100 ml of dimethylformamide and cooled to 0 ° C. 10.32 g (50 mmol) of dicyclohexylcarbodiimide and 7.66 g (50 mmol) of N-hydroxybenzotriazole are added with stirring. The mixture is stirred for 10 minutes at 0 ° C and the precipitated dicyclohexylurea is filtered off.

12.74 g (50 mmol) of H-Trp-OMeHCl are dissolved in 70 ml of dimethylformamide and the solution is cooled to 0 ° C. Triethylamine (6.94 mL, 50 mmol) was added, and after stirring for 5 min, the precipitated triethylamine hydrochloride was filtered off.

The two solutions were combined and stirred at 0 ° C overnight. The next day, the precipitated DCU was filtered off and the solution was evaporated to dryness. The resulting oil was dissolved in ethyl acetate (500 mL) and extracted with ice-cold 1M potassium bisulfate (3 x 100 mL), brine (5 x 100 mL), and 10% sodium chloride (2 x 100 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered and evaporated to dryness. The resulting oily material was pulverized with petroleum ether, filtered and dried. The crystalline material thus obtained can be recrystallized from ethyl acetate with petroleum ether.

Yield: 27.70 g (89%)

119-122 'C [α] g = + 14.1' (c = 1, DMF)

Rf = 0.62; Rf = 0.41

b) H ~ His (DNP) ~ Trp ~ OMe-2HCl

M, 522.49 (free base); 595.41 (-2HCl)

BOC-His (DNP) -Trp-OMe dipeptide (24.9 g, 40 mmol) was dissolved in methanol (100 mL) and 4N HCl in methanol (100 mL) was added. The mixture was allowed to stand at room temperature for 30 minutes while the dipeptide ester hydrochloride crystallized. It was filtered off, washed with ether and dried.

Yield: 21.91 g (92%)

M.p. 198-202 'C [u] ² D ² = 0.49' (c = 1, DMF)

Rf = 0.46; Rf = 0.18

c) Cdp-His- (DNP) -Trp-OMe M: 633.60

4.85 g (36.8 mmol) of L-pyroglutamic acid. 7.58 g of dicyclohexylcarbodiimide and 5.63 g of N-hydroxybenzotriazole are dissolved in 100 ml of dimethylformamide. The mixture was stirred for 10 minutes at 5-10 ° C and the precipitated DCU was filtered off.

H-His (DNP) -TrpOMe-2HCl (20.84 g, 35 mmol) was dissolved in DMF (100 mL) and triethylamine (9.72 mL, 70 mmol) was added. After stirring for 5 minutes, the precipitated triethylamine hydrochloride is filtered off. The crude solution was combined and stirred overnight at room temperature.

190 207

The next day, acetone (90 mL) was added and the precipitated insoluble material was filtered off. Concentration of the solution gave an oily substance; this was triturated with ethyl acetate, filtered and dried. The dry crystalline material was washed three times with 25 ml of water and dried again. The material can be recrystallized from hot ethyl acetate and then cooled.

Yield: 18.42 g (83.1%)

Mp 148-151 'C [<= +5,2' (c = 1, DMF)

Rf = 0.53; RJ = 0.38

d) Glp-His-Trp-OMe M: 466.50

15.84 g (25 mmol) of Glp-His (DNP) -Trp-OMe protected tripeptide are dissolved in a mixture of 100 ml of dimethylformamide and 40 ml of water. 4 ml of mercaptoethanol are added and the solution is adjusted to pH 8 with triethylamine. Allow to stand for 30 minutes at room temperature and then evaporate to dryness in vacuo. The oily material was triturated with ether, filtered and dried. The resulting material was dissolved in a little methanol and recrystallized by addition of ether. The precipitated crystals are filtered off and dried.

Yield: 10.92 g (93.6%)

Mp 228-232 'C [ajjj = +4.04' (c = 0.42, DMF)

RJ = 0.30

e) Glp-His-Trp N ₂ H _J M: 466.51

Glp-His-Trp-OMe tripeptide (9.33 g, 20 mmol) was dissolved in methanol (250 mL). 20 ml of 98% hydrazine hydrate are added. The reaction mixture was stirred at 40 ° C for 3 hours and then at room temperature overnight. The next day, the precipitate was filtered off, washed with cold methanol and dried.

Yield: 7.58 g (81.2%)

M.p. 166-169 'C [?] JJ = - 22.3' (c = 0.5, DMF)

RJ = 0.50; R t = 0.14

f) Glp-His-Trp-Ser-Tyr-OMe M: 716.8

Glp-His-Trp-N ₂ H ₃ (product of Step 5 (e) of Example 5) (7.0 g, 15 mmol) was dissolved in DMF (60 mL). The solution was cooled to 0 ° C and 7.5 ml (45 mmol) of 6N HCl was added with stirring. A concentrated aqueous solution of sodium nitrite (1.035 g, 15 mmol) was slowly added dropwise to the mixture and the mixture was stirred at 0 ° C for an additional 15 minutes. After stirring, a solution of 4.78 g (15 mmol) of H-Ser-TyrOMe HCl in 15 mL of DMF was added. Triethylamine (6.25 mL, 45 mmol) was adjusted to neutral pH. The mixture was stirred overnight at 0-4 ° C, evaporated to dryness in vacuo the next day, and the oil was triturated with ether.

The resulting powdered material weighed 12.1 g (100%); it contains two small amounts of impurities, but can be converted, without purification, to a hydrazide which crystallizes well.

Physical constants for control material recrystallized from methanol:

Mp 188-190 'C [finger; = -3.48 ° (c = 1, DMF)

RJ = 0.58; R f = 0.26

g) Glp-His-Trp-Ser-Tyr-N ₂ H ₃ M: 716.8 g of crude powdered Glp-His-Trp-Ser-Tyr-OMe pentapeptide ester are dissolved in 200 ml of methanol, 10 ml of 98% hydrazine hydrate and the mixture was stirred for 3 hours at 40 ° C. After stirring overnight at room temperature, the precipitated crystals were filtered off and dried in a desiccator over concentrated sulfuric acid. The dried crystalline material was dissolved in 250 ml of 0.5N hydrochloric acid solution, then neutralized to pH 8 with saturated sodium carbonate solution and allowed to stand at 0 ° C for 2 hours. The precipitated crystals are filtered off, washed with ice-cold water and dried.

Yield: 6.12 g (56.9% based on the two steps)

205-206 'C [α] J J = 21.51' (c = 1, DMF)

Rj = 0.39; R t = 0.14

Hydrazine nitrogen:

found: 3.79%, 3.76%, theoretical 3.91%

h) Z-Gln-Pro-NHEt M: 405.4

3.242 g of proline ethyl amide (22.8 mmol of Z-ProNHEt by hydrogenation) are dissolved in 70 ml of tetrahydrofuran. To the solution was added 5.60 g (20 mmol) of Z-Gln-OH and 1.034 g of N-hydroxybenzotriazole. The reaction mixture was cooled in an ice water bath and a solution of dicyclohexylcarbodiimide (5.34 g, 25.9 mmol) in tetrahydrofuran (50 mL) was added with stirring. The reaction mixture was stirred under ice-cooling for 5 hours and then for additional 20 hours at room temperature, then filtered and washed with tetrahydrofuran. The filtrate was concentrated in vacuo and the residue dissolved in chloroform. The chloroform solution was extracted three times with 35 ml of a 1: 5 mixture of ammonium hydroxide-water, twice with 20 ml of water, three times with 35 ml of hydrochloric acid solution and twice with 20 ml of water. The chloroform portion was dried over anhydrous sodium sulfate, filtered, washed with chloroform, and the chloroform solution was evaporated. The residual oil was dissolved in THF (35 mL) and ethyl acetate (5 mL) and filtered. After concentration of the solution, 100 ml of ether are added to the oil obtained. The solid was washed with ether on the filter and dried in vacuo; weight 7.68 g (95%).

Rj: 0.45; Rf: 0.73

i) Z-Leu-Gln-Pro-NHEt M: 518.57

4.5 g (11 mmol) of Z-Gln-Pro-NHE-protected dipeptide ethylamide are dissolved in 30 ml of glacial acetic acid. To the solution was added 55 ml of 4N glacial acetic acid HBr. The reaction mixture was allowed to stand at room temperature for 1.5 hours. To the solution was added 250 ml of absolute ether and the mixture was allowed to stand for 1 hour. The supernatant is decanted from the precipitated material. To the residue was added ether (100 mL), after standing for 15 minutes, filtered and rinsed thoroughly with ether. The hygroscopic solid was dried under vacuum in the presence of phosphorus pentoxide and sodium hydroxide. The material weighed 4.9 g (hygroscopic).

3.8 g (11 mmol) of the dipeptide-ethylamide hydrobromide thus obtained are suspended in 150 ml of chloroform. To the slurry was added 7.0 mL of triethylamine. 6.4 g of the reaction mixture were then added. 190 207

After evaporation, the oil was dissolved in 5 ml of 0.2 N acetic acid and applied to a Sephadex G25 column (2 x 95 cm).

Chromatograph with 0.2 N acetic acid. The appropriate fractions were collected and lyophilized.

Yield: 298 mg (62%)

Rf: 0.74; R _f ⁵ : 0.65; Rf: 0.37; Rf: 0.80

Amino acid analysis: Ser 0.89; Pro 0.97; Leu 1.00; Phe 0.99; Glu 1.98; Gly 1.01; Tyr 1.03; Trp 0.91.

A solution of Z-Leu-pentachlorophenyl ester in 65 ml of chloroform was added and stirred overnight. The next day, the reaction mixture was extracted with 1N hydrochloric acid solution, saturated sodium chloride solution, then 2N ammonium hydroxide solution, and then again with saturated sodium chloride solution. The chloroform solution was dried over anhydrous sodium sulfate and evaporated in vacuo. The residue was triturated with ether to give a solid; the material was filtered off, washed with ether and dried in vacuo. The crude product weighed 4.6 g (81%).

i

j) H-Leu-Gln-Pro-NHEtHBr M: 465.5

The Z-LeuGln-Pro-NHEt tripeptide obtained as described above is deprotected without further purification. Dissolve in 10 ml of glacial acetic acid hydrobromic acid and stir for 1 hour at room temperature. It is pulverized with 100 ml of anhydrous ether, filtered and the light yellow powder is dried in an desiccator. A pale yellow hygroscopic material weighing 3.54 g (76%) is obtained. Rf: 0.42;

k) Boc-D-Phe-Leu-Gln-Pro-NHEt M: 631.74 '

2.33 g (5 mmol) of the H-Leu-Gln-Pro-NHEt.:HBr tripeptide salt are dissolved in 5 ml of dimethylformamide and cooled to 0 ° C and 0.70 ml (5 mmol) of triethylamine and

A solution of Boc-D-Phe-pentachlorophenyl ester (2.57 g, 5 mmol) in dimethylformamide (10 mL) was added. The pH of the solution was then adjusted to 7-8 with triethylamine and the solution was stirred at room temperature for 48 hours while maintaining the pH neutral with triethylamine. The solution was concentrated in vacuo and the residue was dissolved in chloroform (100 mL) and washed with 0.1M ammonia solution (10 mL). The chloroform solution was dried over sodium sulfate, filtered and evaporated. The residual oil can be pulverized with ether. The weight of the filtered and dried crude product was 2.81 g (89%); the protecting group is removed without further purification.

l) H-D-Phe-Leu-Gln-Pro-NHEt-TEA M: 645.65

1.26 g (2 mmol) of Boc-D-Phe-Leu-Gln-Pro-NHEt tetrapeptide are dissolved in 10 ml of trifluoroacetic acid and stirred at room temperature for 20 minutes. The TFA was rapidly removed in vacuo and the residue was triturated with ether and filtered. The dried material weighed 1.19 g (92%).

Rf: 0.72; Rf: 0.9; Rf: 0.65

m) Glp-His-Trp-Ser-Tyr-DPhe-Leu-GlIn-Pro-NHC ₂ H _s M: 1198

286 g (0.4 mmol) of Glp-His-Trp-Ser-Tyr-N ₂ H ₃ pentapeptide hydrazide prepared in Example 5 (g) are dissolved in 10 mL DMF at -10 ° C, cooled and stirred 0.27 ml of 6N hydrochloric acid and then 30.2 mg of a concentrated aqueous solution of sodium nitrite are added dropwise. After 5 minutes, a solution of HD-Phe-Leu-Gln-Gln-Pro-NHEtTFA tetrapeptide (258 mg, 0.4 mmol) in DMF (1 mL) in the presence of TEA (0.27 mL) was added. If necessary, the reaction mixture is neutralized with TEA, stirred for 1 hour at -5 ° C, for another 1 hour at 0 ° C, and then allowed to warm to room temperature. It is then concentrated in vacuo.

Example 6 Preparation of [Phe ⁷ , Leu ⁸ ] gonadoliberin

a) Synthesis

Starting from 0.62 g (0.5 mmol) of benzhydrylamine HCl resin [0.8 meq / g], Glp-His (Tos) -TrpSer (OBzl) -Tyr is synthesized as described in Example 1 (a). (OBzl) -Gly-Phe-Leu-Pro-Gly-NH ₂ decapeptide amide. The resulting peptide resin weighed 1.31 g. AW: 0.69 g (92%) (M protected peptide · 1506)

b) HF cleavage

The procedure is the same as described in Example 1 (b). The 50% acetic acid filtrate was evaporated and then lyophilized to give a dry residue of 510 mg (87%).

(c) Gel chromatography

Gel chromatography was performed on a Sephadex G25 column using the conditions described under le). The fraction collected and lyophilized weighed 363 mg (62%).

d) SiO ₂ chromatography

Silica gel chromatography was performed on a Kieselgel 60 resin column (2 x 100 cm). Equilibration and elution were performed with a mixture of n-butanol-acetic acid-water-ethyl acetate = 1: 1: 1: 1. The content of each fraction was assayed by UV absorbance at 280 nm and TLC in solvent mixture 5 followed by amino acid analysis. The weight of pure [Phe ⁷ , Leu ⁸ ] gonado-liberin was 299 mg (51%).

Rf: 0.58; Rf: 0.39; Rf: 0.62; Rf: 0.73

Amino acid analysis: Ser 0.91; Glu 0.97; Pro 1.07: Gly 2.12; Leu 1.00; Tyr 1.03; Phe 0.94; His 1.05.

Mp: 175-178 ° C.

Example 7 Preparation of [Phe ⁷ , Gln ⁸ ] Gonadoliberin Μ: 1 IS7 'a) Synthesis

1.25 g (1 mmol) of benzhydrylamine HCl resin [0.8 meq / g] in dichloromethane were swollen for two hours and then synthesized in the manner described in Example 1 (a) for Glp-His (Tos) - Trp-Sci (OBzl) -Tyr (OBzl) -Gly-Phe-Gln-Pro-Gly-NH, decapeptamide. The resulting peptide resin weighs 2.9 AW

1.27 g (83%) (Mvcdctt peptide: 1521)

190 207

b) HF cleavage

The procedure is the same as described in Example 1 (b). The weight obtained after lyophilization was 980 mg (82%).

(c) Gel chromatography

Further purification of the crude product was carried out on a Sephadex G25 column using the conditions described under le). Yield: 576 mg (49%).

d) Preparative HPLC

The procedure was carried out in the same manner as in Example 1 (d) except that the equilibrium was adjusted with a mixture of 10% methanol and 90% 20% acetic acid. The material purified by gel chromatography was eluted from the column with a linear gradient of 10% methanol to 90% 20% acetic acid and 25% methanol to 75% 20% acetic acid, followed by 25% methanol to 75% 20% acetic acid and % methanol - 60% 20% acetic acid linear gradient. The volume of the solutions in each case is 150-150 ml. Other conditions for fractionation are the same as described under ld). The weight of the lyophilized final product was 448 mg (38%).

RJ: 0.12; Rj: 0.7; Rj: 0.24; Rj: 0.87

Amino acid analysis: Ser 0.87; Glu 2.05; Pro 1.03; Gly 2.13; Tyr 0.92; Phe 1.00; His 0.95; Trp 0.81.

182 ° C [α] D = -21.0 '(c = 1, 0.2 n acetic acid)

The hydrochloride salt of the product obtained is prepared as follows:

mg (0.05 mmol) of [Phe ⁷ , Gln ⁸ ] -gonadoliberin was dissolved in 2 ml of 0.5 N hydrochloric acid and lyophilized. The dry residue was triturated with ether and filtered. The weight of [Phe ⁷ , Gln ⁸ ] gonadoliberin HCl thus obtained was 62 mg.

M.p. 233 'C (dec.).

Example 8 Preparation of [Trp ⁷ , Gln ⁸ , desGly ¹⁰ ] -Gonadoliberin-ethylamide M: 1220

a) Boc-Gln-Pro-NH-C ₂ H, M: 370

Dissolve 1.0 g (7 mmol) of proline ethylamide in 15 mL of dimethylformamide, adjust the pH to 8 with triethylamine, and then add 3.2 g (8.4 mmol) of Boc-Gln-ONP in 15 mL of dimethylformamide. formamide solution. The reaction mixture was allowed to stand at room temperature with stirring for 48 hours while the pH was controlled and adjusted to 8 if necessary. The reaction mixture was evaporated to dryness, the oily residue was dissolved in water and washed with ether. The aqueous phase was concentrated in vacuo and dried. Yield: 1.93 g (74.5%) Rf: 0.76.

b) H-Gln-Pro-NH-Cfif TFA M: 367 (TFA salt)

1.9 g (7.2 mmol) of Boc-Gln-Pro-NH-C _{? And} H were dissolved in 10 ml of trifluoroacetic acid and maintained at room temperature for 20 minutes with stirring. The solution was then concentrated in vacuo, the residue triturated with ether, filtered and dried in vacuo. This gave 1.52 g (58%) of hygroscopic material.

Rj: 0.45; kRj: 0.43.

Nd ⁼ -34.8 '(c = 1, methanol)

c) Boc-Trp-Gln-Pro-NH-C ₂ H ₅ M: 557

500 mg (1.85 mmol) of k-Hn-Gln-Pro-NHC ₂ H ₅ -TFA salt are dissolved in 10 ml of DmF and the pH is adjusted to 8 with triethylamine. A solution of Boc-Trp (608 mg, 2 mmol) in DMF (10 mL) and Ν, Ν'-diisopropylcarbodiimide (620 µl, 4 mmol) was then added. The reaction mixture was stirred at room temperature for 48 hours and then concentrated in vacuo. The residue was dissolved in ethyl acetate and diluted with ether. The precipitated Boc-TrpGln-Pro-NH-C ₂ H ₅ was filtered off, washed with ether and dried. Yield: 740 mg (72%).

Rf: 0.75; Rf: 0.38.

d) H-Trp-Gln-Pro-NH-C ₂ H / TFA M: 554 (TFA salt)

700 mg (1.25 mmol) of Boc-Trp-Gln-Pro-NHC ₂ H ₅ was dissolved in 15 ml of trifluoroacetic acid and methylene chloride 1: 1 mixture of. The solution was allowed to stand at room temperature for 20 minutes with stirring, then concentrated in vacuo and the residue was triturated with ether. The solid was filtered and dried. The crude product (500 mg) was purified by silica gel chromatography with ethyl acetate-pyridine-acetic acid-water (47: 20: 6: 11).

Under the same conditions as in Example 2 (d). The appropriate fractions were collected and lyophilized. 360 mg (52%) of the peptide are obtained.

Rj: 0.46; Rf: 0.18.

119-123 'C.

[α] D = -4.8 ° (c = 0.1, 0.1 n HCl)

e) Boc-Gly-Trp-Ghi-Pro-HN-C ₂ H _S M: 613.7

H-Trp-Gln-Pro-NH-C ₂ H ₅ (350 mg, 0.76 mmol) was dissolved in DMF and reacted with BocGly (104 mg, 0.8 mmol) as described in (c). This gave 375 mg (81%) of crystalline material.

R _f ³ : 0.88; Rf: 0.82; Rj: 0.61.

f) H-Gly-Trp-Gln-Pro-NH-Cl / TFA M: 609.6 (TFA salt)

Above (375 mg, 0.6 mmol), Boc-Gly-Trp-Gln-Pro-NHC ₂ H ₅ are summarized to the protecting group) is removed by the described conditions. This gives 285 mg (76%) of a white powder.

Rf: 0.08; Rj: 0.16; Rj: 0.67; Rf: 0.28.

g) Glp-His-Trp-Ser-Tvr-Gly-Trp-Gln-Pro-NHCfL M: 1216.4

286 mg (0.4 mmol) of Glp-His-Trp-Ser-Tyr-N ₂ H ₃ pentapeptide hydrazide are reacted with 243.8 mg (0.4 mmol) of H-Gly-Trp-Gln-Pro-NH-C _{2 H5} azide as described in Example 5, part m) manner. The fraction collected and lyophilized after Sephadex chromatography weighed 252.9 mg (52%).

Rj: 0.26; Rf: 0.32.

Amino acid analysis: Ser 0.87; Glu 2.02; Pro 0.91; Gly 1.00; Tyr 1.12; His 1.05; Trp 1.80.

Mp: 179-180'C.

-8I

190 207

9-12. example

Claims (5)

Claims Using the procedure described in Example 1, the following compounds were prepared: The serial number of the example The name of the compound rr Production 9th 10. II 12. [D-Trp ⁶ , Phe ⁷ , Leu ⁸ ] -Gonadoliber 0.69 [D-Ala ⁶ , Gln ⁸ ] -Gonadoliber 0.55 [D-Leu ⁶ , Gln ⁸ ] -Gonadoliber 0.57 [O-tertiary- butyl-D-Ser ⁶ , G [n ^e ] -gonadoliberin ' 32% 43% 47% 37% 9-12. The amino acid analysis of the compounds of Examples 1 to 4 is given in the following table: The number of the example is ___ ι Ser Glu Pro Gly Alá Leu Tyr Phe His Trp 9. 0.87 1.01 1.12 1.04 - 1.00 0.92 0.96 1.02 1.87 10. 0.91 1.90 0.95 1.10 1.07 1.00 1.02 - 0.95 0.82 11. 0.97 2.01 1.07 1.00 - 2.12 1.01 - 0.93 0.91 i 12. 1.87 1.98 1.05 1.07 - 1.00 0.96 - 1.03 0.83 Example 13 Preparation of injection for intramuscular, subcutaneous or intravenous administration a) (I) gonadoliberin derivative is dissolved in a concentration of 1-10 mg / ml of distilled water, physiological saline or buffered aqueous bromide solution ₃₅ toluene layer was separated. The solution is sterile filtered, and the volumes containing 50 to 500 pg are filled into ampoules, lyophilized, and finally sealed. The contents of the ampoules were dissolved material before treatment Fresh 1-10 _4Q ml of distilled water was added to and injected according to the required dose volume. I b) of formula (I) gonadoliberin derivative of general j corresponding derivative saline solution containing from 20 to 500 pg / ml, is filled into ⁴⁵ or dead benzilalko- vials and the vials sealed. The solutions thus obtained are suitable for direct injection. A process for preparing Glp-His-Trp-Ser-Tyr-X, -X ₂ -X ₃ -Pro-X _{4 of} formula (I) - in which formula X 1 is glycyl, D-alanyl, D-leucyl, O-tert-butyl D-seryl, D-tryptophyl or D-phenylalanyl, X ₂ is leucyl, tryptophyl or phenylalanyl; X ₃ is leucyl or glutaminyl, X ₄ is a glycylamide or ethyl amide, with the proviso that when X ₂ is D-alanyl, X ₂ is tryptophyl, X ₃ is leucyl, then X ₄ may be other than ethyl amide only nonapeptide ethyl amide or decapeptide amide and salts thereof, characterized in that a) employing solid phase peptide synthesis, stepwise condensation of the optionally protected amino acids on a solid support in an appropriate order and removing the final product by acidic or basic cleavage and optionally removing the protecting groups either before or after cleavage from the resin. , obsession b) coupling, if desired, protected amino acids using stepwise and / or fragment condensation and optionally deprotecting the resulting product. Process (a) according to claim 1, characterized in that the solid support is a benzhydrylamine resin or a chloromethylated polystyrene resin. 3: Method a) according to claim 1, characterized in that the end-group containing the protecting groups ^. they are removed from the solid support by treatment with hydrogen fluoride and / or ethylammonolysis. Process b) according to claim 1, characterized in that it has a formula (II) Glp-His-Trp-Ser-Tyr-N ₃ (II) Protected Pentapeptide Azide of Formula III X ₁ -X ₂ -X ₃ -Pro-X ₄ (III) are fused to a tetra- or pentapeptide, wherein X _{1 (} X ₂ , X ₃ and X ₄ are as defined in claim 1). A process for the preparation of fish breeding compositions, wherein the compound of formula (I), wherein X ₂ , X ₂ , X ₃ and X ₄ are as defined in claim 1, or an acid thereof is prepared according to claim 1. . its addition salt is mixed with conventional carriers and / or carriers to form a fish breeding composition.