CS232977B1 - Method of gonadoreline making - Google Patents

Abstract

The invention relates to the production of gonadorelin a formula that is also referred to as the pro factor luteinizing and follicle release stimulating hormone or LH / ρτ, etc. The new synthesis tactic is based on a new one reaction scheme 3 + (2 + 5), which was chosen for distinct distinctive character tripeptide 1-3 from the rest of the gonadoree molecule · linu.

Description

The present invention relates to a process for the production of gonadorelin, also referred to as a luteinizing and follicle-stimulating hormone release factor or LH / RH and the like. (Biochem, Biophys. Res. Commun. Li. 393 and 1334, 1971) and proposed the structure of the decapeptidamide of formula I

23456789 10 pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NHg (I), which was confirmed by comparing the biological effects of the synthetic substance to those obtained from a natural material.

Since Schally's discovery, there has been a growing interest in gonadorelin in connection with the expanding possibilities of its practical use in human and veterinary medicine (infertility treatment and diagnostics), but especially in animal production during the synchronization of rut of cattle, pigs and sheep, poultry, etc.

For the preparation of this relatively complicated compound, the most effective syntheses have been sought, as evidenced by many publications in both scientific and patent literature. In particular, two basic synthetic approaches were studied, namely classical classical solution synthesis and various modifications of solid phase synthesis (R. Geiger et al., Biophys. Res. Commun. & (3), 767 (1971) and N. Yanaihara). et al., J. Med Chem J6 (4), 373 (1973);

T. Fuji et al., Bull. Chem. Soc. Jpn. 49, 1595 (1976).

Individual procedures differ mainly by the so-called synthesis strategy or tactics. Some use sequential construction of the peptidic chain, others link peptide fragments according to different schemes, for example 2 + 8, 3 + 7 or 6 + 4 and the like.

The gonadorelin molecule contains six tri-functional amino acids, providing many possibilities for the nature and number of suitable protecting groups in the preparation of the starting amino acid derivatives and the necessary peptide fragments (intermediates). Most of the published procedures are characterized by efforts to minimize the use of protecting groups and thereby simplify synthesis.

A large number of authors prefer solid phase syntheses (H. Sievertson et al., J. Med. Chem. 22 (3). 222 (1972); WH Arnold et al., J. Med. Chem. 16 (9), 1054 (1973)). Some works directly compare the yields and overall efficiency of classical and solid phase synthesis (G. Flouret et al., J. Med. Chem. 16 (4), 369 (1973)). Combination (L. Bilk et al., Mh. Vet.-Med. 34 606 (1979)), for example by splicing the fragments of Scheme 3 + 7, wherein the heptapeptide is obtained by solid phase synthesis. In summary, the literature suggests that the advantages of solid phase synthesis are high time savings and relatively high yields. In contrast, although classical syntheses provide somewhat lower yields, the purity of the final product tends to be higher, so that when processed to a quality usable for pharmaceutical purposes, losses are already relatively lower.

In the search for a new industrial-scale process for the production of gonadorelin, it was necessary to respect the aspect of maximum efficiency. In this respect, all the syntheses so far published have been critically analyzed. On the basis of the selection of the most appropriate knowledge, supplemented by new original processes, a new strategy for the synthesis of gonadorelin has been developed which is the object of the present invention. It is penetratingly more efficient than other previously known syntheses: it is very fast, with low demands on labor and material costs, provides a very pure, homogeneous product that can be easily cleaned to the quality required for pharmaceutical use in a single operation such as preparative HPLC ( chromatographically pure substance). The synthesis can be carried out in conventional equipment, in large batches.

The invention therefore relates to a process for the production of gonadorelin of the above formula I, characterized in that the tripeptide 1-3 in the form of a hydrazide of the formula II

3? -Glu-His-Trp-N ₂ H ₃ (II), reacted with heptapeptidamide 4-10 of formula III

5 6 7 8 9

Ser-Tyr-Gly-Leu-Arg (Tos) -Pro-Gly-NH ₂ (III), prepared by condensation of the pentapeptidamide 6-10 formula IV

7 6 9 9

Gly-Leu-Arg (Tos) -Pro-Gly-NH ₂ (IV), with hydrazide protected dipeptide 4-5 of formula V

Z-Ser-Tyr-KgH 4 wherein Z is a protecting group, for example benzyloxycarbonyl, after which the tosyl protecting group is removed from the obtained protected decapeptidamide.

The synthesis tactics for the production of the gonorelin according to the invention are based on the 3 + (2 + 5) 'reaction scheme, which was chosen mainly because the nature of the tripeptide 1-3 of formula II is clearly different from the rest of the molecule. For its preparation, we managed to work out an originally modified process that provides high yields.

In an effort to do away with minimal protection of the functional groups of delicate amino acids, ie, lilac and tyrosine, it has been attempted to incorporate these acids into the synthetic scheme as late as possible and react with them in as few reaction steps as possible. Thus, the well-characterized dipeptide hydrazine Z-Ser-Tyr-NgH 4 was used, which is condensed in an azide fashion with pentapeptidester or the corresponding amide 6-10. Under these conditions, only minimal functional group protection is required, just tosyl blocking the guanido group of arginine.

AND

Pentapeptidester 6-10 is prepared by known methods by sequential construction from the carboxyl terminus, using simple amino acid derivatives that are readily available.

For azide condensation of pGlu-His-Trp-NgH 2 with HBr / Ser-Tyr-Gly-Leu-Arg (Tos) -Pro-Gly-IIH ₂ . A modified process (under anhydrous conditions) has been developed which provides the corresponding protected decapeptide in high purity and with very good yields.

The tripeptide hydrazide pGlu-His-Trp-NgH 4 is prepared by a novel process, wherein pyroglutamylhistidine methyl ester is obtained by condensation of the pyroglutamic acid dicyclohexylammonium salt with histidine methyl ester dihydrochloride with dicyclohexylcarbodiimide in dimethylformamide. The histidine methyl ester is liberated in situ by the addition of dicyclohexylamine and the reaction is continued for 48 hours with the progressive addition of the condensing agent in excess (1.5 equivalents). The dipeptidester is isolated in a yield of over 90% of theory. The corresponding pyroglutamylhistidine hydrazide is obtained by hydrazinolysis of 80 to 90% hydrazine hydrate in the usual manner in methanol.

232977 4

The preparation of pyroglutamyl-hystidyl-tryptophan methyl ester makes it difficult to dissolve in water and the presence of salts in the reaction of the mixture. The newly accomplished azide condensation under anhydrous conditions with 1.1 equivalents of butyl nitrite in dimethylformamide in the presence of anisole eliminates the difficulties, using dicyclohexylamine to adjust the pH. The yield is in this case more than 90% of theory.

Pyroglutamyl-histidyl-tryptophan hydrazide is obtained by hydrazinolysis of the previous tripeptidester in a conventional manner in methanol or dimethylformamide. The advantage of this modification is the utilization of low solubility of dicyclohexylamine hydrochloride or hydrobromide in dimethylformamide and the resulting easy isolation of the products and their desalting by simple filtration of the reaction mixture. The presence of anisole suppresses the formation of by-products.

The preparation of pentapeptidester 5-10 uses conventional procedures. A condensed anhydride-pivaloyl chloride method in the presence of pyridine was used for the condensation to give high yields of homogeneous and very pure products. The benzyloxycarbonyl group was cleaved off with a solution of hydrogen bromide in glacial acetic acid. The individual steps were performed directly with the appropriate hydrobromides and either @ 3 H-ethylpiperidine, triethylamine or dicyclohexylamine was used to release the peptide in situ (depending on product properties).

The tosyl protecting group can be cleaved from the partially protected decapeptidamide gonadorelin by a variety of known methods. Of these, cleavage by liquid hydrogen fluoride has been found to be the most advantageous and relatively gentle, particularly in the presence of preservatives, for example anisole, thioanisole, thioglycolic acid or mixtures thereof, guaranteeing particularly gentle cleavage without risk of peptide bonds or other side reactions. Free gonadorelin is obtained in high yield and satisfactory purity. Better results were obtained by the original cleavage procedure, which consists in the use of trifluoromethanesulfonic acid in the presence of the aforementioned preservatives (anisole, etc.) and which gives the same product yield as the hydrofluoric acid cleavage, but moreover has a high purity of free gonadorelin. This can be purified by a newly developed procedure in one operation by HPLC to a chromatographically uniform pure substance, containing almost 100% by weight, usable for pharmaceutical purposes.

The process according to the invention is illustrated in more detail by the following non-limiting example.

Pyroglutamyl-histidyl-tryptophan methyl ester pGlu-His-N ₂ H ₃ (4.2 g, mp 242-244 ° C) was dissolved in a mixture of dimethylformamide (170 mL) and 8 N hydrochloric acid in dioxane (5.2 mL). N-Butyl nitrite (1.86 mL) was added dropwise to the solution cooled to -30 ° C. After stirring for 30 minutes with cooling to -15 to -20 ° C, the pH is adjusted to 5 to 5 with dicyclohexylamine and a solution of HCl.Trp-Otoe (3.99 g, mp 218-223 ° C) and anisole are added to the reaction mixture. (1.7 mL) in dimethylformamide. After adjusting the pH to 8-9 with dicyclohexylamine, stirring was continued at 0 ° C overnight. After filtration, the filtrates were evaporated and the residue was crystallized from a mixture of methanol (30 ml), ethyl acetate (300 ml) and ether (100 ml). 5.94 g (85% of theory) of chromatographically pure pGlu-His-Trp-OMe are obtained, mp 225-227 ° C.

Pyroglutamyl - histidyl - tryptophan hydrazole

A solution of pGlu-His-Trp-Otae (4.0 g) in dimethylformamide (50 mL) with the addition of 80% hydrazine hydrate (5 mL) was allowed to stand for 4 h at room temperature and overnight in a refrigerator (0 ° C).

After evaporation of the solvents, methanol (30 ml) was added and the product precipitated by standing in the freezer. Filtration and drying afforded 3.8 g (95% of theory) of pGlu-His-Trp-N4H4 with m.p.

Mp 157-158 ° C; [α] D = -20.7 ^° (c = 0.2, dimethylformamide).

Benzyloxycarbonylseryl - tyrosine methyl ester

To a solution of benzyloxycarbonyl-sulfur (22.2 g, mp 109-111 ° C) in a mixture of dimethylformamide (50 mL) and pyridine (8.2 mL) cooled to -50 ° C was added N-ethylpiperidine (12.7 mL) dropwise. ) and pivaloyl chloride (11.32 mL). After stirring for 10 min, a solution of tyrosine methyl ester hydrochloride (21.4 g, mp 189-194 ° C) in dimethylformamide (50 mL) and N-ethylpiperidine (12.7 mL) was added, the pH was adjusted to 7.5 and the reaction mixture was added. Allow to stand at 0 ° C for 20 h. The solvents were distilled off, the residue was dissolved in methylene chloride (300 ml), shaken with a solution of 1 N hydrochloric acid and 5% sodium bicarbonate solution. Azeotropic distillation with benzene gave 38.6 g (100%) of Z-Ser-Tyr-OMe as a chromatographically pure oil.

Benzyloxycarbonylseryl - tyrosine hydrazide

To a solution of Z-Ser-Tyr-OMe (38.6 g) in methanol (460 mL) was added 80% hydrazine hydrate (40 mL) at room temperature. After 1 h an additional portion of methanol (300 mL) was added and after an additional 3 h the reaction mixture was stored at 0 ° C for 20 h. Filtration and drying afforded 36.67 g (95% of theory) of chromatographically pure Z-Ser-Tyr-NgH4, m.p. Mp 220-225 ° C.

^« Q

Benzyloxycarbonylseryl - tyrosyl - glycyl - leucyl - N - tosyl - arginyl - prolyl - glycine amide

To a solution of chromatographically pure Z-Gly-Leu-Arg (Tos) -Pro-Gly-NH ₂ (17 g) in acetic acid (50 mL) was added a solution of 35% hydrogen bromide in acetic acid (50 mL). The resulting bromobromo-pentapeptidamide was purged with 1 liter of ether after 1 h, filtered and dried. Then, n-butylnitrite (3 mL) was added dropwise to a solution of Z-Ser-Tyr-NgH4 (9 g) in a mixture of dimethylformamide (70 mL) and 7 N hydrogen chloride in dioxane (22.3 mL), cooled to -50 ° C. 66 ml). After stirring at -10 to -30 ° C for 15 min, the reaction mixture was cooled to -50 ° C and a solution of pentapeptidamide hydrobromide in dimethylformamide (70 mL) was added. The pH of the reaction mixture was immediately adjusted to 7.0-7.5 with N-ethylpiperidine and stored at 0 ° C for 48 h. After evaporation of the solvents, the residue was dissolved in methylene chloride (250 ml), shaken with 1 N hydrochloric acid solution and water. Azeotropic distillation with benzene and crystallization of the residue from methanol / ether gave 20.62 g (92% of theory) of Z-Ser-Tyr-Gly-Leu-Arg (Tos) -Pro-Gly-NH ₂ with mp 157 ° C.

Amino Acid Composition: Ser 0.91; Tyr 1.03; Gly 1.93; Arg 1.06; Orn 0.009; For 1.04;

Leu 1,08.

The found values of elemental analysis agree with the values calculated.

Q

Pyroglutamyl - histidyl - tryptophyll - seryl - tyrosyl - glycyl - leucyl - N - tosyl - arginyl - prolyl - glycine amide

To a solution of Z-Ser-Tyr-Gly-Leu-Arg (Tos) -Pro-Gly-NH ₂ (2.2 g) in trifluoroacetic acid (30 mL) was added hydrogen bromide gas at 0 ° C through an anisole-containing washer. Precipitation with ether and drying in a desiccator affords the heptapeptidamide hydrobromide from which the hydrobromide ions are removed on an anion exchange resin in an OH-cycle (100 mL). Elution is performed with methanol. Concentration and azeotropic distillation with benzene gave 2.05 g. Then, to a solution of pGlu-His-Trp-1H (0.665 g) in a mixture of dimethylformamide (7 mL), dimethylsulfoxide (6 mL) and 2.4 N hydrogen chloride in tetrahydrofuran (35 mL), cooled to -20 ° C, a solution of n-butyl nitrite (0.173 mL) in dimethylformamide (1 mL) was added dropwise.

After stirring at -20 ° C for 30 min, the pH was adjusted to 8-9 with triethylamine and a solution of the previously prepared heptapeptidamide in dimethylformamide (6 mL) was added. After stirring for an additional 30 min at -30 ° C, the reaction mixture was stored in a refrigerator at 0 ° C for 20 h. After filtration x, the filtrate was evaporated and the residue was precipitated from a solution in dimethylformamide (3 ml) with ethyl acetate (150 ml). Filtration and drying afforded 2.61 g (92%) of chromatographically pure pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg (Tos) -Pro-Gly-NH ₂ , mp 175-180 ° C.

Amino Acid Composition: Ser 0.91; Tyr 0.95; Olu 1.05; For 1.02; Leu 1,00; Arg 0.95;

Gly 1.95; His 1.01; Trp 0.92.

Gonadorelin

To a solution of protected decapeptidamide (10 g) in trifluoroacetic acid (50 mg) and thioglycolic acid (10 mL) cooled to 0 ° C was added trifluoromethanesulfonic acid (50 mL). After stirring at 0 ° C for 60 min, the reaction mixture was poured into 1 L of ether and stored at -20 ° C for 2 h. Filtration and drying in a desiccator yielded 6.76 g (99%) of gonadiol relin, which was purified by preparative liquid chromatography on a treated silica gel column (1500 g) using a mobile phase containing 40% by volume methanol and 60% v / v. , 2% trifluoroacetic acid. Evaporation by lyophilization from a solution in 50% acetic acid gave 7.96 g (90% of theory) of the purified amphoteric structure. The purity of the substance was verified by liquid chromatography (2 systems) and electrophoresis on paper (2 buffers). Amino Acid Composition: Ser 0.93; Tyr 0.98; Glu 1.03; For 1.02; Leu 1,00; Arg 0.97;

Gly 1.95; His 1.01; Trp 0.96. The found values of elemental analysis agree with the values calculated.

Explanation of the whole PV:

HPLC ........ high pressure liquid chromatography

Claims (2)

A process for producing gonadorelin of formula I PGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NHg characterized in that the tripeptide 1-3 in the form of the hydrazide of formula II 1 2 3 pGlu-β-Trp-NgHj reacts with heptapeptidamide 4-10 of formula III 4 5 6-7 8 912 Ser-Tyr-Gly-Leu-Arg (Tos) -Pro-Gly-KH 2 prepared by condensation of pentapeptidamide 6-10 of Formula IV 678 9 10 Gly-Leu-Arg (Tos) -Pro-Gly-NHg with hydrazide protected dipeptide 4-5 of formula V (II), (III), (IV), 4 5 Z-Ser-Tyr-1H-J (V), wherein Z is a protecting group, e.g., benzyloxycarbonyl, after which the tosyl protecting group is removed from the obtained protected decapeptidamide. 2. Process according to claim 1, characterized in that the tosyl group is removed from the protected decapeptidamide by treatment with trifluoromethanesulfonic acid, in the presence of preservatives, in particular anisole, thianisole, thioglycolic acid or mixtures thereof, at a temperature of 0 to -30 ° C.