CS255616B1 - Method of (2-methyl tyrosine)deamino-1-carbaoxytocin production - Google Patents

Abstract

The present invention relates to the production of (2-O-methylthyrosine) deamino-1-carbaoxytocin. First, a heptapeptide of formula I Z-Ile-Gln-Asn-Cys (Bzl) -Pro-Leu-Gly-O- (7) (I) is prepared by solid-phase synthesis. wherein Z is benzyloxycarbonyl which is converted into solution by cleavage from the support by treatment with methanolic ammonia to form protected heptapeptide II Z-Ile-Gln-Asn-Cys (Bzl) -Pro-Leu-Gly-NH2 (II) t from which the protecting group is cleaved with a solution of sodium in liquid ammonia, the sulfhydril group of cysteine is alkylated with ethyl elné-bromobutyrate and the ester group is hydrolysed with sodium hydroxide solution to prepare free heptapeptidamide III H-Ile-Gln-Asn-Cys (C 3 H 6 COOH) -Pro-Leu-Gly-NH 2 (III), to which a tyrosine derivative IV is fused X-Tyr (Me) -OH (IV), wherein X is a tert-butyloxycarbonyl or o-nitrophenylsulfenyl group to form octapeptidamide V X-Tyr (Me) -Ile-Gln-Asn-Cys (C 3 H 6 COOH) -Pro-Leu-Gly-NH 2 (V), wherein X is as defined above, and the protecting group is cleaved and the free octapeptidamide is cyclized by dissolution in a buffered solution of dimethylformamide with diphenylphosphorazide or N, N * -dicyclohexylcarbodiimide and N-hydroxybenzotriazole. The product is used in veterinary medicine.

Description

The present invention relates to a process for the production of (2-O-methyltyrosine) deamino-1-carbaoxytocin, an analogue of the natural hormone oxytocin, which is more metabolically resistant, i. It has a prolonged effect compared to oxytocin, which is a welcome property in clinical application.

This analog, which has the character of cyclic peptides, is known and its preparation has also been described (U.S. Pat. No. 149,028, U.S. Pat. No. 230,542). More recently, its traditional solution synthesis has been replaced by the so-called solid phase synthesis, i.e.. on a suitable medium (Czech author's certificate no. 237 944). Both of these methods do not provide sufficient yields and, moreover, they are inconveniently laborious when converted to a larger scale. Although the solid support method provides many advantages, including shortening of the individual steps, the yields of the last step, i.e. cyclization of octapeptidamide, are low, the product is not of the required purity, and sufficient purification means losses and delays again, thereby undermining the advantages of the preceding steps.

In an effort to overcome these disadvantages, optimum synthesis conditions (2-O-methyltyrosine) of deamino-1-carbaoxytocin were sought, using the proven advantages of the known methods. Although many structurally close derivatives could be cyclized on a solid support with satisfactory yields, this was not the case with (2-O-methyltyrosine) deamino-1-carbaoxytocin, especially isolation of the end product after cyclization proved to be extremely laborious, time consuming difficult and unprofitable, which were the basic obstacles that prevented its transfer to the desired larger, ie operational scale.

Surprisingly, these disadvantages have been eliminated by the efficient use of both known methods and more recently described preferred cyclizing agents, in particular diphenylphosphorazide.

Accordingly, the present invention provides a process for the preparation of (2-O-methyltyrosine) deamino-1-carbaoxytocin, comprising:

Ζ -Ile-Gln-Asn-Cys (Bzl) -Pro-Leu-Gly-O- wherein Z is benzyloxycarbonyl, Bzl is benzyl and cleavage from the support by treatment with methanolic peptides II

Z-Ile-Gln-Agn-Cys (Bzl) -Pro-Leu-Gly-Ni is synthesized on a solid support by hepta (I), which is a carrier which is taken up in ammonia solution to form protected hepta (II), from which the protecting groups are cleaved with a solution of sodium in liquid ammonia, the sulfhydryl group of the cysteine is alkylated with f-butyric acid ethyl ester, and the ester group is hydrolyzed with sodium hydroxide solution to prepare the free heptapeptidamide III

Η -Ile-Gln-Asn-Cys (C ^HgCOOH) -Pro-Leu-Gly-Ni ^ (III) to which the tyrosine derivative IV is condensed

X-Tyr (Me) -OH (IV), wherein X is a t-butyloxycarbonyl or o-nitrophenylsulfenyl group and Me is methyl, to form the octapeptidamide V

X-Tyr (Me) -Ile-Gln-Asn-Cys (C 4 -COOH) -Pro-Leu-Gly-NH ₂ (V), where X and Me are as previously defined, whereupon the protecting group is removed and the free octapeptidamide is removed. cyclized by dissolving in a buffered dimethylformamide solution with the addition of diphenylphosphorazide, or with N, N'-dicyclohexylcarbodiimide and N-hydroxybenztriazole.

In carrying out the process of the present invention, a protected glycine derivative is first fixed on a suitable carrier, typically a chloromethylated styrene-1% divinylbenzene copolymer, and sequentially coupled with the necessary protected amino acid derivatives to form the heptapeptidamide of formula I. The synthesis works in solution according to any of the known methods. At the conclusion of the synthesis, i.e. cyclization of the octapeptidamide of formula V (after deprotection) to the desired (2-O-methyltyrosine) deamino-1-carbaoxytocin, the use of diphenylphosphorazide has proven advantageous.

As is apparent from the foregoing, the process of the invention combines the advantages of both known methods. The decisive moment was to find a suitable stage where to interrupt the synthesis on a solid support and to work out the remaining stages in the solution, to which systematic comparative experiments contributed. The process according to the invention has resulted in an increased production of the desired peptide while reducing material costs, saving raw materials and labor, and the peptide obtained is of excellent quality.

The process according to the invention is described in more detail in the following examples, which are illustrative but not limiting.

Example 1

(a) Benzyloxycarbonyl-isoleucyl-glutaminyl-asparaginyl-S-benzylcysteinyl-propyl-leucyl-glycine amide (substance II)

100 g of the chloromethylated copolymer (styrene-1% divinylbenzene) to which Boc-Gly was bound at 1 mmol / g are successively condensed with a three molar excess of Ν, Ν'-dicyclohexylcarbodiimide, N-hydroxybenztriazole and a suitable amino acid derivative with the following amino acids: Boc-Leu, Boc-Pro, Boc-Cys (Bzl), Boc-Asn, Boc-Gln and Z-Ile, to give Compound I. A solution of trifluoroacetic acid in methylene chloride and methylene chloride is used to cleave the t-butyloxycarbonyl group. in dimethylformamide.

After completion of the synthesis, the resin was separated, dried and suspended in 3 L of absolute methanol. The suspension is cooled to -10 ° C and saturated with ammonia gas. After standing for 24 h, the resin was filtered off and extracted three times with three liters of dimethylformamide at 80 ° C. The extract was concentrated in vacuo to a volume of about 200 ml and the product precipitated from the residue by adding 2 liters of ether. After filtering and drying, 70 g (72% of theory) of compound II are obtained, which is identical to the product prepared by the solution method. This consistency was confirmed by amino acid composition, thin layer chromatography and high performance liquid chromatography.

b) Isoleucyl-glutaminyl-asparaginyl-β-carboxypropionylcysteinyl-propyl-leucyl-glycine amide (III) g of II is dissolved in a solution of 0.5 g of sodium amide in 6 liters of boiling liquid ammonia and reduced with sodium (about 12 g). until a blue color of the solution is obtained which is stable for 20 seconds. The solution is decolorized by the addition of about 16 g of ammonium chloride and 75 ml (101.3 g) of ethyl-butyric acid ester are added. The ammonia was evaporated in vacuo and the residue was dissolved in 1.5 L of 0.2 N sodium hydroxide. After stirring for 45 minutes, the solution is filtered off, loaded onto a cation exchange column in an H ⁺ -cycle (DOWEX 50) and the product is displaced with a 10% aqueous pyridine solution. The pyridine eluate is evaporated to dryness, the residue is dissolved in 230 ml of 90% aqueous methanol and the product is precipitated with 7.5 liters of ether. After filtration and drying, 44 g (i.e. 85% of theory) of III are obtained, which is identical to the product prepared by the solution method.

(c) O-Nitrophenylsulphenyl-O-methyltyrosyl-isoleucyl-glutaminyl-asparaginyl-S-Υ-carboxypropionyl-prolyl-leucyl-glycine amide (Compound V)

To a solution of 38.3 of III in 500 ml of DMF was added 7.9 g of N-hydroxybenzotriazole and 22.7 g of o-nitrobenzenesulfenyl-O-methyltyrosine p-nitrophenyl ester. After 24 hours, an additional 13.3 g of the active ester is added and allowed to stand the next day. After precipitation of a mixture of ether (2.7 L) with ethyl acetate (1.13 L), 46 g (i.e. 86% of theory) of compound V are obtained, identical to the product prepared by the solution method.

d) (2-O-methyltyrosine) deamino-1-carbaoxytocin g of the substance V is dissolved in 300 ml of dimethylformamide in the heat and cooled to room temperature, whereby the substance V precipitates again from the solution. 50 ml of a 2 N solution of hydrogen chloride in dioxane are added to the suspension. After standing at room temperature for 15 minutes, the resulting solution was poured into 4 L of ether. The precipitate formed is filtered off with suction and dissolved in 4 l of dimethylformamide. The solution is cooled to 0 [deg.] C. and 4 g of potassium hydrogen phosphate and 11.5 g of diphenylphosphorazide are added with stirring. The progress of the reaction was monitored by high performance liquid chromatography and the reaction continued until the starting material disappeared. The reaction mixture was filtered, the filtrate was concentrated in vacuo until most of the diemtylformamide was distilled off. The residue was dissolved in about 1000 mL of 20% acetic acid, filtered and used directly to purify the crude product by high performance liquid chromatography. 8 g of (2-O-methyltyrosine) deamino-1-carbaoxytocin (based on 100% of substance) are obtained, which corresponds to the product obtained by the classical solution synthesis method.

Example 2

The procedure of Example 1 was followed to V, 60 g of V being dissolved in 900 ml of concentrated acetic acid while cooling, cooled to room temperature, and 75 ml of 8N hydrogen chloride in dioxane was added. After standing at room temperature for 20 minutes, the solution was poured into 10 L of absolute ether. The precipitate formed is filtered off with suction, dried and dissolved in 940 ml of dimethylformamide and 670 ml of dioxane, 79 g of N-hydroxybenzotriazole are added and the solution is cooled to -10 ° C. A solution of 86 g of N, N'-dicyclohexylcarbodiimide in 100 ml of dimethylformamide is added and stirred at room temperature for 2 hours. The reaction mixture was then added dropwise to a solution of 150 ml of triethylamine in 6 L of methanol heated to 40 ° C at a rate of about 1 L per hour. The reaction mixture was allowed to stand overnight, acidified with acetic acid and the solvents evaporated in vacuo. The residue was dissolved in 20% acetic acid, filtered and used directly to purify the crude product by high performance liquid chromatography. 10 g of (2-O-methyltyrosine) deamino-1-carbaoxytocin (based on 100% of substance) are obtained, which, according to their properties, corresponds to the product obtained by the classical solution synthesis method.

Example 3

Following the procedure of Examples 1 and 2, the same results were obtained when the tert-butyloxycarbonyl group was used for the octapeptidamide of formula V instead of the o-nitrophenylsulfenyl group.

List of abbreviations used in the text

Z-benzyloxycarbonyl

Boc-t-butyloxycarbonyl group (p'-copolymer (styrene-1% divinylbenzene)

Bzl-benzyl group

Methyl

Tyr tyrosine

Ile isoleucine

Gin glutamine

Asn asparagin

Cys cysteine

For proline

Leu

Gly leucine glycine

-C1 H8 COOH-carboxypropionyl

HPLC high performance liquid chromatography

Claims (1)

(2-O-methyltyrosine) deamino-1-carbaoxytocin, characterized in that a heptapeptide of the formula I is first prepared by solid-phase synthesis Z-Ile-Gln-Asn-Cys (Bzl) -Pro-Leu-Gly-O- (II, where Z is benzyloxycarbonyl, Qp) is a carrier, Bzl is benzyl, which is converted into solution by cleavage from the carrier by methanolic treatment ammonia solution to form the protected heptapeptide II Z-Ile-Gln-Asn-Cys (Bzl) -Pro-Leu-Gly-NH4 (II), where Z and Bzl are as previously defined, from which the protecting groups are cleaved with a solution of sodium in liquid ammonia, the sulfhydril group is alkylated of cysteine with ethyl .beta.-bromobutyrate and the ester group is hydrolyzed with sodium hydroxide solution to prepare the free heptapeptidamide III. Η -Ile-Gln-Asn-Cys (C ^COOH) -Pro-Leu-Gly-NH ₂ (III) to which the tyrosine derivative IV is condensed X-Tyr (Me) -OH (IV) wherein X is a tert-butyloxycarbonyl or o-nitrophenylsulfenyl group and Me is methyl to form the octapeptide V X-Tyr (Me) -Ile-Gln-Asn-Cys (C 4 -COOH) -Pro-Leu-Gly-NH ₂ (V), where X and Me are as previously defined, whereupon the protecting group is removed and the free octapeptidamide is removed. cycling by dissolving in a buffered dimethylformamide solution with the addition of diphenylphosphorazide or with N, Ν'-dicyclohexylcarbodiimide and N-hydroxybenztriazole.