EA023360B1 - Linear acyl azide pegylating agent, method for preparing the same anf method for preparing pegylated interferon - Google Patents

Abstract

The invention relates to pharmaceutical industry, particularly to pegylating agent, capable of binding to free amino groups of proteins with formation of amide bond by reaction of azide condensation, as well as to a method for preparing the same and to a method for pegylation of biologically active proteins, for instance interferons. The invention provides methods for preparing linear acyl azide pegylating agent and respective methods for pegylating interferons (IFN), in particular IFN α-2b. Technical result of the invention includes pegylating agent - PEG-phenylalanine derivative, capable of binding to free amino groups of proteins with formation of amide bond by means of azide condensation, without causing racemisation of substrate, which can be easily qualitatively identified in UV-range by aromatic fragment and quantified.

Description

The invention relates to the pharmaceutical industry, in particular to a pegylating agent capable of attaching to the free amino groups of proteins to form an amide bond through an azide condensation reaction, as well as to a method for its preparation and a method for pegylating biologically active proteins, for example interferons. Pegylated interferons can be used for the preparation of antiviral drugs.

State of the art

Polyethylene oxide (polyethylene glycol, PEG) is a widely used polymer for covalent modification (pegylation) of biological macromolecules, for example polypeptides, which is of great importance in modern medicine. The purpose of pegylation is to prevent protein degradation by proteolytic enzymes and to protect against antigenic and immunogenic epitopes. In addition, pegylation increases the size of the polypeptides, reducing renal filtration, and also changes the volume of biodistribution. All this helps to lengthen the residence time of modified physiologically active proteins in the patient's body, which increases their value as medicines.

The PEG molecule is not able to directly interact with protein molecules, because it does not contain reactive functional groups that can react with any functional groups of proteins. Therefore, it is required to introduce into the molecule of this polymer such reactive groups or their precursors that are converted to the active form shortly before the reaction with proteins.

Of greatest importance during the pegylation of polypeptides is the choice of the pegylating agent and the conjugation method, which determine the type and structure of the final conjugate, and, consequently, its biological activity, stability and circulation time in the blood.

So, for example, the antiviral activity of pegylated interferons (INF) depends to a large extent both on the size and geometry of the PEG molecule, and on the location and method of attachment of the polymer to the protein molecule, i.e. from the INF functional group, with which the corresponding pegylating agent directly interacts (Koiake M.1. e (a1. SNetMgu Toternuy ρτοΐθίη RESU1a1ui. Αάν. Igid Ieenuu Keu, νοί. 54 (2002), p. 459-476).

The introduction of various active functional groups capable of interacting with the functional groups of proteins under mild conditions to obtain the corresponding covalent conjugates is described, in particular, by A. e. (7) (2001), p. 419-29.

From the chemistry of peptides (Greenstein J., Vinitz M. Chemistry of amino acids and peptides. M .: Mir. 1965) it is known that, when an amide bond is formed with optically active α-amino acids, as a rule, racemization of the substrate is observed to one extent or another, moreover to the least extent, racemization is observed using the azide method, which consists in the interaction of the acylazide of the corresponding protected amino acid or peptide with the free amino group of the corresponding amine substrate. The main disadvantage of acyl azides is their low stability, acyl azides are obtained from the corresponding stable hydrazides by treatment with nitrous acid or its esters immediately before use (A. Gershkovich, V. Kibirev. Chemical synthesis of peptides. Kiev: Nukova Dumka, 1992).

At present, a fairly wide range of PEG hydrazide derivatives is known that are used as pegylating agents, while, as a rule, it is in the hydrazide form without conversion to acyl azide and the use of azide condensation.

EP 0 605 963 (published July 13, 1994) discloses, in particular, hydrazide derivatives of PEG with a molar mass of 2 to 12 kDa having structures

Ρ-Ο-αμ-εΟ-ΝΗΝΗι Ρ-ΝΗ-Ο8-ΝΗΝΗ ₂

R-O-CO-ΝΙΙΝΉ, ΡΚΙΚ'Ο-ΝΙΙΝΗΟΟ-ΝΗΝΗ,

Ρ-ΝΙΙΝΗΟΟ-ΝΗΝΙΤ

Ρ-ΝΗΝΗΟ8-ΝΗΝΗ ₂

Ρ-ΝΙΙ-ΤΟ-ΝΙΙΝΙΙ,

RYU-SO-SNGSShch-SO-MLShch ρ-ΝΗ-εο-ε ₆ Η4-ΝΗΝΗ ₂ , where P represents PEG, as well as methods for their preparation and use. These PEG derivatives modify, for example, erythropoietin preoxidized by periodate to form a hydrazone bond between the polymer and protein domains.

Application I8 2005118227 (publ. 02.06.2005) describes hydrazide compounds with angiogenic activity, which are obtained by reacting PEG dihydrazide with the corresponding carboxylic acid derivatives activated, for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. In turn, PEG dihydrazide is obtained by treating polyethylene glycol (molar mass of about 2 kDa) with an excess of phosgene, and then with Ν-hydroxysuccinimide in the presence of triethylamine. The obtained PEG disuccinimidyl carbonate is converted to PEG dihydrazide with excess hydrazine.

In the patent I8 4101380 (publ. 07/18/1978) disclosed pegylating agent, which is able to interact with the protein through azide condensation. For example, such a pegylating agent is a crosslinking agent (molar mass of about 2.5 kDa), which is a polyethylene glycol diacyl azide. The specified agent is obtained immediately before crosslinking of proteins from dihydrazide

- 1 023360

PEG. PEG dihydrazide, in turn, is obtained by treating the polyethylene oxide diglycolate with thionyl chloride, followed by conversion of the resulting diacyl chloride to hydrazide by the action of hydrazine hydrate. This crosslinking agent is used in the preparation of protein membranes.

PEG 4179337 (publ. 12/18/1979) describes PEG azide (molar mass of about 0.75 kDa), which is obtained from PEG hydrazide by treatment with sodium nitrite in a hydrochloric acid solution immediately before conjugation with uricase. The required PEG hydrazide is obtained by reacting polyethylene glycol with chloroacetic acid methyl ester in liquid ammonia in the presence of sodium metal, which leads to the formation of PEG glycolic acid methyl ester, which is then converted to the corresponding hydrazide by hydrazinolysis of the ester group.

As the closest analogue of the present invention, the authors consider PEG hydrazide with a molar mass of 5 kDa, disclosed in the application WITH 1992/016555 (publ. 01.10.1992), which is a derivative of β-alanine. The specified PEG hydrazide is obtained by a process comprising the steps of (a) treating monomethoxypolyethylene glycol with phosgene to form PEG chloroformate, (b) treating the resulting product with an excess of β-alanine ethyl ester hydrochloride in the presence of an excess of triethylamine with the formation of PEG-b-alanine ethyl ester and (c ) treating PEG-β-alanine ethyl ester with hydrazine to form PEG-β-alanine hydrazide cG

Protein pegylation (Prgoet) is carried out by reacting PEG hydrazide with a carboxyl group of a protein activated by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide to form an acylhydrazine linker

or with a carbohydrate fragment of a glycosylated protein (O1usorgo1esh), an oxidized periodate, followed by fixing the formed hydrazone bond by reducing sodium cyanoborohydride to the corresponding amine

The possibility of using this PEG agent in the azide condensation reaction in the known invention is not expected.

The main disadvantages of the pegylating agent under consideration are the presence of a relatively easily hydrolyzable carbamate bond, the use of non-physiological β-amino acid (β-alanine) as a linker, and the use of highly toxic phosgene as one of the intermediate reagents. In addition, the disadvantages include the absence of fragments in its structure, which could easily make a qualitative and quantitative determination of the PEG agent, which greatly simplifies the analysis and purification of both the PEG agent and the reaction mixture during conjugation with proteins.

Thus, the technical objective of the present invention is to develop pegylating agents suitable for the preparation of stable derivatives of proteins and expanding the arsenal of pegylating agents and methods for pegylating biologically active proteins, for example interferons.

The technical result of the invention is a pegylating agent, a derivative of PEG-02 02,360 phenylalanine, which is able to attach to the free amino groups of proteins to form an amide bond by means of azide condensation without causing racemization of the substrate, which can be easily qualitatively identified in the UV region by the aromatic fragment and quantified and methods for its preparation.

SUMMARY OF THE INVENTION

As a result of extensive research, the authors found that the disadvantages of the prior art can be overcome by the creation and use of a linear acyl azide pegylating agent of the general formula (I)

where a is an integer from 100 to 1800 and b is an integer from 1 to 9, having a molecular weight of from 5 to 80 kDa.

The present invention also provides a method for preparing a linear acyl azide pegylating agent of the general formula (I)

ABOUT

where a is an integer from 100 to 1800 and b is an integer from 1 to 9, with a molecular weight of 5 to 80 kDa, characterized in that the monomethoxypolyethylene glycol (a1) is alkylated with the corresponding alkyl ester of (b1) ω-halogen-carboxylic acid in an anhydrous organic solvent selected from the group consisting of benzene, toluene, xylene, tetrahydrofuran, dioxane, anhydrous tertbutanol, in the presence of a base B selected from the group consisting of an alkali metal, alkali metal amide, alkali metal alcohol or butyl lithium, in atmosphere of nitrogen or argon in the temperature range from 0 to 100 ° C or upon boiling of an organic solvent with the formation of the corresponding PEG-carboxyether (a2)

where A1k is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, and the leaving group X is C1, Br or I, and the product is isolated by precipitation with diethyl ether;

ester of PEG-carboxyester (a2) was subjected to alkaline hydrolysis in a nitrogen or argon atmosphere an aqueous solution of alkali selected from the hydroxides of alkaline or alkaline earth metals or mixtures thereof 0A1k ^s ₃ c n (O-CH _z CH _z -) - o- (CHX (a2) H ₃ C- (O-CH-CH ₂ -) - o- (CH ₂ | ^ (abs) the product obtained is acidified to obtain a PEG-carboxylic acid (a4) ₃ n c ^ o-CH-CH ^ otsn, - ^ -% (ab3)

ABOUT /

b he • n ₃ c- (o-cn-cn ₂ -) - o- {cn ₂ X (a4) and the resulting PEG-carboxylic acid is isolated from the aqueous solution by extraction with a chlorine-containing solvent selected from the group consisting of dichloromethane, trichlorethylene, chloroform, carbon tetrachloride, followed by precipitation with diethyl ether;

PEG carboxylic acid (a4) in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C in an anhydrous organic solvent selected from the group consisting of dioxane, tetrahydrofuran, dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, dimethylformamide, ethyl acetate by the action of a hydroxyl-containing compound K-OH selected from идhydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol, in the presence of carbodiimide (CO) selected from cyclohexylcarbodiimide or diisopropyl arbodii- 3 023360 bromide is converted to an activated ester (a5-1) PEG-carboxylic acid ₂ N-CH ₂ s4o -snAo / SNA / ^{k 'it'} s ^and one (a4) - * CH-n_s4o; -snH o4sn ₂ -K ^{ν / a} ' ^{/ b} ok (ab5 "1) which is isolated by precipitation with diethyl ether;

or PEG-carboxylic acid (a4) in an atmosphere of nitrogen or argon at a temperature of from -10 to 100 ° C. by treatment with an excess of thionyl chloride in an organic anhydrous solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, or without it, turn into acid chloride (ab5-2) n ₃ s- ^ o-cn ₂ -sn ₂ ^ -o ^ cn ₂ · ^ ^80C1 '(ab4): - ^ o-cn-cn ₂ ^ -o ^ cn ₂ b C1 which is isolated by precipitation with diethyl ether; phenylalanine ester (ab5-2)

in which A1k 'denotes an aliphatic alkyl selected from methyl or ethyl, in an atmosphere of nitrogen or argon at a temperature of from -10 to 30 ° C in the presence of an organic base selected from the group consisting of triethylamine, Ν-methylmorpholine, in an anhydrous organic solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, ethyl acetate, dimethylformamide, dioxane, tetrahydrofuran, acylated with activated ester (a5-1) of PEG-carboxylic acid, in which K represents the residue a hydroxyl-containing compound selected from Ν-hydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol

and compound (a6) is isolated by precipitation with diethyl ether;

or the corresponding phenylalanine ester in anhydrous dimethylformamide medium in the presence of a base selected from triethylamine, methyldiethylamine or β-methylmorpholine, is acylated with PEG-carboxylic acid chloride (a5-5-2)

and compound (a6) is isolated by precipitation with diethyl ether;

or the corresponding phenylalanine ester in a nitrogen or argon atmosphere in an anhydrous organic solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, ethyl acetate, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran, acetonitrile, at a temperature of from -30 to 30 ° C in the presence of an organic base selected from the group consisting of triethylamine, Ν-methylmorpholine, in the presence of a carbodiimide selected from cyclohexylcarbodiimide or diisopropylcarbodiimide, tsiliruyut PEG-carboxylic acid (a4)

and compound (a6) is isolated by precipitation with diethyl ether;

- 4 023360 PEG-phenylalanine ether (a6) by the action of excess hydrazine hydrate in an alcohol solvent selected from methanol, ethanol, or a mixture thereof, in an atmosphere of nitrogen or argon in the temperature range from 10 to 100 ° C under elevated pressure or at the boiling point of the solvent at atmospheric pressure is converted to compound (II)

ABOUT

which is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, followed by precipitation with diethyl ether; and compound (II) is converted into a linear acyl azide pegylating agent of general formula (I) by treating with an excess of nitrous acid obtained by mixing nitrite of an alkali or alkaline earth metal and an acid selected from the group consisting of sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, citric acid, acetic acid, at a reduced temperature in the range from -5 to 10 ° C with the removal of excess nitrous acid by adding alkali metal azide at the end of the reaction.

The invention further provides a method for preparing a linear acyl azide pegylating agent of general formula (I)

where a is an integer from 100 to 1800 and b is an integer from 1 to 9, with a molecular weight of 5 to 80 kDa, characterized in that the monomethoxypolyethylene glycol (a1) is alkylated with the corresponding alkyl ester (b1) of ω-halogencarboxylic acid in anhydrous medium an organic solvent selected from the group consisting of benzene, toluene, xylene, tetrahydrofuran, dioxane, anhydrous tertbutanol, in the presence of a base B selected from the group consisting of an alkali metal, alkali metal amide, alkali metal alcohol or butyl lithium, and atmosphere of nitrogen or argon in the temperature range from 0 to 100 ° C or upon boiling of an organic solvent with the formation of the corresponding PEG-carboxy ester (aB2) n, c4o-cn, -sn, 4-one + X-HcNZA (a1) ( ^b ) ^{b 0A | to}

- n, c4o-sn — snXo / snZA '' a '' x.

where A1k is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, and the leaving group X is C1, Br or I, and the product is isolated by precipitation with diethyl ether; the ester group of the PEG carboxyether (a2) is subjected to alkaline hydrolysis in an atmosphere of nitrogen or argon with an aqueous alkali solution selected from alkali or alkaline-earth metal hydroxides or mixtures thereof

the resulting product is acidified to obtain PEG-carboxylic acid (aB4) n ₃ s- ^ o-cn-cn ₂ ^ o- (cn ₂ - ^ -4 (a3) ° —► n ₃ s) o-cn ₂ - CH ₂ ) -o (cН ₂ -Л (а4)) ^Н and the resulting PEG-carboxylic acid is isolated from the aqueous solution by extraction with a chlorine-containing solvent selected from the group consisting of dichloromethane, trichlorethylene, chloroform, carbon tetrachloride, followed by precipitation with diethyl ether;

PEG carboxylic acid (a4) in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C in an anhydrous organic solvent selected from the group consisting of dioxane, tetrahydrofuran, dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, dimethylform 023360 amide, ethyl acetate, by the action of a hydroxyl-containing compound K-OH selected from идhydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol, in the presence of carbodiimide (SE) selected from cyclohexylcarbodiimide or diisop opilkarbodiimida, is converted into the activated ester (a5-1) PEG-carboxylic acid, ₃ c n (O-CH-CH4-o4snD- ^ - ^to '° d'^'a''b OH (a4) - · - n ₃ s4o -ch ₂ - cn4-o + cn ₂ A`` · '' he (ab5-1) which is isolated by precipitation with diethyl ether;

or PEG-carboxylic acid (a4) in an atmosphere of nitrogen or argon at a temperature of from -10 to 100 ° C. by treatment with an excess of thionyl chloride in an organic anhydrous solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, or without it, turn into acid chloride (ab5-2)

H ₃ C -> O — CH — CH ₂ ··· —O— ^ CHp ^{Ζ / 50C1r} (aB4) ^b . about

D-o- {sn ₂ -) ^

C- ^ O — CH — C

- ^ o-cn ₂ (ab5-2) which is isolated by precipitation with diethyl ether; phenylalanine in an aqueous or aqueous-organic medium, which is a mixture of an organic medium selected from the group consisting of acetonitrile, methanol, ethanol, isopropanol, with water in any proportions in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C acylates activated ester (aL5-1) of PEG-carboxylic acid, in which K represents the residue of a hydroxyl-containing compound selected from Ν-hydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol

and the product is precipitated with diethyl ether, or phenylalanine in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C in an aqueous, aqueous-organic or organic medium, where the organic medium is selected from the group consisting of dimethylformamide, dimethyl sulfoxide, acetonitrile, methanol , ethanol, isopropanol, and the aqueous-organic medium is a mixture of the organic medium with water in any ratio, in the presence of a base selected from alkali and alkaline earth metal hydroxides, carbonates or bicarbonates, oxide or hydra ksida magnesium, triethylamine or mixtures thereof, is acylated with an acid chloride (a5-2) PEG-carboxylic acid

and the product is precipitated with diethyl ether, compound (a6 ') by the action of an excess of hydrazine hydrate in an aqueous medium in the presence of water-soluble carbodiimide (CE) selected from the group consisting of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, Acyclohexyl-B- (2 -morpholinyl- (4) -ethyl) carbodiimide, Mcyclohexyl-I (4-dimethylaminocyclohexyl) carbodiimide, is converted to compound (II)

which is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, followed by precipitation with diethyl ether, or compound (a6 ') by the action of a hydroxyl-containing compound KOH selected from S-6 023360 hydroxysuccinimide, 4- nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol, in the presence of carbodiimide (CO), such as cyclohexylcarbodiimide or diisopropylcarbodiimide, are converted to the corresponding activated ester (a7-1), in which ohm K is the radical of a hydroxyl-containing compound selected from the Ν-hydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol and pentafluorophenol

and activated ester (ab7-1) is isolated by precipitation with diethyl ether.

or compound (ab6 ') in an atmosphere of nitrogen or argon at a temperature of from -10 to 100 ° C by treatment with an excess of thionyl chloride in an organic anhydrous solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, or without it is converted to acid chloride ( ab7-2)

which is isolated by precipitation with diethyl ether;

Compounds (aL7-1) or (aL7-2) are subjected to hydrazinolysis with an excess of hydrazine hydrate in an aqueous, aqueous, organic or organic medium to form a compound of the general formula (II) n ₃ c4o-cn-ciXo-ynAX V ^- a'a'b _Ν Η— / _ΝΗ (ab7-1)} -► (II) o

or

Compound (II) is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, followed by precipitation with diethyl ether, or by treating compound (a6 ') with excess hydrazine hydrate in an aqueous medium in the presence of water-soluble carbodiimide (CO ) selected from the group consisting of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, ^ cyclohexyl - ^ (2-morpholinyl- (4) -ethyl) carbodiimide, Νcyclohexyl-N (4-dimethylaminocyclohexyl) carbodiimide, give a compound (I I) n ₃ c4o-cn — cnXcTsnD ^ V- he 'a' b ΝΗ-Ζ _ΝΛ (a bb) N -► (II)

O which is isolated by extraction with a chlorinated solvent selected from dichloromethane, chloroform, trichlorethylene, carbon tetrachloride or mixtures thereof, followed by precipitation with diethyl ether; and compound (II) is converted into a linear acyl azide pegylating agent of general formula (I) by treatment with an excess of nitrous acid obtained by mixing nitrite of an alkali or alkaline earth metal and an acid selected from sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid , citric acid, acetic acid, or mixtures thereof, at a reduced temperature in the range from -5 to 10 ° C, removing excess nitrous acid by adding alkali metal azide at the end of the reaction.

The invention also provides a method for the preparation of pegylated interferon a-2b (ΖΡΝ a-2b), characterized in that the method according to claim 2 or 3 prepares a solution of a linear acyl azide pegylating agent of general formula (I);

to a solution of α-2b in a phosphate buffer with a pH of 8.0-8.7 at 0-5 ° C, a 4-5-fold molar excess of a solution of a linear acyl azide pegylating agent of the general formula (I) is added

where a is an integer from 100 to 1800 and

B is an integer from 1 to 9, with a molecular weight of from 5 to 80 kDa; when the degree of conversion of α-2b reaches 70-85%, the reaction is stopped by adding excess glycine; the reaction mixture is diluted 5-15 times, the pH value is set to 4.5-5.5, required for chromatographic separation of products on CM-sepharose, followed by gradient elution from 0.1 to 0.8 M No. C1; fractions are selected that analyze reverse-phase HPLC for the content of mono-PEG-содержание a-2b, according to the results of which the target fractions are determined, which are then purified to a degree sufficient to prepare the drug.

An expression selected from the group consisting of, in addition to individual alternatives, also includes possible and rational combinations of two or more such alternatives. For example, the expression chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride includes, in particular, mixtures of dichloroethane and chloroform, chloroform and carbon tetrachloride, irichlorethylene and carbon tetrachloride.

Next, methods for producing a linear acyl azide pegylating agent of the general formula (I) will be described in detail with reference to synthesis routes and transformation schemes at each stage of the routes. In the context of the present invention, the term synthesis route defines the directions of chemical transformations from starting materials to the final product by means of symbols of intermediate products indicating the stages of synthesis that bind them.

Synthesis route 1.

(ab5-1 [5c] [4a] /

- <ab2)--! - - <aM) Φ2b- <ab4),

A. · * -, / ⁸ [5a] (a6> -!

[56]

- (II)

The legend of the compounds given in parentheses indicates the presence of the polymer fragment - (O-CH ₂ -CH ₂ ) _a - (a), - (CH ₂ ) _b - (b), or both fragments (a b) in their molecules. The number after the letter designates the stage number at which the compound or compounds are the starting materials. The second number, if indicated, defines the variant of the intermediate compound. The designations in square brackets indicate the stages of synthesis.

In accordance with the invention, the pegylating agent of general formula (I) can be obtained from monomethoxypolyethylene glycol using several synthetic approaches. In the first embodiment, a method for producing synthesis starts with alkylation of the hydroxyl group of monomethoxypolyethylene glycol ether corresponding ω-halocarboxylic acid (step [1]) ^{with n} of ^{0 4-CH} ~ ^CH 2K ^{x n +} LR (a1) <L1> ^{0A | a}

Stage [1]

-n ₃ s- (o

-sn — cn ₂ ^ -o bk (ab2) ^b OA! k

In step [1], monomethoxypolyethylene glycol (a1) is treated with the alkyl ester (b1) of the corresponding ω-halogenocarboxylic acid in an anhydrous organic solvent in the presence of base B to form the corresponding PEG carboxyether (aB2).

The base is selected from lithium metal, metallic sodium or metallic potassium, lithium amide, sodium amide or potassium amide, butyl lithium, lithium tert-butylate, sodium tert-butylate or potassium tert-butylate, the leaving group X is C1, Br or I, A1k is selected from methyl, ethyl, propyl , isopropyl, butyl, sec-butyl or tert-butyl.

The molar ratio of base to monomethoxypolyethylene glycol is from 1: 1 to 50: 1. The molar ratio of Ω-halocarboxylic acid to monomethoxypolyethylene glycol is from 1: 1 to 50: 1.

The reaction is carried out in an atmosphere of nitrogen or argon in the temperature range from 0 to 100 ° C or when boiling in an anhydrous organic solvent selected from benzene, toluene, xylene, tetrahydrofuran, dioxane, anhydrous tertbutanol or a mixture thereof. The reaction product is precipitated with diethyl ether.

The number of ester groups contained in the PEG-carboxyether is determined either by microdetermination of the saponification number or spectrophotometrically in the visible region with ReC1 ₃ reagent

- 8 023360 after preliminary conversion to the form of the corresponding hydroxamic acid.

Next, the ester group of the PEG-carboxy-ester (a2) is subjected to alkaline hydrolysis (stage [2]) of n ₃ c- (o-cn ₂ -cn ₂ ) -o (cn ₂ C (a2)

Stage [2]

- N.sfz-CH — ΟΗ.) - θ) θΗ ₂ -Υ (ab3) for the preparation of PEG-carboxylic acid (stage [3]) n _e c- (o-cn ₂ -sn ₂ ^ o- (cn ₂ ^ N ⁺ (ab3)

Stage [3]

H, Tso-CH ₂ - ΟΗ ₂ - | -θ) θΗ, Υ (ab4) ^b

The alkaline hydrolysis of PEG-carboxyether (a2) (stage [2]) is carried out in an atmosphere of nitrogen or argon, with an aqueous solution of an alkali selected from alkali or alkaline-earth metal hydroxides or mixtures thereof.

Subsequent acidification of the reaction mass after completion of stage 2, the corresponding PEG-carboxylic acid (a4) is obtained (stage [3]). For acidification, an aqueous solution of an acid selected from sulfuric acid, hydrochloric acid, perchloric acid, hydrobromic acid, phosphoric acid, citric acid, acetic acid or mixtures thereof is used.

The resulting PEG-carboxylic acid (a4) is isolated from the aqueous solution by extraction with a chlorine-containing solvent selected from dichloromethane, trichlorethylene, chloroform, carbon tetrachloride or mixtures thereof, followed by precipitation with diethyl ether.

The amount of carboxyl groups in (a4) is determined either by potentiometric microtiter with a solution of a strong base, or spectrophotometrically in the visible region in the form of a salt with brilliant green or methylene blue.

Next, PEG-carboxylic acid (a4) is converted into the activated ester (a5-1) of PEG-carboxylic acid (step [4a])

ns- (o-sn ₂ -sk ₂ -) - o ^ sn ₂ Ts

Step [4a] <a4) n, c ^ _o-CH-CH2 ^ -O- [CH ₂ C (a5-1)

OK or acid chloride (ab5-2) (stage [4b]) n ₃ Cso-cn ₂ -sn ₂ ^ o- (cn ₂ -} - ^ 5OCl, (ab4)

Stage [4b] n ₃ Tso-cn — cn ₂ ^ -o) cn ₂ -C (ab5-2)

The conversion of PEG carboxylic acid (a4) to the corresponding activated ester (a5-1) in step 4a is carried out by the action of the hydroxyl-containing compound K.-OH in the presence of carbodiimide (ΟΌ) in an anhydrous organic solvent. The reaction is carried out in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C. An anhydrous organic solvent is selected from dioxane, tetrahydrofuran, dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, dimethylformamide, ethyl acetate or mixtures thereof. Carbodiimide is cyclohexylcarbodiimide or diisopropylcarbodiimide. The hydroxyl-containing compound is selected from Ν-hydroxysuccinimide, 4-nitrophenol, 2,4dinitrophenol, pentachlorophenol or pentafluorophenol.

The molar ratio of the hydroxyl-containing compound K.-OH to PEG-carboxylic acid (a4) is from 1: 1 to 50: 1. The molar ratio of carbodiimide (ΟΌ) to PEG-carboxylic acid (a4) is from 1: 1 to 50: 1.

The activated ester of (a5-1) PEG-carboxylic acid is isolated by precipitation with diethyl ether. The number of ester groups contained in the (a5-1) is set either mikroopredeleniem saponification number or spectrophotometrically in the UV or in the visible range with a reagent ReS1 ₃ after prior conversion to the corresponding hydroxamic acid form.

The preparation of the acid chloride (a5-5-2) of PEG-carboxylic acid is carried out by treating PEG-carboxylic acid with an excess of thionyl chloride in an organic anhydrous solvent or without it. An anhydrous organic solvent is selected from dichloromethane, chloroform, trichlorethylene, tetra-9 023360 carbon chloride or mixtures thereof. The reaction is carried out in an atmosphere of nitrogen or argon at a temperature of from -10 to 100 ° C. The product is isolated by precipitation with diethyl ether.

The number of acid chloride groups contained in the (a5-2), determined either potentiometric or microtiter spectrophotometrically in the visible region with a reagent ReS1 ₃ after prior conversion to the corresponding hydroxamic acid form.

Next, the corresponding phenylalanine ester, in which A1k 'is an aliphatic alkyl selected from methyl or ethyl, is acylated using an activated ester (ab5-1) of PEG-carboxylic acid as an acylating agent, in which K represents the residue of a hydroxyl-containing compound selected from Ν- hydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol (stage [5a])

OA1k ‘

Stage [5a]

The phenylalanine ester is acylated in the presence of a base in an anhydrous organic solvent selected from dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, ethyl acetate, dimethylformamide, dioxane, tetrahydrofuran or mixtures thereof. The reaction is carried out in an atmosphere of nitrogen or argon at a temperature of from -10 to 30 ° C. The organic base is triethylamine, Ν methylmorpholine, or a mixture thereof. The molar ratio of phenylalanine ester to activated (aL5-1) PEG-carboxylic ester is from 1: 1 to 50: 1. The reaction product is isolated by precipitation with diethyl ether.

The number of phenylalanine ester fragments attached to the activated ester (ab5-1) is determined either by microdetermination of the saponification number, or spectrophotometrically in the UV region or in the visible layer with FeCl reagent after preliminary conversion of the corresponding hydroxamic acid into the form.

Alternatively, PEG carboxylic acid chloride (aL5-2) is used as the acylating agent in the presence of an aliphatic amine selected from triethylamine, methyldiethylamine or метил-methylmlrfololine as the base. The reaction is preferably carried out in dimethylformamide medium (step [5c])

Stage [5c]

Isolation and characterization of the product is carried out as described for stage [5A].

Alternatively, the phenylalanine methyl or ethyl ester is acylated directly with PEG-carboxylic acid (a4) in the presence of carbodiimide and an organic base in an anhydrous organic solvent (step [5b])

Stage [5b]

An anhydrous organic solvent is selected from dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, ethyl acetate, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran, acetonitrile, or mixtures thereof. The organic base is triethylamine, Ν-methylmorpholine, or a mixture thereof. Carbodiimide is cyclohexylcarbodiimide or diisopropylcarbodiimide.

The reaction is carried out in an atmosphere of nitrogen or argon at a temperature of from -30 to 30 ° C. The molar ratio of phenylalanine ester to PEG-carboxylic acid (a4) is from 1: 1 to 50: 1. The molar ratio of carbodiimide to PEG-carboxylic acid (aB4) is from 1: 1 to 50: 1. The product is isolated by precipitation with diethyl ether.

- 10 023360

The number of phenylalanine ester fragments attached to PEG-carboxylic acid (aB4) is determined either by microdetermination of the saponification number, or spectrophotometrically in the UV region or in the visible growth area with ReCl reagent after preliminary converting the corresponding hydroxamic acid into the form.

In step [6], the PEG-phenylalanine methyl or ethyl ester (a6) is converted to compound (II) by hydrazinolysis

H ₃ sphe-CH — cpf o- ^ cn (ab6)

Stage [6]

Hydrazinolysis is carried out by the action of an excess of hydrazine hydrate in an alcohol solvent medium selected from methanol, ethanol, or a mixture thereof. The reaction is carried out in an atmosphere of nitrogen or argon in the temperature range from 10 to 100 ° C at elevated pressure or at the boiling point of the solvent at atmospheric pressure. Compound (II) is isolated by extraction with a chlorinated solvent selected from dichloromethane, chloroform, trichlorethylene, carbon tetrachloride or mixtures thereof, followed by precipitation with diethyl ether.

The number of hydrazide groups introduced in (a6) is determined spectrophotometrically in the visible region with 4-dimethylaminobenzaldehyde reagent.

In another alternative embodiment of the process for preparing the compound of general formula (II), the chemical reaction sequence described by synthesis route 2 is used.

Synthesis route 2.

The stages of synthesis [1] - [3], [4a], [4b] and the conditions for their implementation are similar to synthesis route 1, disclosed above.

In the present embodiment of the synthesis method, phenylalanine is directly used as an intermediate, which is acylated with an activated ester of (aB5-1) PEG-carboxylic acid, in which K is the residue of a hydroxyl-containing compound selected from β-hydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol (stage [5g])

IT

Stage [5g]

Acylation is carried out in an aqueous or aqueous-organic medium. An aqueous-organic medium is a mixture of an organic medium selected from acetonitrile, methanol, ethanol, isopropanol or mixtures thereof, with water in any ratio. The molar ratio of phenylalanine to activated PEG-carboxylic acid ester (aL5-1) is from 1: 1 to 50: 1. The reaction is carried out in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C. The product was precipitated with diethyl ether.

Alternatively, phenylalanine is acylated by the action of the acid chloride (ab5-2) of PEGcarboxylic acid in an aqueous, aqueous-organic or organic medium in the presence of a base (stage [5e])

Stage [5d]

Organic media are dimethylformamide, dimethyl sulfoxide, acetonitrile, methanol, ethanol, isopropanol, or mixtures thereof. An aqueous-organic medium is a mixture of an organic medium with water in any ratio. The base is alkali and alkaline earth metal hydroxides, carbonates or bicarbonates, magnesium oxide or hydroxide, triethylamine or mixtures thereof.

The molar ratio of phenylalanine to activated PEG-carboxylic acid ester (aL5-1) is from 1: 1 to 50: 1. The reaction is carried out in an atmosphere of nitrogen or argon in the temperature range from -10 to

- 11 023360

30 ° C. The product was precipitated with diethyl ether.

In both cases, the number of attached phenylalanine-carboxyl groups is determined either by potentiometric microtiter or spectrophotometrically in the visible region in the form of a salt with brilliant green or methylene blue. The presence of an aromatic phenylalanine residue is proved spectrophotometrically by the characteristic absorption in the UV region.

Then, compound (a6 ') is converted to compound (II) by the action of an excess of hydrazine hydrate in the presence of water-soluble carbodiimide (CE) in an aqueous medium (stage [6c])

Stage [6c]

The reaction is carried out in an atmosphere of nitrogen or argon at a temperature of from -10 to 30 ° C. The water-soluble carbodimide is 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, Y-cyclohexyl-K- (2morpholinyl- (4) -ethyl) carbodiimide, Y-cyclohexyl-K- (4-dimethylaminocyclohexyl) carbodiimide or a mixture thereof. The molar ratio of carbodiimide to (ab6 ') is from 1: 1 to 50: 1.

Compound (II) is isolated by extraction with a chlorinated solvent selected from dichloromethane, chloroform, trichlorethylene, carbon tetrachloride or mixtures thereof, followed by precipitation with diethyl ether.

The amount of hydrazide groups introduced in (a6 ') is determined spectrophotometrically in the visible region with 4-dimethylaminobenzaldehyde reagent. The presence of an aromatic phenylalanine residue is proved spectrophotometrically in the UV region by characteristic absorption.

Alternatively, hydrazinolysis is preceded by the conversion of compound (aB6 ') to the corresponding activated ester (aB7-1), in which K is the residue of a hydroxyl-containing compound selected from Ν-hydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol (stage [6a])

Stage [6a] by the action of a hydroxyl-containing compound K-OH selected from Ν-hydroxysuccinimide, 4 nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol, in the presence of carbodiimide (SE) such as cyclohexylcarbodiimide or diisopropylcarbodiimide.

The reaction is carried out in an atmosphere of nitrogen or argon in the temperature range from -10 to 20 ° C in an anhydrous organic solvent selected from dioxane, tetrahydrofuran, dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, dimethylformamide, ethyl acetate or a mixture thereof. The activated PEG-carboxylic acid ester (a7-1) is isolated by precipitation with diethyl ether.

The molar ratio of the hydroxyl-containing compound K — OH to the compound (ab6 ′) is from 1: 1 to 50: 1. The molar ratio of SE carbodiimide to compound (ab6 ') is from 1: 1 to 50: 1.

The number of ester groups contained in the compound (ab7-1) is determined either by microdetermination of the saponification number, or spectrophotometrically in the UV region or in the visible region with ReCl reagent after preliminary converting the corresponding hydroxamic acid into the form.

In another alternative embodiment, the hydrazinolysis is preceded by the conversion of the compound (a6) to the corresponding acid chloride (a7-2) (step [6b])

Step [6b] by treating the compound (a6) with an excess of thionyl chloride in an organic anhydrous solvent or without it. An anhydrous organic solvent is selected from dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, or mixtures thereof. The reaction is carried out in an atmosphere of nitrogen or argon at a temperature of from -10 to 100 ° C. The product is isolated by precipitation with diethyl ether.

The number of acid chloride groups contained in the PEG-phenylalanine acid chloride, or determined by potentiometric microtiter either spectrophotometrically in the visible region with a reagent ReS1 ₃ after prior conversion to the corresponding hydroxamic acid form. The presence of an aromatic phenylalanine residue is proved spectrophotometrically in the UV region by characteristic absorption.

Next, in steps [7a] or [7b] compound (a7-1) or (a7-2) is hydrazinolysable to form a compound of general formula (II) / \ 0 0 N _e c (o-CH-CH-4 _g of - (cn - ^ OK 'a b _ΝΗ - / ν ₂ Η ₄ (a b7-1)} - (II)

ABOUT

Stage [7 a]

Stage [7b]

For this activations, the ester (ab7-1), in which K represents the remainder of the hydroxyl-containing compound selected from гидрок-hydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol, or the acid chloride (ab7-2) is treated with excess hydrazine hydrate in aqueous , water-organic or organic medium.

The reaction is carried out in an atmosphere of nitrogen or argon at a temperature of from -10 to 30 ° C. The organic medium is methanol, ethanol, acetonitrile, dioxane, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, or mixtures thereof. An aqueous-organic medium is a mixture of an organic medium with water in any ratio.

Compound (II) is isolated by extraction with a chlorinated solvent selected from dichloromethane, chloroform, trichlorethylene, carbon tetrachloride or mixtures thereof, followed by precipitation with diethyl ether.

The number of introduced hydrazide groups is determined spectrophotometrically in the visible region with the reagent 4-dimethylaminobenzaldehyde. The presence of an aromatic phenylalanine residue is proved spectrophotometrically in the UV region by characteristic absorption.

Alternatively, compound (II) can be directly prepared by hydrazinolysis of PEG carboxylic acid (a6 ') in the presence of a water-soluble carbodiimide (SI) such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, No.cyclohexyl-] No. (2-morpholinyl- (4 ) -ethyl) carbodiimide, Νcyclohexyl-L- (4-dimethylaminocyclohexyl) carbodiimide, or mixtures thereof, in an aqueous medium (stage [6c]) n ₃ c4o-cn — cnAo + cn _{2 4/7 —} he'a b _ΝΗ -Ζ _{Ν n} (ab) 6 - (II)

ABOUT

Stage [6c]

The reaction is carried out in an atmosphere of nitrogen or argon at a temperature of from -10 to 30 ° C. The molar ratio of carbodiimide to compound (ab6 ') is from 1: 1 to 50: 1. Isolation and characterization of the product is carried out as described for stage (7a) or (7b).

The main objective of the invention is to obtain stably pegylated proteins having useful biological properties, especially interferons, and in particular interferon a-2b (ΖΡΝ aa-2b). For this, compound (II) immediately before pegylation of the protein is converted into a linear acyl azide pegylating agent (I) by treatment with an excess of nitrous acid at a reduced temperature in the range from -5 to 10 ° C. The source of nitrous acid is a mixture of an alkali or alkaline earth metal nitrite and an acid selected from sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, citric acid, acetic acid, or mixtures thereof. At the end of the reaction, the excess nitrous acid is removed by the addition of alkali metal azide, after which the resulting compound (I) is used without isolation for conjugation with protein

Upon reaching the degree of protein conversion of 70-85%, the reaction is stopped by adding excess

- 13 023360 glycine. The reaction mixture is diluted, the pH is set to 4.5-5.5, which is required for the chromatographic separation of products on CI-Sepharose, followed by gradient elution from 0.1 to 0.8 M NaCl. The selected fractions were analyzed by reverse-phase HPLC for the content of mono-PEGΙΡΝ a-2b, based on which the target ones were selected for subsequent purification and preparation of the drug.

Further, the achievement of the technical result of the invention will be shown in the preferred embodiments. All examples were reproduced for two fractions of monomethoxypolyethylene glycol PEG ₂₀ and PEG ₃₀ with molecular weights of 20 and 30 kDa, respectively. The presence of a phenylalanine fragment in compounds (aL5-1), (aL6), (aL6 '), (aL7-1) and (II) was confirmed spectrophotometrically by the presence of an absorption band with maxima at 251, 257 and 263 nm, which has a pronounced vibrational structure whose character is the same as described in the publication 1pd1e PB .. Vi8sy K.SCH., Vi8sy MA S1yga1 aia1u818 Ly tiShuapaYu gedgekkyup toyeNpd about £ 8resDa1 ya1a ikshd sus1oye \ mw dneCh-nights-sotr1ehe8 Me1yoY8 £ og yeYutiptd epapyoteps sotromyop \ νί11ι uagushd Syga1 apa1u1e sopseygayop. Ta1aya, wo1. 75 (2) Arg. 2008, p. 572-584.

Information confirming the possibility of carrying out the invention.

Example 1. Obtaining PEG-carboxyether (stage [1]).

In an argon atmosphere, with heating, 50 μmol of monomethoxypolyethylene glycol is dissolved in 50 ml of toluene. The resulting solution was boiled for 15 minutes and about 25 ml of solvent was distilled off, after which the solution was cooled to room temperature and a solution of 1000 μm potassium tert-butoxide in 20 ml of absolute tert-butanol was poured.

The reaction mixture was stirred for 1 h in an argon atmosphere at room temperature and a solution of 1000 μmol of ω-bromocarboxylic acid methyl ester with the number of L atoms from 1 to 9 in 10 ml of toluene was added and stirred overnight at room temperature. Then, the solution was evaporated under reduced pressure, and the PEG-carboxyether was precipitated with diethyl ether, filtered off and precipitated twice from the dichloromethane solution with diethyl ether, and dried in a vacuum desiccator.

Yield: 94-98%. The content of ester groups in the polymer according to titrimetric and spectrophotometric analyzes is 96-102%.

Example 2. Obtaining PEG-carboxylic acid (stage [2] and [3]).

The PEG carboxyether obtained in Example 1 was dissolved in 50 ml of 2M NaOH and stirred overnight under argon, then acidified with 10% sulfuric acid to pH 1-2 and extracted three times with 20 ml of dichloromethane. The combined extracts were washed with 10% sodium sulfate until neutral and dried over anhydrous sodium sulfate overnight. The dried solution was filtered, evaporated and the PEG-carboxylic acid was precipitated with diethyl ether and filtered.

The resulting precipitate was reprecipitated twice with diethyl ether from a solution in absolute isopropanol and dried in a vacuum desiccator.

Yield: 90-96%. The content of ester groups in the polymer according to titrimetric and spectrophotometric analyzes is 98-103%.

Example 3. Obtaining nitrophenyl ether of PEG-carboxylic acid (stage [4A]).

To a solution of 100 μmol of the PEG-carboxylic acid obtained in Example 3 cooled to 0 ° C, an ethyl acetate solution of 500 μmol of 4-nitrophenol was poured into 80 ml of ethyl acetate, stirred and 500 μmol of dicyclohexylcarbodiimide dissolved in 10 ml of ethyl acetate was added. The reaction mixture was stirred overnight at room temperature under argon.

The solution was filtered and the product was isolated by adding diethyl ether, filtered off, precipitated twice with ether from a solution in dichloromethane and dried in a vacuum desiccator.

Yield: 97-100%. The content of nitrophenyl groups in the polymer according to titrimetric and spectrophotometric analyzes is 93-101%.

Example 4. Obtaining the acid chloride of PEG-carboxylic acid (stage [4B]).

To a solution of 100 μmol of the PEG-carboxylic acid obtained in Example 3 in 50 ml of dichloromethane was added 10 ml of freshly distilled thionyl chloride and stirred in an argon atmosphere overnight at room temperature. Then the reaction mixture was evaporated under reduced pressure, the residue was dissolved in 20 ml of methylene chloride and the product was isolated by the addition of diethyl ether.

PEG carboxylic acid chloride is filtered off, precipitated twice with ether from a solution in dichloromethane and dried in a vacuum desiccator.

Yield: 97-100%. The content of acid chloride groups in the polymer according to titrimetric and spectrophotometric analyzes is 89-94%.

Example 5. Obtaining methyl ester of PEG-phenylalanine (stage [5A]).

100 μmol of PEG-carboxylic acid nitrophenyl ether obtained in Example 3 are dissolved in 40 ml of dichloromethane. In parallel, a solution is prepared containing 1000 μmol of Ζ-phenylalanine and triethylamine methyl ester hydrochloride in 10 ml of dimethylformamide. The solutions are mixed and left overnight in an argon atmosphere.

- 14 023360

The product was precipitated by the addition of diethyl ether, filtered off and precipitated twice with ether from a solution in isopropanol. The resulting PEG-phenylalanine methyl ester was dried in vacuo.

Yield: 92-98%. The content of ether groups in the polymer according to titrimetric and spectrophotometric analyzes is 92-97%.

Example 6. Obtaining methyl ester of PEG-phenylalanine (stage [5B]).

100 μmol of PEG carboxylic acid chloride obtained in Example 4 are dissolved in 40 ml of dichloromethane. In parallel, a solution is prepared containing 1000 μmol of L-phenylalanine methyl ester hydrochloride and triethylamine in 10 ml of dimethylformamide. The solutions are mixed and left overnight in an argon atmosphere.

The product was precipitated by the addition of diethyl ether, filtered off and precipitated twice with ether from a solution in isopropanol. The resulting PEG-phenylalanine methyl ester was dried in vacuo.

Yield: 94-98%. The content of ether groups in the polymer according to titrimetric and spectrophotometric analyzes is 88-95%.

Example 7. Obtaining methyl ester of PEG-phenylalanine (stage [5B]).

To a solution of 100 μmol of PEG-carboxylic acid cooled to 0 ° C. obtained in Example 2, a dimethylformamide solution of 500 μmol of L-phenylalanine methyl ester hydrochloride containing 500 μmol of triethylamine was poured into 50 ml of ethyl acetate, stirred and 500 μmol of dicyclohexylcarbodiimide dissolved in 10 ml of ethyl acetate. The reaction mixture was stirred overnight at room temperature under argon. Then the solution is filtered and the product is isolated by adding diethyl ether, filtered off, precipitated twice with ether from the solution in isopropanol and dried in a vacuum desiccator.

Yield: 95-99%. The content of ether groups in the polymer according to titrimetric and spectrophotometric analyzes is 92-100%.

Example 8. Obtaining PEG-phenylalanine (stage [5g]).

100 μmol of PEG-carboxylic acid nitrophenyl ester obtained in Example 3 was dissolved in 50 ml of an aqueous solution containing 1000 μmol of L-phenylalanine and 1000 μmol triethylamine. The solution was stirred overnight at room temperature under argon. The yellow reaction mixture was then extracted three times with 50 ml of dichloromethane. The combined extracts are washed with 10% sodium sulfate in 1% NaHCO ₃ , then with 10% sodium sulfate, acidified to pH 1-2 with sulfuric acid and finally with pure 10% sodium sulfate until neutral and dried over anhydrous sodium sulfate in overnight.

The dried solution was filtered, evaporated and PEG-phenylalanine was precipitated with diethyl ether and filtered. The resulting polymer precipitate was reprecipitated twice with diethyl ether from a solution in absolute isopropanol and dried in vacuo.

Yield: 87-96%. The content of carboxyl groups in the polymer according to titrimetric and spectrophotometric analyzes is 90-97%.

Example 9. Obtaining PEG-phenylalanine (stage [5d]).

100 μmol of PEG carboxylic acid chloride obtained in Example 4 was dissolved in 40 ml of dichloromethane. In parallel, a solution is prepared containing 1000 μmol of L-phenylalanine and triethylamine in 10 ml of dimethylformamide. The solutions are mixed and left overnight in an argon atmosphere. The product is precipitated by the addition of diethyl ether, filtered off and precipitated twice with ether from the solution to isopropanol. The obtained PEG-phenylalanine is dried in a vacuum desiccator.

Yield: 93-98%. The content of ether groups in the polymer according to titrimetric and spectrophotometric analyzes is 89-94%.

Example 10. Obtaining hydroxysuccinimide ester of PEG-phenylalanine (stage [6A]).

To a solution of 100 μmol of PEG-phenylalanine obtained in Example 8 or 9 cooled to 0 ° C, an ethyl acetate solution of 500 μmol of Г-hydroxysuccinimide was poured into 80 ml of ethyl acetate, stirred and 500 μmol of dicyclohexylcarbodiimide dissolved in 10 ml of ethyl acetate was added. The reaction mixture was stirred overnight at room temperature under argon. Then the solution is filtered, and the product is isolated by adding diethyl ether, filtered off, precipitated twice with ether from the solution in dichloromethane and dried in a vacuum desiccator.

Yield: 96-99%. The content of hydroxysuccinimide groups in the polymer according to titrimetric and spectrophotometric analyzes is 95-103%.

Example 11. Obtaining the acid chloride of PEG-phenylalanine (stage [6B]).

To a solution of 100 μmol of PEG-phenylalanine obtained in Example 8 or 9 in 50 ml of dichloromethane is added 10 ml of freshly distilled thionyl chloride and stirred under argon overnight at room temperature. After the reaction mixture was evaporated under reduced pressure, the residue was dissolved in 20 ml of methylene chloride and the product was isolated by the addition of diethyl ether.

PEG-phenylalanine chloride is filtered off, precipitated twice with ether from a solution in dichloromethane and dried in a vacuum desiccator over solid KOH or conc. H ₂ §O ₄ .

- 15 023360

Yield: 98-100%. The content of acid chloride groups in the polymer according to titrimetric and spectrophotometric analyzes is 88-98%.

Example 12. Obtaining PEG-phenylalanine (II) hydrazide (stage [6]).

To a solution of 100 μmol of PEG-phenylalanine methyl ester obtained in Examples 5, 6 or 7 in 30 ml of methanol is added 10 ml of hydrazine hydrate and refluxed under argon for 24 hours. Then the solution is evaporated under reduced pressure, the residue is dissolved in 30 ml 10% sodium sulfate, acidified with 5% sulfuric acid to a pH of 1-2 and 5 times extracted with dichloromethane in 30 ml portions.

The organic layer was washed with 10% sodium sulfate until neutral and dried over anhydrous sodium sulfate overnight. The dried dichloromethane solution of PEG hydrazide was filtered, evaporated under reduced pressure, and the product was precipitated by the addition of diethyl ether. The precipitate is filtered off and precipitated three times with ether from a solution in isopropanol.

Yield: 91-96%. The content of hydrazide groups in the polymer according to spectrophotometric analysis is 83-92%.

Example 13. Obtaining PEG-phenylalanine (II) hydrazide (stage [7a]).

In 30 ml of a 1% aqueous solution of hydrazine hydrate cooled to 0 ° C, 100 μmol of the PEG-phenylalanine hydroxysuccinimide ester obtained in Example 10 is dissolved. The mixture is stirred for 5 hours under argon atmosphere at room temperature, acidified with 5% sulfuric acid to pH 1 -2 and 5 times extracted with dichloromethane in 30 ml portions.

The organic layer was washed with 10% sodium sulfate until neutral and dried over anhydrous sodium sulfate overnight. The dried dichloromethane solution of PEG hydrazide was filtered, evaporated under reduced pressure, and the product was precipitated by the addition of diethyl ether. The precipitate is filtered off and precipitated three times with ether from a solution in isopropanol.

Yield: 92-95%. The content of hydrazide groups in the polymer according to spectrophotometric analysis is 91-94%.

Example 14. Obtaining PEG-phenylalanine (II) hydrazide (stage [7b]).

In 25 ml of dimethylfluramamide cooled to 0 ° C containing 10 mmol of hydrazine hydrate, 100 μmol of PEG-phenylalanine chloride obtained in Example 11 are dissolved. The mixture is stirred under argon for 2 hours at room temperature, evaporated under reduced pressure, dissolved in 30 ml of water, acidified with 5% sulfuric acid to a pH of 1-2 and 5 times extracted with dichloromethane in 30 ml portions.

The organic layer was washed with 10% sodium sulfate until neutral and dried over anhydrous sodium sulfate overnight. The dried dichloromethane solution of PEG hydrazide was filtered, evaporated under reduced pressure, and the product was precipitated by the addition of diethyl ether. The precipitate is filtered off and precipitated three times with ether from a solution in isopropanol.

Yield: 89-94%. The content of hydrazide groups in the polymer according to spectrophotometric analysis is 86-92%.

Example 15. Obtaining PEG-phenylalanine (II) hydrazide (stage [6c]).

An aqueous solution of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (500 μmol / 10 ml) was poured into 30 ml of an aqueous solution of 100 μmol of PEG-phenylalanine obtained in Example 8 or 9 containing 10 mmol of hydrazine hydrate cooled to 3 ° C.

The mixture was stirred under cooling for 3 hours and then overnight at room temperature in argon atmosphere, acidified with 5% sulfuric acid to pH 1-2 and extracted with 30 ml of 5-fold dichloromethane.

The organic layer was washed with 10% sodium sulfate until neutral and dried over anhydrous sodium sulfate overnight. The dried dichloromethane solution of PEG hydrazide was filtered, evaporated under reduced pressure, and the product was precipitated by the addition of diethyl ether. The precipitate is filtered off and precipitated three times with ether from a solution in isopropanol.

Yield: 92-98%. The content of hydrazide groups in the polymer according to spectrophotometric analysis is 82-89%.

Example 16. Obtaining a pegylating linear acyl azide agent of General formula (II).

To a solution of 5 μmol of PEG-phenylalanine hydrazide obtained in Example 13, 14 or 15 cooled to 0 ° C, a solution of 50 μmol ΝαΝ ₂ in 1 ml of water is poured into 3 ml of 0.5 M HC1. The mixture is stirred while cooling for 20 minutes, then the excess nitrous acid is removed by adding 50 μmol ΝαΝ in 0.5 ml of water and immediately used for conjugation with protein.

Example 17. Obtaining pegylated interferon (PEG-ΙΡΝ a-2b).

A solution of 5 μmol of PEG-phenylalanine azide obtained in Example 16 is added to a solution of 1 μmol Γ a-2b cooled to 0-5 ° C in phosphate buffer with a pH of 8.5. The reaction mixture was stirred for 3 hours while cooling. Next, the reaction was stopped by the addition of excess glycine.

The reaction mixture was diluted 10 times, pH 5.0 was adjusted by adding CH ₃ COOH and applied to C ^ Sepharose, previously equilibrated with 20 mM acetate buffer at pH 5.0. After application

- 16 023360 of the reaction mixture was eluted with a balancing buffer, and then with a gradient of 0.1 to 0.8 M No. C1. Selected fractions were analyzed by reverse phase HPLC for mono-PEG-α2-b content. The desired fractions are combined and reanalyzed to determine the purity and content of mono-PEG-ΙΡΝ α2b.

The yield of purified product (in terms of ΙΡΝ a-2b): for PEG-ΙΡΝ a-2b is 54% with a purity of 95.5%; for PEG, S-a-2b is 42% with a purity of 97.3%.

Claims (9)

CLAIM 1. A linear acyl azide pegylating agent of the general formula (Ι) where a is an integer from 100 to 1800 and B is an integer from 1 to 9, having a molecular weight of from 5 to 80 kDa. 2. A method of producing a linear acyl azide pegylating agent of the general formula (Ι) according to claim 1, characterized in that the monomethoxypolyethylene glycol (a1) is alkylated with the corresponding alkyl ester (b1) of ω-halogencarboxylic acid in an anhydrous organic solvent selected from the group consisting of benzene , toluene, xylene, tetrahydrofuran, dioxane, anhydrous tert-butanol, in the presence of a base B selected from the group consisting of alkali metal, alkali metal amide, alkali metal alcohol or butyl lithium, and atmosphere of nitrogen or argon in the temperature range from 0 to 100 ° C or upon boiling of an organic solvent with the formation of the corresponding PEG carboxyether (a2) where A1k is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, and the leaving group X is C1, Br or Ι, wherein the product is isolated by precipitation with diethyl ether; the ester group of the PEG carboxyether (a2) is subjected to alkaline hydrolysis in an atmosphere of nitrogen or argon with an aqueous alkali solution selected from alkali or alkaline earth metal hydroxides or mixtures thereof, the resulting product is acidified to obtain PEG carboxylic acid (a4), the resulting PEG carboxylic acid is isolated from aqueous the solution by extraction with a chlorine-containing solvent selected from the group consisting of dichloromethane, trichlorethylene, chloroform, carbon tetrachloride, followed by precipitation with diethyl ether; PEG carboxylic acid (aB4) in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C in an anhydrous organic solvent selected from the group consisting of dioxane, tetrahydrofuran, dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, dimethylformamide, ethyl acetate by the action of a hydroxyl-containing compound K-OH selected from идhydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol, in the presence of carbodiimide (SI) selected from cyclohexylcarbodiimide or diisopropyl arbodiimide is converted to an activated ester of (a5-1) PEG-carboxylic acid which is isolated by precipitation with diethyl ether; - 17 023360 phenylalanine ether OA1k in which A1k 'is an aliphatic alkyl selected from methyl or ethyl, in an atmosphere of nitrogen or argon at a temperature of from -10 to 30 ° C in the presence of an organic base selected from the group consisting of triethylamine, Ν-methylmorpholine, in an anhydrous organic medium a solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, ethyl acetate, dimethylformamide, dioxane, tetrahydrofuran is acylated with activated ester (a5-1) of PEG-carboxylic acid, in which K represents the residue a hydroxylic compound selected from the Ν-hydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol, and pentafluorophenol or compound (a6) is isolated by precipitation with diethyl ether; PEG-phenylalanine ether (a6) by the action of excess hydrazine hydrate in an alcohol solvent selected from methanol, ethanol, or a mixture thereof, in an atmosphere of nitrogen or argon in the temperature range from 10 to 100 ° C under elevated pressure or at the boiling point of the solvent at atmospheric pressure, is converted to compound (II) which is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, followed by precipitation with diethyl ether; and compound (II) is converted into a linear acyl azide pegylating agent of general formula (I) by treatment with an excess of nitrous acid obtained by mixing nitrite of an alkali or alkaline earth metal and an acid selected from the group consisting of sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid , citric acid, acetic acid, at a reduced temperature in the range from -5 to 10 ° C with removal of excess nitrous acid by adding alkali metal azide at the end of the reaction; in the above formulas (a1), (b1), (a2) - (a6) and (II), the values of a and b are as defined in paragraph 1. 3. A method of producing a linear acyl azide pegylating agent of general formula (I) according to claim 1, characterized in that the monomethoxypolyethylene glycol (a1) is alkylated with the corresponding alkyl ester (b1) of ω-halogencarboxylic acid in an anhydrous organic solvent selected from the group consisting of benzene , toluene, xylene, tetrahydrofuran, dioxane, anhydrous tert-butanol, in the presence of a base B selected from the group consisting of alkali metal, alkali metal amide, alkali metal alcohol or butyl lithium, and mosfere nitrogen or argon at temperatures ranging from 0 to 100 ° C or at reflux of an organic solvent to form the corresponding PEG-carboxyester (a2) n _e c ^ o-CH-CH ₂ - ^ x- ^ OH + CH ₂ - ^ - A (a1) (M) ^{0A | k} - n ₃ c - ^ o-cn-cn- ₂ -) - o {cn ₂ -) ^ (a2) where A1k is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, and the leaving group X is C1, Br or I, wherein the product is isolated by precipitation with diethyl ether; the ester group of the PEG-carboxyether (a2) is subjected to alkaline hydrolysis in an atmosphere of nitrogen or argon, an aqueous alkali solution selected from alkali or alkaline earth metal hydroxides or mixtures thereof - 18 023360 C -> O - CH — C (- - ₃ s -> oΈΗξ (a2) ZN — CH OA1k (ab3), the resulting product is acidified to obtain PEG-carboxylic acid (a4) ^ - (o-cng-cn ^ o ^ cn ^ (a3) О 'н * —- Н ₃ С-> О-СН — СН ₂ - ^ - О- (сН ₂ А <а4) the resulting PEG-carboxylic acid is isolated from the aqueous solution by extraction with a chlorine-containing solvent selected from the group consisting of dichloromethane, trichlorethylene, chloroform, carbon tetrachloride, followed by precipitation with diethyl ether; PEG carboxylic acid (aB4) in an atmosphere of nitrogen or argon at a temperature of from -10 to 100 ° C by treatment with an excess of thionyl chloride in an organic anhydrous solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, or without it is converted to acid chloride (ab5-2) ZOS1 ₂ he z- ^ o-sn — S N. C-10-CH — CH ₂ - + - o { ^cn 2A (aB4) ^< C- ^ O — CH — CH ₂ - ^ - o {c ⁿ 2A Η, ΟήΟ-ΌΗ — CH ₂ -) - O - (- CH. (A5-5-2) ³ which is isolated by precipitation with diethyl ether; phenylalanine ether in which A1k 'is an aliphatic alkyl selected from methyl or ethyl in anhydrous dimethylformamide in the presence of a base selected from triethylamine, methyldiethylamine or Ν methylmorpholine, acylate with an acid chloride (aL5-2) of PEG-carboxylic acid and compound (a6) is isolated by precipitation with diethyl ether; PEG-phenylalanine ether (a6) by the action of excess hydrazine hydrate in an alcohol solvent selected from methanol, ethanol or a mixture thereof, in an atmosphere of nitrogen or argon in the temperature range from 10 to 100 ° C under elevated pressure or at the boiling point of the solvent at atmospheric pressure, is converted to compound (II) which is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, followed by precipitation with diethyl ether; and compound (II) is converted into a linear acyl azide pegylating agent of general formula (I) by treatment with an excess of nitrous acid obtained by mixing nitrite of an alkali or alkaline earth metal and an acid selected from the group consisting of sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid , citric acid, acetic acid, at a reduced temperature in the range from -5 to 10 ° C with removal of excess nitrous acid by adding alkali metal azide at the end of the reaction; Moreover, in the above formulas (a1), (b1), (ab2) - (ab6) and (II), the values of a and b are as defined in 19 023360 in paragraph 1. 4. A method for producing a linear acyl azide pegylating agent of the general formula (I) according to claim 1, characterized in that the monomethoxypolyethylene glycol (a1) is alkylated with the corresponding alkyl ester (b1) of ω-halogencarboxylic acid in an anhydrous organic solvent selected from the group consisting of benzene , toluene, xylene, tetrahydrofuran, dioxane, anhydrous tert-butanol, in the presence of a base B selected from the group consisting of alkali metal, alkali metal amide, alkali metal alcohol or butyl lithium, and mosfere nitrogen or argon at temperatures ranging from 0 to 100 ° C or at reflux of an organic solvent to form the corresponding PEG-carboxyester (a2) H ₃ C-O-CH ^ -CH ^ OH + X-) -2 cN _r X "(a1) is (S) ^{OA | s} ° ^to OA to? - n ₃ c- (o-cn ₂ -sn ₂ -) - o {cn ₂ (a2) where A1k is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, and the leaving group X is C1, Br or I, wherein the product is isolated by precipitation with diethyl ether; the ester group of the PEG-carboxyether (a2) is subjected to alkaline hydrolysis in an atmosphere of nitrogen or argon with an aqueous alkali solution selected from alkali or alkaline-earth metal hydroxides or mixtures thereof, the resulting product is acidified to obtain PEG-carboxylic acid (a4) H ₃ C -> O — CH — CH ₂ -> - O—> CH ₂ - ^ -b * (a3) ° - - ₃ c4o-cn _{2 -} cnXo / cnA / '' ^a 'he (aB4> the resulting PEG-carboxylic acid is isolated from the aqueous solution by extraction with a chlorine-containing solvent selected from the group consisting of dichloromethane, trichlorethylene, chloroform, carbon tetrachloride, followed by precipitation with diethyl ether; phenylalanine ether in which A1k 'is an aliphatic alkyl selected from methyl or ethyl, in an atmosphere of nitrogen or argon, in an anhydrous organic solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, ethyl acetate, dimethylformamide, dimethyl sulfoxide, dimethyl sulfoxide, dimethyl sulfoxide , tetrahydrofuran, acetonitrile, at a temperature of from -30 to 30 ° C in the presence of an organic base selected from the group consisting of triethylamine, Ν-methylmorpholine, in the presence of a carbodiimide selected from cyclohexylcarbodiimide or diisopropylcarbodiimide is acylated with PEG carboxylic acid (a4) and the compound (a6) is isolated by precipitation with diethyl ether; PEG-phenylalanine ether (a6) by the action of excess hydrazine hydrate in an alcohol solvent selected from methanol, ethanol or a mixture thereof, in an atmosphere of nitrogen or argon in the temperature range from 10 to 100 ° C under elevated pressure or at the boiling point of the solvent at atmospheric pressure, is converted to compound (II) - 20 023360 which is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, followed by precipitation with diethyl ether; and compound (II) is converted into a linear acyl azide pegylating agent of general formula (I) by treatment with an excess of nitrous acid obtained by mixing nitrite of an alkali or alkaline earth metal and an acid selected from the group consisting of sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid , citric acid, acetic acid, at a reduced temperature in the range from -5 to 10 ° C with removal of excess nitrous acid by adding alkali metal azide at the end of the reaction; in the above formulas (a1), (b1), (a2) - (a6) and (II), the values of a and b are as defined in paragraph 1. 5. A method of producing a linear acyl azide pegylating agent of the general formula (I) according to claim 1, characterized in that the monomethoxypolyethylene glycol (a1) is alkylated with the corresponding alkyl ester (b1) of ω-halogencarboxylic acid in an anhydrous organic solvent selected from the group consisting of benzene , toluene, xylene, tetrahydrofuran, dioxane, anhydrous tert-butanol, in the presence of a base B selected from the group consisting of alkali metal, alkali metal amide, alkali metal alcohol or butyl lithium, and nitrogen or argon atmosphere in the temperature range from 0 to 100 ° C or when the organic solvent is boiled to form the corresponding PEG carboxyether (a2) where A1k is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, and the leaving group X is C1, Br or I, and the product is isolated by precipitation with diethyl ether; the ester group of the PEG carboxyether (a2) is subjected to alkaline hydrolysis in an atmosphere of nitrogen or argon with an aqueous alkali solution selected from alkali or alkaline earth metal hydroxides or mixtures thereof, the resulting product is acidified to obtain PEG carboxylic acid (a4) and the resulting PEG carboxylic acid is isolated from aqueous solution by extraction with a chlorine-containing solvent selected from the group consisting of dichloromethane, trichlorethylene, chloroform, carbon tetrachloride, followed by precipitation with diethyl ether; PEG carboxylic acid (a4) in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C in an anhydrous organic solvent selected from the group consisting of dioxane, tetrahydrofuran, dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, dimethylformamide, ethyl acetate by the action of a hydroxyl-containing compound K-OH selected from идhydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol, in the presence of carbodiimide (SE) selected from cyclohexylcarbodiimide or diisopropyl of arbodiimide, is converted to an activated ester of (a5-1) PEG-carboxylic acid - 21 023360 /) ~ o {cn ₂ -} ^a b he <a b 4> —N, c- (o-cn — cn ₂ (a5-1)) which is isolated by precipitation with diethyl ether; phenylalanine in an aqueous or aqueous-organic medium, which is a mixture of an organic medium selected from the group consisting of acetonitrile, methanol, ethanol, isopropanol, with water in any proportions in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C acylates activated PEG-carboxylic acid ester (a5-1) in which K is the residue of a hydroxyl-containing compound selected from из-hydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol and the product is precipitated with diethyl ether, I eat compound (ab6 ') by the action of an excess of hydrazine hydrate in an aqueous medium in the presence of a water-soluble carbodiimide (CU) selected from the group consisting of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, ^ cyclohexyl - ^ (2-morpholinyl- (4 ) -ethyl) carbodiimide, ^ cyclohexyl - ^ (4-dimethylaminocyclohexyl) carbodiimide, is converted to compound (II) which is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, followed by precipitation with diethyl ether, after which with Compound (II) is converted into a linear acyl azide pegylating agent of general formula (I) by treatment with an excess of nitrous acid obtained by mixing nitrite of an alkali or alkaline earth metal and an acid selected from sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, citric acid , acetic acid or mixtures thereof, at a reduced temperature in the range from -5 to 10 ° C with the removal of excess nitrous acid by adding alkali metal azide at the end of the reaction; in the above formulas (a1), (b1), (a2) - (a6 ') and (II) the values of a and b are as defined in paragraph 1. 6. A method of obtaining a linear acyl azide pegylating agent of the general formula (I) according to claim 1, characterized in that the monomethoxypolyethylene glycol (a1) is alkylated with the corresponding alkyl ester (b1) of ω-halogencarboxylic acid in an anhydrous organic solvent selected from the group consisting of benzene , toluene, xylene, tetrahydrofuran, dioxane, anhydrous tert-butanol, in the presence of a base B selected from the group consisting of alkali metal, alkali metal amide, alkali metal alcohol or butyl lithium, and mosfere nitrogen or argon at temperatures ranging from 0 to 100 ° C or at reflux of an organic solvent to form the corresponding PEG-carboxyester (a2). h of ^{He +} -NL ^x <L1) ° n, c ^ o-CH-snDun (a1) H, c ^ o-CH-CH ₂₎ -O (CH _r p / (a2) ° 'where A1k is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, and the leaving group X is C1, Br or I, and the product is isolated by precipitation with diethyl ether; the ester group of the PEG-carboxyether (a2) is subjected to alkaline hydrolysis in an atmosphere of nitrogen or argon with an aqueous alkali solution selected from alkali or alkaline earth metal hydroxides or mixtures thereof - 22 023360 НзС - ^ О-СН— ¹ H ^CH K ^and Ολικ (a2) it is ^- -► H ₃ C- [O-CH-CH ₂ (abs) the product obtained is acidified to obtain a PEG-carboxylic acid (a4) ₃ N c ^ o-CH-CH ₂ ^ -o (-CH ₂ - ^ -b "(a3B) ° —- n, c- ^ o-cn ₂ - cn ₂ ^ -o (cn ₂ -p ((aB4) ° ^H and the resulting PEG-carboxylic acid isolated from an aqueous solution by extraction with a chlorine-containing solvent selected from the group consisting of dichloromethane, trichlorethylene, chloroform, carbon tetrachloride, followed by precipitation with diethyl ether, then PEG-carboxylic acid (a4) in an atmosphere of nitrogen or argon at a temperature of from -10 to 100 ° С processing huts With a solution of thionyl chloride in an organic anhydrous solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, or without it, they are converted into acid chloride (aL5-2) n ₃ c- (о-sn — snXo / snDU - - ^{1; a} '' he (ab4) —n ,, c4o-cn-cnXo ^ cn, a a b C1 (ab5-2) _? which is isolated by precipitation with diethyl ether; phenylalanine in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C in an aqueous, aqueous, or organic medium, where the organic medium is selected from the group consisting of dimethylformamide, dimethyl sulfoxide, acetonitrile, methanol, ethanol, isopropanol, and the aqueous medium is a mixture of organic medium with water in any ratio, in the presence of a base selected from alkali and alkaline earth metal hydroxides, carbonates or hydrogen carbonates, magnesium oxide or hydroxide, triethylamine or mixtures thereof, tsiliruyut chloride (a5-2) PEG-carboxylic acid and the product precipitated with diethyl ether; compound (a6 ') by the action of an excess of hydrazine hydrate in an aqueous medium in the presence of a water-soluble carbodiimide (SI) selected from the group consisting of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, ^ cyclohexyl-N- (2-morpholinyl- (4) - ethyl) carbodiimide, Νcyclohexyl-N- (4-dimethylaminocyclohexyl) carbodiimide, is converted to compound (ΙΙ) which is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, followed by precipitation with diethyl ether; and compound (ΙΙ) is converted into a linear acyl azide pegylating agent of general formula (Ι) by treatment with an excess of nitrous acid obtained by mixing nitrite of an alkali or alkaline earth metal and an acid selected from sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, citric acid, acetic acid or mixtures thereof, at a reduced temperature in the range from -5 to 10 ° C, removing excess nitrous acid by adding alkali metal azide at the end of the reaction; Moreover, in the above formulas (a1), (b1), (a b2) - (a b6 ') and (b), the values of a and b are as defined in paragraph 1. - 23 023360 7. A method of producing a linear acyl azide pegylating agent of the general formula (I) according to claim 1, characterized in that the monomethoxypolyethylene glycol (a1) is alkylated with the corresponding alkyl ester (b1) of ω-halogencarboxylic acid in an anhydrous organic solvent selected from the group consisting of benzene , toluene, xylene, tetrahydrofuran, dioxane, anhydrous tert-butanol, in the presence of a base B selected from the group consisting of alkali metal, alkali metal amide, alkali metal alcohol or butyl lithium, and mosfere nitrogen or argon at temperatures ranging from 0 to 100 ° C or at reflux of an organic solvent to form the corresponding PEG-carboxyester (a2) n s4o _{3-CH 2} -CH ₂ - ^ he ^ + x CH ₂ - ^ -► < a1) (S) ^0A1k n ₃ s- (o-cn ₂ -sn ₂ -) - o {cn ₂ -) ^ ^b OA1k where A1k is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl butyl, and the leaving group X is C1, Br or I, and the product is isolated by precipitation with diethyl ether; the ester group of the PEG-carboxyether (a2) is subjected to alkaline hydrolysis in an atmosphere of nitrogen or argon, with an aqueous solution of an alkali selected from hydroxides of alkali or alkaline earth metals or mixtures thereof n ₃ c- ^ o-cn-cn ₂ - ^ - o- ^ cn ₂ <ab2) n ₃ s- (o-cn ₂ -sn _g -) - o {cn _2- ) (ab) the resulting product is acidified to obtain PEG-carboxylic acid (ab4) n ₃ s- ^ o-cn _g -cn ₂ ^ o {cn ₂ -) ^ - * * (ab3) .0 bn • ₃ c- (o-cn ₂ -cn ₂ -) - o- (cn ₂ -) ^ (ab4) and the resulting PEG- carboxylic acid is isolated from the aqueous solution by extraction with a chlorine-containing solvent selected from the group oyaschey from dichloromethane, trichlorethylene, chloroform, carbon tetrachloride, followed by precipitation with diethyl ether; PEG carboxylic acid (a4) in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C in an anhydrous organic solvent selected from the group consisting of dioxane, tetrahydrofuran, dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, dimethylformamide, ethyl acetate by the action of a hydroxyl-containing compound K-OH selected from идhydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol, in the presence of carbodiimide (CO) selected from cyclohexylcarbodiimide or diisopropyl of arbodiimide, is converted into the activated ester of (aL5-1) PEG-carboxylic acid n ₃ c- (o-cn ₂ -sn ₂ -) - o {cn ₂ (ab4) n ₃ c ^ o-cn-cn- ₂ - ^ - o- £ cn ₂ - | ^ (ab5-1) ° which is isolated by precipitation with diethyl ether; phenylalanine in an aqueous or aqueous-organic medium, which is a mixture of an organic medium selected from the group consisting of acetonitrile, methanol, ethanol, isopropanol, with water in any proportions in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C acylates activated PEG-carboxylic acid ester (aL5-1) in which K is the residue of a hydroxyl-containing compound selected from Ν-hydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol; the product is precipitated with diethyl ether; compound (a6 ') by the action of a hydroxyl-containing compound K-OH selected from β-hydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol, in - 24 023360 in the presence of carbodiimide (SE), such as cyclohexylcarbodiimide or diisopropylcarbodiimide, is transferred to the corresponding activated ester (aB7-1), in which K is the residue of a hydroxyl-containing compound selected from Ν-hydroxysuccinimide, 4-nitrophenol, 2,4-dinitrophenol, pentachlorophenol or pentafluorophenol and activated ether (a7-1) are isolated by precipitation with diethyl ether; compound (a7-1) is subjected to hydrazinolysis with excess hydrazine hydrate in an aqueous, aqueous, or organic medium to form a compound of general formula (II), compound (II) is isolated by extraction with a chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, s subsequent precipitation with diethyl ether; and compound (II) is converted into a linear acyl azide pegylating agent of general formula (I) by treatment with an excess of nitrous acid obtained by mixing nitrite of an alkali or alkaline earth metal and an acid selected from sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, citric acid, acetic acid or mixtures thereof, at a reduced temperature in the range from -5 to 10 ° C, removing excess nitrous acid by adding alkali metal azide at the end of the reaction; in the above formulas (a1), (b1), (a2) - (a7-1) and (II) the values of a and b are as defined in paragraph 1. 8. A method of obtaining a linear acyl azide pegylating agent of the general formula (I) according to claim 1, characterized in that the monomethoxypolyethylene glycol (a1) is alkylated with the corresponding alkyl ester (b1) of ω-halogencarboxylic acid in an anhydrous organic solvent selected from the group consisting of benzene , toluene, xylene, tetrahydrofuran, dioxane, anhydrous tert-butanol, in the presence of a base B selected from the group consisting of alkali metal, alkali metal amide, alkali metal alcohol or butyl lithium, and mosfere nitrogen or argon at temperatures ranging from 0 to 100 ° C or at reflux of an organic solvent to form the corresponding PEG-carboxyester (a2) Η, Ο ^ Ο-ΟΗ, -ΟΗ ^ ΟΗ + X- [cH ₂ -) ® / (a1) (L) n ₃ c- (o-cnX- {cn ₂ -C ' ^a L 0A1k <ab2) where A1k is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, and the leaving group X is C1, Br or I, and the product is isolated by precipitation with diethyl ether; the ester group of the PEG carboxyether (a2) is subjected to alkaline hydrolysis in an atmosphere of nitrogen or argon with an aqueous alkali solution selected from hydroxides of alkali or alkaline earth metals or mixtures thereof n ₃ c- (o-cn ₂ -sn ₂ ^ o {cn ₂ - | - ^ - (a2) -► n ₃ c ^ o-CH-CH ₂ - ^ - o / CH ₂ -q ^ (abs) ° the product obtained is acidified to obtain a PEG-carboxylic acid (a4) - 25 023360; - ^ o-cn _{2 -} cn ₂ ) -c n, c-} o-cn_ cnD-o (cn ₂ (ab3) ° - Н ₃ с4о-СН — SnД-о4снЛ1 \ 'a' X | (a4) and the resulting PEG-carboxylic acid are isolated from the aqueous solution by extraction with a chlorine-containing solvent selected from the group consisting of dichloromethane, trichlorethylene, chloroform, carbon tetrachloride, followed by precipitation with diethyl ether, then PEG-carboxylic acid (a4) in an atmosphere of nitrogen or ar at a temperature of from -10 to 100 ° C, with or without treatment with an excess of thionyl chloride in an organic anhydrous solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride they are added to the acid chloride (aL5-2) n ₃ s- (o-cn ₂ -sn ^ o {cn ₂ - ^ 4 (ab4) -n, c) o-cn-cn ₂ ) -o (cn ₂ ) - ((ab5-2) ' ^{b 01} which is isolated by precipitation with diethyl ether; phenylalanine in an atmosphere of nitrogen or argon in the temperature range from -10 to 30 ° C in an aqueous, aqueous, or organic medium, where the organic medium is selected from the group consisting of dimethylformamide, dimethyl sulfoxide, acetonitrile, methanol, ethanol, isopropanol, and the aqueous medium is a mixture of organic medium with water in any ratio, in the presence of a base selected from alkali and alkaline earth metal hydroxides, carbonates or hydrogen carbonates, magnesium oxide or hydroxide, triethylamine or mixtures thereof, tsiliruyut chloride (a5-2) PEG-carboxylic acid and the product precipitated with diethyl ether; the compound (ab6 ') in an atmosphere of nitrogen or argon at a temperature of from -10 to 100 ° C by treatment with an excess of thionyl chloride in an organic anhydrous solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, or without it is converted to acid chloride (ab7 -2) which is isolated by precipitation with diethyl ether, the compound (a7-2) is subjected to hydrazinolysis with an excess of hydrazine hydrate in an aqueous, aqueous, organic or organic medium to form a compound of the general formula (II), compound (II) is extracted chlorinated solvent selected from the group consisting of dichloromethane, chloroform, trichlorethylene, carbon tetrachloride, followed by precipitation with diethyl ether, which is isolated by extraction with a chlorinated solvent selected from dichloromethane, chloroform, trichlorethylene, carbon tetrachloride or mixtures thereof, followed by precipitation of diethyl ether ; and compound (II) is converted into a linear acyl azide pegylating agent of general formula (I) by treating with an excess of nitrous acid obtained by mixing alkaline nitrate or alkaline earth metal and an acid selected from sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid , citric acid, acetic acid, or mixtures thereof, at a reduced temperature in the range from -5 to 10 ° C, removing excess nitrous acid, adding alkali metal azide at the end of the reaction; in the above formulas (a1), (b1), (a2) - (ab7-2) and (II) the values of a and b are as defined in paragraph 1. 9. A method for the preparation of pegylated interferon a-2b (ΙΡΝ a-2b), characterized in that the method according to any one of claims 2-8, prepares a solution of a linear acyl azide pegylating agent of general formula (I), then to a solution of ΙΡΝ a-2b in phosphate a buffer with a pH of 8.0-8.7 at 0-5 ° C is added a 4-5-fold molar excess of a solution of a linear acyl azide pegylating agent of the general formula (I) where a is an integer from 100 to 1800 and b is an integer from 1 to 9 , with a molecular weight of 5 to 80 kDa, upon reaching the degree of conversion of P-αΝ-2b 70-85%, the reaction is stopped by adding an excess of hl zinc, the reaction mixture is diluted 5-15 times, the pH value is set to 4.5-5.5, required for chromatographic separation of products on CA-Sepharose, after which gradient elution is carried out from 0.1 to 0.8 M No. C1, fractions are taken which analyze reverse-phase HPLC for the content of mono-PEG-IΡN a-2b, according to the results of which the target fractions are determined, which are further purified to a degree sufficient to prepare the drug.