Method for producing activated polyethylene glycol having active group at terminal and activated polyethylene glycol having active group at terminal

Abstract

To obtain an activated polyethylene glycol having a high terminal activation rate by reacting a raw material polyethylene glycol having a carboxy group at the terminal with an activating reagent having an amino group at the terminal.SOLUTION: The method includes a step of reacting a raw material polyethylene glycol having a carboxy group with a compound having an active group and an amino group using a condensing agent to obtain a reaction product containing an activated polyethylene glycol having an active group at the terminal, wherein the terminal activation rate in the reaction product analyzed by ion exchange chromatography is 95 wt.% or more.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing activated polyethylene glycol having an active group at its terminal, and to an activated polyethylene glycol having an active group at its terminal. [Background technology]

[0002] In recent years, in the pharmaceutical field, complexes formed by combining a protein active agent with a water-soluble composition have achieved improved efficacy compared to when the protein itself is injected into the body, due to factors such as an increased half-life and evasion of the immune system.

[0003] Polyethylene glycol is one of the water-soluble compositions used for protein binding. Generally, the formation of an active agent-polyethylene glycol conjugate is achieved by the reaction between the active agent and raw polyethylene glycol. To achieve this reaction, activated polyethylene glycol is used, in which the end of the raw polyethylene glycol is substituted with a reactive functional group (active group). However, if the terminal activation rate of the activated polyethylene glycol is low and a large amount of raw polyethylene glycol remains, more activated polyethylene glycol will be required during pharmaceutical manufacturing, which may increase variable costs. For this reason, it is necessary to minimize the amount of raw polyethylene glycol in the activated polyethylene glycol.

[0004] Various functional groups are used as reactive functional groups in activated polyethylene glycols, including the maleimide group, which is particularly effective in binding to thiol groups on proteins.

[0005] As an approach to producing activated polyethylene glycol having a maleimide group at its terminal, Patent Document 1 describes synthesis via polyethylene glycol having an electrophilic group at its terminal. According to the description in Patent Document 1, activated polyethylene glycol having a maleimide group is obtained by reacting a raw material polyethylene glycol having an electrophilic group containing a carboxyl group with a maleimide compound having a nucleophilic group containing a primary amino group under favorable conditions. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2006-517600 Summary of the Invention [Problem to be solved by the invention]

[0007] Patent Document 1 includes an activated polyethylene glycol having a maleimide group. However, the method described in Patent Document 1 could not increase the terminal activation rate of the activated polyethylene glycol.

[0008] Furthermore, as shown in Comparative Example 1 in Patent Document 1, it has been confirmed that the product obtained by reacting terminal NHS-esterified polyethylene glycol with a maleimide compound having an amino group according to the method of Patent Document 1 has a terminal activation rate of less than 95% by mass.

[0009] An object of the present invention is to obtain activated polyethylene glycol having a high terminal activation rate by reacting a raw material polyethylene glycol having a terminal carboxy group with an activating reagent having an active group and an amino group at the terminals. [Means for solving the problem]

[0010] The present invention includes the following (1) to (8). (1) A method for producing activated polyethylene glycol having an active group at its terminal, comprising the steps of: A method for producing activated polyethylene glycol having an active group at its terminal, comprising the step of reacting a raw material polyethylene glycol having a carboxy group with a compound having an active group and an amino group (sometimes referred to as an "activating reagent") using a condensing agent to obtain a reaction product containing activated polyethylene glycol having an active group at its terminal, wherein the terminal activation rate in the reaction product as analyzed by ion exchange chromatography is 95% by weight or more.

[0011] (2) The method according to (1), wherein the activated polyethylene glycol is represented by the following formula (1): PEG-(C=O)-NH-R...Formula (1) (In formula (1), PEG is a polyethylene glycol moiety, R is an active group.

[0012] (3) The method according to (1) or (2), wherein the active group is a maleimide group.

[0013] (4) The method according to any one of (1) to (3), wherein the condensing agent is a triazine-based condensing agent or a uronium-based condensing agent.

[0014] (5) The method according to any one of (1) to (4), wherein the molecular weight of the raw material polyethylene glycol is 100 to 100,000 daltons.

[0015] (6) The method according to any one of (1) to (5), wherein the starting polyethylene glycol has one or more functional groups selected from the group consisting of a hydroxy group, an azide group, a biotin group, a methoxy group, an acetal-protected aldehyde group, a 9-fluorenylmethyloxycarbonyl-protected amino group, a t-butoxycarbonyl-protected amino group, and a benzyloxycarbonyl-protected amino group.

[0016] (7) An activated polyethylene glycol represented by the following formula (2): 1. An activated polyethylene glycol having an active group at its terminal, characterized in that the terminal activation rate as analyzed by ion exchange chromatography is 95% by weight or more. PEG-(C=O)-NH-R...Formula (2) (In formula (2), PEG is a polyethylene glycol moiety, R is an active group.

[0017] (8) The activated polyethylene glycol according to (7), wherein the active group is a maleimide group. [Effects of the Invention]

[0018] According to the present invention, an efficient reaction between the carboxy group of the raw material polyethylene glycol and the amino group of the activating reagent, which was difficult to achieve with conventional techniques, is realized by selecting a specific condensing agent, and activated polyethylene glycol having a high terminal activation rate is successfully obtained. DETAILED DESCRIPTION OF THE INVENTION

[0019] (activated polyethylene glycol) In the following section, the activated polyethylene glycol (hereinafter sometimes referred to as "activated PEG") produced in the present invention will be described.

[0020] The activated PEG of the present invention has a terminal activation rate of 95% or more. The activated PEG has the following formula (2): PEG-O-(C=O)-OR (2) In formula (2), "PEG-" is a polyethylene glycol moiety having a linear, branched, or multi-arm structure, and R is an active group.

[0021] The activated PEG of the present invention has an active group that reacts with a functional group present in a biofunctional molecule to form a covalent bond, preferably one or more functional groups selected from the group consisting of a maleimide group, an azide group, a biotin group, an active ester group, an acetal-protected aldehyde group, a 9-fluorenylmethyloxycarbonyl-protected amino group, a t-butoxycarbonyl-protected amino group, and a benzyloxycarbonyl-protected amino group.

[0022] The activated PEG may have a terminal other than the activated terminal that reacts with a functional group present in a biofunctional molecule to form a covalent bond, such as a protecting group for the functional group, a hydroxyl group, or a hydrocarbon. The protecting group for the functional group that forms a covalent bond may be one or more functional groups selected from the group consisting of an acetal-protected aldehyde group, a 9-fluorenylmethyloxycarbonyl-protected amino group, a t-butoxycarbonyl-protected amino group, and a benzyloxycarbonyl-protected amino group.

[0023] More specific examples of the activated PEG structure include activated PEGs represented by formulas (3) to (9). Among these, formulas (3) to (5) represent activated PEGs having a linear structure, formulas (6) to (8) represent activated PEGs having a branched structure, and formula (9) represents activated PEGs having a multi-arm structure.

[0024] [ka] [ka]

[0025] where X 1 is an atomic group containing a functional group that reacts with a functional group present in a biofunctional molecule to form a covalent bond, and Y 1 is an atomic group that protects a functional group that reacts with a functional group present in a biofunctional molecule to form a covalent bond, and X 1and the functional group contained in the atomic group Y1 are different from each other; R 1 is a hydrocarbon group having 1 to 7 carbon atoms or a hydrogen atom; n is an integer from 3 to 2300; m is an integer from 1 to 1200; A 1 , A 2 , A 3 , A 4 are each independently -L 1 -(CH2)m1-, -(CH2)m1-L 1 -, -L 1 -(CH2)m1-L 2 represents -(CH2)m2- or a single bond, and L 1 represents an ether bond, an amide bond, a urethane bond, a secondary amino group, or a single bond, and L 2 represents an ether bond, an amide bond, or a urethane bond, and m1 and m2 each independently represent an integer of 0 to 5; B 1 represents a carbon atom or a nitrogen atom.

[0026] [Method of producing activated PEG] The following section describes the preparation of activated PEG with terminal activation. The method of the present invention for producing an activated PEG with activated terminals comprises a step of reacting a raw material polyethylene glycol having a carboxy group with a compound (activating reagent) having an active group and an amino group using a condensing agent to obtain a reaction product containing activated polyethylene glycol having an active group at its terminal.

[0027] Raw polyethylene glycol (sometimes called "raw PEG") is a polymer synthesized by the polymerization reaction of ethylene glycol or ethylene oxide, and has at least a carboxy group.

[0028] Starting PEG has one or more carboxy groups. Starting PEG with one carboxy group results in monovalent activated PEG, while starting PEG with two carboxy groups results in divalent activated PEG. Furthermore, starting PEG with three or more carboxy groups results in trivalent or higher activated PEG. In other words, the number of carboxy groups in the starting PEG determines the valency of the activated PEG. The number of carboxyl groups in the starting PEG is preferably 8 or less, more preferably 4 or less.

[0029] The starting PEG can be monodisperse or polydisperse. As described in Japanese Patent No. 6,638,970, monodisperse PEG is characterized by a purity of 90% or more and an impurity content of 2% or less, and is a PEG with a single molecular weight without a molecular weight distribution. Polydisperse PEG is a polymer of ethylene glycol, and unlike monodisperse PEG, it has a molecular weight distribution. Polydisperse PEG useful as a starting PEG is a polymer whose polydispersity is preferably 1.2 or less, more preferably 1.1 or less, and most preferably 1.03 or less.

[0030] The molecular weight of the starting PEG is preferably 100 to 100,000 daltons, more preferably 2,000 to 80,000 daltons, even more preferably 5,000 to 50,000 daltons, and still more preferably 10,000 to 40,000 daltons.

[0031] The starting PEG is particularly preferably a PEG having a linear structure, a branched structure or a multi-arm structure as shown in formulas (10) to (16).

[0032] [ka] [ka]

[0033] where Y 1 is an atomic group that protects a functional group that reacts with a functional group present in a biofunctional molecule to form a covalent bond; R 1 is a hydrocarbon group having 1 to 7 carbon atoms or a hydrogen atom; n is an integer from 3 to 2300; m is an integer from 1 to 1200; A 1 , A 2 , A 3 , A 4 are each independently -L 1 -(CH2)m1-, -(CH2)m1-L 1 -, -L 1 -(CH2)m1-L 2 represents -(CH2)m2- or a single bond, and L 1 represents an ether bond, an amide bond, a urethane bond, a secondary amino group, or a single bond, and L 2 represents an ether bond, an amide bond, or a urethane bond, and m1 and m2 each independently represent an integer of 1 to 5; B 1 represents a carbon atom or a nitrogen atom.

[0034] Exemplary solvents used in this production method include water, alcoholic solvents such as acetonitrile, DMSO, toluene, benzene, methanol, and ethanol; hydrocarbon solvents such as hexane and heptane; and chlorine-containing hydrocarbon solvents such as chloroform and dichloromethane. From the viewpoint of the solubility of the starting PEG, water, acetonitrile, DMSO, methanol, ethanol, toluene, chloroform, and dichloromethane are particularly preferred, with acetonitrile, methanol, and ethanol being more preferred. The solvent required in the present invention is one in which the starting PEG and activating reagent are partially or completely dissolved and the condensation reaction proceeds.

[0035] The active group of the activating reagent is an active group that reacts with a functional group present in a biofunctional molecule to form a covalent bond, and is preferably one or more functional groups selected from the group consisting of a maleimide group, an azide group, a biotin group, an active ester group, an acetal-protected aldehyde group, a 9-fluorenylmethyloxycarbonyl-protected amino group, a t-butoxycarbonyl-protected amino group, and a benzyloxycarbonyl-protected amino group.

[0036] The condensing agent is a compound that promotes the condensation reaction between the starting polyethylene glycol having a carboxy group and a compound having an active group and an amino group.

[0037] Suitable condensing agents include uronium-based condensing agents and triazine-based condensing agents. Uronium-based condensing agents are condensing agents consisting of a compound containing a uronium structure, and include HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), HBTU (1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate), TATU (1-[bis(dimethylamino)methylene]- Preferred are 1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide tetrafluoroborate), TBTU (1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide tetrafluoroborate), and COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate), with HATU and COMU being particularly preferred. The triazine-based condensing agent is a condensing agent comprising a compound containing a triazine structure, with DMT-MM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride)) and (4,6-dimethoxy-1,3,5-triazin-2-yl)-(2-octoxy-2-oxoethyl)dimethylammonium trifluoromethanesulfonate being particularly preferred, with DMT-MM being particularly preferred.

[0038] In this process, if the activated group of the activating reagent is a functional group that reacts with amino groups, the condensation reaction can be selectively carried out by using an amine hydrochloride activating reagent and adding a base to a solution containing the starting PEG with a carboxyl group and the activating reagent containing the amine hydrochloride. The base used in the reaction can be either an inorganic base or an organic base. However, the base must be selected based on its solubility in the solvent being used.

[0039] In this case, exemplary inorganic bases include sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, and disodium hydrogen phosphate. Among amine compounds commonly used as organic bases, primary and secondary amines may react with the activated PEG having active groups produced in this reaction, resulting in the production of impurities as by-products, so tertiary amines are more preferred. Exemplary tertiary amine bases include triethylamine, N-methylmorpholine, N-phenylmorpholine, N,N-diisopropylethylamine, pyridine, and 2,6-lutidine, with triethylamine and N,N-diisopropylethylamine being preferred. [Example]

[0040] Example 1 Compound (A) (Mw 4500, 500 mg, 0.11 mmol: starting PEG), compound (B) (33 mg, 0.13 mmol: activating reagent), N,N-diisopropylethylamine (22.7 μL, 0.13 mmol), and DMT-MM (85 mg, 0.31 mmol: condensing agent) were dissolved in acetonitrile (2.5 g) to obtain a solution. This solution was stirred for 2 hours at 40 °C under nitrogen. After the reaction, the solution was diluted with ethyl acetate (100 mL), crystallized with hexane (100 mL), and filtered. The resulting solid was dried to synthesize compound (C) (activated PEG). The terminal activation rate of the resulting product was measured by ion exchange chromatography, and confirmed to be 96.6%.

[0041] Here, the terminal activation rate is defined as follows. Terminal activation rate= (Mass of activated PEG in the product / Mass of activated PEG in the product + Mass of starting PEG)

[0042] To measure the terminal activation rate, 20 mg of formula (C) was dissolved in 2 mL of a labeling reagent, mercaptosuccinic acid aqueous solution (15 mg / mL). The mixture was then stirred at room temperature for 2 hours to allow the reaction to proceed. Next, the entire reaction solution was added to a gel filtration column (PD-10 (Amersham Biosciences)) equilibrated with the eluent used in HPLC analysis. Further eluent was added, and the first high molecular weight fraction to elute was collected in an HPLC vial. Finally, HPLC analysis was performed under the following conditions.

[0043] The measurement conditions for measuring the terminal activation rate by ion exchange chromatography are as follows. (Ion exchange chromatography measurement conditions) HPLC equipment Alliance 6890 (Waters) Separation column ES-502N (Asahipak) Eluent: 20 mM ammonium formate buffer (pH 8.0) Column temperature: 30°C Flow rate 1.0mL / min Sample concentration: 10 mg / mL Injection volume 20μL Detector: Differential refractive index detector (RI) (Waters)

[0044] [ka]

[0045] [ka]

[0046] [ka]

[0047] <Example 2> Compound (D) (MW 2000, 300 mg, 0.15 mmol: starting PEG), compound (B) (89 mg, 0.36 mmol: activating reagent), N,N-diisopropylethylamine (61.2 μL, 0.36 mmol), and DMT-MM (228 mg, 0.82 mmol: condensing agent) were dissolved in acetonitrile (1.5 g) to prepare a solution. This solution was stirred for 6 hours at 40°C under nitrogen. After the reaction, the solution was diluted with ethyl acetate (100 mL), crystallized with hexane (100 mL), and filtered. The resulting solid was dried to synthesize compound (E) (activated PEG). The product was analyzed by ion exchange chromatography under the same conditions as in Example 1, confirming a terminal activation rate of 97.5%. However, the concentration of the aqueous mercaptosuccinic acid solution was 30 mg / mL, and the eluent was 80 mM ammonium formate buffer (pH 8.0).

[0048] [ka]

[0049] [ka]

[0050] Example 3 Compound (F) (Mw 5000, 500 mg, 0.10 mmol: starting PEG), compound (B) (30 mg, 0.12 mmol: activating reagent), N,N-diisopropylethylamine (20.4 μL, 0.12 mmol), and DMT-MM (76 mg, 0.27 mmol: condensing agent) were dissolved in acetonitrile (2.5 g) to form a solution. This solution was stirred for 6 hours at 40°C under nitrogen. After the reaction, the solution was diluted with ethyl acetate (100 mL), crystallized with hexane (100 mL), and filtered. The resulting solid was dried to synthesize compound (G) (activated PEG). The product was analyzed by ion exchange chromatography under the same conditions as in Example 1, confirming that the terminal activation rate was 96.1%.

[0051] [ka]

[0052] [ka]

[0053] Example 4 Compound (H) (Mw 10,000, 500 mg, 0.05 mmol: starting PEG), compound (B) (62 mg, 0.25 mmol: activating reagent), N,N-diisopropylethylamine (42.5 μL, 0.25 mmol), and DMT-MM (152 mg, 0.55 mmol: condensing agent) were dissolved in acetonitrile (3.5 g) to prepare a solution. This solution was stirred for 2 hours at 40°C under nitrogen. After the reaction, the solution was diluted with ethyl acetate (100 mL), crystallized with hexane (100 mL), and filtered. The resulting solid was dried to synthesize compound (I) (activated PEG). The product was analyzed by ion exchange chromatography under the same conditions as in Example 1, confirming a terminal activation rate of 95.1%. However, the concentration of the aqueous mercaptosuccinic acid solution was 30 mg / mL, and the eluent was 80 mM ammonium formate buffer (pH 8.0).

[0054] [ka]

[0055] [ka]

[0056] <Example 5> Compound (J) (Mw 10,000, 500 mg, 0.05 mmol: starting PEG), compound (B) (15 mg, 0.06 mmol: activating reagent), N,N-diisopropylethylamine (10.2 μL, 0.06 mmol), and DMT-MM (38 mg, 0.14 mmol: condensing agent) were dissolved in acetonitrile (2.5 g) to prepare a solution. This solution was stirred for 4 hours at 40°C under nitrogen. After the reaction, the solution was diluted with ethyl acetate (100 mL), crystallized with hexane (100 mL), and filtered. The resulting solid was dried to synthesize compound (K) (activated PEG). The product was analyzed by ion exchange chromatography under the same conditions as in Example 1, confirming a terminal activation rate of 95.1%. However, the concentration of the mercaptosuccinic acid solution was 15 mg / mL, and the eluent was 1.5 mM ammonium formate buffer (pH 8.0).

[0057] [ka]

[0058] [ka]

[0059] Example 6 Compound (L) (Mw 40,000, 1.5 g, 0.038 mmol: starting PEG), compound (B) (11 mg, 0.044 mmol: activating reagent), N,N-diisopropylethylamine (42.5 μL, 0.044 mmol), and DMT-MM (30 mg, 0.11 mmol: condensing agent) were dissolved in acetonitrile (7.5 g) to prepare a solution. This solution was stirred for 4 hours at 40°C under nitrogen. After the reaction, the solution was diluted with ethyl acetate (100 mL), crystallized with hexane (100 mL), and filtered. The resulting solid was dried to synthesize compound (M) (activated PEG). The product was analyzed by ion exchange chromatography under the same conditions as in Example 1, confirming a terminal activation rate of 97.1%. However, the concentration of the aqueous mercaptosuccinic acid solution was 15 mg / mL, and the eluent was 1.5 mM ammonium formate buffer (pH 8.0).

[0060] [ka]

[0061] [ka]

[0062] Example 6 Compound (L) (Mw 40,000, 1.5 g, 0.038 mmol: starting PEG), compound (B) (11 mg, 0.044 mmol: activating reagent), triethylamine (6.3 μL, 0.044 mmol), and DMT-MM (29 mg, 0.11 mmol: condensing agent) were dissolved in acetonitrile (7.5 g) to prepare a solution. This solution was stirred for 6 hours at 40°C under nitrogen. After the reaction, the solution was diluted with ethyl acetate (100 mL), crystallized with hexane (100 mL), and filtered. The resulting solid was dried to synthesize compound (M) (activated PEG). The product was analyzed by ion exchange chromatography under the same conditions as in Example 1, confirming a terminal activation rate of 97.4%. However, the concentration of the aqueous mercaptosuccinic acid solution was 15 mg / mL, and the eluent was 1.5 mM ammonium formate buffer (pH 8.0).

[0063] <Comparative Example 1> The compound synthesized in Example 6 was synthesized by the method described in Patent Document 1, and the results are shown below. Compound (L) (Mw 40,000, 1.5 g, 0.038 mmol: starting PEG), N-hydroxysuccinimide (Mw 115.09, 13 mg, 0.11 mmol), and N,N'-dicyclohexylcarbodiimide (Mw 206.33, 16 mg, 0.08 mmol) were dissolved in acetonitrile (7.5 g) to obtain a solution. This solution was stirred for 3 hours under nitrogen at 40 °C to synthesize compound (N). Subsequently, triethylamine (10.5 μL, 0.08 mmol) and a solution of compound (B) (11 mg, 0.044 mmol) were added to the reaction solution and stirred for 3 hours under nitrogen at 40 °C. After the reaction, the solution was diluted with ethyl acetate (100 mL), crystallized with hexane (100 mL), and filtered. The resulting solid was dried to synthesize compound (M) (activated PEG). The product was measured by ion exchange chromatography under the same conditions as in Example 6, and it was confirmed that the terminal activation rate was 92.6%, which was less than 95%.

[0064] [ka]

Claims

1. A method for producing activated polyethylene glycol having an active group at its terminal, comprising the steps of:

1. A method for producing activated polyethylene glycol having an active group at its terminal, comprising: a step of reacting a raw material polyethylene glycol having a carboxy group with a compound having an active group and an amino group using a condensing agent to obtain a reaction product containing activated polyethylene glycol having an active group at its terminal; wherein the reaction product has a terminal activation rate of 95% by weight or more as analyzed by ion exchange chromatography.

2. 2. The method for producing activated polyethylene glycol having an active group at its terminal according to claim 1, wherein the activated polyethylene glycol is represented by the following formula (1): PEG-(C=O)-NH-R...Formula (1) (In formula (1), PEG is a polyethylene glycol moiety; R is an active group.

3. 3. The method for producing activated polyethylene glycol having an active group at its terminal according to claim 1, wherein the active group is a maleimide group.

4. 3. The method for producing activated polyethylene glycol having an active group at its terminal according to claim 1, wherein the condensing agent is a triazine-based condensing agent or a uronium-based condensing agent.

5. 3. The method for producing activated polyethylene glycol having an active group at its terminal according to claim 1, wherein the molecular weight of the raw material polyethylene glycol is 100 to 100,000 daltons.

6. 3. The method for producing activated polyethylene glycol having an active group at its terminal according to claim 1 or 2, wherein the starting polyethylene glycol has one or more functional groups selected from the group consisting of a hydroxy group, an azide group, a biotin group, a methoxy group, an aldehyde group protected with an acetal, an amino group protected with a 9-fluorenylmethyloxycarbonyl group, an amino group protected with a t-butoxycarbonyl group, and an amino group protected with a benzyloxycarbonyl group.

7. An activated polyethylene glycol represented by the following formula (2):

1. An activated polyethylene glycol having an active group at its terminal, characterized in that the terminal activation rate as analyzed by ion exchange chromatography is 95% by weight or more. PEG-(C=O)-NH-R...Formula (2) (In formula (2), PEG is a polyethylene glycol moiety; R is an active group.

8. 8. The activated polyethylene glycol according to claim 7, wherein the active group is a maleimide group.

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