WO2009084833A2

WO2009084833A2 - A new preparing method of benxyloxypolyethyleneglycol and its derivatives

Info

Publication number: WO2009084833A2
Application number: PCT/KR2008/007501
Authority: WO
Inventors: Pyeong-Uk Park; Seong-Nyun Kim; Boo-Gyu Lim; Ik-Gu Hwang
Original assignee: Id Biochem, Inc.
Priority date: 2007-12-29
Filing date: 2008-12-18
Publication date: 2009-07-09
Also published as: KR20090072889A; KR100922113B1; WO2009084833A3

Abstract

The present invention relates to a preparing method of high purity benzyloxypolyethyleneglycol (Benzyl PEG) and its derivatives. More precisely, according to this method, the commercial, general low purity benzyloxypolyethyleneglycol (Benzyl PEG) is reacted to produce benzyloxypolyethyleneglycol-phthalate (Benzyl PEG-phthalate), the high purity intermediate, and then high purity benzyloxypolyethyleneglycol (Benzyl PEG) having the molecular weight of 350 ~ 100,000, the polydispersity (PD) of up to 1.05 and the purity of at least 99% is prepared from the intermediate.

Description

[DESCRIPTION]

[invention Title]

A NEW PREPARING METHOD OF BENXYLOXYPOLYETHYLENEGLYCOL AND ITS DERIVATIVES

[Technical Field]

The present invention relates to a preparing method of benzyloxypolyethyleneglycol (referred as Benzyl PEG hereinafter) and its derivatives with high purity.

[Background Art]

Polyethylene glycol (referred as PEG hereinafter) , along with natural polymers and synthetic polymers, is one of the most representative hydrophilic polymers forming hydrogen-bond with water molecules effectively, .

Besides, PEG can be dissolved in many organic solvents and does no harm to human. PEG exhibits a fully extended structure in water. So, when it is conjugated with other medicinal compounds (protein, peptide, enzyme, gene, etc) , it can reduce toxicity of a medicinal molecule using steric hindrance, suggesting that it provides protection from immune system. Therefore, it can be applied in many medicines by taking advantage of the capability of extending half-life in human blood plasma. PEG can also improve solubility and effect reducing toxicity of a drug having strong toxicity and low solubility despite its excellent medicinal effect by binding to produce PEG-Drug. Traditionally, benzyloxy polyethylene glycol (referred as benzyl PEG hereinafter) was prepared by a classical, general method which is the reaction of ethylene oxide and benzyl alcohol in the presence of an alkali metal catalyst such as Na and K. However, it is very difficult to produce the product with at least 99% purity by this method, because reaction speed decreases as benzyl PEG becomes polymerized and thus reaction time is extended, suggesting that ethylene oxide is decomposed and thus diol level is increased during the reaction . A preparing method of benzyl PEG with high purity has not been reported in any prior art. Only methoxypolyethylene glycol (mPEG) with similarly high purity has been produced as follows. US 6,455,639 Bl describes that mPEG is synthesized with the purity of at least SS% . According to this patent document, moisture included in the reactor and methanol used for the reaction is minimized before polymerization and side reaction is also inhibited during the reaction to give mPEG with high purity. However, to eliminate moisture, a large amount of organic solvent (Toluene, etc) is required, resulting in the increase of PEG, the byproduct generated by- side reaction. Or, when PDI of PEG is increased, recycling might not possible.

In "Journal of Chromatography, 641(1993) 71-79, Barbara Selisko, Rudolf Ehwald", a method for separation and purification of PEG produced during mPEG polymerization is described, in which mPEG is separated and purified with high purity by gel permeation chromatography (GPC) . However, this method is accomplished only when the difference of molecular weights between mPEG and PEG is big enough. So, when they have similar molecular weights, separation is not possible, indicating commercialization is not possible.

US 5,298,410 describes that high purity mPEG is separated and purified from synthesis of mPEG-succinate in the presence of methylenechloride solvent by using Dowex50*8-100H resin.

The succinate bound to mPEG is easily hydrolyzed during the separation process of mPEG-succinate. So, mPEG-succinate can be decomposed during the separation process and a large amount of organic solvent is also required for the separation, making commercialization difficult.

To overcome the said problems, Korean Patent Application No. 2006-112267 and No. 2005-0078619 describe a preparing method of mPEG with high purity using mPEG-phthalate and mPEG- acetate . However, according to the method, it is very difficult to produce high purity product if the compound having the structure in which aromatic ring is included in the end of polyethyleneglycol has much different physical characteristics from the compound having alkyl group in its structure. So, it is required to establish a new separation and purification method of PEG containing aromatic ring in its end.

[Disclosure] [Technical Problem]

The compound having an aromatic ring in the polyethyleneglycol end has comparatively high solubility in an organic solvent, compared with mPEG. But, when it is separated and purified in aqueous solution, phthalate group in the end is hydrolyzed to increase the level of diol, the by-product, in the final product, indicating that it is very hard to give a product with high purity of at least 99%. Even when ether or MTBE (methyl-tert-butyl ether) which is commercially unfavorable for crystallization is used as a solvent, the compound can be easily crystallized. However, when a single solvent such as IPA (isopropyl alcohol) , heptane, or hexane is used, the produced crystal particles are so minute that filtering is difficult. To overcome the said problems, it is an object of the present invention to provide a preparing method of high purity benzyloxypolyethyleneglycol demonstrating strong organic characteristics in an aqueous solution.

It is another object of the present invention to provide benzyloxypolyethyleneglycol (Benzyl PEG) having high purity of at least 99% produced by the above method.

It is also an object of the present invention to provide a preparing method of high purity benzyloxypolyethyleneglycolphthalate (Benzyl PEG-phthalate) as an intermediate for the production of high purity benzyloxypolyethyleneglycol (Benzyl PEG) .

[Technical Solution]

The present invention provides a preparing method of high purity benzyloxypolyethyleneglycol (referred as Benzyl PEG hereinafter) and its derivatives by reacting and purifying the general Benzyl PEG containing impurities .

That is, commercialized low purity Benzyl PEG is reacted to give Benzyl PEG-phthalate, the high purity intermediate represented by formula 2. Then, the produced Benzyl PEG- phthalate is purified to give high purity Benzyl PEG represented by formula 1. [Formula 1]

[ Formula 2 ]

[Wherein, n is an integer of 3 ~ 2500.] The commercialized Benzyl PEG used in this invention has the purity of up to 98%, or 70-98%, the number-average molecular weight of 350 ~ 100,000 and the polydispersity (PD) of 1.05 ~ 1.5. When the commercialized Benzyl PEG is purified by the method of the present invention, high purity Benzyl PEG, specifically having the purity of at least 99% more particularly 99 ~ 99.999%, the number-average molecular weight of 350 ~ 100,000 and the polydispersity (PD) of up to 1.05 more particularly 1.01 ~ 1.05 and its derivatives can be produced. Hereinafter, the present invention is described in detail. The terms used in this invention are well acknowledged in this field, and the informed arts are not described herein.

The preparing method of high purity Benzyl PEG of the present invention is composed of the steps of preparing Benzyl PEG-phthalate, the intermediate, and purifying thereof.

Precisely, the method of the present invention comprises the steps of (i) preparing high purity Benzyl PEG-phthalate from low purity Benzyl PEG; and (ii) preparing high purity Benzyl PEG from high purity Benzyl PEG-phthalate. In step (i) , the low purity Benzyl PEG is reacted with phthalic anhydride to give Benzyl PEG-phthalate. The produced Benzyl PEG- phthalate is purified by column chromatography to give the product with high purity of at least 99%. In step (ii) , the high purity Benzyl PEG-phthalate prepared in step (i) is hydrolyzed in the presence of an alkali to give high purity Benzyl PEG.

Each step is described in detail hereinafter. (i) Preparation of high purity Benzyl PEG-phthalate

The step of preparing high purity Benzyl PEG-phthalate is composed of the following steps,-

1) dissolving benzyloxypolyethyleneglycol in an acid solution, followed by extraction with methylenechloride and then eliminating an alkali metal catalyst used in the polymerization reaction;

2) adding benzyloxypolyethyleneglycol to toluene and then eliminating some of the toluene by atmospheric distillation; 3) cooling the reactant to room temperature and then adding metal alkoxide in the presence of alcohol;

4) adding phthalic anhydride to the reaction solution, followed by reaction; 5) neutralizing the reactant, followed by extraction with methylenechloride;

6) adding a solvent to the methylenechloride layer, followed by crystallization; and

7) preparing the crystallized product in an aqueous solution, followed by purification by column chromatography.

In this invention, the Benzyl PEG used for the preparation of Benzyl PEG-phthalate is the general, commercial Benzyl PEG having the purity of up to 98%, or 70-98%, the number-average molecular weight of 350 ~ 100,000 and the polydispersity (PD) of 1.05 ~ 1.5.

An alkali metal such as Na or K is used for the polymerization to prepare Benzyl PEG as a metal catalyst. To eliminate the alkali metal catalyst, the Benzyl PEG having the purity of up to 98% is dissolved in an acid solution of pH 1 - 2, followed by extraction with methylenechloride. Before step 1), the content of the alkali metal is generally 0.1-1 weight%. After step 1) , up to 100 ppm of the alkali metal catalyst is detected by ICP-MS (Induced coupled plasma-Mass Spectrometer) or AA (Atomic Absorption) analysis. If the process of eliminating the alkali metal catalyst is not performed, high purity Benzyl PEG having the purity of at least 99% cannot be prepared. This is because if such alkali metal catalyst remains, the reaction of step (i) to produce Benzyl PEG-phthalate is not completed and thereby the content of diol is increased.

In step 2) , Benzyl PEG is added to toluene and then some of the toluene is eliminated by atmospheric distillation.

Before reaction, Benzyl PEG absorbs moisture in the air. So, if moisture is not eliminated, it reacts with metal alkoxide to reduce reaction conversion rate. Therefore, if moisture remains, reactions in the following steps of 3) and 4) cannot be accomplished, resulting in low yield.

In step 5) , the reactant is neutralized, and Benzyl PEG- phthalate is extracted with methylenechloride . In step 6), a crystallization solvent is added to the methylenechloride layer wherein Benzyl PEG-phthalate is dissolved, followed by crystallization of Benzyl PEG-phthalate. Before step 6), it is preferably included to wash the methylenechloride layer containing Benzyl PEG-phthalate.

The crystallization solvent can be one or more compounds selected from the group consisting of ethylether, methyl-t- butylether, heptane, hexane, isopropylalcohol, and acrylonitrile. When ether or MTBE (methyl-tert-butylether) is used as a crystallization solvent, crystallization is performed satisfactorily but commercialization is difficult.

In the meantime, when IPA (isopropyl alcohol) , heptane, hexane, or acrylonitrile is used as a single solvent, crystallized particles are so minute that filtering is difficult. Therefore, the mixed solvent of isopropyl alcohol and heptane is preferred because it is not only preferable for commercialization but also capable of producing Benzyl PEG- phthalate with high yield because of bigger crystallized particles and successful crystallization.

In step 7) , the Benzyl PEG-phthalate prepared in step 6) is prepared in an aqueous solution, followed by purification using column chromatography to increase purity. Herein, anion exchange resin activated with borate buffer (pH 8-11) or ammonium buffer is preferably used as the stationary phase of the column chromatography. Q-sepharose FF (Amersham

Biosciences) and QAE-Toyopearl (TOSHO) are the examples. And

0.1 ~ 50 mM NH₄HCO₃ solution is used as a moving phase. By the column chromatography, Benzyl PEG-phthalate having the purity of at least 99%, more particularly 99-99.999% can be prepared.

And polydispersity (PD) of the product is also reduced to up to 1.05, more particularly to 1.01 ~ 1.05.

(ii) Preparation of high purity Benzyl PEG

In step (ii) , high purity Benzyl PEG is prepared from high purity Benzyl PEG-phthalate prepared in step (i) , which comprises the following steps; a) de-esterifying Benzyl PEG-phthalate in the presence of an alkali; b) extracting the reaction product with methylenechloride; and c) crystallizing Benzyl PEG by adding a solvent to the methylenechloride layer.

Benzyl PEG-phthalate is prepared in step (i) , which has the purity of at least 99%. In between step b) and step c) , it is preferably included the step of washing methylenechloride layer. The crystallization solvent herein can be one or more or a mixed solvent of those selected from the group consisting of ethylether, methyl-t-butylether, heptane, hexane, isopropylalcohol, and acrylonitrile . If the mixed solvent of isopropylalcohol and heptane is used, commercialization is easy and the produced crystallized particles are bigger and well crystallized, indicating Benzyl PEG is obtained with high yield. The benzyloxypolyethyleneglycol (Benzyl PEG) prepared by the above method has high purity of at least 99% and polydispersity (PD) of up to 1.05.

[Best Mode] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples .

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

[Example 1] 1) Preparation of Benzyl PEG (30K) phthalate (Step 1)

40 kg of Benzyl PEG (number-average molecular weight: 3OK, purity: 85%, polydispersity (PD): 1.089) was dissolved in 400 L of distilled water, to which cone. HCl was added to regulate pH as 1.5. Extraction was performed with 100 L of methylenechloride (MC) three times to eliminate the alkali metal catalyst used for polymerization. The methylenechloride was completely concentrated to give Benzyl PEG excluding the alkali metal catalyst. 450 L of toluene was added to 40 kg of the Benzyl PEG excluding the alkali metal catalyst, followed by atmospheric distillation to eliminate 130 L of the toluene.

After cooling to 40°C, a mixed solution prepared by mixing 1.12 kg (5 eq) of potassium tert-butoxide, 10.5 kg of t-butanol and 10 kg of toluene was added thereto. After one hour-stirring, 3 kg (10 eq) of phthalic anhydride was added thereto, followed by reaction for 3 hours. Inside temperature was lowered to up to 20^°C, to which 270 kg of HCl solution (pH 3) was added, followed by stirring for 30 minutes to separate layers. 180 kg of methylenechloride was added thereto twice for extraction, followed by washing once. 12 kg of MgSO₄ was added thereto, followed by stirring for 10-20 minutes. After filtering, methylenechloride was concentrated to 180 L.

Crystallization was performed with the mixed solvent of 1.26 m³ of isopropylalcohol and 0.54 m³ of heptane. As a result, the yield was 95% (38kg) and the conversion rate was >99% (HPLC, PD=I.089) .

¹H-NMR (200MHz, CDCl₃, ppm) : 11.0 (s, -CO₂H) , 7.1-8.3 (m, aromatic) , 4.6 (s, Ar-CH₂-) , 4.17 (s, -CH₂-CO-) , 3.25 ~ 4.0(m, PEG backbone) 2) Purification of pure Benzyl PEG (30K) phthalate (step

2)

38 kg of the Benzyl PEG (30K) phthalate prepared in step 1) was dissolved in distilled water to prepare 2wt% Benzyl PEG (30K) phthalate solution. The 2 wt% Benzyl PEG (30K) phthalate solution (4.7 m³) was loaded in separation column filled with 200 L of anion exchange resin (Q Sepharose FF) activated with ammonium buffer (pH 9) over 9 times by using a metering pump. After washing with distilled water thoroughly, the column was washed again with 0.2 mM NH4HCO₃ by 5 CV (column volume) to give 10 m³ of high purity Benzyl PEG (30K) phthalate solution. After acidification (pH=2-2.5) with cone.

HCl, extraction was performed with 3 m³ of methylenechloride, followed by treating with MgSO₄. Crystallization was performed with 3 m³ of the mixed solvent of isopropylalcohol and heptane (volume ratio of 7:3) to give high purity Benzyl PEG (30K) phthalate. As a result, the yield was 70% (26.6 kg), the purity was 99.9% (HPLC), and the polydispersity (PD) was 1.045. 1H-NMR (200MHz, CDCl₃, ppm) : 11.0 (s, -CO₂H) , 7.1-8.3 (m, aromatic) , 4.6 (s, Ar-CH₂-) , 4.17 (s, -CH₂-CO-) , 3.25 - 4.0 (m, PEG backbone)

3) Preparation of pure Benzyl PEG (30K) (step 3)

100 L of 4% NaOH was added to 26.6 kg of the Benzyl PEG (30K) phthalate purified in step 2) , followed by reflux for 1 hour. After cooling to room temperature, extraction was performed with 50 L of methylenechloride two times, followed by treating with MgSO₄. 550 L of isopropylalcohol and 150 L of heptane were added to the extracted methylenechloride layer, followed by crystallization. As a result, the yield was 95% (25.3 kg), the purity was >99% (HPLC), the polydispersity (PD) was 1.045, and the content of diol was 0.56%.

¹H-NMR (200MHz, CDCl₃, ppm): 7.1-7.3 (m, aromatic) , 4.6(s, Ar-CH₂-) , 3.25-4.0 (m, PEG backbone) [Comparative Example 1]

Benzyl PEG was purified by the same manner as described in Example 1 except that the process of eliminating the alkali metal catalyst in step 1) to produce Benzyl PEG (30K) phthalate was not performed. As a result, the yield was 95%, the purity was 96%, the polydispersity (PD) was 1.045, and the content of diol was 3.93%.

¹H-NMR (200MHz, CDCl₃, ppm) : 7.1-7.3 (m, aromatic) , 4.6 (s, Ar-CH₂-) , 3.25-4.0 (m, PEG backbone) From the results of the above Example and Comparative Example, it was confirmed that the preparing method of high purity Benzyl PEG of the present invention reduced diol content significantly so that it can prepare Benzyl PEG with high purity of at least 99% and at the same time reduced polydispersity (PD) during purification.

[industrial Applicability]

Benzyloxypolyethyleneglycol phthalate (Benzyl PEG- phthalate) prepared by the method of the present invention has high purity of at least 99%. And as described hereinbefore, benzyloxypolyethyleneglycol (Benzyl PEG) prepared by the method of the present invention is stable in aqueous solution phase and can be obtained with high purity of at least 99% using water. Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

Claims

[CLAIMS] [Claim l]

A preparing method of benzyloxypolyethyleneglycol phthalate (Benzyl PEG-phthalate) having the purity of at least 99% comprising the following steps:

1) dissolving benzyloxypolyethyleneglycol in an acid solution, followed by extraction with methylenechloride and then eliminating alkali metal catalyst used in the polymerization reaction,- 2) adding benzyloxypolyethyleneglycol to toluene and then eliminating some of the toluene by atmospheric distillation;

3) cooling the reactant to room temperature and then adding metal alkoxide in the presence of alcohol; 4) adding phthalic anhydride to the reaction solution, followed by reaction;

5) neutralizing the reactant, followed by extraction with methylenechloride ;

[Claim 2] The preparing method of benzyloxypolyethyleneglycol phthalate (Benzyl PEG-phthalate) according to claim 1, wherein the stationary phase of column chromatography is an anion exchange resin and the moving phase is 0.1 ~ 50 mM NH₄-HCO₃ solution.

[Claim 3)

The preparing method of benzyloxypolyethyleneglycol phthalate (Benzyl PEG-phthalate) according to claim 2, wherein the solvent used for crystallization is one or more compounds selected from the group consisting of ethylether, methyl-t- butylether, heptane, hexane, isopropylalcohol, and acrylonitrile .

[Claim 4]

The preparing method of benzyloxypolyethyleneglycol phthalate (Benzyl PEG-phthalate) according to claim 3, wherein the solvent used for crystallization is the mixed solvent of isopropylalcohol and heptane.

[Claim 5]

Benzyloxypolyethyleneglycol phthalate (Benzyl PEG- phthalate) represented by formula 2 prepared by one of the methods of claim 1 - claim 4, which has the purity of at least 99% .

[ Formula 2 ]

(Wherein, n is an integer of 3 - 2500 . )

[Claim 6]

A preparing method of benzyloxypolyethyleneglycol (Benzyl PEG) having the purity of at least 99% and the polydispersity (PD) of up to 1.05, prepared by the following steps: a) de-esterifying Benzyl PEG-phthalate which has the purity of at least 99% and is prepared according to claim 5 in the presence of an alkali; b) extracting the reaction product with methylenechloride; and c) crystallizing Benzyl PEG by adding a solvent to the methylenechloride layer.

[Claim 7] The preparing method of benzyloxypolyethyleneglycol (Benzyl PEG) according to claim 6, wherein the solvent used for crystallization is selected from the group consisting of ethylether, methyl-t-butylether, heptane, hexane, isopropylalcohol, acrylonitrile and a mixture thereof.

[Claim 8]

The preparing method of benzyloxypolyethyleneglycol (Benzyl PEG) according to claim 7, wherein the solvent used for crystallization is the mixed solvent of isopropylalcohol and heptane.