JPH1087815A - Improved method for producing polyalkylene oxide carboxylic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a highly pure product by reacting a t-alkyl polyaklylene oxide carboxylate prepared by reacting a polyalkylene oxide with a t-alkyl haloacetate in the presence of a base with an acid. SOLUTION: A specific amount of a distillate obtained by the azeotropic distillation of polyethylene glycol (hereinbelow referred to as PEG) and toluene is cooled, and a t-butanol solution of κ-t-butoxide is added to the cooled distillatic. After this mixture is agitated for about 1hr at room temperature, a t-alkyl haloacetate represented by the formula (wherein X is Cl, Br or I; and R1 to R3 are each a 1-8C alkyl, an aryl or the like)(e.g. t-butyl bromoacetate) is added thereto, and the resulting mixture is agitated for about 18hr at room temperature. The solvent is removed from the solution by distillation, and the residue is recrystallezed from methylene chloride/ethyl ether to obtain t-butyl PEG carboxylate. This ester is dissolved in methylene chloride, and an acid (e.g. trifluoroacetic acid) and a specified amount of water are added to the solution, and the entire mixture is agitated for about 3hr. The solvent is then removed by distillation, and the residue is recrystallized from a solvent to obtain a polyalkylene oxide carboxylic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing an activated polyalkylene oxide. In particular, the present invention relates to a method for producing a polyalkylene oxide carboxylic acid with high purity.

[0002]

BACKGROUND OF THE INVENTION It is known to link water-soluble polyalkylene oxides to therapeutic moieties such as proteins and polypeptides. See, for example, U.S. Patent No. 4,179,337. This disclosure is incorporated herein by reference. The 4,179,337 patent discloses that prolonged in vivo circulation of bioactive polypeptides modified with PEG reduces immunogenicity and antigenicity. To link the polyalkylene oxide, the hydroxyl end groups of the polymer must first be converted to reactive functional groups. This process is often called "activation" and the product is called "activated polyalkylene oxide". Most work has been directed to covalently linking polyalkylene oxides (PAOs) to the ε-amino groups of proteins, enzymes and polypeptides. Covalent attachment of a polyalkylene oxide to a lysine amino group can be carried out using succinoyl-N-hydroxysuccinimide esters (Abuchowski et al., Canc.
er Biochem. Biophys., 7, 175-86 (1984)), azlactones, arylimidates and cyclic imidothiones. For example, U.S. Patent Nos. 5,298,643, 5,321,095,
And 5,349,001. The contents of each of these patents is incorporated herein by reference. PAOs have also been activated with hydrazine groups to couple the polymer to activated carbohydrate groups.

In addition to the above, PAOs such as PEG
Conversion of the terminal hydroxy group to carboxylic acid has also been reported. PEG carboxylic acids are useful in at least two respects. First, the carboxylic acid derivative can be used directly to link to the nucleophile via the available hydroxyl or amino moiety. Second, PAO carboxylic acids can be used as intermediates to generate other types of activated polymers. For example, m-PEG carboxylic acid can be converted to a succinimidyl ester derivative with a condensing agent such as N-hydroxysuccinimide and diisopropylcarbodiimide. By generating a PAO-hydrazide derivative by reacting the active ester with hydrazine, another activated PAO can be produced.

A major drawback in preparing carboxylic acid derivatives of polyalkylene oxides is the difficulty in obtaining pure products in high yields. For example, Journal of
Controlled Release, 10 (1989) 145-154 and Polymer
Bulletin, 18 (1987), 487-493 describes m-PEG-OH
Describes the synthesis of m-PEG carboxylic acid by converting to a ethyl ester and then hydrolyzing with a base catalyst to form a carboxylic acid. Apparently, this classic approach should be going without difficulty. However,
In practice, this method is at most about 90% pure m-P
It only gives the EG carboxylic acid, the main foreign substance of which is the starting material, namely m-PEG-OH. In addition, it is very difficult to separate the desired PEG carboxylic acid from the starting PEG alcohol. Standard laboratory methods such as fractional crystallization or column chromatography have no effect.
J. Polymer Sci. Polymer Chem. Ed., Vol.22, 341-352
(1984). Long-term column ion exchange method or HP
Although LC methods give up to 95% purity, these methods are not suitable for large scale methods.

[0005] Sometimes pegylated prod
uct)
Preparation with a carboxylic acid results in a final product contaminated with m-PEG-OH. For low molecular weight peptides and organic conjugates, removal of the foreign material is very difficult due to the small difference in molecular weight between the foreign material, ie, m-PEG-OH and the desired polymer conjugate. In addition, when a low-purity polymer-carboxylic acid derivative is used, the yield of the target conjugate necessarily decreases, and at the same time, a long and expensive separation step needs to be performed, thereby increasing the production cost.

[0006]

Accordingly, there is a need for an improved process for producing high purity polyalkylene oxide carboxylic acids. The present invention addresses this need.

[0007]

SUMMARY OF THE INVENTION In one aspect,
The present invention includes a method for making a polyalkylene oxide carboxylic acid. These methods involve reacting a polyalkylene oxide with a t-alkyl haloacetate in the presence of a base,
An intermediate t-alkyl ester of a polyalkylene oxide carboxylic acid is formed, followed by reacting the intermediate t-alkyl ester with an acid such as trifluoroacetic acid to form a polyalkylene oxide carboxylic acid.
This method is advantageous because it provides the material in high yield and high purity.

[0008] Within this aspect of the invention, preferred polyalkylene oxides include polyethylene glycol and ω-methoxy polyethylene glycol. Preferred t-alkyl haloacetates include t-butyl bromoacetate and t-butyl chloroacetate and other t-butyl haloacetates.
Alcohol esters are included. Preferred bases used in this method include, for example, potassium t-butoxide,
Butyllithium and the like. This method can be carried out using an alcohol such as t-butanol or another inert solvent such as toluene using metal t-butoxide. This method of the invention can be carried out using approximately equal molar ratios of t-alkyl haloacetate to polyalkylene oxide. However, it is preferred that the t-alkyl haloacetate be present in a greater amount than the polyalkylene oxide, on a molar basis.

In a further aspect of the present invention, there are provided mono- or bis-PEG-amines, PEG-amides and PEGs.
A process for producing high purity α and / or ω-substituted polyalkylene oxides, such as esters. The PEG-ester includes succinimidyl, methyl and ethyl esters. These aspects include converting the polyalkylene oxide carboxylic acids described above to the desired terminally substituted polymer. In yet a further aspect of the invention, a method for making a polyalkylene oxide-bioactive nucleophile conjugate is disclosed. In this aspect of the invention, the polyalkylene oxide carboxylic acid is reacted with a bioactive nucleophile such that an ester linkage is formed between the polymer and the bioactive nucleophile. For example, in this aspect of the invention, taxol-2'PEG-monoester and 20-camptothecin-PEG-ester can be produced.

One of the major advantages of the present invention is that polyalkylene oxide carboxylic acids are produced in high purity. Thus, foreign materials, ie starting materials such as m-PEG-OH, are not found in large amounts, in other words they are found in amounts of less than 8%, preferably less than 1%, most preferably less than 0.5%. It just happens. as a result,
There is no need to separate the desired carboxylic acid from the starting alcohol. Furthermore, the desired carboxylic acid can be obtained without the need for a long-term column method, ion exchange method or HPLC method.

1. Polymer Substituents and Polyalkylene Oxides The carboxylic acid derivatives of the present invention are preferably made from room temperature water soluble poly (alkylene oxide) (PAO), such as polyethylene glycol. This group includes methoxy poly (ethylene glycol) (m-PEG
OH-substituted polyalkylene oxide derivatives such as -OH). Other suitable alkyl-substituted PAO derivatives include those containing mono-terminal C ₁ -C ₄ groups. Linear non-antigenic polymers such as monomethyl PEG homopolymer are preferred. Other polyalkylene oxides are also useful, such as other poly (ethylene glycol) homopolymers, other alkyl-poly (ethylene oxide) block copolymers, and copolymers of block copolymers of poly (alkylene oxide). These polyalkylene oxides are preferably azeotroped before conversion to the carboxylic acid.

[0012] The polymer of the present invention has a content of about 200 to about 10
000 daltons, preferably about 2,000 to about 8
It has a molecular weight of 000 daltons. However,
Most preferred is a molecular weight of about 4,000 to about 50,000 daltons. The method of the present invention can also be performed with another polymeric material that can be functionalized or activated as a mono- or bis-carboxylic acid, such as dextran or other similar non-immunogenic polymers. The foregoing is merely illustrative and is not intended to limit the types of non-antigenic polymers suitable for use herein.

2. Synthesis of carboxylic acid derivative The method of the present invention for producing a polyalkylene oxide carboxylic acid includes the following steps. i) reacting the polyalkylene oxide with a t-alkyl haloacetate, such as t-butyl haloacetate, in the presence of a base,
Forming a t-butyl ester of a polyalkylene oxide carboxylic acid; and ii) reacting the t-butyl ester with an acid to form a polyalkylene oxide carboxylic acid. An important aspect of the process of the present invention is that the polyalkylene oxide can be easily converted to the α- and / or ω-carboxylic acids via the t-butyl ester intermediate with very high product purity. After converting the polyalkylene oxides of various molecular weights as described in Section 1 above to their respective anions with a strong base, they are reacted with a commercially available t-butyl haloacetate, such as t-butyl bromoacetate, to give the t. -Butyl ester can be obtained in high yield. Thereafter, by using an acid such as trifluoroacetic acid, this intermediate can be rapidly converted to a similar carboxylic acid in very high yield. The method is performed at a temperature from about 0C to about 50C.
Within the scope of this aspect of the invention, temperatures from about 10C to about 40C are preferred, and temperatures from about 20C to about 30C are particularly preferred.

A suitable t-butyl haloacetate is of the formula

Embedded image Wherein X is chlorine, bromine or iodine; R ₁ , R ₂
And R ₃ is independently selected from C _1-8 alkyl, substituted alkyl or branched alkyl, aryl such as phenyl or substituted phenyl. Preferred t-butyl haloacetates include t-butyl bromoacetate, t-butyl chloroacetate, and t-butyl iodoacetate. Such t-butyl haloacetates are available, for example, from Sigma Chemical Company, St. Louis, Mo. Also, trityl or substituted aryl esters may be used.

The first step in the preparation of the polyalkylene oxide carboxylic acids of the present invention involves forming an intermediate, a t-butyl ester of the polyalkylene oxide carboxylic acid. This intermediate is formed by reacting a polyalkylene oxide with t-butyl haloacetate as described above in the presence of a base. A preferred base is potassium t
-Butoxide, but alternatives such as butyllithium, sodium amide and sodium hydride can also be used. Additional alternatives include sodium ethoxide and other strong bases. These bases, even when used as solids in the methods described herein, are more preferably t-butanol, benzene, toluene, tetrahydrofuran (T
HF), dimethylformamide (DMF), dimethylsulfoxide (DMSO), hexane and the like, and may be dissolved in a suitable solvent.

To form this intermediate, the polyalkylene oxide is reacted with t-butyl haloacetate in an approximately equimolar ratio (1: 1). Preferably, however, t-butyl haloacetate is present in an excess of moles such that complete conversion of the polyalkylene oxide is achieved. Thus, the molar ratio of t-butyl haloacetate to polyalkylene oxide is preferably greater than 1: 1. Upon formation of the t-butyl ester intermediate, the carboxylic acid derivative of the polyalkylene oxide is increased to greater than 92%, preferably greater than 97%, more preferably 99%.
Above, and most preferably more than 99.5%. Thus, foreign substances, especially with respect to the starting material, for example m-PEG-OH, are only found in minimal amounts. In a preferred aspect of the present invention,
When producing mono- or bis-polyethylene glycol carboxylic acids, the amount of starting foreign material found in the final product is less than 8% by weight, preferably less than 3% by weight, more preferably less than 1% by weight, and most preferably Is less than 0.5% by weight.

In this aspect of the invention, the t-butyl ester intermediate is reacted with at least an equal amount of an acid such as trifluoroacetic acid, optionally in the presence of a small amount of water, to form a carboxylic acid of the terminally substituted PAO. Obtain the acid. Alternatively, dilute hydrochloric acid, that is, about 1N hydrochloric acid, sulfuric acid, phosphoric acid, or the like may be used. In its excess, the t-butyl ester intermediate can be converted to the desired carboxylic acid derivative before negating the buffering capacity of the PEG or related starting polymer material. Furthermore, the intermediate is converted to the final carboxylic acid derivative, preferably in the presence of a 3-5 fold molar excess of water, based on the weight of the t-butyl ester intermediate. This reaction is performed in an inert solvent such as methylene chloride, chloroform, and ethyl acetate.

The desired mono- or biscarboxylic acid derivative is
Obtained after a time sufficient to ensure that the intermediate is converted to the final carboxylic acid derivative. The time is about 3
Could be time. However, this reaction time will vary somewhat depending on the individual reactants and reaction conditions. Further, the reaction for converting the t-butyl intermediate to the desired carboxylic acid can be carried out at room temperature, i.e., 20-30C, although temperatures from about 0C to about 50C can be used. After conversion of this intermediate to the final desired carboxylic acid, the solvent, ie, for example, methylene chloride, is distilled off by methods known to those skilled in the art, such as concentration on a rotary evaporator or the like.
The residue obtained is recrystallized from methylene chloride / ethyl ether, toluene / ethyl ether or toluene / hexane to give the final product. After completion of this novel process, the desired carboxylic acid is obtained in very high purity, ie preferably above 99%, according to the process described here, so that additional purification by conventional methods is not possible. Not necessary.
Thus, if pharmaceutical grade polymers are desired, there are significant savings in time, effort and raw materials.

3. Additional α and / or ω-Terminal Moieties As a further aspect of the invention, mono- or bis-carboxylic acid derivatives can be used to generate other activated polyalkylene oxides. For example, the terminal carboxylic acid groups can be converted to: I. Functional groups capable of reacting with amino groups, such as: a) succinimidyl esters; b) carbonyl imidazole; c) azlactones; d) cyclic imidothiones; e) isocyanates or isothiocyanates; or f) aldehydes Kind.

II. The following functional groups capable of reacting with carboxylic acid groups and reactive carbonyl groups: a) primary amines; or b) hydrazide functional groups such as hydrazine and acyl hydrazides, carbazates, semicarbazates ( semicarbazate), thiocarbazates
or c) alcohols, ie, those derived from ethyl esters. The terminal activating group can include a spacer moiety located closer to the polyalkylene oxide. The spacer moiety can be a heteroalkyl, alkoxy, alkyl containing up to 18 carbon atoms, or even an additional polymer chain. The spacer moieties can be added using standard synthetic techniques.

4. Conversion of Carboxylic Acid Derivatives The present polymeric carboxylic acid derivatives also serve as high purity intermediates that can be used to generate additional polyalkylene oxide derivatives. For example, α and / or ω-PEG
-Carboxylic acid derivatives can be converted to their respective PEG-amine derivatives by converting the PEG-carboxylic acid to an acid chloride intermediate and then generating the amine by Hoffman degradation. Similarly, by condensing high purity amides, hydrazides, other esters and the like with appropriate reagents (amides, hydrazines, etc.) using standard methods, the P
It can be formed from AO carboxylic acid activated N-hydroxysuccinimide ester. The carboxylic acid derivative can be converted to succinimidyl ester by reacting the carboxylic acid with dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide in the presence of a base. These subsequent conversion reactions are essentially standard techniques well known to those skilled in the art. An important aspect of this feature of the invention is the intermediate,
For example, PEG-carboxylic acid is essentially pure (9
9 +%), which guarantees an essentially pure end product.

5. Biologically Active Substances Suitable for Ligation The nucleophiles that link with the present carboxylic acid derivatives are described as "biologically active". However, the term is not limited to biological or pharmacological activities. For example, some nucleophilic conjugates, such as conjugates containing enzymes, can catalyze the reaction in organic solvents. Similarly, some polymer conjugates of the invention are also useful as laboratory diagnostics. A key feature of all conjugates is that at least some of the activity associated with the unmodified bioactive agent is retained. In accordance with the present invention, a nucleophile having an available hydroxyl moiety that can undergo substitution without compromising biological activity is prepared by converting a carboxylic acid derivative of a polymer such as PEG-COOH into an ester between the two substituents. The reaction is performed under conditions sufficient for bond formation to occur. For example, taxol and taxotere (taxotere)
re) The conjugate is shown below.

[0023]

Embedded image Pro _{Taxol: R 1 = C 6 H 5} ; R 2 = CH 3 CO _{Purotakisotere: R 1 = (CH 3)} 3 CO; R 2 = H the corresponding diester is at least about 2 equivalents of the desired equivalents per PAO dicarboxylic acid By reacting the same biologically active substance. Details regarding the production of these conjugates can be found in the examples. However, for purposes of illustration, generally accepted conditions that are sufficient to allow an ester bond to be formed between the non-protein or non-peptidic material and the polymer, the polymer and the target moiety are suitable solvents. Dissolve in the system, ie methylene chloride, around room temperature and add an acid activator such as DCC or diisopropylcarbodiimide and a base such as pyridine. On the other hand, aqueous systems will usually require a targeting moiety that can be modified by an organic system.

In a further aspect of the invention, when the carboxylic acid is converted to another terminal functional group, such as a succinimidyl ester, the linking between the activated polymer and the desired nucleophile is effected on the polymer. By reacting with a biologically active nucleophile containing an available amino group. See also, for example, US Pat. No. 5,122,614. The contents of the disclosure are incorporated herein by reference. Similarly, when using other linking groups as described in Section 3, the PAO conjugate reacts the desired activated polymer with the desired target linking group, ie, a biologically active material containing NH ₂ , COOH, etc. It can be manufactured by the following. It is to be understood that the conditions used to complete these ligation reactions are chosen to maintain the highest biological activity of the conjugate.

The conjugate has many therapeutic uses because it is biologically active. A mammal in need of treatment involving a bioactive agent can be treated by administering an effective amount of the polymer conjugate containing the desired bioactive agent. For example, a mammal in need of enzyme replacement therapy or a blood factor can be provided with a polymer conjugate containing the desired agent. The dosage of such conjugates is that amount sufficient to achieve the desired therapeutic result, which will be known to one of skill in the art based on clinical experience. Biologically active nucleophiles of interest of the present invention include organic molecules of natural and synthetic origin such as proteins, peptides, polypeptides, enzymes, medicinal chemicals and the like, but are not limited to these. It is not limited to.

Enzymes of interest include carbohydrate-specific enzymes, proteolytic enzymes, oxidoreductases, transferases, hydrolases, lytic enzymes, isomerases and ligases. Non-limiting examples of enzymes of interest include, but are not limited to, asparaginase, arginase, arginine deaminase, adenosine deaminase, superoxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine. Includes diphosphatase, tyrosinase and bilirubin oxidase. Carbohydrate-specific enzymes of interest include glucose oxidase, glucosidase,
Galactosidase, glucocerebrosidase, glucouronidase and the like.

[0027] Proteins, polypeptides and peptides of interest include hemoglobin; Factor VII, VI
Serum proteins such as blood factors including factor II and factor IX; immunoglobulins; interleukins, α-, β
-And gamma-interferons such as cytokines; colony stimulating factors including granulocyte colony stimulating factor; platelet-derived growth factor; and phospholipase activating protein (P
LAP), but is not limited thereto. Other proteins of wide biological or therapeutic interest include plant proteins such as insulin, lectins and ricin, tumor necrosis factor, growth factor, tissue growth factor, TGF
α or TGFβ and epidermal growth factor, hormone, somatomedin, erythropoietin, pigmentary hormone, hypothalamic release factor, antidiuretic hormone, prolactin, chorionic gonadotropin, follicle-stimulating hormone, thyroid-stimulating hormone, tissue plasminogen activation Factors and the like are included. Immunoglobulins of interest include IgG, IgE, IgM, IgA, IgD and fragments thereof.

Some proteins, such as interleukins, interferons and colony stimulating factors, also exist in non-glycosylated form, usually as a result of using recombinant methods. These non-glycosylated forms are also one of the biologically active nucleophiles of the present invention. The biologically active nucleophile of the invention comprises
Includes any portion of the polypeptide that exhibits in vivo biological activity. This includes amino acid sequences, antisense moieties and the like, antibody fragments, single-chain antigen binding proteins (see, eg, US Pat. No. 4,946,778;
The disclosures of which are incorporated herein by reference), binding molecules including antibody or fragment fusions, polyclonal antibodies, monoclonal antibodies, catalytic antibodies, nucleotides and oligonucleotides.

The protein or portion thereof can be produced or isolated by using techniques known to those skilled in the art, such as tissue culture, extraction from animal sources, or recombinant DNA methods. Transgenic sources of proteins, polypeptides, amino acid sequences and the like are also contemplated. Such substances are obtained from transgenic animals, ie, mice, pigs, cows, etc., whose proteins are expressed in milk, blood or tissues. Transgenic insects and baculovirus expression systems are also contemplated as sources. Furthermore, mutant forms of the protein, such as mutant TNF and mutant interferon, are also within the scope of the invention.

Other proteins of interest are allergen proteins such as ragweed, antigen E, bee venom, mite allergens, and the like.
Useful biologically active nucleophiles are not limited to proteins and peptides. Essentially any biologically active compound is included within the scope of the present invention. Pharmaceutical chemicals: antitumor, cardiovascular, antineoplastic, anti-infective, anxiolytic,
Digestive, central nervous system agonist, analgesic, ovulation inducer or contraceptive, anti-inflammatory, steroid, anti-urethic
chemotherapeutic molecules such as ti-urecemic agents, vasodilators, vasoconstrictors and the like. In a preferred aspect of the invention, the carboxylic acid derivative is reacted under conditions that result in an ester linkage between the polymer and the chemotherapeutic moiety. Particularly preferred biologically active nucleophiles include taxol, taxane, taxotere, camptothecin, podophyllotoxin, hemoglobin, glucocerebrosidase, galactosidase, arginase, asparaginase, arginine deaminase and superoxide dismutase. The foregoing describes biologically active nucleophiles that are suitable for linking with the polymers of the present invention. Although not specifically mentioned, biologically active materials having suitable nucleophilic groups are also contemplated and are within the scope of the present invention.

6. Synthesis of Bioactive Conjugates One or more polymeric acids can be linked to a bioactive nucleophile by standard chemical reactions. The link is represented by the following formula. _{m-PAO-O-CH 2} -C (O) - (Y- nucleophile) or (nucleophile _{-Y) -C (O) -CH 2} -O- (PAO) -O- -CH 2 - C (O)-(Y-nucleophile) wherein PAO is a water-soluble polyalkylene oxide, m is methyl, and Y is N (L) (L is H,
C _1-12 alkyl or aryl), O or S. The upper limit of PAO linked to the nucleophile is determined by the number of linking sites available when using PAO carboxylic acids, i.e., the number of hydroxyl moieties, and the degree of polymer connectivity required by those skilled in the art. The degree of connectivity can be varied by varying the reaction stoichiometry using known techniques. Conjugates in which more than one polymer is linked to a nucleophile can be obtained by reacting a stoichiometric excess of the activated polymer with a nucleophile.

The reaction of biologically active nucleophiles, such as enzymes, proteins and polypeptides, with the activated polymer in an optionally buffered aqueous reaction medium, depending on the pH requirements of the nucleophile. it can. Optimal pH for reaction
Is generally about 6.5 to about 8.0 for protein / polypeptide material, preferably about 7.4. The organic / chemotherapeutic agent portion is reacted in a non-aqueous system. The optimum reaction conditions, reaction efficiency, etc. for the stability of the nucleophile may be determined by those skilled in the art. The preferred temperature range is 4 to 37 ° C. The temperature of the reaction medium cannot exceed the temperature at which the nucleophile denatures or decomposes. Preferably, the nucleophile is reacted with an excess of the activated polymer. After the reaction, the conjugate is recovered and purified by diafiltration, column chromatography, a combination thereof, or the like.

[0033]

The following non-limiting examples illustrate certain aspects of the present invention. All parts and percentages are by weight unless otherwise indicated and temperatures are given in degrees Celsius. Material methoxy poly (ethylene glycol) (m-PEG-O
H) MW 5,000 Dalton was obtained from Union Carbide; PEG MW 20,000 and 40,000 Dalton were obtained from Serva. Solvents were obtained from Aldrich Chemical, Milwaukee, WI. The structure of each product prepared in Examples 1 to 11 was confirmed by ¹³ CNMR.

Example 1 t-butyl ester of m-PEG (5,000) carboxylic acid
Azeotropically a solution of m-PEG-OH in 75g of toluene ether 750 ml (0.015 mol) was distilled off 150 ml. The reaction mixture was then cooled to 30 ° C. before the addition of potassium tert-butoxide in tert-butanol.
25 ml (0.025 mol) of a 0 molar solution were added. The resulting mixture was stirred at room temperature for 1 hour and then 5.9 g
(0.030 mol) of t-butyl bromoacetate was added.
The resulting cloudy mixture was heated to reflux before removing the heat and stirring at room temperature for 18 hours. The reaction mixture was filtered through celite and the solvent was distilled off on a rotary evaporator. The residue was recrystallized from methylene chloride / ethyl ether to give 75.9 g (98% yield). The purity of this target product was confirmed to be more than 99% by ¹³ C NMR.
-PEG-OH was confirmed to be less than 1.0%. ¹³ CNM
R attribution _{:( C H 3) 3 C,} 27.6ppm; OCH 3,
58.3 ppm; (CH ₃ ) ₃ C , 80.6 ppm;
O, 168.9 ppm.

Example 2 m-PEG (5,000) carboxylic acid 10.0 g (2 mmol) of m-PEG carboxylic acid t-butyl ester in 100 ml of methylene chloride, 50
A solution of ml of trifluoroacetic acid and 0.1 ml of water was stirred at room temperature for 3 hours. The solvent was then distilled off on a rotary evaporator, and the residue was recrystallized from methylene chloride / ethyl ether to give 9.4 g (95% yield) of the product. The purity of this target product was confirmed to be over 99%, and the content of m-PEG-OH was confirmed to be less than 1.0%. ¹³
Assignment of CNMR: OCH ₃ , 58.3; C ， O, 17
0.7 ppm.

Example 3 t-butyl ether of m-PEG (12,000) carboxylic acid
Azeotropically a solution of m-PEG-OH (12,000) in 50 g (4.2 mmol) in toluene ester 750 ml 1
50 ml were distilled off. The reaction mixture is then added to 30
° C, and potassium t- in t-butanol
6.3 ml (6.3 mmol) of a 1.0 molar solution of butoxide were added. The resulting mixture was stirred at room temperature for 1 hour and then 2.3 g (12.0 mmol) of bromoacetic acid t-
Butyl was added. The resulting cloudy mixture was heated to reflux before removing the heat and stirring at room temperature for 18 hours. The reaction mixture was filtered through celite and the solvent was distilled off on a rotary evaporator. The residue was recrystallized from methylene chloride / ethyl ether to give 41.2 g (82% yield). The purity of this target product was confirmed to be slightly more than 99% by ¹³ C NMR, and m-PEG-OH was confirmed to be less than 1.0%. ¹³ attributable :( C H ₃ of CNMR) ₃ C, 27.5pp
m; OCH ₃ , 58.4 ppm; (CH ₃ ) ₃ C , 80.
7 ppm; C = O, 169.0 ppm.

Example 4 m-PEG (12,000) carboxylic acid 10.0 g (0.83 mmol) of m-PEG (12,000) carboxylic acid t- in 100 ml of methylene chloride.
A solution of the butyl ester, 50 ml of trifluoroacetic acid, and 0.1 ml of water was stirred at room temperature for 3 hours. Then, the solvent was distilled off with a rotary evaporator,
Trituration in ethyl ether gave 9.75 g (98% yield) of the product. The purity of the desired product is ¹³ CNM
R confirmed that it was over 99%, and m-PEG-OH contained 1.
It was confirmed to be less than 0%. ¹³ C NMR assignment: OCH ₃ ,
58.3 ppm; C = O, 170.6 ppm.

Examples 5, 6 and 7 Following the procedures of Examples 3 and 4, m-PEG carboxylic acids MW 2,000, 20,000 and 42,000 were also prepared. Each product was found to be> 99% pure and 89% for MW 2,000; MW
52% for 20,000; and MW 42,000
0 was recovered in 76% yield (from alcohol).

Example 8 Di-t-butyl PEG (40,000) dicarboxylic acid
A solution of 50 g (1.3 mmol) of PEG- (OH) ₂ in 750 ml of ester in toluene was azeotroped to distill 150 ml. The reaction mixture was then cooled to 30 ° C. before the addition of 4 ml (4.0 mmol) of a 1.0 molar solution of potassium t-butoxide in t-butanol. The resulting mixture was stirred at room temperature for 1 hour and then 1.6 g
(8.0 mmol) of t-butyl bromoacetate was added.
The resulting cloudy mixture was heated to reflux before removing the heat and stirring at room temperature for 18 hours. The reaction mixture was filtered through celite and the solvent was distilled off on a rotary evaporator. The residue was recrystallized from methylene chloride / ethyl ether to give 45.2 g (86% yield). The purity of the desired product is above 99% and the starting material is 1.0%
% Was found to be present. ¹³ CNMR assignments of _{:( C H 3) 3 C,} 27.7ppm; (CH 3) 3 C, 8
0.9 ppm; C = O, 169.1 ppm.

Example 9 PEG (40,000) dicarboxylic acid 20.0 g (0.5 mmol) of PEG (40,000) carboxylic acid t-butyl ester in 200 ml of methylene chloride, 100 ml of trifluoroacetic acid, and 0.1
The solution of ml of water was stirred at room temperature for 3 hours. The solvent was then distilled off on a rotary evaporator, and the residue was recrystallized from methylene chloride / ethyl ether to give 16.9 g.
(84% yield). The purity of this target product was confirmed to be over 99%. ¹³ C NMR assignment: C
= O, 170.9 ppm.

EXAMPLES 10 AND 11 Following the procedure of Examples 8 and 9, PEG dicarboxylic acids with MW = 6,000 and 35,000 were also prepared. The yield (from the alcohol form) was 77% for the MW 6,000 derivative and 81% for the MW 35,000 derivative. In all cases, the purity of the final product was determined to be just over 99%.

Example 12 (Comparative Example) In this example, m-PEG-COOH was used in order to demonstrate that the method of the present invention produced a large difference in purity.
A sample of (MW 5,000) was prepared using ethyl ester instead of the t-butyl derivative of the present invention. Purity (%) was confirmed by ¹³ C NMR.

Example 12 (a) Ethyl ester of m-PEG (5,000) carboxylic acid 10.0 g (2.0 mmol) of m-PEG-OH in 150 ml of toluene was azeotroped to 75 ml. Was distilled. After cooling the reaction mixture to 40 ° C.,
0.6 g (5.0 mmol) of potassium t-butoxide was added. The resulting mixture was stirred at 40 ° C. for 2 hours, and then 3.3 g (20 mmol) of ethyl bromoacetate was added. The resulting solution was stirred at 40 ° C. for 18 hours,
The mixture was filtered through celite and the filtrate was evaporated on a rotary evaporator. The residue was recrystallized from 2-propanol to give 7.2 g (71% yield). ¹³ C NMR
Of CH ₃ CH ₂ , 13.12 ppm; CH ₃ O,
_{57.83ppm; CH 3 CH 2, 59.47ppm} ;
C = O, 169.18 ppm. Impurities: m-PEG-C
H ₂ OH, 60.20ppm.

Example 12 (b) m-PEG (5,000) carboxylic acid 3.0 g from Example 12 (a) in 25 ml of water
(0.6 mmol) ethyl ester of m-PEG (5,000) carboxylic acid and 0.2 g (5.0 mmol)
Of sodium hydroxide was stirred at room temperature for 18 hours.
The reaction mixture was then acidified to pH 2.0 with hydrochloric acid and extracted with methylene chloride. The combined extract was dried over anhydrous sodium sulfate and filtered, and then the solvent was distilled off from the filtrate using a rotary evaporator. 2 residue
2.4 g (80% yield) recrystallized from propanol
I got ¹³ C NMR assignment: CH ₃ O, 57.91 pp
m; C = O, 170.30 ppm. Impurity: m-PEG
-CH ₂ OH, 60.50ppm. m-PEG found using standard literature preparations of m-PEG carboxylic acid
The average amount of -OH impurities is 17%. In eight experiments performed using the above comparison method, the amount of impurities was 9% to 31%.
Was in the range. These results were then compared with those obtained from the product of Example 2 (m-PEG-COOH) and are shown below.

[0045]

[Table 1] Comparison table 方法 Method Purity (%) ───エ チ ル Ethyl ester (8 preparations) 69-91 t-butyl ester (Example 2) 99+ ｔ As can be seen from the above, t-butyl A great effect can be obtained by synthesizing the present carboxylic acid derivative using an ester. It can also be seen that further purification is required before the ethyl ester-derived PEG carboxylic acid is considered suitable for ligation with a biologically active nucleophile.

[0046]Example 13 Preparation of 20-camptothecin PEG 5,000 ester M-PEG 5,000 carboxylic acid from Example 2 (40
(0 mg, 0.079 mmol) azeotropically with toluene
And then dissolved in 10 ml of anhydrous methylene chloride at room temperature.
To give 1,3-diisopropylcarbodiimide (1
8.1 μl, 0.119 mmol), 4-dimethylamido
Nopyridine (14.5 mg, 0.119 mmol) and
(S)-(+)-camptothecin (41.32 mg,
0.119 mmol) at 0 ° C. The reaction solution
Warm to room temperature over 30 minutes and keep at that temperature for 16 hours
Was. Next, the reaction solution is washed with 0.1N HCl.
It was dried and evaporated to give a dull white solid.
This was chromatographed (silica gel, MeOH / C
H_TwoCl_Two). The purified material is CH_TwoC
l _Two/ Crystallized from ether.

Embodiment 14 A. Taxol-2 'PEG 5,000 monoester
M-PEG 5,000 carboxylic acid from Preparation Example 2 (62
5 mg, 0.125 mmol) was azeotroped and then 20
Dissolved at room temperature in ml of anhydrous methylene chloride. The solution was treated with 1,3-diisopropylcarbodiimide (26 μl).
1,0.17 mmol), 4-dimethylaminopyridine (32 mg, 0.26 mmol) and taxol (14
(6 mg, 0.17 mmol) at 0 ° C. The reaction solution was warmed to room temperature after 30 minutes and kept at that temperature for 16 hours. The reaction mixture was then washed with 0.1N HCl, dried and evaporated to give a white solid. This was crystallized from 2-propanol to give 585 mg (80
% Yield) of pure product.

Having described what is presently considered to be the preferred embodiment of the present invention, those skilled in the art will recognize that changes and modifications may be made without departing from the spirit of the invention. All such changes and modifications are intended to be claimed as falling within the true scope of the invention.

Claims (27)

[Claims] 1. A method for producing a polyalkylene oxide carboxylic acid comprising the following steps. i) reacting the polyalkylene oxide with a t-alkyl haloacetate in the presence of a base to form a t-alkyl ester of a polyalkylene oxide carboxylic acid; and ii) reacting the t-alkyl ester with an acid; The polyalkylene oxide carboxylic acid is formed. 2. The method of claim 1, wherein said polyalkylene oxide is polyethylene glycol. 3. The method of claim 2, wherein said polyethylene glycol is ω-methoxy polyethylene glycol. 4. The method of claim 1, wherein said t-alkyl haloacetate comprises the formula: Embedded image Wherein X is chlorine, bromine or iodine; R ₁ , R ₂
And R ₃ are C _1-8 alkyl, C _1-8 substituted alkyl or C 3
Independently selected from the group consisting of _1-8 branched alkyl and aryl. ) 5. The method according to claim 1, wherein the t-alkyl haloacetate is
5. The method of claim 4, wherein said is -butyl. 6. The t-butyl haloacetate is substituted with t-bromoacetic acid.
6. The method of claim 5, wherein the compound is -butyl. 7. The method according to claim 1, wherein the t-butyl haloacetate is t-chloroacetate.
6. The method of claim 5, wherein the compound is -butyl. 8. The method of claim 1, wherein said base is potassium t-butoxide. 9. The method according to claim 9, wherein the base is selected from the group consisting of butyllithium, sodium amide and sodium hydride.
The method of claim 1. 10. The method of claim 1, wherein the molar ratio of the t-alkyl haloacetate to the polyalkylene oxide is greater than 1: 1. 11. The method of claim 1, wherein said acid is trifluoroacetic acid. 12. The method according to claim 1, wherein said acid is selected from the group consisting of sulfuric acid, phosphoric acid and hydrochloric acid. 13. The method according to claim 1, wherein the reaction step ii) is carried out at about 0 ° C. to about 50 ° C.
The method of claim 1, wherein the method is performed at a temperature of 14. The method according to claim 11, wherein said reaction step ii) is carried out at about 20 ° C. to about 30 ° C.
14. The method according to claim 13, which is performed at a temperature of ° C. 15. The method of claim 1, wherein said reaction step ii) is performed in the presence of water. 16. The method as claimed in claim 16, wherein the polyalkylene oxide comprises about 20
The method of claim 1, having a molecular weight of 0 to about 100,000. 17. The method of claim 17, wherein the polyalkylene oxide is about 2,
17. A polymer having a molecular weight of from 000 to about 80,000.
The described method. 18. The method of claim 18, wherein the polyalkylene oxide is about 4,
18. The composition of claim 17, having a molecular weight of from 000 to about 50,000.
The described method. 19. The method of claim 1, wherein said polyalkylene oxide carboxylic acid has a purity of greater than 92%. 20. The method of claim 19, wherein the purity of the polyalkylene oxide carboxylic acid is greater than 97%. 21. The method of claim 20, wherein said polyalkylene oxide carboxylic acid has a purity of more than 99%. 22. The method of claim 1, further comprising converting the polyalkylene oxide carboxylic acid to an amine-terminated polyalkylene oxide. 23. The method of claim 1, further comprising converting the polyalkylene oxide carboxylic acid to a succinimidyl ester terminated polyalkylene oxide. 24. The polyalkylene oxide carboxylic acid is reacted with a bioactive nucleophile under conditions sufficient to produce a bioactive conjugate, whereby the polyalkylene oxide is converted to the bioactive nucleophile. 2. The method of claim 1, further comprising attaching to the body via an ester bond. 25. The method of claim 24, wherein said biologically active nucleophile is taxol. 26. The method of claim 24, wherein said biologically active nucleophile is camptothecin. 27. The method of claim 24, wherein said biologically active nucleophile is podophyllotoxin.