MX2008008916A - High efficiency method of preparing polyalkylene oxide carboxylic acids - Google Patents

Abstract

A new method of preparing a tertiary alkyl ester of a polyalkylene oxide is provided. The new method employs milder conditions that avoid the back reaction to the starting polyalkylene oxide. The tertiary alkyl ester of a polyalkylene oxide is then reacted with a suitable acid to produce a polyalkylene oxide acid.

Description

HIGH EFFICIENCY METHOD FOR THE PREPARATION OF CARBOXYLIC ACIDS OF POLYALYKYLENE OXIDE FIELD OF THE INVENTION The present invention relates to improved and more efficient methods for the preparation of activated polyalkylene oxide acids and esters of increased purity.

BACKGROUND OF THE INVENTION The conjugation of water-soluble polyalkylene oxide ("PAO") with therapeutic radicals such as proteins and polypeptides is known. See, for example, the U.S. Patent. No. 4,179,337, the description of which is hereby incorporated by reference. The '337 patent discloses that physiologically active polypeptides modified with PEG circulate for prolonged periods in vivo, having immunogenicity and reduced antigenicity. To conjugate PAO with other compounds, the hydroxyl end groups of the polymer must first be converted into reactive functional groups. This process is often referred to as "activation" and the product is called an activated polyalkylene oxide or activated PAO. For the most part, the research has been directed to the union covalent of PAO to amino groups epsilon of proteins, enzymes and polypeptides. The covalent attachment of PAO to amino-lysine groups has been effected by linking groups such as succinoyl-N-hydroxysuccinimide ester, as described by Abuchowski et al., Cancer Biochem. Biophys., 7, 175-86 (1984), aziactones, aryl amidates and cyclic imide thiones. See the patent of E.U.A. Nos. 5,298,643, 5,321, 095, and 5,349,001, for example. The contents of each of the above patents are hereby incorporated herein by reference. The PAOs have also been activated with hydrazine groups in order to couple the polymer to activated carbohydrate groups. In addition, the conversion of terminal hydroxy groups of PAO, such as polyethylene glycol ("PEG"), to carboxylic acids have also been reported. PEG-acids are useful in at least two estimates. First, carboxylic acid derivatives can be used directly to conjugate nucleophiles via available hydroxyl or amino radicals. Second, PEG carboxylic acids can be used as intermediates to form other types of activated polymers. For example, mPEG carboxylic acids can be converted to the succinimidyl ester derivative via N-hydroxysuccinimide and a condensing agent such as carbodiimide diisopropyl. Other activated PAOs can be prepared by reacting the active ester with hydrazine to produce PAO hydrazide derivatives. The Patent of E.U.A. of co-ownership No. 5,605,976 (the '976 patent), incorporated for reference herein, resolves many previous difficulties in the preparation of carboxylic acids of polyalkylene oxide. The '976 patent teaches a process for the preparation of PAO carboxylic acids by reacting a PAO (ie, PAO-OH) with a tertiary alkyl haloacetate in the presence of a base to form a tertiary alkyl ester of PAO, and then the reaction of tertiary alkyl ester PAO with an acid, to form the desired polyalkylene oxide carboxylic acid. At the time when the methods of the '976 patent were developed, a need arose for further improvements. For example, with improvements in NMR instrumentation, it becomes apparent that batches of PEG-acid still contain -5% PEG-OH impurities. In addition, it is determined that levels of contamination with the native PEG-OH tend to increase with the molecular weight of the polymer, and with the use of di-substituted and branched PEG polymers. In addition, the procedures taught by the '976 patent require at least 18 hours of reaction time, as well as reflux and rotary evaporation of the reaction solvent. For at least the foregoing reasons, there remains a long-standing need in the art for faster, and therefore more economical methods for the preparation of PAO carboxylic acids, as well as a need for methods for the production of PAO and intermediate acids. of a much higher purity that are free of any detectable PAO-OH contamination. The present invention addresses these needs.

BRIEF DESCRIPTION OF THE INVENTION In one aspect, the present invention includes methods for the preparation of carboxylic acids of polyalkylene oxide and intermediates related thereto with high purity. The methods include the preparation first of a tertiary alkyl ester of a polyalkylene oxide followed by conversion to the carboxylic acid derivative thereof. The tertiary alkyl ester of the polyalkylene oxide is prepared by the steps of: (a) reacting a polyalkylene oxide with a base, in a first solvent system, for a period of time ranging from about 10 minutes to about 60 minutes; and (b) reacting the product of step (a) in a second solvent system, with a tertiary alkyl haloacetate for a period of less than about 30 minutes. The reaction of steps (a) and (b) are conducted at temperatures of about 10 ° C to about 35 ° C. The resulting tertiary alkyl ester of the polyalkylene oxide is then converted to the corresponding carboxylic acid by reaction of the tertiary alkyl ester of the polyalkylene oxide with an acid to form a carboxylic acid of polyalkylene oxide. This method conveniently provides material with high yield and purity. In this aspect of the invention, preferred polyalkylene oxides include polyethylene glycol and omega-methoxy polyethylene glycol. The Preferred tertiary alkyl haloacetates include t-butyl bromo- or chloro-acetate as well as other tertiary alcohol esters of haloacetic acid. Preferred bases used in the method include, for example, potassium t-butoxide, butyl lithium, and the like. The methods can be carried out using metal t-butoxides in an alcohol such as t-butanol or in other inert solvents such as toluene. The methods of the present invention can be carried out using approximately equimolar ratios of tertiary alkyl haloacetate to polyalkylene oxide. It is preferred, however, that the tertiary alkyl haloacetate be present in an amount that is greater than that of the polyalkylene oxide in a molar base. In further aspects of the invention, methods are provided for the preparation of polyalkylene oxides substituted with alpha and / or omega of high purity such as PEG-hydrazines, PEG-amides and PEG-esters including succinimidyl, methyl and ethyl esters. These aspects include the conversion of the polyalkylene oxide carboxylic acids described above into the desired terminally substituted polymer. In yet a further aspect of the invention, methods for the preparation of biologically active polyalkylene oxide-nucleophile conjugates are described. In this aspect of the invention, the carboxylic acids of polyalkylene oxide react with a biologically active nucleophile such that an ester bond is formed between the polymer and the biologically active nucleophile. For example, in this aspect of the invention, taxol-2 ' PEG-monoesters and 20-camptothecin PEG-esters or diesters using bis-activated PEG can be prepared. One of the main advantages of the present invention is that the resulting polyalkylene oxide carboxylic acids are prepared with high purity, even compared to those made with more recently discovered techniques. In this way, the product contaminants, ie, the starting materials, such as m-PEG-OH, are not in appreciable amounts, that is, they are present in amounts of preferably less than about 2% and preferably less than 1. % and more preferably less than 0.5%. As a result, separation of the desired carboxylic acid derivative from the starting alcohol is not required. In addition, a tedious ion exchange or reverse phase HPLC techniques are not required to provide the desired carboxylic acid derivative.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows NMR spectra corresponding to Example 1. Figure 2 shows NMR spectra corresponding to Comparative Example 4.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides improved processes for preparing carboxylic acids of polyalkylene oxide and synthetic intermediates, such as tertiary alkyl esters of polyalkylene oxides. Broadly, PAO carboxylic acids are prepared by reacting a PAO (ie, a PAO-OH) with a suitable tertiary alkyl haloacetate, in the presence of a suitable base, to form a tertiary alkyl ester PAO, and then the reaction of the alkyl ester tertiary PAO with an acid, to obtain a PAO carboxylic acid. Previous methods employ the reaction under reflux, followed by rotary drying to separate the product from the solvent system. Both of these process steps have now been discovered to allow the tertiary alkyl ester PAO to partially revert to the starting material, ie, back to the PAO-alcohol. This reverse reaction leads to unwanted impurities, such as high molecular weight PEG impurities, and PEG-drug conjugates with different bonds, and results in a slower and less efficient reaction procedure. Thus, contrary to previous methods for the preparation of PAO esters and acids, it has now been unexpectedly discovered that improvements are made when the reactions are conducted in the presence of a base in the lowest practical temperature range, and with the lowest effective base concentrations. The lower limit of the reaction temperature is established by means of the precipitation point of the reagents and product in the selected solvent system. Additional details about the present invention are provided below. 1. Polymeric Substituents and Polyalkylene Oxides The carboxylic acid derivatives of the present invention are preferably prepared from poly (alkylene oxide) (PAO) such as polyethylene glycol which are soluble in water at room temperature. Within this group are the substituted omega polyalkylene oxide derivatives such as methoxypoly (ethylene glycols) (mPEG-OH). Other suitable alkyl-substituted PAO derivatives include those containing mono-terminal d-C4 groups. In one embodiment, non-antigenic straight chain polymers, such as monomethyl PEG homopolymers are preferred. In other embodiments, branched polymers or "U-PEG" are preferably employed, depending on the nature of the agent or medicament to be conjugated to the polymer. Alternate polyalkylene oxides such as other poly (ethylene glycol) homopolymers, other alkyl-poly (ethylene oxide) block copolymers, and poly (alkylene oxide) block copolymers are also useful. The polymers of the present invention have a molecular weight of between about 200 and about 100,000 daltons and preferably between about 2,000 and about 80,000 daltons. Molecular weights of between about 4,000 and about 50,000 daltons, however, are more preferred. For purposes of illustration and not limitation, the waste polyethylene glycol (PEG) can be one of Me-O- (CH2CH20) x-CH2CH2-O-Me-O- (CH2CH20) x-CH2CH2-S- -0-CH2CH2-O- (CH2CH2O) x-CH2CH2-O- , and -S-CH2CH2-0- (CH2CH2O) x-CH2CH2-S-. The degree of polymerization for the polymer represents the number of repeating units in the polymer chain and is dependent on the molecular weight of the polymer. Although substantially non-antigenic polymers, PAO and PEG can vary substantially in weight average molecular weight, preferably, R-i has an average molecular weight weight of about 200 to about 100,000 Daltons in more aspects of the invention. More preferably, the non-antigenic polymer substantially has an average molecular weight weight of from about 2,000 to about 48,000 Daltons. The PEG may also be a "star PEG" or multi-arm PEG such as those described in NOF Corp., 2005, the drug delivery system catalog, the disclosure of which is incorporated herein by reference. Specifically, the PEG can be of the formula: or wherein: j is an integer from about 10 to about 340, preferably providing polymers having a total molecular weight of about 12,000 to about 40,000; and at least 1, up to 3, of the terminal portions of the residue is / are blocked with a methyl or other lower alkyl. Said compounds before conversion to the CO2H derivative include Also contemplated within the scope of the invention, is the formation of other compounds based on PEG that have a terminal CO2H in this, including those branched polymeric residues described in the US patents Nos. 5,605,976, 5,643,575, 5,919,455 and 6.1 13,906 jointly assigned, the description of each one is incorporated here to reference. A representative list of some specific polymers corresponding to formula I include: (2a) , CH- (CH2CH20) z - CH2CH2 OH m-PEG- O- C- 'II H or (2b) and (2c) HO - (CH 2 CH 2 O) x L, -L 2 L 3 (CH 2 CH 2 O) 2 CH 2 CH 2 OH wherein Li, L 2 and L 3 are independently selected functional linkers and L 2 can alternatively be a branched linker group such as an alkyl diamine or waste lysine See, for example, U.S. Patent No. 6,113,906 mentioned above, for example; and z is an integer from 1 to about 120. Bifunctional linking groups are known to those of ordinary experience. In this manner, the L -? - 3 radicals can be independently selected from bifunctional linking groups such as one of the following non-limiting compounds -NH (CH2CH2O) and (CH2) qNR9-, -NH (CH2CH20) and C (O) -, -NH (CR10Rn) qOC (O) -, -C (O) (CR10R ??) qNHC (O) (CRi3Ri2) qNR9-, -C (O) O (CH2) qO-, -C (O) (CR10Rn) qNR9- -C (0) NH (CH2CH20) and (CH2) qNR9-, -C (0) O (CH2CH20) and NR9-, -C (O) NH (CR10Rn) qO-, -C (O) O (CR10Rn) qO-, -C (O) NH (CH2CH2O) y-, wherein R9-? 3 is independently selected from the same group as C-? -6 alkyls, etc., and preferably H or CH3; R-? 4 is selected from the same group as defined by R9-? 3 as well as NO2, haloalkyl of C-? -6 and halogen; q, t and y are each independently selected positive integer numbers such as 1 to about 12. The method of the present invention can also be carried with alternative polymeric substances such as dextrans or other similar non-immunogenic polymers which are capable of being functionalized or activated as mono- or bis-carboxylic acids such as dextran, polyvinyl alcohols, carbohydrate-based polymers, hydroxypropyl-methacrylamide (HPMA), oxides of polyalkylene, and / or its copolymers. See also U.S. Patent. No. 6,153,655 assigned jointly, the contents of which are incorporated herein for reference. The above list is merely illustrative and is not intended to restrict the type of non-antigenic polymers suitable for use herein. 2. Synthesis of tertiary alkyl ester derivatives The methods of the present invention for the preparation of a polyalkylene oxide carboxylic acid first include the preparation of PAO tert.-alkyl ester derivatives, by a process of: (a) reacting an oxide of polyalkylene with a base, in a first solvent system, for a period of time ranging from about 10 minutes to about 60 minutes, or more preferably for a period of time ranging from about 20 minutes to about 40 minutes. (b) reacting the product of step (a) with a tertiary alkyl haloacetate for a period of less than 30 minutes, for example, from about 1 minutes to about 30 minutes, or more preferably for a period of time which varies from about 1 minute to about 15 minutes, in a second solvent system to provide a tertiary alkyl ester of PAO. The solvent system preferably remains the same as that of step (a) to step (b), as does the temperature, which preferably ranges from about 10 ° C to about 35 ° C, or more preferably from about 20 to about 31 ° C. In this way, the first and second solvent systems are usually the same. In order to minimize the inverse reaction of tertiary alkyl ester of PAO, a sufficient dilution of the PAO in the solvent system is necessary. It is preferred that the ratio of PAO to solvent system is from about 1 g of PAO to about 10-25 ml, or more, of solvent system. More preferably, the ratio of PAO to solvent system is about 1 g of PAO to about 15 ml of solvent system. The tertiary alkyl ester reaction product of PAO is precipitated from the solvent system by any method known in the suitable art, such as by the addition of a solvent miscible to the solvent system for which the reaction product is relatively insoluble, and / or by decreasing the temperature of the solvent system. The precipitate is collected, subjected to washing one or more times with a solvent without adequate solubilization, and is further purified, for example, by recrystallization. The solvent system can be any solvent or mixture of suitable solvents known in the art selected to carry out the reactants and reaction products in solution. In certain preferred embodiments, the solvent system is selected to avoid precipitation of reactants and reaction products at low temperatures. As exemplified below, the solvent system comprises toluene, for example, 100% toluene, and the precipitate is ethyl ether. In alternative embodiments, the solvent system optionally comprises toluene ranging in concentration from 99% to 5%, in admixture with one or more additional compatible solvents, such as methylene chloride and / or ethylene chloride. The base is selected from the group consisting of potassium t-butoxide, sodium t-butoxide, butyl lithium, sodium amide, sodium hydride, and combinations thereof. Suitable tertiary alkyl haloattatates are of the formula: wherein X is chlorine, bromine or iodine; R? -3 is independently selected from C-? -8 alkyls, substituted alkyl or branched alkyl, aryls such as phenyl or substituted phenyls. Preferred t-butyl haloacetates include t-butyl bromoacetate, t-butyl chloroacetate and t-butyl iodoacetate. Said t-butyl haloacetates are available, for example, from Sigma Chemical Co., St. Lous, Mo.

Alternatively, trifyl or substituted aryl esters can be used. 3- Synthesis of carboxylic acid derivatives In order to produce a PAO carboxylic acid, the resulting tertiary alkyl ester of PAO is then reacted in a suitable solvent system, in the presence of a suitable acid, to form a carboxylic acid of PAO. The solvent system for the acidification reaction is then exemplified as methylene chloride, although other suitable solvents known in the art are optionally employed such as chloroform or dichloroethane. The acid is any acid known in the art to produce the desired PAO carboxylic acid, for example, trifluoroacetic acid, sulfuric, phosphoric and hydrochloric acids. Trifluoroacetic acid is exemplified and is preferred in some aspects of the invention. The first stage of the preparation of carboxylic acids of PAO of the present invention includes the formation of an intermediate, tert-butyl ester of carboxylic acid of polyalkylene oxide. This intermediate is formed by reacting a PAO with a t-butyl haloacetate as described above in the presence of a base. The preferred base is potassium t-butoxide, although alternatives such as butyl lithium, sodium amide, or sodium hydride may also be used. These bases can be used in the methods described herein as a solid, or more preferably, they are dissolved in a suitable solvent such as t-butanol, benzene, toluene, tetrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide (DMSO), hexane and the like. In order to form the intermediate, the polyalkylene oxide is reacted with the t-butyl haloacetate in an amount which is approximately a molar ratio ranging from about 1: 1 to about 1: 10. Preferably, however, the t-butyl haloacetate is present in a molar ratio of about 1: 8. In addition, the molar ratio of the base to the polyalkylene oxide ranges from about 1: 1 to about 1: 2. Once the t-butyl ester intermediate has been formed, the carboxylic acid derivative of the polyalkylene oxide can be easily supplied in purities exceeding 92%, preferably exceeding 97%, more preferably exceeding 99% and more preferably than They exceed 99.5% purity. In this way, contaminants, particularly with respect to the starting material, for example, mPEG-OH or PEG diol are found in trace amounts only. In preferred aspects of the invention, where mono- or bis-polyethylene glycol carboxylic acids are prepared, the amount of contaminant of starting material found in the final product is less than 1%, as determined by 13 C NMR. In this aspect of the invention, t-butyl ester intermediate is reacted with at least an equivalent amount of an acid such as trifluoroacetic acid in order to provide the terminally substituted carboxylic acid of PAO. Alternatively, dilute hydrochloric acid, that is, about 1 N, sulfuric acid, phosphoric, etc., can be used. The excess amount allows the technician to convert the t-butyl ester intermediate to the desired carboxylic acid derivative and counteract the pH-regulating capacity of PEG or related starting polymeric material. The temperature for the reaction with acid is not critical as for the reaction with the base, and it is generally carried out at room temperature, for example at a temperature ranging from about 18 to about 30 ° C. The desired mono or bis-carboxylic acid derivative is obtained after leaving a sufficient time to ensure the conversion of the intermediate to the final acid derivative, which may be about 2-3 hours. The reaction time, however, will vary depending somewhat on the particular reagents and reaction conditions. After conversion of the intermediate to final desired carboxylic acid, the solvent, ie, methylene chloride, for example, is removed by distillation using techniques known to those of ordinary skill in the art such as rotary evaporation or the like. The resulting residue is recrystallized from methylene chloride / ethyl ether, 2-propanol, dimethylformamide / 2-propanol, toluene (ethyl ether or toluene / hexane to produce the final product) After the completion of the novel method, further purification by conventional methods. it is not required since the methods described herein provide the desired carboxylic acid in very high purity, ie, preferably greater than 99% in this way the technician has significant savings in terms of time, work and materials when the pharmaceutical grade polymer is desired. 4. Additional alpha and / or omeqa terminal radicals As a further aspect of the invention, mono- or bis-carboxylic acid derivatives can be used to form other activated polyalkylene oxides. For example, terminal carboxylic acid groups can be converted to: I. Functional groups capable of reacting with an amino group such as: a) succinimidyl ester; b) carbonyl imidazole; c) aziactones; d) cyclic imides; e) isocyanates or isothiocyanates; or f) aldehydes II. Functional groups capable of reacting with carboxylic acid groups and reactive carbonyl groups such as hydrazine or hydrazide functional groups such as acyl hydrazides, carbazates, semicarbazones, thiocarbazones, etc. The conversion of PEG-C02H to several other leaving groups can be carried out using techniques known to those of ordinary skill in the art without undue experimentation. Reactions Conversion has also been reported in the relevant literature. The terminal activation group may also include a spacer moiety located proximal to the polyalkylene oxide. The spacer radical can be a heteroalkyl, alkoxy, alkyl containing up to 18 carbon atoms or even an additional polymer chain. The spacer radicals can be added using standard synthesis techniques.

. Conversion of carboxylic acid derivatives The polymeric carboxylic acid derivatives also serve as high purity intermediates which can be used to form additional polyalkylene oxide derivatives. For example, high purity amides, hydrazides, other esters and the like can be formed from N-hydroxysuccinimide ester activated with PAO carboxylic acid by condensing with the appropriate reagent (amides, hydrazines, etc.) using standard techniques. Alternatively, the carboxylic acid derivative can be converted to a succinimidyl ester by reacting the carboxylic acid with dicyclohexyl carbodiimide (DCC) or diisopropyl carbodiimide in the presence of base. These consecutive conversion reactions are essentially standard techniques well known to those of ordinary skill in the art. An important aspect of this aspect of the invention is the fact that the intermediate, for example PEG carboxylic acid is essentially pure (99 +%) and thus ensures the technician of a essentially pure final product. 6. - Biologically appropriate active materials for conjugation Nucleophiles conjugated with carboxylic acid derivatives are described as "biologically active". The term, however, is not limited to physiological or pharmacological activities. For example, some nucleophilic conjugates, such as those containing enzymes, are capable of catalyzing reactions in organic solvents. Similarly, some inventive polymer conjugates are also useful as laboratory diagnostics. A key aspect of all conjugates is that at least some portion of the activity associated with the biologically unmodified active material is maintained. According to one aspect of the invention, the CO2H-PEG derivative is reacted with a nucleophile, which has an available hydroxyl radical capable of undergoing a substitution reaction without loss of bioactivity, is reacted with the carboxylic acid derivative of the polymer, such as highly pure PEG-COOH, under conditions sufficient to cause the formation of an ester bond between two substituents. Although not wishing to be bound by any particular aspect in relation to specific conjugation reactions, the prodrugs of the invention are generally prepared by: 1) providing an activated polymer, such as a PEG-acid or PEG- diacid as prepared herein and a biologically active compound having a position therein that will allow a hydrolysable bond to form, and 2) reacting the two substituents in an inert solvent such as methylene chloride, chloroform, toluene or DMF in the presence of a coupling reagent such as 1,3-diisopropyl-carbodiimide (DIPC), 1- (3-dimethyl aminopropyl) 3-ethyl carbodiimide (EDC), any suitable dialkylcarbodiimide, Mukaiyama reagents, (e.g., 2-halo halides -1-alkyl-pyridinium) or cyclic anhydride of propane phosphonic acid (PPACA), etc. which are available, for example from commercial sources such as Sigma Chemical, or synthesized using known techniques and a base such as dimethylaminopyridine (preferred), diisopropylethylamine, pyridine, triethylamine, etc. at a temperature of 0 ° C to 22 ° C (room temperature). An illustrative list of compounds that can be conjugated with the polymers prepared here is shown below Paclitaxel podophyllotoxin camptothecin 7-ethyl-10-hydroxycamptothecin (SN38) lrinotecan (CPT-11) topocetan and any number of small molecules known to those of ordinary experience having an -OH conjugation group with activated polymers described herein. Appropriately protected compounds will be required where more than one OH is available for conjugation using techniques of protection and deprotection recognized in the art. In a further aspect of the invention, when the carboxylic acid has been converted to an alternative terminal functional group, such as a succinimidyl ester, the conjugation of the activated polymer with the desired nucleophile is achieved by reacting the polymer with an active necrylic biologically containing an amino group available. See also, for example, U.S. Patent No. 5,122,614, the disclosure of which is incorporated herein by reference. Similarly, when other linking groups such as those outlined above in section 3 are used, PAO conjugates can be prepared by reacting the desired activated polymer with a biologically active material containing a desired target linking group, i.e. NH2, COOH, etc. It should be understood that the conditions used for the termination of these conjugation reactions are selected to maintain optimal biological activity of the conjugate. The conjugates are biologically active and have numerous therapeutic applications. Mammals in need of treatment that include a biologically active material can be treated by administering an effective amount of a polymer conjugate that contains the desired bioactive material. For example, mammals in need of enzyme replacement therapy or blood factors can be given polymeric conjugates that contain the desired material. The doses of said conjugates are amounts that are sufficient to achieve a desired therapeutic result and will be apparent to those of ordinary experience based on clinical experience. Biologically active nucleophiles of interest of the present invention include, but are not limited to, proteins, peptides, polypeptides, enzymes, organic molecules of natural and synthetic origin such as chemicals medicinal and similar. Enzymes of interest include carbohydrate specific enzymes, proteolytic enzymes, oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Without being limited to particular enzymes, examples of enzymes of interest include asparaginase, arginase, arginine deaminase, adenosine deaminase, superoxide dismutase, endotoxinases, catalases, chymotrypsin, lipases, uricases, adenosine diphosphatase, tyrosinases and bilirubin oxidase. Specific carbohydrate enzymes of interest include glucose oxidases, glucosidases, galactosidases, glucocerebrosidases, glucouronidases, etc. Proteins, polypeptides and peptides of interest include, but are not limited to, hemoglobin, serum proteins such as blood factors including factors VII, VIII, and IX; immunoglobulins, cytokines such as interleukins, a-, β-, and β-interferons, colony stimulation factors including granulocyte colony stimulation factors, platelet-derived growth factors, and phospholipase activation protein (PLAP). Other proteins of general biological or therapeutic interest include insulin, plant proteins such as lectins and ricins, tumor necrosis factors, growth factors, tissue growth factors, TGFα or TGFβ and epidermal growth factors, hormones, somatomedins, erythropoietin, hormones. pigmentary, hypothalamic release factors, anti-diuretic hormones, prolactin, chorionic gonadotropin, follicle stimulating hormone, thyroid stimulating hormone, activator tissue plasminogen, and the like. Immunoglobulins of interest include IgG, IgE, IgM, IgA, IgD and their fragments. Some proteins such as interleukins, interferons and colony stimulation factors also exist in non-glycosylated form, usually as a result of the use of recombinant techniques. Non-glycosylated versions are also among the biologically active nucleophiles of the present invention. The biologically active nucleophiles of the present invention also include any portion of a polypeptide that demonstrates bioactivity in vivo. This includes amino acid sequences, antisense radicals and the like, antibody fragments, single chain antigen binding proteins, see, for example, US Pat. No. 4,946,778, the disclosure of which is incorporated herein by reference, binding molecules including fusions of antibodies or fragments, polyclonal antibodies, monoclonal antibodies, catalytic antibodies, nucleotides and oligonucleotides. The proteins or their portions can be prepared or isolated using techniques known to those of skill in the art such as tissue culture, extraction from animal sources, or by recombinant DNA methodologies. Transgenic sources of proteins, polypeptides, amino acid sequences and the like are also contemplated. Said materials are obtained from transgenic animals, i.e., mice, pigs, cows etc., wherein the proteins are expressed in milk, blood or tissue. Transgenic insects and baculovirus expression systems are also contemplate as sources. In addition, mutant versions of proteins, such as mutant TNF and mutant interferons are also within the scope of the invention. Other proteins of interest are allergen proteins such as ragweed, E antigen, bee venom, tick allergen, 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. Chemotherapeutic molecules such as pharmaceutical chemistries, ie anti-tumor agents, cardiovascular agents, anti-neoplastic agents, anti-infectives, anti-anxiety agents, gastrointestinal agents, central nervous system activation agents, analgesics, fertility agents or contraceptives, anti-inflammatory agents, steroidal agents, anti-urecemic agents, cardiovascular agents, vasodilation agents, vasoconstriction agents and the like are included. In preferred aspects of the invention, the carboxylic acid derivative reacts under conditions which produce an ester linkage between the polymer and the chemotherapeutic radical. Particularly preferred biologically active nucleophiles include taxol, taxanes, taxotere, camptothecin, podophyllotoxin, hemoglobin, glucocerebrosidase, galactosidase, arginase, asparaginase, arginine deaminase and superoxide dismutase. The foregoing is illustrative of biologically active nucleophiles which are suitable for conjugation with the polymers of the invention. HE must understand that those biologically active materials do not specifically mentioned but having suitable nucleophilic groups are also intended and are within the scope of the present invention.

EXAMPLES The following non-limiting examples illustrate certain aspects of the invention. All parts and percentages are by weight unless otherwise defined and all temperatures are in degrees Celsius. t-BuO 30K 30K rn-PEG -OH m-PEG -O > K? toluene Dr t-BuOAc OR CH2CI2 / TFA J! "" m-PEG 3 J0ÜKK.-0- CH ^ 0H EXAMPLE 1 m-PE630k Ester 3 With reference to figure 1, a solution of 31 g (1.03 mol) of m-PEG30k -OH (compound 1), in 600 ml of toluene forms azeotrope with the removal of 130 ml of distillate. The reaction mixture is then cooled to 30 ° C, followed by the addition of 2.1 ml (2.07 mmol) of a 1.0 molar solution of potassium t-butoxide in t-butanol. The resulting mixture is stirred for 10-60 minutes at 30 ° C (production of compound 2), followed by the addition of 1.6 g (8.3 moles) of t-butyl bromoacetate. The resulting cloudy mixture is stirred for 1 hour at 30 ° C. The product (compound 3) is precipitated from the reaction mixture with ether, and collected by filtration and washed with additional ether. This crude product is recrystallized from DMF / IPA 12% to yield 27.8 g (90% yield). The product is confirmed by 13C NMR to be > 99% purity since no peak is found at 60.5 ppm for PEG-OH. See figure 1. 13C NMR (75.4 MHz, CDCI3) d 169.07, 81.01, 71.54-68.62 (PEG), 58.65, 27.82.

EXAMPLE 2 m-PEG30k Acid 4 A solution of 8.7 g (0.13 mmol) of m-PEG30k ester (compound 3) in 90 ml of methylene chloride and 45 ml of TFA is stirred for 3 hours at room temperature, followed by partial removal of the solvent with rotovap, and Precipitation of the product with ether. The solid is collected by filtration, and washed several times with ether, recrystallized from DMF / IPA 12% and dried to yield 8.2 g (94% yield) of product (compound 4). The product is confirmed by 13 C NMR with a purity > 99% 3 C NMR (75.4 MHz, CDCl 3) d 170.90, 71.54-68.18 (PEG), 58.65.

EXAMPLE 3 Preparation of taxol-2 'monoester mPEG30k The 30,000 mPEG acid (3750 mg, 0.125 mmol, compound 4) of Example 2 is azeotroped and then dissolved in 20 mL of anhydrous methylene chloride at room temperature. The above solution is treated with 1,3-diisopropyl-carbodiimide (26 μl, 0.17 mmol), 4-dimethylaminopyridine (32 mg, 0.26 mmol) and taxol (146 mg, 0.17 mmol) at 0 ° C. The reaction solution is heated to room temperature after 30 minutes and maintained at that temperature for 16 hours. The reaction mixture is then washed with 0.1 n HCl, dried and evaporated to yield a white solid which is crystallizes from 2-propanol to provide 3000 mg (80% yield) of pure product 5.

COMPARATIVE EXAMPLE 4 The procedure of Example 1 for making the m-PEG30k ester is followed with a change in a part of the synthesis. After compound 2 is formed, and 1.6 g (8.3 moles) of t-butyl bromoacetate are added, the resulting cloudy mixture 6 is heated to reflux, followed by heat removal, and stirring for 18 hours at room temperature. (production of compound 7). This is to contrast with letting the resulting cloudy mixture stir for 1 hour at 30 ° C. The product is confirmed by 13 C NMR to only have approximately 60% purity as opposed to > 99% purity previously thought because the apparatus used to measure the peak is significantly more sensitive and finds a significant peak at 61.18 ppm for PEG-OH. See figure 2. Observe the impurity found in unknown 200.26-peak. 13 C NMR (75.4 MHz, CDCl 3) d 200.26, 169.00, 80.95, 74.00-68.00 (PEG), 63.00, unknown impurity, 61.18 (starting material- PEG-OH), 58.60, 27.77.

EXAMPLE 5 mPEG .3J0ÜkK RNL 8a aldehyde 9 LINK SCHEME mPEGJ 3U0K RNL 8a m-PEG 3t * ~ 0- CH2 -QK 8 linker m-PEGM) l RNL Sa A solution of 10.0 g (0.33 mmol) of m-PEG30k-acid, 8, 0.15 g (1.0 mmol) of 3,5-dimethyl-4-hydroxybenzaldehyde, and 0.15 g (1.24 mmol) of DMAP in 90 ml of chloride Dry methylene is cooled to 0 ° C in a bath of ice, followed by the addition of 0.19 g (1.0 mmol) of EDC hydrochloride. This mixture is allowed to warm to room temperature overnight. At this time, the solvent is partially removed by rotovap, the product is precipitated with ether, and it is collected and washed with ether. This crude product is recrystallized from DMF / IPA 12% to yield 9.4 g (94% yield). 3 C NMR (75.4 MHz, CDCl 3) d 190.80, 167.26, 152.01, 133.74, 131.02, 129.74, 71.57-67.83 (PEG), 58.68, 16.19.

EXAMPLE 6 mPEG30k RN | _ 8a a | coho | 1 0 A solution of 4.8 g (0.16 mmoles) of mPEG30k RNL 8a aldehyde, 9 in 63 ml of anhydrous methanol is cooled to 15 ° C, followed by the addition of 0.01 g (0.25 mmol) of sodium borohydride. This mixture is stirred at 15-20 ° C for a period of 2 hours, followed by adjustment of the pH to 6.5 with 1 N HCl. The methanol is removed by using a rotary evaporator, and the residue taken up in water. The pH is decreased to 2.0 with 0.5 N HCl, and the product is extracted from the water with methylene chloride. This extract is dried over anhydrous sodium sulfate and filtered after partial removal of the solvent through a rotary evaporator. The product is precipitated with ethyl ether, collected by filtration, and washed with ethyl ether to yield 4.6 g (96% yield) of product. 13 C NMR (75.4 MHz, CDCl 3) d 167.76, 146.25, 138.68, 129.42, 126. 67, 71.55-67.87 (PEG), 63.86, 58.65, 16.11.

EXAMPLE 7 m-PEG30k RNL 8a linker 11 A solution of 1.8 g (0.06 mmol) of m-PEG30k RNL 8a alcohol, 10 in a mixture of 18 ml of methylene chloride and 1.8 ml of DMF is cooled to 0 ° C, followed by the addition of 0.13 g (0.48 mmol). ) of DSC and 0.33 g (0.43 mmol) of pyridine. This mixture is allowed to warm to room temperature overnight. At this time, the solvent is partially removed by rotary evaporator, the product is precipitated with ether, and it is collected and washed with ether. This crude product is recrystallized from DMF / IPA 12% to produce 1.6 (88% yield). 13 C NMR (75.4 MHz, CDCl 3) d 168.14, 167.59, 151.03, 147.76, 130.53, 130.27, 128.50, 72.97-67.83 (PEG), 58.67, 25.17, 16.11. The final product can be used for conjugation to any number of biologically active polypeptides, enzymes, proteins, small molecules, etc. having an available amine or hydroxyl on it for conjugation. Methods for such conjugation reactions have been described, for example, in the U.S. Patent. jointly assigned No. 6,180,095 or Greenwald et al. J. Med. Chem. 1999 Vol. 42, No. 18, 3657-3667, the contents of each of which are incorporated herein for reference.

Claims (23)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for the preparation of a tertiary alkyl ester of a polyalkylene oxide, comprising: (a) the reaction of a polyalkylene oxide with a base, in a first solvent system, for a period of time ranging from about 10 minutes to approximately 60 minutes; and (b) reacting the product of step (a) in a second solvent system, with a tertiary alkyl haloacetate for a period of less than about 30 minutes; wherein the reaction of steps (a) and (b) are conducted at temperatures ranging from about 10 ° C to about 35 ° C.

2. A method for the preparation of a polyalkylene oxide carboxylic acid comprising the reaction of the tertiary alkyl ester of a polyalkylene oxide of claim 1 with an acid to form a carboxylic acid of polyalkylene oxide.

3. The method according to claim 1, further characterized in that the polyalkylene oxide is polyethylene glycol.

4. The method according to claim 1, further characterized in that it additionally comprises the separation of the resulting tertiary alkyl ester from a polyalkylene oxide of the second solvent system.

5. - The method according to claim 3, further characterized in that the polyethylene glycol is omega-methoxypolyethylene glycol.

6. The method according to claim 1, further characterized in that the tertiary alkyl haloacetate comprises the formula: wherein X is chlorine, bromine or iodine; R? -3 is independently selected from the group consisting of C1-8 alkyls, substituted C? -8 alkyls or branched C?? -8 alkyls and aryls.

7. The method according to claim 6, further characterized in that the tertiary alkyl haloacetate is tertiary butyl haloacetate.

8. The method according to claim 1, further characterized in that the t-butyl haloacetate is t-butyl bromoacetate or t-butyl chloroacetate.

9. The method according to claim 1, further characterized in that the molar ratio of the polyalkylene oxide to the base varies from 1: 1 to approximately 1: 2.

10. The method according to claim 1, further characterized in that the base is selected from the group consisting of potassium t-butoxide, butyl lithium, sodium amide, sodium hydride, and combinations thereof.

11. The method according to claim 10, further characterized in that the base is potassium t-butoxide.

12. The method according to claim 2, further characterized in that the acid is selected from the group consisting of trifluoroacetic acid, sulfuric, phosphoric and hydrochloric acids.

13. The method according to claim 2, further characterized in that the acid is trifluoroacetic acid.

14. The method according to claim 1, further characterized in that the reaction of step (a) is conducted for a period of time ranging from about 20 minutes to 40 minutes.

15. The method according to claim 1, further characterized in that the solvent system comprises toluene and the reactions of steps (a) and (b) are carried out at a temperature ranging from about 25 to about 31 °. C.

16. The method according to claim 1, further characterized in that the solvent system comprises toluene and methylene chloride, and the reactions of steps (a) and (b) are carried out at a temperature ranging from about 20 at about 31 ° C.

17. The method according to claim 2, further characterized in that the reaction is conducted at a temperature that it varies from about 18 to about 30 ° C.

18. The method according to claim 1, further characterized in that the ratio of polyalkylene oxide to solvent system is 1 g of polyalkylene oxide to about 15 to about 25 ml of solvent system.

19. The method according to claim 1, further characterized in that the polyalkylene oxide has a molecular weight from about 200 to about 100,000.

20. The method according to claim 19, further characterized in that the polyalkylene oxide has a molecular weight from about 2,000 to about 80,000.

21. The method according to claim 19, further characterized in that the polyalkylene oxide has a molecular weight from about 4,000 to about 50,000.

22. The method according to claim 2, further characterized in that the purity of the produced polyalkylene oxide carboxylic acid is greater than 99% as determined by 13 C NMR.

23. The method according to claim 1, further characterized in that the polyalkylene oxide is either a straight chain or a branched chain.