Polyoxyethylene derivative, method for producing same, and polyoxyethylene conjugate

Abstract

Provided is a polyoxyethylene derivative represented by formula (1). (P1 represents a residue that has a valence of 1-8 and that is obtained by removing a terminal hydroxyl group from a polyoxyethylene compound having a terminal hydroxyl group, W represents the valence of the residue and is 1-8, L1 represents a single bond or a divalent hydrocarbon group having 1-6 carbon atoms, Z1 represents a divalent binding group that exhibits a low hydrolysis reactivity, L2 represents a single bond or a divalent hydrocarbon group having 1-30 carbon atoms and optionally having a heteroatom, and X1 represents a leaving group.)

Description

Polyoxyethylene derivatives, methods for producing the same, and polyoxyethylene conjugates

[0001] This invention relates to polyoxyethylene derivatives, methods for producing the same, and polyoxyethylene conjugates.

[0002] Protein modification with polyethylene glycol (PEG) (PEGization) is an essential technology in the fields of medicine and biotechnology. PEGization improves the water solubility and blood retention of proteins, and suppresses rapid excretion from the kidneys and liver. Furthermore, PEGization improves protein stability and immunogenicity, contributing to enhanced therapeutic effects. Due to these properties, PEG-modified proteins are widely used in the development of pharmaceuticals such as anticancer drugs, antibodies, and enzyme therapies.

[0003] Traditionally, PEGylation often targets lysine or cysteine ​​residues in proteins. However, multiple residues of these types exist within a protein, leading to non-specific PEGylation. This non-specificity poses a risk of PEG binding to the protein's active or functional sites, potentially resulting in decreased activity and product heterogeneity.

[0004] To address the problem of non-specificity in conventional PEGylation, there is a need for the development of new PEGylation technologies that target specific residues, and selective PEGylation technologies using the introduction of unnatural amino acids are attracting particular attention. In recent years, technologies for introducing unnatural amino acids into proteins have advanced remarkably, and methods for introducing functional groups such as alkynes and azides, which are used in click reactions, into proteins are known. Click reactions, especially the reaction between alkynes and azides (azide-alkyne cycloaddition reactions), are rapid, highly efficient, proceed in high yield even under physiological conditions, and produce few byproducts, making them suitable for the modification of biomolecules. By using unnatural amino acids, alkyne groups can be selectively introduced into proteins and reacted with PEG containing azide groups (azide PEG), enabling selective PEGylation of specific sites on proteins (see, for example, Patent Document 1). This method using the azide-alkyne cycloaddition reaction achieves far higher selectivity and specificity compared to conventional PEGylation targeting lysine or cysteine ​​residues.

[0005] As mentioned above, while azide-alkyne cycloaddition reactions are useful, the toxicity of copper catalysts can be a problem. Residual copper ions can be toxic to biomolecules and therefore need to be removed. However, complete removal of copper is technically difficult and leads to increased manufacturing costs and process complexity.

[0006] To address this problem, metal-free click reactions that proceed in a non-toxic environment have been developed. Representative examples include the strain-promoting azide-alkyne cycloaddition reaction (SPAAC reaction) between cyclic alkynes and azides, the strain-promoting reverse electron-demanding Diels-Alder reaction (SPIEDAC reaction) between trans-cyclooctene (TCO) and tetrazine, and the dipolar cycloaddition reaction between norbornene and nitrile oxides. Among these, the reaction between norbornene and nitrile oxides is expected to be useful in bioconjugation because it is rapid, highly efficient, and the synthesis of the resulting compounds is relatively easy. In addition, techniques for introducing norbornene-containing unnatural amino acids into proteins, similar to azide and alkyne groups, have also been established. This technique allows for the introduction of norbornene groups at specific positions in proteins, enabling precise modification via click reactions. Furthermore, nitrile oxides are known to react rapidly and efficiently with alkyne groups.

[0007] Furthermore, hydrogels made from nitrile oxide precursor PEG have been reported (for example, Non-Patent Document 1).

[0008] Japanese Patent Application Publication No. 2012-031421

[0009] Macromolecular Rapid Communications 2015, 36, 1729.

[0010] The example reported in Non-Patent Document 1 is a hydrogel made of nitrile oxide precursor PEG and does not demonstrate the PEGation of proteins. However, the inventors considered that it might be possible to PEGate proteins having norbornene groups or alkyne groups using the nitrile oxide precursor PEG synthesized by the method described in this report.

[0011] Incidentally, Non-Patent Document 1 describes a method for synthesizing nitrile oxide precursor PEG. However, it has been found that nitrile oxide precursor PEG obtained according to the disclosure in Non-Patent Document 1 has problems with long-term stability. In other words, when it is present in the body for a long period of time, it may adversely affect the function and effects of PEG-converted biomolecules. For example, there is concern that the effects of PEG conversion (such as extension of blood half-life and reduction of immunogenicity) may be lost.

[0012] The object of the present invention is to provide a polyoxyethylene derivative in which a polyoxyethylene moiety and a nitrile oxide precursor moiety are linked, and which can improve long-term stability.

[0013] As a result of diligent research, the inventors have discovered a novel polyoxyethylene derivative in which PEG and a nitrile oxide precursor are linked by a linker that is stable for a long period of time under mild conditions, and a method for producing the same.

[0014] In other words, the present invention relates to the following [1] to [6]. [1] A polyoxyethylene derivative characterized by being represented by the following formula (1). (P in formula (1) １ is a residue having a valency of 1 to 8 obtained by removing the terminal hydroxyl group from a polyoxyethylene compound having a terminal hydroxyl group, W is the valency of the residue, which is 1 to 8, and L １ Z is a single bond or a divalent hydrocarbon group having 1 to 6 carbon atoms. １ L is a divalent bonding group with low hydrolysis reactivity. ２ X is a divalent hydrocarbon group having 1 to 30 carbon atoms, which may have single bonds or heteroatoms. １ (This is a leaving group.)

[0015] (2) Z １ A polyoxyethylene derivative of (1), characterized in that it is an amide bond, a carbamate bond, a urea bond, a bond group containing a secondary amino group, a bond group containing a tertiary amino group, an ether bond, an alkylene group, an arylene group, or a heteroarylene group.

[0016] (3) A method for producing the polyoxyethylene derivative according to (1) or (2), characterized by comprising: an oximation step of reacting a polyoxyethylene compound represented by the following formula (2) with hydroxylamine to obtain an oximated derivative represented by formula (3); and a leaving group introduction step of reacting the oximated derivative with a leaving group introduction reagent to obtain the polyoxyethylene derivative represented by formula (1). (In formula (2) and formula (3), P １ is a residue having a valency of 1 to 8 obtained by removing a terminal hydroxyl group from a polyoxyethylene compound having a terminal hydroxyl group, W is the valency of the residue and is 1 to 8, L １ is a single bond or a divalent hydrocarbon group having 1 to 6 carbon atoms, Z １ is a divalent linking group with low hydrolysis reactivity, L ２ is a single bond or a divalent hydrocarbon group having 1 to 30 carbon atoms which may have a hetero atom.)

[0017] (4) The method for producing a polyoxyethylene derivative according to (3), characterized by comprising, after the leaving group introduction step, a removal step of removing impurities derived from the leaving group introduction reagent by at least one of crystallization and water washing.

[0018] (5) A polyoxyethylene conjugate, characterized by being represented by the following formula (4). (In formula (4), P １ is a residue having a valency of 1 to 8 obtained by removing a terminal hydroxyl group from a polyoxyethylene compound having a terminal hydroxyl group, W is the valency of the residue and is 1 to 8, L １ is a single bond or a divalent hydrocarbon group having 1 to 6 carbon atoms, Z １ is a divalent linking group with low hydrolysis reactivity, L ２ is a single bond or a divalent hydrocarbon group having 1 to 30 carbon atoms which may have a hetero atom, R １ and R ２At least one of the two is a residue obtained by removing the norbornene group or the oxanorbornene group from a biofunctional molecule having a norbornene group, R １ and R ２ If only one of the two is the aforementioned residue, then the other is a hydrogen atom, a carboxyl group, a primary amide group, or a secondary amide group, and J is CH ２ (Or it is an oxygen atom.)

[0019] (6) A polyoxyethylene compound characterized by being represented by the following formula (5). (P in formula (5) １ is a residue having a valency of 1 to 8 obtained by removing the terminal hydroxyl group from a polyoxyethylene compound having a terminal hydroxyl group, W is the valency of the residue, which is 1 to 8, and L １ Z is a single bond or a divalent hydrocarbon group having 1 to 6 carbon atoms. １ L is a divalent bonding group with low hydrolysis reactivity. ２ R is a divalent hydrocarbon group having 1 to 30 carbon atoms, which may have single bonds or heteroatoms. ３ and R ４ At least one of the two is a residue obtained by removing the alkyne group from a biofunctional molecule having an alkyne group, R ３ and R ４ If only one of them is the aforementioned residue, the other is a hydrogen atom or a hydrocarbon group.

[0020] The polyoxyethylene derivative of the present invention has a structure in which a functional group that serves as a nitrile oxide precursor is connected to the polyoxyethylene moiety via a binding group that is less hydrolyzable under mild conditions. As a result, long-term stability can be achieved for compounds (polyoxyethylene conjugates) PEG-modified using the polyoxyethylene derivative of the present invention.

[0021] Furthermore, the manufacturing method of the present invention makes it possible to produce a polyoxyethylene derivative in which the functional group that serves as the nitrile oxide precursor is connected to the polyoxyethylene moiety via a bonding group that is less hydrolyzable under mild conditions. In particular, impurities can be easily removed by removing impurities derived from the leaving group introduction reagent in the leaving group introduction step.

[0022] These are the results of a decomposition test at 37°C in a pH 9.2 borate buffer using the compound of formula (22) described in Example 5 and the compound of formula (27) described in Comparative Example 4.

[0023] The present invention will be described in detail below. (Polyoxyethylene derivatives) The polyoxyethylene derivatives of the present invention have a functional group that serves as a nitrile oxide precursor, which is bonded to the end of the polyoxyethylene moiety by a bonding group with low hydrolysis resistance. Furthermore, the polyoxyethylene conjugate produced using the polyoxyethylene derivatives of the present invention is stable against hydrolysis, and is particularly stable for long periods under mild conditions.

[0024] In this specification, "mild conditions" means a pH of 4.5 to 9.5 and a temperature of 18 to 40°C, "long period" means 24 to 168 hours, and "stable" means that the decomposition rate of the polyoxyethylene conjugate is 2% or less.

[0025] Furthermore, the stability of the polyoxyethylene conjugate under mild conditions can be evaluated as the decomposition rate after standing for 72 hours in a borate buffer solution at pH 9.2 and a temperature of 37.3°C. Here, "decomposition rate" represents the value obtained by subtracting the purity of the polyoxyethylene conjugate after standing for a predetermined time from the initial purity of the polyoxyethylene conjugate. In addition, the long-term stability of the polyoxyethylene conjugate can be evaluated by the decomposition rate of the polyoxyethylene conjugate represented by formula (22), which is obtained by reacting the polyoxyethylene derivative represented by formula (10) with 5-norbornene-2-carboxylic acid.

[0026] The inventors conducted the following studies in the examples. Specifically, the inventors produced a polyoxyethylene derivative represented by formula (26) by linking PEG and a nitrile oxide precursor moiety with an ester bond, referring to the manufacturing method described in Non-Patent Document 1. Then, by further reacting it with 5-norbornene-2-carboxylic acid, a polyoxyethylene conjugate represented by formula (27) was produced. The decomposition rate of the polyoxyethylene conjugate represented by formula (27) after 72 hours at pH 9.2 and a temperature of 37.3°C was approximately 74%.

[0027] In response to this, the present inventors have provided a polyoxyethylene derivative in which a functional group that serves as a nitrile oxide precursor is bonded to the terminal end of the PEG moiety by a binding group with low hydrolysis resistance. They have also found that a polyoxyethylene conjugate synthesized using this polyoxyethylene derivative is stable for a long period of time under mild conditions.

[0028] Specifically, a polyoxyethylene derivative represented by formula (10) was produced in which the PEG moiety and a functional group that serves as a nitrile oxide precursor were linked by an ether bond. This derivative was then reacted with 5-norbornene-2-carboxylic acid to produce a polyoxyethylene conjugate represented by formula (22). It was then found that the decomposition rate of the polyoxyethylene conjugate represented by formula (22) was less than 1% after 72 hours at pH 9.2 and a temperature of 37.3°C.

[0029] In other words, the present invention is a polyoxyethylene derivative characterized by being represented by the following formula (1).

[0030] In formula (1), P １ is a residue having a valency of 1 to 8 obtained by removing the terminal hydroxyl group from a polyoxyethylene compound having a terminal hydroxyl group, W is the valency of the residue, which is 1 to 8, and L １ Z is a single bond or a divalent hydrocarbon group having 1 to 6 carbon atoms. １ L is a divalent bonding group with low hydrolysis reactivity. ２ X is a divalent hydrocarbon group having 1 to 30 carbon atoms, which may have single bonds or heteroatoms. １ It is a leaving group.

[0031] In formula (1), X １ is a leaving group. Preferred examples of the leaving group are a chlorine atom, a bromine atom, an iodine atom, a methanesulfonyloxy group, a p-toluenesulfonyloxy group, a trifluoromethanesulfonyloxy group, and a p-nitrobenzenesulfonyloxy group, more preferably a chlorine atom, a bromine atom, and an iodine atom, and even more preferably a chlorine atom.

[0032] In formula (1), L １ L is a single bond or a divalent hydrocarbon group having 1 to 6 carbon atoms. １ If it is a single bond, P １ and Z １ This indicates that the two groups are directly bonded. Furthermore, the divalent hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic group. The number of carbon atoms in the divalent hydrocarbon group is preferably 5 to 6. L １ Suitable examples include single bonds and phenylene groups, with single bonds being particularly preferred.

[0033] In formula (1), L ２ This is a divalent hydrocarbon group having 1 to 30 carbon atoms, which may have single bonds or heteroatoms. ２ If it is a single bond, the nitrile oxide precursor site and Z １ This indicates that they are directly connected. ２ If the group is a hydrocarbon group, the divalent hydrocarbon group may be an aliphatic hydrocarbon group, an aromatic group, or a heteroaromatic group. Furthermore, the number of carbon atoms in the divalent hydrocarbon group is preferably 2 to 10. In addition, the heteroatom referred to here refers to a substituted atom other than carbon and hydrogen, but halogen atoms, oxygen atoms, and nitrogen atoms are preferred. Furthermore, the heteroatom may be embedded in the skeleton of the hydrocarbon group (for example, a heterocyclic group), or it may substitute for a hydrogen atom of the hydrocarbon group.

[0034] L ２ Preferred examples include single bonds, ethylene groups, and phenylene groups, with phenylene groups being particularly preferred.

[0035] In formula (1), Z １This is a divalent bonding group with low hydrolysis properties. The hydrolysis properties of the polyoxyethylene derivative of the present invention are generally due to the bonding group Z. １ It is determined by the hydrolysis properties of the bond group Z. １ The hydrolytic properties of the polyoxyethylene derivative can be evaluated using the hydrolytic properties of the polyoxyethylene derivative as an indicator. Here, the hydrolytic properties of the polyoxyethylene derivative are defined as being "stable" at (pH 4.5 to 9.5, temperature 18 to 40°C) for (24 to 168 hours), that is, the decomposition rate of the polyoxyethylene conjugate is 2% or less. Preferred examples of the binding group include amide bonds, carbamate bonds, urea bonds, binding groups containing secondary amino groups, binding groups containing tertiary amino groups, ether bonds, alkylene groups, arylene groups, and heteroarylene groups. Methylene groups, ethylene groups, propylene groups, and butylene groups are particularly preferred as alkylene groups, 1,4-phenylene groups are particularly preferred as arylene groups, and 2,5-thiophenylene groups are particularly preferred as heteroarylene groups. Here, a binding group containing a secondary amino group is a divalent binding group containing a linear amine, represented as -NH-, and a binding group containing a tertiary amino group is -N(R ５ )- represents a divalent bonding group containing a linear amine, R ５ This is a monovalent hydrocarbon group, such as a methyl group, ethyl group, or phenyl group. The bonding group is preferably an amide bond, a urea bond, or an ether bond, and more preferably an ether bond.

[0036] In formula (1), P １ This is a polyoxyethylene compound having a dehydroxyl group, that is, a polyoxyethylene compound having W hydroxyl groups (P １ -(OH)w) - residue P from which all terminal hydroxyl groups have been removed １ Furthermore, this polyoxyethylene includes both polyoxyethylene with a molecular weight distribution obtained by polymerization of ethylene oxide, and monodisperse polyoxyethylene obtained by coupling oligooxyethylene of a single molecular weight. １ Preferred examples of W are preferably the following residues, depending on the number of Ws.

[0037] When W = 1, the residues are represented by the following equations (p1), (p2), and (p3).

[0038] A １ Z is a hydrocarbon group having 1 to 24 carbon atoms, an amino group protected by a protecting group, or a group that can react with biofunctional molecules. ２ is a divalent spacer or single bond, n and l are independently 3 to 2,000, s is 0 or 1, and t is 2 or 3.

[0039] When W = 2, the residue is represented by the following formula (p4) or formula (p5). (A １ Z ２ n, l, s, and t are the same as above.

[0040] When W = 3, the residue is represented by the following formula (p6). (n is equivalent to the above.)

[0041] When W = 4, the residues are represented by the following equations (p7), (p8), and (p9). (A １ Z ２ n, l, s, and t are the same as above.

[0042] When W = 5, the residue is represented by the following formula (p10). (n is equivalent to the above.)

[0043] When W = 6, the residue is represented by the following formula (p11). (n is equivalent to the above.)

[0044] When W = 8, the residues are represented by the following equations (p12), (p13), and (p14). (n is equivalent to the above.)

[0045] In equations (p1) to (p14), n and l are -(OCH ２ CH ２ The numbers represented by ) - represent the number of moles of oxyethylene groups added, and each is independently 3 to 2,000, preferably 20 to 1,500, and more preferably 40 to 1,000.

[0046] In equations (p1), (p2), (p3), (p5), and (p9), Z ２ It consists of a polyoxyethylene group and A ２ It is a divalent spacer or single bond connecting Z １ Similarly, there are no particular restrictions as long as it is stable against hydrolysis reactions. Preferred examples of divalent spacers include amide bonds, carbamate bonds, urea bonds, bonding groups containing secondary amino groups, bonding groups containing tertiary amino groups, and ether bonds, with ether bonds being more preferred.

[0047] In equations (p1), (p2), (p3), (p5), and (p9), A ２ This refers to a hydrocarbon group having 1 to 24 carbon atoms, an amino group protected by a protecting group, or a group that can react with bioactive molecules.

[0048] A ２ Specific examples of hydrocarbon groups include methyl group, ethyl group, propyl group, isopropyl group, butyl group, t-butyl group, pentyl group, isopentyl group, hexyl group, heptyl group, 2-ethylhexyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group, heneicosyl group, docosyl group, toicosyl group, tetracosyl group, phenyl group, benzyl group, cresyl group, butylphenyl group, dodecylphenyl group, and trityl group. Preferably, the hydrocarbon group has 1 to 10 carbon atoms, more preferably a methyl group or an ethyl group, and even more preferably a methyl group.

[0049] Also, A ２A protecting group is a component that prevents or inhibits the reaction of a specific chemically reactive functional group in a molecule under certain reaction conditions. The type of protecting group varies depending on the type of chemically reactive functional group being protected, the conditions under which it is used, and the presence of other functional groups or protecting groups in the molecule. Specific examples of protecting groups can be found in many general textbooks, for example, in "Wuts, P. G. M.; Greene, T. W. Protective Groups in Organic Synthesis, 4th ed.; Wiley-Interscience: New York, 2007".

[0050] Examples of amino groups protected by protecting groups in the present invention include amino groups protected by acyl protecting groups or carbamate protecting groups, or azide groups. Specific examples of acyl protecting groups or carbamate protecting groups include trifluoroacetyl groups, 9-fluorenylmethyloxycarbonyl groups, and 2-(trimethylsilyl)ethyloxycarbonyl groups.

[0051] Also, A ２ The groups that can react with the bioactive molecules are epoxy groups, maleidyl groups, vinyl sulfone groups, acrylic groups, sulfonyloxy groups, carboxyl groups, dithiopyridyl groups, or α-haloacetyl groups.

[0052] More specifically, functional groups that can react with amino groups of biofunctional molecules to form covalent bonds are epoxy groups, maleidyl groups, vinylsulfone groups, acrylic groups, sulfonyloxy groups, and carboxyl groups. Functional groups that can react with thiol groups of biofunctional molecules to form covalent bonds are formyl groups, epoxy groups, maleidyl groups, vinylsulfone groups, acrylic groups, sulfonyloxy groups, carboxyl groups, dithiopyridyl groups, and α-haloacetyl groups.

[0053] In a preferred embodiment of this design, the group that can react with the bioactive molecule is the group indicated by group (I) or group (II). Note that "**" represents Z ２ This represents the connection point.

[0054] Group (I): Functional groups capable of forming covalent bonds by reacting with amino groups of biomolecules (a), (b), and (e) below. Group (II): Functional groups capable of forming covalent bonds by reacting with thiol groups of biomolecules (a), (b), (c), (d), and (e) below.

[0055]

[0056] In the formula, Y １ Each of these is independently a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms. Specific examples of hydrocarbon groups include the methyl group, ethyl group, propyl group, isopropyl group, butyl group, t-butyl group, and pentyl group. ２ This is a halogen atom selected from chlorine, bromine, and iodine atoms.

[0057] In formulas (p2), (p5), and (p6), s is 0 or 1, t is 2 or 3, and v is 0 or 2. Preferred embodiments are residues represented by the following formulas (p15) to (p19).

[0058] (In formulas (p15) to (p19), A １ Z ２ n and l are the same as described above.

[0059] (Method for producing polyoxyethylene derivatives) Next, the method for producing polyoxyethylene derivatives according to the present invention will be described. To produce polyoxyethylene derivatives according to the present invention, an oximing step is performed in which a polyoxyethylene compound having a formyl group at the terminal end, represented by formula (2), is reacted with hydroxylamine to obtain an oximed derivative represented by formula (3), and a leaving group introduction step is performed in which a leaving group introduction reagent is reacted after the oximing step to obtain a polyoxyethylene derivative represented by formula (1).

[0060] (In equations (2) and (3), P １ , L １ Z １ , L ２ (W is the same as mentioned above.)

[0061] The oximeation step, which involves the reaction of a polyoxyethylene compound of formula (2) having a formyl group at its terminus with hydroxylamine, is a step to obtain the oximeated derivative of formula (3) through the following reaction steps. A purification step may be provided after the oximeation step.

[0062] (Oximetion reaction)

[0063] As the hydroxylamine represented by equation (6) of the oximation reaction, an aqueous solution of hydroxylamine may be used, or free hydroxylamine may be prepared in the reaction system by adding a base to a hydroxylammonium salt consisting of hydroxylamine and an acid and then used.

[0064] When using an aqueous solution of hydroxylamine, there are no particular restrictions on the proportion of the aqueous solution used, but it is preferable to use an equimolar or greater amount relative to the formyl group of the polyoxyethylene compound having a formyl group represented by formula (2).

[0065] There are no restrictions on the type of acid that constitutes the hydroxylammonium salt, but examples include hydrochloride, sulfate, nitrate, or acetate salts, with hydrochloride or sulfate being preferred. The base used to prepare free hydroxylamine from the hydroxylammonium salt can be either an organic or inorganic base, and there are no particular restrictions, but specific examples include organic bases such as triethylamine, tributylamine, N-methylmorpholine, pyridine, or 4-dimethylaminopyridine, or inorganic bases such as sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, lithium carbonate, and cesium carbonate. There are no particular restrictions on the ratio of hydroxylammonium salt and base used, but it is preferable to use an equimolar or greater ratio relative to the formyl group of the polyoxyethylene derivative having a formyl group represented by formula (2).

[0066] After the oximation step, it is preferable to remove impurities produced as by-products of the reaction, as well as compounds and bases that remain unconsumed during the reaction, in a purification step. The purification method is not particularly limited, but can be performed by extraction, recrystallization, adsorption, reprecipitation, column chromatography, supercritical extraction, etc.

[0067] After the oxime formation step, a leaving group introduction step is performed to convert the oxime group into a nitrile oxide precursor. In this leaving group introduction step, the oxime group of the oxime derivative represented by formula (3) is reacted with a leaving group introduction reagent to convert it into a hydroxymoyl chloride group, hydroxymoyl bromide group, hydroxymoyl iodide group, hydroxymoyl mesylate group, hydroxymoyl tosylate group, hydroxymoyl triflate group, hydroxymoyl-p-nitrobenzenesulfonate group, etc. After the oxime formation reaction step, it is preferable to remove impurities produced as by-products of the reaction or reagents that remain unconsumed in the reaction by purification.

[0068] In the leaving group introduction step, the oximed derivative represented by formula (3) and the reagent for introducing the respective leaving group are reacted in an organic solvent. Preferred organic solvents include chloroform, dichloromethane, carbon tetrachloride, tetrahydrofuran, 4-methyltetrahydropyran, acetonitrile, methyl acetate, ethyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, methanol, ethanol, isopropyl alcohol, t-butyl alcohol, dimethylformamide, or dimethylacetamide. There are no particular restrictions on the proportion of the leaving group introduction reagent used, but equimolar or greater relative to the oxime group of the oximed derivative represented by formula (3) is preferred. The leaving group introduction reagent may be a commercially available product or may be manufactured using a known reaction.

[0069] X １ If the atom is a chlorine atom, for example, a leaving group introduction reagent shown in formulas (r1) to (r11) can be used.

[0070]

[0071] Leaving group X １If the atom is a bromine atom, for example, a leaving group introduction reagent shown in formulas (r12) to (r25) can be used.

[0072]

[0073] Leaving group X １ If the atom is an iodine atom, for example, a leaving group introduction reagent shown in formulas (r26) to (r35) can be used.

[0074]

[0075] Leaving group X １ When the group is a methanesulfonyloxy group, a p-toluenesulfonyloxy group, a trifluoromethanesulfonyloxy group, or a p-nitrobenzenesulfonyloxy group, for example, the leaving group introduction reagents listed in Table 1 can be used.

[0076]

[0077] After the leaving group introduction step, it is preferable to remove impurities produced as by-products of the reaction, as well as compounds that remain unconsumed during the reaction, by purification. For purification, it is preferable to use at least one of crystallization and washing with water. Purification may be performed by crystallization and / or washing with water alone, or in combination. Crystallization and washing with water may be repeated multiple times.

[0078] (Polyoxyethylene conjugate) The polyoxyethylene conjugate in the present invention is the nitrile oxide precursor moiety -C(=N-OH)X of the polyoxyethylene derivative. １ It is obtained by reacting it with a norbornene group, oxanorbornene group, or alkyne group contained in a biofunctional molecule, and is represented by the following formula (4) or (5).

[0079]

[0080]

[0081] (In formulas (4) and (5), P １ is a residue having a valency of 1 to 8 obtained by removing the terminal hydroxyl group from a polyoxyethylene compound having a terminal hydroxyl group, W is the valency of the residue, which is 1 to 8, and L １represents a single bond or a divalent hydrocarbon group having 1 to 6 carbon atoms, and Z １ represents a divalent bonding group with low hydrolysis reactivity, and L ２ represents a single bond or a divalent hydrocarbon group having 1 to 30 carbon atoms that may optionally have at least one of an aromatic ring and a hetero atom. These substituents are the same as those exemplified in the aforementioned formulas (1) to (3).

[0082] Further, in formula (4), R １ and R ２ has at least one that is a residue obtained by removing the norbornene group or oxanorbornene group from a biofunctional molecule having a norbornene group or an oxanorbornene group, and R １ and R ２ when only one of them is the aforementioned residue, the other is a hydrogen atom, a carboxyl group, a primary amide group or a secondary amide group, and J is CH ２ or an oxygen atom. Further, in formula (5), R ３ and R ４ has at least one that is a residue obtained by removing the alkyne group from a biofunctional molecule having an alkyne group, and R ３ and R ４ when only one of them is the aforementioned residue, the other is a hydrogen atom or a hydrocarbon group.

[0083] R １ and R ２ when only one of them is a residue obtained by removing the norbornene group or oxanorbornene group from a biofunctional molecule having a norbornene group or an oxanorbornene group, there is no particular limitation on the other; examples include a hydrogen atom, a carboxyl group, a primary amide group, a secondary amide group and the like. The other is preferably a hydrogen atom, a carboxyl group or a secondary amide group, and more preferably a hydrogen atom.

[0084] R ３ and R ４If only one of the residues is a biofunctional molecule having an alkyne group, the other residue is not particularly limited, but can be, for example, a hydrogen atom or a hydrocarbon group, preferably a hydrogen atom. This hydrocarbon group may be an alkyl group or an alkenyl group, and it is preferable that it has 1 to 3 carbon atoms.

[0085] While there are no particular restrictions on biofunctional molecules, they are substances involved in the diagnosis, cure, alleviation, treatment, or prevention of diseases in humans or other animals. Specifically, this includes low-molecular-weight organic compounds, peptides, proteins, natural or synthetic polymers that form hydrogels through crosslinking, nucleic acids, cells, viruses, etc., that exhibit physiological activity, diagnostic or imaging capabilities with a molecular weight of 900 or less.

[0086] There are no particular restrictions on organic compounds exhibiting physiological activity, diagnostic and imaging capabilities with a molecular weight of 900 or less, but examples include doxorubicin and fludeoxyglucose. １８ Examples include F) iohexol.

[0087] There are no particular restrictions on the natural or synthetic polymers that form hydrogels through crosslinking, but examples include hyaluronic acid, chitosan, polyoxyethylene, and polyvinyl alcohol.

[0088] The polyoxyethylene conjugate of the present invention is obtained by reacting a polyoxyethylene derivative represented by formula (1) with a biofunctional molecule having a norbornene group, an oxanorbornene group, or an alkyne group in an organic solvent, water, or a buffer solution with a pH of 4.0 to 11.0. The obtained polyoxyethylene conjugate may be purified by extraction, recrystallization, adsorption treatment, reprecipitation, dialysis, gel filtration, column chromatography, supercritical fluid extraction, or the like.

[0089] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0090] The results obtained in the following examples １¹H-NMR was obtained using JNM-ECP400 or JNM-ECA600 manufactured by JEOL Datum Ltd. A φ5 mm tube was used for the measurement, and the deuterated solvent was a CDClone solution containing tetramethylsilane (TMS) as an internal standard. ３ The following was used. The molecular weight and terminal functional group purity of the obtained polyoxyethylene derivatives were calculated using liquid chromatography (GPC and HPLC). For liquid chromatography, a Prominence system from Shimadzu Corporation was used for GPC, and an Alliance system from Waters Corporation was used for HPLC.

[0091] (Example 1) (Synthesis of Precursor) A 1 L four-necked flask equipped with a thermometer, nitrogen inlet tube, stirrer, Dean-Stark tube, and condenser was charged with methoxypolyethylene glycol 5000 (100 g: 20.0 mmol) and toluene (250 g), and water was removed azeotropically with toluene. After cooling to 40°C, triethylamine (3.24 g: 32.0 mmol) was charged. After stirring for a while, methanesulfonyl chloride (2.75 g: 24.0 mmol) was added dropwise, ensuring that the internal temperature did not exceed 45°C. After reacting at 40°C for 3 hours, ethanol (1.11 g: 24.1 mmol) was added, stirred for 30 minutes, and then filtered by suction. The filtrate was diluted with ethyl acetate (200 g), and hexane (500 g) was added to perform crystallization, after which crystals were obtained by suction filtration. Furthermore, the obtained crystals were dissolved in ethyl acetate (500g), and then crystallized by adding hexane (500g) to remove low molecular weight impurities. The crystals were washed with hexane (400g), filtered, and then dried under reduced pressure to obtain the compound of formula (7) (precursor).

[0092] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.08(3H, s, -OCH2CH2OSO2CH3), 3.38(3H, s, -(OCH2CH2) n OCH3), 3.52-3.80(m, -(OCH2CH2) n -), 4.38(2H, m, -OCH2CH2OSO2CH3) Number average molecular weight (Mn): 5,361

[0093]

[0094] (Synthesis of the polyoxyethylene compound of formula (2) from the precursor) In a 300 mL four-necked flask equipped with a thermometer, nitrogen inlet, stirrer, and condenser, the compound of formula (7) (20.0 g: 4.00 mmol), 4-hydroxybenzaldehyde (1.95 g: 16.0 mmol), and acetonitrile (100 g) were charged and dissolved. After dissolution, potassium carbonate (5.53 g: 40.0 mmol) was added. The reaction was carried out at 80 °C for 6 hours, then cooled to 25 °C, filtered, and the solvent was removed by vacuum distillation. The residue was dissolved in dichloromethane (160 g), and repeatedly washed with 1 wt% potassium carbonate + 10 wt% saline solution to remove some low molecular weight impurities. The organic layer was then dried over magnesium sulfate, filtered, and the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (160 g), and hexane (120 g) was added for crystallization to remove low molecular weight impurities. The crystals were washed with hexane (80 g), filtered, and then dried under reduced pressure to obtain the compound of formula (8). The compound of formula (8) belongs to the polyoxyethylene compound of formula (2).

[0095] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.38(3H, s, -(OCH2CH2) n OCH3), 3.52-3.90(m, -(OCH2CH2) n -), 4.22(2H, t, -OCH2CH2OAr), 7.02(2H, d, arom.H), 7.83(2H, d, arom.H), 9.89(1H, s, -CHO) Number average molecular weight (Mn): 5,240

[0096]

[0097] (Synthesis of the oxime derivative of formula (3)) Compound (8) (2.00 g: 0.4 mmol) and deionized water (10 g) were charged into a 20 mL screw-cap tube. After dissolution, potassium carbonate (207 mg: 1.50 mmol) and hydroxylamine hydrochloride (96.4 mg: 1.39 mmol) were added. The reaction was carried out at 25°C for 6 hours, and then the extraction process with dichloromethane (8 g) was repeated three times. After the organic layers were combined, the organic layers were washed with 10 wt% saline solution (6 g), and the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (12 g), the solution was dried over magnesium sulfate, filtered, and then crystallized with hexane (12 g). The crystals were washed with hexane (8 g), filtered, and dried under vacuum to obtain the oxime derivative of formula (9). The oxime derivative of formula (9) belongs to formula (3).

[0098] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.38(3H, s, -(OCH2CH2) n OCH3), 3.54-3.89(m, -(OCH2CH2) n -), 4.15(2H, t, -OCH2CH2OAr), 6.89(2H, d, arom.H), 7.49(2H, d, arom.H), 8.03(1H, s, -CH=NOH), 8.05(1H, s, -CH=NOH) Number average molecular weight (Mn): 5,573

[0099]

[0100] (Synthesis of polyoxyethylene derivative of formula (1)) In a 4 mL screw tube, the oximed derivative of formula (9) (500 mg: 0.100 mmol) and acetonitrile (625 mg) were charged. After dissolution, N-chlorosuccinimide (14.9 mg: 0.112 mmol), a leaving group introduction reagent, was added. The reaction was carried out at 15°C for 5.5 hours, after which anisole (5.4 μL: 0.0499 mmol) was added and the quench reaction was carried out at 15°C for 3 hours. After dilution with chloroform (3.5 g), succinimide, an impurity derived from N-chlorosuccinimide, was removed by repeated washing with water using 1 mol / L hydrochloric acid (1.5 g) in which 10 wt% sodium chloride was dissolved. The solvent was removed by vacuum distillation, and the obtained residue was dissolved in ethyl acetate (10 g). After filtration, hexane (10 g) was added and crystallization was performed to remove low molecular weight impurities. The crystals were washed with hexane (10 g), filtered, and then dried under reduced pressure to obtain the polyoxyethylene derivative of formula (10).

[0101] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.38(3H, s, -(OCH2CH2) n OCH3), 3.54-3.88(m, -(OCH2CH2) n -), 4.17(2H, t, -OCH2CH2OAr), 6.92(2H, d, arom.H), 7.76(2H, d, arom.H), 9.35(1H, s, -C(Cl)=NOH)

[0102]

[0103] Here, the compound of formula (10) belongs to the polyoxyethylene derivative of formula (1). In formula (1), P １ This is a monovalent residue obtained by removing the terminal hydroxyl group from a polyoxyethylene compound that has a terminal hydroxyl group, where W is 1 and L １ It is a single bond, Z １ This is an ether bond (a divalent bond group with low hydrolysis reactivity), L ２ This is a C6-carbon, divalent aromatic hydrocarbon group (phenylene group), and X １ It is Cl.

[0104] (Example 2) (Synthesis of Precursor) A 500 mL four-necked flask equipped with a thermometer, nitrogen blowing tube, stirrer, Dean-Stark tube, and condenser was charged with polyethylene glycol 3400 (100 g: 29.4 mmol), toluene (250 g), and 2,6-di-tert-butyl-p-cresol (100 mg), and water was removed azeotropically with toluene. After cooling to 40°C, triethylamine (8.92 g: 88.2 mmol) was charged. After stirring for a while, methanesulfonyl chloride (8.09 g: 70.6 mmol) was added dropwise, ensuring that the internal temperature did not exceed 45°C. After reacting at 40°C for 3 hours, the mixture was diluted with chloroform (700 g) containing 2,6-di-tert-butyl-p-cresol (70 mg), and washed with a mixed aqueous solution of 6 mol / L hydrochloric acid (15.6 g) and 5% saline solution (300 g). Low molecular weight impurities were removed by repeated washing with 5% saline solution (300 g), and the mixture was diluted with toluene (300 g), after which the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (650 g), filtered, and the filtrate was added to hexane (500 g) for reprecipitation. The crystals were washed with hexane (400 g), filtered, and dried under vacuum to obtain the compound of formula (11) (precursor).

[0105] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.09(6H, s, -OCH2CH2OSO2CH3), 3.52-3.81(m, -(OCH2CH2) n -), 4.38(4H, m, -OCH2CH2OSO2CH3) Number average molecular weight (Mn): 3,551

[0106]

[0107] (Synthesis of the polyoxyethylene compound of formula (2)) In a 2 L four-necked flask equipped with a thermometer, nitrogen inlet, stirrer, and condenser, the precursor of formula (11) (85.2 g: 25.1 mmol), 4-hydroxybenzaldehyde (12.2 g: 99.9 mmol), and acetonitrile (850 g) were charged and dissolved. After dissolution, potassium carbonate (15.2 g: 110 mmol) was added. The reaction was carried out at 80°C for 6 hours, then cooled to 45°C, filtered, and the solvent was removed by vacuum distillation. The residue was dissolved in dichloromethane (850 g) and repeatedly washed with a 10 wt% saline solution (340 g) adjusted to pH 12 with sodium hydroxide to remove some low molecular weight impurities, after which the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (425 g), filtered, and then crystallized by adding hexane (425 g) to remove low molecular weight impurities. The crystals were washed with hexane (343 g), filtered, and then dried under reduced pressure to obtain the compound of formula (12). The compound of formula (12) belongs to the polyoxyethylene compound of formula (2).

[0108] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.52-3.90(m, -(OCH2CH2) n -), 4.22(4H, t, -OCH2CH2OAr), 7.03(4H, d, arom.H), 7.83(4H, d, arom.H), 9.89(2H, s, -CHO) Number average molecular weight (Mn): 3,533

[0109]

[0110] (Synthesis of the oxime derivative of formula (3)) In a 1 L four-necked flask equipped with a thermometer, nitrogen blowing tube, and stirrer, the precursor of formula (12) (70.0 g: 20.6 mmol) and deionized water (350 g) were charged. After dissolution, potassium carbonate (21.4 g: 155 mmol) and hydroxylamine hydrochloride (7.16 g, 103 mmol) were added. The reaction was carried out at 25 °C for 6 hours, and then the extraction process with dichloromethane (280 g) was repeated twice. After the organic layers were combined, the organic layers were washed with 10 wt% saline solution (210 g), and the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (420 g), the solution was dried over magnesium sulfate, filtered, and then crystallized by adding hexane (420 g). The crystals were washed with hexane (280 g), filtered, and dried under vacuum to obtain the compound of formula (13). The compound of formula (13) belongs to the oxime derivative of formula (3).

[0111] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.45-3.88(m, -(OCH2CH2) n -), 4.15(4H, t, -OCH2CH2OAr), 6.91(4H, d, arom.H), 7.49(4H, d, arom.H), 8.05(2H, s, -CH=NOH), 8.11(2H, s, -CH=NOH) Number average molecular weight (Mn): 3,777

[0112]

[0113] (Synthesis of polyoxyethylene derivative of formula (1)) In a 1 L four-necked flask equipped with a thermometer, nitrogen blowing tube, and stirrer, the oximed derivative of formula (13) (45.1 g: 13.3 mmol) and N,N-dimethylformamide (57.0 g) were charged. After dissolution, N-chlorosuccinimide (4.24 g: 31.8 mmol), a leaving group introduction reagent, was added. After reacting at 25°C for 1 hour, anisole (1.43 mL: 13.2 mmol) was added, and the quench reaction was carried out at 25°C for 2.5 hours. After dilution with dichloromethane (315 g), succinimide, an impurity derived from N-chlorosuccinimide, was removed by repeated washing with water using 1 mol / L hydrochloric acid (135 g) in which 10 wt% sodium chloride was dissolved. The organic layer was dried over magnesium sulfate, filtered, and the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (360 g), filtered, and then crystallized by adding hexane (315 g) to remove low molecular weight impurities. The crystals were washed twice with hexane (180 g), filtered, and dried under reduced pressure to obtain the compound of formula (14).

[0114] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.52-3.88(m, -(OCH2CH2) n -), 4.17(4H, t, -OCH2CH2OAr), 6.92(4H, d, arom.H), 7.76(4H, d, arom.H), 9.36(2H, s, -C(Cl)=NOH)

[0115]

[0116] Here, the compound of formula (14) belongs to the polyoxyethylene derivative of formula (1). In formula (1), P １ This is a divalent residue obtained by removing the terminal hydroxyl group from a polyoxyethylene compound that has a terminal hydroxyl group, where W is 2 and L １ It is a single bond, Z １ This is an ether bond (a divalent bond group with low hydrolysis reactivity), L ２ This is a C6-carbon, divalent aromatic hydrocarbon group (phenylene group), and X １ It is Cl.

[0117] (Example 3) (Synthesis of Precursor) Pentaerythritol-tetra(polyethylene glycol)-average molecular weight 10000 (200g: 20.0 mmol) and toluene (400g) were charged into a 1 L four-necked flask equipped with a thermometer, nitrogen blowing tube, stirrer, Dean-Stark tube, and condenser, and water was removed azeotropically with toluene. After cooling to 40°C, triethylamine (12.1g: 120 mmol) was charged. After stirring for a while, methanesulfonyl chloride (10.2g: 89.0 mmol) was added dropwise so that the internal temperature did not exceed 45°C. After reacting at 40°C for 6 hours, ethanol (4.07g: 88.3 mmol) was added, stirred for 30 minutes, and then filtered by suction. The filtrate was diluted with toluene (100 g) and ethyl acetate (400 g), and then hexane (800 g) was added to perform crystallization. The crystals were then obtained by suction filtration. The crystals were washed twice with hexane (6600 g), filtered, and dried under reduced pressure to obtain the compound (precursor) of formula (15).

[0118] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.09(12H, s, -OCH2CH2OSO2CH3), 3.41(8H, s, -C(CH2O)4), 3.52-3.78(m, -(OCH2CH2) n -), 4.38(8H, m, -OCH2CH2OSO2CH3) Number average molecular weight (Mn): 9,723

[0119]

[0120] (Synthesis of the polyoxyethylene compound of formula (2)) In a 1 L four-necked flask equipped with a thermometer, nitrogen inlet, stirrer, and condenser, the compound of formula (15) (80.0 g: 8.00 mmol), 4-hydroxybenzaldehyde (7.81 g: 64.0 mmol), and acetonitrile (400 g) were charged and dissolved. After dissolution, potassium carbonate (22.0 g: 159 mmol) was added. The reaction was carried out at 80°C for 7 hours, then filtered, and the solvent was removed by vacuum distillation. The residue was dissolved in dichloromethane (800 g), and repeatedly washed with a 10 wt% saline solution (320 g) adjusted to pH 12 with sodium hydroxide to remove some low molecular weight impurities, after which the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (400 g), filtered, and then crystallized by adding hexane (400 g) to remove low molecular weight impurities. The crystals were washed with hexane (320 g), filtered, and then dried under reduced pressure to obtain the polyoxyethylene compound of formula (16). The compound of formula (16) belongs to the polyoxyethylene compound of formula (2).

[0121] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.41(8H, s, -C(CH2O)4), 3.52-3.90(m, -(OCH2CH2) n -), 4.21(8H, t, -OCH2CH2OAr), 7.02(8H, d, arom.H), 7.83(8H, d, arom.H), 9.89(4H, s, -CHO) Number average molecular weight (Mn): 9,510

[0122]

[0123] (Synthesis of the oximed derivative of formula (3)) In a 1 L four-necked flask equipped with a thermometer, nitrogen inlet tube, and stirrer, compound (16) (60.0 g: 6.00 mmol) and methanol (300 g) were charged. After dissolution, triethylamine (9.11 g: 90.0 mmol) and hydroxylamine hydrochloride (4.17 g: 60.0 mmol) were added. The reaction was carried out at 25 °C for 7 hours, after which the solvent was removed by vacuum distillation. Dichloromethane (480 g) was added to the residue and dissolved, and then the mixture was repeatedly washed with 1 mol / L hydrochloric acid (180 g) in which 10 wt% sodium chloride was dissolved to remove some low molecular weight impurities. The organic layer was then repeatedly washed with 10 wt% saline solution (180 g), and the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (300 g), the solution was dried over magnesium sulfate, filtered, and then crystallized by adding hexane (300 g). The crystals were washed with hexane (240 g), filtered, and dried under reduced pressure to obtain the oxime derivative of formula (17). The compound of formula (17) belongs to the oxime derivatives of formula (3).

[0124] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.41(8H, s, -C(CH2O)4), 3.46-3.86(m, -(OCH2CH2) n -), 4.15(8H, t, -OCH2CH2OAr), 6.91(8H, d, arom.H), 7.49(8H, d, arom.H), 8.03(4H, s, -CH=NOH), 8.05(4H, s, -CH=NOH) Number average molecular weight (Mn): 10,082

[0125]

[0126] (Synthesis of polyoxyethylene derivative of formula (1)) In a 300 mL four-necked flask equipped with a thermometer, nitrogen blowing tube, and stirrer, the oximed derivative of formula (17) (45.0 g: 4.50 mmol) and N,N-dimethylformamide (56.6 g) were charged. After dissolution, N-chlorosuccinimide (3.00 g: 22.5 mmol), a leaving group introduction reagent, was added. The reaction was carried out at 25°C for 4.5 hours, then diluted with ethyl acetate (360 g), and crystallization was performed by adding hexane (315 g). Unreacted N-chlorosuccinimide and succinimide, an impurity derived from N-chlorosuccinimide, were removed by repeatedly dissolving the crystals in ethyl acetate (360 g) and adding hexane (315 g) for crystallization. The crystals were washed with hexane (180 g), filtered, and dried under reduced pressure to obtain the compound of formula (18).

[0127] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.41(8H, s, -C(CH2O)4), 3.52-3.88(m, -(OCH2CH2) n -), 4.17(8H, t, -OCH2CH2OAr), 6.92(8H, d, arom.H), 7.76(8H, d, arom.H), 9.41(4H, s, -C(Cl)=NOH)

[0128]

[0129] Here, the compound of formula (18) belongs to the polyoxyethylene derivative of formula (1). In formula (1), P １ This is a tetravalent residue obtained by removing the terminal hydroxyl group from a polyoxyethylene compound that has a terminal hydroxyl group, where W is 4 and L １ It is a single bond, Z １ This is an ether bond (a divalent bond group with low hydrolysis reactivity), L ２ This is a C6-carbon, divalent aromatic hydrocarbon group (phenylene group), and X １ It is Cl.

[0130] (Example 4) (Synthesis of the polyoxyethylene compound of formula (2)) A 100 mL four-necked flask equipped with a thermometer, nitrogen inlet, stirrer, and condenser was charged with the precursor of formula (15) (3.01 g: 0.301 mmol), 4-hydroxy-3,5-diiodobenzaldehyde (900 mg: 2.41 mmol), and toluene (30 g). After dissolution, potassium carbonate (3.65 g: 2.65 mmol) was added. The reaction was carried out at 100 °C for 10 hours, then filtered, and the solvent was removed by vacuum distillation. The residue was dissolved in dichloromethane (30 g), washed with 10 wt% sodium dihydrogen phosphate aqueous solution (12 g) to remove some low molecular weight impurities, and then the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (15 g), filtered, and then crystallized by adding hexane (15 g). Low molecular weight impurities were removed by repeatedly dissolving the crystals in ethyl acetate (15 g) and crystallizing them with the addition of hexane (15 g). The crystals were washed with hexane (24 g), filtered, and dried under reduced pressure to obtain the compound of formula (19). The compound of formula (19) belongs to the polyoxyethylene compound of formula (2).

[0131] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.41(8H, s, -C(CH2O)4), 3.50-4.02(m, -(OCH2CH2) n -), 4.24(8H, t, -OCH2CH2OAr), 8.27(8H, s, arom.H), 9.81(4H, s, -CHO) Number average molecular weight (Mn): 9,156

[0132]

[0133] (Synthesis of the oxime derivative of formula (3)) Compound (1.50 g: 0.150 mmol) of formula (19) and deionized water (7.5 g) were charged into a 20 mL screw-cap tube. After dissolution, potassium carbonate (311 mg: 2.25 mmol) and hydroxylamine hydrochloride (104 mg: 1.50 mmol) were added. The reaction was carried out at 25°C for 2.5 hours, after which dichloromethane (6 g) was added for extraction, and this process was repeated twice. After the organic layers were combined, the organic layers were washed with 10 wt% saline solution (4.5 g), and the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (6 g), the solution was dried over magnesium sulfate, filtered, and then crystallized by adding hexane (9 g). The crystals were washed with hexane (6 g), filtered, and dried under vacuum to obtain the oxime derivative of formula (20). Compound (20) belongs to the oxime derivatives of formula (3).

[0134] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.41(8H, s, -C(CH2O)4), 3.52-3.99(m, -(OCH2CH2) n -), 4.18(8H, t, -OCH2CH2OAr), 7.91(4H, s, -CH=NOH), 7.98(8H, s, arom.H), 8.73(4H, s, -CH=NOH) Number average molecular weight (Mn): 9,194

[0135]

[0136] (Synthesis of polyoxyethylene derivative of formula (1)) The oximed derivative of formula (20) (1.00 g: 0.100 mmol) and acetonitrile (1.25 g) were charged into a 4 mL screw tube. After dissolution, N-chlorosuccinimide (58.8 mg: 0.440 mmol), a leaving group introduction reagent, was added. The reaction was carried out at 40°C for 3 hours, after which anisole (8.6 μL: 0.080 mmol) was added and the quench reaction was carried out at 25°C for 1.5 hours. After dilution with dichloromethane (7 g), succinimide, an impurity derived from N-chlorosuccinimide, was removed by repeated washing with water using 1 mol / L hydrochloric acid (3 g) in which 10 wt% sodium chloride was dissolved. The organic layer was dried over magnesium sulfate, filtered, and the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (8 g), filtered, and crystallized by adding hexane (7 g) to remove low molecular weight impurities. The crystals were washed with hexane (4g), filtered, and then dried under reduced pressure to obtain the polyoxyethylene derivative of formula (21).

[0137] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.41(8H, s, -C(CH2O)4), 3.52-3.99(m, -(OCH2CH2) n -), 4.19(8H, t, -OCH2CH2OAr) 8.23(8H, s, arom.H), 10.08(4H, s, -C(Cl)=NOH) Number average molecular weight (Mn): 8,593

[0138]

[0139] Here, the compound of formula (21) belongs to the polyoxyethylene derivative of formula (1). In formula (1), P １ This is a tetravalent residue obtained by removing the terminal hydroxyl group from a polyoxyethylene compound that has a terminal hydroxyl group, where W is 4 and L １ It is a single bond, Z １ This is an ether bond (a divalent bond group with low hydrolysis reactivity), L ２ This is a C6, divalent aromatic hydrocarbon group (phenylene group) substituted with two iodine (heteroatoms), and X １ It is Cl.

[0140] (Example 5) A 4 mL screw-cap tube was filled with the polyoxyethylene derivative of formula (10) (120 mg: 0.0240 mmol), 5-norbornene-2-carboxylic acid (40 μL: 0.241 mmol), and acetonitrile (1.5 mL). After dissolution, triethylamine (50 μL: 0.36 mmol) was added. The reaction was carried out at 25 °C for 3 hours to prepare the polyoxyethylene conjugate of formula (22) in situ. The polyoxyethylene conjugate of formula (22) belongs to the polyoxyethylene conjugate of formula (4).

[0141] Next, the reaction solution was diluted with 150 μL of pH 9.2 borate buffer and then purified by gel filtration using a PD-10 column to remove acetonitrile and excess 5-norbornene-2-carboxylic acid and triethylamine. The solution after gel filtration was diluted with 5.0 mL of pH 9.2 borate buffer and allowed to stand in an aluminum block constant temperature bath at 37°C, with 1 mL samples taken at arbitrary intervals. The sampled solutions were buffered using a PD-10 column and 5 mM ammonium formate buffer (pH 8.0), and HPLC measurements were performed using a cation exchange column under the following conditions. • HPLC system: Waters Alliance • Column: Asahipak E-502N (7.5 × 100 mm, 9 μm) • Flow rate: 1 mL / min • Analysis time: 20 min • Column temperature: 30 °C • Injection volume: 20 μL • Detector: RI • Mobile phase: 5 mM ammonium formate buffer (pH 8.0)

[0142]

[0143] (Example 6) A 9 mL screw-cap tube was filled with the polyoxyethylene derivative of formula (21) (100 mg: 0.0100 mmol) and acetonitrile (0.5 mL). After dissolution, N,N-dimethylpropargylamine (60 μL: 0.570 mmol) was added. The reaction was carried out at 25 °C for 3 hours, then diluted with toluene (50 mL), filtered, and crystallized by adding hexane (50 mL) to remove low molecular weight impurities. The crystals were washed with hexane (50 mL), filtered, and dried under reduced pressure to obtain the polyoxyethylene derivative of formula (23). The polyoxyethylene conjugate of formula (23) belongs to the polyoxyethylene conjugate of formula (5).

[0144] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 2.86(24H, s, -CH2N(CH3)2), 3.41(8H, s, -C(CH2O)4), 3.52-4.01(m, -(OCH2CH2) n -), 4.21(8H, t, -OCH2CH2OAr), 4.38(8H, s, -CH2N(CH3)2), 7.23(4H, s, Isoxazole C4 CH), 8.48(8H, s, arom.H)

[0145]

[0146] (Comparative Example 1) Methoxypolyethylene glycol 5000 (5.01 g: 1.00 mmol), 4-dimethylaminopyridine (12.2 mg: 0.0999 mmol), and 4-methyltetrahydropyran (20 g) were charged into a 50 mL screw tube. After heating to 40 °C to dissolve, 4-formylbenzoic acid (450 mg: 3.00 mmol) and N,N'-diisopropylcarbodiimide (462 μL) were added. After reacting at 40 °C for 8.5 hours, the mixture was filtered, diluted with ethyl acetate (100 g), and crystallized by adding methyl tert-butyl ether (100 g). Low molecular weight impurities were removed by repeatedly dissolving the crystals in ethyl acetate (100 g) and adding methyl tert-butyl ether (100 g) to crystallize. The crystals were washed with methyl tert-butyl ether (50 g), filtered, and then dried under reduced pressure to obtain the compound of formula (24).

[0147] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.38(3H, s, -(OCH2CH2) n OCH3), 3.54-3.86(m, -(OCH2CH2) n -), 4.52(2H, t, -CH2OC(=O)Ar), 7.96(2H, d, arom.H), 8.22(2H, d, arom.H), 10.11(1H, s, -CHO) Number average molecular weight (Mn): 5,121

[0148]

[0149] (Comparative Example 2) Compound (24) (4.01 g: 0.802 mmol) and dichloromethane (20 g) were charged into a 50 mL screw-cap tube. After dissolution, triethylamine (416 μL: 3.00 mmol) and hydroxylamine hydrochloride (139 mg: 2.00 mmol) were added. The reaction was carried out at 25°C for 6 hours, then diluted with dichloromethane (12 g), and repeatedly washed with 5 wt% sodium dihydrogen phosphate aqueous solution (12 g) to remove some low molecular weight impurities. The organic layer was then repeatedly washed with 10 wt% saline solution (12 g), and the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate (40 g), the solution was dried over magnesium sulfate, filtered, and then crystallized by adding hexane (40 g). The crystals were washed with hexane (16 g), filtered, and dried under vacuum to obtain compound (25).

[0150] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.38(3H, s, -(OCH2CH2) n OCH3), 3.54-3.85(m, -(OCH2CH2) n -), 4.48(2H, t, -CH2OC(=O)Ar), 7.64(2H, d, arom.H), 8.05(2H, d, arom.H), 8.13(1H, s, -CH=NOH), 8.87(1H, s, -CH=NOH) Number average molecular weight (Mn): 5,296

[0151]

[0152] (Comparative Example 3) Compound (25) (3.01 g: 0.602 mmol) and acetonitrile (3.75 g) were charged into a 50 mL screw tube. After dissolution, N-chlorosuccinimide (100 mg: 0.749 mmol), a leaving group introduction reagent, was added. The reaction was carried out at 25°C for 1.5 hours, after which anisole (32.4 μL: 0.300 mmol) was added and the quench reaction was carried out at 25°C for 4 hours. After dilution with chloroform (21 g), succinimide, an impurity derived from N-chlorosuccinimide, was removed by repeated washing with water using 1 mol / L hydrochloric acid (9 g) in which 10 wt% sodium chloride was dissolved. The solvent was removed by vacuum distillation, and the obtained residue was dissolved in ethyl acetate (60 g). After filtration, hexane (60 g) was added and crystallization was performed to remove low molecular weight impurities. The crystals were washed with hexane (60 g), filtered, and then dried under reduced pressure to obtain the compound of formula (26).

[0153] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.38(3H, s, -(OCH2CH2) n OCH3), 3.54-3.87(m, -(OCH2CH2) n -), 4.49(2H, t, -CH2OC(=O)Ar), 7.93(2H, d, arom.H), 8.06(2H, d, arom.H), 10.21(1H, s, -C(Cl)=NOH)

[0154]

[0155] (Comparative Example 4) Compound (26) (120 mg: 0.0240 mmol), 5-norbornene-2-carboxylic acid (40 μL: 0.241 mmol), and acetonitrile (1.5 mL) were charged into a 4 mL screw tube. After dissolution, triethylamine (50 μL: 0.36 mmol) was added. The reaction was carried out at 25 °C for 3 hours to prepare the polyoxyethylene conjugate of formula (27) in situ. Subsequently, 150 μL of pH 9.2 borate buffer was added to the reaction solution to dilute it, and the solution was purified by gel filtration using a PD-10 column to remove acetonitrile and excess 5-norbornene-2-carboxylic acid and triethylamine. After gel filtration, 5.0 mL of pH 9.2 borate buffer was added to the solution to dilute it, and the solution was allowed to stand in an aluminum block constant temperature bath at 37 °C, and 1 mL samples were taken at arbitrary intervals. The sampled solution was buffered using a PD-10 column and 5 mM ammonium formate buffer (pH 8.0), and HPLC measurement was performed using a cation exchange column under the following conditions: • HPLC instrument: Waters Alliance • Column: Asahipak E-502N (7.5 × 100 mm, 9 μm) • Flow rate: 1 mL / min • Analysis time: 20 min • Column temperature: 30 °C • Injection volume: 20 μL • Detector: RI • Mobile phase: 5 mM ammonium formate buffer (pH 8.0)

[0156]

[0157] As a result, as shown in Figure 1, in Comparative Example 4, the decomposition rate of the polyoxyethylene compound represented by formula (27), based on Non-Patent Document 1, at pH 9.2 and a temperature of 37.3°C over 72 hours was approximately 74%.

[0158] On the other hand, it was found that the decomposition rate of the polyoxyethylene compound represented by formula (22) according to Example 5 of the present invention at pH 9.2 and temperature 37.3°C over 72 hours was less than 1%.

[0159] The polyoxyethylene derivatives of the present invention can improve the long-term stability of polyoxyethylene conjugates of biofunctional molecules and improve the pharmacological effects and therapeutic effects of biofunctional molecules modified with polyoxyethylene derivatives.

[0160] Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. This application is based on Japanese Patent Application No. 2025-049271 filed on 25 March 2025, the contents of which are incorporated herein by reference.

Claims

1. A polyoxyethylene derivative characterized by being represented by the following formula (1). (P in formula (1) １ is a residue having a valency of 1 to 8 obtained by removing the terminal hydroxyl group from a polyoxyethylene compound having a terminal hydroxyl group, W is the valency of the residue, which is 1 to 8, and L １ Z is a single bond or a divalent hydrocarbon group having 1 to 6 carbon atoms. １ L is a divalent bonding group with low hydrolysis reactivity. ２ X is a divalent hydrocarbon group having 1 to 30 carbon atoms, which may have single bonds or heteroatoms. １ (This is a leaving group.) 2. Z １ The polyoxyethylene derivative according to claim 1, characterized in that it is an amide bond, a carbamate bond, a urea bond, a bond containing a secondary amino group, a bond containing a tertiary amino group, an ether bond, an alkylene group, an arylene group, or a heteroarylene group.

3. A method for producing the polyoxyethylene derivative according to claim 1 or 2, comprising: an oximation step of obtaining an oximated derivative represented by formula (3) by reacting hydroxylamine with a polyoxyethylene compound represented by the following formula (2); and a leaving group introduction step of obtaining the polyoxyethylene derivative represented by formula (1) by reacting the oximated derivative with a leaving group-introducing reagent, wherein the method is characterized by comprising the above steps. (In formula (2) and formula (3), P １ is a residue having a valency of 1 to 8 obtained by removing a terminal hydroxyl group from a polyoxyethylene compound having a terminal hydroxyl group, W is the valency of the residue and is 1 to 8, L １ is a single bond or a divalent hydrocarbon group having 1 to 6 carbon atoms, Z １ is a divalent linking group having low hydrolysis reactivity, L ２ is a single bond or a divalent hydrocarbon group having 1 to 30 carbon atoms which may optionally contain a hetero atom.) 4. The method for producing a polyoxyethylene derivative according to claim 3, characterized in that, after the leaving group introduction step, a removal step is taken to remove impurities originating from the leaving group introduction reagent by crystallization and washing with water.

5. A polyoxyethylene compound characterized by being represented by the following formula (4). (P in formula (4) １ is a residue having a valency of 1 to 8 obtained by removing the terminal hydroxyl group from a polyoxyethylene compound having a terminal hydroxyl group, W is the valency of the residue, which is 1 to 8, and L １ Z is a single bond or a divalent hydrocarbon group having 1 to 6 carbon atoms. １ L is a divalent bonding group with low hydrolysis reactivity. ２ R is a divalent hydrocarbon group having 1 to 30 carbon atoms, which may have single bonds or heteroatoms. １ and R ２ At least one of the two is a residue obtained by removing the norbornene group or the oxanorbornene group from a biofunctional molecule having a norbornene group, R １ and R ２ If only one of the two is the aforementioned residue, then the other is a hydrogen atom, a carboxyl group, a primary amide group, or a secondary amide group, and J is CH ２ (Or it is an oxygen atom.) 6. A polyoxyethylene compound characterized by being represented by the following formula (5). (P in formula (5) １ is a residue having a valency of 1 to 8 obtained by removing the terminal hydroxyl group from a polyoxyethylene compound having a terminal hydroxyl group, W is the valency of the residue, which is 1 to 8, and L １ Z is a single bond or a divalent hydrocarbon group having 1 to 6 carbon atoms. １ L is a divalent bonding group with low hydrolysis reactivity. ２ R is a divalent hydrocarbon group having 1 to 30 carbon atoms, which may have single bonds or heteroatoms. ３ and R ４ At least one of the two is a residue obtained by removing the alkyne group from a biofunctional molecule having an alkyne group, R ３ and R ４ If only one of them is the aforementioned residue, the other is a hydrogen atom or a hydrocarbon group.

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