Ketal-type releasable polyoxyethylene derivative, method for producing the same and ketal-type releasable polyoxyethylene conjugate

Abstract

To provide a polyoxyethylene derivative having a ketal structure, which converts a functional biomolecule into a prodrug and gradually releases the functional biomolecule under physiological conditions.SOLUTION: There is provided a ketal-type releasable polyoxyethylene derivative which is, for example, represented by a formula (1) and is cleaved under physiological conditions. (B1 represents a hydrogen atom or -C(R1)(R2)OC(O)E1, R1 and R2 each independently represent a hydrocarbon group having 1 to 10 carbon atoms or H, E1 represents a leaving group, P1 represents a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having the terminal hydroxyl group, and w represents an integer of 1 to 8).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a ketal-type releasable polyoxyethylene derivative, a method for producing the same, and a ketal-type releasable polyoxyethylene conjugate which is a conjugate with a biofunctional molecule. [Background technology]

[0002] When drugs using biofunctional molecules such as hormones, cytokines, and enzymes are administered to the body, they are usually quickly excreted from the body by glomerular filtration in the kidneys or uptake by macrophages in the liver and spleen. As a result, their half-life in the blood is short, and it is often difficult to obtain sufficient pharmacological effects. To solve this problem, attempts have been made to chemically modify biofunctional molecules with water-soluble polymers such as polyoxyethylene (PEG). As a result, it is possible to extend the half-life in the blood of biofunctional molecules by increasing the molecular weight and forming a hydration layer. It is also well known that these modifications can reduce the toxicity and antigenicity of biofunctional molecules and improve their aggregation properties.

[0003] However, it is known that biofunctional molecules chemically modified with water-soluble polymers such as PEG can have undesirable effects such as reduced interactions with target endogenous molecules and receptors due to the formation of a hydration layer by the water-soluble polymer and steric shielding effect of the active site, resulting in a reduction in the inherent pharmacological action of the biofunctional molecule and changes in its dynamics in the body and within cells.

[0004] As an approach to the above-mentioned problems, a method is used in which a water-soluble polymer is chemically modified to a biofunctional molecule via a temporary bond, and the temporary bond is cleaved in vivo to release the unmodified biofunctional molecule, i.e., a prodrug method.

[0005] In recent years, for biofunctional molecules modified with water-soluble polymers, the techniques described in Patent Documents 1 and 2, for example, have been known as techniques capable of suppressing the decrease in the inherent pharmacological action of biofunctional molecules and changes in the dynamics in the body and within cells by using a prodrug method. These Patent Documents 1 and 2 report that PEGylated hGH and PEGylated IL-2, which are biofunctional molecules modified with PEG via a linker that decomposes non-enzyme-dependently at an appropriate rate under physiological conditions, i.e., neutral conditions, can improve the decrease in pharmacological action caused by PEGylation by releasing the biofunctional molecule with the decomposition of the linker while extending the half-life in the blood of the biofunctional molecule. For this reason, a prodrug technique that extends the half-life of the biofunctional molecule in the blood and releases the biofunctional molecule at an appropriate rate under physiological conditions in an enzyme-independent manner to express a pharmacological action is important.

[0006] Patent Document 3 reports a compound that non-enzyme-dependently cleaves a temporary bond in a biofunctional molecule that has been chemically modified with PEG, thereby releasing the biofunctional molecule that has not been chemically modified. Specifically, it has been reported that the hydrolysis of a linker having an acetal structure that is hydrolyzed under acidic conditions triggers cleavage of the temporary carbamate bond through 1,4- or 1,6-benzyl elimination, releasing an unmodified biofunctional molecule. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2018-150311 A [Patent Document 2] Patent Publication No. 2022-000043 [Patent Document 3] JP 2018-172648 A Summary of the Invention [Problem to be solved by the invention]

[0008] Incidentally, in the examples of Patent Document 3, specific structures capable of releasing biofunctional molecules in an enzyme-independent manner and under acidic conditions are exemplified, but no prodrug technology is described that releases biofunctional molecules in an enzyme-independent manner at an appropriate rate under physiological conditions, i.e., under neutral conditions around pH 7.4, and exerts a pharmacological action.

[0009] In view of the above problems, the present invention aims to provide a polyoxyethylene derivative having a ketal structure, which is characterized by converting a biofunctional molecule into a prodrug and gradually releasing the biofunctional molecule under physiological conditions, a stable production method for the same, and a ketal-type releasable polyoxyethylene conjugate. [Means for solving the problem]

[0010] As a result of extensive research, the present inventors have discovered a polyoxyethylene derivative having a ketal structure, which is characterized by converting a biofunctional molecule into a prodrug and gradually hydrolyzing the ketal under physiological conditions to induce benzyl elimination, thereby releasing the biofunctional molecule, as well as a method for stably producing the same and a ketal-type releasable polyoxyethylene conjugate.

[0011] That is, the present invention relates to the following [1] to [5]. [1] A ketal-type releasable polyoxyethylene derivative represented by the following formula (1), (2), (3) or (4), which is cleaved under physiological conditions. [ka] [ka] [ka] [ka] (In formula (1), formula (2), formula (3) and formula (4), B 1 is a hydrogen atom or -C(R 1 )(R 2 )OC(O)E 1 and E 1 is a leaving group, P 1 is a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group, w is an integer from 1 to 8; R 1 , R 2 , R 3 , R 4 , R 5 and R 12 are each independently a hydrocarbon group having 1 to 10 carbon atoms or a hydrogen atom, R 6 is an optionally substituted hydrocarbon group having 1 to 24 carbon atoms, R 7 , R 8 , R 9 , R 10 and R 11 are each independently an electron-withdrawing substituent, an electron-donating substituent, or a hydrogen atom; m is 0 or 1.)

[0012] [2] m is 0 and R 1 and R 2 is a hydrogen atom, and R 3 , R 4 , R 5 and R 12 and each independently represent a hydrogen atom or a methyl group.

[0013] [3] A method for producing the ketal-type releasable polyoxyethylene derivative of [1] or [2], comprising the steps of: a coupling step of coupling a polyoxyethylene derivative with an aromatic ketone derivative having a hydroxy group to obtain a coupling product represented by the following formula (5) or (6); a dialkyl ketalization step of reacting the coupling product represented by the following formula (5) or (6) with a monohydric alcohol under acidic conditions to obtain a dialkyl ketal structure represented by the following formula (7) or (8); After the dialkyl ketalization step, the dialkyl ketal structure represented by the following formula (7) or (8) is converted under acidic conditions into a compound having a hydroxymethyl group at the 2-position and a substituent (-CH=CB 1 ) m C(R 1 )(R 2 )-OH(B 1 , m, R 1 , R 2 a ketalization step in which the ketal structure is obtained by reacting the phenol having the formula (I) with the phenol having the formula (II) as described above; After the ketalization step, a leaving group structure (-OC(O)E 1 ) is introduced into the leaving group structure introduction step. A method for producing a ketal-type releasable polyoxyethylene derivative, comprising the steps of: [ka] [ka] (In formula (5) and formula (6), P 1 ,w,R 6 , R 7 , R 8 , R 9 , R 10 and R 11 (as mentioned above) [ka] [ka] (In formula (7) and formula (8), R 13 and R 14 is an alkyl group having 1 to 10 carbon atoms, P 1 ,w,R 6 , R 7 , R 8 , R 9 , R 10 and R 11 (as mentioned above)

[0014] [4] Between the ketalization step and the leaving group structure introduction step, P of the dialkyl ketal structure represented by the formula (7) and the formula (8) 1 a step of deprotecting the amino group protected by a protecting group in A step of introducing a functional group capable of reacting with a biofunctional molecule into the amino group deprotected after the deprotection step. The method for producing a ketal-type releasable polyoxyethylene derivative according to [3],

[0015] [5] A ketal-type releasable polyoxyethylene conjugate represented by the following formula (9), (10), (11) or (12), which is cleaved under physiological conditions: [ka] [ka] [ka] [ka] (In formula (9), formula (10), formula (11) and formula (12), B 2 is a hydrogen atom or -C(R 1 )(R 2 )OC(O)NHD 1 and D 1 is a residue obtained by removing an amino group constituting a carbamate bond from an amino group contained in a biofunctional molecule, P 2 is a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group, or a bond between a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group and a biofunctional molecule, w is an integer from 1 to 8; R 1 , R 2 , R 3 , R 4 , R 5 and R 12 each independently represents a hydrocarbon group having 1 to 10 carbon atoms or a hydrogen atom, R 6 is an optionally substituted hydrocarbon group having 1 to 24 carbon atoms, R 7 , R 8 , R 9 , R 10 and R 11 are each independently an electron-withdrawing substituent, an electron-donating substituent, or a hydrogen atom; m is 0 or 1.) Effect of the Invention

[0016] The ketal-type releasable polyoxyethylene derivative of the present invention converts a biofunctional molecule into a prodrug, and gradually hydrolyzes the ketal under physiological conditions to induce benzyl elimination, gradually releasing the biofunctional molecule, thereby improving the pharmacological action of the biofunctional molecule modified with the polyoxyethylene derivative.

[0017] In the examples of Patent Document 3, a compound having an acetal structure is capable of releasing a biofunctional molecule in an enzyme-independent manner, and a specific structure capable of releasing a biofunctional molecule under acidic conditions is exemplified. However, no specific structure is described that is capable of releasing a biofunctional molecule by benzyl elimination triggered by hydrolysis of an acetal or ketal under physiological conditions, i.e., neutral conditions at about pH 7.4.

[0018] Here, the acetal structure and ketal structure in the compound of Patent Document 3 are generally used as protecting groups for diols or carbonyl groups, and are known to be deprotected under acidic conditions and are stable under neutral or basic conditions. Therefore, the compound of Patent Document 3 does not suggest the creation of a compound that is hydrolyzed at an appropriate rate under physiological conditions, i.e., neutral conditions, to release a physiologically functional molecule.

[0019] Furthermore, the production method of Patent Document 3 describes a step of coupling a phenolic hydroxyl group of a low molecular weight compound having an acetal structure with a hydroxyl group of PEG under basic conditions, but does not describe a specific method for producing the ketal-type releasable polyoxyethylene derivative of the present invention. [Brief description of the drawings]

[0020]

Figure 1

[0021] The present invention will be described in detail below. The present invention is characterized in that, under physiological conditions, the ketal undergoes hydrolysis, followed by benzyl elimination, and the biofunctional molecule is gradually released. In this specification, "under physiological conditions" refers to a pH of 6.0 to 8.0.

[0022] The release rate of a biofunctional molecule under physiological conditions can be evaluated as the half-life of a ketal in a buffer solution of pH 7.4. The half-life of a ketal in a buffer solution of pH 7.4 is preferably 0.5 to 35 days. Here, the "half-life" refers to the time required for a ketal to be hydrolyzed to convert 1 / 2 equivalent to a ketone.

[0023] The above-mentioned ketal half-life under physiological conditions could not be achieved with a compound in which a polyoxyethylene derivative is introduced via an ether bond to the 3-position of a phenyl group bonded to an acetal or ketal, as exemplified in the examples of Patent Document 3. In contrast, the present inventors have found that a compound in which a polyoxyethylene derivative is introduced via an ether bond to the 2- or 4-position of a phenyl group bonded to a ketal can achieve the above-mentioned ketal half-life when applied under physiological conditions.

[0024] That is, the present invention relates to a ketal-type releasable polyoxyethylene derivative represented by the following formula (1), formula (2), formula (3) or formula (4), which is characterized by being cleaved under physiological conditions. [ka] [ka] [ka] [ka]

[0025] In formula (1), formula (2), formula (3) and formula (4), B 1 is a hydrogen atom or -C(R 1 )(R 2 )OC(O)E 1 and E 1 is a leaving group, P1 is a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group, w is an integer from 1 to 8; R 1 , R 2 , R 3 , R 4 , R 5 and R 12 are each independently a hydrocarbon group having 1 to 10 carbon atoms or a hydrogen atom, R 6 is an optionally substituted hydrocarbon group having 1 to 24 carbon atoms, R 7 , R 8 , R 9 , R 10 and R 11 are each independently an electron-withdrawing substituent, an electron-donating substituent, or a hydrogen atom; m is 0 or 1.

[0026] In formula (1), formula (2), formula (3), and formula (4), R 1 , R 2 and E 1 The details of will be described later, but B 1 is a hydrogen atom or -C(R 1 )(R 2 )OC(O)E 1 and preferably a hydrogen atom.

[0027] In formula (1), formula (2), formula (3), and formula (4), E 1 is a leaving group, and preferred examples of the leaving group include succinimidyloxy, phthalimidyloxy, 4-nitrophenoxy, 1-imidazolyl, pentafluorophenoxy, benzotriazol-1-yloxy and 7-azabenzotriazol-1-yloxy groups, more preferably succinimidyloxy, 4-nitrophenoxy, 1-imidazolyl and pentafluorophenoxy groups, and even more preferably succinimidyloxy or 4-nitrophenoxy groups.

[0028] In formula (1), formula (2), formula (3), and formula (4), P1 is a residue obtained by removing the terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group. Specifically, 1 It is the residue obtained by removing the terminal hydroxyl group (OH) that forms an ether bond (-O-) with the phenyl group bonded to the ketal from the aryl group (-OH). Polyoxyethylene includes both polyoxyethylene having a molecular weight distribution obtained by polymerization of ethylene oxide, and monodisperse polyoxyethylene obtained by bonding oligooxyethylenes having a single molecular weight by a coupling reaction. Also, P 1 may have a hydrocarbon group having 1 to 24 carbon atoms, an amino group protected by a protecting group, or a functional group capable of reacting with a biofunctional molecule.

[0029] P 1 Preferred examples of the following residues are preferred depending on the number of W: When w=1, the residue is represented by the following formula (p1) or formula (p2). [ka] (X 1 is a hydrocarbon group having 1 to 24 carbon atoms, an amino group protected by a protecting group, or a functional group capable of reacting with a biofunctional molecule, Z 1 is a divalent spacer or a single bond, n and l each independently represent 3 to 2,000; s is 0 or 1, t is 2 or 3, and v is 0 or 2.

[0030] When w=2, the residue is represented by the following formula (p3) or (p4). [ka] (X 1 , Z 1 , n, l, s and t are as defined above.)

[0031] When w=3, the residue is represented by the following formula (p5). [ka] (n has the same meaning as above.)

[0032] When w=4, the residues are represented by the following formulas (p6), (p7), and (p8). [ka] (X 1 , Z 1 , n, l, s and t are as defined above.)

[0033] When w=5, the residue is represented by the following formula (p9). [ka] (n has the same meaning as above.)

[0034] When w=6, the residue is represented by the following formula (p10). [ka] (n has the same meaning as above.)

[0035] When w=8, the residues are represented by the following formulas (p11), (p12), and (p13). [ka] (n is as defined above, * indicates the point of attachment to the oxygen atom.)

[0036] In the formulae (p1) to (p13), n and l represent the number of moles of oxyethylene groups represented by -(OCH2CH2)- added, and each independently is 3 to 2,000, preferably 20 to 1,500, more preferably 40 to 1,000, and even more preferably 60 to 500. From the number average molecular weight of the polyoxyethylene derivative determined by size exclusion chromatography, mass spectrometry, or the like, -(OCH2CH2) n It can be calculated by subtracting the molecular weight derived from molecules other than the polyoxyethylene chain, represented by -, and dividing the result by the molecular weight of 44 derived from the oxyethylene group.

[0037] In formulas (p1), (p2), (p4) and (p8), Z 1 is a polyoxyethylene group and X 1 The divalent spacer is not particularly limited as long as it is more stable than a ketal structure, but is preferably an ether bond, an ester bond, a carbonate bond, a urethane bond, an amide bond, a secondary amino group, or an alkylene group containing these. The alkylene group has 1 to 24 carbon atoms, and examples of the spacers include spacers (z1) to (z8) described in group (I).

[0038] Group (I) [ka]

[0039] In the formula, q1 and q2 are each independently an integer of 1 to 12. For example, when it is desired to bond the terminal active carbonate group in a hydrophobic environment such as the inside of a protein, it is preferable that q1 and q2 are large, and when it is desired to bond in a hydrophilic environment, it is preferable that q1 and q2 are small. However, Z 1 is an ether bond, an ester bond, a carbonate bond, a urethane bond, an amide bond, a secondary amino group, or an alkylene group containing these, and when a plurality of the same structural units are bonded, the number of said structural units is 2 or less.

[0040] In the formulas (p1), (p2), (p4) and (p8), X 1 is a hydrocarbon group having 1 to 24 carbon atoms, an amino group protected by a protecting group, or a functional group capable of reacting with a biofunctional molecule.

[0041] X 1 Specific examples of the hydrocarbon group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a t-butyl group, a pentyl group, an isopentyl group, a hexyl group, a heptyl group, a 2-ethylhexyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an eicosyl group, a heneicosyl group, a docosyl group, a tricosyl group, a tetracosyl group, a phenyl group, a benzyl group, a cresyl group, a butylphenyl group, a dodecylphenyl group, and a trityl group. A hydrocarbon group having 1 to 10 carbon atoms is preferable, a methyl group or an ethyl group is more preferable, and a methyl group is even more preferable.

[0042] Here, a 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 protecting group varies depending on the type of chemically reactive functional group to be protected, the conditions 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, "Wuts, PGM; Greene, TW Protective Groups in Organic Synthesis, 4th ed.; Wiley-Interscience: New York, 2007". In the present invention, the amino group protected by a protecting group includes, for example, an amino group protected by an acyl-based protecting group or a carbamate-based protecting group, or an azide group, and specific examples of the acyl-based protecting group or the carbamate-based protecting group include a trifluoroacetyl group, a 9-fluorenylmethyloxycarbonyl group, and a 2-(trimethylsilyl)ethyloxycarbonyl group.

[0043] Furthermore, the functional group capable of reacting with a biofunctional molecule is a formyl group, an epoxy group, a maleimidyl group, a vinylsulfone group, an acryl group, a sulfonyloxy group, a carboxy group, a dithiopyridyl group, an α-haloacetyl group, an alkynyl group, an allyl group, a vinyl group, or an azide group.

[0044] More specifically, functional groups capable of reacting with an amino group of a biofunctional molecule to form a covalent bond include formyl, epoxy, maleimidyl, vinylsulfone, acryl, sulfonyloxy, and carboxy groups. Functional groups capable of reacting with a thiol group of a biofunctional molecule to form a covalent bond include formyl, epoxy, maleimidyl, vinylsulfone, acryl, sulfonyloxy, carboxy, dithiopyridyl, α-haloacetyl, alkynyl, allyl, and vinyl groups. Functional groups capable of reacting with an alkynyl group of a biofunctional molecule to form a covalent bond include an azide group. Functional groups capable of reacting with an azide group of a biofunctional molecule to form a covalent bond include an alkynyl group and a triple bond-containing functional group.

[0045] In a preferred embodiment of this aspect, the functional group capable of reacting with a biofunctional molecule is a group represented by group (II), group (III), group (IV) or group (V). 1 Represents the connection point with.

[0046] Group (II): Functional groups capable of reacting with amino groups of biofunctional molecules to form covalent bonds Items (a), (b), (e) and (f) below. Group (III): Functional groups capable of reacting with a thiol group of a biofunctional molecule to form a covalent bond Items (a), (b), (c), (d), (e), (f) and (h) below. Group (IV): Functional groups capable of reacting with an alkynyl group of a biofunctional molecule to form a covalent bond Items (c), (d) and (g) below. Group (V): Functional groups capable of reacting with an azide group of a biofunctional molecule to form a covalent bond (i) and (j) below.

[0047] [ka]

[0048] In formula (a) and formula (h), Y 1 and Y 3 are each independently a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms, and specific examples of the hydrocarbon group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a t-butyl group, and a pentyl group. 2 is a halogen atom selected from a chlorine atom, a bromine atom, and an iodine atom. In formulae (p2), (p4) and (p8), s is 0 or 1, t is 2 or 3, and v is 0 or 2. Preferred embodiments are residues represented by the following formulae (p14) to (p19).

[0049] [ka] In formulas (p14) to (p19), X 1 , Z 1 , n and l are as defined above.

[0050] In formulas (1) to (4), R 1 , R 2 , R 3 , R 4 , R 5 and R 12 are each independently a hydrocarbon group having 1 to 10 carbon atoms or a hydrogen atom. Specific examples of the hydrocarbon group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a t-butyl group, a phenyl group, and a benzyl group, and a hydrogen atom or a methyl group is more preferable.

[0051] In formula (1), formula (2), formula (3), and formula (4), R 6is a hydrocarbon group having 1 to 24 carbon atoms which may be substituted. Specific examples of the hydrocarbon group which may be substituted include a methyl group, a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, a bromomethyl group, a dibromomethyl group, a tribromomethyl group, a chloromethyl group, a dichloromethyl group, a trichloromethyl group, an ethyl group, a propyl group, a isopropyl group, a butyl group, a t-butyl group, a pentyl group, a isopentyl group, a hexyl group, a heptyl group, a 2-ethylhexyl group, an octyl group, a nonyl group, a decyl group, a undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a Examples of such groups include a phenyl group, a nonadecyl group, an eicosyl group, a heneicosyl group, a docosyl group, a tricosyl group, a tetracosyl group, a phenyl group, a benzyl group, a cresyl group, a butylphenyl group, a dodecylphenyl group and a trityl group, of which a methyl group, a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, a bromomethyl group, a dibromomethyl group, a tribromomethyl group, a chloromethyl group, a dichloromethyl group, a trichloromethyl group, an ethyl group or a phenyl group which may be substituted is preferred, and a methyl group, an ethyl group or a phenyl group which may be substituted is more preferred.

[0052] The optionally substituted phenyl group is represented by the following formula (13): [ka]

[0053] In formula (13), R 15 ~R 19are each independently an electron-withdrawing substituent, an electron-donating substituent, or a hydrogen atom. Examples of the electron-withdrawing substituent include an acyl group having 2 to 5 carbon atoms, an alkoxycarbonyl group having 2 to 5 carbon atoms, a carbamoyl group having 2 to 5 carbon atoms, an acyloxy group having 2 to 5 carbon atoms, an acylamino group having 2 to 5 carbon atoms, an alkoxycarbonylamino group having 2 to 5 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, and a cyano group. Preferred examples include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamide group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, and a cyano group.

[0054] The electron-donating substituent is an alkyl group having 1 to 4 carbon atoms, and preferred examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, and a t-butyl group. 16 or R 18 , electron-withdrawing, para and ortho positions, i.e., R 15 , R 17 or R 19 In the above, examples of the electron-donating substituent include an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, and an aryloxy group having 6 to 10 carbon atoms. Preferred examples include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a t-butoxy group, a phenyl group, and a phenoxy group.

[0055] In formula (1), formula (2), formula (3), and formula (4), R 7 , R 8 , R 9 , R 10 and R 11 are each independently an electron-withdrawing substituent, an electron-donating substituent, or a hydrogen atom. For a detailed description of the electron-withdrawing substituent, see R 15 ~R 19 This is equivalent to the explanation above.

[0056] The electron-donating substituent is an alkyl group having 1 to 4 carbon atoms, and preferred examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, and a t-butyl group. 8 or R 9 , electron-withdrawing, para and ortho positions, i.e., R 7 , R 9 or R 11 In the above, examples of the electron-donating substituent include an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, and an aryloxy group having 6 to 10 carbon atoms. Preferred examples include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a t-butoxy group, a phenyl group, and a phenoxy group.

[0057] In a more preferred embodiment of the present invention, m is 0 in formula (1), formula (2), formula (3), and formula (4), and R 1 and R 2 is a hydrogen atom, and R 3 , R 4 , R 5 and R 12 is a hydrogen atom or a methyl group having one carbon atom, and R 3 , R 4 and R 5 is a hydrogen atom, and R 12 More preferably, is a methyl group.

[0058] Next, the method for producing the ketal-type releasable polyoxyethylene derivative of the present invention will be described. The ketal-type releasable polyoxyethylene derivative of the present invention can be produced by a coupling step of coupling a polyoxyethylene derivative with an aromatic ketone derivative to obtain a coupling product represented by the following formula (5) or formula (6), a dialkyl ketalization step of reacting the coupling product represented by the following formula (5) or formula (6) with a monohydric alcohol under acidic conditions after the coupling step to obtain a dialkyl ketal structure represented by the following formula (7) or formula (8), and a dialkyl ketalization step of reacting the dialkyl ketal structure represented by the following formula (7) or formula (8) with a monohydric alcohol under acidic conditions after the dialkyl ketalization step to obtain a dialkyl ketal structure having a hydroxymethyl group at the 2-position and a substituent (-CH=CB) at the 4-position or 6-position. 1 ) m C(R 1 )(R 2 )-OH(B 1 , m, R 1 , R 2 a ketalization step of obtaining a ketal structure by reacting a phenol having the substituent at the 4-position or the 6-position with a phenol having the same structure as described above, and 1 A leaving group structure introduction step is carried out to introduce the

[0059] [ka] [ka] [ka] [ka]

[0060] In formulas (5), (6), (7), (8), R 13 and R 14 is an alkyl group having 1 to 10 carbon atoms, and P 1 ,w,R 6, R 7 , R 8 , R 9 , R 10 and R 11 is as mentioned above.

[0061] The coupling step of a polyoxyethylene derivative and an aromatic ketone derivative is a step of obtaining a coupling product of formula (5) or (6) through a reaction step represented by reaction 1-1 or reaction 1-2, and a purification step may be performed after the reaction step.

[0062] (Reaction 1-1) [ka]

[0063] (Reaction 1-2) [ka]

[0064] The polyoxyethylene derivative used in the coupling reaction is a compound represented by formula (14), in which P 1 The detailed explanation of and w is the same as above, and E 2 is a leaving group or a hydrogen atom. The leaving group is not particularly limited as long as it is a group that has reactivity in a coupling reaction, and examples thereof include a chloro group, a bromo group, an iodine group, a mesylate group, a tosylate group, and a chloromethanesulfonate group. A mesylate group or a tosylate group is preferred, and a mesylate group is more preferred. An aromatic ketone derivative is a compound represented by formula (15) or formula (16), in which R 6 ~R 11 The detailed explanation of is the same as that described above.

[0065] In the formula (14) of reaction 1-1 and reaction 1-2, E 2When is a leaving group, the compound of formula (14) and an aromatic ketone derivative are coupled in an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, t-butyl methyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide, or in the presence of no solvent, an organic base such as triethylamine, N-methylmorpholine, potassium t-butoxide, or sodium hexamethyldisilazide, or an inorganic base such as potassium carbonate, potassium hydroxide, or sodium hydride. The proportions of the aromatic ketone derivative, organic base, and inorganic base used are not particularly limited, but are preferably equimolar or more relative to the chemically reactive functional group of the compound of formula (14). An organic base may also be used as a solvent.

[0066] In the formula (14) of reaction 1-1 and reaction 1-2, E 2 When is a hydrogen atom, it is a coupling reaction of the formula (14) with a hydroxyphenyl ketone derivative in the presence of an azo reagent such as diethyl azodicarboxylate or diisopropyl azodicarboxylate and a phosphine such as triphenylphosphine, tri-n-octylphosphine or tributylphosphine in an organic solvent such as tetrahydrofuran, dioxane or dichloromethane. The ratio of the hydroxyphenyl ketone derivative, azo reagent and phosphine used is not particularly limited, but it is preferably equimolar or more with respect to the chemically reactive functional group of the compound of formula (14).

[0067] After the reaction steps of Reaction 1-1 and Reaction 1-2, impurities produced as by-products in the reaction, compounds remaining in the reaction without being consumed, and the base catalyst are preferably removed in a purification step. The purification method is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0068] The dialkyl ketalization step after the coupling step is a step of reacting the coupling product represented by formula (5) or formula (6) with a monohydric alcohol under acidic conditions to obtain a dialkyl ketal structure represented by formula (7) or formula (8), and a purification step may be performed after the reaction step.

[0069] Monohydric alcohols are R 13 -OH, R 14 -OH or R 13 -OH and R 14 Alcohols containing -OH, R 13 and R 14 is an alkyl group having 1 to 10 carbon atoms, and preferred examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, and a t-butyl group, more preferably a methyl group, an ethyl group, and a propyl group, and even more preferably a methyl group and an ethyl group.

[0070] The reaction of the coupling product represented by formula (5) or (6) with a monohydric alcohol is carried out in an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, t-butyl methyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide, in the presence of an acid catalyst in the monohydric alcohol or without a solvent. The acid catalyst may be either an organic acid or an inorganic acid, and is not particularly limited, but specific examples include p-toluenesulfonic acid, pyridinium p-toluenesulfonate, methanesulfonic acid, 10-camphorsulfonic acid, hydrogen chloride, iodine, ammonium chloride, oxalic acid, and boron trifluoride diethyl ether complex. Furthermore, a dehydrating agent may be added to the reaction system in order to eliminate water molecules produced in the reaction. The type of dehydrating agent is not particularly limited as long as it does not interfere with the reaction, and examples of the dehydrating agent include orthoesters such as trimethyl orthoformate or triethyl orthoformate, sodium sulfate, magnesium sulfate, alumina, silica gel, and molecular sieves, and preferably orthoesters or molecular sieves.

[0071] After the reaction of the coupling product represented by formula (5) or (6) with the monohydric alcohol, it is preferable to remove impurities produced as by-products in the reaction, compounds remaining in the reaction without being consumed, and the base catalyst in a purification step. The purification method is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0072] After the dialkyl ketalization step, the coupling product represented by formula (7) or (8) is reacted under acidic conditions with a ketalyl group having a hydroxymethyl group at the 2-position and a substituent (-CH=CB 1 ) m C(R 1 )(R 2 )-OH(B 1 , m, R 1 , R 2 The ketalization step of reacting a phenol having the formula (I) with a phenol having the formula (II) (wherein the formula (II) is as described above) is a step of obtaining a ketal structure represented by formula (18), (19), (21) or (22) through a reaction step represented by any one of reactions 2-1 to 2-4, and a purification step may be performed after the reaction step.

[0073] (Reaction 2-1) [ka]

[0074] (Reaction 2-2) [ka]

[0075] (Reaction 2-3) [ka]

[0076] (Reaction 2-4) [ka]

[0077] Reactions 2-1 to 2-4 are steps in which a dialkyl ketal structure represented by formula (7) or formula (8) is reacted with the phenol represented by formula (17) or formula (20) in an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, t-butyl methyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide, or in the absence of a solvent, in the presence of an acid catalyst to obtain a ketal structure represented by formula (18), formula (19), formula (21), or formula (22). The acid catalyst may be either an organic acid or an inorganic acid, and is not particularly limited. Specific examples include p-toluenesulfonic acid, pyridinium p-toluenesulfonate, methanesulfonic acid, 10-camphorsulfonic acid, hydrogen chloride, iodine, ammonium chloride, oxalic acid, and boron trifluoride diethyl ether complex.

[0078] Furthermore, a dehydrating agent may be added to the reaction system in order to remove water molecules generated by the reaction. The type of dehydrating agent is not particularly limited as long as it does not interfere with the reaction, and examples of the dehydrating agent include sodium sulfate, magnesium sulfate, alumina, silica gel, and molecular sieves, and preferably molecular sieves.

[0079] After the reaction steps of Reactions 2-1 to 2-4, impurities produced as by-products in the reaction, compounds remaining in the reaction without being consumed, and base catalyst are preferably removed in a purification step. The purification method is not particularly limited, but the product can be purified by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0080] In particular, between the ketalization step and the step of introducing a leaving group structure into the hydroxy group, P of the dialkyl ketal structure in formula (7) and formula (8) may be 1The method may further comprise a step of deprotecting the amino group protected by a protecting group in the amine, and a step of introducing a functional group capable of reacting with a biofunctional molecule into the deprotected amino group after the deprotection step. Conditions for the deprotection step of the protecting group and the step of introducing a functional group capable of reacting with a biofunctional molecule into the deprotected amino group can be found in many general textbooks, and the deprotection step can be carried out, for example, according to "Wuts, PGM; Greene, TW Protective Groups in Organic Synthesis, 4th ed.; Wiley-Interscience: New York, 2007", and the step of introducing a functional group capable of reacting with a biofunctional molecule into the deprotected amino group can be carried out, for example, according to "Greg T. Hermanso, Bioconjugate Techniques, 3rd ed."

[0081] The leaving group structure introduction step of introducing a leaving group structure in place of a terminal hydroxy group after the ketalization step or the step of introducing a functional group capable of reacting with a biofunctional molecule into the deprotected amino group is a step of converting a hydroxy group in the polyoxyethylene derivative represented by formula (18), formula (19), formula (21) or formula (22) into a succinimidyloxycarbonyloxy group, a phthalimidyloxycarbonyloxy group, a 4-nitrophenoxycarbonyloxy group, a 1-imidazolylcarbonyloxy group, a pentafluorophenoxycarbonyloxy group, a benzotriazol-1-yloxycarbonyloxy group or a 7-azabenzotriazol-1-yloxycarbonyloxy group, and a purification step may be performed after the reaction step.

[0082] The conversion of the hydroxyl group is performed by condensing the polyoxyethylene derivative represented by formula (18), formula (19), formula (21), or formula (22) with a reagent for converting the polyoxyethylene derivative into each of the leaving groups shown in Table 1 in an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, t-butyl methyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide, or in the presence of an organic base such as triethylamine, N-methylmorpholine, pyridine, or 4-dimethylaminopyridine, or an inorganic base such as sodium carbonate, sodium hydrogen carbonate, sodium acetate, or potassium carbonate in a solventless solvent. The proportion of the reagent and base catalyst shown in Table 1 is not particularly limited, but is preferably equimolar or more relative to the hydroxyl group of the polyoxyethylene derivative shown in formula (18), formula (19), formula (21), or formula (22). The reagents shown in Table 1 may be commercially available products or may be produced using known reactions.

[0083] [Table 1]

[0084] After the reaction step in the leaving group structure introduction step, impurities produced as by-products in the reaction, compounds remaining in the reaction without being consumed, and a base catalyst are preferably removed in a purification step. The purification method is not particularly limited, and purification can be performed by extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, supercritical extraction, or the like.

[0085] The ketal-type releasable polyoxyethylene conjugate of the present invention is a -OC(O)E conjugate of the ketal-type releasable polyethylene glycol derivative. 1 The compound is obtained by reacting a group with an amino group contained in a biofunctional molecule, and is represented by the following formula (9), formula (10), formula (11), or formula (12).

[0086] [ka] [ka] [ka] [ka]

[0087] In formulas (9), (10), (11) and (12), B 2 is a hydrogen atom or -C(R 1 )(R 2 )OC(O)NHD 1 and D 1 is a residue obtained by removing an amino group constituting a carbamate bond from an amino group contained in a biofunctional molecule, P 2 is a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group, or a bond between a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group and a biofunctional molecule, w is an integer from 1 to 8; R 1 , R 2 , R 3 , R 4 , R 5 and R 12 each independently represents a hydrocarbon group having 1 to 10 carbon atoms or a hydrogen atom, R 6 is an optionally substituted hydrocarbon group having 1 to 24 carbon atoms, R 7 , R 8 , R 9 , R 10 and R 11 are each independently an electron-withdrawing substituent, an electron-donating substituent, or a hydrogen atom; m is 0 or 1.

[0088] In formula (10), formula (11), and formula (12), R 1~R 12 The details of and m are the same as those described above. In formula (10), formula (11), and formula (12), B 2 is a hydrogen atom or -C(R 1 )(R 2 )OC(O)NHD 1 and preferably a hydrogen atom.

[0089] In formula (10), formula (11), and formula (12), D 1 is a residue of amino groups contained in a biofunctional molecule excluding the amino groups constituting a carbamate bond, and the carbamate bond may be formed independently from each of the multiple amino groups contained in the biofunctional molecule. For example, -OC(O)E of the ketal-type releasable polyoxyethylene derivative 1 In the case where the group forms a carbamate bond with one of the amino groups contained in the biofunctional molecule, in the ketal-type releasable polyoxyethylene conjugate, P 2 is one, and when it forms a carbamate bond with two, P 2 There are two.

[0090] Biofunctional molecules are not particularly limited, but are substances involved in the diagnosis, cure, mitigation, treatment or prevention of diseases in humans or other animals. Specific examples include proteins, peptides, nucleic acids, cells, viruses, etc., and suitable proteins or peptides include hormones, cytokines, antibodies, aptamers, enzymes, etc.

[0091] More specifically, cytokines include interferon type I, type II, and type III, which regulate immunity, interleukins, tumor necrosis factors, and their receptor antagonists. Growth factors include erythropoietin, which is a hematopoietic factor, and granulocyte colony-stimulating factor (GCSF), which is a stimulating factor. Blood coagulation factors include factor V, factor VII, factor VIII, factor IX, factor X, and factor XII. Hormones include calcitonin, insulin, its analogs, exenatide, GLP-1, somatostatin, and human growth hormone. Antibodies include full-length antibodies, and antibody fragments such as Fab and svFV. Aptamers include DNA aptamers and RNA aptamers. Enzymes include superoxide dismutase and uricase.

[0092] Suitable proteins include interferons, interleukins, erythropoietin, GCSF, Factor VIII, Factor IX, human growth hormone, antibody fragments, and the like, more preferably human growth hormone, interferon, GCSF, erythropoietin, or antibody fragments (particularly Fabs).

[0093] Suitable peptides include insulin, bivalirudin, teriparatide, exenatide, enfuvirtide, degarelix, mifamurtide, nesiritide, goserelin, glatiramer, octreotide, lanreotide, icatibant, zicotinide, pramlintide, romiplostim, calcitonin, oxytocin, leuprorelin, and glucagon, and more preferably, insulin, exenatide, and calcitonin (especially salmon calcitonin).

[0094] In formula (10), formula (11), and formula (12), P 2 is a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group, or a bond between a biofunctional molecule and a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group.

[0095] For a detailed explanation of the residue obtained by removing the terminal hydroxyl group from the polyoxyethylene derivative having the terminal hydroxyl group, see P of the ketal-type releasable polyoxyethylene derivative. 1 is synonymous with.

[0096] In addition, the term "a conjugate of a biofunctional molecule with a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group" refers to a conjugate of the P 1 X 1 D 2 D 2 is a group formed by the reaction of a functional group capable of reacting with a biofunctional molecule with the biofunctional molecule. The details of the functional group capable of reacting with a biofunctional molecule are as follows: X of the ketal-type releasable polyoxyethylene derivative 1 The term "biofunctional molecule" is synonymous with the description of the above, and is not particularly limited, but is a substance involved in the diagnosis, cure, mitigation, treatment or prevention of a disease in humans or other animals. Specifically, it includes proteins, peptides, nucleic acids, cells, viruses, etc., and suitable proteins or peptides include hormones, cytokines, antibodies, aptamers, enzymes, etc.

[0097] D 2 Suitable examples of biofunctional molecules include target-directed biofunctional molecules, such as antibodies and aptamers. Antibodies include antibody fragments such as Fab, Fab', and F(ab')2, and aptamers include peptide aptamers, RNA aptamers, and DNA aptamers.

[0098] The method for producing the ketal-type releasable polyoxyethylene conjugate of the present invention will be described. To produce the polyoxyethylene conjugate of the present invention, a coupling step is carried out in which the ketal-type polyoxyethylene derivative and a biofunctional molecule are reacted in a neutral or basic buffer solution which may contain a water-soluble organic solvent such as acetonitrile, dimethyl sulfoxide, or N,N-dimethylformamide to obtain the polyoxyethylene conjugate, and a purification step is carried out after the coupling step to remove the unreacted polyoxyethylene derivative, the biofunctional molecule, or by-products under basic conditions. The term "neutral or basic" refers to a pH range of 6.5 to 11.0, preferably a pH range of 7.0 to 10.5, more preferably a pH range of 7.0 to 10.0, and particularly preferably a pH range of 7.0 to 9.0.

[0099] A neutral or basic buffer solution is an aqueous solution that has a buffering effect that mitigates the effects of adding a small amount of acid or base from outside or changing the concentration by diluting it, thereby keeping the pH (hydrogen ion exponent) at a nearly constant neutral or basic level.

[0100] A water-soluble organic solvent can also be added to the buffer solution. In this case, examples of the water-soluble organic solvent include methanol, ethanol, propanol, isopropanol, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, triethylamine, pyridine, and hexamethylphosphoric acid triamide, preferably methanol, ethanol, propanol, isopropanol, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, and N,N-dimethylformamide, and more preferably acetonitrile, dimethyl sulfoxide, and N,N-dimethylformamide.

[0101] Specific examples of the purification method carried out after the coupling step include ion exchange chromatography, gel filtration chromatography, hydrophobic interaction chromatography, reverse phase chromatography, and affinity chromatography.

[0102] In the purification step, the unreacted polyoxyethylene derivative, biofunctional molecule, or by-product can be removed under neutral or basic conditions under which the ketal structure in the ketal-type releasable polyoxyethylene conjugate is not easily hydrolyzed, and the neutral or basic conditions are pH 6.5 to pH 11.0, preferably pH 7.0 to 10.5, more preferably pH 7.0 to 10.0, and particularly preferably pH 7.0 to 9.0. EXAMPLES

[0103] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0104] Obtained in the following examples 1 H-NMR was obtained from JNM-ECZ400 or JNM-ECA600 manufactured by JEOL Ltd. A φ5 mm tube was used for the measurement, and CDCl3 containing tetramethylsilane (TMS) as an internal standard substance was used as the deuterated solvent. The molecular weight and terminal functional group purity of the obtained ketal-type releasable polyoxyethylene conjugate were determined by liquid chromatography (GPC) and 1 The calculation was performed using H-NMR. The liquid chromatography system used was "Prominence" manufactured by Shimadzu Corporation.

[0105] (Example 1-1) The reactor is made by NOF Corp. ME-200MS (α-methyl-ω-[(methylsulfonyl)oxy]poly(oxyethylene), 10 g, 0.5 mmol), 4-hydroxyacetophenone (272 mg, 2 mmol), potassium carbonate (0.69 g, 5 mmol) and acetonitrile (50 g) were charged and reacted at 80°C under a nitrogen atmosphere for 6 hours. After the reaction, the mixture was filtered, concentrated and redissolved in dichloromethane (80 g). Then, an aqueous solution containing 1% by weight of potassium carbonate and 10% by weight of table salt was added and stirred at 25°C under a nitrogen atmosphere for 10 minutes. After stirring, the mixture was left to stand for 10 minutes and the organic layer was recovered. Magnesium sulfate was added to the recovered organic layer and stirred at 25°C under a nitrogen atmosphere for 10 minutes, and then suction filtered. The obtained filtrate was concentrated, dissolved in ethyl acetate, and hexane was added to precipitate crystals at 25°C under a nitrogen atmosphere. The precipitated crystals were collected by suction filtration and dried under reduced pressure to obtain a compound of formula (23).

[0106] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 2.56(3H, s, -C H 3), 3.38(3H, s, -(OCH2CH2) n O.C. H 3), 3.52-4.17(m, -(OC H 2C H 2) n -), 6.95(2H, d, arom.H ), 7.93(2H, d, arom.H ) [ka]

[0107] (Example 1-2) The compound of formula (23) (4.00 g, 0.20 mmol), trimethyl orthoformate (9.55 g, 90 mmol), p-toluenesulfonic acid monohydrate (419 mg, 2.20 mmol), 2,6-di-tert-butyl-p-cresol (BHT) (0.8 mg) and methanol (34.8 mg) were charged into a reactor, and the reaction was carried out at 25°C for 5 hours under a nitrogen atmosphere. N-methylmorpholine (455 mg, 4.4 mmol) was added and stirred at 25°C for 5 minutes under a nitrogen atmosphere, then concentrated and redissolved in dichloromethane (40 g). After washing with 5% by weight aqueous sodium hydrogen carbonate solution and 25% by weight saline, the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. Thereafter, the concentrated and dried solid was dissolved in ethyl acetate containing added BHT, and hexane was added to cause crystallization. The crystallization was then suction filtered and dried under reduced pressure to obtain the compound of formula (24).

[0108] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 1.52(3H, s, -C H 3), 3.17(6H, s, -OC H 3), 3.38(3H, s, -(OCH2CH2) n O.C. H 3), 3.52-4.17(m, -(OC H 2C H 2) n -), 6.88(2H, d, arom.H ), 7.39(2H, d, arom.H ) [ka]

[0109] (Examples 1-3) The compound of formula (24) (1.0 g, 0.05 mmol), 2,6-bis(hydroxymethyl)-p-cresol (336 mg, 2.0 mmol), tetrahydrofuran (5.0 g) and BHT (1.1 mg) were charged into a reactor, and after dissolving, molecular sieve 5A (1.0 g) and p-toluenesulfonic acid monohydrate (11.4 mg, 0.06 mmol) were added, and the reaction was carried out at 40 ° C. for 4 hours under a nitrogen atmosphere. N-methylmorpholine (12.1 mg, 0.12 mmol) was added, and the mixture was stirred at 25 ° C. for 5 minutes under a nitrogen atmosphere, and then filtered. The filtrate was diluted with toluene (9 g), and washed with 20% saline solution (10 g) at pH 12. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The concentrated, dried solid was dissolved in toluene containing BHT (18 mg), and hexane was added to cause crystallization. The crystallization was then carried out by suction filtration to obtain the compound of formula (25).

[0110] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 1.77(3H, s, -C H 3), 2.20(3H, s, -C H 3), 3.38(3H, s, -(OCH2CH2) n O.C. H 3), 3.52-4.17(m, -(OCH2CH2) n -), 4.59(2H, dd, -C H 2OH), 4.80(2H, dq, -C H 2OCH<), ), 6.56-7.31(6H, arom.H ) [ka]

[0111] (Examples 1 to 4) The compound of formula (25) (200 mg, 0.01 mmol), BHT (0.2 mg) and toluene (0.8 g) were charged into a reactor, and after dissolution, triethylamine (15 mg, 0.15 mmol) and p-nitrophenyl chloroformate (20.2 mg, 0.1 mmol) were charged, and the reaction was carried out for 3 hours at 60°C under a nitrogen atmosphere. After the reaction, the mixture was diluted with a mixed solvent of ethyl acetate and acetonitrile to which BHT had been added, and hexane was added to precipitate crystals, which were then filtered by suction to obtain the crystals. The obtained crystals were dried under reduced pressure to obtain the compound of formula (26).

[0112] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 1.78(3H, s, -C H 3), 2.22(3H, s, -C H 3), 3.38(3H, s, -(OCH2CH2) n O.C. H 3), 3.52-4.24(m, -(OC H 2C H 2) n -), 4.62(2H, dd, -C H 2O-), 5.49(2H, dd, -C H 2OCO-), 6.66-7.44(8H, arom.H ), 8.29(2H, d, arom.H ) Number average molecular weight (Mn): 20,269 [ka]

[0113] Example 2 β-alanine was dissolved in 0.1M sodium phosphate buffer solution (pH 8.5) to prepare a 20mg / mL β-alanine solution. The compound of formula (26) (74mg, 0.0037mmol) was dissolved in 20mg / mL β-alanine solution (1.5mL) and reacted for 6 hours at 25°C under a nitrogen atmosphere. After the reaction, the mixture was diluted with 0.1M sodium phosphate buffer solution (pH 8.5) containing 20% ​​by weight of sodium chloride, and extracted with chloroform (3g). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was diluted with ethyl acetate, and hexane was added to perform crystallization. The crystals obtained by filtration were dried under reduced pressure to obtain the compound of formula (27).

[0114] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 1.75(3H, s, -C H 3), 2.20(3H, s, -C H 3), 2.53(2H, t, -C H 2COOH), 3.38(3H, s, -(OCH2CH2) n O.C. H 3), 3.37-3.43(2H, m, -C H 2NH-), 3.52-4.21(m, -(OC H 2C H 2) n -), 4.60(2H, dd, -C H 2O-), 5.07(1H, d, -C H 2OCONH-), 5.38(1H, d, -C H 2OCONH-), 6.59-7.38(6H, arom.H ) [ka]

[0115] (Example 3-1) ME-200MS (α-methyl-ω-[(methylsulfonyl)oxy]poly(oxyethylene), 10 g, 0.5 mmol) manufactured by NOF Corp., 2-hydroxy-4-methoxyacetophenone (332 mg, 2 mmol), potassium carbonate (0.69 g, 5 mmol) and acetonitrile (50 g) were charged into a reactor and reacted at 80°C for 6 hours under a nitrogen atmosphere. After the reaction, the mixture was filtered, concentrated and redissolved in dichloromethane (80 g). Then, an aqueous solution containing 1% by weight of potassium carbonate and 10% by weight of table salt was added and stirred at 25°C for 10 minutes under a nitrogen atmosphere. After stirring, the mixture was left to stand for 10 minutes and the organic layer was recovered. Magnesium sulfate was added to the recovered organic layer and stirred at 25°C for 10 minutes under a nitrogen atmosphere, and then suction filtered. The obtained filtrate was concentrated and then dissolved in ethyl acetate, and hexane was added to precipitate crystals at 25°C under a nitrogen atmosphere. The precipitated crystals were collected by suction filtration and dried under reduced pressure to obtain the compound of formula (28).

[0116] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 2.61(3H, s, -C H 3), 3.38(3H, s, -(OCH2CH2) n O.C. H 3), 3.52-4.17(m, -(OC H 2C H 2) n -), 6.44(1H, d, arom.H ), 6.53(2H, d, arom.H ), 7.84(2H, d, arom.H ) [ka]

[0117] (Example 3-2) The compound of formula (28) (5.00 g, 0.25 mmol), trimethyl orthoformate (11.94 g, 112.5 mmol), p-toluenesulfonic acid monohydrate (523 mg, 2.75 mmol), BHT (1.0 mg) and methanol (43.5 g) were charged into a reactor, and the reaction was carried out at 25 ° C. for 5 hours under a nitrogen atmosphere. N-methylmorpholine (557 mg, 5.5 mmol) was added, and the mixture was stirred at 25 ° C. for 5 minutes under a nitrogen atmosphere, concentrated, and then redissolved in dichloromethane (40 g). After washing with a 5 wt % aqueous sodium hydrogen carbonate solution and a 25 wt % saline solution, the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The concentrated and dried product was then dissolved in ethyl acetate to which BHT had been added, and hexane was added to perform crystallization, followed by suction filtration and drying under reduced pressure to obtain the compound of formula (29).

[0118] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 1.64(3H, s, -C H 3), 3.13(6H, s, -OC H 3), 3.38(3H, s, -(OCH2CH2) n O.C. H 3), 3.52-4.17(m, -(OC H 2C H 2) n -), 6.39-6.48(3H, arom.H ), 7.56(2H, d, arom.H ) [ka]

[0119] (Example 3-3) The compound of formula (29) (2.0g, 0.10mmol), 2,6-bis(hydroxymethyl)-p-cresol (101mg, 6mmol), tetrahydrofuran (10.0g) and BHT (1.1mg) were charged into a reactor, dissolved, molecular sieve 5A (2.0g) and pyridinium p-toluenesulfonate (45.3mg, 0.18mmol) were added, and the reaction was carried out at 40°C for 4 hours under a nitrogen atmosphere. N-methylmorpholine (36.4mg, 0.36mmol) was added, and the mixture was stirred at 25°C for 5 minutes under a nitrogen atmosphere, and then filtered. The filtrate was diluted with toluene (9g), washed with 20% saline solution (10g) at pH 12, and the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The concentrated, dried solid was dissolved in toluene containing BHT (18 mg), and hexane was added to cause crystallization. The crystallization was then carried out by suction filtration to obtain the compound of formula (30).

[0120] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 1.89(3H, s, -C H 3), 2.21(3H, s, -C H 3), 3.38(3H, s, -(OCH2CH2) n O.C. H 3), 3.52-4.17(m, -(OCH2CH2) n -), 4.60(2H, dd, -C H 2OH), 4.76(2H, dq, -C H 2OCH<), ), 6.33-7.24(5H, arom.H ) [ka]

[0121] (Examples 3-4) The compound of formula (30) (200 mg, 0.010 mmol), BHT (0.2 mg) and toluene (0.8 g) were charged into a reactor, and after dissolution, triethylamine (15 mg, 0.15 mmol) and p-nitrophenyl chloroformate (20.2 mg, 0.1 mmol) were charged, and the reaction was carried out for 3 hours at 60°C under a nitrogen atmosphere. After the reaction, the mixture was diluted with a mixed solvent of ethyl acetate and acetonitrile to which BHT had been added, and hexane was added to precipitate crystals, which were then filtered by suction to obtain the crystals. The obtained crystals were dried under reduced pressure to obtain the compound of formula (31).

[0122] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 1.89(3H, s, -C H 3), 2.21(3H, s, -C H 3), 3.38(3H, s, -(OCH2CH2) n O.C. H 3), 3.52-4.17(m, -(OC H 2C H 2) n -), 4.58(2H, dd, -C H 2O-), 5.50(2H, dd, -C H 2OCO-), 6.33-7.24(5H, arom.H ), 8.29(2H, d, arom.H ) Number average molecular weight (Mn): 20,188 [ka]

[0123] Example 4 β-alanine was dissolved in 0.1M sodium phosphate buffer solution (pH 8.5) to prepare a 20mg / mL β-alanine solution. The compound of formula (31) (74mg, 0.0037mmol) was dissolved in 20mg / mL β-alanine solution (1.5mL) and reacted for 6 hours at 25°C under a nitrogen atmosphere. After the reaction, the mixture was diluted with 0.1M sodium phosphate buffer solution (pH 8.5) containing 20% ​​by weight of sodium chloride and extracted with chloroform (3g). The organic layer was dried over anhydrous sodium sulfate and then filtered. The filtrate was diluted with ethyl acetate, and hexane was added to perform crystallization. The crystals obtained by filtration were dried under reduced pressure to obtain the compound of formula (32).

[0124] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 1.89(3H, s, -C H 3), 2.21(3H, s, -C H 3), 2.53(2H, t, -C H 2COOH), 3.38(3H, s, -(OCH2CH2) n O.C. H 3), 3.37-3.43(2H, m, -C H 2NH-), 3.52-4.17(m, -(OC H 2C H 2) n -), 4.60(2H, dd, -C H 2O-), 5.08(1H, d, -C H 2OCONH-), 5.38(1H, d, -C H 2OCONH-), 6.59-7.38(6H, arom.H ) [ka]

[0125] Comparative Example 1 3-Hydroxybenzaldehyde (2.00g, 16.4mmol), trimethyl orthoformate (3.48g, 32.8mmol), and methanol (17g) were charged into a 50mL three-neck flask equipped with a thermometer, a nitrogen inlet tube, a stirrer, and a cooling tube, and p-toluenesulfonic acid monohydrate (0.312mg, 1.64mmol) was added and reacted at 25℃ for 2 hours. Sodium hydroxide was added and stirred for a while, and then the solvent was distilled off under reduced pressure. The residue was dissolved in dichloromethane and washed with 5wt% aqueous sodium bicarbonate solution and 25wt% saline solution in that order, and the organic layer was dried over anhydrous sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the compound of formula (33).

[0126] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.33(6H, s, -OC H 3), 5.35(1H, s, -C H <), 6.81(1H, d, arom.H ), 6.95(1H, d, arom.H ), 7.23-7.26(1H, m, arom.H ) [ka]

[0127] Comparative Example 2 In a 50mL three-neck flask equipped with a thermometer, nitrogen inlet, stirrer and condenser, 2,4-di(hydroxymethyl)phenol (50.0mg, 0.324mmol) synthesized according to the literature (Freeman, JH; JAm. Chem. Soc. 1952, 74, 6257-6260), the compound of formula (33) (217mg, 1.29mmol), 2,6-di-tert-butyl-p-cresol (7.14mg, 0.0324mmol), anhydrous sodium sulfate (1g) and cyclopentyl methyl ether (10g) were charged, and p-toluenesulfonic acid monohydrate (4.10mg, 0.0212mmol) was added and the reaction was carried out at 40°C for 2 hours. N-methylmorpholine was added and stirred for a while, and then filtered. After washing with 10wt% saline, the organic layer was dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure to give the compound of formula (34).

[0128] 1 H-NMR (d6-DMSO, internal standard TMS); δ(ppm): 4.42(2H, d, -C H 2OH), 4.93(1H, d, -C H 2O-), 5.10(1H, t, -CH2O H ), 5.15(1H, d, -C H 2O-), 6.01(1H, s, -C H <), 6.80-7.21(7H, m, arom.H ), 9.53(1H, bs, >CO H ) [ka]

[0129] Comparative Example 3 A 50 mL three-neck flask equipped with a thermometer, a nitrogen inlet tube, a stirrer and a cooling tube was charged with the compound of formula (34) (37.0 mg, 0.141 mmol), ME-200MS (α-methyl-ω-[(methylsulfonyl)oxy]poly(oxyethylene), 705 mg, 0.0353 mmol) manufactured by NOF Corp., potassium carbonate (97.0 mg, 0.705 mmol) and acetonitrile (3.5 g), and the reaction was carried out at 80°C for 4 hours. After filtration, the solvent was distilled off under reduced pressure, and the residue was dissolved in dichloromethane. After washing with 10 wt% saline, the organic layer was dried over anhydrous sodium sulfate. After filtration, the solvent was distilled off under reduced pressure, and the residue was dissolved in toluene (50 g). Hexane (50 g) was added to perform crystallization, and the mixture was filtered and dried under reduced pressure to obtain the compound of formula (35).

[0130] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 3.38(3H, s, -OC H 3), 3.52-4.18(m, -(OC H 2C H 2) n -), 4.62(2H, s, -C H 2OH), 4.98(1H, d, -C H 2O-), 5.18(1H, d, -C H 2O-), 5.95(1H, s, -C H <), 6.87-7.34(7H, m, arom.H ) [ka]

[0131] Comparative Example 4 A 50mL three-neck flask equipped with a thermometer, nitrogen inlet, stirrer and condenser was charged with the compound of formula (35) (300mg, 0.0150mmol), di(N-succinimidyl)carbonate (46.0mg, 0.180mmol), triethylamine (21.0mg, 0.208mmol) and dichloromethane (5g), and reacted at 25°C for 12 hours. After filtration, the mixture was washed with 5wt% saline, and the organic layer solvent was distilled off under reduced pressure. The residue was dissolved in ethyl acetate (6g), dried over anhydrous sodium sulfate, and then filtered. After adding ethyl acetate (44g), hexane (50g) was added to cause crystallization, filtered, and dried under reduced pressure to obtain the compound of formula (36).

[0132] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 2.85(4H, s, -COC H 2C H 2CO-), 3.38(3H, s, -OC H 3), 3.52-4.18(m, -(OC H 2C H 2) n -), 5.00(1H, d, -C H 2OCH<), 5.18(1H, d, -C H 2O-), 5.25(2H, s, -CH2OCO-), 5.97(1H, s, -C H <), 6.96-7.35(7H, m, arom.H ) [ka]

[0133] Comparative Example 5 A 20 mg / mL buffer solution of β-alanine was prepared using 0.1 M sodium phosphate buffer solution (pH 8.5). The compound of formula (36) (143 mg, 0.0072 mmol) and a 20 mg / mL buffer solution of β-alanine (3.0 g) were charged into a 50 mL three-neck flask equipped with a thermometer, a nitrogen inlet tube, and a stirrer, and after dissolution, the reaction was carried out at 25°C for 6 hours. After dissolving salt (750 mg), extraction was carried out using chloroform (4.5 g), and the organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was diluted with ethyl acetate (45 g), and hexane (33 g) was added to cause crystallization, filtered, and dried under reduced pressure to obtain the compound of formula (37).

[0134] 1 H-NMR (CDCl3, internal standard TMS); δ(ppm): 2.47(2H,t,-C H 2COOH), 3.38(3H, s, -(OCH2CH2) n O.C. H 3), 3.37-3.43(2H, m, -C H 2NH-), 3.52-4.21(m, -(OC H 2C H 2) n -), 4.89(1H, d, -C H 2O-), 5.05(1H, d, -C H 2O-), 5.14(2H, dd, -C H 2OCONH-), 6.22(1H, s, -C H <), 6.52-7.57(7H, arom.H ) [ka]

[0135] (Degradability test) The compounds (2 mg) of formula (27), formula (32) and formula (37) were each dissolved in phosphate buffered saline (2 mL) of pH 7.4, and then allowed to stand in a thermostatic bath at 37°C, and HPLC measurement was performed at any timing. Figure 1 shows the amount of β-alanine adduct at any timing when the amount of β-alanine adduct at 0 hours of standing is taken as 100%. The approximation equation of the exponential approximation curve obtained from the graph, with the y-intercept set to 100, is y=100e -ax (-a is the power exponent.)

number

[0136] It was shown that the compound exemplified in Example (7) of Patent Document 3 represented by formula (37) does not have a desired half-life under physiological conditions, whereas the compounds of the present invention represented by formulas (27) and (32) have a desired half-life under physiological conditions.

Claims

1. A ketal-type releasable polyoxyethylene derivative represented by the following formula (1), (2), (3) or (4), which is cleaved under physiological conditions. 【Chemistry 1】 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 (In formula (1), formula (2), formula (3) and formula (4), B １ is a hydrogen atom or -C(R １ ) (R ２ ) O.C.(O)E １ and E １ is a leaving group, P １ is a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group, w is an integer from 1 to 8; R １ , R ２ , R ３ , R ４ , R ５ and R １2 each independently represents a hydrocarbon group having 1 to 10 carbon atoms or a hydrogen atom, R 6 represents an optionally substituted hydrocarbon group having 1 to 24 carbon atoms, R 7 , R 8 , R 9 , R 10 and R １1 are each independently an electron-withdrawing substituent, an electron-donating substituent, or a hydrogen atom; m is 0 or 1.

2. m is 0, R １ and R ２ is a hydrogen atom, R ３ , R ４ , R ５ and R １2 The ketal-type releasable polyoxyethylene derivative according to claim 1 , wherein each of the is independently a hydrogen atom or a methyl group.

3. A method for producing the ketal-type releasable polyoxyethylene derivative according to claim 1 or 2, comprising the steps of: a coupling step of coupling a polyoxyethylene derivative with an aromatic ketone derivative having a hydroxy group to obtain a coupling product represented by the following formula (5) or (6); a dialkyl ketalization step of reacting the coupling product represented by the following formula (5) or (6) with a monohydric alcohol under acidic conditions to obtain a dialkyl ketal structure represented by the following formula (7) or (8); After the dialkyl ketalization step, the dialkyl ketal structure represented by the following formula (7) or (8) is reacted under acidic conditions with a hydroxymethyl group at the 2-position and a substituent (-CH=CB) at the 4-position or 6-position. １ ) ｍ C(R １ ) (R ２ )-OH(B １ , m, R １ , R ２ a ketalization step in which the ketal structure is obtained by reacting the phenol having the formula (I) with the phenol having the formula (II) as described above; After the ketalization step, a leaving group structure (-OC(O)E) is attached to the terminal of the substituent at the 4-position or 6-position. １ A process for introducing a leaving group structure A method for producing a ketal-type releasable polyoxyethylene derivative, comprising the steps of: 【Chemistry 5】 【Chemistry 6】 (In formula (5) and formula (6), P １ , w, R ６ , R ７ , R ８ , R ９ , R １０ and R １１ (as mentioned above) 【Chemistry 7】 【Chemistry 8】 (In formula (7) and formula (8), R １３ and R １４ is an alkyl group having 1 to 10 carbon atoms, P １ , w, R ６ , R ７ , R ８ , R ９ , R １０ and R １１ (as mentioned above)

4. Between the ketalization step and the leaving group structure introduction step, P of the dialkyl ketal structure represented by the formula (7) and the formula (8) １ a step of deprotecting the amino group protected by a protecting group in A step of introducing a functional group capable of reacting with a biofunctional molecule into the amino group deprotected after the deprotection step. The method for producing a ketal-type releasable polyoxyethylene derivative according to claim 3, further comprising the steps of:

5. A ketal-type releasable polyoxyethylene conjugate represented by the following formula (9), (10), (11) or (12), which is cleaved under physiological conditions: 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 (In formulas (9), (10), (11) and (12), B ２ is a hydrogen atom or -C(R １ ) (R ２ )OC(O)NHD １ and D １ is a residue obtained by removing an amino group constituting a carbamate bond from an amino group contained in a biofunctional molecule, P ２ is a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group, or a bond between a residue obtained by removing a terminal hydroxyl group from a polyoxyethylene derivative having a terminal hydroxyl group and a biofunctional molecule, w is an integer from 1 to 8; R １ , R ２ , R ３ , R ４ , R ５ and R １2 are each independently a hydrocarbon group having 1 to 10 carbon atoms or a hydrogen atom, R 6 represents an optionally substituted hydrocarbon group having 1 to 24 carbon atoms, R ７ , R ８ , R ９ , R １０ and R １1 are each independently an electron-withdrawing substituent, an electron-donating substituent, or a hydrogen atom; m is 0 or 1.

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