Hydrophilic polymer derivative having benzylidene acetal linker

Abstract

A hydrophilic polymer derivative having a benzylidene acetal linker whose hydrolysis rate at the pH of a weakly acidic environment in the living body can be accurately controlled. A hydrophilic polymer derivative having a benzylidene acetal linker, which is represented by the following formula (1): ##STR00001##
wherein R.sup.1 is a hydrogen atom or a hydrocarbon group; R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each independently a hydrogen atom or a substituent and at least one of R.sup.3, R.sup.4, and R.sup.5 is an electron-withdrawing substituent or at least one of R.sup.2 and R.sup.6 represents a substituent; X.sup.1 is a chemically reactive functional group; P is a hydrophilic polymer; w is an integer of 1 to 8; and Z.sup.1 and Z.sup.2 are an independently selected divalent spacer.

Claims

1. A hydrophilic polymer derivative having a benzylidene acetal linker, which is represented by the following formula (1): ##STR00051## wherein R.sup.1 is a hydrogen atom or a hydrocarbon group; R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each independently a hydrogen atom or a substituent and at least one of R.sup.3, R.sup.4, and R.sup.5 is an electron-withdrawing substituent or at least one of R.sup.2 and R.sup.6 is a substituent, with the proviso that when R.sup.2 and R.sup.6 are a hydrogen atom, one of R.sup.3 to R.sup.5 is a substituent selected from the group consisting of a trifluoromethyl group, a cyano group, an acetyl group, a methoxycarbonyl group and a nitro group; X.sup.1 is a chemically reactive functional group selected from the group consisting of an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimide group, a vinyl sulfone group, an acryl group, a sulfonyloxy group, a carboxy group, a thiol group, a dithiopyridyl group, an -haloacetyl group, an alkynyl group, an allyl group, a vinyl group, an amino group, an oxyamino group, a hydrazide group, and an azide group; P is a polyethylene glycol having a hydrocarbon group or a chemically reactive functional group different from X.sup.1 at an end; w is an integer of 1 to 8; and Z.sup.1 and Z.sup.2 are an independently selected divalent spacer.

2. The hydrophilic polymer derivative according to claim 1, wherein w is 1 and P is represented by the formula (4): ##STR00052## wherein Y is a hydrocarbon group having 1 to 24 carbon atoms; n is an integer of 3 to 1000; and v is 0 or 2.

3. The hydrophilic polymer derivative according to claim 1, wherein w is 1 and P is represented by the formula (5): ##STR00053## wherein X.sup.2 is a chemically reactive functional group different from X.sup.1; Z.sup.3 is a divalent spacer; n is an integer of 3 to 1000; and v is 0 or 2.

4. The hydrophilic polymer derivative according to claim 1, wherein w is v+2 and P is represented by the formula (6): ##STR00054## wherein X.sup.2 is a chemically reactive functional group different from X.sup.1; Z.sup.3 is a divalent spacer; n is an integer of 3 to 1000; and v is 0 or 2.

5. The hydrophilic polymer derivative according to claim 1, wherein P is a polyethylene glycol having an end number of 2 to 8, all the ends of the polyethylene glycol constituting P are each bound to Z.sup.2, and w is equal to the end number of the polyethylene glycol.

6. The hydrophilic polymer derivative according to claim 5, wherein P is selected from the group consisting of the formula (r), the formula (s), the formula (t), the formula (u), and the formula (v): ##STR00055## wherein the variable w in structure (r) is 2, the variable w in structure (s) is 3, the variable w in structures (t) and (u) is 4, and the variable w in structure (v) is 8, and n is an integer of 3 to 2000.

7. The hydrophilic polymer derivative according to claim 3, wherein X.sup.2 is selected from the group consisting of an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimide group, a vinyl sulfone group, an acryl group, a sulfonyloxy group, a carboxy group, a thiol group, a dithiopyridyl group, -haloacetyl group, an alkynyl group, an allyl group, a vinyl group, an amino group, an oxyamino group, a hydrazide group, and an azide group.

8. The hydrophilic polymer derivative according to claim 4, wherein X.sup.2 is selected from the group consisting of an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimide group, a vinyl sulfone group, an acryl group, a sulfonyloxy group, a carboxy group, a thiol group, a dithiopyridyl group, an -haloacetyl group, an alkynyl group, an allyl group, a vinyl group, an amino group, an oxyamino group, a hydrazide group, and an azide group.

9. The hydrophilic polymer derivative according to claim 3, wherein X.sup.2 is selected from the group consisting of the formula (a), the formula (b), the formula (c), the formula (d), the formula (e), the formula (f), the formula (g), the formula (h), the formula (i), the formula (j), the formula (k), the formula (l), the formula (m), and the formula (n): ##STR00056## wherein R.sup.7 is a hydrogen atom or a sulfo group; R.sup.8 and R.sup.11 are independently a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms; R.sup.9 is a hydrocarbon group having 1 to 10 carbon atoms, which may contain a halogen atom; and R.sup.10 is a halogen atom selected from a chlorine atom, a bromine atom, and an iodine atom.

10. The hydrophilic polymer derivative according to claim 4, wherein X.sup.2 is selected from the group consisting of the formula (a), the formula (b), the formula (c), the formula (d), the formula (e), the formula (f), the formula (g), the formula (h), the formula (i), the formula (j), the formula (k), the formula (l), the formula (m), and the formula (n): ##STR00057## wherein R.sup.7 is a hydrogen atom or a sulfo group; R.sup.8 and R.sup.11 are independently a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms; R.sup.9 is a hydrocarbon group having 1 to 10 carbon atoms, which may contain a halogen atom; and R.sup.10 is a halogen atom selected from a chlorine atom, a bromine atom, and an iodine atom.

11. The hydrophilic polymer derivative according to claim 3, wherein Z.sup.3 is a saturated hydrocarbon group which may have a bond selected from an ether bond, an ester bond, a carbonate bond, a urethane bond, an amide bond, and a secondary amino group.

12. The hydrophilic polymer derivative according to claim 4, wherein Z.sup.3 is a saturated hydrocarbon group which may have a bond selected from an ether bond, an ester bond, a carbonate bond, a urethane bond, an amide bond, and a secondary amino group.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 Results of the hydrolysis test in MES deuterated water buffer at pD 5.5 at 37 C. using the compounds of the formula (26), the formula (29), the formula (30), and the formula (31) described in Examples.

(2) FIG. 2 Results of the hydrolysis test in HEPES deuterated water buffer at pD 7.4 at 37 C. using the compounds of the formula (26), the formula (29), the formula (30), and the formula (31) described in Examples.

MODES FOR CARRYING OUT THE INVENTION

(3) The following will describe the present invention in detail.

(4) The term acetal as used herein means both of an acetal structure derived from an aldehyde and an acetal structure derived from a ketone, i.e., a ketal structure.

(5) R.sup.1 in the formula (1) of the invention is a hydrogen atom or a hydrocarbon group, and the carbon number of the hydrocarbon group is preferably 10 or less and specific hydrocarbon groups include a methyl group, an ethyl group, a propyl group, an isopropyl group, a t-butyl group, a phenyl group, a benzyl group, and the like. A preferred embodiment of R.sup.1 is a hydrogen atom or a methyl group and more preferred is a hydrogen atom.

(6) The benzene ring in the formula (1) of the invention may have a plurality of substituents and, by appropriately selecting the type and position of the substituents on the benzene ring, it is possible to adjust the degree of electron density and steric hindrance around the acetal group, which affects the hydrolysis rate of the acetal linker. This makes it possible to impart a desired hydrolysis rate to the acetal linker.

(7) In the present specification, the substituent(s) on the benzene ring in the formula (1) is described using the substituent constant (), which means the substituent constant in the Hammett's rule that quantifies the effect of the substituent(s) on the reaction rate or equilibrium of benzene derivatives. However, as is known, the Hammett's rule is applied only to para-substituted and meta-substituted benzene derivatives and cannot be applied to ortho-substituted benzene derivatives that are affected by steric hindrance. Therefore, in the case of ortho-substituted benzene derivatives, the substituent constant means the substituent constant in the Taft's equation that extends the above Hammett's rule.

(8) In the above para-substituted and meta-substituted benzene derivatives, the Hammett's rule is represented by the following formula (7).
log(k/k.sub.0)=(7)
wherein k is a rate constant or equilibrium constant in any reactions of para-substituted and meta-substituted benzene derivatives, k.sub.0 is a rate constant or equilibrium constant in the case where the above benzene derivatives do not have any substituent, that is, the substituent is a hydrogen atom, is a reaction constant, and is a substituent constant.

(9) The reaction constant () in the above formula (7) is a constant that is determined depending on reaction conditions such as the type of reaction, temperature, and solvent, and can be calculated from the slope of Hammett plots. In the acid hydrolysis reaction of the hydrophilic polymer derivative having a benzylidene acetal linker of the invention, the constant is calculated as =3.0 from the results of the hydrolysis tests performed for the compounds of the formula (26), the formula (29), the formula (30), and the formula (31).

(10) The substituent constant () in the above formula (7) is a constant that is determined only depending on the type and position of the substituent(s), regardless of the type of reaction. In the case where no substituent is present, that is, the substituent is a hydrogen atom, the constant is 0. The term electron-withdrawing as used herein means the case where is a positive value and the term electron-donating means the case where is a negative value.

(11) As described above, the Hammett's rule is applied only to para-substituted and meta-substituted benzene derivatives and cannot be applied to the case of ortho-substituted benzene derivatives that are affected by steric hindrance. Therefore, the effect of such steric hindrance is introduced as a factor of the position, i.e., a position constant (Es) of the substituent(s), to extend the Hammett's rule so that it can be applied to the case of the ortho-substituted benzene derivatives, and the resultant is the Taft's equation. The Taft's equation is represented by the following formula (8).
log(k/k.sub.0)=**+Es(8)
wherein k is a rate constant or equilibrium constant in any reactions of para-substituted and meta-substituted benzene derivatives, k.sub.0 is a rate constant or equilibrium constant in the case where the benzene derivatives do not have any substituent, that is, the substituent is a hydrogen atom, * is a reaction constant, * is a substituent constant, and Es is a position constant of the substituent(s).

(12) As is known, since the reaction constants () of para-substituted and meta-substituted benzene derivatives and the reaction constant (*) of ortho-substituted benzene derivatives are approximately equal, it is defined herein that and * are the same. Since the substituent constant (*) in the ortho position is similar to the substituent constant in the para position, for example, as described in Charton, M. Can. J. Chem. 1960, 38 2493-2499, a corresponding substituent constant in the para position is applied to the substituent constant in the ortho position herein.

(13) The substituent constants () in the para position and the meta position are described in Hansch, C.; Leo, A.; Taft, R. W. Chem. Rev. 1991, 91, 165-195 and, with regard to a substituent for which the substituent constant () is unknown, the constant can be measured and determined by the method described in Hammett, L. P. Chem. Rev. 1935, 17(1), 125-136. In addition, the position constant (Es) is described in Unger, S. H.; Hansch, C. Prog. Phys. Org. Chem. 1976, 12, 91-118. However, Es as used herein is one where a hydrogen atom is defined as 0.

(14) In the formula (1), in the case where a plurality of substituents are present on the benzene ring, it is defined that additivity is established for the substituent constant () and the position constant (Es) thereof, and the sum of is represented by and the sum of Es is represented by Es.

(15) With regard to a suitable hydrolysis rate of the hydrophilic polymer derivative having a benzylidene acetal linker of the invention, hydrolysis half life (t.sub.1/2) in a buffer at pH 5.5 and 37 C. is in the range of 1 hour to 6 months, more preferably in the range of 1 hour to 1 month, and further preferably in the range of 1 hour to 24 hours. In this specification, using a numerical value derived from the compound of the formula (26) described in Examples, in which t.sub.1/2 under the above hydrolysis conditions is 24 hours, a suitable range of the sum () of substituent constants of the invention is defined. When log(k/k.sub.0) for the compound of the formula (26) is calculated using the Taft's equation (8), the following equation (9) is obtained.
log(k/k.sub.0)=3.00.231.16=1.85(9)
wherein 3.0 represents a reaction constant (), 0.23 represents the sum () of substituent constants, and 1.16 represents the sum (Es) of position constants.

(16) When the sum () of substituent constants that t.sub.1/2 is 24 hours is calculated using the above equations (9) and (7) in the case where R.sup.2 and R.sup.6 in the formula (1) are a hydrogen atom, the following equation (10) is obtained.
log(k/k.sub.0)=3.0=1.85
=0.62(10)

(17) Moreover, in the case where R.sup.2 and R.sup.6 in the formula (1) are a hydrogen atom, when log(k/k.sub.0) is calculated with taking the rate constant at the time when t.sub.1/2 is 1 hour as k, the following equation (11) is obtained.
log(k/k)=log(24k/k)=1.38

(18) When the equation is modified,
log(k/k)=log [(k/k.sub.0)/(k/k.sub.0)]=1.38
log(k/k.sub.0)log(k/k.sub.0)=1.38

(19) When the above equation (9) is substituted,
log(k/k.sub.0)(1.85)=1.38
log(k/k.sub.0)=0.47(11)

(20) Here, when the sum () of the substituent constants is calculated using the above equations (11) and (7), the following equation (12) is obtained.
log(k/k.sub.0)=3.0=0.47
=0.16(12)

(21) From the equation (10) and the equation (12), in the case where R.sup.2 and R.sup.6 in the formula (1) is a hydrogen atom, t.sub.1/2 of the hydrophilic polymer derivative is 1 hour or more and 24 hours or less at the time of 0.160.62. Similarly, the calculation of the ranges of at 1 hourt.sub.1/21 month and 1 hourt.sub.1/126 months results in the following: 0.161.11 at the time of 1 hourt.sub.1/21 month and 0.161.37 at the time of 1 hourt.sub.1/26 months, respectively.

(22) The substituent that can be used in the invention is an electron-withdrawing or electron-donating substituent that do not inhibit the acetalization reaction of the benzylidene acetal linker in the synthetic process of the hydrophilic polymer derivative, the coupling reaction of the benzylidene acetal linker with the polyethylene glycol precursor and the end functional group conversion reaction of the hydrophilic polymer derivative, and further the bond-forming reaction between the hydrophilic polymer derivative and the drug or the like.

(23) The electron-withdrawing substituent includes 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 of 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, and a cyano group, and preferred examples include a trifluoromethyl group, a cyano group, an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamide group, a methoxyarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl group, a phenylsulfonyl group, and a nitro group. The electron-donating substituent is an alkyl group having 1 to 4 carbon atoms and preferred examples include a methyl group, an ethyl group, a propyl group, an isopropyl group, and a t-butyl group. The substituent that is an electron-withdrawing group in the meta-position and an electron-donating group in the para-position is an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, or an aryloxy group having 6 to 10 carbon atoms and preferred examples include a phenyl group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a t-butoxy group, a phenoxy groups, and the like.

(24) In the case where at least one of R.sup.2 and R.sup.6 in the formula (1) is a substituent other than a hydrogen atom, by using the position constant (Es) of the phenyl group that the influence of steric hindrance is largest and the fluorine atom that the influence of steric hindrance is smallest, among the substituents, the calculation of the ranges of in a buffer at pH 5.5 and 37 C. at 1 hourt.sub.1/224 hours, 1 hourt.sub.1/21 month, and 1 hour t.sub.1/2 6 months results in the following: 1.120.45 at the time of 1 hourt.sub.1/224 hours, 1.120.94 at the time of 1 hourt.sub.1/21 month, and 1.121.20 at the time of 1 hourt.sub.1/26 months, respectively.

(25) In the case where R.sup.2 and R.sup.6 in the formula (1) is a hydrogen atom, at least one of R.sup.3 to R.sup.5 is an electron-withdrawing substituent since is a positive value and R.sup.3 to R.sup.5 other than the above may be an electron-donating substituent. For example, in a preferred embodiment which satisfies 0.160.62 at the time of 1 hourt.sub.1/224 hours, one of R.sup.3 to R.sup.5 in the formula (1) is a substituent selected from the group consisting of a trifluoromethyl group, a cyano group, an acetyl group, a methoxycarbonyl group, and a nitro group, and is more preferably a trifluoromethyl group or a cyano group. In another preferred embodiment, one of R.sup.3 to R.sup.5 in the formula (1) is a substituent selected from the group consisting of a trifluoromethyl group, a cyano group, an acetyl group, a methoxycarbonyl group, and a nitro group and one of R.sup.3 to R.sup.5 other than the above is a substituent selected from the group consisting of a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a t-butyl group, a phenyl group, a methoxy group, an ethoxy group, and an acetamide group. More preferably, one of R.sup.3 to R.sup.5 is a trifluoromethyl group, a cyano group, or a nitro group, and one of R.sup.3 to R.sup.5 other than the above is a methyl group, an ethyl group, a propyl group, or a methoxy group. In still another preferred embodiment, two of R.sup.3 to R.sup.5 in the formula (1) are substituents independently selected from the group consisting of a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom and is more preferably substituents independently selected from the group consisting of a chlorine atom, a bromine atom, and an iodine atom.

(26) Moreover, in the case where at least one of R.sup.2 and R.sup.6 in the formula (1) is a substituent other than a hydrogen atom, for example, the following will show preferred embodiment which satisfies 1.120.45 at the time of 1 hourt.sub.1/224 hours. When one of at least one of R.sup.2 and R.sup.6 in the formula (1) is a fluorine atom or a methoxy group and another one is a hydrogen atom, one of R.sup.3 to R.sup.5 is a substituent selected from the group consisting of a trifluoromethyl group, a cyano group, an acetyl group, a methoxycarbonyl group, and a nitro group, and is more preferably a trifluoromethyl group or a cyano group. In the case where at least one of R.sup.2 and R.sup.6 in the formula (1) is a cyano group, one of R.sup.3 to R.sup.5 is a substituent selected from the group consisting of a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a t-butyl group, a phenyl group, a methoxy group, an ethoxy group, and an acetamide group, and is more preferably a methyl group, an ethyl group, or a methoxy group. When R.sup.2 and R.sup.6 in the formula (1) is independently a fluorine atom or a methoxy group, one of R.sup.3 to R.sup.5 is a substituent selected from the group consisting of a trifluoromethyl group, an acetyl group, a methoxycarbonyl group, a phenyl group, a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and is more preferably a trifluoromethyl group, a phenyl group, or a fluorine atom. When one of R.sup.2 and R.sup.6 in the formula (1) is a methyl group or an ethyl group and another one is a hydrogen atom, one of R.sup.3 to R.sup.5 is a substituent selected from the group consisting of an acetyl group, a methoxycarbonyl group, a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and is more preferably a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. When one of R.sup.2 and R.sup.6 in the formula (1) is a chlorine atom, a bromine atom, or an iodine atom and another one is a hydrogen atom, R.sup.3 to R.sup.5 are a hydrogen atom or one of R.sup.3 to R.sup.5 is a phenyl group or an acetamide group, and is more preferably a hydrogen atom. When one of R.sup.2 and R.sup.6 in the formula (1) is a propyl group, an isopropyl group, or a butyl group and another one is a hydrogen atom, one of R.sup.3 to R.sup.5 is a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and is more preferably a chlorine atom, a bromine atom, or an iodine atom. When one of R.sup.2 and R.sup.6 in the formula (1) is a substituent selected from the group consisting of a t-butyl group, a phenyl group, a trifluoromethyl group, and a nitro group and another one is a hydrogen atom, two of R.sup.3 to R.sup.5 are a substituent selected from the group consisting of a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a t-butyl group, a methoxy group, an ethoxy group, and an acetamide group, and more preferably, two of R.sup.3 to R.sup.5 are a substituent selected from the group consisting of a methyl group, an ethyl group, a t-butyl group, a methoxy group, and an acetamide group. When R.sup.2 and R.sup.6 in the formula (1) are independently a substituent selected from the group consisting of a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a chlorine atom, a bromine atom, and an iodine atom, two of R.sup.3 to R.sup.5 are a substituent selected from the group consisting of a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a t-butyl group, a methoxy group, an ethoxy group, and an acetamide group, and more preferably, two of R.sup.3 to R.sup.5 are a substituent selected from the group consisting of a methyl group, an ethyl group, a t-butyl group, a methoxy group, and an acetamide group.

(27) As mentioned above, the type and position of the substituent(s) suitable for imparting desirable hydrolyzability to the hydrophilic polymer derivative having a benzylidene acetal linker of the invention can be reasonably set by performing the aforementioned calculation using the equations (7), (8), and (9).

(28) X.sup.1 in the formula (1) of the invention is not particularly limited as long as it is a functional group that forms a covalent bond through the reaction with a functional group present in each of biofunctional molecules such as physiologically active proteins, peptides, antibodies, nucleic acids, and low-molecular drugs and drug carriers such as liposomes and polymer micelles to be an object of chemical modification. For example, there may be mentioned functional groups described in Harris, J. M. Poly(Ethylene Glycol) Chemistry; Plenum Press: New York, 1992, Hermanson, G. T. Bioconjugate Techniques, 2nd ed.; Academic Press: San Diego, Calif., 2008, and PEGylated Protein Drugs: Basic Science and Clinical Applications; Veronese, F. M., Ed.; Birkhauser: Basel, Switzerland, 2009; and the like.

(29) Preferred examples of X.sup.1 include an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimide group, a vinyl sulfone group, an acryl group, a sulfonyloxy group, a carboxy group, a thiol group, a dithiopyridyl group, an -haloacetyl group, an alkynyl group, an allyl group, a vinyl group, an amino group, an oxyamino group, a hydrazide group, and an azide group. More specifically, the functional group capable of forming a covalent bond through the reaction with an amino group of each of the biofunctional molecules is an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimide group, a vinyl sulfone group, an acryl group, a sulfonyloxy group, or a carboxy group, the functional group capable of forming a covalent bond through the reaction with a thiol group of each of the biofunctional molecules is an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimide group, a vinyl sulfone group, an acryl group, a sulfonyloxy group, a carboxy group, a thiol group, a dithiopyridyl group, an -haloacetyl group, an alkynyl group, an allyl group, or a vinyl group, the functional group capable of forming a covalent bond through the reaction with an aldehyde group or carboxy group of each of the biofunctional molecules is a thiol group, an amino group, an oxyamino group, or a hydrazide group, the functional group capable of forming a covalent bond through the reaction with an alkylyl group of each of the biofunctional molecules is a thiol group or an azide group, and the functional group capable of forming a covalent bond through the reaction with an azide group of each of the biofunctional molecules is an alkynyl group.

(30) Here, the term active ester refers to an activated carboxy group represented by the formula: C(O)-L, and L represents a leaving group. The leaving group represented by L includes a succinimidyloxy group, a phthalimidyloxy group, a 4-nitrophenoxy group, a 1-imidazolyl group, a pentafluorophenoxy group, a benzotriazol-1-yloxy group, a 7-azabenzotriazol-1-yloxy group, and the like. The term active carbonate indicates an activated carbonate group represented by the formula: OC(O)-L, and L represents the same leaving group as described above.

(31) In a preferred embodiment of the invention, X.sup.1 is a group represented by the group (I), the group (II), the group (III), the group (IV), and the group (V). Group (I): functional groups each capable of forming a covalent bond through the reaction with an amino group of each of the biofunctional molecules

(32) The following (a), (b), (c), (d), (e), and (f) Group (II): functional groups each capable of forming a covalent bond through the reaction with a thiol group of each of the biofunctional molecules

(33) The following (a), (b), (c), (d), (e), (f), (g), (h), (i), and (j) Group (III): functional groups each capable of forming a covalent bond through the reaction with an aldehyde group or carboxy group of each of the biofunctional molecules

(34) The following (g), (k), (l), and (m) Group (IV): functional groups each capable of forming a covalent bond through the reaction with an alkynyl group of each of the biofunctional molecules

(35) The following (g), (k), (1), (m), and (n) Group (V): functional groups each capable of forming a covalent bond through the reaction with an azide group of each of the biofunctional molecules

(36) The following (j)

(37) ##STR00009##

(38) In the formula, R.sup.7 is a hydrogen atom or a sulfo group and, as the sulfo group, sodium sulfonate and potassium sulfonate may be mentioned, but preferred is a hydrogen atom. R.sup.8 and R.sup.11 are a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms and specific hydrocarbon groups include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a t-butyl group, a pentyl group, and the like. R.sup.9 is a hydrocarbon group having 1 to 10 carbon atoms which may contain a halogen atom, and specific hydrocarbon groups 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 benzyl group, a 4-methylphenyl group, a trifluoromethyl group, a 2,2,2-trifluoroethyl group, a 4-(trifluoromethoxy)phenyl group, a vinyl group, a chloroethyl group, a bromoethyl group, an iodoethyl group, and the like, and preferred is a methyl group, a vinyl group, a 4-methylphenyl group, or a 2,2,2-trifluoroethyl group. R.sup.10 is a halogen atom selected from a chlorine atom, a bromine atom, and an iodine atom.

(39) Z.sup.1 in the formula (1) of the invention is a divalent spacer between the functional group X.sup.1 and the benzylidene acetal linker and Z.sup.2 is a divalent spacer between the benzylidene acetal linker and the hydrophilic polymer chain. They are composed of covalent bonds and are not particularly limited as long as they are more stable to acid hydrolysis than the benzylidene acetal linker is, and are preferably an alkylene group alone or an alkylene group having bond(s) selected from an ether bond, an ester bond, a carbonate bond, a urethane bond, an amide bond, and a secondary amino group in the alkylene chain or at the end. The number of the carbon atoms of the alkylene group is preferably from 1 to 24. By way of illustration and without limitation, preferable examples of the alkylene group include structures such as (z1). Preferable examples of the alkylene group having an ether bond include structures such as (z2) or (z3). Preferable examples of the alkylene group having an ester bond include structures such as (z4). Preferable examples of the alkylene group having a carbonate bond include structures such as (z5). Preferable examples of the alkylene group having a urethane bond include structures such as (z6). Preferable examples of the alkylene group having an amide bond include structures such as (z7). Preferable examples of the alkylene group having a secondary amino group include structures such as (z8). In a preferred embodiment, p and q are independently an integer of 1 to 12. For example, in the case where it is intended to bind the functional group X.sup.1 in a hydrophobic environment such as the inside of a protein, p and q are preferably large and, in the case where it is intended to bind it in a hydrophilic environment, p and q are preferably small.

(40) ##STR00010##

(41) P in the formula (1) of the invention is a hydrophilic polymer and specific examples thereof include polyalkylene glycols, polyoxazoline, polycarbonate, polyurethane, polyvinyl alcohol, polyacrylates, polymethacrylates, polyacrylamide, polyvinylpyrrolidone, polylactic acid, polyglycolic acid, polyamino acids, copolymers derived from the above polymers, and the like, and P is preferably a polyalkylene glycol, and more preferably polyethylene glycol.

(42) The term polyethylene glycol as used herein means both of a polyethylene glycol having a molecular weight distribution obtained by the polymerization of ethylene oxide and a monodispersed polyethylene glycol resulting from combination of oligoethylene glycol having a single molecular weight by a coupling reaction.

(43) In one aspect of the invention, P in the formula (1) is a linear type polyethylene glycol.

(44) In a preferred embodiment of this aspect, P in the formula (1) is represented by the formula (2).
Y(OCH.sub.2CH.sub.2).sub.n(2)

(45) In the formula, n is the number of repeating units per polyethylene glycol chain and, in the polyethylene glycol having a molecular weight distribution, it is defined that n is calculate by various theoretical calculations based on the number-average molecular weight (Mn) of the compound.

(46) In the formula, Y is a hydrocarbon group having 1 to 24 carbon atoms, specific examples thereof 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 toicosyl group, a tetracosyl group, a phenyl group, a benzyl group, a cresyl group, a butylphenyl group, a dodecylphenyl group, a trityl group, and the like, and preferred is a hydrocarbon group having 1 to 10 carbon atoms, more preferred is a methyl group or an ethyl group, and further preferred is a methyl group.

(47) In another preferred embodiment of this aspect, P in the formula (1) is represented by the formula (3).
X.sup.2Z.sup.3(OCH.sub.2CH.sub.2).sub.n(3)

(48) In the formula, X.sup.2 is a chemically reactive functional group different from X.sup.1, and Z.sup.3 is a divalent spacer between the functional group X.sup.2 and the polyethylene glycol chain. Since the polyethylene glycol derivative has two different reactive functional groups X.sup.1 and X.sup.2, it is possible to provide a polyethylene glycol-drug conjugate having a target directing property, for example, by binding a drug to X.sup.1 and binding a targeting molecule to X.sup.2.

(49) Preferable examples of X.sup.2 include an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimide group, a vinyl sulfone group, an acryl group, a sulfonyloxy group, a carboxy group, a thiol group, a dithiopyridyl group, an -haloacetyl group, an alkynyl group, an allyl group, a vinyl group, an amino group, an oxyamino group, a hydrazide group, and an azide group. More specifically, the functional group capable of forming a covalent bond through the reaction with an amino group of each of the biofunctional molecules is an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimide group, a vinyl sulfone group, an acryl group, a sulfonyloxy group, or a carboxy group, the functional group capable of forming a covalent bond through the reaction with a thiol group of each of the biofunctional molecules is an active ester group, an active carbonate group, an aldehyde group, an isocyanate group, an isothiocyanate group, an epoxy group, a maleimide group, a vinyl sulfone group, an acryl group, a sulfonyloxy group, a carboxy group, a thiol group, a dithiopyridyl group, an -haloacetyl group, an alkynyl group, an allyl group, or a vinyl group, the functional group capable of forming a covalent bond through the reaction with an aldehyde group or carboxy group of each of the biofunctional molecules is a thiol group, an amino group, an oxyamino group, or a hydrazide group, the functional group capable of forming a covalent bond through the reaction with an alkylyl group of each of the biofunctional molecules is a thiol group or an azide group, and the functional group capable of forming a covalent bond through the reaction with an azide group of each of the biofunctional molecules is an alkynyl group.

(50) In a preferred embodiment of the invention, X.sup.2 is a group represented by the group (I), the group (II), the group (III), the group (IV), and the group (V). Group (I): functional groups each capable of forming a covalent bond through the reaction with an amino group of each of the biofunctional molecules

(51) The following (a), (b), (c), (d), (e), and (f) Group (II): functional groups each capable of forming a covalent bond through the reaction with a thiol group of each of the biofunctional molecules

(52) The following (a), (b), (c), (d), (e), (f), (g), (h), (i), and (j) Group (III): functional groups each capable of forming a covalent bond through the reaction with an aldehyde group or carboxy group of each of the biofunctional molecules

(53) The following (g), (k), (l), and (m) Group (IV): functional groups each capable of forming a covalent bond through the reaction with an alkynyl group of each of the biofunctional molecules

(54) The following (g), (k), (l), (m), and (n) Group (V): functional groups each capable of forming a covalent bond through the reaction with an azide group of each of the biofunctional molecules

(55) The following (j)

(56) ##STR00011##

(57) In the formula, R.sup.7 is a hydrogen atom or a sulfo group and, as the sulfo group, sodium sulfonate and potassium sulfonate may be mentioned, but preferred is a hydrogen atom. R.sup.8 and R.sup.11 are a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms, and specific hydrocarbon groups include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a t-butyl group, a pentyl group, and the like. R.sup.9 is a hydrocarbon group having 1 to 10 carbon atoms which may contain a halogen atom, and specific hydrocarbon groups 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 benzyl group, a 4-methylphenyl group, a trifluoromethyl group, a 2,2,2-trifluoroethyl group, a 4-(trifluoromethoxy)phenyl group, a vinyl group, a chloroethyl group, a bromoethyl group, an iodoethyl group, and the like, and preferred is a methyl group, a vinyl group, a 4-methylphenyl group, or a 2,2,2-trifluoroethyl group. R.sup.10 is a halogen atom selected from a chlorine atom, a bromine atom, and an iodine atom.

(58) X.sup.2 should be different from X.sup.1 and, as preferred combinations of X.sup.1 and X.sup.2, when X.sup.1 is an active ester group or an active carbonate group, X.sup.2 is a group selected from a maleimide group, a vinyl sulfone group, an -haloacetyl group, an alkynyl group, and an azide group; when X.sup.1 is an aldehyde group, X.sup.2 is a group selected from a maleimide group, a vinyl sulfone group, an alkynyl group, and an azide group; when X.sup.1 is a maleimide group, a vinyl sulfone group, or an -haloacetyl group, X.sup.2 is a group selected from an active ester group, an active carbonate group, an alkynyl group, and an azide group; when X.sup.1 is an alkynyl group or an azide group, X.sup.2 is a group selected from a maleimide group, a vinyl sulfone group, an -haloacetyl group, an active ester group, an active carbonate group, an amino group, and an oxyamino group; when X.sup.1 is an amino group or an oxyamino group, X.sup.2 is an alkynyl group, an azide group, a thiol group, or a carboxy group; and when X.sup.1 is a thiol group, X.sup.2 is a group selected from an amino group, an oxyamino group, an azide group, and a carboxyl group. More preferably, when X.sup.1 is an active ester or an active carbonate group, X.sup.2 is a group selected from a maleimide group, an -haloacetyl group, an alkynyl group, and an azide group; when X.sup.1 is an aldehyde group, X.sup.2 is a group selected from a maleimide group, an -haloacetyl group, an alkynyl group, and an azide group; when X.sup.1 is a maleimide group or an -haloacetyl group, X.sup.2 is a group selected from an active ester group, an active carbonate group, an alkynyl group, and an azide group; when X.sup.1 is an alkynyl group or an azide group, X.sup.2 is a group selected from a maleimide group, an -haloacetyl group, an active ester group, an active carbonate group, an amino group, and an oxyamino group; when X.sup.1 is an amino group or an oxyamino group, X.sup.2 is an alkynyl group, an azide group or a thiol group; and when X.sup.1 is a thiol group, X.sup.2 is a group selected from an amino group, an oxyamino group, and an azide group.

(59) Z.sup.3 is composed of a covalent bond and is not particularly limited as long as it is more stable to acid hydrolysis than the benzylidene acetal linker is, and is preferably an alkylene group alone or an alkylene group having bond(s) selected from an ether bond, an ester bond, a carbonate bond, a urethane bond, an amide bond, and a secondary amino group in the alkylene chain or at the end. The number of the carbon atoms of the alkylene group is preferably from 1 to 24. By way of illustration and without limitation, preferable examples of the alkylene group include structures such as (z1). Preferable examples of the alkylene group having an ether bond include structures such as (z2) or (z3). Preferable examples of the alkylene group having an ester bond include structures such as (z4). Preferable examples of the alkylene group having a carbonate bond include structures such as (z5). Preferable examples of the alkylene group having a urethane bond include structures such as (z6). Preferable examples of the alkylene group having an amide bond include structures such as (z7). Preferable examples of the alkylene group having a secondary amino group include structures such as (z8). In a preferred embodiment, p and q are independently an integer of 1 to 12. For example, in the case where it is intended to bind the functional group X.sup.2 in a hydrophobic environment such as the inside of a protein, p and q are preferably large and, in the case where it is intended to bind it in a hydrophilic environment, p and q are preferably small.

(60) ##STR00012##

(61) In another aspect of the invention, P in the formula (1) is a branched polyethylene glycol.

(62) In a preferred embodiment of this aspect, P in the formula (1) is represented by the formula (4).

(63) ##STR00013##

(64) wherein Y is a hydrocarbon group having 1 to 24 carbon atoms as mentioned above and v is 0 or 2.

(65) In the case where v is 0, two polyethylene glycol chains are present and, in the case where v is 2, four polyethylene glycol chains are present. In general, in the chemical modification of a bio-related substance with polyethylene glycol, when binding points to the polyethylene glycol are introduced more than necessary, the active sites of the bio-related substance are destroyed to reduce its function, so that an attempt to increase the effect by increasing the molecular weight of the polyethylene glycol has been performed. However, the viscosity increases with the increase in the molecular weight and hence, for example, handling as an aqueous solution preparation such as an injection preparation becomes difficult. Since the polyethylene glycol derivative has a branched structure, it shows low viscosity as compared with a linear polyethylene glycol derivative having the same molecular and thus is useful in applications such as an aqueous solution preparation.

(66) In another preferred embodiment of this aspect, P in the formula (1) is represented by the formula (5).

(67) ##STR00014##

(68) In the formula, X.sup.2 is the aforementioned chemically reactive functional group different from X.sup.1, Z.sup.3 is a divalent spacer as mentioned above, and v is 0 or 2.

(69) The polyethylene glycol derivative has one X.sup.1 and two or four X.sup.2's and, for example, when a drug is bound to X.sup.1 and a targeting molecule is bound to X.sup.2, high target directing performance can be obtained.

(70) In another preferred embodiment of this aspect, P in the formula (1) is represented by the formula (6).

(71) ##STR00015##

(72) In the formula, X.sup.2 is a chemically reactive functional group different from X.sup.1 as mentioned above, Z.sup.3 is a divalent spacer as mentioned above, and v is 0 or 2.

(73) In the antibody-drug conjugate (ADC)-related field, in order to increase drug transportation efficiency, it is preferable to bind a plurality of drugs to an antibody but, when a plurality of binding points are introduced into the antibody, a decrease in the affinity to an antigen becomes a problem. The polyethylene glycol derivative has two or four X.sup.1's and one X.sup.2, and thus, for example, when an anticancer agent is bind to X.sup.1 and an antibody is bound to X.sup.2 in ADC where cancer is targeted, it is possible to improve the transportation efficiency of the anticancer agent without increasing the binding points to the antibody.

(74) In another aspect of the invention, P in the formula (1) is a polyethylene glycol having an end number of 2 to 8, all the ends of the polyethylene glycol constituting P are each bound to Z.sup.2, and w is equal to the end number of the polyethylene glycol.

(75) In a preferred embodiment of this aspect, P in the formula (1) is selected from the group consisting of the formula (r), the formula (s), the formula (t), the formula (u), and the formula (v). w is 2 in the case where P is represented by the formula (r), w is 3 in the case where P is represented by the formula (s), w is 4 in the case where P is represented by the formula (t), w is 4 in the case where P is represented by the formula (u), and w is 8 in the case where P is represented by the formula (v).

(76) ##STR00016##

(77) A preferred range of n in the formulae (2) and (3) of the invention is from 3 to 2000, more preferably from 20 to 1500, more preferably from 40 to 1000, and most preferably from 60 to 500. Furthermore, a preferred range of n in the formula (4), the formula (5), and the formula (6) is from 3 to 1,000, preferably from 10 to 800, more preferably from 20 to 500, and most preferably from 30 to 300.

(78) The hydrophilic polymer derivative having a benzylidene acetal linker of the invention can be synthesized by the coupling reaction of a linker unit, into which a benzylidene acetal group having substituent(s) is introduced, with a hydrophilic polymer precursor. The bond generated by the coupling reaction is determined by a combination of the functional groups used in the reaction, and is the ether bond, the ester bond, the carbonate bond, the urethane bond, the amide bond, or the secondary amino group contained in the above divalent spacer Z.sup.2. In the synthesized hydrophilic polymer derivative, the end functional group of the benzylidene acetal linker is chemically converted as required. As the reaction used for the functional group conversion, a conventionally known method can be used but, conditions that do not decompose the benzylidene acetal group of the formula (1) and the bonds contained in the above divalent spacers Z.sup.2 and Z.sup.1 should be properly selected.

(79) As a typical example of the coupling reaction of the benzylidene acetal linker unit with the hydrophilic polymer precursor and the further chemical conversion of the end functional group, the following steps may be mentioned. Here, polyethylene glycol that is a typical hydrophilic polymer will be described as an example.

(80) (A) Synthesis of Benzylidene Acetal Linker Unit

(81) ##STR00017##
wherein Ar is a benzene ring having substituent(s) and R.sup.1 is a hydrogen atom or a hydrocarbon group.

(82) A 3-aminopropanol derivative whose amino group is protected with a phthalimide group that is a typical protective group is allowed to react with a carbonyl compound of the formula (13) 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 without any solvent in the presence of an acid catalyst to obtain a compound of the following formula (14) having a benzylidene acetal group. The resulting compound may be purified by extraction, recrystallization, adsorbent treatment, column chromatography, or the like. Instead of the carbonyl compound, it is possible to use a corresponding acetal derivative of a lower alcohol. The lower alcohol is preferably an alcohol having 1 to 5 carbon atoms, and more preferably methanol or ethanol. The acid catalyst may be either an organic acid or an inorganic acid and is not particularly limited but specific examples thereof include p-toluenesulfonic acid, pyridinium p-toluenesulfonate, methanesulfonic acid, 10-camphorsulfonic acid, hydrogen chloride, iodine, ammonium chloride, oxalic acid, boron trifluoride-diethyl ether complex, and the like.

(83) ##STR00018##

(84) Here, the protective group is a component that prevents or blocks a reaction of a particular reactive functional group in a molecule under certain reaction conditions. The protective group varies depending on the type of the reactive functional group to be protected, the conditions to be used, and the presence of the other functional group or protective group in the molecule. Specific examples of the protective group can be found in many general books and are described in, for example, Wuts, P. G. M.; Greene, T. W. Protective Groups in Organic Synthesis, 4th ed.; Wiley-Interscience: New York, 2007.

(85) As the protective group in the compound of the formula (14), other protective groups may be used as long as they are stable in the acidic conditions of the acetalization reaction and can be deprotected by a method other than the catalytic reduction by which the benzylidene acetal group is decomposed. When the reactive functional group is an amino group, for example, acyl-based protective groups and carbamate-based protective groups may be mentioned and specifically, a trifluoroacetyl group, a 9-fluorenylmethyloxycarbonyl group, a 2-(trimethylsilyl)ethyloxycarbonyl group, and the like may be mentioned. Furthermore, when the reactive functional group is a hydroxy group, for example, silyl-based protective groups and acyl-based protective groups may be mentioned and specifically, a t-butyldiphenylsilyl group, a t-butyldimethylsilyl group, a triisopropylsilyl group, an acetyl group, a pivaloyl group, and the like may be mentioned.

(86) Then, after the compound of the formula (14) is treated with hydrazine monohydrate that is a deprotecting reagent to deprotect two phthalimide groups, ethyl trifluoroacetate that is a protective reagent is reacted in an amount of 1 molar equivalent to obtain a compound of the formula (15), in which only one of the amino groups is protected with a trifluoroacetyl group. Alternatively, by adjusting the amount of the deprotecting reagent for the compound of the formula (14), it is also possible to obtain a compound analogous to the formula (15), in which only one of the amino groups is protected with a phthalimide group. The obtained compound may be purified by the aforementioned purification methods.

(87) ##STR00019##

(88) As the protective group in the compound of the formula (15), other protecting groups may also be used as long as it is possible to deprotect them by any method other than acidic conditions under which the benzylidene acetal group is decomposed and catalytic reduction, as well as conditions which decompose the bond generated by the coupling reaction of the benzylidene acetal linker unit with the hydrophilic polymer precursor. When the reactive functional group is an amino group, for example, acyl-based protective groups and carbamate-based protective groups may be mentioned and specifically, a phthalimide group, a 9-fluorenylmethyloxycarbonyl group, a 2-(trimethylsilyl)ethyloxycarbonyl group, and the like may be mentioned. Furthermore, when the reactive functional group is a hydroxy group, for example, silyl protective groups and acyl-based protective groups may be mentioned and specifically, a t-butyldiphenylsilyl group, a t-butyldimethylsilyl group, a triisopropylsilyl group, an acetyl group, a pivaloyl group, and the like may be mentioned. Typical deprotection conditions for the protective groups are described in the aforementioned literature and it is possible to select a method suitable for each of the protective groups.

(89) (B) Synthesis of Polyethylene Glycol Precursor

(90) Ethylene oxide is polymerized in an amount of 3 to 2000 molar equivalents to methanol that is an initiator in toluene or with no solvent under alkaline conditions such as metallic sodium, metallic potassium, sodium hydride, or potassium hydride to obtain a polyethylene glycol of the formula (16). The initiator is preferably an alcohol having a hydrocarbon group having 1 to 24 carbon atoms, and specifically methanol, ethanol, propanol, isopropanol, butanol, t-butanol, phenol, benzyl alcohol, and the like may be mentioned.
CH.sub.3(OCH.sub.2CH.sub.2).sub.nOH(16)

(91) The polyethylene glycol of the formula (16) is allowed to react with N,N-disuccinimidyl carbonate 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 with no solvent 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 to obtain a polyethylene glycol precursor of the formula (17). The above organic bases and inorganic bases may not be used. The use ratio of the organic bases or the inorganic base is not particularly limited but is preferably equimolar or more to the hydroxyl group of the polyethylene glycol of the formula (16). Also, it is possible to use an organic base as a solvent. The obtained compound may be purified by a purification method such as extraction, recrystallization, adsorbent treatment, reprecipitation, column chromatography, or supercritical extraction.

(92) ##STR00020##

(93) As the reactive functional group in the polyethylene glycol precursor of the formula (17), other reactive functional groups can be also used. Preferred examples of the reactive functional groups are functional groups where the bond generated by the coupling reaction of the polyethylene glycol precursor with the above benzylidene acetal linker unit becomes the ether bond, the ester bond, the carbonate bond, the urethane bond, the amide bond, or the secondary amino group contained in the divalent spacer Z.sup.2 of the formula (1), and specifically, an active ester, an active carbonate, an aldehyde group, a sulfonyloxy group, an amino group, and the like may be mentioned.

(94) (C) Coupling Reaction of Benzylidene Acetal Linker Unit with Polyethylene Glycol Precursor

(95) The benzylidene acetal linker unit of the formula (15) and the polyethylene glycol precursor of the formula (17) are subjected to a coupling reaction 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 with no solvent 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 to obtain a compound of the formula (18). The above organic bases and inorganic bases may not be used. The use ratio of the organic base or the inorganic base is not particularly limited but is preferably equimolar or more to the reactive functional group of the polyethylene glycol precursor of the formula (17). Also, it is possible to use an organic base as a solvent. The obtained compound may be purified by the aforementioned purification means.

(96) ##STR00021##

(97) The reaction conditions for the coupling reaction are determined depending on the combination of the reactive functional group of the benzylidene acetal linker unit and the reactive functional group of the polyethylene glycol precursor and a conventionally known method may be used. However, it is necessary to select conditions that do not decompose the bonds contained in the benzylidene acetal group of the formula (1) and the above divalent spacers Z.sup.2 and Z.sup.1.

(98) (D) Conversion of End Functional Group of Polyethylene Glycol Derivative Having Benzylidene Acetal Linker

(99) The compound of the formula (18) is treated with a base such as sodium carbonate, sodium hydroxide, potassium carbonate, potassium hydroxide, lithium hydroxide, or ammonia in a protic solvent such as water, methanol, or ethanol or in an aprotic solvent containing them to obtain a compound of the formula (19) in which the trifluoroacetyl group is removed and converted into an amino group. The obtained compound may be purified by the aforementioned purification means.

(100) ##STR00022##

(101) Furthermore, the compound of formula (19) is allowed to react with N-succinimidyl 3-maleimidopropionate 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 with no solvent 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 to obtain a formula (20) in which a maleimide group is introduced into the end. The organic bases and inorganic bases may not be used. The use ratio of the organic base or the inorganic base is not particularly limited but is preferably equimolar or more to the reactive functional group of the compound of the formula (19). Also, it is possible to use an organic base as a solvent. The obtained compound may be purified by the aforementioned purification means.

(102) ##STR00023##

EXAMPLES

(103) The present invention will be further specifically described with reference to Examples but the invention should not be construed as being limited thereto.

(104) In .sup.1H-NMR analysis, JNM-ECP400 or JNM-ECA600 manufactured by JEOL Ltd. DATUM was used. For the measurement, a tube of 5 mm was used and tetramethylsilane (TMS) was used as an internal standard substance in the case where a deuterated solvent was CDCl.sub.3, CD.sub.3CN, or CD.sub.3OD or HDO is used as a standard in the case of D.sub.2O.

(105) In gel permeation chromatography (GPC) analysis, there were used SHODEX GPC SYSTEM-11 as a GPC system, SHODEX RIX8 as a differential refractometer that is a detector, and three columns, i.e., SHODEX KF801L, KF803L, and KF804L (8 mm300 mm) as GPC columns connected in series, and the temperature of the column oven was controlled to 40 C. Measurement was performed while tetrahydrofuran was used as an eluent, the flow rate was controlled to 1 ml/min, the concentration of a sample is controlled to 0.1 wt %, and the injection volume was controlled to 0.1 mL. As a calibration curve, there was used one created using ethylene glycol, diethylene glycol, and triethylene glycol manufactured by Kanto Chemical Co., Ltd. and Polymer Standards for GPC of polyethylene glycol or polyethylene oxide having a molecular weight of 600 to 70,000 manufactured by Polymer Laboratory. For analysis of data, BORWIN GPC calculation program was used. Mn represents number-average molecular weight, Mw represents weight-average molecular weight, and for molecular weight distribution, as Mw/Mn, calculated values thereof were shown.

(106) A deuterated water buffer of MES (2-morpholinoethanesulfonic acid) having pD 5.5 and a deuterated water buffer of HEPES (2-[4-(Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) having pD 7.4 for use in hydrolysis tests were prepared by adding a 0.1M sodium hydroxide deuterated water solution to a 0.1M MES deuterated water solution and a 0.1M HEPES deuterated water solution, respectively, based on the following relational equation described in [Glasoe, P. K.; Long, F. A. J. Phys. Chem. 1960, 64, 188-190].
pD=Measured value on pH meter+0.40

(107) A hydrolysis ratio was evaluated by .sup.1H-NMR and, in the case of the compounds of the formula (26), the formula (29) and the formula (30), the ratio was calculated according to the following calculation equation while an integrated value of the hydrogen of the acetal group was taken as I.sup.1, an integral value of the hydrogen of the aldehyde group to be formed by hydrolysis was taken as I.sup.2.
Hydrolysis ratio (%)=[I.sup.2/(I.sup.1+I.sup.2)]100
In the case of the compound of the formula (31), when an integrated value of the benzene ring bonded to the acetal group was taken as I.sup.3 and an integral value of the benzene ring of acetophenone formed by hydrolysis was taken as I.sup.4, the ratio was calculated according to the following equation.
Hydrolysis ratio (%)=[I.sup.4/(I.sup.3+I.sup.4)]100

Example 1

(108) Into a 300 mL four-necked flask equipped with a thermometer, a nitrogen inlet tube, a stirrer, a Dean-stark tube, and a condenser tube were charged 2-bromo-benzaldehyde dimethyl acetal (2.31 g, 10.0 mmol), N-(3-hydroxypropyl)phthalimide (8.21 g, 40.0 mmol), and toluene (120 g), and water was removed as an azeotrope with toluene. After cooling to room temperature, pyridinium p-toluenesulfonate (0.25 g, 1.0 mmol) was added and reaction was carried out for three hours while methanol formed as a by-product was removed as an azeotrope with toluene. After quenching with triethylamine (10 mL), the whole was diluted with a mixed solution of ethyl acetate (100 mL) and hexane (100 mL). After sequential washing with a saturated aqueous sodium hydrogen carbonate solution (100 mL) and saturated brine (100 mL), the organic layer was dried over anhydrous sodium sulfate. After filtration, the solvent was removed by distillation under reduced pressure and the residue was purified by recrystallization to obtain a compound of the formula (21). .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.92-2.01 (4H, m, CH.sub.2CH.sub.2CH.sub.2O), 3.53-3.67(4H, m, CH.sub.2CH.sub.2CH.sub.2O), 3.75-3.81(4H, m, CH.sub.2CH.sub.2CH.sub.2O), 5.64(1H, s, >CH), 7.08-7.49 (4H, m, arom. H), 7.64-7.81(8H, m, arom. H)

(109) ##STR00024##

Example 2

(110) Into a 100 mL three-necked flask equipped with a thermometer, a nitrogen inlet tube, and a stirrer were charged the compound of the formula (21) (1.00 g, 1.73 mmol), ethanol (36 mL), and hydrazine monohydrate (0.84 mL), and reaction was carried out at 60 C. for 6 hours. After cooling to room temperature and filtration, the filtrate was diluted with dichloromethane (108 mL). After washing with 10% brine (80 mL), the organic layer was dried over anhydrous sodium sulfate. After filtration, the solvent was removed by distillation under reduced pressure to obtain a compound of the formula (22).

(111) .sup.1H-NMR (CD.sub.3OD, internal standard TMS); (ppm): 1.73-1.78(4H, m, CH.sub.2CH.sub.2CH.sub.2O), 2.74(4H, t, CH.sub.2CH.sub.2CH.sub.2O), 3.57-3.70(4H, m, CH.sub.2CH.sub.2CH.sub.2O), 5.67(1H, s, >CH), 7.23-7.61(4H, m, arom. H)

(112) ##STR00025##

Example 3

(113) Into a 100 mL three-necked flask equipped with a thermometer, a nitrogen inlet tube, and a stirrer were charged the compound of the formula (22) (0.30 g, 0.95 mmol) and dry methanol (18 mL), and the whole was stirred while a solution obtained by dissolving ethyl trifluoroacetate (0.13 g, 0.95 mmol) into dry methanol (18 mL) was added dropwise. After dropwise addition, reaction was carried out at 25 C. for 2 hours. The solvent was removed by distillation under reduced pressure and the residue was diluted with t-butyl methyl ether (18 mL). After washing with ion-exchange water (18 mL), the organic layer was dried over anhydrous sodium sulfate. After filtration, the solvent was removed by distillation under reduced pressure to obtain a compound of the formula (23) as a mixture with a compound (contained in a ratio of 40 wt %) in which the amino groups at both ends were protected with a trifluoroacetyl group.

(114) .sup.1H-NMR (CD.sub.3OD, internal standard TMS); (ppm): 1.73-1.78(2H, m, NH.sub.2CH.sub.2CH.sub.2CH.sub.2O), 1.83-1.88(2H, m, CF.sub.3CONHCH.sub.2CH.sub.2CH.sub.2O), 2.74(2H, t, NH.sub.2CH.sub.2CH.sub.2CH.sub.2O), 3.40(2H, t, CF.sub.3CONHCH.sub.2CH.sub.2CH.sub.2O), 3.54-3.70(4H, m, CH.sub.2CH.sub.2CH.sub.2O), 5.68(1H, s, >CH), 7.23-7.63(4H, m, arom. H)

(115) ##STR00026##

Example 4

(116) Into a 300 mL four-necked flask equipped with a thermometer, a nitrogen inlet tube, and a stirrer were charged dry methanol (12.8 g, 0.400 mol), dry toluene (150 g), and metal sodium (0.3 g, 13 mmol), and the whole was stirred until the metal sodium was dissolved while nitrogen was introduced with bubbling. The solution was charged into a 5 L autoclave and, after the inside of the system was substituted with nitrogen, temperature was raised to 100 C. After ethylene oxide gas (1,987 g, 45 mol) was added at 100 to 130 C. under a pressure of 1 MPa or less, the reaction was continued for another 2 hours. After an unreacted ethylene oxide gas was removed under reduced pressure, the whole was cooled to 60 C. and pH was adjusted to 7.5 with an aqueous 85% phosphoric acid solution to obtain a compound of the formula (24).

(117) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 2.68(1H, t, OH), 3.38(3H, s, CH.sub.3O), 3.49-3.85(450H, m, (OCH.sub.2CH.sub.2).sub.n)

(118) GPC analysis; number-average molecular weight (Mn): 5119, weight-average molecular weight (Mw): 5226, polydispersity (Mw/Mn): 1.021
CH.sub.3(OCH.sub.2CH.sub.2).sub.nOH(24)
n=about 113

Example 5

(119) Into a 100 mL three-necked flask equipped with a thermometer, a nitrogen inlet tube, a stirrer, a Dean-stark tube, and a condenser tube were charged the compound of the formula (24) (10.0 g, 2.00 mmol) and toluene (40 g), and water was removed as an azeotrope with toluene. After cooling to 40 C., pyridine (0.71 g, 9.00 mmol) and N,N-disuccinimidyl carbonate (1.54 g, 6.00 mmol) was charged and reaction was carried out at 80 C. for 7 hours. After filtration, the whole was diluted with ethyl acetate (45 g) and crystallization was performed by adding hexane (16 g). After filtration, crystals were dissolved into ethyl acetate (76 g) and crystallization was again performed by adding hexane (24 g). After filtration, crystals were dried under reduced pressure to obtain a compound of the formula (25).

(120) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 2.84(4H, s, succinimide), 3.38(3H, s, CH.sub.3O), 3.48-3.85(448H, m, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2), 4.44-4.48(2H, m, CH.sub.2OCOO-succinimide)

(121) GPC analysis; number-average molecular weight (Mn): 5129, weight-average molecular weight (Mw): 5247, polydispersity (Mw/Mn): 1.023.

(122) ##STR00027##

Example 6

(123) Into a 100 mL three-necked flask equipped with a thermometer, a nitrogen inlet tube, and a stirrer were charged the compound of the formula (25) (1.0 g, 0.20 mmol) and dry dichloromethane (36 g), and the whole was stirred while a dry dichloromethane (4 g) solution of the compound of the formula (23) (purity: 60 wt %) (206 mg, 0.30 mmol) and N,N-diisopropylethylamine (38 mg, 0.30 mmol) was added dropwise. After completion of the dropwise addition, reaction was carried out at 25 C. for 3 hours. The solvent was removed by distillation under reduced pressure, the residue was dissolved in ethyl acetate (25 g), and then crystallization was performed by adding hexane (25 g). After filtration, drying was performed under reduced pressure to obtain a compound of the formula (26).

(124) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.78-1.93(4H, m, CH.sub.2CH.sub.2CH.sub.2O), 3.26-3.37(2H, m, OCONHCH.sub.2CH.sub.2CH.sub.2O), 3.38(3H, s, CH.sub.3O), 3.47-3.81 (454H, m, CF.sub.3CONHCH.sub.2CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2CH.sub.2O, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2), 4.20(2H, t, OCH.sub.2CH.sub.2OCONH), 5.11(1H, brs, OCONH), 5.67(1H, s, >CH), 7.21-7.59(4H, m, arom. H), 7.31(1H, brs, CF.sub.3CONH)

(125) GPC analysis; number-average molecular weight (Mn): 5424, weight-average molecular weight (Mw): 5549, polydispersity (Mw/Mn): 1.023.

(126) ##STR00028##

Example 7

(127) Into a 50 mL three-necked flask equipped with a thermometer, a nitrogen inlet tube, and a stirrer were charged the compound of the formula (26) (0.50 g, 0.10 mmol) and a 1M aqueous potassium carbonate solution (15 g), and reaction was carried out at 25 C. for 3 hours. Extraction was performed with dichloromethane (15 g) and, after the solvent was removed by distillation under reduced pressure, the residue was dissolved in ethyl acetate (25 g) and dried over anhydrous sodium sulfate. After filtration, crystallization was performed by adding hexane (25 g). After filtration, drying was performed under reduced pressure to obtain a compound of the formula (27).

(128) .sup.1H-NMR (CD.sub.3CN, internal standard TMS); (ppm): 1.62-1.68(2H, m, NH.sub.2CH.sub.2CH.sub.2CH.sub.2O), 1.71-1.77(2H, m, OCONHCH.sub.2CH.sub.2CH.sub.2O), 2.67-2.71(2H, m, NH.sub.2CH.sub.2CH.sub.2CH.sub.2O), 3.15-3.20(2H, m, OCONHCH.sub.2CH.sub.2CH.sub.2O), 3.29(3H, s, CH.sub.3O), 3.41-3.69(452H, m, CH.sub.2CH.sub.2CH.sub.2O, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2), 4.08(2H, t, OCH.sub.2CH.sub.2OCONH), 5.62(1H, s, >CH), 5.73(1H, brs, OCONH), 7.24-7.63(4H, m, arom. H)

(129) GPC analysis; number-average molecular weight (Mn): 5408, weight-average molecular weight (Mw): 5532, polydispersity (Mw/Mn): 1.023.

(130) ##STR00029##

Example 8

(131) Into a 50 mL three-necked flask equipped with a thermometer, a nitrogen inlet tube, and a stirrer were charged the compound of the formula (27) (0.20 g, 0.040 mmol) and acetonitrile (10 g), and N-succinimidyl 3-maleimidopropionate (32 mg, 0.048 mmol) was added thereto and reaction was carried out at 25 C. for 3 hours. After filtration, the solvent was removed by distillation under reduced pressure. The residue was dissolved in ethyl acetate (25 g) and then crystallization was performed by adding hexane (25 g). After filtration, drying was performed under reduced pressure to obtain a compound of the formula (28).

(132) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.78-1.83(4H, m, CONHCH.sub.2CH.sub.2CH.sub.2O), 2.44(2H, t, CH.sub.2CH.sub.2-maleimide), 3.27-3.37(4H, m, CONHCH.sub.2CH.sub.2CH.sub.2O), 3.38(3H, s, CH.sub.3O), 3.47-3.83(454H, m, CH.sub.2CH.sub.2CH.sub.2O, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2, CH.sub.2CH.sub.2-maleimide), 4.20(2H, t, OCH.sub.2CH.sub.2OCONH), 5.18(1H, brs, OCONH), 5.65(1H, s, >CH), 6.15(1H, brs, NHCO), 6.70(2H, s, maleimide), 7.20-7.60(4H, m, arom. H)

(133) GPC analysis; number-average molecular weight (Mn): 5561, weight-average molecular weight (Mw): 5694, polydispersity (Mw/Mn): 1.024.

(134) ##STR00030##

Example 9

(135) A compound of the formula (29) was obtained in the same manner as in Examples 1 to 6 using 2-nitro-benzaldehyde dimethyl acetal.

(136) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.83-1.91(4H, m, CH.sub.2CH.sub.2CH.sub.2O), 3.26-3.36(2H, m, OCONHCH.sub.2CH.sub.2CH.sub.2O), 3.38(3H, s, CH.sub.3O), 3.47-3.85(454H, m, CF.sub.3CONHCH.sub.2CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2CH.sub.2O, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2), 4.21(2H, t, OCH.sub.2CH.sub.2OCONH), 5.10(1H, brs, OCONH), 6.00(1H, s, >CH), 7.50-7.86(4H, m, arom. H), 7.27(1H, brs, CF.sub.3CONH)

(137) GPC analysis; number-average molecular weight (Mn): 5391, weight-average molecular weight (Mw): 5510, polydispersity (Mw/Mn): 1.022.

(138) ##STR00031##

Example 10

(139) A compound of the formula (30) was obtained in the same manner as in Examples 1 to 6 using 4-nitro-benzaldehyde dimethyl acetal.

(140) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.80-1.93(4H, m, CH.sub.2CH.sub.2CH.sub.2O), 3.31-3.34(2H, m, OCONHCH.sub.2CH.sub.2CH.sub.2O), 3.38(3H, s, CH.sub.3O), 3.47-3.81(454H, m, CF.sub.3CONHCH.sub.2CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2CH.sub.2O, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2), 4.20(2H, t, OCH.sub.2CH.sub.2OCONH), 5.09(1H, brs, OCONH), 5.56(1H, s, >CH), 7.27(1H, brs, CF.sub.3CONH), 7.62(2H, d, arom. H), 8.24(2H, d, arom. H)

(141) GPC analysis; number-average molecular weight (Mn): 5375, weight-average molecular weight (Mw): 5488, polydispersity (Mw/Mn): 1.021.

(142) ##STR00032##

Example 11

(143) A compound of the formula (31) was obtained in the same manner as in Examples 1 to 6 using 2-nitroacetophenone dimethyl acetal.

(144) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.74-1.88(7H, m, CH.sub.3, CH.sub.2CH.sub.2CH.sub.2O), 3.21-3.33(2H, m, OCONHCH.sub.2CH.sub.2CH.sub.2O), 3.38(3H, s, CH.sub.3O), 3.41-3.81(454H, m, CF.sub.3CONHCH.sub.2CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2CH.sub.2O, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2), 4.21(2H, t, OCH.sub.2CH.sub.2OCONH), 5.06(1H, brs, OCONH), 7.27(1H, brs, CF.sub.3CONH), 7.42-7.55(4H, m, arom. H)

(145) GPC analysis; number-average molecular weight (Mn): 5402, weight-average molecular weight (Mw): 5521, polydispersity (Mw/Mn): 1.022.

(146) ##STR00033##

Example 12

(147) ##STR00034##

(148) A compound of the formula (33) was obtained by removing the t-butyl group from the compound of the formula (32) synthesized by the method described in JP-A-2010-248504 using hydrochloric acid.

(149) .sup.1H-NMR (D.sub.2O, internal standard TMS); (ppm): 3.14(2H, t, NH.sub.2CH.sub.2), 3.40-4.00(452H, m, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2)
NH.sub.2(CH.sub.2).sub.2(OCH.sub.2CH.sub.2).sub.nOH (33)
n=about 113

Example 13

(150) A compound of the formula (34) was obtained by reacting the compound of the formula (33) with 5-azidopentanoic anhydride.

(151) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.60-1.74(4H, m, CH.sub.2CH.sub.2CH.sub.2CH.sub.2N.sub.3), 2.18(2H, t, CH.sub.2CH.sub.2CH.sub.2CH.sub.2N.sub.3), 3.29(2H, t, CH.sub.2CH.sub.2CH.sub.2CH.sub.2N.sub.3), 3.40-3.85(454H, m, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2, CONHCH.sub.2), 6.30(1H, brs, CONH)

(152) ##STR00035##

Example 14

(153) A compound of the formula (35) was obtained by reacting the compound of the formula (34) with N,N-disuccinimidyl carbonate in dichloromethane in the presence of triethylamine.

(154) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.60-1.74(4H, m, CH.sub.2CH.sub.2CH.sub.2CH.sub.2N.sub.3), 2.18(2H, t, CH.sub.2CH.sub.2CH.sub.2CH.sub.2N.sub.3), 2.84(4H, s, succinimide), 3.29(2H, t, CH.sub.2CH.sub.2CH.sub.2CH.sub.2N.sub.3), 3.40-3.85(452H, m, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2, CONHCH.sub.2), 4.44-4.48(2H, m, CH.sub.2OCOO-succinimide), 6.30(1H, brs, CH.sub.2CONH)

(155) ##STR00036##

Example 15

(156) A compound of the formula (36) was obtained by reacting the compound of the formula (35) with the compound of the formula (23) in the same manner as in Example 6.

(157) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.60-1.93(8H, m, CH.sub.2CH.sub.2CH.sub.2CH.sub.2N.sub.3, CH.sub.2CH.sub.2CH.sub.2O), 2.18(2H, t, CH.sub.2CH.sub.2CH.sub.2CH.sub.2N.sub.3), 3.26-3.37(4H, m, OCONHCH.sub.2CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2CH.sub.2CH.sub.2N.sub.3), 3.40-3.85(458H, m, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2, CH.sub.2CONHCH.sub.2, CF.sub.3CONHCH.sub.2CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2CH.sub.2O), 4.20(2H, t, OCH.sub.2CH.sub.2OCONH), 5.11(1H, brs, OCONH), 5.67(1H, s, >CH), 6.30(1H, brs, CH.sub.2CONH), 7.21-7.59(4H, m, arom. H), 7.31(1H, brs, CF.sub.3CONH)

(158) ##STR00037##

Example 16

(159) A compound of the formula (37) was obtained by removing the trifluoroacetyl group from the compound of the formula (36) in the same manner as in Example 7, followed by reaction with iodoacetic anhydride.

(160) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.60-1.93(8H, m, CH.sub.2CH.sub.2CH.sub.2CH.sub.2N.sub.3, CH.sub.2CH.sub.2CH.sub.2O), 2.18 (2H, t, CH.sub.2CH.sub.2CH.sub.2CH.sub.2N.sub.3), 3.26-3.37(6H, m, OCONHCH.sub.2CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2CH.sub.2CH.sub.2N.sub.3, ICH.sub.2CONHCH.sub.2), 3.40-3.85(458H, m, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2, CH.sub.2CH.sub.2CONHCH.sub.2, CH.sub.2CH.sub.2CH.sub.2O, ICH.sub.2CONHCH.sub.2), 4.20(2H, t, OCH.sub.2CH.sub.2OCONH), 5.11(1H, brs, OCONH), 5.67(1H, s, >CH), 6.30(1H, brs, CH.sub.2CH.sub.2CONH), 6.96(1H, brs, ICH.sub.2CONHCH.sub.2), 7.21-7.59(4H, m, arom. H)

(161) GPC analysis; number-average molecular weight (Mn): 5764, weight-average molecular weight (Mw): 5890, polydispersity (Mw/Mn): 1.024.

(162) ##STR00038##

Example 17

(163) ##STR00039##

(164) A compound of the formula (39) was obtained by reacting the compound of the formula (38) synthesized by the method described in JP-A-2004-197077 with N,N-disuccinimidyl carbonate in the same manner as in Example 5.

(165) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 2.84(4H, s, succinimide), 3.38(6H, s, CH.sub.3O), 3.40-4.00(903H, m, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2, (OCH.sub.2CH.sub.2).sub.nOCH<), 4.39(1H, dd, CH.sub.2OCOO-succinimide), 4.49(1H, dd, CH.sub.2OCOO-succinimide)

(166) ##STR00040##

Example 18

(167) A compound of the formula (40) was obtained by reacting the compound of the formula (39) with the compound of the formula (23) in the same manner as in Example 6.

(168) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.78-1.93(4H, m, CH.sub.2CH.sub.2CH.sub.2O), 3.26-3.37(2H, m, OCONHCH.sub.2CH.sub.2CH.sub.2O), 3.38(6H, s, CH.sub.3O), 3.40-4.00(909H, m, CF.sub.3CONHCH.sub.2CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2CH.sub.2O, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2, (OCH.sub.2CH.sub.2).sub.nOCH<), 4.08(1H, dd, CH.sub.2OCONH), 4.19(1H, dd, CH.sub.2OCONH), 5.05(1H, brs, OCONH), 5.67(1H, s, >CH), 7.21-7.59(4H, m, arom. H), 7.31(1H, brs, CF.sub.3CONH)

(169) GPC analysis; number-average molecular weight (Mn): 9697, weight-average molecular weight (Mw): 9920, polydispersity (Mw/Mn): 1.023.

(170) ##STR00041##

Example 19

(171) ##STR00042##

(172) A compound of the formula (42) was obtained by reacting the compound of the formula (41) synthesized by the method described in JP-A-2004-197077 with acetic anhydride in the presence of triethylamine and 4-dimethylaminopyridine.

(173) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 2.08(6H, s, CH.sub.3CO), 3.40-4.00(905H, m, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2, (OCH.sub.2CH.sub.2).sub.nOCH<, CH.sub.2OCH.sub.2Ph), 4.22(4H, t, CH.sub.3COOCH.sub.2), 4.54(2H, s, CH.sub.2OCH.sub.2Ph), 7.27-7.38(5H, m, CH.sub.2OCH.sub.2Ph)

(174) ##STR00043##

Example 20

(175) A compound of the formula (43) was obtained by removing the benzyl group from the compound of the formula (42) by the method described in JP-A-2004-197077 and subsequently reacting the resulting compound with N,N-disuccinimidyl carbonate in the same manner as in Example 5.

(176) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 2.08(6H, s, CH.sub.3CO), 2.84(4H, s, succinimide), 3.40-4.00(901H, m, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2, (OCH.sub.2CH.sub.2).sub.nOCH<), 4.22(4H, t, CH.sub.3COOCH.sub.2), 4.39(1H, dd, CH.sub.2OCOO-succinimide), 4.49(1H, dd, CH.sub.2OCOO-succinimide)

(177) ##STR00044##

Example 21

(178) A compound of the formula (44) was obtained by reacting the compound of the formula (43) with the compound of the formula (23) in the same manner as in Example 6.

(179) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.78-1.93(4H, m, CH.sub.2CH.sub.2CH.sub.2O), 2.08(6H, s, CH.sub.3CO), 3.26-3.37(2H, m, OCONHCH.sub.2CH.sub.2CH.sub.2O), 3.38(6H, s, CH.sub.3O), 3.40-4.00(907H, m, CF.sub.3CONHCH.sub.2CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2CH.sub.2O, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2, (OCH.sub.2CH.sub.2).sub.nOCH<), 4.08(1H, dd, CH.sub.2OCONH), 4.19(1H, dd, CH.sub.2OCONH), 4.22(4H, t, CH.sub.3COOCH.sub.2), 5.05 (1H, brs, OCONH), 5.67(1H, s, >CH), 7.21-7.59(4H, m, arom. H), 7.31(1H, brs, CF.sub.3CONH)

(180) ##STR00045##

Example 22

(181) A compound of the formula (45) was obtained by removing the acetyl group and the trifluoroacetamide group from the compound of the formula (44) using an aqueous sodium hydroxide solution and subsequently reacting the resulting compound with N-succinimidyl 3-maleimidopropionate in the same manner as in Example 8.

(182) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.78-1.83(4H, m, CONHCH.sub.2CH.sub.2CH.sub.2O), 2.44(2H, t, CH.sub.2CH.sub.2-maleimide), 3.27-3.37(4H, m, CONHCH.sub.2CH.sub.2CH.sub.2O), 3.40-4.00(909H, m, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2, (OCH.sub.2CH.sub.2).sub.nOCH<, CH.sub.2CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2-maleimide), 4.08(1H, dd, CH.sub.2OCONH), 4.19(1H, dd, CH.sub.2OCONH), 5.12(1H, brs, OCONH), 5.65(1H, s, >CH), 6.15 (1H, brs, NHCO), 6.70(2H, s, maleimide), 7.20-7.60(4H, m, arom. H)

(183) ##STR00046##

Example 23

(184) A compound of the formula (46) was obtained by reacting the compound of the formula (45) with N,N-disuccinimidyl carbonate in the same manner as in Example 14.

(185) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.78-1.83(4H, m, CONHCH.sub.2CH.sub.2CH.sub.2O), 2.44(2H, t, CH.sub.2CH.sub.2-maleimide), 2.84(4H, s, succinimide), 3.27-3.37(4H, m, CONHCH.sub.2CH.sub.2CH.sub.2O), 3.40-4.00(905H, m, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2, (OCH.sub.2CH.sub.2).sub.nOCH<, CH.sub.2CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2-maleimide), 4.08(1H, dd, CH.sub.2OCONH), 4.19(1H, dd, CH.sub.2OCONH), 4.44-4.48(4H, m, CH.sub.2OCOO-succinimide), 5.12(1H, brs, OCONH), 5.65(1H, s, >CH), 6.15(1H, brs, NHCO), 6.70(2H, s, maleimide), 7.20-7.60(4H, m, arom. H)

(186) GPC analysis; number-average molecular weight (Mn): 10069, weight-average molecular weight (Mw): 10321, polydispersity (Mw/Mn): 1.025.

(187) ##STR00047##

Example 24

(188) ##STR00048##

(189) A compound of the formula (48) was obtained by reacting the compound of the formula (47), which had been synthesized by polymerizing ethylene oxide to pentaerythritol, with N,N-disuccinimidyl carbonate in the same manner as in Example 5.

(190) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 2.84(16H, s, succinimide), 3.47-3.85(1800H, m, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2), 4.39(4H, dd, CH.sub.2OCOO-succinimide), 4.49(4H, dd, CH.sub.2OCOO-succinimide)

(191) ##STR00049##

Example 25

(192) A compound of the formula (49) was obtained by reacting the compound of the formula (48) with the compound of the formula (23) in the same manner as in Example 6.

(193) .sup.1H-NMR (CDCl.sub.3, internal standard TMS); (ppm): 1.78-1.93(16H, m, CH.sub.2CH.sub.2CH.sub.2O), 3.26-3.37(8H, m, OCONHCH.sub.2CH.sub.2CH.sub.2O), 3.47-3.85(1806H, m, CF.sub.3CONHCH.sub.2CH.sub.2CH.sub.2O, CH.sub.2CH.sub.2CH.sub.2O, (OCH.sub.2CH.sub.2).sub.nOCH.sub.2), 4.20(8H, t, OCH.sub.2CH.sub.2OCONH), 5.11(4H, brs, OCONH), 5.67(4H, s, >CH), 7.21-7.59(16H, m, arom. H), 7.31(4H, brs, CF.sub.3CONH)

(194) GPC analysis; number-average molecular weight (Mn): 19393, weight-average molecular weight (Mw): 19897, polydispersity (Mw/Mn): 1.026.

(195) ##STR00050##

Example 26

(196) Each of the compounds (20 mg) of the formula (26), the formula (29), the formula (30), and the formula (31) were dissolved in MES deuterated water buffer (1 mL) of pD 5.5 and HEPES deuterated water buffer (1 mL) of pD 7.4 and allowed to stand in a thermostatic bath at 37 C. FIG. 1 and FIG. 2 show results of measuring hydrolysis ratios at pD 5.5 and pD 7.4, respectively.

(197) As shown in FIG. 1, half lives (t.sub.112) of the hydrolysis ratios of the compounds of the formula (26), the formula (29), the formula (30), and the formula (31) at pD 5.5 at 37 C. were 24 hours, 2.2 months, 18 days, and 36 hours, respectively. Moreover, at pD 7.4 and 37 C., hydrolysis in a degree of about 10% was observed for 18 days for the compounds of the formula (26) and the formula (31) but no hydrolysis was observed even after 18 days for the compounds of the formula (29) and the formula (30).

(198) While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.