JPS6238369B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a highly reactive hydrophilic polymer having a large number of carboxyl groups (or salts thereof) in its side chain and a disulfide bond-containing group in the main chain or at the carboxyl end of the main chain, and a method for producing the same. Regarding. The purpose of the present invention is to manufacture target-directed anti-cancer drugs (anti-tumor drugs) by combining anti-tumor antibodies, etc. that have the ability to bind to targets such as tumor cells, and cytotoxic substances, such as anti-cancer drugs. The object of the present invention is to provide a polymer that can be used to effectively and efficiently combine the two. BACKGROUND ART Conventionally, it has been known to use a reactive polymer as a mediator in order to efficiently bind an antitumor antibody and a cytotoxin to produce a target-directed anticancer agent. For example, in JP-A No. 51-126281, an antitumor immunoglobulin and a polymer carrier (e.g., polyglutamic acid) to which 5 to 500 molecules of an anticancer drug are covalently bound per molecule are bonded to each other through an amide bond. It is disclosed that a tumor agent has been obtained. The antitumor agent obtained by this method is expected to selectively bind to tumor cells and exhibit toxicity to tumor cells, making it a very interesting drug. However, the drawbacks of this known antitumor agent are that
The point is that the binding between the anti-tumor antibody and the cytotoxic substance (polymer carrier bound to the anti-cancer drug) is carried out through an amide bond, that is, through a free amino group or carboxyl group in the anti-tumor antibody. It is. Immunoglobulins also have many amino groups and carboxyl groups in their antigen recognition sites.
Therefore, when a cytotoxin is bound to an antitumor immunoglobulin through an amide bond, the cytotoxin also binds to the antigen recognition site of the antitumor immunoglobulin, and as a result, the resulting antitumor agent The problem arises that the cells no longer have the ability to bind to tumor cells completely or are reduced.
Furthermore, in the method described in JP-A-51-126281, amide bonds are formed within antibody molecules, within polyglutamic acid molecules, or even between molecules of the same type. As a result of the formation of these undesirable amide bonds, the performance of the resulting antitumor agent is reduced, and there is also the problem that it contains high molecular weight substances that are unsuitable for use in tumor treatment. The present inventors have conducted extensive research to solve the drawbacks of the prior art, and have found that if cytotoxic substances are bound using disulfide bonds that exist in limited positions in immunoglobulins, the above-mentioned drawbacks can be overcome. It was discovered that an antitumor agent can be obtained. The present invention provides a hydrophilic polymer that can be optimally used in the production of such antitumor agents or other target-directed drugs. That is, in the present invention, 60 mol% or more of the structural units consist of structural units represented by the formula [], In [Formula [], Z represents a hydrogen atom or a monovalent cation. m represents an integer from 1 to 4. ] When the constituent units of formula [] are not 100 mol%,
The remaining structural units are expressed by the formula [′] [In formula [′], R is -H, -CH ₃ ,

It is a group represented by [Formula] or -CH ₂ OH. ], and has a disulfide bond-containing group represented by the formula [] in the main chain or at the carboxyl terminal of the main chain, In [Formula [], W represents an alkylene group having 1 to 4 carbon atoms, and R ₁ is a hydrogen atom or an alkylene group having 1 to 4 carbon atoms.
~4 alkyl group. R ₂ represents an alkyl group, an aralkyl group, or an aryl group when the group represented by the formula [] is a terminal group of the main chain, and when the group represented by the formula [] is present in the main chain, teeth,

It is a divalent group represented by the formula: However, W′ has the same or different carbon number as W
It is an alkylene group of ~4 and is bonded to S of the formula []. R ₁ ' is the same as or different from R ₁ and represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ] It is a hydrophilic polymer with a degree of polymerization of 5 to 3000. The hydrophilic polymer of the present invention can bind cytotoxic substances such as anticancer drugs to it by utilizing a large number of carboxyl groups (or salts thereof) present in the side chains,
Furthermore, it is possible to bind immunoglobulins such as antitumor antibodies to the disulfide bonds present in the main chain or at one end of the main chain. In formula [], Z is a hydrogen atom or a monovalent cation, such as Na ⁺ , K ⁺ , NH ₄ ⁺ . m is 1
It represents an integer of ~4, but m is preferably 1 or 2. In addition, in the hydrophilic polymer of the present invention, among the structural units represented by the formula [], for example, those with m=1 and those with m=2 may be mixed. These are the total of all constituent units.
The content should be at least 60 mol%, preferably at least 80 mol%. The hydrophilic polymer of the present invention contains 40 of the total structural units.
Constituent units other than those represented by the formula [] may be contained within a range of less than mol%. Examples of these include α-amino acids, such as glycine, alanine, phenylalanine, and serine, which do not have a carboxyl group (or a salt thereof) in the α-position side chain. Such a constituent unit consisting of an α-amino acid does not participate in any way in binding with a cytotoxic substance, but it regulates the water solubility of a hydrophilic polymer and the lipid solubility and water solubility of a polymer obtained by binding a cytotoxic substance. It may be helpful to do so. Therefore, if there is no particular need to adjust fat solubility or water solubility, it is practically advantageous to use a product that does not contain such a constituent unit consisting of an α-amino acid. In formula [], W and W' represent divalent organic groups, and are not particularly limited as long as they are inert groups that do not participate in any reaction during the process of obtaining the hydrophilic polymer of the present invention and the subsequent reaction process. These groups include, for example, straight chain residues such as 2-aminoethanethiol residues (-CH ₂ CH ₂ -), or cysteine benzyl ester residues.

[Formula] and homocysteineben Dyl ester residue

Alkylene group having a side chain such as [Formula], 4-aminothiophenol residue

Phenylene groups having no substituents or having substituents as shown in the formula are exemplified, and alkylene groups having 1 to 4 carbon atoms are particularly preferred. R ₁ and R ₁ ' are hydrogen atoms or carbon atoms 1 to 4
is an alkyl group, and a hydrogen atom is preferred. R ₂ is an alkyl group, an aralkyl group, or an aryl group when the group represented by the formula [] is a terminal group of the main chain. In this case, the hydrophilic polymer of the present invention can be expressed as shown below (however, the structural unit of formula [] is 100 mol%). (n represents the number of structural units.) When the group represented by the formula [] is present in the main chain, _R2 is

[Synthesis of γ-benzyl-L-glutamate N-carboxylic acid anhydride]

A dispersion was prepared by adding 10.0 g of γ-benzyl-L-glutamic acid to 120 ml of anhydrous tetrahydrofuran. Separately, under a nitrogen atmosphere, add 20 ml of trichloromethyl chloroformate to carbon black 10.0
The generated phosgene was gradually added dropwise over 70 minutes to generate phosgene.
The dispersion of L-glutamic acid was sucked under a nitrogen atmosphere. After 70 minutes, the dispersion became a pale yellow transparent liquid, so the phosgene was stopped, and then nitrogen was sucked in for 1.5 hours to remove unreacted phosgene. The solvent was distilled off from the resulting transparent liquid under reduced pressure under a nitrogen stream (140 mmHg, 27°C). 150 ml of anhydrous n-hexane was added to the residue to dissolve it, and the mixture was stirred on an ice bath for 5 minutes to precipitate a white solid. This solid was purified by reprecipitation twice in a system of ethyl acetate-n-hexane (anhydrous) under a nitrogen atmosphere, collected by suction filtration, and dried under reduced pressure to produce γ-benzyl-L-glutamate N-carboxylic anhydride ( It has the following structural formula) Obtained 7.75 g as a white solid. The melting point of this thing is
The temperature was 94.0-94.°C (decomposition), and the yield was 69.8%. Example 1 7.75 g of γ-benzyl-L-glutamate N-carboxylic acid anhydride obtained in Reference Example was dried 1,4-
It was dissolved in 185 ml of dioxane under a nitrogen atmosphere with stirring. To the solution thus obtained, a solution obtained by dissolving 95 mg of cystamine (H ₂ NCH ₂ CH ₂ SSCH ₂ CH ₂ NH ₂ ) in 10 ml of dry dioxane was added and mixed, and the mixture was incubated at room temperature under nitrogen atmosphere for 24 hours. The mixture was stirred to carry out a polymerization reaction. After the reaction, the reaction mixture was added to the isopropyl ether of 4 with stirring to precipitate the produced polymer. When the precipitated white polymer was taken and dried under reduced pressure, the yield was 6.19g.
The yield was 95.9%. The average molecular weight of the obtained polymer was determined by the viscosity method (dichloroacetic acid, 25.0°C) and was found to be 47,300. (P. Doty et al., J. Am.
Chem.Soc., vol. 78, p. 947, 1956). The obtained polymer was determined from the raw materials, initiator, and reaction mechanism used to form a poly-γ-benzyl-L-
It was reasonably estimated that the substance was mainly composed of glutamate, and it was also confirmed by infrared absorption spectra. 3.11 g of poly-γ-benzyl-L-glutamate obtained above was dissolved in a mixed solution of 25.0 ml of trifluoroacetic acid and 4.5 ml of anisole. To the thus obtained solution, 25.0 ml of methanesulfonic acid was added, and the mixture was stirred for 20 minutes on an ice bath under a nitrogen atmosphere, and then stirred for 30 minutes at room temperature to carry out an acid decomposition reaction of the γ-benzyl ester. After the reaction, the reaction mixture
The polymer was precipitated by adding it to 450 ml of isopropyl ether with stirring. The precipitated white polymer was sucked out and suspended in 50 ml of water. Approximately 60 ml of saturated deuterated water was added to this and mixed. The mixture was stirred at room temperature for 30 minutes to carry out a neutralization reaction of carboxyl groups.
Thereafter, the obtained reaction solution was dialyzed against pure water at 4° C. for 3 days using a cellulose tube, and then freeze-dried to obtain 1.91 g of white solid. When the obtained solid was examined by infrared absorption spectrum, it was confirmed that the absorption of benzyl ester had disappeared and that the carboxyl group had become a sodium salt. The yield of poly-L-glutamate as sodium salt was 89.3%. In addition, viscosity method (salt-phosphoric acid buffer mixture, ionic strength 0.11 and
The average molecular weight determined by 1.01, 25.5℃) is 29200
It was hot. (RBHawkins et al., Macromolecules,
5, p. 294, 1972). The obtained polymer is
It is mainly composed of sodium salt of poly-L-glutamate of the following formula. Example 2 5.50 g of γ-benzyl-L-glutamate N-carboxylic acid anhydride obtained in Reference Example was dried 1,4-
It was dissolved in 150 ml of dioxane under a nitrogen atmosphere with stirring. A solution obtained by dissolving 142 g of n-propyl 2-aminoethyl disulfide (CH ₃ CH ₂ CH ₂ SSCH ₂ CH ₂ NH ₂ ) in 10 ml of dry dioxane was added to the solution obtained in this manner, and the mixture was heated under a nitrogen atmosphere. The mixture was stirred at room temperature for 40 hours to carry out a polymerization reaction. . After the reaction, the reaction mixture was added to the isopropyl ether of 4 with stirring to precipitate the produced polymer. When the precipitate was collected and dried under reduced pressure, the yield was 4.41 g, with a yield of 96.3%. Next, 4.00 g of the obtained polymer was dissolved in a mixture of 35 ml of trifluoroacetic acid and 5.0 ml of anisole, and 35 ml of methanesulfonic acid was added, and the mixture was incubated for 30 minutes under ice-cooling under a nitrogen atmosphere and for 30 minutes at room temperature. By stirring, the γ-benzyl ester was acid-decomposed. After the reaction was completed, the reaction mixture was added to 540 ml of isopropyl ether with stirring, and the polymer was precipitated and collected. This was dissolved in 100 ml of 5.0 deuterium soybean water and a carboxyl group neutralization reaction was carried out.
Next, the reaction solution was poured into a cellulose tube.
Dialysis was performed against pure water at 4°C for 3 days. When the obtained solution was freeze-dried, sodium poly-L-
2.33 g (77.4% yield) of glutamate was obtained as a hygroscopic flocculent solid. No benzyl ester absorption was observed in the infrared absorption spectrum of the product, confirming that the carboxyl group had become a sodium salt. The average molecular weight was 16,700 when measured using the same method as above. The obtained polymer is mainly composed of a sodium salt of poly-L-glutamate of the following formula. Example 3 10.0 g of γ-benzyl-L-glutamate N-carboxylic anhydride obtained in Reference Example and 0.23 g of L-alanine N-carboxylic acid anhydride were added to 280 ml of dry 1,4-dioxane under a nitrogen atmosphere. and stirred to dissolve. To the solution thus obtained, 198 mg of 4
A solution obtained by dissolving -aminophenyl disulfide in 10 ml of dry dioxane was added and mixed, and the mixture was stirred at room temperature under a nitrogen atmosphere for 24 hours to carry out a polymerization reaction. After the reaction, the reaction mixture was added to the isopropyl ether in step 4 with stirring, and the resulting polymer was used as a precipitate. The polymer precipitate was collected and dried under reduced pressure to obtain 8.21 g. The yield was 97%.
The obtained polymer is reasonably estimated to be a copolymer of γ-benzyl-L-glutamate and L-alanine as described below from the raw materials, initiator, and reaction mechanism used, and also has an infrared absorption property. It was also confirmed by spectra. 4.0 g of the thus obtained copolymer was dissolved in a mixed solution of 30 ml of trifluoroacetic acid and 5.0 ml of anisole. To the obtained solution, 30 ml of methanesulfonic acid was added, and the mixture was stirred for 20 minutes on an ice bath under a nitrogen atmosphere, and then stirred for 30 minutes at room temperature to carry out an acid decomposition reaction of the γ-benzyl ester. After the reaction, the reaction mixture was added to 600 ml of isopropyl ether with stirring to precipitate the polymer. The precipitated white polymer was suctioned off and suspended in 65 ml of water. Approximately 80 ml of saturated deuterated water was added and mixed to this, and the mixture was stirred at room temperature for 30 minutes to perform a neutralization reaction of carboxyl groups, thereby forming a homogeneous solution. The resulting solution was heated to pure water at 4°C using a cellulose tube.
for 3 days and then lyophilized to obtain 2.45 g of white solid. When the obtained solid was examined by infrared absorption spectrum, it was confirmed that the absorption of benzyl ester had disappeared and that the carboxyl group had become a sodium salt. The yield as sodium salt was 88%. The obtained polymer is mainly composed of a sodium salt of a polymer of L-glutamate and L-alanine represented by the following formula.

Claims (1)

[Claims] 1. 60 mol% or more of the structural units consist of the structural units represented by the formula [], [In the formula [ ], Z represents a hydrogen atom or a monovalent cation. m represents an integer from 1 to 4. ] When the constituent units of formula [] are not 100 mol%,
The remaining structural units are expressed by the formula [′] [In formula [′], R is -H, -CH ₃ ,
It is a group represented by [Formula] or -CH ₂ OH. ], and has a disulfide bond-containing group represented by the formula [] in the main chain or at the carboxyl end of the main chain, In [Formula [], W represents an alkylene group having 1 to 4 carbon atoms. R ₁ is a hydrogen atom or carbon number 1
~4 alkyl group. R ₂ represents an alkyl group, an aralkyl group, or an aryl group when the group represented by the formula [] is a terminal group of the main chain, and when the group represented by the formula [] is present in the main chain, is a divalent group represented by [Formula]. However, W′ has the same or different carbon number as W
It is an alkylene group of ~4 and is bonded to S of the formula []. R ₁ ' is the same as or different from R ₁ and represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ] Hydrophilic polymer with a degree of polymerization of 5 to 3000. 2 60 mol% or more of the structural units consist of the structural units represented by the formula [], In [Formula [], X is a protecting group for a carboxyl group, and represents an alkyl group having 1 to 4 carbon atoms, a benzyl group, or a substituted benzyl group. m represents an integer from 1 to 4. ] When the constituent units of formula [] are not 100 mol%, the remaining constituent units are of formula [′] [In formula [′], R is -H, -CH ₃ ,
It is a group represented by [Formula] or -CH ₂ OH. ], and has a disulfide bond-containing group represented by the formula [] in the main chain or at the carboxyl terminal of the main chain, In [Formula [], W represents an alkylene group having 1 to 4 carbon atoms. R ₁ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ₂ represents an alkyl group, an aralkyl group, or an aryl group when the group represented by the formula [] is a terminal group of the main chain, and when the group represented by the formula [] is present in the main chain, teeth,
It is a divalent group represented by the formula: However, W' is an alkylene group having 1 to 4 carbon atoms, which is the same as or different from W, and is bonded to S in the formula []. R ₁ ' is the same as or different from R ₁ and represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ] It is characterized in that a polymer having a degree of polymerization of 5 to 3000 is decomposed with an acid or alkali to remove the protecting group X of the carboxyl group. 60 mol% or more of the structural units consist of the structural units represented by the formula [], In [Formula [], Z represents a hydrogen atom or a monovalent cation. m represents an integer from 1 to 4. ] When the constituent units of formula [] are not 100 mol%, the remaining constituent units are of formula [′] [In the formula, the definition of -R is the same as in the above formula [']. ], and has a disulfide bond-containing period represented by the formula [] in the main chain or at the carboxyl terminal of the main chain, In [formula [], the definitions of W, R ₁ and R ₂ are the same as in the above formula []. ] A method for producing a hydrophilic polymer having a degree of polymerization of 5 to 3000. 3. The method for producing a hydrophilic polymer according to claim 2, wherein in formula [], X is a methyl group or a benzyl group.