JPS6256898B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a polymer having a large number of protected carboxyl groups 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. 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 an intermediate for producing 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 having 5 to 500 anticancer drug molecules covalently bonded to each molecule, such as polydaltamic acid, are bonded through an amide bond to form an antitumor drug. It is disclosed that it has been obtained. The antitumor agent obtained by this method selectively binds to tumor cells,
It is a drug of great interest as it is expected to exert toxicity on tumor cells. However, a drawback of this known antitumor agent is that the antitumor antibody and the toxic moiety (polymer carrier bound to the antitumor agent) are bound to each other through an amide bond, that is, via a free amino group or carboxyl group in the antitumor antibody. The point is that this is done through a combination of Immunoglobulins also have many amino groups and carboxyl groups in their antigen recognition sites. Therefore, when a cytotoxic substance or toxic moiety is bound to an anti-tumor immunoglobulin through an amide bond, the cytotoxic substance will also bind to the antigen recognition site of the anti-tumor immunoglobulin, and as a result, the resulting Antitumor drugs no longer have the ability to bind to tumor cells at all, or
Otherwise, a problem arises in that it is lowered. In addition, in the method described in JP-A-51-126281, within the antibody molecule and within the polyglutamic acid molecule,
Alternatively, amide bonds are formed between molecules of the same type. And the antitumor agents obtained as a result of the formation of these undesirable amide bonds have reduced performance,
Furthermore, there is the problem that they contain 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 it is possible to bind cytotoxins or toxic moieties using disulfide bonds that exist in limited positions in immunoglobulins. It was discovered that an antitumor agent free from the above-mentioned drawbacks can be obtained. The present invention also provides intermediates for producing reactive polymers that can be optimally used in producing 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 [], X is a protecting group for a carboxyl group, and represents an alkyl group having 1 to 4 carbon atoms or a benzyl group. m represents an integer from 1 to 4. ] Formula [] in the main chain or at the carboxyl terminal of the main chain
It has a disulfide bond-containing group represented by In [Formula [], W represents an alkylene group having 1 to 4 carbon atoms. R ₁ is a hydrogen atom or has 1 or more carbon atoms
4 represents an alkyl group. When the group represented by the formula [] is the terminal group of the main chain, R ₂ represents an alkyl group, an aralkyl group, or an aryl group,
When the group represented by the formula [] is present in the main chain,

It is a divalent group represented by the formula: However, W′ has 1 to 1 carbon atoms, which is the same as or different from W.
4, and is bonded to S in 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 polymer having a disulfide bond in the molecule with a degree of polymerization of 5 to 3000. For example, by subjecting the polymer of the present invention to acid or alkali decomposition, the protecting groups X of the many protected carboxyl groups present in the side chains are removed, and the many reactive carboxyl groups ( or its salt)
is converted into a reactive polymer with Then, using this carboxyl group (or its salt), a cytotoxic substance such as an anticancer drug can be bound to it,
Furthermore, immunoglobulins such as anti-tumor antibodies can be bound to this by utilizing disulfide bonds present in the main chain or at the carboxyl terminus of the main chain. 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, with a benzyl group being particularly preferred. m represents an integer of 1 to 4, preferably m is 1 or 2. In addition, in the polymer of the present invention, among the structural units represented by the formula [], for example, those in which m=1 and those in which m=2 may be mixed. The total amount of these is 60 mol% or more, preferably 80 mol% of the total structural units.
It is sufficient if there is more than that. In the polymer of the present invention, 40 mol% of the total structural units
Constituent units other than those represented by formula [] may be included within the range below. Examples of these include glycine, alanine, which does not have a carboxyl group (or its salt) in the α-position side chain,
There are α-amino acids such as phenylalanine and serine. Such a constituent unit consisting of α-amino acids does not participate in any way in binding with cytotoxins, but is useful in regulating the water solubility of the polymer and the fat solubility and water solubility of the polymer obtained by binding cytotoxins. It may be helpful. 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 polymer of the present invention and the subsequent reaction process. These groups include, for example, 2-aminoethanethiol residue (-
linear or cysteine benzyl ester residues such as CH ₂ CH ₂ −)

[Formula] and homocysteine benzyl 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 a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, preferably a hydrogen atom. When the group represented by the formula [] is the terminal group of the main chain, R ₂ is an alkyl group, and the aralkyl group is an aryl group. In this case, the 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

It is a divalent group represented by the formula: In this case, the polymer of the present invention can be expressed as follows (provided that the structural unit of formula [] is
100 mol%). (p and q each represent the number of structural units.) The polymer of the present invention has a degree of polymerization of 5 to 3000, preferably 10 to 1500. When the degree of polymerization is less than 5, the amount of lipotoxic substances that can bind to the reactive polymer obtained from the polymer of the present invention is small, resulting in efficient reactivity when producing antitumor agents, etc. It does not form a polymer. When the degree of polymerization exceeds 3000, it is not only difficult to produce, but also inconvenient to handle when producing antitumor agents. The polymer of the present invention can be produced by the following method. That is, the formula [] (The definitions of x and m are the same as in the case of the formula [].) Using the N-carboxylic acid anhydride represented by the formula as a polymerization initiator, the formula [] R' ₂ -S-S-W-NHR ₁ ...[ ] [Definitions of W and R ₁ are the same as in formula []. R' ₂ is an alkyl group, an aralkyl group, an aryl group, or -
It is a divalent group represented by W'-NHR' ₁ . W′ and
The definition of R' ₁ is the same as in the case of formula []. ] It can be produced by a method of polymerizing using an amine compound having a disulfide bond in the molecule represented by the following. Examples of the polymerization initiator represented by the formula [] include cystamine (H ₂ NCH ₂ CH ₂ SSCH ₂ CH ₂ NH ₂ ), n-propyl 2
-Aminoethyl disulfide (CH ₃ CH ₂ CH ₂ SSCH ₂ CH ₂ NH ₂ ), 4-aminophenyl disulfide

[Formula] Cystine diesters (

【formula】

[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
was gradually dropped over 70 minutes to generate phosgene. The generated phosgene is the γ-benzyl-
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 (described below). structural formula) Obtained 7.75 g as a white solid. The melting point of this product was 94.0-94.5°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. Example 2 5.50 g of γ-benzyl-L-glutamate N-carboxylic acid anhydride obtained in Reference Example was dried 1.4
-Dissolved in 150 ml of dioxane under nitrogen atmosphere with stirring. A solution obtained by dissolving 142 mg of n-propyl 2-aminoethyl disulfide (CH ₂ 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 stirred 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%. Example 3 10.0 g of γ-benzyl-L-glutamate N-carboxylic acid 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.

Claims (1)

[Scope of Claims] 1 60 mol% or more of the structural units consist of structural units represented by the formula [], [In the formula [], X is a protecting group for a carboxyl group, and represents an alkyl group having 1 to 4 carbon atoms or a benzyl group. 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, _-CH3 ,
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 is a hydrogen atom or has 1 or more carbon atoms
4 represents an alkyl group. R ₂ represents an alkyl group, an aralkyl group, or an aryl group when the group represented by formula [ ] is the terminal group of the main chain, and when the group represented by formula [] is present in the main chain, , is a divalent group represented by [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. ] A polymer having a disulfide bond in the molecule with a degree of polymerization of 5 to 3000. 2. The polymer having a disulfide bond in the molecule according to claim 1, wherein in formula [], X is a benzyl group. 3 formula [] [In the formula [], X is a protecting group for a carboxyl group, and represents an alkyl group having 1 to 4 carbon atoms or a benzyl group. m represents an integer from 1 to 4. ] N-carboxylic acid anhydride represented by, or this product and the formula [′] [In formula ['], R is -H, _-CH3 ,
It is a group represented by [Formula] or -CH ₂ OH. ] The proportion of the N-carboxylic anhydride represented by [] to the N-carboxylic anhydride represented by [ ] is
A mixture in a range of 60 mol% or more is used as a polymerization initiator with the formula [] R ₂ '-S-S-W-NHR ₁ ...[] [In the formula [], W has 1 to 4 carbon atoms. represents an alkylene group. R ₁ is a hydrogen atom or has 1 or more carbon atoms
4 represents an alkyl group. _R2 ' is an alkyl group, an aralkyl group, an aryl group, or a group represented by -W'- _NHR1 '. W' is the same or different from W and is an alkylene group having 1 to 4 carbon atoms, and R ₁ ' is the same or different from R ₁ and represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ] Characterized by polymerization using an amine compound having a disulfide bond in the molecule represented by the formula, 60 mol% or more of the structural units are composed of the structural unit represented by the formula [], [In formula [], the definitions of X and m are the same as in the above formula []. ] When the constituent units of formula [] are not 100 mol%,
The remaining structural units are expressed by the formula [′] [In formula ['], the definition of R is the above formula [']
Same as in the case of ], 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 is a hydrogen atom or has 1 or more carbon atoms
4 represents an alkyl group. R ₂ represents an alkyl group, an aralkyl group, or an aryl group when the group represented by formula [ ] is the terminal group of the main chain, and when the group represented by formula [] is present in the main chain, , is a divalent group represented by [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. ] A method for producing a polymer having a disulfide bond in the molecule and having a degree of polymerization of 5 to 3000.