JPS62138430A - Production of conjugate of methotrexate with protein - Google Patents

Abstract

PURPOSE:To obtain the titled conjugate useful as a remedy for leukemia or solid tumor, etc., by reacting a methotrexate with a dehydrating condensing agent and reacting the reaction product with an alcohol to form an active ester of the methotrexate. CONSTITUTION:Methotrexate expressed by formula I is reacted with a dehydrating condensing agent, e.g. DDC, to form a cyclic intramolecular acid anhydride expressed by formula II (R is formula III), which is then reacted with an alcohol, e.g. N-hydroxysuccinimide, to afford an active ester of the methotrexate expressed by formula IV or V. The resultant active ester is then reacted with a protein, e.g. blood serum albumin, to give the aimed conjugate of the methotrexate with the protein. When an active ester obtained by reacting the methotrexate with an equimolar amount of the dehydrating condensing agent is used, the increase in molecular weight of the protein in the next step is suppressed to afford the aimed conjugate in high yield.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing mentreguisate and protein sulfate, which are therapeutic agents for leukemia, solid food, etc.
More specifically, the present invention relates to a method for producing the conjugate that suppresses the production of JP7 cridate, a polymerized protein LF.

In recent years, it has become known that conjugates of mentrexate and proteins can be useful anti-cancer agents. and exhibits antitumor activity (Barbara C, F.

Chu et al. J. Natl. Cancer In5t.
66, 121 (1981): 5).

Also, mentrexate and blood 7R albumin f411
(A complex in which k molecules are further bound to an anti-tumor antibody can be a selectively acting anti-tumor drug (M, C).

Garnett et al., Int. J. Cancer,
31, 661 (1983)).

A conjugate of menthone xate and an antitumor antibody, transferrin, etc. is also expected to be an antipulmonary agent that selectively acts on tumors.

and the carboxyl group that mentrexate has.

The active ester method is a method for producing a bond between mentrexate and a protein by forming an amide bond between the amino group of the protein.

Carbodiimide method, mixed acid pyrohydride method, etc. are known. The active ester method is the most effective way to produce a bound protein while retaining the activity of methotrexate, since the inherent electrical potential of a protein, such as r in the case of an antibody, does not inhibit its antigen binding ability. It has been reported that it is present immediately (P, N.

Kulkarni et al. + CancerResea
rch, 41, 2700 (1981)).

The Kulkarni active ester method reported above consists of two steps. First, in the first step, equal moles M of each of the kings of mentrexate, N-hydroxysuccinimide (Ho5u), and N,N'-dicyclohexylcarbodiimide (DCC), which is a dehydration condensation agent, are mixed in a sushi pot. Then, in the 2Jl step, the product from the first step is reacted with the antibody protein in water M solution to form the mentrexate antibody conjugate. ing. Since the active ester of mentrexate produced in the tlEl step is highly reactive and unstable, it is reacted in the second step without being isolated and purified.

However, the one-line method cannot be said to be a satisfactory method for the following reasons. The molecule of mentrexate has α-9r-2
Because of the presence of 2 carboxyl groups, in addition to α-2γ-2 types of monoesters, °C diesters are produced during ester formation.

Diesterdino formation is not preferred in the second step of the protein binding reaction. The reason for this is that diester is 2
Because it can react with two different amine groups, it acts as a cross-linking agent to cross-link protein molecules.

This is to promote the generation of polymerized products and aggregates of proteins that are unsuitable for therapeutic agents and Li.
In the first step, an equimolar amount of a dehydration condensation agent is applied to mentrexate at 1. After that, an active ester is produced by reacting with alcohol.

Findings 1: If the product mixture thus obtained is used in the second step, polymerization of the protein can be suppressed and a desirable mentrexate protein '11 conjugate can be obtained.
1. The present invention has been achieved.

That is, in the first step, the present invention reacts a dehydration condensation agent with a dehydration condensation agent, which is represented by the following formula This is a method for producing a conjugate of mentrexate and protein, which is characterized in that the active ester is reacted with a protein in the second step.

In order to further clarify the method of the present invention, a chemical reaction formula will be shown to explain how the polymerization of protein occurs.

1st step 2nd step In the n'cl step of the method of the present invention, mentrexate produces a cyclic intramolecular acid anhydride through the reaction of nontrexate with the dehydration condensation agent in the same molar amount. ,
Next, by allowing an alcohol to act on this product, α-1 or r-ester is produced through a ring-opening reaction.

The proportion of diester in the active ester mixture obtained by the first step in the method of the invention is Ku
It was confirmed by the following experiment that the proportion of dispur in the product of the first step in the method of Lkarni et al. Have difficulty).

The active ester mixture obtained by each method was reacted with a low-molecular amine, and the proportion of diamide in the resulting mixture of 7 amides was analyzed by high performance liquid chromatography. As a result, it was revealed that the reaction mixture obtained by the method of the present invention was smaller than that obtained by the method of Kullcarni et al. The results of high performance liquid chromatography analysis are shown in Table 1.

The dehydration condensation agent used in the first step of the present pruning method is N,N'-dicyclohexylcarbodiimide (DCC
), 1-ethyl-3-(carbodiimides such as 3-dimethylaminopropyl, ethyloxycarbonyl chloride,
carbonic acid monoalkyl ester chloride such as inphthyloxycarbonyl chloride, N-ethoxycarbonyl, 2-ethoxy-1,2-dihydroquinoline, etc., but especially DCC
and EDCI are preferably used.

Alcohol refers to
-Human ρxyl,2,3-benzotriazole, N-humanroxyphthalimide, N-humanroxy5-norhorne-2,3-dicarboximide, p-nitrophenol, 2,4-dinitrophenol, 2,4.5
These include r-lichlorophenol, pentafluorophenol, etc. Among them, N-hydroxysuccinidide is particularly preferably used.

As the solvent used in the first step of the present invention, any solvent can be used as long as it dissolves mentrexate and does not participate in the reaction. For example, dimethylformamide, dimethylsulfoxide, hexamethylphosphorotriamide, etc. are preferable.

The dehydration condensation agent is 0.5 to 1.0% with respect to mentrexate.
It is preferable to use 5 molar equivalents, more preferably 1.
0 molar equivalent is used. The reaction temperature is -800 to 50°C, particularly preferably -30Q to 4o. A time period of 0.5 to 24 hours is sufficient for the reaction with the dehydration condensation agent.

Next, an alcohol is allowed to act, but the amount used is preferably 1 to 2 times the mole of mentrexate. The reaction temperature may be the same as or different from the dehydration condensation reaction, and is -800 to 50
It is ℃. , the reaction time is 0.5 to 24 hours.

The reaction can also be promoted by adding 1.0 to 10.0 equivalents of a base such as triethylamine or pyridine to the furfurs.

In the second step, the above-mentioned reaction solution is used, but if a precipitate is generated at °C, it is preferable to separate it and use it at °C. The protein was prepared at a concentration of o. d of a reaction mixture containing the above mentrexate active ester dissolved in 5 my to 200 μ/7
Add liquid. The concentration of limethotrexate in the solution is determined in advance by measuring ultraviolet absorbance at ℃ (thereby, the amount added can be adjusted. The reaction temperature is 0゛'
~30°C2 The appropriate reaction time is 1 to 24 hours. After the reaction is completed, the reaction solution is purified by gel filtration or dialysis.

Below, 1゜ will be explained using all the examples of the present invention and comparative examples. Example 1゜ Production of Mentrexate Serum Frucumin Binding Book.

(II 1st Step Mentorexe) To a solution of 454 M9 dissolved in 15 m of dimethylform 7mide, 206 g of N,N'-dicyclohexylcarbodiimide was added under ice cooling, and the mixture was heated at 4°C.
The reaction was carried out for 15 hours. Next, to this, N-hydroxysuccinimide 11511i+ and pyridine 158 Q
was added and reacted for 6 hours.

The resulting solid substance was separated into i1, washed with a small amount of dimethylformamide, and added to solution 7 containing mentrexate active ester.
．． I got 6-. The concentration of mentrexate active ester present in this solution was determined by the method of Kullcarni et al. (Cancer Research, 41.2).
700 (1981)), +04
．． , IFFIM was 1, (1j, ν in the above solution
The concentration of nomentrexate was determined by measuring the absorbance at 372 weight m (-72] group-containing Glue Afft-gel 102NicoQ) solution.
714> Add () to react, then wash the gel to recover unreacted mentrexate and determine its amount (
-It is. The difference from the initial value, that is, the concentration of mentrexate active ester, was determined by determining the amount of gel bound to the gel. )(2)
2nd step: Dissolve human serum argumin 17.4Q, 0.14
To a pH 8,000, 1M phosphate buffer containing M sodium chloride (hereinafter abbreviated as buffer A) 0.6-, the first
10 of the nontrexate active ester obtained in the process.
4.4mM dimethylformamide solution 0.2d'&
, Add VC little by little with gentle stirring [4° after -1]
The mixture was reacted for 4 hours (this means that 79 molecules of active ester of mentrexate were added to one molecule of albumin and reacted). After centrifuging the precipitate, the supernatant was diluted with lomM phosphorus #i containing 0.14 M sodium chloride, buffer, pH 7.
Sephadec, Z, G-25 column (0,6X 40crIK
) to remove unreacted mentrexate derivatives and low molecular weight reaction products to obtain the desired mentrexate-bound human serum albumin solution 2.2-.

From the absorbance at 372 nm, the concentration of bound mentrexate residues is 0.9811ST/-;
On the other hand, protein determination by bio-rad protein assay (Analytical Biochemist)
ry, Vol. 72, p. 248, 1976),
Albumin concentration is 5.6711! i/ml. From this, the number of mentrexate residues bound per molecule of human serum albumin was calculated to be 25.2.

Example 2 Manufacture of mentrexate immunoglobulin binding book.

Mentrexate-activated esthetics/l, (/J 9
0.6 mM dimethylborumamide solution? , 4μt,
The mixture was reacted for 4 hours at a pressure of 6≧40 with gentle stirring (this means that 1 molecule of IgG was reacted with 10 molecules of mentrexate derivatives).

After centrifuging the precipitate, the supernatant was applied to a Sephadex G-25 column (0.6 x 40 crn) flattened in buffer B to collect the high molecular weight fraction, and to collect the target substance, the mesothre A-sade bond. Rabbit IgG2.8- was obtained.

At 372 nm? The concentration of bound 1-mentrexate residues was 19.8 µV' from the absorbance at 6 µV, while the IgG C1) concentration was 1.83 ρIy/- from protein quantification by bio-radoprotein assay. became. From this, the number of bonded methotrexate residues per molecule of rabbit IgG was calculated to be 3.6.

Comparative Examples Known methods (Kurekarni et al.'s method, Can
cerResearch, 41, 2700 (19
Using menthone xate active ester prepared according to 81), a reaction with rabbit IgG was carried out, and compared with Example 2, 1-σ containing 10 μ of purified rabbit IgG was prepared.
] 17.1 μL of a 39 mM dimethylformamide solution of mentrexate active ester prepared by the method of Kurekarni et al. was added to buffer A with gentle stirring, and then incubated at 4° for 4 hours. reacted (
This means that 1 molecule of IgG was reacted with 10 molecules of mentrexate derivatives). Hereinafter, the same procedure as in the example was carried out at 10°C. Mentrexate-conjugated rabbit I containing 4.0 methotoxate residues per molecule of rabbit IgG.
1, Comparative Example and Example 20 In the production example of the present invention, IgG 1.85 me was obtained.
Comparative data on the recovery rate and the production rate of polymers that are unsuitable as products are shown in Table 2.1. Ta.

The production rate of the polymer was determined by scanning the electrophoresis pattern obtained by SDS-containing polyacrylamide gel electrophoresis using a chromato scanner (manufactured by Koguruma Seisakusho, dual-wavelength pMado scanner C).
Analysis 1-1 was calculated using 3=930).

As is clear from Table 2, according to the method of the present invention, a high IgG recovery rate and a low polymerization book production rate can be achieved.

Claims (1)

[Claims] 1. In the first step, methotrexate represented by the following formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼...[I] is reacted with a dehydration condensation agent, and then an alcohol A method for producing a conjugate of methotrexate and protein, which comprises reacting to obtain an active ester of methotrexate, and then reacting the active ester with a protein in a second step. 2. The manufacturing method according to claim 1, wherein the dehydration condensation agent is a carbodiimide. 3. The manufacturing method according to claim 1 or 2, wherein the alcohol is N-hydroxysuccinimide or phenol. 4. The manufacturing method according to claim 1, 2, or 3, wherein the protein is serum albumin, immunoglobulin, or transferrin.