JPS60168726A - Cytotoxic substance-bound reactive polymer and its production - Google Patents

Abstract

PURPOSE:To obtain a cytotoxic substance-bound reactive polymer useful as a cytotoxic substance in the production of a target-directing carcinostatic agent, by reacting a specified polysuccinimide with a cytotoxic substance containing an amino or imino group. CONSTITUTION:A polymer of formula I (wherein Y is a water-soluble aliphatic primary amine, W is a lower alkylene, Z is H, a group which, together with the adjacent S, can form an active disulfide group, or a like group, n', m', l', r', p', and q' are each 0 or a natural number, n'+m'>=2, 0.001<=p'+q'/n'+m'+l'+r'+ p'+q'<=0.5). This polymer is reacted with a cytotoxic substance containing an amino or imino group and, optionally, a water-soluble primary aliphatic amine to obtain a polysuccinamide derivative represented by formula II (wherein X is a residue of a cytotoxic substance containing an amino or imino group in the molecule) and having a MW of 2,000-3,000,000.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to reactive polymers bound with cytotoxic substances, intermediates thereof, and methods for their production. More specifically, the present invention provides reactivity that can be used as a cytotoxic agent in the production of a target-directed cancer agent by combining a cytotoxic agent with an anti-tumor antibody that has the ability to bind to a target such as a tumor cell. It relates to polymers, their production methods, their intermediates, and their production methods.

It has been known to produce antitumor agents by binding antitumor antibodies to cells and toxins. Polymer carriers are used to bind a large number of cytotoxic substances to antitumor antibodies without reducing the target binding ability of the antitumor antibodies. A typical polymer used as a polymeric carrier is dextran polyglutamic acid. In the case of dextran, it is mainly oxidized with periodic acid etc. to form an aldehyde derivative and used for C, but it is difficult to obtain a conjugate with good reproducibility because the cytotoxic substance and anti-tumor antibody are reacted using the same functional group. Moreover, the anti-tumor antibody binds with the anti-tumor antibody more than necessary, producing a high molecular weight substance, which tends to reduce the ability to bind to tumor cells and the like. Furthermore, polyglutamic acid is not desirable as a targeting agent because it has a low charge and has the above-mentioned drawbacks, which lowers the binding ability of antibodies and at the same time causes it to be trapped in specific organs in the body.

The present inventors have conducted intensive studies to solve these drawbacks, and as a result, we have developed a polymer carrier that has no charge, is inexpensive, does not easily reduce the activity of anti-tumor antibodies, and has a tumor-controlling effect. This discovery led to the present invention.

The present invention provides reactive surface molecules conjugated with cytotoxic substances that can be used to produce targeted therapeutic agents, and which have the ability to bind anti-tumor antibodies with targeting ability.

That is, the present invention is a reactive polymer represented by formula [1] to which a cytotoxic substance is bound.

[In the formula, X, Y, W and 2 each have the following meanings.

X: Reactive residue or hydroxyl group of a cytotoxic substance having an amino group or imino group in the molecule Y: Water-soluble aliphatic primary amine residue W: Lower alkylene group 2: Hydrogen atom or general formula 1 or -S A group represented by -SZ, where Z' is a group capable of forming an active disulfide bond with the adjacent sulfur atom, and a group is a hydrocarbon (however, if 2 is a hydrogen atom, the 18H group represented by -SZ is a molecule) -5-5-bonds may be formed in equilibrium with other 8H groups within or between molecules;
It shall be expressed as H. ) Further, n, m, l, r, p and 9 are 0 or a natural number and indicate the number of constituent units, and the relationship between them is as follows.

n 10m≧2 0.001≦p+q/n-)-m-H+r+p+q≦0
．． 50) and has a molecular weight of 2000 to 8,000.
,000 polysuccinimide derivative. Kenmei 4□Here, Z in general formula (I) is a hydrogen atom or general formula 2'
2' represents a group capable of forming an active disulfide bond with the adjacent sulfur atom, but the latter (
Z') is, for example, 2-pyridylthio group 8-carboxyl-4-nitrophenylthio-, -)
Low 2- Hi+) Silch, t 7E (o2N-c
! j-s-), and N-phenylamino-N'-phenyl.

W is a lower alkylene group, such as a linear or branched alkylene group having 1 to 4 carbon atoms.

X in formula (1) represents a reactive residue of a cytotoxic substance containing an amino group or an imino group in the molecule or a hydroxyl group.

A cytotoxic substance in the present invention refers to a substance that exhibits toxicity to cells as it is, or a substance that does not exhibit toxicity as it is but can be converted into a substance capable of exhibiting toxicity to cells in vivo.

Examples of these include p-(N,N-bis(2-chloroethyl)]phenylenediamine P-(bis(2-chloroethyl)amino)-L-phenylalanine (melphalan) 2-amino-N-(p-bis( 2-chloroethyl)amine] phenyl-8-hydroxypropionamide 1-(5'-(2-aminoethylphosphoryl)-β-D
-arabinofuranosyl)cytosine 2-amino-N-(
p-bis(2-chloroethyl)aminocophenyl-8-
Hydroxy-2-hydroxymethylpropionamide methotrexate cH, cti.

Adriamycin D mitomycin C x=ii, daunomycin It is not necessary to use it for the purpose of the present invention (
4? +r=0) is introduced for the purpose of improving the water solubility of the reactive polymer.

The water-soluble aliphatic amines used include ethanolamine, aliphatic amines having a hydroxyl group such as 8-amino and -1,2-propanediol, and CHs (CHs).
g"Hz (gt = an integer of 1 to 4), etc., such as aliphatic amines. In particular, ethanolamine having a hydroxyl group, 8-
Amines such as amino-1°2-propanediol are preferred.

n0m is the number of structural units that X is bonded to, 4+r
represents the number of structural units to which the water-soluble aliphatic amine is bonded, and p+q represents the number of structural units to which Z is bonded.

n+m/n+m+6+r+p+q is the number of condensed moles of X per mole of succinimide group, which is a monomer unit.

p+q/n+m+6+r+p+q has the general formula -NH-W per mole of succinimide group, which is a monomer unit.
It represents the number of condensed moles of the group represented by -3-Z.

The molecular weight was expressed as the value obtained by quantifying the terminal amino group.

The arrangement of the structural units in the polymer is arbitrary. That is, although formula [1] is expressed as a block polymer for convenience, it includes, for example, a random copolymer, and is not limited thereto.

The molecular weight is 2.000 to a, o o o, o o o, preferably 2.000 to 8,000,000, and n
Ten meters is more than 2, n+m/n+m-+1! +r+p+q is less than 1 and p+q /n+m+6+r+p+q is 0
．． 001 or more and 0.50 or less, preferably 0.8 or less.

If Ft is less than this, the number of cytotoxic substances bound to one molecule of the reactive polymer decreases, and the amount of cytotoxic substances that can bind to anti-tumor antibodies etc. decreases, which is not preferable. Moreover, if the molecular weight is too large, aggregates of anti-tumor antibodies are likely to be formed, and insolubilized components and large molecular weights are likely to be generated, which is not preferable.

p+q/n+m+6+r+p+q is equal to or greater than o, o o i, and when it is less than this, the binding ratio with anti-tumor antibodies etc. becomes low, which is not preferable. Also, 0.6 or less, preferably 0.8
If the amount is less than or equal to too much, aggregates of anti-tumor antibodies etc. tend to occur (and the insolubilized components and large molecular weight components increase, which is not preferable).

The reactive polymer of the present invention represented by general formula (1) is:
It can be easily derived from polysuccinimide containing the structural unit of the following general formula [2] as a main component.

[In the formula, k represents a natural number. ]

Polysuccinimide is a polymer also known as anhydropyaspartic acid, and can be obtained mainly by thermal condensation of aspartic acid. (J,
Kovacset al, J, Org, Chem
, 26 1084 (1961) iP, Neri
et al, J, Med, Chem, ↓189
8(197B)') As an example of a specific production method, aspartic acid (which may be an optically active form or a DL form) is heated to 16 ml under normal pressure or reduced pressure.
It can be obtained by dehydration polymerization at 0°C or higher and 240°C or lower. The more severe the dehydration conditions are, the faster the polymer can be obtained, but if the temperature is increased above this level, the polymer will decompose, which is undesirable. If the temperature is lower than this, it will take a long time to obtain the polymer, which is not preferable.

After polymerization, the reaction product is dissolved in DMF or DMSO and reprecipitated with water to remove unreacted aspartic acid and low-molecular condensate, followed by filtration through a glass filter and drying. Repeat this operation if necessary.

IRSNMR suggests that the polymer thus obtained is represented by the general formula [2], but even if it partially contains other constituent units, it is substantially the same as the general formula [2]. It is generally accepted that the constituent units are the main components.

The polysuccinimide [2] thus obtained is reacted with a compound having an amino group, such as ethanolamine, and
It is known that the reaction shown below is carried out.

0 [2] C In the formula, k, x and γ represent natural numbers. ] It is known that the following reaction formula can be carried out under alkaline conditions.

[2] [In the formula, x and γ represent natural numbers. ] There are two methods for obtaining the polysuccinimide derivative (1) in the present invention.

The first method is to obtain a reactive polymer [8] in the formula [1] where X, , Of(, and then a reactive polymer [1] in which In other words, it is a method of converting polysuccinimide into a derivative represented by the following general formula [8], further bonding a cytotoxic substance containing an amine group or an imino group in the molecule, and if necessary, converting polysuccinimide into a derivative represented by the following general formula [8]. This method involves reacting with an amine.

The second method is to obtain a polymer [1] in which X = cytotoxic substance, and it is a method that is effective for binding cytotoxic substances having amine groups, and is used to bind cytotoxic substances, antitumor antibodies, etc. A functional group donating compound (NH, -W-8-7,
11) Both water-soluble aliphatic primary amines are directly converted to succinimide [1] using the amine group they have.
This is the way to do it.

As the first method, there are the following methods (11 to (3)).

+11 (al polysuccinimide and (a) general formula) I
! A compound represented by N-W-S-Z (W and 2 are the same as above), after reacting with a water-soluble aliphatic primary amine if necessary, is treated with an aqueous Alekali solution. General formula [
8] intermediate is obtained.

(bl) followed by a method of reacting with a water-soluble aliphatic primary amine (2) Polysuccinimide and (a) (a) General Formula H,
N -W-8-8-R (W is the same as above. K is an alkyl group (for example, a lower alkyl group having 1 to 5 carbon atoms), an aralkyl group (for example, an unsubstituted or substituted phenylalkyl group), or an aryl group (for example, an unsubstituted or substituted phenylalkyl group). (substituted or substituted phenyl group)), (b) If necessary, after reacting with a water-soluble aliphatic-grade amine, it is treated with an aqueous alkali solution to form a compound of the general formula (8) (Z=- 3-R) intermediate is obtained.

(b) Thereafter, (c) the cytotoxic substance containing an amino group or an imino group is reacted with a water-soluble aliphatic primary amine, if necessary.

After the reaction of (a) or lb), if necessary, the disulfide group of the reactive product is reductively cleaved by reacting with a thiol compound or a borohydride compound, etc., so that 2 = hydrogen atom in formula [l]. A polymer of

(8) There is a method of obtaining a thiol-type reactive polymer (in formula (1), Z is a hydrogen atom) obtained by any of the above methods with an active disulfide ratio compound. In 1), a polymer with Z=Z/ is obtained.

Furthermore, as the method of (g $5 and (2) above),
e\ in (a), (b) and (b) in a)
), ←) is a sequential reaction method in which reactions are performed in any order,
There is a simultaneous reaction method in which two components are reacted simultaneously.

As a reaction solvent, DMF or DMSO, which is a good solvent for polysuccinimide, is used in the reactions (a) and (b).

When the reaction of polysuccinimide or (a) or (b) is carried out by a sequential reaction method, the polysuccinimide derivative reacted with (a) or (b) is
W ) % c7) D MF Mata D M SO
One or two components (a), f) in the solvent (o, o per mole of succinimide group in the solution)
ot ~ 1O mol) and 1 min ~ 2 min at -40 ~ 80 °C
It is appropriate to carry out the reaction under stirring for several days. Unreacted components can be removed by dialysis or gel filtration. In order to obtain the intermediate represented by the general formula [8], the above (11,
(It is desirable that there is an unreacted succinimide group in the reaction intermediate of (λ) before treatment with an alkaline aqueous solution in 21. This is because the unreacted succinimide group undergoes hydrolysis to form a 1000H group.

Even when there is no unreacted succinimide group (A) or (01
The reaction residue undergoes hydrolysis to form a 1000H group. However, under such conditions, the main chain is likely to undergo hydrolysis and become a low-molecular compound, so the former is preferable. Conditions that leave unreacted succinimide groups can be obtained by separately increasing the reaction rate between (a) or (cl) and polysuccinimide.

Regarding the number of moles of (a) and (b) used, it is desirable that the total number of moles of (a) or, if necessary, (a) and (b) be less than the total number of moles of succinimide groups used. Carboxyl groups can be easily introduced into the polysuccinimide derivative thus obtained by subjecting it to alkaline conditions. The reaction with an alkaline aqueous solution can be carried out by dropping the alkaline aqueous solution into a DMF or DMSO solution, or by adding the polysuccinimide derivative into an alkaline aqueous solution. The alkaline aqueous solution may have a pH of 3 or more, but a 0.0 IN to 2 N NaOH aqueous solution is suitable.

The reaction of the intermediate represented by the general formula [8] with method (1), (e＼ in 21, ni)) can be carried out in any sequential order by a sequential reaction method, or simultaneously by a simultaneous reaction method. There is a reaction method. For the purpose of the present invention, it is preferable to introduce a large amount of (c), so it is preferable to react 11) and then react 1)).

The reaction is usually carried out in a homogeneous reaction system using water or an organic solvent such as DMF, DMSO, etc. as the reaction solution.

During the reaction, carboxyl groups in the polymer may be activated into a mixed acid anhydride using, for example, l-ethyl-8-(8-dimethylaminopropyl)carbodiimide hydrochloride or dicyclohexylcarbodiimide. The reaction temperature is suitably -40C to 100C, and the reaction time is suitably 10 minutes to 10 days. The ratio of the intermediate used and (c) is 1 of the intermediate
0 to 200%, particularly 100 to 200% is desirable. (c)
If a carboxyl group remains unreacted in the reaction with (2), the carboxyl group is eliminated in the reaction with (2).

In the method (1), Z is preferably a hydrogen atom.

When using a cytotoxic substance containing an amino group, it can be produced by the second, simpler method.

A cytotoxic substance containing an amide 7 group refers to a compound that is cytotoxic and has the ability to form an amide bond by reacting the amino group in the molecule with the succinimide group 0 1 1 of polysuccinimide.

In other words, it must have an amino group that reacts with a succinimide group to form an amide bond.

Examples of these include daunomycin, adriamycin, P-amino-N-(p-bisC2-chloroethyl)
Aminocophenyl-8-hydroxyamide, 2-amino-N-(p-bis(2-chloroethyl)aminocophenyl-8-hydroxy-2-hydroxymethylpropionamide, P-[bis(2-chloroethyl)amino) −
Examples include those having an aliphatic amino group such as L-phenylalanine.

The second method includes methods (4) to (6) below.

(4) Polysuccinimide [2] and (a) a cytotoxic substance having an amino group (b) general formula H2N-W-8-Z (
W, Z are the same as above), and f→
If necessary, it can be obtained by reaction with a water-soluble aliphatic amine.

(5) Polysuccinimide [2] and (a) Cytotoxic substance having an amino group (b) General formula H2N-W-5-8-
A method of reacting a compound represented by R (W, R are the same as above) and GTl) with a water-soluble aliphatic primary amine to obtain a disulfide type polymer, or a subsequent reaction product if necessary. A method of obtaining a thiol type polymer by reacting the disulfide group of a substance with a thiol compound or a borohydride compound and reductively cleaving it.

(6) There is a method of obtaining the thiol-type reactive polymer obtained by any of the above methods (in formula (1), by reacting Z=f() with an active disulfide compound. In this way, Z is A reactive polymer (1) is obtained.

Furthermore, as methods (4) and (5) above, the above (
There are sequential reaction methods in which the reactions with each component (a), (b), and (c) are carried out sequentially in any order, and simultaneous reaction methods in which reactions with multiple components are carried out simultaneously.

When a reaction with a component of mar:eq is required, a two-step method is possible in which the reaction with any one component is carried out alone and the reactions with the remaining two components are carried out simultaneously.

As a reaction solvent, DMF or DMSO, which is a good solvent for polysuccinimide, is used in the first reaction and simultaneous reaction in the sequential reaction or two-step reaction. The second and subsequent reactions in a sequential reaction or two-step reaction can be carried out in an aqueous solvent when DMF or DMSO or the polysuccinimide derivative obtained in the first or second reaction becomes water-soluble. .

In the reaction methods (4) and (5) above, (a) and (b).

The reaction of the component (c) is carried out by the following operation.

Polysuccinimide [2] or the unreacted succinimide group mide derivative after the first or second reaction, 1 to 1g of it
o (w, "w)% of DMF, DMSO or water solvent, one or more of the power components (a), (b), and (c) to be reacted (one per mole of succinimide group in the solution) 0.001 to 10 mol per component) and -40 to
The reaction is carried out at 80° C. for 10 minutes to 20 days with stirring.

Unreacted components can be removed by dialysis or gel filtration.

The desired polysuccinimide derivative (1) can be obtained by distilling off the solvent under reduced pressure or by freeze-drying.

A cytotoxic substance and a compound represented by the general formula H2N-W-5-Z (W and Z are the same as above) or a compound represented by the general formula H2N-W-5-5-R (W and k are the same as above)
The method for controlling the amount of the compound represented by (-) to be introduced is to know in advance the reaction rate with the succinimide group under certain conditions when carrying out the reaction with the - component, and then to control the concentration of the succinimide group in the solution, the temperature, This is possible by determining the reaction time and the amount of addition of the components to be reacted. When carrying out simultaneous reaction with two or more components, this can be done by knowing in advance the relative reaction rate of each component in a mixture of two or more components and adjusting the mixing ratio and amount of the components to be added.

The molecular weight is controlled by selecting the condensation conditions to obtain the polysuccinimide polymer, and the separation of the polymer having the desired molecular weight is carried out using DMF of the polysuccinimide polymer.
Molecular weight fractionation by gel filtration in a solution and molecular weight fractionation by gel filtration in an aqueous solvent after obtaining a polysuccinimide derivative.

In the method (4), Z is preferably a hydrogen atom.

Selection of condensation conditions for obtaining a polysuccinimide polymer in order to improve the yield of the desired molecular weight fraction. Molecular weight fractionation is performed in advance by gel filtration with a DMF solution of polysuccinimide.

In the methods (8) and (6) above, a thiol-type reactive polymer (Z=H in general formula (1)) is reacted with an active disulfide compound to produce a reactive polymer having an active disulfide group (general formula ( The active disulfide compound used in the method for obtaining Z=Z/ ) in 1) includes a disulfide of the group represented by Z' in general formula [1],
Specifically, the disulfide form of the above-mentioned group exemplified as Z', for example, the disulfide form of 2-pyridylthio group 2
-4 which is a disulfide of pyridi4-pyridylthio group
-Pyridyl disulfide (N-s-s)), 5,5'- which is a disulfide of 8-carboxyl-4-nitrophenylthio group.
Dithiobis (2-nitro).

The reaction between the thiol-type reactive polymer and the active disulfide compound is usually carried out in a homogeneous reaction system using water or an organic solvent such as DMF or DMSO as a reaction solvent. Or again,
The reaction can also be carried out using a reaction system in which an active disulfide compound or its acetoid solution or dioxane solution is added and mixed to an aqueous solution of the polymer. Suitable reaction temperature is -5 to 70°C and reaction time is 1 minute to 24 hours.

The reactive polymer bound to the cytotoxic substance of the present invention has a thiol group in the molecule that is cytotoxic and has high reactivity due to the cytotoxic substance, or a disulfide group from which a thiol group can be easily obtained from a ring element, Alternatively, since it contains an active disulfide group, N-succinimidyl 8-(2-pyridyldithio ) Propionate (SPDP
), or N-
(meta-maleimidobenzoyloxy)succinimide (
It is possible to bind to anti-tumor antibodies etc. via the maleimide group introduced from N-hydroxysuccinimide ester (CHM) of N-(4-carboxycyclohexylmethyl)maleimide (MBS) and N-(4-carboxycyclohexylmethyl)maleimide.

As a method of bonding a thiol group derived from a disulfide bond of an anti-tumor antibody with a thiol group derived from a reactive polymer, one maleimide group of a simalimide compound such as 0-phenylene dialeimide is attached to the anti-tumor antibody or a reactive polymer in advance. There is a method in which a maleimide group is introduced by reacting with one thiol group of a polymer, and then reacted with the other thiol group.

Thiol groups derived from disulfide bonds in antitumor antibodies and the like can be obtained by reducing the disulfide bonds of the antibody with a reducing agent such as dithiothreitol or cysteamine.

Further, a dimer of Fab' is obtained by treating the antibody with pepsin, and the disulfide bond possessed by this 24-dimer is cleaved with a reducing agent such as dithiothreitol or cysteamine to obtain Fab' having a thiol group in the molecule. .

The thus obtained reactive polymer [1] and its conjugate with a substance having targeting ability, such as an antitumor antibody, are expected to be effective as selective cancer therapeutics. The present invention will be explained in detail below with reference to Reference Examples and Examples.

Reference Example 1 Polymerization of vorisuccinimide L-aspartic acid 66.5 F to 85% phosphoric acid 20.
6 ml was added and polymerization was carried out under reduced pressure of 10 ffHr for 5.5 hours while heating to 190° C. using a rotary evaporator.

After polymerization, dissolve in DMF and reprecipitate with water. After re-sedimentation, it was passed through a lath filter and vacuum-dried at 80°C for 8 days.

When the infrared absorption of the obtained polymer was measured by the KBr tablet method, absorption peculiar to succinimide groups was observed at 1800.1720°-1660 cyn.

Molecular weight measurement was performed by GPC measurement. GPC for DMF
Using a column (AD-3QM/S-Showa Denko), the obtained polysuccinimide F wo DMF + 0.01 M
It was dissolved in LtBr solvent to a concentration of 0.2%, 260 μl of this solution was injected, and measurement was performed using a differential refractometer using the same solvent as the elution solvent. A calibration curve was created using polyethylene glycol of various molecular weights.

The obtained polysuccinimide had a weight average molecular weight of 8.9 x 1.0 and a number average molecular weight of 1 in terms of polyethylene glycol.
．． It was 5×10.

Example 1 Polysuccinimide 1.56F (obtained in the above reference example)
90 g 1 succinimide group (equivalent to 3 mmoe)
of DMF.

To this solution was added 84.7 μ of cysteamine (0.45 rr-n)
o(1), x)i/-/L/7mi7658vRf(l□
, 7mmod) in 10g/DMF was added, and the reaction was carried out at 4°C for 24 hours. After reaction, add IN to the reaction solution.
Approximately 50 ml of NaOH solution was added dropwise over 1 hour while stirring. After that, dialysis was performed with an aqueous solution having a pH of 4. After concentrating to Lost using Diaflow Membrane 18) (manufactured by Amicon, molecular weight: 10,000), 5ephadex'
G-25 Super Fire (manufactured by Pharmasha) 2.
Gel filtration was performed using a 6 cm x f x 40- column to remove low molecular components.

Freeze-drying was performed to obtain an intermediate having the general formula [8] where Z=hydrogen atom.

When the thiol groups in the intermediate were determined by the method shown in Example 8, 1.5×10' moles of thiol groups were present in the intermediate lf. The amount of carboxyl group was determined by acid-base titration according to a conventional method. As a result, intermediate l
There were 2.8×10 mol of carboxyl groups in f. No unreacted succinimide group was observed from IR and C-NMR measurements.

From the above measurement results, the amount of cysteamine introduced (K mol) and the amount of carboxyl group introduced (7 mol) per mol of succinimide group were determined from the following simultaneous equations.

a(1-x-y)+bx+cy γ a (1-x-y) + bx + c y Here, a, b, c, t, and s are as follows.

Molecular weight of the monomer unit in which the succinimide group reacted with ethanolamine fit a = 158 Molecular weight of the monomer unit in which the succinimide group reacted with cysteamine
1)=174 Molecular weight of the monomer unit in which the succinimide group is hydrolyzed C=115 Intermediate (amount of cysteamine introduced into 811f t=1,
42XlO mol Amount of carboxyl group introduced in intermediate (8) If S=2
．． As a result of 8XIO mole calculation, X = 0.02 mole, y = o, a mole.

The molecular weight was determined by measuring the terminal amine. Intermediate 51NI above was dissolved in O,l M sodium borate solution and then 2.4.6-dolinitrobenzenesulfone m
(hereinafter abbreviated as "lNB5") 82q is 0.1 of LOtxt
0.1 ml of solution dissolved in M sodium borate solution
was added, and the reaction was carried out at 25'C for 45 minutes.
Add 19 rxl of 1M sodium borate solution and add 420
The absorption was measured at nm. The values of molecular extinction coefficients ii and too were obtained from a calibration curve using a known amount of aspartic acid, and the value of the terminal amine was calculated using these molecular extinction coefficients.

As a result, 5 X l O-' moles of amino groups were present in intermediate lv. From this result, the average molecular weight is approximately 20
,000.

From the values of X and γ, the average molecular weight of the monomer unit is 145
．． 4, and the degree of polymerization n' -1-rd + from the molecular weight
IJ' + ” + P' + Q' (7) Average value 1
From t188°x, y, the average value of n'+m' is 41,
The average value of e/+r/ is 94. The average value of p'+a is 8.

Example 2 20 μl of the intermediate obtained in Example 1 was dissolved in 5 vtl of water, 68 μl of 1-ethyl-(8-dimethylaminopropyl)carbodiimide hydrochloride was added, and INHcJ
The pH was adjusted to 5.6. A solution of 20 ml of daunomycin hydrochloride dissolved in 8 d of water was added dropwise to this solution. After carrying out the reaction at room temperature for 1 hour, 1N NaOH solution (pH 9.5) was added (
It was adjusted. 5ephadex @ G-25 Super Fine (
(Manufactured by Pharmasha) After gel filtration with an 8.9 cm x 4.5 cm column to remove low molecular components, 8%
After dialysis with NaCl aqueous solution, 100W of ethanolamine hydrochloride and 200W of l-ethyl-3-(8-dimethylaminopropyl)carbodiimide hydrochloride were added.
NHC #tepi (5,07!: L) The reaction was carried out at room temperature for 1 hour. After that, dialysis was carried out with an 8% NaCl aqueous solution, and freeze-drying was carried out.

Yield 1t18rNi The amount of downmycin introduced into the obtained polysuccinimide derivative was 0.1M O, 1M sodium phosphate-0,
4850m in a solution of 15M saline buffer (PH7,4)
The absorption of 49XlO' moles of daunomycin were introduced into the polysuccinimide derivative lV.

The emplacement of thiol groups was carried out by the method of Example 8. There were 9.8XlO' moles of thiol groups in the polysuccinimide derivative lf. No unreacted carboxyl group was detected by IR and C-NMR.

As a result of the above, 0.02 mole of thiol group and 0.1 mole of daunomycin are bound per mole of succinimide group.

Quantification of the terminal amine 7 groups was carried out in the same manner as in Example 1.

8.58XlO' in polysuccinimide derivative lv
moles of amino groups are present, giving an average molecular weight of 28,000.

From these values, in formula (1), X = daunomycin, 2 is a hydrogen atom, and the degree of polymerization is n+m+6+
Average value of r+p+q 188, average value of n-1-m 1
4. Average value of d-1-r 121, average value of p+q 8
is a polysuccinimide derivative of

Example 8 This example shows a method for converting a thiol type intermediate [8] (Z=hydrogen atom) into an intermediate [8] where Z=Z/, and also A method for quantifying the group is also shown.

Intermediate (8) obtained in Example 1 was heated at 20 mF at 8 rst.
Q, l M lj sodium 7 acid 17 mu 1mME
DTA (P f (7,0)) was dissolved in a buffer solution, dithiothreitol lQmy was added, and a reduction reaction was performed at 80°C for 1 hour. After the reaction, equilibration was performed with the above buffer solution.
oG-25 Super Fine (Pharmacy')' I
lj) 2.0 can x 15 cm and diluted 20 times with the same buffer from which low molecular components had been removed. To 500 μe of this reaction solution, 20 μe of a 5 mM 4-pyridyl disulfide methanol solution was added and reacted at 30° C. for 15 minutes to obtain an intermediate [8] represented by Z = Z II = -8. The 4-thiopyridone produced at the same time was
Measured at 24 nm. When the amount of thiol groups in intermediate [8] (z = hydrogen atom) was calculated using the molecular extinction coefficient of 4-thiopyridone of 1,98 It exists.

Example 4 2 ml of polysuccinimide 86 (corresponding to succinimide group Q, 39 mmol) obtained in the above reference example
of DMF.

Add daunomycin hydrochloride 20my(86μmol) to this solution.
) and then 12μe of triethylamine (86μm
ol) was added and allowed to react at room temperature for 10 days. Then cysteamine s, 7+ da, ethanolamine 0.18 tan/
Add a solution containing DMFL (ml) and react at 4°C for 4 hours.

After dialysis of this reaction solution against water for 2 days, the pore size was 0.45μ.
Remove the insolubilized portion with a membrane (Millipore filter)
The solution was gel-filtered through a 5 ephadex [phase] G-25 Super Fine (manufactured by Pharmasha) 2.6 mm x 40 t-In column to remove low molecular components using distilled water as the eluent. Thereafter, freeze-drying was performed to obtain a polysuccinimide derivative. Yield 4o++v.

No unreacted succinimide group was detected by IR and C-NM measurements.

The introduction density of daunomycin and cysteamine per mo/mo of succinimide group in the obtained polysuccinimide derivative was determined in the same manner as in Example 2. 1.2XlO moles of daunomycin were bound in the polysuccinimide derivative 12.

The amount of bound cysteamine was determined by the method of Example 8.

There were 2.5XlO moles of cysteamine present in polysuccinimide derivative 11.

No unreacted succinimide group was detected from the 111 and "C: -NMR measurements.

From the above values, 0.02 mole of daunomycin and 0.005 mole of cysteamine are bound to 1 mole of succinimide group.

When the terminal amine group was quantified in the same manner as in Example 1, 1.25
l O moles of amino groups were present. Average molecular weight is g
o, o o.

becomes.

From the above results, the average value of the degree of polymerization n + m + g + r + p + q is 478, and n + m (7) The average value is t o ,
A polysuccinimide derivative was obtained in which the average value of a+r was 466° and the average value of P+9 was 2, in which X=downmycin and Z=hydrogen atom in formula (1').

Example 5 2 m of polysuccinimide 86 (corresponding to succinimide group Q, 39 rrmol) obtained in the above reference example
1 of DMF.

Add 40 μm of adriamycin hydrochloride (69μm0) to this solution.
After adding 4), 20 μe of triethylamine was added, and the mixture was allowed to react at room temperature for 10 days. Then cysteamine 6, 7
After adding m9 and stirring at room temperature for 8 hours, ethanolamine 0
．． A solution mixed with 1 ml of 18 tsl'lfeDM F was added, and the reaction was further carried out for 1 hour. This reaction solution was dialyzed against water for 2 days, and the insolubilized portion was removed with a pore size of 0.45 μm.
Remove the r liquid with a membrane of m (millibore filter) and filter it with 5 ephadex G-25 superfine (Pharmasha W) 2.6 (W *
Low molecular components were removed by gel filtration using a 40 Crn column using distilled water as an eluent. Thereafter, freeze-drying was performed to obtain a polysuccinimide derivative. Yield: 401η.

No unreacted succinimide was detected by IR, C-NMR measurements.

The quantification of adriamycin in the obtained polysuccinimide derivative was carried out in the same manner as the quantification of daunomycin in Example 2.

There were 2.24×10 mol of adriamycin bound in lf.

The amount of cysteamine bound was determined by the method of Example 8, based on the quantitative determination of thiol groups. 2.8XlO' moles of cysteamine were bound in 11.

From the above values, 0.04 mole of adriamycin and 0.005 mole of cysteamine were bound to 1 mole of succinimide group.

When the terminal amino groups were quantified in the same manner as in Example 1, it was found that 1.18XlO mol of amine 7 groups were present in 11. The average molecular weight will be 85,000.

From the above results, the average value of the degree of polymerization n+m+g+r+p+q is 472. The average value of n+m(7) is l 9 , (1+T
A polysuccinimide derivative was obtained in which the average value of is 451 and the average value of p+q is 2, in which in formula (1), X=niadriamycin and Z=hydrogen atom.

Example 6 Polysuccinimide 86rNI (obtained in the above reference example)
Succinimide group Q, equivalent to 39 mmoe) is 2
ml of DMF.

Add P-(bis(2-chloroethyl)amino)- to this solution.
L-Phenylalanine (also known as Melphalan) 58FW
After adding (0.1 gmmojlj), 58 μe of triethylamine was added and stirred at 60° C. for 48 hours. Then cysteamine a, 4+9, a-amino-1゜2-propanediol 0.12 ml, DM Fo, 5 m
A mixed solution of 1.1 °C was added and the reaction was carried out at 40 °C for 2 hours. After that, it was dialyzed against distilled water for 2 days, the insoluble part was filtered through a membrane with a pore size of 0.45 μm (Millipore filter), and the Oki solution was
Low molecular components were removed by gel filtration using distilled water as an eluent using an Ephadex"G-25 Super Fine (manufactured by Pharmasha) 2.6 cm column x 40 columns.

Thereafter, freeze-drying was performed to obtain a polysuccinimide derivative. Yield 85■.

No unreacted succinimide group was detected by IR and C-NMR measurements.

The amounts of p-Cbis(2-chloroethyl)amino)-L-phenylalanine and cysteine introduced per mod of succinimide group in the obtained polysuccinimide derivative were determined as follows.

The amount of p-[bis(2-chloroethyl)amino)-L-phenylalanine introduced in the polysuccinimide derivative lv is determined by g of the polysuccinimide derivative, absorption at 258.5 nm in a solution of IMPBS buffer (pH 7,4). from P-[bis(2-chloroethyl)amino)-L-
Measurements were made assuming that the absorbance did not change with phenylalanine. 2.5 X 1 O moles were bound in If.

The amount of cysteamine bound was determined by the method of Example 8, based on the quantitative determination of thiol groups. 2.4XlO' moles of cysteamine were bound in li.

From the above values, p-(bis(2
-chloroethyl)amino]-L-phenylalanine is 0
．． 1 mole and 0.005 mole of cysteamine are bonded.

When the terminal amine 7 groups were quantified in the same manner as in Example 1, it was found that 2.5 x 10 mol of amino groups were present in lt. The average molecular weight will be 40.000.

From the above results, the average value of the degree of polymerization n + m + g + r + p + q is 191, and the average value of n + m c7) is 19, e +
The average value of r is t71. The average value of p+9 is 1, the formula [
1], X, , P-(bis(2-chloroethyl)
amine)-L-phenylalanine, a polysuccinimide derivative in which Z=hydrogen atom, was obtained.

Reference Example 2 The anticancer effect of the daunomycin-bound polysuccinimide derivative obtained in Example 4 was investigated.

5 x 10' cells/tan of P2S5, a mouse leukemia cell
A solution was prepared, a polysuccinimide derivative was added to this solution, cultured for 2 days at 37° C. and 5% CO2, and the number of cells was measured using a Coulter counter.

The results were as follows.

Dose Growth inhibition rate 20 μg/yil 97% 6 89 2 84 It was revealed that the daunomycin-conjugated polysuccinimide derivative obtained in Example 4 has an anticancer effect.

Claims (1)

[Claims] (11 General formula % Formula % [In the formula, X, Y, W and Z each have the following meanings. X: Reaction residue of a cytotoxic substance having an amino group or imino group in the molecule. group or hydroxyl group. Y: Water-soluble aliphatic primary amine residue W: Lower alkylene group Z: Hydrogen atom or group represented by general formula Z' or general formula -S-4, where Z/ is A group that can form an active disulfide group together with the adjacent sulfur atom is a hydrocarbon group (however, when Z is a hydrogen atom, it is represented by -SZ) -SR group is a group that can form an active disulfide group within or between molecules. -5-S- bond can be formed in equilibrium with the group, but including these -5-S- bonds.
It shall be expressed as H. ) Also, n, m, l, y, p and 9 are 0
or a natural number indicating the number of constituent units, and the relationship between these is as follows. n0m≧2 0.001≦p+q/n-+-m+4i'+r+p+q
≦0.50), and the molecular weight is 2000 to a, o
o o, o o. A polysuccinimide derivative. (2) General formula [In the formula, x, y, w and 2 each have the following meanings. x' Reactive residue of a cytotoxic substance having an amino group or imino group in two molecules Y: Water-soluble aliphatic primary amine residue W: Lower alkylene group 2: Hydrogen atom or general formula Z/ or general formula - 5-1
A group represented by, where Z' is a group capable of forming an active disulfide group together with the adjacent sulfur atom, and k is a hydrocarbon (however, when Z is a hydrogen atom, the -5H group represented by -SZ is Although -5-S- bonds can be formed in equilibrium with other 5H groups in molecules, 18H groups containing these
shall be expressed as ) Further, n, m, l, r, p and q are 0 or a natural number and indicate the number of constituent units, and the relationship between them is as follows. n-1-m≧2 0.001≦p+q/n-1-m+6+r+p≦0.5
0 ], the molecular weight is 2000~s, o o
o, o. In producing the polysuccinimide derivative,
General formula [wherein, Y, Z and W are the same as above. n' , In' , /z er'eP'Oyohiq'
Ha. or a natural number indicating the number of constituent units, and the relationship between these is as follows. n'-1-m'≧2 o, oot≦〆+(1'/n'-1-1n'+ 6
'+r'+p'+q'≦o, 50) (a) A cytotoxic substance containing an amino group or an imino group (O) If necessary, react with a water-soluble aliphatic primary amine. 8) A method for producing the polysuccinimide derivative characterized by the general formula [wherein, X, Y and W each have the following meanings. X: Reactive residue of a cytotoxic substance having amine 7 group or imino group in the molecule Y: Water-soluble aliphatic primary amine residue W: Lower alkylene group Also, n, m, g, r, p and q is 0 or a natural number and indicates the number of constituent units, and the relationship between them is as follows. n-4-m≧2 0.001≦p+q/nt-m-H+r+p≦0.5
0) and has a molecular weight of 2000 to 8,000,
000, in the general formula C, Y, Z and W are the same as above. R is a hydrocarbon group. n', m', l', r', p' and q/ are O or a natural number and indicate the number of constituent units, and their relationship is as follows. n'-1-m'≧2 0.001≦〆+q'/n'+rn'+#l+r'+p
'+q'≦o, +s O) is reacted with (a) a cytotoxic substance containing an amino group or an imino group (b) with a water-soluble aliphatic primary amide, if necessary, and then a disulfide bond is formed. Method for producing the above-mentioned borisuccinimide derivative characterized by reductive cleavage (4) General formula [wherein, Xr', Y, W and 2 each have the following meanings. x' Reactive residue or hydroxyl group of a cytotoxic substance having an amino group in two molecules Y: Water-soluble aliphatic primary amine residue W: Lower alkylene group Z: Hydrogen atom or general formula Z/ or general formula - 5-t
The group represented by t, where Z' is a group capable of forming an active disulfide group together with the adjacent sulfur atom, and the group is a hydrocarbon group (however, when Z is a hydrogen atom, the 18H group represented by -SZ is -8-8-bonds may be formed in equilibrium with other -SH groups within or intermolecularly;
It shall be expressed as H. ) Also, n, m, l, r, p and 9 are 0
or a natural number indicating the number of constituent units, and the relationship between these is as follows. n-4-m≧2 o, oot≦p+q/n-1-al-H+r+p+q≦
It is expressed as 0.50'1, and the molecular weight is 20 in terms of globular protein.
00 to 8.000,000, the general formula 0 [wherein k represents a natural number] is used. ] Polysuccinimide represented by (a) A bifunctional cytotoxic substance having an amino group or water (all general formula 82N-W-3-Z (wherein, W and 2 have the same meanings as above). A method for producing the polysuccinimide derivative, characterized in that the compound represented by (7X) and (7X) are reacted with a water-soluble aliphatic amine if necessary. (5) General formula [wherein, The following meanings are shown. r, p and q are 0
or a natural number indicating the number of constituent units, and the relationship between these is as follows. n-1-m≧2 0.001≦p+q /n+m-H+r+p+q≦0.
50 (However, the 18H group can be linked to other 3R groups within or between molecules.
Although it may form a -5-5- bond in equilibrium with the group, it is expressed as -8H including these bonds. )], and the molecular adhesion is 2000~a, o o o, o
o. In producing the polysuccinimide derivative,
General formula [In the formula, k represents a natural number. ] In the polysuccinimide represented by (a) a cytotoxic substance having an amino group or water (b) the general formula H2N-W-3-8-[wherein, W is the same as above, and is a hydrocarbon] Indicates the group. ] and (iii) a water-soluble aliphatic amine if necessary, and then reductively cleaving the disulfide bonds of the reaction product. . (6) General formula [In the formula, X, Y, W and 21 each have the following meanings. X: Reactive residue or hydroxyl group of a cytotoxic substance having an amino group or imino group in the molecule Y: Water-soluble aliphatic primary amine residue W: Lower alkylene group z': Active disulfide group together with the adjacent sulfur atom Also, n, m, l, r, p and q are O
or a natural number indicating the number of constituent units, and the relationship between these is as follows. n-1-m≧2 o, o o i≦p+q /n-)-m-H+r+p+
q≦0.50), and the molecular needle is 2000 to a, o
o o, o o. In producing a polysuccinimide derivative having the general formula [wherein, X, Y, W, n, m, l
, r, p and 9 have the same meanings as above. ] A method for producing a polysucciimide derivative, which comprises reacting a thiol-type polysucciimide derivative represented by the following with an active disulfide compound.