JPS60243121A - Reactive high polymer having bonded cytotoxic substance - Google Patents

Abstract

PURPOSE:The titled high polymer, obtained by introducing a cytotoxic substance having NH2 or NH groups in the molecule thereof and terminal amino active groups into a specific polysuccinimide, useful for preparing target directive carcinostatic agents, etc., and usable as cytotoxins. CONSTITUTION:A reactive high polymer, obtained by reacting a cytotoxic substance having amino or imino groups with a polysuccinimide derivative having n, m and l constituent units of formulas I -III (Y is water-soluble aliphatic primary amine) and r constituent units of formula IV (n, m, l and r are 0 or natural numbers and n+m>=2) and 2,000-3,000,000 molecular weight, and if necessary reacting the resultant reaction product with a water-soluble primary amine, and introducing an active group of formula V [Z is SR (R is hydrocarbon) or a group capable of forming active disulfide together with the adjacent S atom; W is hydrocarbon; A is O, NH, etc.] into the amino group, and consisting of constituent units of formula VI (X is reaction residue of the cytotoxic substance having the NH2 or NH groups in the molecule or OH) and formulas VII, III, and IV.

Description

[Detailed description of the invention]

The present invention relates to reactive polymers bound with cytotoxic substances, intermediates thereof, and methods for producing them. More specifically, the present invention provides a reaction method that can be used as a cytotoxic agent in the production of a target-directed anticancer agent by combining an anti-tumor antibody or the like that has the ability to bind to a target such as a tumor cell with a cytotoxic agent. The present invention relates to polymers and their production methods, as well as their intermediates and their production methods. Conventionally, it has been known to produce an antitumor agent by binding a cytotoxin to an antitumor antibody. When binding anti-tumor antibodies to cytotoxic substances, polymer carriers are used in order to bind a large number of cytotoxic substances without reducing the target binding ability of the anti-tumor antibodies. A typical polymer used as a polymeric carrier is dextran polygly5 tamic acid. In the case of dextran, it is mainly used as an aldehyde derivative by oxidation with periodic acid, etc., but it is difficult to obtain a conjugate with good reproducibility because the cytotoxic substance and antitumor 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 undesirable as a target-directed anticancer agent because it accumulates in the upper part of the body. The present inventors conducted intensive studies to solve these drawbacks, and as a result, we found that a polymer carrier with no electric charge, that is, a reactive polymer that binds a cytotoxic substance and has a functional group that can bind to an antitumor antibody. It was discovered that it could be easily manufactured at low cost, leading to the present invention. The present invention provides a reactive polymer conjugated with a cytotoxic substance that can be used to produce a target-directed therapeutic agent and has the ability to bind to an anti-tumor antibody having targeting ability. . That is, the present invention provides compounds of the following formula [1] to which a cytotoxic substance is bound.
[4] X [1) C2) (In the above formulas [1] to [4], X and Y each have the following meanings. or hydroxyl group Y: water-soluble aliphatic primary amine residue; - each structural unit in the molecule [1]; the number of C2'J, C8J, [4) is n, respectively;
m, t. r, these numbers are 0 or natural numbers, and n+
m≧2. ) The following formula [5] (In the above formula [5], Z, W and A each represent the following meanings 1, Z: hydrogen atom or the general formula Z ' or a group represented by -5-R (where Z' is a group capable of forming an active disulfide group together with the adjacent sulfur atom, and R is a substituted or unsubstituted hydrocarbon group.) W: Substituted or Unsubstituted hydrocarbon group A: has an active group represented by an oxygen atom or NH group or a salt thereof) and has a molecular base of 2.000 to a, ooo, ooo
The present invention relates to a polysuccinimide derivative (hereinafter referred to as polysuccinimide derivative [1]) and a method for producing the same. Here, the group (2') capable of forming an active disulfide bond with the adjacent sulfur atom includes υ 5-nitro-2-pyridylthio group, pyridylthio group or its N-oxide, and 8-carboxy-4-nitrophenylthio group. phenylthio groups,
Further, in the substituted or unsubstituted hydrocarbon group represented by N-phenylamino-N'-phenylamino-W, the hydrocarbon group may be, for example, a linear or branched chain having 1 to 10 carbon plates. Mention may be made of alkylene groups. In formulas [1] and [2], X represents a reactive residue or hydroxyl group of a cytotoxic substance that contains an amino group or an imino group in its molecule, but a cytotoxic substance is a substance that is toxic to cells as it is. or substances that are not toxic as they are, but can be converted in vivo into substances that can be toxic to cells. Examples of these include: p-('N,N-bis(2-chloroethyl)]phenylenediamine p-[bis(2-chloroethyl)amino]-L-phenylalanine (melphalan) 2-amino-N-Cp-bis(2-chloroethyl)amino ] Phenyl-8-hydroxypropionamide H2 1-C5'-(2-aminoethylphosphoryl)-β-
D-arabinofuranosyl)cytosine 2-amino-N-[p-bis(2-chloroethyl)aminocophenyl-8-hydroxy-2-hydroxymethylpropionamide methotrexate CH(CHa)2 CH(CHa)2 CHa CHg Adriamycin D Mitomycin C X=Hridaunomycin (z+r=g) is introduced for the purpose of improving the water solubility of the reactive polymer. The water-soluble aliphatic-class amines used include, for example, ethanolamine, aliphatic primary amines having a hydroxyl group such as 8-amino-1,2-propanediol, CHg (CH2)gNHg(g-1~
aliphatic amines (an integer of 4), etc. In particular, amines having a hydroxyl group such as ethanolamine and 8-amino-1,2-propanediol are preferred. n+m is the number of structural units to which X is bonded, and t+r
is the number of structural units to which water-soluble aliphatic primary amines are bonded, and (n+m)/(n+m+z+r) is the monomer unit l.
Calculate the number of condensed moles of X per mole of succinimide group. The molecular weight is 2.000-a, ooo, ooo, preferably 2.000-aoo, ooo, and n+m is 2 or more. If the molecular weight is less than this, the number of cytotoxic substances bound to one reactive polymer molecule will decrease, and the amount of cytotoxic substances that can bind to anti-tumor antibodies will decrease, resulting in an undesirable increase in molecular weight. If it is too much, aggregates of anti-tumor antibodies are likely to be formed, and insoluble components and large molecular weights are likely to be produced, which is not preferable. The polysuccinimide derivative [1] of the present invention can be easily derived from polysuccinimide having the structure of the following general formula [■]. [In the formula, k represents a natural number. ] Polysuccinimide is also known as anhydropolyasparati.
A polymer also called Tsukuacid, which can be obtained mainly by thermal condensation of aspartic acid (J
, kovacset al, , J, Org, Che
m, 26, 1084 (1961): P, Neri
et al., J.Med.Chem.,16.
898 (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 a polymer with a higher molecular weight can be obtained, but if the temperature is higher than this, decomposition of the polymer will occur, which is not preferable. 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 unskinned aspartic acid and low-molecular condensate, followed by filtration through a glass filter and drying. Repeat this operation if necessary. Although IR and NMR suggest that the polymer thus obtained is represented by the general formula rII),
It is generally accepted that the structural unit of the general formula [n') is substantially the main component even if it partially contains other structural units. It is known that the polysuccinimide thus obtained reacts with a compound having an amino group, such as ethanolamine, to carry out a reaction according to the following reaction formula. 1 [■] NHQ (2CH20H [In the formula, X and y represent natural numbers.] It is also known that the reaction of the following reaction formula is carried out under alkaline conditions. , x and y are natural numbers] There are two methods for obtaining the polysuccinimide derivative of the present invention [■]. The first method is based on the formula [6] ~

[9] H [6] [7] ['8F [9] (In the above formula [8), C9], Y is as described above. -Each structural unit in the molecule [6F
, [7'), C8], and C9'J are each n
, m, t, r, and these numbers are as described above. ) and has a molecular weight of 2.000~
A polysuccinimide derivative consisting of a, ooo, and ooo (hereinafter referred to as polysuccinimide derivative [m). )
, or a polysuccinimide derivative [■] having an active group represented by the above formula [5] at the terminal amino group (i.e., a polysuccinimide derivative [I] in which X, =OH). This is a method to obtain an imide derivative (referred to as IV"J) and then to obtain a polysuccinimide derivative CI'J in which X = cytotoxic substance. The first representative method is as follows (1) to (4). There is a method. (1) Polysuccinimide [■] is (a) reacted with a water-soluble aliphatic primary amine if necessary, and then hydrolyzed to form a polysuccinimide derivative Cm.
), and reacted the amino group terminal of (cl) polysuccinimide derivative Cml with a reagent that introduces a -5-Z group via a spacer to obtain a polysuccinimide derivative [■v] which is a reactive intermediate. get. Thereafter, the polysuccinimide derivative [TV] of the present invention is reacted with a cytotoxic substance containing a amino group or an imino group, and if necessary, a water-soluble aliphatic primary amine. can be manufactured. (2) Polysuccinimide [■] is reacted with (a) a water-soluble aliphatic primary amine if necessary, and then hydrolyzed to form a polysuccinimide derivative [m
), the (Cl) polysuccinimide derivative [m] is reacted with a cytotoxic substance containing an amino group or an imino group, and then e → is reacted with a water-soluble aliphatic primary amine if necessary, and further (2) The polysuccinimide derivative of the present invention can be produced by reacting with a reagent that introduces a -5-Z group into the terminal amino group via a spacer. - (3) (1) above. Or in method (2), z=
When the polysuccinimide derivative of the present invention in which z' or Z=-5-R is obtained, the polysuccinimide derivative is subjected to a reductive cleavage reaction, whereby X=cytotoxic substance, 2=water↓atom A polysuccinimide derivative [I
] can be obtained. Furthermore, in the method (1) above, the above-mentioned polysuccinimate derivative [1] can also be obtained by performing a reductive cleavage reaction after the reaction (b). The reductive cleavage reaction can be carried out, for example, by reacting with a thiol compound or a borohydride compound. (4) By reacting an active disulfide compound with the X-cytotoxic substance obtained by any of the methods (1) to (3) above, a polysuccinimide derivative [■] where Z = hydrogen atom, A polysuccinimide derivative [11] where X=cytotoxic substance and 2=2' can be produced. The first representative methods (1) to (4) above will be explained in more detail below. As the reaction solvent, DMF, DMSO, water, or a mixed solvent thereof can be used. A preferred method for carrying out the reaction with the water-soluble aliphatic primary amine in (a) of (1) and (2) is to prepare polysuccinimide [10] in DMF or DMSO with 1~go(W/
W)%, add 0.01 to 10 moles, preferably 0.62 to 1 mole, of a water-soluble aliphatic primary amine per mole of succinimide group, which is the monomer unit of polysuccinimide rlI), and heat at -40°C. The reaction is allowed to proceed at ~80° C. for 1 minute to 2 days with stirring, followed by hydrolysis. Hydrolysis is carried out in an alkaline aqueous solution. For example, a reactant of polysuccinimide or polysuccinimide with a water-soluble aliphatic primary amine may be dissolved in DMF or DMS.
Either dissolve it in O and add dropwise an alkaline aqueous solution, or add a reactant of polysuccinimide [ID or polysuccinimide [■]] and a water-soluble aliphatic primary amine to an alkaline aqueous solution and heat at -40°C to 80°C. Allow to react for 10 minutes to 2 days, then tighten. The alkaline aqueous solution has a pH of 3 or higher, preferably 0.
．． 1-IN NaOH aqueous solution. -5-
The reagent for introducing the Z group may be any of such known reagents, but for example, it can be represented by the general formula [vIa] (where Ll is a divalent organic group and Ql is an alcohol residue of an imidoester. ) or a salt thereof (eg, HCt salt, HBr salt, etc.) can be used. In the above general formula (Vt a'], the organic group represented by Ll is, for example, a linear or branched alkylene group having 2 to 6 carbon atoms such as ethylene or trimethylene, and an alcohol residue represented by Ql. Examples of the group include alkoxy groups having 1 to 8 carbon atoms such as methoxy and ethoxy.More specifically, examples of the reagent include methyl-8-mercaptopropylimidate H-HCt (H8- CH2-CH2-C-0CHa), methyl-
4-Mercaptobutyrimidate. *f:,・2−4°) f2511>C (inclusive 1・) etc. can also be used as a reagent.Furthermore, the general formula [v■b] [wherein Z and Ll are as described above.Q2 represents the alcohol residue of the active ester.] Compounds represented by the general formula [v■C] [wherein Z'
, Ll and Ql are as described above. ] or a salt thereof (e.g. HCt salt,
HBr salt, etc.), general formula [vId]N)f [wherein Lt and Qt are as described above. ] or a salt thereof (e.g. HCt salt,
HBr salt, etc.), general formula [vIe] [wherein Ll and Q2 are as described above. ] □ A compound represented by the formula [vtb] and S-acetylmercaptosuccinic anhydride may be used. Examples of the reagent represented by the above general formula [vtb] include N-succinimidyl-3-(2-pyridylthio)
Examples of reagents that can be prepared using the general formula (VIC), such as propionate, include 8+4'-dithiopyridyl)mercaptopropioimidate, and the like. ] Examples of reagents that can be used include dithiobis(succinimidyl propionate). It can be carried out as follows: DMF, 1 to 8Q (W/
W)% polysuccinimide or (1) a)
Dissolve the polysuccinimide derivative after the reaction or after the reaction of f→ in (2), and add 1 to 10
Add 0 times the number of moles of a reagent that introduces -5-Z group via a spacer using an amino group, and if necessary, in the presence of a basic substance such as pyridine or triethylamine, -
The reaction is carried out at 40-80° C. for 30 minutes to 2 days with stirring. Unreacted reagents are reprecipitated or dialyzed using a solvent such as ethanol, which is a non-solvent for polysuccinimide or its derivatives.
It can be removed by a method such as filtration. Any conditions for the reaction between a carboxyl group and an amino group in peptide synthesis may be used. As a reaction solvent! Usually, water, DMF, DMSO or a mixed solvent thereof is used. During the reaction, for example, a polysuccinimide derivative having a carboxyl group and a polysuccinimide derivative having an amino group or a limino group are combined in the presence of a condensing agent such as 1-ethyl-8-(8-dimethylaminopropyl)carbodiimide hydrochloride or dicyclohexylcarbodiimide. A method of condensing a cytotoxic substance, a method of activating the carboxyl group of a polysuccinimide derivative, for example, in the form of isochlorobutylformate mixed acid anhydride, and reacting it with a cytotoxic substance having an amino group or an imino group, etc. be. In this reaction, the reaction temperature is -40 to 100°C, and the reaction time is 10 minutes to 10 days. The second method for obtaining the polysuccinimide derivative [I] of the present invention is an effective method for binding a cytotoxic substance having an amino group, and the reaction with the cytotoxic substance and aliphatic primary amine is not possible. using the amino groups they have, and reacting with a reagent that uses the amino group to introduce an -S-Z group via a spacer, using the amino group at the end, where X = cytotoxic substance. This is a method for obtaining a certain polysuccinimide derivative [I]. A cytotoxic substance containing an amino group refers to a compound that has cytotoxicity and the amino group in the molecule has the function of the succinimide group of polysuccinimide. In other words, it must have an amino group that reacts with a succinimide group to form an amide bond. Examples of such cytotoxic substances include daunomycin, adriamycin, p-amino-N-Cp-bis(2
-chloroethyl)amino]phenyl-3-hydroxyamide, 2-ami2N-[p-bis(2-chloroethyl)aminocophenyl-3-hydroxy-2hydroxymethylpropionamide, p-[bis(2-chloroethyl)amino] Examples of the second method include those having an aliphatic amino group such as -L-phenylalanine, which include the following methods (5) to (7). (5) Polysuccinimide [■] a) Reaction with a cytotoxic substance having an amino group (d) If necessary, reaction with a water-soluble aliphatic primary argon compound → Using the amino group, -S- through spacer
A method for obtaining a polysuccinimide conductor [■] in which X = a cytotoxic substance by subjecting it to a reaction with a reagent capable of introducing a Z group. (6) When a polysuccinimide derivative in which z=z' or -5-R is obtained by the method in (5) above, the polysuccinimide conductor is subjected to a reduction reaction in the same manner as in (8) above. A polysuccinimide derivative in which X=cytotoxic substance and Z=hydrogen atom [method for obtaining rIF]. (7) The X-cytotoxic substance obtained by either method (5) or (6) above, a polysuccinimide derivative white in which Z = hydrogen atom] is reacted with an active disulfide compound in the same manner as in (4) above. A method for obtaining a polysuccinimide derivative [I] in which X=cytotoxic substance, zz' by There may be a simultaneous reaction method in which the reactions of (a) and (0) are carried out at the same time.In the simultaneous reaction method, either of the reactions (a), (0) or the reaction n may be carried out first. The preferred implementation method is the bottling reaction method, and in the case of particularly unstable cytotoxic substances, after performing the reaction (c), (b),
It is desirable to perform the steps in the order (0). A reaction in which the reactions of (a) or (1') and (1') are carried out simultaneously is a polysuccinimide or (a). (b), a polysuccinimide derivative subjected to any one of e→ It is appropriate to carry out the reaction in which the polysuccinimide derivative subjected to one of the following reactions is dissolved in a DMF or DMSO solvent.More specifically, for example, The reaction can be carried out as follows: polysuccinimide or an unreacted succinimide group imide derivative is mixed with (a) and/or in 1 to 80<W/W)% of DMF or DMSO solvent. or ((2)
The components to be reacted in the reaction (0.001 to 10 moles per -component per mole of succinimide group in the solution) are reacted with stirring at -40 to 80°C for 10 minutes to 20 days. Unreacted reagents are reprecipitated with non-solvent,
It can be removed by dialysis or gel filtration. The desired polysuccinimide derivative can be obtained by distilling off the solvent under reduced pressure or by freeze-drying. In the reaction (c), polysuccinimide or a polysuccinimide derivative subjected to any one of the reactions in (a) or (iv), or a polysuccinimide derivative subjected to the reactions in (a) and (2) is used. The reaction is carried out in the same manner as in (1) (reaction (2), reaction (2)). The molecular weight is controlled by selecting the condensation conditions to obtain the polysuccinimide polymer [ID] and by selecting various reaction conditions. We separate the polymer having the desired molecular weight by dividing the polysuccinimide polymer by gel filtration in a DMF solution, and after obtaining the polysuccinimide derivative, by gel filtration in an aqueous solvent. Depends on the method of fractionation. It is preferable to perform molecular weight fractionation by gel filtration in an aqueous solvent after obtaining the polysuccinimide derivative. In order to improve the yield of the desired molecular weight fraction, polysuccinimide polymers are Selection of condensation conditions to obtain polysuccinimide and molecular weight fractionation by gel filtration with a DMF solution are carried out in advance. The active disulfide compound used in the method for obtaining a reactive polymer having an active disulfide group is the above-mentioned active disulfide compound with the adjacent sulfur atom, which is exemplified as Z', for example. 5,5'-dithiobis(2-nitrobenzoyl The reaction between a thiol-type reactive polymer and an 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. The reaction can also be carried out using a reaction system in which the compound or its acetone solution or dioxane solution is added and mixed.The reaction temperature is -5 to 70°C, and the reaction time is 1 minute to 24 hours. The bound reactive polymer has cytotoxicity due to a cytotoxic substance and contains a highly reactive thiol group in the molecule, a disulfide group that can easily obtain a thiol group by reduction, or an active disulfide group. So anti-tumor antibodies (anti-tumor immunoglobulin)
An active disulfide bond introduced from, for example, N-succinimidyl 8-(2-pyridyldithio)propionate (SPDP) using a thiol group or an amino group derived from a disulfide bond such as N-(meta-maleimidobenzo iloxy) succinimide (MBS
), N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)maleimide (CH
It can bind to anti-tumor antibodies etc. via the maleimide group introduced from M) etc. As a method for 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 simaleimide compound such as O-phenylene dimaleimide is attached in advance to the anti-tumor antibody or to the reaction of the present invention. There is a method in which a maleimide group is introduced by reacting with one thiol group of a polymer, and then reacting 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. In addition, a Faty dimer is obtained by treating the antibody with pepsin, and the disulfide bond of this 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 [l] and a conjugate with a substance having targeting ability such as an anti-tumor antibody are expected to be effective as selective cancer therapeutics. The invention will be described in detail. Reference Example 1 Polymerization of polysuccinimide with 66.5 N of monoaspartic acid and 20.6 N of 85% phosphoric acid
- was added and polymerization was carried out under a reduced pressure of 10 μHg for 5.5 hours while heating to 190 ''C with a rotary evaporator. After polymerization, it was dissolved in DMF and reprecipitation was performed in water. After reprecipitation It was passed through a glass filter and vacuum-dried at 80°C for 8 days.The obtained polymer was measured for infrared absorption using the KBr tablet method, and it was found to be 1800.1720°1660 cm-'
Absorption characteristic of succinimide groups was observed. Molecular weight measurement was performed by GPC measurement. Using a GPC column for DMF (AD-80M/S - Showa Denko), the obtained polysuccinimide was subjected to DMF
+0. OI MLiBr solvent 0.2% (7)
The solution was dissolved to a desired concentration, 250 μt of the 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 418.9xlO' and a number average molecular weight of 1.5xlO' in terms of polyethylene glycol. Example 1 1.8 g of polysuccinimide obtained in the above reference example (corresponding to 18.6 mmol of succinimide groups) was added to DMF.
0.78 m of ethanolamine in a solution dissolved in 80-
(corresponding to 18 mmol) dissolved in 10-DMF was added, and the reaction was carried out at 4°C for 18 hours. To this solution, 10 ml of 1N-NaOH solution was gradually added dropwise over 1 hour while stirring. 2 for an aqueous solution with a pH of 4.0 after reaction.
Dialysis was performed for several days. After this, 5ephadeX! BG-50 Kara A Tegel filtration was performed, fractionated and freeze-dried. The yield was 1.0 g. The terminal amino group was quantified by the following method. The above lyophilized product (5q) was dissolved in 0.1 M sodium borate solution, and then 8211F (abbreviated as TNBs) was dissolved in 10-0.
After adding 0.1 mm of a 1M sodium borate solution and reacting at 25°C for 46 minutes, 1.9 mm of a 0.1 M sodium borate solution was added and the absorption at 420 nm was measured. The amount of amine in 1 g was 8.6xlO-5M, and the average molecular weight was 2.
It is 8.000. The content of carboxylic acid was determined by acid-base titration. 1
There were 2.07xlO moles of carboxylic acid in 1 g. No unreacted succinimide group was detected by IR and 18C-NMR. From the above results, the molecular weight of the succinimide group monomer unit into which ethanolamine has been introduced is 158, and the molecular weight Ji of the hydrolyzed succinimide group monomer unit is 158.
Using 115, the number of moles (X) of carboxyl groups introduced per mole of succinimide group was calculated using the following formula. The calculation result was 0.8 mol. Dissolve 0.9f of the above freeze-dried product in DMF9-1, N-
101 q of succinimidyl-8-(2-pyridyldithio) propionate (hereinafter abbreviated as 5PDP) and 8.4 q of triethylamine were added, and the reaction was carried out at room temperature for 24 hours. After dialysis against distilled water, it was freeze-dried. Yield 0,
8 f. The reactive polymer 20w9 thus obtained was diluted with 8-0.1
M-sodium phosphate-1mM EDTA (pH 7,0)
Add 10μ of dithiothreitol and heat to 80°C.
Reduction was carried out for 1 hour, and the generated 4-thiopyridone was reduced to 8
The absorption was measured at 48 nm. When quantified using a molecular extinction coefficient of 8.080, 2.45
It was revealed that x 10-5 moles of 5PDP had reacted. An X=OH-Z-reactive polymer was obtained with a yield of 70%. Note that the average value of the degree of polymerization (n + m + z + r) of the reactive polymer obtained from the molecular weight and X obtained above is 198, the amount of introduced carboxyl group (n + m) is 58,
The reaction amount (t+r) of ethanolamine is 185. Example 2 The polysuccinimide obtained in Reference Example 1 was subjected to ethanolamine reaction and then hydrolyzed in the same manner as in Example 1, and dissolved in 0.9fltDMF9- of the lyophilized product to obtain dithiobis(succinimidylpropiodine). Nate) (
Purchased from Pierce) 180 Misaki (8,24xlO'mol),
8.4 Wt of triethylamine was added and the reaction was carried out at room temperature for 24 hours. After dialysis against 2 L of distilled water, 125 m'F of dithiothretol was added and reduced at 30'C for 1 hour. This solution was added to 5ephadexoG-257,-
/, -7fi) (made by 7 Almasya) (8,8onφx1
1.5Lyn) and 0, OIM Sodium Monoate-i
After gel filtration using mM EDTA (pH 7,0) to remove low molecular weight components, 50 d (wet volume) of activated thiopropyl Sepharose 6B resin was mixed with 0.01 M sodium phosphate-1 mM EDTA (pH 7,0) 50- The mixture was added to the dispersion liquid and gently stirred at 4°C for 18 hours. After that, the resin was packed in a column and washed with a solution of 0.01M sodium phosphate-1, mMEDTA (pH 7,0), and dithiothreitol 180rRf was added at 10%.
-0.01M sodium phosphate-t? ? IMEDTA
(pH 7.0) was packed into a column and reduced at room temperature for 80 minutes. Thereafter, 0.01M sodium phosphate-1mM EDTA (1) H7,0) was passed through the column to collect the effluent polymer fraction, which was dialyzed against water and then freeze-dried. Yield: 0.51 The thiol groups in the thus obtained polysuccinimide derivative having terminal thiol groups were determined by the method based on Example 3. The results showed that 8.6 x 10-5 moles of thiol groups were present in 1 g of the polysuccinimide derivative, and this value coincided with the amount of terminal amino groups in the lyophilized product initially used in this example. The average value of molecular weight is 28.000,
X=OH, Z=hydrogen atom, n+m+t+r, n+
A reactive polymer having the same average values of m and t+r as in Example 1 was obtained. Example 3 This example shows a method for converting a thiol-type reactive polymer in which Z = hydrogen atom into a reactive polymer in which Z = Z', and also describes a method for quantifying thiol groups in the thiol-type reactive polymer. The thiol-type reactive polymer 20 obtained in Example 2, which is shown in Fig. 3, was
0.1 M Sodium Phosphate - 1 m M EDT
A (pH 7.0) was dissolved in a buffer solution, 10 μm of dithiothreitol was added, and a reduction reaction was carried out at 30°C for 1 hour. 5ephadexoG equilibrated with the above buffer after reaction
-25 Super Fine (manufactured by Pharmasha) 2. O
After gel filtration with crnφ×15 m to remove low molecular weight components, the mixture was diluted 20 times with the same buffer. This diluted solution 500
20 td of a methanol solution of 5mM 4-pyridyl disulfide was added to μt to obtain a reactive polymer showing a temperature of 80°C. 4-thiopyridone produced at the same time was measured at 824 nm. When the amount of thiol groups in the thiol-type reactive polymer was calculated using the molecular extinction coefficient of 4-thiopyridone of 1.98x104, it was found that 8.5xlO moles of thiol groups were present in intermediate lf. Example 4 The reactive polymer 20 obtained in Example 2 was dissolved in 5-water, and 1-ethyl-(8-dimethylaminopropyl)carbodiimide hydrochloride 68 was added thereto and adjusted to pH 5 with IN HCt. , adjusted to 0. To this liquid, 20 vol of daunomycin hydrochloride dissolved in 13 ml of water was added dropwise. After reacting for 1 hour at room temperature, p
Adjusted to H9.5. After that, equilibration was carried out with a 0.15 N -NaCl aqueous solution.
After gel filtration was performed using a phadex G-25 Super Fine (manufactured by Pharmasha) column of 8.9cWIφX4.5m to remove low molecular components, dialysis was performed with an 8% NaC4 aqueous solution. After dialysis ethanolamine hydrochloride ioo■
, 200q of 1-ethyl-8-(8-dimethylaminopropyl)carbodiimide hydrochloride was added, and the pH was adjusted with INHct.
5,Q and the reaction was carried out at room temperature for 1 hour. Thereafter, dialysis was performed with an 8% NaCt aqueous solution, followed by freeze-drying. Yield 17118-p The amount of daunomycin introduced into the obtained reactive polymer was 0.
4. in a solution of IM-PBS buffer (pH 7,4).
Based on absorption at 85 nm. 4.9 x l0-4 in polysuccinimide derivative IIi
Mol daunomycin had been introduced. No unreacted carboxyl group was detected by IR and 18C-NMR. In the reactive polymer obtained from this value, 0.1 mol of daunomycin is introduced per 1 mol of succinimide group. The average degree of polymerization of the reactive polymer used from Example 2 was 19B.
From the above results, X = cytotoxic substance = daunomycin, Z = hydrogen atom, and the average value of n + m + z + r is 1
98, average value of n + m 19, average value of t + r 168 (
1'), [A reactive polymer having the characteristics of [2F] was obtained. Example 5 1 g of polysuccinimide obtained in Reference Example 1 was dissolved in DMF 10
51q of ditheobis (succinimidyl propionate) was added to the mixture and the mixture was reacted at room temperature for 24 hours. After the reaction, the reaction solution was added dropwise to 100 ml of ethanol with stirring to cause reprecipitation, and the mixture was passed through a glass filter. DM the precipitate again
It was dissolved in F 10 m and reprecipitated with ethanol five times, and then dried in a vacuum dryer. Yield: 0.9 g After drying, 0.5 g was dissolved in 10 #Ig of DMF, 280 m of daunomycin hydrochloride was added to this solution, and then 0.14 m of triethylamine was added and reacted at room temperature for 8 days. Thereafter, a mixture of 0.6-ml ethanolamine and 5 wt of DMF was added, and the mixture was reacted at 4°C for 4 hours. After dialysis of this reaction solution against water for 2 days, the pore size was 0.45.
The insolubilized portion was removed using a μm membrane (Millipore filter), and the 0 solution was gel-filtered through a 5ephadex G-25 Super Fine (manufactured by Pharmasha) 2.6 cmφ x 40m column to remove low molecular components. . Add 180q of dithiothretol to this solution and perform a reduction reaction at 80°C for 1 hour. co solution & 5ephadexoG-252-/f
-Fine (manufactured by Pharmasha) 8.8c1nφX 11
．． 0.01M sodium phosphate-tmM in 5 columns
Low molecular components were removed by gel filtration using ED'rA (pH 7,0), and a thiol group was introduced at the terminal using activated thiopropyl Sepharose 6B resin in the same manner as in Example 2. A polymer was obtained. Yield 0.25f. When thiol groups were quantified using the method of Example 8, 1.26 x 10"" moles of thiol groups were present in 1 g of the reactive polymer. From this value, the average molecular weight was so.
It becomes ooo. The amount of Dautzmailen introduced was determined in the same manner as in Example B. 2.25×10′ moles of daunomycin were incorporated in 1 g of reactive polymer. IR and 18C
- No unreacted succinimide group was detected by NMR. From this measurement, 0.04 mol of daunomycin was introduced per 1 mol of succinimide group. From the above results, X = cytotoxic substance = daunomycin, Z
= hydrogen atom, average degree of polymerization n + m + t + r = 45
8-A reaction polymer having the characteristics of [1) and [2] was obtained, in which the average amount of daunomycin in one molecule was n+m=18 and the average z+r=485. Example 6 Polysuccinimide obtained in Reference Example 1 was subjected to ethanolamine reaction and water-saving decomposition in the same manner as in Example 1, and 10.2f of the lyophilized product was dissolved in DMF 100-, and ethanolamine 4.4- was added, and the mixture was heated to room temperature. The reaction took place for 5 hours. After the reaction, 1N NaOH (50-) was added and the reaction was carried out at room temperature for 1 hour, followed by dialysis against distilled water and freeze-drying. Yield: 8.9
g. 1.6 g of the above hydrolyzed polymer was dissolved in 80% of 5% NaC6 solution, and 2.0'lf of 1-ethyl-(8-dimethylaminopropyl)carbodiimide hydrochloride was added.
Adjust the pH to 5.4 with NHCt, and add 600ml of daunomycin hydrochloride to this solution with 3Qml of 5% NaCt solution:
The solution was added and reacted at room temperature for 18 hours. After reaction, Sephadex G-25■ (manufactured by Pharmasha) 8.8 mm φ
Low molecular weight components were removed using a column of Tokoropolymer Il
There were 8.7×10 −4 moles of daunomycin bound in 1. When the terminal amino groups were quantified in the same manner as in Example 1, it was found that the terminal amino groups were 1 x 10-4 mol in the polymer lf, and the average molecular weight was 10.000. molecular weight 0.18 f) and dimethyl-3,8'-dithiobispropionimidate (
Purchased from Pierce) 121 Misaki and Dithiothreitol 60W
A solution of II dissolved in 6-distilled water was added and reacted at 28°C for 18 hours. Wa, back +17 7', v'l,, -G-25■,
Gel filtration was performed using a 3.8-φxlll:rn column (manufactured by Almasia, Inc.) to remove low-molecular components. When the terminal amino group was measured using this fractionated solution, 9
0% of the amino groups were reacted. After the above reaction, the reactive polymer was cyclized with dithiothreitol, and the thiol groups were quantified in the same manner as in Example 8. As a result, 8.88 x 10-4 moles of thiol groups were present in 1 g, which was 90%. A thiol group could be introduced using the terminal amino group with a yield of . Example 7 The reaction between polysuccinimide and dithiobis(succinimidylpropionate) and the purification of the reactant were carried out under the same conditions as in Example 5. The above reactants o and sy were dissolved in 10-DMF, and 295 μm of adriamycin hydrochloride was added to this solution, followed by 0.14 m+ of triethylamine and allowed to react at room temperature for 10 days. Thereafter, the same reaction as in Example 5 was carried out. After introducing ethanolamine and performing a reduction reaction, a polysuccinimide derivative with a terminal thiol group was obtained using activated thiopropyl sepharose 6B. Yield: 0.259. The amount of adriamycin introduced was determined.The terminal thiol group was
It contains 0' mole and has an average molecular weight of 82.000. Adriamycin is 2.7xlO in 1g of reactive polymer.
0 per mole of succinimide group.
．． This means that 0.5 mole of the amount was introduced. From the above results, X = cytotoxic substance = adriamycin,
Z=hydrogen atom, average degree of polymerization rl+rrl-+-z
+r=450, -C' where the average number of adriamycin bonds in the molecule n+m=22 and the average z+r=428
A reactive polymer having the following characteristics was obtained: l') and [2']. Example 8 0.5f of polysuccinimide obtained in the above reference example was added to 1
Dissolve in 0 m DMF. Add p-(bis(2-chloroethyl)amino) to this solution.
-] L-phenylalanine (also known as melpheran) 815 cape was added, then triethylamine 0.14 m was added to 60
C. Stirring was performed for 48 hours. Then 8-amino-1,2
A mixed solution of -propanediol 1.2- and DMF5- 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 portion was filtered through a membrane with a pore size of 0.45 μm (Millipore filter), and the o liquid was filtered with 5 ephadex.
(2) Low-molecular components were removed by gel filtration using a G-252-Par7 Ian (26 cm φ x 40 cm) using distilled water as an eluent. After this, freeze-drying was performed. Yield 0.2f. Freeze-dried 0.1MPBS Pa Sofa (pH 7.4
) was measured at 258.5 nm tD absorption in a solution of (2-chloroethyl)]-]L
-The amount of phenylalanine introduced was estimated to be 2.9 in lf.
X 10-' moles were introduced. From this value, 0.1 mole of melphalane was introduced per mole of succinimide group. The terminal 72 group was quantified in the same manner as in Example 1, and was found to be present in 1.25 x 10 moles in lf. The average molecular weight is 40,000. Dissolve 0.11 of the above freeze-dried product in 1-DMF and 5P
DP7.8W was added and the reaction was carried out at room temperature for 24 hours. After dialysis against distilled water, lyophilization yielded 80%
q The amount of 5PDP introduced into the terminal amino group of the reactive polymer thus obtained was measured by measuring the amount of 4-thiopyridone produced by reduction in the same manner as in Example 1. Inside is 1.75X10'MoJ and S
PDP was introduced. From the above results, x = cytotoxic substance - melf n + m + z
+r=-t 91, - average number of melphalan in the molecule n
+m=19. A reactive polymer having the characteristics of [11, [2] with an average t + r = 171 was obtained,

Claims (1)

[Claims] (1) Consisting of structural units represented by the following formulas [1] to [4] ['1'] [2) ↓ [81'C4''3 and → each structural unit in calf (1 ), C2J.CB], (4) The following is attached to the amino group poison end of the polymer in which the numbers of n, m, t, and γ are each (these numbers are 0 or a natural number, and n+m≧2). A polysuccinimide derivative having an active group represented by the formula [5] and having a molecular weight of 2.000 to 8,000.000. (2) Consisting of structural units represented by the following formulas [1] to [4] 1 [1] [2] (8) [4) and - each structural unit 11-1') in the molecule, C
2]. [81, the number of [4] is n, m, t, γ (these numbers are 0 or a natural number, and n+m≧2).The lower molecular weight is 2.000 at the amino group end of the polymer. ~a,o
In producing polynosuccinimide derivatives which are oo and ooo, H [6] [7] [8] [9] and - each structure in the molecule have the following structural units [6] to [9]. Unit [6], [7]
. 11:'8], [9) numbers are n, m, L respectively
, r (these numbers are 0 or natural numbers and n+m≧
It is 2. ), and the molecular weight is 2.000~a, o
oo, oo. After (a) reacting a cytotoxic substance containing an amino group or an imino group with a polysuccinimide derivative that is -5 through the spacer
- A method for producing the polysuccinimide rice conductor, characterized by carrying out a reaction with a reagent for introducing a Z group. (8) Consisting of structural units represented by the following formulas [1] to [4] 1 ('I) (2) Y [8] [4] and each structural unit C'l], C2] in one molecule. The numbers of [81, C4] are n, m, t, γ(
These numbers are 0 or natural numbers, and n+m≧2. ) has an active group represented by the following formula [5] at the amino group end of the polymer [5] In producing the z-s-w-c-succinimide derivative, the following [6]
H [6') [71 and each constituent unit in one molecule [61, C73° (8
], [The number of 91 is n, m, and 1.1 (these numbers are 0 or a natural number, and n+m≧2), and the amino group end of the polymer is represented by the following formula [10] A polysuccinimide derivative having an active group and a molecular weight of 2.000 to s, ooo, ooo is reacted with a cytotoxic substance containing an amino group or an imino group, and if necessary, a water-soluble aliphatic primary amine is added. A method for producing the polysuccinimide derivative, which comprises reacting the polysuccinimide derivative. (4) Consisting of structural units represented by the following formulas rl) to [4] [1] [2] Y [3] [4] and each structural unit in one molecule (11, C2''J. [8), An active group represented by the following formula [5] is attached to the amino group terminal of a polymer in which the number of CUJ is n, m, t, and 1 (these numbers are 0 or a natural number, and n+m≧2). In producing a polysuccinimide derivative having a molecular weight of 2.000 to 8.000.000, (a) an amino group is added to the polysuccinimide formed by the general formula [wherein k represents a natural number]. Reaction with a cytotoxic substance or water (b) Using an amino group to react with -5- through a spacer
Reaction with a reagent for introducing a Z group (c) The method for producing the polysuccinimide derivative, characterized in that a reaction with a water-soluble aliphatic amine is carried out if necessary. (5) Consisting of structural units represented by the following formulas [1] to [4] [,11[2F CB '3 C4) and - each structural unit in the molecule [1) , [21゜[8) , C4] having an active group represented by the following formula [5] at the amino group terminal of a polymer whose numbers are n, m, t, and γ, respectively (these numbers are 0 or a natural number, and n+m≧2). In producing a polysuccinimide derivative having a molecular weight of 2.000 to a, ooo, ooo, the following formula [
Consisting of structural units represented by 6] to [9] (:'6) [7) and - each structural unit ('6') in the molecule, C'
l]. [81, The number of (9) is n, m, t, r (these numbers are 0 or a natural number, and n+m≧2). At the amino group end of the polymer, the number is represented by the following formula [10] A method for producing the polysuccinimide derivative, which comprises reacting a polysuccinimide derivative having an active group and a molecular weight of 2.000 to 8.000.000 with an active disulfidated metal. (6) The following method: Consists of structural units represented by formulas [1) to [4] CI) C'21 [8), r41, and each structural unit in one molecule (1'J, [21
゜ [8) , [41 numbers are n, m, t, r, respectively (These numbers are 0 or natural numbers, and n+m≧2.)
The polymer has an active group represented by the following formula [10] at the terminal amino group and has a molecular weight of 2.000 to a, ooo
, ooo, consisting of structural units represented by the following formulas [6] to [9] l [6') [7] (:'81 C9] and each structure in one molecule Unit [6), 'C7]. C8], [9) are respectively n, m, z, r (these numbers are 0 or a natural number, and n+m≧2), and the following formula [11] is added to the amino group terminal of the polymer. The above method is characterized in that the disulfide bond of a polysuccinimide derivative having an active group represented by [11] z2-s-w-c- and a molecular weight of 2.000 to 8.000.000 is reductively cleaved. Method for producing vorisuccinimide derivatives.