JPS60260622A - Reactive polymer bound with bifunctional ligand compound - Google Patents

Abstract

PURPOSE:The titled polymer bound with a physiologically active substance having an ability to form a chelate with a radio-active metal and being useful as a diagnostic agent for particular diseases, prepared by reacting a specified polysuccinimide with an amino group-containing bifunctional ligand compound. CONSTITUTION:A polysuccinimide of formula I (wherein k is a natural number) obtained by polymerizing aspartic acid by dehydrating at 160-240 deg.C at normal or reduced pressure is reacted with an amino group-containing bifunctional ligand compound (e.g., deferoxamine) or an agent for introducing an -SZ group [wherein Z is H, -SR, or Z' (= a group which, together with the adjacent S, can form a reactive disulfide group) and R is W as defined below] through a spacer which is a terminal group - formula II [wherein W is an (un)substituted hydrocarbon group, and A is O, NH, or the like] or a like compound to obtain a polysuccinimide derivative of a MW of 2,000-3,000,000, composed of a polymer comprising structural units of formulas III-VI (wherein X is a residue of a bifunctional ligand compound having amino groups in the molecule) and reactive groups of formula VII bound with the terminal groups of the polymer.

Description

[Detailed description of the invention]

The present invention relates to a reactive polymer bonded with a difunctional ligand compound and a method for producing the same. More specifically, the present invention relates to a reactive polymer useful for producing a radiodiagnostic agent with an i-paired metal label for use in nuclear medicine for the purpose of detecting a specific disease, and a method for producing the same. Attempts have been made to label physiologically active substances consisting of protein substances such as fibrinogen with radioactive substances for the purpose of detecting specific diseases, for example diagnosing the site of thrombosis. One such labeling method involves labeling with a radioactive metal that has physical properties suitable for nuclear medicine diagnosis. At that time, attempts are being made to label physiologically active substances with radioactive metals using bifunctional ligand compounds that can form chelates with the radioactive metals and form stable chemical bonds with the physiologically active substances. . However, when many bifunctional ligand compounds are directly reacted with a physiologically active substance in order to increase specific radioactivity, the physiologically active substance is likely to be denatured. Therefore, it is difficult to obtain a nuclear medicine diagnostic agent that does not cause denaturation of physiologically active substances and has high specific radioactivity. The present inventors conducted intensive studies to solve the above problems, and as a result, the following formulas (1) to [4] (1) (2) (8) (4) (the above formulas (1) to In [4], X and Y each have the following meanings: X: reactive residue of a bifunctional ligand compound having an amino group in the molecule Y: water-soluble aliphatic primary amine residue; The numbers of each structural unit CD (2), (8), and (4) in the molecule are n, m. The following formula [5] 18- [In the above formula [5], Z, W and A each represent the following meanings. 2: Hydrogen an atom or a group represented by the general formula 21 or -3-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: Oxygen atom or NH group or a salt thereof] and has a molecular weight of 2,000 to 8,000,000
A polysuccinimide derivative (hereinafter referred to as polysuccinimide derivative □(I)), which is a highly reactive polysuccinimide derivative that (1) binds a difunctional ligand compound and (2) has a thiol group or a disulfide bond. The present invention was achieved by discovering that the above-mentioned problems can be solved by combining a molecule with a physiologically active substance and further labeling it with a radioactive metal. 14- The present invention provides a reactive polymer for use in such a radiodiagnostic agent. Here, the group (Z') that forms an active disulfide bond with the adjacent sulfur atom includes 5-nitro-2-pyridylthio group, pyridylthio group or its N-oxide, and 8-carboxy-4-nitrophenylthio group. phenylthio groups,
Furthermore, in the substituted or unsubstituted hydrocarbon group represented by N-phenylamino-N/-phenylyW, the hydrocarbon group includes, for example, a linear or branched alkylene group having 1 to 10 carbon atoms. be able to. Bifunctional ligand compound is a term used in nuclear medicine, and it has 1) strong chelate-forming ability for various metals.
...(2) A general name for a compound that has a functional group that can chemically bond with a physiologically active substance. (Rediopharmaceuticals 5tr
ucture-ActivityRelations,
R-fchard P, 5pencer ed. P, 282
) Bifunctional ligand compounds having an amino group include not only those having an amino group as a difunctional ligand compound.
This includes those in which an amino group is newly introduced into a bifunctional ligand compound that does not have an amino group. For example, if the bifunctional ligand compound is a compound having a carboxyl group, a new amino group can be introduced by reacting this carboxyl group with one amino group of a diamino compound (for example, hexamethylene diamine) according to the usual method. obtain. Specific examples of difunctional ligand compounds containing amino groups are:
Deferoxamine, ethylenediamine-N, N-, which is known as a difunctional ligand compound containing an amino group
Examples include cyacetic acid (EDDAχ1-(p-aminoethyl)phenylpropane-1,2-dione-bis(thiosemicarbazone)).Also, bifunctional ligand compounds that can newly introduce an amino group include , Example 17 - For example diethylenetriaminepentaacetic acid (DTPA),
Examples include those in which a 7-mino group is introduced into ethylenediaminetriacetic acid (EDTA) and the like. Of these, deferoxamine contains 67Ga and 1-(p-
Aminoethyl) phenylpropane-1,2-dione-bis(thiosemicarbazone) has a stable ligand ability of 99 mTC and is preferred. In particular, the use of de7eloxamine is particularly costly since the amount of labeling is easily increased. Y in formulas [8] and [4] is a reactive residue of a water-soluble aliphatic primary amine. Although it is not necessarily necessary to use it for the purpose of the present invention (l+r=0), it 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, CH, (CH,)gNH2 (t = (integer from 1 to 4)
aliphatic amines, etc. In particular, amines having a hydroxyl group such as ethanolamine and 3-amino-1,2-propane-18-diol are preferred. n-1-m is the number of structural units to which X is bonded, and e
+r represents the number of structural units to which the water-soluble aliphatic primary amine is bonded. (n+m)/(n+m+e+r) is the number of condensed moles of X per mole of succinimide group, which is a monomer unit. (p+q)/(n+m+6-1-r) represents the number of condensed moles of the group represented by the general formula % with respect to 1 mole of the succinimide group, which is a heptamer unit. The number of molecules of the polysuccinimide derivative (I) of the present invention is 2,
000-8,000,000 preferably 2,000-8
,000,000, n-4-m is 2 or more, (n+
m)/(n+m+g+r) is the final tone, and (p+q
)/(n+m+g+r) is o, oot or more and 0.5 or less, preferably 0.3 or less. If the number of molecules is less than this, the number of bifunctional ligand compounds bonded to one molecule of the reactive polymer will be small, and the amount of bifunctional ligand compounds that can be bonded to the physiologically active substance will be small. This is not desirable. On the other hand, if the molecular acid is too large, aggregates of physiologically active substances tend to form, and insolubilized components and large molecular weight components tend to be generated, which is not preferable. The polysuccinimide derivative CI) of the present invention can be easily derived from polysuccinimide having the structure of general formula (n) below. 1 0 ・115 [1 [In the formula, k represents a natural number. ] Polysuccinimide is a polymer also called anhydropyasparatic acid, and can be obtained mainly by thermal condensation of aspartic acid. (J,
Kovacs etal, J, Org, Chem
, 26.1084 (1961) +P, Neri e
tal,, J, Med, Chem, 16, 89
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% 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, the polymer will decompose, 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 unreacted aspartic acid and low-molecular condensate, followed by filtration through a glass filter and drying. 21- Repeat the tobacco operation if necessary. IR and NMR suggest that the polymer thus obtained is represented by the general formula (II), but even if it partially contains other structural units, it is substantially the same as the general formula (II). It is generally accepted that the structural unit (II) is the main component. 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 22- (In the formula, x and y represent natural numbers.) It is also known to carry out the reaction of the following reaction formula under alkaline conditions. [■] [In the formula, x and y represent natural numbers] There are two methods for obtaining the polysuccinimide derivative of the present invention [■]. The first method is formula [6] ~

[9] H (6) (7) ・, i (In the above formulas (8) and (9), Y is as described in 6 above. Also, - each structural unit (6) in the molecule,
(7), (8);

The numbers of [9] are n', ', l' respectively.
, r'. , these numbers are 0 or natural numbers, and H'-
1-m'≧2. ) Poly+succinimide derivatives (hereinafter referred to as polysuccinimide derivatives ([[
). ), or a polysuccinimide derivative (hereinafter referred to as polysuccinimide derivative 1) having an active group represented by the above formula [6] at the amino group end of the polysuccinimide derivative [nail]. ), and then obtain a polysuccinimide derivative [I] in which X = "bifunctional ligand compound having an amino group in the molecule". As the first representative method, there are methods +11 to (4) below. (1) Polysuccinimide (II) is (a) reacted with a water-soluble aliphatic primary amine if necessary, and then hydrolyzed to obtain a polysuccinimide derivative (([[), 25- ) Polysuccinimide derivative
A reagent for introducing a -Z group is reacted to obtain a polysuccinimide derivative (IV) as a reactive intermediate.゛Thereafter, the polysuccinimide derivative (IV) is reacted with el) a difunctional ligand compound having an amino group, and further) if necessary, a water-soluble aliphatic primary amine to prepare the polysuccinimide derivative (IV) of the present invention. Succinimide derivatives can be produced. (2) Polysuccinimide (II) is reacted with (a) a water-soluble aliphatic primary amine if necessary, and then hydrolyzed to obtain a polysuccinimide derivative ([1
1), (b) react the polysuccinimide derivative [published] with a difunctional ligand compound 26-1 having an amino group, and then react with a water-soluble aliphatic primary amine if necessary. -5-Z to the terminal of the amino group via a spacer.
The polysuccinimide derivative of the present invention can be produced by reacting with a reagent that introduces a group. (8) In the method (1) or (2) above, 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 to form a bifunctional polysuccinimide derivative having X=diamino. sexual ligand compound, Z
- A polysuccinimide derivative (I) which is a hydrogen atom can be obtained. In addition, in the method (11) above, (
The above polysuccinimide derivative (I) 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) The bifunctional ligand compound having X = diamino obtained by any of the methods (11 to (8) above) and the polysuccinimide derivative (I) in which Z = hydrogen atom, an active disulfide By reacting the compounds, a bifunctional ligand compound having an X-amino group, a polysuccinimide derivative CI) in which Z = Z/, 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. The preferred method for carrying out the reaction with the water-soluble aliphatic primary amine in (a) of (1) and (2) is to use polysa
°“)DMFXi”DMSO$ 17. (, to l~8
The water-soluble aliphatic primary amine was dissolved to a concentration of 0 (W/W)%, and the water-soluble aliphatic primary amine was added in an amount of
- to mol, preferably 0.2 to 1 mol, and -40°C to
The reaction is carried out at 80° C. for 1 minute to 2 days with stirring, and then hydrolysis is carried out. Hydrolysis is carried out in an alkaline aqueous solution. For example, the reactant of polysuccinimide (If) or polysuccinimide (II) and a water-soluble aliphatic primary amine is mixed with DMF or DM.
Dissolve in SO and dropwise add alkaline aqueous solution, or add polysuccinimide (If) or a reactant of polysuccinimide ([) and water-soluble aliphatic primary amine to the alkaline aqueous solution and heat at 0°C to 80°C. Allow to react for 10 minutes to 2 days. The alkaline aqueous solution has a pH of 8 or higher, preferably 0.
1-1N NaOH aqueous solution. Using the amino group of (cll in reaction (1) or !F- in reaction (2)), -
The reagent for introducing the 5-Z group may be any of such known reagents, but for example, the general formula (VIa) 29-Nl (where Ll is a divalent organic group and Ql is an imidoester alcohol ) or its salt (for example, HCl salt, HBr salt, etc.) can be used. In the above general formula [■a], the organic group represented by Ll is, for example, ethylene , carbon number 2 such as trimethylene
Examples of the alcohol residue represented by +Qs for the linear or branched alkylene group of ~6 include alkoxy groups having 1 to B carbon atoms such as methoxy and ethoxy. More specifically, the above reagents include, for example, methyl-8-mercaptopropylimidate NH, HCe 1 (H5-C-CH, -C-OCH,), methyl-4-
Examples include mercaptobutyrimidate 80-NH, HCe 11 (H5-CH, -CH2-CH, -C-QC8), and the like. etc. can also be used as a reagent. Furthermore, in the general formula [■b] C, ZI and Ll are as described above. Q represents the alcohol residue of the active ester. ] A compound having the general formula [■C]C, where Z/,
L and Ql are as described above. ] or a salt thereof (e.g. HCe salt,
(HBr salt, etc.), a compound having the general formula id) NH 1 +5-Ll-C-Q, ) 2 (■d) [wherein Ll and Q are as described above] or a salt thereof ( For example, HCe salt,
HBr salt, etc.), general formula [■e] [wherein L and Ql are as described above. ] and S-acetylmercaptosuccinic anhydride. As the reagent represented by the above general formula (Vlb),
For example, as a reagent in which N-succinimidyl-8-(2-pyridylthio)propionate is expressed by the general formula [■C], for example, 8-C41-dithiopyridyl)mercaptoprobioyl (date general formula [■d ] For example, dimethyl-8,81-dithiobispropionimidate
NH, HC/1 ((-5-CH2-CH!-C-OCH,), 1, etc.; examples of reagents expressed by the general formula [■e] include dithiobis(succiniimii-88-dylpropionate), etc. Such a reaction with a reagent for introducing a -5-Z group via a spacer can be carried out, for example, as follows: DMF, DMSO, water or their l~go(W/
Dissolve the polysuccinimide or the polysuccinimide derivative after the reaction of (a) in (1) or after the reaction of rll in (2) so that the terminal amino group is
Add 100 times the number of moles of a reagent for introducing a -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, -40 to 80 C. for 80 minutes to 2 days with stirring. Unreacted reagents can be removed by reprecipitation using a solvent such as ethanol, which is a non-solvent for polysuccinimide or its derivatives, or by dialysis, gel filtration, or the like. The reaction with Hl in (1) (21) with a bifunctional coordination compound having an amino group (initial) may be carried out under any conditions of the reaction between a carboxyl group and an amino group in peptide synthesis. As a reaction solvent This is usually carried out using water, DMF, DMSO or a mixed solvent thereof.In the reaction, a condensing agent such as l-ethyl-8-(8-dimethylaminopropyl)carbodiimide hydrochloride or dicyclohexylcarbodiimide is used. A method of condensing a polysuccinimide derivative having a carboxyl group and a bifunctional ligand compound having an amino group in the coexistence of the polysuccinimide derivative, a method in which the carboxyl group of the polysuccinimide derivative is condensed with inchlorobutyl formate, etc. in the form of a mixed acid anhydride. There is a method in which a bifunctional ligand compound having an amino group is activated and reacted with the difunctional ligand compound.In this reaction, the reaction temperature is -40 to 100°C and the reaction time is 1.
The period is from 0 minutes to 10 days. The second method for obtaining the polysuccinimide derivative CI"l of the present invention is to directly connect the amino group of a bifunctional coordination molecule compound having an amino group with succinimide, and to attach -5-Z group to the terminal amino group via a spacer. React the reagent that introduces the
This is a method for obtaining a polysuccinimide derivative CI) which is a bifunctional ligand compound having X=diamino. The second method includes methods (5) to (7) below. (5) Reaction of polysuccinimide [■] with (a) a difunctional ligand compound having an amino group...(, (b) If necessary, a water-soluble aliphatic primary amine Reaction with el) Utilizing the amino group, -S via a spacer
A method for obtaining a polysuccinimide derivative CI) which is a bifunctional ligand compound having X=diamino by subjecting it to a reaction with a reagent capable of introducing a -Z group. (61 Above (51) Method Te Z=Z/Also Bar S-R Te When a certain polysuccinimide derivative is obtained, the polysuccinimide derivative is subjected to a reduction reaction in the same manner as in (8) above to obtain X - Bifunctional ligand compound having an amino group, polysuccinimide derivative where Z=hydrogen atom (
I) How to obtain. (7) A bifunctional ligand compound having X=diamino obtained by any method of (5) or (6) above, Z-=
By reacting the polysuccinimide derivative CI), which is a hydrogen atom, with an active disulfide compound as in (4) above, a bifunctional ligand compound in which X = diamino, and a polysuccinimide derivative in which z = z' A method for obtaining a succinimide derivative (II). 87- Furthermore, as a method for the above (5), a sequential reaction method in which reactions (a), (b), and (c) are performed sequentially in any order;
There is a simultaneous reaction method in which the reactions of (b) and (b) are carried out simultaneously. In the simultaneous reaction method, either reaction (a), -) or reaction (c) may be performed first. The preferred implementation method is a sequential reaction method, especially in the case of unstable bifunctional ligand compounds, after performing the reaction (c),
It is desirable to perform (a) and (b) in this order. (a) or (0) or the reaction of (a) and (b) at the same time is polysuccinimide or polysuccinimide). Polysuccinimide derivatives subjected to any one of the reactions of (trunk), (c), or t=q or K), (01
It is appropriate to conduct the reaction in a solution in which the polysuccinimide derivative subjected to any one of the reactions is dissolved in a DMF or DMSO solvent. More specifically, the reaction can be carried out, for example, as follows. Polysuccinimide or an unreacted succinimide group 88-0 imide derivative is added to the solvent in an amount of 1 to 80 (W/W)%.
In the DMF or DMSO solvent added so that
0.001 to 10 mol of - component per 1 mol of succinimide group in the solution) is reacted with stirring at -40 to 80°C for 10 minutes to 20 days. Unreacted reagents can be removed by reprecipitation with a nonsolvent, dialysis, or gel filtration. The desired vorisuccinimide derivative can be obtained by distilling off the solvent under reduced pressure or by freeze-drying. The reaction of i/Sai uses polysuccinimide or a polysuccinimide derivative subjected to any one of the reactions (a) or (b), or a polysuccinimide derivative subjected to the reactions (a) and (b). The reaction of (0) in (1),
Carry out the same reaction as in (2). The molecular weight is controlled by selecting the condensation conditions to obtain the polysuccinimide polymer (If) and selecting various reaction conditions.
Separation of a polymer having the desired molecular weight involves molecular weight fractionation by gel filtration of a DMF bath solution of the polysuccinimide polymer and gel filtration in an aqueous solvent after obtaining the polysuccinimide derivative CI). By the method of molecular weight fractionation by oral filtration. After obtaining the polysuccinimide derivative [I], it is desirable to carry out gel filtration in an aqueous solvent to perform molecular weight fractionation. In order to improve the yield of the desired molecular weight fraction, it is necessary to select the condensation conditions to obtain the polysuccinimide polymer (n), and to carefully perform the molecular weight fractionation by gel filtration with a D IVf F solution of polysuccinimide. Let's do it. Said (41, +717) method'? l'arrow SH group ti
The active disulfide compound used in the method of reacting a sulfide compound to obtain a reactive polymer having an active disulfide group refers to the above-mentioned group capable of forming an active disulfide bond with an adjacent sulfur atom ( Z') is a disulfide (do) form, and specific examples of such active disulfide compounds include a disulfide form of the aforementioned group exemplified as ZI, such as a 2-pyridylthio group, which is a disulfide form of a 2-pyridylthio group. 5,51-dithiobis(2-nitrobenzoic acid), which is a disulfide of 8-carboxyl-4-nitrophenylthio group, which is a disulfide of Usually water or DMF or DMSO
The reaction is carried out in a homogeneous reaction system using organic solvents such as Alternatively, the reaction can be carried out using a reaction system in which an active disulfide compound or its acetone solution, dioxane solution, etc. are added and mixed to an aqueous solution of the polymer. The reaction temperature is -5
Appropriate reaction time is 1 minute to 24 hours at ~70°C. The polysuccinimide derivative of the present invention bonded with a bifunctional ligand compound having an amino group has the ability to form a chelate with a radioactive metal and has a highly reactive thiol group or a reduced Since it contains a disulfide group or an active disulfide group from which a thiol group can be easily obtained, it can be used with anti-tumor antibodies (anti-tumor immunoglobulins, physiologically active substances such as fibrinogen, which are protein substances that can detect specific diseases). For example, N-succinimidyl-8-(2 -pyridyldithio)
Active disulfide groups introduced from propionate (SPDP) etc., thiol groups introduced from methyl-8-mercaptopropylimidate etc., e.g. dimethyl-
A thiol group obtained by reacting and then reducing 8,B'-dithiobispropionimidate, or, for example, N
It can be formed via a maleimide group introduced from an N-hydroxysuccinimide ester such as -(meta-maleimidobenzoyloxy)succinimide (MBS) or N-(4-carboxycyclohexylmethyl)maleimide. As a method for bonding a thiol group derived from a physiologically active substance or a thiol group derived from a disulfide bond such as that of an anti-tumor antibody with a thiol group derived from a reactive polymer, one of the cimaleimide compounds such as 0-7 22 dimaleimide can be used. There is a method in which two maleimide groups are reacted in advance with one thiol group of a physiologically active substance or the reactive polymer of the present invention to introduce a maleimide group, and then reacted with the other thiol group. Thiol groups derived from disulfide groups, such as in antitumor antibodies (immunoglobulins), can be obtained by reducing the disulfide bonds of the antibody with a reducing agent such as dithiothreitol or cysteamine. In addition, by treating immunoglobulin with pepsin, Fab
A dimer of ' is obtained, 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 one molecule. The thus obtained conjugate of the polysuccinimide derivative [■] and a physiologically active substance having targeting ability is useful for nuclear medicine for the detection of specific diseases.
L r 9i * b' M n 8 n 6 , ,
, t, The present invention will be described in detail with reference examples and examples below. 0
do. Reference Example 1 Production of vorisuccinimide L-aspartic acid 66.5F to 85% phosphoric acid 20.6
ay/ was added, and while heating to 190° C. using a rotary evaporator, polymerization was carried out under reduced pressure of 10 wsH7 for 5.5 hours. After polymerization, it was dissolved in DMF and reprecipitated in water. After reprecipitation, the resultant was filtered through a lath filter and vacuum-dried at 80° C. for 8 days to obtain polysuccinimide. When the infrared absorption of the obtained polymer was measured using the KBr tablet method, it was found that an absorption characteristic of succinimide groups was present at 1800.1720°1660 cm'' by 8. The molecular weight was measured by GPC measurement.D G P Ch 5 A (A D -8
The obtained polysuccinimite was dissolved in DMF + 0.01 MLiBr solvent to a concentration of 0.2% using a solution (QM/S-Showa Denko), and the solution was diluted with 260 μe.
was injected and the same solvent was used as the elution solvent to perform measurements using a differential refractometer. 46- A calibration curve was created using polyethylene glycol of various molecular weights. The obtained polysuccinimide had a weight average molecular weight of ff18.9×104 in terms of polyethylene glycol,
The number average molecular weight was 1.5×104. Reference Example 2 Polysuccinimide obtained in the above Reference Example 1.8?
Add 10 g of 0.78 wtl (equivalent to 18 mmol) of ethanolamine to a solution of C4t Kushifiimi t' group I B (equivalent to 6 mmol) dissolved in DMF80+/.
Add the solution dissolved in g/g of DIVI P and boil at 4C for 1
The reaction was carried out for 8 hours. This solution (CIN-NaOH
Solution 1 (++/) was gradually added dropwise over 1 hour with stirring. After the reaction, dialysis was performed for 2 days against an aqueous solution with a pH of 4.0. Polysuccinimide derivative (111) was obtained by freeze-drying.The yield was 1.Of.The terminal amino group was quantified by the following method.5 mg of the above freeze-dried product was mixed with 0.0. A solution of 2.4.6-dolinitrobenzenesulfonic acid (hereinafter abbreviated as TNBS) 82~ dissolved in 1 ml of 1M sodium borate solution and 1 ml of 1M sodium borate solution was added.
After adding 1 wt1 and reacting at 25°C for 45 minutes, 1.9 ml of 0.1 M sodium borate solution was added.
Absorption measurements were made at 20 nm. Quantification was performed using a molecular extinction coefficient of 11.100 obtained from a calibration curve obtained using a known amount of aspartic acid. The amount of amine in li is 8.8 x 10-'M and the average molecular weight is 28,000. The content of carboxylic acid was determined by acid-base titration. l
2.07 x 10-'' moles of carboxylic acid were present in V. No unreacted succinimide groups were detected by IR and 1'' C-NMR. From the above results, using a monomer unit of succinimide group into which ethanolamine is introduced, a molecule of 4 feet, ff1158, and a hydrolyzed seven-mer unit of succinimide group, which has a molecular weight of 115, a carboxyl group is introduced per mole of succinimide group. The number of moles (X) of was calculated using the following formula. =2.07X10→158X(1-X)+115Xx The calculated result was 0.8 mol. The above freeze-dried product 0.91 is DMF 9m? dithiobis(succinimidylprobionate) (purchased from Pierce) 180 μ (8,24,× 1.0-' mol) dissolved in
8.4 tnl of triethylamine was added and the reaction was carried out at room temperature for 24 hours. After dialysis against distilled water 2e, dithiothretol 125~ was added and reduced at 80°C for 1 hour. Add this solution to 5ephadex G-25 Super Fine (manufactured by Pharmacia) (8.8 cm d
11.5 cm) F 0.01 ・, after gel filtration using M sodium phosphate-1, IMEDTA (pH 7,0) to remove low molecular components, activated thiopropyl cef 10- Sodium 6B resin 50 tel (wet volume) to 0.01M sodium phosphate-1□MEDTA (pH 7,0) 50
It was added to the liquid dispersed in wtl 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-1rnMEDTA (pH 7, Q), and then 10M of dithiothreitol 180μ was added.
1 of 0.01M sodium phosphate-1mM EDTA (p
Fill a column with a solution dissolved in H7,0') and incubate at room temperature for 8 hours.
The light was on for 0 minutes. Then 0.01M sodium phosphate-1mM EDTA (
pH 7.0) was passed through a column to collect the effluent polymer fraction, dialyzed against water, and then freeze-dried to obtain a polysuccinimide derivative [■] (where Z = hydrogen atom). Ta. Yield 10.5F The thiol group of the thus obtained polysuccinimide derivative having a terminal thiol group was placed in accordance with the method described in Example 8 below. The results are GC8,6 in polysuccinimide derivative IP
X, 10' moles of thiol groups were present, and this value was 28,000 for the lyophilized product used in this reference example. In addition, the average value of the molecular weight obtained above and the degree of polymerization (n'+m'+e'+r') of the reactive polymer mixed with X is r
/) was 185. Example 1 DM polysuccinimide 1.8f obtained in Reference Example 1
Dissolved in SO80d. To this solution, add 2.41 ml of deferoxamine mesylate (purchased by Takeda Yakutama Yori, Tesferal ■) (Iyal) and 0.6 ml of triethylamine.
A solution prepared by dissolving Goji in Drv'I SO was added and stirred at 60° C. for 4 hours. Thereafter, ethanol cyamine was added and the mixture was left at 60° C. for 1 hour and at room temperature overnight. After performing molecular weight fractionation by gel filtration with a 2,6-m x 40 crn column of 5ephadexo G-50 Super Fine (manufactured by Pharmasha), which was dialyzed against water for 2 days and equilibrated with distilled water, Lyophilized. Yield: 1.2f The terminal amino groups of the above freeze-dried product were quantified in the same manner as in Reference Example 2. OX L O-'
There were 7 mol of amino groups present. average molecule ito, ooo
It is. No unreacted succinimide group was detected by IR and 13C-NMR. The amount of deferoxamine introduced into polysuccinimide was measured by the following method. After reaction with ethanolamine, 400μ of reaction solution before dialysis
400 μCi of 67Ga-citrate solution was added to the solution and left to stand for 5 hours to chelate 67Ga to deferoxamine. Thereafter, this reaction solution was separated by cellulose acetate membrane electrophoresis, and deferoxamine introduced into the polysuccinimide and unreacted deferoxamine were completely separated. Based on the radioactivity ratio of each component, the ratio of deferoxamine introduced into polysuccinimide was 78% of the charged deferoxamine. The amount of deferoxamine introduced per mole of succinimide group was calculated from the following formula. [Number of moles of deferoxamine mesylate charged] x [ratio of deferoxamine reacted with polysuccinimide] /
[Number of moles of charged succinimide group] = 9.7 m
mol Xo, 85/18.6mmol = 0.1
7 0.17 mol of defluoroxamine was introduced per mol of succinimide group. b, t, Above, 0.88F of the freeze-dried product was dissolved in 8 txt of DMF', 5PDPI87 and 0.2 ml of triethylamine were added, and the mixture was stirred at room temperature for 24 hours. After the reaction, the mixture was dialyzed against distilled water 2e and freeze-dried. Yield: 0.71 When the introduced 5PDP0)it was reduced in the same manner as in Reference Example 2 and quantified from the amount of 4-thiopyridone produced, 7 X 10-' mol (7) was found in it.
5PDP power reaction source. A polysuccinimide derivative (I) has been obtained having a terminal end. From the above values and the molecular weight of the 7-mer unit of succinimide group introduced with ethanolamine (0) molecule M 1 58, and the molecular weight of the monomer unit of succinimide group introduced with deferoxamine of 667, the average molecular weight of the monomer unit is given by the following formula. 158X0.88+657X0.17#248 From the average molecule i10,000, the average degree of polymerization (n+m+g+r) is n+m+g+r=l O,000+248#4058
− Also, the average binding amount of deferoxamine per molecule n+
m=7 s e+r=a a. Example 2 Polysuccinimide 1.51 obtained in Reference Example 1 was converted to 15
Deferoxamine mesylate (purchased from Takeda Yakutama, Desferal ■ vial) 7,59 f and triethylamine 1,86 x t were dissolved in DMSO and stirred at 60°C for 4 hours. Thereafter, a mixture of 8.06 liters of ethanolamine and 15 ml of DMSO was added, and the mixture was further left at 60° C. for 1 hour and then at room temperature overnight. 5ephadex dialyzed against water for 2 days and equilibrated with distilled water
Molecular weight fractionation was performed by gel filtration using G-50 (manufactured by Lumashiya), followed by freeze-drying. Yield t, or0 Quantification of the terminal amino groups in the freeze-dried product was carried out in the same manner as in Reference Example 2, and it was found that 4.5 x 10-5 moles of amino groups were present in 14. The average molecule is 122,000. No unreacted succinimide group was detected by IR and 13C-NMR. The amount of deferoxamine introduced into polysuccinimide was measured in the same manner as in Example 1. The ratio of introduced de7-eloxamine is 78 to that of the prepared de-7-eloxamine.
%Met. 0.54 mol of deferoxamine was introduced per mol of succinimide group. Dissolve 0.9j' of the above freeze-dried product in 8 ml of DMF, add 160q of dithiobissuccinimidyl propionate (purchased from Pierce), and 8.6 Mt of triethylamine.
was added and the reaction was carried out at room temperature for 24 hours. After dialysis against distilled water 2e, 125 ml of thiothreitol was added and reduced at 80°C for 1 hour. 5ephad of this solution
ex G-25 Super Fine (manufactured by Pharmasha)
Equilibrate with a column of 8.8 mm x 11.5 mm and use elution buffer as 0.01 M sodium phosphate-1-MEDT.
After removing low-molecular components using A (pH 7.0) (hereinafter referred to as buffer), activated thiopropyl ceftonose 6
45 ml (8 wet volumes) of B resin was added to a dispersion prepared by dispersing 45 ml of the buffer solution, and the mixture was gently stirred at 40° C. for 18 hours. Thereafter, the resin was packed into a column and washed with a buffer solution, and then a solution prepared by dissolving 180 mg of dithiothreitol in a 10 Mt buffer solution was packed into the column and exposed to light for 80 minutes at room temperature. Thereafter, elution was performed with a buffer solution, and the effluent polymer fractions were collected, dialyzed against water, and then freeze-dried. Convergence: 0.5f0 The thiol group of the thus obtained polysuccinimide derivative having a thiol group at its terminal was determined by the method based on Example 8. There are 4.5X10-5 moles of thiol groups in lf, -
,. ,picture. i・( From the above results, the average degree of polymerization n + m + e + r = 51
, - average value of the amount of deferoxamine bound in the molecule n+m
A polysuccinimide derivative [■] with =27.5 and e+r =28.5 (where Z = hydrogen atom: r-) was obtained. Example 8 This example shows a method for converting a thiol-type reactive polymer, which is a Z-hydrogen atom, into a reactive polymer with z = z', and
This shows an electromagnetic method for thiol groups in thiol-type reactive polymers. The thiol-type reactive polymer 20q obtained in Example 2 was
0.1 M sodium phosphate in ttt-1mMEDT
It was dissolved in buffer A (pH 7.0), 10 mt of dithiothreitol was added, and a reduction reaction was carried out at 80°C for 1 hour. 5ephadexoG-25x equilibrated with the above buffer after reaction
-Perfine (manufactured by 7 Almasya) 2. Ocm
After gel filtration with ItX 15 cm to remove low molecular weight components, the mixture was diluted 20 times with the same buffer. This diluted solution 500
20 μe of a methanol solution of 5 mM 4-pyridyl disulfide was added to the μe and reacted at 80° C. for 15 minutes, and 4-thiopyridone produced at the same time was measured at 824 nm. 4-
Thiopyridone molecule 1, extinction coefficient 1.98X104
When the thiol groups in the thiol-type reactive polymer were calculated using the formula, it was found that 4.5 X l O-5 moles of thiol groups were present in the intermediate lf. Example 4 1 t of polysuccinimide obtained in Reference Example 1 was DM F 1
0 ml of the solution, 51 ml of dithiobis(succinimidylpropionate) was added, and the reaction was carried out at room temperature for 24 hours. After the reaction, the reaction mixture was added dropwise to 100 ml of ethanol while stirring to perform reprecipitation, and the mixture was passed through a TT glass filter. The precipitate was again dissolved in 10 m/ml of DMF, reprecipitated with ethanol five times, and then dried in a vacuum dryer. Collection to, 9r
. 8.0 mmol of the above dried product (equivalent to 58-5,16 mmol of succinimide groups)
Deferoxamine mesylate (purchased from Takeda Yakutama, Desphere 5-0P'r) was dissolved in DMSO and added to this bath solution.
L) 9601VC1,47 mmol) was added to the mixture at room temperature.
After reacting for 4 hours, 1.20 mmol of n-propyl-2-aminoethyl disulfide was added and reacted for 6 hours at room temperature, and then 10 mmol of 8-amino-1,2-propanediamine was added and reacted at room temperature for 1.20 mmol. A time reaction was performed. After the reaction, the mixture was placed in a dialysis tube and dialyzed against water for 2 days, followed by freeze-drying. 6.0 ml of the polysuccinimide derivative 200'f thus obtained was added to 0.1
M) Lis-hydrochloric acid-1mMEDTA buffer r-(1)
A solution of 0.02M dithiothreitol dissolved in 2.0 ml of the same buffer was added thereto, and a reduction reaction was performed at 50C for 100 minutes. After gelling and filtering this reaction solution with Cef1Dex G-50,
The reactive polymer having terminal thiol groups was separated using 59-activated thiopropyl Sepharose 6B resin in the same manner as in Example 2, followed by dialysis and freeze-drying. Yield 0.2? . The amount of deferoxamine introduced into the above reactive polymer per mole of succinimide groups was measured in the same manner as in Example 1. The amount of deferoxamine introduced per mole of succinimide groups was 0.
25 moles of deferoxamine had been introduced. No unreacted succinimide group was observed by IR and Inc-NMR. When the thiol group was quantified by the method of Example 8, l
There were 8.8 X l O-5 moles of thiol groups in i. The average molecular weight is go, ooo. From the above values, average degree of polymerization n + m + g + r = ゛・
−';j f ′l'l E 4 A 8 tL r:
= F 7"at-,,. A polysuccinimide derivative [I] (wherein 2 = hydrogen atom) with average values of samine n + m = 25, # + r = 74 + (where 2 = hydrogen atom) was obtained. 60 Example 5 Reference Example 2 5 g of the intermediate 20 obtained in step 1 was dissolved in ice water, and 68 rq of 1-ethyl-(8-dimethylaminopropyl)carbodiimide hydrochloride was added to the solution.
g Tep H6, OE adjusted. A solution of deferoxamine mesylate (purchased from Takeda Yakutama, Des7 Eral ■ vial) 2B+9 dissolved in 8 ytl of water was added dropwise to this solution. After reacting for 1 hour at room temperature, p
Adjusted to H9.5. Then 0, 15 N NaCl
5ephade$)G-25x equilibrated with aqueous solution
-Bar -y 1 inch (made by 7 Almasha) 8.9c++
After gel filtration was performed using a +*X4.5 cIn column to remove low molecular weight components, dialysis was performed with an 8% NaC/aqueous solution. After dialysis, 100 q of ethanolamine hydrochloride and 200 q of 1-ethyl-8-(8-dimethylaminopropyl)carbodiimide hydrochloride were added, and the pH was adjusted to 5.0 with INHC6.
The reaction was carried out at room temperature for an hour. Thereafter, the mixture was subjected to 61-fold analysis using an 8% NaCl aqueous solution, and then freeze-dried. After the reaction with deferoxamine mesylate, the amount of deferoxamine introduced per mole of succinimide group was measured in the same manner as in Example 1 using the reaction solution before gel filtration. 0.1 mole of deferoxamine was introduced per mole of succinimide groups. No unreacted carboxyl group was detected by IR and 'C-NMR. The average degree of polymerization of the reactive polymer used from Reference Example 2 was 19B.
, and from the above results, it is a 2-hydrogen atom, and n+
Average value of m + g + r 19B, average value of n-) - m 19,
Polysuccinimide derivative CI with an average e+r value of 168
)was gotten. 162 Completed - Continuation of page 1 [Phase] Inventor Masaaki Yo Tsue 4-chome Takashi, Takarazuka City In-house

Claims (1)

[Scope of Claims] (1) Consisting of structural units represented by the following formulas [1] to [4] (1) (2) (8) ("4), and - each structural unit [1] in the molecule. The numbers of (2), (8), and (4) are n, m, respectively. 6, r (these numbers are 0 or natural numbers, n-)-
m≧2. ) A polysuccinimide derivative having an active group represented by the following formula [5] at the terminal amino group of a polymer having a molecular weight of 2,000 to 8,000,000. (2) Consisting of structural units represented by the following formulas (1) to [4] (8) (4) and - each structural unit [l] in the molecule. (2), (8), and (4) are respectively n, m. 4, r (these numbers are 0 or natural numbers, and n-4-
m≧2. ) has an active group represented by the following formula [5] at the amino group terminal of the polymer (5), and has a molecular weight of 2,000 to a, ooo, ooo. When manufacturing, the following [
H (6) (7) 5- (8) (9) and - each structural unit [6] in the molecule. The numbers of (7), (8), and (9) are n' and m', respectively. 11, r' (these numbers are 0 or natural numbers, n'
-)-m'≧2. ) has an active group represented by the following formula [5] at the amino group end of the polymer [in the above formula [5], Z, W, and A have the same meanings as above. ] A polysuccinimide derivative having a molecular weight of 2,000 to a, o o o, o o o, (a) Reaction of 6- with a difunctional ligand compound having an amino group (a) If necessary, water-soluble The polysuccinimide derivative [I] is obtained by reacting with a primary aliphatic amine.
and when the group represented by (/X)Z is a group represented by -3-R, a disulfide bond (S -SR
) to produce the polysuccinimide derivative CI) in which 2 is a hydrogen atom; A method for producing the polysuccinimide derivative [■], which is characterized in that the polysuccinimide derivative (I"l) is zz' by reacting a disulfide compound. (8) The following formula [1] - It consists of the structural units represented by [4] (1) (2'] (8) (4) and - each structural unit [l] in the molecule. (2), (8], (4"The number of l is respectively n, nl.4, r (these numbers are 0 or natural numbers, n−)−
m≧2. ) A polysuccinimide derivative [■] having an active group represented by the following formula [5] at the amino group end of the polymer and having a molecular weight of 2,000 to a, o o o, o o o is produced. In this regard, the general formula [where k represents a natural number] is used. ] Reaction of polysuccinimide prepared with (a) a bifunctional ligand compound having an amino group A 1 (cll to the terminal amino group, a spacer (-W-C-)
c) If necessary, the polysuccinimide derivative (I) is reacted with a water-soluble aliphatic primary amine.
and further) When the group represented by Z is a group represented by -8-R, a disulfide bond (S-8R,) is added as necessary.
The above borisuccinimide derivative CI) in which Z is a hydrogen atom is produced by reductively cleaving l
O- Also, when the group represented by 1=l Z is a hydrogen atom, an active disulfide compound is reacted as necessary. The method for producing the polysuccinimide derivative CI), characterized in that the polysuccinimide derivative CI) is characterized in that '1=ZI.