Method for evaluating the purity of a drug contained in a complex and method for producing the complex

Abstract

Disclosed is a method that can evaluate the degree of purity of an active pharmaceutical substance contained in a composite body. Also disclosed is a method for producing a composite body in which the degree of purity of an active pharmaceutical substance itself is 95.0% or higher. The method for evaluating purity comprises: a reaction step for reacting, in a polar solvent, a nitrogen-containing nucleophilic agent, such as hydroxylamine, with a composite body in the presence of a protonic acid; and an evaluation step for evaluating, using rapid liquid chromatography, the degree of purity of a reaction mixture obtained in the reaction step. The method for producing a composite body comprises a reaction step for obtaining a composite body by reacting, in a polar solvent, N-(2-hydroxypropyl)methacrylamide polymer with an anthracycline pharmaceutical substance, in the presence of a protonic acid and at 10°C or lower.

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the purity of a drug contained in a complex and a method for producing the complex. [Background technology]

[0002] A conjugate (hereinafter referred to as P-THP) in which pirarubicin (hereinafter referred to as THP), an anthracycline drug, is bound to N-(2-hydroxypropyl)methacrylamide polymer, a biocompatible polymer compound, via a hydrazone bond is a useful compound as an antitumor agent or anticancer agent (Patent Documents 1 and 2).

[0003] As a method for producing P-THP, for example, a method of reacting THP with N-(2-hydroxypropyl) methacrylamide polymer in the presence of acetic acid has been disclosed (Non-Patent Document 1).

[0004] Furthermore, a method for quantifying a complex in which doxorubicin (hereinafter referred to as DOX), an anthracycline drug, is bound to an N-(2-hydroxypropyl) methacrylamide polymer via an oligopeptide has been disclosed, in which hydrochloric acid is added to a mixture of the complex to hydrolyze it (Non-Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5904602 specification [Patent Document 2] International Publication No. 2017 / 191843 [Non-patent literature]

[0006] [Non-Patent Document 1] Etrych et al., European Journal of Pharmaceutical Sciences, 2017, Vol. 106, pp. 10-19 [Non-patent document 2] Fraier et al., Journal of Pharmaceutical and Biomedical Analysis, 1995, Volume 13, p.625-633 Summary of the Invention [Problem to be solved by the invention]

[0007] Pharmaceuticals generally undergo strict purity evaluation to ensure their quality. For example, the purity of DOX hydrochloride, as specified in the Japanese Pharmacopoeia, is defined as 98.0 to 108.0% potency by quantitative assay. On the other hand, in complexes such as P-THP, the active pharmaceutical ingredient (API) is bound to a polymer (e.g., a polymer), making it technically difficult to directly evaluate the purity of the active pharmaceutical ingredient using the complex. Therefore, to evaluate the purity of the active pharmaceutical ingredient, it is necessary to liberate the active pharmaceutical ingredient from the complex. However, when THP is liberated from P-THP using the method described in Non-Patent Document 2, for example, THP decomposes into DOX and other degradation products upon liberation. Therefore, it has been difficult to accurately and precisely evaluate the purity of the active pharmaceutical ingredient contained in the complex. (Hereinafter, the purity of the active pharmaceutical ingredient contained in the complex, i.e., the purity of the active pharmaceutical ingredient bound to the polymer, is also referred to as "bound drug purity.") Furthermore, when the purity of the bound drug of P-THP produced by the method described in Non-Patent Document 1 was evaluated using the purity evaluation method of the present invention described below, it was newly discovered that the P-THP did not meet the THP purity standard (95.0% or more) described in the Japanese Pharmacopoeia, and that P-THP produced by the conventional method has issues with the purity of the bound drug.

[0008] Therefore, an object of the present invention is to provide a method capable of evaluating the purity of the active drug contained in the complex.A further object of the present invention is to provide a method for producing an anthracycline drug-containing complex with a drug purity of 95.0% or more as evaluated by the purity evaluation method of the present invention. [Means for solving the problem]

[0009] As a result of extensive research to solve the above-mentioned problems, the present inventors discovered that by reacting a conjugate having a hydrazone bond with a specific nitrogen-containing nucleophile in a polar solvent in the presence of a protonic acid to convert the anthracycline drug into a stable drug equivalent, it is possible to evaluate the purity of the bound drug, and further that by reacting an anthracycline drug with an N-(2-hydroxypropyl)methacrylamide polymer in a polar solvent in the presence of a protonic acid at 10°C or below, a high-purity conjugate can be produced, thereby completing the present invention.

[0010] That is, the present invention provides the following. [1] a reaction step of reacting a complex represented by general formula (I) or a pharmacologically acceptable salt thereof with at least one nitrogen-containing nucleophile selected from the group consisting of hydroxylamine, O-alkylhydroxylamine, and carboxylic acid hydrazide in a polar solvent in the presence of a protonic acid; an evaluation step of evaluating the purity of the reaction mixture obtained in the reaction step by high performance liquid chromatography; A method for evaluating the purity of a drug contained in a complex represented by general formula (I) or a pharmacologically acceptable salt thereof, comprising:

[0011] [ka]

[0012] [In the formula, A represents a hydrogen atom or an (R)-tetrahydro-2H-pyran-2-yl group, b, c, d, and e each independently represent a positive integer, and the bond depicted with a wavy line represents that it can have either an E-configuration or a Z-configuration.] [2] the polar solvent is an alcohol-based solvent, the nitrogen-containing nucleophile is at least one selected from the group consisting of hydroxylamine and carboxylic acid hydrazide; The method according to [1], wherein the protonic acid is a carboxylic acid. [3] the polar solvent is methanol, the nitrogen-containing nucleophile is at least one selected from the group consisting of hydroxylamine, acetohydrazide, propanohydrazide, butyrohydrazide, and 3-methylbutanohydrazide; The method according to [1] or [2], wherein the protonic acid is acetic acid. [4] The method according to any one of [1] to [3], wherein in the general formula (I), b is an integer of 1 to 10, c is an integer of 30 to 500, d is an integer of 1 to 50, and e is an integer of 1 to 50. [5] A complex represented by general formula (I) or a pharmacologically acceptable salt thereof, wherein the purity of the drug contained in the complex represented by general formula (I) or a pharmacologically acceptable salt thereof is 95.0% or more when evaluated by the method according to any one of [1] to [4].

[0013] [ka]

[0014] [In the formula, A represents a hydrogen atom or an (R)-tetrahydro-2H-pyran-2-yl group, b, c, d, and e each independently represent a positive integer, and the bond depicted with a wavy line represents that it can have either an E-configuration or a Z-configuration.] [6] A is an (R)-tetrahydro-2H-pyran-2-yl group; [5] The complex or pharmacologically acceptable salt thereof according to [5], wherein b is 5. [7] A method for producing a conjugate represented by general formula (I) or a pharmacologically acceptable salt thereof, comprising a reaction step of reacting an anthracycline drug represented by general formula (II) with an N-(2-hydroxypropyl)methacrylamide polymer represented by general formula (III) in the presence of a protonic acid at 10°C or below in a polar solvent to obtain a conjugate represented by general formula (I) or a pharmacologically acceptable salt thereof.

[0015] [ka]

[0016] [ka]

[0017] [ka]

[0018] [In formulas (I) and (II), A represents a hydrogen atom or an (R)-tetrahydro-2H-pyran-2-yl group; in formulas (I) and (III), b, c, d, e, and f each independently represent a positive integer; and in formula (I), the bond depicted with a wavy line indicates that it can have either an E-configuration or a Z-configuration.] [8] the polar solvent is methanol, the protonic acid is acetic acid; The method for producing according to [7], wherein the reaction temperature in the reaction step is -30°C to 10°C. [9] The method according to [7] or [8], wherein in the general formula (I) and the general formula (III), b is an integer of 1 to 10, c is an integer of 30 to 500, d is an integer of 1 to 50, e is an integer of 1 to 50, and f is the sum of d and e. DETAILED DESCRIPTION OF THE INVENTION

[0019] 1. Method for evaluating the purity of drugs contained in the complex The purity assessment method according to the present invention comprises a reaction step of reacting a complex represented by general formula (I) or a pharmacologically acceptable salt thereof with a nitrogen-containing nucleophile selected from the group consisting of hydroxylamine, O-alkylhydroxylamine, and carboxylic acid hydrazide in a polar solvent in the presence of a protonic acid, and an assessment step of assessing the purity of the reaction mixture obtained in the reaction step by high performance liquid chromatography.

[0020] [ka]

[0021] [In the formula, A represents a hydrogen atom or an (R)-tetrahydro-2H-pyran-2-yl group, b, c, d, and e each independently represent a positive integer, and the bond depicted with a wavy line represents that it can have either an E-configuration or a Z-configuration.]

[0022] In this specification and claims, when a chemical formula contains a wavy line, the wavy line indicates that either the E-form or the Z-form may be adopted, and the compound may be either the E-form alone, the Z-form alone, or a mixture of the E-form and the Z-form.

[0023] From the viewpoint of transport efficiency by the transporter, A is preferably an (R)-tetrahydro-2H-pyran-2-yl group.

[0024] Furthermore, b, c, d, and e are not particularly limited as long as they are positive integers, but it is preferable that b is an integer of 1 to 10 (for example, 5), c is an integer of 30 to 500, particularly 50 to 500, d is an integer of 1 to 50, and e is an integer of 1 to 50.

[0025] Here, "conjugate" refers to an anthracycline drug-containing conjugate in which an anthracycline drug represented by general formula (II) and an N-(2-hydroxypropyl)methacrylamide polymer represented by general formula (III) are bound via a hydrazone bond, and the anthracycline drug represented by general formula (II) refers to the active ingredient of the conjugate. Furthermore, the anthracycline drug represented by general formula (II) refers to THP or DOX, a drug in which THP and an N-(2-hydroxypropyl)methacrylamide polymer represented by general formula (III) are bound via a hydrazone bond refers to P-THP, and a drug in which DOX and an N-(2-hydroxypropyl)methacrylamide polymer represented by general formula (III) are bound via a hydrazone bond refers to P-DOX.

[0026] [ka]

[0027] [In the formula, A represents a hydrogen atom or an (R)-tetrahydro-2H-pyran-2-yl group.]

[0028] [ka]

[0029] [In the formula, b, c, and f each independently represent a positive integer.]

[0030] As used herein, "a drug contained in a complex represented by general formula (I) (hereinafter referred to as complex (I)) or a pharmacologically acceptable salt thereof" means an anthracycline drug contained in the complex or a pharmacologically acceptable salt thereof.

[0031] As used herein, "bound drug purity" refers to the estimated purity of the drug contained in complex (I), and refers to the area percentage of the peak to be measured, based on a high-performance liquid chromatography (hereinafter referred to as HPLC) chromatogram obtained according to the following method, where the area of ​​all peaks excluding the blank peak is taken as 100%. The blank peak refers to the peak detected when methanol is measured under the following HPLC measurement conditions. The peak to be measured varies depending on the type of anthracycline drug and the type of nitrogen-containing nucleophile, but refers to the peak of an anthracycline drug equivalent based on molecular weight. When isomer peaks are separated under the following analytical conditions, the sum of the area percentages of the peaks of each isomer is taken as the area percentage of the peak to be measured. The following explanation exemplifies the use of hydroxylamine as the nitrogen-containing nucleophile, methanol as the polar solvent, and acetic acid as the protonic acid, but the conditions, such as the amount added, may be changed appropriately depending on the reagents used.

[0032] (1) Sample preparation To a solution of the complex (10 mg) in methanol (160 μL), add 50 wt% aqueous hydroxylamine solution (32 μL) and acetic acid (27 μL) at 0°C, and stir for 25 hours at 0°C. To the resulting reaction mixture (50 μL), add methanol (450 μL) to prepare a measurement sample.

[0033] (2) HPLC The measurement sample prepared above is analyzed using HPLC under the following measurement conditions. Detector: Photodiode array detector (measurement wavelength: 488 nm) Column: Scherzo SM-C18 (Intact) Column size: 150 x 4.6 mm (3 μm) Column temperature: 30℃ Mobile phase: Solution A: 5mmol / L ammonium formate solution / methanol mixture (95:5) Solution B: Methanol / water / formic acid mixture (90:10:0.1) Deployment conditions: A / B=75 / 25 (0~3 minutes) A / B = 75 / 25-37 / 63 (3-4 min; linear gradient) A / B=37 / 63(4~24 minutes) A / B = 37 / 63 to 20 / 80 (24 to 25 minutes; linear gradient) A / B=20 / 80(25~35 minutes) A / B = 20 / 80 to 0 / 100 (35 to 39 minutes; linear gradient) A / B=0 / 100(39~54 minutes) A / B = 0 / 100 to 75 / 25 (54 to 54.1 min; linear gradient) A / B=75 / 25(54.1~59.5 minutes) Flow rate: 1.0mL / min Injection volume: 20μL Sample cooler temperature: 4℃

[0034] The O-alkylhydroxylamine used in the purity evaluation method according to the present invention is preferably one in which the alkyl group has 1 to 4 carbon atoms. Preferred examples include O-methylhydroxylamine, O-ethylhydroxylamine, and O-propylhydroxylamine. From the viewpoint of suppressing side reactions, O-methylhydroxylamine or O-ethylhydroxylamine is preferred, and O-methylhydroxylamine is more preferred. The O-alkylhydroxylamine used in the present invention may be a single compound, or two or more compounds may be used in combination.

[0035] Examples of the carboxylic acid hydrazide used in the purity evaluation method of the present invention include formhydrazide, acetohydrazide, propanohydrazide, butyrohydrazide, 3-methylbutanohydrazide, 2,2-dimethylpropanohydrazide, cyclohexanecarbohydrazide, and adamantane-1-carbohydrazide. From the viewpoint of suppressing side reactions, at least one selected from the group consisting of acetohydrazide, propanohydrazide, butyrohydrazide, and 3-methylbutanohydrazide is preferred, and acetohydrazide is more preferred. The carboxylic acid hydrazide used in the present invention may be a single compound, or two or more compounds may be used in combination.

[0036] The nitrogen-containing nucleophile is preferably at least one selected from the group consisting of hydroxylamine and carboxylic acid hydrazide, more preferably at least one selected from the group consisting of hydroxylamine, acetohydrazide, propanohydrazide, butyrohydrazide, and 3-methylbutanohydrazide, and even more preferably at least one selected from the group consisting of hydroxylamine and acetohydrazide.

[0037] When a drug is released from a conjugate using the nitrogen-containing nucleophile, the drug is converted into the corresponding drug equivalent and released. Because the released drug equivalent is highly stable under the reaction conditions, this method is suitable for evaluating the purity of the conjugated drug.

[0038] Here, the term "drug equivalent" refers to an anthracycline drug equivalent represented by the following general formula (IV) or general formula (V).

[0039] [ka]

[0040] [In formulas (IV) and (V), A represents a hydrogen atom or an (R)-tetrahydro-2H-pyran-2-yl group, G represents a hydrogen atom or an alkyl group, and the bond depicted with a wavy line indicates that it can have either an E-configuration or a Z-configuration.]

[0041] In general formula (IV) or (V), G is an alkyl group with no particular limitation on the number of carbon atoms, and may be linear, branched, or cyclic. Examples of the alkyl group include alkyl groups having 1 to 10 carbon atoms (e.g., methyl, ethyl, propyl, and adamantyl groups) and alkyl groups having 1 to 4 carbon atoms (e.g., methyl, ethyl, and propyl groups).

[0042] In a preferred embodiment, the anthracycline drug equivalent of general formula (IV) or general formula (V) may be represented by any of the following formulas:

[0043] [ka]

[0044] Examples of protonic acids in the purity evaluation method according to the present invention include inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and hydrobromic acid, carboxylic acids such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, and benzoic acid, sulfonic acids such as methanesulfonic acid and p-toluenesulfonic acid, and ascorbic acid. From the viewpoint of acidity, carboxylic acids are preferred, and acetic acid is more preferred. The protonic acid used may be a single compound or a combination of two or more compounds.

[0045] The amount of the nitrogen-containing nucleophile used is preferably 0.1 to 100 times, and more preferably 0.5 to 50 times, the weight of the complex from the viewpoints of obtaining a sufficient reaction conversion rate and suppressing side reactions. The amount of the protonic acid used is preferably 0.1 to 100 times, and more preferably 0.5 to 50 times, the weight of the complex from the viewpoints of obtaining a sufficient reaction conversion rate and suppressing side reactions.

[0046] Furthermore, examples of polar solvents in the drug purity evaluation method of the present invention include ether solvents such as tetrahydrofuran or dimethoxyethane, nitrile solvents such as acetonitrile or propionitrile, amide solvents such as N,N-dimethylformamide, N,N-dimethylacetamide or N-methylpyrrolidone, sulfoxide solvents such as dimethyl sulfoxide, urea solvents such as 1,3-dimethyl-2-imidazolidinone, alcohol solvents such as methanol, ethanol or 2-propanol, or mixed solvents thereof, but from the viewpoint of dissolving the complex, alcohol solvents such as methanol, ethanol or 2-propanol are preferred, with methanol being more preferred. Furthermore, from the viewpoint of suppressing side reactions, the amount of the solvent used is preferably 3 to 100 times, and more preferably 5 to 20 times, the weight of the complex.

[0047] In the purity evaluation method according to the present invention, preferred embodiments of the nitrogen-containing nucleophile, the protonic acid, and the polar solvent can be combined in any desired manner, such as hydroxylamine, carboxylic acid, and an alcoholic solvent, O-alkylhydroxylamine, carboxylic acid, and an alcoholic solvent, or carboxylic acid hydrazide, carboxylic acid, and an alcoholic solvent, and preferred combinations include hydroxylamine, acetic acid, and methanol, acetohydrazide, acetic acid, and methanol, propanohydrazide, acetic acid, and methanol, butyrohydrazide, acetic acid, and methanol, or 3-methylbutanohydrazide, acetic acid, and methanol.

[0048] The nitrogen-containing nucleophile, protonic acid and polar solvent may be labeled with a radioisotope or may be deuterium-converted.

[0049] Furthermore, from the viewpoint of suppressing side reactions, the reaction temperature in the reaction step in the purity evaluation method according to the present invention is preferably −10° C. to 30° C., more preferably −5° C. to 5° C. Furthermore, the reaction time can be appropriately selected depending on conditions such as the reaction temperature, but from the viewpoint of suppressing side reactions, it is preferably 1 to 40 hours, more preferably 3 to 30 hours.

[0050] 2. Complex (I) or a pharmacologically acceptable salt thereof The conjugate represented by general formula (I) or a pharmacologically acceptable salt thereof according to the present invention has a purity of the drug contained in the conjugate (I) or a pharmacologically acceptable salt thereof of 95.0% or more when evaluated by the above-mentioned purity evaluation method.

[0051] [ka]

[0052] [In the formula, A represents a hydrogen atom or an (R)-tetrahydro-2H-pyran-2-yl group, b, c, d, and e each independently represent a positive integer, and the bond depicted with a wavy line represents that it can have either an E-configuration or a Z-configuration.]

[0053] From the viewpoint of transport efficiency by the transporter, A is preferably an (R)-tetrahydro-2H-pyran-2-yl group.

[0054] Furthermore, b, c, d, and e are not particularly limited as long as they are positive integers, but it is preferable that b is an integer of 1 to 10 (for example, 5), c is an integer of 30 to 500, particularly 50 to 500, d is an integer of 1 to 50, and e is an integer of 1 to 50.

[0055] In a preferred embodiment, the complex represented by general formula (I) or a pharmacologically acceptable salt thereof may be represented by the following general formula (I').

[0056] [ka]

[0057] [In the formula, c, d, and e each independently represent a positive integer, and the bond depicted with a wavy line indicates that it can have either an E-configuration or a Z-configuration.]

[0058] In general formula (I'), c, d, and e are not particularly limited as long as they are positive integers, but it is preferable that c is an integer of 30 to 500, particularly 50 to 500, d is an integer of 1 to 50, and e is an integer of 1 to 50.

[0059] Furthermore, examples of pharmacologically acceptable salts of the complex represented by the above general formula (I) include inorganic acid salts such as hydrochloride, sulfate, phosphate, and hydrobromide, and organic acid salts such as oxalate, malonate, citrate, fumarate, lactate, malate, succinate, tartrate, acetate, trifluoroacetate, maleate, gluconate, benzoate, salicylate, xinafoate, pamoate, ascorbate, adipate, methanesulfonate, p-toluenesulfonate, and cinnamate. Furthermore, these salts may form hydrates, solvates, or crystalline polymorphs.

[0060] In addition, Complex (I) or a pharmacologically acceptable salt thereof may be labeled with a radioisotope or may be a deuterium-converted product.

[0061] The complex (I) or a pharmacologically acceptable salt thereof also includes their hydrates, solvates, crystalline polymorphs, labeled compounds, and mixtures thereof.

[0062] The purity of the bound drug in the complex (I) or a pharmacologically acceptable salt thereof according to the present invention is 95.0% or more, preferably 96.0% or more, and more preferably 97.0% or more, when evaluated by the same method as described in the purity evaluation method for the drug contained in the complex, in accordance with the THP purity standard set forth in the Japanese Pharmacopoeia. Taking into consideration the purity standard for DOX hydrochloride set forth in the Japanese Pharmacopoeia, 98.0% or more is preferred. The closer the bound drug purity is to 100%, the more preferable it is; the manufacturing method of the present invention described below has been able to produce bound drug purities of up to 99.5%.

[0063] Purity assessment is generally performed on pharmaceuticals to confirm their quality. For example, the Japanese Pharmacopoeia specifies that the purity of THP must be 95.0% or higher by quantitative assay, and high-purity pharmaceuticals are considered desirable in terms of quality. On the other hand, when a complex is used as a pharmaceutical, the anthracycline drug released from the complex exerts its therapeutic effect. Therefore, the purity of the drug contained in the complex, i.e., the bound drug purity, corresponds to the purity of a typical pharmaceutical, and evaluation of the bound drug purity is extremely important for pharmaceutical quality control. Therefore, a high bound drug purity is preferable for pharmaceutical quality control. When THP is used as the anthracycline drug, it is necessary to at least meet the purity standard for THP specified in the Japanese Pharmacopoeia. When DOX hydrochloride is used as the anthracycline drug, it is also preferable to meet the purity standard for DOX hydrochloride specified in the Japanese Pharmacopoeia.

[0064] From the viewpoint of drug transport, the bound drug loading of Complex (I) or a pharmacologically acceptable salt thereof is preferably 3 to 20 wt%, more preferably 4 to 19 wt%, and even more preferably 7 to 17 wt%. On the other hand, from the viewpoint of storage stability, the bound drug loading of Complex (I) or a pharmacologically acceptable salt thereof is preferably 4 to 20 wt%, more preferably 10 to 19 wt%, and even more preferably 15 to 19 wt%.

[0065] As used herein, the term "bound drug loading" refers to the estimated amount of drug loaded in Complex (I) or a pharmacologically acceptable salt thereof, and can be calculated by quantitative analysis of the target substance according to the following method, which conforms to the purity evaluation method for the drug contained in Complex (I) or a pharmacologically acceptable salt thereof. Based on the HPLC chromatogram obtained according to the following method, the number of moles of the target substance peak is calculated by quantitative analysis using the HPLC area value of the target substance peak and a calibration curve prepared from the corresponding target substance. The weight of the corresponding active drug substance is calculated from the obtained number of moles, and the bound drug loading is then calculated using the following formula: Bound drug loading (wt%) = weight of active drug (mg) / weight of complex (mg) × 100

[0066] Furthermore, the peak to be measured refers to the peak of an anthracycline drug equivalent based on molecular weight, and when peaks of isomers are separated under the above analytical conditions, the sum of the area percentages of the peaks of each isomer is taken as the HPLC area value of the peak to be measured.

[0067] (1) Sample preparation Accurately weigh the complex (20±0.4 mg) into a 2 mL volumetric flask, add methanol (1 mL) to dissolve, then add 50 wt% hydroxylamine aqueous solution (320 μL) and acetic acid (270 μL), and make the volume to exactly 2 mL using methanol. Stir the resulting solution at 0°C for 25 hours to prepare the measurement sample.

[0068] (2) HPLC The measurement sample prepared above is analyzed using HPLC under the following measurement conditions. Detector: Photodiode array detector (measurement wavelength: 488 nm) Column: Scherzo SM-C18 (Intact) Column size: 150 x 4.6 mm (3 μm) Column temperature: 30℃ Mobile phase: Solution A: 5mmol / L ammonium formate solution / methanol mixture (95:5) Solution B: Methanol / water / formic acid mixture (90:10:0.1) Deployment conditions: A / B=75 / 25 (0~3 minutes) A / B = 75 / 25-37 / 63 (3-4 min; linear gradient) A / B=37 / 63(4~24 minutes) A / B = 37 / 63 to 20 / 80 (24 to 25 minutes; linear gradient) A / B=20 / 80(25~35 minutes) A / B = 20 / 80 to 0 / 100 (35 to 39 minutes; linear gradient) A / B=0 / 100(39~54 minutes) A / B = 0 / 100 to 75 / 25 (54 to 54.1 min; linear gradient) A / B=75 / 25(54.1~59.5 minutes) Flow rate: 1.0mL / min Injection volume: 10μL Sample cooler temperature: 4℃

[0069] Moreover, the weight-average molecular weight of the complex (I) or a pharmacologically acceptable salt thereof is preferably 25,000 to 85,000, more preferably 27,000 to 65,000, and even more preferably 30,000 to 50,000, from the viewpoint of safety.

[0070] In this specification, the term "weight average molecular weight" means a molecular weight calculated according to the following method.

[0071] (1) Sample preparation Methanol (500 μL) is added to the complex (5 mg) to prepare a measurement sample.

[0072] (2) Weight average molecular weight Each measurement sample prepared above is analyzed using HPLC and MALS under the following measurement conditions, and the weight-average molecular weight (Mw) is calculated using analytical software [ASTRA Ver. 7.3.2.17 64-bit (Wyatt Technology)] (refractive index increment: dn / dc = 0.175). HPLC: LC-40 (Shimadzu Corporation) Detector: Photodiode array detector (measurement wavelength: 488 nm) and differential refractometer Column: TSKgel α-M (Tosoh Corporation) TSKgel α-2500 (Tosoh Corporation) and two tubes connected in sequence Column size: TSKgel α-M (300 x 7.8 mm (7 μm)) TSKgel α-2500(300×7.8mm(7μm)) Column temperature: 30℃ Mobile phase: methanol: 0.3 mol / L sodium acetate solution (pH 6.5) =80:20 Flow rate: 0.8mL / min Injection volume: 20μL Sample cooler temperature: 4℃ Injector cleaning solution: methanol:water = 80:20 MALS:DAWN8 (Wyatt technology) Light Scattering Instrument: Calibration constant: 5.2929 / 100000[1 / Vcm] RI Instrument: Refractive constant: 1.338

[0073] In Complex (I) or a pharmacologically acceptable salt thereof, the preferred embodiments of the bound drug purity, the preferred embodiments of the bound drug load, and the preferred embodiments of the weight-average molecular weight can be combined in any manner, such as in Complex (I) or a pharmacologically acceptable salt thereof, in which the bound drug purity is 95.0% or more, the bound drug load is 3 to 20 wt%, and the weight-average molecular weight is 25,000 to 85,000.

[0074] In general, the stability of pharmaceuticals must be evaluated to determine storage conditions and expiration dates. Typical stability tests for pharmaceuticals include stress tests, long-term storage tests, and accelerated tests. For example, stress tests for drug substances are used to identify degradation products that may be generated from the drug substance, confirm the suitability of analytical methods, and predict the stability of the drug substance. A four-week stress test at 60°C under airtight conditions is used to predict stability in the early stages of development, corresponding to a three-year storage period at 25°C under airtight conditions (Yoshioka Sumie, Stability of Pharmaceuticals, Nanzando, 1995, p. 142).

[0075] The complex (I) or a pharmacologically acceptable salt thereof having a bound drug purity of 95.0% or more when evaluated by the above-mentioned purity evaluation method can be obtained by the following production method.

[0076] 3. Method for producing complex (I) or a pharmacologically acceptable salt thereof The method of the present invention for producing a conjugate represented by general formula (I) or a pharmacologically acceptable salt thereof comprises a reaction step of reacting an anthracycline drug represented by general formula (II) with an N-(2-hydroxypropyl)methacrylamide polymer represented by general formula (III) in the presence of a protonic acid in a polar solvent at 10°C or below to obtain a conjugate represented by general formula (I) or a pharmacologically acceptable salt thereof.

[0077] [ka]

[0078] [ka]

[0079] [ka]

[0080] [In formulas (I) and (II), A represents a hydrogen atom or an (R)-tetrahydro-2H-pyran-2-yl group, and in formulas (I) and (III), b, c, d, e, and f each independently represent a positive integer, and the bond depicted with a wavy line indicates that it can have either an E-configuration or a Z-configuration.]

[0081] From the viewpoint of transport efficiency by the transporter, A is preferably an (R)-tetrahydro-2H-pyran-2-yl group.

[0082] Furthermore, b, c, d, and e are not particularly limited as long as they are positive integers, but it is preferable that b is an integer of 1 to 10 (for example, 5), c is an integer of 30 to 500, particularly 50 to 500, d is an integer of 1 to 50, and e is an integer of 1 to 50. Furthermore, f is the sum of d and e.

[0083] The method for producing Complex (I) or a pharmacologically acceptable salt thereof according to the present invention can be used as a method for producing Complex (I) or a pharmacologically acceptable salt thereof having a bound drug purity of 95.0% or more when evaluated by the above-mentioned purity evaluation method.

[0084] Unlike typical low-molecular-weight organic compounds, in the purification of conjugate (I) or its pharmacologically acceptable salt, low-molecular-weight compounds such as unreacted anthracycline drugs and anthracycline drugs whose hydrazone bonds have been cleaved can be removed by purification procedures such as column chromatography, thin-layer chromatography, recrystallization, or reprecipitation, but the purity of the bound drug cannot be improved. That is, even if a partial structure other than the hydrazone bond of the anthracycline drug on the conjugate is decomposed, the decomposition product of the drug will not be separated from the conjugate unless the hydrazone bond is cleaved, and the purity of the bound drug will not be improved. Therefore, the reaction step in producing the conjugate is extremely important in terms of quality control, and the purity of the bound drug is determined by how well the decomposition of the anthracycline drug during the reaction step is suppressed.

[0085] In the method for producing Complex (I) or a pharmacologically acceptable salt thereof, the amount of the N-(2-hydroxypropyl)methacrylamide polymer used in the reaction step can be appropriately selected depending on the desired amount of bound drug to be carried. From the viewpoint of drug transport efficiency, the amount is preferably 1 to 30 times by weight, more preferably 3 to 25 times by weight, and even more preferably 3 to 15 times by weight relative to the anthracycline drug.

[0086] In the method for producing complex (I) or a pharmacologically acceptable salt thereof, the protonic acid used in the reaction step may be, for example, an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, or hydrobromic acid, or an organic acid such as oxalic acid, malonic acid, citric acid, fumaric acid, lactic acid, malic acid, succinic acid, tartaric acid, acetic acid, trifluoroacetic acid, maleic acid, gluconic acid, benzoic acid, salicylic acid, xinafoic acid, pamoic acid, ascorbic acid, adipic acid, methanesulfonic acid, p-toluenesulfonic acid, or cinnamic acid. From the viewpoint of acidity, however, organic acids are preferred, and acetic acid is more preferred. The protonic acid used may be a single compound, or two or more compounds may be used in combination.

[0087] In the method for producing Complex (I) or a pharmacologically acceptable salt thereof, the amount of protonic acid used in the reaction step is preferably 10 to 300 molar equivalents, more preferably 20 to 200 molar equivalents, relative to the anthracycline drug, from the viewpoint of obtaining a sufficient reaction conversion rate. Here, "molar equivalent" refers to the number of moles of protonic acid used per mole of the anthracycline drug.

[0088] In the method for producing Complex (I) or a pharmacologically acceptable salt thereof, examples of polar solvents used in the reaction step include ether solvents such as tetrahydrofuran or dimethoxyethane, nitrile solvents such as acetonitrile or propionitrile, amide solvents such as N,N-dimethylformamide, N,N-dimethylacetamide, or N-methylpyrrolidone, sulfoxide solvents such as dimethyl sulfoxide, urea solvents such as 1,3-dimethyl-2-imidazolidinone, alcohol solvents such as methanol, ethanol, or 2-propanol, or mixed solvents thereof. From the viewpoint of dissolving the complex, alcohol solvents such as methanol, ethanol, or 2-propanol are preferred, with methanol being more preferred. The amount of solvent used is preferably 10 to 200 times, and more preferably 20 to 70 times, the weight of the anthracycline drug, from the viewpoints of facilitating stirring and improving production efficiency per unit volume.

[0089] In the method for producing Complex (I) or a pharmacologically acceptable salt thereof, the reaction temperature in the reaction step is 10° C. or lower from the viewpoint of suppressing side reactions, and from the viewpoint of improving production efficiency per unit time, it is preferably −30° C. to 10° C., particularly −30° C. to 5° C. Furthermore, the reaction time can be appropriately selected depending on conditions such as the reaction temperature, but from the viewpoint of improving production efficiency per unit time, it is preferably 1 to 300 hours, more preferably 10 to 150 hours.

[0090] In the method for producing Complex (I) or a pharmacologically acceptable salt thereof, the preferred polar solvent, the preferred protonic acid, and the preferred reaction temperature can be arbitrarily combined. Examples of such combinations include an alcoholic solvent and an organic acid at 10°C or lower, and methanol and acetic acid at -30°C to 10°C, particularly -30°C to 5°C.

[0091] In the method for producing Complex (I) or a pharmacologically acceptable salt thereof, the order of addition of the polar solvent, the anthracycline drug represented by general formula (II), the N-(2-hydroxypropyl)methacrylamide polymer represented by general formula (III), and the protonic acid used in the reaction step is not particularly limited. However, from the viewpoint of obtaining a sufficient reaction conversion rate and suppressing side reactions, it is preferred that the N-(2-hydroxypropyl)methacrylamide polymer represented by general formula (III) is added and dissolved in the polar solvent, then the anthracycline drug represented by general formula (II) is added, and then the protonic acid is added.

[0092] Commercially available anthracycline drugs represented by general formula (II) can be used as they are. For example, THP (A is an (R)-tetrahydro-2H-pyran-2-yl group) can be purchased from MicroBiopharm Japan (quantitative analysis: 95.0% or higher). DOX hydrochloride (A is a hydrogen atom) can be purchased from MedKoo Biosciences (HPLC purity: 99.0% or higher). N-(2-hydroxypropyl)methacrylamide polymers represented by general formula (III) can be used as they are, and can be purchased, for example, from the Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic or Chemicalsoft.

[0093] The N-(2-hydroxypropyl)methacrylamide polymer represented by general formula (III) is not particularly limited with respect to the position of the hydrazide group, and may be regular or random, for example, two or more monomer units having a hydrazide group may be bonded consecutively. The terminal structure of the N-(2-hydroxypropyl)methacrylamide polymer is considered to be either a saturated or unsaturated terminal structure (disproportionated terminal by hydrogen abstraction) or a dimethylnitrile terminal structure (radical initiator terminal derived from azobisisobutyronitrile).

[0094] The N-(2-hydroxypropyl) methacrylamide polymer represented by general formula (III), which can be purchased from the Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic or Chemicalsoft, has a weight-average molecular weight of 20,000 to 35,000 and a content of hydrazide groups, which are copolymerization components, of 5.0 to 7.0 mol %.

[0095] Complex (I) or a pharmacologically acceptable salt thereof can be purified by methods such as column chromatography, thin-layer chromatography, recrystallization, or reprecipitation. Those skilled in the art can select or combine methods suited to a specific target compound, and can easily optimize the purification method. Complex (I) or a pharmacologically acceptable salt thereof obtained by the production method of the present invention contains few by-products, so it can be obtained by a simple isolation and purification procedure. Furthermore, considering commercial production, recrystallization or reprecipitation using, for example, a mixed solvent of ethyl acetate and methanol is preferred.

[0096] The method for producing Complex (I) or a pharmacologically acceptable salt thereof may include a precipitation step and / or a drying step after the reaction step.

[0097] Examples of the solvent used in the precipitation step include ether solvents such as tetrahydrofuran or dimethoxyethane, nitrile solvents such as acetonitrile or propionitrile, ester solvents such as ethyl acetate or isopropyl acetate, alcohol solvents such as methanol, ethanol or 2-propanol, and mixed solvents thereof. From the viewpoints of high recovery rate of the complex and ease of solvent distillation, a mixed solvent of an alcohol solvent and an ester solvent is preferred, and a mixed solvent of methanol and ethyl acetate is more preferred.

[0098] The amount of the mixed solvent used is preferably 50 to 1000 times, and more preferably 100 to 350 times by weight, the anthracycline drug represented by general formula (II), from the viewpoints of facilitating stirring and improving production efficiency per unit volume. The ratio of the ester solvent in the mixed solvent is preferably 1 to 10 times, and more preferably 2 to 6 times by weight, the alcohol solvent, from the viewpoints of achieving a high recovery rate of the complex and removing unreacted anthracycline drug.

[0099] The stirring temperature in the precipitation step is preferably −20° C. to 50° C., more preferably −10° C. to 40° C., from the viewpoints of suppressing decomposition of the complex and removing unreacted anthracycline drug. The stirring time in the precipitation step is preferably 0.1 to 100 hours, more preferably 0.25 to 50 hours, from the viewpoints of suppressing decomposition of the complex and removing unreacted anthracycline drug.

[0100] The drying temperature in the drying step is preferably -10°C to 50°C, more preferably 0°C to 40°C, from the viewpoint of suppressing decomposition of the complex and quickly removing the residual solvent. The drying method is not particularly limited, but is preferably drying under reduced pressure from the viewpoint of quickly removing the residual solvent. The degree of vacuum in the drying under reduced pressure is preferably 500 Pa or less from the viewpoint of quickly removing the residual solvent. [Example]

[0101] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these. First, the purity evaluation method will be described.

[0102] (1) Sample preparation To a methanol (160 μL) solution of the solid (10 mg) obtained in Examples 2 to 15 and Comparative Examples 1 and 2 described below, a 50 wt % aqueous hydroxylamine solution (32 μL) and acetic acid (27 μL) were added at 0° C., and the mixture was stirred at 0° C. for 25 hours. Methanol (450 μL) was added to the resulting reaction mixture (50 μL) to prepare a measurement sample.

[0103] (2) HPLC Each of the measurement samples prepared above was analyzed using HPLC under the following measurement conditions. HPLC: LC-20AD (Shimadzu Corporation) Detector: Photodiode array detector (measurement wavelength: 488 nm) Column: Scherzo SM-C18 (Intact) Column size: 150 x 4.6 mm (3 μm) Column temperature: 30℃ Mobile phase: Solution A: 5mmol / L ammonium formate solution / methanol mixture (95:5) Solution B: Methanol / water / formic acid mixture (90:10:0.1) Deployment conditions: A / B=75 / 25 (0~3 minutes) A / B = 75 / 25-37 / 63 (3-4 min; linear gradient) A / B=37 / 63(4~24 minutes) A / B = 37 / 63 to 20 / 80 (24 to 25 minutes; linear gradient) A / B=20 / 80(25~35 minutes) A / B = 20 / 80 to 0 / 100 (35 to 39 minutes; linear gradient) A / B=0 / 100(39~54 minutes) A / B = 0 / 100 to 75 / 25 (54 to 54.1 min; linear gradient) A / B=75 / 25(54.1~59.5 minutes) Flow rate: 1.0mL / min Injection volume: 20μL Sample cooler temperature: 4℃ Injector cleaning solution: Acetonitrile / water mixture (60:40)

[0104] (3) Purity of the bound drug Based on the HPLC chromatogram measured above, the area percentage of the target peak was calculated as the bound drug purity, assuming that all peaks excluding the blank peak were 100%. The blank peak refers to the peak detected when methanol is measured under the following HPLC measurement conditions. The target peak refers to the peak of an anthracycline drug equivalent based on molecular weight. When isomer peaks are separated under the above analytical conditions, the sum of the area percentages of the peaks of each isomer was used as the area percentage of the target peak.

[0105] Next, a method for evaluating the amount of bound drug carried will be described.

[0106] (1) Sample preparation The solids (20±0.4 mg) obtained in Examples 2 to 15 and Comparative Examples 1 and 2 described below were accurately weighed into a 2 mL volumetric flask, dissolved in 1 mL of methanol, and then 320 μL of a 50 wt % aqueous hydroxylamine solution and 270 μL of acetic acid were added, and the volume was adjusted to exactly 2 mL using methanol. The resulting solution was stirred at 0°C for 25 hours to prepare a measurement sample.

[0107] (2) HPLC The measurement samples prepared above were analyzed using HPLC under the following measurement conditions. Detector: Photodiode array detector (measurement wavelength: 488 nm) Column: Scherzo SM-C18 (Intact) Column size: 150 x 4.6 mm (3 μm) Column temperature: 30℃ Mobile phase: Solution A: 5mmol / L ammonium formate solution / methanol mixture (95:5) Solution B: Methanol / water / formic acid mixture (90:10:0.1) Deployment conditions: A / B=75 / 25 (0~3 minutes) A / B = 75 / 25-37 / 63 (3-4 min; linear gradient) A / B=37 / 63(4~24 minutes) A / B = 37 / 63 to 20 / 80 (24 to 25 minutes; linear gradient) A / B=20 / 80(25~35 minutes) A / B = 20 / 80 to 0 / 100 (35 to 39 minutes; linear gradient) A / B=0 / 100(39~54 minutes) A / B = 0 / 100 to 75 / 25 (54 to 54.1 min; linear gradient) A / B=75 / 25(54.1~59.5 minutes) Flow rate: 1.0mL / min Injection volume: 10μL Sample cooler temperature: 4℃

[0108] (3) Bound drug loading Based on the HPLC chromatogram measured above, the number of moles of the peak to be measured was calculated by quantitative analysis using the HPLC area value of the peak to be measured and a calibration curve prepared from the corresponding standard sample to be measured.The weight of the corresponding active drug substance was calculated from the obtained number of moles, and the amount of bound drug carried was then calculated using the following formula. Bound drug loading (wt%) = weight of active drug (mg) / weight of complex (mg) × 100

[0109] Furthermore, the peak to be measured refers to the peak of an equivalent of an anthracycline drug based on molecular weight, and when peaks of isomers are separated under the above analytical conditions, the sum of the area percentages of the peaks of each isomer was taken as the HPLC area value of the peak to be measured.

[0110] The above-mentioned sample to be measured can be synthesized by the following method.

[0111] Synthesis Example 1 Synthesis of THP equivalent using hydroxylamine (measurement target): To a suspension of THP (625 mg, Microbiopharm Japan) in methanol (8.0 mL), 50 wt % aqueous hydroxylamine solution (530 μL) and acetic acid (460 μL) were added at 0°C, and the mixture was stirred at 0°C for 25 hours. The resulting reaction mixture was purified by silica gel column chromatography, and then acetonitrile (40 mL) and diisopropyl ether (100 mL) were added and the mixture was stirred at 25°C for 2 hours. The precipitated solid was collected by filtration and dried under reduced pressure at 25°C to obtain a THP equivalent (yield: 506 mg, 79%).

[0112] Synthesis Example 2 Synthesis of DOX equivalent using hydroxylamine (measurement target): To a suspension of DOX hydrochloride (500 mg, MedKoo Biosciences) in methanol (9.1 mL), 50 wt% aqueous hydroxylamine solution (610 μL) and acetic acid (530 μL) were added at 0°C, and the mixture was stirred at 0°C for 25 hours. Methanol (1.0 mL) and acetonitrile (20 mL) were added to the resulting reaction mixture, and the mixture was stirred at 25°C for 2 hours. The precipitated solid was collected by filtration and dried under reduced pressure at 25°C to obtain a DOX equivalent (yield: 464 mg, 90%).

[0113] Next, the method for evaluating the weight average molecular weight will be described.

[0114] (1) Sample preparation Methanol (500 μL) was added to the solids (5 mg) obtained in Examples 2 to 15 and Comparative Examples 1 and 2 described below to prepare measurement samples.

[0115] (2) Weight average molecular weight Each measurement sample prepared above was analyzed using HPLC and MALS under the following measurement conditions, and the weight-average molecular weight (Mw) was calculated using analytical software [ASTRA Ver. 7.3.2.17 64-bit (Wyatt Technology)] (refractive index increment: dn / dc = 0.175). HPLC: LC-40 (Shimadzu Corporation) Detector: Photodiode array detector (measurement wavelength: 488 nm) and differential refractometer Column: TSKgel α-M (Tosoh Corporation) TSKgel α-2500 (Tosoh Corporation) and two tubes connected in sequence Column size: TSKgel α-M (300 x 7.8 mm (7 μm)) TSKgel α-2500(300×7.8mm(7μm)) Column temperature: 30℃ Mobile phase: methanol: 0.3 mol / L sodium acetate solution (pH 6.5) =80:20 Flow rate: 0.8mL / min Injection volume: 20μL Sample cooler temperature: 4℃ Injector cleaning solution: methanol:water = 80:20 MALS:DAWN8 (Wyatt technology) Light Scattering Instrument: Calibration constant: 5.2929 / 100000[1 / Vcm] RI Instrument: Refractive constant: 1.338

[0116] Example 1 Assessment of bound drug purity: The results of the evaluation method for the purity of the bound drug are shown below in Test Examples 1 to 11.

[0117] Test Example 1 Hydrolysis of P-THP (Comparative Example 1) using the method described in Non-Patent Document 2: To the P-THP (20 mg) obtained in Comparative Example 1, 1 mol / L hydrochloric acid (2.0 mL) was added and stirred at 85°C for 20 minutes. The resulting reaction mixture was used as a measurement sample. The obtained measurement sample was subjected to HPLC measurement using the above-mentioned method, and the purity of THP was found to be 1.1%.

[0118] Test Example 2 Hydrolysis of P-THP (Comparative Example 1) according to the method described in Non-Patent Document 2 (reaction temperature: 0°C, hydrochloric acid): To the P-THP (20 mg) obtained in Comparative Example 1, cooled 1 mol / L hydrochloric acid (2.0 mL, 0°C) was added and stirred at 0°C for 1 hour. The resulting reaction mixture was used as a measurement sample. The obtained measurement sample was subjected to HPLC measurement using the above-mentioned method, and the purity of THP was found to be 0.2%.

[0119] Test Example 3 Hydrolysis of P-THP (Comparative Example 1) according to the method described in Non-Patent Document 2 (reaction temperature: 0°C, acetic acid): To the P-THP (20 mg) obtained in Comparative Example 1, a cooled 1 mol / L aqueous acetic acid solution (2.0 mL, 0°C) was added, and the mixture was stirred at 0°C for 2 hours. The resulting reaction mixture was used as a measurement sample. The obtained measurement sample was subjected to HPLC measurement using the above-mentioned method, and the purity of THP was found to be 83.3%.

[0120] Test Example 4 Conversion of THP with hydroxylamine and acetic acid: To a suspension of THP (1.0 mg, Microbiopharm Japan) in methanol (160 μL), 50 wt% aqueous hydroxylamine solution (32 μL) and acetic acid (27 μL) were added at 0°C, and the mixture was stirred at 0°C for 25 hours. Methanol (450 μL) was added to the resulting reaction mixture (50 μL) to prepare a measurement sample. HPLC analysis of the resulting measurement sample using the method described above revealed that the purity of the THP equivalent was 98.9%.

[0121] Test Example 5 THP Purity Assessment: A measurement sample was prepared by adding N,N-dimethylformamide (4.0 mL) to a suspension of THP (5.0 mg, manufactured by Microbiopharm Japan) in methanol (6.0 mL). HPLC analysis of the obtained measurement sample using the method described above revealed that the purity of THP was 99.0%.

[0122] The HPLC analysis results of Test Examples 1 to 5 are shown in Table 1.

[0123] [Table 1]

[0124] As shown in Table 1, the method described in Non-Patent Document 2 resulted in the release of THP from the complex and its decomposition into a substance of unknown structure (Test Example 1). Furthermore, when hydrolysis using hydrochloric acid at a reaction temperature of 0°C in accordance with the method described in Non-Patent Document 2 was performed, THP was released and converted to DOX, making it difficult to calculate the purity of the conjugated drug (Test Example 2). Furthermore, when hydrolysis using acetic acid at a reaction temperature of 0°C in accordance with the method described in Non-Patent Document 2 resulted in a high conversion rate to THP, but the conversion of THP to DOX could not be completely suppressed, making it difficult to calculate the purity of the conjugated drug (Test Example 3). On the other hand, when THP was used instead of the complex and hydroxylamine and acetic acid were used as additives, the conversion of THP to DOX hardly progressed, and a THP equivalent was obtained with a high purity, even when compared with the purity evaluation results of the THP used (Test Examples 4 and 5). From the above results, it was considered that if a similar reaction proceeds when a complex is used under the reaction conditions of Test Example 4, it may be possible to evaluate the purity of the bound drug.

[0125] Test Example 6 Bound Drug Purity Assessment of P-THP (Comparative Example 1) Using Hydroxylamine, Acetic Acid, and Methanol: To a solution of P-THP (10 mg) obtained in Comparative Example 1 in methanol (160 μL), 50 wt% aqueous hydroxylamine solution (32 μL) and acetic acid (27 μL) were added at 0°C, and the mixture was stirred at 0°C for 25 hours. Methanol (450 μL) was added to the resulting reaction mixture (50 μL) to prepare a measurement sample. The obtained measurement sample was subjected to HPLC measurement using the above-mentioned method, and the purity of the bound drug was found to be 94.5%.

[0126] Test Example 7 Bound Drug Purity Assessment of P-THP (Example 7) Using Hydroxylamine, Acetic Acid, and Methanol: To a solution of P-THP (10 mg) obtained in Example 7 in methanol (160 μL), 50 wt% aqueous hydroxylamine solution (32 μL) and acetic acid (27 μL) were added at 0°C, and the mixture was stirred at 0°C for 25 hours. Methanol (450 μL) was added to the resulting reaction mixture (50 μL) to prepare a measurement sample. The obtained measurement sample was subjected to HPLC analysis using the above-mentioned method, and the purity of the bound drug was found to be 98.9%.

[0127] Test Example 8 Conjugated Drug Purity Assessment of P-THP (Example 7) Using Acetohydrazide, Acetic Acid, and Methanol: To a solution of P-THP (10 mg) obtained in Example 7 in methanol (160 μL), acetohydrazide (35 mg) and acetic acid (27 μL) were added at 0°C, and the mixture was stirred at 0°C for 20 hours. Methanol (450 μL) was added to the resulting reaction mixture (50 μL) to prepare a measurement sample. The obtained measurement sample was subjected to HPLC analysis using the above-mentioned method, and the purity of the bound drug was found to be 97.7%.

[0128] Test Example 9 Conjugated Drug Purity Assessment of P-THP (Example 7) Using Propanohydrazide, Acetic Acid, and Methanol: To a solution of P-THP (10 mg) obtained in Example 7 in methanol (160 μL), propanohydrazide (42 mg) and acetic acid (27 μL) were added at 0°C, and the mixture was stirred at 0°C for 20 hours. Methanol (450 μL) was added to the resulting reaction mixture (50 μL) to prepare a measurement sample. The obtained measurement sample was subjected to HPLC analysis using the above-mentioned method, and the purity of the bound drug was found to be 96.0%.

[0129] Test Example 10 Conjugated Drug Purity Assessment of P-THP (Example 7) Using Butyrohydrazide, Acetic Acid, and Methanol: To a solution of P-THP (10 mg) obtained in Example 7 in methanol (160 μL), butyrohydrazide (49 mg) and acetic acid (27 μL) were added at 0°C, and the mixture was stirred at 0°C for 20 hours. Methanol (450 μL) was added to the resulting reaction mixture (50 μL) to prepare a measurement sample. The obtained measurement sample was subjected to HPLC analysis using the above-mentioned method, and the purity of the bound drug was found to be 96.8%.

[0130] Test Example 11 Conjugated Drug Purity Assessment of P-THP (Example 7) Using 3-Methylbutanohydrazide, Acetic Acid, and Methanol: To a solution of P-THP (10 mg) obtained in Example 7 in methanol (160 μL), 3-methylbutanohydrazide (56 mg) and acetic acid (27 μL) were added at 0°C, and the mixture was stirred at 0°C for 20 hours. Methanol (450 μL) was added to the resulting reaction mixture (50 μL) to prepare a measurement sample. The obtained measurement sample was subjected to HPLC analysis using the above-mentioned method, and the purity of the bound drug was found to be 96.6%.

[0131] The results of Test Examples 6 to 11 are shown in Table 2.

[0132] [Table 2]

[0133] As shown in Table 2, when the P-THP obtained in Comparative Example 1 was subjected to the reaction conditions of Test Example 4, the nucleophilic substitution reaction on the conjugate proceeded smoothly, but the P-THP did not exhibit a bound drug purity of 95.0% or more, indicating that P-THP produced by conventional methods has problems with bound drug purity (Test Example 6). On the other hand, when the P-THP obtained in Example 7 was subjected to the same conditions as Test Example 6, it was found to exhibit a high bound drug purity of 98.9% (Test Example 7). Furthermore, when the bound drug purity of the P-THP obtained in Example 7 was evaluated using various carboxylic acid hydrazides, it was found to exhibit good bound drug purity (Test Examples 8 to 11).

[0134] Example 2 A method for producing P-THP under the conditions of 8.8 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 40 molar equivalents of acetic acid, 24 times by weight of methanol, reaction temperature 1 to 2°C, reaction time 15 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (526 mg, manufactured by the Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (1.8 mL), THP (60.0 mg, manufactured by Microbiopharm Japan) and acetic acid (219 μL) were added, and the mixture was stirred at 1-2°C for 15 hours. To the reaction mixture, methanol (3.2 mL) was added. The resulting reaction mixture was added to ethyl acetate (21 mL) and stirred at 33-34°C for 24 hours. The precipitated solid was collected by filtration and dried under reduced pressure at 31-32°C to obtain P-THP (yield: 562 mg, 96.1%).

[0135] Example 3 A method for producing P-THP under the conditions of 9.1 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 32 molar equivalents of acetic acid, 58 times by weight of methanol, reaction temperature 1 to 2°C, reaction time 40 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (200 mg, Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (1.6 mL), THP (22.0 mg, Microbiopharm Japan Co., Ltd.) and acetic acid (65 μL) were added and stirred at 1-2°C for 40 hours. To the reaction mixture, methanol (0.3 mL) was added. The resulting reaction mixture was added to ethyl acetate (7.7 mL) and stirred at 21-22°C for 2 hours. The precipitated solid was collected by filtration. A solution of the resulting solid in methanol (1.4 mL) was added to ethyl acetate (5.7 mL) and stirred at 22-23°C for 2 hours. The precipitated solid was collected by filtration and dried under reduced pressure at 30-31°C to obtain P-THP (yield: 205 mg, 92.8%).

[0136] Example 4 A method for producing P-THP under the conditions of 8.8 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 40 molar equivalents of acetic acid, 24 times by weight of methanol, reaction temperature 1 to 5°C, reaction time 16 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (3.16 g, Chemicalsoft Co., Ltd.) in methanol (10.8 mL), THP (360 mg, MicroBiopharm Japan Co., Ltd.) and acetic acid (1.31 mL) were added and stirred at 1-5°C for 16 hours. Methanol (19.2 mL) was then added to the reaction mixture. The resulting reaction mixture was added to ethyl acetate (126 mL) and stirred at 19-22°C for 2 hours. The precipitated solid was collected by filtration. A solution of the resulting solid in methanol (24.0 mL) was added to ethyl acetate (96.0 mL) and stirred at 17-21°C for 2 hours. The precipitated solid was collected by filtration and dried under reduced pressure at 30-37°C to obtain P-THP (yield 3.43 g, 97.8%).

[0137] Example 5 A method for producing P-THP under the conditions of 8.8 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 100 molar equivalents of acetic acid, 56 times by weight of methanol, reaction temperature 2 to 4°C, reaction time 15 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (3.16 g, Chemicalsoft Co., Ltd.) in methanol (25.2 mL), THP (360 mg, MicroBiopharm Japan Co., Ltd.) and acetic acid (3.28 mL) were added. The mixture was stirred at 2-4°C for 15 hours, and then methanol (2.8 mL) was added to the reaction mixture. The resulting reaction mixture was added to ethyl acetate (126 mL) and stirred at 21-22°C for 2 hours. The precipitated solid was collected by filtration. A solution of the resulting solid in methanol (24.0 mL) was added to ethyl acetate (96.0 mL) and stirred at 21-24°C for 2 hours. The precipitated solid was collected by filtration and dried under reduced pressure at 35-42°C to obtain P-THP (yield: 3.48 g, 99.3%).

[0138] Example 6 A method for producing P-THP under the conditions of 8.8 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 40 molar equivalents of acetic acid, 24 times by weight of methanol, reaction temperature -10 to -9°C, reaction time 38 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (526 mg, Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (1.8 mL), THP (60.0 mg, MicroBiopharm Japan Co., Ltd.) and acetic acid (219 μL) were added and stirred at -10 to -9°C for 38 hours. To the reaction mixture, methanol (3.2 mL) was added. The resulting reaction mixture was added to ethyl acetate (21 mL) and stirred at 1 to 2°C for 1 hour. The precipitated solid was collected by filtration. A solution of the resulting solid in methanol (4.0 mL) was added to ethyl acetate (16.0 mL) and stirred at 1 to 5°C for 1 hour. The precipitated solid was collected by filtration and dried under reduced pressure at 31 to 32°C to obtain P-THP (yield: 541 mg, 92.6%).

[0139] Example 7 A method for producing P-THP under the conditions of 8.8 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 40 molar equivalents of acetic acid, 24 times by weight of methanol, reaction temperature -30 to -29°C, reaction time 112 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (526 mg, manufactured by the Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (1.8 mL), THP (60.0 mg, manufactured by MicroBiopharm Japan) and acetic acid (219 μL) were added, and the mixture was stirred at -30 to -29°C for 112 hours. To the reaction mixture, methanol (3.2 mL) was added. The resulting reaction mixture was added to ethyl acetate (21 mL) and stirred at 22 to 23°C for 0.5 hours. The precipitated solid was collected by filtration and dried under reduced pressure at 32 to 33°C to obtain P-THP (yield: 548 mg, 93.7%).

[0140] Example 8 A method for producing P-THP under the conditions of 8.8 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 40 molar equivalents of acetic acid, 24 times by weight of methanol, reaction temperature 2 to 5°C, reaction time 15 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (13.2 g, manufactured by the Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (45.0 mL), THP (1.50 g, manufactured by Microbiopharm Japan Co., Ltd.) and acetic acid (5.47 mL) were added. The mixture was stirred at 2-5°C for 15 hours, and then methanol (79.5 mL) was added to the reaction mixture. The resulting reaction mixture was added to ethyl acetate (525 mL) and stirred at 20-22°C for 1 hour. The precipitated solid was collected by filtration. A solution of the resulting solid in methanol (97.5 mL) was added to ethyl acetate (390 mL) and stirred at 22-23°C for 1 hour. The precipitated solid was collected by filtration and dried under reduced pressure at 31-32°C to obtain P-THP (yield: 14.2 g, 97.4%).

[0141] Example 9 A method for producing P-DOX hydrochloride under the conditions of 9.8 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 100 molar equivalents of acetic acid, 62 times by weight of methanol, reaction temperature 1 to 4°C, reaction time 46 hours: DOX hydrochloride (20.3 mg, MedKoo Biosciences) and acetic acid (200 μL) were added to a methanol (1.6 mL) solution of N-(2-hydroxypropyl)methacrylamide polymer (200 mg, Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic). The mixture was stirred at 1-4°C for 46 hours, and then methanol (0.3 mL) was added to the reaction mixture. The resulting reaction mixture was added to ethyl acetate (7.7 mL) and stirred at 21-22°C for 1 hour. The precipitated solid was collected by filtration and dried under reduced pressure at 32-33°C to obtain P-DOX hydrochloride (yield: 219 mg, 99.7%).

[0142] Comparative Example 1 A method for producing P-THP under conditions of 9.1 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 32 molar equivalents of acetic acid, 58 times by weight of methanol, a reaction temperature of 29 to 30°C, and a reaction time of 15 hours (see the temperature described in Patent Document 1 and the method described in Non-Patent Document 1): To a solution of N-(2-hydroxypropyl)methacrylamide polymer (200 mg, manufactured by the Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (1.6 mL), THP (22.0 mg, manufactured by Microbiopharm Japan) and acetic acid (65 μL) were added, and the mixture was stirred at 29-30°C for 15 hours in the dark. To the reaction mixture, methanol (0.3 mL) was added. The resulting reaction mixture was added to ethyl acetate (7.7 mL) and stirred at 29-32°C for 24 hours. The precipitated solid was collected by filtration and dried under reduced pressure at 31-34°C to obtain P-THP (yield: 209 mg, 94.3%).

[0143] Comparative Example 2 A method for producing P-THP under the conditions of 8.8 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 40 molar equivalents of acetic acid, 56 times by weight of methanol, reaction temperature 34 to 35°C, reaction time 17 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (3.16 g, manufactured by the Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (25.2 mL), THP (360 mg, manufactured by Microbiopharm Japan Co., Ltd.) and acetic acid (1.31 mL) were added and stirred at 34-35°C for 17 hours. Methanol (4.8 mL) was then added to the reaction mixture. The resulting reaction mixture was added to ethyl acetate (126 mL) and stirred at 19-23°C for 1 hour. The precipitated solid was collected by filtration. A solution of the resulting solid in methanol (24.0 mL) was added to ethyl acetate (96.0 mL) and stirred at 22-23°C for 1 hour. The precipitated solid was collected by filtration and dried under reduced pressure at 31-35°C to obtain P-THP (yield: 3.47 g, 98.9%).

[0144] Example 10 A method for producing P-THP under the conditions of 24.0 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 100 molar equivalents of acetic acid, 56 times by weight of methanol, a reaction temperature of 4 to 7°C, and a reaction time of 20 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (1.20 g, Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (3.5 mL), THP (50 mg, Microbiopharm Japan Co., Ltd.) and acetic acid (0.46 mL) were added and stirred at 4-7°C for 20 hours. To the reaction mixture, methanol (2.0 mL) was added. The resulting reaction mixture was added to ethyl acetate (25 mL) and stirred at 22-23°C for 1 hour. The precipitated solid was collected by filtration. A solution of the resulting solid in methanol (10.0 mL) was added to ethyl acetate (40.0 mL) and stirred at 21-23°C for 1 hour. The precipitated solid was collected by filtration and dried under reduced pressure at 23-24°C to obtain P-THP (yield: 1.11 g, 89.4%).

[0145] Example 11 A method for producing P-THP under the conditions of 13.3 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 100 molar equivalents of acetic acid, 56 times by weight of methanol, a reaction temperature of 2 to 4°C, and a reaction time of 21 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (0.66 g, manufactured by the Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (3.5 mL), THP (50 mg, manufactured by Microbiopharm Japan Co., Ltd.) and acetic acid (0.46 mL) were added, and the mixture was stirred at 2-4°C for 21 hours. To the reaction mixture, methanol (2.0 mL) was then added. The resulting reaction mixture was added to ethyl acetate (25 mL), and the mixture was stirred at 22-23°C for 1 hour. The precipitated solid was collected by filtration and dried under reduced pressure at 22-23°C to obtain P-THP (yield: 0.70 g, 98.4%).

[0146] Example 12 A method for producing P-THP under the conditions of 8.8 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 40 molar equivalents of acetic acid, 24 times by weight of methanol, reaction temperature 1 to 5°C, reaction time 19 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (122.8 g, manufactured by the Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (420 mL), THP (14.0 g, manufactured by Microbiopharm Japan Co., Ltd.) and acetic acid (51.0 mL) were added. The mixture was stirred at 1-5°C for 19 hours, and then methanol (742 mL) was added to the reaction mixture. The resulting reaction mixture was added to ethyl acetate (4900 mL) and stirred at 18-20°C for 1 hour. The precipitated solid was collected by filtration. A solution of the resulting solid in methanol (910 mL) was added to ethyl acetate (3640 mL) and stirred at 18-20°C for 1 hour. The precipitated solid was collected by filtration and dried under reduced pressure at 16-20°C to obtain P-THP (yield: 130.2 g, 95.4%).

[0147] Example 13 A method for producing P-THP under the conditions of 6.7 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 60 molar equivalents of acetic acid, 16 times by weight of methanol, reaction temperature 4 to 7°C, reaction time 22 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (0.67 g, manufactured by the Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (2.0 mL), THP (100 mg, manufactured by Microbiopharm Japan Co., Ltd.) and acetic acid (0.55 mL) were added, and the mixture was stirred at 4-7°C for 22 hours. To the reaction mixture, methanol (2.0 mL) was then added. The resulting reaction mixture was added to ethyl acetate (25 mL), and the mixture was stirred at 22-23°C for 1 hour. The precipitated solid was collected by filtration and dried under reduced pressure at 21-22°C to obtain P-THP (yield: 0.76 g, 98.6%).

[0148] Example 14 A method for producing P-THP under the conditions of 5.0 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 60 molar equivalents of acetic acid, 16 times by weight of methanol, reaction temperature 1 to 5°C, reaction time 20 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (3.00 g, Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (12.0 mL), THP (600 mg, Microbiopharm Japan Co., Ltd.) and acetic acid (3.28 mL) were added. The mixture was stirred at 1-5°C for 20 hours, and then methanol (15.0 mL) was added to the reaction mixture. The resulting reaction mixture was added to ethyl acetate (120 mL) and stirred at 19-21°C for 0.5 hours. The precipitated solid was collected by filtration. A solution of the resulting solid in methanol (27.0 mL) was added to ethyl acetate (120 mL) and stirred at 19-23°C for 1 hour. The precipitated solid was collected by filtration and dried under reduced pressure at 23-24°C to obtain P-THP (yield 3.37 g, 93.5%).

[0149] Example 15 A method for producing P-THP under the conditions of 4.0 times by weight of N-(2-hydroxypropyl) methacrylamide polymer, 40 molar equivalents of acetic acid, 16 times by weight of methanol, a reaction temperature of 1 to 4°C, and a reaction time of 37 hours: To a solution of N-(2-hydroxypropyl)methacrylamide polymer (200 mg, Institute of Polymer Chemistry, Academy of Sciences of the Czech Republic) in methanol (1.0 mL), THP (50 mg, Microbiopharm Japan Co., Ltd.) and acetic acid (0.18 mL) were added. The mixture was stirred at 1-4°C for 37 hours, and then methanol (0.8 mL) was added to the reaction mixture. The resulting reaction mixture was added to ethyl acetate (10 mL) and stirred at 1-5°C for 1 hour. The precipitated solid was collected by filtration. A solution of the resulting solid in methanol (3.0 mL) was added to ethyl acetate (15 mL) and stirred at 22-23°C for 0.5 hours. The precipitated solid was collected by filtration and dried under reduced pressure at 23-24°C to obtain P-THP (yield: 197 mg, 78.9%).

[0150] The results of Examples 2 to 15 and Comparative Examples 1 and 2 are shown in Table 3.

[0151] [Table 3]

[0152] The conjugates obtained in Examples 2-15 and Comparative Examples 1 and 2 were evaluated using hydroxylamine, acetic acid, and methanol. As shown in Table 3, a reaction temperature of 10°C or less resulted in the production of target products with higher conjugated drug purity compared to Comparative Examples 1 and 2 (Examples 2-15). Furthermore, good conjugated drug purity was achieved even when the type of anthracycline drug or the type of N-(2-hydroxypropyl)methacrylamide polymer was varied (Examples 4, 5, and 9). Conjugated drug purity of 98.0% or higher was also achieved when the amount of N-(2-hydroxypropyl)methacrylamide polymer, the molar equivalent of acetic acid, or the amount of methanol used was varied (Examples 3, 5, and 9). This indicates that reaction temperature is important for improving conjugated drug purity. Furthermore, the reaction proceeded well even at reaction temperatures of approximately -10°C or -30°C (Examples 6 and 7), demonstrating the feasibility of scale-up production (Example 8).

[0153] Furthermore, even when the amount of N-(2-hydroxypropyl)methacrylamide polymer used relative to the anthracycline drug was significantly changed, it was found that the bound drug loading amount could be changed while maintaining a bound drug purity of 98.0% or more by setting the reaction temperature at 10°C or less (Examples 10 to 15).Furthermore, it was found that, compared to Comparative Example 1, the bound drug purity further decreased when the reaction temperature was further increased (Comparative Example 2).

[0154] Example 16 Storage stability evaluation under airtight conditions at 60°C (change in weight average molecular weight over time in short-term stress test): The composites (5 mg) obtained in Comparative Example 1 and Examples 12 to 15 were weighed into HPLC vials, which were then capped and placed in an oven at 60°C. After two and four weeks, the vials were removed from the oven and the weight-average molecular weights were evaluated. The weight-average molecular weight at the start of the storage stability evaluation was set as week 0, and the evaluation results after two and four weeks are shown in Table 4.

[0155] [Table 4]

[0156] As shown in Table 4, when the change in weight-average molecular weight over time was compared with the purity of the bound drug, it was found that when the purity of the bound drug was less than 95.0%, the weight-average molecular weight increased significantly, i.e., storage stability was low, whereas when the purity of the bound drug was 95.0% or higher, the increase in molecular weight of the complex was significantly suppressed, resulting in high storage stability (Comparative Example 1, Example 12). Furthermore, when the change in weight-average molecular weight over time was compared with the amount of bound drug carried, it was found that by increasing the amount of bound drug carried to 15 wt% or higher, the increase in molecular weight of the complex was further suppressed, resulting in even higher storage stability (Examples 12 to 15). [Industrial Applicability]

[0157] The method for evaluating the purity of a drug contained in Complex (I) or a pharmacologically acceptable salt thereof according to the present invention makes it possible to evaluate whether the purity of the bound drug satisfies the purity standards for THP and DOX hydrochloride as specified in the Japanese Pharmacopoeia. Furthermore, the production method of the present invention makes it possible to produce Complex (I) with unprecedented high purity.

Claims

1. a reaction step of reacting a complex represented by general formula (I) or a pharmacologically acceptable salt thereof with at least one nitrogen-containing nucleophile selected from the group consisting of hydroxylamine, O-alkylhydroxylamine, and carboxylic acid hydrazide in a polar solvent in the presence of a protonic acid; an evaluation step of evaluating the purity of the reaction mixture obtained in the reaction step by high performance liquid chromatography; The compound of general formula (I): 【Chemistry 1】 [In the formula, A represents a hydrogen atom or an (R)-tetrahydro-2H-pyran-2-yl group, b, c, d, and e each independently represent a positive integer, and the bond depicted with a wavy line represents that it can have either an E-configuration or a Z-configuration.] The complex represented by general formula (I) or a pharmacologically acceptable salt thereof has a purity of 95.0% or more of the drug contained in the complex represented by general formula (I) or a pharmacologically acceptable salt thereof, when evaluated by a method for evaluating the purity of the drug contained in the complex represented by general formula (I) or a pharmacologically acceptable salt thereof.

2. A is an (R)-tetrahydro-2H-pyran-2-yl group, 2. The complex or pharmacologically acceptable salt thereof according to claim 1, wherein b is 5.

3. 3. The complex or pharmacologically acceptable salt thereof according to claim 1 or 2, wherein the amount of the bound drug carried is 4 to 20 wt %.

4. the polar solvent is an alcohol-based solvent, the nitrogen-containing nucleophile is at least one selected from the group consisting of hydroxylamine and carboxylic acid hydrazide; The complex or pharmacologically acceptable salt thereof according to any one of claims 1 to 3, wherein the protonic acid is a carboxylic acid.

5. the polar solvent is methanol; the nitrogen-containing nucleophile is at least one selected from the group consisting of hydroxylamine, acetohydrazide, propanohydrazide, butyrohydrazide, and 3-methylbutanohydrazide; The complex or pharmacologically acceptable salt thereof according to any one of claims 1 to 4, wherein the protonic acid is acetic acid.

6. The complex or pharmacologically acceptable salt thereof according to any one of claims 1 to 5, wherein in the general formula (I), b is an integer of 1 to 10, c is an integer of 30 to 500, d is an integer of 1 to 50, and e is an integer of 1 to 50.

7. A method for producing a conjugate represented by general formula (I) or a pharmacologically acceptable salt thereof, comprising a reaction step of reacting an anthracycline drug represented by general formula (II) with an N-(2-hydroxypropyl)methacrylamide polymer represented by general formula (III) in the presence of a protonic acid at 10°C or lower in a polar solvent to obtain a conjugate represented by general formula (I) or a pharmacologically acceptable salt thereof. 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 [In formulas (I) and (II), A represents a hydrogen atom or an (R)-tetrahydro-2H-pyran-2-yl group, and in formulas (I) and (III), b, c, d, e, and f each independently represent a positive integer, and the bond depicted with a wavy line indicates that it can have either an E-configuration or a Z-configuration.]

8. the polar solvent is methanol; the protonic acid is acetic acid; The method according to claim 7, wherein the reaction temperature in the reaction step is from -30°C to 10°C.

9. The method according to claim 7 or 8, wherein in general formula (I) and general formula (III), b is an integer of 1 to 10, c is an integer of 30 to 500, d is an integer of 1 to 50, e is an integer of 1 to 50, and f is the sum of d and e.

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