Novel drug complex

Abstract

Provided is a novel copolymer that can be used in a drug delivery technique. The present invention relates to a drug complex in which a target recognition molecule is bound to a copolymer X comprising structural units represented by formulae (A), (B), and (C). [In the formula, R1, R2 and R3 may be the same or different and each represents a hydrogen atom or a C1-3 alkyl group; r4 represents a C1-3 alkyl group; r5 represents a hydrogen atom, a C1-18 alkyl group, a 3-to 8-membered cycloalkyl group which may have a substituent, an adamantyl group, a C6-18 aryl group which may have a substituent, or a 5-to 10-membered heteroaryl group which may have a substituent; x1, X2, and X3 may be the same or different and each represents an oxygen atom, a sulfur atom, or N-R7; r6 represents a hydrogen atom, a leaving group or a linking group; r7 represents a hydrogen atom or a C1-3 alkyl group; m represents an integer of 1 to 100; and n represents an integer of 0-3]. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a novel copolymer that can be used in drug delivery technology. More specifically, it relates to a copolymer for drug delivery carrier targeting tumors, a pharmaceutical composition in which the copolymer carries a physiologically active substance such as an anticancer agent, and a pharmaceutical product containing the composition. Background Art

[0002] In recent years, as a technology for efficiently and safely delivering drugs to disease sites, research related to drug delivery systems (DDS) has become popular in the industry. Among them, as a technology that utilizes the structural characteristics of disease sites to improve the selectivity of drug aggregation, the demand for DDS using nanoparticles as drug delivery carriers is growing.

[0003] For example, in solid cancer tissue, compared with normal blood vessels, since the structure of new blood vessels (tumor blood vessels) is not yet mature, a few hundred nm of intercellular spaces are generated in the vascular endothelium, and the permeability of the substance is high. It is known that through this structural feature, high molecular weight bodies containing nanoparticles selectively penetrate tumor blood vessels and accumulate in solid cancer tissues. Furthermore, in solid cancer tissues, the lymphatic system involved in the discharge of macromolecules is dysfunctional, so the infiltrated nanoparticles are continuously retained in the tissue (Enhanced permeability and retention effect, EPR effect). Conventional low molecular weight drugs leak out of the blood vessels due to the membrane penetration of vascular cells, so they are non-selectively distributed in the tissues, and are not accumulated in solid cancer tissues. According to the methodology of the EPR effect, drug delivery using nanoparticles is dominated by the permeability of the intercellular spaces of vascular endothelial cells due to the distribution in the tissue, so the tissue selectivity for solid cancer is improved in the distribution of drugs. Therefore, the EPR effect has become a powerful academic basis for the development of nanotechnology-applied pharmaceuticals (nanomedicine) targeting solid cancer.

[0004] The delivery process of the drug in the EPR effect is considered to be via blood flow, and the extravasation process of the nanoparticles is passively carried out. Therefore, in order to maximize the aggregation of nanoparticles to solid cancer, it is important to give the constituents of the nanoparticles that become drug delivery carriers a molecular design that can tolerate long-term retention in the blood. Therefore, drug delivery carriers require the ability to avoid obstacles such as nonspecific interactions with blood constituents, foreign body recognition using the reticuloendothelial system (RES) in the liver, spleen and lungs, and glomerular filtration in the kidney. In addition, it is known that these obstacles can be overcome by optimizing particle diameter or utilizing particle characteristics such as surface modification of biocompatible polymers. For example, the particle diameter of the drug delivery carrier is preferably greater than about 6nm, which is the threshold value of kidney clearance, and less than 200nm, so that it can be avoided from being recognized by RES.

[0005] In addition, the particle size of known drug delivery carriers can also affect the tissue permeability in the diseased area. For example, when the anti-cancer activity of the drug-enclosed nanoparticles with a particle size of 30nm, 50nm, 70nm and 100nm showing equal retention in the blood is compared and studied, it is known that the drug-enclosed nanoparticles with a particle size of 30nm can reach the depth of the diseased area, thereby showing the highest therapeutic effect (non-patent literature 1). Therefore, it is believed that the particle size of the nanoparticles used in the drug delivery carriers targeting solid cancer is preferably the smallest possible particle size within the range of avoiding kidney clearance.

[0006] The following methods have been developed for the production of nanoparticles as drug delivery carriers: methods using colloidal dispersions such as liposomes, emulsions, or nanoparticles; methods using biogenic raw materials such as albumin; methods using natural polymers such as natural polysaccharides; or methods using synthetic polymers. Among them, synthetic polymers can be used as components of drug delivery carriers because they can produce nanoparticles with precisely controlled particle sizes by appropriately selecting monomers that become components and synthesis methods.

[0007] For example, the utilization method of the amphiphilic block copolymer composed of hydrophilic segments and hydrophobic segments as a drug delivery carrier has been disclosed. The block copolymer uses the hydrophobic interaction between molecules as a driving force, and spontaneously associates in an aqueous medium to form core-shell nanoparticles (polymer micelles). The hydrophobic segments of the known polymer micelles can be encapsulated or combined with low-molecular drugs, and the obtained drug encapsulated polymer micelles show higher stability in blood, and compared to the solution of low-molecular drugs, by the selective aggregation of solid cancers via the EPR effect and produce higher tumor suppressor activity (patent document 1). However, polymer micelles are the associations of multiple molecules, so the lower limit that can be prepared is about 30nm of particle diameter, and it is difficult to control the fine size near the particle diameter 10nm that can avoid the influence of kidney clearance.

[0008] On the other hand, in the nanoparticles formed by synthetic polymers, the particles are formed by chemical crosslinking, hydrophobic interaction, ionic bonding, etc. in a single chain as a driving force (hereinafter referred to as single-chain nanoparticles (SCNP, single chain nanoparticle)). It is known to form nanoparticles with a particle size of less than 20nm (non-patent document 2). Therefore, although the usefulness of SCNP as a drug delivery carrier is expected, the technology that can accurately control its particle size has not yet been found.

[0009] As other drug delivery technologies, there are antibody-drug conjugates (ADCs) that can deliver cytotoxic agents (drugs) to antigen-expressing tumor cells in a targeted manner (non-patent literature 3 to 5). ADC has three components: an antibody (Ab), a linker, and a drug. In order to deliver the drug locally to the target, the drug is linked or conjugated to the antibody. The conjugation method is usually a chemical modification of the amino group of the lysine side chain of the antibody or the cysteine ​​sulfhydryl group obtained by reducing the interchain disulfide bond. The design of ADC is still difficult, and it must control several factors (such as the selection of antibodies, the stability of the linker, the drug / toxin (payload) and its cleavage kinetics, etc.). Among them, the important parameter is the number of payloads for a single antibody (drug-antibody ratio or DAR (Drug Antibody ratio, drug-antibody ratio)). Antibodies bound to a large number of drug / toxin molecules show a binding barrier to the target antigen, or a faster clearance rate from the bloodstream. Usually, one antibody can only bind a limited number of drug / toxin molecules (usually DAR is 4 to 6). As a result, in order to obtain sufficient effectiveness in killing target cells, it is necessary to use highly toxic agents with an IC50 of less than 1 nM (e.g., calicheamicin or auristatin monomethyl ester (MMAE)) (Non-patent Documents 6, 7, etc.). Therefore, if a small portion of the conjugate is off-target, serious adverse effects will occur. As described above, a new method with both high effectiveness and improved tolerability is needed.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: Japanese Patent No. 3270592

[0013] Non-patent literature

[0014] Non-patent document 1: H. Cabral et al., Nat. Nanotechnol. 6 815-823 (2011) Non-patent document 2: Jose A. Pomposo, Single-Chain Polymer Nanoparticles: Synthesis, Characterization, Simulations, and Applications (2017)

[0015] Non-patent document 3: Chari, RV et al., Angew. Chem. Int. Ed. 53, 3796-3827 (2014)

[0016] Non-patent document 4: Jagadeesh, D., Smith, MR, Curr. Treat. options Oncol. 17, 55 (2016)

[0017] Non-patent document 5: Ducry, L., Stump, B., Bioconjugate Chem. 21, 5-13 (2010) Non-patent document 6: Casi, G., Neri, D., J. Controlled Release 161, 422-428 (2012)

[0018] Non-patent document 7: Wu, AM, Senter, PD, Nat. Biotechnol. 23, 1137-1146 (2005) Summary of the invention

[0019] Technical problem to be solved by the invention

[0020] The technical problem of the present invention is to provide a copolymer for drug delivery carriers targeting tumors. More specifically, the technical problem of the present invention is to provide a copolymer for drug delivery carriers that can be used to improve the blood retention and / or tumor accumulation of drugs.

[0021] Technical solutions for solving technical problems

[0022] The inventors of the present invention have conducted in-depth research to solve the above technical problems, and in the process discovered that the terpolymer of acrylic acid derivatives forms SCNPs in water. In addition, they have successfully created a copolymer for drug delivery carriers that can not only achieve precise particle size control of SCNPs to a microscale of about 10nm below 20nm, but also have high tumor aggregation. The drug complex supported or combined with an anticancer agent on the polymer is administered to a subcutaneous transplant model mouse of cancer, and the result is that an excellent anti-tumor effect can be exerted.

[0023] The present invention relates to the following inventions.

[0024] [1] A copolymer in which a target recognition molecule is bonded to a copolymer X having structural units represented by the following formulae (A), (B) and (C),

[0025]

[0026] [Where R 1 , R 2 and R 3 Same or different, indicating hydrogen atoms or C 1-3 Alkyl; R 4 Represents C 1-3 Alkyl; R 5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent; X 1 , X 2 and X 3 The same or different, representing oxygen atoms, sulfur atoms or NR 7 ; R 6 represents a hydrogen atom, a leaving group or a connecting group; R 7 Represents a hydrogen atom or C 1-3 alkyl; m represents an integer from 1 to 100; n represents an integer from 0 to 3].

[0027] [2] The copolymer as described in [1] above, wherein the copolymer X is a copolymer formed by polymerization of three monomers represented by the following general formulae (1) to (3):

[0028]

[0029] [Where R 1 , R 2 and R 3 Same or different, indicating hydrogen atoms or C 1-3 Alkyl; R 4 Represents C 1-3 Alkyl; R 5represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent; X 1 , X 2 and X 3 The same or different, representing oxygen atoms, sulfur atoms or NR 7 ; R 6 represents a hydrogen atom, a leaving group or a connecting group; R 7 Represents a hydrogen atom or C 1-3 alkyl; m represents an integer from 1 to 100; n represents an integer from 0 to 3].

[0030] [3] The copolymer described in [1] or [2] above, wherein R 1 A hydrogen atom.

[0031] [4] The copolymer described in any one of [1] to [3] above, wherein R 2 A hydrogen atom.

[0032] [5] The copolymer described in any one of [1] to [4] above, wherein R 3 A hydrogen atom.

[0033] [6] The copolymer described in any one of [1] to [5] above, wherein R 4 It is methyl.

[0034] [7] The copolymer described in any one of [1] to [6] above, wherein R 5 is a C which may have a substituent 6-18 Aryl.

[0035] [8] The copolymer described in any one of [1] to [7] above, wherein R 5 It is phenyl.

[0036] [9] The copolymer described in any one of [1] to [8] above, wherein R 6 A hydrogen atom.

[0037]

[10] The copolymer described in any one of [1] to [8] above, wherein R 6 The leaving group is a group represented by the following formula (4):

[0038]

[0039]

[11] The copolymer described in any one of [1] to [8] above, wherein R 6 The linking group is a group represented by the following formula (5):

[0040]

[0041] [Where R 8 Represents a hydrogen atom or a drug; Ak 1 Represents C 1-7 Alkylene bond; X 4 represents an oxygen atom, a sulfur atom or -N(R 7 )-(R 7 Represents a hydrogen atom or C 1-3 alkyl)].

[0042]

[12] The copolymer described in any one of [1] to

[11] above, wherein X 1 For oxygen atoms.

[0043]

[13] The copolymer described in any one of [1] to

[12] above, wherein X 2 For oxygen atoms.

[0044]

[14] The copolymer described in any one of [1] to

[13] above, wherein X 3 is an oxygen atom or NH.

[0045]

[15] The copolymer described in any one of [1] to

[14] above, wherein m is an integer of 4 to 22.

[0046]

[16] The copolymer described in any one of [1] to

[15] above, wherein n is 1.

[0047]

[17] The copolymer as described in [1] to

[16] above, wherein the ratio of the structural units (A), (B) and (C) is 0.01 to 100 parts by mass of (B) and 0.1 to 100 parts by mass of (C) per 1 part by mass of (A).

[0048]

[18] The copolymer as described in any one of [2] to

[16] above, which is obtained by polymerizing 0.01 to 100 parts by mass of the monomer (2) and 0.1 to 100 parts by mass of the monomer (3) relative to 1 part by mass of the monomer (1).

[0049]

[19] The copolymer described in any one of [1] to

[18] above, which has a number average molecular weight of 5,000 to 150,000.

[0050]

[20] The copolymer described in any one of [1] to

[19] above, wherein the target recognition molecule is an antibody.

[0051]

[21] The copolymer described in

[20] above, wherein the antibody is an anti-epidermal growth factor receptor (EGFR) antibody, an anti-Her2 antibody, an anti-CD20 antibody, an anti-CD276 antibody, an anti-MUC1 antibody, an anti-PD-L1 antibody, or an anti-TROP-2 antibody.

[0052]

[22] The copolymer according to

[20] , wherein the antibody is cetuximab, panitumumab, necituzumab, amivantamab, panitumumab, trastuzumab, pertuzumab, magituximab, rituximab, ibritumomab tiuxetan, tositumomab, ofatumumab, obituzumab, clivatuzumab, gatipotuzumab, ifinatamab, mirzotamab, vobramitamab, atezolizumab, avelumab, durvalumab, sacituzumab, or functional fragments thereof.

[0053]

[23] A drug complex comprising the copolymer described in any one of [1] to

[22] above and a drug.

[0054]

[24] The drug complex as described in

[23] above, wherein the drug is a metabolic antagonist, an alkylating agent, an anthracycline, an antibiotic, a mitotic inhibitor, a topoisomerase inhibitor, a proteasome inhibitor or an antihormone.

[0055]

[25] The drug complex as described in

[23] above, wherein the drug is DM0, DM1, DM2, DM3, DM4, metansine, auristatin E, auristatin phenylalanine phenylenediamine (AFP), monomethyl auristatin E, monomethyl auristatin D, monomethyl auristatin F, paclitaxel, docetaxel, irinotecan, topotecan, nojitecan, amsacrine, etoposide, teniposide, mitoxantrone (mizantrone), SN-38, exitecan, or derunotecan.

[0056]

[26] The drug complex as described in any one of

[23] to

[25] above, wherein the binding of the target recognition molecule or drug to the copolymer X is covalent binding or non-covalent binding.

[0057]

[27] The drug complex as described in any one of

[23] to

[26] above, wherein the combination of the target recognition molecule or drug and the copolymer X is the following formula (a):

[0058]

[0059] [Where J 1 It is the binding part with target recognition molecule or drug; 2 is the bonding part with copolymer X; Ak 2 ,Ak 3 Each independently represents a single bond, or C 1-7 Alkylene bond; B 1 , B 2 Each independently represents a single bond, an amide group or an ester bond; L 1 Represents a single bond, -(CH2CH2O) o CH2CH2-, phenylene, cyclohexylene, -NH-peptide-CO-, or phenylene-NH-peptide-CO-; o represents an integer from 0 to 100].

[0060]

[28] A single-chain nanoparticle comprising the copolymer or drug conjugate described in any one of [1] to

[27] .

[0061]

[29] A pharmaceutical composition comprising the copolymer or drug complex described in any one of [1] to

[28] above.

[0062] Effects of the Invention

[0063] According to the examples described below, after the SCNP obtained by the self-association of the copolymer of the present invention is loaded or combined with an anticancer agent, a tumor growth inhibition effect is shown in a mouse tumor-bearing model, so it can be used as a therapeutic agent for malignant tumors. After the SCNP obtained by the self-association of the copolymer of the present invention is loaded or combined with an anticancer agent, a higher DAR can be obtained compared to the existing ADC, and a higher tumor growth inhibition effect can be obtained at a low dose, so it can provide a therapeutic agent for malignant tumors that can take into account both the enhancement of pharmacological effects and the inhibition of side effects. In addition, the copolymer of the present invention combined with a target recognition molecule can be effectively used for drug delivery using a target-specific target recognition molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The copolymer obtained in Example 1 was measured by nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0065] Figure 2This is a diagram showing a chromatogram of the copolymer obtained in Example 1 obtained by gel permeation chromatography (GPC).

[0066] Figure 3 It is a graph showing the particle size measurement results (scattering intensity distribution) of the copolymer before DACHPt encapsulation (Example 69) and DACHPt encapsulating SCNP (Example 70) by dynamic light scattering (DLS).

[0067] Figure 4 The N3-poly[(benzyl acrylate)-co-(poly(ethylene glycol)methyl ether acrylate)-co-(acrylic acid)] obtained in Example 71 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0068] Figure 5 This is a diagram showing a chromatogram obtained by gel permeation chromatography (GPC) of N3-poly[(benzyl acrylate)-(poly(ethylene glycol) methyl ether acrylate)-(acrylic acid) copolymer] obtained in Example 71.

[0069] Figure 6 This is a diagram showing the UV spectrum of N3-poly[(benzyl acrylate)-(poly(ethylene glycol) methyl ether acrylate)-(acrylic acid) copolymer]-isobutyronitrile obtained in Example 92 measured using UV (ultraviolet) spectrometry (UV).

[0070] Figure 7 The DM1-cysteamine-binding copolymer obtained in Example 96 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0071] Figure 8The DM1-N-4-APM copolymer obtained in Example 108 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0072] Fig. 9 The 1,4-butanediamine-bonded copolymer obtained in Example 109 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0073] Fig.10 The DM1-MHA-1,4-butanediamine-bound copolymer obtained in Example 117 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0074] Fig.11 The DM1-SPDP-1,4-butanediamine-bound copolymer obtained in Example 118 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0075] Fig.12 The DM1-SMCC-1,4-butanediamine-bound copolymer obtained in Example 122 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0076] Fig.13 The DM1-CL-031-1,4-butanediamine-bound copolymer obtained in Example 124 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0077] Fig.14 The DM1-CL-018-1,4-butanediamine-bound copolymer obtained in Example 125 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0078] Fig.15 The DM1-CL-038-1,4-butanediamine-bound copolymer obtained in Example 126 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0079] Fig.16 The DM1-CL-047-1,4-butanediamine-bound copolymer obtained in Example 127 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0080] Fig.17 The SN-38-CO-1,4-butanediamine-bound copolymer obtained in Example 128 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0081] Fig.18 The results of measuring the delutec-SPDP-1,4-butanediamine copolymer obtained in Example 129 using nuclear magnetic resonance spectroscopy (NMR) are shown in FIG. 1 Figure 1. H-NMR spectrum.

[0082] Fig.19 The 4-hydroxybutylamine-bonded copolymer obtained in Example 130 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0083] Fig. 20 The staurosporine (STS)-bound copolymer obtained in Example 131 was measured using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0084] Fig.21The following is the result of measuring the exitecan-PAB-Cit-Val-Ahx conjugate copolymer obtained in Example 158 using nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1. H-NMR spectrum.

[0085] Fig. 22 This is a graph showing the change in relative tumor volume when an oxaliplatin solution or DACHPt-encapsulated SCNP (Example 70) was administered three times every other day to a mouse colon cancer cell line (C26) subcutaneously transplanted model mouse on the back.

[0086] Fig.23 The graph shows changes in tumor volume when physiological saline or cetuximab-bound micelle drug complex (Example 138) was administered to model mice subcutaneously transplanted with mouse EGFR-positive human colon cancer cell line HT-29 on the right flank.

[0087] Fig.24 The graph shows changes in tumor volume when physiological saline or the trastuzumab-bound micelle drug complex (Example 151) was administered to model mice subcutaneously transplanted with the mouse HER2-positive human gastric cancer cell line NCI-N87 on the right flank.

[0088] Fig.25 The graph shows changes in tumor volume when physiological saline or cetuximab-bound micelle drug complex (Example 160 or Example 161) was administered to right flank subcutaneously transplanted model mice with mouse EGFR-positive human breast cancer cell line MDA-MB-468.

[0089] Fig.26 The graph shows changes in tumor volume when physiological saline or cetuximab-bound micelle drug complex (Example 162 or Example 163) was administered to right flank subcutaneously transplanted model mice with EGFR-positive human colorectal cancer cell line HCT-116 having KRAS mutation (G13D). DETAILED DESCRIPTION

[0090] The terms used in this specification are used with the meanings commonly used in the field unless otherwise specified. Hereinafter, the present invention will be described in further detail.

[0091] In this specification, the term "nanoparticle" refers to a structure having a particle size of 100 nm or less.

[0092] In this specification, "single chain nanoparticles (SCNP)" refer to nanoparticles formed by chemical crosslinking, hydrophobic interaction, ionic bonding, etc. within a single chain as a driving force. In most cases, it means a relatively small particle size among nanoparticles of 20 nm or less.

[0093] In this specification, the "initiator" means an initiator for thermal radical polymerization such as an azo compound or a peroxide.

[0094] In the present specification, the "chain transfer agent" refers to a compound that causes a chain transfer reaction in radical polymerization, and is preferably a compound having a thiocarbonyl group.

[0095] In this specification, the term “C 1-3 The term "alkyl" refers to a linear or branched alkyl group having 1 to 3 carbon atoms, and examples thereof include methyl, ethyl, n-propyl and isopropyl.

[0096] In this specification, the term “C 1-18 The term "alkyl" refers to a linear or branched alkyl group having 1 to 18 carbon atoms, and examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl and octadecyl.

[0097] In the present specification, the term "3- to 8-membered cycloalkyl group which may have a substituent" means a cyclic alkyl group having 3 to 8 carbon atoms, and examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The substituent is not particularly limited, and examples thereof include a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, an amino group, an alkylamino group having 1 to 6 carbon atoms, a dialkylamino group having 1 to 6 carbon atoms in which the alkyl groups are the same or different, a thiol group, an alkylthio group having 1 to 6 carbon atoms, a carboxyl group, an alkoxycarbonyl group having 1 to 6 carbon atoms, and a carbamoyl group.

[0098] In this specification, the term "C 6-18 "Aryl" means a monocyclic or polycyclic aromatic hydrocarbon group, and examples thereof include phenyl, naphthyl, anthracenyl, phenanthrenyl, terphenyl, pyrene, In addition, the so-called "C 6-14The term "aryl" means a monocyclic or polycyclic aromatic hydrocarbon group, and examples thereof include phenyl, naphthyl, anthryl, phenanthryl, and the like. Substituents are not particularly limited, and examples thereof include halogen atoms, alkyl groups having 1 to 6 carbon atoms, alkenyl groups having 2 to 6 carbon atoms, alkynyl groups having 2 to 6 carbon atoms, hydroxyl groups, alkoxy groups having 1 to 6 carbon atoms, amino groups, alkylamino groups having 1 to 6 carbon atoms, dialkylamino groups having 1 to 6 carbon atoms in which the alkyl groups are the same or different, thiol groups, alkylthio groups having 1 to 6 carbon atoms, carboxyl groups, alkoxycarbonyl groups having 1 to 6 carbon atoms, and carbamoyl groups.

[0099] In the present specification, the term "5- to 10-membered heteroaryl group which may have a substituent" means a 5- to 10-membered monocyclic aromatic heterocyclic group or a condensed aromatic heterocyclic group which contains 1 to 4 heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms as atoms constituting the ring in addition to carbon atoms. Examples of the monocyclic aromatic heterocyclic group include furanyl, thienyl, pyrrolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, 1,3,4-thiadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl and tetrazolyl. Examples of the condensed aromatic heterocyclic group include benzofuranyl, benzothienyl, quinoxalinyl, indolyl, isoindolyl, isobenzofuranyl, chromanyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, quinolyl, isoquinolyl, etc. In addition, the so-called "6- to 10-membered heteroaryl group which may have a substituent" means a 6- to 10-membered monocyclic aromatic heterocyclic group or a condensed aromatic heterocyclic group containing 1 to 4 hetero atoms selected from nitrogen atoms, oxygen atoms, and sulfur atoms as atoms constituting the ring in addition to carbon atoms. Examples of the monocyclic aromatic heterocyclic group include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, etc. Examples of the condensed aromatic heterocyclic group include benzofuranyl, benzothienyl, quinoxalinyl, indolyl, isoindolyl, isobenzofuranyl, chromanyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, quinolyl, isoquinolyl, etc. Examples of the substituent include, but are not limited to, a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, an amino group, an alkylamino group having 1 to 6 carbon atoms, a dialkylamino group having 1 to 6 carbon atoms in which the alkyl groups are the same or different, a thiol group, an alkylthio group having 1 to 6 carbon atoms, a carboxyl group, an alkoxycarbonyl group having 1 to 6 carbon atoms, and a carbamoyl group.

[0100] In the present specification, examples of the "halogen atom" include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0101] In this specification, the term “C 1-7The term "alkylene bond" means an optionally substituted straight or branched alkylene group having 1 to 7 carbon atoms, and examples thereof include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, -CH(CH3)-, -C(CH3)2-, -CH(CH2CH3)-, -CH(CH3)CH2-, -CH(CH2CH2CH3)-, -CH(CH2(CH3)2)-, -C(CH3)(CH2CH3)-, -C(CH3)2CH2-, -CH(CH2CH3)CH2-, -CH(CH3)CH(CH3)-, -CH( CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH(CH2CH2CH2CH3)-, -C(CH3)(CH2CH2CH3)-, -C(CH2CH3)2-, -CH(CH2CH2CH3)CH2-, -CH(C H2(CH3)2)CH2-, -C(CH3)(CH2CH3)CH2-, -C(CH3)2CH(CH3)-, -CH(CH2CH3)CH(CH3)-, -C(CH3)2CH2CH2-, -CH(CH2CH3)CH2CH2- , -CH2CH(CH2CH3)CH2- , -CH(CH3)CH(CH3)CH2- , -CH(CH3)CH2CH(CH3)- , -CH(CH2CH2CH2CH2CH3)- , -C(CH3)(CH2CH2CH2CH3)- , -C(CH2CH3)(CH2CH2CH3)- , -C(CH2CH3)(CH2(CH3)2)- , -CH(CH2CH2CH2CH3)CH2-, -CH(CH(CH3)CH2CH3)CH2-, -CH(CH2CH(CH3)CH3)CH2-, -CH(CH2CH2(CH3)2)CH2-, -C(CH3)(CH2CH2CH3)CH2-, -C(CH2CH3)2CH2-, -CH(CH2(CH3)2)CH2-, -C(CH3)(CH2CH3)CH(CH3)-, -CH(CH2CH3)C(CH3)2-, -CH(CH2CH3)CH(CH2CH3)-, -C(CH3)2C(CH3)2- , -CH(CH2CH2CH3)CH2CH2- , -CH(CH2(CH3)2)CH2CH2- , -CH2CH(CH2CH2CH3)CH2- , -CH2CH(CH2(CH3)2)CH2- , -C(CH3)(CH2CH3)CH2CH2-, -CH(CH2CH3)CH(CH3)CH2-, -CH(CH2CH3)CH2CH(CH3)-,-CH2CH(CH2CH3)CH(CH3)-、-CH(CH3)CH(CH3)CH(CH3)- 、 -C(CH3)2CH(CH3)CH2- 、-C(CH3)2CH2CH(CH3)- 、 -CH(CH2CH3)CH2CH2CH2- 、-CH2CH(CH2CH3)CH2CH2- 、 -C(CH3)2CH2CH2CH2- 、-CH(CH3)CH(CH3)CH2CH2-、 -CH(CH3)CH2CH(CH3)CH2-、-CH(CH3)CH2CH2CH(CH3)-、 -CH(CH3)CH2CH2CH2CH2-、-CH2CH(CH3)CH2CH2CH2-、 -CH2CH2CH(CH3)CH2CH2-、-C(CH2CH3)(CH2CH2CH2CH3)- 、 -C(CH2CH2CH3)2- 、-C(CH3)(CH2CH2CH2CH3)CH2-、-C(CH3)(CH(CH3)CH2CH3)CH2-、-C(CH3)(CH2CH(CH3)CH3)CH2-、-C(CH3)(CH2CH2(CH3)2)CH2-、-CH(CH2CH2CH2CH3)CH(CH3)-、-CH(CH(CH3)CH2CH3)CH(CH3)-、-CH(CH2CH(CH3)CH3)CH(CH3)-、-CH(CH2CH(CH3)CH3)CH(CH3)-、-CH(CH2CH2(CH3)2)CH(CH3)-、-C(CH3)(CH2CH2CH3)CH(CH3)-、 -C(CH2CH3)2CH(CH3)-、-CH(CH2(CH3)2)CH(CH3)-、-C(CH3)(CH2CH3)C(CH3)2-、-C(CH3)(CH2CH2CH3)CH2CH2-、-CH(CH 2CH2CH3)CH(CH3)CH2-、-CH(CH2CH2CH3)CH2CH(CH3)-、-CH2CH(CH2CH2CH3)CH(CH3)-、-C(CH3)(CH2CH3)CH(CH3)CH2-、- C(CH3)(CH2CH3)CH2CH(CH3)-、-CH(CH2CH3)C(CH3)2CH2-、-CH(CH2CH3)CH(CH3)CH(CH3)-、-CH(CH2CH3)CHCH(CH3)2-、-C(CH3)2CH(CH2CH3)CH2-、-CH(CH3)C(CH3)(CH2CH3)CH2-、-CH(CH3)CH(CH2CH3)CH(CH3)-、-C(CH3)2CH(CH3)CH(CH3)-、-CH(CH3)C(CH3)2CH(CH3)-, -C(CH3)2CH(CH3)CH2CH2-, -C(CH3)2CH2CH(CH3)CH2-, -C(CH3)2CH2CH2CH(CH3)-, -CH(CH3)C(CH3)2CH2CH2-, -CH(CH3)CH(CH3)CH(CH3)CH2-, -CH(CH3)CH(CH3)CH2CH(CH3)-, -CH(CH3)CH(CH3)CH2CH2CH2-, -CH(CH3)CH2C H(CH3)CH2CH2-, -CH(CH3)CH2CH2CH(CH3)CH2-, -CH(CH3)CH2CH2CH2CH(CH3)-, -C(CH3)2CH2CH2CH2CH2-, or -CH(CH2CH3)CH2CH2CH2CH2-, etc. The substituent is not particularly limited and may replace any hydrogen atom. Examples thereof include a phenoxy group, a carboxylic acid group, a sulfonic acid group, a phosphoric acid group, a hydroxyl group, and a thiol group.

[0102] In this specification, the so-called "target recognition molecule" is a molecule that recognizes a marker or receptor (such as a transmembrane protein, a surface insoluble protein, or a proteoglycan, etc.) expressed on the cell surface and specifically binds to it. It is a molecule that specifically recognizes a target that is unique to or overexpressed by cancer cells required for the growth or metastasis of cancer, and examples thereof include antibodies, lipocalin (Anticalin, etc.), proteins (interferons, lymphokines, growth factors, colony stimulating factors, etc.), peptides (Luteinising-hormone releasing hormone (Luteinising-hormone releasing hormone, LHRH) receptor target peptides, EC-1 peptides, etc.) or peptidomimetics, etc. In addition to having binding specificity, the target recognition molecule may also have specific therapeutic effects such as anti-proliferation (cell proliferation inhibition, and / or cytotoxicity) activity for target cells or pathways. The target recognition molecule can be carried on the copolymer by electrostatic interaction, hydrogen bonding, hydrophobic interaction, or covalent bonding with the copolymer X of the present invention. It can be modified within the range of maintaining binding specificity, and can be bound to the copolymer X via a chemically reactive group (carboxylic acid, primary amine, secondary amine, thiol, etc.), a chemically reactive amino acid residue or its side chain (tyrosine, histidine, cysteine, lysine, etc.). Hereinafter, the copolymer X of the present invention to which the target recognition molecule is bound or carried is sometimes referred to as a "target recognition copolymer".

[0103] In this specification, the so-called "antibody" is a molecule or a functional fragment thereof that has the characteristic of immunospecifically binding to a target antigen and has a sequence from an immunoglobulin such as IgG, IgM, IgA, IgD or IgE. It includes monoclonal antibodies, chimeric antibodies, recombinant antibodies, or humanized antibodies. It only needs to contain a unique type. As antigen-binding fragments, for example, Fab regions, F(ab')2 fragments, pFc' fragments or Fab' fragments can be cited. The Fab region is composed of a constant region and a variable region from the heavy chain and light chain of an antibody. Fc fragments and Fab fragments are fragments from immunoglobulins cut by papain. F(ab')2 fragments and pFc' fragments are fragments from immunoglobulins cut by pepsin. Fab' fragments are fragments formed by reducing F(ab')2 fragments under mild conditions. As a further embodiment, it can also be used as an Fc fusion protein formed by fusing functional proteins such as receptor extracellular domains with the Fc region of immunoglobulins. In addition, it can also be used as a bispecific antibody capable of binding to two antigens or a multispecific antibody with further increased antigen binding sites. Furthermore, chemical or biological modifications can also be implemented. When using genetic recombination technology, as long as the characteristics of the original amino acid sequence are maintained, it can allow the deletion, substitution, insertion or addition of one or more amino acids simultaneously or differently. For example, it is an amino acid sequence with more than 80% homology, preferably more than 90% homology, and more preferably an amino acid sequence with more than 95% homology.

[0104] In this specification, the so-called "pharmaceutical composition" means that the active ingredient (drug, physiologically active substance) that can be used for diagnosis, prevention or treatment of a disease is supported on the copolymer X, target recognition copolymer, or drug complex of the present invention through electrostatic interaction, hydrogen bonding, hydrophobic interaction, covalent bonding, etc. As the supported form, when the copolymer X, target recognition copolymer, or drug complex forms nanoparticles, the form in which the drug is present on the particle surface or the form in which the drug is enclosed in the nanoparticles, or a combination of these forms can be cited.

[0105] One embodiment of the present invention is a copolymer or a drug conjugate in which a target recognition molecule is bonded to a copolymer X having structural units represented by the following formulae (A), (B), and (C).

[0106]

[0107] [Where R 1 , R 2 and R 3 Same or different, indicating hydrogen atoms or C 1-3 Alkyl; R 4 Represents C 1-3 Alkyl; R5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent; X 1 , X 2 and X 3 The same or different, representing oxygen atoms, sulfur atoms or NR 7 ; R 6 represents a hydrogen atom, a leaving group or a connecting group; R 7 Represents a hydrogen atom or C 1-3 alkyl; m represents an integer from 1 to 100; n represents an integer from 0 to 3].

[0108] In the copolymer X of the present invention, the structural unit (A) functions as a unit that imparts hydrophilicity, and the structural unit (B) functions as a unit that imparts hydrophobicity. In addition, the structural unit (C) functions as a scaffold for binding the effective ingredient (drug, physiologically active substance) to the copolymer X or the target recognition copolymer. The copolymer X, the target recognition copolymer or the drug complex of the present invention has the characteristic of forming SCNP in water by having these three structural units, and the formed SCNP can be precisely controlled in a microscopic scale of less than 20 nm, thereby functioning as a drug delivery carrier with high tumor aggregation.

[0109] R in the structural unit (A) 1 Represents a hydrogen atom or C 1-3 The alkyl group is preferably a hydrogen atom or a methyl group, and is preferably a hydrogen atom, an ethyl group or a propyl group, and is more preferably a hydrogen atom.

[0110] X 1 represents an oxygen atom, a sulfur atom or NR 7 , preferably an oxygen atom, a sulfur atom or NH, more preferably an oxygen atom.

[0111] m represents an integer of 1 to 100, preferably an integer of 3 to 100, and is preferably 3 to 80, more preferably 4 to 60, further preferably 4 to 40, and particularly preferably 4 to 22 from the viewpoint of imparting good hydrophilicity.

[0112] R 4 Represents C 1-3 Specifically, the alkyl group is a methyl group, an ethyl group, an n-propyl group or an isopropyl group, preferably a methyl group or an ethyl group, and more preferably a methyl group.

[0113] R in the structural unit (B) 2 Represents a hydrogen atom or C 1-3 The alkyl group is preferably a hydrogen atom or a methyl group, and is preferably a hydrogen atom, an ethyl group or a propyl group, and is more preferably a hydrogen atom.

[0114] X 2 represents an oxygen atom, a sulfur atom or NR 7 , preferably an oxygen atom, a sulfur atom or NH, more preferably an oxygen atom.

[0115] n represents an integer of 0 to 3, preferably an integer of 1 to 3, and more preferably 1.

[0116] R 5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 The aryl group or the 5- to 10-membered heteroaryl group which may have a substituent is preferably C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent, more preferably C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 An aryl group or an optionally substituted 5- to 10-membered heteroaryl group, more preferably a C 1-18 Alkyl, 3-8 membered cycloalkyl, adamantyl or C 6-18 On the other hand, a 3- to 8-membered cycloalkyl group which may have a substituent, an adamantyl group, a C 6-14 The aryl group or the 6- to 10-membered heteroaryl group which may have a substituent is preferably one or more selected from the group consisting of a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, and an alkynyl group having 2 to 6 carbon atoms.

[0117] R in the structural unit (C) 3 Represents a hydrogen atom or C 1-3 The alkyl group is preferably a hydrogen atom or a methyl group, and is preferably a hydrogen atom, an ethyl group or a propyl group, and is more preferably a hydrogen atom.

[0118] X 3 represents an oxygen atom, a sulfur atom or NR 7 , preferably an oxygen atom, a sulfur atom or NH, more preferably an oxygen atom or NH.

[0119] R 6 represents a hydrogen atom, a leaving group or a linking group. The leaving group is a group that can be detached when the structural unit (C) is combined with the drug (physiologically active substance), and the linking group is a group that can be used for cross-linking when the structural unit (C) is combined with the drug (physiologically active substance). As these leaving groups or linking groups, C which may have a substituent is preferred. 1-18Alkyl, 3- to 8-membered cycloalkyl which may have a substituent, C 7-19 Aralkyl. Here, as the substituent, for example, a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, an amino group, an alkylamino group having 1 to 6 carbon atoms, a dialkylamino group having 1 to 6 carbon atoms in which the alkyl groups are the same or different, a thiol group, an alkylthio group having 1 to 6 carbon atoms, a carboxyl group, an alkoxycarbonyl group having 1 to 6 carbon atoms, a carbamoyl group, etc. Among these groups, as the linking group, a group having a functional group such as a hydroxyl group, an amino group, a thiol group, a carboxyl group, etc. as a substituent is preferred.

[0120] As R 6 Preferred specific examples of the leaving group include groups represented by the following formula (4):

[0121]

[0122] As R 6 Preferred specific examples of the linking group include groups represented by the following formula (5):

[0123]

[0124] [Where R 8 Represents a hydrogen atom or a drug; Ak 1 Represents C 1-7 Alkylene bond; X 4 represents an oxygen atom, a sulfur atom or -N(R 7 )-(R 7 Represents a hydrogen atom or C 1-3 More preferably, X in formula (5) 4 represents an oxygen atom, a sulfur atom or NH.

[0125] The copolymer X of the present invention is a copolymer having structural units represented by formula (A), (B) and (C). The copolymer X may be a random copolymer or a block copolymer, preferably a random copolymer. The composition ratio of each structural unit in one molecule is preferably a ratio of 0.01 to 100 parts by mass and 0.1 to 100 parts by mass when (A) is 1 part by mass, more preferably a ratio of 0.05 to 18 parts by mass and 0.1 to 20 parts by mass when (A) is 1 part by mass, and particularly preferably a ratio of 0.05 to 4 parts by mass and 0.1 to 16 parts by mass when (A) is 1 part by mass.

[0126] The degree of polymerization of the copolymer X of the present invention is not particularly limited, but is preferably 5,000 to 150,000, more preferably 8,000 to 150,000 as a number average molecular weight.

[0127] In the copolymer of the present invention, as described above, the monomer represented by the general formula (1) functions as a unit imparting hydrophilicity, and the monomer represented by the general formula (2) functions as a unit imparting hydrophobicity. In addition, the monomer represented by the general formula (3) functions as a scaffold for combining drugs with the copolymer. As monomers that function as hydrophobic units represented by the general formula (2), for example, monomers represented by the following formulas can be exemplified:

[0128]

[0129]

[0130] In the general formula (1), R 1 Represents a hydrogen atom or C 1-3 The alkyl group is preferably a hydrogen atom or a methyl group, and is preferably a hydrogen atom, an ethyl group or a propyl group, and is more preferably a hydrogen atom.

[0131] In the general formula (2), R 2 Represents a hydrogen atom or C 1-3 The alkyl group is preferably a hydrogen atom or a methyl group, and is preferably a hydrogen atom, an ethyl group or a propyl group, and is more preferably a hydrogen atom.

[0132] In the general formula (3), R 3 Represents a hydrogen atom or C 1-3 The alkyl group is preferably a hydrogen atom or a methyl group, and is preferably a hydrogen atom, an ethyl group or a propyl group, and is more preferably a hydrogen atom.

[0133] In the general formula (1), R 4 Represents C 1-3 Specifically, the alkyl group is a methyl group, an ethyl group, an n-propyl group or an isopropyl group, preferably a methyl group or an ethyl group, and more preferably a methyl group.

[0134] In the general formula (1), X 1 represents an oxygen atom, a sulfur atom or NR 7 , preferably an oxygen atom, a sulfur atom or NH, more preferably an oxygen atom.

[0135] In the general formula (1), m represents an integer of 1 to 100, preferably an integer of 3 to 100, and is preferably 3 to 80, more preferably 4 to 60, further preferably 4 to 40, and particularly preferably 4 to 22 from the viewpoint of imparting good hydrophilicity.

[0136] In the general formula (2), R 5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18The aryl group or the 5- to 10-membered heteroaryl group which may have a substituent is preferably C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent, more preferably C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 An aryl group or an optionally substituted 5- to 10-membered heteroaryl group, more preferably a C 1-18 Alkyl, 3-8 membered cycloalkyl, adamantyl or C 6-18 On the other hand, 3- to 8-membered cycloalkyl groups which may have a substituent, adamantyl groups, C 6-14 The aryl group or the 6- to 10-membered heteroaryl group which may have a substituent is preferably one or more selected from the group consisting of a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, and an alkynyl group having 2 to 6 carbon atoms.

[0137] In the general formula (2), X 2 represents an oxygen atom, a sulfur atom or NR 7 , preferably an oxygen atom, a sulfur atom or NH, more preferably an oxygen atom.

[0138] In the general formula (2), n represents an integer of 0 to 3, preferably an integer of 1 to 3, and more preferably 1.

[0139] In the general formula (3), R 6 represents a hydrogen atom, a leaving group or a linking group. As these leaving groups or linking groups, preferably C 1-18 Alkyl, 3- to 8-membered cycloalkyl which may have a substituent, C 7-19 Aralkyl. Here, as the substituent, for example, a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, an amino group, an alkylamino group having 1 to 6 carbon atoms, a dialkylamino group having 1 to 6 carbon atoms in which the alkyl groups are the same or different, a thiol group, an alkylthio group having 1 to 6 carbon atoms, a carboxyl group, an alkoxycarbonyl group having 1 to 6 carbon atoms, a carbamoyl group, etc. Among these groups, as the linking group, a group having a functional group such as a hydroxyl group, an amino group, a thiol group, a carboxyl group, etc. as a substituent is preferred.

[0140] As R 6 Preferred specific examples of the leaving group include groups represented by the following formula (4):

[0141]

[0142] As R 6 Preferred specific examples of the linking group include groups represented by the following formula (5):

[0143]

[0144] [Where R 8 Represents a hydrogen atom or a drug; Ak 1 Represents C 1-7 Alkylene bond, X 4 represents an oxygen atom, a sulfur atom or -N(R 7 )-(R 7 Represents a hydrogen atom or C 1-3 More preferably, X in formula (5) 4 represents an oxygen atom, a sulfur atom or NH.

[0145] In the general formula (3), X 3 represents an oxygen atom, a sulfur atom or NR 7 , preferably an oxygen atom, a sulfur atom or NH, more preferably an oxygen atom or NH.

[0146] The copolymer X of the present invention is formed by copolymerizing three monomers represented by general formulas (1) to (3). The copolymerization may be random copolymerization or block copolymerization, and is preferably formed by random copolymerization. When the mass part of the monomer (1) is set to 1, the mixing ratio of the three monomers is preferably 0.01 to 100 mass parts of the monomer (2) and 0.1 to 100 mass parts of the monomer (3) are polymerized, more preferably 0.05 to 18 mass parts of the monomer (2) and 0.1 to 20 mass parts of the monomer (3) are polymerized, and particularly preferably 0.05 to 4 mass parts of the monomer (2) and 0.1 to 16 mass parts of the monomer (3) are polymerized.

[0147] In addition, "solvates" coordinated with various solvents are also included in the copolymer X of the present invention. In the present specification, examples of "solvates" include hydrates and ethanolates. The solvent may be coordinated with the copolymer X of the present invention in any amount.

[0148] The copolymer X of the present invention can be produced by various known methods. The production method is not particularly limited, and for example, the copolymer can be produced according to the basic polymer synthesis method described below.

[0149]

[0150] [wherein, R' represents a hydrogen atom or C 1-3 Alkyl, R" means the above R 4 , R 5 or R 6 The group represented].

[0151] This reaction represents a step of making a monomer (I) react with a chain transfer agent (II) and an initiator to produce a polymer (III). This reaction can be carried out without a solvent or in the following solvents: alcohols such as methanol, ethanol, 1-propanol, and 2-propanol; ethers such as diethyl ether, tetrahydrofuran, and 1,4-dioxane; aromatic hydrocarbons such as benzene, toluene, and xylene; halogenated hydrocarbons such as dichloromethane, chloroform, and 1,2-dichloroethane; N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, ethyl acetate, etc., preferably aromatic hydrocarbons such as toluene and xylene are used as solvents.

[0152] As the chain transfer agent, 2-(dodecylthiocarbonylthio)-2-methylpropionic acid (DDMAT), dodecyl cyanomethyl trithiocarbonate (CDTTC), 2-cyano-2-propyl dodecyl trithiocarbonate (CPDTTC), 4-cyano-4-[(dodecylthio-carbonylthio)thio]pentanoic acid (CDSPA), 2-(dodecylthiocarbonylthio)-2-methylpropionic acid 3-azido-1-propanol ester (N3-CTA), N3-PEG m Ester-CTA (m is the same as above) such as 2-(2-(2-azidoethoxy)ethoxy)ethyl 2-(dodecylthiothiocarbonylthio)-2-methylpropanoate (N3-PEG2 ester-CTA), 2-(dodecylthiothiocarbonylthio)-2-methylpropanoate 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol ester (N3-PEG5 ester-CTA), 2-(dodecylthiothiocarbonylthio)-2-methylpropanoate 23-azido-3,6,9,12,15,18,21-heptaoxatricosan-1-ol ester (N3-PEG7 ester-CTA), N3-PEG mAmide-CTA (m is the same as above), for example, N-(8-azido-3,6-dioxaoctane-1-yl)-2-(dodecylthiocarbonylthio)-2-methylpropionamide (N3-PEG2amide-CTA), N-(17-azido-3,6,9,12,15-pentaoxaheptadecan-1-yl)-2-(dodecylthiocarbonylthio)-2-methylpropionamide (N3-PEG5amide-CTA), or N-(23-azido-3,6,9,12,15,18,21-heptaoxatricosan-1-yl)-2-(dodecylthiocarbonylthio)-2-methylpropionamide (N3-PEG7amide-CTA), etc., preferably DDMAT or N3-CTA is used, and more preferably N3-CTA. When a chain transfer agent is used for polymerization, the copolymer X of the present invention may have a structure in which a part or all of the structure of the chain transfer agent is partially bonded. When the copolymer X contains the structure of the chain transfer agent, the structure may be removed by an appropriate method.

[0153] As the initiator, an azo polymerization initiator such as 2,2'-azobis-diisobutyronitrile (AIBN), 1,1'-azobis(cyclohexanecarbonitrile) (ACHN), 2,2'-azobis-2-methylbutyronitrile (AMBN), 2,2'-azobis-2,4-dimethylvaleronitrile (ADVN), or 2,2'-azobis(methyl 2-methylpropionate) (MAIB) can be used, and AIBN is preferably used.

[0154] The reaction temperature is 0 to 300° C., preferably 0 to 150° C., more preferably 1 to 100° C., and the reaction time is 1 minute to 48 hours, preferably 5 minutes to 24 hours. In this reaction, a random copolymer X can be produced by reacting in the presence of monomers (I) having different structures.

[0155] For example, when N3-CTA having an azide group is used as a chain transfer agent, a copolymer X having an azide group at the terminal can be obtained, which can be advantageous for the production of a target recognition copolymer by the click reaction described later.

[0156] The target recognition molecule bound to the copolymer X is preferably an antibody that can recognize a marker or receptor expressed on the cell surface, a variant (modified) thereof, or a functional fragment (fragmented antibody). Specifically, for example, B-cell maturation antigen (BCMA), BLyS, cancer antigen (CA)-125, CC chemokine receptor (CCR) 4, cluster of differentiation (CD) 3, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD40L, CD52, CD74, CD79b, carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 5, CEACAM6, CSAp, cytotoxic T-lymphocyte-associated antigen (CTL ... Antigen, CTLA-4), CXCR4, EGFR, EpCAM, ErbB2 (HER2), GD2, gp100, HLA-DR, insulin-like growth factor 1 receptor (IGF-1R), interleukin-6 receptor (IL-6R), cMET, MUC1, adhesion protein-4, programmed cell death protein (PD)-1, PD-L1, platelet-derived growth factor receptor (PDGFR), signaling lymphocytic activation molecule family member (SLAMF) 7, prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein (TAG)-72, tissue factor (TF), tumor necrosis factor (TNF)-1, necrosis factor, TNF)-α, TROP-2 (epithelial glycoprotein,EGP)-1), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR) 1, VEGFR2, α4 integrin, α-fetoprotein (AFP), fibrinogen, carbonic anhydrase IX (CAIX), A33, B7, CA125, CCL19, CD2, CD4, CD8, CD11A, CD14, CD15, CD16, CD18, CD23, CD32b, CD37, CD40, CD44, CD45, CD54, CD55, CD59, CD64, CD66, CD70, CD80, CD95, CD138, CD147, CD154, CD276, EGFRvIII, fibroblast growth factor (FGF), Flt-1, FRα, high mobility group protein (HMG-1 groupbox, HMGB)-1, IL-4R, IL-12, IL-15, IGF-1, IGF-2, Macrophage Migration Inhibitory Factor (MIF), TRAG-3, Monocyte Chemoattractant Protein (MCP)-1, CD67, CD70L, CD79a, CD132, CD133, Cell Division Cycle (CDC) 27, Cyclin-dependent kinases (CDK)-4 / m, CDKN2A, CXCR7, CXCL12, Hypoxia Inducible Factor (HIF)-1α, EGP-2, Insulin-like growth factor (IGF-1α), factor, ILGF)-1R, SAGE, S100, anti-apoptotic protein, anti-apoptotic protein-2B, TAC, tenascin, TRAIL-R, Tn antigen, Thomsen-Friedenreich antigen, WT-1, bcl-2, bcl-6, or Kras as one or more of the antibodies or their variants (modified) or functional fragments.

[0157] Among them, preferred targets include, for example, antibodies or variants (modified) thereof or functional fragments thereof targeting one or more of CD20, CD276, MUC1, EGFR, HER2, PD-L1 and TROP-2.

[0158] Preferred individual antibodies include, for example, alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), ramucirumab (anti-VEGFR2), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), panitumumab (anti-EGFR), nexitumomab (anti-EGFR), ervantumab (anti-EGFR / cMET), rituximab (anti-CD20), ibritumomab tiuxetan (anti-CD20), tositumomab (anti-CD20), ofatumumab (anti-CD20), mosunetuzumab (anti-CD20 / CD3), trastuzumab (anti-ErbB2), pertuzumab (anti-HER 2), magituximab (anti-HER2), patritumab (anti-HER3), lambrolizumab (anti-PD-1), nivolumab (anti-PD-1), pembrolizumab (anti-PD-1), cemiplizumab (anti-PD-1), dostarlimab (anti-PD-1), toripalimab (anti-PD-1), atezolizumab (anti-PD-L1), avelumab (anti-PD-L1), durvalumab (anti-PD-L1), ipilimumab (anti-CTLA-4), avagomomab (anti-CA-125), adelimumab (anti-EpCAM), belantamab ( belantamab (anti-BCMA), mogamulizumab (anti-CCR4), catumaxomab (anti-CD3 / EpCAM), edrecolomab (anti-EpCAM), blinatumomab (anti-CD19 / CD3), tafasitamab (anti-CD19), loncastuximab (anti-CD19), intuzumab (anti-CD22), mositumomab (anti-CD22), brentuximab (anti-CD30), polatuzumab (anti-CD79b), dactuximab (anti-CD22), Monoclonal antibody (anti-GD2), naxitamab (anti-GD2), tebentafusp (anti-gp100 / CD3), enfortumab (anti-fibronectin-4), olaratumab (anti-PDGFR), elotuzumab (anti-SLAMF7), tisotumab (anti-TF), sacizumab (anti-TROP-2), tocilizumab (also known as atlizumab: anti-IL-6 receptor), obinutuzumab (also known as GA101: anti-CD20), CC49 (anti-TAG-72),AB-PG1-XG1-026 (anti-PSMA, U.S. Patent No. 8,114,965, deposited as ATCC PTA-4405 and PTA-4406), D2 / B (anti-PSMA, International Publication No. 2009 / 130575), daclizumab (anti-CD25), muromonab-CD3 (anti-CD3), natalizumab (anti-α4 integrin), infliximab (anti-TNF-α), certuin (anti-TNF-α), adalimumab (anti-TNF-α), pegol-dapirolizumab (anti-TNF-α), and dapirolizumab (anti-CD25). pegol (anti-CD40L), letolizumab (anti-CD40L), rulizumab (anti-CD40L), belimumab (anti-BLyS), 59D8 (anti-fibrinogen), biciromab (also known as T2G1s: anti-fibrinogen), MH1 (anti-fibrinogen), felzartamab (anti-CD38), isatuximab (anti-CD38), daratumumab (anti-CD38), hR1 (anti-I GF-1R, U.S. Publication No. 2010 / 226,884), clituzumab (anti-MUC1), gatuzumab (anti-MUC1), veltuzumab (also known as hA20: anti-CD20, U.S. Patent No. 7,151,164), hA19 (anti-CD19, U.S. Patent No. 7,109,304), hIMMU31 (anti-AFP, U.S. Patent No. 7,300,655), milatuzumab (also known as hLL1 (anti-CD74, U.S. Patent No. 7 , 312,318), epratuzumab (also known as hLL2: anti-CD22, U.S. Patent No. 7,074,403), hMu-9 (anti-CSAp, U.S. Patent No. 7,387,773), hL243 (anti-HLA-DR, U.S. Patent No. 7,612,180), labetuzumab (also known as hMN-14: anti-CEACAM5, U.S. Patent No. 6,676,924), hMN-3 and hMN-15 (anti-CEACAM6, U.S. Patent No. 6,676,924), U.S. Patent No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Patent No. 7,138,496), G250 (anti-CAIX), A33 (anti-A33), galiximab (anti-B7), OC125 (anti-CA125), abagovomab (anti-CA125), CAP-100 (anti-CCL19), TRX-3 (anti-CD2), IT-1208 (anti-CD4), ranolizumab (anti-CD4), crefmirlimab (anti-CD8),Afelimomab (anti-CD11A), Cytolin (anti-CD11A), efalizumab (anti-CD11A), odelimab (anti-CD11A), atibuclimab (anti-CD14), fanolesomab (anti-CD15), GTB-4550 (anti-CD16), ererumab (anti-CD18), odelimab (anti-CD18), rovizumab (anti-CD18), ruximab (anti-CD23), obexelimab (anti-CD32b), BI-1206 (anti-CD32b), HuMax-CD32b (anti-CD32b), N VS-32b (anti-CD32b), NNV-003 (anti-CD37), lilotomab (anti-CD37), K7153A (anti-CD37), iscalimab (anti-CD40), ChiLob7 / 4 (anti-CD40), CDX-1140 (anti-CD40), TNX-1500 (anti-CD40L), TES-23 (anti-CD44), bivatuzumab (anti-CD44), Actimab-B (anti-CD45), BI-505 (anti-CD54), enlimomab (anti-CD54), MOR- 101 (anti-CD54), MOR-102 (anti-CD54), Onyvax-105 (anti-CD55), GB-262 (anti-CD55), PAT-SC1 (anti-CD55), VG-102 (anti-CD55), AR36A36.11.1 (anti-CD59), KNP-302 (anti-CD59), MDX-210 (anti-CD64), MDX-220 (anti-CD64), tinurilimab (anti-CD66c), cusatuzumab (anti-CD70), MDX-1411 (anti-CD70), cosibelimab (anti-PD-L1), Galiximab (anti-CD80), Novotarg (anti-CD95), DOM-1112 (anti-CD138), indatuximab (anti-CD138), gavitumomab (anti-CD147), lelizumab (anti-CD154), ABI-793 (anti-CD154), DOM-0800 (anti-CD154), ifenatumumab (anti-CD276), mizotuzumab (anti-CD276), bolamituzumab (anti-CD276), AMG-596 (anti-EGFRvIII), bemarituzumab (anti-FGF), burosumab (anti-FGF),U3-1784 (anti-FGF), aprutumab (anti-FGF), icrucumab (anti-Flt-1 / VEGFR), farletuzumab (anti-FRα), mirvetuximab (anti-FRα), girentuximab (anti-CAIX), MEDI-541 (anti-HMGB-1), dupilumab (anti-VEGFR), mab (anti-IL-4R), levilimab (anti-IL-6R), SANT-7 (anti-IL-6R), vobarilizumab (anti-IL-6R), sarilumab (anti-IL-6R), clazakizumab (anti-IL-6R), TZLS-501 (anti-IL-6R), ustekinumab (anti-IL-12), briakizumab (anti-IL-6R), numab (anti-IL-12), ordesekimab (anti-IL-15), cixutumumab (anti-IGF-1), figitumumab (anti-IGF-1), teprotumumab (anti-IGF-1), dalotuzumab (anti-IGF-1), ganitumab (anti-IGF-1), rovatumab (anti-IGF-1), AVE1642 (anti-IGF-1), dusitumab (anti-IGF-1 / 2), Estilab (anti-IGF-1), xentuzumab (anti-IGF-1), imalumab (anti-MIF), relatlimab (anti-TRAG-3), carlumab (anti-MCP-1), alacizumab pegol) (anti-VEGFR-2), brolucizumab (anti-VEGFR), gentuximab (anti-VEGFR), olinvacimab (anti-VEGFR-2), etc.

[0159] The linking group bound to the copolymer X may be any group that can link (interact) the copolymer X with the drug or target recognition molecule, and may be an amino acid residue, a bifunctional derivative, a bond utilizing a bioorthogonal reaction, an alkylene bond, a polyethylene glycol (PEG) bond, a disulfide bond, or a thioether bond.

[0160] As a linker having a cleavage site using a protease (e.g., a cathepsin B cleavage site, a cathepsin C cleavage site, or a cathepsin D cleavage site), for example, a peptide whose amino acid residues include alanine, phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, or aspartic acid can be cited. For example, dipeptides, tripeptides, tetrapeptides, or pentapeptides can be natural amino acid residues or amino acid residues that do not exist in nature. As such examples, valine-citrulline (ve or val-cit), valine-alanine (va or val-ala), valine-lysine (val-lys), phenylalanine-alanine (phe-ala), phenylalanine-lysine (fk or phe-lys), phenylalanine-citrulline (phe-cit), phenylalanine-phenylalanine-lysine (phe-phe-lys), alanine-phenylalanine (af or ala-phe), alanine-lysine (ala-lys), glycine-glycine (gly-gly), glycine-alanine-phenylalanine (gly-ala-phe) can be cited. ), glycine-valine-citrulline (gly-val-cit), glycine-glycine-glycine (gly-gly-gly), glycine-phenylalanine-lysine (gly-phe-lys), glycine-phenylalanine-leucine-glycine (gly-phe-leu-gly), glycine-glycine-phenylalanine-glycine (gly-gly-phe-gly), leucine-citrulline (leu-cit), isoleucine-citrulline (ile-cit), tryptophan-citrulline (trp-cit), or alanine-leucine-alanine-leucine (ala-leu-ala-leu), etc.

[0161] Furthermore, examples of linkers such as carbamate groups / carbonate groups formed with drugs or antibodies via peptide-p-aminobenzyl alcohol ("peptide-PAB") include valine-citrulline-p-aminobenzyl carbamate, maleimidocaproyl-p-aminobenzyl carbamate, maleimidocaproyl-phenylalanine-lysine-p-aminobenzyl carbamate, or maleimidocaproyl-valine-citrulline-p-aminobenzyl carbamate.

[0162] Examples of the bifunctional derivatives include N-[β-maleimidopropoxy]succinimide ester (BMPS), [N-ε-maleimidocaproyloxy]succinimide ester (EMCS), N-[γ-maleimidobutyryloxy]succinimide ester (GMBS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), [N-ε-maleimidocaproyloxy]sulfosuccinimide ester (sulfo-EMCS), N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GMBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), N-succinimide -3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP), iminothiolane (IT), imide esters (dimethyl diimide adipate HCl, etc.), active esters (disuccinimidyl suberate, etc.), aldehydes (glutaraldehyde, etc.), bis-azido compounds (bis(p-azidobenzoyl)hexanediamine, etc.), bis-diazonium derivatives (bis-(p-diazoniumbenzoyl)-ethylenediamine, etc.), diisocyanates (toluene 2,6-diisocyanate, etc.), his-active fluorine compounds (1,5-difluoro-2, 4-dinitrobenzene, etc.), dibenzocyclooctyne (DBCO)-N-hydroxysuccinimidyl (NHS) ester, DBCO-C6-NHS ester, DBCO-sulfo-NHS ester, DBCO-PEG4-NHS ester, DBCO-PEG5-NHS ester, sulfoDBCO-tetrafluorophenyl (TFP) ester, sulfoDBCO-PEG4-TFP ester, DBCO-PEG5-TFP ester, DBCO-sulfotetrafluorophenyl (STP) ester, DBCO Acid, DBCO-C6-acid, DBCO-PEG5-acid, DBCO amine, DBCO-PEG4-amine, sulfoDBCO-amine, DBCO maleimide, sulfoDBCO-maleimide, DBCO-PEG4-maleimide, bicyclo[6.1.0]nonyne (BCN)-PEG3-Val-Cit, DBCO-PEG4-Val-Cit-PAB-p-nitrophenol (PNP), or trans-cyclooctene (TCO)-PEG4-Val-Cit-PAB-PNP, etc.

[0163] In addition, for example, triazole or oxime / hydrazone bonds formed by bioorthogonal reactions such as the Huisgen reaction of an azide group and an alkyne can be mentioned.

[0164] Furthermore, an alkylene bond, a polyethylene glycol (PEG) bond, a disulfide bond, a thioether bond, or the like can be used alone or in combination.

[0165] As a preferred combination, for example, the following formula (a) can be cited:

[0166]

[0167] [Where J 1 It is the binding part with the target recognition molecule or drug; 2 is the bonding part with copolymer X; Ak 2 ,Ak 3 Each independently represents a single bond or C 1-7 Alkylene bond; B 1 , B 2 Each independently represents a single bond, an amide group or an ester bond; L 1 Represents a single bond, -(CH2CH2O) o CH2CH2-, phenylene, cyclohexylene, -NH-peptide-CO-, or phenylene-NH-peptide-CO-; o represents an integer from 0 to 100].

[0168] In the general formula (a), J 1 The binding site for the target recognition molecule or drug is -CO-, -S-, -CO-O-, -CO-Ak 4 -O-, or the following formula (a'):

[0169]

[0170] [Where, * 1 Indicates binding to target recognition molecules or drugs, * 2 Indicates that Ak 2 combination of].

[0171] In the general formula (a), Ak 2 ,Ak 3 ,Ak 4 Each independently represents a single bond or C 1-7 Alkylene bond.

[0172] In the general formula (a), B 1 , B 2 Each independently represents a single bond, an amide group, or an ester bond.

[0173] In the general formula (a), L 1 Represents a single bond, -(CH2CH2O) oCH2CH2-, phenylene, cyclohexylene, -NH-peptide-CO-, or phenylene-NH-peptide-CO-, and o represents an integer of 0 to 100. The peptide has 2 to 5 amino acid residues.

[0174] In the general formula (a), J 2 is a bonding part with the copolymer X, and represents a single bond, a triazole-containing bond formed by Huisgen reaction of an azide group and an alkyne-containing functional group, or the following formula (a"):

[0175]

[0176] [Where, * 3 Indicates the combination with copolymer X, * 4 Indicates that Ak 3 combination of].

[0177] As a more preferred combination, the bond between the antibody and the copolymer X includes "a triazole-containing bond formed by Huisgen reaction of an azide group and an alkyne-containing functional group", which can be exemplified by the following formula (b):

[0178]

[0179] [Where J 3 It is the binding part with the target recognition molecule; Ak 6 Represents a single bond, or C 1-7 Alkylene bond; B 3 represents a single bond, an amide group, or an ester bond; J 4 represents a bond portion containing a triazole formed by Huisgen reaction of an azide group and an alkyne-containing functional group; p and q each independently represent an integer of 0 to 100].

[0180] In the general formula (b), J 3 is the linking part with the target recognition molecule and represents -CO-, or the following formula (b'):

[0181]

[0182] [Where, * 5 Indicates binding to target recognition molecules, * 6 Indicates that Ak 6 combination of].

[0183] In the general formula (b), Ak 6 Represents a single bond, or C 1～7 Alkylene bond.

[0184] In the general formula (b), B 3 , B 4 Each independently represents a single bond, an amide group, or an ester bond.

[0185] In the general formula (b), p and q each independently represent an integer of 0-100, preferably an integer of 1-50, and more preferably an integer of 2-25.

[0186] In the general formula (b), J 4 The bonding part with the copolymer X is a triazole bond formed by Huisgen reaction of an azide group and an alkyne-containing functional group, and examples thereof include the following formula (b"):

[0187]

[0188] [Where, * 7 represents the combination with -CO- of general formula (b), * 8 represents the combination with copolymer X].

[0189] Furthermore, more preferred combinations of the target recognition molecule and the copolymer X include, for example, linking groups represented by the following formulae (6) to (13):

[0190]

[0191] [Where, * 7 Indicates binding to target recognition molecules, * 8 represents the combination with copolymer X].

[0192] In addition, as a preferred combination of the drug and the copolymer X, for example, the following formula (c) can be cited:

[0193]

[0194] [Where J 5 J is the bonding part with copolymer X; 6 It is the binding part with the drug; Ak 7 ,Ak 8 Each independently represents a single bond, or C 1-7 Alkylene bond; B 5 represents a single bond, an amide group, or an ester bond; L 2 represents a single bond, phenylene, cyclohexylene, -CO-peptide-NH-, or -CO-peptide-NH-phenylene].

[0195] In the general formula (c), J 5 is the bonding part with the copolymer X, and represents a single bond, or the following formula (c'):

[0196]

[0197] [Where, * 9 Indicates the combination with copolymer X, * 10 Indicates that Ak7 combination of].

[0198] In the general formula (c), Ak 7 ,Ak 8 ,Ak 9 ,Ak 10 Each independently represents a single bond, or C 1-7 Alkylene bond.

[0199] In the general formula (c), B 5 represents a single bond, an amide group, or an ester bond.

[0200] In the general formula (c), L 2 represents a single bond, phenylene, cyclohexylene, -CO-peptide-NH-, or -CO-peptide-NH-phenylene. The peptide has 2 to 5 amino acid residues.

[0201] In the general formula (c), J 6 The binding site with the drug represents -CO-, -S-, -O-CO-, -O-Ak 10 -CO-, or the following formula (c"):

[0202]

[0203] [Where, * 11 Indicates the combination with the drug, * 12 Indicates that Ak 8

[0063] preferably comprises a sulfur atom from the drug and a "disulfide bond".

[0204] Furthermore, more preferred combinations of the drug and the copolymer X include, for example, linking groups represented by the following formulae (14) to (22):

[0205]

[0206]

[0207] [Where, * 13 Indicates the combination with copolymer X, * 14 indicates combination with drug].

[0208] When the compound of the present invention exists in geometrical isomers or optical isomers, mixtures or separated forms of these isomers are also included in the scope of the present invention. Separation of isomers can be carried out by conventional methods.

[0209] The target recognition copolymer of the present invention can be produced by various known methods. The production method is not particularly limited, and for example, it can be produced according to the synthesis method of the click reaction described below.

[0210]

[0211] [wherein, R' represents a hydrogen atom or C 1-3 Alkyl; R" represents the above R 4 , R 5 or R 6 The group represented by [Ab represents the target recognition molecule].

[0212] The SH group of the target recognition molecule is obtained by reducing the disulfide bond between the cysteine ​​residues connecting the chain. As a reducing agent, for example, tricarboxyethylphosphine (TCEP), 2-mercaptoethanol, 2-mercaptoethylamine, cysteine ​​hydrochloride, dithiothreitol, or salts thereof (e.g., hydrochloride) can be cited. This method only requires mixing a solution containing the target recognition molecule with a solution containing a reducing agent. In addition to generating a partially reduced antibody, it can be reacted with a linker having a functional group that reacts with the SH group of the target recognition molecule to generate a linker-bound (modified) target recognition molecule. The concentration of the target recognition molecule in the reaction is, for example, 1 mg / mL to 100 mg / mL. The concentration of the reducing agent is, for example, 1 mM to 100 mM, and can be mixed in an excess amount relative to the target recognition molecule. It can be used in a range of 1 to 50 times molar equivalent, for example, 2 to 30 times molar equivalent, 5 to 20 times molar equivalent, 7 to 13 times molar equivalent or 10 times molar equivalent relative to the target recognition molecule. In addition, the protein can be heated to a degree of denaturation, for example, in a range of 1 to 37°C, and the reaction time can be adjusted according to the amount of the reducing agent. For example, it is about several seconds to 5 minutes, several seconds to 2 minutes, preferably 1 minute to 5 minutes, and more preferably about 1 minute to 2 minutes.

[0213] In the case of implementing the complexation of the target recognition molecule and SCNP using a covalent bond, a functional group for complexation reaction can be introduced to the surface of the SCNP, which is complexed by reacting with the SH group, amino group, or carboxyl group of the end or side chain of the target recognition molecule that can react with the functional group. In addition, a spacer can be introduced to the end or side chain of the target recognition molecule by an appropriate cross-linking reagent (cross-linking agent) to form a covalent bond with the SCNP as a modified target recognition molecule. As such bond formation, for example, the click chemistry of an azide group and an alkyne, the reaction of a sulfhydryl group and a maleimide group, and the reaction of an amino group and a succinimide group can be cited.

[0214] "Click chemistry" is classified as a reaction similar to a natural biochemical reaction with the following properties, which has the following characteristics. It is a very effective reaction that can be carried out quickly to a high yield. In addition, it is a reaction that does not generate byproducts at all (or almost) and allows very high selectivity of multiple functional groups. Furthermore, it is a reaction carried out under mild reaction conditions such as low temperature (or ambient) or in an aqueous solution. This reaction can be carried out in the following solvents: water or alcohols such as methanol, ethanol, 1-propanol, 2-propanol; ethers such as diethyl ether, tetrahydrofuran, 1,4-dioxane; aromatic hydrocarbons such as benzene, toluene, and xylene; halogenated hydrocarbons such as dichloromethane, chloroform, 1,2-dichloroethane; N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, ethyl acetate, etc.; preferably in water or using N,N-dimethylformamide as a solvent. In some embodiments, cyclooctyne is dibenzocyclooctyne (DBCO), difluorobenzocyclooctyne (DIFBO), biaryl azacyclooctyne ketone (BARAC), dibenzocyclooctyne (DIBO), difluorinated cyclooctyne (DIFO), monofluorinated cyclooctyne (MOFO), dimethoxy azacyclooctyne (DIMAC) or aryl-less octyne (ALO), preferably dibenzocyclooctyne (DBCO). In some embodiments, the alkyne is an aliphatic alkyne, and the step of reacting is carried out in the presence of a copper (I) catalyst. In some embodiments, the alkyne is cyclooctyne, and the step of reacting is carried out under copper-free conditions. The reaction temperature is 0 to 300°C, preferably 0 to 150°C, more preferably 1 to 100°C, and the reaction time is 1 minute to 48 hours, preferably 5 minutes to 24 hours.

[0215] The reaction in the above reaction formula is a Huisgen cycloaddition reaction, which is a type of click reaction and is a 1,3-dipolar addition cyclization reaction of an azide group and an alkyne to form a 1,2,3-triazole.

[0216] The polymer X, target recognition copolymer, and target recognition micellar drug complex produced by the present invention can be purified by a separation and purification method of a polymer generally known in the field of polymer chemistry. Specifically, for example, extraction, recrystallization, salting out using ammonium sulfate or sodium sulfate, centrifugal separation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reverse phase chromatography, desalting column chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or a combination of these treatment operations can be cited. In particular, in hydrophobic chromatography, the retention time varies depending on the number of SCNPs bound to the target recognition molecule, so any component that satisfies the DAR can be recovered by fractionation preparation.

[0217] The copolymer X and target recognition copolymer of the present invention can be used as carriers for delivering various physiologically active substances (drugs). For example, the copolymer X of the present invention, the target recognition copolymer, or the pharmaceutical composition formed by carrying (encapsulating) a tumor therapeutic drug in a target recognition micelle drug complex can inhibit the growth of tumors as confirmed in the following test examples, and therefore can be used as a preventive and / or therapeutic agent for various cancer diseases such as colorectal cancer, duodenal cancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer, uterine cancer, and ovarian cancer. In addition, due to the high tumor aggregation ability, it can be used as a diagnostic agent and contrast agent for tumors.

[0218] When the copolymer and target recognition copolymer of the present invention are used as drug delivery carriers, the dosage and number of administrations can be appropriately selected in consideration of the administration form, the patient's age, weight, the nature or severity of the symptoms to be treated, etc. The dosage and number of administrations should not be limited. However, when the polymer encapsulating the drug or the target recognition micelle drug complex is intravenously injected by injection, for example, in one administration, it is preferred to administer 0.12 mg to 12,000,000 mg, more preferably 1.2 mg to 1,200,000 mg, and particularly preferably 12 to 120,000 mg.

[0219] The pharmaceutical composition of the present invention can be manufactured by mixing the copolymer X of the present invention, the target recognition copolymer, or the target recognition micelle drug complex with a drug. Preferably, the copolymer of the present invention, the target recognition copolymer, or the target recognition micelle drug complex is mixed with a drug to manufacture single-chain nanoparticles, or after manufacturing the single-chain nanoparticles of the copolymer X of the present invention, the target recognition copolymer, or the target recognition micelle drug complex. The single-chain nanoparticle can be manufactured by a known method.

[0220] In the pharmaceutical composition of the present invention, the drug may be supported on the copolymer X, the target-recognizing copolymer, or the target-recognizing micelle-drug complex by electrostatic interaction, hydrogen bonding, hydrophobic interaction, covalent bonding, or the like.

[0221] The drug is preferably an anticancer agent, more preferably an anticancer agent that acts on cancer cells to inhibit the growth of cancer cells, and examples thereof include metabolic antagonists, alkylating agents, anthracyclines, antibiotics, mitotic inhibitors, topoisomerase inhibitors, proteasome inhibitors, and antihormonal agents.

[0222] Examples of metabolic antagonists include azathioprine, 6-mercaptopurine, 6-thioguanine, fludarabine, pentostatin, cladribine, 5-fluorouracil (5FU), floxuridine (FUDR), cytarabine, methotrexate, trimethoprim, pyrimethamine, or pemetrexed. Examples of alkylating agents include cyclophosphamide, dichloromethyldiethylamine, uramustine, melphalan, chlorambucil, thiotepa / chlorambucil, ifosfamide, carmustine, lomustine, streptozotocin, busulfan, dibromomannitol, cisplatin, carboplatin, nedaplatin, oxaliplatin, miriplatin, satraplatin, triplatin tetranitrate, procarbazine, hexamethylmelamine, dacarbazine, mitozolamide, trabectedin, or temozolomide. Examples of anthracyclines include daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, aclarubicin, amrubicin, pirarubicin, etc. Examples of antibiotics include actinomycin, bleomycin, mithramycin, anthramycin, streptozotocin, gramicidin D, staurosporine, mitomycins (e.g., mitomycin C), duocarmycins (e.g., CC-1065), and calicheamicins. Examples of mitotic inhibitors include maytansines (e.g., DMO, maytansine (also known as DM1), DM2, DM3, DM4, or maytansine), auristatins (e.g., auristatin E, auristatin phenylalanine phenylenediamine (AFP), monomethyl auristatin E, monomethyl auristatin D, and monomethyl auristatin F), dolastatins, cryptophycins, vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), taxanes (e.g., paclitaxel, docetaxel), and colchicines. Examples of topoisomerase inhibitors include irinotecan, topotecan, noginotecan, amsacrine, etoposide, teniposide, mizantrone, mitoxantrone, SN-38, exitecan, and derutecan.

[0223] Examples of proteasome inhibitors include peptidylboronic acid, carfilzomib, bortezomib, etc. Examples of antihormonal agents include fulvestrant, tamoxifen, toremifene, etc. When these drugs are prepared as the pharmaceutical composition of the present invention, one or more of them may be used, and the drug may be carried on the copolymer in the form of a free body.

[0224] The administration route of the pharmaceutical composition of the present invention is preferably the most effective route for treatment, and can be administered in the form of oral administration preparations, injections or non-oral administration preparations such as transdermal administration preparations, for example, preferably intra-arterial injection, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection and other non-oral administration, more preferably intra-arterial injection and intravenous injection. The number of administrations should not be limited, but for example, an average of 1 to several times a week can be cited.

[0225] Various preparations suitable for the route of administration can be produced by appropriately selecting pharmaceutical additives such as excipients, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, antiseptics, flavoring agents, analgesics, stabilizers, isotonic agents, etc., which are commonly used in preparations, and the like, and by conventional methods.

[0226] The preparation additives that can be included in the above-mentioned various preparations are not particularly limited as long as they are pharmaceutically acceptable substances. Examples of such preparation additives include purified water, water for injection, distilled water for injection, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymers, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerol, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, etc. The additives used can be appropriately selected according to various preparations and used alone or in combination.

[0227] In addition, injections can also be prepared as non-aqueous diluents (e.g., polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), suspending agents or emulsions. The sterilization of injections can be carried out by using a filter for sterilization, a bactericide, etc. In addition, injections can be manufactured in a form prepared for use. That is, a sterile solid composition can be made by freeze drying, etc., and dissolved in water for injection, distilled water for injection, or other solvents before use.

[0228] Example

[0229] The present invention will be further specifically described below by way of examples. These examples are provided for illustration purposes only and are not intended to limit the embodiments of the present invention.

[0230] [Example 1] Preparation of Poly[(Benzyl Acrylate)-(Poly(Ethylene Glycol) Methyl Ether Acrylate)-(1-Ethoxyethyl Acrylate) Copolymer]

[0231] (1) Synthesis of 1-ethoxyethyl acrylate (EEA)

[0232] Under argon atmosphere, weigh ethyl vinyl ether (28.725 mL), add phosphoric acid (50 mg) while cooling in an ice bath. Then, add acrylic acid (17.15 mL) and stir at room temperature for 48 hours. Add hydrotalcite (3 g) and stir for another 2 hours to stop the reaction. After filtering with diatomaceous earth, remove the unreacted ethyl vinyl ether by evaporation. Add phenothiazine as an inhibitor in a manner to 500 ppm, and perform vacuum distillation with calcium hydride to purify (distillation temperature 28-32°C). The obtained 1-ethoxyethyl acrylate is divided into a glass vial and stored at -30°C.

[0233] 13 C NMR (400MHz, CDCl3), δ, ppm: 15.29(-OCH2CH3), 21.16(-COOCH(CH3)), 64.98(-OCH2-), 96.73(-COOCH(CH3)), 128.84(CH2CH-), 131.43(CH2CH-), 166.00(-COO).

[0234] (2) Synthesis of Poly[(Benzyl acrylate)-(Poly(ethylene glycol) methyl ether acrylate)-(1-ethoxyethyl acrylate) Copolymer]

[0235] 100 mg of 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid (DDMAT) was weighed and dissolved in 17.3 mL of toluene to prepare a DDMAT / toluene stock solution (DDMAT concentration was 5.78 mg / mL). Similarly, 22 mg of 2,2'-azobis(2-methylpropionitrile) (AIBN) was weighed and dissolved in 17.3 mL of toluene to prepare an AIBN / toluene stock solution (AIBN concentration was 1.27 mg / mL). In addition, 1.296 g of poly(ethylene glycol) methyl ether acrylate (mPEGA, the average value (n) of the number of repeats of ethylene glycol was 9), 0.394 g of benzyl acrylate (BnA), 0.039 g of 1-ethoxyethyl acrylate, 1.73 mL of the DDMAT / toluene stock solution, and 1.73 mL of the AIBN / toluene stock solution were added, and polymerization was carried out in an oil bath at 70°C. After 90 minutes, the polymerization was stopped, and the reaction solution was then dialyzed against methanol to recover the copolymer. The copolymer obtained was basically a viscous body, so in the reprecipitation method, the reaction solution was added dropwise to a centrifuge tube to which a poor solvent (hexane / ethyl acetate = 7 / 3 [v / v]) was added, and recovered by centrifugal separation (2,000×g, 5 min). This operation was repeated 3 times, and finally vacuum dried to obtain 1.223 g of poly[(benzyl acrylate)-(poly(ethylene glycol) methyl ether acrylate)-(1-ethoxyethyl acrylate) copolymer].

[0236] The obtained copolymer was measured by NMR. 1 The H-NMR spectrum shows the degree of polymerization and number average molecular weight (M n,NMR ) was analyzed, and the results showed that the degree of polymerization of mPEGA (n = 9) was 102, the degree of polymerization of BnA was 94, the degree of polymerization of EEA was 9, and the degree of polymerization of M n,NMR The molecular weight dispersion (M w / M n ) was measured and the result was 1.53.

[0237]

[0238] [Measurement equipment and conditions, etc.]

[0239] (1) 1 H-NMR measurement

[0240] Device: JNM-ECX400 (400MHz) / JEOL

[0241] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0242] Sample concentration: 20mg / mL

[0243] Measurement temperature: 25℃

[0244] Total times: 256 times

[0245] result: Figure 1

[0246] (2) GPC measurement

[0247] Equipment: High performance liquid chromatography (HPLC) - Prominence system / Shimadzu Corporation

[0248] Detector: RID-10A refractive index detector / Shimadzu Corporation

[0249] Chromatographic column: TSKgelα-2500 chromatographic column / Tosoh

[0250] (Column size: 7.8 mm × 300 mm, particle size: 7 μm, exclusion limit molecular weight: 5 × 10 3 )

[0251] TSKgelα-4000 Column / Tosoh

[0252] (Column size: 7.8 mm × 300 mm, particle size: 10 μm, exclusion limit molecular weight: 4 × 10 5 )

[0253] TSKgel Guard Column / Tosoh

[0254] Mobile phase: N,N-dimethylformamide (DMF) containing 10 mmol / L lithium bromide

[0255] Temperature: 40℃

[0256] Flow rate: 0.5mL / min

[0257] Sample concentration: 6mg / mL

[0258] Standard material: Poly(methyl methacrylate) standard ReadyCal set, M p 800-2,200,000Da / SIGMA

[0259] result: Figure 2

[0260] [Table 1]

[0261]

[0262] [Examples 2 to 68]

[0263] The same method as in Example 1 was used by appropriately changing the types, addition amounts, reaction temperatures, and polymerization times of the monomers (mPEGA, BnA, and EEA) used in Example 1 to produce polymers having different composition ratios or average molecular weights as shown in the following table.

[0264] [Table 2]

[0265]

[0266]

[0267] [Table 3]

[0268]

[0269]

[0270] [Example 69]

[0271] Production of poly[(benzyl acrylate)-(poly(ethylene glycol) methyl ether acrylate)-(acrylic acid) copolymer]

[0272] The poly[(benzyl acrylate)-(poly(ethylene glycol) methyl ether acrylate)-(1-ethoxyethyl acrylate) copolymer] obtained in Example 1 was treated with 0.5N HCl at room temperature to remove the ethoxyethyl group, thereby obtaining 1.176 g of a terpolymer having a carboxyl group. The Z average particle size and polydispersity index of the obtained terpolymer in water were measured by dynamic light scattering (DLS), and the results were 8.5 nm (polydispersity index 0.14).

[0273]

[0274] [Measurement equipment and conditions, etc.]

[0275] (1) DLS assay

[0276] Device: Zetasizer NanoZS / Malvern Instruments Ltd.

[0277] Measurement temperature: 25℃

[0278] Sample concentration: 10mg / mL

[0279] result: Figure 3

[0280] [Example 70]

[0281] Method for producing SCNPs encapsulated with (1,2-diaminocyclohexane) platinum (II)

[0282] Dissolve 65.28 mg of the Cl(H2O) form (DACHPt·Cl·H2O) of (1,2-diaminocyclohexane)platinum(II) (hereinafter abbreviated as DACHPt) in 20 mL of purified water and stir at 70°C for 2 hours. To 5 mL of this solution, add 287.4 mg of the terpolymer obtained in Example 69 and stir at 50°C overnight. After the stirring, the reaction solution was dialyzed and purified using purified water as the external liquid to obtain 5 mL of DACHPt-encapsulated SCNPs. The Pt content of the purified DACHPt-encapsulated SCNPs was measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES), which was 720 μg / mL (1.14 mg / mL in terms of DACHPt). In addition, 200 μL of DACHPt-encapsulated SCNPs were freeze-dried, and the solid content concentration was calculated. The ratio with the Pt content was taken to calculate the Pt binding amount relative to the polymer, and the result was 3.4 mol / mol. In addition, the Z average particle size and polydispersity index of the obtained DACHPt-encapsulated SCNPs were measured by dynamic light scattering (DLS), and the result was 8.7 nm (polydispersity index 0.14). The particle size of the SCNPs before and after DACHPt encapsulation is shown in Figure 3 The particle size of SCNPs hardly changed before and after DACHPt inclusion. The results are summarized in the table below.

[0283] [Measurement equipment and conditions, etc.]

[0284] (1) ICP-AES measurement

[0285] Device: Continuous high-frequency plasma luminescence device ICPE-9000 / Shimadzu Corporation

[0286] Pretreatment device: Microwave sample pretreatment device ETHOS EASY / Milestone General

[0287] Measurement wavelength: 214nm

[0288] Standard solution: Platinum standard solution (Pt1000) for ICP analysis / FUJIFILM Wako Pure Chemical

[0289] (2) DLS assay

[0290] Device: Zetasizer NanoZS / Malvern Instruments Ltd.

[0291] Measurement temperature: 25℃

[0292] Sample concentration: 10mg / mL

[0293] result: Figure 3

[0294] [Table 4]

[0295]

[0296] [Example 71]

[0297] Preparation of N3-poly[(benzyl acrylate)-(poly(ethylene glycol) methyl ether acrylate)-(acrylic acid) copolymer]

[0298] N3-CTA was used instead of the chain transfer agent DDMAT used in Example 1, and the synthesis was carried out in the same manner as in Example 1 and Example 69. 100 mg of 2-(dodecylthiocarbonylthiothio)-2-methylpropionic acid 3-azido-1-propanol ester (N3-CTA) was weighed and dissolved in 17.3 mL of toluene to prepare an N3-CTA / toluene stock solution (with an N3-CTA concentration of 5.78 mg / mL). Similarly, 10 mg of 2,2'-azobis(2-methylpropionitrile) (AIBN) was weighed and dissolved in 7.87 mL of toluene to prepare an AIBN / toluene stock solution (with an AIBN concentration of 1.27 mg / mL). Separately, 2.592 g of poly(ethylene glycol) methyl ether acrylate (mPEGA, average value (n) of the number of repeats of ethylene glycol is 9), 0.684 g of benzyl acrylate (BnA), 0.172 g of 1-ethoxyethyl acrylate, 4.15 mL of N3-CTA / toluene stock solution, and 3.46 mL of AIBN / toluene stock solution were added, and polymerization was carried out in an oil bath at 70°C. After 90 minutes, the polymerization was stopped, and then the reaction solution was recovered by reprecipitation or by dialysis against methanol. In the reprecipitation method, the reaction solution was added dropwise to a centrifuge tube to which a poor solvent (hexane / ethyl acetate = 7 / 3 [v / v]) was added, and the copolymer was recovered by centrifugal separation (2,000×g, 5 min). This operation was repeated 3 times, and finally vacuum dried. The obtained copolymer was treated with 0.5N HCl at room temperature to remove the ethoxyethyl group, thereby obtaining 2.455 g of N3-poly[(benzyl acrylate)-(poly(ethylene glycol) methyl ether acrylate)-(acrylic acid) copolymer].

[0299] The obtained copolymer was measured by NMR. 1 The H-NMR spectrum shows the degree of polymerization and number average molecular weight (M n,NMR ) were analyzed, and the results showed that the degree of polymerization of mPEGA (n = 9) was 70, the degree of polymerization of BnA was 56, the degree of polymerization of EEA was 15, and the degree of polymerization of Mn,NMR The molecular weight dispersion (M w / M n ) was measured and the result was 1.32.

[0300]

[0301] [Measurement equipment and conditions, etc.]

[0302] (1) 1 H-NMR measurement

[0303] Device: JNM-ECX400 (400MHz) / JEOL

[0304] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0305] Sample concentration: 20mg / mL

[0306] Measurement temperature: 25℃

[0307] Total times: 256 times

[0308] result: Figure 4

[0309] (2) GPC measurement

[0310] Equipment: HPLC-Prominence system / Shimadzu Corporation

[0311] Detector: RID-10A refractive index detector / Shimadzu Corporation

[0312] Chromatographic column: TSKgelα-2500 chromatographic column / Tosoh

[0313] (Column size: 7.8 mm × 300 mm, particle size: 7 μm, exclusion limit molecular weight: 5 × 10 3 )

[0314] TSKgelα-4000 Column / Tosoh

[0315] (Column size: 7.8 mm × 300 mm, particle size: 10 μm, exclusion limit molecular weight: 4 × 10 5 )

[0316] TSKgel Guard Column / Tosoh

[0317] Mobile phase: N,N-dimethylformamide (DMF) containing 10 mmol / L lithium bromide

[0318] Temperature: 40℃

[0319] Flow rate: 0.5mL / min

[0320] Sample concentration: 6mg / mL

[0321] Standard material: Poly(methyl methacrylate) standard ReadyCal set, M p 800-2,200,000Da / SIGMA

[0322] result: Figure 5

[0323] [Table 5]

[0324]

[0325] [Examples 72 to 85]

[0326] The addition amounts of the monomers (mPEGA, BnA, EEA) used in Example 71 and the polymerization time were appropriately changed, and the same method as in Example 71 was used to produce polymers having different composition ratios or average molecular weights as shown in the table below.

[0327] [Table 6]

[0328]

[0329] [Table 7]

[0330]

[0331] [Examples 86-88]

[0332] N3(PEG m Preparation of poly[(benzyl acrylate)-(poly(ethylene glycol) methyl ether acrylate)-(acrylic acid) copolymer]

[0333] Instead of the chain transfer agent N3-CTA used in Example 71, 2-(2-(2-azidoethoxy)ethoxy)ethyl 2-(dodecylthiothiocarbonylthio)-2-methylpropanoate (N3-PEG2-ester CTA), 2-(dodecylthiothiocarbonylthio)-2-methylpropanoate 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-ol 2-(dodecylthiothiocarbonylthio)-2-methylpropanoate (N3-PEG5-ester CTA), or 2-(dodecylthiothiocarbonylthio)-2-methylpropanoate 23-azido-3,6,9,12,15,18,21-heptaoxatricosan-1-ol 2-(dodecylthiothiocarbonylthio)-2-methylpropanoate (N3-PEG7-ester CTA) was used to synthesize under the same (feed ratio) conditions as in Example 83.

[0334] [Table 8]

[0335]

[0336]

[0337] [Examples 89-91]

[0338] N3(PEG m Preparation of poly(acrylic acid)-poly[(benzyl acrylate)-(poly(ethylene glycol) methyl ether acrylate)-(acrylic acid) copolymer]

[0339] N-(8-azido-3,6-dioxaoctane-1-yl)-2-(dodecylthiocarbonylthio)-2-methylpropionamide (N3-PEG2-amide CTA), N-(17-azido-3,6,9,12,15-pentaoxaheptadecan-1-yl)-2-(dodecylthiocarbonylthio)-2-methylpropionamide (N3-PEG5-amide CTA), or N-(23-azido-3,6,9,12,15,18,21-heptaoxatotricosan-1-yl)-2-(dodecylthiocarbonylthio)-2-methylpropionamide (N3-PEG7-amide CTA) was used instead of the chain transfer agent N3-CTA used in Example 71, and the synthesis was carried out under the same (feed ratio) conditions as in Example 83.

[0340] [Table 9]

[0341]

[0342]

[0343] [Example 92]

[0344] Preparation of N3-poly[(benzyl acrylate)-(poly(ethylene glycol) methyl ether acrylate)-(acrylic acid) copolymer]-isobutyronitrile

[0345] 2.40 g of the copolymer obtained in Example 71 was weighed and dissolved in 32 mL of toluene. 170 mg of AIBN and 62 mg of lauroyl peroxide were added to the solution, and the mixture was stirred in an oil bath at 80°C for 20 hours. After the reaction was stopped by cooling in an ice bath, the reaction solution was recovered by reprecipitation or by dialysis against methanol. The obtained copolymer was basically a viscous body, so in the reprecipitation method, the reaction solution was added dropwise to a centrifuge tube to which a poor solvent (hexane / ethyl acetate = 7 / 3 [v / v]) was added, and the copolymer was recovered by centrifugal separation (2,000 × g, 5 min). This operation was repeated 3 times, and finally vacuum dried to obtain 2.21 g of N3-poly[(benzyl acrylate)-(poly(ethylene glycol) methyl ether acrylate)-(acrylic acid) copolymer]-isobutyronitrile with converted terminal structure. The obtained copolymer was measured using an ultraviolet-visible spectrophotometer, and the residual rate of the terminal structure was evaluated based on the obtained UV spectrum, and the result was 0.0%.

[0346]

[0347] [Measurement equipment and conditions, etc.]

[0348] (1) UV spectrum measurement

[0349] Device: Hitachi Spectrophotometer U-9300 / Hitachi

[0350] Solvent: Purified water

[0351] Sample concentration: 4 mg / mL

[0352] Measurement wavelength: 250~500nm

[0353] result: Figure 6

[0354] [Table 10]

[0355] Example Copolymers used Azo compounds used in the reaction The residual rate of terminal structure (%) 92 Embodiment 71 AIBN 0.0

[0356] [Examples 93 to 95]

[0357] The copolymers obtained in Examples 84 and 85 were prepared in the same manner as in Example 92 by appropriately changing the type and amount of the azo compound added, thereby synthesizing copolymers having different terminal structures as shown in the following table.

[0358] [Table 11]

[0359] Example Copolymers used Azo compounds used in the reaction The residual rate of terminal structure (%) 93 Embodiment 84 AIBN 0.0 94 Embodiment 84 MAIB 0.0 95 Embodiment 85 AIBN 0.0

[0360] [Example 96]

[0361] Preparation of DM1-cysteamine conjugated copolymers

[0362] 200 mg of the copolymer obtained in Example 92 was weighed and dissolved in 2 mL of DMF, 59 mg of (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinylcarbonium hexafluorophosphate (COMU) and 23.2 μL of 2,2,6,6-tetramethylpiperidine (TMP) were added, and the mixture was stirred at 30° C. for 2 hours. In addition, 101 mg of maytansine (DM1) and 18 mg of S-(2-pyridylthio)cysteamine hydrochloride were added, dissolved in 3 mL of THF, 35.9 μL of DIPEA was added, and the mixture was stirred at 30° C. for 2 hours. The reaction solutions were mixed, and the obtained reaction solution was stirred at 30° C. for 24 hours. The reaction solution was dialyzed and purified (dialysis membrane: Spectra / Por Regenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external solution: methanol), and the solvent was removed by vacuum distillation and vacuum drying, and the copolymer was recovered to obtain 222 mg of DM1-cysteamine-bound copolymer. In addition, the Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS), and the result was 11 nm (polydispersity index 0.32).

[0363]

[0364] The DM1-cysteamine-binding copolymer was measured by NMR. 1 The H-NMR spectrum was used to analyze the amount of DM1 introduced per molecule of the copolymer, and the result was 13 mol / mol.

[0365] [Measurement equipment and conditions, etc.]

[0366] (1) 1 H-NMR measurement

[0367] Device: JNM-ECX400 (400MHz) / JEOL

[0368] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0369] Sample concentration: 20mg / mL

[0370] Measurement temperature: 25℃

[0371] Total times: 256 times

[0372] result: Figure 7

[0373] [Table 12]

[0374]

[0375] [Examples 97 to 107]

[0376] The copolymers obtained in Examples 79, 85 to 91 and 93 to 95 were synthesized by appropriately changing the linking group and the amount of DM1 added and using the same method as in Example 96, thereby synthesizing copolymers having different numbers of DM1 introduced per molecule as shown in the table below.

[0377] [Table 13]

[0378]

[0379] [Example 108]

[0380] Preparation of DM1-N-4-APM conjugate copolymer

[0381] Weigh 509 mg of the copolymer obtained in Example 72, dissolve it in 10 mL of DMF, add 84 mg of COMU and 33 μL of TMP, and stir at room temperature for 3 hours. Then, add 55 mg of N-(4-aminophenyl)maleimide (N-4-APM), and stir at 30°C for 3 days. After the reaction solution is dialyzed and purified (dialysis membrane: Spectra / Por Regenerated CelluloseMembrane6, molecular weight cutoff: 3.5 kDa, external solution: methanol), the solvent is removed by vacuum distillation and vacuum drying to obtain 475 mg of N-4-APM-bound copolymer. The obtained 475 mg of N-4-APM-bound copolymer is dissolved in 10 mL of DMF, 102 mg of DM1 is added, and stirred at room temperature for 24 hours. The reaction solution was dialyzed and purified (dialysis membrane: Spectra / PorRegenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external solution: methanol), and the solvent was removed by vacuum distillation and vacuum drying to obtain 458 mg of DM1-N-4-APM conjugated copolymer. The Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS), and the result was 11 nm (polydispersity index 0.19).

[0382]

[0383] The DM1-N-4-APM conjugate copolymer was measured by NMR. 1 The H-NMR spectrum was used to analyze the amount of DM1 introduced per molecule of the copolymer, and the result was 7 mol / mol.

[0384] [Measurement equipment and conditions, etc.]

[0385] (1) 1 H-NMR measurement

[0386] Device: JNM-ECX400 (400MHz) / JEOL

[0387] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0388] Sample concentration: 20mg / mL

[0389] Measurement temperature: 25℃

[0390] Total times: 256 times

[0391] result: Figure 8

[0392] [Table 14]

[0393]

[0394] [Example 109]

[0395] Synthesis of 1,4-Butanediamine-bonded Copolymer

[0396] 810 mg of the copolymer obtained in Example 74 was weighed and dissolved in 16 mL of DMF, and 164.9 mg of COMU and 78 μL of TMP were added, and the mixture was stirred at room temperature for 3 hours. Then, 735 μL of N-(tert-butoxycarbonyl)-1,4-butanediamine was added, and the mixture was stirred at 30°C for 3 days. After the reaction solution was dialyzed and purified (dialysis membrane: Spectra / Por Regenerated CelluloseMembrane 6, molecular weight cutoff: 3.5 kDa, external solution: methanol), the solvent was removed by vacuum distillation and vacuum drying, and the copolymer was recovered. The obtained N-Boc-1,4-butanediamine copolymer was dissolved in 32 mL of a mixed solution of DCM and TFA [DCM / TFA=5 / 3 (v / v)], stirred at room temperature overnight, and deprotected, and then the solvent was removed by vacuum distillation and vacuum drying to obtain 643 mg of a 1,4-butanediamine-bound copolymer.

[0397]

[0398] The 1,4-butanediamine-bonded copolymer was measured by NMR. 1 The H-NMR spectrum was analyzed to determine the amount of 1,4-butanediamine introduced per molecule of the copolymer, which was 12 mol / mol.

[0399] [Measurement equipment and conditions, etc.]

[0400] (1) 1 H-NMR measurement

[0401] Device: JNM-ECX400 (400MHz) / JEOL

[0402] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0403] Sample concentration: 20mg / mL

[0404] Measurement temperature: 25℃

[0405] Total times: 256 times

[0406] result: Fig. 9

[0407] [Table 15]

[0408]

[0409] [Examples 110 to 116]

[0410] For the copolymers obtained in Examples 73, 75 to 78 and 80 to 81, the addition amount of N-(tert-butoxycarbonyl)-1,4-butanediamine was appropriately changed, and the same method as Example 109 was used to synthesize copolymers having different numbers of 1,4-butanediamine introduced per molecule as shown in the table below.

[0411] [Table 16]

[0412]

[0413] [Example 117]

[0414] Preparation of DM1-MHA-1,4-butanediamine-bound copolymer

[0415] Weigh 295 mg of the copolymer obtained in Example 109, dissolve it in 6 mL of DMF, add 59.3 mg of COMU and 28 μL of TMP, and stir at room temperature for 3 hours. Then, add 293 mg of 6-maleimidocaproic acid (MHA), and stir at 30°C for 3 days. After the reaction solution is dialyzed and purified (dialysis membrane: Spectra / Por Regenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external liquid: methanol), the solvent is removed by vacuum distillation and vacuum drying to obtain 313 mg of MHA-1,4-butanediamine-bound copolymer. After dissolving the obtained MHA-1,4-butanediamine-bound copolymer in 10 mL of DMF, add 80 mg of DM1, and stir at 30°C for 24 hours. The reaction solution was dialyzed and purified (dialysis membrane: Spectra / PorRegenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external solution: methanol), and the solvent was removed by vacuum distillation and vacuum drying to obtain 321 mg of DM1-MHA-1,4-butanediamine-bound copolymer. The Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS), and the result was 10 nm (polydispersity index 0.21).

[0416]

[0417] The DM1-MHA-1,4-butanediamine-bound copolymer was measured by NMR. 1 The H-NMR spectrum was used to analyze the amount of DM1 introduced per molecule of the copolymer, and the result was 12 mol / mol.

[0418] [Measurement equipment and conditions, etc.]

[0419] (1) 1 H-NMR measurement

[0420] Device: JNM-ECX400 (400MHz) / JEOL

[0421] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0422] Sample concentration: 20mg / mL

[0423] Measurement temperature: 25℃

[0424] Total times: 256 times

[0425] result: Fig.10

[0426] [Table 17]

[0427]

[0428] [Example 118]

[0429] Preparation of DM1-SPDP-1,4-butanediamine-bound copolymer

[0430] DM1 81 mg and 3-(pyridin-2-yldisulfide) propionic acid 2,5-dioxopyrrolidin-1-yl ester (SPDP) 29 mg were added, dissolved in DCM 1 mL, N,N-diisopropylethylamine (DIPEA) 16 μL, and stirred at 30° C. for 3 hours. 200 mg of the copolymer obtained in Example 110 was dissolved in DCM 4 mL and added to the reaction solution, and the obtained reaction solution was stirred at 30° C. for 24 hours. After the reaction solution was dialyzed and purified (dialysis membrane: Spectra / Por Regenerated CelluloseMembrane 6, molecular weight cutoff: 3.5 kDa, external solution: methanol), the solvent was removed by vacuum distillation and vacuum drying, and the copolymer was recovered to obtain 189 mg of DM1-SPDP-1,4-butanediamine-bound copolymer. The Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS) and the results were 13 nm (polydispersity index: 0.23).

[0431]

[0432] The DM1-SPDP-1,4-butanediamine-bound copolymer was measured by NMR. 1 The H-NMR spectrum was used to analyze the amount of DM1 introduced per molecule of the copolymer, and the result was 18 mol / mol.

[0433] [Measurement equipment and conditions, etc.]

[0434] (1) 1 H-NMR measurement

[0435] Device: JNM-ECX400 (400MHz) / JEOL

[0436] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0437] Sample concentration: 20mg / mL

[0438] Measurement temperature: 25℃

[0439] Total times: 256 times

[0440] result: Fig.11

[0441] [Table 18]

[0442]

[0443] [Examples 119 to 121]

[0444] The copolymers obtained in Examples 112 to 114 were prepared in the same manner as in Example 118 by appropriately changing the amount of DM1 added, thereby synthesizing copolymers having different numbers of DM1 introduced per molecule as shown in the following table.

[0445] [Table 19]

[0446]

[0447] [Example 122]

[0448] Preparation of DM1-SMCC-1,4-butanediamine-bound copolymer

[0449] Add DM1 39mg and N-succinimidyl 4-(N-maleimidomethyl) cyclohexanecarboxylate (SMCC) 15mg, dissolve in DCM 1mL, add DIPEA 7.8μL, and stir at 30°C for 3 hours. Dissolve 96mg of the copolymer obtained in Example 110 in DCM 4mL, add to the reaction solution, and stir the obtained reaction solution at 30°C for 48 hours. After the reaction solution was dialyzed and purified (dialysis membrane: Spectra / Por Regenerated Cellulose Membrane 6, molecular weight cutoff: 3.5kDa, external solution: methanol), the solvent was removed by vacuum distillation and vacuum drying, and the copolymer was recovered to obtain DM1-SMCC-1,4-butanediamine-bound copolymer 122mg. The Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS), and the result was 12nm (polydispersity index 0.29).

[0450]

[0451] The DM1-SMCC-1,4-butanediamine-bound copolymer was measured by NMR. 1 The H-NMR spectrum was used to analyze the amount of DM1 introduced per molecule of the copolymer, and the result was 18 mol / mol.

[0452] [Measurement equipment and conditions, etc.]

[0453] (1) 1 H-NMR measurement

[0454] Device: JNM-ECX400 (400MHz) / JEOL

[0455] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0456] Sample concentration: 20mg / mL

[0457] Measurement temperature: 25℃

[0458] Total times: 256 times

[0459] result: Fig.12

[0460] [Table 20]

[0461]

[0462] [Example 123]

[0463] The copolymer obtained in Example 111 was subjected to the same method as in Example 122 by appropriately changing the amount of DM1 added, thereby synthesizing copolymers having different numbers of DM1 introduced per molecule as shown in the following table.

[0464] [Table 21]

[0465]

[0466] [Example 124]

[0467] DM1-CL-031-1,4-Butanediamine-bonded copolymer manufacturing

[0468] DM1 81 mg and 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldithio) pentanoate (CL-031) 32 mg were added, dissolved in DCM 1 mL, DIPEA 16 μL was added, and stirred at 30° C. for 3 hours. 200 mg of the copolymer obtained in Example 110 was dissolved in DCM 4 mL and added to the reaction solution, and the obtained reaction solution was stirred at 30° C. for 24 hours. The reaction solution was dialyzed and purified (dialysis membrane: Spectra / Por Regenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external solution: methanol), and the solvent was removed by vacuum distillation and vacuum drying, and the copolymer was recovered to obtain 222 mg of DM1-CL-031-1,4-butanediamine-bound copolymer. The Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS), and the result was 13 nm (polydispersity index: 0.34).

[0469]

[0470] The DM1-CL-031-1,4-butanediamine-bound copolymer was measured by NMR. 1 The H-NMR spectrum was used to analyze the amount of DM1 introduced per molecule of the copolymer, and the result was 17 mol / mol.

[0471] [Measurement equipment and conditions, etc.]

[0472] (1) 1 H-NMR measurement

[0473] Device: JNM-ECX400 (400MHz) / JEOL

[0474] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0475] Sample concentration: 20mg / mL

[0476] Measurement temperature: 25℃

[0477] Total times: 256 times

[0478] result: Fig.13

[0479] [Table 22]

[0480]

[0481] [Example 125]

[0482] DM1-CL-018-Manufacture of 1,4-butanediamine-bonded copolymer

[0483] 114 mg of DM1 and 43 mg of 2,5-dioxopyrrolidin-1-yl 3-methyl-3-(pyridin-2-yldisulfide)butyrate (CL-018) were added, dissolved in 1 mL of N,N-dimethylacetamide (DMAC), 8.4 mg of 4-dimethylaminopyridine (DMAP) was added, and stirred at 50°C for 2 hours. 150 mg of the copolymer obtained in Example 115 was dissolved in 4 mL of DMAC and added to the reaction solution, and the obtained reaction solution was stirred at 50°C for 24 hours. After the reaction solution was dialyzed and purified (dialysis membrane: Spectra / Por Regenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external solution: methanol), the solvent was removed by reduced pressure distillation and vacuum drying, and the copolymer was recovered to obtain 156 mg of DM1-CL-018-1,4-butanediamine-bound copolymer. The Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS) and the results were 13 nm (polydispersity index: 0.23).

[0484]

[0485] The DM1-CL-018-1,4-butanediamine-bound copolymer was measured by NMR. 1 The H-NMR spectrum was used to analyze the amount of DM1 introduced per molecule of the copolymer, and the result was 16 mol / mol.

[0486] [Measurement equipment and conditions, etc.]

[0487] (1) 1 H-NMR measurement

[0488] Device: JNM-ECX400 (400MHz) / JEOL

[0489] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0490] Sample concentration: 20mg / mL

[0491] Measurement temperature: 25℃

[0492] Total times: 256 times

[0493] result: Fig.14

[0494] [Table 23]

[0495]

[0496] [Example 126]

[0497] DM1-CL-038-1,4-Butanediamine-bonded copolymer production

[0498] DM1 76 mg and 2,5-dioxopyrrolidin-1-yl 4-methyl-4-(pyridin-2-yldithio)pentanoate (CL-038) 29 mg were added, dissolved in DMAC 1 mL, DMAP 6 mg was added, and stirred at 50°C for 2 hours. 100 mg of the copolymer obtained in Example 115 was dissolved in DMAC 4 mL, added to the reaction solution, and stirred at 50°C for 24 hours. After the reaction solution was dialyzed and purified (dialysis membrane: Spectra / Por Regenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external solution: methanol), the solvent was removed by vacuum distillation and vacuum drying, and the copolymer was recovered to obtain DM1-CL-038-1,4-butanediamine-bound copolymer 56 mg. The Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS), and the result was 11 nm (polydispersity index 0.32).

[0499]

[0500] The DM1-CL-038-1,4-butanediamine-bound copolymer was measured by NMR. 1 The H-NMR spectrum was used to analyze the amount of DM1 introduced per molecule of the copolymer, and the result was 8 mol / mol.

[0501] [Measurement equipment and conditions, etc.]

[0502] (1) 1 H-NMR measurement

[0503] Device: JNM-ECX400 (400MHz) / JEOL

[0504] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0505] Sample concentration: 20mg / mL

[0506] Measurement temperature: 25℃

[0507] Total times: 256 times

[0508] result: Fig.15

[0509] [Table 24]

[0510]

[0511] [Example 127]

[0512] DM1-CL-047-1,4-Butanediamine-bonded copolymer manufacturing

[0513] 21.2 mg of 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldisulfide)toluene (CL-047) was dissolved in 3 mL of DMF, and 100 mg of the copolymer obtained in Example 113 and 10 μL of DIPEA were added, and the mixture was stirred at 30°C for 24 hours. 140 mg of DM was added to the reaction solution, and the mixture was stirred at 30°C for 24 hours. After the reaction solution was dialyzed and purified (dialysis membrane: Spectra / Por Regenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external solution: methanol), the solvent was removed by vacuum distillation and vacuum drying, and the copolymer was recovered to obtain 153 mg of DM1-CL-047-1,4-butanediamine-bound copolymer. The Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS), and the result was 12 nm (polydispersity index 0.18).

[0514]

[0515] The DM1-CL-047-1,4-butanediamine-bound copolymer was measured by NMR. 1 The H-NMR spectrum was used to analyze the amount of DM1 introduced per molecule of the copolymer, and the result was 20 mol / mol.

[0516] [Measurement equipment and conditions, etc.]

[0517] (1) 1 H-NMR measurement

[0518] Device: JNM-ECX400 (400MHz) / JEOL

[0519] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0520] Sample concentration: 20mg / mL

[0521] Measurement temperature: 25℃

[0522] Total times: 256 times

[0523] result: Fig.16

[0524] [Table 25]

[0525]

[0526] [Example 128]

[0527] Preparation of SN-38-CO-1,4-butanediamine-bonded copolymer

[0528] 0.5 g of SN-38 was dissolved in 50 mL of DCM, 353 mg of di-tert-butyl dicarbonate and 3 mL of pyridine were added, and the mixture was stirred at room temperature overnight. The reaction solution was transferred to a separatory funnel, washed three times with 150 mL of 0.5 N HCl aqueous solution, and then washed once with a saturated NaHCO3 aqueous solution. The organic layer was recovered, and DCM was removed by vacuum distillation using an evaporator, followed by vacuum drying to obtain 0.586 g of Boc-SN-38.

[0529] Then, 200 mg of the copolymer obtained in Example 115 was dissolved in benzene and freeze-dried. It was dissolved in THF under an argon atmosphere, and 27.6 mg of Boc-SN-38, 12 mg of 1,1'-carbonyldiimidazole and 15 mg of DMAP were added respectively, and stirred at room temperature for 3 hours. After the reaction solution was dialyzed and purified (dialysis membrane: Spectra / Por Regenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external liquid: methanol), the solvent was removed by vacuum distillation and vacuum drying, and the Boc-SN-38-CO-1,4-butanediamine-bound copolymer was recovered. The obtained Boc-SN-38-CO-1,4-butanediamine-bound copolymer was dissolved in 32 mL of a mixed solution of DCM and TFA [DCM / TFA=5 / 3 (v / v)], stirred at room temperature overnight, and deprotected, and then the solvent was removed by vacuum distillation and vacuum drying to obtain 220 mg of SN-38-CO-1,4-butanediamine-bound copolymer. The Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS), and the result was 8 nm (polydispersity index 0.23).

[0530]

[0531] The SN-38-CO-1,4-butanediamine-bound copolymer was measured by NMR. 1 The H-NMR spectrum was used to analyze the amount of SN-38 introduced per molecule of the copolymer, and the result was 13 mol / mol.

[0532] [Measurement equipment and conditions, etc.]

[0533] (1) 1 H-NMR measurement

[0534] Device: JNM-ECX400 (400MHz) / JEOL

[0535] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0536] Sample concentration: 20mg / mL

[0537] Measurement temperature: 25℃

[0538] Total times: 256 times

[0539] result: Fig.17

[0540] [Table 26]

[0541]

[0542] [Example 129]

[0543] Preparation of Delutec-SPDP-1,4-butanediamine-conjugated copolymer

[0544] 200 mg of the copolymer obtained in Example 116, 14.2 mg of 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfide) propionate (SPDP) and 51.4 mg of derutecan were dissolved in 4 mL of DMF and stirred at 30° C. for 2 hours. Then, 1 mL of an aqueous solution of tri(2-carboxyethyl)phosphine (TCEP) was added dropwise to the reaction solution under stirring, and stirred at 30° C. overnight. The obtained reaction solution was stirred at 30° C. for 24 hours. After the reaction solution was dialyzed and purified (dialysis membrane: Spectra / PorRegenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external liquid: methanol), the solvent was removed by vacuum distillation and vacuum drying, and the copolymer was recovered to obtain 205 mg of derutecan-SPDP-1,4-butanediamine-bound copolymer (sulfide bond). The Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS) and the results were 12 nm (polydispersity index: 0.23).

[0545]

[0546] The delutec-SPDP-1,4-butanediamine copolymer was measured by NMR.1 The H-NMR spectrum was used to analyze the amount of delutec introduced per molecule of the copolymer, and the result was 16 mol / mol.

[0547] [Measurement equipment and conditions, etc.]

[0548] (1) 1 H-NMR measurement

[0549] Device: JNM-ECX400 (400MHz) / JEOL

[0550] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0551] Sample concentration: 20mg / mL

[0552] Measurement temperature: 25℃

[0553] Total times: 256 times

[0554] result: Fig.18

[0555] [Table 27]

[0556]

[0557] [Example 130]

[0558] Preparation of 4-Hydroxybutylamine-Conjugated Copolymer

[0559] 200 mg of the copolymer obtained in Example 82 was weighed and dissolved in 4 mL of DMF, and 64.4 mg of COMU and 31 μL of TMP were added, and stirred at room temperature for 3 hours. Then, 73 μL of 4-amino-1-butanol was added, and stirred at 30°C overnight. The reaction solution was dialyzed and purified (dialysis membrane: Spectra / Por Regenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external solution: methanol), and the solvent was removed by reduced pressure distillation and vacuum drying to obtain 205 mg of a 4-hydroxybutylamine-bound copolymer.

[0560]

[0561] The 4-hydroxybutylamine-bound copolymers were measured by NMR. 1 The H-NMR spectrum was analyzed to determine the amount of 4-amino-1-butanol introduced per molecule of the copolymer, which was 15 mol / mol.

[0562] [Measurement equipment and conditions, etc.]

[0563] (1) 1H-NMR measurement

[0564] Device: JNM-ECX400 (400MHz) / JEOL

[0565] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0566] Sample concentration: 20mg / mL

[0567] Measurement temperature: 25℃

[0568] Total times: 256 times

[0569] result: Fig.19

[0570] [Table 28]

[0571]

[0572] [Example 131]

[0573] Synthesis of Staurosporine (STS)-conjugated copolymers

[0574] 200 mg of the copolymer obtained in Example 130 was dissolved in THF, 7.2 mg of triphosgene and 22.1 mg of DMAP were added, and the mixture was stirred at 10°C for 30 minutes. Then, 28.2 mg of staurosporine was added and stirred at room temperature for 3 hours. After the reaction solution was dialyzed and purified (dialysis membrane: Spectra / Por Regenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external solution: methanol), the solvent was removed by vacuum distillation and vacuum drying to obtain 233 mg of staurosporine-bound copolymer. The Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS), and the result was 10 nm (polydispersity index 0.21).

[0575]

[0576] For STS-bound copolymers, the 1 The H-NMR spectrum was analyzed to determine the number of STS introduced per molecule of the copolymer, which was 13 mol / mol.

[0577] [Measurement equipment and conditions, etc.]

[0578] (1) 1 H-NMR measurement

[0579] Device: JNM-ECX400 (400MHz) / JEOL

[0580] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0581] Sample concentration: 20mg / mL

[0582] Measurement temperature: 25℃

[0583] Total times: 256 times

[0584] result: Fig. 20

[0585] [Table 29]

[0586]

[0587] [Example 132]

[0588] Preparation of Cetuximab-Maleimide-PEG4-DBCO Conjugated Micelle Drug Complex

[0589] In a glass vial containing 3 mL of cetuximab (Erbitux, Merck BioPharma) antibody stock solution (10 mg / mL) and 9 mL of phosphate buffered saline (PBS), 42 μL of 10 mM tris(2-carboxyethyl)phosphine (TCEP) dissolved in PBS was added, and the mixture was stirred at 37°C for 15 minutes. Then, 99 μL of 10 mM DBCO-PEG4-maleimide dissolved in dimethyl sulfoxide (DMSO) was added, and the mixture was stirred at 37°C for 15 minutes to react with the cysteine ​​residues of the antibody. 20 μL of an aqueous solution of L-cysteine ​​(168149, Sigma-Aldrich) (10 mg / mL) dissolved in PBS was added, and the mixture was stirred at 37°C for 1 minute to stop the reaction. The reaction solution was recovered and dialyzed against purified water overnight (Slide-A-Lyzer TM The unreacted modification reagent was removed by ultrafiltration (Vivaspin Turbo 15, molecular weight cutoff: 100K) using a dialysis cassette (molecular weight cutoff: 20K, 7736, ThermoFisher Scientific). The volume was accurately fixed to 5 mL using PBS, and 1 mL of the solution was dispensed into a glass vial. Then, 10 mg of the copolymer obtained in Example 99 was added and the mixture was stirred at 4 °C for 2 h. TMThe mixture was allowed to stand at -20°C for 1 day. After that, the mixture was thawed at 4°C and the obtained solution was recovered. After the copolymer-bound antibody and the non-bound antibody were separated by hydrophobic chromatography using the difference in retention time, the target fractions were recovered separately, and after washing by ultrafiltration (Vivaspin Turbo 15, molecular weight cutoff: 100K) using PBS, the target cetuximab-maleimide-PEG4-DBCO-bound micellar drug complex was obtained.

[0590] The Z average particle size and polydispersity index of the cetuximab-maleimide-PEG4-DBCO conjugated micelle drug complex were measured by dynamic light scattering (DLS). In addition, the number average molecular weight (Mn, MALS) of the complex in water was measured by size exclusion chromatography (SEC) equipped with a multi-angle light scattering (MALS) detector, and the absolute molecular weight was calculated. Based on the molecular weight of the antibody and the molecular weight of the bound copolymer, the number of copolymers bound to each antibody and the antibody drug binding ratio (DAR) were calculated.

[0591]

[0592] [Measurement equipment and conditions, etc.]

[0593] (1) SEC-MALS assay

[0594] Installation: Waters Alliance / Waters

[0595] Detector: 2998PDA detector / Waters

[0596] 2414 Refractive Index Detector / Waters DAWN HELEOSII 8+ / WYATT

[0597] Chromatographic column: TSKgel G-3000PWXL / Tosoh (column size 7.8mm×300mm, particle size 7μm, exclusion limit molecular weight 2×10 5 )

[0598] TSKgel Guard Column / Tosoh

[0599] Mobile phase: 100mmol / L sodium chloride aqueous solution

[0600] Temperature: 25℃

[0601] Flow rate: 0.8 mL / min

[0602] Sample concentration: 10mg / mL

[0603] [Table 30]

[0604]

[0605] [Examples 133-134]

[0606] In Example 133, DBCO-PEG12-maleimide was used instead of DBCO-PEG4-maleimide used in Example 132, and in Example 134, DBCO-PEG24-maleimide was used, and the synthesis was carried out by the same production method as in Example 132.

[0607] [Table 31]

[0608]

[0609] [Examples 135 to 144]

[0610] The copolymers obtained in Examples 83, 96, 100, 101, 120, 121, 122, 124, 125 and 128 were synthesized in the same manner as in Example 132, with appropriate changes in the amount of feed.

[0611] [Table 32]

[0612]

[0613] [Example 145]

[0614] Preparation of Cetuximab-NHS-DBCO Conjugated Micelle Drug Complex

[0615] The copolymer obtained in Example 118 was complexed with cetuximab. In a glass vial with 14.5 mL of PBS solution (2.07 mg / mL) of cetuximab, 0.5 mL of sulfo DBCO-NHS ester dissolved in PBS was added at 1.22 mg / mL and stirred for 3 hours to react with the lysine residue of the antibody. Afterwards, the unreacted modification reagent was removed by dialysis purification (dialysis membrane: Spectra / Por Regenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external liquid: purified water) and ultrafiltration (Vivaspin Turbo 15, molecular weight cutoff: 100K). PBS was used to accurately adjust the volume to 5 mL. 1 mL of the solution was dispensed into a glass vial (6 mg / bottle in terms of antibody) and recovered by freeze drying. Afterwards, the target cetuximab-NHS-DBCO-bound micelle drug complex was recovered by the same operation as in Example 132.

[0616] The Z average particle size and polydispersity index of the cetuximab-NHS-DBCO conjugated micelle drug complex were measured by dynamic light scattering (DLS), and the result was 32 nm (polydispersity index 0.47). In addition, the absolute molecular weight was calculated by MALS measurement, and the number of copolymers bound to each antibody calculated based on the molecular weight of the antibody and the molecular weight of the bound copolymer was 1.9, and the antibody drug binding ratio (DAR) was 34.

[0617] [Table 33]

[0618]

[0619] [Examples 146 to 148]

[0620] Instead of sulfo DBCO-NHS ester used in Example 145, DBCO-NHS ester was used in Example 146, DBCO-PEG5-NHS ester was used in Example 147, and DBCO-PEG13-NHS ester was used in Example 148, and the synthesis was carried out by the same production method as in Example 145.

[0621] [Table 34]

[0622]

[0623] [Examples 149 to 152]

[0624] Preparation of trastuzumab-bound micellar drug complexes

[0625] The same procedure as in Example 132 was performed except that the antibody was changed to trastuzumab (Heaiping, Chugai Pharmaceutical Co., Ltd.).

[0626] [Table 35]

[0627]

[0628] [Example 153]

[0629] Preparation of rituximab-S-binding micellar drug complex

[0630] The same procedure as in Example 132 was performed except that the antibody was changed to rituximab (MabThera, Zenyaku Kogyo Co., Ltd.).

[0631] [Table 36]

[0632]

[0633] [Example 154]

[0634] Preparation of rituximab-NH-conjugated micellar drug complex

[0635] The same procedure as in Example 146 was performed except that the antibody was changed to rituximab (MabThera, Zenyaku Kogyo Co., Ltd.).

[0636] [Table 37]

[0637]

[0638] [Example 155]

[0639] Preparation of panitumumab-bound micellar drug complexes

[0640] The same procedure as in Example 132 was performed except that the antibody was changed to panitumumab (Vectibix, Takeda Pharmaceutical Company).

[0641] [Table 38]

[0642]

[0643] [Example 156]

[0644] Preparation of IgG-binding micellar drug complexes

[0645] The same procedure as in Example 132 was carried out except that the antibody was changed to IgG (normal human IgG, whole molecule, purified product, 143-09501, FUJIFILM Wako Pure Chemical).

[0646] [Table 39]

[0647]

[0648] [Example 157]

[0649] Preparation of Cetuximab-Fab Conjugated Micelle Drug Complex

[0650] The fragment antibody (Fab) of cetuximab was prepared in the same manner as in Example 145 using a commercially available purification kit (Fab Preparation Kit, Pierce, #44985).

[0651] [Table 40]

[0652]

[0653] [Example 158]

[0654] Preparation of Exotecan-PAB-Cit-Val-Ahx Conjugate Copolymer

[0655] Step 1: Synthesis of Boc-Ahx-Val-Cit-PAB-PNP

[0656] 239 mg of Boc-Ahx-Val-Cit-PAB-OH (compound HDP 30.1267 described in International Publication No. 2016 / 142049) and 362 mg of bis(4-nitrophenyl) carbonate were dissolved in 1.5 mL of DMF, 207 μL of N,N-diisopropylethylamine was added, and stirred overnight at room temperature. After the reaction solution was distilled off under reduced pressure by an evaporator, it was purified by silica gel column chromatography (CHCl3 / MeOH=100 / 0→85 / 15) to obtain 241 mg of Boc-Ahx-Val-Cit-PAB-PNP.

[0657]

[0658] 1H NMR (400MHz, DMSO-d6) δ: 10.06 (s, 1H), 8.33-8.29 (m, 2H), 8.11 (d, J = 7.6Hz, 1H), 7.80 (d, J = 8.5Hz, 1H), 7.65 (d, J = 9.0Hz ,2H),7.57(dt,J=10.0,2.7Hz,2H),7.41(d,J=8.8Hz,2H),6.75(t,J=5.6Hz,1H),5.97(t,J=5.8Hz,1H),5.42(s,2H),5.24 (s,2H),4.41-4.34(m,1H),4.20(dd,J=8.8,7.6Hz,1H),3.07-2.92(m,2H),2.90-2.85(m,2H),2.23-2.08(m,2H),2.01-1 .91(m,1H),1.75-1.32(m,17H),1.25-1.17(m,2H),0.87(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H).MS(ESI)m / z:758.2[M+H] +

[0659] Step 2: Synthesis of Boc-Ahx-Val-Cit-PAB-Ixitecan

[0660] 240 mg of Boc-Ahx-Val-Cit-PAB-PNP and 168 mg of exitecan mesylate were dissolved in 6.33 mL of DMSO, 88.2 μL of triethylamine was added, and the mixture was stirred at room temperature overnight. After adding water to the reaction solution, the resulting precipitate was filtered out by suction filtration. The filtrate was purified by silica gel column chromatography (CHCl3 / MeOH=100 / 0→85 / 15) to obtain 202 mg of Boc-Ahx-Val-Cit-PAB-exitecan.

[0661]

[0662] 1H NMR (400MHz, DMSO-d6) δ: 9.98 (s, 1H), 8.09-8.04 (m, 2H), 7.81-7.76 (m, 2H), 7.61 (d, J = 8.5Hz, 2H), 7.36 (d, J = 8.5Hz, 2 H),7.31(s,1H),6.74(t,J=5.6Hz,1H),6.52(s,1H),5.97(t,J=5.8Hz,1H),5.45(s,2H),5.41(s,2H),5.34-5.23(m,3H ),5.08(s,2H),4.40-4.33(m,1H),4.19(dd,J=8.5,6.7Hz,1H),3.30-3.06(m,2H),3.05-2.84(m,4H),2.37(s,3H),2.2 4-2.08(m,4H),2.03-1.80(m,3H),1.74-1.31(m,17H),1.21-1.18(m,2H),0.89-0.82(m,9H).MS(ESI)m / z:1054.5[M+H] +

[0663] Step 3: Synthesis of H-Ahx-Val-Cit-PAB-Ixitecan trifluoroacetate

[0664] 100 mg of Boc-Ahx-Val-Cit-PAB-exitecan was dissolved in 3 mL of DCM, 0.3 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 4 hours. The reaction solution was distilled off under reduced pressure by an evaporator, and then purified by reverse phase chromatography (0.1% TFA aqueous solution / MeCN=100 / 0→60 / 40) using an octadecylsilane (ODS) column to obtain 63.7 mg of H-Ahx-Val-Cit-PAB-exitecan trifluoroacetate.

[0665]

[0666] MS (ESI) m / z: 954.5 [M+H] +

[0667] Step 4: Synthesis of Exotecan-PAB-Cit-Val-Ahx Conjugated Copolymer

[0668] 100 mg of the copolymer obtained by the same preparation method as in Example 72 was dissolved in 1 mL of DMF, and 25.4 mg of H-Ahx-Val-Cit-PAB-exitecan trifluoroacetate, 15.5 μL of N,N-diisopropylethylamine and 11.3 mg of hexafluorophosphate Azabenzotriazole TetramethylUronium (HATU) were added, and stirred at room temperature overnight. The reaction solution was dialyzed and purified (dialysis membrane: Spectra / PorRegenerated Cellulose Membrane 6, molecular weight cutoff: 3.5 kDa, external solution: methanol), and the solvent was removed by vacuum distillation and vacuum drying, and the copolymer was recovered to obtain 106 mg of an exitecan-PAB-Cit-Val-Ahx conjugated copolymer. The Z average particle size and polydispersity index of the obtained copolymer in water were measured by dynamic light scattering (DLS) and the result was 8.5 nm (polydispersity index: 0.129).

[0669]

[0670] The exotecan-PAB-Cit-Val-Ahx conjugate copolymer was measured by NMR. 1 The H-NMR spectrum was used to analyze the amount of exotecan introduced per molecule of the copolymer, and the result was 9.4 mol / mol.

[0671] [Measurement equipment and conditions, etc.]

[0672] (1) 1 H-NMR measurement

[0673] Device: JNM-ECX400 (400MHz) / JEOL

[0674] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0675] Sample concentration: 20mg / mL

[0676] Measurement temperature: 25℃

[0677] Total times: 399 times

[0678] result: Fig.21

[0679] [Examples 159 to 163]

[0680] The copolymer obtained in Example 158 was synthesized by appropriately changing the feed amount in the same manner as in Example 132. In addition, copolymers obtained in the same manner as in Examples 72, 79, 82 and 84 were synthesized by appropriately changing the feed amount in the same manner as in Examples 94, 96 and 132, respectively.

[0681] [Table 41]

[0682]

[0683] [Comparative Example 1]

[0684] Preparation of oxaliplatin solution

[0685] ELPLAT TM 1 mL of intravenous injection solution 50 mg (Yakult Honsha) was added to 5.58 mL of 5.9 (w / v) % glucose solution to prepare a 5 (w / v) % glucose solution containing 760 μg of oxaliplatin.

[0686] [Comparative Example 2]

[0687] Preparation of Cetuximab Solution

[0688] Cetuximab (Erbitux) was diluted with normal saline (Otsuka Naishi Injection) TM Injection 100 mg, Merck BioPharma Company), a 2 mg / mL solution of cetuximab was prepared for intravenous administration (dosage based on 10 mg / kg body weight). In addition, similarly, RPMI1640 (hereinafter referred to as RPMI1640 medium) containing 10% fetal bovine serum (Merck KGaA) was used for dilution, so that the final concentration of cetuximab was 66nmol / L, 20nmol / L, 6.6nmol / L, 2.0nmol / L, 0.66nmol / L, 0.20nmol / L, 0.066nmol / L or 0.020nmol / L, and the anti-cell effect was evaluated.

[0689] [Comparative Example 3]

[0690] Preparation of trastuzumab solution

[0691] Trastuzumab (Hecancer TMInjection 60, Chugai Pharmaceutical Co., Ltd.) 60 mg was dissolved in 3.0 mL of water for injection in accordance with the Japanese Pharmacopoeia to prepare a 20 mg / mL solution. Subsequently, the solution was diluted with RPMI1640 medium, and the final concentration of trastuzumab was 20 nmol / L, 6.8 nmol / L, 2.0 nmol / L, 0.68 nmol / L, 0.20 nmol / L, 0.068 nmol / L, 0.020 nmol / L, 0.0068 nmol / L or 0.0020 nmol / L to evaluate the anti-cell effect.

[0692] [Comparative Example 4]

[0693] Preparation of rituximab solution

[0694] Rituximab (Rituxan) was diluted in RPMI1640 medium. TM 100 mg was injected intravenously (Zen Yao Industry Co., Ltd.), and the final concentration of rituximab was 69 nmol / L, 21 nmol / L, 6.9 nmol / L, 2.1 nmol / L, 0.69 nmol / L, 0.21 nmol / L, 0.069 nmol / L or 0.021 nmol / L, and the anti-cellular effect was evaluated.

[0695] [Test Example 1] Drug efficacy test

[0696] Mouse colon cancer cell line C26 (American Type Culture Collection) was subcutaneously transplanted into female nude mice (BALB / c-nu / nu, 7 weeks old; Charles River Laboratories Japan), and the resulting tumor-bearing model was used for drug efficacy testing.

[0697] Mouse colorectal cancer cell line C26 subcultured in a CO2 incubator was suspended in liquid culture medium (high-glucose Dulbecco's modified Eagle's medium, Sigma-Aldrich) at a cell number of 1×10 per mouse. 6 The nude mice were then subcutaneously injected with 100 μL of the drug. After the nude mice were fed for about a week, the average tumor volume grew to about 30 mm. 3The administration of the drug was started at 1:00 p.m. . DACHPt-encapsulated SCNP (DACHPt-encapsulated SCNP prepared using the copolymer of Example 70) was administered intravenously in the tail vein (3 times every other day), and the anti-tumor effect was evaluated according to the tumor volume (4 to 5 mice in a group). For comparison, oxaliplatin solution (Comparative Example 1) was used and administered in the same manner. Regarding the dosage of each preparation, the maximum dosage of oxaliplatin solution that can be administered is set to 8 mg / kg (3.9 mg / kg in terms of Pt), and the DACHPt-encapsulated SCNP is set to 3 mg / kg in terms of Pt.

[0698] The changes in tumor volume over time are shown in Fig. 22 . In the case of DACHPt containing SCNPs, T / C = 0.4 [T / C: ratio of tumor volume of drug administration group (T) to control group (C)] was obtained 14 days after administration. In the case of oxaliplatin solution (Comparative Example 1), T / C = 1.1 was obtained 14 days after administration. In addition, it was confirmed that DACHPt containing SCNPs significantly inhibited tumor growth compared with the control group 14 days after administration (Student's t test). The above results indicate that DACHPt containing SCNPs has an excellent anti-tumor effect compared with oxaliplatin solution.

[0699] [Test Example 2] Anti-cell test

[0700] RPMI1640 medium was used to make 2.0×10 4 cells / mL and 4.0×10 4 The EGFR antigen-positive human breast cancer cell line MDA-MB-468 (American Type Culture Collection) and the EGFR antigen-negative human breast cancer cell line MDA-MB-453 (RIKEN BRC CELL BANK) were adjusted at 100 μL per well in a 96-well cell culture microplate and cultured overnight. The next day, the evaluation specimen diluted with RPMI medium was added to the microplate at 100 μL per well and cultured at 37°C and 5% CO2 for 4 days. After culture, CellTiter-Glo was added to the microplate. TM Luminescent Cell Viability Assay (Promega Corporation) was used and stirred, and the mixture was allowed to stand at room temperature for 10 minutes, and then the luminescence was measured using a plate reader (Molecular Devices, LLC.) The cell viability was calculated using the following formula.

[0701] Cell survival rate (%) = a ÷ b × 100

[0702] [wherein: a represents the average value of the luminescence amount of the sample wells (n=3), and b represents the average value of the luminescence amount of the wells without adding the sample (n=3)]

[0703] The IC concentration at which cell viability reaches 50% 50 Values ​​using SAS TM 9.4Software (SAS Institute Japan Ltd.) Calculated.

[0704] [Table 42]

[0705]

[0706] [Test Example 3] Anti-cell test

[0707] RPMI1640 medium was used to make 2.0×10 4 The HER2 antigen-positive human gastric cancer cell line NCI-N87 (American Type Culture Collection) or the HER2 antigen-negative human breast cancer cell line MDA-MB-468 was adjusted in the form of cells / mL and added to a 96-well cell culture microplate at 100 μL per well for overnight culture. The next day, the evaluation specimen diluted with RPMI medium was added to the microplate at 100 μL per well and cultured at 37°C and 5% CO2 for 4 days. After culture, CellTiter-Glo was added to the microplate. TM Luminescent Cell Viability Assay (Promega Corporation) was added and stirred, and the cells were allowed to stand at room temperature for 10 minutes before measuring the luminescence using a plate reader (Molecular Devices, LLC.). Cell viability and IC 50 value.

[0708] [Table 43]

[0709]

[0710] [Test Example 4] Anti-cell test

[0711] RPMI1640 medium was used to make 5.0×10 4CD20 antigen-positive Raji cells from human B-cell lymphoma or CD20 antigen-negative MOLT-4 cells from human T-lymphoblastic leukemia were adjusted at 10 cells / mL and added to a 96-well cell culture microplate at 50 μL per well for overnight culture. The next day, the evaluation specimen diluted with RPMI medium was added to the microplate at 50 μL per well and cultured at 37°C and 5% CO2 for 4 days. After culture, CellTiter-Glo was added to the microplate. TM Luminescent Cell Viability Assay (Promega Corporation) was added and stirred, and the cells were allowed to stand at room temperature for 10 minutes before measuring the luminescence using a plate reader (Molecular Devices, LLC.). Cell viability and IC 50 value.

[0712] [Table 44]

[0713]

[0714] [Test Example 5] Antitumor effect confirmation test

[0715] EGFR-positive human colorectal cancer cell line HT-29 was suspended in PBS to prepare 2.5×10 7 The concentration of 5×10 cells / mL was 6 Cells (0.2 mL) were transplanted subcutaneously into the right flank of nude mice (female, 6 weeks old) after the quarantine period. The mice were randomly divided into groups (12 mice per group) 8 days after the subcutaneous transplantation of the cells. Cetuximab-bound micelle drug complex (Example 138, DAR=38) was administered intravenously at a dose of 1.8 and 5.5 mg / kg (1.0 and 3.0 mg antibody / kg of the antibody portion, and 0.18 and 0.55 mg DM1 / kg of the DM1 portion), or DM1-bound micelles (Example 120, antibody unbound) were administered intravenously at a dose of 2.5 mg / kg (0.55 mg DM1 / kg of the DM1 portion). The group administered with normal saline was set as the control group. The results are shown in Fig.23 The evaluation specimens showed an antitumor effect that depended on the dosage. In addition, no weight loss of mice was observed after administration. [Test Example 6] Antitumor effect confirmation test

[0716] The HER2-positive human gastric cancer cell line NCI-N87 was suspended in PBS and prepared into 5×10 7 The concentration of 1×10 cells / mL was 7The cells (0.2 mL) were transplanted subcutaneously into the right flank of nude mice (female, 6 weeks old; Charles River Laboratories Japan).

[0717] Seven days after the subcutaneous transplantation of cells, the mice were randomly divided into groups (12 in each group). The trastuzumab-conjugated micelle drug complex (Example 151, DAR=40) was administered intravenously at a dose of 1.9 and 5.7 mg / kg (1.0 and 3.0 mg antibody / kg of the antibody portion, and 0.20 and 0.60 mg DM1 / kg of the DM1 portion), or the DM1-conjugated micelle (Example 120, antibody unconjugated) was administered at a dose of 0.70 mg / kg (0.20 mg DM1 / kg of the DM1 portion). The group administered with normal saline was set as the control group. The results are shown in Fig.24 The evaluation specimens showed an antitumor effect depending on the dosage. In addition, no weight loss of mice was observed after administration.

[0718] [Test Example 7] Antitumor effect confirmation test

[0719] EGFR-positive human breast cancer cell line MDA-MB-468 was suspended in PBS and prepared into 5×10 7 The concentration of 1×10 cells / mL was 7 The cells (0.2 mL) were transplanted subcutaneously into the right flank of nude mice (female, 6 weeks old; Jackson Laboratory Japan).

[0720] 27 days after subcutaneous cell transplantation, the mice were randomly divided into groups (10 mice per group). Cetuximab-bound micellar drug complex (Example 160, DAR=25) was administered intravenously at a dose of 3.0 mg / kg (1.6 mg antibody / kg based on the amount of the antibody portion, and 0.20 mg DM1 / kg based on the amount of the DM1 portion), or cetuximab-bound micellar drug complex (Example 161, DAR=41) was administered intravenously at a dose of 1.8 mg / kg (1.0 mg antibody / kg based on the amount of the antibody portion, and 0.20 mg DM1 / kg based on the amount of the DM1 portion). The group administered with normal saline was set as the control group. The results are shown in Fig.25 The test specimens were evaluated to have anti-tumor effects. In addition, no weight loss of mice was observed after administration.

[0721] [Test Example 8] Antitumor effect confirmation test

[0722] EGFR-positive human colorectal cancer cell line HCT-116 with KRAS mutation (G13D) was suspended in PBS to prepare 5×10 7 The concentration of 1×10 cells / mL was 7 The cells (0.2 mL) were transplanted subcutaneously into the right flank of nude mice (female, 6 weeks old; Jackson Laboratory Japan).

[0723] Seven days after the subcutaneous transplantation of cells, the mice were randomly divided into groups (10 mice per group). Cetuximab-bound micellar drug complex (Example 162, DAR=20) was administered intravenously at a dose of 5.5 mg / kg (3.1 mg antibody / kg based on the amount of the antibody portion, and 0.30 mg DM1 / kg based on the amount of the DM1 portion), or cetuximab-bound micellar drug complex (Example 163, DAR=43) was administered intravenously at a dose of 2.6 mg / kg (1.4 mg antibody / kg based on the amount of the antibody portion, and 0.30 mg DM1 / kg based on the amount of the DM1 portion). The group administered with normal saline was set as the control group. The results are shown in Fig.26 The test specimens were evaluated to have anti-tumor effects. In addition, no weight loss of mice was observed after administration.

[0724] The long and short diameters of the tumor were measured twice a week using a digital vernier caliper (product number 19975, Shinwa Rules Co., Ltd.), and the tumor volume was calculated according to the following formula.

[0725] Tumor volume (mm 3 )=a×b 2 / 2

[0726] [Wherein, a represents the major diameter (mm), and b represents the minor diameter (mm)].

Claims

1. A copolymer in which a target recognition molecule is bonded to a copolymer X having structural units represented by the following formulae (A), (B) and (C), In the formula, R 1 , R 2 and R 3 Same or different, indicating hydrogen atoms or C 1-3 Alkyl; R 4 Represents C 1-3 Alkyl; R 5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent; X 1 , X 2 and X 3 The same or different, representing oxygen atoms, sulfur atoms or NR 7 ; R 6 represents a hydrogen atom, a leaving group or a connecting group; R 7 Represents a hydrogen atom or C 1-3 An alkyl group; m represents an integer of 1 to 100; and n represents an integer of 0 to 3.

2. The copolymer according to claim 1, characterized in that: The polymer X is a copolymer formed by polymerization of three monomers represented by the following general formulas (1) to (3): In the formula, R 1 , R 2 and R 3 Same or different, indicating hydrogen atoms or C 1-3 Alkyl; R 4 Represents C 1-3 Alkyl; R 5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent; X 1 , X 2 and X 3 The same or different, representing oxygen atoms, sulfur atoms or NR 7 ; R 6 represents a hydrogen atom, a leaving group or a connecting group; R 7 Represents a hydrogen atom or C 1-3 An alkyl group; m represents an integer of 1 to 100; and n represents an integer of 0 to 3.

3. The copolymer according to claim 1 or 2, characterized in that: R 1 A hydrogen atom.

4. The copolymer according to claim 1 or 2, characterized in that: R 2 A hydrogen atom.

5. The copolymer according to claim 1 or 2, characterized in that: R 3 A hydrogen atom.

6. The copolymer according to claim 1 or 2, characterized in that: R 4 It is methyl.

7. The copolymer according to claim 1 or 2, characterized in that: R 5 is a C which may have a substituent 6-18 Aryl.

8. The copolymer according to claim 1 or 2, characterized in that: R 5 It is phenyl.

9. The copolymer according to claim 1 or 2, characterized in that: R 6 A hydrogen atom.

10. The copolymer according to claim 1 or 2, characterized in that: R 6 The leaving group is a group represented by the following formula (4):

11. The copolymer according to claim 1 or 2, characterized in that: R 6 The linking group is a group represented by the following formula (5): In the formula, R 8 Represents a hydrogen atom or a drug; Ak 1 Represents C 1-7 Alkylene bond; X 4 represents an oxygen atom, a sulfur atom or -N(R 7 )-, R 7 Represents a hydrogen atom or C 1-3 alkyl.

12. The copolymer according to claim 1 or 2, characterized in that: X 1 For oxygen atoms.

13. The copolymer according to claim 1 or 2, characterized in that: X 2 For oxygen atoms.

14. The copolymer according to claim 1 or 2, characterized in that: X 3 is an oxygen atom or NH.

15. The copolymer according to claim 1 or 2, characterized in that: m is an integer from 4 to 22.

16. The copolymer according to claim 1 or 2, characterized in that: n is 1.

17. The copolymer according to claim 1 or 2, characterized in that: The ratio of the structural units (A), (B), and (C) is 0.01 to 100 parts by mass of (B) and 0.1 to 100 parts by mass of (C) per 1 part by mass of (A).

18. The copolymer according to claim 2, characterized in that: The monomer (2) is prepared by polymerizing 0.01 to 100 parts by mass of the monomer (2) and 0.1 to 100 parts by mass of the monomer (3) with respect to 1 part by mass of the monomer (1).

19. The copolymer according to claim 1 or 2, characterized in that: The number average molecular weight is 5000 to 150000.

20. The copolymer according to claim 1 or 2, characterized in that: The target recognition molecule is an antibody.

21. The copolymer according to claim 20, characterized in that: The antibody is an anti-EGFR antibody, an anti-Her2 antibody, an anti-CD20 antibody, an anti-CD276 antibody, an anti-MUC1 antibody, an anti-PD-L1 antibody, or an anti-TROP-2 antibody.

22. The copolymer according to claim 20, characterized in that: The antibody is cetuximab, panitumumab, necituzumab, ervantumab, panitumumab, trastuzumab, pertuzumab, makituximab, rituximab, ibritumomab tiuxetan, tositumomab, ofatumumab, obatuzumab, clituzumab, gatuzumab, ifenatumumab, mizotuzumab, bolamituzumab, atezolizumab, avelumab, durvalumab, sacetuzumab, or a functional fragment thereof.

23. A drug complex, characterized in that: The invention comprises the copolymer according to claim 1 or 2 and a drug.

24. The drug complex according to claim 23, characterized in that: The drug is a metabolic antagonist, an alkylating agent, an anthracycline, an antibiotic, a mitotic inhibitor, a topoisomerase inhibitor, a proteasome inhibitor, or an anti-hormone agent.

25. The drug complex according to claim 23, characterized in that: The drug is DM0, DM1, DM2, DM3, DM4, metansine, auristatin E, auristatin phenylalanine phenylenediamine (AFP), monomethyl auristatin E, monomethyl auristatin D, monomethyl auristatin F, paclitaxel, docetaxel, irinotecan, topotecan, nojitecan, amsacrine, etoposide, teniposide, mitoxantrone, SN-38, exitecan, or delunotecan.

26. The drug complex according to any one of claims 23 to 25, characterized in that: The binding of the target recognition molecule or drug to the copolymer X is covalent binding or non-covalent binding.

27. The drug complex according to any one of claims 23 to 26, characterized in that: The combination of the target recognition molecule or drug and the copolymer X is the following formula (a): In the formula, J 1 It is the binding part with the target recognition molecule or drug; 2 is the bonding part with copolymer X; Ak 2 ,Ak 3 Each independently represents a single bond or C 1-7 Alkylene bond; B 1 , B 2 Each independently represents a single bond, an amide group, or an ester bond; L 1 Represents a single bond, -(CH2CH2O) o CH2CH2-, phenylene, cyclohexylene, -NH-peptide-CO-, or phenylene-NH-peptide-CO-; o represents an integer from 0 to 100.

28. A single-chain nanoparticle, characterized in that: The copolymer comprising claim 1 or 2.

29. A single-chain nanoparticle, characterized in that: Comprising the drug complex of claim 23 or 24.

30. A pharmaceutical composition, characterized in that: The copolymer comprising claim 1 or 2.

31. A pharmaceutical composition, characterized in that: Comprising the drug complex of claim 23 or 24.

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