A process for the preparation of an intermediate of a camptothecin derivative

Abstract

The present disclosure relates to a preparation method of an intermediate of camptothecin derivative. Specifically, the present disclosure relates to a preparation method of a compound as shown in formula (II), which comprises the step of reacting a compound as shown in formula (III) with a compound as shown in formula (VII) in the presence of a base. The method has high yield and mild reaction conditions, and is suitable for industrial production.

Description

Technical Field

[0001] This disclosure pertains to the pharmaceutical field and relates to a method for preparing an intermediate of a camptothecin derivative. Background Technology

[0002] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds. This fully leverages the specificity of antibodies in binding to antigens on the surface of normal and tumor cells, as well as the high efficiency of cytotoxins, while avoiding the drawbacks of low efficacy of the former and excessive toxicity of the latter. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can precisely bind to tumor cells and reduce the impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329–332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814).

[0003] Several classes of small molecules with cytotoxicity are used in antibody-drug conjugates, one of which is camptothecin derivatives, which have antitumor effects by inhibiting topoisomerase I. Reports on the application of the camptothecin derivative eczemacon (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione) in antibody-drug conjugates (ADCs) include WO2014057687, WO2020063676, WO2020063673, and CN112125915A.

[0004]

[0005] WO2020063676 discloses the compound icinotecan and its preparation method, in which compounds C and D are important reaction intermediates.

[0006] Summary of the Invention

[0007] The purpose of this disclosure is to provide a new method for preparing intermediates of camptothecin derivatives.

[0008] This disclosure also provides a method for preparing a compound as shown in formula (II), comprising the step of reacting a compound as shown in formula (III) with a compound as shown in formula (VII) under the presence of a base.

[0009]

[0010] The alkali is selected from carbonates, bicarbonates, alkoxides, hydroxides, or hydrides of alkali metals or alkaline earth metals.

[0011] R1 and R2 are each independently selected from hydrogen atoms and C atoms. 1-6 Alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl, or 5-10 membered heteroaryl, wherein the alkyl, cycloalkyl, aryl, or heteroaryl group is optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino, oxo, 3-6 membered cycloalkyl, 6-10 membered aryl, or C1-C6 alkoxy.

[0012] Alternatively, R1 and R2 together with the carbon atom they are attached to form a 3-6 membered cycloalkyl group, optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino, oxo or C1-C6 alkoxy groups.

[0013] R3, R4, and R5 are each independently selected from hydrogen atoms or C atoms. 1-6 alkyl;

[0014] R6 is selected from hydrogen atom, deuterium atom, and C. 1-6 Alkyl, 6-10 aryl or 5-10 heteroaryl, wherein the alkyl, aryl or heteroaryl group is optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino and oxo groups;

[0015] R c Selected from amino groups or amino groups protected by amino protecting groups;

[0016] m is an integer from 0 to 4.

[0017] The solvent used in the reaction can be one or more conventional solvents, such as dimethylformamide, 1-methyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, dioxane, toluene, dimethyl sulfoxide, diethyl ether, isopropyl ether, methyl tert-butyl ether, dichloromethane, chloroform, acetone, acetonitrile, methanol, ethanol, isopropanol, and water, preferably one or more of tetrahydrofuran, ethyl acetate, dioxane, toluene, dimethyl sulfoxide, diethyl ether, isopropyl ether, dichloromethane, chloroform, acetone, acetonitrile, methanol, ethanol, and isopropanol.

[0018] The amino protecting group can be a tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, benzyloxycarbonyl, or other amino protecting groups commonly used in peptide synthesis. Other amino protecting groups include alkanoyl groups such as acetyl; alkanoyl groups such as methoxycarbonyl and ethoxycarbonyl; arylmethoxycarbonyl groups such as p-methoxybenzyloxycarbonyl and p-(or o-)nitrobenzyloxycarbonyl; arylmethyl groups such as benzyl and triphenylmethyl; aryl acyl groups such as benzoyl; arylsulfonyl groups such as 2,4-dinitrobenzenesulfonyl and o-nitrobenzenesulfonyl; preferably 9-fluorenylmethyloxycarbonyl.

[0019] In some embodiments, the alkali is selected from alkali metal carbonates, bicarbonates, or hydroxides, preferably sodium carbonate or potassium carbonate.

[0020] In some embodiments, the molar ratio of the compound represented by formula (III) to the base is 1:0.1 to 1:10, preferably 1:1 to 1:5.

[0021] In some embodiments, R1 is selected from hydrogen atoms, C atoms, and C atoms. 1-6 Alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl, or 5-10 membered heteroaryl, wherein the alkyl, cycloalkyl, aryl, or heteroaryl group is optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino, oxo, 3-6 membered cycloalkyl, 6-10 membered aryl, or C1-C6 alkoxy.

[0022] R2 is selected from a hydrogen atom or optionally from a C1-C6 alkyl group substituted with one or more substituents selected from halogen, hydroxyl, amino, or oxo groups.

[0023] Alternatively, R1 and R2 together with the carbon atom they are attached to form a 3-6 membered cycloalkyl group, optionally substituted with one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino, oxo, or C1-C6 alkoxy groups.

[0024] In some implementations, R1 is selected from C 1-6 Alkyl, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl substituted C 1-6 alkyl, 6-10 aryl substituted C 1-6 Alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl, or 5-10 membered heteroaryl, wherein R2 is a hydrogen atom, or R1 and R2 together with the carbon atom attached to them form a 3-6 membered cycloalkyl; preferably R1 is selected from C. 1-6 Alkyl, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl, where R2 is a hydrogen atom, or R1 and R2 together with the carbon atom attached to them form a 3-6 membered cycloalkyl.

[0025] In some implementations, R3, R4, and R5 are all hydrogen atoms.

[0026] In some embodiments, R6 is selected from hydrogen atoms, C atoms, and C atoms. 1-6 Alkyl, Halogenated C 1-6 Alkyl or hydroxy C 1-6 Alkyl group, more preferably hydrogen atom.

[0027] In some embodiments, the method includes

[0028]

[0029] This disclosure provides a method for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof, including a method for preparing a compound of formula (II) as described in this disclosure.

[0030]

[0031] in,

[0032] R1, R2, R3, R4, R5, R6, m are as described above, and n is an integer from 2 to 8.

[0033] In some embodiments, the compound represented by formula (I) is selected from...

[0034]

[0035] The compound shown in formula (I) can be prepared by using the compound shown in formula (II) as a reactant using methods disclosed in the prior art, such as those disclosed in WO2014057687, WO2020063676, WO2020063673, CN112125915A, etc., which are incorporated herein by reference in their entirety.

[0036] Optional solutions include, for example, Solution 1:

[0037]

[0038] Option 2:

[0039]

[0040] Among them, R1, R2, R3, R4, R5, R6, m, n, R c As mentioned earlier, R d Selected from amino groups or amino groups protected by amino protecting groups.

[0041] This disclosure also provides a method for preparing an antibody-drug conjugate, comprising: the step of preparing the compound shown in formula (I) as described in this disclosure, and the step of reducing Ab and then coupling it with the compound shown in formula (I) to obtain the antibody-drug conjugate shown in formula (X).

[0042]

[0043] Where Ab is an antibody or antigen-binding fragment, k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any value between any two values), and R1, R2, R3, R4, R5, R6, m, n are as described above.

[0044] The reducing agent is preferably TCEP, and in particular, it is preferred to reduce the disulfide bonds on the antibody.

[0045] In some embodiments, the antibody is selected from chimeric antibodies, humanized antibodies, or fully human antibodies; preferably, it is a monoclonal antibody.

[0046] In some embodiments, the antibody or its antigen-binding fragment is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, or anti-Mesothelin antibody or its antigen-binding fragment.

[0047] In some embodiments, the antibody or its antigen-binding fragment is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, Glematumamab, or its antigen-binding fragment.

[0048] In some implementations, k is 2 to 8, preferably 5 to 9. Non-limiting embodiments include 3, 4, 5, 6, 7.2, 7.5, 8, 8.5, and 9.

[0049] In some embodiments, the method includes

[0050]

[0051] Antibody-drug conjugates can be prepared from compounds, antibodies, or antigen-binding fragments of formula (I) using methods disclosed in the prior art, such as those disclosed in WO2014057687, WO2020063676, WO2020063673, CN112125915A, etc., which are incorporated herein by reference in their entirety.

[0052] The "alkyl" referred to in this disclosure is preferably a C1-C6 alkyl.

[0053] The "alkenyl" as described in this disclosure is preferably a C2-C6 alkenyl.

[0054] The "alkynyl group" described in this disclosure is preferably a C2-C6 alkynyl group.

[0055] The "alkylene" described in this disclosure is preferably a C1-C6 alkylene.

[0056] The "sub-alkenyl" described in this disclosure is preferably a C2-C6 sub-alkenyl.

[0057] The "sub-chain alkynyl" described in this disclosure is preferably a C2-C6 sub-chain alkynyl.

[0058] The "alkoxy group" described in this disclosure is preferably a C1-C6 alkoxy group.

[0059] The "alkyl thioether group" described in this disclosure is preferably a C1-C6 alkyl thioether group.

[0060] The "cycloalkyl" in this disclosure is preferably 3 to 12-membered, and more preferably 3 to 6-membered cycloalkyl.

[0061] The "fused cycloalkyl" described in this disclosure is preferably a 6- to 14-membered fused cycloalkyl, more preferably a 7- to 10-membered fused cycloalkyl.

[0062] The "heterocyclic group" described in this disclosure is preferably a 3- to 12-membered heterocyclic group, and more preferably a 3- to 6-membered heterocyclic group.

[0063] The "fused heterocyclic group" described in this disclosure is preferably a 6- to 14-membered fused heterocyclic group, and more preferably a 7- to 10-membered fused heterocyclic group.

[0064] The "aryl" in this disclosure is preferably 6 to 14 methyl groups, and more preferably 6 to 10 methyl groups.

[0065] The "heteroaryl" as described in this disclosure is preferably 5 to 12 quinones, and more preferably 5 to 10 quinones.

[0066] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0067] The term "antibody-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker. In this disclosure, "antibody-drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active glucocorticoid via a stable linker. The antibody or antibody fragment may bind to a glucocorticoid molecule containing a linker via specific groups therein (e.g., interchain disulfide bonds).

[0068] The term "drug loading" refers to the average amount of drug carried by each antibody-drug conjugate molecule in a population of antibody-drug conjugates, and can also be expressed as the ratio of drug amount to antibody amount. The drug loading range can be 1-20, preferably 1-10, glucocorticoids (D) linked to each antibody (Ab). In embodiments of this disclosure, the drug loading is represented by k, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the average of any two values. Preferably 1-10, more preferably 1-8, or 2-8, or 2-7, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or the average of 4-5. The average amount of drug per ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, CE-SDS (monoclonal antibody size variant assay), and HPLC characterization.

[0069] The term "antibody" refers to immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further divide them into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in the constant region. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0070] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0071] The antibodies disclosed herein include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

[0072] The term "mouse antibody" in this disclosure refers to antibodies prepared using mice in accordance with the knowledge and skills in the art. Preparation involves injecting a test subject with a specific antigen, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics.

[0073] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can reduce the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies must first be established. Then, the variable region gene is cloned from the murine hybridoma cells. Next, the constant region gene of the human antibody is cloned as needed. The murine variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

[0074] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., a human germline antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse or reversion mutations to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR. Further literature describing methods that can be used to participate in humanization includes, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and Winter et al. [Jones et al., Nature, 321, 522 (1986), Riechmann et al., Nature, 332, 323-327 (1988), Verhoeyen et al., Science, 239, 1534 (1988)].

[0075] The term "fully human antibody," also known as a "fully human monoclonal antibody," refers to an antibody whose variable and constant regions are both human-derived, eliminating immunogenicity and toxicity. The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. This disclosure pertains to fully human monoclonal antibodies. Related technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

[0076] The term “antigen-binding fragment” refers to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in “antigen-binding fragments” include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bridges on hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains of a single arm of the antibody; (v) single-domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546) consisting of a VH domain; and (vi) separate complementarity-determining regions (CDRs) or (vii) combinations of two or more separate CDRs optionally linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0077] Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by treating IgG antibody molecules with the protease papain (which cleaves the amino acid residue at position 224 of the H chain). Approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

[0078] F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000, possessing antigen-binding activity, and containing two Fab regions connected at the hinge position, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin.

[0079] Fab' is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2.

[0080] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote to express the Fab'.

[0081] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using variants with 1–4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444–6448). Other connectors that may be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0082] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable domain and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable domain. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (see Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, in the classic format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).Combining the CDR definitions from Kabat and Chothia, the CDR is composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL. Following IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). Following IMGT rules, the CDR region of an antibody can be determined using the IMGT / DomainGap Align procedure.

[0083] The term "antibody framework" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0084] The term “epitope” or “antigenic determinant” refers to the site on an antigen where an immunoglobulin or antibody specifically binds. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)).

[0085] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example: approximately less than 10 -8 M, 10 - 9 M or 10 -10 M or lower affinity (KD) binding.

[0086] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0087] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

[0088] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to a non-human CDR region. Human FR germline sequences can be obtained by comparing with the IMGT Human Antibody Variable Region Germline Gene Database and MOE software, from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr, or from the journal Immunoglobulins, 2001 ISBN012441351.

[0089] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.

[0090] The engineered antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or GSepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0091] Amino acid sequence “identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence, after aligning the amino acid sequences and, where necessary, introducing gaps to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0092] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0093] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester.

[0094] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0095] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups. "Carbocyclic" refers to the ring system within the cycloalkyl group.

[0096] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m The heteroatom (where m is an integer from 0 to 2) excluding the ring portion of -OO-, -OS-, or -SS-, with the remaining ring atoms being carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 6 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, imidazoalkyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazoyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably piperidinyl or pyrrolidinyl. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups. "Heterocyclic" refers to the ring system within the heterocyclic group.

[0097] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring. "Aromatic ring" refers to the ring system within the aryl group. Non-limiting examples of aryl groups include:

[0098]

[0099]

[0100] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylic acid ester group, preferably phenyl.

[0101] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 12-membered, such as imidazolyl, furanyl, thiophenel, thiazolyl, pyrazolyl, oxazolyl, pyrrololyl, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl, or thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. "Heteroaryl ring" refers to the ring system within the heteroaryl group. Non-limiting examples of heteroaryl groups include:

[0102]

[0103] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylic acid ester group.

[0104] "Carboxyl protecting group" is a suitable group known in the art for carboxyl protection, see reference ("Protective Groups in Organic Synthesis", 5). Th The carboxyl protecting group in Ed.TWGreene & P.GMWuts, as an example, can be a substituted or unsubstituted C. 1-10 Straight-chain or branched alkyl, substituted or unsubstituted C 2-10 Straight-chain or branched alkenyl or alkynyl, substituted or unsubstituted C 3-8 Cyclic alkyl, substituted or unsubstituted C 5-10 aryl or heteroaryl, or (C 1-8 Alkyl or aryl) 3-silyl, etc.

[0105] "Amino protecting group" is a suitable group known in the art for amino protection, see reference ("Protective Groups in Organic Synthesis", 5). Th The amino protecting group in Ed.TW Greene & P. ​​GMWuts, preferably, is a (C 1-10 Alkyl or aromatic acyl group, such as formyl, acetyl, benzoyl, etc.; can be (C 1-6 Alkyl or C 6-10 aryl)sulfonyl; or (C 1-6 Alkoxy or C 6-10 Aryloxy)carbonyl, such as Boc or Cbz; can also be substituted or unsubstituted alkyl, such as triphenylmethyl (Tr), 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).

[0106] "Optional" or "optionally" means that the event or circumstance described below may, but does not have to, occur. This description includes situations in which the event or circumstance may or may not occur. For example, "optionally alkyl-substituted heterocyclic group" means that an alkyl group may, but does not have to, be present. This description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0107] In the chemical structure of the compounds described in this disclosure, the bonds... The configuration is not specified, meaning that if configurational isomerism exists in the chemical structure, the bond... It can be Or simultaneously include Two configurations. In the chemical structure of the compounds described in this disclosure, the bonds... No configuration is specified, meaning it can be Z configuration, E configuration, or both configurations. Detailed Implementation

[0108] The following detailed explanation of this disclosure will be provided with specific examples to enable those skilled in the art to have a more comprehensive understanding of this disclosure. The specific examples are only used to illustrate the technical solutions of this disclosure and do not limit this disclosure in any way.

[0109] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard.

[0110] MS measurements were performed using a Finnigan LCQAd(ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQadvantage MAX).

[0111] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent HPLC 1200DAD, an Agilent HPLC 1200VWD, and a Waters HPLC e2695-2489 high-performance liquid chromatograph.

[0112] Chiral HPLC analysis was performed using an Agilent 1260DAD high-performance liquid chromatograph.

[0113] High performance liquid chromatography (HPLC) was performed using Waters 2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson-281 preparative chromatographs.

[0114] Chiral preparation was performed using a Shimadzu LC-20AP preparative chromatograph.

[0115] Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15 mm to 0.2 mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4 mm to 0.5 mm.

[0116] Silica gel column chromatography generally uses Yantai Huanghai silica gel with a mesh size of 200-300 as the carrier.

[0117] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0118] Unless otherwise specified in the examples, the reactions can be carried out under an argon or nitrogen atmosphere.

[0119] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0120] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0121] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20℃~30℃.

[0122] Example 1

[0123]

[0124] Compound A (105 g), compound B (100.6 g, prepared according to the method disclosed in WO2020063676), dichloromethane (3.15 L), and methanol (1.05 L) were added to a reaction flask. The mixture was cooled to 0 °C, and DMTMM (87.3 g) and anhydrous potassium carbonate (81.9 g) were added. The mixture was kept warm and stirred. After the reaction was complete, purified water (1.0 L) was added and stirred. Then, dichloromethane (3.1 L) was added and stirred. The mixture was allowed to stand, and the organic phase was collected. The organic phase was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (eluting with dichloromethane:methanol (40:1)) to obtain compound C (154.2 g, yield 92.7%). The purity was determined to be 99.5% by HPLC.

[0125] Example 2

[0126]

[0127] Compound C (10 mg, 11.88 μmol) was placed in a reaction flask, and DCM (2 mL) and diethylamine (870 μg, 11.88 μmol) were added. The mixture was stirred at room temperature under argon protection. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the residue was washed with n-hexane. The supernatant was removed, and the residue was concentrated under reduced pressure to obtain 7.4 mg of compound D. The product was used directly in the next reaction without purification.

[0128] Example 3

[0129]

[0130] Compound D (10.5 mg, 16.9454 μmol) was placed in a reaction flask, DMF was added, and the mixture was cooled in an ice bath. Compound E (8 mg, 16.9316 μmol) was then added while the mixture was in an ice bath, and the mixture was brought to room temperature with stirring. After the reaction was complete, the product was purified by high-performance liquid chromatography (HPLC) (separation conditions: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm column, mobile phase: A-water (5 mmol NH4OAc), B-acetonitrile), yielding a total of 2.7 mg of compound I-1.

[0131] Example 4

[0132] According to the method in Example 1, compound A (105 g) was added, and anhydrous potassium carbonate was replaced with triethylamine (81.9 g) to obtain compound C (142.7 g, yield 85.8%). Its purity was determined to be 98.24% by HPLC.

[0133] Example 5: Stability study of C samples obtained by different processes

[0134] Samples C obtained in Examples 1 and 4 were placed at room temperature and sampled at 0, 7, 15, and 30 days. Related substances were detected by HPLC, and the results are shown in the table below:

[0135] Table 1. Stability study of sample C obtained by different processes

[0136]

[0137]

[0138] Since this disclosure has been described in accordance with its specific implementation, certain modifications and equivalent variations will be apparent to those skilled in the art and are included within the scope of this disclosure.

Claims

1. A method for preparing a compound as shown in formula (II), comprising the step of reacting a compound as shown in formula (III) with a compound as shown in formula (VII) under the presence of a base. in, The alkali is selected from potassium carbonate. R1 is selected from C 1-6 Alkyl, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl substituted C 1-6 alkyl, 6-10 aryl substituted C 1-6 Alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl or 5-10 membered heteroaryl, where R2 is a hydrogen atom; Alternatively, R1 and R2 together with the carbon atoms they are attached to form 3-6 membered cycloalkyl groups; R3, R4, and R5 are each independently selected from hydrogen atoms or C atoms. 1-6 alkyl; R6 is selected from hydrogen atom, deuterium atom, and C. 1-6 Alkyl, 6-10 aryl or 5-10 heteroaryl, wherein the alkyl, aryl or heteroaryl group is optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino and oxo groups; R c Selected from amino groups or amino groups protected by amino protecting groups; m is an integer from 0 to 4.

2. The preparation method according to claim 1, wherein R c Selected from amino groups protected by 9-fluorenylmethyloxycarbonyl groups.

3. The preparation method according to claim 1 or 2, wherein the molar ratio of the compound represented by formula (III) to the base is 1:0.1 to 1:

10.

4. The preparation method according to claim 3, wherein the molar ratio of the compound represented by formula (III) to the base is 1:1 to 1:

5.

5. The preparation method according to claim 1, wherein R1 is selected from C 1-6 Alkyl, C 1-6 Halogenated alkyl, 3-6 membered cycloalkyl, where R2 is a hydrogen atom, or R1 and R2 together with the carbon atom attached to them form a 3-6 membered cycloalkyl.

6. The preparation method according to claim 1, wherein R3, R4, and R5 are all hydrogen atoms.

7. The preparation method according to claim 1, wherein R6 is selected from hydrogen atoms, C 1-6 Alkyl, Halogenated C 1-6 Alkyl or hydroxy C 1-6 alkyl.

8. The preparation method according to claim 7, wherein R6 is selected from hydrogen atoms.

9. The preparation method according to claim 1, wherein the method comprises 。 10. A method for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof, comprising the steps of preparing the compound of formula (II) as described in any one of claims 1-9. in, R1, R2, R3, R4, R5, R6, m as described in claim 1, where n is an integer from 2 to 8.

11. The preparation method according to claim 10, wherein the method further comprises: 。 12. The preparation method according to claim 11, wherein the method further comprises: 。 13. The preparation method according to claim 10, wherein the compound represented by formula (I) is selected from... 。 14. A method for preparing an antibody-drug conjugate, comprising the step of preparing the compound of formula (I) as described in any one of claims 10-13, and the step of reducing Ab and then coupling it with the compound of formula (I) to obtain the antibody-drug conjugate of formula (X). Where Ab is an antibody or antigen-binding fragment, k is 1 to 20, R1, R2, R3, R4, R5, R6, and m are as described in claim 1, and n is an integer from 2 to 8.

15. The preparation method according to claim 14, wherein the antibody is selected from chimeric antibodies, humanized antibodies, or fully human antibodies.

16. The preparation method according to claim 14, wherein the antibody is selected from monoclonal antibodies.

17. The preparation method according to claim 14, wherein the antibody or its antigen-binding fragment is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, or anti-Mesothelin antibody or its antigen-binding fragment.

18. The preparation method according to claim 14, wherein the antibody or its antigen-binding fragment is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, Glematumamab, or its antigen-binding fragment.

19. The preparation method according to claim 14, wherein k is 2 to 8.

20. The preparation method according to claim 14, wherein k is 5 to 9.

21. The preparation method according to claim 14, wherein the method comprises 。

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