Preparation method of camptothecin derivative and intermediate thereof

Abstract

The invention relates to a preparation method of a camptothecin derivative and an intermediate thereof. The preparation method comprises the following steps: preparing a compound shown as a formula (V) from a compound shown as a formula (VI), and preparing the compound shown as the formula (IV) or the salt thereof from the compound shown as the formula (V). The method is high in yield, mild in reaction condition and suitable for industrial production. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to a method for preparing a camptothecin derivative and an intermediate thereof. Background Art

[0002] Antibody drug conjugate ADC ) connects monoclonal antibodies or antibody fragments to biologically active cytotoxins through stable chemical linker compounds, making full use of the antibody's specificity for binding to surface antigens of normal cells and tumor cells and the high efficiency of cytotoxins, while avoiding the defects of the former's low efficacy and the latter's excessive toxic side effects. This means that compared with traditional chemotherapy drugs, antibody-drug conjugates can accurately bind to tumor cells and reduce the impact on normal cells.

[0003] There are several types of cytotoxic small molecules used in antibody-drug conjugates, one of which is camptothecin derivatives, which have antitumor effects by inhibiting topoisomerase I. It is reported that the camptothecin derivative exotecan (chemical name: (1S,9S)-1- Amino The literature on the application of 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 (ADC) includes WO2014057687, WO2020063676, WO2020063673, CN112125915A, CN115925796A, etc. Summary of the invention

[0004] In one aspect, the present disclosure provides a method for preparing a compound of formula (IV) or a salt thereof, comprising: a step of preparing a compound of formula (V) from a compound of formula (VI), and a step of preparing a compound of formula (IV) or a salt thereof from a compound of formula (V), wherein:

[0005] R1 is an amino group or an amino group protected by an amino protecting group,

[0006] R2 is a hydroxyl protecting group,

[0007]

[0008] In some embodiments, the amino protecting group disclosed herein is selected from acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, pivaloyl, formyl, benzoyl, phthaloyl, 9-fluorenylmethoxycarbonyl, tert-butyloxycarbonyl and benzyloxycarbonyl, such as 9-fluorenylmethoxycarbonyl.

[0009] In some embodiments, the hydroxyl protecting group described in the present disclosure is selected from p-nitrobenzyl, acetyl, benzoyl, formyl, trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, isopropyldiethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl, tert-butyloxycarbonyl, methoxymethyl and benzyloxymethyl, such as acetyl or benzoyl.

[0010] In some embodiments, R1 is an amino group protected by an amino protecting group.

[0011] In some embodiments, the compound represented by formula (VI) is reacted with an aldehyde to prepare a compound represented by formula (V). In some embodiments, the molar ratio of the compound represented by formula (VI) to the aldehyde is 1:1-1:5, for example, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4. In some embodiments, the aldehyde may be formaldehyde.

[0012] In some embodiments, R2 is benzoyl, and the reaction for preparing the compound shown in formula (IV) or its salt is reacted in the presence of benzoyl halide and acid binding agent. The acid binding agent is selected from at least one of potassium carbonate, triethylamine, DIPEA, and pyridine, such as triethylamine. In some embodiments, the molar ratio of the compound shown in formula (V) to benzoyl halide is 1:1-1:5, such as 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.7, 1:1.8, 1:2, 1:2.5, 1:3, 1:3.5, 1:4. In some embodiments, the benzoyl halide can be benzoyl chloride.

[0013] In some embodiments, the reaction solvent in each step is independently selected from one or more of water, ethyl acetate, isopropyl acetate, dimethylformamide, 1-methyl-2-pyrrolidone, tetrahydrofuran, methyltetrahydrofuran, dioxane, toluene, xylene, dimethyl sulfoxide, ethyl ether, isopropyl ether, methyl tert-butyl ether, acetonitrile, propionitrile, isopropanol, propanol, ethanol, and methanol.

[0014] In some embodiments, the reaction temperature of each step is -20-100°C, for example, -10°C, 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C.

[0015] In some embodiments, the compound represented by formula (IV) is a compound represented by formula (IV-1), and the method comprises:

[0016]

[0017] In some embodiments, the compound represented by formula (VI-1) is reacted with aldehyde to prepare the compound represented by formula (V-1), and the molar ratio of the compound represented by formula (V-1) to the aldehyde is 1:1-1:5.

[0018] In some embodiments, the reaction for preparing the compound represented by formula (IV) or its salt is carried out in the presence of benzoyl halide and an acid binding agent, and the molar ratio of the compound represented by formula (V-1) to benzoyl halide is 1:1-1:5.

[0019] The present disclosure also provides a method for preparing a compound of formula (III) or a salt thereof, comprising the steps of preparing a compound of formula (IV) or a salt thereof as described in the present disclosure, and the steps of preparing a compound of formula (III) or a salt thereof from a compound of formula (IV) or a salt thereof,

[0020]

[0021] Wherein, R1 is as described above;

[0022] L1 is -(C(R 11 )(R 12 )) n -(CH2) m -, C3-C6 saturated cycloalkyl or 3-6 membered saturated heterocycloalkyl, the C3-C6 saturated cycloalkyl and 3-6 membered saturated heterocycloalkyl are each independently optionally substituted by one or more substituents selected from halogen, hydroxyl, amino and haloalkyl;

[0023] R 11 , R 12 are independently selected from hydrogen atoms, C 1-6 alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl or 5-10 membered heteroaryl, wherein the alkyl, cycloalkyl, aryl or heteroaryl is optionally substituted with one or more substituents selected from C1-C6 alkyl, halogen, hydroxy, amino, oxo, 3-6 membered cycloalkyl, 6-10 membered aryl or C1-C6 alkoxy, or, R 11 , R 12 Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxy, amino, oxo or C1-C6 alkoxy;

[0024] m is 0 or 1, for example 0; n is 1, 2, 3 or 4, for example 1.

[0025] In some embodiments, the compound represented by formula (III) is selected from

[0026] In some embodiments, the method comprises the steps of preparing a compound represented by formula (III') or a salt thereof from a compound represented by formula (IV) or a salt thereof, and preparing a compound represented by formula (III') or a salt thereof by decarboxylating a protecting group of the compound represented by formula (III') or a salt thereof.

[0027]

[0028] Wherein, R3 is a carboxyl group protected by a carboxyl protecting group, such as methyl, substituted methyl, ethyl, 2-substituted ethyl, allyl, tert-butyl, alkoxyalkyl, alkoxyalkoxyalkyl, 2,6-dialkylphenyl, benzyl, substituted benzyl, silyl or stannyl, such as methyl, allyl, tert-butyl, benzyl, 2,4-dimethoxybenzyl, p-methylbenzyl, pentafluorophenyl or methoxyethoxymethyl.

[0029] The present disclosure also provides a method for preparing a compound represented by formula (II) or a salt thereof, comprising the steps of preparing a compound represented by formula (IV) or a salt thereof and / or a compound represented by formula (III) or a salt thereof as described in the present disclosure,

[0030]

[0031] Among them, R1 and L1 are as described above;

[0032] R 13 Selected from hydrogen atoms, deuterium atoms, C 1-6 Alkyl, 6-10 membered aryl or 5-10 membered heteroaryl, wherein the alkyl, aryl and heteroaryl are optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino and oxo.

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

[0034]

[0035] The present disclosure also provides a method for preparing a compound of formula (I) or a salt thereof, comprising at least one of the steps of preparing a compound of formula (II) or a salt thereof, a compound of formula (III) or a salt thereof, and a compound of formula (IV) or a salt thereof as described in the present disclosure,

[0036]

[0037] Among them, R1, L1, R 13 As mentioned above, p is an integer from 2 to 8. In some embodiments, the compound represented by formula (I) is selected from

[0038]

[0039] The present disclosure also provides a method for preparing an antibody-drug conjugate of formula (A), comprising: a step of preparing the compound of formula (I) or a salt thereof as described in the present disclosure, and a step of reducing Ab and then subjecting it to a coupling reaction with the compound of formula (I) to obtain the antibody-drug conjugate of formula (A).

[0040]

[0041] wherein 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 number between any two numbers), L1, R 13 , p as described above.

[0042] In some embodiments, the reducing agent may be TCEP, for example to reduce disulfide bonds on the antibody.

[0043] In some embodiments, the antibody is selected from a chimeric antibody, a humanized antibody, or a fully human antibody; for example, a monoclonal antibody.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof 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 an antigen-binding fragment thereof.

[0045] In some embodiments, the antibody or antigen-binding fragment thereof is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, Glematumamab, or an antigen-binding fragment thereof.

[0046] In some embodiments, k is from 2 to 8, preferably from 5 to 9. Non-limiting examples include 3, 4, 5, 6, 7.2, 7.5, 8, 8.5, 9.

[0047] The compound represented by formula (III), the compound represented by formula (III'), the compound represented by formula (II), the compound represented by formula (I), the compound represented by formula (A) or pharmaceutically acceptable salts thereof can be prepared by referring to the methods disclosed in patents or applications such as CN104755494B, CN111228511A, CN112125915A, CN112512591A, CN115197088A, CN115197234A, CN115925796A, and CN117015549A, which are hereby cited in their entirety.

[0048] In some embodiments, the preparation method of the present disclosure optionally further comprises a purification step, wherein the purification step comprises one or more of column chromatography, solvent slurrying and recrystallization.

[0049] The salts of the compounds described in the present disclosure may be inorganic acid salts and organic acid salts. The inorganic acid salts may be hydrochlorides, sulfates, phosphates, hydrobromides, trifluoroacetates, etc., and the organic acids may be formates, acetates, sulfonates, optionally substituted alkyl sulfonates, succinates, maleates, tartrates, citrates, lactates, oxalates, gluconates, fumarates, malonates, malates, etc.

[0050] Another aspect of the present disclosure provides a compound represented by formula (IV') or a salt thereof,

[0051]

[0052] Wherein, R2 is selected from p-nitrobenzyl, benzoyl, formyl, trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, isopropyldiethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl, tert-butyloxycarbonyl, methoxymethyl and benzyloxymethyl.

[0053] In some embodiments, the compound is

[0054]

[0055] The preparation method of camptothecin derivatives and intermediates disclosed in the present invention avoids the use of lead tetraacetate required in the prior art, reduces environmental pollution and harm to operators. At the same time, the post-treatment step does not require column chromatography, and the purification efficiency is higher. The starting materials are cheap and easy to obtain, the reaction route is short, and it is more suitable for industrial production.

[0056] In the preparation method disclosed in the present invention, the reactions connected by “→” all refer to a one-step reaction to obtain the product.

[0057] The term "antibody-drug conjugate" refers to a ligand connected to a biologically active drug through a stable linking unit. In the present disclosure, "antibody-drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment connected to a biologically active glucocorticoid through a stable linking unit. The antibody or antibody fragment can be combined with a glucocorticoid molecule containing a linker through a specific group therein (e.g., an interchain disulfide bond).

[0058] The term "drug loading" refers to the average number of drugs carried by each antibody-drug conjugate molecule in an antibody-drug conjugate population, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The range of drug loading can be 1-20, preferably 1-10 glucocorticoids (D) connected to each antibody (Ab). In an embodiment of the present disclosure, drug loading is expressed as 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 mean 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 4-5 mean. The average amount of drug per ADC molecule after the conjugation reaction can be identified by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, mAb size variant assays (CE-SDS) and HPLC characterization.

[0059] The term "antibody" covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies may refer to immunoglobulins, which are tetrapeptide chain structures formed by two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the heavy chain of immunoglobulins are different, so their antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains or λ chains by different constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0060] The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0061] The antibodies disclosed herein include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

[0062] The term "murine antibody" in this disclosure refers to antibodies prepared in mice according to the knowledge and skills in the art. When preparing, a test subject is injected with a specific antigen, and then a hybridoma expressing an antibody with the desired sequence or functional characteristics is isolated.

[0063] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region gene from the mouse hybridoma cells, and then clone the constant region gene of the human antibody as needed, connect the mouse variable region gene with the human constant region gene into a chimeric gene, and then insert it into an expression vector, and finally express the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.

[0064] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the mouse CDR sequence into the human antibody variable region framework, i.e., different types of human germline antibody framework sequences. The heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components can be overcome. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk / vbase), and in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies disclosed in the present invention also include humanized antibodies after further affinity maturation of CDR by phage display. References further describing methods for humanizing mouse antibodies include, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and the methods of Winter and colleagues [Jones et al., Nature, 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].

[0065] The terms "fully human antibody", "fully human antibody" or "completely human antibody", also known as "fully human monoclonal antibody", are antibodies whose variable and constant regions are both human, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The present disclosure is a fully human monoclonal antibody. The relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.

[0066] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody. 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 connected by a disulfide bridge on the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody; (v) single domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) combinations of two or more isolated CDRs, optionally connected by synthetic linkers. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, so that they can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called 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 the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies can be of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0067] Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, among fragments obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which about half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.

[0068] F(ab')2 is an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, obtained by digesting the portion below two disulfide bonds in the hinge region of IgG with the enzyme pepsin, and comprising two Fab regions linked at the hinge position.

[0069] Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F(ab')2.

[0070] Furthermore, the Fab' fragment of the antibody can be produced by inserting a DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic organism or a eukaryotic organism to express the Fab'.

[0071] The term "single-chain antibody", "single-chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using variants of 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present 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.

[0072] The term "CDR" refers to one of the six hypervariable regions that mainly contribute to antigen binding in the variable domain of an antibody. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDR is only applied 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 region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of a CDR can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (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 convention (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (see Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, for the classical format, following the Kabat rule, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); 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 the Chothia rule, the CDR amino acid residues 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).By combining the CDR definitions of Kabat and Chothia, CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL. Following the IMGT rules, the CDR amino acid residues in VH are numbered approximately as 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately as 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3). Following the IMGT rules, the CDR regions of antibodies can be determined using the program IMGT / DomainGap Align.

[0073] The term "antibody framework" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

[0074] The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

[0075] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example: approximately less than 10 -8 M, 10 - 9 M or 10 -10 Binds with an affinity (KD) of M or less.

[0076] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is 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 operably linked to the coding sequence.

[0077] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA segments can be connected. In another embodiment, the vector is a viral vector, in which other DNA segments can be connected to the viral genome. The vector disclosed herein can be autonomously replicated in the host cell introduced therein (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome (e.g., non-additional mammalian vectors).

[0078] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art, such as the Cold Spring Harbor Guide to Antibody Experimental Techniques, Chapters 5-8 and 15. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR region. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulins, 2001 ISBN012441351.

[0079] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plants or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, 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 NS0 cells.

[0080] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0081] Amino acid sequence "identity" refers to the percentage of amino acid residues in a first sequence that are identical to the amino acid residues in a second sequence, while aligning the amino acid sequences and, if necessary, introducing gaps to achieve maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be achieved in a variety of ways within the scope of the art, for example, 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 algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0082] The term "alkyl" refers to a saturated straight or branched aliphatic hydrocarbon group, including 1 to 20 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), such as an alkyl group containing 1 to 6 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 and various branched isomers thereof, etc. Alkyl groups can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available point of attachment, such as one or more of the following groups, independently selected from halogen, hydroxy, oxo, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, 3- to 6-membered cycloalkyl or 3- to 6-membered heterocycloalkyl, wherein the alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally substituted by halogen, hydroxy, nitro, cyano or amino.

[0083] The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, oxo, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, 3 to 7-membered cycloalkyl or 3 to 7-membered heterocycloalkyl, wherein the alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally substituted by halogen, hydroxy, nitro, cyano or amino.

[0084] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0085] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc.; polycyclic cycloalkyls include spirocyclic, paracyclic and bridged cycloalkyls. Cycloalkyls may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from halogen, hydroxyl, oxo, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, 3- to 6-membered cycloalkyl or 3- to 6-membered heterocycloalkyl, wherein the alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally substituted by halogen, hydroxy, nitro, cyano or amino.

[0086] The term "heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, but excluding the ring portion of -OO-, -OS- or -SS-, the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 7 ring atoms. Non-limiting examples of "heterocycloalkyl" include:

[0087]

[0088] etc.

[0089] The heterocycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a heterocycloalkyl, non-limiting examples of which include:

[0090] wait.

[0091] The heterocycloalkyl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, oxo, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, 3- to 6-membered cycloalkyl or 3- to 6-membered heterocycloalkyl, wherein the alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally substituted by halogen, hydroxy, nitro, cyano or amino.

[0092] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 12-membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocycloalkyl or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, non-limiting examples of which include:

[0093]

[0094] Aryl may be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, 3- to 6-membered cycloalkoxy, 3- to 6-membered heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, 3 to 6-membered cycloalkoxy, 3 to 6-membered heterocycloalkoxy, 3 to 8-membered cycloalkenyloxy, 5 to 6-membered aryl or heteroaryl are optionally substituted with one or more selected from halogen, hydroxy, cyano, amino, C 1-6 Alkyl or C 1-6 Alkoxy substitution.

[0095] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 6 to 12 members, more preferably 5 or 6 members. For example. Non-limiting examples include: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, wait.

[0096] The heteroaryl ring may be fused to an aryl, heterocycloalkyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:

[0097]

[0098] The heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, cyano, amino, C 1-6 Alkyl or C 1-6 Alkoxy.

[0099] The "hydroxyl protecting group" is a group known in the art and can be used to protect a hydroxyl group. See the literature ("Protective Groups in Organic Synthesis", 5Th Ed. TW Greene & P.GMWuts) for hydroxyl protecting groups. As examples, including but not limited to, as examples, preferably, the hydroxyl protecting group can be a (C1-10 alkyl or aryl)3 silyl group, for example: triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, etc.; it can be a C1-10 alkyl or substituted alkyl group, for example: methyl, tert-butyl, allyl, benzyl, methoxymethyl, ethoxyethyl, 2-tetrahydropyranyl (THP), etc.; it can be a (C1-10 alkyl or aromatic) acyl group, for example: formyl, acetyl, benzoyl, etc.; it can be a (C1-6 alkyl or C6-10 aromatic) sulfonyl group; it can also be a (C1-6 alkoxy or C6-10 aromatic) carbonyl group, which can be acetyl (Ac), 2-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether (MTM).

[0100] "Amino protecting groups" are groups known in the art that can be used to protect amino groups, see amino protecting groups in the literature (Protective Groups in Organic Synthesis, 5Th Ed. TW Greene & P. ​​GMWuts). Examples include, but are not limited to, carbamate protecting groups, such as 2-trimethyl-silylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)-ethoxy-carbonyl (Bpoc), tert-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc) and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl, acetyl, trichloroacetyl, benzoyl and nitrophenylacetyl; sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and imide and cyclic imine protecting groups, such as phthalimido and dithiosuccinyl.

[0101] "Carboxyl protecting group" is a suitable group for protecting carboxyl group known in the art, see the literature ("Protective Groups in Organic Synthesis", 5 Th Ed.TWGreene & P.GMWuts) in the carboxyl protecting group, as an example, the carboxyl protecting group can be a substituted or unsubstituted C 1-10 A straight chain or branched alkyl, substituted or unsubstituted C 2-10 A 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)3silyl, etc.

[0102] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocycloalkyl group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes instances where the heterocycloalkyl group is substituted with an alkyl group and instances where the heterocycloalkyl group is not substituted with an alkyl group.

[0103] In the chemical structures of the compounds disclosed herein, the bond Indicates that the configuration is not specified, that is, if there are chiral isomers in the chemical structure, the bond Can be or include both In the chemical structure of the compounds disclosed in the present invention, the bond No configuration is specified, i.e., the bond The configuration can be E-type or Z-type, or include both E and Z configurations.

[0104] Although all of the above formulae are drawn in certain isomeric forms for simplicity, the present disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers. DETAILED DESCRIPTION

[0105] The present disclosure will be explained in detail below in conjunction with specific examples so that those skilled in the art can have a more comprehensive understanding of the present disclosure. The specific examples are only used to illustrate the technical solutions of the present disclosure and do not limit the present disclosure in any way.

[0106] Example 1

[0107]

[0108] Step 1: Synthesis of compound 1

[0109] The reactant glycine amide (40 g, 1.05 eq.) was suspended in THF (80 mL, 2 V), water (240 mL, 6 V), and ethyl acetate (800 mL, 20 V), and sodium carbonate (44.5 g, 1.2 eq.) and Fmoc-OSu (118 g, 1.0 eq.) were added. The reaction was stirred at 25 °C. After the reaction was completed, petroleum ether (160 mL, 4 V) was added to the reaction solution. After stirring at room temperature for 2 hours, the mixture was filtered, and the filter cake was washed with ethyl acetate and dried to obtain compound 1 (107 g, yield: 100%).

[0110] 1 HNMR (400MHz, DMSO-d6) δ7.90(d,J=7.6Hz,2H),7.72(d,J=7.6Hz,2H),7.45-7.20(m,6H),7.05-6.98(m,1H),4.32-4.18(m,3H),3.60-3.55(m,2H).

[0111] Step 2: Synthesis of Compound 2

[0112] Compound 1 (50 g, 1.0 eq.) was suspended in water (400 mL, 8 V), potassium carbonate (1.17 g, 0.05 eq.) and formaldehyde aqueous solution (27.3 g, 2.0 eq.) were added, and the mixture was heated to an external temperature of 80°C and stirred for reaction. After the reaction was completed, the reaction solution was cooled to room temperature and filtered. The filter cake was washed with water (200 mL, 4 V) and dried to obtain compound 2 (50 g, yield: 91%).

[0113] 1 HNMR (400MHz, DMSO-d6) δ8.48-8.40(m,1H),7.90(d,J=7.6Hz,2H),7.73(d,J=7.6Hz,2H),7.58-7.50(m,1H),7. 45-7.36(m,2H),7.36-7.27(m,2H),5.65-5.55(m,1H),4.55-4.45(m,2H),4.30-4.15(m,3H),3.65-3.57(m,2H).

[0114] Step 3: Synthesis of compound 3

[0115] Compound 2 (20 g, 1.0 eq.) was suspended in dichloromethane (300 mL, 15 V), triethylamine (12.4 g, 2.0 eq.) was added, the temperature was lowered to 0°C, benzoyl chloride (12.9 g, 1.5 eq.) was added dropwise, the temperature was raised to room temperature and stirred for reaction, after the reaction was completed, the reaction liquid was directly filtered, the filter cake was slurried with water (100 mL, 5 V), re-filtered, and the filter cake was dried to obtain compound 3 (18.4 g, yield: 70%).

[0116] 1 HNMR(400MHz,DMSO-d6)δ9.15-9.06(m,1H),7.98-7.85(m,4H),7.75-7.50(m,6H),7.45-7. 37(m,2H),7.37-7.28(m,2H),5.41(d,J=6.8Hz,2H),4.35-4.15(m,3H),3.78-3.65(m,2H).

[0117] Step 4: Synthesis of compound 4

[0118] Compound 3-1 (1.13 g, 1.0 eq.) was dissolved in THF (11 mL, 10 V), cooled to -20 ° C, lithium tert-butoxide (0.4 g, 0.92 eq.) was added, and the reaction was stirred for 30 min; compound 3 (2 g, 0.85 eq.) was suspended in THF (14 mL, 10 V), and slowly added dropwise to the reaction bottle, and the reaction was kept at low temperature. After the reaction was completed, the reaction was quenched with 10% citric acid, extracted with ethyl acetate (50 mL), and concentrated to dryness to obtain a crude product. The crude product was crystallized with ethyl acetate to obtain compound 4 (1.84 g, yield: 65%).

[0119] 1 HNMR (400MHz, DMSO-d6) δ8.72-8.70(m,1H),7.90(d,J=7.6Hz,2H),7.20(d,J=7.6Hz,2H),7.61-7.57(m,1H),7.45-7.25(m ,9H),5.17(q,J=12Hz,2H),4.68-4.55(m,2H),4.32-4.15(m,3H),3.70-3.55(m,3H),1.05-0.95(m,1H),0.50-0.30(m,4H).

[0120] Step 5: Synthesis of Compound 5

[0121] Compound 4 (2 g, 1.0 eq.) was dissolved in THF (4 mL, 2 V), and ethyl acetate (60 mL, 30 V) was added. After the solution was clear, 10% palladium carbon (0.4 g, 0.2 wt) was added; the temperature was lowered to 0°C, and vacuum-hydrogen replacement was performed 3 times. The hydrogen atmosphere was maintained and stirred for 12 h. After the reaction was completed, the system was replaced with nitrogen, and the palladium carbon was removed by filtration with diatomaceous earth. The filter cake was washed with THF, and the filtrate was concentrated to dryness to obtain a crude product, which was crystallized with DCM to obtain compound 5 (1.3 g, yield: 80%).

[0122] 1 HNMR(400MHz, DMSO-d6)δ8.70-8.60(m,1H),7.89(d,J=7.6Hz,2H),7.72(d,J=7.6Hz,2H),7.58-7.55(m,1H),7.45-7.38(m,2H),7.38- 7.28(m,2H),4.65-4.52(m,2H),4.33-4.18(m,3H),3.61(d,J=5.2Hz,2H),3.46(d,J=7.6Hz,1H),1.03-1.00(m,1H),0.52-0.25(m,4H).

[0123] Comparative Example 1

[0124]

[0125] Add 1440mL of tetrahydrofuran to the reaction flask, add 80g of raw material D-1, 480mL of toluene, and 80g of diatomaceous earth. Add lead tetraacetate at one time and stir at room temperature for 10min. Add pyridine and reflux for 3h. Cool to room temperature, filter with diatomaceous earth, wash the filter cake with 400mL of tetrahydrofuran, and combine the filtrate. Concentrate the filtrate to obtain 130g of oil. Add 500mL of anhydrous ethanol to dissolve, transfer to the reactor, continue to add 1100mL of anhydrous ethanol, stir for 24h, filter, wash the filter cake with 500mL of n-heptane, and dry to obtain compound D (28g, yield 33.6%).

[0126] Since the disclosure has been described in terms of specific embodiments thereof, certain modifications and equivalent changes will be apparent to one skilled in the art and are intended to be included within the scope of the disclosure.

Claims

1. A method for preparing a compound represented by formula (IV) or a salt thereof, comprising: The step of preparing the compound represented by formula (V) from the compound represented by formula (VI), and the step of preparing the compound represented by formula (IV) or a salt thereof from the compound represented by formula (V), wherein: R1 is an amino group or an amino group protected by an amino protecting group, R2 is a hydroxyl protecting group, 2. The preparation method according to claim 1, wherein the amino protecting group is selected from acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, pivaloyl, formyl, benzoyl, phthaloyl, 9-fluorenylmethoxycarbonyl, tert-butyloxycarbonyl and benzyloxycarbonyl, preferably 9-fluorenylmethoxycarbonyl.

3. according to the preparation method described in claim 1 or 2, wherein said hydroxyl protecting group is selected from p-nitrobenzyl, acetyl, benzoyl, formyl, trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, isopropyldiethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl, tert-butyloxycarbonyl, methoxymethyl and benzyloxymethyl, preferably acetyl or benzoyl.

4. The preparation method according to any one of claims 1 to 3, wherein the compound represented by formula (VI) reacts with aldehyde to prepare the compound represented by formula (V), and preferably the molar ratio of the compound represented by formula (VI) to the aldehyde is 1:1-1:

5.

5. The preparation method according to any one of claims 1 to 4, wherein R2 is a benzoyl group, and the reaction for preparing the compound represented by formula (IV) or a salt thereof is carried out in the presence of a benzoyl halide and an acid binding agent, wherein the acid binding agent is preferably selected from at least one of potassium carbonate, triethylamine, DIPEA, and pyridine, and the molar ratio of the compound represented by formula (V) to the benzoyl halide is preferably 1:1-1:

5.

6. The preparation method according to any one of claims 1 to 5, wherein the method comprises:

7. A method for preparing a compound of formula (III) or a salt thereof, comprising the step of preparing a compound of formula (IV) or a salt thereof as described in the present disclosure, and the step of preparing a compound of formula (III) or a salt thereof from a compound of formula (IV) or a salt thereof, in, R1 as claimed in claim 1; L1 is -(C(R 11 )(R 12 )) n -(CH2) m -, C3-C6 saturated cycloalkyl or 3-6 membered saturated heterocycloalkyl, the C3-C6 saturated cycloalkyl and 3-6 membered saturated heterocycloalkyl are each independently optionally substituted by one or more substituents selected from halogen, hydroxyl, amino and haloalkyl; R 11 , R 12 are independently selected from hydrogen atoms, C 1-6 alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl or 5-10 membered heteroaryl, wherein the alkyl, cycloalkyl, aryl or heteroaryl is optionally substituted with one or more substituents selected from C1-C6 alkyl, halogen, hydroxy, amino, oxo, 3-6 membered cycloalkyl, 6-10 membered aryl or C1-C6 alkoxy, or, R 11 , R 12 Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxy, amino, oxo or C1-C6 alkoxy; m is 0 or 1, preferably 0; n is 1, 2, 3 or 4, preferably 1.

8. The preparation method according to claim 7, wherein the compound represented by formula (III) is selected from 9. The preparation method according to claim 7 or 8, wherein the method comprises the steps of preparing the compound represented by formula (III') or its salt from the compound represented by formula (IV) or its salt, and preparing the compound represented by formula (III') or its salt by decarboxylating the protecting group of the compound represented by formula (III') or its salt, in, R3 is a carboxyl group protected by a carboxyl protecting group, and the carboxyl protecting group is preferably a methyl group, a substituted methyl group, an ethyl group, a 2-substituted ethyl group, an allyl group, a tert-butyl group, an alkoxyalkyl group, an alkoxyalkoxyalkyl group, a 2,6-dialkylphenyl group, a benzyl group, a substituted benzyl group, a silyl group, and a stannyl group.

10. A method for preparing a compound of formula (II) or a salt thereof, comprising the step of preparing a compound of formula (IV) or a salt thereof according to any one of claims 1 to 6, and / or the step of preparing a compound of formula (III) or a salt thereof according to any one of claims 7 to 9, in, R1, L1 as claimed in claim 7; R 13 Selected from hydrogen atoms, deuterium atoms, C 1-6 Alkyl, 6-10 membered aryl or 5-10 membered heteroaryl, wherein the alkyl, aryl and heteroaryl are optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino and oxo.

11. The preparation method according to claim 10, wherein the compound represented by formula (II) is selected from 12. A method for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof, comprising at least one of the steps of preparing a compound of formula (IV) or a salt thereof according to any one of claims 1 to 6, preparing a compound of formula (III) or a salt thereof according to any one of claims 7 to 9, and preparing a compound of formula (II) according to any one of claims 10 to 11, in, L1, R 13 As described in claim 10, p is an integer from 2 to 8.

13. The preparation method according to claim 12, wherein the compound represented by formula (I) is selected from 14. A method for preparing an antibody-drug conjugate of formula (A), comprising the step of preparing the compound of formula (I) according to any one of claims 12 to 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 (A). Where Ab is an antibody or antigen-binding fragment, k is 1 to 20, L1, R 13 As described in claim 10, p is an integer from 2 to 8.

15. 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.

16. The preparation method according to claim 14 or 15, 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 an antigen-binding fragment thereof.

17. The preparation method according to any one of claims 14 to 16, wherein k is 2 to 8, preferably 5 to 9.

18. A compound represented by formula (IV') or a salt thereof, in, R2 is selected from p-nitrobenzyl, benzoyl, formyl, trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, isopropyldiethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl, tert-butyloxycarbonyl, methoxymethyl and benzyloxymethyl; The compound is preferably

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