Preparation method for camptothecin derivative and intermediate thereof

Abstract

The present disclosure relates to a preparation method for a camptothecin derivative and an intermediate thereof. Specifically, the present disclosure relates to a preparation method for a compound represented by formula (IV) or a salt thereof, comprising: a step of preparing a compound represented by formula (V) from a compound represented by formula (VI), and a step of preparing a compound represented by formula (IV) or a salt thereof from the compound represented by formula (V). The method achieves a high yield and involves mild reaction conditions, and is suitable for industrial production.

Description

Preparation method of camptothecin derivative and intermediate thereof TECHNICAL FIELD

[0001] The present disclosure belongs to the field of medicine and relates to a preparation method of a camptothecin derivative and an intermediate thereof. BACKGROUND

[0002] An antibody drug conjugate (ADC) links a monoclonal antibody or an antibody fragment to a cytotoxic toxin through a stable chemical linker compound, fully utilizes the specificity of the antibody in binding to the surface antigens of normal cells and tumor cells and the high efficiency of the cytotoxic toxin, and avoids the defects of low therapeutic effect of the former and excessive toxicity of the latter. This means that, compared with the traditional chemotherapy drugs, the antibody drug conjugate can precisely bind to tumor cells and reduce the impact on normal cells.

[0003] There are several categories of small molecules with cytotoxicity for antibody drug conjugates, one of which is camptothecin derivatives, which have anti-tumor effects by inhibiting topoisomerase I. There are reports of camptothecin derivatives, such as exatecan (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), which are used in antibody conjugate drugs (ADC) in documents such as WO2014057687, WO2020063676, WO2020063673, CN112125915A, CN115925796A, etc. SUMMARY

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

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

[0006] R2 is a hydroxyl protecting group,

[0007] In another aspect, the present disclosure provides a preparation method of a compound represented by formula (IV) or a salt thereof, comprising: a step of preparing a compound represented by formula (V) or a salt thereof from a compound represented by formula (VI) or a salt thereof, and a step of preparing the compound represented by formula (IV) or a salt thereof from the compound represented by formula (V) or a salt thereof, wherein R1 is amino or amino protected by an amino protecting group, and R2 is a hydroxyl protecting group.

[0008] In some embodiments, the amino protecting group of the present disclosure is selected from acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, pivaloyl, formyl, benzoyl, phthaloyl, 9-fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl, for example 9-fluorenylmethyloxycarbonyl.

[0009] In some embodiments, the hydroxyl protecting group of 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, for example acetyl or benzoyl.

[0010] In some embodiments, R1is amino protected by an amino protecting group.

[0011] In some embodiments, the compound of formula (VI) is reacted with an aldehyde to prepare the compound of formula (V). In some embodiments, the molar ratio of the compound of 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 can be formaldehyde.

[0012] In some embodiments, R2is benzoyl, and the reaction to prepare the compound of formula (IV) or a salt thereof is reacted in the presence of benzoyl halide and an acid binding agent. The acid binding agent is selected from at least one of potassium carbonate, triethylamine, DIPEA, pyridine, for example triethylamine. In some embodiments, the molar ratio of the compound of formula (V) to the benzoyl halide is 1:1-1:5, for example 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-pyrrolidinone, tetrahydrofuran, methyltetrahydrofuran, dioxane, toluene, xylene, dimethyl sulfoxide, diethyl ether, isopropyl ether, methyl tert-butyl ether, acetonitrile, propionitrile, isopropyl alcohol, propyl alcohol, ethanol, methanol.

[0014] In some embodiments, the reaction temperature of each step is independently -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 of Formula (IV) is a compound of Formula (IV-1), and the method comprises:

[0016] In some embodiments, the compound of Formula (VI-1) is reacted with an aldehyde to produce a compound of Formula (V-1) in a molar ratio of 1:1-1:5.

[0017] In some embodiments, the reaction to produce a compound of Formula (IV) or a salt thereof is carried out in the presence of a benzoyl halide and an acid binding agent, and the molar ratio of the compound of Formula (V-1) to the benzoyl halide is 1:1-1:5.

[0018] 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,

[0019] wherein R1is as previously described;

[0020] L1is -(C(R 11 )(R 12 )) n -(CH2) m -, C3-C6 saturated cycloalkyl, or 3-6 membered saturated heterocycloalkyl, each independently optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, and haloalkyl;

[0021] R 11 , R 12 are each independently selected from the group consisting of a hydrogen atom, C 1-6 alkyl, 3-6 membered cycloalkyl, 6-10 membered aryl, or 5-10 membered heteroaryl, wherein said alkyl, cycloalkyl, aryl, heteroaryl is optionally substituted with one or more substituents selected from the group consisting of C1-C6 alkyl, halogen, hydroxyl, 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 they are attached form a 3-6 membered cycloalkyl optionally substituted with one or more substituents selected from the group consisting of C1-C6 alkyl, halogen, hydroxyl, amino, oxo, or C1-C6 alkoxy;

[0022] m is 0 or 1, for example 0; n is 1, 2, 3, or 4, for example 1.

[0023] In some embodiments, the compound of Formula (III) is selected from the group consisting of

[0024] In some embodiments, the method comprises a step of preparing a compound of formula (III') or a salt thereof from a compound of formula (IV) or a salt thereof, and a step of decarboxylating a compound of formula (III') or a salt thereof to prepare a compound of formula (III) or a salt thereof,

[0025] wherein R3 is a carboxyl group protected by a carboxyl protecting group, such as a methyl group, a substituted methyl group, an ethyl group, a 2-substituted ethyl group, an allyl group, a t-butyl group, an alkoxyalkyl group, an alkoxyalkoxyalkyl group, a 2,6-dialkylphenyl group, a benzyl group, a substituted benzyl group, a silyl group, or a stannyl group, such as a methyl group, an allyl group, a t-butyl group, a benzyl group, a 2,4-dimethoxybenzyl group, a p-methylbenzyl group, a pentafluorophenyl group, or a methoxyethoxymethyl group.

[0026] The present disclosure also provides a method for preparing a compound of formula (II) or a salt thereof, comprising a step of preparing a compound of formula (IV) or a salt thereof and / or a compound of formula (III) or a salt thereof as described in the present disclosure,

[0027] wherein R1, L1 are as previously described;

[0028] R 13 selected from a hydrogen atom, a deuterium atom, a C 1-6 alkyl, a 6-10 membered aryl group, or a 5-10 membered heteroaryl group, wherein the alkyl, aryl, and heteroaryl groups are optionally substituted with one or more substituents selected from a C1-C6 alkyl group, a halogen, a hydroxyl group, an amino group, and an oxo group.

[0029] In some embodiments, the compound of formula (II) is selected from

[0030] 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, a compound of formula (IV) or a salt thereof as described in the present disclosure,

[0031] wherein R1, L1, R 13 As previously described, p is an integer from 2 to 8.

[0032] In some embodiments, the compound of formula (I) is selected from

[0033] The present disclosure also provides a method for preparing an antibody-drug conjugate represented by formula (A), comprising: the step of preparing a compound represented by formula (I) or a salt thereof as described in the present disclosure, and the step of coupling reaction with the compound represented by formula (I) after reduction of Ab to obtain the antibody-drug conjugate represented by formula (A),

[0034] wherein Ab is an antibody or an 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 numerical values),

[0035] L1, R 13 , p are as described above.

[0036] In some embodiments, the reducing agent can be TCEP, for example, to reduce the disulfide bond on the antibody.

[0037] In some embodiments, the antibody is selected from a chimeric antibody, a humanized antibody or a fully human antibody; for example, is a monoclonal antibody.

[0038] In some embodiments, wherein the antibody or antigen-binding fragment thereof is selected from an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUC1 antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC44A4 antibody, or an anti-Mesothelin antibody or an antigen-binding fragment thereof.

[0039] In some embodiments, wherein 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.

[0040] In some embodiments, k is 2 to 8, preferably 5 to 9. Non-limiting examples include 3, 4, 5, 6, 7.2, 7.5, 8, 8.5, 9.

[0041] 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 a pharmaceutically acceptable salt thereof can be prepared by referring to the method disclosed in patents or applications such as CN104755494B, CN111228511A, CN112125915A, CN112512591A, CN115197088A, CN115197234A, CN115925796A, CN117015549A, etc., which are hereby incorporated in their entirety.

[0042] In some embodiments, the preparation method of the present disclosure optionally further comprises a purification step comprising one or more of column chromatography, solvent beating and recrystallization.

[0043] The salt of the compound of the present disclosure can be an inorganic acid salt and an organic acid salt, the inorganic acid salt can be a hydrochloride, a sulfate, a phosphate, a hydrobromide, a trifluoroacetate, etc., and the organic acid can be a formate, an acetate, a sulfonate, an optionally substituted alkyl sulfonate, a succinate, a maleate, a tartrate, a citrate, a lactate, an oxalate, a gluconate, a fumarate, a malonate, a malate, etc.

[0044] Another aspect of the present disclosure provides a compound represented by formula (IV') or a salt thereof,

[0045] wherein R2 is selected from the group consisting of p-nitrobenzyl, benzoyl, formyl, trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, isopropyldiethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, t-butyloxycarbonyl, methoxymethyl and benzyloxymethyl.

[0046] In some embodiments, the compound is

[0047] The preparation method of the camptothecin derivative and the intermediate thereof disclosed by the present disclosure avoids the use of lead tetraacetate required in the prior art, reduces environmental pollution and harm to operators. At the same time, the post-processing step does not require column chromatography, and the purification efficiency is higher. The starting material is cheap and easy to obtain, the reaction route is short, and it is more suitable for industrial production.

[0048] In the preparation method disclosed by the present disclosure, the reactions connected by "→" are one-step reactions to obtain the product.

[0049] The term "antibody-drug conjugate" refers to a ligand linked to a biologically active drug through a stable linker. In the present disclosure, "antibody drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active glucocorticoid through a stable linker. The antibody or antibody fragment can be bound to the glucocorticoid molecule comprising the linker through a specific group therein, such as an interchain disulfide bond.

[0050] The term "drug loading" refers to the average number of drugs carried by each antibody-drug conjugate molecule in a population of antibody-drug conjugate molecules, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The drug loading can range from 1 to 20, preferably 1 to 10, glucocorticoids (D) per antibody (Ab). In embodiments of the present disclosure, the drug loading is expressed as k, and exemplary values 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 values between any two values. Preferably, the average value is 1 to 10, more preferably 1 to 8, or 2 to 8, or 2 to 7, or 3 to 8, or 3 to 7, or 3 to 6, or 4 to 7, or 4 to 6, or 4 to 5. The average number of drugs per ADC molecule after conjugation reaction can be identified by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, monoclonal antibody size variant assay (CE-SDS), and HPLC profiling.

[0051] The term "antibody" encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., 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. An antibody can refer to an immunoglobulin, which is a four polypeptide chain structure connected by interchain disulfide bonds, consisting of two heavy chains and two light chains. The immunoglobulin heavy chain constant region has different amino acid compositions and arrangement orders, and thus has different antigenicities. Accordingly, the immunoglobulin can be classified into five types, or called isotypes of immunoglobulin, i.e., IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same type of Ig can be further classified into different subtypes according to the differences in the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds, such as IgG1, IgG2, IgG3, and IgG4. The light chain is classified into κ chain or λ chain through the constant region.

[0052] The sequences of about 110 amino acids near the N-terminus of both the heavy and light chains are highly variable and form the variable region (Fv region); the remaining amino acids at the C-terminus are relatively stable and form the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity-determining region (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of three CDR regions and four FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0053] The antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, preferably humanized antibodies and fully human antibodies.

[0054] The term "murine antibody" in the present disclosure is an antibody prepared from a mouse according to the knowledge and skills in the art. The preparation is performed by injecting a test subject with a specific antigen, and then isolating a hybridoma expressing an antibody having the desired sequence or functional characteristics.

[0055] The term "chimeric antibody" is an antibody in which the variable region of a murine antibody is fused with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the murine hybridoma cells, and the constant region gene of a human antibody is cloned as needed. The murine variable region gene is linked to the human constant region gene to form a chimeric gene, which is then inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.

[0056] The term "humanized antibody" also known as CDR-grafted antibody refers to an antibody produced by grafting murine CDR sequences into a human antibody variable region framework, i.e., different types of human germline antibody framework sequences. This can overcome the heterogeneity reaction induced by chimeric antibodies due to carrying a large number of murine protein components. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. The germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet 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 the decrease in immunogenicity while causing a decrease in activity, the human antibody variable region framework sequence can be subjected to minimal back or reverse mutations to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies further affinity matured by phage display on CDR. Further descriptions of the methods involved in humanizing antibodies using mouse antibodies include, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 88, 2869, 1991 and the methods of Winter and co-workers [Jones et al., Nature, 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].

[0057] The term "fully human antibody", "fully human antibody" or "fully human antibody" also known as "fully human monoclonal antibody" refers to an antibody whose variable region and constant region are both human, removing immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibody, chimeric monoclonal antibody, humanized monoclonal antibody and fully human monoclonal antibody. The present disclosure is a fully human monoclonal antibody. The related 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.

[0058] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain its ability to specifically bind to an antigen. It has been shown that the antigen- binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a single domain or dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which can optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be produced as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., 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 encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those with skill in the art, and are screened for utility in the same fashion as are intact antibodies. Antigen binding moieties can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulin. The antibody can be an antibody of different isotype, e.g., an IgG (e.g., IgGl, IgG2, IgG3, or IgG4 subtypes), IgAl, IgA2, IgD, IgE, or IgM antibody.

[0059] Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen binding activity, which is obtained in a fragment of IgG antibody molecules by treating with a protease papain (cutting amino acid residues at position 224 of H chain), in which about half of the H chain N-terminal side and the entire L chain are bound together by disulfide bonds.

[0060] F(ab')2 is an antibody fragment having a molecular weight of about 100,000 and having antigen binding activity and containing two Fab regions connected at the hinge position, which is obtained by digesting IgG with the enzyme pepsin to digest two disulfide bonds below the hinge region.

[0061] Fab' is a fragment of an antibody having a molecular weight of about 50,000 and having an antigen binding activity, which is obtained by cleaving the disulfide bond of the hinge region of the above F(ab')2.

[0062] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or a eukaryote to express the Fab'.

[0063] 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 can 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 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.

[0064] The term "CDR" refers to one of six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E. A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDRs applies only to CDR1, CDR2, and CDR3 of the light chain variable domain (CDR LI, CDR L2, CDR L3 or L1, L2, L3), and CDR2 and CDR3 of the heavy chain variable domain (CDR H2, CDR H3 or H2, H3). Generally, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3). The amino acid sequence boundaries of the CDRs 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 Ed. 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 M.P., Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), among others. For example, for the canonical format, following the Kabat convention, 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 in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following the Chothia convention, the CDR amino acid residues in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).By combining the CDR definitions of both Kabat and Chothia, CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Following the IMGT rules, the CDR amino acid residue numbering in VH is approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and in VL is approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). Following the IMGT rules, CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0065] The term "antibody framework" refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain. In essence, it is the variable domain without the CDRs.

[0066] The term "epitope" or "antigenic determinant" refers to the site or sites on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes are generally formed both by contiguous amino acids and non-contiguous amino acids juxtaposed in three-dimensional space and epitope regions can have a spatial and / or conformational structure which is recognized by the antibody. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0067] The terms "specifically binds," "selectively binds," "selectively binds to," and "binds specifically to" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, an antibody will bind to its antigen with an affinity (KD) of about less than 10 -7 M, e.g., about less than 10 -8 M, 10 -9 M or 10 -10 M or less.

[0068] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. Nucleic acid molecules are "operably linked" when they are functionally connected to another nucleic acid sequence so that the desired function of the nucleic acid sequence is affected, e.g., expression of a coding sequence is affected when a promoter or enhancer affects the transcription of that coding sequence.

[0069] 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 into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. The vectors disclosed herein can autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or can integrate into the genome of a host cell, after introduction into the host cell, and replicate along with the host genome (e.g., non-episomal mammalian vectors).

[0070] Methods for producing and purifying antibodies and antigen binding fragments are well known in the art, such as in Current Protocols in Protein Science, John Wiley & Sons, Inc., 2002, Vol. 1, pages 10.1-10.20 and 10.28-10.33. Antigen binding fragments can also be prepared using conventional methods. The antibodies or antigen binding fragments described herein are genetically engineered to have one or more human FR regions in the CDR regions from a non-human source. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website at http: / / imgt.cines.fr by alignment of the IMGT human antibody variable germline genes database and MOE software, or from the journal of immunology, 2001 ISBN 012441351.

[0071] 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. Bacteria that are readily transformed include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microbial hosts include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.

[0072] The engineered antibodies or antigen-binding fragments of the present disclosure can be produced and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred prior art, mammalian expression systems result in glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Positive clones are expanded in a bioreactor in serum-free media to produce the antibody. The antibody-secreting culture fluid can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column with a modified buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fractions are detected using SDS-PAGE and collected. The antibody can be concentrated using conventional filtration methods. Soluble aggregates and multimers can also be removed using conventional methods, such as molecular sieving, ion exchange. The resulting product is immediately frozen, such as at -70°C, or lyophilized.

[0073] Amino acid sequence "identity" means the percentage of amino acid residues in a first sequence that are identical with the amino acid residues in a second sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment can be achieved in various ways that are within the skill in 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 appropriate parameters to achieve maximal alignment, including any algorithms necessary to achieve the maximum percent sequence identity.

[0074] The term "alkyl" refers to saturated straight-chain or branched-chain aliphatic hydrocarbon groups 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), for example, alkyl groups containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and various branched isomers of the latter group. The alkyl group can be substituted or unsubstituted, and when substituted, the substituent(s) can be substituted at any available attachment point, for example, one or more 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, the alkyl, alkoxy, cycloalkyl, or heterocycloalkyl being optionally substituted with halogen, hydroxyl, nitro, cyano, or amino.

[0075] The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propyloxy, butyloxy. The alkoxy group can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more 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, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl being optionally substituted with halogen, hydroxy, nitro, cyano, or amino.

[0076] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0077] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring comprising 3 to 20 carbon atoms, preferably comprising 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyl groups include spiro, fused, and bridged cycloalkyl groups. The cycloalkyl group can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available point of attachment, preferably one or more 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, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl being optionally substituted with halogen, hydroxy, nitro, cyano, or amino.

[0078] The term "heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, one or more of which are heteroatoms selected from nitrogen, oxygen, or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, but excluding -O-O-, -O-S-, or -S-S- ring moieties, the remaining ring atoms being carbon. Preferably, the heterocycloalkyl group comprises 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the heterocycloalkyl group comprises 3 to 7 ring atoms. Non-limiting examples of "heterocycloalkyl" groups include: , and the like.

[0079] The heterocycloalkyl ring can be fused to an aryl or heteroaryl ring, wherein the ring that is attached to the parent structure is the heterocycloalkyl group, non-limiting examples of which include:

[0080] , and the like.

[0081] Heterocycloalkyl can be optionally substituted or unsubstituted, when substituted, the substituents are preferably one or more 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, said alkyl, alkoxy, cycloalkyl, or heterocycloalkyl being optionally substituted with halogen, hydroxy, nitro, cyano, or amino.

[0082] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (that is, rings which share adjacent pairs of carbon atoms) ring systems having a conjugated pi-electron system, preferably 6- to 12-membered, such as phenyl and naphthyl. The aryl ring can be fused to a heteroaryl, heterocycloalkyl, or cycloalkyl ring, wherein the ring which is attached to the parent structure is the aryl ring, non-limiting examples of which include:

[0083] Aryl can be substituted or unsubstituted, when substituted, the substituents are preferably one or more groups independently selected from halogen, hydroxy, oxo, nitro, cyano, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, 3- to 6-membered cycloalkyl, 3- to 6-membered heterocycloalkyl, C 3-8 cycloalkenyl, 5- to 6-membered aryl, or heteroaryl, said C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, 3- to 6-membered cycloalkyl, 3- to 6-membered heterocycloalkyl, 3- to 8-membered cycloalkenyl, 5- to 6-membered aryl, or heteroaryl, optionally substituted with one or more groups selected from halogen, hydroxy, cyano, amino, C 1-6 alkyl, or C 1-6 alkoxy.

[0084] The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl is preferably 6- to 12-membered, more preferably 5- or 6-membered. For example. Non-limiting examples include: imidazolyl, furanyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, and the like.

[0085] The heteroaryl ring can be fused to an aryl, heterocycloalkyl, or cycloalkyl ring, wherein the ring which is attached to the parent structure is the heteroaryl ring, non-limiting examples of which include:

[0086] Heteroaryl groups can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from halogen, hydroxyl, cyano, amino, C 1-6 Alkyl or C 1-6 Alkoxy.

[0087] "Hydroxyl protecting groups" are groups known in the art to be useful for protecting hydroxyl groups, see the hydroxyl protecting groups in the text (Protective Groups in Organic Synthesis, 5th Ed. T. W. Greene & P. G. M. Wuts). By way of example, including but not limited to, preferably the hydroxyl protecting group can be (C1-10alkyl or aryl)3silyl, for example: triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and the like; can be C1-10alkyl or substituted alkyl, for example: methyl, tert-butyl, allyl, benzyl, methoxymethyl, ethoxyethyl, 2-tetrahydropyranyl (THP), and the like; can be (C1-10alkyl or aryl)acyl, for example: formyl, acetyl, benzoyl, and the like; can be (C1-6alkyl or C6-10aryl)sulfonyl; and can be (C1-6alkoxy or C6-10aryloxy)carbonyl, can be acetyl (Ac), 2-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether (MTM).

[0088] "Amino protecting groups" are groups known in the art to be useful for protecting amino groups, see the amino protecting groups in the text (Protective Groups in Organic Synthesis, 5th Ed. T. W. Greene & P. G. M. Wuts). By way of example, including but not limited to, carbamate protecting groups such as 2-trimethyl-silyl-ethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)-ethoxycarbonyl (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 imine and cyclic imine protecting groups such as phthalimido and dithiasuccinimido.

[0089] "Carboxyl protecting groups" are groups known in the art to be suitable for protecting carboxyl groups, see the carboxyl protecting groups in the text ("Protective Groups in Organic Synthesis", 5 ThThe 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.

[0090] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes situations in which the event or environment may or may not occur. For example, "optionally alkyl-substituted heterocyclic alkyl group" means that an alkyl group may but does not have to be present, and the description includes cases where the heterocyclic alkyl group is substituted with an alkyl group and cases where the heterocyclic alkyl group is not substituted with an alkyl group.

[0091] In the chemical structure of the compounds described in this disclosure, the bonds... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be or Or simultaneously include and Two configurations. In the chemical structure of the compounds described in this disclosure, the bonds... No configuration was specified, i.e., key The configuration can be E-type or Z-type, or it can contain both E-type and Z-type configurations.

[0092] Although all the above structural formulas are shown in some isomer form for simplicity, this disclosure can include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers. Detailed Implementation

[0093] 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 are not intended to limit this disclosure in any way.

[0094] Example 1

[0095] Step 1: Synthesis of Compound 1

[0096] The reactant glycylamide (40 g, 1.05 eq.) was suspended in THF (80 mL, 2V), water (240 mL, 6V), ethyl acetate (800 mL, 20V), sodium carbonate (44.5 g, 1.2 eq.) was added, Fmoc-OSu (118 g, 1.0 eq.), the reaction was stirred at 25 °C, after the reaction was completed, petroleum ether (160 mL, 4V) was added to the reaction solution, stirred at room temperature for 2 hours, then filtered, the filter cake was washed with ethyl acetate, and dried to obtain compound 1 (107 g, yield: 100%).

[0097] 1 HNMR (400 MHz, DMSO-d6) δ 7.90 (d, J = 7.6 Hz, 2H), 7.72 (d, J = 7.6 Hz, 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).

[0098] Second step: synthesis of compound 2

[0099] Compound 1 (50 g, 1.0 eq.) was suspended in water (400 mL, 8V), potassium carbonate (1.17 g, 0.05 eq.) was added, aqueous formaldehyde (27.3 g, 2.0 eq.), the reaction was stirred at an external temperature of 80 °C, after the reaction was completed, the reaction solution was cooled to room temperature, filtered, the filter cake was washed with water (200 mL, 4V), and dried to obtain compound 2 (50 g, yield: 91%).

[0100] 1 HNMR (400 MHz, DMSO-d6) δ 7.90 (d, J = 7.6 Hz, 2H), 7.72 (d, J = 7.6 Hz, 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).

[0101] Third step: synthesis of compound 3

[0102] Compound 2 (20 g, 1.0 eq.) was suspended in dichloromethane (300 mL, 15V), triethylamine (12.4 g, 2.0 eq.) was added, and the temperature was lowered to 0°C. Benzoyl chloride (12.9 g, 1.5 eq.) was added dropwise, and the reaction was stirred at room temperature after the addition was completed. After the reaction was completed, the reaction solution was directly filtered, the filter cake was slurried with water (100 mL, 5V), and then filtered again. After the filter cake was dried, compound 3 (18.4 g, yield: 70%) was obtained.

[0103] 1 HNMR (400 MHz, DMSO-d6) δ 9.15-9.06 (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 = 6.8 Hz, 2H), 4.35-4.15 (m, 3H), 3.78-3.65 (m, 2H).

[0104] Fourth step: synthesis of compound 4

[0105] Compound 3-1 (1.13 g, 1.0 eq.) was dissolved in THF (11 mL, 10V), and the temperature was lowered to -20°C. Tert-butoxy lithium (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, 10V), and was slowly added dropwise to the reaction bottle while maintaining a 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. Compound 4 (1.84 g, yield: 65%) was obtained by crystallizing the crude product with ethyl acetate.

[0106] 1 HNMR (400 MHz, DMSO-d6) δ 9.15-9.06 (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 = 6.8 Hz, 2H), 4.35-4.15 (m, 3H), 3.78-3.65 (m, 2H).

[0107] Fifth step: synthesis of compound 5

[0108] Compound 4 (2 g, 1.0 eq.) was dissolved in THF (4 mL, 2V), ethyl acetate (60 mL, 30V) was added, and after dissolution, 10% palladium-carbon (0.4 g, 0.2 wt) was added; the temperature was lowered to 0°C, and the system was replaced with hydrogen three times by vacuum-hydrogen replacement, and the reaction was stirred for 12 h under a hydrogen atmosphere; after the reaction was completed, the system was replaced with nitrogen, and the palladium-carbon was removed by filtration over celite, the filter cake was washed with THF, and the filtrate was concentrated to dryness to obtain the crude product, which was crystallized with DCM to obtain compound 5 (1.3 g, yield: 80%).

[0109] 1 HNMR (400 MHz, DMSO-d6) δ 8.70-8.60 (m, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.72 (d, J = 7.6 Hz, 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 = 5.2 Hz, 2H), 3.46 (d, J = 7.6 Hz, 1H), 1.03-1.00 (m, 1H), 0.52-0.25 (m, 4H).

[0110] Comparative Example 1

[0111] A reaction bottle was charged with 1440 mL of tetrahydrofuran, and 80 g of raw material D-1, 480 mL of toluene, and 80 g of celite were added. Tetraacetate was added at one time, and the system was stirred at room temperature for 10 min. Pyridine was added, and the reaction was carried out under reflux for 3 h. The temperature was lowered to room temperature, and the system was filtered over celite, and the filter cake was washed with 400 mL of tetrahydrofuran, and the filtrates were combined. The filtrate was concentrated to obtain 130 g of an oil. 500 mL of anhydrous ethanol was added to dissolve the system, and the system was transferred to a reaction kettle. 1100 mL of anhydrous ethanol was further added, and the system was stirred for 24 h. The system was filtered, the filter cake was washed with 500 mL of n-heptane, and the product was dried to obtain compound D (28 g, yield 33.6%).

[0112] Since the present disclosure has been described in terms of specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are included within the scope of the present disclosure.

Claims

1. A process for preparing a compound represented by the formula (IV) or a salt thereof, comprising: the preparation of a compound of formula (V) or a salt thereof from a compound of formula (VI) or a salt thereof, and the preparation of a compound of formula (IV) or a salt thereof from a compound of formula (V) or a salt thereof, wherein R1is amino or amino protected by an amino protecting group, R2 is a hydroxyl protecting group, 2. The method of preparation according to claim 1, wherein the amino protecting group is selected from the group consisting of acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, pivaloyl, formyl, benzoyl, phthaloyl, 9-fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl and benzyloxycarbonyl, preferably 9-fluorenylmethyloxycarbonyl.

3. The method of preparation according to claim 1 or 2, wherein the hydroxyl protecting group is selected from the group consisting of 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 method of preparation according to any one of claims 1 to 3, wherein the compound of formula (VI) or a salt thereof is reacted with an aldehyde to produce the compound of formula (V) or a salt thereof, preferably the molar ratio of the compound of formula (VI) or a salt thereof to the aldehyde is 1 : 1 to 1 :

5.

5. The method of preparation according to any one of claims 1 to 4, wherein R2is benzoyl, the reaction to produce the compound of formula (IV) or a salt thereof is carried out in the presence of benzoyl halide and an acid binding agent, preferably the acid binding agent is selected from at least one of potassium carbonate, triethylamine, DIPEA, pyridine, preferably the molar ratio of the compound of formula (V) or a salt thereof to the benzoyl halide is 1 : 1 to 1 :

5.

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

7. 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 according to the present disclosure, and preparing a compound of formula (III) or a salt thereof from a compound of formula (IV) or a salt thereof, wherein, R1is as defined in claim 1 ; L1is -(C(R 11 )(R 12 )) n -(CH2) m -; C3-C6saturated cycloalkyl or 3-6 membered saturated heterocycloalkyl, each independently optionally substituted with one or more substituents selected from halogen, hydroxyl, amino, and haloalkyl; R 11 R 12 Each is independently selected from hydrogen atoms, 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, or R 11 R 12 Together with the carbon atom connected thereto, they form a 3-6 membered cycloalkyl group which may be optionally substituted by one or more substituents selected from C1-C6 alkyl, halogen, hydroxyl, amino, oxo and C1-C6 alkoxy groups; m is 0 or 1, preferably 0; n is 1, 2, 3 or 4, preferably 1.

8. The production method according to claim 7, wherein the compound represented by formula (III) is selected from 9. The production method according to claim 7 or 8, wherein the method comprises a step of producing a compound represented by formula (III') or a salt thereof from a compound represented by formula (IV) or a salt thereof, and a step of producing a compound represented by formula (III) or a salt thereof by decarboxylating a protecting group from the compound represented by formula (III') or a salt thereof, wherein R3is carboxyl protected by a carboxyl protecting group, preferably a methyl, substituted methyl, ethyl, 2-substituted ethyl, allyl, tert-butyl, alkoxyalkyl, alkoxyalkoxyalkyl, 2,6-dialkylphenyl, benzyl, substituted benzyl, silyl and stannyl carboxyl protecting group.

10. A process for the preparation of a compound of formula (II) or a salt thereof, comprising the steps of preparing a compound of formula (IV) or a salt thereof according to any one of claims 1 to 6, and / or the steps of preparing a compound of formula (III) or a salt thereof according to any one of claims 7 to 9, wherein, R1, L1are as defined in claim 7; R 13 selected from the group consisting of hydrogen atom, deuterium atom, C 1-6 alkyl, 6-10 membered aryl and 5-10 membered heteroaryl, wherein said alkyl, aryl, heteroaryl are optionally substituted by one or more substituents selected from the group consisting of C1-C6 alkyl, halogen, hydroxy, amino and oxo.

11. The method of manufacturing according to claim 10, wherein the compound of 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, a compound of formula (III) or a salt thereof according to any one of claims 7 to 9, and a compound of formula (II) according to any one of claims 10 to 11, wherein L1, R 13 As recited in claim 10, p is an integer from 2 to 8.

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

15. The method of manufacturing of claim 14, wherein the antibody or antigen-binding fragment thereof is selected from an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUC1 antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC44A4 antibody, or an anti-Mesothelin antibody or antigen-binding fragment thereof.

16. The method of manufacturing of claim 14 or 15, wherein 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.

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

18. A compound represented by the formula (IV') or a salt thereof, wherein, R2is selected from the group consisting of p-nitrobenzyl, benzoyl, formyl, trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, isopropyldiethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, t-butyloxycarbonyl, methoxymethyl, and benzyloxymethyl; The compounds are preferably

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