A method for preparing an eribulin derivative

Abstract

A method for preparing eribulin derivatives is disclosed. Specifically, a method for preparing a compound as shown in formula (I) is disclosed, which has high yield, mild reaction conditions, and is suitable for industrial production.

Description

[0001] This application claims priority to Chinese patent application 202111196863.7, filed on October 14, 2021. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the pharmaceutical field and relates to a method for preparing an eribulin derivative. Background Technology

[0003] Microtubules are powerful, filamentous cytoskeletal proteins involved in a wide range of cellular functions, including intracellular migration and transport, cell signaling, and maintaining cell shape. Microtubules also play a crucial role in mitotic cell division by forming the mitotic spindle, necessary for chromosomes to divide into two daughter cells. The biological function of microtubules in all cells is largely regulated by their polymerization kinetics, which occur through the reversible, non-covalent addition of α- and β-tubulin dimers to the ends of the microtubules. This kinetic behavior and the resulting control over microtubule length are essential for the proper functioning of the mitotic spindle. Even minute changes in microtubule kinetics can involve axon checkpoints, inhibiting cell cycle progression during mitosis and subsequently leading to cell death (Mukhtar et al. (2014) Mol. Cancer Ther. 13: 275-84). Because cancer cells divide rapidly, they are generally more sensitive than normal cells to compounds that bind to tubulin and disrupt its normal function. Therefore, tubulin inhibitors and other microtubule-targeting agents are expected to become a class of drugs for treating cancer (Dumontet and Jordan (2010) Nat. Rev. Drug Discov. 9: 790-803).

[0004] Halichondrin B is a polyether macrocyclic lactone compound containing only C, H, and O atoms, isolated in 1986 by Japanese scientists Hirata and Uemura from the sponge *Halichondria okadai*. It exhibits extremely strong in vitro antitumor activity. Halichondrin B has a very complex molecular structure, containing 32 chiral centers and over 4 billion isomers, making its synthesis extremely difficult. Eribulin is a derivative of halichondrin B and a microtubule inhibitor. Its structure and synthesis method were first disclosed in WO9965894. On November 15, 2010, the FDA approved eribulin mesylate (Halaven) injection for the treatment of metastatic breast cancer patients who have received at least two prior chemotherapy regimens.

[0005] PCT / CN2021 / 073314 relates to a novel eribulin derivative and its drug conjugate. This compound and its drug conjugate exhibit good antitumor activity. The compound structure is shown below:

[0006] Summary of the Invention

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

[0008] This disclosure also provides a method for preparing a compound as shown in formula (I), comprising the steps of preparing a compound of formula (I-1) from a compound of formula (I-2), and the steps of removing a protecting group from a compound of formula (I-1) to prepare a compound of formula (I).

[0009]

[0010] R1 is selected from alkyl groups (such as C12-alkyl groups). 1-6 Alkyl groups, including but not limited to methyl, ethyl, and isopropyl groups, and cycloalkyl groups (such as C14, C24, C34, C4 ... 3-8 Cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl, or cyclohexyl), aryl, and heteroaryl groups, wherein each alkyl, cycloalkyl, aryl, and heteroaryl group is independently selected from alkyl groups (such as C14, C24, C34, C44, C54, C6 ... 1-6 Alkyl groups, including but not limited to methyl, ethyl, and isopropyl groups, and alkoxy groups (such as C14, C24, C34, C4 ... 1-6 The alkoxy group (including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups are substituted with one or more substituents, preferably C10. 1-6 Alkyl or C 3-8 Cycloalkyl, more preferably methyl.

[0011] In some embodiments, R1 is selected from methyl, and the methylating agent used in the step is selected from iodomethane, dimethyl sulfate or trimethyloxonium tetrafluoroborate, preferably trimethyloxonium tetrafluoroborate.

[0012] In some embodiments, the molar ratio of the compound represented by formula (I-2) to the methylating agent is 1:0.1 to 1:10, preferably 1:1 to 1:5, and more preferably 1:1 to 1:3.

[0013] In some embodiments, the reaction temperature is -50 to -50°C, preferably -20 to -5°C.

[0014] The amino protecting group can be removed using methods commonly used in the art. In some embodiments, the reagent for removing the amino protecting group is selected from ammonia, methylamine alcohol solution, ethylenediamine, and hydrazine, preferably methylamine ethanol solution.

[0015] In some embodiments, the method further includes the step of preparing the compound of formula (I-2) from the compound of formula (I-3).

[0016]

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

[0018] This disclosure also provides a method for preparing the compound of formula (I), including the method for preparing the compound of formula (I) described in this disclosure, wherein L is a linker.

[0019]

[0020] In some embodiments, the linker is stable outside the cell, allowing the ADC to remain intact in its extracellular environment but cleave upon internalization in cells, such as cancer cells. In some embodiments, when the ADC enters a cell expressing an antigen specific to the antibody portion of the ADC, the eribulin derivative drug portion cleaves from the antibody portion, releasing the unmodified form of the eribulin derivative. In some embodiments, the linker includes a cleavable portion positioned such that no portion of the linker or the antibody portion remains bound to the eribulin derivative after cleavage.

[0021] In some embodiments, the cleavable portion in the linker is a cleavable peptide portion. In some embodiments, an ADC containing a cleavable peptide portion exhibits lower aggregation levels, improved antibody:drug ratios, increased targeted killing of cancer cells, reduced off-target killing of non-cancer cells, and / or higher drug loading compared to an ADC containing an alternative cleavable portion. In some embodiments, adding a cleavable portion increases cytotoxicity and / or potency compared to a non-cleavable linker. In some embodiments, the increased potency and / or cytotoxicity is in cancers expressing moderate levels of antigens targeted by the antibody portion of the ADC (e.g., moderate FRA expression). In some embodiments, the cleavable peptide portion is enzymatically cleavable, and the linker is an enzyme-cleavable linker. In some embodiments, the enzyme is a cathepsin, and the linker is a cathepsin-cleavable linker. In some embodiments, an enzyme-cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the aforementioned improved properties compared to an alternative cleavage mechanism.

[0022] In some embodiments, the linker comprises an amino acid unit, which preferably comprises a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, more preferably valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lys (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe), or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0023] In some implementations, the connector is selected from:

[0024]

[0025]

[0026] This disclosure also provides a method for preparing an antibody-drug conjugate of formula (ADC-I), comprising the steps of preparing the antibody-drug conjugate of formula (ADC-I) from a compound of formula (LI), and the steps for preparing the compound of formula (LI) as described in this disclosure.

[0027] Ab-(LD) k

[0028] (ADC-I)

[0029] Wherein, Ab represents an antibody or its antigen-binding fragment.

[0030] L is a connector that covalently links Ab to D, as defined in any one of claims 5-7; and k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any value between any two values).

[0031] -D is shown in the following formula:

[0032]

[0033] R1 is selected from alkyl groups (such as C12-alkyl groups). 1-6 Alkyl groups, including but not limited to methyl, ethyl, and isopropyl groups, and cycloalkyl groups (such as C14, C24, C34, C4 ... 3-8 Cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl, or cyclohexyl), aryl, and heteroaryl groups, wherein each alkyl, cycloalkyl, aryl, and heteroaryl group is independently selected from alkyl groups (such as C14, C24, C34, C44, C54, C6 ... 1-6 Alkyl groups, including but not limited to methyl, ethyl, and isopropyl groups, and alkoxy groups (such as C14, C24, C34, C4 ... 1-6 The alkoxy group (including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups are substituted with one or more substituents, preferably C10. 1-6 Alkyl or C 3-8 Cycloalkyl, more preferably methyl.

[0034] In some embodiments, the antibody described in the antibody-drug conjugate (ADC) of this disclosure is selected from murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0035] In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate (ADC-I) 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 antibody, etc. Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-CD79 antibody, anti-TROP-2 antibody, anti-CD79B antibody, anti-Mesothelin antibody, anti-folate receptor α (FRA) antibody or their antigen-binding fragments.

[0036] In some embodiments, the antibody in the antibody-drug conjugate (ADC-I) is a known antibody selected from, but not limited to, trastuzumab, pertuzumab, nimotuzumab, enoblituzumab, emibetuzumab, Inotuzumab, vitin-pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96, farletuzumab, and glematumamab, or an antigen-binding fragment thereof.

[0037] In some embodiments, the antibody-drug conjugate is

[0038]

[0039] Where D is The preferred Ab is farletuzumab, and the preferred k is selected from 2.0 to 2.5 or 2.5 to 3.5.

[0040] The compound shown in formula (LI) can be prepared by using the compound shown in formula (I) as a reactant, or by using the compound shown in formula (LI) as a reactant to prepare the antibody-drug conjugate shown in formula (ADC-I), such as the method disclosed in PCT / CN2021 / 073314, which is incorporated herein by reference in its entirety.

[0041] This disclosure also provides compounds of formula (I-1),

[0042]

[0043] R1 is selected from alkyl groups (such as C12-alkyl groups). 1-6 Alkyl groups, including but not limited to methyl, ethyl, and isopropyl groups, and cycloalkyl groups (such as C14, C24, C34, C4 ... 3-8 Cycloalkyl groups, including but not limited to cyclopropyl, cyclopentyl, or cyclohexyl), aryl, and heteroaryl groups, wherein each alkyl, cycloalkyl, aryl, and heteroaryl group is independently selected from alkyl groups (such as C14, C24, C34, C44, C54, C6 ... 1-6 Alkyl groups, including but not limited to methyl, ethyl, and isopropyl groups, and alkoxy groups (such as C14, C24, C34, C4 ... 1-6 The alkoxy group (including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups are substituted with one or more substituents, preferably C10. 1-6 Alkyl or C 3-8 Cycloalkyl, more preferably methyl.

[0044] The novel method for preparing eribulin derivatives disclosed herein employs a new amino protecting group, avoiding selectivity issues during methylation, significantly reducing the generation of dimethylated impurities, alleviating the difficulty of post-reaction processing, and improving reaction yield, which is beneficial for large-scale industrial production. Existing preparation processes yield products with numerous dimethylated impurities that are difficult to remove, ultimately affecting the quality of the final product.

[0045] The "alkyl" referred to in this disclosure is preferably a C1-C6 alkyl.

[0046] The "alkenyl" as described in this disclosure is preferably a C2-C6 alkenyl.

[0047] The "alkynyl group" described in this disclosure is preferably a C2-C6 alkynyl group.

[0048] The "alkylene" described in this disclosure is preferably a C1-C6 alkylene.

[0049] The "sub-alkenyl" described in this disclosure is preferably a C2-C6 sub-alkenyl.

[0050] The "sub-chain alkynyl" described in this disclosure is preferably a C2-C6 sub-chain alkynyl.

[0051] The "alkoxy group" described in this disclosure is preferably a C1-C6 alkoxy group.

[0052] The "alkyl thioether group" described in this disclosure is preferably a C1-C6 alkyl thioether group.

[0053] The "cycloalkyl" in this disclosure is preferably 3 to 12-membered, and more preferably 3 to 6-membered cycloalkyl.

[0054] The "fused cycloalkyl" described in this disclosure is preferably a 6- to 14-membered fused cycloalkyl, more preferably a 7- to 10-membered fused cycloalkyl.

[0055] The "heterocyclic group" described in this disclosure is preferably a 3- to 12-membered heterocyclic group, and more preferably a 3- to 6-membered heterocyclic group.

[0056] The "fused heterocyclic group" described in this disclosure is preferably a 6- to 14-membered fused heterocyclic group, and more preferably a 7- to 10-membered fused heterocyclic group.

[0057] The "aryl" in this disclosure is preferably 6 to 14 methyl groups, and more preferably 6 to 10 methyl groups.

[0058] The "heteroaryl" as described in this disclosure is preferably 5 to 12 quinones, and more preferably 5 to 10 quinones.

[0059] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0060] The term "drug" refers to cytotoxic drugs or immunomodulators. Cytotoxic drugs are chemical molecules that strongly disrupt the normal growth of tumor cells. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations; however, due to their lack of specificity, they can also induce apoptosis in normal cells while killing tumor cells, leading to serious side effects. The term also includes toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., At). 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 (and radioactive isotopes of Lu), toxic drugs, chemotherapeutic agents, antibiotics, and ribolysins. Immunomodulators are inhibitors of immune checkpoint molecules.

[0061] The terms “linker,” “linker unit,” “connector unit,” “connector,” or “linker fragment” refer to a chemical structural segment or bond that is connected to a ligand at one end and to a drug at the other end. It can also be connected to other linkers before being connected to a drug.

[0062] The linker may comprise one or more linker components. Exemplary linker components include 6-maleiminohexanoyl (MC), maleiminopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with a linker reagent: N-succinimino-4-(2-pyridylthio)valerate (SPP), N-succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylate (SMCC, also referred to herein as MCC), and N-succinimino-(4-iodo-acetyl)aminobenzoate (SIAB). The linker may comprise stretching units, spacer units, amino acid units, and extension units. It can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker may be a “cleavable linker” that facilitates drug release into cells. For example, acid-labile connectors (e.g., hydrazone), protease-sensitive connectors (e.g., peptidase-sensitive), light-labile connectors, dimethyl connectors, or disulfide-containing connectors can be used (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0063] The term "stretching unit" refers to a chemical structural segment that is covalently linked to an antibody at one end via a carbon atom and to an amino acid unit, disulfide moiety, sulfonamide moiety, or non-peptide chemical moiety at the other end.

[0064] The term "spacer unit" is a bifunctional compound structural fragment that can be used to couple amino acid units and cytotoxic drugs to ultimately form antibody-drug conjugates. This coupling method allows cytotoxic drugs to be selectively linked to amino acid units.

[0065] The term "amino acid" refers to an organic compound whose molecular structure contains both an amino group and a carboxyl group, with both groups directly attached to the -CH- structure. The general formula is H₂NCHRCOOH, where R is H, a substituted or unsubstituted alkyl group, etc. Based on the position of the amino group attached to the carbon atom in the carboxylic acid, amino acids can be classified into α, β, γ, δ, ε...-amino acids. In the biological world, the amino acids that constitute natural proteins have specific structural characteristics, namely, their amino groups are directly attached to the α-carbon atom, i.e., α-amino acids, including glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, and proline. Non-natural amino acids, such as citrulline, are also present. As is known to those skilled in the art, non-natural amino acids do not constitute natural proteins and therefore do not participate in the synthesis of the antibodies disclosed herein. The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0066]

[0067]

[0068] In this disclosure, the extension unit is PAB, with a structure like the 4-iminobenzylcarbamoyl segment, as shown in formula (VI), attached to D.

[0069]

[0070] abbreviation

[0071] Connector components include, but are not limited to:

[0072] MC = 6-maleiminohexanoyl, with the following structure:

[0073]

[0074] Val-Cit or "vc" = valine-citrulline (an example dipeptide in a protease-cleavable linker).

[0075] Citrulline = 2-amino-5-ureaporic acid

[0076] PAB = p-Aminobenzyloxycarbonyl (an example of a "self-sacrificing" connector assembly)

[0077] Me-Val-Cit = N-methyl-valine-citrulline (where the linker peptide bond is modified to prevent cleavage by cathepsin B).

[0078] MC(PEG)6-OH = Maleiminohexanoyl-polyethylene glycol (can attach to antibody cysteine)

[0079] SPP = N-succinimino-4-(2-pyridylthio)valerate

[0080] SPDP = N-succinimino-3-(2-pyridyldithio)propionate

[0081] SMCC = succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid ester

[0082] IT = iminothion

[0083] PBS = phosphate buffered saline solution.

[0084] The term "antibody-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker. In this disclosure, "antibody-drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a stable linker.

[0085] The term "drug loading" can be expressed as the ratio of drug amount to antibody amount, i.e., the average number of drugs conjugated to each antibody in an ADC. The drug loading range can be 1-20, preferably 1-10, cytotoxic drugs (D) linked to each antibody (Ab). In embodiments of this disclosure, the 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 average of any two values. Preferably 1-10, more preferably 1-8, or 2-8, or 2-7, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or the average of 4-5. The average number of drugs per ADC molecule after the conjugation reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, monoclonal antibody molecular size variant assay (CE-SDS), and HPLC characterization.

[0086] The present invention discloses a method for determining the molecular size variant of monoclonal antibodies (CE-SDS), which uses sodium dodecyl sulfate capillary electrophoresis (CE-SDS) with ultraviolet detection. Under reducing and non-reducing conditions, the purity of recombinant monoclonal antibody products is quantitatively determined according to molecular weight using capillary electrophoresis (2015 edition of the Chinese Pharmacopoeia 0542).

[0087] The loading of antibody-drug conjugates can be controlled using the following non-restrictive methods, including:

[0088] (1) Control the molar ratio of the ligation reagent and the monoclonal antibody.

[0089] (2) Control the reaction time and temperature.

[0090] (3) Choose different reaction reagents.

[0091] The term "antibody" refers to immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further divide them into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in the constant region. Each of the five classes of Ig can have either a κ chain or a λ chain. The antibodies described in this disclosure are preferably specific antibodies against cell surface antigens on target cells. The sequence of approximately 110 amino acids near the N-terminus of both the heavy and light chains of the antibody varies considerably and is termed the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are termed the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0092] The antibodies disclosed herein include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

[0093] The term "mouse antibody" in this disclosure refers to antibodies prepared using mice in accordance with the knowledge and skills in the art. Preparation involves injecting a test subject with a specific antigen, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics.

[0094] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can reduce the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies must first be established. Then, the variable region gene is cloned from the murine hybridoma cells. Next, the constant region gene of the human antibody is cloned as needed. The murine variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

[0095] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., a human germline antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse or reversion mutations to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR. Further literature describing methods that can be used to participate in humanization includes, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and Winter et al. [Jones et al., Nature, 321, 522 (1986), Riechmann et al., Nature, 332, 323-327 (1988), Verhoeyen et al., Science, 239, 1534 (1988)].

[0096] The term "fully human antibody," also known as a "fully human monoclonal antibody," refers to an antibody whose variable and constant regions are both human-derived, eliminating immunogenicity and toxicity. The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. This disclosure pertains to fully human monoclonal antibodies. Related technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

[0097] The term “antigen-binding fragment” refers to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in “antigen-binding fragments” include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab′)2 fragments, bivalent fragments comprising two Fab fragments connected by disulfide bridges on hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains of a single arm of the antibody; (v) single-domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546) consisting of a VH domain; and (vi) separate complementarity-determining regions (CDRs) or (vii) combinations of two or more separate CDRs optionally connected by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0098] Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by treating IgG antibody molecules with the protease papain (which cleaves the amino acid residue at position 224 of the H chain). Approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

[0099] F(ab′)2 is an antibody fragment with a molecular weight of approximately 100,000, possessing antigen-binding activity, and containing two Fab regions connected at the hinge position, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin.

[0100] Fab′ is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab′)2.

[0101] In addition, Fab′ can be produced by inserting DNA encoding the Fab′ fragment of an antibody into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote to express Fab′.

[0102] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using variants with 1–4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444–6448). Other connectors that may be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immuno 1.31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipfiyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

[0103] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (CDRL1, CDRL2, CDRL3 or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDRH2, CDRH3 or H2, H3) of the heavy chain variable domain. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable domain and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable domain. The amino acid sequence boundaries of the CDR can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (see Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)). Following the IMGT rule, the CDR region of an antibody can be determined using the IMGT / DomainGap Align procedure.

[0104] The term "antibody framework" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0105] The term “epitope” or “antigenic determinant” refers to the site on an antigen where an immunoglobulin or antibody specifically binds. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed (1996)).

[0106] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example: approximately less than 10 -8 M, 10 - 9 M or 10 -10 M or lower affinity (KD) binding.

[0107] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0108] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

[0109] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to a non-human CDR region. Human FR germline sequences can be obtained by comparing with the IMGT Human Antibody Variable Region Germline Gene Database and MOE software, from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr, or from the journal Immunoglobulins, 2001 ISBN012441351.

[0110] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.

[0111] The engineered antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or GSepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0112] Amino acid sequence “identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence, after aligning the amino acid sequences and, where necessary, introducing gaps to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0113] The term "peptide" refers to a compound fragment that lies between amino acids and proteins. It is composed of two or more amino acid molecules linked together by peptide bonds. It is a structural and functional fragment of proteins, such as hormones and enzymes, which are essentially peptides.

[0114] The term "sugar" refers to a biological macromolecule composed of three elements: C, H, and O. It can be classified into monosaccharides, disaccharides, and polysaccharides.

[0115] The term "fluorescent probe" refers to a class of fluorescent molecules that exhibit characteristic fluorescence in the ultraviolet-visible-near-infrared region, and whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime, and polarization, etc.) can be sensitively altered by changes in the properties of their environment, such as polarity, refractive index, and viscosity. These fluorescent probes interact non-covalently with nucleic acids (DNA or RNA), proteins, or other macromolecular structures, causing changes in one or more fluorescence properties. They can be used to study the properties and behavior of macromolecules.

[0116] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0117] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester.

[0118] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0119] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups. "Carbocyclic" refers to the ring system within the cycloalkyl group.

[0120] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m The heteroatom (where m is an integer from 0 to 2) excluding the ring portion of -OO-, -OS-, or -SS-, with the remaining ring atoms being carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 6 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, imidazoalkyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazoyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably piperidinyl or pyrrolidinyl. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups. "Heterocyclic" refers to the ring system within the heterocyclic group.

[0121] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring. "Aromatic ring" refers to the ring system within the aryl group. Non-limiting examples of aryl groups include:

[0122]

[0123] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylic acid ester group, preferably phenyl.

[0124] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 12-membered, such as imidazolyl, furanyl, thiophenel, thiazolyl, pyrazolyl, oxazolyl, pyrrololyl, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl, or thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. "Heteroaryl ring" refers to the ring system within the heteroaryl group. Non-limiting examples of heteroaryl groups include:

[0125]

[0126] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylic acid ester group.

[0127] "Carboxyl protecting group" is a suitable group known in the art for carboxyl protection, see reference ("Protective Groups in Organic Synthesis", 5). Th The carboxyl protecting group in Ed.TWGreene & P.GMWuts, as an example, can be a substituted or unsubstituted C. 1-10 Straight-chain or branched alkyl, substituted or unsubstituted C 2-10 Straight-chain or branched alkenyl or alkynyl, substituted or unsubstituted C 3-8 Cyclic alkyl, substituted or unsubstituted C 5-10 aryl or heteroaryl, or (C 1-8 Alkyl or aryl) 3-silyl, etc.

[0128] "Amino protecting group" is a suitable group known in the art for amino protection, see reference ("Protective Groups in Organic Synthesis", 5). ThThe amino protecting group in Ed.TW Greene & P.GMWuts, preferably, is a (C 1-10 Alkyl or aromatic acyl group, such as formyl, acetyl, benzoyl, etc.; can be (C 1-6 Alkyl or C 6-10 aryl)sulfonyl; or (C 1-6 Alkoxy or C 6-10 Aryloxy)carbonyl, such as Boc or Cbz; can also be substituted or unsubstituted alkyl, such as triphenylmethyl (Tr), 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).

[0129] The term "leaving group" refers to an atom or functional group that detaches from a larger molecule during a chemical reaction. Representative leaving groups include halogens, substituted sulfonyloxy groups, phosphoryloxy groups, amino groups, and (R... j )3N-, cyano, R j S-etc., where R j It is independently selected from hydrogen atoms or C1 to C6 alkyl groups.

[0130] The substituted sulfonyloxy group can be C t ~C6 alkylsulfonyloxy, perfluorinated C1~C6 alkylsulfonyloxy, arylsulfonyloxy, aralkylsulfonyloxy, etc.

[0131] Specific examples of C1-C6 alkylsulfonyloxy groups include C1-C6 straight-chain or branched alkylsulfonyloxy groups, such as methylsulfonyloxy, ethylsulfonyloxy, n-propylsulfonyloxy, isopropylsulfonyloxy, n-butylsulfonyloxy, tert-butylsulfonyloxy, n-pentylsulfonyloxy, and n-hexylsulfonyloxy.

[0132] Specific examples of perfluoroC1–C6 alkylsulfonyloxy groups include C1–C6 straight-chain or branched perfluoroalkylsulfonyloxy groups, such as trifluoromethylsulfonyloxy, 1,1,2,2,2-pentafluoro-1-ethylsulfonyloxy, 1,1,2,2,3,3,3-heptafluoro-1-propylsulfonyloxy, and 1,1,2,2,3,3,4,4,4-nonafluoro-1-butylsulfonyloxy.

[0133] Examples of arylsulfonyloxy groups include phenylsulfonyloxy and naphthylsulfonyloxy groups, which optionally have 1 to 3 substituents on the benzene ring, selected from the group consisting of a C1-C6 straight-chain or branched alkyl group, a C1-C6 straight-chain or branched alkyl group, a nitro group, and a halogen atom. Specific examples of phenylsulfonyloxy groups optionally having substituents include phenylsulfonyloxy, 4-methylphenylsulfonyloxy, 2-methylphenylsulfonyloxy, 4-nitrophenylsulfonyloxy, 4-tolylsulfonyloxy, 2-nitrophenylsulfonyloxy, 3-chlorophenylsulfonyloxy, and so on. Specific examples of naphthylsulfonyloxy groups include α-naphthylsulfonyloxy, β-naphthylsulfonyloxy, and so on.

[0134] Examples of aralkylsulfonyloxy groups include: C1-C6 straight-chain or branched alkylsulfonyloxy groups substituted with a phenyl group (which optionally has 1 to 3 substituents selected from C1-C6 straight-chain or branched alkyl groups, C1-C6 straight-chain or branched alkyl groups, nitro groups, and halogen atoms on the benzene ring); and C1-C6 straight-chain or branched alkylsulfonyloxy groups substituted with a naphthyl group. Specific examples of phenyl-substituted alkylsulfonyloxy groups include benzylsulfonyloxy, 2-phenylethylsulfonyloxy, 4-phenylbutylsulfonyloxy, 4-methylbenzylsulfonyloxy, 2-methylbenzylsulfonyloxy, 4-nitrobenzylsulfonyloxy, 4-methylbenzylsulfonyloxy, 3-chlorobenzylsulfonyloxy, etc. Specific examples of naphthyl-substituted alkylsulfonyloxy groups include α-naphthylmethylsulfonyloxy, β-naphthylmethylsulfonyloxy, etc.

[0135] "Optional" or "optionally" means that the event or circumstance described below may, but does not have to, occur. This description includes situations in which the event or circumstance may or may not occur. For example, "optionally alkyl-substituted heterocyclic group" means that an alkyl group may, but does not have to, be present. This description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0136] In the chemical structure of the compounds described in this disclosure, the bonds... The configuration is not specified, meaning that if configurational isomerism exists in the chemical structure, the bond... It can be or Or simultaneously include and Two configurations. In the chemical structure of the compounds described in this disclosure, the bonds... No configuration is specified, meaning it can be Z configuration, E configuration, or both configurations. Detailed Implementation

[0137] The following detailed explanation of this disclosure will be provided with specific examples to enable those skilled in the art to have a more comprehensive understanding of this disclosure. The specific examples are only used to illustrate the technical solutions of this disclosure and do not limit this disclosure in any way.

[0138] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0139] Unless otherwise specified in the examples, the reactions can be carried out under an argon or nitrogen atmosphere.

[0140] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0141] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0142] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20℃~30℃.

[0143] Example 1

[0144]

[0145] Step 1: Synthesis of Compounds 1-2

[0146] Sodium bicarbonate (2.0 g, 24 mmol) was weighed into a reaction flask, and 10 mL of purified water was added. Compounds 1-3 (4.95 g, 6.00 mmol) were dissolved in 130 mL of purified water and added to the reaction flask. N-ethoxycarbonyl phthalimide (5.3 g, 24 mmol) was weighed into the above reaction solution. After the addition was complete, the mixture was evacuated under argon protection and stirred at room temperature until the reaction was complete. The reaction solution was extracted with DCM, and the aqueous phase was extracted with DCM. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous magnesium sulfate, filtered, concentrated, and dried to obtain the crude product. Column chromatography yielded 4.89 g of compounds 1-2, yield: 93%.

[0147] Step 2: Synthesis of Compound 1-1

[0148] Weigh trimethyloxonium tetrafluoroborate (13.4 g, 90.5 mmol) and 1,8-bis(dimethylaminonaphthalene) (19.4 g, 90.5 mmol) into a reaction flask. After addition, evacuate the flask under argon protection and add 100 mL of anhydrous DCM. Cool to -10°C. Dissolve 1-2 (4.86 g, 5.66 mmol) in 150 mL of DCM and add it to the above reaction solution. Incubate the reaction overnight. Stop the reaction when the reactants have basically reacted completely. Filter the solution and concentrate it to dryness. Add 200 mL of methyl tert-butyl ether, filter, and wash the mother liquor three times with 100 mL of 1M potassium hydrogen sulfate aqueous solution. Wash the aqueous phase twice with methyl tert-butyl ether. Combine the organic phases, dry to anhydrous magnesium sulfate, concentrate, and dry to obtain the crude product. Column chromatography yields 4.74 g of compound 1-1; yield: 96%, HPLC purity: 98%.

[0149] Step 3: Synthesis of Compound 1

[0150] Weigh 6.0 g of compound 1-1 into a reaction flask, add 180 mL of ethanol solution of methylamine, and stir overnight at room temperature under argon protection. Stop the reaction when the reactants have completely reacted. Concentrate to dryness under reduced pressure, extract with 200 mL of DCM and 100 mL of purified water. Wash the organic phase three times with 100 mL of purified water, combine the aqueous phases, extract twice with DCM, combine the organic phases, dry to anhydrous magnesium sulfate, concentrate, and give the crude product. Column chromatography yields 4.2 g of compound 1; overall yield: 86.7%.

[0151] Example 2

[0152]

[0153] Compound 2 (17.6 g, prepared according to PCT / CN2021 / 073314) and 126 ml DMF were added to the reaction flask and stirred to dissolve. Sodium bicarbonate was added, and the mixture was stirred at 20-25 °C. Compound 1 was dissolved in 63 ml DMF and added dropwise to the above reaction solution using a constant pressure dropping funnel. After the addition was complete, the reaction was continued at 20-25 °C for 2.5-3 h. The reaction was confirmed to be complete by TLC. 1134 ml ethyl acetate / isobutanol (v:v = 9:1), 1134 ml drinking water, and 110 ml saturated NaCl were added. The mixture was extracted and separated. The remaining aqueous phase was extracted twice more with ethyl acetate / isobutanol (v:v = 9:1). The combined organic phases were washed with saturated NaCl, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. Column chromatography of the crude product yielded 16.9 g of compound L-1, with a yield of 74%.

[0154] Comparative Example 1

[0155]

[0156] At room temperature, 1-2' (190 mg, 0.179 mmol), trimethyloxytetrafluoroborate (212 mg, 1.433 mmol), and 1,8-bis(dimethylaminonaphthalene) (306 mg, 1.43 mmol) were added to a reaction flask; 4.5 mL of dichloromethane was added, and the mixture was stirred at room temperature under nitrogen balloon protection until the reaction was complete. The mixture was diluted with 20 mL of MTBE, filtered, and the filter cake was washed with about 20 mL of MTBE; the combined filtrates were washed with 1N hydrochloric acid; the organic phase was dried over anhydrous sodium sulfate; the mixture was filtered, and the filtrate was evaporated to dryness under reduced pressure; the product 1-1' (154 mg) was obtained after purification by silica gel column chromatography (PE:EtOAc = 1:1), with a yield of 80% and an HPLC purity of 92%.

[0157] Since this disclosure has been described in accordance with its specific implementation, certain modifications and equivalent variations will be apparent to those skilled in the art and are included within the scope of this disclosure.

Claims

1. A method for preparing a compound as shown in formula (I), comprising the steps of methylating a compound as shown in formula (I-2) to prepare a compound as shown in formula (I-1), deprotecting a compound as shown in formula (I-1) to prepare a compound as shown in formula (I), and preparing a compound as shown in formula (I-2) from a compound as shown in formula (I-3). in, R1 is selected from methyl, and the methylating agent used in the step is selected from trimethyloxonium tetrafluoroborate.

2. The preparation method according to claim 1, wherein the molar ratio of the compound of formula (I-2) to the methylating agent is 1:0.1 to 1:

10.

3. The preparation method according to claim 1, wherein the molar ratio of the compound represented by formula (I-2) to the methylating agent is 1:1 to 1:

5.

4. The preparation method according to claim 1, wherein the molar ratio of the compound represented by formula (I-2) to the methylating agent is 1:1 to 1:

3.

5. A method for preparing a compound of formula (I), comprising the method for preparing a compound of formula (I) according to any one of claims 1-4, wherein L is a linker. 。 6. The preparation method according to claim 5, wherein the linker comprises an amino acid unit, the amino acid unit being selected from peptide residues comprising 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid.

7. The preparation method according to claim 6, wherein the amino acid unit is selected from valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe), or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

8. The preparation method according to claim 5, wherein the linker is selected from: ， ， ， ， or 。 9. A method for preparing an antibody-drug conjugate of formula (ADC-I), comprising the step of preparing the antibody-drug conjugate of formula (ADC-I) from a compound of formula (LI), and the step of preparing the compound of formula (LI) as described in any one of claims 5-8. Ab-(LD) k (ADC-I) in, Ab represents an antibody or its antigen-binding fragment. L is a connector that covalently links Ab to D, as defined in any one of claims 5-7; and k is 1 to 20. -D is shown in the following formula: ， R1 is selected from methyl.

10. The preparation method according to claim 9, wherein the antibody is selected from mouse antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

11. The preparation method according to claim 9 or 10, wherein the antibody or its antigen-binding fragment in the antibody-drug conjugate (ADC-I) 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 antibody, etc. Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-CD79 antibody, anti-TROP-2 antibody, anti-CD79B antibody, anti-Mesothelin antibody, anti-folate receptor α (FRA) antibody or their antigen-binding fragments.

12. The preparation method according to claim 9, wherein the antibody in the antibody-drug conjugate (ADC-I) is a known antibody selected from, but not limited to, trastuzumab, pertuzumab, nimotuzumab, enoblituzumab, emibetuzumab, innotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96, farletuzumab, and glematumamab, or an antigen-binding fragment thereof.

13. The preparation method according to claim 9, wherein the antibody-drug conjugate is... or ， Where D is .

14. The compound shown in formula (I-1), in, R1 is selected from methyl.

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