Bicyclo[6,1,0]nonyne derivative and antibody-drug conjugate thereof, preparation method therefor, and use thereof

Abstract

Provided in the present invention are a bicyclo[6,1,0]nonyne derivative represented by formula (I) and an antibody-drug conjugate thereof, a preparation method therefor, and use thereof. Ab-{Y-X-[Z-(L-D)m]n}q (I)

Description

Bicyclic [6,1,0]nonyne derivatives and their antibody-drug conjugates, their preparation methods and applications

[0001] This application claims priority to Chinese patent application 2025103101553, filed on March 14, 2025; Chinese patent application 2025110462804, filed on July 28, 2025; and Chinese patent application 2026102821937, filed on March 9, 2026. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This disclosure relates to a bicyclic [6,1,0]nonyne derivative and its antibody-drug conjugate, its preparation method and application. Background Technology

[0003] Traditional antibody-drug conjugates (ADCs) typically utilize the amino group of lysine on the antibody or the thiol group of cysteine ​​obtained by breaking interchain disulfide bonds. However, this method suffers from poor uniformity and low stability, severely impacting the efficacy and therapeutic window of ADCs. To overcome these shortcomings, researchers have developed various site-specific conjugation techniques to precisely conjugate cytotoxic drugs or chemotherapeutic agents to specific positions on antibody molecules, thereby obtaining ADCs with superior activity and pharmacokinetic properties.

[0004] Among these, glycan-mediated coupling technology offers a unique site-specific coupling method. By coupling drug linkers to the N297 glycan located in the CH2 domain of an antibody, highly uniform ADC preparation can be achieved. Since the non-reducing ends of glycans contain various monosaccharides (such as fucose, galactose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid (SA), researchers have developed various methods to attach drug linkers to these glycans. For example, galactose or galactose analogs can be introduced into the glycan via galactosyltransferases. Furthermore, strain-promoted cycloaddition reactions of cycloalkynes and azides (SPAAC) have been applied to the GlycoConnect technology (developed by Synaffix). The core of this technology is the enzymatic introduction of azide sugars onto the polysaccharides of natural antibodies. Specifically, in the presence of UDP 6-azido group GalNAc, the antibody converts the natural sugar into a homogeneous, truncated azido-labeled trisaccharide through the action of endoglucosidase and glycosyltransferase (GalNAc-T), and then generates a highly homogeneous ADC through drug linker coupling.

[0005] With the rapid development of chemical biology and medicinal chemistry, bicyclic [6,1,0]nonyne (BCN) derivatives, as a novel type of linker, have shown significant advantages in ADC drug development. BCN derivatives can bind efficiently and specifically to azides via copper-free click chemistry reactions (such as SPAAC), thereby achieving precise conjugation between antibodies and drug molecules. However, despite the numerous advantages of BCN derivatives in ADC development, their practical application still faces a series of challenges and shortcomings. For example, the conjugation efficiency of some BCN derivatives is low, potentially leading to incomplete conjugation; while BCN derivatives can improve the water solubility of linkers, in some cases, their water solubility may still be insufficient, affecting the solubility and distribution of ADC drugs; furthermore, the stability of BCN derivatives in blood circulation needs further optimization to avoid premature degradation or drug release, leading to drug leakage and increased toxicity. Therefore, more efficient and stable BCN linkers need to be developed to achieve efficient and specific conjugation between antibodies and drug molecules, thereby improving the therapeutic efficacy and stability of ADC drugs. Summary of the Invention

[0006] This disclosure provides an antibody-drug conjugate of formula (I), an isomer thereof, or a pharmaceutically acceptable salt thereof: Ab-{YX-[Z-(LD)} m ] n} q (I)

[0007] Wherein, Ab is an antibody or antigen-binding fragment;

[0008] Y is Among them, wavy lines The asterisk (*) represents being connected to Ab, and the asterisk (*) represents being connected to X.

[0009] X is a sugar or a sugar derivative;

[0010] Z is the segment connecting X and L;

[0011] L stands for connector;

[0012] D is a cytotoxic drug;

[0013] t is 0 or 1;

[0014] m is 1 to 2;

[0015] n is 1 to 5;

[0016] q is 1 to 2.

[0017] In some embodiments, X is a disaccharide derivative; preferably, X is... Among them, wavy lines The asterisk (*) represents being connected to Y, and the asterisk (*) represents being connected to Z.

[0018] In some implementations, Z is any one of Z-1 to Z-35: Among them, wavy lines The asterisk (*) represents being connected to X, and the asterisk (*) represents being connected to L.

[0019] Preferably, Z is Z-9;

[0020] Z 1 For O or NH;

[0021] Z 2 For O or NH;

[0022] Z 3 It is a 5-10 membered heteroaryl group, -N=C(NH2)-NH-(CH2) p O-CO- or -N=C(NH2)-NH-(CH2) p -;

[0023] R 1 For H or -(CH2) p N + (CH3)3;

[0024] R 2 For H or -(CH2) p N + (CH3)3;

[0025] R 3 -(CH2CH2O) p -、

[0026] R 4 and R 5 Each is independently -(CH2) p SO3H, -(CH2) p P(=O)(OH)2、-(CH2) p B(OH)2、-(CH2) p COOH,

[0027] p can be 0, 1, 2, 3 or 4; preferably, p is 2.

[0028] In some implementations, Z is any one of Z-36 to Z-40: Among them, wavy lines The asterisk (*) represents being connected to X, and the asterisk (*) represents being connected to L.

[0029] In some implementation schemes, Z 1 It is O.

[0030] In some implementation schemes, Z 2 It is O.

[0031] In some implementation schemes, Z 3 It consists of 5-10 heteroaryl compounds.

[0032] In some implementation schemes, R 1 For H.

[0033] In some implementation schemes, R 2 For H.

[0034] In some implementation schemes, R 3 -(CH2CH2O) p -or Preferably, R 3 -(CH2CH2O) p -

[0035] In some implementation schemes, R 4 and R 5 Each is independently -(CH2) p SO3H or Preferably, R 4 and R 5 Each is independently -(CH2) p SO3H.

[0036] In some implementation schemes, Z 1 For O, Z 2 For O, Z 3 It is a 5-10 quinone heteroaryl group, R 1 For H, R 2 For H, R 3 -(CH2CH2O) p -or R 4 and R 5 Each is independently -(CH2) p SO3H or Preferably, Z 1 For O, Z 2 For O, Z 3 It is a 5-10 quinone heteroaryl group, R 1 For H, R 2 For H, R3 -(CH2CH2O) p -, R 4 and R 5 Each is independently -(CH2) p SO3H.

[0037] In some implementations, Z is any one of Z-1 to Z-20:

[0038] Preferably, Z is Z-9;

[0039] Z 1 For O or NH;

[0040] Z 2 For O or NH;

[0041] Z 3 It is a 5-10 membered heteroaryl group, -N=C(NH2)-NH-(CH2) p O-CO- or -N=C(NH2)-NH-(CH2) p -;

[0042] R 1 For H or -(CH2) p N + (CH3)3;

[0043] R 2 For H or -(CH2) p N + (CH3)3;

[0044] R 3 -(CH2CH2O) p -or

[0045] R 4 -(CH2) p SO3H;

[0046] p is 0, 1, 2, 3 or 4; preferably, p is 2;

[0047] Among them, wavy lines The asterisk (*) represents being connected to X, and the asterisk (*) represents being connected to L.

[0048] In some implementations, Z is any one of Z-36 to Z-40, where the wavy line... The asterisk (*) represents being connected to X, and the asterisk (*) represents being connected to L.

[0049] In some implementation schemes, Z 1 It is O.

[0050] In some implementation schemes, Z 2 It is O.

[0051] In some implementation schemes, Z 3 It consists of 5-10 heteroaryl compounds.

[0052] In some implementation schemes, R 1 For H.

[0053] In some implementation schemes, R 2 For H.

[0054] In some implementation schemes, R 3 -(CH2CH2O) p -

[0055] In some implementation schemes, R 4 and R 5 Each is independently -(CH2) p SO3H.

[0056] In some implementation schemes, Z 1 For O, Z 2 For O, Z 3 It is a 5-10 quinone heteroaryl group, R 1 For H, R 2 For H, R 3 -(CH2CH2O) p -, R 4 and R 5 Each is independently -(CH2) p SO3H.

[0057] In some implementation schemes, Z 3 It is a 5-6 member heteroaryl group.

[0058] In some implementation schemes, Z 3 It is pyridinyl or pyrimidinyl; preferably, Z 3 It is a pyrimidinyl group. In some embodiments, Z is any one of Z-1-1 to Z-1-22:

[0059] An asterisk (*) indicates that it is connected to L; preferably, Z is Z-1-6.

[0060] In some implementations, Z is any one of Z-1-23 to Z-1-31: An asterisk (*) indicates that it is connected to an L.

[0061] In some implementations, Z is any one of Z-1-32 to Z-1-42: An asterisk (*) indicates that it is connected to an L.

[0062] In some implementations, Z is any one of Z-1-43 to Z-1-45: An asterisk (*) indicates that it is connected to an L.

[0063] In some implementations, L is -L 1 -L 2 -L 3 -;

[0064] L 1 for The asterisk * represents L 2 Connected; L 2 A peptide consisting of 2-7 amino acid residues, wherein the amino acid residues are residues formed from valine, citrulline, phenylalanine, alanine, proline, leucine, isoleucine, glycine, lysine, serine, glutamic acid, or aspartic acid; preferably, L 2 for The asterisk * represents L 3 Connected,

[0065] L 3 for Or chemical bond, asterisk * indicates bonded to D;

[0066] In some implementations, L is An asterisk (*) indicates that it is connected to D.

[0067] In some implementations, L is An asterisk (*) indicates that it is connected to D.

[0068] In some implementations, L is -L 1 -L 2 -L 3 -;

[0069] L 1 for The asterisk * represents L 2 Connected;

[0070] In some implementations, L is

[0071] In some implementations, L is -L 1 -L 2 -L 3 -;

[0072] L 1 for The asterisk * represents L 2 Connected;

[0073] L 2 A peptide residue consisting of 2-7 amino acid residues, wherein the amino acid residues are residues formed from valine, citrulline, phenylalanine, alanine, proline, leucine, isoleucine, glycine, valine, lysine, serine, glutamic acid or aspartic acid.

[0074] L 3 for An asterisk (*) indicates that it is connected to D.

[0075] In some implementations, the L 2 for The asterisk * represents L 3 Connected.

[0076] In some implementations, L is

[0077] In some implementations, D is or its derivatives; preferably, D is

[0078] In some implementations, D is

[0079] In some implementations, Y is

[0080] In some implementations, m is 1 or 2.

[0081] In some implementations, n is 1.

[0082] In some implementations, the structural unit -Z-(LD) m for:

[0083] In some implementations, the structural unit -Z-(LD) m for:

[0084] In some implementations, the structural unit -Z-(LD) m for:

[0085] In some implementations, the structural unit -Z-(LD) m for:

[0086] In some implementations, the structural unit -Z-(LD) m for:

[0087] In some implementations, the structural unit -Z-(LD) m for:

[0088] In some embodiments, the antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof is:

[0089] Wherein Ab is an antibody; preferably, Ab is Her2 antibody; more preferably, Ab is trastuzumab; q is 1 to 2; preferably, q is 2.

[0090] In some embodiments, the antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof is: Wherein Ab is an antibody; preferably, Ab is Her2 antibody; more preferably, Ab is trastuzumab; t is 0 or 1; q is 1 to 2; preferably, q is 2.

[0091] In some embodiments, the antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof is: Wherein Ab is an antibody; preferably, Ab is Her2 antibody; more preferably, Ab is trastuzumab; t is 0 or 1; q is 1 to 2; preferably, q is 2.

[0092] This disclosure also provides a pharmaceutical linker of formula (II), an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0093] Among them, Z 4 For Z 4 -1 to Z 4 Any one of -24: The asterisk * indicates that it is connected to L; m is 1 or 2;

[0094] Z 1 Z 2 Z 3 R 1 R 2 R 3 R 4 R 5 L and D are defined as any one of the terms in equation (I).

[0095] This disclosure also provides a pharmaceutical linker of formula (II), an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0096] Among them, Z 4 For Z 4 -1 to Z 4 Any one of -20: The asterisk * indicates that it is connected to L; m is 1 or 2;

[0097] Z 1 Z 2 Z 3 R 1 R 2 R 3 R 4 L and D are defined as in equation (I).

[0098] This disclosure also provides a pharmaceutical linker of formula (II), an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0099] Among them, Z 4 For Z 4 -25 to Z 4 Any one of -30: The asterisk * indicates that it is connected to L; m is 1 or 2;

[0100] Z 1 Z2 Z 3 R 1 R 2 R 3 R 4 R 5 L and D are defined as any one of the terms in equation (I).

[0101] In some implementations, the Z 4 For Z 4 -1-1 to Z 4 Any one of -1-22: An asterisk (*) indicates that it is connected to an L.

[0102] In some implementations, the Z 4 For Z 4 -1-23 to Z 4 Any one of -1-30: An asterisk (*) indicates that it is connected to an L.

[0103] In some implementations, the Z 4 For Z 4 -1-31 to Z 4 Any one of -1-39:

[0104] The L is -L 1 -L 2 -L 3 -;

[0105] L 1 for The asterisk * represents L 2 Connected;

[0106] L 2 A peptide consisting of 2-7 amino acid residues, wherein the amino acid residues are residues formed from valine, citrulline, phenylalanine, alanine, proline, leucine, isoleucine, glycine, lysine, serine, glutamic acid, or aspartic acid; preferably, L 2 for The asterisk * represents L 3 Connected,

[0107] L 3 for Or chemical bond, asterisk * indicates bonded to D;

[0108] In some implementations, L is

[0109] In some implementations, L is

[0110] In some implementations, L is -L 1 -L 2 -L 3 -;

[0111] L 1 for The asterisk * represents L 2 Connected;

[0112] In some implementations, L is

[0113] In some implementations, each L can be independently selected from the above-mentioned groups; when m is 2, each L can be the same or different.

[0114] In some implementations, L is -L 1 -L 2 -L 3 -;

[0115] L 1 for The asterisk * represents L 2 Connected;

[0116] L 2 A peptide residue consisting of 2-7 amino acid residues, wherein the amino acid residues are residues formed from valine, citrulline, phenylalanine, alanine, proline, leucine, isoleucine, glycine, lysine, serine, glutamic acid or aspartic acid.

[0117] L 3 for An asterisk (*) indicates that it is connected to D.

[0118] In some implementations, the L 2 for The asterisk * represents L 3 Connected.

[0119] In some implementations, L is In some implementations, D is or its derivatives.

[0120] In some implementations, D is

[0121] In some implementations, each D may be independently selected from the above-mentioned groups; when m is 2, each D may be the same or different.

[0122] In some embodiments, the drug linker, its isomer, or a pharmaceutically acceptable salt thereof represented by formula (II) is:

[0123] In some embodiments, the drug linker, its isomer, or a pharmaceutically acceptable salt thereof represented by formula (II) is:

[0124] In some embodiments, the drug linker, its isomer, or a pharmaceutically acceptable salt thereof represented by formula (II) is:

[0125] This disclosure also provides compounds as shown in formula Z(I):

[0126] Z 1 Z 2 R 4 As defined by any term in equation (I), R 6 It is H or a leaving group, such as H or Preferably, Z(I) is

[0127] This disclosure also provides compounds as shown in formula Z(II):

[0128] Z 1 Z 2 R 4 As defined by any term in equation (I), R 7 It is H or a leaving group, such as H or Preferably, Z(II) is

[0129] This disclosure also provides a pharmaceutical composition comprising an antibody-drug conjugate of formula (I), an isomer thereof or a pharmaceutically acceptable salt thereof, or a drug linker of formula (II), an isomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier thereof.

[0130] In some embodiments, the amount of the antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is selected from 0.1 mg to 1000 mg.

[0131] In some embodiments, the content of the antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 1%-95%.

[0132] This disclosure also provides the use of an antibody-drug conjugate of formula (I), an isomer thereof or a pharmaceutically acceptable salt thereof, or a drug linker of formula (II), an isomer thereof or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the preparation of a drug for treating tumors.

[0133] This disclosure also provides a method for preventing or treating tumors, comprising administering to a patient an effective preventive or therapeutic dose of the antibody-drug conjugate of formula (I), its isomer or a pharmaceutically acceptable salt thereof, or the drug linker of formula (II), its isomer or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition thereof.

[0134] In some implementations, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, head and neck cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.

[0135] In some implementations, the lung cancer is selected from small cell lung cancer and non-small cell lung cancer; the leukemia is selected from acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia; and the lymphoma is selected from Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma.

[0136] Terminology Explanation

[0137] Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense.

[0138] The term "cytotoxic drug" or "drug" refers to small molecule compounds that can effectively 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.

[0139] The term "antibody-drug conjugate" refers to an antibody linked to a biologically active cytotoxic drug via a linker.

[0140] The term "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains or two identical light chains linked by interchain disulfide bonds.

[0141] The term "antigen-binding fragment" refers to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen.

[0142] The term "linker" refers to a chemical structural fragment that is linked to an antibody or antibody fragment at one end and to a drug at the other end. It can also be linked to other linkers before being linked to a drug.

[0143] The term "sugar" refers to a polysaccharide or an oxidized or unoxidized molecule containing carbohydrates, including but not limited to monosaccharides, disaccharides, trisaccharides, oligosaccharides, or polysaccharides. Non-limiting examples include, but are not limited to, glucose (Glc), galactose (Gal), mannose (Man), and fucose (Fuc). In this disclosure, the terms "sugar" and "glycan" are used interchangeably.

[0144] The term "sugar derivative" refers to a derivative of a sugar, that is, a sugar containing substituents and / or functional groups, for example, by introducing functional groups through azidation, alkynylation, aldehyde hydration, thiolation, etc.

[0145] The term "disaccharide derivative" is a sugar derivative containing at least two monosaccharide units, preferably the two monosaccharide units are linked by a glycosidic bond or a thioether bond.

[0146] The term "glycoside endonuclease" in this disclosure refers to a glycoside hydrolase possessing beta-N-acetylglucosinolate endonuclease activity. Such enzymes typically belong to glycoside hydrolase family 18 or 85. Some glycoside endonucleases known in the art include Endo S, Endo S2, Endo F3, Endo A, Endo D, Endo CC, and their mutants, such as those described in WO2022 / 226420. These enzymes may also possess glycosyltransfer activity, and enzymes with this activity known in the art include Endo S, Endo S2, Endo F3, and their mutants, such as those described in WO2022 / 226420.

[0147] The term "wild-type antibody" as used in this disclosure typically refers to antibodies that are naturally occurring or produced through recombinant expression and have an N-glycosylation site (N-glycan). For example, all antibodies with an N297 site in their Fc region belong to the category of "wild-type antibodies," and the Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

[0148] The term "deglycosylated antibody" in this disclosure refers to an antibody formed by the action of glycoside hydrolases on wild-type antibodies, containing one N-acetylglucosamine or a core fucosylated N-acetylglucosamine.

[0149] The term "drug loading" or "average drug binding" is also known as Drug to Antibiody Ration (DAR), which is the average amount of drug bound to each antibody in an antibody-drug conjugate.

[0150] In the general formula (I) of this disclosure, m, n, and q can be integers or decimals. For example, n is selected from 1 to 5, representing integers or decimals from 1 to 5; preferably, n is 1 to 2.

[0151] When a nitrogen atom in Z is connected to two asterisks *, m is 2; when a nitrogen atom in Z is connected to one asterisk *, m is 1. For example, when Z is selected from... When m is 2;

[0152] When Z is selected When m is 1, this corresponds to the case where m is 1.

[0153] When m is 2, it represents the relationship with Z. 4 Or, the two LDs connected by Z can be the same or different; for example, the structure The corresponding situation is: with Z 4 There are two connected LDs, and these two LDs are different.

[0154] structure The corresponding situation is: with Z 4 There are two connected LDs, and these two LDs are identical.

[0155] The term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in human and animal tissues to the extent of reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0156] The term "pharmaceutically acceptable salt" refers to derivatives obtained from the compounds of this invention prepared with relatively non-toxic acids or bases. These salts can be prepared during the synthesis, isolation, and purification of the compounds, or by reacting the purified free form of the compounds with suitable acids or bases. When the compounds contain relatively acidic functional groups, they react with alkali metal, alkaline earth metal hydroxides, or organic amines to yield base addition salts, including cations based on alkali metals and alkaline earth metals. When the compounds contain relatively basic functional groups, they react with organic or inorganic acids to yield acid addition salts.

[0157] The term "isomer" as used in this disclosure includes geometric isomers and isomers such as blocked trans isomers, cis-trans isomers, enantiomers, diastereomers, tautomers, racemic mixtures thereof, and other mixtures, all of which are within the scope of this disclosure. The term "enantiomer" refers to isomers that are mirror images of each other. The term "tautomer" refers to a functional group isomer that has different hydrogen bonding sites through one or more double bond shifts; for example, a ketone and its enol form are keto-enol tautomers. The term "diastereomer" refers to stereoisomers of molecules having two or more chiral centers and being non-mirror images of each other. The term "cis-trans isomer" refers to different spatial configurations of a molecule where double bonds or single bonds of cyclic carbon atoms cannot rotate freely. The term "blocked trans isomer" refers to stereoisomers that can be separated due to impeded or very slow rotation of single bonds. The stereoisomers of the compounds disclosed herein can be prepared by chiral synthesis or using chiral reagents or other conventional techniques. For example, an enantiomer of a compound disclosed herein can be prepared by asymmetric catalysis or chiral derivative derivatization. Alternatively, a single stereoisomer can be obtained from a mixture using chiral resolution techniques. Alternatively, it can be prepared directly from chiral starting materials. The separation of optically pure compounds in this disclosure is typically accomplished using preparative chromatography, employing a chiral column to achieve the separation of chiral compounds.

[0158] The absolute stereoconfiguration of a compound can be confirmed using conventional techniques in the art. For example, single-crystal X-ray diffraction can be used, or the absolute configuration of the compound can be confirmed by examining the chiral structure of the starting material and the reaction mechanism of asymmetric synthesis. Alternatively, after resolution, the stereoconfiguration can be determined by comparing it with a product whose absolute configuration is known (e.g., by comparing bioactivity). Compounds marked "Absolute configuration unknown / undetermined" in this document are typically obtained by resolving racemic compounds into single isomers via chiral preparative SFC, followed by characterization and testing.

[0159] The term "pharmaceutically acceptable carrier" refers to a medium generally acceptable in the art for delivering a bioactive pharmaceutical agent to animals, particularly mammals. Depending on the route of administration and dosage form, this includes adjuvants, excipients, or excipients such as diluents, preservatives, fillers, flow modifiers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromatizers, antibacterial agents, antifungal agents, lubricants, and dispersants. Pharmaceutically acceptable carriers are formulated based on a multitude of factors, within the scope of those skilled in the art. These include, but are not limited to, the type and nature of the formulated active pharmaceutical agent, the target population to which the composition containing the pharmaceutical agent is to be administered, the intended route of administration of the composition, and the target therapeutic indication. Pharmaceutically acceptable carriers include both aqueous and non-aqueous media, as well as various solid and semi-solid dosage forms. In addition to the active pharmaceutical agent, such carriers include many different components and additives, and other components included in the formulation for various reasons (e.g., stabilizing active pharmaceutical agents, binders, etc.) are well known to those skilled in the art.

[0160] The term "effective preventive or therapeutic dose" refers to a sufficient amount of the compound of the present invention or a pharmaceutically acceptable salt thereof to provide a reasonable benefit / risk ratio for treating any medical condition and / or prevention. However, it should be understood that the total daily dose of the compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof and the composition thereof must be determined by the attending physician within the bounds of reliable medical judgment. For any given patient, the specific effective therapeutic dose level must be determined based on a number of factors, including the condition being treated and its severity; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors known in the medical field. For example, it is practiced in the art to start the dose of the compound below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0161] Unless otherwise specified, "ring" means a saturated, partially saturated or unsaturated monocyclic or polycyclic ring.

[0162] Unless otherwise specified, the term "heterocyclic group" refers to a substituted or unsubstituted saturated or unsaturated non-aromatic ring containing 1-3 heteroatoms selected from N, O, or S. The "heterocyclic group" as described in this disclosure refers to a non-aromatic cyclic group derived by removing a hydrogen atom and containing at least one heteroatom as a ring atom; including saturated or partially saturated monocyclic and polycyclic heterocyclic groups; the heterocyclic group is independent of the bonding position (i.e., it can be bonded by carbon atoms or heteroatoms). The polycyclic heterocyclic groups include fused heterocyclic groups, spirocyclic groups, and bridged heterocyclic groups.

[0163] The term "fused heterocyclic group" refers to a ring structure formed by two or more rings sharing two adjacent ring atoms, wherein at least one ring is a heterocyclic group; the fused heterocyclic group includes a ring structure formed by the fusion of a monoheterocyclic group with a monoheterocyclic group or a cycloalkyl group or an aryl group or a heteroaryl group, and also includes a ring structure formed by the fusion of a heteroaryl group with a cycloalkyl group or a monoheterocyclic group.

[0164] The term "spiro-heterocyclic group" refers to a ring structure formed by two or more rings sharing a ring atom, wherein at least one ring is a heterocyclic ring.

[0165] The term "bridged heterocyclic group" refers to a ring structure formed by two or more rings sharing non-adjacent ring atoms, wherein at least one ring is a heterocyclic group.

[0166] The heterocyclic groups described in this disclosure are preferably 5-10 nucleotide monocyclic groups, 5-10 nucleotide fused heterocyclic groups, 5-10 nucleotide spirocyclic groups, or 5-10 nucleotide bridged heterocyclic groups; further, the heterocyclic groups are preferably 5-6 nucleotide monocyclic groups, 8-10 nucleotide fused heterocyclic groups, 8-10 nucleotide spirocyclic groups, or 7-9 nucleotide bridged heterocyclic groups.

[0167] Unless otherwise specified, the term "aryl" refers to an unsaturated, usually aromatic, hydrocarbon group, which may be a monocyclic or fused rings. Examples of 6-10 membered aryl groups include, but are not limited to, phenyl and naphthyl.

[0168] Unless otherwise specified, the term "heteroaryl" refers to a stable monocyclic or polycyclic aromatic hydrocarbon, preferably comprising a carbon atom and 1, 2, 3, or 4 cyclic heteroatoms independently selected from N, O, and S. Preferably, it is a 5-12-membered heteroaryl, more preferably a 5-8-membered heteroaryl, and even more preferably a 5-6-membered heteroaryl. Specific examples include, but are not limited to, pyrroleyl, furanyl, thiopheneyl, pyrazolyl, imidazolyl, pyrazinyl, pyridazinyl, triazinyl, oxazolyl, isoxazolyl, thiazolyl, isoxazolyl, pyridinyl, and pyrimidinyl.

[0169] Unless otherwise specified, "cycloalkyl" refers to a saturated monocyclic or polycyclic hydrocarbon group. Cycloalkyl is preferably C10. 3-8 cycloalkyl, more preferably C 3-6 Cycloalkyl groups, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0170] Unless otherwise specified, the term "alkyl" is used to denote a straight-chain or branched saturated hydrocarbon group. C is preferred. 1-6 Alkyl groups, more preferably C 1-4 Alkyl groups, examples of which include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, n-hexyl, etc.

[0171] Unless otherwise specified, the term "alkoxy" refers to an alkyl group as defined herein, in which an alkyl group is attached to another group by an oxygen atom, i.e., "alkyl-O-". This includes "C". 1-6 Alkoxy (structure is C) 1-6 alkyl-O-), "C 1-4 "Alkoxy" is a suffix, specifically including but not limited to methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, etc.; preferably, the "alkoxy" in this disclosure is preferably C 1-4 Alkoxy, more preferably C 1-3 Alkyl group.

[0172] Unless otherwise specified, the term "alkenyl" refers to a group derived from a straight-chain or branched alkene (containing at least one carbon-carbon double bond) by removing one hydrogen atom, including "C". 2- 6-alkenyl", C 2-5 "alkenyl", "C" 2-4 "alkenyl", "C" 2-3 "Alkenyl", specific examples include but are not limited to: -CH=CH2, -CH=CHCH3, -C(CH2)=CH2, -CH=CHCH2CH3, -CH2CH=CHCH3, etc.

[0173] Unless otherwise specified, the term "alkynyl" refers to a group derived from a straight-chain or branched alkyne (containing at least one carbon-carbon triple bond) by removing one hydrogen atom, including "C". 2- 5-acetylinyl group, C 2-4 "Alkyne", "C" 2-3 "Alkyne group", specific examples include but are not limited to: -C≡CH, -C≡CCH3, HC≡CCH2-, HC≡CC≡C-, etc.

[0174] Unless otherwise specified, the term "halogen" refers to a fluorine, chlorine, bromine, or iodine atom.

[0175] Specifically, all combinations of substituents and / or their variants are permitted only if such combinations produce stable compounds. A stable compound or stable structure is defined as a compound that is sufficiently stable to withstand chemical reactions, can be isolated in useful purity, and can be formulated into an effective therapeutic agent.

[0176] In the embodiments of this disclosure, if there is a discrepancy between the compound name and the compound structure, the discrepancy can be determined by comprehensively considering relevant information and reaction routes; if it cannot be confirmed by other means, the given compound structural formula shall prevail. The preparation methods for some compounds in this invention reference the preparation methods for the aforementioned similar compounds. Those skilled in the art should understand that when using or referring to the referenced preparation methods, the reactant ratio, reaction solvent, reaction temperature, etc., can be appropriately adjusted according to the different reactants.

[0177] If the compounds disclosed herein form a salt due to the elution solvent system during purification, their reaction pathway or compound structure will be represented in their free form (for example, if a -SO3H group is present in the structure, it may form a -SO3NH4 salt during purification, but the reaction pathway or structure will still be shown in the -SO3H form). Neither the free nor salt form affects their biological activity.

[0178] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention.

[0179] Summary of experimental instruments:

[0180] The structures of the compounds disclosed herein were determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS), or ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR determinations were performed using a Bruker Neo 400M or Bruker Ascend 400 NMR spectrometer, with solvents including deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), and deuterated chloroform (CDCl3), heavy water (D2O), and tetramethylsilane (TMS) as the internal standard.

[0181] The determination was performed using an Agilent 1260-6125B single quadrupole mass spectrometer with a Welch Biomate column (C18, 2.7 μm, 4.6 × 50 mm) or a Waters H-Class SQD2 column with a Welch Ultimate column (XB-C18, 1.8 μm, 2.1 × 50 mm) (ion source: electrospray ionization).

[0182] The determination was performed using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) with a Waters UPLC H-class SQD mass spectrometer (ion source: electrospray ionization).

[0183] HPLC determinations were performed using a Waters e2695-2998 or Waters ARC and an Agilent 1260 or Agilent Poroshell HPH high-performance liquid chromatograph.

[0184] Preparative HPLC was performed using a Waters 2555-2489 (10 μm, ODS 250 cm × 5 cm) or a GILSON Trilution LC column, with a Welch XB-C18 column (5 μm, 21.2 × 150 mm).

[0185] Thin-layer chromatography (TLC) uses GF254 silica gel plates from Yantai Jiangyou Silica Gel Development Co., Ltd. or GF254 silica gel plates from Rushan Shangbang New Materials Co., Ltd. The TLC standard is 0.15mm to 0.20mm, with a preparative type of 20×20cm. Column chromatography generally uses 200-300 mesh silica gel from chemical industry as the carrier.

[0186] The starting materials used in the embodiments of this disclosure are known and commercially available, or can be synthesized using methods known in the art. Unless otherwise specified, all reactions of this invention are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, using a dry solvent, and the reaction temperature is expressed in degrees Celsius or °C. Unless otherwise specified, percentages used herein refer to mass percentages for solid-liquid mixtures and solid-phase mixtures, and volume percentages for liquid-phase mixtures.

[0187] HIC-DAR Analysis Method (DAR Value Determination Using Hydrophobic Chromatography-HIC-HPLC)

[0188] Sample preparation: The sample was diluted to 3.0 mg / mL with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis.

[0189] Chromatographic conditions:

[0190] Chromatographic column: TSKgel Butyl-NPR, 2.5 μm, 4.6 mm × 10 cm; Mobile phase: Mobile phase A: 100 mM PB + 1.2 M (NH4)2SO4 (pH 7.0); Mobile phase B: 100 mM PB (pH 7.0) + 20% isopropanol; Detection wavelength: 280 nm; Flow rate: 0.5 mL / min; Column temperature: 25 ℃; Injection volume: 20 μL

[0191] Gradient elution procedure:

[0192] DAR value calculation formula:

[0193] DAR = Σ(weighted peak area) / 100, that is, DAR = (D0 peak area ratio × 0 + D1 peak area ratio × 1 + D2 peak area ratio × 2 + D3 peak area ratio × 3 + D4 peak area ratio × 4 + D5 peak area ratio × 5 + D6 peak area ratio × 6 + D7 peak area ratio × 7 + D8 peak area ratio × 8) / 100;

[0194] Determination of ADC purity by size exclusion chromatography-HPLC

[0195] Sample preparation: Sample concentration 1.0-5 mg / mL, filtered through a 0.22 μm filter membrane; Instrumentation: Agilent 1260 Infinity II Bio-Inert LC System; Column: TOSOH, TSKgel G3000SWxL, 5 μm, 7.8 mm × 300 mm; Mobile phase: 0.2 M PB, 5-15% isopropanol, pH 7.0; Flow rate: 0.5-1 mL / min; Detection wavelength: 280 nm & 360 nm; Column temperature: room temperature; Sample loading: 30 μg; SEC elution method: isocratic elution.

[0196] In this disclosure, PB refers to sodium phosphate buffer solution with disodium hydrogen phosphate (DHP) as the main component. DHP buffer solutions with different pH values ​​are typically prepared using solutions of sodium hydrogen phosphate and disodium dihydrogen phosphate at the same concentration.

[0197] The abbreviations and their corresponding chemical names used in the embodiments of this disclosure are as follows:

[0198] Example 1: Compound 1

[0199] Reaction route:

[0200] Operating steps:

[0201] Step A: Oxaloyl chloride (1.4 mL, 16.54 mmol) was dissolved in dry DCM (40 mL), cooled to -78 °C, and DMSO (2.4 mL in 40 mL DCM, 33.79 mmol) was slowly added dropwise. The reaction system was stirred at -78 °C for 30 min under argon protection. Then, compound 1-1 (1.0 g in 40 mL DCM, 6.66 mmol) was added dropwise, and the mixture was stirred at -78 °C for 1 hour. TEA (12 mL, 86.57 mmol) was added dropwise, and the mixture was stirred at -78 °C for half an hour. The reaction was then brought to room temperature and continued for another half hour.

[0202] After the reaction was completed by TLC monitoring, the reaction solution was washed with saturated sodium chloride solution, dried and concentrated, and the crude product was purified by column chromatography to obtain compounds 1-2.

[0203] 1 H NMR (400MHz, DMSO-d6) δ9.50 (d, J = 6.3Hz, 1H), 2.34–2.07 (m, 6H), 2.03–1.90 (m, 1H), 1.59–1.51 (m, 1H), 1.33–1.18 (m, 1H).

[0204] Step B: Compounds 1-2 (660 mg, 4.45 mmol) were dissolved in MeOH (80 mL), and ammonium acetate (22.3 g, 288.67 mmol) and sodium cyanoborohydride (333 mg, 5.30 mmol) were added. The reaction system was stirred overnight at room temperature.

[0205] After the reaction was completed as monitored by TLC, the reaction solution was quenched with water (40 mL), and then extracted sequentially with DCM (200 mL) and chloroform (200 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by column chromatography to obtain compounds 1-3.

[0206] LCMS(ESI) m / z: 150.3 [M+H] + .

[0207] Step C: Dissolve compounds 1-4 (4.17 g, 5.43 mmol, Fmoc-Val-Cit-PAB-PNP, purchased from MCE, CAS No.: 863971-53-3) in dry DMF (90 mL), add HOBt (370 mg, 2.72 mmol) and DIEA (1.08 g, 8.36 mmol) sequentially, stir at room temperature for 10 min, then dissolve compound MMAE (3 g, 4.18 mmol) in dry DMF (20 mL) and slowly add it dropwise to the above reaction solution, and stir the reaction at room temperature for 15 hours.

[0208] After the reaction was completed as monitored by LCMS, the reaction solution was purified by reverse-phase flash to obtain compounds 1-5.

[0209] LCMS(ESI) m / z: 1346.6 [M+H] + .

[0210] Step D: Dissolve compounds 1-5 (4.3 g, 0.056 mmol) in DCM (200 mL), then add diethylamine (40 mL). The reaction system is carried out under nitrogen protection at room temperature for 12 hours.

[0211] After the reaction was completed as monitored by LCMS, the reaction solution was concentrated, and the crude product was purified by reverse-phase flash to obtain compounds 1-6.

[0212] LCMS(ESI) m / z: 1124.8 [M+H] + .

[0213] Step E: Sulfonyl chloride (5.53 mL, 68.37 mmol) was dissolved in dry DCM (100 mL). At 0 °C, a DCM solution (50 mL) of commercially available compound 2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)ethylamine (purchased from Shanghai Bid Pharmaceutical, CAS: 215297-17-9) 5 g, 22.79 mmol) and TEA (12.64 mL, 91.16 mmol) was added dropwise. The reaction system was stirred at 0 °C for 2 hours.

[0214] The reaction solution was concentrated at low temperature, and crude products 1-7 were used directly in the next reaction step.

[0215] Step F: Dissolve 2-aminopyrimidine-5-carboxylic acid (7.93 g, 57.00 mmol) in DCM (40 mL), add TEA (8.65 g, 85.5 mmol), and then add crude product 1-7 in DCM (60 mL). Stir the reaction system overnight at room temperature.

[0216] After the reaction was completed as monitored by LCMS, the reaction solution was concentrated, and the crude product was purified by reverse-phase flash to obtain compounds 1-8.

[0217] LCMS(ESI) m / z: 421.3 [M+H] + .

[0218] Step G: Dissolve compounds 1-8 (1.4 g, 3.33 mmol) in MeCN (20 mL), add compounds 1-3 (0.38 g, 2.56 mmol), EDCI (0.61 g, 3.20 mmol), HOPO (0.36 g, 3.20 mmol) and 2,6-dimethylpyridine (1.45 mL, 12.45 mmol), and stir the reaction system at room temperature for 1 hour.

[0219] After the reaction was completed as monitored by LCMS, the reaction solution was concentrated, and the crude product was purified by reverse-phase flash to obtain compounds 1-9.

[0220] LCMS(ESI) m / z: 552.5 [M+H] + .

[0221] Step H: Dissolve compounds 1-9 (560 mg, 1.01 mmol) in THF (20 mL), add triethylamine trifluoride salt (3.29 mL, 20.20 mmol) and triethylamine (2.81 mL, 20.20 mmol), and stir the reaction system at room temperature for 1 hour.

[0222] After the reaction was completed as monitored by LCMS, the reaction solution was concentrated, and the crude product was purified by column chromatography to obtain compounds 1-10.

[0223] LCMS(ESI) m / z: 438.3 [M+H] + .

[0224] Step I: Dissolve compound 1-10 (177 mg, 0.40 mmol) in THF (10 mL), add PNP-Cl (202 mg, 1.00 mmol) and triethylamine (122 mg, 1.20 mmol), and stir the reaction system at room temperature for 3 hours.

[0225] After the reaction is completed as monitored by LCMS, reaction solution 1-11 is not treated and is used directly for the next step.

[0226] LCMS(ESI) m / z: 603.2 [M+H] + .

[0227] Step J: Dissolve diethanolamine (370 mg, 3.53 mmol) in DMF (7 mL), add TEA (789 mg, 7.79 mmol) and the reaction solution 1-11 (20 mL) from the previous step, and stir the reaction system at room temperature for 1 hour.

[0228] After the reaction was completed as monitored by LCMS, the reaction solution was purified by TLC to obtain compounds 1-12.

[0229] LCMS(ESI) m / z: 569.2 [M+H] + .

[0230] Step K: Dissolve compound 1-12 (100 mg, 0.18 mmol) in THF (5 mL), add p-nitrophenyl chloroformate PNP-Cl (181 mg, 0.90 mmol) and triethylamine (146 mg, 1.44 mmol), and stir the reaction system at room temperature for 3 hours.

[0231] After the reaction was completed as monitored by LCMS, the reaction solution was concentrated at low temperature, and the crude product was purified by column chromatography to obtain compound 1-13.

[0232] LCMS(ESI) m / z: 899.2 [M+H] + .

[0233] Step L: Dissolve compound 1-13 (56 mg, 0.05 mmol) in THF (3 mL), add compound 1-6 (132 mg, 0.12 mmol) and triethylamine (22 mg, 0.21 mmol), and stir the reaction system overnight at room temperature.

[0234] After the reaction was completed as monitored by LCMS, compound 1 was obtained by flash purification.

[0235] LCMS(ESI) m / z: 2867.9 [M+H] + .

[0236] 1 H NMR (400MHz, DMSO) δ10.18–9.88(m,2H),8.78(s,2H),8.40–8.26(m,2H),8.19–8.00(m,3H),7.95–7.85(m,1H),7.69–7.53(m,5H),7.42–7.0 8(m,17H),6.76–6.54(m,1H),6.18–5.97(m,2H),5.52–5.32(m,6H),5 .16–4.91(m,5H),4.82–4.58(m,3H),4.53–4.34(m,7H),4.31–4.20(m, 3H),4.12–3.86(m,15H),3.81–3.76(m,2H),3.25–3.17(m,16H),3.13 –3.09(m,4H),3.04–2.91(m,10H),2.89–2.81(m,6H),2.46–2.36(m,3H ),2.35–2.06(m,14H),2.04–1.90(m,5H),1.83–1.67(m,8H),1.60–1.4 3(m,10H),1.37–1.22(m,6H),1.08–0.96(m,14H),0.89–0.73(m,48H).

[0237] Example 2: Compound 2

[0238] Reaction route:

[0239] Operating steps:

[0240] Step A: Compound 1-1 (1.0 g, 6.66 mmol) was dissolved in dry ACN (50 mL), and N,N-disuccinimidyl carbonate (3.4 g, 13.39 mmol) and TEA (2.3 g, 22.30 mmol) were added. The mixture was reacted at room temperature for 1.5 hours.

[0241] After the reaction was completed by TLC monitoring, the reaction solution was concentrated, and the crude product was purified by column chromatography to obtain compound 2-1.

[0242] 1 H NMR (400MHz, CDCl3) δ4.45 (d, J = 8.3Hz, 2H), 2.84 (s, 4H), 2.41–2.14 (m, 6H), 1.70–1.44 (m, 3H), 1.20–0.94 (m, 2H)

[0243] Step B: The compound diethylene glycolamine (1.0 g, 9.51 mmol) was dissolved in water (20 mL), and sodium vinyl sulfonate (1.2 g, 9.51 mmol) was added. The reaction system was refluxed and stirred at 100 °C for 2 hours.

[0244] After the reaction was completed as monitored by LCMS, the reaction solution was directly freeze-dried, and the crude product 2-2 was directly used for the next reaction.

[0245] LCMS(ESI) m / z: 212.1 [M+H] + .

[0246] Step C: Crude product 2-2 (2.0 g) was dissolved in dry DMF (30 mL), and imidazole (1.9 g, 28.14 mmol) and TBDPSCl (28.4 g, 10.32 mmol) were added. The reaction system was stirred at room temperature for 2 hours.

[0247] The reaction solution was purified by reverse-phase flash to obtain compounds 2-3.

[0248] LCMS(ESI) m / z: 450.2 [M+H] + .

[0249] Step D: Dissolve compound 2-3 (786 mg, 1.74 mmol) in DMF (4 mL), add TEA (528 mg, 5.22 mmol), then add compound 2-1 (507 mg, 1.74 mmol), and stir the reaction system at room temperature for 1 hour.

[0250] After the reaction was completed as monitored by LCMS, the reaction solution was concentrated, and the crude product was purified by reverse-phase flash to obtain compounds 2-4.

[0251] LCMS(ESI) m / z: 626.3 [M+H] + .

[0252] Step E: Dissolve compound 2-4 (1.2 g, 1.88 mmol) in THF (20 mL), add compound triethylamine trihydrofluoride (3.0 g, 18.80 mmol) and triethylamine (3.8 g, 37.60 mmol), and stir the reaction system overnight at room temperature.

[0253] After the reaction was completed as monitored by LCMS, the reaction solution was purified by reverse-phase flash to obtain compounds 2-5.

[0254] LCMS(ESI) m / z: 388.1 [M+H] + .

[0255] Step F: Dissolve compound 2-5 (730 mg, 1.87 mmol) in THF / DMF (5 mL / 5 mL), add PNP-Cl (942 mg, 4.68 mmol) and triethylamine (740 mg, 9.35 mmol), and stir the reaction system at room temperature for 3 hours.

[0256] After the reaction was completed as monitored by LCMS, the reaction solution was purified by reverse-phase flash to obtain compounds 2-6.

[0257] LCMS(ESI) m / z: 553.2 [M+H] + .

[0258] Step G: Diethanolamine (240 mg, 2.28 mmol) was dissolved in DMF (7 mL), and TEA (577 mg, 5.70 mmol) and compound 2-6 (633 mg, 0.97 mmol) were added. The reaction system was stirred at room temperature for 1 hour.

[0259] After the reaction was completed as monitored by LCMS, the reaction solution was purified by reverse-phase flash to obtain compounds 2-7.

[0260] LCMS(ESI) m / z: 519.2 [M+H] + .

[0261] Step H: Compound 2-7 (45 mg, 0.086 mmol) was dissolved in ultradry THF (5 mL), and PNP-Cl (87 mg, 0.43 mmol) and triethylamine (51 mg, 0.64 mmol) were added. The reaction system was stirred at room temperature for 2 hours.

[0262] After the reaction was completed as monitored by LCMS, the reaction solution was purified by a second reverse-phase flash process and lyophilized to obtain compounds 2-8.

[0263] LCMS(ESI) m / z: 849.1 [M+H] + .

[0264] Step I: Dissolve compound 2-8 (25 mg, 0.029 mmol) in ultradry DMF (2 mL), add compound 1-6 (82 mg, 0.073 mmol) and triethylamine (13 mg, 0.13 mmol), and stir the reaction system overnight at room temperature.

[0265] After the reaction was completed as monitored by LCMS, the reaction solution was purified by reverse-phase flash to obtain compound 2.

[0266] LCMS(ESI) m / z: 1407.8 [1 / 2M+H] + .

[0267] 1 H NMR(400MHz,DMSO-d6)δ10.21–9.78(m,2H),8.32(brs,1H),8.21–8.01(m,3H) ,7.89(d,J=8.1Hz,1H),7.73–7.54(m,5H),7.47–7.12(m,18H),7.09(s,1H),6. 97(s,1H),6.03(brs,2H),5.60–5.25(m,6H),5.19–4.90(m,5H),4.79–4.70(m, 1H),4.68–4.59(m,1H),4.54–4.36(m,6H),4.31–4.22(m,2H),4.15–3.89(m,14 H),3.81–3.76(m,1H),3.65–3.47(m,13H),3.27–3.17(m,13H),3.15–3.11(m,3 H),3.08–2.93(m,8H),2.91–2.82(m,6H),2.81–2.70(m,3H),2.69–2.64(m,1H) ,2.46–2.37(m,2H),2.35–2.08(m,12H),2.06–1.91(m,5H),1.86–1.64(m,9H), 1.60–1.42(m,9H),1.41–1.23(m,7H),1.12–0.96(m,14H),0.92–0.74(m,48H).

[0268] Example 3: Compound 3

[0269] Reaction route:

[0270] Operating steps:

[0271] Step A: Dissolve (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (800 mg, 5.33 mmol) in tetrahydrofuran (10 mL), add triphenylphosphine (3.08 g, 11.73 mmol) and compound 3-1 (2.21 g, 8.53 mmol), add diisopropyl azodicarbonate (2.37 g, 11.73 mmol) dropwise at 0 °C, and slowly raise the temperature to room temperature in an ice-water bath while stirring for 2 hours.

[0272] The reaction mixture was monitored by LCMS until it was almost completely reacted. Water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, dried, filtered, and evaporated to dryness. The residue was purified by silica gel column chromatography to obtain compound 3-2.

[0273] MS(ESI)M / Z:392.2[M+H] + .

[0274] Step B: Dissolve compound 3-2 (900 mg, 2.29 mmol) in methanol (28 mL), add potassium carbonate (0.32 g, 2.29 mmol) in water (2.8 mL), stir well, add potassium carbonate (1.28 g, 9.16 mmol), and stir at 45 °C for 8 hours.

[0275] LCMS showed that the reaction was complete. The reaction solution was directly evaporated to dryness, diluted with water (20 mL), extracted with ethyl acetate (30 mL × 3), the organic phases were combined, dried and evaporated to dryness, and the residue was purified by silica gel column chromatography to give compound 3-3.

[0276] MS(ESI)M / Z:292.2[M+H] + .

[0277] Step C: Dissolve sulfonyl chloride (3.08 g, 22.79 mmol) in dichloromethane (6 mL). Add compound 3-4 (0.69 g, 3.15 mmol) and triethylamine (4.61 g, 45.58 mmol) in dichloromethane under dry ice bath conditions, and stir in a dry ice bath for 1.5 hours under nitrogen protection. Add a dichloromethane solution (10 mL) of compound 3-3 (0.61 g, 2.14 mmol), stir in a dry ice bath for 0.5 hours, and then allow to rise naturally to room temperature for 2 hours under a small amount of dry ice while stirring.

[0278] LCMS showed that the reaction was complete. The reaction solution was washed with water (20 mL × 3), the organic phase was dried, filtered, concentrated at room temperature, and the residue was purified by silica gel column chromatography to give compounds 3-5.

[0279] MS(ESI)M / Z:573.2[M+H] + .

[0280] Step D: Dissolve triethylamine (2.12 g, 21 mmol) in tetrahydrofuran (40 mL), add triethylamine trihydrofluoride (3.39 g, 21 mmol), stir at room temperature for 0.5 hours, add compound 3-5 (600 mg, 1.05 mmol), and stir at room temperature for 2 hours.

[0281] LCMS showed that the reaction was complete. The reaction solution was concentrated at room temperature, and the residue was purified by silica gel column chromatography to obtain compounds 3-6.

[0282] MS(ESI)M / Z:459.2[M+H] + .

[0283] Step E: Dissolve 2,6-dimethylpyridine (1.77 g, 16.5 mmol) in dichloromethane (22 mL), add trimethylsilyl trifluoromethanesulfonate (2.44 g, 11 mmol), stir at room temperature for 0.5 hours, add compound 3-6 (250 mg, 0.55 mmol), and stir at room temperature for 2 hours.

[0284] LCMS showed that the reaction was complete. The reaction solution was concentrated at room temperature, and the residue was directly purified by high performance liquid chromatography to obtain compounds 3-7.

[0285] MS(ESI)M / Z:359.2[M+H] + .

[0286] Step F: Compounds 3-7 (40 mg, 0.11 mmol) were dissolved in tetrahydrofuran (3 mL). A THF mixture of triethylamine (0.056 g, 0.55 mmol) and p-nitrophenyl chloroformate (0.055 g, 0.28 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in N,N-dimethylformamide (1 mL). Compounds 3-8 (0.12 g, 1.1 mmol) and triethylamine (0.056 g, 0.55 mmol) were added, and the mixture was stirred at room temperature for 2 hours.

[0287] LCMS monitoring showed that the raw materials had basically completed the reaction, and the reaction solution was directly sent to high performance liquid chromatography for purification to obtain compounds 3-9.

[0288] MS(ESI)M / Z:490.3[M+H] + .

[0289] Step G: Dissolve compounds 3-9 (17 mg, 0.035 mmol) in tetrahydrofuran (3 mL), add a mixed solution of p-nitrophenyl chloroformate (0.035 g, 0.18 mmol) and triethylamine (0.028 g, 0.28 mmol) in tetrahydrofuran (1 mL) in an ice-water bath, and stir for 1 hour. Stir overnight at room temperature, then heat to 40 °C and stir for 1 hour.

[0290] LCMS monitoring showed that the reaction of the raw materials was basically complete. The reaction solution was directly evaporated to dryness, and the residue was purified by silica gel column chromatography to obtain compound 3-10.

[0291] MS(ESI)M / Z:820.2[M+H] + .

[0292] Step H: Compound 3-10 (10 mg, 0.012 mmol) was dissolved in N,N-dimethylformamide (2 mL). Compound 1-6 (0.034 g, 0.030 mmol) and triethylamine (0.0061 g, 0.060 mmol) were added at room temperature, and the reaction was continued to be stirred at 20 °C for 16 hours. The temperature was then raised to 40 °C and stirred for 1 hour.

[0293] LCMS monitoring showed that the reaction was complete. The reaction solution was directly purified by high-performance liquid chromatography to obtain compound 3.

[0294] MS(ESI)M / Z:1395.22[1 / 2M+H] + .

[0295] Example 4 Compound 4

[0296] Reaction route:

[0297] Operating steps:

[0298] Steps A and B: Under N2 protection at 0°C, compound [(chlorosulfonyl)imino] methyl ketone (0.26 g, 1.82 mmol) was dissolved in dichloromethane (15 mL). Then, compound 2-(2-[(tert-butyldimethylsilyl)oxy]ethoxy)ethanol-1-ol (4-1) (0.4 g, 1.82 mmol) dissolved in dichloromethane (5 mL) was added to the reaction system, and the reaction system was stirred for 15 minutes. Then, triethylamine (0.18 g, 1.82 mmol) was added, and the reaction was continued for 5 minutes. Then, compound 1-3 (0.22 g, 1.46 mmol) dissolved in dichloromethane (50 mL) and triethylamine (0.18 g, 1.82 mmol) was added dropwise to the reaction system, and the reaction system was stirred for 15 minutes.

[0299] LCMS showed that after the starting material was converted into the product, the reaction solution was quenched with water (20 mL), and the mixture was extracted with dichloromethane (50 mL × 3 times). The organic phase was washed with sodium chloride aqueous solution (20 mL × 2 times). Then, it was dried with anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to give 0.24 g of compound 4-3.

[0300] MS(ESI)M / Z:475.2[M+H]+ .

[0301] Step C: Under nitrogen protection at 0°C, triethylamine trihydrofluoride (1.64 g, 10.2 mmol) was dissolved in tetrahydrofuran (20 mL). Then, triethylamine (1.03 g, 10.2 mmol) was added dropwise to the reaction mixture, and the mixture was stirred for 15 minutes. Compound 4-3 (0.24 g, 0.51 mmol) was then added to the reaction mixture, and the reaction mixture was stirred for another 2 hours.

[0302] LCMS monitoring showed that after the starting material was converted into the product, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give 0.128 g of compound 4-4.

[0303] MS(ESI)M / Z:361.2[M+H] + .

[0304] Step D: Under nitrogen protection at 0°C, compound 4-4 (0.128 g, 0.36 mmol) and triethylamine (0.18 g, 1.80 mmol) were dissolved in dichloromethane (10 mL). Subsequently, 4-nitrophenyl chloroformate (0.15 g, 0.72 mmol) was added to the above reaction solution. The reaction mixture was stirred for 4 hours.

[0305] LCMS monitoring showed that after the raw materials were converted into products, the crude product was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compounds 4-5.

[0306] MS(ESI)M / Z:526.2[M+H] + .

[0307] Step E: Under nitrogen protection at room temperature, compound 2-[(2-hydroxyethyl)amino]ethane-1-ol (0.030 g, 0.29 mmol) and triethylamine (0.029 g, 0.29 mmol) were dissolved in N,N-dimethylformamide (0.5 mL). Subsequently, compounds 4-5 (0.03 g, 0.057 mmol) dissolved in dichloromethane (2 mL) were added to the reaction system. The reaction system was stirred for 12 hours.

[0308] LCMS monitoring showed that after the reactants were converted into products, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by preparative high-performance liquid chromatography to obtain compounds 4-6.

[0309] MS(ESI)M / Z:492.2[M+H] + .

[0310] Step F: Under nitrogen protection at 0°C, compound 4-6 (0.01 g, 0.02 mmol) was dissolved in dichloromethane (1 mL). Subsequently, 4-nitrophenyl chloroformate (0.02 g, 0.1 mmol) was added to the above reaction solution. The reaction system was stirred for 4 hours.

[0311] LCMS monitoring showed that after the raw materials were converted into products, the crude product was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compounds 4-7.

[0312] MS(ESI)M / Z:822.2[M+H] +

[0313] Step G: Under nitrogen protection at room temperature, compounds 4-7 (0.008 g, 0.0097 mmol) and 1-6 (0.027 g, 0.024 mmol) were dissolved in N,N-dimethylformamide (0.5 mL). Triethylamine (0.0098 g, 0.097 mmol) was then added dropwise to the reaction mixture. After reacting at room temperature for 12 hours, the temperature was raised to 40 °C, and stirring was continued for 4 hours.

[0314] LCMS monitoring showed that after the raw materials were converted into products, the reaction solution was separated by preparative high performance liquid chromatography to obtain compound 4.

[0315] MS(ESI)M / Z:931.0[1 / 3M+H] + .

[0316] Example 5: Recombinant expression and purification of EndoSi enzyme, refer to PCT / CN2025 / 071512

[0317] The encoding gene of Endo Si from Streptococcus iniae was cloned into the pET22b vector (GenScript), and its amino acid sequence is positions 34-928 of SEQ ID NO:1.

[0318] SEQ ID NO:1

[0319] The plasmid containing the target gene was transformed into *E. coli* BL21(DE3) and plated on 2×YT agar plates containing 100 μg / mL ampicillin. The plates were incubated overnight at 37°C. Single colonies were picked and inoculated into 4 mL of 2×YT liquid medium containing 100 μg / mL ampicillin, and incubated overnight. 4 mL of the bacterial culture was then inoculated into 1 L of 2×YT broth medium containing 100 μg / mL ampicillin and incubated at 37°C until OD (Organic Growth Rate) was reached.600 The culture was incubated until the concentration reached 0.8-1.0. Then, 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture and incubated at 20°C to induce protein overexpression. After 16 hours, the cells were harvested by centrifugation. The expression was then analyzed using B-PER. TM Bacterial Protein Extraction Reagent (Thermo) was used to lyse the cell pellet according to the manufacturer's instructions. The recombinant Endo Si protein was purified using the cOmplete His-Tag Purification Column (Roche) and the SDA030 protein purification system (Sepure). The protein was concentrated using an Amicon centrifuge filter (30 kDa, Millipore) and further processed through a HiLoad centrifuge. TM 26 / 600 Superdex TM Purification was performed using a 200 prep grade column (Cytiva) via size exclusion. Fractions containing the Endo Si fusion protein were concentrated using an Amicon centrifugal filter (30 kDa, Millipore) and stored in storage buffer (20 mM PB, pH 7.5). Protein purity was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel imaging (Gel Doc EZ Imager, Bio-RAD), and protein concentration was quantified using a spectrophotometer (Nano-300).

[0320] Example 6: Synthesis of compound G7, refer to WO2025148977A1

[0321] Reaction process:

[0322] Reaction steps:

[0323] Step 1: Weigh 26.2 g (100 mmol) of 2-[(azidoacetyl)amino]-2-deoxy-D-glucose and 64.1 g of UDP-Gal (disodium uridine 5′-bisphosphate galactose, 64.1 g, 105 mmol) into a 1000 mL single-necked reaction flask. Add 600 mL of deionized water and stir to dissolve. Add 4 g (42 mmol) of MgCl2 and 0.3 g of β-1,4-galactosyltransferase (NmLgtB-4) to catalyze the reaction. The system pH is 8.0, the reaction temperature is 37 °C, the stirring speed is 200 rpm, and the reaction is carried out overnight. After the reaction is complete, add an equal volume of ethanol to remove the enzyme. After centrifugation, concentrate the supernatant. Purify the concentrate by electrodialysis, concentrate through a membrane, and purify by silica gel column chromatography (dichloromethane:methanol = 10:1). Collect the fraction and concentrate to obtain compound 6-1 (8.2 g, yield 19%).

[0324] Step 2: Add 10 mL of deionized water and compound 6-1 (100 mg, 0.24 mmol) to a 100 mL single-necked reaction flask. Cool the reaction system to approximately 0 °C, add potassium phosphate (610 mg, 2.87 mmol), and then cool to -3 to 0 °C. Add CDMBI (220 mg, 1.01 mmol), and stir the reaction at approximately 0 °C overnight. Detect the reaction endpoint by TLC. After the reaction is complete, filter the reaction solution to remove insoluble matter, and wash the insoluble matter with 3 mL of water. Wash the filtrate three times with 15 mL of toluene. Purify the aqueous phase using a C18 column (deionized water:methanol = 5:1-3:1, containing 0.1% concentrated ammonia). Collect the fraction, concentrate it, and then freeze-dry it to obtain compound G7 (51 mg, yield 52%).

[0325] HRMS calc.for C 14 H 23 N4O 10 [M+H] + m / z=407.14153, found m / z=407.14003.

[0326] 1 HNMR(400MHz,deuterium oxide)δ5.99(d,J=7.2Hz,1H),4.22–4.17(m,2H),4.07–4.05(m,1H),3.96(dd, J=11.2,3.4Hz,1H),3.7(d,J=3.4Hz,1H),3.61–3.38(m,8H),3.27–3.21(m,2H).

[0327] Example 7: Preparation of ADC

[0328] (1) Synthesis of Tmab-G7

[0329] In a pH 7.4 PBS solution, wild-type antibody trastuzumab (Tmab), disaccharide oxazoline (compound G7), and wild-type glycoside endonuclease Endo Si were prepared at concentrations of 10 mg / mL, 1.67 mM (25 equivalents of the antibody concentration), and 0.6 mg / mL, respectively. The pH of the reaction system was adjusted to 7.4, the reaction temperature was 25℃, 800 rpm, and the reaction time was 3 h. The small molecule compounds and glycoside hydrolase were removed by protein A purification to obtain the corresponding transglycosylated antibodies.

[0330] Wild-type trastuzumab (Tmab) was purchased from: Sanyou Biopharmaceuticals (Shanghai) Co., Ltd., and the antibody sequence is as follows:

[0331] The amino acid sequence of the light chain is shown in SEQ ID NO:2:

[0332]

[0333] The amino acid sequence of the heavy chain is shown in SEQ ID NO:3:

[0334]

[0335] (2) Synthesis of ADC

[0336] The prepared transglycosylation antibody Tmab-G7 and drug-linkers (compounds 1, 4, 3, and 2) were prepared in PBS solution at pH 7.4. The concentrations of each compound were adjusted to 5 mg / mL and 0.33 mM, respectively. The reaction system was adjusted to pH 7.4 and incubated overnight at 25°C. The desired sugar-conjugated ADC was obtained by ultrafiltration. The quality control data of the prepared HER2-targeting sugar-conjugated ADC are shown in Table 1. The ADC structure is as follows:

[0337] Table 1. Quality control data of HER2-targeted sugar-based ADC

[0338] Example 9: Compound 9

[0339] Reaction route:

[0340] Operating steps:

[0341] Step A: Dissolve compound N-tert-butoxycarbonyl diethylamine (500 mg, 2.44 mmol) in acetonitrile (15 mL), add compound N,N'-disuccinimidyl carbonate (1.56 g, 6.10 mmol) and triethylamine (1.25 g, 12.2 mmol), and stir at room temperature for 1.5 hours.

[0342] After the reaction was completed by LCMS monitoring, the reaction solution was concentrated, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1-1:2) to obtain 1g of compound 9-1.

[0343] LCMS:(ESI)m / z:388.1[M+H-100] + .

[0344] Step B: Compound 9-2 (5 g, 12.2 mmol) was dissolved in dichloromethane (150 mL) and methanol (75 mL), p-aminobenzyl alcohol (3.06 g, 24.35 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (6.1 g, 24.35 mmol) were added, and the mixture was stirred at room temperature for 15 hours.

[0345] After the reaction was completed as monitored by LCMS, the reaction solution was concentrated, and the crude product was repeatedly slurried with diethyl ether to obtain 7.5 g of compound 9-3.

[0346] LCMS:(ESI)m / z:516.4[M+H] + .

[0347] Step C: Add phenyl p-nitrochloroformate (4.1 g, 20.1 mmol) and pyridine (1.65 mL, 20.4 mmol) to a solution of 9-3 (5.0 g, 9.7 mmol) of tetrahydrofuran (20 mL) and dichloromethane (10 mL), and stir the mixture at room temperature for 15 hours.

[0348] After the reaction was monitored by LCMS, the reaction was quenched by adding 10% citric acid aqueous solution, extracted twice with ethyl acetate, the organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was repeatedly slurried with diethyl ether to obtain 5.6 g of compound 9-4.

[0349] LCMS:(ESI)m / z:681.3[M+H] +

[0350] Step D: Dissolve compounds 9-4 (2.7 g, 3.97 mmol) and 9-5 (2 g, 3.76 mmol) in ultradry N,N-dimethylformamide (30 mL), add diisopropylethylamine (1.9 mL), and stir the reaction system at room temperature for 15 hours.

[0351] After the reaction was completed as monitored by LCMS, the reaction solution was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain 3g of compound 9-6.

[0352] LCMS:(ESI)m / z:977.6[M+H] +

[0353] Step E: Dissolve compound 9-6 (3g, 3.07mmol) in ultradry N,N-dimethylformamide (30mL), add diethylamine (6mL), and stir the reaction system at room temperature for 2 hours.

[0354] After the reaction was completed as monitored by LCMS, the reaction solution was purified by reversed-phase chromatography (tetrahydrofuran / 0.1% formic acid aqueous solution) to obtain 2g of compound 9-7.

[0355] LCMS:(ESI)m / z:755.7[M+H] +

[0356] Step F: Dissolve compound 9-1 (350 mg, 0.72 mmol) in ultradry N,N-dimethylformamide (10 mL), add compound 9-7 (1071 mg, 1.42 mmol) and triethylamine (291 mg, 2.87 mmol), and stir the reaction system overnight at room temperature.

[0357] After the reaction was completed as monitored by LCMS, the reaction solution was concentrated, and the crude product was purified by normal phase (dichloromethane:methanol = 9:1) to obtain 900 mg of compound 9-8.

[0358] LCMS:(ESI)m / z:1767.4[M+H] + .

[0359] Step G: Dissolve compound 9-8 (150 mg, 0.085 mmol) in dichloromethane / methanol (v / v = 9:1, 10 mL), add trifluoroacetic acid (5 mL), and stir the reaction system at room temperature for 2 hours.

[0360] After the reaction was completed as monitored by LCMS, the reaction solution was purified by reversed-phase chromatography (acetonitrile / 0.1% trifluoroacetic acid aqueous solution) to obtain 75 mg of compound 9-9.

[0361] LCMS:(ESI)m / z:1667.6[M+H] + .

[0362] Step H: Dissolve 8-10 (30 mg, 0.054 mmol) in ultradry N,N-dimethylformamide (2 mL), add 9-9 (20 mg, 0.012 mmol) and triethylamine (12 mg, 0.12 mmol), and stir the reaction system overnight at room temperature.

[0363] After the reaction was completed as monitored by LCMS, the reaction solution was purified by passing it through two reversed-phase chromatography columns to obtain 8 mg of compound 9.

[0364] LCMS:(ESI)m / z:1041.8[M / 2+H]+ .

[0365] 1 H NMR (400MHz, DMSO-d6) δ10.07–9.86(m,2H),8.27–8.10(m,2H),8.05(d,J=8.1Hz,2H),7.75(d,J=11.3Hz,2H),7.60(d,J=8.1Hz,4 H),7.35(d,J=8.3Hz,4H),7.30(s,2H),7.24–7.12(m,2H),6.50(s,2H),5.44(s,4H),5.35–5.19(m,6H),5.06(s,4H),4.45–4.34( m,2H),4.12–4.00(m,8H),3.93–3.83(m,2H),3.58–3.54(m,2H),3.50–3.46(m,5H),3.23–3.19(m,2H),3.13–3.04(m,2H),2.70–2 .66(m,2H),2.38–2.31(m,8H),2.22–2.08(m,10H),2.03–1.82(m,8H),1.59–1.41(m,1H),1.31–1.24(m,11H),0.89–0.81(m,19H).

[0366] Example 10: Compound 10

[0367] Reaction route:

[0368] Operating steps:

[0369] Step A: Dissolve compound 8-8 (1510 mg, 7.08 mmol) in dimethyl sulfoxide (10 mL), add triethylamine (1194 mg, 11.80 mmol), and then add compound dibenzocyclooctyne-N-hydroxysuccinimino ester (950 mg, 2.36 mmol). Stir the reaction system overnight at room temperature.

[0370] After the reaction was completed as monitored by LCMS, the reaction solution was purified by reversed-phase chromatography (acetonitrile / water) to obtain 900 mg of compound 10-1.

[0371] LCMS:(ESI)m / z:499.1[MH] - .

[0372] Step B: Dissolve compound 10-1 (500 mg, 1.00 mmol) in anhydrous dichloromethane (10 mL), add triethylamine (506 mg, 5.00 mmol) and phenyl p-nitrochloroformate (670 mg, 3.30 mmol), and stir the reaction system at room temperature for 1 hour.

[0373] After the reaction was completed as monitored by LCMS, the reaction solution was purified by reversed-phase chromatography (acetonitrile / water) to obtain 200 mg of compound 10⁻².

[0374] LCMS:(ESI)m / z:664.3[MH] - .

[0375] Step C: Dissolve compound 10-2 (150 mg, 0.230 mmol) in ultradry N,N-dimethylformamide (7 mL), add 9-9 (90 mg, 0.054 mmol) and triethylamine (78 mg, 0.77 mmol), and stir the reaction system overnight at room temperature.

[0376] After the reaction was completed as monitored by LCMS, the reaction solution was purified by reversed-phase chromatography (acetonitrile / water) to obtain 58 mg of compound 10.

[0377] LCMS:(ESI)m / z:1097.3[M / 2+H] + .

[0378] 1 H NMR (400MHz, DMSO-d6) δ10.00–9.84(m,2H),8.29–8.11(m,2H),8.05(d,J=8.9Hz,2H),7.74(d,J=10.9Hz,3H),7. 60(d,J=7.9Hz,5H),7.46–7.13(m,14H),6.50(s,2H),5.44(s,4H),5.35–5.18(m,6H),5.13–4.97(m,5H),4.52–4 .32(m,2H),4.15–3.95(m,6H),3.92–3.81(m,2H),3.59–3.43(m,8H),3.28–3.17(m,4H),3.13–3.01(m,2H),2.39 –2.32(m,7H),2.26–2.08(m,5H),2.04–1.61(m,9H),1.53–1.40(m,1H),1.37–1.13(m,10H),0.92–0.79(m,18H).

[0379] Example 11: Compound 11

[0380] Reaction route:

[0381] Operating steps:

[0382] Step A: Compound 11-1 (160 mg, 0.61 mmol) was dissolved in dry DMF (10 mL), and then compound 9-7 (1.01 g, 1.34 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (510.27 mg, 1.34 mmol) and 2,6-dimethylpyridine (274.52 mg, 2.56 mmol) were added sequentially. The reaction system was stirred at room temperature for 4 hours under nitrogen protection.

[0383] After the reaction was completed as monitored by LCMS, the reaction solution was purified by reversed-phase chromatography (tetrahydrofuran / 0.1% formic acid aqueous solution) to obtain 900 mg of compound 11-2.

[0384] LCMS:(ESI):m / z 1736.1[M+H] +

[0385] Step B: Dissolve compound 11-2 (420 mg, 0.24 mmol) in dry dichloromethane (15 mL), then add trifluoroacetic acid (1.5 mL, 20.13 mmol). The reaction system is stirred at room temperature for 1 hour under nitrogen protection.

[0386] After the reaction was completed as monitored by LCMS, the solvent was removed by vacuum rotary evaporation under ice-water bath conditions. The crude product was dissolved in N,N-dimethylformamide and purified by preparation (acetonitrile / 0.1% formic acid aqueous solution) to obtain 150 mg of compound 11-3.

[0387] LCMS:(ESI):m / z 1635.1[M+H] +

[0388] Step C: Dissolve compound 11-3 (70 mg, 0.043 mmol) in dry N,N-dimethylformamide (15 mL), then add compound 8-10 (47.69 mg, 0.086 mmol) and triethylamine (21.76 mg, 0.21 mmol) sequentially. The reaction system is stirred at room temperature for 12 hours under nitrogen protection.

[0389] After the reaction was completed as monitored by LCMS, the reaction solution was purified by reverse-phase flash to obtain 37 mg of compound 11.

[0390] LCMS:(ESI):m / z 1026.0[M / 2+H] +

[0391] 1H NMR (400MHz, DMSO) δ9.91(d,J=6.2Hz,2H),8.31–8.14(m,2H),8.10–7.91(m,4H),7.74(d,J=10.9Hz,2H),7.61(d,J=8.4Hz,4H),7.36 (d,J=8.5Hz,4H),7.30(s,2H),6.51(s,2H),5.44(s,4H),5.34–5.17(m,6H),5.15–5.00(m,4H),4.44–4.33(m,2H),4.22–4.12(m,2H) ,4.12–4.01(m,4H),3.60–3.46(m,6H),3.45–3.37(m,4H),3.27–3.18(m,2H),3.15–3.04(m,2H),2.74–2.66(m,2H),2.47–2.39(m,3H ),2.36(s,6H),2.26–2.06(m,10H),2.05–1.76(m,7H),1.59–1.44(m,2H),1.37–1.19(m,8H),1.17–1.02(m,2H),0.92–0.80(m,20H).

[0392] Example 12: Compound 12

[0393] Reaction route:

[0394] Operating steps:

[0395] Step A: Dissolve 12-1 (4.2 g, 39.58 mmol) and 12-2 (5.1 g, 19.58 mmol) in tetrahydrofuran (100 mL), add triphenylphosphine (10.38 g, 39.58 mmol), add diisopropyl azodicarbonate (8 g, 39.73 mmol) dropwise at 0 °C, and slowly raise the temperature to room temperature in an ice-water bath while stirring for 2 hours.

[0396] The reaction mixture was monitored by LCMS until it was almost completely reacted. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, dried, filtered, and evaporated to dryness. The residue was purified by silica gel column chromatography to obtain 3 g of compound 12-3.

[0397] LCMS:(ESI):348.3[M+H] + .

[0398] Step B: Dissolve 12-3 (2.25 g, 6.48 mmol) in dichloromethane (70 mL), add triethylamine (0.32 g, 2.29 mmol) and phenyl p-nitrochloroformate (1.96 g, 9.72 mmol), and stir at 40 °C for 6 hours.

[0399] LCMS showed that the reaction was complete. The reaction solution was directly evaporated to dryness at room temperature. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate containing 1% acetic acid = 1:3) to give 3.1 g of compound 12-4.

[0400] LCMS:(ESI):513.2[M+H] + .

[0401] Step C: Dissolve 12-4 (3.1 g, 6.05 mmol) in dichloromethane (50 mL). Add bis[2-(tert-butyldimethylsiloxy)ethyl]amine (0.065 g, 0.057 mmol) and triethylamine (12 mg, 0.11 mmol) at room temperature, and continue stirring at 20 °C for 16 hours.

[0402] LCMS monitoring showed that the reaction was complete. The reaction solution was evaporated to dryness at room temperature, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 95:5) to give 3g of compound 12-5.

[0403] LCMS:(ESI):708.29[M+H] + .

[0404] Step D: Dissolve 12-5 (3g, 4.24mmol) in methanol (50mL), add potassium carbonate (0.59g, 4.24mmol) in water (4.24mL), stir at 60°C for 5 minutes, add potassium carbonate (2.36g, 16.96mmol), and stir at 60°C for 6 hours.

[0405] LCMS showed that the reaction was complete. The reaction solution was concentrated at room temperature, and the residue was diluted with dichloromethane (100 ml), then washed three times with water (20 ml). The organic phase was dried, filtered, and evaporated to dryness. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 98 / 2) to give 1.8 g of compound 12-6.

[0406] LCMS:(ESI):607.4[M+H] + .

[0407] Step E: Dissolve chlorosulfonyl isocyanate (0.45 g, 3.2 mmol) in dichloromethane (70 mL). Under dry ice and ethanol conditions, slowly add 10 mL of exo-bicyclo[6.1.0]non-4-yn-9-ylmethanol (0.48 g, 3.2 mmol) in dichloromethane. Stir in a dry ice bath under nitrogen protection for 0.5 hours. Under 0°C conditions, slowly add 10 mL of 12-6 (1.94 g, 3.2 mmol) in dichloromethane. Stir at 0°C for 0.5 hours, then allow to rise naturally to room temperature and stir for 2 hours.

[0408] LCMS showed that the reaction was complete. The reaction solution was washed with water (20 mL × 3), the organic phase was dried, filtered, concentrated at room temperature, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 99 / 1) to give 1.45 g of compound 12-7.

[0409] LCMS:(ESI):862.4[M+H] + .

[0410] Step F: Dissolve triethylamine (3.4 g, 33.6 mmol) in tetrahydrofuran (40 mL), add triethylamine trihydrofluoride (5.42 g, 33.6 mmol), stir at room temperature for 0.5 hours, add 12-7 (1450 mg, 1.68 mmol), and stir at room temperature for 2 hours.

[0411] LCMS showed that the reaction was complete. The reaction solution was concentrated at room temperature, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 97 / 3) to give 0.9 g of compound 12-8.

[0412] LCMS (ESI): 634.2 [M+H] + .

[0413] Step G: Dissolve 2,6-dimethylpyridine (4.56 g, 42.6 mmol) in dichloromethane (56 mL), add trimethylsilyl trifluoromethanesulfonate (6.31 g, 28.4 mmol), stir at room temperature for 0.5 hours, add 12-8 (900 mg, 1.42 mmol), and stir at room temperature for 2 hours.

[0414] LCMS showed that the reaction was complete. The reaction solution was concentrated at room temperature, and the residue was directly purified by high performance liquid chromatography (0.1% FA water: acetonitrile = 6:4) to give 20 mg of compound 12-9.

[0415] LCMS:(ESI):535.2[M+H] + .

[0416] Step H: Dissolve 12-9 (20 mg, 0.037 mmol) in tetrahydrofuran (2 mL). Under ice-water bath conditions, add phenyl p-nitrochloroformate (0.037 g, 0.18 mmol) and triethylamine (0.03 g, 0.030 mmol), and continue stirring at 20 °C for 2 hours.

[0417] LCMS monitoring showed that the reaction was complete. The reaction solution was evaporated to dryness at room temperature, and the residue was directly purified by silica gel column chromatography (wet loading) to obtain 20 mg of compound 12-10.

[0418] LCMS:(ESI):865.2[M+H] + .

[0419] Step I: Dissolve 12-10 (20 mg, 0.023 mmol) in N,N-dimethylformamide (1 mL). Add 1-6 (0.065 g, 0.057 mmol) and triethylamine (12 mg, 0.11 mmol) at room temperature, and continue stirring at 20 °C for 16 hours. Increase the temperature to 40 °C and stir for 1 hour.

[0420] LCMS monitoring showed that the reaction was complete. The reaction solution was prepared and purified to give 5 mg of compound 12.

[0421] LCMS:(ESI):m / z 1417.0[1 / 2M+H] + .

[0422] 1 H NMR(400MHz,DMSO-d6)δ10.09–9.96(m,2H),8.46–8.38(m,1H),8.33–8.26(m,1H),8.18–8 .09(m,2H),8.07–8.01(m,1H),7.90–7.86(m,1H),7.62–7.55(m,4H),7.36–7.23(m,11H), 7.21–7.12(m,4H),6.89–6.68(m,3H),6.68–6.61(m,1H),6.06–5.98(m,2H),5.50–5.37(m ,5H),5.36–5.30(m,2H),5.16–4.93(m,5H),4.79–4.58(m,3H),4.52–4.37(m,6H),4.29–4. 23(m,2H),4.15–3.88(m,14H),3.80–3.76(m,1H),3.62–3.54(m,5H),3.49–3.42(m,7H),3 .25–3.22(m,8H),3.21–3.16(m,7H),3.14–3.10(m,3H),3.07–3.00(m,3H),2.99–2.92(m,4 H),2.89–2.82(m,5H),2.44–2.37(m,2H),2.30–2.20(m,3H),2.20–1.92(m,15H),1.84–1. 65(m,8H),1.63–1.40(m,11H),1.37–1.28(m,5H),1.09–0.93(m,13H),0.93–0.58(m,47H).

[0423] Example 13: Compound 13

[0424] Reaction route:

[0425] Operating steps:

[0426] Step A: Dissolve 13-1 (10 g, 80 mmol) in dichloromethane (110 mL), add imidazole (11 g, 160 mmol), and slowly add diphenyl tert-butylsilyl chloride (22 g, 80 mmol) at 0 °C. Stir for 4 hours at room temperature.

[0427] TLC showed that the reaction was complete. The reaction solution was washed with water (30 mL × 3), the organic phase was dried by rotary evaporation, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 95: 5) to give 20 g of compound 13-2.

[0428] LCMS:(ESI)m / z:N / A[M+H] + .

[0429] Step B: Dissolve 13-2 (5g, 13.76mmol) in acetonitrile (50mL), add N-tert-butoxycarbonyl-1,2-ethylenediamine (11g, 68.7mmol), and continue stirring at 80°C for 4 hours.

[0430] LCMS showed that the reaction was complete. The reaction solution was concentrated at room temperature, and the residue was diluted with ethyl acetate (100 mL). The organic phase was washed with water (30 mL × 3), dried by rotary evaporation, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 95:5) to give 5.4 g of compound 13-3.

[0431] LCMS:(ESI)m / z:443.3[M+H] + .

[0432] Step C: Dissolve 13-3 (5 g, 11.3 mmol) in N,N-dimethylformamide (40 mL) and water (4 mL). Add potassium carbonate (4.65 g, 33.9 mmol) at room temperature, heat to 90 °C and stir for 5 minutes. Add sodium 2-bromoethane-1-sulfonate (4.76 g, 22.6 mmol) and potassium iodide (750 mg, 4.52 mmol). Continue stirring at 90 °C for 6 hours.

[0433] LCMS monitoring showed that the reaction was complete. The reaction solution was directly purified by high performance liquid chromatography (water:acetonitrile = 3:7) to give 3.6 g of compound 13-4.

[0434] LCMS:(ESI)m / z:551.2[M+H] + .

[0435] Step D: Dissolve 14-4 (3.8 g, 6.9 mmol) in dichloromethane (250 mL), add trifluoroacetic acid (6.31 g, 350 mmol), stir in an ice-water bath for 5 minutes, and then let it rise naturally to room temperature and stir for 2 hours.

[0436] LCMS showed that the reaction was complete. The reaction solution was concentrated at room temperature, and the residue was directly purified by high performance liquid chromatography (water:acetonitrile = 55:45) to give 1.5 g of compound 13-5.

[0437] LCMS:(ESI)m / z:451.2[M+H] + .

[0438] Step E: Dissolve 13-5 (1.5 g, 3.44 mmol) in N,N-dimethylformamide (15 mL) and water (1.5 mL). Add potassium carbonate (1.5 g, 10 mmol) at room temperature, heat to 90 °C and stir for 5 minutes. Add sodium 2-bromoethane-1-sulfonate (718 mg, 3.44 mmol) and potassium iodide (109 mg, 0.68 mmol). Continue stirring at 90 °C for 6 hours.

[0439] LCMS monitoring showed that the reaction was complete. The reaction solution was directly purified by high-performance liquid chromatography to obtain 600 mg of compound 13-6.

[0440] LCMS:(ESI)m / z:559.2[M+H] + .

[0441] Step F: Dissolve 13-6 (0.55 g, 0.98 mmol) in N,N-dimethylformamide (5 mL). Add 2-1 (0.32 g, 1.18 mmol) and triethylamine (300 mg, 2.94 mmol) at room temperature, and stir at room temperature for 16 hours.

[0442] LCMS monitoring showed that the reaction was complete. The reaction solution was directly purified by high performance liquid chromatography (water:acetonitrile = 7:3) to obtain 700 mg of compound 13-7.

[0443] LCMS:(ESI)m / z:735.5[MH] - .

[0444] Step G: Dissolve triethylamine (2.4 g, 23.76 mmol) in tetrahydrofuran (23 mL), add triethylamine trihydrofluoride (3.08 g, 23.76 mmol), stir at room temperature for 0.5 hours, add 13-7 (700 mg, 0.95 mmol) in tetrahydrofuran (2 mL), and stir at room temperature for 2 hours.

[0445] LCMS showed that the reaction was complete. The reaction solution was concentrated at room temperature, and the residue was purified by high performance liquid chromatography (water:acetonitrile = 75:25) to give 0.28 g of compound 13-8.

[0446] LCMS:(ESI)m / z:495.3[MH] - .

[0447] Step H: Dissolve 13-8 (260 mg, 0.52 mmol) in dichloromethane (6 mL). Under ice-water bath conditions, add phenyl p-nitrochloroformate (0.21 g, 1.04 mmol) and triethylamine (0.21 g, 2.08 mmol), and continue stirring at 20 °C for 2 hours.

[0448] LCMS monitoring showed that the reaction was complete. The reaction solution was rotary evaporated at room temperature to obtain 13-9. The residue was used directly in the next step without purification.

[0449] LCMS:(ESI)m / z:660.4[MH] - .

[0450] Step I: Dissolve 13-9 (333 mg, 0.5 mmol) in N,N-dimethylformamide (5 mL). Add bis[2-(tert-butyldimethylsiloxy)ethyl]amine (0.330 g, 1 mmol) and triethylamine (200 mg, 2 mmol) at room temperature, and continue stirring at 20 °C for 16 hours.

[0451] LCMS monitoring showed that the reaction was complete. The reaction solution was directly purified by high performance liquid chromatography to obtain 200 mg of compound 13-10 and 50 mg of compound 13-10'.

[0452] LCMS:(ESI)m / z:856.7[MH] - .

[0453] Step J: Dissolve triethylamine (0.36 g, 3.6 mmol) in tetrahydrofuran (40 mL), add triethylamine trihydrofluoride (0.58 g, 3.6 mmol), stir at room temperature for 0.5 hours, add 13-10 (150 mg, 0.18 mmol) and 13-10' (50 mg), and stir at room temperature for 2 hours.

[0454] LCMS showed that the reaction was complete. The reaction solution was concentrated at room temperature, and the residue was purified by high performance liquid chromatography (water:acetonitrile = 7:3) to give 0.09 g of compound 13-11.

[0455] LCMS:(ESI)m / z:626.5[MH] - .

[0456] Step K: Dissolve 13-11 (88 mg, 0.14 mmol) in tetrahydrofuran (2 mL). Under ice-water bath conditions, add phenyl p-nitrochloroformate (0.14 g, 0.7 mmol) and triethylamine (0.11 g, 1.12 mmol), and continue stirring at 20 °C for 2 hours.

[0457] LCMS monitoring showed that the reaction was complete. The reaction solution was evaporated to dryness at room temperature, and the residue was purified by high performance liquid chromatography to give 55 mg of compound 13-12.

[0458] LCMS:(ESI)m / z:956.5[MH] - .

[0459] Step L: Dissolve 13-13 (15 mg, 0.016 mmol) in N,N-dimethylformamide (1 mL). Add 1-6 (30 mg, 0.04 mmol) and triethylamine (65 mg, 0.064 mmol) at room temperature, and continue stirring at 20 °C for 16 hours. Increase the temperature to 40 °C and stir for 1 hour.

[0460] LCMS monitoring showed that the reaction was complete. The reaction solution was prepared and purified to give 12 mg of compound 13.

[0461] LCMS:(ESI)m / z:1462.1[1 / 2M-H] - .

[0462] 1H NMR(400MHz,DMSO-d6)δ10.04–9.78(m,3H),8.31–7.92(m,6H),7.91–7.78( m,2H),7.64–7.54(m,5H),7.37–7.21(m,13H),7.20–7.05(m,4H),6.13–5.9 8(m,3H),5.44–5.29(m,6H),5.12–4.93(m,5H),4.77–4.59(m,3H),4.52–4. 34(m,7H),4.30–4.20(m,3H),4.11–3.94(m,12H),3.93–3.85(m,3H),3.81–3 .74(m,2H),3.64–3.52(m,6H),3.21–3.17(m,8H),3.14–3.09(m,4H),3.01– 2.93(m,7H),2.89–2.81(m,8H),2.69–2.64(m,6H),2.43–2.36(m,3H),2.35– 2.23(m,5H),2.22–2.06(m,10H),2.03–1.91(m,5H),1.87–1.62(m,12H),1. 60–1.44(m,9H),1.35–1.28(m,3H),1.07–0.96(m,13H),0.91–0.72(m,47H).

[0463] Example 14: Compound 14

[0464] Reaction route:

[0465] Operating steps:

[0466] Step A: Dissolve 13-12 (15 mg, 0.016 mmol) in N,N-dimethylformamide (1 mL). Add 9-7 (30 mg, 0.04 mmol) and triethylamine (65 mg, 0.064 mmol) at room temperature, and continue stirring at 20 °C for 16 hours. Increase the temperature to 40 °C and stir for 1 hour.

[0467] LCMS monitoring showed that the reaction was complete. The reaction solution was prepared and purified to give 9.1 mg of compound 14.

[0468] LCMS:(ESI)m / z:1093.5[1 / 2M-H] - .

[0469] 1H NMR (400MHz, DMSO-d6) δ10.05–9.61(m,3H),8.28–8.09(m,2H),8.04–7.99(m,1H),7.83–7.49(m,6H),7.43–7.27(m,5H),7.26–7.12(m, 3H),7.11–7.05(m,1H),6.99–6.93(m,1H),6.49–6.45(m,1H),5.48–5.39(m,3H),5.34–5.19(m,5H),5.15–4.96(m,4H),4.44–4.35(m,2H ),4.35–4.23(m,2H),4.17–4.00(m,6H),3.98–3.79(m,4H),3.59–3.41(m,12H),3.21–3.03(m,8H),2.96–2.85(m,3H),2.84–2.74(m,3H) ,2.67–2.64(m,1H),2.39–2.31(m,6H),2.25–2.06(m,9H),1.99–1.79(m,6H),1.56–1.46(m,2H),1.40–1.25(m,6H),1.03–0.58(m,18H).

[0470] Example 15: Compound 15

[0471] Reaction route:

[0472] Operating steps:

[0473] Step A: Under nitrogen protection at room temperature, N-(2-aminoethyl)carbamate tert-butyl ester (9.83 g, 61.35 mmol) was dissolved in acetonitrile (50 mL). Subsequently, compound 15-1 (5 g, 12.27 mmol) was added dropwise to the above reaction solution. The reaction system was heated to 80 °C and stirred for 3 hours.

[0474] LCMS monitoring showed that after the reactants were converted into products, water (40 mL) was added to the reaction solution to quench the reaction. The mixture was extracted with ethyl acetate (20 mL × 3 times), and the organic phases were combined and washed with sodium chloride aqueous solution (20 mL × 2 times). The solution was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (5 g) to give compound 15-2.

[0475] LCMS:(ESI)m / z:487.4[M+H] + .

[0476] Step B: Under nitrogen protection at room temperature, compound 15-2 (3.5 g, 7.19 mmol) and potassium carbonate (2.98 g, 21.57 mmol) were dissolved in N,N-dimethylformamide (20 mL) and water (2 mL). The reaction system was heated to 80 °C and reacted for 15 minutes. Subsequently, potassium iodide (0.16 g, 0.98 mmol) and sodium 2-bromoethane-1-sulfonate (4.55 g, 21.57 mmol) were added to the reaction system, and heating was continued for 6 hours.

[0477] LCMS monitoring showed that after the raw material was converted into the product, the crude product was purified by reversed-phase column chromatography to obtain 1.4 g of compound 15-3.

[0478] LCMS:(ESI)m / z:593.2[MH] - .

[0479] Step C: Under nitrogen protection at room temperature, compound 15-3 (1.3 g, 2.19 mmol) was dissolved in dichloromethane (50 mL). Trifluoroacetic acid (7.49 g, 65.7 mmol) was then added dropwise to the reaction mixture. The reaction system was stirred for 4 hours.

[0480] LCMS monitoring showed that after the starting material was converted into the product, the reaction solution was concentrated under reduced pressure. The resulting 1.6 g of compound 15-4 was used directly in the next step without purification.

[0481] LCMS:(ESI)m / z:493.2[MH] - .

[0482] Step D: Under nitrogen protection at room temperature, compound 15-4 (1.6 g, 2.21 mmol) and potassium carbonate (0.92 g, 6.63 mmol) were dissolved in N,N-dimethylformamide (10 mL) and water (1 mL). The reaction mixture was then heated to 80 °C and stirred for 15 minutes. Potassium iodide (0.082 g, 0.49 mmol) and sodium 2-bromoethane-1-sulfonate (0.70 g, 3.31 mmol) were then added to the mixture. Stirring continued for 4 hours.

[0483] LCMS monitoring showed that after the raw materials were converted into products, the reaction solution was purified by reverse-phase column chromatography to obtain 0.5 g of compound 15-5.

[0484] LCMS:(ESI)m / z:601.2[MH] - .

[0485] Step E: Under nitrogen protection at room temperature, compound 15-5 (0.5 g, 0.83 mmol) and triethylamine (0.42 g, 4.15 mmol) were dissolved in N,N-dimethylformamide (4 mL). Then, compound 2-1 (0.29 g, 1.0 mmol) was added to the above reaction solution. The reaction mixture was stirred for 12 hours.

[0486] LCMS monitoring showed that after the raw material was converted into the product, the crude product was purified by reverse-phase column chromatography to obtain 0.6 g of compound 15-6.

[0487] LCMS:(ESI)m / z:777.2[MH] - .

[0488] Step F: Under nitrogen protection at room temperature, triethylamine trifluoride (2.48 g, 15.4 mmol) and triethylamine (1.56 g, 15.4 mmol) were dissolved in tetrahydrofuran (15 mL). The mixture was stirred for 5 minutes, and then compound 15-6 (0.6 g, 0.77 mmol) was added to the reaction mixture. The reaction mixture was stirred for another 6 hours.

[0489] LCMS monitoring showed that after the reactants were converted into products, the reaction solution was directly concentrated under reduced pressure at 30°C. The resulting residue was purified by reverse-phase column chromatography to give 0.26 g of compound 15-7.

[0490] LCMS:(ESI)m / z:539.0[MH] - .

[0491] Step G: Under nitrogen protection at 0°C, compound 15-7 (0.26 g, 0.48 mmol) and triethylamine (0.39 g, 3.84 mmol) were dissolved in dichloromethane (5 mL). Subsequently, 4-nitrophenyl chloroformate (0.19 g, 0.96 mmol) dissolved in dichloromethane (2 mL) was added to the above reaction solution. The reaction system was then heated to room temperature and stirred for 2 hours.

[0492] LCMS monitoring showed that after the raw materials were converted into products, the reaction solution was purified by reverse-phase column chromatography to obtain 0.19 g of compound 15-8.

[0493] LCMS:(ESI)m / z:704.0[MH] - .

[0494] Step H: Under nitrogen protection at room temperature, compound 16-2 (0.61 g, 1.04 mmol) and triethylamine (0.26 g, 2.6 mmol) were dissolved in N,N-dimethylformamide (1 mL). Subsequently, compound 15-8 (0.18 g, 0.26 mmol) was added to the reaction system. The reaction mixture was heated to 40 °C and stirred for 6 h.

[0495] LCMS monitoring showed that after the raw material was converted into the product, the residue was purified by reversed-phase column chromatography to obtain 0.26 g of compound 15-9.

[0496] LCMS:(ESI)m / z:1148.8[M+H] + .

[0497] Step I: Under nitrogen protection at room temperature, triethylamine trifluoride (0.74 g, 4.60 mmol) and triethylamine (0.47 g, 4.60 mmol) were dissolved in tetrahydrofuran (5 mL). The mixture was stirred for 5 minutes, and then compound 15-9 (0.26 g, 0.23 mmol) was added to the reaction mixture. The reaction mixture was stirred for another 6 hours.

[0498] LCMS monitoring showed that after the reactants were converted into products, the reaction solution was directly concentrated under reduced pressure at 30°C. The resulting residue was purified by reverse-phase column chromatography to give 0.14 g of compound 15-10.

[0499] LCMS:(ESI)m / z:670.4[MH] - .

[0500] Step J: Under nitrogen protection at 0°C, compound 15-10 (140 mg, 0.21 mmol) and triethylamine (0.25 g, 2.52 mmol) were dissolved in dichloromethane (3 mL). Subsequently, 4-nitrophenyl chloroformate (0.21 g, 1.05 mmol) was added to the reaction mixture. The reaction system was then heated to room temperature and stirred for 5 hours.

[0501] LCMS monitoring showed that after the raw material was converted into the product, the residue was purified by reversed-phase column chromatography to obtain 90 mg of compound 15-11.

[0502] LCMS:(ESI)m / z:1000.0[MH] - .

[0503] Step K: Under nitrogen protection at room temperature, compound 9-7 (0.038 g, 0.050 mmol) and triethylamine (0.020 g, 0.20 mmol) were dissolved in N,N-dimethylformamide (0.5 mL). Then, compound 15-11 (0.02 g, 0.02 mmol) was added to the reaction mixture. The reaction system was heated to 40 °C and stirred for 6 hours.

[0504] LCMS monitoring showed that after the raw material was converted into the product, the crude product was purified by high performance liquid chromatography to obtain 0.02 g of compound.

[0505] LCMS:(ESI)m / z:1117.8[1 / 2M+H] + .

[0506] 1 H NMR(400MHz,DMSO-d6)δ10.10–9.90(m,2H),9.71–9.34(m,2H),8.27–8.10(m, 2H),8.08–7.94(m,2H),7.83–7.69(m,2H),7.66–7.52(m,4H),7.38–7.33(m,3 H),7.32–7.26(m,2H),7.25–7.11(m,3H),7.07(s,1H),6.95(s,1H),6.50(s,2 H),5.55–5.37(m,4H),5.36–5.13(m,6H),5.12–4.94(m,4H),4.45–4.33(m,2H ),4.27–3.95(m,8H),3.95–3.84(m,2H),3.84–3.72(m,2H),3.67–3.60(m,2H) ,3.57–3.36(m,10H),3.26–3.17(m,2H),3.15–3.05(m,2H),2.99–2.85(m,2H) ,2.83–2.72(m,2H),2.71–2.64(m,1H),2.41–2.29(m,7H),2.26–2.05(m,9H), 2.03–1.75(m,7H),1.58–1.45(m,2H),1.37–1.25(m,6H),0.97–0.71(m,18H).

[0507] Example 16: Compound 16

[0508] Reaction route:

[0509] Operating steps:

[0510] Step A: Dissolve 16-1 (2 g, 19.01 mmol) in N,N-dimethylformamide (40 mL). Under ice-water bath conditions, add imidazole (2.59 g, 38.04 mmol) and tert-butyldiphenylchlorosilane (10.46 g, 38.04 mmol), and continue stirring at 20 °C for 16 hours.

[0511] LCMS monitoring showed that the reaction was complete. The mixture was diluted with ethyl acetate (150 mL), washed with semi-saturated brine (40 mL × 5), dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography to give 4 g of compound 16-2.

[0512] LCMS:(ESI)m / z:582.3[M+H] + .

[0513] Step B: Dissolve 15-5 (666 mg, 1.1 mmol) in N,N-dimethylformamide (10 mL). Add dibenzocyclooctyne-N-hydroxysuccinimino ester (0.66 g, 3.3 mmol) and triethylamine (400 mg, 4 mmol) at room temperature, and continue stirring at 20 °C for 16 hours.

[0514] LCMS monitoring showed that the reaction was complete. The reaction solution was directly purified by high performance liquid chromatography (water:acetonitrile = 3:7) to obtain 690 mg of compound 16-3.

[0515] LCMS:(ESI)m / z:888.7[MH] - .

[0516] Step C: Dissolve triethylamine (0.79 g, 7.8 mmol) in tetrahydrofuran (8 mL), add triethylamine trihydrofluoride (1.26 g, 7.8 mmol), stir at room temperature for 5 minutes, add 16-3 (347 mg, 0.39 mmol), and stir at room temperature for 2 hours.

[0517] LCMS showed that the reaction was complete. The reaction solution was concentrated at room temperature, and the residue was purified by high performance liquid chromatography (water:acetonitrile = 7:3) to give 200 mg of compound 16-4.

[0518] LCMS:(ESI)m / z:650.4[MH] - .

[0519] Step D: Dissolve 16-4 (190 mg, 0.28 mmol) in dichloromethane (5 mL). Under ice-water bath conditions, add phenyl p-nitrochloroformate (0.11 g, 0.56 mmol) and triethylamine (0.085 g, 0.84 mmol), and continue stirring at 20 °C for 2 hours.

[0520] LCMS monitoring showed that the reaction was complete. The reaction solution was evaporated to dryness at room temperature, and the residue was purified by high-performance liquid chromatography to give 200 mg of compound 16-5.

[0521] LCMS:(ESI)m / z:815.4[MH] - .

[0522] Step E: Dissolve 16-5 (200 mg, 0.24 mmol) in N,N-dimethylformamide (5 mL). Add 16-2 (0.560 g, 0.96 mmol) and triethylamine (120 mg, 1.2 mmol) at room temperature, and continue stirring at 20 °C for 16 hours.

[0523] LCMS monitoring showed that the reaction was complete. The reaction solution was directly purified by high performance liquid chromatography (water:acetonitrile = 25:75) to obtain 189 mg of compound 16-6.

[0524] LCMS:(ESI)m / z:1258.0[MH] - .

[0525] Step F: Dissolve triethylamine (0.32 mg, 3.2 mmol) in tetrahydrofuran (4 mL), add triethylamine trihydrofluoride (0.52 g, 3.2 mmol), stir at room temperature for 5 minutes, add 16-6 (189 mg, 0.15 mmol) of tetrahydrofuran (1 mL), and stir at room temperature for 2 hours.

[0526] LCMS showed that the reaction was complete. The reaction solution was concentrated at room temperature, and the residue was purified by high performance liquid chromatography (water:acetonitrile = 7:3) to give 0.1 g of compound 16-7.

[0527] LCMS:(ESI)m / z:815.5[MH] - .

[0528] Step G: Dissolve 16-7 (100 mg, 0.13 mmol) in dichloromethane (3 mL). Under ice-water bath conditions, add phenyl p-nitrochloroformate (0.21 g, 1.05 mmol) and triethylamine (0.15 g, 1.5 mmol), and continue stirring at 20 °C for 2 hours.

[0529] LCMS monitoring showed that the reaction was complete. The reaction solution was evaporated to dryness at room temperature, and the residue was purified by high performance liquid chromatography (water:acetonitrile = 6:4) to give 70 mg of compound 16-8.

[0530] LCMS:(ESI)m / z:1111.7[MH] - .

[0531] Step H: Dissolve 16-8 (20 mg, 0.018 mmol) in N,N-dimethylformamide (1 mL). Add 9-7 (40 mg, 0.054 mmol) and triethylamine (7.3 mg, 0.072 mmol) at room temperature, and continue stirring at 20 °C for 16 hours. Increase the temperature to 40 °C and stir for 1 hour.

[0532] LCMS monitoring showed that the reaction was complete. The reaction solution was prepared and purified to give 16 mg of compound 16.

[0533] LCMS:(ESI)m / z:1171.50[1 / 2M-H] - .

[0534] 1 H NMR (400MHz, DMSO-d6) δ10.09–9.87(m,2H),9.24–9.00(m,1H),8.27–8.12(m,2H),8.07–8.03(m,1H),7.77–7.71(m,2H),7.64–7.57(m, 4H),7.50–7.40(m,3H),7.38–7.33(m,4H),7.31–7.28(m,2H),7.28–6.91(m,7H),6.60–6.46(m,2H),5.54–5.37(m,4H),5.37–5.17(m,6H ),5.17–4.96(m,5H),4.45–4.33(m,2H),4.18–3.98(m,6H),3.94–3.85(m,2H),3.74–3.49(m,11H),3.28–3.06(m,8H),2.99–2.83(m,3H) ,2.79–2.62(m,6H),2.39–2.31(m,6H),2.27–2.09(m,5H),2.02–1.82(m,6H),1.82–1.67(m,2H),1.45–1.26(m,6H),1.05–0.66(m,18H).

[0535] Example 17: Compound 17

[0536] Reaction route:

[0537] Operating steps:

[0538] Step A: Dissolve 16-8 (25 mg, 0.031 mmol) in N,N-dimethylformamide (1 mL). Add 9-7 (21 mg, 0.028 mmol) and triethylamine (13 mg, 0.12 mmol) at room temperature, and continue stirring at 20 °C for 5 hours.

[0539] LCMS monitoring showed that the reaction was complete. The reaction solution was directly purified by high performance liquid chromatography (water:acetonitrile = 4:6) to obtain 30 mg of compound 17-1.

[0540] LCMS:(ESI)m / z:1727.1[1 / 2M-H] - .

[0541] Step B: Dissolve 17-1 (30 mg, 0.017 mmol) in N,N-dimethylformamide (1 mL). Add 17-2 (15 mg, 0.017 mmol, prepared according to the method in Example 45 of WO2025067221A1) and triethylamine (6.9 mg, 0.068 mmol) at room temperature, and continue stirring at 20°C for 16 hours.

[0542] LCMS monitoring showed that the reaction was complete. The reaction solution was prepared and purified to give 8 mg of compound 17.

[0543] LCMS:(ESI)m / z:1243.0[1 / 2M-H] - .

[0544] 1 H NMR (400MHz, DMSO-d6) δ10.03–9.77(m,2H),9.32–8.76(m,2H),8.25–8.05(m,3H),7.80–7.69(m,2H),7.67–7.58(m,3H),7.58–7 .53(m,1H),7.50–7.39(m,4H),7.38–7.33(m,3H),7.33–7.25(m,3H),7.25–7.12(m,2H),7.06–6.87(m,2H),6.57–6.46(m,1H),5 .50–5.39(m,2H),5.34–5.23(m,3H),5.14–4.99(m,3H),4.81–4.26(m,8H),4.21–3.85(m,12H),3.82–3.58(m,12H),3.28–3.07( m,13H),2.99–2.66(m,12H),2.64–2.59(m,2H),2.40–2.11(m,9H),2.09–1.60(m,12H),1.34–1.09(m,17H),1.03–0.72(m,29H).

[0545] Example 18: Compound 18

[0546] Reaction route:

[0547] Operating steps:

[0548] Step A: Under nitrogen protection at room temperature, compounds 1-6 (0.10 g, 0.090 mmol) and triethylamine (0.030 g, 0.30 mmol) were dissolved in N,N-dimethylformamide (1 mL). Subsequently, compound 15-11 (0.03 g, 0.030 mmol) was added to the above reaction solution. The reaction system was stirred for 12 hours.

[0549] LCMS monitoring showed that after the raw material was converted into the product, the crude product was purified by high performance liquid chromatography to obtain 20 mg of compound 18.

[0550] LCMS:(ESI)m / z:1486.6[1 / 2M+H] + .

[0551] 1 H NMR(400MHz,DMSO-d6)δ10.12–9.86(m,2H),9.78–9.30(m,2H),8.43–8.27(m ,1H),8.23–7.94(m,3H),7.93–7.83(m,1H),7.70–7.49(m,4H),7.35–7.13(m, 12H),7.10–7.03(m,1H),7.01–6.92(m,1H),6.02(s,2H),5.55–5.37(m,4H),5 .37–5.29(m,1H),5.17–4.83(m,4H),4.80–4.58(m,2H),4.51–4.36(m,4H),4. 34–4.19(m,2H),4.17–3.88(m,10H),3.82–3.71(m,2H),3.66–3.38(m,12H), 3.26–3.16(m,10H),3.14–3.09(m,2H),3.05–2.92(m,6H),2.90–2.75(m,6H), 2.70–2.63(m,1H),2.45–2.35(m,2H),2.35–2.04(m,10H),2.04–1.86(m,4H), 1.86–1.65(m,6H),1.64–1.40(m,8H),1.40–1.24(m,4H),1.08–0.68(m,46H).

[0552] Example 19: Compound 19

[0553] Reaction route:

[0554] Operating steps:

[0555] Step A: Under nitrogen protection at room temperature, compound 19-1 (5 g, 29.58 mmol) and 1H-imidazole (3.22 g, 47.33 mmol) were dissolved in dichloromethane (100 mL). Then, tert-butyl(chloro)diphenylsilane (9.76 g, 35.50 mmol) was added to the reaction mixture. The reaction system was stirred for 6 hours.

[0556] LCMS monitoring showed that after the reactants were converted into products, water (40 mL) was added to the reaction solution to quench the reaction, and the mixture was extracted with dichloromethane (50 mL × 3 times). The organic phases were combined and washed with sodium chloride aqueous solution (20 mL × 2 times). Then, the mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 11 g of compound 19-2.

[0557] LCMS:(ESI)m / z:NO Mass.

[0558] Step B: At room temperature, 11.06 g (184.05 mmol) of ethane-1,2-diamine was dissolved in acetonitrile (50 mL). Then, 5 g (12.27 mmol) of compound 19-2 was added to the reaction mixture. The reaction system was heated to 80 °C and stirred for 2 hours.

[0559] LCMS monitoring showed that after the raw materials were converted into products, water (50 mL) was added to the reaction solution to quench the reaction, and the mixture was extracted with dichloromethane (10 mL × 3 times). The organic phases were combined and washed with sodium chloride aqueous solution (20 mL × 2 times). Then, the mixture was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue 19-3 was used directly in the next step.

[0560] LCMS:(ESI)m / z:387.2[M+H] + .

[0561] Step C: Under nitrogen protection at 0°C, compound 19-3 (4 g, 10.35 mmol) was dissolved in tetrahydrofuran (20 mL). Then, ethyl 2,2,2-trifluoroacetate (1.47 g, 10.35 mmol) dissolved in tetrahydrofuran (40 mL) was added to the reaction system, and stirring continued for 1 hour. Subsequently, di-tert-butyl oxalate (2.30 g, 11.38 mmol) dissolved in tetrahydrofuran (20 mL) was added to the system, followed by triethylamine (1.26 g, 12.42 mmol). The reaction system was then brought to room temperature and stirred for 2 hours.

[0562] LCMS monitoring showed that after the starting material was converted into the product, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give 6 g of compound 19-4.

[0563] LCMS:(ESI)m / z:483.2[M-99] + .

[0564] Step D: Under nitrogen protection at room temperature, compound 19-4 (2.4 g, 4.12 mmol) was dissolved in methanol (24 mL). Subsequently, sodium hydroxide (0.24 g, 6.18 mmol) dissolved in water (6 mL) was added to the above reaction solution. The reaction system was stirred for 4 hours.

[0565] LCMS monitoring showed that after the reactants were converted into the product, the reaction solution was concentrated under reduced pressure. The mixture was extracted with dichloromethane (20 mL × 3 times), and the organic phases were combined and washed with sodium chloride aqueous solution (20 mL × 2 times). The solution was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 2.4 g of compound 19-5.

[0566] LCMS:(ESI)m / z:487.3[M+H] + .

[0567] Step E: Under nitrogen protection at room temperature, compound 19-5 (2.4 g, 4.93 mmol) and potassium carbonate (2.04 g, 14.79 mmol) were dissolved in N,N-dimethylformamide (15 mL) and water (1.5 mL). The mixture was then heated to 80 °C and stirred for 15 minutes. Sodium 2-bromoethane-1-sulfonate (2.60 g, 12.32 mmol) and potassium iodide (0.082 g, 0.49 mmol) were then added to the reaction mixture, and stirring continued for 3 hours. Then, sodium 2-bromoethane-1-sulfonate (2.60 g, 12.32 mmol) and potassium carbonate (2.04 g, 14.79 mmol) were added again, and stirring continued for 3 hours.

[0568] LCMS monitoring showed that after the raw materials were converted into products, the reaction solution was purified by reverse-phase column chromatography to obtain 2.1 g of compound 19-6.

[0569] LCMS:(ESI)m / z:701.2[MH] - .

[0570] Step F: Under nitrogen protection at room temperature, compound 19-6 (0.65 g, 0.92 mmol) was dissolved in dichloromethane (50 mL). Trifluoroacetic acid (7.34 g, 64.4 mmol) was then added dropwise to the reaction mixture. The reaction system was stirred for 2 hours.

[0571] LCMS monitoring showed that after the raw materials were converted into products, the reaction solution was concentrated under reduced pressure. The resulting residue 19-7 was used directly in the next step without purification.

[0572] LCMS:(ESI)m / z:601.2[MH] - .

[0573] Step G: Under nitrogen protection at room temperature, compound 19-7 (0.6 g, 1.0 mmol) and triethylamine (1.01 g, 10 mmol) were dissolved in N,N-dimethylformamide (3 mL). Subsequently, compound 2-1 (0.33 g, 1.2 mmol) was added to the above reaction solution. The reaction system was stirred for 4 hours.

[0574] LCMS monitoring showed that after the raw material was converted into the product, the crude product was purified by reverse-phase column chromatography to obtain 0.62 g of compound 19-8.

[0575] LCMS:(ESI)m / z:777.2[MH] - .

[0576] Step H: Under nitrogen protection at room temperature, triethylamine trifluoride (2.58 g, 16 mmol) and triethylamine (1.62 g, 16 mmol) were dissolved in tetrahydrofuran (16 mL). The mixture was stirred for 5 minutes, and then compound 19-8 (0.62 g, 0.80 mmol) was added to the reaction mixture. The reaction system was stirred for another 6 hours.

[0577] LCMS monitoring showed that after the reactants were converted into products, the reaction solution was directly concentrated under reduced pressure at 30°C. The resulting residue was purified by reversed-phase column chromatography to give 0.25 g of compound 19-9.

[0578] LCMS:(ESI)m / z:539.4[MH] - .

[0579] Step I: Under nitrogen protection at 0°C, compound 19-9 (0.25 g, 0.46 mmol) and triethylamine (0.37 g, 3.68 mmol) were dissolved in N,N-dimethylformamide (3 mL). Subsequently, 4-nitrophenyl chloroformate (0.23 g, 1.15 mmol) was added to the above reaction solution. The reaction system was then heated to room temperature and stirred for 5 hours.

[0580] LCMS monitoring showed that after the raw materials were converted into products, the reaction solution was purified by reverse-phase column chromatography to obtain 0.25 g of compound 19-10.

[0581] LCMS:(ESI)m / z:704.4[MH] - .

[0582] Step J: Under nitrogen protection at room temperature, bis(2-((tert-butyldimethylsilyl)oxy)ethyl)amine (0.47 g, 1.4 mmol) and triethylamine (0.28 g, 2.8 mmol) were dissolved in N,N-dimethylformamide (2 mL). Subsequently, compound 19-10 (0.25 g, 0.35 mmol) was added to the reaction system. The reaction mixture was stirred for 2 h.

[0583] LCMS monitoring showed that after the raw material was converted into the product, the residue was purified by reversed-phase column chromatography to obtain 0.22 g of compound 19-11.

[0584] LCMS:(ESI)m / z:899.2[MH] - .

[0585] Step K: Under nitrogen protection at room temperature, triethylamine trifluoride (0.77 g, 4.8 mmol) and triethylamine (0.49 g, 4.8 mmol) were dissolved in tetrahydrofuran (4 mL). The mixture was stirred for 5 minutes, and then compound 19-11 (0.22 g, 0.24 mmol) was added to the reaction mixture. The reaction mixture was stirred for another 6 hours.

[0586] LCMS monitoring showed that after the reactants were converted into products, the reaction solution was directly concentrated under reduced pressure at 30°C. The resulting residue was purified by reversed-phase column chromatography to give 0.16 g of compound 19-12.

[0587] LCMS:(ESI)m / z:670.2[MH] - .

[0588] Step L: Under nitrogen protection at 0°C, compound 19-12 (160 mg, 0.24 mmol) and triethylamine (0.29 g, 2.88 mmol) were dissolved in N,N-dimethylformamide (3 mL). Subsequently, 4-nitrophenyl chloroformate (0.24 g, 1.2 mmol) was added to the reaction mixture. The reaction system was then heated to room temperature and stirred for 5 hours.

[0589] LCMS monitoring showed that after the raw material was converted into the product, the resulting residue was purified by reversed-phase column chromatography to obtain 120 mg of compound 19-13.

[0590] LCMS:(ESI)m / z:1000.0[MH] - .

[0591] Step M: Under nitrogen protection at room temperature, compounds 1-6 (0.056 g, 0.050 mmol) and triethylamine (0.020 g, 0.20 mmol) were dissolved in N,N-dimethylformamide (0.6 mL). Subsequently, compound 19-13 (0.02 g, 0.020 mmol) was added to the above reaction solution. The reaction system was stirred for 12 hours.

[0592] LCMS monitoring showed that after the raw material was converted into the product, the crude product was purified by reverse-phase column chromatography to obtain 20 mg of compound 19.

[0593] LCMS:(ESI)m / z:1486.6[1 / 2M+H] + .

[0594] 1 H NMR (400MHz, DMSO-d6) δ10.16–9.91(m,2H),9.33–8.91(m,2H),8.31(s,1H),8.24–7.95(m,3H),7.94–7.82(m,1H),7.71–7.41(m,4H),7.3 9–6.82(m,14H),6.00(s,2H),5.55–5.22(m,5H),5.18–4.82(m,4H),4.78–4.54(m,2H),4.53–4.35(m,4H),4.32–4.18(m,2H),4.17–3.82( m,10H),3.82–3.71(m,1H),3.67–3.36(m,12H),3.29–3.14(m,10H),3.14–3.09(m,2H),2.97(s,2H),2.89–2.83(m,4H),2.69–2.64(m,1H) ,2.45–2.35(m,2H),2.35–2.05(m,9H),2.05–1.86(m,4H),1.86–1.63(m,6H),1.62–1.21(m,11H),1.08–0.96(m,8H),0.96–0.68(m,32H).

[0595] Example 20: Compound 20

[0596] Reaction route:

[0597] Operating steps:

[0598] Step A: Under nitrogen protection at room temperature, compound 9-7 (0.047 g, 0.063 mmol) and triethylamine (0.025 g, 0.25 mmol) were dissolved in N,N-dimethylformamide (1 mL). Subsequently, compound 19-13 (0.025 g, 0.025 mmol) was added to the above reaction solution, and the reaction system was stirred for 12 hours.

[0599] LCMS monitoring showed that after the raw material was converted into the product, the crude product was purified by reverse-phase column chromatography to obtain 25 mg of compound 20.

[0600] MS(ESI)M / Z:1117.8[1 / 2M+H] + .

[0601] 1 H NMR (400MHz, DMSO-d6) δ10.09–9.84(m,2H),9.37–8.71(m,2H),8.27–8.09(m,2H),8.09–7.93(m,2H),7.82–7.69(m,2H),7. 65–7.50(m,4H),7.43–7.32(m,4H),7.30(s,2H),7.25–6.80(m,6H),6.50(s,2H),5.44(s,4H),5.36–5.15(m,6H),5.15–4.94 (m,4H),4.49–4.33(m,2H),4.33–3.97(m,8H),3.94–3.82(m,2H),3.67–3.34(m,16H),3.30–3.16(m,4H),3.14–3.03(m,2H), 2.89(s,4H),2.42–2.30(m,6H),2.29–2.01(m,10H),2.00–1.77(m,6H),1.52(s,2H),1.35–1.26(m,6H),0.96–0.76(m,18H).

[0602] Example 21: Compound 21

[0603] Reaction route:

[0604] Operating steps:

[0605] Step A: Under nitrogen protection at room temperature, compound 19-3 (0.01 g, 0.010 mmol) and triethylamine (0.010 g, 0.10 mmol) were dissolved in N,N-dimethylformamide (0.6 mL). Then, 9-7 (0.0075 g, 0.010 mmol) was added to the reaction mixture. The reaction system was stirred for 6 hours. LCMS monitoring showed that the starting material was converted to compound 21-1. Then, compound 1-6 (0.0015 g, 0.013 mmol) was added to the reaction system, and stirring was continued for 12 hours.

[0606] LCMS monitoring showed that after the raw material was converted into the product, the crude product was purified by preparative high performance liquid chromatography to obtain 3.17 mg of compound 21.

[0607] LCMS:(ESI)m / z:1302.8[1 / 2M+H] + .

[0608] Example 22 Compound 22

[0609] Reaction route:

[0610] Operating steps:

[0611] Step A: Under nitrogen protection at room temperature, compound 19-7 (0.9 g, 1.49 mmol) and triethylamine (1.51 g, 14.9 mmol) were dissolved in N,N-dimethylformamide (5 mL). Then, dibenzocyclooctyne-N-hydroxysuccinimino ester (0.72 g, 1.79 mmol) was added to the above reaction solution. The reaction mixture was stirred for 24 hours.

[0612] LCMS monitoring showed that after the raw material was converted into the product, the crude product was purified by reversed-phase column chromatography to obtain 0.9 g of compound 22-1.

[0613] LCMS:(ESI)m / z:890.6[M+H] + .

[0614] Step B: Under nitrogen protection at room temperature, triethylamine trifluoride (3.26 g, 20.2 mmol) and triethylamine (2.04 g, 20.2 mmol) were dissolved in tetrahydrofuran (20 mL). The mixture was stirred for 5 minutes, and then compound 22-1 (0.9 g, 1.01 mmol) was added to the reaction mixture. The reaction system was stirred for another 6 hours.

[0615] LCMS monitoring showed that after the reactants were converted into products, the reaction solution was directly concentrated under reduced pressure at 30°C. The resulting residue was purified by reverse-phase column chromatography to give 0.65 g of compound 22-2.

[0616] LCMS:(ESI)m / z:650.4[MH] - .

[0617] Step C: Under nitrogen protection at 0°C, compound 22-2 (0.65 g, 1.0 mmol) and triethylamine (0.81 g, 8 mmol) were dissolved in dichloromethane (15 mL). Subsequently, 4-nitrophenyl chloroformate (0.40 g, 2 mmol) dissolved in dichloromethane (2 mL) was added to the above reaction solution. The reaction system was then heated to room temperature and stirred for 2 hours.

[0618] LCMS monitoring showed that after the raw materials were converted into products, the reaction solution was purified by reverse-phase column chromatography to obtain 0.65 g of compound 22-3.

[0619] LCMS:(ESI)m / z:815.4[MH] - .

[0620] Step D: Under nitrogen protection at room temperature, compound 16-2 (1.28 g, 2.2 mmol) and triethylamine (0.45 g, 4.4 mmol) were dissolved in N,N-dimethylformamide (5 mL). Subsequently, compound 22-3 (0.45 g, 0.55 mmol) was added to the reaction system. The reaction mixture was heated to 40 °C and stirred for 6 h.

[0621] LCMS monitoring showed that after the raw material was converted into the product, the residue was purified by reversed-phase column chromatography to obtain 0.5 g of compound 22-4.

[0622] LCMS:(ESI)m / z:1259.9[M+H] + .

[0623] Step E: Under nitrogen protection at room temperature, triethylamine trifluoride (1.29 g, 8 mmol) and triethylamine (0.81 g, 8 mmol) were dissolved in tetrahydrofuran (2.8 mL). The mixture was stirred for 5 minutes, and then compound 22-4 (0.5 g, 0.40 mmol) was added to the reaction mixture. The reaction system was stirred for another 6 hours.

[0624] LCMS monitoring showed that after the reactants were converted into products, the reaction solution was directly concentrated under reduced pressure at 30°C. The resulting residue was purified by reverse-phase column chromatography to give 0.3 g of compound 22-5.

[0625] LCMS:(ESI)m / z:781.5[MH] - .

[0626] Step F: Under nitrogen protection at 0°C, compound 22-5 (0.2 g, 0.26 mmol) and triethylamine (0.21 g, 2.08 mmol) were dissolved in dichloromethane (4 mL). Subsequently, 4-nitrophenyl chloroformate (0.16 g, 0.78 mmol) dissolved in dichloromethane (2 mL) was added to the above reaction solution. The reaction system was then heated to room temperature and stirred for 2 hours.

[0627] LCMS monitoring showed that after the raw materials were converted into products, the reaction solution was purified by reverse-phase column chromatography to obtain 0.14 g of compound 22-6.

[0628] LCMS:(ESI)m / z:1111.7[MH] - .

[0629] Step G: Under nitrogen protection at room temperature, compound 9-7 (0.051 g, 0.068 mmol) and triethylamine (0.027 g, 0.27 mmol) were dissolved in N,N-dimethylformamide (1 mL). Then, compound 22-6 (0.03 g, 0.027 mmol) was added to the above reaction solution. The reaction mixture was stirred for 12 hours.

[0630] LCMS monitoring showed that after the raw material was converted into the product, the crude product was purified by reverse-phase column chromatography to obtain 13 mg of compound 22.

[0631] LCMS:(ESI)m / z:1171.5[1 / 2M-H] - .

[0632] 1H NMR(400MHz,DMSO-d6)δ10.05–9.86(m,2H),9.17–8.62(m,2H),8.27–8.12(m,2H),8.10–7.96(m,2H),7.81–7.67(m,3H),7 .64–7.52(m,5H),7.49–7.40(m,2H),7.40–6.95(m,14H),6.50(s,2H),5.44(s,4H),5.34–5.13(m,6H),5.12–5.01(m,4H), 4.40(s,2H),4.23–3.96(m,6H),3.93–3.84(m,2H),3.70–3.35(m,14H),3.27–3.15(m,4H),3.14–3.06(m,2H),2.95–2.73( m,4H),2.73–2.59(m,2H),2.43–2.31(m,7H),2.31–2.05(m,6H),2.02–1.78(m,6H),1.39–1.20(m,6H),0.93–0.76(m,16H).

[0633] Example 23: Compound 23

[0634] Reaction route:

[0635] Operating steps:

[0636] Step A: Under nitrogen protection at room temperature, compound 9-7 (0.041 g, 0.054 mmol) and triethylamine (0.055 g, 0.54 mmol) were dissolved in N,N-dimethylformamide (2.5 mL). Then, compound 22-6 (0.06 g, 0.054 mmol) was added to the above reaction solution. The reaction mixture was stirred for 5 hours.

[0637] LCMS monitoring showed that after the raw material was converted into the product, the crude product was purified by preparative high performance liquid chromatography to obtain 40 mg of compound 23-1.

[0638] LCMS:(ESI)m / z:1728.1[M+H] + .

[0639] Step 2: Under nitrogen protection at room temperature, compound 1-6 (0.017 g, 0.17 mmol) was dissolved in N,N-dimethylformamide (1 mL). Subsequently, compound 23-1 (0.03 g, 0.017 mmol) was added to the above reaction solution. The reaction system was stirred for 12 hours.

[0640] LCMS monitoring showed that after the raw material was converted into the product, the crude product was purified by preparative high performance liquid chromatography to obtain 15 mg of compound 23.

[0641] LCMS:(ESI)m / z:1245.2[1 / 2M+H] + .

[0642] 1 H NMR (400MHz, DMSO-d6) δ10.05–9.72(m,2H),9.13–8.59(m,2H),8.32–7.98(m,3H),7.90–7.63(m,2H),7.64– 7.52(m,3H),7.51–6.74(m,11H),6.62–6.36(m,1H),5.58–5.38(m,1H),5.33–5.19(m,2H),5.13–4.99(m,2H) ,4.79–4.26(m,6H),4.17–3.74(m,8H),3.74–3.37(m,12H),3.28–2.97(m,14H),2.92–2.62(m,7H),2.40–2. 09(m,10H),2.04–1.82(m,4H),1.82–1.45(m,4H),1.33–1.25(m,4H),1.23–1.13(m,8H),1.03–0.65(m,20H).

[0643] Example 24:

[0644] According to the preparation method of the ADC in Example 7, a HER2-targeting sugar-based ADC can be obtained, wherein the control compound (drug linker) is obtained by referring to the synthesis method of compound 41 in patent WO2020094670A1. The structures of the disclosed ADC and the control ADC-5 are shown below:

[0645] The obtained ADC quality control data are shown in the table below:

[0646] Table 2 Quality control data for HER2-targeted sugar-based ADCs

[0647] Biological Test Example 1: In vitro efficacy of HER2-targeted sugar-based ADC

[0648] Experimental objective: To detect the in vitro inhibitory activity of ADC compounds against SK-BR-3 (human breast adenocarcinoma cells), NCI-N87 (human gastric cancer cells), and MDA-MB-468 (human breast cancer cells).

[0649] Tumor cells in logarithmic growth phase, SK-BR-3 (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), NCI-N87 (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), or MDA-MB-468 (source: Cell Bank of the Chinese Academy of Sciences), were added to cell culture plates at a density of 2000 cells / well. The cell culture plates were incubated at 37°C in a 5% CO2 cell culture incubator for 12-16 hours. 100 μL of sample (starting at 50 μg / ml, 5-fold dilution, 9 concentrations) was added to each well, gently mixed, and incubated. After 144 hours of incubation, 70 μL of CellTiter-Glo TM (Promega, Catalog No.: G7572) Working solution, gently shake to lyse cells, and read the plate on a microplate reader. Cell proliferation inhibition rate is calculated as: Cell proliferation inhibition rate = (1 - Sample well / Control well) × 100%. A curve is plotted with the sample concentration (Log value) on the x-axis and Cytotoxicity% on the y-axis. Nonlinear regression (curve fit) analysis is performed on the data to obtain the IC50 value for each sample. 50 value.

[0650] Table 3. Inhibitory activity of the compounds of the present invention against cell proliferation

[0651] Conclusion: The ADC disclosed herein exhibits strong in vitro killing effect on KPL-4 / SK-BR-3 / NCI-N87 (Her2-positive cells), but significantly weaker killing effect on MDA-MB-468 (Her2-negative cells). The ADC disclosed herein shows better selectivity for Her2-positive cells.

[0652] Biological test example 2: In vitro plasma stability

[0653] Experimental Methods: Under sterile conditions, ADC samples, healthy human plasma (Liaocheng Second People's Hospital), and SD rat plasma (Suzhou Fangda New Drug Development Co., Ltd.) were filtered through a 0.22 μm filter for sterilization. The drug stock solution was diluted to 150 μg / mL using plasma from different species and incubated in a 37℃ cell culture incubator. The day of incubation was recorded as day 0. Samples were then collected on day 21 for free toxin detection.

[0654] Free toxin detection method: Add 120 μL of prepared biological sample or biological sample of unknown concentration (40 μL) to acetonitrile solution containing internal standard (internal standard: 0.2 ng / mL aprepitant), vortex to mix for 5 min, centrifuge at 6000 g at low temperature (4℃) for 10 min, take 100 μL of supernatant to 100 μL of aqueous solution containing 0.2% formic acid, vortex for 3 min, transfer to a 96-well plate for LC-MS / MS analysis (SCIEX Triple Quad 6500+LC-MS / MS mass spectrometer).

[0655] DAR value detection method: Human plasma samples were captured using human her2 protein beads. PBST (EZ-Buffers E 10X PBST Buffer, manufacturer: Sangon Biotech, catalog number: C520004-0500) was added and incubated at 1000 rpm for 1 h with shaking. The samples were then washed sequentially with 200 μL of PBST, PBS (pH 7.4, manufacturer: Yuanpei, catalog number: B320KJ), ultrapure water, and 10% acetonitrile. The samples were then eluted with 100 μL of 20% acetonitrile (containing 0.5% formic acid) for 5 min. All supernatant was collected, and 20 μL of 100 mM TCEP (manufacturer: Sigma, catalog number: C4706-2G) was added. The mixture was incubated at 1000 rpm for 1 h at room temperature before being loaded for analysis (QTOF-MS) to determine the DAR value loss rate.

[0656] Table 4 Results of in vitro plasma stability test of ADC

[0657] Conclusion: The plasma stability of the ADC disclosed in this study is good.

[0658] Biological Test Example 3: In vivo efficacy of HER2-targeted sugar-based ADC

[0659] 3.1 Low-dose group

[0660] Using BALB / c Nude mice (purchased from Vital River Pharmaceuticals, Beijing) as test animals, the efficacy of anti-HER2-ADC administered via tail vein injection in nude mice with human gastric cancer cell NCI-N87 xenografts was evaluated.

[0661] Mice were subcutaneously inoculated with NCI-N87 cells in the right axilla (Source: ATCC) (5×10 6 / each, with 50% Matrigel), tumors grew for 8 days, reaching an average tumor volume of 150 mm. 3 Animals were randomly grouped according to tumor volume (D8), with 5 animals per group.

[0662] A single dose was administered via tail vein injection. Tumor volume and body weight were measured twice weekly, and data were recorded. Tumor inhibition rate (TGI) (%) = [1 - (T... i -T0) / (V i -V0)]×100,T i T0 and V represent the tumor volume on day i and day i respectively after drug administration in the experimental group. i Vi and V0 represent the tumor volumes of the blank control group (Vehicle, PBS) on day i and day i, respectively, after drug administration. The experimental results at the end of the experiment on day 50 after the start of drug administration are shown in Table 5.

[0663] Table 5. In vivo efficacy evaluation of the ADC compounds disclosed in this paper.

[0664] 3.2 High-dose group

[0665] Using BALB / c Nude mice (purchased from Vital River Pharmaceuticals, Beijing) as test animals, the efficacy of anti-HER2-ADC administered via tail vein injection in nude mice with human gastric cancer cell NCI-N87 xenografts was evaluated.

[0666] Mice were subcutaneously inoculated with NCI-N87 cells in the right axilla (Source: ATCC) (5×10 6 / each, with 50% Matrigel), tumors grew for 9 days, reaching an average tumor volume of 150 mm. 3 Animals were randomly grouped according to tumor volume (D9), with 5 animals per group.

[0667] A single dose was administered via tail vein injection. Tumor volume and body weight were measured twice weekly, and data were recorded. Tumor inhibition rate (TGI) (%) = [1 - (T... i -T0) / (V i -V0)]×100,T i T0 and V represent the tumor volume on day i and day i respectively after drug administration in the experimental group. i Vi and V0 represent the tumor volumes of the blank control group (Vehicle, PBS) on day i and day i, respectively, after drug administration. The experimental results at the end of the experiment on day 61 after the start of drug administration are shown in Table 6.

[0668] Table 6. In vivo efficacy evaluation of the ADC compounds disclosed in this paper.

[0669] Experimental results show that, regardless of whether the dose is low or high, the in vivo efficacy of the disclosed ADC-4 is significantly better than that of the control ADC-5.

Claims

1. The antibody-drug conjugate shown in formula (I), its isomers, or pharmaceutically acceptable salts thereof: Ab-{YX-[Z-(LD)} m ] n } q (I) in, The Ab is an antibody or antigen-binding fragment; Y is Among them, the wavy line The asterisk (*) represents being connected to Ab, and the asterisk (*) represents being connected to X. X is a sugar or a sugar derivative; Z is the segment connecting X and L; L stands for connector; D is a cytotoxic drug; t is 0 or 1; m is 1 to 2; n is 1 to 5; q is 1 to 2.

2. The antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof according to claim 1, wherein X is a disaccharide derivative; preferably, X is... Among them, the wavy line The asterisk (*) represents being connected to Y, and the asterisk (*) represents being connected to Z.

3. The antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein Z is any one of Z-1 to Z-40; preferably, Z is Z-9, wherein the wavy line... The asterisk (*) represents being connected to X, and the asterisk (*) represents being connected to L. Z 1 It is O or NH; preferably, Z 1 It is O; Z 2 It is O or NH; preferably, Z 2 It is O; Z 3 It is a 5-10 membered heteroaryl group, -N=C(NH2)-NH-(CH2) p O-CO- or -N=C(NH2)-NH-(CH2) p -; Preferably, Z 3 It is a 5-10 quinone heteroaryl group; more preferably, Z 3 It is pyridinyl or pyrimidinyl; more preferably, Z 3 It is a pyrimidinyl group; R 1 For H or -(CH2) p N + (CH3)3; preferably, R 1 For H; R 2 For H or -(CH2) p N + (CH3)3; preferably, R 2 For H; R 3 -(CH2CH2O) p -、 Preferably, R 3 -(CH2CH2O) p -or More preferably, R 3 -(CH2CH2O) p -; R 4 and R 5 Each is independently -(CH2) p SO3H, -(CH2) p P(=O)(OH)2、-(CH2) p B(OH)2、-(CH2) p COOH, Preferably, R 4 and R 5 Each is independently -(CH2) p SO3H or More preferably, R 4 and R 5 Each is independently -(CH2) p SO3H; p can be 0, 1, 2, 3 or 4; preferably, p is 2.

4. The antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein Z is any one of Z-1-1 to Z-1-45; preferably, Z is Z-1-6, and the asterisk * represents the conjugate with L.

5. The antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein the L is selected as -L. 1 -L 2 -L 3 -; L 1 for Preferably, L 1 for The asterisk * represents L 2 Connected; L 2 A peptide consisting of 2-7 amino acid residues, wherein the amino acid residues are residues formed from valine, citrulline, phenylalanine, alanine, proline, leucine, isoleucine, glycine, valine, lysine, serine, glutamic acid, or aspartic acid; preferably, L 2 for More preferably, L 2 for The asterisk * represents L 3 Connected, L 3 for Or chemical bond; preferably, L 3 for The asterisk * indicates that it is connected to D; Preferably, L is More preferably, L is 6. The antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, wherein D is... or its derivatives; preferably, D is 7. The antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1-6, wherein the structural unit -Z-(LD) m for:

8. The antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, wherein it satisfies one or more of the following conditions: (1) The Y is (2) The value of m is 1 or 2; (3) The n is 1.

9. The antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, wherein the conjugate is: Wherein Ab is an antibody; preferably, Ab is Her2 antibody; more preferably, Ab is trastuzumab; t is 0 or 1; q is 1 to 2.

10. The drug linker shown in formula (II), its isomer, or a pharmaceutically acceptable salt thereof: Z 4 -(LD)m (II) in, Z 4 For Z 4 -1 to Z 4 Any of -30, where the asterisk * indicates connection to L; m is 1 or 2; Z 1 Z 2 Z 3 R 1 R 2 R 3 R 4 R 5 L and D are as defined in any one of claims 1-7.

11. The drug linker, its isomer, or a pharmaceutically acceptable salt thereof according to claim 10, Z 4 For Z 4 -1-1 to Z 4 Any one of -1-39, with the asterisk * indicating connection to L.

12. The drug linker, its isomer, or a pharmaceutically acceptable salt thereof according to claim 10 or 11, wherein L is -L 1 -L 2 -L 3 -; L 1 for Preferably, L 1 for The asterisk * represents L 2 Connected; L 2 A peptide consisting of 2-7 amino acid residues, wherein the amino acid residues are residues formed from valine, citrulline, phenylalanine, alanine, proline, leucine, isoleucine, glycine, valine, lysine, serine, glutamic acid, or aspartic acid; preferably, L 2 Selected from More preferably, L 2 for The asterisk * represents L 3 Connected, L 3 for Or chemical bond; preferably, L 3 for The asterisk * indicates that it is connected to D; Preferably, L is More preferably, L is 13. The drug linker, its isomer, or a pharmaceutically acceptable salt thereof according to any one of claims 10-12, wherein D is... or its derivatives.

14. The drug linker, its isomer, or a pharmaceutically acceptable salt thereof according to any one of claims 10-13, wherein the linker is:

15. A compound as shown in formula Z(I) or Z(II): Z 1 Z 2 R 4 As defined by any term in equation (I), R 6 It is H or a leaving group, such as H or Preferably, Z(I) is Z 1 Z 2 R 4 As defined by any term in equation (I), R 7 It is H or a leaving group, such as H or Preferably, Z(II) is 16. A pharmaceutical composition comprising an antibody-drug conjugate, an isomer thereof, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-9, or a drug linker, an isomer thereof, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 10-14, and a pharmaceutically acceptable carrier.

17. Use of the antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1-9, or the drug linker, its isomer, or a pharmaceutically acceptable salt thereof according to any one of claims 10-14, or the pharmaceutical composition of claim 16 in the preparation of a medicament for treating tumors.

18. The use according to claim 17, wherein the tumor is selected from breast cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, head and neck cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.

19. A method of preventing or treating tumors, comprising administering to a patient an effective preventive or therapeutic dose of the antibody-drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof, as described in any one of claims 1-9, or the drug linker, its isomer, or a pharmaceutically acceptable salt thereof, as described in any one of claims 10-14, or the pharmaceutical composition of claim 16.

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