Ligand-drug conjugate of auristatin drug and preparation and application of ligand-drug conjugate

Abstract

The invention relates to the technical field of medicines, and relates to an auristatin drug and a conjugate thereof. In particular, the present invention relates to a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, a preparation method and use thereof. The invention also relates to a linker-drug compound or an isomer, a meso-form, a raceme, an enantiomer thereof or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, a method for the production thereof and the use thereof.

Description

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to an application with CN application number 202410042571.5 and a filing date of January 11, 2024, and an application with CN application number 202510016135.5 and a filing date of January 6, 2025. The contents of the above CN applications are hereby incorporated herein by reference in their entirety. Technical field

[0003] The present invention relates to the field of pharmaceutical technologies, and particularly to ligand-drug conjugates, linker-drug compounds, their preparation methods and uses. Background art

[0004] Antibody-drug conjugates (ADCs), as a new type of targeted therapeutic drug, combine the advantages of high selectivity of antibodies and high activity of cytotoxic drugs, and have the advantage of "high efficiency and low toxicity", and have become a research hotspot in tumor targeted therapy. In recent years, ADCs have developed rapidly and have reached the third generation. Currently, 14 ADC drugs have been approved for marketing, namely Mylotarg, Adcetris, Kadcyla, Besponsa, Lumoxiti, Polivy, Enhertu, Padcev, Trodelvy, Blenrep, Zynlonta, Aidixi, Tivdak and Elahere, and more than 200 ADCs are in clinical trials. ADCs are playing an increasingly important role in the field of tumor targeted therapy.

[0005] Antibody-drug conjugates (ADCs) consist of three parts: an antibody (mAb) with high specificity and affinity, a linker with high stability, and a highly efficient small molecule cytotoxic drug (payload / warhead). The antibody part of ADCs is most commonly IgG1, and its warhead part is generally a cytotoxic drug that acts on microtubules, DNA or RNA, such as maytansinoids, auristatins, calicheamicin, camptothecins, pyrrolobenzodiazepines and phallotoxins, etc. Its linkers are mainly divided into two categories, one is a cleavable linker, and the other is a non-cleavable linker.

[0006] Auristatins are a class of tubulin inhibitors that can block the binding of tubulin to GTP and the binding of microtubules to the vinca alkaloid binding site, thereby inducing apoptosis and inhibiting tumor growth. Currently, MMAE (US6884869) and MMAF (US7498298) are the most commonly used ADC warheads, both of which are pentapeptides modified based on dolastatin 10. Both MMAE and MMAF exhibit good anti-tumor activity, but due to factors such as lack of selectivity and low therapeutic index, they cannot be used clinically as monotherapy. However, the high cytotoxicity of MMAE and MMAF makes them ideal warheads for ADCs. MMAE / MMAF has been widely used in the field of ADCs. Currently, 5 ADC drugs with MMAE as the warhead and 1 ADC drug with MMAF as the warhead have been launched, namely Adcetris, Polivy, Padcev, Aidixi, Tivdak and Blenrep. However, these ADCs based on auristatins have some deficiencies. For example, ADCs with MMAE as the toxin have adverse reactions such as neurotoxicity (peripheral neuropathy) and hematotoxicity (thrombocytopenia and neutropenia), and ADCs with MMAF as the toxin have ocular toxicity, etc., and their safety needs to be improved. In addition, most of the linkers used in these ADCs are VClinkers, and the maximum DAR is 4. This is because VClinker has strong hydrophobicity. However, high DAR is likely to cause precipitation and aggregation of ADCs, so the DAR cannot be increased to enhance the drug efficacy.

[0007] Furthermore, currently, for ADCs with MMAE as the warhead, most of them introduce the linker from the secondary amine at the N-terminus of MMAE, and there are few examples of introducing the linker from the hydroxyl group of MMAE. Only Seagen, Technical University of Denmark, Mersana and Shanghai New Ideas Company have reported it.

[0008] In its patent application US2005009751A1, Seagen esterified the hydroxyl group of the MMAE analogue Auristatin E and then introduced a linker with a hydrazone bond. This type of linker with an ester bond and a hydrazone bond is unstable in human plasma and undergoes a certain degree of hydrolysis, resulting in some off-target toxicity (see Nat Biotechnol. 2003 Jul; 21(7): 778-84). In its patent CN105813653, Seagen introduced a self-eliminating group, methylene alkoxy carbamate (MAC), into the hydroxyl group of MMAE and then introduced a β-glucuronide linker. Although the ADC with this type of linker has good stability in human plasma, the release of its warhead involves the catalysis of β-glucuronidase and subsequent self-elimination processes, which is relatively complex.

[0009] In its patent application WO2020260597A1, the Technical University of Denmark introduced a linker onto the hydroxyl group of the MMAE analogue in a similar way to Seagen, that is, by esterification followed by the introduction of the linker. However, after the introduction of the linker, its ester bond is difficult to be hydrolyzed by enzymes and it is not easy to release the warhead. In its patent application CN110234357A, Mersana introduced a linker from the hydroxyl group of the MMAE analogue in a similar way to Seagen, introducing an ester bond or a carbonate structure. However, the ester bond is difficult to be hydrolyzed by enzymes, and the carbonate has poor plasma stability, which will reduce the drug efficacy. In its patent application CN106279352A, Shanghai New Concept Company introduced a carbamate onto the hydroxyl group of the MMAE analogue and then introduced a Mc-Vc-PAB linker. This carbamate has poor stability in plasma and has a potential off-target risk.

[0010] Therefore, developing a new way to introduce the linker to obtain auristatin-based ADC drugs with higher safety and effectiveness has very important clinical significance. Summary of the Invention

[0011] One technical problem to be solved by the present invention is to explore and discover a better anti-tumor auristatin-based ADC drug, making it have higher safety and effectiveness and better meeting the clinical needs.

[0012] In the present invention, auristatin or its derivative is linked to the enzyme-cleavable peptide unit through an aminomethylene structure at the hydroxyl site to form the corresponding linker-payload, which can increase the DAR value of the ADC to 8, have better hydrophilicity at high drug loading, better plasma stability at high drug loading values, and have more advantages in terms of efficacy. Compared with the positive drug (for example, an ADC with Vc-MMAE as the linker-payload), the same tumor suppression effect can be achieved with a lower dose, the MTD is increased, and the neurotoxicity and blood toxicity are reduced. Accordingly, the present application provides the following inventions:

[0013] In a first aspect, the present application provides a ligand-drug conjugate of the general formula I or a pharmaceutically acceptable salt or solvate thereof,

[0014]

[0015] wherein: Ab is an antibody or an antigen-binding fragment thereof that specifically binds to the HER2 antigen;

[0016] wherein the antibody is composed of a light chain and a heavy chain, the light chain contains CDR-L1, CDR-L2, and CDR-L3, and their amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; in certain embodiments, the heavy chain contains CDR-H1, CDR-H2, and CDR-H3, and their amino acid sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively;

[0017] M is a linking unit linked to Ab;

[0018] A is selected from peptide residues composed of 2 to 7 amino acids, wherein, optionally, each of the amino acids is independently substituted by one or more substituents selected from: deuterium atom, halogen, hydroxyl group, cyano group, amino group, nitro group, alkyl group, substituted alkyl group, alkoxy group, cycloalkyl group, substituted cycloalkyl group;

[0019] W represents an aminomethyleneoxy structural unit as shown in formula (i):

[0020]

[0021] wherein:

[0022] The left wavy line represents the connection site of the nitrogen atom in formula (i) to A, and the right wavy line represents the connection site of the oxygen atom in formula (i) to the drug D. The oxygen atom is the common group of the drug D and W;

[0023] R1, R2, and R3 are each independently selected from a hydrogen atom, a deuterium atom, an alkyl group, and a substituted alkyl group;

[0024] p is an integer or a decimal number selected from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20);

[0025] And

[0026] The drug D is an auristatin having the structure shown by formula D, or an isomer, meso form, racemic form, enantiomer or a mixture thereof, or a pharmaceutically acceptable salt thereof,

[0027]

[0028] Wherein:

[0029] R4 and R5 are each independently selected from a hydrogen atom, a deuterium atom, an alkyl group and a deuterated alkyl group, or R4 and R5 are linked together to form the following structure: -(CR 11 R 12 ) n -B-(CR 13 R 14 ) m -, wherein R 11 、R 12 、R 13 and R 14 are selected from a hydrogen atom, a deuterium atom, an alkyl group and a deuterated alkyl group; B is selected from O, NR 15 、CR 16 R 17 , wherein R 15 、R 16 、R 17 are selected from a hydrogen atom, a deuterium atom and an alkyl group; n and m are each independently selected from integers from 0 to 8 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8); the nitrogen atom bonded to R4 and R5 and -(CR 11 R 12 ) n -B-(CR 13 R 14 ) m - together form a ring;

[0030] R6, R7, R8, R9 are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an azide group, an alkyl group and NR 18 R 19 , or any two of R6, R7, R8, R9 together with the atom to which they are bonded form a cycloalkyl group, and the remaining two groups are each independently selected from a hydrogen atom, a halogen, an azide group, an alkyl group and NR 18 R 19 , wherein R 18 、R 19 are selected from a hydrogen atom and an alkyl group;

[0031] R 10 selected from aryl and heteroaryl, wherein the aryl or heteroaryl is optionally substituted with one or more substituents selected from: a hydrogen atom, a halogen, an alkyl, an alkoxy, an amino, and a nitro;

[0032] The wavy line in Formula D represents the connection site of the oxygen atom at position 1 in the structure of Drug D to W, and the oxygen atom is a common group of Drug D and W.

[0033] In certain embodiments, the light chain comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 7.

[0034] In certain embodiments, the light chain further comprises a light chain constant region having the amino acid sequence of SEQ ID NO: 8.

[0035] In certain embodiments, the amino acid sequence of the light chain is SEQ ID NO: 9.

[0036] In certain embodiments, the heavy chain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10.

[0037] In certain embodiments, the heavy chain further comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO: 11.

[0038] In certain embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 12.

[0039] In certain embodiments, the light chain comprises CDR-L1, CDR-L2, and CDR-L3, and their nucleotide sequences are shown as SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively.

[0040] In certain embodiments, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, and their nucleotide sequences are shown as SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0041] In certain embodiments, the light chain comprises a light chain variable region having the nucleotide sequence of SEQ ID NO: 19.

[0042] In certain embodiments, the light chain further comprises a light chain constant region having the nucleotide sequence of SEQ ID NO: 20.

[0043] In certain embodiments, the nucleotide sequence of the light chain is SEQ ID NO: 21.

[0044] In certain embodiments, the heavy chain comprises a heavy chain variable region having the nucleotide sequence of SEQ ID NO: 22.

[0045] In certain embodiments, the heavy chain further comprises a heavy chain constant region having the nucleotide sequence of SEQ ID NO: 23.

[0046] In certain embodiments, the nucleotide sequence of the heavy chain is SEQ ID NO: 24.

[0047] Information on some of the sequences involved in the present invention is provided in the following table.

[0048] Description of the sequence

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] In certain embodiments, each of R4 and R5 is independently selected from a hydrogen atom and a C1-C4 alkyl group.

[0056] In certain embodiments, R4 and R5 are linked to form the following structure: -(CH2)2-B-(CH2)2-, where B is selected from O and NH, and the nitrogen atom bonded to R4 and R5 forms a ring together with -(CH2)2-B-(CH2)2-.

[0057] In certain embodiments, each of R6, R7, R8, and R9 in formula D is a hydrogen atom.

[0058] In certain embodiments, one of R6, R7, R8, and R9 in formula D is selected from a halogen, an azide group, and an amino group, and the remaining three are each hydrogen.

[0059] In certain embodiments, any two of the groups R6, R7, R8, and R9 in formula D together with the atoms to which they are bonded form a cyclopropyl group, and the remaining two groups are each independently a hydrogen atom.

[0060] In certain embodiments, R in formula D 10 is a phenyl group, optionally substituted by one or more of the said substituents. In certain embodiments, the substituents are selected from an amino group and a nitro group.

[0061] In certain embodiments, the drug D is non - restrictively selected from the following compounds:

[0062]

[0063] In the above - mentioned embodiments, the drug D is connected to W through a hydroxyl group.

[0064] In certain embodiments, the linking unit M contains maleimide. In these embodiments, the ligand - drug conjugate can undergo hydrolysis under hydrolyzable conditions, and the hydrolysis site is the maleimide part of the linking unit. When there are multiple linker - drugs in the ligand, the following situations may occur depending on the degree of hydrolysis:

[0065] The maleimide is not hydrolyzed at all, that is, all maleimides are in the closed - ring form

[0066] The maleimide is incompletely hydrolyzed, that is, some maleimides are in the closed - ring form while the other part of the maleimides is in the open - ring form

[0067] The maleimide is completely hydrolyzed, that is, all maleimides are in the open - ring form

[0068] Therefore, when there are multiple linking units M containing maleimide groups in the ADC (i.e., the Ab is connected to multiple drug - linkers containing maleimide groups), these maleimide groups can all be in the closed - ring form, partially in the open - ring form, or all in the open - ring form.

[0069] In each structural formula of the above - mentioned maleimide, the wavy line on the left represents the connection site to the Ab, and the wavy line on the right represents the connection to other structures in M.

[0070] In certain embodiments, the ligand - drug conjugate has the structure shown in Formula Ia:

[0071]

[0072] Wherein: Z is selected from - C1 - C 10 alkylene -, - C3 - C8 carbocycle -, - arylene -, - C1 - C 10 alkylene - arylene -, - arylene - C1 - C 10 - alkylene -, - C1 - C 10 alkylene -(C3 - C8 carbocycle)-, -(C3 - C8 carbocycle)-C1 - C 10 alkylene -, 3 - 8 - membered heterocycle -, - C1 - C 10 alkylene -(3 - 8 - membered heterocycle)-, -(3 - 8 - membered heterocycle)-C1 - C 10Alkylene-, -(CH2CH2O) r -、-(CH2CH2O) r -CH2- or wherein X is selected from -C1-C 10 Alkylene-, -C3-C8 carbocycle-, -Arylene-, -C1-C 10 Alkylene-arylene-, -Arylene-C1-C 10 -Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocycle)-, -(C3-C8 carbocycle)-C1-C 10 Alkylene-, -3-8 membered heterocycle-, -C1-C 10 Alkylene-(3-8 membered heterocycle)-, -(3-8 membered heterocycle)-C1-C 10 Alkylene-, -(CH2CH2O) r -、-(CH2CH2O) r -CH2-; Y is a hydrophilic structure selected from carboxyl, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid or polyethylene glycol (PEG); each of the heterocycles independently contains 1-3 atoms selected from N, O, S; each of the -C1-C 10 Alkylene-, -C3-C8 carbocycle-, heterocycle is independently substituted by one or more substituents selected from deuterium atom, halogen, hydroxyl, cyano, nitro, amino, alkyl, heteroalkyl, substituted alkyl, alkoxy, carboxyl or cycloalkyl;

[0073] The left wavy line in represents the connection site to the N on maleimide, and the right wavy line represents the connection site to the carbonyl; r is an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); q is an integer between 1 and 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8);

[0074] n 1 、n 2 、n 3 are independently selected from integers or decimals between 0 and 20, n 1 、n 2 、n 3 are not simultaneously 0, and n 1 +n 2 +n 3 ≤20, for example 1≤n 1 +n 2 +n 3 ≤2, or 7≤n 1 +n 2 +n 3 ≤8.

[0075] In certain embodiments, n 1 、n 2 、n 3 are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 (e.g., 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10).

[0076] In certain embodiments, A in Formula I or Formula Ia is selected from polypeptide residues formed by 2 - 7 amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), alanine (A), citrulline, serine (S), glutamic acid (E), or aspartic acid (D).

[0077] In certain embodiments, A is a peptide residue formed by 2 - 4 amino acids selected from phenylalanine and glycine. In certain embodiments, A is a tetrapeptide residue consisting of glycine - glycine - phenylalanine - glycine.

[0078] In certain embodiments, A is a dipeptide residue consisting of valine - citrulline.

[0079] In certain embodiments, A is a dipeptide residue consisting of valine - alanine.

[0080] In certain embodiments, Z is selected from -C1 - C 10 alkylene-, e.g., -C4 - C6 alkylene-, e.g., -C5 alkylene-.

[0081] In certain embodiments, Z is wherein q is an integer between 1 - 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8), e.g., 1.

[0082] In certain embodiments, Z is wherein X is a methylene group, which methylene group is optionally substituted with a carboxyl group, and Y is selected from a carboxyl group and a phosphate group; The left wavy line in represents the connection site to the N on the maleimide, and the right wavy line represents the connection site to the carbonyl group.

[0083] In certain embodiments, R1, R2, and R3 are each independently selected from a hydrogen atom, a deuterium atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0084] In certain embodiments, R1, R2, and R3 are simultaneously a hydrogen atom or a deuterium atom.

[0085] In certain embodiments, R1, R2, and R3 are simultaneously a hydrogen atom. In certain embodiments, the ligand-drug conjugate has a structure as shown in formula Ib,

[0086]

[0087] wherein, R4, R5, R6, R7, R8, R9, R 10 , n 1 , n 2 , n 3 as defined above.

[0088] In certain embodiments, the ligand-drug conjugate has a structure as shown in formula Ic,

[0089]

[0090] In certain embodiments, the ligand-drug conjugate has a structure as shown in formula Id,

[0091]

[0092] In certain embodiments, the ligand-drug conjugate has a structure as shown in formula Ie,

[0093] In formula Ic, formula Id, and formula Ie, Ac is each independently a hydrophilic structural unit having the structure shown in formula c:

[0094]

[0095] Ac is connected to the methylene carbon at position 2 indicated in formula Ic, formula Id, or formula Ie through an amino functional group, and X, Y, R4, R5, R6, R7, R8, R9, R 10 , n 1 , n 2 , n3 Each is independently defined as above. In certain embodiments, each of the A's is independently selected from glycine, (D / L)alanine, (D / L)leucine, (D / L)isoleucine, (D / L)valine, (D / L)phenylalanine, (D / L)proline, (D / L)tryptophan, (D / L)serine, (D / L)tyrosine, (D / L)cysteine, (D / L)cystine, (D / L)arginine, (D / L)histidine, (D / L)methionine, (D / L)asparagine, (D / L)glutamine, (D / L)threonine, (D / L)aspartic acid, (D / L)glutamic acid, natural or unnatural amino acid derivatives, or the following structures,

[0096]

[0097] In certain embodiments, the A is selected from -NH-CH2-COOH. The ligand-drug conjugates of the present invention may non-limitingly be selected from the following structures:

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] Among them, the configuration of the 2-bit chiral carbon is of the R type or the S type.

[0119] In a second aspect, the present application provides a linker-drug compound represented by Formula II, or a pharmaceutically acceptable salt or solvate thereof,

[0120]

[0121] wherein:

[0122] Z, A, R1, R2, R3, R4, R5, R 11 、R 12 、R 13 、R 14 、B、R 15 、R 16 、R 17 、n, m, R6, R7, R8, R9, R 18 、R 19 、R 10 are as defined in any one of the above.

[0123] In certain embodiments, the linker-drug compound has the structure shown in Formula IIa,

[0124]

[0125] In certain embodiments, the linker-drug compound has the structure shown in Formula IIb,

[0126]

[0127] In certain embodiments, the linker-drug compound has the structure shown in Formula IIc,

[0128]

[0129] In certain embodiments, the linker-drug compound has the structure shown in Formula IId,

[0130]

[0131] In Formula IIb, Formula IIc, and Formula IId, each Ac is independently a hydrophilic structural unit having the structure shown in Formula c:

[0132]

[0133] Wherein, X and Y are each independently as defined above, and Ac is connected to the 2-position methylene carbon already labeled in Formula IIb, Formula IIc, or Formula IId through -NH-.

[0134] The linker-drug compounds of the present invention can be non-limitingly selected from the following structures:

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] Wherein, the configuration of the 2-position chiral carbon is R or S.

[0146] In a third aspect, the present application also provides the use of the ligand-drug conjugate of the present invention or its pharmaceutically acceptable salt or solvate, or the linker-drug compound of the present invention or its isomer, meso form, racemate, enantiomer or mixture thereof, or its pharmaceutically acceptable salt or solvate for the preparation of a drug for treating or preventing tumors.

[0147] In certain embodiments, the tumor expresses HER2.

[0148] In certain embodiments, the tumor is cancer.

[0149] In certain embodiments, the tumor is selected from solid tumors or non-solid tumors, such as breast cancer (e.g., triple-negative breast cancer), ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer (e.g., gastric adenocarcinoma), endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer (e.g., cutaneous squamous cell carcinoma), thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma, and leukemia.

[0150] In a fourth aspect, the present application also provides a pharmaceutical composition comprising an effective amount of the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound of the present invention or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, optionally further comprising a pharmaceutically acceptable carrier, diluent, or excipient.

[0151] In a fifth aspect, the present application also provides a pharmaceutical formulation comprising an effective amount of the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound of the present invention or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0152] In a sixth aspect, the present application also provides a method for treating or preventing a tumor, comprising administering to a subject in need thereof an effective amount of the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound of the present invention or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, the subject is a mammal, such as a human.

[0153] In a seventh aspect, the present application also provides the use of the linker-drug compound of the present invention or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof for the preparation of a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof. In certain embodiments, the ligand-drug conjugate is selected from the ligand-drug conjugates of the present invention.

[0154] In an eighth aspect, the present application also provides a method for preparing the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, the method comprising: coupling a reduced antibody or an antigen-binding fragment thereof with a linker-drug compound of the present invention or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, to obtain the ligand-drug conjugate. The antibody or an antigen-binding fragment thereof can be reduced by a thiol reducing agent (such as tris(2-carboxyethyl)phosphine (TCEP)).

[0155] Term Definitions

[0156] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used to implement or test the present invention, the preferred methods and materials are described herein. When describing and claiming the present invention, the following terms are used according to the following definitions.

[0157] When a trade name is used in the present invention, the applicant intends to include the formulation of the product under that trade name, the generic drug of that trade name, and the active pharmaceutical ingredient.

[0158] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0159] The term "ligand" is a targeting agent that specifically binds to a target moiety. The ligand is capable of specifically binding to a cellular component or to other target molecules of interest. The target moiety or target is typically on the cell surface. In some aspects, the role of the ligand is to deliver a drug moiety to a specific population of target cells with which the ligand moiety interacts. Ligands include, but are not limited to, proteins, polypeptides and peptides, and non-proteins such as sugars. Suitable ligand moieties include, for example, antibodies, such as full-length (intact) antibodies and antigen-binding fragments thereof. In embodiments where the ligand moiety is a non-antibody targeting reagent, it can be a peptide or polypeptide, or a non-protein molecule. Examples of such targeting reagents include interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient transport molecules, or any other cell-binding molecule or substance. In some embodiments, the linker is covalently linked to a sulfur atom of the ligand. In some aspects, the sulfur atom is the sulfur atom of a cysteine residue that forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is the sulfur atom of a cysteine residue that has been introduced into the ligand moiety and forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is the sulfur atom of a cysteine residue introduced into the ligand moiety by, for example, site-directed mutagenesis or chemical reaction.

[0160] The term "drug" refers to a cytotoxic drug, i.e., a molecule that has a strong ability to disrupt the normal growth of tumor or cancer cells within the cells. In principle, cytotoxic drugs can kill tumor cells at a sufficiently high concentration. However, due to the lack of specificity, while killing tumor or cancer cells, they can also cause apoptosis of normal cells, easily leading to serious side effects.

[0161] The term "ligand-drug conjugate" refers to a molecule formed by linking a ligand to a drug through a stable linker unit. In the present invention, the "ligand-drug conjugate" is preferably an antibody drug conjugate (ADC), which refers to conjugating a monoclonal antibody, a functional antibody fragment, a targeting protein, etc. to a cytotoxic drug through a stable linker unit.

[0162] As used herein, "antibody" or "antibody unit" within its scope includes any part of the antibody structure. This unit can bind, reactively associate, or complex with a receptor, an antigen, or other receptor units possessed by a target cell population. An antibody can be any protein or protein-like molecule that can bind, complex, or react with a part of the cell population to be treated or biotransformed. The antibody constituting the antibody drug conjugate in the present invention retains its antigen-binding ability in its original wild state. Therefore, the antibody in the present invention can specifically bind to an antigen. The antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules related to tissue growth and differentiation (such as those known or predicted to be functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as those known or predicted to be functional). Tumor-associated factors can be cluster of differentiation factors (such as CD proteins).

[0163] Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art and can be prepared by methods and information well known in the art for preparing antibodies. To develop effective cell-level targets for cancer diagnosis and treatment, researchers have sought transmembrane or other tumor-associated polypeptides. These targets are specifically expressed on the surface of one or more cancer cells and are expressed little or not at all on the surface of one or more non-cancer cells. Generally, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. Identifying such tumor-associated factors can greatly improve the specific targeting characteristics of antibody-based cancer treatment. For convenience, antigen-related information well known in the art is labeled below, including the name, other names, and GenBank accession number. The nucleic acid and protein sequences corresponding to the tumor-associated antigens can be found in public databases such as GenBank. Antibodies targeting the corresponding tumor-associated antigens include all amino acid sequence variants and isotypes that have at least 70%, 80%, 85%, 90%, or 95% homology with the sequences identified in the references, or have the exact same biological properties and characteristics as the tumor-associated antigen sequences cited in the references.

[0164] The antibodies of the present invention include, but are not limited to, murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, preferably humanized antibodies and fully human antibodies.

[0165] The term "inhibit" or "inhibition of" means a reduction in the detectable amount or complete prevention.

[0166] The term "cancer" refers to a physiological disorder or disease characterized by dysregulated cell growth. "Tumor" includes cancer cells.

[0167] The term "linker" or "linking fragment" or "linking unit" refers to a chemical structural fragment or bond that is connected to a ligand at one end and a drug at the other end, or can be connected to other linkers and then to the drug.

[0168] Linkers, including spacers, spacers, and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker can be a "cleavable linker" that facilitates the release of the drug in the cell. For example, acid-labile linkers (such as hydrazones), protease-sensitive (such as peptidase-sensitive) linkers, photo-labile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al. Cancer Research 52:127-131, 1992); US Patent No. 5,208,020 can be used.

[0169] According to the mechanism of intracellular drug release, as used herein, a "linker" or "linker of an antibody-drug conjugate" can be classified into two categories: non-cleavable linkers and cleavable linkers. For antibody-drug conjugates containing non-cleavable linkers, the drug release mechanism is as follows: after the conjugate binds to the antigen and is endocytosed into the cell, the antibody is enzymatically degraded in the lysosome, releasing an active molecule composed of a small molecule drug, a linker, and an antibody amino acid residue. The resulting change in the drug molecular structure does not weaken its cytotoxicity, but since the active molecule is charged (amino acid residue), it cannot penetrate adjacent cells. Therefore, such active drugs cannot kill tumor cells (bystander effect) that do not express the target antigen (antigen-negative cells) adjacent to them (Ducry et al., 2010, Bioconjugate Chem. 21:5-13). For antibody-drug conjugates containing cleavable linkers, the drug release mechanism is as follows: after the conjugate binds to the antigen and is endocytosed into the cell, it breaks in the target cell and releases the active ingredient (the small molecule drug itself). Cleavable linkers are mainly divided into: chemically sensitive linkers and enzyme-sensitive linkers. Chemically sensitive linkers can be selectively cleaved due to differences in the properties of plasma and cytoplasm or the tumor microenvironment. Such properties include pH value, glutathione concentration, etc. pH-sensitive linkers are relatively stable in the neutral or weakly alkaline environment of the blood (pH 7.3-7.5), but will be hydrolyzed in the weakly acidic tumor microenvironment (pH 5.0-6.5) and lysosome (pH 4.5-5.0), such as hydrazone, carbonate, acetal, ketal, etc. Due to the limited plasma stability of acid-cleavable linkers, antibody-drug conjugates based on such linkers usually have a short half-life (2-3 days). This short half-life limits to some extent the application of pH-sensitive linkers in the new generation of antibody-drug conjugates. For glutathione-sensitive linkers, also known as disulfide bond linkers. Drug release is caused by the difference in the high concentration of glutathione in the cell (in the millimolar range) and the relatively low concentration of glutathione in the blood (in the micromolar range). This is especially true for tumor cells, whose low oxygen content leads to enhanced activity of reductase, thus resulting in a higher glutathione concentration. Disulfide bonds have thermodynamic stability, so they have good stability in plasma. Enzyme-labile linkers, such as peptide linkers, can better control drug release. Peptide linkers can be effectively cleaved by proteases in the lysosome, such as cathepsin B. This peptide linker is considered to be very stable in plasma circulation because the inappropriate extracellular pH value and serum protease inhibitors cause proteases to usually be inactive outside the cell. Given the high plasma stability and good intracellular cleavage selectivity and effectiveness, enzyme-labile linkers are widely used as cleavable linkers for antibody-drug conjugates.

[0170] The term "antibody-drug conjugate" refers to an antibody linked to a bioactive drug via a stable linker unit. In the present invention, the "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment linked to a bioactive cytotoxic drug via a stable linker unit.

[0171] The three-letter and one-letter codes of amino acids used in the present invention are as described in J. boil. Chem. 1968, 243, 3558.

[0172] The term "natural amino acid" refers to an amino acid that can be biosynthesized. Natural amino acids are generally of the L-form, but there are a few exceptions, such as glycine, including both natural and biosynthetic ones.

[0173] The term "unnatural amino acid" refers to an amino acid that can only be synthesized by artificial methods.

[0174] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched-chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 or 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof. More preferably, it is a lower alkyl group containing 1 to 6 (such as 1 to 4) carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any available attachment point. The substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo group.

[0175] The term "substituted alkyl" means that the hydrogen in the alkyl is replaced by a substituent group. Unless otherwise specified in the text, the substituents of the alkyl can be one or more groups selected from the following group: - halogen, -OR', -NR'R", -SR', -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R", -NR'S(O)2R", -CN and -NO2, and the number of substituents is from 1 to (2m'+1), where m' is the total number of carbon atoms in the group, for example 1, 2, 3, 4, 5 or 6. R', R" and R"' each independently refer to hydrogen, C 1-8 alkyl, aryl, aryl substituted by 1-3 halogens, C 1-8 alkyl, C 1-8 alkoxy or C 1-8 thioalkoxy, or unsubstituted aryl-C 1-4 alkyl. When R' and R" are attached to the same nitrogen atom, they can together with the nitrogen atom form a 3-, 4-, 5-, 6- or 7-membered ring. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

[0176] The term "heteroalkyl" means a group formed by replacing one or more carbons in the alkyl with N, O or S.

[0177] The term "cycloalkyl" means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon group. The ring of the cycloalkyl contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyls include spirocyclic, fused-ring and bridged-ring cycloalkyls.

[0178] The term "alkoxy" means -O-(alkyl) and -O-(cycloalkyl), where the alkyl or cycloalkyl is defined as above. Non-limiting examples of alkoxys include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. The alkoxy can be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0179] The term "heterocyclic" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon containing 3 to 20 ring atoms, wherein one or more (e.g., 1, 2, 3 or 4) ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (where m is 0, 1 or 2), and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, among which 1 to 4 are heteroatoms; more preferably, it contains 3 to 10 or 3 to 8 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups.

[0180] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6-10 membered, such as phenyl. The aryl can be substituted or unsubstituted. When substituted, the substituents can be one or more of the following groups, non-limitingly selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, deuterium atom, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio or heterocycloalkylthio.

[0181] The term "heteroaryl" includes 5- to 8-membered monocyclic heteroaryl and 8- to 12-membered fused heteroaryl.

[0182] The term "5- to 8-membered monocyclic heteroaryl" refers to an aromatic monocyclic ring group containing 5 to 8 ring atoms, at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom. Optionally, the ring atoms (such as carbon atoms, nitrogen atoms, or sulfur atoms) in the cyclic structure can be oxo-substituted. "5- to 8-membered monocyclic heteroaryl" includes, for example, "5- to 7-membered monocyclic heteroaryl", "5- to 6-membered monocyclic heteroaryl", "5- to 6-membered nitrogen-containing monocyclic heteroaryl", "6-membered nitrogen-containing monocyclic heteroaryl", etc. The heteroatoms in the "nitrogen-containing heteroaryl" contain at least one nitrogen atom, for example, only 1 or 2 nitrogen atoms, or, contain one nitrogen atom and 1 or 2 other heteroatoms (such as an oxygen atom and / or a sulfur atom), or, contain 2 nitrogen atoms and 1 or 2 other heteroatoms (such as an oxygen atom and / or a sulfur atom). Specific examples of "5- to 8-membered monocyclic heteroaryl" include, but are not limited to, furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, 2-pyridone, 4-pyridone, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azepinyl, 1,3-diazepinyl, azocinyl, etc.

[0183] The term "8- to 12-membered fused heteroaryl" refers to an unsaturated aromatic cyclic structure formed by two or more cyclic structures sharing two adjacent atoms with each other and containing 8 to 12 ring atoms, at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom. Optionally, the ring atoms (such as carbon atoms, nitrogen atoms, or sulfur atoms) in the cyclic structure can be oxo-substituted. "8- to 12-membered fused heteroaryl" includes "8- to 10-membered fused heteroaryl", "8- to 9-membered fused heteroaryl", etc.; specific examples include, but are not limited to, pyrrolopyrrole, pyrrolofuran, pyrazolopyrrole, pyrazolothiophene, furanothiophene, pyrazolooxazole, benzofuranyl, benzisofuranyl, benzothienyl, indolyl, isoindolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolinyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolinyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, purinyl, naphthyridinyl, etc.

[0184] The term "haloalkyl" refers to an alkyl group substituted by one or more halogen atoms, where the alkyl group is as defined above.

[0185] The term "deuterated alkyl" refers to an alkyl group substituted by one or more deuterium atoms, where the alkyl group is as defined above.

[0186] The term "hydroxyl" refers to the -OH group.

[0187] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0188] The term "amino" refers to -NH2.

[0189] The term "nitro" refers to -NO2.

[0190] The term "derivative" refers to a substance having a chemical structure similar to that of a compound but also containing at least one chemical group not present in the compound and / or lacking at least one chemical group present in the compound. The compound to which the derivative is compared is called the "parent" compound. Generally, a "derivative" can be produced from the parent compound in one or more chemical reaction steps.

[0191] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a drug, a linker-drug compound or a ligand-drug conjugate). The compound or conjugate may contain at least one amino or carboxyl group and can thus form an addition salt with the corresponding acid or base. Exemplary salts include, but are not limited to: sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, salicylate, formate, benzoate, glutamate, mesylate, esylate, benzenesulfonate, p-toluenesulfonate, potassium salt, sodium salt, etc.

[0192] The term "solvate" refers to a linker-drug compound or a ligand-drug conjugate in the present invention formed with one or more solvent molecules, and the solvent molecules include, but are not limited to, water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate, etc.

[0193] The term "pharmaceutical composition" refers to a mixture containing one or more compounds described in the present invention or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and / or excipients. The purpose of the pharmaceutical composition is to facilitate the administration to an organism, favor the absorption of the active ingredient and thus exert its biological activity.

[0194] The term "carrier" refers to a system that can change the way a drug enters the human body, its distribution in the body, control the release rate of the drug and deliver the drug to the target site. The drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects and improve bioavailability.

[0195] The term "excipient" refers to additives or auxiliary materials other than the main drug in pharmaceutical preparations. Such as binders, fillers, disintegrants, lubricants in tablets; matrix backups in semi-solid preparations like ointments and creams; preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, penetration enhancers, osmotic pressure regulators, coloring agents, etc. in liquid preparations can all be referred to as excipients.

[0196] The term "diluent" or "filler" is mainly used to increase the weight and / or volume of the preparation. The addition of diluents not only ensures a certain size but also reduces the dosage deviation of the main ingredient and improves the compression moldability of the drug, etc.

[0197] Beneficial effects

[0198] The present invention provides an auristatin drug conjugate with a highly stable hydrophilic linking unit. The conjugate introduces a linker by forming an ether bond between an aminomethyl group with a highly stable hydrophilic linking unit and a hydroxyl group. It can carry multiple toxins, has good plasma stability, good water solubility, homogeneity, and safety, can specifically bind to receptors highly expressed in tumor cells, and release toxins within tumor cells, having good anti-tumor activity and can be used for preventing or treating tumors and other diseases. Description of the drawings

[0199] Figure 1 It is the LC-MS spectrum of compound LP-1a.

[0200] Figure 2 It is the LC-MS spectrum of VcMMAE.

[0201] Figure 3A It is the RP-HPLC detection result of ADC-1-1a.

[0202] Figure 3B It is the RP-HPLC detection result of ADC-C-1a.

[0203] Figure 3C It is the RP-HPLC detection result of FITC-IgG1-LP-1a.

[0204] Figure 3D It is the RP-HPLC detection result of ADC-Dxd.

[0205] Figure 3E It is the RP-HPLC detection result of ADC-1-1b.

[0206] Figure 3F It is the RP-HPLC detection result of ADC-1-1c.

[0207] Figure 3GRP-HPLC detection results for ADC-1-1d.

[0208] Figure 3H RP-HPLC detection results for ADC-1-1e.

[0209] Figure 3I RP-HPLC detection results for RC-48.

[0210] Figure 4A SEC-HPLC detection results for ADC-1-1a.

[0211] Figure 4B SEC-HPLC detection results for ADC-C-1a.

[0212] Figure 4C SEC-HPLC detection results for RC-48.

[0213] Figure 4D SEC-HPLC detection results for ADC-1-1b.

[0214] Figure 4E SEC-HPLC detection results for ADC-1-1c.

[0215] Figure 4F SEC-HPLC detection results for ADC-1-1d.

[0216] Figure 4G SEC-HPLC detection results for ADC-1-1e.

[0217] Figure 5A In vitro efficacy of HER2-ADC in NCI-N87 cells

[0218] Figure 5B In vitro efficacy of HER2-ADC in SK-OV-3 cells.

[0219] Figure 5C In vitro efficacy of HER2-ADC in Calu-3 cells.

[0220] Figure 5D In vitro efficacy of HER2-ADC in JIMT-1 cells.

[0221] Figure 5E In vitro efficacy of HER2-ADC in A431 cells.

[0222] Figure 5F In vitro efficacy of HER2-ADC in MDA-MB-468 cells.

[0223] Figure 5GFor the in vitro efficacy of HER2-ADC in the mixed tumor cell model (JIMT-1 + MDA-MB-468).

[0224] Figure 6 For the in vivo efficacy of HER2-ADC in the single tumor (JIMT-1).

[0225] Figure 7 For the in vivo efficacy of HER2-ADC in the mixed tumor (JIMT-1 + MDA-MB-468).

[0226] Figure 8A For the in vitro efficacy of ADC-1-1a and RC-48 in NCI-N87 cells.

[0227] Figure 8B For the in vitro efficacy of ADC-1-1a and RC-48 in SK-OV-3 cells.

[0228] Figure 8C For the in vitro efficacy of ADC-1-1a and RC-48 in Calu-3 cells.

[0229] Figure 8D For the in vitro efficacy of ADC-1-1a and RC-48 in JIMT-1 cells.

[0230] Figure 8E For the in vitro efficacy of ADC-1-1a and RC-48 in NCI-H1975 cells.

[0231] Figure 8F For the in vitro efficacy of ADC-1-1a and RC-48 in A431 cells.

[0232] Figure 8G For the in vitro efficacy of ADC-1-1a and RC-48 in MDA-MB-468 cells.

[0233] Figure 8H For the in vitro efficacy of ADC-1-1a and RC-48 in the mixed tumor cell model JIMT-1 + MDA-MB-468.

[0234] Figure 9 For the in vivo efficacy of ADC-1-1a and RC-48 in the single tumor (JIMT-1).

[0235] Figure 10 For the in vivo efficacy of ADC-1-1a and RC-48 in the mixed tumor (JIMT-1 + MDA-MB-468).

[0236] Figure 11In vivo efficacy of ADC-1-1a and RC-48 in xenograft tumors (NCI-N87).

[0237] Figure 12 In vivo efficacy of ADC-1-1a and RC-48 in xenograft tumors (NCI-H1975). Detailed implementation manners

[0238] The following is a further elaboration of the present invention in combination with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, rates, or parts are by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0239] The general steps adopted in the following embodiments of the present invention are as follows:

[0240] General step A

[0241] Coupling to prepare ADC

[0242] After cell expression and purification by Protein A affinity chromatography and molecular sieve chromatography, the antibody is exchanged into a buffer of 20 mM acetate, pH 6.0, and the antibody is concentrated or diluted to a protein concentration of 3 mg / mL. The linker-payload is dissolved to 20 mg / mL with DMA for standby. In order to open the inter-chain disulfide bonds of the antibody, 20-fold of TECP is added according to the molecular ratio and reacted at room temperature for 3 h. Then 20-fold of the linker-payload solution is added according to the molecular ratio and reacted at room temperature for 1 h. After the reaction, ultrafiltration is carried out using a 30 KDa ultrafiltration centrifugal tube to remove the linker-payload that has not been conjugated to the antibody, and the corresponding antibody-drug conjugate (ADC) sample is obtained.

[0243] General step B

[0244] Reverse-phase high performance liquid chromatography (RP-HPLC) for detecting DAR

[0245] Place the sample vial filled with the sample on the sample plate, and set the corresponding positions, injection volumes, injection needle numbers, and injection methods for each sample according to the "Standard Operating Procedure for UPLC". The chromatographic column model is Proteomix RP-1000 (4.6*100 mm, 5 μm,[[]] ),Sepax, product number: 465950-4610.

[0246] The method parameters are as follows:

[0247]

[0248] General step C

[0249] SEC detects the monomer ratio of the antibody or ADC

[0250] Place the sample vial on the sample plate and set the corresponding position, injection volume, number of injection needles, and injection method for each sample according to the "Standard Operating Procedure for UPLC Use".

[0251] Chromatographic column: Biocore SEC-300 5μm, 4.6×300mm

[0252] Manufacturer: NanoChrom, part number: B213-050030-04630S

[0253] Mobile phase: 50mM PB + 300mM NaCl + 200mM Arg + 5% IPA, pH = 6.5 The method parameters are as follows:

[0254] Parameter Setting Flow rate 0.3 mL / min Wavelength 280 nm Column temperature 30℃ Sample tray temperature Room temperature Injection volume 20 μg Maximum pressure 150 bar / 15 MPa / 2175 PSI Gradient Isocratic Run time 20 minutes

[0255] General step D

[0256] Plasma stability study

[0257] Prepare a mixture of ADC and IgG-depleted plasma so that the final concentration of ADC is 0.6 mg / mL, place it in a water bath box in an incubator at 37°C, and set the incubation time to 0 days, 3 days, and 7 days. At the same time, set a plasma non-incubated control. After incubation, the sample is purified and extracted, and the drug-antibody ratio DAR is measured to reflect the stability of ADC in plasma.

[0258] Synthesis of compound 1 in Example 1

[0259]

[0260] In a 50 mL single-necked round-bottom flask, compound Auristatin E (826 mg, 1.128 mmol, 1.0 eq), ki-1 (831.4 mg, 2.256 mmol, 2.0 eq, the synthesis method refers to the synthesis of compound 1 in CN111686259A), zinc acetate (414.2 mg, 5.64 mmol, 2.0 eq) and toluene (15 mL) were added successively. Nitrogen was displaced three times, and the reaction was refluxed at 115 °C for 4 h. The reaction was stopped, cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure, purified by reverse-phase preparative column, and freeze-dried to obtain a white solid (605 mg, 51.55%). LC-MS m / z (ES + ):[M+H] + : 1041.3.

[0261] Synthesis of Compound 3 in Example 2

[0262]

[0263] In a 10 mL EP tube, Z-Gly-Gly-Phe-OH (240 mg, 0.581 mmol, 1.0 eq), HATU (264 mg, 0.697 mmol, 1.2 eq), HOBt (94 mg, 0.697 mmol, 1.2 eq), and DMF (2 mL) were added successively, and stirred at room temperature for standby.

[0264] In another 25 mL single-necked round-bottom flask, compound 1 (605 mg, 0.581 mmol, 1.0 eq) and DMF (8 mL) were added successively. After stirring and dissolving at room temperature, DBU (95.6 μL, 0.64 mmol, 1.1 eq) was added, and the reaction was carried out at room temperature for 0.5 h. Monitored by TLC, the raw materials disappeared, and compound 2 was produced. Then the above mixed solution was added to this flask, and DIEA (96 μL, 0.581 mmol, 1.0 eq) was added. The reaction was carried out at room temperature for 1 h and monitored by HPLC. After the reaction was completed, the white solid product was obtained by purification with a reverse-phase preparative column (487 mg, 69.08%). LC-MS m / z (ES + ):[M+H] + : 1213.6.

[0265] Synthesis of Compound 5 in Example 3

[0266]

[0267] Into a 25 mL single-necked round-bottom flask, compound 3 (487 mg, 0.402 mmol, 1.0 eq), 5% Pd / C (48.7 mg) and DMF (5 mL) were added successively. After replacing the gas with hydrogen three times, the reaction was carried out at room temperature for 1 h. The reaction was monitored by HPLC. The raw material disappeared and a new peak appeared, which was compound 4, denoted as reaction solution ①.

[0268] In another 25 mL single-necked flask, ki-2 (176 mg, 0.442 mmol, 1.1 eq, the synthesis method refers to CN108452321A), pentafluorophenol (81 mg, 0.442 mmol, 1.1 eq), DCC (91 mg, 0.442 mmol, 1.1 eq) and DMF (3 mL) were added. The reaction was carried out at room temperature for 30 min. The reaction was monitored by TLC. After the reaction was completed, ki-3 was obtained, denoted as reaction solution ②.

[0269] Then, reaction solution ① was filtered into a new 25 mL single-necked round-bottom flask. Under an ice-water bath, DIEA (73 μL, 0.442 mmol, 1.1 eq) and the filtrate of reaction solution ② were added. After adding, the temperature was raised to room temperature and the reaction was carried out for 1 h. The reaction was monitored by HPLC. The reaction solution was filtered. The filtrate was added to the reaction solution containing compound 4 under ice-bath stirring. Then, it was added, the ice-bath was removed, and the reaction was carried out at room temperature for 1 h. The reaction was monitored by HPLC. The reaction solution was directly purified by reverse-phase preparation. The preparation solution was freeze-dried to obtain a white solid product (320 mg, 54.53%). LC-MS m / z (ES + ):[M / 2+H] + :730.4。

[0270] Example 4 Synthesis of compound LP-1a

[0271]

[0272] Compound 5 (100 mg, 0.0685 mmol, 1.0 eq) was dissolved in 10 mL of dry dichloromethane and 4 mL of TFA. The reaction was carried out at room temperature for 3 h. The reaction was monitored by HPLC. After the reaction was completed, the solvent was removed by concentration under reduced pressure. It was purified by a reverse-phase preparation column and the preparation solution was freeze-dried to obtain a white solid product (21 mg, 29.78%). LC-MS m / z (ES + ):[M / 2+H] + :652.4. The LC-MS spectrum is shown in Figure 1 。

[0273] Example 5 Synthesis of compound 6

[0274]

[0275] Add MMAE (2.0 g, 2.79 mmol, 1.0 eq) to a 100 mL single-necked flask, (Boc)2O (1.21 g, 5.57 mmol, 2.0 eq), and dissolve it in DCM (20 mL). Add TEA (563 mg, 5.57 mmol, 2.0 eq) under an ice-water bath. After addition, raise the temperature to room temperature and react for 72 h. Monitor by TLC until the raw material MMAE is completely reacted. Post-treatment: Concentrate the reaction solution under reduced pressure and purify it by column chromatography (eluent: DCM / MeOH = 20 / 1) to obtain a white solid (2.28 g, 100%). LC-MS m / z (ES + ):[M+H] + : 818.4。

[0276] Synthesis of Compound 7 in Example 6

[0277]

[0278] Add Compound 6 (2.28 g, 2.79 mmol, 1.0 eq), ki-1 (2.05 g, 5.57 mmol, 2.0 eq), and zinc acetate (1.02 g, 5.57 mmol, 2.0 eq) to a 100 mL single-necked flask in sequence, dissolve them in toluene (30 mL). After replacing with N2 three times, raise the temperature to 115 °C under N2 protection and react for 4 h. Stop the reaction, cool to room temperature, filter, concentrate the filtrate under reduced pressure, purify it by preparative reverse-phase column, and freeze-dry the preparation solution to obtain a white solid product (1.071 g, 34%). LC-MS m / z (ES + ):[M+H] + : 1126.4。

[0279] Synthesis of Compound 9 in Example 7

[0280]

[0281] Add Compound 7 (900 mg, 0.8 mmol, 1.0 eq) and DMF (9 mL) to a 50 mL single-necked flask. After stirring to dissolve, add DBU (134 mg, 0.88 mmol, 1.1 eq) dropwise under an ice-water bath. After addition, raise the temperature to room temperature and react for 30 min. Monitor by TLC until the reaction ends, and record it as Reaction Solution ①;

[0282] In another 50 mL single-necked flask, add Z-Gly-Gly-Phe-OH (364 mg, 0.88 mmol, 1.1 eq), HATU (365 mg, 0.96 mmol, 1.2 eq), HOBt (129.7 mg, 0.96 mmol, 1.2 eq) and DMF (7 mL). After dissolving clearly, dropwise add reaction solution ① and DIEA (103.4 mg, 0.8 mmol, 1.0 eq) successively under an ice-water bath. After adding, raise the temperature to room temperature and react for 1 h, and monitor the reaction by HPLC. The reaction solution is purified by reverse-phase preparation, and the preparation solution is freeze-dried to obtain a white solid (960 mg, 92.4%). LC-MS m / z (ES + ):[M+H] + : 1299.6。

[0283] Synthesis of Compound 11 in Example 8

[0284]

[0285] Add compound 10 (960 mg, 0.74 mmol, 1.0 eq), 5% Pd / C (960 mg) and DMF (10 mL) to a 50 mL single-necked flask. After replacing with H2 three times, react at room temperature for 1 h, and monitor the reaction by HPLC, denoted as reaction solution ①;

[0286] In another 25 mL single-necked flask, add ki-2 (322.7 mg, 0.81 mmol, 1.1 eq), DCC (167 mg, 0.81 mmol, 1.1 eq) and DMF (5 mL). After dissolving clearly, add pentafluorophenol (149 mg, 0.81 mmol, 1.1 eq) under an ice-water bath. After adding, raise the temperature to room temperature and react for 30 min, and monitor the reaction by TLC. After the reaction is complete, obtain ki-3, denoted as reaction solution ②.

[0287] Filter reaction solution ① into a 50 mL single-necked flask, add DIEA (105 mg, 0.81 mmol, 1.1 eq) and the filtrate of reaction solution ② under an ice-water bath. After adding, raise the temperature to room temperature and react for 1 h, and monitor the reaction by HPLC. The reaction solution is purified by reverse-phase preparation, and the preparation solution is freeze-dried to obtain a white solid (895 mg, 78.5%). LC-MS m / z (ES + ):[M / 2+H] + :773.4。

[0288] Synthesis of Compound LP-2a in Example 9

[0289]

[0290] Dissolve compound 12 (400 mg, 0.259 mmol, 1.0 eq) in 20 mL of dry dichloromethane and 8 mL of TFA, react at room temperature for 3 h, and monitor by HPLC. After the reaction is completed, concentrate the solvent under reduced pressure. Purify the crude product by reverse-phase preparative column, freeze-dry the preparation solution to obtain a white solid (248 mg, 74%), LC-MS m / z (ES + ):[M / 2 + H] + :645.3。

[0291] Synthesis of Example 10 VcMMAE

[0292]

[0293] In a 25 mL round-bottom flask, add MMAE (120 mg, 0.167 mmol, 1.0 eq) and MC-VC-PAB-PNP (186 mg, 0.25 mmol, 1.5 eq). After dissolving with DMF (5 mL), sequentially add HOBt (27.1 mg, 0.20 mmol, 1.2 eq) and pyridine (1 mL), stir overnight at room temperature, and monitor by HPLC. After the reaction is completed, purify the crude product by reverse-phase preparative column, freeze-dry the preparation solution to obtain a white solid (158.3 mg, 72%), LC-MS m / z (ES + ):[M / 2 + H] + :659.0. The LC-MS spectrum is shown in Figure 2 .

[0294] Synthesis of Example 11 Compound 13

[0295]

[0296] Referring to the synthesis method of Example 5, use compound 12 (synthesis of compound 17 in patent CN106279352) as the raw material to synthesize compound 13. LC-MS m / z (ES + ):[M + H] + : 873.6

[0297] Synthesis of Example 12 Compound 14

[0298]

[0299] Referring to the synthesis method of Example 6, use compound 13 and ki-1 as the raw materials to synthesize compound 14. LC-MS m / z (ES + ):[M + H] + : 1180.7.

[0300] Synthesis of Example 13 Compound 16

[0301]

[0302] Referring to the synthesis method of Example 7, compound 16 was synthesized using compound 14 as the raw material. LC-MS m / z (ES + ):[M+H] + : 1353.9.

[0303] Synthesis of Compound 18 in Example 14

[0304]

[0305] Referring to the synthesis method of Example 8, compound 18 was synthesized using compound 16 as the raw material. LC-MS m / z (ES + ):[M+H] + : 1600.9.

[0306] Synthesis of Compound LP-3a in Example 15

[0307]

[0308] Referring to the synthesis method of Example 9, compound LP-3a was synthesized using compound 18 as the raw material. LC-

[0309] MS m / z (ES + ):[M+H] + :1344.7.

[0310] Synthesis of Compound 23 in Example 16

[0311]

[0312] Referring to the synthesis method of Example 10, compound 19 was synthesized using compound 12 and MC-VC-PAB-PNP as the raw materials. LC-MS m / z (ES + ):[M+H] + :1371.8.

[0313] Synthesis of Compound 21 in Example 17

[0314]

[0315] Referring to the synthesis method of Example 5, compound 21 was synthesized using compound 20 (synthesized with reference to Patent US2017014524A1) as the raw material. LC-MS m / z (ES + ):[M+H] + : 933.6.

[0316] Synthesis of Compound 22 in Example 18

[0317]

[0318] Referring to the synthesis method of Example 6, compound 22 was synthesized using compound 21 and ki-1 as raw materials. LC-MS m / z (ES + ):[M+H] + : 1241.7.

[0319] Synthesis of Compound 24 in Example 19

[0320]

[0321] Referring to the synthesis method of Example 7, compound 24 was synthesized using compound 22 as a raw material. LC-MS m / z (ES + ):[M+H] + : 1414.8.

[0322] Synthesis of Compound 26 in Example 20

[0323]

[0324] Referring to the synthesis method of Example 8, compound 26 was synthesized using compound 24 as a raw material. LC-MS m / z (ES + ):[M+H] + : 1660.9.

[0325] Synthesis of Compound 27 (LP-3sa) in Example 21

[0326]

[0327] Referring to the synthesis method of Example 9, compound 27 (LP-3sa) was synthesized using compound 26 as a raw material. LC-MS m / z (ES + ):[M+H] + :1304.7.

[0328] Synthesis of Compound 29 in Example 22

[0329]

[0330] Referring to the synthesis method of Example 5, compound 29 was synthesized using compound 28 (synthesized with reference to Patent US2017014524A1) as a raw material. LC-MS m / z (ES + ):[M+H] + : 847.6.

[0331] Synthesis of Compound 30 in Example 23

[0332]

[0333] Referring to the synthesis method of Example 6, compound 30 was synthesized using compound 29 and ki-1 as starting materials. LC-MS m / z (ES + ): [M+H] + : 1155.7.

[0334] Synthesis of Compound 32 in Example 24

[0335]

[0336] Referring to the synthesis method of Example 7, compound 32 was synthesized using compound 30 as the starting material. LC-MS m / z (ES + ): [M+H] + : 1328.8.

[0337] Synthesis of Compound 34 in Example 25

[0338]

[0339] Referring to the synthesis method of Example 8, compound 34 was synthesized using compound 32 as the starting material. LC-MS m / z (ES + ): [M+H] + : 1574.9.

[0340] Synthesis of Compound LP-4a in Example 26

[0341]

[0342] Referring to the synthesis method of Example 9, compound LP-4a was synthesized using compound 34 as the starting material. LC-MS m / z (ES + ): [M+H] + : 1318.7.

[0343] Synthesis of Compound 36 in Example 27

[0344]

[0345] Referring to the synthesis method of Example 5, compound 36 was synthesized using compound 35 (synthesized referring to Patent US20210346523A1) as the starting material. LC-MS m / z (ES + ): [M+H] + : 863.5.

[0346] Synthesis of Compound 37 in Example 28

[0347]

[0348] Refer to the synthesis method of Example 6, and use compound 36 and ki-1 as raw materials to synthesize compound 37. LC-MS m / z (ES + ):[M+H] + : 1171.7.

[0349] Synthesis of Compound 39 in Example 29

[0350]

[0351] Refer to the synthesis method of Example 7, and use compound 37 as the raw material to synthesize compound 39. LC-MS m / z (ES + ):[M+H] + : 1344.7.

[0352] Synthesis of Compound 41 in Example 30

[0353]

[0354] Refer to the synthesis method of Example 8, and use compound 39 as the raw material to synthesize compound 41. LC-MS m / z (ES + ):[M+H] + : 1590.9.

[0355] Synthesis of Compound LP-5a in Example 31

[0356]

[0357] Refer to the synthesis method of Example 9, and use compound 41 as the raw material to synthesize compound LP-5a. LC-MS m / z (ES + ):[M+H] + :1334.7.

[0358] Synthesis of Compound 43 in Example 32

[0359]

[0360] Refer to the synthesis method of Example 6, and use compound 42 (synthesis of compound 17 in Patent US20210346523A1) and ki-1 as raw materials to synthesize compound 43. LC-MS m / z (ES + ):[M+H] + : 1085.6.

[0361] Synthesis of Compound 45 in Example 33

[0362]

[0363] Referring to the synthesis method of Example 7, compound 45 was synthesized using compound 43 as the starting material. LC-MS m / z (ES + ):[M+H] + : 1258.7.

[0364] Synthesis of Compound 47 in Example 34

[0365]

[0366] Referring to the synthesis method of Example 8, compound 47 was synthesized using compound 45 as the starting material. LC-MS m / z (ES + ):[M+H] + : 1504.8.

[0367] Synthesis of Compound LP-6a in Example 35

[0368]

[0369] Referring to the synthesis method of Example 9, compound LP-6a was synthesized using compound 47 as the starting material. LC-MS m / z (ES + ):[M+H] + :1348.7.

[0370] Synthesis of Compound 49 in Example 36

[0371]

[0372] Referring to the synthesis method of Example 6, compound 49 was synthesized using compound 48 (synthesis of compound 6 in Patent CN106279352) and ki-1 as the starting materials. LC-MS m / z (ES + ):[M+H] + : 1082.6.

[0373] Synthesis of Compound 51 in Example 37

[0374]

[0375] Referring to the synthesis method of Example 7, compound 51 was synthesized using compound 49 as the starting material. LC-MS m / z (ES + ):[M+H] + : 1255.7.

[0376] Synthesis of Compound 53 in Example 38

[0377]

[0378] Referring to the synthesis method of Example 8, compound 53 was synthesized using compound 51 as the starting material. LC-MS m / z (ES+ ): [M+H] + : 1501.8。

[0379] Synthesis of Compound LP-7a in Example 39

[0380]

[0381] Referring to the synthesis method of Example 9, Compound LP-7a was synthesized using Compound 53 as the raw material. LC-MS m / z (ES + ):[M+H] + : 1345.7。

[0382] Synthesis of Compound 55 in Example 40

[0383]

[0384] Referring to the synthesis method of Example 5, Compound 55 was synthesized using Compound 54 (synthesis of Compound 1 in Patent CN113121639A) as the raw material. LC-MS m / z (ES + ):[M+H] + : 830.6。

[0385] Synthesis of Compound 56 in Example 41

[0386]

[0387] Referring to the synthesis method of Example 6, Compound 56 was synthesized using Compound 55 and ki-1 as the raw materials. LC-MS m / z (ES + ):[M+H] + : 1138.7。

[0388] Synthesis of Compound 58 in Example 42

[0389]

[0390] Referring to the synthesis method of Example 7, Compound 58 was synthesized using Compound 56 as the raw material. LC-MS m / z (ES + ):[M+H] + : 1311.8。

[0391] Synthesis of Compound 60 in Example 43

[0392]

[0393] Referring to the synthesis method of Example 8, Compound 60 was synthesized using Compound 58 as the raw material. LC-MS m / z (ES + ):[M+H] + : 1557.9。

[0394] Synthesis of Compound LP-8a in Example 44

[0395]

[0396] Referring to the synthesis method of Example 9, Compound LP-8a was synthesized using Compound 60 as the raw material. LC-MS m / z (ES + ):[M+H] + :1301.7。

[0397] Synthesis of Compound 62 in Example 45

[0398]

[0399] Referring to the synthesis method of Example 6, Compound 62 was synthesized using Compound 61 (synthesis of Compound 1 in Patent CN113121639A) and ki-1 as the raw materials. LC-MS m / z (ES + ):[M+H] + :1052.6。

[0400] Synthesis of Compound 63 in Example 46

[0401]

[0402] Referring to the synthesis method of Example 7, Compound 63 was synthesized using Compound 61 as the raw material. LC-MS m / z (ES + ):[M+H] + :1225.7。

[0403] Synthesis of Compound 65 in Example 47

[0404]

[0405] Referring to the synthesis method of Example 8, Compound 65 was synthesized using Compound 63 as the raw material. LC-MS m / z (ES + ):[M+H] + :1471.8。

[0406] Synthesis of Compound LP-9a in Example 48

[0407]

[0408] Referring to the synthesis method of Example 9, Compound LP-9a was synthesized using Compound 65 as the raw material. LC-MS m / z (ES + ):[M+H] + :1315.7。

[0409] Synthesis of Compound 66 in Example 49

[0410]

[0411] Add compound 9 (960 mg, 0.74 mmol, 1.0 eq), 5% Pd / C (960 mg) and DMF (10 mL) into a 50 mL single-necked flask. After replacing with H2 three times, react at room temperature for 1 h and monitor the reaction by HPLC. After the reaction is completed, it is designated as reaction solution ①. Filter reaction solution ① into a 50 mL single-necked flask, and successively add MCOSU (274 mg, 0.89 mmol, 1.2 eq) and DIEA (105 mg, 0.81 mmol, 1.1 eq) under an ice-water bath. After addition, raise the temperature to room temperature and react for 1 h, and monitor the reaction by HPLC. The reaction solution is purified by reverse-phase preparation, and the preparation solution is freeze-dried to obtain a white solid (847.5 mg, 84.3%). LC-MS m / z (ES + ) : [M + H] + : 1358.2.

[0412] Synthesis of Compound LP-10 in Example 50

[0413]

[0414] Referring to the synthesis method of Example 9, synthesize compound LP-10 using compound 66 as the raw material. LC-MS m / z (ES + ) : [M / 2 + H] + : 630.0.

[0415] Synthesis of Compound LP-11 in Example 51

[0416]

[0417] Referring to the synthesis method of Example 49, synthesize compound LP-11 using compound 3 as the raw material. LC-MS m / z (ES + ) : [M + H] + : 1272.8.

[0418] Synthesis of Compound 67 in Example 52

[0419]

[0420] Referring to the synthesis method of Example 49, synthesize compound 67 using compound 28 as the raw material. LC-MS m / z (ES + ) : [M + H] + : 1473.9.

[0421] Synthesis of Compound LP-12 in Example 53

[0422]

[0423] Referring to the synthesis method of Example 9, compound LP-12 was synthesized using compound 67 as the starting material. LC-MS m / z (ES + ):[M+H] + :1273.8。

[0424] Synthesis of Compound 68 in Example 54

[0425]

[0426] Referring to the synthesis method of Example 49, compound 68 was synthesized using compound 36 as the starting material. LC-MS m / z (ES + ):[M+H] + :1387.8。

[0427] Synthesis of Compound LP-13 in Example 55

[0428]

[0429] Referring to the synthesis method of Example 9, compound LP-13 was synthesized using compound 68 as the starting material. LC-MS m / z (ES + ):[M+H] + :1287.8。

[0430] Synthesis of Compound 69 in Example 56

[0431]

[0432] Referring to the synthesis method of Example 49, compound 69 was synthesized using compound 39 as the starting material. LC-MS m / z (ES + ):[M+H] + :1403.8。

[0433] Synthesis of Compound LP-14 in Example 57

[0434]

[0435] Referring to the synthesis method of Example 9, compound LP-14 was synthesized using compound 69 as the starting material. LC-MS m / z (ES + ):[M+H] + :1303.7。

[0436] Synthesis of Compound LP-15 in Example 58

[0437]

[0438] Referring to the synthesis method of Example 49, compound LP-15 was synthesized using compound 45 as the starting material. LC-MS m / z (ES + ):[M+H] + :1403.8。

[0439] Synthesis of Compound LP-16 in Example 59

[0440]

[0441] Referring to the synthesis method of Example 49, compound LP-16 was synthesized using compound 51 as the starting material. LC-MS m / z (ES + ):[M+H] + :1314.8。

[0442] Synthesis of Compound 70 in Example 60

[0443]

[0444] Referring to the synthesis method of Example 49, compound 70 was synthesized using compound 58 as the starting material. LC-MS m / z (ES + ):[M+H] + :1370.8。

[0445] Synthesis of Compound LP-17 in Example 61

[0446]

[0447] Referring to the synthesis method of Example 9, compound LP-17 was synthesized using compound 70 as the starting material. LC-MS m / z (ES + ):[M+H] + :1270.8。

[0448] Synthesis of Compound LP-18 in Example 62

[0449]

[0450] Referring to the synthesis method of Example 49, compound LP-18 was synthesized using compound 63 as the starting material. LC-MS m / z (ES + ):[M+H] + :1284.8。

[0451] Synthesis of Compound 71 in Example 63

[0452]

[0453] In a 50 mL single-necked flask, add compound 9 (960 mg, 0.74 mmol, 1.0 eq), 5% Pd / C (960 mg) and DMF (10 mL). After purging with H2 three times, react at room temperature for 1 h. Monitor the reaction by HPLC. After the reaction is complete, label it as reaction solution ①. Filter reaction solution ① into a 50 mL single-necked flask. Under an ice-water bath, sequentially add compound M6 (512.5 mg, 0.74 mmol, 1.0 eq, for the synthesis of compound M6, refer to compound M6 in CN113827736) and DIEA (105 mg, 0.81 mmol, 1.1 eq). After addition, raise the temperature to room temperature and react for 1 h. Monitor the reaction by HPLC. The reaction solution is purified by reverse-phase preparation, and the preparation solution is freeze-dried to obtain a white solid (860 mg, 70%). LC-MSm / z (ES + ):[M+H] + :1660.1。

[0454] Synthesis of Compound LP-19a in Example 64

[0455]

[0456] Referring to the synthesis method of Example 9, use compound 71 as the raw material to synthesize compound LP-19a. LC-MSm / z (ES + ):[M / 2+H] + :674.4。

[0457] Synthesis of Compound 72 in Example 65

[0458]

[0459] Referring to the synthesis method of Example 63, use compound 3 as the raw material to synthesize compound 72. LC-MSm / z (ES + ):[M / 2+H] + :783.5。

[0460] Synthesis of Compound LP-20a in Example 66

[0461]

[0462] Referring to the synthesis method of Example 9, use compound 72 as the raw material to synthesize compound LP-20a. LC-MSm / z (ES + ):[M / 2+H] + :681.4。

[0463] Synthesis of Compound 73 in Example 67

[0464]

[0465] Referring to the synthesis method of Example 63, compound 73 was synthesized using compound 24 as the starting material. LC-MS m / z (ES + ):[M / 2 + H] + :888.5。

[0466] Synthesis of Compound LP-21a in Example 68

[0467]

[0468] Referring to the synthesis method of Example 9, compound LP-21a was synthesized using compound 73 as the starting material. LC-MS m / z (ES + ):[M + H] + :1362.7。

[0469] Synthesis of Compound 74 in Example 69

[0470]

[0471] Referring to the synthesis method of Example 63, compound 74 was synthesized using compound 32 as the starting material. LC-MS m / z (ES + ):[M / 2 + H] + :845.1。

[0472] Synthesis of Compound LP-22a in Example 70

[0473]

[0474] Referring to the synthesis method of Example 9, compound LP-22a was synthesized using compound 74 as the starting material. LC-MS m / z (ES + ):[M + H] + :1376.8。

[0475] Synthesis of Compound 75 in Example 71

[0476]

[0477] Referring to the synthesis method of Example 63, compound 75 was synthesized using compound 39 as the starting material. LC-MS m / z (ES + ):[M / 2 + H] + :853.1。

[0478] Synthesis of Compound LP-23a in Example 72

[0479]

[0480] Referring to the synthesis method of Example 9, compound LP-23a was synthesized using compound 75 as the starting material. LC-MS m / z (ES+ ):[M+H] + :1392.7。

[0481] Synthesis of Compound 76 in Example 73

[0482]

[0483] Referring to the synthesis method of Example 63, Compound 76 was synthesized using Compound 45 as the raw material. LC-MS m / z (ES + ):[M / 2+H] + :810.1。

[0484] Synthesis of Compound LP-24a in Example 74

[0485]

[0486] Referring to the synthesis method of Example 9, Compound LP-24a was synthesized using Compound 76 as the raw material. LC-MS m / z (ES + ):[M+H] + :1406.8。

[0487] Synthesis of Compound 77 in Example 75

[0488]

[0489] Referring to the synthesis method of Example 63, Compound 77 was synthesized using Compound 51 as the raw material. LC-MS m / z (ES + ):[M / 2+H] + :808.5。

[0490] Synthesis of Compound LP-25a in Example 76

[0491]

[0492] Referring to the synthesis method of Example 9, Compound LP-25a was synthesized using Compound 77 as the raw material. LC-MS m / z (ES + ):[M+H] + :1403.8。

[0493] Synthesis of Compound 78 in Example 77

[0494]

[0495] Referring to the synthesis method of Example 63, Compound 78 was synthesized using Compound 58 as the raw material. LC-MS m / z (ES + ):[M / 2+H] + :836.5。

[0496] Synthesis of Compound LP-26a in Example 78

[0497]

[0498] Referring to the synthesis method of Example 9, compound LP-26a was synthesized using compound 78 as the raw material. LC-MS m / z(ES+): [M+H]+: 1359.7.

[0501] Synthesis of Compound 79 in Example 79

[0502]

[0503] Referring to the synthesis method of Example 63, compound 79 was synthesized using compound 63 as the raw material. LC-MS m / z(ES + ): [M / 2+H] + : 793.5.

[0504] Synthesis of Compound LP-27a in Example 80

[0505]

[0506] Referring to the synthesis method of Example 9, compound LP-27a was synthesized using compound 79 as the raw material. LC-MS m / z(ES + ): [M+H] + : 1373.8.

[0507] Synthesis of Compound 80 in Example 81

[0508]

[0509] Compound 9 (960 mg, 0.74 mmol, 1.0 eq), 5% Pd / C (960 mg) and DMF (10 mL) were added to a 50 mL single-necked flask. After purging with H2 three times, the reaction was carried out at room temperature for 1 h, and the reaction was monitored by HPLC. When the reaction was completed, it was designated as reaction solution ①; reaction solution ① was filtered into a 50 mL single-necked flask, and compound M8 (497.7 mg, 0.74 mmol, 1.0 eq, the synthesis of compound M8 refers to the synthesis of compound M8 in CN113827736) and DIEA (105 mg, 0.81 mmol, 1.1 eq) were successively added under an ice-water bath. After addition, the temperature was raised to room temperature and the reaction was carried out for 1 h, and the reaction was monitored by HPLC. The reaction solution was purified by reverse-phase preparation, and the preparation solution was freeze-dried to obtain a white solid (918 mg, 75%). LC-MS m / z(ES + ): [M / 2+H] + : 827.5.

[0510] Synthesis of Compound LP-28a in Example 82

[0511]

[0512] Referring to the synthesis method of Example 9, compound LP-28a was synthesized using compound 80 as the raw material. LC-MS m / z (ES + ): [M / 2 + H] + : 671.4.

[0513] Synthesis of Compound 81 in Example 83

[0514]

[0515] Referring to the synthesis method of Example 81, compound 81 was synthesized using compound 3 as the raw material. LC-MS m / z (ES + ): [M / 2 + H] + : 784.5.

[0516] Synthesis of Compound LP-29a in Example 84

[0517]

[0518] Referring to the synthesis method of Example 9, compound LP-29a was synthesized using compound 81 as the raw material. LC-MS m / z (ES + ): [M / 2 + H] + : 678.4.

[0519] Synthesis of Compound 82 in Example 85

[0520]

[0521] Referring to the synthesis method of Example 81, compound 82 was synthesized using compound 24 as the raw material. LC-MS m / z (ES + ): [M / 2 + H] + : 885.0.

[0522] Synthesis of Compound LP-30a in Example 86

[0523]

[0524] Referring to the synthesis method of Example 9, compound LP-30a was synthesized using compound 82 as the raw material. LC-MS m / z (ES + ): [M / 2 + H] + : 678.9.

[0525] Synthesis of Compound 83 in Example 87

[0526]

[0527] Refer to the synthesis method of Example 81, and use compound 32 as the raw material to synthesize compound 83. LC-MS m / z (ES + ):[M / 2 + H] + :842.0。

[0528] Synthesis of Compound LP-31a in Example 88

[0529]

[0530] Refer to the synthesis method of Example 9, and use compound 83 as the raw material to synthesize compound LP-31a. LC-MS m / z (ES + ):[M / 2 + H] + :685.9。

[0531] Synthesis of Compound 84 in Example 89

[0532]

[0533] Refer to the synthesis method of Example 81, and use compound 39 as the raw material to synthesize compound 84. LC-MS m / z (ES + ):[M / 2 + H] + :850.1。

[0534] Synthesis of Compound LP-32a in Example 90

[0535]

[0536] Refer to the synthesis method of Example 9, and use compound 84 as the raw material to synthesize compound LP-32a. LC-MS m / z (ES + ):[M / 2 + H] + :693.9。

[0537] Synthesis of Compound 85 in Example 91

[0538]

[0539] Refer to the synthesis method of Example 81, and use compound 45 as the raw material to synthesize compound 85. LC-MS m / z (ES + ):[M / 2 + H] + :806.9。

[0540] Synthesis of Compound LP-33a in Example 92

[0541]

[0542] Referring to the synthesis method of Example 9, compound LP-33a was synthesized using compound 85 as the starting material. LC-MS m / z (ES + ):[M / 2+H] + :700.9。

[0543] Example 93 Synthesis of compound 86

[0544]

[0545] Referring to the synthesis method of Example 81, compound 86 was synthesized using compound 51 as the starting material. LC-MS m / z (ES + ):[M / 2+H] + :805.5。

[0546] Example 94 Synthesis of compound LP-34a

[0547]

[0548] Referring to the synthesis method of Example 9, compound LP-34a was synthesized using compound 86 as the starting material. LC-MS m / z (ES + ):[M / 2+H] + :699.4。

[0549] Example 95 Synthesis of compound 87

[0550]

[0551] Referring to the synthesis method of Example 81, compound 87 was synthesized using compound 58 as the starting material. LC-MS m / z (ES + ):[M / 2+H] + :833.5。

[0552] Example 96 Synthesis of compound LP-35a

[0553]

[0554] Referring to the synthesis method of Example 9, compound LP-35a was synthesized using compound 87 as the starting material. LC-MS m / z (ES + ):[M / 2+H] + :677.4。

[0555] Example 97 Synthesis of compound 88

[0556]

[0557] Referring to the synthesis method of Example 81, compound 88 was synthesized using compound 63 as the starting material. LC-MS m / z (ES+ ): [M / 2 + H] + : 790.5.

[0558] Synthesis of Compound LP-36a in Example 98

[0559]

[0560] Referring to the synthesis method of Example 9, compound LP-36a was synthesized using compound 88 as the raw material. LC-MS m / z (ES + ) : [M / 2 + H] + : 670.4.

[0561] Synthesis of Compound 89 in Example 99

[0562]

[0563] Referring to the synthesis method of Example 49, compound 89 was synthesized using compound 16 as the raw material. LC-MS m / z (ES + ) : [M / 2 + H] + : 707.4.

[0564] Synthesis of Compound LP-37 in Example 100

[0565]

[0566] Referring to the synthesis method of Example 9, compound LP-37 was synthesized using compound 89 as the raw material. LC-MS m / z (ES + ) : [M + H] + : 1313.8.

[0567] Synthesis of Compound 90 in Example 101

[0568]

[0569] Referring to the synthesis method of Example 63, compound 90 was synthesized using compound 16 as the raw material. LC-MS m / z (ES + ) : [M / 2 + H] + : 858.0.

[0570] Synthesis of Compound LP-38a in Example 102

[0571]

[0572] Referring to the synthesis method of Example 9, compound LP-38a was synthesized using compound 90 as the raw material. LC-MS m / z (ES + ) : [M / 2 + H] + : 701.9.

[0573] Synthesis of Compound 91 in Example 103

[0574]

[0575] Referring to the synthesis method of Reference Example 81, Compound 91 was synthesized using Compound 16 as a raw material. LC-MS m / z (ES + ): [M / 2 + H] + : 855.0.

[0576] Synthesis of Compound LP-39a in Example 104

[0577]

[0578] Referring to the synthesis method of Reference Example 9, Compound LP-39a was synthesized using Compound 91 as a raw material. LC-MS m / z (ES + ): [M / 2 + H] + : 698.9.

[0579] Expression and Purification of TA001 Antibody in Example 105:

[0580] Expand the culture of Expi293 suspension cells (Shanghai Opumax Biotech Co., Ltd.). One day before transfection, inoculate the cells at a certain density into OPM-293CD05 Medium (Shanghai Opumax Biotech Co., Ltd.) and culture overnight in a cell culture shaker at 37°C, 5% CO2, and 120 rpm. The next day, transfect the antibody expression plasmid with PEI-MAX. Feed supplements of OPM-293ProFeed (Shanghai Opumax Biotech Co., Ltd.) were added on the first and third days after transfection, and the supernatant was collected by centrifugation on the sixth day after transfection.

[0581] The supernatant was preliminarily purified using a Protein A affinity chromatography column and then finely purified by SEC or CHT to remove impurities such as multimers.

[0582] Antibody TA001:

[0583] Amino acid sequence of the light chain (SEQ ID NO: 9)

[0584] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0585] Among them, the amino acid sequence of the light chain variable region is:

[0586] SEQ ID NO: 7

[0587] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKL LIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT FGQGTKVEIK;

[0588] Heavy chain amino acid sequence (SEQ ID NO: 12)

[0589] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG;

[0590] Among them, the amino acid sequence of the heavy chain variable region is:

[0591] SEQ ID NO: 10

[0592] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY YCSRWGGDGFYAMDYWGQGTLVTVSS。

[0593] The amino acid sequences of CDR-L1, CDR-L2, CDR-L3 and the light chain constant region of TA001, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3 and the heavy chain constant region, and the nucleotide sequences corresponding to each amino acid can be found in the table above.

[0594] Synthesis of Compound 92 in Example 106

[0595]

[0596] Compound 6 (2.28 g, 2.79 mmol, 1.0 eq) was successively added to a 100 mL single-necked flask, followed by ki-4 (2.13 g, 5.57 mmol, 2.0 eq, the synthesis method was referred to the synthesis of compound 4b in WO2020146541), zinc acetate (1.02 g, 5.57 mmol, 2.0 eq). The mixture was dissolved in toluene (30 mL). After three N2 replacements, the reaction was carried out at 115 °C for 4 h under N2 protection. The reaction was stopped, cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The product was purified by reverse-phase preparative column, and the preparative solution was freeze-dried to obtain a white solid product (1.2 g, 37.8%). LC-MS m / z (ES + ) : [M+H] + : 1140.5.

[0597] Synthesis of Compound 93 in Example 107

[0598]

[0599] Compound 92 (1.0 g, 0.877 mmol, 1.0 eq) and DMF (10 mL) were successively added to a 50 mL single-necked round-bottom flask. After stirring and dissolving at room temperature, DBU (144.1 μL, 0.985 mmol, 1.1 eq) was added, and the reaction was carried out at room temperature for 0.5 h. Monitored by TLC, the raw materials disappeared, and compound 93 was produced. The reaction solution was directly purified by reverse-phase preparation, and the preparative solution was freeze-dried to obtain a white solid product (661.0 mg, 82.1%). LC-MS m / z (ES + ) : [M+H] + : 918.6.

[0600] Synthesis of Compound 94 in Example 108

[0601]

[0602] Compound 93 (0.53 g, 0.581 mmol, 1.0 eq) and Fmoc-L-valine (197.2 mg, 0.581 mmol, 1.0 eq) were successively added to a 25 mL single-necked round-bottom flask, dissolved in DMF (5 mL), and then HATU (264 mg, 0.697 mmol, 1.2 eq), HOBt (94 mg, 0.697 mmol, 1.2 eq), and DIEA (288 μL, 1.743 mmol, 3.0 eq) were successively added under ice bath. After addition, the mixture was stirred at room temperature for 1 h and monitored by HPLC. After the reaction was completed, the product was purified by a reverse-phase preparative column to obtain a white solid (540.2 mg, 75.0%). LC-MS m / z (ES + ):[M+H] + : 1239.8.

[0603] Synthesis of Compound 96 in Example 109

[0604]

[0605] KI-2 (115.9 mg, 0.291 mmol, 1.0 eq), HATU (132 mg, 0.349 mmol, 1.2 eq), HOBt (47 mg, 0.349 mmol, 1.2 eq), and DMF (2 mL) were successively added to a 10 mL EP tube and stirred at room temperature for standby.

[0606] In another 10 mL single-necked round-bottom flask, compound 94 (360.1 mg, 0.291 mmol, 1.0 eq) and DMF (2 mL) were successively added, stirred and dissolved at room temperature, then DBU (47.8 μL, 0.32 mmol, 1.1 eq) was added, and the reaction was carried out at room temperature for 0.5 h. The reaction was monitored by HPLC. When the raw materials disappeared, compound 95 was produced. Then the above mixture was added to this flask, and DIEA (48 μL, 0.291 mmol, 1.0 eq) was added. The reaction was carried out at room temperature for 1 h and monitored by HPLC. After the reaction was completed, the product was purified by a reverse-phase preparative column to obtain a white solid (308.6 mg, 76%). LC-MS m / z (ES + ):[M+2H] 2+ :699.4.

[0607] Synthesis of Compound LP-51a in Example 110

[0608]

[0609] Compound 96 (200 mg, 0.143 mmol, 1.0 eq) was dissolved in 10 mL of dry dichloromethane and 4 mL of TFA, and the reaction was carried out at room temperature for 3 h, monitored by HPLC. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The crude product was purified by reverse-phase preparative column, and the preparation solution was freeze-dried to obtain a white solid (127.4 mg, 78%). LC-MS m / z (ES + ) : [M + 2H] 2+ : 571.3.

[0610] Synthesis of Compound 97 in Example 111

[0611]

[0612] Auristatin E (2.04 g, 2.79 mmol, 1.0 eq), ki-4 (2.13 g, 5.57 mmol, 2.0 eq, the synthesis method refers to the synthesis of compound 4b in WO2020146541), and zinc acetate (1.02 g, 5.57 mmol, 2.0 eq) were successively added to a 100 mL single-necked flask, dissolved in toluene (30 mL). After replacing with N2 three times, the reaction was carried out at 115 °C for 4 h under N2 protection. The reaction was stopped, cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure, purified by reverse-phase preparative column, and the preparation solution was freeze-dried to obtain a white solid product (1.3 g, 44.2%). LC-MS m / z (ES + ) : [M + H] + : 1053.7.

[0613] Synthesis of Compound 98 in Example 112

[0614]

[0615] Compound 97 (924.7 mg, 0.877 mmol, 1.0 eq) and DMF (9 mL) were successively added to a 50 mL single-necked round-bottom flask. After stirring and dissolving at room temperature, DBU (144.1 μL, 0.985 mmol, 1.1 eq) was added, and the reaction was carried out at room temperature for 0.5 h, monitored by TLC. When the raw materials disappeared, compound 98 was produced. The reaction solution was directly purified by reverse-phase preparation, and the preparation solution was freeze-dried to obtain a white solid product (622.5 mg, 85.3%). LC-MS m / z (ES + ) : [M + H] + : 832.6.

[0616] Synthesis of Compound 99 in Example 113

[0617]

[0618] To a 25 mL single-necked round-bottom flask were successively added compound 93 (483.5 mg, 0.581 mmol, 1.0 eq) and Fmoc-L-valine (197.2 mg, 0.581 mmol, 1.0 eq). DMF (5 mL) was added to dissolve them. Then, under ice bath, HATU (264 mg, 0.697 mmol, 1.2 eq), HOBt (94 mg, 0.697 mmol, 1.2 eq), and DIEA (288 μL, 1.743 mmol, 3.0 eq) were successively added. After addition, the mixture was warmed to room temperature and stirred for 1 h, and monitored by HPLC. After the reaction was completed, the product was purified by a reversed-phase preparative column to obtain a white solid (517.4 mg, 77.2%). LC-MS m / z (ES + ):[M+H] + : 1153.7.

[0619] Synthesis of Compound 101 in Example 114

[0620]

[0621] To a 10 mL EP tube were successively added ki-2 (115.9 mg, 0.291 mmol, 1.0 eq), HATU (132 mg, 0.349 mmol, 1.2 eq), HOBt (47 mg, 0.349 mmol, 1.2 eq), and DMF (2 mL). The mixture was stirred at room temperature for standby.

[0622] In another 10 mL single-necked round-bottom flask, compound 99 (335.1 mg, 0.291 mmol, 1.0 eq) and DMF (2 mL) were successively added. After stirring and dissolving at room temperature, DBU (47.8 μL, 0.32 mmol, 1.1 eq) was added, and the reaction was carried out at room temperature for 0.5 h. The reaction was monitored by HPLC. When the raw materials disappeared and compound 100 was produced, the above mixture was added to this flask, and then DIEA (48 μL, 0.291 mmol, 1.0 eq) was added. The reaction was carried out at room temperature for 1 h and monitored by HPLC. After the reaction was completed, the product was purified by a reversed-phase preparative column to obtain a white solid (299.1 mg, 78.5%). LC-MS m / z (ES + ):[M+2H] 2+ :656.4.

[0623] Synthesis of Compound LP-52a in Example 115

[0624]

[0625] Dissolve compound 101 (200 mg, 0.152 mmol, 1.0 eq) in 10 mL of dry dichloromethane and 4 mL of TFA, react at room temperature for 3 h, and monitor by HPLC. After the reaction is completed, concentrate under reduced pressure to remove the solvent. The crude product is purified by a reverse-phase preparative column, and the preparation solution is freeze-dried to obtain a white solid (122.3 mg, 69.4%), LC-MS m / z (ES + ):[M+2H] 2+ :578.3。

[0626] Example 116 Synthesis of Compound 102

[0627]

[0628] To a solution of Fmoc-L-citrulline (5 g, 12.58 mmol, 1 eq) and tert-butyl glycinate (2.48 g, 18.87 mmol, 1.5 eq) in DCM (10 mL), add compound DIEA (4.88 g, 37.74 mmol, 3 eq) and HATU (5.74 g, 15.09 mmol, 1.2 eq), and stir the reaction solution at room temperature overnight. LCMS shows that the reaction is complete. Add dichloromethane / methanol (10:1, 150 mL) and water (50 mL) to the reaction solution. The mixture is washed twice with dilute hydrochloric acid (0.5 M), 50 mL each time, washed twice with saturated brine, 50 mL each time, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (eluent: methanol / dichloromethane 0:1 to 1:9) to obtain a white solid product (5.7 g, 88.73%). LC-MS m / z (ES + ):[M+H] + :511.3。

[0629] Example 117 Synthesis of Compound 103

[0630]

[0631] Add TFA (2 mL) to a solution of compound 102 (1.60 g, 3.13 mmol, 1 eq) in DCM (10 mL) at 0 °C, and stir the reaction at room temperature overnight. LCMS shows that the reaction is complete. Concentrate the reaction solution under reduced pressure, and purify the residue by silica gel column chromatography to obtain a white solid product (1.82 g, 63.83%). LC-MS m / z (ES + ):[M+H] + :455.2。

[0632] Example 118 Synthesis of Compound 104

[0633]

[0634] To a solution of Compound 103 (1.01 g, 2.22 mmol, 1 eq) in DMF (20 mL) were added acetic acid (400.35 mg, 382.02 μL, 6.66 mmol, 3 eq), copper(II) acetate (121.09 mg, 113.38 μL, 0.67 mmol, 0.3 eq) and lead(II) acetate (1.97 g, 0.89 mL, 4.45 mmol, 2 eq). The reaction was stirred at 60 °C for 1 hour. LCMS indicated the completion of the reaction. The reaction mixture was concentrated under reduced pressure. Saturated NaHCO3 solution was added to the residue to adjust the pH to 7. DCM / MeOH (10:1, 1 L) was added, and the mixture was extracted three times with DCM / MeOH (10:1), 500 mL each time. The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: methanol / dichloromethane 0:1 to 10:1) to obtain a white solid product (256.5 mg, 24.63%). LC-MS m / z (ES + ):[M+Na] + : 491.3。

[0635] Example 119 Synthesis of Compound 105

[0636]

[0637] Compound 6 (2.12 g, 2.59 mmol, 1.0 eq), Compound 104 (2.77 g, 5.18 mmol, 2.0 eq), and zinc acetate (1.08 g, 5.18 mmol, 2.0 eq) were successively added to a 100 mL single-necked flask and dissolved in toluene (30 mL). After displacing the air with N2 three times, the reaction was carried out at 115 °C for 4 h under N2 protection. The reaction was stopped, cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. It was purified by reverse-phase preparative column and the preparative solution was freeze-dried to obtain a white solid product (1.35 g, 42.4%). LC-MS m / z (ES + ):[M+H] + : 1226.7。

[0638] Example 120 Synthesis of Compound 106

[0639]

[0640] To a 50 mL single-necked round-bottom flask were successively added compound 105 (1.02 g, 0.832 mmol, 1.0 eq) and DMF (9 mL). After stirring and dissolving at room temperature, DBU (136.7 μL, 0.915 mmol, 1.1 eq) was added, and the reaction was carried out at room temperature for 0.5 h. Monitored by TLC, the raw material disappeared and compound 98 was produced. The reaction solution was directly purified by reverse-phase preparation. The preparation solution was freeze-dried to obtain a white solid product (726.6 mg, 87%). LC-MS m / z (ES + ):[M+H] + :1004.6。

[0641] Synthesis of Compound 107 in Example 121

[0642]

[0643] To a 25 mL single-necked round-bottom flask were successively added compound 106 (496.5 mg, 0.494 mmol, 1.0 eq) and Fmoc-L-valine (186.3 mg, 0.494 mmol, 1.0 eq). DMF (5 mL) was added for dissolution, and then successively added HATU (225.6 mg, 0.593 mmol, 1.2 eq), HOBt (80 mg, 0.593 mmol, 1.2 eq), and DIEA (191.6 μL, 1.483 mmol, 3.0 eq) under ice bath. After adding, the mixture was warmed to room temperature and stirred for 1 h, and monitored by HPLC. After the reaction was completed, it was purified by a reverse-phase preparation column to obtain a white solid product (496.8 mg, 75.8%). LC-MS m / z (ES + ):[M+H] + :1325.8。

[0644] Synthesis of Compound 109 in Example 122

[0645]

[0646] To a 10 mL EP tube were successively added ki-2 (115.9 mg, 0.291 mmol, 1.0 eq), HATU (132 mg, 0.349 mmol, 1.2 eq), HOBt (47 mg, 0.349 mmol, 1.2 eq), and DMF (2 mL), and stirred at room temperature for standby.

[0647] In another 10 mL single-necked round-bottom flask, compound 107 (385.8 mg, 0.291 mmol, 1.0 eq) and DMF (2 mL) were successively added. After stirring and dissolving at room temperature, DBU (47.8 μL, 0.32 mmol, 1.1 eq) was added, and the reaction was carried out at room temperature for 0.5 h. The reaction was monitored by HPLC. When the raw materials disappeared, compound 108 was produced. Then the above-mentioned mixed solution was added to this flask, and DIEA (48 μL, 0.291 mmol, 1.0 eq) was added. The reaction was carried out at room temperature for 1 h and monitored by HPLC. After the reaction was completed, the product was purified by a reverse-phase preparative column to obtain a white solid product (331.6 mg, 76.8%). LC-MS m / z (ES + ):[M+2H] 2+ :742.4。

[0648] Synthesis of Compound LP-53a in Example 123

[0649]

[0650] Compound 109 (200 mg, 0.135 mmol, 1.0 eq) was dissolved in 10 mL of dry dichloromethane and 4 mL of TFA, and the reaction was carried out at room temperature for 3 h. The reaction was monitored by HPLC. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The crude product was purified by a reverse-phase preparative column, and the preparation solution was freeze-dried to obtain a white solid (107.9 mg, 65.2%). LC-MS m / z (ES + ):[M+2H] 2+ :614.3。

[0651] Synthesis of Compound 110 in Example 124

[0652]

[0653] Auristatin E (2.3 g, 3.14 mmol, 1.0 eq), compound 104 (2.94 g, 6.28 mmol, 2.0 eq), zinc acetate (1.15 g, 6.28 mmol, 2.0 eq) were successively added to a 100 mL single-necked flask and dissolved in toluene (30 mL). After displacing with N2 three times, the reaction was carried out at 115 °C for 4 h under N2 protection. The reaction was stopped, cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure, and purified by a reverse-phase preparative column. The preparation solution was freeze-dried to obtain a white solid product (1.5 g, 43.2%). LC-MS m / z (ES + ):[M+H] + :1140.7。

[0654] Synthesis of Compound 111 in Example 125

[0655]

[0656] In a 50 mL single-necked round-bottom flask, 110 (918.7 mg, 0.805 mmol, 1.0 eq) and DMF (9 mL) were successively added. After stirring and dissolving at room temperature, DBU (132.4 μL, 0.886 mmol, 1.1 eq) was added, and the reaction was carried out at room temperature for 0.5 h. Monitored by TLC, the raw materials disappeared, and Compound 98 was produced. The reaction solution was directly purified by reverse-phase preparation, and the preparation solution was freeze-dried to obtain a white solid product (623.5 mg, 84.3%). LC-MS m / z (ES + ):[M+H] + :918.6。

[0657] Synthesis of Compound 112 in Example 126

[0658]

[0659] In a 25 mL single-necked round-bottom flask, 111 (481.3 mg, 0.524 mmol, 1.0 eq) and Fmoc-L-valine (177.9 mg, 0.524 mmol, 1.0 eq) were successively added, and DMF (5 mL) was added to dissolve. Then, under ice bath, HATU (239 mg, 0.629 mmol, 1.2 eq), HOBt (85 mg, 0.629 mmol, 1.2 eq), and DIEA (203 μL, 1.572 mmol, 3.0 eq) were successively added. After adding, the temperature was raised to room temperature and stirred for 1 h, and then monitored by HPLC. After the reaction was completed, the product was purified by a reverse-phase preparation column to obtain a white solid product (495.7 mg, 76.3%). LC-MS m / z (ES + ):[M+H] + :1239.7。

[0660] Synthesis of Compound 114 in Example 127

[0661]

[0662] In a 10 mL EP tube, ki-2 (115.9 mg, 0.291 mmol, 1.0 eq), HATU (132 mg, 0.349 mmol, 1.2 eq), HOBt (47 mg, 0.349 mmol, 1.2 eq), and DMF (2 mL) were successively added, and stirred at room temperature for standby.

[0663] In another 10 mL single-necked round-bottom flask, compound 112 (360.7 mg, 0.291 mmol, 1.0 eq) and DMF (2 mL) were successively added. After stirring and dissolving at room temperature, DBU (47.8 μL, 0.32 mmol, 1.1 eq) was added, and the reaction was carried out at room temperature for 0.5 h. The reaction was monitored by HPLC. The raw material disappeared, and compound 113 was produced. Then the above mixture was added to this flask, and DIEA (48 μL, 0.291 mmol, 1.0 eq) was added. The reaction was carried out at room temperature for 1 h and monitored by HPLC. After the reaction was completed, the product was purified by a reverse-phase preparative column to obtain a white solid (314 mg, 77.2%). LC-MS m / z (ES + ):[M + 2H] 2+ :699.4。

[0664] Example 128 Synthesis of Compound LP-54a

[0665]

[0666] Compound 114 (200 mg, 0.143 mmol, 1.0 eq) was dissolved in 10 mL of dry dichloromethane and 4 mL of TFA, and the reaction was carried out at room temperature for 3 h. The reaction was monitored by HPLC. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The crude product was purified by a reverse-phase preparative column, and the preparation was freeze-dried to obtain a white solid (120.6 mg, 67.9%). LC-MS m / z (ES + ):[M + 2H] 2+ :621.3。

[0667] Example 129 Preparation of Antibody-Drug Conjugate ADC-1-1a

[0668] The preparation of ADC-1-1a was carried out by reacting the corresponding linker-payload (LP-1a, the structure of which is shown in the structural formula of LP-1, and the configuration of the chiral carbon at the 2-position is the S configuration) with the antibody TA001 according to the method of General Procedure A. The sequence information of TA001 is shown in Example 105. The RP-HPLC detection results of ADC-1-1a are shown in Figure 3A , and the SEC-HPLC detection results are shown in Figure 4A .

[0669]

[0670] Example 130 Preparation of Antibody-Drug Conjugate ADC-2-1a

[0671] The preparation of ADC-2-1a was obtained by reacting the corresponding linker-payload (LP-2a, whose structure is shown in the structural formula of LP-2, where the configuration of the chiral carbon at the 2-position is of the S type) with antibody TA001 according to the method of General Procedure A. The sequence information of TA001 is shown in Example 105.

[0672]

[0673] Preparation of antibody-drug conjugate ADC-C-1a in Comparative Example 1

[0674] The preparation of ADC-C-1a was obtained by reacting compound VcMMAE with antibody TA001 according to the method of General Procedure A. The RP-HPLC detection results of ADC-C-1a are shown in Figure 3B , and the SEC-HPLC detection results are shown in Figure 4B .

[0675]

[0676] Preparation of antibody-drug conjugate ADC-3-1a in Example 131

[0677] The preparation of ADC-3-1a was obtained by reacting the corresponding linker-payload (LP-10) with antibody TA001 according to the method of General Procedure A.

[0678]

[0679] Preparation of antibody-drug conjugate ADC-4-1a in Example 132

[0680] The preparation of ADC-4-1a was obtained by reacting the corresponding linker-payload (LP-37) with antibody TA001 according to the method of General Procedure A.

[0681]

[0682] Preparation of antibody-drug conjugate ADC-5-1a in Example 133

[0683] The preparation of ADC-5-1a was obtained by reacting the corresponding linker-payload (LP-16) with antibody TA001 according to the method of General Procedure A.

[0684]

[0685] Preparation of antibody-drug conjugate ADC-6-1a in Example 134

[0686] The preparation of ADC-6-1a was obtained by reacting the corresponding linker-payload (LP-11) with antibody TA001 according to the general procedure A.

[0687]

[0688] Example 135 Preparation of Antibody-Drug Conjugate ADC-7-1a

[0689] The preparation of ADC-7-1a was obtained by reacting the corresponding linker-payload (LP-12) with antibody TA001 according to the general procedure A.

[0690]

[0691] Example 136 Preparation of Antibody-Drug Conjugate ADC-8-1a

[0692] The preparation of ADC-8-1a was obtained by reacting the corresponding linker-payload (LP-13) with antibody TA001 according to the general procedure A.

[0693]

[0694] Example 137 Preparation of Antibody-Drug Conjugate ADC-9-1a

[0695] The preparation of ADC-9-1a was obtained by reacting the corresponding linker-payload (LP-14) with antibody TA001 according to the general procedure A.

[0696]

[0697] Example 138 Preparation of Antibody-Drug Conjugate ADC-10-1a

[0698] The preparation of ADC-10-1a was obtained by reacting the corresponding linker-payload (LP-15) with antibody TA001 according to the general procedure A.

[0699]

[0700] Example 139 Preparation of Antibody-Drug Conjugate ADC-11-1a

[0701] The preparation of ADC-11-1a was obtained by reacting the corresponding linker-payload (LP-17) with antibody TA001 according to the general procedure A.

[0702]

[0703] Preparation of Antibody-Drug Conjugate ADC-12-1a in Example 140

[0704] The ADC-12-1a was prepared by reacting the corresponding linker-payload (LP-18) with antibody TA001 according to the method of General Procedure A.

[0705]

[0706] Preparation of Antibody-Drug Conjugate ADC-13-1a in Example 141

[0707] The ADC-13-1a was prepared by reacting the corresponding linker-payload (LP-3a, whose structure is shown in the structural formula of LP-3, and the configuration of the chiral carbon at the 2-position is S) with antibody TA001 according to the method of General Procedure A.

[0708]

[0709] Preparation of Antibody-Drug Conjugate ADC-14-1a in Example 142

[0710] The ADC-14-1a was prepared by reacting the corresponding linker-payload (LP-7a, whose structure is shown in the structural formula of LP-7, and the configuration of the chiral carbon at the 2-position is S) with antibody TA001 according to the method of General Procedure A.

[0711]

[0712] Preparation of Antibody-Drug Conjugate ADC-15-1a in Example 143

[0713] The ADC-15-1a was prepared by reacting the corresponding linker-payload (LP-3sa, whose structure is shown in the structural formula of LP-3s, and the configuration of the chiral carbon at the 2-position is S) with antibody TA001 according to the method of General Procedure A.

[0714]

[0715] Preparation of Antibody-Drug Conjugate ADC-16-1a in Example 144

[0716] The ADC-16-1a was prepared by reacting the corresponding linker-payload (LP-4a, whose structure is shown in the structural formula of LP-4, and the configuration of the chiral carbon at the 2-position is S) with antibody TA001 according to the method of General Procedure A.

[0717]

[0718] Preparation of Antibody-Drug Conjugate ADC-17-1a in Example 145

[0719] The preparation of ADC-17-1a was obtained by reacting the corresponding linker-payload (LP-5a, whose structure is shown in the structural formula of LP-5, where the configuration of the chiral carbon at the 2-position is of the S type) with antibody TA001 according to the method of General Procedure A.

[0720]

[0721] Preparation of Antibody-Drug Conjugate ADC-18-1a in Example 146

[0722] The preparation of ADC-18-1a was obtained by reacting the corresponding linker-payload (LP-6a, whose structure is shown in the structural formula of LP-6, where the configuration of the chiral carbon at the 2-position is of the S type) with antibody TA001 according to the method of General Procedure A.

[0723]

[0724] Preparation of Antibody-Drug Conjugate ADC-19-1a in Example 147

[0725] The preparation of ADC-19-1a was obtained by reacting the corresponding linker-payload (LP-8a, whose structure is shown in the structural formula of LP-8, where the configuration of the chiral carbon at the 2-position is of the S type) with antibody TA001 according to the method of General Procedure A.

[0726]

[0727] Preparation of Antibody-Drug Conjugate ADC-20-1a in Example 148

[0728] The preparation of ADC-20-1a was obtained by reacting the corresponding linker-payload (LP-9a, whose structure is shown in the structural formula of LP-9, where the configuration of the chiral carbon at the 2-position is of the S type) with antibody TA001 according to the method of General Procedure A.

[0729]

[0730] Preparation of Antibody-Drug Conjugate ADC-21-1a in Example 149

[0731] The preparation of ADC-21-1a was obtained by reacting the corresponding linker-payload (LP-28a, whose structure is shown in the structural formula of LP-28, where the configuration of the chiral carbon at the 2-position is of the S type) with antibody TA001 according to the method of General Procedure A.

[0732]

[0733] Preparation of Antibody-Drug Conjugate ADC-22-1a in Example 150

[0734] The preparation of ADC-22-1a was obtained by reacting the corresponding linker-payload (LP-29a, whose structure is shown in the structural formula of LP-29, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0735]

[0736] Preparation of Antibody-Drug Conjugate ADC-23-1a in Example 151

[0737] The preparation of ADC-23-1a was obtained by reacting the corresponding linker-payload (LP-30a, whose structure is shown in the structural formula of LP-30, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0738]

[0739] Preparation of Antibody-Drug Conjugate ADC-24-1a in Example 152

[0740] The preparation of ADC-24-1a was obtained by reacting the corresponding linker-payload (LP-31a, whose structure is shown in the structural formula of LP-31, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0741]

[0742] Preparation of Antibody-Drug Conjugate ADC-25-1a in Example 153

[0743] The preparation of ADC-25-1a was obtained by reacting the corresponding linker-payload (LP-32a, whose structure is shown in the structural formula of LP-32, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0744]

[0745] Preparation of Antibody-Drug Conjugate ADC-26-1a in Example 154

[0746] The preparation of ADC-26-1a was obtained by reacting the corresponding linker-payload (LP-33a, whose structure is shown in the structural formula of LP-33, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0747]

[0748] Example 155 Preparation of Antibody-Drug Conjugate ADC-27-1a

[0749] The preparation of ADC-27-1a was obtained by reacting the corresponding linker-payload (LP-35a, whose structure is shown in the structural formula of LP-35, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0750]

[0751] Example 156 Preparation of Antibody-Drug Conjugate ADC-28-1a

[0752] The preparation of ADC-28-1a was obtained by reacting the corresponding linker-payload (LP-36a, whose structure is shown in the structural formula of LP-36, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0753]

[0754] Example 157 Preparation of Antibody-Drug Conjugate ADC-29-1a

[0755] The preparation of ADC-29-1a was obtained by reacting the corresponding linker-payload (LP-39a, whose structure is shown in the structural formula of LP-39, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0756]

[0757] Example 158 Preparation of Antibody-Drug Conjugate ADC-30-1a

[0758] The preparation of ADC-30-1a was obtained by reacting the corresponding linker-payload (LP-34a, whose structure is shown in the structural formula of LP-34, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0759]

[0760] Preparation of Antibody-Drug Conjugate ADC-31-1a in Example 159

[0761] The preparation of ADC-31-1a was obtained by reacting the corresponding linker-payload (LP-19a, whose structure is shown in the structural formula of LP-19, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0762]

[0763] Preparation of Antibody-Drug Conjugate ADC-35-1a in Example 160

[0764] The preparation of ADC-35-1a was obtained by reacting the corresponding linker-payload (LP-22a, whose structure is shown in the structural formula of LP-22, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0765]

[0766] Preparation of Antibody-Drug Conjugate ADC-39-1a in Example 161

[0767] The preparation of ADC-39-1a was obtained by reacting the corresponding linker-payload (LP-26a, whose structure is shown in the structural formula of LP-26, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0768]

[0769] Preparation of Antibody-Drug Conjugate ADC-45-1a in Example 162

[0770] The preparation of ADC-45-1a was obtained by reacting the corresponding linker-payload (LP-20a, whose structure is shown in the structural formula of LP-20, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0771]

[0772] Preparation of Antibody-Drug Conjugate ADC-48-1a in Example 163

[0773] The preparation of ADC-48-1a was obtained by reacting the corresponding linker-payload (LP-27a, whose structure is shown in the structural formula of LP-27, where the configuration of the chiral carbon at the 2-position is the S configuration) with antibody TA001 according to the method of General Procedure A.

[0774]

[0775] Preparation of Antibody-Drug Conjugate ADC-51-1a in Example 164

[0776] The ADC-51-1a was prepared by reacting the corresponding linker-payload (LP-51a) with the antibody TA001 according to the general procedure A.

[0777]

[0778] Preparation of Antibody-Drug Conjugate ADC-52-1a in Example 165

[0779] The ADC-52-1a was prepared by reacting the corresponding linker-payload (LP-52a) with the antibody TA001 according to the general procedure A.

[0780]

[0781] Preparation of Antibody-Drug Conjugate ADC-53-1a in Example 166

[0782] The ADC-53-1a was prepared by reacting the corresponding linker-payload (LP-53a) with the antibody TA001 according to the general procedure A.

[0783]

[0784] Preparation of Antibody-Drug Conjugate ADC-54-1a in Example 167

[0785] The ADC-54-1a was prepared by reacting the corresponding linker-payload (LP-54a) with the antibody TA001 according to the general procedure A.

[0786]

[0787] Preparation of Antibody-Drug Conjugate FITC-IgG1-LP-1a in Comparative Example 2

[0788] The FITC-IgG1-LP-1a was prepared by reacting the corresponding linker-payload (LP-1a, the structure of which is shown in the structural formula of LP-1, where the configuration of the chiral carbon at position 2 is the S configuration) with the irrelevant antibody FITC-IgG1 (an isotype IgG antibody that does not target any antigen) according to the general procedure A. The RP-HPLC detection results of FITC-IgG1-LP-1a are shown in Figure 3C .

[0789]

[0790] The light chain amino acid sequence of the irrelevant antibody FITC-IgG1 (SEQ ID NO: 25) is as follows:

[0791] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,

[0792] The light chain variable region (SEQ ID NO: 26) is as follows:

[0793] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

[0794] The heavy chain amino acid sequence (SEQ ID NO: 27) is as follows:

[0795] EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,

[0796] The heavy chain variable region (SEQ ID NO: 28) is shown as follows:

[0797] EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKG LEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVED MGIYYCTGSYYGMDYWGQGTSVTVSS。

[0798] Preparation of the antibody-drug conjugate ADC-Dxd of Comparative Example 3

[0799] The ADC-Dxd was prepared by reacting the corresponding linker-payload (LP-55, synthesized according to the method described in Example 58 of the patent application "CN104755494A") with the antibody TA001 according to the method of General Procedure A. The RP-HPLC detection results of ADC-Dxd are shown in Figure 3D .

[0800]

[0801]

[0802] Preparation of the antibody-drug conjugate ADC-1-1b of Comparative Example 4

[0803] The preparation of ADC-1-1b was obtained by reacting the corresponding linker-payload (LP-1a, whose structure is shown in the structural formula of LP-1, where the configuration of the chiral carbon at position 2 is of the S type) with the Anti-HER2 antibody Disitamab according to the method of General Procedure A. The RP-HPLC detection results of ADC-1-1b are shown in Figure 3E , and the SEC-HPLC detection results are shown in Figure 4D .

[0804]

[0805] The light chain amino acid sequence (SEQ ID NO: 29) of the antibody Disitamab is shown as follows:

[0806] DIQMTQSPSSVSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASIRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQFATYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,

[0807] The light chain variable region (SEQ ID NO: 30) is shown as follows:

[0808] DIQMTQSPSSVSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPK LLIYWASIRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQFATYTF GGGTKVEIK,

[0809] The heavy chain amino acid sequence (SEQ ID NO: 31) is shown as follows:

[0810] EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYIHWVQQAPGKGLEWMGRVNPDHGDSYYNQKFKDKATITADKSTDTAYMELSSLRSEDTAVYFCARNYLFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,

[0811] The heavy chain variable region (SEQ ID NO: 32) is shown as follows:

[0812] EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYIHWVQQAPGKGL EWMGRVNPDHGDSYYNQKFKDKATITADKSTDTAYMELSSLRSEDTAV YFCARNYLFDHWGQGTLVTVSS。

[0813] Preparation of the antibody-drug conjugate ADC-1-1c of Comparative Example 5

[0814] The ADC-1-1c was prepared by reacting the corresponding linker-payload (LP-1a, the structure of which is shown in the structural formula of LP-1, where the configuration of the chiral carbon at position 2 is S) with the Anti-HER2 antibody HT-19 according to the method of General Procedure A. The RP-HPLC detection results of ADC-1-1c are shown in Figure 3F and the SEC-HPLC detection results are shown in Figure 4E 。

[0815]

[0816] The amino acid sequence of the light chain of antibody HT-19 (SEQ ID NO: 33) is shown as follows:

[0817] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHHSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,

[0818] The light chain variable region (SEQ ID NO: 34) is as follows:

[0819] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRL LIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHHSPLT FGGGTKVEIK,

[0820] The heavy chain amino acid sequence (SEQ ID NO: 35) is as follows:

[0821] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGHGYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,

[0822] The heavy chain variable region (SEQ ID NO: 36) is as follows:

[0823] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL EWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CARGGHGYFDLWGRGTLVTVSS。

[0824] Preparation of Comparative Example 6 Antibody-Drug Conjugate ADC-1-1d

[0825] The preparation of ADC-1-1d was obtained by reacting the corresponding linker-payload (LP-1a, whose structure is shown in the structural formula of LP-1, where the configuration of the chiral carbon at position 2 is the S configuration) with the Anti-HER2 antibody XMT-1517 according to the method of General Procedure A. The RP-HPLC detection results of ADC-1-1d are shown in Figure 3G , and the SEC-HPLC detection results are shown in Figure 4F .

[0826]

[0827] The amino acid sequence of the light chain of antibody XMT-1517 (SEQ ID NO: 37) is shown below:

[0828] EIVLTQSPGTLSLSPGERATLSCRASQSVSSDYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYVSYWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,

[0829] The light chain variable region (SEQ ID NO: 38) is shown below:

[0830] EIVLTQSPGTLSLSPGERATLSCRASQSVSSDYLAWYQQKPGQAPR LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYVSYW TFGGGTKVEIK,

[0831] The amino acid sequence of the heavy chain (SEQ ID NO: 39) is shown below:

[0832] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEAPYYAKDYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,

[0833] The heavy chain variable region (SEQ ID NO: 40) is shown as follows:

[0834] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKG LEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAKEAPYYAKDYMDVWGKGTTVTVSS。

[0835] Preparation of the antibody-drug conjugate ADC-1-1e of Comparative Example 7

[0836] The preparation of ADC-1-1e was obtained by reacting the corresponding linker-payload (LP-1a, whose structure is shown in the structural formula of LP-1, where the configuration of the chiral carbon at position 2 is the S configuration) with the Anti-HER2 antibody MHES0488A according to the method of General Procedure A. The RP-HPLC detection results of ADC-1-1e are shown in Figure 3H and the SEC-HPLC detection results are shown in Figure 4G 。

[0837]

[0838] The amino acid sequence of the light chain of antibody MHES0488A (SEQ ID NO: 41) is shown as follows:

[0839] DIVMTQSPDSLAVSLGERATINCRASQSVSGSRFTYMHWYQQKPGQPPKLLIKYASILESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSWEIPPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,

[0840] The light chain variable region (SEQ ID NO: 42) is shown as follows:

[0841] DIVMTQSPDSLAVSLGERATINCRASQSVSGSRFTYMHWYQQKPG QPPKLLIKYASILESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHS WEIPPWTFGQGTKVEIK,

[0842] The heavy chain amino acid sequence (SEQ ID NO: 43) is shown as follows:

[0843] EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYWMNWVRQAPGQGLEWIGMIHPLDAEIRANQKFRDRVTITVDTSTSTAYLELSSLRSEDTAVYYCARGTYDGGFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,

[0844] The heavy chain variable region (SEQ ID NO: 44) is as follows:

[0845] EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYWMNWVRQAPGQ GLEWIGMIHPLDAEIRANQKFRDRVTITVDTSTSTAYLELSSLRSEDTAV YYCARGTYDGGFEYWGQGTLVTVSS。

[0846] Example 168 Detection of the DAR of ADC by Reversed-Phase High Performance Liquid Chromatography (RP-HPLC)

[0847] As determined by reversed-phase high performance liquid chromatography using General Procedure B, the average drug / antibody ratio DAR of the corresponding ADCs is shown in Table 1 below. RC-48 was purchased from Rongchang Biologics (batch number: RC48-X1-202302003, the RP-HPLC test results are shown in Figure 3I , and the SEC-HPLC test results are shown in Figure 4C ). For ADC-C-1a and ADC-Dxd, DAR = n, and for the remaining ADCs other than these, DAR = n1 + n2 + n3. As can be seen from Table 1, the ADCs disclosed in the present invention have the excellent property of a high DAR value, and can significantly increase the drug concentration at the target site at the administration dose of the ADC drug at the same dose.

[0848] Table 1 DAR test results of ADCs

[0849]

[0850] Example 169 SEC detection of the monomer ratio of ADC

[0851] The aggregation degree of each ADC can be obtained by General Procedure C, and the SEC-HPLC results are shown in Figure 4A 、 Figure 4B 、 Figure 4C 、 Figure 4D 、 Figure 4E 、 Figure 4F 、 Figure 4G, the data are summarized in Table 2 below. As can be seen from Table 2, for RC-48 using the classical MC linker VcMMAE, its DAR is 4.19, the monomer ratio is greater than 99%, and the degree of aggregation and degradation is low. While for ADC-C-1a using VcMMAE as the linker-payload, its DAR is 7.63, the monomer ratio is 17.59%, and the degree of aggregation is high, indicating that ADCs using the classical MC linker VcMMAE are prone to aggregation when made into high DAR. In addition, as can be seen from the table, ADCs produced by coupling different HER2 antibodies with LP-1a have different degrees of aggregation. The ADC-1-1a obtained by coupling TA001 with LP-1a has the highest monomer ratio, indicating that TA001 is most suitable as the antibody for ADCs, and its properties are more stable than other antibodies. Using it as the antibody can result in low degrees of aggregation and degradation of the obtained ADCs.

[0852] On the other hand, for ADC-1-1b with an acid-stable linker introduced on the hydroxyl group of the toxin, its DAR is 7.48. Compared with RC-48, its DAR is nearly doubled, but the monomer ratio is similar to that of RC-48, and the degrees of aggregation and degradation are low. Compared with ADC-C-1a, ADC-1-1a has a similar DAR, and the monomer ratio is much greater than that of ADC-C-1a. This shows that high-DAR ADCs prepared by introducing an acid-stable linker on the hydroxyl group of the toxin have the characteristics of low degradation rate and low aggregation rate, and excellent properties of high monomer ratio.

[0853] Table 2 Test results of ADC monomer ratio

[0854]

[0855] Example 170 ADC in vitro plasma stability

[0856] The plasma stability study of ADCs was carried out according to General Procedure D, and the results are shown in Table 3 below. The experimental results show that for ADCs with an acid-stable linker introduced on the hydroxyl group of the toxin, less drug loss occurred during plasma incubation. And ADC-1-1a with TA001 as the antibody is the most stable, while for the classical MC linker ADC (ADC-C-1a), the DAR decreased very significantly after 7 days of incubation. The experimental results prove that the ADC drugs disclosed in the present invention with an acid-stable linker introduced on the hydroxyl group of the toxin have good plasma stability, and no large changes in DAR values occurred.

[0857] Table 3 Test results of ADC in vitro plasma stability

[0858]

[0859] Note: Incubation for 0 days means the DAR value measured after starting the purification and extraction steps immediately after adding the ADC to the plasma; incubation for 3 days means the DAR value measured after starting the purification and extraction steps after placing the ADC in the plasma at 37°C for 3 days; incubation for 7 days means the DAR value measured after starting the purification and extraction steps after placing the ADC in the plasma at 37°C for 7 days.

[0860] Example 171 Antitumor Cell Activity Test of ADC 1

[0861] In this example, human tumor cell lines of different cancer types and a mixed tumor cell line (JIMT-1 + MDA-MB-468) were used as experimental models (the cell lines are shown in Table 4) to evaluate the in vitro tumor cell killing activity of antibody-drug conjugates. A certain number of tumor cells were inoculated into 96-well plates, and gradient-diluted test antibodies or ADC drugs were added to the cells. After treatment for 5 days, the Luminescent Cell Viability Assay was used to detect cell viability, and the killing effects of the test antibodies and ADC drugs on the tumor cell lines were evaluated by calculating the half-maximal inhibitory concentration (IC50).

[0862] The starting dose of the antibody drug or ADC drug was 100 nM, and the dilution factor was 10-fold, with a total of 8 detection concentration points. After treatment for 5 days, the cell survival rate = (experimental group - blank) / (control group - blank group) × 100%.

[0863] Table 4 Information Related to Cell Lines

[0864] Cell line name Cancer type HER2 expression level Source NCI-N87 Human gastric adenocarcinoma High expression Shanghai Cell Bank SK-OV-3 Human ovarian cancer High expression Shanghai Cell Bank Clau-3 Human non-small cell lung cancer Medium-high expression Shanghai Tongpai JIMT-1 Human breast cancer Medium expression Shanghai Tongpai A431 Human cutaneous squamous cell carcinoma Low expression Pricella MDA-MB-468 Human triple-negative breast cancer Negative Shanghai Tongpai

[0865] The in vitro mixed tumor cell model of JIMT-1 + MDA-MB-468 (1:2.5) was constructed according to the following protocol:

[0866] Take the test cells in the logarithmic growth phase, discard the culture medium and rinse the cells with sterile 1×PBS. Add an appropriate amount of 0.25% Trpsin-EDTA to digest the cells according to the size of the culture flask. After the cells are digested, resuspended and counted, first inoculate the breast cancer cell line JIMT-1 with medium HER2 expression at a density of 2000 cells / well into 96-well plates, and then inoculate the triple-negative breast cancer cell line MDA-MB-468 with negative HER2 at a density of 5000 cells / well into 96-well plates. The inoculation quantity ratio of the two types of cells per well is 1:2.5. Finally, add 300 μL of sterile 1×PBS or sterile ddH2O to the 96-well plates without adding cell suspension at the edges for sealing, and place them in a CO2 incubator for overnight culture for later use.

[0867] The corresponding in vitro data are shown in Table 5, and Figure 5A 、 Figure 5B 、 Figure 5C 、Figure 5D , Figure 5E , Figure 5F , Figure 5G . As can be seen from Table 5, in the HER2 high-expression, medium-expression, and low-expression cell models, the tumor cell killing activity of ADC-1-1a obtained by conjugating antibody TA-001 with LP-1a was overall superior to that of ADCs obtained by conjugating other Anti-HER2 antibodies with LP-1a. Moreover, in the mixed tumor cell model (JIMT-1 + MDA-MB-468), ADC-1-1a exhibited a more excellent bystander effect and the strongest tumor cell killing activity, indicating that ADC obtained by conjugating TA001 as an antibody with LP-1a has more advantages in vitro anti-tumor activity compared with ADCs obtained by conjugating other Anti-HER2 antibodies with LP-1a.

[0868] In addition, the tumor cell killing activity of ADC-1-1a was superior to that of ADC-Dxd, which indicates that compared with ADCs with Dxd as the toxin, the ADCs disclosed in this application with auristatin as the toxin have advantages in vitro anti-tumor activity.

[0869] Table 5 Cell killing IC50 (nM) of related ADCs

[0870]

[0871] In vivo pharmacodynamic evaluation of HER2-ADC in single tumor (JIMT-1) in Example 172

[0872] In this example, a subcutaneous xenograft tumor model of HER2+ human breast cancer cell JIMT-1 in BALB / c-nu mice was established to evaluate the in vivo pharmacodynamics of HER2-ADC. JIMT-1 (5×10 6 / mouse) cell suspension (0.1 mL / mouse) was subcutaneously injected into the right scapular region of 6- to 7-week-old BALB / c-nu mice. When the average tumor volume of the mice grew to 166 mm 3Around [time], they were randomly divided into 13 groups, namely the vehicle control group (Vehicle), the treatment groups of ADC-1-1a (0.25 mg / kg, 0.5 mg / kg), the treatment groups of ADC-1-1b (0.25 mg / kg, 0.5 mg / kg), the treatment groups of ADC-1-1c (0.25 mg / kg, 0.5 mg / kg), the treatment groups of ADC-1-1d (0.25 mg / kg, 0.5 mg / kg), the treatment groups of ADC-1-1e (0.25 mg / kg, 0.5 mg / kg), the treatment groups of ADC-Dxd (0.25 mg / kg, 0.5 mg / kg), with 6 mice in each group, and drug administration was started (D0). Each treatment group was administered by tail vein injection at 10 mL / kg body weight. Each 7 days was a dosing cycle (QW), and continuous dosing was carried out for 4 cycles. All groups were observed for 28 days (D28) after grouped drug administration to evaluate the inhibitory effect of the test ADC drug on tumor growth.

[0873] After the dosing cycle ended, statistical analysis was performed on the average tumor volume in each treatment group over 28 days. The results are shown in Table 6 and Figure 6 As shown, in the JIMT-1 xenograft tumors with HER2 positive expression, in the treatment groups of ADC-1-1a at 0.25 mg / kg and 5 mg / kg, compared with the ADC treatment groups obtained by conjugating other Anti-HER2 antibodies with LP-1a at the same dose, the average tumor volume decreased. The in vivo antitumor effect of ADC-1-1a was better than that of other HER2-ADCs. This indicates that as an ADC obtained by conjugating an antibody with LP-1a, TA001 has an advantage in the treatment of xenograft tumors compared with the ADCs obtained by conjugating other HER2-targeted antibodies with LP-1a.

[0874] In addition, the antitumor effect of ADC-1-1a was also stronger than that of ADC-Dxd. This indicates that the ADC disclosed in this application, which uses auristatin as the toxin, has an advantage in the treatment of xenograft tumors compared with the ADC using Dxd as the toxin.

[0875] Table 6 Average tumor volume of each group

[0876]

[0877] Example 173 In vivo pharmacodynamic evaluation of HER2-ADC in mixed tumors (JIMT-1 + MDA-MB-468)

[0878] In this example, a subcutaneous mixed xenograft tumor model of HER2+ human breast cancer cell JIMT-1 and HER2- human breast cancer cell MDA-MB-468 in BALB / c-nu mice was established to evaluate the in vivo pharmacodynamics of HER2-ADC. Using JIMT-1 (1×10 6 ) and MDA-MB-468 (2.5×106 ) The cell mixture (0.1 mL / rat) was subcutaneously injected into the right scapular region of BALB / c-nu mice at 6 - 7 weeks of age. When the average tumor volume of the mice grew to about 172 mm 3 , they were randomly divided into 13 groups, namely the vehicle control group (Vehicle), the ADC-1-1a treatment groups (0.25 mg / kg, 0.5 mg / kg), the ADC-1-1b treatment groups (0.25 mg / kg, 0.5 mg / kg), the ADC-1-1c treatment groups (0.25 mg / kg, 0.5 mg / kg), the ADC-1-1d treatment groups (0.25 mg / kg, 0.5 mg / kg), the ADC-1-1e treatment groups (0.25 mg / kg, 0.5 mg / kg), the ADC-Dxd treatment groups (0.25 mg / kg, 0.5 mg / kg), with 6 mice in each group, and drug administration was started (D0). Each treatment group was administered by tail vein injection at 10 mL / kg body weight. Each 7-day period was a dosing cycle (QW), and continuous dosing was carried out for 4 cycles. All groups were observed for 28 days (D28) after grouped dosing to evaluate the inhibitory effect of the test ADC drugs on tumor growth.

[0879] After the dosing cycle ended, the average tumor volume in each treatment group over 28 days was statistically analyzed. The results are shown in Table 7 and Figure 7 as follows. In the mixed tumors of JIMT-1 with positive HER2 expression and MDA-MB-468 without HER2 expression, the tumor inhibitory effects of the ADC-1-1a treatment groups at 0.25 mg / kg and 5 mg / kg were better than those of the ADC treatment groups obtained by conjugating other Anti-HER2 antibodies with LP-1a at the same dose. This indicates that TA001, as an ADC obtained by conjugating an antibody with LP-1a, has an advantage in treating mixed tumors compared to other ADCs targeting the HER2 target.

[0880] In addition, the tumor inhibitory effect of ADC-1-1a was also stronger than that of ADC-Dxd, which indicates that the ADC disclosed in this application, using auristatin as the toxin, has an advantage in treating mixed tumors compared to the ADC using Dxd as the toxin.

[0881] Table 7 Average tumor volume of each group

[0882]

[0883]

[0884] Example 174 ADC anti-tumor cell activity test 2

[0885] In this example, human tumor cell lines of different cancer types and JIMT-1+MDA-MB-468 (1:1) in vitro mixed tumor cells were used as experimental models (the cell lines are shown in Table 8) to evaluate the in vitro tumor cell killing activity of antibody-drug conjugates. A certain number of tumor cells were inoculated into 96-well plates, and gradient-diluted test antibodies or ADC drugs were added to the cells and treated for 5 days. The Luminescent Cell Viability Assay was used to detect cell viability, and the killing effects of the test antibodies and ADC drugs on the tumor cell lines were evaluated by calculating the half-maximal inhibitory concentration (IC50).

[0886] The starting dose of the antibody drug or ADC drug was 100 nM, the dilution factor was 10-fold, and there were a total of 8 detection concentration points, and the treatment was for 5 days. Cell survival rate = (experimental group - blank) / (control group - blank group) × 100%.

[0887] Table 8 Information related to cell lines

[0888] Cell line name Cancer type HER2 expression level Source NCI-N87 Human gastric adenocarcinoma High expression Shanghai Cell Bank SK-OV-3 Human ovarian cancer High expression Shanghai Cell Bank Clau-3 Human non-small cell lung cancer Medium-high expression Shanghai Tongpai JIMT-1 Human breast cancer Medium expression Shanghai Tongpai NCI-H1975 Human non-small cell lung cancer Low expression ATCC A431 Human cutaneous squamous cell carcinoma Low expression Pricella MDA-MB-468 Human triple-negative breast cancer Negative Shanghai Tongpai

[0889] The in vitro mixed tumor cell model of JIMT-1+MDA-MB-468 (1:1) was constructed according to the following protocol:

[0890] Take the test cells in the logarithmic growth phase, discard the culture medium and rinse the cells with sterile 1×PBS. Add an appropriate amount of 0.25% Trpsin-EDTA to digest the cells according to the size of the culture flask. After the cells are digested, resuspended and counted, first inoculate the breast cancer cell line JIMT-1 with medium HER2 expression at a density of 3500 cells / well into 96-well plates, and then inoculate the triple-negative breast cancer cell line MDA-MB-468 with negative HER2 expression at a density of 3500 cells / well into 96-well plates. The inoculation quantity ratio of the two kinds of cells per well is 1:1. Finally, add 300 μL of sterile 1×PBS or sterile ddH2O to the 96-well plates where the cell suspension was not added to the edge for sealing, and place them in a CO2 incubator for overnight culture for standby.

[0891] The corresponding in vitro data are shown in Table 9, and Figure 8A 、 Figure 8B 、 Figure 8C 、 Figure 8D 、 Figure 8E 、 Figure 8F 、 Figure 8G 、 Figure 8HThe results showed that in HER2 highly expressed and moderately expressed cell models, the tumor cell killing activity of ADC-1-1a was stronger than that of FITC-IgG1-LP-1a. The killing effect of FITC-IgG1-LP-1a was poor and it had no targeting ability, indicating that the killing of tumor cells by ADC was due to antibody-mediated ADC. In HER2 highly expressed, moderately expressed and lowly expressed cell models, the tumor cell killing activity of ADC-1-1a was stronger than that of the marketed drug RC-48. In addition, in the mixed tumor cell model (JIMT-1+MDA-MB-468), compared with RC-48, ADC-1-1a showed a stronger bystander effect and stronger killing activity.

[0892] Table 9 Cell killing IC50 (nM) of related ADCs

[0893]

[0894] Example 175 In vivo pharmacodynamic evaluation of ADC-1-1a in a single tumor (JIMT-1)

[0895] In this example, a human tumor cell line (JIMT-1) was subcutaneously inoculated into BALB / c-Nude as an experimental model to evaluate the in vivo pharmacodynamics of ADC-1-1a. A certain number of tumor cell suspensions were inoculated subcutaneously into BALB / c-Nude. When the tumor volume grew to about 170 mm 3 or so, vehicle, naked antibody control (TA001), irrelevant antibody ADC control (FITC-IgG1-LP-1a), payload control (Auristatin E), positive control (RC-48) and the corresponding ADC drug (ADC-1-1a) were injected via the tail vein once a week for four times. Continuous observation was carried out, and the tumor volume and mouse body weight were measured twice a week to evaluate the inhibitory effect of the tested ADC drug on tumor growth. The results are shown in Table 10, Table 11 and Figure 9 as follows. In the HER2-positive JIMT-1 single tumor, 1 mg / kg of ADC-1-1a showed stronger tumor inhibitory effects compared with 1 mg / kg of naked antibody control (TA001), 1 mg / kg of irrelevant antibody ADC control (FITC-IgG1-LP-1a), and 0.039 mg / kg of payload control (Auristatin E); at equimolar doses of payload, 0.25 mg / kg, 0.5 mg / kg and 1 mg / kg of ADC-1-1a showed stronger tumor inhibitory effects compared with 0.5 mg / kg, 1 mg / kg and 2 mg / kg of positive control (RC-48); the mice had good tolerance to ADC-1-1a. This indicates that ADC-1-1a has significant in vivo anti-tumor activity and safety.

[0896] Table 10 Average Tumor Volume of Each Group

[0897]

[0898] Table 11 Average Body Weight of Each Group of Mice

[0899]

[0900]

[0901] In Vivo Pharmacodynamic Evaluation of ADC-1-1a in Hybridoma (JIMT-1 + MDA-MB-468) in Example 176

[0902] In this example, a human tumor cell line (JIMT-1 + MDA-MB-468) was subcutaneously inoculated into BALB / c-Nude as an experimental model to evaluate the in vivo pharmacodynamics of ADC-1-1a. A certain number of tumor cell suspension was inoculated subcutaneously into BALB / c-Nude. When the tumor volume grew to about 160 mm 3 or so, vehicle, naked antibody control (TA001), irrelevant antibody ADC control (FITC-IgG1-LP-1a), payload control (Auristatin E), positive control (RC-48) and the corresponding ADC drug (ADC-1-1a) were injected via the tail vein once a week for four times. Continuous observation was carried out, and the tumor volume and mouse body weight were measured twice a week to evaluate the inhibitory effect of the tested ADC drug on tumor growth. The results are shown in Table 12, Table 13 and Figure 10 as follows. In the hybridoma of HER2-positive-expressing JIMT-1 and HER2-non-expressing MDA-MB-468, ADC-1-1a at 1 mg / kg showed stronger antitumor effects compared with naked antibody control (TA001) at 1 mg / kg, irrelevant antibody ADC control (FITC-IgG1-LP-1a) at 1 mg / kg, and payload control (Auristatin E) at 0.039 mg / kg; at equimolar doses of payload, ADC-1-1a at 0.25 mg / kg, 0.5 mg / kg and 1 mg / kg showed stronger antitumor effects compared with positive control (RC-48) at 0.5 mg / kg, 1 mg / kg and 2 mg / kg; the mice tolerated ADC-1-1a well. This indicates that ADC-1-1a has significant in vivo antitumor activity and safety.

[0903] Table 12 Average Tumor Volume of Each Group

[0904]

[0905]

[0906] Average body weight of mice in each group in Table 13

[0907]

[0908] In vivo pharmacodynamic evaluation of 177ADC-1-1a in xenograft tumors (NCI-N87)

[0909] In this example, a BALB / c-Nude subcutaneous inoculation of a human tumor cell line (NCI-N87) was used as an experimental model to evaluate the in vivo pharmacodynamics of ADC-1-1a. A certain number of tumor cell suspensions were inoculated subcutaneously into BALB / c-Nude. When the tumor volume grew to about 180 mm 3 or so, vehicle, naked antibody control (TA001), irrelevant antibody ADC control (FITC-IgG1-LP-1a), payload control (Auristatin E), positive control (RC-48) and the corresponding ADC drug (ADC-1-1a) were injected via the tail vein once a week for four times. Continuous observation was carried out, and the tumor volume and mouse body weight were measured twice a week to evaluate the inhibitory effect of the tested ADC drug on tumor growth. The results are shown in Table 14, Table 15 and Figure 11 as shown. In the NCI-N87 xenograft tumors with positive HER2 expression, 1 mg / kg of ADC-1-1a showed stronger tumor inhibitory effects compared with 1 mg / kg of naked antibody control (TA001), 1 mg / kg of irrelevant antibody ADC control (FITC-IgG1-LP-1a), and 0.039 mg / kg of payload control (Auristatin E); at equimolar doses of payload, 0.25 mg / kg, 0.5 mg / kg and 1 mg / kg of ADC-1-1a showed stronger tumor inhibitory effects compared with 0.5 mg / kg, 1 mg / kg and 2 mg / kg of positive control (RC-48); the mice tolerated ADC-1-1a well. This indicates that ADC-1-1a has significant in vivo anti-tumor activity and safety.

[0910] Average tumor volume of each group in Table 14

[0911]

[0912] Average body weight of mice in each group in Table 15

[0913]

[0914]

[0915] In Vivo Pharmacodynamic Evaluation of ADC-1-1a in Monoclonal Tumor (NCI-H1975) in Example 178

[0916] In this example, a human tumor cell line (NCI-H1975) was subcutaneously inoculated into BALB / c-Nude as an experimental model to evaluate the in vivo pharmacodynamics of ADC-1-1a. A certain number of tumor cell suspensions were inoculated subcutaneously into BALB / c-Nude. When the tumor volume grew to about 180 mm 3 or so, vehicle, naked antibody control (TA001), irrelevant antibody ADC control (FITC-IgG1-LP-1a), payload control (Auristatin E), positive control (RC-48), and the corresponding ADC drug (ADC-1-1a) were injected via the tail vein once a week for four times. Continuous observation was carried out, and the tumor volume and mouse body weight were measured twice a week to evaluate the inhibitory effect of the tested ADC drug on tumor growth. The results are shown in Tables 16, 17 and Figure 12 as follows. In the NCI-H1975 monoclonal tumor with positive HER2 expression, ADC-1-1a at 1 mg / kg showed stronger tumor inhibitory effects compared to naked antibody control (TA001) at 1 mg / kg, irrelevant antibody ADC control (FITC-IgG1-LP-1a) at 1 mg / kg, and payload control (Auristatin E) at 0.039 mg / kg; at equimolar doses of payload, ADC-1-1a at 0.25 mg / kg, 0.5 mg / kg, and 1 mg / kg showed stronger tumor inhibitory effects compared to positive control (RC-48) at 0.5 mg / kg, 1 mg / kg, and 2 mg / kg; the mice tolerated ADC-1-1a well. This indicates that ADC-1-1a has significant in vivo anti-tumor activity and safety.

[0917] Table 16 Average Tumor Volume of Each Group

[0918]

[0919]

[0920] Note: "-" represents animal death and no data

[0921] Table 17 Average Body Weight of Mice in Each Group

[0922]

[0923] Note: "-" represents animal death and no data

[0924] It is understood that the above specific description of the present invention is only for explaining the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced to achieve the same technical effect; the above-mentioned modifications or equivalent replacements are all within the protection scope of the present invention.

Claims

1. A ligand-drug conjugate of formula I or a pharmaceutically acceptable salt or solvate thereof, wherein: Ab is an antibody or an antigen-binding fragment thereof that specifically binds to the HER2 antigen; wherein the antibody is composed of a light chain and a heavy chain, the light chain contains CDR-L1, CDR-L2 and CDR-L3, and their amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 respectively; preferably, the heavy chain contains CDR-H1, CDR-H2, CDR-H3, and their amino acid sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 respectively; M is a linking unit linked to Ab; A is a peptide residue composed of 2-7 amino acids, wherein optionally each of the amino acids is independently substituted by one or more substituents selected from: deuterium atom, halogen, hydroxyl group, cyano group, amino group, nitro group, alkyl group, substituted alkyl group, alkoxy group, cycloalkyl group, substituted cycloalkyl group; W represents an aminomethyleneoxy structural unit as shown in formula (i): wherein: The left wavy line represents the connection site of the nitrogen atom in formula (i) to A, and the right wavy line represents the connection site of the oxygen atom in formula (i) to the drug D. The oxygen atom is the common group of the drug D and W; R1, R2 and R3 are each independently selected from a hydrogen atom, a deuterium atom, an alkyl group and a substituted alkyl group; p is an integer or a decimal selected from 1-20; and The drug D is an auristatin having the structure shown in formula D, or an isomer, meso form, racemic form, enantiomer or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: R4 and R5 are each independently selected from a hydrogen atom, a deuterium atom, an alkyl group, and a deuterated alkyl group, or R4 and R5 are connected to form the following structure: -(CR 11 R 12 ) n -B-(CR 13 R 14 ) m -, where R 11 , R 12 , R 13 and R 14 are selected from a hydrogen atom, a deuterium atom, an alkyl group, and a deuterated alkyl group; B is selected from O, NR 15 , CR 16 R 17 , where R 15 , R 16 , R 17 are selected from a hydrogen atom, a deuterium atom, and an alkyl group; n and m are each independently selected from integers from 0 to 8; the nitrogen atom bonded to R4 and R5 and -(CR 11 R 12 ) n -B-(CR 13 R 14 ) m - together form a ring; R6, R7, R8, and R9 are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an azide group, an alkyl group, and NR 18 R 19 , or any two of R6, R7, R8, and R9 together with the atom to which they are bonded form a cycloalkyl group, and the remaining two groups are each independently selected from a hydrogen atom, a halogen, an azide group, an alkyl group, and NR 18 R 19 , where R 18 , R 19 are selected from a hydrogen atom and an alkyl group; R 10 selected from aryl, heteroaryl, said aryl or heteroaryl optionally being substituted by one or more substituents selected from: a hydrogen atom, a halogen, an alkyl, an alkoxy, an amino, a nitro; The wavy line in formula D represents the connection site of the oxygen atom at position 1 in the structure of the drug D to W. The oxygen atom is the common group of the drug D and W.

2. The ligand-drug conjugate of claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the light chain contains a light chain variable region with the amino acid sequence of SEQ ID NO: 7; Preferably, the light chain further contains a light chain constant region with the amino acid sequence of SEQ ID NO: 8; Preferably, the amino acid sequence of the light chain is SEQ ID NO: 9; Preferably, the heavy chain contains a heavy chain variable region with the amino acid sequence of SEQ ID NO: 10; Preferably, the heavy chain further contains a heavy chain constant region with the amino acid sequence of SEQ ID NO: 11; Preferably, the amino acid sequence of the heavy chain is SEQ ID NO:

12.

3. The ligand-drug conjugate of claim 1 or 2 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or its antigen-binding fragment is selected from murine antibodies, rabbit antibodies, phage display-derived antibodies, yeast display-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies.

4. The ligand-drug conjugate of any one of claims 1-3 or a pharmaceutically acceptable salt or solvate thereof, R4 and R5 are each independently selected from a hydrogen atom, a C1-C4 alkyl group, or R4 and R5 are linked to form the following structure: -(CH2)2-B-(CH2)2-, where B is selected from O, NH, and the nitrogen atom bonded to R4 and R5 forms a ring together with -(CH2)2-B-(CH2)2-.

5. The ligand-drug conjugate according to any one of claims 1-4, or a pharmaceutically acceptable salt or solvate thereof, In formula D, R6, R7, R8, and R9 are each a hydrogen atom; alternatively, one of R6, R7, R8, and R9 in formula D is selected from a halogen, an azide group, an amino group, and the remaining three are each hydrogen; alternatively, any two of the groups R6, R7, R8, and R9 in formula D together with the atoms to which they are bonded form a cyclopropyl group, and the remaining two groups are each independently a hydrogen atom.

6. The ligand-drug conjugate according to any one of claims 1-5, or a pharmaceutically acceptable salt or solvate thereof, R in formula D 10 is phenyl, optionally substituted by one or more of said substituents; preferably, said substituents are selected from amino, nitro; Preferably, the drug D is selected from the following compounds or their isomers, meso forms, racemates, enantiomers or mixtures thereof in any form, or pharmaceutically acceptable salts thereof:

7. The ligand-drug conjugate according to any one of claims 1-6, or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate has the structure shown in formula Ia: Wherein: Z is selected from -C1-C 10 alkylene-, -C3-C8 carbocycle-, -arylene-, -C1-C 10 alkylene-arylene-, -arylene-C1-C 10 -alkylene-, -C1-C 10 alkylene-(C3-C8 carbocycle)-, -(C3-C8 carbocycle)-C1-C 10 alkylene-, -3-8 membered heterocycle-, -C1-C 10 alkylene-(3-8 membered heterocycle)-, -(3-8 membered heterocycle)-C1-C 10 alkylene-, -(CH2CH2O) r -, -(CH2CH2O) r -CH2- or wherein X is selected from -C1-C 10 alkylene-, -C3-C8 carbocycle-, -arylene-, -C1-C 10 alkylene-arylene-, -arylene-C1-C 10 -alkylene-, -C1-C 10 alkylene-(C3-C8 carbocycle)-, -(C3-C8 carbocycle)-C1-C 10 alkylene-, -3-8 membered heterocycle-, -C1-C 10 alkylene-(3-8 membered heterocycle)-, -(3-8 membered heterocycle)-C1-C 10 alkylene-, -(CH2CH2O) r -, -(CH2CH2O) r -CH2-; Y is a hydrophilic structure selected from carboxyl, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid or polyethylene glycol (PEG); each of the heterocycles independently contains 1-3 atoms selected from N, O, S; the -C1-C 10 alkylene-, -C3-C8 carbocycle-, and the heterocycles are each independently substituted with one or more substituents selected from deuterium atom, halogen, hydroxyl, cyano, nitro, amino, alkyl, heteroalkyl, substituted alkyl, alkoxy, carboxyl or cycloalkyl; The left wavy line in represents the connection site to the N on maleimide, and the right wavy line represents the connection site to the carbonyl; r is an integer between 1 and 10; q is an integer between 1 and 8; n 1 、n 2 、n 3 are independently selected from integers or decimals between 0 and 20, n 1 、n 2 、n 3 are not simultaneously 0, and n 1 +n 2 +n 3 ≤ 20.

8. The ligand-drug conjugate according to any one of claims 1-7, or a pharmaceutically acceptable salt or solvate thereof, A is a polypeptide residue formed by 2-7 amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), alanine (A), citrulline, serine (S), glutamic acid (E), or aspartic acid (D); Preferably, A is a peptide residue formed by 2-4 amino acids selected from phenylalanine and glycine; Preferably, A is a tetrapeptide residue composed of glycine-glycine-phenylalanine-glycine.

9. The ligand-drug conjugate according to claim 7 or 8, or a pharmaceutically acceptable salt or solvate thereof, Z is selected from -C1-C 10 alkylene-, such as -C4-C6 alkylene-, such as -C5 alkylene-; Preferably, Z is wherein q is an integer between 1 and 8.

10. The ligand-drug conjugate according to any one of claims 7-9, or a pharmaceutically acceptable salt or solvate thereof, R1, R2, and R3 are each independently selected from a hydrogen atom, a deuterium atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; Preferably, R1, R2, and R3 are simultaneously a hydrogen atom or a deuterium atom; Preferably, R1, R2, and R3 are simultaneously a hydrogen atom.

11. The ligand-drug conjugate according to any one of claims 1-10, or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate has the structure shown in formula Ib, Among them, R4, R5, R6, R7, R8, R9, R 10 , n 1 , n 2 , n 3 as defined in any one of claims 1 - 7.

12. The ligand-drug conjugate according to any one of claims 1-10, or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate has the structure shown in formula Ic, formula Id, or formula Ie, Among them, Each Ac is independently a hydrophilic structural unit having the structure shown in formula c: Ac is linked to the methylene carbon at position 2 indicated in Formula Ic, Formula Id or Formula Ie through an amino functional group, X and Y are each independently as defined in claim 7; R4, R5, R6, R7, R8, R9, R 10 , n 1 , n 2 , n 3 is as defined in any one of claims 1-7; Preferably, each Ac is independently selected from glycine, (D / L) alanine, (D / L) leucine, (D / L) isoleucine, (D / L) valine, (D / L) phenylalanine, (D / L) proline, (D / L) tryptophan, (D / L) serine, (D / L) tyrosine, (D / L) cysteine, (D / L) cystine, (D / L) arginine, (D / L) histidine, (D / L) methionine, (D / L) asparagine, (D / L) glutamine, (D / L) threonine, (D / L) aspartic acid, (D / L) glutamic acid, natural or unnatural amino acid derivatives or the following structures, 13. The ligand-drug conjugate according to any one of claims 1-12, or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate is selected from the following structures: Among them, The configuration of the chiral carbon at the 2-position is R or S.

14. The linker-drug compound as shown in formula II, or an isomer, meso form, racemate, enantiomer or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, Wherein: Z, A, R1, R2, R3, R4, R5, R 11 , R 12 , R 13 , R 14 , B, R 15 , R 16 , R 17 , n, m, R6, R7, R8, R9, R 18 , R 19 , R 10 As defined in any one of claims 1-10.

15. The linker-drug compound according to claim 14, or an isomer, meso form, racemate, enantiomer or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, The linker-drug compound has the structure shown in formula IIa, 16. The linker-drug compound according to claim 15, or an isomer, meso form, racemate, enantiomer or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, The linker-drug compound has the structure shown in formula IIb, IIc or IId, Among them, Each Ac is independently a hydrophilic structural unit having the structure shown in formula c: Wherein, X and Y are each independently defined as in claim 7, and Ac is connected to the 2-position methylene carbon indicated in formula IIb, IIc or IId through -NH-.

17. The linker-drug compound according to any one of claims 14-16, or an isomer, meso form, racemate, enantiomer or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker-drug compound is selected from the following structures: Among them, The configuration of the chiral carbon at the 2-position is R or S.

18. Use of the ligand-drug conjugate according to any one of claims 1-13, or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound according to any one of claims 14-17, or an isomer, meso form, racemate, enantiomer or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof for the preparation of a drug for the treatment or prevention of tumors; Preferably, the tumor expresses HER2; Preferably, the tumor is cancer; More preferably, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma, and leukemia.

19. A pharmaceutical composition comprising an effective amount of the ligand-drug conjugate of any one of claims 1-13 or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound of any one of claims 14-17 or an isomer, meso form, racemate, enantiomer, or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, optionally further comprising a pharmaceutically acceptable carrier, diluent, or excipient.

20. A pharmaceutical preparation comprising an effective amount of the ligand-drug conjugate of any one of claims 1-13 or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound of any one of claims 14-17 or an isomer, meso form, racemate, enantiomer, or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

21. Use of the linker-drug compound of any one of claims 14-17 or an isomer, meso form, racemate, enantiomer, or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof for the preparation of the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof; Preferably, the ligand-drug conjugate is selected from the ligand-drug conjugates of any one of claims 1-13.

22. A method for preparing a ligand-drug conjugate according to any one of claims 1-13 or a pharmaceutically acceptable salt or solvate thereof, the method comprising: The ligand-drug conjugate is obtained by coupling a reduced antibody or an antigen-binding fragment thereof with the linker-drug compound of any one of claims 14-17 or an isomer, meso form, racemate, enantiomer, or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

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