Anti-CD33 antibodies and anti-CD33 antibody-drug conjugates and uses thereof

Abstract

The present invention discloses anti-CD33 antibodies and anti-CD33 antibody-drug conjugates and uses thereof, particularly pharmaceutical compositions comprising an anti-CD33 antibody and / or antigen-binding fragment thereof, antibody-drug conjugate and / or linker-drug or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, and uses in treating tumors or cancer.

Description

[Technical Field]

[0001] The present invention belongs to the field of biopharmaceuticals and relates to an anti-CD33 antibody, an anti-CD33 antibody-drug conjugate, and uses thereof, and in particular to an antibody-drug conjugate formed from an anti-human CD33 antibody and a drug having cytotoxic activity, as well as a method for producing and using said antibody-drug conjugate. [Background technology]

[0002] CD33 is a 364-amino acid myeloid cell differentiation antigen. It is a type I transmembrane glycoprotein with a molecular weight of 67 kDa that is primarily distributed in myeloid blood cells at the onset of differentiation. CD33 is a member of the immunoglobulin superfamily and contains two immunoglobulin-like extracellular domains (IgV and IgC2) and two tyrosine-dependent signaling motifs (ITIMs) within its intracellular structure, which can function as an inhibitory receptor to control inhibitory signaling processes. For example, during immune responses, CD33 acts as an inhibitory receptor by recruiting cytoplasmic phosphatases and causing their dephosphorylation, thereby preventing signal transduction. At the same time, CD33 is also a member of the sialic acid-binding immunoglobulin-like lectin (SIGLEC) family, which recognizes cell surface glycans by binding to sialic acid, mediating cell-cell interactions and mediating adhesion between leukocytes and endothelial cells. Three alternatively spliced ​​forms of CD33 have been identified: the isoform CD33m, which has a smaller disulfide bond linking the V and C domains; the larger molecular weight variant CD33M; and CD33M, which lacks the IgV structural domain.

[0003] CD33 is a myeloid cell-specific leukemia antigen found on blast cells in over 90% of patients with acute myeloid leukemia (AML). AML is a malignant tumor of the hematopoietic system characterized by intractable treatment, a high incidence of relapse, and a high incidence of fatality during treatment. This fatal disease results in the production of abnormal myeloblasts in the bone marrow, leading to a dramatic decrease in the number of red blood cells, platelets, and white blood cells. Studies have shown that targeted ablation of CD33-positive cells can restore hematopoietic function after culture. Furthermore, CD33 has been shown not to be expressed on hematopoietic stem cells, mature granulocytes, or other tissues, making it an excellent target for specific immunotherapy of AML in clinical settings.

[0004] Gemtuzumab ozogamicin (Mylotarg, GO), a CD33-targeting ADC drug, was approved for marketing by the U.S. Food and Drug Administration (FDA) in 2000. Mylotarg is a conjugated anti-CD33 humanized IgG4 monoclonal antibody and calicheamicin, and is primarily used to treat patients with refractory or relapsed acute granulocytic leukemia. Upon binding to the CD33 antigen, Mylotarg releases calicheamicin within cancer cells, inducing tumor cell apoptosis. However, the drug was voluntarily withdrawn from the market in 2010 due to poor clinical efficacy and side effects, including severe and potentially fatal liver damage. After market withdrawal and adjustment of the dosing regimen, Mylotarg was reapproved by the FDA in 2017 as a first-line treatment for acute myeloid leukemia.

[0005] Among the therapeutic drugs targeting CD33, the humanized IgG1 monoclonal antibody lintuzumab failed to demonstrate superior efficacy in combination with cytarabine compared to cytarabine alone in clinical trials, leading to the discontinuation of development of lintuzumab in 2010. SGN-CD33A, a CD33-targeting ADC clinical candidate developed by Seattle Genetics, was discontinued in 2017 due to side effects such as fatal infections and a high patient mortality rate observed in Phase III trials. Clinical trials of AVE9633, an ADC drug combining an anti-CD33 IgG1 monoclonal antibody and DM-4, ​​were discontinued in 2009 due to low clinical activity. Research is also being conducted to improve tumor targeting and efficacy by conjugating vincristine, an alkaloid extracted from Catharanthus roseus, to CD33 antibody polypeptides via carbodiimide conversion. CD33 is a validated AML target with sufficient mean antigen density, making it the antigen of choice for ADCs and other novel therapies. Currently, CD33-targeted immunotherapies, including monoclonal antibodies, bispecific antibodies, trispecific antibodies, and ADCs, are being introduced by various companies and are undergoing clinical evaluation or preclinical development.

[0006] Antibody-drug conjugates (ADCs) are a new type of biologic drug that combines a monoclonal antibody (mAb) targeting a specific antigen with a potent cytotoxic small molecule drug (payload) via a bioactive linker. ADC drugs are specifically recognized by antibodies, which guide the small molecule drug to cancer cells, where it is internalized and released as a cytotoxic drug, thereby specifically killing the cancer cells. Since their introduction, ADCs have undergone continuous innovation and optimization, resulting in successful tumor treatment. Clinical studies have shown that ADC drugs have high efficacy and can effectively reduce the toxicity of small molecule cytotoxic drugs to healthy tissues. This has made ADCs a hot topic in antitumor drug research and development and an important tool in the field of cancer treatment. CD33 is an excellent target for specific immunotherapy against AML, and ADC drugs have already been approved for sale, making CD33 a promising target for ADC drug development. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides an anti-human CD33 antibody-drug conjugate, which is an antibody-drug conjugate formed by combining a humanized anti-human CD33 antibody with a drug having cytotoxic activity. Compared to existing drugs of the same type, the anti-human CD33 antibody-drug conjugate has better molecular stability and preclinical efficacy, and is expected to have excellent clinical therapeutic effects. [Means for solving the problem]

[0008] Thus, in a first aspect, the present invention provides an antibody-drug conjugate represented by general formula I, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof: JPEG2025533577000001.jpg134128 where, The drug in the antibody-drug conjugate is a camptothecin-based antitumor drug represented by the following formula II or a stereoisomer thereof: JPEG2025533577000002.jpg4247Ab is selected from an anti-CD33 antibody or an antigen-binding fragment thereof; M1, M2, and M3 are each independently JPEG2025533577000003.jpg1328, JPEG2025533577000004.jpg1531, JPEG2025533577000005.jpg1531, and M1, M2, and M3 are different from each other; or M1, M2, and M3 are each independently selected from the structures represented by Formula A1, Formula A2, and Formula A3, and M1, M2, and M3 are different from each other; JPEG2025533577000006.jpg1928 formula A1 JPEG2025533577000007.jpg2223 formula A2 JPEG2025533577000008.jpg1922 formula A3 wherein Y is a skeleton selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl, preferably Y is C1-C6 alkyl, Ac is a hydrophilic structural unit, the position indicated by the wavy line on the left is linked to Ab, the position indicated by the wavy line on the right is linked to B or linked to L; B may be present or absent, and when present, is selected from a modifying unit; L is selected from linker units, preferably peptide-containing linker units; X is selected from -NH-, -O- or -S-, preferably -O-; R1 and R2 are the same or different and are each independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl; R3 and R4 are the same or different and are each independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, C6-C10 arylC1-C6 alkyl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl; preferably, R3 and R4 are each independently selected from hydrogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, or C6-C10 arylC1-C6 alkyl; Alternatively, R3, R4 and the carbon atom to which they are attached form a C3-C8 cycloalkyl, a 3- to 7-membered heterocyclyl, or a substituted 3- to 7-membered heterocyclyl, preferably R3, R4 and the carbon atom to which they are attached form a C3-C8 cycloalkyl; R5 is selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, preferably R5 is selected from hydrogen, C1-C6 alkyl; m is selected from integers from 0 to 5; n1, n2, and n3 are each independently selected from any integer or any decimal number between 0 and 10, and n1, n2, and n3 are not simultaneously 0, and 1≦n1+n2+n3≦10 (for example, n1+n2+n3 is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 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, or, for example, n n1+n2+n3 is 8.01, 8.32, 8.56, 8.63, 8.66 or 9.40, or, for example, n1+n2+n3 is selected from any integer or decimal point of 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, 9 to 9.5 or 9.5 to 10, preferably, for example, n1+n2+n3 is selected from any integer or decimal point of 7 to 10, i.e., 7≦n1+n2+n3≦10, more preferably, for example, n1+n2+n3 is selected from any integer or decimal point of 8 to 10, i.e., 8≦n1+n2+n3≦10; The chiral carbon atom indicated by * has the R or S absolute configuration; M1, M2, and M3 are each independently JPEG2025533577000009.jpg1328, JPEG2025533577000010.jpg1531, If M1, M2, and M3 are different from each other, then B is present and satisfies the condition of being selected from the modification unit.

[0009] In some embodiments, M1, M2, and M3 are each independently: JPEG2025533577000012.jpg1328, JPEG2025533577000013.jpg1531, JPEG2025533577000014.jpg1531, and M1, M2, and M3 are different from each other; or M1, M2, and M3 are each independently selected from the structures represented by Formula A1, Formula A2, and Formula A3, and M1, M2, and M3 are different from each other; Preferably, M1, M2, and M3 are each independently selected from the structures represented by formula A1, formula A2, and formula A3, and M1, M2, and M3 are different from each other; JPEG2025533577000015.jpg1928 formula A1 JPEG2025533577000016.jpg2223 formula A2 JPEG2025533577000017.jpg1922 formula A3 wherein Y is a backbone selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl; preferably, Y is C1-C6 alkyl, more preferably C1-C3 alkyl, and most preferably methylene; The position indicated by * has two chiralities, R absolute configuration or S absolute configuration. The position indicated by the wavy line on the left is linked to Ab, the position indicated by the wavy line on the right is linked to B or linked to L, The hydrophilic structural unit Ac is selected from, but not limited to, a natural or unnatural amino acid, 1-20 polyethylene glycol, a phosphate group, a carboxylic acid group, a sulfonic acid group, a sulfinic acid group, or the following structure: JPEG2025533577000018.jpg918, JPEG2025533577000019.jpg1020, JPEG2025533577000020.jpg1022, JPEG2025533577000021.jpg1217, JPEG2025533577000022.jpg1722, JPEG2025533577000023.jpg1533, JPEG2025533577000024.jpg1235, JPEG2025533577000025.jpg1540, JPEG2025533577000026.jpg1020, JPEG2025533577000027.jpg917, JPEG2025533577000028.jpg1018 or JPEG2025533577000029.jpg928, Here, p is selected from integers of 0 to 10, and the position indicated by the wavy line is linked to the skeleton Y. Preferably, the hydrophilic structural unit Ac is JPEG2025533577000030.jpg918, JPEG2025533577000031.jpg1020, The positions selected from JPEG2025533577000032.jpg1022 and indicated by the wavy lines are concatenated to skeleton Y. More preferably, the hydrophilic structural unit Ac is JPEG2025533577000033.jpg918, and the position indicated by the wavy line is connected to skeleton Y.

[0010] In some embodiments, M1, M2, and M3 are selected from any of the following combinations (1) to (7), and M1, M2, and M3 are different from each other. (1) JPEG2025533577000034.jpg1531, JPEG2025533577000035.jpg1531, JPEG2025533577000036.jpg1531, (2) JPEG2025533577000037.jpg1728, JPEG2025533577000038.jpg2026, JPEG2025533577000039.jpg1523, (3) JPEG2025533577000040.jpg2231, JPEG2025533577000041.jpg2231, JPEG2025533577000042.jpg2233, (4) JPEG2025533577000043.jpg1728, JPEG2025533577000044.jpg2028, JPEG2025533577000045.jpg2030, (5) JPEG2025533577000046.jpg1728, JPEG2025533577000047.jpg1828, JPEG2025533577000048.jpg1728, (6) JPEG2025533577000049.jpg2528, JPEG2025533577000050.jpg2528, JPEG2025533577000051.jpg2228, (7) JPEG2025533577000052.jpg2528, JPEG2025533577000053.jpg2828, JPEG2025533577000054.jpg2528; Preferably, M1, M2, and M3 are selected from any of the following combinations (1) and (2), and M1, M2, and M3 are different from each other. (1) JPEG2025533577000055.jpg1531, JPEG2025533577000056.jpg1531, JPEG2025533577000057.jpg1531, (2) JPEG2025533577000058.jpg1728, JPEG2025533577000059.jpg2026, JPEG2025533577000060.jpg1523; More preferably, M1, M2, and M3 are each independently JPEG2025533577000061.jpg1728, JPEG2025533577000062.jpg2026, JPEG2025533577000063.jpg1523, and M1, M2, and M3 are different from each other. where: The * position is either the R or S absolute configuration. The position indicated by the wavy line on the left is linked to Ab, and the position indicated by the wavy line on the right is linked to the modifying unit B or the linker unit L.

[0011] In some embodiments, modifying unit B may be present or absent, and when present, said modifying unit B is selected from, but is not limited to, structures that increase hydrophilicity or stereoisomers thereof.

[0012] In some embodiments, modifying unit B may be present or absent, and when present, the modifying unit B is selected from, but is not limited to, the hydrophilic enhancing structures shown in the following formula or stereoisomers thereof: JPEG2025533577000064.jpg1028, JPEG2025533577000065.jpg1243, JPEG2025533577000066.jpg2017, JPEG2025533577000067.jpg1522, JPEG2025533577000068.jpg2230, JPEG2025533577000069.jpg1735, JPEG2025533577000070.jpg3041, JPEG2025533577000071.jpg2242, JPEG2025533577000072.jpg1517, JPEG2025533577000073.jpg1420; Preferably, the modifying unit B may be present or absent, and when present, the modifying unit B is selected from the following structures that enhance hydrophilicity, or stereoisomers thereof, but is not limited thereto: JPEG2025533577000074.jpg1028, JPEG2025533577000075.jpg1243, JPEG2025533577000076.jpg1522, JPEG2025533577000077.jpg1735, JPEG2025533577000078.jpg2242; where: R is selected from, but not limited to, hydroxyl, amino, polyethylene glycol, a carboxylic acid group, a sulfonic acid group, a sulfinic acid group, a phosphate group, C1-C6 alkyl, C1-C6 alkoxy, a natural or unnatural amino acid residue, a sugar or a derivative thereof, or a combination thereof. Preferably, R is selected from C1-C6 alkoxy, a carboxylic acid group, and amino. More preferably, R is selected from methoxy, a carboxylic acid group, and amino. The position indicated by the wavy line on the left is connected to M1, M2, and M3. The position indicated by the wavy line on the right is linked to the linker unit L. q is selected from integers of 1 to 10 (preferably integers of 2 to 8), and is preferably selected from 2, 7, and 8. Most preferably, the modifying unit B is selected from the hydrophilic enhancing structures of the following formulae or stereoisomers thereof: JPEG2025533577000079.jpg1228, JPEG2025533577000080.jpg1341, JPEG2025533577000081.jpg1238, JPEG2025533577000082.jpg1521, JPEG2025533577000083.jpg2231, JPEG2025533577000084.jpg3240, JPEG2025533577000085.jpg3445, JPEG2025533577000086.jpg3243 The positions indicated by the wavy lines on the left side are linked to M1, M2, and M3, and the position indicated by the wavy lines on the right side is linked to the linker unit L.

[0013] In some embodiments, the linker unit L is L1-L2, L1 is a peptide residue containing 2 to 10 amino acid residues, and the amino terminus of the peptide residue is linked to a modifying unit B or M1, M2, or M3, and the carbonyl terminus is linked to L2. Preferably, L1 is a peptide residue containing 2 to 4 amino acid residues, the amino terminus of the peptide residue being linked to the modifying unit B or M1, M2, or M3, and the carbonyl terminus being linked to L2. Preferably, the amino acids are selected from valine, alanine, phenylalanine, glycine, lysine, citrulline, serine, glutamic acid, and aspartic acid, more preferably, the amino acids are selected from valine, alanine, phenylalanine, glycine, lysine, and citrulline, and most preferably, the amino acids are selected from valine, phenylalanine, glycine, lysine, and citrulline. More preferably, L1 is selected from the following peptide residues: glycine-glycine-phenylalanine-glycine, valine-citrulline, phenylalanine-lysine, valine-alanine, alanine-alanine-alanine (preferably selected from glycine-glycine-phenylalanine-glycine, valine-citrulline, phenylalanine-lysine), the amino terminus of said peptide residue being linked to modifying unit B or M1, M2, M3, and the carbonyl terminus being linked to L2. Most preferably, L1 is one of the following peptide residues: JPEG2025533577000087.jpg1543, JPEG2025533577000088.jpg2235, JPEG2025533577000089.jpg2531, JPEG2025533577000090.jpg1226, JPEG2025533577000091.jpg1333 (preferably, JPEG2025533577000092.jpg1543, JPEG2025533577000093.jpg2235, JPEG2025533577000094.jpg2531), the amino terminus of the peptide residue is linked to modifying unit B or M1, M2, M3, and the carbonyl terminus is linked to L2. L2 is JPEG2025533577000095.jpg810, JPEG2025533577000096.jpg1828, JPEG2025533577000097.jpg1320, JPEG2025533577000098.jpg1743, The position indicated by the wavy line on the left is linked to L1, and the position indicated by the wavy line on the right is linked to X. Preferably, L2 is JPEG2025533577000100.jpg810, JPEG2025533577000101.jpg1828, JPEG2025533577000102.jpg1320, The position indicated by the wavy line on the left is linked to L1, and the position indicated by the wavy line on the right is linked to X. More preferably, L2 is JPEG2025533577000104.jpg810, The position indicated by the wavy line on the left is linked to L1, and the position indicated by the wavy line on the right is linked to X. Preferably, the linker unit L is selected from, but not limited to, the following structures or stereoisomers thereof: JPEG2025533577000106.jpg2238, JPEG2025533577000107.jpg2251, JPEG2025533577000108.jpg2341, JPEG2025533577000109.jpg1538, JPEG2025533577000110.jpg1551, JPEG2025533577000111.jpg1541, JPEG2025533577000112.jpg1638, JPEG2025533577000113.jpg1651, JPEG2025533577000114.jpg1641, JPEG2025533577000115.jpg1554, JPEG2025533577000116.jpg1341, JPEG2025533577000117.jpg1034 or JPEG2025533577000118.jpg1546; where: r is selected from an integer from 1 to 10; More preferably, the linker unit L is selected from the following structures or stereoisomers thereof: JPEG2025533577000119.jpg1341, JPEG2025533577000120.jpg2351, JPEG2025533577000121.jpg2151; The position indicated by the wavy line on the left side of the linker unit L is linked to the modifying unit B or M1, M2, or M3. The position indicated by the wavy line on the right side of the linker unit L is linked to X.

[0014] In some embodiments, R1 and R2 are the same or different and are each independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl. Preferably, R1 and R2 are the same or different and are each independently selected from halogen, C1-C6 alkyl. More preferably, R1 is selected from C1-C6 alkyl and R2 is selected from halogen. Most preferably, R1 is methyl and R2 is fluorine. R3 and R4 are the same or different and are each independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, C6-C10 arylC1-C6 alkyl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl. Preferably, R3 and R4 are the same or different and each independently selected from hydrogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, or C6-C10 arylC1-C6 alkyl. More preferably, R3 and R4 are the same or different and are each independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkylC1-C6 alkyl, or phenylC1-C6 alkyl. More preferably, R3 and R4 are the same or different and are each independently selected from hydrogen, methyl, ethyl, trifluoromethyl, cyclopropyl, cyclopropylmethyl, and benzyl. Most preferably, either R3 or R4 is selected from hydrogen and methyl, and the other is selected from hydrogen, methyl, ethyl, trifluoromethyl, cyclopropyl, cyclopropylmethyl, and benzyl. Alternatively, R3, R4 and the carbon atom to which they are attached comprise a C3-C8 cycloalkyl, a 3- to 7-membered heterocyclyl, or a substituted 3- to 7-membered heterocyclyl. Preferably, R3, R4 and the carbon atom to which they are attached comprise a C3-C8 cycloalkyl. More preferably, R3, R4 and the carbon atom to which they are attached comprise a C3-C6 cycloalkyl. Most preferably, R3, R4 and the carbon atom to which they are attached comprise cyclopropyl, cyclobutyl, or cyclopentyl. R5 is selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, preferably R5 is selected from hydrogen, C1-C6 alkyl, more preferably R5 is selected from hydrogen, methyl. X is selected from -NH-, -O- or -S-, preferably -O-. m is selected from integers of 0 to 5, and is preferably selected from 0 and 1.

[0015] In some embodiments, the camptothecin-based anti-tumor agent is selected from, but not limited to, the following compounds or stereoisomers thereof: JPEG2025533577000122.jpg2828, JPEG2025533577000123.jpg2928, JPEG2025533577000124.jpg3028, JPEG2025533577000125.jpg3028, JPEG2025533577000126.jpg3028、 JPEG2025533577000127.jpg3028、 JPEG2025533577000128.jpg2928、 JPEG2025533577000129.jpg2928、 JPEG2025533577000130.jpg3128、 JPEG2025533577000131.jpg3128、 JPEG2025533577000132.jpg3128、 JPEG2025533577000133.jpg3128、 JPEG2025533577000134.jpg3028、 JPEG2025533577000135.jpg3028、 JPEG2025533577000136.jpg3028、 JPEG2025533577000137.jpg3028、 JPEG2025533577000138.jpg3028、 JPEG2025533577000139.jpg3028、 JPEG2025533577000140.jpg3128、 JPEG2025533577000141.jpg3128、 JPEG2025533577000142.jpg2828、 JPEG2025533577000143.jpg2928、 JPEG2025533577000144.jpg3028、 JPEG2025533577000145.jpg3028、 JPEG2025533577000146.jpg3028、 JPEG2025533577000147.jpg3028、 JPEG2025533577000148.jpg3028、 JPEG2025533577000149.jpg3028, JPEG2025533577000150.jpg3028, JPEG2025533577000151.jpg3028, JPEG2025533577000152.jpg3128, JPEG2025533577000153.jpg3128, JPEG2025533577000154.jpg2828, JPEG2025533577000155.jpg2928, JPEG2025533577000156.jpg3028, JPEG2025533577000157.jpg3028, JPEG2025533577000158.jpg3028, JPEG2025533577000159.jpg3028, JPEG2025533577000160.jpg2928 or JPEG2025533577000161.jpg2928.

[0016] In some embodiments, the antibody-drug conjugate is selected from, but not limited to, the following structures: JPEG2025533577000162.jpg125128, JPEG2025533577000163.jpg127128, JPEG2025533577000164.jpg104128, JPEG2025533577000165.jpg106128, JPEG2025533577000166.jpg104128, JPEG2025533577000167.jpg106128, JPEG2025533577000168.jpg104128, JPEG2025533577000169.jpg124128, JPEG2025533577000170.jpg104128、 JPEG2025533577000171.jpg119128、 JPEG2025533577000172.jpg106128、 JPEG2025533577000173.jpg122128、 JPEG2025533577000174.jpg103128、 JPEG2025533577000175.jpg104128、 JPEG2025533577000176.jpg104128、 JPEG2025533577000177.jpg104128、 JPEG2025533577000178.jpg106128、 JPEG2025533577000179.jpg106128、 JPEG2025533577000180.jpg111128、 JPEG2025533577000181.jpg112128、 JPEG2025533577000182.jpg111128、 JPEG2025533577000183.jpg109128、 JPEG2025533577000184.jpg109128、 JPEG2025533577000185.jpg109128、 JPEG2025533577000186.jpg101128、 JPEG2025533577000187.jpg106128、 JPEG2025533577000188.jpg104128、 JPEG2025533577000189.jpg104128、 JPEG2025533577000190.jpg109128、 JPEG2025533577000191.jpg106128、 JPEG2025533577000192.jpg99128、 JPEG2025533577000193.jpg109128, JPEG2025533577000194.jpg120128, JPEG2025533577000195.jpg114128, JPEG2025533577000196.jpg119128, JPEG2025533577000197.jpg119128, JPEG2025533577000198.jpg114128, JPEG2025533577000199.jpg122137, JPEG2025533577000200.jpg106128, JPEG2025533577000201.jpg114128, JPEG2025533577000202.jpg114128, JPEG2025533577000203.jpg106128, JPEG2025533577000204.jpg106128, JPEG2025533577000205.jpg111128, JPEG2025533577000206.jpg117128, JPEG2025533577000207.jpg119128, JPEG2025533577000208.jpg117128 or JPEG2025533577000209.jpg126128; where: ANTI-CD33 is an anti-CD33 antibody or an antigen-binding fragment thereof. n1, n2, and n3 are each independently selected from any integer or any decimal number between 0 and 10, and n1, n2, and n3 are not simultaneously 0, and 1≦n1+n2+n3≦10 (for example, n1+n2+n3 is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 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, Alternatively, for example, n1+n2+n3 is 8.01, 8.32, 8.56, 8.63, 8.66, or 9.40; or, for example, n1+n2+n3 is selected from any integer or decimal point between 7 and 7.5, 7.5 and 8, 8 and 8.5, 8.5 and 9, 9 and 9.5, or 9.5 and 10, preferably, for example, n1+n2+n3 is selected from any integer or decimal point between 7 and 10, i.e., 7≦n1+n2+n3≦10, and preferably, n1+n2+n3 is selected from any integer or decimal point between 8 and 10, i.e., 8≦n1+n2+n3≦10.

[0017] In some embodiments, the antibodies of the Ab comprise two light chains and two heavy chains, wherein the light and heavy chains form an IgG with binding specificity for CD33.

[0018] In some embodiments, the light chain variable region of the anti-CD33 antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, and the heavy chain variable region comprises CDR1, CDR2, and CDR3 set forth in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0019] In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof has a light chain variable region set forth in SEQ ID NO:7 and a heavy chain variable region set forth in SEQ ID NO:1.

[0020] In some embodiments, the anti-CD33 antibody comprises a constant region derived from a human immunoglobulin. Preferably, the light chain of the anti-CD33 antibody comprises a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda), and the heavy chain of the antibody comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4). Preferably, the amino acid sequence of the light chain of the anti-CD33 antibody is SEQ ID NO:11, and the amino acid sequence of the heavy chain is SEQ ID NO:5. Preferably, the nucleic acid coding sequence for the light chain of said anti-CD33 antibody is SEQ ID NO:23 and the nucleic acid coding sequence for the heavy chain is SEQ ID NO:17.

[0021] In some embodiments, the anti-CD33 antibody further comprises a cysteine ​​site-directed insertion. Preferably, the site for the cysteine ​​insertion is in the light chain constant region. Preferably, the site of insertion of the cysteine ​​is at position 206 (Kabat numbering) of the kappa light chain constant region. Preferably, the amino acid sequence of the light chain of the anti-CD33 antibody is SEQ ID NO:25, and the amino acid sequence of the heavy chain is SEQ ID NO:5. Preferably, the nucleic acid coding sequence for the light chain of said anti-CD33 antibody is SEQ ID NO:27 and the nucleic acid coding sequence for the heavy chain is SEQ ID NO:17.

[0022] In some embodiments, the pharmaceutically acceptable salts include sodium, potassium, calcium, or magnesium salts formed with acidic functional groups in the structural formula, as well as acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate salts formed with basic functional groups in the structural formula.

[0023] In a second aspect of the present invention, there is provided a linker-drug of Formula III, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof: JPEG2025533577000210.jpg4053where, M' is JPEG2025533577000211.jpg1323 or a concatenation unit represented by the following formula A'. JPEG2025533577000212.jpg1720 Formula A' wherein Y is a skeleton selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl, preferably Y is C1-C6 alkyl, Ac is a hydrophilic structural unit, and the position indicated by the wavy line on the right is connected to B or connected to L; B may be present or absent, and when present, is selected from a modifying unit; L is selected from linker units, preferably peptide-containing linker units; X is selected from -NH-, -O- or -S-, preferably -O-; R1 and R2 are the same or different and are each independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl. Preferably, R1 and R2 are the same or different and are each independently selected from halogen, C1-C6 alkyl. More preferably, R1 is selected from C1-C6 alkyl and R2 is selected from halogen. Most preferably, R1 is methyl and R2 is fluorine. R3 and R4 are the same or different and are each independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, C6-C10 arylC1-C6 alkyl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl. Preferably, R3 and R4 are the same or different and are each independently selected from hydrogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, or C6-C10 arylC1-C6 alkyl; More preferably, R3 and R4 are the same or different and are each independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkylC1-C6 alkyl, or phenylC1-C6 alkyl; More preferably, R3 and R4 are the same or different and are each independently selected from hydrogen, methyl, ethyl, trifluoromethyl, cyclopropyl, cyclopropylmethyl, and benzyl; Most preferably, one of R3 and R4 is selected from hydrogen and methyl, and the other is selected from hydrogen, methyl, ethyl, trifluoromethyl, cyclopropyl, cyclopropylmethyl, and benzyl; or R3, R4 and the carbon atom to which they are attached comprise a C3-C8 cycloalkyl, a 3- to 7-membered heterocyclyl, or a substituted 3- to 7-membered heterocyclyl; Preferably, R3, R4 and the carbon atom to which they are attached form a C3-C8 cycloalkyl; More preferably, R3, R4 and the carbon atom to which they are attached form a C3-C6 cycloalkyl; Most preferably, R3, R4 and the carbon atom to which they are attached comprise cyclopropyl, cyclobutyl, or cyclopentyl; R5 is selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl; preferably, R5 is selected from hydrogen, C1-C6 alkyl; more preferably, R5 is selected from hydrogen, methyl; m is selected from integers of 0 to 5, preferably 0 and 1; The chiral carbon atom indicated by * has the R or S absolute configuration; The following condition is satisfied: M' is If JPEG2025533577000213.jpg1323, then B is present and selected from the modification units.

[0024] In some embodiments, M' is JPEG2025533577000214.jpg1326 or a linking unit represented by the following formula A', preferably a linking unit represented by the following formula A'. JPEG2025533577000215.jpg1720 Formula A' wherein Y is a backbone selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl; preferably, Y is C1-C6 alkyl, more preferably C1-C3 alkyl, and most preferably methylene; The position indicated by * has two chiralities, R absolute configuration or S absolute configuration. The position indicated by the wavy line on the right is connected to B or L, The hydrophilic structural unit Ac is selected from, but not limited to, a natural or unnatural amino acid, 1-20 polyethylene glycol, a phosphate group, a carboxylic acid group, a sulfonic acid group, a sulfinic acid group, or the following structure: JPEG2025533577000216.jpg918, JPEG2025533577000217.jpg1020, JPEG2025533577000218.jpg1022, JPEG2025533577000219.jpg1217, JPEG2025533577000220.jpg1722, JPEG2025533577000221.jpg1533, JPEG2025533577000222.jpg1235, JPEG2025533577000223.jpg1540, JPEG2025533577000224.jpg1020, JPEG2025533577000225.jpg917, JPEG2025533577000226.jpg1018 or JPEG2025533577000227.jpg928 where p is selected from integers from 0 to 10, and the positions indicated by the wavy lines are linked to the skeleton Y. Preferably, the hydrophilic structural unit Ac is JPEG2025533577000228.jpg918, JPEG2025533577000229.jpg1020, The positions selected from JPEG2025533577000230.jpg1022 and indicated by the wavy lines are concatenated to skeleton Y. More preferably, the hydrophilic structural unit Ac is JPEG2025533577000231.jpg918, and the position indicated by the wavy line is connected to skeleton Y.

[0025] In some embodiments, the linking unit M' is selected from, but not limited to, structures shown in the following formulae: JPEG2025533577000232.jpg1329, JPEG2025533577000233.jpg1726, JPEG2025533577000234.jpg2228, JPEG2025533577000235.jpg1726, JPEG2025533577000236.jpg1726, JPEG2025533577000237.jpg2526 or JPEG2025533577000238.jpg2526; Preferably, said linking unit M' is JPEG2025533577000239.jpg1329, Selected from JPEG2025533577000240.jpg1725. More preferably, the linking unit M' is JPEG2025533577000241.jpg1725. where: The * position is either the R or S absolute configuration, The position indicated by the wavy line on the right is linked to the modifying unit B or the linker unit L.

[0026] In some embodiments, modifying unit B may be present or absent, and when present, said modifying unit B is selected from, but is not limited to, structures that increase hydrophilicity or stereoisomers thereof.

[0027] In some embodiments, modifying unit B may be present or absent, and when present, the modifying unit B is selected from, but is not limited to, the hydrophilic enhancing structures shown in the following formula or stereoisomers thereof: JPEG2025533577000242.jpg1028, JPEG2025533577000243.jpg1243, JPEG2025533577000244.jpg2017, JPEG2025533577000245.jpg1522, JPEG2025533577000246.jpg2230, JPEG2025533577000247.jpg1735, JPEG2025533577000248.jpg3041, JPEG2025533577000249.jpg2242, JPEG2025533577000250.jpg1517, JPEG2025533577000251.jpg1420; Preferably, the modifying unit B may be present or absent, and when present, the modifying unit B is selected from the following structures that enhance hydrophilicity, or stereoisomers thereof, but is not limited thereto: JPEG2025533577000252.jpg1028, JPEG2025533577000253.jpg1243, JPEG2025533577000254.jpg1522, JPEG2025533577000255.jpg1735, JPEG2025533577000256.jpg2242; where: R is selected from, but not limited to, hydroxyl, amino, polyethylene glycol, a carboxylic acid group, a sulfonic acid group, a sulfinic acid group, a phosphate group, C1-C6 alkyl, C1-C6 alkoxy, a natural or unnatural amino acid residue, a sugar or a derivative thereof, or a combination thereof. Preferably, R is selected from C1-C6 alkoxy, a carboxylic acid group, and amino. More preferably, R is selected from methoxy, a carboxylic acid group, and amino. The position indicated by the wavy line on the left is connected to the connecting unit M'. The position indicated by the wavy line on the right is linked to the linker unit L. q is selected from integers of 1 to 10 (preferably integers of 2 to 8), and is preferably selected from 2, 7, and 8. Most preferably, the modifying unit B is selected from the hydrophilic enhancing structures of the following formulae or stereoisomers thereof: JPEG2025533577000257.jpg1228, JPEG2025533577000258.jpg1341, JPEG2025533577000259.jpg1239, JPEG2025533577000260.jpg1521, JPEG2025533577000261.jpg2230, JPEG2025533577000262.jpg3241, JPEG2025533577000263.jpg3445, JPEG2025533577000264.jpg3243 The position indicated by the wavy line on the left side is linked to linking unit M′, and the position indicated by the wavy line on the right side is linked to linker unit L.

[0028] In some embodiments, the linker unit L is L1-L2, L1 is a peptide residue containing 2 to 10 amino acid residues, and the amino terminus of the peptide residue is linked to linking unit M' or modifying unit B, and the carbonyl terminus is linked to L2. Preferably, L1 is a peptide residue containing 2 to 4 amino acid residues, the amino terminus of which is linked to linking unit M' or modifying unit B, and the carbonyl terminus of which is linked to L2. Preferably, the amino acids are selected from valine, alanine, phenylalanine, glycine, lysine, citrulline, serine, glutamic acid, and aspartic acid, more preferably, the amino acids are selected from valine, alanine, phenylalanine, glycine, lysine, and citrulline, and most preferably, the amino acids are selected from valine, phenylalanine, glycine, lysine, and citrulline. More preferably, L1 is selected from the following peptide residues: glycine-glycine-phenylalanine-glycine, valine-citrulline, phenylalanine-lysine, valine-alanine, and alanine-alanine-alanine (preferably selected from glycine-glycine-phenylalanine-glycine, valine-citrulline, and phenylalanine-lysine), the amino terminus of which is linked to linking unit M' or modifying unit B, and the carbonyl terminus of which is linked to L2. Most preferably, L1 is one of the following peptide residues: JPEG2025533577000265.jpg1543, JPEG2025533577000266.jpg2236, JPEG2025533577000267.jpg2531, JPEG2025533577000268.jpg1226, JPEG2025533577000269.jpg1333 (preferably, JPEG2025533577000270.jpg1543, JPEG2025533577000271.jpg2236, JPEG2025533577000272.jpg2531). The amino terminus of the peptide residue is linked to linking unit M' or modifying unit B, and the carbonyl terminus is linked to L2. L2 is JPEG2025533577000273.jpg810, JPEG2025533577000274.jpg1828, JPEG2025533577000275.jpg1320, JPEG2025533577000276.jpg1743, The position indicated by the wavy line on the left is linked to L1, and the position indicated by the wavy line on the right is linked to X. Preferably, L2 is JPEG2025533577000278.jpg810, JPEG2025533577000279.jpg1828, JPEG2025533577000280.jpg1320, The position indicated by the wavy line on the left is linked to L1, and the position indicated by the wavy line on the right is linked to X. More preferably, L2 is JPEG2025533577000282.jpg810, The position indicated by the wavy line on the left is linked to L1, and the position indicated by the wavy line on the right is linked to X. Preferably, the linker unit L is selected from, but not limited to, the following structures or stereoisomers thereof: JPEG2025533577000284.jpg2238, JPEG2025533577000285.jpg2251, JPEG2025533577000286.jpg2341, JPEG2025533577000287.jpg1538, JPEG2025533577000288.jpg1551, JPEG2025533577000289.jpg1541, JPEG2025533577000290.jpg1638, JPEG2025533577000291.jpg1651, JPEG2025533577000292.jpg1641, JPEG2025533577000293.jpg1554, JPEG2025533577000294.jpg1341, JPEG2025533577000295.jpg1034 or JPEG2025533577000296.jpg1546; where: r is selected from an integer from 1 to 10; More preferably, the linker unit L is selected from the following structures or stereoisomers thereof: JPEG2025533577000297.jpg1341, JPEG2025533577000298.jpg2351, JPEG2025533577000299.jpg2151; The linker unit L is linked to the linking unit M′ or the modifying unit B at the position indicated by the wavy line on the left side of the linker unit L. The position indicated by the wavy line on the right side of the linker unit L is linked to X.

[0029] In some embodiments, the linker-drug is selected from, but not limited to, the following structures or stereoisomers thereof: JPEG2025533577000300.jpg2867, JPEG2025533577000301.jpg2867, JPEG2025533577000302.jpg2866, JPEG2025533577000303.jpg2866, JPEG2025533577000304.jpg2866, JPEG2025533577000305.jpg2866, JPEG2025533577000306.jpg2866, JPEG2025533577000307.jpg3368, JPEG2025533577000308.jpg2866, JPEG2025533577000309.jpg3368, JPEG2025533577000310.jpg2866, JPEG2025533577000311.jpg3368, JPEG2025533577000312.jpg2866, JPEG2025533577000313.jpg2866, JPEG2025533577000314.jpg2966, JPEG2025533577000315.jpg2966, JPEG2025533577000316.jpg3166, JPEG2025533577000317.jpg3166, JPEG2025533577000318.jpg3166, JPEG2025533577000319.jpg3166, <h2 style=";text-align:left;direction:ltr">JPEG2025533577000320.jpg2867、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000321.jpg2867、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000322.jpg2866、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000323.jpg2867、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000324.jpg2866、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000325.jpg2867、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000326.jpg2966、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000327.jpg2967、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000328.jpg3166、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000329.jpg3167、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000330.jpg2866、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000331.jpg3369、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000332.jpg3183、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000333.jpg34128、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000334.jpg34128、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000335.jpg3876、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000336.jpg40128、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000337.jpg38128、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000338.jpg3566、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000339.jpg3366、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000340.jpg3366、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000341.jpg3578、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000342.jpg3578、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> JPEG2025533577000343.jpg3778、 JPEG2025533577000344.jpg3778 or JPEG2025533577000345.jpg3778.

[0030] In a third aspect, the present invention provides a method for preparing the antibody-drug conjugate according to the first aspect, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, comprising the steps of: JPEG2025533577000346.jpg94128Ab is linked to a linker-drug represented by general formula III to prepare an antibody-drug conjugate represented by general formula I of the first aspect, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof. where: the Ab is selected from an anti-CD33 antibody or an antigen-binding fragment thereof; Preferably, said anti-CD33 antibody is as defined in the first aspect, M1, M2, M3, B, L, X, R1, R2, R3, R4, R5, m, n1, n2, n3 are as defined in the first aspect, and M' is as defined in the second aspect; The chiral carbon atom at the position indicated by * has two chiralities, R absolute configuration or S absolute configuration.

[0031] In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising the antibody-drug conjugate or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the first aspect, or the linker-drug or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the second aspect, and optionally a pharmaceutically acceptable carrier.

[0032] In a fifth aspect of the present invention, there is provided a pharmaceutical formulation comprising the antibody-drug conjugate or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the first aspect, or the linker-drug or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the second aspect.

[0033] A sixth aspect of the present invention provides use of the antibody-drug conjugate or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the first aspect, or the linker-drug or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the second aspect, or the pharmaceutical composition according to the fourth aspect and / or the pharmaceutical formulation according to the fifth aspect in the preparation of a medicament for treating or preventing cancer or tumors.

[0034] In some embodiments, the cancer or tumor expresses CD33.

[0035] In some embodiments, the cancer or tumor is selected from a solid tumor or a hematological tumor, such as adenocarcinoma, 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 (e.g., non-small cell lung cancer), colon cancer, breast cancer (e.g., triple-negative breast cancer), rectal cancer, colorectal cancer, bone cancer, skin cancer (e.g., epidermal cancer), thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, myeloma, leukemia, etc.

[0036] In some embodiments, the cancer or tumor is selected from adenocarcinoma, 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, triple-negative breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, leukemia, or other solid tumors or hematological tumors.

[0037] In a seventh aspect of the present invention, there is provided an antibody-drug conjugate or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the first aspect, or a linker-drug or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to the second aspect, or a pharmaceutical composition according to the fourth aspect and / or a pharmaceutical formulation according to the fifth aspect, for use in treating or preventing cancer or tumors.

[0038] In some embodiments, the cancer or tumor expresses CD33.

[0039] In some embodiments, the cancer or tumor is selected from a solid tumor or a hematological tumor, such as adenocarcinoma, 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 (e.g., non-small cell lung cancer), colon cancer, breast cancer (e.g., triple-negative breast cancer), rectal cancer, colorectal cancer, bone cancer, skin cancer (e.g., epidermal cancer), thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, myeloma, leukemia, etc.

[0040] In some embodiments, the cancer or tumor is selected from adenocarcinoma, 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, triple-negative breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, leukemia, or other solid tumors or hematological tumors.

[0041] In an eighth aspect of the present invention, there is provided a method for treating or preventing cancer or tumor, comprising administering to a subject in need thereof an effective amount of the antibody-drug conjugate or a stereoisomer, pharmaceutically acceptable salt or solvate thereof described in the first aspect, or the linker-drug or a stereoisomer, pharmaceutically acceptable salt or solvate thereof described in the second aspect, or the pharmaceutical composition described in the fourth aspect and / or the pharmaceutical formulation described in the fifth aspect.

[0042] In some embodiments, the cancer or tumor expresses CD33.

[0043] In some embodiments, the cancer or tumor is selected from a solid tumor or a hematological tumor, such as adenocarcinoma, 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 (e.g., non-small cell lung cancer), colon cancer, breast cancer (e.g., triple-negative breast cancer), rectal cancer, colorectal cancer, bone cancer, skin cancer (e.g., epidermal cancer), thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, myeloma, leukemia, etc.

[0044] In some embodiments, the cancer or tumor is selected from adenocarcinoma, 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, triple-negative breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, leukemia, or other solid tumors or hematological tumors. [Brief explanation of the drawings]

[0045] [Figure 1A] 1 shows aggregation of ADC-46 detected by SEC-HPLC. [Figure 1B] Figure 1 shows aggregation of ADC-47 detected by SEC-HPLC. [Figure 1C] Figure 1 shows aggregation of ADC-48 detected by SEC-HPLC. [Figure 1D] Figure 1 shows aggregation of ADC-50 detected by SEC-HPLC. [Figure 1E] 1 shows aggregation of ADC-3 detected by SEC-HPLC. [Figure 1F] Figure 1 shows aggregation of ADC-34 detected by SEC-HPLC. [Figure 1G] Figure 1 shows aggregation of ADC-44 detected by SEC-HPLC. [Figure 1H] 1 shows aggregation of ADC-49 detected by SEC-HPLC. [Figure 2A]The drug-antibody binding ratio (DAR) of ADC-46 detected by RP-HPLC is shown. [Figure 2B] The drug-antibody binding ratio (DAR) of ADC-47 detected by RP-HPLC is shown. [Figure 2C] The drug-antibody binding ratio (DAR) of ADC-48 detected by RP-HPLC is shown. [Figure 2D] The drug-antibody binding ratio (DAR) of ADC-50 detected by RP-HPLC is shown. [Figure 2E] The drug-antibody conjugation ratio (DAR) of ADC-3 detected by RP-HPLC is shown. [Figure 2F] The drug-antibody binding ratio (DAR) of ADC-34 detected by RP-HPLC is shown. [Figure 2G] The drug-antibody binding ratio (DAR) of ADC-44 detected by RP-HPLC is shown. [Figure 2H] The drug-antibody binding ratio (DAR) of ADC-49 detected by RP-HPLC is shown. [Figure 3] The results of measuring the molecular weight of ADC-3 are shown. [Figure 4A] This shows that ADC-3 of the present invention maintains the same affinity for the CD33 antigen as the naked antibody. [Figure 4B] This shows that ADC-48 of the present invention maintains the same affinity for the CD33 antigen as the naked antibody. [Figure 4C] This shows that ADC-46 and ADC-47 of the present invention maintain the same affinity for the CD33 antigen as the naked antibody. [Figure 5A] 1 shows the killing curves of the anti-CD33 of the present invention, ADC-3, ADC-47, and ADC-48 against HL-60 cells. [Figure 5B] 1 shows the killing curves of the anti-CD33 of the present invention, ADC-3, ADC-47, and ADC-48 against HEL92.1.7 cells. [Figure 5C] 1 shows the killing curves of the anti-CD33, ADC-3, ADC-47, and ADC-48 of the present invention against TF-1 cells. [Figure 5D]1 shows the killing curves of MV4-11 cells by the anti-CD33 of the present invention, ADC-3, ADC-47, and ADC-48. [Figure 5E] 1 shows the killing curves of the anti-CD33 of the present invention, ADC-3, ADC-47, and ADC-48 against MOLM-13 cells. [Figure 5F] 1 shows the killing curves of the anti-CD33, ADC-3, ADC-47, and ADC-48 of the present invention against U937 cells. [Figure 5G] 1 shows the killing curves of the anti-CD33, ADC-3, ADC-47, and ADC-48 of the present invention against CMK cells. [Figure 6A] 1 shows the tumor-suppressing effects of anti-CD33 antibodies and antibody-drug conjugates in a mouse HEL92.1.7 cell subcutaneously transplanted tumor model. [Figure 6B] 1 shows the tumor-suppressing effects of anti-CD33 antibodies and antibody-drug conjugates in a mouse MV4-11 cell subcutaneously transplanted tumor model. [Figure 7A] 1 shows the killing curves of the anti-CD33 of the present invention, the small molecule drugs D3, ADC-3, and ADC-34 against HL-60 cells. [Figure 7B] 1 shows the killing curves of the anti-CD33 of the present invention, the small molecule drugs D3, ADC-3, and ADC-34 against JVM-3 cells. [Figure 7C] 1 shows the killing curves of the anti-CD33 of the present invention, the small molecule drugs D3, ADC-3, and ADC-34 against Raji cells. DETAILED DESCRIPTION OF THE INVENTION

[0046] Abbreviations and Definitions Unless otherwise indicated, the following words and phrases as used herein shall have the following meanings: When trade names are used herein, they include formulations, generic drugs, and active ingredients of the trade name products unless the context otherwise requires. Unless stated to the contrary, terms used in the claims and this specification have the following meanings.

[0047] The terms "antibody" or "antigen-binding fragment" include within their scope any portion of an antibody structure. This unit can bind to, reactively associate with, or complex with a receptor, antigen, or other receptor unit on a target cell population. An antibody can be any protein or protein-like molecule capable of binding to, complexing with, or reacting with a portion of a cell population to be treated or biologically modified. In the present invention, the antibody conjugates retain their original, wild-type antigen-binding ability. Thus, the antibodies of the present invention can specifically bind to an antigen. Relevant antigens include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (e.g., those with known or predictable functionality), lymphokines, cytokines, molecules involved in cell cycle control, molecules involved in angiogenesis, and molecules associated with angiogenesis (e.g., those with known or predictable functionality). Tumor-associated factors can be cluster differentiation factors (e.g., CD proteins).

[0048] 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 using antibody preparation methods and information well known in the art. To develop effective cellular targets for cancer diagnosis and treatment, researchers have focused on transmembrane polypeptides and other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells, while being expressed little or not at all on the surface of one or more non-cancerous cells. Typically, such tumor-associated polypeptides are overexpressed on the surface of cancer cells relative to the surface of non-cancerous cells. Identifying such tumor-associated factors can significantly improve the specific targeting properties of antibody-based cancer therapy. For convenience, information related to antigens well known in the art (such as names, other names, and GenBank accession numbers) is provided below. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases such as GenBank. The tumor-associated antigen corresponding to the antibody target includes all amino acid sequence variants and isoforms, and has at least 70%, 80%, 85%, 90% or 95% identity with the sequence identified in the reference, or has biological properties and characteristics that are completely identical to the sequence of the tumor-associated antigen described in the reference.

[0049] In the present invention, the term "antibody" refers to an immunoglobulin having a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds. The antigenicity of immunoglobulins varies due to differences in the amino acid composition and sequence order of the heavy chain constant regions. Therefore, immunoglobulins are classified into five classes (also called immunoglobulin isoforms): IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Even within the same class, Ig is further classified into different subclasses based on differences in the amino acid composition of the hinge region and the number and location of disulfide bonds in the heavy chain. For example, IgG is classified into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ and λ chains based on differences in the constant region. Each of the five Ig classes can have either κ or λ chains. The antibodies described in the present invention are preferably specific for cell surface antigens on target cells, specifically, anti-CD33 antibodies.

[0050] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. Biol. Chem. 1968.243.3558.

[0051] The term "drug" refers to a cytotoxic drug (drug is designated by D), which is a chemical molecule with a strong ability to inhibit the normal growth of cells.

[0052] The term "antibody-drug conjugate" refers to the conjugation of an antibody to a biologically active drug via a linking unit, optionally a modifying unit, and a linker unit.

[0053] The term "linking unit" refers to a chemical structural fragment or bond that is linked at one end to an antibody or antigen-binding fragment thereof and at the other end to a modifying unit or a linker unit, and may be linked to another linking unit and then to a drug.

[0054] The term "linker unit" includes a stretcher, spacer, or amino acid unit, and can be synthesized according to methods known in the art (eg, the method described in US2005-0238649A1).

[0055] Depending on the mechanism of intracellular drug release, the term "linker unit" or "linker" as used herein can be divided into two types: non-cleavable linker units and cleavable linker units. The drug release mechanism of antibody-drug conjugates containing non-cleavable linker units is as follows: After the conjugate binds to an antigen and is taken up by cells, the antibody is enzymatically hydrolyzed in the lysosome, releasing an active molecule consisting of a small molecule drug, a linker unit, and antibody amino acid residues. This change in the drug molecule structure does not reduce its cytotoxicity, but the charged active molecule (amino acid residues) prevents it from penetrating neighboring cells. Therefore, such active drugs cannot kill neighboring tumor cells that do not express the target antigen (antigen-negative cells) (the bystander effect) (Ducry et al., 2010, Bioconjugate Chem. 21:5-13). The linker unit may be a "cleavable linker unit" that facilitates drug release in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, or a disulfide-containing linker (Charliet et al. Cancer Research 52:127-131, 1992; U.S. Patent No. 5,208,020) can be used.

[0056] The term "modifying unit" refers to any one or combination of a chemical bond or stretcher, spacer, amino acid unit having a branched structure, sugar unit, polyethylene glycol unit, carbonyl group, amide group, carboxylic acid group, phosphate group, sulfonic acid group, sulfinic acid group, alkyl group, cycloalkyl group, heterocyclyl group, aryl group, or heteroaryl group, which is linked at one end to a linking unit and at the other end to a linker unit. The role of the modifying unit is to increase the hydrophilicity of the antibody-drug conjugate or to increase the drug loading capacity by providing a branched structure.

[0057] In formula I of the present invention, JPEG2025533577000347.jpg10976B may or may not be present. Those skilled in the art will recognize that when B is present, the structure of Formula I is as described above, and when B is absent, the structure of Formula I is It can be understood that the definition is JPEG2025533577000348.jpg10276. Other similar definitions can be understood by referring to the above content.

[0058] Therefore, in the present invention, the connecting units M1, M2, and M3 each independently: JPEG2025533577000349.jpg1728, JPEG2025533577000350.jpg2026, JPEG2025533577000351.jpg1523, and the position indicated by the wavy line on the right is linked to modifying unit B or linker unit L. A person skilled in the art would understand that if the modifying unit B is present, the position indicated by the wavy line on the right is linked to modifying unit B, and if the modifying unit B is not present, the position indicated by the wavy line on the right is linked to linker unit L. Other similar definitions can be understood by referring to the above content.

[0059] The term "drug loading" or "drug-antibody ratio (DAR)" refers to the average number of cytotoxic drugs loaded onto each antibody in Formula I (e.g., n1+n2+n3 in General Formula I), which can be expressed as the ratio of the amount of drug to the amount of antibody and can be an integer or a decimal. The range of drug loading (e.g., n1+n2+n3 in General Formula I) is such that each antibody can be bound to an average of 1 to 10 cytotoxic drugs (D) (i.e., any integer selected from 1 to 10 (including endpoints 1 and 10), or any decimal number between 1 and 10). Preferably, the range is 8 to 10 cytotoxic drugs (D) (i.e., any integer selected from 8 to 10 (including endpoints 8 and 10), or any decimal number between 8 and 10).

[0060] When preparing the antibody-drug conjugates provided by the present invention, the linker-drug represented by general formula III is easily hydrolyzed under hydrolysis conditions when bound to an Ab, and the hydrolysis site is the maleimide moiety. When an Ab is bound to multiple linker-drugs, the following situations may occur depending on the degree of hydrolysis: (1) The maleimides are not hydrolyzed, i.e., all maleimides are in a closed ring state. (2) Maleimide is incompletely hydrolyzed, i.e., part of the maleimide is in a closed ring state. JPEG2025533577000353.jpg1513, and the other parts of the maleimide are in an open state. JPEG2025533577000354.jpg1315 or (3) The maleimide is completely hydrolyzed, i.e., all the maleimides are in the open ring state. JPEG2025533577000356.jpg1315 or JPEG2025533577000357.jpg1215. Thus, in the antibody-drug conjugate represented by general formula I, n1, n2, and n3 are each independently selected from any integer or decimal number between 0 and 10, and n1, n2, and n3 are not simultaneously 0. At the same time, as described above, those skilled in the art will understand that the number of linker-drugs conjugated to each Ab can be the same or different, and therefore the average number of linker-drugs conjugated to each Ab can be an integer or decimal number. Therefore, when linker-drugs are conjugated to Abs, the maleimide moieties are hydrolyzed under conditions that allow for easy hydrolysis, and the degree of hydrolysis can be the same or different, and therefore n1, n2, and n3 can be integers or decimal numbers.

[0061] The term "pharmaceutically acceptable salt" refers to a salt of an antibody-drug conjugate or linker-drug of the present invention, or a salt of a compound described in the present invention, which is safe and effective when used in mammals and has the desired biological activity. For example, a carboxylic acid group in an antibody-drug conjugate or linker-drug of the present invention may form a salt with a base, non-limiting examples of which include sodium, potassium, calcium, or magnesium salts. An amino group in an antibody-drug conjugate or linker-drug of the present invention may also form a salt with an acid, non-limiting examples of which include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogensulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogenphosphate, dihydrogenphosphate, salicylate, hydrogencitrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate. In some embodiments, "pharmaceutically acceptable salts" of the present invention include sodium, potassium, calcium, or magnesium salts formed with acidic functional groups in the structural formula, as well as acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate salts formed with basic functional groups in the structural formula.

[0062] The term "amino acid residue" refers to an incomplete amino acid structure having an amino terminus and a carbonyl terminus, which remains after one hydrogen atom is lost from the amino group of an amino acid and one hydroxyl group is lost from the carboxyl group. In the present invention, L1 is a peptide residue consisting of 2 to 10 (preferably 2 to 4) amino acid residues, and the types of the 2 to 10 (preferably 2 to 4) amino acids may be the same or different. For example, when L1 is a peptide residue consisting of 4 amino acid residues, and the amino acids are selected from glycine and phenylalanine, L1 may be a peptide residue of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), specifically: It could also be JPEG2025533577000358.jpg2044.

[0063] The term "solvate" refers to a pharmaceutically acceptable solvate formed by an antibody-drug conjugate or linker-drug of the invention and one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate, etc.

[0064] The term "absolute configuration" refers to the actual spatial arrangement of groups in a chiral molecule, i.e., the absolute spatial relationship. In the present invention, a configuration notation characterized in that groups bonded to a chiral carbon atom are arranged in different spatial directions, i.e., the R,S configuration notation, is adopted.

[0065] The term "naturally occurring amino acid" refers to an amino acid that is biologically synthesized. Naturally occurring amino acids are generally L-configured, with some exceptions, such as glycine, including both naturally occurring and biologically synthesized amino acids.

[0066] The term "unnatural amino acid" refers to an amino acid obtained by synthetic means.

[0067] The term "stereoisomer" refers to compounds with the same molecular formula in which the bonding order of atoms or substituents is the same but the spatial arrangement is different, and belongs to the isomerism phenomenon in organic chemistry, similar to structural isomers.

[0068] The term "hydroxy" refers to --OH.

[0069] The term "amino" refers to -NH2.

[0070] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0071] The term "polyethylene glycol" refers to an oligomer or polymer of ethylene oxide.

[0072] The term "carboxylic acid group" refers to -COOH.

[0073] The term "sulfonic acid group" refers to -S(O)2OH.

[0074] The term "sulfinic acid group" refers to -S(O)OH.

[0075] The term "phosphate group" refers to -OP(O)2OH.

[0076] The term "sugar" refers to a polyhydroxy(two or more) aldehyde or ketone compound, or an organic compound that can be converted to any of the above by hydrolysis.

[0077] The term "derivative" refers to a product produced by replacing an atom or group in a compound molecule with another atom or group.

[0078] The term "alkyl" refers to saturated aliphatic hydrocarbon groups, including straight- or branched-chain groups of 1 to 20 carbon atoms (i.e., "C1-C20 alkyl"), preferably alkyl of 1 to 12 carbon atoms (i.e., "C1-C12 alkyl"), more preferably alkyl of 1 to 10 carbon atoms (i.e., "C1-C10 alkyl"), even more preferably alkyl of 1 to 6 carbon atoms (i.e., "C1-C6 alkyl"), even more preferably alkyl of 1 to 4 carbon atoms (i.e., "C1-C4 alkyl"), and most preferably alkyl of 1 to 3 carbon atoms (i.e., "C1-C3 alkyl"). Examples of "C1-C6 alkyl" include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. Examples of "C1-C4 alkyl" include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), etc. Examples of "C1-C3 alkyl" include methyl, ethyl, propyl (e.g., n-propyl, isopropyl), etc.

[0079] The term "substituted" refers to the replacement of a hydrogen atom in a compound with a substituent. Unless otherwise specified herein, the substituent can be a variety of groups selected from -halogen, -OR', -NR'R'', -SR', -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)R', -NH-C(NH)=NH, -NR'C(NH)=NH, -NH-C(NH)=NR', -S(O)R', -S(O)R', -S(O)NR'R'', -NR'S(O)R'', -CN, and -NO. The number of substituents ranges from 1 to (2m'+1), where m' is the total number of carbon atoms in the group. R', R" and R"' each independently represent hydrogen, unsubstituted C1-C6 alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted with 1-3 halogens, unsubstituted C1-C6 alkoxy or C1-C6 thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)C1-C6 alkyl. When R' and R" are attached to the same nitrogen atom, they can be combined with the atom to form a 3-, 4-, 5-, 6- or 7-membered ring.

[0080] The term "halogenated alkyl" refers to a group in which one or more hydrogen atoms in the above alkyl (e.g., C1-C20 alkyl, C1-C12 alkyl, C1-C10 alkyl, C1-C6 alkyl, C1-C4 alkyl, C1-C3 alkyl, etc.) have been substituted with halogen (preferably fluorine). Examples of "halogenated C1-C6 alkyl" include monofluoromethyl, difluoromethyl, difluoroethyl, trifluoromethyl, etc.

[0081] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent. The cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, even more preferably 3 to 8 carbon atoms, and most preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. Polycyclic cycloalkyls include spirocyclic, fused-ring, and bridged-ring cycloalkyls. Examples of "C3-C6 cycloalkyl" include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0082] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms (i.e., a "3- to 20-membered heterocyclyl group"), in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or sulfur (excluding ring moieties of -OO-, -OS-, or -SS-), and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms (i.e., a "3- to 12-membered heterocyclyl"), of which 1 to 4 are heteroatoms. More preferably, the heterocyclyl ring contains 3 to 10 ring atoms (i.e., a "3- to 10-membered heterocyclyl"). Non-limiting examples of monocyclic heterocyclyls (e.g., 3- to 7-membered heterocyclyls) include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyls include spirocyclic, fused-ring, and bridged-ring heterocyclyls.

[0083] The term "C6-C10 aryl" refers to a carbocyclic aromatic group having 6 to 10 carbon atoms, such as benzene, naphthalene, and the like.

[0084] The term "cycloalkylalkyl" refers to an alkyl substituted with one or more cycloalkyls, preferably one cycloalkyl, where alkyl is as defined above and cycloalkyl is as defined above. Examples include C3-C8 cycloalkylC1-C6 alkyl and C3-C6 cycloalkylC1-C6 alkyl. Similarly, the term "C6-C10 arylC1-C6 alkyl" refers to a C1-C6 alkyl substituted with one or more C6-C10 aryls, preferably one C6-C10 aryl, where alkyl is as defined above and C6-C10 aryl is as defined above. A specific example is "phenylC1-C6 alkyl" which means that the C1-C6 alkyl is substituted with one phenyl, and examples include benzyl. Other similar definitions can be understood by referring to the above content.

[0085] The term "alkoxy" refers to -O-(alkyl), where alkyl is as defined above. Non-limiting examples of C-C alkoxy include methoxy, ethoxy, propoxy, and butoxy. The term "cycloalkoxy" refers to -O-(cycloalkyl), where cycloalkyl is as defined above. Non-limiting examples of C-C cycloalkoxy include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. An alkoxy or cycloalkoxy can be optionally substituted or unsubstituted; if substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0086] The term "heteroaryl" refers to an aromatic heterocycle, typically a 5-, 6-, 7-, 8-, 9-, or 10-membered heterocycle having 1 to 3 heteroatoms selected from N, O, or S. Heteroaryl rings may optionally be further fused to or attached to aromatic and non-aromatic carbocyclic and heterocyclic rings.

[0087] In one embodiment of the present invention, the cytotoxic drug is conjugated to the open cysteine ​​thiol-SH of the antibody interchain and / or the thiol-SH of the site-directed mutated cysteine ​​residue via a linker unit, an optional modifying unit, and a linking unit. Generally, the number of drug molecules that can be conjugated to the antibody in a conjugation reaction is less than or equal to the theoretical maximum.

[0088] The loading of the ligand-cytotoxic drug conjugate can be controlled by the following non-limiting methods. (1) Control the molar ratio of linker to monoclonal antibody. (2) Controlling the reaction time and temperature. (3) Select a different reaction reagent.

[0089] The term "pharmaceutical composition" refers to a mixture containing one or more antibody-drug conjugates or stereoisomers, pharmaceutically acceptable salts or solvates thereof, or linker-drugs or stereoisomers, pharmaceutically acceptable salts or solvates thereof, as described herein, together with other chemical components and other ingredients, such as physiologically or pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism and promote absorption of the active ingredient(s), thereby exerting biological activity.

[0090] For the preparation of conventional pharmaceutical compositions, please refer to the Chinese Pharmacopoeia.

[0091] The term "carrier" refers to a system that can modify the uptake and distribution of drugs in the body, control the drug release rate, and deliver drugs to target organs. The release and targeting system of a drug carrier can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymer surfactants that can be used as carriers can self-assemble to form aggregates of various morphologies due to their unique amphiphilic structure. Preferred examples include micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates can encapsulate drug molecules and have good membrane permeability, making them excellent drug carriers.

[0092] The term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic, in terms of partial or complete stabilization or cure of the disease and / or side effects resulting from the disease. As used herein, "treatment" includes any treatment of a disease in a patient, including: (a) preventing the onset of a disease or condition in a patient who is susceptible to, but has not yet been diagnosed with, the disease or condition; (b) suppressing the symptoms of the disease, i.e., arresting its progression; or (c) alleviating the symptoms of the disease, i.e., causing regression of the disease or condition.

[0093] The term "subject" includes humans or non-human animals. Exemplary human subjects include humans (referred to as patients) suffering from a disease (e.g., a disease described herein) or normal individuals. As used herein, the term "non-human animal" includes all vertebrates, e.g., non-mammals (e.g., birds, amphibians, reptiles), as well as mammals such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0094] The term "effective amount" refers to that amount of a drug that, upon administration, relieves to some extent one or more of the symptoms being treated.

[0095] The present invention will be further described below with reference to specific examples. However, it should be understood that these examples are used only to illustrate the present invention and are not intended to limit the scope of the present invention. In the following examples, test methods for which specific conditions are not specified are generally carried out according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all percentages, ratios, proportions, or parts are by weight. [Example]

[0096] Example 1: Synthesis of Compound M1 N-Fluorenylmethoxycarbonyl-glycine-glycine (100 g, 282 mmol, 1.0 eq), lead tetraacetate (175 g, 395 mmol, 1.4 eq), 2000 mL of dry tetrahydrofuran, and 670 mL of toluene were added to a 5000 mL one-neck flask, stirred uniformly under a nitrogen atmosphere, heated to 85°C, and reacted for 2.5 hours. The reaction was monitored by TLC until the reaction of the raw materials was complete. The reaction solution was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain compound M1 (87 g) (LC-MS: [M+NH4] + =386.0).

[0097] Example 2: Synthesis of Compound M3 Compound SM-2 (synthesized according to the method disclosed in patent CN108452321A) (40 g, 96 mmol, 1.0 eq), triethylamine (26.7 mL, 2.0 eq), and toluene (400 mL) were added to a 1000 mL single-neck flask, heated to 120 °C, and refluxed for 2 hours. After confirming completion of the reaction by TLC, the temperature was lowered to 50 °C and the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (150 mL) and water (40 mL). The pH was adjusted to 2-3 with 1 M HCl while stirring in an ice bath, and the layers were separated. The aqueous layer was extracted again with ethyl acetate, and the combined organic layers were dried over anhydrous sodium sulfate. After filtration and concentration, a pale yellow crude product was obtained. The crude product was purified by column chromatography (DCM:MeOH=40:1) to give compound M2 (26.6 g) (LC-MS: [M+H] + =399.3). Compound M2 (26.5 g, 60.5 mmol, 1.0 eq), pentafluorophenol (12.2 g, 66.5 mmol, 1.1 eq), DCC (13.7 g, 66.5 mmol, 1.1 eq), and THF (300 mL) were added to a 1000 mL single-neck flask and reacted at room temperature for 30 minutes (monitored by TLC). Insoluble materials were removed by filtration. The reaction solution was directly purified by preparative HPLC. The preparative solution was concentrated under reduced pressure in a 35 °C water bath using a water pump to remove acetonitrile, and lyophilized to give compound M3 (31.5 g) (yield 64%, LC-MS: [M+H]). + =565.1).

[0098] Example 3: Synthesis of Compound P1 JPEG2025533577000361.jpg94136Step 1: Compound 1a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was cooled to 0 °C with stirring, and benzyl hydroxyacetate (5.4 g, 32.6 mmol) was added dropwise. After the addition, the mixture was allowed to warm to room temperature under TLC monitoring (reaction time: approximately 2-4 h). After completion of the reaction, saturated NaHCO3 solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-1:1) to give 1a (4 g) (yield 52%, LC-MS: [M+H] + =475.18). Step 2: Compound 1b In a 25 mL single-neck flask, 1a (2 g, 4.2 mmol) and 10 mL of DMF were added and stirred at 0°C. DBU (766 mg, 5.04 mmol) was added and the reaction was continued for 1 hour under TLC monitoring. After the Fmoc group was completely deprotected, the reaction was left for use. In a separate 25 mL single-neck flask, M4 (prepared according to the method disclosed in Patent CN111051330A, 1.73 g, 4.2 mmol), PyBOP (2.61 g, 5.04 mmol), HOBt (680 mg, 5.04 mmol), and 10 mL of DMF were added. DIPEA (830 μL, 5.04 mmol) was added under ice-water bath conditions and stirred for 30 minutes. The reaction solution was then added to the reaction flask and allowed to warm to room temperature. After confirming the completion of the reaction by HPLC, the reaction solution was purified by preparative HPLC to obtain a preparative solution of the product. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain solid 1b (1.7 g) (yield 63%, LC-MS: [M+H] + =648.26). Step 3: Compound 1c 1b (900 mg, 1.39 mmol) was placed in a 25 mL single-neck flask and dissolved in 15 mL of DMF. 900 mg of 5% Pd / C was added and the hydrogenation reaction was carried out for 2 hours. After completion of the reaction, the mixture was filtered, and the obtained filtrate was used in the next step without further purification. Step 4: Compound 1d The crude product 1c was placed in an ice-water bath, DIPEA (235 μL, 1.39 mmol) was added, followed by compound M3 (784 mg, 1.39 mmol), and the mixture was warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fractionated solution. The fractionated solution was lyophilized to obtain 1d (504 mg) (LC-MS: [M+H] + =804.4). Step 5: Compound 1e 1d (500 mg, 0.62 mmol), M5 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were added to a 50 mL single-neck flask. DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath, the mixture was warmed to room temperature, and the reaction was carried out for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 1e. The fraction was lyophilized to obtain 1e (210 mg) (LC-MS: [M+H] + =1221.6). Step 6: Compound P1 1e (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain solid compound P1 (60 mg) (LC-MS: [M+H] + =1065.3).

[0099] Example 4: Synthesis of Compound P2 JPEG2025533577000362.jpg94136Step 1: Compound 2a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was cooled to 0 °C with stirring, and benzyl 2-hydroxy-2-methylpropionate (6.3 g, 32.6 mmol) was added dropwise. After the addition, the mixture was allowed to warm to room temperature under TLC monitoring (reaction time: approximately 2-4 h). After completion of the reaction, saturated NaHCO3 solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to give 2a (4.2 g) (yield 52%, LC-MS: [M+H] + =503.3). Step 2: Compound 2b In a 25 mL single-neck flask, 2a (2 g, 4.0 mmol) and 10 mL of DMF were added and stirred at 0 °C. DBU (760 mg, 5.0 mmol) was added and the reaction was continued for 1 h under TLC monitoring. After the Fmoc group was completely deprotected, the mixture was left for use. In a separate 25 mL one-neck flask, M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol), and 10 mL of DMF were added. DIPEA (823 μL, 5.04 mmol) was added under ice-water bath conditions and stirred for 30 minutes. The reaction solution was then added to the reaction flask and allowed to warm to room temperature. After confirming the completion of the reaction by HPLC, the reaction solution was purified by preparative HPLC to obtain a preparative solution of the product. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain solid 2b (1.4 g) (yield 53%, LC-MS: [M+H] + =676.2). Step 3: Compound 2c 2b (700 mg, 1.04 mmol) was added to a 25 mL single-neck flask and dissolved in 10 mL of DMF. 700 mg of 5% Pd / C was added, and the hydrogenation reaction was carried out for 1.5 hours. After completion of the reaction, the mixture was filtered, and the obtained filtrate was used in the next step without further purification. Step 4: Compound 2d The crude product 2c was placed in an ice-water bath, DIPEA (210 μL, 1.25 mmol) was added, followed by compound M3 (704 mg, 1.25 mmol), and the mixture was warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fractionated solution. The fractionated solution was lyophilized to obtain 2d (486 mg) (LC-MS: [M-H] - =830.5). Step 5: Compound 2e 2d (300 mg, 0.36 mmol), M5 (180 mg, 0.36 mmol), PyBOP (260 mg, 0.5 mmol), HOBt (67 mg, 0.5 mmol), and 10 mL of DMF were added to a 50 mL single-neck flask. DIPEA (219.5 μL, 1.33 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 3 hours. After confirming completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 2e. The fraction was lyophilized to obtain 2e (157 mg) (LC-MS: [M+H] + =1249.6). Step 6: Compound P2 2e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain solid compound P2 (64 mg) (LC-MS: [M+H] + =1093.1).

[0100] Example 5: Synthesis of Compound P3 JPEG2025533577000363.jpg86140 Step 1: Compound 3a A 25 mL single-neck flask was charged with M1 (500 mg, 1.4 mmol), p-toluenesulfonic acid monohydrate (26 mg, 0.1 mmol), and 10 mL of THF. The mixture was stirred evenly and then cooled to 0 °C. L-benzyl lactate (1.2 g, 7.0 mmol) was slowly added. After the addition, the mixture was allowed to warm to room temperature and react. After confirming the completion of the reaction by TLC monitoring, saturated NaHCO3 solution was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified using a reverse-phase column to give 3a (400 mg) (LC-MS: [M+NH4] + =506.2). 1 H NMR(400Mz,CDCl3 / CD3OD):1.39(3H,d,J=6.8Hz), 3.78(2H,t,J=4.0Hz), 4.17-4.27(2H,m), 4.42(2H,d,J=4.0Hz), 4.72-4.85(2H,m), 5.11 -5.58(2H,m), 5.43(1H,s), 7.06(1H,t,J=8.0Hz), 7.25-7.33(6H,m), 7.38(2H,t,J=8.0Hz), 7.57(2H,d,J=8.0Hz), 7.75(2H,d,J=8.0Hz). Step 2: Compound 3b Compound 3a (400 mg, 0.8 mmol, 1.0 eq) and 4 mL of DMF were added to a 25 mL single-neck flask and stirred uniformly. The mixture was then cooled to 0°C, and DBU (137 mg, 0.9 mmol, 1.1 eq) was slowly added. After the addition, the mixture was allowed to warm to room temperature and react. The completion of the reaction was confirmed by TLC monitoring, and the reaction solution was recorded as reaction solution (1). Into another 25 mL one-neck flask, M4 (372 mg, 0.9 mmol, 1.1 eq), PyBOP (852 mg, 1.6 mmol, 2.0 eq), and 3 mL of DMF were added and stirred at room temperature for 5 minutes. Reaction solution (1) was added and the reaction was allowed to proceed at room temperature. The reaction was monitored by HPLC. After completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain compound 3b (326 mg) (LC-MS: [M+NH] + =679.2). Step 3: Compound 3c 3b (4.0 g, 6.05 mmol, 1.0 eq) was added to a 100 mL single-neck flask and dissolved in DMF (60 mL). 5% Pd / C (4 g) was added and the reaction was carried out at room temperature for 4 h. (The reaction progress was monitored by HPLC.) The Pd / C was filtered, and the filtrate was placed in an ice-water bath (approximately 0 °C) without concentration for further use. Step 4: Compound 3d The crude product 3c was placed in an ice-water bath, and DIPEA (1.1 mL, 1.1 eq) was added, followed by compound M3 (3.4 g, 6.05 mmol). After the addition, the mixture was warmed to room temperature and reacted for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fractionated solution. The fractionated solution was lyophilized to obtain 3d (3.15 g) (LC-MS: [M-H] - =816.3). Step 5: Compound 3e A 100 mL single-neck flask was charged with 3d (2.07 g, 2.53 mmol, 1.0 eq), M5 (1.35 g, 2.53 mmol, 1.0 eq), PyBOP (1.98 g, 3.79 mmol, 1.5 eq), HOBt (0.51 g, 3.79 mmol, 1.5 eq), and DMF (40 mL). DIPEA (1.05 mL, 1.5 eq) was added under ice-water bath conditions. The mixture was warmed to room temperature and reacted for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative HPLC. The preparative solution was concentrated under reduced pressure in a 35 °C water bath using a water pump to remove acetonitrile, and lyophilized to give compound 3e (1.92 g) (61% yield, LC-MS: [M+H]). + =1235.4). Step 6: Compound P3 Compound 3e (1.0 g, 0.8 mmol, 1.0 eq) and 35 mL of nitromethane were added to a 100 mL single-neck flask. After dissolution, zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added and the mixture was allowed to react for 30 minutes in a 40 °C oil bath (preheated for stabilization). The nitromethane was removed by concentrating in a 45 °C water bath under reduced pressure using a water pump to obtain a yellow solid residue (monitored by HPLC). A preparative solution of compound 5A was obtained by the acid method. The preparative solution was concentrated in a 35 °C water bath under reduced pressure using a water pump to remove acetonitrile, and lyophilized to give compound P3 (786 mg) (yield 90%, LC-MS: [M+H] + =1079.4). 1H NMR(400MHz,d6DMSO) δ9.39-9.02(m,1H), 8.70(t,J=6.5Hz,1H), 8.64(t,J=5.7Hz,1H), 8.56(d,J=8.8Hz,1H), 8.34(t,J=5.7Hz,1H) , 8.16(d,J=8.2Hz,1H), 8.01(t,J=5.5Hz,1H), 7.71(d,J=10.9Hz,1H), 7.30(s,1H), 7.28-7.15(m,4H), 7.14(s ,2H), 5.53(dd,J=14.5,6.4Hz,1H), 5.49-5.34(m,2H), 5.22(d,J=18.8Hz,1H), 5.09(d,J=18.7Hz,1H), 5.03(d d,J=9.6,3.9Hz,1H), 4.73(dd,J=9.9,6.9Hz,1H), 4.59(dd,J=10.1,6.5Hz,1H), 4.49(ddd,J=13.2,8.6,4.4Hz ,1H), 4.14(dd,J=13.3,6.6Hz,2H), 3.93(s,2H), 3.84(dd,J=16.5,6.3Hz,1H), 3.76(dd,J=16.9,5.7Hz,2H), 3 .70(d,J=5.2Hz,2H), 3.60(dd,J=16.7,5.4Hz,1H), 3.52(dd,J=16.4,5.1Hz,1H), 3.45(dd,J=12.8,10.1Hz,1H ), 3.25-3.15(m,1H), 3.14-3.05(m,1H), 3.01(dd,J=13.7,4.1Hz,1H), 2.73(dd,J=13.5,9.8Hz,1H), 2.54-2.4 7(m,1H), 2.33(s,2H), 2.17(d,J=5.5Hz,2H), 1.91-1.79(m,2H), 1.33(d,J=6.6Hz,2H), 0.87(t,J=7.3Hz,2H).

[0101] Example 6: Synthesis of Compound P4 JPEG2025533577000364.jpg2866 Synthesis of Example 5, Compound P4 (468 mg) (LC-MS: [M+H] + =1079.6)をmodulationした.

[0102] Example 7: Synthesis of Compound P5 JPEG2025533577000365.jpg89137Step 1: Compound 5a A 250 mL single-neck flask was charged with M1 (10 g, 27.1 mmol), R-3,3,3-benzyl trifluorolactate (prepared according to the method disclosed in Patent WO2020063673A1) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene. The mixture was heated to 100 °C and reacted for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, insoluble matter was removed by filtration, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 5.15 g of the desired product (yield 35.1%, LC-MS: [M+H] + =543.17). Step 2: Compound 5b In a 50 mL single-neck flask, 5a (5 g, 9.2 mmol) and 15 mL of DMF were added and dissolved. After dissolving, DBU (1.68 g, 11 mmol) was added in an ice-water bath and the mixture was allowed to react for 1 hour. This was recorded as reaction solution (1). In a separate 50 mL single-neck flask, M4 (3.8 g, 9.2 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol), and 10 mL of DMF were added. After dissolution, DIPEA (1.82 mL, 11 mmol) was added in an ice-water bath and the reaction was continued for 30 minutes. Then, reaction solution (1) was added, the mixture was warmed to room temperature, and the reaction was continued for 2 hours. The progress of the reaction was monitored by HPLC. After completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.1 g of a solid (yield 62.3%, LC-MS: [M+H]). + =716.25). Step 3: Compound 5d 5b (900 mg, 1.26 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 900 mg of 5% Pd / C was added, and the hydrogenation reaction was carried out for 2 hours. After completion of the reaction, the mixture was filtered, and the filtrate was placed in an ice-water bath. DIPEA (228 μL, 1.38 mmol) was added, followed by M3 (712 mg, 1.26 mmol). The mixture was warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was lyophilized, and 525 mg of the product was obtained (yield 47.9%, LC-MS: [MH] - =870.33). Step 4: Compound 5e A 50 mL single-neck flask was charged with 5d (500 mg, 0.57 mmol), M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF. DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath, the mixture was warmed to room temperature, and the reaction was carried out for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 5e. The fraction was lyophilized to obtain 150 mg of compound 5e (LC-MS: [M+H] + =1289.46). Step 5: Compound P5 5e (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain 52 mg of a solid (TOF-MS: [M+H] + =1133.3613).

[0103] Example 8: Synthesis of Compound P6 Compound P6 (221 mg) (LC-MS: [M+H] + =1133.5) was prepared.

[0104] Example 9: Synthesis of Compound P7 JPEG2025533577000367.jpg89140 Step 1: Compound 7a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was cooled to 0 °C with stirring, and 2-hydroxy-2-cyclopropylbenzyl acetate (prepared according to the method disclosed in Patent US20050020645A1) (6.3 g, 32.6 mmol) was added dropwise. After the addition, the mixture was allowed to warm to room temperature under TLC monitoring (reaction time: approximately 2-4 h). After completion of the reaction, saturated NaHCO3 solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to give 7a (3.7 g) (yield 45%, LC-MS: [M+H] + =501.5). Step 2: Compound 7b In a 25 mL single-neck flask, 7a (2 g, 4.0 mmol) and 10 mL of DMF were added and stirred at 0°C. DBU (760 mg, 5.0 mmol) was added and the reaction was continued for 1 hour under TLC monitoring. After the Fmoc group was completely deprotected, the reaction was left for use. Into another 25 mL one-neck flask, M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol), and 10 mL of DMF were added. DIPEA (823 μL, 5.04 mmol) was added in an ice-water bath and stirred for 30 minutes. The reaction solution was then added to the reaction flask and allowed to warm to room temperature. After confirming the completion of the reaction by HPLC, the reaction solution was purified by preparative HPLC to obtain a preparative solution of the product. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1.5 g of a solid (yield 56%, LC-MS: [M+H] + =674.7). Step 3: Compound 7c 7b (900 mg, 1.3 mmol) was added to a 25 mL single-neck flask and dissolved in 10 mL of DMF. 900 mg of 5% Pd / C was added, and the hydrogenation reaction was carried out for 1.5 hours. After completion of the reaction, the mixture was filtered, and the obtained filtrate was used in the next step without further purification. Step 4: Compound 7d The crude product 7c was placed in an ice-water bath, DIPEA (223 μL, 1.3 mmol) was added, followed by compound M3 (750 mg, 1.3 mmol). The mixture was warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fractionated solution. The fractionated solution was lyophilized to obtain 7d (529 mg) (LC-MS: [M-H] - =828.4). Step 5: Compound 7e 7d (500 mg, 0.6 mmol), M5 (300 mg, 0.6 mmol), PyBOP (416 mg, 0.8 mmol), HOBt (108 mg, 0.5 mmol), and 15 mL of DMF were added to a 50 mL single-neck flask. DIPEA (351 μL, 2.13 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 3 hours. After confirming completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 7e. The fraction was lyophilized to obtain 7e (257 mg) (LC-MS: [M+H] + =1247.5). Step 6: Compound P7 7e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain solid compound P7 (55 mg) (LC-MS: [M+H] + =1091.3).

[0105] Example 10: Synthesis of Compound P8 JPEG2025533577000368.jpg92145Step 1: Compound 8a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was cooled to 0 °C with stirring, and benzyl 3-hydroxy-2-cyclopropylpropionate (prepared according to the method disclosed in Patent WO2013187496A1, 6.7 g, 32.6 mmol) was added dropwise. After the dropwise addition, the mixture was allowed to warm to room temperature under TLC monitoring (reaction time: approximately 2-4 h). After completion of the reaction, saturated NaHCO3 solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to give 8a (4.9 g) (yield 58%, LC-MS: [M+H] + =515.4). Step 2: Compound 8b In a 25 mL single-neck flask, 8a (4 g, 7.8 mmol) and 10 mL of DMF were added and stirred at 0 °C. DBU (1.2 g, 8.0 mmol) was added and the reaction was continued for 1 h under TLC monitoring. After the Fmoc group was completely deprotected, the reaction was left for use. In a separate 25 mL one-neck flask, M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol), and 10 mL of DMF were added. DIPEA (1.65 mL, 10.1 mmol) was added in an ice-water bath and stirred for 50 minutes. The reaction solution was then added to the reaction flask and allowed to warm to room temperature. After confirming the completion of the reaction by HPLC, the reaction solution was purified by preparative HPLC to obtain a preparative solution of the product. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.3 g of a solid (42% yield, LC-MS: [M+H] + =688.8). Step 3: Compound 8c 8b (1.0 g, 1.45 mmol) was placed in a 25 mL single-neck flask and dissolved in 15 mL of DMF. 1.0 g of 5% Pd / C was added and the hydrogenation reaction was carried out for 1.5 hours. After completion of the reaction, the mixture was filtered, and the filtrate was used in the next step without further purification. Step 4: Compound 8d The crude product 8c was placed in an ice-water bath, DIPEA (258 μL, 1.5 mmol) was added, followed by compound M3 (837 mg, 1.45 mmol), and the mixture was warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fractionated solution. The fractionated solution was lyophilized to obtain 10d (499 mg) (LC-MS: [M-H] - =842.4). Step 5: Compound 8e A 50 mL single-neck flask was charged with 8d (400 mg, 0.48 mmol), M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (104 mg, 0.48 mmol), and 15 mL of DMF. DIPEA (330 μL, 2.0 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 3 hours. After confirming completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 8e. The fraction was lyophilized to obtain 8e (188 mg) (LC-MS: [M+H] + =1261.5). Step 6: Compound P8 8e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain solid compound P8 (61 mg) (LC-MS: [M+H] + =1105.6).

[0106] Example 11: Synthesis of Compound P9 JPEG2025533577000369.jpg89140 Step 1: Compound 9a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was cooled to 0 °C with stirring, and 2-hydroxy-2-cyclobutylbenzyl acetate (synthesized according to the method published in Journal of Medicinal Chemistry, 2013, 56(13), 5541-5552, 6.7 g, 32.6 mmol) was added dropwise. After the addition, the mixture was allowed to warm to room temperature under TLC monitoring (reaction time: approximately 2-4 hours). After the reaction was completed, saturated NaHCO3 solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to give 9a (5.1 g) (yield 62%, LC-MS: [M+H] + =515.7). Step 2: Compound 9b In a 25 mL single-neck flask, 9a (4 g, 7.8 mmol) and 10 mL of DMF were added and stirred at 0 °C. DBU (1.2 g, 8.0 mmol) was added and the reaction was continued for 1 h under TLC monitoring. After the Fmoc group was completely deprotected, the reaction was left for use. Into another 25 mL one-neck flask, M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol), and 10 mL of DMF were added. DIPEA (1.63 mL, 10.0 mmol) was added in an ice-water bath and stirred for 40 minutes. The reaction solution was then added to the reaction flask and allowed to warm to room temperature. After confirming the completion of the reaction by HPLC, the reaction solution was purified by preparative HPLC to obtain a preparative solution of the product. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.3 g of a solid (42% yield, LC-MS: [M+H] + =688.3). Step 3: Compound 9c 9b (2.0 g, 2.9 mmol) was added to a 25 mL single-neck flask and dissolved in 25 mL of DMF. 2.0 g of 5% Pd / C was added and the hydrogenation reaction was carried out for 3 hours. After completion of the reaction, the mixture was filtered, and the filtrate was used in the next step without further purification. Step 4: Compound 9d The crude product 9c was placed in an ice-water bath, and DIPEA (516 μL, 3.0 mmol) was added, followed by compound M3 (1.7 g, 2.9 mmol). After the addition, the mixture was warmed to room temperature and reacted for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fractionated solution. The fractionated solution was lyophilized to obtain 9d (934 mg) (LC-MS: [M-H] - =842.4). Step 5: Compound 9e 9d (800 mg, 0.96 mmol), M5 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol), and 30 mL of DMF were added to a 50 mL single-neck flask. DIPEA (660 μL, 4.0 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 4 hours. After confirming completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 9e. The fraction was lyophilized to obtain 9e (401 mg) (LC-MS: [M+H] + =1261.4). Step 6: Compound P9 9e (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol), and 10 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain solid compound P9 (86 mg) (LC-MS: [M+H] + =1105.5).

[0107] Example 12: Synthesis of Compound P10 JPEG2025533577000370.jpg92145Step 1: Compound 10a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was cooled to 0 °C with stirring, and benzyl 3-hydroxy-2-cyclobutylpropionate (prepared according to the method disclosed in Patent WO2009011285A1, 7.2 g, 32.6 mmol) was added dropwise. After the addition, the mixture was allowed to warm to room temperature under TLC monitoring (reaction time: approximately 2-4 h). After the reaction was completed, saturated NaHCO3 solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to give 10a (4.5 g) (yield 52%, LC-MS: [M+H] + =529.4). Step 2: Compound 10b In a 25 mL single-neck flask, 10a (4 g, 7.6 mmol) and 10 mL of DMF were added and stirred at 0 °C. DBU (1.2 g, 8.0 mmol) was added and the reaction was continued for 1 hour under TLC monitoring. After the Fmoc group was completely deprotected, the mixture was left for use. In a separate 25 mL one-neck flask, M4 (3.2 g, 7.6 mmol), PyBOP (4.7 g, 9.0 mmol), HOBt (1.22 g, 9.0 mmol), and 10 mL of DMF were added. DIPEA (1.49 mL, 0.9 mmol) was added in an ice-water bath and stirred for 30 minutes. The reaction solution was then added to the reaction flask and allowed to warm to room temperature. After confirming the completion of the reaction by HPLC, the reaction solution was purified by preparative HPLC to obtain a preparative solution of the product. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.0 g of a solid (yield 37%, LC-MS: [M+H] + =702.8). Step 3: Compound 10c 1.0 g (1.43 mmol) of 10b was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 1.0 g of 5% Pd / C was added and the hydrogenation reaction was carried out for 1.5 hours. After completion of the reaction, the mixture was filtered, and the filtrate was used in the next step without further purification. Step 4: Compound 10d The crude product 10c was placed in an ice-water bath, DIPEA (258 μL, 1.5 mmol) was added, followed by compound M3 (825 mg, 1.43 mmol). The mixture was warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fractionated solution. The fractionated solution was lyophilized to obtain 10d (522 mg) (LC-MS: [M-H] - =856.4). Step 5: Compound 10e 10d (400 mg, 0.47 mmol), M5 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol), and 15 mL of DMF were added to a 50 mL single-neck flask. DIPEA (330 μL, 2.0 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 3 hours. After confirming completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 10e. The fraction was lyophilized to obtain 10e (198 mg) (LC-MS: [M+H] + =1275.4). Step 6: Compound P10 10e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain solid compound P10 (55 mg) (LC-MS: [M+H] + =1119.5).

[0108] Example 13: Synthesis of Compound P11 JPEG2025533577000371.jpg91140 Step 1: Compound 11a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was cooled to 0 °C with stirring, and 2-hydroxy-2-cyclopentylbenzyl acetate (synthesized according to the method published in Journal of Medicinal Chemistry, 2013, 56(13), 5541-5552) (7.2 g, 32.6 mmol) was added dropwise. After the addition, the mixture was allowed to warm to room temperature under TLC monitoring (reaction time: approximately 2-4 hours). After the reaction was completed, saturated NaHCO3 solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to give 11a (4.6 g) (yield 53%, LC-MS: [M+H] + =529.5). Step 2: Compound 11b In a 25 mL single-neck flask, 11a (4 g, 7.6 mmol) and 10 mL of DMF were added and stirred at 0 °C. DBU (1.17 g, 7.8 mmol) was added and the reaction was continued for 1 h under TLC monitoring. After the Fmoc group was completely deprotected, the reaction was left for use. In a separate 25 mL one-neck flask, M4 (3.14 g, 7.6 mmol), PyBOP (4.42 g, 8.5 mmol), HOBt (1.15 g, 8.5 mmol), and 10 mL of DMF were added. DIPEA (1.39 mL, 0.85 mmol) was added under ice-water bath conditions and stirred for 30 minutes. The reaction solution was then added to the reaction flask and allowed to warm to room temperature. After confirming the completion of the reaction by HPLC, the reaction solution was purified by preparative HPLC to obtain a preparative solution of the product. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.1 g of a solid (39% yield, LC-MS: [M+H] + =702.8). Step 3: Compound 11c 11b (1.5 g, 1.87 mmol) was added to a 25 mL one-neck flask and dissolved in 25 mL of DMF. 1.5 g of 5% Pd / C was added and the hydrogenation reaction was carried out for 3 hours. After completion of the reaction, the mixture was filtered, and the filtrate was used in the next step without further purification. Step 4: Compound 11d The crude product 11c was placed in an ice-water bath, DIPEA (333 μL, 1.93 mmol) was added, followed by compound M3 (1.1 g, 1.87 mmol), and the mixture was warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fractionated solution. The fractionated solution was lyophilized to obtain 11d (519 mg) (LC-MS: [M-H] - =856.6). Step 5: Compound 11e A 50 mL single-neck flask was charged with 11d (400 mg, 0.47 mmol), M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (103 mg, 48 mmol), and 15 mL of DMF. DIPEA (330 μL, 2.0 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 4 hours. After confirming completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 11e. The fraction was lyophilized to obtain 11e (187 mg) (LC-MS: [M+H] + =1275.5). Step 6: Compound P11 11e (100 mg, 0.08 mmol), zinc bromide (355 mg, 0.16 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain solid compound P11 (60 mg) (LC-MS: [M+H] + =1119.7).

[0109] Example 14: Synthesis of Compound P12 JPEG2025533577000372.jpg91145Step 1: Compound 12a A 250 mL single-neck flask was charged with M1 (6 g, 16.3 mmol), 100 mL of THF, and p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol). The mixture was cooled to 0 °C with stirring, and benzyl 3-hydroxy-2-cyclopentylpropionate (synthesized according to the method disclosed in Patent WO2009011285A1) (7.6 g, 32.6 mmol) was added dropwise. After the dropwise addition, the mixture was allowed to warm to room temperature under TLC monitoring (reaction time: approximately 2-4 h). After completion of the reaction, saturated NaHCO3 solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (PE:EA = 10:1-5:1-2:1) to give 12a (4.4 g) (yield 49%, LC-MS: [M+H] + =543.6). Step 2: Compound 12b In a 25 mL single-neck flask, 12a (4 g, 7.4 mmol) and 10 mL of DMF were added and stirred at 0 °C. DBU (1.2 g, 8.0 mmol) was added and the reaction was continued for 1 h under TLC monitoring. After the Fmoc group was completely deprotected, the mixture was left for use. Into another 25 mL one-neck flask, M4 (3.1 g, 7.4 mmol), PyBOP (4.6 g, 8.8 mmol), HOBt (1.19 g, 8.8 mmol), and 10 mL of DMF were added. DIPEA (1.49 mL, 9.0 mmol) was added in an ice-water bath and stirred for 30 minutes. The reaction solution was then added to the reaction flask and allowed to warm to room temperature. After confirming the completion of the reaction by HPLC, the reaction solution was purified by preparative HPLC to obtain a preparative solution of the product. The preparative solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.6 g of a solid (49% yield, LC-MS: [M+H] + =716.4). Step 3: Compound 12c 12b (1.0 g, 1.4 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 1.0 g of 5% Pd / C was added, and the hydrogenation reaction was carried out for 1.5 hours. After completion of the reaction, the mixture was filtered, and the obtained filtrate was used in the next step without further purification. Step 4: Compound 12d The crude product 12c was placed in an ice-water bath, DIPEA (248 μL, 1.5 mmol) was added, followed by compound M3 (808 mg, 1.4 mmol). The mixture was warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fractionated solution. The fractionated solution was lyophilized to obtain 12d (500 mg) (LC-MS: [M-H] - =870.5). Step 5: Compound 12e 12d (400 mg, 0.46 mmol), M5 (235 mg, 0.46 mmol), PyBOP (245 mg, 0.46 mmol), HOBt (99 mg, 0.46 mmol), and 15 mL of DMF were added to a 50 mL single-neck flask. DIPEA (331 μL, 2.0 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 3 hours. After confirming completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 12e. The fraction was lyophilized to obtain 12e (146 mg) (LC-MS: [M+H] + =1289.5). Step 6: Compound P12 12e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain solid compound P12 (52 mg) (LC-MS: [M+H] + =1133.7).

[0110] Example 15: Synthesis of Compound P13 JPEG2025533577000373.jpg89140 Step 1: Compound 13a In a 250 mL one-neck flask, M1 (10 g, 27.1 mmol), benzyl 2-hydroxybutanoate (prepared according to the method published in Chemical Communications, 2019, 55(53), 7699-7702) (10.5 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were added and heated to 100 °C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, insoluble matter was removed by filtration, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 5.67 g of the desired product (yield 42%, LC-MS: [M+H] + =503.5). Step 2: Compound 13b 13a (5 g, 9.95 mmol) and 15 mL of DMF were added to a 50 mL single-neck flask, and after dissolution, DBU (1.68 g, 11 mmol) was added in an ice-water bath and the mixture was allowed to react for 1 hour. This was recorded as reaction solution (1). In a separate 50 mL single-neck flask, M4 (4.1 g, 10.0 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol), and 10 mL of DMF were added. After dissolution, DIPEA (1.82 mL, 11 mmol) was added in an ice-water bath and the reaction was continued for 40 minutes. The reaction solution (1) was then added, and the mixture was warmed to room temperature and reacted for 2 hours. The reaction progress was monitored by HPLC. After completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.6 g of a solid (68% yield, LC-MS: [M+H]). + =676.7). Step 3: Compound 13d 13b (2.0 g, 2.96 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 2.0 g of 5% Pd / C was added, and the hydrogenation reaction was carried out for 2 hours. After completion of the reaction, the mixture was filtered, and the filtrate was placed in an ice-water bath. DIPEA (496 μL, 3.0 mmol) was added, followed by M3 (1.7 g, 2.96 mmol). The mixture was warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was lyophilized to obtain 1120 mg of product (45% yield, LC-MS: [MH]). - =830.3). Step 4: Compounds 13e and 13f A 50 mL single-neck flask was charged with 13d (500 mg, 0.60 mmol), M5 (321 mg, 0.60 mmol), PyBOP (469 mg, 0.90 mmol), HOBt (121 mg, 0.90 mmol), and 15 mL of DMF. DIPEA (446 μL, 2.7 mmol) was added in an ice-water bath, the mixture was warmed to room temperature, and the reaction was carried out for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain fractionated solutions of compound 13e and compound 13f. The fractionated solutions were each lyophilized to obtain 138 mg of compound 13e (LC-MS: [M+H] + =1249.5), 140 mg of compound 13f (LC-MS: [M+H] + =1249.5). Step 5: Compound P13 JPEG2025533577000374.jpg281402 13e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 5 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain 59 mg of a solid (LC-MS: [M+H] + =1093.8).

[0111] Example 16: Synthesis of Compound P14 JPEG2025533577000375.jpg281322 13f (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 5 mL one-neck flask and reacted at 40 °C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain 60 mg of a solid (LC-MS: [M+H] + =1093.7).

[0112] Example 17: Synthesis of Compound P15 JPEG2025533577000376.jpg71140Step 1: Compound 15a A 250 mL single-neck flask was charged with M1 (10 g, 27.1 mmol), 2-cyclopropyl-2-hydroxyl benzyl acetate (prepared according to the method disclosed in Patent WO2020244657A1) (11.2 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene. The mixture was heated to 100 °C and reacted for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, insoluble matter was removed by filtration, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 4.97 g of the desired product (yield 36%, LC-MS: [M+H] + =515.2). Step 2: Compound 15b 15a (4 g, 7.8 mmol) and 10 mL of DMF were added to a 50 mL single-neck flask, and after dissolution, DBU (1.42 g, 9.3 mmol) was added in an ice-water bath and the mixture was allowed to react for 1 hour. This was recorded as reaction solution (1). In a separate 50 mL single-neck flask, M4 (3.2 g, 7.8 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol), and 10 mL of DMF were added. After dissolution, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath and the reaction was continued for 30 minutes. Then, reaction solution (1) was added, the mixture was warmed to room temperature, and the reaction was continued for 2 hours. The progress of the reaction was monitored by HPLC. After completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.2 g of a solid (78% yield, LC-MS: [M+H]). + =688.3). Step 3: Compound 15d 15b (1000 mg, 1.45 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 1000 mg of 5% Pd / C was added, and the hydrogenation reaction was carried out for 2 hours. After completion of the reaction, the mixture was filtered, and the filtrate was placed in an ice-water bath. DIPEA (248 μL, 1.5 mmol) was added, followed by M3 (720 mg, 1.45 mmol). The mixture was warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was lyophilized, and 503 mg of the product was obtained (yield 41%, LC-MS: [MH] - =842.3). Step 4: Compounds 15e and 15f A 50 mL single-neck flask was charged with 15d (500 mg, 0.59 mmol), M5 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol), and 10 mL of DMF. DIPEA (292 μL, 1.77 mmol) was added in an ice-water bath, the mixture was warmed to room temperature, and the reaction was carried out for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain fractionated solutions of compound 15e and compound 15f. The fractionated solutions were each lyophilized to obtain 112 mg of compound 15e (LC-MS: [M+H] + =1261.5), 131 mg of compound 15f (LC-MS: [M+H] + =1261.5). Step 5: Compound P15 JPEG2025533577000377.jpg291322 15e (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain 55 mg of a solid (LC-MS: [M+H] + =1105.5).

[0113] Example 18: Synthesis of Compound P16 JPEG2025533577000378.jpg291322 15f (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain 58 mg of a solid (LC-MS: [M+H] + =1105.6).

[0114] Example 19: Synthesis of Compound P17 JPEG2025533577000379.jpg71140 Step 1: Compound 17a A 250 mL single-neck flask was charged with M1 (10 g, 27.1 mmol), benzyl 2-hydroxycyclopropylpropionate (synthesized according to the method disclosed in Patent WO2020063676A) (12.0 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene. The mixture was heated to 100 °C and reacted for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, insoluble matter was removed by filtration, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 5.09 g of the desired product (LC-MS: [M+H] + =529.2). Step 2: Compound 17b 17a (4 g, 7.6 mmol) and 10 mL of DMF were added to a 50 mL single-neck flask, and after dissolution, DBU (1.39 g, 9.1 mmol) was added in an ice-water bath and the mixture was allowed to react for 1 hour. This was recorded as reaction solution (1). In a separate 50 mL single-neck flask, M4 (3.12 g, 7.6 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol), and 10 mL of DMF were added. After dissolution, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath and the reaction was continued for 30 minutes. Then, reaction solution (1) was added, the mixture was warmed to room temperature, and the reaction was continued for 2 hours. The progress of the reaction was monitored by HPLC. After completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4.5 g of a solid (84% yield, LC-MS: [M+H]). + =702.3). Step 3: Compound 17d 17b (1000 mg, 1.42 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 1000 mg of 5% Pd / C was added, and the hydrogenation reaction was carried out for 2 hours. After completion of the reaction, the mixture was filtered, and the filtrate was placed in an ice-water bath. DIPEA (248 μL, 1.5 mmol) was added, followed by M3 (708 mg, 1.42 mmol). The mixture was warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was lyophilized, and 443 mg of the product was obtained (yield 36%, LC-MS: [MH]). - =856.4). Step 4: Compounds 17e and 17f A 50 mL single-neck flask was charged with 17d (400 mg, 0.47 mmol), M5 exatecan mesylate (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF. DIPEA (248 μL, 1.5 mmol) was added in an ice-water bath, the mixture was warmed to room temperature, and the reaction was carried out for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain fractionated solutions of compound 17e and compound 17f. The fractionated solutions were each lyophilized to obtain 103 mg of compound 17e (LC-MS: [M+H] + =1275.5), 103 mg of compound 17f (LC-MS: [M+H] + =1275.5). Step 5: Compound P17 JPEG2025533577000380.jpg291322 17e (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain 51 mg of a solid (LC-MS: [M+H] + =1119.7).

[0115] Example 20: Synthesis of compound P18 JPEG2025533577000381.jpg291322 17f (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain 47 mg of a solid (LC-MS: [M+H] + =1119.6).

[0116] Example 21: Synthesis of Compound P19 JPEG2025533577000382.jpg89140Step 1: Compound 19a In a 250 mL one-neck flask, M1 (10 g, 27.1 mmol), benzyl 2-hydroxyphenylpropionate (synthesized according to the method published in Nature Communications, 2020.11(1), 56.) (14.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene were added, heated to 100 °C, and reacted for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, insoluble matter was removed by filtration, and the filtrate was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (PE:EA = 10:1-5:1-2:1) to obtain 6.13 g of the desired product (yield 40%, LC-MS: [M+H] + =565.6). Step 2: Compound 19b 19a (5 g, 8.86 mmol) and 15 mL of DMF were added to a 50 mL single-neck flask, and after dissolution, DBU (1.53 g, 10 mmol) was added in an ice-water bath and the mixture was allowed to react for 1 hour. This was recorded as reaction solution (1). In a separate 50 mL single-neck flask, M4 (3.6 g, 8.86 mmol), PyBOP (5.23 g, 10 mmol), HOBt (1.36 g, 10 mmol), and 10 mL of DMF were added. After dissolution, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath and the reaction was continued for 30 minutes. Then, reaction solution (1) was added, the mixture was warmed to room temperature, and the reaction was continued for 2 hours. The progress of the reaction was monitored by HPLC. After completion of the reaction, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 5.0 g of a solid (77% yield, LC-MS: [M+H]). + =738.3). Step 3: Compound 19d 19b (3.0 g, 4.07 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. 3.0 g of 5% Pd / C was added, and the hydrogenation reaction was carried out for 2 hours. After completion of the reaction, the mixture was filtered, and the filtrate was placed in an ice-water bath. DIPEA (744 μL, 4.5 mmol) was added, followed by M3 (2.34 g, 4.07 mmol). The mixture was warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was lyophilized, and 1.2 g of product (33% yield, LC-MS: [MH]) was obtained. - =892.4). Step 4: Compounds 19e and 19f A 50 mL single-neck flask was charged with 19d (500 mg, 0.56 mmol), M5 (300 mg, 0.56 mmol), PyBOP (438 mg, 0.84 mmol), HOBt (113 mg, 0.84 mmol), and 15 mL of DMF. DIPEA (330 μL, 2.0 mmol) was added in an ice-water bath, the mixture was warmed to room temperature, and the reaction was allowed to proceed for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain fractionated solutions of compound 19e and compound 19f. The fractionated solutions were each lyophilized to obtain 156 mg of compound 19e (LC-MS: [M+H] + =1311.6), 150 mg of compound 19f (LC-MS:[M+H] + =1311.7). Step 5: Compound P19 JPEG2025533577000383.jpg281402 19e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 5 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain 43 mg of a solid (LC-MS: [M+H] + =1155.7).

[0117] Example 22: Synthesis of Compound P20 JPEG2025533577000384.jpg281302 19f (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 5 mL one-neck flask and reacted at 40 °C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain 40 mg of a solid (LC-MS: [M+H] + =1155.8).

[0118] Example 23: Synthesis of Compound 14 JPEG2025533577000385.jpg43128Step 1: Synthesis of Compound 4 Exatecan mesylate M5 (15 g, 28 mol, prepared by the method disclosed in patent application EP0737683A1) was added to 400 mL of DMF, cooled to 0°C in an ice-water bath, triethylamine was added dropwise, the pH was adjusted to 7-8, and benzyl bromide (9.6 g, 56 mmol) was added dropwise in an ice-water bath. The mixture was then heated to room temperature (25°C) and reacted for 1 hour. After confirming completion of the reaction by TLC, the reaction solution was concentrated under reduced pressure. The resulting crude product was purified by preparative high-performance liquid chromatography (acetonitrile / pure water system). The target peak was collected, the acetonitrile was removed under reduced pressure, and the mixture was lyophilized to obtain approximately 11 g of compound 4 (yield: approximately 74%, MS m / z: [M+H]). + 526.3) was obtained as a yellow solid. Step 2: Synthesis of compound 6 Compound 4 (11 g, 21 mol) and 120 mL of formic acid were added to a 250 mL single-neck flask at room temperature and dissolved. 30 mL of formaldehyde (40% aqueous solution) was added to the resulting bright yellow solution, and the mixture was heated to 50 °C and reacted for 1 hour. After confirming the completion of the reaction by TLC, the mixture was cooled to room temperature and purified by preparative high-performance liquid chromatography (acetonitrile / pure water system). The target peak was collected, the acetonitrile was removed under reduced pressure, and the mixture was lyophilized to obtain approximately 4.5 g of compound 6 (yield approximately 40%, MS m / z: [M+H]). + 540.6) was obtained as a yellow powdery solid. Step 3: Synthesis of compound 14 Compound 6 (2.3 g, 4.3 mol) was added to a 250 mL single-neck flask at room temperature, and 100 mL of DMF was added to dissolve the compound. 2.3 g of 5% Pd / C was added to the resulting bright yellow solution, and the atmosphere in the system was replaced with hydrogen gas. The reaction was maintained at room temperature for 1.5 hours. After confirming completion of the reaction by HPLC, the Pd / C was removed by filtration. The resulting reaction solution was concentrated and purified by preparative high-performance liquid chromatography (acetonitrile / pure water system). The target peak was collected, the acetonitrile was removed under reduced pressure, and the mixture was lyophilized to obtain approximately 1.0 g of compound 14 (yield approximately 52%, MS m / z: [M+H]). + 450.5) was obtained as a yellow powdery solid.

[0119] Example 24: Synthesis of Compound P21 JPEG2025533577000386.jpg53136Step 1: Compound 21a 1d (500 mg, 0.62 mmol), 14 (279 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were added to a 50 mL single-neck flask. DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath, the mixture was warmed to room temperature, and the reaction was carried out for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was lyophilized to obtain 21a (166 mg) (LC-MS: [M+H] + =1235.6). Step 2: Compound P21 21a (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol), and 10 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain solid compound P21 (43 mg) (LC-MS: [M+H] + =1079.5).

[0120] Example 25: Synthesis of Compound P22 JPEG2025533577000387.jpg2864 According to the route of Example 24, 73 mg of compound P22 (LC-MS: [M+H] + =1107.6) was prepared.

[0121] Example 26: Synthesis of Compound P23 JPEG2025533577000388.jpg53142Step 1: Compound 23a A 100 mL single-neck flask was charged with 3d (1.66 g, 2.02 mmol, 1.0 eq), 14 (0.91 g, 2.02 mmol, 1.0 eq), PyBOP (1.58 g, 3.03 mmol, 1.5 eq), HOBt (0.41 g, 3.03 mmol, 1.5 eq), and DMF (40 mL). DIPEA (0.84 mL, 1.5 eq) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative HPLC. The preparative solution was concentrated under reduced pressure in a 35 °C water bath using a water pump to remove acetonitrile, and lyophilized to give compound 23a (1.21 g) (LC-MS: [M+H] + =1249.4). Step 2: Compound P23 Compound 23a (1.0 g, 0.8 mmol, 1.0 eq) and 35 mL of nitromethane were added to a 100 mL single-neck flask. After dissolution, zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added and the mixture was allowed to react for 30 minutes in a 40 °C oil bath (preheated for stabilization). The nitromethane was removed by concentrating in a 45 °C water bath under reduced pressure using a water pump to obtain a yellow solid residue (monitored by HPLC). A fraction was obtained by the acid method. The fraction was then concentrated in a 35 °C water bath under reduced pressure using a water pump to remove acetonitrile, and lyophilized to obtain compound P23 (786 mg) (LC-MS: [M+H] + =1093.6).

[0122] Example 27: Synthesis of Compound P24 JPEG2025533577000389.jpg2867 Following the route of Example 26, 677 mg of compound P24 (LC-MS: [M+H] + =1093.7) was prepared.

[0123] Example 28: Synthesis of Compound P25 JPEG2025533577000390.jpg48128Step 1: Compound 25a A 50 mL single-neck flask was charged with 5d (500 mg, 0.57 mmol), 14 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF. DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath, the mixture was warmed to room temperature, and the reaction was allowed to proceed for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 25a. The fraction was lyophilized to obtain 313 mg of compound 25a (LC-MS: [M+H] + =1303.6). Step 2: Compound P25 25a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution. The fractionated solution was lyophilized to obtain 49 mg of solid compound P25 (LC-MS: [M+H] + =1147.6).

[0124] Example 29: Synthesis of Compound P26 JPEG2025533577000391.jpg2866 Following the route of Example 28, 89 mg of compound P26 (LC-MS: [M+H] + =1147.7) was prepared.

[0125] Example 30: Synthesis of compounds P27 and P28 JPEG2025533577000392.jpg72130 Step 1: Compounds 27a and 28a A 50 mL single-neck flask was charged with 15d (500 mg, 0.59 mmol), 14 (266 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol), and 10 mL of DMF. DIPEA (292 μL, 1.77 mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain fractionated solutions of compounds 27a and 28a. The fractionated solutions were each lyophilized to obtain 109 mg of compound 27a (LC-MS: [M+H] + =1275.5), 111 mg of compound 28 (LC-MS: [M+H] + =1275.7). Step 2: Compound P27 27a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain 53 mg of a solid (LC-MS: [M+H] + =1119.5). Step 3: Compound P28 28a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain 54 mg of a solid (LC-MS: [M+H] + =1119.4).

[0126] Example 31: Synthesis of compounds P29 and P30 JPEG2025533577000393.jpg74128 Step 1: Compounds 29a and 30a A 50 mL single-neck flask was charged with 17d (400 mg, 0.47 mmol), 14 (211.7 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF. DIPEA (248 μL, 1.5 mmol) was added in an ice-water bath, and the mixture was warmed to room temperature and reacted for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain fractionated solutions of compounds 29a and 30a. The fractionated solutions were each lyophilized to obtain 106 mg of compound 29a (LC-MS: [M+H] + =1289.5), 101 mg of compound 30a (LC-MS: [M+H] + =1289.4). Step 2: Compound P29 29a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain 39 mg of a solid (LC-MS: [M+H] + =1133.4). Step 3: Compound P30 30a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain 35 mg of a solid (LC-MS: [M+H] + =1133.6).

[0127] Example 32: Synthesis of Compound P31 JPEG2025533577000394.jpg51140 Step 1: Compound 31a 9d (800 mg, 0.96 mmol), 14 (432.5 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol), and 30 mL of DMF were added to a 50 mL single-neck flask. DIPEA (660 μL, 4.0 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 4 hours. After confirming completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 67a. The fraction was lyophilized to obtain 31a (402 mg) (LC-MS: [M+H] + =1275.4). Step 2: Compound P31 31a (100 mg, 0.78 mmol), zinc bromide (356 mg, 1.57 mmol), and 10 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain solid compound P31 (47 mg) (LC-MS: [M+H] + =1119.6).

[0128] Example 33: Synthesis of Compound P32 JPEG2025533577000395.jpg51133Step 1: Compound 32a A 50 mL single-neck flask was charged with 10d (400 mg, 0.47 mmol), 14 (211.7 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt (101 mg, 0.47 mmol), and 15 mL of DMF. DIPEA (330 μL, 2.0 mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature and react for 3 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction of compound 32a. The fraction was lyophilized to obtain 32a (177 mg) (LC-MS: [M+H] + =1289.4). Step 2: Compound P32 32a (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain solid compound P32 (45 mg) (LC-MS: [M+H] + =1133.5).

[0129] Example 34: Synthesis of Compound M7 JPEG2025533577000396.jpg46128 Step 1: Compound M6 Compound M3 (11.0 g, 19.5 mmol, 1.0 eq), DIPEA (2.8 g, 21.4 mmol, 1.1 eq), 27-amino-4,7,10,13,16,19,22,25-octaoxaheptacosanoic acid (9.7 g, 20.5 mmol, 1.05 eq), and DMF (60 mL) were added to a 100 mL single-neck flask and reacted at room temperature for 20 min (monitored by TLC). The reaction solution was directly purified by preparative HPLC. The preparative solution was concentrated under reduced pressure in a 35 °C water bath using a water pump to remove acetonitrile, and lyophilized to give compound M6 (13.2 g) (78% yield, LC-MS: [M-H]). - =820.5). Step 2: Compound M7 Compound M6 (13.0 g, 15 mmol, 1.0 eq), pentafluorophenol (3 g, 16.5 mmol, 1.1 eq), DCC (3.4 g, 16.5 mmol, 1.1 eq), and THF (30 mL) were added to a 100 mL single-neck flask and reacted at room temperature for 30 min (monitored by TLC). Insoluble materials were removed by filtration. The reaction solution was directly purified by preparative HPLC. The preparative solution was concentrated under reduced pressure in a water bath at 35 °C using a water pump to remove acetonitrile, and lyophilized to give compound M7 (14.2 g) (yield 92%, LC-MS: [M+H]). + =986.9).

[0130] Example 35: Synthesis of Compound P33 JPEG2025533577000397.jpg84128Step 1: Synthesis of compound 33a M7 (1 g, 0.79 mol) was added to 10 mL of DMF and cooled to 0 °C in an ice-water bath. Compound 1c (334 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added and reacted under these conditions for 1 hour. After confirming the completion of the reaction by TLC, the reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile / pure water system). The target peak was collected, the acetonitrile was removed under reduced pressure, and the mixture was lyophilized to obtain approximately 1.2 g of compound 33a (MS m / z: [M−H] - =1225.8). Step 2: Synthesis of compound 33b A 25 mL single-neck flask was charged with 33a (1.2 g, 0.94 mmol), M5 (500 mg, 0.94 mmol), PyBOP (625 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol), and 15 mL of DMF. DIPEA (310 mg, 2.4 mmol) was added in an ice-water bath, the mixture was warmed to room temperature, and the reaction was carried out for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was lyophilized to obtain 33b (709 mg) (LC-MS: [M+H] + =1644.9). Step 3: Synthesis of compound P33 33b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol), and 10 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain solid compound P33 (88 mg) (LC-MS: [M+H] + =1488.9).

[0131] Example 36: Synthesis of Compound P34 JPEG2025533577000398.jpg84128Step 1: Synthesis of compound 34a M7 (1 g, 0.79 mol) was added to 10 mL of DMF and cooled to 0 °C in an ice-water bath. Compound 3c (345 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added and reacted under these conditions for 1 hour. After confirming the completion of the reaction by TLC, the reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile / pure water system). The target peak was collected, the acetonitrile was removed under reduced pressure, and the mixture was lyophilized to obtain 0.9 g of compound 34a (MS m / z: [M−H] - =1239.8). Step 2: Synthesis of compound 34b A 25 mL single-neck flask was charged with 34a (700 mg, 0.54 mmol), M5 (289 mg, 0.54 mmol), PyBOP (313 mg, 0.6 mmol), HOBt (81 mg, 0.6 mmol), and 10 mL of DMF. DIPEA (155 mg, 1.2 mmol) was added in an ice-water bath, the mixture was warmed to room temperature, and the reaction was carried out for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fraction. The fraction was lyophilized to obtain 34b (304 mg) (LC-MS: [M+H] + =1658.9). Step 3: Synthesis of compound P34 34b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol), and 10 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain solid compound P34 (96 mg) (LC-MS: [M+H] + =1502.8).

[0132] Example 37: Synthesis of Compound P35 JPEG2025533577000399.jpg34128 Following the synthetic route of Example 36, 80 mg of compound P35 (LC-MS: [M+H] + =1502.7) was prepared.

[0133] Example 38: Synthesis of Compound 16 JPEG2025533577000400.jpg37130Step 1: Preparation of compound 16a Compound 16a was prepared according to the method disclosed in Example 76 on page 147 of the specification of patent "EP2907824A1". Step 2: Preparation of Compound 16 Compound 16a (3 g, 6.08 mmol) was added to a 100 mL single-neck flask and dissolved in 50 mL of DCM. The mixture was cooled to 0 °C in an ice-water bath. While maintaining this temperature, DIPEA (2.03 mL, 12.16 mmol) and NPC (3.78 g, 12.16 mmol) were added in that order. The mixture was slowly warmed to room temperature and reacted for 5 h. The end point of the reaction was monitored by TLC. After completion of the reaction, the mixture was concentrated under reduced pressure at 45 °C using a water pump to remove the solvent, yielding a residue. The residue was purified by silica gel column chromatography (DCM / MeOH = 100 / 1 - 50:1 to 10:1) to give compound 16 (3.4 g) (LC-MS: [M+H] + =659.4).

[0134] Example 38: Synthesis of Compound 18 JPEG2025533577000401.jpg58128Step 1: Synthesis of compound 18a Fmoc-VC-PAB-OH (30 g, 49.92 mmol) was added to a 5000 mL single-neck flask, followed by DMF (2 L). After stirring to dissolve, NPC (22.76 g, 74.87 mmol) was added, and DIPEA (9.68 g, 74.87 mmol) was added dropwise. The mixture was stirred at room temperature for 1.5 hours. The reaction was stopped when TLC showed complete reaction of the raw materials. With vigorous stirring, 1.5 L of isopropyl ether was added to the reaction flask, and the mixture was stirred for 2 hours. The upper liquid layer was discarded, and 800 mL of isopropyl ether was added to the residue, and the mixture was stirred vigorously for 1 hour. The mixture was filtered, and the filter cake was added to 600 mL of isopropyl ether. The mixture was stirred overnight, and then filtered to obtain 31.2 g of a tan powdery solid (82% yield, LC-MS: [M+H]). + =767.6). Step 2: Synthesis of compound 18b Compound 18a (31.2 g, 40.73 mmol) and DMF (800 mL) were added to a 5000 mL single-neck flask and stirred to dissolve. tert-Butylmethyl(2-(methylamino)ethyl)aminocarbamate (8.1 g, 36.66 mmol) dissolved in 50 mL of DMF was added and stirred at room temperature for 3.5 hours. The reaction was stopped when TLC showed the reaction was complete. 2 L of isopropyl ether was added to the reaction solution and stirred until a viscous oil precipitated on the flask wall. The supernatant was discarded, followed by the addition of 1 L of isopropyl ether, vigorously stirred, and the supernatant was discarded again. 500 mL of isopropyl ether was added, stirred overnight, and filtered to obtain 27.2 g of a yellow-brown powdery solid (yield 81.9%, LC-MS: [M+H] + =816.7). Step 3: Synthesis of compound 18 Compound 18b (27.2 g) and DCM (200 mL) were added to a 1000 mL single-neck flask and stirred to dissolve. 400 mL of TFA was added and the mixture was stirred at room temperature for 1.5 hours. The mixture was concentrated under reduced pressure in a 45 °C water bath using a water pump to remove TFA and DCM, yielding a yellow liquid residue, which was used directly in the next reaction without further purification.

[0135] Example 39: Synthesis of Compound P36 JPEG2025533577000402.jpg160137Step 1: Preparation of compound 36a Compound 18 (2 g, 2.8 mmol) was added to a 50 mL single-neck flask and dissolved in 30 mL of DCM. The mixture was cooled to 0 °C in an ice-water bath. While maintaining this temperature, DIPEA (2.32 mL, 14 mmol) and NPC (4.32 g, 14 mmol) were added in that order. The mixture was slowly warmed to room temperature and reacted for 5 h. The end point of the reaction was monitored by TLC. After completion of the reaction, the mixture was concentrated under reduced pressure at 45 °C using a water pump to remove the solvent, yielding a residue. The residue was purified by silica gel column chromatography (DCM / MeOH = 100 / 1 - 50:1 to 10:1) to give compound 36a (1.98 g) (LC-MS: [M+H] + =881.6). Step 2: Preparation of compound 36b Compound 36a (1.98 g, 2.24 mmol) and compound 16 (1.11 g, 2.24 mmol) were added to a 50 mL single-neck flask, dissolved in 25 mL of DMF, and cooled to 0 °C in an ice-water bath. DIPEA (0.745 mL, 4.48 mmol) was added, and the mixture was slowly warmed to room temperature and reacted for 1 hour. The end point of the reaction was monitored by HPLC. After completion of the reaction, the mixture was concentrated under reduced pressure in a 45 °C water bath using an oil pump to remove the solvent and obtain a residue. The residue was purified by preparative high-performance liquid chromatography, and the product solution was collected and lyophilized to obtain compound 36b (2.13 g) (LC-MS: [M+H] + =1135.7). Step 3: Preparation of compound 36c Compound 36b (2.1 g, 1.85 mmol) was added to a 50 mL single-neck flask, dissolved in 20 mL of DMF, and cooled to 0 °C in an ice-water bath. Diethylamine (5 mL) was added, and the mixture was allowed to warm to room temperature and react for 4 hours. The end point of the reaction was monitored by HPLC. After completion of the reaction, the mixture was concentrated under reduced pressure in a 45 °C water bath using an oil pump to remove the solvent. The resulting residue was used directly in the next reaction. Step 4: Preparation of compound 36d Crude compound 36c (1.85 mmol, added according to the moles of 36b) and compound M3 (1.05 g, 1.85 mmol) were added to a 50 mL single-neck flask, dissolved in 20 mL of DMF, cooled to 0 °C in an ice-water bath, and DIPEA (615 μL, 3.7 mmol) was added. The mixture was slowly warmed to room temperature and reacted for 2 hours. The end point of the reaction was monitored by HPLC. After completion of the reaction, the mixture was concentrated under reduced pressure in a 45 °C water bath using an oil pump to remove the solvent, yielding a residue. The residue was purified by high-performance liquid chromatography, and the product solution was collected and lyophilized to obtain compound 36d (2.3 g) (LC-MS: [M+H] + =1393.9). Step 4: Preparation of compound P36 Compound 36d (200 mg, 0.144 mmol) and 15 mL of nitromethane were added to a 10 mL single-neck flask. After dissolution, zinc bromide (652 mg, 2.87 mmol) was added and the mixture was allowed to react at room temperature for 30 minutes. The nitromethane was removed by concentration in a water bath at 45 °C under reduced pressure using a water pump, and a yellow solid residue was obtained. The solid was purified by preparative high-performance liquid chromatography, and the solution was lyophilized to obtain compound P36 (172 mg) (LC-MS: [M+H] + =1237.8).

[0136] Example 40: Synthesis of Compound P37 JPEG2025533577000403.jpg84136Step 1: Preparation of compound 37a Compound 6-maleimidohexanoate N-succinimidyl (2 g, 6.8 mmol) was added to a 100 mL single-neck flask and dissolved in 50 mL of DMF. The mixture was cooled to 0 °C in an ice-water bath. While maintaining this temperature, propargylamine (0.44 mL, 6.8 mmol) and DIPEA (2.27 mL, 13.6 mmol) were added in that order. The mixture was slowly warmed to room temperature and reacted overnight. The end point of the reaction was monitored by TLC. After completion of the reaction, the mixture was concentrated under reduced pressure at 45 °C using a water pump to remove the solvent, yielding a residue. The residue was purified by silica gel column chromatography (PE / EA = 50 / 1-20:1-5:1) to give compound 37a (1.3 g) (LC-MS: [M+H] + =249.3). Step 2: Preparation of compound 37b Compound 37a (1.3 g, 5.3 mmol) was placed in a 100 mL single-neck flask and dissolved in 50 mL of ultra-dry, oxygen-free THF. The atmosphere in the system was flushed with nitrogen three times. N-PEG-CHCHCOOH (2.6 g, 5.3 mmol), DIPEA (0.914 mL, 5.5 mmol), and CuI (110 mg, 0.55 mmol) were added sequentially at room temperature. The mixture was slowly warmed to room temperature and reacted for 4 hours. The end point of the reaction was monitored by HPLC. The reaction solution was directly purified by preparative high-performance liquid chromatography (acid method). The fractionated solution was collected and lyophilized to give compound 37b (2.7 g) (LC-MS: [M-H]). - =714.3). Step 3: Preparation of compound P37 Compound 37b (200 mg, 0.285 mmol), compound 36c (289 mg, 0.285 mmol), PyBOP (301 mg, 0.57 mmol), HOBt (78 mg, 0.57 mmol), and DMF (6 mL) were added to a 25 mL single-neck flask, cooled to 0 °C in an ice-water bath, and DIPEA (95 μL, 0.57 mmol) was added. After the addition, the mixture was warmed to room temperature and reacted for 2 h. Completion of the reaction was monitored by HPLC. The reaction solution was directly purified by preparative high-performance liquid chromatography, and the product solution was collected and lyophilized to obtain compound P37 (288 mg) (TOF-MS: [M+H] + =1710.8014).

[0137] Example 41: Synthesis of Compound P38 JPEG2025533577000404.jpg111128Step 1: Preparation of compound 38a Compound 37a (1 g, 4.1 mmol) was placed in a 100 mL single-neck flask and dissolved in 50 mL of ultra-dry, oxygen-free THF. The atmosphere in the system was replaced with nitrogen three times. Azide-ethylene glycol-acetic acid (872 mg, 4.1 mmol), DIPEA (0.75 mL, 4.5 mmol), and CuI (82 mg, 0.41 mmol) were added sequentially at room temperature. The mixture was slowly warmed to room temperature and reacted for 4 hours. The end point of the reaction was monitored by HPLC. The reaction solution was directly purified by preparative high-performance liquid chromatography (acid method). The fraction was collected and lyophilized to obtain compound 38a (1.1 g) (LC-MS: [M-H]). - =436.3). Step 2: Preparation of compound 38c Compound 38b (0.44 g, 1 mmol, prepared according to the synthetic route for compound 12b in Chemistry-A European Journal. 2015.21, pp. 6921-6929), 38a (0.7 g, 1 mmol), PyBOP (1.06 g, 2 mmol), HOBt (273 mg, 2 mmol), and DMF (30 mL) were added to a 100 mL single-neck flask. The mixture was cooled to 0 °C in an ice-water bath, and DIPEA (225 μL, 2 mmol) was added. The mixture was then warmed to room temperature and reacted for 2 h. Completion of the reaction was monitored by HPLC. The reaction solution was directly purified by preparative high-performance liquid chromatography, and the product solution was collected and lyophilized to give compound 38c (521 mg) (LC-MS: [M-Boc+H]). + =1016.6). Step 3: Synthesis of compound 38d Compound 38c (500 mg, 0.5 mmol) was placed in a 25 mL single-neck flask and dissolved in 5 mL of DCM. TFA (10 mL) was added dropwise under ice-water bath cooling. After the addition was complete, the reaction was allowed to proceed for 2 hours while maintaining the temperature at 0°C. The end point of the reaction was monitored by HPLC. The reaction solution was directly concentrated in a 45°C water bath using a water pump, and the resulting yellow oil was used directly in the next reaction. The crude product compound (0.5 mmol, depending on the amount of compound 38c added), compound 7a (324 mg, 0.5 mmol), and DMF (6 mL) were added to a 25 mL single-neck flask, cooled to 0 °C in an ice-water bath, and DIPEA (225 μL, 2 mmol) was added. After the addition, the mixture was warmed to room temperature and reacted for 2 hours. The reaction completion was monitored by HPLC. The reaction solution was directly purified by preparative high-performance liquid chromatography, and the product solution was collected and lyophilized to obtain compound 38d (446 mg) (LC-MS: [M+H] + =1549.6). Step 4: Synthesis of compound P38 Under a nitrogen atmosphere, compound 38d (50 mg, 0.033 mmol), ultra-dry DMF (6 mL), and AcOH (0.2 mL) were added to a 25 mL single-neck flask. Tetrakis(triphenylphosphine)palladium (37 mg, 0.033 mmol) and Bu3SnH (19 mg, 0.066 mmol) were added sequentially at room temperature. After the addition, the mixture was warmed to room temperature and reacted overnight. The reaction completion was monitored by HPLC. The reaction solution was directly purified by preparative high-performance liquid chromatography. The product solution was collected and lyophilized to obtain compound P38 (39 mg) (LC-MS: [M+Na]). + )=1487.8 was obtained.

[0138] Example 42: Synthesis of Compound P39 JPEG2025533577000405.jpg79132Step 1: Preparation of compound 39a Compound 5c (1 g, 3.2 mmol) was added to a 100 mL single-neck flask and dissolved in 30 mL of DMF. Compound 6-maleimidohexanoate N-succinimidyl (1.6 g, 3.2 mmol) and DIPEA (1.06 mL, 6.4 mmol) were added sequentially at room temperature. The reaction was allowed to proceed for 4 hours while maintaining room temperature. The end point of the reaction was monitored by HPLC. The reaction solution was directly purified by preparative high-performance liquid chromatography (acid method). The fractionated solution was collected and lyophilized to obtain compound 39a (1.9 g) (LC-MS: [MH] - =683.2). Step 2: Synthesis of compound P39 Compound 39a (200 mg, 0.29 mmol), M5 exatecan mesylate (155 mg, 0.29 mmol), PyBOP (187 mg, 0.36 mmol), HOBt (48 mg, 0.36 mmol), and DMF (6 mL) were added to a 25 mL single-neck flask, cooled to 0 °C in an ice-water bath, and DIPEA (62 mg, 0.48 mmol) was added. After the addition, the mixture was heated to 20 °C and reacted for 2 h. The reaction completion was monitored by HPLC. The reaction solution was directly purified by preparative HPLC, and the product fraction was collected and lyophilized to obtain compound P39 (156 mg) (LC-MS: [M+H]). + =1102.7).

[0139] Example 43: Synthesis of Compound P40 JPEG2025533577000406.jpg3474 Following the synthetic route of Example 42, 134 mg of compound P40 (LC-MS: [M+H] + =1102.3) was prepared.

[0140] Example 44: Synthesis of Compound P41 JPEG2025533577000407.jpg51136 Step 1: Compound 41a In a 50 mL single-neck flask, 1d (500 mg, 0.62 mmol), M8 (310 mg, 0.62 mmol) (the compound shown in Formula 31 was prepared according to the method disclosed in Patent CN111065621A, with the subsequent preparation method following Example 7-2 of the same document), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol), and 15 mL of DMF were added. DIPEA (378 μL, 2.29 mmol) was added in an ice-water bath, the mixture was warmed to room temperature, and the reaction was carried out for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography to obtain a fractionated solution. The fractionated solution was lyophilized to obtain 41a (210 mg) (LC-MS: [M+H] + =1221.6). Step 2: Compound P41 41a (200 mg, 0.162 mmol), zinc bromide (736 mg, 3.26 mmol), and 10 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain solid compound P41 (120 mg) (LC-MS: [M+H] + =1065.3).

[0141] Example 45: Synthesis of Compound P42 JPEG2025533577000408.jpg51142Step 1: Compound 42a A 100 mL single-neck flask was charged with 3d (1.66 g, 2.02 mmol, 1.0 eq), M8 (1.08 g, 2.02 mmol, 1.0 eq), PyBOP (1.58 g, 3.03 mmol, 1.5 eq), HOBt (0.41 g, 3.03 mmol, 1.5 eq), and DMF (40 mL). DIPEA (0.84 mL, 1.5 eq) was added under ice-water bath conditions. The mixture was warmed to room temperature and reacted for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative HPLC. The preparative solution was concentrated under reduced pressure in a 35 °C water bath using a water pump to remove acetonitrile, and lyophilized to give compound 42a (1.54 g) (61% yield, LC-MS: [M+H]). + =1235.4). Step 6: Compound P42 Compound 42a (1.0 g, 0.8 mmol, 1.0 eq) and 35 mL of nitromethane were added to a 100 mL single-neck flask. After dissolution, zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added and the mixture was allowed to react for 30 minutes in a 40 °C oil bath (preheated for stabilization). The nitromethane was removed by concentrating in a 45 °C water bath under reduced pressure using a water pump to obtain a yellow solid residue (monitored by HPLC). The acid method gave a fractional solution. The fractional solution was concentrated in a 35 °C water bath under reduced pressure using a water pump to remove acetonitrile, and lyophilized to give compound P42 (786 mg) (yield 90%, LC-MS: [M+H] + =1079.8).

[0142] Example 46: Synthesis of Compound P43 JPEG2025533577000409.jpg3366 Following the synthetic route of Example 45, 442 mg of compound P43 (LC-MS: [M+H] + =1079.6) was prepared.

[0143] Example 47: Synthesis of Compound P44 JPEG2025533577000410.jpg112136 Step 1: Preparation of compound 44a Fluorenylmethoxycarbonyl-L-glutamate (4 g, 9.35 mmol) was dissolved in 100 mL of DCM at room temperature. HOSu (1.17 g, 10 mmol) and DCC (2.92 g, 14 mmol) were added sequentially and the mixture was allowed to react at room temperature for 2 h. After confirming the completion of the reaction by TLC, the insoluble white solid was removed by filtration. The filter cake was washed with DCM and the organic phase was dried in a water bath at 45 °C using a water pump. The resulting yellow residue was purified by silica gel column chromatography (PE / EA = 50 / 1-20 / 1) to give 4.1 g of compound 44a. Step 2: Preparation of compound 44b Compound 44a (2 g, 3.83 mmol) and 40 mL of DMF were added to a 100 mL single-neck flask. Under ice-water bath cooling, DIPEA (1.28 mL, 7.66 mmol) and compound 3c (1.7 g, 3.83 mmol) were added. The mixture was slowly warmed to room temperature and reacted for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography. The fraction was lyophilized to obtain 2.8 g of compound 44b (LC-MS: [M-H]). - =844.0). Step 3: Preparation of compound 44c Compound 44b (500 mg, 0.59 mmol) and 25 mL of DMF were added to a 50 mL single-neck flask, and exatecan mesilate (319 mg, 0.59 mmol), PyBOP (625 mg, 1.2 mmol), HOBt (164 mg, 1.2 mmol), and DIPEA (400 μL, 2.4 mmol) were added in this order at room temperature. The mixture was then heated to room temperature and reacted for 5 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography, and the fractions were lyophilized to obtain compound 44c (502 mg) (LC-MS: [M+H] + =1262.5). Step 4: Preparation of compound 44d Compound 44c (500 mg, 0.4 mmol) and 20 mL of DMF were added to a 50 mL single-neck flask, and diethylamine (5 mL) was added under ice-water bath cooling. The mixture was then slowly warmed to room temperature and reacted for 5 hours. After confirming the completion of the reaction by HPLC, the reaction solution was dried in a water bath at 45 °C using an oil pump. The resulting crude product was used directly in the next reaction. The crude product compound (0.4 mmol) and 20 mL of DMF were added to a 50 mL single-neck flask. DIPEA (145 μL, 0.8 mmol) and M3 (227 mg, 0.4 mmol) were added under ice-water bath cooling. The mixture was then warmed to room temperature and reacted for 1 hour. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography, and the fraction was lyophilized to obtain compound 44d (358 mg) (LC-MS: [M+H] + =1420.7). Step 5: Preparation of compound P44 Compound 44d (150 mg, 0.106 mmol), zinc bromide (476 mg, 2.11 mmol), and 5 mL of nitromethane were added to a 25 mL one-neck flask and reacted at 40°C for 1 hour. After confirming the completion of the reaction by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. The crude product was purified by high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was freeze-dried to obtain solid compound P44 (98 mg) (TOF: [M+H] + =1208.4281).

[0144] Example 48: Synthesis of Compound P45 JPEG2025533577000411.jpg85128Step 1: Preparation of compound 45a Fmoc-O-tert-butyl-L-glutamate (4 g, 9.35 mmol) was dissolved in 100 mL of DCM at room temperature. HOSu (1.17 g, 10 mmol) and DCC (2.92 g, 14 mmol) were added sequentially and the mixture was allowed to react at room temperature for 2 hours. After confirming the completion of the reaction by TLC, the insoluble white solid was removed by filtration. The filter cake was washed with DCM and the organic phase was dried in a water bath at 45 °C using a water pump. The pale yellow residue was purified by silica gel column chromatography (PE / EA = 50 / 1-20 / 1) to give 4.12 g of compound 45a. Step 2: Preparation of compound 45b Compound 45a (2 g, 7.66 mmol) and 50 mL of DMF were added to a 100 mL single-neck flask. Under ice-water bath cooling, DIPEA (2.46 mL, 15.32 mmol) and aminooctaglycol monomethyl ether (3.1 g, 7.66 mmol) were added. The mixture was slowly warmed to room temperature and reacted for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography. The fraction was lyophilized to obtain 3.7 g of compound 45b (LC-MS: [M+H] + =791.9). Step 3: Synthesis of compound 45c Compound 45b (3.7 g) and 30 mL of DCM were added to a 100 mL single-neck flask, and TFA (30 mL) was added under ice-water bath cooling. The mixture was allowed to react for 2 hours while maintaining the same temperature. After confirming the completion of the reaction by HPLC, the reaction solution was heated in a water bath at 45 °C under reduced pressure using a water pump. The solvent was spin-dried and used directly in the next step. The crude product was dissolved in 50 mL of DCM at room temperature, and HOSu (2.4 g, 20 mmol) and DCC (4.6 g, 22 mmol) were added in that order. The reaction was allowed to proceed overnight at room temperature. After confirming the completion of the reaction by HPLC, the reaction solution was purified by high-performance liquid chromatography. The fraction was lyophilized to obtain 2.1 g of compound 45c (LC-MS: [M+H] + =833.2). Step 4: Synthesis of compound P45 Starting from compound 45c and compound 3c, 102 mg of compound P45 (LC-MS: [M+H] + =1573.7) was prepared.

[0145] Example 49: Synthesis of Compound 22 JPEG2025533577000412.jpg96138 Step 1: Preparation of compound 22a Boc-L-glutamic acid 5-tert-butyl ester (50 g, 164.8 mmol, 1.0 eq) was dissolved in 250 mL of THF in a 1 L single-neck flask. Propargylamine (9.97 g, 181.3 mmol, 1.1 eq), HOBt (22.25 g, 164.8 mmol, 1.0 eq), DIPEA (54.3 mL, 329.6 mmol, 2.0 eq), and EDCI (37.7 g, 197.7 mmol, 1.2 eq) were added in this order in an ice-water bath. The mixture was warmed to room temperature and incubated for 2 h. The end point of the reaction was monitored by TLC. After completion of the reaction, the reaction solution was poured into 600 mL of ice-water and extracted three times with ethyl acetate (200 mL × 3). The combined organic phases were washed twice with saturated brine (300 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified with petroleum ether / ethyl acetate (2 / 1) to give 35.5 g of compound 22a (LC-MS: [M+H] + =341.2). Step 2: Preparation of compound 22b Compound 22a (20 g) was placed in a 1 L single-neck flask and dissolved in 150 mL of dichloromethane. 150 mL of trifluoroacetic acid was added and the reaction was allowed to proceed at room temperature for 1 hour. The end point of the reaction was monitored by TLC. After the reaction was completed, the reaction solution was concentrated at 45 °C using a water pump to obtain an oily crude product. Methyl tert-ether was added to the crude product to crystallize overnight, followed by filtration. The filter cake was dried by blowing air at 45 °C for 4 hours and then weighed to obtain 7.68 g of the product (LC-MS: [M+H]). + =184.2). Step 3: Preparation of compound 22c Compound 22b (5.0 g, 27.1 mmol) and compound M2 (10.8 g, 27.1 mmol) were added to a 50 mL single-neck flask and dissolved in 20 mL of DMF. EEDQ (13.4 g, 54.2 mmol) was added in an ice-water bath. After the addition, the mixture was warmed to room temperature and reacted for 2 hours. The end point of the reaction was monitored by HPLC. The reaction solution was purified by preparative high-performance liquid chromatography and lyophilized to obtain product 22c (7.84 g) (LC-MS m / z (M+H) + :465.2). Step 4: Preparation of compound 22d Compound 22c (0.85 g, 1.51 mmol) was added to a 25 mL single-neck flask and dissolved in 15 mL of DMF. DCC (1.55 g, 7.56 mmol) and pentafluorophenol (0.31 g, 1.66 mmol) were added in an ice-water bath, and the mixture was heated to room temperature and reacted for 2 hours. The end point of the reaction was monitored by TLC. After completion of the reaction, the mixture was filtered, and the resulting filtrate was spin-dried and used directly in the next reaction. Step 5: Preparation of Compound 22e In an ice-water bath, compound 3c (661 mg, 1.51 mmol) was dissolved in 15 mL of DMF, DIPEA (0.37 mL, 2.26 mmol) was added dropwise, and then 3 mL of the crude product compound in DMF was added. The mixture was warmed to room temperature and reacted for 1 hour, and the end point of the reaction was monitored by HPLC. After completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain 502 mg of a white solid, compound 22e (LC-MS: [M+H] + =985.2). Step 6: Preparation of Compound 22 Compound 22e (500 mg, 0.51 mmol) was added to a 50 mL single-neck flask and dissolved in 10 mL of DMF. PyBOP (396 mg, 0.76 mmol), HOBt (103 mg, 0.76 mmol), and M5 exatecan mesylate (270 mg, 0.51 mmol) were added in this order in an ice-water bath, followed by DIEA (252 μL, 1.52 mmol). The mixture was warmed to room temperature and reacted for 2 hours. The end point of the reaction was monitored by HPLC. After completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized to obtain 500 mg of a white solid compound 22 (LC-MS: [M+H] + =1402.2).

[0146] Example 50: Synthesis of Compound P46 JPEG2025533577000413.jpg69128Step 1: Synthesis of compound 46a Compound 22 (50 mg, 0.036 mmol) and azidooctaethylene glycol monomethyl ether (156 mg, 0.036 mmol) were added to a 25 mL single-neck flask and dissolved in 5 mL of DMF. Subsequently, cuprous iodide (2 mg, 0.007 mmol) and TEA (10 μL, 0.07 mmol) were added. The mixture was heated to 50 °C and reacted for 10 hours. The reaction progress was monitored by HPLC. After completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain 59 mg of compound 46a (TOF-MS: [M+Na] + =1833.9517). Step 2: Synthesis of compound P46 Compound 46a (50 mg, 0.028 mmol) was added to a 25 mL single-neck flask and dissolved in 5 mL of nitromethane. Zinc bromide (130 mg, 0.56 mmol) was then added and the reaction was allowed to proceed at room temperature for 1 hour. The progress of the reaction was monitored by HPLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure at 45 °C using a water pump to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain a preparative solution. The preparative solution was lyophilized and weighed to obtain 33 mg of compound P46 (TOF-MS: [M+H] + =1654.9).

[0147] Example 51: Synthesis of Compound P47 JPEG2025533577000414.jpg3778 Compound N3-PEG8-CH2CH2COOH was used as a starting material, and 42 mg of compound P47 (TOF-MS: [M+H] + =1712.7061) was prepared.

[0148] Example 52: Synthesis of Compound P48 JPEG2025533577000415.jpg3778 Compound O-(2-aminoethyl)-O'-(2-azidoethyl)heptapolyethylene glycol was used as a starting material, and 42 mg of compound P48 (TOF-MS: [M+H] + =1683.7627) was prepared.

[0149] Example 53: Antibody Expression and Purification 1) Transient expression Expi293 suspension cells (Shanghai OPM Biosciences Co., Ltd.) were used to express antibodies targeting CD33. The day before transfection, 0.9 × 10 cells were cultured. 6 The cells were seeded at a density of 100 cells / mL into a 1 L shake flask containing 300 mL of OPM-293 CD05 medium (81075-001, Shanghai OPM Biosciences Co., Ltd.) and cultured overnight at 37°C, 5% CO2, and 120 rpm in a cell culture shaker. The following day, they were transfected with the antibody expression plasmid using PEI-MAX. The mass ratio of plasmid to PEI-MAX was 1:3. On day 1 post-transfection, 5% (v / v) OPM-293 ProFeed was added. On day 3 post-transfection, 5% (v / v) OPM-293 ProFeed was added again. On day 6 post-transfection, the supernatant was collected by centrifugation. 2) Antibody purification The collected cell supernatant was purified using a protein A affinity chromatography column (UniMab 50, Suzhou Nanotechnology Co., Ltd.). The antibody was eluted with 0.05 M sodium acetate (pH 3.6), and the captured antibody was adjusted to pH 7.0 with 1 M Tris-HCl (pH 8.8) (0.7 / 10 (v / v)). Impurities such as polymers were removed by gel filtration chromatography using a SEC column (Superdex 200, GE). Antibody 1: SEQ ID No.1 (heavy chain variable region) EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS SEQ ID No. 2 (heavy chain CDR1) DSNIH SEQ ID No. 3 (heavy chain CDR2) YIYPYNGGTDYNQKFKN SEQ ID No. 4 (heavy chain CDR3) GNPWLAY SEQ ID No. 5 (heavy chain) EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID No.6 (Heavy chain constant region) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID No.7 (Light chain variable region) DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVK SEQ ID No.8 (light chain variable region CDR1) RASESLDNYGIRFLT SEQ ID No. 9 (light chain variable region CDR2) AASNQGS SEQ ID No. 10 (light chain variable region CDR3) QQTKEVPWS SEQ ID No. 11 (light chain) DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVE VKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No.12 (light chain constant region) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 13 (heavy chain variable region - nucleic acid coding sequence) GAGGTGCAGCTGGTGCAGAGTGGAGCTGAGGTGAAGAAGCCAGGCTCCTCTGTGAAGGTGTCCTGCAAGGCCTCTGGCTACACCATCACAGACAGCAACATCCACTGGGTGAGGCAGGCCCCAGGCCAGAGCCTGGAGTGGATTGGCTACATCTACCCCTACAATGGAGGCACA GACTACAACCAGAAGTTCAAGAACAGAGCCACCCTGACTGTGGACAACCCCACCAACACAGCCTACATGGAGCTGAGCTCCCTGAGAAGTGAAGACACAGCCTTCTACTACTGTGTGAATGGGAACCCCTGGCTGGCCTACTGGGGCCAGGGCACCCTGGTGACTGTGTCCTCT SEQ ID No. 14 (heavy chain CDR1 - nucleic acid coding sequence) GACAGCAACATCCAC SEQ ID No. 15 (heavy chain CDR2 - nucleic acid coding sequence) TACATCTACCCCTACAATGGAGGCACAGACTACAACCAGAAGTTCAAGAAC SEQ ID No. 16 (heavy chain CDR3 - nucleic acid coding sequence) GGGAACCCCTGGCTGGCCTAC SEQ ID No. 17 (heavy chain - nucleic acid coding sequence) SEQ ID No. 18 (heavy chain constant region - nucleic acid coding sequence) GCTAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCTCCCTCCTCCAAGAGCACCTCTGGAGGCACAGCTGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCAGAGCCTGTGACTGTGTCCTGGAACTCTGGAGCCCTGACCTCTGGAGTGCACACCTTCCCAGCTGTGCTGCAGAGCTCTGGCCTCTACAGCCTGTCCTCTGTGGTGACTGTGCCCTCCTCCTCCCTGGGAACCCAGACCTACATCTGCAATGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCACCCTGCCCAGCCCCAGAGCTGCTGGGAGGACCCTCTGTGTTCCTCTTTCCACCCAAGCCCAAGGACACCCTGATGATCTCCAGAACCCCAGAGGTGACCTGTGTGGTGGTGGATGTGTCCCATGAAGACCCAGAAGTGAAGTTCAACTGGTATGTGGATGGAGTGGAAGTGCACAATGCCAAGACCAAGCCCAGAGAAGAGCAGTACAACAGCACCTACAGAGTGGTGTCTGTGCTGACTGTGCTGCACCAGGACTGGCTGAATGGCAAAGAGTACAAGTGCAAAGTGTCCAACAAGGCCCTGCCAGCTCCCATTGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGAGAGCCTCAGGTGTACACCCTGCCACCATCCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAAGGCTTCTACCCCTCTGACATTGCTGTGGAGTGGGAGAGCAATGGCCAGCCAGAGAACAACTACAAGACCACACCACCTGTGCTGGACTCTGATGGCAGCTTCTTCCTGTACAGCAAGCTGACTGTGGACAAGAGCAGATGGCAGCAGGGCAATGTCTTCTCCTGCTCTGTGATGCATGAAGCTCTGCACAACCACTACACCCAGAAGAGCCTGTCCCTGTCTCCAGGGTGA SEQ ID No. 19 (light chain variable region - nucleic acid coding sequence) GACATCCAGCTGACCCAGAGCCCCTCCACCCTGTCTGCCTCTGTGGGAGACAGAGTGACCATCACCTGCAGAGCCTCTGAGAGCCTGGACAACTATGGCATCAGATTCCTGACCTGGTTCCAGCAGAGCCAGGGAAAGCCCCCAAGCTGCTGATGTATGCTGCCT CCAACCAGGGCAGTGGAGTGCCCTCCAGATTCTCTGGCTCTGGCTCTGGCACAGAGTTCACCCTGACCATCTCCTCCCTGCAGCCAGATGACTTTGCCACCTACTACTGCCAGCAGACCAAGGAGGTGCCCTGGAGCTTTGGACAGGGCACCAAGGTGGAGGTGAAG SEQ ID No. 20 (light chain variable region CDR1 - nucleic acid coding sequence) AGAGCCTCTGAGAGCCTGGACAACTATGGCATCAGATTCCTGACC SEQ ID No. 21 (light chain variable region CDR2 - nucleic acid coding sequence) GCTGCCTCCAACCAGGGCAGT SEQ ID No. 22 (light chain variable region CDR3 - nucleic acid coding sequence) CAGCAGACCAAGGAGGTGCCCTGGAGC SEQ ID No. 23 (light chain - nucleic acid coding sequence) GACATCCAGCTGACCCAGAGCCCCTCCACCCTGTCTGCCTCTGTGGGAGACAGAGTGACCATCACCTGCAGAGCCTCTGAGAGCCTGACAACTATGGCATCAGATTCCTGACCTGGTTCCAGCAGAAGCCAGGGAAAGCCCCCAAGCTGCTGATGTATGCTGCCTCCAACCAGGGCAGTGGAGTGCCCTCCAGATTCTCTGGCTCTGGCTCTGGCACAGAGTTCACCCTGACCATCTCCTCCCCTGCAGCCAGATGACTTTGCCACCTACTACTGCCAGCAGACCAAGGAGGTGCCCTGGAGCTTTGGACAGGGCACCAAGGTGGAGG TGAAGCGTACGGTGGCTGCTCCTGTGTTTCATCTTCCCACCCTCTGATGAGCAGCTGAAGTCTGGCACAGCCTCTGTGGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTCAGTGGAAAGTGGACAATGCCCTGCAGGTGGGAACAGCCAGGAGAGTGACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTGTCCTCCACCCTGACCCTGTCCAAAGCTGACTATGAGAAGCACAAAAGTGTATGCCTGTGAACCCACAGGGCCTGTCCTCTCCTGTCCAAGAGCTTCAACAGAGGAGAGTGCTGA SEQ ID No.24 CGTACGGTGGCTGCTCCCTCTGTGTTCATCTTCCCACCCTCTGATGAGCAGCTGAAGTCTGGCACAGCCTCTGTGGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAAGTGGACAATGCCCTGCAGAGTGGGAACAGCCAGGAG AGTGTGACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTGTCCTCCACCCTGACCCTGTCCAAAGCTGACTATGAGAAGCACAAAGTGTATGCCTGTGAAGTGACCCACCAGGGCCTGTCCTCTCCTGTGACCAAGAGCTTCAACAGAGGAGAGTGCTGA Antibody 2: Except for the amino acid sequence of the light chain constant region and its nucleic acid coding sequence, and the amino acid sequence of the light chain and its nucleic acid coding sequence, the remaining sequences are the same as those of Antibody 1. SEQ ID No. 25 (light chain) DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVE VKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVCTKSFNRGEC SEQ ID No.26 (light chain constant region) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVCTKSFNRGEC SEQ ID No. 27 (light chain - nucleic acid coding sequence) GACATCCAGCTGACCCAGAGCCCTCCCACCTGTCTGCCCTCTGGGAGACAGAGTGACCATCACCTGCAGAGCCTCTGAGAGCCTGGACACTATGGCATCAGATTCCTGACCTGGTTCCAGCAGAAGCCAGGGAAAGCCCCCAAGCTGCTGATGTATGCTGCC TCCAACCAGGGCAGTGGAGTGCCCTCCAGATTCTCTGGCTCTGGCTCTGGCACAGAGTTCACCCTGACCATCTCCTCCCCTGCAGCCAGATGACTTTGCCACCTACTACTGCCAGCAGACCAAGGAGGTGCCCTGGAGCTTTGGACAGGGCACCAAGGTGGAGGTG AAGCGTACGGTGGCTGCTCCCTCTGTGTTCATCTTCCCACCCTCTGATGAGCAGCTGAAGTCTGGCACAGCCTCTGTGGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAAGTGGACAATGCCCTGCAGAGTGGGAACAGCCAGGAG AGTGTGACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTGTCCTCCACCCTGACCCTGTCCAAAGCTGACTATGAGAAGCACAAAGTGTATGCCTGTGAAGTGACCCACCAGGGCCTGTCCTCTCCTGTGTGCCAAGAGCTTCAACAGAGGAGAGTGCTGA SEQ ID No.28 CGTACGGTGGCTGCTCCCTCTGTGTTCATCTTCCCACCCTCTGATGAGCAGCTGAAGTCTGGCACAGCCTCTGTGGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAAGTGGACAATGCCCTGCAGAGTGGGAACAGCCAGGAGA GTTGACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTGTCCTCCACCCTGACCCTGTCCAAAGCTGACTATGAGAAGCACAAAGTGTATGCCTGTGAAGTGACCCACCAGGGCCTGTCCTCTCCTGTGTGCACCAAGAGCTTCAACAGAGGAGAGTGCTGA

[0150] Example 54: General preparation of antibody-drug conjugates Methods for preparing antibody-drug conjugates After cell expression and purification by protein A affinity chromatography and molecular sieve chromatography, antibody 2 from Example 53 was concentrated or diluted to a protein concentration of 5 mg / mL in 20 mM NaAc-HAc, pH 6.0 buffer. The payload was a white or pale yellow powder and was dissolved in N,N-dimethylacetamide (DMA) at 10 mg / mL for further use. To cleave the interchain disulfide bond of antibody 2, tris(2-carboxyethyl)phosphine (TCEP) was added at a 20x molecular ratio and allowed to react at room temperature for 2 hours. Payload solution was added at a 25x molecular ratio and allowed to react at room temperature for 8 hours. After the reaction was complete, the liquid was exchanged using a 30 kDa ultrafiltration concentration tube, and unbound payload was removed to obtain the antibody 2-drug conjugate sample.

[0151] Example 55: Preparation of ADC-1 JPEG2025533577000416.jpg125128 ADC-1 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0152] Example 56: Preparation of ADC-2 JPEG2025533577000417.jpg122128 ADC-2 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0153] Example 57: Preparation of ADC-3 JPEG2025533577000418.jpg104128 ADC-3 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0154] Example 58: Preparation of ADC-4 JPEG2025533577000419.jpg106128 ADC-4 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0155] Example 59: Preparation of ADC-5 JPEG2025533577000420.jpg104128 ADC-5 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0156] Example 60: Preparation of ADC-6 JPEG2025533577000421.jpg106128 ADC-6 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0157] Example 61: Preparation of ADC-7 JPEG2025533577000422.jpg104128 ADC-7 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0158] Example 62: Preparation of ADC-8 JPEG2025533577000423.jpg124128 ADC-8 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0159] Example 63: Preparation of ADC-9 JPEG2025533577000424.jpg104128 ADC-9 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0160] Example 64: Preparation of ADC-10 JPEG2025533577000425.jpg119128 ADC-10 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0161] Example 65: Preparation of ADC-11 JPEG2025533577000426.jpg106128 ADC-11 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0162] Example 66: Preparation of ADC-12 JPEG2025533577000427.jpg122128 ADC-12 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0163] Example 67: Preparation of ADC-13 JPEG2025533577000428.jpg103128 ADC-13 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0164] Example 68: Preparation of ADC-14 JPEG2025533577000429.jpg104128 ADC-14 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0165] Example 69: Preparation of ADC-15 JPEG2025533577000430.jpg104128 ADC-15 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0166] Example 70: Preparation of ADC-16 JPEG2025533577000431.jpg104128 ADC-16 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0167] Example 71: Preparation of ADC-17 JPEG2025533577000432.jpg106128 ADC-17 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0168] Example 72: Preparation of ADC-18 JPEG2025533577000433.jpg106128 ADC-18 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0169] Example 73: Preparation of ADC-19 JPEG2025533577000434.jpg111128 ADC-19 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0170] Example 74: Preparation of ADC-20 JPEG2025533577000435.jpg112128 ADC-20 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0171] Example 75: Preparation of ADC-21 JPEG2025533577000436.jpg111128 ADC-21 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0172] Example 76: Preparation of ADC-22 JPEG2025533577000437.jpg109128 ADC-22 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0173] Example 77: Preparation of ADC-23 JPEG2025533577000438.jpg109128 ADC-23 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0174] Example 78: Preparation of ADC-24 JPEG2025533577000439.jpg109128 ADC-24 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0175] Example 79: Preparation of ADC-25 JPEG2025533577000440.jpg101128 ADC-25 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0176] Example 80: Preparation of ADC-26 JPEG2025533577000441.jpg106128 ADC-26 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0177] Example 81: Preparation of ADC-27 JPEG2025533577000442.jpg104128 ADC-27 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0178] Example 82: Preparation of ADC-28 JPEG2025533577000443.jpg104128 ADC-28 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0179] Example 83: Preparation of ADC-29 JPEG2025533577000444.jpg109128 ADC-29 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0180] Example 84: Preparation of ADC-30 JPEG2025533577000445.jpg106128 ADC-30 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0181] Example 85: Preparation of ADC-31 JPEG2025533577000446.jpg99128 ADC-31 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0182] Example 86: Preparation of ADC-32 JPEG2025533577000447.jpg109128 ADC-32 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0183] Example 87: Preparation of ADC-33 JPEG2025533577000448.jpg120128 ADC-33 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0184] Example 88: Preparation of ADC-34 JPEG2025533577000449.jpg114128 ADC-34 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0185] Example 89: Preparation of ADC-35 JPEG2025533577000450.jpg119128 ADC-35 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0186] Example 90: Preparation of ADC-36 JPEG2025533577000451.jpg119128 ADC-36 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0187] Example 91: Preparation of ADC-37 JPEG2025533577000452.jpg114128 ADC-37 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0188] Example 92: Preparation of ADC-38 JPEG2025533577000453.jpg122137 ADC-38 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0189] Example 93: Preparation of ADC-39 JPEG2025533577000454.jpg106128 ADC-39 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0190] Example 94: Preparation of ADC-40 JPEG2025533577000455.jpg114128 ADC-40 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0191] Example 95: Preparation of ADC-41 JPEG2025533577000456.jpg114128 ADC-41 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0192] Example 96: Preparation of ADC-42 JPEG2025533577000457.jpg107128 ADC-42 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0193] Example 97: Preparation of ADC-43 JPEG2025533577000458.jpg106128 ADC-43 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0194] Example 98: Preparation of ADC-44 JPEG2025533577000459.jpg111128 ADC-44 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0195] Example 99: Preparation of ADC-45 JPEG2025533577000460.jpg117128 ADC-45 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0196] Example 100: Preparation of ADC-46 JPEG2025533577000461.jpg119128 ADC-46 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0197] Example 101: Preparation of ADC-47 JPEG2025533577000462.jpg117128 ADC-47 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0198] Example 102: Preparation of ADC-48 JPEG2025533577000463.jpg126128. ADC-48 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0199] Example 103: Preparation of ADC-49 (control group) JPEG2025533577000464.jpg96128 The payload of ADC-49 was prepared according to the synthetic route of Example 4-3 on page 148 of the patent "WO2019195665A1", and ADC-49 was prepared according to the general conjugation method of antibody-drug conjugates in Example 54.

[0200] Example 104: Preparation of ADC-50 (control group) JPEG2025533577000465.jpg109128 The payload of ADC-50 was prepared according to the synthetic route for the compound represented by formula (14) on page 47 of patent "CN111065621A," and ADC-50 was prepared according to the general conjugation method for antibody-drug conjugates in Example 54.

[0201] Example 105: Preparation of ADC-51 (control group) JPEG2025533577000466.jpg134128 The payload of ADC-51 was prepared according to the synthetic route of the compound in Example 9 on page 58 of patent "WO2020063676A1," and ADC-51 was prepared according to the general conjugation method of antibody-drug conjugates in Example 54.

[0202] Example 106: Measurement of monomer content by SEC-HPLC method The content of the monomer is measured by SEC-HPLC. Column: Biocore SEC-300 5 μm, 4.6 × 300 mm Manufacturer: NanoChrom, Part Number: B213-050030-04630S Mobile phase: 50mM PB+300mM NaCl+200mM Arg+5% IPA, pH=6.5 Table 1: Method parameters JPEG2025533577000467.jpg96128Table 2. Monomer content data for ligand-drug conjugates (ADCs) of the present disclosure JPEG2025533577000468.jpg62128The results are shown in Table 2 above and Figures 1A-1H. Conclusion: The ADC disclosed in this invention has excellent properties such as low degradation rate, low aggregation rate, and high monomer content.

[0203] Example 107: Drug-Antibody Ratio (DAR) Measurement The drug-antibody ratio (DAR) is measured by RP-HPLC. Column name: Proteomix RP-1000 4.6 x 100 mm 5 μm 1000A Manufacturer: Sepax Part Number: 465950-4610 Table 3: Method parameters JPEG2025533577000469.jpg165146Table 4: Detailed data on binding ratios (DAR) of ligand-drug conjugates (ADCs) of the present disclosure JPEG2025533577000470.jpg6268The results are shown in Table 4 above and Figures 2A-2H. Conclusion: The ADC disclosed in this invention has the excellent property of a high DAR value, which can significantly increase the drug concentration at the target site when the same dose of ADC drug is used.

[0204] Example 108: Hydrolysis analysis of antibody-drug conjugates In the present invention, molecular weight analysis is used to characterize the hydrolysis of antibody-drug conjugates. 1) Purpose and principle of the experiment: The purpose of this experiment was to confirm the intact molecular weight of the ADC by comparing the theoretical molecular weight with the measured molecular weight by LC-MS. Liquid chromatography-mass spectrometry (LC-MS) analysis of intact proteins is an important technical tool for comparing the theoretical average molecular weight of antibodies with the measured average molecular weight. The difference between the theoretical and measured molecular weights indicates a structural change in the antibody-drug conjugate. The payload in the ADC of the present invention readily interacts with water molecules, undergoing a ring-opening reaction, resulting in an 18 Da increase in molecular weight. Comparing the theoretical and measured molecular weights of the ADC allows us to confirm the degree of hydrolysis and ring-opening of the ADC drug. 2) Experimental conditions: Table 5-1 List of equipment and devices JPEG2025533577000471.jpg82148Table 5-2 Reagent list JPEG2025533577000472.jpg38134A. Sample preparation The samples were desalted using Zeba desalting spin columns and replaced with ultrapure water, after which 10 μg of each sample was taken and loaded onto the LC-MS for analysis. B. UPLC Separation Method Setup Mobile phase: 50 mM ammonium acetate aqueous solution Column: ACQUITY UPLC Protein BEH SEC Column, 200Å, 1.7μm, 4.6mm x 150mm Flow rate: 0.3 mL / min, column temperature: 30°C, detection wavelength: 280 nm Table 5-3 Gradient parameters JPEG2025533577000473.jpg55128C. Mass analysis parameter settings Data acquisition: positive ion mode Capillary voltage: 3 kV; cone voltage: 200 V Ion source temperature: 150°C; desolvation gas temperature: 500°C Cone gas flow rate: 50 L / Hr; Desolvation gas flow rate: 600 L / Hr Scan settings: 3.6-7.8 min MS analysis Low mass end (m / z): 500; High mass end (m / z): 7500 Scan time: 1.000 s; Collision energy: 6 eV Lock mass: leucine enkephalin (m / z 556.2766) D. Data Processing: UNIFI Software Setup Data analysis was performed using Waters Biopharmaceutical Platform Solution and UNIFI software (version number: V.1.9.4). MaxEnt1 Input mass-to-charge ratio range: Antibody:4800~6300m / z;ADC:5100~6500m / z Output mass range: Antibody:148000~149000Da;ADC:158000~162000Da MaxEnt1 parameter Output resolution: 1.0Da Minimum intensity ratio (left): 30%; Minimum intensity ratio (right): 30% Automatic noise reduction: On Peak width mode: Manual Initial m / z half-width: 1.0 (antibody or ADC) End m / z half-width: 1.0 (antibody or ADC) Iterations: 20 (antibody); 15 (ADC) During data processing, the structure of the theoretical array is set as follows: For antibodies: All disulfide bonds are linked according to the theoretical linkage scheme, but the light chain cysteine ​​site-specific insertion site is free. For ADC samples: all intrachain disulfide bonds are linked according to the theoretical linkage scheme. Cys corresponding to the interchain disulfide bonds and the light chain cysteine ​​site-specific insertion sites are free to bind the payload. 3) Experimental results: Table 5: Detailed data on hydrolysis of ligand-drug conjugates (ADCs) of the present disclosure JPEG2025533577000474.jpg96148The data disclosed in Table 5 show that the theoretical molecular weight of the naked antibody is 148600.1889 Da, the measured molecular weight is 148610.174 Da, and the deviation is +9.99 Da, which does not exceed the deviation range of the instrument (20 Da). When an antibody is conjugated with 10 payloads, the theoretical molecular weight is 159,398.757 Da. As shown in Figure 3, the measured molecular weights are 159,437.5384 Da, 159,481.1591 Da, and 159,590.7599 Da. When the succinimide linker of the ADC-3 payload is hydrolyzed to 2, 5, and 10 succinimides, the theoretical molecular weights are 159,434.778 Da, 159,488.8239 Da, and 159,578.9003 Da, respectively (see Table 5), which are nearly identical to the measured values, with deviations of +2.76 Da, -7.66 Da, and +11.86 Da, respectively, within the instrument error range. Therefore, the antibody-drug conjugate is partially hydrolyzed.

[0205] Example 109: Plasma stability experiments A mixture of ADC and IgG-free human plasma was prepared to a final ADC concentration of 0.6 mg / mL and incubated in a water bath in a 37°C incubator. The incubation times were set to 0, 3, and 7 days. Simultaneously, a no-incubation, no-extraction control group and a no-incubation, no-extraction control group were also set. After incubation, the samples were purified and extracted, and the drug-antibody ratio (DAR), which reflects the stability of the ADC in human plasma, was measured. Table 6. Plasma stability DAR data for ligand-drug conjugates (ADCs) of the present disclosure JPEG2025533577000475.jpg122148Note: "No incubation / no extraction" refers to the DAR value after the ADC was left to stabilize at 4°C for 0 days. "No incubation / extraction" refers to the DAR value of the ADC after the purification / extraction process without incubation with plasma, primarily to determine whether extraction affects the DAR. "0-day incubation" refers to the DAR value measured after adding the ADC to plasma and initiating the purification / extraction process. "3-day incubation" refers to the DAR value measured after adding the ADC to plasma, incubating at 37°C for 3 days, and then initiating the purification / extraction process. "7-day incubation" refers to the DAR value measured after adding the ADC to plasma, incubating at 37°C for 7 days, and then initiating the purification / extraction process. Conclusion: The ADC disclosed in the present invention has good plasma stability, with no significant changes in DAR value, and its plasma stability is superior to that of the control groups ADC-50 and ADC-51.

[0206] Example 110: Detecting binding of antibody-drug conjugates to recombinant human CD33 protein by ELISA The binding ability of the antibody to the antigen can be determined in advance by ELISA. In this study, recombinant human CD33 protein was immobilized on an enzyme strip and detected by adding a series of CD33 antibodies with a gradient concentration. 1. Coating: CD33 was diluted to 0.5 μg / mL with 1×PBS and coated at 100 μL / well overnight at 4°C. 2. Washing: Washing buffer (1x PBS + 0.05% Tween 20) was added (200 μL / well) and the plate was washed three times, leaving it to stand for 1 minute each time. 3. Blocking: After gently tapping the washing buffer three times to dry, blocking buffer (1x PBS + 0.05% Tween 20 + 1% BSA) was added (200 μL / well) and blocking was carried out at 37°C for 1 hour. 4. Washing: The plate was washed three times with washing buffer (200 μL / well), leaving it for 1 minute each time. 5. Loading of primary antibody: The antibody-drug conjugate was diluted 3-fold starting from 2 μg / mL in sample diluent (1x PBS + 0.05% Tween 20 + 1% BSA). Wash three times with wash buffer (200 μL / well), leaving each time for 1 minute. 6. Secondary antibody loading: Solarbio rabbit anti-human was diluted 1:10000 in assay buffer (1x PBS + 0.05% Tween 20 + 1% BSA) (100 μL / well) and incubated at 37°C for 1 hour. 7. Washing: The plate was washed three times with washing buffer (200 μL / well), leaving it for 1 minute each time. 8. Reading: Add TMB color development solution (50 μL / well), and after 7 minutes, add 2M H2SO4 (50 μL / well) to stop the color development. The plate was read at 450 nm using a microplate reader. Conclusion: As shown in Figures 4A, 4B, and 4C, the conjugated ADC-3, ADC-46, ADC-47, and ADC-48 maintained affinities comparable to those of the naked antibodies, with no significant differences in EC50 values. This indicates that the affinity of the naked antibodies of the present invention for the antigen remains unchanged even after binding to the toxin.

[0207] Example 111 In vitro efficacy testing of antibody-drug conjugates In this study, various human tumor cell lines (HL-60, HEL92.1.7, TF-1, MV4-11, MOLM-13, U937, and CMK) were used as experimental models to evaluate the in vitro efficacy of ADC-conjugated drugs. A fixed number of tumor cell lines were seeded into a 96-well plate, and gradient dilutions of test antibodies and corresponding ADC drugs were added to the cells. After 5 days of treatment, cell viability was detected using Alamar Blue or MTS, and the inhibitory effects of the test antibodies and ADCs on the tumor cell lines were evaluated by calculating the IC50. The antibody drug was initially treated at a concentration of 500 nM and diluted 7-fold for a total of eight concentration points for 5 days. The final algorithm was based on viability = (experimental group - blank group) / (control group - blank group) × 100%, and then curve fitting was performed using GraphPad Prism to calculate the median inhibitory concentration (IC50). Table 7: In vitro efficacy results of test anti-CD33 naked antibodies, ADC-3, ADC-47, and ADC-48, in human tumor cell lines HL-60, HEL92.1.7, TF-1, MV4-11, MOLM-13, U937, and CMK: JPEG2025533577000476.jpg240137JPEG2025533577000477.jpg19137The results are shown in Table 7 above and Figures 5A-5G. Conclusions: In HL-60, HEL92.1.7, TF-1, MV4-11, MOLM-13, U937, and CMK cell models, naked anti-CD33 antibody did not demonstrate tumor cell killing activity. In multiple myeloid tumor cell lines, ADC-3 demonstrated the most potent effect.

[0208] Example 112 In vivo efficacy testing of antibody-drug conjugates In this study, we established a subcutaneously transplanted tumor model of human erythroleukemia cell line HEL92.1.7 in BALB / c nude mice, and evaluated the in vivo efficacy of an ADC-conjugated drug, anti-CD33 mab, ADC-3. 6HEL92.1.7 cells (0.1 mL / mouse) were subcutaneously injected into the right scapula of 5-6 week-old BALB / c nude mice. The average tumor size in the mice was approximately 230-270 mm. 3 At maturity, mice were randomly assigned to a vehicle control group (Vehicle), an anti-CD33 MAb treatment group (10 mg / kg), or an ADC-3 treatment group (5 mg / kg or 10 mg / kg), with five mice per group. Drug administration began (D0). Each group received 10 mL / kg of drug via tail vein injection once weekly for four consecutive weeks (QWx4). Data for all groups were reported on day 24 after treatment (D24) (Table 8, Figure 6A) as the mean tumor volume ± SE. Anti-CD33 at 10 mg / kg and ADC-3 at 5 mg / kg or 10 mg / kg were administered intravenously (QWx4), demonstrating significant tumor-inhibitory effects. Table 8. Efficacy analysis of each group in a subcutaneously transplanted tumor model of human erythroleukemia cells HEL92.1.7 in BALB / c-Nude mice JPEG2025533577000478.jpg104148Notes: 1. Data are expressed as "mean ± standard error." 2. T / C%=T RTV / C RTV ×100%, TGI%=(1-T / C)×100%. 3. P values ​​were obtained by comparing the tumor volume between each treatment group and the vehicle control group on day 24 of administration (24 days after administration). (Analysis was performed using Graphpad Prism 6. If the P value of one-way ANOVA analysis (one-way analysis of variance) was less than 0.05, Tukey's multiple test was used for pairwise comparisons between all groups. If the P value was less than 0.05, it was considered statistically significant.) The results of the comparison between groups are as follows. In this study, a subcutaneously transplanted tumor model of human myelomonocytic leukemia cell MV-4-11 was established in BALB / c nude mice, and the in vivo efficacy of ADC-conjugated drugs, anti-CD33 mab and ADC-3, was evaluated. 6MV4-11 cells (0.1 mL / mouse) were subcutaneously injected into the right scapula of 5-6 week-old BALB / c nude mice. The average tumor size in the mice was approximately 180-200 mm. 3 At 1 year of age, mice were randomly assigned to a vehicle control group (Vehicle), an anti-CD33 MAb treatment group (10 mg / kg), or an ADC-3 treatment group (5 mg / kg or 10 mg / kg), with five mice per group. Drug administration began (D0). Each group received 10 mL / kg of drug via tail vein injection once weekly for four consecutive weeks (QWx4). Data for all groups, observed on day 28 after treatment (D28) (Table 9, Figure 6B), are mean tumor volumes ± SE. Anti-CD33 at 10 mg / kg and ADC-3 at 5 mg / kg or 10 mg / kg were administered intravenously (QWx4), demonstrating significant tumor-inhibitory effects. Table 9. Efficacy analysis of each group in a subcutaneously transplanted tumor model of human myelomonocytic leukemia cell MV4-11 in BALB / c-Nude mice JPEG2025533577000480.jpg104148Notes: 1. Data are expressed as "mean ± standard error." 2. T / C%=T RTV / C RTV ×100%, TGI%=(1-T / C)×100%. 3. P values ​​were obtained by comparing the tumor volume between each treatment group and the vehicle control group on day 24 (24 days after administration). (Analysis was performed using Graphpad Prism 6. If the P value of one-way ANOVA analysis (one-way analysis of variance) was less than 0.05, Tukey's multiple test was used for pairwise comparisons between all groups. If the P value was less than 0.05, it was considered statistically significant.) The results of the comparison between groups are as follows. JPEG2025533577000481.jpg46148

[0209] Example 113: In vitro efficacy of compound P3 1) Experimental materials: Cells: Test cells were obtained from the Cell Bank of the Chinese Academy of Sciences. Cell medium DMEM: Gibco FBS:BIOWEST. 2) Preparation of medium: Growth medium (containing 10% FBS and penicillin / streptomycin (100 U / mL)) Detection medium (containing 1% FBS and penicillin / streptomycin (100 U / mL)). 3) Operation: The UV light in the biosafety cabinet was turned on 30 minutes before the experiment, and then the ventilation was opened for 3 minutes. Growth medium, detection medium, D-PBS, and trypsin were preheated in a 37°C water bath, and the surface was disinfected with alcohol before being placed in the biosafety cabinet. Cells at approximately 80% confluence were placed in the biosafety cabinet, the old medium was removed, rinsed with D-PBS, and discarded. They were then digested with trypsin for 2-3 minutes, neutralized with growth medium, and centrifuged at 1200 rpm for 3 minutes. After centrifugation, the supernatant was removed and mixed with 4 mL of detection medium, and 100 μL was taken for counting (50 μL of cell solution was taken, 50 μL of trypan blue stain was added, and mixed. After mixing, the cells were counted). Cells were seeded at the pre-optimized cell seeding density (80 μL / well) in a 96-well plate. 80 μL of detection medium alone was added to wells E11 and F11, and 150 μL of DPBS was added to the edge wells. 24 hours after seeding, 20uL of diluted antibody was added per well to set up a control group, and 20uL of detection medium alone was added to column 11, setting up two replicate wells for each concentration. After addition, the wells were mixed at 550 rpm for 3 minutes on a cell vortex shaker. Dilution of the solution: Using detection medium, 300 μL of test sample solution with a starting concentration of 5 μM was prepared in column 1 of a V-shaped 96-well plate, and 240 μL of detection medium was added to columns 2 through 10. 60 μL was taken from column 1 and added to column 2, mixed up and down with a pipette 10 times, discarded the pipette tip, and this process was repeated for the next seven concentrations. 4) Detection: After 4 days, the MTS reagent was removed and thawed at room temperature in the dark. After vortexing, 20 μL of CellTiter One Solution Reagen MTS reagent was added for every 100 μL of cell culture along the sidewall of the well in a biosafety cabinet. The plate was gently tapped to mix the MTS solution evenly and then placed in a cell culture incubator for 2 hours in the dark. After the reaction was complete, the 96-well plate was removed and the absorbance at OD 490 nm was measured using a microplate reader. The data was recorded, sorted, analyzed, and stored. 5) Results: Table 10. In vitro inhibitory effects of compound P3 on human non-small cell lung adenocarcinoma cells H1975, human non-small cell lung cancer cells HCC827, human epidermoid carcinoma cells A431, human gastric cancer cells NCI-N87, human pancreatic adenocarcinoma cells BXPC-3, human epidermoid carcinoma cells A431 + human colon cancer cells SW620, human breast cancer cells ZR-75-1, human plasma cell leukemia cells H929, human multiple myeloma cells RPMI8226, human leukemia cells JJN-3, human breast cancer cells MDA-MB-361, and human breast cancer cells SK-BR-3 JPEG2025533577000482.jpg10278Table 10 shows that compound P3 has excellent inhibitory effects on both solid tumor cells and blood tumor cells.

[0210] Example 114: In vitro efficacy testing of naked antibodies, small molecule drugs, and antibody-drug conjugates (ADCs) We evaluated the in vitro efficacy of naked antibodies, small molecule drugs, and ADC-conjugated drugs using experimental models of three human tumor cell lines (HL-60, JVM-3, and Raji). A fixed number of tumor cells were seeded into a 96-well plate, and gradient dilutions of small molecule drugs, test antibodies, and corresponding ADC drugs were added to the cells. After 5 days of treatment, cell viability was detected using Alamar Blue or MTS, and the inhibitory effect of the test drugs on tumor cell lines was evaluated by calculating the IC50. The antibody drug was initially treated at 500 nM, diluted 7-fold, for a total of eight concentration points, for 5 days. The final algorithm was based on viability = (experimental group - blank group) / (control group - blank group) × 100%, and then curve fitting was performed using GraphPad Prism to calculate the median inhibitory concentration (IC50). The results are shown in Table 11 and Figures 7A-7C. Table 11. In vitro efficacy studies of naked antibodies, small molecule drugs, and antibody-drug conjugates (ADCs) JPEG2025533577000483.jpg77148Conclusion: The small molecule drugs and antibody-drug conjugates (ADCs) prepared in this invention showed excellent tumor inhibitory activity in experimental models of three types of human tumor cell lines (HL-60, JVM-3, Raji).

Claims

1. An antibody-drug conjugate represented by general formula I, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein where: The drug in the antibody-drug conjugate is a camptothecin-based antitumor drug represented by the following formula II or a stereoisomer thereof: Ab is selected from an anti-CD33 antibody or an antigen-binding fragment thereof; M １ , M ２ , M ３ are each independently 、 、 and M １ , M ２ , M ３ are different from each other, or M １ , M ２ , M ３ are each independently a group of formula A １ , formula A ２ , formula A ３ and M １ , M ２ , M ３ are different from each other, Formula A １ Formula A ２ Formula A ３ wherein Y is a skeleton selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl, preferably Y is C1-C6 alkyl, Ac is a hydrophilic structural unit, the position indicated by the wavy line on the left is linked to Ab, and the position indicated by the wavy line on the right is linked to B or linked to L; B may be present or absent, and when present, is selected from a modifying unit; L is selected from linker units, preferably peptide-containing linker units; X is selected from —NH—, —O—, or —S—, preferably —O—; R １ and R ２ are the same or different and each independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl; R ３ and R ４ are the same or different and each independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, C6-C10 arylC1-C6 alkyl, 5-10 membered heteroaryl, substituted 5-10 membered heteroaryl, preferably R ３ , R ４ are each independently selected from hydrogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, or C6-C10 arylC1-C6 alkyl; Or, R ３ , R ４ and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl, a 3- to 7-membered heterocyclyl, or a substituted 3- to 7-membered heterocyclyl, preferably R ３ , R ４ and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl; R ５ is selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, preferably R ５ is selected from hydrogen, C1-C6 alkyl, m is selected from integers from 0 to 5; n1, n2, and n3 are each independently selected from any integer or any decimal point in the range of 0 to 10, and n1, n2, and n3 are not simultaneously 0, and 1≦n1+n2+n3≦10; The chiral carbon atom indicated at the * position has the R or S absolute configuration; M １ , M ２ , M ３ are each independently 、 、 is selected from M １ , M ２ , M ３ are different from each other, B is present and is selected from the modifying units; An antibody-drug conjugate or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

2. Said M １ , M ２ , M ３ are each independently 、 、 and M １ , M ２ , M ３ are different from each other, or M １ , M ２ , M ３ are each independently a group of formula A １ , formula A ２ , formula A ３ and M １ , M ２ , M ３ are different from each other, Preferably, M １ , M ２ , M ３ are each independently a group of formula A １ , formula A ２ , formula A ３ and M １ , M ２ , M ３ are different from each other, Formula A １ Formula A ２ Formula A ３ wherein Y is a backbone selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl; preferably, Y is C1-C6 alkyl, more preferably C1-C3 alkyl, and most preferably methylene; The position indicated by * has two chiralities, R absolute configuration or S absolute configuration, The position indicated by the wavy line on the left is linked to Ab, the position indicated by the wavy line on the right is linked to B or to L, The hydrophilic structural unit Ac comprises a natural or unnatural amino acid, 1-20 polyethylene glycol, a phosphate group, a carboxylic acid group, a sulfonic acid group, a sulfinic acid group, or the following structure: 、 、 、 、 、 、 、 、 、 、 or where p is selected from an integer from 0 to 10, and the position indicated by the wavy line is linked to the backbone Y; Preferably, the hydrophilic structural unit Ac is 、 、 and the positions indicated by wavy lines are linked to the backbone Y, More preferably, the hydrophilic structural unit Ac is and the position indicated by the wavy line is linked to the skeleton Y, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to claim 1.

3. M １ , M ２ , M ３ is selected from any of the following combinations (1) to (7), and M １ , M ２ , M ３ are different from each other, （１） 、 、 、 （２） 、 、 、 （３） 、 、 、 （４） 、 、 、 （５） 、 、 、 （６） 、 、 、 （７） 、 、 ； Preferably, M １ , M ２ , M ３ is selected from any of the following combinations (1) to (2), and M １ , M ２ , M ３ are different from each other, （１） 、 、 、 （２） 、 、 、 More preferably, M １ , M ２ , M ３ are each independently 、 、 and M １ , M ２ , M ３ are different from each other, where: The * position is either the R or S absolute configuration, The antibody-drug conjugate, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to claim 1 or 2, wherein the position indicated by the wavy line on the left is linked to Ab, and the position indicated by the wavy line on the right is linked to a modifying unit B or a linker unit L.

4. The antibody-drug conjugate, or stereoisomer thereof, pharmaceutically acceptable salt or solvate thereof, according to any one of claims 1 to 3, wherein modifying unit B may be present or absent, and when said modifying unit B is present, said conjugate comprises a structure that enhances hydrophilicity or a stereoisomer thereof.

5. The modifying unit B may be present or absent. When the modifying unit B is present, it has a hydrophilicity-enhancing structure represented by the following formula or a stereoisomer thereof: 、 、 、 、 、 、 、 、 、 ； Preferably, the modifying unit B may be present or absent, and when the modifying unit B is present, it comprises a hydrophilic enhancing structure represented by the following formula or a stereoisomer thereof: 、 、 、 、 ； where: R comprises any one of or a combination of hydroxyl, amino, polyethylene glycol, a carboxylic acid group, a sulfonic acid group, a sulfinic acid group, a phosphate group, C1-C6 alkyl, C1-C6 alkoxy, a natural or unnatural amino acid residue, a sugar, or a derivative thereof; Preferably, R is selected from C1-C6 alkoxy, a carboxylic acid group, and amino; More preferably, R is selected from methoxy, a carboxylic acid group, and amino; The position indicated by the wavy line on the left is M １ , M ２ , M ３ is connected to The position indicated by the wavy line on the right is linked to the linker unit L, q is selected from the integers 1 to 10, preferably 2, 7, and 8; Most preferably, the modifying unit B is selected from the hydrophilicity-enhancing structures of the following formulae or stereoisomers thereof: 、 、 、 、 、 、 、 、 The position indicated by the wavy line on the left is M １ , M ２ , M ３ and the position indicated by the wavy line on the right is linked to a linker unit L.

6. The linker unit L is L １ -L ２ and L １ is a peptide residue containing 2 to 10 amino acid residues, and the amino terminus of said peptide residue is １ , M ２ , M ３ and the carbonyl end is L ２ is connected to Preferably, L １ is a peptide residue containing 2 to 4 amino acid residues, and the amino terminus of the peptide residue is a modifying unit B or M １ , M ２ , M ３ and the carbonyl end is L ２ wherein preferably said amino acids are selected from valine, alanine, phenylalanine, glycine, lysine, citrulline, serine, glutamic acid, aspartic acid, more preferably said amino acids are selected from valine, alanine, phenylalanine, glycine, lysine, citrulline, most preferably said amino acids are selected from valine, phenylalanine, glycine, lysine, citrulline, More preferably, L １ is selected from the following peptide residues: glycine-glycine-phenylalanine-glycine, valine-citrulline, phenylalanine-lysine, valine-alanine, alanine-alanine-alanine (preferably selected from glycine-glycine-phenylalanine-glycine, valine-citrulline, phenylalanine-lysine), and the amino terminus of said peptide residue is modified by a modifying unit B or M １ , M ２ , M ３ and the carbonyl end is L ２ is connected to Most preferably, L １ is the following peptide residue: 、 、 、 、 (preferably, 、 、 and the amino terminus of said peptide residue is selected from the group consisting of modifying units B and M １ , M ２ , M ３ and the carbonyl end is L ２ is connected to L ２ teeth, 、 、 、 、 The position indicated by the wavy line on the left is selected from L １ The position indicated by the wavy line on the right is connected to X, Preferably, L ２ teeth, 、 、 、 The position indicated by the wavy line on the left is selected from L １ The position indicated by the wavy line on the right is connected to X, More preferably, L ２ teeth, 、 The position indicated by the wavy line on the left is selected from L １ The position indicated by the wavy line on the right is connected to X, Preferably, the linker unit L comprises the following structure or a stereoisomer thereof: 、 、 、 、 、 、 、 、 、 、 、 or ； where: r is selected from an integer from 1 to 10; More preferably, said linker unit L is selected from the following structures or stereoisomers thereof: 、 、 ； The position indicated by the wavy line on the left side of the linker unit L is the position of the modifying unit B or M. １ , M ２ , M ３ is connected to The antibody-drug conjugate, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 1 to 5, wherein the position indicated by the wavy line on the right side of the linker unit L is linked to X.

7. R １ and R ２ are the same or different and each independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl; Preferably, R １ and R ２ are the same or different and each independently selected from halogen and C1-C6 alkyl; More preferably, R １ is selected from C1-C6 alkyl, R ２ is selected from halogens, Most preferably, R １ is methyl, and R ２ is fluorine, R ３ and R ４ are the same or different and each independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, C6-C10 arylC1-C6 alkyl, 5-10 membered heteroaryl, substituted 5-10 membered heteroaryl; Preferably, R ３ , R ４ are the same or different and each independently selected from hydrogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, or C6-C10 arylC1-C6 alkyl; More preferably, R ３ , R ４ are the same or different and each independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkylC1-C6 alkyl, or phenylC1-C6 alkyl; More preferably, R ３ , R ４ are the same or different and are each independently selected from hydrogen, methyl, ethyl, trifluoromethyl, cyclopropyl, cyclopropylmethyl, and benzyl; Most preferably, R ３ , R ４ one of which is selected from hydrogen and methyl, and the other is selected from hydrogen, methyl, ethyl, trifluoromethyl, cyclopropyl, cyclopropylmethyl, and benzyl; Or, R ３ , R ４ and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl, a 3- to 7-membered heterocyclyl, or a substituted 3- to 7-membered heterocyclyl; Preferably, R ３ , R ４ and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl; More preferably, R ３ , R ４ and the carbon atoms to which they are attached constitute a C3-C6 cycloalkyl; Most preferably, R ３ , R ４ and the carbon atom to which they are attached constitute a cyclopropyl, cyclobutyl, or cyclopentyl; R ５ is selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, preferably R ５ is selected from hydrogen, C1-C6 alkyl, more preferably R ５ is selected from hydrogen, methyl, X is selected from —NH—, —O—, or —S—, preferably —O—; The antibody-drug conjugate, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 1 to 6, wherein m is selected from integers of 0 to 5, preferably 0 or 1.

8. The antibody-drug conjugate, or a stereoisomer thereof, a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 7, wherein the camptothecin-based antitumor drug comprises the following compound or a stereoisomer thereof: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or

9. The antibody-drug conjugate comprises the following structure: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or where: ANTI-CD33 is an anti-CD33 antibody or antigen-binding fragment thereof; n1, n2, and n3 are each independently selected from any integer or any decimal point in the range of 0 to 10, and n1, n2, and n3 are not simultaneously 0, and 1≦n1+n2+n3≦10; Preferably, n1, n2, and n3 are each independently selected from any integer or any decimal point between 0 and 10, and n1, n2, and n3 are not simultaneously 0, and 8≦n1+n2+n3≦10. The antibody-drug conjugate, or stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 1 to 8.

10. The antibody-drug conjugate, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 1 to 9, wherein the light chain variable region of the anti-CD33 antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3 shown in SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, and the heavy chain variable region comprises CDR1, CDR2, and CDR3 shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

11. The antibody-drug conjugate, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 1 to 10, wherein the anti-CD33 antibody or antigen-binding fragment thereof has a light chain variable region shown in SEQ ID NO: 7 and a heavy chain variable region shown in SEQ ID NO:

1.

12. the anti-CD33 antibody comprises a constant region derived from a human immunoglobulin; Preferably, the light chain of the anti-CD33 antibody comprises a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda) and the heavy chain of the antibody comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4); Preferably, the amino acid sequence of the light chain of the anti-CD33 antibody is SEQ ID NO: 11 and the amino acid sequence of the heavy chain is SEQ ID NO: 5; The antibody-drug conjugate or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof according to any one of claims 1 to 11, wherein the nucleic acid coding sequence of the light chain of the anti-CD33 antibody is preferably SEQ ID NO: 23, and the nucleic acid coding sequence of the heavy chain of the anti-CD33 antibody is SEQ ID NO:

17.

13. the anti-CD33 antibody further comprises a cysteine ​​site-specific insertion; Preferably, the site for the cysteine ​​insertion is in the light chain constant region; Preferably, the cysteine ​​insertion site is at position 206 (Kabat numbering) of the kappa light chain constant region; Preferably, the amino acid sequence of the light chain of the anti-CD33 antibody is SEQ ID NO: 25 and the amino acid sequence of the heavy chain is SEQ ID NO: 5; The antibody-drug conjugate or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof according to any one of claims 10 to 12, wherein the nucleic acid coding sequence of the light chain of the anti-CD33 antibody is preferably SEQ ID NO: 27, and the nucleic acid coding sequence of the heavy chain of the anti-CD33 antibody is SEQ ID NO:

17.

14. The antibody-drug conjugate, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof according to any one of claims 1 to 13, characterized in that the pharmaceutically acceptable salt includes sodium, potassium, calcium, or magnesium salts formed with acidic functional groups in the structural formula, and acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, hydrogensulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate salts formed with basic functional groups in the structural formula.

15. A linker-drug of formula III, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof: where: M' is or a linking unit selected from the linking units represented by the following formula A': Formula A' wherein Y is a skeleton selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl, preferably Y is C1-C6 alkyl, Ac is a hydrophilic structural unit, and the position indicated by the wavy line on the right is connected to B or connected to L; B may be present or absent, and when present, is selected from a modifying unit; L is selected from linker units, preferably peptide-containing linker units; X is selected from —NH—, —O—, or —S—, preferably —O—; R １ and R ２ are the same or different and each independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 membered heteroaryl, and substituted 5-10 membered heteroaryl; Preferably, R １ and R ２ are the same or different and each independently selected from halogen and C1-C6 alkyl; More preferably, R １ is selected from C1-C6 alkyl, R ２ is selected from halogens, Most preferably, R １ is methyl, and R ２ is fluorine, R ３ and R ４ are the same or different and each independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, 3-7 membered heterocyclyl, substituted 3-7 membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, C6-C10 arylC1-C6 alkyl, 5-10 membered heteroaryl, substituted 5-10 membered heteroaryl; Preferably, R ３ , R ４ are the same or different and each independently selected from hydrogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C6 alkyl, or C6-C10 arylC1-C6 alkyl; More preferably, R ３ , R ４ are the same or different and each independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkylC1-C6 alkyl, or phenylC1-C6 alkyl; More preferably, R ３ , R ４ are the same or different and are each independently selected from hydrogen, methyl, ethyl, trifluoromethyl, cyclopropyl, cyclopropylmethyl, and benzyl; Most preferably, R ３ , R ４ one of which is selected from hydrogen and methyl, and the other is selected from hydrogen, methyl, ethyl, trifluoromethyl, cyclopropyl, cyclopropylmethyl, and benzyl; Or, R ３ , R ４ and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl, a 3- to 7-membered heterocyclyl, or a substituted 3- to 7-membered heterocyclyl; Preferably, R ３ , R ４ and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl; More preferably, R ３ , R ４ and the carbon atoms to which they are attached constitute a C3-C6 cycloalkyl; Most preferably, R ３ , R ４ and the carbon atom to which they are attached constitute a cyclopropyl, cyclobutyl, or cyclopentyl; R ５ is selected from hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, preferably R ５ is selected from hydrogen, C1-C6 alkyl, more preferably R ５ is selected from hydrogen and methyl; m is selected from integers of 0 to 5, preferably 0 and 1; The chiral carbon atom indicated at the * position has the R or S absolute configuration; M' is B is present and is selected from the modifying units Linker - Drug or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

16. The M' is or a linking unit represented by the following formula A', preferably a linking unit represented by the following formula A': Formula A' wherein Y is a backbone selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl; preferably, Y is C1-C6 alkyl, more preferably C1-C3 alkyl, and most preferably methylene; The position indicated by * has two chiralities, R absolute configuration or S absolute configuration, The position indicated by the wavy line on the right is connected to B or L, The hydrophilic structural unit Ac comprises a natural or unnatural amino acid, 1-20 polyethylene glycol, a phosphate group, a carboxylic acid group, a sulfonic acid group, a sulfinic acid group, or the following structure: 、 、 、 、 、 、 、 、 、 、 or where p is selected from an integer from 0 to 10, and the position indicated by the wavy line is linked to the backbone Y; Preferably, the hydrophilic structural unit Ac is 、 、 and the positions indicated by wavy lines are linked to the backbone Y, More preferably, the hydrophilic structural unit Ac is and the position indicated by the wavy line is linked to the skeleton Y.

17. The linking unit M′ comprises a structure represented by the following formula: 、 、 、 、 、 or ； Preferably, said connecting unit M' comprises: 、 is selected from More preferably, the connecting unit M' is and where: The * position is either the R or S absolute configuration, The linker-drug, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, according to claim 15 or 16, characterized in that the position indicated by the wavy line on the right is linked to a modifying unit B or a linker unit L.

18. The linker-drug or its stereoisomer, pharmaceutically acceptable salt or solvate according to any one of claims 15 to 17, wherein modifying unit B may be present or absent, and when said modifying unit B is present, it comprises a structure or a stereoisomer thereof that enhances hydrophilicity.

19. The modifying unit B may be present or absent. When the modifying unit B is present, it has a hydrophilicity-enhancing structure represented by the following formula or a stereoisomer thereof: 、 、 、 、 、 、 、 、 、 ； Preferably, the modifying unit B may be present or absent, and when the modifying unit B is present, it comprises a hydrophilic enhancing structure represented by the following formula or a stereoisomer thereof: 、 、 、 、 ； where: R comprises any one of or a combination of hydroxyl, amino, polyethylene glycol, a carboxylic acid group, a sulfonic acid group, a sulfinic acid group, a phosphate group, C1-C6 alkyl, C1-C6 alkoxy, a natural or unnatural amino acid residue, a sugar, or a derivative thereof; Preferably, R is selected from C1-C6 alkoxy, a carboxylic acid group, and amino; More preferably, R is selected from methoxy, a carboxylic acid group, and amino; The position indicated by the wavy line on the left is connected to the connecting unit M', The position indicated by the wavy line on the right is linked to the linker unit L, q is selected from the integers 1 to 10, preferably 2, 7, and 8; Most preferably, the modifying unit B is selected from the hydrophilicity-enhancing structures of the following formulae or stereoisomers thereof: 、 、 、 、 、 、 、 The linker-drug or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 15 to 18, wherein the position indicated by the wavy line on the left is linked to linking unit M', and the position indicated by the wavy line on the right is linked to linker unit L.

20. The linker unit L is L １ -L ２ and L １ is a peptide residue containing 2 to 10 amino acid residues, the amino terminus of the peptide residue is linked to a linking unit M' or a modifying unit B, and the carbonyl terminus is L ２ is connected to Preferably, L １ is a peptide residue containing 2 to 4 amino acid residues, the amino terminus of the peptide residue is linked to a linking unit M' or a modifying unit B, and the carbonyl terminus is L ２ wherein preferably said amino acids are selected from valine, alanine, phenylalanine, glycine, lysine, citrulline, serine, glutamic acid, aspartic acid, more preferably said amino acids are selected from valine, alanine, phenylalanine, glycine, lysine, citrulline, most preferably said amino acids are selected from valine, phenylalanine, glycine, lysine, citrulline, More preferably, L １ is selected from the following peptide residues: glycine-glycine-phenylalanine-glycine, valine-citrulline, phenylalanine-lysine, valine-alanine, alanine-alanine-alanine (preferably selected from glycine-glycine-phenylalanine-glycine, valine-citrulline, phenylalanine-lysine), the amino terminus of said peptide residue is linked to linking unit M' or modifying unit B, and the carbonyl terminus is L ２ is connected to Most preferably, L １ is the following peptide residue: 、 、 、 、 (preferably, 、 、 the amino terminus of said peptide residue is linked to a linking unit M' or a modifying unit B, and the carbonyl terminus is linked to L ２ is connected to L ２ teeth, 、 、 、 、 The position indicated by the wavy line on the left is selected from L １ The position indicated by the wavy line on the right is connected to X, Preferably, L ２ teeth, 、 、 、 The position indicated by the wavy line on the left is selected from L １ The position indicated by the wavy line on the right is connected to X, More preferably, L ２ teeth, 、 The position indicated by the wavy line on the left is selected from L １ The position indicated by the wavy line on the right is connected to X, Preferably, the linker unit L comprises the following structure or a stereoisomer thereof: 、 、 、 、 、 、 、 、 、 、 、 or ； where: r is selected from an integer from 1 to 10; More preferably, said linker unit L is selected from the following structures or stereoisomers thereof: 、 、 ； The position indicated by the wavy line on the left side of the linker unit L is linked to the linking unit M′ or the modifying unit B, The linker-drug, or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof, according to any one of claims 15 to 19, wherein the position indicated by the wavy line on the right side of the linker unit L is linked to X.

21. The linker-drug or its stereoisomer, pharmaceutically acceptable salt or solvate according to any one of claims 15 to 20, wherein the linker-drug is selected from the following structures or stereoisomers thereof: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or

22. A method for preparing the antibody-drug conjugate or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof according to any one of claims 1 to 14, comprising: The method comprises: and binding Ab to a linker-drug represented by general formula III to prepare an antibody-drug conjugate represented by general formula I according to any one of claims 1 to 14, or a stereoisomer, pharmaceutically acceptable salt or solvate thereof; where: Ab is selected from an anti-CD33 antibody or an antigen-binding fragment thereof; Preferably, the anti-CD33 antibody is as defined in any of claims 10 to 13, M １ , M ２ , M ３ , B, L, X, R １ , R ２ , R ３ , R ４ , R ５ , m, n1, n2, n3 are as defined in any one of claims 1 to 8, and M' is as defined in any one of claims 15 to 17, The chiral carbon atom at the position indicated by * has two chiralities, R absolute configuration or S absolute configuration. method.

23. A pharmaceutical composition comprising the antibody-drug conjugate or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 14, or the linker-drug or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 15 to 21, and optionally a pharmaceutically acceptable carrier.

24. A pharmaceutical formulation comprising the antibody-drug conjugate or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 14, or the linker-drug or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 15 to 21.

25. Use of the antibody-drug conjugate or stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 14, or the linker-drug or stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 15 to 21, or the pharmaceutical composition according to claim 23 and / or the pharmaceutical formulation according to claim 24 in the preparation of a drug for treating or preventing cancer or tumor, Preferably, the cancer or tumor expresses CD33; More preferably, the cancer or tumor is selected from adenocarcinoma, 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 (e.g., non-small cell lung cancer), colon cancer, breast cancer (e.g., triple-negative breast cancer), rectal cancer, colorectal cancer, bone cancer, skin cancer (e.g., epidermal cancer), thyroid cancer, pancreatic cancer, solid tumors or hematological tumors such as melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, myeloma, leukemia, use.

26. used to treat or prevent cancer or tumors, Preferably, the cancer or tumor expresses CD33; More preferably, the cancer or tumor is selected from adenocarcinoma, 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 (e.g., non-small cell lung cancer), colon cancer, breast cancer (e.g., triple-negative breast cancer), rectal cancer, colorectal cancer, bone cancer, skin cancer (e.g., epidermal cancer), thyroid cancer, pancreatic cancer, solid tumors or hematological tumors such as melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, myeloma, leukemia, An antibody-drug conjugate or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 14, or a linker-drug or a stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 15 to 21, or a pharmaceutical composition according to claim 23 and / or a pharmaceutical formulation according to claim 24.

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