Eribulin-based drug conjugate

Abstract

The present invention relates to the pharmaceutical field, specifically to conjugates of eribulin-based drugs. In particular, the present invention relates to a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, a preparation method and use thereof. The present invention also relates to a linker-drug conjugate or an isomer, meso form, racemate, enantiomer or a mixture thereof, a pharmaceutically acceptable salt or solvate thereof, a preparation method and use thereof. Furthermore, the present invention relates to a cytotoxin or an isomer, meso form, racemate, enantiomer or a mixture thereof, a pharmaceutically acceptable salt or solvate thereof, a preparation method and use thereof.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority from Chinese Patent Application No. 202310082754.5 filed on January 17, 2023, Chinese Patent Application No. 202310563948.7 filed on May 18, 2023, and Chinese Patent Application No. 2024100454262 filed on January 11, 2024, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of pharmaceutical technology, in particular to ligand-drug conjugates, linker-drug compounds, and methods for their preparation and use. [Background technology]

[0003] The development of antibody-drug conjugates (ADCs) as specific therapeutic agents capable of targeting cancer cells has made great strides since 2000. Currently, 15 ADC drugs are on the market worldwide, including Mylotarg, Besponsa, Kadcyla, Polivy, Lumoxiti, Enhertu, Adcetris, Padcev, Tivdak, Blenrep, Trodelvy, Akalux, Zynlonta, Aidixi, and Elaher. Their therapeutic areas are primarily focused on hematological and solid tumors. ADCs are composed of three main components: a monoclonal antibody or antibody fragment that selectively binds to an antigen on the surface of tumor cells, a cleavable or non-cleavable linker, and a small molecule cytotoxin. They take full advantage of the binding specificity of antibodies to tumor cell antigens and the high efficacy of small molecule drugs.

[0004] Microtubules are hollow fibers composed of α-tubulin and β-tubulin. They are components of the cytoskeleton and play important roles in cell signaling, intracellular migration and transport, and cell shape maintenance. During mitosis, microtubules rearrange to form the mitotic spindle, which is crucial for sister chromatid movement and separation. Most of the biological functions of microtubules in cells are regulated by polymerization dynamics, and disruption of microtubule dynamics significantly affects mitotic spindle formation and cell division. Due to their rapid division, cancer cells are more sensitive than normal cells to drugs that affect the normal function of tubulin. Currently, important microtubule inhibitors used as payloads in ADCs include auristatin, eribulin, and maytansinoids, which are derived from marine organisms.

[0005] Eribulin is a simplified synthetic derivative of the macrocyclic compound halichondrin B. In November 2010, eribulin (methanesulfonate salt) was first approved by the US FDA for the clinical treatment of patients with metastatic breast cancer who had received at least two prior chemotherapy regimens (including anthracycline and taxane chemotherapy drugs), and its trade name is Halaven. TM Eribulin is a compound developed and marketed by Eisai Pharmaceuticals. Eribulin selectively binds to the (+) end of β-tubulin and inhibits microtubule elongation. In addition to directly killing cancer cells, it can alter the tumor microenvironment and enhance the "bystander effect" of ADCs as a small molecule toxin. Eribulin's most common side effects include fatigue, neutropenia, alopecia, peripheral neuropathy, nausea, and constipation. Currently, there are few studies on ADCs using eribulin as a payload. All studies involve introducing a linker through the amino group at C35 of eribulin; there are no examples of introducing a linker through the hydroxyl group at C34. Furthermore, there are no studies using dihydroxy compounds, eribulin's precursor, as a payload.

[0006] As mentioned above, eribulin is clinically available as a single agent, but designing stable ADC molecules containing eribulin or its derivatives and delivering them to tumor cells may make the treatment safer and more effective and better address clinical needs. Summary of the Invention [Problem to be solved by the invention]

[0007] Based on a comprehensive understanding of ADC drugs, the present inventors have creatively designed and synthesized a series of ADC molecules, including eribulin or its derivatives, and experimental results have shown that the designed ADC molecules exhibit excellent antitumor activity. [Means for solving the problem]

[0008] In a first aspect, the present application relates to a ligand-drug conjugate having the structure shown in Formula I, or a pharmaceutically acceptable salt or solvate thereof: JPEG2026502604000001.jpg1334In formula, Ab is a ligand unit, L is a linker covalently linking Ab to D; n is selected from integers or decimals from 1 to 40; -D is represented by formula II or formula III, JPEG2026502604000002.jpg47148Where W is selected from an oxygen atom or a sulfur atom, R1 and R2 are the same or different and each independently selected from a hydrogen atom, an alkyl, an alkoxy, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -C(O)-Q1-Q2, and -SO2-Q1-Q2, where Q1 is selected from an O, N, or S atom or a chemical bond, and Q2 is selected from an alkyl, a cycloalkyl, a heterocyclyl, a spirocyclyl, a bridged cyclic group, an alkenyl, an aryl, and a heteroaryl, and optionally, the alkyl, alkoxy, alkenyl, cycloalkyl, heterocyclyl, spirocyclyl, a bridged cyclic group, an aryl, and a heteroaryl may each independently be substituted with one or more substituents selected from a hydrogen atom, an alkyl, an alkoxy, a halogen, a deuterium, an amino, a cyano, a hydroxyl, a mercapto, an azide, a nitro, a carboxyl, an acyl, a carbonyl, a hydroxyalkyl, a cycloalkyl, a heterocyclyl, an aryl, and a heteroaryl. Alternatively, R and R together with the nitrogen atom bonded thereto form a 3- to 8-membered heterocyclyl, which may optionally be substituted with one or more substituents selected from hydrogen, alkyl, alkoxy, halogen, deuterium, amino, cyano, hydroxyl, mercapto, azido, nitro, carboxyl, acyl, carbonyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl. R is selected from hydrogen, alkyl, acyl, sulfonyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, which may optionally be substituted with one or more substituents selected from hydrogen, alkyl, alkoxy, halogen, deuterium, amino, cyano, hydroxyl, mercapto, azido, nitro, carboxyl, acyl, carbonyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0009] In some embodiments, L has the structure shown in formula IV: JPEG2026502604000003.jpg2237In formula, M is a linking unit that covalently binds to the Ab, Z is -C1-C10 alkylene-, -C3-C8 carbocycle-, -arylene-, -3-8 membered heterocycle-, -(CH2CH2O) r -, sulfonyl, amide, chemical bond, JPEG2026502604000004.jpg3567, or a combination thereof, wherein X1 and X2 are selected from -C1-C10 alkylene-, -C3-C8 carbocycle-, -arylene-, -3-8 membered heterocycle-, -(CH2CH2O) r wherein R is selected from a hydrogen atom, a deuterium atom, an alkyl, and a substituted alkyl; optionally, the -C-C alkylene-, -C-C carbocycle-, and -3- to 8-membered heterocycle- are each independently substituted with one or more substituents selected from a hydrogen atom, a deuterium atom, a halogen atom, a hydroxyl, a cyano, a nitro, an amino, an alkyl, a heteroalkyl, a substituted alkyl, an alkoxy, a carboxyl, or a cycloalkyl; each heterocycle independently contains 1 to 3 atoms selected from N, O, and S; Y is a hydrophilic structure selected from the group consisting of one or more of carboxyl, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid, and polyethylene glycol (PEG); and Y is selected from the following structures: JPEG2026502604000005.jpg52158r is selected from an integer of 1 to 10, q1 and q2 are selected from integers of 1 to 8, and e is selected from an integer of 1 to 20; A is a peptide residue consisting of 2 to 7 amino acids, G is a spacer unit that binds to D, p is 0 or 1.

[0010] In some embodiments, The wavy line on the left in JPEG2026502604000006.jpg3565 indicates the bond to the binding site of M, and the wavy line on the right indicates the bond to the carbonyl. In some embodiments, the linking unit M has a succinimide structure represented by formula a, or a ring-opened succinimide structure represented by formula b or formula c. JPEG2026502604000007.jpg2685In formula a, formula b or formula c, the wavy line on the left indicates binding to the Ab binding site, and the wavy line on the right indicates binding to the Z binding site.

[0011] In some embodiments, A is a polypeptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, and cysteic acid.

[0012] In some embodiments, the spacer unit G is one or a combination of structures shown in formula Va, Vb, Vc, or Vd. JPEG2026502604000008.jpg23156In the formula, the wavy line on the left indicates the binding site between the nitrogen atom and peptide residue A, the wavy line on the right indicates the binding site between the oxygen atom and drug D, W is selected from an oxygen atom or a sulfur atom and is a covalent group between drug D and spacer unit G, R5, R5', R6, and R7 are each independently selected from a hydrogen atom, a deuterium atom, an alkyl, and a substituted alkyl.

[0013] In some embodiments, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of the present application has the structure shown in Formula VI or Formula VII: JPEG2026502604000009.jpg143128JPEG2026502604000010.jpg145128In the formula, Ab, Z, A, R5, R6, R7, R3, and W are as defined above, and n1, n2, and n3 are independently selected from integers or decimals of 0 to 40 (e.g., 0 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, or 35 to 40), where n1, n2, and n3 are not simultaneously 0 and n1 + n2 + n3 ≦ 40.

[0014] In some embodiments, Z is -C1-C10 alkylene-, -(CH2CH2O) r -, amide, JPEG2026502604000011.jpg3466, or a combination thereof.

[0015] In some embodiments, Z is —C2-alkylene or —C5-alkylene; In some embodiments, Z is JPEG2026502604000012.jpg2320, where q1 is selected from integers from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8).

[0016] In some embodiments, q1 is 1.

[0017] In some embodiments, q1 is 1, and -NH-X1-Y1 constitutes a hydrophilic structural unit Ac1, wherein Ac1 is selected from, but is not limited to, (D / L)alanine, (D / L)leucine, (D / L)isoleucine, (D / L)valine, (D / L)phenylalanine, (D / L)proline, (D / L)tryptophan, (D / L)serine, (D / L)tyrosine, (D / L)cysteine, (D / L)cystine, (D / L)arginine, (D / L)histidine, (D / L)methionine, (D / L)asparagine, (D / L)glutamine, (D / L)threonine, (D / L)aspartic acid, (D / L)glutamic acid, a natural or unnatural amino acid derivative, or the following structures: JPEG2026502604000013.jpg44163

[0018] In some embodiments, q1 is 1, and -NH-X1-Y1 constitutes a hydrophilic structural unit Ac1, wherein Ac1 is selected from the following structures: JPEG2026502604000014.jpg1573

[0019] In some embodiments, Z is JPEG2026502604000015.jpg2652, where q2 is selected from an integer of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8), preferably q2 is 1, and preferably X2 is -(C1-C10 alkylene)-(CH2CH2O) r -(C=O)- or -(C1-C10 alkylene)-(CH2CH2O) r -NR4-.

[0020] In some embodiments, q2 is 1, and -X2-Y2 constitutes a hydrophilic structural unit Ac2, wherein Ac2 is JPEG2026502604000016.jpg66158, r is selected from an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), and e is selected from an integer between 1 and 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

[0021] In some embodiments, q2 is 1, and -X2-Y2 constitutes a hydrophilic structural unit Ac2, wherein Ac2 is selected from the following structures: JPEG2026502604000017.jpg19138

[0022] In some embodiments, A is a polypeptide residue consisting of 2 to 5 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, and cysteic acid.

[0023] In some embodiments, A is a peptide residue formed by 2 to 4 amino acids selected from phenylalanine and glycine.

[0024] In some embodiments, A is a tetrapeptide residue consisting of glycine (G), glycine (G), phenylalanine (F), glycine (G).

[0025] In some embodiments, A is -GGFG-.

[0026] In some embodiments, W is selected from an oxygen atom or a sulfur atom, and R1 and R2 are the same or different and each independently selected from a hydrogen atom, an alkyl, an alkoxy, an alkenyl, a cycloalkyl, -C(O)-Q1-Q2, and -SO2-Q1-Q2, where Q1 is selected from O or a chemical bond, and Q2 is selected from an alkyl, a heterocyclyl, an alkenyl, an aryl, a heteroaryl, and a cycloalkyl, and optionally, the alkyl, heterocyclyl, an alkenyl, an aryl, a heteroaryl, and a cycloalkyl may each independently be substituted with one or more substituents selected from a hydrogen atom, an alkyl, a hydroxyalkyl, a halogen, a deuterium, a mercapto, an amino, a hydroxyl, a cycloalkyl, and a heterocyclyl. Alternatively, R1 and R2 together with the nitrogen atom bonded thereto form a 3- to 6-membered heterocyclyl, optionally substituted with one or more substituents selected from a hydrogen atom, alkyl, alkoxy, halogen, deuterium, amino, aryl, and hydroxyl; and R3 is selected from a hydrogen atom and an alkyl, optionally substituted with one or more substituents selected from a hydrogen atom, alkyl, alkoxy, halogen, deuterium, amino, hydroxyl, cycloalkyl, and heterocyclyl.

[0027] In some embodiments, W is an oxygen atom; R and R are the same or different and are each independently selected from a hydrogen atom, an alkyl, an alkenyl, a haloalkyl, -C(O)-Q-Q, and -SO-Q-Q, where Q is selected from O or a bond and Q is selected from an alkyl, an alkenyl, an aryl, and a 3- to 8-membered cycloalkyl, each of which is optionally substituted with one or more substituents selected from a hydrogen atom, an alkyl, a hydroxyalkyl, a halogen, an aryl, and a hydroxyl; or R and R together with the nitrogen atom bonded thereto form a 3- to 6-membered heterocyclyl, which contains one to two nitrogen atoms and optionally further contains one oxygen atom. Optionally, the 3-6 membered heterocyclyl may be substituted with one or more substituents selected from a hydrogen atom, an alkyl, and a haloalkyl, and R3 is selected from a hydrogen atom and an alkyl, and the alkyl is substituted with one or more substituents selected from a hydrogen atom, an alkyl, a halogen, a deuterium, an amino, and a hydroxyl.

[0028] In some embodiments, W is an oxygen atom, and R1 and R2 are the same or different and are each independently selected from a hydrogen atom, a C1-C4 alkyl (e.g., methyl, ethyl), a C2-C4 alkenyl (e.g., allyl), a C1-C4 haloalkyl (e.g., difluoroethyl), and / or the following structures: JPEG2026502604000018.jpg101156 Alternatively, R1 and R2 together with the nitrogen atom bound thereto form a 3-6 membered heterocyclyl, said 3-6 membered heterocyclyl containing 1 to 2 nitrogen atoms and optionally further containing 1 oxygen atom (e.g., aziridinyl, piperidinyl, piperazinyl, morpholinyl). Optionally, said 3-6 membered heterocyclyl may be substituted with one or more substituents selected from a hydrogen atom, a C1-C4 alkyl (e.g., methyl), and a C1-C4 haloalkyl (e.g., trifluoromethyl), and R3 is selected from a hydrogen atom and methyl.

[0029] In some embodiments, R5, R6, and R7 are each independently selected from hydrogen, deuterium, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl.

[0030] In some embodiments, R5, R6, and R7 are simultaneously hydrogen atoms.

[0031] In some embodiments, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of the present application has the structure shown in Formula VIa or Formula VIIa: JPEG2026502604000019.jpg113132JPEG2026502604000020.jpg110137In the formula, R1, R2, R3, n1, n2, and n3 are as defined above.

[0032] In some embodiments, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of the present application has the structure shown in Formula VIb or Formula VIIb: JPEG2026502604000021.jpg113128 JPEG2026502604000022.jpg110128In the formula, R1, R2, R3, n1, n2, and n3 are as defined above. Ac1 is a hydrophilic structural unit having a structure represented by formula d. JPEG2026502604000023.jpg2513X1, Y1 are as defined above.

[0033] In some embodiments, the Ac1 is selected from, but not limited to, (D / L)alanine, (D / L)leucine, (D / L)isoleucine, (D / L)valine, (D / L)phenylalanine, (D / L)proline, (D / L)tryptophan, (D / L)serine, (D / L)tyrosine, (D / L)cysteine, (D / L)cystine, (D / L)arginine, (D / L)histidine, (D / L)methionine, (D / L)asparagine, (D / L)glutamine, (D / L)threonine, (D / L)aspartic acid, (D / L)glutamic acid, a natural or unnatural amino acid derivative, or the following structures: JPEG2026502604000024.jpg44161

[0034] In some embodiments, Ac1 is selected from the following structures: JPEG2026502604000025.jpg1573

[0035] In some embodiments, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of the present application has the structure shown in Formula VIc: JPEG2026502604000026.jpg113129In the formula, R1, R2, n1, n2, and n3 are as defined above.

[0036] In some embodiments, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of the present application has the structure shown in Formula VId: JPEG2026502604000027.jpg113145In the formula, R1, R2, n1, n2, and n3 are as defined above.

[0037] In some embodiments, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of the present application has the structure shown in Formula VIe: JPEG2026502604000028.jpg118147 wherein R1, R2, n1, n2, and n3 are as defined above; Ac2 is a hydrophilic structural unit composed of -X2-Y2, where X2 and Y2 are as defined above.

[0038] In some embodiments, Ac2 is JPEG2026502604000029.jpg65158, where r is selected from an integer from 1 to 10 and e is selected from an integer from 1 to 20.

[0039] In some embodiments, Ac2 is selected from the following structures: JPEG2026502604000030.jpg18128

[0040] In some embodiments, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of the present application has the structure shown in formula VIf: JPEG2026502604000031.jpg120142 where R1, R2, n1, n2, and n3 are as defined above; Ac1 is a hydrophilic structural unit having a structure represented by formula d, JPEG2026502604000032.jpg2715X1, Y1 are as defined above.

[0041] In some embodiments, Ac1 is selected from, but is not limited to, (D / L) alanine, (D / L) leucine, (D / L) isoleucine, (D / L) valine, (D / L) phenylalanine, (D / L) proline, (D / L) tryptophan, (D / L) serine, (D / L) tyrosine, (D / L) cysteine, (D / L) cystine, (D / L) arginine, (D / L) histidine, (D / L) methionine, (D / L) asparagine, (D / L) glutamine, (D / L) threonine, (D / L) aspartic acid, (D / L) glutamic acid, a natural or unnatural amino acid derivative, or the following structures: JPEG2026502604000033.jpg41158

[0042] In some embodiments, Ac1 is selected from the following structures: JPEG2026502604000034.jpg1573

[0043] In some embodiments, Ac1 is JPEG2026502604000035.jpg1023. Ac2 is a hydrophilic structural unit composed of -X2-Y2, where X2 and Y2 are as defined above.

[0044] In some embodiments, Ac2 is JPEG2026502604000036.jpg65158, where r is selected from an integer from 1 to 10 and e is selected from an integer from 1 to 20.

[0045] In some embodiments, Ac2 is selected from the following structures: JPEG2026502604000037.jpg19138

[0046] In some embodiments, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of the present application has the structure shown in Formula VIg: JPEG2026502604000038.jpg103140, where R1, R2, n1, n2, and n3 are as defined above; Ac1 is a hydrophilic structural unit having a structure represented by formula d, JPEG2026502604000039.jpg2715X1, Y1 are as defined above.

[0047] In some embodiments, Ac1 is selected from, but is not limited to, (D / L) alanine, (D / L) leucine, (D / L) isoleucine, (D / L) valine, (D / L) phenylalanine, (D / L) proline, (D / L) tryptophan, (D / L) serine, (D / L) tyrosine, (D / L) cysteine, (D / L) cystine, (D / L) arginine, (D / L) histidine, (D / L) methionine, (D / L) asparagine, (D / L) glutamine, (D / L) threonine, (D / L) aspartic acid, (D / L) glutamic acid, a natural or unnatural amino acid derivative, or the following structures: JPEG2026502604000040.jpg41158

[0048] In some embodiments, Ac1 is selected from the following structures: JPEG2026502604000041.jpg1573

[0049] In some embodiments, Ac1 is JPEG2026502604000042.jpg1023.

[0050] In some embodiments, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of the present application is selected from the following structures: JPEG2026502604000043.jpg104131JPEG2026502604000044.jpg104131JPEG2026502604000045.jpg104131JPEG2026502604000046.jpg104131JPEG2026502604000047.jpg102136JPEG2026502604000048.jpg104131JPEG2026502604000049.jpg105128JPEG2026502604000050.jpg105128JPEG2026502604000051.jpg105128JPEG2026502604000052.jpg105128JPEG2026502604000053.jpg105143JPEG2026502604000054.jpg105143JPEG2026502604000055.jpg105143JPEG2026502604000056.jpg105143JPEG2026502604000057.jpg105143JPEG2026502604000058.jpg104128JPEG2026502604000059.jpg104128JPEG2026502604000060.jpg104128JPEG2026502604000061.jpg104128JPEG2026502604000062.jpg104128JPEG2026502604000063.jpg104128JPEG2026502604000064.jpg104128JPEG2026502604000065.jpg104128JPEG2026502604000066.jpg104128JPEG2026502604000067.jpg104128JPEG2026502604000068.jpg104128JPEG2026502604000069.jpg104128JPEG2026502604000070.jpg101128JPEG2026502604000071.jpg101128JPEG2026502604000072.jpg101128JPEG2026502604000073.jpg104128JPEG2026502604000074.jpg104128JPEG2026502604000075.jpg104128JPEG2026502604000076.jpg104128JPEG2026502604000077.jpg104128JPEG2026502604000078.jpg104128JPEG2026502604000079.jpg104128JPEG2026502604000080.jpg104128JPEG2026502604000081.jpg104128JPEG2026502604000082.jpg104128JPEG2026502604000083.jpg104128JPEG2026502604000084.jpg104128JPEG2026502604000085.jpg104128. JPEG2026502604000086.jpg104128JPEG2026502604000087.jpg104128JPEG2026502604000088.jpg104128JPEG2026502604000089.jpg104128JPEG2026502604000090.jpg104128 JPEG2026502604000091.jpg104128JPEG2026502604000092.jpg104128JPEG2026502604000093.jpg104128JPEG2026502604000094.jpg104128JPEG2026502604000095.jpg104128 JPEG2026502604000096.jpg104128JPEG2026502604000097.jpg104128JPEG2026502604 000098.jpg104128JPEG2026502604000099.jpg104128JPEG2026502604000100.jpg1041 28JPEG2026502604000101.jpg107128JPEG2026502604000102.jpg107128JPEG20265026 04000103.jpg107128JPEG2026502604000104.jpg100128JPEG2026502604000105.jpg100 128JPEG2026502604000106.jpg100128JPEG2026502604000107.jpg105128JPEG2026502 604000108.jpg105128JPEG2026502604000109.jpg105128JPEG2026502604000110.jpg9 8128JPEG2026502604000111.jpg98128JPEG2026502604000112.jpg98128JPEG20265026 04000113.jpg79128JPEG2026502604000114.jpg79128JPEG2026502604000115.jpg79128 wherein the configurations of the chiral carbons at positions 2 and 3 are independently R or S. In some embodiments, the configuration of the chiral carbon at position 2 is S, and the configuration of the chiral carbon at position 3 is S.

[0051] In the present application, the ligand unit Ab may be selected from an antibody, an antibody fragment, or a protein, and the antibody is preferably selected from a mouse antibody, a rabbit antibody, a phage display antibody, a yeast display antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody, and a multispecific antibody.

[0052] In some embodiments, the antibody is a monoclonal antibody selected non - restrictively from anti - EGFRvIII antibody, anti - PD - 1 antibody, anti - PD - L1 antibody, anti - DLL - 3 antibody, anti - PSMA antibody, anti - CD70 antibody, anti - MUC16 antibody, anti - ENPP3 antibody, anti - TDGF1 antibody, anti - ETBR antibody, anti - MSLN antibody, anti - TIM - 1 antibody, anti - LRRC15 antibody, anti - LIV - 1 antibody, anti - CanAg / AFP antibody, anti - claudin 18.2 antibody, anti - Mesothelin antibody, anti - HER2(ErbB2) antibody, anti - EGFR antibody, anti - c - MET antibody, anti - SLITRK6 antibody, anti - KIT / CD117 antibody, anti - STEAP1 antibody, anti - SLAMF7 / CS1 antibody, anti - NaPi2B / SLC34A2 antibody, anti - GPNMB antibody, anti - HER3(ErbB3) antibody, anti - MUC1 / CD227 antibody, anti - AXL antibody, anti - CD166 antibody, anti - B7 - H3(CD276) antibody, anti - PTK7 / CCK4 antibody, anti - PRLR antibody, anti - EFNA4 antibody, anti - 5T4 antibody, anti - NOTCH3 antibody, anti - Nectin 4 antibody, anti - TROP - 2 antibody, anti - CD142 antibody, anti - CA6 antibody, anti - GPR20 antibody, anti - CD174 antibody, anti - CD71 antibody, anti - EphA2 antibody, anti - LYPD3 antibody, anti - FGFR2 antibody, anti - FGFR3 antibody, anti - FRα antibody, anti - CEACAMs antibody, anti - GCC antibody, anti - Integrin Av antibody, anti - CAIX antibody, anti - P - cadherin antibody, anti - GD3 antibody, anti - Cadherin 6 antibody, anti - LAMP1 antibody, anti - FLT3 antibody, anti - BCMA antibody, anti - CD79b antibody, anti - CD19 antibody, anti - CD33 antibody, anti - CD56 antibody, anti - CD74 antibody, anti - CD22 antibody, anti - CD30 antibody, anti - CD37 antibody, anti - CD47 antibody, anti - CD138 antibody, anti - CD352 antibody, anti - CD25 antibody or anti - CD123 antibody.

[0053] In some embodiments, the antibody is an anti-TROP-2 antibody. In some embodiments, the antibody comprises a light chain and a heavy chain, wherein the light chain comprises CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. In some embodiments, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0054] In some embodiments, the light chain comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the light chain further comprises a light chain constant region having the amino acid sequence of SEQ ID NO: 8. In some embodiments, the amino acid sequence of the light chain is SEQ ID NO: 9.

[0055] In some embodiments, the light chain comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 10. In some embodiments, the light chain further comprises a light chain constant region having the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence of the light chain is SEQ ID NO: 12.

[0056] In some embodiments, the heavy chain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13. In some embodiments, the heavy chain further comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO: 14. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 15.

[0057] In some embodiments, the antibody is an anti-TROP-2 antibody. In some embodiments, the antibody comprises a light chain and a heavy chain, wherein the light chain comprises CDR-L1, CDR-L2, and CDR-L3, the nucleic acid coding sequences of which are set forth in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively. In some embodiments, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, the nucleic acid coding sequences of which are set forth in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively.

[0058] In some embodiments, the light chain comprises a light chain variable region having a nucleic acid coding sequence of SEQ ID NO: 22. In some embodiments, the light chain further comprises a light chain constant region having a nucleic acid coding sequence of SEQ ID NO: 23. In some embodiments, the nucleic acid coding sequence for the light chain is SEQ ID NO: 24.

[0059] In some embodiments, the light chain comprises a light chain variable region having a nucleic acid coding sequence of SEQ ID NO: 25. In some embodiments, the light chain further comprises a light chain constant region having a nucleic acid coding sequence of SEQ ID NO: 26. In some embodiments, the nucleic acid coding sequence for the light chain is SEQ ID NO: 27.

[0060] In some embodiments, the heavy chain comprises a heavy chain variable region having a nucleic acid coding sequence of SEQ ID NO: 28. In some embodiments, the heavy chain further comprises a heavy chain constant region having a nucleic acid coding sequence of SEQ ID NO: 29. In some embodiments, the nucleic acid coding sequence for the heavy chain is SEQ ID NO: 30.

[0061] Some sequence information according to the present invention is shown in Table 1. Table 1: Sequence description JPEG2026502604000116.jpg246164JPEG2026502604000117.jpg254164JPEG2026502604 000118.jpg246164JPEG2026502604000119.jpg254164JPEG2026502604000120.jpg25316 4JPEG2026502604000121.jpg253164JPEG2026502604000122.jpg253164JPEG2026502604 000123.jpg253164JPEG2026502604000124.jpg253164JPEG2026502604000125.jpg17164

[0062] In a second aspect, the present application provides a linker-drug conjugate having the structure shown in Formula VIII or Formula IX, or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof. JPEG2026502604000126.jpg5274JPEG2026502604000127.jpg5084In the formula, M1 is a linker unit, Z, p, A, G, W, R1, R2, and R3 are as defined above.

[0063] In some embodiments, the linker-drug conjugate of the present invention or its isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof has the structure shown in formula VIII-1 or formula IX-1. JPEG2026502604000128.jpg5580JPEG2026502604000129.jpg50128In the formula, Z, A, R1, R2, R3, R5, R6, and R7 are as defined above.

[0064] In some embodiments, the linker-drug conjugate of the present application or its isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof has the structure shown in Formula VIIIa or Formula IXa. JPEG2026502604000130.jpg50128JPEG2026502604000131.jpg46128In the formula, R1, R2, and R3 are as defined above.

[0065] In some embodiments, the linker-drug conjugate of the present application, or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, has the structure shown in Formula VIIIb or Formula IXb. JPEG2026502604000132.jpg52128JPEG2026502604000133.jpg49128In the formula, Ac is a hydrophilic structural unit having a structure represented by formula d, JPEG2026502604000134.jpg2514X, Y are as defined above. In some embodiments, Ac is as defined above.

[0066] In some embodiments, the linker-drug conjugate of the present invention, or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, has the structure shown in Formula VIIIc: JPEG2026502604000135.jpg45128 where R1 and R2 are as defined above.

[0067] In some embodiments, the linker-drug conjugate of the present invention, or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, has the structure shown in Formula VIIId: JPEG2026502604000136.jpg45128 where R1 and R2 are as defined above.

[0068] In some embodiments, the linker-drug conjugate of the present invention, or its isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, has the structure shown in Formula VIIIe: JPEG2026502604000137.jpg50128In the formula, R1 and R2 are as defined above, Ac2 is a hydrophilic structural unit composed of -X2-Y2, and Ac2, X2, and Y2 are as defined above.

[0069] In some embodiments, the linker-drug conjugate of the present invention, or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, has the structure shown in Formula VIIIf: JPEG2026502604000138.jpg58131, where R1 and R2 are as defined above; Ac1 is a hydrophilic structural unit composed of -X1-Y1, where X1 and Y1 are as defined above.

[0070] In some embodiments, Ac1 is selected from, but is not limited to, (D / L) alanine, (D / L) leucine, (D / L) isoleucine, (D / L) valine, (D / L) phenylalanine, (D / L) proline, (D / L) tryptophan, (D / L) serine, (D / L) tyrosine, (D / L) cysteine, (D / L) cystine, (D / L) arginine, (D / L) histidine, (D / L) methionine, (D / L) asparagine, (D / L) glutamine, (D / L) threonine, (D / L) aspartic acid, (D / L) glutamic acid, a natural or unnatural amino acid derivative, or the following structures: JPEG2026502604000139.jpg42159

[0071] In some embodiments, Ac1 is selected from the following structures: JPEG2026502604000140.jpg1573

[0072] In some embodiments, Ac1 is JPEG2026502604000141.jpg1023. Ac2 is a hydrophilic structural unit composed of -X2-Y2, where Ac2, X2, and Y2 are as defined above.

[0073] In some embodiments, the linker-drug conjugate of the present invention, or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, has the structure shown in Formula VIIIg: JPEG2026502604000142.jpg50128, where R1 and R2 are as defined above; Ac1 is a hydrophilic structural unit composed of -X1-Y1, where X1 and Y1 are as defined above.

[0074] In some embodiments, Ac1 is selected from, but is not limited to, (D / L) alanine, (D / L) leucine, (D / L) isoleucine, (D / L) valine, (D / L) phenylalanine, (D / L) proline, (D / L) tryptophan, (D / L) serine, (D / L) tyrosine, (D / L) cysteine, (D / L) cystine, (D / L) arginine, (D / L) histidine, (D / L) methionine, (D / L) asparagine, (D / L) glutamine, (D / L) threonine, (D / L) aspartic acid, (D / L) glutamic acid, a natural or unnatural amino acid derivative, or the following structures: JPEG2026502604000143.jpg41159

[0075] In some embodiments, Ac1 is selected from the following structures: JPEG2026502604000144.jpg1573

[0076] In some embodiments, Ac1 is JPEG2026502604000145.jpg1023. In some embodiments, the linker-drug conjugate of the present application or its isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof is selected from the following structures: JPEG2026502604000146.jpg42128JPEG2026502604000147.jpg42128JPEG2026502604000148.jpg42128JPEG2026502604000149. jpg42128JPEG2026502604000150.jpg38128JPEG2026502604000151.jpg42128JPEG2026502604000152.jpg42128JPEG202650260 4000153.jpg3785JPEG2026502604000154.jpg3785JPEG2026502604000155.jpg3785JPEG2026502604000156.jpg37128JPEG2026 502604000157.jpg37128JPEG2026502604000158.jpg37128JPEG2026502604000159.jpg37128JPEG2026502604000160.jpg37128 JPEG2026502604000161.jpg44128JPEG2026502604000162.jpg44128JPEG2026502604000163.jpg44128JPEG2026502604000164.jpg44128JPEG2026502604000165.jpg44128JPEG2026502604000166.jpg44128JPEG2026502604000167.jpg44128JPEG2026502604000168.jpg44128JPEG2026502604000169.jpg44128JPEG2026502604000170.jpg44128JPEG2026502604000171.jpg44128JPEG2026502604000172.jpg44128JPEG2026502604000173.jpg40128JPEG2026502604000174.jpg40128JPEG2026502604000175.jpg40128JPEG2026502604000176.jpg3783JPEG2026502604000177.jpg3783JPEG2026502604000178.jpg3783JPEG2026502604000179.jpg3783JPEG2026502604000180.jpg3783JPEG2026502604000181.jpg3783JPEG2026502604000182.jpg3783JPEG2026502604000183.jpg3783JPEG2026502604000184.jpg3783JPEG2026502604000185.jpg3783JPEG2026502604000186.jpg3783JPEG2026502604000187.jpg3783JPEG2026502604000188.jpg3783 JPEG2026502604000189.jpg44128JPEG2026502604000190.jpg44128JPEG202650260 4000191.jpg44128JPEG2026502604000192.jpg3782JPEG2026502604000193.jpg3783 JPEG2026502604000194.jpg44128JPEG2026502604000195.jpg44128JPEG202650260 4000196.jpg44128JPEG2026502604000197.jpg3783JPEG2026502604000198.jpg3783 JPEG2026502604000199.jpg44128JPEG2026502604000200.jpg44128JPEG20265026040002 01.jpg44128JPEG2026502604000202.jpg3783JPEG2026502604000203.jpg3783JPEG20265 02604000204.jpg43128JPEG2026502604000205.jpg43128JPEG2026502604000206.jpg431 28JPEG2026502604000207.jpg42128JPEG2026502604000208.jpg42128JPEG2026502604000 209.jpg42128JPEG2026502604000210.jpg40128JPEG2026502604000211.jpg40128JPEG20 26502604000212.jpg40128JPEG2026502604000213.jpg39128JPEG2026502604000214.jpg3 9128JPEG2026502604000215.jpg39128JPEG2026502604000216.jpg31128JPEG2026502604000217.jpg31128JPEG2026502604000218.jpg31128In the formula, the configurations of the chiral carbons at positions 2 and 3 are independently R or S. In some embodiments, the configuration of the chiral carbon at position 2 is S, and the configuration of the chiral carbon at position 3 is S.

[0077] In a third aspect, the present application further provides a compound having the structure set forth in Formula X, or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof. JPEG2026502604000219.jpg5468 wherein W is selected from an oxygen atom or a sulfur atom, preferably W is an oxygen atom; R1 and R8 are each independently selected from hydrogen, alkyl, acyl, sulfonyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and are not simultaneously hydrogen; optionally, the alkyl, acyl, sulfonyl, cycloalkyl, aryl, and heteroaryl may each independently be substituted with one or more substituents selected from hydrogen, alkyl, alkoxy, halogen, deuterium, amino, cyano, hydroxyl, mercapto, azido, nitro, carboxyl, acyl, carbonyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl. Preferably, R1 is alkyl, and R8 is hydrogen. R2 is selected from -C(O)-Q1-Q2 or -SO2-Q1-Q2, where Q1 is selected from an O, N, S atom or a chemical bond; When Q1 is selected from O, N, and S atoms, Q2 is selected from alkyl, cycloalkyl, heterocyclyl, spirocyclyl, bridged cyclic group, alkenyl, aryl, and heteroaryl, and optionally, the alkyl, cycloalkyl, heterocyclyl, spirocyclyl, bridged cyclic group, alkenyl, aryl, and heteroaryl may each independently be substituted with one or more substituents selected from hydrogen atom, alkyl, alkoxy, halogen, deuterium, amino, cyano, hydroxyl, mercapto, azido, nitro, carboxyl, acyl, carbonyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl. When Q1 is a chemical bond, Q2 is selected from alkyl, benzyl, cycloalkyl, heterocyclyl, spirocyclyl, bridged cyclic groups, alkenyl, aryl, and heteroaryl; When Q2 is selected from alkyl, the alkyl is substituted with one or more substituents selected from hydrogen, alkyl, alkoxy, halogen, deuterium, cyano, azido, nitro, carboxyl, acyl, carbonyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl. When Q2 is selected from cycloalkyl, heterocyclyl, spirocyclyl, bridged cyclic group, alkenyl, aryl, and heteroaryl, the cycloalkyl, heterocyclyl, spirocyclyl, bridged cyclic group, alkenyl, aryl, and heteroaryl are each independently substituted with one or more substituents selected from hydrogen atom, alkyl, alkoxy, halogen, deuterium, amino, cyano, hydroxyl, mercapto, azido, nitro, carboxyl, acyl, carbonyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl. When Q2 is cycloalkyl and the substituent is hydroxyl, amino, or mercapto, the position of the substituent is selected from any position other than the carbon atom bonded to -C(O)-. In some embodiments, the compound of formula X or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof is a compound of formula XIa or formula XIb or an isomer, meso form, racemate, enantiomer, or mixture thereof. JPEG2026502604000220.jpg53157, wherein R1 is selected from alkyl, preferably R1 is methyl; Q1 is selected from an O atom or a chemical bond; When Q1 is an O atom, Q2 is selected from alkyl, cycloalkyl, alkenyl, and aryl, and optionally, the alkyl, cycloalkyl, alkenyl, and aryl may each be independently substituted with one or more substituents selected from hydrogen atom, alkyl, halogen, hydroxyl, hydroxyalkyl, and aryl. When Q1 is selected from a chemical bond, Q2 is selected from alkyl, benzyl, cycloalkyl, alkenyl, and aryl; When Q2 is selected from alkyl, said alkyl is substituted with one or more substituents selected from hydrogen, alkyl, halogen, deuterium, azido, nitro, and aryl; When Q2 is selected from cycloalkyl, alkenyl, and aryl, the cycloalkyl, alkenyl, and aryl are each independently substituted with one or more substituents selected from hydrogen atoms, alkyl, halogen, hydroxyl, hydroxyalkyl, and aryl, and when Q2 is cycloalkyl and the substituent is hydroxyl, amino, or mercapto, the position of the substituent is selected from any position other than the carbon atom bonded to -C(O)-.

[0078] In some embodiments, the compound of formula X or its isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof is selected from the following structures: JPEG2026502604000221.jpg229156JPEG2026502604000222.jpg179156

[0079] In some embodiments of the present application, pharmaceutically acceptable salts of a Ligand-Drug conjugate, or a Linker-Drug conjugate, or an isomer, meso-isomer, racemate, enantiomer, or mixture thereof, or a compound of Formula X, or an isomer, meso-isomer, racemate, enantiomer, or mixture thereof, include 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, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate salts formed with basic functional groups in the structural formula.

[0080] In a fourth aspect, the present application provides use of any of the above linker-drug conjugates, or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, or a compound represented by Formula X, or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, as an intermediate for preparing a ligand-drug conjugate of the present application.

[0081] In a fifth aspect, the present application provides a pharmaceutical composition comprising a Ligand-Drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof, or a Linker-Drug conjugate of the present application or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, or a compound represented by Formula X or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, and optionally further comprising a pharmaceutically acceptable carrier.

[0082] In a sixth aspect, the present application provides a pharmaceutical formulation comprising a ligand-drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug conjugate of the present application or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, or a compound represented by Formula X or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0083] In a seventh aspect, the present application provides use of any of the above-mentioned ligand-drug conjugates or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug conjugate or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, or a compound of Formula X or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament for treating or preventing cancer or tumors.

[0084] In an eighth aspect, the present application provides a method for preventing or treating cancer or tumors, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a ligand-drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug conjugate of the present application or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, or a compound represented by Formula X or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0085] In the present application, the cancer or tumor may be a solid tumor or a hematological tumor such as, but not limited to, adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer (e.g., lung adenocarcinoma), colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer (e.g., epidermal cancer), thyroid cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma, leukemia, hypopharyngeal cancer (e.g., hypopharyngeal squamous cell carcinoma).

[0086] Definition of Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0087] When trade names are used herein, applicants intend to encompass formulations of the trade name products, their generic drugs, and the active pharmaceutical ingredients.

[0088] Unless stated to the contrary, terms used in the specification and claims have the following meanings.

[0089] The term "ligand" refers to a targeting agent that specifically binds to a target moiety. The ligand can specifically bind to a cellular component or other target molecule. The target moiety or target is typically located on the cell surface. In some aspects, the role of the ligand is to deliver the drug unit to a specific target cell population that interacts with the ligand unit. Ligands include, but are not limited to, non-proteins such as proteins, polypeptides, peptides, and sugars. Suitable ligand units include, for example, antibodies, such as full-length (intact) antibodies, and antigen-binding fragments thereof. In embodiments that are non-antibody targeting agents, the ligand unit may be a peptide or polypeptide, or a non-protein molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient transport molecules, or any other cell-binding molecule or substance. In some embodiments, the linker is covalently bonded to a sulfur atom of the ligand. In some aspects, the sulfur atom is the sulfur atom of a cysteine ​​residue, forming an interchain disulfide bond in an antibody. In another embodiment, the sulfur atom is the sulfur atom of a cysteine ​​residue into which a Ligand unit has been introduced, forming an interchain disulfide bond of the antibody. In another embodiment, the sulfur atom is the sulfur atom of a cysteine ​​residue into which a Ligand unit has been introduced (e.g., by site-directed mutagenesis or chemical reaction).

[0090] The term "drug" refers to cytotoxic drugs, i.e., molecules with a strong ability to inhibit the normal growth of tumor or cancer cells. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations, but due to lack of specificity, they may also cause apoptosis of normal cells while killing tumor or cancer cells, leading to serious side effects.

[0091] The term "ligand-drug conjugate" refers to a molecule formed by binding a ligand to a drug via a stable linker unit. In the present invention, the "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), which is a monoclonal antibody, a functional antibody fragment, or a target protein bound to a cytotoxic drug via a stable linker unit.

[0092] The term "antibody" or "functional antibody fragment" includes within its scope any part of the antibody structure, which is capable of binding to, reactively associating with, or complexing 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.

[0093] The antibodies of the present invention include, but are not limited to, mouse antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, and are preferably humanized antibodies or fully human antibodies.

[0094] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. Boil. Chem. 1968, 243, 3558.

[0095] The term "naturally occurring amino acid" refers to an amino acid that can be 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.

[0096] The term "unnatural amino acid" refers to an amino acid that can only be synthesized by artificial means.

[0097] The term "alkyl" refers to a saturated aliphatic hydrocarbon group that is a straight or branched chain group of 1 to 20 carbon atoms, preferably an alkyl containing 1 to 12 carbon atoms, more preferably an alkyl containing 1 to 10 carbon atoms, and most preferably an alkyl containing 1 to 6 or 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, Examples include 3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof.More preferred are lower alkyl groups containing 1 to 6 (e.g., 1 to 4) carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available position. 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, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0098] The term "substituted alkyl" refers to an alkyl group in which a hydrogen is replaced with a substituent. Unless otherwise specified in the specification, the substituents on the alkyl group are one or more groups selected from the following group: -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 is 1 to (2m'+1), where m' is the total number of carbon atoms in the group, for example, 1, 2, 3, 4, 5, or 6. R', R'', and R''' are each hydrogen, C1-8 Alkyl, aryl, aryl substituted with 1 to 3 halogens, C substituted with 1 to 3 halogens 1-8 Alkyl, C 1-8 Alkoxy or C 1-8 Thioalkoxy, or unsubstituted aryl-C 1-4 represents alkyl. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.

[0099] The term "heteroalkyl" refers to a group in which one or more carbons on the alkyl group have been replaced with N, O, or S. Preferably, the heteroalkyl contains 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms, and most preferably 1 to 6 or 1 to 4 carbon atoms.

[0100] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon group, wherein the ring of the cycloalkyl group contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. Polycyclic cycloalkyls include spirocyclic, fused, and bridged ring cycloalkyls. The spirocyclic, fused, and bridged rings preferably contain 5 to 12 ring atoms. Optionally, one or more carbon atoms on the cycloalkyl may be substituted with one or more carbonyl groups.

[0101] The term "alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl), where alkyl or cycloalkyl are as defined above. The alkyl preferably contains 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms, and most preferably 1 to 6 or 1 to 4 carbon atoms, and the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. An alkoxy may 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, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, and heterocycloalkylthio.

[0102] The term "heterocycle" refers to a heterocyclic ring containing 3 to 20 ring atoms, one or more (e.g., 1, 2, 3, or 4) of which are nitrogen, oxygen, or S(O). m (where m is 0, 1, or 2), and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms. More preferably, it contains 3 to 10 or 3 to 8 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups. The spirocyclic, fused-ring, and bridged-ring groups preferably contain 5 to 12 ring atoms. Optionally, one or more carbon atoms on the heterocyclic ring may be substituted with one or more carbonyl groups.

[0103] The term "aryl" refers to a 6-14 membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated π-electron system, preferably 6-10 membered, such as phenyl. Aryl can be substituted or unsubstituted. If substituted, the substituents are one or more groups selected from, but not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, deuterium atom, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, or heterocycloalkylthio.

[0104] The term "heteroaryl" includes 5-8 membered monocyclic heteroaryls and 8-12 membered fused heteroaryls.

[0105] The term "5-8-membered monocyclic heteroaryl" refers to an aromatic monocyclic ring group containing 5 to 8 ring atoms (at least one of which is a heteroatom such as a nitrogen atom, oxygen atom, or sulfur atom). Ring atoms (such as carbon atoms, nitrogen atoms, or sulfur atoms) in the ring structure may be optionally oxo-substituted. Examples of "5-8-membered monocyclic heteroaryl" include "5-7-membered monocyclic heteroaryl," "5-6-membered monocyclic heteroaryl," "5-6-membered monocyclic nitrogen-containing heteroaryl," and "6-membered monocyclic nitrogen-containing heteroaryl." The heteroatom in the "nitrogen-containing heteroaryl" includes at least one nitrogen atom, and may include, for example, only one or two nitrogen atoms, or one nitrogen atom and one or two other heteroatoms (e.g., oxygen atoms and / or sulfur atoms), or two nitrogen atoms and one or two other heteroatoms (e.g., oxygen atoms and / or sulfur atoms). Specific examples of "5-8 membered monocyclic heteroaryl" include, but are not limited to, furanyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, 2-pyridononyl, 4-pyridononyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl, azacyclooctatetraenyl, and the like.

[0106] The term "8-12-membered fused heteroaryl" refers to an unsaturated ring structure having aromaticity, which is formed by two or more ring structures sharing two adjacent atoms and contains 8 to 12 ring atoms (at least one of which is a heteroatom such as a nitrogen atom, oxygen atom, or sulfur atom). Ring atoms (e.g., carbon atoms, nitrogen atoms, or sulfur atoms) in the ring structure may be optionally oxo-substituted. The term "8- to 12-membered fused heteroaryl" includes "8- to 10-membered fused heteroaryl" and "8- to 9-membered fused heteroaryl", and specific examples include pyrrolopyrrole, pyrrolofuran, pyrazolopyrrole, pyrazolothiophene, furathiophene, pyrazolooxazole, benzofuranyl, benzisofuranyl, benzothiophenyl, indolyl, isoindolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, 2-quinolinonyl, 4-quinolinonyl, 1-isoquinolinonyl, isoquinolyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, purinyl, naphthyridinyl, and the like, but are not limited to these.

[0107] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, where alkyl is defined above.

[0108] The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, where alkyl is defined above.

[0109] The term "hydroxy" refers to the group --OH.

[0110] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0111] The term "amino" refers to -NH2.

[0112] The term "nitro" refers to -NO2.

[0113] The term "acyl" refers to a group formed by carbonyl and alkyl, alkoxy, or amino, and includes alkylcarbonyl, ester, and amide groups.

[0114] The term "substituted" means that a hydrogen in a group is replaced with a substituent, and "substituted" includes the implicit condition that such substitution is compatible with the substituted atom and the allowed valences of the substituent, and that the substitution results in a stable compound. Substituents are as listed above. Optionally, two substituents on the same substituted atom may form a cyclic group. The cyclic group may be a cycloalkyl or heterocyclyl (such as a lactam group or lactone group).

[0115] The term "derivative" refers to a substance that has a similar chemical structure to a compound, but that contains at least one chemical group that is not present in the compound and / or lacks at least one chemical group that is present in the compound. The compound to which the derivative is compared is called the "parent" compound. Typically, a "derivative" can be produced from the parent compound by one or more chemical reaction steps.

[0116] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a drug, a linker-drug compound, or a ligand-drug conjugate). The compound or conjugate contains at least one amino or carboxyl group and can thereby form an addition salt with a corresponding acid or base. Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, salicylate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, potassium salt, sodium salt, and the like.

[0117] The term "solvate" refers to a linker-drug compound or ligand-drug conjugate of the invention formed with one or more solvent molecules, including, but not limited to, water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate, etc.

[0118] The term "pharmaceutical composition" refers to a mixture containing one or more compounds of the present invention or physiologically / pharmaceutically acceptable salts or prodrugs thereof, together with other chemical components and other ingredients, such as physiologically or pharmaceutically acceptable carriers and / or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living body and promote absorption of the active ingredients, thereby exerting their biological activity.

[0119] The term "carrier" refers to a system that can modify the uptake and distribution of a drug in the body, control the drug release rate, and deliver the drug to a target. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability.

[0120] The term "excipient" refers to additives or auxiliary substances other than the active ingredient in pharmaceutical formulations. For example, adhesives, fillers, disintegrants, and lubricants in tablets, matrix backups in semi-solid preparations such as ointments and creams, and preservatives, antioxidants, flavors, fragrances, solubilizers, emulsifiers, penetration enhancers, osmotic pressure adjusters, and coloring agents in liquid preparations can all be called excipients.

[0121] The term "diluent" or "filler" is primarily used to increase the weight and / or volume of a formulation. The addition of a diluent not only ensures a consistent volume, but also reduces dosage deviations and improves the compressibility of the drug. [Brief explanation of the drawings]

[0122] [Figure 1]Figures 1A, 1B, 1C, 1D, 1E, and 1F show the antitumor effects of the compounds in BxPC-3, SW620, H1975, HCC827, FaDu, and A431 cell models, respectively. [Figure 2] Figures 2A, 2B, 2C, 2D, and 2E show the antitumor effects of the ADC in A431, HCC827, FaDu, N87, and SW620 cell models, respectively. [Figure 3] 1 shows the antitumor effect of ADCs on heterologous tumor cell lines A431+SW620. DETAILED DESCRIPTION OF THE INVENTION

[0123] 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 do not 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. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are for illustrative purposes only.

[0124] The general procedure employed in the following examples of the invention is as follows.

[0125] General Procedure A Preparation of ADCs by conjugation Prepurified antibody molecules with a monomer content of over 95% were transferred to phosphate buffer containing EDTA (10 mg / mL) using an ultrafiltration centrifuge tube. TCEP was added in a 10-fold molar amount relative to the antibody and allowed to react for 6 hours at room temperature. The interchain disulfide bonds of the antibody were cleaved, and the number of free thiols was measured using the Ellman method to determine whether all disulfide bonds had been cleaved. Next, a 10-fold molar amount of payload relative to the antibody was added and allowed to react for 6 hours at room temperature. After the reaction was complete, the solution was transferred to PBS using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa, and unbound payload was removed to obtain the conjugated ADC (DAR = 8).

[0126] General Procedure B Preparation of ADCs by site-specific conjugation Pre-purified antibody molecules with a monomer content of over 95% were transferred to phosphate buffer containing EDTA (10 mg / mL) using an ultrafiltration centrifuge tube. TCEP was added in an amount 8 times the molar amount of antibody, and the mixture was allowed to react at room temperature for 3 hours. The solution was then transferred to phosphate buffer at pH 6.5 using an ultrafiltration centrifuge tube, and DHAA (dehydroascorbic acid) was added in an amount 8 times the molar amount of antibody, and the mixture was allowed to react at room temperature for 3 hours. Next, payload was added in an amount 5 times the molar amount of antibody, and the mixture was allowed to react at room temperature for 3 hours. After the reaction was complete, the solution was transferred to PBS using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa, and unbound payload was removed to obtain a site-specifically conjugated ADC (DAR = 2).

[0127] General Procedure C Preparation of ADCs by conjugation Pre-purified antibody molecules with a monomer content of over 95% were transferred into 20 mM sodium acetate / pH 6.0 buffer (concentration >5 mg / mL) using an ultrafiltration centrifuge tube. To prepare the antibody-drug conjugate, an appropriate amount of the transferred antibody was placed in a reaction tube, and TCEP (0.1 M) was added in a molar equivalent of 3.6 times the antibody. The mixture was then incubated in a 25°C incubator for 2 hours. The reaction tube was then removed, and a 5-fold molar equivalent of linker-drug was added, mixed thoroughly, and incubated in a 25°C incubator for 2 hours. After the reaction was complete, the sample solution was removed and transferred into 20 mM histidine / pH 5.3 storage buffer using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa. Unbound payload was removed, yielding the conjugated ADC (DAR = 4).

[0128] Example 1 Synthesis of Compound D-2 Compound D-1 (80 mg, 0.109 mmol, 1.0 eq) was added to a 25 mL single-neck flask and dissolved in dry dichloromethane (4 mL). 2,4,6-trimethylpyridine (58 μL, 0.438 mmol, 4.0 eq) and pyridine (0.44 μL, 0.005 mmol, 0.05 eq) were added in sequence. The reaction solution was cooled to -10 ° C., and p-toluenesulfonic anhydride (89.3 mg, 0.274 mmol, 2.5 eq) was added under nitrogen protection. The reaction was continued for 20 hours at -10 ° C. Water (310 μL) was then added to the reaction solution, the temperature was raised to room temperature, and the mixture was stirred for 30 minutes. Methylamine (methanol solution, 4 mL) was then added, and the reaction was continued for 46 hours at room temperature. The reaction solution was then concentrated under reduced pressure and purified using a reverse-phase preparative column. The preparative solution was lyophilized to obtain compound D-2 (white solid, 58 mg, 71%) (LC-MS m / z (ES + ):[M+H] + :744.2).

[0129] Example 2 Synthesis of Compound D-3 Compound D-2 (55 mg, 0.074 mmol, 1.0 eq) was added to a 25 mL one-neck flask and dissolved in dry dichloromethane (3 mL). Triethylamine (15.4 μL, 0.111 mmol, 1.5 eq) and di-tert-butyl dicarbonate (20.4 μL, 0.089 mmol, 1.2 eq) were added sequentially and the reaction was allowed to proceed at room temperature for 2 hours. After confirming the completion of the reaction by TLC, the reaction solution was concentrated under reduced pressure and purified using a reverse-phase preparative column. The preparative solution was lyophilized to obtain compound D-3 (white solid, 43 mg, 69%) (LC-MS m / z (ES) + ):[M+H] + :844.2).

[0130] Example 3 Synthesis of Compound D-4 (eribulin) Compound D-1 (80 mg, 0.109 mmol, 1.0 eq) was added to a 25 mL single-neck flask and dissolved in dry dichloromethane (4 mL). 2,4,6-trimethylpyridine (58 μL, 0.438 mmol, 4.0 eq) and pyridine (0.44 μL, 0.005 mmol, 0.05 eq) were added in sequence. The reaction solution was cooled to -10 ° C., and p-toluenesulfonic anhydride (89.3 mg, 0.274 mmol, 2.5 eq) was added under nitrogen protection. The reaction was continued for 20 hours at -10 ° C. Water (310 μL) was then added to the reaction solution, the temperature was raised to room temperature, and the mixture was stirred for 30 minutes. After that, isopropanol (4 mL) and aqueous ammonia (4 mL) were added, and the reaction was continued for 3 days at room temperature. The reaction solution was then concentrated under reduced pressure and purified using a reverse-phase preparative column. The preparative solution was lyophilized to obtain compound D-4 (white solid, 56 mg, 70%) (LC-MS m / z (ES + ):[M+H] + :730.2).

[0131] Example 4 Synthesis of Compound D-5 JPEG2026502604000226.jpg361482 Compound D-1 (80 mg, 0.109 mmol, 1.0 eq) was placed in a 25 mL one-neck flask and dissolved in dry dichloromethane (4 mL). 2,4,6-trimethylpyridine (58 μL, 0.438 mmol, 4.0 eq) and pyridine (0.44 μL, 0.005 mmol, 0.05 eq) were added in that order, and the reaction solution was cooled to -10°C. p-Toluenesulfonic anhydride (89.3 mg, 0.274 mmol, 2.5 eq) was added under nitrogen protection, and the reaction was continued at -10°C for 20 hours. Water (310 μL) was then added to the reaction solution, which was then warmed to room temperature and stirred for 30 minutes. After this, methanol (4 mL) and piperazine (943 mg, 10.9 mmol, 100.0 eq) were added, and the reaction was continued at room temperature for 3 days. The reaction solution was then concentrated under reduced pressure and purified using a reversed-phase preparative column. The preparative solution was lyophilized to obtain compound D-5 (white solid, 66 mg, 75%) (LC-MS m / z (ES + ):[M+H] + :799.2).

[0132] Example 5 Synthesis of Compound D-6 Compound D-1 (80 mg, 0.109 mmol, 1.0 eq) was added to a 25 mL single-neck flask and dissolved in dry dichloromethane (4 mL). 2,4,6-trimethylpyridine (58 μL, 0.438 mmol, 4.0 eq) and pyridine (0.44 μL, 0.005 mmol, 0.05 eq) were added in sequence. The reaction solution was cooled to -10 ° C., and p-toluenesulfonic anhydride (89.3 mg, 0.274 mmol, 2.5 eq) was added under nitrogen protection. The reaction was continued for 20 hours at -10 ° C. Water (310 μL) was then added to the reaction solution, the temperature was raised to room temperature, and the mixture was stirred for 30 minutes. Dimethylamine (methanol solution, 8 mL) was then added, and the reaction was continued for 40 hours at room temperature. The reaction solution was then concentrated under reduced pressure and purified using a reverse-phase preparative column. The preparative solution was lyophilized to obtain compound D-6 (white solid, 60 mg, 72%) (LC-MS m / z (ES + ):[M+H] + :758.4).

[0133] Example 6 Synthesis of Compound D-7 Compound D-2 (20 mg, 0.027 mmol, 1.0 eq) was added to a 25 mL one-neck flask and dissolved in tetrahydrofuran and water (2 mL). Sodium carbonate (28 mg, 0.269 mmol, 10 eq) and allyl chloroformate (5.7 μL, 0.054 mmol, 2.0 eq) 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 reaction solution was purified using a reverse-phase preparative column. The preparative solution was lyophilized to obtain compound D-7 (white solid, 17 mg, 76%) (LC-MS m / z (ES)). + ):[M+H] + :828.4).

[0134] Example 7 Synthesis of Compound D-8 Compound D-8 (LC-MS m / z (ES + ):[M+H] + :878.3) was synthesized.

[0135] Example 8 Synthesis of Compound D-9 Compound D-2 (20 mg, 0.027 mmol, 1.0 eq) was added to a 25 mL one-neck flask and dissolved in dry tetrahydrofuran (2 mL). Triethylamine (7.5 μL, 0.054 mmol, 2.0 eq) and benzoyl chloride (6.2 μL, 0.054 mmol, 2.0 eq) 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 reaction solution was purified using a reverse-phase preparative column. The preparative solution was lyophilized to obtain compound D-9 (white solid, 18 mg, 79%) (LC-MS m / z (ES)). + ):[M+H] + :848.4).

[0136] Example 9 Synthesis of Compound D-10 Compound D-2 (20 mg, 0.027 mmol, 1.0 eq), HATU (20 mg, 0.054 mmol, 2.0 eq), HOBt (7.3 mg, 0.054 mmol, 2.0 eq), and dry DMF (2 mL) were added to a 25 mL single-neck flask and dissolved uniformly. Then, glacial acetic acid (2.3 μL, 0.040 mmol, 1.5 eq) and DIEA (9.4 μL, 0.054 mmol, 2.0 eq) were added sequentially in an ice bath, and the mixture was allowed to warm to room temperature. After 30 minutes, the reaction was confirmed to be complete by HPLC. The reaction solution was purified using a reverse-phase preparative column, and the preparative solution was lyophilized to obtain compound D-10 (white solid, 14 mg, 66%) (LC-MS m / z (ES + ):[M+H] + :886.3).

[0137] Example 10 Synthesis of Compound D-11 Compound D-11 (LC-MS m / z (ES + ):[M+H] + :814.4) was synthesized.

[0138] Example 11 Synthesis of Compound D-12 Compound D-12 (LC-MS m / z (ES + ):[M+H] + :828.3) was synthesized.

[0139] Example 12 Synthesis of Compound D-13 According to the synthesis method of Example 2, compound D-4 was used as a raw material to obtain compound D-13 (LC-MS m / z (ES + ):[M+H] + :830.3) was synthesized.

[0140] Example 13 Synthesis of Compound D-14 Compound D-14 (LC-MS m / z (ES + ):[M+H] + :822.3) was synthesized.

[0141] Example 14 Synthesis of Compound D-15 Compound D-15 (LC-MS m / z (ES)) was synthesized from compound D-2 and difluoroacetic anhydride according to the synthesis method of Example 8. + ):[M+H] + :822.3) was synthesized.

[0142] Example 15 Synthesis of Compound D-16 Compound D-16 (LC-MS m / z (ES)) was synthesized from compound D-4 and cis-3-hydroxycyclobutanecarboxylic acid S1 according to the synthesis method of Example 9. + ):[M+H] + :828.3) was synthesized.

[0143] Example 16 Synthesis of Compound D-17 According to the synthesis method of Example 9, compound D-17 (LC-MS m / z (ES + ):[M+H] + :828.3) was synthesized.

[0144] Example 17 Synthesis of Compound D-18 Compound D-13 (61 mg, 0.074 mmol, 1.0 eq) was weighed into a 25 mL one-neck flask and dissolved in dry dichloromethane (6 mL). 1,8-bis(dimethylamino)naphthalene (proton sponge) (48 mg, 0.222 mmol, 3.0 eq) and trimethyloxonium tetrafluoroborate (33 mg, 0.222 mmol, 3.0 eq) were added sequentially and allowed to react overnight at room temperature. After confirming the completion of the reaction by TLC, the reaction solution was filtered, concentrated under reduced pressure, and purified by TLC preparative separation to give compound D-18 (white foam solid, 56 mg, 90%) (LC-MS m / z (ES)). + ):[M+H] + :844.4).

[0145] Example 18 Synthesis of Compound D-19 Compound D-18 (56 mg, 0.066 mmol, 1.0 eq) was dissolved in dry dichloromethane (4 mL), trifluoroacetic acid (200 μL) was added, and the mixture was allowed to react at room temperature for 40 minutes. After confirming the completion of the reaction by TLC, the reaction solution was adjusted to pH 8 with sodium bicarbonate, extracted with dichloromethane, dried, filtered, and concentrated to obtain crude compound D-19.

[0146] Example 19 Synthesis of Compound D-20 According to the synthesis method of Example 9, compound D-20 (LC-MS m / z (ES + ):[M+H] + :842.3) was synthesized.

[0147] Example 20 Synthesis of Compound S5 JPEG2026502604000242.jpg16129 Step 1: Compound S3 Cis-3-hydroxycyclobutanecarboxylic acid S1 (150 mg, 1.29 mmol, 1.0 eq) and potassium carbonate (536 mg, 3.88 mmol, 3.0 eq) were weighed into a 25 mL one-neck flask, and DMF (3 mL) and benzyl bromide (230 μL, 1.94 mmol, 1.5 eq) were added. The mixture was allowed to react at room temperature for 2 hours. After completion of the reaction was confirmed by TLC, water was added to the reaction solution, which was then extracted with ethyl acetate, dried, filtered, concentrated, and purified by column chromatography (dichloromethane:methanol=30:1) to give compound S3 (yellow oil, 207 mg, 78%). Step 2: Compound S4 Compound S3 (173 mg, 0.839 mmol, 1.0 eq) was weighed into a 25 mL single-neck flask and dissolved in dry dichloromethane (5 mL). 1,8-bis(dimethylamino)naphthalene (539 mg, 2.52 mmol, 3.0 eq) and trimethyloxonium tetrafluoroborate (372 mg, 2.52 mmol, 3.0 eq) were added sequentially and the mixture was allowed to react overnight at room temperature. After confirming the completion of the reaction by TLC, the reaction solution was filtered, concentrated under reduced pressure, and purified by column chromatography (petroleum ether:ethyl acetate = 6:1) to give compound S4 (colorless oil, 168 mg, 91%). Step 2: Compound S5 Compound S4 (6 mg, 0.027 mmol, 1.0 eq) was weighed into a 25 mL single-neck flask and dissolved in absolute ethanol (1 mL). 5% palladium on carbon (3 mg) was added and the mixture was allowed to react at room temperature for 2 hours. After confirming the completion of the reaction by TLC, the reaction solution was filtered and concentrated to obtain crude compound S5.

[0148] Example 21 Synthesis of Compound D-21 According to the synthesis method of Example 9, compound D-21 (LC-MS m / z (ES + ):[M+H] + :842.3) was synthesized.

[0149] Example 22 Synthesis of Compound S7 Compound S6 (2.0 g, 4.84 mmol, 1.0 eq, see Patent CN111051330A for synthesis method) and dry tetrahydrofuran (4 mL) were added to a 25 mL one-neck flask and completely dissolved. After complete dissolution, 5% Pd / C (100 mg) was added and the hydrogenation reaction was carried out overnight. After completion of the reaction, the reaction solution was filtered, and the filtrate was concentrated to obtain compound S7 (white solid, 1.17 g, 87%) (LC-MS m / z (ES + ):[M+H] + :280.1).

[0150] Example 23 Synthesis of Compound S9 Compound S8 (1.0 g, 2.51 mmol, 1.0 eq; see the synthesis of compound M2 in CN113827736A for synthesis method), pentafluorophenol (508 mg, 2.76 mmol, 1.1 eq), DCC (570 mg, 2.76 mmol, 1.1 eq), and DMF (8 mL) were added to a 25 mL one-neck flask and reacted at room temperature for 2 hours. Completion of the reaction was confirmed by TLC. The reaction solution was filtered, and the crude filtrate of compound S9 (LC-MS m / z (ES)) was used for further analysis. + ):[M+H] + :565.1).

[0151] Example 24 Synthesis of Compound S10 Compound S7 (771 mg, 2.76 mmol, 1.1 eq), DMF (8 mL), and DIEA (656 μL, 3.76 mmol, 1.5 eq) were added to a 50 mL one-neck flask, and the crude filtrate of compound S9 was added in an ice bath. The mixture was warmed to room temperature and allowed to react overnight. The reaction solution was purified using a reverse-phase preparative column, and the preparative solution was lyophilized to obtain compound S10 (white solid, 778 mg, 47%) (LC-MS m / z (ES)). + ):[M+H] + :660.2).

[0152] Example 25 Synthesis of Compound S12 Compound D-3 (40 mg, 0.047 mmol, 1.0 eq), compound S11 (52.4 mg, 0.142 mmol, 3.0 eq; see the synthesis of compound 1 in CN111686259A for the synthesis method), zinc acetate (26.1 mg, 0.142 mmol, 3.0 eq), and 4 Å molecular sieves (60 mg) were weighed in a 25 mL single-neck flask. Dry toluene (4 mL) was added and the mixture was purged with nitrogen three times. The mixture was heated and stirred at 100 °C. After 2 h, the reaction was terminated, cooled to room temperature, and filtered. The filtrate was concentrated under reduced pressure and purified using a reverse-phase preparative column. The solution was lyophilized to obtain compound S12 (white solid, 37 mg, 68%) (LC-MS m / z (ES + ):[M+Na] + :1174.2).

[0153] Example 26 Synthesis of Compound S13 Compound S12 (30 mg, 0.026 mmol, 1.0 eq) and dry DMF (1.5 mL) were added to a 25 mL one-neck flask and completely dissolved. After that, DBU (4.3 μL, 0.029 mmol, 1.1 eq) was added and the reaction was carried out at room temperature. After confirming that compound S12 was completely consumed by TLC, the crude product solution of compound S13 (LC-MS m / z (ES + ):[M+Na] + :953.3) was used directly in the next reaction.

[0154] Example 27 Synthesis of Compound S14 In a separate 25 mL one-neck flask, compound S10 (18.9 mg, 0.029 mmol, 1.1 eq), HATU (11.9 mg, 0.031 mmol, 1.2 eq), HOBt (4.2 mg, 0.031 mmol, 1.2 eq), and dry DMF (1.5 mL) were added in this order and dissolved uniformly. Then, the crude solution of compound S13 and DIEA (5.4 μL, 0.031 mmol, 1.2 eq) were added dropwise in an ice bath, and the mixture was allowed to warm to room temperature. After 30 minutes, the reaction was confirmed to be complete by HPLC. The reaction solution was purified using a reverse-phase preparative column, and the preparative solution was lyophilized to obtain compound S14 (white solid, 23 mg, 56%) (LC-MS m / z (ES + ):[M-99] + :1472.2) (fragment ion obtained after removing the Boc protecting group of compound S14 and adding a hydrogen ion).

[0155] Example 28 Synthesis of Compound L1 Compound S14 (20 mg, 0.013 mmol, 1.0 eq) was added to a 25 mL one-neck flask and dissolved in nitromethane (2 mL). Zinc bromide (57 mg, 0.25 mmol, 20.0 eq) was added and reacted at room temperature for 30 minutes. The reaction progress 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 the crude product. The reaction solution was purified using a reverse-phase preparative column, and the fractionated solution was lyophilized to obtain compound L-1 (white solid, 9.2 mg, 55%) and compound L-1' (white solid, 5.2 mg, 31%) (L-1: LC-MS m / z (ES) + ):[M+Na] + : 1337.3, L-1': LC-MS m / z (ES + ):[M+Na] + :1337.3).

[0156] Example 29 Synthesis of Compound S15 Compound D-1 (50 mg, 0.068 mmol, 1.0 eq), compound S11 (27.7 mg, 0.075 mmol, 1.1 eq), zinc acetate (25.1 mg, 0.137 mmol, 2.0 eq), and 4 Å molecular sieves (100 mg) were weighed in a 25 mL single-neck flask. Dry toluene (5 mL) was added and the mixture was purged with nitrogen three times. The mixture was then heated and stirred at 100 °C. After 3 h, the reaction was terminated, cooled to room temperature, and filtered. The filtrate was concentrated under reduced pressure and purified using a reverse-phase preparative column. The solution was lyophilized to give compound S15 (white solid, 42 mg, 59%) (LC-MS m / z (ES) + ):[M+Na] + :1061.3).

[0157] Example 30 Synthesis of Compound S16 Compound S15 (37 mg, 0.036 mmol, 1.0 eq) and dry DMF (1.5 mL) were added to a 25 mL one-neck flask and completely dissolved. After that, DBU (5.9 μL, 0.039 mmol, 1.1 eq) was added and the reaction was allowed to proceed at room temperature. After confirming that compound S15 was completely consumed by TLC, the crude product solution of compound S16 (LC-MS m / z (ES + ):[M+Na] + :839.1) was used directly in the next reaction.

[0158] Example 31 Synthesis of Compound S17 In a separate 25 mL one-neck flask, compound S10 (25.8 mg, 0.039 mmol, 1.1 eq), HATU (16.2 mg, 0.043 mmol, 1.2 eq), HOBt (5.8 mg, 0.043 mmol, 1.2 eq), and dry DMF (1.5 mL) were added in this order and dissolved uniformly. Then, the crude solution of compound S16 and DIEA (7.4 μL, 0.043 mmol, 1.2 eq) were added dropwise in an ice bath, and the mixture was allowed to warm to room temperature. After 30 minutes, the reaction was confirmed to be complete by HPLC. The reaction solution was purified using a reverse-phase preparative column, and the preparative solution was lyophilized to obtain compound S17 (white solid, 26 mg, 50%) (LC-MS m / z (ES + ):[M+H] + :1459.2).

[0159] Example 32 Synthesis of Compound L-2 Compound S17 (21 mg, 0.014 mmol, 1.0 eq) was added to a 25 mL one-neck flask and dissolved in nitromethane (2 mL). Zinc bromide (65 mg, 0.288 mmol, 20.0 eq) was added and reacted at room temperature for 30 minutes. The reaction progress 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 the crude product. The reaction solution was purified using a reverse-phase preparative column, and the fractionated solution was lyophilized to obtain compound L-2 (white solid, 10.7 mg, 57%) and compound L-2' (white solid, 5.4 mg, 29%) (L-2: LC-MS m / z (ES + ):[M+Na] + : 1324.3, L-2': LC-MS m / z (ES + ):[M+Na] + :1324.3).

[0160] Example 33 Synthesis of Compound L-3 According to the synthesis method of Example 27, compound L-3 (LC-MS m / z (ES+ ):[M-99] + :1285.1 (fragment ion obtained after removing the Boc protecting group of compound L-3 and adding a hydrogen ion) was synthesized.

[0161] Example 34 Synthesis of Compound D-22 Compound D-4 (53 mg, 0.073 mmol, 1.0 eq) was added to a 25 mL one-neck flask and dissolved in dry dichloromethane (3 mL). Triethylamine (30 μL, 0.218 mmol, 3.0 eq), DMAP (1.8 mg, 0.015 mmol, 0.2 eq), and di-tert-butyl dicarbonate (42 μL, 0.182 mmol, 2.5 eq) were added in that order and reacted at room temperature for 2 hours. After confirming the completion of the reaction by TLC, the reaction solution was concentrated under reduced pressure and purified using a reverse-phase preparative column. The fractionated solution was lyophilized to obtain compound D-22 (white solid, 36 mg, 53%) (LC-MS m / z (ES)). + ):[M+H] + :930.2).

[0162] Example 35 Synthesis of Compound S19 According to the synthesis method of Example 25, compound S19 (LC-MS m / z (ES + ):[M+Na] + :1260.2) was synthesized.

[0163] Example 36 Synthesis of Compound S20 According to the synthesis method of Example 26, a crude product solution of compound S20 was synthesized from compound S19 as a raw material, and was used in the next reaction without post-treatment (LC-MS m / z (ES + ):[M+Na] + :1038.3).

[0164] Example 37 Synthesis of Compound S21 According to the synthesis method of Example 27, compound S21 (TOF m / z: [M+H] + :1657.5) was synthesized.

[0165] Example 38 Synthesis of Compound L-4 Compound L-4 (LC-MS m / z (ES + ):[M+Na] + :1323.3) was synthesized.

[0166] Example 39 Synthesis of Compound S22 According to the synthesis method of Example 2, compound S22 (LC-MS m / z (ES + ):[M+H] + :898.3) was synthesized.

[0167] Example 40 Synthesis of Compound S23 According to the synthesis method of Example 25, compound S23 (LC-MS m / z (ES + ):[M+Na] + :1229.3) was synthesized.

[0168] Example 41 Synthesis of Compound S24 According to the synthesis method of Example 26, a crude product solution of compound S24 was synthesized from compound S23 as a raw material, and was used in the next reaction without post-treatment (LC-MS m / z (ES + ):[M+Na] + :1007.3).

[0169] Example 42 Synthesis of Compound S25 According to the synthesis method of Example 27, compound S25 (TOF m / z: [M+H] + :1626.5) was synthesized.

[0170] Example 43 Synthesis of Compound L-5 Compound L-5 (LC-MS m / z (ES + ):[M+Na] + :1392.4) was synthesized.

[0171] Example 44 Synthesis of Compound S26 According to the synthesis method of Example 25, compound S26 (LC-MS m / z (ES + ):[M+Na] + :1088.3) was synthesized.

[0172] Example 45 Synthesis of Compound S27 According to the synthesis method of Example 26, compound S26 was used as a raw material to obtain a crude product solution of compound S27 (LC-MS m / z (ES + ):[M+Na] + :866.2) was synthesized and used in the next reaction without post-treatment.

[0173] Example 46 Synthesis of Compound S28 According to the synthesis method of Example 27, compound S27 and compound S10 were used as raw materials to obtain compound S28 (LC-MS m / z (ES + ):[M+Na] + :1485.5) was synthesized.

[0174] Example 47 Synthesis of Compound L-6 Compound L-6 (LC-MS m / z (ES + ):[M+Na] + :1351.3) was synthesized.

[0175] Example 48 Synthesis of Compound L-7 JPEG2026502604000270.jpg30128 Compound L-7 was synthesized according to the synthesis method of compound ER-001235638 in patent CN108883198A.

[0176] Example 49 Synthesis of Compound L-8 JPEG2026502604000271.jpg44128 Compound L-8 was synthesized according to the synthesis method of compound ER-001159569 in patent CN108883198A.

[0177] Example 50 Synthesis of Compound S29 According to the synthesis method of Example 25, compound S29 (LC-MS m / z (ES + ):[M+Na] + :1158.4) was synthesized.

[0178] Example 51 Synthesis of Compound S30 According to the synthesis method of Example 26, a crude product solution of compound S30 was synthesized from compound S29 as a starting material, and was used in the next reaction without post-treatment (LC-MS m / z (ES + ):[M+Na] + :936.5).

[0179] Example 52 Synthesis of Compound S31 According to the synthesis method of Example 27, compound S31 (TOF m / z: [M+H] +:1555.7) was synthesized.

[0180] Example 53 Synthesis of Compound L-9 Compound L-9 (LC-MS m / z (ES + ):[M+Na] + :1421.7) was synthesized.

[0181] Example 54 Synthesis of Compound S32 According to the synthesis method of Example 25, compound S32 (LC-MS m / z (ES + ):[M+Na] + :1152.5) ​​was synthesized.

[0182] Example 55 Synthesis of Compound S33 According to the synthesis method of Example 26, a crude product solution of compound S33 was synthesized from compound S32 as a starting material, and was used in the next reaction without post-treatment (LC-MS m / z (ES + ):[M+Na] + :930.4).

[0183] Example 56 Synthesis of Compound S33 According to the synthesis method of Example 27, compound S33 (TOF m / z: [M+H] + :1549.7) was synthesized.

[0184] Example 57 Synthesis of Compound L-10 Compound L-10 (LC-MS m / z (ES + ):[M+Na] + :1415.6) was synthesized.

[0185] Example 58 Synthesis of Compound S34 According to the synthesis method of Example 25, compound S34 (LC-MS m / z (ES + ):[M+Na] + :1152.4) was synthesized.

[0186] Example 59 Synthesis of Compound S35 According to the synthesis method of Example 26, a crude product solution of compound S35 was synthesized from compound S34 as a starting material, and was used in the next reaction without post-treatment (LC-MS m / z (ES + ):[M+Na] + :930.3).

[0187] Example 60 Synthesis of Compound S36 According to the synthesis method of Example 27, compound S36 (TOF m / z: [M+H] + :1549.6) was synthesized.

[0188] Example 61 Synthesis of Compound L-11 Compound L-11 (LC-MS m / z (ES + ):[M+Na] + :1415.6) was synthesized.

[0189] Example 62 Synthesis of Compound S39 JPEG2026502604000284.jpg21128JPEG2026502604000285.jpg19144Step 1: Synthesis of compound S38 Compound S37 (400 mg, 1.13 mmol, 1.0 eq), pentafluorophenol (228 mg, 1.24 mmol, 1.1 eq), DCC (255 mg, 1.24 mmol, 1.1 eq), and DMF (4 mL) were added to a 25 mL single-neck flask and reacted overnight at room temperature. Completion of the reaction was confirmed by TLC. The reaction solution was filtered to obtain the crude product of compound S38 (for later use). Step 2: Synthesis of compound S39 Compound S7 (377 mg, 1.35 mmol, 1.2 eq), DMF (4 mL), and DIEA (235 μL, 1.35 mmol, 1.2 eq) were added to a 25 mL one-neck flask, and the crude filtrate of compound S38 was added in an ice bath. The mixture was warmed to room temperature and allowed to react overnight. The reaction solution was purified using a reverse-phase preparative column, and the preparative solution was lyophilized to obtain compound S39 (white solid, 543 mg, 78%) (LC-MS m / z (ES)). + ):[M+H] + :617.1).

[0190] Example 63 Synthesis of Compound S44 JPEG2026502604000286.jpg23158Step 1: Synthesis of compound S41 A 2-L single-neck flask was charged with Fmoc-L-glutamic acid 5-tert-butyl ester (S40, 200 g, 470 mmol, 1.0 eq), propargylamine (28.5 g, 517 mmol, 1.1 eq), and 400 mL of DMF. After complete dissolution, the mixture was cooled to approximately 0 °C in an ice-water bath. EDCI (108.2 g, 564 mmol, 1.2 eq), HOBt (76.2 g, 564 mmol), and DIEA (117 mL, 705 mmol, 1.5 eq) were added, the mixture was warmed to room temperature, and the reaction was allowed to proceed for 2 h. The end point of the reaction was monitored by TLC. After completion of the reaction, the reaction solution was poured into 2 L of water and extracted three times with ethyl acetate (500 mL x 3). The organic phases were combined, washed twice with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The sodium sulfate was filtered off, and the filtrate was concentrated to obtain the crude product. The crude product was purified with petroleum ether / ethyl acetate (2 / 1) to give compound S41 (100.9 g, 48% yield) (LC-MS m / z (ES + ):[M+H]+ :463.2). Step 2: Synthesis of compound S42 S41 (100.0 g) was placed in a 1 L single-neck flask and dissolved in 500 mL of dichloromethane. 200 mL of trifluoroacetic acid was added and the reaction was allowed to proceed at room temperature for 2 hours. The end point of the reaction was monitored by TLC. After the reaction was completed, the dichloromethane and trifluoroacetic acid were partially removed by concentration to obtain a crude product. Methyl tert-ether was added to the crude product to precipitate a white solid, which was filtered and dried to obtain compound S42 (77.5 g, 87% yield) (LC-MS m / z (ES - ):[MH] - :405.1). Step 3: Synthesis of compound S43 In a 2 L one-neck flask, S42 (77.0 g) was added and dissolved in 150 mL of DMF. 38 mL of diethylamine was added and the reaction was allowed to proceed at room temperature for 2 hours. The end point of the reaction was monitored by TLC. After the reaction was completed, 800 mL of methyl tert-ether was added to the reaction solution to precipitate a white solid. This solid was filtered and dried to obtain compound S43 (33.7 g, yield 96.6%) (LC-MS m / z (ES) - ):[MH] - :183.1). Step 4: Synthesis of compound S44 Compound S43 (10.02 g, 54.4 mmol, 1.5 eq) and compound S8 (14.48 g, 36.3 mmol, 1.0 eq) were added to a 100 mL single-neck flask and dissolved in 100 mL of DMF. EEDQ (13.5 g, 54.8 mmol, 1.5 eq) was added and the reaction was allowed to proceed at room temperature 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 liquid chromatography to obtain a fractionated solution of the product. The fractionated solution was lyophilized to obtain compound S44 (9.94 g, 48% yield) (LC-MS m / z (ES - ):[MH] - :563.2).

[0191] Example 64 Synthesis of Compound S47 JPEG2026502604000287.jpg2684JPEG2026502604000288.jpg20128JPEG2026502604000289.jpg28161Step 1: Synthesis of compound S45 Compound S44 (230 mg, 0.407 mmol, 1.0 eq), pentafluorophenol (83 mg, 0.448 mmol, 1.1 eq), DCC (93 mg, 0.448 mmol, 1.1 eq), and DMF (3.5 mL) were added to a 25 mL single-neck flask and reacted overnight at room temperature. Completion of the reaction was confirmed by TLC. The reaction solution was filtered to obtain the crude product filtrate of compound S45 (for later use). Step 2: Synthesis of compound S46 Compound S39 (301 mg, 0.489 mmol, 1.2 eq) and dry DMF (3 mL) were added to a 25 mL single-neck flask and completely dissolved. After that, DBU (79 μL, 0.529 mmol, 1.3 eq) was added and the mixture was allowed to react at room temperature. After confirming that compound S39 was completely consumed by TLC, the crude product solution of compound S46 was used directly in the next reaction without any post-reaction treatment. Step 3: Synthesis of compound S47 The crude filtrate of compound S45 and the crude solution of compound S46 were mixed in an ice bath, and DIEA (71 μL, 0.407 mmol, 1.0 eq) was slowly added dropwise. The mixture was warmed to room temperature and reacted for 40 minutes. The reaction solution was purified using a reversed-phase preparative column, and the preparative solution was lyophilized to obtain compound S47 (white solid, 258 mg, 67%) (LC-MS m / z (ES + ):[M+H] + :941.3).

[0192] Example 65 Synthesis of Compound S50 Compound S48 (667 mg, 3.28 mmol, 1.0 eq), S49 (1.58 g, 3.94 mmol, 1.2 eq; see WO0152900A2 for the synthesis of compound 11), EDCI (944 mg, 4.92 mmol, 1.5 eq), HOBt (665 mg, 4.92 mmol, 1.5 eq), and dry dichloromethane (60 mL) were added to a 150 mL single-neck flask in this order and dissolved uniformly. DIEA (1.14 mL, 6.56 mmol, 2.0 eq) was added dropwise in an ice bath, and the mixture was allowed to warm to room temperature and react. After 2 hours, the reaction was confirmed to be complete by TLC monitoring. The reaction solution was quenched with saturated aqueous ammonium chloride, extracted with dichloromethane, dried, filtered, concentrated, and then purified by column chromatography (petroleum ether: ethyl acetate = 30:1) to obtain compound S50 (yellow oil, 1.3 g, 67%) (LC-MS m / z (ES + ):[M+H] + :588.4).

[0193] Example 66 Synthesis of Compound S51 Compound S47 (58 mg, 0.061 mmol, 1.0 eq), compound S50 (36 mg, 0.061 mmol, 1.0 eq), copper sulfate pentahydrate (31 mg, 0.123 mmol, 2.0 eq), sodium ascorbate (24 mg, 0.123 mmol, 2.0 eq), and DMF (2 mL) were added to a 25 mL one-neck flask. Water (1 mL) was added in an ice bath and the reaction was allowed to proceed at room temperature. After 30 minutes, the reaction was confirmed to be complete by HPLC. The reaction solution was purified using a reverse-phase preparative column, and the preparative solution was lyophilized to obtain compound S51 (white solid, 85 mg, 91%) (LC-MS m / z (ES)). + ):[M+2H] 2+ :764.7).

[0194] Example 67 Synthesis of Compound S52 According to the synthesis method of Example 27, compound S52 (LC-MS m / z (ES + ):[M+H+Na] 2+ :1223.8) was synthesized.

[0195] Example 68 Synthesis of Compound L-12 Compound S52 (19 mg, 0.0078 mmol, 1.0 eq) was added to a 25 mL one-neck flask and dissolved in nitromethane (2 mL). Zinc bromide (124 mg, 0.548 mmol, 70.0 eq) was added and reacted at room temperature for 45 minutes. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified using a reverse-phase preparative column, and the preparative solution was lyophilized to obtain compound L-12 (white solid, 9.2 mg, 55%) (L-12: LC-MS m / z (ES) + ):[M+2H] 2+ :1050.3).

[0196] Example 69 Synthesis of Compound S53 According to the synthesis method of Example 27, compound S51 and compound S33 were used as raw materials to obtain compound S53 (LC-MS m / z (ES + ):[M+2H] 2+ :1209.9) was synthesized.

[0197] Example 70 Synthesis of Compound L-13 Compound L-13 (LC-MS m / z (ES + ):[M+2H] 2+ :1047.4) was synthesized.

[0198] Example 71 Synthesis of Compound S54 According to the synthesis method of Example 27, compound S51 and compound S35 were used as raw materials to obtain compound S54 (LC-MS m / z (ES + ):[M+2H+Na] 2+ :1220.8) was synthesized.

[0199] Example 72 Synthesis of Compound L-14 Compound L-14 (LC-MS m / z (ES + ):[M+2H] 2+ :1047.4) was synthesized.

[0200] Example 73 Synthesis of Compound S57 JPEG2026502604000298.jpg2063JPEG2026502604000299.jpg20128Step 1: Synthesis of compound S56 Compound S55 (400 mg, 2.36 mmol, 1.0 eq), pentafluorophenol (479 mg, 2.60 mmol, 1.1 eq), DCC (537 mg, 2.60 mmol, 1.1 eq), and DMF (4 mL) were added to a 25 mL single-neck flask and reacted at room temperature for 4 hours. Completion of the reaction was confirmed by TLC. The reaction solution was filtered to obtain the crude product of compound S56 (for later use). Step 2: Synthesis of compound S57 Compound S43 (436 mg, 2.36 mmol, 1.0 eq) and DMF (7 mL) were added to the crude filtrate of compound S56, and DIEA (412 μL, 2.36 mmol, 1.0 eq) was added in an ice bath. 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 using a reversed-phase preparative column. The preparative solution was lyophilized to obtain compound S57 (white solid, 649 mg, 82%) (LC-MS m / z (ES + ):[M+Na] + :358.2).

[0201] Example 74 Synthesis of Compound S59 JPEG2026502604000300.jpg2083JPEG2026502604000301.jpg21161 According to the synthesis method of Example 64, compound S59 (LC-MS m / z (ES + ):[M+H] + :712.0) was synthesized.

[0202] Example 75 Synthesis of Compound S60 According to the synthesis method of Example 27, compound S60 (LC-MS m / z (ES + ):[M+2H] 2+ :804.4) was synthesized.

[0203] Example 76 Synthesis of Compound S61 Compound S50 (201 mg, 0.342 mmol, 1.0 eq) was dissolved in dry dichloromethane (4 mL), trifluoroacetic acid (800 μL) was added, and the mixture was allowed to react at room temperature. After 30 hours, the reaction solution was concentrated under reduced pressure, and the crude product was purified using a reverse-phase preparative column. The preparative solution was lyophilized to obtain compound S61 (white solid, 98 mg, 69%) (LC-MS m / z (ES)). - ):[MH] - :418.2).

[0204] Example 77 Synthesis of Compound L-15 Compound S60 (25 mg, 0.016 mmol, 1.0 eq), compound S61 (7.8 mg, 0.019 mmol, 1.2 eq), copper sulfate pentahydrate (19 mg, 0.078 mmol, 5.0 eq), sodium ascorbate (15 mg, 0.078 mmol, 5.0 eq), and DMF (2 mL) were added to a 25 mL one-neck flask. Water (1 mL) was added in an ice bath and the reaction was allowed to proceed at room temperature. After 30 min, the reaction was confirmed to be complete by HPLC. The reaction solution was purified using a reverse-phase preparative column, and the preparative solution was lyophilized to obtain compound L-15 (white solid, 21 mg, 66%) (LC-MS m / z (ES)). + ):[M+H+Na] 2+ :1024.9).

[0205] Example 78 Synthesis of Compound S62 According to the synthesis method of Example 27, compound S62 (LC-MS m / z (ES + ):[M+2H] 2+ :801.3) was synthesized.

[0206] Example 79 Synthesis of Compound L-16 Compound L-16 (LC-MS m / z (ES + ):[M+H+NH4] 2+ :1019.5) was synthesized.

[0207] Example 80 Synthesis of Compound S63 According to the synthesis method of Example 27, compound S63 (LC-MS m / z (ES + ):[M+2H] 2+ :801.0) was synthesized.

[0208] Example 81 Synthesis of Compound L-17 Compound L-17 (LC-MS m / z (ES + ):[M+2H] 2+ :1010.9) was synthesized.

[0209] Example 82 Synthesis of Compound S68 Step 1 (Resin Loading): 4.6 g of 2-chlorotrityl chloride resin beads (1.05 mmol / g) were weighed into a resin reactor and allowed to swell in DMF for 1 hour. After swelling, the solvent was filtered off, and a DMF solution (30 mL) of Fmoc-sarcosine (1.5 g, 4.8 mmol, 1.0 eq) and DIEA (1.7 mL, 9.6 mmol, 2.0 eq) was added to the resin, and the resin reactor was shaken at room temperature for 4 hours. After filtration, the resin was blocked with MeOH / DIEA / DMF (5 mL / 2.5 mL / 25 mL) for 30 minutes. The resin was then thoroughly washed eight times with DMF to obtain compound S64. Step 2: Elongation of the polysarcosine compound Step a: The resin was treated with piperidine / DMF (6 mL / 24 mL) twice (20 min each time), and then washed with DMF eight times. Step b: A DMF solution (30 mL) of Fmoc-sarcosine (4.5 g, 14.4 mmol, 3.0 eq), HATU (5.5 g, 14.4 mmol, 3.0 eq), HOBt (1.9 g, 14.4 mmol, 3.0 eq), and DIEA (4.2 mL, 24 mmol, 5.0 eq) was added to the resin, and the resin reactor was shaken at room temperature for 2 h. The resin was then washed eight times with DMF. Steps a and b were repeated until the length of the polysarcosine compound on the resin reached 16 peptides, yielding compound S66. Step 3: Acetylation The resin was treated with piperidine / DMF (6 mL / 24 mL) twice (20 min each time), and then washed with DMF eight times. The resin was then treated twice with acetic anhydride / DIEA / DMF (5 mL / 10 mL / 15 mL) for 1 hour each time, and then washed eight times with DMF to obtain compound S67. Step 4: Cutting the resin The polysarcosine oligomer was cleaved from the resin by treatment with HFIP / CHCl (6 mL / 24 mL) solution for 30 minutes. The resin was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified using a reverse-phase preparative column. The preparative solution was lyophilized to obtain compound S68 (white solid, 1.7 g, 30%) (LC-MS m / z (ES + ):[M+2NH4] 2+ :616.4).

[0210] Example 83 Synthesis of Compound S70 Compound S68 (93 mg, 0.078 mmol, 1.0 eq), S69 (17 mg, 0.078 mmol, 1.0 eq), HATU (45 mg, 0.117 mmol, 1.5 eq), HOBt (16 mg, 0.117 mmol, 1.5 eq), and dry DMF (2.5 mL) were added in order to a 25 mL single-neck flask and dissolved uniformly. DIEA (27 μL, 0.156 mmol, 2.0 eq) was added dropwise in an ice bath, and the mixture was allowed to warm to room temperature. After 1 h, the reaction was confirmed to be complete by HPLC. The reaction solution was purified using a reverse-phase preparative column, and the preparative solution was lyophilized to obtain compound S70 (white solid, 80 mg, 73%) (LC-MS m / z (ES + ):[M+H] + :1397.1).

[0211] Example 84 Synthesis of Compound L-18 According to the synthesis method of Example 77, compound L-18 (TOF m / z: [M+Na] + :3026.5) was synthesized.

[0212] Example 85 Synthesis of Compound L-19 JPEG2026502604000312.jpg28161 According to the synthesis method of Example 77, compound L-19 (TOF m / z: [M+Na] + :3020.4) was synthesized.

[0213] Example 86 Synthesis of Compound L-20 According to the synthesis method of Example 77, compound L-20 (TOF m / z: [M+Na] + :3020.4) was synthesized.

[0214] Example 87 Synthesis of Compound S76 JPEG2026502604000314.jpg66157Step 1 (Filling into resin): 3.7 g (1.05 mmol / g) of 2-chlorotrityl chloride resin beads were weighed into a resin reactor and swollen in DMF for 1 hour. After swelling, the solvent was filtered off, and a DMF solution (30 mL) of Fmoc-L-phenylalanine (1.5 g, 3.9 mmol, 1.0 eq) and DIEA (1.3 mL, 7.7 mmol, 2.0 eq) was added to the resin, and the resin reactor was shaken at room temperature for 4 hours. After filtration, the resin was blocked with MeOH / DIEA / DMF (5 mL / 2.5 mL / 25 mL) for 30 minutes. The resin was then thoroughly washed with DMF eight times to obtain compound S71. Step 2: Peptide chain elongation The resin was treated with piperidine / DMF (6 mL / 24 mL) twice (20 min each time) and then thoroughly washed with DMF eight times. A DMF solution (30 mL) of Fmoc-glycyl-glycine (4.1 g, 11.6 mmol, 3.0 eq), HATU (4.4 g, 11.6 mmol, 3.0 eq), HOBt (1.6 g, 11.6 mmol, 3.0 eq), and DIEA (3.4 mL, 19.4 mmol, 5.0 eq) was added to the resin, and the resin reactor was shaken at room temperature for 2 hours. The resin was then washed eight times with DMF to obtain compound S72. The resin was treated with piperidine / DMF (6 mL / 24 mL) twice (20 min each time) and then thoroughly washed with DMF eight times. A DMF solution (30 mL) of Fmoc-cysteic acid (4.5 g, 11.6 mmol, 3.0 eq), HATU (4.4 g, 11.6 mmol, 3.0 eq), HOBt (1.6 g, 11.6 mmol, 3.0 eq), and DIEA (3.4 mL, 19.4 mmol, 5.0 eq) was added to the resin, and the resin reactor was shaken at room temperature for 4 hours. The resin was then washed eight times with DMF to obtain compound S73. The resin was treated with piperidine / DMF (6 mL / 24 mL) twice (20 min each time), then thoroughly washed with DMF eight times. A DMF solution (30 mL) of compound S37 (4.1 g, 11.6 mmol, 3.0 eq), HATU (4.4 g, 11.6 mmol, 3.0 eq), HOBt (1.6 g, 11.6 mmol, 3.0 eq), and DIEA (3.4 mL, 19.4 mmol, 5.0 eq) was added to the resin, and the resin reactor was shaken at room temperature for 4 hours. The resin was then washed eight times with DMF to obtain compound S74. The resin was treated with piperidine / DMF (6 mL / 24 mL) twice (20 min each time), and then thoroughly washed with DMF eight times. A DMF solution (30 mL) of compound S8 (4.6 g, 11.6 mmol, 3.0 eq) and EEDQ (2.9 g, 11.6 mmol, 3.0 eq) was added to the resin, and the resin reactor was shaken at room temperature for 4 hours. The resin was then washed eight times with DMF to obtain compound S75. Step 3: Cutting the resin The resin was treated with HFIP / CH2Cl2 (6 mL / 24 mL) for 30 minutes. The resin was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by reverse-phase preparative column chromatography. The preparative solution was lyophilized to give compound S76 (white solid, 836 mg, 23%) (LC-MS m / z (ES - ):[MH] - :924.2).

[0215] Example 88 Synthesis of Compound S77 Compound D-4 (50 mg, 0.069 mmol, 1.0 eq), Fmoc-glycine (31 mg, 0.103 mmol, 1.5 eq), HATU (39 mg, 0.103 mmol, 1.5 eq), HOBt (14 mg, 0.103 mmol, 1.5 eq), and dry DMF (3 mL) were added in this order to a 125 mL single-neck flask. After homogeneous dissolution, DIEA (24 μL, 0.137 mmol, 2.0 eq) was added dropwise in an ice bath and the mixture was allowed to warm to room temperature. After 1 h, the reaction was confirmed to be complete by HPLC. The reaction solution was purified using a reverse-phase preparative column, and the preparative solution was lyophilized to obtain compound S77 (white solid, 58 mg, 84%) (LC-MS m / z (ES + ):[M+H] + :1009.2).

[0216] Example 89 Synthesis of Compound S79 JPEG2026502604000316.jpg23128JPEG2026502604000317.jpg23163 According to the synthesis methods of Examples 26 and 27, compound S79 (LC-MS m / z (ES + ):[M-100+2H] 2+ :797.9 (fragment ion response after removing the Boc protecting group of compound S79 and adding a hydrogen ion) was synthesized.

[0217] Example 90 Synthesis of Compound L-21 Compound L-21 (LC-MS m / z (ES + ):[M+2H] 2+ :769.8) was synthesized.

[0218] Example 91 Synthesis of Compound S80 According to the synthesis method of Example 27, compound S80 (LC-MS m / z (ES + ):[M+2H]2+ :911.5) was synthesized.

[0219] Example 92 Synthesis of Compound L-22 Compound L-22 (LC-MS m / z (ES + ):[M+2H] 2+ :833.4) was synthesized.

[0220] Example 93 Synthesis of Compound S81 According to the synthesis method of Example 27, compound S81 (LC-MS m / z (ES + ):[M+2H] 2+ :908.4) was synthesized.

[0221] Example 94 Synthesis of Compound L-23 Compound L-23 (LC-MS m / z (ES + ):[M+2H] 2+ :830.3) was synthesized.

[0222] Example 95 Synthesis of Compound S82 According to the synthesis method of Example 27, compound S82 (LC-MS m / z (ES + ):[M+2H] 2+ :908.3) was synthesized.

[0223] Example 96 Synthesis of Compound L-24 Compound L-24 (LC-MS m / z (ES + ):[M+2H] 2+ :830.3) was synthesized.

[0224] Example 97 Preparation of antibody-drug conjugate ADC-1 Antibody-drug conjugate ADC-1 was prepared using compound L-1 and anti-Trop2 antibody TR001 according to General Procedure A. The sequence information of TR001 is shown in Table 2. JPEG2026502604000325.jpg104128

[0225] Table 2: TR001 sequence description JPEG2026502604000326.jpg196152

[0226] Example 98 Preparation of antibody drug conjugate ADC-2 Antibody-drug conjugate ADC-2 was prepared using compound L-2 and anti-Trop2 antibody TR001 according to General Procedure A. JPEG2026502604000327.jpg101128

[0227] Example 99 Preparation of antibody drug conjugate ADC-3 Antibody-drug conjugate ADC-3 was prepared using compound L-1 and anti-Trop2 antibody TR002 according to General Procedure B. The sequence information of TR002 is shown in Table 3. JPEG2026502604000328.jpg104128

[0228] Table 3: TR002 sequence description JPEG2026502604000329.jpg196152

[0229] Example 100 Preparation of Antibody Drug Conjugate ADC-4 Antibody-drug conjugate ADC-4 was prepared using compound L-2 and anti-Trop2 antibody TR002 according to General Procedure B. JPEG2026502604000330.jpg101128

[0230] Example 101 Preparation of antibody drug conjugate ADC-5 Antibody-drug conjugate ADC-5 was prepared using compound L-3 and anti-Trop2 antibody TR001 according to General Procedure A. JPEG2026502604000331.jpg104131

[0231] Example 102 Preparation of antibody drug conjugate ADC-6 Antibody-drug conjugate ADC-6 was prepared using compound L-7 and anti-Trop2 antibody TR001 according to General Procedure A. JPEG2026502604000332.jpg100134

[0232] Example 103 Preparation of antibody drug conjugate ADC-7 Antibody-drug conjugate ADC-7 was prepared using compound L-8 and anti-Trop2 antibody TR001 according to General Procedure A. JPEG2026502604000333.jpg107134

[0233] Example 104 Preparation of antibody drug conjugate ADC-8 Antibody-drug conjugate ADC-8 was prepared using compound L-9 and anti-Trop2 antibody TR001 according to General Procedure A. JPEG2026502604000334.jpg104128

[0234] Example 105 Preparation of antibody drug conjugate ADC-9 Antibody-drug conjugate ADC-9 was prepared using compound L-10 and anti-Trop2 antibody TR001 according to General Procedure A. JPEG2026502604000335.jpg101128

[0235] Example 106 Preparation of antibody drug conjugate ADC-10 Antibody-drug conjugate ADC-10 was prepared using compound L-11 and anti-Trop2 antibody TR001 according to General Procedure A. JPEG2026502604000336.jpg104128

[0236] Example 107 Preparation of antibody drug conjugate ADC-11 Antibody-drug conjugate ADC-11 was prepared using compound L-1′ and anti-Trop2 antibody TR001 according to General Procedure A. JPEG2026502604000337.jpg104128

[0237] Example 108 Preparation of antibody drug conjugate ADC-12 Antibody-drug conjugate ADC-12 was prepared using compound L-2′ and anti-Trop2 antibody TR001 according to General Procedure A. JPEG2026502604000338.jpg101128

[0238] Example 109 Preparation of antibody drug conjugate ADC-13 Antibody-drug conjugate ADC-13 was prepared using compound L-15 and anti-Trop2 antibody TR001 according to General Procedure C. JPEG2026502604000339.jpg105128

[0239] Example 110 Preparation of antibody drug conjugate ADC-14 Antibody-drug conjugate ADC-14 was prepared using compound L-16 and anti-Trop2 antibody TR001 according to General Procedure C. JPEG2026502604000340.jpg105129

[0240] Example 111 Preparation of antibody drug conjugate ADC-15 Antibody-drug conjugate ADC-15 was prepared using compound L-17 and anti-Trop2 antibody TR001 according to General Procedure C. JPEG2026502604000341.jpg100128

[0241] Example 112 Preparation of antibody drug conjugate ADC-16 Antibody-drug conjugate ADC-16 was prepared using compound L-18 and anti-Trop2 antibody TR001 according to General Procedure C. JPEG2026502604000342.jpg101128

[0242] Example 113 Preparation of antibody drug conjugate ADC-17 Antibody-drug conjugate ADC-17 was prepared using compound L-19 and anti-Trop2 antibody TR001 according to General Procedure C. JPEG2026502604000343.jpg103129

[0243] Example 114 Preparation of antibody drug conjugate ADC-18 Antibody-drug conjugate ADC-18 was prepared using compound L-20 and anti-Trop2 antibody TR001 according to General Procedure C. JPEG2026502604000344.jpg98128

[0244] Example 115 Preparation of antibody drug conjugate ADC-19 Antibody-drug conjugate ADC-19 was prepared using compound L-8 and anti-Trop2 antibody TR001 according to General Procedure C. JPEG2026502604000345.jpg109136

[0245] Example 116 Preparation of antibody drug conjugate ADC-20 Antibody-drug conjugate ADC-20 was prepared using compound L-21 and anti-Trop2 antibody TR001 according to General Procedure C. JPEG2026502604000346.jpg94144

[0246] Example 117 Preparation of antibody drug conjugate ADC-21 Antibody-drug conjugate ADC-21 was prepared using compound L-22 and anti-Trop2 antibody TR001 according to General Procedure C. JPEG2026502604000347.jpg85128

[0247] Example 118 Preparation of antibody drug conjugate ADC-22 Antibody-drug conjugate ADC-22 was prepared using compound L-23 and anti-Trop2 antibody TR001 according to General Procedure C. JPEG2026502604000348.jpg91133

[0248] Example 119 Preparation of antibody drug conjugate ADC-23 Antibody-drug conjugate ADC-23 was prepared using compound L-24 and anti-Trop2 antibody TR001 according to General Procedure C. JPEG2026502604000349.jpg87128

[0249] Example 120: Measurement of Drug-Antibody Ratio (DAR) Detection of DAR by reversed-phase high-performance liquid chromatography (RP-HPLC) Preparation of RP-HPLC mobile phase: RP mobile phase A: 0.1% TFA in water, RP mobile phase B: 0.1% TFA in acetonitrile. Test samples and corresponding antibody controls were diluted to 1 mg / mL in sample diluent. 2 μL of DTT stock solution was added to every 98 μL of diluted sample. A blank control was also prepared with 98 μL of sample diluent and 2 μL of DTT stock solution. After mixing, each sample was heated in a metal bath at 65°C for 30 minutes. After treatment, the samples were centrifuged at 14,000 rpm for 5 minutes or filtered through a 0.22 μm filter to remove large particles. The inner tube was then placed in a sample bottle and capped. The sample bottles containing the samples were placed on the sample plate, and the position, injection volume, injection needle number, and injection method for each sample were set according to the "UPLC standard operating procedure." The chromatography column was a Proteomix RP-1000 (4.6*100mm, 5μm), Sepax. The detection wavelengths were 214 nm and 280 nm. The analytical method was as follows. JPEG2026502604000350.jpg76165Table 4: Detailed data of binding ratios (DAR) of ligand-drug conjugates (ADCs) of the present disclosure: JPEG2026502604000351.jpg208147

[0250] Example 121 In vitro drug activity screening of compounds In the present invention, various human tumor cell lines (BxPC-3 (human pancreatic adenocarcinoma cells), SW620 (human colon cancer cells), H1975 (human lung adenocarcinoma cells), HCC827 (human lung adenocarcinoma cells), FaDu (human pharyngeal squamous cell carcinoma cells), and A431 (human epidermal carcinoma cells)) were used as experimental models to evaluate the cytotoxic activity of compounds against tumor cell lines. A certain number of tumor cells were seeded into a 96-well plate. After the cells adhered to the wells, serially diluted test compounds were added to the test cells and incubated at 37°C under 5% CO2 for 5 days. Cell viability was detected using MTS, and IC 50 The inhibitory effects of the control and test compounds on tumor cell lines were evaluated based on the IC values. The initial concentration of the compound was 4000 nM, and the dilution ratio was 7 times, for a total of eight concentration points. Finally, the viability was calculated based on the survival rate = (experimental group - blank group) / (control group - blank group) × 100%, and the median inhibitory concentration (IC) was calculated by fitting the curve using a four-parameter model in Graph Pad Prism. 50 ) was calculated. Table 5: In vitro efficacy results of control and test compounds in human tumor cell lines BxPC-3, SW620, and H1975: JPEG2026502604000352.jpg134146Table 6: In vitro efficacy results of control and test compounds in human tumor cell lines HCC827, FaDu, and A431: JPEG2026502604000353.jpg134147The results are shown in the table above and Figures 1A-1F. Conclusion: In BxPC-3, SW620, H1975, HCC827, FaDu, and A431 cell models, various compounds synthesized in this invention showed significant antitumor effects, among which compounds D-3, D-7, D-8, D-9, D-12, and D-13 showed the most excellent effects.

[0251] Example 122 Evaluation of the stability of antibody-drug conjugates The ADC was diluted with sterile human plasma to prepare samples with a drug concentration of 0.6 mg / ml, which were then incubated in a 37°C water bath for 0, 3, and 7 days. The samples incubated in plasma for 0, 3, and 7 days, as well as a control sample diluted with PBS to a final drug concentration of 0.6 mg / ml, were then purified and extracted to prepare the ADC. Finally, the purified and extracted ADC was analyzed for stability in plasma. Determination of monomer content 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 The method parameters are: JPEG2026502604000354.jpg76128Table 7: Plasma stability assessment of test ADCs of the invention: JPEG2026502604000355.jpg93163Conclusion: The above results indicate that the antibody-drug conjugate synthesized in this invention has good stability in human plasma.

[0252] Example 123 In vitro efficacy testing of antibody-drug conjugates - single tumor model In this study, various human tumor cell lines (A431, HCC827, N87 (human gastric cancer cells), SW620, and FaDu) were used as experimental models to evaluate the in vitro efficacy of antibody-drug conjugates (ADCs). A certain number of tumor cells were seeded in a 96-well plate, and serially diluted test antibodies and corresponding ADC drugs were added to the cells. After 5 days of treatment, cell viability was detected by MTS, and IC 50The inhibitory effects of the test antibodies and ADCs on tumor cell lines were evaluated using IC values. The initial concentration of the antibody and ADC drug was 500 nM, diluted 7 times, for a total of eight concentration points, and the treatment was carried out for five days. The final algorithm was based on survival rate = (experimental group - blank group) / (control group - blank group) × 100%, and then curve fitting was performed using Graph Pad Prism to determine the median inhibitory concentration (IC). 50 ) was calculated. Table 8: In vitro efficacy results of tested ADCs in human tumor cell lines A431, HCC827, and FaDu: JPEG2026502604000356.jpg101146Table 9: In vitro efficacy results of the tested ADCs in human tumor cell lines N87 and SW620: JPEG2026502604000357.jpg43128The results are shown in the table above and Figures 2A-2E. Conclusion: In A431, HCC827, N87, SW620, and Fadu cell models, the naked antibody TR001 did not exhibit tumor cell killing activity, while the antibody-drug conjugate synthesized in this invention exhibited significant antitumor effects.

[0253] Example 124 In vitro efficacy testing of antibody-drug conjugates - xenogeneic tumor model In this study, heterologous tumor cell lines (A431 + SW620) were used as an experimental model to evaluate the in vitro efficacy of antibody-drug conjugates (ADCs). A certain number of tumor cells were seeded in a 96-well plate, and serially diluted test ADC drugs were added to the cells. After 5 days of treatment, cell viability was detected by MTS, and IC 50 The inhibitory effects of the test antibodies and ADCs on tumor cell lines were evaluated using IC values. The initial concentration of the antibody and ADC drug was 500 nM, diluted 7 times, for a total of eight concentration points, and the treatment was carried out for five days. The final algorithm was based on survival rate = (experimental group - blank group) / (control group - blank group) × 100%, and then curve fitting was performed using Graph Pad Prism to determine the median inhibitory concentration (IC). 50 ) was calculated. Table 10: In vitro efficacy results of tested ADCs in heterologous tumor cell lines A431+SW620: JPEG2026502604000358.jpg5983The results are shown in the table above and in Figure 3. Conclusion: In the A431+SW620 xenogeneic tumor cell model, the antibody-drug conjugate synthesized in this invention exhibited significant antitumor effects, which were superior to the control antibody-drug conjugate ADC-19.

[0254] Example 125 In vivo efficacy testing of antibody-drug conjugates In this study, the in vivo efficacy of ADC was evaluated using a BALB / c-nu subcutaneously inoculated human tumor cell line (A431) as an experimental model. A certain number of tumor cell suspensions were subcutaneously inoculated into BALB / c-nu, and tumors with a volume of approximately 180 mm were observed. 3 Once the mice reached 100 mm HCC, vehicle and ADC-5 were administered via tail vein injection once a week for a total of three doses. The tumors were continuously monitored, and tumors were measured and weighed twice a week to assess the inhibitory effect of the ADC drug on tumor growth. The results are shown in the table below. Table 11: Efficacy of tested ADCs against A431 mouse xenograft tumors: JPEG2026502604000359.jpg37160T / C%=T RTV / C RTV × 100%; TGI% = (1 - T / C) × 100% Table 12: Effect of test ADCs on body weight of A431 implanted mice: JPEG2026502604000360.jpg31162Conclusion: The antibody-drug conjugate synthesized in this invention showed significant anti-tumor effects in mice bearing A431 monomer tumors.

[0255] The above examples are merely illustrative of the technical means of the present invention, and do not limit the present invention. Any modifications or equivalent replacements of the technical means of the present invention without departing from the spirit and scope of the technical means of the present invention are included in the protection scope of the present invention.

Claims

1. A ligand-drug conjugate having the structure shown in Formula I, or a pharmaceutically acceptable salt or solvate thereof: During the ceremony, Ab is a ligand unit; L is a linker covalently linking Ab to D; n is selected from integers or decimals from 1 to 40; -D is represented by formula II or formula III, wherein W is selected from an oxygen atom or a sulfur atom; R １ , R ２ are the same or different and each independently represent a hydrogen atom, an alkyl, an alkoxy, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, or —C(O)—Q １ -Q ２ , and -SO ２ -Q １ -Q ２ where Q is selected from １ is selected from an O, N, S atom or a chemical bond, and Q ２ is selected from alkyl, cycloalkyl, heterocyclyl, spirocyclyl, bridged cyclic group, alkenyl, aryl, and heteroaryl, and optionally said alkyl, alkoxy, alkenyl, cycloalkyl, heterocyclyl, spirocyclyl, bridged cyclic group, aryl, and heteroaryl are each independently substituted with one or more substituents selected from hydrogen atom, alkyl, alkoxy, halogen, deuterium, amino, cyano, hydroxyl, mercapto, azido, nitro, carboxyl, acyl, carbonyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or R １ , R ２ together with the nitrogen atom attached thereto form a 3- to 8-membered heterocyclyl, optionally substituted with one or more substituents selected from hydrogen, alkyl, alkoxy, halogen, deuterium, amino, cyano, hydroxyl, mercapto, azido, nitro, carboxyl, acyl, carbonyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; R ３ is selected from a hydrogen atom, an alkyl, an acyl, a sulfonyl, a cycloalkyl, a heterocyclyl, an aryl, and a heteroaryl, and optionally, said alkyl, acyl, sulfonyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl may each be independently substituted with one or more substituents selected from a hydrogen atom, an alkyl, an alkoxy, a halogen, a deuterium, an amino, a cyano, a hydroxyl, a mercapto, an azido, a nitro, a carboxyl, an acyl, a carbonyl, a hydroxyalkyl, a cycloalkyl, a heterocyclyl, an aryl, and a heteroaryl; A ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof.

2. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the ligand unit Ab is selected from an antibody, an antibody fragment, or a protein, and the antibody is preferably selected from a mouse antibody, a rabbit antibody, a phage-displayed antibody, a yeast-displayed antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody, and a multispecific antibody.

3. The antibodies include anti-EGFRvIII antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, and anti-ENPP3 antibody. body, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg / AFP antibody, anti-claudin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-G PNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibodies, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-Integrin antibody Av antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD47 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody, or anti-CD123 antibody,

4. L has the structure shown in Formula IV: During the ceremony, M is a linking unit that covalently binds to Ab; Z is -C1-C10 alkylene-, -C3-C8 carbocyclic ring-, -arylene-, -3-8 membered heterocyclic ring-, -(CH ２ CH ２ O) ｒ -, sulfonyl, amide, chemical bond, or a combination thereof, wherein X １ and X ２ is -C1-C10 alkylene-, -C3-C8 carbocyclic ring-, -arylene-, -3-8 membered heterocyclic ring-, -(CH ２ CH ２ O) ｒ -, -NR ４ - or carbonyl, or combinations thereof, wherein R ４ is selected from a hydrogen atom, a deuterium atom, an alkyl, and a substituted alkyl, and optionally, the -C1-C10 alkylene-, -C3-C8 carbocycle-, and -3-8 membered heterocycle- may each independently be substituted with one or more substituents selected from a hydrogen atom, a deuterium atom, a halogen atom, a hydroxyl, a cyano, a nitro, an amino, an alkyl, a heteroalkyl, a substituted alkyl, an alkoxy, a carboxyl, or a cycloalkyl, and the heterocycles each independently contain 1 to 3 atoms selected from N, O, and S, and Y １ is a hydrophilic structure selected from the group consisting of one or more of carboxyl, phosphoric acid, polyphosphoric acid, phosphorous acid, sulfonic acid, sulfinic acid, and polyethylene glycol (PEG); Y ２ is selected from the following structures: r is selected from an integer from 1 to 10; １ and q ２ is selected from integers of 1 to 8, e is selected from integers of 1 to 20, A is a peptide residue consisting of 2 to 7 amino acids, G is a spacer unit that binds to D; p is 0 or 1; The ligand-drug conjugate according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

5. The linking unit M has a succinimide structure represented by formula a, or a ring-opened succinimide structure represented by formula b or formula c, In Formula a, Formula b, or Formula c, the wavy line on the left indicates binding to the Ab binding site, and the wavy line on the right indicates binding to the Z binding site. The ligand-drug complex according to claim 4, or a pharmaceutically acceptable salt or solvate thereof.

6. The ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 4 or 5, wherein A is a polypeptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, and cysteic acid.

7. the spacer unit G is one of the structures represented by formula Va, Vb, Vc or Vd, or a combination thereof; In the formula, the wavy line on the left indicates the binding site between the nitrogen atom and peptide residue A, the wavy line on the right indicates the binding site between the oxygen atom and drug D, W is selected from an oxygen atom or a sulfur atom and is a covalent group between drug D and spacer unit G, R ５ , R ５ ', R ６ , and R ７ are each independently selected from a hydrogen atom, a deuterium atom, alkyl, and substituted alkyl; The ligand-drug conjugate according to any one of claims 4 to 6, or a pharmaceutically acceptable salt or solvate thereof.

8. having a structure shown in Formula VI or Formula VII, During the ceremony, Ab, Z, A, R ５ , R ６ , R ７ , R ３ , W is as defined in any one of claims 1 to 7, and n １ , n ２ , n ３ are independently selected from integers or decimals from 0 to 40; １ , n ２ , n ３ is not 0 at the same time, and n １ +n ２ +n ３ ≦40, The ligand-drug conjugate according to any one of claims 1 to 7, or a pharmaceutically acceptable salt or solvate thereof.

9. Z is -C1-C10 alkylene-, -(CH ２ CH ２ O) ｒ -, amide, or a combination thereof; Preferably, Z is —C2-alkylene or —C5-alkylene; Preferably, Z is where q １ is selected from integers from 1 to 8, preferably q １ is 1, Preferably, q １ is 1, and —NH—X １ -Y １ is a hydrophilic structural unit Ac １ and the Ac １ is selected from, but not limited to, (D / L) alanine, (D / L) leucine, (D / L) isoleucine, (D / L) valine, (D / L) phenylalanine, (D / L) proline, (D / L) tryptophan, (D / L) serine, (D / L) tyrosine, (D / L) cysteine, (D / L) cystine, (D / L) arginine, (D / L) histidine, (D / L) methionine, (D / L) asparagine, (D / L) glutamine, (D / L) threonine, (D / L) aspartic acid, (D / L) glutamic acid, a natural or unnatural amino acid derivative, or the following structures: Preferably, q １ is 1, and —NH—X １ -Y １ is a hydrophilic structural unit Ac １ and the Ac １ is selected from the following structures: Preferably, Z is where q ２ is selected from integers from 1 to 8, preferably q ２ is 1, and preferably, X ２ is -(C1-C10 alkylene)-(CH ２ CH ２ O) ｒ -(C=O)- or -(C1-C10 alkylene)-(CH ２ CH ２ O) ｒ -NR ４ - and Preferably, q ２ is 1, and -X ２ -Y ２ is a hydrophilic structural unit Ac ２ and the Ac ２ teeth, wherein r is selected from an integer from 1 to 10, and e is selected from an integer from 1 to 20; Preferably, q ２ is 1, and -X ２ -Y ２ is a hydrophilic structural unit Ac ２ and the Ac ２ is selected from the following structures: The ligand-drug conjugate according to any one of claims 4 to 8, or a pharmaceutically acceptable salt or solvate thereof.

10. A is a polypeptide residue consisting of 2 to 5 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, and cysteic acid; Preferably, A is a peptide residue formed by 2 to 4 amino acids selected from phenylalanine and glycine, More preferably, A is a tetrapeptide residue consisting of glycine (G), glycine (G), phenylalanine (F), glycine (G); More preferably, A is -GGFG-. The ligand-drug conjugate according to any one of claims 4 to 9, or a pharmaceutically acceptable salt or solvate thereof.

11. W is selected from an oxygen atom or a sulfur atom, and R １ , R ２ are the same or different and each independently represent a hydrogen atom, an alkyl, an alkoxy, an alkenyl, a cycloalkyl, or —C(O)—Q １ -Q ２ , and -SO ２ -Q １ -Q ２ where Q is selected from １ is selected from O or a chemical bond, and Q ２ is selected from alkyl, heterocyclyl, alkenyl, aryl, heteroaryl, and cycloalkyl, and optionally, said alkyl, heterocyclyl, alkenyl, aryl, heteroaryl, and cycloalkyl are each independently substituted with one or more substituents selected from hydrogen, alkyl, hydroxyalkyl, halogen, deuterium, mercapto, amino, aryl, hydroxyl, or R １ , R ２ together with the nitrogen atom to which they are attached form a 3-6 membered heterocyclyl, optionally substituted with one or more substituents selected from hydrogen atoms, alkyl, alkoxy, halogen, deuterium, amino, hydroxyl, cycloalkyl, and heterocyclyl; R ３ is selected from a hydrogen atom and an alkyl, optionally wherein the alkyl is substituted with one or more substituents selected from a hydrogen atom, an alkyl, an alkoxy, a halogen, a deuterium, an amino, a hydroxyl, a cycloalkyl, and a heterocyclyl; Preferably, W is an oxygen atom and R １ , R ２ are the same or different and each independently represent a hydrogen atom, an alkyl, an alkenyl, a haloalkyl, —C(O)—Q １ -Q ２ , and -SO ２ -Q １ -Q ２ where Q is selected from １ is selected from O or a chemical bond, and Q ２ is selected from alkyl, alkenyl, aryl, and 3- to 8-membered cycloalkyl, and optionally, said alkyl, alkenyl, aryl, and cycloalkyl may each be independently substituted with one or more substituents selected from hydrogen atom, alkyl, hydroxyalkyl, halogen, aryl, hydroxyl, or R １ , R ２ together with the nitrogen atom bound thereto form a 3-6 membered heterocyclyl, said 3-6 membered heterocyclyl containing 1 to 2 nitrogen atoms and optionally further containing 1 oxygen atom, said 3-6 membered heterocyclyl being optionally substituted with one or more substituents selected from a hydrogen atom, an alkyl, and a haloalkyl; R ３ is selected from a hydrogen atom and an alkyl, wherein the alkyl is substituted with one or more substituents selected from a hydrogen atom, an alkyl, a halogen, a deuterium, an amino, and a hydroxyl; The ligand-drug conjugate according to any one of claims 1 to 10, or a pharmaceutically acceptable salt or solvate thereof.

12. W is an oxygen atom, and R １ , R ２ are the same or different and each independently represent a hydrogen atom, C １ -C ４ Alkyl (e.g., methyl, ethyl), C ２ -C ４ Alkenyl (e.g., allyl), C １ -C ４ haloalkyl (e.g., difluoroethyl) or selected from the following structures: or R １ , R ２ together with the nitrogen atom to which they are attached form a 3-6 membered heterocyclyl, said 3-6 membered heterocyclyl containing 1 to 2 nitrogen atoms and optionally further containing 1 oxygen atom (e.g., aziridinyl, piperidinyl, piperazinyl, morpholinyl), and optionally said 3-6 membered heterocyclyl containing a hydrogen atom, C １ -C ４ Alkyl (e.g., methyl), C １ -C ４ haloalkyl (e.g., trifluoromethyl), R ３ is selected from a hydrogen atom and methyl; The ligand-drug conjugate according to any one of claims 1 to 11, or a pharmaceutically acceptable salt or solvate thereof.

13. R ５ , R ６ , and R ７ are each independently selected from a hydrogen atom, a deuterium atom, an alkyl, a haloalkyl, a deuterated alkyl, and a hydroxyalkyl; Preferably, R ５ , R ６ , and R ７ is also a hydrogen atom, The ligand-drug complex according to any one of claims 7 to 12, or a pharmaceutically acceptable salt or solvate thereof.

14. having a structure of Formula VIa or Formula VIIa, During the ceremony, R １ , R ２ , R ３ , n １ , n ２ , n ３ is as defined in any of claims 1 to 13, The ligand-drug conjugate according to any one of claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof.

15. having a structure of Formula VIb or Formula VIIb, During the ceremony, R １ , R ２ , R ３ , n １ , n ２ , n ３ is as defined in any one of claims 1 to 13, A.C. １ is a hydrophilic structural unit having a structure represented by formula d, X １ , Y １ is as defined in claim 4, Preferably, the Ac １ is selected from, but not limited to, (D / L) alanine, (D / L) leucine, (D / L) isoleucine, (D / L) valine, (D / L) phenylalanine, (D / L) proline, (D / L) tryptophan, (D / L) serine, (D / L) tyrosine, (D / L) cysteine, (D / L) cystine, (D / L) arginine, (D / L) histidine, (D / L) methionine, (D / L) asparagine, (D / L) glutamine, (D / L) threonine, (D / L) aspartic acid, (D / L) glutamic acid, a natural or unnatural amino acid derivative, or the following structures: Preferably, Ac １ is selected from the following structures: The ligand-drug conjugate according to any one of claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof.

16. having a structure shown in formula VIc: During the ceremony, R １ , R ２ , n １ , n ２ , n ３ is as defined in any of claims 1 to 13, The ligand-drug conjugate according to any one of claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof.

17. having a structure of formula VId: During the ceremony, R １ , R ２ , n １ , n ２ , n ３ is as defined in any of claims 1 to 13, The ligand-drug conjugate according to any one of claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof.

18. having a structure of formula VIe: In the formula, R １ , R ２ , n １ , n ２ , n ３ is as defined in any one of claims 1 to 13, A.C. ２ is -X ２ -Y ２ is a hydrophilic structural unit composed of X ２ , Y ２ is as defined in claim 4, Preferably, the Ac ２ teeth, where r is selected from an integer from 1 to 10 and e is selected from an integer from 1 to 20; Preferably, Ac ２ is selected from the following structures: The ligand-drug conjugate according to any one of claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof.

19. having a structure shown in formula VIf, In the formula, R １ , R ２ , n １ , n ２ , n ３ is as defined in any one of claims 1 to 13, A.C. １ is a hydrophilic structural unit having a structure represented by formula d, X １ , Y １ is as defined in claim 4, Preferably, Ac １ is as defined in claim 15, A.C. ２ is -X ２ -Y ２ is a hydrophilic structural unit composed of X ２ , Y ２ is as defined in claim 4, Preferably, the Ac ２ teeth, where r is selected from an integer from 1 to 10 and e is selected from an integer from 1 to 20; Preferably, Ac ２ is selected from the following structures: The ligand-drug conjugate according to any one of claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof.

20. having a structure of formula VIg: In the formula, R １ , R ２ , n １ , n ２ , n ３ is as defined in any one of claims 1 to 13, A.C. １ is a hydrophilic structural unit having a structure represented by formula d, X １ , Y １ is as defined in claim 4, Preferably, Ac １ is as defined in claim 15 The ligand-drug conjugate according to any one of claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof.

21. selected from the following structures: wherein the configurations of the chiral carbons at the 2- and 3-positions are independently R or S, and preferably the configuration of the chiral carbon at the 2-position is S, and the configuration of the chiral carbon at the 3-position is S. The ligand-drug conjugate according to any one of claims 1 to 20, or a pharmaceutically acceptable salt or solvate thereof.

22. the ligand unit Ab is an antibody, and the antibody is an anti-TROP-2 antibody; Preferably, the antibody comprises a light chain and a heavy chain, the light chain comprising CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; Preferably, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; Preferably, the light chain comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 7, and more preferably, the light chain further comprises a light chain constant region having the amino acid sequence of SEQ ID NO: 8; Preferably, the amino acid sequence of the light chain is SEQ ID NO: 9, Preferably, the light chain comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 10, and more preferably, the light chain further comprises a light chain constant region having the amino acid sequence of SEQ ID NO: 11; Preferably, the amino acid sequence of the light chain is SEQ ID NO: 12; Preferably, the heavy chain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13, and more preferably, the heavy chain further comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO: 14; Preferably, the amino acid sequence of the heavy chain is SEQ ID NO:

15.

22. The ligand-drug conjugate according to any one of claims 1 to 21, or a pharmaceutically acceptable salt or solvate thereof.

23. A linker-drug conjugate having a structure represented by Formula VIII or Formula IX, or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof: During the ceremony, M １ is a linker unit, Z, p, A, G, W, R １ , R ２ , R ３ is as defined in any of claims 1 to 13, A linker-drug conjugate or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

24. having a structure of formula VIII-1 or formula IX-1, During the ceremony, Z, A, R １ , R ２ , R ３ , R ５ , R ６ , R ７ is as defined in any of claims 1 to 13, 24. The linker-drug conjugate according to claim 23, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

25. having a structure of Formula VIIIa or Formula IXa, During the ceremony, R １ , R ２ , R ３ is as defined in any of claims 1 to 13, 25. The linker-drug conjugate according to claim 23 or 24, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

26. having a structure of formula VIIIb or formula IXb, During the ceremony, R １ , R ２ , and R ３ is as defined in any one of claims 1 to 13, A.C. １ is a hydrophilic structural unit having a structure represented by formula d, X １ , Y １ is as defined in claim 4, Preferably, Ac １ is as defined in claim 15 25. The linker-drug conjugate according to claim 23 or 24, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

27. having a structure shown in Formula VIIIc: During the ceremony, R １ , R ２ is as defined in any of claims 1 to 13, 25. The linker-drug conjugate according to claim 23 or 24, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

28. having a structure shown in formula VIIId: During the ceremony, R １ , R ２ is as defined in any of claims 1 to 13, 25. The linker-drug conjugate according to claim 23 or 24, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

29. having a structure shown in Formula VIIIe: During the ceremony, R １ , R ２ is as defined in any one of claims 1 to 13, A.C. ２ is -X ２ -Y ２ is a hydrophilic structural unit composed of X ２ , Y ２ is as defined in claim 4, Preferably, Ac ２ is as defined in claim 9 or claim 18, 25. The linker-drug conjugate according to claim 23 or 24, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

30. having a structure shown in formula VIIIf: In the formula, R １ , R ２ is as defined in any one of claims 1 to 13, A.C. １ is -X １ -Y １ is a hydrophilic structural unit composed of X １ , Y １ is as defined in claim 4, Preferably, Ac １ is as defined in claim 15, A.C. ２ is -X ２ -Y ２ is a hydrophilic structural unit composed of X ２ , Y ２ is as defined in claim 4, Preferably, Ac ２ is as defined in claim 9 or claim 18, 25. The linker-drug conjugate according to claim 23 or 24, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

31. having a structure shown in Formula VIIIg: In the formula, R １ , R ２ is as defined in any one of claims 1 to 13, A.C. １ is -X １ -Y １ is a hydrophilic structural unit composed of X １ , Y １ is as defined in claim 4, Preferably, Ac １ is as defined in claim 15 25. The linker-drug conjugate according to claim 23 or 24, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

32. selected from the following structures: wherein the configurations of the chiral carbons at the 2- and 3-positions are independently R or S, and preferably the configuration of the chiral carbon at the 2-position is S, and the configuration of the chiral carbon at the 3-position is S.

32. The linker-drug conjugate according to any one of claims 23 to 31, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

33. A compound having the structure of Formula X, or an isomer, meso form, racemate, enantiomer, or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, Formula X wherein W is selected from an oxygen atom or a sulfur atom, preferably W is an oxygen atom; R １ , R ８ are each independently selected from a hydrogen atom, alkyl, acyl, sulfonyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and are not simultaneously hydrogen; optionally, said alkyl, acyl, sulfonyl, cycloalkyl, aryl, and heteroaryl may each independently be substituted with one or more substituents selected from a hydrogen atom, alkyl, alkoxy, halogen, deuterium, amino, cyano, hydroxyl, mercapto, azido, nitro, carboxyl, acyl, carbonyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and preferably R １ is alkyl, and R ８ is hydrogen, R ２ is -C(O)-Q １ -Q ２ or -SO ２ -Q １ -Q ２ where Q is selected from １ is selected from an O, N, S atom or a chemical bond; Q １ When is selected from O, N, and S atoms, Q ２ is selected from alkyl, cycloalkyl, heterocyclyl, spirocyclyl, bridged cyclic group, alkenyl, aryl, and heteroaryl, and optionally said alkyl, cycloalkyl, heterocyclyl, spirocyclyl, bridged cyclic group, alkenyl, aryl, and heteroaryl are each independently substituted with one or more substituents selected from hydrogen atom, alkyl, alkoxy, halogen, deuterium, amino, cyano, hydroxyl, mercapto, azido, nitro, carboxyl, acyl, carbonyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; Q １ is a chemical bond, Q ２ is selected from alkyl, benzyl, cycloalkyl, heterocyclyl, spirocyclyl, bridged cyclic groups, alkenyl, aryl, and heteroaryl; Q ２ is selected from alkyl, said alkyl is substituted with one or more substituents selected from hydrogen, alkyl, alkoxy, halogen, deuterium, cyano, azido, nitro, carboxyl, acyl, carbonyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; Q ２ is selected from cycloalkyl, heterocyclyl, spirocyclyl, bridged ring group, alkenyl, aryl, and heteroaryl, said cycloalkyl, heterocyclyl, spirocyclyl, bridged ring group, alkenyl, aryl, and heteroaryl are each independently substituted with one or more substituents selected from hydrogen atom, alkyl, alkoxy, halogen, deuterium, amino, cyano, hydroxyl, mercapto, azido, nitro, carboxyl, acyl, carbonyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and Q ２ is cycloalkyl and the substituent is hydroxyl, amino or mercapto, the position of the substituent is selected from any position other than the carbon atom bonded to —C(O)—; A compound or its isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

34. A compound of formula XIa or XIb, or an isomer, meso form, racemate, enantiomer or mixture thereof, In the formula, R １ is selected from alkyl, preferably R １ is methyl, Q １ is selected from an O atom or a chemical bond; Q １ is an O atom, Q ２ is selected from alkyl, cycloalkyl, alkenyl, and aryl, and optionally, said alkyl, cycloalkyl, alkenyl, and aryl are each independently substituted with one or more substituents selected from hydrogen atoms, alkyl, halogen, hydroxyl, hydroxyalkyl, and aryl; Q １ When is selected from a chemical bond, Q ２ is selected from alkyl, benzyl, cycloalkyl, alkenyl, and aryl; Q ２ is selected from alkyl, said alkyl being substituted with one or more substituents selected from hydrogen, alkyl, halogen, deuterium, azido, nitro, and aryl; Q ２ is selected from cycloalkyl, alkenyl, and aryl, each of which is independently substituted with one or more substituents selected from hydrogen, alkyl, halogen, hydroxyl, hydroxyalkyl, and aryl; and Q ２ is cycloalkyl and the substituent is hydroxyl, amino or mercapto, the position of the substituent is selected from any position other than the carbon atom bonded to —C(O)—; 34. A compound of formula X according to claim 33, or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

35. 35. A compound of formula X according to claim 33 or claim 34, selected from the following structures: or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

36. Use of a linker-drug conjugate according to any one of claims 23 to 32, or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, or a compound according to any one of claims 33 to 35, or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, as an intermediate for preparing a ligand-drug conjugate according to any one of claims 1 to 22.

37. 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. A ligand-drug conjugate according to any one of claims 1 to 22, or a pharmaceutically acceptable salt or solvate thereof; a linker-drug conjugate according to any one of claims 23 to 32, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof; or a compound according to any one of claims 33 to 35, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof,

38. A pharmaceutical composition comprising the ligand-drug conjugate according to any one of claims 1 to 22, or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug conjugate according to any one of claims 23 to 32, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, or the compound according to any one of claims 33 to 35, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, and optionally further comprising a pharmaceutically acceptable carrier.

39. A pharmaceutical formulation comprising the ligand-drug conjugate according to any one of claims 1 to 22, or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug conjugate according to any one of claims 23 to 32, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, or the compound according to any one of claims 33 to 35, or an isomer, meso form, racemic form, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.

40. Use of a ligand-drug conjugate according to any one of claims 1 to 22, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug conjugate according to any one of claims 23 to 32, or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, or a compound according to any one of claims 33 to 35, or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament for treating or preventing cancer or tumor, Preferably, the cancer or tumor is a solid tumor or a hematological tumor; Preferably, the cancer or tumor is selected from adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma, leukemia, hypopharyngeal cancer, use.

41. 1. A method for preventing or treating cancer or tumors, comprising: Administering to a subject in need thereof a prophylactically or therapeutically effective amount of a ligand-drug conjugate according to any one of claims 1 to 22, or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug conjugate according to any one of claims 23 to 32, or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, or a compound according to any one of claims 33 to 35, or an isomer, meso form, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, Preferably, the cancer or tumor is a solid tumor or a hematological tumor; Preferably, the cancer or tumor is selected from adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma, leukemia, hypopharyngeal cancer, method.

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