Ligand-drug conjugate and linker for conjugation

Abstract

Provided are a ligand-drug conjugate having a novel structure and a linker for conjugation. Specifically, provided are a ligand-drug conjugate having a structural general formula of Pc-(La-D)n or Pc-(Lb-D)n, a corresponding linker for conjugation, a pharmaceutical composition containing the conjugate, and a use of the conjugate as an anti-tumor drug.

Description

Ligand-drug conjugates and their conjugated linkers

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority and related rights and interests of Chinese Patent Application No. 202310924391.5 filed on July 25, 2023, and the entire contents of the above-mentioned Chinese patent application are incorporated herein by reference. Technical Field

[0003] The present disclosure belongs to the field of biomedicine and relates to a class of ligand-drug conjugates with novel structures and their coupling linkers, a pharmaceutical composition containing the conjugates, and their use as anti-tumor drugs. Background Art

[0004] Antibody-drug conjugates (ADCs) are new targeted drugs that link monoclonal antibodies that specifically bind to tumor cell surface antigens with biologically active toxin molecules. The concept of ADC drugs was first proposed by Paul Ehrlich in 1913. After 87 years of research and exploration, the first ADC drug, Mylotarg, was launched in 2000. With the gradual deepening of ADC drug research, ADC technology has achieved many new major breakthroughs in recent years. Currently, 15 ADC drugs have been approved for marketing worldwide. Most of these ADCs are conjugated via non-site-specific conjugation, primarily through lysine residues of the antibody or cysteine ​​residues between antibody chains.

[0005] Summary of the Invention

[0006] The present disclosure provides a novel linker technology that can achieve site-specific coupling between drugs and ligands, and the ligand-drug conjugates thereof have better stability and / or anti-tumor efficacy and / or drug release efficiency and / or drug uniformity.

[0007] In a first aspect, the present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, the general structural formula of which is Pc-(L a -D) n ,

[0008] in,

[0009] Pc is the ligand unit;

[0010] L a is a connecting subunit of the following structure,

[0011] in,

[0012] L a The b end of is connected to D;

[0013] R 1 、R 2 Each independently selected from Where a end and L 1 or L 2 Connected to the other end and connected to the PC;

[0014] L 1 , L 2 , L 3 Each independently selected from C1-C6 alkylene, -C(=O)NR e - and any combination thereof;

[0015] R e Selected from H or C1-C6 alkyl, the C1-C6 alkyl optionally substituted with one or more halogen, CN, ═O, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2;

[0016] L 4 selected from cleavable units;

[0017] D is the drug unit;

[0018] n is a real number from 1 to 8.

[0019] In some embodiments, L 1 , L 2 Each is independently selected from C1-C6 alkylene.

[0020] In some embodiments, L 1 , L 2 are all selected from -CH2CH2-.

[0021] In some embodiments, L 3 Selected from -CH2- or

[0022] In some embodiments, L 4 Selected from Where W 1 selected from the group consisting of 2 to 8 amino acid residues, for example selected from the group consisting of 2 to 5 amino acid residues, which are optionally substituted by halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl, is substituted with one or more substituents.

[0023] In some embodiments, L 4 Selected from Where W 1is selected from peptide residues consisting of 2 to 5 amino acids, the peptide residues being optionally is substituted with one or more substituents.

[0024] In some embodiments, L 4 Selected from Where W 1 A peptide residue selected from Val-Cit, Gly-Gly-Phe-Gly or Gly-Gly-Gly-Phe-Gly, said peptide residue being optionally replace.

[0025] In some embodiments, L 4 Selected from Where W 1 Selected from peptide residues consisting of 2 to 8 amino acids.

[0026] In some embodiments, L 4 Selected from W 1 Selected from Val-Cit.

[0027] In some embodiments, L a Selected from

[0028] In some embodiments, the drug unit D is selected from a tubulin inhibitor, a DNA damaging agent or a topoisomerase inhibitor, wherein the tubulin inhibitor includes but is not limited to dolastatin, auristatin, maytansine, tubulysins and cryptomycins, the DNA damaging agent includes but is not limited to PBD drugs, and the topoisomerase inhibitor includes but is not limited to topoisomerase I inhibitors, such as camptothecin drugs.

[0029] In some embodiments, the Drug unit D is selected from a topoisomerase I inhibitor or an auristatin.

[0030] In some embodiments, the drug unit D is selected from camptothecin drugs, MMAE and MMAE derivatives. In some embodiments, the drug unit D is selected from

[0031] In some embodiments, the drug unit D is selected from

[0032] In some embodiments, the drug unit D is selected from auristatins.

[0033] In some embodiments, the Drug unit D is selected from MMAE and MMAE derivatives.

[0034] In some embodiments, D is selected from

[0035] In some embodiments, the present disclosure has the general formula Pc-(L a -D) n The ligand-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following compounds of formula (Ia) or a pharmaceutically acceptable salt thereof:

[0036] Pc, R 1 、R 2 、L 1 、L 2 、L 3 、L 4 , D, n are as defined above.

[0037] In some embodiments, the present disclosure is of the formula Pc-(L a -D) n The ligand-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0038] In some embodiments, the Ligand unit Pc can be selected from a polypeptide, an antibody, or an antigen-binding fragment thereof.

[0039] In some embodiments, the Ligand unit Pc can specifically bind to one or more antigens selected from the group consisting of HER2 (ErbB2), p95HER2, HER3 (ErbB3), CD3, CD16, DLL3, CDH6, CDH11, CDH17, CD5, CD20, BCMA, EGFR, VEGF, Trop-2, Claudin6, Claudin18.2, c-MET, CD30, MUC1, Nectin-4, CD22, CD74, CD19, CD79b, MSLN, TOPO2, EpCAM, CEACAM-5, Mesothelin, PD-L1, PSMA, ROR1, ROR2, CD25, CD33, CD70, CD123, FLT3, CD174, CD166, CD326, CD71, LIV-1, MUC16, MUC17, ENPP3, TDGF1, ETBR, TIM1, TIM3, LRRC15, CanAg / AFP, FAP, SLITRK6, KIT / CD117 , STEAP1, SLAMF7 / CS1, NaPi2B / SLC34A2, GPNMB, AXL, ALPP / ALPPL2, ADAM9, B7-H3(CD276), B7H4, PTK7 / CCK4, PRLR, EFNA4, 5T4, LRG5, NOTCH3, CD142, CA6, GPR20, GPC-1, Eph A2, LYPD3, ITGB6, FGFR2, FGFR3, FRα, CEACAMs, GUCY2C, SEZ6, IntegrinAv, CAIX, P-cadherin, GD3, cadherin6, LAMP1, KLK2, PD-L1, CD56, CD37, CD47, CD138, CD228 or CD352.

[0040] In some embodiments, the ligand unit Pc can specifically bind to one or more antigens selected from the group consisting of HER2, p95HER2, HER3, CD3, CD16, ROR1, DLL3, CDH6, CD70, CD5, CD20, BCMA, EGFR, VEGF, Trop-2, Claudin18.2, c-MET, CD30, MUC1, Nectin-4, CD22, FR-a, CD74, CD19, CD79b, MSLN, TOPO2, EpCAM, CEACAM-5, Mesothelin, PD-L1, PSMA, ROR1, ROR2, CD25, CD33, CD123, FLT3, CD174, CD166, CD326, CD71, and LIV-1.

[0041] In some embodiments, the Ligand unit Pc can specifically bind to one or more antigens selected from the group consisting of HER2, p95HER2, ROR1, CDH6, and LIV-1.

[0042] In some embodiments, the ligand unit Pc is an antibody or antigen-binding fragment thereof that specifically binds to HER2, CDH6 or LIV-1; the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) or / and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, or / and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the HCDR1-3 and / or the LCDR1-3 are selected from the following:

[0043] (1) the HCDR1-3 are SEQ ID NOs: 14-16; or / and the LCDR1-3 are SEQ ID NOs: 17-19;

[0044] (2) the HCDR1-3 are SEQ ID NOs: 20-22; or / and the LCDR1-3 are SEQ ID NOs: 23-25;

[0045] (3) the HCDR1-3 are SEQ ID NOs: 26-28; or / and the LCDR1-3 are SEQ ID NOs: 29-31;

[0046] (4) the HCDR1-3 are SEQ ID NOs: 34-36; or / and the LCDR1-3 are SEQ ID NOs: 37-39;

[0047] (5) the HCDR1-3 are SEQ ID NOs: 40-42; or / and the LCDR1-3 are SEQ ID NOs: 43-45;

[0048] or,

[0049] (6) The HCDR1-3 and / or the LCDR1-3 have an amino acid sequence that is at least 80% identical to the corresponding CDR in any one of the HCDR1-3 and / or LCDR1-3 in groups (1) to (5), or has at most three insertion, deletion or substitution mutations; preferably, the HCDR1-3 and / or the LCDR1-3 have an amino acid sequence that is at least 80% identical to the corresponding CDR in any one of the HCDR1-3 and / or LCDR1-3 in groups (1) to (5); more preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0050] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the heavy chain variable region and / or the light chain variable region are selected from the following:

[0051] (1) the heavy chain variable region is the sequence shown in SEQ ID NO: 1, or / and the light chain variable region is the sequence shown in SEQ ID NO: 2;

[0052] (2) the heavy chain variable region is the sequence shown in SEQ ID NO: 3, or / and the light chain variable region is the sequence shown in SEQ ID NO: 4;

[0053] (3) the heavy chain variable region is the sequence shown in SEQ ID NO: 7, or / and the light chain variable region is the sequence shown in SEQ ID NO: 8;

[0054] (4) the heavy chain variable region is the sequence shown in SEQ ID NO: 9, or / and the light chain variable region is the sequence shown in SEQ ID NO: 10;

[0055] (5) the heavy chain variable region is the sequence shown in SEQ ID NO: 11, and / or the light chain variable region is the sequence shown in SEQ ID NO: 12; or,

[0056] The heavy chain variable region and / or the light chain variable region have a sequence that is at least 80% identical to the heavy chain variable region and / or the light chain variable region in any one of the above groups (1) to (5), or a sequence in which at most three insertion, deletion or substitution mutations occur; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0057] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region sequence and / or a light chain constant region sequence, optionally, the heavy chain constant region and / or the light chain constant region are selected from a complete constant region sequence or a fragment thereof, the constant region fragment comprising CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region is selected from human or mouse kappa constant region or lamda constant region; optionally, the antibody or antigen-binding fragment comprises a complete heavy chain and a light chain, the heavy chain is composed of the VH and the heavy chain constant region, the heavy chain constant region has the sequence shown in SEQ ID NO: 5 or 13, and the light chain is composed of the VL and the light chain constant region, the light chain constant region has the sequence shown in SEQ ID NO: 6.

[0058] In some embodiments, the antibody or antigen-binding fragment is:

[0059] (1) Chimeric antibodies or fragments thereof;

[0060] (2) humanized antibodies or fragments thereof; and / or,

[0061] (3) fully human antibodies or fragments thereof;

[0062] Preferably, the antibody or antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, a diabody or a single domain antibody.

[0063] In some embodiments, the antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody and antibody minimal recognition unit.

[0064] In some embodiments, n is selected from a real number of 1-6, for example, n is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0; preferably, n is selected from a real number of 1-5, more preferably, n is selected from a real number of 2-4.

[0065] In the second aspect, the present disclosure provides a formula (L a ) is used to connect the ligand unit and the drug unit to obtain a ligand-drug conjugate,

[0066] Among them, R 1 、R 2 、L 1 、L 2 、L 3 、L 4 As defined in any one of the first aspects above, L a The b-end is connected to the drug unit, R 1 、R 2 The end is connected to the ligand unit.

[0067] In a third aspect, the present disclosure provides a drug-linker compound represented by formula (IIa) or a pharmaceutically acceptable salt thereof,

[0068] Among them, L 1 、L 2 、L 3 、L 4 , D is as defined in any one of the first aspects above.

[0069] In some embodiments, the drug-linker compound represented by formula (IIa) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0070] In a fourth aspect, the present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, the general structural formula of which is Pc-(L b -D) n ,

[0071] in,

[0072] Pc is the ligand unit;

[0073] L b is a connecting subunit of the following structure,

[0074] in,

[0075] L b The b end of is connected to D;

[0076] R 3 、R 4 Each independently selected from Where a end and R 5 or R 6 The connected secondary carbon atoms are connected, and the other end is connected to Pc;

[0077] R 5 、R 6 Each is independently selected from C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl, wherein the C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted with one or more halogen, CN, ═O, C1-C3 alkyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2;

[0078] L 7 、L 8 、L 9 Each is independently selected from C1-C6 alkylene, the C1-C6 alkylene being optionally substituted with one or more halogen, CN, ═O, C1-C3 alkyl, OH, NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2;

[0079] L 10 is a cleavable unit;

[0080] D is the drug unit;

[0081] n is a real number from 1 to 8.

[0082] In some embodiments, R 5 、R 6 Each is independently selected from C1-C3 alkyl, said C1-C3 alkyl being optionally substituted with one or more halogen, CN, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2.

[0083] In some embodiments, R 5 、R 6 are all selected from CH2NH2.

[0084] In some embodiments, L 7、L 8 、L 9 Each is independently selected from C1-C6 alkylene.

[0085] In some embodiments, L 7 、L 8 All selected from CH2, L 9 Selected from CH2CH2.

[0086] In some embodiments, L 10 Selected from Where W 2 Selected from peptide residues consisting of 2 to 8 amino acids.

[0087] In some embodiments, L 10 Selected from Where W 2 Selected from Val-Cit.

[0088] In some embodiments, L b Selected from

[0089] In some embodiments, D is selected from the class of auristatins.

[0090] In some embodiments, D is selected from MMAE and MMAE derivatives.

[0091] In some embodiments, D is selected from

[0092] In some embodiments, the present disclosure is of the formula Pc-(L b -D) n The ligand-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following compounds of formula (Ib) or a pharmaceutically acceptable salt thereof:

[0093] Among them, Pc, R 3 、R 4 、R 5 、R 6 、L 7 、L 8 、L 9 、L 10 , D, n are as defined above.

[0094] In some embodiments, the present disclosure is of the formula Pc-(L b -D) n The ligand-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0095] In some embodiments, the Ligand unit Pc can be selected from a polypeptide, an antibody, or an antigen-binding fragment thereof.

[0096] In some embodiments, the ligand unit Pc can specifically bind to one or more antigens selected from the group consisting of HER2, p95HER2, HER3, CD3, CD16, ROR1, DLL3, CDH6, CD70, CD5, CD20, BCMA, EGFR, VEGF, Trop-2, Claudin18.2, c-MET, CD30, MUC1, Nectin-4, CD22, FR-a, CD74, CD19, CD79b, MSLN, TOPO2, EpCAM, CEACAM-5, Mesothelin, PD-L1, PSMA, ROR1, ROR2, CD25, CD33, CD123, FLT3, CD174, CD166, CD326, CD71, and LIV-1.

[0097] In some embodiments, the Ligand unit Pc can specifically bind to one or more antigens selected from the group consisting of HER2, p95HER2, ROR1, CDH6, and LIV-1.

[0098] In some embodiments, the Pc is an antibody or antigen-binding fragment thereof that specifically binds to HER2, CDH6 or LIV-1; the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) or / and a light chain variable region (VL), the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, or / and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the HCDR1-3 and / or the LCDR1-3 are selected from the following:

[0099] (1) the HCDR1-3 are SEQ ID NOs: 14-16; or / and the LCDR1-3 are SEQ ID NOs: 17-19;

[0100] (2) the HCDR1-3 are SEQ ID NOs: 20-22; or / and the LCDR1-3 are SEQ ID NOs: 23-25;

[0101] (3) the HCDR1-3 are SEQ ID NOs: 26-28; or / and the LCDR1-3 are SEQ ID NOs: 29-31;

[0102] (4) the HCDR1-3 are SEQ ID NOs: 34-36; or / and the LCDR1-3 are SEQ ID NOs: 37-39;

[0103] (5) the HCDR1-3 are SEQ ID NOs: 40-42; or / and the LCDR1-3 are SEQ ID NOs: 43-45;

[0104] or,

[0105] (6) The HCDR1-3 and / or the LCDR1-3 have an amino acid sequence that is at least 80% identical to the corresponding CDR in any one of the HCDR1-3 and / or LCDR1-3 in groups (1) to (5), or has at most three insertion, deletion or substitution mutations; preferably, the HCDR1-3 and / or the LCDR1-3 have an amino acid sequence that is at least 80% identical to the corresponding CDR in any one of the HCDR1-3 and / or LCDR1-3 in groups (1) to (5); more preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0106] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the heavy chain variable region and / or the light chain variable region are selected from the following:

[0107] (1) the heavy chain variable region is the sequence shown in SEQ ID NO: 1, or / and the light chain variable region is the sequence shown in SEQ ID NO: 2;

[0108] (2) the heavy chain variable region is the sequence shown in SEQ ID NO: 3, or / and the light chain variable region is the sequence shown in SEQ ID NO: 4;

[0109] (3) the heavy chain variable region is the sequence shown in SEQ ID NO: 7, or / and the light chain variable region is the sequence shown in SEQ ID NO: 8;

[0110] (4) the heavy chain variable region is the sequence shown in SEQ ID NO: 9, or / and the light chain variable region is the sequence shown in SEQ ID NO: 10;

[0111] (5) the heavy chain variable region is the sequence shown in SEQ ID NO: 11, and / or the light chain variable region is the sequence shown in SEQ ID NO: 12;

[0112] or,

[0113] The heavy chain variable region and / or the light chain variable region have a sequence that is at least 80% identical to the heavy chain variable region and / or the light chain variable region in any one of the above groups (1) to (5), or a sequence in which at most three insertion, deletion or substitution mutations occur; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0114] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region sequence and / or a light chain constant region sequence, optionally, the heavy chain constant region and / or the light chain constant region are selected from a complete constant region sequence or a fragment thereof, and the constant region fragment comprises CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region is selected from human or mouse kappa constant region or lamda constant region; optionally, the antibody or antigen-binding fragment comprises a complete heavy chain and a light chain, the heavy chain is composed of the VH and the heavy chain constant region, and the heavy chain constant region has the sequence shown in SEQ ID NO: 5 or 13, and the light chain is composed of the VL and the light chain constant region, and the light chain constant region has the sequence shown in SEQ ID NO: 6.

[0115] In some embodiments, the antibody or antigen-binding fragment is:

[0116] (1) Chimeric antibodies or fragments thereof;

[0117] (2) humanized antibodies or fragments thereof; and / or,

[0118] (3) fully human antibodies or fragments thereof;

[0119] Preferably, the antibody or antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, a diabody or a single domain antibody.

[0120] In some embodiments, the antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody and antibody minimal recognition unit.

[0121] In some embodiments, n is selected from a real number of 1-6, for example, n is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0; preferably, n is selected from a real number of 1-5, more preferably, n is selected from a real number of 2-4.

[0122] In a fifth aspect, the present disclosure provides a formula (L b ) is used to connect the ligand unit and the drug unit to obtain a ligand-drug conjugate,

[0123] Among them, R 3 、R 4 、R 5 、R 6 、L 7 、L 8 、L 9 、L 10 As defined in any one of the fourth aspects above, L b The b-end is connected to the drug unit, R 3 、R 4 The end is connected to the ligand unit.

[0124] In a sixth aspect, the present disclosure provides a drug-linker compound represented by formula (IIb) or a pharmaceutically acceptable salt thereof,

[0125] Among them, R 5 、R 6 、L 7 、L 8 、L 9 、L 10 , D is as defined in any one of the fourth aspects above.

[0126] In some embodiments, the drug-linker compound represented by formula (IIb) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0127] On the other hand, the present disclosure provides a pharmaceutical composition comprising the aforementioned general formula of the present disclosure Pc-(L a -D) n or Pc-(Lb -D) n ligand-drug conjugate or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0128] On the other hand, the present disclosure provides a method for treating mammalian tumors, comprising administering to a mammal in need of such treatment, preferably a human, a therapeutically effective amount of the aforementioned compound of the general formula Pc-(L a -D) n or Pc-(L b -D) n The invention relates to a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the ligand-drug conjugate or a pharmaceutically acceptable salt thereof.

[0129] On the other hand, the present disclosure provides the aforementioned general formula Pc-(L a -D) n or Pc-(L b -D) n Use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the ligand-drug conjugate or a pharmaceutically acceptable salt thereof, in preparing a drug for treating tumors.

[0130] On the other hand, the present disclosure provides the aforementioned general formula Pc-(L a -D) n or Pc-(L b -D) n Use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the ligand-drug conjugate or a pharmaceutically acceptable salt thereof in treating tumors.

[0131] On the other hand, the present disclosure provides the aforementioned general formula Pc-(L a -D) n or Pc-(L b -D) n The invention relates to a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the ligand-drug conjugate or a pharmaceutically acceptable salt thereof.

[0132] In another aspect, the present disclosure provides a a -D) n or Pc-(L b -D) n A method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof comprises the step of coupling the drug-linker compound of the aforementioned general formula (IIa) or (IIb) disclosed herein with the aforementioned ligand, optionally, the ligand is an antibody or an antigen-binding fragment thereof.

[0133] In another aspect, the present disclosure provides a a -D) n or Pc-(L b -D) n The preparation method of the ligand-drug conjugate or a pharmaceutically acceptable salt thereof comprises the steps of connecting the aforementioned drug unit D of the present disclosure with the aforementioned ligand unit Pc; optionally, through the aforementioned linker unit L a or L b Connection; optionally, the ligand is an antibody or an antigen-binding fragment thereof.

[0134] The ligand-drug conjugates or pharmaceutically acceptable salts thereof provided by the present disclosure have significant anti-tumor activity and / or reduced toxic side effects and / or improved product uniformity. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] FIG1 is an overlay of HIC-HPLC chromatograms of naked antibody and ADC-10 in Example 6.

[0136] FIG2 is a graph showing the efficacy of the antibody-drug conjugate in Example 10 in the NCI-H838 subcutaneous tumor model.

[0137] Definitions and Explanations of Terms

[0138] Unless otherwise indicated, the terms used in this disclosure have the following meanings. The definitions of groups and terms described in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions described in tables, and definitions of specific compounds in the examples, may be combined and coupled with each other in any manner. A particular term should not be considered as undefined or unclear unless specifically defined, but should be understood according to its ordinary meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0139] The term "ligand" refers to a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell. The role of the ligand is to present the drug to the target cell population bound to the ligand. These ligands include but are not limited to protein hormones, lectins, growth factors, antibodies or other molecules that can bind to cells. In the embodiments of the present disclosure, the ligand or ligand unit is represented by Pc, and the ligand can form a connection bond with the linker unit through a heteroatom on the ligand. In some embodiments of the present disclosure, the ligand unit Pc may include a heteroatom group (such as an S atom or an N atom) on the ligand for connection to the linker unit, or may not include the above-mentioned heteroatom group for connection to the linker unit. In some embodiments of the present disclosure, the ligand is selected from an antibody or an antigen-binding fragment, and the antibody is selected from a chimeric antibody, a humanized antibody, a fully human antibody or a mouse antibody; in some embodiments of the present disclosure, the antibody is a monoclonal antibody.

[0140] The term "linker" or "linker unit" refers to a chemical structure fragment or chemical bond that is connected to the ligand at one end and to the drug at the other end.

[0141] The term "drug" refers to a small molecule compound that has biological activity in an organism. In some embodiments of the present disclosure, the drug is a glucocorticoid receptor agonist or its corresponding phosphate ester molecule with anti-inflammatory function.

[0142] The term "ligand-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker unit. In some embodiments of the present disclosure, the "ligand-drug conjugate" is an antibody-drug conjugate (ADC), which is a monoclonal antibody or antibody fragment linked to a biologically active drug via a stable linker unit.

[0143] The term "DAR" or "drug-to-antibody ratio" refers to the average number of small molecule glucocorticoid receptor agonist drugs attached to each antibody molecule. In the antibody-drug conjugates of the present disclosure, DAR is defined by the variable "n," which can be either an integer or a decimal.

[0144] The term "antibody" is used in the broadest sense to refer to a polypeptide or combination of polypeptides that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0145] The term "antibody" herein includes a typical "tetrabodies," which are immunoglobulins composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain consisting, from the N-terminus to the C-terminus, of a heavy chain variable region (VH), a heavy chain constant region CHI domain, a hinge region (HR), a heavy chain constant region C2 domain, and a heavy chain constant region C3 domain. Furthermore, when the full-length antibody is of the IgE isotype, it optionally includes a heavy chain constant region C4 domain. The light chain is a polypeptide chain consisting, from the N-terminus to the C-terminus, of a light chain variable region (VL) and a light chain constant region (CL). Heavy chains are linked to each other and to each other through disulfide bonds, forming a "Y"-shaped structure. Due to the different amino acid composition and arrangement order of the constant regions of the heavy chains of immunoglobulins, their antigenicity also varies. Based on this, the "immunoglobulins" referred to herein can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE. Their corresponding heavy chains are μ, δ, γ, α, and ε, respectively. Igs of the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0146] The "antibodies" herein also include antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanacos (Lama guanicoe) and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0147] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0148] "Antibodies" herein include, but are not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, whole antibodies, fragments of whole antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0149] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variants (e.g., containing naturally occurring mutations or generated during the production of the preparation, such variants typically being present in small amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" herein should not be interpreted as requiring the antibody or antigen-binding molecule to be produced by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including but not limited to hybridoma technology, recombinant DNA methods, phage library display technology, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus and other methods known in the art.

[0150] The term "natural antibody" refers to an antibody produced and paired by the immune system of a multicellular organism. The term "engineered antibody" herein refers to a non-natural antibody obtained by techniques such as genetic engineering and antibody engineering. For example, "engineered antibodies" include humanized antibodies, small molecule antibodies (e.g., scFv), bispecific antibodies, and the like.

[0151] The term "HER2," also known as "human epidermal growth factor receptor 2," is a cellular oncogene and a transmembrane glycoprotein with tyrosine kinase activity that activates intracellular tyrosine kinase-regulated signal transduction systems. HER2 gene copy number amplification and protein overexpression lead to overactivation of signaling, a biological function closely associated with the development, malignant proliferation, invasion and metastasis, and resistance to chemotherapy and radiotherapy in various cancers. Both the oncogene and its protein product (P185) are overexpressed and amplified in a variety of tumors. Positive expression of HER2 has been observed in various cancers, including breast, ovarian, gastric, esophageal, salivary gland, lung, bile duct, bladder, prostate, and colorectal cancers.

[0152] The term "CDH6" herein is also known as "cadherin 6", which refers to a cell adhesion molecule, which is a member of the cadherin family of cell-cell adhesion molecules. CDH6 is a single transmembrane protein consisting of 790 amino acids, which is classified as a type II cadherin family, and the protein has an N-terminal extracellular and C-terminal intracellular domain. The CDH6 protein used in the present invention can be directly purified from cells expressing CDH6 of humans or non-human mammals (such as rats, mice or monkeys), and can be used subsequently, or the cell membrane fraction of the above-mentioned cells can be prepared and can be used as CDH6 protein. Alternatively, CDH6 can also be obtained by allowing host cells to produce CDH6 by in vitro synthesis or by genetic manipulation. According to this genetic manipulation, CDH6 protein can be obtained, specifically, CDH6 cDNA is incorporated into a vector capable of expressing CDH6 cDNA, and then CDH6 is synthesized in a solution containing enzymes, substrates and energy materials required for transcription and translation, or by transforming host cells of other prokaryotes or eukaryotes, thereby allowing them to express CDH6. CDH6-expressing cells or CDH6-expressing cell lines based on the above genetic manipulations can also be used to present CDH6 protein. Alternatively, an expression vector incorporating CDH6 cDNA can be directly administered to an animal to be immunized, and CDH6 can be expressed in the animal thus immunized.

[0153] The term "LIV-1" herein, also referred to as Zip6, refers to a zinc transporter belonging to the subfamily of ZIP zinc transporters known as LZTs. In GeneCard, LIV-1 is also referred to as SLC39A6 (solute carrier protein family 39 (zinc transporter), member 6). Although for the sake of brevity, the present invention is primarily exemplified by LIV-1, it should be understood that the definitions and embodiments relating to LIV-1 are also applicable to the other zinc transporters disclosed herein.

[0154] The term "monospecific" is intended to mean having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.

[0155] The term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody / antigen-binding molecule can bind.

[0156] The term "valent" refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0157] "Full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.

[0158] "Antigen-binding fragment" and "antibody fragment" herein are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH, and scFv.

[0159] Papain digestion of intact antibodies generates two identical antigen-binding fragments, called "Fab" fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising a light chain fragment comprising the VL domain and constant domain (CL) of the light chain, and a VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domain carry a free thiol group. Pepsin treatment produces a F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0160] "Fv fragment" is the smallest fragment produced by IgG and IgM that contains a complete antigen binding site. The Fv fragment has the same binding properties and similar three-dimensional binding properties as Fab. The VH and VL chains of the Fv fragment are bound together by non-covalent interactions.

[0161] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of the scFv to form a disulfide-linked Fv (dsFv).

[0162] The term "diabody" refers to a antibody whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0163] The term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0164] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., FR and / or constant region in the variable region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0165] The term "fully human antibody" refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. "fully human antibody" herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibody" herein does not include antibodies in which the CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0166] The term "variable region" refers to the region of an antibody heavy or light chain that binds to the antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or light chain variable region (VL). This region is also called the complementarity determining region because it can form precise complementarity with the antigen epitope in terms of spatial structure. The heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR. The terms "framework region" or "FR region" are used interchangeably herein and refer to those amino acid residues in the heavy chain variable region or light chain variable region of an antibody other than the CDR. Typically, a typical antibody variable region consists of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0167] The "CDRs" herein can be annotated and defined by methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and the tool websites used include but are not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). The CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0168] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in antibody-antigen binding but exhibits effector functions, such as interactions with Fc receptors. It has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant regions" include "full-length heavy chain constant regions" and "heavy chain constant region fragments." The former has a structure substantially similar to that of a native antibody constant region, while the latter only comprises "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. For example, a typical "heavy chain constant region fragment" can be selected from the CH1, Fc, or CH3 domains.

[0169] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0170] The term "Fc" herein refers to the antibody carboxyl terminal portion formed by papain hydrolysis of intact antibodies, typically comprising the CH3 and CH2 domains of an antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at Cys226 position or from Pro230 to its carboxyl terminal. The C-terminal lysine (residue 447 according to the Kabat numbering system) in the Fc region can, for example, be present in the production or purification process of an antibody, or by removing the nucleic acid recombinant engineering of an encoding antibody heavy chain. Therefore, the Fc region may include or may not include Lys447.

[0171] The term "identity" herein can be calculated in the following manner: to determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0172] The percent identity between the two sequences will vary depending on the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0173] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at www.gcg.com), is used with a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program in the GCG software package (available at www.gcg.com) is used with a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0174] Herein, “n is a real number of 1-8” means that n is any real number greater than or equal to 1 and less than or equal to 8.

[0175] In this article Indicates the attachment site.

[0176] The diagrammatic representations of racemates or enantiomerically pure compounds herein are from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, wedge and dotted wedge keys are used. To indicate the absolute configuration of a stereocenter, use black real and imaginary bonds. Indicates the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

[0177] The term "tautomer" refers to functional group isomers resulting from the rapid shift of an atom in a molecule between two positions. The compounds of the present disclosure may exhibit tautomerism. Tautomeric compounds may exist as two or more interconvertible species. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually result in a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates, while in phenols, the enol form predominates. The present disclosure encompasses all tautomeric forms of the compounds.

[0178] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0179] The compounds of the present invention may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so that the compounds of the present invention may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the definition of the compounds of the present invention and mixtures thereof. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms may be present in substituents such as alkyl groups, and all of which are within the definition of the compounds of the present invention and mixtures thereof. Compounds of the present disclosure containing an asymmetric atom can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or reagents.

[0180] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a particular atom by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo (i.e., =O), it means that two hydrogen atoms are replaced. Oxo does not occur on aromatic groups.

[0181] The term "optional" or "optionally" refers to that the event or situation described subsequently may or may not occur, and the description includes that the event or situation occurs and that the event or situation does not occur. For example, an ethyl group is "optionally" substituted with halogen, meaning that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, any sterically impossible and / or incomposable replacement or substitution pattern will not be introduced.

[0182] When any variable (such as R a 、R b ) appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, if a group is represented by two R bis replaced, then each R b There are independent options.

[0183] When the number of a linking group is 0, such as -(CH2)0-, it means that the linking group is a chemical bond.

[0184] When one of the variables is selected from a chemical bond or does not exist, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a bond, it means that the structure is actually AZ.

[0185] When the linking group mentioned in this article does not specify its connection direction, its connection direction is arbitrary. When X is selected from "C1-C3 alkylene-O", X can connect ring A and ring B from left to right to form "ring A-C1-C3 alkylene-O-ring B", or can connect ring A and ring B from right to left to form "ring A0-C1-C3 alkylene-ring B".

[0186] In this article, C m -C n , means having an integer number of carbon atoms in the range of mn.

[0187] The term "alkyl" refers to a group of the formula C n H 2n+1 The term "C1-C6 alkyl" is understood to mean a straight-chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl group includes, but is not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.; the term "C1-C3 alkyl" refers to an alkyl group containing 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl.

[0188] The "C1-C6 alkyl group" described herein may further include a "C1-C3 alkyl group".

[0189] The term "cycloalkyl" refers to a fully saturated carbocyclic ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. The term "C3-C6 cycloalkyl" should be understood to mean a saturated monocyclic, fused, spirocyclic, or bridged ring having 3 to 6 carbon atoms. Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0190] The term "heterocyclyl" refers to a fully saturated or partially saturated monocyclic, fused, spiro or bridged ring group, which contains 1 to 5 heteroatoms or heteroatom groups (i.e., heteroatom-containing atomic groups) in its ring atoms, wherein the "heteroatoms or heteroatom groups" include, but are not limited to, nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH- or -NHC(=O)NH-, etc. The term "4-7 membered heterocyclyl" refers to a heterocyclyl group having 4, 5, 6 or 7 ring atoms, and containing 1 to 3 heteroatoms or heteroatom groups independently selected from the above-mentioned heteroatoms or heteroatom groups in its ring atoms. Examples of 4-membered heterocyclic groups include, but are not limited to, azetidinyl and oxetanyl; examples of 5-membered heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazole, or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; examples of 7-membered heterocyclic groups include, but are not limited to, diazepanyl. Preferably, "4-7-membered heterocyclic group" may include "4-7-membered heterocycloalkyl," "5-6-membered heterocyclic group," "5-6-membered heterocycloalkyl," and the like.

[0191] The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0192] The term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as the progression of cancer, autoimmune diseases and viral infections. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, weakening of the disease extent, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Objects in need of treatment include objects already suffering from a disease or disease, objects susceptible to a disease or disease, or objects intended to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, alleviating, etc., their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0193] The term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0194] The term "subject" refers to an organism that is being treated for a particular disease or condition as described herein. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals, being treated for a disease or condition.

[0195] The amount of a compound of the disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art based on their own knowledge and this disclosure.

[0196] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0197] The term "pharmaceutically acceptable salt" refers to a salt of a pharmaceutically acceptable acid or base, including a salt formed between a compound and an inorganic acid or organic acid, and a salt formed between a compound and an inorganic base or an organic base.

[0198] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or their salts and a pharmaceutically acceptable excipient. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present disclosure to an organism.

[0199] The term "pharmaceutically acceptable excipient" refers to an excipient that is non-irritating to organisms and does not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0200] The word "comprise" or "comprises" and its English variations such as comprises or comprising are to be understood as having an open and non-exclusive meaning, ie, "including but not limited to".

[0201] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0202] Certain isotopically labeled compounds of the present disclosure (e.g., 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron emitting isotopes, such as 15 O. 13 N. 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0203] The pharmaceutical compositions of the present disclosure may be suitable for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in suitable unit dosage forms. For example, the pharmaceutical compositions of the present disclosure may be in the form of sterile aqueous injection solutions for intramuscular or subcutaneous administration. The pharmaceutical compositions of the present disclosure may be administered in other solvents or media, such as water, Ringer's solution, or isotonic sodium chloride solution.

[0204] In all administration methods of the compounds described herein, the daily dosage is 0.001 mg / kg to 600 mg / kg body weight, preferably 0.05 mg / kg to 200 mg / kg body weight, more preferably 0.1 mg / kg to 100 mg / kg body weight, in single or divided doses.

[0205] The compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining the same with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples disclosed herein.

[0206] The chemical reactions of the embodiments of the present disclosure are carried out in a suitable solvent that is compatible with the chemical transformations of the present disclosure and the reagents and materials required. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0207] An important consideration in synthetic route planning in the art is the selection of appropriate protecting groups for reactive functional groups (e.g., amino and carboxyl groups in the present disclosure). For example, reference may be made to Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in the present disclosure are hereby incorporated into the present disclosure in their entirety.

[0208] The present invention uses the following abbreviations: DIPEA or DIEA represents diisopropylethylamine; DMF represents N,N-dimethylformamide; TFA represents trifluoroacetic acid; HATU represents O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DMA represents N,N-dimethylacetamide. DETAILED DESCRIPTION

[0209] The present disclosure is described in detail below by way of examples, but is not intended to limit the present disclosure in any way. While the present disclosure has been described in detail herein, including specific embodiments thereof, it will be apparent to those skilled in the art that various modifications can be made to the specific embodiments without departing from the spirit and scope of the present disclosure. All reagents used in the present disclosure are commercially available and used without further purification.

[0210] Unless otherwise specified, the ratios expressed for mixed solvents are volume ratios.

[0211] Unless otherwise stated, % refers to wt%.

[0212] Compounds are manually or The software named the commercially available compounds using the supplier's catalog name.

[0213] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The units of NMR shifts are 10 -6 (ppm). The solvents for NMR determination are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS); "IC 50 " refers to the half-maximal inhibitory concentration, which is the concentration at which half of the maximum inhibitory effect is achieved. 50 "half maximal effect concentration" refers to the concentration that causes 50% of the maximum effect.

[0214] Example 1: 4-((2S,5S,16S)-17-amino-12-(((S)-3-amino-2-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)propionylamino)methyl)-16-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-4,7,10,15-tetracarbonyl-2-(3-ureidopropyl)-3,6,11,14-tetraazaheptadecanoylamino)benzyl ((S)-1-(((S)-1 -(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-carbonylpropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-carbonylheptan-4-yl)(methyl)amino)-3-methyl-1-carbonylbutan-2-yl)amino)-3-methyl-1-carbonylbutan-2-yl)(methyl)carbamate (Drug-Linker 1)

[0215] Step 1: Synthesis of intermediate 1-2

[0216] Dihydrofuran-2,5-dione (160 mg) was added to a DMF solution of starting material 1-1 (1.5 g) and DIEA (2.2 mL) at room temperature. After stirring for 30 minutes, TFA was added to adjust the pH to 7. The residue was purified by HPLC (column model: YMC-Actus Triart C18 column, 5 μm silica, 30 mm diameter, 150 mm length; eluent: decreasingly polar mixtures of water (containing 0.1% TFA) and acetonitrile; acetonitrile gradient: 50% over 10 minutes) to afford the title compound (1.4 g).

[0217] MS m / z(ESI):[M+H] + =1223.7.

[0218] Step 2: Synthesis of Intermediates 1-4

[0219] A solution of intermediate 1-2 (550 mg) and HATU (205.2 mg) in DMF (6 mL) was stirred at 0°C for 10 minutes, and a solution of DIEA (174 mg) and intermediate 1-3 (195 mg) in DMF was slowly added. The mixture was stirred at room temperature for 30 minutes, and the residue was purified by HPLC (column model: YMC-Actus Triart C18 column 5 μm silica, 30 mm diameter, 150 mm length; decreasingly polar mixtures of water (containing 0.1% TFA) and acetonitrile as eluent; acetonitrile gradient ratio 65-70%, elution time 16 minutes) to give the title compound (0.4 g).

[0220] MS m / z(ESI):1494.9[M+H] + .

[0221] Step 3 Synthesis of Intermediate 1-5

[0222] A solution of intermediate 1-4 (0.2 g) in dichloromethane (1 mL) was cooled to 0°C, trifluoroacetic acid (TFA, 1 mL) was added, and the mixture was stirred at room temperature for 20 minutes. After completion of the reaction, the reaction solution was purified by preparative HPLC (column model: YMC-Actus Triart C18 column 5 μm silica, 30 mm diameter, 150 mm length) using decreasingly polar mixtures of water (containing 0.1% TFA) and acetonitrile as eluents; acetonitrile gradient ratio 50-60%, elution time 10 minutes) to give the title compound (160 mg).

[0223] MS m / z(ESI):1294.8[M+H] + .

[0224] Step 4: Synthesis of Intermediate 1-7

[0225] A DMF solution of Intermediate 1-5 (0.16 g) and HATU (95.8 mg) was stirred at 0°C for 10 minutes. DIEA (0.3 mL) and a DMF solution of Intermediate 1-6 (72 mg) were then added. The mixture was stirred at room temperature for 1 hour. The reaction solution was purified by preparative HPLC (column model: YMC-Actus Triart C18 column, 5 μm silica, 30 mm diameter, 150 mm length; eluent: decreasingly polar mixtures of water (containing 0.1% TFA) and acetonitrile; acetonitrile gradient from 50% to 60% over 10 minutes) to afford the title compound (90 mg).

[0226] MS m / z(ESI):1827.1[M+H]+ .

[0227] Step 5: Synthesis of Drug-Linker 1

[0228] To a solution of intermediate 1-7 (90 mg) in dichloromethane (1 mL) was added TFA (1 mL) at 0°C and stirred for 1 h. The reaction mixture was purified by preparative HPLC (Ultimate XB-C18 column, 10 μm, 50 mm diameter, 250 mm length; eluent: decreasingly polar mixtures of water (containing 0.1% TFA) and acetonitrile; acetonitrile gradient 25-55% over 10 minutes) to afford the title compound (35 mg).

[0229] MS m / z(ESI):1626.9[M+H] + .

[0230] 1 H NMR(400MHz,DMSO)δ9.99(s,1H),8.41-8.06(m,5H),8.02-7.89(m,8H),7.70-7.45(m,4H),7.3 8-7.22(m,7H),7.20-7.13(m,5H),6.10-6.00(m,1H),5.53-5.39(m,2H),5.13-4.90(m,2H),4.8 1-4.78(m,3H),4.51-4.20(m,7H),4.08-3.72(m,5H),3.23-2.80(m,20H),2.48-2.23(m,4H),2 .20-1.96(m,4H),1.90-1.70(m,4H),1.60-1.25(m,6H),1.10-0.97(m,7H),0.90-0.76(m,24H).

[0231] Synthesis of intermediates 1-9 and 1-6

[0232] Synthesis of (S,Z)-4-((2-((tert-butoxycarbonyl)amino)-1-carboxyethyl)amino)-4-carbonylbut-2-enoic acid (Intermediate 1-9)

[0233] The starting material 1-8 (2.448 g) and maleic anhydride (1.176 g) were dissolved in acetic acid (12 mL), and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was concentrated under reduced pressure, and then 40 mL of dichloromethane was added. The mixture was stirred for 20 minutes and filtered to obtain the title compound (2.4 g).

[0234] MS m / z(ESI):303.3[M+H] + .

[0235] Synthesis of (S)-3-((tert-Butoxycarbonyl)amino)-2-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (Intermediate 1-6)

[0236] Intermediate 1-9 (1.9 g), TEA (1.7 mL), and molecular sieves (6 g) were added sequentially to a mixture of toluene (30 mL) and DMA (0.6 mL). The reaction mixture was heated to 110°C and stirred for 6 hours. After completion, the reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by preparative HPLC (column model: YMC-Actus Triart C18 column, 5 μm silica, 30 mm diameter, 150 mm length; eluent: decreasingly polar mixtures of water (containing 0.1% TFA) and acetonitrile; acetonitrile gradient ratio 20-30%, elution time 6 minutes) to obtain the title compound (1.3 g).

[0237] MS m / z(ESI):285.3[M+H] + .

[0238] Example 2: 4-((S)-2-((S)-2-(4-((2-(N,N-bis(2-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)ethyl)sulfamoyl)ethyl)amino)-4-carbonylbutyrylamino)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1 R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-carbonylpropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-carbonylheptan-4-yl)(methyl)amino)-3-methyl-1-carbonylbutan-2-yl)amino)-3-methyl-1-carbonylbutan-2-yl)(methyl)carbamate (Drug-Linker 2)

[0239] Step 1: Synthesis of 2-((tert-butoxycarbonyl)amino)ethane-1-sulfonic acid tetrabutylammonium salt (Intermediate 2-2)

[0240] The starting material 2-1 (2.5 g) was dissolved in 20 mL of water, followed by the addition of a 40% tetrabutylammonium hydroxide solution (13 mL, 40% solution content), followed by a solution of Boc anhydride (4.36 g) in acetone (60 mL). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure, and the residue was extracted three times with dichloromethane (50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (5.6 g).

[0241] Step 2: Synthesis of tert-butyl (2-(chlorosulfonyl)ethyl)carbamate (Intermediate 2-3)

[0242] Intermediate 2-2 (5.6 g) was dissolved in dry dichloromethane (60 mL), and DMF (0.3 mL) and triphosgene (1.43 g) were added sequentially. The reaction mixture was allowed to react at room temperature for 2 h. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 5:1) to obtain the title compound (1.6 g).

[0243] 1 H NMR (400MHz, CDCl3) δ3.97-3.82 (m, 4H), 1.50 (s, 9H).

[0244] Step 3: Synthesis of tert-butyl (2-(N,N-bis(2-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)ethyl)sulfamoyl)ethyl)carbamate (Intermediate 2-5)

[0245] Intermediate 2-4 (0.3 g) and DIEA (0.4 mL) were stirred at 0°C for 5 minutes. A solution of intermediate 2-3 (0.35 g) in dichloromethane (5 mL) was slowly added dropwise, and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC (YMC-Actus Triart C18 column, 5 μm silica, 30 mm diameter, 150 mm length; eluent: a decreasingly polar mixture of water and acetonitrile; acetonitrile gradient ratio 40-50%, elution time 6 minutes) to obtain the title compound (100 mg).

[0246] MS m / z(ESI):471.1[M+H] + .

[0247] Step 4: Synthesis of 2-amino-N,N-bis(2-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)ethyl)ethane-1-sulfonamide (Intermediate 2-6)

[0248] At 0°C, TFA (3 mL) was added dropwise to a dichloromethane (3 mL) solution of intermediate 2-5 (100 mg). The mixture was stirred at room temperature for 30 minutes. After the reaction was completed, the reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (90 mg).

[0249] MS m / z(ESI):371.1[M+H] + .

[0250] Step 5: Synthesis of Drug-Linker 2

[0251] Intermediate 1-2 (264 mg) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 102.6 mg) were added to DMF (4 mL) at 0°C and stirred for 10 minutes. N,N-diisopropylethylamine (DIEA, 0.279 g) and a solution of Intermediate 2-6 (90 mg) in DMF (4 mL) were then slowly added and stirred at room temperature for 1 hour. The reaction mixture was then purified by preparative HPLC (Ultimate XB-C18 column, 10 μm, 50 mm diameter, 250 mm length; eluent: a decreasingly polar mixture of water (containing 0.1% TFA) and acetonitrile; acetonitrile gradient from 35% to 65% over 12 minutes) to afford the title compound (73 mg).

[0252] MS m / z(ESI):1575.8[M+H] + .

[0253] 1 H NMR(400MHz,DMSO)δ9.80(s,1H),8.30-7.80(m,5H),7.65-7.57(m,2H),7. 35-7.12(m,7H),7.02(s,4H),5.12-4.95(m,3H),4.55-4.12(m,10H),4.09- 3.90(m,2H),3.65-3.55(m,5H),3.38-2.81(m,24H),2.45-2.22(m,6H),2.1 8-1.92(m,4H),1.90-1.25(m,11H),1.10-0.97(m,6H),0.90-0.76(m,24H).

[0254] Synthesis of intermediate 2-9

[0255] The starting material 2-7 (4.0 g) was dissolved in MeOH (40 mL), and the starting material 2-8 (6.72 g) and solid NaHCO3 (8.3 g) were added thereto. The reaction solution was stirred at room temperature for 2 h. The reaction solution was concentrated to dryness under reduced pressure, and ethyl acetate (50 ml) and water (50 ml) were added to the residue and the extracts were combined and washed with water. The washed organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain the title compound (4 g).

[0256] 1 H NMR (400MHz, CDCl3) δ6.82 (d, J = 16Hz, 4H), 3.81-3.78 (m, 4H), 3.55-3.48 (m, 4H), 1.50 (s, 9H).

[0257] Synthesis of intermediate 2-4

[0258] Intermediate 2-9 (4.0 g) was added to a dioxane hydrochloride solution (40 mL, 4 M), and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then concentrated to dryness under reduced pressure to obtain the title compound (2.6 g).

[0259] 1 H NMR (400MHz, D2O) δ7.08 (s, 4H), 3.73-3.70 (m, 4H), 3.17-3.10 (m, 4H).

[0260] Example 3: 4-((S)-2-((S)-2-(2-(N,N-bis(2-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)ethyl)sulfamoyl)acetylamino)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3 -(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-carbonylpropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-carbonylheptan-4-yl)(methyl)amino)-3-methyl-1-carbonylbutan-2-yl)(methyl)carbamate (Drug-Linker 3)

[0261] Step 1: Synthesis of 2-(chlorosulfonyl)acetyl chloride (Intermediate 3-2)

[0262] The starting material 3-1 (5.0 g) was added to a 250 mL single-necked bottle, and POCl3 (50 mL) was added thereto. The reaction solution was refluxed at 110 ° C for 6 h under a nitrogen atmosphere, and then purified by distillation. The middle fraction was collected at 92-93 ° C to obtain the title compound (1.2 g).

[0263] 1 H NMR (400 MHz, D2O) δ 5.07 (s, 2H)

[0264] Step 2: Synthesis of tert-butyl 2-(chlorosulfonyl)acetate (Intermediate 3-3)

[0265] Intermediate 3-2 (1 g) was added to a 50 mL three-necked flask, THF (10 mL) was added, and tert-butanol (2.1 g) was added under nitrogen protection. The reaction solution was reacted at 25°C for 2 h, and then the reaction solution was concentrated under reduced pressure (water bath at 30°C) to obtain the title compound (1.2 g).

[0266] Step 3: Synthesis of tert-butyl 2-(N,N-bis(2-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)ethyl)sulfamoyl)acetate (Intermediate 3-4)

[0267] Intermediate 3-3 (0.512 g) was added to a 50 mL three-necked flask, followed by dichloromethane (10 mL). TEA (1.9 g) was added under nitrogen protection, and intermediate 2-6 (0.5 g) was added at 0°C. The mixture was reacted for 4 h. The reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by preparative HPLC (Ultimate XB-C18, 50*250 mm, 10 μm, using decreasingly polar mixtures of water (containing 0.1% FA) and acetonitrile as eluents; acetonitrile gradient ratio 35-65%, elution time 30 min) to give the title compound (8 mg).

[0268] MS m / z(ESI):386.0[Mt-Bu] + .

[0269] Step 4: Synthesis of 2-(N,N-bis(2-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrol-1-yl)ethyl)sulfonamido)acetic acid (Intermediate 3-5)

[0270] Intermediate 3-4 (8 mg) was added to a 25 mL single-necked bottle, and dichloromethane (2 mL) and TFA (0.5 mL) were added. The reaction solution was stirred at 25°C for 2 h, and then the reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (8 mg).

[0271] MS m / z(ESI):408.0[M+Na] + .

[0272] Step 5: Synthesis of Drug-Linker 3

[0273] Intermediate 3-5 (5 mg) was placed in a 25 mL three-necked flask, and DMF (0.5 mL) was added. After dissolution, starting material 1-1 (14.6 mg) was added under a nitrogen atmosphere. DIEA (4.2 mg) and HATU (6.4 mg) were added to the reaction mixture at 0°C, and the reaction mixture was stirred for 2 h. The reaction mixture was then purified by preparative HPLC (Ultimate XB-C18, 50*250 mm, 10 μm; eluent: decreasingly polar mixtures of water (containing 0.1% TFA) and acetonitrile; acetonitrile gradient ratio 45-75% over 40 minutes) to obtain the title compound (9 mg).

[0274] MS m / z(ESI):1491.6[M+H] + .

[0275] 1H NMR (400MHz, DMSO-d6) δ9.95 (s, 1H), 8.20 (d, J = 8.4Hz, 2H), 8.03-7.94 (m, 1H), 7. 82(d,J=8.7Hz,1H),7.53-7.46(m,2H),7.32-7.12(m,7H),7.12-7.05(m,1H),6.9 6(s,4H),5.99-5.83(m,1H),5.10-4.82(m,3H),4.70-4.53(m,1H),4.46-4-40(m, 1H),4.38-4.27(m,2H),4.24-4.15(m,2H),4.11-4.03(m,1H),4.01-3.97(m,1H), 3.96-3.90(m,2H),3.74-3.66(m,1H),3.62-3.53(m,5H),3.20-3.14(m,4H),3.13 -3.06(m,4H),3.05–3.01(m,2H),3.00-2.93(m,2H),2.92-2.85(m,2H),2.83-2.7 5(m,3H),2.38-2.30(m,1H),2.25-2.15(m,1H),2.09-1.83(m,5H),1.79-1.59(m, 4H),1.54-1.21(m,7H),1.20-1.05(m,2H),1.01-0.89(m,7H),0.81-0.67(m,24H).

[0276] Comparative Example 1: Preparation / Source of Control Drugs - Linkers 4 and 5

[0277] ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl- Synthesis of 4-((S)-2-((S)-2-(4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutyrylamino)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl ester (Drug-Linker 4)

[0278] Step 1: Synthesis of 4-(bis(2-((tert-butoxycarbonyl)amino)ethyl)amino)-4-oxobutanoic acid (Intermediate 4-2)

[0279] Dissolve compound 4-1 (2 g) in anhydrous dichloromethane (30 mL) and add dihydrofuran-2,5-dione (2 g). Stir the mixture at 20°C for 4 h. After the reaction, separate the mixture by column chromatography (tetrahydrofuran / petroleum ether, 0-50%) to obtain the title compound (2.6 g).

[0280] MS m / z(ESI):426.2[M+Na] + .

[0281] Step 2: Synthesis of 4-(bis(2-aminoethyl)amino)-4-oxobutanoic acid (Intermediate 4-3)

[0282] Intermediate 4-2 (2.6 g) was dissolved in hydrochloric acid-dioxane (100 mL, 4 M) solution and the reaction mixture was stirred at 25°C for 16 h. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain the title compound (1.6 g).

[0283] 1 H NMR (400MHz, DMSO-d6) δ = 3.63 (t, J = 6.9 Hz, 2H), 3.53 (t, J = 6.1 Hz, 2H), 3.01 (d, J = 5.3 Hz, 2H), 2.92 (d, J = 5.5 Hz, 2H), 2.64-2.58 (m, 2H), 2.48 (s, 2H).

[0284] Step 3: Synthesis of 4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutanoic acid (Intermediate 4-4)

[0285] A mixture of Intermediate 4-3 (100 mg) in saturated sodium bicarbonate (3 mL) and tetrahydrofuran (3 mL) was cooled to 0°C, and Intermediate 2-8 (152.64 mg) was added portionwise. The mixture was reacted at 0°C for 10 minutes and then at 25°C for 2 hours. After completion of the reaction, the pH was adjusted to 2-3 with hydrochloric acid, and the mixture was extracted with ethyl acetate (10 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by reverse phase chromatography (column: Spherical C18 20-35 μm 100A 20 g; mobile phase: [A: water, B: acetonitrile]; B%: 5%-30%, 20 mL / min) to obtain the title compound (54 mg).

[0286] MS m / z(ESI):364.1[M+H] + .

[0287] Step 4: Synthesis of drug-linker 4 compounds

[0288] Intermediate 4-4 (32.34 mg), intermediate 1-1 (100 mg), HATU (43.66 mg), and N,N-diisopropylethylamine (155.04 μL) were dissolved in N,N-dimethylformamide (1 mL). The atmosphere was purged with nitrogen three times, and the reaction mixture was stirred at 25°C under nitrogen for 1 hour. After completion of the reaction, the pH was adjusted to 5-6 with acetic acid and purified by HPLC (column: Welch Xtimate C18 150*30mm*5μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 40%-60%, 10 min) to obtain the title compound (23 mg).

[0289] MS m / z(ESI):735.1[(M+2H) / 2] + .

[0290] 1 H NMR (400MHz, DMSO-d6) δ = 9.75 (s, 1H), 8.32 (s, 1H), 8.11-8.03 (m, 1H), 8.02-7. 93(m,2H),7.90(d,J=8.6Hz,1H),7.67-7.51(m,3H),7.35-7.22(m,6H),7.21-7 .12(m,1H),7.01(s,2H),6.96(s,2H),5.97(t,J=5.6Hz,1H),5.46-5.33(m,3H) ,5.14-4.91(m,2H),4.79-4.57(m,1H),4.53-4.31(m,3H),4.30-4.21(m,1H),4 .15(t,J=6.9Hz,1H),4.07-3.91(m,2H),3.78(d,J=9.4Hz,1H),3.61-3.37(m,8 H),3.27-3.17(m,8H),3.12(s,2H),3.06-2.91(m,4H),2.86(d,J=14.4Hz,3H), 2.45-2.33(m,4H),2.28-2.26(m,1H),2.17-1.91(m,5H),1.76(d,J=5.1Hz,4H) ,1.65-1.40(m,4H),1.38-1.23(m,1H),1.08-0.95(m,6H),0.92-0.72(m,24H).

[0291] The structure of drug-linker 5 is shown below, and it was purchased from MedChemExpress (MCE) with the product number HY-15575.

[0292] Example 4: Preparation of drug-linker 6:

[0293] Step 1: Synthesis of di-tert-butyl (((vinylsulfonyl)aminodiyl)bis(ethane-2,1-diyl))dicarbamate (Intermediate 6-2)

[0294] Intermediate 6-1 (23 g) and triethylamine (23 g) were dissolved in anhydrous dichloromethane (300 mL). 2-Chloroethanesulfonyl chloride (13.6 g) was added at 0°C. The reaction was stirred at 0°C for 2 h. The mixture was then washed with water (100 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the title compound (5.7 g).

[0295] MS:m / z(ESI):394.2[M+H] +

[0296] Step 2: Synthesis of di-tert-butyl ((((2-aminoethyl)sulfonyl)aminodiyl)bis(ethane-2,1-diyl))dicarbamate (Intermediate 6-3)

[0297] Intermediate 6-2 (5.7 g) was dissolved in 7 M methanolic ammonia solution (60 mL). The reaction was stirred at 20°C for 4 h and then concentrated under reduced pressure to obtain the title compound (5.7 g).

[0298] MS m / z(ESI):411.3[M+H] +

[0299] Step 3: Synthesis of 4-((2-(N,N-bis(2-((tert-butoxycarbonyl)amino)ethyl)sulfamoyl)ethyl)amino)-4-oxobutanoic acid (Intermediate 6-4)

[0300] Intermediate 6-3 (5.7 g) and succinic anhydride (6.9 g) were dissolved in anhydrous dichloromethane (30 mL), and triethylamine (5.8 mL) was added. The reaction was stirred at 25°C for 2 h. The reaction solution was then added directly to water (50 mL) and extracted three times with dichloromethane (50 mL). The combined organic phases were dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain the title compound (7 g).

[0301] MS m / z(ESI):511.3[M+H] +

[0302] Step 4: Synthesis of 4-((2-(N,N-bis(2-aminoethyl)sulfamoyl)ethyl)amino)-4-oxobutanoic acid (Intermediate 6-5)

[0303] Intermediate 6-4 (2.7 g) was dissolved in 2M dioxane hydrochloride (30 mL), and the reaction mixture was stirred at 25°C for 16 h. Afterwards, the mixture was concentrated under reduced pressure and stirred with petroleum ether / ethyl acetate (5 / 1, 10 mL) at 25°C for 1 h. The liquid was filtered to obtain the title compound (1.9 g).

[0304] MS m / z(ESI):311.2[M+H] +

[0305] Step 5: Synthesis of 4-((2-(N,N-bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)sulfamoyl)ethyl)amino)-4-oxobutanoic acid (Intermediate 6-6)

[0306] A mixed solution of intermediate 6-5 (1.9 g) in saturated sodium bicarbonate (25 mL) and methanol (3 mL) was cooled to 0°C, and compound 6-5A (152.64 mg) was added portionwise. The mixture was reacted at 0°C for 10 minutes and then at 25°C for 1 hour. The pH was then adjusted to 2-3 with hydrochloric acid, and the mixture was extracted with ethyl acetate (10 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified via reverse-phase column chromatography to obtain the title compound (600 mg).

[0307] MS m / z(ESI):471.1[M+H] +

[0308] Step 6: Synthesis of (S)-5-allyloxycarbonyl-1-(9H-fluoren-9-yl)-8,11,14,17,20,23,26,29,32-nonamethyl-3,7,10,13,16,19,22,25,28,31-decaoxo-2-oxa-4,8,11,14,17,20,23,26,29,32-decaazatetratriacontane-34-carboxylic acid (Intermediate 6-7)

[0309] The compound was synthesized by peptide solid phase synthesis method, the steps are as follows:

[0310] 1) Under nitrogen, dichloromethane was added to a mixture of CTC resin (0.64 mmol / g, 12.5 g) and N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine (2.50 g);

[0311] 2) Diisopropylethylamine (DIEA) (6.2 g) was added dropwise and the mixture was stirred for 2 h;

[0312] 3) Methanol (13 mL) was added and the mixture was stirred for 0.5 h;

[0313] 4) Filter and wash three times with DMF;

[0314] 5) Add 20% piperidine / DMF solution and react for 30 min;

[0315] 6) Filter and wash five times with DMF;

[0316] 7) Add the materials listed in the "Raw Materials" column in the table below and mix for 30 seconds. Then add the materials listed in the "Reagents" column in the table below. The reaction is carried out under nitrogen bubbling for 1 hour.

[0317] 8) Repeat steps 4-7 to complete the synthesis of the desired peptide.

[0318] After the solid phase was prepared, a 20% hexafluoroisopropanol / dichloromethane solution was added to the resulting mixture and stirred for 1.5 h. The mixture was then filtered, the filtrate collected, and concentrated. The resulting mixture was purified by preparative liquid chromatography (A: 0.075% TFA in H2O, B: acetonitrile) to afford the title compound (2.15 g).

[0319] MS m / z(ESI):1035.6[M+H] + .

[0320] Step 7: Synthesis of (S)-30-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-amino-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-3,6,9,12,15,18,21,24,27-nonaazatriacontane-31-carboxylic acid allyl ester (Intermediate 6-8)

[0321] Intermediate 6-7 (1 g), ammonium chloride (155.09 mg), diisopropylethylamine (499.44 mg), HATU (728.95 mg) were dissolved in DMF (5 mL), and the atmosphere was replaced with nitrogen three times. The reaction was stirred at 25 ° C for 3 h. After the reaction was completed, the crude product was purified by reverse phase high performance liquid chromatography ( 80 g C 18 Column, mobile phase gradient 0-40% acetonitrile / water, flow rate 40 mL / min) to obtain the title compound (800 mg).

[0322] MS m / z(ESI):1034.6[M+H] + .

[0323] Step 8: Synthesis of (S)-30-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-amino-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-3,6,9,12,15,18,21,24,27-nonaazatriacontane-31-carboxylic acid (Intermediate 6-9)

[0324] Intermediate 6-8 (800 mg), tetrakistriphenylphosphine palladium (201.84 mg), 1.3-dimethylbarbituric acid (108.71 mg) were dissolved in DMF (5 mL), and the atmosphere was replaced with nitrogen three times. The reaction was stirred at 25°C for 4 h. After the reaction was completed, the crude product was purified by reverse phase high performance liquid chromatography ( 80g C18 Column, mobile phase gradient 0-40% acetonitrile / water, flow rate 40mL / min) to obtain the title compound (700mg).

[0325] MS m / z(ESI):994.9[M+H] + .

[0326] Step 9: Synthesis of resin-supported (2S,10S,19S)-19-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-48-amino-10-benzyl-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetraazatetraoctadecanoic acid (Intermediate 6-11)

[0327] Intermediate 6-9 (700 mg) was added to DMF (10 mL), followed by Intermediate 6-10 (synthesized according to the method reported in patent WO2023217227, with a total weight of 400 mg of resin and substrate, and an amount of substrate of approximately 500 μmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (336.58 mg), and diisopropylethylamine (205.62 mg). The reaction mixture was nitrogen-purged three times and shaken on a shaker at 25°C for 1 h. After completion of the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, filtered to dryness, and dried under vacuum to obtain the title compound (420 mg).

[0328] MS m / z(ESI):1461.4[M+Na] + .

[0329] Step 10: Synthesis of resin-supported (2S,10S,19S)-19,48-diamino-10-benzyl-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetraazatetraoctadecanoic acid (Intermediate 6-12)

[0330] Intermediate 6-11 (420 mg) was dissolved in DMF (5 mL) and piperidine (1.25 mL) was added. The reaction mixture was shaken at 25°C for 1 hour. After the reaction, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) three times, followed by filtration to dryness and drying under vacuum to obtain the title compound (400 mg).

[0331] MS m / z(ESI):1217.4[M+H] + .

[0332] Step 11: Synthesis of resin-supported (2S,10S,19S)-48-amino-10-benzyl-19-(4-((2-(N,N-bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)sulfamoyl)ethyl)amino)-4-oxobutyrylamino)-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetraazatetradecanoic acid (Intermediate 6-13)

[0333] Under a nitrogen atmosphere, DMF (10 mL) and DIEA (295 μL) were added to a mixture of Intermediate 6-12 (850 mg), Intermediate 6-6 (397 mg), and benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate (286 mg). The resulting mixture was shaken on a shaker at 20°C for 1 hour. The resin was then washed three times with methanol (20 mL) and dichloromethane (20 mL). The resin was then filtered to dryness and dried under vacuum to obtain the title compound (1 g).

[0334] LC-MS: m / z(ESI):1670.5[M+H] +

[0335] Step 12: Synthesis of (2S,10S,19S)-48-amino-10-benzyl-19-(4-((2-(N,N-bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)sulfamoyl)ethyl)amino)-4-oxobutyrylamino)-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetraazatetraoctadecanoic acid (Intermediate 6-14)

[0336] Intermediate 6-13 (1 g) was dissolved in dichloromethane (20 mL) and hexafluoroisopropanol (5 mL). The resulting mixture was shaken at 25°C for 0.5 hr. The reaction mixture was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure. The product was purified by HPLC (column: Welch Xtimate C18 150*30mm*5µm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 16%-46%, 15 min) to yield the title compound (57 mg).

[0337] LC-MS: m / z(ESI):1691.4[M+Na] +

[0338] Step 13: Synthesis of Drug-Linker 6

[0339] Under a nitrogen atmosphere, DMF (1 mL) was added to a mixture of intermediate 6-14 (25 mg), intermediate 6-15 (synthesized according to the method reported in patent WO2023217227, 7 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6 mg), 1-hydroxybenzotriazole (4 mg), and pyridine (4 μL). The resulting mixture was stirred at room temperature for 1 hour. The pH was then adjusted to 6-7 with acetic acid, and the reaction mixture was directly purified by HPLC (column: Welch Xtimate C18 150*30mm*5μm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 11%-41%, 20 min) to obtain the title compound (12.1 mg).

[0340] LC-MS:m / z(ESI):1038.0[(M+2H) / 2] +

[0341] 1H NMR (400MHz, DMSO-d6)δ=8.68-8.62(m,1H),8.60-8.52(m,1H),8.29-8.18(m,2H),8.13-8.08(m,1H),8.05-7.91(m,3H) ,7.79(s,1H),7.52(s,1H),7.25(s,1H),7.25-7.19(m,4H),7.19-7.13(m,1H),7.01(s,4H),6.48(s,1H),5.49-5.38(m,4 H),4.84-4.70(m,2H),4.67-4.65(m,1H),4.62-4.44(m,2H),4.44-4.15(m,9H),4.15-3.82(m,10H),3.70-3.44(m,18H) ,3.09-2.71(m,36H),2.31-2.27(m,2H),1.93-1.79(m,2H),1.00-0.92(m,1H),0.87(t,J=7.3Hz,3H),0.41-0.26(m,4H).

[0342] Example 5: Preparation of drug-linker 7:

[0343] Step 1: Synthesis of 2,5-dioxopyrrolidin-1-yl 4-((2-(N,N-di(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)sulfamoyl)ethyl)amino)-4-oxobutanoate (Intermediate 7-1)

[0344] Intermediate 6-6 (30 mg) was dissolved in DMF (1 mL), and N-hydroxysuccinimide (8.8 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (14.7 mg) were added sequentially. The reaction mixture was stirred at 25°C for 1 hour to obtain a solution containing the title compound, which was used directly in the next reaction.

[0345] LC-MS: m / z(ESI):568.1[M+H] +

[0346] Step 2: Synthesis of (2S,10S)-16-amino-10-benzyl-2-cyclopropyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecanoic acid (Intermediate 7-2)

[0347] Intermediate 6-10 (50 mg) was dissolved in dichloromethane (4 mL) and hexafluoroisopropanol (1 mL). The resulting mixture was shaken at 25°C for 0.5 hr. The reaction mixture was then filtered to remove the resin, and the filtrate was concentrated under reduced pressure and lyophilized to yield the title compound (10 mg).

[0348] LC-MS: m / z(ESI):464.3[M+H] +

[0349] Step 3: Synthesis of (2S,10S)-10-benzyl-24-(N,N-di(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)sulfamoyl)-2-cyclopropyl-6,9,12,15,18,21-hexaoxo-3-oxa-5,8,11,14,17,22-hexaazatetracosanoic acid (Intermediate 7-3)

[0350] Intermediate 7-1 (30 mg) was dissolved in N,N-dimethylformamide (1 mL), and intermediate 7-2 (24.5 mg) was added. The reaction mixture was stirred at 25°C for 1 hour to obtain a solution containing the title compound, which was used directly in the next reaction.

[0351] LC-MS: m / z (ESI): 771.2 [fragment molecular weight M] +

[0352] Step 4: Synthesis of drug-linker 7

[0353] Intermediate 7-3 (48 mg) and intermediate 6-15 (24.4 mg) were dissolved in DMF (1 mL). n-Butylphosphoric anhydride (50% in ethyl acetate) (75.5 mg) and DIEA (36.5 μL) were added, and the reaction mixture was stirred at 25°C for 1 hour. The pH of the reaction mixture was then adjusted to 6 with acetic acid and purified by HPLC (column: C18 150×40 mm; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 11%-51%, 9 min) to yield the title compound (8 mg).

[0354] LC-MS: m / z(ESI):1321.3[M+H] +

[0355] 1H NMR (400MHz, DMSO-d6) δ = 8.66 (t, J = 5.6Hz, 1H), 8.58 (t, J = 6.4Hz, 1H), 8.27 (t, J = 5.4Hz, 1H), 8.16 (t, J = 5.6Hz, 1H), 8.09-7.99 (m, 3H) ,7.79(s,1H),7.51(s,1H),7.25(s,1H),7.24-7.20(m,3H),7.18-7.13(m,1H),7.01(s,4H),5.43(d,J=9.1Hz,4H),4.85-4.70(m,2H), 4.68-4.64(m,1H),4.49-4.47(m,1H),4.41-4.38(m,1H),3.76-3.50(m,10H),3.07-3.00(m,4H),2.79-2.77(m,1H),2.35-2.29(m,4H) ,1.92-1.79(m,2H),1.53-1.41(m,3H),1.34-1.30(m,3H),0.99-0.92(m,1H),0.90-0.82(m,5H),0.39-0.34(m,2H),0.34-0.28(m,2H).

[0356] Example 6: Preparation of Antibody-Drug Conjugates

[0357] 6.1 Antibodies:

[0358] 6.1.1 Construction and Production of Anti-Human HER2 Monoclonal Antibodies

[0359] The sequences of the anti-human HER2 monoclonal antibodies are shown in Table 1 below (antibody sequence sources: US Pat. No. 5,821,337A and US Pat. No. 9,815,904B2). The heavy and light chain nucleotide sequences were cloned into the pTT5 vector (purchased from Ubiquitin Biosciences). Plasmids were prepared according to established standard molecular biology methods. For details, see Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). The expression vector and transfection reagent PEI (Polysciences, Catalog No. 24765-1) were added to OPTI-MEM (Gibco, Catalog No. 11058021), mixed, and allowed to stand for 15 minutes. The cells were then added to Expi293F cells (Thermofisher, Catalog No. A14527) and cultured in a shaking incubator at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No.: F081918-001) and 6 g / L glucose (Manufacturer: Sigma, Catalog No.: G7528) were added. On the sixth day of transfection, the cell supernatant was collected.

[0360] Table 1 Sequence information and CDR sequences of anti-human HER2 antibodies trastuzumab and pertuzumab (according to Kabat classification)

[0361] 6.1.2 Construction and Production of Anti-Human CDH6 Antibodies

[0362] The anti-human CDH6 antibody CDH6-Ab-1 was produced by immunizing mice. The immunogens were human CDH6-hFc protein and HEK293T cells overexpressing human CDH6. Hybridoma cells were prepared from splenic lymphocytes of mice with high antibody titers in serum. Positive hybridoma clones were screened using conventional methods in the art, such as ELISA and FACS. Antibodies were further prepared using serum-free cell culture, purified, and sequenced to obtain the murine anti-human CDH6 antibody and its variable region sequence. By comparing the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database and MOE (Molecular Operating Environment) software, we selected heavy and light chain variable region germline genes with high homology to the mouse antibody as templates. The mouse antibody CDRs were transplanted into the corresponding human templates, forming a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on this, back mutations were performed as needed to maintain the original affinity and / or hotspot mutations were performed to eliminate the risk of molecular modification. The final optimized humanized antibody CDH6-Ab-1 corresponding VH and VL sequences and CDR sequences classified according to Kabat format are shown in Table 2.

[0363] Table 2 Sequence information of anti-human CDH6 antibody CDH6-Ab-1 and its CDR sequence (according to Kabat classification)

[0364] The nucleic acid sequences encoding the antibody VH and VL were recombined into the expression vector pTT5 containing a signal peptide (MGWSWILLFLLSVTAGVHS, SEQ ID NO: 46) and heavy chain constant region / light chain constant region sequences to generate a recombinant plasmid expressing VH-CH1-Fc / VL-CL. The plasmid and transfection reagent PEI (Polysciences, Catalog No.: 24765-1) were added to OPTI-MEM (Gibco, Catalog No.: 11058021), mixed, and allowed to stand for 15 minutes. The cells were then added to Expi293F cells (Manufacturer: Thermofisher, Catalog No.: A14527) and cultured in a shaking incubator at 37°C with 5% CO2 and 120 rpm. The day after transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No.: F081918-001) and 6 g / L glucose (Manufacturer: Sigma, Catalog No.: G7528) were added. On the sixth day after transfection, the cell culture supernatant secreting antibodies was obtained.

[0365] 6.1.3 Construction and Production of Anti-Human LIV-1 Monoclonal Antibodies

[0366] The sequence of the anti-human LIV-1 monoclonal antibody ladiratuzumab (antibody sequence source: US20200165335A) is shown in Table 3 below. The nucleic acid sequences encoding the antibody VH and VL were recombined into the expression vector pTT5 with a signal peptide and heavy chain constant region (SEQ ID NO: 13) / light chain constant region sequence (SEQ ID NO: 6) to obtain a recombinant plasmid expressing ladiratuzumab. After sequencing verification, the plasmid was extracted.

[0367] The anti-human LIV-1 monoclonal antibody LIV1-Ab-020 was produced by immunizing mice. The immunogen was the human LIV1-hFc protein. Hybridoma cells were prepared from splenic lymphocytes of mice with high antibody titers in serum. Positive hybridoma clones were screened using conventional methods in the art, such as ELISA and FACS. Antibodies were further produced using serum-free cell culture, purified, and sequenced to obtain the murine anti-human LIV-1 antibody and its variable region sequences. By comparing the IMGT (http: / / imgt.cines.fr) database of human antibody heavy and light chain variable region germline genes and using MOE (Molecular Operating Environment) software, germline heavy and light chain variable region genes with high homology to the murine antibody were selected as templates. The CDRs of the murine antibody were transplanted into the corresponding human templates, resulting in a variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Back mutations were then performed to maintain the original affinity and / or hotspot mutations were performed to eliminate the risk of molecular modification. The VH and VL corresponding to the final optimized humanized antibody LIV1-Ab-020 are shown in Table 3 (SEQ ID NOs: 11 and 12). The nucleic acid sequences encoding the VH and VL of the antibody were recombined into the expression vector pTT5 containing a signal peptide and heavy chain constant region (SEQ ID NO: 13) / light chain constant region sequence (SEQ ID NO: 6) to generate a recombinant plasmid expressing LIV1-Ab-020. After sequencing verification, the plasmid was extracted.

[0368] Plasmid and transfection reagent PEI (Polysciences, Catalog No. 24765-1) were added to OPTI-MEM (Gibco, Catalog No. 11058021), mixed, and allowed to stand for 15 minutes. The cells were then added to Expi293 cells (Thermofisher, Catalog No. A14527) and incubated in a shaking incubator at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No. F081918-001) and 6 g / L glucose (Sigma, Catalog No. G7528) were added. On the sixth day of transfection, the cell supernatant was collected.

[0369] Table 3 Sequence information and CDR sequences of anti-human LIV-1 antibodies Ladiratuzumab and LIV1-Ab-020 (according to Kabat classification)

[0370] 6.1.4 Construction and Production of Anti-Human FITC Monoclonal Antibodies

[0371] The sequence of the anti-human FITC monoclonal antibody (anti-FITC-hIgG1) is shown in Table 4 below. The nucleic acid sequences encoding the antibody VH and VL were recombined into the expression vector pTT5 containing a signal peptide and heavy chain constant region (sequence) / light chain constant region sequence to obtain a recombinant plasmid expressing anti-FITC. After sequencing verification, the plasmid was extracted. The plasmid and transfection reagent PEI (Polysciences, Catalog No.: 24765-1) were added to OPTI-MEM (Gibco, Catalog No.: 11058021) and mixed. The mixture was allowed to stand for 15 minutes and then added to Expi293 cells (Manufacturer: Thermofisher, Catalog No.: A14527). The cells were cultured in a shaking incubator at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No.: F081918-001) and 6 g / L glucose (Manufacturer: Sigma, Catalog No.: G7528) were added. On the sixth day after transfection, the cell supernatant was collected.

[0372] Table 4 Anti-human FITC antibody anti-FITC-hIgG1 sequence (according to Kabat classification)

[0373] Note: The underline represents the CDR sequence divided by Kabat.

[0374] 6.2 Antibody Purification

[0375] Purify the above-mentioned antibodies from cell culture supernatant using Protein A affinity chromatography (GE, Catalog No. 17549802). Wash the Protein A affinity column with 6M guanidine hydrochloride for 3-5 column volumes, followed by 3-5 column volumes with purified water. Equilibrate the column with a buffer system such as 1× PBS (pH 7.4) for 3-5 column volumes. Load the cell supernatant for binding at a low flow rate, controlling the flow rate to achieve a retention time of approximately 1 minute or longer. After binding, wash the column with 1× PBS (pH 7.4) for 3-5 column volumes until UV absorbance returns to baseline. Elute the sample with 0.02M citrate buffer, pH 3.5. Collect the elution peak under UV monitoring. Adjust the pH of the eluted product to 5-6 using 1M Tris-HCl (pH 8.0, Sinopharm, Catalog No. 30188336) for storage. The eluted product can be subjected to solvent exchange using methods well known to those skilled in the art, such as ultrafiltration and concentration using ultrafiltration tubes and solvent exchange into the desired buffer system, or using molecular exclusion chromatography, such as G-25 desalting, to replace the desired buffer system, or using a high-resolution molecular exclusion column such as Superdex 200 to remove aggregates from the eluted product to improve sample purity. After purification, proteins that meet purity requirements are sterile filtered using a 0.22 μm filter (Millipore, Catalog No. SLGVR13SL). After passing SEC-HPLC purity testing, the samples are aliquoted and stored at -80°C until ready for use.

[0376] 6.3 Preparation of Antibody-Drug Conjugates

[0377] Add the antibodies (as shown in Table 1-4) to the Thermo-Slide-A-Lyzer TM G3 Dialysis cassette (Thermo Scientific TM, Cat. No. 88252) was dialyzed to 20 mM PB, 150 mM NaCl, 1 mM EDTA solution (pH 6.5). 2-10 molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) were added to a 2-10 mg / ml antibody solution (1× PBS, 1 mM EDTA, pH 7.4). The mixture was incubated at 37°C for 2 hours. Then, 5-10 molar equivalents of the drug-linker compound (e.g., drug-linkers 1-7) were added. The reaction mixture was mixed and incubated at 25°C for 16 hours. After the reaction, the solution was replaced with PBS buffer by ultrafiltration three times to remove the residual unreacted free small molecules to obtain an antibody drug conjugate. The representative structures of the antibody drug conjugates are as follows (Formula i was prepared using drug-linker 2, Formula ii was prepared using drug-linker 3, Formula iii was prepared using drug-linker 1, Formula iv was prepared using drug-linker 7, and Formula v was prepared using drug-linker 6):

[0378] Among them, Pc is Pertuzumab, Trastuzumab, Ladiratuzumab, LIV1-Ab-020, CDH6-Ab-1 and anti-FITC-hIgG1.

[0379] The purity of the antibody drug conjugate was detected by SEC-HPLC, and the DAR value of the antibody drug conjugate was detected by hydrophobic high performance liquid chromatography (HIC-HPLC).

[0380] 6.4 Antibody Drug Conjugate Purity Testing (SEC-HPLC)

[0381] The purity of the antibody-drug conjugate prepared above was analyzed by SEC-HPLC. The HPLC used in this method was Agilent 1260, the chromatographic column was TSKgel G3000SWXL from Tosoh Bioscience, the mobile phase was 200mM phosphate buffer pH 7.0 containing 10% isopropanol, the detection temperature was 25°C, the flow rate was 0.5mL / min, the detection wavelength was 280nm, the target protein load was 50μg, and the analysis time was 40 minutes. For the SEC-HPLC data, the chromatogram was analyzed by manual integration, and the protein purity was calculated according to the area normalization method. The main peak was considered to be a monomer, the chromatographic peak before the main peak was called an aggregate, and the chromatographic peak after the main peak was called a fragment. The test results are shown in Table 5.

[0382] 6.5 DAR Value Detection of Antibody-Drug Conjugates (HIC-HPLC)

[0383] The HIC-HPLC method was used to analyze the ADC samples to characterize their DAR values. The HPLC used in this method was an Agilent 1260, with a TSKgel Butyl-NPR column from Tosoh Bioscience. Mobile phase A consisted of 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0, and mobile phase B consisted of 25 mM phosphate buffer, pH 7.0, containing 20% ​​isopropanol. The detection temperature was 25°C, the flow rate was 0.5 mL / min, and the detection wavelengths were 280 nm and 250 nm. The sample was diluted to 0.5 mg / ml with 2.0 M ammonium sulfate, 100 mM phosphate buffer, pH 7.0, with a final ammonium sulfate concentration of 0.8 M. The sample was mixed thoroughly, centrifuged, and the supernatant was collected. The protein loading amount was 40 μg. The mobile phase gradient was increased from 0% to 100% mobile phase B over 50 minutes, and the analysis time was 70 minutes.

[0384] There are reports in the literature that the dual-wavelength HIC-HPLC technique was used to directly determine the DAR value of ADC (Clin Cancer Res. 2004 Oct 15; 10(20): 7063-70; MAbs. 2011 Mar-Apr; 3(2): 161-72.). The experimental principle is that antibodies and compounds have different light absorption intensities at 280nm and 248nm. When the amount of antibody is the same, the 248nm light absorption intensity is positively correlated with the DAR value. The stronger the 248nm light absorption signal, the higher the DAR value of the ADC. Based on this principle, the peak height of each chromatographic peak at 250nm was normalized by the peak height at 280nm. Quantitative analysis found that the normalized peak height at 250nm of each chromatographic peak was × 250nm The DAR values ​​of each HIC chromatographic peak were confirmed by showing regular changes. As shown in Figure 1 and Table 5-1, ADC-10 molecules detected 5 chromatographic peaks in HIC analysis. After normalizing the peak height of each chromatographic peak at 250nm with the peak height at 280nm, the peak height of peak 1 at 250nm was compared with the DAR0 of the naked antibody peak. 250nm Peak 2 increased by about 15.6 units and was confirmed as DAR2. Peak 3 increased by about 23.6 units and was confirmed as DAR3. Peak 4 increased by about 31.2 units and was confirmed as DAR4. Peak 5 increased by about 39.1 units and was confirmed as DAR5. It can be seen that the normalized peak height of ADC-10 molecule at 250 nm is X 250nm For every approximately 7.8 units increase, the DAR value increases by 1. Finally, the HIC-HPLC chromatograms were analyzed using manual integration. The area percentage of each peak at 280 nm was calculated using area normalization. The average DAR was calculated by multiplying the peak area percentage by the DAR value for each peak. Using this calculation method, the DAR values ​​for other ADCs were calculated as shown in Table 5-3.

[0385] HIC-HPLC analysis of the control ADC2 molecule showed that Peak 1 increased by approximately 30.51 units, confirming DAR4; Peak 2 increased by approximately 38.2 units, confirming DAR5; and Peak 3 increased by 43.68 units, confirming DAR6. The calculated average DAR value for the control ADC2 was 4.33. The test results are shown in Table 5-2.

[0386] Validation was performed using mass spectrometry using the following conditions: AB Sciex TripleTOF 7600; mobile phase: 20 mM ammonium acetate; detection range: m / z 1000-8000; output range: 140,000-170,000 Da. The molecular weights of peak 1 and peak 2 components of the control ADC2 were calculated based on the corresponding mass spectrometric response values ​​of the drug-linker in the ADC sample. Mass spectrometry analysis revealed that the molecular weights of peak 1 and peak 2 components of the control ADC2 were 151,762.06 (DAR 4) and 153,248.65 (DAR 5), respectively, consistent with the DAR values ​​determined by HIC-HPLC.

[0387] Table 5-1 Hydrophobic interaction chromatography (HIC-HPLC) analysis results of ADC-10

[0388] Table 5-2 Hydrophobic interaction chromatography (HIC-HPLC) analysis results of ADC2

[0389] Table 5-3 Preparation, DAR value and SEC purity of antibody-drug conjugates

[0390] Example 7: Biological activity and related properties testing

[0391] 7.1 Antibody-drug conjugate binding activity assay

[0392] 7.1.1 Flow cytometry (FACS) assay for binding activity of anti-HER2 antibody-drug conjugates to SKBR3 tumor cells

[0393] In order to detect the binding activity of anti-HER2 antibody drug conjugates with human HER2 full-length protein, breast cancer cells SKBR3 (from ATCC) that highly express human HER-2 protein were expanded and cultured in T-75 cell culture flasks to the logarithmic growth phase, the culture supernatant was discarded by centrifugation, and the cell pellet was washed twice with PBS. The sample to be tested was added with gradient dilutions and incubated at 4°C for 1 hour. After washing twice with ice PBS, Alexa Fluor-647 goat anti-human Fc secondary antibody (JacksonImmuno, 109-605-098) was added and incubated at 4°C for 1 hour. After washing twice with ice PBS, the cells were resuspended and analyzed by flow cytometry (BD FACSCanto TM II) Analyze the mean fluorescence intensity (MFI). Graphpad Prism software was used to perform four-parameter curve fitting analysis on the data to obtain the EC 50 The detection results are shown in Table 6.

[0394] Table 6. FACS detection of binding activity of anti-HER2-ADC to SKBR3 tumor cells

[0395] “\” represents no combination

[0396] 7.1.2 Flow cytometry (FACS) assay to detect the binding activity of anti-CDH6-ADC to ovarian cancer cells

[0397] The FACS method in Example 7.1.1 was used to detect the binding ability of anti-CDH6-ADC to ovarian cancer cells OVCAR3 (purchased from ATCC) with high CDH6 expression and ovarian teratoma cell line PA-1 (purchased from Kebai Bio) with medium to high CDH6 expression. The results are shown in Tables 7 and 8.

[0398] Table 7. FACS detection of binding activity of anti-CDH6-ADC to ovarian cancer cell OVCAR3

[0399] Table 8. FACS detection of binding activity of anti-CDH6-ADC to ovarian cancer cell PA-1

[0400] 7.1.3 Flow cytometry (FACS) assay to detect the binding activity of anti-LIV-1 ADC to NCI-H838 tumor cells

[0401] The FACS method described in Example 7.1.1 was used to detect the binding ability of the anti-LIV-1 ADC to the lung cancer cells NCI-H838 (purchased from the cell bank of the Chinese Academy of Sciences) that highly express LIV-1. The results are shown in Table 9.

[0402] Table 9. FACS detection of binding activity of anti-LIV-1-ADC to lung cancer cells NCI-H838

[0403] 7.2 Antibody-drug conjugate endocytosis capacity testing

[0404] 7.2.1 Flow cytometry (FACS) assay to detect the endocytosis of anti-HER2 ADC in SKBR3 tumor cells

[0405] SKBR3 tumor cells in the logarithmic growth phase were collected and cultured at a density of 1-2 × 10 5 Cells were seeded in a 96-well plate at a density of 100 μg / well, washed once with PBS, and then antibodies or ADCs were added (final concentration was 100 nM) and incubated at 4°C for 1 hour. The control group was placed at 4°C, and the endocytosis group was placed at 37°C. After incubation for 2 hours, the cells were washed three times with PBS, and secondary antibodies were added: Alexa Fluor Cells were incubated with 647AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (Jackson Immuno, Cat. No. 109-605-098) at 4°C for 1 hour, washed twice with PBS, and analyzed by FACS (FACS Canto™, BD Biosciences). Endocytosis efficiency = (control group MFI - endocytosis group MFI) / control group MFI * 100%. The results are shown in Table 10.

[0406] Table 10. FACS detection of endogenous cell endocytosis efficiency of anti-HER2-ADC in SKBR3 cells

[0407] 7.2.2 Flow cytometry (FACS) assay to detect the endocytosis of anti-CDH6 ADC

[0408] In order to detect the endocytosis effect of anti-CDH6 ADC in PA-1 cells, the FACS endocytosis detection method in Example 7.2.1 was used for detection. The detection results are shown in Table 11.

[0409] Table 11. FACS detection of endocytic effect of anti-CDH6 ADC (PA-1)

[0410] 7.2.3 Flow cytometry (FACS) assay to detect the endocytic effect of anti-LIV-1 ADC on NCI-H838 tumor cells

[0411] In order to detect the endocytic effect of anti-LIV-1 ADC on NCI-H838 cells, the FACS endocytic detection method in Example 7.2.1 was used. The detection results are shown in Table 12.

[0412] Table 12. FACS detection of endocytosis of anti-LIV1 ADC in NCI-H838 cells

[0413] 7.3 Anti-proliferative activity testing of antibody-drug conjugates against tumor cells

[0414] 7.3.1. Anti-proliferative activity test of anti-Her2-ADC on tumor cells

[0415] Materials and instruments: Human breast cancer cell line SKBR3 was purchased from ATCC, and human gastric cancer cell line NCI-N87 cells were purchased from Cobioer. RPMI-1640 medium was purchased from BasalMedia (L240KJ), McCoy's 5A (Modified) medium was purchased from BasalMedia (L630KJ), and fetal bovine serum was purchased from Proteintech (PM00011). 0.25% Trypsin-EDTA was purchased from BasalMedia (S310KJ), and PBS was purchased from BasalMedia (B320KJ). 96-well plates were purchased from Cave Bio (#062096). Luminescent Cell Viability Detection Kit was purchased from VKEY-BIO (A2010003N). The automated cell counter was Beckman, model Vi-cell XR. The multifunctional microplate reader was PerkinElmer, model Envision 2105.

[0416] Cell culture: SKBR3 cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum, and NCI-N87 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0417] Cell proliferation activity detection: using The Luminescent Cell Viability Detection Kit was used to detect the inhibitory activity of ADC on the proliferation of SKBR3 and N87 cell lines. The SKBR3 and NCI-N87 cells were digested and blown away from the cell culture flask, resuspended with the corresponding fresh culture medium, and the cell density was adjusted after counting with a cell counter. Both SKBR3 and NCI-N87 cells were 4000 cells / 90μL / well and cultured overnight at 37°C and 5% CO2. The ADC concentration was diluted to 1000nM with complete culture medium, and a 5-fold gradient dilution was performed for a total of 9 concentration gradients. Then 10μL of the ADC dilution solution was transferred to a 96-well plate, that is, the initial and final concentration of ADC was 100nM. The 96-well plate was cultured at 37°C and 5% CO2 for 5 days. After 5 days, 50μL of ADC was added to each well. Luminescent Cell Viability Detection Kit was used, and the fluorescence signal was read using a microplate reader.

[0418] A negative control group (bottom) and a positive control group (top) were set up. The negative control group did not contain cells, but only the same volume of culture medium. All other procedures were the same as those for the experimental group. The positive control group did not contain the test antibody. All other procedures were the same as those for the experimental group.

[0419] Data Analysis:

[0420] Calculate the inhibition percentage (% Inhibition) and fit to obtain the IC of the compound 50 .

[0421] Percent inhibition (% Inhibition) = 1-100%*(Signal-Bottom) / (Top-Bottom).

[0422] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0423] Experimental results:

[0424] The experimental results are shown in Table 13.

[0425] Table 13-1. Anti-proliferative activity test data of anti-Her2-ADC on human breast cancer cell line SKBR3

[0426] Table 13-2. Anti-proliferative activity test data of anti-Her2-ADC on human breast cancer cell line SKBR3 and human gastric cancer cell line NCI-N87

[0427] Under the experimental conditions, the anti-Her2-ADC disclosed herein exhibited strong proliferation inhibitory activity against the Her2-highly expressing human breast cancer cell line SKBR3 and / or the Her2-highly expressing human gastric cancer cell line NCI-N87.

[0428] 7.3.2. Anti-proliferative activity test of anti-CDH6-ADC on tumor cells

[0429] Cells and materials: Human ovarian cancer cell line OVCAR3 was purchased from ATCC, 1640 medium (Gibco#A10491-01), penicillin-streptomycin (Gibco#15140-122) and 0.25% Trypsin-EDTA (Gibco#25200-056) were purchased from Gibco (USA), bovine insulin (Solarbio#I8040) was purchased from Solarbio, 96-well plates (Greiner Bio-one#655098) were purchased from Corning (USA), and Cell-Titer Glo reagent (Promega#G7568) was purchased from Promega (USA).

[0430] Cell culture: OVCAR3 cells were cultured in 1640 medium containing 20% ​​fetal bovine serum, 2 μg / mL bovine insulin, and 1% penicillin-streptomycin at 37°C in 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0431] Cell proliferation activity assay: The inhibitory activity of ADCs against OVCAR3 cell proliferation was assessed using Cell-Titer Glo reagent. OVCAR3 cells were dissociated from the cell culture flask and resuspended in fresh culture medium. The cell density was adjusted to 5000 cells / 90 μL / well. The cells were seeded in a 96-well plate and incubated overnight at 37°C, 5% CO2. The ADC was diluted to a concentration of 1000 nM using complete culture medium and serially diluted three-fold for a total of nine concentrations. Then, 10 μL of the diluted ADC solution was transferred to a 96-well plate, resulting in a starting and ending ADC concentration of 100 nM. The 96-well plate was incubated at 37°C, 5% CO2 for 5 days. Cell-Titer Glo reagent was then added to assess cell viability.

[0432] A negative control group (bottom) and a positive control group (top) were set up. The negative control group did not contain cells, but only the same volume of culture medium. All other procedures were the same as those for the experimental group. The positive control group did not contain the test drug. All other procedures were the same as those for the experimental group.

[0433] Data Analysis:

[0434] Calculate the inhibition percentage (% Inhibition) and fit to obtain the IC of the compound 50 .

[0435] Percent inhibition (% Inhibition) = 1-100%*(Signal-Bottom) / (Top-Bottom).

[0436] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0437] The experimental results are shown in Table 14.

[0438] Table 14. Anti-proliferative activity test data of anti-CDH6-ADC on human ovarian cancer cell line OVCAR3

[0439] Note: “ / ” indicates IC cannot be fitted 50 .

[0440] Under the experimental conditions, the CDH6-ADC disclosed herein exhibited strong proliferation inhibitory activity against the human ovarian cancer cell line OVCAR3.

[0441] 7.3.3 Anti-LIV-1 ADC Antiproliferative Activity Test on Tumor Cells

[0442] Cells and materials: LIV-1-expressing human ovarian cancer cell line OVCAR3 and LIV-1-overexpressing human non-small cell lung cancer cell line NCI-H838 were purchased from ATCC, 1640 culture medium (Gibco#A10491-01), penicillin-streptomycin (Gibco#15140-122) and 0.25% Trypsin-EDTA (Gibco#25200-056) were purchased from Gibco (USA), bovine insulin (Solarbio#I8040) was purchased from Solarbio, 96-well plates (Greiner Bio-one#655098) were purchased from Corning (USA), and Cell-Titer Glo reagent (Promega#G7568) was purchased from Promega (USA).

[0443] Cell culture: OVCAR3 cells were cultured in 1640 medium containing 20% ​​fetal bovine serum, 2 μg / mL bovine insulin, and 1% penicillin-streptomycin at 37°C and 5% CO2. H838 cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0444] Cell proliferation activity assay: The inhibitory activity of ADCs against OVCAR3 and NCI-H838 cell lines was assessed using Cell-Titer Glo reagent. OVCAR3 or NCI-H838 cells were dissociated from the cell culture flask and resuspended in fresh culture medium. The cell density was adjusted to 1500 cells / 90 μL / well for OVCAR3 cells and 450 cells / 90 μL / well for NCI-H838 cells. The cells were seeded in a 96-well plate and incubated overnight at 37°C, 5% CO2. The ADC was diluted to 100 nM in complete culture medium and serially diluted three-fold for a total of eight dilutions. Then, 10 μL of the diluted ADC solution was transferred to a 96-well plate, giving a starting and ending ADC concentration of 100 nM. The 96-well plate was incubated at 37°C, 5% CO2 for 5 days. Cell-Titer Glo reagent was then added, and cell viability was assessed.

[0445] A negative control group (bottom) and a positive control group (top) were set up. The negative control group did not contain cells, but only the same volume of culture medium. All other procedures were the same as those for the experimental group. The positive control group did not contain the test antibody. All other procedures were the same as those for the experimental group.

[0446] Data Analysis:

[0447] Calculate the inhibition percentage (% Inhibition) and fit to obtain the IC of the compound 50 .

[0448] Percent inhibition (% Inhibition) = 1-100%*(Signal-Bottom) / (Top-Bottom).

[0449] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0450] The experimental results are shown in Table 15.

[0451] Table 15. Anti-proliferative activity test data of anti-LIV-1 ADC against NCI-H838 and OVCAR3

[0452] Under the experimental conditions, the anti-LIV-1 ADC disclosed in the present invention exhibited strong proliferation inhibitory activity against human ovarian cancer cell line OVCAR3 and human non-small cell lung cancer cell line H838. Compared with the control ADC3, the IC inhibitory activity of ADC-7, ADC-8, and ADC-9 against NCI-H838 and OVCAR3 was 50 Smaller, indicating stronger tumor-suppressing activity.

[0453] Example 8: Antibody Drug Conjugate Drug Release Efficiency Test

[0454] 8.1 Release Efficiency Test of Antibody Drug Conjugates in Tumor Tissue Homogenate

[0455] The purpose of this experimental study was to compare the cleavage efficiency of different antibody-drug conjugates in tumor tissue.

[0456] 1. Take NCI-H838 tumor tissue blocks, mince them on ice, weigh them, transfer them to a grinding tube, add grinding steel beads, and grind them in 50mM acetic acid-sodium acetate (pH 5.5) and 1mM ethylenediaminetetraacetic acid (EDTA) buffer at a ratio of 1:4 (w / v) to prepare a tissue homogenate for later use.

[0457] 2. Add 3 μg of ADC sample to 100 μl of homogenate and incubate at 37°C for 0, 2, 4, 8, and 24 hours. After each sample reaction, add 1 ml of acetonitrile precipitant and vortex for 5 minutes to terminate the reaction. Centrifuge and store the supernatant at -80°C.

[0458] 3. Detect the free toxin in the sample by LC-MS / MS and calculate the toxin release ratio.

[0459] 4. Toxin release ratio (%) = (MMAE concentration in supernatant) / ((ADC concentration * MMAE molecular weight * DAR) / ADC molecular weight)

[0460] The test results are shown in Table 16: The following ADC molecules all effectively released the toxin-loaded MMAE into tumor homogenates, with release efficiency increasing with prolonged incubation. Compared to the control molecules (control ADC1, control ADC2, control ADC3, and control ADC4), ADC-2, ADC-5, ADC-8, and ADC-11 demonstrated higher release efficiency at all time points.

[0461] Table 16. Release efficiency test of antibody drug conjugates in tumor tissue homogenate

[0462] 8.2 Antibody Drug Conjugate Cleavage Efficiency Test by Human Cathepsin B

[0463] The purpose of this study was to compare the cleavage efficiency of human cathepsin B on different antibody-drug conjugates.

[0464] 1. Add 50 μg of ADC sample to 100 μl of enzyme digestion buffer (50 mM acetic acid-sodium acetate pH 5.5, 1 mM ethylenediaminetetraacetic acid (EDTA)). Add cathepsin B (Human Cathepsin B, Acro brand, cat. no. CTB-H5222) at a molar ratio of enzyme:substrate = 1:100. Incubate at 37°C for 0, 2, 4, and 8 hours.

[0465] 2. After the reaction is completed, add cysteine ​​protease inhibitor E-64 (MCE brand, product number HY-15282) at an equal molar ratio to the enzyme, and store the sample at -80°C.

[0466] 3. Detect the free toxin in the sample by LC-MS / MS and calculate the toxin release ratio.

[0467] 4. Toxin release ratio (%) = (MMAE concentration in supernatant) / ((ADC concentration * MMAE molecular weight * DAR) / ADC molecular weight)

[0468] The test results are shown in Table 17: The following ADC molecules can effectively release the toxin payload MMAE under enzymatic digestion by human cathepsin B, and the release efficiency gradually increases with prolonged incubation time. Compared with the control molecules (control ADC1, control ADC2, control ADC3, and control ADC4), ADC-2, ADC-5, ADC-8, and ADC-11 showed higher release efficiency at all time points.

[0469] 17. Test of the enzymatic cleavage efficiency of antibody drug conjugates in human cathepsin B

[0470] Example 9: In vitro plasma stability of antibody drug conjugates

[0471] A certain amount of ADC sample was added to sterile IgG-depleted human plasma to a final ADC concentration of 0.4 mg / ml. The samples were incubated at 37°C for 0, 1, 3, and 7 days. After incubation, the samples were stored in a -80°C freezer. 50 μL of Protein A filler (MabSelect SuRe) was added to each tube and adsorbed using a shaker for 1 hour. After washing, elution, and Tris-HCl neutralization, the incubated ADC samples were obtained. Their DAR values ​​were determined by HIC-HPLC to determine the plasma stability of the samples. The test results are shown in Table 18.

[0472] Table 18. In vitro plasma stability of antibody drug conjugates

[0473] The experimental results show that the DAR value of the ADC of the present invention does not change significantly during the plasma incubation process, and has good stability in plasma.

[0474] Example 10: Evaluation of the efficacy of antibody drug conjugates in the NCI-H838 subcutaneous tumor model

[0475] Human lung cancer NCI-H838 cells: Kebai

[0476] RPMI-1640 culture medium: Gbico; Cat No.: 61870-036

[0477] Fetal bovine serum: Gibco; Cat No.: 10099-141C

[0478] 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072

[0479] D-PBS (Calcium- and magnesium-free phosphate buffered saline): Hyclone, Cat. No.: SH30256.01

[0480] Matrigel:Corning,Cat.No.:356237

[0481] Experimental methods:

[0482] Animal information: B-NDG mice, female, 5-6 weeks old, weighing approximately 14-20 g, were purchased from Biocytogen. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to standard certified commercial laboratory diet and free drinking water.

[0483] Cell Culture: Human lung cancer NCI-H838 cells were cultured in RPMI-1640 (cell culture medium) supplemented with 10% fetal bovine serum and 1% Pen-Strep in a 37°C, 5% CO2 incubator. Cells were routinely digested and passaged twice weekly using 0.25% trypsin-EDTA. Cells were harvested and counted when cell saturation reached 80%-90% and the required number of cells was reached.

[0484] Cell inoculation: 0.1 ml (containing 1×107) NCI-H838 cell suspension (RPMI-1640:Matrigel, volume ratio 1:1) was subcutaneously inoculated into the axilla of each mouse. On day 21 after cell inoculation, mice were randomly divided into groups based on tumor volume for dosing, with grouping designated Day 0.

[0485] Administration: The dose of ADC-16, ADC-17, ADC-18, ADC-19, and the control ADC-20 was 3 mg / kg, administered intraperitoneally once every 8 days, for a total of three doses during the experimental period. Each group consisted of 6 mice.

[0486] Tumor measurements and experimental parameters:

[0487] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5 axb 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of mice was measured twice a week.

[0488] The anti-tumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume of a treatment group at the end of dosing - mean tumor volume of the treatment group at the start of dosing) / (mean tumor volume of the solvent control group at the end of treatment - mean tumor volume of the solvent control group at the start of treatment)] x 100%.

[0489] Experimental results:

[0490] In the mouse subcutaneous transplant tumor NCI-H838 model, the conjugates ADC-16, ADC-17, ADC-18, and ADC-19 of the present application all had a significant inhibitory effect on tumor growth at a dose of 3 mpk (P < 0.0001). The efficacy of each ADC-16, ADC-17, and ADC18 molecule was similar, and all significantly superior to the ADC-19 molecule (P < 0.0001). The isotype control ADC-20 had no significant tumor inhibitory effect. The examples of the present invention were not found to affect mouse body weight at the doses tested, nor did they cause any mouse deaths, indicating that the mice were well tolerated. See Table 19 and Figure 2 for the results.

[0491] Table 19. Efficacy evaluation of antibody drug conjugates in the NCI-H838 subcutaneous tumor model

Claims

1. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the general structural formula is Pc-(L a -D) n ,in, Pc is the ligand unit; L a is a connecting subunit of the following structure, in, L a The b end of is connected to D; R 1 , R 2 Each independently selected from The a end and L 1 or L 2 Connected to the other end, and the other end is connected to the PC; L 1 , L 2 , L 3 Each independently selected from C1-C6 alkylene, -C(=O)NR e - and any combination thereof; R e Selected from H or C1-C6 alkyl, the C1-C6 alkyl is optionally substituted by one or more halogen, CN, =O, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2; L 4 selected from cleavable units; D is the drug unit; n is a real number ranging from 1 to 8.

2. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein L 1 , L 2 Each independently selected from C1-C6 alkylene; or L 1 , L 2 Are selected from -CH2CH2-.

3. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein L 3 Selected from -CH2- or 4. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein L 4 Selected from Where W 1 The peptide residues are selected from 2 to 8 amino acids, for example selected from 2 to 5 amino acids, which are optionally substituted by halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl, is substituted with one or more substituents; Preferably, W 1 A peptide residue selected from Val-Cit, Gly-Gly-Phe-Gly or Gly-Gly-Gly-Phe-Gly, the peptide residue being optionally is substituted with one or more substituents.

5. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein L a Selected from 6. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the drug unit D is selected from a tubulin inhibitor, a DNA damaging agent or a topoisomerase inhibitor; or The drug unit D is selected from topoisomerase I inhibitors or auristatins; or The drug unit D is selected from MMAE and MMAE derivatives; or The drug unit D is selected from 7. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein the ligand-drug conjugate is selected from the following compounds of formula (Ia): Pc, R 1 , R 2 , L 1 , L 2 , L 3 , L 4 , D, n as defined in any one of claims 1-6.

8. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein the ligand-drug conjugate is selected from the following compounds:

9. A formula (L a ) is used to connect the ligand unit and the drug unit to obtain a ligand-drug conjugate, in, R 1 , R 2 , L 1 , L 2 , L 3 , L 4 As defined in any one of claims 1 to 5, L a The b-end of the drug unit is connected to the drug unit, R 1 , R 2 The end is connected to the ligand unit.

10. A drug-linker compound represented by formula (IIa) or a pharmaceutically acceptable salt thereof, in, L 1 , L 2 , L 3 , L 4 , D as defined in any one of claims 1-6.

11. The drug-linker compound or a pharmaceutically acceptable salt thereof according to claim 10, wherein the drug-linker compound is selected from the following compounds:

12. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the general structural formula is Pc-(L b -D) n ,in, Pc is the ligand unit; L b is a connecting subunit of the following structure, in, L b The b end of is connected to D; R 3 , R 4 Each independently selected from The a end and R 5 or R 6 The connected secondary carbon atom is connected to the other end and connected to Pc; R 5 , R 6 Each is independently selected from C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl, wherein the C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted with one or more halogen, CN, =O, C1-C3 alkyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2; L 7 , L 8 , L 9 Each is independently selected from C1-C6 alkylene, the C1-C6 alkylene is optionally substituted by one or more halogen, CN, =O, C1-C3 alkyl, OH, NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2; L 10 is a cleavable unit; D is the drug unit; n is a real number ranging from 1 to 8.

13. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 12, wherein R 5 , R 6 Each is independently selected from C1-C3 alkyl, the C1-C3 alkyl being optionally substituted by one or more halogen, CN, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2; or R 5 , R 6 All are selected from CH2NH2.

14. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 12 or 13, wherein L 7 , L 8 , L 9 Each independently selected from C1-C6 alkylene; or L 7 , L 8 All selected from CH2, L 9 Selected from CH2CH2.

15. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 12 to 14, wherein L 10 Selected from W 2 selected from peptide residues consisting of 2 to 8 amino acids; or L 10 Selected from Where W 2 Selected from Val-Cit.

16. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 12 to 15, wherein L b Selected from 17. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 12 to 16, wherein D is selected from auristatins; or The D is selected from MMAE and MMAE derivatives; or The D is selected from 18. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 12 to 17, wherein the ligand-drug conjugate is selected from the following compounds of formula (Ib): in, Pc, R 3 , R 4 , R 5 , R 6 , L 7 , L 8 , L 9 , L 10 , D, n as defined in any one of claims 12-17.

19. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 12 to 18, wherein the ligand-drug conjugate is selected from the following compounds:

20. A formula (L b ) is used to connect the ligand unit and the drug unit to obtain a ligand-drug conjugate, in, R 3 , R 4 , R 5 , R 6 , L 7 , L 8 , L 9 , L 10 As defined in any one of claims 12 to 16, L b The b-end of the drug unit is connected to the drug unit, R 3 , R 4 The end is connected to the ligand unit.

21. A drug-linker compound of formula (IIb) or a pharmaceutically acceptable salt thereof, in, R 5 , R 6 , L 7 , L 8 , L 9 , L 10 , D as defined in any one of claims 12-17.

22. The drug-linker compound or a pharmaceutically acceptable salt thereof according to claim 21, wherein the drug-linker compound is selected from the following compounds:

23. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-8, 12-19, wherein the ligand unit Pc is selected from a polypeptide, an antibody or an antigen-binding fragment thereof.

24. The ligand-drug conjugate according to claim 23, or a pharmaceutically acceptable salt thereof, wherein the ligand unit Pc specifically binds to one or more antigens selected from the group consisting of HER2, p95HER2, HER3, CD3, CD16, ROR1, DLL3, CDH6, CD70, CD5, CD20, BCMA, EGFR, VEGF, Trop-2, Claudin18.2, c-MET, CD30, MUC1, Nectin-4, CD22, FR-a, CD74, CD19, CD79b, MSLN, TOPO2, EpCAM, CEACAM-5, Mesothelin, PD-L1, PSMA, ROR1, ROR2, CD25, CD33, CD123, FLT3, CD174, CD166, CD326, CD71, and LIV-1; or The ligand unit Pc specifically binds to one or more antigens selected from the following: HER2, p95HER2, ROR1, CDH6 and LIV-1; or The ligand unit Pc is an antibody or an antigen-binding fragment thereof that specifically binds to HER2, CDH6 or LIV-1; the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) or / and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, or / and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: The HCDR1-3 and / or the LCDR1-3 are selected from the following: (1) the HCDR1-3 are SEQ ID NOs: 14-16; or / and the LCDR1-3 are SEQ ID NOs: 17-19; (2) the HCDR1-3 are SEQ ID NOs: 20-22; or / and the LCDR1-3 are SEQ ID NOs: 23-25; (3) the HCDR1-3 are SEQ ID NOs: 26-28; or / and the LCDR1-3 are SEQ ID NOs: 29-31; (4) the HCDR1-3 are SEQ ID NOs: 34-36; or / and the LCDR1-3 are SEQ ID NOs: 37-39; (5) the HCDR1-3 are SEQ ID NOs: 40-42; or / and the LCDR1-3 are SEQ ID NOs: 43-45; or, (6) The HCDR1-3 and / or the LCDR1-3 have an amino acid sequence that is at least 80% identical to the corresponding CDR in any one of the HCDR1-3 and / or LCDR1-3 in groups (1) to (5), or has at most 3 insertion, deletion or substitution mutations; preferably, the HCDR1-3 and / or the LCDR1-3 have an amino acid sequence that is at least 80% identical to the corresponding CDR in any one of the HCDR1-3 and / or LCDR1-3 in groups (1) to (5); more preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

25. The ligand-drug conjugate according to claim 23 or a pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the heavy chain variable region and / or the light chain variable region are selected from the following: (1) the heavy chain variable region is the sequence shown in SEQ ID NO: 1, and / or the light chain variable region is the sequence shown in SEQ ID NO: 2; (2) the heavy chain variable region is the sequence shown in SEQ ID NO: 3, and / or the light chain variable region is the sequence shown in SEQ ID NO: 4; (3) the heavy chain variable region is the sequence shown in SEQ ID NO: 7, and / or the light chain variable region is the sequence shown in SEQ ID NO: 8; (4) the heavy chain variable region is the sequence shown in SEQ ID NO: 9, or / and the light chain variable region is the sequence shown in SEQ ID NO: 10; (5) the heavy chain variable region is the sequence shown in SEQ ID NO: 11, and / or the light chain variable region is the sequence shown in SEQ ID NO: 12; or, The heavy chain variable region and / or the light chain variable region have a sequence that is at least 80% identical to the heavy chain variable region and / or the light chain variable region in any one of the above groups (1) to (5), or a sequence in which at most three insertion, deletion or substitution mutations occur; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

26. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-8, 12-19, wherein n is selected from a real number of 1-6; preferably, n is selected from a real number of 1-5; more preferably, n is selected from a real number of 2-4.

27. A pharmaceutical composition comprising the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-8, 12-19, 23-26 and a pharmaceutically acceptable excipient.

28. Use of the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-8, 12-19, 23-26, or the pharmaceutical composition according to claim 27 in the preparation of a drug for treating tumors.

29. A method for preparing the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-8, 12-19, 23-26, comprising the step of coupling the drug-linker compound according to any one of claims 10-11, 21-22 with a ligand, wherein the ligand is an antibody or an antigen-binding fragment.

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