Antibody-drug conjugate, and preparation method therefor and use thereof

Abstract

Provided in the present application are an antibody-drug conjugate, and a preparation method therefor and the use thereof. The antibody-drug conjugate has a structure as represented by the following formula (III) or formula (IV). The drug is selected from a DNA topoisomerase inhibitor, a DNA intercalator, an RNA polymerase inhibitor, and a PARP inhibitor. The prepared antibody-drug conjugate exhibits a relatively good drug-to-antibody ratio and good targeted killing effects on breast cancer, gastric cancer, and lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma).

Description

Antibody drug conjugates, and methods of making and using the same

[0001] This application is based on and claims priority to CN application No. 202411376020.9, filed on September 29, 2024, the entire contents of which are hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the field of medicine, and relates to antibody drug conjugates, and methods of making and using the same. BACKGROUND

[0003] Since the 20th century, biological macromolecular drugs (such as therapeutic antibodies or antibody fragments) and targeted small molecule ligands have been used for the development of anti-tumor drugs and tumor targeted therapy, and breakthrough progress has been made. However, biological macromolecular drugs have limited therapeutic effect on solid tumors, although they have strong targeting properties; and bioactive molecules have high killing efficiency on cancer cells, but often lack targeting properties, often mistakenly harming normal cells, thereby causing serious side effects.

[0004] In recent years, it has been found that therapeutic antibodies can be connected to bioactive molecules to form antibody drug conjugates (ADC). ADC combines the targeting effect of antibodies and the activity of bioactive molecules. Antibodies guide ADC to bind to target cells, and then are internalized by cells to release drugs to kill cells and treat diseases. Because antibodies have specificity and targeting properties for tumor cell-related targets, their application value not only lies in treatment, but also makes them ideal carriers for drug targeted delivery, reducing the side effects of drugs.

[0005] PARP inhibitors are a class of cancer therapies that target the enzyme poly ADP-ribose polymerase (PARP). The mechanism of action of PARP inhibitors is "synthetic lethality", which means that when two different genes or proteins are mutated at the same time, the cell dies, but if only one of the two genes / proteins is mutated, the cell does not die. The role of PARP1 is to bind to DNA damage sites (mostly single-strand DNA breaks) and catalyze the synthesis of poly ADP-ribose chains on protein substrates. Through this catalytic action, PARP1 is able to recruit other DNA repair proteins to the damage site to repair the DNA damage. PARP inhibitors bind to the catalytic site of PARP1 or PARP2, preventing the PARP protein from dissociating from the DNA damage site. The PARP protein, which is bound to the DNA, causes the DNA replication fork to stall and the DNA replication to fail. At this point, the cell usually triggers a repair method called homologous recombination repair (HRR) to repair the error. BRCA1, BRCA2 and other proteins called "BRCAness" play an important role in HRR, and when these proteins are damaged, causing HRR dysfunction, the cell usually uses other DNA repair methods that can introduce large-scale genomic rearrangements, leading to cell death.

[0006] It should be noted that although PARP inhibitors are used to treat BRCA-mutated tumors and successfully apply the concept of synthetic lethality to the treatment of tumors, the current application of PARP inhibitors is not limited to BRCA-mutated tumors. Tumors with HRR function loss also benefit from PARP inhibitors. For tumor cells that do not carry BRCA mutations, mutations in other DNA repair processes can also cause HRR function loss in cells, making them sensitive to PARP inhibitors. This will greatly expand the scope of PARP inhibitors. Studies have shown that some patients with advanced ovarian cancer, advanced prostate cancer and metastatic pancreatic cancer may benefit from PARP inhibitor therapy.

[0007] Although the initial purpose of developing PARP inhibitors is to make tumor cells more sensitive to chemotherapy that causes DNA damage, the clinical performance of PARP inhibitors combined with chemotherapy drugs is uneven, mainly because the toxic side effects of chemotherapy on healthy cells often limit the dose of the drug, and the use of PARP inhibitors usually enhances the toxic side effects on healthy cells.

[0008] There is a report that the ADC drug IMMU-132 has a combined damage effect of Topo I inhibition activity and PARP inhibitor to destroy the DNA repair of triple negative breast cancer (TNBC). The results show that IMMU-132 combined with all three different PARP inhibitors can synergistically inhibit tumor growth, increase dsDNA breaks and cell accumulation in the cell cycle regardless of the BRCA1 / 2 status. In BRCA1 / 2 mutant HCC1806 TNBC tumor mice, IMMU-132 combined with olaparib or talazoparib can significantly improve the anti-tumor effect and delay tumor progression time compared with single drug treatment. In addition, in mice carrying BRCA1 / 2 wild-type tumors (MDA-MB-468 or MDA-MB-231), IMMU-132 combined with olaparib has a significant anti-tumor effect and survival benefit than single drug treatment. Most importantly, this combination therapy is well tolerated, and there is no significant change in hematological parameters. These data show that in TNBC, regardless of the status of BRCA1 / 2, the combination of Topo I inhibition mediated by IMMU-132 and the synthetic lethality provided by PARP inhibitors achieves a synergistic effect, thus supporting the theoretical basis of this combination in the clinic. (Clin Cancer Res; 23(13); 3405-15.)

[0009] Based on the targeting and effectiveness of antibody drug conjugates, mounting the same or different proportions of small molecule PARP inhibitors and DNA intercalators, DNA topoisomerase inhibitors or RNA polymerase inhibitors and other cytotoxic drugs on a single antibody through appropriate linking methods is an innovative attempt that is expected to further improve the precision, effectiveness and safety of chemotherapy drugs and PARP inhibitors in treating tumors, and achieve a synergistic therapeutic effect. SUMMARY

[0010] The present application finds a novel drug linker and its antibody drug conjugate, which simultaneously mounts small molecule PARP inhibitors and DNA topoisomerase inhibitors, DNA intercalators or RNA polymerase inhibitors and other cytotoxic drugs on a single antibody through a branched linker, or mounts the same or different proportions of small molecule PARP inhibitors and DNA topoisomerase inhibitors, DNA intercalators or RNA polymerase inhibitors and other cytotoxic drugs on a single antibody through different linking methods, improving the targeting of PARP inhibitors and cytotoxic drugs, and playing a synergistic role.

[0011] Drug linker

[0012] In one aspect, the application provides a drug-linker that is a compound of the following formula (I) or formula (II), or a pharmaceutically acceptable salt thereof:

[0013] wherein:

[0014] M a is the structure before attachment to an antibody or antigen binding fragment thereof;

[0015] B is a moiety linking M a and L and L';

[0016] L and L' are moieties linking B and E, E' between M a and E;

[0017] E and E' are moieties linking L and L' to D and D';

[0018] D and D' are each independently a moiety of a pharmaceutically active molecule.

[0019] The application also provides a drug-linker that is a compound of the following formula (II') or a pharmaceutically acceptable salt thereof:

[0020] M a -L'-E'-D'

[0021] formula (II')

[0022] wherein: M a , L', E' and D' are as described above.

[0023] In some embodiments, the pharmaceutically active molecule is selected from a cytotoxic drug or a PARP inhibitor.

[0024] In some embodiments, D is a moiety of a pharmaceutically active molecule that is a cytotoxic drug.

[0025] In some embodiments, the cytotoxic drug is a DNA intercalator, a DNA topoisomerase inhibitor, or an RNA polymerase inhibitor.

[0026] In some embodiments, D' is a moiety of a pharmaceutically active molecule that is a PARP inhibitor.

[0027] In some embodiments, M a is selected from the following structures at each occurrence:

[0028] wherein R represents hydrogen, C 1-6alkyl or -(CH2CH2O)p-alkyl, wherein p is, at each occurrence, independently selected from an integer from 1 to 12. p -alkyl, wherein p is, at each occurrence, independently selected from an integer from 1 to 12.

[0029] In some embodiments, M a is independently selected from the following structures:

[0030] In some embodiments, M a is selected from the following structures:

[0031] In some embodiments, R is selected from hydrogen and methyl.

[0032] In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

[0033] In some embodiments, M a is

[0034] In some embodiments, M a is

[0035] In some embodiments, M a is R is hydrogen.

[0036] In some embodiments, B is selected from the following structures:

[0037] wherein q is, at each occurrence, independently selected from an integer from 1 to 24.

[0038] In some embodiments, B is selected from the following structures and combinations thereof:

[0039] wherein q is, at each occurrence, independently selected from an integer from 1 to 24.

[0040] In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0041] In some embodiments, B is

[0042] In some embodiments, L and L' are each independently selected from one or more substituted or unsubstituted structural fragments that are divalent and are selected from the group consisting of C 1-6alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 5-12 membered heterocyclyl, -N(R')-, carbonyl, -0-, a natural amino acid or a non-natural amino acid and analogs thereof (such as Ala, Arg, Asn, Asp, Cit, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2) r OCH3)), Lys(R'), a short peptide consisting of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG, SEQ ID NO: 44), Gly-Gly-Val-Ala (GGVA, SEQ ID NO: 45), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO: 46), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO: 47), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO: 48), Ala-Ala-Glu), EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, NOPO,

[0043] wherein said Ra is EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, or NOPO; R' represents hydrogen, C 1-6 alkyl or -(CH2CH2O) r r, at each occurrence, is independently selected from an integer between 1 and 12.

[0044] In some embodiments, L and L' are each independently selected from one or more substituted or unsubstituted structural fragments consisting of C 1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 5-12 membered heterocyclyl, -N(R')-, carbonyl, -O-, a natural amino acid or a non-natural amino acid and analogs thereof (such as Ala, Arg, Asn, Asp, Cit, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2) r OCH3)), Lys(R'), a short peptide consisting of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG, SEQ ID NO: 44), Gly-Gly-Val-Ala (GGVA, SEQ ID NO: 45), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO: 46), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO: 47), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO: 48), Ala-Ala-Glu), EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, NOPO,

[0045] wherein said Ra is EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, or NOPO; R' represents hydrogen, C 1-6 alkyl or -(CH2CH2O) ralkyl of the formula: -CnH(2n+1); r is, at each occurrence, independently selected from an integer between 1 and 12.

[0046] In some embodiments, "DOTA" refers to

[0047] In some embodiments, Lys(R') refers to

[0048] In some embodiments, L and L' are each independently selected from one or more (1, 2, 3, 4, or 5) substituted or unsubstituted structural fragments consisting of:

[0049] Ala, Arg, Cit, Gly, Lys, Phe, Val, wherein r is, at each occurrence, independently selected from an integer between 1 and 12; n is, at each occurrence, independently selected from an integer between 1 and 20.

[0050] In some embodiments, r is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. In some embodiments, r is selected from 3, 5, 8, 11.

[0051] In some embodiments, each of L and L' independently contains 1 or 2 structural fragments of the following:

[0052] In some embodiments, L and L' are each independently selected from the following structures: wherein n is selected from an integer between 1 and 20.

[0053] In some embodiments, n is selected from an integer between 1 and 12. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In some embodiments, n is 3, 5, 8, 10, 11.

[0054] In some embodiments,

[0055] In some embodiments,

[0056] In some embodiments,

[0057] ​​​In some embodiments, L and L' are each independently selected from the following structures:

[0058] In some embodiments, L and L' are each independently selected from the following structures:

[0059] In some embodiments, L and L' are each independently selected from the following structures:

[0060] In some embodiments, L and L' are each independently selected from the following structures:

[0061] In some embodiments, E and E' are each independently selected from a single bond, -NH-CH2-, or from the following structures: wherein n is, at each occurrence, independently selected from an integer from 1-20.

[0062] In some embodiments, E and E' are each independently selected from a single bond, -NH-CH2-, or from the following structures: wherein n is, at each occurrence, independently selected from an integer from 1-20.

[0063] In some embodiments, n is, at each occurrence, independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0064] In some embodiments, E and E' are each independently selected from a single bond, -NH-CH2-,

[0065] In some embodiments, E and E' are each independently selected from a single bond, -NH-CH2-, and

[0066] In some embodiments, E and E' are each independently selected from -NH-CH2-, and

[0067] In some embodiments, E and E' are each independently selected from a single bond, and -NH-CH2-.

[0068] In some embodiments, the pharmaceutically active molecule is selected from the group consisting of an anti-tubulin agent, a DNA intercalator, a DNA topoisomerase inhibitor, an RNA polymerase inhibitor, and a PARP inhibitor. In some embodiments, the tubulin inhibitor is a maytansinoid or a dolastatin. In some embodiments, the DNA intercalator is a pyrrolobenzodiazepine (PBD). In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., a camptothecin, such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is triptolide, a-amanitin, and pharmaceutically acceptable salts, esters, and analogs thereof. In some embodiments, the PARP inhibitor is olaparib, niraparib, pamiparib, talazoparib, rucaparib, veliparib, or talazopanib. In some embodiments, the PARP inhibitor can also be AZ9482.

[0069] The pharmaceutically active molecules disclosed in this application typically contain various functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), secondary amine (-NR1H), tertiary amine (-NR2R3), where R1, R2, R3 here represent only non-hydrogen substituents on N, or thiol (-SH), which can be linked to the rest of the conjugate through chemical reactions.

[0070] In some embodiments, the pharmaceutically active molecules are each independently linked to E or E' in the antibody drug conjugate through an -OH, a primary amino group, a secondary amine group, a tertiary amine group, or a -SH on it.

[0071] In some embodiments, the pharmaceutically active molecule is selected from the group consisting of the following compounds:

[0072] In some embodiments, the pharmaceutically active molecule is selected from the group consisting of the following compounds:

[0073] In some embodiments, the pharmaceutically active molecule is selected from the group consisting of the following compounds:

[0074] In some embodiments, the cytotoxic drug is selected from the group consisting of the following compounds:

[0075] In some embodiments, the PARP inhibitor is selected from the following compounds:

[0076] In some embodiments, D and D' are each independently selected from the following structures:

[0077] In some embodiments, D and D' are each independently selected from the following structures:

[0078] In some embodiments, D is selected from the following structures:

[0079] In some embodiments, D is selected from the following structures:

[0080] In some embodiments, D' is selected from the following structures:

[0081] In some embodiments, D' is

[0082] In some embodiments, the "drug-linker" in free form is selected from the following A-1 ~ A-13, B-1 ~ B-8, C-1 ~ C-4, D-1 ~ D-3 and F-1 ~ F-3:

[0083] Antibody drug conjugate

[0084] In another aspect, the present application provides an antibody drug conjugate having

[0085] wherein B, L, E, D, L', E', D' are as described in any one of the above,

[0086] Ab is an antibody or an antigen binding fragment thereof;

[0087] M and M' are each independently M as described in any one of the above a a structural form after covalently linked to an antibody or an antigen binding fragment thereof; x and y are each independently selected from 1 to 10.

[0088] In the antibody drug conjugate, D and D' can be linked through a linker (such as shown in the present application "M-L-E", "M'-L'-E'" fragment) is linked to the antibody or antigen-binding fragment thereof.

[0089] In some embodiments, M and M' are independently selected from the following structures:

[0090] wherein R represents hydrogen, C 1-6 alkyl or -(CH2CH20)p- containing alkyl, wherein p is selected from an integer between 1 and 12. p

[0091] In some embodiments, M and M' are independently selected from the following structures:

[0092] In some embodiments, R is selected from hydrogen and methyl.

[0093] In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

[0094] In some embodiments, M and M' are independently In some embodiments, M and M' are independently In some embodiments, M and M' are independently R is hydrogen. In some embodiments, selected from the following structures:

[0095] wherein n is selected from an integer between 0 and 20.

[0096] In some embodiments, selected from the following structures:

[0097] wherein M is the structural form after covalent linkage to the antibody or antigen-binding fragment thereof; n is selected from an integer between 0 and 20. In some embodiments, selected from the following structures:

[0098] In some embodiments, selected from the following structures:

[0099] wherein M, M' are the structures after linkage to the antibody or antigen-binding fragment thereof; n is selected from an integer between 0 and 20. ​

[0100] In some embodiments, selected from the following structures:

[0101] wherein M, M' are the structure after the antibody or antigen-binding fragment thereof is linked; n is selected from an integer from 0-20.

[0102] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that specifically binds to a member of the ErbB family of receptor tyrosine kinases, epidermal growth factor receptor 2 (Her2); or is an antibody or antigen-binding fragment thereof that binds to human trophoblast cell-surface antigens 2 (Trop-2).

[0103] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that specifically binds to a member of the ErbB family of receptor tyrosine kinases, epidermal growth factor receptor 2 (Her2).

[0104] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody against Her 2, such as trastuzumab, Pertuzumab, or a monoclonal antibody against Trop-2, such as hRS7.

[0105] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0106] (1) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0107] (1a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or,

[0108] (1 b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0109] wherein the variant of any one of (1 a), (1 b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions, or additions relative to the sequence from which it is derived; preferably said substitution is a conservative substitution;

[0110] or,

[0111] (2) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined by the AbM numbering system:

[0112] (2a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, CDR-L3 of SEQ ID NO: 10 or a variant thereof; or,

[0113] (2b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0114] wherein the variant of any one of (2a), (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution;

[0115] or,

[0116] (3) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the Kabat numbering system:

[0117] (3a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, CDR-L3 of SEQ ID NO: 10 or a variant thereof; or,

[0118] (3b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0119] wherein the variant of any one of (3a), (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; preferably, said substitution is a conservative substitution;

[0120] or,

[0121] (4) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the IMGT numbering system:

[0122] (4a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or,

[0123] (4b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0124] wherein the variant of any one of (4a), (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived or has one or several (e.g., 1, 2, or 3) amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

[0125] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0126] (1) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0127] (1a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or,

[0128] (1b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; and, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0129] or,

[0130] (2) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system:

[0131] (2a) a heavy chain variable region (VH) comprising CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, and CDR-H3 of SEQ ID NO: 7, and a light chain variable region (VL) comprising CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or,

[0132] (2b) a heavy chain variable region (VH) comprising CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, and CDR-H3 of SEQ ID NO: 22, and a light chain variable region (VL) comprising CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0133] or,

[0134] (3) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the Kabat numbering system:

[0135] (3a) a heavy chain variable region (VH) comprising CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 7, and a light chain variable region (VL) comprising CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or,

[0136] (3b) a heavy chain variable region (VH) comprising CDR-H1 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 27, and CDR-H3 of SEQ ID NO: 22, and a light chain variable region (VL) comprising CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25.

[0137] or,

[0138] (4) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0139] (4a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 10; or,

[0140] (4b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 28, CDR-H2 of SEQ ID NO: 29, and CDR-H3 of SEQ ID NO: 30; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 31, CDR-L2 of SEQ ID NO: 32, and CDR-L3 of SEQ ID NO: 25.

[0141] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0142] (a) a VH of SEQ ID NO: 1 or a variant thereof, and / or, a VL of SEQ ID NO: 2 or a variant thereof; or

[0143] (b) a VH of SEQ ID NO: 3 or a variant thereof, and / or, a VL of SEQ ID NO: 4 or a variant thereof;

[0144] wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or the variant has one or several (e.g. 1, 2, 3, 4 or 5) amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

[0145] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0146] (a) a VH as set forth in SEQ ID NO: 1, and, a VL as set forth in SEQ ID NO: 2; or

[0147] (b) a VH as set forth in SEQ ID NO: 3, and, a VL as set forth in SEQ ID NO: 4.

[0148] In some embodiments, the antibody or antigen-binding fragment thereof further comprises:

[0149] (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more substitutions, deletions, or additions of amino acids as compared to the wild type sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions); and

[0150] (b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more substitutions, deletions, or additions of amino acids as compared to the wild type sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions).

[0151] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgGl, IgG2, IgG3, or IgG4 heavy chain constant region, e.g., a human IgGl heavy chain constant region or a human IgG4 heavy chain constant region.

[0152] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35 or a variant thereof having up to 20 conservative substitutions of amino acids as compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids).

[0153] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 41 or a variant thereof having up to 20 conservative substitutions of amino acids as compared to SEQ ID NO: 41 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids).

[0154] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 41 and a light chain constant region (CL) as set forth in SEQ ID NO: 36.

[0155] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35 and a light chain constant region (CL) as set forth in SEQ ID NO: 36.

[0156] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 41 and a light chain constant region (CL) as set forth in SEQ ID NO: 36.

[0157] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0158] (1) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, and, a light chain comprising a VL as set forth in SEQ ID NO: 2 and a light chain constant region (CL) as set forth in SEQ ID NO: 36;

[0159] (2) a heavy chain comprising a VH as set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, and, a light chain comprising a VL as set forth in SEQ ID NO: 4 and a light chain constant region (CL) as set forth in SEQ ID NO: 36; or

[0160] (3) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 41, and, a light chain comprising a VL as set forth in SEQ ID NO: 2 and a light chain constant region (CL) as set forth in SEQ ID NO: 36; or

[0161] (4) a heavy chain comprising a VH as set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 41, and, a light chain comprising a VL as set forth in SEQ ID NO: 4 and a light chain constant region (CL) as set forth in SEQ ID NO: 36.

[0162] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0163] (1) a heavy chain comprising the sequence set forth in SEQ ID NO: 37, and, a light chain comprising the sequence set forth in SEQ ID NO: 38;

[0164] (2) a heavy chain comprising the sequence set forth in SEQ ID NO: 39, and, a light chain comprising the sequence set forth in SEQ ID NO: 40;

[0165] (3) a heavy chain comprising the sequence set forth in SEQ ID NO: 42, and, a light chain comprising the sequence set forth in SEQ ID NO: 38; or

[0166] (4) a heavy chain comprising the sequence set forth in SEQ ID NO: 43, and, a light chain comprising the sequence set forth in SEQ ID NO: 40.

[0167] In certain embodiments of the antibodies or antigen-binding fragments disclosed herein, the heavy chain constant domain can comprise a C-terminal lysine or lack a C-terminal lysine or lack a C-terminal glycine-lysine dipeptide. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof can be cyclized to pyroglutamic acid.

[0168] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0169] In certain embodiments, provided herein are compositions comprising the antibodies or antigen-binding fragments disclosed herein, where the various antibodies or antigen-binding fragments can independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid, N-terminal amino acid cyclization to pyroglutamic acid, or N-terminal amino acid cyclization to pyroglutamate.

[0170] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein comprise antibodies or antigen-binding fragments that specifically bind to an antigen, and can include post-translational modifications thereof (e.g., C-terminal lysine clipping in the heavy chain, N-terminal glutamine or glutamic acid conversion to pyroglutamic acid or pyroglutamate in the heavy chain or light chain), which can occur upon recombinant expression in a host cell (e.g., a CHO cell) or during purification / storage.

[0171] In certain embodiments, the N-terminal glutamine of the VH set forth in SEQ ID NO: 1 or 3 or a variant thereof or the heavy chain set forth in SEQ ID NO: 37, 39, 42, or 43 or a variant thereof is cyclized to form pyroglutamic acid or pyroglutamate.

[0172] In certain embodiments, the heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 35 or 41 or a variant thereof or the heavy chain of the sequence set forth in SEQ ID NO: 37, 39, 42 or 43 or a variant thereof lacks the C-terminal lysine.

[0173] In some embodiments, Ab is selected from an antibody or antigen-binding fragment thereof that specifically binds to a member of the ErbB family of receptor tyrosine kinases, epidermal growth factor receptor 2 (Her2).

[0174] In some embodiments, Ab is selected from Trastuzumab, Pertuzumab, a Trastuzumab mutant and a Pertuzumab mutant, or is a biparatopic antibody constructed from Trastuzumab and Pertuzumab or an antigen-binding fragment thereof.

[0175] In some embodiments, Ab is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which is assigned the query accession number (IMGT / mAb-DB ID) 97 in the IMGT database, or the amino acid sequence of which is assigned the query accession number (IMGT / mAb-DB ID) 80 in the IMGT database, or an antigen-binding fragment thereof.

[0176] Those skilled in the art will appreciate that the antibody drug conjugates described herein can be prepared modularly. For example, a "drug-linker" in free form as described in any one of the above (e.g. M a -L-E-D, wherein M a is the structural form of M before covalent attachment to the antibody or antigen-binding fragment thereof, and then covalently attaching it to the antibody or antigen-binding fragment thereof to obtain the antibody drug conjugate described herein. Accordingly, M a by substitution reaction (e.g. removal of -SO2Me, pentafluorophenol or -Br, etc. structures thereon) or by addition reaction, etc. to one or more sulfhydryl (-SH) or amino (-NH2) groups on the antibody or antigen-binding fragment thereof.

[0177] In some embodiments, the antibody drug conjugate is selected from the group consisting of ADC A-1 to ADC A-13, ADC B-1 to ADC B-8, ADC C-1 to ADC C-4, ADC D-1 to ADC D-3, ADC F-1 to ADC F-3, ADC E-1 to ADC E-13:

[0178] wherein x, y are each independently 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0179] wherein the HA-(S- and -S)-HA-(NH- in ADC A-1 to ADC A-13, ADC C-1 to ADC C-4, ADC D-1 to ADC D-3, and ADC F-1 to ADC F-3 are selected from any one of the antibodies or antigen binding fragments thereof described above. the HA-(NH- in ADC B-1 to ADC B-8, and -S)-HA-(NH- in ADC E1-E13 are selected from trastuzumab or pertuzumab.

[0180] In some embodiments, the antibody or antigen binding fragment thereof comprises an antibody or antigen binding fragment thereof comprising a VH as set forth in SEQ ID NO: 1 and a VL as set forth in SEQ ID NO: 2.

[0181] In some embodiments, the antibody or antigen binding fragment thereof comprises an antibody or antigen binding fragment thereof comprising a VH as set forth in SEQ ID NO: 3 and a VL as set forth in SEQ ID NO: 4.

[0182] wherein the HA-(S- and -S)-HA-(NH- in ADC A-1 to ADC A-13, ADC C-1 to ADC C-4, ADC D-1 to ADC D-3, and ADC F-1 to ADC F-3 are selected from any one of the antibodies or antigen binding fragments thereof described above. the HA-(NH- in ADC B-1 to ADC B-8, and -S)-HA-(NH- in ADC E1-E13 are selected from trastuzumab or pertuzumab.

[0183] wherein, denotes the specific linkage of a thiol or amine group in the antibody or antigen binding fragment thereof to the linker.

[0184] In some embodiments, x and y are each independently 1 to 10. In some embodiments, x and y are each independently 1 to 8, or x and y are each independently 1 to 4.

[0185] Compositions

[0186] In another aspect, the present application provides a composition of an antibody drug conjugate (ADC) as described herein. Such a composition can comprise a plurality of ADCs described herein, wherein each ADC is conjugated by a drug-linker to an antibody, wherein x is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug-linkers. Thus, the composition is characterized by a drug-antibody ratio (DAR) in the range of about 1 to about 10. Methods to determine DAR are well known to the skilled person, including methods using reverse phase chromatography or HPLC-MS.

[0187] For example, in any embodiment, the ADC composition described herein has a DAR of about 1 to about 10, or any sub-range therebetween, for example: about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10, or about 9 to 10.

[0188] In certain embodiments, the DAR of the ADC compositions described herein is about 1 to 8, for example, about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 3.5 to 7.0, about 3.5 to 7.5, about 3.5 to 8.0, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 7.5, about 4.0 to 8.0, about 4.5 to 5.0, about 4.5 to 5.5, about 4.5 to 6.0, about 4.5 to 6.5, about 4.5 to 7.0, about 4.5 to 7.5, about 4.5 to 8.0, about 5.0 to 5.5, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.0, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.0, about 7.0 to 7.5, about 7.0 to 8.0, about 7.5 to 8.0.

[0189] In certain embodiments, the DAR of the ADC compositions described herein is about 4.0 to 8.0.

[0190] In certain embodiments, the DAR of the ADC compositions described herein is about 6.0 to 8.0.

[0191] In certain embodiments, the DAR of the ADC compositions described herein is about 7.0 to 7.5.

[0192] In certain embodiments, the DAR of the ADC compositions described herein is about 2.5 to 8.0.

[0193] Pharmaceutical compositions

[0194] In another aspect, the present application provides a pharmaceutical composition comprising the antibody drug conjugate or composition of any of the preceding, and one or more pharmaceutically acceptable excipients.

[0195] The antibody drug conjugates described herein are typically formulated in unit injectable form in a parenterally acceptable diluent, carrier, vehicle or stabilizer (Remington's Pharmaceutical Sciences (1980) 16th Edition, Mack Publishing Company, Easton, PA) with a parenterally acceptable gntestinal vehicle, such as a sterile pyrogen free diluent. Optionally, the antibody drug conjugate is in unit dosage form with a preservative to prevent growth on the conjugate. The preparations can be formulated so as to provide quickly, sustained or delayed release of the active ingredient after administration. By way of example, the following methods of administration and compositions are contemplated. th(ed., Osol, A.Ed.). The antibody-drug conjugates described herein or pharmaceutical compositions containing the antibody-drug conjugates may be administered via any route appropriate for the individual to be treated.

[0196] application

[0197] The antibody-drug conjugates, drug-linkers, ADC compositions, or drug compositions thereof described herein can be used to treat a variety of diseases or conditions, such as Her2-expressing cancers, including solid tumors or hematologic malignancies such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

[0198] Therefore, this application provides the use of any of the antibody-drug conjugates, drug-linkers, ADC compositions, or pharmaceutical compositions containing the thereof described in the foregoing in the preparation of a medicament for treating Her2-expressing cancers.

[0199] In addition, this application provides any of the antibody-drug conjugates, drug-linkers, ADC compositions, or pharmaceutical compositions containing the thereof described above for the treatment of Her2-expressing cancers.

[0200] Additionally, this application provides a method for treating Her2-expressing cancer, comprising administering to a subject in need an effective amount of any of the preceding antibody-drug conjugates, drug-linkers, ADC compositions, or pharmaceutical compositions containing the conjugates.

[0201] In some embodiments, the antibody-drug conjugate, drug-linker, ADC composition, or drug composition is sufficient (e.g., in a subject):

[0202] (1) Inhibits the proliferation of cells (such as tumor cells);

[0203] (2) Inhibits tumor growth;

[0204] (3) Inducing and / or increasing antibody-dependent cytotoxic activity;

[0205] (4) Inhibit HER2-mediated signal transduction;

[0206] (5) Prevention and / or treatment of HER2-mediated diseases / disorders; or

[0207] (6) Any combination of (1)-(5) above.

[0208] In some implementations, the cancer or tumor is selected from solid tumors and hematologic malignancies, such as colon cancer, stomach cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

[0209] Definitions

[0210] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. References to techniques employed herein are intended to refer to the techniques as commonly understood by those skilled in the art to which this application pertains, including variations and / or modifications well known to those of ordinary skill in the art. Also, the practice of the present application employs, unless otherwise indicated, conventional methods of genomic science, nucleic acid chemistry, molecular biology, etc., which are well within the purview of the skilled artisan. Although the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the application.

[0211] The term "antibody" refers to an immunoglobulin molecule comprised of two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a different class of antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains exhibit less inter-chain variability than the variable domains, but still exhibit some inter-chain variability, and can be further subdivided into regions of even greater sequence homogeneity, referred to as "microdomains." The variable domains of the heavy and light chains, which specifically interact with an antigen, are referred to as "antigen combining sites" or "antigen binding sites." The VHand VLregions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" (CDRs), interspersed with regions that are more conserved, termed "framework regions" (FRs). Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains, VHand VL, contribute to the specificity of the antibody for its antigen. The assignment of amino acids to each region or domain can follow various numbering systems known in the art.

[0212] The term "complementarity determining region" or "CDR" refers to amino acid residues within the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the variable regions of the heavy and light chains, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, e.g., as defined by the Rabat numbering system (Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, one of skill in the art will readily be able to identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0213] In the present application, the CDRs contained by an antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art, e.g., by the Rabat, Chothia, IMGT, or AbM numbering system. In certain embodiments, the CDRs contained by an antibody or antigen-binding fragment thereof are defined by the Chothia numbering system.

[0214] The term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody that are not CDR residues as defined above.

[0215] The term "antigen binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, e.g., a polypeptide of a fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide stabilized Fv proteins ("dsFv"), single domain antibodies (sdAb, nanobodies), and polypeptides comprising at least a portion of an antibody that is sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0216] The term "Fd" means an antibody fragment consisting of a VH and CHI domain; the term "dAb fragment" means an antibody fragment that consists of a VH domain (Ward et al., Nature 341 :544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of a VL, VH, CL and CHI domain; the term "F(ab')2 fragment" means an antibody fragment that comprises two Fab fragments linked by disulfide bridges on the hinge region; the term "Fab' fragment" means the fragment obtained by reducing the disulfide bonds of a F(ab')2 fragment, consisting of an intact light chain and a Fd fragment of a heavy chain (consisting of a VH and CHI domain).

[0217] The term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is commonly considered the smallest fragment of an antibody that is capable of forming a complete antigen binding site. It is generally considered that the six CDRs confer antigen binding specificity to an antibody. However, even a single variable domain (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although it may do so at a lower affinity than the entire binding site.

[0218] The term "Fc" means an antibody fragment formed by disulfide bonds between the second, third constant regions of the first heavy chain and the second, third constant regions of the second heavy chain of an antibody. The Fc fragment of an antibody has various diverse functions, but is not involved in antigen binding.

[0219] The term "scFv" refers to a single polypeptide chain comprising a VL and a 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, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS (SEQ ID NO: 49) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4(SEQ ID NO: 50) 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 application 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 be present between the VHand VLof the scFv. In certain embodiments, the VHand VLdomains can be positioned relative to each other in any suitable arrangement. For example, scFv comprising NH2-VH-VH-COOH, NH2-VH-VL-COOH, NH2-VL-VH-COOH, or NH2-VL-VL-COOH are contemplated. 2- VL-VL-COOH.

[0220] The term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art and refers to an antibody fragment consisting of a single monomeric variable antibody domain, such as a single heavy chain variable region, that retains the ability to specifically bind the same antigen to which a full-length antibody binds (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

[0221] Each of the above antibody fragments maintains the ability to specifically bind to the same antigen bound by the full-length antibody from which they are derived, and / or competes with the full-length antibody for specific binding to the antigen.

[0222] In the present context, the term "antibody" when used in reference to a term "antibody" includes not only intact antibodies, but also antigen binding fragments of antibodies, unless otherwise clearly indicated by context.

[0223] Antigen binding fragments of antibodies (e.g., the above antibody fragments) can be obtained using conventional techniques known to those of skill in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) from a given antibody (e.g., an antibody provided herein) and screened for specificity in the same manner as is used for intact antibodies.

[0224] The term "murine antibody" refers to an antibody obtained by fusing B cells from an immunized mouse with myeloma cells, screening for murine hybridoma cells that both proliferate indefinitely and secrete antibody, followed by screening, antibody production, and antibody purification, or refers to an antibody secreted by a plasma cell that developed from a B cell that proliferated in response to an antigen invading a mouse.

[0225] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered to have an amino acid sequence that is modified to increase homology to the sequence of a human antibody. Typically, a humanized antibody has all or a portion of the CDR regions from a non-human antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., variable region FR and / or constant region) from a human immunoglobulin (acceptor antibody). A humanized antibody typically retains the desired properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance an immune response, and the like. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody that has the desired properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity, and / or ability to enhance an immune response).

[0226] The term "identity" is used in reference to the matching of sequences between two polypeptides or between two nucleic acids. When a position in each sequence that is being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules that is occupied by adenine, or a position in each of two polypeptides that is occupied by lysine), then the molecules are identical at that position. The "percentage of identity" between two sequences is the function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions in two sequences are matched then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are matched). Typically, the comparison is made over the full length of the two sequences. Such a comparison can be conveniently accomplished by use of the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, as implemented in the computer program Align (DNAstar, Inc.), or by use of the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as integrated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) as implemented in the GAP program, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length weight of 1, 2, 3, 4, 5, or 6, as incorporated into the GCG software package (available at www.gcg.com).

[0227] The term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with residues having similar side chains, e.g., substitutions that take place within a family of amino acid residues that are physicochemically or functionally similar, e.g., substitutions having similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a preferred substitution is one in which the replaced amino acid residue is replaced with another amino acid residue from the same side chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0228] The nomenclature used herein for the twenty conventional amino acids follows the conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present application, the amino acids are generally represented by the one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0229] In some embodiments of the disclosure, the anti-Trop-2 antibody targeting moiety is RS7 described in U.S. Patent No. 7,517,964; and hRS7 described in US2012 / 0237518. The heavy chain sequence and light chain amino acid sequence of the hRS7 antibody can be found in, for example, SEQ ID NO: 51 and SEQ ID NO: 52, respectively. The heavy chain of SEQ ID NO: 51 ends with a K (or lys) which is easily deleted, but such deletion does not affect the biological activity, see Dick, L.W. et al., Biotechnol. Bioeng., 100: 1132-1143.

[0230] Monoclonal antibodies useful in the present disclosure can be produced by a number of methods. For example, monoclonal antibodies useful in the present disclosure can be obtained by the hybridoma method using a number of species, including mouse, hamster, rat, and human cells (see, e.g., Kohler et al., 1975, Nature, 256:495), or made by recombinant DNA techniques (see, e.g., US 4,816,567), or isolated from phage antibody libraries (see, e.g., Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, Journal of Molecular Biology, 222:581-597). Monoclonal antibodies useful in the present disclosure include, but are not limited to, anti-Her 2 monoclonal antibodies, such as trastuzumab, pertuzumab, or anti-Trop-2 monoclonal antibodies, such as hRS7 antibody (i.e., Sacituzumab antibody).

[0231] The terms "comprise", "comprising", "have", "having", "include", "including", "contain", "containing", or "involve" and other variants thereof are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0232] The term "alkyl" denotes a straight-chain or branched hydrocarbon group, having the indicated number of carbon atoms, which is derived from a hydrocarbon by the removal of one hydrogen atom, e.g., "C 1-20 The term "alkyl" denotes a straight-chain or branched hydrocarbon group, having the indicated number of carbon atoms, which is derived from a hydrocarbon by the removal of one hydrogen atom, e.g., "C 1-10 The term "alkyl" denotes a straight-chain or branched hydrocarbon group, having the indicated number of carbon atoms, which is derived from a hydrocarbon by the removal of one hydrogen atom, e.g., "C 1-6 The term "alkyl" denotes a straight-chain or branched hydrocarbon group, having the indicated number of carbon atoms, which is derived from a hydrocarbon by the removal of one hydrogen atom, e.g., "C 1-4 The term "alkyl" denotes a straight-chain or branched hydrocarbon group, having the indicated number of carbon atoms, which is derived from a hydrocarbon by the removal of one hydrogen atom, e.g., "C 1-3"alkyl" and the like, specific examples include, but are not limited to: methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, i-hexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, and the like.

[0233] If a functional group or structure is described as being "substituted or unsubstituted," that functional group or structure can be (1) unsubstituted or (2) substituted.

[0234] The term "substituted" means that one or more (e.g., 1, 2, 3, 4, or 5) hydrogens on the designated compound or structure are replaced with a substituent, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds. By way of example, and without limitation, the substituents can each independently be selected from one or more of the following groups: NR 8 R 9 , -O-, -S-, -NR'-, halogen, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -CN, =O, C 1-6 (alkyl)2N-, (alkyl)3N+, -O-alkyl, -S-alkyl, -NR'-alkyl, -O-aryl, -S-aryl, -NR'-aryl, -O-heteroaryl, -S-heteroaryl, -NR'-heteroaryl, -O-heterocyclyl, -S-heterocyclyl, -NR'-heterocyclyl, -halo, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -C(O)O-, -C(O)NR'-, -C(O)N(R')2-, -C(O)NH(R')2-, -C(O)NH2-, -C(O)NH-, -C(O)N-, 1-6 haloalkyl, C 1-6 haloalkenyl, C 2-6 haloalkynyl, C 2- (alkyl)2N-, (alkyl)3N+, -O-alkyl, -S-alkyl, -NR'-alkyl, -O-aryl, -S-aryl, -NR'-aryl, -O-heteroaryl, -S-heteroaryl, -NR'-heteroaryl, -O-heterocyclyl, -S-heterocyclyl, -NR'-heterocyclyl, -halo, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -C(O)O-, -C(O)NR'-, -C(O)N(R')2-, -C(O)NH(R')2-, -C(O)NH2-, -C(O)NH-, -C(O)N-, 3-8 (alkyl)2N-, (alkyl)3N+, -O-alkyl, -S-alkyl, -NR'-alkyl, -O-aryl, -S-aryl, -NR'-aryl, -O-heteroaryl, -S-heteroaryl, -NR'-heteroaryl, -O-heterocyclyl, -S-heterocyclyl, -NR'-heterocyclyl, -halo, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -C(O)O-, -C(O)NR'-, -C(O)N(R')2-, -C(O)NH(R')2-, -C(O)NH2-, -C(O)NH-, -C(O)N-, 6-10 (alkyl)2N-, (alkyl)3N+, -O-alkyl, -S-alkyl, -NR'-alkyl, -O-aryl, -S-aryl, -NR'-aryl, -O-heteroaryl, -S-heteroaryl, -NR'-heteroaryl, -O-heterocyclyl, -S-heterocyclyl, -NR'-heterocyclyl, -halo, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -C(O)O-, -C(O)NR'-, -C(O)N(R')2-, -C(O)NH(R')2-, -C(O)NH2-, -C(O)NH-, -C(O)N-, 8 , R 9 are each independently selected from H, C 1-6 alkyl, C 3-6 cycloalkyl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl, C 6-10 aryl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, halo, hydroxyl, carboxyl, and ester (e.g., -C 1-6 alkylene-C(=O)-OC 1- 6alkyl), R' is as previously defined. For example, the substituents can be suitable substituents. If a substituent is described as being "independently selected from a group of functional groups," each substituent is selected independently of the other(s). Thus, each substituent can be the same or different from each other substituent.

[0235] As used herein, the term "suitable substituent" refers to modifications of a compound that can be made by those skilled in the art to suit the needs of the compound's substituents. "Suitable substituents" include oxo (=O), halogen, cyano, and NR. 8 R 9 Carboxyl, thiol, hydroxyl, ester group (e.g., -C) 1-6 Alkylene-C(=O)-OC 1-6 Alkyl), C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkyl-OC 1-6 Alkyl, C 1- 6-Hydroalkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-8 membered heterocyclic, 5-10 membered heteroaryl, C 6-10 Aryl, benzyl, hydroxy-substituted benzyl, indolylmethylene and C 1-6 Halogenated alkoxy groups.

[0236] As used herein, the term “one or more” means one or more under reasonable conditions, such as two, three, four, five or ten.

[0237] Unless otherwise specified, as used herein, the connection point of a substituent may be located at any suitable position of the substituent.

[0238] The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0239] Solid lines may be used in this article. solid wedge Or virtual wedge Carbon-carbon bonds of the compounds of the present application are depicted. The use of a solid line to depict a bond to an asymmetric carbon atom is intended to indicate that all possible stereoisomers (e.g., particular enantiomers, racemic mixtures, etc.) at that carbon atom are included. The use of a solid or dashed wedge to depict a bond to an asymmetric carbon atom is intended to indicate that the depicted stereoisomer is present. Unless otherwise indicated, the compounds of the present application are intended to exist in stereoisomeric forms, which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof. The compounds of the present application can exhibit more than one type of isomerism, and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0240] The term "N-oxide" refers to a compound containing an amine oxide moiety (i.e., an oxide of a tertiary amine group) formed by the oxidation of at least one nitrogen atom in the compound. Those skilled in the art will appreciate that not all nitrogen-containing heterocycles are capable of forming N-oxides, as the nitrogen requires an available lone pair of electrons to oxidize to an oxide; those skilled in the art will recognize nitrogen-containing heterocycles capable of forming N-oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for preparing N-oxides of nitrogen-containing heterocycles and tertiary amines are well known to those skilled in the art, for example, a nitrogen atom (e.g., a trivalent nitrogen) can be converted to the corresponding N-oxide form by known methods such as treatment with an oxidizing agent.

[0241] The present application encompasses all possible crystalline forms or polymorphs of the compounds of the present application, which can be a single polymorph or a mixture of more than one polymorph in any ratio.

[0242] It is also to be understood that certain compounds of the present application can exist in free form for treatment, or as appropriate, in the form of a pharmaceutically acceptable derivative thereof. In the present application, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites, or prodrugs, which upon administration to a patient in need thereof, are capable of providing (directly or indirectly) a compound of the present application or a metabolite or residue thereof. Accordingly, as used herein, reference to a "compound of the present application" is intended to encompass also the various derivative forms of the compound.

[0243] Pharmaceutically acceptable salts of the compounds of the present application include both acid and base addition salts. Suitable acid addition salts are formed from pharmaceutically acceptable acids including aspartic acid, fumaric acid, glucoheptonic acid, glycolic acid, glycerophosphonic acid, glyceric acid, glutamic acid, lactic acid, 2-naphthalene sulfonic acid, succinic acid, sulfuric acid and the like. Suitable base salts are formed from pharmaceutically acceptable bases including aluminum hydroxide, arginine, choline, N, N'- dibenzylethylenediamine, N-methylglucamine, lysine, sodium hydroxide, triethylamine, and the like. A review of suitable salts is found in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing the pharmaceutically acceptable salts of the compounds of the present application are known to those skilled in the art.

[0244] The term "ester" means an ester derived from the various generic compounds of the present application and includes physiologically hydrolysable esters (which can be hydrolyzed under physiological conditions to release the free acid or alcohol form of the compounds of the present application). The compounds of the present application can also be esters themselves.

[0245] The compounds of the present application can exist in solvate (preferably hydrate) form, wherein the compound of the present application contains a polar solvent, particularly, for example, water, methanol or ethanol, as a structural element of the crystal lattice of said compound. The amount of polar solvent, particularly water, can be present in stoichiometric or non-stoichiometric amounts.

[0246] Metabolites of the compounds of the present application, i.e., species derived from the compounds of the present application in vivo, are also within the scope of the present application. Such products can result, for example, from the oxidation, reduction, hydrolysis, amidation, demidation, esterification, deesterification, enzymatic cleavage, and the like, of the administered compound. Accordingly, the present application includes metabolites of the compounds of the present application, including those produced by the ingestion of the compounds of the present application by a mammal.

[0247] The present application further includes within its scope prodrugs of the compounds of the present application. In general, such prodrugs will be derivatives of the compounds of the present application which are readily convertible in vivo into the desired therapeutically active compound. Thus, in these cases, the term "administering" shall include the

[0248] The present application further includes, within its scope, isotopically-labeled compounds of the present application which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having the atomic mass or mass number different from the predominant atomic mass or mass number in nature.

[0249] The present application also encompasses compounds of the present application that contain protecting groups. During any of the processes for preparation of the compounds of the present application, it can be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules concerned, as described in Protective Groups in Organic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W. Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which are incorporated herein by reference. The protecting groups can be removed at a suitable subsequent stage using methods known from the art.

[0250] As used herein, the term "DAR" or "drug antibody ratio" means: (a) the number of linker / drug moieties attached to an antibody in a single antibody-drug conjugate molecule, which is an integer from 0 to 10, e.g., an integer from 1 to 10; or (b) the average number of linker / drug moieties attached to an antibody in a composition comprising more than one antibody-drug conjugate molecule, which is an integer or decimal number from 0 to 10, e.g., an integer or decimal number from 1 to 10. Methods for determining DAR are well known to the skilled person, including methods using reverse phase chromatography or HPLC-MS.

[0251] Whether explicitly indicated or not, the numerical values of the present application are modified by the term "about". The term "about" means within ±10% of the stated value, preferably within ±5%, more preferably within ±2%. BRIEF DESCRIPTION OF DRAWINGS

[0252] Figure 1 shows the efficacy results of anti-human HER2 antibody drug conjugates in a JIMT-1 cell subcutaneous tumor bearing mouse model.

[0253] Figure 2 shows the body weight changes of mice in each group in a JIMT-1 cell subcutaneous tumor bearing mouse model. DETAILED DESCRIPTION

[0254] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic ideas and scope of the invention.

[0255] Information about the sequences involved in this invention is described in the table below:

[0256] The abbreviations used in this article have the following meanings:

[0257] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0258] Nuclear magnetic resonance (¹H NMR) measurements were performed using a Bruker 400MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0259] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0260] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0261] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0262] I. Compound Preparation Examples

[0263] Example 1 of intermediate preparation: N 6 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (INT-1)

[0264] (2S,5S)-5-(9H-fluoren-9-ylmethyl)-2-(2-(methylsulfonyl)pyrimidin-5-yl)pyrrolidine-1,2-dicarboxamide (INT-2-1) was prepared according to the following scheme: 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine (1 g, 2.07 mmol) was dissolved in DMF (10 mL) and DIPEA (803.65 mg, 6.22 mmol, 1.08 mL) was added dropwise with stirring. The reaction was stirred for 1 hour and then concentrated. The residue was purified by reverse phase column (acetonitrile / 0.05% formic acid in water = 0% to 50%) and lyophilized to give the title solid (937 mg, 1.51 mmol).

[0265] The structural characterization data thereof are as follows:

[0266] MS m / z (ESI): 619.4 [M+H] +

[0267] Intermediate Preparation Example Two: Preparation of (S)-2-amino-N-((S)-1-(((S)-1-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxoprop-2-yl)amino)-1-oxopropan-2-yl)propanamide (INT-2)

[0268] Step One: Preparation of (9H-fluoren-9-yl)methyl ((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecyl)carbamate (INT-2-2)

[0269] (5S,8S,11S)-1-(9H-fluoren-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13- tetraazahexadecan-17-oic acid (657 mg, 1.22 mmol) and (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4- methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13- dione (500 mg, 1.11 mmol) were dissolved in N,N-dimethylformamide (10 mL), followed by the addition of HATU (630.67 mg, 1.66 mmol) and N,N-diisopropylethylamine (428 mg, 3.32 mmol), and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high-performance liquid chromatography and freeze-dried to obtain 700 mg of the title compound.

[0270] The structure thereof was characterized as follows:

[0271] ESI-MS (m / z): 974.3 [M+H] + .

[0272] The preparation method thereof is as follows:

[0273] Column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm)

[0274] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0275] Step two: preparation of (S)-2-amino-N-((S)-1-(((S)-1-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)amino)-2-oxoethoxy)methyl)amino)-1-oxoprop-2-yl)amino)-1-oxoprop-2-yl)propanamide (INT-2)

[0276] (9H-fluoren-9-yl)methyl ((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11- triazatetradecyl)carbamate (500 mg, 0.513 mmol) was dissolved in N,N- dimethylformamide (2 mL), diethylamine (75.05 mg, 1.03 mmol) was added, and the reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain 307 mg of the title compound.

[0277] The structure thereof was characterized as follows:

[0278] ESI-MS (m / z): 752.3 [M+H] + .

[0279] The preparation method thereof is as follows:

[0280] Column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm)

[0281] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0282] Intermediate Preparation Example Three: 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4 -((S)-1-(((S)-1-(((S)-1-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)- L-asparagine (INT-3)

[0283] (S)-2-amino-N-((S)-1-(((S)-1-(((2-(((1S,9R)-5-chloro-9-ethyl-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1- oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (120 mg, 159.53 μmol) and (9H-fluoren-9-yl)methyl (S)-(2,5-dioxotetrahydrofuran-3-yl)carbamate (56.50 mg, 167.51 μmol) were dissolved in DMF (3 mL), after adding DIPEA (20.62 mg, 159.53 μmol), the reaction was stirred at room temperature for 2 hours, the reaction solution was directly purified by reverse phase column (acetonitrile / 0.05% formic acid aqueous solution = 0% ~ 90%), and then freeze-dried to obtain the title compound (124 mg, 113.81 μmol).

[0284] The structural characterization data thereof are as follows:

[0285] MS m / z (ESI): 1089.4 [M+H] +

[0286] Preparation of intermediate Example Four: Preparation of (S)-2-amino-N-((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrano[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (INT-4)

[0287] Step One: Preparation of (9H-fluoren-9-yl)methyl (S)-(1-((chloromethyl)amino)-1- oxopropan-2-yl)carbamate (INT-4-2)

[0288] To 1,2-dichloroethane (4 mL) was added (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl acetate (50 mg, 130.75 μmol), and then TMSCl (142.05 mg, 1.31 mmol) was added dropwise. After addition, the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to obtain the crude product of the title compound (46 mg, 128.20 μmol), which was used directly in the next step without purification.

[0289] Step two: Preparation of (9H-fluoren-9-yl)methyl ((S)-l-((((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-l-oxopropan-2-yl)carbamate (INT-4-3)

[0290] (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-2,7,8,9- tetrahydro-3H-pyrido[4,3,2-de]phthalazine-3-one (30 mg, 78.87 μmol) was dissolved in THF (1.5 mL) and cooled to -78 °C under nitrogen protection. Then NaHMDS (78.87 μmol, 2M in THF) was added dropwise, the system turned yellow, and the reaction was stirred for 20 minutes. Then (9H-fluoren-9-yl)methyl (S)-(l-((chloromethyl)amino)-l-oxopropan-2-yl)carbamate (42.45 mg, 118.31 μmol) in THF (1.5 mL) was added dropwise. After the addition was completed, the reaction was stirred for 20 minutes after warming to -10 °C, and then stirred for 1 hour. After the reaction was completed, water was added to the reaction solution, and ethyl acetate was added for extraction. The organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (70 mg, 69.73 μmol). Without purification, the product was directly used in the next reaction.

[0291] The structural characterization data thereof are as follows:

[0292] MS m / z (ESI): 703.3 [M+H] +

[0293] Step three: Preparation of (S)-2-amino-N-(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l- methyl-lH-l,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine- 2(7H)-yl)methyl)propanamide (INT-4-4)

[0294] (9H-fluoren-9-yl)methyl ((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl- 1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl) amino)-1-oxopropan-2-yl)carbamate (70 mg, 69.73 μmol) was dissolved in DMF (1 mL), diethylamine (0.2 mL) was added, and the reaction was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was purified by reverse-phase column chromatography (acetonitrile / 0.05% aqueous ammonium bicarbonate solution = 0-95%) and then freeze-dried to obtain the title compound (20 mg, 41.63 μmol).

[0295] The structural characterization data thereof are as follows:

[0296] MS m / z (ESI): 481.2 [M+H] +

[0297] Step four: Preparation of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((S)-1-((((8S,9R)-5- fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1- oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (INT-4-5)

[0298] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (596.93 mg, 1.56 mmol) was dissolved in DMF (10 mL), HATU (593.17 mg, 1.56 mmol) and DIPEA (403.48 mg, 3.12 mmol) were added, and the reaction was stirred at room temperature for 0.5 hours. Then (S)-2-amino-N-(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)propanamide (500 mg, 1.04 mmol) was added, and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was used as it was in the next reaction without treatment.

[0299] The structural characterization data thereof are as follows:

[0300] MS m / z (ESI): 845.4 [M+H] +

[0301] Step five: Preparation of (S)-2-amino-N-((S)-l-(((S)-l-((((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-l-oxopropan-2-yl)amino)- 1-oxopropan-2-yl)propanamide (INT-4)

[0302] To the reaction solution of step four, diethylamine (1 mL) was added and stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was freeze-dried after removing part of the solvent with a freeze dryer and purified by reverse phase (acetonitrile-0.05% aqueous ammonium bicarbonate solution = 0-90%) and freeze-dried to obtain the title compound (700 mg, 1.01 mmol).

[0303] The structural characterization data thereof are as follows:

[0304] MS m / z (ESI): 623.4 [M+H] +

[0305] Intermediate Preparation Example Five: Preparation of 3-(3-(3-((l,3-dihydroxy-2- (hydroxymethyl)propan-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((l,3-dihydroxy-2- (hydroxymethyl)propan-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propanoic acid (INT-5)

[0306] Step one: Preparation of 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2- ((2-carboxyethoxy)methyl)propane-l,3-diyl)bis(oxy))dipropionic acid (INT-5-2)

[0307] 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-l,3-diyl)bis(oxy))dipropionic acid (0.94 g, 4.71 mmol) and 2-benzyloxy-l-methylpyridinium trifluoromethanesulfonate (1.65 g, 4.71 mmol) were reacted at 80-85 °C for 16 hours, and the reaction solution was directly purified by a C18 reverse phase column (acetonitrile / 0.05% formic acid aqueous solution = 10-70%) and freeze-dried to obtain the title compound (0.78 g, 1.51 mmol).

[0308] The structural characterization data thereof are as follows:

[0309] ESI-MS (m / z): 515.2 (M+H) +

[0310] Step two: Preparation of 3-(3-(3-((1,3-dihydroxy-2- (hydroxymethyl)propan-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2- (hydroxymethyl)propan-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propanoic acid (INT-5-3)

[0311] Dissolve 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2- carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (0.78 g, 1.51 mmol), 2-amino-2-(hydroxymethyl)propane-1,3-diol (0.82 g, 6.80 mmol) in DMF (15 mL), add DIPEA (1.17 g, 9.07 mmol), add HATU (2.59 g, 6.80 mmol) portionwise, react at 25 °C for 2 h, purify the reaction mixture directly by C18 reverse phase column (acetonitrile / 0.05% aqueous formic acid = 10-70%), lyophilize to give the title compound (0.74 g, 0.90 mmol).

[0312] The structural characterization data thereof are as follows:

[0313] ESI-MS (m / z): 824.4 (M+H) +

[0314] Step three: Preparation of 3-(3-(3-((1,3-dihydroxy-2- (hydroxymethyl)propan-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2- (hydroxymethyl)propan-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propanoic acid (INT-5)

[0315] Dissolve 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3- oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3- oxopropoxy)methyl)propoxy)propanoic acid benzyl ester (0.74 g, 0.90 mmol) in ethanol (30 mL), add 10% palladium on carbon (0.15 g), acetic acid (0.14 g, 2.42 mmol), evacuate air, introduce hydrogen, warm to 40 °C, react for 4 h, filter off the palladium on carbon, concentrate the filtrate, dissolve the residue in water and acetonitrile, clarify, lyophilize to give the title compound (0.59 g, 0.80 mmol).

[0316] The structural characterization data thereof are as follows:

[0317] ESI-MS (m / z): 734.3 (M+H) +

[0318] Preparation of intermediate: 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6- pentyloxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6- pentyloxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propanoic acid (INT-6)

[0319] Step one: Preparation of benzyl 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6- pentyloxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6- pentyloxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propanoate (INT-6-1)

[0320] Benzyl 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2- carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate (1.07 g, 2.08 mmol), (2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentol (1.62 g, 8.32 mmol) were dissolved in DMF (15 mL), DIPEA (1.34 g, 10.40 mmol) was added, HATU (3.56 g, 9.36 mmol) was added portionwise, and the reaction was stirred at 25 °C for 1 h. The reaction mixture was directly purified by C18 reverse phase column (acetonitrile / 0.05% formic acid in water = 10-70%), and lyophilized to give the title compound (0.66 g, 0.63 mmol).

[0321] The structural characterization data thereof are as follows:

[0322] ESI-MS (m / z): 1046.5 (M+H) +

[0323] Step two: Preparation of 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6- pentyloxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6- pentyloxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propanoic acid (INT-6)

[0324] Benzyl 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3- oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)- 3-oxopropoxy)methyl)propoxy)propanoate (0.66 g, 0.63 mmol) was dissolved in ethanol (14 mL) and water (7 ml), 10% palladium on carbon (0.13 g), acetic acid (0.10 g, 1.70 mmol) were added, the air was removed and hydrogen was bubbled through the solution, the temperature was raised to 40 °C and the reaction was stirred for 4 hours, the palladium on carbon was filtered off, the filtrate was concentrated, water and acetonitrile were added to dissolve the residue and the solution was clarified, then the title compound was obtained by lyophilization (0.54 g, 0.56 mmol).

[0325] The structural characterization data thereof are as follows:

[0326] ESI-MS (m / z): 956.4 (M+H) +

[0327] Preparation Example Seven: Preparation of N 6 -((allyloxy)carbonyl)-N2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L- lysine (INT-7)

[0328] Step One: Preparation of N 6 -((allyloxy)carbonyl)-L-lysine (INT-7-2)

[0329] N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 6 -((allyloxy)carbonyl)-L-lysine (1.00 g, 2.21 mmol) was dissolved in DMF (6 mL), DBU (504 mg, 3.31 mmol) was added, and the reaction was stirred at 25 °C for 1 h. The reaction solution was used directly in the next step.

[0330] Step Two: Preparation of N 6 -((allyloxy)carbonyl)-N 2 -((allyloxy)carbonyl)-N

[0331] 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-ynoic acid (590 mg, 2.20 mmol) was dissolved in DMF (5 mL), DIPEA (426 mg, 3.30 mmol), HATU (919 mg, 2.42 mmol) were added, and the reaction was stirred at 25 °C for 0.5 h. N 6- ((allyloxy)carbonyl)-L-lysine (506 mg, 2.20 mmol) (reaction solution from previous step) was reacted at room temperature for 0.5 h and the reaction solution was directly purified by C18 reverse phase column (ACN / 0.05% formic acid in water = 10-70%) and lyophilized to give the title compound (492 mg, 1.02 mmol).

[0332] The structural characterization data thereof are as follows:

[0333] MS m / z (ESI): 481.2 [M+H] +

[0334] Intermediate Preparation Example Eight: Preparation of (S)-3-(((S)-2-((S)-2-((S)-2-aminopropionamido)propionamido)propionamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)thiazolidine-4-carboxamide (INT-8)

[0335] Step One: Preparation of (9H-fluoren-9-yl)methyl ((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropan-2-yl)carbamate (INT-8-2)

[0336] (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)thiazolidine-4-carboxamide (70 mg, 127.13 μmol) was dissolved in THF (10 mL), after three times of nitrogen substitution, lithium hydroxide monohydrate (32.01 mg, 762.80 μmol) was added under ice-bath, after stirring at room temperature for 10 minutes, (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl acetate (97.23 mg, 254.27 μmol) was added, after addition, the reaction was stirred at 22 °C for 5 hours. To the reaction solution, saturated aqueous ammonium chloride solution 20 mL was added, extracted with dichloromethane for 3 times (10 mL x 3), washed with brine 10 mL, dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain the crude product of the title compound, which was purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (70 mg, 80.19 μmol).

[0337] The structural characterization data thereof are as follows:

[0338] MS m / z (ESI): 873.3 [M+H] +

[0339] The preparation method thereof is as follows:

[0340] Column: Waters XBridge Prep C18 OBD (5 μm*19 mm*150 mm)

[0341] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0342] Step two: preparation of (S)-3-(((S)-2-aminopropanamido)methyl)-N-((1S,9S)-9- ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)thiazolidine-4-carboxamide (INT-8-3)

[0343] (9H-fluoren-9-yl)methyl ((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1- oxopropan-2-yl)carbamate (40 mg, 45.82 µmol) was dissolved in DMF (2 mL), after adding diethylamine (16.76 mg, 229.11 µmol), the reaction was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the trifluoroacetate salt of the title compound (34 mg, 44.46 µmol).

[0344] The structural characterization data thereof are as follows:

[0345] MS m / z (ESI): 651.2 [M+H] +

[0346] The preparation method thereof is as follows:

[0347] Column: Waters XBridge Prep C18 OBD (5 µm*19 mm*150 mm)

[0348] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

[0349] Step three: preparation of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((S)-1-((((S)-4- (((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro- 1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)thiazolidin- 3-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan- 2-yl)carbamate (INT-8-4):

[0350] (S)-3-(((S)-2-aminopropanoylamino)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9- hydroxy-4-methyl-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)thiazolidine-4- carboxamide trifluoroacetate (2.7 g, 3.53 mmol, TFA) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (1.62 g, 4.24 mmol) were dissolved in DMF (15 mL), DIPEA (1.83 g, 14.12 mmol) was added, followed by DMTMM (2.08 g, 7.06 mmol), and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to remove the solvent. The crude product was dissolved in water and purified by reverse phase column chromatography (acetonitrile-0.05% trifluoroacetic acid aqueous solution = 0-90%) to obtain the title compound (2.8 g, 2.76 mmol) after freeze-drying.

[0351] The structural characterization data thereof are as follows:

[0352] MS m / z (ESI): 1032.4 [M+18] +

[0353] Step four: preparation of (S)-3-(((S)-2-((S)-2-((S)-2-aminopropanoylamino)propanoylamino)propanoylamino)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)thiazolidine-4-carboxamide (INT-8)

[0354] To (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5- fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)thiazolidin-3- yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (2.8 g, 2.76 mmol) was added DMF (10 mL), then diethylamine (403.49 mg, 5.52 mmol), and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to remove the solvent. The crude product was dissolved in water and purified by reverse phase column chromatography (acetonitrile-0.05% aqueous formic acid solution = 0-90%) to give the trifluoroacetate salt of the title compound (2.3 g, 2.54 mmol) after freeze-drying.

[0355] The structural characterization data thereof are as follows:

[0356] MS m / z (ESI): 793.2 [M+H] +

[0357] Intermediate Preparation Example Nine: Preparation of N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N4-((S)-1-(((S)-1-(((S)-1-((((S)-4-(((1S,9S)-9- ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)thiazolidin-3- yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-L-asparagine (INT-9)

[0358] Step One: Preparation of N 2Preparation of (S)-3-(((S)-2-((S)-2-((S)-2-aminopropanamido)propanamido)methyl)- N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)thiazolidine- 4-carboxamide (200 mg, 0.22 mmol, trifluoroacetate salt) was dissolved in DMF (3 mL), (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutanoic acid (99.0 mg, 0.25 mmol), DIPEA (68 mg, 0.52 mmol) was stirred for 1 min, then HATU (120 mg, 0.31 mmol) was added, the reaction was stirred at room temperature for 1 h, the reaction was directly purified by C18 reverse column (ACN / 0.05% formic acid in water = 20-80%), lyophilized to give the title compound (212 mg, 0.18 mmol).

[0359] The structural characterization data thereof are as follows:

[0360] MS m / z (ESI): 1170.5 [M+H] +

[0361] MS m / z (ESI): 1170.5 [M+H] +

[0362] Step 2: Preparation of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N4-((S)-1-(((S)-1-(((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-L-asparagine (INT-9)

[0363] Under nitrogen protection, N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N4-((S)-1-(((S)-1-(((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropyl-2-yl)amino) 1-(oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-L-asparaginic acid allyl ester (212 mg, 0.18 mmol) was dissolved in DMF (3 mL), and 1,3-dimethylbarbituric acid (51 mg, 0.33 mmol) and tetrakis(triphenylphosphine)palladium (38 mg, 0.033 mmol) were added. The mixture was heated to 35 °C and reacted for 2 hours. The reaction solution was directly purified by C18 reverse-phase column chromatography (ACN / 0.05% formic acid aqueous solution = 10–70%), and lyophilized to obtain the title compound (127 mg, 0.11 mmol).

[0364] Its structural characterization data are as follows:

[0365] MS m / z (ESI): 1130.4 [M+H] +

[0366] Example 10: Preparation of (S)-3-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-4-(((S)-6-(allyloxy)-5-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-6-oxohexyl)amino)-4-oxobutyric acid (INT-10)

[0367] Step 1: N 6 -(tert-Butoxycarbonyl)-N 2Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L- lysine allyl ester (INT-10-2):

[0368] N 6 -(tert-butoxycarbonyl)-L-lysine allyl ester hydrochloride (1 g, 3.10 mmol) was dissolved in DMF (10 mL), DIPEA (1.00 g, 7.74 mmol) was added dropwise, HATU (1.24 g, 3.25 mmol) was added portionwise, and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was extracted with water and ethyl acetate, and the organic phase was washed with brine and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate = 0-100%) and concentrated under reduced pressure again to obtain the title compound (1.63 g, 3.04 mmol).

[0369] The structural characterization data thereof are as follows:

[0370] ESI-MS (m / z): 437.2 (M-100+H) +

[0371] Step two: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L- lysine allyl ester (INT-10-3):

[0372] N 6 -(tert-butoxycarbonyl)-N 2 -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-lysine allyl ester (1.8 g, 3.35 mmol, FR) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (3 mL) was added dropwise at room temperature, and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude trifluoroacetate salt of the title compound (2.18 g, 3.28 mmol).

[0373] The structural characterization data thereof are as follows:

[0374] ESI-MS (m / z): 437.3 (M+H) +

[0375] Step three: Preparation of N 6 -((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4- oxobutanoic acid (INT-10-5): 2

[0376] ​To a solution of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L- lysine allyl ester bis trifluoroacetate salt (2.13 g, 3.21 mmol) in DMF (15 mL) was added (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert- butoxy)-4-oxobutanoic acid (1.32 g, 3.21 mmol) followed by PyBOP (2.00 g, 3.85 mmol) and then DIPEA (1.66 g, 12.82 mmol) and stirred at room temperature for 1 h. After completion of the reaction, the reaction was diluted with water and ethyl acetate and the organic layer was washed with brine, dried and concentrated under reduced pressure. The crude was purified by silica gel column chromatography (petroleum ether-ethyl acetate = 0-100%) and concentrated under reduced pressure to afford the title compound (3.25 g, 3.13 mmol, 80% purity).

[0377] The structural characterization data thereof are as follows:

[0378] ESI-MS (m / z): 830.4 (M+H) +

[0379] Step four: Preparation of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4- (((S)-6-(allyloxy)-5-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yn-1- ylamino)-6-oxohexyl)amino)-4-oxobutanoic acid (INT-10)

[0380] To a solution of N 6 -((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4- oxobutryoyl)-N 2 -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-lysine allyl ester (3.25 g, 3.13 mmol, 80% purity) was dissolved in dichloromethane (10 mL) and trifluoroacetic acid (10 mL) was added and stirred at room temperature for 1 h. After completion of the reaction, the reaction was concentrated under reduced pressure and the crude was lyophilized from water to afford the title compound (1.77 g, 2.29 mmol).

[0381] The structural characterization data thereof are as follows:

[0382] ESI-MS (m / z): 775.2 (M+H) +

[0383] Preparation of Intermediate: (S)-2-amino-N-((S)-l-(((S)-8-ethyl-8-hydroxy-9, 12-dioxo- 1, 2, 8, 9, 12, 14-hexahydro-l lH-cyclopenta[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-3- yl)amino)-l-oxopropan-2-yl)-3-methylbutanamide (INT-11)

[0384] Step one: Preparation of tert-butyl ((S)-l-(((S)-8-ethyl-8-hydroxy-9, 12-dioxo- 1, 2, 8, 9, 12, 14-hexahydro-l lH-cyclopenta[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-3- yl)amino)-l-oxopropan-2-yl)carbamate (INT-11-2)

[0385] Tert-butyl (L)-alaninate (262.38 mg, 1.39 mmol) and (S)-3-amino-8-ethyl-8-hydroxy- 2,8,11,14-tetrahydro-12H-cyclopenta[de]pyrano[3',4':6,7]indolizino[l,2-b]quinoline-9,12(lH)- dione hydrochloride (270.00 mg, 693.37 μmol) were dissolved in DMF (3 mL), DIPEA (358.44 mg, 2.77 mmol, 483.08 μL) was added, followed by HATU (579.66 mg, 1.53 mmol), and the reaction was allowed to proceed at room temperature for 16 hours. After the reaction was completed, the reaction solution was added dropwise into water (100 mL) with stirring, and a large amount of solid precipitated, which was filtered, the filter cake was washed with an appropriate amount of water and MTBE, and dried to obtain the crude product of the title compound (687 mg, 674.01 μmol, 55% purity).

[0386] The structural characterization data thereof are as follows:

[0387] MS m / z (ESI): 561.3 [M+H] +

[0388] Step two: Preparation of (S)-2-amino-N-((S)-8-ethyl-8-hydroxy-9, 12-dioxo- 1, 2, 8, 9, 12, 14-hexahydro-l lH-cyclopenta[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-3- yl)propanamide (INT-11-3)

[0389] To the crude of ((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14- hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3-yl)amino)- 1-oxopropan-2-yl)carbamic acid tert-butyl ester (687 mg, 674.01 μmol) was added dichloromethane (4 mL) and trifluoroacetic acid (4 mL), the reaction was stirred at room temperature for 1 hour. The reaction was concentrated under reduced pressure, the crude was purified by reverse phase column chromatography (acetonitrile-0.05% trifluoroacetic acid in water = 0-90%) and then freeze-dried to give the trifluoroacetate salt of the title product (270 mg, 469.97 μmol).

[0390] The structural characterization data thereof are as follows:

[0391] MS m / z (ESI): 461.2 [M+H] +

[0392] Step three: preparation of ((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo- 1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 3-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamic acid tert- butyl ester (INT-11-4):

[0393] To (tert-butoxycarbonyl)-L-valine (20 mg, 92.06 μmol) and the trifluoroacetate salt of (S)-2-amino-N-((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14- hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3-yl)propanamide (30 mg, 52.22 μmol, TF) were dissolved in DMF (2 mL), DIPEA (32 mg, 247.60 μmol) was added, followed by the addition of HATU (23 mg, 60.53 μmol), the reaction was stirred at room temperature for 1.5 hours. After the reaction was completed, the reaction was extracted with water and ethyl acetate, the organic phase was washed with brine, dried and concentrated under reduced pressure to give the crude of the title compound (34 mg, 51.54 μmol), which was used directly in the next reaction without purification.

[0394] The structural characterization data thereof are as follows:

[0395] MS m / z (ESI): 660.3 [M+H] +

[0396] Step four: Preparation of (S)-2-amino-N-((S)-l-(((S)-8-ethyl-8-hydroxy-9,12- dioxo-l,2,8,9,12,14-hexahydro-l lH-cyclopenta[de]pyrano[3',4':6,7]indolizino[l,2- b]quinolin-3-yl)amino)-l-oxopropan-2-yl)-3-methylbutanamide (INT-11)

[0397] To the crude of tert-butyl ((S)-l-(((S)-l-(((S)-8-ethyl-8-hydroxy-9,12-dioxo- 1,2,8,9,12,14-hexahydro-l lH-cyclopenta[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-3- yl)amino)-l-oxopropan-2-yl)amino)-3-methyl-l-oxobutan-2-yl)carbamate (34 mg, 51.54 μmol) was added dichloromethane (4 mL), then trifluoroacetic acid (2 mL), stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to remove the solvent. The crude product was dissolved in water and purified by reverse phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%) and then freeze-dried to obtain the trifluoroacetate salt of the title compound (27 mg, 40.08 μmol).

[0398] The structural characterization data thereof are as follows:

[0399] MS m / z (ESI): 560.3 [M+H] +

[0400] Intermediate Preparation Example Twelve: N 6 -(Diphenyl(p-tolyl)methyl)-N 2 Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-lysine (INT-12)

[0401] Step one: N 6 Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-lysine (INT-12)

[0402] N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 6 -(Diphenyl(p-tolyl)methyl)-L-lysine (150.0 mg, 0.24 mmol) was dissolved in DMF (2 mL), diethylamine (182.3 mg, 2.5 mmol) was added, and stirring was performed at room temperature for 1 hour. After the reaction was completed, recrystallization was performed using a mixed solvent of (EA / PE = 1 / 3) 10 mL for 1 hour, and filtration was performed to obtain the title compound (90.1 mg, 0.22 mmol).

[0403] Step two: N 6 -(Diphenyl(p-tolyl)methyl)-N 2 Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-lysine (INT-12)

[0404] N 6 -(Diphenyl(p-tolyl)methyl)-L-lysine (90.1 mg, 0.22 mmol) was dissolved in DMF (3 mL), DIPEA (86.3 mg, 0.66 mmol) was added, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (160.6 mg, 0.44 mmol) was added, after the reaction was completed, purified by flash column chromatography (C18, water / acetonitrile = 0.5) and freeze-dried to obtain the title compound (75.2 mg, 0.11 mmol).

[0405] The structural characterization data thereof are as follows:

[0406] MS m / z (ESI): 653.2 [M+H] +

[0407] Intermediate Preparation Example Thirteen: N 6 -(Diphenyl(p-tolyl)methyl)-N 2 Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-lysine (INT-13)

[0408] Step one: Preparation of (9H-fluoren-9-yl)methyl (S)-(1-(((4-(3-(4-(3-cyanopyridin-2- yl)piperazine-1-carbonyl)benzyl)-1-oxo-phthalazin-2(1H)-yl)methyl)amino)-1- oxopropan-2-yl)carbamate (INT-13-2)

[0409] Dissolve 2-(4-(3-((4-oxo-3,4-dihydrophthalazine-l-yl)methyl)benzoyl)piperazin-l- yl)nicotinonitrile (200 mg, 443.96 μmol) in THF (1.5 mL) and cool to -78 °C under nitrogen protection. Then drop in NaHMDS (532.75 μmol, 2M in THF), the system turns yellow, and stir the reaction for 20 minutes. Then drop in (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl acetate (220.71 mg, 577.15 μmol) in THF (1.5 mL). After adding, cool the reaction to -10 °C and stir for 1 hour. After the reaction is completed, add water to the reaction and extract with ethyl acetate. Wash the organic phase with brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the crude title compound (70 mg, 69.73 μmol). Purify the reaction liquid by C18 reverse column (ACN / 0.05% FA aqueous solution = 10-70%), and freeze-dry to obtain the title compound (266 mg, 344.18 μmol).

[0410] The structural characterization data thereof are as follows:

[0411] MS m / z (ESI): 773.4 [M+H] +

[0412] Step two: preparation of (S)-2-amino-N-((4-(3-(4-(3-cyanopyridin-2-yl)piperazine-l- carbonyl)benzyl)-l-oxophthalazin-2(lH)-yl)methyl)propanamide (INT-13-3)

[0413] Dissolve (9H-fluoren-9-yl)methyl (S)-(l-(((4-(3-(4-(3-cyanopyridin-2-yl)piperazine-l- carbonyl)benzyl)-l-oxophthalazin-2(lH)-yl)methyl)amino)-l-oxopropan-2-yl)carbamate (266 mg, 344.18 μmol) in DMF (2 mL), add diethylamine (0.5 mL), and react at 25 °C for 1 h. Freeze-dry the reaction liquid to obtain the crude title compound (0.16 g, 290.59 μmol).

[0414] The structural characterization data thereof are as follows:

[0415] ESI-MS (m / z): 551.4 (M+H) +

[0416] Step 3: Preparation of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((4-(3-(4-(3- cyano pyridin-2-yl)piperazine-1-carbonyl)benzyl)-1-oxophthalazin-2(1H)-yl)methyl)amino)- 1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (INT-13-4)

[0417] (S)-2-amino-N-((4-(3-(4-(3-cyano pyridin-2-yl)piperazine-1-carbonyl)benzyl)-1- oxophthalazin-2(1H)-yl)methyl)propanamide (0.16 g, 290.59 μmol), (((9H-fluoren-9-yl)methoxy) carbonyl)-L-alanyl-L-alanine (133.35 mg, 348.70 μmol) were dissolved in DMF (3 mL), DIPEA (112.68 mg, 871.77 μmol) was added and stirred for 1 min, then HATU (121.47 mg, 319.65 μmol) was added, the reaction was carried out at 25 °C for 0.5 h, the reaction solution was directly purified by C18 reverse phase column (ACN / 0.05% formic acid aqueous solution = 10-70%), and then lyophilized to obtain the title compound (0.21 g, 229.51 μmol).

[0418] The structural characterization data thereof are as follows:

[0419] ESI-MS (m / z): 916.3 (M+H) +

[0420] Step 4: Preparation of (S)-2-amino-N-((S)-1-(((S)-1-(((4-(3-(4-(3-cyano pyridin-2- yl)piperazine-1-carbonyl)benzyl)-1-oxophthalazin-2(1H)-yl)methyl)amino)-1-oxopropan-2- yl)amino)-1-oxopropan-2-yl)propanamide (INT-13)

[0421] (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((4-(3-(4-(3-cyano pyridin-2-yl)piperazine-1- carbonyl)benzyl)-1-oxophthalazin-2(1H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1- oxopropan-2-yl)carbamate (0.21 g, 229.51 μmol) was dissolved in DMF (2 mL), diethylamine (0.5 mL) was added, the reaction was carried out at 25 °C for 1 h, and the reaction solution was lyophilized to obtain the crude title compound (0.14 g, 202.09 μmol).

[0422] The structural characterization data thereof are as follows:

[0423] ESI-MS (m / z): 694.2 (M+H) +

[0424] Example 14 of intermediate preparation: N 6 -(diphenyl(p-tolyl)methyl)-N 2 Preparation of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-14)

[0425] Step 1: N 2 -((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 Preparation of 1-(diphenyl(p-tolyl)methyl)-L-lysine (INT-14-1)

[0426] N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (2 g, 4.97 mmol) and 2,5-dioxopyrrolidone-1-yl(((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine ester (2.39 g, 5.47 mmol) were dissolved in N,N-dimethylacetamide (20 mL), and N,N-diisopropylethylamine (1.28 g, 9.94 mmol) was added. The reaction mixture was reacted at 20 °C for 2 hours. The reaction solution was subjected to rapid column chromatography (dichloromethane / methanol = 10 / 1) and then evaporated to dryness to give the title compound (2.4 g, 3.23 mmol).

[0427] Its structural characterization data are as follows:

[0428] MS m / z(ESI): 724 [M+H] +

[0429] Step Two: N 2 -(L-valine)-N 6 Preparation of 1-(diphenyl(p-tolyl)methyl)-L-lysine (INT-14-2)

[0430] N 2 -((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (2.4 g, 3.32 mmol) was dissolved in N,N-dimethylacetamide (20 mL), and diethylamine (484.95 mg, 6.63 mmol) was added. The reaction was carried out at 20 °C for 2 hours. After the reaction was completed, the reaction solution was subjected to rapid column chromatography (C18, water / acetonitrile = 2 / 1) and evaporated to dryness to give the title compound (1.98 g, 3.32 mmol).

[0431] The structural characterization data thereof are as follows:

[0432] MS m / z (ESI): 502 [M+H] +

[0433] Step three: N 6 -(diphenyl(p-tolyl)methyl)-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (INT-14) was prepared

[0434] N 2 -(L-valyl)-N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (1.98 g, 3.25 mmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate were dissolved in N,N-dimethylacetamide (20 mL), and DIPEA (1.26 g, 9.76 mmol) was added. The reaction was carried out at 20 °C for 2 hours. After the reaction was completed, the reaction solution was diluted with water and extracted with ethyl acetate. The combined organic phase was dried and rotary evaporated to give the title compound (2.4 g, 3.03 mmol).

[0435] The structural characterization data thereof are as follows:

[0436] MS m / z (ESI): 752 [M+H] +

[0437] Intermediate Preparation Example Fifteen: (S)-3-((2-aminoacetylamino)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)thiazolidine-4-carboxamide (INT-15) was prepared

[0438] Step one: (9H-fluoren-9-yl)methyl (2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamate (INT-15-1) was prepared

[0439] (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)thiazolidine-4-carboxamide (240 mg, 435.89 μmol) was dissolved in THF (10 mL), after nitrogen substitution for three times, lithium hydroxide monohydrate (109.74 mg, 2.62 mmol) was added under ice-bath, after stirring at room temperature for 10 minutes, (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methyl acetate (642.29 mg, 1.74 mmol) was added, after addition, the reaction was stirred at 22 °C for 5 hours. To the reaction solution, saturated aqueous ammonium chloride solution 20 mL was added, extracted with dichloromethane for three times (10 mL x 3), washed with brine 10 mL, dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain the crude product of the title compound, which was purified by preparative high performance liquid chromatography, and freeze-dried to obtain the title compound (100 mg, 116.42 μmol).

[0440] The structural characterization data thereof are as follows:

[0441] MS m / z (ESI): 859.3 [M+H] +

[0442] The preparation method thereof is as follows:

[0443] Column: Waters XBridge Prep C18 OBD (5 μm*19 mm*150 mm)

[0444] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0445] Step two: preparation of (S)-3-((2-aminoacetylamino)methyl)-N-((1S,9S)-9- ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)thiazolidine-4-carboxamide (INT-15)

[0446] (9H-fluoren-9-yl)methyl (2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamate (40 mg, 46.57 μmol) was dissolved in DMF (2 mL), after adding diethylamine (17.03 mg, 232.85 μmol), the reaction was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (12 mg, 18.85 μmol).

[0447] The structural characterization data thereof are as follows:

[0448] MS m / z (ESI): 637.3 [M+H] +

[0449] The preparation method thereof is as follows:

[0450] Column: Waters XBridge Prep C18 OBD (5 μm*19 mm*150 mm)

[0451] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

[0452] II. Preparation Example of Compound

[0453] Example 1: Preparation of 2,2',2"-(10-((4S,7S,10S,13S,20S)-20-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl amino)-3,6,9,12,19,22-hexaoxo-2,5,8,11,18,21-hexaazatricosan-23-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-1)

[0454] Step 1: Preparation of (9H-fluoren-9-yl)methyl ((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl amino)-3,6,9,12-tetraoxo-2,5,8,11-tetraazahexadecan-17-yl)carbamate (A-1-1)

[0455] (S)-2-amino-N-((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (50 mg, 80.31 µmol) and N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-lysine (49.68 mg, 80.31 μmol) was dissolved in DMF (1 mL), HATU (30.52 mg, 80.31 μmol) and DIPEA (20.76 mg, 160.61 μmol) were added, the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was used directly in the next step without treatment.

[0456] The structural characterization data thereof are as follows:

[0457] MS m / z (ESI): 1223.5 [M+H] +

[0458] Step two: Preparation of N-((4S,7S,10S,13S)-17-amino-1-((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2- de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazheptadec- 13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic amide (A-1-2)

[0459] Directly to the theoretical containing (9H-fluoren-9-yl)methyl ((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9- dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic amide)-3,6,9,12-tetraoxo-2,5,8,11- tetraazheptadec-17-yl)carbamate (98 mg, 80.11 μmol) and DMF (1 mL) of the previous reaction solution was added diethylamine (70.70 mg, 966.69 μmol), the reaction was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was freeze-dried after removing part of the solvent with a freeze dryer, and then purified by reversed phase (acetonitrile-0.05% aqueous ammonium bicarbonate solution = 0-90%) and freeze-dried to obtain the title compound (35 mg, 34.96 μmol).

[0460] The structural characterization data thereof are as follows:

[0461] MS m / z (ESI): 1001.4 [M+H] +

[0462] Step three: Preparation of (9H-fluoren-9-yl)methyl ((4S,7S,10S,13S,20S,24S,27S,30S)-36-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)oxazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10,24,27,30-hexamethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12,19,22,25,28,31,36-decaoxa-34-oxa-2,5,8,11,18,23,26,29,32-nonazatetratriacontan-20-yl)carbamate (A-1-3)

[0463] N-((4S,7S,10S,13S)-17-amino-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-3,6,9,12-tetraoxa-2,5,8,11-tetraazahexadecyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (35 mg, 34.96 μmol) was dissolved in DMF (3 mL), HATU (15.94 mg, 41.96 μmol) and DIPEA (18.07 mg, 139.85 μmol) were added, the reaction was stirred at room temperature for 0.5 hours, then N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4- ((S)-1-(((S)-1-(((S)-1-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)- 1-oxopropan-2-yl)-L-asparagine (35 mg, 34.96 μmol), stirred at room temperature for 1.5 hours. Add PyBOP (27.29 mg, 52.44 μmol) and DIPEA (18.07 mg, 139.85 μmol), stir the reaction at room temperature for 1 hour. After the reaction is completed, the reaction solution is directly used in the next step reaction as a solution.

[0464] The structural characterization data thereof are as follows:

[0465] MS m / z (ESI): 1037.0 [M / 2+H] +

[0466] Step four: (S)-2-amino-N 4 - ((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10- dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecan-13-yl)-N 1 - ((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)- 3-oxo-8,9-dihydro-3H-pyrano[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazahexadecan- 17-yl)succinamide (A-1-4)

[0467] To the reaction solution of (9H-fluoren-9-yl)methyl ((4S,7S,10S,13S,20S,24S,27S,30S)-36-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-1-yl)oxazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10,24,27,30-hexamethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12,19,22,25,28,31,36-decaoxa-34-oxa-2,5,8,11,18,23,26,29,32-nonazatetratriacontan-20-yl)carbamate (72 mg, 34.74 μmol) and DMF (4 mL) was added diethylamine (50.81 mg, 694.78 μmol) and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was removed part of the solvent by freeze dryer, then added 1 drop of formic acid, and then purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (21 mg, 11.07 μmol).

[0468] The structural characterization data thereof are as follows:

[0469] MS m / z (ESI): 1852.5 [M+H] +

[0470] The preparation method thereof is as follows:

[0471] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0472] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0473] Step five: preparation of 2,2',2"-(10-((4S,7S,10S,13S,20S)-20-((7S,10S,13S)-1-(((1S,9S)-5- chloro-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10,13- trimethyl-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-1-((8S,9R)- 5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin- 5-yl)hex-5-ynoyl)-3,6,9,12,19,22-hexaoxo-2,5,8,11,18,21-hexaazatricosan-23-yl)-1,4,7- triazacyclododecane-1,4,7-triyl)triacetic acid (A-1)

[0474] (S)-2-amino-N 4 -((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10- dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecan-13-yl)-N 1- ((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10- trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12-tetraoxo-2,5,8,11- tetraazahexadec-17-yl)succinamide (20.49 mg, 11.07 μmol) was dissolved in DMF (2 mL), 2,2',2”-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7- triyl)triacetic acid (11.11 mg, 22.15 μmol) was added, followed by the addition of DIPEA (8 mg, 61.90 μmol), and the reaction was stirred at room temperature for 1.5 hours. 2,2',2”-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7- triyl)triacetic acid (16.66 mg, 33.22 μmol) and DIPEA (16 mg, 123.80 μmol) were added, and the reaction was stirred for another 1.5 hours. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (6.63 mg, 2.84 μmol).

[0475] The structural characterization data thereof are as follows:

[0476] MS m / z (ESI): 1119.3 [M / 2+H] +

[0477] The preparation method thereof is as follows:

[0478] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0479] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0480] Example 2: Preparation of 2,2',2"-(10-((7S,10S,13S,16S,23S)-1-(((1S,9S)-5-chloro-9- ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-23-((4S,7S,10S)-1-((8S,9R)- 5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-3,6,9,12-tetraoxo-2,5,8,11- tetraazatridecan-13-yl)-7,10,13-trimethyl-16-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5- enoylamino)-1,6,9,12,15,22,25-heptaoxo-3-oxa-5,8,11,14,21,24-hexaazahexacosan-26-yl)- 1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-2)

[0481] Step One: Preparation of (9H-fluoren-9-yl)methyl ((7S,10S,13S,16S)-1-(((1S,9S)-5-chloro-9- ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-16-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-enoylamino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14- tetraazaeicosan-20-yl)carbamate (A-2-1)

[0482] N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2(S)-2-amino-N-((S)-1-(((S)-1-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxoprop-2-yl)amino)-1- oxopropan-2-yl)propanamide (32.25 mg, 40.41 μmol) was added after stirring for 15 minutes in DMF (2 mL), and the reaction was stirred at room temperature for 45 minutes. After the reaction was completed, the reaction solution was used directly in the next reaction as a solution.

[0483] The structural characterization data thereof are as follows:

[0484] MS m / z (ESI): 1353.7 [M+H] +

[0485] Step two: Preparation of N-((7S,10S,13S,16S)-20-amino-1-(((1S,9S)-5-chloro-9- ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl- 1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazaeicosan-16-yl)-6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic amide (A-2-2)

[0486] To the reaction solution of (9H-fluoren-9-yl)methyl ((7S,10S,13S,16S)-1-(((1S,9S)-5- chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-16-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14- tetraazaeicosan-20-yl)carbamate (54 mg, 39.91 μmol) and DMF (2 mL) was added diethylamine (106.05 mg, 1.45 mmol) and stirred at room temperature for 0.5 h. After the reaction was completed, the reaction solution was purified by reverse phase column chromatography (acetonitrile-0.05% aqueous formic acid = 0-90%) and freeze-dried to obtain the title compound (26 mg, 22.10 μmol).

[0487] The structural characterization data thereof are as follows:

[0488] MS m / z (ESI): 1130.5 [M+H] +

[0489] Step three: Preparation of N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4 -((S)-1-(((S)-1-(((S)-1-(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1- oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-L-asparagine (A-2-3)

[0490] (S)-2-amino-N-((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl- 1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (30 mg, 48.18 μmol) was dissolved in DMF (1 mL), DIPEA (16 mg, 123.80 μmol) was added, then (9H-fluoren-9-yl)methyl (S)-(2,5-dioxotetrahydrofuran-3-yl)carbamate (17.88 mg, 53.00 μmol) was added, the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was directly purified by reverse phase column chromatography (acetonitrile-0.05% aqueous formic acid solution = 0-90%) and freeze-dried to obtain the title compound (40 mg, 39.76 μmol).

[0491] The structural characterization data thereof are as follows:

[0492] MS m / z (ESI): 960.3 [M+H] +

[0493] Step four: Preparation of (9H-fluoren-9-yl)methyl ((4S,7S,10S,14S,21S,24S,27S,30S)-36-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)oxazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10,24,27,30-hexamethyl-21-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12,15,22,25,28,31,36-decaoxo-34-oxa-2,5,8,11,16,23,26,29,32-nonazatetratriaconta-14-yl)carbamate (A-2-4)

[0494] N-((7S,10S,13S,16S)-20-amino-l-(((lS,9S)-5-chloro-9-ethyl-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)amino)-7,10,13-trimethyl-l,6,9,12,15- pentoaoxo-3-oxa-5,8,11,14-tetraazaeicosan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynamide (25 mg, 21.25 μmol) and N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4 -((S)-l-(((S)-l-(((S)-l-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)- 1-oxopropan-2-yl)amino)-l-oxopropan-2-yl)amino)-l-oxopropan-2-yl)-L-asparagine (23.51 mg, 23.37 μmol) was dissolved in DMF (2 mL), DIPEA (16 mg, 123.80 μmol) was added, after stirring at room temperature for 10 minutes, PyBOP (13.27 mg, 25.50 μmol) was added, and the stirring was continued for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (13 mg, 6.27 μmol).

[0495] The structural characterization data thereof are as follows:

[0496] MS m / z (ESI): 1036.6 [M / 2+H] +

[0497] The preparation method thereof is as follows:

[0498] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0499] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0500] Step five: (S)-2-amino-N 1- ((7S,10S,13S,16S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 1-yl)amino)-7,10,13-trimethyl-16-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)- 1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazaeicosan-20-yl)-N 4 - ((S)-1-(((S)-1-(((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl- 1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl) amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2- yl)succinamide (A-2-5)

[0501] (9H-fluoren-9-yl)methyl ((4S,7S,10S,14S,21S,24S,27S,30S)-36-(((1S,9S)-5-chloro-9- ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano [3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)- 9-(1-methyl-1H-1,2,4-triazol-5-yl)oxazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2- de]phthalazine-2(7H)-yl)-4,7,10,24,27,30-hexamethyl-21-(6-(2-(methylsulfonyl)pyrimidin- 5-yl)hex-5-ynamido)-3,6,9,12,15,22,25,28,31,36-decaoxo-34-oxa-2,5,8,11,16,23,26,29,32- ena-14-yl)carbamate (13 mg, 6.27 μmol) was dissolved in DMF (2 mL), diethylamine (14.14 mg, 193.34 μmol) was added, and the reaction was stirred at room temperature for 1 h. After the reaction was completed, the solvent was removed directly by lyophilization, and the product was used directly in the next step without further purification.

[0502] The structural characterization data thereof are as follows:

[0503] MS m / z (ESI): 935.5 [(M+H2O) / 2+H]+

[0504] Step six: Preparation of 2,2',2"-(10-((7S,10S,13S,16S,23S)-1-(((1S,9S)-5-chloro-9- ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-23-((4S,7S,10S)-1- ((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9- dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-3,6,9,12- tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-7,10,13-trimethyl-16-(6-(2-(methylsulfonyl) pyrimidin-5-yl)hex-5-ynoyl)-1,6,9,12,15,22,25-heptaoxo-3-oxa-5,8,11,14,21,24- hexaazahexacosan-26-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-2)

[0505] directly to a theoretical mixture of (S)-2-amino-N 1 -((7S,10S,13S,16S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 1-yl)amino)-7,10,13-trimethyl-16-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)- 1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazaeicosan-20-yl)-N 4- ((S)-1-(((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H- 1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl) amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2- yl)succinamide (12 mg, 6.49 μmol) was added to the reaction mixture from the previous step, which was removed from the lyophilizer. DMF (1 mL) was added, followed by DIPEA (16 mg, 123.80 μmol) and 2,2',2”-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetic acid (20 mg, 39.88 μmol). The reaction was stirred at room temperature for 3 hours. After the reaction was complete, the reaction mixture was lyophilized to remove some of the solvent and purified by preparative high performance liquid chromatography and lyophilized to yield the title compound (6.09 mg, 2.59 μmol).

[0506] The structural characterization data thereof are as follows:

[0507] MS m / z (ESI): 1119.1 [M / 2+H] + ; 746.4 [M / 3+H] +

[0508] The preparation method thereof is as follows:

[0509] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0510] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0511] Example Three: Preparation of N-((S)-1-(((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2- de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2- yl)amino)-1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoyl amide (D-1)

[0512] (6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynyl)-L-alanine-L-alanine (17.09 mg, 41.63 μmol) was dissolved in DMF (1 mL), HATU (17.41 mg, 45.79 μmol) and DIPEA (16.14 mg, 124.88 μmol) were added, the reaction was stirred at room temperature for 0.5 hours, then (S)-2-amino-N-(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl))-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazin-2(7H)-yl)methyl)propanamide (20 mg, 41.63 μmol) was added, the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (11.37 mg, 12.19 μmol).

[0513] The structural characterization data thereof are as follows:

[0514] MS m / z (ESI): 873.7 [M+H]+; 437.5 [M / 2+H]+

[0515] The preparation method thereof is as follows:

[0516] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0517] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0518] Example Four: Preparation of N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxo-5,8,11-triazatetradecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (C-1)

[0519] Step one: Preparation of (9H-fluoren-9-yl)methyl [(7S,10S,13S)-1-(((9S)-5-chloro-9- ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino]-7,10-dimethyl-1,6,9,12- tetraoxo-3-oxa-5,8,11-triazahexadecan-13-yl]carbamate (C-1-2)

[0520] (5S,8S,11S)-1-(9H-fluoren-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13- tetraazahexadecan-17-oic acid (657 mg, 1.22 mmol) and (9S)-1-amino-5-chloro-9-ethyl-9- hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinoline-10,13-dione (500 mg, 1.11 mmol) were dissolved in N,N-dimethylformamide (10 mL), followed by the addition of HATU (630.67 mg, 1.66 mmol) and N,N- diisopropylethylamine (428 mg, 3.32 mmol), and stirring at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high-performance liquid chromatography and freeze-dried to obtain 700 mg of the title compound.

[0521] Preparation method thereof is as follows:

[0522] Column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm)

[0523] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0524] Step two: Preparation of (2S)-2-amino-N-((2S)-1-(((2S)-1-(((2-(((9S)-5-chloro-9-ethyl-9- hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxoprop-2-yl)amino)- 1-oxoprop-2-yl)propanamide (C-1-3)

[0525] (9H-fluoren-9-yl)methyl ((7S,10S,13S)-1-(((9S)-5-chloro-9-ethyl-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11- triazatetradecan-13-yl)carbamate (500 mg, 0.513 mmol) was dissolved in N,N- dimethylformamide (2 mL), diethylamine (75.05 mg, 1.03 mmol) was added, and the reaction was allowed to proceed at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high-performance liquid chromatography and freeze-dried to obtain 307 mg of the title compound.

[0526] The preparation method thereof is as follows:

[0527] Column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm)

[0528] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0529] Step three: preparation of N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11- triazatetradecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic amide (C-1)

[0530] (2S)-2-amino-N-((2S)-1-(((2S)-1-(((2-(((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (170 mg, 0.226 mmol), 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (90.83 mg, 0.249 mmol, FR) were dissolved in N,N-dimethylformamide (10 mL). N,N-diisopropylethylamine (29.21 mg, 0.226 mmol) was added to the reaction. The reaction was replaced with nitrogen three times and stirred at room temperature for 16 hours. After the reaction was completed, the reaction was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (50.56 mg).

[0531] The structural characterization data thereof are as follows:

[0532] MS m / z (ESI): 1002.4 [M+H]+

[0533] The preparation method thereof is as follows:

[0534] Column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm)

[0535] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0536] Example Five: Preparation of 2,2',2"-(10-((2S,5S,27S,34S)-27-(3,5-bis(2- (methylsulfonyl)pyrimidin-5-yl)benzamido)-l-(((S)-9-ethyl-9-hydroxy-10,13-dioxo- 2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin- 4-yl)amino)-34-((4S,7S,10S)-l-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH- 1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10- trimethyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-5-isopropyl-2-methyl-l,4,7,26,33,36-hexaoxo-10,13,16,19,22-pentaoxa-3,6,25,32,35-pentaazatetracosan-37-yl)- 1,4,7,10-tetraazacyclododecane-l,4,7-triyl)triacetic acid (A-3)

[0537] Step One: Preparation of (9H-fluoren-9-yl)methyl ((20S,23S)-24-(((S)-9-ethyl-9- hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-4-yl)amino)-20-isopropyl-23-methyl-18,21,24-trioxo-3,6,9,12,15- pentaaoxa-19,22-diazatetracosyl)carbamate (A-3-2)

[0538] Step 1: Preparation of (S)-2-amino-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (A-3-1)

[0539] The structural characterization data thereof are as follows:

[0540] MS m / z (ESI): 1087.6 [M+H] +

[0541] Step 2: Preparation of 1-amino-N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13- dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15- pentoaoctadecan-18-amide (A-3-3)

[0542] ((20S,23S)-24-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-20-isopropyl-23-methyl- 18,21,24-trioxo-3,6,9,12,15-pentoaocta-19,22-diazatetracosyl)carbamate (116 mg, 106.69 μmol) was dissolved in DMF (2 mL), diethylamine (1 mL) was added, and the reaction was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the title compound (92.29 mg, 106.69 μmol).

[0543] Its structural characterization data are as follows:

[0544] MS m / z (ESI): 865.3 [M+H] +

[0545] Step 3: Preparation of ((2S,5S,27S)-27-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,26-tetraoxo-10,13,16,19,22-pentaoxa-3,6,25-triazatrione-31-yl)tert-butyl carbamate (A-3-4)

[0546] N 2 -(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-N 6 -(tert-Butoxycarbonyl)-L-lysine (37.93 mg, 57.23 μmol) was dissolved in DMF (1 mL), DIPEA (20.13 mg, 156.06 μmol, 27.50 μL) was added, and then HATU (23.72 mg, 57.23 μmol) was added. The mixture was stirred at room temperature for 20 minutes. Then, 1-amino-N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-3,6,9,12,15-pentaoctadecane-18-amide (45 mg, 52.02 μmol) was added, and the mixture was stirred at room temperature for 100 minutes. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (50 mg, 33.12 μmol).

[0547] Its structural characterization data are as follows:

[0548] MS m / z (ESI): 1509.7 [M+H] +

[0549] Step four: Preparation of N-((2S,5S,27S)-31-amino-1-(((S)-9-ethyl-9-hydroxy- 10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo[de]pyrano[3', 4':6, 7]indolizino[1, 2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-1, 4, 7, 26-tetraoxo- 10, 13, 16, 19, 22-pentaoxa-3, 6, 25-triazatriacontan-27-yl)-3, 5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (A-3-5)

[0550] tert-Butyl ((2S,5S,27S)-27-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-5-isopropyl-2- methyl-1,4,7,26-tetraoxo-10,13,16,19,22-pentaoxa-3,6,25-triazatriacontan-31- yl)carbamate (25 mg, 33.12 μmol) was dissolved in DCM (2 mL), TFA (1 mL) was added, and the reaction was stirred at room temperature for 1 h. After the reaction was completed, it was concentrated under reduced pressure to give the title compound (20 mg, 14.19 μmol).

[0551] The structural characterization data thereof are as follows:

[0552] MS m / z (ESI): 1409.6 [M+H2O] +

[0553] Step five: Preparation of (9H-fluoren-9-yl)methyl ((4S,7S,10S,14S,21S,43S,46S)-21-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-47-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-43-isopropyl-4,7,10,46-tetramethyl-3,6,9,12,15,22,41,44,47-nonaoxo-26,29,32,35,38-pentaoxa-2,5,8,11,16,23,42,45-octazatetracontan-14-yl)carbamate (A-3-6)

[0554] N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4-((2S,5S,27S)-31-amino-l-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15- hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-4-yl)amino)- 5-isopropyl-2-methyl-l,4,7,26-tetraoxo-10,13,16,19,22-pentaoxa-3,6,25- triazatriacontan-27-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (20 mg, 14.19 μmol) was added, and the reaction was stirred at room temperature for 100 min. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (15 mg, 6.38 μmol).

[0555] The structural characterization data thereof are as follows:

[0556] MS m / z (ESI): 1176.1 [M / 2+H] +

[0557] Step six: (S)-2-amino-N 1 -((2S,5S,27S)-27-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-l-(((S)-9- ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH, 12H-benzo[de]pyrano[3',4':6,7] indolizino[l,2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-l,4,7,26-tetraoxo- 10,13,16,19,22-pentaoxa-3,6,25-triazatriacontan-31-yl)-N 4Preparation of ((S)-1-(((S)-1-(8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1- oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl) succinamide (A-3-7)

[0558] (9H-fluoren-9-yl)methyl ((4S,7S,10S,14S,21S,43S,46S)-21-(3,5-bis(2-(methylsulfonyl)pyrimidin-5- yl)benzamido)-47-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-4-yl)amino)-1-((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine- 2(7H)-yl)-43-isopropyl-4,7,10,46-tetramethyl-3,6,9,12,15,22,41,44,47-nonaoxo-26,29,32,35,38- pentoaoxa-2,5,8,11,16,23,42,45-octazatetracontan-14-yl)carbamate (15 mg, 6.38 µmol) was dissolved in DMF (1 mL), diethylamine (0.5 mL) was added, and the reaction was stirred at room temperature for 0.5 hours. After the reaction was completed, the title compound (10 mg, 4.70 µmol) was obtained by concentration under reduced pressure.

[0559] The structural characterization data thereof are as follows:

[0560] MS m / z (ESI): 1064.6 [M / 2+H] +

[0561] Step seven: Preparation of 2,2',2"-(10-((2S,5S,27S,34S)-27-(3,5-bis(2- (methylsulfonyl)pyrimidin-5-yl)benzamido)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 4-yl)amino)-34-((4S,7S,10S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H- 1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10- trimethyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-5-isopropyl-2-methyl-1,4,7,26,33,36-hexaoxo-10,13,16,19,22-pentaoxa-3,6,25,32,35-pentaazahexatriacontan- 37-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-3)

[0562] (S)-2-amino-N 1 -((2S,5S,27S)-27-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-1-(((S)-9- ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-1,4,7,26-tetraoxo-10,13,16,19,22-pentaoxa-3,6,25-triazahentriacontan-31-yl)-N 4-((S)-1-(((S)-1-(8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)- 3-oxo-8,9-dihydro-3H-pyrano[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan- 2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)succinamide (10 mg, 4.70 μmol) was dissolved in DMF (1 mL), DIPEA (1.81 mg, 14.1 μmol) was added, followed by 2,2',2”-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetic acid (7.06 mg, 14.1 μmol), and stirred at 25 °C for 1 h. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (2.43 mg, 0.95 μmol).

[0563] The structural characterization data thereof are as follows:

[0564] MS m / z (ESI): 1257.7 [M / 2+H] +

[0565] The preparation method thereof is as follows:

[0566] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0567] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0568] Example Six: (S)-N 4 -((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H,12H-benzo[de]pyranopyrazino[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10- dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradodecan-13-yl)-N 1Preparation of (S)-N-((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1- methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)- yl)-4,7,10-trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylaminyl)-3,6,9,12- tetraoxo-2,5,8,11-tetraazahexadec-17-yl)-2-(3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6- pentyloxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6- pentyloxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propanoylaminyl)succinamide (A-7)

[0569] To A-1-14 (11 mg, 5.80 μmol) and 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6- pentyloxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6- pentyloxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propanoic acid (6.16 mg, 5.80 μmol) was added DMF (1 mL), DIPEA (749.67 μg, 5.80 μmol) was added dropwise, then HATU (2.20 mg, 5.80 μmol) was added, and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (7.36 mg, 2.59 μmol, 98% purity).

[0570] The structural characterization data thereof are as follows:

[0571] MS m / z (ESI): 1395.0 [M / 2+H] + ; 930.3 [M / 3+H] +

[0572] The preparation method thereof is as follows:

[0573] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0574] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0575] Example Seven: (S)-N4 - ((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b] quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11- triazatetradodecyl)-2-(3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl) amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2- (hydroxymethy l)propan-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propanamido)- N 1 - ((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10- trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamido)-3,6,9,12- tetraoxo-2,5,8,11-tetraazahexadec-17-yl)succinamide (A-8)

[0576] To A-1-14 (11 mg, 5.80 μmol) and 3-(3-(3-((1,3-dihydroxy-2- (hydroxymethyl)propan-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2- (hydroxymethyl)propan-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propanoic acid (4.73 mg, 5.80 μmol) was added DMF (1 mL), DIPEA (749.67 μg, 5.80 μmol) was added dropwise, followed by HATU (2.20 mg, 5.80 μmol), and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (8.83 mg, 3.37 μmol, 98% purity).

[0577] The structural characterization data thereof are as follows:

[0578] MS m / z (ESI): 1283.1 [M / 2+H] + ; 856.2 [M / 3+H] +

[0579] The preparation method thereof is as follows:

[0580] Column: Waters Sunfire Prep C18 OBD (5 pm * 19 mm * 150 mm)

[0581] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0582] Example Eight: (S)-N 4 -((S)-1-(((S)-1-(((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)carbamateacyl)thiazolidin-3-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2- yl)-N 1 -((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5- yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylaminyl)-3,6,9,12-tetraoxo-2,5,8,11- tetraazahexadec-17-yl)-2-(3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)- 3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3- oxopropoxy)methyl)propoxy)propanamido)succinamide (A-9)

[0583] Step One: Preparation of ((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1- methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)- 4,7,10-trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylaminyl)-3,6,9,12- tetraoxo-2,5,8,11-tetraazahexadec-17-yl)carbamic acid allyl ester (A-9-1)

[0584] (S)-2-amino-N-((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H- 1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)- 1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (100 mg, 0.15 mmol), N- ((allyloxy)carbonyl)-L-glutamic acid (50.0 mg, 0.17 mmol) were dissolved in DMF (3 mL), DIPEA (39.4 mg, 0.31 mmol) was added and stirred for 1 min, then HATU (87.0 mg, 0.23 mmol) was added, and the reaction was stirred at 25 °C for 0.5 h. The reaction solution was directly purified by C18 reverse phase column (ACN / 0.05% formic acid aqueous solution = 10-70%), and then lyophilized to give the title compound (91 mg, 0.084 mmol). 6 -((allyloxy)carbonyl)-N 2 -((allyloxy)carbonyl)-N

[0585] The structural characterization data thereof are as follows:

[0586] ESI-MS (m / z): 1085.4 (M+H) +

[0587] Step two: Preparation of N-((4S,7S,10S,13S)-17-amino-1-((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2- de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazheptadec- 13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-9-2)

[0588] A-9-1 (91 mg, 0.084 mmol) was dissolved in DMF (2 mL) under nitrogen protection, 1,3-dimethylbarbituric acid (23.6 mg, 0.15 mmol), and tetrakis triphenylphosphine palladium (17.4 mg, 0.015 mmol) were added, and the reaction was stirred at 35 °C for 2 h. The reaction solution was directly purified by C18 reverse phase column (ACN / 0.05% TFA aqueous solution = 10-70%), and then lyophilized to give the title compound (74 mg, 0.066 mmol, trifluoroacetate salt).

[0589] The structural characterization data thereof are as follows:

[0590] ESI-MS (m / z): 1001.5 (M+H) +

[0591] Step three: Preparation of (9H-fluoren-9-yl)methyl ((4S,7S,10S,14S,21S,24S,27S,30S)-1-((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)thiazolidin-3-yl)-33-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10,24,27,30-hexamethyl-21-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-3,6,9,12,15,22,25,28,31-nonaoxo-2,5,8,11,16,23,26,29,32-nonazatricosan-14-yl)carbamate (A-9-3)

[0592] A-9-2 (69 mg, 0.062 mmol, trifluoroacetate salt) and INT-9 (69.93 mg, 0.062 mmol) were dissolved in DMF (3 mL), DIPEA (18.0 mg, 0.14 mmol) was added and stirred for 1 min, then HATU (42.4 mg, 0.11 mmol) was added, and the reaction was stirred at 25 °C for 0.5 h. The reaction solution was directly purified by C18 reverse column (ACN / 0.05% formic acid aqueous solution = 20-80%), and lyophilized to give the title compound (105 mg, 0.049 mmol).

[0593] The structural characterization data thereof are as follows:

[0594] ESI-MS (m / z): 1057.4 (M / 2+H) +

[0595] Step four: Preparation of (S)-2-amino-N 4-((S)-1-(((S)-1-(((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo[de]pyrano[3', 4':6, 7]indolizino[1, 2-b]quinolin-1-yl) carbamoyl) thiazolidin-3-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-N 1 -((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8, 9-dihydro-3H-pyrrolo[4, 3, 2-de]phthalazine-2(7H)-yl)-4, 7, 10- trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3, 6, 9, 12-tetraoxo-2, 5, 8, 11- tetraazahexadec-17-yl) succinamide (A-9-4)

[0596] A-9-3 (105 mg, 0.049 mmol) was dissolved in DMF (2 mL), diethylamine (32.7 mg, 0.45 mmol) was added, and the reaction was allowed to react at 25 °C for 1 h. The reaction solution was directly purified by a C18 reverse column (ACN / 0.05% trifluoroacetic acid aqueous solution = 20-90%), and lyophilized to obtain the title compound (75 mg, 0.037 mmol).

[0597] The structural characterization data thereof are as follows:

[0598] ESI-MS (m / z): 1890.7 (M+H) +

[0599] Step five: (S)-N 4 -((S)-1-(((S)-1-(((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo[de]pyrano[3', 4':6, 7]indolizino[1, 2-b]quinolin-1-yl) carbamoyl) thiazolidin-3-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-N 1Preparation of ((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1- methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine- 2(7H)-yl)-4,7,10-trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl- amino)-3,6,9,12-tetraoxo-2,5,8,11-tetraazahexadec-17-yl)-2-(3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3- oxopropoxy)methyl)propoxy)propanamido)succinamide (A-9)

[0600] A-9-4 (20.0 mg, 0.010 mmol) was dissolved in DMF (1 mL), 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propanoic acid (14.3 mg, 0.015 mmol), DIPEA (3.5 mg, 0.027 mmol) were stirred for 1 min, HATU (6.8 mg, 0.018 mmol) was added, the reaction was stirred at room temperature for 0.5 h, the reaction solution was directly purified by preparative high performance liquid chromatography and lyophilized to obtain the title compound (14.0 mg, 0.005 mmol).

[0601] The structural characterization data thereof are as follows:

[0602] MS m / z (ESI): 1415.0 [M / 2+H] +

[0603] The preparation method thereof is as follows:

[0604] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0605] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0606] Example Nine: (S)-2-(3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3- oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3- oxopropoxy)methyl)propoxy)propanamido)-N 4 -((S)-1-(((S)-1-(((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13- dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-N 1 -((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)- 3-oxo-8,9-dihydro-3H-pyrano[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazahexadecan- 17-yl)succinamide (A-10)

[0607] (S)-2-amino-N 4 -((S)-1-(((S)-1-(((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13- dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-N 1- ((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10- trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12-tetraoxo-2,5,8,11- tetraazahexadec-17-yl) succinamide (20.0 mg, 0.010 mmol) was dissolved in DMF (1 mL), 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3-oxopropoxy)-2,2- bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3-oxopropoxy)methyl)propoxy) propanoic acid (11.0 mg, 0.015 mmol), DIPEA (3.5 mg, 0.027 mmol) was stirred for 1 min, HATU (6.8 mg, 0.018 mmol) was added, the reaction was stirred at room temperature for 0.5 h, the reaction was purified by preparative high performance liquid chromatography directly and lyophilized to give the title compound (8.20 mg, 0.003 mmol).

[0608] The structural characterization data thereof are as follows:

[0609] MS m / z (ESI): 1303.9 [M / 2+H] +

[0610] The preparation method thereof is as follows:

[0611] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0612] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0613] Example Ten: (S)-2-(3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3- oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3- oxopropoxy)methyl)propoxy)propanoylamino)-N 4- ((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9, 12-dioxo-1, 2, 8, 9, 12, 14-hexahydro- 11H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3-yl)amino)-1- oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-N 1 - ((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10- trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12- tetraoxo-2,5,8,11-tetraazaheneicos-17-yl) succinamide (A-11)

[0614] Step one: N 6 - ((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10- trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12- tetraoxo-2,5,8,11-tetraazaheneicos-17-yl) succinamide (A-11) 2 - ((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10- trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12- tetraoxo-2,5,8,11-tetraazaheneicos-17-yl) succinamide (A-11) 4 - ((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9, 12-dioxo-1, 2, 8, 9, 12, 14-hexahydro- 11H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3-yl)amino)-1- oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-L-aspartyl)-N 2 - ((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10- trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12- tetraoxo-2,5,8,11-tetraazaheneicos-17-yl) succinamide (A-11)

[0615] Trifluoroacetate salt of (S)-2-amino-N-((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo- 1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline- 3-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (60 mg, 89.07 μmol) and (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(((S)-6-(allyloxy)-5-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-yn-1-ylamino)-6-oxohexyl)amino)-4- oxobutanoic acid (68.93 mg, 89.07 μmol) were dissolved in DMF (2 mL), DIPEA (34.53 mg, 267.21 μmol) was added dropwise, HATU (37.23 mg, 97.98 μmol) was added portionwise, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was added dropwise to water with stirring, and a light yellow solid was precipitated. The solid was filtered, washed with water, and dried to obtain the title compound (114 mg, 86.66 μmol).

[0616] The structural characterization data thereof are as follows:

[0617] ESI-MS (m / z): 1315.5 (M+H) +

[0618] Step two: N 6 -(N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4 -((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H- cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3-yl)amino)-1-oxopropan-2- yl)amino)-3-methyl-1-oxobutan-2-yl)-L-asparaginyl)-N 2 Preparation of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(((S)-6-(allyloxy)- 5-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yn-1-ylamino)-6-oxohexyl)amino)-4- oxobutanoic acid (A-11-2)

[0619] To A-11-1 (114 mg, 86.66 μmol) was added DMF (4 mL), followed by 1,3-dimethylbarbituric acid (54.13 mg, 346.65 μmol) and tetrakis triphenylphosphine palladium (15.02 mg, 13.00 μmol). After nitrogen substitution, it was stirred at room temperature for 1.5 hours. After the reaction was completed, the reaction solution was directly purified by reverse phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%) and freeze-dried to obtain the title compound (70 mg, 54.89 μmol).

[0620] The structural characterization data thereof are as follows:

[0621] ESI-MS (m / z): 1467.3 (M+H) +

[0622] Step three: Preparation of (9H-fluoren-9-yl)methyl ((4S,7S,10S,13S,20S,24S,27S)-28-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3-yl)amino)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)-24-isopropyl-4,7,10,27-tetramethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-3,6,9,12,19,22,25,28-octaoxo-2,5,8,11,18,23,26-heptazanonacosan-20-yl)carbamate (A-11-3)

[0623] A-11-2 (65 mg, 50.97 μmol) and (S)-2-amino-N-((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (31.73 mg, 50.97 μmol) were dissolved in DMF (4 mL), DIPEA (13.17 mg, 101.93 μmol) was added dropwise, and HATU (19.37 mg, 50.97 μmol) was added, and stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was added dropwise into water with stirring, and a solid was precipitated, which was filtered and dried to obtain the title compound (87 mg, 46.28 μmol).

[0624] The structural characterization data thereof are as follows:

[0625] ESI-MS (m / z): 940.8 (M / 2+H) +

[0626] Step four: (S)-2-amino-N 4 -((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H- cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3-yl)amino)-1-oxopropan-2- yl)amino)-3-methyl-1-oxobutan-2-yl)-N 1 -((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-trans-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrano[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10- trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12-tetraoxo-2,5,8,11- tetraazahexadecan-17-yl)succinamide (A-11-4)

[0627] To the crude A-11-3 (80 mg, 42.55 μmol) was added DMF (3 mL), diethylamine (31.12 mg, 425.53 μmol) was added dropwise, and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was directly purified by reverse phase column chromatography (acetonitrile-0.05% trifluoroacetic acid aqueous solution = 0-90%) and freeze-dried to obtain a crude product, which was further purified by preparative high performance liquid chromatography and freeze-dried to obtain the trifluoroacetate salt of the title compound (14 mg, 7.90 μmol).

[0628] The structural characterization data thereof are as follows:

[0629] MS m / z (ESI): 829.4 [M / 2+H] + ; 553.3 [M / 3+H] +

[0630] The preparation method thereof is as follows:

[0631] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0632] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

[0633] Step five: (S)-2-(3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3- oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3- oxopropoxy)methyl)propoxy)propanamido)-N 4 -((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H- cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3-yl)amino)-1-oxopropan-2- yl)amino)-3-methyl-1-oxobutan-2-yl)-N 1 -((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)- 3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazahexadecan- 17-yl)succinamide (A-11) was prepared

[0634] A-11-4 (7 mg, 3.95 μmol) and 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)- 3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3- oxopropoxy)methyl)propoxy)propanoic acid (5.80 mg, 7.90 μmol) were dissolved in DMF (1 mL), DIPEA (510.62 μg, 3.95 μmol) was added dropwise, HATU (3.00 mg, 7.90 μmol) was added, and stirring was performed at room temperature for 1.5 hours. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (4.87 mg, 2.01 μmol, 98% purity).

[0635] The structural characterization data thereof are as follows:

[0636] MS m / z (ESI): 1187.4 [M / 2+H] +

[0637] The preparation method thereof is as follows:

[0638] Column: Waters Sunfire Prep C18 OBD (5 pm * 19 mm * 150 mm)

[0639] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% formic acid)

[0640] Example XI: (S)-N 4 -((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H- cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3-yl)amino)-1-oxopropan-2- yl)amino)-3-methyl-1-oxobutan-2-yl)-N 1 -((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)- 3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylaminyl)-3,6,9,12-tetraoxo-2,5,8,11- tetraazaheneicosyl-17-yl)-2-(3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)- 3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3- oxopropoxy)methyl)propoxy)propanamido)succinamide (A-12)

[0641] A-11-4 (7 mg, 3.95 pmol) and 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)- 3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3- oxopropoxy)methyl)propoxy)propanoic acid (7 mg, 7.32 pmol) were dissolved in DMF (1 mL), DIPEA (510.62 pg, 3.95 pmol) was added dropwise, HATU (3.00 mg, 7.90 pmol) was added, and stirring was performed at room temperature for 1.5 hours. After the reaction was completed, the reaction solution was directly purified by preparative high-performance liquid chromatography and freeze-dried to obtain the title compound (4.33 mg, 1.63 pmol, 98% purity).

[0642] The structural characterization data thereof are as follows:

[0643] MS m / z (ESI): 1298.2 [M / 2+H] +

[0644] Preparation method as follows:

[0645] Column: Waters Sunfire Prep C18 OBD (5 pm*19 mm*150 mm)

[0646] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% formic acid)

[0647] Example Twelve: (S)-N 4 -((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazahexadecan-13-yl)-N 1 -((4S,7S,10S,13S)-1-(4-(3-(4-(3-cyanopyridin-2-yl)piperazine-1-carbonyl)benzyl)- 1-oxophthalazine-2(1H)-yl)-4,7,10-trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoylaminol-3,6,9,12-tetraoxo-2,5,8,11-tetraazahexadecan-17-yl)-2-(3-(3-(3- (methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2- bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl) propoxy)propanamido)succinamide (A-13)

[0648] Step One: Preparation of N-((4S,7S,10S,13S)-1-(4-(3-(4-(3-cyanopyridin-2-yl)piperazine- 1-carbonyl)benzyl)-1-oxophthalazine-2(1H)-yl)-4,7,10-trimethyl-3,6,9,12-tetraoxo- 19,19-diphenyl-19-(p-tolyl)-2,5,8,11,18-pentaazanonadecan-13-yl)-6-(2-(methylsulfonyl) pyrimidin-5-yl)hex-5-ynoylamine (A-13-1)

[0649] (S)-2-amino-N-((S)-1-(((S)-1-(((4-(3-(4-(3-cyanopyridin-2-yl)piperazin-1-carbonyl)benzyl)-1-oxophthalazin-2(1H)-yl)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (0.14 g, 202.09 μmol) and N 6 -(diphenyl(p-tolyl)methyl)-N 2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (171.50 mg, 262.72 μmol) was dissolved in DMF (3 mL), DIPEA (78.35 mg, 606.27 μmol) was added and stirred for 1 minute, followed by the addition of HATU (92.15 mg, 242.51 μmol). The reaction was carried out at 25 °C for 0.5 hours. The reaction solution was directly purified by C18 reverse-phase column chromatography (ACN / 0.05% formic acid aqueous solution = 20-80%), and lyophilized to obtain the title compound (255 mg, 192.08 μmol).

[0650] Its structural characterization data are as follows:

[0651] ESI-MS (m / z): 1327.5 (M+H) +

[0652] Step 2: Preparation of N-((4S,7S,10S,13S)-17-amino-1-(4-(3-(4-(3-cyanopyridin-2-yl)piperazin-1-carbonyl)benzyl)-1-oxophthalazin-2(1H)-yl)-4,7,10-trimethyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamide (A-13-2)

[0653] N-((4S,7S,10S,13S)-1-(4-(3-(4-(3-cyanopyridin-2-yl)piperazin-1-carbonyl)benzyl)-1-oxophthalazin-2(1H)-yl)-4,7,10-trimethyl-3,6,9,12-tetraoxo-19,19-diphenyl-19-(p-tolyl)-2,5,8,11,18-pentazanonadecan-13-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetyleneamide (100 mg, 75.33 μmol) was dissolved in FA (2 mL) and reacted at 25 °C for 1 h. The reaction solution was directly purified by C18 reverse column chromatography (ACN / 0.05% TFA solution = 20-90%) and lyophilized to obtain the title compound (70 mg, 62.65 μmol, trifluoroacetate).

[0654] Its structural characterization data are as follows:

[0655] ESI-MS (m / z): 1071.4 (M+H) +

[0656] Step three: Preparation of (9H-fluoren-9-yl)methyl ((4S,7S,10S,13S,20S,24S,27S,30S)- 36-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1- (4-(3-(4-(3-cyanopyridin-2-yl)piperazine-1-carbonyl)benzyl)-1-oxophthalazine-2(1H)-yl)- 4,7,10,24,27,30-hexamethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)- 3,6,9,12,19,22,25,28,31,36-decaoxo-34-oxa-2,5,8,11,18,23,26,29,32-nonazatetratriacontan- 20-yl)carbamate (A-13-3)

[0657] N-((4S,7S,10S,13S)-17-amino-1-(4-(3-(4-(3-cyanopyridin-2-yl)piperazine-1- carbonyl)benzyl)-1-oxophthalazine-2(1H)-yl)-4,7,10-trimethyl-3,6,9,12-tetraoxo-2,5,8,11- tetraazahexadec-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (50 mg, 46.68 μmol, trifluoroacetate salt) was dissolved in DMF (1 mL) and N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4-((S)-1-(((S)-1-(((2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2- yl)-L-asparagine (50.86 mg, 46.68 μmol), DIPEA (18.10 mg, 140.03 μmol) was stirred for 1 min, HATU (17.74 mg, 46.68 μmol) was added, the reaction was stirred at room temperature for 0.5 h, the reaction solution was directly purified by C18 reverse column (ACN / 0.05% TFA solution = 20-90%), and lyophilized to give the title compound (52 mg, 16.99 μmol).

[0658] The structural characterization data thereof are as follows:

[0659] MS m / z (ESI): 1170.5 [M / 2+H] +

[0660] Step four: (S)-2-amino-N 4 -((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecan-13-yl)-N 1 Preparation of (S)-2-amino-N

[0661] (9H-fluoren-9-yl)methyl ((4S,7S,10S,13S,20S,24S,27S,30S)-36-(((1S,9S)-5-chloro-9- ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-(4-(3-(4-(3- cyanopyridin-2-yl)piperazine-1-carbonyl)benzyl)-1-oxophthalazine-2(1H)-yl)-4,7,10,24,27,30-hexamethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)- 3,6,9,12,19,22,25,28,31,36-decaoxa-34-oxa-2,5,8,11,18,23,26,29,32- nonazatetratriacontan-20-yl)carbamate (20 mg, 6.53 μmol) was dissolved in DMF (1 mL), diethylamine (0.3 mL) was added, and the reaction was stirred at 25 °C for 1 h. The reaction solution was lyophilized to give the title compound as a crude product (10 mg, 5.21 μmol).

[0662] The structural characterization data thereof are as follows:

[0663] ESI-MS (m / z): 1921.5 (M+H) +

[0664] Step five: (S)-N 4 -((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10- dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecyl)-N 1Preparation of ((4S,7S,10S,13S)-1-(4-(3-(4-(3-cyanopyridin-2-yl)piperazine-1- carbonyl)benzyl)-1-oxophthalazine-2(1H)-yl)-4,7,10-trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoyl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazahexadecan-17-yl)-2-(3-(3-(3-(methyl((2S,3R,4R,5R)- 2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6- pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propanamido)succinamide (A-13)

[0665] (S)-2-amino-N 4 -((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12- tetraoxo-3-oxa-5,8,11-triazatetradecan-13-yl)-N 1 -((4S,7S,10S,13S)-1-(4-(3-(4-(3-cyanopyridin-2-yl)piperazine-1-carbonyl)benzyl)-1-oxophthalazine- 2(1H)-yl)-4,7,10-trimethyl-13-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-3,6,9,12-tetraoxo- 2,5,8,11-tetraazahexadecan-17-yl)succinamide (10 mg, 5.21 μmol), 3-(3-(3-(methyl((2S,3R,4R,5R)- 2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6- pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propanoic acid (14.93 mg, 15.62 μmol), and DIPEA (2.02 mg, 15.62 μmol) were stirred for 1 min, HATU (3.96 mg, 10.41 μmol) was added, the reaction was stirred at room temperature for 0.5 h, and the reaction solution was directly purified by preparative high performance liquid chromatography and lyophilized to give the title compound (4.34 mg, 1.47 μmol).

[0666] The structural characterization data thereof are as follows:

[0667] MS m / z (ESI): 1429.7 [M / 2+H] +

[0668] It is prepared as follows:

[0669] Column: Waters Sunfire Prep C18 OBD (5 pm*19 mm*150 mm)

[0670] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% formic acid)

[0671] Example Thirteen: Preparation of (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)-3-((4S,7S,10S)-4,7,10-trimethyl-17-(2-(methylsulfonyl) pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazahexadecan-16-yn-1-yl)thiazolidine- 4-carboxamide (F-1)

[0672] (S)-3-(((S)-2-aminopropanamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)thiazolidine-4-carboxamide (40 mg, 52.31 pmol) and (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-alanyl-L-alanine (25.76 mg, 62.77 pmol) were dissolved in DMF (2 mL), after adding DIPEA (27.04 mg, 209.22 pmol) and HATU (39.75 mg, 104.61 pmol), the reaction was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (41 mg, 38.52 pmol).

[0673] The structural characterization data thereof are as follows:

[0674] MS m / z (ESI): 1043.3 [M+H] +

[0675] It is prepared as follows:

[0676] Column: Waters XBridge Prep C18 OBD (5 pm*19 mm*150 mm)

[0677] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% Formic acid)

[0678] Example Fourteen: Preparation of 3,3'-((2-((11S,14S)-11-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamoyl)-14-isopropyl-21-(2-(methylsulfonyl)pyrimidin-5-yl)-5,13,16-trioxo-2-oxa-6,12,15-triazahenicosan-20- yl)-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propanamide) (F-2)

[0679] Step One: Preparation of (S)-3-((7S,10S)-7-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazahexadecan-16- yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)thiazolidine-4-carboxamide (F-2-1)

[0680] (S)-3-((2-aminoacetylamino)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)thiazolidine-4-carboxamide (100 mg, 133.21 pmol) and N 6 -(diphenyl(p-tolyl)methyl)-N2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valyl)-L-lysine (110.18 mg, 146.53 μmol) was dissolved in DMF (2 mL), after the addition of DIPEA (68.86 mg, 532.82 μmol) and HATU (119.37 mg, 314.12 μmol), the reaction was stirred at 22 °C for 1 h. The reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (60 mg, 43.78 μmol).

[0681] The structural characterization data thereof are as follows:

[0682] MS m / z (ESI): 1370.5 [M+H] +

[0683] The preparation method thereof is as follows:

[0684] Column: Waters XBridge Prep C18 OBD (5 μm*19 mm*150 mm)

[0685] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0686] Step two: preparation of (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2- (methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazahexadec-16- en-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)thiazolidine- 4-carboxamide (F-2-2)

[0687] (S)-3-((7S,10S)-7-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-10- isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11- tetraazahexadecan-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl)thiazolidine-4-carboxamide (80 mg, 58.37 μmol) was dissolved in DCM (5 mL), after the addition of formic acid (1 mL), the reaction was stirred at 22 °C for 4 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (11 mg, 8.51 μmol, purity 95%).

[0688] The structural characterization data thereof are as follows:

[0689] MS m / z (ESI): 1114.4 [M+H] +

[0690] The preparation method thereof is as follows:

[0691] Column: Waters XBridge Prep C18 OBD (5 μm*19 mm*150 mm)

[0692] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

[0693] Step 3: Preparation of 3,3'-((2-((11S,14S)-11-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamoyl)-14-isopropyl-21-(2-(methylsulfonyl)pyrimidin-5-yl)-5,13,16-trioxo-2-oxa-6,12,15-triazahenicos-20-yn-1-yl)-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propanamide) (F-2)

[0694] (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazahexadec-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)thiazolidine-4-carboxamide (20 mg, 16.28 µmol) and 3-(3-(3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)-2,2-bis((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propoxy)propanoic acid (20.24 mg, 21.17 µmol) were dissolved in DMF (2 mL), after the addition of DIPEA (8.42 mg, 65.13 µmol) and HATU (12.37 mg, 32.57 µmol), the reaction was stirred at 22 °C for 1 h. The reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (8.8 mg, 4.07 µmol, purity 95%).

[0695] The structural characterization data thereof are as follows:

[0696] MS m / z (ESI): 1026.9 [M / 2+H] +

[0697] The preparation method is as follows:

[0698] Column: Waters XBridge Prep C18 OBD (5 pm*19 mm*150 mm)

[0699] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0700] Example Fifteen: Preparation of N-((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12- dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-3-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (F-3)

[0701] (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine (8.95 mg, 24.37 pmol) and (S)-2-amino-N-((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14- hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-3-yl)propanamide trifluoroacetate salt (20 mg, 24.37 pmol) were dissolved in DMF (1 mL), PyBOP (15.22 mg, 29.24 pmol) was added, followed by the addition of DIPEA (16 mg, 123.80 pmol), and stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was purified by twice high performance liquid chromatography preparation and freeze-dried to obtain the title compound (9.62 mg, 11.64 pmol).

[0702] The structural characterization data thereof are as follows:

[0703] MS m / z (ESI): 810.3 [M+H] +

[0704] The first preparation method is as follows:

[0705] Column: Waters Sunfire Prep C18 OBD (5 pm*19 mm*150 mm)

[0706] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

[0707] The second preparation method is as follows:

[0708] Column: Waters Xbridge Prep C18 OBD (5 pm*19 mm*150 mm)

[0709] Mobile phase A: acetonitrile; mobile phase B: water (0.05% ammonium bicarbonate)

[0710] II. Preparation of antibody drug conjugates

[0711] 1. Preparation of Trastuzumab A-1

[0712] Take 1.736 mL of Trastuzumab antibody (14.4 mg / mL), dilute with 87 uL of 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl) phosphine, 43 uL, pH 7.6) solution and mix, and let stand at room temperature for 1.5 h. Add A-1 solution dissolved in dimethyl sulfoxide (97 uL, 10 mM, 5.5 molar equivalents of antibody), mix, and let stand at room temperature for 2 h after mixing. After completion, replace the buffer with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., Trastuzumab A-1). Mass spectrometry determines the DAR value to be 3.5.

[0713] 2. Preparation of Trastuzumab A-2

[0714] Take 1.736 mL of Trastuzumab antibody (14.4 mg / mL), dilute with 87.6 uL of 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl) phosphine, 43.0 uL, pH 7.6) solution and mix, and let stand at room temperature for 1.5 h. Add A-2 solution dissolved in dimethyl sulfoxide (108.8 uL, 10 mM, 6 molar equivalents of antibody), mix, and let stand at room temperature for 2 h after mixing. After completion, replace the buffer with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., Trastuzumab A-2). Mass spectrometry determines the DAR value to be 3.5.

[0715] 3. Preparation of Trastuzumab A-2’ 3. Preparation of Trastuzumab A-2’

[0716] Take 0.393 mL Trastuzumab antibody (24.4 mg / mL), dilute with 45.0 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 36.4 uL, pH 7.6) solution and mix well, and stand at room temperature for 1.5 h. Add A-2 solution dissolved in dimethyl sulfoxide (83.4 uL, 10 mM, 12 molar equivalents of the antibody), mix well, and stand at room temperature for 2 h after mixing. After completion, replace the buffer with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., Trastuzumab A-2’). The DAR value is 8.0 as determined by mass spectrometry.

[0717] 4. Preparation of Trastuzumab A-3

[0718] Take 0.393 mL Trastuzumab antibody (24.4 mg / mL), dilute with 45.0 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 36.4 uL, pH 7.6) solution and mix well, and stand at room temperature for 1.5 h. Add A-2 solution dissolved in dimethyl sulfoxide (83.4 uL, 10 mM, 12 molar equivalents of the antibody), mix well, and stand at room temperature for 2 h after mixing. After completion, replace the buffer with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., Trastuzumab A-2’). The DAR value is 8.0 as determined by mass spectrometry.

[0719] 5. Preparation of Trastuzumab C-1

[0720] Take 4.276 mL Trastuzumab antibody (16.2 mg / mL), dilute with 213.8 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 262.5 uL, pH 7.6) solution and mix, and let stand at room temperature for 1.5 h. Add C-1 solution dissolved in dimethyl sulfoxide (487.6 uL, 10 mM, 10-fold molar equivalent of antibody), mix after mixing, and let stand at room temperature for 2 h. After completion, replace the buffer with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., Trastuzumab C-1). The DAR value is 7.9 as determined by mass spectrometry.

[0721] 6. Preparation of Trastuzumab D-1

[0722] Take 4.276 mL Trastuzumab antibody (16.2 mg / mL), dilute with 213.8 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 262.5 uL, pH 7.6) solution and mix, and let stand at room temperature for 1.5 h. Add C-1 solution dissolved in dimethyl sulfoxide (487.6 uL, 10 mM, 10-fold molar equivalent of antibody), mix after mixing, and let stand at room temperature for 2 h. After completion, replace the buffer with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., Trastuzumab C-1). The DAR value is 7.9 as determined by mass spectrometry.

[0723] 7. Preparation of hRS7 A-7

[0724] Take 4.276 mL Trastuzumab antibody (16.2 mg / mL), dilute with 213.8 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 262.5 uL, pH 7.6) solution and mix, and let stand at room temperature for 1.5 h. Add C-1 solution dissolved in dimethyl sulfoxide (487.6 uL, 10 mM, 10-fold molar equivalent of antibody), mix after mixing, and let stand at room temperature for 2 h. After completion, replace the buffer with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., Trastuzumab C-1). The DAR value is 7.9 as determined by mass spectrometry.

[0723] 7. Preparation of hRS7 A-7

[0724] Take 4.276 mL Trastuzumab antibody (16.2 mg / mL), dilute with 213.8 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 262.5 uL, pH 7.6) solution and mix, and let stand at room temperature for 1.5 h. Add C-1 solution dissolved in dimethyl sulfoxide (487.6 uL, 10 mM, 10-fold molar equivalent of antibody), mix after mixing, and let stand at room temperature for 2 h. After completion, replace the buffer with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., Trastuzumab C-1). The DAR value is 7.9 as determined by mass spectrometry.

[0725] 8. Preparation of hRS7 A-8

[0726] Take 1.572 mL hRS7 antibody (15.9 mg / mL), dilute with 78.6 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 50.6 uL, pH 7.6) solution and mix well, and let stand at room temperature for 1.5 h. Add A-8 solution dissolved in dimethyl sulfoxide (103.3 uL, 10 mM, 6 molar equivalents of antibody), mix well, and let stand at room temperature for 2 h after mixing. After completion, replace the buffer with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., hRS7 A-8). The DAR value is 3.4 as determined by mass spectrometry.

[0727] 9. Preparation of Trastuzumab A-7

[0728] Take 0.847 mL Trastuzumab antibody (17.7 mg / mL), dilute with 42.4 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 27.4 uL, pH 7.6) solution and mix well, and let stand at room temperature for 1.5 h. Add A-7 solution dissolved in dimethyl sulfoxide (72.4 uL, 10 mM, 7 molar equivalents of antibody), mix well, and let stand at room temperature for 2 h after mixing. After completion, replace the buffer with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., Trastuzumab A-7). The DAR value is 3.1 as determined by mass spectrometry.

[0729] 10. Preparation of Trastuzumab A-8

[0730] Take 0.847 mL Trastuzumab antibody (17.7 mg / mL), dilute with 42.4 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 27.4 uL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Add A-8 solution dissolved in dimethyl sulfoxide (72.4 uL, 10 mM, 7-fold molar equivalent of antibody), mix well, and let stand at room temperature for 2 h after mixing. After completion, replace the buffer with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., Trastuzumab A-8). The DAR value is determined by mass spectrometry to be 3.5.

[0731] 11. Preparation of hRS7 C-1

[0732] Take 2.516 mL hRS7 antibody (15.9 mg / mL), dilute with 126.0 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 148.5 uL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Add C-1 solution dissolved in dimethyl sulfoxide (275.6 uL, 10 mM, 10-fold molar equivalent of antibody), mix well, and let stand at room temperature for 2 h after mixing. After completion, replace the buffer with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., hRS7 C-1). The DAR value is determined by mass spectrometry to be 7.1.

[0733] 12. Preparation of hRS7 D-1

[0734] Take 2.516 mL hRS7 antibody (15.9 mg / mL), dilute with 126.0 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 148.5 uL, pH 7.6) solution, mix well, and let stand at room temperature for 1.5 h. Add D-1 solution dissolved in dimethyl sulfoxide (275.6 uL, 10 mM, 10-fold molar equivalent of antibody), mix well, and let stand at room temperature for 2 h after mixing. After completion, replace the buffer with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., hRS7 D-1). The DAR value is determined by mass spectrometry to be 7.8.

[0735] 13. Preparation of Trastuzumab C-1 '

[0736] Take 0.847 mL Trastuzumab antibody (17.7 mg / mL), dilute with 42.4 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris (2-carboxyethyl) phosphine, 27.4 uL, pH 7.6) solution and mix well, and stand at room temperature for 1.5 h. Add C-1 solution dissolved in dimethyl sulfoxide (51.7 uL, 10 mM, 5 molar equivalents of the antibody), mix well, and stand at room temperature for 2 h after mixing. After completion, replace the buffer with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (Trastuzumab C-1 '). The DAR value is 3.05 as determined by mass spectrometry.

[0737] 14. Preparation of Trastuzumab A-11

[0738] Take 0.789 mL Trastuzumab antibody (19.0 mg / mL), dilute with 39.5 uL 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris (2-carboxyethyl) phosphine, 31.0 uL, pH 7.6) solution and mix well, and stand at room temperature for 1.5 h. Add A-11 solution dissolved in dimethyl sulfoxide (63.3 uL, 10 mM, 6 molar equivalents of the antibody), mix well, and stand at room temperature for 2 h after mixing. After completion, replace the buffer with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (Trastuzumab A-11). The DAR value is 3.97 as determined by mass spectrometry.

[0739] 15. Preparation of Trastuzumab F-3

[0740] Take 1.699 mL of Trastuzumab antibody (20.6 mg / mL), dilute with 85.0 uL of 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4solution, add 10 mM TCEP (tris (2-carboxyethyl) phosphine, 72.3 uL, pH 7.6) solution and mix well, and stand at room temperature for 1.5 h. Add F-3 solution dissolved in dimethyl sulfoxide (147.6 uL, 10 mM, 6 molar equivalents of the antibody), mix well, and stand at room temperature for 2 h after mixing. After completion, replace the buffer with a 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e., Trastuzumab F-3). The DAR value is determined by mass spectrometry to be 4.3.

[0741] III. Biological Examples

[0742] 1. Pharmacodynamic detection of antibody drug conjugates in JIMT-1 model

[0743] The ADCs of the present application are diluted with 0.9% NaCl injection solution to prepare the administration solution. 0.9% NaCl injection solution is used as the vehicle control (Vehicle).

[0744] JIMT-1 cells (from Nanjing Kebai) are cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2. Exponentially growing JIMT-1 cells are collected, resuspended to an appropriate concentration with PBS containing 50% Matrigel, and inoculated subcutaneously into female NOD SCID mice to establish a breast cancer model. When the average tumor volume is about 150 mm 3 left and right, according to the tumor size, randomly divided into groups in turn: vehicle control group (i.e., negative control, Vehicle group), Trastuzumab A-2 6 mg / kg group of the present application, Trastuzumab A-1 6 mg / kg group, Trastuzumab C-1 3 mg / kg and Trastuzumab D-1 3 mg / kg combination group.

[0745] Each group is injected intravenously (i.v.), and Trastuzumab A-2, Trastuzumab A-1, Trastuzumab C-1 and Trastuzumab D-1 of the present application are administered on Day 0, a total of 1 time.

[0746] The tumor diameter is measured once a week after administration with a vernier caliper, and the tumor volume is calculated according to the following formula: V = 0.5a x b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. The animal death is observed and recorded daily.

[0747] The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

[0748] V T末 >V T0 TGI(%) = [1 - (V) T末 -V T0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 TGI(%) = [1 - (V) T末 -VT0) / VT0]*100%.

[0749] Where V T末 Mean tumor volume at the end of the experiment in the treatment group;

[0750] V T0 Mean tumor volume at the start of treatment in the treatment group;

[0751] V C末 Mean tumor volume at the end of the experiment in the negative control group;

[0752] V C0 Mean tumor volume at the start of drug administration in the negative control group;

[0753] The tumor relative proliferation rate (T / C%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

[0754] T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 )*100%.

[0755] Table 1. JIMT-1CDX model of human breast cancer cells.

[0756] Table 1 and Figures 1 and 2 above show that the ADC of the present invention significantly inhibited tumor growth in the JIMT-1 breast cancer xenograft model. Compared with the Vehicle group, the tumor growth inhibition rate (TGI) of the Trastuzumab A-2, Trastuzumab A-1, Trastuzumab C-1 and Trastuzumab D-1 combination groups were 96.78%, 114.12% and 91.24%, respectively. During the administration period, there were no animal deaths or significant weight loss in any of the treatment groups, and no obvious drug toxicity was observed. Mice tolerated the ADC of the present invention well during the treatment period.

[0757] Therefore, it can be seen that the ADC of the present invention has a significant inhibitory effect on the growth of tumor cells.

[0758] 2. Evaluate the inhibitory effect of antibody-drug conjugates on tumor growth in a mouse subcutaneous xenograft model.

[0759] The formulation containing the ADC of the present invention was administered to a CDX mouse model of subcutaneous transplantation of human breast cancer cells MX-1 via tail vein injection. Tumor volume and animal weight changes were measured once a week to calculate the tumor-suppressing efficacy of the ADC of the present invention in tumor-bearing mice.

[0760] test drug

[0761] Take an appropriate amount of ADC and administer it at 3 mg / kg or 1.5 mg / kg, as detailed below. Dilute the stock solution to the administration solution using 0.9% NaCl injection. Use 0.9% NaCl injection as a solvent control (Vehicle).

[0762] Laboratory animals and cell lines

[0763] Balb / c Nude mice (Sichuan Vital River Laboratory Animal Technology Co., Ltd.)

[0764] Human breast cancer cell line MX-1 (Nanjing Kebai)

[0765] Experimental grouping and evaluation methods

[0766] Select tumors with an average volume of approximately 150 mm. 3 Tumor-bearing mice were randomly assigned to groups (the number of groups was determined based on the sample size). Each group was administered either 0.9% NaCl injection (hereinafter referred to as solvent control, Vehicle) or ADC, with the dosing frequency as detailed in the specific examples. The administration method was tail vein injection, with a volume of 10 ml / kg. Tumor diameter was measured weekly using calipers, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

[0767] The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

[0768] V T末 >V T0 TGI(%) = [1 - (V) T末 -V T0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 TGI(%) = [1 - (V) T末V0 / V0)*100%.

[0769] wherein V T末 : mean tumor volume at the end of the experiment for the treatment group;

[0770] V T0 : mean tumor volume at the start of dosing for the treatment group;

[0771] V C末 : mean tumor volume at the end of the experiment for the negative control group;

[0772] V C0 : mean tumor volume at the start of dosing for the negative control group;

[0773] The relative tumor proliferation rate T / C (%) was calculated using the following formula to evaluate the anti-tumor efficacy of the ADCs:

[0774] T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 )*100%.

[0775] (1) Anti-human Her2 ADC efficacy in MX-1 model

[0776] MX-1 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2. Exponentially growing MX-1 cells were collected and resuspended in PBS containing 50% Matrigel to an appropriate concentration, and inoculated subcutaneously into female NOD SCID mice to establish a breast cancer model. When the average tumor volume was about 150 mm 3 around, the mice were randomly divided into groups according to the tumor size, and the groups were as follows: vehicle control (i.e. negative control, Vehicle group), hRS7 A-7 (3 mg / kg), hRS7 A-8 (3 mg / kg), hRS7 C-1 (1.5 mg / kg), hRS7 D-1 (1.5 mg / kg), hRS7 C-1 + hRS7 D-1 (1.5+1.5 mg / kg). The mice were dosed on Day 0 and Day 7, for a total of 2 doses.

[0777] The ADC of the present application has a significant tumor growth inhibition effect on MX-1 breast cancer transplanted tumor model. Compared with the Vehicle group, the tumor growth inhibition rates (TGI) of hRS7 A-7 (3 mg / kg), hRS7 A-8 (3 mg / kg), hRS7 C-1 (1.5 mg / kg), hRS7 D-1 (1.5 mg / kg), hRS7 C-1 + hRS7 D-1 (1.5 + 1.5 mg / kg) are 181.21%, 168.88%, 103.65%, -13.17% and 115.84%, respectively. On Day 34, no animal died and no significant animal weight loss was observed in each treatment group, and no obvious drug toxicity reaction was observed, and the mice tolerated the ADC of the present application well during the treatment period. The specific results are shown in Table 2.

[0778] Table 2 Human breast cancer cell MX-1 CDX model

[0779] 3. Evaluation of the tumor growth inhibition effect of the antibody drug conjugate on the mouse subcutaneous transplanted tumor model

[0780] The formulation containing the ADC of the present application was administered to the mouse CDX model of subcutaneously transplanted human breast cancer cell MX-1 by tail vein injection, respectively, the tumor volume and animal weight change were measured once a week, and the tumor inhibition effect of the ADC of the present application on tumor-bearing mice was calculated.

[0781] Test drug

[0782] An appropriate amount of ADC was administered at 3 mg / kg, and the specific administration dose is shown below. The mother solution was diluted to the administration solution using 0.9% NaCl injection solution. 0.9% NaCl injection solution was used as the vehicle control (Vehicle).

[0783] Experimental animals and cell lines

[0784] Balb / c Nude mice (Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd.)

[0785] Human breast cancer cell MX-1 (Nanjing Kebai)

[0786] Experimental grouping and evaluation method

[0787] The tumor-bearing mice with an average tumor volume of about 150 mm 3 were randomly grouped (the number of groups was determined according to the number of samples). According to the groups, 0.9% NaCl injection solution (hereinafter referred to as vehicle control, Vehicle) and ADC were administered, the administration frequency is shown in the specific examples, the administration mode was tail vein injection, and the administration volume was 10 ml / kg. The tumor diameter was measured once a week after administration using a vernier caliper, and the tumor volume was calculated according to the following formula: V = 0.5a x b2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

[0788] The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

[0789] V T末 >V T0 TGI(%) = [1 - (V) T末 -V T0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 TGI(%) = [1 - (V) T末 -VT0) / VT0]*100%.

[0790] Where V T末 Mean tumor volume at the end of the experiment in the treatment group;

[0791] V T0 Mean tumor volume at the start of treatment in the treatment group;

[0792] V C末 Mean tumor volume at the end of the experiment in the negative control group;

[0793] V C0 Mean tumor volume at the start of drug administration in the negative control group;

[0794] The tumor relative proliferation rate (T / C%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

[0795] T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 )*100%.

[0796] (1) Pharmacodynamic assay of anti-human Her2 antibody-drug conjugate in MX-1 model

[0797] MX-1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. MX-1 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and subcutaneously inoculated into female Balb / c Nude mice to establish a breast cancer model. The tumors were cultured until the average volume reached approximately 150 mm². 3The mice were randomly grouped according to the tumor size, and sequentially were: vehicle control group (i.e. negative control, Vehicle group), Trastuzumab A-8 3 mg / kg, Trastuzumab C-1' 3 mg / kg, administered at Day 0 and Day 7, for a total of 2 administrations.

[0798] The ADC of the present application significantly inhibited the tumor growth in the MX-1 breast cancer xenograft model. Compared with the Vehicle group, the tumor growth inhibition rates (TGI) of the Trastuzumab A-8 3 mg / kg and Trastuzumab C-1' 3 mg / kg groups were 74.26% and 53.88%, respectively. No animal death and no significant animal weight loss were observed in each treatment group at Day 44, and no obvious drug toxicity was observed. The mice tolerated the ADC of the present application well during the treatment period. The specific results are shown in Table 3.

[0799] Table 3 Human breast cancer cell MX-1 CDX model

[0800] 4. Evaluation of the tumor growth inhibition effect of the antibody drug conjugate on a mouse subcutaneous xenograft model

[0801] The formulation containing the ADC of the present application was administered to the mouse CDX model of subcutaneously transplanted human breast cancer cell MX-1 by tail vein injection, respectively. The tumor volume and animal weight change were measured once a week, and the tumor inhibition effect of the ADC of the present application on tumor-bearing mice was calculated.

[0802] Test drug

[0803] An appropriate amount of ADC was administered at 2 mg / kg, and the specific administration dose is shown below. The 0.9% NaCl injection was used to dilute the mother liquor to the administration solution. The 0.9% NaCl injection was used as the vehicle control (Vehicle).

[0804] Experimental animals and cell lines

[0805] Balb / c Nude mice (Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd.)

[0806] Human breast cancer cell MX-1 (Nanjing Kebai)

[0807] Experimental grouping and evaluation method

[0808] The tumor average volume was about 150 mm 3Tumor-bearing mice were randomly assigned to groups (the number of groups was determined based on the sample size). Each group was administered either 0.9% NaCl injection (hereinafter referred to as solvent control, Vehicle) or ADC, with the dosing frequency as detailed in the specific examples. The administration method was tail vein injection, with a volume of 10 ml / kg. Tumor diameter was measured weekly using calipers, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

[0809] The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

[0810] V T末 >V T0 TGI(%) = [1 - (V) T末 -V T0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 TGI(%) = [1 - (V) T末 -VT0) / VT0]*100%.

[0811] Where V T末 Mean tumor volume at the end of the experiment in the treatment group;

[0812] V T0 Mean tumor volume at the start of treatment in the treatment group;

[0813] V C末 Mean tumor volume at the end of the experiment in the negative control group;

[0814] V C0 Mean tumor volume at the start of drug administration in the negative control group;

[0815] The tumor relative proliferation rate (T / C%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

[0816] T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 )*100%.

[0817] (1) Pharmacodynamic assay of anti-human Her2 antibody-drug conjugate in MX-1 model

[0818] MX-1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in 5% CO2. Exponential growth phase MX-1 cells were collected, resuspended with PBS to an appropriate concentration, and inoculated subcutaneously in female Balb / c Nude mice to establish a breast cancer model. When the average tumor volume was about 150mm3 3 When the average tumor volume was about 150mm3, the mice were randomly divided into groups according to tumor size, in the order of: vehicle control (i.e., negative control, Vehicle group), Trastuzumab F-3 2mg / kg, Trastuzumab A-11 2mg / kg, dosed at Day 0, for a total of 1 dose.

[0819] The ADC of the present application significantly inhibited tumor growth in the MX-1 breast cancer xenograft model. Compared with the Vehicle group, the tumor growth inhibition rates (TGI) of the Trastuzumab F-3 2mg / kg and Trastuzumab A-11 2mg / kg groups were 98.46% and 133.91%, respectively. No animal deaths or significant animal weight loss were observed in the treatment groups on Day 29, and no obvious drug toxicity was observed. The mice tolerated the ADC of the present application well during the treatment period. The specific results are shown in Table 4.

[0820] Table 4 Human breast cancer cell MX-1 CDX model

[0821] Finally, it should be noted that the above examples are merely intended to illustrate the technical solutions of the present application and not to limit the same; although the present application has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the specific embodiments of the present application can be modified or some technical features can be replaced by equivalents; without departing from the spirit of the technical solutions of the present application, they should all be included in the technical solution range of the present application claimed.

Claims

1. A compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, said compound having the structure shown in formula (I) or formula (II): wherein: M a is the structure before linkage to the antibody or antigen binding fragment thereof; B is the linker M a and the moiety between L and L'; L and L' are moieties linking B and E, E' between M a and E; E and E' are moieties connecting L and L' to D and D', respectively; D and D' are each independently a pharmaceutically active molecule moiety; Preferably, the pharmaceutically active molecule is selected from a cytotoxic drug or a PARP inhibitor.

2. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, M a selected from the following structures: Where R represents hydrogen, C 1-6 Alkyl groups or containing -(CH2CH2O) p - alkyl groups, wherein p, each time it appears, is independently selected from an integer from 1 to 12; preferably, M a Selected from the following structures: Preferably, M a selected from the following structures:

3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, B is selected from the following structures: wherein q is, at each occurrence, independently selected from an integer from 1 to 24; Preferably, B is selected from the following structures or combinations thereof: wherein q is, at each occurrence, independently selected from an integer from 1 to 24.

4. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, L and L' are each independently selected from one or more substituted or unsubstituted structural fragments consisting of C 1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 5-12 membered heterocyclyl, -N(R')-, carbonyl, -0-, a natural amino acid or a non-natural amino acid and analogs thereof (such as Ala, Arg, Asn, Asp, Cit, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2) r OCH3)), Lys(R'), a short peptide consisting of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Ala-Ala-Glu), EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, NOPO, wherein said Ra is EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA or NOPO; R' represents hydrogen, C 1-6 alkyl or -(CH2CH20) r - containing alkyl; r is, at each occurrence, independently selected from an integer between 1 and 12; Preferably, L and L' are each independently selected from the following structures:

5. The compound of any one of claims 1-4, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, E and E' are each independently selected from a single bond, -NH-CH2-, or from the following structures: Preferably, E and E' are each independently selected from a single bond, -NH-CH2-, 6. The compound of any one of claims 1-5, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, D and D' are each independently selected from a DNA topoisomerase inhibitor, a DNA intercalator, an RNA polymerase inhibitor, and a PARP inhibitor; Preferably, the DNA intercalator is a pyrrolobenzodiazepine (PBD); Preferably, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., a camptothecin, such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); Preferably, the RNA polymerase inhibitor is triptolide, a-amanitin, and pharmaceutically acceptable salts, esters, and analogs thereof; Preferably, the PARP inhibitor is olaparib, niraparib, pamiparib, talazoparib, rucaparib, veliparib, talazoparib, or AZ9482; Preferably, D and D' are connected to E and E', respectively, through -OH, primary amino, secondary amine, or -SH on D and D', respectively.

7. The compound of any one of claims 1-6, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein the compound is selected from the following A-1 to A-13, B-1 to B-8, C-1 to C-4, D-1 to D-3, and F-1 to F-3: A-1： A-2： A-3： A-4： A-5： A-6： A-7： A-8： A-9： A-10： A-11： A-12： A-13: B-1: B-2: B-3: B-4: B-5: B-6: B-7: B-8: C-1: C-2: C-3: C-4: D-1: D-2: D-3: F-1: F-2: F-3:

8. The compound of any one of claims 1-7, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein the compound is conjugated to an antibody or antigen-binding fragment thereof by a substitution reaction (e.g., removal of -SO2Me or pentafluorophenol, etc.) or by an addition reaction.

9. An antibody drug conjugate, the conjugate having the structure: wherein B, L, E, D, L', E', D' are as described in any one of claims 1-8, M and M' are each independently said M a the structure after linkage to the antibody or antigen-binding fragment thereof; Ab is an antibody or antigen-binding fragment thereof; x and y are each independently selected from 1 to 10; Preferably, the antibody or antigen-binding fragment thereof specifically binds to the ErbB family receptor tyrosine kinase member human epidermal growth factor receptor 2 (Her2).

10. The antibody drug conjugate of claim 9, wherein, The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (1a) a heavy chain variable region (VH) comprising 3 CDRs as follows: CDR-H1 of SEQ ID NO:5 or a variant thereof, CDR-H2 of SEQ ID NO:6 or a variant thereof, CDR-H3 of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs as follows: CDR-L1 of SEQ ID NO:8 or a variant thereof, CDR-L2 of SEQ ID NO:9 or a variant thereof, CDR-L3 of SEQ ID NO: 10 or a variant thereof; or, (1b) a heavy chain variable region (VH) comprising 3 CDRs as follows: CDR-H1 of SEQ ID NO:20 or a variant thereof, CDR-H2 of SEQ ID NO:21 or a variant thereof, CDR-H3 of SEQ ID NO:22 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs as follows: CDR-L1 of SEQ ID NO:23 or a variant thereof, CDR-L2 of SEQ ID NO:24 or a variant thereof, CDR-L3 of SEQ ID NO:25 or a variant thereof; wherein the variant of any one of (1a), (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or several (e.g., 1, 2, or 3) amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution; or, (2) a heavy chain variable region (VH) and / or a light chain variable region (VL) wherein the CDRs are defined by the AbM numbering system: (2a) a heavy chain variable region (VH) comprising 3 CDRs as follows: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, CDR-H3 of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs as follows: CDR-L1 of SEQ ID NO:8 or a variant thereof, CDR-L2 of SEQ ID NO:9 or a variant thereof, CDR-L3 of SEQ ID NO: 10 or a variant thereof; or, (2b) a heavy chain variable region (VH) comprising 3 CDRs as follows: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, CDR-H3 of SEQ ID NO:22 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs as follows: CDR-L1 of SEQ ID NO:23 or a variant thereof, CDR-L2 of SEQ ID NO:24 or a variant thereof, CDR-L3 of SEQ ID NO:25 or a variant thereof. (2b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, CDR-L3 of SEQ ID NO: 25 or a variant thereof; wherein the variant of any one of (2a), (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived or has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; preferably, said substitution is a conservative substitution; or, (3) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the Kabat numbering system: (3a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, CDR-L3 of SEQ ID NO: 10 or a variant thereof; or, (3b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, CDR-L3 of SEQ ID NO: 25 or a variant thereof; wherein the variant of any one of (3a), (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived or has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; or, (4) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (4a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or, (4b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof; wherein the variant of any one of (4a), (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived or has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

11. The antibody drug conjugate of claim 9 or 10, wherein, the antibody or antigen-binding fragment thereof comprises: (a) a VH of SEQ ID NO: 1 or a variant thereof, and / or, a VL of SEQ ID NO: 2 or a variant thereof; or (b) a VH of SEQ ID NO: 3 or a variant thereof, and / or, a VL of SEQ ID NO: 4 or a variant thereof; wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably the substitutions are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof comprises: (a) a VH as depicted in SEQ ID NO: 1, and, a VL as depicted in SEQ ID NO: 2; or (b) a VH as depicted in SEQ ID NO: 3, and, a VL as depicted in SEQ ID NO: 4; Preferably, the antibody or antigen-binding fragment thereof further comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or several amino acid substitutions, deletions or additions compared to the wild type sequence from which it is derived (e.g. up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); and (b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or several amino acid substitutions, deletions or additions compared to the wild type sequence from which it is derived (e.g. up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); Preferably, the heavy chain constant region is an IgG heavy chain constant region, e.g. an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, e.g. a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as depicted in SEQ ID NO: 35 or a variant thereof having up to 20 conservative substitutions compared to SEQ ID NO: 35 (e.g. up to 15, up to 10, or up to 5 conservative substitutions; e.g. 1, 2, 3, 4 or 5 conservative substitutions); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as depicted in SEQ ID NO: 41 or a variant thereof having up to 20 conservative substitutions compared to SEQ ID NO: 41 (e.g. up to 15, up to 10, or up to 5 conservative substitutions; e.g. 1, 2, 3, 4 or 5 conservative substitutions); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35 and a light chain constant region (CL) as set forth in SEQ ID NO: 36; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35 and a light chain constant region (CL) as set forth in SEQ ID NO: 36; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 41 and a light chain constant region (CL) as set forth in SEQ ID NO: 36; Preferably, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, and a light chain comprising a VL as set forth in SEQ ID NO: 2 and a light chain constant region (CL) as set forth in SEQ ID NO: 36; (2) a heavy chain comprising a VH as set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, and a light chain comprising a VL as set forth in SEQ ID NO: 4 and a light chain constant region (CL) as set forth in SEQ ID NO: 36; or (3) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 41, and a light chain comprising a VL as set forth in SEQ ID NO: 2 and a light chain constant region (CL) as set forth in SEQ ID NO: 36; or (4) a heavy chain comprising a VH as set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 41, and a light chain comprising a VL as set forth in SEQ ID NO: 4 and a light chain constant region (CL) as set forth in SEQ ID NO: 36; Preferably, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a sequence as set forth in SEQ ID NO: 37, and a light chain comprising a sequence as set forth in SEQ ID NO: 38; (2) a heavy chain comprising a sequence as set forth in SEQ ID NO: 39, and a light chain comprising a sequence as set forth in SEQ ID NO: 40; (3) a heavy chain comprising a sequence as set forth in SEQ ID NO: 42, and a light chain comprising a sequence as set forth in SEQ ID NO: 38; or (4) a heavy chain comprising a sequence as set forth in SEQ ID NO: 43, and a light chain comprising a sequence as set forth in SEQ ID NO:

40.

12. The antibody drug conjugate of any one of claims 9-11, M a to a thiol (-SH) or an amino (-NH2) group on Ab.

13. The antibody drug conjugate of any one of claims 9-12, wherein, the antibody or antigen-binding fragment thereof is selected from the group consisting of the antibodies or antigen-binding fragments thereof of claim 11 ; or M a -L-E-D is selected from the group consisting of the compounds of claim 7; x and y are each independently 1 to 10; preferably, x and y are each independently 1 to 8, or x and y are each independently 1 to 4.

14. The antibody drug conjugate of any one of claims 9-13, which is selected from the group consisting of: ADC A-1 to ADC A-13 and ADC E-1 to ADC E-13: ADC A-1: ADC A-2: ADC A-3: ADCA-4: ADCA-5: ADCA-6: ADCA-7: ADCA-8: ADC A-9: ADCA-10: ADCA-11: ADCA-12: ADCA-13: ADC E-1: ADC E-2: ADC E-3: ADC E-4: ADC E-5: ADC E-6: ADC E-7: ADC E-8: ADC E-9: ADC E-10: ADC E-11: ADC E-12: ADC E-13: wherein HA in each antibody drug conjugate represents an antibody or an antigen binding fragment thereof comprising a VH as set forth in SEQ ID NO: 1 and a VL as set forth in SEQ ID NO: 2, for example trastuzumab, patitumumab, etc; wherein each of x, y is independently 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein represents the specific linkage mode of the thiol or amine group in the antibody or antigen binding fragment thereof to the linker.

15. A composition of antibody drug conjugates, said composition comprising one or more antibody drug conjugates according to any one of claims 9-14; preferably, the DAR value (drug antibody ratio) of said composition is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-6, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5-6.0, 4.0-4.5, 4.0-5.0, 4.0-5.5, 4.0-6.0, 4.5-5.0, 4.5-5.5, 4.5-6.0, 5.0-5.5, 5.0-6.0, 5.5-6.0, 6.0-8.0, 6.5-8.0, 7.0-8.0, 7.5-8.0 or 7.5-8.

5.

16. A pharmaceutical composition comprising an antibody drug conjugate according to any one of claims 9-14 or a pharmaceutically acceptable salt or stereoisomer thereof, or a composition according to claim 15, and one or more pharmaceutical excipients.

17. Use of a compound according to any one of claims 1-8 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, or an antibody drug conjugate according to any one of claims 9-14, a composition according to claim 15, or a pharmaceutical composition according to claim 16, for the manufacture of a medicament for the treatment of cancer; preferably, the cancer is a HER2 expressing cancer.

18. The use according to claim 17, wherein the cancer disease is selected from solid tumors and hematological malignancies; for example from breast cancer, gastric cancer, lung cancer (for example, non-small cell lung cancer, in particular lung adenocarcinoma), colon cancer and lymphoma.

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