Drug linker, and antibody-drug conjugate thereof, preparation method therefor and use thereof

Abstract

A drug linker, and an antibody-drug conjugate thereof, a preparation method therefor and the use thereof. The drug linker and the antibody-drug conjugate have structures as represented by formula (I) and formula (II), respectively. The prepared antibody-drug conjugate has a targeted killing effect on breast cancer, gastric cancer, lung cancer (for example, non-small cell lung cancer, and specifically, lung adenocarcinoma), colon cancer, or lymphoma.

Description

Drug linkers, antibody-drug conjugates, preparation methods and uses

[0001] This application is based on and claims priority to Chinese patent applications No. 202411732932.5, filed on November 29, 2024; No. 202510783419.7, filed on June 12, 2025; and No. 202511203174.2, filed on August 26, 2025, the disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0002] This application belongs to the pharmaceutical field and relates to drug linkers, antibody-drug conjugates, their preparation methods, and uses. Background Technology

[0003] Significant progress has been made in the development of antitumor drugs and targeted tumor therapy using biopharmaceuticals (such as therapeutic antibodies or antibody fragments) and targeted small molecule ligands. However, while biopharmaceuticals are highly targeted, their therapeutic effects on solid tumors are limited; and while bioactive molecules have high killing power against cancer cells, they often lack targeting and frequently damage normal cells, leading to serious toxic side effects.

[0004] Recent studies have found that therapeutic antibodies can be linked to bioactive molecules to form antibody-drug conjugates (ADCs). ADCs combine the targeting properties of antibodies with the activity of bioactive molecules. Antibodies guide ADCs to bind to target cells, where they are subsequently internalized, releasing the drug to kill cells and treat the disease. Because antibodies are specific and targeted to tumor cell-related targets, their application value lies not only in treatment but also in serving as ideal carriers for targeted drug delivery, reducing drug side effects.

[0005] With the widespread use of ADC drugs in clinical treatment, drug resistance has become a potential problem. The mechanisms of ADC drug resistance may be related to reduced antigen expression, decreased endocytosis, toxin efflux, and target mutations. To address ADC drug resistance and improve their efficacy, combination therapy and multiple loading methods have emerged as new directions for development.

[0006] To address drug resistance in ADC drugs, attaching drugs with different mechanisms of action and synergistic effects to antibodies via appropriate linking methods is an innovative approach that is expected to improve the effectiveness and safety of cancer treatment. Summary of the Invention

[0007] This application provides novel dual (multi)-loaded drug linkers and their antibody-drug conjugates. The dual (multi)-loaded drugs are mounted on the antibody via irreversible covalent binding and in the same or different proportions. The antibody conjugates exhibit a significant synergistic effect.

[0008] Drug linker

[0009] On the one hand, this application provides a drug linker, wherein the drug linker is a compound with the structure shown in Formula I or a drug-acceptable salt thereof:

[0010] in:

[0011] …represents L a Existence or non-existence;

[0012] Q is a structure used to link with antibody or antigen-binding fragments;

[0013] B connects to Q, L, or L. a Part of;

[0014] L and L a It is a connector sub-part;

[0015] E is the self-eliminating part;

[0016] D represents the bioactive molecule portion;

[0017] n is selected from 2-10;

[0018] Each L may be the same or different; each E may be the same or different; each D may be the same or different.

[0019] In some implementations, n is selected from 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0020] In some embodiments, the compound of formula I has the structure shown in formula (IA), formula (IB), formula (IC), or formula (ID):

[0021] in:

[0022] Q is a structure used to link with antibody or antigen-binding fragments;

[0023] L 1 L 2 L 3 L 4 and L a It is a connector sub-part;

[0024] E 1 E 2 E 3 and E4 It is the self-eliminating part;

[0025] D 1 D 2 D 3 and D 4 It is the bioactive molecular part;

[0026] n is selected from 2-10;

[0027] L 1 L 2 L 3 and L 4 They may be the same or different; E 1 E 2 E 3 and E 4 They are the same or different; D 1 D 2 D 3 and D 4 They may be the same or different.

[0028] In some implementations, Q is selected individually from the following structures each time it appears:

[0029] Each time p appears, it is independently selected from an integer between 1 and 12.

[0030] In some implementations, Q is selected from the following structures:

[0031] Each time p appears, it is independently selected from an integer between 1 and 12.

[0032] In some implementations, Q is selected from the following structures:

[0033] In some implementations, Q is selected from...

[0034] In some implementations, B is selected from the following structures or any combination thereof:

[0035] and -NR 0 -C 1-10 Alkyl; the alkyl group may optionally be substituted with one or more OH groups; R 0 Selected from H or C 1-6Alkyl group; q is independently selected from an integer from 1 to 24 each time it appears (e.g., an integer from 1 to 10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0036] In some implementations, B is selected from the following structures or any combination thereof:

[0037] Each time q appears, it is independently selected from an integer from 1 to 24 (e.g., an integer from 1 to 10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0038] In some implementations, B is selected from the following structures:

[0039] In some implementations, B is selected from the following structures:

[0040] In some implementation schemes, L, L 1 L 2 L 3 and L 4 A bivalent structure composed of one or more substituted or unsubstituted structural segments, each independently selected from the following: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, 5-12 heterocyclic, -N(R')-, carbonyl, -O-, glycosyl, tromethamine, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)) r OCH3)) and Lys(R') rAnd short peptides composed 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,

[0041] Wherein Ra is EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, or NOPO; R′ represents hydrogen, C 1-6 Alkyl groups, polyhydroxy fragments, glycosyl groups, polyethylene glycol-containing fragments, -(CH2CH2O) r -C 1-6 Alkyl group, -C(=O)-(CH2CH2O) r -C 1-6 Alkyl, polysarcosine, -(C(=O)-CH2N(Me)) r -C 1-6 Alkyl groups, carboxylic acid-containing fragments, tetracarboxylic acid residues and their derivatives, EDTA and its derivatives, or DOTA and its derivatives; r is independently selected from integers 1-12 each time it appears.

[0042] In some implementation schemes, L, L 1 L 2 L 3 and L 4Each is independently selected from the following structures:

[0043] In some implementation schemes, L, L 1 L 2 L 3 and L 4 Each is independently selected from the following structures:

[0044] In some implementation schemes, L, L 1 L 2 L 3 and L 4 Each is independently selected from the following structures:

[0045] In some implementations, L a Select from the following structures or any combination thereof:

[0046] Each time r appears, it is independently selected from an integer between 0 and 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0047] In some implementations, L a Select from the following structures or any combination thereof:

[0048] Each time r appears, it is independently selected from an integer from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0049] In some implementations, L a Selected from the following structures:

[0050] Each time r appears, it is independently selected from an integer from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0051] In some implementations, L a Selected from the following structures:

[0052] Each time r appears, it is independently selected from an integer from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0053] In some implementation schemes, E, E 1 E 2 E 3 and E 4 Each is independently selected from a single bond, -NH-CH2-.

[0054] In some implementation schemes, E, E 1 E 2 E 3 and E 4 Each is independently selected from a single bond, -NH-CH2- or

[0055] In some implementation schemes, E, E 1 E 2 E 3 and E 4 Each is independently selected from a single key.

[0056] In some implementation schemes, E, E 1 E 2 E 3 and E 4 Each is independently selected from -NH-CH2-.

[0057] In some implementation schemes, E, E 1 E 2 E 3 and E 4 Each independently selected

[0058] The bioactive molecules disclosed in this application typically contain a variety of functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), secondary amino (-NR1H), and tertiary amino (-NR2R3), where R1, R2, and R3 represent only non-hydrogen substituents on N or mercapto (-SH). These functional groups can be linked to the remaining parts through chemical reactions.

[0059] In some embodiments, the bioactive molecules are each independently associated with the E, E' group via -OH, primary amino group, secondary amino group, tertiary amino group, or -SH group thereon. 1 E 2 E 3 or E 4 connect.

[0060] In some embodiments, the bioactive molecules are each independently selected from antitumor drugs or compounds with antitumor effects.

[0061] In some embodiments, the bioactive molecules are each independently selected from cytotoxic compounds, antimetabolites, cell cycle regulators, apoptosis regulators, synthetic lethal agents, kinase inhibitors, or protein degraders.

[0062] In some embodiments, the cytotoxic compound is a DNA damaging agent, a topoisomerase inhibitor, an RNA polymerase inhibitor, or a microtubule inhibitor.

[0063] In some embodiments, the antimetabolite is a nucleoside compound or a folic acid analogue.

[0064] In some embodiments, the cell cycle regulator is a CDK inhibitor.

[0065] In some embodiments, the apoptosis regulator is a Bcl-2 inhibitor or a Bcl-xL inhibitor.

[0066] In some embodiments, the synthetic lethal agent is a poly(ADP-ribose) polymerase (PARP) inhibitor, PRMT5 inhibitor, ATR inhibitor, ATM inhibitor, WEE1 inhibitor, WRN inhibitor, PARG inhibitor, Polθ inhibitor, or KIF18A inhibitor.

[0067] In some embodiments, the kinase inhibitor is an EGFR inhibitor or a VEGFR inhibitor.

[0068] In some embodiments, the protein degrading agent is PROTAC or a molecular gel compound.

[0069] In some embodiments, the DNA damaging agent is selected from pyrrolobenzodiazepines, carzimidoxones, pyroximide, or anthracyclines.

[0070] In some embodiments, the topoisomerase inhibitor is selected from camptothecin compounds, anthracycline compounds, and etoposide compounds.

[0071] In some embodiments, the RNA polymerase inhibitor is selected from sucrose compounds and α-amaminoid compounds.

[0072] In some embodiments, the microtubule inhibitor is selected from olistatin compounds, maytansine compounds, leucospirin compounds, taxane compounds, vincristine compounds, and hammetrine compounds.

[0073] In some embodiments, the nucleoside compound is selected from pyrimidine nucleoside compounds or purine nucleoside compounds.

[0074] In some embodiments, the bioactive molecule is selected from the following compounds:

[0075] In some embodiments, the bioactive molecule is selected from the following compounds:

[0076] In some implementations, the D, D 1 D 2 D 3 and D 4 Each is independently selected from the following structures:

[0077] In some embodiments, the "drug linker" is selected from the following structures:

[0078] In some embodiments, the drug linker described above may optionally be replaced by one or more suitable substituents.

[0079] Antibody-drug conjugates

[0080] In another aspect, this application provides an antibody-drug conjugate having the structure shown in Formula II:

[0081] Where B, L a L, E, D, and n are as described in any of the above.

[0082] Ab represents an antibody or its antigen-binding fragment;

[0083] M is the structural form of Q covalently linked to an antibody or its antigen-binding fragment as described in any of the above items;

[0084] x are each independently selected from 1 to 10.

[0085] In some embodiments, the antibody-drug conjugate has the structure shown in formula (IIA), formula (IIB), formula (IIC), or formula (IID):

[0086] Among them, B and L a D 1 To D 4 L 1 To L 4 E 1 To E 4 As stated in any of the above,

[0087] Ab represents an antibody or its antigen-binding fragment;

[0088] M is the structural form of Q covalently linked to an antibody or its antigen-binding fragment as described in any of the above items;

[0089] x are each independently selected from 1 to 10.

[0090] In some embodiments, in the antibody-drug conjugate, D, D 1 D 2 D 3 Or D 4 It can be conjugated to the antibody or its antigen-binding fragment via a linker.

[0091] In some implementations, M is selected from the following structures:

[0092] Where p is an integer selected from 1 to 12.

[0093] In some implementations, M is selected from the following structures:

[0094] In some implementations, M is selected from the following structures:

[0095] In some implementations, M is selected from the following structures:

[0096] In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase, or an antibody or its antigen-binding fragment that specifically binds to human trophoblast surface antigen 2 (TROP2).

[0097] In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0098] In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that specifically binds to human trophoblast surface antigen 2 (TROP2).

[0099] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0100] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

[0101] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0102] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0103] (1c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:56 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:57 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:58 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:59 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:60 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:61 or a variant thereof;

[0104] Wherein, the variant described in any one of (1a), (1b) or (1c) 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 respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0105] or,

[0106] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0107] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0108] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0109] (2c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:68 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:69 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:58 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:59 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:60 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:61 or a variant thereof;

[0110] Wherein, the variant described in any one of (2a), (2b) or (2c) 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 respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0111] or,

[0112] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

[0113] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0114] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0115] (3c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:62 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:63 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:58 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:59 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:60 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:61 or a variant thereof;

[0116] Wherein, the variant described in any one of (3a), (3b) or (3c) 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 respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0117] or,

[0118] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0119] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0120] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0121] (4c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:64 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:65 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:66 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:67 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:61 or a variant thereof;

[0122] Wherein, the variant described in any one of (4a), (4b) or (4c) 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 respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0123] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0124] (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

[0125] (1a) A heavy chain variable region (VH) comprising the following 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 the following 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,

[0126] (1b) A heavy chain variable region (VH) comprising the following 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 the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25; or

[0127] (1c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:56, CDR-H2 of SEQ ID NO:57, and CDR-H3 of SEQ ID NO:58; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:59, CDR-L2 of SEQ ID NO:60, and CDR-L3 of SEQ ID NO:61;

[0128] or,

[0129] (2) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0130] (2a) A heavy chain variable region (VH) comprising the following three CDRs: 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 the following 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,

[0131] (2b) A heavy chain variable region (VH) comprising the following three CDRs: 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 the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0132] (2c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:68, CDR-H2 of SEQ ID NO:69, and CDR-H3 of SEQ ID NO:58; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:59, CDR-L2 of SEQ ID NO:60, and CDR-L3 of SEQ ID NO:61;

[0133] or,

[0134] (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

[0135] (3a) A heavy chain variable region (VH) comprising the following three CDRs: 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 the following 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,

[0136] (3b) A heavy chain variable region (VH) comprising the following three CDRs: 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 the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0137] (3c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:62, CDR-H2 with sequence SEQ ID NO:63, and CDR-H3 with sequence SEQ ID NO:58; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:59, CDR-L2 with sequence SEQ ID NO:60, and CDR-L3 with sequence SEQ ID NO:61;

[0138] or,

[0139] (4) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0140] (4a) A heavy chain variable region (VH) comprising the following three 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 the following three CDRs: CDR-L1 of SEQ ID NO:16, CDR-L2 of SEQ ID NO:17, and CDR-L3 of SEQ ID NO:10; or,

[0141] (4b) A heavy chain variable region (VH) comprising the following three 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 the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25;

[0142] (4c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:64, CDR-H2 with sequence SEQ ID NO:65, and CDR-H3 with sequence SEQ ID NO:66; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:67, CDR-L2 with sequence SEQ ID NO:17, and CDR-L3 with sequence SEQ ID NO:61.

[0143] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0144] (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or

[0145] (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4; or

[0146] (c) VH or a variant thereof shown in SEQ ID NO: 51, and / or VL or a variant thereof shown in SEQ ID NO: 52;

[0147] 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 originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

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

[0149] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0150] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4; or

[0151] (c) VH shown in SEQ ID NO: 51 and VL shown in SEQ ID NO: 52.

[0152] In some embodiments, the antibody or its antigen-binding fragment further comprises:

[0153] (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and

[0154] (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

[0155] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.

[0156] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35.

[0157] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 41.

[0158] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36.

[0159] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 53 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 53.

[0160] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36.

[0161] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 and a light chain constant region (CL) as shown in SEQ ID NO: 36.

[0162] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 53 and a light chain constant region (CL) as shown in SEQ ID NO: 36.

[0163] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0164] (1) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36;

[0165] (2) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0166] (3) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 41, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0167] (4) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 51 and the heavy chain constant region (CH) shown in SEQ ID NO: 41, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0168] (5) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 52 and the heavy chain constant region (CH) shown in SEQ ID NO: 53, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0169] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0170] (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38;

[0171] (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40;

[0172] (3) The heavy chain comprising the sequence shown in SEQ ID NO: 42, and the light chain comprising the sequence shown in SEQ ID NO: 38;

[0173] (4) The heavy chain comprising the sequence shown in SEQ ID NO: 43, and the light chain comprising the sequence shown in SEQ ID NO: 40; or

[0174] (5) The heavy chain comprising the sequence shown in SEQ ID NO: 54, and the light chain comprising the sequence shown in SEQ ID NO: 55.

[0175] In some embodiments of the antibody or antigen-binding fragment disclosed herein, the heavy chain constant domain may contain a C-terminal lysine residue or may lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid.

[0176] As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0177] In some embodiments, compositions comprising antibody or antigen-binding fragments disclosed herein are provided, wherein the various antibody or antigen-binding fragments may 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, an N-terminal amino acid cyclized to pyroglutamic acid or an N-terminal amino acid cyclized to pyroglutamate salt.

[0178] In some embodiments, the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0179] In some embodiments, the N-terminal glutamine of the VH or variant thereof, as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof, as shown in SEQ ID NO:37, 39, 42 or 43, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0180] In some embodiments, the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or 41 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37, 39, 42 or 43 or a variant thereof, lacks a C-terminal lysine residue.

[0181] In some implementations, the Ab is selected from antibodies or antigen-binding fragments thereof that specifically bind to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0182] In some implementations, Ab is selected from trastuzumab, pertuzumab, trastuzumab mutant, pertuzumab mutant, or a biepisode antibody or antigen-binding fragment thereof constructed from trastuzumab and pertuzumab.

[0183] In some embodiments, the antibody or its antigen-binding fragment is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 97 and the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 80.

[0184] In some implementations, the Ab is selected from antibodies or antigen-binding fragments thereof that specifically bind to human trophoblast surface antigen 2 (TROP2).

[0185] In some embodiments, the antibody or its antigen-binding fragment is selected from Sacituzumab (i.e., Isactuzumab or hRS7 antibody).

[0186] Those skilled in the art will understand that the antibody-drug conjugates described in this application can be prepared in a modular manner. For example, first obtain the free form of the "drug linker" as described in any of the above claims ( Wherein Q is the structural form of M before covalently linking it to the antibody or its antigen-binding fragment, and then covalently linking it to the antibody or its antigen-binding fragment yields the antibody-drug conjugate described in this application. Accordingly, in the free form of the "drug linker", Q is linked to one or more thiol (-SH) or amino (-NH2) groups on the antibody or its antigen-binding fragment through a substitution reaction (e.g., removal of -SO2Me or -Br structures) or an addition reaction.

[0187] In some embodiments, the antibody-drug conjugate is selected from the following structures:

[0188] Where x is selected from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0189] In some embodiments, the antibody or its antigen-binding fragment comprises an antibody or its antigen-binding fragment as shown in SEQ ID NO:1 (VH) and as shown in SEQ ID NO:2 (VL).

[0190] In some embodiments, the antibody or its antigen-binding fragment comprises an antibody or its antigen-binding fragment as shown in SEQ ID NO:3 (VH) and as shown in SEQ ID NO:4 (VL).

[0191] In some embodiments, the antibody or its antigen-binding fragment comprises an antibody or its antigen-binding fragment of VH as shown in SEQ ID NO:51 and VL as shown in SEQ ID NO:52.

[0192] In some implementation schemes, Ab' is selected from trastuzumab or pertuzumab.

[0193] In some implementation schemes, Ab' is selected from Sacituzumab monoclonal antibody.

[0194] in, This indicates the specific connection method between the antibody or its antigen-binding fragment and the antibody-drug conjugate.

[0195] Composition

[0196] On the other hand, this application provides compositions of antibody-drug conjugates (ADCs) as described herein. Such compositions may comprise a plurality of ADCs as described herein, wherein each ADC comprises a drug linker as described herein, wherein x independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug linkers. Therefore, the compositions are characterized by a drug-to-antibody ratio (DAR) in the range of about 1 to about 40. Methods for determining DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0197] For example, in any embodiment, the ADC composition described herein has a DAR of about 1 to about 40 or any subrange therebetween, such as: about 1 to 10, about 1 to 15, about 1 to 20, about 1 to 25, about 1 to 30, about 1 to 35, about 1 to 40, about 5 to 10, about 5 to 15, about 5 to 20, about 5 to 25, about 5 to 30, about 5 to 35, about 5 to 40, about 10 to 15, about 10 to 20, about 10 to 25, about 10 to 30, about 10 to 35, about 10 to 40, about 15 to 20, about 15 to 25, about 15 to 30, about 15 to 35, about 15 to 40, about 20 to 25, about 20 to 30, about 20 to 35, about 20 to 40, about 25 to 30, about 25 to 35, about 25 to 40. Approximately 30 to 35, approximately 30 to 40, approximately 35 to 40, approximately 1 to 2, approximately 1 to 3, approximately 1 to 4, approximately 1 to 5, approximately 1 to 6, approximately 1 to 7, approximately 1 to 8, approximately 1 to 9, approximately 1 to 10, approximately 2 to 3, approximately 2 to 4, approximately 2 to 5, approximately 2 to 6, approximately 2 to 7, approximately 2 to 8, approximately 2 to 9, approximately 2 to 10, approximately 3 to 4, approximately 3 to 5, approximately 3 to 6, approximately 3 to 7 Approximately 3 to 8, Approximately 3 to 9, Approximately 3 to 10, Approximately 4 to 5, Approximately 4 to 6, Approximately 4 to 7, Approximately 4 to 8, Approximately 4 to 9, Approximately 4 to 10, Approximately 5 to 6, Approximately 5 to 7, Approximately 5 to 8, Approximately 5 to 9, Approximately 5 to 10, Approximately 6 to 7, Approximately 6 to 8, Approximately 6 to 9, Approximately 6 to 10, Approximately 7 to 8, Approximately 7 to 9, Approximately 7 to 10, Approximately 8 to 9, Approximately 8 to 10, Approximately 9 to 10.

[0198] In some embodiments, the DAR of the ADC compositions described herein is about 10, about 10.1, about 10.2, about 10.3, about 10.4, about 10.5, about 10.6, about 10.7, about 10.8, about 10.9, about 11, about 11.1, about 11.2, about 11.3, about 11.4, about 11.5, about 11.6, about 11.7, about 11.8, about 11.9, about 12, about 12.1, about 12.2, about 12.3, about 12.4, about 12.5, about 12.6, about 12.7, about 12.8, about 12.9, about 13, about 13.1, about 13.2, about 13.3, about 13.4, about 13.5, about 13.6, about 13.7, about 13.8. Approximately 13.9, approximately 14, approximately 14.1, approximately 14.2, approximately 14.3, approximately 14.4, approximately 14.5, approximately 14.6, approximately 14.7, approximately 14.8, approximately 14.9, approximately 15, approximately 15.1, approximately 15.2, approximately 15.3, approximately 15.4, approximately 15.5, approximately 15.6, approximately 15.7, approximately 15.8, approximately 15.9, approximately 16, approximately 16.1, approximately 16.2, approximately 16.3, approximately 16.4, approximately 16.5, approximately 16.6, approximately 16.7, approximately 16.8, approximately 16.9, approximately 17, approximately 17.1, approximately 17.2, approximately 17.3, approximately 17.4, approximately 17.5, approximately 17.6, approximately 17.7, approximately 17.8, approximately 17.9, approximately 18, approximately 18.1, Approximately 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 2 0.3, approximately 20.4, approximately 20.5, approximately 20.6, approximately 20.7, approximately 20.8, approximately 20.9, approximately 21, approximately 21.1, approximately 21.2, approximately 21.3, approximately 21.4, approximately 21.5, approximately 21.6, approximately 21.7, approximately 21.8, approximately 21.9, approximately 22, approximately 22.1, approximately 22.2, approximately 22.3, approximately 22.4 Approximately 22.5, Approximately 22.6, Approximately 22.7, Approximately 22.8, Approximately 22.9, Approximately 23, Approximately 23.1, Approximately 23.2, Approximately 23.3, Approximately 23.4, Approximately 23.5, Approximately 23.6, Approximately 23.7, Approximately 23.8, Approximately 23.9, Approximately 24, Approximately 24.1, Approximately 24.2, Approximately 24.3, Approximately 24.4, Approximately 24.5, Approximately 24.6, approximately 24.7, approximately 24.8, approximately 24.9, approximately 25, approximately 25.1, approximately 25.2, approximately 25.3, approximately 25.4, approximately 25.5, approximately 25.6, approximately 25.7, approximately 25.8, approximately 25.9, approximately 26, approximately 26.1, approximately 26.2, approximately 26.3, approximately 26.4, approximately 26.5, approximately 26.6, approximately 26.7, approximately 26.8, approximately 26.9, approximately 27, approximately 27.1, approximately 27.2, approximately 27.3, approximately 27.4, approximately 27.5, approximately 27.6, approximately 27.7, approximately 27.8, approximately 27.9, approximately 28, approximately 28.1, approximately 28.2, approximately 28.3, approximately 28.4, approximately 28.5, approximately 28.6, approximately 28.7, approximately 28.8, approximately 28.9, approximately 29, approximately 29.1, approximately 29.2, approximately 29.3, approximately 29.4, approximately 29.5, approximately 29.6, approximately 29.7, approximately 29.8, approximately 29.9, approximately 30, approximately 30.1, approximately 30.2, approximately 30.3, approximately 30.4, approximately 30.5, approximately 30.6, approximately 30.7, approximately 30.8, approximately 30.9, approximately 31, approximately 31.1, approximately 31.2, approximately 31.3, approximately 31.4, approximately 31.5, approximately 31.6, approximately 31.7, approximately 31.8, approximately 31.9, approximately 32, approximately 32.1, approximately 32.2, approximately 32.3, approximately 32.4, approximately 32.5, approximately 32.6, approximately 32.7, approximately 32.8, approximately 32.9, approximately 33, approximately 33.1, approximately 33.2, approximately 33.3, approximately 3 3.4, approximately 33.5, approximately 33.6, approximately 33.7, approximately 33.8, approximately 33.9, approximately 34, approximately 34.1, approximately 34.2, approximately 34.3, approximately 34.4, approximately 34.5, approximately 34.6, approximately 34.7, approximately 34.8, approximately 34.9, approximately 35, approximately 35.1, approximately 35.2, approximately 35.3, approximately 35.4, approximately 35.5, approximately 35.6, approximately 35.7, approximately 35.8, approximately 35.9, approximately 36, approximately 36.1, approximately 36.2, approximately 36.3, approximately 36.4, approximately 36.5, approximately 36.6, approximately 36 7, approximately 36.8, approximately 36.9, approximately 37, approximately 37.1, approximately 37.2, approximately 37.3, approximately 37.4, approximately 37.5, approximately 37.6, approximately 37.7, approximately 37.8, approximately 37.9, approximately 38, approximately 38.1, approximately 38.2, approximately 38.3, approximately 38.4, approximately 38.5, approximately 38.6, approximately 38.7, approximately 38.8, approximately 38.9, approximately 39, approximately 39.1, approximately 39.2, approximately 39.3, approximately 39.4, approximately 39.5, approximately 39.6, approximately 39.7, approximately 39.8, approximately 39.9, approximately 40.

[0199] In some embodiments, the composition contains the following antibody-drug conjugates:

[0200] x is selected from 1 to 10, for example 1-8, 1-6, 2-5, 2-4;

[0201] Ab' is Trastuzumab;

[0202] Preferably, the composition has a DAR of 7.0.

[0203] In some embodiments, the composition contains the following antibody-drug conjugates:

[0204] Ab' is Trastuzumab;

[0205] x is selected from 1 to 10, for example 1-8, 1-6, 2-5; preferably, the DAR of the composition is 7.0;

[0206] Alternatively, x is selected from 1 to 10, for example 2-10, 4-10, 5-9; preferably, the DAR of the composition is 16.0.

[0207] In some embodiments, the composition contains the following antibody-drug conjugates:

[0208] x is selected from 1 to 10, for example 1-8, 1-6, 2-5; Ab' is Trastuzumab; preferably, the DAR of the composition is 6.2;

[0209] Alternatively, x is selected from 1 to 10, for example 1-8, 1-6, 2-5; Ab' is Sacituzumab; preferably, the DAR of the composition is 5.8.

[0210] In some embodiments, the composition contains the following antibody-drug conjugates:

[0211] x is selected from 1 to 10, for example 1-8, 1-6, 2-5; Ab' is Trastuzumab; preferably, the DAR of the composition is 7.0;

[0212] Alternatively, x is selected from 1 to 10, for example 1-8, 1-6, 2-5; Ab' is Sacituzumab; preferably, the DAR of the composition is 6.8.

[0213] In some embodiments, the composition contains the following antibody-drug conjugates:

[0214] x is selected from 1 to 10, for example 2-10, 4-10, 5-9;

[0215] Ab' is Trastuzumab;

[0216] Preferably, the DAR of the composition is 31.32.

[0217] Connector

[0218] In another aspect, this application provides a compound having the following structure or any combination thereof:

[0219] and -NR 0 -C 1-10 Alkyl; the alkyl group may optionally be substituted with one or more OH groups; R 0 Selected from H or C 1-6 Alkyl group; q is independently selected from an integer from 1 to 24 each time it appears (e.g., an integer from 1 to 10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0220] In some embodiments, the compound contains the following structure:

[0221] In another aspect, this application provides a compound comprising the following structures or any combination thereof:

[0222] Each time r appears, it is independently selected from an integer between 0 and 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0223] In some embodiments, the compound contains the following structure:

[0224] Each time r appears, it is independently selected from an integer from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0225] On the other hand, this application provides a connector having the following structure: Among them, B and L a L, E, and n are as described in any of the preceding texts.

[0226] In some embodiments, the connector has the following structure:

[0227] In another aspect, this application provides a compound comprising a linker with the following structure: Among them, B, La, L, E and n are as described in any of the preceding items.

[0228] In some implementations, position 1 of the connector is connected to Q or M, and position 2 of the connector is connected to D, D 1 D 2 D 3 Or D 4 Connections, namely Q, M, D, D 1 D 2 D 3 and D 4 As stated in any of the preceding items.

[0229] Preparation method

[0230] In another aspect, this application provides a method for preparing the drug linker described above, comprising:

[0231] Q-Ag and Pg-BL 1 -E 1 -D 1 Coupling yields QB(Pg)-L 1 -E 1 -D 1 ;

[0232] QB(Pg)-L 1 -E 1 -D 1 With HL 2 -E 2 -D 2 Coupling yields the compound shown in formula IA; optionally, removal of QB(Pg)-L is further performed prior to coupling. 1 -E 1 -D 1 Steps for protecting the middle group;

[0233] Wherein Ag is a carboxyl activating group, including halogens (e.g., Cl), acid anhydrides (e.g., RC(O)O) - Or RC(NR')O - R is selected from H and C. 1-6 Alkyl, C2-6 alkenyl, C 2-6 alkynyl group, C 6-10 aryl, wherein the alkyl, alkenyl, alkynyl or aryl group is optionally substituted with one or more halogens, amino groups, or nitro groups; R' is selected from H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 6-10 Aryl), alkoxy (e.g., 2,5-dioxopyrrolidone-1-yl), etc.;

[0234] Pg is an amino or carboxyl protecting group, such as hydroxyl groups (e.g., methyl, ethyl, allyloxy, benzyloxy, etc.), TBS, TBDPS, THP, perfluorophenoxy, MTT, trityl, Fmoc, Boc, etc.

[0235] The remaining groups are as defined in any of the preceding items.

[0236] In some embodiments, the method further includes further reacting the compound of formula IA with L a The steps for obtaining the compound shown in formula IB by coupling with Ag;

[0237] Wherein, Ag is a carboxyl activating group, including halogens (e.g., Cl), acid anhydrides (e.g., RC(O)O) - Or RC(NR')O - ), alkoxy groups (e.g., 2,5-dioxopyrrolidone-1-yl), etc.; the remaining groups are as defined in any of the preceding items.

[0238] In another aspect, this application provides yet another method for preparing the drug linker described above, comprising:

[0239] HL 1 -E 1 -D 1 HL 2 -E 2 -D 2 Coupling with B'-Pg yields B'(Pg)(L 1 -E 1 -D 1 (L) 2 -E 2 -D 2 );

[0240] QB”-L 3 -E 3 -D 3 With B'(Pg)(L 1 -E 1 -D 1 (L) 2 -E 2-D 2 Coupling yields the compound shown in formula IC; optionally, the removal of B'(Pg)(L) is further performed prior to coupling. 1 -E 1 -D 1 (L) 2 -E 2 -D 2 The steps for protecting the base in )

[0241] Wherein, B' and B” are fragments of B as described in any of the preceding items, and after coupling, B' and B” are connected to form B as described in this article;

[0242] Pg is an amino or carboxyl protecting group, such as hydroxyl groups (e.g., methyl, ethyl, allyloxy, benzyloxy, etc.), TBS, TBDPS, THP, perfluorophenoxy, MTT, trityl, Fmoc, Boc, etc.

[0243] The remaining groups are as defined in any of the preceding items.

[0244] In some implementation schemes, HL 1 -E 1 -D 1 and HL 2 -E 2 -D 2 Same or different.

[0245] In another aspect, this application provides yet another method for preparing the drug linker described above, comprising:

[0246] Q-B'-L 1 -E 1 -D 1 and B (Pg) n1 Coupling yields QB(Pg). n1 -L 1 -E 1 -D 1 ;

[0247] QB(Pg) n1 -L 1 -E 1 -D 1 The reaction with HLED yields the compound shown in Formula I; optionally, the removal of QB(Pg) is further performed prior to coupling. n1 -L 1 -E 1 -D 1 Steps for protecting the middle group;

[0248] Where n1 is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9;

[0249] Pg is an amino or carboxyl protecting group, such as hydroxyl groups (e.g., methyl, ethyl, allyloxy, benzyloxy, etc.), TBS, TBDPS, THP, perfluorophenoxy, MTT, trityl, Fmoc, Boc, etc.

[0250] The remaining groups are as defined in any of the preceding items.

[0251] In some implementations, when n1 = 3, the compound shown in formula ID can be prepared according to the above method.

[0252] In some embodiments, prior to any of the steps described above, the method includes a step of protecting functional groups at non-target reaction sites with a suitable protecting group chemistry.

[0253] In some embodiments, prior to any step of any of the above aspects, the method includes a step of removing a protecting group from the functional group of the target reactive site.

[0254] Pharmaceutical Composition

[0255] In another aspect, this application provides a pharmaceutical composition comprising any of the antibody-drug conjugates described above, and one or more pharmaceutical excipients.

[0256] The antibody-drug conjugates described herein are typically formulated in a single injectable form with a pharmaceutically acceptable parenteral medium for parenteral use, such as bolus injection, intravenous injection, or intratumoral injection. Optionally, antibody-drug conjugates of desired purity are mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers in the form of lyophilized or solution formulations (Remington's Pharmaceutical Sciences (1980) 16). 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.

[0257] application

[0258] 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 or TROP2-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.

[0259] 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 medicaments for treating Her2-expressing cancers or TROP2-expressing cancers.

[0260] 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 or TROP2-expressing cancers.

[0261] Additionally, this application provides a method for treating Her2-expressing cancer or TROP2-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.

[0262] In some embodiments, the antibody-drug conjugate, drug-linker, ADC composition, or drug composition is sufficient (e.g., in a subject):

[0263] (1) Inhibits cell (such as tumor cells) proliferation;

[0264] (2) Inhibits tumor growth;

[0265] (3) Inducing and / or increasing antibody-dependent cytotoxic activity;

[0266] (4) Inhibit HER2 / TROP2-mediated signal transduction;

[0267] (5) Prevention and / or treatment of HER2 / TROP2-mediated diseases / disorders; or

[0268] (6) Any combination of (1)-(5) above.

[0269] In some implementations, the cancer or tumor is selected from solid tumors or hematologic malignancies, such as colon cancer, stomach cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma. Detailed Implementation

[0270] definition

[0271] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques obvious to those skilled in the art. Furthermore, laboratory procedures used herein, such as those related to genomics, nucleic acid chemistry, and molecular biology, are standard procedures widely used in their respective fields. While it is believed that the following terms will be readily understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0272] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The amino acid distribution in each region or domain can follow various numbering systems known in the art.

[0273] The term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat 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, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0274] In this invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined using the Chothia numbering system.

[0275] The term "framework region" or "FR" residues refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0276] The term "antigen-binding fragment" in antibody refers to a fragment of the antibody polypeptide, such as a fragment of the full-length antibody polypeptide, which 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; it is also referred to as the "antigen-binding moiety". See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology 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 peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0277] The term "Fd" refers to an antibody fragment composed of VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).

[0278] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0279] The term "Fc" refers to an antibody fragment formed by disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0280] The term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:49) amino acid sequence or a variant 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 that can be used in this invention 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 may also exist between VH and VL of scFv. In some implementations, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, domains containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH of scFv.

[0281] The term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

[0282] Each of the above antibody fragments 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.

[0283] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0284] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0285] The term "mouse antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies, and then screening, preparing and purifying the antibodies; or it refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells after the antigen enters the mouse body.

[0286] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance the immune response. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and / or ability to enhance the immune response).

[0287] The term "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods conveniently performed, for example, by computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0288] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using 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 amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with 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 and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, 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).

[0289] The twenty common amino acids discussed herein are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0290] The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0291] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, such as "C". 1-20Alkyl", C 1-10 Alkyl", C 1-6 Alkyl", C 1-4 Alkyl", C 1-3 Alkyl groups, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 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, etc.

[0292] 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.

[0293] Information about the sequences involved in this invention is described in the table below:

[0294] The abbreviations used in this article have the following meanings:

[0295] 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).

[0296] 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).

[0297] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0298] 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.

[0299] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0300] 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)

[0301] 2,5-Dioxopyrrolidone-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (757.30 mg, 2.07 mmol), N 6 -(((9H-fluorene-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 continued for 1 hour. The reaction solution was concentrated and purified by reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0%–50%), and then freeze-dried to obtain the title solid (937 mg, 1.51 mmol).

[0302] Its structural characterization data are as follows:

[0303] MS m / z (ESI): 619.4 [M+H] +

[0304] Example 2 of intermediate preparation: 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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (INT-2)

[0305] Step 1: Preparation of (9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)carbamate (INT-2-2)

[0306] (5S,8S,11S)-1-(9H-fluorene-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecane-17-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-1 OH,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (500 mg, 1.11 mmol) was 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 the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give 700 mg of the title compound.

[0307] Its structural characterization is as follows:

[0308] ESI-MS (m / z): 974.3 [M+H] + .

[0309] Its preparation method is as follows:

[0310] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0311] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0312] Step 2: 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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (INT-2)

[0313] (9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)carbamate (500 mg, 0.513 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (75.05 mg, 1.03 mmol) was added. The mixture was reacted at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain compound INT-2 307 mg.

[0314] Its structural characterization is as follows:

[0315] ESI-MS (m / z): 752.3 [M+H] + .

[0316] Its preparation method is as follows:

[0317] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0318] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0319] Example 3 of intermediate preparation: Preparation of (S)-2-amino-N-((S)-1-(((S)-1-(((((2S,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propionamide (INT-3)

[0320] Step 1: Preparation of (9H-fluorene-9-yl)methyl[(S)-1-(((((2S,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropane-2-yl]carbamate (INT-3-2)

[0321] Gemcitabine (3 g, 11.40 mmol) and (S)-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionamido)methyl ester (4.36 g, 11.40 mmol) were dissolved in NMP (55 mL) and reacted dropwise with HCl / 1,4-dioxane (4 M, 5.70 mL) at 0 °C for 10 hours at room temperature. Water (100 mL) and ethyl acetate (100 mL) were added to the system for extraction. The aqueous phase was collected, purified by reversed-phase chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0%-50%), and lyophilized to give the title compound (2.0 g, 3.24 mmol).

[0322] Its structural characterization is as follows:

[0323] ESI-MS (m / z): 586.2 [M+H] + .

[0324] Step 2: Preparation of (S)-2-amino-N-((((2S,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)propionamide (INT-3-3)

[0325] (9H-fluorene-9-yl)methyl[(S)-1-(((((2S,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropane-2-yl]carbamate (2.0 g, 3.24 mmol) was dissolved in DMF (10 mL) and then added dropwise to diethylamine (1.19 g, 16.22 mmol, 1.68 mL). The reaction was carried out at room temperature for 1 hour. The reaction solution was concentrated under vacuum to obtain the crude product (1.18 g, 3.18 mmol).

[0326] Its structural characterization is as follows:

[0327] ESI-MS (m / z): 364.3 [M+H] + .

[0328] Step 3: Preparation of (9H-fluorene-9-yl)methyl((6S,9S,12S)-1-((2S,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)carbamate (INT-3-4)

[0329] (S)-2-amino-N-((((2S,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)propionamide (1.18 g, 3.18 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (1.46 g, 3.82 mmol) were dissolved in DMF (8.34 mL), and then DIPEA (1.23 g, 9.55 mmol, 1.66 mL) and HATU (1.45 g, 3.82 mmol) were added and reacted at room temperature for two hours. The crude product was purified by reversed-phase chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0%-50%) and lyophilized to give the title compound (2.0 g, 2.47 mmol).

[0330] Its structural characterization is as follows:

[0331] ESI-MS (m / z): 728.3 [M+H] + .

[0332] Step 4: Preparation of (S)-2-amino-N-((S)-1-(((S)-1-(((((2S,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propionamide (INT-3)

[0333] (9H-fluorene-9-yl)methyl((6S,9S,12S)-1-((2S,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)carbamate (380 mg, 522.19 μmol) was dissolved in DMF (3 mL), and then diethylamine (522.19 μmol, 1 mL) was added. The mixture was reacted at room temperature for one hour. The system was then directly concentrated under vacuum to give the crude title compound (263 mg, 509.90 μmol).

[0334] Its structural characterization is as follows:

[0335] ESI-MS (m / z): 506.3 [M+H] + .

[0336] Example 4 of intermediate preparation: Preparation of (S)-2-amino-N-((S)-1-(((S)-1-(((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propionamide (INT-4)

[0337] Step 1: Preparation of (9H-fluorene-9-yl)methyl[(S)-1-(((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropane-2-yl]carbamate (INT-4-2)

[0338] 5-fluoro-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-dione (4 g, 16.25 mmol) and (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)methyl ester (6.21 g, 16.25 mmol, FR) were dissolved in NMP (72.03 mL) and HCl / 1,4-dioxane (4 M, 8.12 mL) was added dropwise at 0 °C. The mixture was purified by reversed-phase chromatography (acetonitrile / 0.05% ammonium bicarbonate aqueous solution = 0%-40%), lyophilized, and then purified by silica gel chromatography (methanol / dichloromethane = 0%-10%) to obtain the title compound (2.83 g, 4.97 mmol).

[0339] Its structural characterization is as follows:

[0340] ESI-MS (m / z): 569.3 [M+H] + .

[0341] Step 2: Preparation of (S)-2-amino-N-((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)propionamide (INT-4-3)

[0342] (9H-fluorene-9-yl)methyl[(S)-1-(((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropane-2-yl]carbamate was dissolved in DMF (10 mL), and then diethylamine (1.84 g, 24.85 mmol) was added. The mixture was reacted at 25 °C for one hour. The system was concentrated under vacuum to give the crude title compound (1.72 g, 4.72 mmol).

[0343] Its structural characterization is as follows:

[0344] ESI-MS (m / z): 347.3 [M+H] + .

[0345] Step 3: Preparation of (9H-fluorene-9-yl)methyl((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)carbamate (INT-4-4-4)

[0346] (S)-2-amino-N-((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)propionamide (1.72 g, 4.72 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (1.80 g, 4.72 mmol) were dissolved in DMF (10 mL), and then DIPEA (1.83 g, 14.15 mmol) and HATU (1.79 g, 4.72 mmol) were added and reacted at room temperature for two hours. The crude product was purified by reversed-phase chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0%-60%) and lyophilized to give the title compound (2.82 g, 3.97 mmol).

[0347] Its structural characterization is as follows:

[0348] ESI-MS (m / z): 711.3 [M+H] + .

[0349] Step 4: Preparation of (S)-2-amino-N-((S)-1-(((S)-1-(((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propionamide (INT-4)

[0350] (9H-fluorene-9-yl)methyl((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)carbamate (285 mg, 401.01 μmol) was dissolved in DMF (2 mL), and then diethylamine (0.5 mL) was added. The mixture was reacted at room temperature for two hours. The system was then concentrated under vacuum to give the crude title compound (195 mg, 379.25 μmol).

[0351] Its structural characterization is as follows:

[0352] ESI-MS (m / z): 489.3 [M+H] + .

[0353] Example 5 of intermediate preparation: 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-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (INT-5)

[0354] Step 1: N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 4 Preparation of -((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]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-L-asparagine (INT-5-1)

[0355] (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]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (120 mg) Dissolved (159.53 μmol) and (9H-fluorene-9-yl)methyl(S)-(2,5-dioxotetrahydrofuran-3-yl)carbamate (56.50 mg, 167.51 μmol) in DMF (3 mL), added DIPEA (20.62 mg, 159.53 μmol), and stirred at room temperature for 2 hours. The reaction solution was directly purified by reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0%–90%) and then freeze-dried to give the title compound (124 mg, 113.81 μmol).

[0356] Its structural characterization data are as follows:

[0357] MS m / z (ESI): 1089.4 [M+H] +

[0358] Step 2: Preparation of (9H-fluorene-9-yl)methyl(S)-(1-((chloromethyl)amino)-1-oxopropane-2-yl)carbamate (INT-5-3)

[0359] (S)-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propamido)methyl acetate (50 mg, 130.75 μmol) was added to 1,2-dichloroethane (4 mL), followed by the dropwise addition of TMSCl (142.05 mg, 1.31 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 hour. After the reaction was complete, 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 reaction without purification.

[0360] Step 3: Preparation of (9H-fluorene-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-oxopropane-2-yl)carbamate (INT-5-4)

[0361] (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one (30 mg, 78.87 μmol) was dissolved in THF (1.5 mL), and the mixture was cooled to -78 °C under nitrogen protection. Then, NaHMDS (78.87 μmol, 2 M in THF) was added dropwise, and the system turned yellow. The mixture was kept at this temperature and stirred for 20 minutes. Then, a THF (1.5 mL) solution of (9H-fluorene-9-yl)methyl(S)-(1-((chloromethyl)amino)-1-oxopropane-2-yl)carbamate (42.45 mg, 118.31 μmol) was added dropwise. After the addition was complete, the mixture was kept at this temperature for 20 minutes, and then the temperature was raised to -10 °C and stirred for 1 hour. After the reaction was complete, water and ethyl acetate were added to the reaction solution 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), which was used directly in the next reaction without purification.

[0362] Its structural characterization data are as follows:

[0363] MS m / z (ESI): 703.3 [M+H] +

[0364] Step 4: Preparation of (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)propionamide (INT-5-5)

[0365] (9H-fluorene-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-oxopropane-2-yl)carbamate (70 mg, 69.73 μmol) was dissolved in DMF (1 mL), and diethylamine (0.2 mL) was added. The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the reaction solution was directly purified by reversed-phase column chromatography (acetonitrile / 0.05% ammonium bicarbonate aqueous solution = 0–95%) and then freeze-dried to obtain the title compound (20 mg, 41.63 μmol).

[0366] Its structural characterization data are as follows:

[0367] MS m / z (ESI): 481.2 [M+H] +

[0368] Step 5: Preparation of (9H-fluorene-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-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (INT-5-6)

[0369] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (596.93 mg, 1.56 mmol) was dissolved in DMF (10 mL), and HATU (593.17 mg, 1.56 mmol) and DIPEA (403.48 mg, 3.12 mmol) were added. The mixture 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)propionamide (500 mg, 1.04 mmol) was added, and the mixture was stirred at room temperature overnight. After the reaction was complete, the reaction solution was used directly as a solution for the next reaction without further treatment.

[0370] Its structural characterization data are as follows:

[0371] MS m / z (ESI): 845.4 [M+H] +

[0372] Step Six: 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-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (INT-5)

[0373] In step five, DMF (10 mL) and diethylamine (1 mL) were added to the reaction solution, and the mixture was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was partially purified by lyophilization (acetonitrile-0.05% ammonium bicarbonate aqueous solution = 0-90%) and then freeze-dried to obtain the title compound (700 mg, 1.01 mmol).

[0374] Its structural characterization data are as follows:

[0375] MS m / z (ESI): 623.4 [M+H] +

[0376] Example 6 of intermediate preparation: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-6)

[0377] Step 1: Preparation of 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxo))dipropionic acid (INT-6-2)

[0378] 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (0.94 g, 4.71 mmol) and 2-benzyloxy-1-methylpyridine trifluoromethanesulfonate (1.65 g, 4.71 mmol) were reacted at 80–85 °C for 16 hours. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10–70%) and lyophilized to give the title compound (0.78 g, 1.51 mmol).

[0379] Its structural characterization data are as follows:

[0380] ESI-MS (m / z): 515.2 (M+H) +

[0381] Step 2: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)benzyl propionate (INT-6-3)

[0382] 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) and 2-amino-2-(hydroxymethyl)propane-1,3-diol (0.82 g, 6.80 mmol) were dissolved in DMF (15 mL), DIPEA (1.17 g, 9.07 mmol) was added, and HATU (2.59 g, 6.80 mmol) was added in portions. The reaction was carried out at 25 °C for 2 hours. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10-70%) and lyophilized to give the title compound (0.74 g, 0.90 mmol).

[0383] Its structural characterization data are as follows:

[0384] ESI-MS (m / z): 824.4 (M+H) +

[0385] Step 3: Preparation of 3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (INT-6)

[0386] Benzyl 3-(3-(((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propoxy)propionate (0.74 g, 0.90 mmol) was dissolved in ethanol (30 mL), 10% palladium on carbon (0.15 g) and acetic acid (0.14 g, 2.42 mmol) were added, the air was removed, hydrogen was introduced and the temperature was raised to 40 °C for 4 hours, palladium on carbon was filtered off, the filtrate was concentrated, the residue was dissolved in water and acetonitrile and clarified, and lyophilized to give the title compound (0.59 g, 0.80 mmol).

[0387] Its structural characterization data are as follows:

[0388] ESI-MS (m / z): 734.3 (M+H) +

[0389] Example 7 of intermediate preparation: Preparation of 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)propionic acid (INT-7)

[0390] Step 1: Preparation of 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)benzyl propionate (INT-7-1)

[0391] 3,3'-((2-((3-(benzyloxy)-3-oxopropoxy)methyl)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (1.07 g, 2.08 mmol) and (2R,3R,4R,5S)-6-(methylamino)hexane-1,2,3,4,5-pentaol (1.62 g, 8.32 mmol) were dissolved in DMF (15 mL), DIPEA (1.34 g, 10.40 mmol) was added, and HATU (3.56 g, 9.36 mmol) was added in portions. The reaction was carried out at 25 °C for 1 hour. The reaction solution was directly purified by C18 reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 10-70%) and lyophilized to give the title compound (0.66 g, 0.63 mmol).

[0392] Its structural characterization data are as follows:

[0393] ESI-MS (m / z): 1046.5 (M+H) +

[0394] Step 2: Preparation of 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)propionic acid (INT-7)

[0395] 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)propoxy)propionate (0.66 g, 0.63 mmol) was dissolved in ethanol (14 mL) and water (7 mL). 10% palladium on carbon (0.13 g) and acetic acid (0.10 g, 1.70 mmol) were added. The air was removed, and hydrogen was introduced to raise the temperature to 40 °C and react for 4 hours. The palladium on carbon was filtered off, the filtrate was concentrated, and the residue was dissolved in water and acetonitrile to clarify. The solution was lyophilized to give the title compound (0.54 g, 0.56 mmol).

[0396] Its structural characterization data are as follows:

[0397] ESI-MS (m / z): 956.4 (M+H) +

[0398] Example 8 of intermediate preparation: Preparation of (S)-5-(((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxopentanoic acid (INT-8)

[0399] Step 1: Preparation of (S)-4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-(tert-butoxy)-3-methyl-1-oxobutane-2-yl)amino)-5-oxopentanoic acid allyl ester (INT-8-2):

[0400] (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxovaleric acid (3 g, 7.33 mmol) and L-valine tert-butyl hydrochloride (1.54 g, 7.33 mmol) were dissolved in DMF (30 mL), and DIPEA (2.84 g, 21.98 mmol) was added dropwise. HATU (2.92 g, 7.69 mmol) was added in portions, and the mixture was stirred at room temperature for 1.5 hours. The reaction mixture was poured into 200 mL of water with stirring, extracted with ethyl acetate, washed with organic phase brine, and concentrated under reduced pressure to obtain the crude product of the title compound (6.55 g, 6.96 mmol, 60% purity), which was used directly in the next reaction without purification.

[0401] Its structural characterization data are as follows:

[0402] MS m / z (ESI): 565.3 [M+H] +

[0403] Step 2: Preparation of (S)-4-amino-5-(((S)-1-(tert-butoxy)-3-methyl-1-oxobutane-2-yl)amino)-5-oxopentanoic acid allyl ester (INT-8-3):

[0404] DMF (20 mL) was added to (S)-4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(((S)-1-(tert-butoxy)-3-methyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid allyl ester (6.55 g, 6.96 mmol), followed by diethylamine (2.55 g, 34.80 mmol, 3.60 mL). The mixture was stirred at room temperature for 1 hour. The reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, diluted with dichloromethane, and purified by normal-phase silica gel column chromatography (petroleum ether-ethyl acetate = 0-100%). The solution was then concentrated again under reduced pressure to give the title compound (2.3 g, 6.72 mmol).

[0405] Its structural characterization data are as follows:

[0406] MS m / z(ESI): 343.3 [M+H] +

[0407] Step 3: Preparation of (S)-5-(((S)-1-(tert-butoxy)-3-methyl-1-oxobutan-2-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-5-oxopentanoic acid allyl ester (INT-8-4):

[0408] 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynic acid (626.79 mg, 2.34 mmol) and (S)-4-amino-5-(((S)-1-(tert-butoxy)-3-methyl-1-oxobutane-2-yl)amino)-5-oxopentanoic acid allyl ester (0.8 g, 2.34 mmol) were dissolved in DMF (10 mL), and DIPEA (603.88 mg, 4.67 mmol) was added dropwise. Then, HATU (932.16 mg, 2.45 mmol) was added in portions. After the addition was complete, the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate. The organic phase was washed with brine and concentrated under reduced pressure to obtain the crude product of the title compound (1.8 g, 2.28 mmol, 75% purity), which was used directly in the next reaction without purification.

[0409] Its structural characterization data are as follows:

[0410] MS m / z (ESI): 537.2 [M+H-56] +

[0411] Step 4: Preparation of ((S)-5-(allyloxy)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxovaleryl)-L-valine (INT-8-5):

[0412] Crude (S)-5-(((S)-1-(tert-butoxy)-3-methyl-1-oxobutan-2-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxopentanoic acid allyl ester (1.8 g, 2.28 mmol, 75% purity) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (5 mL) was added. The mixture was stirred at room temperature for 1.5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent. The crude product was dissolved in water and purified by reversed-phase column chromatography (acetonitrile-0.05% trifluoroacetic acid aqueous solution = 0-90%), followed by freeze-drying to give the title compound (705 mg, 1.31 mmol).

[0413] Its structural characterization data are as follows:

[0414] MS m / z (ESI): 537.2 [M+H] +

[0415] Step 5: Preparation of (S)-5-(((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxopentanoic acid allyl ester (INT-8-6):

[0416] Add 6 mL of DMF, 53.99 mg of DIPEA, and 417.75 μmol of HATU to trifluoroacetate of (S)-2-amino-N-((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentano[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-3-yl)propionamide (60 mg, 104.44 μmol) and ((S)-5-(allyloxy)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-5-oxopentanoyl)-L-valine (60 mg, 111.82 μmol). Add DIFEA (53.99 mg, 417.75 μmol) and HATU (43.66 mg, 114.88 μmol). Stir the mixture at room temperature for 1 hour. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, filtered, and the filter cake was dried to obtain the product. The filtrate was purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid = 0-90%) and concentrated under reduced pressure. The solid and filter cake were combined to obtain the product (110 mg, 101.12 μmol, 90% purity).

[0417] Its structural characterization data are as follows:

[0418] MS m / z (ESI): 979.3 [M+H] +

[0419] Step Six: Preparation of (S)-5-(((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5-oxopentanoic acid (INT-8)

[0420] The (S)-5-(((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-4-( Crude 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-5-oxopentanoic acid allyl ester (110.00 mg, 101.12 μmol) was mixed with 4 mL of DMF and 63.15 mg of 1,3-dimethylbarbituric acid (404.47 μmol). Tetraphenylphosphine palladium (23.37 mg, 20.22 μmol) was added under nitrogen protection. After nitrogen purging, the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the solvent was partially removed by lyophilization. The remaining reaction solution was purified by high-performance liquid chromatography and then lyophilized to obtain the title compound (60 mg, 62.62 μmol, 98% purity).

[0421] Its structural characterization data are as follows:

[0422] MS m / z (ESI): 939.3 [M+H] +

[0423] Its preparation method is as follows:

[0424] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0425] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0426] Example 9 of intermediate preparation: Synthesis of ((S)-2-amino-N-((S)-1-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide)(INT-9):

[0427] Step 1: Preparation of 4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one (INT-9-2):

[0428] 4-Amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one hydrochloride (2 g, 6.67 mmol) was dissolved in DMF (10 mL), and triethylamine (1.35 g, 13.35 mmol) was added dropwise. Imidazole (1.82 g, 26.70 mmol) was then added, followed by tert-butyldimethylchlorosilane (4.02 g, 26.70 mmol). The mixture was heated to 50 °C and stirred for 3 hours, then cooled to room temperature and reacted overnight. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate. The solution was washed with organic phase brine and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate = 0-100%) and concentrated again under reduced pressure to give the title compound (3.3 g, 6.58 mmol, 98% purity).

[0429] Its structural characterization data are as follows:

[0430] MS m / z(ESI): 492.3 [M+H] +

[0431] Step 2: Preparation of 2,5-dioxopyrrolidone-1-yl((benzyloxy)carbonyl)-L-alanine ester (INT-9-4):

[0432] 1-Hydroxypyrrolidine-2,5-dione (386.67 mg, 3.36 mmol) and ((benzyloxy)carbonyl)-L-alanine (0.5 g, 2.24 mmol) were reacted with THF (10 mL), EDCI (644.08 mg, 3.36 mmol), and dichloromethane (10 mL) at room temperature for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was diluted with dichloromethane, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the title compound (710 mg, 2.22 mmol).

[0433] Its structural characterization data are as follows:

[0434] MS m / z (ESI): 343.1 [M+Na] +

[0435] Step 3: Preparation of ((benzyloxy)carbonyl)-L-alanyl-L-alanyl-L-alanine (INT-9-5):

[0436] L-alanyl-L-alanine (322.69 mg, 2.01 mmol) and 2,5-dioxopyrrolidone-1-yl((benzyloxy)carbonyl)-L-alanine ester (710 mg, 2.22 mmol) were mixed with DMF (5 mL), DIPEA (578.63 mg, 4.48 mmol), and water (3 mL) and stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was directly purified by reversed-phase column chromatography (acetonitrile-0.05% trifluoroacetic acid aqueous solution = 0-100%) and then freeze-dried to give the title compound (589 mg, 1.61 mmol).

[0437] Its structural characterization data are as follows:

[0438] MS m / z(ESI): 366.1 [M+H] +

[0439] Step 4: Preparation of benzyl((S)-1-(((S)-1-(((S)-1-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (INT-9-6):

[0440] ((benzyloxy)carbonyl)-L-alanyl-L-alanyl-L-alanine (430 mg, 1.18 mmol) and 4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one (578.68 mg, 1.18 mmol) were dissolved in DMF (5 mL), and DIPEA (176 mg, 1.36 mmol) was added dropwise. HATU (491.93 mg, 1.29 mmol) was added in portions, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate = 0-100%) and then concentrated under reduced pressure to obtain the title compound (0.79 g, 941.50 μmol).

[0441] Its structural characterization data are as follows:

[0442] MS m / z (ESI): 839.3 [M+H] +

[0443] Step 5: Preparation of benzyl ((S)-1-(((S)-1-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (INT-9-7):

[0444] Benzyl((S)-1-(((S)-1-(((S)-1-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (0.79 g, 941.50 μmol) was mixed with DMF (5 mL) and pyridine hydrofluoric acid (746.50 mg, 7.53 mmol) at room temperature for 16 hours. After the reaction was completed, the reaction solution was directly purified by reversed-phase column chromatography (acetonitrile-0.05% trifluoroacetic acid aqueous solution = 0-90%) and then freeze-dried to obtain the title compound (418 mg, 684.61 μmol).

[0445] Its structural characterization data are as follows:

[0446] MS m / z (ESI): 611.2 [M+H] +

[0447] Step Six: Preparation of (S)-2-amino-N-((S)-1-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (INT-9):

[0448] Benzyl((S)-1-(((S)-1-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (418 mg, 684.61 μmol) was dissolved in 20 mL of ethanol by sonication, followed by the addition of palladium on carbon (40 mg, 684.61 μmol), followed by hydrogen purging, and the reaction was carried out at room temperature for 5 hours. After the reaction was completed, the reaction solution was filtered, the filter cake was washed with methanol and concentrated under reduced pressure to obtain the title compound (320 mg, 671.66 μmol).

[0449] Its structural characterization data are as follows:

[0450] MS m / z (ESI): 477.1 [M+H] +

[0451] Example 10: Preparation of intermediates: N 5 -((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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (INT-10)

[0452] Step 1: Allyl N 5 Preparation of -((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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-L-glutamine (INT-10-1)

[0453] (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoic acid (293.92 mg, 717.88 μmol), (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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (540 mg, 717.88 μmol) was dissolved in DMF (7 mL), HATU (327.35 mg, 861.46 μmol) was added, and DIPEA (278.34 mg, 2.15 mmol) was added dropwise. The mixture was stirred for 1 hour. Water was added and stirred to precipitate a solid. The solid was filtered, washed with water, and dried under vacuum. The solid was dissolved in DMF (8 mL), and diethylamine (0.8 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. After vacuum evacuation to remove most of the solvent, the compound was purified by C18 reversed-phase column (eluent: 0-35% acetonitrile / 0.05% formic acid water) and freeze-dried to obtain the title compound (428 mg, 442.42 μmol).

[0454] Its structural characterization data are as follows:

[0455] MS m / z (ESI): 921.4 [M+H] +

[0456] Step 2: Allyl N 5 -((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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (INT-10-2)

[0457] (2,5-dioxopyrrolidone-1-yl)6-(2-methylsulfonylpyrimidin-5-yl)hex-5-acetylacetate (83.09 mg, 227.41 μmol), allyl N 5-((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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-L-glutamine (200 mg, 206.74 μmol) was dissolved in DMF (4 mL), and diisopropylethylamine (80.16 mg, 620.21 μmol) was added dropwise. The mixture was stirred and reacted for 1 hour. The title compound (120 mg, 102.42 μmol) was obtained by preparative high performance liquid chromatography purification followed by freeze drying.

[0458] Its structural characterization data are as follows:

[0459] MS m / z (ESI): 1172.3 [M+H] +

[0460] Its preparation method is as follows:

[0461] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0462] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0463] Step 3: N 5 -((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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (INT-10)

[0464] Allyl N 5-((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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-N 2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (120 mg, 102.42 μmol) was dissolved in DMF (4 mL), and tetrakis(triphenylphosphine)palladium (23.67 mg, 20.48 μmol) and 1,3-dimethylbarbituric acid (23.99 mg, 153.63 μmol) were added. The mixture was stirred for 1 hour under nitrogen purging and protection. After purification by C18 reversed-phase column chromatography (eluent: 0-35% acetonitrile / 0.05% formic acid water), the compound was lyophilized to give the title compound (101 mg, 89.25 μmol).

[0465] Its structural characterization data are as follows:

[0466] MS m / z(ESI): 1132.3 [M+H] +

[0467] Example 11: Preparation of intermediate (S)-2-amino-N-((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (INT-11)

[0468] Step 1: Preparation of ((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)tert-butyl carbamate (INT-11-2)

[0469] (tert-Butoxycarbonyl)-L-alanine (79.97 mg, 422.67 μmol) and (S)-3-amino-8-ethyl-8-hydroxy-2,8,11,14-tetrahydro-12H-cyclopentanopyrano[3',4':6,7]indolo[1,2-b]quinoline-9,12(1H)-dione hydrochloride (30 mg, 70.45 μmol) were dissolved in DMF (3 mL), DIPEA (91.04 mg, 704.45 μmol) was added, followed by HATU (160.61 mg, 422.67 μmol). The reaction was heated to 50 °C and reacted for 48 hours. After the reaction was complete, the reaction solution was added dropwise to water with stirring, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (39 mg, 69.57 μmol).

[0470] Its structural characterization data are as follows:

[0471] MS m / z (ESI): 561.3 [M+H] +

[0472] Step 2: Preparation 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]indolo[1,2-b]quinoline-3-yl)propionamide (INT-11-3)

[0473] Dichloromethane (2 mL) and dioxane hydrochloride solution (4 M, 4 mL) were added to crude ((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)carbamate tert-butyl ester (39 mg, 69.57 μmol) and stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by reversed-phase column chromatography (acetonitrile-0.05% trifluoroacetic acid aqueous solution = 0-90%) and freeze-dried to obtain the trifluoroacetate of the title product (35 mg, 70.43 μmol).

[0474] Its structural characterization data are as follows:

[0475] MS m / z (ESI): 461.2 [M+H] +

[0476] Step 3: Preparation of ((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate tert-butyl carbamate (INT-11-4):

[0477] (tert-Butoxycarbonyl)-L-valine (20 mg, 92.06 μmol) and trifluoroacetate (30 mg, 52.22 μmol, TF) 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]indolo[1,2-b]quinoline-3-yl)propionamide were dissolved in DMF (2 mL), DIPEA (32 mg, 247.60 μmol) was added, followed by HATU (23 mg, 60.53 μmol), and the reaction was stirred at room temperature for 1.5 hours. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried, and concentrated under reduced pressure to obtain the crude product of the title compound (34 mg, 51.54 μmol), which was used directly in the next reaction without purification.

[0478] Its structural characterization data are as follows:

[0479] MS m / z (ESI): 660.3 [M+H] +

[0480] Step 4: Preparation of (S)-2-amino-N-((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadien[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (INT-11)

[0481] Dichloromethane (4 mL) was added to crude ((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentadieno[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate (34 mg, 51.54 μmol) (4 mL), followed by trifluoroacetic acid (2 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was directly concentrated under reduced pressure to remove the solvent. The crude product was dissolved in water, purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%), and then freeze-dried to give the trifluoroacetate of the title compound (27 mg, 40.08 μmol).

[0482] Its structural characterization data are as follows:

[0483] MS m / z (ESI): 560.3 [M+H] +

[0484] Example 1: (S)-N1-((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)-N5-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9- Preparation of ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)pentadiamide (N-1)

[0485] Step 1: Preparation of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N5-((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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-L-glutamic acid allyl ester (N-1-1)

[0486] N-fluorenemethoxycarbonyl-L-glutamic acid 1-allyl ester (209.56 mg, 511.82 μmol), (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 Indo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (385 mg, 511.82 μmol) was dissolved in DMF (5 mL), HATU (233.39 mg, 614.19 μmol) was added, and DIPEA (198.44 mg, 1.54 mmol) was added dropwise. The mixture was stirred for 1 hour. The reaction solution was poured into water to precipitate a solid, which was filtered, washed with water, and dried to obtain the crude product (580 mg, 507.16 μmol), which was used directly in the next reaction.

[0487] Its structural characterization data are as follows:

[0488] MS m / z(ESI): 1144.4 [M+H]+

[0489] Step 2: Preparation of N5-((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]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)-L-glutamic acid allyl ester (N-1-2)

[0490] The N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N5-((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]) Indorazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-L-glutamic acid allyl ester (580 mg, 507.16 μmol) was dissolved in DMF (5 mL), and diethylamine (0.5 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. The reaction solution was directly purified by C18 reverse-phase preparative column (0-30% acetonitrile / formic acid water) and lyophilized to give the title compound (270 mg, 279.09 μmol).

[0491] Its structural characterization data are as follows:

[0492] MS m / z(ESI): 922.4 [M+H]+

[0493] Step 3: Preparation of N5-((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]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamic acid allyl ester (N-1-3)

[0494] (2,5-dioxopyrrolidone-1-yl)6-(2-methylsulfonylpyrimidin-5-yl)hex-5-acetylacetate (101.97 mg, 279.09 μmol), N5-((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]pyran) [3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-L-glutamic acid allyl ester (270 mg, 279.09 μmol) was dissolved in DMF (5 mL), and DIPEA (108.21 mg, 837.28 μmol) was added dropwise. The reaction mixture was stirred for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (115 mg, 98.15 μmol).

[0495] Its structural characterization data are as follows:

[0496] MS m / z(ESI): 1172.3 [M+H]+

[0497] Its preparation method is as follows:

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

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

[0500] Step 4: Preparation of N5-((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]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (N-1-4):

[0501] The N5-((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]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1- (Oxopropane-2-yl)amino)-1-oxopropane-2-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamic acid allyl ester (115 mg, 98.15 μmol) was dissolved in DMF (4 mL), and tetrakis(triphenylphosphine)palladium (22.68 mg, 19.63 μmol) and 1,3-dimethylbarbituric acid (22.99 mg, 147.23 μmol) were added. The mixture was stirred for 1 hour under nitrogen purging and protection. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (93 mg, 82.18 μmol).

[0502] Its structural characterization data are as follows:

[0503] MS m / z(ESI): 1131.3 [M+H]+

[0504] Its preparation method is as follows:

[0505] Column: Sunfire XBridge Prep C18 OBD (5μm*19mm*150mm)

[0506] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0507] Step 5: Preparation of (9H-fluorene-9-yl)methyl((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)carbamate (N-1-5):

[0508] 3-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)ethoxy)propionic acid (297.77 mg, 712.21 μmol), (S)-2-amino-N-((S)-1-(((S)-1-(((((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methyl (Oxy)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propionamide (300 mg, 593.50 μmol) was dissolved in DMF (5 mL), and DIPEA (230.11 mg, 1.78 mmol, 310.13 μL) was added dropwise with stirring. HATU (338.30 mg, 890.26 μmol) was then added, and the reaction was continued for 1 hour. The reaction solution was directly purified by a C18 reverse-phase preparative column (0-30% acetonitrile / formic acid aqueous solution) and lyophilized to give the title compound (337 mg, 399.84 μmol).

[0509] Its structural characterization data are as follows:

[0510] MS m / z(ESI): 843.3 [M+H ]+

[0511] Step Six: Preparation of N-((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)-3-(2-aminoethoxy)propionamide (N-1-6):

[0512] (9H-fluorene-9-yl)methyl((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)carbamate (100 mg, 118.65 μmol) was dissolved in DMF (1 mL), and diethylamine (0.1 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. After removing the solvent under vacuum, the crude product was obtained and directly proceeded to the next step of the reaction.

[0513] Its structural characterization data are as follows:

[0514] MS m / z (ESI): 621.3 [M+H] +

[0515] Step 7: (S)-N1-((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)-N5-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl Preparation of 9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)pentadiamide (N-1):

[0516] N5-((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]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (30mg) 26.51 μmol of N-((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)-3-(2-aminoethoxy)propionamide (16.45 mg, 26.51 μmol) was dissolved in DMF (2 mL). HATU (15.11 mg, 39.77 μmol) was added with stirring, followed by dropwise addition of DIPEA (10.28 mg, 79.53 μmol). The reaction was continued for 1 hour. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (6.08 mg, 3.47 μmol).

[0517] Its structural characterization data are as follows:

[0518] MS m / z(ESI):1735.6[M+H]+,867.9[1 / 2M+H]+

[0519] Its preparation method is as follows:

[0520] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0521] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0522] Example 2: (S)-N5-((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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13- Preparation of 5-(6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)pentadiamide (N-2)

[0523] Step 1: Preparation of (9H-fluorene-9-yl)methyl((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)carbamate (N-2-1):

[0524] 3-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)ethoxy)propionic acid (328.68 mg, 786.13 μmol), (S)-2-amino-N-((S)-1-(((S)-1-(((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2- (320 mg, 655.11 μmol)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propamide (5 mL) was dissolved in DMF, and DIPEA (254.00 mg, 1.97 mmol, 342.32 μL) was added dropwise. HATU (373.41 mg, 982.67 μmol) was then added, and the mixture was stirred for 1 hour. The reaction solution was directly purified by a C18 reverse-phase preparative column (0-30% acetonitrile / formic acid water) and lyophilized to give the title compound (412 mg, 498.89 μmol).

[0525] Its structural characterization data are as follows:

[0526] MS m / z(ESI): 843.3 [M+18]+

[0527] Step 2: Preparation of 3-(2-aminoethoxy)-N-((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)propionamide (N-2-1):

[0528] (9H-fluorene-9-yl)methyl((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)carbamate (100 mg, 121.09 μmol) was dissolved in DMF (1 mL), and diethylamine (0.1 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. After removing the solvent under vacuum, the crude product was obtained and directly proceeded to the next step of the reaction.

[0529] Its structural characterization data are as follows:

[0530] MS m / z (ESI): 604.4 [M+H]+

[0531] Step 3: (S)-N 5-((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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)-N 1 Preparation of -((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamide)pentadiamide (N-2):

[0532] N 5 -((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]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)-N 2 -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (30 mg, 26.51 μmol) was dissolved in DMF (1.5 mL), HATU (15.11 mg, 39.77 μmol) was added, and the mixture was stirred for 10 minutes. Then, 3-(2-aminoethoxy)-N-((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-yl)-L-glutamine (30 mg, 26.51 μmol) was added dropwise. A solution of 20.80 mg (34.46 μmol) of dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)propionamide in DMF (0.5 mL) was added dropwise, followed by stirring and reaction for 1 hour. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (14.88 mg, 8.58 μmol).

[0533] Its structural characterization data are as follows:

[0534] MS m / z(ESI):1717.6[M+H]+,859.0[1 / 2M+H]+

[0535] Its preparation method is as follows:

[0536] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0537] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0538] Example 3: N-((6S,9S,12S,22S,34S,37S,40S)-46-(((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]indolazino[1,2-b]quinoline-1-yl)amino)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4) Preparation of -dihydropyrimidindol-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12,34,37,40-hexamethyl-5,8,11,14,21,28,32,35,38,41,46-undecoxo-2,17,30,44-tetraoxa-4,7,10,13,20,27,33,36,39,42-decazahexadecane-22-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (N-3)

[0539] Step 1: Preparation of (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]indolo[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentoxo-3,17-dioxa-5,8,11,14-tetraazanonadecan-19-acid (N-3-1):

[0540] The (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]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl) (Amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (230 mg, 305.77 μmol), diethylene glycol (82.00 mg, 611.53 μmol), and HATU (174.29 mg, 458.65 μmol) were dissolved in DMF (10 mL), and DIPEA (118.55 mg, 917.30 μmol) was added dropwise. The mixture was stirred for 1 hour. The reaction solution was directly purified by C18 reverse-phase preparative column (0-35% acetonitrile / formic acid water) and lyophilized to give the title compound (172 mg, 198.09 μmol).

[0541] Its structural characterization data are as follows:

[0542] MS m / z(ESI): 868.3 [M+18]+

[0543] Step 2: Preparation of (9H-fluorene-9-yl)methyl((6S,9S,12S,22S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-22-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5,8,11,14,21-pentoxo-2,17-dioxa-4,7,10,13,20-pentazahexacosane-26-yl)carbamate (N-3-2):

[0544] N6-(((9H-fluorene-9-yl)methoxy)carbonyl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (141.45 mg, 228.63 μmol), 3-(2-aminoethoxy)-N-((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)- 3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)propionamide (138 mg, 228.63 μmol) and HATU (130.32 mg, 342.94 μmol) were dissolved in DMF (3 mL), and DIPEA (88.64 mg, 685.89 μmol) was added dropwise. The mixture was stirred for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (145 mg, 120.40 μmol).

[0545] Its structural characterization data are as follows:

[0546] MS m / z (ESI): 1205.4 [M+H]+

[0547] Its preparation method is as follows:

[0548] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0549] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0550] Step 3: Preparation of N-((6S,9S,12S,22S)-26-amino-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14,21-pentoxo-2,17-dioxa-4,7,10,13,20-pentazahexacosane-22-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylenamide (N-3-3):

[0551] (9H-fluorene-9-yl)methyl((6S,9S,12S,22S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-22-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-5,8,11,14,21-pentoxo-2,17-dioxa-4,7,10,13,20-pentazahexacosane-26-yl)carbamate (32 mg, 26.57 μmol) was dissolved in DMF (2 mL), and diethylamine (0.2 mL) was added dropwise with stirring. The reaction was stirred for 0.5 hours. After removing the solvent under vacuum, the crude product was obtained and directly proceeded to the next step of the reaction.

[0552] Its structural characterization data are as follows:

[0553] MS m / z(ESI): 983.3 [M+H]+

[0554] Step 4: N-((6S,9S,12S,22S,34S,37S,40S)-46-(((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]indolazino[1,2-b]quinoline-1-yl)amino)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-) Preparation of dihydropyrimidindol-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12,34,37,40-hexamethyl-5,8,11,14,21,28,32,35,38,41,46-undecoxo-2,17,30,44-tetraoxa-4,7,10,13,20,27,33,36,39,42-decazahexadecane-22-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (N-3)

[0555] N-((6S,9S,12S,22S)-26-amino-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14,21-pentoxo-2,17-dioxa-4,7,10,13,20-pentazahexacosane-22-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide (26 mg, 26.48 μmol), (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]indolo[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentoxo-3,17-dioxa-5,8,11,14-tetraazanonadecano-19-acid (22.99 mg, 26.48 μmol) was dissolved in DMF (2 mL), HATU (15.09 mg, 39.71 μmol) was added, and DIPEA (10.27 mg, 79.43 μmol) was added dropwise. The reaction mixture was stirred for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (5.87 mg, 3.04 μmol).

[0556] Its structural characterization data are as follows:

[0557] MS m / z(ESI):1832.8[M+H]+,917.0[1 / 2M+H]+

[0558] Its preparation method is as follows:

[0559] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0560] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0561] Example 4: N-((6S,9S,12S,22S,34S,37S,40S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-46-(((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', Preparation of [4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-6,9,12,34,37,40-hexamethyl-5,8,11,14,21,28,32,35,38,41,46-undecoxo-2,17,30,44-tetraoxa-4,7,10,13,20,27,33,36,39,42-decaza-hexadecane-22-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide (N-4)

[0562] Step 1: Preparation of N-((6S,9S,12S,22S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14,21-pentoxo-28,28-diphenyl-28-(p-tolyl)-2,17-dioxa-4,7,10,13,20,27-hexaazaoctacosan-22-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylenamide (N-4-1):

[0563] N6-(diphenyl(p-tolyl)methyl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (92.14 mg, 141.15 μmol), (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-benzene) [de]pyrano[3',4′:6,7]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (73 mg, 117.63 μmol) was dissolved in DMF (2 mL), HATU (67.05 mg, 176.44 μmol) was added, and DIPEA (45.61 mg, 352.88 μmol) was added dropwise. The mixture was stirred for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (63 mg, 50.18 μmol).

[0564] Its structural characterization data are as follows:

[0565] MS m / z (ESI): 1256.5 [M+H] +

[0566] Its preparation method is as follows:

[0567] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0568] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0569] Step 2: Preparation of N-((6S,9S,12S,22S)-26-amino-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14,21-pentoxo-2,17-dioxa-4,7,10,13,20-pentazahexacosane-22-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamide (N-4-2):

[0570] N-((6S,9S,12S,22S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14,21-pentoxo-28,28-diphenyl-28-(p-tolyl)-2,17-dioxa-4,7,10,13,20,27-hexaazaoctacosan-22-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylenamide (20 mg, 15.93 μmol) was dissolved in acetonitrile (1 mL), and formic acid (0.5 mL) was added dropwise with stirring. The reaction was continued for 48 hours. After removing the solvent under vacuum, the crude product was obtained and directly proceeded to the next step of the reaction.

[0571] Its structural characterization data are as follows:

[0572] MS m / z(ESI): 999.4 [M+H]+

[0573] Step 3: N-((6S,9S,12S,22S,34S,37S,40S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-46-(((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] Preparation of [6,7]indolazino[1,2-b]quinoline-1-yl)amino)-6,9,12,34,37,40-hexamethyl-5,8,11,14,21,28,32,35,38,41,46-undecoxo-2,17,30,44-tetraoxa-4,7,10,13,20,27,33,36,39,42-decazatetrazol-22-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide (N-4)

[0574] (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]indolo[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentoxo-3,17-dioxa-5,8,11,14-tetraazanonadecano-19-acid (15 mg, 17.28 μmol) was dissolved in DMF (2 mL), and HATU (9.85 mg, 25.91 μmol) was added with stirring. The reaction was continued for 10 minutes. Add DIPEA (6.70 mg, 51.83 μmol) dropwise, then add 2 mL of DMF solution containing N-((6S,9S,12S,22S)-26-amino-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14,21-pentoxo-2,17-dioxa-4,7,10,13,20-pentazahexadecane-22-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamide (18.05 mg, 17.28 μmol), and continue the reaction for 1 hour. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (5.63 mg, 3.01 μmol).

[0575] Its structural characterization data are as follows:

[0576] MS m / z(ESI):1849.4[M+H]+,925.3[1 / 2M+H]+

[0577] Its preparation method is as follows:

[0578] Column: Waters XBridge Prep C18 OBD (5μm*19mm*150mm)

[0579] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0580] Example 5: 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]indolo[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-1-( Preparation of (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-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-3,6,9,12,19,22-hexaoxo-2,5,8,11,18,21-hexaazatrisane-23-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-14)

[0581] Step 1: Preparation of (9H-fluorene-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]phthalazin-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-17-yl)carbamate (A-14-1)

[0582] (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-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propamide (50 mg, 80.31 μmol) and N 6 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (49.68 mg, 80.31 μmol) was dissolved in DMF (1 mL), and HATU (30.52 mg, 80.31 μmol) and DIPEA (20.76 mg, 160.61 μmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution did not require further treatment and was used directly as a solution for the next reaction.

[0583] Its structural characterization data are as follows:

[0584] MS m / z (ESI): 1223.5 [M+H] +

[0585] Step 2: 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-tetraazaheptadecane-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamide (A-14-2)

[0586] DMF (1 mL) and diethylamine (70.70 mg, 966.69 μmol) were added to the reaction solution from the previous step, and the mixture was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was partially purified by lyophilization (acetonitrile-0.05% ammonium bicarbonate aqueous solution = 0-90%) and then freeze-dried to obtain the title compound (35 mg, 34.96 μmol).

[0587] Its structural characterization data are as follows:

[0588] MS m / z (ESI): 1001.4 [M+H] +

[0589] Step 3: (9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)amino)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2) Preparation of ,4-trifluoromethyl)azol-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-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-3,6,9,12,19,22,25,28,31,36-decaoxo-34-oxa-2,5,8,11,18,23,26,29,32-nonazahexahexadecane-20-yl)carbamate (A-14-3)

[0590] 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-Tetraazaheptadecane-13-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide (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, and the mixture was stirred at room temperature for 0.5 hours. Then N was added. 2 -(((9H-fluorene-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]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-L-asparagine (35 mg, 34.96 μmol), stirred at room temperature for 1.5 hours. PyBOP (27.29 mg, 52.44 μmol) and DIPEA (18.07 mg, 139.85 μmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was used directly as a solution for the next reaction.

[0591] Its structural characterization data are as follows:

[0592] MS m / z(ESI): 1037.0 [M / 2+H] +

[0593] Step 4: (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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)-N 1 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-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-17-yl)succinamide (A-14-4)

[0594] DMF (4 mL) and diethylamine (50.81 mg, 694.78 μmol) were added to the reaction solution from the previous step, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was partially desolvated using a lyophilizer, 1 drop of formic acid was added, and the solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (21 mg, 11.07 μmol).

[0595] Its structural characterization data are as follows:

[0596] MS m / z (ESI): 1852.5 [M+H] +

[0597] Its preparation method is as follows:

[0598] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0599] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0600] Step 5: 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]indolo[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-1-( Preparation of (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-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-3,6,9,12,19,22-hexaoxo-2,5,8,11,18,21-hexaazatrisane-23-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-14)

[0601] (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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-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-pyrido[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-17-yl)succinamide (20.49 mg, 11.07 μmol) The compound was dissolved in DMF (2 mL), and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (27.77 mg, 55.37 μmol) was added. Then DIPEA (24 mg, 185.7 μmol) was added, and the mixture was stirred at room temperature. 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 (6.63 mg, 2.84 μmol).

[0602] Its structural characterization data are as follows:

[0603] MS m / z (ESI): 1119.3 [M / 2+H] +

[0604] Its preparation method is as follows:

[0605] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0606] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0607] Example 6: 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]indolo[1,2-b]quinoline-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 Preparation of 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-tetraazatridecane-13-yl)-7,10,13-trimethyl-16-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,6,9,12,15,22,25-heptaoxo-3-oxa-5,8,11,14,21,24-hexaazahexacosane-26-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-15)

[0608] Step 1: Preparation of (9H-fluorene-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]indolo[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-16-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazaeicosaecan-20-yl)carbamate (A-15-1)

[0609] N 6 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (25 mg, 40.41 μmol) was dissolved in DMF (2 mL), HATU (16.89 mg, 44.45 μmol) and DIPEA (15.67 mg, 121.22 μmol) were added, and the mixture was stirred for 15 minutes. Then (S)-2-amino-N-((S)-1-(((S)-1-(((2-(((1S,9S)-5)-L-lysine) was added. 32.25 mg, 40.41 μmol of 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]indolo[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (32.25 mg, 40.41 μmol) was stirred at room temperature for 45 minutes. After the reaction was complete, the reaction solution was used directly in the next step of the reaction.

[0610] Its structural characterization data are as follows:

[0611] MS m / z (ESI): 1353.7 [M+H] +

[0612] Step 2: 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]indolo[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazaeicosaecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (A-15-2)

[0613] DMF (2 mL) and diethylamine (106.05 mg, 1.45 mmol) were added to the reaction solution from the previous step, and the mixture was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%) and then freeze-dried to obtain the title compound (26 mg, 22.10 μmol).

[0614] Its structural characterization data are as follows:

[0615] MS m / z (ESI): 1130.5 [M+H] +

[0616] Step 3: N 2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N 4 Preparation of -((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-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-L-asparagine (A-15-3)

[0617] (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-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (30 mg, 48.18 μmol) was dissolved in DMF (1 mL), DIPEA (16 mg, 123.80 μmol) was added, and then (9H-fluorene-9-yl)methyl(S)-(2,5-dioxotetrahydrofuran-3-yl)carbamate (17.88 mg, 53.00 μmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was directly purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%) and then freeze-dried to obtain the title compound (40 mg, 39.76 μmol).

[0618] Its structural characterization data are as follows:

[0619] MS m / z (ESI): 960.3 [M+H] +

[0620] Step 4: (9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)amino)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2) Preparation of ,4-trifluoromethyl)azol-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-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-3,6,9,12,15,22,25,28,31,36-decaoxo-34-oxa-2,5,8,11,16,23,26,29,32-nonazahexahexadecane-14-yl)carbamate (A-15-4)

[0621] 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]indolo[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazaeicosano-16-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (25 mg, 21.25 μmol) and N 2 -(((9H-fluorene-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-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-L-asparagine (23.51 mg, 23.37 μmol) was dissolved in DMF (2 mL), DIPEA (16 mg, 123.80 μmol) was added, and the mixture was stirred at room temperature for 10 minutes. Then, PyBOP (13.27 mg, 25.50 μmol) was added, and the reaction was continued with stirring for 1 hour. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (13 mg, 6.27 μmol).

[0622] Its structural characterization data are as follows:

[0623] MS m / z (ESI): 1036.6 [M / 2+H] +

[0624] Its preparation method is as follows:

[0625] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0626] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0627] Step 5: (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]indolo[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-16-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazaeicosano-20-yl)-N 4Preparation of -((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-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)succinamide (A-15-5)

[0628] The (9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)amino)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-trifluoromethyl)azol-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-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-3,6,9,12,15,22,25,28,31,36-decaoxo-34-oxa-2,5,8,11,16,23,26,29,32-nonazahexahexadecane-14-yl)carbamate (13 mg, 6.27 μmol) was dissolved in DMF (2 mL), and diethylamine (14.14 mg, 193.34 μmol) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was directly lyophilized to remove the solvent and used directly for the next reaction without further treatment.

[0629] Its structural characterization data are as follows:

[0630] MS m / z(ESI):935.5[(M+H2O) / 2+H] +

[0631] Step Six: 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]indolo[1,2-b]quinoline-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, Preparation of 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-tetraazatridecane-13-yl)-7,10,13-trimethyl-16-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)-1,6,9,12,15,22,25-heptaoxo-3-oxa-5,8,11,14,21,24-hexaazahexacosane-26-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-15)

[0632] DMF (1 mL) was added to the reaction solution from the previous step, followed by DIPEA (16 mg, 123.80 μmol) and 2,2',2”-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (20 mg, 39.88 μmol). The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was partially desolventized using a lyophilizer, purified by preparative high-performance liquid chromatography, and then freeze-dried to obtain the title compound (6.09 mg, 2.59 μmol).

[0633] Its structural characterization data are as follows:

[0634] MS m / z (ESI): 1119.1 [M / 2+H] + 746.4 [M / 3+H] +

[0635] Its preparation method is as follows:

[0636] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0637] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0638] Example 7: (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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)-N1-((4S,7S,10S,13S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo Preparation of 8,9-dihydro-3H-p-pyrimidino[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamidinamide)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-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)propamidyl)succinamide (A-16)

[0639] (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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-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-pyrido[4,3,2-de]phthalazine-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-17-yl)succinamide formate (11 mg, 5.80 μmol) and 3 DMF (1 mL) was added to 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)propionic acid (6.16 mg, 5.80 μmol), followed by the addition of DIPEA (749.67 μg, 5.80 μmol), and then HATU (2.20 mg, 5.80 μmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was purified directly by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (7.36 mg, 2.59 μmol, 98% purity).

[0640] Its structural characterization data are as follows:

[0641] MS m / z(ESI): 1395.0 [M / 2+H] + 930.3 [M / 3+H] +

[0642] Its preparation method is as follows:

[0643] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0644] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0645] Example 8: (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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)-2-(3-(3-(3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis( ... Preparation of hydroxy-2-(hydroxyoxymethyl)prop-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propamidyl)-N1-((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-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-17-yl)succinamide (A-17)

[0646] (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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-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-pyrido[4,3,2-de]phthalazin-2(7H)-yl)-4,7,10-trimethyl-13-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-17-yl)succinamide formate (11 mg, DMF (1 mL) was added to 5.80 μmol of 1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)-2,2-bis((3-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)-3-oxopropoxy)methyl)propoxy)propionic acid (4.73 mg, 5.80 μmol), followed by the addition of DIPEA (749.67 μg, 5.80 μmol), and then HATU (2.20 mg, 5.80 μmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was purified directly by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (8.83 mg, 3.37 μmol, 98% purity).

[0647] Its structural characterization data are as follows:

[0648] MS m / z (ESI): 1283.1 [M / 2+H] + 856.2 [M / 3+H] +

[0649] Its preparation method is as follows:

[0650] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0651] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0652] Example 9: (S)-N 1-((6S,9S,12S,22S,34S,37S,40S)-1,45-bis((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12,34,37,40-hexamethyl-5,8,11,14,21,25,32,35,38,41-decaoxo-2,17,29,44-tetraoxa-4,7,10,13,20,26,33,36,39,42-decaazatrapentadecane-22-yl)-N 5 Preparation of -((7S,10S,13S)-1-(((1R,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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamido)pentadiamide (O-1)

[0653] Step 1: Preparation of (9H-fluorene-9-yl)methyl((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)carbamate (O-1-1)

[0654] (S)-2-amino-N-((S)-1-(((S)-1-(((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propionamide (INT-4) (206 mg, 421.73 μmol) and 3-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)ethoxy)propionic acid (194.84 mg, 548.25 μmol) were dissolved in DMF (2 mL), and then DIPEA (163.51 mg, 1.27 mmol) and HATU (208.46 mg, 548.25 μmol) were added and reacted at room temperature for two hours. After the reaction was completed, the reaction solution was purified by reversed-phase column chromatography (acetonitrile-0.05% ammonium bicarbonate aqueous solution = 0-60%) and then freeze-dried to obtain the title compound (254 mg, 292.19 μmol).

[0655] Its structural characterization data are as follows:

[0656] MS m / z (ESI): 826.3 [M+H] +

[0657] Step 2: Preparation of 3-(2-aminoethoxy)-N-((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)propionamide (O-1-2)

[0658] (9H-fluorene-9-yl)methyl((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)carbamate (254 mg, 292.19 μmol) was dissolved in DMF (2 mL), and then diethylamine (292.19 μmol, 0.5 mL) was added. The mixture was reacted at room temperature for one hour. After the reaction was complete, the reaction solution was freeze-dried to obtain the crude product of the title compound.

[0659] Its structural characterization data are as follows:

[0660] MS m / z (ESI): 602.3 [MH] +

[0661] Step 3: Preparation of (9H-fluorene-9-yl)methyl((6S,9S,12S,22S,34S,37S,40S)-1,45-bis((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12,34,37,40-hexamethyl-5,8,11,14,21,25,32,35,38,41-decaoxo-2,17,29,44-tetraoxa-4,7,10,13,20,26,33,36,39,42-decaazatriapentadecan-22-yl)carbamate (O-1-3)

[0662] Crude 3-(2-aminoethoxy)-N-((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)propionamide and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-glutamic acid (53.85 mg, 145.79 μmol) were dissolved in DMF, and then DIPEA (113.05 mg, 874.76 μmol) and HATU (110.87 mg, 291.59 μmol) were added and reacted at room temperature for two hours. After the reaction was completed, the reaction solution was purified by reversed-phase column chromatography (acetonitrile-0.05% ammonium bicarbonate aqueous solution = 0-50%) and then freeze-dried to obtain the title compound (154 mg, 94.97 μmol).

[0663] Its structural characterization data are as follows:

[0664] MS m / z (ESI): 1539.7 [MH] +

[0665] Step 4: (S)-2-amino-N 1 N 5 Preparation of bis((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)pentanediamide (O-1-4)

[0666] The (9H-fluorene-9-yl)methyl((6S,9S,12S,22S,34S,37S,40S)-1,45-bis((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12,34,37,40-hexamethyl-5,8,11,14,21, 25,32,35,38,41-decoxo-2,17,29,44-tetraoxa-4,7,10,13,20,26,33,36,39,42-decazatriapentadecane-22-yl)carbamate (30 mg, 19.47 μmol) was dissolved in DMF (2 mL), and then diethylamine (7.12 mg, 97.37 μmol) was added. The mixture was reacted at room temperature for one hour. After the reaction was complete, the reaction solution was freeze-dried to give the crude title compound.

[0667] Its structural characterization data are as follows:

[0668] MS m / z (ESI): 1317.5 [MH] +

[0669] Step 5: (S)-N 1 -((6S,9S,12S,22S,34S,37S,40S)-1,45-bis((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12,34,37,40-hexamethyl-5,8,11,14,21,25,32,35,38,41-decaoxo-2,17,29,44-tetraoxa-4,7,10,13,20,26,33,36,39,42-decaazatrapentadecane-22-yl)-N 5 Preparation of -((7S,10S,13S)-1-(((1R,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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamido)pentadiamide (O-1)

[0670] (S)-2-amino-N 1 N 5-Bis((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)pentanediamide (O-1-4) crude product and N 5 -((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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-N 2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (25.75 mg, 22.76 μmol) was dissolved in DMF (2 mL), and DIPEA (7.35 mg, 56.89 μmol) was added and stirred for five minutes. Then HATU (8.65 mg, 22.76 μmol) was added and the mixture was reacted at room temperature for two hours. 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 (14.01 mg, 5.53 μmol).

[0671] Its structural characterization data are as follows:

[0672] MS m / z (ESI): 1216.4 [M / 2+H] +

[0673] Its preparation method is as follows:

[0674] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0675] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0676] Example 10: (S)-N 1 N 5-bis((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)-2-((7S,10S,13S,18S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl) Preparation of methylethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-18-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazanonadecan-19-amido)pentadiamide (O-2)

[0677] Step 1: Preparation of (9H-fluorene-9-yl)methyl((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)carbamate (O-2-1)

[0678] 3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)ethoxy]propionic acid (297.77 mg, 712.21 μmol), (S)-2-amino-N-((S)-1-(((S)-1-(((((2S,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl (300 mg, 593.50 μmol)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propamide (5 mL) was dissolved in DMF. Diisopropylethylamine (230.11 mg, 1.78 mmol, 310.13 μL) was added dropwise with stirring, followed by HATU (338.30 mg, 890.26 μmol). The reaction was continued for 1 hour. The solution was purified by C18 reversed-phase column chromatography (eluent: 0-40% acetonitrile / 0.05% formic acid aqueous solution) and lyophilized to give the title compound (337 mg, 399.84 μmol).

[0679] Its structural characterization data are as follows:

[0680] MS m / z (ESI): 843.3 [M+H] +

[0681] Step 2: Preparation of N-((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)-3-(2-aminoethoxy)propionamide (O-2-2)

[0682] (9H-fluorene-9-yl)methyl((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)carbamate (80 mg, 94.92 μmol) was dissolved in DMF (1 mL), and diethylamine (0.1 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. Water and ethyl acetate were added and stirred. The mixture was allowed to stand and separated. The aqueous phase was freeze-dried to give the crude product of the title compound (51 mg, 82.18 μmol).

[0683] Its structural characterization data are as follows:

[0684] MS m / z (ESI): 621.3 [M+H] +

[0685] Step 3: Preparation of (9H-fluorene-9-yl)methyl((6S,9S,12S,22S,34S,37S,40S)-1,45-bis((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12,34,37,40-hexamethyl-5,8,11,14,21,25,32,35,38,41-decaoxo-2,17,29,44-tetraoxa-4,7,10,13,20,26,33,36,39,42-decaazatriapentadecan-22-yl)carbamate (O-2-3)

[0686] N-((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)-3-(2-aminoethoxy)propionamide (50.24 mg, 80.95 μmol) and (2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)glutaric acid (13 mg, 35.20 μmol) were dissolved in DMF (2 mL), HATU (40.12 mg, 105.59 μmol) was added, and DIPEA (4.55 mg, 35.20 μmol) was added dropwise. The mixture was stirred and reacted for 1 hour. After removing the solvent under vacuum, the compound was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (25 mg, 15.88 μmol).

[0687] Its structural characterization data are as follows:

[0688] MS m / z (ESI): 1575.5 [M+H] +

[0689] Its preparation method is as follows:

[0690] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0691] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0692] Step 4: Preparation of (S)-2-amino-N1,N5-bis((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)pentanediamide (O-2-4)

[0693] The (9H-fluorene-9-yl)methyl((6S,9S,12S,22S,34S,37S,40S)-1,45-bis((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12,34,37,40-hexamethyl-5,8,11,14, 21,25,32,35,38,41-decazo-2,17,29,44-tetraoxa-4,7,10,13,20,26,33,36,39,42-decazatrapentadecane-22-yl)carbamate (25 mg, 15.88 μmol) was dissolved in DMF (1 mL), and diethylamine (0.1 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. After removing the solvent under vacuum, the next reaction was carried out directly.

[0694] Its structural characterization data are as follows:

[0695] MS m / z (ESI): 1352.3 [M+H] +

[0696] Step 5: (S)-N 1 N 5 -bis((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)-2-((7S,10S,13S,18S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl) Preparation of methylethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-18-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazanonadecan-19-amido)pentadiamide (O-2)

[0697] (S)-2-amino-N1,N5-bis((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazanonadecan-19-yl)pentanediamide (21 mg, 15.53 μmol), N5-((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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-N2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (17.57 mg, 15.53 μmol), HATU (7.08 mg, 18.63 μmol) were dissolved in DMF (1 mL), and diisopropylethylamine (6.02 mg, 46.59 μmol) was added dropwise. The mixture was stirred and reacted for 1 hour. The pH was adjusted to 5 by adding 0.5N hydrochloric acid aqueous solution, and the solvent was removed by vacuum extraction. After purification by preparative high performance liquid chromatography, the compound was freeze-dried to obtain the title compound (5.45 mg, 2.17 μmol).

[0698] Its structural characterization data are as follows:

[0699] MS m / z (ESI): 1233.5 [1 / 2M+H] + 822.6 [1 / 3M+H] +

[0700] Its preparation method is as follows:

[0701] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0702] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0703] Example 11: (S)-N 1-((2S,5S,8S,18S,30S,33S,36S)-1,37-bis((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2,5,8,30,33,36-hexamethyl-1,4,7,10,17,21,28,31,34,37-decaoxy-13,25-dioxa-3,6,9,16,22,29,32,35-octaazaheptadecane-18-yl)-N 5 Preparation of -((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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)pentadiamide (O-3)

[0704] Step 1: Preparation of (9H-fluorene-9-yl)methyl((2S,5S,8S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2,5,8-trimethyl-1,4,7,10-tetraoxo-13-oxa-3,6,9-triazapentadecan-15-yl)carbamate (O-3-1)

[0705] 3-[2-(9H-fluorene-9-ylmethoxycarbonylamino)ethoxy]propionic acid (84.18 mg, 165.82 μmol), (S)-2-amino-N-((S)-1-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (79 mg, 165.82 μmol, FR) were dissolved in DMF (2 mL), HATU (94.52 mg, 248.72 μmol) was added, and diisopropylethylamine (64.29 mg, 497.45 μmol, 86.64 μL) was added dropwise. The mixture was stirred and reacted for 1 hour. The title compound (77 mg, 94.62 μmol) was obtained by preparative high performance liquid chromatography purification followed by freeze drying.

[0706] Its structural characterization data are as follows:

[0707] MS m / z (ESI): 814.3 [M+H] +

[0708] Its preparation method is as follows:

[0709] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0710] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0711] Step 2: Preparation of 3-(2-aminoethoxy)-N-((S)-1-(((S)-1-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (O-3-2)

[0712] (9H-fluorene-9-yl)methyl((2S,5S,8S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2,5,8-trimethyl-1,4,7,10-tetraoxo-13-oxa-3,6,9-triazapentadecan-15-yl)carbamate (77 mg, 94.62 μmol) was dissolved in DMF (1 mL), and diethylamine (0.1 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. The solvent was removed under vacuum, water and ethyl acetate were added and stirred, and the mixture was allowed to stand and separated. The aqueous phase was freeze-dried to obtain the crude product of the title compound (55 mg, 92.97 μmol).

[0713] Its structural characterization data are as follows:

[0714] MS m / z(ESI): 592.2 [M+H] +

[0715] Step 3: Preparation of (9H-fluorene-9-yl)methyl((2S,5S,8S,18S,30S,33S,36S)-1,37-bis((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2,5,8,30,33,36-hexamethyl-1,4,7,10,17,21,28,31,34,37-decaoxo-13,25-dioxa-3,6,9,16,22,29,32,35-octaazaheptadecane-18-yl)carbamate (O-3-3):

[0716] 3-(2-aminoethoxy)-N-((S)-1-(((S)-1-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionyl Amine (55 mg, 92.97 μmol) and (2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)glutaric acid (15 mg, 40.61 μmol, FR) were dissolved in DMF (1 mL), HATU (46.30 mg, 121.83 μmol) was added, and diisopropylethylamine (15.75 mg, 121.83 μmol, 21.22 μL) was added dropwise. The mixture was stirred and reacted for 1 hour. After purification by preparative high-performance liquid chromatography, the compound was freeze-dried to obtain the title compound (30 mg, 19.78 μmol).

[0717] Its structural characterization data are as follows:

[0718] MS m / z (ESI): 1517.4 [M+H] + 758.9 [1 / 2M+H] +

[0719] Its preparation method is as follows:

[0720] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0721] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0722] Step 4: (S)-2-amino-N 1 N 5Preparation of bis((2S,5S,8S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2,5,8-trimethyl-1,4,7,10-tetraoxo-13-oxa-3,6,9-triazapentadecan-15-yl)pentanediamide (O-3-4):

[0723] (9H-fluorene-9-yl)methyl((2S,5S,8S,18S,30S,33S,36S)-1,37-bis((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2,5,8,30,33,36-hexamethyl- 1,4,7,10,17,21,28,31,34,37-decaoxo-13,25-dioxa-3,6,9,16,22,29,32,35-octaazaheptadecane-18-yl)carbamate (30 mg, 19.78 μmol) was dissolved in DMF (1 mL), and diethylamine (0.1 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. The solvent was removed under vacuum, and water and ethyl acetate were added and stirred. The mixture was allowed to stand and separated. The aqueous phase was freeze-dried to give the crude product of the title compound (25 mg, 19.32 μmol).

[0724] Its structural characterization data are as follows:

[0725] MS m / z(ESI): 1294.2 [M+H] + 647.8 [1 / 2M+H] +

[0726] Step 5: (S)-N 1 -((2S,5S,8S,18S,30S,33S,36S)-1,37-bis((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2,5,8,30,33,36-hexamethyl-1,4,7,10,17,21,28,31,34,37-decaoxy-13,25-dioxa-3,6,9,16,22,29,32,35-octaazaheptadecane-18-yl)-N 5Preparation of -((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]indolazino[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)pentadiamide (O-3)

[0727] (S)-2-amino-N 1 N 5 -Bis((2S,5S,8S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2,5,8-trimethyl-1,4,7,10-tetraoxo-13-oxa-3,6,9-triazapentadecan-15-yl)pentanediamide (25 mg, 19.32 μmol), N 5 -((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]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)-N 2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-glutamine (21.86 mg, 19.32 μmol) was dissolved in DMF (1 mL), HATU (8.81 mg, 23.18 μmol) was added, and diisopropylethylamine (7.49 mg, 57.95 μmol, 10.09 μL) was added dropwise. The mixture was stirred for 1 hour. After purification by preparative high-performance liquid chromatography, the compound was freeze-dried to obtain the title compound (9.65 mg, 3.93 μmol).

[0728] Its structural characterization data are as follows:

[0729] MS m / z (ESI): 803.3 [1 / 3M+H] + 1204.0 [1 / 2M+H] + 1605.8 [2 / 3M+H] +

[0730] Its preparation method is as follows:

[0731] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0732] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0733] Example 12: (S)-N5-((6S,9S,12S,26S,29S,32S)-1,37-bis((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-19-((6S,9S,12S)-1-((2R,3R,5) R)-5-(4-amino-2-oxo)limidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazaoctadecane-18-yl)-6,9,12,26,29,32-hexamethyl-5 ,8,11,14,24,27,30,33-octaoxo-2,17,21,36-tetraoxa-4,7,10,13,25,28,31,34-octaazaheptadecane-19-yl)-N1-((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,1 Preparation of 2,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolazido[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)pentanediamide (P-1)

[0734] Step 1: Preparation of (2S,5S,8S)-13,13-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-olate (P-1-1):

[0735] The (S)-5-(((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]indolazino[1,2-b]quinoline-3-yl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-4-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5 5-oxopentanoic acid (13 mg, 13.84 μmol) and di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxo))dipropionate (8 mg, 15.82 μmol) were dissolved in DMF (1 mL), and DIPEA (4 mg, 30.95 μmol) and HATU (5.79 mg, 15.23 μmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was added dropwise to water with stirring. A solid precipitated out, and the solid was filtered and dried to obtain the title compound (19 mg, 13.32 μmol).

[0736] Its structural characterization data are as follows:

[0737] MS m / z (ESI): 1427.6 [M+H] +

[0738] Step 2: Preparation of (2S,5S,8S)-13,13-bis((2-carboxyethoxy)methyl)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-acid (P-1-2):

[0739] (2S,5S,8S)-13,13-bis((3-(tert-butoxy)-3-oxopropoxy)methyl)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-oic acid tert-butyl ester (19 mg, 13.32 μmol) was dissolved in trifluoroacetic acid (2 mL) and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove trifluoroacetic acid, and then purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%) and freeze-dried to obtain the title compound (16 mg, 12.72 μmol).

[0740] Its structural characterization data are as follows:

[0741] MS m / z (ESI): 1259.4 [M+H] +

[0742] Step 3: (S)-N5-((6S,9S,12S,26S,29S,32S)-1,37-bis((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-19-((6S,9S,12S)-1-((2R,3R,5R) )-5-(4-amino-2-oxo)limidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9,12-trimethyl-5,8,11,14-tetraoxo-2,17-dioxa-4,7,10,13-tetraazaoctadecane-18-yl)-6,9,12,26,29,32-hexamethyl-5, Preparation of 8,11,14,24,27,30,33-octaoxo-2,17,21,36-tetraoxa-4,7,10,13,25,28,31,34-octaazaheptadecane-19-yl)-N1-((S)-1-(((S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentanopyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamino)pentanediamide (P-1)

[0743] (S)-2-amino-N-((S)-1-(((S)-1-(((((2S,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propionamide (18 mg, 36.62 μmol) and (2S,5S,8S)-13,13-bis((2-carboxyethoxy)methyl)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14) -Hexahydro-11H-cyclopentanopyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-acid (9 mg, 7.15 μmol) was dissolved in DMF (1 mL), DIPEA (3.71 mg, 28.71 μmol) was added dropwise, and HATU (10 mg, 26.32 μmol) was added in portions. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the solvent was removed by a freeze dryer, the reaction solution was purified by preparative high performance liquid chromatography, and then freeze-dried. Then, 0.05% trifluoroacetic acid aqueous solution was added and freeze-dried again to obtain the title compound (13.43 mg, 4.44 μmol).

[0744] Its structural characterization data are as follows:

[0745] MS m / z (ESI): 1361.1 [M / 2+H] + 907.7 [M / 3+H] +

[0746] Its preparation method is as follows:

[0747] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0748] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0749] Example 13: (S)-N5-((2S,5S,8S,22S,25S,28S)-1,29-bis((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-15-((7S,10S,13S)-14-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-7,10,13-trimethyl-5,8,11,14-tetraoxo-2-oxa-6,9,12-triazatetradecyl)-2,5,8,22,2 Preparation of 5,28-hexamethyl-1,4,7,10,20,23,26,29-octaoxo-13,17-dioxa-3,6,9,21,24,27-hexaazanonadecan-15-yl)-N1-((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]indolazino[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)pentadiamide (P-2)

[0750] Step 1: Preparation of (2S,5S,8S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4′:6,7]indolazino[1,2-b]quinoline-3-yl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,4,7,11-tetraoxo-13,13-bis((3-oxo-3-(perfluorophenoxy)propoxy)methyl)-15-oxa-3,6,12-triazaoctadecane-18-acid pentafluorophenol ester (P-2-1):

[0751] The (2S,5S,8S)-13,13-bis((2-carboxyethoxy)methyl)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin) A mixture of 10 mg (7.95 μmol) of pyridin-5-hexyl-5-acetylamidoyl-1,4,7,11-tetraoxo-15-oxa-3,6,12-triazaoctadecane-18-acid and 2,3,4,5,6-pentafluorophenol (43.88 mg, 238.42 μmol) was dissolved in DMF (1 mL), and EDCI (45.70 mg, 238.42 μmol) was added. The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction solution was purified directly by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (5 mg, 2.85 μmol).

[0752] Its structural characterization data are as follows:

[0753] MS m / z (ESI): 1756.2 [M+H] +

[0754] Its preparation method is as follows:

[0755] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0756] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0757] Step 2: (S)-N5-((2S,5S,8S,22S,25S,28S)-1,29-bis((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-15-((7S,10S,13S)-14-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-7,10,13-trimethyl-5,8,11,14-tetraoxo-2-oxa-6,9,12-triazatetradecyl)-2,5,8,22,25 Preparation of ,28-hexamethyl-1,4,7,10,20,23,26,29-octaoxo-13,17-dioxa-3,6,9,21,24,27-hexaazanonadecan-15-yl)-N1-((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]indolazino[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)pentadiamide (P-2):

[0758] (S)-2-amino-N-((S)-1-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (8.14 mg, 17.08 μmol) and perfluorophenyl (2S,5S,8S)-1-(((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentanamide)[de] Pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3-yl)amino)-5-isopropyl-2-methyl-8-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)-1,4,7,11-tetraoxo-13,13-bis((3-oxo-3-(perfluorophenoxy)propoxy)methyl)-15-oxa-3,6,12-triazaoctadecane-18-ester (5 mg, 2.85 μmol, FR) was dissolved in DMF (1 mL), and DIPEA (2.21 mg, 17.08 μmol) was added dropwise. The mixture was stirred at room temperature for 5 hours. After the reaction was complete, the reaction solution was purified twice by high performance liquid chromatography and then freeze-dried to obtain the title compound (3.11 mg, 1.06 μmol, 90% purity).

[0759] Its structural characterization data are as follows:

[0760] MS m / z (ESI): 1317.7 [M / 2+H] + 878.6 [M / 3+H] +

[0761] The first preparation method is as follows:

[0762] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0763] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0764] The second preparation method is as follows:

[0765] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0766] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0767] Comparative Example 1: 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]pyran[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxo-5,8,11-triazatetradecane-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-amide (S-3)

[0768] INT-2 (170 mg, 0.226 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (90.83 mg, 0.249 mmol) were dissolved in N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (29.21 mg, 0.226 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was directly purified by preparative high-performance liquid chromatography and then freeze-dried to give 50.56 mg of the title compound.

[0769] Its structural characterization data are as follows:

[0770] MS m / z (ESI): 1002.4 [M+H] + .

[0771] The separation and purification methods are as follows:

[0772] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0773] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0774] 1H NMR (400MHz, DMSO) δ9.11(s,2H),8.68(t,J=6.4Hz,1H),8.49(d,J=8.8Hz,1H),8.16(s,1H),8.10(d,J=7.2H z,1H),8.01(d,J=7.2Hz,1H),7.91(d,J=6.8Hz,1H),7.31(s,1H),6.55(s,1H),5.65-5.55(m,1H),5.43(s,2H ),5.21(s,2H),4.67-4.55(m,2H),4.29-4.15(m,3H),3.98(s,2H),3.41(s,3H),3.25-3.15(m,2H),2.57-2.5 6(m,2H),2.35-2.27(m,2H),2.22-2.12(m,2H),1.91-1.75(m,4H),1.23-1.09(m,9H),0.87(t,J=7.2Hz,3H).

[0775] Comparative Example 2: 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-oxopropane-2-yl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-acetylamide (S-4)

[0776] (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), and HATU (17.41 mg, 45.79 μmol) and DIPEA (16.14 mg, 124.88 μmol) were added. The mixture 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]phthalazin-2(7H)-yl)methyl)propionamide (20 mg, 41.63 μmol) was added. The mixture 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 then freeze-dried to obtain the title compound (11.37 mg, 12.19 μmol).

[0777] Its structural characterization data are as follows:

[0778] MS m / z (ESI): 873.7 [M+H] + 437.5 [M / 2+H] +

[0779] Its preparation method is as follows:

[0780] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0781] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0782] Comparative Example 3: Preparation of N-((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetamide (S-1)

[0783] (S)-2-amino-N-((((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)propionamide H-1-3 (119 mg, 327.54 μmol) and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine INT-1 (201.65 mg, 491.31 μmol) were dissolved in DMF (3 mL), and then HATU (186.81 mg, 491.31 μmol) and DIPEA (126.99 mg, 982.61 μmol) were added and reacted at room temperature for one hour. The solvent was removed under vacuum to obtain the crude product, which was then purified by high-performance preparative chromatography and freeze-dried to obtain the title compound (86.11 mg, 111.66 μmol).

[0784] Its structural characterization data are as follows:

[0785] MS m / z (ESI): 756.3 [M+H] +

[0786] Its preparation method is as follows:

[0787] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% formic acid aqueous solution)

[0788] Comparative Example 4: Preparation of N-((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecane-12-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamide (S-2)

[0789] (S)-2-amino-N-((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)propionamide (100.74 mg, 245.44 μmol) and (5-(2-(methanesulfonyl)pyrimidin-5-yl)pent-4-ynyl)-L-alanyl-L-alanine (85 mg, 245.44 μmol) were dissolved in DMF (2 mL), followed by the addition of HATU (111.99 mg, 294.53 μmol), and finally DIPEA (95.16 mg, 736.33 μmol). The mixture was stirred at room temperature for two hours. After the reaction was complete, the reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (108.21 mg, 142.08 μmol).

[0790] Its structural characterization data are as follows:

[0791] MS m / z (ESI): 761.3 [M+Na] +

[0792] Its preparation method is as follows:

[0793] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0794] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0795] II. Examples of Antibody-Drug Conjugate Preparation

[0796] 1. Preparation of Trastuzumab A-14

[0797] 1.736 mL of Trastuzumab antibody (14.4 mg / mL) was diluted with 87 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 43 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was incubated at room temperature for 1.5 h. Then, A-14 solution (97 μL, 10 mM, 5.5 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and incubated at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab A-14). The DAR value was determined to be 7.0 by mass spectrometry.

[0798] 2. Preparation of Trastuzumab A-15

[0799] 1.736 mL of Trastuzumab antibody (14.4 mg / mL) was diluted with 87.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 43.0 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was incubated at room temperature for 1.5 h. Then, A-15 solution (108.8 μL, 10 mM, 6 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and incubated at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab A-15). The DAR value was determined by mass spectrometry to be 7.0.

[0800] 3. Preparation of Trastuzumab A-15'

[0801] 0.393 mL of Trastuzumab antibody (24.4 mg / mL) was diluted with 45.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 36.4 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, A-2 solution (83.4 μL, 10 mM, 12 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab A-15'). The DAR value was determined by mass spectrometry to be 16.0.

[0802] 4. Preparation of Trastuzumab A-16

[0803] 0.847 mL of Trastuzumab antibody (17.7 mg / mL) was diluted with 42.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 27.4 μL, pH 7.6) solution was added and mixed. The mixture was incubated at room temperature for 1.5 h. Then, A-16 solution (72.4 μL, 10 mM, 7 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added and mixed. The mixture was incubated at room temperature for 2 h. After incubation, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab A-16). The DAR value was determined to be 6.2 by mass spectrometry.

[0804] 5. Preparation of Trastuzumab A-17

[0805] 0.847 mL of Trastuzumab antibody (17.7 mg / mL) was diluted with 42.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 27.4 μL, pH 7.6) solution was added and mixed, and the mixture was allowed to stand at room temperature for 1.5 h. Then, A-17 solution (72.4 μL, 10 mM, 7 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added and mixed, and the mixture was allowed to stand at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab A-17). The DAR value was determined by mass spectrometry to be 7.0.

[0806] 6. Preparation of hRS7 A-16

[0807] 1.572 mL of hRS7 antibody (15.9 mg / mL) was diluted with 78.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 50.6 μL, pH 7.6) solution was added and mixed. The mixture was allowed to stand at room temperature for 1.5 h. Then, A-16 solution (103.3 μL, 10 mM, 6 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added and mixed. The mixture was allowed to stand at room temperature for 2 h. After this, the buffer solution was replaced 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-16). The DAR value was determined by mass spectrometry to be 5.8.

[0808] 7. Preparation of hRS7 A-17

[0809] Take 1.572 mL of hRS7 antibody (15.9 mg / mL), dilute with 78.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 50.6 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Add A-17 solution dissolved in dimethyl sulfoxide (103.3 μL, 10 mM, 6 molar equivalents of the antibody) and mix well. Incubate at room temperature for 2 h. 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-17). The DAR value was determined by mass spectrometry to be 6.8.

[0810] 8. Preparation of hRS7 S-3

[0811] Take 2.516 mL of hRS7 antibody (15.9 mg / mL), dilute with 126.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 148.5 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Add S-3 solution dissolved in dimethyl sulfoxide (275.5 μL, 10 mM, 10 molar equivalents of the antibody) and mix well. Incubate at room temperature for 2 h. 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 S-3). The DAR value was determined by mass spectrometry to be 7.1.

[0812] Preparation of 9.hRS7 S-4

[0813] Take 2.516 mL of hRS7 antibody (15.9 mg / mL), dilute with 126.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 148.5 μL, pH 7.6) solution and mix well. Incubate at room temperature for 1.5 h. Add S-4 solution dissolved in dimethyl sulfoxide (275.5 μL, 10 mM, 10 molar equivalents of the antibody) and mix well. Incubate at room temperature for 2 h. 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 S-4). The DAR value was determined by mass spectrometry to be 7.8.

[0814] 10. Preparation of Trastuzumab P-1

[0815] 0.971 mL of Trastuzumab antibody (20.6 mg / mL) was diluted with 48.5 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.6) solution was added and mixed. The mixture was incubated at room temperature for 1.5 h. Then, 199.0 μL of P-1 solution (10 mM, 13 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed, and incubated at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab P-1). The DAR value was determined by mass spectrometry to be 31.32.

[0816] 11. Preparation of Trastuzumab INT-8

[0817] 0.857 mL of Trastuzumab antibody (17.5 mg / mL) was diluted with 42.9 μL of 20 mM PB + 0.1 M EDTA (pH 7.6), and then the pH was adjusted to 7.6 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 56.8 μL, pH 7.6) solution was added and mixed thoroughly. The mixture was incubated at room temperature for 1.5 h. Then, 105.5 μL of INT-8 solution (10 mM, 10 molar equivalents of the antibody) dissolved in dimethyl sulfoxide was added, mixed thoroughly, and incubated at room temperature for 2 h. Finally, the buffer solution was replaced with 20 mM histidine buffer solution at pH 5.5 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab INT-8). The DAR value was determined to be 8.0 by mass spectrometry.

[0818] III. Biological Examples

[0819] 1. Evaluate the inhibitory effect of antibody-drug conjugates on tumor growth in a mouse subcutaneous xenograft model.

[0820] The MX-1 human breast cancer cell CDX model was administered via tail vein injection to the mice. Tumor volume and animal weight changes were measured weekly to calculate the tumor-suppressing efficacy of ADC in tumor-bearing mice.

[0821] test drug

[0822] Take an appropriate amount of ADC and dilute the stock solution to the drug solution using 0.9% NaCl injection. Use 0.9% NaCl injection as a solvent control (Vehicle).

[0823] Laboratory animals and cell lines

[0824] Balb / c Nude mice (Sichuan Vital River Laboratory Animal Technology Co., Ltd.)

[0825] Human breast cancer cell line MX-1 (Nanjing Kebai)

[0826] Experimental grouping and evaluation methods

[0827] MX-1 cells were cultured in DMEM 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 containing 50% matrix gel to a suitable concentration, and subcutaneously inoculated into female Balb / c Nude mice to establish a breast cancer model. Tumors with an average volume of approximately 150 mmHg were selected. 3 Tumor-bearing mice were randomly assigned to groups and administered the drug twice daily on Day 0 and Day 7 according to the dosages listed in Table 1. The administration route 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.

[0828] The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the antitumor efficacy of ADCs:

[0829] V T末 >V T0 At that time, TGI(%) = [1-(V) T末 -V T0 ) / (V C末 -V C0 )]*100%;

[0830] V T末 ≤V T0 At that time, TGI(%) = [1-(V) T末 -V T0 ) / V T0 ]*100%.

[0831] Where V T末 Mean tumor volume at the end of the experiment in the treatment group;

[0832] V T0 Mean tumor volume at the start of treatment in the treatment group;

[0833] V C末 Mean tumor volume at the end of the experiment in the negative control group;

[0834] V C0 Mean tumor volume at the start of drug administration in the negative control group;

[0835] The tumor relative proliferation rate (T / C%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

[0836] T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 )*100%.

[0837] Compared with the Vehicle group, on day 34, the tumor growth inhibition rate (TGI) of hRS7 A-16 (3 mg / kg), hRS7 A-17 (3 mg / kg), hRS7 S-3 (1.5 mg / kg), hRS7 S-4 (1.5 mg / kg), and hRS7 S-3+hRS7 S-4 (1.5+1.5 mg / kg combination therapy) were 181.21%, 168.88%, 103.65%, -13.17%, and 115.84%, respectively. This indicates that the ADCs of the present invention (e.g., hRS7 A-16, hRS7 A-17) have a significant inhibitory effect on tumor growth in the MX-1 breast cancer xenograft model. On day 34, no animal deaths or significant weight loss were observed in any of the treatment groups, and no obvious drug toxicity was observed. The mice tolerated the ADCs of the present invention well during the treatment period. Specific results are shown in Table 1.

[0838] Table 1. Human breast cancer cell MX-1CDX model

[0839] 2. Evaluate the inhibitory effect of antibody-drug conjugates on tumor growth in a mouse subcutaneous xenograft model.

[0840] Laboratory animals and cell lines

[0841] Balb / c Nude mice (Sichuan Vital River Laboratory Animal Technology Co., Ltd.)

[0842] Calu-3 cells, a type of human non-small cell lung cancer (Nanjing Kebai).

[0843] Experimental grouping and evaluation methods

[0844] Calu-3 cells were cultured in MEM medium containing 1% NEAA, 1% sodium pyruvate, and 10% fetal bovine serum at 37°C and 5% CO2. Calu-3 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 non-small cell lung cancer cell model. The tumors were cultured until the average volume reached approximately 150 mmHg. 3At approximately 10:00 AM, tumors were randomly assigned to three groups based on size: a solvent control group (i.e., negative control, Vehicle group), a Trastuzumab P-1 (0.5 mpk) group, and a Trastuzumab INT-8 (0.5 mpk) group. Administration was performed on Day 0, with a single dose administered via tail vein injection. 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.

[0845] The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the antitumor efficacy of ADCs:

[0846] V T末 >V T0 At that time, TGI(%) = [1-(V) T末 -V T0 ) / (V C末 -V C0 )]*100%;

[0847] V T末 ≤V T0 At that time, TGI(%) = [1-(V) T末 -V T0 ) / V T0 ]*100%.

[0848] Where V T末 Mean tumor volume at the end of the experiment in the treatment group;

[0849] V T0 Mean tumor volume at the start of treatment in the treatment group;

[0850] V C末 Mean tumor volume at the end of the experiment in the negative control group;

[0851] V C0 Mean tumor volume at the start of drug administration in the negative control group;

[0852] The tumor relative proliferation rate (T / C%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

[0853] T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 )*100%.

[0854] Compared with the Vehicle group, the tumor growth inhibition rate (TGI) of the Trastuzumab P-1 (0.5 mpk) group and the Trastuzumab INT-8 (0.5 mpk) group were 97.35% and 83.31%, respectively (see Table 2 for details). This indicates that the ADC of the present invention has a significant inhibitory effect on tumor growth in the Calu-3 non-small cell lung cancer xenograft model. On Day 28, there were no animal deaths or significant weight loss in any group, and no obvious drug toxicity was observed. The mice tolerated the ADC of the present invention well during the treatment period.

[0855] Table 2. Calu-3 CDX model of human non-small cell lung cancer cells.

[0856] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

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): in: …represents L a Existence or non-existence; Q is a structure used to link with antibody or antigen-binding fragments; B connects to Q, L, or L. a Part of; L and L a It is a connector sub-part; E is the self-eliminating part; D represents the bioactive molecule portion; n is selected from 2-10; Each L may be the same or different; each E may be the same or different; each D may be the same or different; Preferably, the compound of formula I has the structure shown in formula (IA), formula (IB), formula (IC), or formula (ID): in: Q is a structure used to link with antibody or antigen-binding fragments; L 1 L 2 L 3 L 4 and L a It is a connector sub-part; E 1 E 2 E 3 and E 4 It is the self-eliminating part; D 1 D 2 D 3 and D 4 It is the bioactive molecular part; n is selected from 2-10; L 1 L 2 L 3 and L 4 They may be the same or different; E 1 E 2 E 3 and E 4 They are the same or different; D 1 D 2 D 3 and D 4 They may be the same or different. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, Q is selected independently from the following structures: Each time p appears, it is independently selected from an integer between 1 and 12; Preferably, Q is selected from the following structures: Each time p appears, it is independently selected from an integer between 1 and 12; Preferably, Q is selected from the following structures: 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 or any combination thereof: and -NR 0 -C 1-10 Alkyl; the alkyl group may optionally be substituted with one or more OH groups; R 0 Selected from H or C 1-6 Alkyl group; q is independently selected from an integer from 1 to 24 each time it appears (e.g., an integer from 1 to 10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Preferably, B is selected from the following structures or any combination thereof: Each time q appears, it is independently selected from an integer from 1 to 24 (e.g., an integer from 1 to 10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Preferably, B is selected from the following structures: The compound of any one of claims 1-3 or its pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug, wherein, L, L 1 L 2 L 3 and L 4 A bivalent structure composed of one or more substituted or unsubstituted structural segments, each independently selected from the following: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, 5-12 heterocyclic, -N(R')-, carbonyl, -O-, glycosyl, tromethamine, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)) r OCH3)) and Lys(R') r And short peptides composed 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, Wherein Ra is EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, or NOPO; R′ represents hydrogen, C 1-6 Alkyl groups, polyhydroxy fragments, glycosyl groups, polyethylene glycol-containing fragments, -(CH2CH2O) r -C 1-6 Alkyl group, -C(=O)-(CH2CH2O) r -C 1-6 Alkyl, polysarcosine, -(C(=O)-CH2N(Me)) r -C 1-6 Alkyl groups, carboxylic acid-containing fragments, tetracarboxylic acid residues and their derivatives, EDTA and its derivatives, or DOTA and its derivatives; r is independently selected from integers 1-12 each time it appears; Preferably, L, L 1 L 2 L 3 and L 4 Each is independently selected from the following structures: Preferably, L, L 1 L 2 L 3 and L 4 Each is independently selected from the following structures: Preferably, L, L 1 L 2 L 3 and L 4 Each is independently selected from the following structures: 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, L a Select from the following structures or any combination thereof: Each time r appears, it is independently selected from an integer between 0 and 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); Preferably, L a Select from the following structures or any combination thereof: Each time r appears, it is independently selected from an integer from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); Preferably, L a Selected from the following structures: (For example, )， (For example, )， Each time r appears, it is independently selected from an integer from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). 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, E, E 1 E 2 E 3 and E 4 Each is independently selected from a single bond, -NH-CH2-. Preferably, E, E 1 E 2 E 3 and E 4 Each is independently selected from a single bond, -NH-CH2- or Preferably, E, E 1 E 2 E 3 and E 4 Each is independently selected from -NH-CH2- or 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 bioactive molecules are selected from antitumor drugs or compounds with antitumor effects; Preferably, the bioactive molecule is selected from cytotoxic compounds, antimetabolites, cell cycle regulators, apoptosis regulators, synthetic lethal agents, kinase inhibitors, or protein degraders; Preferably, the cytotoxic compound is a DNA damaging agent, a topoisomerase inhibitor, an RNA polymerase inhibitor, or a microtubule inhibitor; Preferably, the antimetabolite is a nucleoside compound or a folic acid analogue; Preferably, the cell cycle regulator is a CDK inhibitor; Preferably, the apoptosis agent is a Bcl-2 inhibitor or a Bcl-xL inhibitor; Preferably, the synthetic lethal agent is a poly(ADP-ribose) polymerase (PARP) inhibitor, PRMT5 inhibitor, ATR inhibitor, ATM inhibitor, WEE1 inhibitor, WRN inhibitor, PARG inhibitor, Polθ inhibitor, or KIF18A inhibitor. Preferably, the kinase inhibitor is an EGFR inhibitor or a VEGFR inhibitor; Preferably, the protein degrading agent is PROTAC or a molecular gel compound; Preferably, the DNA damaging agent is selected from pyrrolobenzodiazepines, carzimidoxones, pyroximide compounds, or anthracycline compounds; Preferably, the topoisomerase inhibitor is selected from basal compounds, anthracycline compounds, and etoposide compounds; Preferably, the RNA polymerase inhibitor is selected from sucrose compounds and α-amaminoid compounds; Preferably, the microtubule inhibitor is selected from olistatin compounds, maytansine compounds, leucocele compounds, taxane compounds, vincristine compounds, and hammetrine compounds; Preferably, the nucleoside compound is selected from pyrimidine nucleoside compounds or purine nucleoside compounds; Preferably, the bioactive molecule is associated with E, E', E'' through its -OH, primary amino, secondary amino, or -SH groups, respectively. 1 E 2 E 3 or E 4 connect. 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 bioactive molecule is selected from the structures shown below: Preferably, the bioactive molecule is selected from the following compounds: Preferably, the D, D 1 D 2 D 3 and D 4 Each is independently selected from the following structures: The compound of any one of claims 1-8, 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 structures shown below: The compound of any one of claims 1-9 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 fragment by a substitution reaction (e.g., removal of -SO2Me) or by an addition reaction. Antibody-drug conjugates having the structure shown in Formula II: Where B, L a L, E, D and n are as described in any one of claims 1-9. Ab represents an antibody or its antigen-binding fragment; M is the structure formed by covalently linking Q according to any one of claims 1-9 with the antibody or its antigen-binding fragment; x are each independently selected from 1 to 10; Preferably, the antibody-drug conjugate has the structure shown in formula (IIA), formula (IIB), formula (IIC), or formula (IID): in, B, L a D 1 To D 4 L 1 To L 4 E 1 To E 4 As described in any one of claims 1-9, Ab represents an antibody or its antigen-binding fragment; M is the structure formed by covalently linking Q according to any one of claims 1-9 with the antibody or its antigen-binding fragment; x are each independently selected from 1 to 10; Preferably, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase, or an antibody or its antigen-binding fragment that specifically binds to human trophoblast surface antigen 2 (TROP2). Preferably, M is selected from the following structures: Where p is selected from integers from 1 to 12; Preferably, M is selected from the following structures: The antibody-drug conjugate of claim 11, wherein, The antibody or its antigen-binding fragment comprises: (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system: (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or, (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof; (1c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:56 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:57 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:58 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:59 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:60 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:61 or a variant thereof; Wherein, the variant described in any one of (1a), (1b) or (1c) 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 respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions; or, (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system: (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or, (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; (2c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:68 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:69 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:58 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:59 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:60 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:61 or a variant thereof; Wherein, the variant described in any one of (2a), (2b) or (2c) 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 respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions; or, (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system: (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or, (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; (3c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:62 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:63 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:58 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:59 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:60 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:61 or a variant thereof; Wherein, the variant described in any one of (3a), (3b) or (3c) 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 respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions; or, (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system: (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or, (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; (4c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:64 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:65 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:66 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:67 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:61 or a variant thereof; Wherein, the variant described in any one of (4a), (4b) or (4c) 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 respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions. The antibody-drug conjugate of claim 11 or 12, wherein, The antibody or its antigen-binding fragment comprises: (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4; or (c) VH or a variant thereof shown in SEQ ID NO: 51, and / or VL or a variant thereof shown in SEQ ID NO: 52; 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 its source sequence, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to its source sequence; preferably, the substitutions are conservative substitutions; Preferably, the antibody or its antigen-binding fragment comprises: (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4; or (c) VH shown in SEQ ID NO: 51, and VL shown in SEQ ID NO: 52; Preferably, the antibody or its antigen-binding fragment further comprises: (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids). Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region. Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35; The antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, 10, or 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 41; or The antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 53 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 53; Preferably, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36; The antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36; The antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 and a light chain constant region (CL) as shown in SEQ ID NO: 36; or The antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 53 and a light chain constant region (CL) as shown in SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises: (1) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; (2) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36; or (3) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 41, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or (4) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 41, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36; or (5) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 51 and the heavy chain constant region (CH) shown in SEQ ID NO: 53, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 52 and the light chain constant region (CL) shown in SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises: (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38; (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40; (3) The heavy chain comprising the sequence shown in SEQ ID NO: 42, and the light chain comprising the sequence shown in SEQ ID NO: 38; (4) The heavy chain comprising the sequence shown in SEQ ID NO: 43, and the light chain comprising the sequence shown in SEQ ID NO: 40; or (5) The heavy chain comprising the sequence shown in SEQ ID NO: 54, and the light chain comprising the sequence shown in SEQ ID NO:

55. The antibody-drug conjugate according to any one of claims 11-13 is selected from the following structures: in, This indicates the manner in which the antibody or its antigen-binding fragment is connected to the remaining portion of the antibody-drug conjugate; x is independently 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, Ab' in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof containing VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2, or Ab' represents an antibody or antigen-binding fragment thereof containing VH as shown in SEQ ID NO:3 and VL as shown in SEQ ID NO:4, or Ab' represents an antibody or antigen-binding fragment thereof containing VH as shown in SEQ ID NO:51 and VL as shown in SEQ ID NO:

52. Preferably, Ab' represents trastuzumab, pertuzumab, or goxatuzumab. A composition of an antibody-drug conjugate, the composition comprising one or more antibody-drug conjugates according to any one of claims 11-14; preferably, the composition has a DAR value (drug-antibody conjugate ratio) of 1-40, for example 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 15 to 20, 15 to 25, 15 to 30, 15 to 35, 15 to 40, 20 to 25, 20 to 30, 20 to 35, 20 to 40, 25 to 30, 25 to 35, 25 to 40, 30 to 35, 30 to 40, 35 to 40. A pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt or stereoisomer of any one of claims 11-14, the composition of claim 15, and one or more pharmaceutical excipients. Use of the compound of any one of claims 1-10, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug of any one of claims 11-14, the composition of claim 15, or the pharmaceutical composition of claim 16, in the preparation of a medicament for treating cancer; preferably, the cancer is a cancer expressing HER2 or TROP2. The use as described in claim 17, wherein the cancer is selected from solid tumors or hematologic malignancies; for example, selected from breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), colon cancer, and lymphoma. The compound of any one of claims 1-10 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, or the antibody-drug conjugate of any one of claims 11-14, the composition of claim 15, or the pharmaceutical composition of claim 16, is used to treat cancer; preferably, the cancer is a cancer expressing HER2 or TROP2. The compound, drug conjugate, composition, or pharmaceutical composition of claim 19, wherein the cancer is selected from solid tumors or hematologic malignancies; for example, selected from breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), colon cancer, and lymphoma. A method for treating Her2-expressing cancer or TROP2-expressing cancer, comprising administering to a subject in need an effective amount of the compound of any one of claims 1-10 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, or an antibody-drug conjugate of any one of claims 11-14, the composition of claim 15, or the pharmaceutical composition of claim 16. The method of claim 21, wherein the cancer is selected from solid tumors or hematologic malignancies; for example, selected from breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), colon cancer, and lymphoma.

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