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

Abstract

Provided are a drug-linker, an antibody-drug conjugate thereof, a preparation method therefor and the use thereof. The drug-linker has a structure as represented by formula (I), and the antibody-drug conjugate has a structure as represented by formula (II). The drug can be selected from a topoisomerase inhibitor, a DNA damage agent, a microtubule inhibitor, an RNA polymerase inhibitor, and an antimetabolic compound. The prepared antibody-drug conjugate exhibits a significant synergistic effect, and achieves a targeted killing effect on breast cancer, gastric cancer, lung cancer, colorectal cancer and lymphoma.

Description

Drug linkers and their antibody-drug conjugates, preparation methods and uses Technical Field

[0001] This application belongs to the pharmaceutical field and relates to drug linkers and their antibody-drug conjugates, preparation methods and uses. Background Technology

[0002] 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.

[0003] 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.

[0004] 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.

[0005] 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

[0006] This application provides a novel dual-load drug linker and its antibody-drug conjugate. The dual-load drugs are mounted on the antibody via irreversible covalent binding and in the same or different proportions. The antibody-drug conjugate exhibits a significant synergistic effect.

[0007] Drug linker

[0008] 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:

[0009] in:

[0010] Q is the structure before it is linked to the antibody or antigen-binding fragment;

[0011] L is the connector sub-part;

[0012] E is the self-eliminating part;

[0013] D represents the bioactive molecule portion;

[0014] n is selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10);

[0015] In some embodiments, the bioactive molecules are each independently selected from antitumor drugs or compounds with antitumor effects.

[0016] In some embodiments, the bioactive molecules are each independently selected from cytotoxic compounds or antimetabolites.

[0017] In some embodiments, the cytotoxic compound is a DNA damaging agent, a topoisomerase inhibitor, an RNA polymerase inhibitor, or a microtubule inhibitor.

[0018] In some embodiments, the antimetabolite compound (antimetabolite drug) is a nucleoside compound or a folic acid analog.

[0019] In some embodiments, the DNA damaging agent is selected from pyrrolobenzodiazepines (PBDs), kazimidoxams, pyroximide, or anthracyclines.

[0020] In some embodiments, the topoisomerase inhibitor is selected from camptothecin compounds, anthracycline compounds, and etoposide compounds.

[0021] In some embodiments, the (DNA) topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecins, including but not limited to hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, or rubotecan, etc.) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, docalmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide).

[0022] In some embodiments, the RNA polymerase inhibitor is selected from sucrose compounds and α-amaminoid compounds.

[0023] In some embodiments, the microtubule inhibitor is selected from olistatin compounds, maytansine compounds, leucospirin compounds, taxane compounds, vincristine compounds, and hammetrine compounds.

[0024] In some embodiments, the nucleoside compound is selected from pyrimidine nucleoside compounds or purine nucleoside compounds.

[0025] In some embodiments, the bioactive molecules are each independently selected from epomycin compounds, mitomycin compounds, and platinum compounds.

[0026] In some implementations, Q is selected individually from the following structures each time it appears:

[0027] Where R is H or C 1-6 Alkyl; p is independently selected from an integer from 1 to 12 each time it appears, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0028] In some implementations, Q is selected from the following structures:

[0029] R is as described above.

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

[0031] In some implementations, Q is selected from the following structures:

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

[0033] In some implementations, Q is selected from the following structures:

[0034] In some implementations, Q is

[0035] In some implementations, Q is

[0036] In some implementations, Q is

[0037] In some implementations, Q is

[0038] In some implementations, Q is

[0039] In some implementations, Q is

[0040] In some implementations, L is selected from a divalent structure composed of one or more substituted or unsubstituted structural segments: 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, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:44), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:45), Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:46), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:47), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO: 48), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO: 49), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO: 50), 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, 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 from 1 to 20 each time it appears, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0042] In some implementations, L is selected from the following structures:

[0043] Each time r appears, it is independently selected from an integer between 1 and 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0044] In some implementations, L is selected from the following structures:

[0045] Each time r appears, it is independently selected from an integer between 1 and 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0046] In some implementations, L is selected from the following structures:

[0047] In some implementations, L is selected from the following structures:

[0048] Each time r appears, it is independently selected from an integer between 1 and 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0049] In some implementations, L is selected from the following structures:

[0050] Each time r appears, it is independently selected from an integer between 1 and 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0051] In some implementations, E is selected from a single bond, -NH-CH2-.

[0052] In some implementations, E is selected from single bonds, -NH-CH2- or

[0053] In some implementations, E is selected from a single bond.

[0054] In some implementations, E is selected from -NH-CH2-.

[0055] In some implementations, E is selected from

[0056] In some implementations, n in equation (I) is 1, and -ELQ is selected from the following structures:

[0057] Each time r appears, it is independently selected from an integer between 1 and 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[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 linked to the E via -OH, primary amino, secondary amino, tertiary amino, or -SH groups.

[0060] In some embodiments, the bioactive molecule is selected from the following compounds:

[0061] In some embodiments, the bioactive molecule is selected from the following compounds:

[0062] In some embodiments, the bioactive molecule is selected from the following compounds:

[0063] In some embodiments, the bioactive molecule is selected from the following compounds:

[0064] In some implementations, the -D is selected from the following structures:

[0065] In some implementations, the -D is selected from the following structures:

[0066] In some implementations, the -D is selected from the following structures:

[0067] In some embodiments, the bioactive molecule is selected from the following compounds:

[0068] In some embodiments, the bioactive molecule is selected from the following compounds:

[0069] In some implementations, the -D is selected from the following structures:

[0070] In some embodiments, the "drug linker" is selected from the following structures:

[0071] Antibody-drug conjugates

[0072] In another aspect, this application provides an antibody-drug conjugate having the structure shown in formula (II):

[0073] Where L' and L” are each independently described as any one of L above;

[0074] E' and E” are each independently described as any one of the above E items;

[0075] D' and D” are each independently described as any one of the D items above;

[0076] n1 and n2 are each independently as described in any of the above items n;

[0077] A represents an antibody or its antigen-binding fragment;

[0078] Q' and Q” are the structural forms of Q covalently linked to an antibody or its antigen-binding fragment as described above; x and y are each independently selected from 1 to 10.

[0079] In some implementations, x and y are the same, and each is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0080] In some implementations, x and y are different and are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0081] In some embodiments, the antibody-drug conjugate has the structure shown in formula (III):

[0082] L', L”, E', E”, D', D”, A, Q', Q” are each independently described as any one of the items above.

[0083] In the antibody-drug conjugate, D' and D” can be conjugated to the antibody or its antigen-binding fragment via linkers.

[0084] In some implementations, Q' and Q” are each independently selected from the following structures:

[0085] In some implementations, Q' and Q” are individually selected from the following structures:

[0086] 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.

[0087] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0088] (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:

[0089] (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,

[0090] (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;

[0091] Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with 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;

[0092] or,

[0093] (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:

[0094] (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,

[0095] (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;

[0096] Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with 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;

[0097] or,

[0098] (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:

[0099] (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,

[0100] (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;

[0101] Wherein, the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with 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;

[0102] or,

[0103] (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:

[0104] (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,

[0105] (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;

[0106] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with 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.

[0107] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0108] (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

[0109] (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,

[0110] (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;

[0111] or,

[0112] (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:

[0113] (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,

[0114] (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;

[0115] or,

[0116] (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

[0117] (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,

[0118] (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;

[0119] or,

[0120] (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:

[0121] (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,

[0122] (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;

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

[0124] (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

[0125] (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;

[0126] 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.

[0127] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0128] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0129] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment further comprises:

[0130] (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

[0131] (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).

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0139] (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;

[0140] (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

[0141] (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

[0142] (4) A heavy chain comprising the VH 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 of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

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

[0144] (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;

[0145] (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;

[0146] (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; or

[0147] (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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] In some embodiments, A is selected from 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 family.

[0155] In some implementations, A is selected from trastuzumab, pertuzumab, trastuzumab mutant, pertuzumab mutant, or a biepisode antibody or its antigen-binding fragment constructed from trastuzumab and pertuzumab.

[0156] 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.

[0157] 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, a free form of the "drug-linker" (QLED, where Q is the structural form of Q' or Q" before covalently linking it with an antibody or its antigen-binding fragment) described above can be obtained first, and then covalently linked to the antibody or its antigen-binding fragment to obtain the antibody-drug conjugates 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 the -SO2Me or -Br structure) or an addition reaction.

[0158] In some embodiments, the present invention provides, for example (For example: The antibody-drug conjugate shown in the figure is as follows: D, E, L, Q', n and A are as described in any of the above.

[0159] In some embodiments, the antibody-drug conjugate is selected from the following structures:

[0160] Among them, the -S) in each antibody-drug conjugate x -Ab' or -NH)x -Ab' represents any of the antibodies or antigen-binding fragments described above. This indicates the specific linkage between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment. This indicates the specific connection between the amino and carbonyl groups in the antibody or its antigen-binding fragment, where x is selected from 1 to 10.

[0161] In some embodiments, the antibody-drug conjugate is selected from the following structures:

[0162] Among them, the -S) in each antibody-drug conjugate x -Ab' or -NH) x -Ab' represents any of the antibodies or antigen-binding fragments described above. This indicates the specific linkage between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment. This indicates the specific connection between the amino and carbonyl groups in the antibody or its antigen-binding fragment, where x is selected from 1 to 10.

[0163] In some implementation schemes, Selected from the following structures:

[0164] In some implementation schemes, the -S in each antibody-drug conjugate x -Ab- y (NH-, -NH) x -Ab- y (S-, or -S) x -Ab- y (S- represents any of the antibodies or antigen-binding fragments described above) This indicates the specific linkage between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment. This indicates the specific connection between the amino and carbonyl groups in the antibody or its antigen-binding fragment, where x and y are each independently selected from 1 to 10.

[0165] In some implementations, x and y are the same, and each is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0166] In some implementations, x and y are different and are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0167] In some implementation schemes, the -S in each antibody-drug conjugate x -Ab- y (NH-, -NH) x -Ab- y (S-, or -S) x -Ab- y (S- represents any of the A mentioned above.)

[0168] In some implementation schemes, the -S in each antibody-drug conjugate x -Ab- y (NH-, -NH) x -Ab- y (S-, or -S) x -Ab- y (S- represents 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;

[0169] in, This indicates the specific linkage between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment;

[0170] This indicates the specific linkage between the amino and carbonyl groups in the antibody or its antigen-binding fragment.

[0171] In some implementation schemes, This indicates the specific linkage between the thiol group and the pyrimidine group of cysteine ​​in the antibody or its antigen-binding fragment.

[0172] This indicates the specific linkage between the amino and carbonyl groups of lysine in the antibody or its antigen-binding fragment.

[0173] Connector

[0174] On the other hand, the present invention provides a connector with the structural formula -E'-L'). x -Q'- or -E”-L”) y -Q”-; wherein, E', L', Q', E”, L”, Q”, x and y are as described in any of the above.

[0175] In some embodiments, E' is used to link with D' as described in any one of the present applications, E” is used to link with D” as described in any one of the present applications, and Q' or Q” is used to link with the antibody or its antigen-binding fragment as described in any one of the present applications.

[0176] In some embodiments, the connector structure is as follows:

[0177] In some embodiments, the present invention provides a connector, the connector having the structural formula -EL). n -Q; wherein E, L, Q and n are as described in any of the above.

[0178] In some embodiments, E is used to connect with D as described in any one of the claims of this application.

[0179] In some embodiments, the connector structure is as follows:

[0180] Bioactive compounds

[0181] On the other hand, this application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite, or prodrug thereof, wherein the compound has the structure shown in formula (III):

[0182] in,

[0183] Xp is selected from -CH2-, -O-, -S- and -N(R6)-;

[0184] R4 is selected from hydrogen, halogens, and C. 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, cyano, HO-C 1-6 Alkylene-OC 1-6 Alkylene-, H2N-C 1-6 Alkylene-OC 1-6 Alkylene-, HO-C 1-6 Alkylene-, H2N-C 1-6 Alkylene-, HS-C 1-6 alkylene, aryl, benzyl, heteroaryl, and heterocyclic groups, wherein C 1-6 Alkyl, C 1-6 Alkylene, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 The alkynyl, aryl, benzyl, heteroaryl, and heterocyclic groups may optionally be selected from one or more halogens, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Substituents include alkylamine, hydroxyl, hydroxyalkyl, amino, and aminoalkyl groups;

[0185] R5 is selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, haloalkyl, amino, aminoalkyl, hydroxyalkyl, HO-(CH2) n-O-(CH2) n - and H2N-(CH2) n -O-(CH2) n -;

[0186] n is independently selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10);

[0187] R6 is selected from H and C. 1-6 Alkyl, haloalkyl, and hydroxyl; or,

[0188] When X is selected from -N(R6)-, R6 can also form a ring with R4 and the atoms attached to it.

[0189] In some implementations, -Xp-R4 is selected from: -C 1-6 Alkylene -OH, -N(R6)-C 3-6 Cycloalkyl-SH, -N(R6)-C 1-6 Alkylene -NH2, -SC 1-6 Alkylene -OH, -N(R6)-C 1-6 Alkylene -SH or -SC 1-6 Alkylene-NH2; preferably, the -Xp-R4 is selected from: -N(R6)-C 1-6 Alkylene -SH or -SC 1-6 Alkylene-NH2.

[0190] In some implementations, -Xp-R4 is selected from: -C 1-6 Alkylene -OH, -N(R6)-C 1-6 Alkylene -NH2, -SC 1-6 Alkylene -OH, -N(R6)-C 1-6 Alkylene -SH or -SC 1-6 Alkylene-NH2; preferably, the -Xp-R4 is selected from: -N(R6)-C 1-6 Alkylene -SH or -SC 1-6 Alkylene-NH 2。

[0191] In some embodiments, the compound has the following structure:

[0192] In some embodiments, the compound has the following structure:

[0193] intermediate

[0194] In another aspect, the present invention provides the following intermediate compounds and their acceptable pharmaceutical salts:

[0195] In some embodiments, the intermediate compound has the following structure:

[0196] Wherein, PG is an amino protecting group, such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl (or ethoxycarbonyl).

[0197] In some implementations, PG is phosphooxycarbonyl (Fmoc).

[0198] In some embodiments, the intermediate compound has the following structure:

[0199] In some embodiments, the intermediate compound has the following structure:

[0200] Wherein, PG is as defined in any of the above; preferably, PG is phosphonomethyloxycarbonyl (Fmoc) or allyloxycarbonyl (Alloc).

[0201] Composition

[0202] 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 contains a drug-linker as described herein, wherein x and y are each independently 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, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 drug-linkers. The compositions are characterized by a drug-antibody ratio (DAR) in the range of about 1 to about 50. Methods for determining the DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0203] For example, in any embodiment, the ADC composition described herein has a DAR of about 1 to about 50 or any subrange therebetween, such as: about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 1 to 11, about 1 to 12, about 1 to 13, about 1 to 14, about 1 to 15, about 1 to 16, about 1 to 17, about 1 to 18, about 1 to 19, about 1 to 20, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 2 to 11, about 2 to 12, about 2 to 13, about 2 to 14, about 2 to 15, about 2 to 16, about 2 to 17, about 2 to 18, about 2 to 19, about 2 Up to 20, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 3 to 11, about 3 to 12, about 3 to 13, about 3 to 14, about 3 to 15, about 3 to 16, about 3 to 17, about 3 to 18, about 3 to 19, about 3 to 20, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9. Approximately 4 to 10, approximately 4 to 11, approximately 4 to 12, approximately 4 to 13, approximately 4 to 14, approximately 4 to 15, approximately 4 to 16, approximately 4 to 17, approximately 4 to 18, approximately 4 to 19, approximately 4 to 20, approximately 5 to 6, approximately 5 to 7, approximately 5 to 8, approximately 5 to 9, approximately 5 to 10, approximately 5 to 11, approximately 5 to 12, approximately 5 to 13, approximately 5 to 14, approximately 5 to 15, approximately 5 to 16, about 5 to 17, about 5 to 18, about 5 to 19, about 5 to 20, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 6 to 11, about 6 to 12, about 6 to 13, about 6 to 14, about 6 to 15, about 6 to 16, about 6 to 17, about 6 to 18, about 6 to 19, about 6 to 20, about 7 to 8, about 7 to 9, about 7 to 10, about 7 to 11, approximately 7 to 12, approximately 7 to 13, approximately 7 to 14, approximately 7 to 15, approximately 7 to 16, approximately 7 to 17, approximately 7 to 18, approximately 7 to 19, approximately 7 to 20, approximately 8 to 9, approximately 8 to 10, approximately 8 to 11, approximately 8 to 12, approximately 8 to 13, approximately 8 to 14, approximately 8 to 15, approximately 8 to 16, approximately 8 to 17, approximately 8 to 18, approximately 8 to 19, approximately 8 to 20, or about 9 to 10, about 9 to 11, about 9 to 12, about 9 to 13, about 9 to 14, about 9 to 15, about 9 to 16, about 9 to 17, about 9 to 18, about 9 to 19, about 9 to 20, or about 10 to 11, about 10 to 12, about 10 to 13, about 10 to 14, about 10 to 15, about 10 to 16, about 10 to 17, about 10 To 18, about 10 to 19, about 10 to 20, or about 11 to 12, about 11 to 13, about 11 to 14, about 11 to 15, about 11 to 16, about 11 to 17, about 11 to 18, about 11 to 19, about 11 to 20, or about 12 to 13, about 12 to 14, about 12 to 15, about 12 to 16, about 12 to 17, about 12 to 18,Around 12 to 19, around 12 to 20, or around 12 to 13, around 12 to 14, around 12 to 15, around 12 to 16, around 12 to 17, around 12 to 18, around 12 to 19, around 12 to 20, or around 13 to 14, around 13 to 15, around 13 to 16, around 13 to 17, around 13 to 18, around 13 to 19, around 13 to 20, or around 14 to 15, around 14 to 16, around 14 to 17, around 14 to 18, around 14 to 19. Approximately 14 to 20, or approximately 15 to 16, approximately 15 to 17, approximately 15 to 18, approximately 15 to 19, approximately 15 to 20, approximately 16 to 17, approximately 16 to 18, approximately 16 to 19, approximately 16 to 20, or approximately 17 to 18, approximately 17 to 19, approximately 17 to 20, approximately 18 to 19, approximately 18 to 20, approximately 19 to 20, approximately 10 to 50, approximately 15 to 50, approximately 15 to 45, approximately 15 to 40, approximately 15 to 35, or approximately 15 to 30.

[0204] In some embodiments, the DAR of the ADC composition described herein is about 1 to 20. In some embodiments, the DAR of the ADC compositions described herein is about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 3.5 to 7.0, about 3.5 to 7.5, about 3.5 to 8.0, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 7.5, about 4.0 to 8.0, about 4.5 to 5.0, about 4.5 to 5.5, about 4. .5 to 6.0, approximately 4.5 to 6.5, approximately 4.5 to 7.0, approximately 4.5 to 7.5, approximately 4.5 to 8.0, approximately 5.0 to 5.5, approximately 5.5 to 6.0, approximately 5.5 to 6.5, approximately 5.5 to 7.0, approximately 5.5 to 7.5, approximately 5.5 to 8.0, approximately 6.0 to 6.5, approximately 6.0 to 7.0, approximately 6.0 to 7.5, approximately 6.0 to 8.0, approximately 6.5 to 7.0, approximately 6.5 to 7.5, approximately 6.5 to 8.0, approximately 7.0 to 7.5, approximately 7.0 to 8.0, approximately 7.5 to 8.0, approximately 7.5 to 8.5, approximately 7.5 to 9.0, approximately 7.5 to 9.5, approximately 8.0 to 8.5, approximately 8.0 to 9.0, approximately 8.0 to 9.5, approximately 8.0 to 10.0, approximately 8.5 to 9.0, approximately 8.5 to 9.5, approximately 8.5 to 10.0, approximately 8.5 to 10.5, approximately 9.0 to 9.5, approximately 9.0 to 10.0, approximately 9.0 to 10.5, approximately 9.0 to 11.0, approximately 9.5 to 10.0, approximately 9.5 to 10.5, approximately 9.5 to 11.0, approximately 9.5 to 11.5, approximately 9.5 to 12.0, approximately 9.5 to 13.5, approximately 9.5 to 14.0, approximately 9.5 to 15.5, approximately 9.5 to 16.0, approximately 9.5 to 16.5, approximately 9.5 to 17.0, approximately 9.5 to 17.5, approximately 9.5 to 18.0, approximately 9.5 to 18.5, approximately 9.5 to 19.0, approximately 9.5 to 1 9.5, approximately 9.5 to 20.0, approximately 10.0 to 10.5, approximately 10.0 to 11.0, approximately 10.0 to 11.5, approximately 10.0 to 12.0, approximately 10.0 to 13.5, approximately 10.0 to 14.0, approximately 10.0 to 15.5, approximately 10.0 to 16.0, approximately 10.0 to 16.5, approximately 10.0 to 17.0, approximately 10.0 to 17.5, approximately 10.0 to 18.0, approximately 10.0 to 18.5, approximately 10.0 to 19.0, approximately 10.0 to 19.5, approximately 10.0 to 20.0, approximately 10.5 to 11.0, approximately 10.5 to 11.5, approximately 10.5 to 12.0, approximately 10.5 to 13.5, approximately 10.5 to 14.0, approximately 10.5 to 15.5, approximately 10.5 to 16.0, approximately 10.5 to 16.5, approximately 10.5 to 17.0, approximately 10.5 to 17.5, approximately 10.5 to 18.0, approximately 10.5 to 18.5, approximately 10.5 to 19.0, approximately 10.5 to 19.5, approximately 10.5 to 20.0, approximately 11.0 to 11.5, approximately 11.0 to 12.0, approximately 11.0 to 13.5, approximately 11.0 to 14.0, approximately 11.0 to 15.5, approximately 11.0 to 16.0, approximately 11.0 to 16.5, approximately 11.0 to 17. 0, approximately 11.0 to 17.5, approximately 11.0 to 18.0, approximately 11.0 to 18.5, approximately 11.0 to 19.0, approximately 11.0 to 19.5, approximately 11.0 to 20.0, approximately 11.5 to 12.0, approximately 11.5 to 13.5, approximately 11.5 to 14.0, approximately 11.5 to 15.5, approximately 11.5 to 16.0, approximately 11.5 to 16.5, approximately 11.5 to 17.0, approximately 11.5 to 17.5, approximately 11.5 to 18.0, approximately 11.5 to 18.5, approximately 11.5 to 19.0, approximately 11.5 to 19. 5, approximately 11.5 to 20.0, approximately 12.0 to 13.5, approximately 12.0 to 14.0, approximately 12.0 to 15.5, approximately 12.0 to 16.0, approximately 12.0 to 16.5, approximately 12.0 to 17.0, approximately 12.0 to 17.5, approximately 12.0 to 18.0, approximately 12.0 to 18.5, approximately 12.0 to 19.0, approximately 12.0 to 19.5, approximately 12.0 to 20.0, approximately 12.5 to 10.0, approximately 12.5 to 10.5, approximately 12.5 to 11.0, approximately 12.5 to 11.5, approximately 12.5 to 12. 0, approximately 12.5 to 13.5, approximately 12.5 to 14.0, approximately 12.5 to 15.5, approximately 12.5 to 16.0, approximately 12.5 to 16.5, approximately 12.5 to 17.0, approximately 12.5 to 17.5, approximately 12.5 to 18.0, approximately 12.5 to 18.5, approximately 12.5 to 19.0, approximately 12.5 to 19.5, approximately 12.5 to 20.0, approximately 10.5 to 50, approximately 15.5 to 50.0, approximately 15.5 to 45.5, approximately 15.5 to 40.5, approximately 15.5 to 35.5, or approximately 15.5 to 30.5.

[0205] In some embodiments, the DAR of the ADC composition described herein is about 8.0 to 12.0.

[0206] In some embodiments, the DAR of the ADC composition described herein is about 9.0 to 11.0.

[0207] In some embodiments, the DAR of the ADC composition described herein is about 10.0 to 10.5, for example, 10.2.

[0208] In some embodiments, the DAR of the ADC composition described herein is from about 10.0 to 30.0, for example 17.7, 16.8, 25.5, 25.0, 17.6, 17.1, 24.2, or 22.6.

[0209] In some embodiments, the ADC compositions described herein comprise a plurality of ADCs described herein, wherein each ADC comprises two different drug-linkers described herein, namely a first drug-linker and a second drug-linker. The compositions are characterized in that the ratio of drug to antibody in the first drug-linker is in the range of about 1 to about 30, and the ratio of drug to antibody in the second drug-linker is in the range of about 1 to about 30. The ratio of drug to antibody in the first drug-linker and the ratio of drug to antibody in the second drug-linker can also be determined using the method for determining DAR described above.

[0210] In some embodiments, the ratio of drug to antibody in the first drug conjugate is about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 3.5 to 7.0, about 3.5 to 7.5, about 3.5 to 8.0, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 7.5, about 4.0 to 8.0, about 4.5 to 5.0, about 4.5 to 5.5, about 4.5 to 6. .0, approximately 4.5 to 6.5, approximately 4.5 to 7.0, approximately 4.5 to 7.5, approximately 4.5 to 8.0, approximately 5.0 to 5.5, approximately 5.5 to 6.0, approximately 5.5 to 6.5, approximately 5.5 to 7.0, approximately 5.5 to 7.5, approximately 6.0 to 8.0, approximately 6.0 to 6.5, approximately 6.0 to 7.0, approximately 6.0 to 7.5, approximately 6.0 to 8.0, approximately 6.5 to 7.0, approximately 6.5 to 7.5, approximately 6.5 to 8.0, approximately 7.0 to 7.5, approximately 7.0 to 8.0, approximately 7.5 to 8.0, approximately 7.5 to 8.5, approximately 7.5 to 9.0, approximately 7.5 to 9.5, approximately 8.0 to 8.5, approximately 8.0 to 9.0, approximately 8.0 to 9.5, approximately 8.0 to 10.0, approximately 8. 5 to 9.0, approximately 8.5 to 9.5, approximately 8.5 to 10.0, approximately 8.5 to 10.5, approximately 9.0 to 9.5, approximately 9.0 to 10.0, approximately 9.0 to 10.5, approximately 9.0 to 11.0, approximately 9.5 to 10.0, approximately 9.5 to 10.5, approximately 9.5 to 11.0, approximately 9.5 to 11.5, approximately 9.5 to 12.0, approximately 9.5 to 13.5, approximately 9.5 to 14.0, approximately 9.5 to 15.5, approximately 9.5 to 16.0, approximately 9.5 to 16.5, approximately 9.5 to 17.0, approximately 9.5 to 17.5, approximately 9.5 to 18.0, approximately 9.5 to 18.5, approximately 9.5 to 19.0, approximately 9.5 to 19.5, approximately 9.5 to 20.0, approximately 10.0 to 10.5, approximately 10.0 to 11.0, approximately 10.0 to 11.5, approximately 10.0 to 12.0, approximately 10.0 to 13.5, approximately 10.0 to 14.0, approximately 10.0 to 15.5, approximately 10.0 to 16.0, approximately 10.0 to 16.5, approximately 10.0 to 17.0, approximately 10.0 to 17.5, approximately 10.0 to 18.0 Approximately 10.0 to 18.5, approximately 10.0 to 19.0, approximately 10.0 to 19.5, approximately 10.0 to 20.0, approximately 10.5 to 11.0, approximately 10.5 to 11.5, approximately 10.5 to 12.0, approximately 10.5 to 13.5, approximately 10.5 to 14.0, approximately 10.5 to 15.5, approximately 10.5 to 16.0, approximately 10.5 to 16.5, approximately 10.5 to 17.0, approximately 10.5 to 17.5, approximately 10.5 to 18.0, approximately 10.5 to 18.5, approximately 10.5 to 19.0, approximately 10.5 to 19.5, approximately 10.5 to 20.0, approximately 11.0 to 11.5, approximately 11.0 to 12.0, approximately 11.0 to 13.5, approximately 11.0 to 14.0, approximately 11.0 to 15.5, approximately 11.0 to 16.0, approximately 11.0 to 16.5, approximately 11.0 to 17.0, approximately 11.0 to 17 .5, approximately 11.0 to 18.0, approximately 11.0 to 18.5, approximately 11.0 to 19.0, approximately 11.0 to 19.5, approximately 11.0 to 20.0, approximately 11.5 to 12.0, approximately 11.5 to 13.5, approximately 11.5 to 14.0, approximately 11.5 to 15.5, approximately 11.5 to 16.0, approximately 11.5 to 16.5, approximately 11.5 to 17.0, approximately 11.5 to 17.5, approximately 11.5 to 18.0, approximately 11.5 to 18.5, approximately 11.5 to 19.0, approximately 11.5 to 19.5, approximately 11.5 to 20.0, approximately 12.0 to 13.5, approximately 12.0 to 14.0, approximately 12.0 to 15.5, approximately 12.0 to 16.0, approximately 12.0 to 16.5, approximately 12.0 to 17.0, approximately 12.0 to 17.5, approximately 12.0 to 18.0, approximately 12.0 to 18.5, approximately 12.0 to 19.0, approximately 12.0 to 19.5, approximately 12.0 to 20.0, approximately 12.5 to 10.0, approximately 12.5 to 10.5, approximately 12.5 Up to 11.0, approximately 12.5 to 11.5, approximately 12.5 to 12.0, approximately 12.5 to 13.5, approximately 12.5 to 14.0, approximately 12.5 to 15.5, approximately 12.5 to 16.0, approximately 12.5 to 16.5, approximately 12.5 to 17.0, approximately 12.5 to 17.5, approximately 12.5 to 18.0, approximately 12.5 to 18.5, approximately 12.5 to 19.0, approximately 12.5 to 19.5, approximately 12.5 to 20.0, approximately 10.5 to 30, or approximately 15.5 to 30.0.

[0211] In some embodiments, the ratio of drug to antibody in the second drug linker is about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 3.5 to 7.0, about 3.5 to 7.5, about 3.5 to 8.0, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 7.5, about 4.0 to 8.0, about 4.5 to 5.0, about 4.5 to 5.5, about 4.5 to 6. .0, approximately 4.5 to 6.5, approximately 4.5 to 7.0, approximately 4.5 to 7.5, approximately 4.5 to 8.0, approximately 5.0 to 5.5, approximately 5.5 to 6.0, approximately 5.5 to 6.5, approximately 5.5 to 7.0, approximately 5.5 to 7.5, approximately 6.0 to 8.0, approximately 6.0 to 6.5, approximately 6.0 to 7.0, approximately 6.0 to 7.5, approximately 6.0 to 8.0, approximately 6.5 to 7.0, approximately 6.5 to 7.5, approximately 6.5 to 8.0, approximately 7.0 to 7.5, approximately 7.0 to 8.0, approximately 7.5 to 8.0, approximately 7.5 to 8.5, approximately 7.5 to 9.0, approximately 7.5 to 9.5, approximately 8.0 to 8.5, approximately 8.0 to 9.0, approximately 8.0 to 9.5, approximately 8.0 to 10.0, approximately 8. 5 to 9.0, approximately 8.5 to 9.5, approximately 8.5 to 10.0, approximately 8.5 to 10.5, approximately 9.0 to 9.5, approximately 9.0 to 10.0, approximately 9.0 to 10.5, approximately 9.0 to 11.0, approximately 9.5 to 10.0, approximately 9.5 to 10.5, approximately 9.5 to 11.0, approximately 9.5 to 11.5, approximately 9.5 to 12.0, approximately 9.5 to 13.5, approximately 9.5 to 14.0, approximately 9.5 to 15.5, approximately 9.5 to 16.0, approximately 9.5 to 16.5, approximately 9.5 to 17.0, approximately 9.5 to 17.5, approximately 9.5 to 18.0, approximately 9.5 to 18.5, approximately 9.5 to 19.0, approximately 9.5 to 19.5, approximately 9.5 to 20.0, approximately 10.0 to 10.5, approximately 10.0 to 11.0, approximately 10.0 to 11.5, approximately 10.0 to 12.0, approximately 10.0 to 13.5, approximately 10.0 to 14.0, approximately 10.0 to 15.5, approximately 10.0 to 16.0, approximately 10.0 to 16.5, approximately 10.0 to 17.0, approximately 10.0 to 17.5, approximately 10.0 to 18.0 Approximately 10.0 to 18.5, approximately 10.0 to 19.0, approximately 10.0 to 19.5, approximately 10.0 to 20.0, approximately 10.5 to 11.0, approximately 10.5 to 11.5, approximately 10.5 to 12.0, approximately 10.5 to 13.5, approximately 10.5 to 14.0, approximately 10.5 to 15.5, approximately 10.5 to 16.0, approximately 10.5 to 16.5, approximately 10.5 to 17.0, approximately 10.5 to 17.5, approximately 10.5 to 18.0, approximately 10.5 to 18.5, approximately 10.5 to 19.0, approximately 10.5 to 19.5, approximately 10.5 to 20.0, approximately 11.0 to 11.5, approximately 11.0 to 12.0, approximately 11.0 to 13.5, approximately 11.0 to 14.0, approximately 11.0 to 15.5, approximately 11.0 to 16.0, approximately 11.0 to 16.5, approximately 11.0 to 17.0, approximately 11.0 to 17 .5, approximately 11.0 to 18.0, approximately 11.0 to 18.5, approximately 11.0 to 19.0, approximately 11.0 to 19.5, approximately 11.0 to 20.0, approximately 11.5 to 12.0, approximately 11.5 to 13.5, approximately 11.5 to 14.0, approximately 11.5 to 15.5, approximately 11.5 to 16.0, approximately 11.5 to 16.5, approximately 11.5 to 17.0, approximately 11.5 to 17.5, approximately 11.5 to 18.0, approximately 11.5 to 18.5, approximately 11.5 to 19.0, approximately 11.5 to 19.5, approximately 11.5 to 20.0, approximately 12.0 to 13.5, approximately 12.0 to 14.0, approximately 12.0 to 15.5, approximately 12.0 to 16.0, approximately 12.0 to 16.5, approximately 12.0 to 17.0, approximately 12.0 to 17.5, approximately 12.0 to 18.0, approximately 12.0 to 18.5, approximately 12.0 to 19.0, approximately 12.0 to 19.5, approximately 12.0 to 20.0, approximately 12.5 to 10.0, approximately 12.5 to 10.5, approximately 12.5 Up to 11.0, approximately 12.5 to 11.5, approximately 12.5 to 12.0, approximately 12.5 to 13.5, approximately 12.5 to 14.0, approximately 12.5 to 15.5, approximately 12.5 to 16.0, approximately 12.5 to 16.5, approximately 12.5 to 17.0, approximately 12.5 to 17.5, approximately 12.5 to 18.0, approximately 12.5 to 18.5, approximately 12.5 to 19.0, approximately 12.5 to 19.5, approximately 12.5 to 20.0, approximately 10.5 to 30, or approximately 15.5 to 30.0.

[0212] Pharmaceutical Composition

[0213] In another aspect, this application provides a pharmaceutical composition comprising any of the antibody-drug conjugates described above, and one or more pharmaceutical excipients.

[0214] 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.

[0215] application

[0216] The antibody-drug conjugates, drug-linkers, ADC compositions, or drug compositions thereof described herein can be used to treat a variety of diseases or conditions, such as Her2-expressing cancers, including solid tumors or hematologic malignancies such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

[0217] Therefore, this application provides the use of any of the antibody-drug conjugates, drug-linkers, ADC compositions, or pharmaceutical compositions containing the thereof described in the foregoing in the preparation of a medicament for treating Her2-expressing cancers.

[0218] 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.

[0219] Additionally, this application provides a method for treating Her2-expressing cancer, comprising the step of administering an effective amount of any of the preceding antibody-drug conjugates, drug-linkers, ADC compositions, or pharmaceutical compositions containing the conjugates to a subject in need thereof.

[0220] In some embodiments, the antibody-drug conjugate, drug-linker, ADC composition, or drug composition is sufficient (e.g., in a subject):

[0221] (1) Inhibits the proliferation of cells (such as tumor cells);

[0222] (2) Inhibits tumor growth;

[0223] (3) Inducing and / or increasing antibody-dependent cytotoxic activity;

[0224] (4) Inhibit HER2-mediated signal transduction;

[0225] (5) Prevention and / or treatment of HER2-mediated diseases / disorders; or

[0226] (6) Any combination of (1)-(5) above.

[0227] 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.

[0228] This application provides the use of any of the compounds described above or their pharmaceutically acceptable salts, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, antibody-drug conjugates, compositions or pharmaceutical compositions in the preparation of medicaments for the treatment or prevention of cancer.

[0229] In addition, this application provides any of the compounds described above or their pharmaceutically acceptable salts, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites or prodrugs, antibody-drug conjugates, compositions or pharmaceutical compositions for the treatment or prevention of cancer.

[0230] Meanwhile, this application provides a method for treating or preventing cancer, comprising the step of administering to a subject in need an effective amount of any of the preceding compounds or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, antibody-drug conjugate, composition or pharmaceutical composition thereof.

[0231] In some implementations, the cancer is a cancer that expresses HER2. In some implementations, the cancer is selected from solid tumors or hematologic malignancies; for example, breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), colon cancer, and lymphoma.

[0232] 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.

[0233] Additionally, this application provides a method for treating Her2-expressing cancer, comprising the step of administering an effective amount of any of the preceding antibody-drug conjugates, drug-linkers, ADC compositions, or pharmaceutical compositions containing the conjugates to a subject in need thereof.

[0234] definition

[0235] 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.

[0236] 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.

[0237] 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).

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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).

[0242] 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.

[0243] 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.

[0244] 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:51) amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO:52) 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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).

[0251] 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.

[0252] 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).

[0253] 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.

[0254] 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.

[0255] 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.

[0256] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight-chain or branched hydrocarbon group, such as "C". 1-20 Alkylene, C 1-10 Alkylene, C 3-10 Alkylene, C 5-8 Alkylene, C 1-6 Alkylene, C 1-4 Alkylene, C 1-3 "alkylene", etc., specific examples include but are not limited to: methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene, etc.

[0257] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C..." 2-6 "alkenyl", "C" 2-4 Examples of these include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc.

[0258] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon triple bond. This includes, for example, "C..." 2-6 "Alkyne", "C" 4-6 Examples of "alkynyl" include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butyrynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentyrynyl, 1,4-pentyrynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4-hexadiynyl, etc.

[0259] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, including but not limited to monocycloalkyl and bicycloalkyl (such as spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl). The term "C"3-6 "Cycloalkyl" refers to a cycloalkyl group having 3 to 6 cyclic carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., which may optionally be substituted by one or more (such as 1, 2 or 3) suitable substituents, such as methyl-substituted cyclopropyl.

[0260] The term "heterocyclic group" or "heterocycle" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic cyclic structure whose ring atoms consist of carbon atoms and at least one (e.g., 1, 2, or 3) heteroatoms selected from nitrogen, oxygen, and sulfur. The heterocyclic group can be connected to the rest of the molecule through any one ring atom, provided that valence requirements are met. The heterocyclic group in this invention is preferably a 3-6 membered heterocyclic group. The term "3-6 membered heterocyclic group" as used in this invention refers to a heterocyclic group having 3 to 6 ring atoms, including 3-membered, 4-membered, 5-membered, and 6-membered heterocyclic groups, including nitrogen-containing heterocyclic groups and oxygen-containing heterocyclic groups, such as 4-6 membered heterocyclic groups, for example, 4-6 membered nitrogen-containing heterocyclic groups and 4-6 membered oxygen-containing heterocyclic groups. Common heterocyclic groups include (but are not limited to) azetidinyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, and morpholinyl. The heterocyclic groups in this invention may optionally be substituted with one or more of the substituents described herein. The heterocyclic groups in this invention may optionally be fused with one or more aromatic or non-aromatic rings.

[0261] The term "heteroaromatic ring" refers to an aromatic ring structure containing at least one ring member selected from N, O, and S. Specific examples include, but are not limited to, 5-6 membered aromatic heterocycles, 5-6 membered nitrogen-containing aromatic heterocycles, and 5-6 membered oxygen-containing aromatic heterocycles, such as furan, thiophene, pyrrole, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, oxadiazole, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,3,5-triazine, 1,2,4,5-tetraazine, etc.

[0262] The term "alkoxy" refers to a group having an "alkyl-O-" structure, where alkyl is defined as described above. For example, C 1-6 Alkoxy, C 1-4 Alkoxy, C 1-3Alkoxy or C 1-2 Alkoxy groups, etc. Common alkoxy groups include (but are not limited to) methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups in this invention are optionally substituted by one or more substituents described in this invention.

[0263] The term "halogenated" or "halogenated" is defined as including F, Cl, Br, or I.

[0264] The term "stereoisomer" refers to isomers formed due to the presence of at least one asymmetric center in a compound. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, mixtures of diastereomers, and individual diastereomers can be produced. Specific individual molecules may also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0265] This invention covers all possible crystalline forms or polymorphs of the compounds of this invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion.

[0266] The compounds of the present invention can exist as solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, can be stoichiometric or non-stoichiometric.

[0267] Those skilled in the art will understand that not all nitrogen-containing heterocycles can form N-oxides because nitrogen requires available lone pairs of electrons to be oxidized into oxides; those skilled in the art will identify nitrogen-containing heterocycles that can form N-oxides. Those skilled in the art will also recognize that tertiary amines can form N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art, including the oxidation of heterocycles and tertiary amines with peroxy acids such as peracetic acid and m-chloroperoxybenzoic acid (MCPBA), hydrogen peroxide, alkyl peroxides such as tert-butyl peroxide, sodium perborate, and dioxiranes such as dimethyldioxirane. These methods for preparing N-oxides have been extensively described and reviewed in the literature, see, for example: T.L. Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp. 748-750; A.R. Katritzky and A.J. Boulton, Eds., Academic Press; and G.W. H. Heeseman and E.S. G. Wierstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp. 390-392, A.R. Katritzky and A.J. Boulton, Eds., Academic Press.

[0268] The term "isotopically labeled compound" means that the compound is structurally identical to the compound of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number than the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g.) 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); and isotopes of sulfur (e.g. 35 S).

[0269] The scope of this invention also includes metabolites of the compounds of this invention, i.e., substances formed in the body when the compounds of this invention are administered. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of this invention, including compounds obtained by methods that expose the compounds of this invention to mammals for a time sufficient to produce their metabolites.

[0270] This invention further includes, within its scope, prodrugs of the compounds of the invention, which are certain derivatives of the compounds of the invention that may themselves have little or no pharmacological activity, and which, when administered to or onto the body, can be converted, for example, by hydrolysis and cleavage into the compounds of the invention having the desired activity. Typically, such prodrugs are functional group derivatives of the compounds that are readily converted in vivo into the compounds with the desired therapeutic activity. Further information regarding the use of prodrugs can be found in “Pro-drugs as Novel Delivery Systems,” Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella) and “Bioreversible Carriers in Drug Design,” Pergamon Press, 1987 (EB Roche, editor, American Pharmaceutical Association). The prodrugs of the invention can be prepared, for example, by replacing suitable functional groups present in the compounds of the invention with certain portions known to those skilled in the art as “pro-moiety” (e.g., as described in “Design of Prodrugs,” H. Bundgaard (Elsevier, 1985)).

[0271] In any process of preparing the compounds of the present invention, protection of sensitive or reactive groups on any relevant molecule may be necessary and / or desired, thereby forming a form of chemical protection for the compounds of the present invention. This can be achieved by conventional protecting groups, for example, those described in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P. ​​G.W. Uts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which are incorporated herein by reference. Protecting groups can be removed at appropriate subsequent stages using methods known in the art.

[0272] If a functional group or structural segment is described as “substituted or unsubstituted”, then the functional group or structural segment may be (1) unsubstituted or (2) substituted.

[0273] As used herein, the term "one or more" means one or more under reasonable conditions, such as two, three, four, five, or ten.

[0274] Unless otherwise specified, as used herein, the connection point of a substituent may be derived from any suitable location of the substituent.

[0275] Pharmaceutically acceptable salts of the compounds of the present invention include their acid addition salts and base addition salts. Suitable acid addition salts are formed by acids that form pharmaceutically acceptable salts, including aspartate, fumarate, glucohepanoate, glucuronide, glucuronide, hexafluorophosphate, etc. Suitable base addition salts are formed by bases that form pharmaceutically acceptable salts, including aluminum salts, arginine salts, choline salts, diethylamine salts, etc. For a review of suitable salts, see Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0276] Whether explicitly stated or not, all numerical values ​​in this application are modified by the term "about". The term "about" means within ±20%, ±10%, preferably ±5%, and more preferably ±2% of the stated numerical value.

[0277] 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.

[0278] Information about the sequences involved in this invention is described in the table below:

[0279] The abbreviations used in this article have the following meanings:

[0280] 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).

[0281] 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).

[0282] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0283] 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.

[0284] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0285] I. Preparation of intermediates

[0286] Example 1 of intermediate preparation: Preparation of 25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynic acid (INT-1)

[0287] Step 1: Preparation of 1-amino-3,6,9,12,15-pentaoxoctadecane-18-acid (INT-1-2)

[0288] 0.50 g (1.22 mmol) of 2,2-dimethyl-4-oxo-3,8,11,14,17,20-hexaoxa-5-azatriane-23-acid was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (3 mL) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was directly concentrated under reduced pressure to give the title compound as crude trifluoroacetate (650 mg, 1.21 mmol).

[0289] Its structural characterization data are as follows:

[0290] MS m / z (ESI): 310.2 [M+H] +

[0291] Step 2: Preparation of 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynical acid (INT-1)

[0292] 1-Amino-3,6,9,12,15-pentoxaoctadecanoic acid trifluoroacetate (650 mg, 1.21 mmol) was dissolved in DMF (5.0 mL), and DIPEA (781.61 mg, 6.05 mmol) was added. Then, 2,5-dioxopyrrolidone-1-yl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (441.91 mg, 1.21 mmol) was added in portions, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by reversed-phase column chromatography (acetonitrile / 0.05% ammonium bicarbonate aqueous solution = 0–60%) and then freeze-dried to give the title compound (646 mg, 1.15 mmol).

[0293] Its structural characterization data are as follows:

[0294] MS m / z (ESI): 560.3 [M+H] +

[0295] Example 2 of intermediate preparation: Preparation of (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (INT-2)

[0296] Step 1: Preparation of L-alanyl-L-alanine (INT-2-1)

[0297] (tert-butoxycarbonyl)-L-alanyl-L-alanine (100 mg, 384.19 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added. The reaction was carried out at 20 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, yielding the pale yellow title compound (160 mg, 370.93 μmol).

[0298] Its structural characterization data are as follows:

[0299] MS m / z (ESI): 161.1 [M+H] +

[0300] Step 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (INT-2)

[0301] L-alanyl-L-alanine (160 mg, 412.14 μmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetyl ester (150.58 mg, 412.14 μmol) were dissolved in N,N-dimethylacetamide (2 mL), and N,N-diisopropylethylamine (266.33 mg, 2.06 mmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction solution was subjected to rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to give the title compound (125 mg, 304.55 μmol).

[0302] Its structural characterization data are as follows:

[0303] MS m / z (ESI): 428.1 [M+H2O] +

[0304] Example 3 of intermediate preparation: Preparation of (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycine (INT-3)

[0305] Glycylglycine (1 g, 7.57 mmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (2.77 g, 7.57 mmol) were dissolved in dimethyl sulfoxide (10 mL). The reaction mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (2.52 g, 6.59 mmol).

[0306] Its structural characterization data are as follows:

[0307] MS m / z (ESI): 383.2 [M+H] +

[0308] Example 4 of intermediate preparation: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (INT-4)

[0309] Step 1: Preparation of glycyl-glycyl-L-phenylalanine (INT-4-2)

[0310] ((benzyloxy)carbonyl)glycylglycyl-L-phenylalanine (200 mg, 483.77 μmol) was dissolved in methanol (8 mL). After purging with nitrogen, palladium on carbon (20 mg) was added. The gas was purged three times with hydrogen, and the reaction was stirred at room temperature for 3 hours. After the reaction was complete, palladium on carbon was removed by filtration. The filtrate was then concentrated to remove the solvent, giving the title compound (135 mg, 483.37 μmol).

[0311] Its structural characterization data are as follows:

[0312] MS m / z (ESI): 280.2 [M+H] +

[0313] Step 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (INT-4)

[0314] Glycylglycyl-L-phenylalanine (130 mg, 465.46 μmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (170.06 mg, 465.46 μmol) were dissolved in N,N-dimethylformamide (8 mL), and N,N-diisopropylethylamine (120.32 mg, 930.93 μmol) was added. The reaction was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (60 mg, 0.11 mmol).

[0315] Its structural characterization data are as follows:

[0316] MS m / z (ESI): 530.3 [M+H] +

[0317] Example 5 of intermediate preparation: Preparation of (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-5)

[0318] Step 1: Preparation of 2-hydroxymethyl-N-methyl-5-nitrobenzamide (INT-5-2)

[0319] 50.0 g (279 mmol) of 6-nitroisobenzofuran-1(3H)-one was added to a tetrahydrofuran solution of methylamine (2 M, 500 mL), and the mixture was heated to 75 °C and stirred for 5 hours. The reaction solution was directly concentrated to obtain the crude title compound (58.0 g), which was used directly in the next step without purification.

[0320] Its structural characterization data are as follows:

[0321] ESI-MS (m / z): 211.0 [M+H] + .

[0322] Step 2: Preparation of (2-((methylamino)methyl)-4-nitrophenyl)methanol (INT-5-3)

[0323] 2-Hydroxymethyl-N-methyl-5-nitrobenzamide (25.0 g, 119 mmol) was dissolved in tetrahydrofuran (500 mL). After cooling to 0 °C, a borane-dimethyl sulfide solution (10 M, 89.3 mL) was added dropwise to the reaction system. After the addition was complete, the temperature was raised to 70 °C and stirred for 5 hours. The temperature was lowered to 0 °C again, and a hydrogen chloride-methanol solution (2 M, 100 mL) was added dropwise to the above reaction solution. The temperature was raised again to 65 °C and stirred for 12 hours. The reaction solution was filtered, and the filtrate was directly concentrated to obtain the crude title compound (44.3 g), which was used directly in the next step without purification.

[0324] Its structural characterization data are as follows:

[0325] ESI-MS (m / z): 197.0 [M+H] + .

[0326] Step 3: Preparation of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethylamine (INT-5-4)

[0327] (2-((methylamino)methyl)-4-nitrophenyl)methanol (40.3 g, 205 mmol) was dissolved in dichloromethane (800 mL), cooled to 0 °C, and then imidazole (55.9 g, 822 mmol) and tert-butyldiphenylchlorosilane (84.0 g, 308 mmol, 78.8 mL) were added. The mixture was then restored to 25 °C and stirred for 1 hour. The reaction was quenched with water (200 mL), and the mixture was extracted three times with dichloromethane (300 mL x 3). The combined organic phases were washed with saturated sodium chloride aqueous solution (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After purification by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 5 / 1), the product was concentrated again to obtain the title compound (59.7 g, 137 mmol).

[0328] Its structural characterization data are as follows:

[0329] ESI-MS (m / z): 435.1 [M+H] + .

[0330] Step 4: Preparation of (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (INT-5-5)

[0331] 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethylamine (54.9 g, 126 mmol) was dissolved in dichloromethane (550 mL), cooled to 0 °C, and DIPEA (48.9 g, 379 mmol, 66.0 mL) and allyl chloroformate (30.5 g, 253 mmol, 26.8 mL) were added. The mixture was then restored to 25 °C and stirred for 1 hour. The reaction was quenched with water (300 mL), and the mixture was extracted three times with dichloromethane (200 mL x 3). The combined organic phases were washed with saturated sodium chloride aqueous solution (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After purification by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1 to 3 / 1), the product was concentrated again to obtain the title compound (65.5 g, 126 mmol).

[0332] Its structural characterization data are as follows:

[0333] ESI-MS (m / z): 519.1 [M+H] + .

[0334] Step 5: Preparation of (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-5)

[0335] (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (62.8 g, 121 mmol) was dissolved in a mixed solvent of ethanol (300 mL) and water (300 mL). Iron powder (33.8 g, 605 mmol) and ammonium chloride (64.8 g, 1.21 mol) were added, and the mixture was heated to 80 °C and stirred for 2 hours. The reaction solution was filtered, and the filtrate was extracted three times with dichloromethane (100 mL x 3). The combined organic phases were washed with a saturated sodium chloride aqueous solution (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (49.3 g).

[0336] Its structural characterization data are as follows:

[0337] ESI-MS (m / z): 511.7 [M+H] + .

[0338] Example 6: Preparation of intermediate (5-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6)

[0339] Step 1: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamidyl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-1)

[0340] (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (150.0 mg, 0.31 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (115.1 mg, 0.37 mmol) were dissolved in DCM (8 mL) and MeOH (2 mL). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (229.7 mg, 0.93 mmol) was added, and the mixture was stirred at room temperature for 15 hours. The solution was concentrated under reduced pressure to obtain the crude product, which was then purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0–90%) and concentrated again under reduced pressure to obtain the title compound (194.2 mg, 0.25 mmol).

[0341] Its structural characterization data are as follows:

[0342] MS m / z (ESI): 782.2 [M+H] +

[0343] Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (INT-6-2)

[0344] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (194.2 mg, 0.25 mmol) was dissolved in DMF (5 mL), and pyridine hydrofluoric acid salt (390.2 mg, 3.93 mmol) was added. The mixture was stirred at room temperature for 15 hours. After the reaction was complete, 20 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL x 3), washed with 10 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound. The crude product was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to obtain the title compound (109.3 mg, 0.21 mmol).

[0345] Its structural characterization data are as follows:

[0346] MS m / z (ESI): 566.1 [M+Na] +

[0347] Step 3: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-3)

[0348] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (109.3 mg, 0.21 mmol) was dissolved in DMF (5 mL), DIPEA (81.3 mg, 0.63 mmol) was added, followed by p-nitrophenyl chloroformate (50.8 mg, 0.25 mmol). The mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (134.50 mg, 0.19 mmol).

[0349] Its structural characterization data are as follows:

[0350] MS m / z (ESI): 731.2 [M+Na] +

[0351] Step 4: Preparation of (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7] indenyl[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-4)

[0352] Allyl carbamate (134.50 mg, 0.19 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H 165.50 mg, 0.38 mmol of benzo[de]pyran[3',4':6,7]-indo[1,2-b]quinoline-10,13-dione was dissolved in DMF (5 mL), and 1-hydroxybenzotriazole (30.80 mg, 0.23 mmol), pyridine (16.5 mg, 0.21 mmol), and DIPEA (73.5 mg, 0.57 mmol) were added. The mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.7) and lyophilized to give the title compound (114.70 mg, 0.12 mmol).

[0353] Its structural characterization data are as follows:

[0354] MS m / z (ESI): 1005.2 [M+H] +

[0355] Step 5: Preparation of (5-((S)-2-aminopropamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-5)

[0356] Dissolve (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7] indene[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (114.70 mg, 0.12 mmol) in DMF (3 mL), add diethylamine (87.8 mg, 1.2 mmol), and stir at room temperature for 1 hour. After the reaction was complete, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.7) after most of the solvent was removed, and then freeze-dried to obtain the title compound (62.6 mg, 0.08 mmol).

[0357] Its structural characterization data are as follows:

[0358] MS m / z(ESI): 783.1 [M+H] +

[0359] Step Six: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-6)

[0360] (5-((S)-2-aminopropamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl) Allyl carbamate (1.0 g, 1.28 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (476.90 mg, 1.41 mmol) were dissolved in DMF (5 mL). HATU (631.06 mg, 1.66 mmol) and DIPEA (495.29 mg, 3.83 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was rapidly purified and then freeze-dried to give the title compound (950 mg, 860.37 μmol).

[0361] Its structural characterization data are as follows:

[0362] MS m / z (ESI): 1104.5 [M+H] +

[0363] Its preparation method is as follows:

[0364] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0365] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0366] Step 7: Preparation of (5-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6)

[0367] (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]) Quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)methyl)carbamate (950 mg, 860.37 μmol) was dissolved in DMF (3.5 mL), and diethylamine (860.37 μmol) was added. The mixture was stirred at room temperature for 20 min. The reaction solution was concentrated and then added to 100 mL of a mixed solvent (EA / PE = 1 / 3) and stirred overnight. The mixture was filtered and dried to obtain the title compound (750 mg, 850.4 μmol).

[0368] Its structural characterization data are as follows:

[0369] MS m / z (ESI): 882.5 [M+H] +

[0370] Example 7 of intermediate preparation: Preparation of (S)-6-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-methylsulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatridodecane-31-alkynic acid (INT-7)

[0371] Step 1: Preparation of 3-((tert-Butoxycarbonyl)amino)propionate allyl ester (INT-7-2)

[0372] 3-((tert-Butoxycarbonyl)amino)propionic acid (2.3 g, 12.16 mmol) and 3-bromopropene (1.62 g, 13.37 mmol) were dissolved in DMF (10 mL), and potassium carbonate (5.04 g, 36.47 mmol) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was filtered, and the filtrate was directly concentrated to obtain the crude title compound. After purification by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1 to 5 / 1), the crude compound was concentrated again to obtain the title compound (2.2 g, 9.6 mmol).

[0373] Its structural characterization data are as follows:

[0374] ESI-MS (m / z): 252.1 [M+Na] + .

[0375] Step 2: Preparation of allyl 3-aminopropionate (INT-7-3)

[0376] Allyl 3-((tert-Butoxycarbonyl)amino)propionate (2.2 g, 9.6 mmol) was dissolved in dichloromethane (10 mL), followed by the addition of trifluoroacetic acid (5 mL). The mixture was stirred at room temperature for 2 hours. The reaction solution was directly concentrated to give the crude title compound (2.3 g), which was used directly in the next step without purification.

[0377] Its structural characterization data are as follows:

[0378] ESI-MS (m / z): 130.1 [M+H] + .

[0379] Step 3: Preparation of (S)-3-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexamamide)allyl propionate (INT-7-4)

[0380] Allyl 3-aminopropionate (2.0 g, 8.22 mmol) and N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6-(diphenyl(p-tolyl)methyl)-L-lysine (5.14 g, 8.22 mmol) was dissolved in DMF (20 mL), and DIPEA (5.31 g, 41.12 mmol, 7.38 mL) and HATU (6.25 g, 16.45 mmol) were added. The mixture was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (4.5 g, 6.11 mmol).

[0381] Its structural characterization data are as follows:

[0382] ESI-MS (m / z): 436.4 [M+H] + .

[0383] Step 4: Preparation of (S)-3-(2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexamethylene)propionate (INT-7-5)

[0384] (S)-3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)hexamethylene)propionate (3.0 g, 4.08 mmol) was dissolved in DMF (10 mL), and diethylamine (596.30 mg, 8.15 mmol, 828.20 μL) was added. The mixture was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and freeze-dried to give the title compound (1.8 g, 3.5 mmol).

[0385] Its structural characterization data are as follows:

[0386] ESI-MS (m / z): 514.3 [M+H] + .

[0387] Step 5: Preparation of (S)-6-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-(methylsulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatridodecane-31-alkynyl ester (INT-7-6)

[0388] (S)-3-(2-amino-6-((diphenyl(p-tolyl)methyl)amino)hexamamide)propionate (1.8 g, 3.5 mmol) and 25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azatetradecane-24-alkynic acid (1.69 g, 3.01 mmol) were dissolved in DMF (5 mL). DIPEA (1.85 g, 14.34 mmol, 2.57 mL) and HATU (2.18 g, 5.74 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (960 mg, 909.71 μmol).

[0389] Its structural characterization data are as follows:

[0390] MS m / z (ESI): 1055.5 [M+H] +

[0391] Step Six: Preparation of (S)-6-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-methylsulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatridodecane-31-alkynic acid (INT-7)

[0392] (S)-6-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-(2-(methanesulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triaza-tetane-31-alkynyl ester (960 mg, 909.71 μmol) was dissolved in DMF (5 mL), purged three times with nitrogen, and then tetratetraphenylphosphine palladium (525.61 mg, 454.85 μmol) and 1,3-dimethylbarbituric acid (710.21 mg, 4.55 mmol) were added. The mixture was stirred at room temperature for 1 hour. The reaction solution was purified by preparative high-performance liquid chromatography and then freeze-dried to give the title compound (390.0 mg, 384.15 mmol).

[0393] Its structural characterization data are as follows:

[0394] MS m / z (ESI): 1015.4 [M+H] +

[0395] Its preparation method is as follows:

[0396] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0397] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0398] Example 8 of intermediate preparation: Preparation of (5-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8)

[0399] Step 1: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8-1)

[0400] (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (1.0 g, 2.05 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-citrulline (1.22 g, 3.07 mmol) were dissolved in DCM (28 mL) and MeOH (7 mL). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.52 g, 6.15 mmol) was added, and the mixture was stirred at room temperature for 15 hours. The solution was concentrated under reduced pressure to obtain a crude product, which was then purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0–90%) and concentrated again under reduced pressure to obtain the title compound (1.07 g, 1.23 mmol, 90% purity).

[0401] Its structural characterization data are as follows:

[0402] MS m / z (ESI): 868.2 [M+H] +

[0403] Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(hydroxymethyl)benzyl)(methyl)carbamate (INT-8-2)

[0404] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (1.07 g, 1.23 mmol, 90% purity) was dissolved in DMF (8 mL), and pyridine hydrofluoric acid salt (585.2 mg, 5.9 mmol) was added. The mixture was stirred at room temperature for 15 hours. After the reaction was complete, 50 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (30 mL x 3), washed with 30 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound. The crude product was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to obtain the title compound (418.5 mg, 0.67 mmol, 92% purity).

[0405] Its structural characterization data are as follows:

[0406] MS m / z (ESI): 652.1 [M+Na] +

[0407] Step 3: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8-3)

[0408] (S)-(5-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(hydroxymethyl)benzyl)(methyl)carbamate (418.5 mg, 0.67 mmol, 92% purity) was dissolved in DMF (5 mL), DIPEA (172.3 mg, 1.34 mmol) was added, and p-nitrophenyl chloroformate (161.8 mg, 0.81 mmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 3 / 2) and then freeze-dried to give the title compound (416.10 mg, 0.52 mmol, 90% purity).

[0409] Its structural characterization data are as follows:

[0410] MS m / z (ESI): 817.2 [M+Na] +

[0411] Step 4: Preparation of (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8-4)

[0412] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (416.10 mg, 0.52 mmol, 90% purity), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10 H,13H-benzo[de]pyran[3',4':6,7] indo[1,2-b]quinoline-10,13-dione (331.50 mg, 0.78 mmol) was dissolved in DMF (8 mL), and 1-hydroxybenzotriazole (70.20 mg, 0.52 mmol), pyridine (61.5 mg, 0.78 mmol), and DIPEA (201.5 mg, 1.57 mmol) were added. The mixture was stirred at room temperature for 2 hours. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.7), and lyophilized to give the title compound (357.10 mg, 0.33 mmol, 89% purity).

[0413] Its structural characterization data are as follows:

[0414] MS m / z (ESI): 1091.2 [M+H] +

[0415] Step 5: Preparation of (5-((S)-2-amino-5-ureidopentanamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8-5)

[0416] (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (357.10 mg, 0.33 mmol, 89% purity) was dissolved in DMF (3 mL), and diethylamine (73.8 mg, 1.0 mmol) was added. The mixture was stirred at room temperature for 1 hour. After removing most of the solvent from the reaction solution, it was purified by rapid column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to obtain the title compound (242.5 mg, 0.28 mmol, 95% purity).

[0417] Its structural characterization data are as follows:

[0418] MS m / z (ESI): 869.1 [M+H] +

[0419] Step Six: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8-2)

[0420] (5-((S)-2-amino-5-ureidopentanamide)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)methyl)allyl)carbamate (242.5) (mg, 0.28mmol, 95% purity) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (113.6mg, 0.34mmol) were dissolved in DMF (3mL), and HATU (160.3mg, 0.42mmol) and DIPEA (108.4mg, 0.84mmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, 80mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (30mL x 3). The mixture was washed with 50mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound. After purification by rapid column chromatography (silica gel column, dichloromethane / methanol = 10 / 1), the crude product of the title compound (266.6mg, 0.23mmol, 89% purity) was obtained by concentration.

[0421] Its structural characterization data are as follows:

[0422] MS m / z(ESI): 1190.9 [M+H] +

[0423] Step 7: Preparation of (5-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (INT-8)

[0424] (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (266.6 mg, 0.23 mmol, 89% purity) was dissolved in DMF (3 mL), and diethylamine (50.4 mg, 0.69 mmol) was added. The mixture was stirred at room temperature for 1 hour. After removing most of the solvent from the reaction solution, it was purified by rapid column chromatography (C18, water / acetonitrile = 0.6) and freeze-dried to obtain the title compound (211.5 mg, 0.22 mmol, 92% purity).

[0425] Its structural characterization data are as follows:

[0426] MS m / z (ESI): 968.3 [M+H] +

[0427] Example 9 of intermediate preparation: N 6 -(diphenyl(p-tolyl)methyl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (INT-9)

[0428] Step 1: N 6 Preparation of 1-(diphenyl(p-tolyl)methyl)-L-lysine (INT-9-2)

[0429] N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (150.0 mg, 0.24 mmol) was dissolved in DMF (2 mL), and diethylamine (182.3 mg, 2.5 mmol) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, 10 mL of a mixed solvent (EA / PE = 1 / 3) was added for recrystallization, and the solution was filtered to give the title compound (90.1 mg, 0.22 mmol).

[0430] Step Two: N 6 -(diphenyl(p-tolyl)methyl)-N 2 Preparation of -(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (INT-9)

[0431] N6-(diphenyl(p-tolyl)methyl)-L-lysine (90.1 mg, 0.22 mmol) was dissolved in DMF (3 mL), DIPEA (86.3 mg, 0.66 mmol) was added, followed by 2,5-dioxopyrrolidone-1-yl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylacetate (160.6 mg, 0.44 mmol). After the reaction was complete, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 0.5) and then freeze-dried to obtain the title compound (75.2 mg, 0.11 mmol).

[0432] Its structural characterization data are as follows:

[0433] MS m / z (ESI): 653.2 [M+H] +

[0434] Example 10: Preparation of intermediate 3,6,9,12,15,18,21,24,27,30-decamethyl-36-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,10,13,16,19,22,25,28,31-decaoxo-3,6,9,12,15,18,21,24,27,30-decaazahexahexadecyl-35-alkynical acid (INT-10)

[0435] Step 1: Preparation of 5,8,11,14,17,20,23,26,29-nonamethyl-4,7,10,13,16,19,22,25,28-nonaoxo-2,5,8,11,14,17,20,23,26,29-decaazatrione-31-acid (INT-10-2)

[0436] 1-(9H-fluorene-9-yl)-4,7,10,13,16,19,22,25,28,31-decamethyl-3,6,9,12,15,18,21,24,27,30-decaoxo-2-oxa-4,7,10,13,16,19,22,25,28,31-decaazatritane-33-acid (1.0 g, 1.05 mmol) was dissolved in DMF (5.0 mL), and diethylamine (0.5 mL) was added. The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the solvent was removed using a lyophilizer, and 50 mL of ethyl acetate / petroleum ether (1 / 3) was added and stirred for 1 hour. The solvent was then removed to obtain the title compound (688.9 mg, 0.95 mmol, 85% purity).

[0437] Its structural characterization data are as follows:

[0438] MS m / z (ESI): 729.2 [M+H] +

[0439] Step 2: Preparation of 3,6,9,12,15,18,21,24,27,30-decamethyl-36-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,10,13,16,19,22,25,28,31-decaoxo-3,6,9,12,15,18,21,24,27,30-decaazahexadecane-35-alkynical acid (INT-10)

[0440] 5,8,11,14,17,20,23,26,29-nonamethyl-4,7,10,13,16,19,22,25,28-nonaoxo-2,5,8,11,14,17,20,23,26,29-decazaneco-31-acid (688.9 mg, 0.95 mmol, 85% purity) was dissolved in DMF (8 mL), and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetic acid ester (520.2 mg, 1.43 mmol) and DIPEA (367.7 mg, 2.9 mmol) were added. The mixture was stirred at room temperature for 2 hours under nitrogen protection. After the reaction was completed, the product was rapidly purified and then freeze-dried to obtain the title compound (465.1 mg, 0.48 mmol, 98.0% purity).

[0441] Its structural characterization data are as follows:

[0442] MS m / z (ESI): 979.3 [M+H] +

[0443] Its preparation method is as follows:

[0444] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0445] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0446] Example 11: Preparation of intermediate (25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynyl)-L-valine (INT-11)

[0447] Step 1: Preparation of (25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-ynyl)-L-valine tert-butyl ester (INT-11-1)

[0448] 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynyl acid (0.23 g, 0.41 mmol) and L-valine tert-butyl ester (0.95 g, 0.45 mmol) were dissolved in DMF (3 mL), and HATU (305.3 mg, 1.23 mmol) and DIPEA (106.2 mg, 0.82 mmol) were added. The mixture was stirred at room temperature for 3 hours. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (142.0 mg, 198.6 μmol).

[0449] Its structural characterization data are as follows:

[0450] MS m / z (ESI): 715.8 [M+H] +

[0451] Its preparation method is as follows:

[0452] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0453] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0454] Step 2: Preparation of (25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecosano-24-ynyl)-L-valine (INT-11)

[0455] (25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-ynyl)-L-valine tert-butyl ester (142.0 mg, 198.6 μmol) was dissolved in DCM (5.0 mL), and TFA (5.0 mL) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was directly concentrated to obtain a crude product, which was purified by reversed-phase column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to obtain the title compound (60 mg, 91.08 mmol).

[0456] Its structural characterization data are as follows:

[0457] MS m / z (ESI): 659.7 [M+H] +

[0458] Example 12 of intermediate preparation: Preparation of (S)-6-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butyl)-32-(2-(methanesulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triaza-tetane-31-alkynic acid (INT-12):

[0459] Step 1: Preparation of tert-butyl N6-(((9H-fluorene-9-yl)methoxy)carbonyl)-N2-(25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-aza-peco-24-acetylacetyl)-L-lysine ester (INT-12-1)

[0460] 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-alkynic acid (800 mg, 1.43 mmol), N6-(((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine tert-butyl ester (724.90 mg, 1.57 mmol), DIPEA (739 mg, 5.72 mmol), and HATU (815.26 mg, 2.14 mmol) were added to DMF (2 mL). After addition, the mixture was stirred at 25 °C for 3 h. The reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.5) and then freeze-dried to give the title compound (1.3 g, 1.14 mmol, 85% purity).

[0461] Its structural characterization data are as follows:

[0462] MS m / z (ESI): 966.4 [M+H] +

[0463] Step 2: Preparation of N6-(((9H-fluorene-9-yl)methoxy)carbonyl)-N2-(25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapecopentane-24-acetylacetyl)-L-lysine (INT-12-2)

[0464] 1.3 g (1.14 mmol, 85% purity) of tert-butyl N6-(((9H-fluorene-9-yl)methoxy)carbonyl)-N2-(25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-penta-19-aza-pecapone-24-acetylacetyl)-L-lysine ester was added to DCM (10 mL) and TFA (5 mL). After addition, the mixture was stirred at 25 °C for 3 h. The reaction solution was directly evaporated to dryness to give the crude title compound (1.2 g).

[0465] Its structural characterization data are as follows:

[0466] MS m / z (ESI): 910.3 [M+H] +

[0467] Step 3: Preparation of tert-butyl(S)-6-(4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)butyl)-32-(2-(methanesulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatricarbon-31-acetylacetate (INT-12-3):

[0468] To DMF (2 mL), 600 mg of crude N6-(((9H-fluorene-9-yl)methoxy)carbonyl)-N2-(25-(2-(methanesulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-penta-19-aza-peco-24-acetylacetyl)-L-lysine, tert-butyl 3-aminopropionate hydrochloride (143.7 mg, 791.8 μmol), DIPEA (426.05 mg, 3.30 mmol), and HATU (426.14 mg, 1.12 mmol) were added. The mixture was stirred at 25 °C for 4 h. The reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.5) and then freeze-dried to give the title compound (332 mg, 294.48 μmol).

[0469] Its structural characterization data are as follows:

[0470] MS m / z (ESI): 1037.4 [M+H] +

[0471] Step 4: Preparation of (S)-6-(4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)butyl)-32-(2-(methanesulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatridodecane-31-alkynic acid (INT-12)

[0472] 332 mg (320.09 μmol) of tert-butyl(S)-6-(4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)butyl)-32-(2-(methanesulfonyl)pyrimidin-5-yl)-5,8,27-trioxo-11,14,17,20,23-pentaoxa-4,7,26-triazatricarbon-31-acetylacetate was added to DCM (5 mL) and TFA (3 mL). After the addition was complete, the mixture was stirred at 25 °C for 2 h. The reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 0.5) and then freeze-dried to give the title compound (250 mg, 234.43 μmol).

[0473] Its structural characterization data are as follows:

[0474] MS m / z (ESI): 981.5 [M+H] +

[0475] Example 13 of intermediate preparation: N 6 -(diphenyl(p-tolyl)methyl)-N 2 Preparation of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-13)

[0476] Step 1: N 6 Preparation of 1-(diphenyl(p-tolyl)methyl)-L-lysine (INT-13-1)

[0477] N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (10 g, 16.01 mmol) was dissolved in DMF (100 mL), and diethylamine (10 g, 16.01 mmol) was added. The reaction was carried out at 20 °C for 1 hour. After the reaction was completed, the reaction solution was diluted with water and extracted with ethyl acetate. The combined organic phases were dried and concentrated under reduced pressure to remove the solvent, giving the pale yellow title compound (6.44 g, 14.16 mmol).

[0478] Its structural characterization data are as follows:

[0479] MS m / z(ESI): 403 [M+H] +

[0480] Step Two: N 2 -((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 Preparation of 1-(diphenyl(p-tolyl)methyl)-L-lysine (INT-13-2)

[0481] N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (2 g, 4.97 mmol) and 2,5-dioxopyrrolidone-1-yl(((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine ester (2.39 g, 5.47 mmol) were dissolved in N,N-dimethylacetamide (20 mL), and N,N-diisopropylethylamine (1.28 g, 9.94 mmol) was added. The reaction mixture was reacted at 20 °C for 2 hours. The reaction solution was subjected to rapid column chromatography (dichloromethane / methanol = 10 / 1) and then evaporated to dryness to give the title compound (2.4 g, 3.23 mmol).

[0482] Its structural characterization data are as follows:

[0483] MS m / z(ESI): 724 [M+H] +

[0484] Step 3: N 2 -(L-valine)-N 6 Preparation of 1-(diphenyl(p-tolyl)methyl)-L-lysine (INT-3)

[0485] N 2 -((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (2.4 g, 3.32 mmol) was dissolved in N,N-dimethylacetamide (20 mL), and diethylamine (484.95 mg, 6.63 mmol) was added. The reaction was carried out at 20 °C for 2 hours. After the reaction was completed, the reaction solution was subjected to rapid column chromatography (C18, water / acetonitrile = 2 / 1) and evaporated to dryness to give the title compound (1.98 g, 3.32 mmol).

[0486] Its structural characterization data are as follows:

[0487] MS m / z(ESI): 502 [M+H] +

[0488] Step 4: N 6 -(diphenyl(p-tolyl)methyl)-N 2 Preparation of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-13)

[0489] N 2 -(L-valine)-N 6-(diphenyl(p-tolyl)methyl)-L-lysine (1.98 g, 3.25 mmol), 2,5-dioxopyrrolidone-1-yl6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetyl ester, and N,N-dimethylacetamide (20 mL) were dissolved in the solution, and DIPEA (1.26 g, 9.76 mmol) was added. The reaction was carried out at 20 °C for 2 hours. After the reaction was completed, the reaction solution was diluted with water, extracted with ethyl acetate, and the combined organic phases were dried and then evaporated to dryness to give the title compound (2.4 g, 3.03 mmol).

[0490] Its structural characterization data are as follows:

[0491] MS m / z(ESI): 752 [M+H] +

[0492] Example 14: Preparation of (S)-3-(6-((diphenyl(p-tolyl)methyl)amino)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-yneamide)hexamide)propionic acid (INT-14)

[0493] N 6 -(diphenyl(p-tolyl)methyl)-N 2 -(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (1.15 g, 1.76 mmol), DIPEA (683.02 mg, 5.28 mmol), and HATU (736.83 mg, 1.94 mmol) were added to DMF (20 mL), and the mixture was stirred at 25 °C for 30 minutes. Then, 3-aminopropionic acid (235.42 mg, 2.64 mmol, FR) was added to the reaction mixture, and the mixture was stirred at 25 °C for 2.5 hours. The reaction mixture was concentrated to dryness and lyophilized to give the title compound (170 mg, 234.85 μmol).

[0494] Its structural characterization data are as follows:

[0495] MS m / z(ESI): 724.1 [M+H] +

[0496] Its preparation method is as follows:

[0497] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0498] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0499] Example 15 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-15)

[0500] 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-15-2)

[0501] 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).

[0502] Its structural characterization data are as follows:

[0503] ESI-MS (m / z): 515.2 (M+H) +

[0504] 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-15-3)

[0505] 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).

[0506] Its structural characterization data are as follows:

[0507] ESI-MS (m / z): 824.4 (M+H) +

[0508] Step 3: Preparation of 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-15)

[0509] 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 gas (balloon) was introduced, the temperature was raised to 40 °C and reacted for 4 hours, palladium on carbon was filtered off, the filtrate was concentrated, the residue was dissolved in water (15 mL) and acetonitrile (15 mL) and clarified, and lyophilized to give the title compound (0.59 g, 0.80 mmol).

[0510] Its structural characterization data are as follows:

[0511] ESI-MS (m / z): 734.3 (M+H) +

[0512] Example 16: Preparation of intermediate 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-16)

[0513] 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-16-1)

[0514] 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).

[0515] Its structural characterization data are as follows:

[0516] ESI-MS (m / z): 1046.5 (M+H) +

[0517] 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-16)

[0518] 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 gas (in a balloon) was introduced. The mixture was heated to 40 °C and reacted for 4 hours. The palladium on carbon was filtered off, the filtrate was concentrated, and the residue was dissolved and clarified in water (15 mL) and acetonitrile (15 mL). The solution was lyophilized to give the title compound (0.54 g, 0.56 mmol).

[0519] Its structural characterization data are as follows:

[0520] ESI-MS (m / z): 956.4 (M+H) +

[0521] Example 17: Preparation of intermediate 3,3'-((2-((2-carboxyethoxy)methyl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)propane-1,3-diyl)bis(oxy))dipropionic acid (INT-17)

[0522] Step 1: Preparation of di-tert-butyl 3,3'-((2-((3-(tert-butoxy)-3-oxopropoxy)methyl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)propane-1,3-diyl)bis(oxo))dipropionate (INT-17-2)

[0523] Di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxo))dipropionate (1.0 g) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (975.47 mg, 2.67 mmol) were dissolved in DMF (3.81 mL), and then DIPEA (575.09 mg, 4.45 mmol, 775.06 μL) was added and stirred at 25 °C 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 (0.62 g, 820.20 μmol).

[0524] Its structural characterization is as follows:

[0525] ESI-MS (m / z): 756.3 [M+H] + .

[0526] Step 2: Preparation of 3,3'-((2-((2-carboxyethoxy)methyl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)propane-1,3-diyl)bis(oxy))dipropionic acid (INT-17)

[0527] Di-tert-butyl 3,3'-((2-((3-(tert-butoxy)-3-oxopropoxy)methyl)-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)propane-1,3-diyl)bis(oxo))dipropionate (0.62 g, 820.20 μmol) was dissolved in DCM (2 mL), and TFA (93.52 mg, 820.20 μmol) was added. The mixture was 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 (450 mg, 689.25 μmol).

[0528] Its structural characterization is as follows:

[0529] ESI-MS (m / z): 588.2 [M+H] + .

[0530] Example 18 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-18):

[0531] 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-18-2):

[0532] 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).

[0533] Its structural characterization data are as follows:

[0534] MS m / z(ESI): 492.3 [M+H] +

[0535] Step 2: Preparation of 2,5-dioxopyrrolidone-1-yl((benzyloxy)carbonyl)-L-alanine ester (INT-18-4):

[0536] 1-Hydroxypyrrolidine-2,5-dione (386.67 mg, 3.36 mmol) and ((benzyloxy)carbonyl)-L-alanine (0.5 g, 2.24 mmol) were added to THF (10 mL), followed by EDCI (644.08 mg, 3.36 mmol), but the mixture could not be completely dissolved. 10 mL of dichloromethane was added, and the mixture dissolved completely. The reaction was carried out 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).

[0537] Its structural characterization data are as follows:

[0538] MS m / z (ESI): 343.1 [M+Na] +

[0539] Step 3: Preparation of ((benzyloxy)carbonyl)-L-alanyl-L-alanyl-L-alanine (INT-18-5):

[0540] DMF (5 mL) was added to 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), followed by the addition of DIPEA (578.63 mg, 4.48 mmol) and water (3 mL). The mixture was 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 obtain the title compound (589 mg, 1.61 mmol).

[0541] Its structural characterization data are as follows:

[0542] MS m / z(ESI): 366.1 [M+H] +

[0543] 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-18-6):

[0544] ((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).

[0545] Its structural characterization data are as follows:

[0546] MS m / z (ESI): 839.3 [M+H] +

[0547] 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-18-7):

[0548] 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 dissolved in DMF (5 mL), and pyridine hydrofluoric acid (746.50 mg, 7.53 mmol) was added dropwise. The mixture was stirred 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).

[0549] Its structural characterization data are as follows:

[0550] MS m / z (ESI): 611.2 [M+H]+

[0551] 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-18):

[0552] 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 reaction at room temperature for 5 hours. After the reaction was complete, 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).

[0553] Its structural characterization data are as follows:

[0554] MS m / z (ESI): 477.1 [M+H] +

[0555] Example 19 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-19)

[0556] 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-19-1)

[0557] 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 HCl / 1,4-dioxane (4 M, 5.70 mL) was added dropwise at 0 °C. The reaction was carried out at room temperature for 10 hours. Water (100 mL) and ethyl acetate (100 mL) were added to the system for extraction. The aqueous phase was collected and 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).

[0558] Its structural characterization is as follows:

[0559] ESI-MS (m / z): 586.2 [M+H] + .

[0560] 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-19-2)

[0561] (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).

[0562] Its structural characterization is as follows:

[0563] ESI-MS (m / z): 364.3 [M+H] + .

[0564] 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-19-3)

[0565] (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).

[0566] Its structural characterization is as follows:

[0567] ESI-MS (m / z): 728.3 [M+H] + .

[0568] 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-19)

[0569] (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).

[0570] Its structural characterization is as follows:

[0571] ESI-MS (m / z): 506.3 [M+H] + .

[0572] Example 20 of intermediate preparation: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R))-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamate (INT-20)

[0573] Step 1: Preparation of 4-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl))amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (INT-20-1)

[0574] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (1.0 g, 1.41 mmol) and MMAE (CAS: 474645-27-7; 1.52 g, 2.12 mmol) were dissolved in DMF (6 mL), and DIPEA (820.6 mg) and 1-hydroxybenzotriazole (190.6 mg, 1.41 mmol) were added. After the addition was complete, the reaction was carried out at 25 °C for 2 hours. After the reaction was completed, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 0.6) and then freeze-dried to give the title compound (1.18 g, 0.92 mmol).

[0575] Its structural characterization data are as follows:

[0576] MS m / z (ESI): 1287.3 [M+H] +

[0577] Step 2: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-aminopropamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamate (INT-20-2)

[0578] 4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl))amino)-1-methoxy-2-methyl-3- (Oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohep-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamate (1.18 g, 91.65 μmol) was dissolved in DMF (10 mL), and diethylamine (201.6 mg) was added. After the addition was complete, the reaction was carried out at 25 °C for 0.5 h. After the reaction was complete, the solvent was removed, and the solution was added to an ethyl acetate / petroleum ether mixture (100 mL, ethyl acetate / petroleum ether = 1 / 3). The mixture was stirred for 2 h, filtered, and dried to obtain the title compound (878.5 mg, 0.83 mmol).

[0579] Its structural characterization data are as follows:

[0580] MS m / z (ESI): 1065.3 [M+H] +

[0581] Step 3: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R))-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamate (INT-20)

[0582] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-aminopropionamido)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolid-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (878.5 mg, 82.5 μmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (308.1 mg, 90.75 μmol) were dissolved in DMF (5 mL), followed by the addition of DIPEA (320 mg) and HATU (470 mg). The reaction was carried out at 25 °C for 2 hours after the addition was complete. The mixture was purified by rapid column chromatography (C18, water / acetonitrile = 0.7) and then freeze-dried to give the title compound (800.2 mg, 0.58 mmol).

[0583] Its structural characterization data are as follows:

[0584] MS m / z (ESI): 1386.9 [M+H] +

[0585] Example 21 of intermediate preparation: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamate (INT-21)

[0586] Step 1: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-21-1)

[0587] (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate allyl ester (800 mg, 1.64 mmol) and (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidovalerate (649.2 mg, 1.64 mmol) were dissolved in DCM (30 mL) and MeOH (6 mL). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.22 g, 4.92 mmol) was added, and the reaction was carried out at 25 °C for 16 hours. After the reaction was complete, the mixture was purified by rapid column chromatography (silica gel, ethyl acetate / petroleum ether = 1) and then concentrated again to give the title compound (995.5 mg, 1.15 mmol).

[0588] Its structural characterization data are as follows:

[0589] MS m / z (ESI): 885.3 [M+H2O] +

[0590] Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (INT-21-2)

[0591] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (995.5 mg, 1.15 mmol) was dissolved in DMF (10 mL), and pyridinium hydrofluoride (3.5 mL) was added dropwise under ice bath conditions. After the addition was complete, the reaction was carried out at 25 °C for 16 hours. After the reaction was completed, the mixture was purified by rapid column chromatography (silica gel, ethyl acetate / petroleum ether = 1) and then concentrated again to give the title compound (506.5 mg, 0.81 mmol).

[0592] Its structural characterization data are as follows:

[0593] MS m / z (ESI): 647.3 [M+H2O] +

[0594] Step 3: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)(methyl)carbamate (INT-21-3)

[0595] (S)-(5-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-ureidopentamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (506.5 mg, 0.81 mmol) was dissolved in DMF (3 mL), followed by the addition of DIPEA (209 mg) and bis(4-nitrophenyl) carbonate (295.5 mg, 0.972 mmol). The reaction was carried out at 25 °C for 16 hours. After the reaction was completed, the mixture was purified by rapid column chromatography (silica gel, ethyl acetate / petroleum ether = 1) and then concentrated again to give the title compound (515.2 mg, 0.65 mmol).

[0596] Its structural characterization data are as follows:

[0597] MS m / z (ESI): 795.3 [M+H] +

[0598] Step 4: Preparation of 4-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-ureidopentamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamate (INT-21-4)

[0599] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)(methyl)carbamate (515.2 mg, 0.65 mmol) and MMAE (CAS: 474645-27-7; 699.1 mg, 0.98 mmol) were dissolved in DMF (3 mL), and DIPEA (251.6 mg) and 1-hydroxybenzotriazole (87.8 mg, 0.65 mmol) were added. After the addition was complete, the reaction was carried out at 25 °C for 2 hours. After the reaction was completed, the product was purified by rapid column chromatography (C18, water / acetonitrile = 0.6) and then freeze-dried to give the title compound (624.7 mg, 0.46 mmol).

[0600] Its structural characterization data are as follows:

[0601] MS m / z (ESI): 1373.8 [M+H] +

[0602] Step 5: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-amino-5-ureidopentamido)benzyl(1-((1-(((3R,5S)-1)-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamate (INT-21-5)

[0603] 4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl) (-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohep-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamate (624.7 mg, 0.46 mmol) was dissolved in DMF (5 mL), and diethylamine (120 mg) was added. After the addition was complete, the reaction was carried out at 25 °C for 0.5 h. After the reaction was complete, the solvent was removed, and a mixed solution of ethyl acetate / petroleum ether (60 mL, ethyl acetate / petroleum ether = 1 / 3) was added and stirred for 2 h. The mixture was filtered and dried to give the title compound (450.1 mg, 0.39 mmol).

[0604] Its structural characterization data are as follows:

[0605] MS m / z (ESI): 1151.8 [M+H] +

[0606] Step Six: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(1-((1-(((3R,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobut-2-yl)amino)-3-methyl-1-oxobut-2-yl)(methyl)carbamate (INT-21)

[0607] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-amino-5-ureidopentanamido)benzyl(1-((1-(((3R,5S)-1)-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolid-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl) (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (450.1 mg, 0.39 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (164.8 mg, 0.47 mmol) were dissolved in DMF (5 mL), and DIPEA (151 mg) and HATU (223.1 mg) were added sequentially. After the addition was complete, the reaction was carried out at 25 °C for 1 hour. After purification by rapid column chromatography (C18, water / acetonitrile = 0.8), the compound was freeze-dried to give the title compound (459.3 mg, 0.31 mmol).

[0608] Its structural characterization data are as follows:

[0609] MS m / z (ESI): 1472.9 [M+H] +

[0610] Example 22 of intermediate preparation: 2-((((4-(methoxycarbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (INT-22)

[0611] Step 1: Preparation of methyl 1-(N-((2-(benzyloxy)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylic acid (INT-22-2)

[0612] Chlorosulfonyl isocyanate (500 mg, 3.53 mmol, 307 μL) was dissolved in dichloromethane (10.0 mL) at 0 °C. Benzyl glycolate (533 mg, 3.21 mmol, 455 μL) was added, and the mixture was stirred for 1 hour. Then, a solution of methyl 4-piperidincarnate (459 mg, 3.21 mmol) and triethylamine (974 mg, 9.63 mmol, 1.34 mL) in dichloromethane (5.00 mL) was added to the above reaction mixture, and the mixture was heated to 25 °C and stirred for 1 hour. Water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with dichloromethane (50.0 mL x 3). The organic phases were combined, washed with cooled 1N dilute hydrochloric acid (20.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (1.30 g, 3.14 mmol), which was used directly in the next step without purification.

[0613] The structural characterization data are as follows:

[0614] ESI-MS (m / z): 414.9 [M+H] +

[0615] Step 2: Preparation of 2-((((4-(methoxycarbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (INT-22)

[0616] Under a nitrogen atmosphere, 10% Pd / C (0.70 g) was added to a methanol (20.0 mL) solution of compound INT-22-2 (700 mg, 1.69 mmol), and the mixture was purged with hydrogen three times and reacted at 25 °C for 3 hours (15 PSI). The reaction solution was filtered, and the filter cake was washed three times with methanol (100 mL x 3). The filtrate was concentrated to give the crude title compound (590 mg).

[0617] The structural characterization data are as follows:

[0618] ESI-MS (m / z): 325.1 [M+H] +

[0619] Example 23 of intermediate preparation: 2-((((4-(((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (INT-23)

[0620] Step 1: Preparation of 1-(N-((2-(tert-butoxy)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylic acid allyl ester (INT-23-2)

[0621] Chlorosulfonyl isocyanate (1.76 g, 12.41 mmol) was dissolved in acetonitrile (80.0 mL) at 0 °C, and tert-butyl glycolate (1.56 g, 11.82 mmol, 455 μL) was added. The mixture was stirred for 1 hour. Then, allyl 4-piperidincarnate (2 g, 11.82 mmol) and pyridine (1.12 g, 14.18 mmol) were added to the above reaction solution, and the mixture was heated to 25 °C and stirred for 1 hour. Water (100 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (25.0 mL x 3). The organic phases were combined, washed with cooled 1N dilute hydrochloric acid (20.0 mL) and saturated sodium chloride aqueous solution (30.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude title compound (2.3 g, 5.66 mmol), which was used directly in the next step without purification.

[0622] Its structural characterization data are as follows:

[0623] ESI-MS (m / z): 407.1 [M+H]+

[0624] Step 2: Preparation of 2-((((4-(((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (INT-23)

[0625] Compound INT-23-2 (2.3 g, 5.66 mmol) was dissolved in dichloromethane (10 mL), and TFA (2 mL) was added. The mixture was reacted at 25 °C for 2 hours. The reaction solution was concentrated, and the pH was adjusted to 8 with 1 N sodium bicarbonate aqueous solution. The mixture was extracted twice with ethyl acetate (25.0 mL x 2). The aqueous phases were combined, and the pH was adjusted to 3 with 1 N dilute hydrochloric acid. The mixture was then extracted three times with ethyl acetate (25.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product of the title compound (1.6 g, 4.57 mmol).

[0626] Its structural characterization data are as follows:

[0627] ESI-MS (m / z): 351.1 [M+H] +

[0628] Example 24 of intermediate preparation: Preparation of (9H-fluorene-9-yl)methyl[(S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxobutyl-2-yl)carbamate (INT-24)

[0629] Step 1: Preparation of (9H-fluorene-9-yl)methyl[(S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino]-3-methyl-1-oxobutyl-2-yl]carbamate (INT-24-2)

[0630] ((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanine (1.0 g, 2.44 mmol) and p-aminobenzyl alcohol (285.1 mg, 2.31 mmol) were dissolved in dichloromethane (15 mL) and methanol (5 mL), and then 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.81 g, 7.31 mmol) was added and reacted at 25 °C for 15 hours. The reaction solution was concentrated to obtain the crude product, which was purified by silica gel column chromatography (dichloromethane / methanol = 0%-15%) and concentrated again to obtain the title compound (300 mg, 0.58 mmol).

[0631] Its structural characterization is as follows:

[0632] ESI-MS (m / z): 516.1 [M+H]+ .

[0633] Step 2: Preparation of (9H-fluorene-9-yl)methyl[(S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxobut-2-yl)carbamate (INT-24)

[0634] (9H-fluorene-9-yl)methyl[(S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino]-3-methyl-1-oxobutyl-2-yl]carbamate (200 mg, 387.9 μmol) was dissolved in DMF (3 mL), and then DIPEA (100.3 mg, 0.78 mmol) and bis(4-nitrophenyl) carbonate (177.1 mg, 0.58 mmol) were added. The mixture was reacted at 25 °C for three hours. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%-60%) and then concentrated again to give the title compound (170 mg, 249.7 μmol).

[0635] Its structural characterization is as follows:

[0636] ESI-MS (m / z): 703.3 [M+Na] + .

[0637] Example 25 of intermediate preparation: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-25)

[0638] Step 1: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-aminopropamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-25-1)

[0639] INT-6-3 (6.18 g, 8.72 mmol) and (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (1.50 g, 2.18 mmol, 80% purity) were dissolved in DMF (20 mL) and pyridine (10 mL). 1-hydroxybenzotriazole (1.18 g, 0.93 mmol) was added, and the mixture was stirred at 85 °C for 1 hour. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to prepare the title compound (328 mg, 324.11 μmol).

[0640] Its structural characterization data are as follows:

[0641] MS m / z (ESI): 898.3 [M+H] +

[0642] Its preparation method is as follows:

[0643] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

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

[0645] Step 2: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-25-2)

[0646] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-aminopropamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazole Alkyl-3-carboxylic acid ester (225 mg, 222.33 μmol) was dissolved in DMF (20 mL), and N,N-diisopropylethylamine (172.41 mg, 1.33 mmol), Fmoc-L-valine (264.10 mg, 778.17 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (262.12 mg, 889.34 μmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to prepare the title compound (125 mg, 102.51 μmol).

[0647] Its structural characterization data are as follows:

[0648] MS m / z (ESI): 1219.4 [M+H] +

[0649] Spectral column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0650] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0651] Step 3: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-25-3)

[0652] 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (125 mg, 102.51 μmol) was dissolved in DMF (6 mL), and DBU (46.82 mg, 307.54 μmol) was added. The mixture was stirred at room temperature for 20 minutes. After the reaction was complete, the compound was purified by rapid column chromatography (C18, water / acetonitrile = 0-50%, 0.05% TFA) and then freeze-dried to obtain the title compound (91 mg, 81.90 μmol).

[0653] Its structural characterization data are as follows:

[0654] MS m / z(ESI): 998.0 [M+H]+

[0655] Step 4: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-25-4)

[0656] 2-(((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminomethyl Acyl)thiazolidin-3-carboxylic acid ester (55 mg, 49.50 μmol) was dissolved in DMF (5 mL), and N,N-diisopropylethylamine (31.99 mg, 247.50 μmol), 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-ethynic acid (26.56 mg, 99.00 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (43.77 mg, 148.50 μmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (45 mg, 36.08 μmol).

[0657] Its structural characterization data are as follows:

[0658] MS m / z (ESI): 1247.4 [M+H] +

[0659] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0660] Step 5: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-25)

[0661] 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ [de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazoline-3-carboxylic acid ester (45 mg, 35.72 μmol) was dissolved in DMF (6 mL), followed by the addition of 1,3-dimethylbarbituric acid (27.88 mg, 178.58 μmol) and tetrakis(triphenylphosphine)palladium (20.64 mg, 17.86 μmol). The mixture was evacuated, purged with nitrogen three times, and stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was analyzed by high performance liquid chromatography to obtain the title compound (29 mg, 24.68 μmol).

[0662] Its structural characterization data are as follows:

[0663] MS m / z (ESI): 1163.3 [M+H] +

[0664] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0665] Example 26 of intermediate preparation: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-26)

[0666] Step 1: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-26-1)

[0667] Add (6.11 g) of crude (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate crude product and (S)-N-((1S,9S)-9-ethyl- 5-Fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (1 g, 1.45 mmol) and HOBt (785.30 mg, 5.81 mmol) were added, and the mixture was heated to 85 °C under nitrogen protection and stirred for 2 hours. The reaction solution was purified by rapid column chromatography (C18, 0.5% formic acid aqueous solution / acetonitrile = 10%–95%) and then freeze-dried to give the crude title compound (2.8 g, purity 15%, 321.73 μmol).

[0668] Its structural characterization data are as follows:

[0669] ESI-MS (m / z): 1306.4 [M+H] + .

[0670] Step 2: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-26-2)

[0671] Add 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)-5-ureidopentamido)-2-((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxane to DMF (3 mL) Crude 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (1 g, 15% purity, 114.90 μmol) and diethylamine (25.51 mg, 344.71 μmol) were added, and the mixture was stirred at 30 °C for 3 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to give the trifluoroacetate of the title compound (84 mg, 70.16 μmol).

[0672] Its structural characterization data are as follows:

[0673] MS m / z (ESI): 1083.4 [M+H] +

[0674] Its preparation method is as follows:

[0675] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0676] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0677] Step 3: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-26-3)

[0678] Add 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1, [2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (42 mg, 35.08 μmol), 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-alkynic acid (11.29 mg, 42.10 μmol), DIPEA (22.67 mg, 175.41 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (19.37 mg, 70.16 μmol) were added, and the mixture was stirred at 30 °C for 2 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (39 mg, 29.25 μmol).

[0679] Its structural characterization data are as follows:

[0680] MS m / z (ESI): 1334.4 [M+H] +

[0681] Its preparation method is as follows:

[0682] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0683] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0684] Step 4: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-26)

[0685] Under nitrogen protection, 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,1 0,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (30 mg, 22.50 μmol), tetraphenylphosphine palladium (12.99 mg, 11.25 μmol), and 1,3-dimethylbarbituric acid (5.27 mg, 33.75 μmol) were added, and the mixture was stirred at 30 °C for 2 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the formate salt of the title compound (13 mg, 10.04 μmol).

[0686] Its structural characterization data are as follows:

[0687] MS m / z (ESI): 1250.4 [M+H] +

[0688] Its preparation method is as follows:

[0689] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0690] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0691] Example 27 of intermediate preparation: 2-(((S)-1-(((S)-1-((4-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline Preparation of lin-1-yl)carbamoyl)thiazolyl-3-carbonyl)oxy)methyl)-3-((N-methyl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamino)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-N,N,N-trimethyl-2-oxoethane-1-ammonium (INT-27)

[0692] Step 1: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-27-1)

[0693] 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((allyloxy)carbonyl)(methyl)amino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12) H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (155.00 mg, 127.12 μmol) was dissolved in DMF (3 mL), and tetrakis(triphenylphosphine)palladium (73.45 mg, 63.56 μmol) and 1,3-dimethylbarbituric acid (99.24 mg, 635.59 μmol) were added. The reaction mixture was stirred at 25 °C for 2 hours. The reaction solution was directly dried by removing the solvent and then frozen to give the title compound (80.00 mg, 70.47 μmol).

[0694] Its structural characterization data are as follows:

[0695] ESI-MS (m / z): 1436.3 (M+H) + .

[0696] The purification method is as follows:

[0697] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0698] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[0699] Step 2: Preparation of 4-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-((N-methyl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamido)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-27-2)

[0700] 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-((methylamino)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline- 1-(carbamoyl)thiazolidin-3-carboxylic acid ester (80.00 mg, 70.47 μmol) was dissolved in DMF (3 mL), and N,N-diisopropylethylamine (27.32 mg, 211.41 μmol), 6-(2-methylsulfonylpyrimidin-5-yl)hex-5-ethynic acid (20.80 mg, 77.52 μmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (39.00 mg, 140.94 μmol) were added. The reaction mixture was stirred at 25 °C for 2 hours. The reaction solution was directly dried by removing the solvent and then frozen to obtain the crude product of the title compound (72.00 mg, 51.97 μmol).

[0701] Its structural characterization data are as follows:

[0702] ESI-MS(m / z):1386.6(M+H)+.

[0703] Step 3: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-2-((N-methyl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (INT-27)

[0704] (4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)propamido)-2-((N-methyl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamido)methyl)benzyl(S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzene) Crude [de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-carboxylic acid ester (72.00 mg, 51.97 μmol) was dissolved in DMF (3 mL), and diethylamine (9.50 mg, 129.91 μmol) was added. The reaction mixture was stirred at 25 °C for 2 hours. After the solvent was directly removed from the reaction solution, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (45.00 mg, 33.47 μmol).

[0705] Its structural characterization data are as follows:

[0706] ESI-MS (m / z): 1163.4 (M / 2) + .

[0707] The purification method is as follows:

[0708] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0709] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% TFA)

[0710] Example 28: Preparation of (S)-2-acetamido-5-(allyloxy)-5-oxovaleric acid (INT-28)

[0711] (S)-5-(allyloxy)-2-amino-5-oxovaleric acid (277.00 mg, 1.48 mmol) was dissolved in DMF (3 mL), and DIPEA (764.98 mg, 5.92 mmol) and 2,5-dioxopyrrolidine-1-ylacetate (232.51 mg, 1.48 mmol) were added. The reaction mixture was stirred at 25 °C for 2 hours. After the solvent was directly removed from the reaction solution, the reaction solution was subjected to high performance liquid chromatography to obtain the title compound (70.00 mg, 57.41 μmol).

[0712] Its structural characterization data are as follows:

[0713] ESI-MS (m / z): 230.3 (M+H)+

[0714] The purification method is as follows:

[0715] Column: Agilent Prep C18 OBD 19*150mm*5μm

[0716] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% FA)

[0717] Example 29: Preparation of N-((S)-1-(((S)-1-((4-(chloromethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (INT-29)

[0718] Step 1: Preparation of (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (INT-29-2)

[0719] Add (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (500.00 mg, 969.75 μmol) and diethylamine (212.30 mg, 2.91 mmol) to DMF (4 mL). After addition, stir at 30 °C for 2 h. After the reaction is complete, the reaction solution is purified by C18 reverse-phase column chromatography (ACN / H2O = 10–90%, 0.05% ammonium bicarbonate) and lyophilized to give the title compound (260.00 mg, 886.62 μmol).

[0720] Its structural characterization is as follows:

[0721] ESI-MS (m / z): 294.2 (M+H) + .

[0722] Step 2: Preparation of N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (INT-29-3)

[0723] (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (260.00 mg, 886.62 μmol), 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ethynic acid (237.78 mg, 886.62 μmol), DIPEA (458.17 mg, 3.55 mmol), and DMTMM (485.68 mg, 1.77 mmol) were added sequentially to DMF (4 mL). After the addition was complete, the mixture was stirred at 30 °C for 2 h. The reaction solution was purified by C18 reverse-phase column chromatography (ACN / H2O = 10–80%, 0.05% ammonium bicarbonate) and lyophilized to give the title compound (370.00 mg, 578.51 μmol, 85% purity).

[0724] Its structural characterization is as follows:

[0725] ESI-MS (m / z): 526.2 (M-18+H) + .

[0726] Step 3: Preparation of N-((S)-1-(((S)-1-((4-(chloromethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (INT-29)

[0727] N-((S)-1-(((S)-1-((4-(chloromethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide (70.00 mg, 128.76 μmol) and thionyl chloride (45.96 mg, 386.29 μmol) were added to acetonitrile (3 mL). After the addition was complete, the mixture was stirred at 0 °C for 3 h. The reaction solution was evaporated to dryness to obtain the crude product (60.00 mg, 96.07 μmol), which was used directly in the next step.

[0728] II. Compound Preparation Examples

[0729] Preparation Example 1: Preparation of (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (MP-5)

[0730] Step 1: Preparation of (2S,4R)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (MP-5-2)

[0731] 4,5-Dimethoxy-2-nitrobenzoic acid (5 g, 22.01 mmol) and (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester hydrochloride (4.4 g, 24.21 mmol) were dissolved in DMF (80 mL), and HATU (8.78 g, 23.11 mmol) and DIPEA (8.53 g, 66.03 mmol) were added. The reaction mixture was reacted for 16 hours. After the reaction was complete, the reaction solution was concentrated, saturated brine (100 mL) was added, and the mixture was extracted twice with ethyl acetate (100 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (methanol / dichloromethane = 0%–30%) and concentrated again to obtain the title compound (3.6 g, 10.16 mmol).

[0732] Its structural characterization data are as follows:

[0733] ESI-MS (m / z): 355.1 [M+H] +

[0734] Step 2: Preparation of (2R,11aS)-2-hydroxy-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (MP-5-3)

[0735] Methyl (2S,4R)-1-(4,5-dimethoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid (3.6 g, 10.16 mmol) was dissolved in methanol (60 mL), and zinc powder (6.6 g, 101.6 mmol) and saturated ammonium chloride aqueous solution (20 mL) were added. The mixture was heated to 80 °C and reacted for 16 hours. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography (methanol / dichloromethane = 0%–30%). The solution was then concentrated again to obtain the title compound (2.1 g, 7.18 mmol).

[0736] Its structural characterization data are as follows:

[0737] ESI-MS (m / z): 293.1 [M+H] +

[0738] Step 3: Preparation of (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (MP-5-4)

[0739] (2R,11aS)-2-hydroxy-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (2.1 g, 7.18 mmol) was dissolved in DMF (30 mL), and imidazole (2.45 g, 35.92 mmol) and tert-butyldimethylchlorosilane (2.38 g, 15.81 mmol) were added. The mixture was stirred for 16 hours. After the reaction was complete, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The solution was purified by silica gel column chromatography (methanol / dichloromethane = 0%–15%) to give the title compound (1 g, 2.46 mmol).

[0740] Its structural characterization data are as follows:

[0741] ESI-MS (m / z): 407.2 [M+H] +

[0742] Step 4: Preparation of (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (MP-5-5)

[0743] Under nitrogen protection, NaH (147.58 mg, 3.69 mmol) was dissolved in 30 mL of DMF at 0 °C. (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (1 g, 2.46 mmol) was added and reacted for 30 minutes. Then, 2-(trimethylsilyl)ethoxymethyl chloride (616.17 mg, 2.46 mmol) was added and the reaction was carried out for 1 hour. After the reaction was complete, water was added to quench the reaction, and the system was poured into ice water. A solid precipitated, and the crude product (1.3 g, 2.42 mmol) was obtained by filtration and used directly in the next reaction step.

[0744] Its structural characterization data are as follows:

[0745] ESI-MS (m / z): 537.3 [M+H] +

[0746] Step 5: Preparation of (2R,11aS)-2-hydroxy-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (MP-5-6)

[0747] (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (1.3 g, 2.42 mmol) was dissolved in tetrahydrofuran (20 mL), and tetrabutylammonium fluoride (1.26 g, 4.84 mmol) was added. The reaction was allowed to proceed for 2 hours. Saturated brine was added, and the mixture was extracted three times with ethyl acetate (30 mL x 3). The organic phases were combined, dried, and concentrated. The solution was purified by silica gel column chromatography (methanol / dichloromethane = 0%–10%) to give the title compound (0.8 g, 1.89 mmol).

[0748] Its structural characterization data are as follows:

[0749] ESI-MS (m / z): 423.1 [M+H] +

[0750] Step Six: Preparation of (S)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2,5,11(3H,10H)-trione (MP-5-7)

[0751] (2R,11aS)-2-hydroxy-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (0.8 g, 1.89 mmol) was dissolved in dichloromethane (20 mL), and Dys-Martin oxidant (2.01 g, 4.73 mmol) was added. The reaction was allowed to proceed for 1 hour. The reaction was quenched with saturated sodium bicarbonate solution, and the mixture was extracted three times with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The crude product was used directly in the next step of the reaction (0.65 g, 1.55 mmol).

[0752] Its structural characterization data are as follows:

[0753] ESI-MS (m / z): 421.1 [M+H] +

[0754] Step 7: Preparation of (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yltrifluoromethanesulfonate (MP-5-8)

[0755] (S)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2,5,11(3H,10H)-trione (0.65 g, 1.55 mmol) was dissolved in dichloromethane (20 mL) under nitrogen protection. After cooling to -40 °C, 2,6-dimethylpyridine (1.32 g, 12.37 mmol) and trifluoromethanesulfonic anhydride (3.05 g, 10.82 mmol) were added, and the reaction was carried out at -40 °C for 1 hour. After the reaction was completed, the reaction was quenched with water, extracted three times with dichloromethane (20 mL x 3), the organic phases were combined, washed with saturated brine, dried, and concentrated. Silica gel column purification (methanol / dichloromethane = 0%–5%) yielded the title compound (0.6 g, 1.09 mmol).

[0756] Its structural characterization data are as follows:

[0757] ESI-MS (m / z): 553.3 [M+H] +

[0758] Step 8: Preparation of (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (MP-5-9)

[0759] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yltrifluoromethanesulfonate (0.6 g, 1.09 mmol) and (E)-(4-aminostyryl)boronic acid (210.88 mg, 1.29 mmol) were dissolved in toluene (20 mL), ethanol (5 mL), and water (5 mL). Tetra(triphenylphosphine)palladium (62.95 mg, 54.5 μmol) and sodium carbonate (346.62 mg, 3.27 mmol) were added, and the mixture was then heated to 80 °C for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated, saturated saline (30 mL) was added, and the mixture was extracted twice with ethyl acetate (20 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (0.35 g, 670.91 μmol).

[0760] Its structural characterization data are as follows:

[0761] ESI-MS (m / z): 521.2 [M+H] +

[0762] Step Nine: Preparation of (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (MP-5)

[0763] (S,E)-2-(4-aminostyryl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (0.35 g, 670.91 μmol) was dissolved in dry tetrahydrofuran (10 mL). The mixture was reacted under nitrogen protection at -78 °C with triethyllithium borohydride (1 M in tetrahydrofuran) (1.34 mL, 1.34 mmol) for 30 min. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The solution was purified by reversed-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution) and freeze-dried to give the title compound (7.3 mg, 19.25 μmol).

[0764] Its structural characterization data are as follows:

[0765] ESI-MS (m / z): 376.2 [M+H] +

[0766] 1H NMR (400MHz, CDCl3) δ7.88(d,J=3.8Hz,1H),7.52(s,1H),7.24(s,2H),7.09(s,1H),6.83(s,1H),6.81(d,J=18.2Hz,1H),6.66(d,J =8.2Hz,2H),6.34(d,J=16.0Hz,1H),4.41-4.34(m,1H),3.97(s,3H),3.95(s,3H),3.49-3.41(m,1H),3.27(dd,J=16.0,4.4Hz,1H).

[0767] Its preparation method is as follows:

[0768] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0769] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0770] Preparation Example 2: Preparation of (S,E)-7,8-dimethoxy-2-styryl-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (MP-1)

[0771] Step 1: Preparation of (E)-4,4,5,5-tetramethyl-2-styryl-1,3,2-dioxaborane (MP-1-2)

[0772] Phenylacetylene (2 g, 19.58 mmol) was dissolved in pinacolborane (10 mL) and reacted at 110 °C for 16 h under nitrogen protection. After the reaction was complete, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (100 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–30%) yielded the title compound (3.77 g, 16.38 mmol).

[0773] Its structural characterization data are as follows:

[0774] ESI-MS (m / z): 231.2 [M+H] +

[0775] Step 2: Preparation of (S,E)-7,8-dimethoxy-2-styryl-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (MP-1-3)

[0776] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yltrifluoromethanesulfonate (120.07 mg, 217.29 μmol) and (E)-4,4,5,5-tetramethyl-2-styryl-1,3,2-dioxaborane (50 mg, 217.29 μmol) were dissolved in 1,4-dioxane (4 mL) and water (1 mL). DPPF palladium dichloride (15.90 mg, 21.73 μmol) and potassium carbonate (90.09 mg, 651.86 μmol) were added, and the mixture was then heated to 80 °C for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated, saturated saline (10 mL) was added, and the mixture was extracted twice with ethyl acetate (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (60 mg, 118.42 μmol).

[0777] Its structural characterization data are as follows:

[0778] ESI-MS (m / z): 507.2 [M+H] +

[0779] Step 3: Preparation of (S,E)-7,8-dimethoxy-2-styryl-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (MP-1)

[0780] (S,E)-7,8-dimethoxy-2-styryl-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (60 mg, 118.42 μmol) was dissolved in dry tetrahydrofuran (5 mL). Under nitrogen protection, triethyllithium borohydride (1 M in tetrahydrofuran) (236.84 μL, 236.84 μmol) was added at -78 °C and reacted for 30 min. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The solution was purified by reversed-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution) and freeze-dried to give the title compound (4.15 mg, 11.40 μmol).

[0781] Its structural characterization data are as follows:

[0782] ESI-MS (m / z): 361.2 [M+H] +

[0783] 1 H NMR (400MHz, CDCl3) δ7.90 (d, J = 4.0Hz, 1H), 7.57-7.50 (m, 1H), 7.46-7.36 (m, 3H), 7.37-7.31 (m, 2H), 7.26-7.22 (m, 1H), 7.1 7(s,1H),7.03-6.97(m,1H),6.85(s,1H),4.44-4.37(m,1H),3.97(s,3H),3.95(s,3H),3.52-3.41(m,1H),3.34-3.26(m,1H).

[0784] Its preparation method is as follows:

[0785] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0786] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0787] Preparation Example 3: Preparation of (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (MP-6)

[0788] Step 1: Preparation of 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)naphthyl-2-amine (MP-6-2)

[0789] 100 mg (270.81 μmol) of tert-butyl (6-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)naphth-2-yl)carbamate was dissolved in 1 mL of dichloromethane, and 1 mL of TFA was added. The reaction was carried out for 1 hour. After the reaction was completed, the crude product of the title compound (72.89 mg, 270.81 μmol) was concentrated.

[0790] Its structural characterization data are as follows:

[0791] ESI-MS (m / z): 270.1 [M+H] +

[0792] Step 2: Preparation of (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (MP-6-3)

[0793] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yltrifluoromethanesulfonate (149.65 mg, 270.82 μmol) and 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)naphthyl-2-amine (72.89 mg, 270.81 μmol) were dissolved in 1,4-dioxane (4 mL) and water (1 mL). DPPF palladium dichloride (7.64 mg, 27.08 μmol) and potassium carbonate (112.29 mg, 812.46 μmol) were added, and the mixture was then heated to 80 °C for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated, saturated saline (10 mL) was added, and the mixture was extracted twice with ethyl acetate (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (110 mg, 201.58 μmol).

[0794] Its structural characterization data are as follows:

[0795] ESI-MS (m / z): 546.3 [M+H] +

[0796] Step 3: Preparation of (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (MP-6)

[0797] (S)-2-(6-aminonaphthyl-2-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (110 mg, 201.58 μmol) was dissolved in dry tetrahydrofuran (5 mL). Under nitrogen protection, triethyllithium borohydride (1 M in tetrahydrofuran) (403.15 μL, 403.15 μmol) was added at -78 °C and reacted for 30 min. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The solution was purified by reversed-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution) and freeze-dried to give the title compound (7.6 mg, 18.84 μmol).

[0798] Its structural characterization data are as follows:

[0799] ESI-MS (m / z): 400.3 [M+H] +

[0800] 1 H NMR (400MHz, CDCl3) δ7.94(d,J=4.0Hz,1H),7.63(d,J=8.6Hz,1H),7.62-7.44(m,5H),7.01-6.93(m, 2H),6.85(s,1H),4.50-4.43(m,1H),3.99(s,3H),3.96(s,3H),3.74-3.67(m,1H),3.55-3.48(m,1H).

[0801] Its preparation method is as follows:

[0802] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0803] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0804] Preparation Example 4: Preparation of (S)-2-(7-aminoquinolin-3-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (MP-7)

[0805] Step 1: Preparation of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)quinoline-7-amine (MP-7-2)

[0806] 3-Bromoquinoline-7-amine (100.00 mg, 448.29 μmol) and pinacol diborate (227.68 mg, 896.58 μmol) were dissolved in 1,4-dioxane (5 mL), and DPPF palladium dichloride (32.80 mg, 44.83 μmol) and potassium acetate (131.99 mg, 1.34 mmol) were added. The mixture was then heated to 90 °C for 2 hours under nitrogen protection. After the reaction was complete, the mixture was allowed to return to room temperature before proceeding to the next step.

[0807] Its structural characterization data are as follows:

[0808] ESI-MS (m / z): 271.2 [M+H] +

[0809] Step 2: Preparation of (S)-2-(7-aminoquinoline-3-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (MP-7-3)

[0810] (S)-7,8-dimethoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2-yltrifluoromethanesulfonate (149.65 mg, 270.82 μmol), DPPF palladium dichloride (7.64 mg, 27.08 μmol), potassium carbonate (112.29 mg, 812.46 μmol), and water (1 mL) were added to the reaction system of the previous step, and then the mixture was heated to 80 °C for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated, saturated saline (10 mL) was added, and the mixture was extracted twice with ethyl acetate (10 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0%–50%) and concentrated again to obtain the title compound (132 mg, 144.87 μmol).

[0811] Its structural characterization data are as follows:

[0812] ESI-MS (m / z): 548.3 [M+H] +

[0813] Step 3: Preparation of (S)-2-(7-aminoquinolin-3-yl)-7,8-dimethoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (MP-7)

[0814] (S)-2-(7-aminoquinoline-3-yl)-7,8-dimethoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H)-dione (132 mg, 144.87 μmol) was dissolved in dry tetrahydrofuran (5 mL). Under nitrogen protection, triethyllithium borohydride (1 M in tetrahydrofuran) (403.15 μL, 403.15 μmol) was added at -78 °C and reacted for 30 min. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The solution was purified by reversed-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution) and freeze-dried to give the title compound (5.3 mg, 12.97 μmol).

[0815] Its structural characterization data are as follows:

[0816] ESI-MS (m / z): 401.2 [M+H] +

[0817] 1 H NMR (400MHz, CDCl3) δ7.96 (d, J = 4.0Hz, 1H), 7.63 (m, 3H), 7.55-7.52 (m, 2H), 7.00 (s, 2H), 6.8 6(s,1H),4.50-4.43(m,1H),3.99(s,3H),3.96(s,3H),3.74-3.67(m,1H),3.55-3.48(m,1H).

[0818] Its preparation method is as follows:

[0819] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0820] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0821] III. Examples of Drug Linker Compound Preparation

[0822] Example 1: 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 (H-1)

[0823] (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).

[0824] Its structural characterization data are as follows:

[0825] MS m / z (ESI): 756.3 [M+H] +

[0826] Its preparation method is as follows:

[0827] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% formic acid aqueous solution)

[0828] Example 2: 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 (H-2)

[0829] Step 1:

[0830] 5-Fluoro-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-dione (500.00 mg, 2.03 mmol) and (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)methyl ester (776.65 mg, 2.03 mmol) were dissolved in DMF (20 mL), and then HCl / 1,4-dioxane (2 M, 2.03 mL) was added. The mixture was reacted overnight at room temperature. After the reaction was complete, the system was extracted with saturated sodium chloride aqueous solution and ethyl acetate. The organic phase was collected, dried, and concentrated to obtain the crude product. After the reaction was complete, the product was purified by silica gel column chromatography (methanol / dichloromethane = 0%-10%, 40 min) to obtain compound H-2-2 (140 mg, 246.24 μmol).

[0831] Step Two:

[0832] (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 (120 mg, 211.06 μmol) was dissolved in DMF (2 mL), and then diethylamine (1 mL) was added. The reaction was carried out at room temperature for two hours. After the reaction was completed, the solvent was removed to obtain crude H-2-2. The crude product was then rapidly proceeded to the next step without purification.

[0833] Step 3:

[0834] (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).

[0835] Its structural characterization data are as follows:

[0836] MS m / z (ESI): 761.3 [M+Na] +

[0837] Its preparation method is as follows:

[0838] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0839] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0840] Example 3: 1-(N-((S)-1-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo[1,2-b]quinoline-4-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylic acid pentafluorophenol ester (A-1)

[0841] Step 1:

[0842] Chlorosulfonyl isocyanate (2.14 g, 15.13 mmol) was added to dichloromethane (50 mL), and the mixture was cooled in an ice-water bath for 10 minutes. Then, tert-butyl 2-hydroxyacetate (2 g, 15.13 mmol) was added to the reaction mixture, and the mixture was stirred in an ice-water bath for 2 hours. Piperidine-4-carboxylic acid allyl ester hydrochloride (3.74 g, 18.16 mmol) and triethylamine (4.59 g, 45.40 mmol) were added to the reaction mixture, and the mixture was removed from the ice-water bath and allowed to return to room temperature naturally before stirring for 3 hours. The reaction was quenched with water (100 mL), and the mixture was extracted three times with ethyl acetate (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After purification by silica gel column chromatography (DCM / PE = 0-100%), the product was concentrated again to obtain compound A-1-2 (1.87 g, 4.19 mmol).

[0843] Its structural characterization data are as follows:

[0844] 1H NMR (400MHz, DMSO) δ11.64(s,1H),6.06-5.79(m,1H),5.35-5.17(m,2H),4.61-4.48(m,4H),4.03(q,J=7.1Hz,1H),3.5 9(dt,J=12.4,3.3Hz,2H),3.04-2.88(m,2H),1.93(dd,J=13.5,3.2Hz,2H),1.59(dd,J=13.4,3.6Hz,2H),1.43(s,9H).

[0845] Step Two:

[0846] 1-(N-((2-(tert-butoxy)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylic acid allyl ester (I-50-2) (1.75 g, 4.31 mmol) was added to a mixed solvent of trifluoroacetic acid (5 mL) and dichloromethane (10 mL) and reacted at 25 °C for 2 hours. The reaction mixture was concentrated to dryness, diluted with ethyl acetate, and the pH was adjusted to approximately 8 with sodium bicarbonate aqueous solution. Impurities were removed by extraction, and the pH was adjusted to approximately 3 with 3N dilute hydrochloric acid. The mixture was extracted three times with ethyl acetate (30 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound A-1-3 (1.4 g, 4.00 mmol).

[0847] Its structural characterization data are as follows:

[0848] ESI-MS (m / z): 351.1 [M+H] + .

[0849] Step 3:

[0850] 2-((((4-(((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (I-50-3) (18.32 mg, 52.30 μmol), (S)-2-amino-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indene[1,2-b]quinoline-4-yl)amino)-1-oxopropane- 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (23.85 mg, 62.76 μmol) and DIPEA (20.28 mg, 156.89 μmol) were dissolved in DMF (1 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 1 / 2) and then freeze-dried to obtain compound A-1-4 (40 mg, 44.15 μmol).

[0851] Its structural characterization data are as follows:

[0852] MS m / z (ESI): 906.4 [M+H] +

[0853] Step Four:

[0854] Dissolve 1-((2-(((S)-1-(((S)-1-(((S)-1-((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indo[1,2-b]quinoline-4-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidin-4-carboxylic acid allyl ester (I-50-4) (40 mg, 44.15 μmol) in DMF (1 mL), add tetrakis(triphenylphosphine)palladium (5.1 mg, 4.41 μmol), add 15 μL formic acid, 30 μL N-methylmorpholine was stirred at room temperature for 1 hour under nitrogen protection. After the reaction was completed, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 3 / 1) and then freeze-dried to obtain compound A-1-5 (20 mg, 23.10 μmol).

[0855] Its structural characterization data are as follows:

[0856] MS m / z (ESI): 867.4 [M+H] +

[0857] Step 5:

[0858] The 1-(N-((2-(((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indene[1,2-b]quinoline-4-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-2-oxoethoxy)carbonyl)aminosulfonyl Piperidine-4-carboxylic acid (I-50-5) (20 mg, 23.10 μmol) was dissolved in DMF (1 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (27.72 mg, 144.36 μmol) and pentafluorophenol (15.94 mg, 86.61 μmol) were added. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain compound A-1 (12.53 mg, 12.02 μmol).

[0859] Its structural characterization data are as follows:

[0860] MS m / z (ESI): 1033.4 [M+H] +

[0861] Its preparation method is as follows:

[0862] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0863] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0864] Example 4: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholine)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-N-(4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentoxa-3,6,25-triazatridodecane-30-acetylamidamide)benzyl)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxamide (K-4)

[0865] Step 1: Preparation of (S)-(4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamidyl)benzyl)tert-butyl carbamate (K-4-2):

[0866] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (700.30 mg, 2.25 mmol) and tert-butyl 4-aminobenzylcarbamate (0.5 g, 2.25 mmol, FR) were dissolved in DMF (12 mL), and DIPEA (581.43 mg, 4.50 mmol, 783.60 μL) was added. Then, HATU (940.24 mg, 2.47 mmol) was added in portions, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was added dropwise to water with stirring. A large amount of solid precipitated out. The solid was filtered, and the filter cake was dried to obtain the title compound (1.04 g, 2.02 mmol).

[0867] Its structural characterization data are as follows:

[0868] MS m / z(ESI): 460.2 [M+H-56] +

[0869] Step 2: Preparation of (S)-(4-(2-aminopropionamido)benzyl)carbamate tert-butyl ester (K-4-3):

[0870] DMF (6 mL) was added to (S)-(4-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propamido)benzyl)tert-butyl carbamate (0.51 g, 989.14 μmol), followed by the addition of diethylamine (723.42 mg, 9.89 mmol, 1.02 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the solvent was removed by lyophilization. The crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate = 0-100%; ethyl acetate-methanol = 0-20%) and concentrated under reduced pressure to obtain the title compound (266 mg, 906.73 μmol).

[0871] Its structural characterization data are as follows:

[0872] MS m / z(ESI): 588.4 [2M+H] +

[0873] Step 3: Preparation of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butoxycarbonyl)amino)amino)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate (K-4-4):

[0874] (S)-(4-(2-aminopropamido)benzyl)carbamate tert-butyl ester (133 mg, 453.37 μmol) was dissolved in DMF (3 mL), followed by the addition of DIPEA (117.19 mg, 906.73 μmol), and then (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (169.25 mg, 498.70 μmol). The mixture was stirred until dissolved. Then, HATU (189.51 mg, 498.70 μmol) was added, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was complete, the reaction solution was used directly as the theoretical amount (270 mg, 439.22 μmol) for the next step of deprotection without any further treatment.

[0875] Its structural characterization data are as follows:

[0876] MS m / z (ESI): 1251.5 [2M+Na] +

[0877] Step 4: Preparation of tert-butyl(4-((S)-2-((S)-2-amino-3-methylbutamido)propionamido)benzyl)carbamate (K-4-5):

[0878] Diethylamine (321.23 mg, 4.39 mmol) was added dropwise to the previous reaction solution, which theoretically contained (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-((((tert-butoxycarbonyl)amino)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate (270 mg, 439.22 μmol) and DMF (3 mL). The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the reaction solution was directly concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (ethyl acetate-methanol = 0-30%) and concentrated under reduced pressure to give the title compound (158 mg, 402.56 μmol).

[0879] Its structural characterization data are as follows:

[0880] MS m / z(ESI): 393.3 [M+H] +

[0881] Step 5: Preparation of (4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triaza-tetane-30-acetylamidamide)benzyl)tert-butyl carbamate (K-4-6):

[0882] 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-penta-19-aza-peco-24-alkynic acid (90.30 mg, 145.23 μmol) and (4-((S)-2-((S)-2-amino-3-methylbutamido)propamido)benzyl)carbamate tert-butyl carbamate (57 mg, 145.23 μmol) were dissolved in DMF (2 mL), DIPEA (48 mg, 371.40 μmol) was added dropwise, and then HATU (60.70 mg, 159.75 μmol) was added in portions. 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, washed with organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (150 mg, 144.52 μmol, 90% purity), which was used directly in the next reaction without purification.

[0883] Its structural characterization data are as follows:

[0884] MS m / z (ESI): 934.4 [M+H] +

[0885] Step Six: Preparation of N-((S)-1-(((S)-1-((4-(aminomethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-1-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-3,6,9,12,15-pentoxooctadecane-18-amide (K-4-7):

[0886] In the next step, crude tert-butyl carbamate (4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-penta-3,6,25-triaza-tetane-30-acetylamidamide)benzyl)carbamate (150 mg, 144.52 μmol, 90% purity) was reacted with dichloromethane (3 mL) followed by trifluoroacetic acid (1.5 mL) 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 and acetonitrile, purified by reversed-phase column chromatography (acetonitrile-0.05% trifluoroacetic acid aqueous solution = 0-90%), and then freeze-dried to obtain the trifluoroacetate of the title compound (85 mg, 89.66 μmol).

[0887] Its structural characterization data are as follows:

[0888] MS m / z (ESI): 834.4 [M+H] +

[0889] Step 7: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholine)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-N-(4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatridodecane-30-acetylamide)benzyl)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxamide (K-4)

[0890] Trifluoroacetate of N-((S)-1-(((S)-1-((4-(aminomethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-1-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-3,6,9,12,15-pentoxooctadecane-18-amide (35 mg, 41.97 μmol) and (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5 -Hydroxy-4-((S)-2-methoxymorpholine)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxylic acid (66.06 mg, 41.97 μmol, 40% purity) was dissolved in DMF (3 mL), followed by the addition of DIPEA (32.54 mg, 251.80 μmol) and then HATU (23.92 mg, 62.95 μmol). 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 obtain the title compound (13.99 mg, 9.48 μmol, 98% purity).

[0891] Its structural characterization data are as follows:

[0892] MS m / z (ESI): 1446.5 [M+H] +

[0893] Its preparation method is as follows:

[0894] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0895] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0896] Example 5: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholine)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-7-methoxy-N-(4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxamide (K-2)

[0897] Step 1: Preparation of (4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)tert-butyl carbamate (K-2-1)

[0898] (S)-(4-(2-aminopropamido)benzyl)carbamate tert-butyl ester (133 mg, 453.37 μmol) was dissolved in DMF (3 mL), DIPEA (256 mg, 1.98 mmol) was added, followed by trifluoroacetate of (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (266.77 mg, 498.70 μmol), and the mixture was stirred until dissolved. HATU (189.51 mg, 498.70 μmol) was then added, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was complete, the reaction mixture was extracted with water and ethyl acetate, washed with organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (290 mg, 451.18 μmol), which was used directly in the next reaction without purification.

[0899] Its structural characterization data are as follows:

[0900] MS m / z (ESI): 1285.4 [2M+H] + 543.3 [M+H-100] +

[0901] Step 2: Preparation of N-((S)-1-(((S)-1-((4-(aminomethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (K-2-2)

[0902] Dichloromethane (6 mL) was added to crude tert-butyl carbamate (4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)carbamate (6 mL), followed by trifluoroacetic acid (3 mL). The mixture was stirred at room temperature for 1 hour. 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 obtain the trifluoroacetate salt of the title compound (176 mg, 268.02 μmol).

[0903] Its structural characterization data are as follows:

[0904] MS m / z (ESI): 543.3 [M+H] +

[0905] Step 3: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholine)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-7-methoxy-N-(4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)propionamide)benzyl)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxamide (K-2)

[0906] The trifluoroacetate of N-((S)-1-(((S)-1-((4-(aminomethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (35 mg, 53.30 μmol) and (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methyl) (Oxymorpholine)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxylic acid (83.89 mg, 53.30 μmol, 40% purity) was dissolved in DMF (3 mL), DIPEA (41.33 mg, 319.79 μmol) and HATU (30.38 mg, 79.95 μmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate. The organic phase was filtered to remove flocculent matter, washed with brine, and concentrated under reduced pressure. The crude product was purified by preparative high performance liquid chromatography and freeze-dried to give the title compound (11.83 mg, 10.04 μmol, 98% purity).

[0907] Its structural characterization data are as follows:

[0908] MS m / z (ESI): 1154.4 [M+H] +

[0909] Its preparation method is as follows:

[0910] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0911] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0912] Example 6: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholine)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-N-(4-((2S,5S)-5-isopropyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triazatridodecane-30-acetylamide)benzyl)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxamide (K-3)

[0913] Step 1: Preparation of (S)-(4-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-ureidopentamido)benzyl)tert-butyl carbamate (K-3-1)

[0914] (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (590.0 mg, 1.48 mmol) and tert-butyl 4-aminobenzylcarbamate (300.1 mg, 1.35 mmol) were dissolved in dichloromethane (12 mL) and methanol (4 mL), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.0 g, 4.05 mmol) was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the system was evaporated to dryness to obtain a crude product. The crude product was purified by silica gel column chromatography (dichloromethane-methanol = 0-35%) and concentrated under reduced pressure to obtain the title compound (550 mg, 914.1 μmol).

[0915] Its structural characterization data are as follows:

[0916] MS m / z (ESI): 602.2 [M+H] +

[0917] Step 2: Preparation of (S)-(4-(2-amino-5-ureidopentamido)benzyl)tert-butyl carbamate (K-3-2):

[0918] DMF (3 mL) was added to (S)-(4-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-ureidopentamido)benzyl)tert-butyl carbamate (550 mg, 914.1 μmol), followed by the addition of diethylamine (0.3 mL). The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the solvent was removed by lyophilization, and 30 mL of petroleum ether / ethyl acetate (35%) was added and stirred for 1 hour. The mixture was then filtered to obtain the title compound (340 mg, 894.7 μmol).

[0919] Its structural characterization data are as follows:

[0920] MS m / z(ESI): 380.2 [2M+H] +

[0921] Step 3: Preparation of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-((((tert-butoxycarbonyl)amino)amino)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (K-3-3):

[0922] (S)-(4-(2-amino-5-ureidopentamido)benzyl)tert-butyl carbamate (340 mg, 894.7 μmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (308.5 mg, 909.2 μmol) were dissolved in DMF (3 mL), followed by the addition of DIPEA (352.5 mg, 273.1 μmol) and then HATU (414.6 mg, 1.09 mmol). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, 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 (320 mg, 456.5 μmol).

[0923] Its structural characterization data are as follows:

[0924] MS m / z (ESI): 701.1 [M+H] +

[0925] Step 4: Preparation of (4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl)tert-butyl carbamate (K-3-4):

[0926] DMF (3 mL) was added to (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-((((tert-butoxycarbonyl)amino)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (320 mg, 456.5 μmol), followed by the addition of diethylamine (0.3 mL). The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the solvent was removed by lyophilization, and 30 mL of petroleum ether / ethyl acetate (35%) was added and stirred for 1 hour. The mixture was then filtered to obtain the title compound (288 mg, 602.5 μmol).

[0927] Its structural characterization data are as follows:

[0928] MS m / z(ESI): 479.2 [2M+H] +

[0929] Step 5: Preparation of (4-((2S,5S)-5-isopropyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triaza-tetane-30-acetylamidamide)benzyl)tert-butyl carbamate (K-3-5):

[0930] 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-penta-19-aza-pecapone-24-alkynyl acid (50.10 mg, 104.5 μmol) and tert-butyl(4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl)carbamate (76.1 mg, 135.8 μmol) were dissolved in DMF (2 mL), and DIPEA (41 mg, 313.40 μmol) was added dropwise. Then, HATU (59.60 mg, 156.75 μmol) was added in portions, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%) and then freeze-dried to give the title compound (65 mg, 63.5 μmol).

[0931] Its structural characterization data are as follows:

[0932] MS m / z (ESI): 1020.2 [M+H] +

[0933] Step Six: Preparation of N-((S)-1-(((S)-1-((4-(aminomethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)-3,6,9,12,15-pentoxooctadecane-18-amide (K-3-6)

[0934] Dichloromethane (3 mL) was added to (4-((2S,5S)-5-isopropyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triaza-tetane-30-acetylamidamide)benzyl)tert-butyl carbamate (65 mg, 63.5 μmol), followed by trifluoroacetic acid (1.5 mL). The mixture was stirred at room temperature for 1 hour. 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 acetonitrile and then freeze-dried to obtain the trifluoroacetate salt of the title compound (33 mg, 32.1 μmol).

[0935] Its structural characterization data are as follows:

[0936] MS m / z (ESI): 920.2 [M+H] +

[0937] Step 7: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholine)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-N-(4-(((2S,5S)-5-isopropyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-2-(3-ureopropyl)-10,13,16,19,22-pentaoxa-3,6,25-triaza-tetane-30-acetylamide)benzyl)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxamide (K-3)

[0938] N-((S)-1-(((S)-1-((4-(aminomethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-3,6,9,12,15-pentoxooctadecane-18-amide (33 mg, 32.1 μmol) and (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)- 5-Hydroxy-4-((S)-2-methoxymorpholine)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxylic acid (50.4 mg, 32.1 μmol, 40% purity) was dissolved in DMF (3 mL), followed by the addition of DIPEA (24.84 mg, 192.60 μmol) and then HATU (24.6 mg, 38.4 μmol). 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 obtain the title compound (15.5 mg, 9.83 μmol, 97% purity).

[0939] Its structural characterization data are as follows:

[0940] MS m / z (ESI): 1531.8 [M+H] +

[0941] Its preparation method is as follows:

[0942] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0943] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0944] Example 7: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholine)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-7-methoxy-N-(4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)benzyl)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxamide (K-1)

[0945] Step 1: Preparation of (4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)butyramide)-5-ureidopentanamide)benzyl)tert-butyl carbamate (K-1-1)

[0946] (4-((S)-2-((S)-2-amino-3-methylbutamido)-5-ureidopentamido)benzyl)carbamate tert-butyl ester (50 mg, 104.5 μmol) was dissolved in DMF (2 mL), DIPEA (40.5 mg, 0.31 mmol) was added, followed by 2,5-dioxopyrrolidone-1-yl6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (45.8 mg, 125.30 μmol). After the reaction was complete, the reaction solution was purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%) and then freeze-dried to give the title compound (36 mg, 49.4 μmol).

[0947] Its structural characterization data are as follows:

[0948] MS m / z (ESI): 729.3 [M+H] +

[0949] Step 2: Preparation of N-((S)-1-(((S)-1-((4-(aminomethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (K-1-2)

[0950] Dichloromethane (2 mL) was added to (36 mg, 49.4 μmol) tert-butyl carbamate (4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)butyramide)-5-ureidopentanamide)benzyl)carbamate. The mixture was stirred at room temperature for 1 hour. 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 acetonitrile and then freeze-dried to obtain the trifluoroacetate salt of the title compound (31 mg, 49.4 μmol).

[0951] Its structural characterization data are as follows:

[0952] MS m / z (ESI): 629.2 [M+H] +

[0953] Step 3: Preparation of (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholine)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-7-methoxy-N-(4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)butyramide)-5-ureidopentanamide)benzyl)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxamide (K-1)

[0954] N-((S)-1-(((S)-1-((4-(aminomethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide (31 mg, 49.4 μmol) and (2S,4S)-2,5,12-trihydroxy-4-(((2R,4S,5S,6S)-5-hydroxy-4-((S)-2- Methoxymorpholine-6-methyltetrahydro-2H-pyran-2-yl)oxy-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxylic acid (75.1 mg, 49.4 μmol, 40% purity) was dissolved in DMF (3 mL), and DIPEA (38.2 mg, 296.2 μmol) and HATU (21.76 mg, 57.26 μmol) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate. The organic phase was filtered to remove flocculent matter, washed with brine, and concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography and freeze-dried to obtain the title compound (5.23 mg, 4.08 μmol, 96% purity).

[0955] Its structural characterization data are as follows:

[0956] MS m / z(ESI): 1240.8 [M+H] +

[0957] Its preparation method is as follows:

[0958] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[0959] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0960] Example 8: Preparation of S-((S)-10-benzyl-23-(2-(methanesulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentoxo-3-oxa-5,8,11,14,17-pentazatrisane-22-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-1)

[0961] Step 1: Preparation of (9H-fluorene-9-yl)methyl(2-(((2-iodoethoxy)methyl)amino)-2-oxoethyl)carbamate (J-1-2)

[0962] (2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (500 mg, 1.36 mmol) was dissolved in THF (5.00 mL), followed by the addition of 2-iodoethanol (479.62 mg, 2.79 mmol), and then p-toluenesulfonic acid monohydrate (51.64 mg, 271.46 μmol). The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was dissolved in dichloromethane and purified by silica gel column chromatography (petroleum ether-ethyl acetate = 0-100%), followed by further concentration under reduced pressure to obtain the title compound (400 mg, 832.82 μmol).

[0963] Its structural characterization data are as follows:

[0964] MS m / z (ESI): 498.1 [M+H2O] +

[0965] Step 2: Preparation of S-(1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-1-4)

[0966] Directly to the theoretically contained (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene The reaction mixture of 2-thiocarboxylic acid (78.35 mg, 163.13 μmol), excess sodium hydrosulfide (16 mg, 285.39 μmol), and DMF (3 mL) from the previous step was reacted with (9H-fluorene-9-yl)methyl(2-(((2-iodoethoxy)methyl)amino)-2-oxoethyl)carbamate (78.35 mg, 163.13 μmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was extracted with water and ethyl acetate, washed with organic phase brine, dried over anhydrous sodium sulfate, 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 (70 mg, 70.28 μmol).

[0967] Its structural characterization data are as follows:

[0968] MS m / z (ESI): 996.2 [M+H] + ; 1019.2 [M+Na] +

[0969] Step 3: Preparation of S-(2-((2-aminoacetamido)methoxy)ethyl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-1-5)

[0970] S-(1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (20 mg, 20.08 μmol) was dissolved in DMF (1 mL), and piperidine (17.10 mg, 200.80 μmol) was added dropwise. The system turned black instantly, and the mixture was stirred at room temperature for 15 minutes. After the reaction was completed, the reaction solution was diluted with ethyl acetate and washed with plenty of water, followed by washing with organic phase brine, drying with anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain the crude product of the title compound (15 mg, 19.38 μmol), which was used directly in the next reaction without purification.

[0971] Its structural characterization data are as follows:

[0972] MS m / z (ESI): 774.3 [M+H] +

[0973] Step 4: Preparation of S-((S)-10-benzyl-23-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentoxo-3-oxa-5,8,11,14,17-pentazatrisane-22-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-1)

[0974] S-(2-((2-aminoacetamido)methoxy)ethyl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene 2-Thiocarboxylic acid ester (15 mg, 19.38 μmol) and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (10.27 mg, 19.38 μmol) were dissolved in DMF (2 mL), DIPEA (5.01 mg, 38.77 μmol) was added, followed by PyBOP (12.11 mg, 23.26 μmol), and the mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (3.63 mg, 2.77 μmol, 98% purity).

[0975] Its structural characterization data are as follows:

[0976] MS m / z (ESI): 1286.4 [M+H] +

[0977] Its preparation method is as follows:

[0978] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0979] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0980] Example 9: Preparation of S-((7S,10S,13S)-7,10,13-trimethyl-20-(2-(methanesulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosico-19-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-2)

[0981] Step 1: Preparation of (9H-fluorene-9-yl)methyl(S)-(1-(((2-iodoethoxy)methyl)amino)-1-oxopropane-2-yl)carbamate (J-2-2)

[0982] (S)-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propamido)methyl ester (1 g, 2.62 mmol) was dissolved in THF (10 mL), followed by the addition of 2-iodoethanol (924.06 mg, 5.37 mmol), and then p-toluenesulfonic acid monohydrate (99.48 mg, 523.00 μmol). The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried over anhydrous sodium sulfate, 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 (928 mg, 1.88 mmol).

[0983] Its structural characterization data are as follows:

[0984] MS m / z (ESI): 512.1 [M+H2O] +

[0985] Step 2: Preparation of S-((S)-1-(9H-fluorene-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-2-3)

[0986] Directly to the theoretically contained (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thio A mixture of substituted carboxylic acid (127 mg, 197.31 μmol), DMF (5 mL), and excess sodium hydrosulfide (28.80 mg, 513.71 μmol) from the previous reaction was added to (9H-fluorene-9-yl)methyl(S)-(1-(((2-iodoethoxy)methyl)amino)-1-oxopropane-2-yl)carbamate (100 mg, 202.30 μmol). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was used directly in the next reaction at the theoretical yield (127 mg, 197.31 μmol) without any treatment.

[0987] Its structural characterization data are as follows:

[0988] MS m / z (ESI): 644.2 [M+H] +

[0989] Step 3: Preparation of S-(2-(((S)-2-aminopropamido)methoxy)ethyl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thioester (J-2-4)

[0990] S-((S)-1-(9H-fluorene-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4, 5]Oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (40 mg, 27.72 μmol) was dissolved in DMF (2.00 mL), and piperidine (17.24 mg, 202.47 μmol) was added dropwise. The system turned black instantly, and the reaction was stirred at room temperature for 15 minutes. After the reaction was complete, the reaction solution was diluted with ethyl acetate, washed with plenty of water, washed with organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (20 mg, 25.39 μmol), which was used directly in the next reaction without purification.

[0991] Its structural characterization data are as follows:

[0992] MS m / z(ESI): 788.3 [M+H] +

[0993] Step 4: Preparation of S-((7S,10S,13S)-7,10,13-trimethyl-20-(2-(methanesulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosico-19-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-2)

[0994] (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (10.42 mg, 25.39 μmol) and S-(2-(((S)-2-aminopropionamido)methoxy)ethyl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4'] The crude product of [3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thioester (20 mg, 25.39 μmol) was dissolved in DMF (2 mL), followed by the addition of DIPEA (16 mg, 123.80 μmol) and then PyBOP (15.85 mg, 30.46 μmol). The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (6.65 mg, 5.35 μmol, 95% purity).

[0995] Its structural characterization data are as follows:

[0996] MS m / z (ESI): 1181.4 [M+H] +

[0997] Its preparation method is as follows:

[0998] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0999] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[1000] Example 10: Preparation of S-((10S,13S)-10-(4-aminobutyl)-13-isopropyl-20-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosico-19-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aR,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-3)

[1001] Step 1: S-((10S,13S)-10-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-13-isopropyl-20-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosoe-19-yn-1-yl)(2S,4S)-2,5,12-trihydroxy- Preparation of 7-methoxy-4-(((1S,3R,4aR,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-3-1)

[1002] N 6 -(diphenyl(p-tolyl)methyl)-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (29.54 mg, 39.29 μmol) and S-(2-((2-aminoacetamido)methoxy)ethyl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aR,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3': The crude product of [4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thioester (38 mg, 49.11 μmol) was mixed with 3 mL of DMF, followed by dropwise addition of DIPEA (19.04 mg, 147.32 μmol), and then BOP condensing agent (21.72 mg, 49.11 μmol). The mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate and washed several times with water. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane-(dichloromethane:methanol = 3:1) = 0-50%) and concentrated again under reduced pressure to obtain the title compound (43 mg, 28.52 μmol).

[1003] Its structural characterization data are as follows:

[1004] MS m / z (ESI): 1507.5 [M+H] +

[1005] Step 2: Preparation of S-((10S,13S)-10-(4-aminobutyl)-13-isopropyl-20-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosico-19-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aR,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-3)

[1006] S-((10S,13S)-10-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-13-isopropyl-20-(2-(methanesulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosoe-19-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aR,9S) Dichloromethane (2 mL) was added to 43 mg (28.52 μmol) of 9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester, followed by formic acid (0.2 mL). The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure at room temperature to remove the solvent. The crude product was dissolved in acetonitrile, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (1.35 mg, 0.99 μmol, 95% purity).

[1007] Its structural characterization data are as follows:

[1008] MS m / z (ESI): 1251.4 [M+H] + 626.4 [M / 2+H] +

[1009] Its preparation method is as follows:

[1010] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[1011] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[1012] Example 11: 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 (G-1)

[1013] 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 (G-1-2)

[1014] (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.

[1015] Its structural characterization is as follows:

[1016] ESI-MS (m / z): 974.3 [M+H] + .

[1017] Its preparation method is as follows:

[1018] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1019] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1020] 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 (G-1-3)

[1021] (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 purified by preparative high performance liquid chromatography and then freeze-dried to obtain 307 mg of compound G-1-3.

[1022] Its structural characterization is as follows:

[1023] ESI-MS (m / z): 752.3 [M+H] + .

[1024] Its preparation method is as follows:

[1025] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1026] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1027] Step 3: 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 (G-1)

[1028] G-1-3 (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.

[1029] Its structural characterization data are as follows:

[1030] MS m / z (ESI): 1002.4 [M+H] + .

[1031] The separation and purification methods are as follows:

[1032] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[1033] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1034] Example 12: Preparation of N-((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)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (G-7)

[1035] Step 1: Preparation of ((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)tert-butyl carbamate (G-7-2)

[1036] (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).

[1037] Its structural characterization data are as follows:

[1038] MS m / z (ESI): 561.3 [M+H] +

[1039] 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 (G-7-3)

[1040] 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 give the hydrochloride salt of the title product (35 mg, 70.43 μmol).

[1041] Its structural characterization data are as follows:

[1042] MS m / z (ESI): 461.2 [M+H] +

[1043] Step 3: Preparation of N-((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)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (G-7)

[1044] Dissolve (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine (8.95 mg, 24.37 μmol) and (S)-2-amino-N-((S)-8-ethyl-8-hydroxy-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopentanopyrano[3',4':6,7]indolo[1,2-b]quinoline-3-yl)propionamide trifluoroacetate (20 mg, 24.37 μmol) in DMF (1 mL), add PyBOP (15.22 mg, 29.24 μmol), then add DIPEA (16 mg, 123.80 μmol), and stir at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography twice and then freeze-dried to obtain the title compound (9.62 mg, 11.64 μmol, 98% purity).

[1045] Its structural characterization data are as follows:

[1046] MS m / z (ESI): 810.3 [M+H] +

[1047] The first preparation method is as follows:

[1048] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[1049] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1050] The second preparation method is as follows:

[1051] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[1052] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[1053] Example 13: Preparation of (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-3-((4S,7S,10S)-4,7,10-trimethyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)thiazolidin-4-carboxamide (G-5)

[1054] Step 1: Preparation of (9H-fluorene-9-yl)methyl((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropane-2-yl)carbamate (G-5-1)

[1055] (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (70 mg, 127.13 μmol) was dissolved in THF (10 mL). After purging with nitrogen three times, lithium hydroxide monohydrate (32.01 mg, 762.80 μmol) was added under ice bath conditions. After stirring at room temperature for 10 minutes, (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)methyl ester (97.23 mg, 254.27 μmol) was added. After the addition was complete, the reaction was stirred at 22 °C for 5 hours. Add 20 mL of saturated ammonium chloride aqueous solution to the reaction solution, extract three times with dichloromethane (10 mL x 3), wash with 10 mL of brine, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the crude product of the title compound. After purification by preparative high performance liquid chromatography, freeze-dry to obtain the title compound (70 mg, 80.19 μmol).

[1056] Its structural characterization data are as follows:

[1057] MS m / z (ESI): 873.3 [M+H]+

[1058] Its preparation method is as follows:

[1059] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1060] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1061] Step 2: Preparation of (S)-3-(((S)-2-aminopropamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (G-5-2)

[1062] (9H-fluorene-9-yl)methyl((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropane-2-yl)carbamate (40 mg, 45.82 μmol) was dissolved in DMF (2 mL), and diethylamine (16.76 mg, 229.11 μmol) was added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (34 mg, 44.46 μmol).

[1063] Its structural characterization data are as follows:

[1064] MS m / z (ESI): 651.2 [M+H] +

[1065] Its preparation method is as follows:

[1066] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1067] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1068] Step 3: Preparation of (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-3-((4S,7S,10S)-4,7,10-trimethyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)thiazolidin-4-carboxamide (G-5)

[1069] (S)-3-(((S)-2-aminopropamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (40mg, 52mg) (31 μmol) and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (25.76 mg, 62.77 μmol) were dissolved in DMF (2 mL), and DIPEA (27.04 mg, 209.22 μmol) and HATU (39.75 mg, 104.61 μmol) were added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (41 mg, 38.52 μmol).

[1070] Its structural characterization data are as follows:

[1071] MS m / z (ESI): 1043.3 [M+H] +

[1072] Its preparation method is as follows:

[1073] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1074] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1075] Example 14: Preparation of pentafluorophenyl 1-(N-((2-(((S)-1-(((S)-1-(((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)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylic acid ester (A-4)

[1076] Step 1: 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 (A-4-1):

[1077] (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.

[1078] Its structural characterization data are as follows:

[1079] MS m / z (ESI): 660.3 [M+H] +

[1080] Step 2: 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]indolazino[1,2-b]quinoline-3-yl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (A-4-2)

[1081] 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).

[1082] Its structural characterization data are as follows:

[1083] MS m / z (ESI): 560.3 [M+H] +

[1084] Step 3: Preparation of 1-(N-((2-(((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)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylic acid allyl ester (A-4-3)

[1085] (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 trifluoroacetate (27 mg, 40.08 μmol) 1) and 2-((((4-(((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (18.17 mg, 44.09 μmol, 85% purity) were dissolved in DMF (3 mL), and DIPEA (16 mg, 123.80 μmol) was added dropwise. Then HATU (18.28 mg, 48.10 μmol) was added in portions, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (50 mg, 39.24 μmol, 70% purity), which was used directly in the next reaction without purification.

[1086] Its structural characterization data are as follows:

[1087] MS m / z (ESI): 892.2 [M+H] +

[1088] Step 4: Preparation of 1-(N-((2-(((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)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylic acid (A-4-4)

[1089] 1-(N-((2-(((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) Crude 2-oxoethoxy)carbonyl)aminosulfonyl)piperidin-4-carboxylic acid allyl ester (50.00 mg, 39.24 μmol, 70% purity) was dissolved in DMF (2 mL), followed by the addition of tetrahydropyrrole (17.04 mg, 239.59 μmol, 20 μL) and tetratetraphenylphosphine palladium (9.07 mg, 7.85 μmol). After nitrogen purging, the mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. After the reaction was complete, 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 (20 mg, 23.48 μmol).

[1090] Its structural characterization data are as follows:

[1091] MS m / z (ESI): 852.2 [M+H] +

[1092] Step 5: Preparation of pentafluorophenyl 1-(N-((2-(((S)-1-(((S)-1-(((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)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylic acid ester (A-4)

[1093] 1-(N-((2-(((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)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxyl) An acid (20 mg, 23.48 μmol) and 2,3,4,5,6-pentafluorophenol (21.61 mg, 117.39 μmol) were dissolved in DMF (2 mL). EDCI (22.50 mg, 117.39 μmol) was added, and the mixture was stirred at room temperature for 1 hour. Then, 2,3,4,5,6-pentafluorophenol (21.61 mg, 117.39 μmol) and EDCI (22.50 mg, 117.39 μmol) were added, and the reaction was continued for another 0.5 hours. After the reaction was complete, the reaction solution was directly purified by high-performance liquid chromatography and then freeze-dried to obtain the title compound (9.89 mg, 9.42 μmol, 97% purity).

[1094] Its structural characterization data are as follows:

[1095] MS m / z (ESI): 1018.2 [M+H] +

[1096] Its preparation method is as follows:

[1097] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[1098] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1099] Example 15: Preparation of perfluorophenyl 1-(N-((4S,7S,10S)-1-((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)-4,7,10-trimethyl-3,6,9,12-tetraoxo-14-oxa-2,5,8,11-tetraazapentadecan-15-acyl)aminosulfonyl)piperidine-4-carboxylic acid ester (A-10)

[1100] Step 1: Preparation of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (A-10-1):

[1101] The trifluoroethyl (S)-3-(((S)-2-aminopropamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide The acid salt (2.7 g, 3.53 mmol, TF) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (1.62 g, 4.24 mmol) were dissolved in DMF (15 mL), DIPEA (1.83 g, 14.12 mmol) was added, followed by DMTMM (2.08 g, 7.06 mmol), and the 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% trifluoroacetic acid aqueous solution = 0-90%), and then freeze-dried to give the title compound (2.8 g, 2.76 mmol).

[1102] Its structural characterization data are as follows:

[1103] MS m / z(ESI): 1032.4 [M+18] +

[1104] Step 2: Preparation of (S)-3-(((S)-2-((S)-2-((S)-2-aminopropamido)propamido)propamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (A-10-2)

[1105] To (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((S)-1-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2- [b] Quinolin-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (2.8 g, 2.76 mmol) was added to DMF (10 mL), followed by diethylamine (403.49 mg, 5.52 mmol). 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 and purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%), followed by freeze-drying to obtain the trifluoroacetate of the title compound (2.3 g, 2.54 mmol).

[1106] Its structural characterization data are as follows:

[1107] MS m / z(ESI): 793.2 [M+H] +

[1108] Step 3: Preparation of 1-(N-((4S,7S,10S)-1-((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)-4,7,10-trimethyl-3,6,9,12-tetraoxo-14-oxa-2,5,8,11-tetraazapentadecan-15-acyl)aminosulfonyl)piperidine-4-carboxylic acid allyl ester (A-10-3)

[1109] The (S)-3-(((S)-2-((S)-2-((S)-2-aminopropamido)propamido)propamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)thiazolyl-4-carboxyl The trifluoroacetate of the amine (800 mg, 882 μmol) and 2-((((4-(((allyloxy)carbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (1.77 g, 5.04 mmol) were dissolved in DMF (10 mL), and DIPEA (391.21 mg, 3.03 mmol) was added dropwise. Then, DMTMM (594.78 mg, 2.02 mmol) was added in portions, and the mixture was stirred at room temperature for 2 hours. 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 and purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%), followed by freeze-drying to give the title compound (850 mg, 755.42 μmol).

[1110] Its structural characterization data are as follows:

[1111] MS m / z (ESI): 1125.3 [M+H] +

[1112] Step 4: Preparation of 1-(N-((4S,7S,10S)-1-((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)-4,7,10-trimethyl-3,6,9,12-tetraoxo-14-oxa-2,5,8,11-tetraazapentadecan-15-acyl)aminosulfonyl)piperidine-4-carboxylic acid (A-10-4)

[1113] 1-(N-((4S,7S,10S)-1-((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)-4,7,10-trimethyl-3,6,9,1 2-Tetraoxo-14-oxa-2,5,8,11-tetraazapentadecan-15-acyl)aminosulfonyl)piperidin-4-carboxylic acid allyl ester (850 mg, 755.42 μmol) was dissolved in DMF (10 mL), followed by the addition of 1,3-dimethylbarbituric acid (176.77 mg, 1.13 mmol) and tetratetraphenylphosphine palladium (261.75 mg, 226.63 μmol). After nitrogen purging, the mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. After the reaction was complete, 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 (550 mg, 458.65 μmol).

[1114] Its structural characterization data are as follows:

[1115] MS m / z (ESI): 1085.2 [M+H] +

[1116] Step 5: Preparation of perfluorophenyl 1-(N-((4S,7S,10S)-1-((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)-4,7,10-trimethyl-3,6,9,12-tetraoxo-14-oxa-2,5,8,11-tetraazapentadecan-15-acyl)aminosulfonyl)piperidine-4-carboxylic acid ester (A-10)

[1117] 1-(N-((4S,7S,10S)-1-((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)-4,7,10-trimethyl -3,6,9,12-tetraoxo-14-oxa-2,5,8,11-tetraazapentadecan-15-acyl)aminosulfonyl)piperidine-4-carboxylic acid (300 mg, 250.17 μmol) and 2,3,4,5,6-pentafluorophenol (138.14 mg, 750.52 μmol) were dissolved in DMF (10 mL), and EDCI (95.92 mg, 500.35 μmol) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was directly purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (68 mg, 51.09 μmol, 94% purity).

[1118] Its structural characterization data are as follows:

[1119] MS m / z (ESI): 1251.2 [M+H] +

[1120] Its preparation method is as follows:

[1121] Column: Waters Sunfire Prep C18 OBD (5μm*19mm*150mm)

[1122] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1123] Example 16: Preparation of (S)-3-((S)-7-benzyl-20-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosico-19-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (G-6)

[1124] Step 1: Preparation of (9H-fluorene-9-yl)methyl(2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamate (G-6-1)

[1125] (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (240 mg, 435.89 μmol) was dissolved in THF (10 mL). After purging with nitrogen three times, lithium hydroxide monohydrate (109.74 mg, 2.62 mmol) was added under ice bath conditions. After stirring at room temperature for 10 minutes, (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (642.29 mg, 1.74 mmol) was added. After the addition was complete, the mixture was stirred at 22 °C for 5 hours. Add 20 mL of saturated ammonium chloride aqueous solution to the reaction solution, extract three times with dichloromethane (10 mL x 3), wash with 10 mL of brine, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the crude product of the title compound. After purification by preparative high performance liquid chromatography, freeze-dry to obtain the title compound (100 mg, 116.42 μmol).

[1126] Its structural characterization data are as follows:

[1127] MS m / z (ESI): 859.3 [M+H] +

[1128] Its preparation method is as follows:

[1129] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1130] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1131] Step 2: Preparation of (S)-3-((2-aminoacetamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (G-6-2)

[1132] (9H-fluorene-9-yl)methyl(2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamate (40 mg, 46.57 μmol) was dissolved in DMF (2 mL), and diethylamine (17.03 mg, 232.85 μmol) was added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (12 mg, 18.85 μmol).

[1133] Its structural characterization data are as follows:

[1134] MS m / z (ESI): 637.3 [M+H] +

[1135] Its preparation method is as follows:

[1136] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1137] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1138] Step 3: Preparation of (S)-3-((S)-7-benzyl-20-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosico-19-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (G-6)

[1139] (S)-3-((2-aminoacetamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (12 mg, 18 mg). 85 μmol) and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (11.98 mg, 22.62 μmol) were dissolved in DMF (2 mL), and DIPEA (9.74 mg, 75.39 μmol) and HATU (14.32 mg, 37.69 μmol) were added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (18.25 mg, 15.42 μmol).

[1140] Its structural characterization data are as follows:

[1141] MS m / z (ESI): 1148.3 [M+H] +

[1142] Its preparation method is as follows:

[1143] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1144] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1145] Example 17: Preparation of (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (G-19)

[1146] Step 1: Preparation of (S)-3-((7S,10S)-7-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (G-19-1)

[1147] (S)-3-((2-aminoacetamido)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (100 mg, 133.21 μmol) and N 6 -(diphenyl(p-tolyl)methyl)-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (110.18 mg, 146.53 μmol) was dissolved in DMF (2 mL), and DIPEA (68.86 mg, 532.82 μmol) and HATU (119.37 mg, 314.12 μmol) were added. The mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (60 mg, 43.78 μmol).

[1148] Its structural characterization data are as follows:

[1149] MS m / z (ESI): 1370.5 [M+H] +

[1150] Its preparation method is as follows:

[1151] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1152] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1153] Step 2: Preparation of (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (G-19)

[1154] (S)-3-((7S,10S)-7-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- Methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (80 mg, 58.37 μmol) was dissolved in DCM (5 mL), and formic acid (1 mL) was added. The mixture was stirred at 22 °C for 4 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (11 mg, 8.51 μmol, purity 95%).

[1155] Its structural characterization data are as follows:

[1156] MS m / z (ESI): 1114.4 [M+H] +

[1157] Its preparation method is as follows:

[1158] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1159] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[1160] Example 18: (17S,20S)-6,6-bis((2-carboxyethoxy)methyl)-17-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6, Preparation of [7]indo[1,2-b]quinoline-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamoyl)-20-isopropyl-27-(2-(methylsulfonyl)pyrimidin-5-yl)-11,19,22-trioxo-4,8-dioxa-12,18,21-triazaheptadecane-26-alkynic acid (G-20)

[1161] The (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methylsulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]in Dolino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (20 mg, 16.28 μmol) and 3,3'-((2,2-bis((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionic acid (27.64 mg, 65.13 μmol) were dissolved in DMF (2 mL). DIPEA (8.42 mg, 65.13 μmol) and HATU (13.64 mg, 35.90 μmol) were added, and the mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (5.0 mg, 3.09 μmol, purity 94%).

[1162] Its structural characterization data are as follows:

[1163] MS m / z (ESI): 1520.4 [M+H] +

[1164] Its preparation method is as follows:

[1165] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1166] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1167] Example 19: 3,3'-((2-((3-((1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl)amino)-3-oxopropoxy)methyl)-2-((11S,14S)-11-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indole) Preparation of [1,2-b]quinolin-1-yl)carbamoyl)thiazolidin-3-yl)methyl)amino)-2-oxoethyl)carbamoyl)-14-isopropyl-21-(2-(methylsulfonyl)pyrimidin-5-yl)-5,13,16-trioxo-2-oxa-6,12,15-triazacotetraane-20-yn-1-yl)propane-1,3-diyl)bis(oxy))bis(N-(1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl)propamide) (G-21)

[1168] (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)thiazolidin-4-carboxamide Dissolve 6 mg (4.85 μmol) and 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 (5.93 mg, 7.27 μmol) in DMF (1 mL), add 2,6-dimethylpyridine (2.08 mg, 19.38 μmol) and HATU (3.69 mg, 9.69 μmol), and stir at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (3.18 mg, 1.65 μmol, purity 95%).

[1169] Its structural characterization data are as follows:

[1170] MS m / z(ESI): 1829.7 [M+H] +

[1171] Its preparation method is as follows:

[1172] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1173] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1174] Example 20: 3,3'-((2-((11S,14S)-11-((2-((((S)-4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-1-yl)carbamoyl)thiazolyl-3-yl)methyl)amino)-2-oxoethyl)carbamoyl)-14-isopropyl-2 Preparation of 1-(2-(methylsulfonyl)pyrimidin-5-yl)-5,13,16-trioxo-2-oxa-6,12,15-triazacotetradecane-20-yn-1-yl)-2-((3-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))bis(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)propionamide)(G-22)

[1175] (S)-3-((7S,10S)-7-(4-aminobutyl)-10-isopropyl-17-(2-(methanesulfonyl)pyrimidin-5-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazaheptadecane-16-yn-1-yl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)thiazolidin-4-carboxamide (20 mg, 16.28 μL) 20.24 mg (21.17 μmol) of methylpropoxy ((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 (20.24 mg, 21.17 μmol) was dissolved in DMF (2 mL), followed by the addition of DIPEA (8.42 mg, 65.13 μmol) and HATU (12.37 mg, 32.57 μmol), and the mixture was stirred at 22 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (8.8 mg, 4.07 μmol, purity 95%).

[1176] Its structural characterization data are as follows:

[1177] MS m / z (ESI): 1026.9 [M / 2+H] +

[1178] Its preparation method is as follows:

[1179] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[1180] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1181] Example 21: Preparation of 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-oxopropane-2-yl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (I-1)

[1182] Step 1: Preparation of tert-butyl((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-oxopropane-2-yl)carbamate (I-1-1)

[1183] 4-Amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one (200 mg, 0.41 mmol) and (tert-butyloxycarbonyl)-L-alanine (92.3 mg, 0.49 mmol) were dissolved in acetonitrile (6 mL), and tetramethylchlorourea hexafluorophosphate (182.5 mg, 0.65 mmol) and N-methylimidazolium (100.1 mg, 1.22 mmol) were added. The mixture was stirred at room temperature for 15 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 give the title compound (210 mg, 0.32 mmol).

[1184] Its structural characterization data are as follows:

[1185] MS m / z (ESI): 663.3 [M+H] +

[1186] Step 2: Preparation of (S)-2-amino-N-(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)propionamide (I-1-2)

[1187] Dichloromethane (5 mL) was added to tert-butyl((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-oxopropane-2-yl)carbamate (210 mg, 0.32 mmol), followed by the addition of trifluoroacetic acid (1 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 purified by reversed-phase column chromatography (acetonitrile-0.05% formic acid aqueous solution = 0-90%) and then freeze-dried to give the title compound (45 mg, 0.08 mmol).

[1188] Its structural characterization data are as follows:

[1189] MS m / z (ESI): 562.2 [M+H] +

[1190] Step 3: Preparation of N-((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...

Claims

1. A compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, said compound having the structure shown in formula (I): in: Q is the structure before it is linked to the antibody or antigen-binding fragment; L is the connector sub-part; E is the self-eliminating part; D represents the bioactive molecule portion; n is selected from 1-10.

2. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, Q is selected independently from the following structures: Where R is H or C 1-6 Alkyl group; p is selected independently from integers 1-12 each time it appears; Preferably, Q is selected from the following structures: Where R is H or C 1-6 alkyl; Preferably, Q is selected from the following structures:

3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, L is a divalent structure composed of one or more of the following substituted or unsubstituted structural segments: 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, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:44), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:45), Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:46), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:47), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO: 48), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO: 49), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO: 50), 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, 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 from 1 to 20 each time it appears, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Preferably, L is selected from the following structures: Each time r appears, it is independently selected from an integer between 1 and 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

4. The compound according to any one of claims 1-3, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, E is selected independently from single bonds. -NH-CH2-, Preferably, E is selected from single bonds, -NH-CH2- or Preferably, E is selected from single bonds; Preferably, E is selected from -NH-CH2-.

5. The compound of any one of claims 1-4, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, The bioactive molecules are each independently selected from antitumor drugs or compounds with antitumor effects; Preferably, each of the bioactive molecules is independently selected from cytotoxic compounds or antimetabolites; Preferably, the cytotoxic compound is a DNA damaging agent, a topoisomerase inhibitor, an RNA polymerase inhibitor, or a microtubule inhibitor; Preferably, the antimetabolite compound is a nucleoside compound or a folic acid analogue; Preferably, the DNA damaging agent is selected from pyrrolobenzodiazepines, carzimidoxamines, pyroximide compounds, and anthracyclines; Preferably, the topoisomerase inhibitor is selected from camptothecin compounds, anthracycline compounds, and etoposide compounds; Preferably, the topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecins, including but not limited to hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, or rubotecan, etc.) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, docalmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); 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, D is connected to E via -OH, a primary amino group or a secondary amino group, or -SH, respectively; Preferably, the bioactive molecule is selected from the following compounds:

6. The compound of any one of claims 1-5, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein the compound is selected from the structures shown below:

7. The compound of any one of claims 1-6 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, wherein the compound is coupled to an antibody or antigen fragment by a substitution reaction (e.g., removal of -SO2Me or pentafluorophenol) or by an addition reaction.

8. An antibody-drug conjugate, said conjugate having the following structure: Wherein L' and L” are each independently described as L in claim 1 or 3; E' and E” are each independently as described in claim 1 or 4 of E; D' and D” are each independently as described in claim 1 or 5 of D; n1 and n2 are each independently as described in n of claim 1; A represents an antibody or its antigen-binding fragment; Q' and Q” are the structural forms of Q according to claim 1 or 2 after covalently linking it with an antibody or its antigen-binding fragment; x and y are each independently selected from 1 to 10; Preferably, in the antibody-drug conjugate, D' and D” are conjugated to the antibody or its antigen-binding fragment via linkers; Preferably, the antibody or its antigen-binding fragment is capable of specifically binding to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

9. The antibody-drug conjugate of claim 8, wherein, Q' and Q" are selected individually from the following structures: Preferably, Q' and Q” are individually selected from the following structures:

10. The antibody-drug conjugate of claim 8 or 9, 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; Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with 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; Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with 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; Wherein, the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with 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; Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with 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.

11. The antibody-drug conjugate according to any one of claims 8-10, 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; 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; 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; Preferably, 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; 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; Preferably, 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; Preferably, 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; 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 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 of the sequence shown in SEQ ID NO: 4 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; or (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.

12. The antibody-drug conjugate according to any one of claims 8-11, wherein Q is linked to a thiol group (-SH) or an amino group (-NH2) on A.

13. The antibody-drug conjugate according to any one of claims 8-12, wherein, The antibody or its antigen-binding fragment is selected from the antibody or its antigen-binding fragment as described in claim 11; The compound is selected from the compound of claim 6; x and y are each independently 1 to 10; preferably, x and y are each independently 1 to 8, or one of x and y is selected from 1 to 4 and the other is selected from 4 to 8.

14. Antibody-drug conjugates, the structure of which is shown in the following formula: in, The x, D, E, L, Q' and A are as described in any one of claims 1-13; Preferably, the antibody-drug conjugate is selected from the following structures:

15. The antibody-drug conjugate according to any one of claims 8-13, wherein the conjugate is selected from: Preferably, the -S) in each antibody-drug conjugate x -Ab- y (NH-, -NH) x -Ab- y (S-, or -S) x -Ab- y (S- 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; wherein x and y are each independently 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; in, This indicates the specific linkage between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment; This indicates the specific linkage between the amino and carbonyl groups in the antibody or its antigen-binding fragment; -NH in various antibody-drug conjugates x -Ab'- y (S-、-NH) x -Ab' or -S) x -Ab' 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; wherein x and y are each independently 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; in, This indicates the specific linkage between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment; This indicates the specific linkage between the amino and carbonyl groups in the antibody or its antigen-binding fragment.

16. A composition of an antibody-drug conjugate, said composition comprising one or more antibody-drug conjugates according to any one of claims 8-15; preferably, the composition has a DAR (drug-antibody conjugate ratio) of 1-30, for example: about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 1 to 11, about 1 to 12, about 1 to 13, about 1 to 14, about 1 to 15, about 1 to 16, about 1 to 17, about 1 to 18, about 1 to 19, about 1 to 20, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 2 to 11, about 2 to 12, about 2 to 13, about 2 to 30. 14, about 2 to 15, about 2 to 16, about 2 to 17, about 2 to 18, about 2 to 19, about 2 to 20, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 3 to 11, about 3 to 12, about 3 to 13, about 3 to 14, about 3 to 15, about 3 to 16, about 3 to 17, about 3 to 18, about 3 To 19, about 3 to 20, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 4 to 11, about 4 to 12, about 4 to 13, about 4 to 14, about 4 to 15, about 4 to 16, about 4 to 17, about 4 to 18, about 4 to 19, about 4 to 20, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 1 0, approximately 5 to 11, approximately 5 to 12, approximately 5 to 13, approximately 5 to 14, approximately 5 to 15, approximately 5 to 16, approximately 5 to 17, approximately 5 to 18, approximately 5 to 19, approximately 5 to 20, approximately 6 to 7, approximately 6 to 8, approximately 6 to 9, approximately 6 to 10, approximately 6 to 11, approximately 6 to 12, approximately 6 to 13, approximately 6 to 14, approximately 6 to 15, approximately 6 to 16, approximately 6 to 17, Approximately 6 to 18, approximately 6 to 19, approximately 6 to 20, approximately 7 to 8, approximately 7 to 9, approximately 7 to 10, approximately 7 to 11, approximately 7 to 12, approximately 7 to 13, approximately 7 to 14, approximately 7 to 15, approximately 7 to 16, approximately 7 to 17, approximately 7 to 18, approximately 7 to 19, approximately 7 to 20, approximately 8 to 9, approximately 8 to 10, approximately 8 to 11, approximately 8 to 12, approximately 8 to 13, approximately 8 To 14, about 8 to 15, about 8 to 16, about 8 to 17, about 8 to 18, about 8 to 19, about 8 to 20, or about 9 to 10, about 9 to 11, about 9 to 12, about 9 to 13, about 9 to 14, about 9 to 15, about 9 to 16, about 9 to 17, about 9 to 18, about 9 to 19, about 9 to 20, or about 10 to 11, about 10 to 12, about 10 Up to 13, about 10 to 14, about 10 to 15, about 10 to 16, about 10 to 17, about 10 to 18, about 10 to 19, about 10 to 20, or about 11 to 12, about 11 to 13, about 11 to 14, about 11 to 15, about 11 to 16, about 11 to 17, about 11 to 18, about 11 to 19, about 11 to 20, or about 12 to 13.Approximately 12 to 14, approximately 12 to 15, approximately 12 to 16, approximately 12 to 17, approximately 12 to 18, approximately 12 to 19, approximately 12 to 20, or approximately 12 to 13, approximately 12 to 14, approximately 12 to 15, approximately 12 to 16, approximately 12 to 17, approximately 12 to 18, approximately 12 to 19, approximately 12 to 20, or approximately 13 to 14, approximately 13 to 15, approximately 13 to 16, approximately 13 to 17, approximately 13 to 18, approximately 13 to 19, approximately 13 to 20, or about 14 to 15, about 14 to 16, about 14 to 17, about 14 to 18, about 14 to 19, about 14 to 20, or about 15 to 16, about 15 to 17, about 15 to 18, about 15 to 19, about 15 to 20, about 16 to 17, about 16 to 18, about 16 to 19, about 16 to 20, or about 17 to 18, about 17 to 19, about 17 to 20, about 18 to 19, about 18 to 20, or about 19 to 20.

17. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 8-15, the composition of claim 16, and one or more pharmaceutical excipients.

18. Use of 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 of the same, or an antibody-drug conjugate of any one of claims 8-15, a composition of claim 16, or a pharmaceutical composition of claim 17 in the preparation of a medicament for treating or preventing cancer; preferably, the cancer is a HER2-expressing cancer.

19. The use according to claim 18, 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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