Camptothecin derivative, antibody, drug conjugate, composition comprising same and use thereof

Abstract

Disclosed in the present invention are a camptothecin derivative, an antibody, a drug conjugate, a composition comprising same, and a use thereof. Specifically provided is a compound as shown in formula (I) or a pharmaceutically acceptable salt thereof. The compound of the present invention has one or more of the following advantages: (1) high cell activity, (2) broad sensitivity, (3) excellent in vitro tumor cell proliferation inhibition effect, (4) excellent in vivo tumor inhibition effect, (5) capabilities to enhance DNA damage activity and reduce the ability of tumor cells to repair DNA damage, (6) a capability to enhance the activity of inducing tumor cell apoptosis, (7) good in vivo safety, (8) high DAR value uniformity of ADC containing the compound, (9) good in vivo stability, (10) wide therapeutic window, (11) simple preparation, and (12) a novel toxin delivery and release mechanism.

Description

Camptothecin derivatives, antibodies, drug conjugates, and their combinations and applications

[0001] This application claims priority to Chinese patent application 2024108016800, filed on June 20, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to a camptothecin derivative, an antibody, a drug conjugate, a combination thereof, and its applications. Background Technology

[0003] DNA topoisomerases are located in the cell nucleus, and their substrate is DNA. They participate in cell replication, transcription, and mitosis. Camptothecin inhibits nuclear topoisomerase I, inducing double-strand DNA breaks during the S phase of mitosis in affected cells. Topoisomerases are divided into topoisomerase I (TopoI) and topoisomerase II (TopoII). Inhibition of topoisomerases leads to the accumulation of large amounts of broken DNA in tumor cells, thereby inducing tumor cell death. DNA topoisomerase I inhibitors include camptothecin and its derivatives, which have been used clinically to treat malignant tumors, such as irinotecan.

[0004] Camptothecin, first isolated from the camptotheca tree, possesses strong cytotoxicity and shows good efficacy against malignant tumors such as gastrointestinal cancer (gastric cancer, colon cancer, rectal cancer), liver cancer, breast cancer, bladder cancer, and leukemia. The main drawbacks of camptothecin are its poor solubility and stability, as well as its high toxicity, which limits its clinical application. Camptothecin derivatives can be improved by introducing water-soluble groups or preparing prodrugs, thereby enhancing its clinical applicability. Several camptothecin derivatives with significantly improved solubility have been approved, including topotecan and its carbamate precursor irinotecan.

[0005] Besides being used as chemotherapeutic agents in cancer treatment, camptothecin derivatives are also used as the effective payload for antibody-drug conjugates (ADCs). ADCs combine antibodies and small-molecule toxins, possessing the specificity of antibody binding to tumor cell surface antigens and the high activity of cytotoxic drugs in inhibiting and killing tumor cells. Compared to traditional chemotherapeutic drugs, ADCs can kill tumor cells more precisely and have less impact on normal cells.

[0006] In recent years, significant progress has been made in ADCs (anti-addictive drugs) using camptothecin derivatives as small molecule toxins. Daiichi Sankyo's first ADC, DS-8201a (Trastuzumab Deruxtecan), has been approved for marketing in Japan. This ADC uses deruxtecan, a derivative of exatecan, as its small molecule toxin, and features a self-destructive linker structure and a GGFG (SEQ ID NO:13) tetrapeptide that can be hydrolyzed by cathepsin B in tumor cells. Another ADC using the camptothecin derivative SN-38 as its payload, Sacituzumab Govitecan, has also been approved for marketing. When the ADC is internalized, the use of a moderately stable linker (carbonate linker) allows for the release of SN-38 in the acidic environment of tumor cells and their microenvironment, thereby contributing to a bystander effect on neighboring cancer cells.

[0007] However, using camptothecin as a small molecule toxin in ADCs typically requires a large DAR (drug irradiation rate) because it is not a highly toxic drug, has difficult manufacturing processes, and often makes the ADC unstable. Therefore, novel camptothecin derivatives with higher activity and broader sensitivity in tumor cells have specific and broad application potential as payloads for ADCs. Summary of the Invention

[0008] This invention addresses the shortcomings of existing camptothecin derivatives in terms of cell activity and / or sensitivity by providing a camptothecin derivative, its drug conjugate, its pharmaceutical composition, and its applications. The camptothecin derivative of this invention has at least one or more of the following advantages: (1) high cell activity, (2) broad sensitivity, (3) superior in vitro tumor cell proliferation inhibition, (4) superior in vivo tumor suppression, (5) enhanced DNA damage activity and reduced tumor cell repair capacity, (6) enhanced tumor cell apoptosis induction activity, (7) good in vivo safety, (8) high uniformity of DAR values ​​in ADCs containing it, (9) good in vivo stability, (10) wide therapeutic window, (11) simple preparation, and (12) novel toxin delivery and release mechanism.

[0009] The present invention mainly solves the above-mentioned technical problems through the following technical means.

[0010] This invention provides a compound of formula I or a pharmaceutically acceptable salt thereof.

[0011] Among them, R 1 It can be F, Cl, or CH3;

[0012] R 2 For F;

[0013] X is C; Y is NH, O, or α-NHCOC(CR) a R b Ob; end a is connected to the ring, and end b is connected to H;

[0014] R 3 and R 4 Independently, it is F, C1-C5 alkyl, or -OC1-C5 alkyl; and R 3 and R 4 Not both H; or, R 3 and R 4 Together with the attached carbon, it forms a cyclopropyl or cyclobutyl group;

[0015] R a and R b It is independently H, C1-C5 alkyl, or cyclopropyl;

[0016] or,

[0017] X is O; R 3 and R 4 It does not exist; Y is O or a-OCOC(CR) a R b Ob; end a is connected to the ring, and end b is connected to H.

[0018] In certain preferred embodiments of the present invention, certain groups in the compound represented by formula (I) or its pharmaceutically acceptable salt are defined as follows, and groups not mentioned are as described in any embodiment of the present invention (hereinafter referred to as "in some preferred embodiments").

[0019] In some preferred embodiments, R 3 and R 4 Independently, it is H, F, C1-C5 alkyl or -OC1-C5 alkyl; and R 3 and R 4 Not both H; or, R 3 and R 4 Together with the attached carbon, it forms a cyclopropyl or cyclobutyl group.

[0020] In some preferred embodiments, R 3 and R 4 Independently, it is H, F, C1-C5 alkyl or -OC1-C5 alkyl; and R 3 and R 4 They are not both H.

[0021] In some preferred embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is a compound represented by formula (I-1), formula (I-2), formula (I-3), or formula (I-4) or a pharmaceutically acceptable salt thereof:

[0022] In equations (I-1), (I-2), (I-3), and (I-4), R 1 R 2 R 3 R 4 X and Y are defined as described in any of the schemes.

[0023] In some preferred embodiments, R 1 It can be Cl or CH3; CH3 is preferred.

[0024] In some preferred embodiments, R 3 and R 4 Independently, it is F.

[0025] In some preferred embodiments, R 3 and R 4 Together with the attached carbon, it forms a cyclopropyl or cyclobutyl group; preferably a cyclopropyl group.

[0026] In some preferred embodiments, the C1-C5 alkyl group and the -OC1-C5 alkyl group are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl; preferably methyl.

[0027] In some preferred embodiments, R a For H, R b It is H or cyclopropyl.

[0028] In some preferred embodiments, X is C; Y is NH, O, or α-NHCOCCH2O-b, α-NHCOCH(cyclopropyl)Ob (preferred). ); End a is connected to the ring, and end b is connected to H.

[0029] In some preferred embodiments, X is O; Y is O or a-OCOCCH2O-b.

[0030] In some preferred embodiments, the compound represented by formula (I) has any of the following structures:

[0031] In some preferred embodiments, the pharmaceutically acceptable salt of the compound represented by formula (I) has any of the following structures:

[0032] trifluoroacetate, Trifluoroacetate.

[0033] This invention provides a ligand-drug conjugate, or a pharmaceutically acceptable salt thereof.

[0034] Pc-(LD)p,

[0035] Wherein, D is the structural segment shown in the following formula D;

[0036] Among them, R 1 R 2 R 3 R 4 The definitions of X and Y are as described in any of the schemes; terminal 1 is connected to L;

[0037] L is the connector (or connector body) that connects Pc and D;

[0038] Pc is the ligand or target portion that binds to the target.

[0039] p represents the average number of drug ligands connected, and p is selected from an integer or decimal from 1 to 20.

[0040] In certain preferred embodiments of the present invention, certain groups in the ligand-drug conjugate or its pharmaceutically acceptable salt are defined as follows, and groups not mentioned are as described in any embodiment of the present invention (hereinafter referred to as "in some preferred embodiments").

[0041] In some preferred embodiments, the compound represented by formula D has any of the following structures:

[0042] In some preferred embodiments, the compound as shown in Formula D is

[0043] In a preferred embodiment of the present invention, p is a decimal number between 1, 2, 3, 4, 5, 6, 7, 8, or any number in between.

[0044] In a preferred embodiment of the present invention, p is a decimal number between 1, 2, 3, 4, or any number in between.

[0045] In a preferred embodiment of the present invention, p is 3, 4, or a decimal number between them; preferably 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0, and even more preferably 3.82 or 3.85.

[0046] In a preferred embodiment of the present invention, L is -AL. 1 -L 2 -,

[0047] in;

[0048] A is the connector segment that connects to Pc;

[0049] L 1 It is a polypeptide linker;

[0050] L 2 This refers to the connector segment for the D-type connection.

[0051] In a preferred embodiment of the present invention,

[0052] A is Where R A It is H or C1-C5 alkyl;

[0053] n1, n2 and n3 are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16;

[0054] Preferred, And the best

[0055] n1 can be 0, 1, 2 or 3 independently; preferably 0;

[0056] n2 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; preferably 6 or 12;

[0057] n3 can be 0, 1, 2, 3, 4, 5 or 6 independently; preferably 3 or 6;

[0058] Preferred Terminal 1 is connected to Pc, and terminal 2 is connected to L. 1 Connected.

[0059] In a preferred embodiment of the present invention,

[0060] A is

[0061] In a preferred embodiment of the present invention, A is Preferred

[0062] In a preferred embodiment of the present invention,

[0063] L 1 It is an amino acid residue or a short peptide composed of 2-10 amino acid residues; the amino acid residues are natural amino acid residues or non-natural amino acid residues.

[0064] In a preferred embodiment of the present invention, L 1 It is a short peptide composed of amino acid residues Val, D-Val, Cit, Phe, Lys, Lys(Ac), Leu, Gly, Ala, Asn, Asp, Arg, or 2-10 amino acid residues selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Asp.

[0065] In a preferred embodiment of the present invention, L 1 For Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys ( Ac), Phe-Lys, Phe-Lys(Ac), Ala-Ala, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Asp, Lys-Ala-Ala-Asn (SEQ ID NO:11), Lys-(Ala)2-Asp (SEQ ID NO:12), Gly-Gly-Gly, Gly-Phe-Gly, (Gly)2-Phe-Gly (SEQ ID NO: 13), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Gly-Gly-Phe, Val-Lys-Gly, Val-Lys-(Gly)2 (SEQ ID NO: 14), Val-Lys or Lys-Ala-Asn.

[0066] In a preferred embodiment of the present invention, L 1 It is a short peptide composed of Val, D-Val, Cit, Phe, Lys, Lys(Ac), Leu, Gly, Ala, Asn, Asp, Arg, or 2-10 amino acid residues selected from Val, Cit, Phe, Lys, Leu, Gly, Ala, Asn, and Asp.

[0067] Preferred L 1 is Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val-Cit, Gly-Lys, Ala-Ala, Ala-Ala-Asn, Gly-Phe-Gly, (Gly)2-Phe-Gly (SEQ ID NO: 13), Val-Lys, Glu-Val-Ala, Ala-Ala-Asn, Val-Lys-(Gly)2 (SEQ ID NO: 14), Val-Lys or Lys-Ala-Asn.

[0068] In a preferred embodiment of the present invention, L 1 for (-(G)2FG-)(SEQ ID NO:13); End 1 is connected to A, and end 2 is connected to L. 2 Connected.

[0069] In a preferred embodiment of the present invention,

[0070] L2 for R l1 H or C1-C5 alkyl; R l2 and R l3 Independently, it is H, C1-C5 alkyl, or C3-C6 cycloalkyl; or, R l2 and R l3 Together with the attached C, they form C3-C6 cycloalkyl groups; the C1-C5 alkyl groups and C3-C6 cycloalkyl groups are optionally substituted with one or more D or halogens.

[0071] In a preferred embodiment of the present invention, the halogen may be F or Cl.

[0072] In a preferred embodiment of the present invention, the C1-C5 alkyl group may be methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl.

[0073] In a preferred embodiment of the present invention, the C3-C6 cycloalkyl group may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; preferably cyclopropyl, cyclobutyl or cyclopentyl.

[0074] In a preferred embodiment of the present invention,

[0075] L 2 for R l1 H or C1-C5 alkyl; R l2 and R l3 It can be independently H, C1-C5 alkyl or C3-C6 cycloalkyl.

[0076] In a preferred embodiment of the present invention, L 2 for 1 end and L 1 Connected, with terminals 2 connected to D.

[0077] In a preferred embodiment of the present invention, L 2 for 1 end and L 1 Connected, with ends 2 connected to D; preferred

[0078] In a preferred embodiment of the present invention, L 2 for

[0079] In a preferred embodiment of the present invention, L has the following structure:

[0080] In a preferred embodiment of the present invention, L has the following structure:

[0081] Preferred,

[0082] In a preferred embodiment of the present invention, Pc is an antibody (Ab).

[0083] In a preferred embodiment of the present invention, Pc is an antibody (Ab) or its antigen-binding fragment, wherein the antibody (Ab) or its antigen-binding fragment specifically binds to the target antigen expressed on the cell surface;

[0084] The Pc is an antibody or its antigen-binding fragment that targets tumor-specific surface antigens, immunosuppressive receptors or immune checkpoint molecules, tumor-associated neoantigens and tissue-specific antigens.

[0085] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment includes a constant region, wherein the amino acid residue in the constant region corresponding to position N297 of the IgG antibody constant region is Q.

[0086] In a preferred embodiment of the present invention, the tumor-specific surface antigen is selected from: HER2, Claudin 18.2, EGFR, TROP2 and Nectin-4.

[0087] In a preferred embodiment of the present invention, the immunosuppressive receptor or immune checkpoint molecule is selected from: LILRB2, PD-L1, TIGIT, VISTA, B7-H3 and TIM-3.

[0088] In a preferred embodiment of the present invention, the tumor-associated neoantigen and tissue-specific antigen are selected from MUC1, Mesothelin, CD138 and GPC3.

[0089] In a preferred embodiment of the present invention, the antibody has endocytosis capability, thereby enabling the release of drug from the ADC.

[0090] In a preferred embodiment of the present invention, the antibody comprises a variable region and a constant region, wherein the amino acid residue in the constant region corresponding to position N297 of the constant region of the IgG antibody is Q.

[0091] In a preferred embodiment of the present invention, the constant region further includes amino acid residues A at positions L234 and L235 corresponding to the constant region of the IgG antibody, and / or amino acid residues Q at position P329.

[0092] In a preferred embodiment of the present invention, the constant region of the antibody includes CH2 and CH3.

[0093] In a preferred embodiment of the present invention, the constant region of the antibody further includes CH1 and / or CH4.

[0094] In a preferred embodiment of the present invention, the antibody is IgG, IgA, IgM, IgE, IgD or its isotype antibody.

[0095] In a preferred embodiment of the present invention, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0096] In a preferred embodiment of the present invention, the variable region of the antibody targets one or more immune checkpoints and / or tumor antigens.

[0097] In a preferred embodiment of the present invention, the antibody is a monoclonal antibody or a bispecific antibody.

[0098] In a preferred embodiment of the present invention, the immune checkpoints include SIPRα, VISTA, TIM-3, TIGIT, LILRB2, LILRB4, Siglec-7, LAG-3, PD-1 / PD-L1, and BTLA.

[0099] In a preferred embodiment of the present invention, the immune checkpoint includes B7-H3.

[0100] In a preferred embodiment of the present invention, the tumor antigen includes tumor-specific antigen (TSA) or tumor-associated antigen (TAA).

[0101] In a preferred embodiment of the present invention, the tumor antigens include: Claudin18.2, MART-1 / MelanA (MART-1), gp 100 (Pmel17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, CEA, p53, Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR, Epstein-Barr virus antigen EBVA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, Alpha-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA15-3, CA27.29, BCAA, CA195, CA242, CA50, CAM43, CD68, p1, CO-O29, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV 18. NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilic protein C-related protein, TAAL6, TAG72, TLP, HER-3, B7H3, TROP2, EGFR and TPS.

[0102] In a preferred embodiment of the present invention, the tumor antigen is Nectin-4.

[0103] In a preferred embodiment of the present invention, the tumor antigen is: MUC1, Mesothelin, CD138, GPC3.

[0104] In a preferred embodiment of the present invention, the antibody targets LILRB2.

[0105] In a preferred embodiment of the present invention, the antibody comprises a heavy chain variable region and a light chain variable region of an antibody targeting LILRB2.

[0106] In a preferred embodiment of the present invention, the antibody is an antibody targeting LILRB2, such as that in US2024 / 01099962A1.

[0107] In a preferred embodiment of the present invention, the antibody comprises a heavy chain variable region and a light chain variable region of an antibody targeting LILRB2. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0108] In a preferred embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8.

[0109] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0110] In a preferred embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having 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 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity with SEQ ID NO:7.

[0111] In a preferred embodiment of the present invention, the light chain variable region comprises an amino acid sequence having 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 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity with SEQ ID NO:8.

[0112] In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:9, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:10.

[0113] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain is shown in SEQ ID NO:9, and the amino acid sequence of the light chain is shown in SEQ ID NO:10.

[0114] In a preferred embodiment of the present invention, the heavy chain comprises an amino acid sequence having 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 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity with SEQ ID NO:9.

[0115] In a preferred embodiment of the present invention, the light chain comprises an amino acid sequence having 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 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity with SEQ ID NO:10.

[0116] The antibody or antigen-binding fragment of the Ab can be prepared by various methods known in the art, such as by genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the disclosed antibody can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the disclosed antibody.

[0117] In a preferred embodiment of the present invention, the ligand-drug conjugate is as shown in formulas X-1, X-2, X-3, and X-4.

[0118] R 1 R 2 R 3 R 4 X, Ra, R b The definitions of , p, and Ab are as described in any of the schemes.

[0119] In a preferred embodiment of the present invention, the ligand-drug conjugate has any of the following structures:

[0120] Wherein, n2 is 3, 6, or 12; preferably 6;

[0121] p is 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0;

[0122] Ab is an anti-LILRB2 antibody, the amino acid sequence of the heavy chain variable region of the anti-LILRB2 antibody is as shown in SEQ ID NO:7 or has at least 90% sequence identity with SEQ ID NO:7, and the amino acid sequence of the light chain variable region of the anti-LILRB2 antibody is as shown in SEQ ID NO:8; or has at least 90% sequence identity with SEQ ID NO:8.

[0123] In a preferred embodiment of the present invention, the ligand-drug conjugate has any of the following structures:

[0124] The asterisk (*) indicates that the atom at that location is either R or S chiral.

[0125] Ab is an anti-LILRB2 antibody, wherein the amino acid sequence of the heavy chain of the anti-LILRB2 antibody is as shown in SEQ ID NO:9 or has at least 90% sequence identity with SEQ ID NO:9, and the amino acid sequence of the light chain of the anti-LILRB2 antibody is as shown in SEQ ID NO:10 or has at least 90% sequence identity with SEQ ID NO:10.

[0126] In a preferred embodiment of the present invention, the ligand-drug conjugate has any of the following structures:

[0127] In a preferred embodiment of the present invention, the ligand-drug conjugate has any of the following structures:

[0128] The asterisk (*) indicates that the atom at that location is either R or S chiral.

[0129] The present invention also relates to a compound as shown in Formula II or a pharmaceutically acceptable salt thereof.

[0130] HAL 1 -L 2 -D,

[0131] A, L 1 L 2 The definitions of D are as described in any of the schemes.

[0132] In a preferred embodiment of the present invention, the compound represented by Formula II has any of the following structures:

[0133] R 1 R 2 R 3 R 4 X, R a R bThe definitions of , p, and Ab are as described in any of the schemes.

[0134] In a preferred embodiment of the present invention, the compound represented by Formula II has any of the following structures:

[0135] The present invention also relates to an antibody or antigen-binding fragment thereof for preparing antibody-drug conjugates (ADCs), said antibody or antigen-binding fragment thereof being capable of specifically binding to a target antigen expressed on the cell surface;

[0136] The target antigen is selected from tumor-specific surface antigens, immunosuppressive receptors or immune checkpoint molecules, tumor-associated neoantigens, and tissue-specific antigens.

[0137] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment includes a constant region, wherein the amino acid residue in the constant region corresponding to position N297 of the IgG antibody constant region is Q.

[0138] In a preferred embodiment of the present invention, the tumor-specific surface antigen is selected from: HER2, Claudin 18.2, EGFR, TROP2 and Nectin-4.

[0139] In a preferred embodiment of the present invention, the immunosuppressive receptor or immune checkpoint molecule is selected from: LILRB2, PD-L1, TIGIT, VISTA, B7-H3 and TIM-3.

[0140] In a preferred embodiment of the present invention, the tumor-associated neoantigen and tissue-specific antigen are selected from MUC1, Mesothelin, CD138 and GPC3.

[0141] In a preferred embodiment of the present invention, the antibody has endocytosis capability, thereby enabling the release of drug from the ADC.

[0142] The present invention also relates to an anti-LILRB2 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a constant region, wherein the amino acid residue in the constant region corresponding to the N297 position of the constant region of an IgG antibody is Q.

[0143] In a preferred embodiment of the present invention, the constant region further includes amino acid residues A at positions L234 and L235 corresponding to the constant region of the IgG antibody, and / or amino acid residues Q at position P329.

[0144] In a preferred embodiment of the present invention, the constant region is a constant region derived from IgG, IgA, IgM, IgE, IgD or their isotype antibodies.

[0145] In a preferred embodiment of the present invention, the constant region is a constant region derived from IgG1, IgG2, IgG3 or IgG4 antibodies.

[0146] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0147] In a preferred embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8.

[0148] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0149] In a preferred embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having 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 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity with SEQ ID NO:7.

[0150] In a preferred embodiment of the present invention, the light chain variable region comprises an amino acid sequence having 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 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity with SEQ ID NO:8.

[0151] In a preferred embodiment of the present invention, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:9, and the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:10.

[0152] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain is shown in SEQ ID NO:9, and the amino acid sequence of the light chain is shown in SEQ ID NO:10.

[0153] In a preferred embodiment of the present invention, the heavy chain comprises an amino acid sequence having 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 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity with SEQ ID NO:9.

[0154] In a preferred embodiment of the present invention, the light chain comprises an amino acid sequence having 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 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity with SEQ ID NO:10.

[0155] This invention also relates to an antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

[0156] The antibody-drug conjugate comprises the following fragments: an antibody or its antigen-binding fragment as described above, a linker unit, and a cytotoxic drug; the antibody or its antigen-binding fragment is an anti-LILRB2 antibody or its antigen-binding fragment; the antibody or its antigen-binding fragment contains a constant region, wherein the amino acid residue at position N297 of the constant region corresponding to the IgG antibody constant region is Q.

[0157] In a preferred embodiment of the present invention, the constant region further includes amino acid residues A at positions L234 and L235 corresponding to the constant region of the IgG antibody, and / or amino acid residues Q at position P329.

[0158] In a preferred embodiment of the present invention, the constant region is a constant region derived from IgG, IgA, IgM, IgE, IgD or their isotype antibodies.

[0159] In a preferred embodiment of the present invention, the constant region is a constant region derived from IgG1, IgG2, IgG3 or IgG4 antibodies.

[0160] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0161] In a preferred embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8.

[0162] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0163] In a preferred embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having 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 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity with SEQ ID NO:7.

[0164] In a preferred embodiment of the present invention, the light chain variable region comprises an amino acid sequence having 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 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity with SEQ ID NO:8.

[0165] In a preferred embodiment of the present invention, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:9, and the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:10.

[0166] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain is shown in SEQ ID NO:9, and the amino acid sequence of the light chain is shown in SEQ ID NO:10.

[0167] In a preferred embodiment of the present invention, the heavy chain comprises an amino acid sequence having 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 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity with SEQ ID NO:9.

[0168] In a preferred embodiment of the present invention, the light chain comprises an amino acid sequence having 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 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, or at least 99.8% sequence identity with SEQ ID NO:10.

[0169] The anti-LILRB2 antibody and its antigen-binding fragment of the present invention can specifically bind to antigens on macrophages, effectively blocking LILRB2-mediated inhibitory signals. Ligand-drug conjugates based on the aforementioned anti-LILRB2 antibody and its antigen-binding fragment retain the specific binding ability and signal pathway blocking ability of the antibody or its antigen-binding fragment, and can release cytotoxic drugs in macrophages, achieving a synergistic effect. Macrophages and tumor cells have different encounter probabilities with drugs; some tumor microenvironment macrophages can bind to antibody-drug conjugates more effectively and mediate a strong bystander killing effect.

[0170] In this invention, the antibody-drug conjugate may mediate a strong bystander killing effect by expressing toxins in tumor cells and immune cells through LILRB2 expression.

[0171] In a preferred embodiment of the present invention, the connector unit is defined as L in the ligand-drug conjugate as described above.

[0172] In a preferred embodiment of the present invention, the DAR is defined as p in the ligand-drug conjugate as described above.

[0173] In a preferred embodiment of the present invention, the cytotoxic drug is camptothecin and its derivatives; preferably, camptothecin (CPT), Dxd, Exatecan (DX-8951), SN-38, or a compound of formula (I) as described above, or a pharmaceutically acceptable salt thereof, as defined herein.

[0174] On the other hand, the present invention relates to a pharmaceutical composition comprising (therapeuticly effective amounts) of a compound or a pharmaceutically acceptable salt thereof as described in any one of the present invention, a ligand drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the present invention, or an antibody drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the present invention, and a pharmaceutical excipient (or a pharmaceutically acceptable carrier).

[0175] This invention further relates to the use of substance X in the preparation of a pharmaceutical for treating diseases associated with abnormal cellular activity (such as cancer).

[0176] The substance X is a compound as described in any one of the present invention or a pharmaceutically acceptable salt thereof, a ligand drug conjugate as described in any one of the present invention or a pharmaceutically acceptable salt thereof, a pharmaceutical composition as described in any one of the present invention, or an antibody drug conjugate as described in any one of the present invention or a pharmaceutically acceptable salt thereof;

[0177] The cancer mentioned can be breast cancer (e.g., triple-negative breast cancer, MDA-MB-231 cells).

[0178] In a preferred embodiment of the present invention, the cancer is melanoma.

[0179] The present invention further relates to the preparation of ligand-drug conjugates using compounds as described in any one of the present invention or pharmaceutically acceptable salts thereof, or anti-LILRB2 antibodies as described in any one of the present invention.

[0180] In a preferred embodiment of the present invention, the ligand-drug conjugate is an antibody-drug conjugate (ADC). For example, the ligand / antibody-drug conjugate described in any of the embodiments of this application, or a pharmaceutically acceptable salt thereof.

[0181] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all standard procedures widely used in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0182] As used herein, examples of the term "pharmaceutically acceptable salt" are organic acid adduct salts formed from organic acids that form pharmaceutically acceptable anions.

[0183] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting an adequate amount of a basic compound with a suitable acid that provides a pharmaceutically acceptable anion.

[0184] In this disclosure, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and the formulation of prescriptions. They are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical preparation. Besides acting as a formifier, carrier, and improving stability, pharmaceutical excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of pharmaceuticals. Based on their origin, they can be classified into natural substances, semi-synthetic substances, and fully synthetic substances. Based on their function and use, pharmaceutical excipients can be classified as follows: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. Based on their route of administration, they can be classified as oral, injection, mucosal, transdermal or local, nasal or oral inhalation, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses.

[0185] As used herein, the term “pharmaceutically acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives.

[0186] The pharmaceutical composition can be formulated into various suitable dosage forms depending on the route of administration. Examples include tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder inhalers, and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the compound disclosed herein or its pharmaceutically acceptable salts or conjugates, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, more preferably 0.5-350 mg, and particularly preferably 1-250 mg.

[0187] The pharmaceutical composition can be administered in injectable form, including injection solutions, sterile powders for injection, and concentrated solutions for injection. Suitable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspension media.

[0188] As used herein, the term “treatment” generally refers to achieving the desired pharmacological and / or physiological effect. This effect may be preventative based on the complete or partial prevention of the disease or its symptoms; and / or therapeutic based on the partial or complete stabilization or cure of the disease and / or side effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a patient’s disease, including: (a) prevention of the disease or symptoms occurring in a patient who is susceptible to the disease or its symptoms but has not yet been diagnosed with the disease; (b) suppression of the symptoms of the disease, i.e., prevention of its progression; or (c) relief of the symptoms of the disease, i.e., causing the disease or its symptoms to regress.

[0189] In this disclosure, the term "individual" includes humans or non-human animals. Exemplary human individuals include human individuals suffering from a disease (such as the disease described herein) (referred to as patients) or normal individuals. The term "non-human animal" in this disclosure includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0190] As used herein, the term "subject" refers to a mammal, such as a primate, a non-human primate, or a human. In some embodiments, the subject (e.g., a human) has a tumor and an infectious disease, or is at risk of having such a disease.

[0191] In this disclosure, the term "effective amount" means an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing disease (e.g., cancer and infectious diseases) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer and infectious diseases); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0192] In this disclosure, the term "ligand-drug conjugate" refers to a substance obtained by linking a bioactive molecule (drug molecule) to a target moiety. In some embodiments of this disclosure, the bioactive molecule and the target moiety are linked via a linker. The linker is capable of cleavage under specific conditions (e.g., hydrolytic enzymes and / or low pH environments within a tumor) or under specific influences (e.g., the action of lysosomal proteases), thereby separating the bioactive molecule from the target moiety. In some embodiments of this disclosure, the linker comprises cleavable or cleavable units, such as peptides or disulfide bonds. In some embodiments of this disclosure, the bioactive molecule and the target moiety are directly linked by a covalent bond, which is capable of cleavage under specific conditions or influences, thereby separating the bioactive molecule from the target moiety. In some embodiments of this disclosure, the ligand-drug conjugate comprises a target moiety, a linker, and a fragment of a compound of formula II of this disclosure.

[0193] In this disclosure, the terms "bioactive substance," "bioactive molecule," or "drug molecule" refer to substances that inhibit or prevent cell function and / or cause cell death or damage. In some embodiments of this disclosure, the bioactive substance, bioactive molecule, or drug molecule in the conjugate is a molecule with antitumor biological activity.

[0194] In this disclosure, the term "linker (or linker)" refers to a segment that connects a bioactive molecule (drug molecule) to a target moiety.

[0195] In this disclosure, the term "targeting portion" refers to a portion of the conjugate that can specifically bind to a target (or a portion of the target) on the cell surface. Through the interaction between the targeting portion and the target, the conjugate can be delivered to a specific cell population.

[0196] In this disclosure, when the target portion of the conjugate is an antibody, the conjugate may be referred to as a "drug-antibody conjugate".

[0197] In this disclosure, antibodies include derivatized antibodies or antigen-binding fragments thereof, such as antibodies or antigen-binding fragments having a thiol group, wherein the derivatization imparts to the antibody a group or ability to react with a drug linker conjugate. The thiol-SH group can be obtained by derivatization by opening disulfide bonds (e.g., by reduction with the reducing agent TCEP).

[0198] The terms “cancer” and “tumor” are used in this article with the same meaning.

[0199] The term "Ab" is an abbreviation for "antibody" and is used interchangeably with "antibody".

[0200] The terms “peptide” and “protein” are used interchangeably in this article.

[0201] The term "cell" as used in this article also includes cells within an animal individual and cultured cells.

[0202] KD refers to the dissociation constant derived from the ratio of Kd (the dissociation rate of the specific bound molecule-target protein interaction) to Ka (the binding rate of the specific bound molecule-target protein interaction) (or Kd / Ka, expressed as molar concentration (M)). KD values ​​can be determined using methods well-established in the art. A preferred method for determining the KD of bound molecules is through the use of surface plasmon resonance, such as a biosensor system like the Biacore™ (GE Healthcare Life Sciences) system.

[0203] In this disclosure, the term "antibody" is used in its broadest sense to include intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, provided they possess the desired biological activity. In this disclosure, "antibody" and "immunoglobulin" are used interchangeably. As used herein, "antibody molecule" or "antibody" refers to an immunoglobulin molecule and the immunologically active portion of an immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that specifically binds to an antigen. Therefore, the term antibody broadly encompasses not only intact antibody molecules but also fragments of said antibodies and variants (including derivatives) of said antibodies and antibody fragments. When "antibody molecule" or "antibody" is used in the same context as an antigen-binding fragment, "antibody molecule" or "antibody" refers to an intact antibody molecule or a full-length antibody. The term antibody molecule as used in this specification includes, but is not limited to, single-chain Fv (scFv), Fab fragments, Fab' fragments, F(ab')2, disulfide-linked Fv (sdFv), Fv, and intact or full-length antibodies. The term “single-chain Fv” or “scFv” refers to a polypeptide containing an antibody’s VL domain linked to the antibody’s VH domain. For example, an antibody that specifically binds to B7H3 may cross-react with other antigens. Preferably, an antibody that specifically binds to B7H3 does not cross-react with other antigens. An antibody that specifically binds to B7H3 can be identified, for example, by immunoassay or other methods known to those skilled in the art. An “intact” antibody or “full-length” antibody refers to a protein comprising two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, the protein comprising: (1) with respect to the heavy chain, a variable region (hereinafter abbreviated as “VH”) and a heavy chain constant region containing three domains CH1, CH2, CH3; and (2) with respect to the light chain, a light chain variable region (hereinafter abbreviated as “VL”) and a light chain constant region containing one domain CL. The antibodies disclosed herein include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab′) fragments, anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies of the antibodies disclosed herein), and epitope-binding fragments of any of the above antibodies. The immunoglobulin molecules disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin. Preferably, the antibodies disclosed herein comprise or consist of a VH domain, a VH CDR (often referred to herein as HCDR), a VL domain, or a VL CDR (often referred to herein as LCDR) having any of the amino acid sequences or fragments or variants thereof described in the Sequence and Specific Information Table.

[0204] In this disclosure, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the antibodies constituting the cluster are identical except for a small number of possible naturally occurring mutations. Monoclonal antibodies possess high specificity against a single determinant (epitope) of an antigen, while polyclonal antibodies, in contrast, comprise different antibodies targeting different determinants (epitopes). In addition to specificity, monoclonal antibodies have the advantage of being synthesized without contamination from other antibodies. The modifier "monoclonal" here indicates that the antibody is characterized by originating from a substantially homogeneous group of antibodies, and should not be construed as requiring special methods for preparation.

[0205] In some embodiments of this disclosure, monoclonal antibodies further include chimeric antibodies, i.e., a portion of the heavy chain and / or light chain is identical or homologous to one, a class, or a subclass of antibody, while the remainder is identical or homologous to another, a different class, or a different subclass of antibody, provided they possess the desired biological activity (see, for example, US 4,816,567; and Morrison et al., 1984, PNAS, 81:6851-6855). Chimeric antibodies that can be used in this disclosure include primatized antibodies, which comprise a variable region antigen-binding sequence from a non-human primate (e.g., ancient monkey, chimpanzee, etc.) and a human constant region sequence.

[0206] The term "antigen-binding fragment" refers to a portion of an antibody, preferably an antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fd, Fv, dAb, and complementarity-determining region fragments, diabody, linear antibody, and single-chain antibody molecules. As used herein, the term "antigen-binding fragment" refers to a partial fragment of an antibody that has antigen-binding activity, wherein the fragment has complete or partial function of the antibody, including, but not limited to, single-chain Fv(scFv), Fab, Fab′, F(ab′)2, disulfide-linked Fv(sdFv), Fv, di-scFv, etc. The term also includes Fab′, which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab′)2 under reducing conditions. However, the term is not limited to these molecules, as long as the fragment has binding affinity for the antigen. Furthermore, these functional fragments include not only fragments obtained by treating the full-length molecule of an antibody protein with a suitable enzyme, but also proteins produced in appropriate host cells using genetically modified antibody genes.

[0207] As used herein, the term "Fab'" refers to a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions as described above. However, Fab' in this disclosure also includes Fab' produced using genetically modified antibody genes.

[0208] The twenty common amino acids referred to 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 document, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. And in this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine may be represented by A or Ala; arginine by R or Arg; glycine by G or Gly; and glutamine by Q or Gln.

[0209] As used herein, the term "prevention" refers to methods implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., tumors and infectious diseases) in a subject. As used herein, the term "treatment" refers to methods implemented to achieve a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0210] Unless otherwise stated, this invention employs traditional methods of mass spectrometry and elemental analysis, and the steps and conditions can be referred to conventional operating procedures and conditions in the field.

[0211] Unless otherwise specified, this invention employs standard nomenclature and standard laboratory procedures and techniques of analytical chemistry, organic synthetic chemistry, and optics. In some cases, standard techniques are used in chemical synthesis and chemical analysis.

[0212] In this article, unless otherwise explicitly stated, the descriptive phrases “each…independently selected” and “…independently selected” used throughout the article are interchangeable and should be interpreted broadly. They can mean that the specific options expressed by the same or different symbols in different groups do not affect each other, or that the specific options expressed by the same or different symbols in the same group do not affect each other.

[0213] In this specification, groups and their substituents may be selected by those skilled in the art to provide stable structural moieties and compounds. When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes chemically equivalent substituents obtained when the structural formula is written from right to left.

[0214] Certain chemical groups defined in this document are preceded by simplified symbols to indicate the total number of carbon atoms present in the group. For example, C1-C4 alkyl or C 1-4 Alkyl refers to an alkyl group having a total of 1, 2, 3, or 4 carbon atoms as defined below. The total number of carbon atoms in the simplified symbol does not include carbons that may be present in substituents of the group.

[0215] In this paper, the numerical ranges defined in the substituents, such as 0 to 10, 1-6, 1-3, etc., indicate the integers within that range. For example, 1-6 represents 1, 2, 3, 4, 5, and 6.

[0216] The term "optionally by one or more R" a "Replaced" indicates that it was not replaced by R a Replaced and by one or more R a Replaces both scenarios.

[0217] The term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention, but does not exclude other aspects.

[0218] The terms “substituted” or “replaced” refer to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the substituted compound is stable.

[0219] Generally, the terms "substituted" or "substituted" indicate that one or more hydrogen atoms in a given structure are substituted by a specific substituent. Further, when the group is substituted by more than one of the substituents, the substituents are independent of each other; that is, the more than one substituent can be different or the same. Unless otherwise indicated, a substituent group can be substituted at each substituted position of the substituted group. When more than one position in a given structural formula can be substituted by one or more substituents selected from a specific group, the substituents can be substituted at the same or different positions.

[0220] In various parts of this specification, the substituents of the compounds disclosed herein are disclosed according to the type or scope of the groups. In particular, this invention includes every independent secondary combination of the respective members of these group types and scopes. The term "C" x -C y Alkyl or C x-y "Alkyl" refers to a straight-chain or branched saturated hydrocarbon containing x to y carbon atoms. For example, the terms "C1-C6 alkyl" or "C 1-6 "alkyl" specifically refers to independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl; "C" 1-4"Alkyl" specifically refers to independently disclosed methyl, ethyl, C3 alkyl (i.e. propyl, including n-propyl and isopropyl), and C4 alkyl (i.e. butyl, including n-butyl, isobutyl, sec-butyl, and tert-butyl).

[0221] The terms “part,” “structural part,” “chemical part,” “group,” and “chemical group” used in this article refer to specific segments or functional groups within a molecule. A chemical part is generally considered to be a chemical entity embedded in or attached to a molecule.

[0222] When the listed substituents do not specify which atom they are attached to in the general chemical formula (including but not specifically mentioned compounds), such substituents may be bonded to any of their atoms. Combinations of substituents and / or their variants are permitted only if such combinations produce stable compounds.

[0223] When any variable (e.g., R) 1-a When a variable appears multiple times in the definition of a compound, the definition at each position is independent of the definitions at the other positions; their meanings are independent and do not affect each other. Therefore, if a group is surrounded by one, two, or three R... 1-a Group substitution, meaning that the group can be replaced by up to 3 R groups. 1-a Replacement, where a certain position R 1-a Definition and other positions R 1-a The definitions are independent of each other. Furthermore, combinations of substituents and / or variables are only permitted if the combination produces a stable compound.

[0224] When a listed group does not explicitly indicate that it has a substituent, the group refers only to the unsubstituted group. For example, when "C 1-6 When "alkyl" is not specified as "substituted or unsubstituted," it refers only to "C". 1-6 "alkyl" itself or "unsubstituted C" 1-6 alkyl".

[0225] Linking substituents are described in various parts of this invention. When the structure clearly requires a linking group, the Markush variable listed for that group should be understood as the linking group. For example, if the structure requires a linking group and the Markush group definition for that variable lists "alkyl", then it should be understood that "alkyl" represents a linked alkylene group.

[0226] In some specific structures, when the alkyl group is clearly indicated as a linking group, then the alkyl group represents a linked alkylene group, for example, the group "halogenated-C". 1-6 C in alkyl- 1-6 Alkyl should be understood as C 1-6 Alkylene.

[0227] In this invention, the structural segments This refers to the structural segment being connected to the rest of the molecule via this bond. For example, It refers to cyclopropyl.

[0228] In this invention, the "-" at the end of a group indicates that the group is connected to the rest of the molecule through that site. For example, -OH refers to a hydroxyl group.

[0229] Those skilled in the art will understand that, according to the conventions used in the art, the structural formulas of the descriptive groups described in this application... This refers to the corresponding group passing through this It can be linked to other fragments or groups in a compound; for example, a racemic mixture.

[0230] Those skilled in the art will understand that, according to the conventions used in the art, the structural formulas of the descriptive groups described in this application... This refers to the corresponding group passing through this It can be linked to other fragments or groups in a compound.

[0231] It should be understood that the singular form used in this invention, such as "a," includes plural references unless otherwise specified.

[0232] The terms "one or more" or "one or two or more" refer to 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. For example, 1, 2 or 3.

[0233] In this invention, the term "B replaced by one or more A" means that when B is replaced by "multiple" A's, the A's are the same or different.

[0234] In this invention, the term "halogen" refers to fluorine, chlorine, bromine or iodine, especially F, Cl or Br.

[0235] In various parts of this specification, the substituents of the compounds disclosed herein are disclosed according to the type or range of groups. In particular, this disclosure includes every independent secondary combination of the respective members of these group types and ranges. For example, the term "C..." 1-6 "Alkyl" specifically refers to independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0236] In this article, the term "C" 1-6 "Alkyl" refers to a straight-chain or branched alkyl group containing 1-6 carbon atoms, including, for example, "C". 1-3 "alkyl" or "C" 1-4 Alkyl, methyl, ethyl, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl.

[0237] In this article, the term "C" 1-5 "Alkyl" refers to a straight-chain or branched alkyl group containing 1-5 carbon atoms, including, for example, "C". 1-3 Alkyl, methyl, ethyl, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.

[0238] In this application, as part of a group or other group, the term "alkylene" refers to a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a saturated straight-chain or branched hydrocarbon; that is, one hydrogen atom of the alkyl group is substituted, and the definition of alkyl is as described above. Examples of alkylene groups include methylene (-CH2-), ethylene {including -CH2CH2- or -CH(CH3)-}, isopropylene {including -CH(CH3)CH2-, -CH2CH(CH3)- or -C(CH3)2-}, and so on.

[0239] In this application, as a group or part of other groups, the term "cycloalkyl" means a saturated carbocyclic substituent that is connected to the rest of the molecule via a single bond through any suitable carbon atom; such as a 3- to 6-membered cycloalkyl group having 3 to 6 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0240] The term "drug-to-antibody ratio" or "DAR" refers to the amount of drug, such as a small molecule toxin attached to an antibody-drug conjugate (ADC). The DAR of an ADC can range from 1 to 16, but higher loadings (e.g., 20) are possible depending on the number of binding sites on the antibody. The term DAR can be used when referring to the amount of drug loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a group of ADCs.

[0241] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0242] The reagents and raw materials used in this invention are all commercially available.

[0243] The positive and progressive effects of this invention are as follows: This invention provides a new class of camptothecin derivatives, which have significantly improved cell activity compared with known compounds such as deruxtecan, which is of great significance for the development of novel antitumor drugs and ADCs. Attached Figure Description

[0244] Figure 1 shows the evaluation of the antitumor activity of AL-20 and its control in the PBMC humanized A375 xenograft mouse model. Detailed Implementation

[0245] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0246] The sequence of the anti-LILRB2 antibody used in this embodiment is shown below:

[0247] Example 1: Synthesis of compounds 11, 12, and 13

[0248] Synthesis route:

[0249] The * position indicates that the atom at that location is either R or S chiral, and the compound is a pure isomer.

[0250] Step 1: Synthesis of Compound 2

[0251] At room temperature, 7-fluoro-8-methyl-5-nitro-1,2,3,4-tetrahydronaphthyl-1-one (compound 1, 31 g, 139 mmol) was dissolved in bis(2-methoxyethyl)aminosulfur trifluoride (bast, 103 mL, 556 mmol), and methanol (1 mL) was slowly added dropwise. The reaction mixture was stirred at 85 °C for 24 hours. TLC showed complete disappearance of the starting material and detection of a new principal spot. The reaction mixture was then quenched with saturated potassium carbonate solution (200 mL) and extracted with dichloromethane (200 mL x 3). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated by filtration. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 20:1) to give 24 g of a yellow oil, 1,1,7-trifluoro-8-methyl-5-nitro-1,2,3,4-tetrahydronaphthyl (compound 2), in 71% yield.

[0252] 1 H NMR (400MHz, CDCl3) δ7.67(d,J=8.3Hz,1H),2.98(s,2H),2.59(s,3H),2.31(d,J=5.3Hz,2H),1.92(s,2H).

[0253] Step 2: Synthesis of Compound 3

[0254] At room temperature, 1,1,7-trifluoro-8-methyl-5-nitro-1,2,3,4-tetrahydronaphthalene (compound 2, 24 g, 98 mmol) was dissolved in ethanol (400 mL) and water (100 mL). Iron powder (55 g, 983 mmol) and ammonium chloride (52 g, 983 mmol) were added to the reaction solution, respectively. The reaction solution was stirred vigorously at 90 °C for 1 hour, and TLC showed that the reaction was complete. The reaction solution was filtered through diatomaceous earth, and the filtrate was extracted with dichloromethane (200 mL x 3). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give 21 g of a yellow oily substance, 3,5,5-trifluoro-4-methyl-5,6,7,8-tetrahydronaphthalene-1-amine (compound 3), with a yield of 99%.

[0255] ESI-MS m / z[M+H] + 215.85.

[0256] 1 H NMR (400MHz, CDCl3) δ6.55(d,J=10.5Hz,1H),4.92–3.97(m,1H),2.53(d,J=39.0Hz,2H),2.23(dd,J=54.0,42.1Hz,4H),2.09(s,2H).

[0257] Step 3: Synthesis of Compound 4

[0258] At room temperature, 3,5,5-trifluoro-4-methyl-5,6,7,8-tetrahydronaphthyl-1-amine (compound 3, 21 g, 98 mmol) and triethylamine (34 mL, 244 mmol) were dissolved in ethyl acetate (200 mL), and acetic anhydride (13.7 mL, 146 mmol) was added to the reaction solution. The reaction solution was stirred at 50 °C for 3 hours, and TLC showed complete disappearance of the starting material. The reaction solution was washed with 1M hydrochloric acid (100 mL × 2). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and then filtered and concentrated. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 4:1) to give 23 g of white solid N-(3,5,5-trifluoro-4-methyl-5,6,7,8-tetrahydro-1-naphthyl)acetamide (compound 4), with a yield of 93%.

[0259] ESI-MS m / z[M+H] + 257.90.

[0260] Step 4: Synthesis of Compound 5

[0261] At 0 °C, N-(3,5,5-trifluoro-4-methyl-5,6,7,8-tetrahydro-1-naphthyl)acetamide (compound 4, 23 g, 91 mmol) and magnesium sulfate (13 g, 109 mmol) were dissolved in acetone (405 mL) and water (45 mL), and potassium permanganate (72 g, 453 mmol) was added in portions. The reaction mixture was stirred at room temperature for 3 hours, and TLC analysis showed that the reaction was complete. A saturated sodium thiosulfate solution (200 mL) was added to the reaction mixture, and the mixture was filtered through diatomaceous earth. The filtrate was extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was slurried with petroleum ether (200 mL) and filtered to obtain 22 g of white solid N-(3,5,5-trifluoro-4-methyl-8-oxo-5,6,7,8-tetrahydro-1-naphthyl)acetamide (compound 5), with a yield of 90%.

[0262] ESI-MS m / z[M+H] + 271.80.

[0263] 1 H NMR (400MHz, CDCl3) δ8.72(d,J=12.6Hz,1H),2.95–2.85(m,2H),2.72–2.56(m,2H),2.45(dd,J=5.7,3.1Hz,3H),2.26(s,3H).

[0264] Step 5: Synthesis of Compound 6

[0265] At 0 °C, N-(3,5,5-trifluoro-4-methyl-8-oxo-5,6,7,8-tetrahydro-1-naphthyl)acetamide (compound 5, 22 g, 81 mmol) and potassium hydroxide (14 g, 243 mmol) were dissolved in methanol (400 mL), and iodophenyldiacetic acid (PIDA, 31 g, 97 mmol) was added in portions. The reaction mixture was stirred at room temperature for 2 hours, and TLC analysis showed no starting material remaining. The reaction mixture was adjusted to pH 4 with 1 N hydrochloric acid and extracted with dichloromethane (200 mL x 3). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 4:1) to give 19 g of white solid N-(3,5,5-trifluoro-7-hydroxy-4-methyl-8-oxo-5,6,7,8-tetrahydro-1-naphthyl)acetamide (compound 6), with a yield of 81%.

[0266] ESI-MS m / z[M+H] + 288.10.

[0267] 1 H NMR (400MHz, CDCl3) δ8.73(d,J=12.5Hz,1H),4.60(ddd,J=13.6,5.6,1.3Hz,1H),3.77(s,1H),3.21–3.05(m,1H),2.57–2.42(m,4H),2.29(s,3H).

[0268] Step 6: Synthesis of Compound 7

[0269] At 0 °C, N-(3,5,5-trifluoro-7-hydroxy-4-methyl-8-oxo-5,6,7,8-tetrahydro-1-naphthyl)acetamide (compound 6, 19 g, 65 mmol) and silver oxide (46 g, 196 mmol) were dissolved in THF (200 mL), and benzyl bromide (39 mL, 327 mmol) was slowly added. The reaction mixture was stirred at room temperature for 3 hours, and TLC analysis showed no residual starting material. The reaction mixture was filtered through diatomaceous earth to remove silver oxide and concentrated under reduced pressure at low temperature. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to give 12 g of white solid N-[7-(benzyloxy)-3,5,5-trifluoro-4-methyl-8-oxo-5,6,7,8-tetrahydro-1-naphthyl]acetamide (compound 7), with a yield of 50%.

[0270] ESI-MS m / z[M+H] + 378.15.

[0271] 1 H NMR (400MHz, CDCl3) δ12.11(s,1H),9.00(d,J=12.4Hz,1H),7.96–7.60(m,5H),5.30(d,J=11.5Hz,1H),5 .02(d,J=11.5Hz,1H),4.65(d,J=12.0Hz,1H),3.24(m,1H),3.03–2.89(m,1H),2.73(s,3H),2.59(s,3H).

[0272] Step 7: Synthesis of Compound 8

[0273] At 0 °C, N-[7-(benzyloxy)-3,5,5-trifluoro-4-methyl-8-oxo-5,6,7,8-tetrahydro-1-naphthyl]acetamide (compound 7, 12 g, 33 mmol) was dissolved in methanol (60 mL), and concentrated hydrochloric acid (40 mL, 480 mmol) was added. The reaction mixture was stirred at 50 °C for 1.5 h, and TLC analysis showed no reactants remaining. The reaction was then quenched with saturated potassium carbonate solution (200 mL) and extracted with dichloromethane (200 mL x 3). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated by filtration. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 5:1) to give 9.5 g of white solid 8-amino-2-(benzyloxy)-4,4,6-trifluoro-5-methyl-1,2,3,4-tetrahydronaphthyl-1-one (compound 8), with a yield of 86%.

[0274] ESI-MS m / z[M+H] + 336.05.

[0275] Step 8: Synthesis of Compound 10

[0276] At room temperature, 8-amino-2-(benzyloxy)-4,4,6-trifluoro-5-methyl-1,2,3,4-tetrahydronaphthyl-1-one (compound 8, 15 mg, 0.03 mmol), (4S)-4-ethyl-4-hydroxy-3,4,6,7,8,10-hexahydro-1H-pyrano[3,4-f]indoleazine-3,6,10-trione (compound 9, 8.5 g, 32.2 mmol), and 4-methylbenzenesulfonic acid (0.4 mL, 2.5 mmol) were dissolved in toluene (200 mL). The reaction mixture was heated to 130 °C and stirred for 3 hours. TLC analysis showed the formation of blue product spots. The reaction mixture was concentrated under reduced pressure and stirred with silica gel. The crude product was purified by column chromatography (dichloromethane:methanol = 100:1) to give 4 g of a white solid (9S)-1-benzyloxy-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-1,2,3,9,10,12,13,15-octahydrocyclohexano[1,2,3-de]pyrano[3',4':6,7]indolazano[1,2-b]quinoline-10,13-dione (compound 10), with a yield of 29%.

[0277] ESI-MS m / z[M+H] + 563.25.

[0278] Step 9: Synthesis of Compound 11

[0279] At room temperature, (9S)-1-benzyloxy-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-1,2,3,9,10,12,13,15-octahydrocyclohexano[1,2,3-de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (compound 10, 4.0 g, 7.1 mmol) and palladium on carbon (0.78 mL, 0.75 mmol) were dissolved in methanol (40 mL). The reaction mixture was stirred vigorously overnight under a hydrogen balloon atmosphere. TLC analysis showed no remaining starting material and the formation of distinct new blue spots. The reaction solution was filtered and concentrated to obtain a black oily substance, which was then slurried with methyl tert-butyl ether (150 mL) to give 1.7 g of a yellow solid (9S)-9-ethyl-3,3,5-trifluoro-1,9-dihydroxy-4-methyl-1,2,3,9,10,12,13,15-octahydrocyclohexano[1,2,3-de]pyrano[3',4':6,7]indolazano[1,2-b]quinoline-10,13-dione (compound 11), with a yield of 51%.

[0280] ESI-MS m / z[M+H] + 473.10.

[0281] 1 H NMR(400MHz,DMSO-d6)δ8.12(d,J=10.6Hz,1H),7.45–7.25(m,1H),6.64–6.37(m,2H),5 .36(d,J=46.0Hz,4H),2.95(s,1H),2.59(s,3H),1.96–1.72(m,2H),0.87–0.82(m,3H).

[0282] Step 10: Synthesis of compounds 12 and 13

[0283] (9S)-9-ethyl-3,3,5-trifluoro-1,9-dihydroxy-4-methyl-1,2,3,9,10,12,13,15-octahydrocyclohexano[1,2,3-de]pyrano[3',4':6,7]indolazano[1,2-b]quinoline-10,13-dione (compound 11, 1.0 g) was dissolved in ethanol and separated by chiral HPLC to give compound 12 (383 mg, 38% yield) and compound 13 (419 mg, 42% yield).

[0284] Chiral separation method: Chromatographic column (Chromatographic column: IE, 10 μm, 30 x 250 mm); mobile phase: [n-hexane-ethanol]; B%: 30 / 70, 30 min). (Compound 12, RT = 13.7 min) and (Compound 13, RT = 26.2 min).

[0285] Compound 12:

[0286] 1 H NMR (400MHz, DMSO-d6) δ8.02(d,J=10.6Hz,1H),7.27(s,1H),6.51(s,2H),5.36(d,J=15.6Hz,3H),5.22(d,J=19.5Hz ,2H),2.93(t,J=6.9Hz,1H),2.73(dt,J=18.1,9.6Hz,1H),2.56(s,3H),1.83(p,J=6.8Hz,2H),0.84(t,J=7.3Hz,3H).

[0287] ESI-MS m / z[M+H] + 473.10.

[0288] Compound 13:

[0289] 1 H NMR (400MHz, DMSO-d6) δ8.06(d,J=10.6Hz,1H),7.29(s,1H),6.60–6.37(m,2H),5.38(d,J=19.0Hz,3H),5.27( d,J=19.3Hz,2H),2.94(dt,J=10.8,5.3Hz,1H),2.75(s,1H),2.56(s,3H),1.85(m,2H),0.86(t,J=7.3Hz,3H).

[0290] ESI-MS m / z[M+H] + 473.10.

[0291] Example 2: Synthesis of compounds 19 and 20

[0292] Synthesis route:

[0293] The * position indicates that the atom at that location is either R or S chiral, and the compound is a pure isomer.

[0294] Step 1: Synthesis of Compound 14

[0295] At -10 °C, N-(3,5,5-trifluoro-4-methyl-8-oxo-5,6,7,8-tetrahydro-1-naphthyl)acetamide (compound 5, 31 g, 114 mmol) and isoamyl nitrite (40 g, 343 mmol) were dissolved in tetrahydrofuran (150 mL) and tert-butanol (150 mL), and potassium tert-butoxide (12.8 g, 114 mmol) was added to the reaction solution in portions. The reaction solution was stirred at -10 °C for 1 hour, and TLC showed that the reaction was complete. The reaction was quenched with 1N hydrochloric acid and extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was pulped with petroleum ether and ethyl acetate (20:1) and filtered to obtain 24.5 g of yellow solid N-[(7Z)-3,5,5-trifluoro-7-(hydroxyimino)-4-methyl-8-oxo-5,6,7,8-tetrahydro-1-naphthyl]acetamide (compound 14), with a yield of 72%.

[0296] ESI-MS m / z [M+Na] + 305.05.

[0297] 1 H NMR (400MHz, CDCl3) δ12.00 (s, 1H), 8.66 (d, J = 12.0Hz, 1H), 6.01 (d, J = 48.0Hz, 1H), 4.05(t,J=16.0Hz,1H),2.76(dd,J=48.0Hz,16.0Hz,1H),2.37(s,3H),2.26(s,3H).

[0298] Step 2: Synthesis of Compound 15

[0299] At room temperature, N-[(7Z)-3,5,5-trifluoro-7-(hydroxyimino)-4-methyl-8-oxo-5,6,7,8-tetrahydro-1-naphthyl]acetamide (compound 14, 1 g, 3.3 mmol) was dissolved in tetrahydrofuran (10 mL), and acetic acid (0.4 mL, 6.7 mmol) and palladium on carbon (350 mg, 0.15 mmol) were added to the reaction system. The reaction was vigorously stirred for 1 hour under a hydrogen balloon at one atmosphere. After the reaction was completed, the reaction solution was filtered and concentrated under reduced pressure to give crude N-(7-amino-3,5,5-trifluoro-4-methyl-8-oxo-5,6,7,8-tetrahydro-1-naphthyl)acetamide (954 mg).

[0300] Under argon protection, benzyloxyacetyl chloride (615 mg, 3.3 mmol) was added to a suspension of N-(7-amino-3,5,5-trifluoro-4-methyl-8-oxo-5,6,7,8-tetrahydro-1-naphthyl)acetamide (954 mg) in tetrahydrofuran (10 mL). The reaction system was stirred at room temperature for 2 hours. After the reaction was completed, the mixture was filtered and concentrated. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 10:1-5:1) to give 480 mg of yellow solid N-[8-(acetamido)-4,4,6-trifluoro-5-methyl-1-oxo-1,2,3,4-tetrahydro-2-naphthyl]-2-(benzyloxy)acetamide (compound 15), with a yield of 30%.

[0301] ESI-MS m / z [M+Na] + 457.10.

[0302] 1 H NMR (500MHz, DMSO-d6) δ11.65(s,1H),8.56(d,J=12.8Hz,1H),8.24(d,J=8.0Hz,1H),7.47–7.38(m,4H),7.34(t,J=7.1Hz,1H), 5.02–4.92(m,1H),4.68–4.59(m,2H),4.06(s,2H),3.11–2.93(m,1H),2.86(dd,J=20.4,9.1Hz,1H),2.37(s,3H),2.20(s,3H).

[0303] Step 3: Synthesis of Compound 16

[0304] At room temperature, N-[8-(acetamido)-4,4,6-trifluoro-5-methyl-1-oxo-1,2,3,4-tetrahydro-2-naphthyl]-2-(benzyloxy)acetamide (compound 15, 480 mg, 1.1 mmol) was dissolved in methanol (12 mL) and concentrated hydrochloric acid (6 mL), and the mixture was heated to 50 °C and stirred for 1.5 hours. After the reaction was complete, the pH was adjusted to 8 with saturated sodium bicarbonate solution, and then extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 10:1-5:1) to give 340 mg of a yellow oil N-(8-amino-4,4,6-trifluoro-5-methyl-1-oxo-1,2,3,4-tetrahydro-2-naphthyl)-2-(benzyloxy)acetamide (compound 16), with a yield of 71%.

[0305] ESI-MS m / z [MH] - 391.00.

[0306] Step 4: Synthesis of Compound 17

[0307] At room temperature, N-(8-amino-4,4,6-trifluoro-5-methyl-1-oxo-1,2,3,4-tetrahydro-2-naphthyl)-2-(benzyloxy)acetamide (compound 16, 340 mg, 0.9 mmol), (4S)-4-ethyl-4-hydroxy-3,4,6,7,8,10-hexahydro-1H-pyrano[3,4-f]indoleazine-3,6,10-trione (compound 9, 228 mg, 0.87 mmol), and 4-methylbenzenesulfonic acid (15 mg, 0.09 mmol) were dissolved in toluene (14 mL). The reaction mixture was placed in an oil bath at 130 °C and then stirred vigorously for 5 hours. After the reaction was completed, the toluene was removed by concentration under reduced pressure, and the mixture was stirred with silica gel. The crude product was purified by column chromatography (dichloromethane / methanol = 100:1) to give 340 mg of a yellow oily substance, 2-(benzyloxy)-N-[(9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,12,15-hexahydro-1H-cyclohexano[1,2,3-de]pyrano[3',4':6,7]indolazano[1,2-b]quinoline-1-yl]acetamide (compound 17), with a yield of 44%.

[0308] ESI-MS m / z[M+H] + 620.20.

[0309] Step 5: Synthesis of Compound 18

[0310] At room temperature, 2-benzyloxy-N-[(9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,12,15-hexahydro-1H-cyclohexano[1,2,3-de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl]acetamide (compound 17, 340 mg, 0.55 mmol) and palladium on carbon (150 mg) were dissolved in methanol (3 mL). The reaction mixture was stirred at room temperature for 6 hours under a hydrogen balloon atmosphere. After the reaction was completed, the mixture was filtered and concentrated. The crude product was purified by column chromatography (dichloromethane / methanol = 30:1) to give 70 mg of a yellow solid N-[(9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,12,15-hexahydro-1H-cyclohexano[1,2,3-de]pyrano[3',4':6,7]indolazano[1,2-b]quinolin-1-yl]-2-hydroxyacetamide (compound 18), with a yield of 22%.

[0311] ESI-MS m / z[M+H] +530.20.

[0312] Step 6: Synthesis of compounds 19 and 20

[0313] N-[(9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,12,15-hexahydro-1H-cyclohexano[1,2,3-de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl]-2-hydroxyacetamide (70 mg, 0.13 mmol) was purified by preparative thin-layer chromatography (DCM / MeOH = 20:1) to give a yellow solid N-((9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13) 10 mg of 1,5-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)-2-hydroxyacetamide (compound 19), in 13% yield, was obtained as a yellow solid. 10 mg of N-((9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)-2-hydroxyacetamide (compound 20), in 13% yield, was also obtained.

[0314] Compound 19:

[0315] 1 H NMR (400MHz, DMSO-d6) δ8.57(d,J=9.0Hz,1H),8.16(d,J=10.6Hz,1H),7.31(s,1H),6.53(s,1H),5.74(dt,J=23.5,7.1Hz,2H),5.4 0(s,2H),5.30–5.10(m,2H),3.98(dd,J=5.4,3.3Hz,2H),3.05–2.76(m,2H),2.61(s,3H),1.89–1.77(m,2H),0.83(d,J=7.0Hz,3H).

[0316] ESI-MS m / z[M+H] + 530.20.

[0317] Compound 20:

[0318] 1H NMR (400MHz, DMSO-d6) δ8.62(d,J=9.1Hz,1H),8.16(d,J=10.6Hz,1H),7.33(s,1H),6.54(s,1H),5.84–5.70(m,2H),5.42(d,J=3.9H z,2H),5.35–5.10(m,2H),4.01(dd,J=5.6,1.6Hz,2H),3.06–2.78(m,2H),2.62(d,J=3.4Hz,3H),1.85(m,2H),0.86(t,J=7.4Hz,3H).

[0319] ESI-MS m / z[M+H] + 530.20.

[0320] Example 3 Synthesis of compounds 26 and 27

[0321] Synthesis route:

[0322] The * position indicates that the atom at that location is either R or S chiral, and the compound is a pure isomer.

[0323] Step 1: Synthesis of Compound 22

[0324] At room temperature, N-(3,5,5-trifluoro-7-(hydroxyimino)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (compound 14, 14 g, 34.5 mmol) and Pd / C (4.2 g, 30% wt) were dissolved in tetrahydrofuran (462 mL), and 9-fluorenylmethyl-N-succinimidyl carbonate (compound 21, 11.6 g, 34.5 mmol) was added. The reaction mixture was stirred vigorously for 1 hour under a hydrogen atmosphere, palladium on carbon was removed by filtration, and tetrahydrofuran was removed by concentration under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to give 14 g of a yellow solid 9H-fluorene-9-ylmethyl{[8-(acetamido)-4,4,6-trifluoro-5-methyl-1-oxo-1,2,3,4-tetrahydro-2-naphthyl]amino}carbamate (compound 22), with a yield of 77%.

[0325] ESI-MS m / z [M+Na] + :531.10.

[0326] 1H NMR(400MHz, CDCl3)δ11.57(s,1H),8.70(d,J=12.4Hz,1H),7.70(m,4H),7.45–7.30(m,4H),5.75 (s,1H),4.73(s,1H),4.53(m,2H),4.26(t,J=6.7Hz,1H),3.26(s,1H),2.43(m,4H),2.24(s,3H).

[0327] Step 2: Synthesis of Compound 23

[0328] At room temperature, 9H-fluorene-9-ylmethyl{[8-(acetamido)-4,4,6-trifluoro-5-methyl-1-oxo-1,2,3,4-tetrahydro-2-naphthyl]amino}carbamate (compound 22, 13.5 g, 26.5 mmol) was dissolved in a mixture of methanol (70 mL) and tetrahydrofuran (210 mL), and concentrated hydrochloric acid (200 mL, 2.4 mol) was added. The reaction mixture was stirred in an oil bath at 55 °C for 3 hours, quenched with saturated sodium carbonate aqueous solution (1 L), and the organic phase was extracted with ethyl acetate (500 mL x 2). The combined organic phases were washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 4:1) to give 12 g of a yellow solid 9H-fluorene-9-ylmethyl[(8-amino-4,4,6-trifluoro-5-methyl-1-oxo-1,2,3,4-tetrahydro-2-naphthyl)amino]carbamate (compound 23), with a yield of 97%.

[0329] ESI-MS m / z[M+H] + 467.10.

[0330] 1 H NMR (400MHz, CDCl3) δ7.78(d,J=7.5Hz,2H),7.63(d,J=7.5Hz,2H),7.44–7.31(m,4H),6.46(d,J=11.2Hz ,3H),5.87(s,1H),4.64(s,1H),4.45(d,J=7.1Hz,2H),4.26(t,J=6.9Hz,1H),3.26(s,1H),2.33(m,4H).

[0331] Step 3: Synthesis of Compound 24

[0332] At room temperature, 23.6 g (13 mmol) of 9H-fluorene-9-ylmethyl[(8-amino-4,4,6-trifluoro-5-methyl-1-oxo-1,2,3,4-tetrahydro-2-naphthyl)amino]carbamate was dissolved in toluene (60 mL), and (4S)-4-ethyl-4-hydroxy-3,4,6,7,8,10-hexahydro-1H-pyrano[3,4-f]indoleazine 3,6,10-trione (9, 5.1 g, 19 mmol) and TsOH (0.22 g, 1.3 mmol) were added separately. The reaction mixture was transferred to an oil bath at 130 °C and stirred vigorously for 5 hours. After the reaction was completed by TLC, the solution was concentrated under reduced pressure and stirred with silica gel. The crude product was purified by column chromatography (dichloromethane:methanol = 100:1) to obtain 8.3 g of crude yellow solid 9H-fluorene-9-ylmethyl{[(9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,12,15-hexahydro-1H-cyclohexano[1,2,3-de]pyrano[3',4':6,7]indolazano[1,2-b]quinoline-1-yl]amino}carbamate (compound 24), with a yield of 93%. 1.7 g of unreacted compound 23 was recovered.

[0333] ESI-MS m / z[M+H] + 694.20

[0334] Step 4: Synthesis of Compound 25

[0335] At 0 °C, 24 g (8.3 g, 12 mmol) of 9H-fluorene-9-ylmethyl{[(9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,12,15-hexahydro-1H-cyclohexano[1,2,3-de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl]amino}carbamate was dissolved in DMF (238 mL), followed by the slow addition of piperidine (5.9 mL, 60 mmol). The reaction mixture was stirred vigorously at 0 °C for 30 minutes and then concentrated under reduced pressure. The crude product was slurried with a mixed solvent of methyl tert-butyl ether and methanol (15:1) to give 3.6 g of a gray solid (9S)-1-amino-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (compound 25), with a yield of 64%.

[0336] 1H NMR(400MHz, DMSO-d6)δ8.12(d,J=10.6Hz,1H),7.78–7.59(m,1H),7.33(s,1H),6.54(s,1H),5.81–5.60(m,1H),5.41(m,3H),4.51(s,1H),4.22 (t,J=6.5Hz,1H),2.87(m,1H),2.67(s,1H),2.62(s,3H),1.87(m,J=7.2 Hz,2H),1.63(q,J=6.9Hz,1H),1.37(q,J=7.4Hz,1H),0.92–0.86(m,3H).

[0337] ESI-MS m / z[M+H] + 472.20.

[0338] Step 5: Synthesis of compounds 26 and 27

[0339] (9S)-1-amino-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (630 mg, 1.3 mmol) was purified by high performance liquid chromatography to obtain solid (9S)-1-amino-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (630 mg, 1.3 mmol). Trifluoroacetate 26 of indolinazino[1,2-b]quinoline-10,13-dione (262 mg, RT = 7.88 min, yield 42%) and (9S)-1-amino-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolinazino[1,2-b]quinoline-10,13-dione (238 mg, RT = 6.50 min, yield 38%).

[0340] Compound 26:

[0341] ESI-MS m / z[M+H] + 472.27.

[0342] 1H NMR(500MHz,DMSO-d6)δ8.74(s,3H),8.29(d,J=10.3Hz,1H),7.38(s,1H),6.60(s,1H),5.80(d,J=19.5Hz,1 H),5.57–5.38(m,4H),3.23–2.97(m,2H),2.68(s,3H),1.89(dt,J=12.6,6.9Hz,2H),0.88(t,J=7.3Hz,3H).

[0343] Compound 27:

[0344] ESI-MS m / z[M+H] + 472.27.

[0345] 1 H NMR(500MHz,DMSO-d6)δ8.68(s,3H),8.30(d,J=10.4Hz,1H),7.39(s,1H),6.60(s,1H),5.79(d,J=1 9.5Hz,1H),5.57–5.37(m,4H),3.21–2.99(m,2H),2.68(s,3H),1.88(m,2H),0.87(t,J=7.3Hz,3H).

[0346] Chromatographic column: Welch Xtimate C18, 21.2 × 150 mm, 5 μm; Mobile phase A: 0.1% TFA aqueous solution; Mobile phase B: ACN

[0347] Example 4 Synthesis of Compound 38

[0348] Synthesis route:

[0349] Step 1: Synthesis of Compound 29

[0350] At 0°C, 3-fluoro-2-methyl-5-nitrophenol (compound 28, 6 g, 35 mmol) was added to a DMF (100 mL) suspension of NaH (1.7 g, 42 mmol). After stirring for 20 minutes, 18-crown-6-ether (0.46 g, 1.7 mmol) and 3-chloropropane-1-ol (3.5 mL, 42 mmol) were added sequentially to the reaction system. The reaction mixture was transferred to an oil bath at 80°C and stirred for 48 hours. After the reaction was completed, water (200 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL × 3). The combined organic phases were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 3:1) to give 4.8 g of a yellow solid 3-[(3-fluoro-2-methyl-5-nitrophenyl)oxy]propane-1-ol (compound 29), with a yield of 61%.

[0351] 1 H NMR(400MHz,DMSO-d6)δ7.72(dd,J=9.2,2.0Hz,1H),7.63(s,1H),4.61(t,J=5.2Hz,1H),4 .22(t,J=6.4Hz,2H),3.59(q,J=6.0Hz,2H),2.16(d,J=2.0Hz,3H),1.92(t,J=6.0Hz,2H).

[0352] Step 2: Synthesis of Compound 30

[0353] At 0°C, 3-[(3-fluoro-2-methyl-5-nitrophenyl)oxy]prop-1-ol (compound 29, 4.8 g, 21 mmol) was dissolved in acetone (50 mL), and a mixed solution of chromium trioxide (6.4 g, 64 mmol), concentrated sulfuric acid (5 mL), and water (30 mL) was slowly added dropwise to the above system. After stirring at room temperature for 15 minutes, the mixture was poured into water, filtered under reduced pressure, and the filter cake was dried to obtain 3.5 g of crude white solid 3-[(3-fluoro-2-methyl-5-nitrophenyl)oxy]propionic acid (compound 30). The crude product was used directly in the next step without further purification.

[0354] Step 3: Synthesis of Compound 31

[0355] At room temperature, iron powder (8.0 g, 144 mmol) and ammonium chloride (7.7 g, 144 mmol) were added sequentially to a solution of 3-[(3-fluoro-2-methyl-5-nitrophenyl)oxy]propionic acid (compound 30, 3.5 g, 14.4 mmol) in ethanol (80 mL) and water (20 mL). The reaction mixture was transferred to an oil bath at 90 °C and stirred vigorously for 1 hour. After the reaction was completed by TLC, the insoluble matter was filtered through diatomaceous earth and extracted with dichloromethane (100 mL x 3). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 3 g of yellow solid 3-[(5-amino-3-fluoro-2-methylphenyl)oxy]propionic acid (compound 31), with a yield of 98%.

[0356] ESI-MS m / z[M+H] + 213.90.

[0357] 1 H NMR (400MHz, DMSO-d6) δ12.45(s,1H),7.75(d,J=2.0Hz,1H),7.72(s,1H),4.35(t,J=6.0Hz,2H),2.75(t,J=6.4Hz,2H),2.12(s,3H).

[0358] Step 4: Synthesis of Compound 32

[0359] At room temperature, 3-[(5-amino-3-fluoro-2-methylphenyl)oxy]propionic acid (compound 31, 3.0 g, 14 mmol) was added in portions to concentrated sulfuric acid (30 mL). The reaction flask was transferred to an oil bath at 80 °C and the reaction was stirred vigorously for 2 hours. After the reaction was completed, the mixture was poured into ice water and filtered under reduced pressure to obtain a filter cake. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 3:1) to give 2.2 g of a yellow solid 5-amino-7-fluoro-8-methyl-3,4-dihydro-2H-benzopyran-3-one (compound 32), with a yield of 79%.

[0360] ESI-MS m / z[M+H] + 196.10.

[0361] 1 H NMR (400MHz, DMSO-d6) δ7.41 (s, 1H), 6.05 (d, J = 12.4Hz, 1H), 4.44 (t, J = 6.4Hz, 2H), 2.68 (t, J = 6.4Hz, 2H), 1.89 (s, 3H).

[0362] Step 5: Synthesis of Compound 33

[0363] At room temperature, 5-amino-7-fluoro-8-methyl-3,4-dihydro-2H-benzopyran-4-one (compound 32, 2.2 g, 11.2 mmol) was dissolved in pyridine (20 mL), and Ac₂O (1.6 mL, 16.8 mmol) and DMAP (0.14 g, 1.1 mmol) were added sequentially. The reaction solution was heated to 60 °C and stirred vigorously for 2 days. After the reaction was completed by TLC, the reaction solution was concentrated under reduced pressure and mixed with silica gel. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 3:1) to give 1.0 g of yellow solid N-(7-fluoro-8-methyl-4-oxo-3,4-dihydro-2H-benzopyran-5-yl)acetamide (compound 33), with a yield of 38%.

[0364] ESI-MS m / z[M+H] + 253.85.

[0365] 1 H NMR (400MHz, DMSO-d6) δ11.69(s,1H),7.95(d,J=12.8Hz,1H),6.12(d,J=5.1 Hz,1H),4.53(dd,J=10.7,4.4Hz,1H),4.32(m,2H),2.15(s,3H),2.00(s,3H).

[0366] Step 6: Synthesis of Compound 34

[0367] At 0 °C, N-(7-fluoro-8-methyl-4-oxo-3,4-dihydro-2H-benzopyran-5-yl)acetamide (compound 33, 986 mg, 4.2 mmol) was dissolved in methanol (20 mL), followed by the sequential addition of potassium hydroxide (2.6 g, 45.7 mmol) and iodophenyl diacetic acid (PIDA, 1.3 g, 4.2 mmol). The reaction mixture was stirred vigorously at room temperature for 2 hours. After TLC confirmation of the reaction completion, 1 N hydrochloric acid (20 mL) was added, and the mixture was stirred for 20 minutes. The organic phase was extracted with dichloromethane (200 mL x 3), washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and mixed with silica gel. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 4:1) to give 799 mg of white solid N-(7-fluoro-3-hydroxy-8-methyl-4-oxo-3,4-dihydro-2H-benzopyran-5-yl)acetamide (compound 34), with a yield of 76%.

[0368] ESI-MS m / z[M+H] + 253.85.

[0369] 1H NMR (400MHz, DMSO-d6) δ11.69(s,1H),7.95(d,J=12.8Hz,1H),6.12(d,J=5.1 Hz,1H),4.53(dd,J=10.7,4.4Hz,1H),4.32(m,2H),2.15(s,3H),2.00(s,3H).

[0370] Step 7: Synthesis of Compound 35

[0371] At 0 °C, N-(7-fluoro-3-hydroxy-8-methyl-4-oxo-3,4-dihydro-2H-benzopyran-5-yl)acetamide (compound 34, 292 mg, 1.2 mmol) was dissolved in tetrahydrofuran (5 mL), and silver oxide (534 mg, 2.3 mmol) and benzyl bromide (690 μL, 5.8 mmol) were added sequentially to the above reaction system. The reaction system was heated to room temperature and stirred for 3 hours. After the reaction was completed, the mixture was filtered through diatomaceous earth, concentrated under reduced pressure, and mixed with silica gel. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 4:1) to give 295 mg of white solid N-[3-(benzyloxy)-7-fluoro-8-methyl-4-oxo-3,4-dihydro-2H-benzopyran-5-yl]acetamide (compound 35), with a yield of 74%.

[0372] ESI-MS m / z [M+Na] + 366.05.

[0373] Step 8: Synthesis of Compound 36

[0374] At room temperature, concentrated hydrochloric acid (1 mL, 12 mmol) was added to a dioxane (1 mL) solution of N-[3-(benzyloxy)-7-fluoro-8-methyl-4-oxo-3,4-dihydro-2H-benzopyran-5-yl]acetamide (compound 35, 295 mg, 0.86 mmol). The reaction mixture was heated to 50 °C and stirred vigorously for 1.5 hours. After the reaction was completed, the mixture was quenched with saturated potassium carbonate aqueous solution (200 mL), the organic phase was extracted with dichloromethane (20 mL x 3), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and mixed with silica gel. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 5:1) to give 208 mg of 5-amino-3-(benzyloxy)-7-fluoro-8-methyl-3,4-dihydro-2H-benzopyran-4-one (compound 36), with a yield of 80%.

[0375] ESI-MS m / z[M+H] + 302.05.

[0376] Step 9: Synthesis of Compound 37

[0377] At room temperature, 4-methylbenzenesulfonic acid (13 mg, 0.08 mmol) was added to a toluene solution (10 mL) of 5-amino-3-(benzyloxy)-7-fluoro-8-methyl-3,4-dihydro-2H-benzopyran-4-one (compound 36, 239 mg, 0.8 mmol) and (4S)-4-ethyl-4-hydroxy-3,4,6,7,8,10-hexahydro-1H-pyrano[3,4-f]indoleazine-3,6,10-trione (compound 9, 209 mg, 0.79 mmol). The reaction system was heated to 130 °C and then stirred vigorously for 3 hours. After the reaction was completed, the mixture was concentrated under reduced pressure and stirred with silica gel. The crude product was purified by column chromatography (dichloromethane:methanol = 100:1) to give 132 mg of a white solid (9S)-1-(benzyloxy)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,9,10,12,13,15-hexahydro-1H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (compound 37), with a yield of 19%.

[0378] ESI-MS m / z[M+H] + 529.25.

[0379] Step 10: Synthesis of Compound 38

[0380] Palladium on carbon (60 mg) was added to a methanol (2 mL) solution of (9S)-1-(benzyloxy)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,9,10,12,13,15-hexahydro-1H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (compound 37, 132 mg, 0.08 mmol). The reaction was stirred overnight at room temperature under a hydrogen balloon atmosphere. TLC showed that the reaction was complete. The reaction solution was filtered through diatomaceous earth and concentrated under reduced pressure. The crude product was slurried with methyl tert-butyl ether (150 mL) to give 7 mg of a yellow solid (9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,9,10,12,13,15-hexahydro-1H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (compound 38), with a yield of 21%.

[0381] ESI-MS m / z[M+H] + 439.15.

[0382] 1H NMR (500MHz, DMSO-d6) δ7.55(d,J=10.8Hz,1H),7.32(s,1H),6.55(s,1H),6.38(dd,J=6.3,2.7Hz,1H),5.49–5.26( m,5H),4.61(dd,J=10.9,4.6Hz,1H),4.23(dd,J=10.9,8.1Hz,1H),2.29(s,3H),1.88(m,2H),0.89(t,J=7.3Hz,3H).

[0383] Example 5 Synthesis of Compound 42

[0384] Synthesis route:

[0385] Step 1: Synthesis of Compound 41

[0386] At 0 °C, N-{2-[(2-aminoacetyl)amino]acetyl}-L-phenylpropionate benzyl ester (39 g, 4.1 g, 11 mmol) and 1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapecopentane-25-acid (40 g, 4.9 g, 11 mmol) were dissolved in DMF (60 mL). Subsequently, HATU (8.4 g, 22 mmol) and DIPEA (5.5 mL, 33 mmol) were added sequentially to the above system. The reaction mixture was heated to room temperature and stirred vigorously for 3 hours. TLC analysis showed that the reaction was complete. The reaction mixture was concentrated under vacuum and purified by silica gel column chromatography (dichloromethane:methanol = 80:1-60:1-40:1) to give 7.1 g of a white solid (benzyl(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azatetradecane-25-acyl)glycylglycyl-L-phenylalanine ester (compound 41), with a yield of 81%).

[0387] ESI-MS m / z[M+H] + 927.3.

[0388] Step 2: Synthesis of Compound 42

[0389] At room temperature, (benzyl(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapecopentane-25-acyl)glycylglycyl-L-phenylalanine ester (41 g, 7.1 g, 9.0 mmol) and Pd / C (1.4 g, 20%) were added to a mixed solvent of MeOH (50 mL) and EtOAc (50 mL), respectively. Wt.). The reaction solution was vigorously stirred for 1 hour under a hydrogen balloon atmosphere. After the reaction was completed, the reaction solution was filtered through diatomaceous earth and concentrated under vacuum to obtain 3.9 g of (1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapecopentane-25-acyl)glycylglycyl-L-phenylalanine (compound 42), with a yield of 78%.

[0390] ESI-MS m / z[M+H] + 837.3.

[0391] 1 H NMR (400MHz, DMSO-d6) δ12.76 (s, 1H), 8.21–8.10 (m, 2H), 8.00 (t, J = 5.8Hz, 1H), 7.90 (d, J=7.5Hz,2H),7.70(d,J=7.5Hz,2H),7.47–7.39(m,2H),7.37–7.24(m,5H),7.24–7.18(m ,3H),4.42(m,1H),4.30(d,J=6.9Hz,2H),4.21(m,1H),3.78–3.64(m,4H),3.60(t,J=6.5 Hz,2H),3.49(m,18H),3.41(m,4H),3.14(m,2H),3.05(m,1H),2.89(m,1H),2.39(m,2H).

[0392] Example 5 Synthesis of Compound 47

[0393] Synthesis route:

[0394] The * position indicates that the atom at that location is either R or S chiral, and the compound is a pure isomer.

[0395] Step 1: Synthesis of Compound 44

[0396] Under N2 protection, 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-acid (43,816 mg, 2.1 mmol) was dissolved in DMF (10 mL), and EDCI (814 mg, 4.3 mmol) and N-hydroxysuccinimide (733 mg, 6.4 mmol) were added separately. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was completed by TLC, the reaction mixture was washed with water (50 mL) to remove EDCI and NHS, and extracted with ethyl acetate (50 mL × 2). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum to give the active ester intermediate. At room temperature, the intermediate was redissolved in DMF (60 mL) and (9S)-1-amino-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (25,500 mg, 1.1 mmol) was added. The reaction mixture was vigorously stirred at room temperature for 18 hours and purified by silica gel column chromatography (dichloromethane:methanol = 30:1) to give 712 mg of (9H-fluorene-9-yl)methyl(2-(((2-(((9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]inazinyl[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (compound 44), with a yield of 80%.

[0397] ESI-MS m / z [M+Na] + 860.30.

[0398] 1 H NMR(500MHz,DMSO-d6)δ8.87(s,1H),8.68(d,J=9.1Hz,1H),8.16(d,J=10.5Hz,1H) ,7.86(d,J=7.7Hz,2H),7.67(d,J=7.5Hz,2H),7.41–7.27(m,5H),6.56(s,1H),5.7 4(m,2H),5.40(s,2H),5.32–5.08(m,2H),4.68(d,J=6.9Hz,2H),4.28–4.08(m,4H) ,3.63(s,2H),2.86(m,2H),2.62(s,3H),1.93–1.76(m,2H),0.84(t,J=7.2Hz,3H).

[0399] Step 2: Synthesis of Compound 45

[0400] At 0 °C, (9H-fluorene-9-yl)methyl(2-(((2-(((9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]inazinyl[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (44,712 mg, 0.85 mmol) was dissolved in DMF (19 mL), and piperidine (420 μL, 4.3 mmol) was slowly added dropwise. The reaction mixture was stirred vigorously at the same temperature for 1 hour. After the reaction was completed by TLC analysis, the mixture was concentrated under vacuum and purified by silica gel column chromatography (dichloromethane:methanol = 7:1, 1% NH4OH) to give 265 mg of 2-amino-N-((2-(((9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]inazinyl[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)acetamide (compound 45), with a yield of 51%.

[0401] ESI-MS m / z[M+H] + 616.25.

[0402] 1 H NMR(500MHz,DMSO-d6)δ8.93(s,1H),8.71(s,1H),8.19(d,J=10.4Hz,1H),7.35(s,1H),6.58(s,1H),5.75(s,1H),5.44 (s,2H),5.33–5.14(m,2H),4.70(s,2H),4.09(s,2H),3.28(s,2H),2.89(m,4H),2.64(s,3H),1.87(m,2H),0.87(m,3H).

[0403] Step 3: Synthesis of Compound 46

[0404] Under N2 protection, N-[1-(9H-fluorene-9-yl)-3,25,28,31-tetraoxo-2,7,10,13,16,19,22-heptaoxa-4,26,29-triazahexadecane-31-yl]-L-phenylalanine (42,693 mg, 0.83 mmol) was dissolved in DMF (4 mL), and EDCI (318 mg, 1.7 mmol) and N-hydroxysuccinimide (286 mg, 2.5 mmol) were added separately. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was completed by TLC, the reaction mixture was washed with water (10 mL) to remove EDCI and NHS, and extracted with ethyl acetate (20 mL × 2). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum to give the active ester intermediate. At room temperature, the intermediate was redissolved in DMF (4 mL), and 2-amino-N-((2-(((9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]inazinyl[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)acetamide (45,255 mg, 0.4 mmol). The reaction mixture was stirred vigorously at room temperature for 3 hours and purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to give 389 mg of 9H-fluorene-9-ylmethyl{[(10S)-10-benzyl-1-{[(9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,12,15-hexahydro-1H-cyclohexa[1,2,3-de]pyrano[3',4':6,7]inzidinyl[1,2-b]quinoline-1-yl]amino}-1,6,9,12,15,18-hexaoxo-3,21,24,27,30,33,36-heptaoxa-5,8,11,14,17-pentazocanoic-38-yl]amino}carbamate (compound 46), with a yield of 66%.

[0405] ESI-MS m / z[M+H] + 1456.75.

[0406] 1H NMR (400MHz, DMSO-d6) δ8.70(s,2H),8.32(s,1H),8.18–8.14(m,2H),8.00(t,J=6.2Hz,1H),7.41(d,J=5.0Hz,2H),7.36 –7.31(m,5H),7.24(ddd,J=9.9,6.9,2.8Hz,6H),6.56(s,1H),5.76(s,1H),5.33–5.13(m,2H),4.69(d,J=6.8Hz,2H),4. 47(s,1H),4.12–4.04(m,2H),3.74(m,3H),3.68(m,3H),3.60(m,2H),3.57(m,2H),3.49(m,20H),3.45(s,2H),3.05–2.9 5(m,2H),2.90(m,2H),2.83–2.74(m,2H),2.63(s,3H),2.42–2.35(m,3H),1.87(t,J=7.7Hz,2H),0.87(t,J=7.3Hz,3H).

[0407] Step 4: Synthesis of Compound 47

[0408] At 0℃, 9H-fluorene-9-ylmethyl{[(10S)-10-benzyl-1-{[(9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,12,15-hexahydro-1H-cyclohexa[1,2,3-de]pyrano[3',4':6,7]indazinyl[1,2-b]quinoline- 1-[yl]amino}-1,6,9,12,15,18-hexaoxo-3,21,24,27,30,33,36-heptaoxa-5,8,11,14,17-pentazocanoic acid-38-yl]carbamate (46,678 mg, 0.47 mmol) was dissolved in DMF (10 mL), and piperidine (233 μL, 2.4 mmol) was slowly added dropwise. The reaction mixture was stirred vigorously at the same temperature for 1 hour. After the reaction was analyzed by TLC, the mixture was concentrated under vacuum and purified by Pre-HPLC to obtain 51 mg of N-[(9S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,12,15-hexahydro-1H-cyclohexa[1,2,3-de]pyran[3',4':6,7]inazinyl[1,2-b]quinoline-1-yl]-2-{[(29S)-1-amino-29-benzyl-21,24,27,30,33-pentoxo-3,6,9,12,15,18-hexaoxa-22,25,28,31,34-pentazolidinyl-35-yl]oxy}acetamide (compound 47), with a yield of 18%.

[0409] ESI-MS m / z[M+H] + :1212.70.

[0410] 1H NMR (400MHz, DMSO-d6) δ8.81–8.72(m,2H),8.40(d,J=4.3Hz,2H),8.25–8.15(m,3H),8.07(s,1H),7.35(s,1 H),7.21(m,5H),5.75(d,J=7.1Hz,1H),5.43(s,2H),5.34–5.15(m,2H),4.69(d,J=6.7Hz,2H),4.46(m,1H),4 .08(d,J=6.7Hz,2H),3.75(d,J=5.6Hz,2H),3.68(d,J=5.7Hz,2H),3.62–3.49(m,26H),3.02(m,2H),2.93–2 .75(m,5H),2.66–2.60(m,3H),2.38(t,J=6.6Hz,2H),1.95–1.77(m,2H),1.24(s,2H),0.87(t,J=7.2Hz,3H).

[0411] Chromatographic column: WATERS XBridge C18, 19×150mm, 5μm; Mobile phase A: 0.1% formic acid; Mobile phase B: acetonitrile

[0412] Example 6 Synthesis of Compound 52

[0413] Synthesis route:

[0414] The * position indicates that the atom at that location is either R or S chiral, and the compound is a pure isomer.

[0415] Step 1: Synthesis of Compound 49

[0416] At room temperature, (9S)-9-ethyl-3,3,5-trifluoro-1,9-dihydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (compound 13, 529 mg, 1.1 mmol) and 9H-fluorene-9-ylmethyl[(2,6-dioxy-5-aza-3-oxahept-7-yl)amino]carbamate (compound 48, 2.3 g, 6.4 mmol) were dissolved in a mixed solution of N,N-dimethylformamide (6 mL) and tetrahydrofuran (1 mL). 4-Methylbenzenesulfonic acid (55 mg, 0.32 mmol) was added to the above reaction system, and the mixture was stirred vigorously at 50 °C for 2 hours. After the reaction was complete as shown by TLC, the mixture was quenched with saturated sodium bicarbonate aqueous solution (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (dichloromethane:methanol = 40:1) to give 300 mg of a yellow solid (9H-fluorene-9-yl)methyl(2-(((((9S)-9-ethyl-3,3,5-trifluoro-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)oxy)methyl)amino)-2-oxoethyl)carbamate (compound 49), with a yield of 60%.

[0417] ESI-MS m / z [M+Na] + 803.30.

[0418] Step 2: Synthesis of Compound 50

[0419] At room temperature, diethylamine (3 mL, 29 mmol) was added to a solution of (9H-fluorene-9-yl)methyl(2-(((((9S)-9-ethyl-3,3,5-trifluoro-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)oxy)methyl)amino)-2-oxoethyl)carbamate (compound 49, 300 mg, 0.38 mmol) in dichloromethane (12 mL). The reaction mixture was stirred at room temperature for 1.5 h. After the reaction was complete as shown by TLC, the reaction solution was concentrated under reduced pressure and stirred with silica gel. The crude product was purified by column chromatography (dichloromethane:methanol = 6:1) to obtain 60 mg of a yellow solid 2-amino-N-((9S)-9-ethyl-3,3,5-trifluoro-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)oxy)methyl)acetamide (compound 50), with a yield of 28%.

[0420] ESI-MS m / z[M+H] + 559.25.

[0421] Step 3: Synthesis of Compound 51

[0422] At 0 °C, 2-amino-N-((9S)-9-ethyl-3,3,5-trifluoro-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)oxy)methyl)acetamide (compound 50, 60 mg, 0.11 mmol) and (1-(9H-fluorene-9-yl)-3-oxo)acetamide were subjected to oxidation. To a solution of 2,7,10,13,16,19,22-heptoxy-4-azapecopentane-25-yl)glycylglycyl-L-phenylalanine (108 mg, 0.13 mmol) in N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (53 μL, 0.32 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (82 mg, 0.22 mmol) were added sequentially. The reaction mixture was stirred vigorously at room temperature for 2 hours. After the reaction was complete as indicated by TLC, the reaction mixture was concentrated under reduced pressure and stirred with silica gel. The crude product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain 140 mg of a yellow solid (9H-fluorene-9-yl)methyl((7S)-7-benzyl-1-(((9S)-9-ethyl-3,3,5-trifluoro-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)oxy)-3,6,9,12,15-pentoxo-18,21,24,27,30,33-hexaoxa-2,5,8,11,14-pentazatritrisaccharide-35-yl)carbamate (compound 51), with a yield of 95%.

[0423] ESI-MS m / z [M+Na] + 1399.80.

[0424] Step 4: Synthesis of Compound 52

[0425] At room temperature, diethylamine (2 mL, 19 mmol) was slowly added dropwise to a solution of (9H-fluorene-9-yl)methyl((7S)-7-benzyl-1-(((9S)-9-ethyl-3,3,5-trifluoro-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)oxy)-3,6,9,12,15-pentoxo-18,21,24,27,30,33-hexaoxa-2,5,8,11,14-pentazatritrisaccharide-35-yl)carbamate (compound 51, 200 mg, 0.15 mmol) in dichloromethane (8 mL). The reaction solution was vigorously stirred at room temperature for 3 hours. After the reaction was complete as shown by TLC, the reaction solution was concentrated under reduced pressure and stirred with silica gel. The crude product was purified by column chromatography (dichloromethane:methanol = 3:1) to obtain a yellow solid 1-amino-N-((7S)-7-benzyl-1-(((9S)-9-ethyl-3,3,5-trifluoro-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)oxy)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatetrahexacyclobutane-13-yl)-3,6,9,12,15,18-hexaoxane-21-amide (compound 52) 98 mg, with a yield of 52%.

[0426] ESI-MS m / z[M+H] + :1155.60.

[0427] 1HNMR(400MHz,DMSO d6)δ9.00(t,J=6.7Hz,1H),8.43(t,J=5.8Hz,1H),8.21–8.14(m,3H),8.03(t,J=5.7Hz,1H),7.80–7.62(m,2H),7.35 (s,1H),7.25–7.17(m,5H),6.58(s,1H),5.44–5.39(m,2H),5.38–5.31(m,2H),4.94(dd,J=6.8,4.6Hz,1H),4.51(m,1 H),3.83(d,J=5.8Hz,2H),3.70–3.67(m,2H),3.57–3.54(m,6H),3.53–3.44(m,20H),3.03–2.87(m,5H),2.83(s,1H) ,2.80–2.72(m,2H),2.63(d,J=2.8Hz,2H),2.38(m,3H),2.01–1.96(m,1H),1.85–1.79(m,1H),0.84(d,J=7.2Hz,3H).

[0428] Example 7: Biological Experiment Evaluation Section

[0429] I. Tumor Cell Proliferation Inhibition Experiment

[0430] The strength of the inhibitory activity of the test compound on the proliferation of the breast cancer cell line MDA-MB-231 was determined.

[0431] 1. Cell Culture Methods

[0432] Table 1. Cells and Culture Methods

[0433] 2. Experimental Reagents

[0434] Table 2. Reagent Information

[0435] 3. Experimental Apparatus

[0436] Table 3. Instrument Information

[0437] 4 Experimental Methods

[0438] (1) Cell plating

[0439] MDA-MB-231 cells were cultured in the appropriate culture medium, digested with trypsin, centrifuged, resuspended and counted, and then plated in 96-well plates at an appropriate concentration. The cells were then cultured at 37°C in a 5% CO2 cell culture incubator for 24 hours.

[0440] (2) In vitro cell viability detection

[0441] Replace the old medium with 100 μL of fresh Opti medium containing different concentrations (0, 0.1, 1, 5, 10, 20, 50, 100 nM) of the test compound. After culturing the 96-well cell plate back in the incubator for 60 h, add 10 μL of CCK-8 solution to each well and incubate for 4 h. Measure the absorbance at 450 nm using a microplate reader.

[0442] 5. Data Analysis and Results

[0443] Using the Origin analysis results, the half-maximal inhibitory concentration (IC50) of the compound was calculated. 50 The test results are shown in Table 4.

[0444] Table 4. Inhibitory activity of MDA-MB-231 cells on proliferation

[0445] The data in Table 4 show that the compound of this invention has better inhibitory activity on the proliferation of MDA-MB-231 cells than the control compound Dxd.

[0446] II. Antibody-Drug Conjugation Assay

[0447] 2.1. Preparation of AL-13, an ADC drug example

[0448] The N297Q mutant anti-LILRB2 human IgG antibody (6.4 mg / mL, 4.0 mL) was replaced with 50 mM phosphate buffer (pH 7.4) by high-speed centrifugation. The antibody was mixed with 50 molar amounts of compound 52 (25 mM, 333 μL, dissolved in DMSO), and microbial transglutaminase (mTG, 62.5 U, Nanjing Duli) was added. The mixture was reacted at 37°C for 24 hours. The product was desalted and purified using a PD-10 column (Sephadex G25, Cytiva) to obtain an ADC with a final concentration of 2.7 mg / mL and a total volume of 6.7 mL.

[0449] The conjugates were characterized by SEC-HPLC and UHPLC-HRMS. The polymer content was 1.06%, the weighted average DAR value was 3.82, and the mass increase was approximately 4551 Da.

[0450] 2.2. Preparation of AL-26, an ADC drug example

[0451] The same antibody (3.8 mg / mL, 530 μL) was reacted with compound 47 (25 mM, 27 μL, dissolved in DMSO) at a 50-fold molar ratio. The amount of mTG added was 5 U. The reaction conditions and purification methods were the same as above, and the final ADC was obtained with a concentration of 1.48 mg / mL and a total volume of 1.0 mL.

[0452] Characterization results by SEC-HPLC and UHPLC-HRMS showed that the polymer ratio was 2.23%, the weighted average DAR value was 3.85, and the mass increase was approximately 4784 Da.

[0453] 2.3. Preparation of TL-13, an ADC drug example

[0454] The commercially available humanized anti-HER2 antibody Trastuzumab (Henlius Biotech) The 6.0 mg / mL, 5.0 mL solution was replaced with 50 mM phosphate buffer (pH 7.4) using ultrafiltration. 50 molar amounts of mTG enzyme substrate compound 52 (25 mM, prepared in DMSO) were added, followed by microbial transglutaminase (mTG, 50 U, Nanjing Duli Biotechnology). The mixture was incubated at 37°C for 24 hours. After the reaction, the solution was desalted and purified using a PD-10 column (Sephadex G25, Cytiva) with 50 mM phosphate buffer (pH 7.4) as the eluent, yielding an ADC candidate with a final concentration of 2.5 mg / mL and a total volume of 6.2 mL.

[0455] Aggregate content was analyzed using SEC-HPLC, and coupling efficiency was analyzed using high-resolution mass spectrometry (UHPLC-HRMS). The results are as follows: aggregate percentage: 3.2%, weighted average drug-to-analyte ratio (DAR): 3.96, and average molecular weight increase of the coupling product: ~4550 Da.

[0456] III. Intra-antibody drug conjugate pharmacological experiments

[0457] 3.1. ADC Drug Example: In Vivo Pharmacodynamic Study of AL-26

[0458] Experimental model:

[0459] This experiment used a humanized PBMC-A375 xenograft mouse model (Hu-PBMC / A375) to evaluate the antitumor activity of the anti-LILRB2 ADC drug AL-26. A375 melanoma cells (4 × 10⁻⁶) were used. 6 (1 cell / mouse) was mixed with human peripheral blood mononuclear cells and subcutaneously injected into the right back of SCID immunodeficient mice. The number of animals in each group was n=6.

[0460] Drug treatment and dosing regimen:

[0461] When the tumor volume reaches approximately 100–150 mm 3 Drug intervention was initiated at that time. Four treatment groups were established: a PBS control group, a homotype IgG control group, an anti-isotype-Topo1 (Topo1-conjugated homotype IgG) group, and an anti-LILRB2-Topo1 (AL-26) group. All ADC formulations were administered via tail vein injection at a dose of 10 mg / kg, on days 10, 13, 17, and 20 post-tumor inoculation, for a total of four times.

[0462] Results analysis:

[0463] Tumor volume was measured with calipers and recorded every 2–3 days, and calculated using the following formula:

[0464] Tumor volume=(length×width2) / 2

[0465] As shown in Figure 1, the anti-LILRB2-Topo1 (AL-26) group exhibited significant tumor inhibition during the dosing cycle, and the tumor volume in this group remained close to the initial level throughout the observation period, with no significant growth trend. In contrast, the PBS group and the isotype control group showed rapid tumor growth, while the anti-isotype-Topo1 group, although showing some degree of slowing, was not as effective as the anti-LILRB2-Topo1 group.

[0466] Statistical analysis:

[0467] Tumor volume was assessed at the end of day 24. The anti-LILRB2-Topo1 group showed significant differences compared with all other groups (p<0.01), indicating that the ADC drug has excellent anti-tumor activity in this model.

[0468] in conclusion:

[0469] These results indicate that AL-26, as a Topo1-conjugated anti-LILRB2 ADC drug, can significantly inhibit tumor growth in the PBMC humanized A375 model, demonstrating its potential to activate tumor killing in the immune microenvironment.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, in, R 1 It can be F, Cl, or CH3; R 2 For F; X is C; Y is NH, O, or α-NHCOC(CR) a R b Ob; end a is connected to the ring, and end b is connected to H; R 3 and R 4 Independently, it is H, F, C1-C5 alkyl or -OC1-C5 alkyl; and R 3 and R 4 Not both H; R a and R b It is independently H, C1-C5 alkyl, or cyclopropyl; or, X is O; R 3 and R 4 It does not exist; Y is O or a-OCOC(CR) a R b Ob; end a is connected to the ring, and end b is connected to H.

2. The compound of formula (I) as claimed in claim 1, characterized in that, The compound represented by formula (I) or a pharmaceutically acceptable salt thereof is a compound represented by formula (I-1), formula (I-2), formula (I-3) or formula (I-4) or a pharmaceutically acceptable salt thereof: In equations (I-1), (I-2), (I-3), and (I-4), R 1 R 2 R 3 R 4 The definitions of X and Y are as described in claim 1.

3. The compound of formula (I) as claimed in claim 1, characterized in that, It satisfies at least one of the following conditions: (1)R 1 It is Cl or CH3; CH3 is preferred; (2)R 3 and R 4 Independently defined as F; (3) The C1-C5 alkyl group and -OC1-C5 alkyl group are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl; preferably methyl; (4)R a For H, R b It is H or cyclopropyl; (5) X is C; Y is NH, O or a-NHCOCCH2O-b, a-NHCOCH(cyclopropyl)Ob; the a end is connected to the ring, and the b end is connected to H; or, X is O; Y is O or a-OCOCCH2O-b.

4. The compound of formula (I) as claimed in claim 1, characterized in that, The compound represented by formula (I) has any of the following structures: The pharmaceutically acceptable salt of the compound represented by formula (I) has any of the following structures: trifluoroacetate, Trifluoroacetate.

5. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, Pc-(LD)p, in, D is the structural segment shown in the following formula D; Among them, R 1 R 2 R 3 R 4 The definitions of X and Y are as described in any one of claims 1-4; terminal 1 is connected to terminal L; L is the connector that connects Pc and D; Pc is an antibody or its antigen-binding fragment; p represents the average number of drug ligands connected, and p is selected from an integer or decimal from 1 to 20.

6. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 5, characterized in that, It satisfies at least one of the following conditions: (1) The compound shown in formula D has any of the following structures: (2) p is a decimal between 1, 2, 3, 4, 5, 6, 7, 8, or any number in between; (3) L is -AL 1 -L 2 -, in; A is the connector segment that connects to the PC, as shown below: n1, n2 and n3 are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; L 1 It is a polypeptide linker; it is an amino acid residue or a short peptide composed of 2-10 amino acid residues; the amino acid residues are natural amino acid residues or non-natural amino acid residues; L 2 The connector segment for the D-type connection is shown below: R l1 It is an H or C1-C5 alkyl group; (4) The Pc mentioned is an antibody or its antigen-binding fragment that targets tumor-specific surface antigens, immunosuppressive receptors or immune checkpoint molecules, tumor-associated neoantigens and tissue-specific antigens.

7. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 6, characterized in that, It satisfies at least one of the following conditions: (1) A is And the best (2) n1 can be 0, 1, 2 or 3 independently; preferably 0; (3) n2 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; preferably 6 or 12; (4) n3 can be 0, 1, 2, 3, 4, 5 or 6 independently; preferably 3 or 6; (5)L 1 It is a short peptide composed of amino acid residues Val, D-Val, Cit, Phe, Lys, Lys(Ac), Leu, Gly, Ala, Asn, Asp, Arg, or 2-10 amino acid residues selected from Val, Cit, Phe, Lys, Leu, Gly, Ala, Asn, and Asp; preferably L 1 Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val-Cit, Gly-Lys, Ala-Ala, Ala-Ala-Asn, Gly-Phe-Gly, (Gly)2-Phe-Gly (SEQ ID NO:13), Val-Lys, Glu-Val-Ala, Ala-Ala-Asn, Val-Lys-(Gly)2 (SEQ ID NO:14), Val-Lys, or Lys-Ala-Asn; preferably... Terminal 1 is connected to A, and terminal 2 is connected to L. 2 Connected; (6)L 2 for 1 end and L 1 Connected, with ends 2 connected to D; preferred (7) p is a decimal between 1, 2, 3, 4, or 1; (8) The antibody comprises a variable region and a constant region, wherein the amino acid residue at position N297 of the constant region corresponding to the constant region of the IgG antibody is Q; preferably, the constant region further comprises amino acid residues at positions L234 and L235 corresponding to the constant region of the IgG antibody being A, and / or amino acid residues at position P329 being Q; (9) The tumor-specific surface antigens are selected from: HER2, Claudin 18.2, EGFR, TROP2 and Nectin-4; (10) The immunosuppressive receptor or immune checkpoint molecule is selected from: LILRB2, PD-L1, TIGIT, VISTA, B7-H3 and TIM-3; (11) The tumor-associated neoantigens and tissue-specific antigens mentioned are selected from MUC1, Mesothelin, CD138 and GPC3; (12) The antibody has endocytosis capability, which causes the drug of the ADC to be released.

8. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 7, characterized in that, It satisfies at least one of the following conditions: (1) L has the following structure: Preferred, (2) p is a decimal between 3 and 4; (3) The antibody comprises a heavy chain variable region and a light chain variable region of an antibody targeting LILRB2; preferably, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.

9. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 8, characterized in that, The ligand drug conjugate is Wherein, n2 is 3, 6, or 12; preferably 6; p is 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0; Ab is an anti-LILRB2 antibody, the amino acid sequence of the heavy chain variable region of the anti-LILRB2 antibody is as shown in SEQ ID NO:7 or has at least 90% sequence identity with SEQ ID NO:7, and the amino acid sequence of the light chain variable region of the anti-LILRB2 antibody is as shown in SEQ ID NO:8; or has at least 90% sequence identity with SEQ ID NO:

8.

10. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 9, characterized in that, The ligand-drug conjugate is: Preferred, The asterisk (*) indicates that the atom at that location is either R or S chiral. Ab is an anti-LILRB2 antibody, wherein the amino acid sequence of the heavy chain of the anti-LILRB2 antibody is as shown in SEQ ID NO:9 or has at least 90% sequence identity with SEQ ID NO:9, and the amino acid sequence of the light chain of the anti-LILRB2 antibody is as shown in SEQ ID NO:10 or has at least 90% sequence identity with SEQ ID NO:

10.

11. A compound as shown in Formula II or a pharmaceutically acceptable salt thereof, H-A-L 1 -L 2 -D, A, L 1 L 2 The definition of D is as described in any one of claims 1-5; Preferably, the compound represented by Formula II has any of the following structures: More preferably, the compound shown in Formula II has any of the following structures:

12. An antibody or its antigen-binding fragment for preparing antibody-drug conjugates, characterized in that, The antibody or its antigen-binding fragment can specifically bind to the target antigen expressed on the cell surface; The target antigen is selected from tumor-specific surface antigens, immunosuppressive receptors or immune checkpoint molecules, tumor-associated neoantigens, and tissue-specific antigens. The antibody or its antigen-binding fragment includes a constant region, wherein the amino acid residue in the constant region at position N297 corresponding to the constant region of the IgG antibody is Q.

13. The antibody or its antigen-binding fragment as described in claim 12, characterized in that, It satisfies at least one of the following conditions: (1) The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively; preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8; or, the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:7; the light chain variable region comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

8. (2) The constant region also includes amino acid residues A at positions L234 and L235 corresponding to the constant region of IgG antibody, and / or amino acid residues Q at position P329; (3) The constant region is a constant region derived from IgG, IgA, IgM, IgE, IgD or their isotype antibodies; preferably a constant region of IgG1, IgG2, IgG3 or IgG4 antibodies; (4) The tumor-specific surface antigens are selected from: HER2, Claudin 18.2, EGFR, TROP2 and Nectin-4; (5) The immunosuppressive receptor or immune checkpoint molecule is selected from: LILRB2, PD-L1, TIGIT, VISTA, B7-H3 and TIM-3; (6) The tumor-associated neoantigens and tissue-specific antigens mentioned are selected from MUC1, Mesothelin, CD138 and GPC3; (7) The antibody has endocytosis capability, which causes the drug of the ADC to be released.

14. The antibody or its antigen-binding fragment as described in claim 13, characterized in that, It satisfies at least one of the following conditions: (1) The heavy chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:9, and the heavy chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:10; or, the heavy chain contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO:9, and the light chain contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

10. (2) The antibody or its antigen-binding fragment is an anti-LILRB2 antibody or its antigen-binding fragment.

15. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that, The antibody-drug conjugate comprises the following fragments: an antibody or its antigen-binding fragment as described in any one of claims 12 to 14, a linker unit, and a cytotoxic drug.

16. The antibody-drug conjugate as claimed in claim 15, or a pharmaceutically acceptable salt thereof, characterized in that, It satisfies at least one of the following conditions: (1) The connector unit is as defined in L in any one of claims 5-10, in the ligand-drug conjugate or a pharmaceutically acceptable salt thereof; (2) Its DAR is as defined in the ligand-drug conjugate or its pharmaceutically acceptable salt as described in any one of claims 5-10; (3) The cytotoxic drug is camptothecin and its derivatives; preferably, camptothecin, Dxd, eczema, SN-38 or the ligand drug conjugate or its pharmaceutically acceptable salt as defined in any one of claims 5-10, or the structural fragment of formula D shown in the definition.

17. A pharmaceutical composition comprising a compound as described in any one of claims 1-4 or a pharmaceutically acceptable salt thereof, a ligand drug conjugate as described in any one of claims 5-10 or a pharmaceutically acceptable salt thereof, or an antibody drug conjugate as described in claim 15 or 16 or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

18. Use of a substance in the preparation of a medicament for treating diseases associated with abnormal cell activity; wherein the substance is a compound as described in any one of claims 1-4 or a pharmaceutically acceptable salt thereof, a ligand-drug conjugate as described in any one of claims 5-10 or a pharmaceutically acceptable salt thereof, a pharmaceutical composition as described in claim 12, or an antibody-drug conjugate as described in claim 15 or 16 or a pharmaceutically acceptable salt thereof; The disease associated with the abnormal cell activity is preferably cancer, more preferably melanoma or breast cancer, and the breast cancer is more preferably triple-negative breast cancer.

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