Ligand-drug conjugate of avixatecan analogue as well as preparation method and application of ligand-drug conjugate

Abstract

The invention relates to a ligand-drug conjugate of an avixetecan analogue as well as a preparation method and application of the ligand-drug conjugate. Specifically, the invention provides a ligand-drug conjugate with a structure as shown in a formula (-D), a preparation method thereof, a pharmaceutical composition containing the ligand-drug conjugate, and application of the ligand-drug conjugate in preparation of drugs for treating cancers through receptor regulation. Wherein each substituent group in the general formula (-D) is as defined in the specification. # imgabs0 #

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201980049841.2, application date September 25, 2019, and invention name “Ligand-drug conjugates of isotecan analogs and preparation methods and applications thereof”. Technical Field

[0002] The present disclosure relates to a ligand-drug conjugate of a novel structure of exotecan (or exatecan) analogs. Specifically, the present disclosure relates to an antibody-drug conjugate of an exotecan analog containing a structural unit Y, a preparation method thereof, a pharmaceutical composition containing the conjugate, and the use of the conjugate or the pharmaceutical composition. Background Art

[0003] Chemotherapy remains one of the most important anti-cancer methods, including surgery, radiotherapy, and targeted therapy. Although there are many types of highly effective cytotoxins, there is little difference between tumor cells and normal cells, which limits the widespread clinical application of these anti-tumor compounds due to toxic side effects. Anti-tumor monoclonal antibodies are specific to tumor cell surface antigens, and antibody drugs have become the frontline drugs for anti-tumor treatment, but when antibodies are used alone as anti-tumor drugs, the efficacy is often unsatisfactory.

[0004] Antibody drug conjugates (ADCs) connect monoclonal antibodies or antibody fragments to biologically active cytotoxins through stable chemical linker compounds, making full use of the specificity of antibodies for binding to surface antigens of normal cells and tumor cells and the high efficiency of cytotoxins, while avoiding the defects of the former's low efficacy and the latter's excessive toxic side effects. This means that compared with traditional chemotherapy drugs, antibody drug conjugates can accurately bind to tumor cells and reduce the impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12: 329-332; DiJoseph JF, Armellino DC, (2004) Blood, 103: 1807-1814).

[0005] In 2000, the first antibody-drug conjugate, Mylotarg (gemtuzumab ozogamicin, Wyeth Pharmaceuticals Co., Ltd.), was approved by the U.S. FDA for the treatment of acute myeloid leukemia (Drugs of the Future (2000) 25 (7): 686; US4970198; US 5079233; US 5585089; US 5606040; US5693762; US 5739116; US 5767285; US 5773001).

[0006] In August 2011, Adcetris (brentuximab vedotin, Seattle Genetics) passed the US FDA fast-track review channel for the treatment of Hodgkin's lymphoma and relapsed anaplastic large cell lymphoma (Nat. Biotechnol (2003) 21 (7): 778-784; WO2004010957; WO2005001038; US7090843A; US7659241; WO2008025020). It is a new type of targeted ADC drug that can directly act on the target CD30 on lymphoma cells and then cause endocytosis, thereby inducing apoptosis of tumor cells.

[0007] Mylotarg and Adcetris are both targeted therapies for blood tumors, which have a relatively simpler tissue structure than solid tumors. In February 2013, Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the US FDA for the treatment of HER2-positive advanced or metastatic breast cancer patients who are resistant to trastuzumab (Tratuzumab, trade name: Herceptin) and paclitaxel (WO2005037992; US8088387). Kadcyla is the first ADC drug approved by the US FDA for the treatment of solid tumors.

[0008] There are several types of cytotoxic small molecules used in antibody-drug conjugates, one of which is camptothecin derivatives, which have antitumor effects by inhibiting topoisomerase I. Literature reporting the application of the camptothecin derivative exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione) in antibody-drug conjugates (ADCs) includes WO2014057687; Clinical Cancer Research (2016) 22(20): 5097-5108; Cancer Sci (2016) 107: 1039-1046. However, further development of ADC drugs with better efficacy is still needed. Summary of the invention

[0009] In order to improve the coupling effect of ligands, especially antibodies and drugs, the present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a structure shown in formula (-D):

[0010]

[0011] in:

[0012] Y is selected from -O-(CR a R b )m-CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b )m-、-O-CR 1 R 2 -、-NH-(CR a R b )m-CR 1 R 2 -C(O)- or -S-(CR a R b )m-CR 1 R 2 -C(O)-;

[0013] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group;

[0014] Or, R a and Rb Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0015] R 1 is selected from halogen, deuterated alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, aryl or heteroaryl;

[0016] R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group;

[0017] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0018] Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0019] Wherein, the wavy line in or -D represents a hydrogen atom, or is covalently linked to the linker unit or to the antibody that binds to the antigen expressed by the target cell;

[0020] m is an integer of 0-4.

[0021] In some embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, which is a compound represented by the general formula (-D1) or a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof:

[0022]

[0023] in:

[0024] R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0025] R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0026] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0027] The wavy line in Formula-D1 represents a hydrogen atom, or is covalently linked to a linker unit or to an antibody that binds to an antigen expressed by a target cell;

[0028] m is 0 or 1.

[0029] In some embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, which is a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof represented by the general formula (Pc-LY-Dr):

[0030]

[0031] in:

[0032] Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -、-O-CR 1 R 2 -、-NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-;

[0033] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group;

[0034] Or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0035] R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl;

[0036] R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group;

[0037] Or, R 1 and R 2Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0038] Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0039] m is an integer from 0 to 4;

[0040] n is 1 to 10 and can be an integer or a decimal;

[0041] Pc is a ligand; L is a linker unit.

[0042] In some embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided.

[0043] Where -Y- is -O-(CR a R b )m-CR 1 R 2 -C(O)-;

[0044] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group;

[0045] R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0046] R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0047] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0048] m is 0 or 1.

[0049] In some embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein the structural unit -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0050] R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0051] R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C3-6 Cycloalkyl;

[0052] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0053] m is 0 or 1.

[0054] In some embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein the structural unit -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0055] R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0056] R 2 is a hydrogen atom;

[0057] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0058] m is 0 or 1.

[0059] In some embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein the structural unit -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0060] R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0061] R 2 is a hydrogen atom;

[0062] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0063] m is 0.

[0064] In other embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein the structural unit -Y- is selected from:

[0065]

[0066]

[0067] In other embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the O end of -Y- is connected to the linker unit L.

[0068] In other embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided, which is a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof represented by the general formula (Pc-L-D1):

[0069]

[0070] in:

[0071] R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0072] R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0073] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0074] m is 0 or 1;

[0075] n is 1 to 10 and can be an integer or a decimal;

[0076] Pc is a ligand; L is a linker unit.

[0077] In another preferred embodiment of the present invention, according to the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of the present invention, n is 2 to 8, which can be an integer or a decimal; preferably 3 to 8, which can be an integer or a decimal.

[0078] In another preferred embodiment of the present invention, according to the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0079] L 1 Selected from -(succinimidyl-3-yl-N)-WC(O)-, -CH2-C(O)-NR3 -WC(O)- or -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0080] L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20; preferably a chemical bond;

[0081] L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0082] L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - or a chemical bond, wherein t is an integer from 1 to 6; preferably -NR 5 (CR 6 R 7 )t-;

[0083] R 3 , R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group;

[0084] R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group.

[0085] In other embodiments of the present disclosure, the ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound, wherein the linker unit L 1 Selected from -(succinimidyl-3-yl-N)-(CH2)s 1 -C(O)-, -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-, -(succinimidyl-3-yl-N)-(CH2CH2O)s 2 -CH2CH2-C(O)-, -CH2-C(O)-NR 3 -(CH2)s 3 -C(O)- or -C(O)-(CH2)s 4 C(O)-, where s 1 is an integer from 2 to 8, s 2 is an integer from 1 to 3, s 3 is an integer from 1 to 8, s 4 is an integer from 1 to 8; s 1 Preferably 5.

[0086] In other embodiments of the present disclosure, the ligand-drug conjugate or its pharmaceutically acceptable salt or solvent compound, wherein the linker unit L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 An integer from 6 to 12.

[0087] In other embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein L 4 Selected from -NR 5 (CR 6 R 7 )t-,R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, t is 1 or 2, preferably 2; L 4 Preferably, it is -NR 5 CR 6 R 7 -;L 4 More preferred is -NHCH2-.

[0088] In other embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0089] L 1 for s 1 is an integer from 2 to 8;

[0090] L 2 is a chemical bond;

[0091] L 3 is a tetrapeptide residue;

[0092] L 4 For-NR 5 (CR 6 R 7 )t-,R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, and t is 1 or 2.

[0093] In other embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0094] L 1 is -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-;

[0095] L 2 For-NR 4 (CH2CH2O)9CH2C(O)-;

[0096] L 3 is a tetrapeptide residue;

[0097] L 4 For-NR 5 (CR 6 R 7 )t-,R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, and t is 1 or 2.

[0098] In some other embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein the L 3The peptide residue is an amino acid residue formed by one, two or more amino acids selected from phenylalanine (E), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E), and aspartic acid (N); preferably, it is an amino acid residue formed by one, two or more amino acids selected from phenylalanine and glycine; more preferably, it is a tetrapeptide residue; and most preferably, it is a tetrapeptide residue of GGFG (glycine-glycine-phenylalanine-glycine).

[0099] In other embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein the linker unit -L-, wherein L 1 The end is connected to the ligand, L 4 Connect the Y end.

[0100] In other embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the -LY- is:

[0101]

[0102] L 1 Selected from -(succinimidyl-3-yl-N)-(CH2)s 1 -C(O)- or -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-;

[0103] L 2 For-NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 is an integer from 6 to 12;

[0104] L 3 is the tetrapeptide residue of GGFG;

[0105] R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0106] R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0107] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0108] R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0109] s 1 is an integer from 2 to 8; preferably 5;

[0110] m is an integer of 0-4.

[0111] In other embodiments of the present disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the -LY- is:

[0112]

[0113] Preferably:

[0114]

[0115] L 2 For-NR 4 (CH2CH2O)9CH2C(O)-;

[0116] L 3 is the tetrapeptide residue of GGFG;

[0117] R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0118] R 2 is selected from a hydrogen atom, a haloalkyl group or a C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0119] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0120] R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0121] m is an integer of 0-4.

[0122] In other embodiments of the present disclosure, the ligand-drug conjugate provided by the general formula (Pc-LY-Dr) or a pharmaceutically acceptable salt or solvent compound thereof, wherein -LY- is

[0123]

[0124] L 2 is a chemical bond;

[0125] L 3 is the tetrapeptide residue of GGFG;

[0126] R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0127] R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0128] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0129] R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0130] s 1 is an integer from 2 to 8; preferably 5;

[0131] m is an integer of 0-4.

[0132] Another aspect of the present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a structure represented by the formula (-LY-):

[0133]

[0134] It can be used to obtain a ligand-drug conjugate by connecting a drug to a ligand via a linker fragment;

[0135] in:

[0136] L 1 Selected from -(succinimidyl-3-yl-N)-(CH2)s 1 -C(O)- or -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-;

[0137] L 2 For-NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 is an integer from 1 to 20;

[0138] L 3 is the tetrapeptide residue of GGFG;

[0139] R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0140] R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0141] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0142] R 5 , R 6 or R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0143] s 1 is an integer from 2 to 8;

[0144] m is an integer of 0-4.

[0145] Another aspect of the present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a structure represented by the formula (-LY-):

[0146]

[0147] in:

[0148] L 2 is a chemical bond;

[0149] L 3 is the tetrapeptide residue of GGFG;

[0150] R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0151] R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0152] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0153] R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7are the same or different and are each independently a hydrogen atom or an alkyl group;

[0154] s 1 is an integer from 2 to 8;

[0155] m is an integer of 0-4.

[0156] In other embodiments of the present disclosure, a ligand-drug conjugate of the general formula (Pc-LY-Dr) or a pharmaceutically acceptable salt or solvent compound thereof is provided, which is a ligand-drug conjugate of the general formula (Pc-LY-Dr) a -Y-Dr) ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof:

[0157]

[0158] in:

[0159] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0160] L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 is an integer from 1 to 20;

[0161] L 3 is a peptide residue consisting of 2 to 7 amino acids, the amino acids may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available attachment point, the substituent being one or more independently selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0162] R 1 is selected from halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; preferably cycloalkylalkyl or cycloalkyl; more preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0163] R2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl; preferably hydrogen; or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0164] R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group;

[0165] R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group;

[0166] m is an integer from 0 to 4;

[0167] n is a non-zero integer or decimal between 0 and 10, preferably an integer or decimal between 1 and 10;

[0168] Pc is a ligand.

[0169] In other embodiments of the present disclosure, a ligand-drug conjugate of the general formula (Pc-La-Y-Dr) or a pharmaceutically acceptable salt or solvent compound thereof is provided, which is the general formula (Pc-L b -Y-Dr) ligand-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof:

[0170]

[0171] in:

[0172] s 1 is an integer from 2 to 8; preferably 5;

[0173] Pc, R 1 , R 2 , R 5 ~R 7 , m and n are as defined in the general formula (Pc-La-Y-Dr).

[0174] The linking unit -LY- of the ligand-drug conjugates disclosed herein includes, but is not limited to:

[0175]

[0176]

[0177] The ligand-drug conjugates shown in the general formula (Pc-LY-Dr) disclosed herein include, but are not limited to:

[0178]

[0179]

[0180]

[0181]

[0182] wherein Pc and n are as defined in the general formula (Pc-La-Y-Dr).

[0183] In other embodiments of the present disclosure, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the Pc is an antibody or an antigen-binding fragment thereof, the antibody is selected from a chimeric antibody, a humanized antibody or a fully human antibody; preferably a monoclonal antibody.

[0184] In other embodiments of the present disclosure, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or its antigen-binding fragment is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody or anti-Mesothelin antibody or its antigen-binding fragment.

[0185] In other embodiments of the present disclosure, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or its antigen-binding fragment is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 and Glematumamab, or an antigen-binding fragment thereof.

[0186] The ligand-drug conjugates represented by the general formula (Pc-LY-Dr) disclosed herein include, but are not limited to, the following structural formulas:

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] Wherein, n is a non-zero integer or decimal from 0 to 10, preferably an integer or decimal between 1 and 10; more preferably, it is from 2 to 8, which can be an integer or a decimal; and most preferably, it is from 3 to 8, which can be an integer or a decimal.

[0193] Another aspect of the present disclosure provides a compound represented by general formula (D) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

[0194]

[0195] in:

[0196] Y is selected from -O-(CR a R b )m-CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b )m-、-O-CR 1 R 2 -、-NH-(CR a R b )m-CR 1 R 2 -C(O)- or -S-(CR a R b )m-CR 1 R 2 -C(O)-;

[0197] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group;

[0198] Or, R a and R bTogether with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0199] R 1 is selected from halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl;

[0200] R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group;

[0201] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0202] Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0203] m is an integer of 0-4.

[0204] In a preferred embodiment of another aspect of the present disclosure, a compound represented by the general formula (D) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof is provided, which is a compound represented by the general formula (D1) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

[0205]

[0206] Where: R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0207] R 2 is selected from a hydrogen atom, a haloalkyl group or a C 3-6 Cycloalkyl;

[0208] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0209] m is 0 or 1.

[0210] The compounds represented by the general formula (D) described in the present disclosure include, but are not limited to:

[0211]

[0212]

[0213]

[0214]

[0215] or its tautomer, meso racemate, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt.

[0216] A preferred embodiment of another aspect of the present disclosure provides a general formula (L a -Y-Dr) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

[0217]

[0218] in

[0219] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0220] L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 An integer from 1 to 20;

[0221] L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl, and when substituted, the substituents may be substituted at any available attachment point, and the substituents are one or more independently selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0222] R 1 is selected from halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl;

[0223] R 2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl or heteroaryl; or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0224] R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group;

[0225] R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group;

[0226] m is an integer of 0-4.

[0227] A preferred embodiment of another aspect of the present disclosure provides a general formula (L a -Y-Dr) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, which is a compound represented by the general formula (L b -Y-Dr) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

[0228]

[0229] Where R 1 , R 2 , R 5 ~R 7 、s 1 and m as in the general formula (L a -Y-Dr) as defined.

[0230] The general formula (L a -Y-Dr) includes, but is not limited to:

[0231]

[0232]

[0233]

[0234]

[0235]

[0236] or its tautomer, meso racemate, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt.

[0237] Another aspect of the present disclosure provides a method for preparing a compound represented by general formula (D1) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0238]

[0239] The general formula (Y1) and the general formula (Dr) are condensed to obtain a compound represented by the general formula (D1).

[0240] Where: R 1 , R 2 and m are as defined in the general formula (D1).

[0241] Another aspect of the present disclosure provides a method for preparing a b -Y-Dr) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0242]

[0243] The general formula (IA) and the general formula (IB) are condensed to obtain the general formula (L b -Y-Dr) represented by the compound,

[0244] Where: R 1 , R 2 , R 5 ~R 7 、s 1 and m as in the general formula (L b -Y-Dr) as defined.

[0245] Another aspect of the present disclosure provides a method for preparing a b -Y-Dr) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0246]

[0247] The general formula (IA) and the general formula (IB) are subjected to condensation reaction to obtain the general formula (L b -Y-Dr) represented by the compound,

[0248] Where: R 1 , R2 , R 5 ~R 7 、s 1 and m as in the general formula (L b -Y-Dr) as defined.

[0249] Another aspect of the present disclosure provides a method for preparing a compound of the general formula (Pc-L a -Y-Dr) or a pharmaceutically acceptable salt or solvate thereof, comprising the following steps:

[0250]

[0251] After reducing Pc, a -Y-Dr) coupling reaction to obtain the general formula (Pc-L a -Y-Dr); the reducing agent is preferably TCEP;

[0252] in:

[0253] Pc is the ligand;

[0254] W.L. 2 , L 3 , R 1 , R 2 , R 5 ~R 7 , m and n are as in the general formula (Pc-L a -Y-Dr) as defined.

[0255] Another aspect of the present disclosure further relates to a pharmaceutical composition, which contains a therapeutically effective amount of the ligand-drug conjugate or compound as described in the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0256] Another aspect of the present disclosure further relates to a pharmaceutical composition, which contains a compound represented by the general formula (D) according to the present disclosure or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0257] Another aspect of the present disclosure further relates to a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, comprising a ligand and a drug connected to the ligand, wherein the drug is selected from the compound represented by the general formula (D) described in the present disclosure, the compound represented by the general formula (L a-Y-Dr), or its tautomer, mesomer, racemate, enantiomer, diastereoisomer, or mixture thereof, or its pharmaceutically acceptable salt, preferably the drug is connected to the ligand through a linker, and preferably the ligand is a monoclonal antibody.

[0258] Another aspect of the present disclosure further relates to a method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, comprising: treating a compound represented by the general formula (D) of the present disclosure, a compound represented by the general formula (L a -Y-Dr), or its tautomer, mesomer, racemate, enantiomer, diastereoisomer, or mixture thereof, or its pharmaceutically acceptable salt is connected to a ligand, preferably through a linker, and the ligand is preferably a monoclonal antibody.

[0259] Another aspect of the present disclosure further relates to the ligand-drug conjugate or compound described in the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof, for use as a drug.

[0260] Another aspect of the present disclosure further relates to the use of the ligand-drug conjugate or compound described in the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof in the preparation of a medicament for treating or preventing a tumor; preferably, wherein the tumor is a cancer associated with the expression of HER2, HER3 or EGFR.

[0261] Another aspect of the present disclosure further relates to the use of the ligand-drug conjugate or compound described in the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition in the preparation of a drug for treating and / or preventing cancer, wherein the cancer is preferably selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, prostate cancer or lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma or relapsed anaplastic large cell lymphoma).

[0262] Another aspect of the present disclosure further relates to a method for treating and / or preventing tumors, which comprises administering to a patient in need thereof a therapeutically effective dose of the ligand-drug conjugate or compound described in the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same; preferably, wherein the tumor is a cancer associated with HER2, HER3 or EGFR expression.

[0263] Another aspect of the present disclosure further relates to a method for treating or preventing cancer, the method comprising administering to a patient in need thereof a therapeutically effective dose of a ligand-drug conjugate or compound of the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same; the cancer is preferably selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer or lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma or relapsed anaplastic large cell lymphoma).

[0264] The active compound may be formulated for administration by any appropriate route, preferably in a unit dosage form, or in a form in which the patient can self-administer a single dose. The unit dosage form of the compound or composition of the present invention may be tablets, capsules, cachets, bottled liquids, powders, granules, lozenges, suppositories, reconstituted powders or liquid preparations.

[0265] The dosage of the compound or composition used in the treatment method of the present invention will generally vary with the severity of the disease, the patient's weight and the relative efficacy of the compound. However, as a general guide, a suitable unit dosage may be 0.1 to 1000 mg.

[0266] The pharmaceutical composition of the present invention may contain one or more excipients in addition to the active compound, and the excipients are selected from the following ingredients: fillers (diluents), binders, wetting agents, disintegrants or excipients, etc. Depending on the administration method, the composition may contain 0.1 to 99% by weight of the active compound.

[0267] The pharmaceutical composition containing the active ingredient may be in a form suitable for oral administration, such as tablets, troches, lozenges, water or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Oral compositions may be prepared according to any known method for preparing pharmaceutical compositions in the art, such compositions may contain binders, fillers, lubricants, disintegrants or pharmaceutically acceptable wetting agents, etc., and such compositions may also contain one or more ingredients selected from the following: sweeteners, flavoring agents, colorants and preservatives to provide pleasing and palatable pharmaceutical preparations.

[0268] Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Aqueous suspensions may also contain one or more preservatives, one or more colorants, one or more flavoring agents and one or more sweetening agents.

[0269] Oil suspensions can be prepared by suspending the active ingredient in a vegetable oil. The oil suspension may contain a thickener. The above-mentioned sweeteners and flavoring agents may be added to provide a palatable preparation.

[0270] The pharmaceutical composition can also be provided as dispersible powders and granules for preparing aqueous suspensions, wherein the active ingredient is mixed with one or more of a dispersant, a wetting agent, a suspending agent or a preservative by adding water. Other excipients such as sweeteners, flavoring agents and coloring agents may also be added. These compositions may be preserved by adding antioxidants such as ascorbic acid.

[0271] The pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions.

[0272] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable solvents or solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerol and processed to form a microemulsion. The injection or microemulsion may be injected into the patient's bloodstream by local mass injection. Alternatively, it is preferred that the solution and microemulsion be administered in a manner that maintains a constant circulating concentration of the disclosed compound. To maintain such a constant concentration, a continuous intravenous drug delivery device may be used. An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous injection pump.

[0273] The pharmaceutical composition can be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration. The suspension can be prepared by known techniques using suitable dispersants or wetting agents and suspending agents as described above. Sterile injection preparations can also be sterile injection solutions or suspensions prepared in parenteral acceptable nontoxic diluents or solvents. In addition, sterile fixed oils can be conveniently used as solvents or suspension media.

[0274] The disclosed compounds may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions may be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid in the rectum and thus dissolves in the rectum to release the drug. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights, and mixtures of fatty acid esters of polyethylene glycol.

[0275] As is well known to those skilled in the art, the dosage of a drug depends on a variety of factors, including but not limited to the following factors: the activity of the specific compound used, the age of the patient, the weight of the patient, the health status of the patient, the behavior of the patient, the diet of the patient, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, etc.; in addition, the optimal treatment method such as the mode of treatment, the daily dosage of the compound of formula (I) or the type of pharmaceutically acceptable salt can be verified according to traditional treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0276] Figure 1A : The plasma stability experimental results of ADC-19 disclosed in the present invention.

[0277] Figure 1B : The plasma stability experimental results of ADC-18 disclosed in the present invention.

[0278] Figure 1C : The plasma stability experimental results of ADC-20 disclosed in the present invention.

[0279] Figure 2 : The present invention discloses the efficacy evaluation of ADC-21 and ADC-24 on JIMT-1 tumor-bearing mice.

[0280] Figure 3 : Evaluation of the efficacy of the disclosed ADC on nude mice transplanted with human breast cancer cells SK-BR-3.

[0281] Figure 4 : The plasma stability experimental results of ADC-25 disclosed in the present invention.

[0282] Figure 5 :The efficacy of the disclosed ADC on human brain astroglioma U87MG nude mouse transplanted tumors.

[0283] Figure 6 : The efficacy of the disclosed ADC on Detroit 562 nude mouse transplanted tumors of human pharyngeal carcinoma pleural effusion metastatic cells.

[0284] Figure 7 :The efficacy of the ADC disclosed herein on human glioma U87MG nude mouse transplanted tumors. DETAILED DESCRIPTION

[0285] Detailed description of the invention

[0286] Unless otherwise defined, all technical and scientific terms used herein are consistent with the common understanding of one of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein may be used to implement or test the present disclosure, preferred methods and materials are described herein. When describing and claiming the present disclosure, the following terms are used in accordance with the following definitions.

[0287] When a trade name is used in this disclosure, Applicants intend to include formulations of the trade name product, generic drugs, and the active drug portion of the trade name product.

[0288] Unless stated otherwise, the terms used in the specification and claims have the following meanings.

[0289] The term "ligand" is a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell. The role of a ligand is to present the drug to a target cell population that binds to the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells. In the embodiments of the present disclosure, the ligand is represented by Pc, and the ligand can form a connecting bond with the connecting unit through a heteroatom on the ligand, preferably an antibody or an antigen-binding fragment thereof, wherein the antibody is selected from a chimeric antibody, a humanized antibody, a fully human antibody, or a mouse antibody; preferably a monoclonal antibody.

[0290] The term "drug" refers to cytotoxic drugs, which are represented by Dr. They are chemical molecules that have a strong ability to destroy the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells in principle at sufficiently high concentrations, but due to the lack of specificity, while killing tumor cells, they can also cause apoptosis of normal cells, leading to serious side effects. The term includes toxins, such as small molecule toxins or enzyme-active toxins from bacteria, fungi, plants or animals, radioactive isotopes (such as At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 and radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.

[0291] The term "linker unit" or "linker fragment" or "linker unit" refers to a chemical structure fragment or bond that is connected to a ligand at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug. The preferred embodiment of the present disclosure is represented by L and L 1 To L 4 , where L 1 The end is connected to the ligand, L 4 The end is connected to the structural unit Y and then connected to the drug (Dr).

[0292] The linker, including the extension, the spacer and the amino acid unit, can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker can be a "cleavable linker" that facilitates the release of the drug in the cell. For example, an acid-labile linker (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).

[0293] The term "ligand-drug conjugate" refers to a ligand connected to a biologically active drug via a stable linker. In the present disclosure, "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment connected to a biologically active toxic drug via a stable linker.

[0294] The three letter codes and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0295] The term "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain respectively. The same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains or λ chains according to the difference in constant regions. Each of the five classes of Ig can have κ chains or λ chains. The antibodies disclosed herein are preferably specific antibodies against cell surface antigens on target cells, and non-limiting examples thereof are the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis antibody Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody or anti-Mesothelin antibody or more; preferably Trastuzumab (Trastuzumab, trade name Herceptin), Pertuzumab (Pertuzumab, also known as 2C4, trade name Perjeta), Nimotuzumab (Nimotuzumab, trade name Taixinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 and Glematumamab.

[0296] The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0297] The antibodies disclosed herein include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

[0298] The term "murine antibody" in this disclosure refers to antibodies prepared in mice according to the knowledge and skills in the art. When preparing, a test subject is injected with a specific antigen, and then a hybridoma expressing an antibody with the desired sequence or functional characteristics is isolated.

[0299] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region gene from the mouse hybridoma cells, and then clone the constant region gene of the human antibody as needed, connect the mouse variable region gene with the human constant region gene into a chimeric gene, and then insert it into an expression vector, and finally express the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.

[0300] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline antibody framework sequences. It can overcome the heterologous response induced by chimeric antibodies due to carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (on the Internet). www.mrccpe.com.ac.uk / vbaseAvailable), and in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies after CDR affinity maturation is further performed by phage display. Literature further describing methods for humanization using mouse antibodies includes, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and Winter and colleagues [Jones et al., Nature, 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].

[0301] The terms "fully human antibody", "fully human antibody" or "completely human antibody", also known as "fully human monoclonal antibody", are antibodies whose variable and constant regions are both human, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The present disclosure is a fully human monoclonal antibody. The relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.

[0302] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody. Examples of binding fragments included in "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge on the hinge region, (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody; (v) single domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) combinations of two or more isolated CDRs, optionally connected by synthetic linkers. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, so that they can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies can be of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0303] Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, among fragments obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which about half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.

[0304] F(ab')2 is an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, obtained by digesting the portion below two disulfide bonds in the hinge region of IgG with the enzyme pepsin, and comprising two Fab regions linked at the hinge position.

[0305] Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F(ab')2.

[0306] Furthermore, the Fab' fragment of the antibody can be produced by inserting a DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic organism or a eukaryotic organism to express the Fab'.

[0307] The term "single-chain antibody", "single-chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using variants of 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0308] The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDR applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain.

[0309] The term "antibody framework" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

[0310] The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-continuous amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0311] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than about 10 -8 M, 10 -9 M or 10 -10 Binds with an affinity (KD) of M or less.

[0312] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.

[0313] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA segments can be connected. In another embodiment, the vector is a viral vector, in which other DNA segments can be connected to the viral genome. The vector disclosed herein can be autonomously replicated in the host cell introduced therein (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome (e.g., non-additional mammalian vectors).

[0314] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art, such as the Cold Spring Harbor Guide to Antibody Experimental Techniques, Chapters 5-8 and 15. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR region. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulins, 2001 ISBN012441351.

[0315] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plants or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary Cell Line) and NS0 cells.

[0316] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0317] The term "peptide" refers to a compound fragment between amino acids and proteins. It is composed of two or more amino acid molecules connected to each other by peptide bonds. It is a structural and functional fragment of protein. Hormones, enzymes, etc. are essentially peptides.

[0318] The term "sugar" refers to a biological macromolecule composed of three elements: C, H, and O, which can be divided into monosaccharides, disaccharides, and polysaccharides.

[0319] The term "fluorescent probe" refers to a class of fluorescent molecules that have characteristic fluorescence in the ultraviolet-visible-near infrared region, and whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime and polarization, etc.) can change sensitively with the properties of the environment, such as polarity, refractive index, viscosity, etc. They non-covalently interact with nucleic acids (DNA or RNA), proteins or other macromolecular structures to change one or several fluorescence properties, and can be used to study the properties and behaviors of macromolecules.

[0320] The term "toxic drug" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. It includes toxins and other compounds that can be used for tumor treatment.

[0321] The term "toxin" refers to any substance that can have a harmful effect on the growth or proliferation of cells, and can be a small molecule toxin and its derivatives from bacteria, fungi, plants or animals, including camptothecin derivatives such as isatecan, maytansinoids and their derivatives (CN101573384) such as DM1, DM3, DM4, auristatin F (AF) and its derivatives, such as MMAF, MMAE, 3024 (WO 2016 / 127790 A1, compound 7), diphtheria toxin, exotoxin, ricin A chain, abrin A chain, modeccin, α-sarcin, Aleurites fordii toxin, dianthin toxin, Phytolaca americana toxin (PAPI, PAPII and PAP-S), Momordica charantia (Momordica charantia) toxin, and safflower toxin. charantia inhibitors, curcin, crotin, sapaonaria officinalis inhibitors, gelonin, mitogellin restrictocin, phenomycin, enomycin and the trichothecenes.

[0322] The term "chemotherapeutic agent" is a chemical compound that can be used to treat tumors. The definition also includes anti-hormonal agents that act to modulate, reduce, block or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or systemic treatment. They can themselves be hormones. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa; cyclosphamide (CYTOXAN); TM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benaodopa, carboquone, meturedopa and uredopa; aziridines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustards mustards such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, oxazolidinone hydrochloride; melphalan, novembichin, cholesteryl phenylacetic acid mustard, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclarubicin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, chromomycin, carzinophilin, chromomycin, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin;Streptozocin, tuberculin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; pteridine analogs such as fludarabine, 6-mercaptophenone, thioguanpterin; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxitluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanol propionate, propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglucose; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; biasntrene; edatraxate; defofamine; colchicine; diaziquone; elfomithine; elliptinium acetate; acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pintostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tricholorthosporic acid; triazoquinone; 2,2',2"-trichlorrotriethylamine; urethan; vinblastine amide; dacarbazine; mannitol mustard; mitobronitol; dibromodulanol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel ( Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel ( Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfant; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above substances. This definition also includes antihormonal agents that can modulate or inhibit the effects of hormones on tumors, such as antiestrogens including tamoxifen, raloxifene, the aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and fareston; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0323] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof. More preferred are lower alkyl groups having 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0324] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.

[0325] The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of a parent alkane by removing two hydrogen atoms, which is a straight or branched group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, and more preferably an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-), etc. The alkylene group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0326] The term "alkoxy" refers to-O-(alkyl) and-O-(non-substituted cycloalkyl), wherein the definition of alkyl or cycloalkyl is as described above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or non-substituted, and when substituted, substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0327] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyls include spirocyclic, fused and bridged cycloalkyls.

[0328] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O). m(wherein m is an integer from 0 to 2) heteroatoms, but does not include -OO-, -OS- or -SS- ring parts, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups.

[0329] The term "spiro heterocyclyl" refers to a polycyclic heterocyclic group in which the monocyclic rings of 5 to 20 members share one atom (called a spiro atom), wherein one or more of the ring atoms is selected from nitrogen, oxygen or S(O) m (wherein m is an integer 0 to 2) heteroatom, and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of shared spiral atoms between rings, the spiral heterocyclic group is divided into a single spiral heterocyclic group, a double spiral heterocyclic group or a multi-spiro heterocyclic group, preferably a single spiral heterocyclic group and a double spiral heterocyclic group. More preferably, it is a 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6 yuan single spiral heterocyclic group. Non-limiting examples of spiral heterocyclic groups include:

[0330]

[0331] The term "fused heterocyclyl" refers to a polycyclic heterocyclic group of 5 to 20 members, each ring in the system shares a pair of adjacent atoms with other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. Preferably 6 to 14 members, more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic group, preferably a bicyclic or tricyclic, more preferably a 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic group. Non-limiting examples of fused heterocyclic groups include:

[0332]

[0333] The term "bridged heterocyclic group" refers to a polycyclic heterocyclic group of 5 to 14 members, any two rings sharing two atoms that are not directly connected, which may contain one or more double bonds, but none of the rings has a completely conjugated π-electron system, wherein one or more of the ring atoms is selected from nitrogen, oxygen or S(O) m(wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. Preferably 6 to 14 members, more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

[0334]

[0335] The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:

[0336]

[0337] The heterocyclic group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0338] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, non-limiting examples of which include:

[0339]

[0340] The aryl group may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0341] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl is preferably 5 to 10 members, more preferably 5 or 6 members, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:

[0342]

[0343]

[0344] The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0345] The term "amino protecting group" is to protect the amino group with a group that is easily removed in order to keep the amino group unchanged when other parts of the molecule react. Non-limiting examples include 9-fluorenylmethoxycarbonyl, tert-butyloxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl. These groups can be optionally substituted with 1-3 substituents selected from halogen, alkoxy or nitro. The amino protecting group is preferably 9-fluorenylmethoxycarbonyl.

[0346] The term "cycloalkylalkyl" refers to an alkyl group substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein alkyl is as defined above, and wherein cycloalkyl is as defined above.

[0347] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0348] The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

[0349] The term "hydroxy" refers to an -OH group.

[0350] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0351] The term "amino" refers to -NH2.

[0352] The term "nitro" refers to -NO2.

[0353] The term "amido" refers to -C(O)N(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0354] The term "carboxylate" refers to -C(O)O(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0355] The present disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of compounds of formula (I) with reference to relevant literature. Commercially available deuterated starting materials can be used when preparing deuterated forms of compounds of formula (I), or they can be synthesized using deuterated reagents using conventional techniques, and deuterated reagents include but are not limited to deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide, etc.

[0356] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0357] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and the skilled person can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (e.g. olefinic) bonds.

[0358] The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0359] The term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" refers to a salt of the ligand-drug conjugate of the present disclosure, or a salt of a compound described in the present disclosure. Such salts are safe and effective when used in mammals and have the desired biological activity. The antibody-antibody drug conjugate compound of the present disclosure contains at least one amino group and can therefore form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0360] The term "solvate" or "solvate compound" refers to a ligand-drug conjugate compound of the present disclosure that forms a pharmaceutically acceptable solvate with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate.

[0361] The term "drug loading" refers to the average number of cytotoxic drugs loaded on each ligand in the molecule of formula (I), and can also be expressed as the ratio of the amount of drug to the amount of antibody. The range of drug loading can be 0-12, preferably 1-10 cytotoxic drugs (D) connected to each ligand (Pc). In an embodiment of the present invention, the drug loading is expressed as n, which can be exemplified by the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The average number of drugs per ADC molecule after the coupling reaction can be identified by conventional methods such as UV / visible light spectroscopy, mass spectrometry, ELISA test and HPLC characteristics.

[0362] In one embodiment of the present disclosure, the cytotoxic drug is coupled to the N-terminal amino group of the ligand and / or the ε-amino group of the lysine residue through a linker. Generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum value.

[0363] The loading of the ligand cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:

[0364] (1) Control the molar ratio of the linking reagent and the monoclonal antibody,

[0365] (2) Control the reaction time and temperature,

[0366] (3) Select different reaction reagents.

[0367] The preparation of conventional pharmaceutical compositions is described in the Chinese Pharmacopoeia.

[0368] The term "carrier" is used for the drugs disclosed herein and refers to a system that can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the targeted organ. The drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure, and preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, and can be used as excellent drug carriers.

[0369] The term "excipient" refers to the additives in pharmaceutical preparations other than the main drug, which can also be called excipients. For example, the binders, fillers, disintegrants, and lubricants in tablets; the matrix part in semi-solid preparations such as ointments and creams; the preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid preparations can all be called excipients.

[0370] The term "diluent" is also called filler, and its main purpose is to increase the weight and volume of the tablet. The addition of diluent not only ensures a certain volume, but also reduces the dosage deviation of the main ingredients and improves the compression molding of the drug. When the drug in the tablet contains oily components, an absorbent needs to be added to absorb the oily substance and keep it in a "dry" state to facilitate tableting. Such as starch, lactose, inorganic salts of calcium, microcrystalline cellulose, etc.

[0371] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Among the acceptable solvents and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerol and processed to form a microemulsion. The injection or microemulsion may be injected into the patient's bloodstream by local mass injection. Alternatively, it is preferred that the solution and microemulsion be administered in a manner that maintains a constant circulating concentration of the disclosed compound. To maintain such a constant concentration, a continuous intravenous drug delivery device may be used. An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous injection pump.

[0372] The pharmaceutical composition can be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration. The suspension can be prepared according to known techniques with suitable dispersants or wetting agents and suspending agents as described above. Sterile injection preparations can also be sterile injection solutions or suspensions prepared in non-toxic parenterally acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. In addition, sterile fixed oils can be conveniently used as solvents or suspension media. For this purpose, any blended fixed oils including synthetic mono- or diglycerides can be used. In addition, fatty acids such as oleic acid can also be used to prepare injections.

[0373] The present disclosure relates to a type of cleavable linker with a specific structure and an active substance with a specific structure, and an antibody-drug conjugate (ADC) composed of the linker, the active substance and the antibody. This type of ADC is a complex formed by connecting a toxic substance to an antibody via a spacer. The antibody-drug conjugate (ADC) is degraded in the body to release active molecules, thereby playing an anti-tumor role.

[0374] Synthesis method disclosed herein

[0375] In order to achieve the synthesis purpose of the present disclosure, the present disclosure adopts the following synthesis technology scheme:

[0376] Option 1:

[0377] The present invention discloses a method for preparing a compound represented by general formula (D1) or a pharmaceutically acceptable salt or solvate thereof, the method comprising:

[0378]

[0379] The compound of the general formula (Y1) and the compound of the general formula (Dr) react in the presence of a condensing agent, optionally under alkaline conditions, to obtain the compound of the general formula (D1),

[0380] Where: R 1 , R 2 and m are as defined in the general formula (D1).

[0381] Reagents providing alkaline conditions include organic bases and inorganic bases. The organic bases include but are not limited to triethylamine, diethylamine, N-methylmorpholine, pyridine, piperidine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide. The inorganic bases include but are not limited to sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide.

[0382] The condensing agent can be selected from 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N , N',N'-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, preferably 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride or 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0383] Option 2:

[0384] The present disclosure generally has the formula (L b -Y-Dr) or a pharmaceutically acceptable salt or solvate thereof, the method comprising:

[0385]

[0386] In the first step, the compound of the general formula (IB-1) and exotecan mesylate (1b) are reacted in the presence of a condensing agent, optionally under alkaline conditions, to obtain a compound of the general formula (IB-2);

[0387] The second step is to remove the protecting group of the compound of the general formula (IB-2) to obtain the compound of the general formula (IB);

[0388] In the third step, the compound of formula (IA) and the compound of formula (IB) are reacted in the presence of a condensing agent, optionally under alkaline conditions, to obtain a compound of formula (L b -Y-Dr),

[0389] in:

[0390] R c is an amino protecting group; preferably 9-fluorenylmethoxycarbonyl (Fmoc);

[0391] R 1 , R 2 , R 5 ~R 7 、s 1 and m as in the general formula (L b -Y-Dr) defined.

[0392] Reagents providing alkaline conditions include organic bases and inorganic bases. The organic bases include but are not limited to triethylamine, diethylamine, N-methylmorpholine, pyridine, piperidine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide. The inorganic bases include but are not limited to sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide.

[0393] The condensing agent is selected from 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methyl morpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N, N',N'-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, preferably 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride or 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0394] Option 3:

[0395] The present disclosure has the general formula (Pc-L a -Y-Dr) shown in the method, which comprises the following steps:

[0396]

[0397] After reducing Pc, a -Y-Dr) reaction to obtain the general formula (Pc-L a -Y-Dr); the reducing agent is preferably TCEP, in particular, preferably reducing the disulfide bonds on the antibody;

[0398] in:

[0399] Pc is the ligand;

[0400] W.L. 2 , L 3 , R 1 , R 2 , R 5 ~R 7 , m and n are as in the general formula (Pc-L a -Y-Dr) as defined.

[0401] The present disclosure is further described below in conjunction with embodiments, but these embodiments are not intended to limit the scope of the present disclosure.

[0402] The experimental methods without specific conditions in the examples disclosed herein are usually carried out under conventional conditions or under conditions recommended by raw material or product manufacturers. Reagents without specific sources are conventional reagents purchased from the market.

[0403] Example

[0404] The structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR was measured using a Bruker AVANCE-400 NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD) as the solvent, tetramethylsilane (TMS) as the internal standard, and chemical shifts at 10 -6 (ppm) is given as the unit.

[0405] MS was measured using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQadvantage MAX).

[0406] UPLC was measured using a Waters Acquity UPLC SQD liquid spectrometer.

[0407] HPLC analysis was performed using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150×4.6 mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150×4.6 mm column).

[0408] UV-HPLC was measured using a Thermo nanodrop 2000 ultraviolet spectrophotometer.

[0409] Proliferation inhibition rate and IC 50 The values ​​were determined using PHERA starFS microplate reader (BMG, Germany).

[0410] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The silica gel plate used in thin layer chromatography (TLC) adopts a specification of 0.15mm-0.2mm, and the specification of the thin layer chromatography separation and purification product adopts a silica gel plate of 0.4mm-0.5mm.

[0411] Column chromatography generally uses Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0412] The known starting materials disclosed herein can be synthesized by methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organnics, Aldrich Chemical Company, AccelaChemBio Inc, Darui Chemicals, and other companies.

[0413] Unless otherwise specified in the examples, all reactions were carried out under argon or nitrogen atmosphere.

[0414] Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1L.

[0415] Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

[0416] The pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or a HC2-SS hydrogenator.

[0417] The hydrogenation reaction is usually carried out by evacuating the vacuum, filling with hydrogen, and repeating the operation three times.

[0418] The microwave reaction was carried out using a CEM Discover-S 908860 microwave reactor.

[0419] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0420] Unless otherwise specified in the examples, the reaction temperature is room temperature.

[0421] Room temperature is the most suitable reaction temperature, and the temperature range is 20℃~30℃.

[0422] Preparation of PBS buffer with pH=6.5 in the embodiment: put 8.5g KH2PO4, 8.56g K2HPO4.3H2O, 5.85g NaCl and 1.5g EDTA in a bottle, dilute to 2L, dissolve completely by ultrasonication and shake well.

[0423] The eluent system for column chromatography and the developing solvent system for thin layer chromatography used for purifying compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, C: petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine and acidic or alkaline reagents can also be added for adjustment.

[0424] Some compounds of the present disclosure were characterized by Q-TOF LC / MS, which used an Agilent 6530 accurate mass quadrupole-time of flight mass spectrometer and an Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 μm, 2.1×75 mm column).

[0425] Example 1

[0426] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzene

[0427] [de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide 1

[0428]

[0429] 1 mL of N,N-dimethylformamide was added to exitecan mesylate 1b (2.0 mg, 3.76 μmol, prepared by the method disclosed in patent application "EP0737686A1"), cooled to 0-5°C in an ice-water bath, and a drop of triethylamine was added, and stirred until the reaction liquid became clear. 1-Hydroxycyclopropylcarboxylic acid 1a (1.4 mg, 3.7 μmol, prepared by the known method "Tetrahedron Letters, 25 (12), 1269-72; 1984") and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (3.8 mg, 13.7 μmol) were added to the reaction liquid in sequence, and the reaction was stirred at 0-5°C for 2 hours. 5 mL of water was added to the reaction solution to quench the reaction. The reaction solution was extracted with ethyl acetate (8 mL×3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL×2). The organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 1 (1.6 mg, yield: 82.1%).

[0430] MS m / z(ESI):520.2[M+1]

[0431] 1 H NMR (400MHz, CDCl3): δ7.90-7.84(m,1H),7.80-7.68(m,1H),5.80-5.70(m,1H),5.62-5.54(m,2H),5.44-5.32(m,2H),5.28-5.10(m,2H) ,3.40-3.15(m,3H),2.44(s,3H),2.23(t,1H),2.06-1.75(m,2H),1.68-1.56(m,1H),1.22-1.18(m,2H),1.04-0.98(m,2H),0.89(t,3H).

[0432] Example 2

[0433] (S)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide 2-A

[0434] (R)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-

[0435] Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[0436] 2-B

[0437]

[0438] Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to 1b (4 mg, 7.53 μmol), replace the argon three times, cool to 0-5 ° C in an ice-water bath, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared by the method disclosed in patent application "WO2013106717"), 1-hydroxybenzotriazole (3 mg, 22.4 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol) to the reaction solution in sequence, stir at 0-5 ° C for 1 hour after addition. Remove the ice-water bath, heat to 30 ° C and stir for 2 hours. The reaction solution was concentrated under reduced pressure, and the crude compound 2 was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridgePrep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product (2-A: 1.5mg, 2-B: 1.5mg).

[0439] MS m / z(ESI):534.0[M+1].

[0440] Single configuration compound 2-B (short retention time)

[0441] UPLC analysis: retention time 1.06 minutes, purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0442] 1H NMR (400MHz, DMSO-d6): δ8.37(d,1H),7.76(d,1H),7.30(s,1H),6.51(s,1H) ,5.58-5.56(m,1H),5.48(d,1H),5.41(s,2H),5.32-5.29(m,2H),3.60(t,1H) ,3.19-3.13(m,1H),2.38(s,3H),2.20-2.14(m,1H),1.98(q,2H),1.87-1.83( m,1H),1.50-1.40(m,1H),1.34-1.28(m,1H),0.86(t,3H),0.50-0.39(m,4H).

[0443] Single configuration compound 2-A (longer retention time)

[0444] UPLC analysis: retention time 1.10 minutes, purity: 86% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0445] 1 H NMR (400MHz, DMSO-d6): δ8.35(d,1H),7.78(d,1H),7.31(s,1H),6.52(s,1H ),5.58-5.53(m,1H),5.42(s,2H),5.37(d,1H),5.32(t,1H),3.62(t,1H),3. 20-3.15(m,2H),2.40(s,3H),2.25-2.16(m,1H),1.98(q,2H),1.87-1.82(m ,1H),1.50-1.40(m,1H),1.21-1.14(m,1H),0.87(t,3H),0.47-0.35(m,4H).

[0446] Example 3

[0447] (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropanamide 3-A

[0448] (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropanamide 3-B

[0449]

[0450] Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to 1b (5.0 mg, 9.41 μmol), cool to 0-5°C in an ice-water bath, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, supplier Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol) to the reaction solution in sequence. After the addition is complete, stir at 0-5°C for 10 minutes. Remove the ice-water bath, heat to 30°C and stir for 8 hours. The reaction solution was concentrated under reduced pressure, and the obtained crude compound 3 was purified by HPLC (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product (1.5mg, 1.5mg).

[0451] MS m / z(ESI):561.9[M+1].

[0452] Single configuration compound (short retention time)

[0453] UPLC analysis: retention time 1.11 minutes, purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0454] 1H NMR (400MHz, DMSO-d6): δ8.94(d,1H),7.80(d,1H),7.32(s,1H),7.20(d,1H) ,6.53(s,1H),5.61-5.55(m,1H),5.45-5.23(m,3H),5.15-5.06(m,1H),4.66- 4.57(m,1H),3.18-3.12(m,1H),2.40(s,3H),2.26-2.20(m,1H),2.16-2.08( m,1H),2.02-1.94(m,1H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H).

[0455] Single configuration compound (longer retention time)

[0456] UPLC analysis: retention time 1.19 minutes, purity: 90% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0457] 1 H NMR (400MHz, DMSO-d6): δ8.97(d,1H),7.80(d,1H),7.31(s,1H),7.16(d,1H),6.53(s,1H),5.63-5.55(m,1H),5.45-5.20(m,3H),5.16-5.07(m,1 H),4.66-4.57(m,1H),3.18-3.12(m,1H),2.40(s,3H),2.22-2.14(m,1H ),2.04-1.95(m,2H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H).

[0458] Example 4

[0459] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzene

[0460] [de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopentane-1-carboxamide

[0461]

[0462] Add 1 mL of N,N-dimethylformamide to 1b (3.0 mg, 5.64 μmol), cool to 0-5°C in an ice-water bath, add a drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-hydroxy-cyclopentanecarboxylic acid 4a (2.2 mg, 16.9 μmol, prepared by the method disclosed in patent application "WO2013106717") and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) to the reaction solution in sequence, and stir at 0-5°C for 1 hour after the addition. 5 mL of water was added to the reaction solution to quench the reaction. The reaction solution was extracted with ethyl acetate (10 mL×3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL×2). The organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 4 (2.5 mg, yield: 80.9%).

[0463] MS m / z(ESI):548.0[M+1].

[0464] 1 H NMR (400MHz, CDCl3): δ7.73-7.62(m,2H),5.75-5.62(m,1H),5.46-5.32(m,2H),5.26-5.10(m,1H),3.30-3.10 (m,1H),2.43(s,3H),2.28-2.20(m,2H),2.08-1.84(m,8H),1.69-1.58(m,2H),1.04-1.00(m,2H),0.89(t,3H).

[0465] Example 5

[0466] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro

[0467] -1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclopropane-1-

[0468] Formamide 5

[0469]

[0470] Add 1 mL of N,N-dimethylformamide to 1b (2.0 mg, 3.76 μmol), cool to 0-5°C in an ice-water bath, add a drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-(Hydroxymethyl)-cyclopentanecarboxylic acid 5a (0.87 mg, 7.5 μmol, prepared by the method disclosed in patent application "WO201396771") and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (2 mg, 7.24 μmol) to the reaction solution in sequence, and stir at 0-5°C for 2 hours after the addition. 5 mL of water was added to the reaction solution to quench the reaction. The reaction solution was extracted with ethyl acetate (8 mL×3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL×2). The organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 5 (1.0 mg, yield: 50%).

[0471] MS m / z(ESI):533.9[M+1].

[0472] 1 H NMR (400MHz, CDCl3): δ8.07(s,1H),7.23-7.18(m,2H),6.71-6.64(m,1H),6.55-6.51(m,1H),5.36-5.27(m,2H),4.67-4.61(m,2H),3.53-3.48 (m,1H),3.30-3.22(m,2H),3.18-3.13(m,1H),2.71-2.61(m,2H),2.35 -2.28(m,1H),2.04-1.91(m,4H),1.53-1.40(m,3H),0.91-0.75(m,4H).

[0473] Example 6

[0474] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclobutane-1-carboxamide 6

[0475]

[0476] Add 1 mL of N,N-dimethylformamide to 1b (3.0 mg, 5.64 μmol), cool to 0-5°C in an ice-water bath, add a drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-(Hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 μmol; prepared by the method disclosed in the literature "Journal of the American Chemical Society, 2014, vol. 136, #22, p. 8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) to the reaction solution in sequence, and stir at 0-5°C for 1 hour after the addition is complete. 5 mL of water was added to the reaction solution to quench the reaction. The reaction solution was extracted with ethyl acetate (10 mL×3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL×2). The organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 6 (2.1 mg, yield: 67.9%).

[0477] MS m / z(ESI):548.0[M+1].

[0478] 1 H NMR (400MHz, DMSO-d6): δ7.85-7.62(m,1H),6.88(br,1H),5.87-5.48(m,2H),5.47-5.33(m,1H),5.31-5.06(m,1H),4.25-3.91(m,2H), 3.25(br,1H),2.60-2.32(m,3H),2.23(t,1H),2.15-1.95(m,3H),1.70-1.56(m,2H),1.41-1.17(m,9H),1.03(s,1H),0.95-0.80(m,2H).

[0479] Example 7

[0480] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzene

[0481] [de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclobutane-1-carboxamide 7

[0482]

[0483] Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to 1b (3.0 mg, 5.64 μmol), cool to 0-5°C in an ice-water bath, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 1-hydroxycyclobutanecarboxylic acid 7a (2.0 mg, 17.22 μmol, supplier Yao Shi), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol) to the reaction solution in sequence. After the addition, stir at 0-5°C for 10 minutes. Remove the ice-water bath and stir at room temperature for 2 hours. The reaction solution is concentrated under reduced pressure, and the residue is purified by thin layer chromatography with developing solvent system B to obtain the title product 7 (2.5 mg, yield: 83.1%).

[0484] MS m / z(ESI):534.0[M+1].

[0485] 1 H NMR (400MHz, DMSO-d6): δ8.28(d,1H),7.75(d,1H),7.29(s,1H),6.51(s,1H) ,6.12(s,1H),5.59-5.51(m,1H),5.41(s,2H),5.20-5.01(m,2H),3.27-3.17( m,1H),3.15-3.05(m,1H),2.71-2.63(m,1H),2.37(s,3H),2.12-2.05(m,1H) ,2.03-1.94(m,2H),1.92-1.78(m,4H),1.50-1.42(m,1H),0.90-0.83(m,4H).

[0486] Example 8

[0487] 1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaeicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-yl)-

[0488] cyclopropane-1-carboxamide 8

[0489]

[0490]

[0491] first step

[0492] Benzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropane-1-carboxylate 8c

[0493] 1-Hydroxycyclopropane-1-carboxylic acid benzyl ester 8a (104 mg, 0.54 mmol; prepared by the method disclosed in patent application "US2005 / 20645") and 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate 8b (100 mg, 0.27 mmol; prepared by the method disclosed in patent application "CN105829346A") were added to the reaction bottle, 5 mL of tetrahydrofuran was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, potassium tert-butoxide (61 mg, 0.54 mmol) was added, the ice bath was removed, the temperature was warmed to room temperature and stirred for 10 minutes, 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL×2) and chloroform (5 mL×5), and the organic phases were combined and concentrated. The obtained residue was dissolved in 3 mL of 1,4-dioxane, 0.6 mL of water was added, sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (70 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. 20 mL of water was added, and the mixture was extracted with ethyl acetate (8 mL×3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with developing solvent system B to obtain the title product 8c (100 mg, yield: 73.6%).

[0494] MS m / z(ESI):501.0[M+1].

[0495] Step 2

[0496] 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropane-1-carboxylic acid 8d

[0497] 8c (50 mg, 0.10 mmol) was dissolved in 3 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V=2:1), palladium carbon (25 mg, 10% content) was added, hydrogen was replaced three times, and the reaction was stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to obtain the title product 8d (41 mg, yield: 100%). MS m / z (ESI): 411.0 [M+1].

[0498] Step 3

[0499] (9H-fluoren-9-yl)methyl (2-(((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropyloxy)methyl)amino)-2-oxoethyl)carbamate 8e

[0500] 1b (7 mg, 0.013 mmol) was added to the reaction flask, 1 mL of N,N-dimethylformamide was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, a drop of triethylamine was added, 8d (7 mg, 0.017 mmol) in 0.5 mL of N,N-dimethylformamide solution was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (7 mg, 0.026 mmol) was added, and the reaction was stirred in an ice bath for 35 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL×3), the organic phase was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 8e (8.5 mg, yield 78.0%).

[0501] MS m / z(ESI):828.0[M+1].

[0502] Step 4

[0503] 1-((2-aminoacetylamino)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 8f

[0504] 8e (4 mg, 4.84 μmol) was dissolved in 0.2 mL of dichloromethane, 0.1 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure, and the reaction was repeated twice. 3 mL of n-hexane was added to make a pulp, and the upper n-hexane was poured out, and the reaction was repeated three times. The crude title product 8f (2.9 mg) was obtained and the product was used directly in the next step without purification.

[0505] MS m / z(ESI):606.0[M+1].

[0506] Step 5

[0507] 1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaeicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 8

[0508] The crude product 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL N,N-dimethylformamide, replaced with argon three times, cooled to 0-5°C in an ice-water bath, and a solution of (S)-2(-2-(-2-(6-(2,5-dioxo-1H-pyrrol-1-yl)hexanoylamino)acetylamino)acetylamino)-3-phenylpropionic acid 8 g (2.7 mg, 5.80 μmol, prepared by the method disclosed in patent application "EP2907824") in 0.3 mL N,N-dimethylformamide was added, and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol) was added. The reaction was stirred in an ice bath for 30 minutes, the ice bath was removed, and the mixture was heated to room temperature and stirred for 15 minutes. The reaction solution was purified by HPLC (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding fractions were collected and concentrated under reduced pressure to give the title product 8 (2mg, yield: 39.0%).

[0509] MS m / z(ESI):1060.0[M+1].

[0510] 1H NMR (400MHz, DMSO-d6): δ9.01(d,1H),8.77(t,1H),8.21(t,1H),8.08-7.92(m,2H),7.73(d,1H),7.28(s,1H), 7.24-7.07(m,4H),6.98(s,1H),6.50(s,1H),5.61(q,1H),5.40(s,2H),5.32(t,1H),5.12(q,2H),4.62(t,1H) ,4.52(t,1H),4.40-4.32(m,1H),3.73-3.47(m,8H),3.16-3.04(m,2H),2.89(dd,1H),2.69-2.55(m,2H),2.37 -2.23(m,4H),2.12-1.93(m,4H),1.90-1.74(m,2H),1.52-1.38(m,4H),1.33-1.11(m,5H),0.91-0.81(m,4H).

[0511] Example 9

[0512] N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro

[0513] -1H-pyrrol-1-yl)hexanamide 9-A

[0514] N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro

[0515] -1H-pyrrol-1-yl)hexanamide 9-B

[0516]

[0517] first step

[0518] Benzyl 2-cyclopropyl-2-hydroxyacetate 9a

[0519] 2a (1.3 g, 11.2 mmol; prepared by the method disclosed in patent application "WO2013 / 106717") was dissolved in 50 mL of acetonitrile, and potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol) and tetrabutylammonium iodide (413 mg, 1.1 mmol) were added in sequence. The reaction solution was stirred at room temperature for 48 hours, filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate (10 ml), the combined filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with developing solvent system C to obtain the title product 9a (2 g, yield: 86.9%).

[0520] Step 2

[0521] 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-oic acid benzyl ester 9b

[0522] 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) were added to the reaction flask, 4 mL of tetrahydrofuran was added, argon was replaced three times, the temperature was cooled to 0-5 ° C in an ice-water bath, potassium tert-butoxide (109 mg, 0.98 mmol) was added, the ice bath was removed, the temperature was raised to room temperature and stirred for 40 minutes, 10 mL of ice water was added, and ethyl acetate (20 mL×2) and chloroform (10 mL×5) were extracted, and the organic phases were combined and concentrated. The obtained residue was dissolved in 4 mL of dioxane, 2 mL of water was added, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 20 mL of water was added, and ethyl acetate (10 mL×3) was extracted. The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using a developing solvent system C to give the title product 9b (48 mg, yield: 19%). MS m / z (ESI): 515.0 [M+1].

[0523] Step 3

[0524] 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid 9c

[0525] 9b (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), palladium carbon (12 mg, 10% content, dry type) was added, hydrogen was replaced three times, and the reaction was stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain a crude title product 9c (13 mg), which was directly used for the next step without purification.

[0526] MS m / z(ESI):424.9[M+1].

[0527] Step 4

[0528] (9H-fluoren-9-yl)methyl (2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate 9d

[0529] 1b (10 mg, 18.8 μmol) was added to the reaction bottle, 1 mL of N,N-dimethylformamide was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, a drop of triethylamine was added, crude product 9c (13 mg, 30.6 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (16.9 mg, 61.2 μmol) was added, and the reaction was stirred in an ice bath for 40 minutes. 10 mL of water was added, extracted with ethyl acetate (10 mL×3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 9d (19 mg, yield: 73.6%).

[0530] MS m / z(ESI):842.1[M+1].

[0531] Step 5

[0532] 2-((2-aminoacetylamino)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline

[0533] -1-yl)acetamide 9e

[0534] 9d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, 1 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 1 mL of toluene was added and concentrated under reduced pressure, and the mixture was repeated twice. 3 mL of n-hexane was added to the residue for pulping, and the supernatant was poured out after standing to retain the solid. The solid residue was concentrated under reduced pressure and the crude title product 9e (17 mg) was obtained by oil pump drying. The product was used directly in the next step without purification. MS m / z (ESI): 638.0 [M+18].

[0535] Step 6

[0536] N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro

[0537] -1H-pyrrol-1-yl)hexanamide 9-A

[0538] N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro

[0539] -1H-pyrrol-1-yl)hexanamide 9-B

[0540] The crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL N,N-dimethylformamide, replaced with argon three times, cooled to 0-5°C in an ice-water bath, 8 g (21.2 mg, 44.8 μmol) of 0.3 mL N,N-dimethylformamide solution was added, and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol) was added. The reaction was stirred in an ice bath for 10 minutes, the ice bath was removed, the temperature was raised to room temperature and stirred for 1 hour to generate compound 9. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product (9-A: 2.4mg, 9-B: 1.7mg).

[0541] MS m / z(ESI):1074.4[M+1].

[0542] Single configuration compound 9-A (shorter retention time):

[0543] UPLC analysis: retention time 1.14 minutes, purity: 85% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0544] 1H NMR (400MHz, DMSO-d6): δ8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H) ,7.31(s,1H),7.26-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,1 H),4.54-4.40(m,2H),3.76-3.64(m,4H),3.62-3.48(m,2H),3.20-3.07(m,2H),3.04-2.94(m,1H),2.80-2.62(m,1H),2.45-2.30(m, 3H),2.25-2.15(m,2H),2.15-2.04(m,2H),1.93-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.87(t,3H),0.64-0.38(m,4H).

[0545] Single configuration compound 9-B (longer retention time):

[0546] UPLC analysis: retention time 1.16 minutes, purity: 89% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0547] 1H NMR (400MHz, DMSO-d6): δ8.68-8.60(m,1H),8.58-8.50(m,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H), 7.31(s,1H),7.26-7.13(m,3H),6.99(s,1H),6.55-6.48(m,1H),5.60- 5.50(m,1H),5.41(s,2H),5.35-5.15(m,2H),4.78-4.68(m,1H),4.60- 4.40(m,2H),3.76-3.58(m,4H),3.58-3.48(m,1H),3.20-3.10(m,2H) ,3.08-2.97(m,2H),2.80-2.72(m,2H),2.45-2.30(m,3H),2.25-2.13( m,2H),2.13-2.04(m,2H),2.03-1.94(m,2H),1.91-1.78(m,2H),1.52- 1.39(m,3H),1.34-1.12(m,4H),0.91-0.79(m,3H),0.53-0.34(m,4H).

[0548] Example 10

[0549] N-((2S,10S)-10-benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-A

[0550] N-((2R,10S)-10-benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-B

[0551]

[0552] first step

[0553] Benzyl 3,3,3-trifluoro-2-hydroxypropionate 10a

[0554] 3a (1.80 g, 12.5 mmol) was dissolved in 100 mL of acetonitrile, and potassium carbonate (5.17 g, 37.5 mmol), benzyl bromide (4.48 mL, 37.5 mmol) and tetrabutylammonium iodide (231 mg, 0.63 mmol) were added in sequence. The reaction solution was heated to 60°C and stirred for 5 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a developing solvent system C to obtain the title product 10a (980 mg, yield: 33.5%).

[0555] 1 H NMR (400MHz, CDCl3): δ7.43-7.36(m,5H),5.34(s,2H),4.53(s,1H),3.44(s,1H).

[0556] Step 2

[0557] 1-(9H-fluoren-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester

[0558] 10b

[0559] 8b (63 mg, 0.17 mmol) and 10a (80 mg, 0.34 mmol) were added to a reaction flask, 3 mL of tetrahydrofuran was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, potassium tert-butoxide (38 mg, 0.34 mmol) was added, the ice bath was removed, the temperature was warmed to room temperature and stirred for 20 minutes, 10 mL of ice water was added, and the mixture was extracted with ethyl acetate (20 mL×2) and chloroform (10 mL×5), the organic phases were combined and concentrated, the residue was dissolved in 2 mL of dioxane, 0.4 mL of water was added, sodium bicarbonate (19 mg, 0.23 mmol) and 9-fluorenylmethyl chloroformate (49 mg, 0.19 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Add 20 mL of water, extract with ethyl acetate (10 mL×3), wash the organic phase with saturated sodium chloride solution (20 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the residue by silica gel column chromatography with developing solvent system C to give the title product 10b (51 mg, yield: 55.3%).

[0560] MS m / z(ESI):559.9[M+18].

[0561] Step 3

[0562] 1-(9H-fluoren-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazaundecane-11-oic acid 10c

[0563] 10b (15 mg, 0.28 mmol) was dissolved in 3 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V=2:1), palladium carbon (15 mg, 10% content) was added, hydrogen was replaced three times, and the reaction was stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to obtain the crude title product 10c (13 mg). MS m / z (ESI): 452.9 [M+1].

[0564] Step 4

[0565] (9H-fluoren-9-yl)methyl (2-((((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,1,1-trifluoro-3-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate 10d

[0566] 1b (10 mg, 18.8 μmol) was added to the reaction bottle, 1 mL of N, N-dimethylformamide was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, a drop of triethylamine was added, 0.5 mL of N, N-dimethylformamide solution of 10c (13 mg, 28.7 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (11 mg, 39.7 μmol) was added, and the reaction was stirred in an ice bath for 30 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL×3), the organic phases were combined, the organic phases were washed with saturated sodium chloride solution (10 mL×2), the organic phases were dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 10d (16 mg, yield 97.8%).

[0567] MS m / z(ESI):870.0[M+1].

[0568] Step 5

[0569] 2-((2-aminoacetylamino)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo

[0570] -2,3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-

[0571] (2-(4-(4-(4-(4-fluoropropionamide)))-3,3,3-trifluoropropionamide 10e

[0572] 10d (16 mg, 18.4 μmol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 2 mL of toluene was added and concentrated under reduced pressure, and the process was repeated twice. 3 mL of n-hexane was added to the residue for pulping, and the supernatant was poured out after standing, and the solid was retained; the process was repeated three times. The solid residue was concentrated under reduced pressure and the oil pump was used to dryness to obtain the crude title product 10e (12 mg), which was used directly in the next step without purification.

[0573] MS m / z(ESI):647.9[M+1].

[0574] Step 6

[0575] N-((2S,10S)-10-benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-A

[0576] N-((2R,10S)-10-benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-B

[0577] The crude product 10e (12 mg, 18.5 μmol) was dissolved in 1.0 mL N,N-dimethylformamide, replaced with argon three times, cooled to 0-5°C in an ice-water bath, 8 g (14 mg, 29.6 μmol) of 0.3 mL N,N-dimethylformamide solution was added, and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (15 mg, 54.2 μmol) was added. The reaction was stirred in an ice bath for 30 minutes, the ice bath was removed, the temperature was raised to room temperature and stirred for 1 hour to generate compound 10. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um19*250mm; mobile phase: A-water (10mmol NH4OAc) B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product (2.7mg, 2.6mg).

[0578] MS m / z(ESI):1102.0[M+1].

[0579] Single configuration compound (short retention time):

[0580] UPLC analysis: retention time 1.18 minutes, purity: 91% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0581] 1 H NMR (400MHz, DMSO-d6): δ8.97(d,1H),8.85-8.76(m,1H),8.37-8.27(m,1H),8.12-8.02(m,1H),8.02-7.95(m,1H),7.80(d,1 H),7.31(s,1H),7.26-7.10(m,4H),6.99(s,1H),6.66(br,1H),6.52(s,1H),5.65-5.54(m,1H),5.41(s,1H),5.37-5.25(m,3H ),5.23-5.13(m,1H),4.81-4.68(m,2H),4.51-4.41(m,1H),3.78-3.45(m,6H),3.21-3.13(m,1H),3.02-2.93(m,1H),2.77-2. 63(m,2H),2.45-2.29(m,3H),2.24-2.05(m,3H),2.04-1.93(m,5H),1.90-1.75(m,2H),1.52-1.38(m,4H),0.90-0.78(m,5H).

[0582] Single configuration compound (longer retention time):

[0583] UPLC analysis: retention time 1.23 minutes, purity: 90% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0584] 1 H NMR (400MHz, DMSO-d6): δ9.05(d,1H),8.97-8.88(m,1H),8.35-8.27(m,1H),8.11-8.03(m,1H),8.02-7.95(m,1H),7.80(d,1 H),7.34(s,1H),7.29-7.13(m,4H),6.99(s,1H),6.66(br,1H),6.54(s,1H),5.64-5.55(m,1H),5.43(s,1H),5.36-5.20(m,3H ),4.92-4.85(m,1H),4.82-4.72(m,2H),4.52-4.42(m,1H),3.77-3.48(m,6H),3.21-3.14(m,1H),3.03-2.95(m,1H),2.79-2. 65(m,2H),2.47-2.28(m,3H),2.25-2.05(m,3H),2.05-1.94(m,5H),1.91-1.76(m,2H),1.52-1.37(m,4H),0.92-0.77(m,5H).

[0585] Embodiment 11

[0586] 1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaeicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-yl)-

[0587] 1-(2-(2-yl)cyclobutane-1-carboxamide 11

[0588]

[0589] first step

[0590] Benzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclobutane-1-carboxylate 11b

[0591] 1-Hydroxycyclobutane-carboxylic acid benzyl ester 11a (167 mg, 0.81 mmol, prepared by the method disclosed in the document "Journal of Medicinal Chemistry, 2013, vol.56, #13, p.5541-5552") and 8b (150 mg, 0.41 mmol) were added to a reaction flask, 5 mL of tetrahydrofuran was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, potassium tert-butoxide (92 mg, 0.82 mmol) was added, the ice bath was removed, the temperature was warmed to room temperature and stirred for 10 minutes, 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL×2) and chloroform (5 mL×5), the organic phases were combined and concentrated, the residue was dissolved in 3 mL of dioxane, 0.6 mL of water was added, sodium bicarbonate (41 mg, 0.48 mmol) and 9-fluorenylmethyl chloroformate (105 mg, 0.41 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Add 20 mL of water, extract with ethyl acetate (8 mL×3), wash the organic phase with saturated sodium chloride solution (20 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the residue by silica gel column chromatography with developing solvent system C to give the title product 11b (37 mg, yield: 17.6%).

[0592] MS m / z(ESI):514.6[M+1].

[0593] Step 2

[0594] 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclobutane-1-carboxylic acid 11c

[0595] 11b (37 mg, 71.9 μmol) was dissolved in 3 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V=2:1), palladium carbon (15 mg, content 10%) was added, hydrogen was replaced three times, and the reaction was stirred at room temperature for 2 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to obtain the title product 11c (35 mg, yield: 82%), which was directly carried out to the next step.

[0596] Step 3

[0597] (9H-fluoren-9-yl)methyl (2-(((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclobutyloxy)methyl)amino)-2-oxoethyl)carbamate 11d

[0598] 1b (10 mg, 0.018 mmol) was added to the reaction flask, 1 mL of N, N-dimethylformamide was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, a drop of triethylamine was added, 11c (13 mg, 0.031 mmol) in 0.5 mL of N, N-dimethylformamide solution was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (25 mg, 0.091 mmol) was added, and the reaction was stirred in an ice bath for 40 minutes. 8 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL×3), the organic phase was washed with saturated sodium chloride solution (8 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system A to obtain the title product 11d (19 mg, yield 73.9%).

[0599] MS m / z(ESI):842.3[M+1].

[0600] Step 4

[0601] 1-((2-aminoacetylamino)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutane-1-carboxamide 11e

[0602] 11d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, 1 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and 1 mL of toluene was added and concentrated under reduced pressure, and the reaction was repeated twice. 4 mL of n-hexane was added to slurry, and the upper n-hexane was poured out, and the reaction was repeated three times. The crude title product 11e (15 mg) was obtained, and the product was used directly in the next step without purification.

[0603] Step 5

[0604] 1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaeicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-yl)-

[0605] 1-(2-(2-yl)cyclobutane-1-carboxamide 11

[0606] The crude product 11e (2 mg, 3.22 μmol) was dissolved in 0.5 mL N, N-dimethylformamide, replaced with argon three times, cooled to 0-5°C in an ice-water bath, 8 g (1.5 mg, 3.17 μmol) of 0.3 mL N, N-dimethylformamide solution was added, and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol) was added, and stirred at room temperature for 30 minutes. The reaction solution was dried with an oil pump, DMF was removed, and the residue was dissolved in DCM and directly purified by thin layer chromatography twice (developing solvent polarity: DCM / MeOH=10 / 1) to obtain the title product 11 (1 mg, yield: 28.8%).

[0607] MS m / z(ESI):1073.6[M+1].

[0608] 1 H NMR (400MHz, CDCl3): δ8.70-8.60(m,1H),8.28-8.19(m,1H),8.13-7.91(m,3H),7.79-7.71(d,1H),7.29(s,1H),7.25-7. 09(m,4H),6.98(s,1H),6.71-6.62(m,1H),6.55-6.47(m,1H),5.64-5.54(m,2H),5.40(s,1H),5.35-5.27(t,2H),5.17-5 .10(m,2H),4.60-4.51(m,1H),4.51-4.35(m,2H),3.93-3.78(m,3H),3.71-3.59(m,3H),3.01-2.88(m,3H),2.70-2.64(m ,2H),2.44-2.30(m,3H),2.28-2.14(m,3H),2.11-1.92(m,6H),1.90-1.76(m,3H),1.51-1.39(m,4H),0.92-0.75(m,6H).

[0609] Example 12

[0610] (S)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 12-A (R)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 12-B

[0611]

[0612] first step

[0613] 3-Cyclopropyl-2-hydroxypropionic acid 12b

[0614] 12a (0.5 g, 3.87 mmol, supplier Adamas) was dissolved in a mixed solvent of 35 mL of water and acetic acid (V:V=4:1), cooled to 0-5°C in an ice-water bath, and a 2M aqueous solution of sodium nitrite (0.53 g, 7.74 mmol) was added dropwise, and the mixture was stirred at room temperature for 3 hours. Solid sodium chloride was added to the reaction solution to saturate the aqueous phase, and the mixture was extracted with ethyl acetate (8 mL×8), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the title product 12b (0.45 g, yield: 89.3%).

[0615] Step 2 (S)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 12-A (R)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 12-B

[0616] 1.5 mL of ethanol and 1.5 mL of N,N-dimethylformamide were added to 1b (45 mg, 0.085 mmol), the argon gas was replaced three times, 0.1 mL of N-methylmorpholine was added dropwise, and the reaction solution was stirred until it became clear. 12b (90 mg, 0.691 mmol), 1-hydroxybenzotriazole (34 mg, 0.251 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (49 mg, 0.256 mmol) were added to the reaction solution in sequence, and the reaction was stirred at room temperature for 3 hours after the addition was completed. The reaction solution was concentrated under reduced pressure, and the crude compound 12 was purified by high performance liquid chromatography (separation conditions: chromatographic column: Sharpsil-T C18 5um 21.2*250 mm; mobile phase: A-water (10 mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL / min) to obtain the title product (7 mg, 15 mg).

[0617] MS m / z(ESI):547.9[M+1].

[0618] Single configuration compound (short retention time)

[0619] UPLC analysis: retention time 1.345 minutes, purity: 72% (chromatographic column: ZORBAX Ecliphase Plus C18 1.8um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0620] 1 H NMR (400MHz, DMSO-d6): δ8.42(d,1H),7.78(d,1H),7.30(s,1H),6.51(s,1H ),5.60-5.50(m,2H),5.42(s,1H),5.19(q,2H),4.02-4.00(m,1H),3.21-3. 11(m,2H),2.39(s,3H),2.21-2.07(m,2H),2.05-1.95(m,1H),1.92-1.68(m ,4H),1.53-1.41(m,1H),0.87(t,3H),0.48-0.34(m,2H),0.14-0.01(m,2H).

[0621] Single configuration compound (longer retention time)

[0622] UPLC analysis: retention time 1.399 minutes, purity: 88% (chromatographic column: ZORBAX Ecliphase Plus C18 1.8um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0623] 1 H NMR (400MHz, DMSO-d6): δ8.36(d,1H),7.77(d,1H),7.31(s,1H),6.51(s,1H),5.58- 5.51(m,1H),5.48(d,1H),5.42(s,1H),5.20(q,2H),4.09-4.02(m,1H),3.22-3.11( m,2H),2.39(s,3H),2.27-2.06(m,2H),2.05-1.95(m,1H),1.93-1.81(m,2H),1.65- 1.43(m,2H),1.32-1.21(m,1H),0.87(t,3H),0.48-0.33(m,2H),0.14-0.01(m,2H).

[0624] Example 13 (reference example)

[0625] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro

[0626] -1H,12H-Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[0627]

[0628] The title compound 13 was prepared according to the method disclosed in "Example 76 on page 147 of the specification of EP2907824A1".

[0629] Embodiment 14

[0630] N-((2R,10S)-10-benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-

[0631] -2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-A

[0632] N-((2S,10S)-10-benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-

[0633] -2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-B

[0634]

[0635]

[0636] first step

[0637] Benzyl 3-cyclopropyl-2-hydroxypropionate 14a

[0638] 12b (200 mg, 1.54 mmol) was dissolved in 20 mL of acetonitrile, and potassium carbonate (1.06 g, 7.68 mmol), benzyl bromide (0.16 mL, 1.34 mmol) and tetrabutylammonium iodide (28 mg, 0.07 mmol) were added in sequence. The reaction solution was stirred at room temperature for 48 hours, filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate (10 ml), the combined filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with developing solvent system C to obtain the title product 14a (140 mg, yield: 41.3%).

[0639] Step 2

[0640] 10-(Cyclopropylmethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester 14b

[0641] 14a (94 mg, 0.427 mmol) and 8b (130 mg, 0.353 mmol) were added to a reaction flask, 10 mL of tetrahydrofuran was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, potassium tert-butoxide (79 mg, 0.704 mmol) was added, the ice bath was removed, the temperature was warmed to room temperature and stirred for 10 minutes, 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (10 mL×4), the organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with a developing solvent system C to obtain the title product 14b (50 mg, yield: 26.8%).

[0642] MS m / z(ESI):529.2[M+1].

[0643] Step 3

[0644] 10-(Cyclopropylmethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid 14c

[0645] 14b (27 mg, 0.051 mmol) was dissolved in 3 mL of ethyl acetate, palladium carbon (7 mg, 10% content, dry type) was added, hydrogen was replaced three times, and the reaction was stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude title product 14c (23 mg), which was directly used for the next step without purification.

[0646] MS m / z(ESI):439.1[M+1].

[0647] Step 4

[0648] (9H-fluoren-9-yl)methyl (2-((((3-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate 14d

[0649] 1b (22 mg, 42.38 μmol) was added to the reaction flask, 3 mL of N,N-dimethylformamide was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, triethylamine (4.3 mg, 42.49 μmol) was added dropwise, crude product 14c (23 mg, 51.1 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 μmol) was added, and the reaction was stirred in an ice bath for 40 minutes. 15 mL of water was added, extracted with ethyl acetate (8 mL×3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 14d (29 mg, yield: 79.9%).

[0650] MS m / z(ESI):856.1[M+1].

[0651] Step 5

[0652] 2-((2-aminoacetylamino)methoxy)-3-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline

[0653] -1-yl) propanamide 14e

[0654] 14d (29 mg, 33.9 μmol) was dissolved in 0.8 mL of dichloromethane, 0.4 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, 1 mL of toluene was added and concentrated under reduced pressure, and the reaction was repeated twice. 3 mL of n-hexane was added to the residue for pulping, and the supernatant was poured out after standing, and the reaction was repeated three times. The residue was concentrated under reduced pressure and the oil pump was used to dry the crude title product 14e (22 mg), which was used directly in the next step without purification.

[0655] MS m / z(ESI):634.1[M+1].

[0656] Step 6

[0657] N-((2R,10S)-10-benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-

[0658] -2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-A

[0659] N-((2S,10S)-10-benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-

[0660] -2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-B

[0661] The crude product 14e (22 mg, 33.9 μmol) was dissolved in 2.5 mL N,N-dimethylformamide, replaced with argon three times, cooled to 0-5°C in an ice-water bath, and 8 g (24 mg, 50.8 μmol) and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (14 mg, 50.6 μmol) were added in sequence, the ice bath was removed, the mixture was heated to room temperature and stirred for 1 hour to react to generate compound 14. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18 mL / min) to obtain the title product (2 mg, 2 mg).

[0662] MS m / z(ESI):1088.4[M+1].

[0663] Single configuration compound (short retention time):

[0664] UPLC analysis: retention time 1.18 minutes, purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0665] Single configuration compound (longer retention time):

[0666] UPLC analysis: retention time 1.23 minutes, purity: 96% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0667] Embodiment 15

[0668] 1-((S)-9-Benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa-4,7,10,13,16-pentaazacosyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 15

[0669]

[0670]

[0671] first step

[0672] Benzyl 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylate 15b

[0673] 8b (500 mg, 1.35 mmol) was added to a reaction flask, and 6 mL of tetrahydrofuran was added. 1-hydroxymethylcyclopropane-1-carboxylic acid benzyl ester 15a (233 mg, 1.13 mmol; prepared by the method disclosed in "Example 22-2 on page 262 of the specification of patent application EP2862856A1") was added to the flask, and the atmosphere was replaced with argon three times. The temperature was cooled to 0-5°C in an ice-water bath, and sodium hydride (54 mg, 1.35 mmol) was added. The ice bath was removed, and the mixture was heated to room temperature and stirred for 40 minutes; the temperature was lowered to zero and 20 mL of ice water was added. The mixture was extracted with ethyl acetate (5 mL×2) and chloroform (5 mL×5). The organic phases were combined, washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a developing solvent system B to obtain the title product 15b (15 mg, yield: 2.5%).

[0674] MS m / z(ESI):515.2[M+1].

[0675] Step 2

[0676] 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylic acid 15c

[0677] 15b (15 mg, 0.029 mmol) was dissolved in 2 mL of ethyl acetate, palladium carbon (3 mg, 10% content, dry type) was added, hydrogen was replaced three times, and the reaction was stirred at room temperature for 4.5 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the title product 15c (11 mg, yield: 89%).

[0678] MS m / z(ESI):425.2[M+1].

[0679] Step 3

[0680] (9H-fluoren-9-yl)methyl (2-((((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropyl)methoxy)methyl)amino)2-oxoethyl)carbamate 15d

[0681] 1b (10 mg, 0.021 mmol) was added to the reaction flask, 1 mL of N,N-dimethylformamide was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, a drop of triethylamine was added, 15c (11 mg, 0.026 mmol) was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (10.7 mg, 0.039 mmol) was added, and the reaction was stirred at room temperature for 60 minutes after the addition was complete. 10 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL×3), and the organic phase was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 15d (19 mg, yield 87.0%).

[0682] MS m / z(ESI):842.2[M+1].

[0683] Step 4

[0684] 1-(((2-aminoacetamido)methoxy)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12Hbenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 15e

[0685] 15d (19 mg, 22.56 μmol) was dissolved in 2 mL of dichloromethane, and 1 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure at 0°C, and 1 mL of toluene was added to concentrate under reduced pressure, and the process was repeated twice; 3 mL of n-hexane was added to slurry, and the upper layer of n-hexane was poured out, and the process was repeated three times; the crude title product 15e (13.9 mg) was obtained by concentration under reduced pressure, and the product was used directly in the next step without purification.

[0686] MS m / z(ESI):620.1[M+1].

[0687] Step 5

[0688] 1-((S)-9-Benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa-4,7,10,13,16-pentaazacosyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 15

[0689] The crude product 15e (13.9 mg, 22.4 μmol) was dissolved in 1 mL N,N-dimethylformamide, replaced with argon three times, cooled to 0-5°C in an ice-water bath, 8 g (15.8 mg, 33.4 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (9.3 mg, 33.6 μmol) was added, and the mixture was stirred at room temperature for 60 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um19*250mm; mobile phase: A-water (10 mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18 mL / min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product 15 (2.5 mg, yield: 10.3%).

[0690] MS m / z(ESI):1074.2[M+1].

[0691] 1 H NMR (400MHz, DMSO-d6): δ8.51-8.37(m,1H),8.22(t,1H),8.14-8.02(m,2H),8.011-7.94(m,1H),7.82-7.73(m,1H),7.29(s,1H),7.26-7.1 0(m,3H),6.98(s,1H),6.53-6.47(m,1H),5.62-5.50(m,1H),5.45-5. 36(m,1H),5.35-5.23(m,2H),5.13-5.02(m,2H),4.61-4.50(m,2H),4 .42-4.28(m,2H),3.76-3.61(m,3H),3.60-3.45(m,3H),3.27-3.23(m ,1H),3.20-2.81(m,7H),2.75-2.61(m,3H),241-2.28(m,3H),2.23-2 .13(m,2H),2.11-2.01(m,1H),2.03-1.94(m,1H),1.90(s,1H),1.87- 1.74(m,2H),1.53-1.36(m,3H),1.29-1.08(m,4H),0.90-0.68(m,4H).

[0692] Example 16

[0693] 1-((S)-9-Benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa-4,7,10,13,16-pentaazacosyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutane-1-carboxamide

[0694]

[0695] first step

[0696] 1-(Hydroxymethyl)cyclobutane-1-carboxylic acid 16b

[0697] Ethyl 1-(hydroxymethyl)cyclobutanecarboxylate 16a (250 mg, 1.58 mmol, supplier Alfa) was dissolved in methanol (2 mL) and water (1 mL), sodium hydroxide (126 mg, 3.15 mmol) was added, the temperature was raised to 40 ° C, and the reaction was stirred for 3 hours. After cooling to room temperature, the organic solvent was removed by concentration under reduced pressure, and the organic solvent was stripped with ether (10 mL), and the aqueous phase was collected. The aqueous phase was adjusted to pH 3-4 with 6N hydrochloric acid aqueous solution and concentrated under reduced pressure to obtain a solid. 3 mL of toluene was added, and the mixture was concentrated under reduced pressure and dried by spin drying, and repeated three times. The crude title product 16b (206 mg) was obtained by oil pump drying, and the product was directly used in the next step without purification.

[0698] MS m / z (ESI, NEG): 129.2 [M-1].

[0699] Step 2

[0700] Benzyl 1-(Hydroxymethyl)cyclobutane-1-carboxylate 16c

[0701] The crude product 16b (206 mg, 1.58 mmol) was dissolved in acetonitrile (15 mL), anhydrous potassium carbonate (1.09 g, 7.90 mmol) and tetrabutylammonium iodide (29 mg, 78.51 umol) were added, and benzyl bromide (216 mg, 1.26 mmol) was added, and the mixture was stirred at room temperature overnight. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a developing solvent system C to obtain the title product 16c (112 mg, yield: 32.1%).

[0702] MS m / z(ESI):221.1[M+1].

[0703] Step 3

[0704] Benzyl 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylate 16d

[0705] 16c (77 mg, 0.35 mmol) and 8b (100 mg, 0.27 mmol) were added to the reaction flask, 3 mL of tetrahydrofuran was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, potassium tert-butoxide (61 mg, 0.54 mmol) was added, and the mixture was stirred in an ice bath for 10 minutes. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL) and chloroform (5 mL×5), and the organic phases were combined and concentrated. The residue was dissolved in 3 mL of 1,4-dioxane, 0.5 mL of water was added, sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (71 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Add 20 mL of water, extract with ethyl acetate (10 mL×3), wash the organic phase with saturated sodium chloride solution (20 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the residue by silica gel column chromatography with developing solvent system C to give the title product 16d (24 mg, yield: 16.7%).

[0706] MS m / z(ESI):551.3[M+23].

[0707] Step 4

[0708] 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylic acid 16e

[0709] 16d (12 mg, 22.7 μmol) was dissolved in 1.5 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V=2:1), palladium carbon (5 mg, 10% content) was added, hydrogen was replaced three times, and the reaction was stirred at room temperature for 2 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated under reduced pressure to obtain a crude title product 16e (10 mg), which was directly used in the next step without purification.

[0710] Step 5

[0711] (9H-fluoren-9-yl)methyl (2-((((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclobutyl)methoxy)methyl)amino)-2-oxoethyl)carbamate 16f

[0712] 1b (7.5 mg, 0.014 mmol) was added to the reaction flask, 1 mL of N,N-dimethylformamide was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, a drop of triethylamine was added, a solution of crude product 16e (10 mg) in 0.5 mL of N,N-dimethylformamide was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (6 mg, 0.026 mmol) was added, and the reaction was stirred in an ice bath for 30 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL×3), the organic phase was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 16f (10.6 mg, yield 87.8%).

[0713] MS m / z(ESI):856.2[M+1].

[0714] Step 6

[0715] 1-(((2-aminoacetylamino)methoxy)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]

[0716] Quinolin-1-yl)cyclobutane-1-carboxamide 16g

[0717] 16f (10.6 mg, 12.4 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added to concentrate under reduced pressure, and the process was repeated twice; 3 mL of n-hexane was added to slurry, and the upper layer of n-hexane was poured out, and the process was repeated three times. The crude title product 16 g (8 mg) was obtained by concentration under reduced pressure, and the product was used directly in the next step without purification.

[0718] MS m / z(ESI):634.1[M+1].

[0719] Step 7

[0720] 1-((S)-9-Benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa-4,7,10,13,16-pentaazacosyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-

[0721] 1-(2-(2-yl)cyclobutane-1-carboxamide 16

[0722] The crude product 16g (8mg) was dissolved in 1mL N,N-dimethylformamide, 8g (8.8mg, 18.6μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (5.2mg, 18.8μmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min) to obtain the title product 16 (1.0mg, yield: 7.2%).

[0723] MS m / z(ESI):1088.0[M+1].

[0724] Embodiment 17

[0725] (1r,4r)-N-((S)-7-benzyl-1-(1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropyloxy)-3,6,9,12,15-pentaoxo-17,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,14-pentaazatricarboxylic-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17

[0726]

[0727]

[0728] first step

[0729] 1-phenyl-2,5,8,11,14,17,20,23,26,29-decaoxahistriacont-31-oic acid tert-butyl ester 17b

[0730] 1-Phenyl-2,5,8,11,14,17,20,23,26-nonaoxaoctacos-28-ol 17a (0.34 g, 0.67 mmol, supplier: Bidex) was dissolved in 10 mL of dichloromethane, and silver oxide (0.24 g, 1.01 mmol), tert-butyl bromoacetate (0.16 g, 0.81 mmol) and potassium iodide (0.07 g, 0.40 mmol) were added in sequence, and the mixture was stirred at room temperature for 3 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a developing solvent system B to obtain the title product 17b (0.42 g, yield: 100%).

[0731] MS m / z(ESI):636.3[M+18].

[0732] Step 2

[0733] 29-Hydroxy-3,6,9,12,15,18,21,24,27-nonaoxaconicotin-1-oic acid tert-butyl ester 17c

[0734] 17b (417 mg, 0.67 mmol) was dissolved in 15 mL of tetrahydrofuran, palladium carbon (110 mg, 10% content, dry type) was added, hydrogen was replaced three times, and the temperature was raised to 60°C and stirred for 3 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to obtain the crude title product 17c (357 mg), which was directly used for the next step without purification.

[0735] MS m / z(ESI):546.2[M+18].

[0736] Step 3

[0737] 29-Azido-3,6,9,12,15,18,21,24,27-nonaoxaconicotin-1-oic acid tert-butyl ester 17d

[0738] 17c (357 mg, 0.675 mmol) was dissolved in 10 mL of toluene, and diphenylphosphoryl azide (279 mg, 1.014 mmol) and 1,8-diazabicycloundec-7-ene (206 mg, 1.353 mmol) were added. The argon atmosphere was replaced three times, and the reaction was stirred at room temperature for 2 hours, and then the temperature was raised to 105°C for 19 hours. The reaction solution was cooled to room temperature, concentrated, 20 mL of water was added, and extracted with ethyl acetate (10 mL×4). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with developing solvent system B to obtain the crude title product 17d (412 mg).

[0739] MS m / z(ESI):571.3[M+18].

[0740] Step 4

[0741] 29-Amino-3,6,9,12,15,18,21,24,27-nonaoxaconicotin-1-oic acid tert-butyl ester 17e

[0742] 17d (230 mg, 0.415 mmol) was dissolved in 8 mL of tetrahydrofuran, palladium carbon (58 mg, 10% content, dry type) was added, hydrogen was replaced three times, and the reaction was stirred at room temperature for 2 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to obtain the crude title product 17e (220 mg), which was directly used for the next step without purification.

[0743] MS m / z(ESI):528.2[M+1].

[0744] Step 5

[0745] 1-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl)-1-oxo

[0746] -5,8,11,14,17,20,23,26,29-nonaoxa-2-azahexatin-31-oic acid tert-butyl ester 17f

[0747] Dissolve (1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylic acid (98.5 mg, 0.415 mmol) in 10 mL of dichloromethane, add 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (190 mg, 0.500 mmol) and N,N-diisopropylethylamine (162 mg, 1.253 mmol), replace with argon three times, add crude product 17e (220 mg, 0.417 mmol), stir at room temperature for 1 hour. Add 15 mL of water, extract with dichloromethane (8 mL×3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (15 mL), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system B to obtain the title product 17f (122 mg, yield: 39.2%).

[0748] MS m / z(ESI):747.2[M+1].

[0749] Step 6

[0750] 1-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl)-1-oxo

[0751] -5,8,11,14,17,20,23,26,29-nonaoxa-2-azahexatin-31-oic acid 17g

[0752] 17f (122 mg, 0.163 mmol) was dissolved in 0.8 mL of dichloromethane, and 0.4 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 1 hour. 15 mL of dichloromethane was added for dilution and concentrated under reduced pressure; 10 mL of n-hexane was added and concentrated under reduced pressure, and the mixture was repeated twice; 10 mL of toluene was added and concentrated under reduced pressure; 10 mL of a mixed solvent of n-hexane: ether = 5:1 was used for slurrying three times until the pH was close to 7, and the mixture was concentrated and pumped dry with an oil pump to obtain the title product 17g (98 mg, yield: 86.8%).

[0753] MS m / z(ESI):691.2[M+1].

[0754] Step 7

[0755] 2,4-Dimethoxybenzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropyl-1-carboxylate 17h

[0756] 8d (164 mg, 0.40 mmol) was dissolved in dichloromethane (5 mL), and 2,4-dimethoxybenzyl alcohol (81 mg, 0.48 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (115 mg, 0.60 mmol) and 4-dimethylaminopyridine (5 mg, 0.041 mmol) were added in sequence. After the addition was completed, the mixture was stirred at room temperature for 1 hour. 20 mL of water was added, and the layers were separated after shaking. The aqueous phase was extracted with dichloromethane (8 mL × 3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with developing solvent system C to obtain the title product 17h (124 mg, yield: 55.4%).

[0757] MS m / z(ESI):583.1[M+23].

[0758] Step 8

[0759] 2,4-dimethoxybenzyl (S)-1-((11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa

[0760] -4,7,10,13,16-pentaazahepta-17-yl)oxy)cyclopropyl-1-carboxylate 17j

[0761] 17h (39 mg, 69.6 umol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added to concentrate under reduced pressure, and the mixture was repeated twice; 3 mL of n-hexane was added to slurry, and the upper n-hexane was poured out, and the mixture was repeated three times, and the mixture was concentrated under reduced pressure. The obtained crude product was dissolved in 2 mL of N,N-dimethylformamide, (((9H-fluorene-9-yl)methoxy)carbonyl)glycyl-L-phenylalanine 17i (35 mg, 69.8 μmol, prepared by the method disclosed in "Example 7-12 on page 13 of the specification of patent application CN108853514A"), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (23 mg, 83.1 μmol) was added, and the mixture was stirred at room temperature for 1 hour. Add 10 mL of water, extract with ethyl acetate (10 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (10 mL × 2), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the resulting residue by thin layer chromatography with developing solvent system B to obtain the title product 17j (48 mg, yield: 83.9%).

[0762] MS m / z(ESI):822.0[M+1].

[0763] Step 9 (S)-1-((11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecyl)

[0764] -17-yl)oxy)cyclopropane-1-carboxylic acid 17k

[0765] 17j (48 mg, 58.4 μmol) was dissolved in 1.4 mL of 3% (v / v) dichloroacetic acid in dichloromethane solution, cooled to 0-5°C in an ice-water bath, triethylsilane (21 mg, 180.6 μmol) was added, and the mixture was stirred in an ice bath for 3 hours. The mixture was concentrated under reduced pressure in an ice bath to remove half of the organic solvent, 5 mL of ether was added, and the mixture was naturally warmed to room temperature and slurried to precipitate a white solid, which was filtered, the filter cake was collected, and the mixture was pumped dry with an oil pump to obtain the title product 17k (33 mg, yield: 84.1%).

[0766] Step 10: (9H-fluoren-9-yl)methyl((S)-7-benzyl-1-(1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropyloxy)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridec-13-yl)carbamate 171

[0767] Add 1b (20 mg, 42.4 μmol) to the reaction bottle, add 1 mL of 10% (v / v) methanol in dichloromethane solution, replace the argon three times, cool to 0-5°C in an ice-water bath, add a drop of triethylamine, and stir until 1b is dissolved. Dissolve 17k (33 mg, 49.1 μmol) in 1 mL of 10% (v / v) methanol in dichloromethane solution, then add dropwise to the above reaction solution, and then add 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 μmol). Warm to room temperature and stir for 1 hour. Add 10 mL of dichloromethane and 5 mL of water, stir for 5 minutes, let stand and separate, collect the organic phase; extract the aqueous phase with dichloromethane (10 mL×3), and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (10 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 171 (37 mg, yield: 80.2%).

[0768] MS m / z(ESI):1090.1[M+1].

[0769] Step 11

[0770] (1r,4r)-N-((S)-7-benzyl-1-(1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropyloxy)-3,6,9,12,15-pentaoxo-17,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,14-pentaazatricarboxylic-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17

[0771] Dissolve 17l (15.5 mg, 14.23 μmol) in 0.6 mL of dichloromethane, add 0.3 mL of diethylamine, and stir at room temperature for 1.5 hours. Concentrate the reaction solution under reduced pressure, add 2 mL of toluene and concentrate under reduced pressure, repeat twice; add 3 mL of n-hexane to slurry, pour out the upper n-hexane, repeat three times. Concentrate under reduced pressure, and then pump dry with an oil pump. Dissolve the obtained crude product in 1 mL of N,N-dimethylformamide, add 17g (11 mg, 15.92 μmol), add 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (6.0 mg, 21.68 μmol), replace with argon three times, and stir at room temperature for 30 minutes. The reaction solution was purified by HPLC (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmolNH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding fractions were collected and concentrated under reduced pressure to give the title product 17 (6 mg, yield: 27.4%).

[0772] MS m / z(ESI):1556.4[M+18].

[0773] 1 H NMR (400MHz, DMSO-d6): δ8.98(d,1H),8.76(s,1H),8.20(br,1H),8.12-7.95(m,3H),7.93-7.76(m,2H),7.75-7.66(m,2H),7.24(s,1H),7.20- 7.05(m,6H),6.97(s,1H),6.64(br,1H),6.55(d,1H),6.47(s,1H),5.61 -5.52(m,2H),5.37(s,1H),5.33-5.23(m,2H),5.18(s,1H),5.13(s,1H) ,5.05(s,1H),5.00(s,1H),4.65-4.55(m,2H),4.53-4.45(m,1H),4.38 -4.28(m,2H),3.84(s,2H),3.67(d,3H),3.60-3.40(m,33H),3.18(d,1H ),3.15-3.08(m,3H),2.28(s,3H),2.00-1.92(m,3H),1.85(s,2H),1.82 -1.73(m,2H),1.68-1.52(m,4H),1.29-1.15(m,3H),0.86-0.76(m,5H).

[0774] Embodiment 18

[0775] (1r,4r)-N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline -1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaazatetrahexa-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18

[0776]

[0777] first step

[0778] (R)-2-Cyclopropyl-2-hydroxybenzyl acetate 18a

[0779] (S)-2-Cyclopropyl-2-hydroxybenzyl acetate 18b

[0780] 2a (7.4 g, 63.7 mmol) was dissolved in 200 mL of acetonitrile, and potassium carbonate (35 g, 253.6 mmol), benzyl bromide (9.3 g, 54.4 mmol) and tetrabutylammonium iodide (500 mg, 1.36 mmol) were added in sequence. The reaction solution was stirred at room temperature for 16 hours, filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate (10 ml), the combined filtrate was concentrated under reduced pressure, and the obtained residue 4.1 g was purified by silica gel column chromatography with developing solvent system C, and further chiral separation and purification were obtained to obtain the title products 18a (1.1 g) and 18b (1.2 g).

[0781] Step 2

[0782] (R)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester

[0783] 18c

[0784] Dissolve 8b (3.1 g, 8.41 mmol) in tetrahydrofuran (55 mL), add 18a (2.0 g, 9.70 mmol), cool to 0-5°C in an ice-water bath, add potassium tert-butoxide (1.89 g, 16.84 mmol), and stir for 10 minutes in an ice-water bath. Add ethyl acetate (30 mL) and water (20 mL), let stand to separate, extract the aqueous phase with chloroform (30 mL×5), and combine the organic phases. The organic phase is concentrated under reduced pressure, and the resulting residue is dissolved in 1,4-dioxane (32 mL) and water (8 mL), and sodium carbonate (1.78 g, 16.79 mmol) and 9-fluorenylmethyl chloroformate (2.18 g, 8.42 mmol) are added, and stir at room temperature for 2 hours. Add water (30 mL) to the reaction solution, extract with ethyl acetate (50 mL×3), and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography using developing solvent System C to obtain the title product 18c (1.3 g, yield: 30.0%).

[0785] MS m / z(ESI):515.2[M+1].

[0786] Step 3

[0787] (R)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid 18d

[0788] 18c (1.29 g, 2.51 mmol) was dissolved in ethyl acetate (15 mL), palladium carbon (260 mg, 10% content, dry type) was added, hydrogen was replaced three times, and the reaction was stirred at room temperature for 5 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate (20 mL) and methanol (20 mL), and the filtrate was concentrated to obtain the crude title product 18d (980 mg), which was directly used for the next step without purification.

[0789] MS m / z(ESI):425.1[M+1].

[0790] Step 4

[0791] 2,4-Dimethoxybenzyl (R)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-ol 18e

[0792] The crude product 18d (980 mg, 2.31 mmol) was dissolved in dichloromethane (15 mL), and 2,4-dimethoxybenzyl alcohol (777 mg, 4.62 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (664 mg, 3.46 mmol) and 4-dimethylaminopyridine (28 mg, 0.23 mmol) were added, and stirred at room temperature for one hour. The organic solvent was removed by concentration under reduced pressure, 20 mL of water was added, and the mixture was extracted with ethyl acetate (50 mL×3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (30 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with a developing solvent system C to obtain the title product 18e (810 mg, yield: 61.1%).

[0793] MS m / z(ESI):575.0[M+1].

[0794] Step 5

[0795] 2,4-Dimethoxybenzyl (R)-2-((2-aminoacetamido)methoxy)-2-cyclopropyl acetate 18f

[0796] 18e (33 mg, 57.4 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure, and the reaction was repeated twice; 3 mL of n-hexane was added to slurry, and the upper n-hexane was poured out, and the reaction was repeated three times, and the crude title product 18f (21 mg) was obtained by concentration under reduced pressure, and the product was used directly in the next step without purification.

[0797] Step 6

[0798] 2,4-Dimethoxybenzyl (11S, 19R)-11-benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo

[0799] -2,18-dioxa-4,7,10,13,16-pentaazaeicosyl-20-acid ester 18g

[0800] The crude product 18f (21 mg, 57.4 μmol) was dissolved in 3 mL of N,N-dimethylformamide, 17i (29 mg, 57.8 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (19 mg, 68.7 μmol) was added, and the mixture was stirred at room temperature for 1 hour. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL×3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 18g (37 mg, yield: 77.1%).

[0801] MS m / z(ESI):853.0[M+18].

[0802] Step 7

[0803] (11S,19R)-11-Benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa

[0804] -4,7,10,13,16-pentaazaicosanoic acid-20-oic acid 18h

[0805] 18g (37mg, 44.3μmol) was dissolved in 1.4mL of 3% (v / v) dichloroacetic acid in dichloromethane solution, cooled to 0-5°C in an ice-water bath, triethylsilane (15.4mg, 132.4μmol) was added, and the mixture was stirred in an ice bath for 3 hours. Half of the organic solvent was removed by vacuum concentration under an ice bath, 5mL of ether was added, and the mixture was naturally warmed to room temperature and slurried to precipitate a white solid, which was filtered, the filter cake was collected, and the mixture was pumped dry with an oil pump to obtain the title product 18h (24mg, yield: 79.1%). MS m / z (ESI): 708.2 [M+23].

[0806] Step 8 (9H-fluoren-9-yl)methyl ((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl)

[0807] 1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexa-16-yl)carbamate 18i

[0808] Add 1b (30 mg, 63.6 μmol) to the reaction bottle, add 1 mL of 10% (v / v) methanol in dichloromethane solution, replace the argon three times, cool to 0-5°C in an ice-water bath, add a drop of triethylamine, and stir until 1b is dissolved. Dissolve 18h (65 mg, 94.8 μmol) in 1 mL of 10% (v / v) methanol in dichloromethane solution, then add dropwise to the above reaction solution, and then add 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (27 mg, 97.6 μmol). Warm to room temperature and stir for 1 hour. Add 10 mL of dichloromethane and 5 mL of water, stir for 5 minutes, let stand and separate, collect the organic phase; extract the aqueous phase with dichloromethane (10 mL×3), and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (10 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 18i (25 mg, yield: 35.6%).

[0809] MS m / z(ESI):1104.4[M+1].

[0810] Step 9 (S)-2-(2-(2-aminoacetylamino)acetylamino)-N-(2-((((R)-1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethoxy)-3-phenylpropanamide 18j

[0811] 18i (12 mg, 10.9 μmol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure, and the reaction was repeated twice. 3 mL of n-hexane was added for pulping, and the upper n-hexane was poured out, and the reaction was repeated three times. The crude title product 18j (10 mg) was obtained and the product was used directly in the next step without purification.

[0812] MS m / z(ESI):881.0[M+1].

[0813] Step 10: (1r,4r)-N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b] Quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaazatetrahexa-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18

[0815] The crude product 18j (10 mg) was dissolved in 1 mL of N,N-dimethylformamide, 17 g (8.5 mg, 12.3 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (4.6 mg, 16.6 μmol) was added, and stirred at room temperature for 30 minutes. The reaction solution was filtered and purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD5um 19*250mm; mobile phase: A-water (10 mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18 mL / min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product 18 (9.5 mg, yield: 56.2%).

[0816] MS m / z(ESI):1570.2[M+18].

[0817] 1H NMR (400MHz, DMSO-d6): δ8.77(d,1H),8.59-8.55(m,1H),8.42(d,1H),8.37-8.28(m,1H),8.25-8.06(m,2H),7.96-7.86(m,1H),7. 86-7.70(m,2H),7.32-7.28(m,1H),7.25-7.14(m,3H),6.67(m,1H),5.96(s,1H),5.80-5.72(m,1H),5.62-5.52(m,2H),5.43-5.30 (m,3H),5.28-5.17(m,2H),5.12-5.08(m,1H),4.72-4.35(m,8H),3.95-3.70(m,13H),3.35-3.22(m,14H),2.42-2.32(m,3H),2.05 -1.98(m,4H),1.88-1.82(m,12H),1.47-1.39(m,3H),1.32-1.18(m,11H),0.90-0.80(m,4H),0.52-0.37(m,3H),0.32-0.18(m,2H).

[0818] Embodiment 19

[0819] (1r,4r)-N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b] Quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaazatetrahexa-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide

[0820]

[0821] first step

[0822] (S)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester 19a

[0823] 18b (252 mg, 1.22 mmol) was added to the reaction flask, 4 mL of dichloromethane was added, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, lithium tert-butoxide (98 mg, 1.22 mmol) was added, the reaction was stirred for 15 minutes in an ice-water bath, and 8b (300 mg, 814.3 μmol) was added, and the reaction was stirred for 2.5 hours in an ice-water bath. Water (10 mL) was added, and the liquid was separated. The aqueous phase was extracted with dichloromethane (8 mL × 2), and the organic phases were combined and washed with water (10 mL × 1), washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The residue was purified by silica gel column chromatography with developing solvent system C to obtain the title product 19a (282 mg, yield: 67.2%).

[0824] Step 2

[0825] (S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid 19b

[0826] 19a (280 mg, 0.554 mmol) was dissolved in 8 mL of ethyl acetate, palladium carbon (84 mg, 10% content, dry type) was added, hydrogen was replaced three times, and the reaction was stirred at room temperature for 3 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude title product 19b (230 mg), which was directly used for the next step without purification.

[0827] Step 3

[0828] 2,4-Dimethoxybenzyl (S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oate 19c

[0829] The crude product 19b (230 mg, 541.8 μmol) was dissolved in 7 mL of dichloromethane, and 2,4-dimethoxybenzyl alcohol (136.7 mg, 812.7 μmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (155 mg, 808.5 μmol) and 4-dimethylaminopyridine (6.6 mg, 53.5 μmol) were added in sequence, and stirred at room temperature for 16 hours. The reaction solution was diluted with 10 mL of dichloromethane, washed with water (10 mL × 1), washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. The residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 19c (159 mg, yield: 51.0%)

[0830] Step 4

[0831] 2,4-Dimethoxybenzyl (S)-2-((2-aminoacetamido)methoxy)-2-cyclopropyl acetate 19d

[0832] 19c (60 mg, 104.4 μmol) was dissolved in 1 mL of dichloromethane, and 0.5 mL of diethylamine was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure, and the reaction was repeated twice; 3 mL of n-hexane was added to slurry, and the upper n-hexane layer was poured out, and the reaction was repeated three times, and the crude title product 19d (21 mg) was obtained after concentration under reduced pressure, and the product was used directly in the next step without purification.

[0833] Step 5

[0834] 2,4-Dimethoxybenzyl (11S, 19S)-11-benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo

[0835] -2,18-dioxa-4,7,10,13,16-pentaazaicosyl-20-oic acid ester 19e

[0836] The crude product 19d (36 mg, 102.2 μmol) was dissolved in 4 mL of N,N-dimethylformamide, 17i (52 mg, 103.6 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (34.6 mg, 125.0 μmol) was added, and the mixture was stirred at room temperature for 1 hour. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL×3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 19e (70 mg, yield: 80.2%).

[0837] Step 6

[0838] (11S,19S)-11-Benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa

[0839] -4,7,10,13,16-pentaazaicos-20-oic acid 19f

[0840] 19e (70 mg, 83.7 μmol) was dissolved in 2.5 mL of 3% (v / v) dichloroacetic acid in dichloromethane solution, cooled to 0-5°C in an ice-water bath, triethylsilane (29 mg, 249.4 μmol) was added, and the mixture was stirred in an ice bath for 3 hours. The mixture was concentrated under reduced pressure in an ice bath to remove half of the organic solvent, 5 mL of ether was added, and the mixture was naturally warmed to room temperature and slurried to precipitate a white solid, which was filtered, the filter cake was collected, and the mixture was pumped dry with an oil pump to obtain the title product 19f (57 mg, yield: 99.2%).

[0841] Step 7: (9H-fluoren-9-yl)methyl((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexa-16-yl)carbamate 19g

[0842] 1b (30 mg, 63.6 μmol) was added to the reaction flask, and 1 mL of 10% (v / v) methanol in dichloromethane solution was added. The argon atmosphere was replaced three times, and the temperature was cooled to 0-5°C in an ice-water bath. A drop of triethylamine was added and stirred until 1b was dissolved. 19f (57 mg, 83.1 μmol) was dissolved in 1 mL of 10% (v / v) methanol in dichloromethane solution, and then added dropwise to the above reaction solution, and then 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (26 mg, 93.9 μmol) was added. The mixture was heated to room temperature and stirred for 1 hour. 10 mL of dichloromethane and 5 mL of water were added, and the mixture was stirred for 5 minutes. The organic phase was collected; the aqueous phase was extracted with dichloromethane (10 mL×3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography using developing solvent system B to obtain 19 g (56 mg, yield: 79.8%) of the title product.

[0843] MS m / z(ESI):1103.1[M+1].

[0844] Step 8 (S)-2-(2-(2-aminoacetylamino)acetylamino)-N-(2-((((S)-1-cyclopropyl-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12Hbenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide 19h

[0845] 19g (4.6mg, 4.16μmol) was dissolved in 1.5mL of dichloromethane, 0.75mL of diethylamine was added, and the mixture was stirred at room temperature for 1.6 hours. The reaction solution was concentrated under reduced pressure, and 2mL of toluene was added and concentrated under reduced pressure, and the reaction was repeated twice. 3mL of n-hexane was added for pulping, and the upper n-hexane was poured out, and the reaction was repeated three times. The crude title product 19h (4.0mg) was obtained, and the product was used directly in the next step without purification.

[0846] Step 9 (1r,4r)-N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b] Quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaazatetrahexa-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 19

[0847] The crude product 19h (4.0 mg) was dissolved in 1 mL of N,N-dimethylformamide, 17 g (2.9 mg, 4.2 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (1.5 mg, 5.4 μmol) was added, and the mixture was stirred at room temperature for 40 minutes. The reaction solution was filtered and purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um19*250mm; mobile phase: A-water (10 mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18 mL / min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product 19 (2.1 mg, yield: 32.4%).

[0848] 1H NMR (400MHz, DMSO-d6): δ8.71-8.62(m,1H),8.59-8.51(m,1H),8.34-8.26( m,1H),8.14-8.02(m,2H),7.95-7.86(m,1H),7.83-7.69(m,2H),7.35-7.31 (m,1H),7.29-7.11(m,3H),7.01(s,1H),6.72-6.50(m,3H),5.59-5.50(m,2 H),5.42(s,2H),5.38-5.18(m,3H),4.79-4.69(m,2H),4.61-4.42(m,3H),3 .91(s,2H),3.79-3.65(m,4H),3.63-3.44(m,13H),3.41-3.30(m,2H),3.26 -3.09(m,5H),3.08-2.84(m,4H),2.81-2.64(m,3H),2.42-2.28(m,3H),2.2 4-2.12(m,2H),2.05-1.93(m,4H),1.89-1.77(m,2H),1.72-1.56(m,3H),1. 53-1.38(m,3H),1.34-1.10(m,11H),0.94-0.78(m,5H),0.52-0.35(m,3H).

[0849] Example 20 (reference example)

[0850]

[0851] The title compound 20 was synthesized according to the method provided in "Example 58 on page 163 of the specification of CN104755494A".

[0852] The following antibodies are prepared according to conventional antibody methods, for example, after constructing a vector, they can be transfected into eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019), purified and expressed.

[0853] The following is the sequence of Trastuzumab:

[0854] Light chain

[0855] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFL

[0856] YSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT

[0857] VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES

[0858] VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0859] SEQ ID NO.1 Heavy chain

[0860] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYP

[0861] TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY

[0862] AMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT

[0863] VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT

[0864] KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV

[0865] DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN

[0866] GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV

[0867] KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

[0868] FSCSVMHEALHNHYTQKSLSLSPGK

[0869] SEQ ID NO.2 The following is the sequence of Pertuzumab:

[0870] Light chain

[0871] DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYR

[0872] YTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRT

[0873] VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES

[0874] VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0875] SEQ ID NO.3 heavy chain

[0876] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADV

[0877] NPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSF

[0878] YFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

[0879] SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK

[0880] VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD

[0881] VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG

[0882] KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK

[0883] GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF

[0884] SCSVMHEALHNHYTQKSLSLSPGK

[0885] SEQ ID NO.4 The following is the sequence of B7H3 antibody 1F9DS:

[0886] Light chain

[0887] DTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSHYPSWYQQTPGQAPRMLIYNTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCAIHVDRDIWVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC

[0888] SEQ ID NO.5

[0889] Heavy chain

[0890] QVQLVQSGGGVVQPGTSLRLSCAASGFIFSSSAMHWVRQAPGKGLEWVAVISYDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSARLYASFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0891] SEQ ID NO.6

[0892] Example 21 ADC-1

[0893]

[0894] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to a PBS buffer solution of the antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 2.5 ml, 9.96 mg / ml, 0.168 μmol), and the solution was placed in a water bath oscillator and oscillated at 37°C for 3 hours to stop the reaction; the reaction solution was cooled to 25°C with a water bath, diluted to 5.0 mg / ml, and 2.0 ml of the solution was taken out for further reaction.

[0895] Compound 10-short retention time compound (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL DMSO, added to the above 2.0 ml solution, placed in a water bath shaker, shaken at 25° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula FADC-1 in PBS buffer (5.0 mg / mL, 1.1 mL), which was stored frozen at 4° C.

[0896] UV-HPLC calculated average value: n=5.09.

[0897] Example 22 ADC-2

[0898]

[0899] At 37°C, to the PBS buffered aqueous solution of antibody Trastuzumab (0.05M PBS buffered aqueous solution at pH = 6.5; 2.5 ml, 9.96 mg / ml, 0.168 μmol), add the prepared aqueous solution of tri(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol), place in a water bath oscillator, oscillate at 37°C for 3 hours, and stop the reaction; cool the reaction solution to 25°C with a water bath, dilute to 5.0 mg / ml, and take out 2.0 ml of the solution for further reaction.

[0900] Compound 10 (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL DMSO, added to the above 2.0 ml solution, placed in a water bath shaker, shaken at 25° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the FADC-1 general formula, ADC-2, in PBS buffer (4.95 mg / mL, 1.1 mL), which was stored frozen at 4° C.

[0901] UV-HPLC calculated average value: n=7.39.

[0902] Example 23 ADC-3

[0903]

[0904] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to a PBS buffer solution of the antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 2.5 ml, 9.96 mg / ml, 0.168 μmol), and the solution was placed in a water bath oscillator and oscillated at 37°C for 3 hours to stop the reaction; the reaction solution was cooled to 25°C with a water bath, diluted to 5.0 mg / ml, and 2.0 ml of the solution was taken out for further reaction.

[0905] Compound 8 (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL DMSO, added to the above 2.0 ml solution, placed in a water bath shaker, shaken at 25° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the FADC-3 general formula, ADC-3, in PBS buffer (5.24 mg / mL, 1.1 mL), which was stored frozen at 4° C.

[0906] UV-HPLC calculated average value: n=7.36.

[0907] Example 24 ADC-4

[0908]

[0909] At 37°C, to the PBS buffered aqueous solution of antibody Trastuzumab (0.05M PBS buffered aqueous solution at pH=6.5; 3.74 ml, 13.38 mg / ml, 0.338 μmol), add the prepared aqueous solution of tri(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol), place in a water bath oscillator, oscillate at 37°C for 3 hours, and stop the reaction; cool the reaction solution to 25°C with a water bath, dilute to 6.7 mg / ml, and take out 1.3 ml of the solution for further reaction.

[0910] Compound 9-short retention time compound 9-A (1.0 mg, 0.93 μmol) was dissolved in 0.10 mL DMSO, added to the above 1.3 ml solution, placed in a water bath shaker, shaken at 25° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product ADC-4 of the general formula FADC-4A in PBS buffer (1.72 mg / mL, 2.36 mL), which was stored frozen at 4° C.

[0911] UV-HPLC calculated average value: n=7.39.

[0912] Example 25 ADC-5

[0913]

[0914] At 37°C, to the PBS buffered aqueous solution of antibody Trastuzumab (0.05M PBS buffered aqueous solution at pH = 6.5; 3.0 ml, 6.70 mg / ml, 0.136 μmol), add the prepared aqueous solution of tri(2-carboxyethyl)phosphine (10 mM, 0.067 mL, 0.67 μmol), place in a water bath oscillator, oscillate at 37°C for 3 hours, and stop the reaction; cool the reaction solution to 25°C with a water bath, and take out 0.614 ml of the solution for further reaction.

[0915] Compound 9-short retention time compound 9-A (0.5 mg, 0.42 μmol) was dissolved in 0.031 mL DMSO, added to the above 0.614 ml solution, placed in a water bath shaker, shaken at 25° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product ADC-5 of the general formula FADC-4A in PBS buffer (3.08 mg / mL, 0.82 mL), which was stored frozen at 4° C.

[0916] UV-HPLC calculated average value: n=3.16.

[0917] Example 26 ADC-6

[0918]

[0919] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to a PBS buffered aqueous solution of the antibody Trastuzumab (0.05 M PBS buffered aqueous solution at pH = 6.5; 3.74 ml, 13.38 mg / ml, 0.338 μmol), and the solution was placed in a water bath oscillator and oscillated at 37°C for 3 hours to stop the reaction; the reaction solution was cooled to 25°C with a water bath, diluted to 6.7 mg / ml, and 0.75 ml of the solution was taken out for further reaction.

[0920] Compound 9-longer retention time compound 9-B (0.68 mg, 0.63 μmol) was dissolved in 0.10 mL DMSO, added to the above 0.75 ml solution, placed in a water bath shaker, shaken at 25° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5, containing 0.001 M EDTA) to obtain PBS buffer (1.78 mg / mL, 1.78 mL) of the exemplary product ADC-6 of the general formula of FADC-4B, which was stored frozen at 4° C.

[0921] UV-HPLC calculated average value: n=3.94.

[0922] Example 27 ADC-7

[0923]

[0924] At 37°C, a prepared aqueous solution of tri(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to a PBS buffer solution of the antibody Pertuzumab (0.05 M PBS buffer solution at pH = 6.5; 5.0 ml, 10 mg / ml, 0.338 μmol), and the solution was placed in a water bath oscillator and oscillated at 37°C for 3 hours to stop the reaction; the reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg / ml, and 1.0 ml of the solution was taken out for further reaction.

[0925] Compound 8 (0.65 mg, 0.6 μmol) was dissolved in 0.1 mL DMSO, added to the above 1.0 ml solution, placed in a water bath shaker, shaken at 25° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula of FADC-7 in PBS buffer (1.42 mg / mL, 2.15 mL), which was stored frozen at 4° C.

[0926] UV-HPLC calculated average value: n=6.91.

[0927] Example 28 ADC-8

[0928]

[0929] At 37°C, a prepared aqueous solution of tri(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to a PBS buffer solution of the antibody Pertuzumab (0.05 M PBS buffer solution at pH = 6.5; 5.0 ml, 10 mg / ml, 0.338 μmol), and the solution was placed in a water bath oscillator and oscillated at 37°C for 3 hours to stop the reaction; the reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg / ml, and 1.6 ml of the solution was taken out for further reaction.

[0930] Compound 10 (1.04 mg, 1.0 μmol) was dissolved in 0.1 mL DMSO, added to the above 1.6 ml solution, placed in a water bath shaker, shaken at 25° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula FADC-8 in PBS buffer (2.14 mg / mL, 2.31 mL), which was stored frozen at 4° C.

[0931] UV-HPLC calculated average value: n=6.58.

[0932] Example 29 ADC-9

[0933]

[0934] At 37°C, a prepared aqueous solution of tri(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to a PBS buffer solution of the antibody Pertuzumab (0.05 M PBS buffer solution at pH = 6.5; 5.0 ml, 10 mg / ml, 0.338 μmol), and the solution was placed in a water bath oscillator and oscillated at 37°C for 3 hours to stop the reaction; the reaction solution was cooled to 25°C with a water bath, diluted to 5.0 mg / ml, and 0.8 ml of the solution was taken out for further reaction.

[0935] Compound 9-short retention time compound 9-A (0.55 mg, 0.5 μmol) was dissolved in 0.1 mL DMSO, added to the above 0.8 ml solution, placed in a water bath shaker, shaken at 25° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula FADC-9A, ADC-9 in PBS buffer (2.27 mg / mL, 1.11 mL), which was stored frozen at 4° C.

[0936] UV-HPLC calculated average value: n=3.16.

[0937] Example 30 ADC-10

[0938]

[0939] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 19.76uL, 197.6nmol) to the PBS buffer solution of antibody Trastuzumab (0.05M PBS buffer solution at pH=6.5; 10.0mg / ml, 0.574mL, 38.78nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0940] Compound 14 (0.64 mg, 588 nmol) was dissolved in 40 ul DMSO, added to the above reaction solution, placed in a water bath shaker, and oscillated at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula FADC-10 in PBS buffer (5.48 mg / mL, 1.03 mL), which was stored frozen at 4°C.

[0941] UV-Vis calculated average value: n=6.25.

[0942] Example 31 ADC-11

[0943]

[0944] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 22.24uL, 222.4nmol) to the PBS buffer solution of antibody Trastuzumab (0.05M PBS buffer solution at pH=6.5; 10.0mg / ml, 0.646mL, 43.64nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0945] Compound 14 (0.72 mg, 662 nmol) was dissolved in 40 ul DMSO, added to the above reaction solution, placed in a water bath shaker, and oscillated at 25° C. for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5, containing 0.001 M EDTA) to obtain PBS buffer (2.13 mg / mL, 1.87 mL) of the exemplary product ADC-11 of the general formula of FADC-10, which was stored frozen at 4° C.

[0946] UV-Vis calculated average value: n=7.03.

[0947] Example 32 ADC-12

[0948]

[0949] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 25.0uL, 250.0nmol) to the PBS buffer solution of antibody Trastuzumab (0.05M PBS buffer solution at pH=6.5; 10.0mg / ml, 0.726mL, 49.05nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0950] Compound 15 (0.81 mg, 754 nmol) was dissolved in 40 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula FADC-12 in PBS buffer (3.34 mg / mL, 1.45 mL), which was stored frozen at 4°C.

[0951] UV-Vis calculated average value: n=6.93.

[0952] Example 33 ADC-13

[0953]

[0954] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 9.88uL, 98.8nmol) to the PBS buffer solution of antibody Trastuzumab (0.05M PBS buffer solution at pH=6.5; 10.0mg / ml, 0.287mL, 19.39nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0955] Compound 16 (0.32 mg, 294 nmol) was dissolved in 20 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula FADC-13 in PBS buffer (2.37 mg / mL, 0.88 mL), which was stored frozen at 4°C.

[0956] UV-Vis calculated average value: n=6.53.

[0957] Example 34 ADC-14

[0958]

[0959] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 uL, 203.8 nmol) to the PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / ml, 0.592 mL, 40.0 nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0960] Compound 17 (0.92 mg, 598 nmol) was dissolved in 40 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula of FADC-14, ADC-14 in PBS buffer (0.30 mg / mL, 12.0 mL), which was stored frozen at 4°C.

[0961] UV-Vis calculated average value: n=7.61.

[0962] Example 35 ADC-15

[0963]

[0964] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 uL, 203.8 nmol) to the PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / ml, 0.592 mL, 40.0 nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0965] Compound 18 (0.93 mg, 599 nmol) was dissolved in 40 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula of FADC-15, ADC-15 in PBS buffer (0.32 mg / mL, 11.8 mL), which was stored frozen at 4°C.

[0966] UV-Vis calculated average value: n=7.89.

[0967] Example 36 ADC-16

[0968]

[0969] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 18.25uL, 182.5nmol) to the PBS buffer solution of antibody Trastuzumab (0.05M PBS buffer solution at pH=6.5; 10.0mg / ml, 0.53mL, 35.8nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0970] Compound 19 (0.83 mg, 534 nmol) was dissolved in 35 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula of FADC-16, ADC-16 in PBS buffer (0.32 mg / mL, 12.0 mL), which was stored frozen at 4°C.

[0971] UV-Vis calculated average value: n=7.43.

[0972] Example 37 ADC-17

[0973]

[0974] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 43.2uL, 432nmol) to the PBS buffer solution of antibody Trastuzumab (0.05M PBS buffer solution at pH=6.5; 10.0mg / ml, 2.0mL, 135.12nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0975] Compound 9-short retention time compound 9-A (2.22 mg, 2067 nmol) was dissolved in 175ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to obtain PBS buffer (1.32 mg / mL, 12.0 mL) of the exemplary product ADC-17 of the general formula of FADC-4A, which was stored frozen at 4°C.

[0976] UV-Vis calculated average value: n=5.42.

[0977] Example 38 ADC-18 (reference example)

[0978]

[0979] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 51.7uL, 517nmol) to the PBS buffer solution of antibody Trastuzumab (0.05M PBS buffer solution at pH=6.5; 10.0mg / ml, 1.5mL, 101.3nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0980] Compound 20 (2.0 mg, 1934 nmol) was dissolved in 100 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula of FADC-18, ADC-18 in PBS buffer (0.79 mg / mL, 13.0 mL), which was stored frozen at 4°C.

[0981] UV-Vis calculated average value: n=7.23.

[0982] Example 39 ADC-19

[0983]

[0984] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 46.9uL, 469nmol) to the PBS buffer solution of antibody Trastuzumab (0.05M PBS buffer solution at pH=6.5; 10.0mg / ml, 1.36mL, 91.9nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0985] Compound 9-short retention time compound 9-A (2.0 mg, 1862 nmol) was dissolved in 100 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain PBS buffer (0.73 mg / mL, 13.0 mL) of the exemplary product ADC-19 of the general formula of FADC-4A, which was stored frozen at 4°C.

[0986] UV-Vis calculated average value: n=6.26.

[0987] Example 40 ADC-20

[0988]

[0989] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 51.7uL, 517nmol) to the PBS buffer solution of antibody Trastuzumab (0.05M PBS buffer solution at pH=6.5; 10.0mg / ml, 1.5mL, 101.3nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0990] Compound 10 (2.0 mg, 1815 nmol) was dissolved in 100 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the FADC-1 general formula, ADC-20, in PBS buffer (0.73 mg / mL, 13.0 mL), which was stored frozen at 4°C.

[0991] UV-Vis calculated average value: n=7.43.

[0992] Example 41 ADC-21 (reference example)

[0993]

[0994] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 63.9uL, 639nmol) to the PBS buffer solution of antibody Trastuzumab (0.05M PBS buffer solution at pH=6.5; 10.0mg / ml, 1.86mL, 125.4nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0995] Compound 20 (2.07 mg, 2001 nmol) was dissolved in 150 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain PBS buffer (2.91 mg / mL, 4.44 mL) of the exemplary product ADC-21 of the general formula of FADC-18, which was stored frozen at 4°C.

[0996] UV-Vis calculated average value: n=7.23.

[0997] Example 42 ADC-22

[0998]

[0999] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 64.9uL, 649nmol) to the PBS buffer solution of antibody Trastuzumab (0.05M PBS buffer solution at pH=6.5; 10.0mg / ml, 1.88mL, 127.2nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[1000] Compound 9-short retention time compound 9-A (2.1 mg, 1955 nmol) was dissolved in 150ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH 6.5, containing 0.001M EDTA) to obtain PBS buffer (3.56 mg / mL, 3.98 mL) of the exemplary product ADC-22 of the general formula of FADC-4A, which was stored frozen at 4°C.

[1001] UV-Vis calculated average value: n=6.79.

[1002] Example 43 ADC-23 (reference example)

[1003]

[1004] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.89 mL, 118.9 umol) to the PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / ml, 345 mL, 23.31 umol), place in a water bath shaker, shake at 37°C for 3.5 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[1005] Compound 20 (362 mg, 350 umol) was dissolved in 7.12 ml MeCN and 3.56 mL DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified by ultrafiltration membrane using PBS buffer solution (0.05 M PBS buffer solution at pH = 6.5) containing 2% (v / v) MeCN and 1% (v / v) DMSO, and succinic acid buffer solution (0.01 M succinic acid buffer solution at pH = 5.3), and then sucrose was added to 60 mg / mL and Tween 20 to 0.2 mg / mL. After lyophilization in bottles, a freeze-dried powder sample of ADC-23, an exemplary product of the general formula of FADC-18, was obtained and stored frozen at 4° C.

[1006] UV-Vis calculated average value: n=7.05.

[1007] Example 44 ADC-24

[1008]

[1009] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.44 mL, 114.4 umol) to the PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / ml, 332 mL, 22.43 umol), place in a water bath shaker, shake at 37°C for 3.5 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[1010] Compound 9-short retention time compound 9-A (241 mg, 224 umol) was dissolved in 13.76 ml MeCN and 6.88 mL DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25° C. for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified by ultrafiltration membrane using PBS buffer solution (0.05 M PBS buffer solution at pH = 6.5) and succinic acid buffer solution (0.01 M succinic acid buffer solution at pH = 5.3), and then sucrose was added to 60 mg / mL and Tween 20 to 0.2 mg / mL. After lyophilization in bottles, a freeze-dried powder sample of the exemplary product ADC-24 of the general formula of FADC-4A was obtained, which was frozen and stored at 4° C.

[1011] UV-Vis calculated average value: n=7.07.

[1012] Example 45 ADC-25

[1013]

[1014] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 73.7uL, 740nmol) to the PBS buffer solution of antibody B7H3 antibody 1F9DS (pH=6.5 0.05M PBS buffer solution; 10.0mg / ml, 2.14mL, 144.60nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[1015] Compound 9-short retention time compound 9-A (3.0 mg, 2793 nmol) was dissolved in 150 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula FADC-25 in PBS buffer (1.28 mg / mL, 13.0 mL), which was stored frozen at 4°C.

[1016] UV-Vis calculated average value: n=6.87.

[1017] Example 46 ADC-26 (reference example)

[1018]

[1019] At 37°C, add the prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 30.1 uL, 300 nmol) to the PBS buffer solution of antibody B7H3 antibody 1F9DS (pH=6.5 0.05 M PBS buffer solution; 10.0 mg / ml, 0.89 mL, 60.14 nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[1020] Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula FADC-26 in PBS buffer (1.61 mg / mL, 4.0 mL), which was stored frozen at 4°C.

[1021] UV-Vis calculated average value: n=6.15.

[1022] Example 47 ADC-27

[1023]

[1024] At 37°C, add the prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 30.1 uL, 300 nmol) to the PBS buffer solution of antibody B7H3 antibody 1F9DS (pH=6.5 0.05 M PBS buffer solution; 10.0 mg / ml, 0.89 mL, 60.14 nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[1025] Compound 9-short retention time compound 9-A (1.02 mg, 950 nmol) was dissolved in 100 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain PBS buffer (1.94 mg / mL, 3.5 mL) of the exemplary product ADC-27 of the general formula of FADC-25, which was stored frozen at 4°C.

[1026] UV-Vis calculated average value: n=6.11.

[1027] Example 48 ADC-28 (reference example)

[1028]

[1029] At 37°C, add the prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 81.3 uL, 810 nmol) to the PBS buffer solution of antibody B7H3 antibody 1F9DS (pH=6.5 0.05 M PBS buffer solution; 10.0 mg / ml, 2.36 mL, 159.47 nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[1030] Compound 20 (3.0 mg, 2901 nmol) was dissolved in 150 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain PBS buffer (1.29 mg / mL, 13.0 mL) of the exemplary product ADC-28 of the general formula of FADC-26, which was stored frozen at 4°C.

[1031] UV-Vis calculated average value: n=7.46.

[1032] Example 49 ADC-29

[1033]

[1034] At 37°C, add the prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 28.6 uL, 290 nmol) to the PBS buffer solution of antibody B7H3 antibody 1F9DS (pH=6.5 0.05 M PBS buffer solution; 10.0 mg / ml, 0.80 mL, 50.06 nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[1035] Compound 9-short retention time compound 9-A (1.29 mg, 1201 nmol) was dissolved in 100 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain PBS buffer (2.63 mg / mL, 2.4 mL) of the exemplary product ADC-29 of the general formula of FADC-25, which was stored frozen at 4°C.

[1036] UV-Vis calculated average value: n=7.24.

[1037] Example 50 ADC-30 (reference example)

[1038]

[1039] At 37°C, add the prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 29.1 uL, 290 nmol) to the PBS buffer solution of antibody B7H3 antibody 1F9DS (pH=6.5 0.05 M PBS buffer solution; 10.0 mg / ml, 0.86 mL, 58.4 nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[1040] Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain PBS buffer (1.61 mg / mL, 4.0 mL) of the exemplary product ADC-30 of the general formula of FADC-26, which was stored frozen at 4°C.

[1041] UV-Vis calculated average value: n=6.15.

[1042] Example 51 ADC-31

[1043]

[1044] At 37°C, add the prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 30.1 uL, 300 nmol) to the PBS buffer solution of antibody B7H3 antibody 1F9DS (pH=6.5 0.05 M PBS buffer solution; 10.0 mg / ml, 0.89 mL, 60.14 nmol), place in a water bath shaker, shake at 37°C for 3 hours, and stop the reaction. Cool the reaction solution to 25°C in a water bath.

[1045] Compound 8 (1.0 mg, 943 nmol) was dissolved in 100 ul DMSO, added to the above reaction solution, placed in a water bath shaker, shaken at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the general formula FADC-31 in PBS buffer (1.47 mg / mL, 4.5 mL), which was stored frozen at 4°C.

[1046] UV-Vis calculated average value: n=6.33.

[1047] ADC stock solution drug loading analysis

[1048] Experimental purpose and principle

[1049] ADC stock solution is a kind of antibody cross-linked drug. Its mechanism of treating diseases is to rely on the targeting of antibodies to transport toxin molecules into cells, thereby killing cells. The drug loading plays a decisive role in the efficacy. The drug loading of ADC stock solution was measured using the UV method.

[1050] Experimental methods

[1051] Place the cuvettes filled with sodium succinate buffer in the reference absorption cell and the sample measurement absorption cell respectively, deduct the solvent blank, and then place the cuvette filled with the test solution in the sample measurement absorption cell to measure the absorbance at 280nm and 370nm.

[1052] Result calculation: The ADC stock solution loading was determined by ultraviolet spectrophotometry (using instrument: Thermo nanodrop2000 ultraviolet spectrophotometer). The principle is that the total absorbance of the ADC stock solution at a certain wavelength is equal to the sum of the absorbance of the cytotoxic drug and the monoclonal antibody at that wavelength, that is:

[1053] (1)A 280nm =ε mab-280 B mab +ε Drug-280 B C Drug

[1054] ε Drug-280 : The average molar extinction coefficient of the drug at 280nm is 5100;

[1055] C Drug : drug concentration;

[1056] ε mab-280 : The average molar extinction coefficient of trastuzumab or pertuzumab at 280 nm is 214600;

[1057] C mab : The concentration of trastuzumab antigen solution or pertuzumab antigen solution;

[1058] b: The optical path length is 1 cm.

[1059] Similarly, the total absorbance equation of the sample at 370nm can be obtained:

[1060] (2)A 370nm =ε mab-370 B C mab +ε Drug-370 B Drug

[1061] ε Drug-370 : The average molar extinction coefficient of the drug at 370nm is 19000;

[1062] C Drug : drug concentration;

[1063] ε mab-370 : The extinction coefficient of the trastuzumab antigen solution or the pertuzumab antigen solution at 370 nm is 0;

[1064] C mab : The concentration of trastuzumab antigen solution;

[1065] b: The optical path length is 1 cm.

[1066] The drug loading can be calculated by combining equations ⑴ and ⑵ with the extinction coefficients and concentration data of the monoclonal antibody and drug at two detection wavelengths.

[1067] Drug loading = C Drug / C mab .

[1068] Biological evaluation

[1069] Test Example 1: Test on the inhibition of tumor cell proliferation in vitro by the compound of formula (D)

[1070] 1. Test Purpose

[1071] The purpose of this experiment is to detect the inhibitory activity of the drug compound of general formula (D) of the present invention on the proliferation of U87MG cells (Cell Bank of the Chinese Academy of Sciences, Catalog #TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, Catalog No. HTB-30) in vitro. The cells were treated with different concentrations of the compound in vitro and cultured for 6 days. CTG (CellTiter- The cell proliferation was detected by using Luminescent Cell Viability Assay (Promega, Catalog No.: G7573), and the in vitro activity of the compound was evaluated based on the IC50 value.

[1072] 2. Experimental Methods

[1073] The following is an example of the in vitro proliferation inhibition test method for U87MG cells to illustrate the method of testing the in vitro proliferation inhibition activity of the compound of the present invention on tumor cells. This method is also applicable to, but not limited to, in vitro proliferation inhibition activity tests on other tumor cells.

[1074] 1. Cell culture: U87MG and SK-BR-3 cells were cultured with EMEM medium containing 10% FBS (GE, catalog number SH30024.01) and McCoy's 5A medium containing 10% FBS (Gibco, catalog number 16600-108), respectively.

[1075] 2. Cell preparation: Take U87MG and SK-BR-3 cells in the logarithmic growth phase, wash once with PBS (phosphate buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.), add 2-3 ml of trypsin (0.25% Trypsin-EDTA (1x), Gibico, Life Technologies) to digest for 2-3 minutes. After the cells are completely digested, add 10-15 ml of cell culture medium to wash the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 ml of cell culture medium to resuspend the cells to make a single cell suspension.

[1076] 3. Cell plating: Mix the single cell suspension of U87MG and SK-BR-3, and adjust the live cell density to 2.75×10 3 cells / ml and 8.25×10 3 cells / ml, mix the cell suspension after density adjustment, and add 180μl / well to a 96-well cell culture plate. Add only 200ul of culture medium to the peripheral wells of the 96-well plate. Incubate the culture plate in an incubator for 24 hours (37°C, 5% CO2).

[1077] 4. Compound preparation: Dissolve the compound in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) and prepare a storage solution with an initial concentration of 10 mM.

[1078] The initial concentration of the small molecule compound was 500 nM, and the preparation method was as follows.

[1079] Add 30μl of different samples to be tested to the first column of a 96-well U-bottomed drug plate, with a sample concentration of 100uM; add 20ul DMSO to each well from the 2nd to the 11th columns. Take 10ul of the sample from the first column and add it to the 20ul DMSO in the second column, mix well, take 10ul to the third column, and so on to the 10th column. Take 5ul of the drug from each well of the drug plate and add it to 95ul EMEM culture medium, mix well, and set aside.

[1080] The starting concentration of ADC was 10 nM or 500 nM, and the preparation method was as follows.

[1081] Add 100μl of different samples to be tested in the first column of the 96-well plate, with a sample concentration of 100nM or 5uM; add 100ul PBS to each well in columns 2 to 11. Take 50ul of the sample in the first column and add it to 100ul PBS in the second column, mix well, take 50ul to the third column, and so on, dilute 3 times to the 10th column.

[1082] 5. Sample addition operation: Add 20 μl of the sample to be tested of different concentrations to the culture plate, with two replicate wells for each sample. Incubate the culture plate in an incubator for 6 days (37° C., 5% CO2).

[1083] 6. Color development operation: Take out the 96-well cell culture plate, add 90 μl CTG solution to each well, and incubate at room temperature for 10 minutes.

[1084] 7. Plate reading operation: Take out the 96-well cell culture plate, place it in an ELISA reader (BMG labtech, PHERAstar FS), and measure the chemiluminescence using the ELISA reader.

[1085] 3. Data Analysis

[1086] The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5. The experimental results are shown in the table below.

[1087] Table 1 IC values ​​of the small molecule fragments disclosed in the present invention for inhibiting proliferation of SK-BR-3 cells and U87 cells in vitro 50 value.

[1088]

[1089]

[1090] Conclusion: The small molecule fragments disclosed in the present invention have obvious proliferation inhibitory activity on SK-BR-3 cells and U87 cells, and the chiral center has a certain influence on the inhibitory activity of the compound.

[1091] Test Example 2: In vitro proliferation inhibition test of HER2-targeted tumor cells by the disclosed antibody-drug conjugate

[1092] The purpose of this experiment is to detect the inhibitory activity of the disclosed antibody-drug conjugate targeting HER2 on the in vitro proliferation of SK-BR-3 (human breast cancer cells, ATCC, Catalog No. HTB-30) and MDA-MB-468 (human breast cancer cells, ATCC, Catalog No. HTB-132). The cells were treated with different concentrations of the compound in vitro, and after 6 days of culture, the proliferation of the cells was detected using CTG reagent. According to the IC 50 The values ​​were used to evaluate the in vitro activity of the compound.

[1093] According to the test method of Test Example 1, the test cells are SK-BR-3 and MDA-MB-468, and the cell culture medium is McCoy's 5A medium (Gibco, Catalog No. 16600-108) containing 10% FBS, EMEM medium (GE, Catalog No. SH30024.01) containing 10% FBS, and L-15 medium (ThermoFisher, Catalog No. 11415-114) containing 10% FBS. The three cell lines were adjusted to a viable cell density of 8.33×10 3 cells / ml, 8.33×10 3 cells / ml and 1.39×10 4 cells / ml, mix the cell suspension after density adjustment, and add 180μl / well to a 96-well cell culture plate. The relevant compounds were tested and the results are shown in the table below.

[1094] Table 2 discloses the IC values ​​of antibody-drug conjugates for in vitro proliferation inhibition of HER2-targeted tumor cells 50 value.

[1095]

[1096] Conclusion: The antibody-drug conjugates targeting HER2 target disclosed in the present invention have obvious proliferation inhibition activity on HER2-positive cells SK-BR-3; at the same time, they have weak proliferation inhibition activity on HER2-negative cells MDA-MB-468; and have good selectivity.

[1097] Test Example 3: Her2-ADC plasma stability experiment

[1098] ADC-19 samples, ADC-18 samples, ADC-20 samples, human plasma, monkey plasma (Shanghai Medicilon Biopharmaceutical Co., Ltd.), and 1% BSA (Sigma) PBS solution (Shanghai Biotech) were sterilized by filtration with a 0.22 μm filter. ADC-19, ADC-18, and ADC-20 were added to the above sterile plasma or 1% BSA PBS solution at a final concentration of 200 ug / ml, respectively, and incubated in a 37°C cell culture incubator. The day of incubation was recorded as day 0, and then samples were taken out on days 7, 14, and 21 for free toxin detection.

[1099] Take 25 μl of sample into a 96-well plate; add 50 μl of internal standard working solution (100 ng / mL camptothecin acetonitrile solution) and 150 μl of acetonitrile; vortex mix for 5 minutes, centrifuge for 10 minutes (4000 rpm), and perform LC / MS / MS (Applied Biosystems, USA) analysis on 5 ul.

[1100] The results showed that ADC-19 was quite stable in human and monkey plasma, as well as in 1% BSA PBS solution. The release rate of free toxin did not exceed 2.1% at most, and it tended to be stable on the 14th day. Figure 1A .

[1101] ADC-18 has poor stability in human and monkey plasma, with the highest free toxin release rates of 14.5% and 8.10%, respectively. It is relatively stable in 1% BSA PBS solution. Figure 1B .

[1102] ADC-20 has poor stability in human plasma, monkey plasma and 1% BSA PBS solution, with the highest release rates of free toxin being 21.7%, 29.7% and 21.7%, respectively. In 1% BSA PBS solution, it is always in a degradation state. Figure 1C .

[1103] Test Example 4: Evaluation of drug efficacy in JIMT-1 tumor-bearing mice

[1104] 1. Purpose of the test

[1105] Nunu mice were used as test animals to evaluate the efficacy of Her2-ADC antibodies T-DM1, ADC-21, and ADC-24 after intraperitoneal injection on nude mice with trastuzumab-resistant human breast cancer cell line JIMT-1 xenografts.

[1106] 2. Test drugs and materials

[1107] 1. Test drug

[1108] T-DM1 (prepared with reference to US20050169933)

[1109] ADC-21: 3 mg / kg

[1110] ADC-21: 10 mg / kg

[1111] ADC-24: 3 mg / kg

[1112] ADC-24: 10 mg / kg

[1113] Blank control: PBS

[1114] 2. Preparation method: All preparations were diluted with PBS.

[1115] 3. Experimental animals

[1116] Nunu mice were purchased from Beijing Weitonglihua.

[1117] 3. Test methods

[1118] JIMT-1 cells (Nanjing Kebai) (5×10 6 / , with 50% artificial basement membrane), the tumor grew for 8 days and grew to 203.09±11.94mm 3 The animals were then randomly divided into groups (d1), 8 animals per group, for a total of 6 groups.

[1119] The drug was administered by intraperitoneal injection for 2 times in total. The tumor volume and body weight were measured twice a week and the data were recorded.

[1120] Data statistics were calculated using Excel 2003 statistical software: the mean was calculated as avg; the SD was calculated as STDEV; the SEM was calculated as STDEV / SQRT; and the P value of the difference between groups was calculated as TTEST.

[1121] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[1122] Relative volume (RTV) = V T / V0

[1123] Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%)

[1124] Where V0, V T These are the tumor volumes at the beginning and end of the experiment, respectively. RTV , T RTV These are the relative tumor volumes of the blank control group (Vehicle, PBS) and the experimental group at the end of the experiment.

[1125] 4. Test results

[1126] The experimental results are as follows Figure 2 The results showed that the drug was intraperitoneally injected twice and the experiment was terminated on the 34th day. T-DM1 (10 mg / kg) had no inhibitory effect on tumors; ADC-21, 3 mg / kg had an inhibitory rate of 46.22% (P < 0.01); ADC-21, 10 mg / kg had an inhibitory rate of 56.77% (P < 0.001); ADC-24, 3 mg / kg had an inhibitory rate of 62.77% (P < 0.001); ADC-24, 10 mg / kg had an inhibitory rate of 76.32% (P < 0.001). Under the same dosage, the inhibitory effect of ADC-24 was significantly better than that of ADC-21.

[1127] Test Example 5: Evaluation of drug efficacy in SK-BR-3 tumor-bearing mice

[1128] 1. Purpose of the test

[1129] Nunu mice were used as test animals to evaluate the efficacy of Her2-ADC antibodies ADC-21 and ADC-22 on nude mice bearing human breast cancer cell SK-BR-3 xenografts after intraperitoneal injection.

[1130] 2. Test drugs and materials

[1131] 1. Test drug

[1132] ADC-21: 1 mg / kg

[1133] ADC-21: 6 mg / kg

[1134] ADC-22: 1 mg / kg

[1135] ADC-22: 6 mg / kg

[1136] Blank control: PBS.

[1137] 2. Preparation method: All preparations were diluted with PBS.

[1138] 3. Experimental animals

[1139] Nunu mice were purchased from Beijing Weitonglihua.

[1140] 3. Test methods

[1141] SK-BR-3 cells (ATCC) (5×10 6 / mouse, with 50% artificial basement membrane), the tumor grew for 20 days, and when it grew to 153.34±11.73mm3, the animals were randomly divided into groups (d0), 8 animals / group, for a total of 5 groups.

[1142] The drug was administered once by intraperitoneal injection. The tumor volume and body weight were measured twice a week and the data were recorded.

[1143] Data statistics were calculated using Excel 2003 statistical software: the mean was calculated as avg; the SD was calculated as STDEV; the SEM was calculated as STDEV / SQRT; and the P value of the difference between groups was calculated as TTEST.

[1144] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[1145] Relative volume (RTV) = V T / V0

[1146] Tumor inhibition rate (%) = (C RTV -T RTV ) / CRTV (%)

[1147] Where V0, V T These are the tumor volumes at the beginning and end of the experiment, respectively. RTV , T RTV They are the relative tumor volumes of the blank control and experimental groups at the end of the experiment.

[1148] 4. Test results

[1149] The experimental results are as follows Figure 3 The results showed that after intraperitoneal injection once, the experiment was terminated on the 28th day. The tumor inhibition rate of ADC-21 1mg / kg was 15.01%; the tumor inhibition rate of ADC-21 6mg / kg was 77.4%, and there was a very significant difference compared with the blank control (P < 0.001). The tumor inhibition rate of ADC-22 1mg / kg was 19.82%; the tumor inhibition rate of ADC-22 6mg / kg was 98.38% (P < 0.001). At the same dose of 6mg / kg, the tumor inhibition effect of ADC-22 was also significantly better than that of ADC-21.

[1150] Test Example 6: Plasma Stability

[1151] The sample ADC-25 was mixed evenly with human plasma, monkey plasma, and 1% BSA PBS solution at a final concentration of 100 μg / ml, filtered and sterilized, and incubated in a 37°C water bath. The day of incubation was recorded as day 0, and samples were then taken out on days 7, 14, and 21 for free toxin detection.

[1152] After taking out the samples at different time points, bring them to room temperature and vortex mix; take 25 μl of sample into a 96-well plate; add 50 μL of internal standard working solution (100 ng / mL camptothecin acetonitrile solution) and 150 μl of acetonitrile; vortex mix for 5 minutes, centrifuge for 10 minutes (4000 rpm), and take 5 μl of the supernatant for LC / MS / MS analysis.

[1153] The results are as follows Figure 4 The results showed that ADC-25 was quite stable in human and monkey plasma, as well as 1% BSA PBS solution. The release rate of free toxin did not exceed 2% at most and tended to be stable on the 14th day.

[1154] Test Example 7: Evaluation of the efficacy of ADC on human brain glioblastoma U87MG nude mouse transplanted tumors I. Purpose of the test

[1155] In this experiment, BALB / cA-nude mice were used as test animals to evaluate the efficacy of the disclosed ADC compound on human brain astroglioma U87MG nude mouse transplanted tumors.

[1156] 2. Test drugs and materials

[1157] 1. Test drug

[1158] ADC-27 (3 mg / kg)

[1159] ADC-26 (3 mg / kg)

[1160] Blank control: PBS buffer at pH 7.4.

[1161] 2. Preparation method: PBS buffer at pH 7.4.

[1162] 3. Experimental animals

[1163] BALB / cA-nude mice were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.

[1164] 3. Test methods

[1165] The experiment used female BALB / cA-nude mice, 6-7 weeks old, subcutaneously inoculated with human astrocytic glioblastoma U87MG cells (human astrocytic glioblastoma, Chinese Academy of Sciences Cell Bank, Catalog #TCHu138). On the tenth day after cell inoculation, the animals were randomly divided into groups (D0), 8 in each group, and intraperitoneal injection was started once a week for a total of 3 times. The tumor volume and body weight were measured 2-3 times a week and the data were recorded. The formula for calculating tumor volume (V) is:

[1166] V=1 / 2×a×b 2

[1167] Where a and b represent length and width respectively.

[1168] Relative volume (RTV) = V T / V0

[1169] Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%)

[1170] Where V0, V T These are the tumor volumes at the beginning and end of the experiment, respectively. RTV , T RTV They are the relative tumor volumes of the control group (blank) and the experimental group at the end of the experiment.

[1171] 4. Test results

[1172] The drug was administered intraperitoneally (ip) once a week for a total of 3 times. On the 22nd day, the tumor inhibition rate of ADC-27 3 mg / kg reached 63.3% (P<0.0001); the inhibition rate of ADC-26 3 mg / kg reached 49.1%. ADC-27 showed a stronger anti-tumor effect than ADC-26.

[1173] The body weight of animals in each group was normal during the administration process, indicating that ADC had no obvious toxic side effects. The test results are shown in Table 3 and Figure 5 As shown in the figure, the detected antibody can effectively inhibit the growth of U87MG transplanted tumors in tumor-bearing nude mice in a dose-dependent manner.

[1174] Table 3 The efficacy of the administered antibody on human brain glioblastoma U87MG nude mouse transplanted tumors (D22)

[1175]

[1176] Test Example 8: Evaluation of the efficacy of ADC on nude mouse transplanted tumors of Detroit 562 cells of human pharyngeal carcinoma pleural effusion metastasis

[1177] 1. Purpose of the test

[1178] In this experiment, BALB / cA-nude mice were used as test animals to evaluate the efficacy of the disclosed ADC compound on Detroit 562 nude mouse transplanted tumors of human pharyngeal carcinoma pleural effusion metastatic cells.

[1179] 2. Test drugs and materials

[1180] 1. Test drug

[1181] ADC-29 (3 mg / kg)

[1182] ADC-28 (3 mg / kg)

[1183] Negative control ADC (3 mg / kg): ligand-toxin conjugate formed by coupling non-B7H3 target with compound 20.

[1184] 2. Preparation method: All preparations were diluted with PBS.

[1185] 3. Experimental animals

[1186] BALB / cA-nude mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

[1187] 3. Test methods

[1188] The experimental BALB / cA-nude nude mice, female, 6-7 weeks old, were subcutaneously inoculated with human pharyngeal carcinoma pleural effusion metastatic cells Detroit 562 cells (ATCC, Catalog # CCL-138 TM ). On the tenth day after cell inoculation, the animals were randomly divided into groups (D0), with 8 animals in each group, and intraperitoneal injection was started once a week for a total of 3 times. The tumor volume and body weight were measured 2-3 times a week and the data were recorded. The formula for calculating tumor volume (V) is:

[1189] V=1 / 2×a×b 2

[1190] Where a and b represent length and width respectively.

[1191] Relative volume (RTV) = V T / V0

[1192] Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%)

[1193] Where V0, V T These are the tumor volumes at the beginning and end of the experiment, respectively. RTV , T RTV They are the relative tumor volumes of the control group (negative control) and the experimental group at the end of the experiment.

[1194] 4. Test results

[1195] The intraperitoneal injection was administered once a week for a total of 3 times. On the 28th day, the tumor inhibition rates of the tested ADCs were: ADC-29 3mg / kg (3mpk) tumor inhibition rate reached 72.27% (P<0.001); ADC-28 3mg / kg (3mpk) tumor inhibition rate reached 56.2% (P<0.001). ADC-29 showed stronger anti-tumor efficacy than ADC-28.

[1196] The body weight of animals in each group was normal during the administration process, indicating that ADC had no obvious toxic side effects. The test results are shown in Table 4 and Figure 6 As shown in the figure, the detected antibody can effectively inhibit the growth of Detroit 562 transplanted tumors in nude mice bearing tumors, and it is dose-dependent.

[1197] Table 4. Efficacy of the administered antibody on Detroit 562 xenograft tumors in nude mice (D28)

[1198]

[1199]

[1200] Test Example 9: Evaluation of drug efficacy in U87-MG tumor-bearing mice

[1201] 1. Purpose of the test

[1202] Balb / c nude mice were used as test animals to evaluate the efficacy of B7H3-antibody drug conjugates after intraperitoneal injection in their human glioma cell U87MG transplant tumor model.

[1203] 2. Test drugs and materials

[1204] 1. Test drug

[1205] ADC-30 1mg / kg

[1206] ADC-30 3mg / kg

[1207] ADC-31 1mg / kg

[1208] ADC-31 3mg / kg

[1209] Blank control: PBS

[1210] 2. Preparation method: All preparations were diluted with PBS.

[1211] 3. Experimental animals

[1212] BALB / cA-nude mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd.

[1213] 3. Test methods

[1214] U87MG cells (human astrocytoblastoma, Cell Bank of the Chinese Academy of Sciences, Catalog#TCHu138) (2.5×10 6 After the tumor grew for 14 days and reached 167.49 mm3, the animals were randomly divided into groups (d1), 8 animals per group, for a total of 5 groups.

[1215] The drug was administered by intraperitoneal injection once a week for a total of 3 times. The tumor volume and body weight were measured twice a week and the data were recorded.

[1216] Data statistics were calculated using Excel 2003 statistical software: the mean was calculated as avg; the SD was calculated as STDEV; the SEM was calculated as STDEV / SQRT; and the P value of the difference between groups was calculated as TTEST.

[1217] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[1218] Relative volume (RTV) = V T / V0

[1219] Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%)

[1220] Where V0, V T These are the tumor volumes at the beginning and end of the experiment, respectively. RTV , T RTV They are the relative tumor volumes of the blank control group (Vehicle) and the experimental group at the end of the experiment.

[1221] 4. Test results

[1222] The experimental results are as follows Figure 7 The results showed that the ADCs were injected intraperitoneally once a week for a total of 3 times. On the 18th day, the tumor inhibition rates of the tested ADCs were as follows: ADC-30 1mg / kg had a tumor inhibition rate of 0.31%; ADC-30 3mg / kg had a tumor inhibition rate of 45.23% (P<0.0001); ADC-31 1mg / kg had a tumor inhibition rate of 39.22% (P<0.01); and ADC-31 3mg / kg had a tumor inhibition rate of 80.24% (P<0.0001). Under the same dosage, the tumor inhibition effect of ADC-31 was significantly better than that of ADC-30.

Claims

1. A pharmaceutical composition comprising a therapeutically effective amount of a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a structure represented by formula (-D), and a pharmaceutically acceptable carrier, diluent or excipient, wherein the composition may contain 0.1 to 99% by weight of an active compound: in: Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -,-NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group; Or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 1 is selected from halogen, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl; R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; Wherein, the wavy line in Formula-D represents a hydrogen atom, or is covalently linked to a linker unit or to an antibody that binds to an antigen expressed by a target cell; m is an integer of 0-4.

2. The pharmaceutical composition according to claim 1, wherein the ligand-drug conjugate comprises a structure represented by formula (-D1): in: R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl; R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; preferably a hydrogen atom; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; Wavy lines represent hydrogen atoms, either covalently linked to the linker unit or to an antibody that binds to an antigen expressed by a target cell; m is 0 or 1.

3. The pharmaceutical composition according to claim 1, wherein the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof represented by the general formula (Pc-LY-Dr): in: Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group; Or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl; R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; m is an integer from 0 to 4; n is 1 to 10 and can be an integer or a decimal; Pc is a ligand; L is a linker unit.

4. The pharmaceutical composition according to claim 3, in: Y is -O-(CR a R b )m-CR 1 R 2 -C(O)-; R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group; R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl; R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; preferably a hydrogen atom; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; m is 0 or 1.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein Y is selected from:

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the O end of Y is connected to the linker unit L.

7. The pharmaceutical composition according to claim 1, wherein the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof represented by the general formula (Pc-L-D1): in: R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl; R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; preferably a hydrogen atom; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; m is 0 or 1; n is 1 to 10 and can be an integer or a decimal; Pc is a ligand; L is a linker unit.

8. The pharmaceutical composition according to any one of claims 3 to 7, wherein n is 2 to 8, which can be an integer or a decimal; preferably 3 to 8, which can be an integer or a decimal.

9. The pharmaceutical composition according to any one of claims 3 to 8, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -, L 1 Selected from -(succinimidyl-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20; preferably a chemical bond; L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - or a chemical bond, wherein t is an integer from 1 to 6; preferably -NR 5 (CR 6 R 7 )t-; R 3 , R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group.

10. The pharmaceutical composition according to claim 9, wherein L 1 Selected from -(succinimidyl-3-yl-N)-(CH2)s 1 -C(O)-, -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-, -(succinimidyl-3-yl-N)-(CH2CH2O)s 2 -CH2CH2-C(O)-, -CH2-C(O)-NR 3 -(CH2)s 3 -C(O)- or -C(O)-(CH2)s 4 C(O)-, where s 1 is an integer from 2 to 8, s 2 is an integer from 1 to 3, s 3 is an integer from 1 to 8, s 4 An integer from 1 to 8.

11. The pharmaceutical composition according to claim 9 or 10, wherein L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 An integer from 6 to 12.

12. The pharmaceutical composition according to any one of claims 9 to 11, wherein L 4 Selected from -NR 5 (CR 6 R 7 ) t -, R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group; and t is 1 or 2, preferably 2.

13. The pharmaceutical composition according to any one of claims 9 to 12, wherein L 3 It is a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid; preferably a tetrapeptide residue; more preferably a tetrapeptide residue of glycine-glycine-phenylalanine-glycine.

14. The pharmaceutical composition according to any one of claims 3 to 13, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -, L 1 for s 1 is an integer from 2 to 8; L 2 is a chemical bond; L 3 is a tetrapeptide residue; L 4 For-NR 5 (CR 6 R 7 ) t -, R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, and t is 1 or 2.

15. The pharmaceutical composition according to any one of claims 3 to 13, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -, L 1 is -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-; L 2 For-NR 4 (CH2CH2O)9CH2C(O)-; L 3 is a tetrapeptide residue; L 4 For-NR 5 (CR 6 R 7 )t-,R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, and t is 1 or 2.

16. The pharmaceutical composition according to any one of claims 9 to 15, wherein the linker unit -L-, wherein L 1 The end is connected to the ligand, L 4 Connect the Y end.

17. The pharmaceutical composition according to any one of claims 3 to 16, wherein -LY- is: Where L 1 Selected from -(succinimidyl-3-yl-N)-(CH2)s 1 -C(O)- or -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-; L 2 For-NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 is an integer from 6 to 12; L 3 is the tetrapeptide residue of GGFG; R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl; R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; R 5 , R 6 or R 7 are the same or different and are each independently a hydrogen atom or an alkyl group; s 1 is an integer from 2 to 8; m is an integer of 0-4.

18. The pharmaceutical composition according to claim 17, wherein the -LY- is: L 2 For-NR 4 (CH2CH2O)9CH2C(O)-; L 3 is the tetrapeptide residue of GGFG; R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl; R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; R 5 , R 6 or R 7 are the same or different and are each independently a hydrogen atom or an alkyl group; m is an integer of 0-4.

19. The pharmaceutical composition according to claim 17, wherein the -LY- is L 2 is a chemical bond; L 3 is the tetrapeptide residue of GGFG; R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl; R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group; s 1 is an integer from 2 to 8; preferably 5; m is an integer of 0-4.

20. The pharmaceutical composition according to any one of claims 1 to 4, wherein the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is of the general formula (Pc-L a -Y-Dr) or a pharmaceutically acceptable salt or solvate thereof: in: W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 is an integer from 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; R 1 is selected from halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; R 2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; m is an integer from 0 to 4; n is 1 to 10 and can be an integer or a decimal; Pc is a ligand.

21. The pharmaceutical composition according to claim 20, wherein the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is of the general formula (Pc-L b -Y-Dr) or a pharmaceutically acceptable salt or solvate thereof: in: s 1 is an integer from 2 to 8; preferably 5; Pc, R 1 , R 2 , R 5 ~R 7 , m and n are as defined in claim 20.

22. The pharmaceutical composition according to any one of claims 3 to 19, wherein -LY- is selected from:

23. The pharmaceutical composition according to any one of claims 1 to 22, wherein the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is selected from the following structural formula: in: n is 1 to 10 and can be an integer or a decimal; Pc is a ligand.

24. The pharmaceutical composition according to any one of claims 3 to 23, wherein the Pc is an antibody or an antigen-binding fragment thereof, wherein the antibody is selected from a chimeric antibody, a humanized antibody and a fully human antibody.

25. The pharmaceutical composition of claim 24, wherein the antibody or antigen-binding fragment thereof is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-Mesothelin antibody or an antigen-binding fragment thereof.

26. The pharmaceutical composition of claim 24, wherein the antibody or antigen-binding fragment thereof is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 and Glematumamab, or an antigen-binding fragment thereof.

27. The pharmaceutical composition according to any one of claims 3 to 26, wherein the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is selected from the following structural formula: wherein n is as defined in claim 3.

28. A pharmaceutical composition comprising a therapeutically effective amount of a compound represented by general formula (D): or its tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers, diluents or excipients, wherein the composition may contain 0.1 to 99% by weight of the active compound, wherein: Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group; Or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 1 is selected from halogen, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl; R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; m is an integer of 0-4.

29. The pharmaceutical composition according to claim 28, wherein the compound represented by general formula (D) is a compound represented by general formula (D1): or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, in: R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl; R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 Cycloalkyl; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; m is 0 or 1.

30. The pharmaceutical composition according to claim 28 or 29, wherein the compound represented by general formula (D) is selected from:

31. A pharmaceutical composition comprising a therapeutically effective amount of a compound of the general formula (L a -Y-Dr) represented by: or its tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers, diluents or excipients, wherein the composition may contain 0.1 to 99% by weight of the active compound, in: W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 An integer from 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; R 1 is selected from halogen, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl; R 2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl or heteroaryl; or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; m is an integer of 0-4.

32. The pharmaceutical composition according to claim 31, wherein the general formula (L a -Y-Dr) is a compound represented by the general formula (L b -Y-Dr) shown in the compound: or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or its pharmaceutically acceptable salt, Where R 1 , R 2 , R 5 ~R 7 、s 1 and m as defined in claim 31.

33. The general formula (L) according to claim 31 or 32 a -Y-Dr) is a pharmaceutical composition selected from:

34. Use of the pharmaceutical composition according to any one of claims 1 to 33 in the preparation of a medicament for treating or preventing a tumor.

35. The use according to claim 34, wherein the tumor is a cancer associated with the expression of HER2, HER3, B7H3 or EGFR.

36. The use of a pharmaceutical composition according to any one of claims 1 to 33 in the preparation of a medicament for treating and / or preventing cancer, wherein the cancer is preferably selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

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