A pharmaceutical composition containing an antibody-drug conjugate and its use

Abstract

A pharmaceutical composition comprises an antibody-drug conjugate in a buffer, wherein the antibody-drug conjugate has a structure as shown in the general formula (Pc-L-Y-D). The pharmaceutical composition further comprises a sugar and a surfactant.

Description

[0001] This application claims priority to the Chinese patent application filed on March 25, 2020 (application number CN 202010219601.7) and the Chinese patent application filed on March 17, 2021 (application number CN 202110287012.7). Technical Field

[0002] The present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition containing an antibody-drug conjugate, and its use as an anti-cancer drug. Background Art

[0003] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[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] 2000 The first antibody-drug conjugate (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; US5079233; US5585089; US5606040; US5693762; US5739116; US5767285; US5773001).

[0006] August 2011, (brentuximab vedotin, Seattle Genetics) has 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] and Both are targeted therapies for blood tumors, which have a relatively simpler structure than solid tumors. (ado-trastuzumab emtansine, T-DM1) has been approved by the US FDA for the treatment of patients with HER2-positive advanced or metastatic breast cancer that is resistant to trastuzumab (Tratuzumab, trade name: Herceptin) and paclitaxel (WO2005037992; US8088387). It 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 anti-tumor effects by inhibiting topoisomerase I. Literature reporting the application of the camptothecin derivative exitecan (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, there is still a need to further develop ADC drugs with better efficacy.

[0009] However, ADCs have a more complex and heterogeneous structure than antibodies, thus posing greater challenges to ADC formulation for therapeutic purposes. Summary of the invention

[0010] The present disclosure provides a pharmaceutical composition comprising an antibody drug conjugate and a buffer, wherein the antibody drug conjugate has a structure as shown in the general formula (Pc-LYD):

[0011]

[0012] in:

[0013] 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)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-;

[0014] 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 and a heterocyclic group;

[0015] Or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0016] R 1 is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl;

[0017] R 2 is selected from the group consisting of a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;

[0018] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0019] Or, Ra and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group;

[0020] m is an integer from 0 to 4;

[0021] n is 1 to 10, and n is a decimal or an integer;

[0022] L is the joint unit;

[0023] Pc is an antibody or an antigen-binding fragment thereof;

[0024] The composition has a pH of from about 4.5 to about 6.0, preferably from about 4.8 to about 5.3, more preferably from about 5.0 to about 5.1.

[0025] In an optional embodiment, the pH value of the buffer in the pharmaceutical composition is about 4.5 to about 6.0, non-limiting examples include about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, preferably about 4.8 to about 5.3, more preferably about 5.0 to about 5.1. In some embodiments, the pH of the pharmaceutical composition is 4.5 to 5.2, preferably 4.8 to 5.2, more preferably 5.0 to 5.1. In some embodiments, the pH of the pharmaceutical composition is 5.0.

[0026] In an optional embodiment, the pharmaceutical composition further comprises a surfactant, which may be selected from polysorbate, polysorbate 20, polysorbate 80, polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-saccharide Betaine, cetyl-betaine, lauryl amidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, methyl cocoyl sodium, methyl oleyl taurate sodium, polyethylene glycol, polypropylene glycol, copolymer of ethylene and propylene glycol, etc. Preferred surfactants are polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

[0027] In an optional embodiment, the concentration of the surfactant in the pharmaceutical composition is about 0.01 mg / mL to about 1.0 mg / mL. In an optional embodiment, the concentration of the surfactant in the pharmaceutical composition is about 0.05 mg / mL to about 0.5 mg / mL, preferably about 0.1 mg / mL to about 0.3 mg / mL, about 0.2 mg / mL to about 0.6 mg / mL, about 0.2 mg / mL to about 0.5 mg / mL or about 0.2 mg / mL to about 0.3 mg / mL, more preferably about 0.2 mg / mL, non-limiting examples include 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.6 mg / mL.

[0028] In an optional embodiment, the aforementioned pharmaceutical composition further comprises sugar. The "sugar" disclosed herein comprises a conventional composition (CH2O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. The sugar may be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, etc. The preferred sugar is a non-reducing disaccharide, more preferably trehalose or sucrose, and most preferably sucrose.

[0029] In an optional embodiment, the concentration of sugar in the aforementioned pharmaceutical composition is about 60 mg / mL to about 90 mg / mL, non-limiting examples include 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, preferably 80 mg / mL. In some embodiments, the concentration of sugar is 70 mg / mL to 90 mg / mL.

[0030] In alternative embodiments, the concentration of the antibody drug conjugate in the pharmaceutical composition is about 1 mg / mL to about 100 mg / mL, non-limiting examples include 1 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, preferably about 10 mg / mL to about 30 mg / mL; more preferably about 20 mg / mL to about 22 mg / mL. Specific, non-limiting examples include 20.1 mg / mL, 20.2 mg / mL, 20.3 mg / mL, 20.4 mg / mL, 20.5 mg / mL, 20.6 mg / mL, 20.7 mg / mL, 20.8 mg / mL, 20.81 mg / mL, 20.82 mg / mL, 20.83 mg / mL, 20.84 mg / mL, 20.85 mg / mL, 20.86 mg / mL, In an alternative embodiment, the concentration of the antibody drug conjugate in the pharmaceutical composition is about 10 mg / mL to about 30 mg / mL, preferably about 20 mg / mL, based on the naked antibody (i.e., the antibody portion of the ADC).

[0031] In an optional embodiment, the buffer in the aforementioned pharmaceutical composition is selected from the group consisting of histidine salt buffer, succinate buffer and citrate buffer, preferably succinate buffer, more preferably succinic acid-sodium succinate buffer.

[0032] In an optional embodiment, the concentration of the buffer in the pharmaceutical composition is about 5 mM to about 50 mM, non-limiting examples include 1 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 30 mM, 40 mM, 50 mM, preferably about 5 mM to about 20 mM; most preferably about 10 mM.

[0033] In an alternative embodiment, the drug loading (n) can range from 3 to 8, 4 to 8, 5 to 7, more preferably 5.3 to 6.1, 5.7 cytotoxic drugs per antibody or antigen-binding fragment thereof (Pc). n is a decimal or an integer.

[0034] In an alternative embodiment, the pharmaceutical composition comprises:

[0035] (a) about 10 mg / mL to about 30 mg / mL of the antibody drug conjugate, (b) about 0.05 mg / mL to about 0.5 mg / mL of a polysorbate, (c) about 60 mg / mL to about 90 mg / mL of a sugar, and (d) about 5 mM to about 20 mM of a buffer; the pH of the pharmaceutical composition is about 4.8 to about 5.3;

[0036] In an alternative embodiment, the pharmaceutical composition comprises:

[0037] (a) about 10 mg / mL to about 30 mg / mL of the antibody drug conjugate, (b) about 0.05 mg / mL to about 0.5 mg / mL of polysorbate, (c) about 60 mg / mL to about 90 mg / mL of a sugar, and (d) about 5 mM to about 20 mM of a buffer; the pH of the pharmaceutical composition is 4.8 to 5.2;

[0038] In an alternative embodiment, the pharmaceutical composition comprises:

[0039] (a) about 20 mg / mL to about 22 mg / mL of the antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) 80 mg / mL of sucrose, and (d) 10 mM succinate buffer, the pH of the pharmaceutical composition being about 5.0 to about 5.1. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 5.1.

[0040] In an optional embodiment, in the aforementioned antibody drug conjugate,

[0041] -Y-for-O-(CR a R b )m-CR 1 R2 -C(O)-;

[0042] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen and an alkyl group;

[0043] R 1 C 3-7 Cycloalkylalkyl or C 3-7 Cycloalkyl;

[0044] R 2 is selected from hydrogen atom, halogenated alkyl and C 3-7 Cycloalkyl; preferably a hydrogen atom;

[0045] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl;

[0046] m is 0 or 1.

[0047] In an optional embodiment, in the aforementioned antibody drug conjugate,

[0048] -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0049] R 1 C 3-7 Cycloalkylalkyl or C 3-7 Cycloalkyl;

[0050] R 2 is selected from hydrogen atom, halogenated alkyl and C 3-7 Cycloalkyl;

[0051] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl;

[0052] m is 0 or 1.

[0053] In an optional embodiment, in the aforementioned antibody drug conjugate,

[0054] -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0055] R 1 C 3-7 Cycloalkylalkyl or C 3-7 Cycloalkyl;

[0056] R2 is a hydrogen atom;

[0057] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl;

[0058] m is 0 or 1.

[0059] In an optional embodiment, in the aforementioned antibody drug conjugate,

[0060] -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0061] R 1 C 3-7 Cycloalkylalkyl or C 3-7 Cycloalkyl;

[0062] R 2 is a hydrogen atom;

[0063] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl;

[0064] m is 0.

[0065] In an optional embodiment, in the aforementioned antibody drug conjugate,

[0066] -Y-Selected from:

[0067]

[0068] In an optional embodiment, in the aforementioned antibody drug conjugate,

[0069] The O end of -Y- is connected to the linker unit L.

[0070] In an optional embodiment, in the aforementioned antibody drug conjugate,

[0071] -Y-Selected from:

[0072]

[0073] In an optional embodiment, the aforementioned antibody drug conjugate has a structure as shown in the general formula (Pc-L-D1):

[0074]

[0075] in:

[0076] R1 is cycloalkylalkyl or cycloalkyl; preferably C 3-7 Cycloalkylalkyl or C 3-7 Cycloalkyl;

[0077] R 2 is selected from hydrogen atom, halogenated alkyl and C 3-7 Cycloalkyl; preferably a hydrogen atom;

[0078] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl;

[0079] m is 0 or 1;

[0080] n is 1 to 10 and can be an integer or a decimal;

[0081] Pc is an antibody or an antigen-binding fragment thereof; and L is a Linker unit.

[0082] In an optional embodiment, in the aforementioned antibody-drug conjugate, n is 2 to 8, which can be an integer or a decimal; preferably, it is 3 to 8, which can be an integer or a decimal.

[0083] In an optional embodiment, in the aforementioned antibody drug conjugate, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,in:

[0084] L 1 Selected from -(succinimidyl-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl and straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said 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;

[0085] L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1C(O)- and chemical bonds, where p 1 is an integer from 1 to 20; L 2 Preferably a chemical bond;

[0086] 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;

[0087] L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - and a chemical bond, wherein t is an integer from 1 to 6; L 4 Preferably -NR 5 (CR 6 R 7 )t-;

[0088] 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;

[0089] 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.

[0090] In an optional embodiment, in the aforementioned antibody drug conjugate, 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)- and -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.

[0091] In an optional embodiment, in the aforementioned antibody drug conjugate, the linker unit L 2 For-NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 An integer from 6 to 12.

[0092] In an optional embodiment, in the aforementioned antibody drug conjugate, L 4 For-NR 5 (CR 6 R 7 )t-,R 5 is 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 -NR 5 CR 6 R 7 -, more preferably -NHCH2-.

[0093] In an optional embodiment, in the aforementioned antibody drug conjugate, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0094] L 1 for s 1 is an integer from 2 to 8;

[0095] L 2 is a chemical bond;

[0096] L 3 is a tetrapeptide residue;

[0097] L 4 For-NR 5 (CR 6 R 7 )t-,R 5 is 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 an optional embodiment, in the aforementioned antibody drug conjugate, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0099] L 1 is -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-;

[0100] L 2 For-NR 4 (CH2CH2O)9CH2C(O)-;

[0101] L 3 is a tetrapeptide residue;

[0102] L 4 For-NR 5 (CR 6 R 7 )t-,R 5 is 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.

[0103] In an optional embodiment, in the aforementioned antibody drug conjugate, the L 3 The 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).

[0104] In an optional embodiment, in the aforementioned antibody drug conjugate, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -, its L 1 The end is connected to the antibody or its antigen-binding fragment, L 4 Connect the Y end.

[0105] In an optional embodiment, in the aforementioned antibody drug conjugate, the -LY- is:

[0106]

[0107] L 1 -(succinimidyl-3-yl-N)-(CH2)s 1 -C(O)- or -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-;

[0108] L 2 For-NR4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 is an integer from 6 to 12; R 4 is selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0109] L 3 is the tetrapeptide residue of GGFG;

[0110] R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-7 Cycloalkylalkyl or C 3-7 Cycloalkyl;

[0111] R 2 is selected from hydrogen atom, halogenated alkyl and C 3-7 Cycloalkyl; preferably a hydrogen atom;

[0112] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl;

[0113] 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;

[0114] s 1 is an integer from 2 to 8; preferably 5;

[0115] m is an integer of 0-4.

[0116] In an optional embodiment, in the aforementioned antibody drug conjugate, the -LY- is:

[0117]

[0118] Preferably:

[0119]

[0120] L 2 For-NR 4 (CH2CH2O)9CH2C(O)-;R 4 is selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0121] L 3 is the tetrapeptide residue of GGFG;

[0122] R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-7 Cycloalkylalkyl or C3-7 Cycloalkyl;

[0123] R 2 is selected from hydrogen atom, halogenated alkyl and C 3-7 Cycloalkyl; preferably a hydrogen atom;

[0124] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl;

[0125] 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;

[0126] m is an integer of 0-4.

[0127] In an optional embodiment, in the aforementioned antibody drug conjugate, the -LY- is:

[0128]

[0129] L 2 is a chemical bond;

[0130] L 3 is the tetrapeptide residue of GGFG;

[0131] R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-7 Cycloalkylalkyl or C 3-7 Cycloalkyl;

[0132] R 2 is selected from hydrogen atom, halogenated alkyl and C 3-7 Cycloalkyl; preferably a hydrogen atom;

[0133] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl;

[0134] 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;

[0135] s 1 is an integer from 2 to 8; preferably 5;

[0136] m is an integer of 0-4.

[0137] In an optional embodiment, in the aforementioned antibody drug conjugate, wherein the -LY- is:

[0138]

[0139] in:

[0140] L 1 -(succinimidyl-3-yl-N)-(CH2)s 1 -C(O)- or -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-;

[0141] L 2 For-NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 is an integer from 1 to 20; R 4 is selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0142] L 3 is the tetrapeptide residue of GGFG;

[0143] R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-7 Cycloalkylalkyl or C 3-7 Cycloalkyl;

[0144] R 2 is selected from hydrogen atom, halogenated alkyl and C 3-7 Cycloalkyl; preferably a hydrogen atom;

[0145] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl;

[0146] R 5 , R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0147] s 1 is an integer from 2 to 8;

[0148] m is an integer of 0-4.

[0149] In an optional embodiment, in the aforementioned antibody drug conjugate, the -LY- is:

[0150]

[0151] in:

[0152] L2 is a chemical bond;

[0153] L 3 is the tetrapeptide residue of GGFG;

[0154] R 1 is cycloalkylalkyl or cycloalkyl; preferably C 3-7 Cycloalkylalkyl or C 3-7 Cycloalkyl;

[0155] R 2 is selected from hydrogen atom, halogenated alkyl and C 3-7 Cycloalkyl; preferably a hydrogen atom;

[0156] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl;

[0157] 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;

[0158] s 1 is an integer from 2 to 8;

[0159] m is an integer of 0-4.

[0160] In an optional embodiment, the antibody drug conjugate has the general formula (Pc-L a -YD) shown in the structure:

[0161]

[0162] in,

[0163] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-C 3-7 Cycloalkyl and straight chain heteroalkyl of 1 to 8 atoms, the straight chain heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, C 3-7 Cycloalkyl and linear heteroalkyl are each independently optionally further selected from halogen, hydroxy, cyano, amino, C 1-6 Alkyl, Chloro C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-7 The cycloalkyl group is substituted with one or more substituents;

[0164] L 2 Selected from -NR 4(CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- and chemical bonds, where p 1 is an integer from 1 to 20; R 4 is selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0165] L 3 The peptide residue is composed of 2 to 7 amino acid residues, wherein the amino acid residues are selected from the group consisting of phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q), and aspartic acid (D), and are optionally further selected from halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, Chloro C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-7 The cycloalkyl group is substituted by one or more substituents;

[0166] R 1 Halogenated C 1-6 Alkyl or C 3-7 Cycloalkyl;

[0167] R 2 Selected from hydrogen atoms, halogenated C 1-6 Alkyl and C 3-7 Cycloalkyl;

[0168] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl;

[0169] R 5 Selected from hydrogen atoms, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl and hydroxyl C 1-6 alkyl;

[0170] R 6 and R 7 are the same or different and are each independently selected from hydrogen atoms, halogens, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl and hydroxyl C 1-6 alkyl;

[0171] m is 0 or 1;

[0172] n is a decimal or integer from 3 to 8;

[0173] Pc is an antibody or an antigen-binding fragment thereof.

[0174] In an optional embodiment, the antibody drug conjugate has the general formula (Pc-L b -YD) shown in the structure:

[0175]

[0176] in:

[0177] s 1 is an integer from 2 to 8;

[0178] Pc, R 1 , R 2 , R 5 , R 6 , R 7 , m and n are as in the general formula (Pc-L a -YD) is defined.

[0179] In an optional embodiment, in the aforementioned antibody drug conjugate, the -LY- includes, but is not limited to:

[0180]

[0181]

[0182] In an optional embodiment, the aforementioned antibody drug conjugate has the following structure:

[0183]

[0184]

[0185]

[0186]

[0187] wherein Pc and n are as defined in the general formula (Pc-La-YD).

[0188] In an optional embodiment, the antibody drug conjugate has a structure as shown in the following formula:

[0189]

[0190] in:

[0191] n is 3 to 8, and n is a decimal or an integer;

[0192] Pc is an antibody or an antigen-binding fragment thereof.

[0193] In an optional embodiment, the Pc is an antibody or an antigen-binding fragment thereof, wherein the antibody is selected from a chimeric antibody, a humanized antibody or a fully human antibody; preferably a monoclonal antibody.

[0194] In an optional embodiment, the Pc 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 and anti-Mesothelin antibody, or an antigen-binding fragment thereof.

[0195] In an optional embodiment, the antibody or its antigen-binding fragment in the antibody-drug conjugate is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 and Glematumamab, or an antigen-binding fragment thereof.

[0196] In an optional embodiment, the antibody drug conjugate has a structure as shown in the following formula:

[0197]

[0198]

[0199]

[0200]

[0201]

[0202] 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.

[0203] In an optional embodiment, the antibody-drug conjugate in the pharmaceutical composition has a structure as shown in the following formula:

[0204]

[0205] Here, n is 3 to 8 and is a decimal or an integer.

[0206] The present disclosure provides a pharmaceutical composition comprising: (a) about 10 mg / mL to about 30 mg / mL of the antibody drug conjugate, (b) about 0.05 mg / mL to about 0.5 mg / mL of polysorbate, (c) about 60 mg / mL to about 90 mg / mL of sugar, and (d) about 5 mM to about 20 mM of buffer; the pH of the composition is 4.8 to 5.2;

[0207] Wherein, the antibody-drug conjugate has a structure as shown in the following formula:

[0208]

[0209] Here, n is 3 to 8 and is a decimal or an integer.

[0210] The present disclosure provides a pharmaceutical composition comprising: (a) about 20 mg / mL to about 22 mg / mL of an antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 80 mg / mL of sucrose, and (d) about 10 mM succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 5.1;

[0211] Wherein, the antibody-drug conjugate has a structure as shown in the following formula:

[0212]

[0213] Here, n is 3 to 8 and is a decimal or an integer.

[0214] The present disclosure also provides a lyophilized preparation containing an antibody drug conjugate, characterized in that the preparation can form the pharmaceutical composition as described above after reconstitution.

[0215] The present disclosure also provides a method for preparing a lyophilized formulation containing an antibody drug conjugate, which comprises the step of freeze-drying the pharmaceutical composition as described above.

[0216] In an optional embodiment, the freeze drying in the method for preparing a lyophilized formulation comprising an antibody drug conjugate comprises the steps of pre-freezing, primary drying and secondary drying in sequence. Freeze drying is performed by freezing the formulation and then sublimating water at a temperature suitable for primary drying. Under this condition, the product temperature is lower than the low eutectic point or collapse temperature of the formulation. Typically, the temperature range for primary drying is about -30 to 25°C (assuming that the product remains frozen during the primary drying process). The size and type of the formulation, the container (e.g., a glass vial) holding the sample, and the volume of the liquid determine the time required for drying, which can range from a few hours to a few days (e.g., 40-60 hours). The secondary drying stage can be performed at about 0-40°C, which depends primarily on the type and size of the container and the type of protein used. The secondary drying time is determined by the desired residual moisture level in the product, and generally requires at least about 5 hours. Typically, the water content of the lyophilized formulation is less than about 5%, preferably less than about 3%. The pressure may be the same as the pressure applied in the primary drying step, preferably, the pressure of the secondary drying is lower than that of the primary drying. Freeze drying conditions may vary with the formulation and vial size.

[0217] In an optional embodiment of the present disclosure, 5 mL of the pharmaceutical composition stock solution is lyophilized, and the lyophilization procedure is as follows: the pre-freezing temperature is -5°C or -45°C, the primary drying temperature is -20°C, the vacuum degree is 10Pa, and the secondary drying temperature is 25°C, and the vacuum degree is 1Pa.

[0218] In some embodiments, the lyophilized formulation is stable at 2-8° C. for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40° C. for at least 7 days, at least 14 days, or at least 28 days.

[0219] The present disclosure also provides a lyophilized preparation comprising an antibody drug conjugate, wherein the preparation is obtained by freeze-drying the pharmaceutical composition comprising the anti-HER2 antibody drug conjugate as described above.

[0220] The present disclosure also provides a reconstituted solution containing an antibody drug conjugate, characterized in that the reconstituted solution is obtained by reconstituted the lyophilized preparation as described above.

[0221] The present disclosure also provides a method for preparing the above-mentioned reconstituted solution, which includes the step of reconstituted the above-mentioned lyophilized preparation, and the solution used for reconstitution is selected from but not limited to water for injection, physiological saline or glucose solution.

[0222] In an optional embodiment, the reconstitution solution comprises the following components:

[0223] (a) about 10 mg / mL to 30 mg / mL of the antibody drug conjugate, (b) about 0.05 mg / mL to 0.5 mg / mL of polysorbate, (c) about 60 mg / mL to 90 mg / mL of a sugar, and (d) about 5 mM to 20 mM of a buffer; the pH of the reconstituted solution is 4.8-5.2.

[0224] In an optional embodiment, the reconstitution solution comprises the following components:

[0225] (a) about 20 mg / mL to 22 mg / mL of an antibody drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 80 mg / mL of sucrose, and (d) about 10 mM succinate buffer, wherein the pH of the reconstituted solution is 5.0-5.1.

[0226] The present disclosure also provides a product, which includes a container, wherein the pharmaceutical composition, lyophilized preparation or reconstituted solution as described above is contained in the container. In some embodiments, the container is a neutral borosilicate glass tube injection bottle.

[0227] The present disclosure also provides the use of the aforementioned pharmaceutical composition or lyophilized preparation or reconstituted solution or product in the preparation of a drug for treating or preventing tumors.

[0228] The present disclosure also provides a method for treating a disease, comprising providing the aforementioned pharmaceutical composition or lyophilized preparation or reconstituted solution or product.

[0229] The present disclosure also provides the aforementioned pharmaceutical composition, or lyophilized preparation, or reconstituted solution, or product as a drug. Preferably, the drug is used to treat or prevent tumor diseases.

[0230] In an alternative embodiment, the disease or tumor is a cancer associated with HER2, HER3, B7H3 or EGFR expression.

[0231] In an optional embodiment, the cancer is 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.

[0232] As is well known to those skilled in the art, one, some or all of the features of the various embodiments described in this disclosure may be further combined to form other embodiments of the present disclosure. The above embodiments of the present disclosure and other embodiments obtained by combination are further described by the following detailed description.

[0233] The present disclosure provides a pharmaceutical composition that is more conducive to production and administration and has stable performance. Specifically, the pharmaceutical composition disclosed herein comprises an antibody drug conjugate and a buffer.

[0234] the term

[0235] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0236] This disclosure incorporates all the contents of application PCT / CN2019 / 107873 (WO2020 / 063676) into this application.

[0237] "Antibody drug conjugate (ADC)" is to connect antibodies or antibody fragments with biologically active cytotoxins or small molecule drugs with cell-killing activity through stable chemical linker compounds, making full use of the antibody's specificity for tumor cell-specific or highly expressed antigens and the high efficiency of cytotoxins to avoid toxic side effects on normal cells. Compared with traditional chemotherapy drugs, antibody drug conjugates can accurately bind to tumor cells and reduce the impact on normal cells.

[0238] "Buffer" refers to a buffer that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that control pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0239] "Histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate and the like, preferably histidine-acetate buffer, histidine-acetate buffer is prepared from histidine and acetic acid, histidine-hydrochloride buffer is prepared from histidine and hydrochloric acid.

[0240] "Citrate buffer" is a buffer including citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

[0241] "Succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate. Exemplarily, the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0242] "Phosphate buffer" is a buffer including phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0243] "Acetate buffer" is a buffer including acetate ions. Examples of acetate buffers include acetate-sodium acetate, acetate-histidine salt, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, etc. A preferred acetate buffer is acetate-sodium acetate.

[0244] "Pharmaceutical composition" means a mixture containing one or more antibody drug conjugates described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to maintain the stability of the antibody active ingredient, promote administration to the organism, and facilitate the absorption of the active ingredient to exert biological activity.

[0245] In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0246] The pharmaceutical compositions described in the present disclosure are in the form of solutions, and unless otherwise specified, the solvents therein are all water.

[0247] "Lyophilized preparation" refers to a pharmaceutical composition in liquid or solution form or a preparation or pharmaceutical composition obtained after a liquid or solution preparation has been subjected to a vacuum freeze-drying step.

[0248] As used herein, the terms "about" and "approximately" refer to values ​​within an acceptable error range for a specific value determined by a person of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation in each practice in the art. Alternatively, "about" or "substantially including" can mean a range of up to 20%. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about" or "substantially including" should be assumed to be within an acceptable error range for the specific value.

[0249] The pharmaceutical composition disclosed herein can achieve a stable effect: the antibody drug conjugate therein substantially retains its physical stability and / or chemical stability and / or biological activity of the pharmaceutical composition after storage, preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are currently a variety of analytical techniques for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0250] A stable formulation is one in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. In addition, a stable liquid formulation includes a liquid formulation that exhibits desired characteristics after being stored at a temperature including 25°C for a period of 1 month, 3 months, and 6 months. A typical example of stability: measured by SEC-HPLC, usually no more than about 10%, preferably no more than about 5%, of antibody monomers aggregate or degrade. By visual analysis, the formulation is a pale yellow, nearly colorless, clear liquid or colorless, or clear to slightly milky white. The concentration, pH, and weight-gram molecular osmotic pressure concentration of the formulation have no more than ±10% variation. A reduction of no more than about 10%, preferably no more than about 5%, is usually observed. Aggregation of no more than about 10%, preferably no more than about 5%, is usually formed.

[0251] The antibody drug conjugate "retains its physical stability" in the pharmaceutical formulation if it shows no significant increase in aggregation, precipitation and / or denaturation as measured after visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS). Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0252] An antibody drug conjugate "retains its chemical stability" in a pharmaceutical formulation if the antibody drug conjugate does not show significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0253] An antibody drug conjugate "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody drug conjugate at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared.

[0254] 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).

[0255] The "antibody" described in this disclosure refers to immunoglobulin, and a complete antibody 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 its antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, 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 the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain by the difference in the constant region. Each of the five types of Ig can have κ chain or λ chain. 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.

[0256] 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 framework regions (FRs) with relatively conservative sequences. 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. The CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments described herein conform to the known Kabat numbering convention (LCDR1-3, HCDR1-3) in terms of number and position.

[0257] In the present disclosure, the antibody light chain described in the present disclosure may further comprise a light chain constant region, wherein the light chain constant region comprises a human or murine κ, λ chain or a variant thereof.

[0258] In the present disclosure, the antibody heavy chain described in the present disclosure may further comprise a heavy chain constant region, wherein the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0259] The antibodies disclosed herein include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

[0260] 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.

[0261] 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 insert it into a human vector, and finally express the chimeric antibody molecule in a eukaryotic or prokaryotic industrial system.

[0262] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the mouse CDR sequence into the human antibody variable region framework, i.e., different types of human germline antibody framework sequences. The strong heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components can be overcome. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, germline DNA sequences of human heavy chain and light chain variable region genes can be obtained in the "VBase" human germline sequence database, 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 further affinity maturation of CDR by phage display.

[0263] The term "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel.

[0264] The "antigen-binding fragment of an antibody" described in the present disclosure may refer to a Fab fragment, a Fab' fragment, a F(ab')2 fragment having antigen-binding activity, and an Fv fragment scFv fragment that binds to an antigen. The Fv fragment contains the variable regions of the heavy and light chains of an antibody, but has no constant region, and is the smallest antibody fragment having all antigen-binding sites. Generally, an Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming a structure required for antigen binding. Two antibody variable regions may also be connected into one polypeptide chain using different connectors, which is called a single chain antibody or single chain Fv (sFv).

[0265] The term "antigen binding site" of the present disclosure refers to a three-dimensional site on an antigen, whether continuous or discontinuous, that is recognized by the antibody or antigen-binding fragment of the present disclosure.

[0266] The "ADCC" mentioned in this disclosure, i.e., antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of target cells coated with antibodies by cells expressing Fc receptors through recognition of the Fc segment of antibodies. The ADCC effector function of antibodies can be reduced or eliminated by modifying the Fc segment on IgG. The modification refers to mutations in the heavy chain constant region of antibodies, such as N297A, L234A, L235A selected from IgG1; IgG2 / 4 chimeras, and F234A / L235A mutations of IgG4.

[0267] "Mutations" in the mutant sequences described in the present disclosure include, but are not limited to, "back mutations", "conservative modifications" or "conservative substitutions or replacements". "Conservative modifications" or "conservative substitutions or replacements" described in the present disclosure refer to replacement of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, replacement of amino acids with similar structures or functions is unlikely to destroy biological activity.

[0268] The "mutated sequence" described in the present disclosure refers to a nucleotide sequence and / or amino acid sequence having different percentage sequence identity with the nucleotide sequence and / or amino acid sequence disclosed herein, obtained by appropriate substitution, insertion or deletion of the nucleotide sequence and / or amino acid sequence disclosed herein. The sequence identity described in the present disclosure may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. The sequence comparison and identity percentage determination between two sequences can be performed by the default settings of the BLASTN / BLASTP algorithm available on the website of the National Center For Biotechnology Institute.

[0269] The term "linker unit" or "linking fragment" or "linking unit" refers to a chemical structure fragment or bond that is connected to an antibody or its antigen-binding fragment at one end and to a drug at the other end, and can also be connected to other linkers before being connected to an antibody or 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 antibody, L 4 The end is connected to the structural unit Y and then connected to the compound or toxin.

[0270] 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).

[0271] 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 -70°C, or lyophilized.

[0272] 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.

[0273] 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.

[0274] The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having a residue derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane, 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.

[0275] 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.

[0276] 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 7 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.

[0277] 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.

[0278] 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:

[0279]

[0280] 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:

[0281]

[0282] The term "bridged heterocyclyl" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share 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, and 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:

[0283]

[0284] 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:

[0285] wait.

[0286] 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.

[0287] 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:

[0288]

[0289]

[0290] 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.

[0291] 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:

[0292]

[0293] 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.

[0294] The term "amino protecting group" is to protect the amino group with a group that is easily removed so that the amino group remains unchanged when other parts of the molecule react. Non-limiting examples include 9-fluorenylmethyloxycarbonyl, 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-fluorenylmethyloxycarbonyl.

[0295] 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.

[0296] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0297] The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

[0298] The term "hydroxy" refers to an -OH group.

[0299] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0300] The term "amino" refers to -NH2.

[0301] The term "nitro" refers to -NO2.

[0302] "Optional" or "optionally" means that the event or environment described later can but need not occur, and the description includes occasions where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a specific sequence can but does not have to be present.

[0303] "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.

[0304] The term drug loading or "drug loading" refers to the average number of cytotoxic drugs loaded on each antibody or antigen-binding fragment thereof in an antibody-drug conjugate molecule, 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, more preferably 3-8, and most preferably 5.3-6.1 cytotoxic drugs connected to each antibody or antigen-binding fragment thereof (Pc). In the embodiments disclosed herein, the drug loading is expressed as n, which can also be referred to as the DAR (Drug-antibody Ratio) value, and can be exemplified as an average value 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 ultraviolet-visible spectroscopy (UV-Vis), hydrophobic chromatography (HIC) mass spectrometry, ELISA test, and HPLC characteristics.

[0305] The loading of cytotoxic drugs can be controlled by the following non-limiting methods, including:

[0306] (1) Control the molar ratio of the linking reagent and the monoclonal antibody,

[0307] (2) Control the reaction time and temperature,

[0308] (3) Select different reaction reagents.

[0309] The preparation of conventional pharmaceutical compositions is described in the Chinese Pharmacopoeia.

[0310] 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.

[0311] "Administering" and "treating" when applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administering" and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid. "Administering" and "treating" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, a diagnostic, a combination composition, or by another cell. "Treatment" when applied to humans, veterinary medicine, or research subjects refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0312] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds disclosed herein, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in the treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptom has been alleviated can be evaluated by any clinical test method commonly used by physicians or other professional health care personnel to evaluate the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., treatment methods or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0313] An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's general health, the method, route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0314] "Replacement" refers to the replacement of the solvent system for dissolving the antibody protein, for example, using the buffer system of the stable preparation to replace the high-salt or hypertonic solvent system containing the antibody protein by physical manipulation, so that the antibody protein is present in the stable preparation. The so-called physical manipulation includes but is not limited to ultrafiltration, dialysis or redissolution after centrifugation. BRIEF DESCRIPTION OF THE DRAWINGS

[0315] Figure 1A :The present invention discloses the plasma stability experimental results of ADC-19.

[0316] Figure 1B : The present invention discloses the plasma stability experimental results of ADC-18.

[0317] Figure 1C : The present invention discloses the plasma stability experimental results of ADC-20.

[0318] Figure 2 : This invention discloses the efficacy evaluation of ADC-21 and ADC-24 on JIMT-1 tumor-bearing mice.

[0319] Figure 3 :The efficacy of the disclosed ADC on nude mice transplanted with human breast cancer cells SK-BR-3 was evaluated.

[0320] Figure 4 : The present invention discloses the plasma stability experimental results of ADC-25.

[0321] Figure 5 :The present invention discloses the efficacy of ADC on human brain astrocytoma U87MG nude mouse transplanted tumors.

[0322] Figure 6 :The present invention discloses the efficacy of ADC on Detroit 562 nude mouse transplanted tumors of human pharyngeal carcinoma pleural effusion metastatic cells.

[0323] Figure 7 :The present invention discloses the efficacy of ADC on human glioma U87MG nude mouse transplanted tumors.

[0324] Figure 8 : Prescription screening experiment fitting trend chart, where the unit of PS80 is 10 -4 g / mL; the unit of ADC-32 protein concentration (based on naked antibody concentration) is mg / mL. DETAILED DESCRIPTION

[0325] The present disclosure is further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods in the examples of the present disclosure that do not specify specific conditions are usually performed under conventional conditions, such as the Antibody Technology Laboratory Manual and the Molecular Cloning Manual published by the Cold Spring Harbor Laboratory; or under the conditions recommended by the raw material or product manufacturer. Reagents that do not specify specific sources are conventional reagents purchased from the market.

[0326] 1. Antibody-drug conjugates

[0327] 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.

[0328] MS was measured using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQadvantage MAX).

[0329] UPLC was measured using a Waters Acquity UPLC SQD liquid spectrometer.

[0330] 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).

[0331] UV-HPLC was measured using a Thermo nanodrop 2000 ultraviolet spectrophotometer.

[0332] Proliferation inhibition rate and IC 50 The values ​​were determined using PHERA starFS microplate reader (BMG, Germany).

[0333] 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.

[0334] Column chromatography generally uses Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0335] 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, Accela ChemBio Inc, Darui Chemicals, and other companies.

[0336] Unless otherwise specified in the examples, all reactions were carried out under argon or nitrogen atmosphere.

[0337] Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1L.

[0338] Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a volume of about 1L.

[0339] The pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or a HC2-SS hydrogenator.

[0340] The hydrogenation reaction is usually carried out by evacuating the vacuum, filling with hydrogen, and repeating the operation three times.

[0341] The microwave reaction was carried out using a CEM Discover-S 908860 microwave reactor.

[0342] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0343] Unless otherwise specified in the examples, the reaction temperature is room temperature.

[0344] Room temperature is the most suitable reaction temperature, and the temperature range is 20℃~30℃.

[0345] 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.

[0346] 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.

[0347] Some compounds disclosed herein were characterized by Q-TOF LC / MS, which was performed using 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).

[0348] Example 1-1

[0349] 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-hydroxycyclopropane-1-carboxamide

[0350]

[0351] 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%).

[0352] MS m / z(ESI):520.2[M+1]

[0353] 1H 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).

[0354] Example 1-2

[0355] (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

[0356] (R)-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-B

[0357]

[0358] Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to compound 1b (4 mg, 7.53 μmol), replace the gas with 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, and 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: 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 (2-A: 1.5mg, 2-B: 1.5mg).

[0359] MS m / z(ESI):534.0[M+1].

[0360] Single configuration compound 2-B (shorter retention time):

[0361] 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).

[0362] 1 H 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).

[0363] Single configuration compound 2-A (longer retention time):

[0364] UPLC analysis: retention time 1.10 minutes, purity: 86% (chromatographic column: ACQUITY UPLC BEHC18 1.7um2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0365] 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).

[0366] Examples 1-3

[0367] (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-hydroxypropanoyl

[0368] Amine 3-A

[0369] (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

[0370]

[0371]

[0372] Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to compound 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 and react 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).

[0373] MS m / z(ESI):561.9[M+1].

[0374] Single configuration compound (short retention time):

[0375] UPLC analysis: retention time 1.11 minutes, purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7um2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0376] 1 H 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).

[0377] Single configuration compound (longer retention time):

[0378] UPLC analysis: retention time 1.19 minutes, purity: 90% (chromatographic column: ACQUITY UPLC BEHC18 1.7um2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0379] 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).

[0380] Examples 1-4

[0381] 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-hydroxycyclopentane-1-carboxamide

[0382]

[0383] Add 1 mL of N,N-dimethylformamide to compound 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%).

[0384] MS m / z(ESI):548.0[M+1].

[0385] 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).

[0386] Examples 1-5

[0387] 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)cyclopropane-1-carboxamide 5

[0388]

[0389] Add 1 mL of N,N-dimethylformamide to compound 1b (2.0 mg, 3.76 μmol), cool to 0-5°C in an ice-water bath, add one 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%).

[0390] MS m / z(ESI):533.9[M+1].

[0391] 1H 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).

[0392] Examples 1-6

[0393] 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

[0394]

[0395]

[0396] Add 1 mL of N,N-dimethylformamide to compound 1b (3.0 mg, 5.64 μmol), cool to 0-5°C in an ice-water bath, add one 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 document "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%).

[0397] MS m / z(ESI):548.0[M+1].

[0398] 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).

[0399] Examples 1-7

[0400] 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-hydroxycyclobutane-1-carboxamide 7

[0401]

[0402]

[0403] Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to compound 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 is complete, 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%).

[0404] MS m / z(ESI):534.0[M+1].

[0405] 1H 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).

[0406] Examples 1-8

[0407] 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

[0408]

[0409]

[0410] first step

[0411] Benzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropane-1-carboxylate 8c

[0412] 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%).

[0413] MS m / z(ESI):501.0[M+1].

[0414] Step 2

[0415] 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropane-1-carboxylic acid 8d

[0416] Compound 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%).

[0417] MS m / z(ESI):411.0[M+1].

[0418] Step 3

[0419] (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

[0420] Compound 1b (7 mg, 0.013 mmol) was added to a 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 0.5 mL N,N-dimethylformamide solution of compound 8d (7 mg, 0.017 mmol) 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 a 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 a developing solvent system B to obtain the title product 8e (8.5 mg, yield 78.0%).

[0421] MS m / z(ESI):828.0[M+1].

[0422] Step 4

[0423] 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

[0424] Compound 8e (4 mg, 4.84 μmol) was dissolved in 0.2 mL of dichloromethane, and 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 for slurrying, and the upper n-hexane layer was poured out, and the reaction was repeated three times. The crude title product 8f (2.9 mg) was obtained and the product was directly used in the next step without purification.

[0425] MS m / z(ESI):606.0[M+1].

[0426] Step 5

[0427] 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

[0428] 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%).

[0429] MS m / z(ESI):1060.0[M+1].

[0430] 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).

[0431] Examples 1-9

[0432] 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-1H-pyrrol-1-yl)hexanamide 9-A

[0433] 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-1H-pyrrol-1-yl)hexanamide 9-B

[0434]

[0435]

[0436] first step

[0437] Benzyl 2-cyclopropyl-2-hydroxyacetate 9a

[0438] Compound 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%).

[0439] Step 2

[0440] 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-oic acid benzyl ester 9b

[0441] Compound 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, ethyl acetate (20 mL×2) and chloroform (10 mL×5) were extracted, 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 the mixture was extracted with ethyl acetate (10 mL×3). The organic phase was washed with a 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%).

[0442] MS m / z(ESI):515.0[M+1].

[0443] Step 3

[0444] 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid 9c

[0445] Compound 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.

[0446] MS m / z(ESI):424.9[M+1].

[0447] Step 4

[0448] (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

[0449] Compound 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 with 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%).

[0450] MS m / z(ESI):842.1[M+1].

[0451] Step 5

[0452] 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]quinolin-1-yl)acetamide 9e

[0453] Compound 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 oil pump was used to dry the crude title product 9e (17 mg), which was used directly in the next step without purification.

[0454] MS m / z(ESI):638.0[M+18].

[0455] Step 6

[0456] 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-1H-pyrrol-1-yl)hexanamide 9-A

[0457] 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-1H-pyrrol-1-yl)hexanamide 9-B

[0458] 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).

[0459] MS m / z(ESI):1074.4[M+1].

[0460] Single configuration compound 9-A (shorter retention time):

[0461] UPLC analysis: retention time 1.14 minutes, purity: 85% (chromatographic column: ACQUITY UPLC BEHC18 1.7um2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0462] 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).

[0463] Single configuration compound 9-B (longer retention time):

[0464] UPLC analysis: retention time 1.16 minutes, purity: 89% (chromatographic column: ACQUITY UPLC BEHC18 1.7um2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0465] 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).

[0466] Examples 1-10

[0467] 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

[0468] 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

[0469]

[0470]

[0471] first step

[0472] Benzyl 3,3,3-trifluoro-2-hydroxypropionate 10a

[0473] Compound 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%).

[0474] 1 H NMR (400MHz, CDCl3): δ7.43-7.36(m,5H),5.34(s,2H),4.53(s,1H),3.44(s,1H).

[0475] Step 2

[0476] 1-(9H-fluoren-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester 10b

[0477] Compound 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%).

[0478] MS m / z(ESI):559.9[M+18].

[0479] Step 3

[0480] 1-(9H-fluoren-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazaundecane-11-oic acid 10c

[0481] Compound 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 a crude title product 10c (13 mg).

[0482] MS m / z(ESI):452.9[M+1].

[0483] Step 4

[0484] (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

[0485] Compound 1b (10 mg, 18.8 μmol) was added to a 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%).

[0486] MS m / z(ESI):870.0[M+1].

[0487] Step 5

[0488] 2-((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)-3,3,3-trifluoropropanamide 10e

[0489] Compound 10d (16 mg, 18.4 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 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 crude title product 10e (12 mg) was obtained by oil pump drying, and the product was used directly in the next step without purification.

[0490] MS m / z(ESI):647.9[M+1].

[0491] Step 6

[0492] 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

[0493] 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

[0494] 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).

[0495] MS m / z(ESI):1102.0[M+1].

[0496] Single configuration compound (short retention time):

[0497] UPLC analysis: retention time 1.18 minutes, purity: 91% (chromatographic column: ACQUITY UPLC BEHC18 1.7um2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0498] 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).

[0499] Single configuration compound (longer retention time):

[0500] 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).

[0501] 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).

[0502] Examples 1-11

[0503] 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)cyclobutane-1-carboxamide

[0504]

[0505]

[0506] first step

[0507] Benzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclobutane-1-carboxylate 11b

[0508] 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%).

[0509] MS m / z(ESI):514.6[M+1].

[0510] Step 2

[0511] 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclobutane-1-carboxylic acid 11c

[0512] Compound 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.

[0513] Step 3

[0514] (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

[0515] Compound 1b (10 mg, 0.018 mmol) was added to a 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 0.5 mL N,N-dimethylformamide solution of compound 11c (13 mg, 0.031 mmol) 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 a 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 a developing solvent system A to obtain the title product 11d (19 mg, yield 73.9%).

[0516] MS m / z(ESI):842.3[M+1].

[0517] Step 4

[0518] 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

[0519] Compound 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, 1 mL of toluene was added and concentrated under reduced pressure, the reaction mixture was repeated twice, 4 mL of n-hexane was added for slurrying, the n-hexane layer was poured out, the reaction mixture was repeated three times, and the crude title product 11e (15 mg) was obtained, which was used directly in the next step without purification.

[0520] Step 5

[0521] 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)cyclobutane-1-carboxamide

[0522] 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%).

[0523] MS m / z(ESI):1073.6[M+1].

[0524] 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).

[0525] Examples 1-12

[0526] (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

[0527] (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

[0528]

[0529] first step

[0530] 3-Cyclopropyl-2-hydroxypropionic acid 12b

[0531] Compound 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%).

[0532] Step 2

[0533] (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

[0534] (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

[0535] Add 1.5 mL of ethanol and 1.5 mL of N,N-dimethylformamide to compound 1b (45 mg, 0.085 mmol), replace the gas with argon three times, add 0.1 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add compound 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) to the reaction solution in sequence, and stir at room temperature for 3 hours after the addition. The reaction solution is concentrated under reduced pressure, and the crude compound 12 obtained is 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).

[0536] MS m / z(ESI):547.9[M+1].

[0537] Single configuration compound (short retention time):

[0538] 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).

[0539] 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).

[0540] Single configuration compound (longer retention time):

[0541] 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).

[0542] 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).

[0543] Example 1-13 (reference example)

[0544] 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

[0545]

[0546] The title compound 13 was prepared according to the method disclosed in "Example 76 on page 147 of the specification of EP2907824A1".

[0547] Examples 1-14

[0548] 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-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-A

[0549] 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-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-B

[0550]

[0551]

[0552] first step

[0553] Benzyl 3-cyclopropyl-2-hydroxypropionate 14a

[0554] Compound 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%).

[0555] Step 2

[0556] 10-(Cyclopropylmethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester 14b

[0557] Compound 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 raised 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 a 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%).

[0558] MS m / z(ESI):529.2[M+1].

[0559] Step 3

[0560] 10-(Cyclopropylmethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid 14c

[0561] Compound 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.

[0562] MS m / z(ESI):439.1[M+1].

[0563] Step 4

[0564] (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

[0565] Compound 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 a 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 a developing solvent system B to obtain the title product 14d (29 mg, yield: 79.9%).

[0566] MS m / z(ESI):856.1[M+1].

[0567] Step 5

[0568] 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]quinolin-1-yl)propanamide 14e

[0569] Compound 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 mixture 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 mixture was repeated three times. The residue was concentrated under reduced pressure, and the crude title product 14e (22 mg) was obtained by oil pump drying. The product was used directly in the next step without purification.

[0570] MS m / z(ESI):634.1[M+1].

[0571] Step 6

[0572] 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-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-A

[0573] 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-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-B

[0574] 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 (10 mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18 mL / min) to obtain the title product (2 mg, 2 mg).

[0575] MS m / z(ESI):1088.4[M+1].

[0576] Single configuration compound (short retention time):

[0577] UPLC analysis: retention time 1.18 minutes, purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7um2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0578] Single configuration compound (longer retention time):

[0579] UPLC analysis: retention time 1.23 minutes, purity: 96% (chromatographic column: ACQUITY UPLC BEHC18 1.7um2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0580] Examples 1-15

[0581] 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

[0582]

[0583] first step

[0584] Benzyl 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylate 15b

[0585] Compound 8b (500 mg, 1.35 mmol) was added to a reaction flask, followed by 6 mL of tetrahydrofuran, and 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, argon was replaced three times, the temperature was cooled to 0-5°C in an ice-water bath, sodium hydride (54 mg, 1.35 mmol) was added, the ice bath was removed, the temperature was raised to room temperature and stirred for 40 minutes; the temperature was lowered to zero and 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, 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%).

[0586] MS m / z(ESI):515.2[M+1].

[0587] Step 2

[0588] 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylic acid 15c

[0589] Compound 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%).

[0590] MS m / z(ESI):425.2[M+1].

[0591] Step 3

[0592] (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

[0593] Compound 1b (10 mg, 0.021 mmol) was added to a 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, compound 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 completed. 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 15d (19 mg, yield 87.0%).

[0594] MS m / z(ESI):842.2[M+1].

[0595] Step 4

[0596] 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

[0597] Compound 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.

[0598] MS m / z(ESI):620.1[M+1].

[0599] Step 5

[0600] 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

[0601] The crude product 15e (13.9 mg, 22.4 μmol) was dissolved in 1 mL N,N-dimethylformamide, argon was replaced three times, and the temperature was cooled to 0-5°C in an ice-water bath, 8 g (15.8 mg, 33.4 μmol) was added, and 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%).

[0602] MS m / z(ESI):1074.2[M+1].

[0603] 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).

[0604] Examples 1-16

[0605] 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

[0606]

[0607]

[0608] first step

[0609] 1-(Hydroxymethyl)cyclobutane-1-carboxylic acid 16b

[0610] 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.

[0611] MS m / z (ESI, NEG): 129.2 [M-1].

[0612] Step 2

[0613] Benzyl 1-(Hydroxymethyl)cyclobutane-1-carboxylate 16c

[0614] 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 μmol) 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%).

[0615] MS m / z(ESI):221.1[M+1].

[0616] Step 3

[0617] Benzyl 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylate 16d

[0618] Compound 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 obtained 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%).

[0619] MS m / z(ESI):551.3[M+23].

[0620] Step 4

[0621] 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylic acid 16e

[0622] Compound 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.

[0623] Step 5

[0624] (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

[0625] Compound 1b (7.5 mg, 0.014 mmol) was added to a 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 0.5 mL N,N-dimethylformamide solution of crude product 16e (10 mg) 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%).

[0626] MS m / z(ESI):856.2[M+1].

[0627] Step 6

[0628] 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]quinolin-1-yl)cyclobutane-1-carboxamide 16g

[0629] Compound 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.

[0630] MS m / z(ESI):634.1[M+1].

[0631] Step 7

[0632] 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

[0633] 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%).

[0634] MS m / z(ESI):1088.0[M+1].

[0635] Examples 1-17

[0636] (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

[0637]

[0638]

[0639] first step

[0640] 1-phenyl-2,5,8,11,14,17,20,23,26,29-decaoxahistriacont-31-oic acid tert-butyl ester 17b

[0641] 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%).

[0642] MS m / z(ESI):636.3[M+18].

[0643] Step 2

[0644] 29-Hydroxy-3,6,9,12,15,18,21,24,27-nonaoxaconicotin-1-oic acid tert-butyl ester 17c

[0645] Compound 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.

[0646] MS m / z(ESI):546.2[M+18].

[0647] Step 3

[0648] 29-Azido-3,6,9,12,15,18,21,24,27-nonaoxaconicotin-1-oic acid tert-butyl ester 17d

[0649] Compound 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 gas 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).

[0650] MS m / z(ESI):571.3[M+18].

[0651] Step 4

[0652] 29-Amino-3,6,9,12,15,18,21,24,27-nonaoxaconicotin-1-oic acid tert-butyl ester 17e

[0653] Compound 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.

[0654] MS m / z(ESI):528.2[M+1].

[0655] Step 5

[0656] 1-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azahexatin-31-oic acid tert-butyl ester 17f

[0657] 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%).

[0658] MS m / z(ESI):747.2[M+1].

[0659] Step 6

[0660] 1-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azahexatin-31-oic acid 17 g

[0661] Compound 17f (122 mg, 0.163 mmol) was dissolved in 0.8 mL of dichloromethane, and 0.4 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 hour. 15 mL of dichloromethane was added for dilution, and the mixture was concentrated under reduced pressure; 10 mL of n-hexane was added, and the mixture was concentrated under reduced pressure, and the mixture was repeated twice; 10 mL of toluene was added, and the mixture was 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 value was close to 7, and the mixture was concentrated and pumped dry with an oil pump to obtain the title product 17 g (98 mg, yield: 86.8%).

[0662] MS m / z(ESI):691.2[M+1].

[0663] Step 7

[0664] 2,4-Dimethoxybenzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropyl-1-carboxylate 17h

[0665] Compound 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%).

[0666] MS m / z(ESI):583.1[M+23].

[0667] Step 8

[0668] 2,4-Dimethoxybenzyl (S)-1-((11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazahepta-17-yl)oxy)cyclopropyl-1-carboxylate 17j

[0669] Compound 17h (39 mg, 69.6 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 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 make pulp, 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%).

[0670] MS m / z(ESI):822.0[M+1].

[0671] Step 9

[0672] (S)-1-((11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazahepta-17-yl)oxy)cyclopropane-1-carboxylic acid 17k

[0673] Compound 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%).

[0674] Step 10

[0675] (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-tetraazatride-13-yl)carbamate 17l

[0676] Compound 1b (20 mg, 42.4 μmol) was added to the reaction flask, and 1 mL of 10% (v / v) methanol in dichloromethane solution was added. The argon gas was replaced three times, and the mixture was cooled to 0-5°C in an ice-water bath. A drop of triethylamine was added and stirred until compound 1b was dissolved. Compound 17k (33 mg, 49.1 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 (17.6 mg, 63.6 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 mixture was allowed to stand for stratification, and 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 the title product 171 (37 mg, yield: 80.2%).

[0677] MS m / z(ESI):1090.1[M+1].

[0678] Step 11

[0679] (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

[0680] Compound 17l (15.5 mg, 14.23 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 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. The mixture was concentrated under reduced pressure, and then pumped dry with an oil pump. The obtained crude product was dissolved in 1 mL of N,N-dimethylformamide, compound 17g (11 mg, 15.92 μmol) was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (6.0 mg, 21.68 μmol) was added, and the mixture was replaced with argon three times. The mixture was stirred 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 (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 17 (6 mg, yield: 27.4%).

[0681] MS m / z(ESI):1556.4[M+18].

[0682] 1H 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).

[0683] Examples 1-18

[0684] (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

[0685]

[0686] first step

[0687] (R)-2-Cyclopropyl-2-hydroxybenzyl acetate 18a

[0688] (S)-2-Cyclopropyl-2-hydroxybenzyl acetate 18b

[0689] Compound 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).

[0690] Step 2

[0691] (R)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester 18c

[0692] Compound 8b (3.1 g, 8.41 mmol) was dissolved in tetrahydrofuran (55 mL), compound 18a (2.0 g, 9.70 mmol) was added, cooled to 0-5°C in an ice-water bath, potassium tert-butoxide (1.89 g, 16.84 mmol) was added, and stirred for 10 minutes in an ice-water bath. Ethyl acetate (30 mL) and water (20 mL) were added, and the layers were allowed to stand. The aqueous phase was extracted with chloroform (30 mL×5), and the organic phases were combined. The organic phase was concentrated under reduced pressure, and the obtained residue was dissolved in 1,4-dioxane (32 mL) and water (8 mL), sodium carbonate (1.78 g, 16.79 mmol) and 9-fluorenylmethyl chloroformate (2.18 g, 8.42 mmol) were added, and stirred at room temperature for 2 hours. Water (30 mL) was added to the reaction solution, and it 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 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%).

[0693] MS m / z(ESI):515.2[M+1].

[0694] Step 3

[0695] (R)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid 18d

[0696] Compound 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.

[0697] MS m / z(ESI):425.1[M+1].

[0698] Step 4

[0699] 2,4-Dimethoxybenzyl (R)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-ol 18e

[0700] 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%).

[0701] MS m / z(ESI):575.0[M+1].

[0702] Step 5

[0703] 2,4-Dimethoxybenzyl (R)-2-((2-aminoacetamido)methoxy)-2-cyclopropyl acetate 18f

[0704] Compound 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.

[0705] Step 6

[0706] 2,4-Dimethoxybenzyl (11S, 19R)-11-benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaicos-20-oic acid ester 18g

[0707] The crude product 18f (21 mg, 57.4 μmol) was dissolved in 3 mL of N,N-dimethylformamide, compound 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%).

[0708] MS m / z(ESI):853.0[M+18].

[0709] Step 7

[0710] (11S,19R)-11-benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaicos-20-oic acid 18h

[0711] Compound 18g (37 mg, 44.3 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 (15.4 mg, 132.4 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 18h (24 mg, yield: 79.1%).

[0712] MS m / z(ESI):708.2[M+23].

[0713] Step 8

[0714] (9H-fluoren-9-yl)methyl ((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-tetraazahexa-16-yl)carbamate 18i

[0715] Compound 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 gas 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 compound 1b was dissolved. Compound 18h ​​(65 mg, 94.8 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 (27 mg, 97.6 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, stirred for 5 minutes, and 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 the title product 18i (25 mg, yield: 35.6%).

[0716] MS m / z(ESI):1104.4[M+1].

[0717] Step 9

[0718] (S)-2-(2-(2-aminoacetamido)acetamido)-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

[0719] Compound 18i (12 mg, 10.9 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 1.5 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.

[0720] MS m / z(ESI):881.0[M+1].

[0721] Step 10

[0722] (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]quinoline 1-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide)-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

[0723] The crude product 18j (10 mg) was dissolved in 1 mL of N,N-dimethylformamide, compound 17g (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 the mixture was stirred at room temperature for 30 minutes. The reaction solution was filtered and purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge PrepC18 OBD 5um 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%).

[0724] MS m / z(ESI):1570.2[M+18].

[0725] 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,1 4H),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).

[0726] Examples 1-19

[0727] (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]quinoline 1-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide)-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

[0728]

[0729] first step

[0730] (S)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester 19a

[0731] Compound 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 with 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%).

[0732] Step 2

[0733] (S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid 19b

[0734] Compound 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 a crude title product 19b (230 mg), which was directly used for the next step without purification.

[0735] Step 3

[0736] 2,4-Dimethoxybenzyl (S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oate 19c

[0737] 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%)

[0738] Step 4

[0739] 2,4-Dimethoxybenzyl (S)-2-((2-aminoacetamido)methoxy)-2-cyclopropyl acetate 19d

[0740] Compound 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 was poured out, and the reaction was repeated three times, and the crude title product 19d (21 mg) was obtained by concentration under reduced pressure, and the product was used directly in the next step without purification.

[0741] Step 5

[0742] 2,4-Dimethoxybenzyl (11S, 19S)-11-benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaicos-20-oate 19e

[0743] The crude product 19d (36 mg, 102.2 μmol) was dissolved in 4 mL of N,N-dimethylformamide, compound 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 a 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 a developing solvent system B to obtain the title product 19e (70 mg, yield: 80.2%).

[0744] Step 6

[0745] (11S,19S)-11-Benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaicos-20-oic acid 19f

[0746] Compound 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%).

[0747] Step 7

[0748] (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

[0749] Compound 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 gas 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 compound 1b was dissolved. Compound 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, stirred for 5 minutes, and 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.

[0750] MS m / z(ESI):1103.1[M+1].

[0751] Step 8

[0752] (S)-2-(2-(2-aminoacetamido)acetamido)-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

[0753] Compound 19g (4.6 mg, 4.16 μmol) was dissolved in 1.5 mL of dichloromethane, and 0.75 mL 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 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 layer of n-hexane was poured out, and the reaction was repeated three times. The crude title product 19h (4.0 mg) was obtained, and the product was used directly in the next step without purification.

[0754] Step 9

[0755] (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]quinoline 1-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide)-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

[0756] The crude product 19h (4.0 mg) was dissolved in 1 mL of N,N-dimethylformamide, compound 17g (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 PrepC18 OBD 5um 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 19 (2.1 mg, yield: 32.4%).

[0757] 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).

[0758] Example 1-20 (reference example)

[0759]

[0760] The title compound 20 was synthesized according to the method provided in "Example 58 on page 163 of the specification of CN104755494A".

[0761] The following antibodies are prepared according to conventional antibody methods, for example, after constructing a vector, transfecting eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019), and obtaining them after expression and purification.

[0762] The following is the sequence of Trastuzumab:

[0763] Light chain

[0764]

[0765] Heavy chain

[0766]

[0767] The following is the sequence of Pertuzumab:

[0768] Light chain

[0769]

[0770] Heavy chain

[0771]

[0772] The following is the sequence of the B7H3 antibody 1F9DS:

[0773] Light chain

[0774]

[0775] Heavy chain

[0776]

[0777] Example 1-21 ADC-1

[0778]

[0779] 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.

[0780] 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 buffered aqueous 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 at 4° C.

[0781] UV-HPLC calculated average value: n=5.09.

[0782] Example 1-22 ADC-2

[0783]

[0784] 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.

[0785] 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, and oscillated for 3 hours at 25° C. 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 FADC-1 general formula, ADC-2, in PBS buffer (4.95 mg / mL, 1.1 mL), which was stored at 4° C.

[0786] UV-HPLC calculated average value: n=7.39.

[0787] Example 1-23 ADC-3

[0788]

[0789] 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.

[0790] 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 buffer 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 at 4° C.

[0791] UV-HPLC calculated average value: n=7.36.

[0792] Example 1-24 ADC-4

[0793]

[0794] 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 1.3 ml of the solution was taken out for further reaction.

[0795] 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 buffered aqueous 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 at 4° C.

[0796] UV-HPLC calculated average value: n=7.39.

[0797] Example 1-25 ADC-5

[0798]

[0799] 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.

[0800] 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 buffered aqueous 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 at 4° C.

[0801] UV-HPLC calculated average value: n=3.16.

[0802] Example 1-26 ADC-6

[0803]

[0804] 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.

[0805] 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 buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product ADC-6 of the general formula FADC-4B in PBS buffer (1.78 mg / mL, 1.78 mL), which was stored at 4° C.

[0806] UV-HPLC calculated average value: n=3.94.

[0807] Example 1-27 ADC-7

[0808]

[0809] 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 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 1.0 ml of the solution was taken out for further reaction.

[0810] 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 at 4° C.

[0811] UV-HPLC calculated average value: n=6.91.

[0812] Example 1-28 ADC-8

[0813]

[0814] 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 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.

[0815] Compound 10-short retention time compound (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 FADC-8 general formula, ADC-8, in PBS buffer (2.14 mg / mL, 2.31 mL), which was stored at 4° C.

[0816] UV-HPLC calculated average value: n=6.58.

[0817] Example 1-29 ADC-9

[0818]

[0819] 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 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.

[0820] 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 buffered aqueous 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 at 4° C.

[0821] UV-HPLC calculated average value: n=3.16.

[0822] Example 1-30 ADC-10

[0823]

[0824] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 19.76 μL, 197.6 nmol) to the PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 0.574 mL, 38.78 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.

[0825] Compound 14 (0.64 mg, 588 nmol) was dissolved in 40 μL 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 at 4° C.

[0826] UV-Vis calculated average value: n=6.25.

[0827] Example 1-31 ADC-11

[0828]

[0829] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 22.24 μL, 222.4 nmol) was added to a PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 0.646 mL, 43.64 nmol), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0830] Compound 14 (0.72 mg, 662 nmol) was dissolved in 40 μL 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, ADC-11, in PBS buffer (2.13 mg / mL, 1.87 mL), which was stored at 4° C.

[0831] UV-Vis calculated average value: n=7.03.

[0832] Example 1-32 ADC-12

[0833]

[0834] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.0 μL, 250.0 nmol) to the PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 0.726 mL, 49.05 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.

[0835] Compound 15 (0.81 mg, 754 nmol) was dissolved in 40 μL 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 buffered aqueous 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 at 4°C.

[0836] UV-Vis calculated average value: n=6.93.

[0837] Example 1-33 ADC-13

[0838]

[0839] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 9.88 μL, 98.8 nmol) to the PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 0.287 mL, 19.39 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.

[0840] Compound 16 (0.32 mg, 294 nmol) was dissolved in 20 μL 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 at 4°C.

[0841] UV-Vis calculated average value: n=6.53.

[0842] Example 1-34 ADC-14

[0843]

[0844] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol) was added to a 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), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0845] Compound 17 (0.92 mg, 598 nmol) was dissolved in 40 μL 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 in PBS buffer (0.30 mg / mL, 12.0 mL), which was stored at 4°C.

[0846] UV-Vis calculated average value: n=7.61.

[0847] Example 1-35 ADC-15

[0848]

[0849] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol) was added to a 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), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0850] Compound 18 (0.93 mg, 599 nmol) was dissolved in 40 μL 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-15 in PBS buffer (0.32 mg / mL, 11.8 mL), which was stored at 4°C.

[0851] UV-Vis calculated average value: n=7.89.

[0852] Example 1-36 ADC-16

[0853]

[0854] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 18.25 μL, 182.5 nmol) to the PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 0.53 mL, 35.8 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.

[0855] Compound 19 (0.83 mg, 534 nmol) was dissolved in 35 μL 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-16 in PBS buffer (0.32 mg / mL, 12.0 mL), which was stored at 4°C.

[0856] UV-Vis calculated average value: n=7.43.

[0857] Example 1-37 ADC-17

[0858]

[0859] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 43.2μL, 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.

[0860] Compound 9-short retention time compound 9-A (2.22 mg, 2067 nmol) was dissolved in 175 μL 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 at 4°C.

[0861] UV-Vis calculated average value: n=5.42.

[0862] Example 1-38 ADC-18 (reference example)

[0863]

[0864] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 51.7μL, 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.

[0865] Compound 20 (2.0 mg, 1934 nmol) was dissolved in 100 μL 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 of FADC-18 in PBS buffer (0.79 mg / mL, 13.0 mL), which was stored at 4° C.

[0866] UV-Vis calculated average value: n=7.23.

[0867] Example 1-39 ADC-19

[0868]

[0869] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 46.9 μL, 469 nmol) was added to a PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 1.36 mL, 91.9 nmol), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0870] Compound 9-short retention time compound 9-A (2.0 mg, 1862 nmol) was dissolved in 100 μL 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-4A formula, ADC-19, in PBS buffer (0.73 mg / mL, 13.0 mL), which was stored at 4°C.

[0871] UV-Vis calculated average value: n=6.26.

[0872] Example 1-40 ADC-20

[0873]

[0874] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10mM, 51.7μL, 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.

[0875] Compound 10 (2.0 mg, 1815 nmol) was dissolved in 100 μl 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 FADC-1 general formula, ADC-20, in PBS buffer (0.73 mg / mL, 13.0 mL), which was stored at 4° C.

[0876] UV-Vis calculated average value: n=7.43.

[0877] Example 1-41 ADC-21 (reference example)

[0878]

[0879] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 63.9 μL, 639 nmol) was added to a PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 1.86 mL, 125.4 nmol), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0880] Compound 20 (2.07 mg, 2001 nmol) was dissolved in 150 μL 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 at 4°C.

[0881] UV-Vis calculated average value: n=7.23.

[0882] Example 1-42 ADC-22

[0883]

[0884] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 64.9 μL, 649 nmol) was added to a PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 1.88 mL, 127.2 nmol), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0885] Compound 9-short retention time compound 9-A (2.1 mg, 1955 nmol) was dissolved in 150 μL 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 at 4°C.

[0886] UV-Vis calculated average value: n=6.79.

[0887] Example 1-43 ADC-23 (reference example)

[0888]

[0889] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.89 mL, 118.9 μmol) was added to a PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 345 mL, 23.31 μmol), placed in a water bath shaker, and oscillated at 37°C for 3.5 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0890] Compound 20 (362 mg, 350 μmol) 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) and succinic acid buffer solution (0.01 M succinic acid buffer solution at pH = 5.3), followed by sucrose 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-23 of the general formula of FADC-18 was obtained and stored at 4° C.

[0891] UV-Vis calculated average value: n=7.05.

[0892] Example 1-44 ADC-24

[0893]

[0894] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.44 mL, 114.4 μmol) was added to a PBS buffer solution of antibody Trastuzumab (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg / mL, 332 mL, 22.43 μmol), placed in a water bath shaker, and oscillated at 37°C for 3.5 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0895] Compound 9-short retention time compound 9-A (241 mg, 224 μmol) 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) containing 4% (v / v) MeCN and 2% (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, and the lyophilized powder sample of the exemplary product ADC-24 of the general formula of FADC-4A was obtained after lyophilization in bottles, and stored at 4° C.

[0896] UV-Vis calculated average value: n=7.07.

[0897] Example 1-45 ADC-25

[0898]

[0899] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 73.7 μL, 740 nmol) was added to a PBS buffer solution of antibody B7H3 antibody 1F9DS (pH=6.5 0.05 M PBS buffer solution; 10.0 mg / mL, 2.14 mL, 144.60 nmol), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0900] Compound 9-short retention time compound 9-A (3.0 mg, 2793 nmol) was dissolved in 150 μL 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 an exemplary product of the general formula FADC-25 in PBS buffer (1.28 mg / mL, 13.0 mL), which was stored at 4°C.

[0901] UV-Vis calculated average value: n=6.87.

[0902] Example 1-46 ADC-26 (reference example)

[0903]

[0904] At 37°C, add the prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 30.1 μL, 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.

[0905] Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μL 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-26 in PBS buffer (1.61 mg / mL, 4.0 mL), which was stored at 4° C.

[0906] UV-Vis calculated average value: n=6.15.

[0907] Example 1-47 ADC-27

[0908]

[0909] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) was added to a 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), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0910] Compound 9-short retention time compound 9-A (1.02 mg, 950 nmol) was dissolved in 100 μL 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 at 4°C.

[0911] UV-Vis calculated average value: n=6.11.

[0912] Example 1-48 ADC-28 (reference example)

[0913]

[0914] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 81.3 μL, 810 nmol) was added to a 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), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0915] Compound 20 (3.0 mg, 2901 nmol) was dissolved in 150 μL 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 at 4° C.

[0916] UV-Vis calculated average value: n=7.46.

[0917] Example 1-49 ADC-29

[0918]

[0919] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 28.6 μL, 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.

[0920] Compound 9-short retention time compound 9-A (1.29 mg, 1201 nmol) was dissolved in 100 μL 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 (2.63 mg / mL, 2.4 mL) of the exemplary product ADC-29 of the general formula of FADC-25, which was stored at 4°C.

[0921] UV-Vis calculated average value: n=7.24.

[0922] Example 1-50 ADC-30 (reference example)

[0923]

[0924] At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 29.1 μL, 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.

[0925] Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μL 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-26, ADC-30, in PBS buffer (1.61 mg / mL, 4.0 mL), which was stored at 4° C.

[0926] UV-Vis calculated average value: n=6.15.

[0927] Example 1-51 ADC-31

[0928]

[0929] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) was added to a 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), and the solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0930] Compound 8 (1.0 mg, 943 nmol) was dissolved in 100 μL 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-31 in PBS buffer (1.47 mg / mL, 4.5 mL), which was stored at 4° C.

[0931] UV-Vis calculated average value: n=6.33.

[0932] Example 1-52 ADC-32

[0933]

[0934] At 25°C, in a buffer (pH 5.6) containing 20 mM histidine-hydrochloric acid and 2.5 mM EDTA, 5.0 g of trastuzumab stock solution (34.44 μmol, 20 mM histidine-hydrochloric acid buffer diluted trastuzumab to a final antibody concentration of 15 mg / mL) and 34.64 mg of tris(2-carboxyethyl)phosphine hydrochloride (reducing agent TCEP, Sigma, 120.84 μmol) were stirred in a constant temperature water bath for 3 hours to generate an intermediate I solution.

[0935] Compound 9-A (406.2 mg, 378.17 μmol), which has a shorter retention time, was dissolved in 9.98 mL DMSO to generate a DMSO solution of compound 9-A. 23.42 mL DMSO was pre-added to the intermediate I solution, and the DMSO solution of compound 9-A was added to the intermediate I solution pre-added with DMSO, and the reaction was stirred in a water bath at 25°C for 1 hour, and cysteine ​​was added to quench the reaction, and the reaction was filtered. At 25°C, the reaction solution was passed through an ultrafiltration membrane (30 kd) and replaced with 20 mM histidine-hydrochloric acid and 2.5 mM EDTA buffer aqueous solution (pH = 6.0) containing 10% (v / v) DMSO, and 10 mM histidine-hydrochloric acid buffer aqueous solution (pH = 5.5) by 10 times and 16 times of equal volume ultrafiltration to remove small molecules and residual solvents, and obtain the exemplary product ADC-32 of the general formula of FADC-4A.

[0936] The average value calculated by HIC method: n=6.0.

[0937] Example 1-53 ADC-33

[0938]

[0939] At 37°C, in a buffer (pH 6.0) containing 20 mM histidine-hydrochloric acid and 2.5 mM EDTA, 8.0 g of trastuzumab stock solution (55.11 μmol, trastuzumab diluted to a final antibody concentration of 15 mg / mL in 20 mM histidine buffer) and 123.95 mg of tris(2-carboxyethyl)phosphine hydrochloride (reducing agent TCEP, Sigma, 432.41 μmol) were stirred in a constant temperature water bath for 5 hours to generate an intermediate I solution.

[0940] Compound 9-A (754.7 mg, 702.63 μmol), which has a shorter retention time, was dissolved in 15.99 mL DMSO to generate a DMSO solution of compound 9-A. 37.34 mL DMSO was pre-added to the intermediate I solution, and then the DMSO solution of compound 9-A was added to the intermediate I solution pre-added with DMSO, and the reaction was stirred in a water bath at 37° C. for 1 hour, and the reaction was stopped and filtered. At 25° C., the reaction solution was passed through an ultrafiltration membrane (30 kd) and replaced with 20 mM histidine-hydrochloric acid and 2.5 mM EDTA buffer aqueous solution (pH=6.0) containing 10% (v / v) DMSO, and then 10 mM histidine-hydrochloric acid buffer aqueous solution (pH=6.0) by 8-fold and 16-fold equal volume ultrafiltration to remove small molecules and residual solvents, and obtain the exemplary product ADC-33 of the general formula of FADC-4A.

[0941] The average value calculated by HIC method was: n=7.15.

[0942] ADC stock solution drug loading analysis

[0943] Experimental purpose and principle

[0944] ADC is an 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 drug efficacy. The drug loading of ADC stock solution was measured using ultraviolet-visible spectrophotometry (UV-Vis) or hydrophobic chromatography (HIC).

[0945] 1. UV-Vis Spectrophotometry

[0946] 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.

[0947] 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:

[0948] (1)A 280nm =ε mab-280 B C mab +ε Drug-280 B C Drug

[0949] ε Drug-280: The average molar extinction coefficient of the drug at 280nm is 5100;

[0950] C Drug : drug concentration;

[0951] ε mab-280 : The average molar extinction coefficient of trastuzumab or pertuzumab at 280 nm is 214600;

[0952] C mab : The concentration of trastuzumab antigen solution or pertuzumab antigen solution;

[0953] b: The optical path length is 1 cm.

[0954] Similarly, the total absorbance equation of the sample at 370nm can be obtained:

[0955] (2)A 370nm =ε mab-370 B C mab +ε Drug-370 B C Drug

[0956] ε Drug-370 : The average molar extinction coefficient of the drug at 370nm is 19000;

[0957] C Drug : drug concentration;

[0958] ε mab-370 : The extinction coefficient of the trastuzumab antigen solution or the pertuzumab antigen solution at 370 nm is 0;

[0959] C mab : The concentration of trastuzumab antigen solution;

[0960] b: The optical path length is 1 cm.

[0961] 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.

[0962] Drug loading n = C Drug / C mab .

[0963] 2. Hydrophobic Chromatography

[0964] 2.1 HPLC analysis was performed under the following measurement conditions:

[0965] HPLC system: Agilent high performance liquid chromatography HPLC system

[0966] Detector: DAD detector (measurement wavelength: 280nm)

[0967] Chromatographic column: TSKgel Butyl-NPR (4.6mm×100mm, 2.5μm)

[0968] Column temperature: 30°C

[0969] Flow rate: 0.5ml / min

[0970] Sample chamber temperature: 4°C

[0971] Mobile phase A: pH 7.00 aqueous solution containing 1.5 M ammonium sulfate ((NH4)2SO4) and 20 mM disodium hydrogen phosphate (Na2HPO4).

[0972] Mobile phase B: a mixed solution at pH 7.00 containing 75% 20 mM disodium hydrogen phosphate (Na2HPO4) and 25% isopropanol.

[0973] Gradient program: 0.0% B-0.0% B (0 min-3.0 min), 0.0% B-100.0% B (3.0 min-30.0 min), 100.0% B-

[0974] 100.0%B (30.0min-33.0min), 0.0%B-0.0%B (33.1min-40.0min),

[0975] Injection sample volume: 50 μg

[0976] 2.2 Data Analysis

[0977] Based on the current data, due to the characteristics of the column, the antibody drug conjugates are eluted in the order of increasing number of bound drugs based on the difference in salt concentration, so the distribution of the number of conjugated drugs is obtained by measuring the area value of each peak. The peaks are D0 (antibody without drug linker), D2, D4, D6 and D8 in the elution order, and the drug loading DAR (n) is calculated as follows:

[0978] Table 1. Calculation table of drug loading in hydrophobic chromatography

[0979] name Number of connected drugs Weighted peak area percentage D0 0 D0 peak area*number of connected drugs D2 2 D2 peak area*number of connected drugs D4 4 D4 peak area*number of connected drugs D6 6 D6 peak area*number of connected drugs D8 8 D8 peak area*number of connected drugs

[0980] Drug loading n = Σ(weighted peak area) / 100

[0981] Biological evaluation

[0982] Test Example 1-1: Test on the inhibition of tumor cell proliferation in vitro by the disclosed compounds

[0983] 1. Test Purpose

[0984] The purpose of this experiment is to detect the inhibitory activity of the disclosed drug compounds on the in vitro proliferation of U87MG cells (Chinese Academy of Sciences Cell Bank, Catalog #TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, Catalog No. HTB-30). The cells were treated with different concentrations of the compounds in vitro and cultured for 6 days. 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.

[0985] 2. Experimental Methods

[0986] The following is an example of the in vitro proliferation inhibition test method for U87MG cells to illustrate the method for testing the in vitro proliferation inhibition activity of the disclosed compounds on tumor cells in the present disclosure. The present method is also applicable to, but not limited to, in vitro proliferation inhibition activity tests on other tumor cells.

[0987] 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.

[0988] 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 2-3mL trypsin (0.25% Trypsin-EDTA (1x), Gibico, Life Technologies) to digest for 2-3 minutes. After the cells are completely digested, add 10-15mL cell culture medium to wash the digested cells, centrifuge at 1000rpm for 5 minutes, discard the supernatant, and then add 10-20mL cell culture medium to resuspend the cells to make a single cell suspension.

[0989] 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 180 μL / well to a 96-well cell culture plate. Add only 200 μL 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).

[0990] 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.

[0991] The initial concentration of the small molecule compound was 500 nM, and the preparation method was as follows.

[0992] Add 30 μL of different samples to be tested to the first column of a 96-well U-bottomed drug plate, with a sample concentration of 100 μM; add 20 μL of DMSO to each well in columns 2 to 11. Take 10 μL of the sample in the first column and add it to 20 μL of DMSO in the second column, mix well, take 10 μL to the third column, and so on to column 10. Take 5 μL of the drug in each well of the drug plate and add it to 95 μL of EMEM culture medium, mix well, and set aside.

[0993] The starting concentration of ADC was 10 nM or 500 nM, and the preparation method was as follows.

[0994] Add 100 μL of different samples to be tested to the first column of the 96-well plate, with a sample concentration of 100 nM or 5 uM; add 100 μL of PBS to each well in columns 2 to 11. Take 50 μL of the sample in the first column and add it to 100 μL of PBS in the second column, mix well, take 50 μL to the third column, and so on, dilute 3 times to the 10th column.

[0995] 5. Sample addition operation: Add 20 μL of the sample to be tested with 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).

[0996] 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.

[0997] 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.

[0998] 3. Data Analysis

[0999] The data were processed and analyzed using Microsoft Excel and Graphpad Prism 5. The experimental results are shown in Table 2 below.

[1000] Table 2. IC values ​​of the small molecule fragments disclosed herein for inhibiting proliferation of SK-BR-3 cells and U87 cells in vitro 50 value

[1001]

[1002] Conclusion: The small molecule fragment disclosed herein has 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.

[1003] Test Example 1-2: In vitro proliferation inhibition test of HER2-targeted tumor cells by the disclosed antibody-drug conjugate

[1004] 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 cell proliferation was detected using CTG reagent. According to the IC 50 The values ​​were used to evaluate the in vitro activity of the compound.

[1005] 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, the cell suspension after density adjustment was mixed and added to a 96-well cell culture plate at 180 μL / well. The relevant compounds were tested and the results are shown in Table 3 below.

[1006] Table 3. IC values ​​of the disclosed antibody-drug conjugates for inhibiting the proliferation of HER2-targeted tumor cells in vitro 50 value

[1007]

[1008] Conclusion: The antibody-drug conjugates targeting HER2 disclosed herein have significant proliferation inhibitory activity against HER2-positive cells SK-BR-3; at the same time, they have weak proliferation inhibitory activity against HER2-negative cells MDA-MB-468 and have good selectivity.

[1009] Test Example 1-3: Her2-ADC plasma stability experiment

[1010] 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 μg / 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.

[1011] 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 μL.

[1012] 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 .

[1013] 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 .

[1014] 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 .

[1015] Test Example 1-4: Evaluation of drug efficacy in JIMT-1 tumor-bearing mice

[1016] 1. Purpose of the test

[1017] 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.

[1018] 2. Test Drugs and Materials

[1019] 1. Test drug

[1020] T-DM1 (prepared with reference to US20050169933)

[1021] ADC-21: 3 mg / kg

[1022] ADC-21: 10 mg / kg

[1023] ADC-24: 3 mg / kg

[1024] ADC-24: 10 mg / kg

[1025] Blank control: PBS

[1026] 2. Preparation method: All preparations were diluted with PBS.

[1027] 3. Experimental animals

[1028] Nunu mice were purchased from Beijing Weitonglihua.

[1029] 3. Test methods

[1030] 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.

[1031] 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.

[1032] 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.

[1033] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[1034] Relative volume (RTV) = V T / V0

[1035] Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%)

[1036] 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.

[1037] 4. Test results

[1038] 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.

[1039] Test Example 1-5: Evaluation of drug efficacy in SK-BR-3 tumor-bearing mice

[1040] 1. Purpose of the test

[1041] 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.

[1042] 2. Test Drugs and Materials

[1043] 1. Test drug

[1044] ADC-21: 1 mg / kg

[1045] ADC-21: 6 mg / kg

[1046] ADC-22: 1 mg / kg

[1047] ADC-22: 6 mg / kg

[1048] Blank control: PBS.

[1049] 2. Preparation method: All preparations were diluted with PBS.

[1050] 3. Experimental animals

[1051] Nunu mice were purchased from Beijing Weitonglihua.

[1052] 3. Test methods

[1053] 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.

[1054] The drug was administered once by intraperitoneal injection. The tumor volume and body weight were measured twice a week and the data were recorded.

[1055] 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.

[1056] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[1057] Relative volume (RTV) = V T / V0

[1058] Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%)

[1059] 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.

[1060] 4. Test results

[1061] 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.

[1062] Test Example 1-6: Plasma Stability

[1063] 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.

[1064] 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 supernatant for LC / MS / MS analysis.

[1065] The results are as follows Figure 4 The results showed that ADC-25 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% at most and tended to be stable on the 14th day.

[1066] Test Example 1-7: Evaluation of the efficacy of ADC on human brain glioblastoma U87MG nude mouse transplanted tumors

[1067] 1. Purpose of the test

[1068] 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.

[1069] 2. Test Drugs and Materials

[1070] 1. Test drug

[1071] ADC-27 (3 mg / kg)

[1072] ADC-26 (3 mg / kg)

[1073] Blank control: PBS buffer at pH 7.4.

[1074] 2. Preparation method: PBS buffer at pH 7.4.

[1075] 3. Experimental animals

[1076] BALB / cA-nude mice were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.

[1077] 3. Test methods

[1078] 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:

[1079] V=1 / 2×a×b2

[1080] Where a and b represent length and width respectively.

[1081] Relative volume (RTV) = V T / V0

[1082] Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%)

[1083] 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.

[1084] 4. Test results

[1085] 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.

[1086] 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 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.

[1087] Table 4. Efficacy of the administered antibodies on human brain glioblastoma U87MG nude mouse transplanted tumors (D22)

[1088]

[1089] ***P<0.001

[1090] Test Example 1-8: Evaluation of the efficacy of ADC on nude mouse transplanted tumors of human pharyngeal carcinoma pleural effusion metastatic cells Detroit 562

[1091] 1. Purpose of the test

[1092] 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.

[1093] 2. Test Drugs and Materials

[1094] 1. Test drug

[1095] ADC-29 (3 mg / kg)

[1096] ADC-28 (3 mg / kg)

[1097] Negative control ADC (3 mg / kg): an antibody-drug conjugate formed by coupling a non-B7H3 target antibody with compound 20.

[1098] 2. Preparation method: All preparations were diluted with PBS.

[1099] 3. Experimental animals

[1100] BALB / cA-nude mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

[1101] 3. Test methods

[1102] 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:

[1103] V=1 / 2×a×b 2

[1104] Where a and b represent length and width respectively.

[1105] Relative volume (RTV) = V T / V0

[1106] Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%)

[1107] 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.

[1108] 4. Test results

[1109] 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.

[1110] 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 5 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.

[1111] Table 5. Efficacy of the administered antibodies on Detroit 562 xenografts in nude mice (D28)

[1112]

[1113] ***P<0.001

[1114] Test Example 1-9: Evaluation of drug efficacy in U87-MG tumor-bearing mice

[1115] 1. Purpose of the test

[1116] 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.

[1117] 2. Test Drugs and Materials

[1118] 1. Test drug

[1119] ADC-30 1mg / kg

[1120] ADC-30 3mg / kg

[1121] ADC-31 1mg / kg

[1122] ADC-31 3mg / kg

[1123] Blank control: PBS

[1124] 2. Preparation method: All preparations were diluted with PBS.

[1125] 3. Experimental animals

[1126] BALB / cA-nude mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd.

[1127] 3. Test methods

[1128] U87MG cells (human astrocytoblastoma, cell bank of 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.

[1129] 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.

[1130] 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.

[1131] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[1132] Relative volume (RTV) = V T / V0

[1133] Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%)

[1134] 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.

[1135] 4. Test results

[1136] 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.

[1137] 2. Preparation

[1138] The equipment used in the preparation and testing process and the calculation method of the results are as follows:

[1139] SEC Size Exclusion Chromatography:

[1140] An analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecule.

[1141] SEC% (SEC monomer content percentage) = A monomer / A total * 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas.)

[1142] SEC measurement instrument: Agilent 1260; column: waters, XBrige SEC (300×7.8mm3.5μm)

[1143] CE capillary gel electrophoresis:

[1144] A method of electrophoresis in which the gel is moved into a capillary tube as a supporting medium and the samples are separated according to their molecular weight at a certain voltage.

[1145] Reduction CE purity percentage = A main peak / A total * 100% (A main peak is the peak area of ​​the light chain main peak + the heavy chain main peak in the sample, and A total is the sum of all peak areas.

[1146] CE measurement instrument: Beckman model plus800

[1147] Turbidity measurement:

[1148] The degree to which light is obstructed when passing through the water layer indicates the ability of the water layer to scatter and absorb light, which is not only related to the content of suspended matter, but also to the particle composition, size, shape and reflective properties of the surface. By comparing the absorption values ​​of the same protein sample with the same concentration and the same wavelength (near ultraviolet and visible light wavelength region), the greater the absorption value, the greater the turbidity, and the more obvious the aggregation tendency of protein molecules in the sample. The measuring instrument is a multifunctional microplate reader (Molecular Devices M5), and the same volume of sample is added to a 96-well plate to read the absorbance value.

[1149] Osmolality determination:

[1150] The freezing point method is used to determine osmotic pressure. It is based on the fact that the freezing point depression value is proportional to the molar concentration of the solution. It uses a highly sensitive temperature sensing element to measure the freezing point of the solution and convert the electricity into osmotic pressure. Instrument manufacturer: Loser, model OM815.

[1151] Protein concentration determination:

[1152] Because the toxin in the antibody drug conjugate absorbs at 280nm, the protein concentration is corrected using the following formula:

[1153] A280=Cd*ε280d+Cmab*ε280mab

[1154] A370=Cd*ε370d

[1155] Cd represents the concentration of toxin, Cmab represents the concentration of protein, ε280d represents the extinction coefficient of toxin at 280nm, ε280mab represents the extinction coefficient of protein at 280nm, ε370d represents the extinction coefficient of toxin at 370nm, ε280mab = 1.49mg-1*cm-1*ml, ε280d = 5000 (molar extinction coefficient of toxin at 280nm) / 1074.13 (molecular weight of toxin) = 4.65mg-1*cm-1*mL, ε370d = 19000 (molar extinction coefficient of toxin at 370nm) / 1074.13 (molecular weight of toxin) = 17.69mg-1*cm-1*mL, the above extinction coefficients are mass extinction coefficients.

[1156] Protein concentration determination instrument: UV-visible spectrophotometer, model: Nano Drop oneC, optical path length: 1 mm.

[1157] For example, the naked antibody has a molecular weight of 145.181 kDa and the toxin has a molecular weight of 1074 Da. If calculated based on the average DAR value of 5.7, the ADC molecular weight is 151.317 kDa, which is 1.042 times that of the naked antibody. For example, if the ADC DAR value is 5.7 and the concentration of the naked antibody protein is 20.00 mg / mL, then the concentration of the ADC is 20.84 mg / mL.

[1158] Example 2-1. Screening of formulation buffer system and pH value

[1159] The following different buffer systems were used to prepare preparations with an ADC-33 protein concentration of 20 mg / mL (based on naked antibody concentration) (except for the following buffer systems, the preparations did not contain sugars, surfactants, and other buffers):

[1160] 1) 10 mM histidine-sodium acetate, pH 5.0

[1161] 2) 10 mM histidine-sodium acetate, pH 5.5

[1162] 3) 10 mM histidine-sodium acetate, pH 6.0

[1163] 4) 10 mM histidine-sodium acetate, pH 6.5

[1164] 5) 10 mM His-Histidine HCl, pH 5.5

[1165] 6) 10 mM succinic acid-sodium succinate, pH 5.5

[1166] 7) 10 mM citric acid-sodium citrate, pH 5.5

[1167] Samples were taken for high temperature stability study (40°C) and shaking study (25°C, 300rpm). The shaking sample contained 0.1mg / mL polysorbate 80 (PS80), and the other samples contained 0.4mg / mL PS80. Each formulation was filtered, filled, stoppered, and capped. The samples were subjected to the above forced degradation experiment to examine appearance, SEC, and turbidity.

[1168] The results are shown in Table 6. The results show that with histidine-sodium acetate as buffer, as the pH increases in the range of pH 5.0-6.5, the appearance of the sample gradually deteriorates, the turbidity increases, and the SEC monomer ratio decreases. At pH 5.5, the appearance and SEC% of the samples using 10mM histidine-sodium acetate and 10mM histidine hydrochloride are similar, and are better than 10mM succinic acid-sodium succinate and 10mM citrate-sodium citrate, respectively. Surprisingly, the turbidity of the preparation containing 10mM succinic acid-sodium succinate after the shaking study is significantly better than that of the other groups of preparations.

[1169] Table 6. pH and buffer system stability results

[1170]

[1171] Note: "D" in the table means day, for example, D12 means 12 days; D0 means the beginning of the experiment, the same below.

[1172] Example 2-2 Screening of surfactant types

[1173] Preparations containing different polysorbate surfactants, all containing 10 mM succinic acid-sodium succinate (pH 5.0) buffer, 80 mg / mL sucrose, and ADC-32 with a protein concentration of 20 mg / mL (based on naked antibody concentration) were prepared. Each preparation was filtered, filled, stoppered, and capped. The samples were subjected to high temperature stability studies (40°C) and shaking studies (25°C, 300 rpm), and the appearance, SEC, and reduced CE were examined.

[1174] The results are shown in Table 7. There are no significant differences in appearance, SEC, and reduced CE under different conditions for the formulations containing PS20 (polysorbate 20) and PS80, indicating that both PS20 and PS80 can effectively stabilize ADC-32.

[1175] Table 7. Polysorbate type screening results

[1176]

[1177]

[1178] Example 2-3 Surfactant concentration screening

[1179] Samples with PS80 concentrations of 0, 0.1, 0.2, 0.4, and 0.6 mg / mL were prepared, each sample also containing 10 mM succinic acid-sodium succinate (pH 5.0), 80 mg / mL sucrose, and ADC-32 with a protein concentration of 20 mg / mL (based on naked antibody concentration). The samples were sterilized and filtered to remove particles, and diluted with 0.9% NaCl. The ADC-32 concentration after dilution was 0.2 mg / mL, and the appearance of the diluted solution and insoluble particles were examined.

[1180] The results are shown in Table 8. When the PS80 concentration in the prescription was 0-0.2 mg / mL, as the concentration increased, the increase in insoluble particles in the dilution decreased and the visible particles became fewer and fewer. When the concentration was 0.2-0.6 mg / mL, there was no significant difference in the insoluble particles in the dilution and the appearance was good.

[1181] Table 8. PS80 concentration screening results

[1182]

[1183] Example 2-4 Sugar Type Screening

[1184] Prepare different samples containing 80 mg / mL sucrose, trehalose, and mannitol. Each sample also contains 10 mM succinic acid-sodium succinate (pH 5.0), 0.2 mg / mL PS80, and ADC-32 with a protein concentration of 20 mg / mL (based on naked antibody concentration). Filter sterilize and remove particles. Each preparation is filtered, filled, stoppered, and capped. The samples are subjected to high temperature stability studies (40°C) and -35°C / 4°C freeze-thaw cycle studies, and the appearance, SEC, and reduced CE are examined.

[1185] The results are shown in Table 9. In terms of appearance under freeze-thaw conditions, sucrose is superior to trehalose and mannitol. SEC results show that sucrose and trehalose are superior to mannitol. CE results at 40°C show that sucrose is slightly superior to trehalose.

[1186] Table 9. Sugar type screening results

[1187]

[1188]

[1189] Example 2-5 pH, antibody drug conjugate concentration and polysorbate screening experiment

[1190] Using 10 mM succinic acid-sodium succinate as buffer and 80 mg / mL sucrose as stabilizer, the formulation was designed according to pH, ADC-32 protein concentration (based on naked antibody) and polysorbate concentration, as shown in Table 10. High temperature stability study (40°C), illumination study (4°C, 4500Lx) and -35°C / 4°C freeze-thaw cycle study were carried out, and SEC and reduced CE were used as evaluation indicators, and the least squares method was used to perform statistical analysis on the results.

[1191] The results are shown in Table 11 and Figure 8 , the preparation disclosed in the present invention shows a high degree of pH dependence. Under the conditions of illumination, shaking and 40°C, as the pH increases, the SEC decrease value increases; similarly, under the conditions of illumination, the monomer peak decrease value of reduced CE increases with the increase of pH; suggesting that the preparation disclosed in the present invention should adopt low pH conditions to improve stability. Under shaking conditions, as the concentration of PS80 increases, the SEC decrease value tends to decrease, and there is no difference in the effect of PS80 on SEC and reduced CE under other conditions. As the concentration of ADC-32 protein increases, the SEC monomer peak decreases more at 40°C, and there is no obvious trend in the effect of ADC-32 protein concentration on SEC and reduced CE under other conditions.

[1192] Table 10. Screening experiment formulation design

[1193]

[1194] Table 11. Prescription screening test results

[1195]

[1196]

[1197] Example 2-6 Freeze-drying process development experiment

[1198] The stock solution was prepared according to 10mM succinic acid-sodium succinate pH5.0, 80mg / mL sucrose, 0.2mg / mL PS80 and 20mg / mL (protein concentration, calculated as naked antibody) ADC-33. After filtering and filling, it was freeze-dried according to freeze-drying process parameter 1 (see Table 12). The freeze-dried sample was a white powder with a smooth surface and a slight collapse in the middle of the bottom of the powder. The moisture content was 1.05%. The pH was measured after reconstitution with water for injection. The results are shown in Table 13. The ionic strength ≥10mM can meet the buffering effect. The sucrose in this prescription is 80mg / mL to meet the isotonicity.

[1199] Table 12. Freeze-drying process parameters 1

[1200] Freeze-drying process parameters Set temperature(℃) Setting time (min) Holding time (h) Vacuum degree(Pa) Pre-freeze 5 10 1 N / A Pre-freeze -45 50 2 N / A One-time drying -25 120 40 10 Secondary drying 25 60 8 1

[1201] Table 13. Final formulation pH and osmotic pressure results

[1202] Buffer pH Osmotic pressure of stock solution pH of stock solution pH after reconstitution 5.00 298mosm 5.06 5.06

[1203] The stock solution was prepared according to 10 mM succinic acid-sodium succinate pH 5.0, 80 mg / mL sucrose, 0.2 mg / mL PS80 and 20 mg / mL (protein concentration, based on naked antibody) ADC-33. After filtering and filling, it was freeze-dried according to freeze-drying process parameter 2 (see Table 14). The surface of the powder was flat without shrinkage or collapse, and the moisture content was 0.89%. The freeze-drying process could meet the product quality requirements.

[1204] Table 14. Freeze-drying process parameters 2

[1205] Freeze-drying process parameters Set temperature(℃) Setting time (min) Holding time (h) Vacuum degree(Pa) Pre-freeze -5 10 1 N / A Pre-freeze -45 40 2 N / A

[1206] One-time drying -20 120 35 10 Secondary drying 25 60 8 1

[1207] The stock solution was prepared according to 10mM succinic acid-sodium succinate pH5.0, 80mg / mL sucrose, 0.2mg / mL PS80 and 20mg / mL (protein concentration, calculated as naked antibody) ADC-32. After filtering and filling, it was freeze-dried according to freeze-drying process parameter 3 (see Table 15). The surface of the powder cake was flat without collapse, the bottom edge of the powder cake was slightly shrunk, and the moisture content was 1.17%. The freeze-drying process basically met the product quality requirements.

[1208] Table 15. Freeze-drying process parameters 3

[1209]

[1210] Example 2-7 Long-term stability data

[1211] The stock solution was prepared according to 10mM succinic acid-sodium succinate pH5.0, 80mg / mL sucrose, 0.2mg / mL PS80 and protein concentration, 20mg / mL (based on naked antibody) ADC-32. After filtration and filling, the pilot batch of finished stock solution was prepared according to the freeze-drying process parameter 2. It was stored for a long time at 2-8°C and tested after reconstitution. The results are shown in Table 16. Compared with D0, SEC, reduced CE and free toxin were all within the acceptable range at 2-8°C M3 (SEC% ≥93%; reduced CE% ≥95%; free toxin ≤330ppm). Except for a slight decrease of 0.5% in SEC, there was no change in reduced CE and free toxin.

[1212] Table 16. Long-term stability data results

[1213]

[1214] Note: "M" in the table stands for month, for example, M3 stands for 3 months.

Claims

1. A pharmaceutical composition comprising an antibody drug conjugate and a buffer, wherein the buffer is selected from a histidine buffer, a succinate buffer and a citrate buffer, and the concentration of the buffer is 5 mM to 50 mM, wherein the concentration of the antibody drug conjugate is 1 mg / mL to 100 mg / mL; the antibody drug conjugate has a structure as shown in the general formula (Pc-LYD): in: Y is selected from -O-(CH2)m-CR 1 R 2 -C(O)-; R 1 C 3-7 Cycloalkyl or C 3-7 Cycloalkyl C 1-6 alkyl; R 2 is a hydrogen atom; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-7 Cycloalkyl; m is 0 or 1; n is 1 to 10, and n is a decimal or an integer; L is the joint unit; 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 the tetrapeptide residue of GGFG; L 4 For-NR 5 (CR 6 R 7 )t-,R 5 A hydrogen atom or C 1-6 Alkyl, R 6 and R 7 are the same or different and are each independently a hydrogen atom or a C 1-6 Alkyl, t is 1 or 2; Pc is Trastuzumab; The pH of the pharmaceutical composition is 4.5 to 5.2, The pharmaceutical composition further comprises a surfactant, wherein the surfactant is polysorbate, and the concentration thereof is 0.01 mg / mL to 1.0 mg / mL; The pharmaceutical composition also comprises sugar at a concentration of 60 mg / mL to 90 mg / mL.

2. The pharmaceutical composition according to claim 1, wherein the pH is 4.8 to 5.

2.

3. The pharmaceutical composition according to claim 2, wherein the pH is 5.0 to 5.

1.

4. The pharmaceutical composition according to claim 1, wherein the surfactant is polysorbate 80 or polysorbate 20.

5. The pharmaceutical composition according to claim 4, wherein the surfactant is polysorbate 80. The pharmaceutical composition according to claim 1 , wherein the concentration of the surfactant is 0.05 mg / mL to 0.5 mg / mL.

7. The pharmaceutical composition according to claim 6, wherein the concentration of the surfactant is 0.2 mg / mL.

8. The pharmaceutical composition according to claim 1, wherein the sugar is selected from sucrose, mannitol, sorbitol and trehalose.

9. The pharmaceutical composition according to claim 8, wherein the sugar is sucrose.

10. The pharmaceutical composition according to claim 1, wherein the concentration of the sugar is 80 mg / mL.

11. The pharmaceutical composition of claim 1, wherein the concentration of the antibody drug conjugate is 10 mg / mL to 30 mg / mL.

12. The pharmaceutical composition according to claim 11, wherein the concentration of the antibody drug conjugate is 20 mg / mL to 22 mg / mL.

13. The pharmaceutical composition of claim 1, wherein the buffer is succinate buffer.

14. The pharmaceutical composition according to claim 13, wherein the buffer is succinic acid-sodium succinate buffer.

15. The pharmaceutical composition according to claim 1, wherein the concentration of the buffer is 5 mM to 20 mM. The pharmaceutical composition according to claim 15 , wherein the concentration of the buffer is 10 mM.

17. A pharmaceutical composition according to claim 1, comprising the following components: (a) 10 mg / mL to 30 mg / mL of the antibody drug conjugate, (b) 0.05 mg / mL to 0.5 mg / mL of polysorbate, (c) 60 mg / mL to 90 mg / mL of a sugar, and (d) 5 mM to 20 mM of a buffer, wherein the buffer is selected from a histidine buffer, a succinate buffer, and a citrate buffer; the pH of the pharmaceutical composition is 4.8-5.

2.

18. The pharmaceutical composition according to claim 1, comprising the following components: (a) 20 mg / mL to 22 mg / mL of the antibody drug conjugate, (b) 0.2 mg / mL of polysorbate 80, (c) 80 mg / mL of sucrose, and (d) 10 mM succinate buffer, the pH of the composition being 5.0-5.

1.

19. The pharmaceutical composition according to claim 1, wherein the antibody conjugate has a structure as shown in the following formula: in, n is 3 to 8, and n is a decimal or an integer.

20. A pharmaceutical composition comprising an antibody drug conjugate having a structure as shown in the following formula: in, n is 3 to 8, and n is a decimal or an integer; Furthermore, the pharmaceutical composition comprises: (a) 10 mg / mL to 30 mg / mL of the antibody drug conjugate, (b) 0.05 mg / mL to 0.5 mg / mL of polysorbate, (c) 60 mg / mL to 90 mg / mL of a sugar, and (d) 5 mM to 20 mM of a buffer, wherein the buffer is selected from a histidine buffer, a succinate buffer, and a citrate buffer; the pH of the pharmaceutical composition is 4.8 to 5.

2.

21. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition comprises: (a) 20 mg / mL to 22 mg / mL of the antibody drug conjugate, (b) 0.2 mg / mL of polysorbate 80, (c) 80 mg / mL of sucrose, and (d) 10 mM succinate buffer, and the pH of the pharmaceutical composition is 5.0 to 5.

1.

22. A lyophilized preparation comprising an antibody drug conjugate, characterized in that The preparation can be reconstituted to form the pharmaceutical composition according to any one of claims 1 to 21.

23. A method for preparing a lyophilized formulation comprising an antibody drug conjugate, comprising the step of freeze-drying the pharmaceutical composition according to any one of claims 1 to 21.

24. A lyophilized preparation comprising an antibody drug conjugate, the preparation being obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 21.

25. A reconstitution solution comprising an antibody drug conjugate, characterized in that The reconstituted solution is obtained by reconstituted the lyophilized preparation according to claim 22 or 24; The reconstitution solution comprises the following components: (a) 10 mg / mL to 30 mg / mL of the antibody drug conjugate, (b) 0.05 mg / mL to 0.5 mg / mL of polysorbate, (c) 60 mg / mL to 90 mg / mL of sugar, and (d) 5 mM to 20 mM buffer, wherein the buffer is selected from a histidine buffer, a succinate buffer, and a citrate buffer; the pH of the reconstituted solution is 4.8 to 5.

2.

26. The reconstitution solution according to claim 25, wherein the reconstitution solution comprises the following components: (a) 20 mg / mL to 22 mg / mL of antibody drug conjugate, (b) 0.2 mg / mL of polysorbate 80, (c) 80 mg / mL of sucrose, and (d) 10 mM succinate buffer, wherein the pH of the reconstituted solution is 5.0 to 5.

1.

27. A product comprising a container containing the pharmaceutical composition according to any one of claims 1 to 21, the lyophilized preparation according to claim 22 or 24, or the reconstituted solution according to claim 25 or 26.

28. Use of the pharmaceutical composition of any one of claims 1 to 21, the lyophilized preparation of claim 22 or 24, the reconstituted solution of claim 25 or 26, or the preparation of claim 27 in the preparation of a medicament for treating cancer, wherein the cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

29. The use according to claim 28, wherein the cancer is selected from endometrial cancer, colon cancer and rectal cancer.

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