Method for preparing antibody-drug conjugates

Abstract

The present disclosure relates to methods for preparing antibody-drug conjugates, including synthesis and purification steps.

Description

[Technical Field]

[0001] This application claims priority to a Chinese patent application filed on March 25, 2020 (application number CN 202010219311.2) and a Chinese patent application filed on March 19, 2021 (application number CN 202110297397.5). The present disclosure relates to methods for preparing antibody-drug conjugates, and in particular to the synthesis and purification steps of the methods for preparing antibody-drug conjugates. [Background technology]

[0002] Nothing herein necessarily constitutes prior art, but rather merely provides background information relevant to the present disclosure.

[0003] Chemotherapy remains one of the most important anti-cancer treatments, including surgery, radiation therapy, and targeted therapy. Although there are many effective cytotoxic drugs, the limited differentiation between tumor and normal cells, as well as toxicity and side effects, limit the widespread clinical application of these anti-tumor compounds. However, because anti-tumor monoclonal antibodies have specificity for tumor cell surface antigens, antibody drugs have become cutting-edge drugs for anti-tumor therapy. However, when antibodies are used alone as anti-tumor drugs, the therapeutic effect is often insufficient.

[0004] Antibody drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxic drugs via stable chemical linker compounds, taking advantage of the specificity of antibodies to bind to surface antigens on normal and tumor cells and the high efficacy of cytotoxic drugs, while avoiding the relatively low therapeutic efficacy of the former and the excessive toxicity and side effects of the latter. This means that, compared with traditional chemotherapy drugs, ADCs can precisely bind to tumor cells and reduce their impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329-332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814).

[0005] In 2000, the first antibody-drug conjugate, Mylotarg (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), was approved for marketing by the US FDA for use in the treatment of acute myeloid leukemia (Drugs of the Future (2000) 25(7):686; US4970198; US 5079233; US 5585089; US 5606040; US 5693762; US 5739116; US 5767285; US 5773001).

[0006] In August 2011, Adcetris (R) (brentuximab vedotin, Seagen) has been approved under accelerated review by the US FDA for the treatment of Hodgkin's lymphoma and recurrent anaplastic large cell lymphoma (Nat. Biotechnol (2003) 21(7):778-784; WO2004010957; WO2005001038; US7090843; US7659241; WO2008025020). (R)is a novel ADC drug that can induce apoptosis of tumor cells by directing the drug to target CD30 on lymphoma cells followed by endocytosis.

[0007] Both Mylotarg and Adcetris are targeted therapies for hematological malignancies, which have a simpler tissue structure than solid tumors. In February 2013, Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the US FDA for the treatment of patients with advanced or metastatic breast cancer that is HER2-positive and resistant to trastuzumab (Herceptin) and Taxol (WO2005037992, US8088387). Kadcyla is the first ADC drug approved by the US FDA for the treatment of solid tumors.

[0008] Several types of cytotoxic small molecules are used in antibody-drug conjugates (ADCs). One of these is a camptothecin derivative, exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3,4:6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione), which has been reported for use in antibody-drug conjugates (ADCs) in literature, including WO2014057687, Clinical Cancer Research (2016) 22(20):5097-5108, and Cancer Sci (2016) 107:1039-1046. However, further development of ADCs with better therapeutic effects is still needed. Summary of the Invention

[0009] The present disclosure provides a method for preparing an antibody-drug conjugate, wherein the structure of the antibody-drug conjugate is represented by the general formula (Pc-L a -YD), that is, [ka] is shown by Among them, W is C 1-8 Alkyl group, C 1-8 Alkyl-C 3-7 A cycloalkyl group or a linear heteroalkyl group having 1 to 8 atoms, said linear heteroalkyl group containing 1 to 3 heteroatoms selected from N, O and S, among which the above C 1-8 Alkyl group, C 3-7 The cycloalkyl group and the straight chain heteroalkyl group may each independently optionally further comprise a halogen, a hydroxy group, a cyano group, an amino group, a C 1-6 Alkyl group, chloro C 1-6 Alkyl groups, deuterated C 1-6 Alkyl group, C 1-6 Alkoxy groups and C 3-7 substituted with one or more substituents selected from cycloalkyl groups; L 2 Ha-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)- and chemical bonds, among which p 1 is an integer between 1 and 20, L 3 is a peptide residue consisting of 2 to 7 amino acid residues, among which the amino acid residues are amino acid residues formed from amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and optionally further containing halogen, hydroxy group, cyano group, amino group, C 1-6 Alkyl group, chloro C 1-6 Alkyl groups, deuterated C 1-6 Alkyl group, C 1-6 Alkoxy groups and C 3-7 substituted with one or more substituents selected from cycloalkyl groups; R1 is halogenated C 1-6 Alkyl group or C 3-7 is a cycloalkyl group, R 2 is a hydrogen atom, halogenated C 1-6 Alkyl groups and C 3-7 cycloalkyl groups, Or, R 1 and R 2 C together with the carbon atoms connected to them 3-7 forming a cycloalkyl group, R 5 is a hydrogen atom, C 1-6 Alkyl groups, halogenated C 1-6 Alkyl groups, deuterated C 1-6 Alkyl and hydroxy C 1-6 selected from alkyl groups, R 6 and R 7 are the same or different and each independently represent a hydrogen atom, a halogen, or C 1-6 Alkyl groups, halogenated C 1-6 Alkyl groups, deuterated C 1-6 Alkyl and hydroxy C 1-6 selected from alkyl groups, m is 0 or 1; n is 3 to 8, and n is a decimal or an integer; Pc is an antibody or an antigen-binding fragment thereof; The above preparation method comprises: Step (a) of reacting the antibody or antigen-binding fragment thereof with a reducing agent at a reaction temperature of about 1°C to about 36°C; The product of step (a) and the compound of formula (La-YD): [ka] and (b) reacting a compound represented by the formula: Among them, W and L 2 , L 3 , R 1 , R 2 , R 5 , R 6 , R 7and m is as defined above.

[0010] In another aspect, the present disclosure provides a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate has the following formula: [ka] and having a structure represented by Wherein, n is 4 to 8, and n is a decimal or an integer; The above preparation method comprises: Step (a) of reacting the antibody or antigen-binding fragment thereof with a reducing agent at a reaction temperature of about 1°C to about 36°C; the product of step (a) and a compound of the formula: [ka] and (b) reacting the compound represented by the formula:

[0011] In alternative embodiments, the reaction temperature conditions in step (a) are from about 4°C to about 30°C, preferably from about 20°C to about 30°C, and more preferably 25°C, with non-limiting examples including about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, and about 30°C. In some embodiments, the reaction temperature conditions are from 13°C to 28°C or from 13°C to 25°C.

[0012] In alternative embodiments, the reaction in step (a) is carried out at a pH of about 4.5 to about 6.5, preferably at a pH of about 5.0 to about 6.0, and more preferably at a pH of about 5.6. In non-limiting examples, the reaction is carried out at a pH of 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, or about 6.0.

[0013] In an alternative embodiment, the reaction in step (a) is carried out in a buffer, and in non-limiting examples, the buffer is selected from a histidine salt buffer, a phosphate buffer, and an acetate buffer.

[0014] In an alternative embodiment, the reaction in step (a) is carried out in a buffer, and in a non-limiting example, the buffer is a histidine-hydrochloride buffer.

[0015] In alternative embodiments, the buffer is selected from a histidine salt buffer containing EDTA and a histidine-hydrochloride buffer containing EDTA. For example, the histidine salt buffer has a concentration of 1 mM to 100 mM, 10 mM to 50 mM, 20 mM, 30 mM, or 40 mM, and the EDTA has a concentration of 1 mM to 10 mM, 2 mM to 5 mM, 2.5 mM, 3 mM, or 4 mM. In some embodiments, the buffer contains 10 mM to 50 mM histidine salt buffer and 1 mM to 10 mM EDTA. In some embodiments, the buffer contains 20 mM histidine-hydrochloride buffer and 2.5 mM EDTA. EDTA refers to ethylenediaminetetraacetic acid.

[0016] In an alternative embodiment, the reducing agent in step (a) is selected from suitable reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) or a salt thereof, 1,4-dithiothreitol (DTT) and β-mercaptoethanol (β-ME), preferably TCEP or a salt thereof, more preferably tris(2-carboxyethyl)phosphine hydrochloride.

[0017] In alternative embodiments, the molar ratio of reducing agent to antibody or antigen-binding fragment thereof (Pc) in step (a) is 2-10:1, 2.6-7:1, 2.9-3.7:1, 3.2-3.4:1, or 3.3:1.

[0018] In alternative embodiments, step (b) is carried out in an organic solvent, preferred organic solvents include dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), acetonitrile, or mixtures thereof. In a non-limiting example, step (b) comprises dissolving a compound of formula (La-YD) in DMSO and combining the product of step (a) with the DMSO solution of a compound of formula (La-YD).

[0019] In an alternative embodiment, the preparation method further comprises step (c), which comprises purifying the product of step (b). The purification can be performed by cation chromatography or affinity column chromatography, and the packing material for the cation chromatography is selected from Capto S Impact and Poros XS, preferably Capto S Impact. In some embodiments, the Capto S Impact is Capto TM In some embodiments, the Poros XS is Poros S Impact. TM It's XS.

[0020] In alternative embodiments, the drug loading (n) ranges such that each antibody or antigen-binding fragment thereof (Pc) may be conjugated to 3 to 8, 4 to 8, 5 to 7, preferably 5.3 to 6.1, and more preferably 5.7 cytotoxic drugs. n is a decimal or an integer. In some embodiments, n is 5.3, 5.4, 5.5, 5.6, or 5.7.

[0021] In an alternative embodiment, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains bound to four drugs is 4% or less, preferably 4% or less, and the proportion of antibody heavy chains that are unbound to any drug is 6% or less. In non-limiting examples, the proportion of antibody heavy chains that are bound to four drugs is 4% or less, 3% or less, 2% or less, or 1% or less, and the proportion of antibody heavy chains that are unbound to any drug is 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Alternatively, in the antibody light chain group, the proportion of antibody light chains that are bound to one drug is 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that bind to four drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to any drugs is 5% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that bind to four drugs is 1% or less, and the proportion of antibody heavy chains that do not bind to any drugs is 4% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that bind to four drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to any drugs is 5% or less, and the proportion of antibody light chains that bind to one drug is 65% or more. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains bound to four drugs is 1% or less, the proportion of antibody heavy chains that are unbound to any drug is 4% or less, and in the antibody light chain group, the proportion of antibody light chains that are bound to one drug is 70% or more. In some embodiments, the proportions of antibody heavy chains and / or antibody light chains are measured by reverse-phase chromatography.

[0022] In an alternative embodiment, the above preparation method is applied to large-scale preparation, and the dosage of trastuzumab in the above preparation method is 100 mg or more, preferably 1 g or more, more preferably 10 g or more, and most preferably 100 g or more.

[0023] In alternative embodiments, the antibody-drug conjugate has the general formula (Pc-L b -YD), that is, [ka] and having a structure represented by Among them, s 1 is an integer from 2 to 8, Pc, R 1 , R 2 , R 5 , R 6 , R 7 , m and n are as defined above; The above preparation method comprises: Step (a) of reacting the antibody or antigen-binding fragment thereof with a reducing agent at a reaction temperature of about 1°C to about 36°C; The product of step (a) and the compound of formula (L b -YD), that is, [ka] and (b) reacting a compound represented by the formula: Among them, s 1 , R 1 , R 2 , R 5 , R 6 , R 7 and m is as defined above.

[0024] In an alternative embodiment, the antibody drug conjugate has the following structure: [Table 1-1] [Table 1-2] [Table 1-3] and Among them, Pc and n are as defined in the general formula (Pc-La-YD).

[0025] In alternative embodiments, the Pc is an antibody or antigen-binding fragment thereof, the antibody being selected from a chimeric antibody, a humanized antibody, and a fully human antibody. In some embodiments, the antibody is a monoclonal antibody.

[0026] In alternative embodiments, the antibody or antigen-binding fragment thereof is selected from an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC44A4 antibody, and an anti-Mesothelin antibody, or an antigen-binding fragment thereof; Preferably, the antibody or antigen-binding fragment thereof is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumamab, or an antigen-binding fragment thereof.

[0027] In an alternative embodiment, the antibody conjugate has the following formula: [ka] and having a structure represented by In this case, n is 4 to 8, and n is a decimal or an integer.

[0028] In another aspect, the present disclosure provides a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate has the following formula: [ka] and step (b) reacting a compound represented by the formula: Wherein, n is 4 to 8, and n is a decimal or an integer; The above preparation method comprises: Step (a) of reacting trastuzumab with TCEP at a reaction temperature of about 4°C to about 30°C and a pH of about 4.5 to about 6.5; the product of step (a) and a compound of the formula: [ka] and (b) reacting the compound represented by the formula:

[0029] In an alternative embodiment, the antibody drug conjugate has the following formula: [ka] and having a structure represented by Wherein, n is 4 to 8, and n is a decimal or an integer; The above preparation method comprises: Step (a) of reacting Trastuzumab with TCEP at a reaction temperature of about 25°C and a pH of about 5.6, wherein the reaction is carried out in a histidine-hydrochloride buffer containing EDTA; the product of step (a) and a compound of the formula: [ka] and step (b) reacting a compound represented by the formula: and step (c) purifying the product of step (b) using a cation chromatography column or an affinity chromatography column. In some embodiments, the histidine-HCl buffer containing EDTA contains 20 mM histidine-HCl buffer and 2.5 mM EDTA.

[0030] The present disclosure further provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the structure of the antibody-drug conjugate is represented by the general formula (Pc-L a -YD), that is, [ka] is shown by Among them, W is C 1-8 Alkyl group, C 1-8 Alkyl-C 3-7 A cycloalkyl group or a linear heteroalkyl group having 1 to 8 atoms, said linear heteroalkyl group containing 1 to 3 heteroatoms selected from N, O and S, among which the above C 1-8 Alkyl group, C 3-7 The cycloalkyl group and the straight chain heteroalkyl group may each independently optionally further comprise a halogen, a hydroxy group, a cyano group, an amino group, a C 1-6 Alkyl group, chloro C 1-6 Alkyl groups, deuterated C 1-6 Alkyl group, C 1-6 Alkoxy groups and C 3-7 substituted with one or more substituents selected from cycloalkyl groups; L 2 Ha-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)- and chemical bonds, among which p 1 is an integer between 1 and 20, L 3is a peptide residue consisting of 2 to 7 amino acid residues, among which the amino acid residues are amino acid residues formed from amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and optionally further containing halogen, hydroxy group, cyano group, amino group, C 1-6 Alkyl group, chloro C 1-6 Alkyl groups, deuterated C 1-6 Alkyl group, C 1-6 Alkoxy groups and C 3-7 substituted with one or more substituents selected from cycloalkyl groups; R 1 is halogenated C 1-6 Alkyl group or C 3-7 is a cycloalkyl group, R 2 is a hydrogen atom, halogenated C 1-6 Alkyl groups and C 3-7 cycloalkyl groups, Or, R 1 and R 2 C together with the carbon atoms connected to them 3-7 forming a cycloalkyl group, R 5 is a hydrogen atom, C 1-6 Alkyl groups, halogenated C 1-6 Alkyl groups, deuterated C 1-6 Alkyl and hydroxy C 1-6 selected from alkyl groups, R 6 and R 7 are the same or different and each independently represent a hydrogen atom, a halogen, or C 1-6 Alkyl groups, halogenated C 1-6 Alkyl groups, deuterated C 1-6 Alkyl and hydroxy C 1-6 selected from alkyl groups, m is 0 or 1; n is 4 to 8, and n is a decimal or an integer; Pc is an antibody or an antigen-binding fragment thereof; The drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains bound to four drugs is 4% or less, preferably 4% or less, and the proportion of antibody heavy chains that are unbound to any drugs is 6% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that are bound to four drugs is 4% or less, and the proportion of antibody heavy chains that are unbound to any drugs is 5% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that are bound to four drugs is 1% or less, and the proportion of antibody heavy chains that are unbound to any drugs is 4% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that are bound to four drugs is 4% or less, and the proportion of antibody heavy chains that are unbound to any drugs is 5% or less, and the proportion of antibody light chains that are unbound to any drugs is 65% or more. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that bind to four drugs is 1% or less and the proportion of antibody heavy chains that do not bind to any drugs is 4% or less, and in the antibody light chain group, the proportion of antibody light chains that bind to one drug is 70% or more.

[0031] The present disclosure further provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, the antibody-drug conjugate being prepared by the above-described method for preparing an antibody-drug conjugate, and the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains bound to four drugs is 4% or less, preferably 4% or less, and the proportion of antibody heavy chains that are unbound to any drugs is 6% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that are bound to four drugs is 4% or less, and the proportion of antibody heavy chains that are unbound to any drugs is 5% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that are bound to four drugs is 1% or less, and the proportion of antibody heavy chains that are unbound to any drugs is 4% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that in the antibody heavy chain group, the proportion of antibody heavy chains bound to four drugs is 4% or less and the proportion of antibody heavy chains that are unbound to any drugs is 5% or less, and in the antibody light chain group, the proportion of antibody light chains that are bound to one drug is 65% or more. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that in the antibody heavy chain group, the proportion of antibody heavy chains that are bound to four drugs is 1% or less and the proportion of antibody heavy chains that are unbound to any drugs is 4% or less, and in the antibody light chain group, the proportion of antibody light chains that are bound to one drug is 70% or more.

[0032] The present disclosure further provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has the following formula: [ka] and having a structure represented by Wherein, n is 4 to 8, and n is a decimal or an integer; The antibody-drug conjugate is prepared by the above-mentioned method for preparing an antibody-drug conjugate, and the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that bind to four drugs is 4% or less, preferably 4% or less, and the proportion of antibody heavy chains that do not bind to any drugs is 6% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that bind to four drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to any drugs is 5% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that bind to four drugs is 1% or less, and the proportion of antibody heavy chains that do not bind to any drugs is 4% or less. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that in the antibody heavy chain group, the proportion of antibody heavy chains bound to four drugs is 4% or less and the proportion of antibody heavy chains that are unbound to any drugs is 5% or less, and in the antibody light chain group, the proportion of antibody light chains that are bound to one drug is 65% or more. In some embodiments, the drug loading distribution of the antibody-drug conjugate is such that in the antibody heavy chain group, the proportion of antibody heavy chains that are bound to four drugs is 1% or less and the proportion of antibody heavy chains that are unbound to any drugs is 4% or less, and in the antibody light chain group, the proportion of antibody light chains that are bound to one drug is 70% or more. Details of the invention

[0033] The present disclosure provides a preparation method that is more advantageous for large-scale production, specifically, the product obtained by the preparation method has a narrower drug loading distribution, a lower content of free toxins, and a higher yield. term

[0034] In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0035] The present disclosure is incorporated herein by reference in its entirety.

[0036] Antibody drug conjugates (ADCs) are compounds that link antibodies or antibody fragments to biologically active cytotoxins or small molecule drugs with cytocidal activity via stable chemical linker compounds, taking advantage of the specificity of antibodies to tumor cells or highly expressing antigens and the high efficacy of cytotoxins, while avoiding toxicity and side effects on normal cells. Compared with traditional chemotherapy drugs, antibody drug conjugates can bind precisely to tumor cells and reduce the impact on normal cells.

[0037] "Buffer" refers to a buffer that resists changes in pH by the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0038] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, and histidine-sulfate, with histidine-hydrochloride being preferred. Histidine-acetate buffers are prepared from histidine and acetic acid, and histidine-hydrochloride buffers are prepared from histidine and hydrochloric acid or histidine and histidine hydrochloride.

[0039] A "phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, etc. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0040] An "acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-histidine salt, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, etc. A preferred acetate buffer is acetic acid-sodium acetate.

[0041] As used herein, the terms "about" and "approximately" mean that a numerical value is within an acceptable error range of a specific value as determined by one of ordinary skill in the art, depending on how the numerical value is measured or determined (i.e., the limitations of the measurement system). For example, in the practice of this art, "about" may mean within or more than a standard deviation of 1. Alternatively, "about" or "essentially comprising" may mean a range of at most 20%. Moreover, particularly with respect to biological systems or processes, the terms may mean at most one order of magnitude or at most 5 times the numerical value. Unless otherwise stated, when specific values ​​appear in this application and claims, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range of the specific value.

[0042] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0043] The term "antibody" as used herein refers to immunoglobulins. A complete antibody has a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in the amino acid composition and sequence order of their heavy chain constant regions, resulting in different antigenicities. Therefore, immunoglobulins can be divided into five types, or so-called immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses based on differences in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains can be divided into κ or λ chains depending on the constant region. Each of the five types of Ig may have either κ or λ chains. The antibodies described in the present disclosure are preferably antibodies specific to cell surface antigens on target cells, and non-limiting examples thereof include anti-HER2 (ErbB2) antibodies, anti-EGFR antibodies, anti-B7-H3 antibodies, anti-c-Met antibodies, anti-HER3 (ErbB3) antibodies, anti-HER4 (ErbB4) antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD44 antibodies, anti-CD56 antibodies, anti-CD70 antibodies, anti-CD73 antibodies, anti-CD105 antibodies, anti-CEA antibodies, anti-A33 antibodies, anti-Cripto antibodies, anti-EphA2 antibodies, anti-G250 antibodies, anti-MUCl antibodies, and anti-Lewis antibodies. The antibody is one or more of 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, 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.

[0044] In antibody heavy and light chains, approximately 110 amino acids near the N-terminus are highly variable and form the variable region (Fv region), while the remaining amino acid sequence near the C-terminus is relatively stable and forms the constant region. The variable region contains three hypervariable regions (HVRs) and four framework regions (FRs) whose sequences are relatively conserved. The three hypervariable regions determine the specificity of the antibody and are also called 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 following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain are LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments described in this disclosure conform in number and position to the known Kabat numbering convention (LCDR1-3, HCDR1-3).

[0045] In the present disclosure, the antibody light chain described in the present disclosure may further comprise a light chain constant region, and the light chain constant region comprises a human or mouse kappa or lambda chain or a variant thereof.

[0046] In the present disclosure, the antibody heavy chain described in the present disclosure may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0047] Antibodies of the present disclosure include murine antibodies, chimeric antibodies and humanized antibodies, preferably humanized antibodies.

[0048] The term "murine antibody" as used in this disclosure refers to an antibody prepared in mice according to the knowledge and skill in the art, in which a specific antigen is injected into a test subject, and hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated.

[0049] The term "chimeric antibody" refers to an antibody in which the variable region of a mouse antibody is fused with the constant region of a human antibody, and can reduce the immune response elicited by mouse antibodies. To prepare a chimeric antibody, first, hybridomas secreting mouse-specific monoclonal antibodies are prepared, and then the variable region genes are cloned from the mouse hybridoma cells. If necessary, human antibody constant region genes are further cloned, and the mouse variable region genes and human constant region genes are ligated to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic cell system.

[0050] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting mouse CDR sequences onto a human antibody variable region framework, i.e., a framework sequence of a different human germline antibody. This overcomes the strong heterologous reactivity induced by chimeric antibodies containing a large amount of mouse protein components. Such framework sequences can be obtained from consensus DNA databases containing germline antibody gene sequences or from published references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database and in Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity associated with reduced immunogenicity, activity can be maintained by performing minimal back mutations or reverse mutations on the human antibody variable region framework sequences. The humanized antibodies of the present disclosure also include phage-displayed humanized antibodies that have undergone affinity maturation of their CDRs.

[0051] The term "naked antibody" refers to an antibody that is not conjugated to a heterologous module (eg, a cytotoxic module) or a radioactive marker.

[0052] The term "antigen-binding fragment of an antibody" as used herein may refer to a Fab fragment, a Fab' fragment, or a F(ab')2 fragment that has antigen-binding activity, as well as an Fv fragment or scFv fragment that binds to an antigen. An Fv fragment contains the antibody heavy chain variable region and light chain variable region but lacks the constant region and contains the minimum antibody fragment containing all of the antigen-binding site. Generally, an Fv antibody further contains a polypeptide linker between the VH and VL domains, enabling it to form the structure necessary for antigen binding. Two antibody variable regions may be linked by a different linker to form a single polypeptide chain called a single-chain antibody or single-chain Fv (sFv).

[0053] The term "antigen-binding site" of the present disclosure refers to the three-dimensional spatial region, contiguous or discontinuous, on an antigen that is recognized by an antibody or antigen-binding fragment of the present disclosure.

[0054] As used herein, "ADCC," or antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the Fc portion of the antibody. Modifications to the Fc portion of IgG can reduce or eliminate the ADCC effector function of the antibody. The modifications refer to mutations in the heavy chain constant region of the antibody selected from N297A, L234A, and L235A in IgG1, IgG2 / 4 chimera, and F234A / L235A in IgG4.

[0055] The term "mutation" in the mutation sequences described in this disclosure includes, but is not limited to, a "back mutation," a "conservative modification," or a "conservative substitution or substitution." A "conservative modification" or "conservative substitution or substitution" as used in this disclosure refers to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, rigidity, etc.), often allowing for alteration without altering the biological activity of the protein. Those skilled in the art generally understand that single amino acid substitutions in non-essential regions of a polypeptide do not fundamentally alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of amino acids with similar structures or functions are unlikely to destroy biological activity.

[0056] As used herein, a "variant sequence" refers to a nucleotide sequence and / or amino acid sequence that has a different percentage of sequence identity from the nucleotide sequence and / or amino acid sequence of the present disclosure when appropriate mutations, such as substitutions, insertions, or deletions, are made to the nucleotide sequence and / or amino acid sequence of the present disclosure. The sequence identity described herein 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%, and 100%. Sequence comparison and percent identity determination between two sequences can be performed using the default settings of the BLASTN / BLASTP algorithm, available from the website of the National Center for Biotechnology Institute.

[0057] The term "linker unit" or "linking fragment" or "linking unit" refers to a chemical structural fragment or bond that is linked at one end to an antibody or antigen-binding fragment thereof and at the other end to a drug, and may be linked to another linker and then to the antibody or drug.

[0058] The linker may comprise an extender, a spacer, and an amino acid unit and may be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker may be a "cleavable linker" that facilitates drug release in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0059] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. A preferred conventional technique is the mammalian expression system, which induces antibody glycosylation, particularly at the highly conserved N-terminal site of the Fc region. Positive clones are cultured in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibodies can be purified by conventional techniques, for example, using an A or G Sepharose FF column containing a conditioned buffer. Nonspecifically bound components are washed away. The bound antibodies are then eluted using a pH gradient, and the antibody fragments are detected and collected by SDS-PAGE. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and multimers may be removed by conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, for example at -70°C, or lyophilized.

[0060] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group that is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group containing from 1 to 12 carbon atoms, more preferably an alkyl group containing from 1 to 10 carbon atoms, and most preferably an alkyl group containing from 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, and the like. Silyl group, 2,3-dimethylpentyl group, 2,4-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 2-ethylpentyl group, 3-ethylpentyl group, n-octyl group, 2,3-dimethylhexyl group, 2,4-dimethylhexyl group, 2,5-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 2 n-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 containing 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 linkage site, and the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0061] The term "heteroalkyl group" refers to an alkyl group containing one or more heteroatoms selected from N, O and S, wherein alkyl group is as defined above.

[0062] The term "alkylene group" refers to a saturated, straight- or branched-chain aliphatic hydrocarbon group having two residues derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane, and is an alkylene group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH-), 1,1-ethylidene (-CH(CH)-), 1,2-ethylidene (-CHCH)-, 1,1-propylidene (-CH(CHCH)-), 1,2-propylidene (-CHCH(CH)-), 1,3-propylidene (-CHCHCHCH-), 1,4-butylidene (-CHCHCHCHCH-), and 1,5-butylidene (-CHCHCHCHCHCH-). The alkylene group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available linkage site, and preferably the substituent is independently optionally substituted with one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0063] The term "alkoxy group" refers to -O-(alkyl group) and -O-(unsubstituted cycloalkyl group), where alkyl group and cycloalkyl group are defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy. An alkoxy group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio groups.

[0064] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, where 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 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like, and polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0065] The term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, of which one or more ring atoms is nitrogen, oxygen, or S(O). m(wherein m is an integer of 0 to 2), but does not include the -OO-, -OS-, or -SS- ring moiety, with the remaining ring atoms being carbon. Preferably, the cycloalkyl group contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms, and more preferably, the cycloalkyl group ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclyl groups include spirocyclic, fused-ring, and bridged-ring heterocyclyl groups.

[0066] The term "spiroheterocyclyl group" refers to a polycyclic heterocyclyl group in which 5- to 20-membered monocyclic rings share one atom (called a spiroatom), in which one or more ring atoms is nitrogen, oxygen, or S(O). m (wherein m is an integer of 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but there is no ring with a completely conjugated π-electron system. It is preferably 6 to 14-membered, more preferably 7 to 10-membered. Depending on the number of spiro atoms shared by the rings, spiroheterocyclyl groups are classified as monospiroheterocyclyl groups, bisspiroheterocyclyl groups, or polyspiroheterocyclyl groups, and preferred are monospiroheterocyclyl groups and bisspiroheterocyclyl groups. More preferred are 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospiroheterocyclyl groups. Non-limiting examples of spiroheterocyclyl groups are: [ka] Includes:

[0067] The term "fused heterocyclyl group" refers to a 5- to 20-membered polycyclic heterocyclyl group in which each ring in the system shares an adjacent pair of atoms with another ring in the system, one or more rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system, and one or more ring atoms is nitrogen, oxygen, or S(O) m(wherein m is an integer of 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6 to 14-membered, more preferably 7 to 10-membered. Depending on the number of rings, it can be classified as a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl group, preferably a bicyclic or tricyclic group, more preferably a 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclyl group. Non-limiting examples of fused heterocyclyl groups are: [ka] Includes:

[0068] The term "bridged heterocyclyl group" refers to a 5- to 14-membered polycyclic heterocyclyl group in which any two rings share two non-directly linked atoms and may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system, and in which one or more ring atoms is nitrogen, oxygen, or S(O). m (wherein m is an integer of 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6 to 14-membered, more preferably 7 to 10-membered. Depending on the number of rings constituting it, it can be classified as a bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclyl group, preferably a bicyclic, tricyclic or tetracyclic group, more preferably a bicyclic or tricyclic group. Non-limiting examples of bridged heterocyclyl groups are: [ka] Includes:

[0069] The heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclyl group, non-limiting examples of which are: [ka] Includes:

[0070] The heterocyclyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably independently one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogen atoms, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0071] The term "aryl group" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl groups, preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, in which the ring connected to the parent structure is an aryl ring, non-limiting examples of which are: [ka] Includes:

[0072] The aryl group may be substituted or unsubstituted. When the aryl group is substituted, the substituents are preferably independently one or more groups selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen atom, a mercapto group, a hydroxy group, a nitro group, a cyano group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocycloalkoxy group, a cycloalkylthio group, and a heterocycloalkylthio group.

[0073] The term "heteroaryl group" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl groups are preferably 5 to 10-membered, more preferably 5 or 6-membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, and tetrazolyl. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, where the ring connected to the parent structure is the heteroaryl ring, non-limiting examples of which are: [ka] Includes:

[0074] The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably independently one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0075] The term "amino-protecting group" refers to a group that protects an amino group with an easily removable group so that the amino group is not altered when other sites on the molecule react. Non-limiting examples include 9-fluorenylmethyloxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl. These groups can be optionally substituted with 1 to 3 substituents selected from halogen, alkoxy, or nitro. The amino-protecting group is preferably 9-fluorenylmethyloxycarbonyl.

[0076] The term "cycloalkylalkyl group" refers to an alkyl group substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, wherein the alkyl group is as defined above and wherein the cycloalkyl group is as defined above.

[0077] The term "haloalkyl group" refers to an alkyl group substituted with one or more halogens, wherein alkyl group is as defined above.

[0078] The term "deuterated alkyl group" refers to an alkyl group substituted with one or more deuterium atoms, wherein the alkyl group is as defined above.

[0079] The term "hydroxy group" refers to an --OH group.

[0080] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0081] The term "amino group" refers to -NH2.

[0082] The term "nitro group" refers to -NO2.

[0083] The term "cyano" refers to -CN.

[0084] "Optional" or "optionally" means that the subsequently described events or circumstances may occur, but are not necessarily required, and this description includes cases where the events or circumstances occur or do not occur. For example, "optionally comprising 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may be present, but are not necessarily required.

[0085] The term "substituted" means that one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, are independently replaced with a corresponding number of substituents. Of course, substituents are only located at chemically feasible positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino group or a hydroxy group having free hydrogen may be unstable if it is bonded to a carbon atom having an unsaturated (e.g., olefinic) bond.

[0086] The term "drug loading" or "drug amount" (DAR) refers to the average quantity of cytotoxic drug loaded onto each antibody or antigen-binding fragment thereof in an ADC, and may be expressed as a ratio of drug amount to antibody amount, which may be an integer or decimal. In embodiments of the present disclosure, the drug loading is represented by n, which may be an average value of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The average amount of drug on each ADC molecule after the coupling reaction can be determined by conventional methods, such as UV / visible spectroscopy, mass spectrometry, ELISA testing, and HPLC.

[0087] The term "drug loading distribution" refers to the distribution of antibodies linked to different numbers of drugs in an antibody-drug conjugate group, e.g., the distribution of antibodies linked to 0, 2, 4, 6, and 8 drugs in a group. It should be noted that due to the potential for degradation products, DARs of 1, 3, 5, and 7 may also be present in a mixture. In the present disclosure, antibody drug loading distribution can be expressed by antibody heavy chains linked to different numbers of drugs, e.g., H0 indicates a heavy chain that does not bind to a drug, H1 indicates a heavy chain that binds to one drug, H2 indicates a heavy chain that binds to two drugs, H3 indicates a heavy chain that binds to three drugs, and H4 indicates a heavy chain that binds to four drugs. For example, a 4% proportion of H3 indicates that 4% of the heavy chains in the antibody-drug conjugate group are linked to three drugs. Accordingly, the antibody drug loading distribution in the present disclosure can also be represented by antibody light chains that bind different quantities of drugs, where L0 represents an antibody light chain that does not bind a drug and L1 represents an antibody light chain that binds one drug.

[0088] "Giving," "administration," and "treatment," when applied to an animal, human, or experimental subject, cell, tissue, organ, or biological fluid, refer to contact of an exogenous agent, therapeutic agent, or diagnostic agent or composition with an animal, human, or subject, cell, tissue, organ, or biological fluid. "Giving," "administration," and "treatment" can refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with a cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Giving," "administration," and "treatment" also refer to ex vivo and in vitro treatment of, for example, cells with a reagent, diagnostic, or binding composition, or via another cell. "Treatment," when applied to a human, veterinary, or research subject, can refer to therapeutic treatment, preventative or prophylactic measures, and research and diagnostic applications.

[0089] "Treatment" refers to administering an oral or topical therapeutic agent, for example, comprising a composition of any one of the binding compounds disclosed herein, to a patient suffering from one or more disease symptoms for which the known therapeutic agent has a therapeutic effect. Typically, the patient or group receiving treatment is administered an amount of therapeutic agent that effectively alleviates one or more disease symptoms, thereby inducing the resolution of those symptoms or inhibiting the progression of those symptoms to any clinically measurable extent. The amount of therapeutic agent that effectively alleviates any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on several factors, including the patient's disease state, age, and weight, and the ability of the agent to produce the desired therapeutic effect in the patient. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians and other professional health care providers to assess the severity or progression of the condition. Although embodiments of the present disclosure (e.g., treatment methods or products) may be ineffective in alleviating each target disease symptom, they can reduce the target disease symptom in a statistically significant number of patients as determined by any statistical testing method known in the art, such as Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0090] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disorder. Effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used in a particular patient or veterinary subject can vary depending on factors such as the condition being treated, the patient's overall health, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0091] "Replacement" refers to the replacement of a solvent system that dissolves the antibody protein, for example, replacing a high-salt or high-osmolarity solvent system containing the antibody protein with a buffer system that stabilizes the formulation by physical manipulation methods, including, but not limited to, ultrafiltration, dialysis, or centrifugation followed by reconstitution, so that the antibody protein is in a stable formulation. [Brief explanation of the drawings]

[0092] [Figure 1A] 1 shows the results of a plasma stability study of ADC-19 of the present disclosure. [Figure 1B] 1 shows the results of a plasma stability study of ADC-18 of the present disclosure. [Figure 1C] 1 shows the results of a plasma stability study of ADC-20 of the present disclosure. [Figure 2] 1 shows an evaluation of the efficacy of ADC-21 and ADC-24 of the present disclosure on JIMT-1 tumor-bearing mice. [Figure 3] 1 shows an evaluation of the therapeutic effect of the ADC of the present disclosure on nude mice bearing tumors transplanted with human breast cancer cells SK-BR-3. [Figure 4] 1 shows the results of a plasma stability study of ADC-25 of the present disclosure. [Figure 5] 1 shows the therapeutic effect of the ADC of the present disclosure on human brain glioblastoma U87MG tumors xenografted in nude mice. [Figure 6] 1 shows the therapeutic effect of the ADC of the present disclosure on Detroit 562 tumors, a human pharyngeal carcinoma pleural effusion metastasis cell line, transplanted into nude mice. [Figure 7] 1 shows the therapeutic effect of the ADC of the present disclosure on human glioblastoma U87MG tumors xenografted in nude mice. DETAILED DESCRIPTION OF THE INVENTION

[0093] The present invention will be further described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0094] Experimental methods in the examples of this disclosure for which specific conditions are not specified generally follow common conditions or conditions recommended by the manufacturers of raw materials or products. Reagents for which specific sources are not specified are commonly available commercially.

[0095] 1. Preparation, properties and biological testing of antibody-drug conjugates The structures of the compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker AVANCE-400 nuclear magnetic spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d), deuterated chloroform (CDCl), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). The chemical shifts were 10 -6 Expressed in ppm.

[0096] For MS measurements, a FINNIGAN LCQAd(ESI) mass spectrometer (manufacturer: Thermo, model number: Finnigan LCQ advantage MAX) was used.

[0097] For UPLC measurements, a Waters Acquity UPLC SQD liquid chromatograph mass spectrometer was used.

[0098] For HPLC measurements, an Agilent 1200DAD high performance liquid chromatograph (Sunfire C18 150×4.6 mm column) and a Waters 2695-2996 high performance liquid chromatograph (Gimini C18 150×4.6 mm column) were used.

[0099] A Thermo nanodrop 2000 ultraviolet spectrophotometer was used for UV-HPLC measurements.

[0100] Growth inhibition rate and IC 50 The values ​​were measured using a PHERA starFS microplate reader (BMG, Germany).

[0101] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates are used as silica gel plates for thin layer chromatography (TLC). The silica gel plate specifications for thin layer chromatography (TLC) are 0.15 mm to 0.2 mm, while the silica gel plate specifications for separating and purifying products by thin layer chromatography are 0.4 mm to 0.5 mm.

[0102] Column chromatography generally used Yantai Huanghai 200-300 mesh silica gel as the vector.

[0103] Known starting materials according to the present disclosure may be synthesized by adopting or following methods known in the art, or may be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc.), and Darui Chemical.

[0104] In the examples, unless otherwise specified, all reactions were carried out in an argon or nitrogen atmosphere.

[0105] An argon or nitrogen atmosphere refers to an argon or nitrogen balloon with a volume of approximately 1 L connected to the reaction flask.

[0106] A hydrogen atmosphere refers to a hydrogen balloon with a volume of approximately 1 L attached to the reaction flask.

[0107] For the pressurized hydrogenation reaction, a Parr 3916EKX hydrogenation apparatus and a Seiran QL-500 hydrogen generator or an HC2-SS hydrogenation apparatus are used.

[0108] The hydrogenation reaction is usually carried out by repeating the procedure of evacuating and refilling with hydrogen three times.

[0109] For microwave reactions, a CEM Discover-S 908860 microwave reactor is used.

[0110] In the examples, unless otherwise specified, the solutions used in the reactions refer to aqueous solutions.

[0111] In the examples, unless otherwise specified, the reaction temperature is room temperature.

[0112] The optimum reaction temperature is room temperature, with a temperature range of 20°C to 30°C.

[0113] Preparation of PBS buffer solution with pH 6.5 in the example: 8.5 g of KH2PO4, 8.56 g of K2HPO4.3H2O, 5.85 g of NaCl, and 1.5 g of EDTA were taken in a flask, and the volume was adjusted to 2 L. The solution was completely dissolved by ultrasonication and then shaken to obtain the solution.

[0114] The eluent system for column chromatography and the developing solvent system for thin-layer chromatography used to purify the compound include A: dichloromethane and isopropyl alcohol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system, and the volume ratio of the solvents may be adjusted depending on the polarity of the compound, or may be adjusted by adding a small amount of triethylamine and an acidic or basic reagent, etc.

[0115] Some of the compounds of the present disclosure were characterized by Q-TOF LC / MS, which utilized 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).

[0116] Example 1-1 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 1 [ka]

[0117] Exatecan mesylate 1b (2.0 mg, 3.76 μmol, prepared by the method disclosed in patent application EP0737686A1) was added to 1 mL of N,N-dimethylformamide and cooled to 0-5°C in an ice-water bath. One drop of triethylamine was added and the reaction mixture was stirred until the solution became clear. 1-Hydroxycyclopropylformate 1a (1.4 mg, 3.7 μmol, prepared by the known method disclosed in Tetrahedron Letters, 25(12), 1269-72, 1984) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (3.8 mg, 13.7 μmol) were added sequentially to the reaction mixture. After the addition was complete, the mixture was stirred at 0-5°C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction mixture, and the reaction mixture 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 phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 1 (1.6 mg, yield: 82.1%). MS m / z (ESI): 520.2 [M+1] 1 H NMR (400 MHz, CDCl3): δ 7.90-7.84 (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).

[0118] Example 1-2 (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 (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 [ka]

[0119] To 1b (4 mg, 7.53 μmol), 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide were added. The mixture was purged with argon gas three times and cooled to 0-5°C in an ice-water bath. 0.3 mL of N-methylmorpholine was added dropwise and the reaction mixture was stirred until clear. 2-Cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared according to 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) were added sequentially. After the addition was complete, the mixture was stirred at 0-5°C for 1 hour. The ice-water bath was removed, and the mixture was heated to 30°C and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product, compound 2, was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A-water (10 mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding components were collected and concentrated under reduced pressure to give the title products (2-A: 1.5 mg, 2-B: 1.5 mg). MS m / z (ESI): 534.0 [M+1].

[0120] Single configuration compound 2-B (relatively short retention time) UPLC analysis: retention time: 1.06 minutes, purity: 88% (chromatogram: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A-water (5 mmol NH4OAc), B-acetyl nitrile). 1 H NMR (400 MHz, DMSO-d6): δ 8.37 (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).

[0121] Single configuration compound 2-A (relatively long retention time) UPLC analysis: retention time: 1.10 minutes, purity: 86% (chromatogram: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A-water (5 mmol NH4OAc), B-acetyl triol). 1H NMR (400 MHz, DMSO-d6): δ 8.35 (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).

[0122] Examples 1-3 (S)-N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropanamide 3-A (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 [ka] [ka]

[0123] To 1b (5.0 mg, 9.41 μmol), 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide were added, cooled to 0-5°C in an ice-water bath, and 0.3 mL of N-methylmorpholine was added dropwise. The reaction mixture was stirred until clear. 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, supplied by Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol) were added sequentially. After the addition was complete, the mixture was stirred at 0-5°C for 10 minutes. The ice-water bath was removed, and the mixture was heated to 30°C and stirred for 8 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product, compound 3, was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A-water (10 mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding components were collected and concentrated under reduced pressure to give the title product (1.5 mg, 1.5 mg). MS m / z (ESI): 561.9 [M+1].

[0124] Single-configuration compounds (relatively short retention times) UPLC analysis: Retention time: 1.11 min, Purity: 88% (Column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, Mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile). 1H NMR (400 MHz, DMSO-d6): δ 8.94 (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).

[0125] Single configuration compound (relatively long retention time) UPLC analysis: retention time: 1.19 minutes, purity: 90% (chromatogram: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A-water (5 mmol NH4OAc), B-Acetonitrile). 1 H NMR (400 MHz, DMSO-d6): δ 8.97 (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, 1H), 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).

[0126] Example 1-4 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 4 [ka]

[0127] 1 mL of N,N-dimethylformamide was added to 1b (3.0 mg, 5.64 μmol), cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added and stirred until the reaction mixture became clear. 1-Hydroxycyclopentaneformic acid 4a (2.2 mg, 16.9 μmol, prepared according to the method disclosed in patent application WO2013106717) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) were added sequentially to the reaction mixture. After the addition was complete, the mixture was stirred at 0-5°C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction mixture, and the reaction mixture 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 phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 4 (2.5 mg, yield: 80.9%). MS m / z (ESI): 548.0 [M+1]. 1 H NMR (400 MHz, CDCl3): δ 7.73-7.62 (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).

[0128] Examples 1-5 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 [ka]

[0129] 1 mL of N,N-dimethylformamide was added to 1b (2.0 mg, 3.76 μmol), cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added and stirred until the reaction mixture became clear. 1-(hydroxymethyl)-cyclopentaneformic acid 5a (0.87 mg, 7.5 μmol, prepared according to the method disclosed in patent application WO201396771) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2 mg, 7.24 μmol) were added sequentially to the reaction mixture. After the addition was complete, the mixture was stirred at 0-5°C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction mixture, and the reaction mixture 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 phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 5 (1.0 mg, yield: 50%). MS m / z (ESI): 533.9 [M+1]. 1H NMR (400 MHz, CDCl3): δ 8.07 (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).

[0130] Examples 1-6 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 [ka]

[0131] 1 mL of N,N-dimethylformamide was added to 1b (3.0 mg, 5.64 μmol), cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added. The reaction mixture was stirred until clear. 1-(hydroxymethyl)cyclobutane-1-formic acid 6a (2.2 mg, 16.9 μmol, prepared according to the method disclosed in "Journal of the American Chemical Society, 2014, Vol. 136, #22, pp. 8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) were added sequentially to the reaction mixture. After the addition was complete, the mixture was stirred at 0-5°C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction mixture, and the reaction mixture 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 phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 6 (2.1 mg, yield: 67.9%). MS m / z (ESI): 548.0 [M+1]. 1 H NMR (400 MHz, DMSO-d6): δ 7.85-7.62 (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).

[0132] Examples 1-7 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 [ka]

[0133] To 1b (3.0 mg, 5.64 μmol), 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide were added, cooled to 0-5°C in an ice-water bath, and 0.3 mL of N-methylmorpholine was added dropwise. The reaction mixture was stirred until clear. 1-Hydroxycyclobutaneformic acid 7a (2.0 mg, 17.22 μmol, supplied by Yakuseki), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol) were added sequentially to the reaction mixture. After the addition was complete, the mixture was stirred at 0-5°C for 10 min. The ice-water bath was removed, and the mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by thin-layer chromatography using developing solvent system B to obtain the title product 7 (2.5 mg, yield: 83.1%). MS m / z (ESI): 534.0 [M+1]. 1 H NMR (400 MHz, DMSO-d6): δ 8.28 (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).

[0134] Examples 1-8 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-pentaazaicosyl)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 [ka]

[0135] Step 1 Benzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropane-1-carboxylate 8c

[0136] Benzyl 1-hydroxycyclopropane-1-carboxylate 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)acetylamino)methyl acetate 8b (100 mg, 0.27 mmol, prepared by the method disclosed in patent application CN105829346A) were added to a reaction flask, 5 mL of tetrahydrofuran was added, the mixture was purged with argon gas three times, and the temperature was lowered to 0-5°C in an ice-water bath. Potassium tert-butoxide (61 mg, 0.54 mmol) was added, the ice-water bath was removed, the mixture was warmed to room temperature, and the mixture was stirred for 10 min. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The combined organic phases were concentrated. The resulting residue was dissolved in 3 mL of 1,4-dioxane, 0.6 mL of water was added, and sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (70 mg, 0.27 mmol) were added. 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 resulting residue was purified by silica gel column chromatography using developing solvent system B to give the title product 8c (100 mg, yield: 73.6%). MS m / z (ESI): 501.0 [M+1].

[0137] Step 2 1-((2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropane-1-carboxylic acid 8d

[0138] 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 on carbon (25 mg, 10% content) was added, and the mixture was purged with hydrogen gas three times and reacted at room temperature with stirring for 1 hour. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to give the title product 8d (41 mg, yield: 100%). MS m / z (ESI): 411.0 [M+1].

[0139] Step 3 (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)cyclopropoxy)methyl)amino)-2-oxoethyl)carbamate 8e

[0140] 1b (7 mg, 0.013 mmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, and the mixture was purged with argon gas three times. The mixture was cooled to 0-5 °C in an ice-water bath, and one drop of triethylamine was added. 8d (7 mg, 0.017 mmol) in 0.5 mL of N,N-dimethylformamide was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (7 mg, 0.026 mmol). The mixture was stirred in an ice bath for 35 min. 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 resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 8e (8.5 mg, yield: 78.0%). MS m / z (ESI): 828.0 [M+1].

[0141] Step 4 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

[0142] 8e (4 mg, 4.84 μmol) was dissolved in 0.2 mL of dichloromethane, 0.1 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was poured off three times. The mixture was then concentrated under reduced pressure to give the crude product 8f (2.9 mg), which was used directly in the next step without further purification. MS m / z (ESI): 606.0 [M+1].

[0143] Step 5 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-pentaazaicosyl)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

[0144] The crude product 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, purged with argon gas three times, and cooled to 0-5°C in an ice-water bath. A 0.3 mL N,N-dimethylformamide solution of (S)-2-(-2-(-2-(6-(-2,5-dioxo-1H-pyrrol-1-yl)hexaneamino)acetamino)acetamino)-3-phenylpropionic acid 8g (2.7 mg, 5.80 μmol, prepared by the method disclosed in patent application "EP2907824") was added, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol). The reaction was carried out in an ice bath with stirring for 30 minutes. The ice bath was removed, the temperature was raised to room temperature, and the reaction was stirred for 15 minutes. The reaction mixture was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, 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 give the title product 8 (2 mg, yield 39.0%). MS m / z (ESI): 1060.0 [M+1]. 1H NMR (400 MHz, DMSO-d6): δ 9.01 (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).

[0145] Examples 1-9 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B [ka] [ka]

[0146] Step 1 2-Cyclopropyl-2-hydroxybenzyl acetate 9a

[0147] 2a (1.3 g, 11.2 mmol, prepared according to 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 sequentially. The reaction mixture was stirred at room temperature for 48 hours, filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate (10 mL). The combined filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent system C to give the title product 9a (2 g, 86.9% yield).

[0148] Step 2 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate benzyl ester 9b

[0149] 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) were added to a reaction flask, 4 mL of tetrahydrofuran was added, and the mixture was purged with argon gas three times. The mixture was cooled to 0-5 °C in an ice-water bath, potassium tert-butoxide (109 mg, 0.98 mmol) was added, the ice-water bath was removed, the mixture was warmed to room temperature, and stirred for 40 min. 10 mL of ice-water was added, and the mixture was extracted with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). The combined organic phase was concentrated. The resulting 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 h. After adding 20 mL of water, the mixture was extracted with ethyl acetate (10 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 resulting residue was purified by silica gel column chromatography using developing solvent system C to give the title product 9b (48 mg, yield: 19%). MS m / z (ESI): 515.0 [M+1].

[0150] Step 3 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oic acid 9c

[0151] 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), and palladium on carbon (12 mg, 10% dry) was added. The mixture was purged with hydrogen gas three times and stirred at room temperature for 1 hour. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to give the crude title product 9c (13 mg), which was used directly in the next step without further purification. MS m / z (ESI): 424.9 [M+1].

[0152] Step 4 (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

[0153] 1b (10 mg, 18.8 μmol) was placed in a reaction flask, 1 mL of N,N-dimethylformamide was added, and the mixture was purged with argon gas three times. The mixture was cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added. Crude product 9c (13 mg, 30.6 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (16.9 mg, 61.2 μmol) were added, and the mixture was stirred in an ice bath for 40 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 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 resulting residue was purified by thin-layer chromatography using developing solvent system B to obtain the title product 9d (19 mg, yield: 73.6%). MS m / z (ESI): 842.1[M+1].

[0154] Step 5 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

[0155] 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 mixture was concentrated under reduced pressure, and 1 mL of toluene was added and the mixture was concentrated under reduced pressure twice. 3 mL of n-hexane was added to the residue and triturated. After standing, the supernatant was poured off and the solid was retained. The solid residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude title product 9e (17 mg), which was used directly in the next step without further purification. MS m / z (ESI): 638.0[M+18].

[0156] Step 6 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

[0157] The crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, purged with argon gas three times, and cooled to 0-5°C in an ice-water bath. A solution of 8g (21.2 mg, 44.8 μmol) in 0.3 mL of N,N-dimethylformamide was added, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol) was added. The reaction was carried out with stirring in an ice bath for 10 minutes, the ice bath was removed, the temperature was raised to room temperature, and the reaction was carried out with stirring for 1 hour to produce compound 9. The reaction mixture was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, 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 give the title products (9-A: 2.4 mg, 9-B: 1.7 mg). MS m / z (ESI): 1074.4 [M+1].

[0158] Single-configuration compound 9-A (relatively short retention time): UPLC analysis: Retention time: 1.14 min, Purity: 85% (Column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, Mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile). 1H NMR (400 MHz, DMSO-d6): δ 8.60 (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, 1H), 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).

[0159] Compound 9-B in a single configuration (comparatively long retention time): UPLC analysis: retention time: 1.16 minutes, purity: 89% (Kura: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A-water (5 mmol NH4OAc), B-アセトニトリル). 1H NMR (400 MHz, DMSO-d6): δ 8.68-8.60 (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).

[0160] Examples 1-10 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-A 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-B [ka] [ka]

[0161] Step 1 Benzyl 3,3,3-trifluoro-2-hydroxypropionate 10a

[0162] 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 that order. The reaction mixture was heated to 60 °C and stirred for 5 hours. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to give the title product 10a (980 mg, yield: 33.5%). 1 H NMR (400 MHz, CDCl3): δ 7.43-7.36 (m, 5H), 5.34 (s, 2H), 4.53 (s, 1H), 3.44 (s, 1H).

[0163] Step 2 1-(9H-Fluoren-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazaundecan-11-oate benzyl ester 10b

[0164] 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, and the mixture was purged with argon gas three times. The mixture was cooled to 0-5°C in an ice-water bath, potassium tert-butoxide (38 mg, 0.34 mmol) was added, the ice-water bath was removed, the mixture was warmed to room temperature, and stirred for 20 min. 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 resulting 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 h. 20 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 (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 developing solvent system C to give the title product 10b (51 mg, yield: 55.3%). MS m / z (ESI): 559.9 [M+18].

[0165] Step 3 1-(9H-Fluoren-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazaundecan-11-oic acid 10c

[0166] 10b (15 mg, 0.28 mmol) was dissolved in 3 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (15 mg, 10% content) was added. The mixture was purged with hydrogen gas three times and allowed to react at room temperature with stirring for 1 hour. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to give the crude title product 10c (13 mg). MS m / z (ESI): 452.9 [M+1].

[0167] Step 4 (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

[0168] 1b (10 mg, 18.8 μmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, the atmosphere was purged with argon gas three times, the temperature was lowered to 0-5°C in an ice-water bath, one drop of triethylamine was added, a 0.5 mL N,N-dimethylformamide solution of 10c (13 mg, 28.7 μmol) was added, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (11 mg, 39.7 μmol) was added, and the reaction was carried out with stirring 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, washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 10d (16 mg, yield: 97.8%). MS m / z (ESI): 870.0[M+1].

[0169] Step 5 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

[0170] 10d (16 mg, 18.4 μmol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure twice. The residue was triturated with 3 mL of n-hexane, and after standing, the supernatant was poured off and the solid was retained. This process was repeated three times. The solid residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude title product 10e (12 mg), which was used directly in the next step without further purification. MS m / z (ESI): 647.9 [M+1].

[0171] Step 6 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-A 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-B

[0172] The crude product 10e (12 mg, 18.5 μmol) was dissolved in 1.0 mL of N,N-dimethylformamide, purged with argon gas three times, and cooled to 0-5°C in an ice-water bath. A solution of 8g (14 mg, 29.6 μmol) in 0.3 mL of N,N-dimethylformamide was added, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (15 mg, 54.2 μmol). The mixture was stirred in an ice bath for 30 minutes, the ice bath was removed, the mixture was warmed to room temperature, and the mixture was stirred for 1 hour to produce compound 10. The reaction mixture was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, 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 give the title products (2.7 mg, 2.6 mg). MS m / z (ESI): 1102.0 [M+1].

[0173] Single configuration compounds (relatively short retention times): UPLC analysis: Retention time: 1.18 min, Purity: 91% (Column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, Mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile). 1H NMR (400 MHz, DMSO-d6): δ 8.97 (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, 1H), 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).

[0174] Single configured compound (comparatively long retention time): UPLC analysis: retention time: 1.23 minutes, purity: 90% (Kura: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A-water (5 mmol NH4OAc), B-アセトニトリル). 1H NMR (400 MHz, DMSO-d6): δ 9.05 (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, 1H), 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).

[0175] Examples 1-11 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-pentaazaicosyl)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 11 [ka] [ka]

[0176] Step 1 Benzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclobutane-1-carboxylate 11b

[0177] 1-Hydroxycyclobutane-benzyl carboxylate 11a (167 mg, 0.81 mmol, prepared by the method disclosed in the literature "Journal of Medicinal Chemistry, 2013, vol. 56, # 13, pp. 5541-5552") and 8b (150 mg, 0.41 mmol) were added to a reaction flask, 5 mL of tetrahydrofuran was added, the atmosphere was replaced with argon gas three times, the temperature was lowered 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 raised to room temperature, and the mixture was stirred for 10 min. 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 resulting residue was dissolved in 3 mL of dioxane, 0.6 mL of water was added, and sodium bicarbonate (41 mg, 0.48 mmol) was added. The mixture was added with 105 mg (0.41 mmol) of 9-fluorenylmethyl chloroformate (105 mg, 0.41 mmol) and 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 resulting residue was purified by silica gel column chromatography using developing solvent system C to give the title product 11b (37 mg, yield: 17.6%). MS m / z (ESI): 514.6 [M+1].

[0178] Step 2 1-((2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclobutane-1-carboxylic acid 11c

[0179] 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 on carbon (15 mg, 10% content) was added, and the mixture was purged with hydrogen gas three times and stirred at room temperature for 2 hours. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to give the title product 11c (35 mg, yield: 82%), which was directly carried on to the next step.

[0180] Step 3 (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)cyclopropoxy)methyl)amino)-2-oxoethyl)carbamate 11d

[0181] 1b (10 mg, 0.018 mmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, the atmosphere was purged with argon gas three times, and the temperature was lowered to 0-5 °C in an ice-water bath. 1 drop of triethylamine was added, and a solution of 11c (13 mg, 0.031 mmol) in 0.5 mL of N,N-dimethylformamide was added. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (25 mg, 0.091 mmol) was added, and the mixture was stirred in an ice bath for 40 min. 8 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL × 3). The organic phase was washed with saturated sodium chloride solution (8 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system A to give the title product 11d (19 mg, yield: 73.9%). MS m / z (ESI): 842.3 [M+1].

[0182] Step 4 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

[0183] 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 mixture was concentrated under reduced pressure, and 1 mL of toluene was added and concentrated under reduced pressure twice. 4 mL of n-hexane was added and triturated, and the upper layer of n-hexane was poured off three times. The mixture was then concentrated under reduced pressure to give the crude product 11e (15 mg), which was used directly in the next step without further purification.

[0184] Step 5 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-pentaazaicosyl)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 11

[0185] Crude product 11e (2 mg, 3.22 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, purged with argon gas three times, and cooled to 0-5 °C in an ice-water bath. A solution of 8 g (1.5 mg, 3.17 μmol) in 0.3 mL of N,N-dimethylformamide was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol) and stirring at room temperature for 30 min. The reaction mixture was dried on an oil pump rotary evaporator to remove DMF. The residue was dissolved in DCM and directly purified twice by thin-layer chromatography (eluent polarity: DCM / MeOH = 10 / 1) to give the title product 11 (1 mg, yield: 28.8%). MS m / z (ESI): 1073.6 [M+1]. 1 H NMR (400 MHz, CDCl3): δ 8.70-8.60 (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).

[0186] Examples 1-12 (S)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 12-A (R)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 12-B [ka] [ka]

[0187] Step 1 3-Cyclopropyl-2-hydroxypropionic acid 12b

[0188] 12a (0.5 g, 3.87 mmol, supplier: Adamas) was dissolved in 35 mL of a mixed solvent of water and acetic acid (V:V = 4:1), cooled to 0-5 °C in an ice-water bath, and a 2 M aqueous solution of sodium nitrite (0.53 g, 7.74 mmol) was added dropwise. The mixture was allowed to warm to room temperature and reacted with stirring for 3 h. Solid sodium chloride was added to the reaction mixture 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 give the title product 12b (0.45 g, yield: 89.3%).

[0189] Step 2 (S)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 12-A (R)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropanamide 12-B

[0190] To 1b (45 mg, 0.085 mmol), 1.5 mL of ethanol and 1.5 mL of N,N-dimethylformamide were added, and the mixture was purged with argon gas three times. 0.1 mL of N-methylmorpholine was added dropwise and stirred until the reaction mixture became clear. 12b (90 mg, 0.691 mmol), 1-hydroxybenzotriazole (34 mg, 0.251 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (49 mg, 0.256 mmol) were added in that order. After the addition was complete, the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product, Compound 12, was purified by high-performance liquid chromatography (separation conditions: column: Sharpsil-T C18 5 μm 21.2*250 mm, mobile phase: A-water (10 mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL / min) to give the title product (7 mg, 15 mg). MS m / z (ESI): 547.9 [M+1].

[0191] Single-configuration compounds (relatively short retention times) UPLC analysis: Retention time: 1.345 min, Purity: 72% (Column: ZORBAX Ecliphase Plus C18 1.8 μm 2.1*50 mm, Mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile). 1 H NMR (400 MHz, DMSO-d6): δ 8.42 (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).

[0192] Single configuration compound (relatively long retention time) UPLC analysis: retention time: 1.399 minutes, purity: 88% (chromatogram: ZORBAX Ecliphase Plus C18 1.8 μm 2.1*50 mm, mobile phase: A-water (5 mmol NH4OAc), B-acetyl triol). 1 H NMR (400 MHz, DMSO-d6): δ 8.36 (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).

[0193] Example 1-13 (reference example) 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 [ka] The title compound 13 was prepared and obtained by the method disclosed in patent "Example 76 on page 147 of the specification in EP2907824A1".

[0194] Examples 1-14 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-A 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-B [ka] [ka]

[0195] Step 1 Benzyl 3-cyclopropyl-2-hydroxypropionate 14a

[0196] 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 sequentially. The reaction mixture was stirred at room temperature for 48 hours, filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate (10 mL). The combined filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to give the title product 14a (140 mg, yield: 41.3%).

[0197] Step 2 10-(Cyclopropylmethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate benzyl ester 14b

[0198] 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, and the mixture was purged with argon gas three times. The mixture was cooled to 0-5 °C in an ice-water bath, potassium tert-butoxide (79 mg, 0.704 mmol) was added, the ice-water bath was removed, the mixture was warmed to room temperature, and stirred for 10 min. 20 mL of ice-water was added, and the mixture was extracted with ethyl acetate (10 mL × 4). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to give the title product 14b (50 mg, yield: 26.8%). MS m / z (ESI): 529.2 [M+1].

[0199] Step 3 10-(Cyclopropylmethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oic acid 14c

[0200] 14b (27 mg, 0.051 mmol) was dissolved in 3 mL of ethyl acetate, and palladium-carbon (7 mg, 10% dry) was added. The mixture was purged with hydrogen gas three times and stirred at room temperature for 1 hour. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to give the crude title product 14c (23 mg), which was used directly in the next step without further purification. MS m / z (ESI): 439.1 [M+1].

[0201] Step 4 (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

[0202] 1b (22 mg, 42.38 μmol) was placed in a reaction flask, 3 mL of N,N-dimethylformamide was added, and the mixture was purged with argon gas three times. The mixture was cooled to 0-5°C in an ice-water bath, triethylamine (4.3 mg, 42.49 μmol) was added dropwise, and crude product 14c (23 mg, 51.1 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 μmol) were added. The mixture was stirred in an ice bath for 40 minutes. 15 mL of water was added, and the mixture was extracted with ethyl acetate (8 mL × 3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 14d (29 mg, yield: 79.9%). MS m / z (ESI): 856.1[M+1].

[0203] Step 5 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

[0204] 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 mixture was concentrated under reduced pressure, and 1 mL of toluene was added and concentrated under reduced pressure twice. The residue was triturated with 3 mL of n-hexane, and the supernatant was poured off after standing. This process was repeated three times. The residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude title product 14e (22 mg), which was used directly in the next step without further purification. MS m / z (ESI): 634.1[M+1].

[0205] Step 6 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-A 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-B

[0206] Crude product 14e (22 mg, 33.9 μmol) was dissolved in 2.5 mL of N,N-dimethylformamide, purged with argon gas three times, and cooled to 0-5°C in an ice-water bath. 8g (24 mg, 50.8 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (14 mg, 50.6 μmol) were added, the ice bath was removed, the mixture was warmed to room temperature, and the mixture was stirred for 1 hour to produce compound 14. The reaction mixture was purified by high-performance liquid chromatography (HPLC) (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A - water (10 mmol NHOAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min) to give the title product (2 mg, 2 mg). MS m / z (ESI): 1088.4 [M+1].

[0207] Single configuration compounds (relatively short retention times): UPLC analysis: Retention time: 1.18 min, Purity: 88% (Column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, Mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0208] Single configuration compounds (relatively long retention time): UPLC analysis: Retention time: 1.23 min, Purity: 96% (Column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, Mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile).

[0209] Examples 1-15 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-pentaazadocosane)-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 [ka]

[0210] Step 1 Benzyl 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylate 15b

[0211] 8b (500 mg, 1.35 mmol) was placed in a reaction flask, 6 mL of tetrahydrofuran was added, and 1-hydroxymethylcyclopropane-1-benzyl formate 15a (233 mg, 1.13 mmol, prepared by the method disclosed in Example 22-2 on page 262 of the patent application "EP2862856A1") was placed in the flask, and the mixture was purged with argon gas three times. The mixture 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 mixture was warmed to room temperature, and stirred for 40 minutes. The mixture was then cooled to 0°C, 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 resulting residue was purified by silica gel column chromatography using developing solvent system B to give the title product 15b (15 mg, yield: 2.5%). MS m / z (ESI): 515.2 [M+1].

[0212] Step 2 1-(10-(9H-Fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylic acid 15c

[0213] 15b (15 mg, 0.029 mmol) was dissolved in 2 mL of ethyl acetate, and palladium-carbon (3 mg, 10% dry) was added. The mixture was purged with hydrogen gas three times and stirred at room temperature for 4.5 hours. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to give the title product 15c (11 mg, 89% yield). MS m / z (ESI): 425.2 [M+1].

[0214] Step 3 (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

[0215] 1b (10 mg, 0.021 mmol) was placed in a reaction flask, 1 mL of N,N-dimethylformamide was added, the mixture was purged with argon gas three times, and the temperature was lowered to 0-5 °C in an ice-water bath. One drop of triethylamine was added, followed by 15c (11 mg, 0.026 mmol), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (10.7 mg, 0.039 mmol). After the addition was complete, the mixture was stirred at room temperature for 60 min. 10 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL x 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 resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 15d (19 mg, yield: 87.0%). MS m / z (ESI): 842.2 [M+1].

[0216] Step 4 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 15e

[0217] 15d (19 mg, 22.56 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 mixture was concentrated under reduced pressure at 0°C, and 1 mL of toluene was added and concentrated under reduced pressure twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was poured off three times. The mixture was then concentrated under reduced pressure to give the crude product 15e (13.9 mg), which was used directly in the next step without further purification. MS m / z (ESI): 620.1 [M+1].

[0218] Step 5 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-pentaazadocosane)-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

[0219] Crude product 15e (13.9 mg, 22.4 μmol) was dissolved in 1 mL of N,N-dimethylformamide, purged with argon gas three times, and cooled to 0-5 °C in an ice-water bath. 8g (15.8 mg, 33.4 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (9.3 mg, 33.6 μmol) were added, and the mixture was warmed to room temperature and stirred for 60 h. The reaction mixture was purified by high-performance liquid chromatography (HPLC) (column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A - water (10 mmol NHOAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding components were collected and concentrated under reduced pressure to give the title product 15 (2.5 mg, 10.3% yield). MS m / z (ESI): 1074.2 [M+1]. 1 H NMR (400 MHz, DMSO-d6): δ 8.51-8.37 (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), 7.26-7.10 (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).

[0220] Examples 1-16 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-pentaazadocosane)-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 16 [ka] [ka]

[0221] Step 1 1-(Hydroxymethyl)cyclobutane-1-carboxylic acid 16b

[0222] Ethyl 1-(hydroxymethyl)cyclobutanecarboxylate 16a (250 mg, 1.58 mmol, supplier: Alfa) was dissolved in methanol (2 mL) and water (1 mL), and sodium hydroxide (126 mg, 3.15 mmol) was added. The mixture was heated to 40 °C and stirred for 3 h. The mixture was cooled to room temperature and concentrated under reduced pressure to remove the organic solvent. The mixture was back-extracted with ether (10 mL), and the aqueous phase was collected. The pH of the aqueous phase was adjusted to 3-4 with 6 N aqueous hydrochloric acid and concentrated under reduced pressure to give a solid. 3 mL of toluene was added, and the mixture was concentrated under reduced pressure and rotary dried three times. The crude product, titled product 16b (206 mg), was obtained by drying on an oil pump. This product was used directly in the next step without further purification. MS m / z (ESI,NEG):129.2 [M-1].

[0223] Step 2 1-(Hydroxymethyl)cyclobutane-1-carboxylate benzyl 16c

[0224] Crude product 16b (206 mg, 1.58 mmol) was dissolved in acetonitrile (15 mL), and anhydrous potassium carbonate (1.09 g, 7.90 mmol) and tetrabutylammonium iodide (29 mg, 78.51 μmol) were added. Benzyl bromide (216 mg, 1.26 mmol) was added, and the mixture was stirred at room temperature overnight. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using developing solvent system C to give the title product 16c (112 mg, yield: 32.1%). MS m / z (ESI): 221.1 [M+1].

[0225] Step 3 Benzyl 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylate 16d

[0226] 16c (77 mg, 0.35 mmol) and 8b (100 mg, 0.27 mmol) were placed in a reaction flask, 3 mL of tetrahydrofuran was added, and the mixture was purged with argon gas three times. The mixture 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 min. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL) and chloroform (5 mL × 5). The organic phases were combined and concentrated. The resulting residue was dissolved in 3 mL of 1,4-dioxane, 0.5 mL of water was added, and sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (71 mg, 0.27 mmol) were added. The mixture was stirred at room temperature for 1 h. 20 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 (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 developing solvent system C to give the title product 16d (24 mg, yield: 16.7%). MS m / z (ESI): 551.3 [M+23].

[0227] Step 4 Benzyl 1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylate 16e

[0228] 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), and palladium on carbon (5 mg, 10% content) was added. The mixture was purged with hydrogen gas three times and allowed to react at room temperature for 2 hours with stirring. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated under reduced pressure to give the crude title product 16e (10 mg), which was used directly in the next step without further purification.

[0229] Step 5 (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

[0230] 1b (7.5 mg, 0.014 mmol) was placed in a reaction flask, 1 mL of N,N-dimethylformamide was added, and the mixture was purged with argon gas three times. The mixture was cooled to 0-5 °C in an ice-water bath, and one drop of triethylamine was added. Crude product 16e (10 mg) in 0.5 mL of N,N-dimethylformamide was added, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (6 mg, 0.026 mmol) was added. The mixture was stirred in an ice bath for 30 min. 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 resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 16f (10.6 mg, yield: 87.8%). MS m / z (ESI): 856.2 [M+1].

[0231] Step 6 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

[0232] 16f (10.6 mg, 12.4 μmol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was poured off. This process was repeated three times. Concentration under reduced pressure gave the crude title product 16g (8 mg), which was used directly in the next step without further purification. MS m / z (ESI): 634.1 [M+1].

[0233] Step 7 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-pentaazadocosane)-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 16

[0234] The crude product 16g (8 mg) was dissolved in 1 mL of N,N-dimethylformamide, and 8g (8.8 mg, 18.6 μmol) was added. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (5.2 mg, 18.8 μmol) was added and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was purified by high-performance liquid chromatography (HPLC) (column: XBridge Prep C18 OBD 5 μm 19*250 mm, mobile phase: A - water (10 mmol NHOAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min) to give the title product 16 (1.0 mg, yield: 7.2%). MS m / z (ESI): 1088.0 [M+1].

[0235] Examples 1-17 (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 (yl)cyclopropyloxy)-3,6,9,12,15-pentaoxo-17,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,14-pentaazatritetracontan-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17 [ka] [ka]

[0236] Step 1 tert-Butyl 1-phenyl-2,5,8,11,14,17,20,23,26,29-decaoxahentriacontan-31-oate 17b

[0237] 1-Phenyl-2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-ol 17a (0.34 g, 0.67 mmol, supplier: BiDe) 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 sequentially. The mixture was stirred at room temperature for 3 h. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using eluent system B to give the title product 17b (0.42 g, 100% yield). MS m / z (ESI): 636.3 [M+18].

[0238] Step 2 29-hydroxy-3,6,9,12,15,18,21,24,27-nonaoxamontan-1-oic acid tert-butyl ester 17c

[0239] 17b (417 mg, 0.67 mmol) was dissolved in 15 mL of tetrahydrofuran, palladium on carbon (110 mg, 10% dry) was added, and the mixture was purged with hydrogen gas three times. The mixture was heated to 60°C and stirred for 3 hours. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to give the crude title product 17c (357 mg), which was used in the next step without further purification. MS m / z (ESI): 546.2 [M+18].

[0240] Step 3 29-azido-3,6,9,12,15,18,21,24,27-nonaoxamontan-1-oate tert-butyl ester 17d

[0241] 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 mixture was purged with argon gas three times and stirred at room temperature for 2 hours. The mixture was then heated to 105 °C and reacted for 19 hours. The reaction mixture was cooled to room temperature, concentrated, and 20 mL of water was added. The mixture was extracted with ethyl acetate (10 mL × 4). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system B to give the crude title product 17d (412 mg). MS m / z (ESI): 571.3 [M+18].

[0242] Step 4 29-amino-3,6,9,12,15,18,21,24,27-nonaoxamontan-1-oic acid tert-butyl ester 17e

[0243] 17d (230 mg, 0.415 mmol) was dissolved in 8 mL of tetrahydrofuran, palladium on carbon (58 mg, 10% dry content) was added, and the mixture was purged with hydrogen gas three times. The mixture was stirred at room temperature for 2 hours. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to give the crude product 17e (220 mg). This product was directly used in the next step without further purification. MS m / z (ESI): 528.2 [M+1].

[0244] Step 5 tert-Butyl 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-azahentriacontan-31-oate 17f

[0245] (1r,4r)-4-((2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylic acid (98.5 mg, 0.415 mmol) was dissolved in 10 mL of dichloromethane, and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (190 mg, 0.500 mmol) and N,N-diisopropylethylamine (162 mg, 1.253 mmol) were added. The mixture was purged with argon gas three times, and crude product 17e (220 mg, 0.417 mmol) was added. The mixture was stirred at room temperature for 1 hour. 15 mL of water was added, and the mixture was extracted with dichloromethane (8 mL × 3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The 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%). MS m / z (ESI): 747.2[M+1].

[0246] Step 6 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-aza-31-oic acid 17g

[0247] 17f (122 mg, 0.163 μmol) was dissolved in 0.8 mL of dichloromethane, 0.4 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 hour. The mixture was diluted with 15 mL of dichloromethane, concentrated under reduced pressure, and then 10 mL of n-hexane was added and concentrated under reduced pressure twice. 10 mL of toluene was added and concentrated under reduced pressure. The mixture was triturated three times with 10 mL of a 5:1 mixture of n-hexane and ether until the pH reached approximately 7. The mixture was concentrated and dried on an oil pump to give the title product 17g (98 mg, yield: 86.8%). MS m / z (ESI): 691.2[M+1].

[0248] Step 7 2,4-Dimethoxybenzyl-1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropyl-1-carboxylic acid ester 17h

[0249] 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)carbodihydrochloride (115 mg, 0.60 mmol), and 4-dimethylaminopyridine (5 mg, 0.041 mmol) were added in that order. After the addition was complete, the mixture was stirred at room temperature for 1 h. 20 mL of water was added, and the mixture was shaken and separated. The aqueous phase was extracted with dichloromethane (8 mL x 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 resulting residue was purified by silica gel column chromatography using developing solvent system C to give the title product 17h (124 mg, yield: 55.4%). MS m / z (ESI): 583.1 [M+23].

[0250] Step 8 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-pentaazaheptadecan-17-yl)oxy)cyclopropyl-1-carboxylate 17j

[0251] 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 mixture was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure twice. 3 mL of n-hexane was added and the mixture was triturated, and the upper layer of n-hexane was poured off. This was repeated three times, and the mixture was concentrated under reduced pressure. The resulting crude product was dissolved in 2 mL of N,N-dimethylformamide, and (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-phenylalanine 17i (35 mg, 69.8 μmol, prepared by the method disclosed in Examples 7-12 on page 13 of the patent application "CN108853514A") was added. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (23 mg, 83.1 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). 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 resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 17j (48 mg, yield: 83.9%). MS m / z (ESI): 822.0[M+1].

[0252] Step 9 (S)-1-((11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16-pentaazaheptadecan-17-yl)oxy)cyclopropane-1-carboxylic acid 17k

[0253] 17j (48 mg, 58.4 μmol) was dissolved in 1.4 mL of a 3% (v / v) dichloroacetic acid solution in dichloromethane, 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, and 5 mL of ether was added. The mixture was allowed to warm to room temperature and then beaten to precipitate a white solid. The solid was filtered, and the filter cake was collected and dried in an oil pump to give the title product 17k (33 mg, yield: 84.1%).

[0254] Step 10 (9H-Fluoren-9-yl)methyl ((S)-7-benzyl-1-(1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropyloxy)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)carbamate 17l

[0255] 1b (20 mg, 42.4 μmol) was placed in a reaction flask, 1 mL of a 10% (v / v) methanol / dichloromethane solution was added, and the mixture was purged with argon gas three times. The mixture was cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added dropwise. 17k (33 mg, 49.1 μmol) was dissolved in 1 mL of a 10% (v / v) methanol / dichloromethane solution and added dropwise to the reaction mixture. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 μmol) was then added. The mixture was warmed to room temperature and stirred for 1 hour. 10 mL of dichloromethane and 5 mL of water were added, stirred for 5 minutes, and allowed to separate. The organic phase was collected, and the aqueous phase was extracted with dichloromethane (10 mL x 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 resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 17l (37 mg, yield: 80.2%). MS m / z (ESI): 1090.1[M+1].

[0256] Step 11 (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 (yl)cyclopropyloxy)-3,6,9,12,15-pentaoxo-17,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,14-pentaazatritetracontan-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17

[0257] 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 mixture was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure twice. 3 mL of n-hexane was added, triturated, and the upper layer of n-hexane was poured off three times. The mixture was concentrated under reduced pressure and dried on an oil pump. The resulting crude product was dissolved in 1 mL of N,N-dimethylformamide, and 17g (11 mg, 15.92 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (6.0 mg, 21.68 μmol) were added. The mixture was purged with argon gas three times and allowed to react at room temperature for 30 minutes with stirring. The reaction mixture was purified by high-performance liquid chromatography (column: XBridge Prep C18 OBD 5 μm 19*250 mm, 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 give the title product 17 (6 mg, yield 27.4%). MS m / z (ESI): 1556.4 [M+18]. 1H NMR (400 MHz, DMSO-d6): δ 8.98 (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).

[0258] Examples 1-18 (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 -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-pentaazatetrahexadecan-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18 [ka] [ka]

[0259] Step 1 (R)-2-Cyclopropyl-2-hydroxybenzyl acetate 18a (S)-2-Cyclopropyl-2-hydroxybenzyl acetate 18b

[0260] 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 that order. The reaction mixture was stirred at room temperature for 16 h, filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate (10 mL). The combined filtrate was concentrated under reduced pressure. The resulting residue (4.1 g) was purified by silica gel column chromatography using developing solvent system C and further purified by chiral separation to give the title products 18a (1.1 g) and 18b (1.2 g).

[0261] Step 2 (R)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate benzyl ester 18c

[0262] 8b (3.1 g, 8.41 mmol) was dissolved in tetrahydrofuran (55 mL), 18a (2.0 g, 9.70 mmol) was added, and the mixture was cooled to 0-5 °C in an ice-water bath. Potassium tert-butoxide (1.89 g, 16.84 mmol) was added, and the mixture was stirred in an ice-water bath for 10 min. Ethyl acetate (30 mL) and water (20 mL) were added, and the mixture was allowed to stand for separation. 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 resulting 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 the mixture was stirred at room temperature for 2 h. Water (30 mL) was added to the reaction mixture, which was then 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 resulting residue was purified by column chromatography using developing solvent system C to give the title product 18c (1.3 g, yield: 30.0%). MS m / z (ESI): 515.2[M+1].

[0263] Step 3 (R)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oic acid 18d

[0264] 18c (1.29 g, 2.51 mmol) was dissolved in ethyl acetate (15 mL), palladium on carbon (260 mg, 10% dry), and the mixture was purged with hydrogen gas three times. The mixture was stirred at room temperature for 5 hours. The reaction mixture was filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate (20 mL) and methanol (20 mL). The filtrate was concentrated to give the crude product 18d (980 mg), which was used in the next step without further purification. MS m / z (ESI): 425.1 [M+1].

[0265] Step 4 2,4-Dimethoxybenzyl (R)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-ester 18e

[0266] 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)carbodihydrochloride (664 mg, 3.46 mmol), and 4-dimethylaminopyridine (28 mg, 0.23 mmol) were added. The mixture was stirred at room temperature for 1 h. The organic solvent was removed by concentration under reduced pressure, and 20 mL of water was added. 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 resulting residue was purified by column chromatography using developing solvent system C to give the title product 18e (810 mg, yield: 61.1%). MS m / z (ESI): 575.0[M+1].

[0267] Step 5 2,4-Dimethoxybenzyl (R)-2-((2-aminoacetylamino)methoxy)-2-cyclopropylacetate 18f

[0268] 18e (33 mg, 57.4 μmol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was poured off three times. The mixture was then concentrated under reduced pressure to give the crude product 18f (21 mg), which was used directly in the next step without further purification.

[0269] Step 6 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-pentaazaeicosan-20-oic acid ester 18g

[0270] Crude product 18f (21 mg, 57.4 μmol) was dissolved in 3 mL of N,N-dimethylformamide, and 17i (29 mg, 57.8 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (19 mg, 68.7 μmol) were added. The mixture was stirred at room temperature for 1 h. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). 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 resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 18g (37 mg, yield: 77.1%). MS m / z (ESI): 853.0[M+18].

[0271] Step 7 (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-pentaazaeicosan-20-oic acid 18h

[0272] 18g (37 mg, 44.3 μmol) was dissolved in 1.4 mL of a 3% (v / v) dichloroacetic acid solution in dichloromethane, 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, and 5 mL of ether was added. The mixture was allowed to warm to room temperature and then beaten to precipitate a white solid. The solid was filtered, and the filter cake was collected and dried in an oil pump to obtain the title product 18h (24 mg, yield: 79.1%). MS m / z (ESI): 708.2[M+23].

[0273] Step 8 (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-tetraazahexadecan-16-yl)carbamate 18i

[0274] 1b (30 mg, 63.6 μmol) was placed in a reaction flask, 1 mL of a 10% (v / v) methanol / dichloromethane solution was added, and the mixture was purged with argon gas three times. The mixture was cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added dropwise. 18h (65 mg, 94.8 μmol) was dissolved in 1 mL of a 10% (v / v) methanol / dichloromethane solution and added dropwise to the reaction mixture. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (27 mg, 97.6 μmol) was then added. The mixture was warmed to room temperature and stirred for 1 hour. 10 mL of dichloromethane and 5 mL of water were added, stirred for 5 minutes, and allowed to stand for separation. The organic phase was collected, and the aqueous phase was extracted with dichloromethane (10 mL x 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 resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 18i (25 mg, yield: 35.6%). MS m / z (ESI): 1104.4[M+1].

[0275] Step 9 (S)-2-(2-(2-aminoacetylamino)acetylamino)-N-(2-((((R)-1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethoxy)-3-phenylpropanamide 18j

[0276] 18i (12 mg, 10.9 μmol) was dissolved in 0.6 mL of dichloromethane, 0.3 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours. The reaction mixture was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was poured off three times. The mixture was then concentrated under reduced pressure to give the crude product 18j (10 mg), which was used directly in the next step without further purification. MS m / z (ESI): 881.0 [M+1].

[0277] Step 10 (1r,4r)-N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]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-pentaazatetrahexadecan-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18

[0278] Crude product 18j (10 mg) was dissolved in 1 mL of N,N-dimethylformamide, and 17g (8.5 mg, 12.3 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.6 mg, 16.6 μmol) were added. The mixture was stirred at room temperature for 30 min. The reaction mixture was filtered and purified by high-performance liquid chromatography (HPLC) (column: XBridge Prep C18 OBD 5 μm 19 × 250 mm, mobile phase: A - water (10 mmol NHOAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding components were collected and concentrated under reduced pressure to give the title product 18 (9.5 mg, yield: 56.2%). MS m / z (ESI): 1570.2 [M+18]. 1 H NMR (400 MHz, DMSO-d6): δ 8.77 (d, 1H), 8.59-8.55 (m, 1H), 8.42 (d, 1H), 8.37-8.28 (m, 1H), 8.25-8.06 (m, 2H), 7.96-7.86 (m, 1H), 7.86-7.70 (m, 2H), 7.32-7.28 (m, 1H), 7.25-7.14 (m, 3H), 6.67 (m, 1H), 5.96 (s, 1H), 5.80-5.72 (m, 1H), 5.62-5.52 (m, 2H), 5.43-5.30 (m, 3H), 5.28-5.17 (m, 2H), 5.12-5.08 (m, 1H), 4.72-4.35 (m, 8H), 3.95-3.70 (m, 13H), 3.35-3.22 (m, 14H), 2.42-2.32 (m, 3H), 2.05-1.98 (m, 4H), 1.88-1.82 (m, 12H), 1.47-1.39 (m, 3H), 1.32-1.18 (m, 11H), 0.90-0.80 (m, 4H), 0.52-0.37 (m, 3H), 0.32-0.18 (m, 2H).

[0279] Examples 1-19 (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 -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-pentaazatetrahexadecan-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 19 [ka]

[0280] Step 1 (S)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate benzyl ester 19a

[0281] 18b (252 mg, 1.22 mmol) was placed in a reaction flask, 4 mL of dichloromethane was added, and the mixture was purged with argon gas three times. The mixture was cooled to 0-5 °C in an ice-water bath. Lithium tert-butoxide (98 mg, 1.22 mmol) was added and the mixture was stirred in an ice-water bath for 15 min. When the mixture became clear, 8b (300 mg, 814.3 μmol) was added and the mixture was stirred in an ice-water bath for 2.5 h. Water (10 mL) was added, and the mixture was separated. The aqueous phase was extracted with dichloromethane (8 mL × 2). The combined organic phase was washed with water (10 mL × 1), saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The resulting residue was purified by silica gel column chromatography using eluent C to give the title product 19a (282 mg, yield: 67.2%).

[0282] Step 2 (S)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oic acid 19b

[0283] 19a (280 mg, 0.554 mmol) was dissolved in 8 mL of ethyl acetate, and palladium-carbon (84 mg, 10% dry) was added. The mixture was purged with hydrogen gas three times and stirred at room temperature for 3 hours. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to give the crude title product 19b (230 mg), which was used directly in the next step without further purification.

[0284] Step 3 2,4-Dimethoxybenzyl (S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oic acid ester 19c

[0285] 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)carbodihydrochloride (155 mg, 808.5 μmol), and 4-dimethylaminopyridine (6.6 mg, 53.5 μmol) were added sequentially. The mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with 10 mL of dichloromethane, washed with water (10 mL × 1), saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 19c (159 mg, yield: 51.0%).

[0286] Step 4 2,4-Dimethoxybenzyl (S)-2-((2-aminoacetylamino)methoxy)-2-cyclopropylacetate 19d

[0287] 19c (60 mg, 104.4 μmol) was dissolved in 1 mL of dichloromethane, 0.5 mL of diethylamine was added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was poured off three times. The mixture was then concentrated under reduced pressure to give the crude product 19d (21 mg), which was used directly in the next step without further purification.

[0288] Step 5 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-pentaazaeicosan-20-oic acid ester 19e

[0289] Crude product 19d (36 mg, 102.2 μmol) was dissolved in 4 mL of N,N-dimethylformamide, and 17i (52 mg, 103.6 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (34.6 mg, 125.0 μmol) were added. The mixture was stirred at room temperature for 1 h. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). 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 resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 19e (70 mg, yield: 80.2%).

[0290] Step 6 (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-pentaazaeicosan-20-oic acid 19f

[0291] 19e (70 mg, 83.7 μmol) was dissolved in 2.5 mL of a 3% (v / v) dichloroacetic acid solution in dichloromethane, 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, and 5 mL of ether was added. The mixture was allowed to warm to room temperature and then beaten to precipitate a white solid. The solid was filtered, and the filter cake was collected and dried in an oil pump to give the title product 19f (57 mg, yield: 99.2%).

[0292] Step 7 (9H-Fluoren-9-yl)methyl ((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)carbamate 19g

[0293] 1b (30 mg, 63.6 μmol) was placed in a reaction flask, 1 mL of a 10% (v / v) methanol / dichloromethane solution was added, and the mixture was purged with argon gas three times. The mixture was cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added dropwise. 19f (57 mg, 83.1 μmol) was dissolved in 1 mL of a 10% (v / v) methanol / dichloromethane solution and added dropwise to the reaction mixture. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (26 mg, 93.9 μmol) was then added. The mixture was warmed to room temperature and stirred for 1 hour. 10 mL of dichloromethane and 5 mL of water were added, stirred for 5 minutes, and allowed to separate. The organic phase was collected, and the aqueous phase was extracted with dichloromethane (10 mL x 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 resulting residue was purified by thin-layer chromatography using developing solvent system B to obtain the title product 19g (56 mg, yield: 79.8%). MS m / z (ESI): 1103.1[M+1].

[0294] Step 8 (S)-2-(2-(2-aminoacetylamino)acetylamino)-N-(2-((((S)-1-cyclopropyl-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide 19h

[0295] 19g (4.6 mg, 4.16 μmol) was dissolved in 1.5 mL of dichloromethane, 0.75 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.6 hours. The reaction mixture was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure twice. 3 mL of n-hexane was added and triturated, and the upper layer of n-hexane was poured off three times. The mixture was then concentrated under reduced pressure to give the crude product 19h (4.0 mg), which was used directly in the next step without further purification.

[0296] Step 9 (1r,4r)-N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]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-pentaazatetrahexadecan-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 19

[0297] Crude product 19h (4.0 mg) was dissolved in 1 mL of N,N-dimethylformamide, and 17g (2.9 mg, 4.2 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.5 mg, 5.4 μmol) were added. The mixture was stirred at room temperature for 40 min. The reaction mixture was filtered and purified by high-performance liquid chromatography (HPLC) (column: XBridge Prep C18 OBD 5 μm 19 × 250 mm, mobile phase: A - water (10 mmol NHOAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding components were collected and concentrated under reduced pressure to give the title product 19 (2.1 mg, yield: 32.4%). 1H NMR (400 MHz, DMSO-d6): δ 8.71-8.62 (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, 2H), 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), 2.24-2.12 (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).

[0298] Examples 1-20 (Reference Examples) [ka] The title compound 20 was synthesized by the method provided in Example 58 on page 163 of the specification of patent CN104755494A.

[0299] The following antibodies may be prepared by conventional antibody methods, for example, by constructing a vector, transfecting eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019), expressing the antibody, and purifying it.

[0300] Below is the sequence of Trastuzumab. Light chain [ka] heavy chain [ka]

[0301] Below is the sequence of Pertuzumab. Light chain [ka] heavy chain [ka]

[0302] Below is the sequence of the B7H3 antibody 1F9DS. Light chain [ka] heavy chain [ka]

[0303] Example 1-21 ADC-1 [ka]

[0304] At 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to an aqueous PBS buffer solution of the antibody trastuzumab (2.5 mL of 0.05 M PBS buffer solution at pH 6.5, 9.96 mg / mL, 0.168 μmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours. The reaction was stopped, and the reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg / mL. 2.0 mL of the solution was removed and allowed to continue the reaction.

[0305] Compound 10—a compound with a relatively short retention time (2.1 mg, 2.02 μmol)—was dissolved in 0.10 mL of DMSO, added to the 2.0 mL of the above solution, placed in a water bath shaker, and reacted with shaking at 25° C. for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-1 (5.0 mg / mL, 1.1 mL) in PBS buffer, which is an example of the general formula of FADC-1, and stored at 4° C.

[0306] The mean value was calculated by UV-HPLC: n=5.09.

[0307] Example 1-22 ADC-2 [ka]

[0308] At 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to an aqueous PBS buffer solution of the antibody trastuzumab (2.5 mL of 0.05 M PBS buffer solution at pH 6.5, 9.96 mg / mL, 0.168 μmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours. The reaction was stopped, and the reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg / mL. 2.0 mL of the solution was removed and allowed to continue the reaction.

[0309] Compound 10—a compound with a relatively long retention time (2.1 mg, 2.02 μmol)—was dissolved in 0.10 mL of DMSO, added to the 2.0 mL of the above solution, placed in a water bath shaker, and reacted with shaking at 25° C. for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-2 (4.95 mg / mL, 1.1 mL) in PBS buffer, which is an example of the general formula of FADC-1, and stored at 4° C.

[0310] The mean value was calculated by UV-HPLC: n=7.39.

[0311] Example 1-23 ADC-3 [ka]

[0312] At 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to an aqueous PBS buffer solution of the antibody trastuzumab (2.5 mL of 0.05 M PBS buffer solution at pH 6.5, 9.96 mg / mL, 0.168 μmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours. The reaction was stopped, and the reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg / mL. 2.0 mL of the solution was removed and allowed to continue the reaction.

[0313] Compound 8 (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO and added to the above 2.0 mL of solution. The mixture was placed in a water bath shaker and reacted with shaking at 25° C. for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution with a pH of 6.5, containing 0.001 M EDTA) to obtain the product ADC-3 (5.24 mg / mL, 1.1 mL) in PBS buffer, which is an example of the general formula of FADC-3, and stored at 4° C.

[0314] The mean value was calculated by UV-HPLC: n=7.36.

[0315] Example 1-24 ADC-4 [ka]

[0316] At 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to an aqueous PBS buffer solution of the antibody trastuzumab (0.05 M PBS buffer solution, pH 6.5, 3.74 mL, 13.38 mg / mL, 0.338 μmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours. The reaction was stopped, and the reaction solution was cooled to 25°C in a water bath and diluted to 6.7 mg / mL. 1.3 mL of the solution was removed and allowed to continue the reaction.

[0317] Compound 9 - Relatively Short Retention Time Compound 9-A (1.0 mg, 0.93 μmol) was dissolved in 0.10 mL of DMSO and added to the above 1.3 mL solution. The solution was placed in a water bath shaker and reacted with shaking at 25 ° C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution with a pH of 6.5, containing 0.001 M EDTA) to obtain the exemplary product ADC-4 of the general formula FADC-4A in PBS buffer (1.72 mg / mL, 2.36 mL), which was then stored at 4 ° C.

[0318] The mean value was calculated by UV-HPLC: n=7.39.

[0319] Example 1-25 ADC-5 [ka]

[0320] At 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.067 mL, 0.67 μmol) was added to an aqueous PBS buffer solution of the antibody trastuzumab (0.05 M PBS buffer solution, pH 6.5, 3.0 mL, 6.70 mg / mL, 0.136 μmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours. The reaction was then stopped, and the reaction solution was cooled to 25°C in a water bath. 0.614 mL of the solution was removed and allowed to continue the reaction.

[0321] Compound 9 - Relatively Short Retention Time Compound 9-A (0.5 mg, 0.42 μmol) was dissolved in 0.031 mL of DMSO and added to the above 0.614 mL solution. The solution was placed in a water bath shaker and shaken at 25 ° C for 3 hours to stop the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-5 (3.08 mg / mL, 0.82 mL) in PBS buffer, which is an example of the general formula of FADC-4A, and stored at 4 ° C.

[0322] The mean value was calculated by UV-HPLC: n=3.16.

[0323] Example 1-26 ADC-6 [ka]

[0324] At 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to an aqueous PBS buffer solution of the antibody trastuzumab (0.05 M PBS buffer solution, pH 6.5, 3.74 mL, 13.38 mg / mL, 0.338 μmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours. The reaction was stopped, and the reaction solution was cooled to 25°C in a water bath and diluted to 6.7 mg / mL. 0.75 mL of the solution was removed and allowed to continue the reaction.

[0325] Compound 9 - Relatively Long Retention Time Compound 9-B (0.68 mg, 0.63 μmol) was dissolved in 0.10 mL of DMSO and added to the above 0.75 mL solution. The solution was placed in a water bath shaker and shaken at 25 ° C for 3 hours to stop the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the 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.

[0326] The mean value was calculated by UV-HPLC: n=3.94.

[0327] Example 1-27 ADC-7 [ka]

[0328] At 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to an aqueous PBS buffer solution of the antibody pertuzumab (0.05 M PBS buffer solution with a pH of 6.5, 5.0 mL, 10 mg / mL, 0.338 μmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours. The reaction was stopped, and the reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg / mL. 1.0 mL of the solution was removed and allowed to continue the reaction.

[0329] Compound 8 (0.65 mg, 0.6 μmol) was dissolved in 0.1 mL of DMSO and added to the above 1.0 mL of solution. The mixture was placed in a water bath shaker and reacted with shaking at 25° C. for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution with a pH of 6.5, containing 0.001 M EDTA) to obtain the product ADC-7 (1.42 mg / mL, 2.15 mL) in PBS buffer, which is an example of the general formula of FADC-7, and stored at 4° C.

[0330] The mean value was calculated by UV-HPLC: n=6.91.

[0331] Example 1-28 ADC-8 [ka]

[0332] At 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to an aqueous PBS buffer solution of the antibody pertuzumab (0.05 M PBS buffer solution with a pH of 6.5, 5.0 mL, 10 mg / mL, 0.338 μmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours. The reaction was stopped, and the reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg / mL. 1.6 mL of the solution was removed and allowed to continue the reaction.

[0333] Compound 10 (a compound with a relatively short retention time) (1.04 mg, 1.0 μmol) was dissolved in 0.1 mL of DMSO and added to the above 1.6 mL solution. The solution was placed in a water bath shaker and reacted with shaking at 25° C. for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution with a pH of 6.5, containing 0.001 M EDTA) to obtain the product ADC-8 (2.14 mg / mL, 2.31 mL) in PBS buffer, which is an example of the general formula of FADC-8, and stored at 4° C.

[0334] The mean value was calculated by UV-HPLC: n=6.58.

[0335] Example 1-29 ADC-9 [ka]

[0336] At 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to an aqueous PBS buffer solution of the antibody pertuzumab (0.05 M PBS buffer solution with a pH of 6.5, 5.0 mL, 10 mg / mL, 0.338 μmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours. After stopping the reaction, the reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg / mL. 0.8 mL of the solution was removed and allowed to continue the reaction.

[0337] Compound 9 - Relatively Short Retention Time Compound 9-A (0.55 mg, 0.5 μmol) was dissolved in 0.1 mL of DMSO and added to the above 0.8 mL solution. The solution was placed in a water bath shaker and shaken at 25 ° C for 3 hours to stop the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the exemplary product ADC-9 of the general formula FADC-9A in PBS buffer (2.27 mg / mL, 1.11 mL), which was stored at 4 ° C.

[0338] The mean value was calculated by UV-HPLC: n=3.16.

[0339] Example 1-30 ADC-10 [ka]

[0340] At 37°C, an aqueous solution of the antibody trastuzumab (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 0.574 mL, 38.78 nmol) was mixed with an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 19.76 μL, 197.6 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0341] Compound 14 (0.64 mg, 588 nmol), a compound with a relatively short retention time, was dissolved in 40 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours, after which the reaction was stopped. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-10 (5.48 mg / mL, 1.03 mL) in PBS buffer, which is an example of the general formula of FADC-10. The product was stored at 4° C.

[0342] The mean value was calculated by UV-Vis: n = 6.25.

[0343] Example 1-31 ADC-11 [ka]

[0344] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 0.646 mL, 43.64 nmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 22.24 μL, 222.4 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0345] Compound 14 (a compound with a relatively long retention time) (0.72 mg, 662 nmol) was dissolved in 40 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours, after which the reaction was stopped. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain ADC-11 (2.13 mg / mL, 1.87 mL) as an example of the general formula of FADC-10 in PBS buffer. The product was stored at 4° C.

[0346] The mean value was calculated by UV-Vis: n=7.03.

[0347] Example 1-32 ADC-12 [ka]

[0348] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 0.726 mL, 49.05 nmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.0 μL, 250.0 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0349] Compound 15 (0.81 mg, 754 nmol) was dissolved in 40 μL of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-12 (3.34 mg / mL, 1.45 mL) in PBS buffer, which is an example of the general formula of FADC-12, and stored at 4° C.

[0350] The mean value was calculated by UV-Vis: n=6.93.

[0351] Example 1-33 ADC-13 [ka]

[0352] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 0.287 mL, 19.39 nmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 9.88 μL, 98.8 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0353] Compound 16 (0.32 mg, 294 nmol) was dissolved in 20 μL of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-13 (2.37 mg / mL, 0.88 mL) in PBS buffer, which is an example of the general formula of FADC-13, and stored at 4° C.

[0354] The mean value was calculated by UV-Vis: n=6.53.

[0355] Example 1-34 ADC-14 [ka]

[0356] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 0.592 mL, 40.0 nmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0357] Compound 17 (0.92 mg, 598 nmol) was dissolved in 40 μL of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-14 (0.30 mg / mL, 12.0 mL) in PBS buffer, which is an example of the general formula of FADC-14, and stored at 4° C.

[0358] The mean value was calculated by UV-Vis: n = 7.61.

[0359] Example 1-35 ADC-15 [ka]

[0360] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 0.592 mL, 40.0 nmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0361] Compound 18 (0.93 mg, 599 nmol) was dissolved in 40 μL of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-15 (0.32 mg / mL, 11.8 mL) in PBS buffer, which is an example of the general formula of FADC-15, and stored at 4° C.

[0362] The mean value was calculated by UV-Vis: n = 7.89.

[0363] Example 1-36 ADC-16 [ka]

[0364] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 0.53 mL, 35.8 nmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 18.25 μL, 182.5 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0365] Compound 19 (0.83 mg, 534 nmol) was dissolved in 35 μL of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-16 (0.32 mg / mL, 12.0 mL) in PBS buffer, which is an example of the general formula of FADC-16, and stored at 4° C.

[0366] The mean value was calculated by UV-Vis: n = 7.43.

[0367] Example 1-37 ADC-17 [ka]

[0368] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 2.0 mL, 135.12 nmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 43.2 μL, 432 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0369] Compound 9 - Relatively short retention time Compound 9-A (2.22 mg, 2067 nmol) was dissolved in 175 μL of DMSO and added to the reaction solution. The solution was placed in a water bath shaker and shaken at 25 ° C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-17 (1.32 mg / mL, 12.0 mL) in PBS buffer, which is an example of the general formula of FADC-4A, and stored at 4 ° C.

[0370] The mean value was calculated by UV-Vis: n = 5.42.

[0371] Example 1-38 ADC-18 (Reference Example) [ka]

[0372] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 1.5 mL, 101.3 nmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0373] Compound 20 (2.0 mg, 1934 nmol) was dissolved in 100 μL of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-18 (0.79 mg / mL, 13.0 mL) in PBS buffer, which is an example of the general formula of FADC-18, and stored at 4° C.

[0374] The mean value was calculated by UV-Vis: n = 7.23.

[0375] Example 1-39 ADC-19 [ka]

[0376] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 1.36 mL, 91.9 nmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 46.9 μL, 469 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0377] Compound 9 - Relatively short retention time Compound 9-A (2.0 mg, 1862 nmol) was dissolved in 100 μL of DMSO and added to the reaction solution. The reaction was then placed in a water bath shaker and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-19 (0.73 mg / mL, 13.0 mL) in PBS buffer, which is an example of the general formula of FADC-4A, and stored at 4 °C.

[0378] The mean value was calculated by UV-Vis: n = 6.26.

[0379] Example 1-40 ADC-20 [ka]

[0380] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 1.5 mL, 101.3 nmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0381] Compound 10 (a compound with a relatively long retention time) (2.0 mg, 1815 nmol) was dissolved in 100 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours, after which the reaction was stopped. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain ADC-20 (0.73 mg / mL, 13.0 mL) as an example of the general formula of FADC-1 in PBS buffer, which was then stored at 4° C.

[0382] The mean value was calculated by UV-Vis: n = 7.43.

[0383] Example 1-41 ADC-21 (Reference Example) [ka]

[0384] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 1.86 mL, 125.4 nmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 63.9 μL, 639 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0385] Compound 20 (2.07 mg, 2001 nmol) was dissolved in 150 μL of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain ADC-21 (2.91 mg / mL, 4.44 mL) as an exemplary product of the general formula of FADC-18 in PBS buffer, which was stored at 4° C.

[0386] The mean value was calculated by UV-Vis: n = 7.23.

[0387] Example 1-42 ADC-22 [ka]

[0388] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 1.88 mL, 127.2 nmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 64.9 μL, 649 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0389] Compound 9 - Relatively short retention time Compound 9-A (2.1 mg, 1955 nmol) was dissolved in 150 μL of DMSO and added to the reaction solution. The reaction was then placed in a water bath shaker and shaken at 25 ° C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-22 (3.56 mg / mL, 3.98 mL) in PBS buffer, which is an example of the general formula of FADC-4A, and stored at 4 ° C.

[0390] The mean value was calculated by UV-Vis: n = 6.79.

[0391] Example 1-43 ADC-23 (Reference Example) [ka]

[0392] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 345 mL, 23.31 μmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.89 mL, 118.9 μmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3.5 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0393] Compound 20 (362 mg, 350 μmol) was dissolved in 7.12 mL of MeCN and 3.56 mL of DMSO, added to the reaction solution, and placed on a water bath shaker for 3 hours at 25 ° C. The reaction was then stopped by shaking. The reaction solution was passed through an ultrafiltration membrane bag and desalted and purified sequentially with PBS buffer solution (0.05 M PBS buffer solution, pH 6.5) containing 2% (v / v) MeCN and 1% (v / v) DMSO, and then with succinic acid buffer solution (0.01 M succinic acid buffer solution, pH 5.3). Sucrose was added to 60 mg / mL and Tween 20 to 0.2 mg / mL, and the mixture was placed in a flask and lyophilized to obtain a lyophilized powder sample of ADC-23, an exemplary product of the general formula of FADC-18, which was then stored at 4 ° C.

[0394] The mean value was calculated by UV-Vis: n=7.05.

[0395] Example 1-44 ADC-24 [ka]

[0396] At 37°C, an aqueous solution of the antibody trastuzumab in PBS buffer (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 332 mL, 22.43 μmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.44 mL, 114.4 μmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3.5 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0397] Compound 9 - Relatively Short Retention Time Compound 9-A (241 mg, 224 μmol) was dissolved in 13.76 mL of MeCN and 6.88 mL of DMSO, added to the reaction solution, placed in a water bath shaker, and shaken at 25 ° C for 3 hours, after which the reaction was stopped. The reaction solution was passed through an ultrafiltration membrane bag and desalted and purified sequentially with PBS buffer solution (0.05 M PBS buffer solution, pH = 6.5) containing 4% (v / v) MeCN and 2% (v / v) DMSO, and then succinic acid buffer solution (0.01 M succinic acid buffer solution, pH = 5.3). Sucrose was added to 60 mg / mL and Tween 20 to 0.2 mg / mL, and the mixture was placed in a flask and lyophilized to obtain a lyophilized powder sample of ADC-24, an exemplary product of the general formula FADC-4A, which was then stored at 4 ° C.

[0398] The mean value was calculated by UV-Vis: n=7.07.

[0399] Example 1-45 ADC-25 [ka]

[0400] At 37°C, an aqueous solution of antibody B7H3 and antibody 1F9DS in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 2.14 mL, 144.60 nmol) was added to a solution of tris(2-carboxyethyl)phosphine (TCEP) in water (10 mM, 73.7 μL, 740 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to terminate the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0401] Compound 9 - Relatively short retention time Compound 9-A (3.0 mg, 2793 nmol) was dissolved in 150 μL of DMSO and added to the reaction solution. The reaction was then placed in a water bath shaker and shaken at 25 ° C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-25 (1.28 mg / mL, 13.0 mL) in PBS buffer, which is an example of the general formula of FADC-25, and stored at 4 ° C.

[0402] The mean value was calculated by UV-Vis: n = 6.87.

[0403] Example 1-46 ADC-26 (Reference Example) [ka]

[0404] At 37°C, an aqueous solution of antibody B7H3 and antibody 1F9DS in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 0.89 mL, 60.14 nmol) was added to a solution of tris(2-carboxyethyl)phosphine (TCEP) in water (10 mM, 30.1 μL, 300 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to terminate the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0405] Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μL of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-26 (1.61 mg / mL, 4.0 mL) in PBS buffer, which is an example of the general formula of FADC-26, and stored at 4° C.

[0406] The mean value was calculated by UV-Vis: n = 6.15.

[0407] Example 1-47 ADC-27 [ka]

[0408] At 37°C, an aqueous solution of antibody B7H3 and antibody 1F9DS in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 0.89 mL, 60.14 nmol) was added to a solution of tris(2-carboxyethyl)phosphine (TCEP) in water (10 mM, 30.1 μL, 300 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to terminate the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0409] Compound 9 - Relatively short retention time Compound 9-A (1.02 mg, 950 nmol) was dissolved in 100 μL of DMSO and added to the reaction solution. The solution was placed in a water bath shaker and shaken at 25 ° C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-27 (1.94 mg / mL, 3.5 mL) in PBS buffer, which is an example of the general formula of FADC-25, and stored at 4 ° C.

[0410] The mean value was calculated by UV-Vis: n = 6.11.

[0411] Example 1-48 ADC-28 (Reference Example) [ka]

[0412] At 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 81.3 μL, 810 nmol) was added to an aqueous PBS buffer solution of antibody B7H3 and antibody 1F9DS (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 2.36 mL, 159.47 nmol), and the mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to terminate the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0413] Compound 20 (3.0 mg, 2901 nmol) was dissolved in 150 μL of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain ADC-28 (1.29 mg / mL, 13.0 mL) as an exemplary product of the general formula of FADC-26 in PBS buffer, which was then stored at 4° C.

[0414] The mean value was calculated by UV-Vis: n = 7.46.

[0415] Example 1-49 ADC-29 [ka]

[0416] At 37°C, an aqueous solution of antibody B7H3 and antibody 1F9DS in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 0.80 mL, 50.06 nmol) was added to a solution of tris(2-carboxyethyl)phosphine (TCEP) in water (10 mM, 28.6 μL, 290 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to terminate the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0417] Compound 9 - Relatively short retention time Compound 9-A (1.29 mg, 1201 nmol) was dissolved in 100 μL of DMSO and added to the reaction solution. The reaction was then placed in a water bath shaker and shaken at 25 ° C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-29 (2.63 mg / mL, 2.4 mL) in PBS buffer, which is an example of the general formula of FADC-25, and stored at 4 ° C.

[0418] The mean value was calculated by UV-Vis: n = 7.24.

[0419] Example 1-50 ADC-30 (Reference Example) [ka]

[0420] At 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 29.1 μL, 290 nmol) was added to an aqueous PBS buffer solution of antibody B7H3 and antibody 1F9DS (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 0.86 mL, 58.4 nmol), and the mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to terminate the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0421] Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μL of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain ADC-30 (1.61 mg / mL, 4.0 mL) as an exemplary product of the general formula of FADC-26 in PBS buffer, which was stored at 4° C.

[0422] The mean value was calculated by UV-Vis: n = 6.15.

[0423] Example 1-51 ADC-31 [ka]

[0424] At 37°C, an aqueous solution of antibody B7H3 and antibody 1F9DS in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 0.89 mL, 60.14 nmol) was added to a solution of tris(2-carboxyethyl)phosphine (TCEP) in water (10 mM, 30.1 μL, 300 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to terminate the reaction. The reaction solution was then cooled to 25°C in a water bath.

[0425] Compound 8 (1.0 mg, 943 nmol) was dissolved in 100 μL of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and shaken at 25° C. for 3 hours to terminate the reaction. The reaction solution was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the product ADC-31 (1.47 mg / mL, 4.5 mL) in PBS buffer, which is an example of the general formula of FADC-31, and stored at 4° C.

[0426] The mean value was calculated by UV-Vis: n=6.33.

[0427] Analysis of drug loading in ADC stock solutions Experimental objectives and principles ADC stock solution is a type of antibody crosslinker drug, and its mechanism of treatment is to deliver toxin molecules to cells through the targeting of antibodies, which then kills the cells. The drug loading plays a crucial role in the efficacy of the drug. The drug loading of ADC stock solution was measured using an ultraviolet method.

[0428] Experimental Method The cuvettes containing the sodium succinate buffer solution were placed in the reference absorption cell and the sample measurement absorption cell, respectively, and after subtracting the solvent blank, the cuvette containing the sample solution was placed in the sample measurement absorption cell, and the absorbance at 280 nm and 370 nm was measured.

[0429] Calculation of results: The loading amount of the ADC stock solution was measured by ultraviolet spectrophotometry (instrument used: Thermo nanodrop 2000 ultraviolet spectrophotometer). The principle is that the total absorbance value of the ADC stock solution at a certain wavelength is equal to the sum of the absorbance values ​​of the cytotoxic drug and the monoclonal antibody at the same wavelength, i.e. (1) A 280nm =ε mab-280 bC mab +ε Drug-280 bC Drug ε Drug-280 : the drug has an average molar extinction coefficient of 5100 at 280 nm; C Drug : drug concentration, ε mab-280 The average molar extinction coefficient at 280 nm of the trastuzumab stock solution or the pertuzumab stock solution is 214600, C mab : Concentration of trastuzumab stock solution or pertuzumab stock solution, b: The optical path length is 1 cm.

[0430] Similarly, the total absorbance equation for the sample at 370 nm can be obtained, i.e., (2) A 370nm =ε mab-370 bC mab +ε Drug-370 bC Drug ε Drug-370 : the drug has an average molar extinction coefficient of 19000 at 370 nm; C Drug : drug concentration, ε mab-370 The extinction coefficient at 370 nm of the trastuzumab stock solution or the pertuzumab stock solution is 0, C mab : Concentration of trastuzumab stock solution, b: The optical path length is 1 cm.

[0431] The drug loading can be calculated using the two equations (1) and (2) in combination with the extinction coefficients and concentration data of the monoclonal antibody and drug at the two detection wavelengths. Drug loading = C Drug / C mab .

[0432] Biological evaluation Test Example 1-1: In vitro growth inhibition test of tumor cells by compounds of the present disclosure

[0433] 1. Purpose of the test This experiment aims to detect the in vitro growth inhibitory activity of the drug compounds disclosed herein on U87MG cells (Cell Bank of the Chinese Academy of Sciences, Catalog # TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, Product No. HTB-30). After treating the cells with different concentrations of the compounds in vitro and culturing for 6 days, the cells were analyzed by CTG (CellTiter-Glo). (R) The cell proliferation was detected using a Luminescent Cell Viability Assay (Promega, product number: G7573) reagent, and the in vitro activity of the compounds was evaluated based on IC50 values.

[0434] 2. Experimental Method Hereinafter, the method for testing the in vitro growth inhibitory activity of the compounds of the present disclosure on tumor cells will be described by taking the in vitro growth inhibitory activity test method on U87MG cells as an example. This method can be similarly applied to, but is not limited to, the in vitro growth inhibitory activity test on other tumor cells. 1. Cell culture: U87MG and SK-BR-3 cells were cultured in EMEM medium containing 10% FBS (GE, product number SH30024.01) and McCoy's 5A medium containing 10% FBS (Gibco, product number 16600-108), respectively. 2. Cell preparation: Logarithmic growth phase U87MG and SK-BR-3 cells were collected and washed once with PBS (phosphate buffer solution, Shanghai Yuanpei Biotechnology Co., Ltd.). After that, 2-3 mL of trypsin (0.25% Trypsin-EDTA (1x), Gibico, Life Technologies) was added and digested for 2-3 minutes. After the cells were completely digested, 10-15 mL of cell culture medium was added to elute the digested cells. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and 10-20 mL of cell culture medium was added to resuspend the cells to prepare a single-cell suspension. 3. Seeding cells onto plates: Single cell suspensions of U87MG and SK-BR-3 were mixed uniformly and cultured in cell culture medium until the viable cell density reached 2.75 × 10 3 cells / mL and 8.25 × 10 3 The density-adjusted cell suspension was adjusted to 180 cells / mL, mixed evenly, and placed in a 96-well cell culture plate at 180 μL per well. 200 μL of medium alone was added to the outer wells of the 96-well plate. The culture plate was then incubated in an incubator (37°C, 5% CO2) for 24 hours. 4. Preparation of compounds: The compounds were dissolved in DMSO (dimethyl sulfoxide, Shanghai Taitan Technology Co., Ltd.) to prepare a stock solution with an initial concentration of 10 mM. The initial concentration of the small molecule compound was 500 nM, and the drug preparation method was as follows. 30 μL of each test sample was added to the first column of a 96-well U-bottom formulation plate, resulting in a sample concentration of 100 μM. 20 μL of DMSO was added to each well of columns 2 to 11. 10 μL of the sample from column 1 was added to 20 μL of DMSO in column 2, mixed evenly, and 10 μL was added to column 3, and so on up to column 10. 5 μL of the drug in the formulation plate was added to 95 μL of EMEM medium per well, mixed evenly, and then prepared for use. The initial concentration of ADC was 10 nM or 500 nM, and the drug preparation method was as follows: 100 μL of each test sample was added to the first column of a 96-well plate, with the sample concentration set to 100 nM or 5 μM. 100 μL of PBS was added to each well of columns 2 to 11. 50 μL of the sample from column 1 was added to 100 μL of PBS in column 2, mixed evenly, and 50 μL was added to column 3. In this way, 3-fold dilutions were made up to column 10. 5. Sample addition: 20 μL of samples to be measured at different concentrations were added to the culture plate so that each sample was in two duplicate wells. The culture plate was incubated in an incubator (37°C, 5% CO2) for 6 days. 6. Color development procedure: A 96-well cell culture plate was taken out, and 90 μL of CTG solution was added to each well and incubated at room temperature for 10 minutes. 7. Plate reading procedure: The 96-well cell culture plate was taken out and placed on a microplate reader (BMG labtech, PHERAstar FS), and the chemiluminescence was measured by the microplate reader.

[0435] 3. Data analysis Data was processed and analyzed using Microsoft Excel and Graphpad Prism 5. The experimental results are shown in Table 1 below.

[0436] [Table 2]

[0437] Conclusion: The small molecule fragments in this disclosure have significant growth inhibitory activity against SK-BR-3 and U87 cells, and the chiral center has a certain effect on the inhibitory activity of the compounds.

[0438] Test Example 1-2: In vitro growth inhibition test of HER2-targeted tumor cells by the antibody-drug conjugate of the present disclosure

[0439] This experiment aimed to detect the in vitro growth inhibitory activity of the antibody-drug conjugate of the present disclosure targeting HER2 on SK-BR-3 (human breast cancer cells, ATCC, Product No. HTB-30) and MDA-MB-468 (human breast cancer cells, ATCC, Product No. HTB-132). The cells were treated in vitro with different concentrations of the compound and cultured for 6 days, after which cell proliferation was detected using a CTG reagent and IC 50 Based on these values, the compounds were evaluated for their in vitro activity.

[0440] Using the test method of Test Example 1, the test cells were SK-BR-3 and MDA-MB-468, and the cell culture media were McCoy's 5A medium (Gibco, product number 16600-108) containing 10% FBS, EMEM medium (GE, product number SH30024.01) containing 10% FBS, and L-15 medium (ThermoFisher, product number 11415-114) containing 10% FBS, respectively. Each of the three cell lines was cultured in the cell culture media until the viable cell density reached 8.33 × 10 3 cells / mL, 8.33 x 10 3 cells / mL and 1.39 x 10 4 The density-adjusted cell suspension was adjusted to cells / mL, and the adjusted cell suspension was mixed uniformly and placed in a 96-well cell culture plate at 180 μL / well. Related compounds were tested, and the results are shown in Table 2 below.

[0441] [Table 3]

[0442] Conclusion: The antibody-drug conjugate of the present disclosure targeting HER2 has significant growth inhibitory activity against the HER2-positive cell line SK-BR-3, and weak growth inhibitory activity against the HER2-negative cell line MDA-MB-468, demonstrating good selectivity.

[0443] Test Example 1-3: Plasma stability experiment of Her2-ADC

[0444] ADC-19, ADC-18, ADC-20 samples, human plasma, monkey plasma (Shanghai Metix Biopharmaceutical Co., Ltd.), and 1% BSA (Sigma) in PBS (Shanghai Biotechnology Co., Ltd.) were each sterilized by filtration through a 0.22 μm filter. ADC-19, ADC-18, and ADC-20 were added to the above sterilized plasma or 1% BSA in PBS at a final concentration of 200 μg / mL, respectively, and incubated in a 37°C cell incubator. The day of incubation was designated day 0, and samples were subsequently removed on days 7, 14, and 21 for detection of free toxin.

[0445] A 25 μL sample was taken and placed in a 96-well plate, and 50 μL of the internal standard working solution (100 ng / mL camptothecin acetonitrile solution) and 150 μL of acetonitrile were added. The mixture was vortexed for 5 minutes, centrifuged for 10 minutes (4000 rpm), and 5 μL was analyzed by LC / MS / MS (Applied Biosystems, USA).

[0446] As shown in Figure 1A, ADC-19 was highly stable in both human and monkey plasma and in a 1% BSA PBS solution, with the maximum release rate of free toxin not exceeding 2.1% and tending to stabilize by day 14.

[0447] ADC-18 was less stable in human and monkey plasma, with a maximum release rate of 14.5% and 8.10%, respectively, but was relatively stable in a 1% BSA PBS solution, as shown in Figure 1B.

[0448] ADC-20 was poorly stable in human plasma, monkey plasma, and 1% BSA PBS solution, with the maximum release rates of free toxin being 21.7%, 29.7%, and 21.7%, respectively. Furthermore, as shown in Figure 1C, ADC-20 was always degraded in 1% BSA PBS solution.

[0449] Test Example 1-4: Evaluation of drug efficacy in JIMT-1 tumor-bearing mice

[0450] 1. Purpose of the test Using nunu nude mice as test animals, the therapeutic effects of Her2-ADC antibodies T-DM1, ADC-21, and ADC-24 administered intraperitoneally were evaluated on nude mice bearing tumors transplanted with the trastuzumab-resistant human breast cancer cell line JIMT-1 (Herceptin).

[0451] 2. Test drug and materials 2-1. Test drug T-DM1 (prepared with reference to US20050169933) ADC-21: 3 mg / kg ADC-21: 10 mg / kg ADC-24: 3 mg / kg ADC-24: 10 mg / kg Blank control (Blank): PBS 2-2. Preparation method: All were prepared by diluting with PBS. 2-3. Test animals Nunu nude mice purchased from Beijing Weitong Lihua.

[0452] 3. Test method JIMT-1 cells (Nanjing Kebai) (5 × 10 6 1000 x 1000 cells / mouse with 50% Matrigel) and allowed to grow for 8 days, resulting in tumors measuring 203.09 ± 11.94 mm 3 After reaching maturity, the animals were randomly divided into six groups (8 animals per group) (d1). The mice were administered intraperitoneally twice in total. Tumor volume and body weight were measured twice a week and the data were recorded. Data statistics were calculated using Excel 2003 statistical software, with the average value calculated as avg, the SD value calculated as STDEV, the SEM value calculated as STDEV / SQRT, and the P value for intergroup differences calculated as TTEST. Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 2 Relative volume (RTV) = V T / V0 Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%) Among them, V0, V T are the tumor volumes at the start and end of the experiment, respectively. RTV , T RTV are the relative tumor volumes of the blank control group (Vehicle, PBS) and the experimental group at the end of the experiment, respectively.

[0453] 4. Test results The experimental results are shown in Figure 2. Two intraperitoneal injections were administered, and the experiment was terminated on day 34. T-DM1 (10 mg / kg) had no tumor inhibitory effect. ADC-21 inhibited tumor growth at 3 mg / kg (46.22%; P<0.01) and 10 mg / kg (56.77%; P<0.001). ADC-24 inhibited tumor growth at 3 mg / kg (62.77%; P<0.001) and 10 mg / kg (76.32%; P<0.001). At comparable doses, the tumor inhibitory effect of ADC-24 was significantly greater than that of ADC-21.

[0454] Test Example 1-5: Evaluation of drug efficacy in SK-BR-3 tumor-bearing mice

[0455] 1. Purpose of the test Using nunu nude mice as test animals, the therapeutic effects of Her2-ADC antibodies ADC-21 and ADC-22 on nude mice bearing human breast cancer cell SK-BR-3 tumors after intraperitoneal administration were evaluated.

[0456] 2. Test drug and materials 2-1. Test drug ADC-21: 1 mg / kg ADC-21: 6 mg / kg ADC-22: 1 mg / kg ADC-22: 6 mg / kg Blank control (Blank): PBS. 2-2. Preparation method: All were prepared by diluting with PBS. 2-3. Test animals Nunu nude mice purchased from Beijing Weitong Lihua.

[0457] 3. Test method SK-BR-3 cells (ATCC) (5 × 10 6 1000 x g / mouse with 50% Matrigel) and allowed to grow tumors for 20 days, reaching a size of 153.34 ± 11.73 mm 3 After reaching maturity, the animals were randomly divided into 5 groups, each with 8 animals (d0). The mice were administered a single intraperitoneal injection. Tumor volume and body weight were measured twice a week and the data were recorded. Data statistics were calculated using Excel 2003 statistical software, with the average value calculated as avg, the SD value calculated as STDEV, the SEM value calculated as STDEV / SQRT, and the P value for intergroup differences calculated as TTEST. Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 2 Relative volume (RTV) = V T / V0 Tumor inhibition rate (%)=(C RTV -T RTV ) / C RTV (%) Among them, V0, V T are the tumor volumes at the start and end of the experiment, respectively. RTV , T RTV are the relative tumor volumes of the blank control and experimental groups at the end of the experiment, respectively.

[0458] 4. Test results The experimental results are shown in Figure 3. ADC-21 was administered intraperitoneally once, and observation was completed on day 28. ADC-21 inhibited tumor growth by 15.01% at 1 mg / kg and 77.4% at 6 mg / kg, demonstrating significant differences compared to the blank control (P<0.001). ADC-22 inhibited tumor growth by 19.82% at 1 mg / kg and 98.38% at 6 mg / kg (P<0.001). At the same dose of 6 mg / kg, the tumor-inhibiting effect of ADC-22 was significantly better than that of ADC-21.

[0459] Test Example 1-6: Plasma stability

[0460] Sample ADC-25 was mixed uniformly with human plasma, monkey plasma, and 1% BSA PBS solution at a final concentration of 100 μg / mL, filtered to sterilize, and then incubated in a water bath at 37°C. The day of incubation was recorded as day 0, and samples were then taken out on days 7, 14, and 21, respectively, to detect free toxin.

[0461] After removing the samples at different time points, they were left at room temperature and mixed uniformly by vortexing. 25 μL of the sample was taken and placed in a 96-well plate. 50 μL of the internal standard working solution (100 ng / mL camptothecin acetonitrile solution) and 150 μL of acetonitrile were added, vortexed for 5 minutes, centrifuged for 10 minutes (4000 rpm), and 5 μL of the supernatant was taken and analyzed by LC / MS / MS.

[0462] As a result, as shown in Figure 4, ADC-25 was very stable in both human and monkey plasma and in 1% BSA PBS solution, with the maximum release rate of free toxin not exceeding 2% and tending to stabilize on day 14.

[0463] Test Example 1-7: Evaluation of the therapeutic effect of ADC on human brain astrocytoma U87MG tumors transplanted into nude mice

[0464] 1. Purpose of the test In this experiment, the therapeutic effect of the ADC compound of the present disclosure on human brain glioblastoma U87MG tumors xenografted in nude mice was evaluated using BALB / cA-nude mice as test animals.

[0465] 2. Test drug and materials 2-1. Test drug ADC-27 (3 mg / kg) ADC-26 (3 mg / kg) Blank control (Blank): PBS buffer at pH 7.4. 2-2. Preparation method: PBS buffer solution, pH 7.4. 2-3. Test animals BALB / cA-nude mice: purchased from Shanghai Jie Si Jie Laboratory Animal Co., Ltd.

[0466] 3. Test method Six- to seven-week-old female BALB / cA nude mice were subcutaneously inoculated with human brain astrocytic glioblastoma U87MG cells (human brain astrocytic glioblastoma, Cell Bank of the Chinese Academy of Sciences, Catalog # TCHu138). Ten days after cell inoculation, animals were randomly assigned to groups of eight (D0) and began receiving intraperitoneal injections once a week for a total of three injections. Tumor volume and body weight were measured two to three times weekly and recorded. The tumor volume (V) was calculated as follows: V=1 / 2×a×b 2 In these, a and b indicate the length and width, respectively. Relative volume (RTV) = V T / V0 Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%) Among them, V0, V T are the tumor volumes at the start and end of the experiment, respectively. RTV , T RTV are the relative tumor volumes of the control group (blank) and the experimental group at the end of the experiment, respectively.

[0467] 4. Test results The tumors were administered intraperitoneally (ip) once a week for a total of three doses. After observation for 22 days, the tumor inhibition rate of ADC-27 at 3 mg / kg reached 63.3% (P<0.0001), and the tumor inhibition rate of ADC-26 at 3 mg / kg reached 49.1%. ADC-27 demonstrated a stronger antitumor effect than ADC-26. During administration, the body weights of the animals in each group were normal, indicating that the ADC had no obvious toxicity or side effects. The detection results are shown in Table 3 and Figure 5. The detected antibody was able to effectively inhibit the growth of U87MG xenograft tumors in tumor-bearing nude mice, and demonstrated dose-dependence.

[0468] [Table 4]

[0469] Test Example 1-8: Evaluation of the therapeutic effect of ADC on Detroit 562 human pharyngeal carcinoma pleural effusion metastasis tumors transplanted into nude mice

[0470] 1. Purpose of the test In this experiment, the therapeutic effect of the ADC compound of the present disclosure on tumors transplanted into nude mice, which are human laryngeal carcinoma pleural effusion metastatic cells, Detroit 562, was evaluated using BALB / cA-nude nude mice as test animals.

[0471] 2. Test drug and materials 2-1. Test drug ADC-29 (3mg / kg) ADC-28 (3 mg / kg) Negative control ADC (3 mg / kg): An antibody-drug conjugate formed by conjugating a non-B7H3-targeting antibody with compound 20. 2-2. Preparation method: All were prepared by diluting with PBS. 2-3. Test animals BALB / cA-nude mice: purchased from Changzhou Kawensi Laboratory Animal Co., Ltd.

[0472] 3. Test method Six to seven week old female BALB / cA nude mice were transfected with Detroit 562 cells (ATCC, Catalog #ATCC) containing human laryngeal carcinoma pleural effusion metastasis cells. (R) CCL-138 TM ) was subcutaneously inoculated. 10 days after cell inoculation, the animals were randomly divided into groups of 8 animals per group (D0), and began receiving intraperitoneal injections once a week for a total of three doses. Tumor volume and body weight were measured two to three times weekly and the data were recorded. The formula for calculating tumor volume (V) is as follows: V=1 / 2×a×b 2 In these, a and b indicate the length and width, respectively. Relative volume (RTV) = V T / V0 Tumor inhibition rate (%)=(C RTV -T RTV ) / C RTV (%) Among them, V0, V T are the tumor volumes at the start and end of the experiment, respectively. RTV , T RTV are the relative tumor volumes of the control group (negative control) and the experimental group at the end of the experiment, respectively.

[0473] 4. Test results The tumor inhibition rates for the tested ADCs were 72.27% (P<0.001) at 3 mg / kg (3 mpk) for ADC-29 and 56.2% (P<0.001) at 3 mg / kg (3 mpk) for ADC-28. Both ADC-29 and ADC-28 demonstrated stronger antitumor effects than ADC-28. During administration, the body weights of the animals in each group were normal, indicating that the ADC had no obvious toxicity or side effects. The detection results are shown in Table 4 and Figure 6. The detected antibody was able to effectively inhibit the growth of Detroit 562 transplanted tumors in tumor-bearing nude mice, and demonstrated dose-dependence.

[0474] [Table 5]

[0475] Test Example 1-9: Evaluation of drug efficacy in U87-MG tumor-bearing mice

[0476] 1. Purpose of the test Balb / c nude mice were used as test animals, and the therapeutic effect of B7H3-antibody drug conjugates was evaluated after intraperitoneal injection in a tumor model implanted with human glioma cells U87MG.

[0477] 2. Test drug and materials 2-1. Test drug ADC-30 1 mg / kg ADC-30 3 mg / kg ADC-31 1 mg / kg ADC-31 3 mg / kg Blank control (Blank): PBS 2-2. Preparation method: All were prepared by diluting with PBS. 2-3. Test animals BALB / cA-nude mice: purchased from Shanghai Slice Laboratory Animal Co., Ltd.

[0478] 3. Test method U87MG cells (human cerebral astrocytoma, Cell Bank of the Chinese Academy of Sciences, Catalog # TCHu138) (2.5 × 10 6 The tumors were grown for 14 days and reached 167.49 mm 3 After reaching maturity, the animals were randomly divided into 5 groups, each with 8 animals (d1). The mice were given intraperitoneal injection once a week for a total of three times. Tumor volume and body weight were measured twice a week and the data were recorded. Data statistics were calculated using Excel 2003 statistical software, with the average value calculated as avg, the SD value calculated as STDEV, the SEM value calculated as STDEV / SQRT, and the P value for intergroup differences calculated by TTEST. Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短2 Relative volume (RTV) = V T / V0 Tumor inhibition rate (%) = (C RTV -T RTV ) / C RTV (%) Among them, V0, V T are the tumor volumes at the start and end of the experiment, respectively. RTV , T RTV are the relative tumor volumes of the blank control group (Vehicle) and the experimental group at the end of the experiment, respectively.

[0479] 4. Test results The experimental results, as shown in Figure 7, were obtained by intraperitoneal injection once a week for a total of three doses. After observation for 18 days, the tumor inhibition rates of the tested ADCs were 0.31% at 1 mg / kg for ADC-30, 45.23% at 3 mg / kg for ADC-30 (P<0.0001), 39.22% at 1 mg / kg for ADC-31 (P<0.01), and 80.24% at 3 mg / kg for ADC-31 (P<0.0001). At comparable doses, the tumor inhibition effect of ADC-31 was significantly better than that of ADC-30.

[0480] 2. Optimization of the preparation process Exemplary products of the following examples have the structures shown in the formulas below: [ka]

[0481] Example 2-1 An antibody stock solution containing 61.71 mg of trastuzumab (0.0004251 mmol; trastuzumab was diluted with 20 mM histidine-HCl buffer to a final antibody concentration of 15 mg / mL) and 0.7311 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.002550 mmol) were reacted in a 20 mM histidine-HCl buffer (pH 5.6) containing 2.5 mM EDTA at 0°C with stirring in a constant-temperature water bath for 3 hours to produce a solution of intermediate I.

[0482] Compound 9 exhibited a relatively short retention time. Compound 9-A (3.196 mg, 0.002975 mmol) was dissolved in 0.2062 mL of DMSO to produce a DMSO solution of compound 9-A. 0.2052 mL of DMSO was added to the above-mentioned intermediate I solution, and the DMSO solution of compound 9-A was then added to the intermediate I solution to which DMSO had been added. The reaction was carried out with stirring in a water bath at 25°C for 1 hour, and the reaction was quenched by adding excess cysteine ​​to obtain product ADC-2-1, an example of the general formula of FADC-4A.

[0483] The mean value was calculated by reversed-phase chromatography-mass spectrometry: n=5.45.

[0484] Example 2-2 An antibody stock solution containing 61.71 mg of trastuzumab (0.0004251 mmol; trastuzumab was diluted with 20 mM histidine-HCl buffer to a final antibody concentration of 15 mg / mL) and 0.5118 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001785 mmol) were reacted in a 20 mM histidine-HCl buffer (pH 5.6) containing 2.5 mM EDTA at 13°C with stirring in a constant-temperature water bath for 3 hours to produce a solution of intermediate I.

[0485] Compound 9 had a relatively short retention time. Compound 9-A (3.196 mg, 0.002975 mmol) was dissolved in 0.2062 mL of DMSO to produce a DMSO solution of compound 9-A. 0.2052 mL of DMSO was added to the above-mentioned intermediate I solution, and the DMSO solution of compound 9-A was then added to the intermediate I solution to which DMSO had been added. The reaction was carried out with stirring in a water bath at 25 °C for 1 hour, and the reaction was quenched by adding excess cysteine. ADC-2-2, an exemplary product of the general formula FADC-4A, was obtained.

[0486] The mean value was calculated by reversed-phase chromatography-mass spectrometry: n=5.49.

[0487] Example 2-3 An antibody stock solution containing 61.71 mg of trastuzumab (0.0004251 mmol; trastuzumab was diluted with 20 mM histidine-HCl buffer to a final antibody concentration of 15 mg / mL) and 0.4021 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001403 mmol) were reacted in a 20 mM histidine-HCl buffer (pH 5.6) containing 2.5 mM EDTA at 25°C with stirring in a constant-temperature water bath for 3 hours to produce a solution of intermediate I.

[0488] Compound 9-A (3.196 mg, 0.002975 mmol) with a relatively short retention time was dissolved in 0.2062 mL of DMSO to produce a DMSO solution of compound 9-A. 0.2052 mL of DMSO was added to the above-mentioned intermediate I solution, and the DMSO solution of compound 9-A was then added to the intermediate I solution to which DMSO had been added. The reaction was carried out with stirring in a water bath at 25 °C for 1 hour, and the reaction was quenched by adding excess cysteine. ADC-2-3, an exemplary product of the general formula FADC-4A, was obtained.

[0489] The mean value was calculated by reversed-phase chromatography-mass spectrometry: n=5.39.

[0490] Examples 2-4 An antibody stock solution containing 61.71 mg of trastuzumab (0.0004251 mmol; trastuzumab was diluted with 20 mM histidine-HCl buffer to a final antibody concentration of 15 mg / mL) and 0.3899 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001360 mmol) were reacted in a 20 mM histidine-HCl buffer (pH 5.6) containing 2.5 mM EDTA at 28°C with stirring for 3 hours in a constant-temperature water bath to produce a solution of intermediate I.

[0491] Compound 9-A (3.196 mg, 0.002975 mmol), which has a relatively short retention time, was dissolved in 0.2062 mL of DMSO to produce a DMSO solution of compound 9-A. 0.2052 mL of DMSO was added to the above-mentioned intermediate I solution, and the DMSO solution of compound 9-A was then added to the intermediate I solution to which DMSO had been added. The reaction was carried out with stirring in a water bath at 25°C for 1 hour, and excess cysteine ​​was added to quench the reaction. ADC-2-4, an exemplary product of the general formula FADC-4A, was obtained.

[0492] The mean value was calculated by reversed-phase chromatography-mass spectrometry: n=5.44.

[0493] Examples 2-5 An antibody stock solution containing 61.71 mg of trastuzumab (0.0004251 mmol; trastuzumab was diluted with 20 mM histidine-HCl buffer to a final antibody concentration of 15 mg / mL) and 0.3778 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001318 mmol) were reacted in a 20 mM histidine-HCl buffer (pH 5.6) containing 2.5 mM EDTA at 37°C with stirring for 3 hours in a constant-temperature water bath to produce a solution of intermediate I.

[0494] Compound 9-A (3.196 mg, 0.002975 mmol) with a relatively short retention time was dissolved in 0.2062 mL of DMSO to produce a DMSO solution of compound 9-A. 0.2052 mL of DMSO was added to the above-mentioned intermediate I solution, and the DMSO solution of compound 9-A was then added to the intermediate I solution to which DMSO had been added. The reaction was carried out with stirring in a water bath at 25 °C for 1 hour, and the reaction was quenched by adding excess cysteine. ADC-2-5, an exemplary product of the general formula FADC-4A, was obtained.

[0495] The mean value was calculated by reversed-phase chromatography-mass spectrometry: n=5.46.

[0496] Examples 2-6 An antibody stock solution containing 55.02 mg of trastuzumab (0.0003790 mmol; trastuzumab was diluted with 50 mM PBS buffer to a final antibody concentration of 15 mg / mL) and 0.4563 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001592 mmol) were reacted in 50 mM PBS buffer (pH 6.5) containing 2.5 mM EDTA at 13°C with stirring for 3 hours in a constant-temperature water bath to produce Intermediate I solution.

[0497] Compound 9-A (2.850 mg, 0.002653 mmol) with a relatively short retention time was dissolved in 0.1839 mL of DMSO to produce a DMSO solution of compound 9-A. 0.1829 mL of DMSO was added to the above-mentioned intermediate I solution, and the DMSO solution of compound 9-A was then added to the intermediate I solution to which DMSO had been added. The reaction was carried out with stirring in a water bath at 25 °C for 1 hour, and the reaction was quenched by adding excess cysteine. ADC-2-6, an exemplary product of the general formula FADC-4A, was obtained.

[0498] The mean value was calculated by reversed-phase chromatography-mass spectrometry: n=5.39.

[0499] Examples 2-7 An antibody stock solution containing 55.02 mg of trastuzumab (0.0003790 mmol; trastuzumab was diluted with 50 mM PBS buffer to a final antibody concentration of 15 mg / mL) and 0.3477 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001213 mmol) were reacted in 50 mM PBS buffer (pH 6.5) containing 2.5 mM EDTA at 25°C with stirring for 3 hours in a constant-temperature water bath to produce Intermediate I solution.

[0500] Compound 9-A (2.850 mg, 0.002653 mmol) with a relatively short retention time was dissolved in 0.1839 mL of DMSO to produce a DMSO solution of compound 9-A. 0.1829 mL of DMSO was added to the above-mentioned intermediate I solution, and the DMSO solution of compound 9-A was then added to the intermediate I solution to which DMSO had been added. The reaction was carried out with stirring in a water bath at 25 °C for 1 hour, and the reaction was quenched by adding excess cysteine. ADC-2-7, an exemplary product of the general formula FADC-4A, was obtained.

[0501] The mean values ​​were calculated by reversed-phase chromatography-mass spectrometry: n=5.50.

[0502] Examples 2-8 An antibody stock solution containing 55.02 mg of trastuzumab (0.0003790 mmol; trastuzumab was diluted with 50 mM PBS buffer to a final antibody concentration of 15 mg / mL) and 0.3259 mg of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 0.001137 mmol) were reacted in 50 mM PBS buffer (pH 6.5) containing 2.5 mM EDTA at 37°C with stirring for 3 hours in a constant-temperature water bath to produce a solution of intermediate I.

[0503] Compound 9-A (2.850 mg, 0.002653 mmol) with a relatively short retention time was dissolved in 0.1839 mL of DMSO to produce a DMSO solution of compound 9-A. 0.1829 mL of DMSO was added to the above-mentioned intermediate I solution, and the DMSO solution of compound 9-A was then added to the intermediate I solution to which DMSO had been added. The reaction was carried out with stirring in a water bath at 25 °C for 1 hour, and the reaction was quenched by adding excess cysteine. ADC-2-8, an exemplary product of the general formula FADC-4A, was obtained.

[0504] The mean value was calculated by reversed-phase chromatography-mass spectrometry: n=5.47.

[0505] Examples 2-9 An antibody stock solution containing 127.4 g of trastuzumab (0.88 mmol; trastuzumab was diluted with 20 mM histidine-HCl buffer to a final antibody concentration of 15 mg / mL) and 0.83 g of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 2.90 mmol) were reacted in a 20 mM histidine-HCl buffer (pH 5.6) containing 2.5 mM EDTA at 25°C with stirring in a constant-temperature water bath for 3 hours to produce an intermediate I solution.

[0506] Compound 9-A (6.6 g, 6.14 mmol) was dissolved in 0.43 L of DMSO to produce a DMSO solution of compound 9-A. 0.43 L of DMSO was added to the intermediate I solution, and the DMSO solution of compound 9-A was added to the intermediate I solution to which DMSO had been added. The reaction was carried out with stirring in a water bath at 25°C for 1 hour, and then the reaction was stopped.

[0507] The reaction mixture was purified using a Capto S Impact (GE) cation chromatography column. It was washed with 9 or more column volumes of 0.05 M acetate buffer (pH 5.5) containing 10% (v / v) DMSO and 6 column volumes of 0.05 M acetate buffer (pH 5.5), respectively, and then eluted with 0.05 M acetate buffer (pH 5.5, containing 0.39 M sodium chloride) to remove free toxins and residual solvents. The cation eluent was replaced with 0.01 M succinate buffer (pH 5.0) by 7-fold equal-volume ultrafiltration (using a 30 kD ultrafiltration membrane package) at 25 °C to obtain ADC-A1, an exemplary product of the general formula FADC-4A. Four lots of samples were prepared using the above method.

[0508] The drug loading of each of the four batches was determined to be 5.7 by reversed-phase chromatography-mass spectrometry. The yields of the four batches were 100.8%, 98.9%, 97.4%, and 99.0%, respectively.

[0509] Example 2-10 At 37°C, a PBS buffer solution of trastuzumab (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 164 mL, 11.08 μmol) was added to an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 3.55 mL, 35.5 μmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3.5 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0510] Compound 9 exhibited a relatively short retention time. Compound 9-A (185 mg, 172 μmol) was dissolved in 3.88 mL of acetonitrile and 1.94 mL of DMSO, added to the reaction mixture, and placed in a water bath shaker for 3 hours at 25 °C. The reaction mixture was then quenched by shaking. The reaction mixture was passed through an ultrafiltration membrane bag and desalted and purified sequentially with PBS buffer (0.05 M PBS buffer, pH 6.5) containing 2% (v / v) acetonitrile and 1% (v / v) DMSO, and then succinic acid buffer (0.01 M succinic acid buffer, pH 5.3) to remove small molecules. A sample of the product ADC-B14, an exemplary product of the general formula FADC-4A, was obtained and stored at 4 °C.

[0511] The mean value was calculated by reversed-phase chromatography-mass spectrometry: n=5.3.

[0512] Test Example 2-1 Drug Load Distribution Test 1. RP-DAR measurement method 1.1. RP-DAR analysis was carried out under the following measurement conditions: UPLC system: Waters H-Class ultra-high performance liquid chromatograph UPLC system Detector: TUV detector (measurement wavelength: 280 nm) Column: Waters ACQUITY UPLC Protein BEH C4 (2.1 mm x 150 mm, 1.7 μm) Column temperature: 80℃ Flow rate: 0.3 mL / min Sample chamber temperature: 20℃ Operating time: 25 min Mobile phase A: 0.1% difluoroacetic acid (DFA) in water Mobile phase B: 0.1% DFA in acetonitrile Gradient program: 27.0%B to 44.0%B (0.00 min to 12.00 min), 44.0%B to 100%B (12.00 min to 13.00 min), 100%B to 100%B (13.00 min to 20.00 min), 100%B to 27.0%B (20.00 min to 20.04 min), 27.0%B to 27.0%B (20.04 min to 25 min) Injection sample volume: 1.0 μL Data Analysis Compared with the light chains (L0) and heavy chains (H0) of antibodies that do not bind to drugs, the hydrophobicity of the light chains that bind to one drug (L1) and the heavy chains that bind to four drugs (H1, H2, H3, and H4) increases in proportion to the number of drug bonds, and the retention time increases. Therefore, the elution order was L0, L1, H0, H1, H2, H3, and H4. Because the drug linker absorbs UV light, the peak area obtained was corrected for the number of drugs attached using the molar extinction coefficients of the light chain, heavy chain, and drug linker according to the following formula: Light chain (εLC-280) / (εLC-280 + number of linked drugs × εdrug-280) Heavy chain (εHC-280) / (εHC-280 + number of linked drugs × εdrug-280) Note: εLC-280: Molar extinction coefficient of the light chain at 280 nm, εHC-28: molar extinction coefficient of the heavy chain at 280 nm, ε drug-280: Molar extinction coefficient of the toxin at 280 nm.

[0513] [Table 6]

[0514] 2.Measurement results [Table 7]

[0515] As shown by the results, for samples prepared using the same buffer system but different reduction reaction temperatures, the uniformity of the drug loading distribution in the samples increased significantly as the reduction reaction temperature decreased. For samples prepared using different buffer systems but the same reduction reaction temperature, the drug loading distribution in the samples prepared using the histidine-HCl buffer system was more uniform.

[0516] Test Example 2-2: Free toxin test 1.Method for measuring free toxins 1.1. HPLC analysis was carried out under the following conditions: HPLC system: Waters H-Class ultra-high performance liquid chromatograph UPLC system Detector: TUV detector (measurement wavelength: 370 nm) Column: Waters ACQUITY UPLC Petide BEH C18 (130 Å, 2.1 mm x 150 mm, 1.7 μm) Column temperature: 40℃ Flow rate: 0.3 mL / min Sample chamber temperature: 10℃ Mobile phase A: 0.1% trifluoroacetic acid (TFA) in water Mobile phase B: 0.1% TFA in acetonitrile Gradient program: 25.0%B to 25.0%B (0.00 min to 1.00 min), 25.0%B to 55.0%B (1.00 min to 17.00 min), 55.0%B to 25.0%B (17.00 min to 17.10 min), 25.0%B to 25.0%B (17.10 min to 20.00 min) Injection sample volume: 5.0 μL Data Analysis The formula for calculating the LOD limit for a toxin is as follows: Toxin limit (ppm)=0.1×4×1000 / C Note: a) 0.1 is the toxin LOD solution concentration (μg / mL), 4 is the dilution factor during sample pretreatment, 1000 is the unit conversion coefficient, and C is the protein concentration of the measurement sample (mg / mL). b) If the peak area of ​​the free toxin in the sample is smaller than the peak area of ​​the LOD solution, it is judged to be below the limit or undetected.

[0517] 2.Measurement results The results of detecting free toxins for ADC-A1 (lots 1 to 4) and ADC-B14 (see Table 7) showed that the use of cation column chromatography is not only applicable to large-scale preparation, but also significantly reduces free toxins.

[0518] [Table 8] Furthermore, the present invention includes the following aspects. [Aspect 1] A method for preparing an antibody-drug conjugate, the structure of which is represented by the general formula (Pc-L a -YD), that is, [ka] is shown by Among them, W is C 1-8 Alkyl group, C 1-8 Alkyl-C 3-7 A cycloalkyl group or a linear heteroalkyl group of 1 to 8 atoms, said linear heteroalkyl group containing 1 to 3 heteroatoms selected from N, O and S, among which the C 1-8 Alkyl group, C 3-7 The cycloalkyl group and the straight chain heteroalkyl group may each independently optionally further comprise a halogen, a hydroxy group, a cyano group, an amino group, a C 1-6 Alkyl group, chloro C 1-6 Alkyl groups, deuterated C 1-6 Alkyl group, C 1-6 Alkoxy groups and C 3-7 substituted with one or more substituents selected from cycloalkyl groups; L 2 Ha-NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 )p 1 C(O)- and chemical bonds, among which p 1 is an integer between 1 and 20, L 3 is a peptide residue consisting of 2 to 7 amino acid residues, among which the amino acid residues are amino acid residues formed from amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and optionally further containing halogen, hydroxy group, cyano group, amino group, C 1-6 Alkyl group, chloro C 1-6 Alkyl groups, deuterated C 1-6 Alkyl group, C 1-6 Alkoxy groups and C 3-7 substituted with one or more substituents selected from cycloalkyl groups; R 1 is halogenated C 1-6 Alkyl group or C 3-7 is a cycloalkyl group, R 2 is a hydrogen atom, halogenated C 1-6 Alkyl groups and C 3-7 cycloalkyl groups, Or, R 1 and R 2 C together with the carbon atoms connected to them 3-7 forming a cycloalkyl group, R 5 is a hydrogen atom, C 1-6 Alkyl groups, halogenated C 1-6 Alkyl groups, deuterated C 1-6 Alkyl and hydroxy C 1-6 selected from alkyl groups, R 6 and R 7 are the same or different and each independently represent a hydrogen atom, a halogen, or C 1-6 Alkyl groups, halogenated C 1-6 Alkyl groups, deuterated C 1-6 Alkyl and hydroxy C 1-6 selected from alkyl groups, m is 0 or 1; n is 3 to 8, and n is a decimal or an integer; Pc is an antibody or an antigen-binding fragment thereof; The preparation method comprises: Step (a) of reacting the antibody or antigen-binding fragment thereof with a reducing agent at a reaction temperature of about 1°C to about 36°C; The product of step (a) and the compound of formula (La-YD) below, i.e.

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Claims

1. A method for preparing an antibody-drug conjugate, the structure of which is represented by the general formula (Pc-L ａ -Y-D), that is, 【Chemical 1】 is shown by Among them, W is C １－８ Alkylene group, C １－８ Alkylene-C ３－７ a cycloalkylene group or a linear heteroalkylene group of 1 to 8 atoms, said linear heteroalkylene group containing 1 to 3 heteroatoms selected from N, O and S, among which the C １－８ Alkylene group, C ３－７ The cycloalkylene group and the straight-chain heteroalkylene group may each independently optionally further comprise a halogen, a hydroxy group, a cyano group, an amino group, a C １－６ Alkyl group, chloro C １－６ Alkyl groups, deuterated C １－６ Alkyl group, C １－６ Alkoxy group and C ３－７ substituted with one or more substituents selected from cycloalkyl groups; L ２ is a chemical bond, L ３ is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are amino acid residues formed from amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and optionally further containing halogen, hydroxy group, cyano group, amino group, C １－６ Alkyl group, chloro C １－６ Alkyl groups, deuterated C １－６ Alkyl group, C １－６ Alkoxy group and C ３－７ substituted with one or more substituents selected from cycloalkyl groups; R １ is halogenated C １－６ Alkyl group or C ３－７ is a cycloalkyl group, R ２ is a hydrogen atom, halogenated C １－６ Alkyl group and C ３－７ cycloalkyl groups, Or, R １ and R ２ are C together with the carbon atoms connected to them. ３－７ forming a cycloalkylene group, R ５ is a hydrogen atom, C １－６ Alkyl group, halogenated C １－６ Alkyl groups, deuterated C １－６ Alkyl group and hydroxy group １－６ selected from alkyl groups, R ６ and R ７ are the same or different and each independently represent a hydrogen atom, a halogen, C １－６ Alkyl group, halogenated C １－６ Alkyl groups, deuterated C １－６ Alkyl group and hydroxy group １－６ selected from alkyl groups, m is 0 or 1; n is 3 to 8, and n is a decimal or an integer; Pc is an antibody or an antigen-binding fragment thereof; The preparation method comprises: (a) reacting an antibody or an antigen-binding fragment thereof with a reducing agent at a reaction temperature of 1°C to 36°C; The product of step (a) and a compound of the formula (La-Y-D): 【Chemistry 2】 and (b) reacting a compound represented by the formula: Among them, W and L ２ , L ３ , R １ , R ２ , R ５ , R ６ , R ７ and m is as defined above. Methods for preparing antibody-drug conjugates.

2. 2. The method for preparing an antibody-drug conjugate according to claim 1, wherein the reaction temperature condition in step (a) is 4°C to 30°C.

3. 2. The method for preparing an antibody-drug conjugate according to claim 1, wherein the reaction temperature condition in step (a) is 20°C to 30°C.

4. The method for preparing an antibody-drug conjugate according to any one of claims 1 to 3, wherein the reaction in step (a) is carried out under conditions of pH 4.5 to 6.

5.

5. The method for preparing an antibody-drug conjugate according to any one of claims 1 to 4, wherein the reaction in step (a) is carried out under conditions of pH 5.0 to 6.

0.

6. 6. The method for preparing an antibody-drug conjugate according to claim 1, wherein the reaction in step (a) is carried out in a buffer selected from the group consisting of a histidine salt buffer, a phosphate buffer, and an acetate buffer.

7. 7. The method for preparing an antibody-drug conjugate according to claim 1, wherein the reducing agent in step (a) is selected from tris(2-carboxyethyl)phosphine or a salt thereof, 1,4-dithiothreitol, and β-mercaptoethanol.

8. The method for preparing an antibody-drug conjugate according to any one of claims 1 to 7, further comprising step (c) of purifying the product of step (b) by cation column chromatography or affinity column chromatography.

9. 9. The method for preparing an antibody-drug conjugate according to claim 8, wherein step (c) comprises subjecting the product of step (b) to cation chromatography, and the cation chromatography packing material is selected from Capto S Impact and Poros XS.

10. The antibody is trastuzumab. A method for preparing the antibody-drug conjugate of claim 1.

11. The antibody-drug conjugate has the following formula: 【Chemistry 3】 and having a structure represented by wherein n is 4 to 8, and n is a decimal or an integer; A method for preparing the antibody-drug conjugate according to any one of claims 1 to 10.

12. The method for preparing an antibody-drug conjugate according to any one of claims 1 to 11, wherein n is a decimal or integer of 5 to 7.

13. 2. The method for preparing an antibody-drug conjugate according to claim 1, wherein the antibody-drug conjugate has the following formula: 【Chemistry 4】 and having a structure represented by wherein n is 4 to 8, and n is a decimal or an integer; The preparation method comprises: Step (a) of reacting trastuzumab with tris(2-carboxyethyl)phosphine hydrochloride at a reaction temperature of 4°C to 30°C and a pH of 5.0 to 6.0; the product of step (a) and a compound of the formula: 【Chemistry 5】 and (b) reacting a compound represented by the formula: Methods for preparing antibody-drug conjugates.

14. 2. The method for preparing an antibody-drug conjugate according to claim 1, wherein the antibody-drug conjugate has the following formula: 【Chemistry 6】 and having a structure represented by wherein n is 4 to 8, and n is a decimal or an integer; The preparation method comprises: Step (a) reacting trastuzumab with tris(2-carboxyethyl)phosphine hydrochloride at a reaction temperature of 25°C and a pH of 5.6, wherein the reaction is carried out in a histidine-hydrochloride buffer containing EDTA; the product of step (a) and a compound of the formula: 【Chemistry 7】 and step (b) reacting a compound represented by the formula: and step (c) comprising purifying the product of step (b) by a cation chromatography column. Methods for preparing antibody-drug conjugates.

15. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has the following formula: 【Chemistry 8】 and having a structure represented by wherein n is 4 to 8, and n is a decimal or an integer; the antibody-drug conjugate has a drug loading distribution in which, in a group of antibody heavy chains, the proportion of antibody heavy chains that bind to four drugs is 4% or less, and the proportion of antibody heavy chains that do not bind to any drug is 5% or less; An antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

16. 16. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 15, wherein the drug loading distribution of the antibody-drug conjugate is such that, in the antibody heavy chain group, the proportion of antibody heavy chains that bind to four drugs is 4% or less and the proportion of antibody heavy chains that do not bind to any drugs is 5% or less, and, in the antibody light chain group, the proportion of antibody light chains that bind to one drug is 65% or more.

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